Effect of Psidium cattleianum leaf extract on enamel demineralisation and dental biofilm composition in situ

Effect

of

Psidium

cattleianum

leaf

extract

on

enamel

demineralisation

and

dental

biofilm

composition

in

situ

Fernanda

Lourenc¸a˜

o

Brighenti,

Elerson

Gaetti-Jardim

Jr.,

Marcelle

Danelon,

Gustavo

Vaz

Evangelista,

Alberto

Carlos

Botazzo

Delbem

*

Arac¸atubaDentalSchool,UNESP–UnivEstadualPaulista,R:Jose´ Bonifa´cio1193,CEP:16015-050,Arac¸atuba-SP,Brazil

1.

Introduction

Dental caries is one of the most common chronic and infectious diseases worldwide.1 _{This disease results from}

theinteractionofspecificbacteriawithconstituentsofadiet inasusceptiblehost.2_{Itisassociatedwiththepresence of}

biofilms, which are microbial communities embedded in a

polymericmatrixattachedtothesurfaceofthetooth.1,2_The

disease appears as a result of a breakdown of biofilm homeostasis, which is related to frequent sugar exposure andalowbiofilmpH.1_{Naturalproductshavebeenstudiedfor}

theirabilitytochemicallycontroldentalbiofilms.Kooetal.3

show that a mouthwash with propolis is able to reduce supragingivalplaqueformationandinsolublepolysaccharide formation. Smullen et al.4 _{demonstrate the antibacterial}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Accepted6February2012

Keywords:

Antibacterialagents Dentalcaries Plantextract

a

b

s

t

r

a

c

t

Objective: PreviousevaluationsofPsidiumcattleianumleafextractwerenotdonein condi-tionssimilartotheoralenvironment.Theaimofthisstudywastoevaluatetheeffectof P.cattleianumleafextractonenameldemineralisation,extracellularpolysaccharide forma-tion,andthemicrobialcompositionofdentalbiofilmsformedinsitu.

Design: Tenvolunteers took partin thiscrossover study.Theywore palatalappliances containing4enamelblocksfor14days.Eachvolunteerdripped20%sucrose8timesperday on the enamel blocks. Twice a day, deionised water (negative control), extract, or a commercial mouthwash (activecontrol) wasdripped after sucroseapplication. Onthe 12thand13thdaysoftheexperiment,plaqueacidogenicitywasmeasuredwitha micro-electrode,andthepHdropwascalculated.Onthe14thday,biofilmswereharvestedand totalanaerobicmicroorganisms(TM),totalstreptococci(TS),mutansstreptococci(MS),and extracellularpolysaccharides(EPS)wereevaluated.Enameldemineralisationwasevaluated by the percentage change of surface microhardness (%DSMH) and integrated loss of subsurfacehardness(DKHN).Theresearcherwasblindedtothetreatmentsduringdata collection.

Results: TheextractgroupshowedlowerTM,TS,MS,EPS,%DSMH,andDKHNvaluesthan thenegativecontrolgroup.Therewerenodifferencesbetweentheactiveandnegative controlgroupsregardingMSandEPSlevels.TherewerenodifferencesinpHdropbetween theextractandactivecontrolgroups,althoughtheyweresignificantlydifferentfromthe negativecontrolgroup.Forallotherparameters,theextractdifferedfromtheactivecontrol group.

Conclusion: Psidiumcattleianumleafextractexhibitsapotentialanticariogeniceffect. #2012ElsevierLtd.Allrightsreserved.

*Correspondingauthor.Tel.:+551836363235;fax:+551836363332. E-mailaddress:adelbem@foa.unesp.br(A.C.B.Delbem).

Available

online

at

www.sciencedirect.com

journalhomepage:http://www.elsevier.com/locate/aob

effectsofawidevarietyofextracts,includingteas,redand greengrapeextracts, andcocoa onStreptococcus mutans.To date,moststudiesofnaturalproductsagainstoralpathogens attempttoverifytheir antibacterialpropertiesand mecha-nismsofactioninvitro.4–7

Once the therapeutic properties ofa medicinal plant is empiricallyknown,theWorld HealthOrganizationinitiates the pharmacological evaluation of phytotherapeutic drugs fromsuchsources.8_{Despitecurrentinterestfortheirusein}

medicine,less than 15% of medicinal plants from tropical areasthathavethepotentialfortherapeuticusehavebeen studied.9

Psidium cattleianum is commonly known as strawberry guava. Itbelongs tothe family Myrtaceaeand is nativeto tropicalAmerica.PlantsofPsidiumspecies(Psidiumspp.)are traditionallyusedtotreatseveraldiseasesworldwide,10_and

the antibacterial effects of these plants have been tested againstbacteriathatcausediarrhoeaoropportunistic infec-tions.11,12_{Previousstudiesshowthattheleafextractsofplants}

fromthisfamily,suchasP.guajava,arealsoabletoreduce gingivitis and halitosis.13,14 _{Brighenti et al.}15 _previously

demonstratedtheefficacyofP.cattleianumleafextractagainst S. mutans biofilms and elucidated its action mechanism. However,this extracthasnotbeen evaluatedinconditions similar to the oral environment. The evaluation of dental productsusingin situcariesmodels helpspresumeclinical outcomes and allows the evaluation of many features of dentalbiofilmandhardtissues.

The aim of this study was to evaluate the effect of P. cattleianumleafextractonenameldemineralisationandassess itseffectonthebiochemicalandbiologicalcompositionoforal biofilmusinganinsitudentalcariesmodel.

2.

Materials

and

methods

2.1. Extractpreparation

TheharvestingofP.cattleianumleaveswasauthorisedbythe Brazilian Environment and Natural Resources Institute (IBAMA,permit #0129208). Avoucherherbariumspecimen was depositedat theCollection ofAromaticand Medicinal Plants(CPMA:Colec¸a˜odePlantasMedicinaiseAroma´ticas)atthe UniversityofCampinas(UNICAMP)(voucher#1299).

Onlyhealthyleaves(i.e.,those withoutsignsofnecrosis andwithtypicalcolourandshape)wereselectedforextract preparation.Theleavesweredriedat378Cfor1weekafter beingwashedintap anddeionisedwater.Theleaveswere thengroundinablenderuntilathinpowderwasachieved. Aqueousextractwasobtainedbydecoctionindeionisedwater (100g/600mL)for5minat1008Candat558Cforanadditional 1h.Thesolutionwasthenfiltersterilisedwith0.22mmmixed celluloseestermembranes(MilliporeTM_{;Billerica,MA,USA).} Theextractwasstoredindarkbottlesat 208Cuntilfurther use.15

2.2. Enamelblockpreparationandanalysis

Enamelblocksmeasuring4mm4mmwereobtainedfrom bovine incisor teeth previously storedin 2% formaldehyde

solution(pH7.0)for1month16_{andhadtheirsurfacesserially}

polished.Measurementsofsurfacemicrohardness(SMH)and cross-sectionalenamelmicrohardness(kgmm 2_)weremade usingaShimadzuHMV-2000microhardnesstester(Shimadzu Corp.,Kyoto,Japan).ForbaselineSMH(SMH1),5indentations spaced100mmapartweremadewitha25-gloadfor10sinthe centreoftheenamelblockasdescribedbyVieiraetal.17

Aftertheexperimentalphase,SMHwasmeasuredagain (SMH2).Fiveindentationsspaced100mmapartandfromthe baselineweremade.ThepercentagechangeofSMH(%DSMH) wascalculatedasfollows:%DSMH=100(SMH2 SMH1)/SMH1. Toperformcross-sectionalmicrohardnesstests,theblocks werelongitudinallysectionedthroughthecentre.Oneofthe halves was embedded in acrylic resin with the cut face exposedandgraduallypolished.

Three rowsof9indentationsspaced100mmapartwere made10,30,50,70,90,110,170,220,and330mmfromtheouter enamelsurfaceundera25-gloadfor10s.Themeanvaluesat all3measuringpointsateachdistancefromthesurfacewere thenaveraged.

The integrated hardness (Knoop hardness number; KHNmm)ofthelesioninto soundenamelwascalculated andsubtractedfromtheintegratedhardnessofsoundenamel toobtaintheintegratedlossofsubsurfacehardness(DKHN).18

DKHNdenotestheenamelsubsurfacedemineralisationarea aftertheinsituexperiment.

2.3. Experimentaldesign

This studywas previously approved bythe human ethical committee from Arac¸atuba Dental School (protocol #2005-02188).Tenhealthyvolunteersaged23–34yearswereselected. Thevolunteersusednonfluoridateddentifrice1weekbefore andthroughouttheexperiment.19,20

The crossover in situ experiment was composed of 3 phasesof14dayseachaccordingtothetreatmentsolution: deionised water (negative control), extract, or Original ListerineAntisepticTM_{(Johnson&Johnson,NewBrunswick,} NJ,USA;activecontrol).Thestudywasnotblindduringthe in situ phase because of the physical differences of the solutionsused(i.e.,colour,smell,andtaste).However,itwas possibletomakethestudyblindduringtheanalysisofthe crossover study. The enamel blocks were coded after the experimental phase, and the researcher who carried outtheanalysiswasblindedtowhichenamelblockbelonged towhichgroup.

The volunteers wore palatal appliances containing 4 enamel bovine blocks covered by plastic mesh to enable biofilmaccumulationandprotectitfromdisturbances.21_One

millimetrewasleftbetweentheenamelandtheplasticmesh toallowbiofilmgrowth.

Enamelblockswererandomisedaccordingtothemeanof theSMHfromallblocksanditsconfidenceintervals.Microsoft Excel1_{2003was usedtocalculate theconfidenceintervals.}

Thelevel ofsignificancewas setat p<0.05 forthis

Thevolunteersremovedthepalatalappliancefromtheir oralcavityandplacedonedropof20%sucroseineachenamel block 8 times a day. The volunteers waited 5min before returningtheappliancetotheiroralcavity.22_Duringthe4th

and 8th sucrose applications, deionised water, extract, or Original Listerine AntisepticTM _{was dripped 1min after} sucrose application twice a day and the appliance was replacedintheoralcavity4minlater.Awashoutperiodof 7dayswasallowedbetweeneachphase.21,23_{Thevolunteers}

receivedoralandwritteninstructiontoweartheapplianceall thetimeandremoveit duringmeals ororalhygiene.They werenotallowedtouseanyantimicrobialorfluorideproducts duringtheexperiment.

2.4. Assessmentofbiofilmacidogenicity

pH was measured according to Pecharki et al.,22 _{using a}

palladiummicroelectrode (BeetrodeTM_NMPH3;World Preci-sion Instruments Inc., Sarasota, FL, USA) with biofilms at overnight fasting toassure thatno bacterialcarbohydrates were stored24 _{and to evaluate the residual effect of the}

treatments.Asaltbridgebetweenthereferenceelectrodeand thevolunteer’sfingerwascreatedwith3molL 1_KCl.25

TwoconditionswereevaluatedduringpHmeasurements. First, on the 12th day,only sucrosewas applied (‘‘sucrose alone’’ measurement). The pH was measured 5min after sucrose applicationwith the appliances placed in the oral cavity.Thiswasdonetoevaluatethepossibleresidualeffectof the treatment solutions and detect whether pH variations wereduetoastructuralchangeinthebiofilminducedbythe treatmentsolutions. Second,on the 13thday,sucrosewas applied and the treatmentsolutions weredripped (‘‘sucro-se+treatment’’ measurement) after 1min. Four minutes later, the appliances were replaced in the mouth and pH was measured. This was done to evaluate the immediate effectofthetreatmentsolutions.

pH was measured twice: before dripping any solutions (baselinemeasurement)(pH1)andafterdrippingsucrosealone (atthe12thday)orsucrose+treatmentsolution(atthe13th day) (pH2). Next, pH variation was calculated as follows:

DpH=pH1 pH2.

2.5. Analysisofdentalbiofilmcomposition

Attheendoftheexperiment,theplasticmeshwasremoved and biofilms were harvested and weighed. Around 5mg biofilmwasdiluted inphosphate-bufferedsaline(PBS:8.0g NaCl, 0.2g KCl, 1.0g Na2HPO4, and 0.2g KH2PO4 per litre, adjustedtopH7.4;1mLmg 1_{biofilm)andsonicatedonicein} anultrasoniccelldisruptor(XL;MisonixInc.,Farmingdale,NY, USA)for69.9swithanamplitudeof40W.26The suspen-sionsweredilutedinPBSandplatedinduplicateinbrainheart infusionagar,Mitissalivariusagar,orMitissalivariussucrose bacitracinagartoanalyse totalanaerobic microorganisms (TM),totalstreptococci(TS),andmutansstreptococci(MS), respectively.27_{ThepresenceofTSandMSwasconfirmedby}

Gram staining. Colony-forming units (CFU) were counted after incubation for 72h at 378C in an anaerobic jar (AnaerojarTM_+AnaerogenTM_{; Oxoid, Cambridge, UK). The} resultsareexpressedinlogCFUmg 1_wetweight.

Theremainingbiofilmwasdriedwithphosphorus pentox-ide(VetecQuı´micaFinaLtda.,DuquedeCaxias,RJ,Brazil).22

Extracellularpolysaccharides(EPS)wereextractedbyadding 1.0molL 1_NaOH(10mLmg 1_{dryweight)tothebiofilm.The} sampleswerevortexedfor1min;after3hunderagitationat room temperature, they were centrifuged for 1min at 11,000gatroomtemperature.28Supernatantswere precipi-tated overnight with 75% cooled ethanol, centrifuged, and resuspended in 1.0molL 1 _NaOH.29 _{Carbohydrate analysis}

wasperformedusingthephenol–sulphuricacidprocedure.30

Theresultsareexpressedasmgmg 1_dryweight.

2.6. Statisticalanalysis

Our hypothesis is that treatment with P. cattleianum leaf extractdecreasesmicrobialviabilityinbiofilmsandenamel demineralisation.Themeansoftheresultsfromthe4enamel blocksofeachparticipantforeachexperimentalphasewere taken. Statistical analysis was carried out using GraphPad PrismVersion3.02(GraphPadSoftwareInc.,SanDiego, CA, USA).Datafor%DSMH,DKHN,EPS,DpHandmicroorganism counts(i.e.,TM,TS,andMS)exhibitedequalityofvariances (Bartlett’stest)andnormaldistribution(Kolmogorov–Smirnov test); theyweresubmittedtoanalysisofvariance(ANOVA) followedbyTukey’smultiplecomparison test.Data ofDpH betweenthe12thand13thdayswereanalysedbytwo-tailed pairedt-test.Thesignificancelimitwassetat5%.Correlations betweentheanalysedparametersweredetermined.

3.

Results

P. cattleianum leaf extract had an effect on microorganism counts(Fig.1).Thereweresignificantlylessviablebacteriain the biofilms after treatment with the extract than after treatmentwithwateroractivecontrolgroups(p<0.05)for

TM, TS and MS. There were no statistically significant differenceswithrespecttoMSviabilityaftertreatmentwith waterortheactivecontrol(p>0.05).Statisticallysignificant

differences were found between water and active control groupsforTMandTS.TMwasmoresusceptiblethanTSorMS, asshowedbythedifferenceinlogreductionobserved(6,1and 1for,respectivelyTM,TSandMS).

P.cattleianumleafextractandtheactivecontrol significant-lyreduced%DSMHandDKHNcomparedtothewatergroup.In addition, theextractexhibitedbetterperformance thanthe activecontrol(p<0.05)(Fig.2).

Comparedtowater,DpHwassignificantlylowerwiththe extract and active control in both sucrose alone and sucrose+treatmentsolutions;however,therewereno signifi-cantdifferencesinpHdropinthese2conditions(p>0.05)

(Fig.3).Theuseoftheextractalsosignificantlydecreasedthe amount ofEPScomparedtotheactiveor negativecontrols (p<0.05). No significant differences in EPS were observed

aftertreatmentwithwaterortheactivecontrol(p>0.05).

4.

Discussion

The present study evaluated the anticaries effect of P. cattleianum leaf extract. The compounds present in P. cattleianum with antibacterial activity are all phenolic compounds: 3 are flavonoids (kaempferol, quercetin, and cyanidin)and1isatannin(ellagicacid).31_{Thestudiedextract}

doesnotcontainconsiderableamountsoffluoride,calcium,or phosphate(datanotshownbecausethelevelswerebelowthe

detectionlimit).Theaimofusingfluoride-freedentifricewas nottooverestimate theefficacyofthetestedextractbutto evaluatethepotentialeffectoftheextractalone.Thisallowed abetterscientificcontrolofthestudyandisconcordantwith theliterature.32,33_{Furtherstudiesareneededtoevaluatethe}

antibacterial effect of the extract in compared to fluoride products.

Althoughthisisthefirsttimeanantibacterialsubstance hasbeentestedusinganinsitucariesmodel,thismodelhas beenextensivelystudiedtoevaluatethemicrobiologicaland Control

Extract Water

-100 -75 -50 -25 0

%

Δ

SMH

*

+

Extract

Water Control

0 10000 20000 30000 40000

*

Δ

KH

N (

K

HN

x

μ

m)

+

Fig.2–Effectofdifferenttreatmentgroupsonpercentagechangeofsurfacemicrohardness(%DSMH)andintegratedlossof

subsurfacehardness(DKHN)(meanWSE,n=10).Distinctsymbolsshowstatisticaldifferencebetweengroups(ANOVA,

p<0.05).

Control Extract Water

0 50 100 150

*

Treatments

µg

⋅

mg

-1 dry weight

Sucrose alone Sucrose + treatment 0.0

0.5 1.0 1.5 2.0

Water

Extract Control *

*

Δ

pH

* *

Fig.3–Effectofdifferenttreatmentsolutionsonalkali-solublecarbohydrates(mgmgS1_{dryweight)andpHvariation(DpH)}

(meanWSE,n=10).Distinctsymbolsshowstatisticaldifferencebetweentreatmentgroups(ANOVA,p<0.05)andbetween

differencesonthepHmeasurements(sucrosealoneorsucrose+treatment)(unpairedt-test,p<0.05).‘‘Sucrosealone’’

standsforDpHafterdrippingonlysucrose,withoutanytreatment.‘‘Sucrose+treatment’’representsDpHafterdripping

sucroseandthetreatmentsolutions.

Total microorganism

Control Extract Water 1.0×1000

1.0×1005

1.0×1010

1.0×1015

*

Treatments

log CFU

. mg

-1 wet weight

+

Total streptococci

Control Extract Water 1.0×1000

1.0×1004

1.0×1008

*

Treatments

log CFU/mg wet weight

+

Mutans streptococci

Control Extract Water

1.0×1000

1.0×1003

1.0×1006

1.0×1009

Treatments

LOG CFU/mg wet weight

*

Fig.1–Viabilityoftotalanaerobicmicroorganism,totalstreptococciandmutansstreptococciafterexposuretodifferent

biochemicalcompositionofbiofilms34–36_{;microbialvirulence}

factors37,38_{; the cariogenicity of foods, sugars, and other}

substances39–41_{; andthe protectiveeffectsofmilkproducts}

andchewinggum.42–44_{Thesestudiessuggestthatthismodel}

issuitableforevaluatingantibacterialsubstances.Moreover, this in situ model was chosen because it promotesa high cariogenicchallenge,leadingtoenameldemineralisation;this wasveryusefulfortestingtheextractafterthepreviousinvitro study15,45_{highlightedthetendencyoftheextracttodecrease}

acidproduction.

A previous study by our research group shows that P. cattleianumleafextractcaninhibittheexpressionofproteins involvedingeneralmetabolism,especiallythoseinvolvedin the carbohydratemetabolism ofS. mutans biofilms.15 _More

recently,Menezesetal.45_{confirmedtheanticariogenicaction}

ofthe extract using acaries ratmodel. Thepresent study representsanintermediatestagebetweeninvitroandinvivo studies.Insituexperimentsallowtheexplorationoflaboratory findingsinasituationthatmimicsthedevelopmentofnatural carieswhilstusingsensitivedetectionmethods.46

ThelowtoxicityofPsidiumspp.extractisdescribedinthe literature. TheLD50 of P.guajava leafextract is morethan 5gkg 1_.47_{Teixeiraetal.}48_{demonstratethatP.guajavainfusion}

doesnotalterchromosomesorthecellcyclebothinvitroand invivo.Costaetal.49_{showthatP.cattleianumleafextractdoes}

nothave any genotoxic ormutagenic effects on somecell typesofmice,suggestingthatthisextractcanbeusedasasafe therapeuticagent.

pHexpressestheacidityoralkalinityofasolutionandis widelyusedtoevaluatetheacidogenicityofbiofilms.22,36_The

lowerDpHvaluesandlowerenameldemineralisationfoundin thisinsituexperimentareconcordantwithpreviousinvitro andinvivoobservations.Brighentietal.15_{foundadecreasein}

S. mutans proteins from carbohydrate metabolism and a reductioninpHdropafterexposuretotheextract.Menezes etal.45_{observedthattheextractisabletoreduceS.mutans}

viabilityandenameldemineralisationinratssubmittedtoa cariogenicchallenge.

On the other hand, the decrease in enamel deminer-alisation observed in the extract group may possibly be attributed to membrane-associated proteins such as glycosyltransferases (GTF) in addition tothe inhibition of theseproteins.5_{Thus,thedecreaseinextracellular}

polysac-charideformation observedafter sucroseapplication may also be relatedto the inhibitionof GTF activity in dental biofilms as was demonstrated by Al-Hebshi et al.50 _after

testingatannin-containingextract.However, theeffectof P. cattleianum leaf extract on GTF activity should be evaluatedinfurtherdetail.

Besidesthereductioninbacterialviability,thecontrolof biofilmmatrixformationfoundaftertheuseoftheextract may also control the pathogenicity of dental biofilm by alteringitsthicknessandporosity.21,51_{Inthepresentstudy,}

thepositivecorrelationsfoundbetweenEPSandbothTMand TS, which are associated with the reduction on enamel demineralisationfoundwiththeextractandactivecontrol, demonstrate the importance of controlling not only the presenceofmicroorganismsbutalsopolysaccharide accu-mulation, which may be an important approach for the prevention of biofilm-associated diseases. The lack of

correlationfoundbetweenMSandtheanalysedparameters iscorroboratedbytheliterature.33,52

ThenumbersofTM,TS,andMSweresignificantlyreduced aftertheuseoftheextractcomparedtotheactiveornegative controls.However,theextract’seffectonbiofilmecologyshould beevaluatedingreaterdetailusingmolecularmethods.The reductioninTMalsoindicatesthatbeneficialspeciesmayalso bereduced.Thissuggeststhattheextractshouldbeusedwith caution and should not be incorporated into oral hygiene productsonadailybasis.Theeffectoftheextractonbeneficial speciesshouldbeevaluatedinfurtherdetail.

AhigherproportionofMSinbiofilmwasfoundinthegroup treatedwiththeactivecontrolfollowedbythegrouptreated withtheextractcomparedwiththewater-treatedgroup. Al-Hebshi et al.53 _{also found similar selective antimicrobial}

propertiesaftertestingatannin-containingplantextract.On theotherhand,boththeextract-andactivecontrol-treated biofilmsshowedlowerEPSandlowerenamel demineralisa-tionthanthewatergroup,suggestingthatthesesolutionsare abletoreducethemetabolismofMS;thisisalsoevidencedby thelackofcorrelationbetweenEPSandMS,whichmeansthat an increase in MS viability does not necessarily mean an increaseinEPSformation.Theextractalsoreducedbiofilm acidogenicitycomparedtotheactivecontrol.Al-Hebshietal.50

previouslydemonstratedaninhibitoryeffectoncell metabo-lism beforecellviabilityisaffectedinwhichGTFactivityis inhibited with low concentrations of a tannin-containing extractwhilst biofilmformationisminimallyinfluencedby suchconcentrations.

The active control was composed of a combination of purifiedessentialoilsandhastheAmericanDentalAssociation sealofapproval.54_{Itcontainscleaningagents,surfactants,and}

preservatives that synergistically inhibit microorganism growth.Inthepresentstudy,theextractexhibitedeitherthe same(i.e.,DpH)orbettereffects(i.e.,TM,TS,MS,%DSMH,DKHN, andEPS)comparedtotheactivecontrol.Thefactthattheextract usedinthepresentstudyisacrudeconcentrateobtainedby decoctionoftheleavesmakesthefindingsevenstronger.This maybeduetosynergisticeffectsbetweendifferentcompounds foundintheplantextractasdescribedpreviously.55,56

Inconclusion,theresultsofthepresentstudyshowthat P.cattleianumleafextractmightreduceenamel demineralisa-tion, acidogenic potential, microorganism viability, and extracellular polysaccharide production. However, it is im-portanttonotethatsincethisinsitustudywasdonewithonly 10 volunteers, the resultsshould be interpretedwith care. Having clearly demonstrated the potential activity of P. cattleianum leaf extract to interfere in in situ biofilm pathogenicity, the effect of this plant extract on biofilm formationinvivoshouldbeevaluatedinthefuture.

Acknowledgements

Funding:ThisstudywassupportedbyCAPES(DSandPDEE –4446/05),CNPq(471634/2007-7)andFAPESP(06/00726-8).

Competinginterests:Therearenoconflictofinterestsin thecurrentmanuscript.

Ethicalapproval:Thisstudywas previouslyapprovedby theHumanEthicalCommittee(protocol#2005-02188)andisin accordancewiththeprincipleslaiddownintheDeclarationof Helsinki;Recommendationsguidingphysiciansinbiomedical researchinvolvinghumansubjects.

r

e

f

e

r

e

n

c

e

s

1. MarshPD.Aredentaldiseasesexamplesofecological catastrophes?Microbiology2003;149:279–94.

2. KooH,JeonJG.Naturallyoccurringmoleculesasalternative therapeuticagentsagainstcariogenicbiofilms.AdvDentRes 2009;21_:63–8.

3. KooH,CuryJA,RosalenPL,AmbrosanoGMB.Effectofa mouthrinsecontainingselectedpropolison3-daydental plaqueaccumulationandpolysaccharideformation.Caries Res2002;36_:445–8.

4. SmullenJ,KoutsouGA,FosterHA,Zumbe´ A,StoreyDM.The antibacterialactivityofplantextractscontaining

polyphenolsagainstStreptococcusmutans.CariesRes 2007;41_:342–9.

5. DuarteS,GregoireS,SinghAP,VorsaN,SchaichK,Bowen WH,etal.Inhibitoryeffectsofcranberrypolyphenolson formationandacidogenicityofStreptococcusmutansbiofilms. FEMSMicrobiolLett2006;257:50–6.

6. PercivalRS,DevineDA,DuggalMS,ChartronS,MarshPD. Theeffectofcocoapolyphenolsonthegrowth,metabolism, andbiofilmformationbyStreptococcusmutansand

Streptococcussanguinis.EurJOralSci2006;114_:343–8. 7. YanagidaA,KandaT,TanabeM,MatsudairaF,Oliveira

CordeiroJG.Inhibitoryeffectsofapplepolyphenolsand relatedcompoundsoncariogenicfactorsofmutans streptococci.JAgricFoodChem2000;48_:5666–71.

8. WorldHealthOrganization.WHOTraditionalMedicineStrategy 2002–2005.Geneva:WorldHealthOrganization;2002. 9. PlottikinMJ.Traditionalknowledgeofmedicinalplants–the

searchfornewjunglemedicines.In:AkerleO,HeywoodO, DyngeH,editors.Conservationofmedicinalplants.Cambridge: CambridgeUniversityPress,Cambridge;1991.p.53–63. 10.JaiarjP,KhoohaswanP,WongkrajangY,PeungvichaP,

SuriyawongP,SarayaML,etal.Anticoughandantimicrobial activitiesofPsidiumguajavaLinnleafextract.J

Ethnopharmacol1999;67:203–12.

11.GnanSO,DemelloMT.InhibitionofStaphylococcusaureusby aqueousGoiabaextracts.JEthnopharmacol1999;68:103–8. 12.VoravuthikunchaiS,LortheeranuwatA,JeejuW,SririrakT,

PhongpaichitS,SupawitaT.Effectivemedicinalplants againstenterohaemorrhagicEscherichiacoliO157:H7. JEthnopharmacol2004;94_:49–54.

13.KraivaphanV,BoonyamanoundL,AmornchatC,Triratana T,KraivaphanP.Theeffectofamouthrinsecontaining Psidiumguajavaleafextractongingivitis.JDentalAssoc Thailand1991;41_:323–8.

14.PongboonrodS.Mai-Tet-MuangThai.Bangkok:Kasem Bannakichapud;1979.p.354.JaiarjP,WongkrajangY, ThongpraditchoteS,PeungvichaP,BunyapraphatsaraN, OpartkiattikulN.Guavaleafextractandtopical

haemostasis.PhytotherRes2000;14:388–91. 15.BrighentiFL,LuppensSBI,DelbemACB,DengDM,

HoogenkampMA,Gaetti-JardimJrEetal.EffectofPsidium cattleianumleafextractonStreptococcusmutansviability,

proteinexpressionandacidproduction.CariesRes 2008;42_:148–54.

16.WhiteDJ,FeatherstoneJDB.Alongitudinalmicrohardness analysisoffluoridedentifriceeffectsonlesionprogression invitro.CariesRes1987;21_:502–12.

17.VieiraAE,DelbemAC,SassakiKT,RodriguesE,CuryJA, CunhaRF.FluoridedoseresponseinpH-cyclingmodels usingbovineenamel.CariesRes2005;39_:514–20.

18.TakeshitaEM,CastroLP,SassakiKT,DelbemACB.Invitro evaluationofdentifricewithlowfluoridecontent supplementedwithtrimetaphosphate.CariesRes 2009;43:50–6.

19.HannigM,FiebigerM,Gu¨ntzerM,Do¨bertA,ZimehlR, NekrashevychY.Protectiveeffectoftheinsituformed short-termsalivarypellicle.ArchOralBiol2004;49:903–10. 20.ThomasRZ,vanderMeiHC,vanderVeenMH,deSoetJJ,

HuysmansMC.Bacterialcompositionandredfluorescence ofplaqueinrelationtoprimaryandsecondarycariesnext tocomposite:aninsitustudy.OralMicrobiolImmunol 2008;23_:7–13.

21.CuryJA,RebeloMAB,DelBelCuryAA,DerbyshireMTVC, TabchouryCPM.Biochemicalcompositionandcariogenicity ofdentalplaqueformedinthepresenceofsucroseor glucoseandfructose.CariesRes2000;34_:491–7.

22.PecharkiGD,CuryJA,PaesLemeAF,TabchouryCP,DelBel CuryAA,RosalenPL,etal.Effectofsucrosecontainingiron (II)ondentalbiofilmandenameldemineralizationinsitu. CariesRes2005;39:123–9.

23.CuryJA,RebelloMAB,DelBelCuryAA.Insiturelationship betweensucroseexposureandthecompositionofdental plaque.CariesRes1997;31:356–60.

24.DenepitiyaL,KleinbergI.Acomparisonoftheacid-baseand aciduricpropertiesofvariousserotypesofthebacterium Streptococcusmutansassociatedwithdentalplaque.ArchOral Biol1984;29_:385–93.

25.Lingstro¨mP,BirkhedD.PlaquepHandoralretentionafter consumptionofstarchysnackproductsatnormalandlow salivarysecretionrate.ActaOdontolScand1993;51_:379–88. 26.BowenWH,PearsonS,YoungD,ThibodeauE.Theeffectof

partialdesalivationoncoronalandrootsurfacecariesinthe rat.In:LeachSA,editor.Factorsrelatingtodemineralization andremineralizationoftheteeth.Oxford:IRLPress;1986.p. 243–50.

27.GoldOG,JordanHV,vanHouteJ.Aselectivemediumfor Streptococcusmutans.ArchOralBiol1973;18:1357–64. 28.NobredosSantosM,MelodosSantosL,FranciscoSB,Cury

JA.Relationshipamongdentalplaquecomposition,daily sugarexposureandcariesintheprimarydentition.Caries Res2002;36:347–52.

29.Ccahuana-Va´squezRA,TabchouryCP,TenutaLM,DelBel CuryAA,ValeGC,CuryJA.Effectoffrequencyofsucrose exposureondentalbiofilmcompositionandenamel demineralizationinthepresenceoffluoride.CariesRes 2007;41_:9–15.

30.DuboisM,GrillesKA,HamiltonJK,RebersPA,SmithF. Colorimetricmethodfordeterminationofsugarsand relatedsubstances.AnalChem1956;28_:350–6. 31.UnitedStatesDepartmentofAgriculture,Agricultural

ResearchService,NationalGeneticResourcesProgram. PhytochemicalandEthnobotanicalDatabases.[OnlineDatabase]. Beltsville,Maryland:NationalGermplasmResources Laboratory;15May2005.

32.CuryJA,MarquesAS,TabchouryCP,DelBelCuryAA. Compositionofdentalplaqueformedinthepresenceof sucroseandafteritsinterruption.BrazDentJ2003;14: 147–52.

enameldemineralizationandplaquecompositioninsitu. BrazOralRes2003;17_:326–31.

34.ValeGC,TabchouryCP,ArthurRA,DelBelCuryAA,Paes LemeAF,CuryJA.Temporalrelationshipbetween sucrose-associatedchangesindentalbiofilmcompositionand enameldemineralization.CariesRes2007;41_:406–12. 35.AiresCP,DelBelCuryAA,TenutaLM,KleinMI,KooH,

DuarteS,etal.Effectofstarchandsucroseondentalbiofilm formationandonrootdentinedemineralization.CariesRes 2008;42:380–6.

36.TenutaLM,CenciMS,CuryAA,Pereira-CenciT,Tabchoury CP,MoiGP,etal.Effectofacalciumglycerophosphate fluoridedentifriceformulationonenameldemineralization insitu.AmJDent2009;22:278–82.

37.ZeroDT,vanHouteJ,RussoJ.Theintra-oraleffecton enameldemineralizationofextracellularmatrixmaterial synthesizedfromsucrosebyStreptococcusmutans.JDentRes 1986;65_:918–23.

38.MacphersonLMD,MacFarlaneTW,StephenKW.An intra-oralappliancestudyoftheplaquemicrofloraassociatedwith earlyenameldemineralization.JDentRes1990;69_:1712–6. 39.ZauraE,vanLoverenC,tenCateJM.Efficacyoffluoride

toothpasteinpreventingdemineralizationofsmoothdentin surfacesandnarrowgroovesinsituunderfrequent exposurestosucroseorbananas.CariesRes2005;39_:116–22. 40.deMazerPapaAM,TabchouryCP,DelBelCuryAA,Tenuta LM,ArthurRA,CuryJA.Effectofmilkandsoy-basedinfant formulasoninsitudemineralizationofhumanprimary enamel.PediatrDent2010;32:35–40.

41.Gonc¸alvesNC,DelBelCuryAA,Simo˜esGS,HaraAT,Rosalen PL,CuryJA.Effectofxylitol:sorbitolonfluorideenamel demineralizationreductioninsitu.JDent2006;34:662–7. 42.SilvaMF,JenkinsGN,BurgessRC,SandhamHJ.Effectsof

cheeseonexperimentalcariesinhumansubjects.CariesRes 1986;20_:263–9.

43.ReynoldsEC.Thepreventionofsub-surface

demineralizationofbovineenamelandchangesinplaque compositionbycaseininanintra-oralmodel.JDentRes 1987;66_:1120–7.

44.SchirrmeisterJF,SegerRK,AltenburgerMJ,LussiA,Hellwig E.Effectsofvariousformsofcalciumaddedtochewinggum oninitialenamelcariouslesionsinsitu.CariesRes

2007;41_:108–14.

45.MenezesTEC,DelbemACB,BrighentiFL,OkamotoAC, Gaetti-JardimJrE.ProtectiveefficacyofPsidiumcattleianum (Sabine)andMyracrodruonurundeuva(Allemao)leafextracts againstcariesdevelopmentinrats.PharmBiol2010;48_:300–5. 46.ZeroDT.Insitucariesmodels.AdvDentRes1995;9_:214–30. 47.Gutie´rrezRM,MitchellS,SolisRV.Psidiumguajava:areview

ofitstraditionaluses,phytochemistryandpharmacology. JEthnopharmacol2008;117_:1–27.

48.CostaTD,VieiraS,AndradeSF,MaistroEL.Absenceof mutagenicityeffectsofPsidiumcattleyanumSabine (Myrtaceae)extractonperipheralbloodandbonemarrow cellsofmice.GenetMolRes2008;7:679–86.

49.TeixeiraRO,CamparotoML,MantovaniMS,VicentiniVEP. Assessmentoftwomedicinalplants,PsidiumguajavaL.and AchilleamillefoliumL.,invitroandinvivoassays.GenetMolBiol 2003;26:551–5.

50.Al-hebshiN,Al-haroniM,SkaugN.Invitroantimicrobial andresistance-modifyingactivitiesofaqueouscrudekhat extractsagainstoralmicroorganisms.ArchOralBiol 2006;51_:183–8.

51.DibdinGH,ShellisRP.Physicalandbiochemicalstudiesof Streptococcusmutanssedimentssuggestnewfactorslinking thecariogenicityofplaquewithitsextracellular

polysaccharidecontent.JDentRes1988;67_:890–5. 52.vanRuyvenFOJ,Lingstro¨mP,vanHouteJ,KentR.

Relationshipamongmutansstreptococci,‘‘low-pH’’ bacteria,andiodophilicpolysaccharideproducingbacteria indentalplaqueandearlyenamelcariesinhumans.JDent Res2000;79:778–84.

53.Al-HebshiNN,NielsenO,SkaugN.Invitroeffectsofcrude khatextractsonthegrowth,colonization,and

glucosyltransferasesofStreptococcusmutans.ActaOdontol Scand2005;63:136–42.

54.CouncilonDentalTherapeutics.CouncilDental TherapeuticsacceptsListerine.JAmDentAssoc 1988;117_:515–6.

55.KooH,HayacibaraMF,SchobelBD,CuryJA,RosalenPL, ParkYK,etal.InhibitionofStreptococcusmutansbiofilm accumulationandpolysaccharideproductionbyapigenin andtt-farnesol.JAntimicrobChemother2003;52_:782–9. 56.MartiniND,EloffJN.Thepreliminaryisolationofseveral