Rev. bras. farmacogn. vol.25 número1

w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Hyptis

pectinata

gel

prevents

alveolar

bone

resorption

in

experimental

periodontitis

in

rats

Mônica

S.

Paixão

_,

_Mônica

_S.

_Melo

_,

_Nicole

_P.

_Damascena

_,

_Adriano

_A.S.

_Arau´jo

_,

_Andrea

_F.

_Soares

_,

Denise

V.A.

Oliveira

_,

_Jeison

_S.

_Oliveira

_,

_Fabrício

_T.C.

_Almeida

_,

_Francilene

_S.

_Amaral

_,

Brancilene

S.

Araújo

_,

_Charles

_S.

_Estevam

_,

_Marco

_A.

_Botelho

_,

_Lucindo

_J.

_{Quintans-Júnior}

a_{DepartmentofOdontology,FederalUniversityofSergipe,Av.MarechalRondons/n,RosaElze,49100-000SãoCristóvão,SE,Brazil} b_{DepartmentofPhysiology,FederalUniversityofSergipe,Av.MarechalRondons/n,RosaElze,49100-000SãoCristóvão,SE,Brazil} c_{DepartmentofPharmacy,FederalUniversityofSergipe,Av.MarechalRondons/n,RosaElze,49100-000SãoCristóvão,SE,Brazil} d_{DepartmentofMorphology,FederalUniversityofSergipe,Av.MarechalRondons/n,RosaElze,49100-000SãoCristóvão,SE,Brazil} e_{TheNortheastBiotechnologyNetwork,UniversityofPotiguar,Natal,RioGrandedoNorte,Brazil}

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t

i

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l

e

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n

f

o

Articlehistory:

Received22July2014

Accepted7December2014

Availableonline12February2015

Keywords: Hyptispectinata Periodontitis

Boneloss

Doxycycline

a

b

s

t

r

a

c

t

Hyptispectinata(L.)Poit.,Lamiaceae,isanaromatic,abundantandbroadlyusedplantspeciesinSergipe totreatoralandgastrointestinalpainandinflammation.Theaimofthepresentstudywastoanalyzethe relationbetweenperiodontitisandchangesinthecorporalmassandalveolarbonestructureafter induc-tionofexperimentalperiodontaldiseaseinrattreatedornottreatedwithH.pectinatagelat5%(GS5%) and10%(GS10%),comparingtheireffectswithdoxycyclinegelat10%(D10%,positivecontrol),vehicle gel(negativecontrol)andagroupwithexperimentalperiodontaldisease,butnon-treated.Thegelswere locallyappliedinthegingivalregionimmediatelyaftertheexperimentalperiodontaldiseaseinduction byligature(3×/day,11days).Bonedestructionwasdeterminedthroughclinicalexam,histopathological analysisandconebeamcomputedtomographyoftheexperimentalanimals(n=36).After11daysof peri-odontitisinduction,allgroupsthatreceivedligaturepresentedadecreaseinthecorporalmass,except tothenaïvegroup(withoutexperimentalperiodontaldisease)(p<0.05).Computedtomographyresults haveshownhealthybonestructureinthegroupIandboneresorptionforthetestgroups. Histopathologi-calanalysisconfirmedthehealthybonestructurefornaïvegroupanimals,whilethetestgroupsexhibited bonelossinseveraldegrees.Inparticular,thenon-treatedgroupanimalshadanintenseinflammatory process.WhentheperiodontiumoftheanimalstreatedwithGS10%washistopathologicallyanalyzed, insertionperiodontiumwaspreserved.Theresultsforthesegroupsweresignificantlydifferentofthe vehiclegroup(p<0.05).Accordingtotheresults,thegelbasedintheaqueousextractofH.pectinataat 10%canpreventbonelossinexperimentalperiodontaldiseasesimilarlytodoxycycline10%.

©2014SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Pathologicalcasesofgingivitisandperiodontitis,theoral ill-nessesmoreprevalentinBrazilaretargetofspecialattentionin needycommunities,especiallyintheBrazilianNortheast,where theindividualswiththesepathologiesnormallyhaveevolvedcases, whichareaccompaniedwiththedeteriorationofthedentalunitas wellastheirbonesupport(Botelhoetal.,2007).Theyarefrequently linkedtooralbacteria,whichareassociatedtomanysystemic dis-easessuchaspneumoniaandcardiovasculardiseases.Therefore,it

∗ Correspondingauthor.

E-mail:lucindo@pq.cnpq.br(L.J.Quintans-Júnior).

hasbeenemphasizedtheneedtooralcareinthesystemiccontrol ofhealth(Takaradaetal.,2004).

Periodontitis, an important cause of teeth loss in adults, is a chronic inflammatory disease characterized by located bone resorption(ChambroneandChambrone,2006;Botelhoetal.,2010). Recentresearcheshaveindicatedthelocalutilizationof doxycy-clinegelsasanisolatedtreatment,whichpreventtoothscalingand rootplanningandareeffectiveincombatingtheperiodontal dis-ease(TinocoandTinoco,2000).Inaddition,ithasbeensuggested thattheassociationofnaturalproductswithpreventivecarecan reducethehighincidenceofdiseasesthataffectthedental ele-mentastheperiodontaldisease(Juizetal.,2010;Siqueiraetal., 2010;Guimarãesetal.,2013).Recently,ourgrouphas develop-ingstudyaboutthepharmacologicaluseofnaturalproducts,as

http://dx.doi.org/10.1016/j.bjp.2014.12.002

medicinalplants,totreatorofacialdisroders(Quintans-Júnioretal., 2010;Bonjardimetal.,2011;Siqueira-Limaetal.,2014;Quintans etal.,2014).

Hyptisspeciesarearomaticplantsthatpresent pharmacolog-icallyactivesubstances withantimicrobial,antifungal,anti-HIV, analgesicand antiinflammatory activities, and cytotoxicity and insecticide properties (Bispo et al., 2001; Santos et al., 2008; Oliveiraetal.,2011;Francoetal.,2011;Paixãoetal.,2013).H. pecti-nata(L.)Poit.,Lamiaceae,anaromaticperennialbushspecieswith lowfoliardensityanderectbranches,iswidelyspreadinthestates ofSergipeandAlagoas.Theplantcanbeeasilyfoundaroundcities tobeusedfortreatingoral,gastricdisordersandfever(Martinez, 1989;Paixãoetal.,2013).Itsaqueousextracthaspreviouslyshown antinociceptiveandantiedematogeniceffectsbyreducing symp-tomsupto54%at400mg/kg(Bispoetal.,2001).Sincetheplantleaf crudeaqueousextractshowedorofacialantinociception (Paixão etal.,2013),itwasfoundnecessarytofurtherinvestigateitsaction againstotherorofacialdiseasessuchasperiodontitis.Therefore, theaimofthepresentstudywastoanalyzetherelationbetween periodontitisandchangesinthecorporalmassandalveolarbone structureafterinductionofexperimentalperiodontaldisease(EPD) inrattreatedornottreatedwithH.pectinatagelscomparedwith thestandarddrugdoxycycline.

Materialsandmethods

Plantmaterial

Hyptis pectinata (L.) Poit., Lamiaceae, leaves were collected duringthe florescence period in themetropolitan areaof Ara-caju,Sergipe,Brazil(255m,10◦₅₅′_56.1′′_South,37◦₀₆′_34.7′′_West).

Theplantmaterial wasidentified anda voucher specimenwas depositedintheherbariumoftheDepartmentofBiologyinthe FederalUniversityofSergipeunderthenumberASE-19005.

Aqueousextractpreparation

Leavesweredriedinanovenat37◦_{Cwithairrenewaland}

circu-lationfor48huntilcompletedehydration.Afterwards,theywere trituratedtogiveafinepowder(2kg),whichwasextractedwith distilledwater(3:10,w/v)underconstantagitationfor4hat35◦_C.

Afterfiltration,thesupernatant waslyophilizedtogivethedry crudeaqueousextract(CAE,32g).CAEwasdistributedinseven potsandkeptinadesiccatorintheLaboratoryofNaturalProduct Pre-clinicalPharmacologyoftheFederalUniversityofSergipetobe usedinthesubsequentexperiments,whentheywereredissolved inwatertogivespecificconcentrations.

Gelpreparation

CAEwas maintainedunder dark to avoid the possible light

degradation of some of the compounds present in it. For the

gelbaseproduction,0.2%Nipagim®_{(BatchGBG0020625,Pharma} Special),0.05%EDTAdisodium(Batch090707#2,DEG)and puri-fiedwater weremeasuredand heatedtogetherina water-bath

up to 70◦_{C. Whenthe temperature was reached, the mixture}

wasremovedfromthewater-bathandslowlyaddedto

hydrox-yethylcellulose,whichwasstirreduntilcompletehomogenization and solubilizationof the polymer.When this previousmixture reached40◦_C,Germal®_{previouslysolubilizedinwaterwasadded} tocompletehomogenizationuntilthegelformation.Afterwards, triethanolaminewasaddedforpHadjustmenttotherange5.0–6.0 atenvironmenttemperature.H.pectinataextractwasmixedwith propilenoglicol(Batche10030930C,PharmaNostra)and incorpo-ratedinthegelat5%or10%consideringgelmass.

The gelswerestored in white polyethylene containers kept hermeticallysealedat8◦_{Cuntiluse.Stabilitystudiestoevaluate}

gelconsistency ona 2-monthperiod wereperformedwiththe

formulationsbeing keptin differentconditions (4◦_{C, 37}◦_{C and}

environmenttemperature)andthegelformulationviscositywas measuredinregularintervals.Thestudyindicatedthatgel viscos-ity,colorandconsistencydidnotchangesignificantlyduringthe periodanalyzedinthespecifiedcircumstances.

Animals

Aprevioustrainingwiththeresearchteamonanesthesia pro-cedures, adaptation and handling of theanimals and gels, and convenientplacementofthebondageswasdonebeforethe begin-ningoftheexperiment.Theanimalswerepositionedonthesurgery tabletoallowtheratoralopeningmaintenance,facilitatingthe accesstothemaxillaposteriorregionoftheirteeth.Previoustothe experiment,36maleWistarrats(100–200g)werehousedin tem-peraturecontrolledroomsandreceivedwaterandfoodadlibitum. Allexperimentswereconductedinaccordancewithlocal guide-linesonthewelfareofexperimentalanimalsandwiththeapproval oftheCommitteeonEthicsinAnimalResearchoftheFederal Uni-versityofSergipe(#47/09).

Theexperimentalgroupsweredividedinnaïve(animals non-treatedandnotsubjectedtoEPD),non-treatedgroup(NT,animals subjectedtoEPD,butwithouttreatment),vehicle-treatedgroup(V, animalssubjectedtoEPDandtreatedwithvehiclegel),GS5%group (animalssubjectedtoEPDandtreatedwithlocalH.pectinatagelat 5%,w/w),GS10%group(animalssubjectedtoEPDandtreatedwith localH.pectinatagelat10%,w/w)andD10%group(animals sub-jectedtoEPDandtreatedwithlocaldoxycyclinegelat10%,w/w). Doxycyclinewasutilizedasareferencedrug.Thelocaltreatment withthegelwasfirstperformedimmediatelyafterthesurgical pro-cedureandthenthreetimes/dayuntiltheanimalsacrificesonthe 11thday.

Theanimalsinthetestgroupswithperiodontaldiseases(n=6, eachgroup)receivedaligature,whiletheanimalsinthecontrol group(naïve,n=6)werenotinducedforperiodontitis,although theyweresubmittedtocapture,hold,dailyweighingandsacrifice similarlytothetestgroups.

Inductionofexperimentalperiodontaldisease(EPD)

TheEPDprotocolusedinpresentstudywasperformedsimilarly toBotelhoetal.(2007,2010).Asterilizednylon(3.0)threadligature wasplacedaroundthecervixofthesecondupper-leftmolarofrats anesthetizedwith10%ketamina(0.08ml/100g,i.p.)andxilazyne chlorhydrate(0.04ml/100g).Theligaturewasknottedonthe buc-calsideofthetooth,resultinginsubgingivalpositionpalatinally andinsupragingival positionorally.Thecontralateralrightside wasusedastheunligatedcontrol.Animalswereweighteddaily.

Histopathologicalanalysis

After sacrifice under anesthesia, animals had their maxillae excised.Thespecimenswerefixedin10%neutralbuffered forma-linanddemineralizedin7%nitricacid.Thesespecimenswerethen dehydrated,embeddedinparaffinandsectionedalongthemolars inamesio-distalplaneforhematoxylinandeosinstaining. Sec-tionsof6␮mthickness,whichincludedtherootsofthefirstand

andrestricted totheregionof themarginalgingival)with pre-servedalveolarprocessandcementum;score1,moderatecellular infiltration(inflammatorycellularinfiltrationpresentalloverthe gingivalinsert)withsomeminoralveolarresorptionprocessand intactcementum;score2,accentuatedcellularinfiltration (inflam-matorycellularinfiltrationpresentinbothgingivalandperiodontal ligament)withaccentuateddegradationofthealveolarprocessand partialdestructionofcementum;andscore3,accentuatedcellular infiltrate,completeresorptionofthealveolarprocessandsevere destructionofcementum.

Weightlossanalysis

Alltheanimalshadtheircorporalmassmeasuredbeforethe surgeryandinthe11subsequentdays,whichwasthe experimen-talperiod.Valuesobservedwereexpressedasthevariationofthe corporalmass(g)regardingtheinitialmass.

Cone-beamcomputedtomography

Priorthe cone-beamcomputed tomography (CBCT),animals

weresacrificedandtheirhemiarcadesweredissected.These hemi-arcadesweresubmittedtoCBCTusingatomographyscannermodel i-Cat(ImagingSciencesInternational)withanexpositionareaof 6cmandexpositiontimeof40swithvoxelof0.2mm(maximum resolution).Thedigitalimageswereanalyzedusingtheiraxial, sag-italandcoronalcutsintheprogrami-CatCone-Beam3DDental ImagingSystem,version3.1.62.Resultswereexpressedasscores accordingtoscoresused intheLaboratory ofPre-clinical Phar-macologyofNaturalProductstoFederalUniversityofSergipe:0, absenceordiscreetbonereabsorption;1,bonereabsorption mod-erated;and2,severebonereabsorption.

Statisticalanalysis

Thedataarepresentedasthemean±SEM.Differencesbetween groupswereevaluatedbyanalysisofvariance(ANOVA)followedby Dunnett’sorBonferroni’stestsusingGraphPadPrismav5.0when meanswerecompared.Statisticaldifferenceswereconsideredto besignificantatp<0.05.

Results

Macroscopicanalysis

Beforethehistopathologicalanalysis,gingivaalterationswere evaluated withan odontological magnifying glass Bioart 4× to notemacroscopictexturemodifications,absenceofinterproximal papillaandchangesinthepositionoroutlineofgingivainthe ani-malsofthegroupswithinducedperiodontaldisease(NT,V,GS5%, GS10%andD10%),whichwerenotpresentinthenaïvegroup.

Gingivaoftheuppersecondmolarteethinvestibular,palatine andinterproximalregionsofthenaïveanimalshadsimilarclinical aspects.Itroundedthecervicalportionoftheteethandfinishedin apointedformsimilaraknifeedge.Interdentalpapillasweresharp andfinishedintriangularform.However,whentheanimalsinthe groupswithinducedperiodontaldisease(NT,V,GS5%,GS10%and D10%)wereevaluated,itwasshownthattheyhadflaccidgingiva intheregionoftheupperfirstmolarwithapicalmigrationof gin-givalmarginandabsenceofinterdentalpapillas,characterizingan inflammatoryprocessinthegingivalvestibularandpalatinefaces.

Histopathologicalanalysis

Thehistopathologicalanalysisoftheregionbetweenthefirst andsecondmolarsofthenormalperiodontiumshowsperiodontal

ligament(pl),alveolarbone(ab),dentin(d),cementumandgingiva (Fig.1A).Thehistopathologyoftheperiodontiumoftheanimals subjectedtoperiodontitisintheNTandVgroupsrevealedintense inflammatorycellinfiltrationcoupledwithcementumdestruction andcompletealveolarprocessresorption(Fig.1BandC),whose medianreceivedscore3.GS5%producednosignificantchangeas demonstratedonFig.1D.However,areductionofinflammatorycell infiltrationwithapartialpreservationofthecementumand alveo-larprocesswasfoundintheperiodontiumofanimalsinGS10%and D10%(Fig.1EandF,respectively),whosemediansreceivedscore 1.Thesevalueswerestatisticallysignificant(p<0.05,respectively) whencomparedtotheVandNTgroup(Fig.2).

Weightlossanalysis

Asofgeneral,allanimalsintheexperimentalgroupspresented variationintheirweightaftertheanalysisperiod.Animalsinthe experimentalgroupstreatedwithH.pectinataanddoxycyclinegels hadanincreasedweightafter11days(p<0.05),whileadecrease inthecorporealbodymasswasobservedforanimalsintheNTand Vgroups(Fig.3).However,animalsinthenaïvegroup,whichdid nothaveperiodontaldisease,showedthehighestincreaseinthe bodyweight(Fig.3).

CBCT

CBCTimagesoftheanimalhemiarcadesshowedalveolarbone lossesinvarieddegrees(Fig.4)whencomparedtothenaïvegroup, after 11 days of experiment. Severe alveolar bone losses were observedintheanimalhemiarcadesoftheNTand Vgroups.In theotherhand,thehemiarcadesobtainedfromtheanimalstreated withH.pectinatagels(GS5%andGS10%)hadasignificantlylower boneloss(p<0.05),whichwassimilartotheoneobservedforthe D10%group(Fig.5).

Discussion

Thisstudydemonstratesthatthelocalapplicationofadental gelbased onH.pectinatahasanti-inflammatory effectand also preventsalveolarboneresorptionandweightlossinanimalswith periodontitis,whichisstillconsideredthemainoralhealthproblem inmostofthedevelopingcountries,affecting60–90%ofchildren inschoolsaswellasadults(PetersenandYamamoto,2005).Asit isfrequentlyseeminthesecountries,wherenaturalproductsare usedtotreatseveralillnesses,ithasbeensuggestedtheuseofgels basedinnaturalproductsinlocalapplicationassupportinginthe periodontaltherapy(Paietal.,2004).Inthiscontext,previous stud-ieshaveshownthatsomenaturalproductssuchaspropolis(richin flavonoids)andessentialoilshavecariostaticactivitybyinhibiting theenzymesthatfacilitatemicrobialaggregationandbiofilm pro-ductionaswellasthegrowthofStreptococcusmutans,whichwill preventperiodontitis(Chungetal.,2006;Duarteetal.,2006).

A

B

D

C

E

F

g

pl d

c

a

lr

lr

Fig.1.Histopatologicalimagesofthenaïve(A),NT(B),V(C),GS5%(D),GS10%(E)andD10%(F)gelgroups.Mesialregionoftheleftseconduppermolarafter11daysofEPD

induction.(A)Interproximalregionshowsanormalperiodontalligament(pl),alveolarbone(ab),dentin(d),cementum(c)andgingiva(g)withhealthsulcularandjunctional

epitheliainnormalconformation(HE100×).(B)and(C)Intenseinsertionlossandboneresorption(arrow)intheinterproximalandfurcationregions(HE40×).(D)Lossof

insertionandboneresorptionintheinterproximalregion(Ir)withformationofbonesequestration(arrow)involvedwithinflammatoryinfiltrate(HE40×).(E)and(F)Flat

gingivalpapillawithapicalmigrationofthejunctionalepitheliumanddiscreetresorptionofinsertionperiodontetotheligatureregion(lr)(HE100×).

3

2

1

0

Scores

***

** **

Naiv

e _NT V

GS5% _GS10% D10%

Fig.2. EffectofthetreatmentwithV,GS5%,GS10%andD10%gelontheEPD.

Sig-nificantdifferencesbetweenmeanswerefoundincomparisonwiththeVgroupby

ANOVAfollowedbytheBonferroni’stest.*p<0.05,**p<0.01and***p<0.001.

0 1 2 3 4 5 6 7 8 9 10 11 Time (days)

300

270

240

210

180

150

V

ar

iation of the bob

y

w

eight (g)

Naive NT V *** GS5% GS10% D10%

Fig.3.EffectoftheV,GS5%,GS10%andD10%inthevariationofthebodyweight

ofratssubmittedtoEPDafter11daysofitsinduction.Datavaluesareexpressedas

mean±SEM(n=6)andweremeasuredeachdaybeforeandaftertheEPDinduction

during11days.Significantdifferencesbetweenmeanswerefoundincomparison

withthenaïvegroupbyANOVAfollowedbytheDunnett’stest.*p<0.05,**p<0.01

A

A

P

B

Fig.4.Cone-beamcomputedtomographyimagesanalyzedthroughthreecuts:axial,sagitalandcoronal.(A)Sagitalviewevidencingthetomographyofthesixpiecesof

theNTgroup.Thepresenceofboneresorptionintheligatureregioncanbeseen(arrow).(B)MPRscreenoftheclassicali-Catscanner(ImagingSciencesInternational).An

expositionareaof6cmandexpositiontimeof40swithvoxelof0.2mm(maximumresolution)wasused.ImagesfortheNTgroup.

etal.,2003,2004).Thisperiodontaldiseasemodelischaracterized byaprogressiveaccumulationofbacterialbiofilmintheligature withconsequentincreaseoftheinflammatoryinfiltrate,whichwill destroytheperiodontaltissue(conjunctiveandbonetissue).

Thestructureandtheorganizationoftheperiodontaltissuein themolartoothregioninrats,includingoralgingivalepithelium, oralsulcularepithelium,junctionalepithelium,collagen periodon-talfibers,cellularandacellularcementumandalveolarbone,are verysimilartothehumanteeth.Thebiggestdifferenceconsistsin thefactthatthegingivalsulcularepitheliumofratsiskeratinized, butstudieshaveshownthatthegingivalfunctionalbarrierisnot differentinratsandhumans,althoughtheextensionoftheaffected areaislargerinthelater.Thus,themodelusedinthepresentstudy isabletoreproducethemaincharacteristicsfoundinhuman peri-odontitisandassuchisappropriatedforthestudyofthisdisease (Botelhoetal.,2007).

*** ***

** *

Scores

Naiv

e _NT V

GS5% _GS10% D10% 2.0

1.5

1.0

0.5

0.0

Fig.5. EffectofV,GS5%,GS10%andD10%gelsintheboneresorptioncausedby

theEPD.Significantdifferencesbetweenmeanswerefoundincomparisonwith

theVgroupbyANOVAfollowedbytheBonferroni’stest.*p<0.05,**p<0.01and

***p<0.001.

Ligatureplacementaroundthefirstuppermolarintheanimals oftheexperimentalgroupsprovidedaninitialgingivallesion char-acterizedbyamicrovasculardilationoftheregionwithanincrease onthehydrostaticspressureandfluidpermeability,besides leuko-cytedisplacementtothefurrow(Botelhoetal.,2010).Inthisstudy, thegroupsthatdidnotreceivetreatmentwithH.pectinataor doxy-cyclinegelsbehavedaccordingly,asdemonstratedbytheNTandV groups,whichwerenotstatisticallydifferentwhencomparedwith eachotherandpresentedthesymptomsofanacutelesioninthe periodontaltissues(Holzhausen,2002).Themacroscopicresults presentedheresuggestthattheligatureplacementaroundthe ani-malteethcausedsignificantalterationsinthegingivalmacroscopic characteristics,like redness,bleeding and curved papilla.These findingscorroboratewithHolzhausen(2002),whichaffirmedthat inthedevelopmentoftheinducedinflammatoryperiodontal dis-ease,biofilmaccumulationinthedentalsurfacesnearthegingiva isthedirectcauseoftheinflammation.

Oneofthepossibleregulatorypathwaysfortheinflammatory responseinperiodontaldiseasescouldbecytokineproductionby thedifferentsub-populationsofauxiliaryTlymphocytes(Thelper, Th),whichwouldattenuateorincreasetheinflammatoryreaction intheperiodontaltissues,anddeterminetheactivityorlatencyof theperiodontaldisease(GemmelandSeymour,2006).Cytokines aremediatorsthatactonseveralcelltypestostimulate,inhibit ordestroytheperiodontaltissues(Lindhe,1999)asdemonstrated bythehistopathologicalanalysisoftheanimalsinthegroupswith EPD(NT,V,GS5%,GS10%andD10%),wherealveolarbonelosses invarieddegrees werepresent, whilethenaïvegroup(without EPD)didnotshowanysignofboneloss.Theseresults corrobo-ratewithNassaretal.(2003,2004)andBotelhoetal.(2007).Itis believedthatinnormalphysiologicsituations,boneformationand resorptionprogressionareinanequilibriumthatisregulatedso thatboneresorptionbyosteoclastsprecedesnewboneformation byosteoblasts(Cueto-Manzanoetal.,1999).

populationhastothisnaturalproductaswellastheeasinessby whichitcanbemanipulatedfortopicadministration.Doxycycline waschosennotonlyduetoitsantimicrobialaction,butalsodue toitsanti-collagenolyticeffect.Severalauthorsdemonstratedthat theuseofthisdrugisefficientassupportingagentinthe periodon-titistreatmentbecause itactsby inhibitingmetalloproteinases, consequently,reducingthedestructionoftheperiodontaltissues. However,itiswell-knownthatveryhighdosesofthisdrugdo lit-tleagainstthealterationsinthesusceptibilityoftheperiodontal microflora,whichinturnreducestheoccurrenceofbacterial resis-tance(Bezerraetal.,2000;Catonetal.,2001;Golubetal.,2001). Nonetheless,thetopicapplicationofthedoxycyclinegelwas pre-viouslyperformedtotreatperiodontitis(TomasiandWennstrom, 2004;Machionetal.,2006;Botelho etal.,2007).Thedrugwas provedeffectivebyreducingboneresorption,boneturnoverand number of microorganismsin theaffected sites as wellas the osteoclastactivity.Thus,doxycyclinewasusedaspositivecontrol. AdministrationofH.pectinataat10%(GS10%)immediatelyafterthe EPDinductionsignificantlypreventedthealveolarboneresorption similarlytothedoxycyclinegel(D10%).

HistopathologicalanalysisandCBCT imagesevidencedtissue alterationsandboneresorptioninallgroupswithperiodontal dis-ease,althoughanimalsinGS5%,GS10%andD10%groupsshowed mildalterationswhencomparedtoNTandVgroups,which pre-sentedintensecellinflammatory infiltrateafter 11days ofEPD inductionwithtotaldestructionofthealveolarbone,whichwas previouslyfoundbyBezerraetal.(2000),Limaetal.(2000)and

Menezesetal.(2005).Inastudyevaluatingtheperiodontal

destruc-tion due to the ligature placement when used together with

antibioticsforthecontrolofthebacterialfilm,theinhibitionofthe alveolarbonelossandtheinfectiousfeatureoftheexperimental periodontitisweredemonstrated(Sallayetal.,1982;Botelhoetal., 2010).ThefindingsinthepresentstudysuggestthatH.pectinatagel maybeinhibitingbonelossprobablybycontrollingtheformation ofbacterialfilm.

Severalstudieshavebeendoneontheuseofmedicinalplants associatedwiththeconventionalperiodontaltreatmentsothatthe anti-inflammatoryeffectcan beobservedas wellasthe reduc-tioninthecountingofthemicroorganismsresponsiblefortissue destructionof thedental element (Lee and Chung, 2004;Bakri and Douglas,2005;Feres et al.,2005).In this context,a previ-ousstudyinvolvingH.pectinatashowedtheplantaqueousextract hasmonoterpenes, sesquiterpenes, sesquiterpenic lactones and triterpeneswithimportantpharmacologicalactivities.The hemo-staticeffectoftheplantfreshleavesaswellastheantimicrobial activity against Escherichia coli, Staphylococus aureus, Klebsiella pneumoniae,Pseudomonas aeruginosa and Streptococus sp. were demonstrated(Basílioetal.,2006).AccordingtoJuizetal.(2010), plantswithactivityagainstperiodontopathogenswouldbemore effectiveiftheyalsoinhibitedS.mutansgrowthbecausethis organ-ismfacilitatesthebacterialbiofilminstallation.Nascimento(2005)

evaluatedtheinvitroantimicrobialpotentialofH.pectinata essen-tialoilandfoundthatitsantimicrobialactivityagainstS.mutans

wasequivalenttochlorhexidine.Moreover,theantinociceptiveand antiedematogeniceffectsoftheH.pectinataleafaqueousextract withlowextracttoxicitywasalsoshown(Bispoetal.,2001;Paixão etal.,2013).

Thus,consideringthe resultsof studiesontheantimicrobial andanti-inflammatorypropertiesofH.pectinata,thefindingshere presentedshowedthatH.pectinataCAEgelsdevelopedinthe Lab-oratoryofPharmacotechniqueoftheFederalUniversityofSergipe decreasetheinitialinflammatoryresponse,whichreflectedinthe posteriorconsequencesoftheperiodontaldiseasesuchasthebone resorption.Thisaccentuatedreductionoftheneutrophill inflam-matoryinfiltrateinducedbythetreatmentwithGS5%,GS10%and D10%intopicalformcouldbetterexplainthepreservationofthe

collagenfibersintheperiodontalligament,aswellastheminor resorptionofthealveolarbone.

Asignificantlossofbodyweightwasobservedfortheanimals intheNTandVgroups,whichwasprobablycausedbythesurgery traumaandacuteinflammatoryprocessthatfollowedit, compli-catinganimalchewingandfeeding.However,treatmentwithH. pectinataanddoxycyclinereducedorpreventedtheinflammatory process,which inturn reducedthetime itseffectlasted inthe animals,whichwereabletochewandeatfairlynormallywhen comparedwiththeNTandVgroups.AnimalstreatedwithGS5%, GS10%andD10%progressivelygainedbodyweightfromthe6th dayafterEPDinduction.Thesefindingsareinaccordancewiththe studyofLimaetal.(2000),wheretheanimallostaround10%oftheir weightinthefirstdayafterEPDinduction,butreturnedtotheir initialweightupontreatment.AccordingtoBotelhoetal.(2007, 2010),thegeltopicapplicationhavebenefitsbydecreasingboth thepainandthebacterialaccountaswellaspromotingoral con-ditionsforabetterfeedingoftheratstreatedwithgelsbasedin naturalproducts.

Conclusions

Thisstudydemonstratesthatthelocalapplicationofadentalgel basedonH.pectinataat10%hasanti-inflammatoryeffectandalso preventsalveolarboneresorptionandweightlossinanimalswith experimentalperiodontitis.Thestudyrevealedthegreatpotential ofthegeltocombatperiodontaldiseases,althoughfurtherstudies areneededtoclarifyitsusefulnessinclinicalsituations.

Conflictofinterest

Thereisnoconflictofinterest.

Authors’contributions

MSP,MSM,NPD,MGBO,AFS,DVAO,JSO,FTCA,FSAand

LJQ-Jcontributed withpharmacological tests.AASA, BSA,CSE, MAB contributed withplantobtention andaqueous extract prepara-tion.AASA,BSA,CSE,MABandLJQ-Jcontributedwithstudydesign, analysisofresults,preparationofthepaper.

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