Rev. bras. farmacogn. vol.26 número2

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Original

Article

Evaluation

of

the

orofacial

antinociceptive

profile

of

the

ethyl

acetate

fraction

and

its

major

constituent,

rosmarinic

acid,

from

the

leaves

of

Hyptis

pectinata

on

rodents

Rosângela

E.A.

Falcão

a

_,

_Silvana

_A.

_de

_Souza

a

_,

_Celso

_A.

_Camara

a

_,

_Jullyana

_S.S.

_Quintans

b

_,

Priscila

L. Santos

b

_,

_Maria

_Tereza

_S.

_Correia

c

_,

_Tania

_M.S.

_Silva

c

_,

_Adley

_A.N.

_Lima

d

_,

Lucindo

J. Quintans-Júnior

b

_,

_Adriana

_G.

_Guimarães

e,∗

a_Laboratório_de_{Bioprospecc¸}_ão_{Fitoquímica,}_Departamento_de_Ciências_Moleculares,_Universidade_Federal_Rural_de_Pernambuco,_Recife,_PE,_Brazil b_Departamento_de_Fisiologia,_Universidade_Federal_de_Sergipe,_São_Cristóvão,_SE,_Brazil

c_Laboratório_de_{Glicoproteínas,}_Departamento_de_Bioquímica,_Universidade_Federal_Pernambuco,_Recife,_PE,_Brazil d_Departamento_de_Farmácia,_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_RN,_Brazil

e_Departamento_de_Educac¸_ão_em_Saúde,_Universidade_Federal_Sergipe,_Lagarto,_SE,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5June2015 Accepted19July2015

Availableonline31December2015

Keywords: Hyptispectinate

Rosmarinicacid Orofacialpain Nociception

a

b

s

t

r

a

c

t

Hyptispectinata(L.)Poit.,Lamiaceae,popularlyknownas“sambacaitá,”isanaromaticshrublargely

grownintheBraziliannortheastern.Weinvestigatedtheantinociceptiveeffectsoftheethylacetate fractionobtainedfromtheleavesofH.pectinataandofitsmainconstituentrosmarinicacid,on for-malin(2%)-,glutamate(25␮M)-andcapsaicin(2.5␮g)-inducedorofacialnociceptioninrodents.Male micewerepretreatedwithethylacetatefraction(100,200or400mg/kg,p.o.),rosmarinicacid(10or 20mg/kg,p.o.),morphine(5mg/kg,i.p.),orvehicle(distilledwater+0.2%Tween80).Ethylacetate frac-tionreducedthenociceptiveface-rubbingbehaviorduringthetwophaseoftheformalintest,whereas pretreatmentwithrosmarinicaciddecreasedthepainbehaviorinthesecondphase.Ethylacetate frac-tionproducedsignificantantinociceptiveeffectsinthecapsaicinandglutamatetests.Thisstudyshowed thatoraladministrationofethylacetatefractionproducedpotentantinociceptiveeffectscomparedto treatmentwithrosmarinicacid.

Introduction

Painintheoralandcraniofacialsystemrepresentsamajor med-icalandsocialproblem(Hargreaves,2011).Indeed,areportfrom theU.S.SurgeonGeneralonorofacialhealthconcludesthat,“...oral

health means much more than healthy teeth. It means being

freeofchronicoral-facialpainconditions...”(NationalInstitutes

ofHealth,2000).Moreover,orofacialpainisderivedfrommany uniquetargettissues,suchasthemeninges,cornea,toothpulp, oral/nasalmucosa,andtemporomandibularjoint,andthushas sev-eraluniquephysiologiccharacteristicscomparedwiththespinal nociceptivesystem(Bereiteretal.,2008;Hargreaves,2011).Thus, themanagement ortreatmentof orofacialpainconditions rep-resentsasignificanthealthcareproblemandachallengeforthe pharmaceuticalindustry.

∗ Correspondingauthor.

E-mail:adrianagibara@pq.cnpq.br(A.G.Guimarães).

Inthelastcoupleofdecades,importantprogresshasbeenmade regardingthedevelopmentofnaturaltherapies.However,there isanurgentneedtodiscovereffectiveandsafeanalgesicagents (Calixtoetal.,2000)andnaturalproductshavebeenshowntobe strongcandidatesfordevelopmentofnewdrugsforpaincontrol (Quintansetal.,2014;Siqueira-Limaetal.,2014).Besides,acurrent approach istodevelop newbiological compoundsfromnatural productsthatmanageorofacialpainwithenhancedefficacyand minimalsideeffects;thesecompoundsarederivedfrom medici-nalplantsortheirsecondarymetabolites(Bonjardimetal.,2012; Guimarãesetal.,2013;Quintans-Júnioretal.,2010;Venâncioetal., 2011).

Hyptis pectinata (L.) Poit., Laminaceae, is a medicinal plant knownas“sambacaitá”or“canudinho”innortheasternBrazilthat iswidelyusedtotreatgastrointestinaldisorders,skininfections, nasalcongestion,fever,cramps,inflammationandpain(Bispoetal., 2001;Raymundoetal.,2011).Somestudieshavedemonstrated thatH.pectinatapossessesantinociceptiveandanti-inflammatory activities(Bispoetal.,2001;Lisboaetal.,2006;Raymundoetal.,

http://dx.doi.org/10.1016/j.bjp.2015.07.029

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0.0 5.0 10.0 15.0 20.0 25.0 min 0

100 200 300

mAU

320 nm, 4 nm (1.00)

1

2 3

4

6 5

7

8 ₉

10 11

Fig.1. ChromatogramfromHPLC-DADanalysisandthecompoundsisolatedfromtheEtOAcfractionoftheleavesofHyptispectinata.

2011).Nascimentoetal.(2008)describedhowH.pectinataleaves areusedastreatmentforseveraloraldiseases,suchasdentalcaries andorofacialpain.Thispharmacologicalprofilewascorroborated byPaixãoetal.(2013,2015),whodemonstratedanimportant neu-rogenicandinflammatoryorofacialantinociceptiveprofileofthe crudeaqueousextractobtainedfromH.pectinataleavesandits possibleapplicationagainstotherorofacialdiseasessuchas peri-odontitis.

Besides, Arrigoni-Blank et al. (2005) demonstrated anti-edematogeniceffectoftheaqueousextractofH.pectinata.Hasanein andMohammadZaheri(2014)showedthatrosmarinicacid(1),an esterofcaffeicacidfoundinHyptisspecies,reducesnociception inpainfuldiabeticneuropathymodel.Nevertheless,littleisknown howH.pectinataextractmodulatesorofacialpaintransmission.

Thepresentstudyproposedtoverifytheantinociceptive prop-ertiesoftheethylacetatefraction(EtOAc)ofH.pectinataleavesand, inparticular,rosmarinicacid(1)(RA),whichwasthemajor com-poundisolatedfromthisfraction.Theantinociceptiveproperties ofEtOAcfractionweretestedonmicefollowingorofacial nocicep-tioninducedbyformalin,capsaicinandglutamate,threealgogens agentsthatpromotepainthroughdifferentmechanismsandalso byactivationofdifferentneuronalpopulations.

Materialsandmethods

Plantmaterial

Plantmaterialwasobtainedandextractedaccordingto proto-colsdescribedinFalcãoetal.(2013).Voucherspecimen(88157)is depositedattheInstitutoAgronômicodePernambuco,Recife,PE. Briefly,theleavesweredried,crushedandsuccessivelyextracted withEtOHtoobtain7gdry extract.This extractwasdissolved in MeOH:H2O (1:1) and successively fractionated withhexane

andEtOAc.AportionoftheEtOAcfraction(3.5g)wassubjected

tochromatographyonaSephadexLH-20column,andthe

com-pounds were purified using a semi-preparative HPLC column.

Thisfractionationresultedintheisolationofsambacaitaricacid (2), 3-O-methyl-sambacaitaricacid(3),rosmarinicacid(1),3-O -methyl-rosmarinicacid(4),ethylcaffeoate(5),nepetoidinA(6), nepetoidinB(7),cirsiliol(8),cirsimaritin(9),7-O-methylluteolin (10)andgenkwanin(11)(Falcãoetal.,2013).Rosmarinicacidwas themajorcompoundisolatedfromtheEtOAcfraction,asshownin thechromatogram(Fig.1)obtainedbyHPLC-DADanalysis.

Animals

Male Swiss mice (28–34g), 2–3 months of age, were used

throughout this study. The animals were randomly housed in

appropriatecagesat22±2◦_C_on_a₁₂_h_light/dark_cycle_(lights_on

between6amand6pm)withfreeaccesstofoodandwater.All experimentswerecarriedoutbetween9amand2pminaquiet room.Allnociceptiontestswerecarriedoutbythesamevisual observer,andbehavioraltestswereperformedunderblind condi-tions.ExperimentalprotocolswereapprovedbytheAnimalCare andUseCommitteeattheFederalUniversityofSergipe(CEPA/UFS #10/11).

Drugandtreatments

Morphinehydrochloride(UniãoQuímica,Brazil),37%

formalde-hyde (Vetec, Brazil), diazepam (Roche, Brazil), Tween 80

(polyoxyethylene-sorbitanmonooleate),glutamate,capsaicinand rosmarinicacid(RA)werepurchasedfromSigma(USA).Duringthe nociceptiontests,theextractoragentwasadministratedbyoral gavage(p.o.,peros)orintraperitoneally(i.p.)atadosevolumeof 0.1ml/10g,withtheexceptionofthenociceptioninducingagents, suchasformalin,glutamateand capsaicin,whichwereinjected subcutaneously(s.c.)intotherightupperlip.

0 25 50 75 100

* **

**

Vehicle 100 200 400 MOR

EtOAc (mg/kg)

A

Face rubbing (s)

10 20

RA (mg/kg)

50 100 150

Fa

ce

ru

bb

ing (s)

B

0 Vehicle

*

100 200

EtOAc (mg/kg)

** **

400 *

10

RA (mg/kg)

*

20 **

**

MOR

(3)

RO

RO O

OH OH

OH

OH R OH

OH

1 R=H

4 R=OCH₃

H₃CO

R₁

2 R=H

3 R=CH₃

8 R=OCH₃; R₁=OH

9 R=OCH₃; R₁=H

10 R=H; R₁=OH

11 R=R₁=H

O

O O O

6 7

5

O O

CO₂H _CO

2H

HO

HO HO

Nociceptionstudies

Formalintest

Orofacialnociceptionwasinducedinmicebys.c.injectionof 20␮l2%formalinintotherightupperlip(perinasalarea),using a 27-gauge needle(Luccariniet al.,2006; Pelissier etal., 2002) accordingtopreviouslyreportedmethodswithchanges (Quintans-Júnioretal.,2010).Thisvolumeandconcentrationofformalinwas selectedduring ourpilotstudies becauseit showed anintense nociception-relatedbiphasic behavioral response (face-rubbing) atperiodsof0–5min(firstphase)and15–40min(secondphase) (Quintans-Júnioretal.,2010).Nociceptivebehaviorwasquantified duringtheseperiodsbymeasuringthetime(s)thatthemicespent rubbingtheirfaceattheinjectionareawithitsforeorhindpaws (Luccarinietal.,2006).Toassesstheeffectsofthetestdrugs,groups ofmice(n=8pergroup)werepretreatedsystemicallywitha vehi-clecontrol(distilledwater+0.2%Tween80),EtOAc(100,200or 400mg/kg,p.o.),orRA(10or20mg/kg,p.o.)1hbeforethe injec-tionofformalin.Morphine(MOR,5mg/kg,i.p.)wasadministered 1hbeforeofnociceptiveagent,asapositivecontrol.

Capsaicinandglutamatetests

Theorofacialnociceptioninducedbycapsaicinorglutamatein rodentswasperformedasdescribedpreviouslybyQuintans-Júnior etal.(2010).Mice(n=8pergroup)wereinjectedwithcapsaicin (20␮l,2.5mg)orglutamate(40␮l,25␮Mpreparedinphosphate bufferedsaline)subcutaneouslyintotherightupperlip(perinasal area),usinga27-gaugeneedle.Capsaicinwasdissolvedinethanol, dimethylsulfoxideanddistilledwater(1:1:8).Anadditionalgroup receiveda similarvolume of thecapsaicinvehicle.Nociception quantificationwasperformedbymeasuringthetime(s)thatthe animalsspentrubbingtheorofacialregionwithgreaterintensity foraperiodof42and30minaftertheinjectionofcapsaicinand glu-tamate,respectively.TheEtOAcfraction(100,200or400mg/kg)or vehicle(distilledwater+0.2%Tween80)wasgivenbyoralgavage toanimals1hbeforetheinjectionofcapsaicinorglutamate,similar totheformalintest.MORwasusedasapositivecontrolandwas administered(5mg/kg, i.p.)1h beforethenociception-inducing agent.

Evaluationofmotoractivity

Inordertoevaluateapossiblenon-specificmuscle-relaxantor sedativeeffectoftheextract,miceweresubmittedtotheRota-rod apparatus,asdescribedbyQuintans-Júnioretal.(2010).Initially, 24hbeforethetest,themicethatwereabletoremainonthe Rota-rodapparatus(AVS®_,_Brazil)_longer_than₁₈₀_s,_at_a_speed_of₉_rpm, wereselected.MiceweretreatedwiththeEtOAcfraction(100,200

or400mg/kg,p.o.),vehicleordiazepam(3mg/kg,i.p.)1,2and4h beforebeingplacedontheRota-rod;eachanimalwastestedonthe Rota-rodapparatus,andthetime(s)thattheanimalremainedon thebarforupto180swasrecorded.

Statisticalanalysis

Dataobtainedfromtheanimalexperimentswereexpressedas themean±thestandarderrorofthemean(mean±S.E.M.). Signif-icantdifferencesbetweenthetreatedandthecontrolgroupswere evaluatedusingtheANOVAandTukeytests.Differenceswere con-sideredtobestatisticallysignificantwhenp<0.05.Allstatistical analyseswereperformedusingGraphPadPrism5(GraphPadPrism SoftwareInc.,SanDiego,CA,USA).

Results

The chemical analysis of the EtOAc fraction from H. pecti-nataleavesshowedthepresenceofelevencompoundsand the

chromatogram (HPLC-DAD) is shown in Fig. 1. As seen in the

chromatogram,rosmarinicacid(1)wasthemaincomponentof thisfraction.Fouroftheelevenisolatedcompoundsarederived fromrosmarinicacid,andcompounds2(sambacaitaricacid)and 3(3-O-methylsambacaitaricacid)wereidentifiedasnewnatural compounds.

Acute treatment of mice withthe EtOAc fraction (100, 200 or 400mg/kg, p.o.)produced a significantreduction (p<0.05 or

p<0.001) in the face-rubbing behavior (Fig.2A and B) in both phasesof formalin-inducednociception.Additionally, treatment withrosmarinicacid(10or20mg/kg,p.o.)alonewaseffectivein sig-nificantlyreducingofthenociceptivebehaviorsduringthesecond phase,p<0.05orp<0.001respectively.

AsshowninFig.3A,pretreatmentwiththeEtOAcfraction(100, 200or400mg/kg,p.o.)significantlyreduced(p<0.001)the face-rubbingbehaviorinducedbycapsaicintreatment.Asexpected,the groupthatreceivedthevehicletreatment(ethanol,dimethyl sul-foxideanddistilledwater1:1:8)didnotpresentanynociceptive behavior(datanotshown).Besides,acutetreatmentwiththeEtOAc fractionproducedasignificantreduction(p<0.05orp<0.001)of glutamate-inducednociceptionwhencomparedwiththecontrol group(Fig.3B).

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0 25 50 75

EtOAc (mg/kg)

A

Face rubbing (s) ** ** **

**

0 50 150

100 200

EtOAc (mg/kg)

B

Face rubbing (s)

*

**

Fig.3. EffectsofEtOAc(Hyptispectinata)on(A)capsaicin-or(B)glutamate-induced orofacialnociceptioninmice.Vehicle(control),EtOAc(100,200or400mg/kg,p.o.), orMOR(5mg/kg,i.p.)wasadministered1hbeforecapsaicinorglutamateinjection. Eachcolumnrepresentsthemean±S.E.M.(n=8pergroup).*p<0.05or**p<0.001

vs.thecontrol(ANOVAfollowedbyTukey’stest).

Discussion

InfolkmedicineusedinnortheasternBrazil,theH.pectinata

plant(“sambacaita”)isusedinthetreatmentofseveralorofacial pathological disorders, includingorofacialpain. However, there arenopharmacologicalstudiesthatreportthiseffect.Forthefirst time,wedemonstratethatoraladministrationoftheEtOAcfraction fromH.pectinataleavesexertsprotectiveeffectsagainst formalin-,capsaicin-,and glutamate-induced orofacialpainin mice; this

effectmayberelatedtothepresenceofrosmarinicacidin this fraction.

Inthisstudy,theelevencompoundsoftheEtOAcfractionfrom

H.pectinataleaveswereisolatedandidentifiedaccordingtothe protocolsdescribedbyFalcãoetal.(2013).Rosmarinicacid(RA),a polyphenolcommonlyfoundinotherspeciesofthisgenusasHyptis atrorubens,wasidentifiedasthemaincomponentofthisfraction.

Duringtheformalintest,thepresenceoftwodistinctphases isatleastpartiallyduetothemechanismsofnociception.Thefirst phaseisassociatedwiththedirectstimulationoftheC-nociceptors

bymechanismsdependentonTRPA1,whereasthesecondphase

reflectstheintegrationbetweentheperipheral(nociceptors)and central (spinal/brainstem) signaling pathways (Capuano et al., 2009;Dalleletal.,1995;McNamaraetal.,2007).Alltesteddosesof EtOAcproducedantinociceptioninboththefirstandsecondphase oftheformalintestcomparedtothevehiclecontrol.Synergistic effectofcompoundsintheextract,aspolyphenolsandflavonoids, certainlycontributedtothereductionofformalin-induced noci-ceptionduringthefirstphase, sinceflavonoidsand phenolsare amongthemainnaturalproductsstudiedpain,asdemonstrated byQuintansetal.(2014).

Interestingly,Bispoetal.(2001)demonstratedthatpretreating micewiththeaqueousextractofH.pectinataatthedosesof200 and400mg/kghadnosignificanteffectduringthefirstphaseofthe formalintest,whenthepainfulagentwasappliedinthepaw. How-ever,Paixãoetal.(2013)showedamarkedantinociceptiveeffectof theaqueousextractofH.pectinatainbothphasesoftheorofacial formalintestandattributedthisprofiletoapossibleantioxidant effectoftheextract.ThediscrepanciesintheresultsofBispoetal. (2001)andPaixãoetal.(2013)maybeduetothegreatersensitivity oftheorofacialregion,theinvolvementofthetrigeminalpathway (Sessle, 2011),orduethechemical variationofplantsecondary metabolitesgeneratedbyenvironmentalandgenetic(Mooreetal., 2014).

RAreducedonlylatephaseofformalintest.Apreviousstudy showed that theadministration of RAat higher doses reduces bothphasesofformalin-inducednociception,withtheinvolvement ofopioid mechanisms(Boonyarikpunchai etal.,2014;Hasanein andMohammadZaheri,2014).Furthermore,thiscompoundand someanalogscouldbeusefulinthepreventionandtreatmentof inflammatorydiseases,andinhibitinflammatoryresponses,such asneutrophilicmigrationandedemainthelungsofmice(Gamaro etal.,2011).

Capsaicin, an active ingredient in hot chili peppers,directly stimulatestransientreceptorpotentialcation channelsubfamily

0 50 100 150 200

*

Vehicle 100 200 400 DZP

1h 2h 4h

*

EtOAc (mg/kg)

Time (s) on Rota-rod

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Vmember1(TRPV1),whichareinvolvedinthetransmissionand modulationofnociceptiveactivity,throughselectiveactions on unmyelinatedC-fibersandthinlymyelinatedAprimarysensory neurones(Dray,1992).AntagonistsofTRPV1receptorshavebeen reportedtoexhibitapain-relievingactivity(Trevisani,2013)and wereeffectiveinreducingnociceptionfrominflammatoryaswell asneuropathicpainmodelsinrats(Khairatkar-JoshiandSzallasi, 2009). Based on our finding, the oral administration of EtOAc producedaneurogenicinhibitionagainstcapsaicin-induced noci-ceptionindicating itsability toinhibitnociceptivetransmission initiatedbyTRPV1activation.

When glutamate is injected into the right upper lip (peri-nasalarea)elicitsanoxiousstimuluscharacterizedbyabehavioral responseof rubbingtheorofacialregion(Quintans-Júnioretal., 2010;Siqueiraetal.,2010).Noxiousstimulationofprimaryafferent fibersresultsinthereleaseofglutamatefromtheperipheraland centralterminalsofthetrigeminalandspinalafferentfibers(Keast andStephensen,2000;Lametal.,2005).Ourresultsdemonstrated thatEtOAcfractionreducedorofacialpaininducedbyglutamate. Thus,suggestthatthesuppressionofglutamate-induced nocicep-tionbyEtOActreatmentcanbeassociatedtoitsinteractionwith theglutamatergicsystem.Theseresultsaresimilartothosefound byGuginskietal.(2009),whodemonstratedthatrosmarinicacid blocksthepaininducedbyglutamateonthemousepaw.

Insummary,wedemonstratedtheEtOAceffectonorofacialpain inducedbythreealgogensagents,whichpromotenociceptionby differentmechanismsasTRPA1,TRPV1andglutamatergic recep-tors(Caterinaetal.,1997;McNamaraetal.,2007;Willcocksonand Valtschanoff,2008)withthepossibleparticipationofpeptidergic andnon-peptidergic primaryafferentneurons(Kobayashietal., 2005;WillcocksonandValtschanoff,2008).Additionally,previous studyhasshowntheCNSactionoftheaqueousextractfromH. pectinataleavesonrodentcentralnervoussystem(Buenoetal., 2006),withpossibleinvolvementofopioid system(Bispoetal., 2001).Thus,itissuggestedthatEtOAchascentralantinociceptive actionsandmaybeactingonperceptionofnociceptivestimuli, signaltransductionoritsconductiontothecentralnervoussystem (CNS).

InthissensedrugsthatactontheCNS,asdiazepamor mor-phine,canimpairmotorcoordinationandgenerateconfusionin analysisofresultsofnociceptivebehavioral tests,suchasthose usedhere(Quintans-Júnioretal.,2010).Therefore,weevaluated whetherpretreatmentwithEtOAccanimpairthemotor coordina-tionofmiceusingaRota-rodapparatus.However,alltesteddosesof EtOAcwereineffectiveinproducingchangesinmotorcoordination. Takenaltogether,ourdataledtothehypothesisthatEtOAchad aprotectiveroleinorofacialnociceptioninmice.Theseeffectsmay berelatedtothepresenceofrosmarinicacidandothercompounds. ThesefindingsalsosupportthehypothesisthatH.pectinatahas potentialtherapeuticuseforpainfulfacialand,perhaps,dental dis-orders.Nevertheless,furtherstudiesareneededtodeterminethe precisemechanismofaction.

Takenaltogether,ourdataledtothehypothesisthatEtOAchad aprotectiveroleinorofacialnociceptioninmice.Theseeffectsmay berelatedtothepresenceofrosmarinicacidandothercompounds. ThesefindingsalsosupportthehypothesisthatH.pectinatahas potentialtherapeuticuseforpainfulfacialand,perhaps,dental dis-orders.Nevertheless,furtherstudiesareneededtodeterminethe precisemechanismofaction.

Authors’contributions

REAF,SASandCACcontributedincollectingplantsampleand identification, confection of herbarium, running the laboratory work,analysisofthedataanddraftedthepaper.JSSQ,PLS,andAANL

contributedtobiologicalstudies.REAF,SASandCACcontributedin plantidentification,herbariumconfectionandcontributedto chro-matographicanalysis.JSSQ,LJQJ,MTSC,TMSScontributedtocritical readingofthemanuscript.AGGandLJQJdesignedthestudy, super-visedthelaboratoryworkandcontributed tocritical readingof themanuscript.Alltheauthorshavereadthefinalmanuscriptand approvedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

We would like to thank the CNPq/Brazil – grant number

555076/2010-5 and FACEPE (grant number PRONEM

APQ-1232.1.06/10)forfinancialsupport.

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