Rev. bras. farmacogn. vol.26 número4

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Antinociceptive

effect

of

semi-purified

petroleum

ether

partition

of

Muntingia

calabura

leaves

Zainul

Amiruddin

Zakaria

a,c,∗

_,

_Mohd

_Hijaz

_Mohd

_Sani

a

_,

_Arifah

_Abdul

_Kadir

b

_,

_Teh

_Lay

_Kek

c,d

_,

Mohd

Zaki

Salleh

c,d

a_Department_of_Biomedical_Sciences,_Faculty_of_Medicine_and_Health_Science,_Universiti_Putra_Malaysia,_Serdang,_Malaysia

b_Department_of_Veterinary_Preclinical_Sciences,_Faculty_of_Veterinary_Medicine,_Universiti_Putra_Malaysia,_Serdang,_Malaysia

c_Integrative_{Pharmacogenomics}_Institute,_Universiti_Teknologi_MARA,_Selangor,_Malaysia

d_Faculty_of_Pharmacy,_Universiti_Teknologi_MARA,_Selangor,_Malaysia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23August2015 Accepted7December2015 Availableonline2April2016

Keywords:

Muntingiacalabura

Petroleumetherpartition Antinociceptiveactivity Mechanismsofantinociception

a

b

s

t

r

a

c

t

MuntingiacalaburaL.,Muntingiaceae,isamedicinalplantforvariouspain-relateddiseases.Theaimsof thepresentstudyweretodeterminetheantinociceptiveprofileandtoelucidatethepossible mech-anismsofantinociceptionofpetroleumetherpartitionobtainedfromcrudemethanolextractofM. calaburaleavesusingvariousanimalmodels.Theantinociceptiveprofileofpetroleumetherfraction (givenoral;100,250and500mg/kg)wasestablishedusingtheinvivochemicals(aceticacid-induced abdominalconstrictionandformalin-inducedpawlickingtest)andthermal(hotplatetest) models ofnociception.Theroleofglutamate,TRPV1receptor,bradykinin,proteinkinaseC,potassium chan-nels,andvariousopioidandnon-opioidreceptorsinmodulatingthepartition’santinociceptiveactivity wasalsodetermined.Theresultsobtaineddemonstratedthatpetroleumetherpartitionexerted signif-icant(p<0.05)antinociceptioninallthechemicals-,thermal-,capsaicin-,glutamate-,bradykinin,and phorbol12-myristate13-acetate(PMA)-inducednociceptionmodels.Theantinociceptiveactivitywas reversedfollowingpretreatmentwithopioidantagonists(i.e.naloxone,␤-funaltrexamine,naltrindole andnor-binaltorphimine),andthenon-opioidreceptorantagonists(i.e.pindolol(a␤-adrenoceptor), haloperidol(anon-selectivedopaminergic),atropine(anon-selectivecholinergicreceptor),caffeine(a non-selectiveadenosinergicreceptor),andyohimbine(an␣2-noradrenergic)).Inaddition,pretreatment withl_-arginine_(a_nitric_oxide_(NO)_donor),_NG_-nitro-l_-arginine_methyl_esters₍l_-NAME;_an_inhibitor_of

NOsynthase(NOS)),methyleneblue(MB;aninhibitorofcyclic-guanosinemonophosphate(cGMP) path-way),ortheircombinationfailedtoinhibitpetroleumetherpartition’santinociception.Inconclusion, petroleumetherpartitionexertsantinociceptiveactivityattheperipheralandcentrallevelsviathe mod-ulationof,partly,theopioid(i.e.␮,␬and␦)andseveralnon-opioids(i.e.␤-adrenergic,dopaminergic, cholinergic,adenosinergic,and␣2-noradrenergic)receptors,glutamatergic,TRPV1receptors,PKCand K+_channels_systems,_but_notl_-arg/NO/cGMP_pathway.

Introduction

Variousneurotransmittersand receptorsystemstakepartin themodulationofpainprocessesinthecentralnervoussystem (CNS)andperipheralnervoussystem(PNS),suchasthevanilloid (Cui et al., 2006), opioid (Craig and Sorkin, 2011), glutamate (Osikowiczetal.,2013),proteinkinaseC(PKC;Velázquezetal., 2007),potassiumion(K+₎_channels₍_Ocana_et_al.,₂₀₀₄_),_and_nitric

oxide/cyclic guanosine monophosphate (NO/cGMP) pathways

∗ Correspondingauthor.

E-mail:[email protected](Z.A.Zakaria).

(Schmidtko etal.,2009).Despitetherapidadvancementsinthe developmentofvarioustreatmentsforpain,theclinicalefficacy andtolerabilityofconventionalanalgesicscanbeovershadowed byitsunwantedadverseeffects.Somepatientshavetaken alterna-tiveapproachtotreatpain,includingcomplementaryalternative medicine(CAM)inordertoavoidthesesideeffects(Astin,1998). Theuseofnaturalproductasalternativetherapiesisan increas-inglypopularmethodtotreatsuchdiscomfort,eitheraloneoras acomplementtoconventionalmedicalapproaches(Wirthetal., 2005).HoltandChandra(2002)categorized naturalproductsas herbs,herbalconcoctions,traditionalChinesemedicines,dietary supplements and alternative medicines. The research in natu-ral product based on its ethnopharmacological knowledge has

http://dx.doi.org/10.1016/j.bjp.2015.12.007

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provided substantial contributions to drug innovation through the discovery of novel chemical structure and/or mechanism of actions (Rates, 2001). Muntingia calabura L., Muntingiaceae, the sole species in the genus, has been traditionally used in the Southeast Asia and tropical America (Kaneda et al., 1991; Nshimoet al., 1993)to treat headaches and gastric ulcer, and asan emmenogogue, antidyspeptic,antispasmodic, diaphoretic, tranquillizerandtonic(Kanedaetal.,1991;Perez-Arbelaez,1975). Various medicinal properties have been scientifically reported from M. calabura leaves, including anti-tumor (Kaneda et al., 1991;Suetal.,2003),hypotensive(Shihetal.,2006),antibacterial (Zakaria etal.,2006a,b;Zakariaetal.,2010;Sufianetal.,2013; Isnariantietal.,2013),antiplateletaggregation(Chenetal.,2007), anti-inflammatory,antipyreticandantinociceptive(Zakariaetal., 2007a,b), anti-myocardial infarction (Nivethetha et al., 2009), antihypertensive(Shih,2009),antioxidant(Siddiquaetal.,2010; Zakariaet al.,2011; ArunaSindhe etal., 2013)cytotoxic(Chen etal.,2005;Sufianetal., 2013), antiproliferative(Zakariaetal., 2011), antihyperglycemic (Aruna Sindhe et al., 2013), antiulcer (Balanetal.,2014;Zakariaetal.,2014a,b),andhepatoprotective (Mahmood et al., 2014) activities. Other parts of M. calabura have also been reported to exert insecticidal (Neto Bandeira et al., 2013), anti-inflammatory (Preethi et al., 2012; Gomathi et al., 2013), antioxidant (Preethi et al., 2010; Gomathi et al., 2013) activities. With regards to the antinociceptive studies, themethanolextractofM.calaburahasbeenreportedtoshow antinociceptive activity (Mohd Sani et al., 2012), which was later shown to involve activation of the non-selective opioid (i.e. ␮-, ␦- and ␬-opioid) and non-opioid (i.e. adenosinergic,

␣2-noradrenergic, and ␤-adrenergic) receptors, modulation of the ATP-sensitive K+ _channel, _and _inhibition _of _bradikinin _and

proteinkinaseCactions(Zakariaet al.,2014a,b).Moreover,the authorsalsosuggestedthattheobservedantinociceptiveactivities anditsmechanismofactionswereresultedfromthesynergistic effectofthebioactivecompounds,flavonoids, saponins,tannins andsteroids, possessedbytheplantextracts.Recentlaboratory researchprovedthatthecrude methanolextractofM.calabura (MEMC)leavespossessesgoodtherapeuticeffectinreducing noci-ceptiveresponse(MohdSanietal.,2012;Zakariaetal.,2014a,b) andfurtherstudybyMohamad Yusofetal. (2013)leadstothe isolationoffourflavonoid-basedantinociceptive-bearingbioactive compounds,ofwhichoneisanewcompoundcalledcalaburone (8-hydroxy-6-methoxyflavone)and threewereknowncompounds, namely 5-hydroxy-3,7,8-trimethoxyflavone,3,7-dimethoxy-5-hydroflavone and 2′_,4′_-dihydroxy-3′_{-methoxychalcone.} _In _this study,we furtherevaluate thepossible antinociceptiveactivity and its mechanism of action of the petroleum ether partition extractedfromMEMC.

Materialsandmethods

Plantcollection

TheleavesofMuntingiacalaburaL.,Muntingiaceae,were col-lectedfromitsnaturalhabitatinShahAlam,Selangor,Malaysia. Plantidentificationhasbeenmadeearlier(Balanetal.,2014)and avoucherspecimen(SK964/04)hasbeendepositedatthe Herbar-iumofInstituteofBiosciences,UniversitiPutraMalaysia,Serdang, Selangor,Malaysia.

PreparationofmethanolextractofM.calabura(MEMC)and itspetroleumetherfraction(PEMC)

ThemethodforpreparationofMEMCwasdescribedindetail byBalanet al.(2014). TheobtainedMEMCwasthen extracted

subsequentlywithpetroleumetherusingthemethoddescribed in detail by Mohamad Yusof et al. (2013). MEMC (2g) dis-solvedinasolventwitharatioof1:7:2whichconsistofMeOH (100ml),petroleumether (700ml)anddistilled water (200ml). Themixturewasrigorouslyshakenusingaseparatoryfunneland thenlefttoseparate.Thesupernatantwascollectedandfiltered whiletheresidue wassoakedagainwithpetroleumetheruntil thesupernatantbecomecolorless.Thecollectedpetroleumether supernatantwasalsosubjectedtorotaryevaporatorat40◦_C_under reduced pressuretoobtaina concentratedextractofpetroleum etherofM.calabura(PEMC).

Experimentalanimals

MaleICRmice(25–30g)andSpragueDawleyrat(150–180g) wereobtainedfroma privatesupplier (ChenurSupplier, Selan-gor, Malaysia) and acclimatized for three days in the Animal Holding Unit, Faculty of Medicine and Health Sciences, UPM. Details on the handled and cared of these animals have been described by Balan et al. (2014) and was in compliance with currentUPMguidelinesforthecareoflaboratoryanimals (Ethi-calapprovalno.:UPM/FPSK/PADS/BR-UUH/00404)andtheethical guidelinesforinvestigationsofexperimentalpaininconscious ani-mals(Zimmermann,1983).Allexperiments(n=6)wereconducted between09:00and16:00htominimizetheeffectofenvironmental changes.

Drugsandchemicals

Capsaicin,glutamate,naloxone,␤-funaltrexamine,naltrindole, nor-binaltorphimine, apamin, charybdotoxin, tetraethylammon-ium,atropine,haloperidol,pindolol,yohimbine,caffeine, gliben-clamide were purchased from Sigma–Aldrich (USA) while bradykininwaspurchasedfromTocrisBioscience,UK.Theywere preparedatthedesireddosebydissolvingthemindistilledwater (dH2O). Acetyl salicylic acid (ASA), morphine and capsezepine

(Sigma–Aldrich,USA)weredissolvedin10%DMSOwhilephorbol 12-myristate 13-acetate (PMA; Sigma–Aldrich, USA) was dis-solved in PBS solution.Acetic acid, petroleum ether, methanol anddimethylsulfoxide(DMSO)werepurchasedfromFisher Sci-entific (U.K.). All drugs and PEMC were given in a 10ml/kg basis.

Antinociceptiveanalysis

Aceticacid-inducedabdominalconstrictiontest

Theaceticacid-inducedabdominalconstrictionwascarriedout withslightmodifications accordingtothemethoddescribed in detailbyMohdSanietal.(2012).Themicewerepretreatedorally with10%DMSO(vehicle,negativecontrol),100mg/kgASA (posi-tivecontrol)orPEMC(100,250and500mg/kg)and60minlater subjectedtotheabdominalconstrictiontest.

Hotplatetest

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Formalintest

Theformalintestwascarriedoutasdescribedindetailin previ-ousresearchbyMohdSanietal.(2012).Ratswereadministered orallywiththetestsolutions (i.e.10%DMSO(negativecontrol), 100mg/kgASA(peripherallyactingpositivecontrol),5mg/kg mor-phine(centrallyactingpositivecontrol)orPEMC (100,250and 500mg/kg))60minpriortotheformalininjection.The nocicep-tiveresponsedescribedastheearlyphaseandthelatephasewas recordedattherespective0–5minand15–30minfollowingthe formalinadministration.

Determinationofthemotorcoordinationandsedative effectsofPEMC

EffectofPEMConmotorcoordination

Rota-rodtest

Arota-rodtreadmilldevice(Ugo Basil,Italy)wasemployed toassesstheeffectofPEMConmotorcoordination(Meloetal., 2011)withslightmodifications. In thebeginning,micecapable ofremainingontherota-rodapparatuslongerthan210s(9rpm) wereselected12and24hpriortothetest.Thirtyminutesafterthe administrationofeither10%DMSO(negativecontrol),diazepam (2.5mg/kg, i.p.) or PECM (100, 250 and 500mg/kg, p.o.), each mousewastestedontherota-rodapparatusat0.5,1and2h post-treatment.Thetime(s)themicewereabletoremainontopof thebarwasrecordedforupto210s.Decreaseinfallofftimeis suggestiveofdepressionofthecentralnervoussystem(CNS).

SedativeeffectofPEMC

Sodiumpentobarbital-inducedsleepingtimetest

ThesedativeeffectofPEMCincombinationwithsodium pen-tobarbital(SP)wasevaluated(Martínez-Vázquezetal.,2012).For thispurpose,atotalof30miceweredividedintofivegroups(n=6) andorallyadministeredwitheither10%DMSO(negativecontrol), diazepam(2.5mg/kg,i.p.)orPECM(100,250and500mg/kg,p.o.) 30minbeforetheintraperitonealadministrationofsodium pento-barbital(42mg/kg,i.p.).Eachmousewasplacedonawarmtable andcautiouslymonitoredfortheonsetofuncoordinated move-mentssubsequenttothesedativephaseofthetest. Lossofthe rightingreflexrelatedtothehypnosisphaseandthedurationof sleepwerealsoobserved.Theeffectswererecordedasfollows: timepassedbetweentheadministrationsofpentobarbitaluntilloss ofrightingreflexwasrecordedasoftheonsetofsleep,whilstthe timefromthelosstorecoverywasconsideredasthedurationof sleep.

Investigationonthepossiblemechanismsofantinociceptionof PEMC

Capsaicin-inducedpawlickingtest

AntinociceptiveactionofPEMConvanilloidreceptors(TRPV1) were investigated using the proceduredescribed by Goncalves etal.(2005)withslightmodifications.Ratswerepretreatedorally withvehicle,capsazepine(0.17mmol/kg)orPEMC(100,250and 500mg/kg)60minbeforecapsaicininjection(1.6␮g/paw,50␮l) intothei.pl.regionof therat’srighthindpaw. Theinducedrat individuallyobservedinatransparentglasschamberfor5min,and theamountoftimetheanimalsspentlickingtheinjectedpawwas recordedwithachronometer.

Glutamate-inducedpawlickingtest

AslightmodificationfromtheproceduredescribedbyBeirith etal.(2002)wasusedtostudytheroleofglutamatergicsystem

inthemodulationofPEMCantinociceptiveaction.Ratswere pre-treatedorallywithvehicle,ASA(100mg/kg)orPEMC(100,250and 500mg/kg)60minpriortoglutamateinjection.Avolumeof50␮l ofglutamate(10␮mol/paw)wasinjectedintothei.pl.regionon therighthindpawofeachrat.Immediatelyafterthephlogistic agentadministration,theinducedratwereplacedinatransparent glasscageobservationchamberandindividuallyobservedfrom0 to15min.Theamountoftimetheanimalsspentlickingorbiting theinjectedpawwasrecordedwithachronometer.

InvolvementofproteinkinaseC

The experiment was conducted based on the previously describedmethodbySavegnagoetal.(2007).Avolumeof50␮l ofPMA(aproteinkinaseCactivator)solution(0.05␮g/paw)was injectedintotheventralsurfaceoftherighthindpawoftherat 60minaftertheoraladministrationofvehicle,ASA(100mg/kg)or PEMC(100,250and500mg/kg).Theanimalswereobserved indi-viduallyfromminute15to45following PMAinjectionandthe amountoftimetheratspentlickingtheinjectedpawwasrecorded usingachronometer.

Bradykinin-inducednociception

Based onthemethodpreviously described byFerreiraet al. (2004),bradykinin(10nmol/pawin50␮l)wasinjectedintothe intraplantarsurfaceoftherighthindpawofeachrat60minafter theoraladministrationofvehicle,ASA(100mg/kg)orPEMC(100, 250and 500mg/kg). The bradykinin-inducedrat was observed individuallyfor10min,andtheamountoftimetheyspentlicking theinjectedpawwasrecorded.

Involvementofl-arginine/nitricoxide/cyclicguanosine

monophosphatepathway

To determine the role of nitric oxide/cyclic-guanosine monophosphate (NO/cGMP) pathway in the modulation of PEMCantinociceptiveactivitythemethoddescribedindetailby

Zakariaetal.(2006a,b)wasadopted.Micewerepretreatedwith 20mg/kgl_-arginine,l_-NAME,_MB_or_their_respective_combination

(l-argininewithl-NAMEorl-argininewithMB)followed5min

laterbypretreatmentwithvehicleorPEMC(500mg/kg), respec-tively.Sixtyminutesaftertheadministrationoftestsolutions,the miceweresubjectedtotheabdominalconstrictiontest.

Involvementofpotassiumchannels

Todeterminetheeffectof PEMC-inducedantinociception on K+_channels,_a_method_previously_described_by_Alves_and_Duarte (2002)wasused.Rats werepre-treated withglibenclamide(an ATPsensitiveK+_channel_inhibitor;₁₀_mg/kg,_i.p_.),_apamin_(small

conductanceCa2+_-activated_K+_channels,_0.04_mg/kg,_i.p_.),

charyb-dotoxin (an inhibitor of large conductance Ca2+_-activated _K+

channels,0.02mg/kg, i.p.)and tetraethylammonium chloride (a non-selective voltage dependant K+ _channel _inhibitor, ₄_mg/kg, i.p.)15minbeforeoraladministrationofeithervehicleorPEMC (500mg/kg).Sixtyminuteslater,painwasinducedusing0.6%acetic acid.Thenumberofwrithingwillberecordedfor25min,5min followingaceticacidinjection.

EffectofvariousreceptorantagonistsonPEMC-induced antinociception

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pre-treatedwithcaffeine(3mg/kg,i.p.),atropine(10mg/kg,i.p.), haloperidol(20mg/kg,i.p.),pindolol(1mg/kg.i.p.),oryohimbine (0.15mg/kg,i.p)15minbeforetheoraladministrationofvehicleor PEMC(500mg/kg).Sixtyminuteslateraftertheadministrationof PEMCorvehicle,themiceweresubjectedtotheabdominal con-strictiontest.

Analysisofopioidreceptorsubtypes

Evaluationontheinvolvementofopioidreceptorsubtypewas doneusingtheabdominalconstrictiontestwhichissimilarwith previouslydescribed.Thepreparation oftheopioidantagonists’ dosesandtimingofadministrationwerebasedonpreviously con-ductedstudiesbyChoietal.(2003),Reetaetal.(2006)andZakaria etal.(2014a,b).The␮opioidantagonist,␤-funaltraxamine(␤-FNA; 10mg/kg, i.p.), ␦opioid receptor antagonist, naltrindole (NALT; 1mg/kg.i.p.)or␬opioidreceptorantagonist,nor-binaltorphimine (nor-BNI; 1mg/kg, i.p.) were administered 90min, 15min and 30min respectively, beforeoral administration of500mg/kg of PEMC.Thenociceptivestimuluswasinjected60minafterPEMC administration.

HPLCprofileofPEMCatvariouswavelengths

The HPLCprofile of PEMC wasestablished according tothe methods described in detail for MEMC by Mohd Sani et al. (2012).Inaddition,PEMCwasalsospikedwithalistof flavonoid-based compounds, namely pinostrobin, hesperetin, flavanone, 4′_{,5,7-trihydroxy}_flavanone,_2,4,4′_-trihydroxy_chalcone,_quercitrin, dihydroquercitin,fisetin,quercetin,rutin,quercitrin,naringenin, silibinin,andgenistein,todeterminetheirpresenceintheextract. The HPLC analysis was carried out in the Laboratory of Phy-tomedicine,MedicinalPlantsDivision,ForestResearchInstituteof Malaysia(FRIM),Kepong,Malaysia.

Statisticalanalyses

Theresultsare presented as mean±standard errorof mean (SEM).Theone-wayANOVAtestwithDunnett’smultiple compar-isonandtwo-wayANOVAwithBonferroniposthoctestwereused toanalyzeandcomparethedata.p<0.05wassetasthelimitof significance.

Result

AntinociceptiveprofileofPEMC

Fig.1 showsthat orallyadministered PEMCproduced a sig-nificant(p<0.001)anddose-dependentantinociceptiveeffectin micesubjectedtotheaceticacid-inducedabdominalconstriction test.Thepercentageanalgesiaproducedrangedbetween26and 70%,while thepositive controltreatment(100mg/kg ASA,p.o.) producedananalgesiceffectof59.31%.

Fig.2showsthecentrallyandperipherallymediated antinoci-ceptiveprofileof orallyadministeredPEMCas indicatedbythe formalintest.Theextractexertedsignificant(p<0.05)and dose-dependentantinociceptiveactivityinboththeearlyandlatephases oftheassay.Theantinociceptiveactivity,inbothphases,waslower thanthatof5mg/kgmorphine.

ThecentralantinociceptiveprofileofPEMC,performedusing hotplatetest,athermal-inducednociceptionmodelwasshownin

Fig.3.PEMCexertedsignificant(p<0.001)antinociceptiveactivity onlybythe500mg/kgwhichisthehighestdosetested.However, theantinociceptioninducedbyPEMCwasagainlowerthanthat inducedby5mg/kgmorphineadministeredviai.p.injection.

0 20 40 60 80 100

26.05% ∗∗∗

49.05% ∗∗∗

70.15% ∗∗∗

59.31% ∗∗∗

ASA 500 250 100 C

PEMC (mg/kg)

Number of constrictions

Fig.1.Numberofabdominalconstrictionsinmiceduringtheaceticacid-induced abdominalconstrictiontestaftertreatmentwithPEMC,acetylsalicylicacid(ASA; positivecontrol),orvehicle([C]negativecontrol).Aceticacidwasadministered intraperitoneally60minbeforeoraladministrationoftreatment.n=6ineachgroup. Numbersabovethebarsindicatethepercentageofanalgesia.***p<0.001as com-paredtonegativecontroltreatmentbyone-wayanalysisofvariancefollowedby Dunnett’sposthoctest.

MotorcoordinationandsedativeeffectsofPEMC

EffectofPEMConmotorcoordination

Intherota rodtest, alldoses ofPECMdidnotimpairmotor coordinationat0.5,1and2hpost-administrationincontrastto 2.5mg/kgdiazepam,whichcausedasignificant(p<0.05)decrease intimethattheanimalsstayedontherota-rodapparatuscompared tothecontrolgroup(Table1).

A

0 30 60 90 120

∗∗∗ ∗∗∗

∗∗

Mor 500

250 100

C ASA

PEMC (mg/kg)

13.94%

32.36%

53.35% 7.57%

82.77%

Paw licking time (s)

B

0 60 120 180

∗∗∗ ∗∗∗

∗∗

Mor 500

250 100

C ASA

∗∗∗ ∗

PEMC (mg/kg)

26.43%

37.63%

53.52%

82.98%

96.44%

Fig.2.EffectofPEMCinformalin-inducedpawlickingtest.Datarepresentsthe mean±SEMofsixrats.Theratswerepretreatedwithvehicle(10%DMSO),PEMC (100,250,and500mg/kg,p.o.),acetylsalicylicacid(ASA,p.o.),ormorphine(5mg/kg,

p.o.),60minbeforei.pl.injectionofformalin.Theasterisksdenotethesignificance

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∗∗∗ ∗∗∗

∗∗∗

∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

∗∗∗ ∗

20

18

16

14

12

10

8

Latency time (s)

Time (min) 6

4

2

0

0 60 90 120 150 180

Vehicle (10% DMSO)

PEMC 100

PEMC 250

PEMC 500

Morphine

210

Fig.3. AntinociceptiveprofileofPEMCassessedusingthehot-platetestinmice.*p<0.05whencomparedwiththecontrolgroupatsametherespectiveinterval.Dataare themean±SEM;n=6micepergroup.

Table1

EffectofPEMConmotorcoordinationassessedbytherota-rodtest.

Treatment Dose(mg/kg) Time(s)onRota-rod/210sa

30min 60min 90min

10%DMSO – 210.0±0.0 207.18±2.11 210.0±0.0

Diazepam 3 41.15±6.32a _28.18

±7.35a _56.92

±7.43a

PEMC

100 205.49±4.26 204.75±3.64 210.0±0.0

250 202.35±6.14 207.03±2.08 206.47±2.52

500 208.08±1.12 205.87±3.02 210.00±0.00

Dataarethemean±SEM;n=6micepergroup.Theratswerepretreatedwith10%DMSO(negativecontrol),2.5mg/kgdiazepam(i.p.)orPEMC(100,250,and500mg/kg, p.o.)30minbeforesubjectiontothetest.

a_{Significantly}_differed_at_P_<_0.05_when_compared_to_the_control_group_as_assessed_by_the_one-way_ANOVA_followed_by_Dunnett’s_post_hoc_test.

Table2

EffectofPEMConmicesleepingactivityassessedusingthesodiumpentobarbital-inducedsleepingtest.

Treatment Dose(mg/kg) Onsetofsleep(min) Durationofsleep(min)

10%DMSO – 3.48±0.41 41.12±0.33

Diazepam 1 1.72±0.28a _94.33_±_0.66a

PEMC

100 3.08±0.17 68.66±0.47a

250 3.26±0.39 71.66±0.49a

500 2.98±0.32 88.83±0.55a

Dataarethemean±SEM;n=6micepergroup.

a_{Significantly}_differed_at_P_<_0.05_when_compared_to_the_control_group_at_the_same_respective_interval_as_assessed_by_the_one-way_ANOVA_followed_by_Dunnett’s_post_hoc test.

Sodiumpentobarbital-inducedsleepingtimetest

AdministrationofPEMCdidnotcausechangesinneitherthe latencysedationnorthesleepingtimeinducedbysodium pento-barbitalsuggestingthatPEMCdidnotexertsedativeeffectsatany ofthedosestested(Table2).

RoleofTRPV1,glutamatergic,B2receptorsandPKCpathway

TheabilityofPEMCtoinhibitthevanilloidreceptor-induced nociceptivetransmissionisshowninFig.4.The250and500mg/kg PEMC significantly (p<0.001) attenuated the capsaicin-induced pawlickingtestinadose-dependentmannerwiththepercentage ofanalgesiarecordedrangingbetween30and64%,respectively. Theinhibitionpercentageof63.52%producedby500mg/kgPEMC washigherthanthepositivecontrolcapsazepine(59.72%).

Fig. 5 shows the antinociceptive profile of PEMC against glutamate-induced paw licking test. PEMC significantly (p<0.001) attenuated the nociceptive effectassessed using the

0 20 40 60

Vehicle Capz

PEMC (mg/kg)

500 250 100

63.52% ∗∗∗

59.72% ∗∗∗ 30.74%

∗∗∗ 12.72%

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0 50 100 150 200

Vehicle ASA

PEMC (mg/kg)

500 250 100

69.43% ∗∗∗ 72.27%_∗∗∗ 45.31%

∗∗∗ 31.55%

∗∗∗

Fig.5.EffectofPEMConglutamate-inducedpawlickingtestinrats.Eachcolumn representsthemean±SEMofsixrats.Theratswerepretreatedwithvehicle (con-trol,10%DMSO)orPEMC(100,250,and500mg/kg,p.o.)60minbeforeinjectionof glutamate(10␮mol/paw,50␮l,i.pl.).Theasterisksdenotethesignificancelevelsas comparedtocontrol,***p<0.001byone-wayANOVAfollowedbyDunnett’spost hoctest.

glutamate-inducedpawlickingtestinadose-dependentmanner. Thepercentageofanalgesiaobtainedfromtheadministrationof 100,250and500mg/kgPEMCrangingbetween32and69%.

Fig.6showsthattheoraladministrationof250and500mg/kg PEMCproducedsignificant(p<0.001)inhibitionofPMA-induced nociception in rat. Interestingly, the 500mg/kg PEMC exerted slightlybetterantinociceptiveactivitycomparedto100mg/kgASA indicatedbythecalculatedpercentage ofanalgesia(66.38%and 55.36%respectively).

AsseeninFig.7,PEMCgivenorallyexhibitssignificant inhi-bitioninadose-dependentmanneronthenociceptioncausedby intra-plantarinjectionofbradykinininrat.Themaximalinhibition observedwas53.92%fordose500mg/kg.Similarinhibitoryeffect wasobservedfor100mg/kgaspirin.

Evaluationofl_{-arginine/NO/cGMP}_pathway

Fig.8aandbshowtheroleoftheabdominalconstrictiontests incorporatingl_-arginine,l_-NAME_and_MB_to_explore_the_role_of_the l-arginine/NO/cGMPpathwayinthemodulationofPEMC-induced

antinociceptiveactivity.InFig.8a,pretreatmentwith20mg/kgl

-argininedidnotproduceanysignificanteffectwhencomparedto thenegativecontrol.Meanwhile,pre-treatmentwith20mg/kgl

-NAMEaloneexertedasignificantantinociceptiveeffect.However, thepositiveeffectwasdiminishedwhenl-NAMEgivenin

combina-tionwithl-arginine.InthegroupstreatedwithPEMC,pretreatment

withl-NAMEdidnot affecttheextractantinociceptive activity,

whereaspretreatmentwithl-arginine significantlyreduced,but

0 20 40 60 80

ASA 500 250 100 Vehicle

PEMC (mg/kg)

36.52% ∗∗∗

66.38% ∗∗∗

55.36%

∗∗∗ 9.57%

Fig.6.EffectofPEMConnociceptioninducedbyphorbol12-myristate13-acetate (PMA)onrats.Eachcolumnrepresentsthemean±SEMtimespentonpaw-licking behaviorofsixanimals.Animalswereadministeredwithvehicle(negativecontrol), PEMC(100,250,and500mg/kg,p.o.)oracetylsalicylicacid(ASA;100mg/kg,p.o.) 60minbeforeinjectionofPMA(50mlsolutioncontaining0.05mgPMA/paw)inthe righthindpaw.Numbersabovebarsindicatepercentageofanalgesia.***p<.001 ascomparedtothecontrolgroupbyone-wayanalysisofvariancefollowedby Dunnett’sposthoctest.

0 20 40 60

Vehicle

PEMC (mg/kg) 500 250

100 ASA

23.82% ∗

53.92% ∗∗∗

51.1% ∗∗∗ 8.16%

Fig.7. TheantinociceptiveeffectofPEMCagainstbradykinin-inducedpawlicking. Eachcolumnrepresentsthemean±S.E.M.ofsixrats.Vehicle(negativecontrol; 10%DMSO,p.o.),PEMC(100,250and500mg/kg,p.o.)andacetylsalicylicacid(ASA: 100mg/kg,p.o.).***p<0.001whencomparedtocontrolgroup.

didnotcompletelyinhibit,theantinociceptiveeffectsofPEMC. Pre-treatmentwithacombinationofl-arginineandl-NAMEalsofailed

toaffecttheintensityofPEMCantinociception.

Pretreatment with 20mg/kg MB alone (Fig. 8b) exerted significant (p<0.05) antinociceptive activity and co-treatment with 20mg/kg l-arginine failed to reverse MB antinociception.

Pre-treatment with MB significantly (p<0.05) enhanced PEMC antinociceptionwhileco-treatmentwithMBandl-argininefailed

toaffecttheantinociceptiveintensityofPEMC.

Involvementofpotassiumchannels,non-opioidandopioid receptorsonPEMC-inducedantinociception

Fig. 9 shows the involvement of potassium channelsin the modulation of PEMC-induced antinociceptive activity. Pretreat-mentwithglibenclamide(10mg/kg,i.p.),Apamin(0.04mg/kg,i.p.), charybdotoxin(0.02mg/kg,i.p.)andtetraethylammoniumchloride (4mg/kg,i.p.),significantly(p<0.01)reversedtheantinociceptive activity of PEMC (500mg/kg) when assessed using acetic acid-inducedabdominalwrithingtest.

The effect of antinociceptive activity of PEMC was signifi-cantly (p<0.05) reversed following intraperitoneal administra-tion of several receptor antagonists (Fig. 10) namely caffeine (3mg/kg),atropine(10mg/kg),yohimbine(0.15mg/kg), haloperi-dol (0.2mg/kg), pindolol (0.2mg/kg), or naloxone, naltrindole, nor-binaltorphimineand␤-funaltrexamine(Fig.11).

HPLCprofileofPEMC

TheHPLCanalysisofPEMCwasmeasuredatvariousdifferent wavelengths(e.g.210,254,280,300,330and366nmandthebest separationwasobtainedat300nm(Fig.12a).Fourmajorpeaks wereseparatedatthesewavelengths,whichwerelabeledasP1 (RT13.772min),P2(RT20.171min),P3(RT21.592min),andP4(RT

28.144min).Furtheranalysisdemonstratedthatthesefourpeaks showedmax values in theregionof 211.9–272.0, 234.3–308.8,

240.1–270.8,241.3–292.2nm,respectively(Fig.12b),suggesting, in part, the presence of flavonoid-based compounds. Compari-sonbetweenchromatogram ofPEMCand chromatogramof the standard compoundsdemonstrated thepresenceof pinostrobin andflavanone(Fig.12c).

Discussion

Thepresentstudywasthecontinuationofpreviousstudiesby

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0 30 60 90 120

A

B

L-arginine + L-NAME Control (10% DMSO)

PEMC

L-arginine L-arginine + PEMC

L-NAME L-NAME + PEMC

L-arginine + L-NAME + PEMC

a

a, b a

a

a c

0 30 60 90 120

Control (10% DMSO)

PEMC MB L-arginine

MB + L-arginine

MB + PEMC

MB + L-arginine + PEMC

a

a _a

a, b a #

Fig.8.(A)Effectsofl-arginine,l-NAME,andtheircombinationonPEMCantinociceptionasassessedbyaceticacid-inducedabdominalconstrictiontest.Thelettersrepresent thesignificancelevelsbetweencomparedgroups;a_p_<_0.001_when_compared_with_control_group;b_p_<_0.001_when_compared_with_PEMC-treated_group;c_p_<_0.001_when comparedwithl-NAME-treatedgroup,byone-wayANOVAfollowedbyDunnett’sposthoctest.(B)Effectsofl-arginine,methyleneblue,andtheircombinationonPEMC antinociceptionasassessedbyaceticacid-inducedabdominalconstrictiontest.Thelettersandsymbolabovethecolumnsrepresentsignificancelevelsbetweencompared groups;a_p_<_0.001_when_compared_with_control_group,b_p_<_0.05_when_compared_with_MB-treated_group,#_p_<_0.05_when_compared_between_selected_group.

ofM.calabura(MEMC).Recentlaboratorystudyconducted focus-ingontheantinociceptivepotential ofpartition extractedfrom theMEMC,wherethecrude extractpartitionedusingthree sol-ventwithdifferentpolaritynamelypetroleumether,ethylacetate andaqueoususingthemethoddescribedabove.Usingacetic acid-induced abdominal writhing test to screen for antinociceptive activity,petroleumether partitionproduced significantlybetter activity(datanotshown).Hence,thePEMCselectedtobefurther testedforitsantinociceptivepropertiesaswellasthemechanism ofactions.

OurdatashownthePEMCattenuatedthenociceptiveresponse inducedbychemicalandthermalstimuli,suggestingthatthe com-poundexertsperipheralandcentralantinociceptiveactivities.Its abilitytoinfluencebothperipheraland centralnociception was furtherconfirmedbyourobservationofitsantinociceptiveeffects in bothphases of theformalin test.Overall, theseobservations suggestthattheantinociceptiveactivityofPEMCresemblesthat ofopioid analgesics,suchas morphine.Moreover,theabilityof PEMCtoattenuatenociception inboth theabdominal constric-tionandhotplatetestssuggeststhat thecompoundiseffective

0 20 40 60 80 100

dH2O GLIB APA CHAR TEA dH2O GLIB APA CHAR TEA PEMC

Vehicle

∗∗ ∗

∗∗

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0 20 40 60 80 100

V PE P P + PE H H + PE A A + PE C C + PE

a

a, b

a

a, b

Y + PE Y

a, b

Fig.10.Theinvolvementofseveralnon-opioidreceptorantagonistsinPEMC-inducedantinociceptionagainstaceticacid-inducedabdominalwrithingtestinmice.Vehicle (V:10%DMSO,p.o.),PEMC(PE:500mg/kg,p.o.),atropine(A:10mg/kg,i.p.)haloperidol(H:20mg/kg,i.p.),pindolol(P:1mg/kg,i.p.),caffeine(C:3mg/kg,i.p.),yohimbine(Y: 0.15mg/kg,i.p.).a_p_<_0.001_{significantly}_different_when_compared_to_{vehicle-treated}_group;b_p_<_0.001_{significantly}_different_when_compared_to_PEMC-treated_group.

withbothinflammatory-inducedandnon-inflammatory-induced nociception,respectively(Zakariaetal.,2007a,b),whichalso sup-ported byourdata fromtheformalin-induced pawlicking test wherebothphases,theearly(neurogenic)andlate(inflammatory) phase,attenuatedbyPEMCtreatment.

Theaceticacid-inducedabdominalconstrictiontest,described asatypicalmodelforinflammatory pain,haslongbeenwidely usedasatooltoscreenforanalgesicoranti-inflammatory prop-ertiesof newagents(Collier etal.,1968; Vongtauet al.,2004). Theacetic acid-inducedabdominalconstriction testreflectsthe inflammatory-mediatednociceptiveresponse (MohdSani etal., 2012)where theinjected acetic acidinto theperitoneal cavity increases thelevel of cyclooxygenase(COX) and lipooxygenase (LOX)intheperitonealfluid andindirectly leadstotherelease ofendogenousmediatorssuchasprostaglandinE2(PGE2),PGF2␣,

serotonin,histamine,cytokines,andeicosanoids. These endoge-nousmediators,inturn,excitetheperipheralnociceptiveneurons withintheperitonealcavitythataresensitivetonon-steroidal anti-inflammatory drugs (NSAID) (Ikeda et al., 2001). Theincreased levelsofPGE2 duetoprolongirritationofperitonealcavitylead

toenhancedcapillarypermeability(Deraedtetal.,1980;Vogeland Vogel,1997)aswellasthereleaseofglutamateandsubstanceP fromperipheralafferentfiber(Millan,1999),indicatingthe stimu-lationofperipheralnociceptivemechanisms.TheabilityofPEMC toinhibitaceticacid-inducednociceptionsuggeststhatthe com-poundattenuates the levelsof peripheralCOXand LOX, which indirectlydecreasesPGE2 synthesisand obstructsthe

transduc-tionofpaininprimaryafferentnociceptors.However,theacetic

acid-inducedabdominalconstructiontestisnotspecific,thus fur-therstudiesusingothernociceptivemodels,namelythehotplate andformalintestsarerequiredtodeterminewhetherthe antinoci-ceptiveeffectsofPEMCarecentrallyorperipherallymediated.

The hot plate test, predominantly a spinal reflex test that involvesactivationofthesupraspinalnociceptiveprocessing,has beenappliedin variousresearches toshowthat onlythe anal-gesicactingcentrally,butnotperipherally,haveaneffectonthis test (Chen et al.,1995; Le Barset al., 2001; Hosseinzadeh and Younesi,2002;Giglioetal.,2006).TheabilityofPEMCtoinhibit boththeearlyandlatephasesoftheformalintestinthepresent studyconfirmedtheextract’sabilitytoexertcentrallyand periph-erally mediatedantinociceptive effects(MohdSani etal.,2012; MohamadYusofetal.,2013).Theearlyphase,classifiedasa neuro-genicpain,isanacutereaction,lastedfor5minwhichimmediately occuraftertheinjectionofformalinwhereitdirectlyactivatethe nociceptors.Thelatephase,appearsbetween15and30min fol-lowingformalininjection,isclassifiedasaninflammatorypainas itiscausedbyinflammatorymediatorsreleasedsuchashistamine, serotonin,bradykininandPGE2 (Vermaetal.,2005).Ourresults

inthepresentstudyindicatethatPEMCpossessesmildcentrally mediatedantinociceptivecharacteristicsasseeninboth thehot platetestandearlyphaseofformalintest.

Overthepastdecade,tremendousresearchbeingdevotedin developing thebestmedicationforpain reliever,and extensive studieshasbeenperformtoexploretheirprecisemechanismof actioninordertounderstandandlimitanyunwantedsideeffect. However,theneuronalbasisofpaintransmissionandmodulation

0 20 40 60 80 100

PEMC + NALT NALT

PEMC

C β-FNA PEMC + β-FNA PEMC + nor- ASA

BNI nor-BNI

∗∗∗

∗

PEMC + NLX NLX

∗∗∗

∗

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AU

0.010

A

B

C

254 nm

300 nm

366 nm

300 nm

366 nm 0.000

0.010

0.000

10.00

10.00 0.010

0.005

0.000 0.000

20.00

Minutes

30.00

10.00 20.00 30.00

20.00

Minutes

13.772extracted

211.9

272.0

234.3 308.8

240.1270.8

Pinostrobin

Flavanone

241.3 292.2

20.171 extracted 21.592 extracted 28.144 extracted

210 nm

254 nm

280 nm

300 nm

330 nm

366 nm 30.00

10.00 20.00 30.00

20.00 30.00

0.005

0.000

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Kv channel

Nociceptor terminal

Nitric oxide + L-citrulline

NOS PKC Post-synaptic nerve cell

Noxious stimulus

5-HTR

Opioid receptor

TRPV1

L-arginine

AMPA

NMDA SP

NO

PGE2 EP1

A1R

Glut

Ca2+

α₂R

A1R

PGE2

B₂

Primary afferent fiber

Opioid receptor KATP channel

s_K Ca channel

Fig.13.SchematicdiagramoftheproposedmechanismsofantinociceptiveactiondemonstratedbyPEMC.PEMCexertsantinociceptiveactivityatperipheralandcentral levelsviamodulationofseveralreceptorsandneurotransmitterssystems.Ca2+_:_calcium_ion;_cGMP:_cyclic_guanosine_{monophosphate;}_GC:_guanylate_cyclase;_GTP:_guanosine triphosphate;Kv:voltage-gatedpotassiumchannel;KATP:ATP-sensitivepotassiumchannel;S_KCa:_small_conductance_{calcium-activated}_potassium_channel;_5-HTR: sero-tonergicreceptor;␣2R:␣2-adrenergicreceptor;A1R:adenosinereceptor;Glut:glutamate;PGE2:prostaglandinE2;EP1:prostaglandinreceptor;B2:bradykininreceptor; NMDA:N-methyl-d-aspartate;PKC:proteinkinaseCenzyme;NOS:nitricoxidesynthase;NO:nitricoxide;TRPV1:transientreceptorpotentialcationchannelsubfamilyV member1.

isstillnotcompletelyunderstood.Numerouschemicalsandfactors suchasopioids,substanceP,glutamate,nitricoxide,serotonin,and otherinflammatorymediators,hasbeendocumentedtoengaged intheactivationofthePNSandCNSnociceptivepathways(Mohd Sanietal.,2014).Theexistencedescriptionofthesemodulatory mechanismssuchastheroleofvanilloidreceptors, glutamater-gicsystem,opioidandnon-opioidreceptors,nitricoxide,PKCas wellasK+_channels_pathways,_have_benefited_and_provide_guidance

forresearchersinthedevelopmentofnewanalgesiccandidates (Rodellaetal.,2010).

Our previous study (Mohd Sani et al., 2012; Zakaria et al., 2014a,b)usingthecrudemethanolextractofM.calabura(MEMC) revealedtheparticipationof mostthemodulatorymechanisms. Inthepresentstudy,weemployedthesameapproachtoexplore thepossiblemechanismsof actionresponsiblefortheobserved antinociceptiveactivityofPEMC.Basedonourfindings,PEMC;(1) attenuatethenociceptioninducedbycapsaicinindicatingpossible involvementofvanilloidreceptors,(2)inhibitglutamate-induced nociceptionwhichinturninhibitthereleaseofNOandNO-related substance (Beirith et al., 2002), (3) significantly inhibit PMA-inducednociceptionsuggestingitsinvolvementinPKC-activated phosphorylationofvanilloidreceptorpathway(Vellanietal.,2001), (4)inhibitbradykinin-inducednociceptionshowingitsabilityto blockB2receptorfrombeingactivated,(5)affectedbyl-arginine,

but not l-NAME and MB,suggesting PEMC antinociception did

notinvolvemodulationviathel-arginine/NO/cGMPpathway,(6)

involved in the activation of ATP-sensitive K+ _channels, _small

conductanceCa2+_-activated_K+_channels_and_{non-selective}

voltage-dependantK+_channels,_but_not_large_conductance_Ca2+_-activated

K+_channels,₍₇₎_play_significant_role_in_{antinociception}_modulated

byadenosinergic,␣2-noradrenergic,cholinergicand␤-adrenergic

receptors,(8)involvedintheactivationofopioidreceptorsubtypes asPEMCantinociceptiveeffectwassignificantlyreducedbythe pretreatmentof␮,␦and␬opioidreceptorsubtypeinhibitors.

Ourfindings,apartfromthepossibleparticipationof choliner-gicreceptorandinabilityofPEMCtoactivatelargeconductance Ca2+_-activated_K+_channels,_were_in_line_with_our_results_obtained

fromthecrudemethanolextractofM.calabura.Theinvolvementof cholinergicreceptorseeninPEMC-inducedantinociceptivemight probably due tohigher purity of theexpressed bioactive com-poundsextractedfromthecrudeextract.

EarlierstudiesimpliedthatthedepressionofCNSandtheeffect of nonspecific muscle relaxation can diminish the response of motorcoordinationand,accordingly,maynullifythe antinocicep-tiveresults(Quintans-Júnioretal.,2010).Interestingly,thepresent findingsdemonstratedthatPEMC-treated(100,250and500mg/kg, orally)miceexertednoperformancealterationsinthe rota-rod-andpentobarbital-inducedsleeping-testindicatingthatPEMCdid notinterfereinthemiceanxietybehaviororinthemotorfunction, whencomparedtothecontrolgroup.Incontrast,diazepaminduced anxietyreductiondemonstratedbyanincreaseinthenumberof visitsandtimespentintheopenarmsandbyanincreaseinthetotal numberofentries.Overall,thesefindingssuggestedthatthereisno alterationinmotorcoordinationamongthetreatedanimals, there-fore,eliminatinganonspecificmusclerelaxationaswellassedative effectsofPEMCatthedosesused.Theseresultssuggestthatthe antinociceptiveeffectofPEMCisnotrelatedtosedativeeffectorto alterationinmotorfunction(deVasconcelosetal.,2011).

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Furtheranalysisoftheidentifiedpeaksbasedontherecorded UVspectrarevealedthatthefourpeaksfallsbetweenthemax

of 210and 310nm suggesting thepresence of flavonoid-based bioactivecompoundsasreportedbyAznametal.(2012).There arefivemajorsubgroupsofflavonoidsnamelyflavones,flavonols, flavanonols,flavanonesanddihydroflavonols(Tsimogiannisetal., 2007).Thepresenceofflavanones-basedbioactivecompounds,in particular,canbesuggestedbasedontherecordedUV–Visspectra obtainedforeachofthepeak(Aznametal.,2012).Accordingto

Aznametal.(2012),severalflavonoidsthathave methoxyland hydroxylgroups,detectedattheUVspectrathatrangebetween 210and290,havebeenisolatedfromfractionsofmethanolextract ofKaempferiarotunda. Recentstudiesby MohamadYusofet al. (2013)tendtosupportthepresenceofflavanonesor methoxyl-andhydroxyl-containedflavonoidsinPEMCastheauthorshave successfully isolatedseveralantinociceptive-bearing flavanones, namely 5-hydroxy-3,7,8-trimethoxyflavone, 3,7-dimethoxy-5-hydroyflavone, 2′_,4′_-dihydroxy-3′_{-methoxychalcone,} _and 8-hydroxy-6-methoxyflavone(calaburone).

Itisreasonabletoconcludefromourfindingsinthepresent studythattheantinociceptivepropertiesofPEMCoccurvia interac-tionofavarietyofphysiologicalpathwayswithinthePNSandCNS. PEMCappearstoactasanagonistforpotassiumchannels,opioid, cholinergic,adenosinergic,adrenergic,andserotonergicreceptors, whileatthesametimeinhibitingthePKC,vanilloidreceptorsand glutamatergicsystem.Fig.13representsamodelofthe mecha-nismsof PEMC’santinociceptiveactionbased onthesefindings. Theseactivitiescouldbeduetothesynergisticeffectofflavonoids, triterpenes,tanninsandsteroids.Theknowledgegainedfromsuch studiesinthepresentandfuturewillbeusefulforthedesignor dis-coveryofnewchemicalentitiesthatexhibitsimilarantinociceptive activity.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contribution

ZAZconceivedofthestudy,participatedinitsdesignandhelped todraftthemanuscript.Allauthorsreadandapprovedthefinal manuscript.MHMScarriedouttheexperimentsanddraftedthe manuscript. AKA participated in the experimental design, and helpedtodraftthemanuscript.TLKandMZSinvolvedinthe statis-ticalanalysisandmanuscriptpreparation.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This study was supported by the Science Fund Research Grant (Reference no. 06-01-04-SF1127) awarded by the Min-istryofScienceTechnologyandInnovation(MOSTI),Malaysiaand

theResearch UniversityGrantScheme(Referenceno. 04-02-12-2019RU)fromtheUniversitiPutraMalaysia,Malaysia.Theauthors thankedtheFaculty ofMedicineandHealthSciences,Universiti PutraMalaysia,Malaysiaforproviding thefacilitiestocarryout thisstudy.

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