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
Musanga
cecropioides
leaf
extract
exhibits
anti-inflammatory
and
anti-nociceptive
activities
in
animal
models
Abimbola
Sowemimo
a,∗,
Eboji
Okwuchuku
a,
Fageyinbo
Muyiwa
Samuel
b,
Olowokudejo
Ayoola
a,
Ibrahim
Mutiat
aaDepartmentofPharmacognosy,FacultyofPharmacy,UniversityofLagos,CollegeofMedicineCampus,Idi-Araba,Lagos,Nigeria
bDepartmentofPharmacology,TherapeuticsandToxicology,FacultyofBasicMedicalSciences,UniversityofLagos,CollegeofMedicineCampus,Idi-Araba,Lagos,Nigeria
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received23February2015 Accepted13July2015
Availableonline3September2015
Keywords:
Musangacecropioides
Anti-inflammatory Anti-nociception Phenol Flavonoid Mineralcontent
a
b
s
t
r
a
c
t
ExtractobtainedfromtheleavesofMusangacecropioidesR.Br.exTedlie,Urticaceae,atreegrowingin Africa,isusedtraditionallyinthetreatmentofedemaandrheumatism.Theanti-inflammatoryand anti-nociceptivepropertiesofethanolextractwerestudiedusingthecarrageenan,histamine,serotoninand xylene-inducededematestsaswellastheformalin,mousewrithingandtailcliptests.Significantdose dependentinhibitionwasobservedinthecarrageenanmodelwithpeakinhibitionat150mg/kg(71.43%, 90min,p<0.001).Inthehistamineandserotoninmodels,theextractcausedsignificantinhibitionof 83.33%(p<0.05)and45%(p<0.01)at120minrespectively.Forthexylenemodel,theextractshowed maximuminhibition(59.25%)at200mg/kg.Also,M.cecropioidesproducedsignificantanti-nociceptive activityinthemousewrithing(55.12%,p<0.01),formalin(81.88%,p<0.01)andtailclip(11.78%,p<0.001) testsat200mg/kgrespectively.Theresultsobtainedinthisstudydemonstratedthattheethanolicleaf extractofM.cecropioidespossessesanti-inflammatoryeffectpossiblymediatedviahistaminergicand serotonergicinhibitionandanti-nociceptiveeffectmediatedviaperipheralmechanismwithmildcentral involvement.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
MusangacecropioidesR.Br.exTedlie,Urticaceae,isa
decidu-ousorevergreen,dioeciousmedium-sizedtreeofupto30mtall
withanumbrella-shapedcrown.Traditionally,theleavesareused
toprepareavaginaldoucheforpainfulmenstruationandalsoused
inthetreatmentofgonorrheaandcough(Burkill,1985;Ayinde
etal.,2003).Thebarkdecoctionistakentotreatarterial
hyper-tension,constipation,painduringchildbirth,cough,diabetesand
schizophrenia.Thestemsapisusedtotreatdysmenorrhoeaandas
galactagogue,whiletherootsapisusedtotreatstomachspasms,
diarrhea,gonorrhea,pulmonarycomplaints,trypanosomiasis,skin
diseases,otitis,rheumatism,edema, epilepsy,andtoease
child-birth.Therootbarkiseatenwithkolanuttocurecough,andthe
barkistiedonwoundswhereitissaidtoeffectacure(Adejuwon,
2001).Thesheath-likestipulesareappliedasemmenagogueand
oxytocic,andtotreatstomachcomplaints,hiccoughandwounds
(Burkill,1985).
∗ Correspondingauthor.
E-mail:asowemimo@unilag.edu.ng(A.Sowemimo).
Pharmacologicalactivitiessuchastheuterotonic,antidiabetic,
hypotensiveandhypoglycemicpropertiesoftheleafandstembark
aswellastheantimicrobialactivityoftherootsapofM.cecropioides
havebeenreported(Kamanyietal.,1992,1996;Dongmoetal.,
1996;Ayindeetal.,2003,2006;Adeneyeetal.,2006,2007;Senjobi etal.,2012;Uwahetal.,2013).Thereis,however,norecordofthe
anti-inflammatoryoranalgesicactivityinliterature;hence,theaim
ofthisworkistoinvestigatetheanti-inflammatoryandanalgesic
propertiesoftheplantwithaviewtovalidatingitsethno-medicinal
use.
Materialsandmethods
Plantcollectionandextraction
TheleavesofMusangacecropioidesR.Br.exTedlie,Urticaceae,
leaveswerecollectedatAbatadu,Oke-ode,avillageabout3kmto
Ikere,OsunState,Nigeria(7◦30′N4◦30′E),inFebruary,2013.The
plantspecimenwasidentifiedandauthenticatedattheHerbarium
oftheDepartmentofBotanyandMicrobiology,UniversityofLagos,
Akoka,Lagos,Nigeriawhereavoucherspecimen(LUH5637)was
deposited.Theleaveswerepulverizedandmaceratedwithabsolute
http://dx.doi.org/10.1016/j.bjp.2015.07.022
ethanolatroomtemperature.Theextractwasfilteredand
concen-tratedusingtherotaryevaporator(Buchi,Switzerland).Theyield
was4.58%(w/w).
Animals
Wistar rats (100–200g) and Swiss albino mice (18–30g) of
eithersexusedinthisstudywerepurchasedfromtheAnimalHouse
oftheNationalAgencyforFoodandDrugAdministrationand
Con-trol(NAFDAC),Lagos.Theanimalsweremaintainedunderstandard
laboratoryconditions(12hlight/darkcycleat22±2◦C)andfed
standardrodent pellets(LivestockFeedPLC,Lagos,Nigeria)and
wateradlibitum.Theprotocolwasapprovedbythe
Experimen-tationEthicsCommitteeoftheCollegeofMedicine,Universityof
Lagos(CM/COM/08).
Acutetoxicity
Acuteoraltoxicityassaywasperformedonthreegroupsofsix
miceeachfastedfor12hpriortotheexperiment.Theplantextract
wasadministeredatdosesof1,2and3g/kg(p.o.).Theanimalswere
observedforimmediatesignsoftoxicityandmortalityfor24hand
furtherobservedforsevendaysforsignsofdelayedtoxicity.
Pharmacologicalstudies
Invivoanti-inflammatoryactivity
Carrageenan-inducedpawedema. Increaseintherathindpaw
lin-earcircumferenceinducedbyplantarinjectionofthephlogistic
agentwasusedasthemeasureofacuteinflammation(Henriques
etal.,1987).Therats(n=6)wereadministeredtheplantextract
(50,100,150and200mg/kg,p.o.)whilethecontrolratsreceived
indomethacin(10mg/kg p.o.)and distilledwater(10ml/kg,p.o.)
respectively.Onehouraftertreatment,0.1mlofcarrageenan(1%,
w/v in water) was administered into the sub-plantartissue of
therighthindpaw.Thelinearpawcircumferencewasmeasured
immediatelybeforeinjectionofthephlogisticagentsandat30min
intervalfor 3husingthecottonthreadmethod(Bamgboseand
Naomesi,1981).
Anti-inflammatory activity is determined by analyzing the
reductioninedemasizeandcalculating%inhibitionofedema.A
meanreduction inedema when comparedwithcontrol andan
increase%inhibitioninthetreatedgroupsisanindicationof
anti-inflammatoryactivity.
Serotonin and histamine induced rat paw edema. In order to
elucidatethemechanismofactionofM.cecropioides,selected
anti-inflammatorymediators(histamineandserotonin)wereused.The
dosewhichgavemaximuminhibitioninthecarrageenanassayfor
wasusedforthetests.
Adult rats (100–200g) fasted overnight were divided into
three groups ofsix animalseach. Distilled water,10ml/kgwas
administeredtogroupone(control),grouptworeceived10mg/kg
indomethacinandgroupthree,150mg/kgM.cecropioides.All
treat-mentswere doneorally. One hour posttreatment, edema was
inducedbyinjectionof0.1mlserotoninorhistamine(10−3mg/ml)
intothesub-plantartissueoftherighthindpaw.Thelinear
circum-ferencesofthepawsweremeasuredusingcottonthreadmethod.
Measurementsweremadeat0minandthereafteratanintervalof
30minfor3h.Themeanofthepawsizewerecomputedand
per-centageinhibitionswerecalculated(AgbajeandFageyinbo,2011).
Xylene-inducedearedema. Adultmice(18–30g)fastedovernight
weredividedintosixgroupsofsixanimalseachandweretreated
asfollows:M.cecropioides(50,100,150and200mg/kg,p.o.),
dexa-methasone(1.0mg/kgp.o.)anddistilledwater(10ml/kgp.o.).Ear
edema wasinduced byapplying0.03ml ofxylene totheinner
surfaceoftherightear.Theleftearwasconsideredascontrol.
Fif-teenminutesaftertheapplicationofxylene,themicewerekilled
underetheranesthesiaandbothearswereremovedandweighed.
Increaseinweightcausedbytheirritantwasmeasuredby
sub-tractingtheweightoftheuntreatedleftearsectionfromthatof
thetreatedrightearsections(Nù ˜nezGuillénetal.,1997).
Anti-nociceptiveactivity
Mousewrithingtest.Miceusedforthisexperimentweredivided
intofivegroupsofsixanimalseach.GroupIwasgivendistilled
water(10ml/kg,p.o.);GroupIIreceivedthestandarddrug
acetyl-salicylicacid(100mg/kg,p.o.)whiletheremainingGroupsIII,IV
and V were given the plant extract (50mg/kg, 100mg/kg and
200mg/kg,p.o.)respectively.Sixtyminutesaftertreatment,acetic
acid(0.6%v/vinsaline,10ml/kgi.p.)wasadministered.Thenumber
ofwrithes(characterizedbycontractionoftheabdominal
muscula-tureandextensionofthehindlimbs)wascountedfor30min(Singh
andMajumdar,1995;MbagwuandAnene,2007).
Formalintest.Inthisstudy,theanimals(n=6)werearrangedinto
fivegroupswhichreceiveddistilledwater(10ml/kg),extract(50,
100and200mg/kg)andmorphine(10mg/kgs.c.)respectively.For
theinductionofpain,formalin(20lof1%solution)wasinjected
intosub-plantartissueoftherighthindpawofeachmouse60min
afteradministrationfortheoralrouteand30minforthe
subcuta-neousroute.Thenociceptiveresponsewasconsideredasthetime
spentinlickingandbitingoftheinjectedpaw.Theresponsesofthe
micewereobservedfor5min(firstphase)and15–30min(second
phase)postformalininjection(Shibataetal.,1989;Viannaetal.,
1998).
Haffner’s tail clip test. Mice used in this experiment were
pre-screenedbyplacingametalarteryclip1in.fromthebaseofthe
tailandanimalswhichdidnotrespondtotheclipplacementby
turningorbitingattheclipwithin10swerediscarded.Eligible
miceweredividedintofivegroupsoffiveanimalseach.The
pre-treatmentreactiontimeofallmicetoclipwasdeterminedafter
whichtheanimalsweretreatedasfollows:Group1:distilledwater
(10ml/kg),Groups 2, 3 and 4 weretreated withthe extractat
50mg/kg,100mg/kgand200mg/kgrespectively,Group5:
Mor-phine10mg/kg s.c.Thepresence orabsenceofanti-nociceptive
activity was determined 60min after drug administration for
oraladministrationand30minfor subcutaneousadministration
(Adeyemietal.,2004).Apost-treatmentcut-offtimeof30swas
used(Adeyemietal.,2004;AgbajeandAdeneye,2008).
Quantitativeanalysis
Determinationoftotalphenolcontent
Thetotalphenolic contentoftheextractwasdeterminedby
the modified Folin-Ciocalteu method (Wolfe et al., 2003).
Gal-lic acid was usedas a standard with a concentration range of
0.01–0.05mg/mlpreparedinmethanol(FolinandCiocalteu,1927).
Theextract(0.5ml)(0.1mg/ml)togetherwiththegallicacidwas
mixedwith2.5mlFolin–Ciocalteureagent(previouslydilutedwith
distilledwater1:10,v/v)and2ml(75g/l)ofsodiumcarbonate.The
mixtureswerevortexedfor15sandallowedtostandfor30minat
roomtemperaturebeforetheabsorbancewasmeasuredat765nm
usingaspectrophotometer.Alldeterminationswereperformedin
triplicates.Thetotalphenoliccontentwasexpressedasmggallic
acidequivalent(GAE)pergramofsample.
Determinationoftotalflavonoidcontent
TotalflavonoidwasestimatedusingthemethodofMiliauskas
thesample.Toobtainthecalibrationcurve,aconcentrationrange
of0.01–0.05mg/mlwasusedforquercetin.Theabsorbancewas
measuredat420nmafter60minatroomtemperature.Thetotal
flavonoidcontentwascalculatedasmilligramquercetinequivalent
(QE)pergramofsample.
Determinationofmineralcontent
Powderedleafsample (1g) wasdigestedusing 10ml of1M
HNO3.Thesolutionwasthenfilteredandmadeupto50mlwith
distilledwater. Concentrationsofsodium,potassium, zinc,
cop-per,calcium,magnesium,ironandmanganeseweredeterminedby
atomicabsorptionspectrophotometry(Analyst200,PerkinElmer,
Waltham,MA,USA).
Statisticalanalysis
All values were expressed as means±SEM. Results were
analyzedbyone-wayANOVAfollowedbyDunnett’smultiple
com-parisonorbytwo-wayANOVAfollowedbyBonferronitestusing
GraphPadPrism6.Resultswereconsideredstatisticallysignificant
atp<0.05,p<0.01,p<0.001.
Results
Acutetoxicitytest
TheethanolleafextractofM.cecropioideswasfoundsafeatthe
dosestested(1,2,3g/kg).Duringthesevendaysassessmenttime,
thetestanimalswerefoundnormalwithnovisiblesignsofdelayed
toxicity.
Anti-inflammatoryactivity
Carrageenan-inducedratpawedematest
TheleafextractofM.cecropioides(50,100,150and200mg/kg)
produced a significant(p<0.05, p<0.01, p<0.001) inhibition of
edemarelativetocontrol(10ml/kgdistilledwater)inadose
inde-pendentmanner.Thehighestinhibitionofedema wasobtained
with150mg/kgdose(71.43%)oftheextractat90min(Table1).
Theextractcomparedeffectivelywithstandarddrugindomethacin
(10mg/kg)usedinthisstudy,whichproducedapeakinhibitionof
edema(68.47%)at30min.
Inflammatorymediators
Serotonin-inducedandhistamine-inducedratpawedema
Intheserotoninmodel,theextractofM.cecropioidesatadoseof
150mg/kggaveamildinhibitoryeffect(45.0%)at120minwhilethe
standardproducedamorepronouncedinhibitory(64.52%)effect
at150min(Fig.1A).Forthehistaminemodel,atthesamedoseof
extract,therewasapeakinhibitoryeffect(83.33%)relativetothe
control(distilledwater10ml/kg)observedat120min.Theextract
wasmore effectivethan thestandard (indomethacin 10mg/kg)
whichalsoproducedapeakinhibitoryeffect(57.14%)atthesame
timewiththeextract(Fig.1B).
Xylene-inducedearedema
Animalspre-treatedwithethanolicleafextractofM.
cecropi-oides(50,100,150and200mg/kg)producedasignificant(p<0.05)
inhibition of edema (Table 2). The effect produced was dose
dependentandcomparedeffectivelywiththestandarddrug
dexa-methasone(1mg/kg)usedinthestudy. Table
80
A
70
60
50
40
% inhibition 30
20
10
0
T30 min
Serotonin + Indomethacin Serotonin + Musanga T60 min T90 minT120 minT150 minT180 min
80 100
90
B
70
60
50
40
% inhibition
30
20
10
0
T30 min
Histamine + Indomethacin Histamine + Musanga T60 min T90 min T120 min T150 min T180 min
Fig.1. TheeffectsofMusangacecropioides(MC)andindomethacinonrathindpaw edemainducedby(A)serotoninand(B)histamine.Datarepresentedasmean±SEM (n=6).*p<0.05,**p<0.01and***p<0.001comparedtothecontrol(one-wayANOVA followedbyDunnett’smultiplecomparison).
Table2
EffectofMusangacecropioidesonxyleneinducedearedema.
Treatment Dose(mg/kg) Increaseinearweight
(mg)
%Inhibition
M.cecropioides 50 12.83±0.003 34.63
M.cecropioides 100 13.00±0.004 33.79
M.cecropioides 150 11.00±0.006 43.96
M.cecropioides 200 8.00±0.002* 59.25
Dexamethasone 1 4.80±0.002* 75.55
Distilledwater 10ml/kg 19.63±0.004 –
Valuesareexpressedasmean±SEM(n=6).
*p<0.05vs.control(one-wayANOVA,Dunnett’smultiplecomparisontest).
Anti-nociceptiveactivity
Mousewrithingtest
Writhingreflexwasproducedbyintraperitonealinjectionof
aceticacidincontrolanimalswith107.3±14numberofwrithes
countedin 30min. Theplant extract (50,100, 200mg/kg)
pro-ducedasignificant(p<0.01)reductioninthenumberofwrithes
inadosedependentmanner.Thepeakinhibitoryeffect(55.12%)
wasproducedatthedoseof200mg/kg.Thestandarddrug(Aspirin,
100mg/kg)alsoproducedasignificant(73.30%,p>0.01)reduction
inthenumberofwritheswithagreaterinhibitioncomparedtothe
extract(Fig.2).
120
100
80
60
Number of writhes
40
20
0
Control Aspirin MC 50 mg/kg
MC 100 mg/kg
MC 200 mg/kg
Fig.2.EffectofMusangacecropioides(MC)onmousewrithingtestinmice.Values areexpressedasmean±SEM(n=6).*p<0.01comparedtothecontrol(one-way ANOVAfollowedbyDunnett’smultiplecomparison).
120 140 160 180
100
80
60
Licking time (s)
40
20
0
Control Morphine MC 50 mg/kg
MC 100 mg/kg
MC 200 mg/kg
First phase Second phase
Fig.3. EffectofMusangacecropioides(MC)onformalininducedreflexinmice.Data representedasmean±SEM(n=6).*p<0.05,**p<0.01and***p<0.001comparedto thecontrol(two-wayANOVAfollowedbyBonferronimultiplecomparison).
Formalintest
Injectionof formalininto thesub-plantartissue of theright
hind pawof control micein thefirst phase produced
nocicep-tiveresponse ofbitingand lickingof thepawwithdurationof
116±26.98s.Theplantextract(50,100,200mg/kg)produceda
statisticallysignificant(p<0.05,p<0.01,p<0.001)dose
depend-entinhibitionofnociceptivereactionwithpeakeffectproduced
(66.81%) ata doseof 200mg/kg. Morphineproduced a greater
inhibitoryresponse(100%).Inthesecondphase,thedurationof
thenociceptivereactioninthecontrolgroupwas151.8±41.3.The
extract,inadosedependentmanner,significantlyinhibited
noci-ceptivereactionwithpeak effect(81.88%)at200mg/kg.In this
phase,theeffectproducedbytheextractcomparedeffectivelywith
morphine(96.4%)(Fig.3).
Haffner’stailcliptest
Applicationofthemetalarteryclipuntothetailofanimalsinthe
controlgroupelicitedreactionstowardclipremovalwiththe
post-treatmentlatencybeing3.18±0.86s,5.04±0.63sand1.17±1.11s
measuredat60,90,and120min,respectively,withapre-treatment
latency of 3.17±0.52s. The ethanolic leaf extract of M.
cecro-pioides(200mg/kg) produced a significant(p<0.01) increase in
Table3
TheeffectsofMusangacecropiodesontailclipreflexinmice.
Treatment Dose(mg/kg) Pre-treatmentlatencyperiod Posttreatmentlatencyperiod
60min 90min 120min
M.cecropiodes 50 3.061±0.84 6.14±0.64(5.418) 8.74±2.02(9.97) 6.25±0.79(5.56)
M.cecropiodes 100 3.26±0.65 8.77±1.51(9.71) 10.26±0.78(12.35) 7.49±1.03(7.45)
M.cecropiodes 200 2.94±0.36 10.84±1.60(13.86)* 12.65±2.27(17.02)* 9.66±0.89(11.78)*
Morphine 10 1.95±0.48 48.41±8.48(80.03)**** 45.75±9.88(75.45)**** 49.22±9.05(81.43)****
Distilledwater 10ml/kg 3.17±0.52 3.18±0.86 5.04±0.63 1.17±1.11
Valuesareexpressedasmean±SEM(n=6).Figuresinparenthesisrepresentpercentageinhibition.
* p<0.01vs.controlgroup(two-wayANOVAfollowedbyBonferronimultiplecomparisontest). ****p<0.001vs.controlgroup(two-wayANOVAfollowedbyBonferronimultiplecomparisontest).
Table4
MineralcompositionofMusangacecropioidesleaves(mg/gofdrymatter).
S/n Element Concentration(mg/g)
1 Sodium(Na) 1.08
2 Potassium(K) 0.85
3 Zinc(Zn) 0.31
4 Copper(Cu) 0.0075
5 Calcium(Ca) 1.47
6 Magnesium(Mg) 1.06
7 Iron(Fe) 0.94
8 Manganese(Mn) 0.35
post-treatment.Thiseffectwasmildcomparedtotheeffect
pro-ducedbymorphine(81.43%inhibition)at120min(Table3).
Quantitativeanalysis
Thetotalphenolicandflavonoidcontentsoftheextractwere
385.46±7.44(GAE/gofdriedextractmg/g)and255.86±12.53(QE
mg/g)respectively.
Mineralcomposition
The mineralcomposition of the leaves of M. cecropioides is
presentedinTable4.Ofallfourmacro-elements(Na,K,Ca,Mg)
investigated,calciumoccurredinthelargestamountandforthe
micro-elements (Zn, Cu, Mn, Fe), iron occurred in the largest
amount.
Discussion
Theuseofplantsasmedicinespredateswrittenhumanhistory.
Plantsareabletosynthesizeawidevarietyofchemicalcompounds
whichhavebeneficialeffectsonlong-termhealthwhenconsumed
byhumans,andcanbeusedtoeffectivelytreathumandiseases.
This study investigated the anti-inflammatory and
anti-nociceptive activities of M. cecropioides. Carrageenan, xylene,
serotonin and histamine tests were used to screen the
anti-inflammatoryactivitywhilemousewrithing,formalinandtailclip
testswereusedtoevaluatetheanti-nociceptiveactivity.
The paw edema induced by carrageenan involves several
chemical mediators such as histamine, serotonin, bradykinin,
andprostaglandins(Vinegaretal.,1987).M.cecropioidesextract
significantly(p<0.001)inhibitedtheedema size.Theeffect
pro-ducedbytheextractcomparedeffectivelywiththestandarddrug
(indomethacin)withapeakinhibitoryeffectat150mg/kgobserved
at90min.ThisresultsuggeststhattheextractofM.cecropioidesis
effectiveintheearlyphaseofinflammationwhichisprimarilydue
tothereleaseofhistamineandserotonin.
Fortheinflammatorymediators,theplantextract(150mg/kg)
showeda significant(p<0.05)inhibitionof serotonincompared
withthe control.The peak inhibition wasobtained at 120min
postinduction.Ontheotherhand,thesamedoseoftheextract
(150mg/kg)showedasignificant(p<0.05)inhibitionofhistamine
whencomparedwiththecontrol(10ml/kgdistilledwater).The
peakinhibitionwasobtainedat120min.Thesedataindicatethat
the plant extract is likely to interfere with histamine release
at the initial phase of inflammation. Thus, it can be
specu-lated that the inhibition of edema by the extract at the early
phaseofinflammation-inducedbycarrageenanmaybemediated
possiblythroughtheinhibitionofhistamine.However,the
differ-enceinthetimeofpeakinhibitionobservedinthecarrageenan
and histamine/serotonin models maybe due to interference of
othermediatorssuchasbradykinin,andprostaglandinswhichare
presentwhencarrageenanisusedinedemainduction.
Thexylene-inducedearedematestisusefulfortheevaluation
ofanti-inflammatorysteroids.Theswellinginducedbyxylenecan
causeanacuteinflammatoryresponsewhichmayleadtosevere
vasodilationandplasmaextravasations.Thismodelislinkedwith
phospholipase A2 which is involved in thepathophysiology of
inflammationdue to xylene(Zaninir etal., 1992; Akindeleand
Adeyemi,2007).In this study,dexamethasone,asteroidal
anti-inflammatory agent, produced significant reduction in the ear
edema.TheextractofM.cecropioidesalsosignificantly(p<0.05)
inhibitededemainducedbyxyleneandthissuggestsasteroidal
anti-inflammatoryactivityoftheplantpossiblymediatedbythe
inhibitionofphospholiphaseA2.
Intraperitonealinjectionofaceticacidelicitsaresponse
charac-terizedbyawaveofabdominalmusculaturecontractionfollowed
byextensionof thehindlimb(writhing).Thisresponse isused
toestablishtheperipheraland non-steroidalaction of an
anal-gesicdrugandisthoughttoinvolvelocalperitonealreceptorsat
thesurfaceofthecellliningtheperitonealcavity(Bentleyetal.,
1983; Berkenkopfand Weichmann, 1988;Nù ˜nez Guillén et al., 1997; Zakaria et al., 2008). The agentreducing the number of
writhingwillrenderanti-nociceptiveeffectpreferablyby
inhibi-tionofprostaglandinsynthesis,aperipheralmechanismofpain
inhibition(Duarteetal.,1988;Ferdousetal.,2008).Peripherally
actingdrugssuchasaspirinhavebeenreportedtoexhibit
anti-nociceptiveactivityinthewrithingtestonly(Zakariaetal.,2008).
M.cecropioidesextractproducedasignificantreductioninnumber
ofwrithesinthisstudyandthissuggestsaperipheralmechanism
ofactioninvolvingdirectactiononnociceptors,directinhibitionof
prostaglandinactionorindirectinhibitionofprostaglandin
synthe-sisbyinhibitionofcyclo-oxygenase(COX)activity(Franzottietal.,
2000).
Biphasic nociceptive response is produced by subcutaneous
injectionof1%formalinintothemicerighthindpaw.Thefirst
tran-sientphasewhichisshortlived,involvesdirecteffectofformalin
onsensoryC-fiberswhilethesecondprolongedphaseis
associ-atedwiththedevelopmentoftheinjuryinducedspinalsensation
whichisresponsibleforfacilitatedpainprocessing.Thisisacentral
sensitizationofthedorsalhornneuron,aprocesswhichoccurs
dur-inginflammatorypain(Rezayatetal.,1999;Ashoketal.,2006;Da
Rochaetal.,2011).Changesinthedorsalhornofthespinalcordare
etal.,1992).Centrallyactinganalgesicdrugs suchasmorphine
inhibitboth phaseswhileperipherallyactingdrugs inhibitonly
thelatterphase(Santosetal.,1994;Chenetal.,1995;Agbajeand
Adeneye,2008).Studieshaveshowntheinvolvementofserotonin,
histamine,substanceP,excitatoryaminoacidandprostaglandin
inthelatephaseofformalintestwithbradykininaffectingboth
phases(Tjølsenetal.,1992;DoakandSawynok,1997;Rezayatetal.,
1999).M.cecropioidesinhibitedboththeearlyandthelatephaseof
formalininducedpainbutgreaterinhibitionwasobservedinthe
latephasewhichcomparedfavorablywiththestandarddrug
(mor-phine)usedinthestudy.Thus,suggestingmoreperipheralaction
thancentraleffect.
Tofurtherconfirmtheinvolvementofthecentralmechanism
intheanti-nociceptiveeffectofM.cecropioides,thetailcliptest
wasused.Inthismodel,increaseinthepainreactiontime(latency
period)indicatesthelevelofanti-nociceptioninducedbythedrug
orextract. Theextract ofM.cecropioides produced a significant
(p<0.01)dosedependentincreaseinpainthresholdinmice.This
effectwashowevermildwhencomparedwiththestandarddrug
(morphine).Maximuminhibitionoftheextractwasobservedat
90minwhilethatofthestandardwasseenat120min.Thisfurther
confirmedthattheplantextracthasverymildcentralactivity.
Thisisthefirstreportoftheanti-inflammatoryand
antinoci-ceptive effects of M. cecropioides in literature. However, the
anti-inflammatoryeffect ofpomolic acidisolated fromCecropia
pachystachya, a member of the family Cecropiaceae has been
reported(Schinellaetal.,2008).
Phenoliccompoundsaresecondarymetabolitesfoundinplants.
Invitroandanimal studieshaveshown thatpolyphenolic
com-poundssuchasflavonoidsandphenolicacidscanexertmultiple
activitiessuchasanti-inflammatoryandanalgesiceffects(Larrosa
et al., 2010). Flavonoids have been reported to effect
anti-nociceptiveactivitybytargetingprostaglandins(Raoetal.,1998;
Rajannarayanaetal.,2001).Theresultsfromthisstudysuggestthat
theextractofM.cecropioidesisrichinpolyphenolsandthesemay
beresponsiblefortheobservedbiologicalactivities.
Theresultsfromthemineralcontentdeterminationbyatomic
absorptionspectrophotometryrevealed thepresenceof
magne-sium,manganese,copper,zinc, sodium,potassium,calciumand
iron.Theroleofmagnesiumininflammationhasbeenreported.
Magnesium deficiency in rats is said to lead to the activation
ofmacrophagesand elevationofplasmaconcentrationof
inter-leukin (IL) 6, which is a known mediator of the acute phase
response(Maieretal., 1997,2005).Manganeseisreportedasa
remedyforstrains, sprainsandinflammationasit hasthe
abil-itytoincreasetheleveloractivityofsuperoxidedismutase(SOD)
therebyincreasingantioxidantactivity(Murray,1996).Magnesium
andmanganeseblockcalciumchannels,thereby preventing
cal-ciumuptakeatthepresynapticterminalandsubsequentrelease
ofsynaptictransmitterhenceinhibitingnociception(Abdel-Azim,
2001;Giniatullinetal.,2003).Coppersupplementationhasbeen
reportedtohaveanti-inflammatoryeffectand thisis relatedto
itsabilitytoformcomplexesthatserveasselectiveantioxidants
(DiSilvestroandMarten,1990).Deficiencyofzinchasbeenreported
indiseases associated withchronicinflammationand oxidative
stresssuchasrheumatoidarthritis,diabetes,andcancers(Prasad,
2009).Ithasalsobeenreportedtoincreaseconcentrationof
inflam-matorycytokinesandoxidativestressandinduceapoptosisand
endothelial cell dysfunction(Brown et al., 2002; Prasad, 2008;
Overbeck et al., 2008). Reports suggest that calcium decreases
tumor-promotingpro-inflammatorymarkersintheplasmaof
spo-radiccolorectaladenomapatients.C-reactiveprotein(CRP),tumor
necrosisfactor-␣(TNF-␣)and interleukin-6(IL-6), specific
pro-inflammatorymarkersarereportedtobeelevatedinpatientswith
inflammatoryboweldisease(Kimetal.,2008;Groblewskaetal.,
2008).Calcium issaidtobindtobile acidsandfreefattyacids,
precipitatingthem fromsolutionin thecolonthereby reducing
oxidative stress and inflammation in the colon (Newmark and
Lipkin,1992).Itisalsoreportedtoactivatethecalciumsensing
receptorinvolvedincell-cycleeventsanddifferentiation
promot-ingcell-cellandcell-matrixadhesion(LamprechtandLipkin,2001).
ThepresenceoftheseelementsintheextractofM.creopioides
maycontributetoanti-inflammatoryandanti-nociceptiveeffects
observed.
Conclusively, the extract of M. cecropioides has shown
anti-inflammatoryactionmediatedpossiblythroughtheinhibitionof
histamineandanti-nociceptiveactionmediatedthrough
periph-eral mechanism with a mild central involvement. This study
justifiesthetraditionaluseoftheplantinthetreatmentof
inflam-mationandpainfulconditions.
Authors’contributions
Plantsamplecollection,identificationandthelaboratorywork
werecarriedoutbyEOandOA.FMSand IMcontributedtothe
biologicalstudiesandanalysisofthedata.SAdesignedthestudy,
supervisedtheworkandwrotethemanuscript.Alltheauthorshave
readthefinalmanuscriptandapprovedsubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
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