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
Anti-inflammatory
action
of
Justicia
acuminatissima
leaves
Maria
Christina
S.
Verdam
a,
Fernanda
Guilhon-Simplicio
a,∗,
Gleyce
S.
Barbosa
a,
Alcineide
L.
Magalhães
b,
Cinthya
I.F.B.
Oliveira
b,
Patrícia
D.O.
Almeida
a,
Tallita
M.
Machado
a,
Marne
C.
Vasconcellos
a,
Emerson
S.
Lima
a,
Débora
T.
Ohana
a,
Maria
M.
Pereira
a aFaculdadedeCiênciasFarmacêuticas,UniversidadeFederaldoAmazonas,Manaus,AM,BrazilbInstitutodeCiênciasBiológicas,UniversidadeFederaldoAmazonas,Manaus,AM,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received24January2015 Accepted16May2015 Availableonline11June2015
Keywords: Justicia Inflammation Nitricoxide COX-2
a
b
s
t
r
a
c
t
InAmazonasState(Brazil),Justiciaacuminatissima(Miq.)Bremek.,Acanthaceae,leafteasareusedin
folkmedicinetotreatseveralinflammatoryillnesses.Inordertovalidatethismedicinalapplication,
weanalyzedtheacutetoxicityandantioxidant,antiedematogenicandantinociceptivepotentialsofan
aqueousextractofthisspecies,usingculturecellsandanimalmodels.Theaqueousextractdidnotcause
toxiceffectsonhumanlymphocytesinhighconcentration(400g/ml),neitheronmicetreatedwithhigh
doses(5000mg/kg)inanacutetoxicityanalysisbyoralroute,andalsodidnotcauselesionsinthegastric
mucosaofanimalstreatedwith300mg/kg,whichwasthemaximaldoseusedintheanti-inflammatory
screening.Theaqueousextractcausedinhibitionofinflammatorypaininformalin-inducedpawlicking
testwithalltesteddoses,30,100and300mg/kg,andantiedematogenicactivityat100and300mg/kg.
Additionally,theaqueousextractpresentedstatisticallysignificantactiononthereleaseofnitricoxideby
lipopolysaccharide-activatedmacrophages.Theseresultsandotherpreliminarystudiessupportthefolk
useofthisspecies,andfurtherinvestigationofitsactionmechanismbyinhibitionofCOX-2orrelated
metabolitewouldbeinteresting.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Inflammationplays a crucial role in thepathogenic mecha-nismsofvariousdiseases,sinceitistriggeredbyvariousstimuli ofphysical, chemical andbiological origin(Ganesh, 2014).Two of the most prominent molecular mechanisms implied in the inflammatoryresponse,amongmanyothers,aretheproductionof nitricoxide(NO)byinducibleNOsynthase(iNOS)andtherelease of prostaglandins by cyclooxygenases (COX). While the use of iNOSinhibitorsasanti-inflammatorymedicinesstillrequiresmore studies(Rochetteet al., 2013), non-steroidal anti-inflammatory drugs(NSAIDs)areanimportantgroupofdrugs largelyutilized intreatmentofinflammatorydiseases,actingbyinhibitionofCOX enzymes(Blackleretal.,2014).However,theuseofthesemedicines isfrequentlyrelatedwithdisordersongastricmucosa,aswellas therenalandcardiovascularsystems(Harirforooshetal.,2013).
Plantextractsarevaluableleadsformulti-targetdrugsduethe interplayoftheirmultiplecomponents.Itbecomesmoreevident that,forcomplexdiseaseslikeinflammation,aninterferencewith
∗ Correspondingauthor.
E-mail:guilhonsimplicio@ufam.edu.br(F.Guilhon-Simplicio).
multipletargetsissuperiortotargetingasinglekeyfactorregarding drugefficiency,sideeffectsandadversecompensatorymechanisms (KoeberleandWerz,2014).Indeed,variousresearchgroupshave studieddifferentplantsusedintraditionalmedicineforthe dis-coveryofnewanti-inflammatorydrugs,whicharemoresafeand efficient.
Intraditionalmedicinearoundtheworld,thelargestgenusof Acanthaceae family, Justicia, is usedfor treating various health complications,includingtoothache,heartdiseases, gastrointesti-naldisturbances and even AIDS. In Brazil, differentspecies are usedfortreatingpain,fever,respiratoryproblemsandheadaches, andmanypharmacologicalpropertieswerescientificallyproven, includinganti-inflammatoryactivity(CôrreaandAlcântara,2012). Ontheotherhand,leafteaofJusticiaacuminatissima(Miq.) Bre-mek.,anativesubshruboftheBrazilianAmazonrainforest,known as“sara-tudo”,isusuallyusedinfolkmedicinetotreat inflamma-toryillnessintheAmazonasState,althoughitspharmacological potentialwasnotknownuntilrecently.
Astudyinvestigatedphytochemicallyandpharmacologicallya few organicextracts andsubstances obtainedfromthis species (Côrrea,2013).However,theanti-inflammatorypotentialandthe possibletoxiceffectsof anaqueousextractafteroral consump-tion, mimicking traditional use, remained unknown. Therefore,
http://dx.doi.org/10.1016/j.bjp.2015.05.002
thisstudyaimstoinvestigatetheanti-inflammatorypotentialofJ.
acuminatissimaaqueousextractbyoralrouteinordertovalidateits
popularuse.Weanalyzedcytotoxiceffectsonhumanlymphocytes, acutetoxicityonexperimentalanimalsandapossibleulcerogenic effectafteroraladministrationtoevaluatethesafeofitsuse.We analyzedits inhibition capacityof therelease of NOby human macrophagesstimulatedbylipopolysaccharides,andalsothe anal-gesicandantiedematogenicactivitiesinexperimentalanimalsin ordertoformulatethepreliminaryconsiderationsaboutitsalleged anti-inflammatorypotential.
Materialsandmethods
Plantmaterial
The collection of samples was authorized by the Managing CounciloftheGeneticPatrimony(CGEN),chairedbytheMinistry ofEnvironment(Brazil),undertheregistration034/2008.Leavesof
Justiciaacuminatissima(Miq.)Bremek.,Acanthaceae,werecollected
atCareiroCastanho,100kmsouthofManaus,capitalofAmazonas state,Brazil.Avoucherspecimen(224414)wasdepositedatthe HerbariumofInstitutoNacionaldePesquisasdaAmazônia,after identificationandtaxonomicauthenticationbyJoséLima.
Preparationoftheextract
Theleavesweredriedat40◦Cinastovefor72h(WHO,2003), andthenscrapedandpulverizedusingamechanicalgrinder.The powderwasmaceratedwithdistilledwaterunderagitationfor72h (10%aqueousextract).Theextract,namedPMFAinthiswork,was thenvacuum-filtered,lyophilizedandconserved2–8◦Cforfurther use.
Cellculture
HumanlymphocyteswereisolatedusingagradientofFicoll– Histopaque(1blood:1ficoll)fromperipheralvenousblood(5ml) collectedinheparinizedvialsfromonenormal,healthydonor,with nohistoryofsmoking/drinkingorchronicuseofmedication.J774 murinemacrophagecellswereobtainedfromRiodeJaneiroCell Bank,RiodeJaneiro,Brazil,andculturedat37◦Cinahumidified incubatorwith5%CO2inRPMI-1640mediumcontaining10%fetal bovineserum(FBS),50U/mlpenicillinand50g/mlstreptomycin (Sigma).
Animals
Male and female Swiss mice (30±5g) and Wistar rats (160–220g)weremaintainedina temperature-controlledroom (23±2◦C) with a 12/12h light–dark cycle and acclimatized in theexperimentalenvironmentfor atleast24hbeforethetests. Waterandabalanceddietwerecontinuallyprovidedadlibitum, butfoodwaswithdrawn12hbeforethetests.Thetestswere per-formedinaccordancewiththeConselhoNacionaldeControleda Experimentac¸ãoAnimalandwereauthorizedbytheEthical Com-mitteeontheUseofAnimalsofUniversidadeFederaldoAmazonas, underregistration002/2015.
Cytotoxicityonhumanlymphocytes
Peripheral blood lymphocytes(PBL) wereseeded in 96-well plates (105 cells per well), and were treated with PMFA, and theAlamarBlueTMassaywasperformedonthem(Ahmedetal., 1994).PMFA (400–6.25g/ml) wasdissolved in dimethyl sulf-oxide(DMSO),added toeach well,and wasincubatedfor72h. Doxorubicinwasusedaspositivecontrol.Controlgroupsreceived
thesameamountofDMSO(0.1%).Fourhoursbeforetheendof theincubations,10lofAlamarBlueTMwasaddedtoeachwell. Thefluorescentsignalwasmonitoredusingamultiplatereader using 530–560nm excitation wavelength and 590nm emission wavelength.Thefluorescentsignalgeneratedfromtheassaywas proportionaltothenumberoflivingcellsinthesample.
Acutetoxicity
Six mice (three male and three female) received orally the maximal doseof5000mg/kgof PMFAdissolvedin freshwater. Twoanimals(1maleand1female) wereusedasnegative con-trolandreceived10ml/kgoffreshwater.Abnormalmorphological andbehaviorsignsoftoxic/pharmacological effects(Hippocratic screening)wereobservedduringthefirst4hafteradministration ofextractandatevery24hfor14days(MaloneandRobichaud, 1962).
Ulcerogeniceffect
Threedifferentgroupsofsixrats(threemaleandthreefemale) receivedorally300mg/kgofPMFA,10mg/kgofindomethacin (pos-itivecontrol)and10ml/kgoffreshwater(vehicle,negativecontrol). Theanimalswerethenanesthetizedandsacrificedbycervical dis-location.Theirstomachswereremovedandopenedalongthegreat curvature,andtheirinnersurfacewasexaminedinordertolook forgastriclesions(Lapaetal.,2008).
NOproductionassay
After the pre-incubation of J774 murine macrophages (1×105cells/ml) with different concentrations of PMFA for 2h,thecellswerewashedandincubatedfor24heitherwithor without bacterial lipopolysaccharide (LPS, 1g/ml) at 37◦C in a 5%CO2 incubator.NO wasmeasuredasnitrite(NO2−),in the culturesupernatantbyreactionwithGriessreagent,followedby measuringat532nmusingamicroplatereader(DTX800,Beckman Coulter®,California,EUA).Sodiumnitritewasusedasastandard tocalculatenitriteconcentrations(Vermaetal.,2012).
Formalin-inducedpawlickingtest
Groupsofsixmice(threemaleandthreefemale)received30, 100and300mg/kgofPMFA,10mg/kgofindomethacin(positive control1),50l/kgoffentanyl(positivecontrol2)and10ml/kg offreshwater(vehicle,negativecontrol),allorallyadministered. Onehourafter,formalinat2.0%wasinjectedintherighthindpaw ofalltheanimals,andthedurationofpawlickingwascountedin periodsof0–5min(earlyphase)and15–30min(latephase).The resultswerecomparedwiththenegativecontrol(Lapaetal.,2008).
Carrageenan-inducedpawedema
Dosesof30,100and300mg/kgofPMFAinfreshwaterwere administered orally in different groups of six rats (three male and three female). Twoother groups receivedindomethacinat 10mg/kg(positivecontrol)andwater(negativecontrol).After1h, 0.3mlof-carrageenan 2%in normalsalinewasinjectedinthe righthindpawofallanimals,andthepawedemaprogressionwas measuredusingadigitalplethysmometer,inhourlyintervals,for 5h(Winteretal.,1962).
Statisticalanalysis
15
10
5
0
Gastr
ic lesion inde
x
A
C
*
B
Water control10 ml/kg
PMF A
300 ml/kg Indomentaci
on
10 ml/kg
Fig.1. EffectofaqueousextractofJusticiaacuminatissimaonthegastricmucosaofrats.(A)Gastricmucosadamageafterindomethacinadministrationat10mg/kg.(B)Gastric mucosaafteradministrationoftheextractPMFAat300mg/kg.(C)ComparisonofthehighestconcentrationofPMFAanalyzedandpositiveandnegativecontrolsanalyzed byone-wayanalysisofvariance(ANOVA)/Dunnett’stest,p-valuesversusnegativecontrol:*p<0.05.
(ANOVA)usingGraphPadPrism6®software(SanDiego,California, USA),consideringvaluesofp<0.05statisticallysignificant.
Resultsanddiscussion
Wedidnotobserveanycytotoxiceffectsinhumanlymphocytes exposedtomaximaldoseof400g/mlofPMFAduring72h.In addi-tion,theanimalsexposedtoveryhighdoseofPMFA(5000mg/kg p.o.)didnotpresenttoxicitysignsandnodeathoccurredduring Hippocraticscreening.TheseresultssuggestedthattheuseofJ.
acuminatissimaleafteabyoralrouteissafe,althoughtoxicityafter
longtimeexpositionshouldbefurtherinvestigated.Thus, three spaceddoses,30,100and300mg/kg,wereselectedforanalysisof theiranti-inflammatorypotential.
Considering that ulceration is one of the more deleterious collateraleffectsoftraditionalanti-inflammatorymedicines,the ulcerogenicpotentialofPMFAwasevaluatedafterthe adminis-trationofthehigherdosetestedintheanti-inflammatoryactivity study.InthisassayweobservedthatPMFAat300mg/kgdidnot inducestomachulcer,whereas indomethacin10mg/kg induced asignificantulceration,withp<0.05(one-wayANOVA/Dunnett’s test)incomparisontonegativecontrol(Fig.1).
Traditional NSAIDs inhibit both isoforms of COX and their adversegastrointestinaltoxicitiesarerelatedtotheinhibitionof gastroprotectiveprostaglandins,producedbyCOX-1pathway.The resultsofevaluationofgastriclesionsafterexpositiontohighdose ofPMFAareinagreementwithclinicalstudieswherenon-selective NSAIwascompared withCOX-2inhibitors andthe ulcerindex wasobserved,suggestingthatPMFAmightactbya mechanism independentofCOX-1inhibition(Kangwanetal.,2014). Empha-sizingthis,inapreviousstudywedemonstratedthatPMFAhasno effectonplateletaggregationinvitro(Verdametal.,2009),which isdependentonactionofthisenzyme(Dovizioetal.,2014).
NO released by activated macrophages has a critical role in the inflammatory response, including activation of the COX enzymes,resultinginexacerbatedproductionofpro-inflammatory prostaglandins.Furthermore,NOisapotentvasodilatorandhad theabilitytoincreasevascularpermeabilityandedemathrough changesinlocalblood flow(Gamperand Ooi,2014).PMFA sig-nificantlyinhibittheformationofNO2−,anoxidizedformofNO releasedbyLPS-activatedmacrophages.SincethisNOproduction alsoreflectstheactivityofiNOS(Vermaetal.,2012),thiseffectcan beduetoradicalscavengingactivityorinhibitoryeffectonenzyme (Fig.2).
Inbiologicalmedium,theoxidationofNOtocytotoxicradical anionperoxynitrite(ONOO−),bycombinationwithanion super-oxideradical(O2•−),isanimportanteventintheaggravationof inflammatoryprocesses,includingthoseinducedbycarrageenan (Salveminietal.,1996a),sinceithasalreadybeendemonstrated that inhibition of excessive production of NO reduces edema (Salveminietal.,1996b).Ontheotherhand,evidencesshowedthat NOandONOO−facilitatePGE2-inducedhyperalgesia,buthaslower hyperalgesicactionwhenactingalone(Ndengeleetal.,2008).
Theinjectionofdiluteformalinproducesapersistentbiphasic nociceptivestimulationnoted bythebehavioral painresponses of licking, flexing and jerking of the injected paw (Lariviere etal., 2011).Theearlyphase ofresponse (within 0–5minafter injection) comprises the neurogenic pain, while thelate phase (within 15–60min after injection) comprises the inflammatory pain(hyperalgesia)(Lapaetal.,2008).Inourstudy,PMFAdidnot inhibittheearlyphase ofthepain,indicatingthattheextractis noteffectivetocontrolneurogenicpain.However,PMFApresented anti-hyperalgesicactivitystatistically significantwithall tested doses(Fig.3),withoutdifferencebetweenthem,whichindicates, inadvance,thesignificantanti-inflammatorypotentialofJ.
acumi-natisimaandreinforcesthehypothesisofapossibleactionofPMFA
onCOX-2oraderivate.
–2 0 2 4 6 8
Nitr
ite dosage (
μ
mol/l)
Negativ e control
Positiv
e control PMF A 5
PMF A 10
PMF A 25
PMF A 50
μg/ml
0 50 100 150 200
Lic
king time (s)
Early phase Late phase
Negative control (water) PMFA 30 mg/kg
PMFA 100 mg/kg PMFA 300 mg/kg Fentanil 50 mg/kg
Indometacin 10 mg/kg
Fig.3. EffectofaqueousextractofJusticiaacuminatissimap.o.(30,100and300mg/kg),onthecontroloftheneurogenic(earlyphase)andinflammatorypain(latephase)of theformalintest.Two-wayANOVA/Bonferroni,p-valuesversusnegativecontrol:*p<0.05.
Edema is another important sign of inflammation. In the carrageenan-induced paw edema model, rats paw volume is recordedatdifferenttimepointsusingaplethysmometer,andthis parametermaybeusedforinferenceonanti-inflammatoryactivity ofvarioussamples(Winter etal.,1962).Twophasesof inflam-matoryresponsecausedbycarrageenanarewellestablished.The firstphaseoccursonehourafterpolysaccharideinjection,andis mainlyrelatedtothereleaseofhistamine,serotonin,bradykinin, plateletactivatingfactorandleukotrienes.Thesecondphase occur-ringwithinthefirstand thirdhours(peakof theinflammatory response)isduetotheproductsreleasefromCOX-1andCOX-2 metabolism(Vinegaretal.,1969).PGssuchasPGE2andPGI2 pro-ducedviatheCOX-2pathwaymagnifythedegreeofinflammation initiatedbyothermediatorsofinflammationsuchashistamineand bradykininleadingtoincreasedvascularpermeabilityandedema (Kawaharaetal.,2014).
PMFA presented statistically significant antiedematogenic activityinboth phasesaftercarrageenan injection(Fig.4), pre-sentingnodifferenceinrelationtopositivecontrol(indomethacin at 10mg/kg weight). This result, however, reaffirms the anti-inflammatory potential of the studied species. Comparing the resultsoftheevaluationoftheanti-hyperalgesicand antiedemato-genicactivitiesofPMFA,itcanbeinferredthatthepopularreports ofhealingofinflammatoryprocessesbyusersoftheJ. acuminatis-simaleafteasaremoredirectlyrelatedtopainrelief,whichisthe mosttroublesomesymptomofinflammation,sincealltesteddoses wereactive.
Wepreviouslyshowedthatcondensedtannins,coumarinsand saponins form the majority of the secondary metabolites in J.
acuminatissimaleaves(Verdametal.,2009).Thesaponinsinhibit
0.0 0.2 0.4 0.6 0.8
Edema v
o
lume (ml)
Early phase Late phase
Negative control (water) PMFA 100 mg/kg PMFA 300 mg/kg PMFA 30 mg/kg
Indometacin 10 mg/kg
Fig. 4.Effect of aqueous extract of Justiciaacuminatissima p.o. (30,100 and 300mg/kg)intheearlyandlatephasesofthecarrageenan-inducedpawedematest. Two-wayANOVA/Bonferroni,p-valuesversusnegativecontrol:*p<0.05,**p<0.01.
theglucocorticoidreceptors,whicharerelated toinflammatory response(Augustinetal.,2011).Thepharmacologicalinterestby coumarins is due, particularly, totheir bacteriostatic and anti-tumoractivitiesandtheircapacityofundoinghighproteinedemas (JainandJoshi,2011),whichmayalsocontributeforthe antiede-matogenic effect of PMFA. In turn, the presence of condensed tanninsin J.acuminatissima,thatare easilyextracted bywater, suggestsgoodantioxidantpotentialforPMFA,asshownintheNO productionassay.
Côrrea and colleagues (2014b) investigated the anti-inflammatorypropertiesofotherorganicextractsandcompounds isolated from J.acuminatissima, gettinggood results. They also isolatedfourluteolinheterosidesofanaqueousextractofJ.
acumi-natissima leaves, and confirmed its anti-inflammatory activity
(Côrrea et al., 2014a). Interestingly, these researchers used a modelwheretheinflammatoryresponsewasinflictedbyphysical damage(contusiononhindrightpaw)andthesubstances were administeredtopically.Here,weevaluatethecapacityofJ.
acumi-natissimatoinhibittheinflammatoryresponsecausedbychemical
stimuli (formalin and carrageenan) after oral administration, andtheresultshavebeenpromising.Thus,theanti-inflammatory potentialofJ.acuminatissimawasdemonstratedbydifferentroutes andstimuli,preliminarilybybasingitonitsethnopharmacological employment.
Inordertoaddmoreinformationtotheknowledgeofthe chem-icalcompositionoftheaqueousextractofJ.acuminatissima,we investigated thepresence oftwelve substances (friedelin,gallic acid,1,8-hydroxiquinone,lupeol,rutin,-stigmasterol,quercetin, p-cumaricacid,kaempferol,naringenin,scopoletinand umbillifer-one)inourextractbyHPLC–DAD.Noneofthesubstancesscreened were detectedby this method, however, thesubstances previ-ouslyidentifiedintheaqueousextractsupporttheobservedresults (Côrreaetal.,2014a).
WiththeresultsofstudieswithJ.acuminatissimauntilnow, we can conclude that the species is rich in biologically active compoundsandhasgoodanti-inflammatorycapacity,beingable tosignificantlyinhibitinflammationcausedbydifferenttypesof stimuli,especiallychemicalandphysical,withoutcriticaleffects afteracuteexposuretohighdoses.Thus, theseresultscan con-tributetothevalidationofthemainpopularuseofthespecies. Asnotedinourwork,itisinterestingtoinvestigateinan upcom-ingstudywhetherPMFAinterfereswiththeactionofPGE2orother relatedmediatorsinvolvedinthephysiologyofinflammatorypain.
Authors’contributions
ofthedata.FGScontributedinanalysisofthedataanddraftedthe paper.GSB,ALM,CIFBO,PDOAandMCVcontributedtobiological studies.TMMcontributedtochromatographicanalysisandanalysis ofthedata.ESMcontributedtocriticalreadingofthemanuscript. DTOandMMPdesignedthestudy,supervisedthelaboratorywork andcontributedtocriticalreadingofthemanuscript.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Theauthorsare gratefultoFundac¸ãodeAmparoàPesquisa doEstadodoAmazonas(FAPEAM)andCNPq(CT-AMAZONIA)for financialsupportofthisresearch.ESLisamemberofNational Insti-tuteofSciencesandTechnology–INCTRedoxoma.
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