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Heliopsis longipes: anti-arthritic activity evaluated in a Freund's adjuvant-induced model in rodents

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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

Heliopsis

longipes:

anti-arthritic

activity

evaluated

in

a

Freund’s

adjuvant-induced

model

in

rodents

Carolina

Escobedo-Martínez

a,∗

,

Silvia

Laura

Guzmán-Gutiérrez

b

,

María

de

los

Milagros

Hernández-Méndez

a

,

Julia

Cassani

c

,

Alfonso

Trujillo-Valdivia

a

,

Luis

Manuel

Orozco-Castellanos

a

,

Raúl

G.

Enríquez

d

aDepartamentodeFarmacia,DivisióndeCienciasNaturalesyExactas,UniversidaddeGuanajuato,Guanajuato,Mexico

bCatedráticaCONACyT,DepartamentodeInmunología,InstitutodeInvestigacionesBiomédicas,UniversidadNacionalAutónomadeMéxico,México,DF,Mexico cDepartamentodeSistemasBiológicos,UniversidadAutónomaMetropolitanaUnidadXochimilco,México,DF,Mexico

dInstitutodeQuímica,UniversidadNacionalAutónomadeMéxico,México,DF,Mexico

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received26June2016 Accepted5September2016 Availableonlinexxx Keywords: Heliopsislongipes Affinin Anti-arthritic

CompleteFreundadjuvant Spilanthol

a

b

s

t

r

a

c

t

Thisstudyassessestheanti-arthriticeffectoftheaffinin-enriched(spilanthol,mainalkamide)hexane extractfromtherootsofHeliopsislongipes(A.Gray)S.F.Blake,Asteraceae,onaFreundadjuvant-induced arthritismodelinrodents.Theextractwasorallyadministeredatadoseof2,6.6,or20mg/kg;asignificant edema-inhibitoryactivityintheacuteandchronicphaseswasobservedwithadoseof2and20mg/kg, respectively.Theextractshowedhigheranti-inflammatoryandanti-arthriticeffectsthanthereference drugphenylbutazone(80mg/kg).Moreover,theextractpreventedtheoccurrenceofsecondarylesions associatedtothispharmacologicalmodel.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Rheumatoidarthritis (RA)isanidiopathicauto-immune dis-ease,characterizedbysymmetricalsynovitis in largeand small jointsthatmayleadtoprogressivearticulardamageanddisability (Mendoza-Vázquezetal.,2013).Analgesicandanti-inflammatory drugs,includingsteroids,areusedtosuppressthesymptoms.New therapies, like those targeting the tumor necrosis factor alpha (infliximab)ortheanti-CD20therapy(rituximab),whichinhibit theunderlyingimmuneprocess,havebeenproposed.However,all thesedrugshaveseveralundesiredeffects.Recentresearchaimsto discoverlong-actinganti-inflammatorydrugswithminimalside effects(Ekambarametal.,2010).Anumberofanimalmodelsare usedtoevaluatepotentiallyusefulcompoundsagainstRA,andthe choiceofaparticularmodeldependsontheimmunological prop-erties beingobserved in themodel and theirrelationwiththe humandisease.Amongtheavailablemodelsiscollagen-induced oradjuvant-induced(e.g.,Freund’sadjuvant)arthritisinrodents, spontaneousarthritisinducedbyTNF-␣orgeneticmodels,likethe transgenicanimalmodel.WhilenoRAmodelcanbeconsidered

∗ Correspondingauthor.

E-mail:[email protected](C.Escobedo-Martínez).

ideal,Freund’sadjuvant-inducedratarthritissharesseveraltraits withhumanarthritis,anditssensitivitytoevaluateanti-arthritic agentswasthebasisforitschoiceasthemodeltoassessthe activ-ityoftheHeliopsislongipes(A.Gray)S.F.Blake,Asteraceae,hexane extract(Ekambarametal.,2010;TanushreeandSaikat,2013).

RAprevalenceindevelopedcountriesis0.5–2%,withanannual incidenceof12–1200 per100,000inhabitants.Thefemale:male ratio is 2–3:1,and the peakage range of onset is30–55 years old,but itcouldoccuratanyage.InMexico,RAaffects1.6%of generalpopulation,beingthemainreasonforconsultationatthe Rheumatology Service (Mendoza-Vázquez et al., 2013; Guía de PrácticaClínica,DiagnósticoyTratamientodeArtritisReumatoide delAdulto,México:SecretaríadeSalud,2009).

H.longipesisaperennialherb,endemicinaregioncomprising partsofSierradeAlvarezandSierraGorda,nearthetripleborderof SanLuisPotosi,GuanajuatoandQueretaro,inCentralMexico.The commonnamesforthisplantarechilcuague,pelitre,goldenroot, andAztecroot(Cilia-Lópezetal.,2008).Theparalyzingandtoxic actionagainstfliesand otherinsects ofH.longipesrootextracts wasdiscoveredassimilarasthatofpyrethrinsduringWorldWar II(Acreeetal.,1945).

Fortunately,chilcuaguedidnotbecomeextinctafterthe expor-tationboom,eventhoughitswildpopulationwasseverelyreduced. Todate,landplotssolelydedicatedtothecultivationofthisspecies, http://dx.doi.org/10.1016/j.bjp.2016.09.003

0102-695X/©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

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disseminatedeitherbyseedorbycutting,canbefoundbetween GuanajuatoandQueretaro;therootisfullydevelopedafter2–3 years (García-Chávezet al., 2004).It is the most economically importantspeciesin itsgenus;itsrootis usedintheregionas a cookingcondiment,and it is added toalcoholicbeverages to improvetaste.Intraditionalmedicine,chilcuaguerootisusedto treatrespiratorydiseases,asananesthetictotreattoothand mus-cularaches,totreatbuccallesions,andasananti-parasitic(Cilia López,2007).SeveralalkamideshavebeenisolatedfromH.longipes, butaffinin,alsoknownasspilanthol ((2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide),wasidentifiedasthemainalkamideintheplant (Molina-Torresetal.,1996;García-Chávezetal.,2004;Hernández etal.,2009).Theoilysubstancecausesintenselipnumbnessand tinglingwithinafew minutesafterplacedinthemouth,and it alsostimulatessalivation(Correaetal.,1971).H.longipesisalso reportedtoproduceanalgesia andanti-inflammation inhuman dentalandoralpathologies(Colvardetal.,2006).Additionally,it hasbeenreportedthatH.longipesdichloromethaneextractshowed analgesicactivity,asdemonstratedinvitrobyreleasingof gamma-aminobutyricacid(GABA)inmousebrainslices;theincreasein GABArelease in a region of cerebral cortex leadsto sustained analgesia,favoringadescendentinhibitionofnociceptivespinal neurons(Ríosetal.,2007).

With regard to anti-inflammatory activity, Hernández et al. (2009)evaluatedH.longipesethanolicextractandpurifiedaffinin on mouse ear edema induced by either arachidonic acid (AA) orphorbol12-myristate13-acetate(PMA),demonstrating a sig-nificantanti-inflammatory effect.DE50values of0.8mg/earand 1.2mg/earwereobtained,respectively,intheAA-inducededema model; nimesulide (1mg/ear) was used as reference drug. In thePMA-inducededemamodel,theethanolicextractandaffinin showedadose-dependentanti-inflammatoryeffectwithDE50 val-uesof 2mg/earand 1.3mg/ear,respectively, usingindometacin (3mg/ear)asreferencedrug.

Ontheotherhand,Cilia-Lópezetal.(2010)evaluatedthe anal-gesicactivityofH.longipesethanolicextractandaffininandtheir effectonthecentralnervoussystemonamousemodel;bothaffinin (1mg/kg)andH.longipesrootethanolicextract(10mg/kg)showed ananalgesicactionsimilartoketorolac(6mg/kg),asassessedby paininductionwithaceticacidandthermalstimulation(hotplate); also,H.longipesextractandaffininshowedastimulanteffecton thecentralnervoussystemcomparabletocaffeine,asmeasuredby theIrwintest.Cari ˜no-Cortésetal.(2010)evaluatedthecytotoxic potentialofH. longipesethanolic extract byrecording the vari-abilityinthecountofmicronucleatedpolychromaticerythrocytes inducedby theextract andthe ratioof polychromatic erythro-cyteswithrespecttonormochromaticerythrocytes;nosignificant cytotoxiceffectwasobserved.However,brainhistopathological studiesshowednecroticchangesinthegraymatter,describedas polioencephalomalaciaandneurophagy,atadoseof1000mg/kg of ethanolic extract. No damage or histopathological change wasobserved in other organs (liver, heart, kidney, spleen, and lung).

AmongtherecentstudiesonH.longipesistheassessmentof ethanolicextractandaffininaspotentialanti-mutagenicand anti-carcinogenicagents.Theanti-mutagenicpropertiesofaffininwere evaluatedbyaddingittoseveralmutagens,eitherwithorwithout S9metabolicactivation,inSalmonellatyphimurium(TA98,TA100, and TA102 strains) cultures. Affinin significantly decreased the pointmutationsinducedby2-aminoanthracene(2AA)(40%)and decreasedtheDNAoxidativedamageinducedbynorfloxacin(NOR) (37–50%).Additionally,itshowedantioxidantpropertiescapable of reducing the rate of 2AA- and NOR-induced mutation in S. typhimuriumTA98andTA102,respectively,whichcouldbe rele-vanttotreatsomepainsymptomsrelatedtoanti-radicalactivity (Arriga-Albaetal.,2013).

Materialsandmethods Plantmaterial

Heliopsislongipes(A.Gray).S.F.Blake,Asteraceae,rootswere col-lectedonFebruary16,2014,2:14:59pm,withintheboundariesof alandplotinBeltrancommunity(N21◦16.427,W100◦0.2672)at 1780mabovesealevel,Xichumunicipality,inGuanajuato,Mexico. Afterproperidentification,fiveplantspecimensweredepositedin theIsidroPalaciosherbarium(SLPM),attheUniversidadAutónoma deSanLuis(No.048150).

Extraction

Dry,groundH.longipesroots(1285g)weremaceratedforthree consecutive times: First component extraction was performed with2.5 lof hexane for 2h; then, the recovered extract solu-tion(1750ml)wasevaporated,yieldingadarkyellow,viscousoil (26.386g,labeledasHl-A).Asecondmacerationwasperformedon thesameplantmaterialafterthefirstextraction,adding1.250lof hexaneandleftfor24h;extractsolution(1l)wasrecovered,and afterevaporationityielded12.29gofviscousoil,labeledasHl-B.A thirdandlastmacerationwasperformedasdescribedabove,using 1.5lofhexanefor4h,recoveringanextractsolution(900ml)and yielding3.469gofviscousoil,labeledasHl-C.Eachmacerationwas monitoredbythin-layerchromatography.TheHl-Asampleyielded 26.386gofaffinin(1),witha95%purity.

HPLCandNMRanalysis

Highperformanceliquidchromatography(HPLC)analysisofthe Hl-AsamplewasperformedinaWaters1525BinaryHPLCpump equipment,controlledbyBreezesoftware.A15cm×4.6mm,C18 Kromasil100-5columnwasused,coupledtoadiodearraydetector (maximumabsorbancewassetto230nm).Awater-acetonitrile gradientfrom60:40to30:70in25minwasusedforelution,ata flowrateof1ml/min(Fig.1).

Magneticnuclearresonance(NMR)measurementsforthe Hl-AsamplewereacquiredinanAgilent600MHz,DD2,Oneprobe equipmentfor1Hy13CinCDCl

3solution,usingtetramethylsilane asinternalstandard(Tables1and2).Chemicalshiftreadingswere comparedwiththosereportedintheliterature(Correaetal.,1971). Pharmacologicalassay

Animals

All experiments were performed in accordance with eth-ical standards for experimental pain research in animals (Zimmermann, 1983)andtheMexicanOfficialStandardfor ani-malcareand handling(NOM-062-ZOO-1999). Theexperimental protocol(CIBIUG-P01-2016)wasapproved andoverseenbythe Institutional Ethics Committee for Care and Use of Laboratory Animals of the Universidad de Guanajuato. Inbred Wistar rats (250–300g) and Balb/c mice(20–25g), obtainedfromthe Nat-uraland ExactScienceVivarium atUniversidad deGuanajuato, were used. Animals were housedin groups of six, under con-trolled temperature (23±2◦C) and humidity (55±10%), with a 12-h light/darkness cycle, and were familiarized with the

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2.50 2.00 1.50 AU 14 046 1.00 0.50 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Minutes 14.00 16.00 18.00 20.00 22.00 24.00

Fig.1.ChromatogramoftheHl-Asampleaffinin(1),injectedataconcentrationof1mg/mlandreadatawavelengthof230nm.

Table1

1HNMRspectraldataofHl-Acomponent(affinin)(600MHz).a

Protona Chemical

shift

Multiplicity Area Coupling constant(Hz) CH3-3andCH3-4 0.92 d 6 J=6.8 CH3-10 1.77 d 3 J=7.2 H-2 1.80 m 1 J=6.0 CH2-4andCH2-5 2.24,2.32 dc 4 J=18.0,6.0 CH2-1 3.13 t 2 J=6.9 H-6 5.26 dt 1 J=12.0,6.0 H-9 5.69 dc 1 J=13.7,6.7 H-2 5.86 d 1 J=15.0 H-7 5.96 t 1 J=11.0 H-8 6.28 ddbroadsignal 1 J=18.0,12.0 H-3 6.82 dt 1 J=15.2,6.7 N-H 6.0 Broadsignal 1 aDatarecordedinCDCl

3.Chemicalshifts(ı)areexpressedinppmwithrespectto

tetramethylsilane(TMS).Couplingpatternsarelabeledas:d,doublet;dd,doubletof doublets;dt,doubletoftriplets;t,triplet;dc,doubletofquartets;m,multiplet.All assignmentswerebasedonthefollowingexperiments:1H-1D,13C-1D,COSY

(corre-lationspectroscopy),HSQC(heteronuclearsinglequantumcoherence;couplingof

1H 13Ctoabond),andHMBC(heteronuclearmultiplebondcorrelation;coupling

of1H 13Cto2and3bonds).

Table2

13CNMRspectraldataofHl-Acomponent(affinin)(125.7MHz).a

Carbona Chemicalshift

C-10 18.30 C-3andC-4 20.16 C-5 26.42 C-2 28.58 C-4 32.12 C-1 46.91 C-6 124.26 C-8 126.70 C-7 127.63 C-2 129.43 C-3 129.89 C-9 143.36 C-1 166.17 aDatarecordedinCDCl

3.Chemicalshifts(ı)areexpressedinppmwithrespectto

tetramethylsilane(TMS).Allassignmentswerebasedonthefollowingexperiments:

1H-1D,13C-1D,COSY,HSQC,andHMBC.

environment one week before the experiments. Animals were allowedfoodandwateradlibitum.Immediatelyafterthe exper-iments,allanimalsweresacrificedinaCO2chamber.

Acutetoxicityassays

TheDL50 value peroswasdeterminedas reportedbyLorke

(1983). Mice were treated with Hl-A doses of 10, 100, or 1000mg/kg. Animalswere maintained under close observation overa14-dayperiod.Theweightoftheanimalswasmonitored throughouttheexperiments,andthedeathorsurvivalofthe ani-malswasregistered.

Anti-inflammatoryactivity

Arthritissyndromewasinducedbyaplantarintradermic injec-tion(througha20-gaugeneedle)intherighthindpawof0.05mlof completeFreund’sadjuvant(SigmaAldrich).Thismethod,reported byNewbould(1963),hasbeenwidelyusedtodetectpotential anti-inflammatorydrugswithanti-arthriticactivity.Amongtheseveral adjuvanttypesavailable,themostcommonlyusedin experimen-talanimalsisFreund’sadjuvant;twovarietiesofFreund’sadjuvant areused:IncompleteFreundAdjuvant(IFA)andCompleteFreund Adjuvant(CFA).IFAconsistsinamixtureofsurfactantandmineral oil,usuallyArlacelandDrakeol;CFAalsocontainskilled mycobacte-ria,usuallyMycobacteriumbovisBCGataconcentrationof1mg/ml orless(Rojas-Espinosa,2006).

Edema degree was evaluated by the volume displacement method,usingaPANLABdigitalplethysmometer.Volume displace-mentwasrecorded24hbeforeand 8,24, 48,72,and96hafter CFAinjection.Tocomplete theassessment ofactivityon arthri-tisinduction,volumevariationsinthehindpawwererecorded up to 25 days after injection. Phenylbutazone (reference drug, SigmaAldrich,80mg/kg)ortheHl-Asample(inadoseof2,6.6, or20mg/kg)wereadministeredp.o.24hbeforeCFAinjectionand dailyfor14daysafter.Theseverityandprogressionofsecondary lesionsintestanimalswerealsocompared.

Thepercentageofedemainhibitionwascalculatedforall ani-mals ineach treatmentgroupwithrespecttoavehicle-treated controlgroup.Differencesbetweencontrolandtreatmentgroups were analyzed by Tukey’s test. A p-value lowerthan 0.05 was regardedasstatisticallysignificant.

Results

Acutetoxicityassays

TheDL50valuep.o.fortheHl-Asamplewas775mg/kg. Anti-inflammatoryactivity

ProgressionofFreund’sadjuvant-inducedarthritisinrats

CFAinjectionintherighthindpawcausedinflammation,which peakedwithinthefirst8h.Then,inflammationslowlydecreased untilday6 post-injection,hadaslightincreaseafterday7,and showedasustainedincreaseuntiltheendoftheexperiment.At day16post-CFA-injectionanduntiltheendoftheperiodofstudy, inflamed lesions(secondary lesions)in the lefthind paw were detected,aswellasdeformedfrontpawsandincreasingthickness inearsandtail(Fig.2).

Dosing

EachtestedconcentrationoftheHl-Asampleandof phenylbu-tazone was administered daily for 15 days. The first dose was

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Fig.2.Secondarylesionsobservedontheday22afterplantarinjectionofcompleteFreund’sadjuvantinaratfromthecontrol,untreatedgroup.

Table3

EffectofHl-AonFreund’sadjuvant-inducededemainrats.

Time(h) Sample Dose(mg/kg) %Inhibition±SEM

8 Phenylbutazone 80 46.5±6.8* 8 Hl-A 2 18.9±7.5 8 Hl-A 6.6 14.5±8.1 8 Hl-A 20 17.5±8.6 24 Phenylbutazone 80 47.8±5.6* 24 Hl-A 2 42.3±4.2* 24 Hl-A 6.6 29.7±11.3 24 Hl-A 20 31.7±10.3

* DatawereanalizedbyANOVAfollowedbyTuke’stest.p<0.05.

administered1daybeforeCFAinjectionintherighthindpawpad (Newbould,1963).Frequentmeasurementsinthehindpawswith aplethysmometershowedthatthetreatmentwith phenylbuta-zoneandwiththetestedHl-Aconcentrations(2,6.6,or20mg/kg) inhibitedinflammationinthehindpawduringthedosingperiod. AsshowninFig.4,prophylactictreatmentwiththereferencedrug (phenylbutazone,80mg/kg)significantlyinhibitedthe inflamma-tory effect of CFA injection 8h after the stimulus, during the acute inflammation phase. This was observed again 24h after injection;additionally, administration p.o. of Hl-A at a dose of 2mg/kginhibitedtheprogressionofacute-phaseedemaby42.3% (F4,20=4.7)(Table3,Fig.3).

Duringinflammationchronicphase,significantvaluesof inhi-bitionpercentage after administrationp.o. of Hl-A at a doseof 20mg/kgwereobservedonlyondays19,20,23,and25afterCFA injection,withinhibitionpercentagesof31.1(F4,20=5.012),36.8 (F4,20=3.17),34.2(F4,20=3.56),and31.3(F4,20=4.30),respectively (Table4andFig.4).Ontheotherhand,Hl-Aadministrationatthe samedoseshowedahigherinhibitionpercentagethan phenylbu-tazoneinallchronic-phasedays.Secondarylesionsdidnotoccur duringthetreatmentperiodatallHl-Adoses(Fig.5),andratsfrom Hl-A-treatedgroupsshowedabetterjointmobility.

No significant difference was observed in corporal weight betweenphenylbutazone-treatednorHl-A-treatedrat groupsat anydosewithrespecttothecontrol,untreatedgroup.

Discussion

Anumberofanimal modelshavebeenproposedtoevaluate anti-inflammatoryactivity,liketheuseofsubstancessuchas for-malinofdextran,whichcauseacuteinflammationwheninjected inrathind paws.However,thesemodelsrequireadministering highdosesofanti-inflammatorydrugslikephenylbutazone,often neartoxicvalues,tosignificantlyinhibitinflammation(Domenjoz, 1960).Anothermethodisthesubcutaneousimplantofcotton pel-lets,amodelthatismoresensitivetosteroidanti-inflammatory drugsbutisrelativelyinsensitivetonon-steroidanti-inflammatory

1.2 1.0 0.8 ∗ ∗ ∗ 0.6 0.4 Change in f oot v olume (ml) 0.2 0.0 8h 24h Treatment mg/kg Vehicle Phen yl 80 H.longipes 2 H.longipes 6.6H.longipes 20 Vehicle Phen yl 80 H.longipes 2 H.longipes 6.6H.longipes 20

Fig.3.Acuteeffectofadministration(p.o.)ofphenylbutazone(80mg/kg)or Heliop-sislongipes Hl-Asample(2,6.6, and20mg/kg)on Freund’sadjuvant-induced inflammationinthehindpawoftherodentmodel(rat).Eachbarshowsthemeanof 5lectures±SEM.DatawereanalyzedbyANOVAfollowedbyTukey’stest.p<0.05. drugslikephenylbutazone(Winderetal.,1962).Themethodused inthisstudyisbasedonasyndromemoresimilartorheumatoid arthritis,whichiscapableofdetectinganti-inflammatoryactivity inawiderangeofdrugtypesusefultotreatrheumatoidarthritisin humans(Pearsonetal.,1961;PearsonandWood,1963).

Phenylbutazone,previouslyusedinothermodels,wasasuitable referencedrugtoinhibitinflammationcausedbyCFAinjectionin rathindpaw;noweight-losswasobservedasreportedwithother drugs(Newbould,1963).Inourstudy,phenylbutazoneinhibited CFA-inducedinflammationby46.5%8hafterinjection(Table3), averysimilarresulttothatreportedbyAbadetal.(1996),who recordedaninhibitionrateof40%24hafterstimulus,increasing toover70%at72handdecreasingagainto40%at96h,remaining constantuntiltheday15.Inourstudy,thecontroldrugwas dis-continuedonthechronicphase,butinhibitionrateremainedina rangeof23–27%,indicatingalong-termeffect.Ontheotherhand, aninhibitionrangeof27.2–36.8%wasobservedintheHl-A-treated group(20mg/kg),higherthanthatofthephenylbutazone-treated animals.

The process underlyingthe occurrence of secondary lesions in themodel hereinused isnon-infectious, and it is suggested

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0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 26 24 22 20 18 16 14 Change in foot volume (ml) Day

Vehi

cle

Phenyl

butazone 80 mg/kg

H. longipes 2 mg/kg

H. longipe

s 6.6 mg/k

g

Fig. 4.Effect administration (p.o.) of phenylbutazone (80mg/kg) or Heliopsis longipesHl-Asample(2,6.6,and20mg/kg)onthelong-term,Freund’s adjuvant-inducedinflammationphaseinthehindpawoftherodentmodel(rat).Eachpoint intime-courseshowsthemeanoffivelectures±SEM.DatawereanalyzedbyANOVA followedbyTukey’stest.p<0.05.

Table4

EffectofHl-AonFreund’sadjuvant-inducededemainrats.

Time(days) Sample Dose(mg/kg) %Inhibition±SEM

16 Phenylbutazone 80 23.8±3.0 16 Hl-A 2 25.8±3.0 16 Hl-A 6.6 17.9±6.8 16 Hl-A 20 29.6±5.9 17 Phenylbutazone 80 20.5±0.8 17 Hl-A 2 15.8±2.7 17 Hl-A 6.6 16.8±4.8 17 Hl-A 20 25.2±6.2 18 Phenylbutazone 80 22.4±3.4 18 Hl-A 2 26.0±2.9 18 Hl-A 6.6 13.5±3.5 18 Hl-A 20 30.3±5.3 19 Phenylbutazone 80 20.6±3.2* 19 Hl-A 2 31.9±4.0* 19 Hl-A 6.6 17.39±2.5 19 Hl-A 20 31.1±6.3* 20 Phenylbutazone 80 25.8±3.2 20 Hl-A 2 27.8±5.5 20 Hl-A 6.6 29.9±3.9 20 Hl-A 20 36.8±7.2* 21 Phenylbutazone 80 23.8±4.4 21 Hl-A 2 20.5±2.1 21 Hl-A 6.6 21.0±5.1 21 Hl-A 20 30.9±2.7 22 Phenylbutazone 80 27.41±4.5 22 Hl-A 2 8.6±3.1 22 Hl-A 6.6 19.0±3.4 22 Hl-A 20 27.2±5.4 23 Phenylbutazone 80 27.9±4.6 23 Hl-A 2 21.5±3.4 23 Hl-A 6.6 26.9±3.6 23 Hl-A 20 34.2±4.1* 24 Phenylbutazone 80 25.5±6.1 24 Hl-A 2 19.2±4.9 24 Hl-A 6.6 25.3±4.9 24 Hl-A 20 30.1±3.7 25 Phenylbutazone 80 26.0±5.7* 25 Hl-A 2 18.3±3.8 25 Hl-A 6.6 25.0±3.8 25 Hl-A 20 31.3±3.2*

*DatawereanalizedbyANOVAfollowedbyTuke’stest.p<0.05.

Fig.5.Statusoftherighthindpawofasubjectratonday22afterprophylactic treatmentwithHl-A(p.o.)atadoseof20mg/kg.

to be result of a generalized immune response to the compo-nentsoftuberclebacilli,disseminatedafterlocaladministration (WarsmanandSharp,1960).Neitherphenylbutazone-treatednor Hl-A-treatedratsatanydose(2,6.6,or 20mg/kg)showed sec-ondarylesions,incontrastwithcontrol,untreatedanimals(Fig.5); thisindicatesthattreatmentsmodifytheimmuneresponse.

Ontheotherhand,thehighestextractdose(20mg/kg)wassetto muchlowervaluethantheDL50valuehereinreported(775mg/kg, p.o.), which allows us to regard it as a low-toxicity substance (Lorke,1983).Extractaccumulationduetoadailyadministration for15daysfailedtocauseanytoxicitysignintheperiodofstudy. Therefore,hereinweprovideinformationsupportingthepotential anti-inflammatory usefulnessof H.longipes hexane extract (Hl-A),whichhasaffininasitsmainconstituentasshownbyHPLC andNMRanalysis.Thepotentialusefulnessasanti-inflammatory agentof theextractata low dose(2mg/kg)wasdemonstrated in the acute-phase inflammation of the model, while its anti-inflammatoryandanti-arthriticactivityathigherbutsecuredoses (20mg/kg)wasdemonstratedinthechronic-phaseinflammation ofthemodel,withnooccurrenceofsecondarylesions.

Conclusions

ThehexaneextractofH.longipes,ofwhichaffininisthemain constituent,haspotentialusefulnessasananti-arthriticagent.This studycontributestoprovideinformationsupportingthetraditional useofthisplantspecies.Itisnoteworthythattheavailable infor-mationaboutthisspecieswouldsufficetocreateamonographand suggest theproductionof a phytopharmaceutical,which would helpencouraginga standardized,high-qualitycultivationofthis plantspeciesandimprovetheeconomyofpeasantsinSierraGorda, Mexico.

Ethicaldisclosures

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

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

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Authors’contributions

CEMdesignedthestudy,supervisedthelaboratoryanddrafted thepaper.SLGGanalyzeddataandcontributedtodraftthepaper. MMHMevaluatedtheAnti-arthriticactivity,runningthe labora-torywork.JCcontributedtorecordNMRspectra.ATVandLMOC contributedtocriticalreadingofthemanuscript.RGEcontributed tothechromatographicanalysisofplantmaterialandthe isola-tionandidentificationofaffinin.Alltheauthorshavereadthefinal manuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest. Acknowledgments

Thefundingof14-IJDPP-Q182-44,Convocatoria“Investigadores Jóvenes2014”; CONCYTEG,401/2014,ConvocatoriaInstitucional deApoyoalaInvestigaciónCientífica2014–UG;UGTO-PTC-382, ConvocatoriaApoyoalaIncorporacióndeNuevosPTC-PRODEP-14; and1088/2016,Convocatoria InstitucionaldeApoyoala Inves-tigaciónCientífica2016-2017–UG,isthankfullyacknowledged. ThankstoTax.JoséD.GarcíaPérez(UASL)forspecimen identifica-tion,andDr.MuraliVenkatBasavanagforNMRspectrarecording. AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.09.003.

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