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
daDepartamentodeFarmacia,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-arthriticCompleteFreundadjuvant 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/).
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
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
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
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.
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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