Rev. bras. farmacogn. vol.25 número2

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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

Toxicity

and

antinociceptive

effects

of

Hamelia

patens

Angel

Josabad

Alonso-Castro

a

_,

_Sarahi

_{Balleza-Ramos}

b

_,

_Alejandro

_{Hernández-Morales}

b

_,

Juan

Ramón

Zapata-Morales

a

_,

_Marco

_Martin

_{González-Chávez}

c

_,

_Candy

_{Carranza-Álvarez}

b,∗

a_Departamento_de_Farmacia,_División_de_Ciencias_Naturales_y_Exactas,_Universidad_de_Guanajuato,_Campus_Guanajuato,_Guanajuato,_Mexico b_Unidad_Académica_{Multidisciplinaria}_Zona_Huasteca,_Universidad_Autónoma_de_San_Luis_Potosí,_Ciudad_Valles,_San_Luis_Potosí,_Mexico c_Facultad_de_Ciencias_Químicas,_Universidad_Autónoma_de_San_Luis_Potosí,_San_Luis_Potosí,_Mexico

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6February2015 Accepted15March2015 Availableonline31March2015

Keywords: Hameliapatens Antinociceptive Toxicity

Gaschromatography–massspectrometry Traditionalmedicine

a

b

s

t

r

a

c

t

Manymedicinalherbsareusedinfolkmedicinewithouttakingintoaccounttheirtoxicity.Hameliapatens

Jacq.(Rubiaceae),aMexicanendemicspecies,isusedfortheempiricaltreatmentofpain.Theaimofthis workwastoevaluatethetoxicityandantinociceptiveeffectsofethanolicextractsofH.patensleaves. ThetoxicityofH.patensleaves(500–5000mg/kg)wasevaluatedinacute(14days)andsubacute(28 days)assays.Inthesubacuteassay,abloodanalysis(bothhematologyandchemistry)wascarriedout. TheantinociceptiveeffectsofH.patensleaves(50–200mg/kg)wereevaluatedusingthermal-induced nociception(hotplate)andthechemical-inducednociceptivetests(acidaceticandformalin).Inthe acutetoxicitytest,theLD50estimatedforH.patensleaveswas2964mg/kgi.p.and>5000mg/kgp.o., whereasinthesubacutetestHPEdidnotaffecthematologicalorbiochemicalparameters.In chemical-inducednociceptionmodels,H.patens(100and200mg/kgp.o.)showedantinociceptiveeffectswith similaractivitythan100mg/kgnaproxen.Inthehotplatetest,HPEat100mg/kg(17%)and200mg/kg (25%)showedmoderateantinociceptiveeffects.HPEcouldbeagoodsourceofantinociceptiveagents becauseofitsgoodactivityandlowtoxicity.

Introduction

Worldwide, thereis an increasinginterest forusing medici-nalplants for the preventionand treatmentof many diseases. Thegeneralpopulationperceivesherbalmedicinesassafebecause oftheirnaturalorigin,theirefficacyandtheirlongusein tradi-tionalmedicine.Nevertheless,manyplantextractsareusedinfolk medicinewithouttakingintoaccounttheirtoxicityaspect.

Hamelia patens Jacq., (Rubiaceae), commonly known as “bayetilla,”“trompetilla,” “coralillo,” or “hierbacoral, is a bush widelydistributed in tropical areasof the American continent. H. patens has shown antidiarrheal (Pérez et al., 1996), anti-inflammatory (Sosaet al.,2002), cytotoxic(Taylor etal., 2013), woundhealing(Gomez-Belozetal.,2003),antifungal(Abubacker etal.,2013),antibacterial(Camporeseetal.,2003)andvasorelaxant (Reyes-Chilpaetal.,2004)properties.Thechemicalconstituents isolated from this plant are pteropodine, isopteropodine, iso-maruquine, maruquine, narirutin, palmirine, rosmarinic acid, rumberine,(−)-hameline,tetrahydroalstonine,aricine,uncarineF,

∗ _{Corresponding}_author.

E-mail:candy.carranza@uaslp.mx(C.Carranza-Álvarez).

stigmast-4-ene-3,6-dioneand5,7,2′_,5′_{-tetrahydroxyflavanone}

7-rutiroside(Aquinoetal.,1990;Ahmadetal.,2012;Paniagua-Vega etal.,2012).

In traditionalmedicine,H. patensis usedasdiureticand for theempiricaltreatmentofpain,inflammation,rheumatism, dia-betes,woundhealing,gastritis,stomachache,snakeandscorpion bites,feverandothers(CoeandAnderson,1999;Leontietal.,2001; Andrade-Cetto,2009;Ahmadetal.,2012).Nevertheless,the toxic-ityandantinociceptiveeffectsofH.patensremaintobestudied.

In Mexican traditional medicine there are many plants that remaintobestudiedin terms oftheirchemical, biological and pharmacologicalproperties.Aspartofourvalidationof pharma-cologicaleffectsinMexicanmedicinalplants,thisstudypresents thatH.patensshowslowtoxicityandexertsantinociceptiveeffects.

Materialsandmethods

Reagents

NaproxensodiumwasobtainedfromTripharma(Distrito Fed-eral,Mexico).N,O-bis(trimethylsilyl)trifluoroacetamide(BSTFA) wasacquiredfromSigmaAldrich(StLouis,MO,USA). Buprenor-phine(BNP)wasfromScheringPloughMexico(DistritoFederal,

http://dx.doi.org/10.1016/j.bjp.2015.03.007

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A.J.Alonso-Castroetal./RevistaBrasileiradeFarmacognosia25(2015)170–176

Mexico). Ketamine and xylazine were obtained from Pisa Agropecuaria(Atitalaquia,Hidalgo,Mexico).

Plantmaterial

HameliapatensJacq.,Rubiaceae,wascollectedinthe municipal-ityofAxtladeTerrazas,locatedat21◦₂₆′_north_latitude_and₉₈◦₅₂′

westlongitudeinthesoutheasternportionofthestateofSanLuis Potosi,Mexico.Avoucherspecimen(HPHP-SP-102)wasdeposited atCentrodeInvestigacióndelareservadelabiosferadeMapimi.

PreparationofethanolextractofH.patensleaves(HPE)

PowdereddryleavesofH.patens(20g)wereextracted with ethanol usinga Soxthermapparatus(Soxtherm automatic, Ger-hadt,Germany) for4h.The extractwasfilteredby anacrodisk nylonmembrane0.45mmporesize(GelmanSciences, Northamp-ton,UK).Thesolventwasevaporatedtodrynessinavaccumrotator evaporatorbelow60◦_C._The_residue_was_protected_from_light.

Samplepreparationforgaschromatography–massspectrometry (GC–MS)analysis

Approximately10mgofHPEwastransferredtopolyethylene tubeanddissolvedwith2mlisooctane. Forsilylation,100␮lof BSTFAwasaddedtothesamplesandincubatedat100◦_C_for₁₀_min

inaCEMDiscovermicrowaveequipmentat150W,290psi.

GC–MSanalysis

AnalysisoftheHPEwasperformedongaschromatograph6890 (AgilentTechnology,SantaClara,CA,USA)withaselectivemass detector5973.DB-5HTcolumn(15m×0.25mmID,0.10␮mfilm thickness)wasusedfortheanalysis.Theoperatingconditionsof thecolumnwereasfollows:oventemperatureprogrammedfrom 100◦_C₍₃_min)_to₃₂₀◦_C_at₁₅◦_C/min,_and₂_min_hold._The_injector

temperaturewasmaintainedat320◦_C_and_the_volume_of_injected

samplewas1␮l.TheMSraninelectronimpactat71eVandMass spectraldata wereacquiredinthescan modeinthem/zrange 33–800.Theidentificationofcompoundswasperformedby com-paringtheirmassspectrawithdatafromNIST11(NationalInstitute ofStandardsandTechnology,USA),WILEY09.

Animals

Balb/corC57BL/6malemiceweighing25–30gorWistarmale ratsweighing150–200g,fromtheUniversidadAutónomadeSan LuisPotosianimalfacility,werehousedinisolatedcagesat24◦_C

under a light–dark cycle of 12:12. The animals were supplied withfoodandwateradlibitum.Theexperimentswerecarriedout accordingtoOfficialMexicanNormNOM062-ZOO-1999 (techni-calspecificationsfor theproduction, careand useof laboratory animals). The research also followed the Guidelines on Ethi-calStandardsforInvestigationsofExperimentalPaininAnimals (Zimmerman,1983).

Acutetoxicitytest

Six weeks old C57BL/6 mice were used. The acute toxicity test of theHPE, i.p. orp.o. administration, and theselection of doseswasperformedaccordingtothemethoddescribedbyLorke (1983).Mice(n=5pergroup)receivedHPEatdosesof500,1000. 2500,3700and5000mg/kgandthecontrolgroupreceivedsaline solution.Micewereobserveddailyduring14daysformortality, behavioralchangesandothertoxicsigns.Bodyweightofmicewas recordedatthebeginningandend oftheexperiment. Thedose

leadingto50%death(LD50)inmicewascalculatedbyregression

analysis(percentdeathvs.logdose).

Subacutetoxicitytest

EightweekoldWistarrats(n=10pergroup)wereused.HPE wasadministered orallyatdoses of500, 1000,2500,3700and 5000mg/kgdailyduring28days.Thecontrolgroupconsistedof ratsadministeredwithsalinesolution.Bodyweightofratswas recordedthroughoutthestudy.Attheendoftheexperiment,rats werefastedovernightandanesthetized witha single intraperi-tonealapplicationofketamine(50mg/kg)andxylazine(12mg/kg), forbloodcollectionbycardiacpuncture.

Bloodanalysis

Blood samples were collected into heparinized and non-heparinizedcentrifugetubes.Abloodanalysis(bothhematology and chemistry) was carried out. The heparinized blood was used for a hematological study which included the estimation of white blood cells (WBC) and differential leukocyte counts, platelet,hematocrit andhemoglobin (Hb).Thenon-heparinized blood was allowed to coagulate before being centrifuged and theserumwasseparated.Hematologicalanalysiswasperformed using an automatic hematological analyzer (XT-1800i, Sysmex, Mundelein,IL,USA).Theparametersofbloodsampleswere:red blood cells (RBC), hematocrit (HCT), mean corpuscular volume (MCV),meancorpuscularhemoglobin(MCH),andmean corpuscu-larhemoglobinconcentration(MCHC),neutrophils,lymphocytes, monocytes, eosinophils and basophils. Biochemical analysis of serumsampleswasperformedusinganautomaticchemistry ana-lyzer(Vitros250,OrthoClinicalDiagnostics,Rochester,NY,USA). Thebiochemicalparametersmeasuredwere:glucose,creatinine, blood urea nitrogen(BUN), alkalinephosphatase (ALP),alanine aminotransferase(ALT),aspartateaminotransferase(AST),glucose, creatinineandglycatedhemoglobin(HbA1c).

Antinociceptiveassays

Hotplate

For antinociceptive tests,Balb/c mice(n=8 per group) were used.Thehotplatetestwasconductedonathermostatically con-trolledheatedmetalplateatatemperatureof55±1◦_C,_according

to the method of Turner (1965). The time (in s) that elapsed betweenplacingthemouseonthehotplateandthe manifesta-tionofsignsofacutediscomfortsuchaslickingofthehindpaw orjumpinginanattempttoescapefromtheheatwastakenas thereactiontimeorlatency.Miceexhibitinglatencytimebetween 10and20swerechosen.Thelatencytimewasdeterminedat60, 120and240minaftertheapplicationofthefollowingtestsamples (n=8eachgroup):vehicle(salinesolution),BNP(1mg/kgi.p.)or HPE(50,100and200mg/kgp.o.).Acutofftimeof45swasallowed toavoidthermalinjurytothepaws.Theantinociceptiveresponse wascalculatedaccordingtothefollowingformula:

% Antinociceptiveresponse

= Reactiontime−basallatency Cutofflatency−basallatency×100

Formalin

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Table1

EffectofHPEonhematologicalparametersonsubacutetoxicitytestinrats.

Parameter Control HPEmg/kg

500 1000 2500 3700 5000

RBC(106_/ml) _9.62_±_0.54 _9.97_±_0.22 _9.11_±_0.92 _9.45_±_0.27 _8.82_±_0.20 _8.84_±_0.14

Hb(g/dl) 17.93±0.93 17.47±0.47 16.17±1.17 16.47±0.38 15.40±0.30 15.43±0.38

HCT(%) 48.13±2.73 49.00±1.45 48.53±4.37 47.80±0.60 45.17±0.87 44.13±1.0

MCV(fL) 50.03±0.67 49.13±0.59 53.30±0.70 50.57±0.95 51.23±0.31 49.93±1.31

MCH(pg) 17.93±0.32 17.50±0.26 17.87±0.29 17.43±0.12 17.47±0.23 17.50±0.46

MCHC(g/dl) 35.80±0.20 35.63±0.25 33.47±0.59 34.43±0.42 34.10±0.26 35.0±0.10

Platelets(103_/_␮_l) ₁₀₇₄_±_50.48 _977.7_±_27.06 _1022.3_±_81.8 _870.3_±_21.2 _847.6_±_93.4 _757.8_±_84.4

WBC(106₎ _2.61_±_0.49 _2.65_±_0.80 _5.39_±_0.24* _3.22_±_1.89 _2.05_±_0.58 _2.04_±_0.21

Neutrophils(%) 19.67±4.50 23.23±5.06 15.00±2.77 14.03±4.37 33.83±5.25* 25.53±2.58

Lymphocytes(%) 72.93±2.92 71.60±6.01 78.90±6.60 72.50±15.23 63.30±6.60 70.37±3.74

Monocytes(%) 2.70±0.35 4.37±1.99 3.23±2.67 3.03±2.35 1.87±1.15 3.50±1.18

Eosinophils(%) 1.13±0.21 0.97±0.74 1.53±0.43 1.17±0.50 0.43±0.40 0.47±0.06

Basophils(%) 0.23±0.11 0.23±0.11 0.33±0.13 0.17±0.15 0.57±0.17 0.23±0.13

RBC,redbloodcells;Hb,hemoglobin;HCT,hematocrit;MCV,meancorpuscularvolume;MCH,meancorpuscularhemoglobin;MCHC,meancorpuscularhemoglobin concentration;WBC,whitebloodcells.

* _{Statistically}_different_from_the_control_group_(ANOVA_followed_by_Tukey,_p_≤_0.05).

injectedwith30␮lof1%formalin(in0.9%saline)intothe subplan-tarspaceoftherighthindpaw,andindividuallyplacedintoglass cylinders.Thedurationofpawlickingwasrecordedat0–15min (firstphase)and15–45min(secondphase)afterformalininjection.

Aceticacid-inducedwrithing

TheaceticacidmethodwascarriedoutasdescribedbyKoster etal.(1959).Onehourpriortoaceticacidinjection,mice(n=8per group)orallyreceived100mg/kgNPX,HPEatdosesof50,100and 200mg/kgandsalinesolution(thevehiclegroup).Eachgroupwas administered10ml/kgbodyweight(i.p.)ofanaqueoussolution ofaceticacid(1.0%).Themicewereindividuallyplacedintoglass cylindersandobservedforthenumberofabdominalconstrictions andstretching,countedoveraperiodof0–30min.

Statisticalanalysis

All experimental values are expressed as the mean±the standarddeviationofatleasttwoindependentexperiments. Statis-ticallysignificantdifferencesfromthevehiclegroupwereidentified byStudent’st-testorANOVAwithposthocTukeytestforpaired data.Thelevelofp≤0.05wasusedtodeterminestatistical signifi-cance.AllcalculationswereperformedusingtheGraphPadPrism V.3softwaresystem(GraphPadSoftware,SanDiego,CA,USA).

Results

ChemicalcompositionofHPE

ThechromatogramofHPEusingGC–MSshowedthepresence ofseveralcomponents(Fig.1A),includingpolyphenol(catechin), hydroxycinnamicacid(caffeicacid),cyclohexanecarboxylicacids

(shikimic acid and quinic acid),diterpene (phytol), phytosterol (␤-sitosterol),triterpenes(squalene,oleanolicacid,ursolicacid), tocopherolsandalkaloid(mitraphylline)(Fig.1B).Themost abun-dantcomponentwasursolicacid(40%)(Fig.1B).

Acutetoxicity

TheLD50estimatedbytheLorkemethod(1983)was2964mg/kg

i.p.and>5000mg/kgp.o.Atdosesof2500mg/kg(i.p.orp.o.)or higher,neurologicaldeficitwasobservedinthemiceonlyinthe first4haftertreatment.Thesymptomsincludedimmobilityand sedation.HPEtestedatanyconcentration,administeredorallyor intraperitoneally,lackedoftoxiceffectsinbodyweightofmice.An autopsyattheendoftheexperimentalperiodrevealednoapparent changesinorgans.

Subacutetoxicity

Hematologicalparameters

HPE at dose of 1000mg/kg increased significantly (p≤0.05) theWBC count.Similarly, 3700mg/kg HPEcaused a significant (p≤0.05) increasein theneutrophilcount.Otherhematological parameterswerenotchangedbytheapplicationofHPE(Table1).

Biochemicalparameters

ThedataindicatedthatBUNdecreased(p≤0.05)inratstreated with5000mg/kgHPE.ThelevelsofASTdecreased(p≤0.05)inrats treatedwith3700mg/kgHPE,whereasthelevelsofALPincreased (p≤0.05) in the group of 2500mg/kg HPE. Other biochemical parameterswerenotalteredbytheadministrationofHPE(Table2).

Table2

EffectofHPEonbiochemicalparametersonsubacutetoxicitytestinrats.

Control HPE(mg/kg)

500 1000 2500 3700 5000

Glucose(mg/dl) 132.67±11.93 110±17.69 171.67±43.36 128.33±42.71 104.67±12.22 155.57±82.97

HbA1c(%) 3.2±0.20 3.17±0.06 3.50±0.00 3.40±0.00 3.26±0.00 3.36±0.00

BUN(mg/dl) 63.20±5.11 54.20±4.53 54.23±7.25 58.37±3.55 65.20±9.43 36.67±4.35*

Creatinine(mg/dl) 0.27±0.11 0.23±0.06 0.40±0.00 0.33±0.06 0.40±0.00 0.60±0.35

AST(U/l) 103.00±31.18 114.33±28.59 90.43±7.02 147.67±18.56 124.00±15.62 103.00±17.28

ALT(U/l) 116.33±53.25 83.00±13.0 47.00±16.64 87.67±17.74 20.33±2.89* 30.33±1.53

ALP(U/l) 137.77±34.27 144.67±19.50 188.67±29.67 229.00±29.72* 150.23±31.37 118.67±19.01

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A

B

Constituents Retention

time

%

glycerol 1.61 2.25

malic acid 4.44 0.11

shikimic acid 7.40 0.63

hexadecanoic acid 8.71 1.16

caffeic acid 9.33 0.17

phytol 9.51 0.48

α-linolenic acid 9.72 1.66

octadecanoic acid 9.89 0.47

dehydroabietic acid 10.57 0.40

bis (3-tert-butyl-2-hydroxy-5-methylphenyl) methane 10.74 0.29

squalene 12.79 4.52

catechin 13.19 3.02

-tocopherol 13.59 0.25

mitraphylline 13.65 4.02

-tocopherol 14.17 1.29

campesterol 14.57 2.67

-sitosterol 14.92 2.92

1-triacontanol 15.00 0.59

oleanolic acid 15.78 6.58

ursolic acid 15.97 40.59

1500000

1400000

1300000

1200000

1100000

1000000

900000

800000

700000

600000

500000

400000

300000

200000

100000

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Fig.1.GC–MSchromatogram(A)andchemicalconstituents(B)ofethanolextractofH.patensleaves(HPE).

HPEexertsantinociceptiveeffects

HPEshowedgoodantinociceptiveactivityintwoofthethree invivomodels.Inthehotplatetestat60minoftreatment,HPE showedthehighestantinociceptiveeffectsby6%(50mg/kg),17% (100mg/kg)and 25% (200mg/kg),incomparison tothevehicle group(Fig.2A).After120min,theantinociceptive effectofHPE

decreased. On the contrary, the highest antinociceptive effects (70%)ofBNP(1mg/kg)wereshownat120minoftreatment.These effectsremaineduptotheendoftheexperiment(Fig.2A).

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A

Time after administration (min)

0 60 120 180 240

Latency time (seconds)

0 10 20 30 40 Vehicle BNP 1 mg/kg HPE 50 mg/kg HPE 100 mg/kg HPE 200 mg/kg

**

Licking time (seconds)

0 100 200 300

400 Phase 1

Phase 2

50 100 200

HPE Vehicle

**

NPX 100 mg/kg

**

Treatments

B

**

0 10 20 30 40 50 60 70

50 100 200

HPE Vehicle

**

NPX 100 mg/kg

Number of writhings

Treatments

C

**

Fig.2. HPEexerts antinociceptive effects. Theantinociceptive effects ofHPE (50–200mg/kg)wereevaluatedusingthermal-inducednociceptionhotplate(A), andthechemical-inducednociceptivetestsformalin(B)andacidacetic(C).The positivecontrolBNP(1mg/kg)wasusedforthehotplatetest,whereasthepositive controlNPX(100mg/kg)wasusedfortheformalinandacidacetictests.Results representthemean±standarddeviation(SD).**denotesp≤0.05,comparedtothe

vehiclegroup.

(50mg/kg),30% (100mg/kg) and 39%(200mg/kg),respectively, whereasin thephase 2theantinociceptiveeffects ofHPEwere 60%(50mg/kg),69%(100mg/kg)and75%(200mg/kg)(Fig.2B).The antinociceptiveeffectsofHPEwerecomparablewiththosefound bythepositivecontrol100mg/kgNPX,whichshowed antinocicep-tiveeffectsby48%(phase1)and74%(phase2)(Fig.2B).

Intheacidacetictest,HPEdecreasedsignificantly(p≤0.05)the numberofwrithingsinadosedependentmanner,comparedto thevehiclegroup(Fig.2C).Themaximumpercentageinhibitionof aceticacid-inducedabdominalconstrictionsbyHPEwasfoundat 100mg/kg(57%)and200mg/kg(65%).Thiseffectwascomparable to100mg/kgNPX(70%)(Fig.2C).

Discussion

HameliapatensJacq.,(Rubiaceae),isusedintraditionalmedicine for theempirical treatment of pain(Ahmad et al., 2012). Nev-ertheless, the antinociceptive effects of H. patens as well as its toxicity remain to be studied. Toxicity tests are commonly used in the preliminary studies of medicinal plants to detect pharmacologicalactivities(MaloneandRobichaud,1962).Inthis study,thetoxicityofH.patensinacuteandsubacuteassayswas recorded.

Inourknowledge,thisisthefirstreportthatindicatesthe chem-icalcomposition of ethanolextract fromH. patensleavesusing GC–MS. Thecompoundsfoundin HPE hadnot beenpreviously reported.

Intheacutetoxicitytest,ifnomortalityisobservedatthedose of5.0g/kg,thedrugtestedcanbeconsideredasnottoxic(Hayes, 1989).TheresultsindicatedthatHPEorallyadministeredcouldbe consideredasnon-toxic.Nevertheless,HPEadministered intraperi-toneallycouldbeconsideredasmoderatetoxic.Thereductionin bodyweightisasimpleandsensitiveindexoftoxicity.Thedata indicatedthattherewerenochangesofbodyweightinmicetreated withHPE.

Severalhematologicalandbiochemicalparameterswere esti-matedinthesubacutetoxicitytestinratstoevaluatethesafeuse ofH.patens.TheresultsindicatedthatHPEdidnotinduceanemia ordisturbancesintheerythrocytesorinthehemoglobin produc-tion.AlthoughtherewasanincreaseinWBC(1000mg/kgHPE)and neutrophils(3700mg/kgHPE),thevaluesareinthephysiological limitsdescribedforthespecies(HarknessandWagner,1993).The increaseofneutrophilsmaybetheresultoftheadministrationof HPEasaphysiologicalresponseagainstforeigncompounds,which mightbeanormalresponsefromratorganism.Inratstreatedwith HPEtherewerenochangesintheotherhematologicparameters. ThesefindingssuggestthatH.patensdidnotinducehematologic alterations.

Thelevelsofserumglucoseandglycatedhemoglobinwerealso evaluated.Thesetwoparameterswerenotsignificantlychanged inallthegroups.Therefore,HPEdoesnotaffectthecarbohydrate metabolism.

Renalfunctionwasevaluatedbymeansofureaandcreatinine serumlevels.Renaldamageisconsideredwhenbothureaand cre-atinineareincreasedconcomitantly(Satyanarayanaetal.,2001). Thelevelofureadecreasedin theratstreatedwith5000mg/kg HPE.However,thevaluesareinthephysiologicallimitsofratspecie (HarknessandWagner,1993).

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Together,theresultsindicatethatH.patenscouldbe consid-eredasanon-toxicplant.Nevertheless,otherstudiesincludingthe mutagenic,genotoxicand carcinogeniceffectsofHPE shouldbe performed.Inaddition,asubchronictoxicitytestshouldbe con-ductedtoestablishtheadverseeffectsofarepeatedresponsetoH. patens.

Nociceptionisamechanismbywhichnoxiousstimuliare trans-mittedtothecentralnervoussystem(CNS).Thenociceptorsare pain-sensitiveneuronslocatedintheskin,vessels,muscles, fas-cia, jointsand viscera(Calixtoet al., 2000).In spitethe useof manyanalgesics,it isneededtofindnewantinociceptivedrugs withhigherorsimilarefficacyandpotencythancurrentanalgesics butwithlesstoxiceffects.Threemodelswereusedinthisstudy toevaluatetheantinociceptiveactions ofH.patens. Onemodel of thermal-induced nociception (hot plate) and two models of chemical-inducednociception(acidaceticandformalin).Thedoses ofHPEusedtoevaluateitsantinociceptiveeffectswereselected basedonthefollowingaspects: (1)theirlackof toxiceffectsas evaluated in acute and subacute toxicity tests, (2) on prelimi-narystudiescarriedoutinourlaboratoryand(3)toanalyzethe antinociceptiveactivityofHPE(50–200mg/kg)comparedtoNPX (100mg/kg).

Thehot-platetestisapredominantlyaspinalreflexthat eval-uatescentrallyactingantinociceptiveagents(Turner,1965).The resultssuggestthatHPEmightnotinducecentralantinociceptive effectsbecauseHPEincreasedmoderatelythelatencytimeinthis model.

Theformalintestisconsideredamodelofpersistentpain pro-ducedintwophases.Thefirstphase(0–15min)ischaracterized byneurogenicpainandthesecondphase(15–45min)is charac-terizedbyinflammatorypain(HunskaarandHole,1987).Inthis model,centrallyactingdrugsinhibitedbothphases,while periph-eralactingdrugsinhibitedmainlythesecondphase.HPEdecreased thepainmainlyinthesecondphase.

Acetic acid is an inducer of abdominal contractions and twisting of abdominal muscles by the increase in the level of pro-inflammatoryagents(interleukin-1␤,tumornecrosis factor-␣and interleukin-8) inthe peripheraltissue fluid(Ikeda etal., 2001).Thispain model is used for evaluatingperipherally act-ing antinociceptive agents (Ikeda et al., 2001). In this study, HPEatthedosesof50,100and200mg/kgsignificantlyreduced the number of writhing episodes in mice, indicating the inhi-bitionof acetic acid-inducedvisceralnociception. These results suggestthatHPE mightexertperipheralantinociceptive effects. In the chemical-induced nociceptive tests, H. patens showed antinociceptive effects with similar activity than 100mg/kg NPX.

Ursolicacid,themostabundantcomponent(40%)foundinHPE, hasbeenshowntoexertantinociceptiveeffectsindifferentmodels (Tapondjouetal.,2003;González-Trujanoetal.,2012;Rodrigues etal.,2012).ThegoodantinociceptiveeffectsofHPEcouldbedue thepresenceofursolicacid.Nevertheless,furtherstudiesare neces-sarytoevaluatetheelectrophysiological,anatomicalandmolecular mechanismsbywhichH.patensexertsitsantinociceptiveeffects. TheresultsherepresentedvalidatethetraditionaluseofH.patens forthetreatmentofpain.

Conclusions

HPEshowslowtoxicityinacuteandsubacutetests.H.patens exertsantinociceptiveeffectswithsimilaractivitythan100mg/kg NPXinchemical-inducednociceptionmodels.Therefore,thisplant couldbeagoodsourceofantinociceptiveagentsbecauseofitsgood activityandlowtoxicity.Inaddition,itisinterestingtoevaluatethe antinociceptiveeffectofH.patensconstituents.

Authorcontribution

AJAC,SBRandJRZMcarriedoutthepharmacologicalstudies. AJACdraftedthemanuscript.AHMparticipatedinthedesignof thestudy.MMGCperformedthephytochemicalstudies.CCA con-ceivedthestudy,andparticipatedinitsdesignandcoordination andhelpeddraftthemanuscript.Allauthorsreadandapproved thefinalmanuscript.

Conflictofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwaspartiallysupportedbyJovenesInvestigadores programfromCONCYTEG(providedtoAJAC).

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