Rev. bras. farmacogn. vol.26 número2

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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

Anti-inflammatory

activity

and

acute

toxicity

studies

of

hydroalcoholic

extract

of

Herissantia

tiubae

Ana

L.A.

Lima

a

_,

_Adriano

_F.

_Alves

a

_,

_Aline

_L.

_Xavier

a

_,

_Talissa

_{Mozzini-Monteiro}

a

_,

Theresa

R.R.

Oliveira

b

_,

_Fagner

_C.

_Leite

a

_,

_Wemerson

_N.

_Matias

a

_,

_Marianna

_V.S.C.

_Branco

a

_,

Maria

F.V.

Souza

a

_,

_Marcia

_R.

_Piuvezam

a,∗

a_Programa_de_{Pós-graduac¸}_ão_em_Produtos_Naturais_e_Sintéticos_Bioativos,_Centro_de_Ciências_da_Saúde,_Universidade_Federal_da_Paraíba,_João_Pessoa,_PB,_Brazil b_Departamento_de_Imunologia,_Instituto_de_Ciências_Biomédicas,_Universidade_de_São_Paulo,_São_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1October2015 Accepted9November2015 Availableonline19December2015

Keywords:

Malvaceae

Herrisantiatiubae

Acuteinflammation Acutetoxicity

Anti-inflammatoryactivity Mice

a

b

s

t

r

a

c

t

HydroalcoholicextractofaerialpartsofHerissantiatiubae(K.Schum.)Brizicky,Malvaceae,was

eval-uatedinexperimentalmodelsofinflammationandtoxicity.Fortoxicityassays,maleandfemaleSwiss

micewereorallytreatedwithhydroalcoholicextractofH.tiubae(2000mg/kg)andanalyzedby

consump-tionofwaterandfood,bodyweight,mortalityandratesofmajororganweights,aswellasbiochemical

andhematologicalindexes.Foranti-inflammatoryeffect,phlogisticagentssuchascarrageenanoracetic

acidwereusedtoevaluatepawedema,cellmigrationandcytokineproduction.Itwasalsoinvestigated

thehydroalcoholicextractofH.tiubaeinRAW264.7macrophagelineagebynitricoxideandcytokine

productions.SwissmicetreatedwithhydroalcoholicextractofH.tiubaeshowedlowtoxicityand(50or

100mg/kg)wasabletoreducesignificantly(p<0.01,p<0.001)polymorphonuclearcellmigration,TNF-␣

andIL-1␤productioninthecarrageenan-inducedperitonitis.HoweverthehydroalcoholicextractofH.

tiubae(50,100or200mg/kg)didnotreducecarrageenan-inducedpawedema.Additionally,

hydroal-coholicextractofH.tiubaedidnotpresentcytotoxicityatconcentrationsof6.25,12.5,25or50␮g/ml

butinducedsignificantlydecreaseofNO,TNF-␣andIL-6productioninmacrophagelineage.Thisstudy

suggeststhathydroalcoholicextractofH.tiubaehasanti-inflammatoryactivitybyinhibitingcell

migra-tionmainlybydecreasingtheinflammatorycytokinelevelsattheinflamedsiteindependentlyofthe

anti-edematogeniceffect.

Introduction

Malvaceae family hasa wide variety of natural compounds withpharmacologicalpropertiessuchasanti-inflammatory, anal-gesic, anti-rheumatic, among others (Falcão-Silva et al., 2009). HerissantiatiubaeK.Schum.)Brizickyisoneofthespeciesofthis botanicfamilylargely foundin tropicalregions ofSouth Amer-ica,especiallyinnortheasternBrazilanditispopularlyknownas “mela-bode”or“lava-prato”.Theplantisusedinfolkmedicineto treatinfluenzaandfever(Albuquerqueetal.,2007).Phytochemical investigationofH.tiubaedemonstratedthepresenceof polyoxy-genflavonoids,triterpenes,steroid,phenoliccompoundsandtwo glycosylated flavonoids (kaempferol 7-O-␣-l-rhamnopyranoside and 4′_{,5-dihydroxy-3,6,7,8,3}′_{-pentamethoxyflavone)}_(Silva _et_al.,

∗ Correspondingauthor.

E-mail:mrpiuvezam@ltf.ufpb.br(M.R.Piuvezam).

2009). Therefore, the aim of this study was to investigate the anti-inflammatoryactivityofthehydroalcoholicextractoftheaero partsoftheH.tiubae(HtE)anditstoxicityusingdifferent experi-mentalmodels.

Materialandmethods

Animals

Male and female Swiss mice (n=6/per group, 6–8 weeks, 25–30g)wereusedthroughoutthestudy.Theanimalswere pro-videdfromProf.ThomasGeorgeVivariumof theBiotechnology Center (CBiotec)from Federal University ofParaíba (UFPB), PB, Brazil.Allexperimentalprotocolswereapprovedandperformedin accordancewiththerecommendationsofCommissionofEthicsfor UseofAnimals(CEUA)fromUFPB,whichwasrecordedunder num-ber0508/12.Animalswerekeptinpolypropylenecage,atroom

http://dx.doi.org/10.1016/j.bjp.2015.11.001

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temperature(25±2◦_C),_under₁₂_h_light/dark_cycle,_and_free_access tofoodandwater.

Plantmaterialandpreparationofhydroalcoholicextract

AerialpartsofHerissantiatiubae(K.Schum.)Brizicky,Malvaceae, werecollectedinJanuary2010inthecityofJuazeirinho,Paraiba, Brazil.ItwasidentifiedbyDr.MariadeFatimaAgrafromUFPB.A voucherspecimen(n◦₂₄₃₄₎_is_deposited_in_the_Herbarium_Lauro PiresXavier– JPBatthesameUniversity.TheaerialpartsofH. tiubae(1kg)weredriedat40◦_C_in_a_circulating_air_oven_for₉₆_h andgroundtopowder.Driedandpowderedplantmaterialwas sub-mittedtoextractionbymacerationwithethanol–water(70:30)as asolventatroomtemperaturefor72h.Theratioofplant mate-rial:solventwas20:80(w/v)and atthefinal extractionprocess thematerialwasfilteredandconcentratedinrotaevaporator,thus obtainingtheHtE.

ChromatographyoftheHtE

HtEwassuccessivelypartitionedwithhexane,CHCl3,EtOAcand butanol.Kaempferolwasisolatedfromtheethylacetateextract andsubjectedtoaSephadexLH-20gelcolumnelutedwithMeOH. Kaempferolwasquantifiedby meansofHigh Performance Liq-uidChromatography(HPLC)withultravioletdetection.Calibration curvestokaempferolwereconstructedbyusingthestandard addi-tionmethod.Theseparationofkaempferolwasachievedusinga ProminenceChromatographicSystem(Shimadzu®_,_Tokyo,_Japan) equippedwithLC-20ATmultisolventdeliverysystem,degassing systemDGU-20A5, autoinjectorSIL-20A, ovenCTO-20A column anddetectionbyelectronspectroscopyintheultraviolet-visible regionwithdiodearraySPD-M20AUV-VIS.Data werecollected andintegratedthroughsoftwareClassVPV6.14SP1.Themobile phaseconsistedofamixtureofmethanol:water:H3PO4(1:1:0.01, v/v)pHcontrolledat3.1andtheflowrateof1.2ml/mininthe gradi-entmode,wheretheproportionoftheorganicphaseconstitutions by47%for18min,from80%in23minandreturningto47%after 28min.Toperformthechromatographicruns,weusedaC18 col-umn(Phenomenex®₎_dimensions₂₅_cm

×4.6mm×5␮m,theUV detectorwiththewavelengthof351nm,injectionvolumeof10␮l temperature50◦_C.

TreatmentwithHtE

Forinvivoexperiments,theHtEin2%Tween20(Vetec®₎_and distilledwater(vehicle)wasorally(p.o.)administeredatdosesof 50,100or 200mg/kg. The untreatedcontrol groupreceivedan equalvolumeofthevehicle.Forinvitroexperiments,theHtEwas dissolvedindimethylsulfoxide(DMSO),thestocksolutionwas ster-ilizedusingadisposablefilterunitof0.22mminporosity(Millipore MillexTM₎_and_used_in_the_follow_range_of_{concentrations}_0,_6.25, 12.5,25,50,100,200or400␮g/ml.

Acutetoxicologicaltest

Groupsofmaleandfemale(n=6)Swissmiceweretreatedorally withHtE(2000mg/kg)orvehicle.Theanimalswereobservedfor signsof generaltoxicity in intervalsof 0, 15,30 and 60min,4 and24hlateranddailyfor14 days(Hibbsetal.,1988).During thesetimes,occurrenceof centralnervoussystemchangeswas analyzed:hyperactivity,irritability,aggressiveness,tremors, con-vulsions,catatonia,analgesia,anesthesia,ptosis,decreasedtouch response,ambulation,cleaningcapacity,raise,andautonomic ner-voussystemchanges:diarrhea,constipation,defecation,urination, muscletone,amongothers(Almeidaetal.,1999).Throughoutthe experiment,theconsumptionofwaterandfoodintakeandweight

gainwereobserved. Onday14th_,_the_treated_animals_and non-treatedanimalswereeuthanizedbyanesthetic:sodiumthiopental (ThiopentaxR,Cristalia–PharmaceuticalChemicals)andorgans wereremoved:heart,liver,kidneys,spleenandthymusto deter-mineitsindexes.Theweightgainforeachanimalwasdetermined usingtheformula:

%ofweightgain=

_animal_weight_on_first_challenge

animalweightonlastchallenge

−1×100 Theindexoftheweightorganswascalculatedfollowingthe formulabelow:

Index= organweight(mg)

animalweight(g)

Evaluationofbiochemicalandhematologicalparameters

Onthe14th_day_animals_fasted_for₆_h_were_anesthetized_with sodiumthiopentalandorbitalsinusbloodwascollectedusinga heparinizedPasteurpipetteandtransferredintotubes(Eppendorf). Thebloodwasanalyzedforhematological(erythrocyteand leuko-cytecounts) and biochemicalparameters (urea, creatinine,uric acid,alaninetransaminase-ALT,aspartatetransaminase-AST, albu-min,totalprotein,triacylglycerides,glucoseandtotalcholesterol).

Carrageenan-inducedmicepawedema

GroupsofSwissmice(n=6)weretreated(p.o.)withvehicle, indomethacin(10mg/kg-Roche®₎_or_HtE_(50,₁₀₀_or₂₀₀_mg/kg) 1hbeforeadministrationofcarrageenanat2.5%(Sigma-Aldrich®₎ injectedsubcutaneouslyintotheplantarregionofthelefthindpaw andphosphatebuffersaline(PBS)inrighthindpaw.Negative con-trolgroupreceived20␮lPBSinjectionsinbothpaws.Pawdiameter wasmeasuredwithadigitalmicrometerat1,2,3,4,6and24h afterstimulation.Resultswereexpressedasdifferencebetween thediameterofleftandrightpaws(DeVasconcelosetal.,2011).

Carrageenaninducedperitonitis

Mice(n=6)wereorallytreated withHtE(50or 100mg/kg), indomethacin 10mg/kg or vehicle 1hbefore carrageenan (1%) intraperitoneal injection. The basal groupreceivedsaline. After 4htheanimalswereeuthanizedbyxylazineandketamine over-doseandtheperitonealcavitywashed with2ml ofsterilecold PBS,followedby aone-minmassageand collectionofthefluid (Guerra et al., 2011;Pinheiro et al.,2013).Exudates were cen-trifuged(10min,266g,4◦_C)_and_the_pellet_of_cells_resuspended in1mlofPBS(4◦_C),_diluted_in_Turk_solution_in_the_ratio_of_1:40 andtotalcellswerecountedinatNeubauercameraunderoptical microscope(NikonE200,Melville,NY–EUA).Differentialcell mea-surementwasmadeincytocentrifuge–254×g,15min(Cytospin –BioResearch,Washington–USA),slidestainedinFastPanoptic (RenyLab)andcountedunderopticalmicroscope(100×objective). For eachslide aminimum of100cells werecountedinoptical microscopeunder1000magnification(Sousaetal.,2010).

Cytotoxicassay

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Minutes

50000

B

40000

30000

20000

10000

0

0 10 20

Kaempferol

30

mA

U

A

40000

30000

20000

10000

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0 10 20

Kaempferol

30 40

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

0.5 1.0 2.0 3.0 4.0 5.0 4.5 3.5 2.5 1.5 0.0 15.25 15.50 15.75 mn0.0

1.0 2.0 3.0 4.0 5.0 mAU 0.7

0.6 0.5 0.4 0.3 0.2 0.1 0.0

7 8 6 5 4 3 2 1 0

15.25 15.50 15.75 16.00 mn 15.25 15.50 15.75 16.00 mn

17.25 17.50 17.75 18.00 mn 0.0

2.5 5.0 7.5 mAU

Minutes

mA

U

Fig.1.Chromatographicprofileat370nmof(A)samplesolutionofkaempferolstandardUSPandyourDADspectrumand(B)sampleofthehydroalcoholicextractofthe aeropartsoftheHerissantiatiubae(HtE)andyourDADspectrum.

Table1

EffectofhydroalcoholicextractoftheaeropartsoftheHerissantiatiubae(HtE)ontoxicalparametersofmaleandfemaleSwissmice.

Parameter(unit) Male(control) Male(HtE) Female(control) Female(HtE)

Feedintake(g) 41.88±2.98 38.13±1.15 37.08±2.73 42.79±1.36 Waterconsumption(ml) 67.50±3.89 59.29±3.05 40.00±4.42 52.29±2.56**

Initialweight(g) 28.60±1.26 25.50±1.29 30.05±0.98 28.17±1.01

Finalweight(g) 38.05±3.22 39.98±1.27 33.14±2.31 33.15±1.07

Weightgain(%) 41.06±6.15 50.19±5.03 14.43±3.46 17.79±1.48

Indexheart(mg/g) 3.93±0.08 4.01±0.17 4.14±0.18 4.08±0.16

Indexliver(mg/g) 61.27±3.71 56.89±1.11 56.53±2.15 56.25±1.78

Indexkidneys(mg/g) 12.66±1.22 11.86±0.99 11.09±0.24 10.70±0.32

Indexspleen(mg/g) 5.59±0.21 6.45±0.53 7.56±0.59 6.97±0.62

Indexthymus(mg/g) 2.94±0.34 2.84±0.24 3.33±0.23 3.89±0.27

Dataareexpressedasmean±S.E.M.andweresubjectedtoanalysisofvarianceofStudent’st-test.

**_p_<_0.01_when_compared_to_female_control_group._The_data_are_{representative}_of_two_experiments_with_n₌₆_(per_group).

25,50,100,200or400␮g/ml)at37◦_C_in_5%_CO2 _for₂₄_h._After 24h,100␮lof5mg/mlMTTsolution(Sigma-Aldrich®₎_was_added toeachwell,followedbyincubationfor4h.Themediumwas aspi-rated,andtheformazancrystalsweredissolvedin100␮lofDMSO for15min.Theopticaldensityofeachwellwasmeasuredat570nm inamicroplatereader(Bio-Radmodel550, Japan).Treatedcells werecomparedtonon-treatedcells.

NOproduction

TheproductionofNOwasdeterminedbymeasuringthe accu-mulatedlevelofnitriteonRAW264.7macrophagesupernatants. Afterpre-incubationofcells(2×105_cells/well)_at₃₇◦_C_in_5%_CO2 for4h,theplantextract(0,6.25,12.5,25or50␮g/ml)wasadded andco-stimulatedor notwithlipopolysaccharide-LPS (1␮g/ml) plusINF-␥(10ng/ml).Thecellswerefurtherincubatedfor24h. AmountsofnitriteweremeasuredusingGriessreagent. Briefly, 50␮lof cell culture medium was mixed with100␮l of Griess reagent.Subsequently,themixturewasincubatedatroom tem-peraturefor10minandtheabsorbanceat540nmwasmeasured inamicroplatereader(Bio-Radmodel550,Japan).Thequantityof nitritewasdeterminedfromasodiumnitritestandardcurve(Green etal.,1982).

Cytokineassays

IL-1␤,TNF-␣and IL-6from cellsupernatantsand peritoneal lavagesweremeasuredbyELISA,usingtherecommended proto-colfromthe antibodies’suppliers.Antibodypairs and standard recombinant cytokines for ELISA assay were purchased from eBiosciences.

Statisticalanalysis

Data were analyzedby Student’s t-test, ANOVAfollowed by Tukeypost-test usingsoftwareGraphPad Prism(GraphPad,San Diego, CA). Valueswere expressedas mean±standard errorof mean (S.E.M.), and results were considered significant when p<0.05.

Resultsanddiscussion

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Table2

EffectofhydroalcoholicextractoftheaeropartsoftheHerissantiatiubae(HtE)onbiochemicalandhematologicalparametersofmaleandfemaleSwissmice.

Parameters(unit) Male(control) Male(HtE) Females(control) Females(HtE)

Biochemical

Glucose(mg/dl) 123.00±18.61 87.17±7.99 149.00±6.37 150.50±16.76 Urea(mg/dl) 59.18±4.423 50.88±6.22 54.10±3.78 44.42±1.75 Creatinine(mg/dl) 0.46±0.01 0.50±0.07 0.45±0.08 0.37±0.01 TotalCholesterol(mg/dl) 88.02±4.86 98.48±1.06 83.20±3.05 64.84±16.47 Triacylglycerides(mg/dl) 82.00±10.45 97.4±10.25 64.50±5.58 55.00±16.02 Uricacid(mg/dl) 3.43±0.54 2.45±0.65 2.08±0.19 2.00±0.27 AST(U/l) 175.40±19.56 139.0±14.52 184.90±14.16 195.00±7.82 ALT(U/l) 79.66±2.44 115.1±30.80 103.3±12.25 84.12±8.53 Totalproteins(g/dl) 5.04±0.30 4.99±0.18 4.99±0.24 5.22±0.21 Albumin(g/dl) 2.53±0.10 2.31±0.19 2.79±0.08 2.52±0.18

Hematological

Redblood(106_/mm3₎ _9.28_±_0.78 _8.90_±_0.43 _8.15_±_0.32 _8.77_±_0.14

Hemaglobin(g/dl) 13.77±0.85 13.98±0.23 12.82±0.45 13.67±0.24 Hematrocit(%) 43.30±2.82 44.54±1.48 39.56±1.72 43.38±0.49 VCM(fm3₎ _47.00

±0.96 48.80±2.31 48.40±0.40 49.80±0.58 HCM(pg) 14.98±0.43 15.96±0.38 15.78±0.14 15.58±0.15 CHCM(g/dl) 31.85±0.35 31.72±0.35 32.30±0.53 31.22±0.19 Leukocytes(103_/mm3₎ _3.73_±_0.32 _3.76_±_0.38 _4.15_±_0.93 _4.28_±_0.69

Neutrophil(%) 35.67±6.87 21.60±6.95 18.40±4.69 30.00±1.78 Lymphocytes(%) 58.67±6.08 73.00±6.77 74.60±4.93 65.33±1.33 Monocytes(%) 5.66±1.80 4.20±0.73 6.80±1.39 7.16±1.56

Dataareexpressedasmean±S.E.M.andweresubjectedtoanalysisofvarianceofStudent’st-test.Thedataarerepresentativeoftwoexperimentswithn=6(pergroup). Band,basophilandeosinophilcellswerenotfoundinthedifferentialcount.

ofinformationaboutpre-clinicaltoxicityofthisplant,preliminary toxicologicalevaluationwascarriedout.HtE(2000mg/kg)acute oraltreatmentdidnotinducesignsofgeneraltoxicity(Table1). However,itwasobservedasignificantincrease(p<0.01)inwater consumptioninthefemaletreatedgroupascomparedtothefemale controlgroup.Thisresultmaybeassociatedwithsusceptibility anddifferentialsensitivitytodrugsbetweengenders(Anderson, 2008). In addition, no significant changes in biochemical and

hematologicalparameterswereobservedinthetreatedanimals ascomparedwiththecontrolanimals,asshowninTable2.

Wealsoevaluatetheanti-inflammatoryeffectofHtE consid-eringitsuseinfolkmedicinetotreatdiseaseswithinflammatory characteristics(Albuquerqueetal.,2007).Thepawedemainduced bycarrageenan is widelyused todetermineanti-edematogenic activityofcompounds. Inthis model,carrageenan promotesan immediatephase(rangefrom0through6h)thatinvolvesseveral

0.0 0.5 1.0 1.5

###

***

A

Paw edema (mm)

0.0 0.5 1.0 1.5

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**

B

0.0 0.5 1.0 1.5

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C

0.0 0.5 1.0 1.5

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Paw edema (mm)

0.0 0.5 1.0 1.5

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Negative control Carrageenan HtE 50 HtE 100 HtE 200 Indomethacin

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###

** _***

***

A

Negative control

HtE 50

Indomethacin HtE

100 Carrageenan 1%

Total cells/ml x 10

6

0 2 4 6

###

** ***

***

B

Negative control

HtE 50

Indomethacin HtE

100 Carrageenan 1%

PMN cells/ml x 10

6

0 1 2 3 4

C

Negative control

HtE 50

Indomethacin HtE

100 Carrageenan 1%

MN cells/ml x 10

6

Fig.3.EffectofhydroalcoholicextractoftheaeropartsoftheHerissantiatiubae(HtE)inthecarrageenan-inducedperitonitismodel.Determinationofthetotalcellularity (A),polymorphonuclearcells(PMN)(B)andmononuclearcells(MN)(C).Thedatarepresentthemean±S.E.M.oftotalanddifferentialcellcounts.###_p_<_0.001_vs_negative group,**p<0.01and***p<0.001vspositivecontrolgroup(carrageenan)afteranalysisbyANOVAonewayfollowedbyTukeypost-test.Thedataarerepresentativeoftwo experimentwithn=6(pergroup).

0 100 200 300 400

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Negative control

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Indomethacin HtE

100 Carrageenan 1%

TNF-α

pg

/m

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HtE 50

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IL

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Negative control

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Indomethacin HtE

100 Carrageenan 1%

IL

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Control 6.25 12.5

25 50 ₁₀₀ ₂₀₀ ₄₀₀

0 1 2 3 4

***

*** ***

HtE μg/ml

Absorbance (A)

Fig.5.EffectofhydroalcoholicextractoftheaeropartsoftheHerissantiatiubae(HtE) onRAW264.7macrophages.Thedatarepresentsmean±S.E.M.opticaldensities ofcelllysateaccordingtothetreatments.***p<0.001vscontrolafteranalysisby ANOVAonewayfollowedbyTukeypost-test.Thedataarerepresentativeoftwo independentexperiments.

mediators(histamine,cytokinesandNO)and,alatephase(range from6through96h)whereitisobservedtheleukocytemigration (Posadasetal.,2004).HtE(50,100,or200mg/kg)didnotreducethe edemaatbothphasesdemonstratingnoanti-edematogeniceffect (Fig.2A–F).

InordertostudytheHtEeffectoncellmigrationtotheinflamed site, the carrageenan-induced peritonitis was performed. The mechanismofactionbywhichcarrageenaninducesthe inflamma-toryprocessesisasynergismamongseveralmediators(bradykinin, serotonin,prostaglandins,leukotrieneB4)(Pinheiroetal.,2013). Asshown in Fig.3A–C,theHtE (50or 100mg/kg)significantly reduced(p<0.01and p<0.001,respectively)theleukocytesand polymorphonuclearcell(PMN)numbersintotheperitonealbutdid notchangemononuclearcellnumber(MN).Thesefindings sug-gested that HtEpresents anti-inflammatoryeffect byinhibiting cellmigrationtotheinflamedsitewithoutdecreasestheedema process.Similarresultsweredescribed byPaiva andcolleagues (2013)wherePseudobombaxmarginatum,Malvaceae,extractalso inhibitedthemigrationofPMNwithoutaffecttheMNcell migra-tion.

Control Control

INF-γ+ LPS

6.25 12.5 25 50 6.25 12.5 25 50

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HtE μg/ml

B

NO

–2 (

μ

M)

NO

–2 (

μ

M)

Fig.6. EffectofhydroalcoholicextractoftheaeropartsoftheHerissantiatiubae(HtE)onNOproductionbyRAW164.7macrophagesexposed(A)ornot(B)toINF-␥

(10ng/ml)+LPS(1␮g/ml).Thedatarepresentmean±S.E.M.concentrationsofnitrite.Thedatarepresenttwoindependentexperiments.***p<0.001and**p<0.01vsthe INF-␥+LPSgroup,###_p_<_0.001_vs_control_group_after_analysis_by_ANOVA_one_way_followed_by_Tukey_post-test._The_data_are_{representative}_of_two_experiments_in_triplicate.

0 2000 4000 6000 8000

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Control

INF-γ+ LPS 6.25

12.5 25 50 _Control

INF-g+ LPS

6.25 12.5 25 50

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Thecarrageenan-inducedperitonitisalsoinvolvesincreasesof TNF-␣,IL-1␤andIL-6levelsattheperitonealfluid,whichpresents akeyroleininflammatoryprocesses(Lorametal.,2007).Inthis regard,weanalyzedtheHtEeffectonthecytokinelevelsatthe peritonealexudateinducedbycarrageenaninjection.Fig.4AandC showstheHtE(50or100mg/kg)decreasedsignificantly(p<0.001) thelevelsofTNF-␣withoutreducingIL-6,respectively.However, only HtEat doseof 100mg/kg was ableto reducethe amount (p<0.01)ofIL-1␤inperitonealfluid(Fig.4B).ThereductionofIL-1

␤andTNF-␣levelsbyHtEmayberesponsiblefortheinhibitionof leukocytemigration,sincethesepro-inflammatorycytokines pro-moteexpressionofendotheliumadhesionmoleculesinvolvedin permeabilityandleukocytetransendothelialmigration(Schmidt etal.,2013).

Tobetterunderstandtheanti-inflammatoryeffectoftheextract anditsmechanismsofactionweusedinvitroanti-inflammatory assaybymeasuringtheproductionofNOandcytokinesinRAW 264.7 macrophages. First of all, we demonstrated (Fig. 5) that HtE wasnot toxic for the cells at concentrations rangingfrom 6.25to 50␮g/ml. As shown in Fig.6, the HtE (6.25–50␮g/ml) significantly (p<0.01 – p<0.001) decreased NO production by activatedcells(Fig.6A).Inaddition,macrophagesinpresenceof HtEdidnotproduceNO(Fig.6B),demonstratingtheabsenceof endotoxinsorotheragentscapableofinitiatinganinflammatory response.AsshowninFig.7,theHtE(12.5or50␮g/ml)wasable toinhibitsignificantly(p<0.001–p<0.01)theproductionof

TNF-␣(Fig.7A)andIL-6(Fig.7B).TheHtEat6.25␮g/mlwasableto reducesignificantly (p<0.001) onlyTNF-␣production (Fig.7A). ThesedatacorroboratewiththeinvivoresultsandwithParkand colleagues(2012)resultswheretheWerckleainsignis,Malvaceae, extractalsoreducedtheinflammatorycytokines(IL-6,IL-1␤and TNF-␣).

In addition, species in the Malvaceae family are known to producephenolic compoundswhich exhibit antioxidant action, thereforetheycanbeusedtotreatseveraldiseaseswith inflam-matorycharacteristics(Oliveiraetal.,2012)andkaempferolwhich hasbeendescribedtopossesspotentanti-inflammatory proper-ties (Devi et al., 2015; Kadiogluet al., 2015). Therefore, these phyto-constituentspresentinHtEcouldbecontributingto anti-inflammatoryactivitypresentedinthisstudy.

Insummary,weshowedforthefirsttimethatHtEpresented lowtoxicityfollowingoraladministrationatadoseof2000mg/kg andalsopresentedanti-inflammatoryactivity(50and100mg/kg) bymodulatinginflammatorycellsinvivoandinvitro.Thus,these resultscancontributetothemainpopularuseoftheH.tiubae. However,themechanismsofactionofcompoundsofHtEon inflam-matoryprocesseswillbeexploredinfuturestudies.

Authors’contributions

ALAL carried out the study, participated in the toxico-logical and anti-inflammatory assays and wrote the paper; MFVS provided the plant; WNM has performed the chem-ical studies; ALX, TMM and MVSCB provided assistance in the acute toxicity; FCL, AFA, TRRO participated in the anti-inflammatory experiments; MRP supervised the work andcorrectedthemanuscriptforpublication.Alltheauthorshave readthefinalmanuscriptandapprovedthesubmission.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith

thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This study was financially supported by INCT for Cancer Control, CNPq573806/2008-0,FAPERJE26/170.026/2008, CNPq-Universal14/2012-472853/2012-0andCAPES/Brazil.

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