isolated from digenea simplex inhibit inflammatory and nociceptive responses

ContentslistsavailableatScienceDirect

Carbohydrate

Polymers

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Polysaccharides

isolated

from

Digenea

simplex

inhibit

inflammatory

and

nociceptive

responses

Juliana

G.

Pereira

_,

_Jacilane

_X.

_Mesquita

_,

_Karoline

_S.

_Aragão

_,

_Álvaro

_X.

_Franco

_,

Marcellus

H.L.P.

Souza

_,

_Tarcisio

_V.

_Brito

_,

_Jordana

_M.

_Dias

_,

_Renan

_O.

_Silva

_,

Jand-Venes

R.

Medeiros

_,

_Jefferson

_S.

_Oliveira

_,

_Clara

_Myrla

_W.S.

_Abreu

_,

Regina

Célia

M.

de

Paula

_,

_André

_Luiz

_R.

_Barbosa

_,

_Ana

_Lúcia

_P.

_Freitas

a_{LaboratoryofProteinsandCarbohydratesofMarineAlgae,DepartmentofBiochemistryandMolecularBiology,FederalUniversityofCeará,Fortaleza,}

Brazil

b_{LAFICA–LaboratoryofPharmacologyofInflammationandCancer,DepartmentofPhysiologyandPharmacology,FederalUniversityofCeará,Fortaleza,}

CE,Brazil

c_{LAFFEX–LaboratoryofExperimentalPhysiopharmacology,BiotechnologyandBiodiversityCenterResearch(BIOTEC),FederalUniversityofPiauí,}

Parnaiba,Piauí,Brazil

d_{PolymerLaboratory,DepartmentofOrganicandInorganicChemistry,FederalUniversityofCeará,Fortaleza,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5December2013

Receivedinrevisedform20January2014 Accepted22January2014

Availableonline12March2014

Keywords:

Polysaccharides Anti-inflammatory Antinociceptive

a

b

s

t

r

a

c

t

Polysaccharides(PLS)havenotablydiversepharmacologicalproperties.Inthepresentstudy,we inves-tigatedthepreviouslyunexploredanti-inflammatoryandantinociceptiveactivitiesofthePLSfraction isolatedfromthemarineredalgaDigeneasimplex.WefoundthatthePLSfractionreduced carrageenan-inducededemainadose-dependentmanner,andinhibitedinflammationinducedbydextran,histamine, serotonin,andbradykinin.Thefractionalsoinhibitedneutrophilmigrationintobothmousepawand per-itonealcavity.ThiseffectwasaccompaniedbydecreasesinIL1-␤andTNF-␣levelsintheperitonealfluid. Pre-treatmentofmicewithPLS(60mg/kg)significantlyreducedaceticacid-inducedabdominalwrithing. ThissamedoseofPLSalsoreducedtotallickingtimeinbothphasesofaformalintest,andincreased latencyinahotplatetest.Therefore,weconcludethatPLSextractedfromD.simplexpossess anti-inflammatoryandantinociceptiveactivitiesandcanbeusefulastherapeuticagentsagainstinflammatory diseases.

©2014ElsevierLtd.Allrightsreserved.

1. Introduction

Presently,about25–30%ofallactivecompoundsthatareused astherapeutictreatmentsarederivedfromnaturalproducts(Silva, Moura,Oliveira,Diniz,&Barbosa-Filho,2003),andnaturalmarine products have been the focus for the efforts to discover new molecules of pharmacological and biomedical interest (Cabrita, Vale,&Rauter,2010;Iannitti&Palmieri,2010)Marinealgaehave receivedspecialattentionsincetheyhavebeenshowntobe valu-ablesourcesofstructurallydiversebioactivecompounds,suchas polyphenols,carotenoids,pigments,enzymes,andpolysaccharides (PLS)(Kusaykinetal.,2008;Wijesekara,Pangestuti,&Kim,2010).

∗Correspondingauthor.Tel.:+558699131650.

∗∗Correspondingauthor.Tel.:+558533669819.

E-mailaddresses:andreluiz@ufpi.edu.br(A.L.R.Barbosa),pfreitas@ufc.br

(A.L.P.Freitas).

Manyspeciesofseaweed(marinemacroalgae)areusedasfood andintraditionalmedicinebecauseoftheirperceivedhealth ben-efits.RedSeaweedsaresourcesofPLS,includingsomethathave becomevaluableadditivesinthefoodindustrybecauseoftheir rhe-ologicalproperties(Kusaykinetal.,2008;Wijesekaraetal.,2010). Inaddition,thesePLShaveanumberofbiologicalactivities, includ-inganticoagulant,antiviral,gastroprotective,antinociceptive,and anti-inflammatoryproperties(Britoetal.,2013;Chavesetal.,2013; Cumashietal.,2007;Silvaetal.,2011).

TheRedSeaweedDigeneasimplex(Wulfen)C.Agardh,a mem-beroftheRhodomelaceaefamily,isusedextensivelyinJapanas a parasiticide, and considereda good source of agar(El-Sayed, 1983;Tomoda,Nakatsuka,&Minami,1972).Inapreviousstudy, thegalactancontentinthePLSofD.simplexwasinvestigatedby ionexchange chromatography,massspectrometry,andinfrared analysisandwasfoundtoberichincommonrepeatinggalactan sulfatebackbones(Takano,Shiomoto,Kamei,Hara,&Hirase,2003). However,nostudydemonstratingthechemicalcharacteristicsof

http://dx.doi.org/10.1016/j.carbpol.2014.01.105

thepolysaccharidefractionofthisalgawithhabitatinBrazilwas performedpreviously.

The inflammatory process is a temporally controlled phe-nomenon involving the participation of diverse mediators including histamine, serotonin, bradykinin, TNF-␣, IL-1␤, and prostaglandins,andisassociatedwithintensemigration of neu-trophilsfromthebloodintoinflamedtissues(Carvalhoetal.,1996; Hajareetal.,2001;Srinivasanetal.,2001).Thebiochemical medi-atorstogetherstimulateasequenceofmolecularevents,aswellas inflammationandnociception(Déciga-Campos,Palacios-Espinosa, Reyes-Ramírez,&Mata,2007;Moncada&Higgs,1993).Itisclear thatthereisastrongassociationbetweentheinflammatoryprocess andthedevelopmentofpain.Inflammatorypain,producedbythe actionofinflammatorymediators,isaccompaniedbytheincreased excitabilityofperipheralnociceptivesensoryfibers(Linley,Rose, Ooi,&Gamper,2010).Interestingly,therearenomarine-derived anti-inflammatorynaturalproductsinclinicaldevelopment cur-rently(Mayeretal.,2010).

Thus, the aim of the present study was to investigate the antinociceptiveand anti-inflammatoryactivitiesof a previously characterizedPLSfractionwasisolatedfromthemarineredalgaD. simplexbyusingexperimentalmodelsofinflammationand noci-ception.

2. Experimental

2.1. Extractionofpolysaccharide(PLS)

TheextractionofthepolysaccharideofGracilariabirdiaewas accomplishedattheLaboratoryofBiochemistryofSeaAlgaeatthe DepartmentofBiochemistryandMolecularBiologyoftheFederal UniversityofCeará.TheRedSeaweedwasharvestedatFlexeiras Beach,Trairí,Ceará,Brazil,inDecember1991,geographical local-ization:03◦₁₃′₂₅′′_{Sand 39}◦₁₆′₆₅′′_{W.A voucher specimen(No.}

4693)wasdepositedintheHerbariumPriscoBezerra,Federal Uni-versityofCeará,Brazil.Samplescleanedofepiphyteswerewashed withdistilled waterand thensubmittedtoextractionand frac-tionationin ordertoobtainof PLSusingexperimentalprotocol previouslydescribed(Takanoetal.,2003).Thedriedtissue(5g) wasmilledandsuspendedin250mLof0.1Msodiumacetatebuffer (pH5.0)containing30mgofpapain(E.Merck),5mMEDTA,5mM cysteineandincubatedat60◦_{Cfor6h.Theresiduewasremoved}

byfiltrationandcentrifugedat2725×gfor30minat4◦_C.ThePLS

wereprecipitatedbytheadditionof48mLof10%cetylpyridinium chloride(CPC,SigmaChemical).Themixturewascentrifugedat 2725×gfor30minat4◦_{C.Thepolysaccharidesinthepelletwere}

washedwith200mLof0.05%cetylpyridiniumchloridesolution, andthenprecipitatedwith200mLofethanol(v/v),for24hat4◦_C.

Afterfurthercentrifugation(2725×gfor30minat4◦_C),the

pre-cipitatewaswashedtwicewith200mLof80%ethanolanddried withacetoneunderhotairflow(60◦_C).

2.2. Infraredspectroscopy

Fouriertransforminfrared(FT-IR)spectraofKBrpelletsofthe polysaccharideswererecordedinaShimadzuIRspectrophotomer (model8300)scanningbetween400and4000cm−1_.

2.3. Nuclearmagneticresonancespectroscopy

13_{CNMRspectraof2.5%w/vsolutionsinD}

2Owererecordedat

353KonaFouriertransformBrukerAvanceDRX500spectrometer withaninversemultinucleargradientprobe-headequippedwith z-shieldedgradientcoils,andwithSiliconGraphics.Acetonewas usedastheinternalstandard(31.07ppmfor13_C).

2.4. Animals

Male Swiss mice weighing 20–25g were used. The animals werehousedintemperature-controlledroomsandreceivedfood andwateradlibitum.Allexperimentswereconductedin accor-dancewiththecurrentlyestablishedprinciplesforthecareanduse ofCOBEA(ColégioBrasileirodeExperimentac¸ãoAnimal),Brazil. TheAnimalStudiesCommitteeofUniversidadeFederaldoCeará approvedtheexperimentalprotocol.

2.5. Carrageenan-inducedpawedema

The animals were randomly divided into 6 groups (n=5), and edemawas inducedbythe injectionof 50␮L ofa suspen-sionofcarrageenan (500␮g/paw)in0.9% sterilesalineintothe right hind paw (group I). The mice were pretreated intraperi-toneally(i.p.)witheither0.9%NaCl(groupII,untreatedcontrol), 10mg/kg indomethacin(group III,reference control),or10, 30, or60mg/kgofPLS(groupsIV,V,andVI,respectively)1hbefore carrageenaninjection.Pawvolumewasmeasuredwitha plethys-mometer (Panlab, Barcelona, Spain) immediately before (Vo), and at 1, 2, 3, and 4h after carrageenan treatment (Vt) as previously described (Winter, Risley, &Nuss, 1962).The effect of pre-treatment was calculated as the percentage of inhibi-tionof edema relativetothepawvolume ofthesaline-treated controlsas previously described (Lanhers, Fleurentin, Dorfman, Mortier,&Pelt,1991)accordingtothefollowingformula:% inibi-tion of edema=(Vt−Vo)“Control”−(Vt−Vo)“Treated”)/(Vt−Vo)

“Control”)×100.

2.6. Pawedemainducedbydifferentinflammatoryagents

To induce paw edema with different inflammatory agents, theanimalswereadministered50-␮Linjectionsofdextran(DXT,

500␮g/paw),serotonin(Ser,1%,w/v),histamine(Hist,1%,w/v), or bradykinin (Bk, 6nmol) into theright hindpaw. Onegroup received50␮Lof0.9%sterilesalineandservedasanuntreated con-trolgroup.PLS(60mg/kg)orindomethacin(10mg/kg,reference control)wereinjectedi.p.30minbeforeintraplantarinjectionsof phlogisticagents.Pawvolumewasmeasuredimmediatelybefore, andatselectedintervaloftime.

2.7. Determinationofmyeloperoxidaseactivity

Theextentofneutrophilaccumulationinthemousepawwas measuredusingamyeloperoxidase(MPO)assay.ToevaluateMPO activity,carrageenanwasinjectedintotherightplantarsurfaceof themicepre-treatedwithsaline,carrageenan,indomethacin,orPLS (60mg/kg).Fourhoursaftercarrageenaninjection,wemeasured theMPOconcentrationintherighthindpaw.Briefly,50–100mg ofhindpawtissuewashomogenizedin1mLofpotassiumbuffer with0.5%hexadecyltrimethylammoniumbromide(HTAB)foreach 50mgoftissue.Thehomogenatewascentrifugedat40,000×gfor 7minat4◦_{C.MPOactivityintheresuspendedpelletwasassayed}

by measuringthechange inabsorbance at 450nm by usingo -dianisidinedihydrochlorideand1%hydrogenperoxide.Theresults arereportedasMPOunits/mgtissue.AunitofMPO(UMPO)activity wasdefinedasthatconverting1mmolhydrogenperoxidetowater over1minat22◦_C.

2.8. Peritonitismodel

Mice were injected orally with 250␮L of sterile saline,

Fig.1. FT-IRspectra(A)inKBrpellets;NMRspectrainD2O,(B)13C-NMRspectrum

s,(C)DEPTspectrumofPLSfromD.simplex.

cavitywaswashedwith1.5mLofheparinizedphosphate-buffered saline(PBS)toharvestthecellscontainedintheperitonealfluid. TotalcellcountswereperformedusingaNeubauerchamber,and differentialcellcounts(100cellstotal)werecarriedoutusing cyto-centrifugeslidesstainedwithhematoxylinandeosin.Theresults

arepresentedasthenumberoftotalleucocytesorneutrophilsper milliliterofperitonealexudates.

2.9. MeasurementofIL-1ˇandTNF-˛

Aftertheperitonitisassay,samplesofperitonealfluidwere col-lectedandthelevelsofIL-1␤andTNF-␣wereevaluatedusinga sandwichenzyme-linkedimmunosorbentassay(ELISA).ELISAkits forIL-1␤wereobtainedfromtheNationalInstitutefor Biologi-calStandardsandControl(PottersBar,UK).Thekitsconsistently showedIL-1␤andTNF-␣levelstobeover4000pg/mLandthatthey didnotcross-reactwithothercytokines.Theresultsareexpressed aspg/mLofeachcytokineperperitonealcavitywashed.

2.10. Aceticacid-inducedwrithingtest

Theacetic-acidwrithingtest wasusedtoevaluatethe anal-gesicactivity(Collier,Dinneen,Johnson,&Schneider,1968)ofPLS. The mice(n=6 per group) wereinjected (i.p.)with 0.6%acetic acid(10mL/kgbodyweight),andtheintensityofnociceptionwas quantifiedbycountingthetotalnumberofwrithesoveraperiod of20min,andincludedabdominalmusclecontractionsandhind pawextensions(Koster,Anderson,&De-Beer,1959).Theanimals receivedPLS(60mg/kg,i.p.)orsterilesaline(controlgroup,0.9%, w/v)30minbeforeaceticacidinjection.Morphine(5mg/kg,s.c.) wasadministered30minbeforeaceticacidasareferencecontrol.

2.11. Formalintest

Thistest,whichresultsinalocaltissueinjurytothepaw,has beenusedasamodelfortonicpainandlocalizedinflammatorypain (Hunskaar,Fasmer,&Hole,1985).Twentymicrolitersof2.5% for-malinwasadministered(s.c.)intotherighthindpaw.Theamount oflickingtimewasrecordedfrom0to5min(phase1,neurogenic) andfrom20to25min(phase2,inflammatory)afterformalin injec-tion (Hunskaar &Hole, 1987).Themice (n=6 per group) were thentreatedwithPLS(60mg/kg,i.p.)orsterilesaline(0.9%)30min beforeformalininjection.Morphine(5mg/kg,s.c.)wasalso admin-istered30minbeforeformalininjectionandusedasareference compound.

2.12. Hot-platetest

Thehotplatetestisanothercommonlyusedmethodtomeasure analgesicactivity(Eddy&Leinback,1953).Eachmousewasplaced twiceontoaheatedplate(51±1◦_{C),separatedbya30-min}

inter-val.Thefirsttrialfamiliarizedtheanimalwiththetestprocedure, andthesecondtrialservedasthecontrolforthereactiontime (lick-ingthepaworjumping).Animalsshowingareactiontimegreater than20swereexcluded.Afterthesecondtrial(controlreaction time),groupsofanimals(n=6)receivedsterilesaline(0.9%,i.p.), PLS(60mg/kg,i.p.),ormorphine(5mg/kg,s.c.;referencedrug).The reactiontimesweremeasuredattimezero(0time)andat30,60, 90,and120minafterthecompoundswereadministered,witha cut-offtimeof45stoavoidpawlesions.

2.13. Statisticalanalysis

Theresultsareshownasthemeans±S.E.M.Forthe

0 1 2 3 4 0.00

0.04 0.08

0.12 Sal

Cg

PLS 10 mg/kg + Cg PLS 30 mg/kg + Cg PLS 60 mg/kg + Cg Indo 10 mg/kg + Cg

*

*

*

*

*

_*

*

*

*

*

*

*

A

Time (h)

Change in volume (ml)

0 1 2 3 4

0.00 0.02 0.04 0.06 0.08

Sal Dxt

PLS 60 mg/kg + Dx Indo 10 mg/kg + Dx

*

*

*

*

*

*

*

_*

B

Time (h)

Change in volume (ml)

Fig.2.EffectofPLSfromD.simplexonpawedemainducedbycarragenan(A)anddextran(B).Pawedemawasinducedbycarragenan(Cg:500␮g/paw;PanelA)ordextran (Dx:500␮g/paw;PanelB)injectionsintotheplantarsurfaceoftherightpaw.Thechangeinpawvolumewasmeasuredattheindicatedtimeintervals.Miceweretreated withpolysaccharide(PLS:10,30and60mg/kg;i.p.)andindomethacin(Indo:10mg/kg,i.p.;positivecontrol).Thevaluesgivenaremeans±S.E.M.(n=6).*Statisticaldifference (p<0.05)comparedtocarragenan(PanelA)ordextran(PanelB)(one-wayANOVAfollowedbyNewman–Keulspost-test).

3. Resultsanddiscussion

3.1. StructuralanalysisofthePLSfromD.simplex

TheFT-IRspectrumofsolublepolysaccharidefromD.simplex

isdepictedinFig.1A.Thebandsintheregionof1400–700cm−1

arecharacteristicofagarocolloids(Chopin&Whalen,1993;Lahaye &Yaphe,1988;Maciel etal., 2008;Melo, Feitosa,Freitas, &de Paula,2002;Mollet,Rahaoui,&Lemoine,1998;Prado-Fernandez, Rodriguez-Vazquez, Tojo, & Andrade, 2003; Rochas, Lahaye, & Yaphe,1986).Thebandat1253and931cm−1_{canbeattributed}

to the S O vibration of the sulphate groups C O C of 3,6-anhydrogalactoserespectively.Theregionat800–850cm−1_isused

for algal polysaccharides to characterize the sulfate pattern of agarocolloids.Thepresenceofthelowintensitybandsat850and 820cm−1_{maysuggestasmalldegreeofsulfatesubstitutionatC-4}

andC-6ofgalactose.

Theanomericregionof13_{CNMR(Fig.1B)showstwomain}

sig-nals,whichwereassignedbasedonliteraturedata(Lahaye,Yaphe, Viet&Rochas,1989;Miller&Furneaux,1997;Usov,Yarotsky& Shashkov,1980; Valiente, Fernandez,Perez, Marquina,&Velez, 1992)asC-1of␤-d-galactoselinkedto3,6-␣-l-anhydrogalactose

atı102.6andC-1of3,6-anhydro-␣-l-galactopyranoseat_ı98.4.

ADEPT135◦ _{experimentwasusedtoinvestigatethepresenceof}

CH2groups,consideringthatthepulsesequencesignalsofthe

car-bonsbearingtwoprotonshaveoppositeamplitudetotheCHand CH3carbons.TheDEPT135◦spectrumofD.simplex(Fig.1C)shows

twointenseCH2 signals atı61.5andı69.5attributedtoC-6of

␤-d-galactose and 3,6-_␣-l-anhydrogalactose, respectively. The

lowerintenseCH2 signalobservedatı65.7maybeattributedas

C-6of␣-l-galactose-6sulfate.

Thetwelvemainsignalsobservedinthe13_{CNMRspectrumcan}

beattributedbasedintheliteraturedata(Lahayeetal.,1989;Maciel etal.,2008;Meloetal.,2002;Miller&Furneaux,1997;Usovetal., 1980;Valienteetal.,1992)toC-1–C-6of␤-d-galactose(102.5,70.3,

82.3,68.9,75.5and61.5)andof3,6-␣-l-anhydrogalactose(98.4,

69.9,80.2,77.4,75.7and69.5).

The13_{C-NMRandFT-IRspectraindicatethatthemain}

polysac-charide fraction extracted from D. simplex is a agarocolloid, corroboratingwithpreviousstudies(El-Sayed,1983;Tomodaetal., 1972).

3.2. Theanti-inflammatoryactivityofPLSonpawedemainduced bycarrageenananddextran

SincePLSderivedfromalgaerepresentanimportantcandidate therapeuticforthetreatmentofinflammation(Chen,Wu,&Wen, 2008;Groth,Grunewald,&Alban,2009),theanti-inflammatory activityofPLSextractedfromD.simplexwasassessedusing clas-sical experimental models of inflammation. Fig. 2 summarizes theeffectsofPLSonpawedemainexperimentalanimals. Treat-mentofmicewithcarrageenan(500␮g/paw;PanelA)ordextran (500␮g/paw;PanelB)inducedasignificantincreaseinpawvolume

thefourthhourafterinflammagenadministration(p<0.05).The maximumeffect induced byPLSadministration at dosesof 10, 30and60mg/kgwasobservedatthethirdhour,withinhibition rates of65.0%, 66.0% and 87.5%,respectively, compared to ani-malsthatreceivedcarrageenanalone.TheinhibitoryeffectofPLS onpawedemainducedbycarrageenanwasdose-dependent.The mosteffectivedosewas60mg/kgandthisdosewasthuschosenfor furtherevaluation,includingthatofitseffectsondextran-induced pawedema.WesubsequentlyobservedthatPLSalsoinhibitedthe edemainducedbydextran.Aninhibitionrateof80.6%wasobserved afterthefirsthour,comparedtothefindingobservedinthe con-trolgroup treated withdextranalone. The anti-oedematogenic effect of PLS against dextran-induced inflammation was main-taineduntilthefourthhourofanalysisinwhichtheedemawas almostcompletelyabsent(95.6%inhibition).Itiswell-documented thatinflammatoryeventstriggeredbydextranadministrationlead todevelopmentof edemaviaresident celldegranulation,while thoseinducedbycarrageenaninvolvecellmigration(Lo,Almeida, &Beaven,1982).Ourdataareinagreementwiththoseofother studiesinwhichanti-inflammatoryeffectsofPLSextractedfrom seaweedagainstcarrageenan-or dextran-inducedinflammation were reported (Chaves et al., 2013; Damasceno et al., 2013). Furthermore,PLS(60mg/kg)wasasefficaciousasindomethacin (p>0.05),acommercialdrugusedasananti-inflammatoryagent, reinforcingthepharmacologicpotentialofPLSextractedfromthis algalspecies.

3.3. EffectofPLSonpawedemainducedbyvariousinflammagens

Comparedtoinjectionofthesalinecontrol,theinjectionof var-iousinflammagensintothesubplantarsurfaceofmousehindpaws producedamarkedincreaseinpawvolume(p<0.05)(Fig.3).The injectionof PLS(60mg/kg, i.p.)significantly reducedtheedema induced byphlogistic agents during alltested time courses.At 30min,thetimepointofthepeakeffectofthetestedagents,the edemavolumeinthePLSgroupwas0.022±0.004mLcomparedto

0.117±0.017mLinthehistaminegroup,correspondingto80.7%

inhibition (Fig.3A).PLSwasmore effectivethan indomethacin, which produced only a 58.9% inhibition. PLS also significantly inhibitedtheincreaseinpawvolumeofanimalstreatedwith sero-toninorbradykininby38.0%and42.8%,respectively(Fig.3BandC). Itiswellknownthatchemicalmediatorsincludinghistamine, sero-tonin,and bradykininareinvolved incarrageenan-induced paw edema(Chen,Tsai,&Wu,1995;Vinegaretal.,1987).Inthismodel, theedema isbelievedtobebiphasic,withthefirstphasebeing mediatedbythereleaseofhistamineandserotonin,followedbythe subsequentreleaseofbradykininandthelateedemaphasebeing dependentoncytokineproductionbyresidentcellsandneutrophil infiltration(Barbosaetal.,2009;Kulkarni,Mehta,&Kunchandy, 1986; Vinegar, Schreiber, & Hugo, 1969). In contrast, dextran-inducedpawedemaismediatedbyincreasedvascularpermeability inducedby mast celldegranulation of histamine andserotonin (Metcalfe, 2008).Theoedematousfluid formedbecauseof dex-traninjectioncontainslittleproteinandfewneutrophils(Gupta etal.,2003).Therefore,wecaninferthattheanti-oedematogenic actionofPLSmaybebecauseofthedifferentialinhibitionofspecific inflammatorymediatorsandmodulationofneutrophilinfiltration intheinflamedplantartissue.

3.4. EffectofPLSoncarrageenan-inducedMPOactivityinmouse pawtissue

AsshowninFig.4A,at4haftertreatmentwithinflammatory stimuli,a significantincrease inMPOactivitywasfoundinthe carrageenan-treatedanimals(15.23±3.45UMPO/mgplantar

tis-sue)comparedtothosetreatedwithsaline(0.68±0.11UMPO/mg

A

0 1 2 3 4

0.00 0.07 0.14 0.21

Sal Hist PLS + Hist Indo + Hist

*

*

*

*

*

*

_*

*

*

*

*

*

*

Time (h)

Change in volume (ml)

B

0 30 60 90 120

0.00 0.03 0.06 0.09 0.12

Sal Ser PLS + Ser Indo + Ser

*

*

*

*

*

*

*

_*

Time (min)

Change in volume (ml)

C

0 30 60 90 120

0.00 0.03 0.06 0.09

Sal Bk PLS + Bk

Indo + Bk

*

*

_*

*

*

_*

*

*

*

*

*

*

Time (min)

Change in volume (ml)

Fig.3.EffectofPLSfromD.simplexonpawedemainducedbyvarious inflamma-gens.Pawedemawasinducedby(A)histamine(Hist:500␮gperpaw),(B)serotonin (Ser:500␮gperpaw),or(C)bradykinin(Bk:6nmol,w/v,perpaw)injectionsintothe plantarsurfaceoftherightpaw.Thechangeinpawvolumewasmeasuredatthe indi-catedtimeintervals.Animalswerepretreatedwithpolysaccharide(PLS:60mg/kg; i.p.)andindomethacin(Indo:10mg/kg,i.p.)wasusedasapositivecontrol.The valuesgivenaremeans±S.E.M.(n=5).*Statisticaldifference(p<0.05)compared toinflammatorystimulitreatment(one-wayANOVAfollowedbyNewman–Keuls post-test).

plantar tissue). Pre-treatment withPLS (60mg/kg) consistently reducedMPOactivityinpawtissue(4.17±0.71UMPO/mgof

tis-sue);theeffectwassimilartothatofindomethacin(5.03±1.48

A

Sal - PLS Indo

0 5 10 15

20 #

*

*

Carrageenan (500 g per paw)

UMPO/mg of tissue

B

C

Sal - PLS Indo

0 2000 4000 6000 8000

10000 #

*

*

Carrageenan (500 g/cavity)

Total leucocytes × 10

3 ml/cavity

Sal - PLS Indo

0 2000 4000 6000 8000 10000

#

*

*

Carrageenan (500 g/cavity)

Neutrophils × 10

3ml/cavity

Fig.4.TheeffectsofPLSfromD.simplexonmyeloperoxidase(MPO)activityinpawtissueandcellmigrationintheperitonealcavity.Animalsreceived polysaccha-ride(PLS:60mg/kg;i.p.)30minbeforecarrageenanadministration,andmyeloperoxidaseactivityorneutrophilmigrationwereevaluated4hlater.Thevaluesgivenare means±S.E.M.(n=5).Indomethacin(Indo:10mg/kg,i.p.)wasusedasapositivecontrol.#_{p<0.05vs.salinegroup;*p<0.05vs.carrageenangroup(one-wayANOVAfollowed}

byNewman–Keulspost-test).

3.5. Anti-inflammatoryeffectofPLSoncarrageenan-induced peritonitisinmice

The hypothesis that the anti-oedematogenic effect of PLS againstcarrageenan-inducedinflammationwasrelatedtoa reduc-tion in neutrophil infiltration to the site of inflammation was

further investigated in an experimental mouse model of peri-tonitis. This model allows quantification of cells and levels of several inflammatory mediators and is a well-characterized pharmacological tool for theexamination of neutrophil migra-tion(Montanher,Zucolotto,Schenkel,&Fröde,2007).Compared to the animals treated with saline alone, those treated with

A

B

Sal

-

PLS

Indo

0

500

1000

1500

*

*

#

Carrageenan (500 g/cavity)

(pg /ml)

Sal - PLS Indo

0 200 400 600 800

#

*

*

Carrageenan (500 g/cavity)

(pg/ml)

Fig.5. EffectofPLSfromD.simplexoncarrageenan-inducedcytokineproductioninperitonitis.Animalswereinjectedwithpolysaccharide(PLS:60mg/kg)30minbefore carrageenanadministration,and4hlaterthelevelsofIL-1␤(A)andTNF-␣(B)weremeasuredintheperitonealfluid.Thevaluesgivenaremeans±S.E.M.(n=5).Indomethacin (Indo:10mg/kg)wasusedasapositivecontrolforanti-inflammatoryactivity.#,_{*Statisticaldifference(p<0.05)comparedtosalineandcarrageenan,respectively(one-way}

Table1

AntinociceptiveeffectofPLSfromD.simplexonaceticacidinducedwrithes.

Treatment Dose

(mg/kg)

Numberofabdominal constrictions(20min)

Inhibition(%)

Control(aceticacid) – 47.0±5.6 –

Morphine 5 0.0±0.0a 100

PLS 60 10.8±3.6a 77.0

Valuesaregivenasthemeans±S.E.M.offiveanimals,asanalyzedbyone-way

ANOVAfollowedbyNewman–Keulstest(p<0.05).PLS:sulphatedpolysaccharides fraction.

a_{Comparedwithaceticacidcontrolgroup.}

intraperitoneal administration of carrageenan showed a sig-nificant increase in total leukocyte and neutrophil counts in the peritoneal fluid (1525±185.4×103 total leukocytes/mL

vs. 9050±429.1×103 total leukocytes/mL; 370.6±42.9×103

neutrophils/mL vs. 7789±459.1×103 neutrophils/mL; p<0.05) (Fig. 4B and C). Pre-treating theanimals with 60mg/kg of PLS reduced the total leukocyte (4913±668.4×103cells/mL) and

neutrophilmigration(3804±1228×103cells/mL)tolevels

com-parableto thecorresponding levelsobserved in the salineand indomethacingroups(p>0.05).

3.6. EffectofthePLSfractiononcarrageenan-inducedcytokine productioninperitonealexudates

Theinflammatoryresponseinducedbycarrageenaninthe peri-tonitismodelwasassociatedwithanincreaseinthelevelsofIL1-␤

andTNF-␣(Fig.5AandB).ThelevelsofIL1-␤andTNF-␣inthe per-itonealfluidofthesalinecontrolgroupwere155.4±19.7pg/mL

and 249.5±91.5pg/mL, respectively; the corresponding levels

were higher in the carrageenan-treated animals (IL1-␤ level: 1187±39.8pg/mL and TNF-␣ level: 564.9±52.3pg/mL).

Car-rageenaninducesneutrophilmigrationintotheperitonealcavity through an indirect mechanism that involvesthe activation of macrophagesandreleaseofpro-inflammatorycytokines,suchas IL-1␤ and TNF-␣ (Lo et al., 1982). Such increases in cytokine levels might result in plasma protein extravasation and cel-lular infiltration into the site of inflammation (Rosenbaum & Boney,1991;Thorlacius,Lindbom,&Raud1997).Inthepresent study, compared to the carrageenan-treated group, the PLS-treated animals exhibited a significant reduction of both IL1-␤

andTNF-␣levelsinperitonealexudates(330.9±45.1pg/mLand

81.1±4.3pg/mL, respectively) (p<0.05). TNF-␣ and IL-1␤ are potentpro-inflammatorycytokinesthatpossessmultipleeffects, includingtheactivationofinflammatorycells,inductionofseveral inflammatoryproteins, edemaformation,andneutrophil migra-tion(Haddad,2002;Hopkins,2003).Onthebasisofthisfinding, we propose thatPLS treatmentdecreased neutrophilmigration by decreasing the production and release of pro-inflammatory cytokines.

3.7. AntinociceptiveeffectofPLSonaceticacid-inducedwrithing

There is a strong association between the development of pain and the inflammatory process (Bitencourt et al., 2008). It was previously demonstrated that inhibition of neutrophil migrationreduceshypernociceptioninducedbydifferent inflam-matory stimuli (Hopkins, 2003; Tjølsen &Hole, 1997). Several reports havedemonstrated that PLSextractedfromalgae show promiseasantinociceptiveagents(Britoetal.,2013;Chavesetal., 2013; Damasceno et al., 2013). Thus, in thepresent study the

0 20 40 60 80

Sal Formalin PLS + Formalin Morphine +Formalin

1st Phase 2nd Phase

# #

*

*

*

*

Licking time (s)

A

0 30 60 90 120

0 10 20 30 40

Sal PLS Morphine

*

*

_*

#

#

#

B

latency (s)

Fig.6. AntinociceptiveeffectofPLSfromD.simplexinformalin-inducedpawlicking(A)andhotplatetest(B).AnimalsreceivedPLS(60mg/kg,i.p.)ormorphine(5mg/kg, sc)30minbefore2.5%formalinadministrationbytheintraplantarroute.Lickingtimewasrecordedinthefirst5min(1stphase)andafter20min(2ndphase)during5min. InthehotplatetesttheanimalsreceivedthesamedosesofPLSormorphinetoevaluatedreactiontimestothermalstimuli.Thevaluesgivenaremeans±S.E.M.(n=6).Panel (A)#,_{*Statisticaldifference(p<0.05)comparedtosalineandformalin,respectively.Panel(B)*},#_{Statisticaldifference(p<0.05)comparedtosaline(one-wayANOVAfollowed}

antinociceptive potentialof PLSfrom D. simplex wasalso eval-uated using three well-accepted murine pain models. Table 1

showstheantinociceptiveeffectofPLSonabdominalcontractions inducedbyaceticacid. Asexpected,morphineshowedapotent analgesicresponsecomparedtothatobservedincontrolanimals (p<0.05). Theadministration of PLS(60mg/kg)30minprior to painfulstimulisignificantlyreducedaceticacid-induced abdom-inal writhing (77.0% inhibition) compared to control (p<0.05). Thewrithing test is commonly used for screening peripherally activeanalgesiccompounds.Algogenicagents,suchasaceticacid, provokeastereotypicalbehaviorinmicecharacterizedby abdom-inalcontractions,movementsofthebodyasawhole,andtwisting ofdorsalabdominalmuscles(Bars,Gozariu,&Cadden,2001).This modelinvolvesdifferentnociceptivemechanisms,suchasrelease of biogenic amines (histamine and serotonin), bradykinin, and PGE2(Collieret al.,1968; Duarte,Nakamura, &Ferreira,1988). Furthermore,itiswellestablishedthatthenociceptiveresponse causedby aceticacidisalsodependentontherelease ofsome cytokines,suchasTNF-␣andIL-1␤viamodulationofmacrophages and mastcells localized totheperitonealcavity (Ribeiroet al., 2000).Thus,PLSmayreducewrithingbyinhibitionoftherelease ofnociceptivemediatorsinresponsetoaceticacid.

3.8. EffectofPLSintheformalintest

Fig.6Ashowsthatintraplantarinjectionofformalin(2.5%/paw) significantlyincreasedthetotallickingtimeinthefirstand sec-ondphases compared to salinetreatment(p>0.05).This event wasstronglyinhibitedinbothphasesbypre-treatmentwiththe PLSfraction(60mg/kg).Weobservedthatthelickingtimeof PLS-treatedanimalswasinhibitedby60.5%and61.7%inthefirstand secondphases,respectively, comparedtothefindings observed in theformalin group. The inhibitoryeffect of PLS wassimilar tothat seen in the morphine group (Firstand secondphases). Formalin-inducedpersistent pain in mouse paws involves two distinctphases.Thefirstphase (neurogenic)ischaracterizedby directchemicalstimulationofnociceptors,andthesecondphaseis accompaniedbythereleaseofinflammatorymediators,such neu-ropeptides,prostaglandins,serotonin,histamine,and bradykinin (Hunskaaretal.,1985;Hunskaar&Hole,1987;Murray,Porreca, &Cowan,1988).SincePLSexhibitedactivityinbothphases,itis suspectedthatitactscentrally,andapossibleinteractionwith opi-oidreceptorsisalsoindicated(Shibata,Ohkubo,Takahashi,&Inoki, 1989).

3.9. EffectofPLSinthehotplatetest

TheeffectofintraperitonealadministrationofPLS(60mg/kg) onanimalsevaluatedinthehotplatetestvariedaccordingtothe observationtimeused(Fig.6B).Attimezeroand120min,no sig-nificantantinociceptiveeffectwasobservedcomparedtocontrols (p>0.05).At30,60,and90min,PLSincreasedthereactiontimesof miceby34.4%,122.9%,and160.5%,respectively.Thereferencedrug morphine,anopioidreceptoragonist,inducedasignificantincrease inlatency,asexpected.Thehot-platetestisawell-knownmodel foracutethermalnociception.Increasesinreactiontimeindicate analgesiceffectsviasupraspinalandspinalreceptors(Nemirovsky, Chen,Zelman,&Jurna,2001).Furthermore,thetestspecifically helps evaluatecentral nociception (Vilela, Padilha, DosSantos, Silva,&Paiva,2009)andmeasurecomplexresponsesto inflam-mationand nociception promotedby opioid agents (Bhandare, Kshirsagar,Vyawahare,Hadambar,&Thorve,2010).Onthebasis ofourresults,weconcludedthatPLScaninhibitnociceptionby eitherperipheralorcentralmechanisms.

References

Barbosa,A.L.,Pinheiro,C.A.,Oliveira,G.J.,Moraes,M.O.,Ribeiro,R.A.,Vale,M.L., etal.(2009).Tumorbearingdecreasessystemicacuteinflammationinrats-role ofmastcelldegranulation.InflammationResearcher,58,235–240.

Bars,D.,Gozariu,M.,&Cadden,S.W.(2001).Animalmodelsofnociception.

Phar-macologicalReviews,53,597–652.

Bhandare,A.M.,Kshirsagar,A.D.,Vyawahare,N.S.,Hadambar,A.A.,&Thorve,V. S.(2010).Potentialanalgesic,anti-inflammatoryandantioxidantactivitiesof hydroalcoholicextractofArecacatechuL.nut.FoodandChemicalToxicology,48, 3412–3417.

Bitencourt,F.S.,Figueiredo,J.G.,Mota,M.R.L.,Bezerra,C.C.R.,Silvestre,P.P.,Vale, M.R.,etal.(2008).Antinociceptiveandanti-inflammatoryeffectsofa mucin-bindingagglutininisolatedfromtheredmarinealgaHypneacervicornis.

Naunyn-Schmiedeberg’sArchivesofPharmacology,377,139–148.

Bradley,P.P.,Priebat,D.A.,Christensen,R.D.,&Rothstein,G.(1982).Measurement ofcutaneousinflammation:Estimationofneutrophilcontentwithanenzyme marker.JournalofInvestigativeDermatology,78,206–209.

Brito,T.V.,Prudêncio,R.S.,Sales,A.B.,Júnior,F.C.V.,Candeira,S.J.N.,Franco,A. X.,etal.(2013).Anti-inflammatoryeffectofsulfacted-polyssacaridefractionof extractedfromredAlgaeHypneamusciformisviathesuppressionneutrophil migrationbyNOsignalingpathway.JournalofPharmacyandPharmacology,65, 724–733.

Cabrita,M.T.,Vale,C.,&Rauter,A.P.(2010).Halogenatedcompoundsfrommarine algae.MarineDrugs,8,2301–2317.

Carvalho,J.T.C.,Teixeira,J.R.M.,Souza,P.J.C.,Bastos,J.K.,Filho,D.S.,&Sarti,S. J.(1996).Preliminarystudiesofanalgesicandanti-inflammatorypropertiesof

Caesalpineaferreacrudeextract.JournalofEthnopharmacology,53,175–178.

Chaves,L.S.,Nicolau,L.A.D.,SILVA,R.O.,Barros,F.C.N.,Freitas,A.L.P.,Aragão,K.S., etal.(2013).Anti-inflammatoryandantinociceptiveeffectsinmiceofasulfated polysaccharidefractionextractedfromthemarineredalgaeGracilariacaudata.

ImmunopharmacologyandImmunotoxicology,35,93–100.

Chen,D.,Wu,X.Z.,&Wen,Z.Y.(2008).Sulfatedpolysaccharidesandimmune response:Promoterorinhibitor?PanminervaMedica,50,177–183.

Chen,Y.F.,Tsai,H.Y.,&Wu,T.S.(1995).Anti-inflammatoryandanalgesicactivities fromrootsofAngelicapubescens.PlantaMedica,61,2–8.

Chopin,T.,&Whalen,E.(1993).Anewandrapidmethodforcarrageenan identifi-cationbyFTIRdiffuse-reflectancespectroscopydirectlyondried,groundalgal material.CarbohydrateResearch,246,51–59.

Collier,H.O.J.,Dinneen,J.C.,Johnson,C.A.,&Schneider,C.(1968).The abdomi-nalconstrictionresponseanditssuppressionbyanalgesicdrugsinthemouse.

BritishJournalofPharmacologyandChemotherapy,32,295–310.

Cumashi,A.,Ushakova,N.A.,Preobrazhenskaya,M.E.,D’Incecco,A.,Piccoli,A., Totani,L.,etal.(2007).Acomparativestudyoftheanti-inflammatory, antico-agulant,antiangiogenic,andantiadhesiveactivitiesofninedifferentfucoidans frombrownseaweeds.Glycobiology,17,541–552.

Damasceno,S.R.B.,Rodrigues,J.C.,Silva,R.O.,Nicolau,L.A.D.,Chaves,L.S.,Freitas, A.L.P.,etal.(2013).RoleoftheNO/KATPpathwayintheprotectiveeffectof asulfatedpolysaccharidefractionfromthealgaeHypneamusciformisagainst ethanolinducedgastricdamageinmice.RevistaBrasileiradeFarmacognosia,23, 320–328.

DeSmet,P.A.(1997).Theroleofplant-deriveddrugsandherbalmedicinesin healthcare.Drugs,54,801–840.

Déciga-Campos,M.,Palacios-Espinosa,J.F.,Reyes-Ramírez,A.,&Mata,R.(2007).

Antinociceptiveand anti-infammatoryeffectsof compoundsisolatedfrom

ScaphyglottislividaandMaxillariadensa.JournalofEthnopharmacology,114,

161–168.

Duarte,I.D.G.,Nakamura,M.,&Ferreira,S.H.(1988).Participationofthe sympa-theticsysteminaceticacid-inducedwrithinginmice.BrazilianJournalofMedical

andBiologicalResearch,21,341–343.

Eddy,N.B.,&Leinback,D.(1953).Syntheticanalgesics.II.Dithienyl-butenyland dithienylbutylamines.JournalofPharmacologyandExperimentalTherapeutics, 107,385–393.

El-Sayed,M.M.(1983).PurificationandcharacterizationofagarfromDigenea

sim-plex.CarbohydrateResearch,118,119–126.

Groth,I.,Grunewald,N.,&Alban,S.(2009).Pharmacologicalprofilesofanimal-and nonanimal-derivedsulfatedpolysaccharides—Comparison of unfractionated heparin,thesemisyntheticglucansulfatePS3,andthesulfatedpolysaccharide fractionisolatedfromDelesseriasanguinea.Glycobiology,19,408–417.

Gupta,M.,Mazumdar,U.K.,Sivakumar,T.,Vamsi,M.L.,Karki,S.S.,Sambathkumar, R.,etal.(2003).Evaluationofanti-inflammatoryactivityofchloroformextract

ofBryonialaciniosainexperimentalanimalmodels.Biological&Pharmaceutical

Bulletin,26,1342–1344.

Haddad,J.J.(2002).Cytokinesandrelated-receptormediatedsignallingpathways.

BiochemicalandBiophysicalResearchCommunications,297,700–713.

Hajare,S.W.,Chandra,S.,Charma,J.,Tandan,S.K.,Lal,J.,&Telang,A.G.(2001).

Anti-inflammatoryactivityofDalbergiasissooleaves.Fitoterapia,72,131–139.

Hopkins,S.J.(2003).Thepathologicalroleofcytokines.LegalMedicine,5,45–47.

Hunskaar,S.,&Hole,K.(1987).Theformalintestinmice:Dissociationbetween inflammatoryandnon-inflammatorypain.Pain,30,103–104.

Hunskaar,S.,Fasmer,O.B.,&Hole,K.(1985).Formalintestinmice,ausefultechnique forevaluatingmildanalgesics.JournalofNeuroscienceMethods,14,69–76.

Iannitti,T.,&Palmieri,B.(2010).Anupdateonthetherapeuticroleofalkylglycerols.

MarineDrugs,8,2267–2300.

Koster,R.,Anderson,M.,&De-Beer,E.J.(1959).Aceticacidforanalgesicscreening.

Kulkarni,S.K.,Mehta,A.K.,&Kunchandy,J.(1986).Anti-inflammatoryactionsof clonidine,guanfacineandB-HT920againstvariousinflammagen-inducedacute pawoedemainrats.ArchivesInternationalesdePharmacodynamieetdeThérapie, 279,324–334.

Kusaykin,M.,Bakunina,I.,Sova,V.,Ermakova,S.,Kuznetsova,T.,Besednova,N.,etal. (2008).Structure,biologicalactivity,andenzymatictransformationoffucoidans fromthebrownseaweeds.BiotechnologyJournal,3,904–915.

Lahaye,M.,&Yaphe,W.(1988).Effectsofseasonsonthechemicalstructureandgel strengthofGracilariapseudoverrucosaagar(Gracilariales,Rhodophyta).

Carbo-hydratePolymers,8,285–301.

Lahaye,M.,Yaphe,W.,Viet,M.T.P.,&Rochas,C.(1989).13_{CNMRspectroscopic}

investigationofmethylatedandchargedagaroseoligosaccharidesand polysac-charides.CarbohydrateResearch,190,249–265.

Lanhers,M.C.,Fleurentin,J.,Dorfman,P.,Mortier,F.,&Pelt,J.M.(1991).Analgesic, antipyreticandanti-inflammatorypropertiesofEuphorbiahirta.PlantaMedica, 57,225–231.

Linley,J.E.,Rose,K.,Ooi,L.,&Gamper,N.(2010).Understandinginflammatorypain: Ionchannelscontributingtoacuteandchronicnociception.EuropeanJournalof

Physiology,459,657–669.

Lo,T.N.,Almeida,A.P.,&Beaven,M.A.(1982).Dextranandcarrageeninevoke differ-entinflammatoryresponseinratwithrespecttocompositionofinfiltratesand effectofindomethacin.JournalofPharmacologyandExperimentalTherapeutics, 221,261–267.

Maciel,J.S.,Chaves,L.S.,Souza,B.W.S.,Teixeira,D.I.A.,freitas,A.L.P.,feitosa,J. P.A.,etal.(2008).Structuralcharacterizationofcoldextractedfractionof sol-ublesulfatedpolysaccharidefromRedSeaweedGracilariabirdiae.Carbohydrate

Polymers,71,559–565.

Mayer,A.M.S.,Glaser,K.B.,Cuevas,C.,Jacobs,R.S.,Kem,W.,Little,R.D.,etal.(2010).

Theodysseyofmarinepharmaceuticals:Acurrentpipelineperspective.Trends

inPharmacologicalSciences,31,255–265.

Melo,M.R.S.,Feitosa,J.P.A.,Freitas,A.L.P.,&dePaula,R.C.M.(2002).Isolation andCharacterizationofsolublesulfatedpolysaccharidefromtheRedSeaweed

Gracilariacornea.CarbohydratePolymers,49,491–498.

Metcalfe,D.D.(2008).Mastcellsandmastocytosis.Blood,15,946–956.

Miller,I.J.,&Furneaux,R.H.(1997).Thestructuraldeterminationoftheagaroid polysaccharidefromfourNewZealandalgaeintheorderCeramialesbymeans ofC-13NMRspectroscopy.BotanicaMarina,40,333–339.

Mollet,J.-C.,Rahaoui,A.,&Lemoine,Y.(1998).Yield,chemicalcompositionandgel strenghofagarocolloidsofGracilariagracilis,Gracilariopsislongissimaandthe newlyreportedGracilariacf.vermiculophyllafromRoscoff(Brittany,France).

JournalofAppliedPhycology,10,59–66.

Moncada,S.,&Higgs,A.(1993).Thel-arginine-nitricoxidepathway.TheNewEngland

JournalofMedicine,329,2002–2012.

Montanher,A.B.,Zucolotto,S.M.,Schenkel,E.P.,&Fröde,T.S.(2007).Evidenceof anti-inflammatoryeffectsofPassifloraedulisinaninflammationmodel.Journal

ofEthnopharmacology,109,281–288.

Murray,C.W.,Porreca,F.,&Cowan,A.(1988).Methodologicalrefinementstothe mousepawformalintest.Ananimalmodeloftonicpain.Journalof

Pharmaco-logicalMethods,20,175–186.

Nemirovsky,A.,Chen,L.,Zelman,V.,&Jurna,I.(2001).Theantinociceptiveeffectof thecombinationofspinalmorphinewithsystemicmorphineorbuprenorphine.

AnesthesiologyAnalgesic,93,197–203.

Prado-Fernandez,J.,Rodriguez-Vazquez,J.A.,Tojo,E.,&Andrade,J.M.(2003). Quan-titationof␬-,␫-and␭-carrageenansbymid-infraredspectroscopyandPLS regression.AnalyticaChimicaActa,480,23–37.

Ribeiro,R.A.,Vale,M.L.,Thomazzi,S.M.,Paschoalato,A.B.,Poole,S.,Ferreira,S.H., etal.(2000).Involvementofresidentmacrophagesandmastcellsinthewrithing nociceptiveresponseinducedbyzymosanandaceticacidinmice.European

JournalofPharmacology,387,111–118.

Rochas,C.,Lahaye,M.,&Yaphe,W.(1986).Sulfatecontentofcarrageenanandagar determinedbyInfraredSpectroscopy.BotanicaMarina,29,335–340.

Rosenbaum,J.T.,&Boney,R.S.(1991).Useofasolubleinterleukin-1receptorto inhibitocularinflammation.CurrentEyeResearch,10,1137–1139.

Shibata,M.,Ohkubo,T.,Takahashi,H.,&Inoki,R.(1989).Modifiedformalintest: Characteristicbiphasicpainresponse.Pain,38,347–352.

Silva,J.S.E.,Moura,M.D.,Oliveira,R.A.G.,Diniz,M.F.F.M.,&Barbosa-Filho,J. M.(2003).Naturalproductsinhibitorsofovarianneoplasia.Phytomedicine,10, 221–232.

Silva,R.O.,Santos,G.M.P.,Nicolau,L.A.D.,Lucetti,L.T.,Santana,A.P.M.,Chaves,L.S., etal.(2011).Sulfated-polysaccharidefractionfromredalgaeGracilariacaudate protectsmicegutagainstethanol-induceddamage.MarineDrugs,9,2188–2200.

Souza,G.E.,Cunha,F.Q.,Mello,R.,&Ferreira,S.H.(1988).Neutrophilmigration inducedbyinflammatorystimuliisreducedbymacrophagedepletion.Agents

Actions,24,377–380.

Srinivasan,K.,Muruganandan,S.,Lal,J.,Chandra,S.,Tandan,S.K.,&Prakash,V.R. (2001).Evaluationofanti-inflammatoryactivityofPongamiapinnataleavesin rats.JournalofEthnopharmacology,78,151–157.

Takano, R.,Shiomoto, K., Kamei, K.,Hara, S.,& Hirase,S.(2003).Occurrence of carrageenan structurein an agarfrom the Red SeaweedDigenea sim-plex(Wulfen)C.agardh(Rhodomelaceae,Ceramiales)withashortreviewof Carrageenan–AgarocolloidhybridintheFlorideophycidae.BotanicaMarina,46, 142–150.

Thorlacius,H.,Lindbom,L.,&Raud,J.(1997).Cytokine-inducedleukocyterollingin mousecremastermusclearteriolesinP-selectindependent.AmericanJournalof

Physiology,272,1725–1729.

Tjølsen,A.,&Hole,K.(1997).Animalmodelsofanalgesia.InA.Dickenson,&J.Besson (Eds.),Thepharmacologyofpain(130)(pp.1–20).SpringerVerlag.

Tomoda,M.,Nakatsuka,S.,&Minami,E.(1972).Plantmucilages.IV.Mainstructural featuresofthemucouspolysaccharideisolatedfromDigeneasimplex.Chemical

andPharmaceuticalBulletin,20,953–957.

Usov,A.I.,Yarotsky,S.V.,&Shashkov,A.S.(1980).13_{C-NMRspectroscopyofred}

algalgalactans.Biopolymers,19,977–990.

Valiente,O.,Fernandez,L.E.,Perez,R.M.,Marquina,G.,&Velez,H.(1992).Agar PolysaccharidesfromRedSeaweedsGracilariadomingensisSonderexKutzing

andGracilariamammilaris(Montagne)Howe.BotanicaMarina,35,77–81.

Vilela,F.C.,Padilha,M.M.,DosSantos-E-Silva,L.,Alves-da-Silva,G.,&Giusti-Paiva,A. (2009).EvaluationoftheantinociceptiveactivityofextractsofSonchusoleraceus L.inmice.JournalofEthnopharmacology,124,306–310.

Vinegar,R.,Schreiber,W.,&Hugo,R.(1969).Biphasicdevelopmentofcarrageenan edemain rat. Journalof Pharmacology andExperimental Therapeutics, 166, 96–103.

Vinegar,R.,Truax,J.F.,Selph,J.L.,Johnston,P.R.,Venable,A.L.,&McKenzie,K.K. (1987).Pathwaytocarrageenaninducedinflammationinthehindlimbofthe

rat.FederationProceedings,46,118–126.

Wijesekara,I.,Pangestuti,R.,&Kim,S.K.(2010).Biologicalactivitiesandpotential healthbenefitsofsulfatedpolysaccharidesderivedfrommarinealgae.

Carbohy-dratePolymers,11,14–21.

Winter,C.A.,Risley,E.A.,&Nuss,G.W.(1962).Carrageenin-inducededemainhind pawoftheratasanassayforanti-inflammatorydrugs.ProceedingsoftheSociety