Sulfated from the marine algae Hypnea musciformis inhibits TNBSinduced intestinal damage in rats

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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

Sulfated

polysaccharide

from

the

marine

algae

Hypnea

musciformis

inhibits

TNBS-induced

intestinal

damage

in

rats

Tarcisio

V.

Brito

_,

_Francisco

_C.N.

_Barros

_,

_Renan

_O.

_Silva

_,

_Genilson

_J.

_Dias

_Júnior

_,

José

Simião

C.

Júnior

_,

_Álvaro

_X.

_Franco

_,

_Pedro

_M.G.

_Soares

_,

_Luciano

_S.

_Chaves

_,

Clara

M.W.S.

Abreu

_,

_Regina

_C.M.

_de

_Paula

_,

_Marcellus

_H.L.P.

_Souza

_,

_Ana

_Lúcia

_P.

_Freitas

_,

André

Luiz

R.

Barbosa

a_{LAFFEX—LaboratoryofExperimentalPhysiopharmacology,BiotechnologyandBiodiversityCenterResearch(BIOTEC),FederalUniversityofPiauí,}

Parnaíba,64202-020PI,Brazil

b_{LaboratoryofProteinsandCarbohydratesofMarineAlgae,DepartmentofBiochemistryandMolecularBiology,FederalUniversityofCeará,Fortaleza,}

60455-760CE,Brazil

c_{LEFFAG—LaboratoryofPhysiopharmacologyStudyofGastrointestinalTract,FederalUniversityofCeará,Fortaleza,CE,Brazil}

d_{DepartmentofMorphology,MedicalSchool,FederalUniversityofCeara,RuaDelmirodeFariass/n,RodolfoTeofilo,Fortaleza,CECEP60416-030,Brazil} e_{FederalInstituteofEducation,ScienceandTechnologyofCeará,JuazeirodoNorte,CE,Brazil}

f_{LaboratoryofPolymers,DepartmentofOrganicandInorganicChemistry,FederalUniversityofCeará,Fortaleza,60455-760CE,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15February2016

Receivedinrevisedform21April2016 Accepted11June2016

Availableonline14June2016

Keywords:

Sulfatedpolysaccharide Marinealgae Colitis

Inflammatorydisease

a

b

s

t

r

a

c

t

Sulfatedpolysaccharidesextractedfromseaweedhaveimportantpharmacologicalproperties.Thus,the aimofthisstudywastocharacterizethesulfatedpolysaccharide(PLS)fromthealgaeHypneamusciformis

andevaluateitsprotectiveeffectincolitisinducedbytrinitrobenzenesulfonicacidinrats.Thesulfated polysaccharidepossessahighmolecularmass(1.24×105gmol−1_{)andiscomposedofa␬-carrageenan,as}

depictedbyFT-IRandNMRspectroscopicdata.PLSwasadministeredorally(10,30,and60mg/kg,p.o.)for threedays,startingbeforeTNBS(trinitrobenzenesulfonicacid)instillation(day1).Theratswerekilledon daythree,theportionofdistalcolon(5cm)wasexcisedandevaluatedmacroscopicscoresandwetweight. Then,samplesoftheintestinalwereusedforhistologicalevaluationandquantificationofglutathione, malonyldialdehydeacid,myeloperoxidase,nitrate/nitriteandcytokines.OurresultsdemonstratethatPLS reducedthecolitisandallanalyzedbiochemicalparameters.Thus,weconcludedthatthePLSextracted fromthemarinealgaeH.musciformisreducedthecolitisinanimalmodelandmayhaveanimportant promisingapplicationintheinflammatoryboweldiseases.

©2016ElsevierLtd.Allrightsreserved.

1. Introduction

Marinealgaeareknownforproducinglargeamountsofsulfated polysaccharides,asacomponentoftheircellwalls,widelyusedin thefoodandpharmaceuticalindustrybecauseoftheir rheologi-calpropertiesasgellingandthickeningagents(Barrosetal.,2013; Kravchenkoetal.,2014).Thesepolymerspresentinredalgaeare galactansconsistingofgalactoseormodifiedgalactoseunits clas-sifiedasagaransandcarrageenansbasedonthestereochemistry ofthe4-linkedresidue,whichbelongstotheseries“L”inagarans

∗Correspondingauthorat:BIOTEC/LAFFEX/UFPI,Av.SãoSebastião,n◦_2819,

Par-naíba,CEP64202-020PI,Brazil.

E-mailaddress:[email protected](A.L.R.Barbosa).

and“D”incarrageenans(Cosenza,Navarro,Fissore,Rojas,&Stortz, 2014;Manivasagan&Oh,2016;Usov,1998).

Carrageenansareafamilyofsulfatedgalactansformedbya lin-earchainofalternatingresiduesof3-linked␤-d-galactopyranose

(G-units)and4-linked␣-d-galactopyranose(D-units)or4-linked

3,6-anhydro-␣-d-galactopyranose(DA-units)(Cosenzaetal.,2014;

VandeVelde,Knutsen,Usov,Rollema,&Cerezo,2002).The let-ter codes usedherebyare basedonnomenclatureproposedby

Knutsen, Myslabodski,Larsen,and Usov(1994).The most com-monly used carrageenansare normallyclassified as , and

formsaccordingtotheirsulfationpatternsandtheexistenceof 3,6-anhydro-␣-d-galactopyranose(3,6-AG,symbolizedasDA)(Liang,

Mao,Peng,&Tang,2014;Recaldeetal.,2016).

Marinealgaehave alsoproventoberichsources of biologi-callyactivemolecules,includingtheirpolysaccharides(Ioannou& Roussis,2009;Wang,Ooi,&Ang,2008).Inthiscontext,H.

T.V.Britoetal./CarbohydratePolymers151(2016)957–964

formisisaredalgaethatproducesseveralsulfatedpolysaccharides (PLS)withbiomedicalpotential.Recently,ourresearchgrouphas shownthatthePLSextractedfromHypneamusciformishas mod-ulated theacute inflammatory response mediated primordially byneutrophilmigration (Britoet al., 2013), hascaused protec-tionofgastricmucosaagainstaggressivestimuli,suchasethanol (Damascenoetal.,2013)hasproducedanti-diarrhealactivityin acute,inflammatory,andsecretorydiarrheamodels(Sousaetal., 2016), but untilnowanystudyhasdemonstratedtheaction of thePLSfromHypneaagainstintestinalinjuryintheTNBS-induced colitis.

Inflammatoryboweldiseases(IBD) arechronicinflammatory diseases characterized by inadequate activation of the intesti-nalandsystemicimmunesystemthatcausesadysregulationof mucosalimmunology (decreaseof immune response) and pro-gressiveinflammationonthecolonandgastrointestinaltract.In thiscontext,thetwomajorclinicallydefinedformsof inflamma-toryboweldiseasearetheulcerativecolitisandCrohn’sdisease (Hollander, 1988; Kaser, Zeissig, & Blumberg, 2010; Weber & Turner,2007).

Currently available treatments for IBD are effective only in ameliorating the disease symptoms while having many con-comitantdisadvantages.Antibiotics,one ofthecommonly used therapies,couldadverselychangetheenvironmentalconditionsof microfloraandtriggerresistance.Moreover,immunosuppressant andanti-inflammatorydrugs(suchascorticosteroids)havemany undesirablesideeffects(Sartor,2004).

KnowingthatPLSextractedfromHypneamusciformishave a potentialpharmacologicaleffectsagainstinflammatoryconditions (systemicinflammationanddiarrhea)andgastricdamage,theaim ofthepresentstudywastocharacterizethechemical structure ofthesulfatedpolysaccharideextractedfromHypneamusciformis

andinvestigatewhetherthisgalactancanmodulatetheintestinal damageduringTNBS-inducedexperimentalcolitisinrats.

2. Experimental

2.1. Algaesamples

ThemarineredalgaeH.musciformiswerecollectedfromthe AtlanticcoastatNortheastofBrazil(Flecheirasbeach,Trairí,Ceará, Brazil),atgeographicallocalization:03◦_{13’25”Sand39}◦_16’65”

W.AspecimenwasdepositedontheFicologicalHerbariumofthe SeaScienceInstituteatFederalUniversityofCeará–Fortaleza-CE, Brazil(ExsicateN◦_{2165).Thesampleswerecleanedofepiphytes,}

washedwithdistilledwater,andstoredat−20◦C.

2.2. Extractionofsulfatedpolysaccharides

Thesulfatedpolysaccharides(PLS)wereextractedaspreviously described(Farias,Valente,Pereira,&Mourão,2000)withminor changes.Driedtissue(5g)wereflouredandsuspendedin250mLof 0.1Msodiumacetatebuffer(pH5.0)containing5mMEDTA,5mM cysteinewithsubsequentadditionof510mgofpapain(E.Merck). Themixturewasincubatedat60◦_{Cfor6h.Residuewasremoved}

byfiltrationandcentrifugationat2725gfor30minat25◦_C.ThePLS

wereprecipitatedbyadditionof16mLof10%cetylpyridinium chlo-ride(CPC,SigmaChemical)andincubationfor24h.Themixture wasthen centrifugedat 8000g for30minat 4◦_{C. The}

polysac-charides in thepelletwere washed with500mLof 0.05% CPC, centrifugedatthesameconditionsandtheprecipitatesuspended in175mLofaNaCl2M:Ethanolsolution(100:15,v/v).The polysac-charideswereallowedtoprecipitatewith300mLethanolandthe pelletwaswashedtwicewith250mLethanol80%(v/v),oncewith

150mLofabsoluteethanolandthreetimeswithacetone,followed bydryingunderhotairflowat60◦_C.

2.3. GeneralmethodsforthechemicalanalysisofPLS

Total sugar content was determined by the sulfuric acid-UV technique (Albalasmeh, Berhe, & Ghezzehei, 2013), using

d-galactoseasstandard.Nitrogenandcarboncontentwere

deter-mined by elemental microanalysis (Perkin Elmer CHN 2400). ProteincontentwascalculatedfromN%usingthecorrectionfactor of6.25,asproposedbyMarks,Buchsbaum,andSwain(1985).

2.4. Molarmassdistribution

To estimate the peak molar mass (Mpk) of PLS a High-Performance Size-Exclusion Chromatography(HPSEC) was con-duced in a Shimadzu system equippedwith a refractive index detector(Model RID-10A)at roomtemperature usingan ultra-hydrogellinearcolumn(7.8×300mm),flowrateof0.5mL/min,

witha0.5%polysaccharidesolutionand0.1MNaNO₃ assolvent andeluant.Acalibrationcurvewasobtainedbyusingpullulans (ShodexDenko)ofdifferentmolecularweightsrangingfrom103_to

106_gmol−1_{.Theequationobtainedfromthiscalibrationplotwas:}

LogMw =146,827−106,967Ve (1)

whereVeistheelutionvolumeinmL.Thelinearcorrelation coef-ficientwas0.99.

2.5. Infrared(FT-IR)andnuclearmagneticresonance(NMR) spectroscopy

The Fourier transform infrared spectra (FT-IR) of PLS were recorded witha Shimadzu IRspectrophotometer (model 8300) scanningbetween400and4000cm−1_{.Thesampleswereanalyzed}

asKBrpellets.13_Cand1_{HNMRspectraof2.5%(w/v)solutionsof}

PLSinD2Owererecordedat353KonaFouriertransformBruker

AvanceDRX500spectrometerwithaninversemultinuclear gradi-entprobe-headequippedwithz-shieldedgradientcoils,andwith Silicon Graphics. Sodium 2,2-dimethylsilapentane-5-sulphonate (DSS)wasusedastheinternalstandard(0.00ppmfor1_H).

2.6. Animals

MaleWistarrats(180–200g)werederivateoftheFederal Uni-versityofPiauí(UFPI).Theanimalswerehousedinplasticcageat 25±2◦Cundera12/12hlight/darkcycle.Allratsweredeprived

offoodfor18–20hpriortotheexperimentalprocedurebutwere allowedfreeaccesstowater.Experimentswereconductedin accor-dance withcurrent established principles for thecare and use ofresearchanimals(NationalInstitutesofHealthguidelines)and approvedbyEthicsCommitteeoftheFederalUniversityofPiauí (ProtocolN◦_036/12).

2.7. Inductionofcolitis

Colitiswasinducedbyasingleintracolonicadministrationof 20mgof trinitricbenzenesulfonicacid(TNBS)dissolvedin50% ethanolsolutionintotheeachcolonofratatvolumeof800␮L.A

T.V.Britoetal./CarbohydratePolymers151(2016)957–964

2.8. Assessmentofcolitis

Theanimalsweredividedinsixgroups(n=5–7rats/group):a negativecontrolgroupthatreceivedintracolonicsaline(GroupI), thecontrolgroupreceivedonlyTNBS(GroupII)intothecolon,the groupsremainingwerepretreatedwithPLS(GroupIII,IVandV:10, 30and60mg/kg,p.o.)ordexamethasone(GroupVI:1mg/kg,s.c.) 1hbeforeofthecolitisinduction.Theanimalsweretreatedeach daywithPLSfractionordexamethasoneinthesecondandthird day.Theratswerekilledonthethirdday,1hafteroftreatment, abdomenswerethen openedandaftertheidentificationofthe intestine,theportionofdistalcolonwasexcised,washedwith0.9% salineandevaluatedthewetweightusingananalyticalbalance, afterthecolonwaspinnedontoawaxblockfortheevaluationof macroscopicscoresbymodifyingthecriteriapreviouslydescribed (Morrisetal.,1989).Then,samplesweretakenforbiochemistry assays.

2.9. Histologicalanalysis

Samplesintestinalwerefixedin10%formalinsolutionfor24h. Then,samplesweretransferredtoasolutionof70%alcohol.The materialwasthenembeddedinparaffinandsectioned;4-␮m-thick sectionsandstainedwithhematoxylin/eosinandevaluatedbyan experiencedpathologist(P.M.G.S).Histologicalcriteriaevaluated included:mucosalarchitectureloss(0–3),cellularinfiltrate(0–3), musclethickening(0–3),cryptabscess(0,absent;1,present),and gobletcelldepletion(0,absent;1,present).

2.10. Myeloperoxidase(MPO)activity

Briefly,thetissuewashomogenizedin1mLofpotassiumbuffer with0.5%ofhexadecitrimetilamônio(HTAB).Then, homogenate wascentrifuged at 40.000g for 10min at 4◦_{C. MPOactivity in}

the resuspended pellet was assayed by measuring the change inabsorbanceat450nmusingo-dianisidinedihydrochlorideand 1%hydrogenperoxide(Bradley,Priebat,Christensen,&Rothstein, 1982).TheresultswerereportedastheMPOunitspermgoftissue.

2.11. TNF-˛andIL-1ˇlevels

Level of IL-1␤ and TNF-␣ were evaluated using sandwich Enzyme-Linked Immunoabsorbent Assay (ELISA). ELISA kits for IL-1␤andTNF-␣werefromtheNationalInstitutefor Biological StandardsandControl(PottersBar,UK).Theresultswereexpressed aspicogramspermilliliter.

2.12. Glutathione(GSH)levels

Samples were homogenized in cold 0.02M EDTA solution (1mL/100mg oftissue).Aliquots(400␮L)oftissue homogenate weremixedwith320␮Lofdistilledwaterand80␮Lof50%(w/v) trichloroaceticacidinglasstubesandcentrifugedat3000rpmfor 15min.Next,400␮Lofeachsupernatantwasmixedwith800␮L ofTrisbuffer(0.4M,pH8.9),and20␮Lof0.01M 5,5-dithio-bis(2-nitrobenzoicacid).Aftershakingthepreparation,absorbancewas measured at 412nm on spectrophotometer (Sedlak & Lindsay, 1968).TheresultsareexpressedasmicrogramsofGSHpergramof tissue.

2.13. Malondialdehyde(MDA)concentration

ToquantifythelevelsofMDAcolonsampleproceededas fol-lows:sampleofthecolonwerehomogenizedwithcold1.15%KCl toprepare10%homogenates,immediatelyafter,250␮L ofeach homogenatewasaddedto1.5mLof1%H3PO4and0.5mLof0.6%

tert-butylalcohol(aqueoussolution).Then,thismixturewasstirred and heatedin a boilingwaterbath for45min.Thepreparation wasthencooledimmediatelyin anicewaterbath,followedby theadditionof2mLofn-butanol.Thismixturewasshakenand thebutanol layerwasseparated bycentrifugation at1200g for 10min.Absorbancewasdeterminedtobe520and535nm,andthe inferencebetweenthe2determinationswascalculated(Mihara& Uchiyama,1978).MDAconcentrationsareexpressedasmillimoles pergramoftissue.

2.14. NO3−_/NO2−_levels

Homogenateofintestinaltissueoftheanimalswasincubated ina microplatewithnitratereductase(0.016U/well)for12hto convertNO3−toNO2−.Nitricoxideproductionwasdetermined

bymeasuringnitriteconcentrations in anELISAplatereaderat 540nmusingtheGriessmethod(Greenetal.,1982).Resultswere expressed asmicromoles of nitrite using the internal standard curve.

2.15. Statisticalanalysis

Data were described as either means±SEM or median, as

appropriate.AnalysisofVariance(ANOVA),followedby Student-Newman-Keuls test, was usedto compare means;p<0.05 was definedasstatisticallysignificant.Thehistopathological parame-terswereanalyzedusingtheKruskal–Wallisnonparametrictest, followedbyamultiplecomparisonsDunn’stest.p<0.05was con-sideredstatisticallysignificant.

3. Resultsanddiscussion

3.1. ChemicalanalysisofHypneamusciformispolysaccharide

The Hypnea musciformis polysaccharide (PLS) was obtained by aqueous extraction under digestion by protease, yielding 31.8% of recovery from the algae dry weight. From a solu-tion of 1.0mgmL−1 _{of PLS (dissolved in distilled water)it was}

detected 0.97±0.03mgmL−1 _{of total soluble carbohydrate by}

the UV/sulfuric acid method (Albalasmeh et al., 2013). The homogeneityofthepolysaccharidewasconfirmedbyelemental microanalyses,whichshowedanitrogencontentof0.16±0.03%,

correspondingto1.00±0.18%of proteinresidues.These results

indicatesthattheprocedureusedtoextractedandisolatePLSwas suitabletoyieldacompoundfreeofundesiredmoleculeswhich couldinterfereinthesubsequentexperiments.

The molarmass distributionof Hypnea musciformis polysac-charide(PLS)wasestimatedbyHigh-PerformanceSize-Exclusion Chromatography. The HPSEC chromatogram of PLS (Fig. 1A) showeda singleand narrow peakat 8.97mL, correspondingto a peakmolarmass (Mpk)of 1.24×105gmol−1.High molecular

masses, such as the one described here, are common in car-rageenans (Campo,Kawano,da Silva,&Carvalho, 2009; Pomin, 2010). Cosenza et al. (2014) and Alves et al. (2012) evalu-ated the molar mass of Hypnea musciformis from Natal coast (Brazil).The molarmass obtainedfromAlveset al.(2012) was 1.47×105gmol−1 while Cosenza et al. (2014) obtained molar

massesrangingfrom0.88to2.87×105dependingonextraction

andpurificationmethodologies.Ourresultsareinthesamerange ofthosepreviouslyobtainedforthisalgaespecies.

FT-IRandNMRspectroscopictechniqueswereusedtoanalyze thechemical structure ofPLS. TheIRspectrum ofPLS(Fig.1B) showedbroadbandsat1230and1261cm−1_{,characteristicof}

T.V.Britoetal./CarbohydratePolymers151(2016)957–964

Fig.1.HPSECchromatogramofHypneamusciformispolysaccharideon ultrahydro-gelcolumn(A)andFT-IRspectruminKBrpellets(B).Wavenumbersrangefrom 1400to700cm−1_.

1068cm−1_{(Barrosetal.,2013;Sekkal&Legrand,1993).The}

posi-tionofsulfategroupscanbeinferredbybandsat800–850cm−1_.

Intense signals at 930cm−1 _{and 848cm}−1 _{were attributed to}

3,6-anhydro-galactopyranose(DA-units) and galactose-4-sulfate (G4S-units),respectively,indicatingthepresenceof-carragenan. Alowintensebandat804cm−1_{,describedforasulfategroupat}

C-2of3,6-anhydro-galactopyranose(DA2S-units),wassuggestive ofasmallamountof-carrageenan, whilenospecificbandsfor -carrageenanwerefound.(Kravchenkoetal.,2014;Villanueva& Montano,2003).

1_{H and} 13_{C NMR spectra are presented in Fig. 2.} 1_{H NMR}

spectrum (Fig. 2A) shows protonfrom anomeric carbon of 3,6 anhydrogalactose(DA)atı5.1andofgalctose-4-sulfate(G4S)at

ı4.63,characteristicof␬-carrageenan (Kravchenkoetal.,2014; Villanueva&Montano,2003).Alowintensesignalatı5.3isdueto 3,6anhydrogalactose-2sulfate(Cosenzaetal.,2014)indicatingthe presenceofverysmallamountof-carrageenan(7.3%oftotal anhy-drogalactose),incorroborationwithFT-IRresults.Table1shows theattributionofH.musciforms1HNMRsignalsbasedonliterature data(Cosenzaetal.,2014;Kravchenkoetal.,2014;VandeVelde etal.,2002;Villanueva&Montano,2003).TheG4S/DAratiowas calculatedfromH-1protonfromDA(ı5.1)andH-4signalofG4S (ı4.83)duetooverlappingofH-1ofG4SandH-4ofDAsignals. TheratioofG4S/DAwas1.15,veryclosetotheideal␬-carrageenan

G4S/DAratio.

13_{C NMR spectrum of purified H.musciforms polysaccharide}

(Fig.2B)showsawellresolvedspectrum,indicatingagood

chemi-Fig.2.1H(A)and13_{CNMR(B)spectraofHypneamusciformispolysaccharideinD2O} 2.5%(w/v)recordedat353K.

Table1

1_Hand13_{CNMRchemicalshiftsforHypneamusciformispolysaccharide.}

Unita 1_{Hchemicalshift(ppm)}

H-1 H-2 H-3 H-4 H-5 H-6

G4S 4.64 3.60 3.98 4.83 3.80 3.80 DA 5.10 4.13 4.51 4.62 4.62b _4.04b

Unita 13_{Cchemicalshift(ppm)}

C-1 C-2 C-3 C-4 C-5 C-6

G4S 102.60 69.72 78.88 74.23 74.91 61.42 DA 95.28 70.02 79.34 78.43 76.91 70.02

a_{DiadnomenclatureproposedbyKnutsenetal.(1994).} b_Notdetected.

calhomogeneity.TheH.musciformsspectrumshowstwoanomeric carbonsintherangeofı95toı105,characteristicofC-1ofG-4S atı102.6and atı95.28due toC-1ofDA.Thisdatacorroborate withourpreviouslydescribedresultsindicatingthatthesulfated polysaccharideextracted from H.musciforms collectedat Ceará state(Brazil)isessentiallya␬-carrageenan.Theassignmentsof13_C

NMRspectrumofH.musciformsaredepictedinTable1,basedon literaturereports (Cosenzaetal.,2014;Kravchenkoetal.,2014; VandeVeldeetal.,2002;Villanueva&Montano,2003).

Thesulfatedpolysaccharidedescribedhereindiffersfromthe onecharacterizedbyAlvesetal.(2012)forthesamealgalspecies collectedindifferentgeographicareasinBrazil,whichpresented a 13_{C NMR spectrum with four anomeric carbons, depicting a}

largefractionof-carrageenanintheirsample.Dependingonthe

fractionationmethodologyused,theauthorsalsofounda larger complexity of polysaccharides, with d- and l-galactans or

T.V.Britoetal./CarbohydratePolymers151(2016)957–964

Fig.3. Sulfatedpolysaccharide(PLS)extractedfromHypneamusciformisreducesmacroscopicscores(A)andwetweight(B)ofthecolonsoftheratswithcolitisinducedby TNBS.

Fig.4. Sulfatedpolysaccharide(PLS)extractedfromHypneamusciformisreduceshistologicaldamageoftissueofthecolonsoftheratswithcolitisinducedbyTNBS.Saline (A),TNBS(B),TNBS+PLS(C)andTNBS+DEXA(D).

thechemicalstructureofcarrageenans,suchasalgalsourceand extractionprocedures.Indeed,unlikeproteinsandnucleicacids, carbohydrates are not synthesized through a template-driven mechanism,butratheradirectproductfromthelevelsof expres-sion,activityandsubstrate-specificityofrelatedanabolicenzymes togetherwiththeproperavailabilityofsubstratesforbiosynthesis (Pomin,2016;Varki&Sharon,2008).Thus,asthestructural fea-turesofsulftatedpolysaccharidesvaryasamultifactorialprocess, theirchemicalanalysisbecomesindispensablepriortoa pharma-cologicalormedicinalapproach.

3.2. PolysaccharidefromH.musciformispreventsTNBS-induced intestinaldamage

The search for compounds extracted from natural products withpharmacologicalpropertieshassignificantlycontributedto thediscovery of molecules withimportant biomedical applica-tions(Corrêa, Melo, &Costa, 2008; Sousa et al., 2008).In this context, marinealgae are valuable sources of diversebioactive molecules such as polyphenols, enzymes, and diverse polysac-charides(Karnjanapratum&You,2010).Inthiscontext,thePLS fromHypnea musciformisis an importantpharmacological tool, duetoitsspecificstructurewithhighcontentofradicalsulfates, topresentantioxidantactivity(Alvesetal.,2012), gastroprotec-tiveeffect(Damascenoetal.,2013)andanti-inflammatoryactions (Brito etal.,2013;Sousaet al.,2016).Therefore, inthepresent study,weinvestigatedtheprotectiveeffectofthepolysaccharide (PLS)extractedfromthemarineredalgaeH.musciformisagainst TNBS-inducedintestinaldamageinrats.

TNBS-inducedcolitisisa modelofchronicinflammationand ulcerationintheratcolonthat manifestsmanyof the histolog-icaland clinicalfeaturesofcolonic inflammatorybowel disease inhuman(Morrisetal.,1989),suchasexcessiveoxidativestress, enhancedvascularpermeability,prolongedneutrophilinfiltration andincreasedproductionofinflammatorymediators,accompanied ofasignificantincreaseofthecolonweight.

Inthepresentstudyweshowedclearlythattheintracolonic administrationofTNBS presentedanintensemacroscopic dam-age(18.00±1.7scoresoflesion)accompaniedbymucosalnecrosis

extending along the colon, bowel wall thickening and hyper-emia, showing scores of lesion very high (18.00±1.7 scores),

andincreasedwetweightofthecolon(1.03±0.03g). However,

pretreatment with PLS at the doses of (10, 30 and 60mg/kg p.o.)significantly(p<0.05)reducedinadose-dependentmanner themacroscopicboweldamage(10mg/kg:9.25±2.13;30mg/kg:

5.75±1.84;60mg/kg:5.00±1.87)(Fig.3A)anddecreasedthewet

weightofcolon(60mg/kg:0.45±0.09g)(Fig.3B)afterintracolonic

administrationofTNBS.Dexamethasone(1mg/kg,s.c.),areference drugfor treatmentofthe IBD,alsoreduced macroscopiclesion (5.50±1.65scores)andweightofthecolon(0.61±0.10g).

T.V.Britoetal./CarbohydratePolymers151(2016)957–964

Fig.5. Sulfatedpolysaccharide(PLS)extractedfromHypneamusciformisreduces myeloperoxidaseactivity(A),IL-1␤(B)andTNF-␣l(C)levelsofthecolonsofthe ratswithcolitisinducedbyTNBS.

3.3. PolysaccharidefromH.musciformisreducesneutrophil infiltrationandcytokinelevelsintheTNBS-inducedcolitis

Toreinforcethis data wemeasuredmyeloperoxidase (MPO) activity,anenzymeproducedmainlybypolymorphnuclear(PMN) leukocytes.MPOactivityisassociatedwithneutrophilinfiltration intissuesandhasbeenwidelyusedtodetectandmonitor intesti-nalinflammation(Halliwell,1996;Yamada,Marshall,Specian,& Grisham,1992).

OurresultsshowthatTNBSadministrationpromotedamarked increaseincolonicMPOlevels(10.03±2.55UMPO/mgoftissue),

ascomparedtosalinegroup(1.22±0.04UMPO/mgoftissue).On

theotherhand,pretreatmentwithPLS(60mg/kg,p.o.)significantly (p<0.05)preventedtheincreaseofcolonicMPOlevels(0.83±0.05

UMPO/mgoftissue)associatedwithTNBSadministration(Fig.5A). Thus,accordingtothesefindings,wecansuggestthatPLSproduced protectiveeffectintheTNBS-inducedcolitismodelbymodulation oftheneutrophilmigrationintotheinflamedcolon.

Theneutrophilmigration intothecolon duringthecolitis is accompanied by production of several inflammatory products, suchasfreeradicalsgenerationandreleasingofpro-inflammatory cytokines(Katz, Itoh, & Fiocchi, 1999). Cytokines as IL-1␤ and

Table2

EffectofPLSonGSH,MDA,andNO2/NO3levelsinTNBS-inducedcolitis.

Experimentalgroup GSH(␮g/gtissue) MDA(nmol/gtissue) NO2/NO3(␮M)

Saline 240.2±18.5 65.0±17.9 0.08±0.01

TNBS 91.5±14.9# 162.5±25.6# 0.22±0.03# PLS+TNBS 225.4±32.4* 76.2±16.3* 0.10±0.01*

Resultsareexpressedasthemeans±S.E.M.of5–6ratspergroup. #_{P<0.05,whencomparedwithsalinegroup.}

*_{P<0.05,whencomparedwithTNBSgroup.}

TNF-␣areincreasedininflamedtissueandresponsiblefor recruit-mentofneutrophilsandmononuclearcellsmediatedinpartbythe up-regulationofadhesionmoleculestowardsvascularendothelial cells.

Fig. 5 shown that TNBS instillation significantly (p<0.05) increased the IL-1␤ (Fig. 5B; 478.7±139.8pg/mL) and TNF-␣ (Fig. 5C; 99.29±18.42pg/mL) levels, when compared

to saline group (IL-1␤: 125.8±12.44pg/mL and TNF-␣: 41.23±8.595pg/mL, respectively). In addition, the treatment

with PLS (60mg/kg, p.o.) significantly reduced the concentra-tionsof thesecytokines (IL-1␤: 108.8±31.03pg/mL and TNF-␣

27.20±2.779pg/mL, respectively) in the inflamed intestinal

mucosa.

3.4. PolysaccharidefromH.musciformisreducesoxidativestress intheTNBS-inducedcolitis

Duringthedevelopmentofcolitis,freeradicalgenerationhas beenproposedasplayinganimportantearlyroleininstallationof thispathogenesis(Pavlicketal.,2002).Thisphenomenonhasan importantrelationshiptotheinitialneutrophilinfiltrationinthe inflamedcolonicmucosa.Therecruitmentandactivationofthese cellsbyreleasingofcytokinesresultinincreaseofthefree radi-calproductionthatoverwhelmsthetissue’santioxidantprotective mechanisms, resulting in a situation of oxidative stress, which definitivelyperpetuatescolonicinflammation(Grisham,1994).

To clarify this point in our experimental model, we inves-tigated two oxidative stress markers, malondialdehyde (MDA) concentrationandglutathione(GSH)levels.MDAisaproductof lipoperoxidativeprocessesthattakeplaceasaconsequenceofthe colonicoxidativeinsult(Loguercioetal.,1996).Studieshaveshown thatcolitiscanincreaseMDAlevelsinratsand,conversely,that variousagentsusedinthetreatmentofthediseasecandecrease thisparameter(Cetinkayaetal.,2006;Girisetal.,2007).Onthe otherhand,GSH;an endogenousantioxidant, protectsthecells againstoxidativedamage,keepingthesulfhydrylgroups(-SH)of proteinsreduced and preventing them fromreacting withfree radicals(Amirshahrokhi,Bohlooli,&Chinifroush,2011). Concen-trationsofendogenous antioxidantssuchas GSHare decreased significantlyinpatientswithinflammatoryboweldiseaseandin experimentalmodelsofcolitis(Spitz,Azzan,&Gius,2004;Tahan etal.,2011).

In this point, our results showed that TNBS promoted a significant (p<0.05) consumption of GSH levels (Table 2: 91.56±14.96mg/g of tissue) and produced an increase in

the MDA concentration (Table 2: 162.5±25.63nmol/g of

tis-sue) in the colonic mucosa, when compared to saline group (240.2±18.55mg/g oftissue and 65.07±17.93nmol/gof tissue,

respectively).However,pretreatmentwithPLSrestoredtheGSH levels(225.4±32.47mg/goftissue)anddecreasedMDA

concentra-tions(76.27±16.30nmol/goftissue)ininflamedtissues(Table2).

T.V.Britoetal./CarbohydratePolymers151(2016)957–964

wecaninferthatPLSdecreasedthemucosadamageactinginthe productionandactionofendogenousantioxidants.

Inaddition,inthepresent studywe showedthat damagein intestinalmucosawasaccompaniedwithhighlevelsofradicals derivedfromnitricoxide(NO).NO₃−_/NO2−_{isafreeradicalfrom}

nitricoxidewithmoderatereactivitybutwithitsoverproductionby upregulationofinduciblenitricoxidesynthase(iNOS)caninhibit importantenzymesinthemitochondrialresponsibleforcell respi-ration,electrontransportandcausedamageintissuedirectlybythe peroxynitriteformationafterreactionwithsuperoxide(Kaulersch, Fiocchi,&Waldmann,1988;Kolios,Valatas,&Ward,2004).

The Table 2 shows that TNBS group increased level of NO3−/NO2− (0.22±0.03␮M) in colon tissue, when compared to saline group (0.08±0.01␮M). However, treatment with PLS significantly (p<0.05) reduces NO₃−_/NO2− _{concentration}

(0.10±0.00␮M) in damage intestinal tissue caused by TNBS administration.

4. Conclusions

Inconclusion,ourresultsrevealedthatPLSextractedfromthe marine algae H. musciformis is essentially composedof a high molecularmass␬-carrageenan.Inaddition,PLSreducedthecolonic

inflammatoryresponseinducedbyadministrationofTNBSthrough inhibitionofcellmigrationintotheinflamedtissueandreduction ofoxidativeprocess.Apossiblemechanismforthisprotectiveeffect mayinvolvethedownregulationoftheinflammatoryresponseby inhibitingthesynthesisandreleasingofproductsofinflammation, suchaspro-inflammatorycytokines.

Conflictsofinterest

Theauthorsdeclarenoconflictofinterest.

Acknowledgments

Theauthorsgratefullyacknowledgethefinancialsupportfrom NationalCounselofTechnologicalandScientific Development– CNPq(Brazil)andResearchfoundationfromthestateofPiauí– FAPEPI.TheauthorsalsowishtoacknowledgeCENAUREMNfor recordingtheNMRspectra.

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