ContentslistsavailableatScienceDirect
Carbohydrate
Polymers
jo u r n al h om ep age :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
fraction
from
marine
algae
Solieria
filiformis
:
Structural
characterization,
gastroprotective
and
antioxidant
effects
Willer
M.
Sousa
a,
Renan
O.
Silva
b,
Francisco
F.
Bezerra
a,
Rudy
D.
Bingana
b,
Francisco
Clark
N.
Barros
a,c,
Luís
E.C.
Costa
a,
Venicios
G.
Sombra
d,
Pedro
M.G.
Soares
b,e,
Judith
P.A.
Feitosa
d,
Regina
C.M.
de
Paula
d,
Marcellus
H.L.P.
Souza
b,
André
Luiz
R.
Barbosa
f,∗,
Ana
Lúcia
P.
Freitas
aaLaboratoryofProteinsandCarbohydratesofMarineAlgae,DepartmentofBiochemistryandMolecularBiology,FederalUniversityofceará,Fortaleza,
60455-760,CE,Brazil
bLEFFAG–LaboratoryofPhysiopharmacologyStudyofGastrointestinalTract,DepartmentofPhysiologyandPharmacology,FederalUniversityofCeará,
Fortaleza,CE,Brazil
cFederalInstituteofEducation,ScienceandTechnologyofCeará,JuazeirodoNorte,CE,Brazil
dLaboratoryofPolymers,DepartmentofOrganicandInorganicChemistry,FederalUniversityofCeará,Fortaleza,60455-760,CE,Brazil
eDepartmentofMorphology,MedicalSchool,FederalUniversityofCeara,RuaDelmirodeFariass/n,RodolfoTeofilo,Fortaleza,CECEP60416-030,Brazil fLAFFEX–LaboratoryofExperimentalPhysiopharmacology,BiotechnologyandBiodiversityCenterResearch(BIOTEC),FederalUniversityofPiauí,
Parnaíba,64202-020,PI,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received9March2016
Receivedinrevisedform21June2016 Accepted28June2016
Availableonline29June2016
Keywords: Solieriafiliformis Polysaccharides Ethanol Antioxidant
a
b
s
t
r
a
c
t
Asulfatedpolysaccharide(SFP)fractionfromthemarinealgaSolieriafiliformiswasextractedand submit-tedtomicroanalysis,molarmassestimationandspectroscopicanalysis.Weevaluateditsgastroprotective potentialinvivoinanethanol-inducedgastricdamagemodelanditsinvitroantioxidantproperties (DPPH,chelatingferrousabilityandtotalantioxidantcapacity).Itschemicalcompositionrevealedto beessentiallyaniota-carrageenanwithamolarmassof210.9kDaandhighdegreeofsubstitution forsulfategroups(1.08).Invivo,SFPsignificantly(P<0.05)reduced,inadosedependentmanner,the ethanol-inducedgastricdamage.SFPpreventsglutathioneconsumeandincreaseofmalondialdehyde andhemoglobinlevels.SFPpresentedanIC50of1.77mg/mLinscavengingDPPH.Thechelatingferrous abilitywas38.98%,andthetotalantioxidantcapacitywas2.01mg/mL.Thus,SFPpreventsthe devel-opmentofethanol-inducedgastricdamagebyreducingoxidativestressinvivoandpossessesrelevant antioxidantactivityinvitro.
©2016ElsevierLtd.Allrightsreserved.
1. Introduction
Naturalmarineproductshavebeenthefocusofmanystudies todiscovernewcompoundswithpharmacologicaleffects(Cabrita, Vale,&Rauter,2010;Iannitti&Palmieri,2010).Marinealgaehave receivedspecial attentionbecausetheyhave beenshowntobe valuablesourcesofstructurallydiversebioactivesubstances,such asenzymes,andsulfatedpolysaccharides(Kusaykinetal.,2008; Wijesekara,Pangestuti,&Kim,2010).
Sulfatedpolysaccharidesarecomplexmacromoleculesthatcan interactwithawidevarietyofmatrixandcellularproteinsdueto
∗ Correspondingauthorat:BIOTEC/LAFFEX/UFPI,Av.SãoSebastião,n2819,CEP 64202-020,Parnaíba,PI,Brazil.
E-mailaddress:[email protected](A.L.R.Barbosa).
theirchemicalstructure,whichisrichinpolyanions.Inred sea-weed,thesecompoundsexistmainlyasgalactans(Fonsecaetal., 2008;Shanmugam&Mody,2000)suchascarrageenansandagars (Stortz&Cerezo,2000).Thesemacromoleculeshavedemonstrated adiverserangeofbiologicalfunctions(Jiao,Yu,Zhang,&Ewart, 2011).Theirbiological activities,suchasantioxidant (Prajapati, Maheriya,Jani,&Solanki,2014)andprotectiveeffectsinethanol andNSAIDgastricdamagemodels(Damascenoetal.,2013;Silva etal., 2012), representa newapproach forinhibiting theharm causedbyexcessiveproductionoffreeradicals.
Solieriafiliformis(Kützing)P.W.Gabrielson(Gigartinales, Solier-aceae) is an abundant species of alga which synthesizes high levels of sulfated polysaccharides, composed mainly of car-rageenans. S. filiformis carrageenans are composed of a linear backboneof 3-linked-d-galactopyranose-4-sulfate(G4S-units)
and 4-linked3,6-anhydro-␣-d-galactopyranose-2-sulfate
units)or 3,6-anhydro-␣-d-galactopyranose(DA-units) (Murano,
Toffanin,Cecere,Rizzo,&Knutsen,1997).
Recent studies have shown that sulfated polysaccharides extractedfromthemarinealgaeSolieriafiliformisdemonstrated considerableanti-leishmania(Piresetal.,2013),anti-inflammatory andanti-nociceptiveactivities(Araújoetal.,2011)andvascular alterations(Assreuyetal.,2010).However,studiescorrelatingthe antioxidantactivityandgastricprotectionofthispolysaccharide arescarceintheliterature.
Ethanol, when ingested in excess, promotes gastric injury characterized by edema of mucosa, hemorrhage, and cellular exfoliation—mechanismsthatinvolveoxygenradicals,withlipid peroxidationandthedepletionofanti-oxidantdefense(Ko,Cho,& Lam,2004).Oxidativestressincludestheproductionoffree radi-cals,whichpromotetheoxidationofmembranelipidsleadingto animbalancebetweenaggressive(suchashydrochloricacidand pepsin)andantioxidantsystems(likesulfhydrylcompounds)that cancausecelland/ortissuedamagedue totheirhighreactivity (Schneider&Oliveira,2004).Thus,disordersofthegastrointestinal tractwithanintenseproductionofreactiveoxygenspecies(ROS), culminatingingastricdysfunction,havebecomeaglobalproblem. Manyresearchgroupshavebeenattemptingtoelucidatethe rele-vantpathologicalmechanisms,withtheintentiontofindeffective therapeuticapproaches.
Thus,thepresentstudyaimstocharacterizeasulfated polysac-charide from the marine red alga S. filiformis, investigate its gastroprotectivepotentialinethanol-inducedgastric damagein mice,anddepictitsantioxidantactivityinvitro.
2. Experimental
2.1. Marinealga
SpecimensoftheredmarinealgaeSolieriafiliformiswere col-lected in June 2014 in the Atlantic coast, northeast of Brazil (FlexeirasBeach,Trairi–Ceará).Aftercollection,thesampleswere cleanedofepiphytes,washed,andstoredat−20◦C.Avoucher
spec-imen(no.56148)wasdepositedintheHerbariumPriscoBezerra, FederalUniversityofCeará,Brazil.
2.2. ExtractionofSFP
Theextractionofthesulfatedpolysaccharidefractionfromthe marinealgaeS.filiformis(referredtoas“SFP”inthismanuscript) wasperformedasdescribedpreviously(Farias,Valente,Pereira,& Mourão),withmodifications.Thedriedtissue(5g)wasmacerated inliquidnitrogenandsuspendedinasodiumacetatebuffer(0.1M, pH5.0)containing5mM EDTA,5mMcysteine and papain,and incubatedfor6hat60◦C.Then,thematerialwasfilteredand
cen-trifugedat7965gfor20minat25◦C,andprecipitatedbyaddingof
10%cetylpyridiniumchloride(CPC).Theprecipitatewasdissolved inNaCl:ethanol(2M,100:15,v/v)andagainprecipitatedby addi-tionofethanolfor24hat4◦C.Finally,SFPwasdialyzedextensively
usingdistilledwater,andlyophilized.
2.3. BiochemicalcharacterizationofSFP
2.3.1. Chemicalcomposition
Carbonandsulfatecontentweredeterminedbymicroanalysis usingaPerkinElmer2400SeriesIICHNSanalyzer(PerkinElmer, Waltham,MA,USA).Thetotalsugarcontentwasdeterminedbya sulfuricacid-UVtechnique(Albalasmeh,Berhe,&Ghezzehei,2013), usingd-galactoseasthestandard.Theproteincontentwas mea-suredusingtheBradfordmethod(Bradford,1976), withbovine serumalbumin(BSA)asthestandard.
2.3.2. High-performancesize-exclusionchromatography(HPSEC)
Thepeakmolarmass(Mpk)of0.5%SFPfractionin0.1MNaNO3
wasestimatedbyHPSECusingaShimadzuLC-20ADpump (Shi-madzu Co., Kyoto,Japan) at 25◦C. In this protocol we usedan
ultrahydrogellinear column(7.8×300mm),witha flowrateof 0.5mL/min,a refractiveindexdetector,andanultraviolet spec-trophotometerat254nm.Theelutionvolumewascorrectedfor theinternalmarkerofethyleneglycolto11.25mL.Acalibration curveofdifferentmolecularweights(range:103–106g/mol)was
obtainedusingPullalan(ShodexDenko). Theequationobtained fromthiscalibrationplotwas:
LogMw=14.6827−1.06967Ve (1)
whereVeistheelutionvolumeinmL.Thelinearcorrelation coef-ficientwas0.99.
2.3.3. Fouriertransforminfrared(FT-IR)spectroscopy
FT-IRspectraofKBrpelletsoftheSFPfractionwererecordedin aShimadzuIRspectrophotomer(model8300)scanningbetween 400and4000cm−1.
2.3.4. Nuclearmagneticresonance(NMR)spectroscopy
1H and 13C NMR spectra of 2.5% (w/v) solutions in D 2O
were obtained using a Bruker Avance DRX 500 spectrometer. The analysis was performed at 60◦C for 12h using sodium
2,2-dimethylsilapentane-5-sulphonate(DSS)asinternalstandard (0.00ppmfor1H)andacetone(31.07ppmfor13C).A
Heteronu-clearSingleQuantumCoherence(HSQC)spectroscopyexperiment wasalsocarriedout.
2.4. Evaluationofgastroprotectiveeffects
2.4.1. Animals
MiceSwiss(25–30g)werehousedinatemperature-controlled roomandreceivedfoodandwateradlibitum.However,theanimals fastedfor18hbeforeexperiments.Allanimaltreatmentsand sur-gicalprocedureswereperformedinaccordancewiththeGuidefor CareandUseofLaboratoryAnimals(NationalInstituteofHealth, Bethesda, MD, USA)and approved bythe Ethics Committeeon AnimalUse,intheCollegeIntegralDifferential(FACID/Teresina-PI, protocoln◦017/13).
2.4.2. Ethanol-inducedgastricdamage
Mice were pretreated with SFP (0.3, 1, 3, and 10mg/kg, p.o.).After30min,gastric damagewasinducedby 50%ethanol (0.5mL/25g) administration by gavage. The controls groups receivedonlysterilesalineor50%ethanol.Onehourlater,the ani-malsweresacrificedandtheirstomachsremoved,opened,washed with sterile saline, and photographed. Gastric damage (mm2)
wasmeasuredusingacomputerplanimetryprogram(ImageJ®) (Medeirosetal.,2009).Onesamplewasremovedandfixedin10% formalinforhistologicalanalysis.Otherssampleswerecollected for determinationof glutathione(GSH) levels,malondialdehyde (MDA)concentrationandhemoglobin(Hb)levels.
2.4.3. Histopathologicalanalysis
Stomachsampleswerefixedin10%formalinsolution,sectioned, andembeddedinparaffin.Four-micrometer-thicksectionswere deparaffinized,stained with hematoxylin and eosin (H&E), and thenexaminedundera microscopebyanexperienced patholo-gist(Soares,PMG).Thespecimenswereassessedaccordingtothe criteriaaspreviouslydescribed(Laine&Weinstein,1988).
2.4.4. GSHlevels
distilledwaterand80Loftrichloroaceticacid(50%,w/v)and cen-trifugedat3000rpmfor15min.Next,400Lofsupernatantwas mixedwith800LofTrisbuffer(0.4M,pH8.9)and20Lof0.01M DTNBwasadded.Subsequently,thesampleswereshakenfor3min andabsorbancemeasuredat412nmusingaspectrophotometer. TheresultsareexpressedasgofGSH/gtissue(Sedlak&Lindsay, 1968).
2.4.5. MDAconcentration
Stomachsampleswerehomogenizedin1.15%KCl(1mL/100mg tissue).Briefly,250Lofhomogenatewasaddedto1.5mLof1% H3PO4and0.5mLof0.6%tert-butylalcohol.Then,thismixturewas
stirredandheatedinaboilingwaterbathfor45min.The prepa-rationwasthencooledimmediatelyinanicewaterbath,followed bytheadditionof2mLofn-butanol.Thismixturewasstirredand thebutanolremovedviacentrifugationat1200rpmfor10min,and absorbancemeasuredat520and535nm.Theresultsareexpressed asnmolofMDA/gtissue(Mihara&Uchiyama,1978).
2.4.6. Hemoglobinconcentration
Thehemoglobin(Hb)concentrationinthegastricmucosawas determinedby a colorimetric method (LABTEST,Diagnostic SA, MinasGerais,Brazil).Stomachsampleswerehomogenizedincolor reagent(1mL/100mgtissue),centrifugedat10,000rpmfor10min. Then,thesupernatantswasremoved,filteredusinga0.22mmfilter andcentrifugedat10,000rpmfor10min.Absorbancewas mea-suredat540nm,andtheHbconcentrationwasexpressedasmgof Hb/gtissue(Medeirosetal.,2008).
2.5. Determinationofantioxidantactivityinvitro
2.5.1. DPPHscavengingactivity
The scavenging capacity of SFP on 1,1-diphenyl-2-picrylhydrazyl (DPPH) was performed as described previously (Blois,1958),withmodifications.Initially,differentconcentrations ofSFP(0.025at4mg/mL)wereaddedtoa3mLmethanolsolution ofDPPH(75M).After30min,theabsorbancewasmeasuredat 517nm.Allreactionswereperformedintriplicatesandbutylated hydroxytoluene(BHT)wasusedasacontrol.TheDPPHscavenging activitywascalculatedusingthefollowingequation:scavenging activity (%)=[A0−(A−Ab)/A0]×100, where A0=DPPH without
sample;A=sample+DPPH;andAb=samplewithoutDPPH.
2.5.2. Chelatingferrousability
Thechelatingferrousability wasanalyzed asdescribed pre-viously (Chew, Lim, Omar, & Khoo, 2008), with modifications. Initially, different concentrations of SFP (0.025 at 4mg/mL) weremixed with 1mL of ferrous sulfate (FeSO4; 0.1mM), and
1mL of ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′
-disulfonicacid;0.25mM).Then,thetubeswereagitatedfor1min andincubatedfor10minatroomtemperature,andabsorbance measuredat562nm.Allreactionswereperformedintriplicates andEDTAwasusedasacontrol.Resultsareexpressedasa percent-ageofchelatingactivityaccordingasfollowingformula:Chelating activity(%)=[A0−(A−Ab)/A0]×100,whereA0=FeSO4+Ferrozine
withoutsample; A=sample+FeSO4+Ferrozine; and Ab=sample
withoutFeSO4+Ferrozine.
2.5.3. Totalantioxidantcapacity
Total antioxidant capacity was performed by formation of thecomplexphosphomolybdate,asdescribedpreviously(Prieto, Pineda,&Aguilar,1999).Briefly,300LofSFPfractions(from0.025 to4mg/mL)wasaddedtoa3mLsolutioncontainingammonium molybdatesolution(4mM),sulfuricacid(0.6M)andsodium phos-phate(28mM),andincubatedat95◦Cfor90min.Aftercooling,the
absorbancewasmeasuredat695nm.Allreactionswereperformed
intriplicateandBHTwasusedasacontrol.Asampleof200g/mL ofascorbicacidwasusedasreferencesubstanceandconsidered as100%antioxidantactivity.Resultswereexpressedaspercentage ofchelatingactivitybythefollowingformula:Totalantioxidant capacity(%)=[(Asample−Ablank)/(Aascorbicac−Ablank)]×100
2.6. Statisticalanalysis
Data are described as either means±SEM or median when appropriate. An analysis of variance (ANOVA) followed by a Student-Newman-Keuls test was used to compare means, and Kruskal-WallisnonparametrictestfollowedbyDunn’stestto com-paremedians.P<0.05wasdefinedasstatisticallysignificant.
3. Resultsanddiscussion
3.1. ExtractionandcharacterizationoftheSFPfraction
ThepolysaccharideextractedfromSolieriafiliformis,by enzy-matic digestion with papain, yielded 21.3%. SFP fraction had 66.0%totalsugar,andonlytracesofproteinweredetected.The weightpercentage ofcarbonandsulfur,determinedby elemen-talmicroanalysis,were27.22%and6.50%,respectively.Usingthe methodology proposed by Melo, Feitosa, Freire, and de Paula (2002),thedegreeofsubstitution(DS)ofsulfategroupsper disac-charideunitesofalgaepolysaccharidescanbeobtainedusingthe followingequation:
DS=4.5
%S %C(2)
TheDSofSFPfractionwas1.08.
The HPSEC chromatogram showed a singlepeak at 8.75mL (Fig. 1A).Howeverthepeak isbroad indicating a high polydis-persivemolar mass. Thisbehavior is frequently foundin algae polysaccharide,suchastheisolatedonesfromthemarinealgae
Gracilariacaudata(Barroset al.,2013)andMastocarpus stellatus
(Gómez-Ordó ˜nez,Jiménez-Escrig,&Rupérez,2012).HPSECof -and -carrageenanalsoshow largermolarmassdistributionas observedintheSFPfraction(Villanueva,Mendonza,Rodrigueza, Romero,&Montano,2004).Usingthecalibrationcurve(Eq.(1)), thepeakmolarmasscanbeestimatedat210.4kDa.Highervalues ofmolecularweightarecommoninsulfatedpolysaccharidesfrom marinealgae,duetothegroupingofpolysaccharidechains(Pomin, 2010).
TheFT-IRspectrumoftheSFPfraction((Fig.1B))showedan intensebandat1258cm−1,characteristicofasymmetric
stretch-ingoftheS Obandduetosulfate groups(Araújoet al.,2011; Muranoetal.,1997;Prado-Fernández,Rodriguez-Vázquez,Tojo, &Andrade,2003;Rochas,Lahaye,&Yaphe,1986).Thespectrum alsodisplayedbandsat935,902,850and805cm−1,indicatingthe
presence of 3,6-anydrogalactose, -d-galactose-6-sulfate (G6S),
-d-galactose-4-sulfate (G4S)and 3,6-anydrogalactose-2-sulfate
(DA2S), respectively (Gómez-Ordó ˜nez & Rupérez, 2011; Araújo etal.,2011).ThesebandsaresimilartothoseobservedbyMurano etal.(1997)fortheS.filiformispolysaccharideobtainedfrommar Piccolo,withoutproteasetreatment,andalsofortheS.filiformis
polysaccharideextractedfromtheAtlanticcoastofBrazil(Araújo etal.,2011).TheFT-IRspectrumofSFPfractionischaracteristic ofcarrageenan-typepolysaccharides(Araújoetal.,2011;Pereira, Gheda,&Ribeiro-Claro,2013).
The13C NMRspectroscopy of thesulfatedpolysaccharideof
S.filiformis,extractedunderproteolyticaction(Fig.2A)show sig-nalcharacteristicsoftheiota-carrageenandisacchariderepeating units(-d-galactose-4-sulfatelinkedto3,6-anydro-␣-d
(C-Fig.1.GPCcurveandFT-IRspectraofSolieriafiliformissulfatedpolysaccharide(SFP).(A)GPCcurve;(B)FT-IRspectrainKBrpellets.
1),ı69.4(C-2),ı76.8(C-3),ı72.2(C-4),ı74.8(C-5)andı61.4(C-6). The3,6-anydro-␣-d-galactose-2-sulfate(DA2S)showssignalsatı
92.2(C-1),ı75.2(C-2),ı77.8(C-3),ı78.2(C-4),ı77.1(C-5)andı
69.9(C-6)(Muranoetal.,1997;Batistaetal.,2014;VanDeVelde, Knutsen,Usov,Rollema,&Cerezo,2002).Differingfrompreviously publishedmaterialbyMuranoetal.(1997),SFPshowsthepresence ofO-methylgroupsatı59.00,probablyduetothe6-O-methyl-
-d-galactose-4-sulfate.Thismethylatedunitwasobservedinother
Solieriaspecie,suchasSolieriachordalis(Bondu,Deslandes,Fabre, Berthou,&Guangli,2010).TheDEPT135experiment(Fig.2B), con-firmstheassignmentofı69.9andı61.8asprimarycarbons(C-6) ofDA2SandG4Srespectively,asthesesignalsappearsinopposite positionstoothercarbonsintheDEPT135spectrum.
2D1H13CHSQCspectrum(Fig.2C)oftheS.filiformis
polysac-charidefractionshowscorrelationsofanomericcarbonswiththeir respectiveproton(H-1/C-1)atı4.62/102.2andı5.29/92.2, respec-tively,forG4SandDA2Sunits.TheabsenceofH-1chemicalshiftat
ı5.1indicatedtheabsencesof-carrageenaninSFPfraction.The crosspeakinHSQCatı3.41/58.9wasattributedtoanO-methyl H/Cfrom6-O-methylgalactose-4sulfate.Table1showsalltheH/C correlationsofH/CforeachunitoftheSFPfraction.
TheFT-IRandNMRresultsindicatethatthepolysaccharide frac-tionextractedfromS.filiformisisrichiniota-carrageenan.
3.2. SFPpreventethanol-inducedgastricdamage
Thepathogenesisofethanol-inducedgastricdamageisa mul-tifactorialprocess,whichdependsonuncontrolledaggressiveand
protectivefactors,whichoccurdirectlyorindirectlythrough inter-mediaries, suchasfree radicalsand lipoxygenase(Abdel-Salam, Czimmer,Debreceni,Szolcsányi,&Mózsik,2001).Duetoseveral factorsthatcancausegastricinjury,suchasethanol,thesearchfor bioactivecompoundswithgastroprotectiveactivity,andthe inter-estofthepharmaceuticalindustry,hasgrowninrecentyears(Yang etal.,2012).
Inthepresentstudy,weevaluatedthegastroprotectiveeffect of anSFP fractionextractedfrommarine S.filiformis,using the ethanol-inducedgastricdamagemodelinmice.Ourresultsshowed that treatment with 50% ethanol promoted the formation of extensivemacroscopiclesionsin themucosa(66.58±5.6mm2),
compared to the saline group. In addition, pretreatment with SFPsignificantly(P<0.05)reducedthemacroscopicgastriclesions induced,causedby50%ethanoladministration,atalldosestested (0.3,1,3,10mg/kg).Thedosetestedwiththehighesteffectonthe preventionofgastriclesionswas10mg/kg(5.9±1.8mm2),
reduc-ing91.1%gastriclesionsintheulceratedarea(Fig.3.).Therefore, thisdosewasselectedforthestudyasthepossiblemechanism involvedingastricprotection.Inaddition,50%ethanol adminis-trationinducedgastricmucosadamagewithalossofepithelial cells(blackarrow),edema(bluearrow),andintensehemorrhage (redarrow).However,inmicepretreatedwithSFP(10mg/kg)these alterationsweresignificantlyprevented(Table2andFig.4).
Severalstudies can befound in theliterature regarding the gastroprotectiveeffectofsulfatedpolysaccharidesfromseaweeds. Amongthese,thesulfatedpolysaccharidesfromHypneamuciformis
Fig.2.NMRspectraofSFPinD2O.(A)13CNMRspectrum;(B)DEPT-135NMRspectrum;(C)2D1H,13CHSQCNMRspectrum.
Table1
Chemicalshift(ppm)assignmentsoftheNMRspectrafromS.filiformispolysaccharide.
RepetingUnitsa Chemicalshift(ppm)
H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6/C-6
G4S 4.63/102.2 3.62/69.4 3.98/76.1 4.88/72.2 3.81/74.8 3.81/61.4
DA2S 5.29/92.2 4.66/75.2 4.83/77.8 4.68/78.2 4.65/77.1 4.25/69.94.10/69.9
aNomenclatureproposedbyKnutsen,Myslabodski,Larsen,andUsov(1994).
Fig.3.SFPpreventethanol-inducedgastricdamageinmice.Theresultsareexpressedasmean±SEM(6–7animalspergroup)#P<0.05vs.salinegroup;*P<0.05vsethanol group;ANOVAandNewman-Keulstest.
by ethanol. The authors suggest that the mechanism of action issecondarytotheantioxidantactivityandourresultsare con-sistentwiththis (Damasceno etal.,2013; Silvaet al.,2011).In addition,sulfatedpolysaccharidesextractedfromGracilariabirdiae
dam-Table2
PolysaccharidefromS.filiformisreducesethanol-inducedmicroscopicgastricdamage.
Experimentalgroup Hemorrhagicdamage(score,0–4) Edema(score,0–4) Epithelialcellloss(score,0–3) Inflammatorycells(score,0–3) Total(score,0–14)
Saline 1(0–1) 0(0–0) 1(1–2) 1(0–1) 3(1–4)
50%Ethanol 3(3–4)* 3(1–4)* 3(3–3)* 2(2–3)* 11(10–14)*
SFP+50%Ethanol 1(1–3)# 0(0–0)# 1(1–3)# 1.5(0–2) 4(2–6)#
Resultsareexpressedasthemeans±S.E.M.of6–7micepergroup. *P<0.05,whencomparedwithsalinegroup.
#P<0.05,whencomparedwith50%Ethanolgroup.
Fig.4. Photomicrographsofgastricmucosa(Magnification,100×).(A)Salinecontrol;(B)50%ethanol,showingdisruptionofthesuperficialregionofthegastricglandwith epithelialcelllossandintensehemorrhage;(C)SFP(10mg/kg)+50%ethanol,showingpreservationofthegastricmucosa.
age,the next step of our workwas to evaluatethe important parametersrelatedtotheredoxbalance,suchastheGSHlevels andMDAconcentrationingastricmucosa.
3.3. SFPreducesoxidativestressintheethanol-inducedgastric damage
Glutathioneisatripeptidepresentinhighconcentrationsinthe cells ofthegastric mucosa,actingasa blocker ofreactive oxy-genspecies(ROS)productionand asasubstrateforglutathione peroxidase, metabolizing hydrogen peroxide (H2O2) and other
hydroperoxidesinthecytosolandmitochondria(Fesharakietal., 2006;Mitobe,Hiraichi, Sasai,Shimada,&Terano,2000).In this study,50%ethanoladministrationsignificantly(P<0.05)reduced theGSHlevels(25.1±11.1mg/gtissue)inthegastricmucosa,as comparedtothesalinegroup(135.2±12.8mg/gtissue).However, pretreatmentwithSFP(10mg/kg)preventstheGSHconsumption promotedbyethanoladministration,andisthereforeeffectiveat maintaininghighGSHlevelsinthestomach(Fig.5A).Thiseffectcan beexplainedinpartbythehighsulfatecontent,sinceithasbeen shownthatthisaspectisofparamountimportancetoits antioxi-danteffectandthuspromotescytoprotectionbyinactivatingfree radicals(Qietal.,2005).
Subsequently,weevaluatedfreeradicalproductionbymeans of anindirectindicatorof lipidperoxidation, theMDA concen-tration(Gaweł,Wardas,Niedworok,&Wardas,2003).Thelipid peroxidationresults from ROS reactionsagainst the cell mem-brane,consequentlyyieldingproductsthatcauseoxidativegastric damage (Kwiecie ´n, Brzozowski, Konturek, & Konturek, 2002). MDA, a product of lipid peroxidation, has been considered as themain indicatorof lipoperoxidative processes (LPO) (Dursun et al., 2009). Therefore, MDA levels are used as an important markerofoxidative damage,andits quantificationcanbeused toshowpossibleantioxidativeactivity(Kanter,Demir,Karakaya, & Ozbek, 2005). Our resultsshow that mice treated with 50% ethanol(224.2±21.1nmol/gtissue)showedasignificantincrease (P<0.05)intheMDAconcentration,comparedtothesalinegroup (119.7±17.7nmol/gtissue),indicatingthatlipidperoxidationby ROS was produced by ethanol administration. However,
pre-treatment withSFP (10mg/kg)prevented theincrease in MDA concentration(103.9±10.2nmol/gtissue)inducedby50%ethanol administration(Fig.5B).
Ourresearchgroupshowedthatsulfatedpolysaccharide frac-tionextractedfrommarinealgaeGracilariacaudataandHypnea musciformispresentedgastroprotectiveeffectsthroughasecondary effect,decreasingtheproductionoffreeradicals,thusincreasing GSHlevelsandindicating possibleantioxidantactivityby these compounds(Damascenoetal.,2013;Silvaetal.,2011).Inaddition, SFPhaveantioxidantactivityandthismechanismplaysan impor-tantroleinthepreventionofoxidativestress(Costaetal.,2010; Tariqetal.,2015).
3.4. SFPreduceshemoglobin(Hb)levelsingastricmucosa
Ethanolisthemainfactorthatleadstointensedamagetothe gastricmucosaandinducesmultiplebleedingredbandsof vary-ingsizesintheglandularstomachaxis(Mincis,Chebli,Khouri,& Mincis,1995).Additionally,gastricstasis inmicrovascularblood flow canleadtoaltercations suchashemorrhagesand necrotic tissue lesions(Szabo, Trier,Brown,&Schnoor,1985).The mea-surementofhemoglobinconcentrationsinstomachmucosacan beused asan indirectmarkerof hemorrhagic lesionextension in ethanol-inducedgastric injury (Medeirosetal., 2008).In the presentstudy,administrationof50%ethanol(11:31±0:59mg/g tissue)showedasignificant(P<0.05)increaseinthehemoglobin concentration(Hb),comparedtothesalinegroup(7.85±0.18mg/g tissue),indicating thatbleedingoccurredinthegastricmucosa. However,pretreatmentwithSFP(10mg/kg)significantly(P<0.05) preventedtheincreasedHbconcentrations(9.25±0.17mg/g tis-sue) (Fig.5C).Thus, measurementsof Hbconcentrations in the stomachconfirmedthegastricprotection,againstethanol-induced hemorrhagicgastricdamage,thatarosefrompretreatmentwith SFP.
3.5. SFPpossessantioxidantactivityinvitro
Fig.5.SFPreducesoxidativestressandhemorrhageinthegastricmucosaofmicetreatedwith50%ethanol.(A)Glutathione(GSH)levels,(B)Malondialdehyde(MDA) concentration,and(C)Hemoglobin(Hb)levels.Resultsareexpressedasmean±SEM(6–7animalspergroup)#P<0.05vs.salinegroup;*P<0.05vsethanolgroup;ANOVA andNewman-Keulstest.
Fig.6.SFPpossessesantioxidantactivityinvitro.(A)Scavenging 1,1-diphenyl-2-picrylhydrazyl(DPPH)radicals,(B)chelatingferrousability,and(C)molybdateion reductionassay(C).Valuesaremeans±SD.#P<0.05vs.salinegroup;*P<0.05vs ethanolgroup;ANOVAandNewman-Keulstest.
fromplants(Martinezetal.,2012)andmarinealgae(Wang,Zhang, Zhang, Song,& Li,2010), analyzed by scavengingthe hydroxyl radical through hydrogen donation. Our results show that SFP promotedasignificantinhibitoryeffectagainsttheformationof hydroxylradicals(IC50=1.77mg/mL).Thescavengingactivityof
theSFPincreasedsignificantly(P<0.05),withmaximalefficacyat 4.0mg/mL (88.93%)(Fig. 6A).Similarresults(IC50=1.62mg/mL)
were foundwith another SFP extracted from Gracilaria birdiae
(Souzaetal.,2012).Inaddition,BHT,thecompoundusedasthe control,obtainedanIC50valueof0.47mg/mL.Thisconcentrationis
muchlowerthantheSFP.ThesyntheticcompoundBHT,usedoften inthepharmaceuticalindustry,haslimitationsandissuspectedto causedamagetolivercells,andisapotentialcarcinogen(Cheng etal.,2013; Panicker,George,&Krishna,2014).Thus,there isa searchfornewcompoundswithantioxidantpropertiesthathave lowtoxicity.LipidperoxidationandexcessiveproductionofROS, especiallyhydroxylradicals,playanimportantroleinthe patho-genesisofethanol-inducedgastriclesions(Panetal.,2008;Park &Oh,2011).Therefore,compoundsthatarecapableofcapturing oxygenfreeradicalsarepotentialanti-ulcerogenicagents(LaCasa, Villegas,DeLaLastra,Motilva,&Calero,2000).Thus,ourresults suggestthatthegastroprotectiveeffectofSFPisdue,atleastin part,toitsantioxidantproperties.
We tested thechelating ferrousability, viaan evaluation of the ability to capture metal ions (like ferrous) present in the medium.Thispreventsreactionswithlipids,proteins,and cellu-larcomponents(Smith,Halliwell, &Aruoma,1992; Wang,Mao, &Wei,2012).Ferrousisanimportantioninpre-oxidizinglipid peroxidation, which occurs according to the Fenton reaction (Fe2++H
2O2→Fe3++OH−+• OH), because of its high reactivity
(Chun-hui,Chang-hai,Zhi-liang,&Yi,2007).Inbiologicalsystems, metalionscanactcausingoxidativedamagebycatalyzing unfa-vorablereactionsto thebody, like theinactivation ofenzymes (Chewetal.,2008).OurresultsdemonstratedthatSFPpromoted thechelatingferrousabilityinaconcentration-dependentmanner withmaximumactivityat4mg/mL(38.98%chelation).EDTA,used ascontrol,obtainedanIC50valueof0.86mg/mL (Fig.6B).Thus,
ourresultssuggestthatSFPcanserveasasecondaryantioxidant, becauseofitsabilitytoreducetheredoxpotential,thusstabilizing theoxidizedformofferrousions.
Furthermore,weevaluatedthetotalantioxidantcapacityofSFP byassessingtheformationofthecomplexphosphomolybdate.The formationofthephosphomolybdenumcomplexisperformedby reducing molybdenum(Mo6+
→Mo5+)bya reducing agent,and
complexationofmolybdenumtophosphorusinanacidmedium (formingbluecolorphosphomolybdenum)(Prietoetal.,1999).Our resultsshowedthatSFPpresentedactivityintheformationof com-plexphosphomolybdates inaconcentration-dependent manner, withanIC50valueof2.01mg/mL.Themaximumconcentration
ana-lyzedwas4mg/mL,whichobtaineda62.4%antioxidantcapacity. BHT,usedasthecontrol,promoted100%antioxidanteffectsatdose of2mg/mL(Fig.6C).OurresultssuggestthatSFPcanserveasa reducingagent.Thus,theresultsofantioxidantactivityinvitroof SFPcorroboratewiththereductionofoxidativestress,presented inthegastricinjurymodelinducedbyethanol.
4. Conclusion
substitution for sulfate and was effective at gastric protection againstethanol-inducedinjuryandhasrelevantantioxidant activ-ity.Theexactmechanismofgastroprotectionremainsunknown; however,ourresultssuggestthattheincreaseofGSHlevels,and decreaseinMDAconcentration,ispertinenttotheprotectionof thegastricmucosa.Inaddition,thiseffectcanalsobecausedby asecondary antioxidant,withferrousionsandhydroxylradical chelatingactivity.Thus,theseproperties,combinedwiththe pro-ductivefavorablefeaturesofthemarinealgaeS.filiformis,makeita promisingsourceofpolysaccharidewithgastroprotectivepotential andapossibleapplicationasanewnaturaltoolagainstoxidative dysfunctioninthegastrointestinaltract.
Acknowledgements
The authors gratefully acknowledge the financial support fromNational Counsel of Technologicaland Scientific Develop-ment/CNPq(Brazil)andtechnical assistanceofMaria Silvandira FreireFranc¸a.TheauthorsalsowishtoacknowledgeCENAUREMN forrecordingtheNMRspectra.
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