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Carbohydrate Polymers
j ou rn a l h o m e pa 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
Protective effect of fucoidan against AAPH-induced oxidative stress in zebrafish model
Eun-A Kim
a, Seung-Hong Lee
b, Chang-ik Ko
c, Seon-Heui Cha
d, Min-Cheol Kang
a, Sung-Myung Kang
e, Seok-Chun Ko
f, Won-Woo Lee
a, Ju-Young Ko
a, Ji-Hyeok Lee
a, Nalae Kang
a, Jae-Young Oh
a, Ginnae Ahn
g, Young Heun Jee
h, You-Jin Jeon
a,∗aDepartmentofMarineLifeSciences,JejuNationalUniversity,Jeju690-756,RepublicofKorea
bDivisionofFoodBioscience,KonkukUniversity,Chungju,Chungbuk380-701,RepublicofKorea
cResearchInstituteofProcessingfromJejuFisherFood,ChoungRyongFisheriesCo.,Ltd.,Jeju697-943,RepublicofKorea
dDepatmentofPharmacology,SchoolofMedicine,AjouUniversitySuwon,RepublicofKorea
ePediatricOncologyExperimentalTherapeuticsInvestigatorsConsortium(POETIC)LaboratoryforPre-ClinicalandDrugDiscoveryStudies,Universityof Calgary,Calgary,AB,Canada
fDepartmentofFoodandNutrition,SungshinWomen’sUniversity,Seoul136-742,RepublicofKorea
gLaboratoryofComparativeAnimalMedicine,DivisionofAnimalLifeScience,InstituteofAgriculture,TokyoUniversityofAgricultureandTechnology, Tokyo183-8509,Japan
hDepartmentofVeterinaryMedicine,AppliedRadiologicalScienceResearchInstitute,JejuNationalUniversity,Jeju690-756,RepublicofKorea
a r t i c l e i n f o
Articlehistory:
Received14December2012
Receivedinrevisedform31October2013 Accepted20November2013
Available online 27 November 2013
Keywords:
Fucoidan Zebrafish AAPH Anti-oxidant
a b s t r a c t
Fucoidan,extractedfromEckloniacava,hasbeenextensivelystudiedbecauseofitswidebiologicalactiv- ities.However,antioxidativeactivitieshavenotbeenyetexamined.Thereforeweevaluatedinvitroand invivostudiesonantioxidativeactivitiesofE.cavafucoidan(ECF).ECFexhibitedmoreprominenteffects inperoxylradicalscavengingactivity,comparedtotheotherscavengingactivities.Thus,ECFwasfur- therevaluatedforitsprotectiveabilityagainst2,2-azobisdihydrochlorideinducedoxidativestressin VerocellsandECFstronglyreducedtheAAPH-inducedoxidativedamagethroughscavengingintracel- lularreactiveoxygenspecies.Furthermore,weevaluatedprotectiveeffectofECFagainstAAPH-induced oxidativestressinzebrafishmodel.ECFsignificantlyreducedROSgeneration,lipidperoxidationandcell deathinzebrafishmodel.ThesefindingsindicatethatECFhasantioxidantactivitiesinvitroVerocells andinvivozebrafishmodel,eventhoughECFisnotapolyphenolorflavonoidcompoundanddoesnot containbenzeneringsorconjugatedstructures.
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Reactiveoxygenspecies (ROS)or highlevelsoffree radicals causeoxidativestressleadingtodegradationofDNA,cellmem- branes, proteins and other cellular constituents (Fang, Yang, &
Wu,2002;Lopaczynski&Zeisei,2001).Asaresult,differentkinds ofserioushumandiseasesarecreatedincludingatherosclerosis, rheumatoidarthritis,musculardystrophy,cataracts,someneuro- logical disorders, sometypesof cancers and aging(Kovatcheva etal.,2001;Ruberto,Baratta,Biondi,&Amico,2001).
Zebrafish(Danioreiro)havedefiniteadvantages.Forexample, their comparatively small size makes them easier to adminis- ter in largenumbers in a laboratory environment (Kishiet al., 2003). Furthermore zebrafish have a very short development, as the basic body plan is laid out 24h post-fertilization (hpf),
∗Correspondingauthor.Tel.:+82647543475;fax:+82647563493.
E-mailaddresses:[email protected],[email protected](Y.-J.Jeon).
embryos hatch approximately 2–3 days post-fertilization (dpf) and theyattain maturity at about 3 months. Additionally, one female can spawn about 100 eggs per day, which are fertil- izedbyspermreleasedintothewaterbymales(Scholz,Fischer,
& Gundel, 2008). Zebrafish have beentraditionally used in the fieldsofmoleculargeneticsanddevelopmentbiology,asamodel organismfordrugdiscoveryandtoxicologicalstudiesbecauseof theirphysiological similaritytomammals (Drieveret al.,1996;
Hertog, 2005; Kimmel, 1989; Pichler et al., 2003). Moreover, zebrafish have been used as a model for human disease and developmentand have beenthetargetof a large-scalefunding program undertaken by the European Union (Zebrafish Model for Human Development and Disease ZF-MODELS) (Bradbury, 2004).
Seaweeds arecomposedof a varietyof bioactivesubstances (polysaccharides,pigments,minerals,peptidesandpolyphenols) withvaluable pharmaceuticaland biomedical potential.In par- ticularbrownseaweedscontainvariousbiologicalbenefitssuch asantioxidant,anticoagulant,antihypertension,antibacterial,and 0144-8617/$–seefrontmatter© 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.11.022
antitumoractivities(Athukorala&Jeon,2005;Heo,Park,Lee,&
Jeon,2005;Kotake-Nara,Asai,&Nagao,2005;Mayer&Hamann, 2004;Nagayama,Iwamura,Shibata,Hirayama,&Nakamura,2002).
Eckloniacava (class,Phaeophyceae; family, Lessoniaceae; order, Laminariales, E.cava)contains a variety of unique compounds including sulfated polysaccharides and phlorotannins with dif- ferent biological activities (Heo et al., 2009). In recent years, sulfatedpolysaccharidesandisolatedcompoundsfromseaweeds have been demonstrated to be potential ROS scavengers, and to possess anticoagulant, antithrombotic, antiviral, anticancer, anti-inflammation and antioxidant activities (Hayashi, Nakano, Hashimoto,Kanekiyo,&Hayashi,2008;Leeetal.,2012;Lietal., 2009;Shi, Nie, Chen,Liu, &Tap, 2007; Wang,Zhang, Zhang,&
Li, 2008). Nevertheless, the antioxidative activities of polysac- charides such as fucoidans have not been evaluated properly, because they do not have benzene rings or conjugated struc- tureswhich aregenerally thefunctional groupsfor antioxidant activity.
In this study, therefore, the antioxidative activities of E. cava fucoidan (ECF) were evaluated against 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stressinvitrocellexperimentsandinvivozebrafishmodel.
2. Materialsandmethods
2.1. Materials
Brownseaweed,E.cavawascollectedfromthecoastofJeju Island,SouthKorea,betweenFebruaryandApril2011.Thesam- plewas rinsedcarefully withfresh water and freezedried. The driedseaweedsamplewasgroundandshiftedthrougha50mesh standardtesting sieve.Allchemicalsand reagentsusedwereof analyticalgradeandobtainedfromcommercialsources(Leeetal., 2012).
2.2. ExtractionoffucoidanfromE.cava
Fucoidanwasextractedandfractionatedaccordingtothepre- viously reported methods withslight modifications (Lee et al., 2012).Tengramssampleoftheground,driedE.cavapowderwas homogenizedwith1000mLofdistilledwater(dH2O)andmixed with100LofCelluclast(NovoNordisk,Bagsvaerd,Denmark).This reactionwascontinuedfor24hat50◦Candthenthedigestwas boiledfor 10minat100◦C toinactivate theenzyme.Theprod- uctwasclarifiedbycentrifugation(3000×gfor20min)toremove theunhydrolyzedresidue.ThentheoptimalpHfortheenzymatic hydrolysiswasadjustedto7.0andthecrudECFwasasadditionof precipitatedwith3volumesofethanolafterthehydrolyticreac- tion.Subsequentlythecentrifugationat10,000×gfor20minat 4◦C, theprecipitate wasre-dissolved in dH2Oand sequentially treatedwith4MCaCl2 forpurification.Theresultingprecipitate wasremovedbycentrifugationandtheresultantsupernatantwas treatedwithcetylpyridinumchloride.Thepyridiniumsaltswere solubilizedwith3MCaCl2 andreprecipitatedwithethanol.The precipitatewasre-dissolvedindH2O,dialyzed(MwCO,10–12kDa) againstwaterat4◦Cfor72h,andthenlyophilized;thelyophilized samplewasusedascrudefucoidansample.Thefucoidanconsisted of mostly carbohydrates (51.8%), sulfates (20.1%), uronic acid (11.3%)andsmallamountsofprotein(8.7%).Monosaccharidecom- positionanalysis showed that fucose(61.1%), galactose (27.2%) werethemajorsugarsin thefucoidan,withminoramounts of xylose (7.0%), rhamnose (3.9%) and glucose (0.8%) (Lee et al., 2012).
2.3. Radicalscavengingassaysbyusingelectronspinresonance (ESR)spectrometer
2.3.1. DPPHradicalscavengingassay
Thisassayisbasedonthescavengingabilityof stableDPPH radicals by the radical scavenging constituentsin the extracts.
MethoddescribedbyNanjoetal.(1996)wasusedtoinvestigate theDPPHscavengingactivityby ESRspectrometer(JES-FAESR, JEOL,Tokyo,Japan).DPPHwaspreparedinMeOHattheconcen- trationof60M.A60LofECFwasaddedtothesamevolume offreshlypreparedDPPHsolution.Then,thereactantswerethor- oughly mixedand transferredto50L glasscapillary tubeand fittedintotheESRspectrometer.Thespinadductwasmeasured after2min.Themeasurementconditionswereasfollows;central field 2min.The measurement conditionswere asfollows; cen- tralfield3475G,modulationwidth0.8mT,amplitude500mT,scan width10mT,microwavepower5mW.
2.3.2. Peroxylradicalscavengingassay
PeroxylradicalsweregeneratedbyAAPHandtheirscavenging effectswereinvestigatedbythemethoddescribedbyHiramoto, Johkoh,Sako,andKikugawa(1993).Thereactionmixturecontain- ing20Lofdistilledwater,20Loftheextract,40mMAAPHand 20Lof40mMPOBNwasincubatedat37◦Cfor30min.Thespin adductwasrecordedonJES-FAESRspectrometer.Themeasure- mentconditionswereasfollows;centralfield3475G,modulation width0.2mT,amplitude 500mT, scan width10mT,microwave power8mW.
2.3.3. Hydrogenperoxidescavengingassay
TheabilityofECFtoscavengehydrogenperoxidescavenging activitywasdeterminedaccordingtothemethodofMüller(1985).
A100Lof0.1Mphosphatebuffer(pH5.0)andthesamplesolution weremixedina96-wellplate.A20Lofhydrogenperoxidewas addedtothemixture,andthenincubatedat37◦Cfor5min.After theincubation,30Lof1.25mMABTSand30Lof peroxidase (1unit/mL)wereaddedtothemixture,andthenincubatedat37◦C for10min.TheabsorbancewasreadwithanELISAreader405nm.
2.4. Invitrocellexperiment 2.4.1. Cellculture
Themonkeykidneyfibroblastcellline(Vero,KCKBNo.10081) was maintained at 37◦C in an incubator with a humidified atmosphereof5%CO2.ThecellswereculturedinDulbecco’smod- ifiedEagle‘smediumcontaining10%heat-inactivatedferalbovine serum,streptomycin(100g/mL),andpenicillin(100unit/mL).
2.4.2. Intracellularreactiveoxygenspecies(ROS)measurement invitroVerocells
ForthedetectionofintracellularROS,Verocellswereseededin 96-wellplatesataconcentrationof1×105cells/mL.After16h,the cellsweretreatedwithvariousconcentrationsofECFthenincu- batedat37◦Cunderahumidifiedatmosphere.After1h,AAPHwas addedatafinalconcentrationof10mM,andthecellswereincu- batedforanadditional30minat37◦C.Finallytheconcentration of 5g/mLof 2,7-dichlorodihydrofluoresceindiacetate (DCFH- DA)wasintroducedtothecells,andDCFH-DAfluorescencewas detectedatanexcitationwavelengthof485nmandanemission wavelengthof535nm,usingaPerkin-ElmerLS-5Bspectrofluo- rometer(LS-5B,Perkin-Elmer,CT,USA).
0 20 40 60 80 100
0.25 0.5 1
DPPH radicalscavenging activity (%)
0 20 40 60 80 100
0.25 0.5 1
Peroxylradical scavenging activity (%)
0 20 40 60 80 100
0.25 0.5 1
Hydrogen peroxide scavengingactivity (%)
ECF (mg/mL)
IC50: 0.78 mg/mL IC50: 0.48 mg/mL IC50> 1 mg/mL
ECF (mg/mL) ECF (mg/mL)
A B C
Fig.1.MeasurementoffreeradicalscavengingactivitiesofECFbyESR.(A)DPPHscavengingactivity;(B)peroxylradicalscavengingactivityand(C)hydrogenperoxide scavengingactivity.Valuesaremeans±SDoftriplicateexperiments.
2.5. Invivozebrafishmodel
2.5.1. Originandmaintenanceofparentalzebrafish
Adultzebrafishwereobtainedfromacommercialdealer(Seoul aquarium,Seoul,Korea)and15 fisheswerekeptin3.5Lacrylic tankwiththefollowingconditions;28.5±1◦C,andwerefedtwice timesaday(TetraGmgHD-49304MelleMadeinGermany)with a14/10hlight/darkcycle.Thedaybefore,breeding1femaleand 2malesinterbreed.Inthemorning(onsetoflight),embryoswere obtainedfromnaturalspawningcollectionofembryoswerecom- pletedwithin30mininpetriDishes(containingmedia).
2.5.2. WaterborneexposureofembryostoECFandAAPH
Theembryos(n=15)weretransferredtoindividualwellsof12- wellplatescontaining900Lembryomediafromapproximately 7–9hpf,ECFwasaddedtothewells. Aftertheincubation1h, a 15mMAAPHsolutionwastreatedtotheembryoexposedwithECF forupto24hpf.Then,embryoswererinsedusingfreshembryo media.
Fluorescence intensity Intracellular radical scavenging activity (%)
*
0 20 40 60 80 100
0 5000 10000 15000 20000 25000
Control 0 25 50 100 200
ECF (µg/mL) + AAPH 10 mM
*
*
*
*
Fig.2.EffectofECFonintracellularreactiveoxygenspecies(ROS)scavengingactiv- ity.IntracellularROSweredetectedbyDCFH-DAmethod.Thebar-graphshows fluorescenceintensityandthelinegraphexhibitsintracellularradicalscavenging activity.Experimentswereperformedintriplicateanddataaremean±SE.*p<0.01.
2.5.3. Measurementofheart-beat
Theheartbeatrateofbothatriumandventriclewasrecordedat 2dpffor1minunderthemicroscope.
2.5.4. Measurementofoxidativestress-inducedintracellularROS generationandimageanalysis
GenerationofROSproductionofzebrafishwasanalyzedusing anoxidation-sensitivefluorescentprobedye,DCFH-DA.At3dpf, a zebrafish larva was transferredto one well of 96-well plate, treatedwithDCFH-DAsolution(20g/mL)andincubatedfor1h inthedarkat28.5±1◦C.Aftertheincubation,thezebrafishlar- vae were rinsed by fresh embryo media and anaesthetizedby 2-phenoxyethanol(1/500dilutionsigma)beforeobservationand photographedunder themicroscopeCoolSNAP-Pro colordigital camera(Olympus, Japan).A fluorescenceintensityof individual larvawasquantifiedusingtheimageJprogram.
2.5.5. Measurementofoxidativestress-inducedlipidperoxidation generationandimageanalysis
Lipidperoxidationwasmeasuredtoassessthemembranedam- age in zebrafish model. Diphenyl-l-pyrenylphosphine (DPPP) is fluorescentprobefordetectionofcellmembranelipidperoxida- tion.DPPP is non-fluorescent,but it becomes fluorescentwhen oxidized.At3dpf,azebrafish larvawastransferredtoonewell of 96-well plates, treated with DPPP solution (25g/mL) and incubatedfor1hinthedarkat28.5±1◦C.Aftertheincubation, thezebrafishlarvaewererinsedusingfreshembryomediaand anaesthetizedby2-phenoxyethanol(1/500dilutionsigma)before observationandphotographedunderthemicroscopeCoolSNAP- Procolordigitalcamera(Olympus,Japan).Afluorescenceintensity ofindividualzebrafishlarvawasquantifiedusingtheimageJpro- gram.
2.5.6. Measurementofoxidativestress-inducedcelldeathand imageanalysis
Celldeathwasdetectedinliveembryosusingacridineorange staining.Acridineorangestaincellswithdisturbedplasmamem- branepermeability,therefore,itpreferentiallystainsnecroticor verylateapoptoticcells.At3dpf,azebrafishlarvawastransferred toonewellof96-wellplates,treatedwithacridineorangesolution (7g/mL)andincubatedfor30minunderthedarkat28.5±1◦C.
Afterthe incubation, the zebrafish larvaewere rinsed by fresh embryo mediaand anaesthetizedby 2-phenoxy ethanol (1/500 dilutionsigma)beforeobservationandphotographedunderthe
microscopeCoolSNAP-Procolordigitalcamera(Olympus,Japan).A fluorescenceintensityofindividualzebrafishlarvawasquantified usingtheimageJprogram.
2.6. Statisticalanalysis
Thedataareexpressedasthemean±standarderror(S.E.)and one-wayANOVAtest(usingSPSS11.5statisticalsoftware)wasused tocomparethemeanvaluesofeachtreatment.Significantdiffer- encesbetweenthemeansofparametersweredeterminedbythe Student’st-test(p<0.05,p<0.01).
3. Resultsanddiscussion
3.1. RadicalscavengingactivitiesofECF
ESR spin trapping provides a sensitive, direct and accurate methodtomonitorreactivespecies(Guoetal.,1999).Weused theESR technique tomeasure DPPH and peroxylradical scav- engingactivitiesofECF.HydrogenperoxideandDPPHhavebeen widely utilized to evaluate the antioxidant activity of natural compounds (Kang, Heo, Kim, Lee, & Jeon, 2012). The percent- agescavengingactivityofECFagainstDPPHisshowninFig.1A.
According to the results, ECF had an IC50value of 0.73mg/mL for DPPH radical scavengingactivity. Asshown in Fig. 1B, ECF reducedtheESRsignalintensitytoscavengetheperoxylradical in a dose-dependent manner.Peroxyl radical scavengingactiv- ityofECF(IC50value,0.48mg/mL)wasthehighestamongother radicalscavengingactivities.Further,Fig.1CshowsthatECFhad concentration-dependent scavenging activity against hydrogen peroxide.Howeverthehydrogenperoxidescavengingactivityof ECFwasverylow.TheseresultsindicatethatECFhasremarkable scavengingactivityontheperoxylradical.Theantioxidantability ofECFagainsttheperoxylradicalsuggestsapossiblebeneficialrole preventingvariouschronicdiseasesthatarelinkedwithoxidative stress.
3.2. MeasurementofintracellularROSinVerocellsinvitro
ECFstronglyscavengedperoxylradicalamongotherradicals.
ThuswedecidedtoinduceoxidativedamagetothecellswithAAPH.
ThelevelofROSproductionincellswasdetectedviathefluorescent
probeDCFtomeasurewhetherECFcouldpreventAAPH-induced ROSgenerationandtheresultingoxidativestressors(Kangetal., 2012).TheintracellularROSscavengingactivityofECFafteradmin- istration of AAPHto Vero cells is shown in Fig.2. The results showedthatintracellularradicalscavengingof thecells treated withECFat different concentrations(25–200g/mL) decreased withanincreasein ECFconcentration.In particularthehighest concentration(200g/mL)ofECFrevealedthestrongestROSscav- engingactivity,andtheIC50valuewas75g/mL.Previousstudies havereportedthathighsulfateandfucoseinalgalfucoidansare relatedtoradicalscavengingeffectswithgreaterpotentialforpre- ventingfreeradical-mediateddisease(Li,Lu,Wei,&Zhan,2008;
Vo&Kim,2012;Wangetal.,2008;Zhang,Yu,Zhou,Li,&Xu,2003).
Inthepresentstudy,ECFappearedtobeapotentialperoxylradi- calscavengeronROSgeneration.AccordinglyECFcouldprotectthe humanbodyagainstoxidativestress.
3.3. MeasurementofECFagainstAAPH-inducedoxidativestress invivozebrafishmodel
Zebrafish have becomea popularmodel in pharmacological studiessuchaschemical toxicity anddrugdiscovery (Ko etal., 2011).We observedthesurvival rateand heart-beating ratein zebrafishtodetermineAAPHtoxicityinzebrafishco-treatedwith ECF. The yolk is quickly exhausted after this point, with total absorptionoccurringby7hpf(Jardine&Litvak,2003).Therefore, wedecidedthat7dpfwastheendpointofthisexperimenttoassess survivalrateduetoECFtoxicity.Survivalratewas83%intheAAPH- treatedzebrafishcomparedtothecontrol(withoutECFandAAPH) group(Fig.3A).However,survivalrateswere91%and97%at100 and200g/mLECF,respectively.Forthisresult,itisprovedthatECF canprotectthezebrafishfromdamageinducedbyAAPH.Zebrafish larvaetypicallyhatchwithin2–3dpfat28.5◦C(Scholzetal.,2008).
Thereforetheheartbeatingwasmeasuredassoonasthezebrafish hatchedat2dpf.Heart-beatingwasobservedduringthefirstmin aszebrafish layona glassplates.The heart-beatingrateofthe AAPH-treatedzebrafishincreasedto114%,comparedwiththecon- trolgroup(Fig.3B).However,thetreatedconcentrationofECFat 100and200g/mLintheAAPH-treatedzebrafishreducedto106%
and103%inheart-beatingrate,respectively.Thus,survivalrateand heartbeatrateofECF-treatedzebrafishweresimilartothecontrol group.Therefore,ECFcouldbeconsideredalatentperoxylradical scavengingagent.
Survival rate (%)
ECF (µg/mL) + AAPH 15 mM
Heart Beating rate (%)
B
* * *
*
50 60 70 80 90 100 110
Control 0 100 200
50 60 70 80 90 100 110 120
Control 0 100 200
A
ECF (µg/mL) + AAPH 15 mM
Fig.3.MeasurementofAAPHtoxicityandAAPH-cotreatedwithECFonsurvivalandheartbeatingrates.(A)Survivalrateand(B)heartbeatingrate.Experimentswere performedintriplicateanddataaremean±SE.*p<0.01.
Fig.4.ProtectiveeffectofECFonAAPH-treatedreactiveoxygenspecies(ROS)productioninzebrafish.(A)ROSlevelsweremeasuredbyimageanalysisandfluorescence microscope.(i)Control(withoutECFandAAPH);(ii)AAPHonly;(iii)AAPH-cotreatedwith100g/mlECFand(iv)AAPH-cotreatedwith200g/mlECF.(B)ROSlevelswere measuredbyImageJ.Experimentswereperformedintriplicateanddataaremean±SE.*p<0.01.
We tested the capacity of ECF to detect changes in a physiological state using DCFH-DA to assess the accumula- tion of ROS, DPPP to assess lipid peroxidation and acridine orange to assess cell death caused by the AAPH treatment.
DCFH-DAisoxidation-dependentlyconvertedtonon-fluorescent 2,7-dichlorodihydrofluorescein(DCFH2)tothefluorescent2,7- dichlorofluorescein(DCF); thus fluorescence intensityincreases withROSproduction(Walkeretal.,2012).ROSlevelwas130%in AAPH-treatedzebrafishcomparedtothecontrolgroup.Incontrast, zebrafishexposedtoAAPHandECFatdifferentconcentrations(100 and200g/mL)showedsignificantlyreducedlevelsofROSpro- duction(106%and103%)(Fig.4).Thisresultsuggestsareduction ofROSgenerationbyECFtreatment.Diphenylpyrenylphosphine (DPPP) is a probe that detects hydroperoxides and DPPP oxide isstronglyfluorescent(Akasaka,Suzuki,Ohrui,&Meguro,1987).
AAPH-inducedlipidperoxidationbyDPPPfluorescentdyeisshown
inFig.5.TheAAPH-treatedzebrafishrevealed137%oflipidperoxi- dation,whereas,zebrafishgroupstreatedwith100and200g/mL ECFshoweddramaticallydecreasedlipidperoxidationto105%and 103%,respectively.Thisresultindicatesareductioninlipidperox- idationproductionbyECFtreatment.Acridineorangeisanucleic acidselectivefluorescentcationicdyeusefulfor apoptoticcells.
Lastly,Fig.6showsthatthecelldeathinducedbyAAPHtreatment wasconfirmedviaacridineorangeasfluorescenceintensity.The AAPH-inducedcelldeathinzebrafishwasmeasuredto118%com- paredtothecontrolgroup.However,celldeathwasreduced(101%
and 98%)by adding ECF(100 and 200g/mL) toAAPH-treated zebrafish.WhenzebrafishweretreatedwithECFpriortoAAPH treatment,adramaticdecreaseincelldeathwasobserved.These resultsindicatethatzebrafishcouldbeagoodanimalmodelfor antioxidantmaterial screening.Collectively,ourresultsdemon- stratethatECFhasexcellentantioxidantpropertiesinvitroand
Fig.5. ProtectiveeffectofECFonAAPH-treatedlipidperoxidationproductioninzebrafish.(A)Lipidperoxidationlevelsweremeasuredbyimageanalysisandfluorescence microscope.(i)Control(withoutECFandAAPH);(ii)AAPHonly;(iii)AAPH-cotreated100g/mlECFand(iv)AAPH-cotreated200g/mlECF.(B)Lipidperoxidationlevels weremeasuredbyImageJ.Experimentswasperformedintriplicateanddataaremean±SE.*p<0.01.
Fig.6.ProtectiveeffectofECFonAAPH-treatedcelldeathinzebrafish.(A)Celldeathlevelsweremeasuredbyimageanalysisandfluorescencemicroscope.(i)Control (withoutECFandAAPH);(ii)AAPHonly;(iii)AAPH-cotreatedwith100g/mlECFand(iv)AAPH-cotreatedwith200g/mlECF.(B)Celldeathlevelsweremeasuredby ImageJ.Experimentswasperformedintriplicateanddataaremean±SE.*p<0.05.
invivo.Therefore,ECFcanbeapotentialcandidateforfuturether- apeuticapplications.
4. Conclusion
ECFexhibited strongperoxyl radical scavenging activityvia ESRandintracellularROSscavengingactivityinVerocellsinvitro induced by AAPH. In addition, antioxidant ability was verified throughROSproduction,lipidperoxidationproduction, andcell deathintheAAPH-inducedoxidativestresszebrafishinvivomodel.
Inconclusion,thefucoidanisolatedfromE.cava(ECF)doesnot include a polyphenol or flavonoid,and does not have benzene ringsor conjugatedstructures.Nevertheless,ourstudiesclearly showthatECFpossessesantioxidantverifiedactivitiesusinginvivo zebrafishmodel.Therefore,zebrafishcouldbeaneffectiveinvivo modelandECFmaybeavaluablenaturalanti-oxidant.
Acknowledgment
ThisresearchwassupportedbyBasicScienceResearchProgram throughtheNationalResearchFoundationofKorea(NRF)funded bytheMinistryofEducation(2013-0611).
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