Fucose-containing sulfated polysaccharides from brown seaweed: Extraction technology and biological activity assessment 0DUFHO7XWRU$OH PhDGLVVHUWDWLRQ2 FucoseͲcontainingsulfated polysaccharidesfrombrownseaweed: Extractiontechnologyandbiological activityassessment FucoseͲcontainingsulfated polysaccharidesfrombrownseaweed: Extractiontechnologyandbiological activityassessment MarcelTutorAle PhDThesis 2012 MarcelTutorAle PhDThesis 2012 1 Copyright©: Marcel Tutor Ale Copyright©: Marcel Tutor Ale February 2012 February 2012 Center For Bioprocess Engineering Center For Bioprocess Engineering Department of Chemical and Biochemical Engineering Department of Chemical and Biochemical Engineering Technical University of Denmark Technical University of Denmark Søltofts Plads, Building 229 Søltofts Plads, Building 229 DK-2800 Kgs Lyngby DK-2800 Kgs Lyngby Denmark Denmark Phone: +45 4525 2800 Phone: +45 4525 2800 Web: www.bioeng.kt.dtu.dk Web: www.bioeng.kt.dtu.dk J&R Frydenberg A/S Print: J&R Frydenberg A/S Print: ISBN: København København May 2012 May 2012 978-87-92481-70-2 ISBN: 978-87-92481-70-2 WƌĞĨĂĐĞ WƌĞĨĂĐĞ Preface Preface Enormousamountsofseaweedresourcestillremainunexploited.Examiningnewapplicationsof Enormousamountsofseaweedresourcestillremainunexploited.Examiningnewapplicationsof thisunexploitedseaweedbydevelopingstateͲofͲtheͲartsolutions;andinnovationofseaweed thisunexploitedseaweedbydevelopingstateͲofͲtheͲartsolutions;andinnovationofseaweed productsaretheprimaryinterestandmotivationofthisPhDstudy. productsaretheprimaryinterestandmotivationofthisPhDstudy. ThePhDworkwasinitiatedattheDepartmentofSystemBiology,TechnicalUniversityofDenmark ThePhDworkwasinitiatedattheDepartmentofSystemBiology,TechnicalUniversityofDenmark (DTU)andwascompletedatCenterforBioprocessEngineering(BioEng),DepartmentofChemical (DTU)andwascompletedatCenterforBioprocessEngineering(BioEng),DepartmentofChemical andBiochemicalEngineeringSystemBiology,TechnicalUniversityofDenmark(DTU),Lyngby andBiochemicalEngineeringSystemBiology,TechnicalUniversityofDenmark(DTU),Lyngby Denmark.ThemainresearchandexperimentalactivitieswereperformedinBioEnglaboratory Denmark.ThemainresearchandexperimentalactivitieswereperformedinBioEnglaboratory facilitieslocatedatBldg.227DTUSøltoftPlads,Kgs.Lyngby.InfulfillmentofthePhDprogramme facilitieslocatedatBldg.227DTUSøltoftPlads,Kgs.Lyngby.InfulfillmentofthePhDprogramme variouscourses,seminars,conferenceparticipationsandresearchactivitiesweretakenboth variouscourses,seminars,conferenceparticipationsandresearchactivitiesweretakenboth locallyandinternationally.ExternalresearchactivitywasalsoaccomplishedatSchoolofAllied locallyandinternationally.ExternalresearchactivitywasalsoaccomplishedatSchoolofAllied HealthSciences,KitasatoUniversity,Sagamihara,Kanagawa,Japan. HealthSciences,KitasatoUniversity,Sagamihara,Kanagawa,Japan. InthisPhDthesis,publishedworksarecitedandthechaptersarebasedonpublishedresearchand InthisPhDthesis,publishedworksarecitedandthechaptersarebasedonpublishedresearchand scientificpaperscompletedbytheauthorduringthePhDstudy: scientificpaperscompletedbytheauthorduringthePhDstudy: Chapter1isanintroductiontothestateͲofͲtheͲartFCSPmethods.Italsooutlinestheproblems, Chapter1isanintroductiontothestateͲofͲtheͲartFCSPmethods.Italsooutlinestheproblems, hypotheses,andcoreobjectivesofthisPhDstudy.Thischapterprovidesanoveralloutlineof hypotheses,andcoreobjectivesofthisPhDstudy.Thischapterprovidesanoveralloutlineof differentscientificinvestigationsandresearchactivitiesintheformofprojectphases. differentscientificinvestigationsandresearchactivitiesintheformofprojectphases. Chapter2isbasedonPaper1,whichhighlightsFCSPstructureͲfunctionrelationshipsand Chapter2isbasedonPaper1,whichhighlightsFCSPstructureͲfunctionrelationshipsand extractionmethods.Italsoincludesanoverviewofseaweedpotentialsbasedonrecent extractionmethods.Italsoincludesanoverviewofseaweedpotentialsbasedonrecent developmentandreportsfrompublishedjournals. developmentandreportsfrompublishedjournals. Chapter3isaboutoptimizedsingleͲstepextractionofFCSPsbasedonPaper2.Thischapter Chapter3isaboutoptimizedsingleͲstepextractionofFCSPsbasedonPaper2.Thischapter providesaninͲdepthinvestigationoftheinfluenceofdifferentextractionparametersonthe providesaninͲdepthinvestigationoftheinfluenceofdifferentextractionparametersonthe chemicalnatureandstructuralfeaturesofFCSPs. chemicalnatureandstructuralfeaturesofFCSPs. Chapter4showstheFCSPbioactivity.TheantiͲproliferativeandimmuneresponseactivityof Chapter4showstheFCSPbioactivity.TheantiͲproliferativeandimmuneresponseactivityof fucoidanextractedfromSargassumsp.usingminimalprocessesbasedonPapers3and4isalso fucoidanextractedfromSargassumsp.usingminimalprocessesbasedonPapers3and4isalso presented. presented. i i WŚdŚĞƐŝƐϮϬϭ2 WŚdŚĞƐŝƐϮϬϭ2 3 WƌĞĨĂĐĞ WƌĞĨĂĐĞ Chapter5isbasedonPaper5.Successfulexploitationofseaweedforcommercialapplicationsis Chapter5isbasedonPaper5.Successfulexploitationofseaweedforcommercialapplicationsis accomplishedprovidedthatgrowthparametersareoptimized;hence,growthandnutrient accomplishedprovidedthatgrowthparametersareoptimized;hence,growthandnutrient assimilationmonitoringofseaweedhasalsobeenthesubjectofthisstudyusingU.lactuca assimilationmonitoringofseaweedhasalsobeenthesubjectofthisstudyusingU.lactuca seaweedasamodel. seaweedasamodel. Chapter6containsthefinalremarksaboutthepresentwork.Italsogivessomefuture Chapter6containsthefinalremarksaboutthepresentwork.Italsogivessomefuture perspectivesandprospectiveresearchareas. perspectivesandprospectiveresearchareas. ThiswholePhDstudywasundertakenwithsuperiorguidanceandsupervisionofProf.AnneS. ThiswholePhDstudywasundertakenwithsuperiorguidanceandsupervisionofProf.AnneS. Meyer,headofCenterforBioprocessEngineeringandtirelessencouragementofProf.Jørn Meyer,headofCenterforBioprocessEngineeringandtirelessencouragementofProf.Jørn DalgaardMikkelsen,CenterforBioprocessEngineeringascoͲsupervisor.Moreover,thediligent DalgaardMikkelsen,CenterforBioprocessEngineeringascoͲsupervisor.Moreover,thediligent assistanceandsupervisionofDr.HirokoMaruyamaandDr.HidezakuTamauchiduringmyresearch assistanceandsupervisionofDr.HirokoMaruyamaandDr.HidezakuTamauchiduringmyresearch workinKitasatoUniversity,KanagawaJapanwassobeneficialfortheadvancementofthisPhD workinKitasatoUniversity,KanagawaJapanwassobeneficialfortheadvancementofthisPhD study. study. ThePhDprojectwasfullyfinancedbytheTechnicalUniversityofDenmark(DTU)foraperiodfrom ThePhDprojectwasfullyfinancedbytheTechnicalUniversityofDenmark(DTU)foraperiodfrom March2008toOctober2011. March2008toOctober2011. ThisthesisissubmittedforthefulfillmentofthePhDdegreerequirementsatTechnicalUniversity ThisthesisissubmittedforthefulfillmentofthePhDdegreerequirementsatTechnicalUniversity ofDenmark. ofDenmark. MarcelTutorAle TechnicalUniversityofDenmark(DTU) February2012 MarcelTutorAle TechnicalUniversityofDenmark(DTU) February2012 ii ii WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 4 ďƐƚƌĂĐƚ A ďƐƚƌĂĐƚ A Abstract Abstract Marineseaweedthatiswasheduponthecoastlineisanuisanceasitsdegradationproducesafoul Marineseaweedthatiswasheduponthecoastlineisanuisanceasitsdegradationproducesafoul asmellandgenerateswasteproblems.ExploitationofcoastlineͲpollutingseaweedssuchas asmellandgenerateswasteproblems.ExploitationofcoastlineͲpollutingseaweedssuchas Sargassumsp.,Ulvasp.,andotherbeachͲcastseaweedspeciesforvariouscommercial Sargassumsp.,Ulvasp.,andotherbeachͲcastseaweedspeciesforvariouscommercial applicationswillgeneratenewvaluableproductsthatmayhelplessencoastalpollutionby applicationswillgeneratenewvaluableproductsthatmayhelplessencoastalpollutionby seaweedsandcreatenewseaweedͲbasedresources.Thus,utilizationofthesenaturalresourcesis seaweedsandcreatenewseaweedͲbasedresources.Thus,utilizationofthesenaturalresourcesis ofgreatimportance.TheobjectivesofthisPhDstudyweretodevelopatechnologytoextract ofgreatimportance.TheobjectivesofthisPhDstudyweretodevelopatechnologytoextract bioactivecompoundsfromnuisancebrownseaweeds,andinvestigatetheirbioactivity.Tothis bioactivecompoundsfromnuisancebrownseaweeds,andinvestigatetheirbioactivity.Tothis effect,designedoptimizedextractionoffucoseͲcontainingsulfatedpolysaccharides(FCSPs)and/or effect,designedoptimizedextractionoffucoseͲcontainingsulfatedpolysaccharides(FCSPs)and/or crudefucoidanfrombrownseaweedwereperformed,andthebioactivityoftheisolatedFCSPs crudefucoidanfrombrownseaweedwereperformed,andthebioactivityoftheisolatedFCSPs wasinvestigated.Moreover,toassessthepotentialofseaweedtoassimilatenitrogenͲbased wasinvestigated.Moreover,toassessthepotentialofseaweedtoassimilatenitrogenͲbased nutrients,atechnologyforaccuratemonitoringofdifferentialseaweedgrowthresponsesto nutrients,atechnologyforaccuratemonitoringofdifferentialseaweedgrowthresponsesto nutrientassimilationwasalsodeveloped. nutrientassimilationwasalsodeveloped. Fucoidanisatermusedtodescribeaclassofsulfatedpolysaccharidesextractedfrombrown Fucoidanisatermusedtodescribeaclassofsulfatedpolysaccharidesextractedfrombrown seaweed,whichcontainssubstantialamountsoffucose;varyingamountsofgalactose,xylose,and seaweed,whichcontainssubstantialamountsoffucose;varyingamountsofgalactose,xylose,and glucuronicacid;anddifferingglycosidiclinkages,andarevariouslysubstitutedwithsulfateand glucuronicacid;anddifferingglycosidiclinkages,andarevariouslysubstitutedwithsulfateand acetylgroupsandsidebranchescontainingfucoseorotherglycosylunits.TheseFCSPsprincipally acetylgroupsandsidebranchescontainingfucoseorotherglycosylunits.TheseFCSPsprincipally consistofabackboneof(1o3)Ͳand/or(1o4)ͲlinkedɲͲLͲfucopyranoseresiduesthatmaybe consistofabackboneof(1o3)Ͳand/or(1o4)ͲlinkedɲͲLͲfucopyranoseresiduesthatmaybe substitutedwithsulfate(SO3Ͳ)onCͲ2,CͲ3,orCͲ4andacetylgroupsatCͲ4onthemainchainor substitutedwithsulfate(SO3Ͳ)onCͲ2,CͲ3,orCͲ4andacetylgroupsatCͲ4onthemainchainor mayhaveshortfucosidesidechainsthatareusuallylinkedfromtheOͲ4ofoneorseveralofthe mayhaveshortfucosidesidechainsthatareusuallylinkedfromtheOͲ4ofoneorseveralofthe fucopyranosebackboneresidues.FCSPsareknowntoexhibitcrucialbiologicalactivitiesincluding fucopyranosebackboneresidues.FCSPsareknowntoexhibitcrucialbiologicalactivitiesincluding antiͲtumoractivity.Althoughdifferentlyextracted,purified,fucoseͲrich,modifiedfucoidanshave antiͲtumoractivity.Althoughdifferentlyextracted,purified,fucoseͲrich,modifiedfucoidanshave beenreportedtoexertbioactivepropertiessuchasantiͲcoagulantandenhanceimmuneresponse beenreportedtoexertbioactivepropertiessuchasantiͲcoagulantandenhanceimmuneresponse activity,fewstudieshaveinvestigatedthebioactivityofunfractionatedFCSPs,notablyFCSPs activity,fewstudieshaveinvestigatedthebioactivityofunfractionatedFCSPs,notablyFCSPs extractedusingmilderandfewerprocessingsteps.CrudefucoidanfromSargassumsp.andFucus extractedusingmilderandfewerprocessingsteps.CrudefucoidanfromSargassumsp.andFucus vesiculosuswereexaminedfortheirbioactivityagainstlungandskincancercelllinesinbothin vesiculosuswereexaminedfortheirbioactivityagainstlungandskincancercelllinesinbothin vitroandinvivostudies.ThisstudyshowedthatunfractionatedFCSPshindertheinvitro vitroandinvivostudies.ThisstudyshowedthatunfractionatedFCSPshindertheinvitro proliferationofLewislungcarcinomaandmelanomaB16celllinesbyinductionofapoptosis. proliferationofLewislungcarcinomaandmelanomaB16celllinesbyinductionofapoptosis. Moreover,theantiͲtumoractivityofcrudefucoidanseemstobeassociatedwithanenhanced Moreover,theantiͲtumoractivityofcrudefucoidanseemstobeassociatedwithanenhanced immuneresponseasdepictedbyanincreaseinnaturalkillercellactivityinmice. immuneresponseasdepictedbyanincreaseinnaturalkillercellactivityinmice. iii P iii WŚdŚĞƐŝƐϮϬϭ2 P WŚdŚĞƐŝƐϮϬϭ2 5 ďƐƚƌĂĐƚ ďƐƚƌĂĐƚ TheclassicalextractionofFCSPsinvolvinglong,repetitive,multiͲstepacidandalkalinetreatments TheclassicalextractionofFCSPsinvolvinglong,repetitive,multiͲstepacidandalkalinetreatments isdetrimentaltoitsstructuralproperties,yield,andcompositionalattributes.Inthisstudy, isdetrimentaltoitsstructuralproperties,yield,andcompositionalattributes.Inthisstudy, statisticallydesigned,optimizedextractionofasingleͲstepextractionofFCSPsfromSargassumsp. statisticallydesigned,optimizedextractionofasingleͲstepextractionofFCSPsfromSargassumsp. wascarriedout.Theeffectsofthedifferentextractionparametersonthenaturalchemical wascarriedout.Theeffectsofthedifferentextractionparametersonthenaturalchemical compositionoftheisolatedsulfatedpolysaccharideswerealsoinvestigated.Thedatashowedthat compositionoftheisolatedsulfatedpolysaccharideswerealsoinvestigated.Thedatashowedthat classicalmultiͲstepextractionusingш0.2MHClatelevatedtemperatureandextendedtimehada classicalmultiͲstepextractionusingш0.2MHClatelevatedtemperatureandextendedtimehada detrimentaleffectontheFCSPsyield,asthistreatmentapparentlydisruptedthestructural detrimentaleffectontheFCSPsyield,asthistreatmentapparentlydisruptedthestructural integrityofthepolymerandevidentlydegradedcarbohydratechainsoffucoseresiduesduring integrityofthepolymerandevidentlydegradedcarbohydratechainsoffucoseresiduesduring extraction.TheresultsalsorevealedamaximalFCSPsyieldofapproximately7%dryweightwith extraction.TheresultsalsorevealedamaximalFCSPsyieldofapproximately7%dryweightwith Sargassumsp.using0.03MHClat90°Cand4Ͳhextractionconditions. Sargassumsp.using0.03MHClat90°Cand4Ͳhextractionconditions. Accuratemonitoringofthedifferentialgrowthresponseofseaweedtodifferentnutrient Accuratemonitoringofthedifferentialgrowthresponseofseaweedtodifferentnutrient assimilationiscrucialtoexplorevariousapplicationsofseaweedresources,suchasbiomassfor assimilationiscrucialtoexplorevariousapplicationsofseaweedresources,suchasbiomassfor bioenergyproductionandsourceoffunctionalhealthycomponentsandbioactivecompounds.A bioenergyproductionandsourceoffunctionalhealthycomponentsandbioactivecompounds.A majorprerequisiteforthesuccessfulexploitationofcultivatedseaweedlikeUlvalactucafor majorprerequisiteforthesuccessfulexploitationofcultivatedseaweedlikeUlvalactucafor commercialpurposesisthatthegrowthrateandyieldsshouldbeoptimized.Inthisstudy,the commercialpurposesisthatthegrowthrateandyieldsshouldbeoptimized.Inthisstudy,the growthresponseofU.lactucatoammoniumandnitrateassimilationwasinvestigatedusinga growthresponseofU.lactucatoammoniumandnitrateassimilationwasinvestigatedusinga photoscanningtechniquetomonitorthegrowthkineticsinU.lactuca.Photoscanningimages photoscanningtechniquetomonitorthegrowthkineticsinU.lactuca.Photoscanningimages revealeddifferentialincreasesinthesurfaceareaofU.lactucadiscsovertimeinresponseto revealeddifferentialincreasesinthesurfaceareaofU.lactucadiscsovertimeinresponseto differentnitrogenͲbasednutrientsources.Theresultsalsoshowedafavorablegrowthresponseto differentnitrogenͲbasednutrientsources.Theresultsalsoshowedafavorablegrowthresponseto ammoniumasanitrogensource,andthepresenceofammoniumdiscriminatedthenitrateuptake ammoniumasanitrogensource,andthepresenceofammoniumdiscriminatedthenitrateuptake byU.lactucauponexposuretoammoniumnitrate.Thisstudyexhibitstheapplicabilityofa byU.lactucauponexposuretoammoniumnitrate.Thisstudyexhibitstheapplicabilityofa photoscanningapproachforacquiringprecisequantitativegrowthdataforU.lactuca. photoscanningapproachforacquiringprecisequantitativegrowthdataforU.lactuca. Inconclusion,wedemonstratedthatnuisanceseaweedcanbeapotentialsourceofbiomassand Inconclusion,wedemonstratedthatnuisanceseaweedcanbeapotentialsourceofbiomassand bioactivecompoundnotablyFCSPs.Thisstudyprovedthehypothesesthatdifferentextraction bioactivecompoundnotablyFCSPs.Thisstudyprovedthehypothesesthatdifferentextraction conditionshavecrucialinfluencedtothechemicalnatureofFCSPs.Thestudyalsodemonstrated conditionshavecrucialinfluencedtothechemicalnatureofFCSPs.Thestudyalsodemonstrated thatunfractionatedFCSPsareabletoexertbioactiveactionssuchasantiͲtumorandimmuneͲ thatunfractionatedFCSPsareabletoexertbioactiveactionssuchasantiͲtumorandimmuneͲ modulatingpropertiesinbothinvitroandinvivostudies.Thisstudyillustratestheimportanceofa modulatingpropertiesinbothinvitroandinvivostudies.Thisstudyillustratestheimportanceofa precisemonitoringtechniqueofthegrowthofU.lactucainordertosuccessfullyexploititfor precisemonitoringtechniqueofthegrowthofU.lactucainordertosuccessfullyexploititfor commercialapplication. commercialapplication. iv iv WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 6 ĂŶƐŬ^ĂŵŵĞŶĨĂƚŶŝŶŐ ĂŶƐŬ^ĂŵŵĞŶĨĂƚŶŝŶŐ DanskSammenfatning DanskSammenfatning Tangpåstrandenerenplage,førstogfremmestpågrundafdelugtgener,somkommernår Tangpåstrandenerenplage,førstogfremmestpågrundafdelugtgener,somkommernår tangengåriforrådnelse.Enudnyttelseafdettetang,såsomSargassumsp.,Ulvasp.,ogandre tangengåriforrådnelse.Enudnyttelseafdettetang,såsomSargassumsp.,Ulvasp.,ogandre lignendekystͲnỉretyperaftang,kanfrembringeheltnyevỉrdifuldeprodukterogmåskesamtidig lignendekystͲnỉretyperaftang,kanfrembringeheltnyevỉrdifuldeprodukterogmåskesamtidig mindskedeùnskedelugtgenerfratangsomskylleroppåstranden.Udnyttelseaftangsom mindskedeùnskedelugtgenerfratangsomskylleroppåstranden.Udnyttelseaftangsom resourceerudgangspunktetfordettePhDstudium.PhDstudietsformålharværetdelsatudvikle resourceerudgangspunktetfordettePhDstudium.PhDstudietsformålharværetdelsatudvikle enekstraktionsmetodetilatisolerebioaktiveprodukterfrabrunttang,delsatundersøgedisse enekstraktionsmetodetilatisolerebioaktiveprodukterfrabrunttang,delsatundersøgedisse produktersbioaktivitet.ForatopfyldedetteformålblevderiPhDarbejdetudvikletenstatistisk produktersbioaktivitet.ForatopfyldedetteformålblevderiPhDarbejdetudvikletenstatistisk designet,optimeretekstraktionsmetodetilekstraktionafsåkaldtefukoseͲindeholdende designet,optimeretekstraktionsmetodetilekstraktionafsåkaldtefukoseͲindeholdende sulfateredepolysakkarider(eng.fucoseͲcontainingsulfatedpolysaccharides),forkortetFCSPs, sulfateredepolysakkarider(eng.fucoseͲcontainingsulfatedpolysaccharides),forkortetFCSPs, henholdsvisgrovfucoidanfrabruntang,primærtSargassumsp.,ogbioaktivitetenaf henholdsvisgrovfucoidanfrabruntang,primærtSargassumsp.,ogbioaktivitetenaf ekstraktionsproduktetblevundersøgt.Forydermereatvurderetangsevnetilatassimilere,og ekstraktionsproduktetblevundersøgt.Forydermereatvurderetangsevnetilatassimilere,og dermedvoksepånitrogenͲholdigesalte,blevderudvikletenteknologitilatmonitorere dermedvoksepånitrogenͲholdigesalte,blevderudvikletenteknologitilatmonitorere differentialvækstafUlvalactuca,påforskelligenæringsstofferivandet. differentialvækstafUlvalactuca,påforskelligenæringsstofferivandet. Fucoidanerenbetegnelse,somdækkeroverengruppeafsulfaterede,fukoseͲholdige, Fucoidanerenbetegnelse,somdækkeroverengruppeafsulfaterede,fukoseͲholdige, polysakkariderfratang.Udoverfukoseindeholderfucoidanforkelligemængdergalaktose,xylose, polysakkariderfratang.Udoverfukoseindeholderfucoidanforkelligemængdergalaktose,xylose, glukuronsyre,somerforbundetviaforskelligetyperglykosidbindinger,ogsomderudoverer glukuronsyre,somerforbundetviaforskelligetyperglykosidbindinger,ogsomderudoverer substitueretiforskelliggradmedsulfatogacetylͲgrupperogsomkanhavesidekæder substitueretiforskelliggradmedsulfatogacetylͲgrupperogsomkanhavesidekæder indeholdendefukoseellerandreglykosylͲsubstituenter.DisseFCSPsbestårprincipieltafen indeholdendefukoseellerandreglykosylͲsubstituenter.DisseFCSPsbestårprincipieltafen rygrad,ellerenhovedkæde,af(1o3)Ͳog/eller(1o4)ͲlinkedeɲͲLͲfucopyranoseenheder,somkan rygrad,ellerenhovedkæde,af(1o3)Ͳog/eller(1o4)ͲlinkedeɲͲLͲfucopyranoseenheder,somkan væresubstitueretmedsulfate(SO3Ͳ)påCͲ2,CͲ3,ellerCͲ4forudenacetylgrupperpåCͲ4påfucoseͲ væresubstitueretmedsulfate(SO3Ͳ)påCͲ2,CͲ3,ellerCͲ4forudenacetylgrupperpåCͲ4påfucoseͲ enhenderneihovedkæden,og/ellersomharkortefukoseͲkæder,dernormalterbundetviaOͲ4 enhenderneihovedkæden,og/ellersomharkortefukoseͲkæder,dernormalterbundetviaOͲ4 fraenellerflerefukoseͲenhederihovedkæden.Deterkendt,atFCSPsisoleretfratanghar fraenellerflerefukoseͲenhederihovedkæden.Deterkendt,atFCSPsisoleretfratanghar forskelligegavnlige,bioaktiveeffekter,herunderantiͲtumoraktivitet.Selvomdetharværet forskelligegavnlige,bioaktiveeffekter,herunderantiͲtumoraktivitet.Selvomdetharværet rapporteret,atoprensede,fucoseͲrige,modificeredefucoidanͲprøverharbioaktiveeffekter, rapporteret,atoprensede,fucoseͲrige,modificeredefucoidanͲprøverharbioaktiveeffekter, såsomantiͲkoagulerende,ogimmunͲresponsforøgendeegenskaber,erbioaktivitetenafmere såsomantiͲkoagulerende,ogimmunͲresponsforøgendeegenskaber,erbioaktivitetenafmere grove,ufraktioneredeFCSPs–ekstraheretmedmildereogfærreekstraktionstrinͲikkeundersøgt. grove,ufraktioneredeFCSPs–ekstraheretmedmildereogfærreekstraktionstrinͲikkeundersøgt. GroftoprensedefucoidanͲprøverfraSargassumsp.ogFucusvesiculosusblevidettePhDarbejde GroftoprensedefucoidanͲprøverfraSargassumsp.ogFucusvesiculosusblevidettePhDarbejde undersøgtforderesbioaktivitetmodlungeͲoghudcancercellelinjervækstbådeinvitrooginvivo. undersøgtforderesbioaktivitetmodlungeͲoghudcancercellelinjervækstbådeinvitrooginvivo. v v WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 7 ĂŶƐŬ^ĂŵŵĞŶĨĂƚŶŝŶŐ ĂŶƐŬ^ĂŵŵĞŶĨĂƚŶŝŶŐ Arbejdetviste,atufraktioneredeFCSPsekstrakterhindrerproliferationinvitroafLewislung Arbejdetviste,atufraktioneredeFCSPsekstrakterhindrerproliferationinvitroafLewislung carcinomaogmelanomaB16cellelinjerviainduktionafapoptosis.Desudenblevdetvist,at carcinomaogmelanomaB16cellelinjerviainduktionafapoptosis.Desudenblevdetvist,at denneantiͲtumoraktivitetafgrovfucoidan,tilsyneladendeerassocieretmedetøget denneantiͲtumoraktivitetafgrovfucoidan,tilsyneladendeerassocieretmedetøget immunrespons,måltsometforøgetniveauafnaturlige”killer”cellersaktivitetimus. immunrespons,måltsometforøgetniveauafnaturlige”killer”cellersaktivitetimus. KlassiskekstraktionafFCSPsfratanginvolvereradskilligelangsommelige,behandlingermedsyre KlassiskekstraktionafFCSPsfratanginvolvereradskilligelangsommelige,behandlingermedsyre ogbase,hvilketerødelæggendeforderesspecifikkestrukturogsammensætning.IdetePhD ogbase,hvilketerødelæggendeforderesspecifikkestrukturogsammensætning.IdetePhD arbejdeblevderudvikletenstatistiskdesignet,optimeretenkeltͲtrinsekstraktionsmetodetilat arbejdeblevderudvikletenstatistiskdesignet,optimeretenkeltͲtrinsekstraktionsmetodetilat udtrækkeFCSPsfraSargassumsp Effektenafdeforskelligeekstraktionsparametrepå udtrækkeFCSPsfraSargassumsp Effektenafdeforskelligeekstraktionsparametrepå sammensætningenafdeekstraheredepolysacchariderbelvogsåvurderet.Resultaterneviste,at sammensætningenafdeekstraheredepolysacchariderbelvogsåvurderet.Resultaterneviste,at klassisk,multiͲtrinsekstraktionvedbrugafш0.2MHClvedhøjtemperaturehavdeen klassisk,multiͲtrinsekstraktionvedbrugafш0.2MHClvedhøjtemperaturehavdeen ødelæggendeeffectpåudbyttetafFCSPsogvistedesudenatensådanbehandlingtilsyneladende ødelæggendeeffectpåudbyttetafFCSPsogvistedesudenatensådanbehandlingtilsyneladende ødelagdepolysaccharidstrukturenogatfukoseͲkæderneblevnedbrudtunderbehandlingen. ødelagdepolysaccharidstrukturenogatfukoseͲkæderneblevnedbrudtunderbehandlingen. Resultaternevisteogså,atetmaximalFCSPSudbytte,påca.7%aftørvægtenforSargassumsp. Resultaternevisteogså,atetmaximalFCSPSudbytte,påca.7%aftørvægtenforSargassumsp. kunneopnåsvedetͲtrinsekstraktionmed0.03MHClved90°Ci4timer. kunneopnåsvedetͲtrinsekstraktionmed0.03MHClved90°Ci4timer. Nøjagtigmonitoreringafvækstresponsaftangpåforskelligenæringsstoffererafgørendeforat Nøjagtigmonitoreringafvækstresponsaftangpåforskelligenæringsstoffererafgørendeforat undersøgeforskelligeanvendelseraftang,primærtbiomassevæksttilbioenergiͲproduktionogtil undersøgeforskelligeanvendelseraftang,primærtbiomassevæksttilbioenergiͲproduktionogtil udnyttelseaftangsomkildetilproduktionaff.eks.funktionellefødevarekomponentereller udnyttelseaftangsomkildetilproduktionaff.eks.funktionellefødevarekomponentereller bioaktivestoffer.EnvigtigforudsætningforkommercieludnyttelseafkultiverettangsåsomUlva bioaktivestoffer.EnvigtigforudsætningforkommercieludnyttelseafkultiverettangsåsomUlva lactucaer,atvækstratenogudbyttetoptimeres.IdettestudieblevvækstresponsafU.lactucaI lactucaer,atvækstratenogudbyttetoptimeres.IdettestudieblevvækstresponsafU.lactucaI forholdtilammoniumognitratͲassimilationundersøgtvedhjælpafenfotoͲscanningsteknik.FotoͲ forholdtilammoniumognitratͲassimilationundersøgtvedhjælpafenfotoͲscanningsteknik.FotoͲ scannedebillederafsløredeforskelligevækstͲinkrementerudframålingafdetfotograferedeareal scannedebillederafsløredeforskelligevækstͲinkrementerudframålingafdetfotograferedeareal afudstukneskiverafU.lactucaovertidsomresponspåforskelligenitrogenͲbaserede afudstukneskiverafU.lactucaovertidsomresponspåforskelligenitrogenͲbaserede næringskilder.Resultaternevisteogsåenfavorabelvækstøgningpåammoniumsom næringskilder.Resultaternevisteogsåenfavorabelvækstøgningpåammoniumsom nitrogenkilde,ogdesudenattilstedeværelsenafammoniumdiskrimineredeU.lactucasnitratͲ nitrogenkilde,ogdesudenattilstedeværelsenafammoniumdiskrimineredeU.lactucasnitratͲ optagiforholdtiloptagetafammoniumnitrat.StudietvisteanvendelighedenafphotͲscanningtil optagiforholdtiloptagetafammoniumnitrat.StudietvisteanvendelighedenafphotͲscanningtil præciskvantitativvækstͲmonitoreringafU.lactuca. præciskvantitativvækstͲmonitoreringafU.lactuca. Densameldekonklusienersåledesatvidemonstrerede,attangkanværeenpotentialkildetil Densameldekonklusienersåledesatvidemonstrerede,attangkanværeenpotentialkildetil biomasseogbioaktivekomponenter,isærFCSPs.PhDstudietvistedesudenathypotesen,at biomasseogbioaktivekomponenter,isærFCSPs.PhDstudietvistedesudenathypotesen,at forskellgieekstraktionsbetingelserharafgørendeindflydelsepådenkemiskenaturafFCSPser forskellgieekstraktionsbetingelserharafgørendeindflydelsepådenkemiskenaturafFCSPser sand.Studietdemonstreredeogså,atufraktioneredeFCSPsharbioaktiveegenskabersåsomantiͲ sand.Studietdemonstreredeogså,atufraktioneredeFCSPsharbioaktiveegenskabersåsomantiͲ tumorogimmunͲmodulerendeeffecter,vistibådeinvitrooginvivo.Studietilustrererdesuden tumorogimmunͲmodulerendeeffecter,vistibådeinvitrooginvivo.Studietilustrererdesuden vigtighedenafenpræcismonitoreringsmetodetilmålingafU.lactucavækst. vigtighedenafenpræcismonitoreringsmetodetilmålingafU.lactucavækst. vi vi WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 8 >ŝƐƚŽĨWƵďůŝĐĂƚŝŽŶƐ >ŝƐƚŽĨWƵďůŝĐĂƚŝŽŶƐ Listofpublications Listofpublications ThePhDthesisisbasedontheworkpresentedinthefollowingpapers: ThePhDthesisisbasedontheworkpresentedinthefollowingpapers: I II III IV Importantdeterminantsforfucoidanbioactivity:AcriticalreviewofstructureͲfunction relationsandextractionmethodsforfucoseͲcontainingsulfatedpolysaccharidesfrom brownseaweed AleMT,MikkelsenJDandMeyerAS MarineDrugs(2011)ϵ͕ϮϭϬϲͲϯϬ͕DOI:10.3390/md9ϭϬϮϭϬϲ, Publishedonline DesignedoptimizationofasingleͲstepextractionoffucoseͲcontainingsulfated polysaccharidesfromSargassumsp. AleMT,MikkelsenJDandMeyerAS JournalofAppliedPhycology(2011) DOI:10.1007/s1081Ͳ011Ͳ9690Ͳ3,Publishedonline FucoidanfromSargassumsp.andFucusvesiculosusreducescellviabilityoflungcarcinoma andmelanomacellsinvitroandactivatesnaturalkillercellsinmiceinvivo AleMT,MaruyamaH,TamauchiH,MikkelsenJDandMeyerAS InternationalJournalofBiologicalMacromolecules49(2011)331–336 DOI:10.1016/j.ibiomac.2011.05.009,Publishedonline FucoseͲcontainingsulfatedpolysaccharidesfrombrownseaweedinhibitproliferationof melanomacellsandinduceapoptosisbyactivationofcaspaseͲ3invitro AleMT,MaruyamaH,TamauchiH,MikkelsenJDandMeyerAS MarineDrugs(2011)ϵ͕ϮϲϬϱͲϮϭDOI:10.3390/mdϵϭϮϮϲϬϱ,Published online I II III IV Importantdeterminantsforfucoidanbioactivity:AcriticalreviewofstructureͲfunction relationsandextractionmethodsforfucoseͲcontainingsulfatedpolysaccharidesfrom brownseaweed AleMT,MikkelsenJDandMeyerAS MarineDrugs(2011)ϵ͕ϮϭϬϲͲϯϬ͕DOI:10.3390/md9ϭϬϮϭϬϲ, Publishedonline DesignedoptimizationofasingleͲstepextractionoffucoseͲcontainingsulfated polysaccharidesfromSargassumsp. AleMT,MikkelsenJDandMeyerAS JournalofAppliedPhycology(2011) DOI:10.1007/s1081Ͳ011Ͳ9690Ͳ3,Publishedonline FucoidanfromSargassumsp.andFucusvesiculosusreducescellviabilityoflungcarcinoma andmelanomacellsinvitroandactivatesnaturalkillercellsinmiceinvivo AleMT,MaruyamaH,TamauchiH,MikkelsenJDandMeyerAS InternationalJournalofBiologicalMacromolecules49(2011)331–336 DOI:10.1016/j.ibiomac.2011.05.009,Publishedonline FucoseͲcontainingsulfatedpolysaccharidesfrombrownseaweedinhibitproliferationof melanomacellsandinduceapoptosisbyactivationofcaspaseͲ3invitro AleMT,MaruyamaH,TamauchiH,MikkelsenJDandMeyerAS MarineDrugs(2011)ϵ͕ϮϲϬϱͲϮϭDOI:10.3390/mdϵϭϮϮϲϬϱ,Published online V DifferentialgrowthresponseofUlvalactucatoammoniumandnitrateassimilation AleMT,MikkelsenJDandMeyerAS JournalofAppliedPhycology(2011)23:345–351,Published V DifferentialgrowthresponseofUlvalactucatoammoniumandnitrateassimilation AleMT,MikkelsenJDandMeyerAS JournalofAppliedPhycology(2011)23:345–351,Published vii vii WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 9 10 10 dĂďůĞŽĨŽŶƚĞŶƚƐ dĂďůĞŽĨŽŶƚĞŶƚƐ TableofContents TableofContents Preface i Preface i Abstract .iii Abstract .iii DanskSammenfatning v DanskSammenfatning v Listofpublications .vii Listofpublications .vii TableofContents ix TableofContents ix 1Introduction 1 1Introduction 1 1.1Problemsstatement .3 1.1Problemsstatement .3 1.2Hypotheses 4 1.2Hypotheses 4 1.3Coreobjectivesandprojectphases 5 1.3Coreobjectivesandprojectphases 5 2Seaweedpotentials:ageneraloverview 9 2Seaweedpotentials:ageneraloverview 9 2.1Bioactiveseaweedcompounds:sulfatedpolysaccharides 10 2.1Bioactiveseaweedcompounds:sulfatedpolysaccharides 10 2.1.1FCSPsstructure 10 2.1.1FCSPsstructure 10 2.1.2AntiͲtumorbioactivityofFCSPs 13 2.1.2AntiͲtumorbioactivityofFCSPs 13 2.2FCSPsextractionandchemicalcomposition:pastandpresent 15 2.2FCSPsextractionandchemicalcomposition:pastandpresent 15 2.2.1FCSPscompositionstudies,1930–1950 17 2.2.1FCSPscompositionstudies,1930–1950 17 2.2.2LabͲscaleextractionoffucoidan,1950s 18 2.2.2LabͲscaleextractionoffucoidan,1950s 18 2.2.3FCSPsextraction,1970s–present 19 2.2.3FCSPsextraction,1970s–present 19 2.3Seaweedproductsandbiomasspotential 19 2.3Seaweedproductsandbiomasspotential 19 2.3.1Phycocolloids .20 2.3.1Phycocolloids .20 2.3.2Seaweedbiomassforbioenergyproduction 21 2.3.2Seaweedbiomassforbioenergyproduction 21 2.4Seaweedproduction .21 2.4Seaweedproduction .21 2.5Paper1:Importantdeterminantsforfucoidanbioactivity:acriticalreviewofstructureͲ functioncorrelationsandextractionmethodsforFCSPsfrombrownseaweeds 22 2.5Paper1:Importantdeterminantsforfucoidanbioactivity:acriticalreviewofstructureͲ functioncorrelationsandextractionmethodsforFCSPsfrombrownseaweeds 22 3ExtractionofFCSPs 4ϵ 3ExtractionofFCSPs 4ϵ 3.1Extractionmethods .4ϵ 3.1Extractionmethods .4ϵ 3.2SingleͲstepextractionofFCSPs .ϱϬ 3.2SingleͲstepextractionofFCSPs .ϱϬ ix ix WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 11 11 dĂďůĞŽĨŽŶƚĞŶƚƐ dĂďůĞŽĨŽŶƚĞŶƚƐ 3.2.1Relevance ϱϬ 3.2.1Relevance ϱϬ 3.2.2Hypothesesandobjectives ϱϬ 3.2.2Hypothesesandobjectives ϱϬ 3.2.3Resulthighlights ϱϭ 3.2.3Resulthighlights ϱϭ 3.2.4Considerationandjustification ϱϭ 3.2.4Considerationandjustification ϱϭ 3.3Paper2:DesignedoptimizationofsingleͲstepextractionofFCSPsfromSargassumsp. .5Ϯ 3.3Paper2:DesignedoptimizationofsingleͲstepextractionofFCSPsfromSargassumsp. .5Ϯ 4BioactivityofFCSPs 6ϱ 4BioactivityofFCSPs 6ϱ 4.1AntiͲtumoractivityofFCSPs 6ϲ 4.1AntiͲtumoractivityofFCSPs 6ϲ 4.2AntiͲproliferativeandimmuneͲresponseactivities 6ϲ 4.2AntiͲproliferativeandimmuneͲresponseactivities 6ϲ 4.2.1Relevance 6ϳ 4.2.1Relevance 6ϳ 4.2.2Hypothesesandobjectives 6ϳ 4.2.2Hypothesesandobjectives 6ϳ 4.2.3Resulthighlights 6ϴ 4.2.3Resulthighlights 6ϴ 4.2.4Considerationandjustification 6ϵ 4.2.4Considerationandjustification 6ϵ 4.3Paper3:FucoidanfromSargassumsp.andFucusvesiculosusreducescellviabilityoflung carcinomaandmelanomacellsinvitroandactivatesnaturalkillercellsinmiceinvivo ϳϬ 4.3Paper3:FucoidanfromSargassumsp.andFucusvesiculosusreducescellviabilityoflung carcinomaandmelanomacellsinvitroandactivatesnaturalkillercellsinmiceinvivo ϳϬ 4.4Paper4:Fucosecontainingsulfatedpolysaccharidesinhibitstheproliferationofmelanoma cellsandinducesapoptosisbyactivationofcaspaseͲ3invitro .ϳϬ 4.4Paper4:Fucosecontainingsulfatedpolysaccharidesinhibitstheproliferationofmelanoma cellsandinducesapoptosisbyactivationofcaspaseͲ3invitro .ϳϬ 5Seaweednutrientassimilationandgrowthresponse .9ϱ 5Seaweednutrientassimilationandgrowthresponse .9ϱ 5.1Evaluationofgrowthandassimilation 9ϲ 5.1Evaluationofgrowthandassimilation 9ϲ 5.2GrowthresponseofU.lactuca .9ϲ 5.2GrowthresponseofU.lactuca .9ϲ 5.2.1Relevance 9ϲ 5.2.1Relevance 9ϲ 5.2.2Hypothesesandobjectives 9ϳ 5.2.2Hypothesesandobjectives 9ϳ 5.2.3Resulthighlights 9ϴ 5.2.3Resulthighlights 9ϴ 5.2.4Considerationandjustification 9ϴ 5.2.4Considerationandjustification 9ϴ 5.3Paper5:DifferentialgrowthresponseofUlvalactucatoammoniumandnitrateassimilation 9ϵ 5.3Paper5:DifferentialgrowthresponseofUlvalactucatoammoniumandnitrateassimilation 9ϵ 6Conclusionsandperspectives 10ϵ 6Conclusionsandperspectives 10ϵ 6.1Futureperspectives 1ϭϮ 6.1Futureperspectives 1ϭϮ 7References 11ϱ 7References 11ϱ x x WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 12 12 ϭ/ŶƚƌŽĚƵĐƚŝŽŶ ϭ/ŶƚƌŽĚƵĐƚŝŽŶ 1Introduction 1Introduction Inthevastcoastalareasworldwide,seaweedhasplayedamajorroleinmaintainingtheecological Inthevastcoastalareasworldwide,seaweedhasplayedamajorroleinmaintainingtheecological balanceoftheenvironmentandmarinebioͲecosystem.Seaweeds,includingvariousbrown balanceoftheenvironmentandmarinebioͲecosystem.Seaweeds,includingvariousbrown seaweedssuchasUndariaandLaminariaspp.,arepartofthefoodcultureinmanyAsian seaweedssuchasUndariaandLaminariaspp.,arepartofthefoodcultureinmanyAsian countries,notablyJapan,thePhilippines,andKorea,andseaweedextractshavealsobeenusedas countries,notablyJapan,thePhilippines,andKorea,andseaweedextractshavealsobeenusedas remediesintraditionalmedicine.Inrecentyears,harvestingandmonoculturefarmingofcertain remediesintraditionalmedicine.Inrecentyears,harvestingandmonoculturefarmingofcertain seaweedspecieshavebecomeanimportantlivelihoodforfishermeninSoutheastAsiaowingto seaweedspecieshavebecomeanimportantlivelihoodforfishermeninSoutheastAsiaowingto theprogressivelyincreasingdemandforrawseaweedworldwide.Manyseaweedspecies,notably theprogressivelyincreasingdemandforrawseaweedworldwide.Manyseaweedspecies,notably beachͲcastseaweeds,stillneedtobeexaminedfortheircharacteristicsandproperties,whichwill beachͲcastseaweeds,stillneedtobeexaminedfortheircharacteristicsandproperties,whichwill determinetheircommercialapplications,beforetheycanberecognizedasimportant determinetheircommercialapplications,beforetheycanberecognizedasimportant commodities. commodities. Seaweedthatwashesuponthecoastlineoftengenerateswasteproblemsforpopulationsresiding Seaweedthatwashesuponthecoastlineoftengenerateswasteproblemsforpopulationsresiding seasideowingtomicrobialaccumulationandunpleasantodors.UtilizationofbeachͲcastseaweeds seasideowingtomicrobialaccumulationandunpleasantodors.UtilizationofbeachͲcastseaweeds suchasUlvasp.,Sargassumsp.,andothernuisanceseaweedspeciesforadvantageous suchasUlvasp.,Sargassumsp.,andothernuisanceseaweedspeciesforadvantageous applicationsmayalleviatetheseproblemsandcreatevaluableseaweedͲbasedproducts.Itis applicationsmayalleviatetheseproblemsandcreatevaluableseaweedͲbasedproducts.Itis widelyknownthatseaweedmaycontainuniquecomponentsthathavepotentialcommercial widelyknownthatseaweedmaycontainuniquecomponentsthathavepotentialcommercial applications;however,fewseaweedspeciesarecommerciallyutilizedandothersremain applications;however,fewseaweedspeciesarecommerciallyutilizedandothersremain unexploited.Someseaweedspeciescouldbepotentialsourcesoffunctionaldietaryfiberand unexploited.Someseaweedspeciescouldbepotentialsourcesoffunctionaldietaryfiberand polysaccharideswithbioactiveproperties(Lahaye1991;Takahashi1983).Recentdevelopmentsin polysaccharideswithbioactiveproperties(Lahaye1991;Takahashi1983).Recentdevelopmentsin seaweedutilizationincludeapplicationsinvolvingnaturallyderivedseaweedextractsandbioactive seaweedutilizationincludeapplicationsinvolvingnaturallyderivedseaweedextractsandbioactive compoundssuchasfucoseͲcontainingsulfatedpolysaccharides(FCSPs)and/orfucoidaninsome compoundssuchasfucoseͲcontainingsulfatedpolysaccharides(FCSPs)and/orfucoidaninsome cosmeticproductsandfoodsupplements. cosmeticproductsandfoodsupplements. Fucoidanisatermusedtodescribedaclassofsulfatedpolysaccharideextractedfromthe Fucoidanisatermusedtodescribedaclassofsulfatedpolysaccharideextractedfromthe seaweedclassPhaeophyceae,whichconsistalmostentirelyoffucoseandestersulfate(Percival seaweedclassPhaeophyceae,whichconsistalmostentirelyoffucoseandestersulfate(Percival andMcDowell1967).ThisFCSPprincipallyconsistsofabackboneof(1o3)Ͳand/or(1o4)Ͳlinked andMcDowell1967).ThisFCSPprincipallyconsistsofabackboneof(1o3)Ͳand/or(1o4)Ͳlinked ɲͲLͲfucopyranoseresiduesthatmaybeorganizedinstretchesof(1o3)ͲDͲfucanorwithalternating ɲͲLͲfucopyranoseresiduesthatmaybeorganizedinstretchesof(1o3)ͲDͲfucanorwithalternating D(1o3)ͲandD(1o4)ͲbondedLͲfucopyranoseresidues.TheLͲfucopyranoseresiduesmaybe D(1o3)ͲandD(1o4)ͲbondedLͲfucopyranoseresidues.TheLͲfucopyranoseresiduesmaybe substitutedwithsulfate(SO3Ͳ)onCͲ2orCͲ4(rarelyonCͲ3)singleLͲfucosylresiduesand/orshort substitutedwithsulfate(SO3Ͳ)onCͲ2orCͲ4(rarelyonCͲ3)singleLͲfucosylresiduesand/orshort fucoside(fucoͲoligosaccharide)sidechains.Ifpresent,thefucosidesidechainsareusuallyOͲ4, fucoside(fucoͲoligosaccharide)sidechains.Ifpresent,thefucosidesidechainsareusuallyOͲ4, linkedtotheDͲLͲfucopyranosebackboneresidues.Apartfromvariationsinthesulfatecontentand linkedtotheDͲLͲfucopyranosebackboneresidues.Apartfromvariationsinthesulfatecontentand 1 1 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 13 13 ϭ/ŶƚƌŽĚƵĐƚŝŽŶ ϭ/ŶƚƌŽĚƵĐƚŝŽŶ substitutions,themonosaccharidecompositionofFCSPsvariesamongdifferentspeciesofbrown substitutions,themonosaccharidecompositionofFCSPsvariesamongdifferentspeciesofbrown seaweeds.Hence,inadditiontofucose,differenttypesofFCSPsmayalsocontaingalactose, seaweeds.Hence,inadditiontofucose,differenttypesofFCSPsmayalsocontaingalactose, mannose,xylose,glucose,and/orglucuronicacid,usuallyinminoramounts(Percivaland mannose,xylose,glucose,and/orglucuronicacid,usuallyinminoramounts(Percivaland McDowell,1967;BilanandUsov,2008).ItappearsthatFCSPscoverabroaderrangeofcomplex McDowell,1967;BilanandUsov,2008).ItappearsthatFCSPscoverabroaderrangeofcomplex polysaccharidesthanthosehavingonlyfucanbackbones.Fucoidan—ormorecorrectly,FCSPs polysaccharidesthanthosehavingonlyfucanbackbones.Fucoidan—ormorecorrectly,FCSPs extractedfrombrownseaweedslikeSargassumsp.andFucusvesiculosus—weredocumentedto extractedfrombrownseaweedslikeSargassumsp.andFucusvesiculosus—weredocumentedto haveawiderangeofbiologicalactivitiesincludinganticoagulant(Nardellaetal.,1996);antiͲ haveawiderangeofbiologicalactivitiesincludinganticoagulant(Nardellaetal.,1996);antiͲ inflammatory(Blondinetal.,1994);antiviral(Adhikarietal.,2006;Trincheroetal.,2009);and, inflammatory(Blondinetal.,1994);antiviral(Adhikarietal.,2006;Trincheroetal.,2009);and, notably,antiͲtumoraleffects(Zhuangetal.,1995;Aleetal.,2011). notably,antiͲtumoraleffects(Zhuangetal.,1995;Aleetal.,2011). TypicalextractionofFCSPsfrombrownseaweedinvolvesaharshprocessingconditionandseveral TypicalextractionofFCSPsfrombrownseaweedinvolvesaharshprocessingconditionandseveral purificationsteps.PurificationofFCSPsbycolumnchromatographywaseffectiveforisolating purificationsteps.PurificationofFCSPsbycolumnchromatographywaseffectiveforisolating polysaccharidefractionsastheyhadhigherfucosecontentsthanthoseFCSPsobtainedusing polysaccharidefractionsastheyhadhigherfucosecontentsthanthoseFCSPsobtainedusing minimalprocessingsteps(Lietal.,2006;Aleetal.,2011).TheconditionsforobtainingFCSPsfrom minimalprocessingsteps(Lietal.,2006;Aleetal.,2011).TheconditionsforobtainingFCSPsfrom brownseaweedgenerallyconsistofmultiple,long,repetitivestepsusingacid(e.g.,HCl)andother brownseaweedgenerallyconsistofmultiple,long,repetitivestepsusingacid(e.g.,HCl)andother solventsatelevatedtemperatures(Chizhovetal.,1999;Bilanetal.,2002).Theinfluenceofthe solventsatelevatedtemperatures(Chizhovetal.,1999;Bilanetal.,2002).Theinfluenceofthe extractionmethodsonthechemicalnatureofsulfatedpolysaccharidehasalreadybeen extractionmethodsonthechemicalnatureofsulfatedpolysaccharidehasalreadybeen demonstratedbyBlacketal.(1952).Ontheotherhand,theFCSPyieldofF.evanescensextracted demonstratedbyBlacketal.(1952).Ontheotherhand,theFCSPyieldofF.evanescensextracted 4timesusing2%CaCl2solutionat85°Cfor5hwas12.9%dryweight(DW)(Bilanetal.,2002), 4timesusing2%CaCl2solutionat85°Cfor5hwas12.9%dryweight(DW)(Bilanetal.,2002), whileextractionat25°Cusing0.4%HClfor5hyielded12.0%DW(Zvyagintsevaetal.,1999). whileextractionat25°Cusing0.4%HClfor5hyielded12.0%DW(Zvyagintsevaetal.,1999). DespitetheexistenceofearlyseminalstudiesaboutFCSPorfucoidanextraction,thereisonly DespitetheexistenceofearlyseminalstudiesaboutFCSPorfucoidanextraction,thereisonly limitedevidenceabouttheinfluencesandapparentlycomplexinteractionsofextraction limitedevidenceabouttheinfluencesandapparentlycomplexinteractionsofextraction parameters,suchasacidsolvents,temperature,andtime,onFCSPyieldandcomposition.FCSP parameters,suchasacidsolvents,temperature,andtime,onFCSPyieldandcomposition.FCSP extractionprocedureswithfewerstepsaremilderonthebrownseaweedsthanareotherthough extractionprocedureswithfewerstepsaremilderonthebrownseaweedsthanareotherthough theymayyieldaheterogeneoussulfatedpolysaccharideproduct.Nevertheless,fewersteps theymayyieldaheterogeneoussulfatedpolysaccharideproduct.Nevertheless,fewersteps extractionapproachminimizesthestructuralalterationofalgalͲsulfatedpolysaccharidesand, extractionapproachminimizesthestructuralalterationofalgalͲsulfatedpolysaccharidesand, thereby,maintainsthenaturalbioactivecharacteristicsofFCSPs. thereby,maintainsthenaturalbioactivecharacteristicsofFCSPs. AlthoughdifferentlyextractedandpurifiedFCSPshavebeenreportedtoexertbioactivity AlthoughdifferentlyextractedandpurifiedFCSPshavebeenreportedtoexertbioactivity (Holtkampetal.,2009),unfractionatedFCSPshasalsobeenfoundtoreducecellproliferationof (Holtkampetal.,2009),unfractionatedFCSPshasalsobeenfoundtoreducecellproliferationof lungcarcinomaandmelanomacells,exertimmunopotentiatingeffectsintumorͲbearinganimals, lungcarcinomaandmelanomacells,exertimmunopotentiatingeffectsintumorͲbearinganimals, andtoactivatenaturalkiller(NK)cellsinmice,leadingtoantiͲtumoractivityefficacy(Takahashi, andtoactivatenaturalkiller(NK)cellsinmice,leadingtoantiͲtumoractivityefficacy(Takahashi, 1983;Aleetal.,2011;Foleyetal.,2011).Kimetal.(2010)appliedacrudepolysaccharide 1983;Aleetal.,2011;Foleyetal.,2011).Kimetal.(2010)appliedacrudepolysaccharide composedpredominantlyofsulfatedfucosefromF.vesiculosustohumancoloncancercellsin composedpredominantlyofsulfatedfucosefromF.vesiculosustohumancoloncancercellsin 2 2 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 14 14 ϭ/ŶƚƌŽĚƵĐƚŝŽŶ ϭ/ŶƚƌŽĚƵĐƚŝŽŶ vitroandconcludedthatthispolysaccharidefrombrownseaweedinducesapoptosis.Moreover, vitroandconcludedthatthispolysaccharidefrombrownseaweedinducesapoptosis.Moreover, commerciallyavailablecrudefucoidan(SigmaInc.)wastestedonhumanlymphomaHSͲSultancell commerciallyavailablecrudefucoidan(SigmaInc.)wastestedonhumanlymphomaHSͲSultancell linesandwasfoundtoinhibitproliferationandinduceapoptosisbyactivatingcaspaseͲ3(Aisaet linesandwasfoundtoinhibitproliferationandinduceapoptosisbyactivatingcaspaseͲ3(Aisaet al.,2005).ItwasreportedrecentlythatFCSPsfromSargassumsp.andcrudefucoidan(SigmaInc.) al.,2005).ItwasreportedrecentlythatFCSPsfromSargassumsp.andcrudefucoidan(SigmaInc.) fromF.vesiculosusinducedapoptosisinmelanomacells(Aleetal.,2011). fromF.vesiculosusinducedapoptosisinmelanomacells(Aleetal.,2011). Besidesthebioactivecompounds,seaweedsalsopossessothervaluablecomponents,suchas Besidesthebioactivecompounds,seaweedsalsopossessothervaluablecomponents,suchas solubledietaryfibersandcarbohydratesforhydrocolloidapplications.Thegreenseaweed(Ulva solubledietaryfibersandcarbohydratesforhydrocolloidapplications.Thegreenseaweed(Ulva species)areparticularlyrichinrarecellwallpolysaccharidesandhavebeenproposedasbeing species)areparticularlyrichinrarecellwallpolysaccharidesandhavebeenproposedasbeing importantsourcesofdietaryfiber,mainlysolublefiber(Lahaye1991;LahayeandAxelos1993). importantsourcesofdietaryfiber,mainlysolublefiber(Lahaye1991;LahayeandAxelos1993). Furthermore,U.lactucaisalsoagoodsourceofvitaminsA,B2,B12,andCandisrichinJͲ Furthermore,U.lactucaisalsoagoodsourceofvitaminsA,B2,B12,andCandisrichinJͲ tocopherol(AbdElͲBakyetal.,2008;Ortizetal.,2006).Ithasbeenshownthatseaweed,notably tocopherol(AbdElͲBakyetal.,2008;Ortizetal.,2006).Ithasbeenshownthatseaweed,notably U.lactuca,wassuitableforpropagationundercontrolledconditions(VermaatandSandͲJensen, U.lactuca,wassuitableforpropagationundercontrolledconditions(VermaatandSandͲJensen, 1987;Lee,2000;Satoetal.,2006).Forthisreason,U.lactucacultivationintanksforeithercrude 1987;Lee,2000;Satoetal.,2006).Forthisreason,U.lactucacultivationintanksforeithercrude biomassproductionforbioenergyorfortheproductionofbiologicallyactivecompoundsis biomassproductionforbioenergyorfortheproductionofbiologicallyactivecompoundsis currentlyreceivingincreasedattention(HiraokaandOka,2008).However,amajorprerequisitefor currentlyreceivingincreasedattention(HiraokaandOka,2008).However,amajorprerequisitefor thesuccessfulexploitationofcultivatedU.lactucaforcommercialapplicationsisoptimizationof thesuccessfulexploitationofcultivatedU.lactucaforcommercialapplicationsisoptimizationof growthratesandyields.Thisinturnrequiresbothanunderstandingoftheinfluenceofdifferent growthratesandyields.Thisinturnrequiresbothanunderstandingoftheinfluenceofdifferent nutrientsonthegrowthresponseandaprecisemethodologytomeasurethegrowth.Nuisance nutrientsonthegrowthresponseandaprecisemethodologytomeasurethegrowth.Nuisance greenseaweedslikeU.lactucashowedbioremediationabilityinnitrogenͲandphosphateͲrich greenseaweedslikeU.lactucashowedbioremediationabilityinnitrogenͲandphosphateͲrich wastewater(Copertinoetal.,2008).Nevertheless,limitedinformationisknownaboutthegrowth wastewater(Copertinoetal.,2008).Nevertheless,limitedinformationisknownaboutthegrowth responseandnutrientuptakeassimilationofU.lactucawhenexposetocombineconcentrationsof responseandnutrientuptakeassimilationofU.lactucawhenexposetocombineconcentrationsof ammoniumandnitrate. ammoniumandnitrate. ThisPhDthesisdeliversthemostrecentstudyinvolvingFCSPsextractiontechnologyandevaluates ThisPhDthesisdeliversthemostrecentstudyinvolvingFCSPsextractiontechnologyandevaluates FCSPsbiologicalactivity.AsingleͲstepextractionmethodfortheremovalofFCSPsfromSargassum FCSPsbiologicalactivity.AsingleͲstepextractionmethodfortheremovalofFCSPsfromSargassum sp.wasdevelopedinthecourseofthisstudy.FCSPsbioactivitystudieshavebeenconductedin sp.wasdevelopedinthecourseofthisstudy.FCSPsbioactivitystudieshavebeenconductedin lungandskincancercellmodelsinvitroandimmuneresponseactivityinvivo.Inadditionto lungandskincancercellmodelsinvitroandimmuneresponseactivityinvivo.Inadditionto extractionandbioactivitystudies,evaluationofthegrowthresponseofU.lactucatonutrients extractionandbioactivitystudies,evaluationofthegrowthresponseofU.lactucatonutrients suchasNH4andNO3wasalsoperformed. suchasNH4andNO3wasalsoperformed. 1.1Problemsstatement 1.1Problemsstatement PurifiedfractionsfromFCSPsarecommonlyusedforstructuralandbioactivityanalysis.These PurifiedfractionsfromFCSPsarecommonlyusedforstructuralandbioactivityanalysis.These samplescontainhighfucoseͲsulfatelevelsandarefreefromothercontaminantsaccharide samplescontainhighfucoseͲsulfatelevelsandarefreefromothercontaminantsaccharide 3 3 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 15 15 ϭ/ŶƚƌŽĚƵĐƚŝŽŶ ϭ/ŶƚƌŽĚƵĐƚŝŽŶ residues.Hence,manyseminalstudieshaveshownthatpurifiedfucoidanhashighbioactivityasa residues.Hence,manyseminalstudieshaveshownthatpurifiedfucoidanhashighbioactivityasa resultofmultiͲstepextractionandfurtherpurificationandfractionation. resultofmultiͲstepextractionandfurtherpurificationandfractionation. CananunpurifiedFCSPsproductextractedusingminimalstepsexertbioactivity? CananunpurifiedFCSPsproductextractedusingminimalstepsexertbioactivity? WhatistheeffectofunpurifiedFCSPsagainstcertaincancercelllinesandwhatisits WhatistheeffectofunpurifiedFCSPsagainstcertaincancercelllinesandwhatisits influenceonimmuneresponseactivity? influenceonimmuneresponseactivity? ThepresentextractiontechnologyusingaqueousͲalkalisolutionordiluteacidatambientor ThepresentextractiontechnologyusingaqueousͲalkalisolutionordiluteacidatambientor slightlyelevatedtemperaturehasalwaysbeenthemostconvenientmethodtoproducedFCSPs. slightlyelevatedtemperaturehasalwaysbeenthemostconvenientmethodtoproducedFCSPs. Woulddifferentextractionparameters,i.e.,acid,temperature,andtime,influencethe Woulddifferentextractionparameters,i.e.,acid,temperature,andtime,influencethe structuralfeaturesandchemicalnatureofFCSPs? structuralfeaturesandchemicalnatureofFCSPs? Whataretheeffectsoftheinteractionsofdifferentextractiontreatments? Whataretheeffectsoftheinteractionsofdifferentextractiontreatments? Exploitationofseaweedresourceshasrecentlyreceivedspecialattentionforitspotentialforboth Exploitationofseaweedresourceshasrecentlyreceivedspecialattentionforitspotentialforboth theproductionofbioactivecompoundsandasabiomasssourceforbioenergyproduction.To theproductionofbioactivecompoundsandasabiomasssourceforbioenergyproduction.To successfullyexploitseaweedforcommercialapplications,thegrowthrateandyieldsmustbe successfullyexploitseaweedforcommercialapplications,thegrowthrateandyieldsmustbe optimized.Hence,aprecisemonitoringtechnologytoevaluatetheseaweedgrowthresponseis optimized.Hence,aprecisemonitoringtechnologytoevaluatetheseaweedgrowthresponseis required. required. WhatarethedifferentmonitoringtechniquesthatareusedtoevaluatetheU.lactuca WhatarethedifferentmonitoringtechniquesthatareusedtoevaluatetheU.lactuca growthresponsetonutrientassimilation?Howdoesitaffectthemeasurements’ growthresponsetonutrientassimilation?Howdoesitaffectthemeasurements’ precision? precision? HowisthegrowthofU.lactucainfluencedbytheassimilationofdifferentnutrients(NH4 HowisthegrowthofU.lactucainfluencedbytheassimilationofdifferentnutrients(NH4 andNO3)? andNO3)? 1.2Hypotheses 1.2Hypotheses Nuisancemarineseaweedthathaswasheduponthecoastlinesisapotentialstartingmaterialfor Nuisancemarineseaweedthathaswasheduponthecoastlinesisapotentialstartingmaterialfor producingbioactivecompoundslikefucoidanorFCSPs.Thepreciseassessmentofseaweedgrowth producingbioactivecompoundslikefucoidanorFCSPs.Thepreciseassessmentofseaweedgrowth iscrucialinourunderstandingofseaweednutrientassimilationmechanisms.Inthisthesis,some iscrucialinourunderstandingofseaweednutrientassimilationmechanisms.Inthisthesis,some specifichypotheseswereoutlined: specifichypotheseswereoutlined: MinimalextractionstepofFCSPswillpreservethepolysaccharides’structuralintegrity MinimalextractionstepofFCSPswillpreservethepolysaccharides’structuralintegrity and,thus,increasetheyieldandimproveitsbiologicalproperties and,thus,increasetheyieldandimproveitsbiologicalproperties 4 4 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 16 16 ϭ/ŶƚƌŽĚƵĐƚŝŽŶ ϭ/ŶƚƌŽĚƵĐƚŝŽŶ FCSPproductsfromasingleͲstepextractionprocess(i.e.,crudefucoidan)frombrown FCSPproductsfromasingleͲstepextractionprocess(i.e.,crudefucoidan)frombrown seaweedcanexertbioactivityagainstcertaintypesofcancercelllines(e.g.,byinducing seaweedcanexertbioactivityagainstcertaintypesofcancercelllines(e.g.,byinducing apoptosis)andcanpromoteimmuneresponses apoptosis)andcanpromoteimmuneresponses ClassicalextractionofFCSPsinvolvinglong,repetitive,multiͲstepacidandalkaline ClassicalextractionofFCSPsinvolvinglong,repetitive,multiͲstepacidandalkaline treatmentscanbedetrimentaltoitsstructuralmakeup,yield,andcompositional treatmentscanbedetrimentaltoitsstructuralmakeup,yield,andcompositional attributesand,thus,mayinfluenceitsbioactiveproperties attributesand,thus,mayinfluenceitsbioactiveproperties Toexploitmarineseaweedforcommercialapplications,growthmonitoringiscrucialto Toexploitmarineseaweedforcommercialapplications,growthmonitoringiscrucialto theaccurateevaluationofdifferentialgrowthresponsesandnutrient(NH4orNaNO3) theaccurateevaluationofdifferentialgrowthresponsesandnutrient(NH4orNaNO3) assimilations assimilations 1.3Coreobjectivesandprojectphases 1.3Coreobjectivesandprojectphases Seaweedresourcesgenerallycoverwidepotentialapplicationsfordifferentindustriesincluding Seaweedresourcesgenerallycoverwidepotentialapplicationsfordifferentindustriesincluding foodandnutrition,pharmaceuticals,cosmetics,andbioenergy.Therefore,theirstudyrequires foodandnutrition,pharmaceuticals,cosmetics,andbioenergy.Therefore,theirstudyrequires extensiveinͲdepthresearchinvolvinganinterdisciplinaryapproachandaconsiderableamountof extensiveinͲdepthresearchinvolvinganinterdisciplinaryapproachandaconsiderableamountof timetoaccomplishcertainachievableobjectives.ThisPhDstudynarrowsthesubjectsintomore timetoaccomplishcertainachievableobjectives.ThisPhDstudynarrowsthesubjectsintomore focusedareaswithveryrealisticaimsorspecificobjectivestoproducenoveltechnologywhile focusedareaswithveryrealisticaimsorspecificobjectivestoproducenoveltechnologywhile attainingbasicscientificunderstanding. attainingbasicscientificunderstanding. Totestthehypotheses,variousspecificobjectiveswereappliedinthisstudy.Thecoreobjectives Totestthehypotheses,variousspecificobjectiveswereappliedinthisstudy.Thecoreobjectives wereprimarilyconcentratedontheinvestigationofdifferentFCSPsextractionmethodsand wereprimarilyconcentratedontheinvestigationofdifferentFCSPsextractionmethodsand developinginnovativeFCSPsextractiontechnology;thepotentialofseaweedsasasourceof developinginnovativeFCSPsextractiontechnology;thepotentialofseaweedsasasourceof bioactivecompounds,notablyFCSPs;andtoassessthebioactivityandmechanismofFCPS bioactivecompounds,notablyFCSPs;andtoassessthebioactivityandmechanismofFCPS productsagainstcertaintypesofcancer.Furthermore,weevaluateseaweeds’differentialgrowth productsagainstcertaintypesofcancer.Furthermore,weevaluateseaweeds’differentialgrowth responsestonutrientassimilation.Alongwiththecoreobjectives,thespecificaimsofthestudy responsestonutrientassimilation.Alongwiththecoreobjectives,thespecificaimsofthestudy wereasfollows: wereasfollows: DevelopmentofanewprocessforproducingbioactivecompoundlikefucoseͲcontaining DevelopmentofanewprocessforproducingbioactivecompoundlikefucoseͲcontaining sulfatedpolysaccharidesfrombrownseaweedbyoptimizeddesignedextraction sulfatedpolysaccharidesfrombrownseaweedbyoptimizeddesignedextraction parametersusingofstateͲofͲtheͲartanalyticalmethodsandquantificationanalyses parametersusingofstateͲofͲtheͲartanalyticalmethodsandquantificationanalyses InvestigationofthebioactivityofsingleͲstepextractedunpurifiedFCSPsproducts(i.e., InvestigationofthebioactivityofsingleͲstepextractedunpurifiedFCSPsproducts(i.e., crudefucoidan)incancercelllinesusinginvitroandinvivoexperiments crudefucoidan)incancercelllinesusinginvitroandinvivoexperiments Evaluationoftheseaweedgrowthresponsetonutrientassimilation(i.e.,NH4andNO3) Evaluationoftheseaweedgrowthresponsetonutrientassimilation(i.e.,NH4andNO3) 5 5 WŚdŚĞƐŝƐϮϬϭ2 WŚdŚĞƐŝƐϮϬϭ2 17 17 ϭ/ŶƚƌŽĚƵĐƚŝŽŶ ϭ/ŶƚƌŽĚƵĐƚŝŽŶ Toensurethattheworkwasconductedintherightdirection,workloadswereorganizedand Toensurethattheworkwasconductedintherightdirection,workloadswereorganizedand narroweddowntospecificscopesinanumberofprojectphases: narroweddowntospecificscopesinanumberofprojectphases: Phase1:Surveyofseaweedbioactivecompoundstructureandbioactivity Phase1:Surveyofseaweedbioactivecompoundstructureandbioactivity Aliteratureresearchandgatheringofreviewarticleswasconductedwiththeaimofgainingan Aliteratureresearchandgatheringofreviewarticleswasconductedwiththeaimofgainingan overallperspectiveofthesubject.Specialfocuswasplacedontheavailabletechnicalandscientific overallperspectiveofthesubject.Specialfocuswasplacedontheavailabletechnicalandscientific informationregardingbioactivecompounds’structuralmakeupandcomposition,theinfluenceof informationregardingbioactivecompounds’structuralmakeupandcomposition,theinfluenceof theextractionprocess,andfactorsaffectingbioactivity. theextractionprocess,andfactorsaffectingbioactivity. Phase2:Optimizedextractionandhydrolysisofseaweedpolysaccharides(i.e.,FCSPs) Phase2:Optimizedextractionandhydrolysisofseaweedpolysaccharides(i.e.,FCSPs) FucoidancanbeobtainedbyextractionfrombrownseaweedslikeSargassumsp.Thetypical FucoidancanbeobtainedbyextractionfrombrownseaweedslikeSargassumsp.Thetypical extractionofFCSPsinvolvesmultiͲstepextractionusingdifferenttemperaturesandacidand extractionofFCSPsinvolvesmultiͲstepextractionusingdifferenttemperaturesandacidand alkalineconcentrationsinanextendedperiod.Inthisstudy,itwashypothesizedthatharsh alkalineconcentrationsinanextendedperiod.Inthisstudy,itwashypothesizedthatharsh extractiontreatmentisdetrimentaltotheintegrityofthechemicalnatureofthese extractiontreatmentisdetrimentaltotheintegrityofthechemicalnatureofthese polysaccharides;thus,preservationofitsstructureduringextractioncanonlybedoneusingamild polysaccharides;thus,preservationofitsstructureduringextractioncanonlybedoneusingamild extractiontechnique.Themainobjectiveofthisprojectwastodesignanoptimizedextractionof extractiontechnique.Themainobjectiveofthisprojectwastodesignanoptimizedextractionof FCSPsusingaminimalͲstepmethodcomparedtoexistingclassicalextractionmethodswithspecial FCSPsusingaminimalͲstepmethodcomparedtoexistingclassicalextractionmethodswithspecial attentionplacedontheinfluenceonchemicalcomposition. attentionplacedontheinfluenceonchemicalcomposition. Phase3:AssessmentofFCSPbioactivity Phase3:AssessmentofFCSPbioactivity FSCPsareknowntopossessbioactivepropertiessuchasantiͲtumoractivity,apoptosisinductionin FSCPsareknowntopossessbioactivepropertiessuchasantiͲtumoractivity,apoptosisinductionin cancercells,andimmunepotentiation.TheefficacyofFCSPsderivedfrombrownseaweedremains cancercells,andimmunepotentiation.TheefficacyofFCSPsderivedfrombrownseaweedremains amatterofdebate;nevertheless,severalreportsattributeitsefficacytoitsstructuralmakeup, amatterofdebate;nevertheless,severalreportsattributeitsefficacytoitsstructuralmakeup, substitutions,andcontentofsulfate.ItwashypothesizedthatmildextractedFCSPsmustpossess substitutions,andcontentofsulfate.ItwashypothesizedthatmildextractedFCSPsmustpossess highbioactivepropertiesbecausetheirstructuralmakeupremainsintact.Theobjectiveofthis highbioactivepropertiesbecausetheirstructuralmakeupremainsintact.Theobjectiveofthis studywastoexaminethedifferentcontributingfactorsinfluencingFCSPbioactivityonsome studywastoexaminethedifferentcontributingfactorsinfluencingFCSPbioactivityonsome cancercelllines.ThebioactivitiesofFCSPsextractedfromnuisanceseaweedwereevaluatedusing cancercelllines.ThebioactivitiesofFCSPsextractedfromnuisanceseaweedwereevaluatedusing cancercelllinesinvitroandinvivoexperiments. cancercelllinesinvitroandinvivoexperiments. Phase4:Seaweedgrowthresponsetonutrientassimilation Phase4:Seaweedgrowthresponsetonutrientassimilation Theproliferationofseaweedismostlyinfluencedbyphotosynthesisandavailablenutrientsthat Theproliferationofseaweedismostlyinfluencedbyphotosynthesisandavailablenutrientsthat canbeutilizedandassimilated.Seaweedgrowthistantamounttoincreasedbiomassproduction. canbeutilizedandassimilated.Seaweedgrowthistantamounttoincreasedbiomassproduction. Seaweedcanpotentiallybeutilizedfortheproductionofbioactivecompoundsandhealthy Seaweedcanpotentiallybeutilizedfortheproductionofbioactivecompoundsandhealthy 6 6 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 18 18 ϭ/ŶƚƌŽĚƵĐƚŝŽŶ 1 ϭ/ŶƚƌŽĚƵĐƚŝŽŶ 1 components.Monitoringtheseaweedgrowthandnutrientuptakeisacrucialstepforevaluating components.Monitoringtheseaweedgrowthandnutrientuptakeisacrucialstepforevaluating theseaweedgrowthrate,nutrientuptakerate,andbiomassyield.Thus,developingamethodto theseaweedgrowthrate,nutrientuptakerate,andbiomassyield.Thus,developingamethodto properlymonitorseaweedgrowthwillcontributetoourunderstandingoftheseaweedgrowth properlymonitorseaweedgrowthwillcontributetoourunderstandingoftheseaweedgrowth responsetonutrientandassimilationpatterns.TheobjectiveofthisstudyistoexaminetheU. responsetonutrientandassimilationpatterns.TheobjectiveofthisstudyistoexaminetheU. lactucagrowthresponsetonutrients,i.e.,NH4orNaNO3,withthespecialaimofdevelopinga lactucagrowthresponsetonutrients,i.e.,NH4orNaNO3,withthespecialaimofdevelopinga growthmonitoringtechniquetoaccuratelyevaluatethedifferentialgrowthresponseandnutrient growthmonitoringtechniquetoaccuratelyevaluatethedifferentialgrowthresponseandnutrient assimilationandtoanalyzedifferentbiomassaccumulationͲrelatedparameters. assimilationandtoanalyzedifferentbiomassaccumulationͲrelatedparameters. 7 7 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 19 19 20 20 Seaweed Ɖotentials Seaweed Ɖotentials 2Seaweedpotentials:ageneraloverview 2Seaweedpotentials:ageneraloverview Marineseaweedsareclassifiedaccordingtotheirmorphologyandtaxonomiccharacteristicinto3 Marineseaweedsareclassifiedaccordingtotheirmorphologyandtaxonomiccharacteristicinto3 groups:thegreen(Chlorophyta),red(Rhodophyta),andbrownalgae(Phaeophyta).Thegreen groups:thegreen(Chlorophyta),red(Rhodophyta),andbrownalgae(Phaeophyta).Thegreen algaearedistributedworldwide(Lahaye&Robic,2007),andthemostcommongreenseaweed algaearedistributedworldwide(Lahaye&Robic,2007),andthemostcommongreenseaweed speciesinthetemperatezonesisUlvalactuca(Fig.2.1).Itisharvestednaturallyorcultivatedin speciesinthetemperatezonesisUlvalactuca(Fig.2.1).Itisharvestednaturallyorcultivatedin tanksforfoodconsumptionorbiomassproductionforbioenergy(Lahaye&Robic,2007).Natural tanksforfoodconsumptionorbiomassproductionforbioenergy(Lahaye&Robic,2007).Natural habitatsofredseaweedarefoundatintertidalandsubtidaldepths.Inrecentyears,fishermenin habitatsofredseaweedarefoundatintertidalandsubtidaldepths.Inrecentyears,fishermenin theSoutheastAsiancountriesengagedinthefarmingoftheredseaweedsKappaphycusand theSoutheastAsiancountriesengagedinthefarmingoftheredseaweedsKappaphycusand Eucheuma(Fig.2.1)fortheproductionofcarrageenan.CarrageenanisagelͲforming,viscosifying Eucheuma(Fig.2.1)fortheproductionofcarrageenan.CarrageenanisagelͲforming,viscosifying polysaccharidethatiscommerciallyexploitedforfoodproductsandcosmeticproducts(DeReiter polysaccharidethatiscommerciallyexploitedforfoodproductsandcosmeticproducts(DeReiter andRudolph,1997).Thebrownseaweedsareusuallyfoundintidalsplashzonesorrockpools,and andRudolph,1997).Thebrownseaweedsareusuallyfoundintidalsplashzonesorrockpools,and certainspeciessuchasSargassumarefoundfloatingontheshorelines(Fig.2.1).Brownalgaeare certainspeciessuchasSargassumarefoundfloatingontheshorelines(Fig.2.1).Brownalgaeare typicallyusedfortheproductionofalginate,whichiscommerciallyusedasaningredientfor typicallyusedfortheproductionofalginate,whichiscommerciallyusedasaningredientfor differentindustrial,biotechnology,andfoodapplications.FCSPsfrombrownseaweed,notably differentindustrial,biotechnology,andfoodapplications.FCSPsfrombrownseaweed,notably fucoidan,havebeenknowntoexertbioactiveproperties. fucoidan,havebeenknowntoexertbioactiveproperties. Fig.2.1Photographsofdifferentseaweedspeciesthatareutilizedfordifferentcommercial applications.a.Ulvalactucaasbulkingagentforfeeds.b.Kappaphycussp.forcarrageenan production.c.Sargassumsp.asapotentialsourceofbioactivefucoseͲcontainingsulfated polysaccharides,particularlyfucoidancompound.(Source:www.algaebase.org) Fig.2.1Photographsofdifferentseaweedspeciesthatareutilizedfordifferentcommercial applications.a.Ulvalactucaasbulkingagentforfeeds.b.Kappaphycussp.forcarrageenan production.c.Sargassumsp.asapotentialsourceofbioactivefucoseͲcontainingsulfated polysaccharides,particularlyfucoidancompound.(Source:www.algaebase.org) SeveralstudieshaveindicatedthatFCSPsfrombrownseaweedsarehighlypotentantiͲcancer SeveralstudieshaveindicatedthatFCSPsfrombrownseaweedsarehighlypotentantiͲcancer agents,tumorcellgrowthinhibitors,andimmunesystemstrengtheners(Jiaoetal.,2011;Pereira agents,tumorcellgrowthinhibitors,andimmunesystemstrengtheners(Jiaoetal.,2011;Pereira 9 WŚdŚĞƐŝƐϮϬϭ2 9 WŚdŚĞƐŝƐϮϬϭ2 21 21 Ϯ^ĞĂǁĞĞĚƉŽƚĞŶƚŝĂůƐ Ϯ^ĞĂǁĞĞĚƉŽƚĞŶƚŝĂůƐ etal.,1999).IthasbeensuggestedthatthelowerbreastcancerandobesityratesinJapanmaybe etal.,1999).IthasbeensuggestedthatthelowerbreastcancerandobesityratesinJapanmaybe relatedtoregularintakeofbrownseaweed(Teas1983).Brownseaweedsareveryabundantand relatedtoregularintakeofbrownseaweed(Teas1983).Brownseaweedsareveryabundantand constituteunexploitedresourcesworldwide,thusmakingthemviablesourcesofpolysaccharides, constituteunexploitedresourcesworldwide,thusmakingthemviablesourcesofpolysaccharides, especiallythosewithbioactivepropertiessuchasfucoidanorotherFCSPs.Nevertheless,thered especiallythosewithbioactivepropertiessuchasfucoidanorotherFCSPs.Nevertheless,thered andgreenseaweedsarealsogoodsourcesoffunctionalfoodingredients,includingphycocolloids andgreenseaweedsarealsogoodsourcesoffunctionalfoodingredients,includingphycocolloids anddietaryfibers. anddietaryfibers. 2.1Bioactiveseaweedcompounds:sulfatedpolysaccharides 2.1Bioactiveseaweedcompounds:sulfatedpolysaccharides Sulfatedpolysaccharidesareafamilyofcompoundscontainingestersulfategroupsintheirsugar Sulfatedpolysaccharidesareafamilyofcompoundscontainingestersulfategroupsintheirsugar residues.ThesepolysaccharidesarecommonlyfoundinthemarinealgalgroupsPhaeophyta, residues.ThesepolysaccharidesarecommonlyfoundinthemarinealgalgroupsPhaeophyta, Rhodophyta,andChlorophyta.Fucansisthegeneraltermforsulfatedpolysaccharidesthatare Rhodophyta,andChlorophyta.Fucansisthegeneraltermforsulfatedpolysaccharidesthatare presentintheclassPhaeophyta,whichincludesFCSPs.Thesulfatedpolysaccharidesintheclass presentintheclassPhaeophyta,whichincludesFCSPs.Thesulfatedpolysaccharidesintheclass Rhodophytaaregalactansconsistingentirelyofgalactoseormodifiedgalactoseunitssuchasagar Rhodophytaaregalactansconsistingentirelyofgalactoseormodifiedgalactoseunitssuchasagar andcarrageenans.ThemajorpolysaccharidesintheclassChlorophytaarepolydisperse andcarrageenans.ThemajorpolysaccharidesintheclassChlorophytaarepolydisperse heteropolysaccharidesknownasulvans. heteropolysaccharidesknownasulvans. Ulva,thegreenseaweedspeciesoftheclassChlorophyta,areparticularlyrichinrarecellwall Ulva,thegreenseaweedspeciesoftheclassChlorophyta,areparticularlyrichinrarecellwall polysaccharidesandhavebeenproposedasbeinganimportantsourceofdietaryfiber,mainly polysaccharidesandhavebeenproposedasbeinganimportantsourceofdietaryfiber,mainly solublefiber,whichcouldbeapotentialprebioticsubstrate(Lahaye1991;LahayeandAxelos solublefiber,whichcouldbeapotentialprebioticsubstrate(Lahaye1991;LahayeandAxelos 1993).Brownseaweedisasourceofauniquecompounds,notablyFCSPs,whichhaveshownhigh 1993).Brownseaweedisasourceofauniquecompounds,notablyFCSPs,whichhaveshownhigh potencyagainstcertaincancersinsomeinvitrostudiesandexhibitedimmuneresponseactivityin potencyagainstcertaincancersinsomeinvitrostudiesandexhibitedimmuneresponseactivityin vivo(Cumashietal.,2007;Maruyamaetal.,2003).Redseaweed,ontheotherhand,isknownfor vivo(Cumashietal.,2007;Maruyamaetal.,2003).Redseaweed,ontheotherhand,isknownfor itshydrocolloidcharacteristics;however,publishedreportshaveshownthatsulfated itshydrocolloidcharacteristics;however,publishedreportshaveshownthatsulfated polysaccharidesfromredseaweedalsopossessedsomebioactiveproperties(Pereiraetal.,2005; polysaccharidesfromredseaweedalsopossessedsomebioactiveproperties(Pereiraetal.,2005; Talaricoetal.,2007). Talaricoetal.,2007). 2.1.1FCSPsstructure 2.1.1FCSPsstructure Fucoidan,anFCSPthatisextractedfrombrownalgae,maycontaindifferingglycosidiclinkagesand Fucoidan,anFCSPthatisextractedfrombrownalgae,maycontaindifferingglycosidiclinkagesand isvariouslysubstitutedwithacetateandsidebranchescontainingfucoseorotherglycosylunits. isvariouslysubstitutedwithacetateandsidebranchescontainingfucoseorotherglycosylunits. BrownseaweedintheorderofFucalessuchasF.evanescensandF.serratuspossessalarge BrownseaweedintheorderofFucalessuchasF.evanescensandF.serratuspossessalarge proportionofbothD(1o3)ͲandD(1o4)ͲlinkedLͲfucopyranoseresiduesmaybesubstitutedwith proportionofbothD(1o3)ͲandD(1o4)ͲlinkedLͲfucopyranoseresiduesmaybesubstitutedwith sulfate(SO3Ͳ)onCͲ2andCͲ4(Fig.2.2;Bilanetal.,2002,2006;Cumashietal.,2007).Thealga sulfate(SO3Ͳ)onCͲ2andCͲ4(Fig.2.2;Bilanetal.,2002,2006;Cumashietal.,2007).Thealga Ascophyllumnodosum(Fucales)hasapredominantrepeatingstructureɲ(1o3)ͲLͲfucopyranose Ascophyllumnodosum(Fucales)hasapredominantrepeatingstructureɲ(1o3)ͲLͲfucopyranose residueswithsulfateatCͲ2positionͲlinkedɲ(1o4)ͲLͲfucopyranoseresidueswithdisulfateatCͲ2 residueswithsulfateatCͲ2positionͲlinkedɲ(1o4)ͲLͲfucopyranoseresidueswithdisulfateatCͲ2 10 10 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 22 22 Seaweed Ɖotentials Seaweed Ɖotentials positionͲlinkedɲ(1o3),thesamestructuralelementsthatarealsoinFCSPsfromF.vesiculosus positionͲlinkedɲ(1o3),thesamestructuralelementsthatarealsoinFCSPsfromF.vesiculosus (Fig.2.2;Chevolotetal.,1999). (Fig.2.2;Chevolotetal.,1999). OH H 3C O OSO 3- -O SO O H 3C O H 3C O HO O O OH O H3C Chorda filum OSO 3- H3C -O3SO O OH H3C O O H 3C O O OH H3C OSO 3- H3C H3C HO -O 3SO O OH H3C O HO OSO 3- O O H3C O H3C O O H3C O HO HO COOOH O OH O -O 3SO O Cladosiphon okamuranus HO O O O OSO 3- O H3C O OSO 3- OSO 3OH OSO 3- OH H3C H3C H3C HO OH OSO 3- O H3C O HO OSO 3- Fucus evanescens C.Ag O H3C O O HO H3C Cladosiphon okamuranus O O HO H3C COOOH O OH O -O 3SO O O OSO 3- O Ascophyllum nodosum Fucus vesiculosus OSO 3- O H3C OH OH -O SO O OSO 3- OSO 3- OSO 3- HO O HO OSO 3- OSO 3- O O O O H3C H 3C O HO O OH H 3C O O OSO 3- Laminaria saccharina O OSO 3- H3C HO O OSO 3- H 3C OSO 3- Fucus serratus L HO HO O OH O -O SO O O OH O O OSO 3- HO -O 3SO O O O OH -O SO O H 3C O H3C O O -O 3SO Ascophyllum nodosum Fucus vesiculosus O H3C H3C -O 3SO OH HO O Chorda filum OH H 3C OSO 3- H3C OH O OSO 3- OSO 3- OSO 3- H 3C O O O -O3SO O OSO 3- O H 3C OSO 3- O H3C H 3C O O OH O OSO 3- HO O OH H 3C O O OSO 3- Laminaria saccharina O O H3C HO O O O H3C HO OH O O H3C O O -O SO O OH HO HO O O O -O 3SO O OSO 3- O -O SO O H3C Fucus serratus L H 3C -O 3SO O H 3C OSO 3- OH H3C OSO 3- -O SO O OSO 3- HO O OSO 3- -O SO OSO 3- O -O 3SO O OH H 3C H 3C O OH O -O SO O H3C H 3C O H 3C O O O OH -O 3SO O H 3C H3C O O -O SO OH OH H3C HO O O OSO 3- O OH OSO 3- OSO 3- Fucus evanescens C.Ag HO OSO 3- O Fig.2.2StructuralmotifsoffucoseͲcontainingsulfatedpolysaccharidesobtainedfromdifferent brownseaweedspeciesoftheorderLaminariales,Chordariales,andFucales(Chizhovetal.,1999; Usovetal.,1998;Nagaokaetal.,1999;Chevolotetal.,2001;Bilanetal.,2002;2006). Fig.2.2StructuralmotifsoffucoseͲcontainingsulfatedpolysaccharidesobtainedfromdifferent brownseaweedspeciesoftheorderLaminariales,Chordariales,andFucales(Chizhovetal.,1999; Usovetal.,1998;Nagaokaetal.,1999;Chevolotetal.,2001;Bilanetal.,2002;2006). SeveralstructuresoffucoidansoftheorderofLaminarialeswerereportedtocontained SeveralstructuresoffucoidansoftheorderofLaminarialeswerereportedtocontained monosulfatecomponentsthatmainlyconsistofɲ(1o3)ͲlinkedLͲfucopyranoseresidueswith monosulfatecomponentsthatmainlyconsistofɲ(1o3)ͲlinkedLͲfucopyranoseresidueswith sulfatesattheCͲ2position(BerteauandMulloy,2003;Anastyuketal.,2009).Fucoidanisolated sulfatesattheCͲ2position(BerteauandMulloy,2003;Anastyuketal.,2009).Fucoidanisolated fromChordafilum(Laminariales)hasastructureofpolyͲɲ(1o3)ͲlinkedLͲfucopyranosebackbone fromChordafilum(Laminariales)hasastructureofpolyͲɲ(1o3)ͲlinkedLͲfucopyranosebackbone andresiduesaresulfatedmainlyatCͲ4andsometimesCͲ2position,whereassomeɲ(1o3)Ͳlinked andresiduesaresulfatedmainlyatCͲ4andsometimesCͲ2position,whereassomeɲ(1o3)Ͳlinked fucoseresidues(Fig.2.2)tobe2ͲOͲacetylated(Chizhovetal.,1999).SimilarstructureofL. fucoseresidues(Fig.2.2)tobe2ͲOͲacetylated(Chizhovetal.,1999).SimilarstructureofL. 11 11 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 23 23 Ϯ^ĞĂǁĞĞĚƉŽƚĞŶƚŝĂůƐ Ϯ^ĞĂǁĞĞĚƉŽƚĞŶƚŝĂůƐ saccharina(Laminariales)composedofɲ(1o3)ͲlinkedLͲfucopyranosewithsulfateatCͲ4(Fig.2.2) saccharina(Laminariales)composedofɲ(1o3)ͲlinkedLͲfucopyranosewithsulfateatCͲ4(Fig.2.2) waspreviouslyreportedbyUsovetal.,(1998).AstructuralstudyoffucoidanfromCladosiphon waspreviouslyreportedbyUsovetal.,(1998).AstructuralstudyoffucoidanfromCladosiphon okamuranus(Chordariales)showedalinearbackboneofɲ(1o3)ͲlinkedLͲfucopyranosewitha okamuranus(Chordariales)showedalinearbackboneofɲ(1o3)ͲlinkedLͲfucopyranosewitha portionoffucoseresiduesthatwasOͲacetylatedandsulfatesubstitutionattheCͲ4position(Fig. portionoffucoseresiduesthatwasOͲacetylatedandsulfatesubstitutionattheCͲ4position(Fig. 2.2).ItalsocontainedɲͲglucuronicacidat2positionsoffucosethatwerenotsubstitutedbya 2.2).ItalsocontainedɲͲglucuronicacidat2positionsoffucosethatwerenotsubstitutedbya sulfategroup(Nagaokaetal.,1999). sulfategroup(Nagaokaetal.,1999). Fig.2.3ProbablestructureoffucoidanfractionfromHizikiafusiforme(Fucales)suggestedbyLietal. (2006);structuralcompositionwastypicalforSargassumspecies(Duarteetal.,2001). Fig.2.3ProbablestructureoffucoidanfractionfromHizikiafusiforme(Fucales)suggestedbyLietal. (2006);structuralcompositionwastypicalforSargassumspecies(Duarteetal.,2001). 12 12 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 WŚdŚĞƐŝƐϮϬϭ2 PhDThesis2011 24 24 ... productsaretheprimaryinterestandmotivationofthis PhD study. productsaretheprimaryinterestandmotivationofthis PhD study. The PhD workwasinitiatedattheDepartmentofSystemBiology,TechnicalUniversityofDenmark The PhD workwasinitiatedattheDepartmentofSystemBiology,TechnicalUniversityofDenmark... mindskedeùnskedelugtgenerfratangsomskylleroppåstranden.Udnyttelseaftangsom resourceerudgangspunktetfordette PhD studium. PhD studietsformålharvỉretdelsatudvikle resourceerudgangspunktetfordette PhD studium. PhD studietsformålharvỉretdelsatudvikle... workinKitasatoUniversity,KanagawaJapanwassobeneficialfortheadvancementofthis PhD workinKitasatoUniversity,KanagawaJapanwassobeneficialfortheadvancementofthis PhD study. study. The PhD projectwasfullyfinancedbytheTechnicalUniversityofDenmark(DTU)foraperiodfrom