OMWW中活性物质抗氧化及其他作用

OMWW中活性物质抗氧化及其他作用 J.Agric.FoodChem.1999,47,3397−34013397 AntioxidantandOtherBiologicalActivitiesofOliveMillWasteWaters FrancescoVisioli,*,†AnnalisaRomani,‡NadiaMulinacci,‡SimonaZarini,†DavideConte,† FrancoF.Vincieri,‡andClaudioGalli† InstituteofPharmacologicalSciences,ViaBalzaretti9,20133Milan,Italy,andDepartmentofPharmaceuticalSciences,UniversityofFlorence,Florence,Italy Duringoliveoilproduction,largevolumesofwateraregeneratedandsubsequentlydiscarded.Olivescontainavarietyofbioactivecomponents,andsomeofthem,accordingtotheirpartitioncoefficients,endupinthewaterphase.Thecurrentinvestigationaimedatcomparingdifferentmethodsfortheextractionofbiologicallyactivecomponentsoftheolivemillwastewaters(OMWW)andevaluatingtheinvitroantioxidantandanti-inflammatoryactivitiesoftheresultingextracts.TheresultsindicatethatOMWWextractsareabletoinhibithumanLDLoxidation(aprocessinvolvedinthepathogenesisofatherosclerosis)andtoscavengesuperoxideanionsandhypochlorousacidatconcentrationsaslowas20ppm.Finally,twoofthethreeextractsalsoinhibitedtheproductionofleukotrienesbyhumanneutrophils.Thepotencyoftheextractsdependedontheirdegreeofrefinement:extractscontainingonlylowmolecularweightphenolswerethemosteffective.Keywords:Antioxidants;wastewater;oliveoil;freeradicals;polyphenols INTRODUCTION Thereisgrowinginterestinnovelsourcesofnaturalantioxidants,duetotherecognizedinvolvementofreactiveoxygenspeciesintheonsetofseveralhumandiseases(Aruoma,1998)andintheoxidativedegrada-tionoffood,animalfeed,andothergoodssuchascosmetics(Aruoma,1997). Olivesandoliveoilcontainphenoliccompounds(Tsimidou,1992,1998)that,invitro,havebeenshowntoexertpotentbiologicalactivitiesincluding,butnotlimitedto,antioxidantactions(VisioliandGalli,1998a,b).Itisnoteworthythat,duringoliveoilproduction,aconsiderableamountofwaterisemployedduringthemalaxationprocess,thatis,thecontinuouswashingoftheolivepastewithwarmwaterpriortotheprocedureofseparationoftheoilfromthepaste(Boskou,1996;Kiritsakis,1991).Thiswater,inadditiontothatendog-enouslycontainedintheolives,makesupto50%ofthetotalyieldoftheolivepaste(oliveoilamountsto∼15%)andisnamed“wastewater”.Infact,duetothefailuretodevelopasuitableend-of-pipetreatmenttechnology,olivemillwastewaters(OMWW)arecurrentlydis-cardedbyoliveoilmanufacturersandrepresentasignificantburdentothemills’economy.PreviousworkcarriedoutinourlaboratoryhasshownthatOMWWcontainaconsiderableamountofphenoliccompoundsendowedwithpotentantioxidantproperties(Visiolietal.,1998).Thus,inviewofthecurrentneedforupgrad-ingbyproductsatallstagesoftheoliveoilindustry(DemicheliandBontoux,1996),aprojectsupportedbytheEuropeanCommunitywasundertakentoinvesti-gatedifferentproceduresfortherecoveryoftheactive *Authortowhomcorrespondenceshouldbeaddressed(telephone+390220488217;fax+390229404961;e-mailfrancesco.visioli@unimi.it). †InstituteofPharmacologicalSciences.‡UniversityofFlorence. componentsofOMWWandtocomparetheantioxidantandotherbiologicalactivitiesofthevariousextracts. EXPERIMENTALPROCEDURES PreparationofOMWWExtracts.OliveswerecollectedfromdifferentlocationsinItaly,Spain,andFranceandwerefrozenandshippedtotheUniversityofFlorence.OMWWwereobtainedbyemployingabenchtopmill,andtheresultingsampleswerefractionatedbyapplyingaliquid-solidextrac-tion(LSE)andfurtherprocessedinthreedifferentwaystoobtainextractswithincreasingdegreesofpuritythatwereanalyzedbyreverse-phaseHPLC.EECregulationsprohibitthefulldescriptionofthemethodology,whichiscurrentlyunderpatentapplicationbutabriefdescriptionfollows. Thefirstextract(hereinafterreferredtoasextract1)wasobtainedbyafractionationoflyophilizedOMWWonanXAD1180resincolumnandbysubsequentelutionswithwaterandethanol.Theethanolicfractionwasthenlyophilizedandemployedinbiologicaltests.Thesecondextract(hereinafterreferredtoasextract2)wasobtainedbyaliquid-liquidextraction(LLE)oftheOMWW.Briefly,afteradefattingwithn-hexane,LLEextractionswithethylacetatewereperformed,andtheextractswerebroughttodryness,resuspendedinethanol,andemployedinbiologicaltests.TheethylacetateLLEisselectiveforlowandmediummolecularweightphenols;itdoesnotextractheaviermoleculesthatremaininthewaterphase.Thethirdextract(hereinafterreferredtoasextract3)wasobtainedfollowingafractionationofextract2onaSephadexLH-20column.Itwasthenbroughttodryness,analyzed,andemployedinbiologicaltests. AnalysesoftheExtracts.HPLC/DADanalyseswereperformedwithanHP1090LliquidchromatographequippedwithanHP1040ADADdetector(Hewlett-Packard,PaloAlto,CA).Thecolumnwasa6.6×250mmLiChrosorbRP18,5µm(Merck),anditstemperaturewasmaintainedat26°C.ThemobilephasewasH2O(adjustedtopH3.2withH3PO4)andCH3.Afour-steplinearsolventgradientwasemployed,startingfrom100%H2Oto100%CH3overa106minperiod,ataflowrateof1mLmin-1,aspreviouslydescribed(Romanietal.,1996). QuantitationofthePhenolicComponentsofOMWWExtracts.Quantitationofindividualphenoliccompoundswas 10.1021/jf9900534CCC:$18.00©1999AmericanChemicalSociety PublishedonWeb07/28/1999 3398J.Agric.FoodChem.,Vol.47,No.8,1999 carriedoutbyusingfour-pointregressioncurves(r2)0.99)obtainedbyemployingauthenticstandards(ExtrasyntheseS.A.,Lyon,France).Tyrosolandhydroxytyrosolandoleuropeinanditsderivativeswerequantifiedat280nm.Elenolicacidwasquantifiedat254nmbyemployingoleuropeinasthereferencecompound.Theappropriatecorrectionsbasedontheindividualmolecularweightswereappliedtocorrectlyquantifyindividualcompounds. EvaluationofOMWWAntioxidantCapacity.Humanlow-densitylipoproteins(LDL,d)1.021-1.063)wereisolatedbysequentialultracentrifugationfromplasmaobtainedfromhealthy,normolipidemicvolunteers.Beforeinitiationoftheexperiments,LDLsamplesweredesaltedbysizeexclusionchromatographyandtheirproteincontentsdeterminedac-cordingtothemethodofLowryetal.(1951).LDLsamplesweredilutedwithPBSto200µg/mL,andoxidationwasstartedbytheadditionofeither5µMcoppersulfateorthefreeradicalgenerator2,2′-azobis(2-amidinopropane)dihydrochloride(AAPH)atconcentrationsof5and10mM.Controlsampleswereaddedwith5µLofethanol,whichwasthevehicleemployedtodissolveOMWWextracts.Incubationswerecarriedoutat37°Cinashakingbath,andaliquotswerewithdrawnatdifferenttimesfortheanalysisofvariousoxidationmarkers(VisioliandGalli,1997).Lipidhydroperoxideandmalondialdehydeforma-tionswereevaluatedaccordingtotheprocedureofBallaetal.(1991),andtheresultsareexpressedasnanomolesofeitherLOOHorTBARSpermilligramofLDL. Superoxideanionwasgeneratedbythexantine/xantineoxidasesystem,anditsrateofformationwasevaluatedbyspectrophotometryat550-540nm(Visiolietal.,1998)andcalculatedaftertheadditionofsuperoxidedismutase. Reactionswithhypochlorousacidwereevaluatedbyassess-ingtheprotectiveactivityofOMWWextractsversusHOCl-inducedinactivationofcatalase,asdescribedbyMarshalletal.(1996)andAruomaandHalliwell(1987). TheDPPH-scavengingpropertiesofOMWWwereevaluatedasdescribedinVisioliandGalli(1998b),andEC50valueswerecalculatedbyemployingMacALLFITsoftware. EvaluationofLeukotrieneB4(LTB4)ProductionbyHumanNeutrophils.HumanneutrophilswereisolatedfrombloodaccordingtothemethodofSalaetal.(1996).Samplesof5×106cellswereincubatedat37°Cinashakingbathinthepresenceorabsencetotheindicatedextracts.Cellularactivationwastriggeredbytheadditionof5µMcalciumionophoreA23187(finalconcentration).After10minofincubation,thereactionwasstoppedbytheadditionofanequalvolumeofchilledmethanol.LeukotrieneswereseparatedbyHPLC(Salaetal.,1996)andquantifiedbyemployingPGB2asinternalstandard.RESULTS Threefinalextractswereobtainedfromwastewaterspreparedasdescribedabove.Figure1reportsthechromatographicprofileofthethreeextracts.Extract1exhibitsacomplexchromatographicprofile,duetothepresenceofpolymersthatareresponsibleforthehighbackgroundseenat254nm,thatis,thewavelengthinwhichphenolicpolymersyieldtheirmaximumabsorp-tion.Extract2mainlycontainslowandmediummo-lecularweightphenols(elenolicacidistheprincipalconstituent),andextract3ismadeofhydroxytyrosol,tyrosol,andastillunidentifiedderivativeoftheformeronly.AquantitationoftheextractsisgiveninTable1.AsolutionofthestablefreeradicalDPPHcanbedecolorizedaccordingtothescavengingpotencyofantioxidantmolecules.LowamountsofOMWWextractswereabletoremoveDPPHfromtheethanolicsolution,withEC50valuesof9.42,3.12,and1.83ppmforextracts1,2,and3,respectively(Table2). TheantioxidantactivitiesofOMWWextractsweretestedbyemployingbothmetalions,thatis,5µM Visiolietal. Figure1.HPLCchromatogramsofOMWWextracts.TheextractswereobtainedasdescribedunderExperimentalProcedures,andsomecompoundswereidentifiedasfollows:(1)hydroxytyrosolderivative;(2)hydroxytyrosol;(3)tyrosol;(4)elenolicacid;(5)oleuropeinderivative;(6)luteolin7-glu-coside;(7and8)cinnamicacidderivatives;(9)quercetin. Table1.HPLCEvaluationofMajorPhenolic CompoundsIdentifiedinOMWWExtracts(Gramsper100gofDryMatter)a compound extract1extract2extract3hydroxytyrosol1.561.29.79tyrosol 0.850.454.72elenolicacid 4.3112.62 oleuropeinderivatives0.5luteolin7-glucoside0.220.67quercetin 0.130.77cinnamicacidderivatives0.551.47 hydroxytyrosolderivatives5.65 totalpolyphenols 8.11 15.56 22.78 a Molecularweightsemployedforcalculationsareasfollows:hydroxytyrosol,154;tyrosol,138;elenolicacid,242;oleuropeinderivative,378;luteolin7-glucoside,448;quercetin,302;cinnamicacidderivatives,180;hydroxytyrosolderivative,154.Table2.DPPHTesta extractEC50(ppm) 19.4223.123 1.83 a ThemethodologyemployedforthistestisdescribedinVisioliandGalli(1998b)andEC50valueswerecalculatedbyemployingMacALLFITsoftware. coppersulfateandthemetal-independentfreeradicalgeneratorAAPH.AsshowninFigure2,thecoppersulfate-inducedproductionofthiobarbituricacid-react-ingsubstances(TBARS),mostlymadeofshort-chainaldehydessuchasmalondialdehydeand4-hydroxynon-enaloriginatingfromtheoxidativedegradationoflipidhydroperoxides,wasgreatlyreducedbythecoincubationofLDLsampleswithOMWWextractsattheconcentra-tionof20ppm.After6hofincubation,extract1lostpartofitsantioxidantcapacity,whereastheothertwoextractswerestillabletoinhibitlipid peroxidation. BioactiveCompoundsinOliveMillWasteWatersFigure2.TBARSlevelsinLDLsamplesoxidizedwith5µMCuSO4andcoincubatedwith20ppmofOMWWextracts.ControlsampleswereaddedwiththesameamountofethanolemployedtodissolveOMWWextracts.Valuesaremeansofduplicateanalysesthatdidnotdifferbymorethan 5%. Figure3.LOOHlevelsinLDLsamplesoxidizedwith5µMCuSO4andcoincubatedwith20ppmofOMWWextracts.ControlsampleswereaddedwiththesameamountofethanolemployedtodissolveOMWWextracts.Valuesaremeansofduplicateanalysesthatdidnotdifferbymorethan 5%. Theseresultswereconfirmedbytheevaluationoflipidperoxide(LOOH)production(Figure3),whichshowedthatOMWWextractswereabletoinhibitLOOHproductionthroughouttheexperiment. WhenAAPHwasemployedtotriggeroxidativestress,aslightlylowerantioxidanteffectofOMWWextractswasnoted,dependingontheconcentrationofAAPHemployed.Inparticular,whenAAPHwasadoptedattheconcentrationof5mM(Figures4and5,upperpanels);asignificantdegreeofprotectionofLDLsamplesfromoxidationwasobserveduntilterminationoftheexperiment,whereaswhenoxidationwasinducedby10mMAAPH(Figures4and5,lowerpanels),asimilardegreeofefficacywasnoted. Thesuperoxide-scavengingpropertiesofOMWWex-tractsweretestedinacell-freesystemandrevealedthat,atEC50values<10ppm,theextractswereabletoremoveO2•-fromthereactionmedium(Table3),unlikevitaminEandBHT(notshown)thatwerefoundtobeineffective. TheantioxidantactivitiesofOMWWextractswerealsotestedagainstHOCl,whichcouldbeconsideredasasourceofreactivechlorinespecies.TheabsorptionspectraofacatalasesolutionwereevaluatedaftertheadditionofHOCl(Figure6),whichcausedadegradationofthehemegroupoftheenzymeandamodificationofitsabsorptionspectrum.CoincubationofthecatalasesolutionwithOMWWextractsaffordedprotectionofthecatalasehemegroup:extract1waslesseffectivethanextracts2and3,whichwerefoundtobeequipotent. J.Agric.FoodChem.,Vol.47,No.8,19993399 Figure4.TBARSlevelsinLDLsamplesoxidizedwith5or10mMAAPH)andcoincubatedwith20ppmofOMWWextracts.ControlsampleswereaddedwiththesameamountofethanolemployedtodissolveOMWWextracts.Valuesaremeansofduplicateanalysesthatdidnotdifferbymorethan 5%. Figure5.LOOHlevelsinLDLsamplesoxidizedwith5or10mMAAPHandcoincubatedwith20ppmofOMWWextracts.ControlsampleswereaddedwiththesameamountofethanolemployedtodissolveOMWWextracts.Valuesaremeansofduplicateanalysesthatdidnotdifferbymorethan5%. Table3.ScavengingEffectsofOMWWExtractsontheRateofFormationofSuperoxideAniona compoundEC50(ppm) extract18.46extract25.32extract3 2.87 a Therateofformationofsuperoxideanion(∆absorbance/min)wascalculatedaftertheadditionofanappropriateamountofsuperoxidedismutase,asdescribedinVisiolietal.(1998). LTB4productionbyactivatedhumanneutrophils(Figure7)waspotentlyinhibitedbyextracts2and3,whereasextract1wasfoundtobeineffective.TheamountsofLTB4thatwereformedbyneutrophilswerecalculatedbycomparisonwiththeinternalstandardprostaglandinB2,andtheresultingEC50valuesindicatethattheinhibitionofcellularactivationbyextracts2and3takesplaceatverylowconcentrations(Table 4). 3400J.Agric.FoodChem.,Vol.47,No.8,1999Figure6.Representativeabsorptionprofileofacatalasesolutionincubatedwith80µMHOClandwithOMWWextractsasdescribedunderExperimental Procedures.DISCUSSION Theinvestigationreportedinthispaperwasunder-takentocomparethebiologicalactivitiesofOMWWextractsthatweresubjectedtodifferentextractionandpurificationmethods.OMWWarethemajorbyproductofoliveoilproductionandaregeneratedinlargeamounts(∼800000tons/yearinItalyalone)atolivemillsthat,atpresent,mustdiscardthem,thusincreas-ingthecostsofwastedispositionandraisingecologicalissuesduetothehypothesizedsoilcontamination.AnalysisofOMWWshowsthattheircontentinphenoliccompoundsfluctuatesfrom0.5to1.8%ofphenolics(Table5).Othercomponentssuchasflavonoids,antho-cyanins,andtanninsareofpotentialbiologicalinterestduetotheirantioxidantactivities(VinsonandHontz,1995;Vinsonetal.,1995). Fromacommercialpointofview,themostwidelyemployedantioxidantsarethoseindigenoustofoods,thewater-solubleascorbate(E)300)andthelipid-solublebutylatedhydroxytoluene(BHT;E)321),butylatedhydroxyanisole(BHA;E)320),theestersof3,4,5-trihydroxybenzoicacids(E)310,311,and312),andvitaminE(mostlydl-R-tocopherols,E)307).Plantextractsarealsoinuse,andtheirshareoftheantioxi-dantmarketisexpectedtogrowby∼15%bytheyear Visiolietal. Figure7.HPLCprofilesshowingtheproductionofLTB4anditsµ1Mmetabolitesppmcalciumbyhumanneutrophilschallengedwiththe5ofextractionophore2(B)A23187or3(C).forProstaglandin10minandcoincubatedBwith2(PGB2)wasemployedasinternalstandardtocalculatetherelativeEC50values(seeTable3). Table4.InhibitionofLeukotrieneB4andRelated MetaboliteFormationinHumanNeutrophilsbyOMWWExtracts compoundEC50(ppm)extract1ineffectiveextract21.26extract3 8.74 Table5.AverageCompositionofOMWWwater(%) 83minerals(carbonates,phosphates,potassiumandsodiumsalts,etc.)(%)∼2organiccompounds(%)∼15sugars ∼2-8proteins,pectins,macromolecules,etc.∼1.2-5polyphenols ∼0.5-1.8 2005(KrishnakumarandGordon,1996).Hydroxytyro-sol,themostactivecomponentofOMWWextracts,isofparticularinterestbecauseitisamphiphilicandthusitactsattheoil-waterinterfaceandinsystemswherebothoilandwaterphasesarepresent,suchasemul-sions(Aruoma,1997). ThedatareportedinthispaperindicatetheOMWWextractscontainpotentantioxidants.Duetothediffer-entdegreesofpurityofthethreeextracts(extract1 is BioactiveCompoundsinOliveMillWasteWatersthelessrefinee,whereasextract3isthemostpurified),adiversedegreeofactivitywasnoted.Thefirstextractis,infact,lesspotentininhibitingLDLoxidation,whereastheothertwoextracts,containingonlylowmolecularweightphenolssuchashydroxytyrosol,pre-ventedLDLoxidationwhenitwasinducedbyeithermetalionsorametal-independentfreeradicalgenera-tor.TheslightlylowerefficacyofOMWWextractsnotedwhenAAPHwasemployedasoxidantsuggeststhattheextractsactmostlyasionchelatorsbutarealsoabletoscavengepreformedradicals,asalsoindicatedbytheDPPHtest,inwhichtheextractsremovedthestablefreeradicalatlowconcentrations. TheabilityofOMWWextractstoscavengesuperox-ide,alreadyreportedforhydroxytyrosolandoleuropein(Visiolietal.,1998),issuggestiveofapotentialuseofOMWWextractsinenvironmentsinwhichFentonandHaber-Weissreactionstakeplaceandinwhichtheconcomitantproductionofsuperoxideandnitricoxidewouldyieldthepowerfuloxidantperoxinitrite.ItisnoteworthythattheestablishedantioxidantsvitaminEandBHTdonotscavengesuperoxide,andthusOMWWextractsmayaddstabilitytoproductsexposedtohighO2•-levels. Theprotectionofhypochlorousacid-induceddamageofcatalase(Figure6)isofbiologicalsignificanceduetothewell-knownprotein-damagingactivityofHOCl,whichisproduced,inbiologicalsystems,atthesiteofinflammationbyactivatedneutrophilsthroughtheenzymemyeloperoxidase(AruomaandHalliwell,1987).Also,becausefoodsoftencomeintocontactwithchlorine-basedbleaches,employedasdisinfectantsinfoodplants,theuseofHOClscavengersmayprovideadditionalprotectionagainstreactivechlorinespecies. Finally,thepotentinhibitionofcalciumionophore-stimulatedproductionofLTBsuggeststhat4anditsmetabolitesbyhumanneutrophilsOMWWextractsexertbiologicaleffectsbeyondtheirantioxidantcapacities.Theactivityofseveralenzymes,includingthosein-volvedintheproductionofeicosanoids,forexample,phospholipasesandoxygenases,ismodulatedbytheintracellularperoxidetone.Thus,byscavengingreactiveoxygenspecies,OMWWextractscouldlowertheactivityofsuchenzymesand,inturn,decreasetheproductionofpro-inflammatoryfactors.Additionalstudiesareneededtoverifyifsuchanti-inflammatoryeffectscouldalsotakeplaceinvivoandtheexactenzymatictargetofthebioactivecompounds. Inconclusion,thesedatashowthatOMWWarerichinantioxidantcompoundsthatcouldberecoveredfromthematrixandemployedbothinpreservativechemistryand,followingappropriatetrialstoevaluatetheirsafetyandefficacy,asprophylacticagentsinthepreventionofcertainradical-inducedhumandiseases. 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VisioliF.;Galli,C.Theeffectofminorconstituentsofoliveoiloncardiovasculardisease:newfindings.Nutr.Rev.1998b,56,142-147. Visioli,F.;Bellomo,G.;Galli,C.Freeradical-scavengingofoliveoilphenols.Biochem.Biophys.Res.Commun.1998,247,60-64.ReceivedforreviewJanuary15,1999.RevisedmanuscriptreceivedJuly1,1999.AcceptedJuly6,1999.SupportedbyEECGrantFAIRCT973039.JF9900534