BIOTECHNOLOGICALPRODUCTSANDPROCESSENGINEERING
Chun-LinLee.Tsung-YuTsai.Jyh-JyeWang.Tzu-MingPan
InvivohypolipidemiceffectsandsafetyoflowdosageMonascuspowderinahamstermodelofhyperlipidemia
Received:10May2005/Revised:10August2005/Accepted:12August2005/Publishedonline:11November2005#Springer-Verlag2005
AbstractMonascusormorecommonlyknownasredmoldriceisfermentedriceonwhichMonascuspurpureushasbeengrown.IthasbeenatraditionalChinesefoodadditiveforthousandsofyearsinChina.Secondaryme-taboliteproductofMonascus,monacolinK,hasbeenproventhatitcouldbeusedasanantihypercholesterolemicagent.Inthisstudy,M.purpureusNTU568mutatedandselectedfromamonacolinKproductivitystrain—M.purpureusHM105producedhighquantitiesofmonacolinKatalevelof9,500mgkg−1.ThisresearchfocusedontheeffectofaddingredmoldricepowderofM.purpureusNTU568toahamsterdietontotalcholesterol(TC),triglyceride(TG),high-densitylipoproteincholesterol,andlow-densitylipoproteincholesterol(LDL-C).Intheresults,theoraladministrationofMonascuspowderinhyper-lipidemiahamsterwasindeedproventodecreaseTC,TG,andLDL-Clevels.PlasmaTClevelsinhamsterfedwithMonascuspowderatonefolddosage[10.78mg(day100gbw)−1]for4and8weeksweresignificantlylower(31.2and22.0%,respectively)thanthatinhyperlipidemiahamster.PlasmaTG(30.1and17.9%)andLDL-Clevels(36.0and20.7%)werealsosignificantlyloweredbyfeedingMonascuspowderatonefolddosagefor4and8weekscomparedtohyperlipidemiahamster.Inaddition,examinationsofliverTCandTGlevelsofhyperlipidemiahamsterwerealsoperformedandshowedsimilareffectsonlipid-loweringactionbyoraladministrationofMonascuspowder.Sincecitrininisamycotoxinthatpossessesneph-rotoxicandhepatoxiceffects,ithasanegativeimpacton
C.-L.Lee.T.-Y.Tsai.T.-M.Pan(*)InstituteofMicrobiologyandBiochemistry,NationalTaiwanUniversity,1,Sec.4,RooseveltRoad,Taipei,106217,Taiwane-mail:tmpan@ntu.edu.twTel.:+886-2-33664519Fax:+886-2-23627044J.-J.Wang
DepartmentofBiotechnology,TajenInstituteofTechnology,PingTung,Taiwan
thesafetyofredmoldriceforpeople.Thisstudyexaminedtheliversomaticindex[plasmaglutamyloxaloacetictrans-aminase(GOT)andglutamylpyruvictransaminase(GPT)levels]andliverbiopsytoinvestigatewhetherMonascuspowderinduceddamageinliver.ItwasfoundthattheplasmaGOTandGPTlevelswerenotsignificantlyin-creasedbyfeedingMonascuspowder.Therewasnodif-ferenceintheresultsoftheliverbiopsybetweentheMonascuspowder-treatedgroupsandthecontrolgroup.
Introduction
MonascusorredmoldriceisfermentedriceonwhichMonascuspurpureus,aredmoldspecies,hasbeengrown.IthasbeenatraditionalChinesefoodadditiveforthou-sandsofyearsinChina.ItsspecialeffectsandapplicationonfoodhavebeenrecordedinancientChineserecords.ThetypesofsecondarymetabolitesproducedfromtheMonascusspeciesinclude(1)agroupofpigments(yellowpigment,ankaflavinandmonascin;orangepigment,mon-ascorubrinandrubropunctanin;redpigment,monascoru-bramineandrubropuctamine(WongandKoehler1981),(2)agroupofantihypercholesterolemicagentsincludingmonacolinKandhypotensiveagent,γ-aminobutyricacid(GABA)(Suetal.2003),(3)antioxidantcompoundsincludingdimerumicacid(Aniyaetal.1999)and3-hy-doxy-4-methoxy-benzoicacid(WuandWu2000),and(4)anantibacterialactivitycompoundincludingpigmentandcitrinin(alsoknownasmonascidin)(Blancetal.1995a,b).SincetheMonascusspeciescontainsmultifunctionalcompounds,itbecomesanimportanttopicinthefieldoffunctionalfood.Inrecentyears,cardiovasculardiseasehasbecomeoneofthemostdifficultproblemstohandleinmanycountries.Thisiswhyprophylaxisandtherapyarebecomingmoreandmoreimportantsubjectsformanyre-searches.MonacolinK(alsoknownaslovastatin,me-vinolin,mevacor)isapotentcompetitiveinhibitorof3-hydroxy-3-methylglutarylcoenzymeAreductase(HMG-CoAreductase),therate-limitingenzymeincholesterolbiosynthesis(Albertetal.1980;Endo1979).Itcannotonly
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inhibitcholesterolbiosynthesis,butalsolowerbloodcho-lesterollevelinbothhumanandanimals.MonacolinKandlovastatinarethesamechemicalcompoundsthatinhibitHMG-CoAreductaseinthecholesterolsynthesispathway(Endo1979).
Redmoldrice,aChinesetraditionalfood,hasbeenusedasadietsupplementorremedyinChinaandJapanforthousandsofyears.Redmoldriceisstillrestrictedandnotacceptedinmanycountries,andcausesmuchcontroversy,becausemycotoxin–citrininisoftensecretedbyMonascusspp.M.purpureusThisfact,provenandMonascusbyBlancruberetal.(produces1995a)indicatesanantibac-thatterialcompound,monascidin,whichhasbeenproventobethesamecompoundascitrinin.Citrininisapotentrenaltoxinandhepatotoxin,whichcausesfunctionalandstruc-turalkidneydamageandalterationsinlivermetabolism(DaLozzoetal.1998).Itinhibitsseveralenzymeslinkedtotherespiratorychainofthekidneycortexandlivermito-chondria,aswellasmalateandglutamatedehydrogenasesandtheATP-synthetasecomplex(DaLozzoetal.1998).Citrininwasoftenfoundinsolidandsubmergedculturedproductsof−1Monascus,anditwasdetectedfrom0.2to122mgkg(Blancetal.1995a;HsiehandPan2002).ManyresearcherssuggestthatMonascusproductshavebeenproventocontainmultifunctionalcompoundsandthat,therefore,thefunctionandapplicationofMonascusspeciesshouldnotbeannulledbecauseitpossessesci-trinin.Inaddition,Monascuswouldbesafeandharmlessforthedailydietifthecontentofcitrininwaslessthanthelevelthatinducesinvivodamage.
AlthoughmanyMonascusspeciescouldsynthesizemonacolinK,theproductionofmonacolinKwaslessthan500mgkg−1(Endoetal.1985;Manzonietal.1999;Wangetal.1998,2003;Laietal.2003;Schneweisetal.2001;Changetal.2002;CasasLópezetal.2003).However,therecommendeddailyallowanceofmonacolinKforadultswassuggestedtobeatleast10mgperday(Haveletal.1987).AdosageofgeneralMonascuspowderagainstcholesterolbiosynthesisforahumanadultthereforeneededatleast10gperday.Therefore,MonascusspecieswithlowmonacolinKproductivitywouldbecomemoreandmoredifficulttodevelopandapplyonfunctionalfoodwithcholesterol-loweringeffect,soitisimportanttoincreasethemonacolinKproductionoftheMonascusspecies.Inourpreviousstudy,M.purpureusNTU568wasselectedfromthemutantsofM.purpureusHM105inviewofthefactthatitcouldrenderahighmonacolinKproductionat9,500mgkg−1.Atthesametime,thecitrininproductionwasfoundtobedecreasedfrom2,500to939μgkg−1[Leeetal.2005,submittedtoJournalofAgricultureandFoodChemistry(JAFC)].
BecauseredmoldricefermentedbyM.purpureusNTU568hasahighmonacolinKproduction,weexpectthatthesupplementationofalowdosageofMonascuspowderwillshowacholesterol-loweringeffect.Inthisstudy,weusedalowdosageofMonascuspowderasanexperimentalsampletoinvestigatethehypolipidemicef-fectinahamstermodelofhyperlipidemia.Therefore,thereferencedosageofMonascuspowderforahumanadult
waschosenat1gadayinthisstudy,whichisatleasttentimeslessthantheefficientdosageofgeneralMonascuspowder.M.purpureusInaddition,NTU5681gofcontainsMonascusamonacolinpowderfermentedKlevelbyof9.5mg,whichisclosetotherecommendeddailyallowanceofmonacolinKforahumanadult.However,theMonascusproducthasbeencategorizedasafunctionalfoodaccord-ingtothefunctionalsecondarymetabolitemonacolinKandGABA.Therefore,thepurposeanddesignofthisstudyshoulddifferfromthatofanefficiencyevaluationstudyfordrugs.High-cholesterol(HChol)dietandMonascuspow-derwerefedsimultaneouslytotwogroupsofhamstersafteranobservedstageof4weekstosimulateanassess-mentmodelforfunctionalfood,similartothatforpeoplesupplementingMonascuspowderacholesterol-richforitshypolipidemicdailydieteffects.
withabitofThisresearchfocusedontheeffectsoforaladministra-tionM.purpureusofasmallamountNTU568offorMonascushyperlipidemiapowderfermentedhamstersbyontotalcholesterol(TC),triglyceride(TG),high-densitylipoproteincholesterol(HDL-C),andlow-densitylipopro-teincholesterol(LDL-C)levelsinbloodorliver.Thisstudyexaminedtheliversomaticindex[serumglutamyloxalo-acetictransaminase(GOT)andglutamylpyruvictransam-inaseMonascus(GPT)powderlevels]inducesandliverdamagebiopsytoininvestigateliverwhenwhethertheci-trininformationofM.purpureusNTU568hasbeensup-pressedbymutationand,therefore,ismuchlessthanthatofthewildtype.
Materialsandmethods
Microorganismandseedcultures
ThereusmicroorganismM.purpureusNTU568.HM105ThisusedstraininisolatedwasthisfrommutatedstudyincludesredmoldandselectedM.purpu-rice.Thefromcul-turestrainwasmaintainedonpotatodextroseagar(PDA)slantedat10°Candtransferredmonthly.SeedcultureswerepreparedbytransferringaloopfulofsporefromthePDAagarslantedintoa500-mlHintonflaskcontaining100mlbasalmedium(100gdextrose,10gpeptone,2gKNO3,2gNH4H2PO4,0.5gMgSOmldistilledwater,4·7HpH6.0).2O,0.1gCaClmadeupin1,000Thecultures2wereincubatedat30°Cfor3daysat110rpm.Afterthat,5%inoculum(volume/weight)wastransferredforsolidstatefermentation.Redmoldricepreparation
Thelong-grainrice(Ipomoeabatatas)waspurchasedfromalocalsupermarketinTaiwanandwasusedforredmoldmetabolitesproductionundersolidstatecultivation.Thestrainswereincubatedonmoisturedriceina“kojidish”(thekojidishwasmadeofwoodandmeasured30×20×5cmhigh).Thepreparationofthericemediumforthesolidculturewasasfollows:dehulledrice(500g)wassoakedin
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distilledwaterfor8h.Afterthat,theexcesswaterwasremovedwithasieve.Thesoakedricewasthenautoclavedfor20minat121°Cinakojidish.Afterbeingcooled,thesteamedrice(500g)wasinoculatedwith25mlsporesuspension.Theinoculatedsubstratewascultivatedat30°Cfor10days.Afterfermentation,Monascusfermentedricewasdriedbyheatingat50°Cfor24handthencrushedtopowder.ThemoisturecontentofMonascuspowderismeasuredat14to17%.Thecrushedanddriedsubstratecontainingthemoldwasusedfortheexperiments(Suetal.2003).
Animalsanddiets
OnehundredandfourmaleGoldenSyrianhamstersweighing100to120gwerehousedinindividualplasticscagesandsubjectedtoa12-hlight/darkcyclewithamaintainedrelativehumidityof60%andatemperatureat25°C.Theanimalsweregivenfreeaccesstoregularrodentchowandwaterfor4weeks.Eighthamsterswerekilledandexaminedforplasmaandliverlipidlevelstoestablishabaseline.Theotherswereweighedandrandomlyas-signedintosixgroupsof16animalseachbeforecom-mencingthestudy.Dosageandgrouping
ThedosageofMonascuspowderwascalculatedinaccord-ancewithBoyd’sformulaofbodysurfaceareaasre-commendedbytheFoodandDrugAdministration(FDA)(Boyd1935).Monascuspowderandprobucolarerespect-ivelyrecommendedtosupplementthedailydietat1gand100mgforanadultwithaweightof70kgandaheightof170cm.Thesedosageswereusedasaframeofreferencefortheconversionofthedosageintoahamstermodel.Therefore,feedinghamsterwithMonascuspowderatahalf-folddosageperdaycorrespondstosupplementingthedailydietwithMonascuspowderat0.5gforahumanadult.Monascuspowderorprobucolwererespectivelysuspendedin1mlwaterandthenfeddailytohamsters,usingoraladministration.ThedosageofMonascuspowderandprobucolwereadjustedweeklyaccordingtotheave-ragebodyweightofthehamster.
ExperimentaldietswereprovidedinaccordancewithAmericanInstituteofNutrition(AIN)-76dietformulation(AmericanInstituteofNutrition1977),withmodification.ThecontrolgroupwasfedanormaldietviaAIN-76formulation,andtheHCholgroupwasgivenahigh-cholesteroldietthatcontained0.1%cholesterol(Table1).Thefoodintakewasrecordeddaily,andanimalswereweightedweekly.HChol-M1/2,HChol-M1,andHChol-M5werefedthehigh-cholesteroldietandwereorallygivenahalf-folddosage(0.5×mgkg−1bwperday)ofMonascuspowder(fermentedbyM.purpureusNTU568andincludingmonacolinKof9,500mgkg−1andcitrininof0.939mgkg−1),onefolddosage(1×mgkg−1bwperday)ofMonascuspowder,andafivefolddosage(5×mgkg−1bw
Table1Compositionoftheexperimentaldieta(gkg−1diet)CompositionCaseinCornstarchCelluloseSoybeanoilMineralVitaminL-cysteine
CholinebitartrateCholesterolCholicacid
aNormaldietb2006505050351032––
High-cholesteroldietc140680508035101211
BasedonAIN-76dietformula(AmericanInstituteofNutrition1977)bThedailydietofthecontrolgroupcThedailydietoftheHCholgroup,HChol–probucolgroup,HChol-M1/2group,HChol-M1group,andtheHChol-M5group
perday)ofMonascuspowder,respectively.Inaddition,theHChol–probucolgroup,whichwasregardedasthepositivecontrolgroup,wasfedthehigh-cholesteroldietandgivenorallyonefolddosage(1×mgkg−1bwperday)ofpro-bucol.Thehamsterswerefedtheseexperimentaldietsfor4and8weeks,respectively.
Twentyfourhoursbeforekilling,allfoodwasremoved.Animalswereanesthetizedandkilledbycarbondioxideinhalation,andbloodandliversampleswerecollectedforassay.Plasmaandredbloodcellswereseparatedandstoredat−20°C.Partoftheliverwascarefullyremovedandimmersedinformalinstock,andthesubsequentlyremain-ingliverwasrinsedfrequentlywith0.8%sodiumchloridesolutionforeliminatinganyblood.Thelivertissuewasexaminedfordamagebymicroscopicexaminationofaliverbiopsy.TheexperimentwasreviewedandapprovedbytheAnimalCareandResearchEthicsCommitteeoftheNationalTaiwanUniversity.
Plasmaandliverlipidanalysis
PlasmaandliverTCandTGlevelsaswellasserumHDL-CandLDL-Clevelsweremeasuredintriplicateusingcommercialenzymatickits.Thesekitswereasfollow:TCassaykit(Partno.MP2-35,JonhsonandJonhson,NJ,USA),TGassaykit(PartNo.MP2-19,JonhsonandJonhson),LDL-Cassaykit(Cat.no.1.14992.0001,MerckCo.,Darmstadat,Germany),andHDL-Cassaykit(Cat.no.1.14210.0001,MerckCo.).Plasmaliverindexanalysis
PlasmaGOTandGPTlevelsweremeasuredintriplicateusingcommercialenzymatickits.Thekitswereasfollow:GOTassaykit(PartNo.MP2-113,JonhsonandJonhson),GPTassaykit(PartNo.MP2-36,JonhsonandJonhson).
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Table2ThebodyweightanddailyfeedintakeofexperimentalhamstersGroup
Bodyweight(g)0thweek
ControlHChol
HChol–probucolHChol-M1/2HChol-M1HChol-M5
82.1±8.12a79.5±8.15a82.3±7.52a77.1±8.84a79.9±9.57a82.3±9.65a4thweek93.6±9.5a87.0±7.43a87.1±7.50a90.6±9.01a87.3±8.40a91.4±7.35a8thweek107±8.48a99.4±9.32a99.2±7.80a103±8.87a105±10.0a104±8.32aDailyfeedintake(gday−1)4thweek6.78±0.50a6.64±0.45a6.75±0.69a6.73±0.74a6.84±0.75a6.92±0.83a8thweek7.73±0.78a7.23±0.92a7.52±1.03a7.22±0.76a7.23±0.60a7.54±0.84aControl,normaldiet(0%cholesterol);HChol,high-cholesteroldiet;HChol–probucol,probucolandhigh-cholesteroldiet;HChol-M1/2,Monascuspowder[0.5×,5.39mg(day100gbw)−1]andhigh-cholesteroldiet;HChol-M1,Monascuspowder[1×,10.78mg
(day100gbw)−1]andhigh-cholesteroldiet;HChol-M5,Monascuspowder[5×,53.9mg(day100gbw)−1]andhigh-cholesteroldietDataarepresentedasmeans±SD(n=8).Meanvalueswithineachcolumnwithdifferentsuperscriptsaresignificantlydifferent(p<0.05)
Statisticalanalysis
Plasmaandliversamplesweremeasuredintriplicateusingcommercialkits.Themaintreatmenteffectswereanalyzedbyone-wayanalysisofvariance(ANOVA)usingthegene-rallinearmodelprocedureofSASsoftware(SASInstituteInc.,Cary,NC,USA).SignificantdifferencesamongdietarytreatmentswereanalyzedbythemethodofPDiffMatrixofleastsquaresmeansafterasignificantmaineffectbyone-wayANOVA.Thesignificancelevelwassetatp<0.05.Datawereexpressedasmeans±SD.
anddailyintakeofthehamstersincreasednormallyanddidnotdifferamongthevariousgroupsduringtheperiodofthepracticalexperiment.Inaddition,theexternalsandhealthofallexperimentalanimalshadanormalexpression.Animalswereanesthetizedandkilledbycarbondioxideinhalationatthe4thandthe8thweek,respectively,andbloodandliversampleswerecollectedandexaminedforestimatingthehypolipidemiceffectsofMonascuspowder.EffectofMonascuspowderonplasmaTCandTGlevels
TheredmoldricepowderfermentedbyM.purpureusNTU568wasusedasahypolipidemicsampletobeusedinthisstudy.Table3showsthechangeofplasmaTCandTGlevels.AsisevidentfromthedataoftheplasmaTClevelsatthe4thweek,thecontrolgroupregistered91.8mgdl−1,andtheHCholgroupfedwithhigh-cholesteroldiethadextremelyhighlevelsat170mgdl−1.TheplasmaTClevelsoftheHCholgroupweremaintainedatover170mgdl−1for8weeksbythefeedingofthehigh-cholesteroldiet.Theseresultsprovedthatthesupplementationofacho-lesteroldietresultsinasignificantincreaseintheplasmaTClevelsincontrastwiththecontrolgroups,andthatit
Results
Thechangeofbodyweightanddailyintakebyhamsters
Inthisstudy,weselectedhamsterasanexperimentalanimalmodelforhypolipidemiceffectsofMonascuspowder.Experimentalanimalsweregivenfreeaccesstoregularrodentchowandwaterfortheobservationstageof4weeks,andthenthe8-weekstudycommenced.TheaveragebodyweightanddailyintakeofhamstersareshowninTable2.Theresultsindicatethatthebodyweight
Table3EffectofMonascuspowderonexperimentalhamsterperformanceplasmaTCandTGlevelsGroup
0thweekCholesterol(mgdl−1)
ControlHChol
HChol–probucolHChol-M1/2HChol-M1HChol-M5
89.7±13.7–––––
Triglyceride(mgdl−1)98.5±10.5–––––
4thweekCholesterol(mgdl−1)91.8±5.37d170±15.3a108±18.7bc149±15.3b117±10.6b104±16.1cdTriglyceride(mgdl−1)104±7.06e186±21.1a165±15.3ab150±12.8b130±14.4c113±7.16d8thweekCholesterol(mgdl−1)89±9.4c173±12.7a124±17.6b140±16.5b135±19.4b134±15.3bTriglyceride(mgdl−1)120±17.7c168±12.9a133±27.9bc146±25.3b138±12.4bc134±15.3bcControl,normaldiet(0%cholesterol);HChol,high-cholesteroldiet;HChol–probucol,probucolandhigh-cholesteroldiet;HChol-M1/2,Monascuspowder[0.5×,5.39mg(day100gbw)−1]andhigh-cholesteroldiet;HChol-M1,Monascuspowder[1×,10.78mg
(day100gbw)−1]andhigh-cholesteroldiet;HChol-M5,Monascuspowder[5×,53.9mg(day100gbw)−1]andhigh-cholesteroldietDataarepresentedasmeans±SD(n=8).Meanvalueswithineachcolumnwithdifferentsuperscriptsaresignificantlydifferent(p<0.05)
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canbeappliedasananimalmodelforhyperlipidemia.Probucol,acholesterol-loweringdrug,wasorallyfedtotheHChol–probucolgrouptoestablishapositivecontrolgroup.Theresultsafterthe4thandthe8thweekshowedthatprobucolsignificantlyloweredtheplasmaTClevelsofhyperlipidemiahamster(p<0.05).
AsshowninTable3,theamountofplasmaTCwassignificantlylessintheMonasucspowder-fedgroups(HChol-M1/2,HChol-M1,andHChol-M5)thanintheHCholgroup.Fromthisresult,weconcludethatMonascuspowderfermentedbyM.purpureusNTU568haswithoutdoubtproventoreduceplasmaTClevels.FeedingMonascuspowder(onefolddosage)for4and8weekswillcausea31.2and22.0%decreaseintheplasmaTClevels,respectively,comparedtotheHCholgroup.TheMonascuspowderdosageofHChol-M1/2,HChol-M1,andHChol-M5werehalf-fold,onefold,andfivefold,respec-tively.AdoseresponsewasfoundbetweenthefeedingdosageofMonascuspowderandthereducedrateofplasmaTClevels,andtherewasasignificantdecreaseinplasmaTClevelscomparedtotheHCholgroupbyfeedingMonascuspowderathalf-folddosage.
AsfarastheplasmaTGlevelsareconcerned,theplasmaTGlevelsoftheHCholgroupatthe4thandthe8thweekweremeasuredat186and168mgdl−1,respectively,whichwerebothsignificantlyhigherthanthatofthecontrolgroup.However,theresultsindicatedthattheplasmaTGsecretionofhamsterwasinhibitedbyfeedingatleastahalf-folddosageofMonascuspowderdailyfor4and8weeks,respectively,althoughthehamsterswerefedahigh-cholesteroldiet.TheHChol-M1grouphadaplasmaTGlevelsat130and138mgdl−1,whichwerealsosignif-icantlylessthanthatoftheHCholgroupby30.1and17.9%,respectively(p<0.05).TheplasmaTG-loweringeffectdependedonthedosageofMonascuspowder,andtherewereremarkabledifferencesintheplasmaTGlevelsamongtheMonascuspowder-treatedgroups.TheresultsindicatedthatMonascuspowderfermentedbyM.purpu-reusNTU568positivelyprovedtoreduceplasmaTGlevelsofhyperlipidemiahamster.
EffectofMonascuspowderonserumHDL-CandLDL-Clevels
TheeffectsofMonascuspowderontheserumHDL-CandLDL-ClevelsofhamsterareshowninTable4.TheHDL-ClevelsweresignificantlyhigherintheHCholgroupthaninthecontrolgroup,becauseHDL-CandLDL-Clevelsarebothtypicalofcholesterol,whichwillsignificantlyincreasebyfeedingofahigh-cholesteroldiet.TheresultsobtainedfromTable3demonstratedtheeffectofMonascuspowderofloweringplasmaTCandTGlevels.However,LDL-Cisoneoftheriskfactorsofatherosclerosis,and,therefore,theinfluenceofMonascuspowderontheLDL-C-loweringeffectneedstobefurtherinvestigated.TheresultsshowninTable4agreedapproximatelywiththeexpectation.ThemajortypeofcholesteroldecreasedbyMonascuspowderwasLDL-C.Fortunately,theHDL-Clevelsdonotdependonthecholesterol-loweringeffectasdoesthedecreaseinLDL-Clevel.Asexpected,theHCholgrouphadhigherLDL-Clevelsthanthecontrolgroupatthe4thand8thweek.InhibitionofLDL-Csecretionde-pendedonthedosageoftheMonascuspowder.Monascuspowderfeedingataonefolddosageatthe4thand8thweekresultedinasignificantdecreaseintheserumLDL-Clevels,comparedtothoseoftheHCholgroup,by36.0and20.7%,respectively(p<0.05).LDL-Chasbeenproventoinducetheriskofatherosclerosis.However,anefficientLCL-C-loweringeffectbyMonascuspowderfermentedbyM.purpureusNTU568willreversethisresult.
TheratioofLDL-CtoHDL-Cwasanothercriterionforevaluatingtheefficiencyofhypolipidemic.Iftheratiowaslow,thenthecontentofHDL-Chadamuchhigherper-centageinTClevels,whileonthecontrary,theathero-scleroticriskfactorLDL-Cwaslowered.TheresultsinTable4indicatethatfeedingthecholesteroldietfor4and8weeksledtoasignificantincreaseintheratioofLDL-CtoHDL-Ccomparedtothatofthecontrolgroup.TheresultsindicatedthathamstersfedwithMonascuspowderwouldincreasetheirHDL-ClevelsandsignificantlydecreasetheirLDL-Clevelatthesametime.Therefore,theresultsobtainedbyastatisticalanalysisshowsthattheratioof
Table4EffectofMonascuspowderonexperimentalhamsterperformanceserumHDLandLDLlevelsGroup
0thweek
HDL-CLDL-C
−1(mgdl)(mgdl−1)
ControlHChol
HChol–probucolHChol-M1/2HChol-M1HChol-M5
58.8±6.76–––––
30.15±5.61–––––
LDL-C/HDL-C0.51±0.084–––––
4thweekHDL-C(mgdl−1)61.8±2.24d80.4±10.4ab67.7±5.78c74.3±6.74bc89.7±12.7a89.5±12.8aLDL-C(mgdl−1)31.7±1.8b59.2±8.6a36.0±8.1b56.3±8.1a37.9±10.5b32.6±8.1bLDL-C/HDL-C0.51±0.042b0.74±0.096a0.53±0.107b0.77±0.165a0.45±0.146bc0.37±0.116c8thweekHDL-C(mgdl−1)59.3±6.16d81.0±8.47b79.0±10.3bc71.7±6.36c89.2±11.0ab96.8±14.9aLDL-C(mgdl−1)30.9±3.7c61.4±5.8a44.4±7.0b52.4±10.5ab48.7±10.2b45.0±9.4bLDL-C/HDL-C0.52±0.059b0.77±0.114a0.57±0.101b0.74±0.172a0.56±0.155b0.47±0.095bControl,normaldiet(0%cholesterol);HChol,high-cholesteroldiet;HChol–probucol,probucolandhigh-cholesteroldiet;HChol-M1/2,Monascuspowder[0.5×,5.39mg(day100gbw)−1]andhigh-cholesteroldiet;HChol-M1,Monascuspowder[1×,10.78mg
(day100gbw)−1]andhigh-cholesteroldiet;HChol-M5,Monascuspowder[5×,53.9mg(day100gbw)−1]andhigh-cholesteroldietDataarepresentedasmeans±SD(n=8).Meanvalueswithineachcolumnwithdifferentsuperscriptsaresignificantlydifferent(p<0.05)
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Table5EffectofMonascuspowderonexperimentalhamsterperformanceliverTCandTGlevelsGroup
0thweekCholesterol(mgdl−1)
ControlHChol
HChol–probucolHChol-M1/2HChol-M1HChol-M5
108±20.5–––––
Triglyceride(mgdl−1)50.0±9.22–––––
4thweekCholesterol(mgdl−1)103±12.8d227±27.7a148±29.2c183±27.4b175±35.8bc150±30.9cTriglyceride(mgdl−1)52.7±5.27c66.8±5.25a51.7±6.52c56.1±6.69bc57.4±6.20bc59.6±4.27b8thweekCholesterol(mgdl−1)96.3±15.1e222±12.8a189±28.3b163±19.5c140±22.4d156±31.2cTriglyceride(mgdl−1)55.1±7.18c67.7±5.80a58.6±10.1b60.3±5.83b59.2±5.10b57.8±4.32bControl,normaldiet(0%cholesterol);HChol,high-cholesteroldiet;HChol–probucol,probucolandhigh-cholesteroldiet;HChol-M1/2,Monascuspowder[0.5×,5.39mg(day100gbw)−1]andhigh-cholesteroldiet;HChol-M1,Monascuspowder[1×,10.78mg
(day100gbw)−1]andhigh-cholesteroldiet;HChol-M5,Monascuspowder[5×,53.9mg(day100gbw)−1]andhigh-cholesteroldietDataarepresentedasmeans±SD(n=8).Meanvalueswithineachcolumnwithdifferentsuperscriptsaresignificantlydifferent(p<0.05)
LDL-CtoHDL-CwaslessintheHChol-M1andHChol-M5groupsthanintheHCholgroup(p<0.05).
EffectofMonascuspowderonliverTCandTGlevelsTheeffectofMonascuspowderonloweringTCandTGlevelsofliverisshowninTable5.Asexpected,hamsterstreatedwithhigh-cholesteroldietfor4and8weeksshowedamarkedincreaseinliverTCandTGlevelscomparedtothecontrolgroup.LiverTCandTGlevelsinallhamstersfedwithMonascuspowderfor4and8weekswerebothsignificantlylowerthanthoseinhyperlipidemiahamsters(p<0.05).
Plasmaliverindexanalysisandliverbiopsy
ManycommercialMonascusproductscontainhighlevelsofcitrinin,whichisatypeofmycotoxincausingliverandkidneydamage.Althoughthemutationtreatmentinpre-viousstudyshowedamarkeddecreasefrom2,500to939μgkg−1incitrininproductionofM.purpureusNTU568comparedtothewildtypestrain(submittedto
JAFC),thesafetyofMonascuspowderneedstobeeval-uated.PlasmaGOTandplasmaGPTlevelswereassayedtoevaluatewhetherornottheMonascuspowdercausesliverdamagetothehamsters.BothGOTandGPTexistsinthelivercells,whichisreleasedintothebloodwhenalivercellisdamaged.AsshowninTable6,plasmaGOTandGPTlevelsinhyperlipidemiahamsterswerenotincreasedbyfeedingthemMonascuspowderascomparedwiththeHCholgroup.However,theHCholgrouphadhigherplasmaGOTandGPTlevelsthanthecontrolgroupafterthe4thweek.
Sacrificingthehamstersafter8thweek,livertissuewasremoved,collected,andthenabiopsywasdone.Thephotoobtainedbymicroscopicexaminationduringtheliverbiopsy(Fig.1)didnotshowanysignificantdamageinlivertissueofMonascuspowdergroupscomparedwithcontrolgroupandHCholgroup.
Discussion
Endo(1979)demonstratedthatMonascussp.formedacholesterol-loweringagent,monacolinK,whichwasproventopossesstheidenticalstructurewithlovastatin.ConversionofHMG-CoAtomevalonatebyHMG-CoA
Table6EffectofMonascuspowderonexperimentalhamsterperformanceplasmaGPTandGOTlevelsGroup
0thweekGPT(Udl−1)
ControlHChol
HChol–probucolHChol-M1/2HChol-M1HChol-M5
70.1±12.9–––––
GOT(Udl−1)66.4±17.6–––––
4thweekGPT(Udl−1)68.3±13.6c109±10.8a92.5±17.0b73.7±15.6c93.3±9.5b76.5±13.1cGOT(Udl−1)64.2±18.4b86.6±21.7a67.8±13.9b64.0±14.6b74.0±14.0ab66.3±17.9ab8thweekGPT(Udl−1)112±12.6a104±18.9ab100±10.2ab94±14.5bc86±11.3cd74±11.2dGOT(Udl−1)70±18.3a68±11.4a64±17.3a61±6.2a57±19.5a58±14.1aControl,normaldiet(0%cholesterol);HChol,high-cholesteroldiet;HChol–probucol,probucolandhigh-cholesteroldiet;HChol-M1/2,Monascuspowder[0.5×,5.39mg(day100gbw)−1]andhigh-cholesteroldiet;HChol-M1,Monascuspowder[1×,10.78mg
(day100gbw)−1]andhigh-cholesteroldiet;HChol-M5,Monascuspowder[5×,53.9mg(day100gbw)−1]andhigh-cholesteroldietDataarepresentedasmeans±SD(n=8).Meanvalueswithineachcolumnwithdifferentsuperscriptsaresignificantlydifferent(p<0.05)
539
Fig.1Themicroscopyphotos(400×and100×)ofliverbiopsyofexperimentalhamster.aControlgroupbHCholgroupcHChol–probucolgroupdHChol-M1/2groupeHChol-M1groupfHChol-M5group
reductaseisarate-limitingstepincholesterolbiosynthesis.LovastatinisaclinicallyusedHMG-CoAreductasein-hibitorknowntolowerserumcholesterollevels.Ingeneral,lovastatinisobtainedviathepurificationofthecrudefermentedproductbyAspergillusterreus(ManzoniandRollini2002).A.terreusisnotastrainofGRAS(generallyregardedassafe),and,furthermore,thepurificationoflov-astatinhastobecarriedoutthroughmanychemicalpro-cesses,solovastatinisnormallyusedasaclinicaldrug.AlthoughredmoldricefermentedbyMonascusisregardedasafunctionalfoodhavingacholesterol-loweringeffect,sincemonacolinKisidenticaltolovastatin,monacolinKproductionismuchlowerinmanyMonascusspeciesthaninA.terreus,whichmakesitverydifficulttouseaMon-ascusproductforcholesterol-loweringaction.
Asshownintheresults,MonascuspowderofM.pur-pureusNTU568significantlyreducedTC,TG,andLDL-CwithoutchangingtheHDL-Clevels.TheresultsofthepresentstudyshowedthatMonascuspowdersignificantlydecreasedTCandTGlevelsatthehalf-folddose.FeedinghamsterwithMonascuspowderathalf-folddosagecor-respondstosupplementingadailydietwithMonascuspowderat0.5gforpeoplewithabodyweightof70kgandaheightof170cm.MonascuspowderfermentedbyM.purpureusNTU568andadministeredatalowerdosagedidindeedhavearemarkableeffectofcholesterol-loweringactionascomparedwiththeeffectual(andhigher)dosageofotherMonascuspowder.Inaddition,thelipidlevelsweregraduallyreducedasthedosageofMonascuspowderofM.purpureusNTU568wasincreased.
AsfarasHDL-Clevelswasconcerned,theserumHDL-ClevelswereincreasedcomparedwiththeHCholgroupandthecontrolgroup,althoughnottoastatisticallysig-nificantlevel.ThequestionofwhethermoreMonascuspowdercanincreaseHDL-ClevelsmorethanlessMon-ascuspowder,orifprobucolwasmoreeffective,wasraisedaswell.ItiswellestablishedthatincreasedHDL-Clevelsarebeneficialtohumanhealthbyreducingtheriskfordevelopingcardiovasculardisease.TheelevatedHDL-ClevelsobtainedbytheuseofMonascuspowder,especiallywhenenhancedtoafivefolddosage,maybenefitthecardiovascularsystem.However,moreinvestigationsareneededtoascertainthiseffect.
Probucolhadbeenproventoreducecholesterollevelsinapreviousstudyandisusedasaclinicaldrug(Notoetal.2003;Michiharaetal.2003).Forthisreason,itwasusedasthepositivecontroloncholesterol-loweringactionasasubstituteforMonascuspowder.Asshownintheresults,hamstersfedwithprobucolandhigh-cholesteroldiethadasignificantdecreaseintheplasmaTClevelsatthe4thand8thweekcomparedwiththeHCholgroup.ThedecreaseinplasmaTGlevelswerenotsignificantinhyperlipidemiahamstersfedwithprobucolfor4weeksascomparedwiththeHCholgroup,butoraladministrationofprobucolfor8weeksresultedinasignificantdecreaseinplasmaTGlevels.Probucolisusedasacholesterol-loweringmedicineasaclinicalremedy,althoughitsmechanismofcholester-ol-loweringactionhadnotbeendemonstrated.Conse-quently,plasmaTGlevelsmightnotbeimmediatelydecreasedbyoraladministrationofprobucolascomparedwithhyperlipidemiahamsters.
ProtectionagainstliverdamagebyaMonascusproducthadbeenreportedinpreviousstudy(Aniyaetal.1999).Aniyaetal.demonstratedthatsomestrainsofMonascusankawouldprotectliveragainstchemicaldamagebyleadingtoanincreaseintheactivityofglutathione-s-transferaseandaspartateaminotransferase(Aniyaetal.1999).Inaddition,feedinghypercholesterolemicrabbitswith0.2to0.8gperdayofMonascuspowderwouldreducenotonlytheriskfordevelopingcardiovasculardiseasebutalsotheriskfordevelopinghepaticfibrosisandhepatomegaly(Lietal.1998).Inthisstudy,plasmaGOTandGPTlevelsshowedmorevariationamonghamstersin
540
thesamegroup,and,therefore,thesignificanteffectamongthevariousgroupswashardtoobservebystatisticalanal-ysis.GOTandGPTbothexistinthelivercell,whichisreleasedintothebloodwhenthelivercellisdamaged.Theresultsindicatedthatfeedinghamsterswithahigh-cho-lesteroldietmayresultinanincreaseinplasmaGOTandGPTlevelscomparedwithcontrolgroup,displayingaremarkableincreaseafterthe4thweek.Itishighlyprob-ablethatamassiveamountofcholesterolsupplementwillleadtoaburdenonthelivermetabolitesandsimultaneous-lyincreasetheriskfordevelopinghepaticfibrosis(Jeongetal.2005;Aguileraetal.2005;Papadiaetal.2004).However,thereweretendenciestowardloweringoftheplasmaGOTandGPTlevelsinhyperlipidemiahamsterswhentheywereorallyadministeredMonascuspowderfor4and8weeks,althoughithadnostatisticallysignificanteffect.Inaddition,citrinin,formedfromMonascus,istyp-icalofthemycotoxinresultingindamagetotheliver.AlthoughMonascuspowderfermentedbyM.purpureusNTU568contained939μgkg−1ofcitrinin,itdidnotleadtodamageoftheliver,aswasevidentwhenexaminingGOTandGPTlevelsaswellasbytheliverbiopsy.ItisworthnotingthattheproductofM.purpureusNTU568wasproventopossessgoodantioxidantactivityon1,1-diphenyl-2-picrylhydrazyl(DPPH)radicalscavengingac-tivity,conjugatedienesinhibition,andreducingpower(Leeetal.2005,submittedtoJAFC).Therefore,wesup-posethatanantioxidantoranothercompoundisformedbyM.purpureusNTU568notonlytorepaircitrinin-inducedliverdamagebutalsotoreducetheriskfordevelopinghepaticfibrosis.
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