天然产物化学英文版副本汇总教学文案.docx
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天然产物化学英文版副本汇总教学文案.docx
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天然产物化学英文版副本汇总教学文案
EffectofOryzanolandFerulicAcidontheGlucoseMetabolismofMiceFedwithaHigh-FatDiet
MyoungJinSon,CatherineW.Rico,SeokHyunNam,andMiYoungKang
Abstract:
Theeffectsoforyzanolandferulicacidontheglucosemetabolismofhigh-fat-fedmicewereinvestigated.MaleC57BL/6Nmicewererandomlydividedinto4groups:
NCgroupfedwithnormalcontroldiet;HFgroupfedwithhigh-fat(17%)diet;HF-Ogroupfedwithhigh-fatdietsupplementedwith0.5%oryzanol;andHF-FAgroupfedwithhigh-fatdietsupplementedwith0.5%ferulicacid.Allanimalswereallowedfreeaccesstotheexperimentaldietsandwaterfor7wk.Attheendoftheexperimentalperiod,theHF-OandHF-FAgroupsexhibitedsignificantlylowerbloodglucoselevelandglucose-6-phosphatase(G6pase)andphosphoenolpyruvatecarboxykinase(PEPCK)activities,andhigherglycogenandinsulinconcentrationsandglucokinase(GK)activitycomparedwithNCandHFgroups.Theresultsofthisstudyillustratethatbothoryzanolandferulicacidcouldreducetheriskofhigh-fatdiet-inducedhyperglycemiaviaregulationofinsulinsecretionandhepaticglucose-regulatingenzymeactivities.
Keywords:
diabetes,ferulicacid,high-fat-fedmice,hypoglycemiceffect,oryzanol
Introduction
Chronicconsumptionofahigh-fatdiethasbeenassociatedwiththedevelopmentofobesityandtype2diabetesmellitus(Hillandothers1992;Brayandothers2004).Scientificstudieshaveshownthatexcessiveintakeofdietaryfatresultsinincreasedbodyweightandpoorglucoseregulation(AlsaifandDuwaihy2004;Petroandothers2004;Messierandothers2007).Diabetesischaracterizedbyhyperglycemiathatresultsinthegenerationoffreeradicalsleadingtooxidativestress(West2000).Duetochangesinlifestylepatterns,particularlypooreatinghabitandsedentarylifestyle,theincidenceofdiabeteshasrapidlyincreasedinepidemicproportions.Around171millioncasesofdiabetesworldwidewerereportedin2001anditwasprojectedthatby2030,366millionpeoplewillhavediabetes(Wildandothers2004).Withthisincreasingglobalprevalenceofdiabetes,theneedfortherapeuticmeasuresagainstthediseasehasbecomestrongerandmoreurgent.Awiderangeoforalmedicinesarecurrentlybeingusedfortreatingdiabetes.However,variousadverseeffectsandhighratesofsecondaryfailureshavebeenassociatedwiththeavailableantidiabeticmedicines(Inzucchi2002).Thus,findingnaturaldrugswithhypoglycemicactivityhasnowbecomethefocusofscientistsandresearchers.
Atpresent,thereisaconsiderablepublicandscientificinterestinutilizingphytochemicalsforthetreatmentandpreventionofvariousdiseases.Naturallyoccurringphenoliccompounds,suchasoryzanolandferulicacid,areknowntohavestrongantioxidantactivities(Wangandothers2002;Srinivasanandothers2007).Oryzanolisamixtureofferulicacid(4-hydroxy-3-methoxycinnamicacid)esterswithphytosterols(Lerma-Garciaandothers2009)andprimarilyextractedfromricebran.Ferulicacidiscommonlyfoundinfruitsandvegetables,includingbanana,broccoli,ricebran,andcitrusfruits(ZhaoandMoghadasian2008).Bothoryzanolandferulicacidpossessseveralphysiologicalproperties,suchasreductionofserumcholesterollevels(Wilsonandothers2007),inhibitionoftumorpromotion(Yasukawaandothers1998),andprotectiveactionagainstliverinjury(Choti-markornandUshio2008).Oxidativestressisregardedasthekeyfactorinthedevelopmentofdiabetesanditsassociatedhealthdisorders.Thehigh-fatdietfedC57BL/6mousemodelhaslongbeenusedbyresearchersininvestigatingthepathophysiologyofimpairedglucosetoleranceandtype2diabetesandforthedevelopmentofnewtreatments(Surwitandothers1988;Surwitandothers1991;Schreyerandothers1998;WinzellandAhren2004).Sincediabetesisafreeradicalmediateddisease,thestrongantioxidantactivityoforyzanolandferulicacidmaybeusefulinpreventingthedevelopmentofdiabetichyperglycemiaunderahigh-fatdiet.Therearelimitedreportsonthephysiologicalfunctionsofthesephenoliccompoundsinrelationtoglucosemetabolisminanimalmodels.Thus,thisstudywasconductedtoinvestigatetheeffectsofdietaryfeedingoforyzanolandferulicacidontheglucosemetabolisminhigh-fat-fedC57BL/6mice.
1.MaterialsandMethods
1.1Animalsanddiets
Twenty-fourmaleC57BL/6Nmiceof4wkofage,weighing12g,wereobtainedfromOrientInc.(Seoul,Korea).Theywereindividuallyhousedinstainlesssteelcagesinaroommaintainedat25◦Cwith50%relativehumidityand12/12hlight/darkcycleandfedwithapelletizedchowdietfor2wkafterarrival.Themicewerethenrandomlydividedinto4dietarygroups(n=6).The1stand2ndgroupswerefedwithanormalandhigh-fat(17%,w/w)diets,respectively,whiletheother2groupswerefedwithhigh-fatdietsupplementedwitheither0.5%oryzanolor0.5%ferulicacid(>98%pure,Tsuno,Osaka,Japan).Thecompositionoftheexperimentaldiet(Table1)wasbasedontheAIN-76semisyntheticdiet.
Themicewerefedfor7wkandallowedfreeaccesstofoodandwaterduringtheexperimentalperiod.Thebodyweightgainwasmeasuredweekly.Attheendoftheexperimentalperiod,themicewereanaesthetizedwith60-μLKetamine-HClfollowinga12hfastandsacrificed.Bloodsampleswerecollectedandcentrifugedat1000×gfor15minat4◦Ctoobtaintheplasma.Theliverswereremoved,rinsedwithphysiologicalsaline,andstoredat−70◦Cuntilanalysis.ThecurrentstudyprotocolwasapprovedbytheEthicsCommitteeofKyungpookNatl.Univ.foranimastudies.
1.2Measurementofbloodglucoselevel
ThebloodglucoselevelinmicewasmeasuredusingAccu-ChekActiveBloodGlucoseTestStrips(RocheDiagnosticsGmbH,Germany).Bloodsamplesweredrawnfromthetailveinofthemicebeforeandafter3and7wkoffeedingtheanimalswithexperimentaldiets.
1.3Determinationofglycogenandinsulinlevels
TheglycogenconcentrationinliverwasdeterminedusingthemethoddescribedbySeifterandothers(1950)。
Freshliver(100mg)wasmixedwith30%KOHandheatedat100◦Cfor30min.Themixturewasthenaddedwith1.5mLethanol(95%)andkeptovernightat4◦C.Thepelletwasmixedwith4mLdistilledwater.A500μLofthemixturewasaddedwith0.2%anthrone(in95%H2SO4)andtheabsorbanceofthesamplesolutionwasmeasuredat620nm.Theresultswerecalculatedonthebasisofastandardcalibrationcurveofglucose.Theinsulincontentwasmeasuredusingenzyme-linkedimmunosorbentassay(ELISA)kits(TMBMouseInsulinELISAkit,Sibayagi,Japan).
1.4Measurementofhepaticglucose-regulatingenzymeactivities
ThehepaticenzymesourcewaspreparedaccordingtothemethoddevelopedbyHulcherandOleson(1973).Theglucokinase(GK)activitywasdeterminedbasedfromthemethodofDavidsonandArion(1987)withslightmodification.A0.98mLofthereactionmixturecontaining50mMHepes-NaOH(pH7.4),100mMKCl,7.5mMMgCl2,2.5mMdithioerythritol,10mg/mLalbumin,10mMglucose,4unitsofglucose-6-phosphate(G6pase)dehydrogenase,50mMNAD+,and10μLcytosolwaspreincubatedat37◦Cfor10min.Thereactionwasinitiatedwiththeadditionof10μLof5mMATPandthemixturewasincubatedat37◦Cfor10min.TheG6paseactivitywasmeasuredusingthemethoddescribedbyAlegreandothers(1988).Thereactionmixturecontained765μLof131.58mMHepes-NaOH(pH6.5),100μLof18mMEDTA(pH6.5),100μLof265mMG6pase,10μLof0.2MNADP+,0.6IU/mLmutarotase,and0.6IU/mLglucosedehydrogenase.themixturewasaddedwith5μLmicrosomeandincubatedat37◦Cfor4min.Thechangeinabsorbanceat340nmwasmeasured.Thephosphoenolpyruvatecarboxykinase(PEPCK)activitywasdeterminedbasedfromthemethoddevelopedbyBentleandLardy(1976).Thereactionmixtureconsistedof72.92mMsodiumHepes(pH7.0),10mMdithiothreitol,500mMNaHCO3,10mMMnCl2,25mMNADH,100mMIDP,200mMPEP,7.2unitofmalicdehydrogenase,and10μLcytosol.Theenzymeactivitywasdeterminedbasedfromthedecreaseintheabsorbanceofthemixtureat350nmat25◦C.在25◦C350nm。
1.5Statisticalanalysis
Alldataarepresentedasthemean±SE.Thedatawereevaluatedby1-wayANOVAusingaStatisticalPackageforSocialSciencessoftwareprogram(SPSSInc.,Chicago,Ill.,U.S.A.)andthedifferencesbetweenthemeanswereassessedusingDuncan’smultiplerangetest.StatisticalsignificancewasconsideredatP<0.05.
2.Results
2.1Bodyweightgain
Therewasnosignificantdifferenceinthebodyweightamongtheanimalgroupspriortofeedingthemicewiththeexperimentaldiets(Table2).
。
Thedailyfoodintakeofmicewasconstant(3g/d)throughoutthestudy.Attheendoftheexperimentalperiod,however,asignificantincreasewasobservedinanimalsfedwithhigh-fatdiet(HFgroup)relativetothatofthecontrolmice(NCgroup)。
Whilethemicefedwithhigh-fatdietsupplementedwithoryzanol(HF-Ogroup)orferulicacid(HF-FAgroup)alsoshowedhigherweightgaincomparedwiththatoftheNCgroup,theirbodyweightgainwassignificantlylowerthanthatoftheHFgroup.BetweenHF-OandHF-FAgroups,thelatterexhibitedlowerfinalbodyweight.
2.2Bloodglucoselevels
Theinitialbloodglucoselevelsinmicepriortofeedingwithexperimentaldietsdidnotsignificantlydifferamongthegroups(Figure1).
However,high-fatfeedingresultedinasignificantincreaseintheglucoselevelofmiceafter3wk.TheNCgroupalsoshowedanincreaseinglucosecontentsimilartothatoftheHFmice.Onthefinalwk,theHF-OandHF-FAmiceexhibitedsubstantiallylowerglucoselevelcomparedwiththeHFandcontrolgroups.
2.3Glycogenandinsulinlevels
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