口服舞茸子实体提取物表现出的抗肿瘤活性研究.docx
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口服舞茸子实体提取物表现出的抗肿瘤活性研究.docx
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口服舞茸子实体提取物表现出的抗肿瘤活性研究
声明:
本文摘自Chem.Pharm.Bull.,全称Chemical and Pharmaceutical Bulletin ,中文名《化学与药学通报》,由日本药学院(PSJ)出版,1953年创刊,全年12期,发表生物分析化学、生物化学、药理学、毒理学和生物药学方面的研究论文及报告,用英文出版。
为方便广大读者阅读,对部分内容进行了中文翻译,译文仅供参考,如有不足之处,敬请指正。
-----本文摘自舞茸D-fraction官方网站,转载请注明
【参考译文】
AntitumorActivityExhibitedbyOrallyAdministeredExtractfromFruitBodyofGrifolafrondosa(Maitake)
口服舞茸子实体提取物表现出的抗肿瘤活性研究
从舞茸子实体提取的酸不溶性、碱溶性、热水溶性高分子聚合物(一种约含30%蛋白的多糖;D-fraction)在小鼠口服试验中表现出抗同种肿瘤和同系肿瘤活性。
Winn的实验结果显示出了舞茸D-fraction口服给药对肿瘤生长的抑制作用。
这一事实表明舞茸D-fraction可激活荷瘤状态的免疫系统。
因此,证明了舞茸D-fraction的抗肿瘤细胞活性与免疫反应是相互关联的。
巨噬细胞或T细胞的细胞溶解活性和白细胞介素的生产能力所展现出的抗原特异性细胞毒性有所提高。
此外,舞茸D-fraction被发现可以增强与抑制肿瘤生长相关的迟发型超敏反应。
关键词:
舞茸;抗肿瘤活性;β-葡聚糖(D-fraction);迟发型超敏反应;口服给药
【论文原文】
Chem.Pharm.Bull.
36(5),1988,1819-1827
AntitumorActivityExhibitedbyOrallyAdministeredExtractfromFruit
BodyofGrifolafrondosa(Maitake)
IKUKOHISHIDA,HIROAKINANBA*andHISATORA KURODA
LaboratoryofMicrobiology,KobeWomen'sCollegeofPharmacy,
Motoyama,Higashinada,Kobe658,Japan
(ReceivedSeptember14,1987)
Theacid-insoluble,alkali-soluble,hot-water-extractablepolymer(apolysaccharidecontainingapproximately30%ofprotein;D-fraction)obtainedfromthefruitbodiesofGrifolafrondosa
(maitake)exhibitedantitumoractivitiesagainstallogenicandsyngeneictumorsonoraladministrationtomice.Winnassayconductedtoexaminethetumorgrowth-suppressingeffectrevealedacompleteinhibitionofthetumorbytheoraladministrationofD-fraction.Thisfactindicatesthatstimulationoftheimmuneresponsesystemtriggeredbythetumor-bearingstateisactivatedbyD-fraction.Consequently,theactivityofD-fractiononcellsassociatedwithimmuneresponsewasexamined.Thecytolyticactivityandinterleukin-1productivitypfmacrophagesorTcellswhichexhibitantigen-specificcytotoxicitywereenhanced.D-fractionwasfoundtopotentiatethedelayed-typehypersensitivityresponsewhichisassociatedwithtumorgrowthsuppression.
Keywords——Grifolafrondosa;antitumoractivity;β-glucan(D-fraction);delayed-typehypersensitivity(DTH);oraladministration
Manyantitumor-activepolysaccharidesderivedfrombasidiomyceteshavebeenreported,includinglentitan,shizophyllan,PSKandothers,mostofwhichprincipallyconsistofsimilarβ-glucans.Mostofthesepolysaccharides,excludingPSK,exhibitantitumoractivityviatheactivationofhostimmunesystemswhenadministeredintravenouslyorintraperitoneally.However,theyhavebeenreportedtobeineffectivewhengivenorally.TheauthorshaveisolatedfromthefruitbodyofGrifolafrondosa(maitake),aβ-glucan(MT-2)whichcarriesmany1,3-linkedoligosacchariedsidechainsbranchingfromthe1,6-linkedmainchain.Theauthorsreportedpreviously1,2)thatMT-2exhibitedantitumoractivitybystimulatingtheimmunesystemofmice,whenadministeredintraperitoneally.Miyazaki3)andMizunoetal.4)obtainedalentinan-likepolysaccharidefromthisfruitbodiesandreportedthatwasinactivewhenadministeredorally,whereasanantitumoreffectwasexhibitedwhenitwasadministeredintraperitoneallyorintravenously.Incontrast,theauthor5,6)haveobservedthattheimmunesystemsinmicewerestimulated,leadingtotheregressionoftumors,whenpowderedfruitbodyofLentinusedodes(shiitake)wasorallyadministeredtomice.Moreover,Ohkumaetal.,7)reportedthatstimulationoftheimmunesystemoccuredwhenaprotein-poly-saccharidecomplexextractedwithhotwaterfromFlammulinavelutipeswasadministeredorallytotumor-bearingmice.Inthiscontext,wehaveexaminedtheantitumoractivityofextractsfromthebodiesofmaitake.
MaitakeandMethods
(1) PreparationofExtractableMaitake——TheprocessusedforextractingmaterialsfromthepowderedfruitbodyofGrifolafrondosa,suppliedbytheMushroomResearchInstituteofJapan(KiryuGumma),wasbasedonthedescribedbyChiharaeral.fortheextractionoflentinanfromshiitake.8)AsshowninFig.1,thepowder(600g)wasmixedwith3000mlofdeionizedwater,andheatedat100℃for10h.Aftercentrifugationat6000rpmfor10min,thesupernatantwasusedaspre-Afraction.Anequalamountof99%EtOHwasaddedtothispre-AfractionandleftatdriedfruitbodiesofG.Frondosa(200g)
4℃for24h.TheprecipitateA-fractionwasseparatedanddissolvedindeionizedwater.Then,25%(CTA-OH)(cetyltrimethylammoniumhydroxide)wasaddeddropwisewithstirringuntilthepHexceeded12andthemixturewasallowedtostandat4℃for12htogiveaprecipitated.Thispelletwastreatedwith20-50%aceticacidtoremovetheacid-solublefraction,andtheresidualfractionwasobtainedasB-fraction.Thisfractionwastreatedwith6%NaOHtoseparatethealkali-solublefraction.Thissolutionwascombinedwith4volumesofEtOHtoobtaintheprecipitatepolymer.Thispellet(C-fraction)wasdissolvedinanequalamountofdistilledwateranddeproteinizedwithCHCl3-MtOH(9:
1).Thesolutionwasagaintreatedwith4volumesofEtOH.Theobtainedprecipitatepellet(D-fraction)wasalightbrownandamorphouspower,whichwaspositiveintheanthronetest,andcontainedapproximately30%proteinasdeterminebyLowry'smethod.TheD-fractionwaschargedonadiethylaminoethyl(DEAE)-Sepharosecolumn(5×40cm),andelutedwith0.01MTris-HClbuffercontaining0.1or0.5MNaCl(pH7.2)togiveE-fractionandF-fractionsuccessively.
(2)MiceandTumors-Sarcoma-180wastransplantedintoICRmice,MM-46carcinomaandMH-134hepatomaintoC3H/HeN(C3H)mice,IMCcarcinomaintoCDF1miceandB-16,melanomaintoC57BL/6mice.
(3)TumorGrowth-InhibitoryEffect-Tumorcells(1×106)wereimplantedsubcutaneouslyintherightaxillaryregionofmice,andfrom24hafteradministration,0.5mlofeachfraction,adjustedtoasugarconcentrationof3.0mg/ml,wasorallyadministeredonalternatedays.Afterthecompletionoftheadministration,thesolidtumorswereextirpatedandweighedtoobtainthetumorgrowthinhibitionrate(TIR),whichwascalculatedaccordingtothepreviouspater.1)
(4)WinnAssay9)-MM-46tumorcells(1×106)wereinoculatedintheaxillaryregion,andfrom24hafterinoculation,0.5mlofD-fraction,adjustedtoasugarconcentrationof1.5mg/ml,wasorallyadministered10timesonalternatedays.Afterthecompletionoftheadministration,thespleenwasextirpatedfromtheseD-fraction-administeredmice(hereaftertermedsimplyD-mice).Theobtained5×106spleencellsweremixedwith1×106MM-46Tumorcells.ThemixedcellsuspensionwasimplantedintotherightaxillaryregionofnormalC3Hmice(recipients),andafter14d,solidtumorswereextirpatedandweighed.Ontheotherhand,twoexperimentalsystemswereemployedtocomparetumorgrowthinhibitioninD-mice.Inthefirst,thetumorwasextirpatedfromC3HmiceinoculatedwithamixtureofMM-46tumorcellsandthespleencellsofMM-46tumor-bearingC3Hmicewhichhadbeengivensalineorally.Inthesecond,thegrpwingtumorwasisolatedfromC3HmiceimplantedwithonlyMM-46tumorcellsascontrolmice.
(5)PreparationofMacrophages(MΦ)-Five-week-oldC3H(orCDF1)malemicewerekilledbydislocatingcervicalvertebraeandperitonealcellswereobtainedbywashingwithHanks'solution.Aftercentrifugationat1200rpmfor5min,theprecipitatedcellswerecollectedandadjustedwithRPMI-1640mediumto1×105cells/ml.Thesecellswereincubatedfor45minat37℃inanatmospherecontaining5%CO2gas,andnon-adherentcellswereeliminatedbywashingwithHanks'solution.MΦwhichwereadsorbedselectivelyontheplatewallwerecollected.
(6) PreparationofWholeSpleenCells-TheextirpatedspleenfromC3Hmalemice(5weeksold)waswashedanddisintegratedsufficientlyinRPMI1640medium.Then,thecellswerepassedthrougha40-meshstainlesssteelscreen.Thecellswerecollectedbycentrifugationat1200rpmfor10minand0.2mlofa10-folddilutionofEagle'sMEMwasaddedtolysecontaminatingerythrocyteshypotonicallyfor10s,then2mlofEagle'sMEM(2-foldconcentration)wasaddedpromptlyandmixed.Thewholewascentrifugedat1200rpmfoe10min,andtheobtainedcellswereadjustedto1×107cells/mlandusedasthewholespleencellsuspension.
(7)PreparationofTCells -Wholespleencellsuspensionwaschargedonanylonwoolcolumn(1×15cm)moistenedwithRPMI-1640medium.Afterincubationfor60minat37℃inahumidifiedatmosphereof5%CO2inair,theTcellswerecollectedbycentrifugationat1200rpmfor5min.
(8)PreparationofLyt-1+TCells(10)-Anti-Lyt-2.1monoclonalantibody(50μl)wasmixedwith1×106 Tcell/ml.Afterincubationat37℃for60mininanatmosphereof5%CO2,inair,thesuspensionwaswashedwithRPMI-1640mediumbycentrifugation.Thecellsobtainedwereincubatedwith50μlofLow·Tox-Mrabbitcomplementfor60minat37℃.Thencellsobtainedbysubsequentcentrifugationwerestainedwithtrypanblue,andapreparationcontaining1×106 viablecellswasmade.
(9)LabelingoftheTargetCells-Asdescribedinthepreviouspaper,2)2×106tumorcellsweremixed50μlof40μCi/ml3H-uridineandafterincubationfor60minat37℃inanatmospherecontaining5%CO2gas,free3H-uridinewassufficientlyrinsedoutwithRPMI-1640medium.Thesamemediumwasfurtheraddedtoprepareasuspensioncontaining1×104cells/ml.
(10)CytotoxicityTest -TheeffectorcellsobtainedfromD-
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- 关 键 词:
- 口服 舞茸子 实体 提取物 表现出 肿瘤 活性 研究