Crude Polysaccharides Extracted From Several Medicinal Fungi Exhibit Hypoglycemic Functions in Mice.docx

Crude Polysaccharides Extracted From Several Medicinal Fungi Exhibit Hypoglycemic Functions in Mice.docx

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CrudePolysaccharidesExtractedFromSeveralMedicinalFungiExhibitHypoglycemicFunctionsinMice

CrudePolysaccharidesExtractedFromSeveralMedicinalFungiExhibitHypoglycemicFunctionsinMice

　　Abstract:

Thehypoglycemiceffectsofcrudepolysaccharidesextractedfromfivemedicinalfungiwereevaluatedusingalloxan-induceddiabeticmice.High（1g/kg）andlow（0.5g/kg）dosesofpolysaccharidesextractedfromCordycepstaiiandC.memorabiliscausedsignificant（P<0.01）decreasesinmicebloodsugarlevels.PolysaccharidesfromGanodermalucidumandC.militarisfruitbodiesbothexhibitedweakhypoglycemiceffectsatsignificancelevelsofP<0.01andP<0.05forhighandlowdoses,respectively.PolysaccharideextractedfrommyceliumofaPhellinussp.wassignificantly（P<0.05）hypoglycemicathigherdoselevelsbutlowerdoseshadnoeffect.

　　Keywords:

Cordyceps;Phellinus；Ganoderma；polysaccharides;hypoglycemiceffect

　　MedicinalfungiareaveryimportantpartoftraditionalChineseherbalmedicine.Extensiveresearchonthestructureandbioactivityoffungalcomponentshasindicatedthatfungalpolysaccharidesinparticulararehighlyeffectiveinmaintaininggeneralgoodhealth,andinthetreatmentofcertaindiseasessuchashyperglycemia.Forexample,polysaccharidesextractedfromGanodermalucidum,Cordycepsspp,Coriulusversicolor,Auriculariapolytricha,A.auricula,Tremellafuciformis,T.aurantialba,Hericiumerinaceum,Grifolafrondosa,ArmillariellamelleaandAgrocybepraecoxincreasedtherateofinsulinsecretionandloweredbloodglucoselevels［1,2］.WenowreporthypoglycemicfunctionsassociatedwithcrudepolysaccharidepreparationsfromfruitbodiesofG.lucidumandCordycepsmilitaris,andfromvegetativemyceliaofCordycepstaii,CordycepsmemorabilisandPhellinussp.

　　1MaterialsandMethods

　　1.1Strainsandtestsample

　　AllfungalstrainswereprovidedbytheGuangdongInstituteofMicrobiology（GIM）.Phellinussp.GIMYY0512wasisolatedfromafruitbodycollectedfromthewildatPreahVihear,Cambodia,andwasidentifiedasanewspeciesofPhellinusthroughITSsequenceanalysis（ITSsequencedepositedinGenbankunderaccessionNo.EU826013）;CordycepstaiiGIMYY0508andCordycepsmemorabilisGIMYY0607wereisolatedfromfruitbodiescollectedfromthewildatHenyang,HunanProvinceandDinghu,GuangdongProvince,respectively.FruitbodiesofGanodermalucidumGIMYY0403andCordycepsmilitarisGIMYY0610werecultivatedinourlaboratory.

　　1.2Testmice

　　Specificpathogen-free（SPF）NIHmice（maleandfemale,18～22g）weresuppliedbytheGuangdongMedicalandLaboratoryAnimalCenter,LicenceNos.SCXK（Yue）2003-002and2007A004.

　　1.3Reagentsandmedia

　　AlloxanwasfromAcrosOrganics,NewJersey,USA,Lot:

A011257301；metforminhydrochloridewasfromShenzhenChinaAssociatedPharmaceutical,Lot:

0610304；Glucosemono-reagentwasfromShanghaiKexinBio-technologyInstitute,Lot:

20070202.

　　Liquidmedium1（%w/v）：

glucose5,peptone1，KNO30.2，MgSO40.2，CaCl20.01,and（NH4）?

?

3PO4?

3H2O0.2;pHadjustedto7.0with1mol/LNaOH.

　　Liquidmedium2（%w/v）：

wheatbran3，glucose3，peptone0.3，yeastextract0.1,K?

?

2HPO40.1，andMgSO40.05;pHadjustedto6.0with1mol/LNaOH.

　　1.4Cultivationoffungalmycelia

　　Liquidmedium1（200mL）in500mLflaskswasinoculatedseparatelywith2%～5%（w/v）C.taiiGIMYY0508orC.memorabilisGIMYY0607myceliumandincubatedat26℃for5dwithshaking（120r/min）.Myceliawereharvestedbyfiltrationanddriedat50℃.

　　Liquidmedium2（200mL）in500mLflaskswasinoculatedwith15%～20%（w/v）Phellinussp.GIMYY0512myceliumandincubatedat28℃for7~10dwithshaking（120r/min）.Myceliawereharvestedbyfiltrationanddriedat50℃.

　　1.5Preparationofcrudepolysaccharides

　　Dryfruitbodiesormyceliaweregroundseparatelyintoapowderandsuspendedin30volwater.Afterextractionfor2hat100℃,thesuspensionwascentrifuged（1400g,10min）andthesolidresiduere-suspendedin20volswaterandextractedagainusingthesameprocedure.Combinedsupernatantswereconcentrated10-foldundervacuum,fourvolumesof90%（v/v）ethanolwereadded,andthemixturewasmaintainedat4℃overnighttoallowcompleteprecipitationofthepolysaccharides.Theprecipitateofcrudepolysaccharidewascollectedbycentrifugation（1400g,15min）anddriedat50～60℃.Thecarbohydratecontentwasdeterminedusingthephenol-sulfuricacidmethod［3］,andthepolysaccharideyield（%）=weightofcrudepolysaccharide/dryweightoffruitbodiesormycelia.

　　1.6Preparationofdiabeticmice

　　Micewerefedanormaldietfor3daysandthengivenonlywaterfor24hours.Alloxan（0.02g/mL）wasinjectedintotheabdominalcavityatadosageof200mg/kg.Bloodsugarlevelsweredetermined5dayslaterbeforethemicehadbeenfed,andanimalswithavalue>10.0mmol/Lwereselectedasdiabeticmice.

　　1.7Micetreatmentgroups

　　Tenuntreatedmiceweredesignatedasthe‘normal’group.Diabeticmiceweredividedrandomlyinto12groups（10miceineachgroup）thatincludedanegativecontrolgroup（administered10mL/kgsaline）,apositivecontrolgroup（administered250mg/kgmetforminhydrochloride）,andtentestgroupsadministeredeitherhigh（1g/kg）orlow（0.5g/kg）dosesofthedifferentcrudepolysaccharidepreparations.Stocksolutions（preparedinsaline）containingeither1.0g/10mLor0.5g/10mLofcrudepolysaccharidewereusedtoadministerhighandlowdoses,respectively.Treatmentswereadministeredthroughstomachfilling（10mL/kg）every24hfor7daysalongwithnormalfeedandwater.Afterthefinalstomachfilling,micewerefastedfor24handbloodsamplesweretakenfromeyeveins.Serumwasseparatedandbloodsugarvaluesweredeterminedusingtheglucoseoxidasemethodaccordingtothemanufacturer?

?

sinstructions.

　　1.8Dataanalysis

　　AllexperimentaldataweresubjectedtostatisticalanalysisbyExcelsoftwareandthet-test.

　　2Resultsandanalysis

　　2.1Polysaccharideyields

　　PolysaccharideyieldsfromdifferentfungiareshowninTable1（seetheChineseversion）.Highestpolysaccharideyields（8.1%）wereobtainedfromC.memorabilismyceliumwhereaslowestyields（2.3%）werefromG.lucidumfruitbodies.

　　2.2Effectofcrudepolysaccharidepreparationsondiabeticmice

　　TheeffectsofthedifferentpolysaccharidetreatmentsondiabeticmicearesummarizedinTable2（seetheChineseversion）.Allofthesamplesexhibitedhypoglycemicfunctionsthatweremorepronouncedinthehigherdosagegroups.CrudepolysaccharideextractedfrommyceliaofC.taiiandC.memorabilisweremosteffectiveinloweringserumglucoselevels.TheimpactsofbothhighandlowdosagesweresignificantatP<0.01,andglucoselevelsinboththehigherdosagegroupsweresimilartothelevelobservedinthepositivecontrolgrouptreatedwithmetformin,acommonmedicinefordiabetics.Incomparison,relativelyweakeffectswereobservedwithcrudepolysaccharideextractedfromfruitbodiesofG.lucidumandC.militaris;theimpactofhighandlowdoseswassignificantatP<0.01andP<0.05,respectively.NosignificanteffectcomparedwithnegativecontrolswasobservedwiththetestgroupgivenlowdosesofcrudepolysaccharideextractedfrommyceliumofPhellinussp.althoughtherewasasignificantimpact（P<0.05）inthegroupadministeredhigherdosesofthismaterial.

　　3Discussion

　　Thecytotoxinalloxandamagespancreaticβ-cellsandiswidelyusedtoinduceexperimentaldiabetesinanimalsinordertotestdifferentcompoundsforhypoglycemicfunctions.Inthepresentstudy,lowerdosages（0.5g/kg）ofcrudepolysaccharideextractedfromG.lucidumloweredbloodglucoselevelsby9.3%inalloxan-induceddiabeticmicecomparedwithcontrolsafteroneweekoftreatment.ThesefindingsareinagreementwithpreviousreportsbyCHENetal［4］,andbyZHANGandFENG［5］.However,ourfindingthatcrudepolysaccharidefromC.militarisatdosesof1.0g/kgloweredglucoselevelsby11.3%didnotcomplywiththeresultsofZHANGetal.［6］,althoughthismayhavebeenduetodifferencesinthefungalstrainand/ortheanimalmodeluseintheirinvestigation.

　　C.taii,whichcontainsvariouschemicalcomponentsincommonwithC.sinensis［7~9］,haspreviouslybeenreportedtopossessimmunomodulatoryfunctions［10,11］,

　　whereasC.memorabilishasbeenassignednervecellprotectingproperties［12,13］,aswellasantisepticandcytotoxicactivity［14］.However,asfarasweareaware,thisisthefirstreportofhypoglycemicfunctionsassociatedwithcrudepolysaccharidematerialextractedfromthesetwofungi.BothMCTandMCMexhibitedgoodhypoglycemiceffectswhenusedathigherdosages（1g/kg）.Ourdatashowedthatbloodglucosewasloweredtosimilarlevelsobservedinmicegivenmetforminhydrochloride,andsuggestthatthesematerialsareworthyoffurtherinvestigation.

　　References

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