Carbohydrate PolymersWord文档格式.docx
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Carbohydrate PolymersWord文档格式.docx
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13CNMRspectra,monosaccharidecompositionandmethylationanalyses,and
P.eryngii
wasselectedforfurthercontrolledSmithdegradation,andDEPTand
1H(obs.),
13CHMQCspectroscopy.Itwasabranchedβ-glucan,withamainchainof(1
→
3)-linked-Glcp
residues,substitutedatO-6bysingle-unitβ-Glcp
side-chains,onaveragetoeverythirdresidueofthebackbone,asinscleroglucan.
Keywords
∙Polysaccharides;
∙Ediblemushrooms;
∙Pleurotus
spp.;
∙β-Glucans
1.Introduction
Mushroomsareknownfortheirnutritionalandmedicinalvalueandthediversityoftheirbioactivecomponents(Ng,1998).Theseorganismshavelongbeenvaluedashighlytastyandnutritionalfoodsbymanysocietiesthroughouttheworld.Manyworldwidecultures,especiallyintheOrient,recognizethatextractsfromcertainmushroomscanhaveprofoundhealthpromotingbenefits.Ediblemushrooms,whichdemonstratemedicinalorfunctionalproperties,includespeciesofthegenera
Lentinus,
Hericium,
Grifola,
Flammulina,
Pleurotus,and
Tremella
(
Kü
es&
Liu,2000).
Pleurotus
spp.occursthroughoutthehardwoodforestsoftheworldthatincludethemostdiverseclimates(Gunde-Cimerman,1999
Rosadoetal.,2002).Theproductionof
hasbeenincreasingatarapidrate.Thesemushroomshaveattractedmuchattentionowingtothembeingagoodsourceofnon-starchycarbohydrates,withahighcontentofdietaryfiber,moderatequantitiesofproteinswithmostoftheessentialaminoacids,minerals,andvitamins(
Croan,2004).Theyhavebeenshowntomodulatetheimmunesystem,havehypoglycemicactivityandtoinhibittumorgrowth(
Gunde-Cimerman,1999
Wasser,2002).
Polysaccharidesrepresentastructurallydiverseclassofmacromoleculesofwidespreadoccurrenceinnatureandofferthehighestcapacityforcarryingbiologicalinformationbecausetheyhavethegreatestpotentialforstructuralvariability.Themonosaccharideunitsinoligosaccharidesandpolysaccharidescaninterconnectatseveralpointstoformawidevarietyofbranchedorlinearstructures(Ooi&
Liu,2000).Thisenormouspotentialvariabilitygivesthenecessaryflexibilityforpreciseregulatorymechanismsofvariousinteractionsinhigherorganisms.
Recentadvancesinchemicaltechnologyhaveallowedtheisolationandpurificationofsomecompounds,especiallypolysaccharideswhichpossesstrongimmunomodulationandanti-canceractivities.Theyareusedasbiologicalresponsemodifiers(Rout,Mondal,Chakraborty,Pramanik,&
Islam,2005).Thepolysaccharidesisolatedfrommushroomfruitingbodiesareeitherwatersolubleor/andinsolubleglucansandheteropolysaccharideswithdifferentmain-andside-chains.Thereisgreatinterestonthesemoleculesbecausetheycanactasbiologicalresponsemodifiers(Gonzagaetal.,2005,
Lavietal.,2006
Smithetal.,2002).
Wenowdescribetheisolationandchemicalcharacterizationofaβ-glucanfromthefruitingbodiesofPleurotuseryngii
andPleurotusostreatoroseus.
2.Materialsandmethods
2.1.Generalexperimentalprocedures
Allsolutionswereevaporatedat<
40
°
Cunderreducedpressure.Centrifugationwascarriedoutat9000
rpmfor15
min,at25
C.AlditolacetatemixturesformedfrompolysaccharideswereanalyzedbyGC–MSusingaVarianmodel3300gaschromatographlinkedtoaFinniganIon-Trap,model810-R12massspectrometer,usingaDB-23capillarycolumn(30
m
×
0.25
mmi.d.)programmedfrom50to220
Cat40
C/min,thenhold.PartiallyO-methylatedalditolacetatemixturesweresimilarlyanalyzed,butwithaprogramfrom50to215
C/min,thenhold.
2.2.Polysaccharideextractionandpurification
Extractionandpurificationoftheβ-glucansfromthefruitingbodiesofthetwospeciesof
wereprocessedaccordingto
Fig.1.Powdered-milledfruitingbodies(P.eryngii,64
g;
P.ostreatoroseus,66
g)wereextractedwith2:
1(v/v)CHCl3–MeOHat60
Cfor3
h(3×
350
mLeach)andthenwith4:
1(v/v)MeOH–H2Oat60
mLeach),toremovelow-molecular-weightmaterial.Theresiduewassubmittedtoextractionwithwaterat100
Cfor6
h(6×
800
mLeach).Thecombinedaq.extractswereevaporatedtoasmallvolumeandpolysaccharideprecipitatedbyadditiontoexcessEtOH(3:
1).Theprecipitatesfrom
(EPW-PE)and
P.ostreatoroseus
(EPW-PO)weredialyzedagainsttapwaterfor48
h,concentratedunderreducedpressuretosmallvolumes,whichwerefreeze-dried.EPW-PEandEPW-POwerethendissolvedinwaterandthesolutionssubmittedtofreezingfollowedbymildthawingat4
C,whichfurnishedsoluble(SEPW-PEandSEPW-PO)andinsolublegel-likefractions(IEPW-PEandIEPW-PO),whichwereseparatedbycentrifugation.
Fig.1.
Schemeofextractionandpurificationoftheβ-glucanfromthe
(PE)and
(PO).
Figureoptions
2.3.Monosaccharidecomposition
Eachfraction(1
mg)washydrolyzedwith2
MTFAat100
Cfor8
h,followedbyevaporationtodryness.TheresiduewassuccessivelyreducedwithexcessofNaBH4
and/orNaB2H4
andacetylatedwithAc2O–pyridine(1:
1,v/v;
2
mL)atroomtemperaturefor12
h(WolfromandThompson,1963a
WolfromandThompson,1963b).TheresultingalditolacetateswereanalyzedbyGC–MSasindicatedaboveandidentifiedbytheirtypicalretentiontimesandelectronimpactprofiles.
2.4.Methylationanalysis
Per-O-methylationoftheisolatedpolysaccharides(10
mgeach)wascarriedoutusing40%aq.NaOH(3
mL)andMe2SO4
(2
mL),addeddropwise(Haworth,1915).Theprocess,afterisolationoftheproductsbyneutralization,dialysis,andevaporationwasrepeated,andthemethylationwasfoundtobecomplete.Theproductsweretreatedwith50%v/vaq.H2SO4
(0.5
mLv/v,1
h,0
C),followedbyadilutionuntilitreached5.5%(additionof4.0
mLofdistilledwater).Thesolutionwaskeptat100
Cfor18
h(Saeman,Moore,Mitchell,&
Millet,1954),andwasneutralizedwithBaCO3,filtered,andthefiltrateevaporatedtodryness.TheresidueswereconvertedintopartiallyO-methylatedalditolacetates,andanalyzedbyGC–MS(asdescribedabove).
2.5.NMRanalyses
13CDEPTand
1H(obs.),
13CHMQCdeterminationswerecarriedoutusinga400
MHzBrukermodelDRXAvancespectrometerincorporatingFourierTransform.SamplesweredissolvedinMe2SO-d6
andexaminedat50or70
C.Chemicalshiftsareexpressedinppm(δ)relativetoresonanceofMe2SO-d6
at
δ
39.70(13C)and2.40(1H)forsamplesexaminedinthissolvent.
2.6.ControlledSmithdegradation
IEPW-PE(300
mg)wassubmittedtooxidationwith0.05
Maq.NaIO4
(20mL)for72
hat25
Cinthedark.Sampleswasthendialyzedagainsttapwaterfor48
handtreatedwithNaBH4
(pH9–10)for∼20
h(Goldstein,Hay,Lewis,&
Smith,2005).Thesolutionsweredialyzedandfreeze-dried.
Theproductswerethensubmittedtopartialacidhydrolysis(TFA,pH2.0,30
min,100
C)(Gorin,Horitsu,&
Spencer,1965)anddialyzedagainsttapwaterusingmembraneswithasizeexclusionof2
kDaandretainedmaterial(SM-PE,97
mg)wasfreeze-dried.
3.Resultsanddiscussion
Asshownin
Fig.1,
wereextractedwithCHCl3–MeOHandthenMeOH–H2Otoremovelow-molecularweightcompounds.Eachresultingresiduewassubmittedtoaqueousextractionsat100
C,andtheextractedpolysaccharideswererecoveredbyethanolprecipitation(fractionsEPW-PEandEPW-POfor
P.ostreatoroseus,respectively)weredialyzedagainsttapwater,andthesolutionfreeze-dried(EPW-PE,7.8%yield;
EPW-PO,7.7%yield).
EPW-PEandEPW-PObothcontainedglucoseastheirmaincomponent,besidesmannose,galactoseand3-O-methyl-galactose(
Table1).Thepresenceof3-O-methylgalactosewasconfirmedbyGC–MSionsatm/z
130and190afterNaBD4
reductionandacetylation.
Table1.
Monosaccharidecompositionandyieldsoffractionsobtainedfrom
P.ostreatoroseus
Fractions
Yieldsa
(%)
Monosaccharidesb
Man
3-O-MeGal
Gal
Glc
P.eryngii
EPW-PE
7.8
2
3
5
90
SEPW-PE
5.3
12
13
28
47
IEPW-PE
2.5
Tr.
0.5
99
EPW-PO
7.7
4
91
SEPW-PO
5.0
23
6.5
23.5
IEPW-PO
2.7
Tr.
⩽
0.5%.
a
Yieldsbasedondryfungi.
b
Alditolacetatesobtainedonsuccessivehydrolysis,NaBH4
reduction,andacetylation,analyzedbyGC–MS.
Tableoptions
Forpurification,EPW-PEandEPW-POweresubmittedtoseveralfreeze-thawingproceduresuntilnomoreprecipitationoccurred(Fig.1).Aftercentrifugationofthefractions,cold-watersolubleSEPW-PE(5.3%yield)andSEPW-PO(5.0%yield)andinsolublePEPW-PE(2.5%yield)andPEPW-PO(2.7%yield)subfractionswereisolated(Fig.1).
Table1
showsbothcoldwater-solublefractions(SEPW-PEandSEPW-PO)tocontainglucose,mannose,galactoseand3-O-methyl-galactose,whiletheinsolublefractions(IEPW-PEandIEPW-PO)showglucoseasmainmonosaccharidecomponents,consistentwithapredominantglucan.
Inordertoelucidatethelinkagetypeofglucans,IEPW-PE
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