Carbohydrate Polymers.docx

Carbohydrate Polymers.docx

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CarbohydratePolymers

CarbohydratePolymers

Volume66,Issue2,27October2006,Pages252–257

Aβ-glucanfromthefruitbodiesofediblemushrooms Pleurotuseryngii and Pleurotusostreatoroseus

∙ElaineR.Carbonero, 

∙AnaHelenaP.Gracher, 

∙FhernandaR.Smiderle, 

∙FábioR.Rosado, 

∙GuilhermeL.Sassaki, 

∙PhilipA.J.Gorin, 

∙MarcelloIacomini, 

 Showmore

DOI:

10.1016/j.carbpol.2006.03.009

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Abstract

Theglucansofbasidiomycetesareanimportantclassofpolysaccharideswithpotentialbiologicalactivities.Inthiswork,theβ-glucanswereisolatedfromthefruitingbodiesofediblemushrooms, Pleurotuseryngii andPleurotusostreatoroseus,viaextractionwithhotwater,andthenfractionationbyfreeze-thawing.Theinsolubleglucansgavesimilar 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 （ Kües&Liu,2000）.

Pleurotus spp.occursthroughoutthehardwoodforestsoftheworldthatincludethemostdiverseclimates（Gunde-Cimerman,1999 and Rosadoetal.,2002）.Theproductionof Pleurotus hasbeenincreasingatarapidrate.Thesemushroomshaveattractedmuchattentionowingtothembeingagoodsourceofnon-starchycarbohydrates,withahighcontentofdietaryfiber,moderatequantitiesofproteinswithmostoftheessentialaminoacids,minerals,andvitamins（ Croan,2004）.Theyhavebeenshowntomodulatetheimmunesystem,havehypoglycemicactivityandtoinhibittumorgrowth（ Gunde-Cimerman,1999 and 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 and 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 °Cat40 °C/min,thenhold.

2.2.Polysaccharideextractionandpurification

Extractionandpurificationoftheβ-glucansfromthefruitingbodiesofthetwospeciesof Pleurotus 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 °Cfor3 h（3×,350 mLeach）,toremovelow-molecular-weightmaterial.Theresiduewassubmittedtoextractionwithwaterat100 °Cfor6 h（6×,800 mLeach）.Thecombinedaq.extractswereevaporatedtoasmallvolumeandpolysaccharideprecipitatedbyadditiontoexcessEtOH（3:

1）.Theprecipitatesfrom P.eryngii （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 Pleurotuseryngii （PE）and P.ostreatoroseus （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 and 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, P.eryngii and P.ostreatoroseus wereextractedwithCHCl3–MeOHandthenMeOH–H2Otoremovelow-molecularweightcompounds.Eachresultingresiduewassubmittedtoaqueousextractionsat100 °C,andtheextractedpolysaccharideswererecoveredbyethanolprecipitation（fractionsEPW-PEandEPW-POfor P.eryngii and 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.eryngii and 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.

Tr.

0.5

99

P.ostreatoroseus

EPW-PO

7.7

4

Tr.

4

91

SEPW-PO

5.0

23

6.5

23.5

47

IEPW-PO

2.7

Tr.

Tr.

Tr.

99

Tr. ⩽ 0.5%.

a

Yieldsbasedondryfungi.

b

Alditolacetatesobtainedonsuccessivehydrolysis,NaBH4 and/orNaB2H4 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