Carbohydrate Polymers.docx

1、Carbohydrate PolymersCarbohydrate PolymersVolume 66, Issue 2, 27 October 2006, Pages 252257A -glucan from the fruit bodies of edible mushroomsPleurotus eryngiiandPleurotus ostreatoroseus Elaine R. Carbonero, Ana Helena P. Gracher, Fhernanda R. Smiderle, Fbio R. Rosado, Guilherme L. Sassaki, Philip A

2、.J. Gorin, Marcello Iacomini,Show moreDOI: 10.1016/j.carbpol.2006.03.009Get rights and contentAbstractThe glucans of basidiomycetes are an important class of polysaccharides with potential biological activities. In this work, the -glucans were isolated from the fruiting bodies of edible mushrooms,Pl

3、eurotus eryngiiandPleurotus ostreatoroseus, via extraction with hot water, and then fractionation by freeze-thawing. The insoluble glucans gave similar13C NMR spectra, monosaccharide composition and methylation analyses, andP. eryngiiwas selected for further controlled Smith degradation, and DEPT an

4、d1H (obs.),13C HMQC spectroscopy. It was a branched -glucan, with a main chain of (13)-linked-Glcpresidues, substituted at O-6 by single-unit -Glcpside-chains, on average to every third residue of the backbone, as in scleroglucan.Keywords Polysaccharides; Edible mushrooms; Pleurotusspp.; -Glucans1.

5、IntroductionMushrooms are known for their nutritional and medicinal value and the diversity of their bioactive components (Ng, 1998). These organisms have long been valued as highly tasty and nutritional foods by many societies throughout the world. Many worldwide cultures, especially in the Orient,

6、 recognize that extracts from certain mushrooms can have profound health promoting benefits. Edible mushrooms, which demonstrate medicinal or functional properties, include species of the generaLentinus,Hericium,Grifola,Flammulina,Pleurotus, andTremella(Kes & Liu, 2000).Pleurotusspp. occurs througho

7、ut the hardwood forests of the world that include the most diverse climates (Gunde-Cimerman, 1999andRosado et al., 2002). The production ofPleurotushas been increasing at a rapid rate. These mushrooms have attracted much attention owing to them being a good source of non-starchy carbohydrates, with

8、a high content of dietary fiber, moderate quantities of proteins with most of the essential amino acids, minerals, and vitamins (Croan, 2004). They have been shown to modulate the immune system, have hypoglycemic activity and to inhibit tumor growth (Gunde-Cimerman, 1999andWasser, 2002).Polysacchari

9、des represent a structurally diverse class of macromolecules of widespread occurrence in nature and offer the highest capacity for carrying biological information because they have the greatest potential for structural variability. The monosaccharide units in oligosaccharides and polysaccharides can

10、 interconnect at several points to form a wide variety of branched or linear structures (Ooi & Liu, 2000). This enormous potential variability gives the necessary flexibility for precise regulatory mechanisms of various interactions in higher organisms.Recent advances in chemical technology have all

11、owed the isolation and purification of some compounds, especially polysaccharides which posses strong immunomodulation and anti-cancer activities. They are used as biological response modifiers (Rout, Mondal, Chakraborty, Pramanik, & Islam, 2005). The polysaccharides isolated from mushroom fruiting

12、bodies are either water soluble or/and insoluble glucans and heteropolysaccharides with different main- and side-chains. There is great interest on these molecules because they can act as biological response modifiers (Gonzaga et al., 2005,Lavi et al., 2006andSmith et al., 2002).We now describe the

13、isolation and chemical characterization of a -glucan from the fruiting bodies ofPleurotus eryngiiandPleurotus ostreatoroseus.2. Materials and methods2.1. General experimental proceduresAll solutions were evaporated at 40C under reduced pressure. Centrifugation was carried out at 9000rpm for 15min, a

14、t 25C. Alditol acetate mixtures formed from polysaccharides were analyzed by GCMS using a Varian model 3300 gas chromatograph linked to a Finnigan Ion-Trap, model 810-R12 mass spectrometer, using a DB-23 capillary column (30m0.25mm i.d.) programmed from 50 to 220C at 40C/min, then hold. Partially O-

15、methylated alditol acetate mixtures were similarly analyzed, but with a program from 50 to 215C at 40C/min, then hold.2.2. Polysaccharide extraction and purificationExtraction and purification of the -glucans from the fruiting bodies of the two species ofPleurotuswere processed according toFig. 1. P

16、owdered-milled fruiting bodies (P. eryngii, 64g;P. ostreatoroseus, 66g) were extracted with 2:1 (v/v) CHCl3MeOH at 60C for 3h (3, 350mL each) and then with 4:1 (v/v) MeOHH2O at 60C for 3h (3, 350mL each), to remove low-molecular-weight material. The residue was submitted to extraction with water at

17、100C for 6h (6, 800mL each). The combined aq. extracts were evaporated to a small volume and polysaccharide precipitated by addition to excess EtOH (3:1). The precipitates fromP. eryngii(EPW-PE) andP. ostreatoroseus(EPW-PO) were dialyzed against tap water for 48h, concentrated under reduced pressure

18、 to small volumes, which were freeze-dried. EPW-PE and EPW-PO were then dissolved in water and the solutions submitted to freezing followed by mild thawing at 4C, which furnished soluble (SEPW-PE and SEPW-PO) and insoluble gel-like fractions (IEPW-PE and IEPW-PO), which were separated by centrifugat

19、ion.Fig. 1.Scheme of extraction and purification of the -glucan from thePleurotus eryngii(PE) andP. ostreatoroseus(PO).Figure options2.3. Monosaccharide compositionEach fraction (1mg) was hydrolyzed with 2M TFA at 100C for 8h, followed by evaporation to dryness. The residue was successively reduced

20、with excess of NaBH4and/or NaB2H4and acetylated with Ac2Opyridine (1:1, v/v; 2mL) at room temperature for 12h (Wolfrom and Thompson, 1963aandWolfrom and Thompson, 1963b). The resulting alditol acetates were analyzed by GCMS as indicated above and identified by their typical retention times and elect

21、ron impact profiles.2.4. Methylation analysisPer-O-methylation of the isolated polysaccharides (10mg each) was carried out using 40% aq. NaOH (3mL) and Me2SO4(2mL), added dropwise (Haworth, 1915). The process, after isolation of the products by neutralization, dialysis, and evaporation was repeated,

22、 and the methylation was found to be complete. The products were treated with 50% v/v aq. H2SO4(0.5mL v/v, 1h, 0C), followed by a dilution until it reached 5.5% (addition of 4.0mL of distilled water). The solution was kept at 100C for 18h (Saeman, Moore, Mitchell, & Millet, 1954), and was neutralize

23、d with BaCO3, filtered, and the filtrate evaporated to dryness. The residues were converted into partially O-methylated alditol acetates, and analyzed by GCMS (as described above).2.5. NMR analyses13C DEPT and1H(obs.),13C HMQC determinations were carried out using a 400MHz Bruker model DRX Avance sp

24、ectrometer incorporating Fourier Transform. Samples were dissolved in Me2SO-d6and examined at 50 or 70C. Chemical shifts are expressed in ppm () relative to resonance of Me2SO-d6at39.70 (13C) and 2.40 (1H) for samples examined in this solvent.2.6. Controlled Smith degradationIEPW-PE (300mg) was subm

25、itted to oxidation with 0.05M aq. NaIO4(20 mL) for 72h at 25C in the dark. Samples was then dialyzed against tap water for 48h and treated with NaBH4(pH 910) for 20h (Goldstein, Hay, Lewis, & Smith, 2005). The solutions were dialyzed and freeze-dried.The products were then submitted to partial acid

26、hydrolysis (TFA, pH 2.0, 30min, 100C) (Gorin, Horitsu, & Spencer, 1965) and dialyzed against tap water using membranes with a size exclusion of 2kDa and retained material (SM-PE, 97mg) was freeze-dried.3. Results and discussionAs shown inFig. 1,P. eryngiiandP. ostreatoroseuswere extracted with CHCl3

27、MeOH and then MeOHH2O to remove low-molecular weight compounds. Each resulting residue was submitted to aqueous extractions at 100C, and the extracted polysaccharides were recovered by ethanol precipitation (fractions EPW-PE and EPW-PO forP. eryngiiandP. ostreatoroseus, respectively) were dialyzed a

28、gainst tap water, and the solution freeze-dried (EPW-PE, 7.8% yield; EPW-PO, 7.7% yield).EPW-PE and EPW-PO both contained glucose as their main component, besides mannose, galactose and 3-O-methyl-galactose (Table 1). The presence of 3-O-methyl galactose was confirmed by GCMS ions atm/z130 and 190 a

29、fter NaBD4reduction and acetylation.Table 1.Monosaccharide composition and yields of fractions obtained fromP. eryngiiandP. ostreatoroseusFractionsYieldsa(%)Monosaccharidesb(%)Man3-O-MeGalGalGlcP. eryngiiEPW-PE7.823590SEPW-PE5.312132847IEPW-PE2.5Tr.Tr.0.599P. ostreatoroseusEPW-PO7.74Tr.491SEPW-PO5.0

30、236.523.547IEPW-PO2.7Tr.Tr.Tr.99Tr.0.5%.aYields based on dry fungi.bAlditol acetates obtained on successive hydrolysis, NaBH4and/or NaB2H4reduction, and acetylation, analyzed by GCMS.Table optionsFor purification, EPW-PE and EPW-PO were submitted to several freeze-thawing procedures until no more pr

31、ecipitation occurred (Fig. 1). After centrifugation of the fractions, cold-water soluble SEPW-PE (5.3% yield) and SEPW-PO (5.0% yield) and insoluble PEPW-PE (2.5% yield) and PEPW-PO (2.7% yield) subfractions were isolated (Fig. 1).Table 1shows both cold water-soluble fractions (SEPW-PE and SEPW-PO) to contain glucose, mannose, galactose and 3-O-methyl-galactose, while the insoluble fractions (IEPW-PE and IEPW-PO) show glucose as main monosaccharide components, consistent with a predominant glucan.In order to elucidate the linkage type of glucans, IEPW-PE