The aim of this paper was to perform a comprehensive characterization of polysaccharides isolated from the interior (IOI) and exterior (IOE) parts of the fungus Inonotus obliquus. Pre-extraction with DCM and MeOH, followed by water and alkali extraction and ethanol precipitation gave two water extracts and two alkali extracts. Neutral and acidic polysaccharide fractions were obtained after anion-exchange chromatography of the water extracts. The neutral polysaccharides (60-73 kDa) were heterogeneous and branched and consisted of a (1→3)-linked β-Glc backbone with (1→6)-linked kinks in the chain at approximately every fifth residue, with branches of (1→6)-linked β-Glc in addition to substantial amounts of (1→6)-linked α-Gal with 3-O-methylation at about every third Gal residue. The acidic polysaccharide fractions (10-31 kDa) showed similar structural motifs as the neutral fractions differing mainly by the presence of (1→4)-linked α-GalA and α-GlcA. β-Xyl, α-Man and α-Rha were also present in varying amounts in all fractions. No major structural differences between the IOI and IOE fractions were observed. An alkaline polysaccharide fraction (>450 kDa) was obtained from the IOI alkali extract, and consisted mainly of (1→3)- and (1→6)-linked β-Glc and (1→4)-linked β-Xyl. Several of the fractions showed in vitro immunomodulatory effect by increasing NO production in the murine macrophage and dendritic cell lines J774.A1 and D2SC/1. Most fractions managed to increase NO production only at the highest concentration tested (100 μg/ml), while the neutral fraction IOE-WN activated potent NO production at 10 μg/ml and was considered the most promising immunomodulating fraction in this study.

NMR, polysaccharides, galactoglucan, Structural characterization, fungi, chaga

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
6,3,3,3	bDGlcp	102.48	73.17	84.59	68.17	75.44	60.80
6,3,3	bDGlcp	102.48	73.17	84.59	68.17	75.44	60.80
6,3	bDGlcp	102.48	73.17	84.59	68.17	75.44	60.80
6	bDGlcp	102.40	72.58	84.79	68.11	75.44	60.80
	bDGlcp	102.92	73.35	75.42	69.62	74.59	68.85


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
6,3,3,3	bDGlcp	4.87	3.65	3.85	3.61	3.60	3.83-4.03
6,3,3	bDGlcp	4.87	3.65	3.85	3.61	3.60	3.83-4.03
6,3	bDGlcp	4.87	3.65	3.85	3.61	3.60	3.83-4.03
6	bDGlcp	4.63	3.61	3.84	3.60	3.58	3.83-4.03
	bDGlcp	4.80	3.47	3.62	3.51	3.80	3.96-4.31


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
6,3,3,3	bDGlcp	102.48/4.87	73.17/3.65	84.59/3.85	68.17/3.61	75.44/3.60	60.80/3.83-4.03
6,3,3	bDGlcp	102.48/4.87	73.17/3.65	84.59/3.85	68.17/3.61	75.44/3.60	60.80/3.83-4.03
6,3	bDGlcp	102.48/4.87	73.17/3.65	84.59/3.85	68.17/3.61	75.44/3.60	60.80/3.83-4.03
6	bDGlcp	102.40/4.63	72.58/3.61	84.79/3.84	68.11/3.60	75.44/3.58	60.80/3.83-4.03
	bDGlcp	102.92/4.80	73.35/3.47	75.42/3.62	69.62/3.51	74.59/3.80	68.85/3.96-4.31

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O[C@@H]2[C@@H](O)[C@H](O[C@@H]3[C@@H](O)[C@H](O[C@H]4[C@H](O)[C@@H](CO)O[C@@H](OC[C@H]5O[C@@H](*)[C@H](O)[C@@H](O)[C@@H]5O)[C@@H]4O)O[C@H](CO)[C@H]3O)O[C@H](CO)[C@H]2O)[C@@H]1O

810.705 g/mol (C₃₀H₅₀R₂O₂₅, R = next and previous repeats, not counted in mol weight)

The aim of this paper was to perform a comprehensive characterization of polysaccharides isolated from the interior (IOI) and exterior (IOE) parts of the fungus Inonotus obliquus. Pre-extraction with DCM and MeOH, followed by water and alkali extraction and ethanol precipitation gave two water extracts and two alkali extracts. Neutral and acidic polysaccharide fractions were obtained after anion-exchange chromatography of the water extracts. The neutral polysaccharides (60-73 kDa) were heterogeneous and branched and consisted of a (1→3)-linked β-Glc backbone with (1→6)-linked kinks in the chain at approximately every fifth residue, with branches of (1→6)-linked β-Glc in addition to substantial amounts of (1→6)-linked α-Gal with 3-O-methylation at about every third Gal residue. The acidic polysaccharide fractions (10-31 kDa) showed similar structural motifs as the neutral fractions differing mainly by the presence of (1→4)-linked α-GalA and α-GlcA. β-Xyl, α-Man and α-Rha were also present in varying amounts in all fractions. No major structural differences between the IOI and IOE fractions were observed. An alkaline polysaccharide fraction (>450 kDa) was obtained from the IOI alkali extract, and consisted mainly of (1→3)- and (1→6)-linked β-Glc and (1→4)-linked β-Xyl. Several of the fractions showed in vitro immunomodulatory effect by increasing NO production in the murine macrophage and dendritic cell lines J774.A1 and D2SC/1. Most fractions managed to increase NO production only at the highest concentration tested (100 μg/ml), while the neutral fraction IOE-WN activated potent NO production at 10 μg/ml and was considered the most promising immunomodulating fraction in this study.

NMR, polysaccharides, galactoglucan, Structural characterization, fungi, chaga

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
6,6,3	33%Me	56.02
6,6	aDGalp	97.72	67.42	78.77	65.10	68.77	66.76
6	bDGlcp	102.72	73.05	75.62	69.52	74.82	68.85
	bDGlcp	102.72	73.05	75.62	69.52	74.82	68.85


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
6,6,3	33%Me	3.53
6,6	aDGalp	5.07	3.97	3.63	4.38	4.27	3.78-4.01
6	bDGlcp	4.61	3.42	3.59	3.55	3.72	3.96-4.31
	bDGlcp	4.61	3.42	3.59	3.55	3.72	3.96-4.31


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
6,6,3	33%Me	56.02/3.53
6,6	aDGalp	97.72/5.07	67.42/3.97	78.77/3.63	65.10/4.38	68.77/4.27	66.76/3.78-4.01
6	bDGlcp	102.72/4.61	73.05/3.42	75.62/3.59	69.52/3.55	74.82/3.72	68.85/3.96-4.31
	bDGlcp	102.72/4.61	73.05/3.42	75.62/3.59	69.52/3.55	74.82/3.72	68.85/3.96-4.31

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

Warning = sub-repeat ending at bDGlcp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at aDGalp: unknown unit count was assumed to be 3
CO[C@H]1[C@@H](O)[C@@H](CO)O[C@H](OC[C@H]2O[C@H](OC[C@H]3O[C@H](OC[C@H]4O[C@@H](OC[C@H]5O[C@@H](OC[C@H]6O[C@@H](OC[C@H]7O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]7O)[C@H](O)[C@@H](O)[C@@H]6O)[C@H](O)[C@@H](O)[C@@H]5O)[C@H](O)[C@@H](O)[C@@H]4O)[C@H](O)[C@@H](OC)[C@H]3O)[C@H](O)[C@@H](OC)[C@H]2O)[C@@H]1O

1195.083 g/mol (C₄₅H₇₈O₃₆)

Agaricus blazei Murill is an edible and medicinal mushroom favored in many countries, by virtue of both its delicious taste and its potential health benefits such as its purported anticancer activity. A neutral α-glucan (ABM401) with a carbohydrate content of 96% was purified from the high-speed shearing homogenization extracts of A. Blazei Murill by ethanol precipitation and column chromatography. Methylation analysis along with nuclear magnetic resonance spectroscopy revealed that ABM40-1 was an α-(1→4)-D-glucopyranan with O-6 position occasionally occupied with α-Glcp-(1→ or α-Glcp-(1→6)-β-Glcp-(1→ side chains. A weight-average molecular weight of 7.34 x 10(6) Da was determined for ABM40-1 and its chain in solution was revealed as a compact sphere by size exclusion chromatography (SEC) coupled with a laser light scattering. This spherical conformation was also further confirmed by Congo red test and using atom force microscopy. These results suggest it would be worthwhile to further study the potential bioactivities of ABM40-1.

structure, polysaccharide, Chain conformation, Agaricus blazei Murill, high-speed shearing homogenization

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
	aDGlcp	99.73	71.85	73.41	77.09	70.21	68.97
6,6	aDGlcp	100.05	71.76	73.06	69.65	72.19	60.43
6	3%bDGlcp	102.98	73.13	75.70	69.75	74.87	68.97
6	7%aDGlcp	100.05	71.76	73.06	69.65	72.19	60.43


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
	aDGlcp	5.41	3.65	3.97	3.68	3.94	3.87-4.24
6,6	aDGlcp	5.41	3.59	3.73	3.43	3.68	3.78-3.86
6	3%bDGlcp	4.54	3.34	3.50	3.47	3.64	3.85-4.24
6	7%aDGlcp	5.41	3.59	3.73	3.43	3.68	3.78-3.86


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
	aDGlcp	99.73/5.41	71.85/3.65	73.41/3.97	77.09/3.68	70.21/3.94	68.97/3.87-4.24
6,6	aDGlcp	100.05/5.41	71.76/3.59	73.06/3.73	69.65/3.43	72.19/3.68	60.43/3.78-3.86
6	3%bDGlcp	102.98/4.54	73.13/3.34	75.70/3.50	69.75/3.47	74.87/3.64	68.97/3.85-4.24
6	7%aDGlcp	100.05/5.41	71.76/3.59	73.06/3.73	69.65/3.43	72.19/3.68	60.43/3.78-3.86

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@@H](CO)O[C@H](*)[C@H](O)[C@H]1O

162.141 g/mol (C₆H₁₀R₂O₅, R = next and previous repeats, not counted in mol weight)