Notes
Structural Characterization of a Heterogalactan from Antler-shaped Ganoderma lucidum
2015 年 62 巻 4 号 p. 149-151
詳細
2015 年 62 巻 4 号 p. 149-151
A heterogalactan fraction, accounting for approximately 0.5% of the dry weight of the fruit body, was derived from a water-extracted polysaccharide fraction from antler-shaped Ganoderma lucidum. The heterogalactan fraction was characterized by sugar composition and sugar linkage composition analyses. The results suggest that the heterogalactan fraction consists of a fucomannogalactan (a linear (1→6)-D-galactan substituted at the O-2 position with non-reducing fucosyl and mannosyl residues), or a mixture of fucogalactan (a linear (1→6)-D-galactan substituted at the O-2 position with non-reducing fucosyl residues) and mannogalactan (a linear (1→6)-D-galactan substituted at the O-2 position with non-reducing mannosyl residues). The molecular weight of the heterogalactan fraction was estimated to be 1.0 × 104 by gel filtration on Sepharose CL-6B.
Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Glc, glucose; Gal, galactose; AS, antler-shaped.
It is well known that many medicinal and therapeutic properties are attributed to polysaccharides derived from various kinds of mushrooms.1) 2) 3) Although the chemical structure of some polysaccharides in the fruit body of Ganoderma lucidum (Reishi) have been investigated, little is known regarding antler-shaped G. lucidum (AS G. lucidum) polysaccharides. To elucidate the constituents of the polysaccharides in the fruit body of AS G. lucidum, we investigated the influence of a culture medium on the composition of water-soluble polysaccharides (WSPs) in the fruit body of AS G. lucidum, using a medium of pruned persimmon, mulberry, Japanese cherry, Japanese plum, apple, Japanese oak, or grape branches.4) Based on the neutral sugar composition and sugar linkage composition analyses, it was found that the kind and composition ratio of water-soluble polysaccharides in the fruit body of AS G. lucidum was dependent on the culture medium (i.e., the type of branches used). Although there was a difference in the quantity, heterogalactan-like polysaccharides and β-glucan-like polysaccharides were found in all WSPs. Interestingly, the WSPs of AS G. lucidum from the persimmon and mulberry branches were rich in Ara- and Xyl-containing polysaccharides.4) Many previous studies have been conducted on the biological functions of β-glucans,1) however, the biological function of heterogalactan remains unknown. Recently, studies on the anti-inflammatory and antinociceptive effects of fucogalactans from Agaricus brasiliensis5) and Agaricus bisporus var. hortensis,5) fucomannogalactans from Lentinus edodes,6) and mannogalactans from Pleurotus pulimonarius7) have been carried out. We thought it would be of interest to determine whether or not the WSPs in the fruit body of AS G. lucidum contain fucogalactan and/or fucomannogalactan. This report deals with the structural characterization of a heterogalactan isolated from the WSPs of AS G. lucidum grown on apple branches (apple-WSPs).
The apple-WSPs (yield: 52.1 mg/10 g dry weight of fruit body. Fuc:Ara:Rha:Gal:Glc:Xyl:Man = 4.1:4.3:2.9:36.6:20.8:4.2:27.1) used in this study are described in a previous paper.4) Unless otherwise stated, all materials and experimental procedures used in this study were as described in a previous paper.4)
To separate neutral polysaccharides from acidic polysaccharides, a portion of the apple-WSPs was subjected to DEAE-Sephadex A-25 chromatography (acetate form). WSP was resolved into five fractions, I‒V (Fig. 1). Table 1 shows the yield and neutral sugar composition of fractions I‒V obtained from apple-WSPs. Fractions I and II (neutral polysaccharide fractions), which were eluted with a sodium acetate buffer from the DEAE-Sephadex column, and which accounted for about 28.5 and 29.5% of the recovered carbohydrates, mainly consisted of Fuc, Man, and Gal residues. Fractions III, IV, and V (acidic polysaccharide fractions), which were eluted with NaCl in a sodium acetate buffer, mainly consisted of Gal, Man, and Glc residues. Small amounts of Ara, Xyl, and Glc residues discovered in a major neutral polysaccharide fraction, WSP-I, were considered to be derived from arabinoxylan-like and β-glucan-like polysaccharides, which were presumably admixed in the heterogalactan. Thus, the isolated WSP-I accounted for approximately 23% of the total WSPs and for about 0.5% of the total dry weight of the fruit body.
DEAE-Sehadex A-25 chromatography of apple-WSPs.
Apple-WSPs (50 mg in 10 mL of water) were applied to a column (φ3.5 × 20 cm) pre-equilibrated with 50 mM sodium acetate buffer (pH 5.5). The column was washed with the same buffer (400 mL). Absorbed materials were eluted, stepwise, with 0.5 M NaCl (400 mL) and 1.0 M NaCl (400 mL) in 50 mM sodium acetate buffer: 10-mL fractions were collected and assayed for carbohydrates by the phenol-sulfuric acid method.8) Tubes 3-9 (I), 10-17 (II), 46-51 (III), 52-56 (IV), and 86-93 (V) were separately combined, dialyzed and concentrated.
Yields and neutral sugar composition of fractions obtained from apple-WSPs by DEAE-Sephadex A-25 chromatography (Fig. 1).
Apple-WSP-I (1 mg) was chromatographed in a calibrated Sepharose CL-6B column (φ1.0 cm × 45 cm) equilibrated with 0.1 M NaOH. It gave a single peak, and its average molecular weight was estimated to be approximately 1.0×104 (Fig. 2). Apple-WSP-I was methylated and hydrolyzed, and the sugar linkage composition was analyzed by gas-liquid chromatography of the resulting alditol acetates. The non-reducing terminal of Fuc (10.8%), the non-reducing terminal of Man (4.5%), the 6-linked Gal (61.3%), and the 2,6-linked Gal (17.6%) residues were shown to be prominent structural units. In a previous sugar linkage analysis of apple-WSPs, 6-linked Gal, 3,4-linked Gal, 2,4-linked Gal, and 3,6-linked Gal residues were identified as the structural units of Gal residues, and the 2,6-linked Gal residue was not identified4) (Table 4 in the reference). The unidentified peak No. 22 in a previous study4) (Table 4 in the reference) may correspond to the 2,6-linked Gal residue from the data on retention time.
Sepharose CL-6B chromatography of apple-WSP-I.
Apple-WSP-I (1.0 mg in 0.5 mL of 0.1 M NaOH) was applied to a column (φ1.0 cm × 45 cm) pre-equilibrated with 0.1 M NaOH, followed by filtration through the column with 0.1 M NaOH. Each tube was assayed for carbohydrates by the phenol-sulfuric acid method.8) The arrows indicate the elution positions of the dextrans of mol wt 5.0 × 105 and 1.0 × 104, respectively.
Although some possible structures can be deduced from the present data, reference to previous works2) 3) 9) 10) 11) would suggest that a heterogalactan fraction (WSP-I) of AS G. lucidum consists of a fucomannogalactan (a linear (1→6)-D-galactan substituted at the O-2 position with non-reducing fucosyl and mannosyl residues), or a mixture of fucogalactan (a linear (1→6)-D-galactan substituted at the O-2 position with non-reducing fucosyl residues) and mannogalactan (a linear (1→6)-D-galactan substituted at the O-2 position with non-reducing mannosyl residues).
Most heterogalactans have a main chain composed of (1→6)-linkedα-D-galactopyranosyl units, with fucose and/or mannose as the main substituents. Heterogalactans, especially those that contain fucose, exhibit biological activities.5) 6) 7) Usui et al.10) and Mizuno et al.11) reported, respectively, that fucogalactan and mannofucogalactan were isolated from the hot-water extract of the fruit body of Fomitopsis pinicola and G. lucidum by a combination of fractionation procedures, including precipitation with ethanol and cetyltrimethylammonium bromide and chromatography on a column of DEAE-cellulose, Sephadex G-100 and concanavalin A-Sepharose CL 4B. A heterogalactan of AS G. lucidum may be fractionated into fucogalactan and mannofucogalactan. It is of interest to clarify whether or not the AS G. lucidum heterogalactan has some medicinal and therapeutic properties.
We would like to thank Mr. S. Torigata for his technical assistance.
