Systematical Analysis Of The Polysaccharides From Bulgaria Inquinans (Fries) And Their Biological Activities

The non-lichenized ascomycete Bulgaria inquinans (Fries) is an edible wood-inhabiting ascomycete that grows on freshly felled oak and commonly found in the Changbai Mountain area of China. It has been used as food and folk antitumor medicine for a long time. Pharmacological research indicates Bulgaria inquinans (Fries) has various biological activities. Its main nutritional ingredient is carbohydrate, but there is no systematically study on structure and pharmacological activity of polysaccharide from it. In the present paper, the extraction, fractionation, structural characterization and biological activity evaluation of polysaccharides from B. inquinans (Fries) have been systematically studied for the first time.The fruit bodies of B. inquinans (Fries) were extracted successively with hot water, 2%, 10%, and 30% KOH to generate four crude polysaccharide fractions. The crude polysaccharides were deproteinated by Sevag method and dialyzed against water, giving rise to water-soluble fraction BIW and water-insoluble fractions BIK2, BIK10, BIK30. BIW exhibited a wide molecular weight distribution (from 3 kD to 50 kD) and contained mannose (27.8%), glucose (39.8%) and galactose (32.4%). BIK2, BIK10 and BIK30 were all mainly composed of glucose (> 92%) and a trace amount of mannose and galactose, and respectively exhibited a single and symmetrical peak on Sepharose CL-6B gel filtration chromatography, indicating their homogeneities.BIW was fractionated by freeze-thawing process to give two fractions, BIWS (supernatant, 63%) and BIWP (precipitate, 19%). BIWP contained 95.2% of glucose, 2.1% of mannose and 2.7% of galactose, and purified by Sephadex G-75 gel filtration chromatography to give a homogeneous polysaccharide BIWP2. BIWP2 contained exclusively glucose. Its molecular weight was estimated to be 2.6 KD and the polydispersity index (Mw/Mn) was calculated to be 1.39.BIWS consisted of glucose, mannose, and galactose in a molar ratio of 1:1:1, and exhibits a wide molecular weight distribution from 15 kD to 50 kD. It was fractionated by gradient ethonal precipitation, Fehling reagent precipitation and cetrimide precipitation, respectively.BIWS was fractionated by gradient precipitation in 30%, 50%, 70%, and 90% ethanol, which resulted in the fractions BIWS-1, BIWS-2, BIWS-3, and BIWS-4, respectively. BIWS-1, BIWS-2, and BIWS-3 showed wide distributions on Sepharose CL-6B or Sephadex G-75 elution profiles, while BIWS-4 was purified by Sephadex G-75 gel filtration chromatography to give a homogeneous polysaccharide BIWS-4b. BIWS-4b was composed of mannose (27.2%), glucose (15.5%) and galactose (57.3%). Its molecular weight was estimated to be 7.4 kD and the polydispersity index (Mw/Mn) was calculated to be 1.35.BIWS was fractionated by Fehling reagent to give two fractions, BIWS-Fs (supernatant, 87.2%) and BIWP-Fp (precipitate, 1.4%). The results of GPC and monosaccharide composition analysis indicated BIWS-Fs and BIWP-Fp have nearly the same monosaccharide compositions and molecular weight distributions, so the method of Fehling reagent precipitation is unsuitable for fractionating BIWS.BIWS was repeatedly fractionated by cetrimide, and then purified by gel permeation chromatography to give ten homogeneous fractions. Base on their monosaccharide compositions, they were divided into four groups: (1) Man>Gal>>Glc, BIWS-pH10P-1, BIWS-pH10P-2, BIWS-S-pH10P–P; (2) Gal>Man>>Glc. BIWS-S-pH10P-S, BIWS-S-pH12P-2, BIWS-pH12P-S, BIWS-pH12P–P; (3) Glc>Man>>Gal. BIWS-S-S-S; (4) Man:Gal:Glc≈1:1:1, BIWS-S-S-pH12p, BIWS-S-pH12p-1.Finally, we obtained 15 homogenous polysaccharide fractions from the fruit bodies of B. inquinans (Fries), following the above extraction, prufication and fractionation procedure. Subsequently, the compositions and structural features of polysaccharides from B. inquinans (Fries) were studied deeply and systematically by HPLC, FT-IR, periodate oxidation, methylation, partial hydrolysis and NMR analyses. The results indicated that polysaccharides from B. inquinans (Fries) consisted ofβ-1,6-glucans and heteropolysaccharides containing same mannan cores, such as galactomannan, glucomannan and glucogalactomannan.β-1,6-glucans contained low molecular weight linearβ-1,6-glucan (BIWP2), and high molecular weightβ-1,6-glucan with singalβ-Glcp residue branches (BIK2, BIK10 and BIK30, which degrees of branch were 0.01, 0.17 and 0.25, respectively). Moreover, BIWP2 was the smallest linearβ-1,6-glucan from fungi by now.Structural analyses indicated that BIWS mainly contained (1→6)-linked, (1→5)-linked and (1→5,6)-linkedβ-Galf units, (1→4)-linked and non-reducing terminalβ-Glcp units, and (1→2)-linked, (1→6)-linked, (1→2,6)-linked and non-reducing terminalα-Manp units. The mannan core containedα-(1→6)-D-mannan backbones, andα-(1→2)-D-mannan or singalα-Manp branches which attached to the backbones at O-2 position. Galactans, glucans or glucogalactans, composed ofβ-Galf andβ-Glcp, might be attached to the O-2 positions ofα-Manp residues in mannan cores viaβ-Galf, which generated a series of galactomannans, glucomannans and glucogalactomannans.The immunological tests showed that all the fourβ-(1→6)-D-glucans from B. inquinans (Fries) could significantly increase the ConA or LPS-induced lymphocytes proliferation in vivo. Moreover, the lymphocytes proliferation activity increased along with the molecular weight and branching ratio ofβ-(1→6)-D-glucan.When examined by the 4-day method and in a prophylactic assay in mice, BIWS-4b exhibited markedly suppressive activity against malaria while enhancing the activity of artesunate. Immunological tests indicated that BIWS-4b significantly enhanced macrophage phagocytosis and splenic lymphocyte proliferation in malaria-bearing mice and normal mice. The results of antimalarial and immunological tests of BIWS-4b were consistent with those of BIWS in our previously study. Thus, we deduced that the anti-malarial activity of heteropolysaccharides from B. inquinans (Fries) might be intermediated by enhancing immune competence and restoring artesunate-suppressed immune function.Moreover, the results of antioxidant testing clearly demonstrated that BIWS and BIWS-4b had strong antioxidant activity according to the in vitro evaluation of their free radicals and H2O2 scavenging activities, ferrous ion-chelating abilities and reducing power. Moreover, the antioxidant activity increased along with the molecular weight of heteropolysaccharides.Summarizing the above results, polysaccharides from B. inquinans (Fries) have extensively pharmacological activities, and might be explored as immunomodulator, antioxidant agent and adjuvant of anti-malarial drugs. Therefore, polysaccharides from B. inquinans (Fries) are greatly potential medicinal resources.