Preparation, Structure, And Solution Properties Of A Novel Antioxidant Exopolysaccharide From Cordyceps Sinensis

Cordyceps sinensis (Berk.) Sace., generally called Cordyceps or Dongchongxiacao (winter worm-summer grass) in Chinese, is a rare medicinal fungus in China and has a wide range of physiological activities and unique medicinal values. Polysaccharides are regarded as the one of major active ingredients. The aim of this research project was to isolate exopolysaccharides from mycelial fermentation of a Cordyceps sinensis fungus Cs-HK1 and to determine the optimal condition for extraction and isolation of exopolysaccharides. Various exopolysacharide fractions which were isolated and fractionated from crude exopolysaccharides were subjected to compare the antioxidant activities in vitro and mechanism, and the best antioxidant activity of polysaccharide fractions were determined and further purified. Physicochemical properties, chemical structure, solution properties and molecular chain conformation in aqueous solution of purified exopolysaccharide owned better antioxidant activity were analyzed. The results are as follows:Ethanol precipitation was used to isolate the exopolysaccharides from mycelial fermentation of cultured Cordyceps sinensis (Cs-HK1), and the extraction conditions were concentration of ethanol 95% (v/v), multiples of ethanol 4.3, sedimentation time 10.5 h, and pH of fermentation broth 6.8, and the yield of crude exopolysaccharide was about 4.27 g/L. This is the highest level in extraction of exopolysaccharides from reported various Cordyceps. Chemical constituents were total sugar 50.1%, uronic acid 13.6%, and protein 28.4%.Removals of protein, decolorization, dialysis and ion-exchange column chromatography were used for isolation and fractionation of crude exopolysaccharides to yield two fractions, namely EPS-1 and EPS-2. In vitro trials of antioxidant activities were chosen to evaluate the free-radical scavenging activity. The results showed that exopolysaccharides EPS, EPS-1, and EPS-2 could moderately scavenge O₂^·-,·OH, and ABTS·+ radicals by the hydrogen atom transfer reaction. Amongst, EPS-1 had stronger effects on free radicals than EPS and EPS-2's, and had significant free-radical scavenging activity on·OH. The IC₅₀ value of EPS-1 on·OH was closed to the vitamin C (Vc)'s, and was regarded as the major antioxidant component in exopolysaccharide of Cordyceps.EPS-1 was subjected to gel filter chromatography to yield a purified exopolysaccharide EPS-1A. Analytical results of UV spectrum, specific rotation and gel permeation chromatography (GPC) suggested that EPS-1A was a relatively homogeneous component, neutral sugar content of 99.0%, the weight-average molecular weight (Mw) of 4.0×10⁴ Da and the specific rotation of +6.5o (c 0.26, H₂O), a non-starch water-soluble and neutral polysaccharide which did not contain monosaccharides, uronic acids, protein, nucleic acids and polyphenols.The structural characteristics of EPS-1A were elucidated with acid hydrolysis, methylation, periodate oxidation, Smith degradation, gas chromatography (GC), gas chromatography-mass spectrograph (GC-MS), infrared spectroscopy (IR), 1D and 2D NMR, etc. EPS-1A was composed of D-glucose, D-mannose and D-galactose at 15.2:3.6:1.0, its backbone was composed of (1→6)-α-D-glucose and (1→6)-α-D-mannose, and branching occurred at O-3 position of (1→6)-α-D-mannose residue of the backbone with (1→6)-α-D-glucose, (1→6)-α-D-mannose and terminated withβ-D-galactose. This is a new structure of repeat units found in mycelial fermentation of Cordyceps sinensis (Cs-HK1).Congo red reaction revealed that EPS-1A was a random coil chain in different aqueous solutions. Changes of intrinsic viscosity, specific rotation and particle size distribution in various aqueous solutions suggested that EPS-1A was a flexible random coil chain in aqueous solutions and formed aggregates at higher concentrations. Factors including temperature, metallic ion, denaturing agent and Congo red could not alter the chain conformation in aqueous solution, but influence the inter- or intra-molecular hydrogen bond interactions of polysaccharide molecules in some extent, so that polysaccharides became loose and disordered.Viscosity method and static and dynamic laser light scattering technology were used to determine intrinsic viscosity ([η]), weight-average molecular weight (Mw), radii of gyration (2>^1/2), hydrodynamic radium (Rh) and structural parameter (ρ) of the EPS-1A, and the obtained results were 6.02 dL/g, 5.03×10⁴g/mol, 133.80nm, 145.35nm, and 0.92 respectively, suggesting that EPS-1A exhibited a flexible random coil"soft sphere"chain conformation in aqueous solution, and the hardness of"soft sphere"was slightly greater than dextran's. The chain conformation of EPS-1A was observed by atomic force microscopy (AFM) showing spherical structure of different sizes which was similar to dextran's, and the height and diameter of the spheres ranged from 10 to 50 nm and 2 to 3 nm, respectively. The solid-state morphology of EPS-1A was also observed by scanning electron microscopy (SEM) showing a flexible network structure with a high degree of branching.