The Study Of The Structure And Bioactivity Of The Polysaccharides From Inonotus Obliquus

Inonotus obliquus (Pers.:Fr.) Pilat is well known as one of the most popular medicinal species for its therapeutic effect, which is a white rot fungus belonging to the family Hymenochaetacea. It is mainly found at Changbai Mountain and Heilong River in China.The polysaccharide distilled from I. obliquus has the functions of immunomodulation, anti-tumor, antioxidant and anti-inflammation and has no poisonous effect. But the studies on polysaccharides from Inonotus obliquus have focused on crude polysaccharide, there is still a lack of understanding of all of the Inonotus obliquus polysaccharides. So far there is not any information published about chemical structures on the homgerous component from the polysaccharide.Therefore, The present paper was concerned with the isolation, chemical characterization, evaluation of the immunomodulative and antitumor activities of a polysaccharide from the fruiting body of Inonotus obliquus. The investigation results are as follows:1. Development of extract technique for polysaccharide from the fruiting body of Inonotus obliquus.Through single-factor experiments of orthognal experiments, it was found that best yield could be obtained. Polysaccharides of Inonotus obliquus were extracted by hot water and its residue extracted by alkali. The optimal extracting conditions of water soluble polysaccharides from Inonotus obliquus are as follows:the adding ratio of water is 1:20,the temperature of extraction is 90℃, the time of extraction is 1 hours,The extracting ratio of water soluble polysaccharides from Inonotus obliquus were 0.992%.Extracted solution was concentrated, then precipitated after incubation for 4 with 3volumes ethanol.The optimal extracting conditions of alkali extractional polysaccharide from Inonotus obliquus are as follows:the adding ratio of alkali is 1:20, the concentration of NaOH is 0.5 moL/L, the time of extraction is 1 hour, the concentration of ethanol is 85%.The extracting ratio of alkali soluble polysaccharides from Inonotus obliquus were 0.659%.2. Extraction and purification of polysaccharides from IOP and AIOP.The water-soluble polysaccharides were extracted from Inonotus obliquus with hot water, precipitated by ethanol and deproteinated using the Sevag method. The polysaccharide mixture, referred to as IOP, was obtained with a yield of 2.67% (w/w). IOP contained 37.2% total carbohydrates and more than 17.8% protein. Sugar composition analysis by GC chromatography indicated that IOP consisted of Rhamnose (7.59%),Glucose (50.87%), Xylose (10.63%),Galactose (14.96%) and Mannose (15.95%).The alkali-soluble polysaccharides were extracted from its residue with hot water, precipitated by ethanol and deproteinated using the Sevag method. The polysaccharide mixture, referred to as AIOP, was obtained with a yield of 2.14 (w/w). IOP contained 31.4% total carbohydrates and more than 16.3% protein. Sugar composition analysis by GC chromatography indicated that AIOP consisted of Rhamnose (8.9%),Mannose (5.21%), Galactose (13.58%), Glucose (62.54%), Xylose (6.71%), and Glucturonic acid (3.06%). IOP was separated on a preparative DEAE-Cellulose chromatographic fractionating, an unbound fraction (IOPN) by water elution and two bound fraction (IOPS-I、IOPS- II) by 1.0 M NaCl elution. Then, IOPN、IOPS-I、IOPS- II and AIOP were separated on Sephades G-100 column into seven fractions IOPN-I、IOPN-II、IOPS-I-A、IOPS-I-B、IOPS-II-A.、AIOP-I and AIOP-II respectively. These seven fractions were homogenous polysaccharides, theirs molecular weights approximately ranging from 3.3×10~3 to 5.9×10~3.3. Structural analysis of homogeneous component from IOPN-I.The structural features of IOPN-I were elucidated using partial acid hydrolys, methylation, periodate oxidation, Smith degradationand and 2D NMR analysis. The results show that the IOPN-I mainly formβglycosidic bond configuration, but also a small amount of a glycosidic bond configuration. IOPN-I had a backbone composed of (1→3)-linked-β-D-Mannopyranos -yl, (1→3,6)-linked-β-D-glucopyranosyl, (1→6)-linked-3-O-Me-β-D-galactopyranosyl, (1→3)-linked-α-D-glucopyranosyl, (1→6)-linked-β-D-glucopyranosyl residues in the ratio of 1:1:2:2:1, and terminated with a single non-reducing terminal (1→)-β-D-glucopyranosyl residues at the C-3 position of (1→3,6)-linked-β-D-glucopyranosyl, along the main chain.4. Study of bioactivity of the polysaccharides from the fruiting body of Inonotus obliquus.The immunological activity of Inonotus obliquus polysaccharides were studied in mice. IOPN、IOPS and AIOP stimulated the B spleen lymphocyte proliferation, increase the production of TNF-αin macrophages, and dose-dependently stimulate macrophages to produce NO through the up-regulation of inducible NO synthase (iNOS) activity. Pharmacological studies revealed IOPN、IOPS and AIOP could inhibit the growth of Sarcoma 180 tumor remarkably in vivo. Polysaccharides from Inonotus obliquus increased in the relative spleen and thymus weight, expression of tumor necrosis factor-alpha (TNF-α) in serum. However, IOPN、IOPS and AIOP at the concentration up to 300μg/ml did not affect the growth of S180、Hela、BGC-830 and HepG2 tumor cells in vitro. The neutral Inonotus obliquus polysaccharide IOPN had best immunomodulative activities in the acidic polysaccharide IOPS and the alkali-soluble polysaccharides AIOP. These results indicated that the tumor inhibitory activity of the three fractions may be related to immunomodulation.