| Object: To separate the polysaccharides from the fruiting body of Phellinus linteus,and choose the optimum exatraction condition. Then separate the polysaccharides with uniformity of molecular weight and study the chemical structures of the parts. To study the anti-tumor activity of the crude polysaccharides, and prepare a kind of modern praeparatum with convenience and activity.Methods: According to the property of polysaccharides can dissolve in hot water, the fruit body were comminuted and extracted in hot water. There were polysaccharides, monosaccharide, floating protein in the extractum. The floating protein and monosaccharide were removed by precipitated with ethanol, and detect the result with HPLC. In order to extract the polysaccharides thoroughly and save manufacture cost, choose the optimum extraction condition through L9(34) orthogonal test based on the results of single factor tests, and the optimun concentration of ethanol.The study to the microstructure of the polysaccharides is difficult because of their complicated structures, thereby limited the clinical application. In order to research the characterization, polysaccharides with uniform molecular weight should be separated and purified. We use DEAE-cellulose and gel-filtration chromatograph to separate the polysaccharides, and remove the salt by ultra filtration.Polysaccharides are compounds with macromolecule, so the purity should not be judged by the purity rubri of micromolecule compounds. Because microcosmic can not be uniform even the sterling. So we consider the polusaccharides with special molecular weight are sterling. The purification result and the molecular weigh are detected with HPLC with dextran contrast.The polysaccharides content were determined with sulphuric acid-phenol method. The reaction was carried out at high temperature, and cooled down quickly, then metered volume with concentrated sulfuric acid, these measures improved the stability, and made the result accurate. PL-B showed absorption at 280nm, so some protein existed in PL-B. we used three methods to determine the protein content, including Lowry,BCA,Bradford, and chose the correct Lowry at last. The reducing sugar content was determined with DNS.In order to study the saccharides group of the polysaccharides, we need to break the chain, then use TLC, HPLC, and GC to determine the monosaccharides. TLC could not separate the different monosaccharides, but could distinguish uronic acid and monosaccharides. There was not uronic acid either in PL-A or PL-B. HPLC could separate the different monosaccharides easily, while GC need derivatization and need more time. In this study, we use DABS Cl as derivatization reagent, and use C18 to determined the amino acid composition by HPLC.Because of the microheterogeneity of the chain, the configuration research is difficult. Physics method, chemistry method, and biological method are commonly used. In this study, we used IR, 1H-NMR, 13C-NMR, periodate oxidation, and Smith degradation to determine the chain configuration of PL-A, PL-B.The anti-tumor activity of the crude polysaccharides was tested by animal experiment. We chose Kunming species mouse to set up S180 tumor model, all mice were divided into 5 random groups. (1) Placebo control group: 0.5% CMC-Na intragastric administration. (2) Positive control group: 0.25% cyclophosphamide hypodermic injection. (3) Low dose group: 250mg/kg crude polysaccharides intragastric administration. (4) Middle dose: 500mg/kg crude polysaccharides intragastric administration. (5) High dose: 1000mg/kg crude polysaccharides intragastric administration. To investigate the tumor weight, inhibition rate of tumor growth, spleen index, lymphocyte transformation test, NK cell function in bearing cancer mice.At last, we completed initial investigate about tablet preparation, including screen adjuvant and prescription predesign. The prescription was detected up to standard. The next work is to choose optimization prescription with scientific methods, and find suitable coating material to give film coat. Normal cellulose material could interfere with the polysaccharides content test, so we are considering studying the character and effect of opadry membrane.Results: The optimum extraction condition according the L9(34) orthogonal test were obtained as 100℃,8 hours every time, ratio of solid-liquid 1:15, then precipitated in 80% ethanol, and repeated 3 times. The crude polysaccharides yield was 3.26%. Polysaccharide PL-A and proteoglycan PL-B were obtained. The molecular weight of PL-A was about 1.42×104 and that of PL-B was 2.22×104. The proteoglycan PL-B consisted of 71% polysaccharide and 6.3% protein. The sugar of PL-A and PL-B were both composed of most glucose and little mannose, and some rhamnose existed in PL-B besides. The amino acid pattern showed that PL-B contained vlaine, lysine, aspartic acid, and glycine. Both structures were complex hetero- polysaccharides withβ-(1→3) chain. Kawagishi considered that polysaccharides withβ-D-glucan structure showed anti-tuomr activity. Hongying Ma considered that polysaccharides with anti-tuomr activity were always composed by glucose, and must withβ-(1→3) link in backbone,β-(1→6) link in branched chain. Our results coincided with these inferences. The pharmacodynamics test results suggested that the crude polysaccharides could produce anti-tumor and immunomodulatory activities in vivo. The tablets were qualified in weight difference and dissolution.Conclusion: The crude polysaccharides extracted from the fruiting body of Phellinus linteus showed antimumor activity. The two kinds of polysaccharides, PL-A and PL-B, are new compounds that have not been reported. The study is confirmed valuable and deserves more investigation.
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