Chemical And Pharmacological Study Of Polysaccharides In Crude And Processed Rhizoma Dioscoreae

In this thesis,we investigated the difference of polysaccharides in crude and processed Rhizoma Dioscoreae.After being processed with bran,the content of polysaccharides in Rhizoma Dioscoreae was increased,and there’s some difference in the monosaccharide composition.The Pharmacological studies showed that the effect of the content of polysaccharides in processed Rhizoma Dioscoreae was better than that of crude one.Our studies were performed in three aspects as following:1.Chemical researchesUsing Phenol-H₂SO₄ method to determine the content of crude and refined polysaccharides in Rhizoma Dioscoreae before and after being processed with bran,we found that while all the contents of refined polysaccharides in crude and processed Rhizoma Dioscoreae could reach above 90%,the content of crude polysaccharides in crude Rhizoma Dioscoreae was 72.63%,and the yield was 4.91%,the content of crude polysaccharides in processed one was 76.44%,and the yield was 5.73%.Using Coomassie brilliant blue G-250 to determine the content of proteins in crude polysaccharides,we found that the content of proteins in crude Rhizoma Dioscoreae was 0.23%,and the content of proteins in processed one was 0.07%.Using HPLC-ELSD to determine the monosaccharides composition of crude and refined polysaccharides.The results showed that the polysaccharides in crude and processed Rhizoma Dioscoreae were all composed by rhamnose and glucose,and there were some quite difference in the molar ratios of monosaccharides composition of crude polysaccharides and refined polysaccharides from DEAE-52 water eluant part in crude and processed Rhizoma Dioscoreae.The molar ratio of monosaccharides composition of crude polysaccharides in crude Rhizoma Dioscoreae was Rha:Glu=1:2.22,and that in processed one was Rha:Glu=1:3.27,the molar ratio of monosaccharides composition of refined polysaccharides from DEAE-52 water eluant part in crude Rhizoma Dioscoreae was Rha:Glu=1:2.04,and that in processed one was Rha:Glu=1:3.47.Through isolation and purification by Cellulose DEAE-52 and Sephadex G-100,we obtained a homogeneous polysaccharide（SYS） from crude Rhizoma Dioscoreae,and its molecular mass was 10,992dalton by the determination of HPLC-ELSD.The homogeneous polysaccharide（SYS） was not obtained from processed Rhizoma Dioscoreae in same condition.Using IR、¹H-NMR、¹³C-NMR to analyze the structure of SYS,and it was estimated that SYS might beα-D-glucosan with 1→4 chain element.2.Pharmacological researches in vivoTo investigate the effect of immuno regulation of polysaccharides in crude and processed Rhizoma Dioscoreae through nonspecific immunity、specific immunity and cell immunity.We found the content of crude polysaccharides in Rhizoma Dioscoreae before and after being processed with bran can enhance the immune function of mice with immune deficiency caused by cyclophosphamide.And the experiments of serum haemolysin and spleen T lymphocyte proliferation showed that the effect of processed Rhizoma Dioscoreae was better than that of crude one.We carried a preliminary study for investigating the effect on gastrointestinal function of mice with spleen deficiency cause by rhubarb.The result showed that the polysaccharides from crude and processed Rhizoma Dioscoreae can significantly inhibit the gastric emptying rate and intestinal propulsive rate of model mice,also spleen index and thymus index were all significantly increased.It’s suggested that the polysaccharides in Rhizoma Dioscoreae have some effects of invigorating the spleen and strengthening the stomach,and the function of immunoregulation might be one of its possible mechanisms.It was the first time to compare and study the effet of T lymphocyte subset of peripheral blood of immuno- deficiency mice caused by cyclophosphamide by using flow cytometry technique with crude polysaccharides in Rhizoma Dioscoreae before and after being processed with bran.3.Pharmacological researches in vitroIt was the first time to compare and study the effet of crude and refined polysaccharides in crude and processed Rhizoma Dioscoreae to mice mononuclear-macrophage Raw264.7.The results showed that the crude and refined polysaccharides of Rhizoma Dioscoreae had strong proliferative effects to mice mononuclear-macrophage Raw264.7,and the effect of processed Rhizoma Dioscoreae was better than that of crude one.Cell cycle analysis showed crude and refined polysaccharides in Rhizoma Dioscoreae can increase the content of S phase in mice macrophage induced by LPS.It’s suggested that the effect of cell proliferation of polysaccharides in Rhizoma Dioscoreae could be reached by the increase of the content of S phase.The further experimental results of T-SOD、MDA、NO and IL-1βshowed that polysaccharides in Rhizoma Dioscoreae could significantly increase the activity of T-SOD in supernatant of mice macrophage and significantly decrease the content of MDA,and meantime it could also improve the ability of NO and IL-1βproduced by the organism in some degree,and thereby to keep the cell of the organism away from injury and to reinforce the immunity of the organism.And generally the effect of processed Rhizoma Dioscoreae was better than that of crude one.The preliminary cell proliferation test was performed in the first time for investigating the effet of the polysaccharides in crude and processed Rhizoma Dioscoreae to astrocyte. The result showed that the polysaccharides in crude and processed Rhizoma Dioscoreae have an obviously protective effect for astrocyte injuried by H₂O₂.