Study On Preparation And Physicochemical Characteristics Of Schizophyllan And Its Chemical Modification

Schizophyllan is one kind of biological response modifiers. It shows good antitumor activity in clinical application. Schizophyllan also shows bright future in the application of food, drug carrier, cosmetics for its particular physical and chemical characteristics. While few research were carried out for the fundamental study about application and chemical modification of Schizophyllan, these factors have an enormous restriction to the further application of Schizophyllan. The culture of Schizophyllum commune, the purification method and typical physical and chemical characteristcs of Schizophyllan were systematically researched and its chemical modification and the relationship of structural and function were discussed. It was aimed to set theory ground for the development of Schizophyllan and improving the use value of polysaccharide product. The main contents and results are as follows:1. Morphology variation of Schizophyllum commune and secreted rules of SchizophyllanThe morphology variation of Schizophyllum commune under different cultured conditions were researched by direct observation and micrographs. Combined with the determination of fermentation parameters such as the content of polysaccharide, reduced sugars and biomass, it was indicated that the synthesis of extracelluar polysaccharide in Schizophyllum Commune is with growth coupling and the yield of Schizophyllan is related with the morphology of mycelial. The yield of Schizophyllan can be improved with more"efficient growing points"by the control of dissolved oxygen and agitation.2. Rapid purification method of gram scale Schizophyllan and its structural identificationHomogeneous polysaccharide can be obtained by following procedures involved centrifugation of suspensions from fermentation, followed by decolouring with activated carbon, removing protein by Savage method, precipitating with ethanol, dialysing,concentrating by 50kD ultramembrane and freeze-drying. This process is a rapid way for the purification of Schizophyllan at gram scale. In addition, two feasible purified processes for the kg scale product of Schizophyllan in liquid or powder were put up based on experimental work.The structure of Schizophyllan secreted from the strain of Sc1 was identified as a 1,3-β-D-glucopyranoses with single 1,6-β-D-glucopyranose branch at about every third glucose, using the analysis with UV, IR, HPLC, GC-MS, 1H NMR and 13C NMR. The weight-averaged molecular weight and number-averaged molecular weight of Schizophyllan determined by GPC were 2.5×107 and 1.2×107, respectively. These can be used as fundamental data for further application.3. Physicochemical characteristics of SchizophyllanUbbelohde-type viscometer, rotating cylinder viscometer and GPC were used to research the determination method of molecular weight and the relationship between viscosity and conformation of Schizophyllan. The formula to determine the relative molecular weight of Schizophyllan by viscosimeter was supplemented with the data from literature.Controling the humidity in desiccator, moisture absorption rate and moisture retaining rate of Schizophyllan were studied, while the thermodynamic parameters of Schizophyllan were determined by DSC. The results indicated that these characteristics are at good with Schizophyllan and showed Schizophyllan is a natural humectant with enormous potential to be exploitated.It was found that there is a linear relationship between the fluorescence intensity and the concentration of Schiphyllan in the concentration of 2.6×10-6～6.5×10-4g/L performed by a FLUOstar Galaxy microplate reader set at 390nm excitation and 520nm emission, when super pure water was used as the blank.The elution profiles of the polysaccharide fractions of two Schizophyllan samples with different purity for the non-typical absorption at 350nm were similar with the typical absorption at 490nm by sulfuric-phenol method. It showed the wavelength at 350nm can be used for monitoring the elution curve and optimizing the purification condition.4. Acetylation and carboxymethylation of SchizophyllanAcetylated Schizophyllan and carboxymethyl Schizophyllan with different DS were synthesized and their structures were identified with UV, IR, 1H NMR or 13C NMR. The factors affected the DS of carboxymethyl Schizophyllan were discussed and a method to determine the DS was established by IR method. Acetylated Schizophyllan is hardly dissoluble in water, while carboxymethyl Schizophyllan is easily dissoluble.5. Antitumor activity of carboxymethyl SchizophyllanSchzophyllan and three kinds of CM-SPG with different DS showed no inhibition effect on GLC-82 tumor cell under the concentratin of 3.3,16.7 and 33.3μg/mL by MTT method in vitro. In vivo antitumor test, two CM-SPG samples with DS of 0.67 and 0.93 respectively showed significant tumor inhibition activity after injected intraperitoneally once daily for 10d at the dose of 10mg/kg and the inhibition ratio were 41.3% and 37.9% respectively. The CM-SPG with DS of 1.14 showed no tumor inhibition activity under the test. It was indicated there is a close correlation for the antitumor acitivity of CM-SPG with its DS. The histological observation of the tumor showed CM-SPG with DS of 0.67 can cause the H22 tumor cell death or apoptosis and cause lymphocyte to infiltrate tumor base.