Preparation Of Chlorella Pyrenoidosa Polysaccharides And Effects On Enhance Of Immunomodulation In Mice

The paper mainly studied the extraction, isolation and purification of polysaccharides from Chlorella pyrenoidosa applying the new technology of membrane separation. Two different polysaccharides were obtained using sepharose 6B column chromatography (CC). Furthermore, relative molecular weight (MW), monosaccharides composition, the bond linkages and functional groups of the polysaccharides were measured by High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and Fourier-transform infrared spectra (FT-IR) respectively. After administration of polysaccharides orally to stomach in the mice, nonspecific immune response, cellular immune response and humoral immune response were also investigated in the paper to determine its immunomodulation activity. The results were as follows:Through an orthogonal design of L16 (45), the highest yield was achieved when the sample was applied with 1000W of ultrasound for 800 seconds and then followed by incubation in water bath at 100℃ for 4 hours in the presence of 80% ethanol. Under the best extraction condition, the highest purity of the polysaccharide could be obtained by adding 3% of trichloroacetic acid (TCA) to remove the proteins. Microfiltration (MF) membrane with pore size of 0.1 um was used first (with pressure of 0 psi and temperature within 25 and 40 ℃). The permeate was fed to ultrafiltration (UF) membrane with molecule weight cut off (MWCO) of 3KDa (temperature within 25 and 40℃, feeding pressure 15 psi, retentate pressure 11 psi) and the retentate was precipitated with 80% ethanol. The polysaccharides with yield of 4.76% and purity of 60.03% were then obtained. Further purification by sepharose 6B column chromagraphy could gain two different molecule weight polysaccharides, CPS1 and CPS2 respectively.Determined by HPLC, MWs of CPS1 and CPS2 were 81877Da and 1749Da, respectively. GC traces of the hydrolyzed polysaccharides showed that majority of monosaccharides from both fractions were mannose (relative mass 78.0% from CPSl and 76.5% from CPS2). The FT-IR of CSP1 and CSP2 revealed typical characteristics of polysaccharides composed of α-Pyranoses.The immunomodulation activities of polysaccharides were determined by administration of 0.4mL of different concentrations of polysaccharides, 100mg mL-1, 50mg mL-1 and 25mg mL-1 respectively orally to the stomach in mice for 30 consecutive days. And then the body weight, relative organ weight, delayed-type hypersensitivity response (DTH), antibody titre level and phagocytosis were measured. The results showed that in the dosage of 100mg mL-1 and 50mg mL-1, phagocytic rate and phagocytic index, ear thickness rate and antibody titre were significantly different from the negative control (P<0.05), whereas in the dosage of 25mg mL-1, only phagocytic rate and phagocytic index and antibody titre exhibited significantly difference. These results indicated polysaccharides from Chlorella pyrenoidosa had dose-dependent immunomodulation activity in mice.