Preparation, Characterization And Biological Activities Of Capsule Polysaccharides From Streptococcus Zooepidemicus

Streptococcus zooepidemicus also named as Streptococcus equi subsp. zooepidemicus. It has been reported that the main component of capsule polysaccharides from S. zooepidemicus is hyaluronic acid (HA). HA have antioxidant, immunomodulation, wound healing, angiogenesis angiogenesis and antitumor activities. So far the studies on the capsule polysaccharides of S. zooepidemicus have been focused on HA. However, there are few reports regarding the antioxidant activity and immunoregulation activities of HA. There are no reports regarding chemical structures and biological activities on the minor components from capsule polysaccharides. The present studied the isolation, purification, chemical structural characterizations and pharmacological activities of capsule polysaecharide from S. zooepidemicus.This dissertation consists of five chapters, which are Fermentation condition of capsule polysaccharides from S. zooepidemicus, purification and structure determination, antioxidant activities, immunoregulation activities of capsule polysaccharides, antioxidant activities and immunoregulation activities of low molecular wieght HA. The results are listed as follows:1 Fermentation conditions for the products of capsule polysaccharides from S. zooepidemicusThrough single-factor experiments, it was found that S. zooepidemicus C55129 was cultivated in medium containing glucose, yeast extract and peptone (2:1), NaN03, MgSO4.7H2O, K2HPO4, and NaH2PO4-2H2O. And inoculation volume for 10%, then the culture was operated at 33℃, pH 6.5 with a shaking at 200 rpm for 18 h. By analyzing, the concentration of glucose, fermentation time and temperature are found to be the most important factors for S. zooepidemicus C55129 produced HA.Based on the results of single-factor experiments, central composite design was employed in the present study to optimize the levels of the chosen variables, which significantly affect the yield of capsule polysaccharides in fermentation using S. zooepidemicus C55129, i.e. the concentration of glucose, fermentation time and temperature. By analysis of the response surface contour plots generated from the quadratic regression equation, the effects of the individual variable as well as the mutual interactions between variables were extensively studied. The results suggest that the concentration of glucose, fermentation time and temperature were decided. The production of HA increase 1.45 times, for 1.473 g/L by the optimization. Experiment value is accord to the predictive value.2 Purification and structural characterization of capsule polysaccharides from S. zooepidemicusBy ethanol precipitation, crude capsule polysaccharides (CCP) from S. zooepidemicus C55129 were separated. CCP were partially purified through an anion-exchange column chromatography to afford partially purified capsule polysaccharides (PCP). The main component of CCP and PCP was HA. CCP also were separated and sequentially purified through a column of DEAE-cellulose 52 and a column of sephadex G-100 to afford three fractions of homogeneous capsule polysaccharide, the recovery rates of PCP-Ⅰ, PCP-Ⅱand PCP-Ⅲwere 5.989% 5.079% and 48.932%, respectively. PCP-Ⅰ, PCP-Ⅱand PCP-Ⅲare a white solid, all are soluble in water, having the molecular weight about 0.0292×103kDa, 0.891×103kDa and 1.338×103 kDa respectively.Monosaccharide composition and molar ratio of PCP-Ⅰand PCP-Ⅱwere analyzed by Gas chromatography(GC). They were arabinose:mannose:glucose: galactose=0.99:27.03:11.84:1.00, and mannose:glucose:galactose=6.87:4.65: 1.00, respectively. Determined by HPLC measurement after complete acid hydrolysis, PCP-Ⅲwas consist of GlcA and GlcNAc, with the percent contents of 43.75% and 40.31% respectively.The major structural features of PCP-Ⅰand PCP-Ⅱwere elucidated using partial acid hydrolysis, periodate oxidation, smith degradation, IR, 1H and 13C NMR, methylation. On the base of the results obtained above, it was possible to conclude that the repeated unit of PCP-Ⅰcontained a backbone composed of 1→6-linked-mannose and glucose, which attached to galactose and arabinose. PCP-Ⅱcontained a backbone composed of 1→2-linked-mannose and glucose, which attached to galactose and GlcA. PCP-Ⅲis HA, which made of repeating units of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) which is bounds by alternatingβ-(1→4)-andβ-(1→3)-bonds.3 Antioxidant activities of capsule polysaccharides from S. zooepidemicus In the present study, the antioxidant properties of capsule polysaccharides from S. zooepidemicus C55129 were demonstrated by using a variety of testing systems in vitro and by a D-galactose induced aging mice model in vivo. In vitro antioxidant assay, CCP, PCP-Ⅰ, PCP-Ⅱand PCP-Ⅲshowed strong inhibition of lipid peroxidation and moderate 1,1-diphenyl-2-picryldydrazyl radical, superoxide radical and hydroxyl radical scavenging activity in the same concentration. Moreover, PCP-Ⅰand the CCP has strong reducing power and chelating ability to metal ions, and the reducing power and chelating metal ions of PCP-Ⅰwas stronger than those of CCP, PCP-Ⅱand PCP-Ⅲ. It can be seen that the capsular polysaccharide showed strong antioxidant activity in vitro, PCP-Ⅰ, PCP-Ⅱand PCP-Ⅲhave different levels of antioxidant activity.For antioxidant testing in vivo, CCP and PCP were orally administrated over a period of 15 days in a D-galactose induced aged mice model. As results, administration of capsule polysaccharides inhibited significantly the formation of malondialdehyde in mice livers and serums and raised the activities of antioxidant enzymes and total antioxidant capacity in a dose-dependent manner. However, the antioxidant activity of CCP was lower than that of PCP. The results suggest that the capsule polysaccharides from S. zooepidemicus C55129 have direct and potent antioxidant activities.4 Immunoregulation activities of capsule polysaccharides from S. zooepidemicusThe immunomodulation activity of PCP was studied by delayed type hypersensi-tivity (DTH) induced by 2,4-dinitro-fluorobenzene (DNFB) and serum lysozyme in immunosuppressed mice induced by a cyclophosphamide (CY). The indexes of thymus gland and spleen were also studied. The results show that the PCP could enhance DTH and increase the level of serum lysozyme. The thymus and spleen indices of the immunosuppressed mice treated with PCP increased in a dose-dependent manner. The results show that the PCP has a better ability of immunostimulation and can improve immune suppression of the immunosuppressed mice.The immuno-stimulating assay in vitro showed that capsule polysaccharides could activate macrophages and stimulate splenocyte proliferation. The effect of PCP-Ⅱand PCP-Ⅲon phagocytosis of peritoneal macrophage was much higher than PCP-Ⅰand CCP. The results indicate that the capsule polysaccharides could enhance nonspecific immunity and specific immunity, and the effect of PCP-Ⅱand PCP-Ⅲ were more significant. It can be seen that the capsular polysaccharide showed strong immunostimulation activity, PCP-Ⅰ, PCP-Ⅱand PCP-Ⅲhave a certain degree of nonspecific immunity and specific immunity function.5 Antioxidant activities and immunoregulation activities of low molecular wieght HALow molecular weight HA (LMWHA), with a molecular weight of 1.302×105 Da, was prepared from high molecular weight HA. In the present study, the antioxidant properties of LMWHA were demonstrated by a CY induced immunosuppressed mice model in vivo and by using a variety of testing systems in vitro. In vivo antioxidant testing, LMWHA were orally administrated over a period of 15 days in a CY induced immunosuppressed mice model. As results, administration of LMWHA inhibited significantly the formation of malondialdehyde in mice livers and serums and raised the activities of antioxidant enzymes and total antioxidant capacity in a dose-dependent manner. In vitro antioxidant assay, LMWHA showed strong inhibition of lipid peroxidation and hydroxyl radical scavenging activity and moderate 1,1-diphenyl-2-picryldydrazyl radical and superoxide radical scavenging activity. In addition, LMWHA exhibited much stronger reductive power and chelating ability on ferrous ions than PCP-Ⅲ, and the antioxidant activity of LMWHA was higher than that of PCP-Ⅲ. The results suggest that the LMWHA has direct and potent antioxidant activities, may play an important role in prevention of oxidative damage in the tissues and organs of immunosuppressed mice.The immunomodulation activity of LMWHA was studied by DTH induced by. DNFB and serum hemolysin in immunosuppressed mice induced by a CY. The indexes of thymus gland and spleen were also studied. The results show that LMWHA has stronger immune stimulating effect. The immuno-stimulating assay in vitro showed that LMWHA could activate macrophages and stimulate spleen lymphocyte proliferation.