Sulfated Modification And Cellular Immunemodulatory Activity Of Polysaccharides From Pinctada Martensii

Aims:Chlorosulfonic acid-pyridine(CSA-Pyr) method has been designed to sulfated modification of polysaccharides from Pinctada Martensii(PMPS) extracted by enzymatic hydrolysis in this paper. The cellular immune-modulatory activity of PMPS and its sulfate derivatives were evaluated in vitro. The relation of chemical structure and bioactivity of PMPS was discussed.Methods:1. The degrees of substitution(DS) and total sugar content of PMPS was studied and the optimal process conditions were confirmed by the single-factor experiments and response surface method(RSM), including the reaction temperature, the reaction time, the volumetric ratio of CSA to Pyr and the volumetric ratio of CSA-Pyr to polysaccharides solution.2. Purified PMPS was purified by DEAE-52 column chromatography, The relative molecular weight and purity of PMPS-1, PMPS-1-S was further confirmed by HPLC;The components of PMPS and its sulfate derivatives were analyzed by IR, UV and NMR.3. The proliferation of spleen lymphocyte from mice was evaluated by MTT method in vitro. The phagocytic activity of mouse peritoneal macrophages was tested by neutral red method in vitro.Results:1. The results of single-factor experiments showed that reaction temperature, the reaction time, the volumetric ratio of CSA to Pyr and the volumetric ratio of CSA-Pyr to polysaccharides solution affected the DS trend, except for total sugar content. The optimum conditions of PMPS sulfated were reaction temperature of 70 ℃, reaction time of 4.6 h, volumetric ratio of CSA to Pyr of 1:4, volumetric ratio of CSA-Pyr to polysaccharides solution of 6:5. In this condition, DS and total sugar content of products reached 1.44 and 76.41%, respectively.2. Results of DEAE-52 column chromatography showed that three independent elution peaks(PMPS-1, PMPS-2 and PMPS-3) were obtained. Relative molecular weight of PMPS-1 and PMPS-1-S were 1.69×104Da, 2.98×106Da respectively. characteristicabsorptions of polysaccharides were observed in B( DS 0.26), C(DS 0.42), D(DS 0.56)and E(DS 1.14) with different DS. characteristic absorptions of polysaccharides were also observed in PMPS-1 and PMPS-1-S. There were pyranose rings in PMPS-1 and PMPS-1-S, and PMPS-1-S was sulfate substituted at C-6 position.3.(1) The effect of PMPS and its sulfate derivatives on proliferation of spleen lymphocyte from mice Results of sulfate derivatives with different DS showed that the five kinds of polysaccharides sulfate all had the ability of promoting lymphocyte proliferation and their ability were E>D>C>B>A, and the same result as that in Con A/LPS induced module. PMPS-1 and PMPS-1-S all had the ability of promoting lymphocyte proliferation and their ability were PMPS-1-S>PMPS-1, and the same result as that in Con A/LPS induced module.(2) The effect of PMPS and its sulfate derivatives on phagocytic activity of mouse peritoneal macrophages B( DS 0.26), C(DS 0.42), D(DS 0.56), E(DS 1.14), PMPS-1 and PMPS-1-S could all enhance ability of devouring neutral red of macrophages with a dose-response relationship. In the same dose, E(DS 1.14) and PMPS-1-S had higher ability of devouring neutral red of mouse peritoneal macrophages than others. A(DS 0.12) also could enhance ability of devouring neutral red of mouse peritoneal macrophages with no statistical significance.Conclusion:(1) Optimum conditions of PMPS sulfate were reaction temperature of 70 ℃, reaction time of 4.6 h, volumetric ratio of CSA to Pyr of 1:4, volumetric ratio of CSA-Pyr to polysaccharides solution of 6:5. In this condition, DS and total sugar content of products reached 1.44 and 76.41%, respectively.(2) B( DS 0.26), C(DS 0.42), D(DS 0.56) and E(DS 1.14) could have different DS;PMPS-1-S was sulfate substituted at C-6 position with two substituted signal.(3) PMPS and its sulfate derivatives could enhance cellular immune-modulatory activity,and the same result as that in Con A/LPS induced module. PMPS sulfate could improve cellular immunemodulatory activity markedly.