Extraction And Isolation Of Polysaccharide From Portula Caoleracea And Evaluation Of Its Therapeutic Effect On Ulcerative Colitis And Its Mechanisms

Objective To extract and isolate polysaccharide from Portulaca oleracea by traditional process combined with membrane separation.And to study its therapeutic effect on ulcerative colitis and its molecular mechanisms.Methods 1.Orthogonal experiment design L₉?4³?was employed to optimize the traditional extraction process.Specifications of membrane with different apertures was used to purify the crude polysaccharide.investigated The processes of deproteinization and depigmentation.And the anti-oxidant activity of the polysaccharide was also evaluated by Fenton reaction and autoxidation of pyrogallol.2.DSS was used to establish the UC mice model.H&E staining was employed to observe pathological changes of the colonic mucosa after the treatment of polysaccharide with high,medium and low dose.The expression level of TNF-?,IL-6,NO and IL-10 were measured by Enzyme Linked Immunosorbent Assay.3.Immunohistochemical staining was used to detect the expression of NF-?B/P65,p-STAT3 and COX-2 in the colonic mucosa.And the protein expression level of NF-?B/P65,IKB?,STAT3 and p-STAT3 were detected by Western blotting.Results 1.The polysaccharide was extracted at 100?for 3 h with four times repeated to give the highest extraction rate.The recycling rates of thicloroacetic acid method to remove protein was 9.66%,The recycling rates of H₂O₂ to remove pigment was 17.09%.The result of membrane separation study showed that The recycling rates of purified purslane polysaccharide was 61.63%with apertures of 0.8?m.The clearance rate of polysaccharide scavenge hydroxyl radical and superoxide radical were 58.89%,and 38.89%.2.The H&E staing results showed that after the treatment with high,medium and low dose of polysaccharide,the cell morphology of colonic mucosal epithelial cells were recovered and arranged orderly,.Compared with the control group,the levels of TNF-?,IL-6 and NO in serum were significantly increased in model group?p<0.01,p<0.05?,and the level of IL-10 was significantly decreased in model group?p<0.01?.Conversely,after the treatment with polysaccharide,the level of TNF-?,IL-6 and NO were decreased and the expression of IL-10 was increased?p<0.01,p<0.05?.Furtherly,there was no significant difference in the expression level of TNF-?,NO and IL-10 in the high and middle dose polysaccharide groups?p>0.05?comparing with positive group.And the expression level of IL-6 was no notable difference in middle dose polysaccharide groups?p>0.05?comparing with positive group.3.The rsults of Immunohistochemical staining exhibited that the protein exression level of NF-?B/P65,p-STAT3,and COX-2 were significantly increased in model group?p<0.01?.After the treatment with media polysaccharide,the protein expression levels of NF-?B/P65,p-STAT3,and COX-2were also increased largely?p<0.01?.Comparied with the positive group,the expression levels of NF-?B/P65 and p-STAT3 showed no significant difference.The results of Western blot gave the same changes of the expression level of these proteins.In deeply,comparied with control group,in model group,the expression levels of IKB?and NF-?B/P65 in cytoplasm were decreased and the protein level of NF-?B/P65 in nucleus was decreased.Intreastingly,the oppositive results were observed in the group treated with polysaccharide.The phospherlation level of STAT3was increased significantly in model group comparing with control.And the oppositive results were observed in the group treated with polysaccharide.Conclusion The combination of traditional process with membrane separation were employed to isolation and purification of polysaccharide from Portulaca oleracea,And the therapeutic effect of polysaccharide on UC was also investigated in our work.The results showed that the expression levels of TNF-?and IL-6 were increased substantially in UC.And furtherly,TNF-?/NF-?B andIL-6/STAT3 signal pathways played important roles in UC progression,and polysaccharide may exert its therapeutic effect on UC via regulating these two key signal pathway.