Study On The Preparation Of Radix Hedysari Polysaccharide With Different Molecular Weights And Its Effect On UC Mice

Objective:Ulcerative colitis is a chronic inflammatory bowel disease with high clinical incidence.Immune factors and the mechanism of intestinal mucosal oxidative stress injury play an important role in the pathogenesis of ulcerative colitis.Radix Hedysari is a genuine medicinal material in Gansu Province,which contains polysaccharides,saponins,flavones and other effective ingredients.Hedysari Polysaccharides has good anti-inflammatory,anti-oxidation,immunoregulation and other pharmacological effects.Our research group has previously reported that Radix Hedysari had a therapeutic effect on ulcerative colitis,which was likely attributed to its polysaccharide components.Moreover,studies been shown that the biological activities of Traditional Chinese medicine polysaccharides of different molecular weights may be different.Therefore,starting from the separation and purification of Hedysari Polysaccharides of different molecular weights,this thesis work continued to explore the effects of different molecular-weight Hedysari Polysaccharides on ulcerative colitis in mice,to provide theoretical basis for the treatment of ulcerative colitis by Hedysari Polysaccharides.Methods:1 Hedysari Polysaccharides HG-2,HG-3 and HG-4 were prepared by classical water extraction and alcohol precipitation,Sevage method for protein removal,Sephadex G-25,Sephadex G-100 and Sephadex G-75 column separation,respectively.2 The purity of Hedysari Polysaccharides HG-2,HG-3,and HG-4 were identified by chemical identification,specific rotation,polyacrylamide gel electrophoresis,UV spectrophotometry,and gel permeation chromatography(GPC),respectively.3 The monosaccharide compositions of Hedysari Polysaccharides HG-2,HG-3 and HG-4were analyzed by TLC,PMP-HPLC and GC-MS,respectively,and the molar ratios of each monosaccharide was calculated.The contents of Hedysari Polysaccharides HG-2,HG-3 and HG-4 were determined by phenol-concentrated sulfuric acid method and GC-MS.4 The functional groups and microstructures of Hedysari Polysaccharides HG-2,HG-3and HG-4 were characterized by infrared spectroscopy,scanning electron microscopy and Congo red test.5 The scavenging ability of Hedysari Polysaccharides HG-2,HG-3 and HG-4 on DPPH free radical was determined by enzyme plate analyzer,and the results were compared with those measured by UV spectrophotometer.6 The mice model of ulcerative colitis was established with 2,4,6-trinitrobenzenesulfonic acid.The pathological changes of colon were evaluated by hematoxylin-eosin staining,and the comprehensive score was made.The levels of tumor necrosis factorα(TNF-α),interleukin-6(IL-6)and interleukin-10(IL-10)in serum were determined by enzyme linked immunosorbent assay(ELISA).Result:1 The extraction rates of Hedysari Polysaccharides HG-2,HG-3 and HG-4 were 31.46%,20.05%,22.56%and 24.44%,respectively,by water extraction,alcohol precipitation,deproteinization and Sephadex series column separation.2 The results of either ninhydrin or iodide tests of Hedysari Polysaccharides HG-2,HG-3and HG-4 was negative.The specific rotation of each polysaccharide was the same at 40%and 80%alcohol concentration.Meanwhile,a single band was observed in polyacrylamide gel electrophoresis,and no obvious absorption was detected at 260-280 nm.All these results collectively indicated that the three kinds of polysaccharides were all of high purity.The molecular weights of the Hedysari Polysaccharides measured using GPC were as follows:HG-2:M_n:3710,M_W:32700,M_Z:452000,M_W/M_n:8.83;HG-4:M_n:5440,M_W:32000,M_Z:168000,M_W/M_n:5.89.The results of Hedysari Polysaccharides HG-3 were consistent with our previous study.3 The results of TLC,PMP-HPLC and GC-MS showed that Hedysari Polysaccharides HG-2 was composed of glucose,arabinose,galactose,rhamnose and xylose;Hedysari Polysaccharides HG-3 was composed of glucose;Hedysari Polysaccharides HG-4 was composed of glucose,galactose,and arabinose.The content of Hedysari Polysaccharides HG-4 was 96.87%by phenol-concentrated sulfuric acid method,while those of Hedysari Polysaccharides HG-2 and HG-3 were 95.97%and 96.79%,respectively,which were consistent with the previous studies of our research group.The contents of Hedysari Polysaccharides HG-2,HG-3 and HG-4 determined by GC-MS were 78.65%,69.71%and88.29%,respectively,which were all lower than those determined by phenol-concentrated sulfuric acid method.4 Infrared spectra indicated that Hedysari Polysaccharides HG-2,HG-3 and HG-4 were allβ-pyran type sugars.SEM showed that with the increase of separation and purification degree of Hedysari Polysaccharides,the surface of Hedysari Polysaccharides became denser.Congo red assay showed that Hedysari Polysaccharides HG-2 and HG-4 did not have triple helix structure,while Hedysari Polysaccharides HG-3 exhibited triple helix structure.5 The results of DPPH radical scavenging ability showed that the maximum scavenging rates of Hedysari Polysaccharides HG-2,HG-3 and HG-4 were 44.5%,40.9%and 46.2%,respectively,and they could scavenge DPPH free radicals at low concentrations compared with total Hedysari Polysaccharides.The results showed that the stability and repeatability of the enzyme plate analyzer were better than that of the ultraviolet spectrophotometer,and the enzyme plate analyzer was more accurate,rapid and simple.6 Pharmacological test results showed that total Hedysari Polysaccharides as well as Hedysari Polysaccharides HG-2,HG-3,HG-4 could significantly inhibit the abnormal weight loss of ulcerative colitis mice,improve fecal characters and macro colon lesions,relieve colon pathological changes,reduce the water content of colon and serum TNF-αand IL-6 level,and increase the serum IL-10 level(P<0.05,P<0.01).Compared with HG-2 and HG-3,the above effects of HG-4 showed an increased trend,among which the effects of HG-4 on the colonic water content and IL-10 were statistically significant(P<0.05).These results indicate that Hedysari Polysaccharides with different molecular weights(HG-2,HG-3 and HG-4)have therapeutic effect on ulcerative colitis,which is related to the alleviation of abnormal changes in TNF-α,IL-6 and IL-10.Conclusion:Hedysari Polysaccharides HG-2,HG-3 and HG-4 were separated and purified by Sephadex G-25,Sephadex G-100 and Sephadex G-75 chromatographic columns with high purity.The monosaccharide composition,molecular weight and microstructure of the three Hedysari Polysaccharides were different.Hedysari Polysaccharides HG-2,HG-3 and HG-4with different molecular weights have therapeutic effect on ulcerative colitis,and this effect is related to the alleviation of abnormal changes in TNF-α,IL-6 and IL-10.This study can provide theoretical basis and clinical reference for the treatment of ulcerative colitis with Hedysari Polysaccharides.