| Hyperuricemia is a disease caused by abnormal purine metabolism and abnormal transport system of uric acid.Hyperuricemia is the basis of the onset of gout,and also related to many cardiovascular diseases.Although many types of western medicines have been marketed,they will cause serious side effects.Therefore,natural products with small side effects have potential to develop uric acid lowering drugs.In the previous experiments,we found that water extract of Glycyrrhiza uralensis had a good effect in lowering uric acid.In this study,the extraction,purification and structure analysis of Glycyrrhiza uralensis polysaccharides(GUP),and their uric acid-lowering effects,mechanisms and preparations were studied to provide references for the development of natural anti-hyperuricemia drugs.The full text can be divided into six chapters:ChapterⅠ:ReviewThis chapter introduced the research situation of polysaccharide,a biological macromolecule,and also introduced the extraction,purification,structure analysis and formulation-related research of polysaccharide from Glycyrrhiza uralensis,then expounds the harm of hyperuricemia and the problems in its treatment.This part lays a theoretical foundation for the research of the experimental part.ChapterⅡ:Research on Extraction Process of Glycyrrhiza uralensisPolysaccharideIn this chapter,hot water extraction method was used to extract polysaccharides from Glycyrrhiza uralensis.Combined with single factor experiments and orthogonal experiments,the effects of extraction time,extraction temperature,extraction frequency and material to liquid ratios on the extraction rate of crude polysaccharides were investigated.The optimum extraction process were:extraction time:3.5 h,extraction temperature:90℃,the ratio of material to liquid:1:20,and extraction times:2 times.The yield of crude polysaccharide was 5.67%under the extraction process.The optimal extraction process greatly improved the yield of crude polysaccharide and saved the extraction cost.ChapterⅢ:Isolation and Purification of Glycyrrhiza uralensisPolysaccharideIn this chapter,the Sevag method was used to remove the proteins,and dialysis method was used to remove low-molecular impurities.Bradford method was used to determine the content of protein in Glycyrrhiza uralensis polysaccharide(GUP).After removing proteins,the protein content of GUP was 0.58%,which showed that the Sevag method was reliable.GUP was purified by DEAE-52 column chromatography and Sephadex G-200 column chromatography to obtain two polysaccharide components,GUP-1 and GUP-2.High performance gel filtration chromatography(HPGFC)showed that GUP-1 and GUP-2 were polysaccharides with uniform molecular weight distribution.Element analysis showed that GUP-1 and GUP-2 did not contain S element and contained little N element.The total sugar contents of GUP-1 and GUP-2 were 98.58%and 98.39%,respectively,and their protein contents were 0.13%and 0.09%.ChapterⅣ:Structural Analysis of Homogeneous Polysaccharidesfrom Glycyrrhiza uralensisThis chapter analyzed the structure of Glycyrrhiza uralensis polysaccharides GUP-1and GUP-2.HPGFC,pre-column derivatization HPLC,periodate oxidation,Infrared,~1H-NMR and Congo red experiments were used to conduct the main structural information.Structural analysis showed that GUP-1 and GUP-2 had different structures.The molecular weight of GUP-1 and GUP-2 were 294373 Da and 17416Da,respectively.The content of glucuronic acid in GUP-1 and GUP-2 was 12.2%and1.66%.GUP-1 mainly consisted of glucuronic acid,galactose and arabinose,while GUP-2 was composed of glucose.The proportion of glycosidic bond was also different,GUP-2 had a higher ratio of 1→3 glycosidic bonds.GUP-1 had a triple helix structure,and GUP-2 did not have a riple helix structure.ChapterⅤ:Hypouricemic activities of Glycyrrhiza uralensispolysaccharides in acute hyperuricemic ratsIn this chapter,we established an HPLC method to analyze uric acid in rats.The methodological validation meet the requirements of analysis and determination.Acute hyperuricemic rats model was established and the uric acid lowing effect of Glycyrrhiza uralensis polysaccharides was studied.All Glycyrrhiza uralensis polysaccharides components were able to reduce the serum uric acid value in a dose-dependent manner,and GUP-2 had the strongest ability to reduce the blood uric acid value among the Glycyrrhiza uralensis polysaccharides groups.Study of the effects of Glycyrrhiza uralensis polysaccharides components on serum and liver xanthine oxidase showed that all Glycyrrhiza uralensis polysaccharides groups could inhibit the XOD activity in serum and liver in a dose-dependent manner.The pathological observation of the renal tissue in each group of acute hyperuricemic rats was measured.In model group,the edema and necrosis of renal tubule epithelial cells and renal interstitium was observed.The renal lesion in allopurinol group,high dose GUP group and high dose GUP-2 group decreased obviously.The pathological observation of the liver tissue in each group of acute hyperuricemic rats was measured.In model group,most of the liver cells had vesicular degeneration,the nuclei became spindle type,and the cell morphology was abnormal.The pathological changes of allopurinol group and high dose GUP-2 group were not obvious,and the cell morphology was basically normal.The above results showed that in acute hyperuricemic rats model,the uric acid lowing effect of GUP-2 was significantly higher than that of GUP-1.The mechanism of uric acid lowering activity of the Glycyrrhiza uralensis polysaccharides could be attributed to the inhibition of xanthine oxidase.Chapter Ⅵ:Research on the Molding Process of Glycyrrhiza uralensis Polysaccharide GranulesIn this chapter,according to the properties of Glycyrrhiza uralensis polysaccharides and the advantages of granules,GUP-2,which had the best effect of lowering uric acid,was selected to prepare Glycyrrhiza uralensis polysaccharide granules.Glycyrrhiza uralensis polysaccharide granules were prepared by wet granulation method.The prescriptions were investigated by single factor and orthogonal experiments.The best prescription was composed of 15.5%GUP-2,18.6%mannitol,64.9%erythritol,1%citric acid.The quality of granules was evaluated.Content,appearance,particle size,moisture content and solubility were detected as evaluation indexes.The results showed that the content of GUP-2 in granules was 15.46%±0.026%,the size of granules was uniform,the size of granularity was in conformity with the requirement,the water content was less than 5%,and granules could be dissolved within two minutes.All evaluation indexes were in accordance with the requirements.An acute hyperuricemia rat model was established to evaluate the efficacy of Glycyrrhiza uralensis polysaccharide granules.The results showed that the blood uric acid value of the GUP-2 group and the Glycyrrhiza uralensis polysaccharide granule group decreased significantly(P<0.01),and there was no difference in the efficacy of GUP-2 and Glycyrrhiza uralensis polysaccharide granule group,indicating that the preparation of granules did not affect the efficacy of GUP-2.Glycyrrhiza uralensis polysaccharide granules was easier to carry and more convenient for taken. |
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