| The main utilization of medicinal plant Chrysanthemum morifolium Ramat.is mainly focus on,the flavonoids,volatile oil and organic acids in its inflorescence.The research on macromolecules,such as polysaccharides,mainly focusing on the extraction,isolation,preliminary purification and biological activities which is relatively shallow.While the research about the chrysanthemum polysaccharides in non-medicinal parts of is more limited.In this study,we mainly discussed the biological activity and its related mechanism of polysaccharides from the root,stem and leaf of Chrysanthemum morifolium,and tried to characterize the structure of polysaccharides.This dissertation was divided into four chapters.Part ?.Literature researchOn the basis of summarizing the relevant literature,the structural characteristics,separation and identification methods and the research status of the biological activity of chrysanthemum polysaccharides were sorted in order to provide scientific reference for carrying out the research about biological activity and structure of the chrysanthemum polysaccharides in non-medicinal parts.At the same time,intervention and treatment of related diseases caused disorders of intestinal barrier function were summarized for providing theoretical support for the study biological effects and mechanism of polysaccharide from Chrysanthemum's Root,Stem and Leaf.Part ?.Experimental researchThis part was devided into two chapters.Firstly,biological effects of polysaccharide from Chrysanthemum's Root,Stem and Leaf.In Section 1,immunosuppressive cyclophosphamide was used to replicate the immunoregulatory model of mice in order to investigate the immunoregulatory effect of polysaccharides from chrysanthemum on the immunosuppressive mice induced by chrysanthemum.The results showed that JFP could be significantly enhanced the thymus index and spleen index of immunocompromised mice significantly enhanced the phagocytic activity of macrophages in mice,and accelerated the removal rate of carbon particles per unit time.It indicated that JFP immunized mice immunized with cyclophosphamide Function has a certain degree of protection and improvement.The MTT results showed that JFP had a certain inhibitory effect on the growth of LOVO,HCT-116,SW480 and CAC02 in human colorectal cancer cells,and further confirmed that the positive effect of JFP on the improvement of inflammatory bowel disease was the future development and utilization of chrysanthemum Polysaccharides to provide scientific basis.In Section 2,acute colitis successfully induced in the rats by rectal administration of TNBS mixed with a certain percentage of ethanol through a special catheter which showed increasingly severe symptoms,including serious diarrhea,obvious rectal bleeding and notable body weight loss.The histopathological characteristics of colon tissue samples from each group were evaluated by H&E staining,the results showed that there were severe pathological changes in the colonic tissues of TNBS treated rats,including mucosal lesion and necrosis as well as infiltration of inflammatory cells such as monocytes and neutrophils.The MPO activity was significantly increased than the normal control group.Treatments with low and middle dose of Chrysanthemum polysaccharides and SASP significantly reduced disease activity index,alleviated the situation of colitis lesions and significantly decreased the activity of MPO.To further evaluate the association between the alleviation of TNBS-induced colitis and inflammatory factors/cytokines in colitis rats,we quantified the levels of these factors/cytokines in colon epithelial tissue samples.In the TNBS-induced colitis,the levels of many cytokines including IL-6,IL-17,IL-23,TNF-?,IFN-? and IL-1? were increased,while IL-13,IL-10 and IL-4 were decreased.However,following low and middle dose of Chrysanthemum polysaccharides treatment,the levels got back.The results suggested that different doses of polysaccharide on the efficacy of colitis is not the same,the low and middle dose which consistent with the positive group is better than the higher.In Section 2,a colitis mouse model was established and successfully used to assess the therapeutic effect of Chrysanthemum polysaccharides different dosage and different ways of administration.Mice were administered with 3%DSS,which directly caused damage to the colon mucosa and thus led to inflammatory bowel disease.From the fifth of administration,mice began to show increasingly severe symptoms,including serious diarrhea,obvious rectal bleeding and notable body weight loss.H&E staining showed that there were severe pathological changes in the colonic tissues of DSS treated mice,including mucosal lesion and necrosis as well as infiltration of inflammatory cells such as monocytes and neutrophils.Compared to the DSS-treated group,daily administration of polysaccharides dramatically attenuated the body weight loss,reduced the rectal bleeding,stool consistency and DAI.Polysaccharides also showed dose-dependent protection against the tissue injuries.Polysaccharides at low and middle dose which decreased the activity and of MPO,NO and SOD in colonic tissue is better than the higher.The above results indicated that Chrysanthemum polysaccharides had a strong protective effect on DSS induced colitis.Secondly,the mechanism of biological effects of polysaccharide from Chrysanthemum's root,stem and leaf on ulcerative colitis.In Section 1,based on intestinal microecology,the mechanism of biological effects of polysaccharide from Chrysanthemum's root,stem and leaf on ulcerative colitis were carried out.Based on the bacterial 16S V3-V4 variable region,high-throughput sequencing and bioinformatics analysis were carried out using the Illumina/Miseq second-generation sequencing technology platform to study the changes of the whole flora structure of polysaccharide from Chrysanthemum's root,stem and leaf on TNBS-induced colitis rats.The results showed that the composition of the intestinal microflora in the model group showed a series of changes,and the proportion of Firmicutes/Bacteroidetes was significantly lower than that of the normal group.The intake of polysaccharide significantly improved the trend,which increased the number of Firmicutes,maintained the ecological balance of intestinal flora.The abundance of beneficial microorganisms,Butyricicoccus,Clostridium,Lachnospiraceae,Rikenellaceae,Lactobacillus and Bifidobacterium were increased in varying degrees,which the relative abundance of pathogenic microorganisms,Escherichia,Enterococcus and prevotella were reduced.In the study of the correlation between intestinal flora and biochemical factors,it was found that the beneficial bacteria such as Butyricicoccus,Clostridium,Lachnospiraceae,Rikenellaceae,Lactobacillus and Bifidobacterium have positive correlation with the content of antiinflammatory factors IL-4,IL-10,IL-11 in intestinal epithelial cells.The Pretotella,Ruminococcus,Bacteroides,Escherichia-Shigella,akkermansia,Turicibacter and other harmful bacteria were positive associated with IL-23,TNF-?,IL-1?,IL-6,IF-17,IFN-?.At the same time,the biochemical function of intestinal microbes was predicted based on COG database and KEGG database.The function of intestinal microflora was related to amino acid metabolism,cell movement,signal transduction,energy metabolism,dextran biosynthesis and metabolism,lipid metabolism,Cell metabolism,coenzyme and vitamin metabolism,nervous system,transcription,cell growth and apoptosis.In the above functions,the normal and model groups were involved in energy metabolism,coenzyme transport and metabolism,transcription,cell wall/membrane synthesis,Signal transduction and other aspects of a more significant difference,after the intake of polysaccharides,these different physiological functions are moving towards the normal group close to the trend,especially in the low dose group is more obvious.In this study,we investigated the effects and the mechanism of biological effects of polysaccharide from Chrysanthemum' s root,stem and leaf on ulcerative colitis.The polysaccharide from Chrysanthemum' s root,stem and leaf could be a potential drugs in the field of treatment of inflammatory bowel disease which provide a new perspective and direction in-depth study of colitis.The content of SCFAs in the intestinal contents of colitis rats was determined by GC-FID.The results showed that polysaccharides increased the content of SCFAs in the intestinal tract of colitis rats in different degrees,especially acetic acid,propionic acid,butyric acid and butyric acid were the most obvious.This result was consistent with the results of the diversity of the flora.By improving the intestinal tract flora imbalance in colitis rats,to restore the metabolic function of intestinal bacteria,and enhance the ability of bacteria to produce SCFAs,and then alleviating and improving the symptoms of colitis.In Section 2,it was confirmed that epithelial barrier dysfunction,leading to intestinal bacteria and other antigen penetration into the intestinal mucosa is a major factor in the pathogenesis of ulcerative colitis.In this study,Claudin,occludin and ZO-1 were choosed as the object proteins to study their expression in rat colonic mucosa.The results showed that the expression of Claudin-1,Occludin and ZO-1 was significantly decreased after TNBS-ethanol induction compared with normal group.That was indicated that TNBS treatment inhibited the expression of the epithelial tight junction protein in intestinal mucosa.However,the expression of Claudin-1,occludin and ZO-1 in protein and mRNA level was increased,to a certain extent,after intaking chrysanthemum polysaccharide,especially in the low dose group,even better than the positive drug SASP.Combining the results suggested that chrysanthemum polysaccharide may improve diversity of intestinal microflora,restore the protective function of intestinal barrier,some of the tight junction protein expression level of intestinal mucosal epithelial cells returned to normal,and then improve the intestinal mucosal barrier permeability,to alleviate and improve colitis in rats TNBS induced symptom.A large number of studies have confirmed that the breakage of intestinal microbial ecological balance is closely related to the occurrence and development of colitis.Colitis patients are always with intestinal flora imbalance.Under the stimulation of various microbial antigens,the immune system is activated which leading to imbalance of cytokines,activation of various inflammatory cells,and then produce chronic inflammation of the intestinal tissue.In this study,the expression levels of NF-?B and TLR4 were detected from the gene and protein levels by using qPCR and westernblot techniques around the TLR4/NF-?B signaling pathway.The results of showed that the expression levels of NF-?B and TLR4 in colonic mucosa of colitis rats were significantly higher than those of normal rats which were reduced by Chrysanthemum polysaccharides treatment.Western blot results showed that the expression levels of NF-?Bp65 phosphorylated pp65 and TLR4 in colonic mucosa of colitis rats were significantly higher than those in normal rats.After intake Chrysanthemum polysaccharides,the expression of NF-KBp65 and its phosphorylated pp65 expression levels have different degree of callback,especially NF-?Bp65 protein expression.It was suggested that the polysaccharides of chrysanthemum could inhibit the activity of TLR4 and NF-?B by blocking the TLR4/NF-?B pathway,and then alleviate the intestinal inflammatory response.The IL-6/STAT3 pathway was also studied in this study.The results showed that the mRNA expression levels of IL-6,JAK2 and STAT3 in colonic mucosa of colitis rats were significantly higher than those of normal rats.The mRNA expression of IL-6 was significantly decreased after administration of polysaccharides,the JAK2 and STAT3 mRNA showed no significant difference but at low dose.However,in protein levels,the expression of STAT3,phosphorylated protein p-STAT3 and p-JAK2 in colonic tissue of colitis rats were significantly higher than those in normal rats.The protein expression level was not consistent with the previous gene expression,indicating that the IL-6/STAT3 pathway only responds to the polysaccharide after protein translation.LPS-induced TLR4/NF-?B,IL-6/STAT3 pathway was over activated and the inflammatory response was enlarged,leading to the development of colitis.Therefore,maintaining the intestinal microecological balance,inhibiting or blocking TLR4-NF-?B,IL-6/STAT3 signaling pathway,will become the new targets to control and prevent ulcerative colitis.Metabolomics was used to study the overall changes of metabolites in plasma and urine in model rats,to explore potential biomarkers in plasma and urine,and then the metabolic disturbance and the change of metabolic phenotype after intake of polysaccharides were studied.The results showed that linoleic acid,arachidonic acid,taurodeoxycholic acid,cholic acid and chenodeoxycholic acid were down-regulated in plasma of colitis rats while the leukotriene A4,glycine,dihydro,Cholesterol were upward.Metabolic pathways include bile acid biosynthesis,retinol metabolism,arachidonic acid metabolism,linoleic acid metabolism,tryptophan metabolism,unsaturated fatty acid biosynthesis,amino acid metabolism,glycerophospholipid metabolism,steroid biosynthesis,which arachidonic acid metabolism,bile acid biosynthesis,glycerol phospholipid metabolism metabolic pathway impact value is relatively high.In the urine of the colitis rats,nicotinamide,glycerophosphorylcholine,urocanic acid,nicotinic acid single nucleotide,dihydropyrimidine and phosphorylcholine were down,D-glucuronic acid-6-trihydrolactone,methyl imidazoleacetic acid,2-oxo-4-methylthiobutyric acid,showed an uptake trend.The metabolic pathways involved include nicotine and nicotinamide metabolism,histidine metabolism,lysine degradation,glycerophospholipid metabolism,lysine biosynthesis,pantothenic acid and CoA biosynthesis,beta-alanine metabolism,starch and sucrose metabolism,Cysteine and methionine metabolism,inositol phosphate metabolism,pyrimidine and purine metabolism.Analysis of metabolic pathways,ascorbic acid metabolism,nicotine and nicotinamide metabolism,glycerol phospholipid metabolism,cysteine and methionine metabolism and histidine metabolism is relatively high.Chrysanthemum polysaccharides affected the metabolic pathway in different degrees,the level of the above markers have different degrees of callback,to the normal state of change,that chrysanthemum polysaccharide can improve and alleviate the symptoms of colitis rats.Thirdly,Structural characterization of polysaccharides from root,stem and leaf of Chrysanthemum.In Section 1,the dried root,stem and leaf of C.morifolium were defatted twice with petroleum benzin,the residue was then dried at room temperature and extracted with boiling water two times.The extracts were filtered,combined,concentrated and ethanol precipitation,yielding the crude polysaccharides.The contents of neutral polysaccharides,acid polysaccharides and protein in crude polysaccharides were determined.The results showed that the content of neutral polysaccharide was 42.67±2.94%,much higher than the acid polysaccharide(18.36±1.09%),and the protein was 6.17±1.02%.In Section 2,the crude polysaccharides were decolored and removed protein by trichloroacetic acid,and then was separated by DEAE-52 cellulose column,Sephadex G-200.The samples were purified,eluted,collected,concentrated under reduced pressure,dialyzed and lyophilized.The relatively homogeneous polysaccharide were obtained,JFP1-1-2(1.6%)and JFP1-2-2(0.4%).The structures of homogeneous polysaccharides JFP1-1-2 and JFP1-2-2 were deduced by IR,acid hydrolysis,methylation,13C-NMR and 1H-NMR.The results showed that JFP1-2-2 was mainly composed of mannose,glucose and galactose at a molar ratio of 4.53:3.06:1.00.JFP1-2-2 was mainly composed of mannose,galactose,glucose,xylose and galacturonic acid Molar ratio of 3.01:2.36:1.25:1.00:1.02.After being hydrolyzed by GC/MS,the results showed that JFP1-1-2 contained 5,6-?-Manp,1,3,5-?-D-Manp,1,3,5-?-D-Manp,1,3-?-D-Manp,1,5-?-D-Galp,2,4-?-D-Galp,1,3,5-?-D-Glcp?2,3,4-?-D-Glcp,the molar ratio of which is 13.41:3.39:30.30:1.87:18.01:6.22:15.10:7.01:4.69.JFP1-2-2 contained T-?-D-Manp,5,6-?-D-Manp,1,4-?-D-Manp,1,3,5-?-D-Manp,2,3,5-?-D-Manp,1,5-?-D-Galp,2,4-?-D-Galp,1,3,5-?-D-Glcp,T-?-D-Xyl p the molar ratio of which is2.31:0.44:1.12:2.6:2.86:5.87:3.33:4.56:4.80.The above results can be seen,whether it is neutral polysaccharide JFP 1-1-2,or with the acidic group of JFP 1-2-2,the sugar structure of mannose content of the largest proportion.Studies have shown that mannose receptor MR is a pattern recognition receptor present on the surface of macrophages and dendritic cell membranes and can specifically recognize mannose,N-acetylglucosamine or fucose Ligands and bind them.MR by identifying,combined with these carbohydrates to mediate phagocytic pathogenic microorganisms,and then play an immune regulation.Guo Zhenjun showed that 49.07%mannose in the rhubarb polysaccharide RTP-2 which had the potential to bind to MR and had therapeutic effect on experimental TNBS replicated colitis rats.In this study,the structure of polysaccharides from root,stem and leaf of C.morifolium,contained a large amount of mannose,which is likely to be a mannose receptor that specifically binds to the surface of macrophages and dendritic cells,phagocytosis of pathogenic microorganisms,Ecological balance,induced immune regulation,and thus improve colitis.This hypothesis will continue to be explored and confirmed in subsequent experimental studies. |
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