| Scutellaria baicalensis Georgi is a dried root of Scutellaria baicalensis Georgi.It processes the role of clearing away heat and promoting Diuresis,clearing away toxic material.The main active components of Scutellaria baicalensis Georgi are flavonoids and polysaccharides.It was reported that plant polysaccharides have the activities of immune regulation,anti-inflammatory,regulation of intestinal flora,regulation of blood sugar level and blood fat,antitumor,and so on.Besides,Scutellaria baicalensis Georgi is associated with a variety of polysaccharides activities in the treatment of chronic inflammation and ulcerative diseases.Therefore,the study of polysaccharides is very important to explain the active function of Scutellaria baicalensis Georgi.According to the literatures at home and abroad,the exploration of polysaccharide from Scutellaria baicalensis Georgi lags far behind that of flavone.The reports of polysaccharide from Scutellaria baicalensis Georgi are limited to studies on the extraction process and the antioxidant activity in vivo and in vitro.There are no studies on the separation,purification,structure analysis,anti-inflammatory and regulation of intestinal flora diversity and function.Therefore,this study optimized the extraction process,isolated and purified the polysaccharide,studied its physical and chemical properties and preliminarily characterized its structure,explored its possible mechanism of anti-ulcerative colitis,and laid a foundation for the in-depth study of polysaccharide from Scutellaria baicalensis Georgi.This paper was divided into five chapters.Part 1 IntroductionThis part inquired and summarized the literatures related to polysaccharides,systematically summarized the methods of purification,structure analysis and pharmacological activities of polysaccharides,in order to provide reference for the studies on the structure and activity of polysaccharide from Scutellaria baicalensis Georgi.At the same time,the relationship between intestinal flora and drug absorption and metabolism was summarized to guide the future research direction of this subject.Part 2 Extraction and purification,physical and chemical properties and preliminary structure characterization of polysaccharide from Scutellaria baicalensis Georgi1 Extraction and purification of polysaccharide from Scutellaria baicalensis Georgi1.1 The optimal extraction process of polysaccharide from Scutellaria baicalensis Georgi was 4.3 h,the extraction temperature was 93℃ and the ratio of liquid to material was 24:1.1.2 After extraction,the crude polysaccharides were deproteinized by Sevag method,decolorized by 3%H2O2 solution,dialysed,alcohol precipitation,and freeze-dried,through DEAE-52 cellulose ion exchange column chromatography,and Sephadex G-100 gel column chromatography,and obtained five polysaccharides:SP1-1,SP2-1,SP2-2,SP3-1,and SP3-2.2 The physical and chemical properties and preliminary structure characterization of polysaccharide from Scutellaria baicalensis Georgi2.1 The physical and chemical properties of five polysaccharides were studied.The total amount of polysaccharides was high and the content was above 80%.The protein content was less than 0.5%.The content of uronic acid:SP3-1,SP3-2>SP2-1,SP2-2>SP1-1.The content of sulfuric acid in SP2-1 and SP2-2 is low.2.2 The high performance gel permeation chromatography(HPGPC)showed that the five polysaccharides from Scutellaria baicalensis Georgi were all single peaks.The Mw was calculated by GPC software,and their Mw was 455980 g/mol,3717767 g/mol,1641491 g/mol,4464034 g/mol and 2181324 g/mol,respectively.2.3 The polysaccharide from Scutellaria baicalensis Georgi was reacted with PMP to produce the PMP derivatives that were analyzed by HPLC.The result showed that SP1-1 was mainly composed of Man,Rib,GluA,Glc,Xyl and Ara with molar ratios of 2.14:3.61:1:2.86:5.98:36.39,SP2-1 was mainly composed of Man,Rib,Rha,GlcUA,Glc,Xyl,Ara and Fuc with molar ratios of 5.06:21.24:1.00:20.25:3.49:50.90:228.77:2.40.SP2-2 was mainly composed of Man,Rib,Rha,GlcUA,GalN,Glc,Xyl,Ara and Fuc with molar ratios of 6.83:7.73:1.00:77.61:2.49:2.20:3.56:19.10:4.79.SP3-1 was mainly composed of Man,Rib,Rha,GlcUA,Glc,Xyl,Ara and Fuc with molar ratios of 4.57:26.90:1.00:30.27:1.42:24.29:197.16:1.56.SP3-2 was mainly composed of Man,Rib,GlcUA,GalN,Glc,Xyl,Ara and Fuc with molar ratios of 6.58:9.02:1.00:78.72:1.51:2.40:5.14:4.23.2.4 The morphology of polysaccharide observed by scanning electron microscopy indicated that SP1-1 presented a honeycomb structure,SP2-1 and SP3-1 presented irregular shapes,SP2-2 and SP3-2 presented a lamellar structure.2.5 Infrared spectroscopy analysis showed that the five polysaccharides had characteristic absorption peaks of polysaccharide.SP1-1,SP2-2,SP3-1 and SP3-2 were acetylated polysaccharides,and the main chain of SP2-2 was β-glycosidic bond.Ultraviolet spectrum analysis showed that the five polysaccharides had no absorption peaks at about 260nm and 280nm,indicating that they did not contain nucleic acids and proteins.Part 3 SP1-1 ameliorated colitis via suppressing NF-κB signaling and NLRP3 inflammasome activation1 The model of acute ulcerative colitis of C57BL/6 mice was successfully established by drinking 3%DSS solution for one week.The effect and mechanism of SP1-1 was studied.The results indicated that SP1-1 decreased DAI,histological score,and MPO activity in UC mice,as well as the expression levels of inflammatory factors such as IL-1β,IL-18,and TNF-α in serum and colon tissues.SP1-1 alleviated macrophage infiltration and reduced cleaved caspase-1 expression in peritoneal macrophages induced by DSS in UC mice.2 SP1-1 inhibited NF-κB signaling activation:SP1-1 dose-dependently reduced the amount of NF-κB p65 in the nucleus.In the THP-1-derived macrophages,LPS increased the expression levels of p-IκBα,p-IKKα and p-IKKβ,and decreased the expression levels of IKBα,IKKα and IKKβ.However,SP1-1 could significantly reverse the effect of LPS.After LPS stimulation,the expression level of p-NF-kB p65 in the nucleus was increased and that in the cytoplasm was decreased.However,SP1-1 inhibited the nuclear localization of p-NF-kB p653 SP1-1 inhibited NLRP3 inflammidosome activation.Compared with the control group,the expression levels of NLRP3 and ASC proteins in the model group were significantly increased.However,SP1-1 reduced the expression levels of NLRP3 and ASC proteins.In addition,SP1-1 significantly reduced the expression levels of cleaved caspase-1,cleaved IL-1β and cleaved IL-18.Immunoprecipitation analysis showed that SP1-1 blocked the binding of ASC to NLRP3 or pro-caspase-1.Part 4 SP1-1 regulated intestinal flora diversity and SCFAs content1 SP1-1 regulated the intestinal flora diversity of UC miceBased on bacterial 16S rDNA sequence,Illumina’s Miseq PE300 platform was used for sequencing to study the effect of SP1-1 on the intestinal flora diversity of UC mice.SP1-1 increased the intestinal flora diversity and richness of ulcerative colitis mice.Venn plot analysis of species showed that compared with the model group,the intestinal flora structure of SP1-1 high-dose group and SP1-1 low-dose group was closer to that of control group.SP1-1 recovered F/B value to make it closer to normal,indicating that SP1-1 has the function of regulating intestinal community structure.According to the β diversity analysis,the composition of intestinal flora in model group was significantly different from that in control group.The separation of intestinal flora in SP1-1 high-dose group and SP1-1 low-dose group was not obvious,and the intestinal floras in the two groups deviated from the model group and were more inclined to the blank group.SP1-1 increased the abundance of beneficial bacteria,such as Lactobacillus,Ruminococcaceae,Lachnospiraceae,Bifidobacterium,Roseburia and Rikenellaceae,decreased the abundance of harmful bacteria,such as Enterococcus,Streptococcus,Turicibacter and Parasutterella,which was beneficial to relieve the symptoms of UC mice induced by DSS and restore the intestinal health.2 SP1-1 regulated the SCFAs content of UC miceThe content of SCFAs in mice was measured by GC-MS method.The content of SCFAs was increased to a certain extent by SP1-1 high dose and SP1-1 low dose.The change of content was more obvious in acetic acid,propionic acid and butyric acid.Part 5 SP1-1 regulated intestinal flora function of UC miceThe metagenomic technique was used to explore the effect of SP1-1 on intestinal flora function in UC mice.PCA analysis and NMDS analysis in EggNOG,KEGG and CAZy databases of samples from control group,model group,SP1-1 low-dose group and SP1-1 high-dose group indicated that the functional genes in the model group were far from that in the control group.The SP1-1 high dose group and SP1-1 low dose group had no obvious boundary,but both groups were more inclined to the control group than the model group.The main gene types in the model group,which were mainly different from other groups,were cellular processes and signaling.The main gene types in SP1-1 intervention groups,which were mainly different from other groups,were metabolism,information storage and processing,cellular processes and signaling.Compared with the KEGG database,the gene types mainly enriched in the model group were those related to metabolism,genetic information processing,cellular processes and organismal system.The gene types mainly enriched in the SP1-1 intervention groups were metabolism,environmental information processing,cellular processes and human diseases.Compared with the CAZy database,the core enzymes in the model group were glycoside hydrolases and glycosyl transferases.The core enzymes in the SP1-1 intervention groups were glycoside hydrolases,glycosyl transferases and carbohydrate-binding modules.The stimulation of DSS disturbed the function of intestinal flora,but the intervention of SP1-1 could alleviate the disorder of intestinal flora function and make it more normal. |
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