| Polysaccharide is a long-chain polymer formed by connecting more than 10monosaccharide molecules by glycosidic bonds.It is a functionally active macromolecular substance with a complex structure.Natural polysaccharides have shown potentials for medical applications with the advantages of biological activity and little side effect.Ulcerative colitis(UC)is a chronic idiopathic inflammatory bowel disease(IBD)with an increasing incidence in Asia.As a relapse-remitting disease,UC seriously affects the life quality of patients.Previous studies reported that polysaccharides have the efficacy of preventing and improving UC.In natural plants,polysaccharides have a wide range of sources and varieties.Therefore,finding polysaccharides with anti-inflammatory activity from natural plants is of great significance.China has abundant bamboo shoot resources,with Phyllostachys edulis(P.edulis)being the most widely available bamboo shoot.P.edulis are rich in soluble dietary fiber(mainly polysaccharide).Whether bamboo shoot polysaccharide has the effect of alleviating UC still need further study.According to our knowledge,little studies have been published about the isolation,purification,structural analysis,and anti-inflammatory activity of polysaccharide from P.edulis.Therefore,this study chose polysaccharide from P.edulis as the research object.P.edulis polysaccharide was isolated,purified,and its structure was characterized by multiple methods.Then,an in vitro digestion and fermentation simulation system was established to explore the changes in the purified polysaccharide fraction(BSDF-1)of P.edulis,imitating the gastrointestinal tract digestion process and the fecal microbiota of healthy human individuals.In addition,in vitro fermentation and in vivo combination study were carried out to investigate the possible mechanism of BSDF-1 in improving colitis by dextran sulfate sodium(DSS)induced colitis mouse models.Finally,cellular inflammation model(RAW 264.7 macrophages)was used to study the anti-inflammatory effect of BSDF-1.The main results were as follows:(1)Polysaccharides were isolated from P.edulis,and their structures were characterized.The BSDF-1 polysaccharide fraction was obtained from P.edulis by two-step water extraction and alcohol precipitation,DEAE anion exchange column chromatography,and Sephadex G-100 Sephadex column chromatography.The total sugar content of BSDF-1 polysaccharide was 90.23%,and its molecular weight was80.8 kDa,and it was mainly composed of Glc,Gal,and Ara,with molar percentages of63.14%,26.77%,and 10.09%,respectively.Scanning electron microscopy results showed that BSDF-1 had a lamellar and filamentous structure with relatively smooth surface and thus good solubility.Methylation and NMR analysis results indicated that the main chain of BSDF-1 was composed of→4)-β-Galp-(1→4)-β-Glcp-(1→3)-β-Glcp-(1→glycosidic linkage,while the end groupα-Araf-(1→5)-α-Araf-(1→fragment is attached to the main chain.(2)In vitro digestion and fermentation characteristics of BSDF-1 were studied.During gastric digestion,the molecular weight of BSDF-1 decreased from 80.8 kDa to58.4 kDa,and the reducing sugar(C_R)content increased significantly(p<0.05).During the whole digestion process,the digestibility of BSDF-1 showed an increasing trend,and the total sugar showed a decreasing trend.In addition,the monosaccharide composition of BSDF-1 was not affected by digestive liquid,but the molar ratio of monosaccharide was changed.Moreover,the characteristics of in vitro fermentation of BSDF-1 were studied by using human fecal microbiota.After 48 h of fermentation,BSDF-1 degradation rate reached 40.29%and the p H of the fermentation broth decreased from 6.89 to 4.57.The production of acetic acid,propionic acid,valeric acid and butyric acid were significantly increased(p<0.05).16S r RNA sequencing results indicated the abundance of Firmicutes and Actinobacteria were significantly increased in BSDF-1 fermentation group(p<0.05).While the abundance of Bacteroides and Fusobacteria were significantly decreased.GC-MS results showed that after 48 h of fermentation by human microbiota,the concentration of carbonyl compounds in the metabolites of BSDF-1 was significantly decreased,while the concentration of fatty acid were significantly increased(p<0.05).These results suggested that P.edulis polysaccharide has beneficial effects on intestinal health.(3)Exploration of the anti-inflammatory effect and the mechanism of BSDF-1 in sodium dextran sulfate(DSS)induce mice.The experiment was divided into 6 groups:normal group,model group,mesalamine group,low-dose BSDF-1 group,medium-dose BSDF-1 group,and high-dose BSDF-1 group.Oral gavage was carried out continuously for 7 days.Results showed that BSDF-1 significantly improved the weight loss,colonic shortening,colonic tissue destruction,colonic mucosal damage and inflammatory cell infiltration in mice with colitis.High-dose BSDF-1(400 mg/kg)treatment significantly decreased the m RNA expression levels of pro-inflammatory factors of IL-1β,IL-6,IL-18 and TNF-α,as well as the concentrations of MDA,MPO and NO in colonic tissues.The concentrations of IL-10,T-SOD and lipocalin-2concentrations were also significantly(p<0.05)increased.Activation of colonic inflammatory signaling pathways were significantly inhibited and the m RNA expression of linker proteins were upregulated.There was significant reduction in the abundance levels of intestinal microbiota Parabacteroides,Mucispirillum,Helicobacter,Bacteroides,and Streptococcus,and significant increases in those of Prevotella,Alistipes,Anaerostipes,Odoribacter Bifidobacterium,Butyricimonas and Lactobacillus.Then,the effect of BSDF-1 on colitis during in vitro fermentation was analyzed by 16S r RNA and untargeted metabolomics.After adding BSDF-1 to the feces of normal mice for 48 h fermentation,the p H value of the fermentation solution was decreased from 6.91 to 5.49,the degradation rate of BSDF-1 was 39.79%,and the total SCFA content increased significantly from 8.61±0.38 mmol/L to 26.74±0.90 mmol/L.After adding BSDF-1 to the feces of colitis model mice for 48 h,the p H value of the fermentation solution decreased from 7.08 to 6.10,the degradation rate of BSDF-1 was26.59%,and the total SCFA content increased significantly from 5.51±0.79 mmol/L to 16.46±0.71 mmol/L.BSDF-1 could alter the intestinal microbiota composition by increasing the relative abundance of Alistipes,Lactobacillus spp.and decreasing the relative abundance of Escherichia-Shigella,Enterococcus and Proteus spp.compared with the Mod group.UHPLC-MS/MS results showed that after 48 h of fermentation,BSDF-1 altered the colonic fecal content of 17 metabolites,including ornithine,creatinine,leucylproline,N-acetylornithine,N-acetyl-L-histidine and creatine,compared with the Mod group.These results indicated that P.edulis polysaccharide BSDF-1 had the effect of alleviating colitis symptoms.(4)The anti-inflammation effect of BSDF-1 was studied by in vitro LPS-induced RAW 264.7 macrophages system.Results confirm that BSDF-1 significantly decreased pro-inflammatory cytokine levels and increased anti-inflammatory cytokine levels,also significantly altered the proportion of macrophages in LPS-stimulated RAW 264.7 cells.RT-q PCR assays showed that 20μg/mL of BSDF-1 significantly increased the m RNA expression levels of IL-10,Arg1 and CD206,and decreased the m RNA expression levels of IL-6,TNF-α,IL-1β,IL-18,IL-23,IL-10,CD86 and i NOS.The western blot analysis results indicated that 20μg/mL of BSDF-1 significantly increased Arg1,AMPK,p-PI3K and p-Akt protein levels,and significantly decreased i NOS and p-p65protein levels.In addition,AMPKαsi RNA827 was found to be effective in silencing AMPK,while 20μg/mL of BSDF-1 increased the AMPK activation,which in turn affected the PI3K/Akt and NF-κB pathways.These results indicated that the P.edulis polysaccharide BSDF-1 had a particular anti-inflammatory effect.In summary,this paper determines the main chain BSDF-1 is composed of the main chain by→4)-β-Galp-(1→4)-β-Glcp-(1→3)-β-Glcp-(1→glycosidic bond of connection and contains a small number of branched polysaccharides.In vitro digestion and fermentation results confirmed that BSDF-1 can significantly increase the concentration of fatty acid,which is beneficial to intestinal health.The effects of BSDF-1 on improving cell inflammation and colitis were systematically studied in this study.Animal experiments showed that a high dose(400 mg/kg)BSDF-1 treatment significantly regulated intestinal microbiota,improved intestinal barrier function,and reduced oxidative stress response.In vitro cell study results showed that BSDF-1 had effective anti-inflammatory activity,could significantly increase the m RNA expression levels of IL-10,Arg1,and CD206,and the protein levels of Arg1,AMPK,P-PI3K,and p-Akt.The m RNA expression levels of IL-6,TNF-α,IL-1β,IL-18,IL-23,IL-10,CD86,and i NOS,and the protein levels of i NOS and p-p65 were significantly decreased.The results showed that BSDF-1 has potential as an anti-inflammatory food fortifier and food additive. |
PDF Full Text Request