Study On Structural Characterization,In Vitro Digestion And Fermentation Characteristics Of Schisandrae Chinensis Fructus Polysaccharides And Its Mechanism Of Improving Alzheimer’s Disease

Schisandra chinensis is a functional resource widely cultivated in northeastern China for both medicinal and food purposes,and has been shown to exhibit good neuroprotective activity.As one of the main active components in Schisandra chinensis,polysaccharides have been shown to have various bioactive effects such as antioxidant,immunomodulatory and neuroprotective effects.However,the structural characteristics of Schisandra chinensis polysaccharides and their mechanisms in neuroprotection are not well studied.Based on this,this paper investigates the fraction purification,antioxidant activity,structural characterization and in vitro digestion and fermentation characteristics of Schisandra chinensis polysaccharides;based on the theory of"microbe-gut-brain"axis combined with metabolomics and intestinal microbiomics,the protective effect and potential mechanism of its effect on Aβ_25-35-induced Alzheimer’s disease（AD）rats were systematically investigated.The main research contents and results are as follows:（1）Extraction separation and purification study of Schisandra chinensis polysaccharidesThe crude polysaccharide（SCP）of Schisandra chinensis was prepared by traditional hot water extraction and alcoholic precipitation after defatting with petroleum ether.The analyzed results showed that the total sugar content of SCP was76.69%,the protein content was 12.57%and the glyoxylate content was 10.79%;SCP was separated by DEAE-52 column chromatography and four polysaccharide fractions（SCP1,SCP2,SCP3 and SCP4）were obtained;further purification by Sephadex G-100column chromatography showed that all four purified polysaccharides were single symmetric peaks;Ultraviolet spectrum（UV）scanning results showed that the purified polysaccharides had no impurities such as nucleic acid and protein.The results of high performance gel chromatography（HPGPC）showed a single symmetric peak for all the fractionated polysaccharides and the dispersion was close to 1,indicating that the homogeneity of the fractionated polysaccharides was good and could be used for subsequent structural analysis and activity studies.（2）In vitro activity study of the purified polysaccharides of Schisandra chinensisThe in vitro antioxidant activity and neuroprotective activity of each purified polysaccharide was investigated by in vitro antioxidant assay and PC12 cells.The results showed that the purified polysaccharides had certain scavenging ability against superoxide anion(O^2-·)radicals,DPPH radicals and hydroxyl radicals（·OH）.Among them,SCP2 and SCP4 had better antioxidant activity,SCP1 had poorer O^2-·scavenging ability,while SCP3 had weaker scavenging ability of DPPH radicals;the in vitro AD model was established by Aβ_25-35 induced PC12 cells,and the results of MTT experiments showed that all purified polysaccharides significantly increased the survival rate of Aβ_25-35-induced PC12 cells,and the medium and high concentrations of all levels of fractionated purified polysaccharide（50,100mg/m L）significantly reduced the content of NO and MDA compared with the model group（P<0.01）.The above results indicated that all purified polysaccharides had certain in vitro antioxidant activities and had a beneficial effect on Aβ_25-35-induced PC12 cells.（3）Structural characterization of SCP2 and SCP4The structural analysis of SCP2 and SCP4 fractions with good in vitro antioxidant activity was performed by chemical and instrumental analysis.The results of infrared spectroscopy showed that SCP2 and SCP4 exhibited typical polysaccharide characteristic absorption peaks;the results of monosaccharide composition showed that SCP2 was a homogeneous polysaccharide consisted of glucose only;SCP4 was consisted of xylose,mannose,arabinose,galactose,galacturonic acid,and glucose.The molar ratio was 0.028:0.051:0.087:0.136:0.163:0.535,which is an acidic polysaccharide;the methylation and NMR analysis showed that the glycosyl type of SCP2 was mainly Glcp-（1→,→4）-Glcp-（1→and→3,4）-Glcp-（1→,while SCP4mainly contained→4）-α-D-Gal A-6-OMe-（1→,→4）-α-D-Glcp-（1→and→4）-α-D-Gal A-(1→,indicating that SCP4 is a pectin-like polysaccharide.（4）In vitro simulated digestion and fermentation of all purified polysaccharidesThe in vitro digestion characteristics of each fraction of purified polysaccharides were investigated using in vitro simulated saliva,gastric and intestinal fluid models.The results showed that all the purified polysaccharides had certain stability in simulated saliva,gastric fluid and intestinal fluid.Among them,the molecular weight and reducing sugar content of SCP1 and SCP2 changed more significantly than that of SCP3 and SCP4 in the simulated digestion process,and the stability was slightly poor.The change of peak shape during digestion of SCP2 may be caused by the hydrolysis ofα-（1→4）glycosidic bond by amylase;The in vitro fermentation experiments demonstrated that the total carbon content of each fractional polysaccharide gradually declined during fermentation process,while the total sugar content of crude polysaccharide SCP did not change significantly;meanwhile,each fractional polysaccharide and SCP could significantly reduce the p H and increase the content of SCFAs during fermentation;the results of intestinal microbial analysis showed that each fractional polysaccharide could regulate the structure of intestinal microflora,promote the production of beneficial bacteria and reduce the relative abundance of pathogenic bacteria to play the role of Prebiotic effect.Among them,SCP2 could significantly increase the species diversity and community diversity of intestinal microorganisms.（5）Pharmacodynamic evaluation of purified polysaccharides on Aβ_25-35 induced AD rats and fecal metabolomics and microbiome analysis of SCP2The AD rat model was established by Aβ_25-35 induction,and the pharmacodynamics was evaluated by the intervention of purified polysaccharide in AD rats.The results of pharmacodynamic experiments showed that all of the purified polysaccharides could improve the learning memory ability,reduce neuroinflammation and restore the integrity of intestinal barrier in AD rats to different degrees.Among them,the SCP2 group had the best efficacy;fecal metabolomics results showed that SCP2 could significantly regulate 19 endogenous metabolites in the feces of AD rats,mainly involving four metabolic pathways:linoleic acid metabolism,arachidonic acid metabolism,α-linolenic acid and TCA cycle;16S r RNA sequencing results showed that Aβ_25-35-induced intestinal microflora dysbiosis in AD rats,and the microbial diversity and SCFAs levels were lower than those of Sham group,while SCP2 intervention can reverse the intestinal microflora disorder and improve the level of SCFAs.The results of the"microbiota-SCFAs-biomarkers"correlation analysis suggested that SCP2 may play a neuroprotective role by improving gut microbial disorders,promoting SCFAs and regulating inflammatory metabolic pathways in AD rats.（6）Serum and urine metabolomics as well as related mechanisms of SCP2 in the treatment of AD ratsA holistic metabolomic study of serum and urine samples from AD rats was performed by UPLC-Q-TOF/MS technique.The results showed that the therapeutic effect of SCP2 on Aβ_25-35 induced AD rats was mainly through the regulation of the levels of 21 endogenous metabolites in serum and 19 endogenous metabolisms in urine.These metabolites were mainly involved inα-linolenic acid metabolism,linoleic acid metabolism and arachidonic acid metabolic pathway.It is hypothesized that SCP2 was involved in regulating unsaturated fatty acids by promoting the levels ofα-linolenic acid and Docosahexaenoic acid（DHA）,which in turn exert neuroprotective effects by improving neuroinflammation.Western blotting results showed that the protein expressions of TLR4,IKB-α,NF-κB p65,NLRP3,ASC,Pro-caspase1,Caspease-1,and TNF-αin rat hippocampal tissues were significantly reduced under the intervention of SCP2,indicating that SCP2 could improve neuroinflammation and exert neuroprotective activity by inhibiting the TLR4/NF-κB/NLRP3 pathway.In summary,this paper systematically investigated the isolation and purification,structural characterization,antioxidant activity and in vitro digestion and fermentation characteristics of Schisandra chinensis polysaccharides.Based on the multi-omics analysis of metabolomics and intestinal microbiomics,combined with traditional pharmacological evaluation methods,a comprehensive analysis network of"microbiota-SCFAs-biomarkers"was established to systematically elucidate the potential mechanism of neuroprotection of Schisandra chinensis polysaccharides and reveal the regulatory role of Schisandra chinensis polysaccharides in the"brain-gut"axis,which provides data reference for promoting the deep processing of Schisandra chinensis polysaccharides and promoting its in-depth development and application.