Study On The Preparation Of Astragalus Polysaccharide APS-IA And Its Anti-aging Activity By Regulating Gut Microbiota

Objective:The aim of this study was to extract,isolate and purify astragalus polysaccharides with anti-aging activity from Astragalus membranaceus and analyze its structure,to explore its effect on various aging phenotypes of natural aging mice,and to clarify its anti-aging mechanism by regulating intestinal flora.It provides more scientific basis for the research and development of anti-aging drugs.Methods:1.By comparing four extraction routes of Astragalus polysaccharides(APS)from Astragalus membranaceus,the optimal extraction process was selected based on the extraction rate of polysaccharides,polysaccharide content and the results of experiments on the lifespan of Caenorhabditis elegans;Using Design Expert software for response surface methodology experimental design,optimizing process parameters for the optimal route to select the most stable and feasible process extraction parameters.2.The crude APS was extracted from Astragalus membranaceus by green and pollution-free hot water extraction and three-step purification(macroporous resin/ion exchange chromatography/gel chromatography)and separated and purified to obtain the purified components of APS.The purified components of APS were scanned by UV full wavelength scanner,and the contents of protein and nucleic acid were determined;The molecular weight of APS purified components was determined by HPLC;The monosaccharide composition of APS purified components was determined by ion chromatography;The primary structure of purified APS was characterized by methylation analysis,infrared spectroscopy,one-dimensional and two-dimensional nuclear magnetic resonance;Using scanning electron microscope to observe the appearance and morphological characteristics of APS purified components;Detecting the anti-aging activity of APS purified components through the lifespan experiment of Caenorhabditis elegans,for subsequent in vivo and in vitro activity experiments.3.The anti-aging activity of purified APS in vivo was studied by using a 22 month old natural aging mouse model:(1)Using morris water maze test to detect the learning and memory ability of mice;(2)Using he staining to observe the morphological and pathological changes of liver tissue,hippocampus and spleen tissue in mice;(3)The levels of SOD,cat,GSH Px,T-AOC and MDA in serum,brain and liver tissues of mice were measured by oxidative stress kit;(4)The levels of IL-2,IL-6,ages and SIRT1 in serum of mice were determined by ELISA kit;(5)He staining was used to observe the ileum structure of mice;(6)We use ab-pas staining to observe the number of goblet cells in ileum and colon of mice;(7)Immunohistochemical staining and Western blotting were used to observe expression levels of the intestinal epithelial tight junction proteins ZO-1,Occluding,cloud-1,mucin MUC2,inflammatory factor TNF-α,myosin light chain kinase MLCK and phosphorylated myosin light chain P-MLC in mouse colon tissue.4.The influence of the changes of intestinal flora abundance in mice was observed and analyzed by using metagenomics.By using the redundancy analysis(RDA)method,the correlation between the main changes of intestinal flora in each group of mice and the related aging biological indicators in liver,brain,serum and intestinal tissues of mice was analyzed;Serum metabonomics was used to detect and analyze the metabolites in the blood of naturally aging mice;The content of short chain fatty acids(SCFAs)in the intestines of mice in each group was detected,and the correlation analysis was carried out with the main changes of bacteria in the intestines of mice by RDA method.5.Using the model of Caenorhabditis elegans fedding with mouse fecal extract supernatant culture medium,observing the integrity of the intestinal barrier,accumulation of lipofuscin,mitochondrial content,and muscle fiber quality of the nematode;Caco-2model of intestinal epithelial cells was used,and APS purified component was added to the supernatant of mouse feces for culture.QRT PCR and Western blotting were used to observe the gene and protein expression changes of related factors in mouse intestinal tissue.Results: 1.Through the comparison of different extraction methods of Astragalus polysaccharides,the extraction rate of polysaccharides,polysaccharide content and the life test results of Caenorhabditis elegans were as conditions to screen preparation process route.Finally,the water decoction method and macroporous resin impurity removal route were determined as the best extraction methods of Astragalus polysaccharides.Response surface methodology(RSM)was successfully used to optimize the optimal extraction process parameters of Astragalus polysaccharides.The optimal water extraction process conditions were determined as follows: extraction temperature 75 ℃,extraction time 3h,solid-liquid ratio 1:22,and extraction times 2.2.The crude polysaccharide extract of Astragalus membranaceus was successfully obtained by macroporous resin column method.After freeze-drying,it was yellow brown rough powder,and the yield was 5.23%;The polysaccharide was separated and purified by DEAE-52 cellulose column and gel chromatography column,and two homogeneous polysaccharide components were obtained,namely APS-IA(weight average molecular weight 4915da)and ASP-IB(weight average molecular weight 2749da);The monosaccharide of APS-IA was only composed of glucose,while the monosaccharide composition of APS-IB was 1.21% arabinose,0.7% galactose,91.37% glucose,and 6.72%fructose;Combining with monosaccharide composition,methylation and NMR,the primary structures of APS-IA and APS-IB were obtained;Scanning electron microscopy(SEM)results showed that APS-IA was irregular columnar,spherical at both ends,flat and slender in the middle,while APS-ib was spherical and flaky in different sizes,and gathered together;The longevity test of Caenorhabditis elegans showed that the anti-aging activity of APS-IA was significantly better than that of APS-ib,so APS-IA was selected as the follow-up efficacy and mechanism study.3.The results of anti-aging activity of APS-IA in natural aging mice:(1)Morris water maze test showed that the learning and cognitive ability of the aged model group mice was decreased compared with the young control group.After APS-IA treatment,the learning and memory ability of the aged model group mice was improved;(2)The results of HE staining showed that APS-IA significantly improved the morphology of liver tissue,hippocampus tissue and spleen tissue in the aged model group;(3)Compared with the young control group,the activities of SOD,cat,GSH PX and T-AOC in the liver and brain of the aged model group showed a downward trend,while the activities of three kinds of free radical dismutase and total antioxidant factors were increased in the APS-IA high,medium and low dose groups,and the high dose group had the best effect,in which the regulating effect of T-AOC activity in the liver was higher than that in the brain.Compared with the young control group,the old model group had a large amount of MDA deposition in the liver and brain.After APS-IA treatment,the content of MDA deposition decreased significantly;(4)The expression of IL-2 and SIRT1 in serum of aged model group decreased,but increased after APS-IA treatment.The levels of IL-6 and ages in serum of aged mice increased and decreased after APS-IA treatment;(5)He staining showed that the ileal villi in the old model group were shorter and the crypts became shallower compared with the young control group.After APS-IA treatment,the villi length increased and the crypts deepened;(6)Ab-pas staining showed that compared with the young control group,the number of goblet cells in the ileum and colon of the aged model group decreased,and increased after APS-IA treatment;(7)Immunohistochemical staining and Western blotting showed that compared with the young control group,the expression levels of tight junction proteins ZO-1,Occluding,cloud-1 and MUC2 in the colon tissue of the aged model group were decreased,and APS-IA could reverse this situation;At the same time,APS-IA also reduced the protein expression of the inflammatory factor TNF-α,myosin light chain kinase MLCK and phosphorylated myosin light chain P-MLC.in the intestinal tissue of aged model mice4.Effects of APS-IA on intestinal homeostasis and serum metabolites in naturally aging mice:(1)the results of alpha diversity showed that the ace and Chao indexes of the aged model group were lower than those of the young control group and APS-IA group(p<0.05),indicating that compared with the young control group and APS-IA group,the bacterial abundance of the aged group decreased slightly.The Shannon and Simpson indexes of the elderly model group were not significantly different from those of the young control group and APS-IA group,and the diversity of surface flora had no significant change;(2)At the level of species composition,the relative abundance of Firmicutes in the old model group was higher than that in the young control group,while the relative abundance of bacteroidea was lower,and the F/B ratio was 10.1.Compared with the elderly model group,the relative abundance of Firmicutes in APS-IA group decreased,while the relative abundance of Bacteroides increased,and the F/B ratio was 5.2;(3)At the genus level,APS-IA increased the abundance of beneficial bacteria such as ocillibacter,acetofactor,Bacteroides,Roseburia and Eubacterium in the intestines of elderly mice.Compared with the young control group,the relative abundance of Clostridium in the elderly model group increased.With the increase of age,the disorder of intestinal microbiota and immune dysfunction lead to the gradual increase of the incidence and mortality related to Clostridium infection(CDI).Compared with the elderly model group,the relative abundance of Clostridium in APS-IA group was lower;(4)APS-IA increased the content of SCFAs in intestinal tract of naturally aging mice;(5)Redundancy analysis(RDA)showed that the abundance of beneficial bacteria in the intestines of APS-IA mice was positively correlated with antioxidant factors SOD,cat,GSH PX and T-AOC,serum IL-2,SIRT1 factor,short chain fatty acids,tight junction protein ZO-1,locking,cloud-1,etc;(6)Serum metabonomics results showed that after APS-IA treatment,beneficial metabolites such as amino acid derivatives such as arginine,ornithine,leucine,and tryptophan such as indole products in the blood of elderly mice increased.5.The results of the model experiment of Caenorhabditis elegans showed that the supernatant culture medium extracted from the feces of APS-IA group mice repaired the integrity of the intestinal barrier of elderly nematodes,reduced the accumulation of lipofuscin,and enhanced the quality of intestinal mitochondria and muscle fibers;The results of intestinal epithelial cell Caco-2 model showed that the supernatant culture medium extracted from the feces of mice in APS-IA group reduced the expression of TNF-α,MLCK and P-MLC,Whlie enhanced the expression levels of tight junction proteins ZO-1 and Occluding.Conclusion:1.The optimal extraction process of Astragalus polysaccharides was screened,and the optimal process parameters were obtained by response surface methodology;APS-IA,a low molecular weight astragalus polysaccharide component,was successfully obtained,and its physicochemical properties and structure were analyzed.2.APS-IA has obvious anti-aging activity on the natural aging mouse model: APS-IA can delay the aging degree of brain,improve the morphology of hippocampal tissue,and improve the learning and memory ability of aged mice;APS-IA can alleviate oxidative damage and inflammatory reaction in aged mice;APS-IA can improve the ileal structure of elderly mice,reduce the expression of inflammatory factors in intestinal tissue,and increase the expression of tight junction protein through MLCK/P-MLC pathway,so as to enhance the intestinal barrier function.3.APS-IA can improve the structure of intestinal flora and the content of metabolites in elderly mice.APS-IA can improve the proportion of bacteroidea and Firmicutes,increase the content of short chain fatty acids,and enhance the function and integrity of intestinal barrier;APS-IA increased the abundance of beneficial bacteria and decreased the abundance of harmful bacteria in the intestines of aged mice;APS-IA increased the expression of beneficial metabolites in the blood of aged mice.4.The role of APS-IA in regulating gut microbiota homeostasis was once again confirmed through the model of Caenorhabditis elegans;Through the Caco-2 cell model,it was confirmed at the cellular level that APS-IA regulates the gut microbiota of naturally aging mice and enhances intestinal barrier integrity through the MLCK/P-MLC pathway.