Study On Structural Characterization Of Laminaria Japonica Polysaccharide And Its Beneficial Effects On Dyslipidemias-associated Intestinal Bacteria

In recent years,cardiovascular disease has become the leading cause of death in China and even the world,with economic transformation,industrialization,urbanization and globalization bringing about new lifestyle changes.Cardiovascular disease seriously affects the individual,family and society,and cardiovascular disease clinical care is expensive and time-consuming.Thereby,looking for cheap edible plant extracts as essential auxiliary treatment and prevention of cardiovascular disease could reduce the risk of the disease occurrence and development in the early stage of cardiovascular disease.At the same time,cheap edible plant extracts as essential auxiliary treatment could help solve all kinds of problems faced by national population ageing,reduce the medical burden and save social resources.Dyslipidemias is the main cause of cardiovascular diseases,and the adjuvant therapeutic effect of Laminaria japonica polysaccharide from staple agricultural products on dyslipidemias has been proved by numerous published papers by using animal experiments,human experiments and in vitro cell experiments.However,the mechanism of the relationship between structure and activity of Laminaria japonica polysaccharide on dyslipidemia are still unclear.And Laminaria japonica polysaccharide,as an auxiliary treatment for dyslipidemia,is mostly administered by oral administration.Therefore,further exploration is needed to explore the mechanism of its digestion in gastrointestinal tract and related animal experiments.In this paper,the effects of extraction methods on the structure of polysaccharides were discussed by using different extraction methods to extract Laminaria japonica polysaccharides,and the optimal extraction method was selected based on the in vitro bile acid binding ability.On this basis,the structure-activity relationship between the structure and in vitro bile acid binding ability was preliminarily explored.Three homogeneous fragments,LP-A4,LP-A6 and LP-A8,were obtained by using acid extraction to prepare the Laminaria japonica polysaccharide.The structure of the three polysaccharide fragments was analyzed accurately,and their simulated digestion characteristics,bile acid binding capacity and human intestinal fermentaion characteristics were explored.Finally,the intestinal functional characteristics of LP-A4?MA?and LP-A8?FS?with huge structural differences were compared in ApoE^-/-hyperlipidemia mouse model.The main research contents and results of this paper are as follows:?1?The structural characterization and bile acid-binding capacity of Laminaria japonica polysaccharides?LP?,obtained by seven different extraction methods,were investigated.The results indicated that extraction methods exhibited significant effects on extraction yield,molecular weight,monosaccharides composition and the content of neutral sugar,fucose,uronic acid and sulfate of LP.The bile acid-binding capacity of acid assisted extracted LP?LP-A?was significantly higher than other LP samples tested,which was probably ascribed to its highly branched structure,low molecular weight,low viscosity and abundant uronic acid and fucose in total monosaccharides.The present study provides scientific evidences in the relationship between structure characterization and bile acid-binding capacity of L.japonica polysaccharides.?2?We further purified the crude LP-A by using ion-exchange chromatography and gel filtration chromatography and conducted a comparison study on the structure characterization of three LP-A fractions?LP-A4,LP-A6 and LP-A8?by using monosaccharide composition analysis,glycosidic linkage analysis and nuclear magnetic resonance?NMR?spectroscopy.The results indicated that LP-A4,LP-A6 and LP-A8,characterized as mannoglucan,fucomannoglucan and fucogalactan,had significantly different structure characterization.Furthermore,the bile acid-binding capacity of LP-A8 was obviously higher than the other fractions,which may be attributed to its highly branched structure??1?2,3,4?linked?-D-ManpA?,abundant sulfate,fucose and galactose in chemical composition and denser interconnected macromolecule network in molecular morphology,indicating the possible correlation between structural characterization and bile acid-binding capacity.?3?The results of in vitro simulated digestion indicated that the LP-As couldn't be entirely digested by gastrointestinal digestion.After simulated salivary and gastric digestion,the molecular weight distribution of the three polysaccharide fractions did not change.After simulated intestinal digestion,the average molecular weight of LP-A6 and LP-A8 decreased slightly,but the molecular weight distribution of LP-A4 did not change significantly during intestinal digestion.The reason might be that LP-A6 and LP-A8 would be easier to trap the enzyme molecules in pancreatin and expose more cutting site for the enzyme digestion according to their denser interconnected macromolecule network with more branches,compared with LP-A4.?4?In addition,fermentation of LP-As by human gut microbiota,especially for LP-A8,in both hyper-lipid and normal-lipid control groups generated large amount of SCFAs compared with the blank,which might change the microbiota composition and have positive impact on lipid metabolism.Acetate and butyrate were the most abundant SCFAs in group NL,and their generation was upregulated sharply by LP-A8,which might reveal that LP-A8 have some beneficial effects on the host lipid metabolism.The relative abundance of Lachnoclostridium and Eubacterium in group HL could be notably increased by LP-A6 and LP-A8?p<0.01?.The findings from the SCFAs production and taxonomic composition collectively suggest that the notable increasing of butyrate and total SCFAs in group HL by the LP-As treatment could be attributed to higher abundance of Firmicutes compared with the blank.Functional analysis revealed that the increased metabolic activities of glycerophospholipid metabolism,ether lipid metabolism and fatty acid metabolism,as well as the decreased fatty acid biosynthesis and fatty acid elongation,induced by LP-A8 treatment in group HL were closely associated with metabolic syndromes and hyperlipidemia.?5?Two purified L.japonica polysaccharides,LP-A4?mannoglucan,MA?and LP-A8?fucogalactan,FS?,have significant different structure and acticities in animal experiment.Biochemical analysis and pathological analysis showed that FS could effectively alleviate the obesity of ApoE^-/-mice caused by high-sugar,high-fat and high-cholesterol diet,and it might be related to the functional mechanism of FS.After FS and MA dietary intervention,the accumulation of fat in liver tissue can be reduced,the lesion of colon epithelial tissue can be relieved,and the formation of early atherosclerotic plaque can be prevented.Mice gut microbiota analysis showed that Bacteroidaceae was the characteristic bacterium in the intestinal tract of ApoE^-/-mice,while Helicobacteraceae occupied a considerable proportion in the intestinal tract of healthy mice.Bacteroidales_S24-7_group and Prevotellaceae with relatively high contents are the main characteristic bacteria in the intestinal tract of healthy mice that distinguish healthy mice from ApoE^-/-mice.In ApoE^-/-mice,there is no significant difference in gut microbiota between the group FS,MA,CT and MC.LefSe analysis showed that most of the different species in healthy mice came from Bacteroidales,and the most significant species was Bacteroidales_S24-7_group.However,Blautia,Mucispirillum,Tyzzerella and Streptococcaceae were the most significant bacteria in group MC,MA,FS and CT,respectively.Analysis of intestinal bacterial metabolites-SCFA showed that acetic acid,propionic acid and butyric acid were the most abundant SCFA in the intestinal tract of healthy mice,and the concentration of these three SCFA in the intestinal tract of ApoE^-/-mice could be significantly up-regulated by FS,which indicated that the dietary intake of FS could be digested by gut microbiota and produce SCFA to do beneficial effects to host health.