Effects Of Lycium Barbarum Oligosaccharides From Saline-alkali Land On Nonalcoholic Fatty Liver And Liver Fibrosis In Mice

Lycium barbarum is a traditional medicinal and food plant.Lycium barbarum polysaccharide（LBP）is an important active substance in the fruit of Lycium barbarum,which has various physiological functions such as antioxidant,blood sugar and blood lipid reduction,and liver protection.The biological activity of Lycium barbarum oligosaccharide（LBO）prepared by hydrolysis of LBP was superior to that of LBP.The biological activity of oligosaccharides is influenced by the structure,and the growth environment can have an impact on the LBO structure.In addition,LBO cannot be digested in the gastrointestinal tract and can act as a prebiotic for intestinal flora.Therefore,in this study,LBO was prepared by a combination of acid and enzymatic degradation of LBP using Lycium barbarum fruit from the Yellow River Delta,Gansu and Shaanxi,and the differences in the structure and biological activity of LBO from the three origins were analyzed.And the effects of LBO on non-alcoholic fatty liver and liver fibrosis were investigated in depth by 16S r RNA gene sequencing and fecal metabolomics techniques.The main research contents and results of this thesis are as follows:（1）LBP was extracted from Lycium barbarum in the Yellow River Delta,Gansu and Shaanxi,and LBO was prepared by a combination of enzymatic and acidic methods to degrade LBP,and the structure of LBO was characterized.The molecular weights of LBOs in the Yellow River Delta,Gansu and Shaanxi were 743.95 Da,775.42 Da and 740.86 Da,respectively.All three origins of LBO consisted of D-mannose,glucose,arabinose and fucose,and the D-mannose and arabinose contents of Yellow River Delta and Gansu LBO were significantly higher than those of Shaanxi LBO,indicating that the growth environment affected the monosaccharide content of LBO.The three origins of LBO were mainly in the form ofɑ-pyranose and all were non-crystalline.（2）The differences in the biological activity of LBO from different origins were analyzed by measuring in vitro simulated digestion,in vitro antioxidant and proliferative probiotic capacity.In vitro simulated gastrointestinal digestion results showed that all three origins of LBO were not hydrolyzed in the gastrointestinal digestive system and could be used as prebiotics to regulate the intestinal flora.The results of antioxidant in vitro showed that the three origins of LBO had strong antioxidant activity,and the DPPH scavenging rate and total reducing power of LBO in the Yellow River Delta were significantly higher than those of LBO in Gansu and Shaanxi when the LBO concentration reached 2 mg/m L.When the LBO concentration was in the range of 0.6 mg/m L～1 mg/m L,the scavenging rate of ABTS was significantly higher for the Yellow River Delta LBO and Gansu LBO than for Shaanxi LBO.Probiotic proliferation experiment showed that Yellow River Delta LBO showed better proliferation ability to Lactobacillus rhamnosus.In conclusion,Yellow River Delta LBO had stronger biological activity and could be used as raw material for subsequent in vivo animal experiments to further analyze the physiological functions of LBO.（3）The non-alcoholic fatty liver model was constructed using a high-fat diet feeding method,and the effects of LBO on non-alcoholic fatty liver were investigated by analyzing liver tissue structure,detecting fatty liver-related indicators and using 16S r RNA gene sequencing and fecal metabolomics techniques.The results showed that LBO could effectively alleviate the organic liver damage caused by high-fat diet,restore the abnormal liver triglyceride（TG）,total superoxide disumutase（T-SOD）and serum total cholesterol（TC）,TG,alanine aminotransferase（ALT）,alkaline phosphatase（ALP）,T-SOD levels caused by high-fat diet,and reduce liver lipid accumulation.The 16S r RNA gene sequencing results showed that LBO regulated the high-fat diet-induced gut microbial imbalance by promoting the growth of Lactobacillus,Dubosiella,and Tyzzerella and decreasing the proportion of Oscillibacter,Faecalibaculum,and Alistipes.Fecal metabolomics results showed that LBO significantly altered the levels of metabolites associated with bile acid secretion,primary bile acid biosynthesis and cholesterol metabolic pathways.LBO can alleviate fatty liver by regulating intestinal flora and altering metabolic characteristics.（4）A liver fibrosis model was constructed using carbon tetrachloride（CCl4）injection,and the effect of LBO on liver fibrosis was investigated by analyzing liver and colon tissue structures,detecting liver fibrosis-related indicators and using 16S r RNA gene sequencing and fecal metabolomics techniques.The results showed that LBO could alleviate the abnormal liver structure and liver collagen deposition caused by carbon tetrachloride（CCl₄）in mice,significantly reduce hydroxyproline（Hyp）content in liver and serum of liver fibrosis mice,and restore liver collagen tissue metabolism.In addition,LBO significantly reduced serum ALT,aspartate aminotransferase（AST）,and liver malondialdehyde（MDA）levels.In conclusion,LBO could improve CCl₄-induced liver injury and oxidative stress and alleviate the process of liver fibrosis.The effect of LBO on intestinal barrier function showed that LBO treatment reduced serum DAO and _D-LA levels,decreased intestinal permeability,and restored intestinal barrier integrity.The 16S r RNA gene sequencing results showed that LBO increased the abundance of beneficial bacteria such as Parvibacter,Alloprevotella,Lactobacillus,Butyrivibrio and Tyzzerella,decreased the abundance of pathogenic bacteria such as Blautia,Faecalibaculum and Ileibacterium,and regulated the composition of the intestinal flora.Fecal metabolomics showed that LBO significantly altered the levels of metabolites associated withβ-alanine metabolism and the digestion and absorption of vitamins.LBO could influence the liver fibrosis process by restoring intestinal barrier function and regulating the composition of intestinal flora and metabolite levels.In conclusion,Yellow River Delta LBO could regulate the host intestinal flora composition and metabolite levels and alleviate nonalcoholic fatty liver and liver fibrosis in mice.This study not only provides a theoretical basis for developing a dietary strategy to alleviate liver injury with LBO as the main component,but also has some scientific guidance and application value for promoting the development and utilization of Lycium barbarum.