CN111388488A - Application of galactooligosaccharide and derivatives thereof in preparation of medicines for preventing and treating non-alcoholic fatty liver disease - Google Patents

Abstract

The invention belongs to the field of marine medicines, and in particular relates to the application of an oligosaccharide containing D-galactose and L-galactose and derivatives thereof as medicines for preventing and treating non-alcoholic fatty liver disease. Using red algae polysaccharide containing D-/L-galactose as raw material, controlled degradation is carried out by physical method, chemical method, biological enzymatic method or any combination of the above methods to prepare galacto-oligosaccharides with different degrees of polymerization and derivatives thereof D-galactose and L-galactose and their derivatives are contained in their molecular backbone. The raw material of the product of the invention is derived from red algae polysaccharide, which has the advantages of abundant resources, simple preparation process, high safety, and easy industrialization. Non-alcoholic fatty liver disease caused by high-fat diet has broad application prospects in the fields of liver protection, lipid-lowering, weight loss and special medical food development.

Description

Galacto-oligosaccharides and their derivatives in the prevention and treatment of non-alcoholic fatty liver disease application

technical field

The invention belongs to the field of marine medicines, in particular to the application of an oligosaccharide containing D-galactose and L-galactose and derivatives thereof as medicines for preventing and treating non-alcoholic fatty liver.

Background technique

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and is the most common chronic liver disease worldwide. NAFLD is a clinicopathological syndrome characterized by abnormal accumulation of hepatic fat that excludes excessive alcohol consumption, viral infection, or other liver diseases. NAFLD consists of a spectrum of liver diseases, including simple hepatic steatosis, nonalcoholic steatohepatitis (NASH), and NASH-related cirrhosis and hepatocellular carcinoma. With the global obesity epidemic, the incidence of NAFLD continues to rise and it is now the most common chronic liver disease worldwide. NAFLD is a liver disease associated with insulin resistance and genetic susceptibility, the exact mechanism of which is not well understood. Day et al. proposed the "second hit" theory, where the first hit is an increase in triglyceride (TG) accumulation in the liver, while the second hit is lipid oxidative stress-induced inflammation of the liver parenchyma, which ultimately leads to NASH. The researchers also proposed the "multifactorial co-hit" hypothesis, that is, inflammation may work together with multiple factors such as lipotoxicity, oxidative stress, and mitochondrial dysfunction to promote the occurrence of NASH before or after simple steatosis. To date, no pharmaceutical preparations have been licensed for specific NAFLD treatment. Recommended NAFLD treatment remains limited to lifestyle changes, including dietary modifications and appropriate physical activity. Therefore, it is of great clinical significance to explore the exact mechanism of NAFLD pathogenesis and identify new targets for NAFLD treatment.

The gut microbiota is a group of commensal microbiota that cooperate with the host. The intestinal flora contains more than 1x10 ¹⁴ cells, more than 1000 different bacterial species, and the genome number is as high as 300,000 genes, which is 150 times the number of the human genome, and is called "the second genome of the human body". The intestinal flora in the human body is not fixed, and it is easily affected by various factors such as heredity, diet, drugs and sanitary environment. Liu et al. found that the induction of NAFLD by high-fat and high-sugar diet was not related to caloric intake, but was related to changes in intestinal flora by giving SD rats different types of diets, suggesting that intestinal flora may play a role in the pathogenesis of NAFLD. play an important role in. Since the liver and gut are connected by the portal vein, the liver is more exposed to translocated bacteria, bacterial products, lipopolysaccharides and inflammatory mediators. Under certain pathological conditions, disruption of the gut barrier can lead to the translocation of bacteria and their metabolites and abnormal activation of the immune system, triggering liver inflammation and damage. The gut-liver axis refers to the interaction between the gut and the liver, which, as an important structure connecting the gut and the liver, plays a key role in the pathogenesis of NAFLD.

Probiotics are live microorganisms that are beneficial to the health of the host. Biochemical and histological improvements in hepatic steatosis following intervention with Lactobacillus rhamnosus and Lactobacillus casei strains in mouse and rodent models reveal protection of gut probiotics in diet-induced NAFLD effect. Intestinal flora can promote the pathogenesis of NAFLD through various mechanisms such as affecting energy metabolism, inducing endotoxemia, and regulating bile acid metabolism. It can be seen that intestinal flora plays an important role in the pathogenesis of NAFLD. The study found that NAFLD patients will have small intestinal bacterial overgrowth and intestinal dysbiosis. More and more studies have confirmed the close association between NAFLD and gut microbiota, which also makes gut microbes a potential marker for early diagnosis of NAFLD. For example, Loomba et al. analyzed the alterations of gut microbiota in NAFLD patients with fibrosis by metagenomic analysis, and the results showed that the abundance of Escherichia coli and Bacteroides vulgatus was significantly increased in NAFLD patients with fibrosis. Prebiotics are a group of non-digestible food components that selectively alter the growth or activity of certain bacteria in the colon, resulting in health benefits. Animal studies have shown that dietary fructooligosaccharide supplementation normalizes gastrointestinal microbiota and intestinal epithelial barrier function and reduces steatohepatitis in a methionine-choline deficient mouse model. This indicates that prebiotics can alleviate NAFLD by regulating intestinal flora, and provide a new direction and approach for the clinical treatment of NAFLD.

The team's research has shown that acidic polysaccharides and oligosaccharides (such as chondroitin sulfate and its oligosaccharides, keratan sulfate, fucoidan sulfate and prolifera polysaccharides (publication number CN108440681A), etc.) can be used as prebiotics to improve intestinal flora Disorders, and then play the role of lowering blood sugar, lowering blood lipids, anti-inflammatory and improving metabolic syndrome. The team also found that agarose oligosaccharide (publication number CN105168232A) has activities such as lowering blood lipids, fucoidan sulfate inhibits α-glucosidase activity (publication number CN103288978A), and alginate oligosaccharide and its derivatives can improve insulin. Resistance and hypoglycemic activity (publication number CN101649004A, publication number CN101691410A), etc., but so far there is no report that containing red algae-derived galacto-oligosaccharide and its derivatives can improve the intestinal flora disorder in non-alcoholic fatty liver mice. The galactan derived from red algae mainly has three structural series, namely carrageenan series, agar gum series and laver series. Among them, the polysaccharides and oligosaccharides of carrageenan series are composed of D-galactose and its sulfate derivatives. (Publication No. CN1513880A, Publication No. CN101012249A, Publication No. CN101279991A), while the polysaccharides and oligosaccharides of the agar and laver series are composed of D-galactose and L-galactose residues and sulfate derivatives. The difference between agar gum and laver is that the former contains more L-AnG, while the latter contains more 6-sulfate-L-Gal(Gal6S). Different types of monosaccharides have different physicochemical properties and biological functions. For example, although carrageenan and its oligosaccharides have antiviral activity as sprays (publication number CN102516323A, publication number CN104546895A), oral administration has certain potential safety hazards (Shang Q., Toxicol Let., 2017, 279:87-95 ), agar and laver and their oligosaccharides are highly safe when taken orally, and are high-quality raw materials for the development of marine medicines and functional foods. Due to the poor solubility and unclear structural sequence of galactan, its quality is difficult to control, so the preparation and application of oligosaccharides are the focus of research and development in recent years. In terms of preparation technology of agar oligosaccharide, there are mainly acid method, enzymatic method and chemical method for degradation. Different methods can obtain oligosaccharides with different structures and activities. For example, acid degradation can obtain odd-numbered agar oligosaccharides (publication number CN1513860A). Enzymatic degradation obtains even-numbered new agarose oligosaccharides (publication number CN102827899A; publication number CN109576328A), reducing acid degradation to obtain even-numbered sugar alcohols (publication number CN100999537A), and free radical degradation obtains mixed agarose oligosaccharides (publication number CN109400756A); Laveran was degraded by acid method (Liu Y., et al, Mar Drugs. 2018, 16(3).pii: E82) or enzymatic degradation (Zhang Y., et al, J. Agr. Food Chem. 2019, 67 , 9307-9313) can obtain laver oligosaccharide and so on. On the basis of the existing degradation technology, the present invention further conducts directional reduction and oxidation reactions on the prepared various oligosaccharides to obtain new oligosaccharide derivatives with different structures and sequences and containing sugar alcohol or sugar acid structures at the reducing end. And through experiments, it has been proved that these galacto-oligosaccharides and their derivatives can be used as prebiotics, have the activity of treating non-alcoholic fatty liver, and can be used to prepare drugs for the prevention and treatment of NAFLD, insulin resistance, anti-hyperlipidemia and anti-metabolic syndrome and other related diseases. Drugs and their functional products.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of galacto-oligosaccharide and its derivatives in the prevention and treatment of non-alcoholic fatty liver drug application, obtain a series of galacto-oligosaccharides and derivatives from marine red algae polysaccharide, and prove that it has improved Gut microbial disorders have broad application prospects in the prevention and treatment of non-alcoholic fatty liver disease and other related diseases.

For realizing the above-mentioned purpose of the invention, the present invention adopts following technical scheme:

The application of a galacto-oligosaccharide and its derivatives as a medicine for preventing and treating non-alcoholic fatty liver disease, the general structural formula of the galac-oligosaccharide and its derivatives is as follows:

In the formula, R=-H or -SO ₃ Na, n=0～30;

The application of the galacto-oligosaccharide and its derivatives as a drug for preventing and treating non-alcoholic fatty liver disease is based on the red algae polysaccharide rich in D-galactose/L-galactose and its derivatives as raw materials, and is subjected to physical degradation. , chemical degradation, enzymatic degradation, one or a combination of two or more degradation methods to prepare oligosaccharides and their derivatives with different degrees of polymerization, and the prepared compounds contain β-1,3-D-galactose ( D-Gal) residues and α-1,4-L-galactose (L-Gal) residues, or both D-Gal and α-1,4-L-3,6-lactone (L-Gal) residues -AnG) residue; the C6 hydroxyl group of D-Gal and L-Gal sugar residues contains different degrees of sulfate groups (Gal6S); the non-reducing end of the prepared oligosaccharide is Gal, Gal6S or AnG, and the reducing end is Gal or sugar alcohol (Gal-OH) and sugar acid (Gal-OOH), or AnG sugar alcohol (AnG-OH), or Gal6S and its sugar alcohol (Gal6S-OH) and sugar acid (Gal6S-OOH).

The application of described galacto-oligosaccharide and its derivatives as medicines for preventing and treating non-alcoholic fatty liver, the galac-oligosaccharide and its derivatives adopt the following preparation process:

Dissolve red algae-derived agarose (Agarose) in hot water at 60 °C, prepare a 10 mg/mL solution with buffer, place it in a 30 °C water bath, add β-agarase (CAS#37288-57-6) and Stir and degrade for 4 hours, centrifuge after cooling, collect the supernatant, add 3 times the volume of 95% medical ethanol at 4°C overnight, centrifuge, collect the precipitate and dissolve it with water, use a 200Da dialysis bag to dialysis and desalt, and concentrate the inner liquid by rotary evaporation. and freeze-dried to obtain a new agar oligosaccharide mixture, which is further reduced with sodium borohydride to obtain a new agar oligosaccharide alcohol, or oxidized with Benedict's reagent to obtain a new agar oligosaccharide acid; or, heat the agarose at 60°C. Dissolve in water, use 0.1M dilute hydrochloric acid to make a 10mg/mL solution, stir and degrade at 80°C for 0.5 hours, neutralize with 2M NaOH aqueous solution after cooling, collect the supernatant by centrifugation, and then add 2 times the volume of 95% Medical ethanol was kept at 4°C overnight, and the precipitate was collected by centrifugation. After dissolving the precipitate in water, dialysis and desalting were carried out with a 200Da dialysis bag. The oligosaccharide mixture was obtained by rotary evaporation, concentration and freeze-drying. Agaropectin is obtained by oxidation with Benedict’s reagent; alternatively, sulfur agar (Agaropectin) is prepared into a 10 mg/mL aqueous solution with 0.1M dilute sulfuric acid, heated to 60°C, stirred and degraded for 1.5 hours, and cooled with molar concentration Neutralize with 2M NaOH aqueous solution, collect the supernatant by centrifugation, then add 3 times the volume of 95% medical ethanol at 4 °C overnight, collect the precipitate by centrifugation, dissolve the precipitate in water, use a 200Da dialysis bag for dialysis and desalting, and then concentrate by rotary evaporation. Freeze-dry to obtain sulfur agar oligosaccharide mixture, and then further reduce it with sodium borohydride to obtain sulfur agar oligosaccharide alcohol, or use Benedict's reagent to oxidize to obtain sulfur agar oligosaccharide acid; Dilute sulfuric acid with a concentration of 0.1M was made into a 10mg/mL aqueous solution, heated to 80 °C, stirred and degraded for 2 hours, neutralized with 2M NaOH aqueous solution after cooling, centrifuged to collect the supernatant, and then added 3 times the volume of 95% medical ethanol to 4°C overnight, centrifuge to collect the precipitate, dissolve the precipitate with water, use a 200Da dialysis bag to dialysis and desalt, then rotate to concentrate and freeze-dry to obtain an oligosaccharide mixture, which is further reduced with sodium borohydride to obtain laver oligosaccharide alcohol, or Benny Oxidation with Dick's reagent yields laver oligosaccharide acid.

The application of the galacto-oligosaccharide and its derivatives as medicines for preventing and treating non-alcoholic fatty liver, the galac-oligosaccharide and its derivatives play the role of preventing and treating non-alcoholic fatty liver as prebiotics and functional factors, and have these structural characteristics The galacto-oligosaccharide and its derivatives can effectively improve the intestinal disorders of patients with non-alcoholic fatty liver disease. By adjusting the intestinal flora, increasing the proportion of beneficial bacteria and reducing the proportion of harmful bacteria, it can reduce liver fat accumulation, anti-oxidation, anti-inflammatory, and relieve Liver fibrosis, reduce liver cell damage, as a drug or health care product for the prevention and treatment of non-alcoholic fatty liver and related diseases.

The application of the galacto-oligosaccharide and its derivatives as medicines for preventing and treating non-alcoholic fatty liver, the galac-oligosaccharide and its derivatives significantly increase the cecum Bacteroidete and Verrucomicrobia of non-alcoholic fatty liver mice at the gate level. At the genus level, this galactooligosaccharide and its derivatives significantly increased Akkermansia, Parabacteroides, Alloprevotella and Clostridium XIVa in the cecum of NAFLD mice, and decreased Helicobacter, Relative abundances of Mucispirillum and Barnesiella, restoring normal levels of gut microbiota.

Application of the galacto-oligosaccharide and its derivatives as medicines for preventing and treating non-alcoholic fatty liver, the galac-oligosaccharide and its derivatives can significantly reduce oxidative stress, inflammation, accumulation of liver lipids and fibrosis , used to prepare anti-alcoholic fatty liver, liver protection, insulin resistance, anti-metabolic syndrome, anti-hyperlipidemia or hypolipidemic drugs.

The application of the galacto-oligosaccharide and its derivatives as medicines for preventing and treating non-alcoholic fatty liver, the galacto-oligosaccharide and its derivatives are used for anti-fatty liver, liver protection or lipid-lowering health care products; It can be used in beverages, beer, dietary supplements, or in combination with other hepatoprotective drugs, or in combination with blood lipid-lowering drugs; or a compound preparation containing the galacto-oligosaccharide and its derivatives; or with the galactooligosaccharide The derivatives prepared from sugar and its derivatives are used in anti-fatty liver, anti-insulin resistance, anti-metabolic syndrome, anti-hyperlipidemia drugs, functional foods or biological products.

The application of the galacto-oligosaccharide and its derivatives as medicines for preventing and treating non-alcoholic fatty liver, the galacto-oligosaccharides and their derivatives are combined with ursodeoxycholic acid, vitamin E, pioglitazone, metformin or related clinical drugs Form a compound formulation.

The advantages and beneficial effects of the present invention are:

1. The oligosaccharides containing D- and L-galactose residues of the present invention and their derivatives can improve the disturbance of intestinal flora in mice with non-alcoholic fatty liver disease.

2. The oligosaccharides containing D- and L-galactose residues and their derivatives of the present invention have significant effects of reducing liver lipid accumulation, reducing liver oxidative stress and inflammation and liver fibrosis, and have a good protective effect on the liver , can be used for the prevention and treatment of fatty liver and hyperlipidemia.

3. The raw material of the product of the present invention is derived from marine red algae polysaccharide, which has the advantages of abundant resources, simple preparation process, good product stability, easy industrialization, high safety, unique effect, etc., and is used to improve non-alcoholic fatty liver disease caused by Disturbance of intestinal flora has broad development and application prospects in the prevention and treatment of non-alcoholic fatty liver disease and in the development of new drugs and special medical foods such as liver protection, lipid-lowering and metabolic syndrome.

4. In the present invention, a series of galacto-oligosaccharides prepared by using a non-alcoholic fatty liver animal model induced and constructed by high-fat diet are used to evaluate related functions such as relieving NAFLD. The results showed that oligosaccharides containing D- and L-galactose residues and their derivatives could significantly improve the disturbance of intestinal flora in mice with high-fat diet-induced non-alcoholic fatty liver disease, and then significantly reduce hepatic oxidative stress, Inflammation, fibrosis and lipid accumulation, which in turn have a good protective effect on the liver, and have the effect of treating non-alcoholic fatty liver and hyperlipidemia.

Description of drawings

Figure 1A, B, C and D are the high-resolution mass spectra and structural formulas of trisaccharides, pentasaccharides, heptasaccharides and nonasaccharides of porphyrin oligosaccharides (PYOs), respectively. In the figure, the abscissa m/z represents the mass-to-charge ratio, and the ordinate Relative Abundance represents the relative abundance.

2A, B, and C are the high-resolution mass spectra and structural formulas of the new agartetraose and its sugar alcohol and sugar acid, respectively. In the figure, the abscissa m/z represents the mass-to-charge ratio, and the ordinate Relative Abundance represents the relative abundance.

Figure 3 is a graph showing the regulatory effect of phylum-level PYOs on the structure of cecal microbiota in NAFLD mice. PYOs stands for laver oligosaccharides. Control represents the low-fat diet group, Model represents the high-fat diet group, Metf represents the high-fat diet plus metformin treatment group, PYOs-L represents the high-fat diet plus 100mg/kg/d PYOs treatment group, PYOs-H represents the high-fat diet plus 300mg /kg/dPYOs treatment group. *P<0.05, compared with Control group; #P<0.05, compared with Model group; &P<0.05, PYOs-H group compared with PYOs-L group. Picture A is the PCoA map of the cecal flora of mice in different groups; Picture B is the overall change of the level of intestinal flora in mice in different treatment groups; Picture C, D, E, and F represent the intestinal flora of mice, respectively The relative abundances of the tract flora at the phylum level were Firmicutes, Bacteroidetes, Verrucomicrobia, Deferribacteres, Candidatus saccharibacteria.

Figure 4 is a graph showing the regulatory effect of genus-level PYOs on the structure of cecal flora in NAFLD mice. Control represents the low-fat diet group, Model represents the high-fat diet group, Metf represents the high-fat diet plus metformin treatment group, PYOs-L represents the high-fat diet plus 100mg/kg/d PYOs treatment group, PYOs-H represents the high-fat diet plus 300mg /kg/d PYOs treatment group. *P<0.05, compared with Control group; #P<0.05, compared with Model group; &P<0.05, PYOs-H group compared with PYOs-L group. Among them, picture A is the overall change chart of the level of intestinal flora of mice in different treatment groups; picture B, picture C, picture D, picture E, picture F are the relative grading of Helicobacter, Akkermansia, Alloprevotella, Mucispirillum and Barnesiella, respectively Figure, where the abscissa represents different groups, and the ordinate represents Relative abundance (%) represents relative indexing.

Figure 5 is a graph showing the effect of PYOs on weight loss in NAFLD mice induced by high-fat diet. Control represents the low-fat diet group, Model represents the high-fat diet group, Metf represents the high-fat diet plus metformin treatment group, PYOs-L represents the high-fat diet plus 100mg/kg/d PYOs treatment group, PYOs-H represents the high-fat diet plus 300mg /kg/d PYOs treatment group. *P<0.05, compared with the Control group; #P<0.05, compared with the Model group. Among them, picture A is the body diagram of the mice at the end of the test; picture B is the body weight change chart of the mice, the abscissa represents the different treatment groups, the ordinate Body weight (g) represents the body weight of the mice, and the non-slash group represents the body weight before modeling , the slash group represents the body weight at the end of the experiment; Figure C is the result of weight gain of mice during the experiment, the horizontal axis represents different treatment groups, and the vertical axis Weight gain (g) represents the weight gain of mice; Figure D is the body weight of mice Index, abscissa represents different treatment groups, ordinate BMI (kg/m ² ) represents body mass index; Figure E is the liver index map of each group, abscissa represents different treatment groups, ordinate Liver index (mg/kg) represents liver index.

Figure 6 is a graph showing the effect of PYOs on inhibiting liver lipid accumulation in NAFLD mice. Control represents the low-fat diet group, Model represents the high-fat diet group, Metf represents the high-fat diet plus metformin treatment group, PYOs-L represents the high-fat diet plus 100mg/kg/d PYOs treatment group, PYOs-H represents the high-fat diet plus 300mg /kg/d PYOs treatment group. *P<0.05, compared with Control group; #P<0.05, compared with Model group; &P<0.05, PYOs-H group compared with PYOs-L group. Among them, picture A shows the appearance of the livers of mice in different treatment groups; picture B shows the results of H&E staining of the livers of mice in different treatment groups; picture C shows the results of Oil Red O staining of the livers of mice in different treatment groups; pictures D, E, Figure F shows the results of liver triglyceride, free fatty acid and cholesterol content of mice in different treatment groups. The horizontal axis represents different treatment groups, the vertical axis TG (mg/g liver) represents triglyceride content, FFA (μmol/g liver) ) represents the free fatty acid content, and Cholesterol (mg/g liver) represents the cholesterol content.

Figure 7 shows the results of PYOs alleviating liver oxidative stress in NAFLD mice. Control represents the low-fat diet group, Model represents the high-fat diet group, Metf represents the high-fat diet plus metformin treatment group, PYOs-L represents the high-fat diet plus 100mg/kg/d PYOs treatment group, PYOs-H represents the high-fat diet plus 300mg /kg/d PYOs treatment group. *P<0.05, compared with Control group; #P<0.05, compared with Model group; &P<0.05, PYOs-H group compared with PYOs-L group. Among them, picture A is the level of reactive oxygen species (ROS) in the liver of different treatment groups; picture B, picture C, picture D, picture E are the levels of glutathione, malondialdehyde, catalase and superoxide dismutase in the liver, respectively Content map, abscissa represents different treatment groups, ordinate GSH (nmol/mg) represents glutathione content, MDA (nmol/mg) represents malondialdehyde content, CAT (U/mg) represents catalase content, SOD (U/mg) represents superoxide dismutase content.

Figure 8 is a graph showing the results of PYOs alleviating liver inflammation in NAFLD mice. Control represents low-fat diet group, Model represents high-fat diet group, Metf represents high-fat diet plus metformin treatment group, PYOs-L represents high-fat diet plus 100mg/kg/dPYOs treatment group, PYOs-H represents high-fat diet plus 300mg/dPYOs kg/d PYOs treatment group. *P<0.05, compared with Control group; #P<0.05, compared with Model group; &P<0.05, PYOs-H group compared with PYOs-L group. Among them, Figure A, Figure B, and Figure C are the levels of liver interleukin-6, tumor necrosis factor α and monocyte chemoattractant protein-1 in different treatment groups, respectively, the abscissa represents different treatment groups, and the ordinate IL-6 (nmol/mg) represents the content of interleukin-6, TNFα (pg/mL) represents the content of tumor necrosis factor α, and MCP-1 (pg/mL) represents the content of monocyte chemoattractant protein-1.

Figure 9 is a graph showing the improvement effect of PYOs on liver fibrosis in NAFLD mice. Control represents the low-fat diet group, Model represents the high-fat diet group, Metf represents the high-fat diet plus metformin treatment group, PYOs-L represents the high-fat diet plus 100mg/kg/d PYOs treatment group, PYOs-H represents the high-fat diet plus 300mg /kg/d PYOs treatment group. Among them, picture A is the result of Masson's trichrome staining of the liver, and the yellow arrows in the figure represent liver fibers; picture B is the immunohistochemical results of liver α-SMA, and the yellow arrows in the figure represent α-SMA positive; picture C, D, E and F are the western blot results of TGF-β, FN, ColIII and ColIV in different treatment groups, respectively. Among them, TGF-β stands for transforming growth factor-β, FN stands for fibronectin, ColIII stands for collagen type III, and ColIV stands for collagen type IV.

Figure 10 is a graph showing the protective effect of PYOs on liver cells of NAFLD mice. Control represents the low-fat diet group, Model represents the high-fat diet group, Metf represents the high-fat diet plus metformin treatment group, PYOs-L represents the high-fat diet plus 100mg/kg/d PYOs treatment group, PYOs-H represents the high-fat diet plus 300mg /kg/d PYOs treatment group. *P<0.05, compared with Control group; #P<0.05, compared with Model group; &P<0.05, PYOs-H group compared with PYOs-L group. Among them, Figure A, Figure B, and Figure C are the serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activity maps of different treatment groups, respectively, the abscissa represents different treatment groups, and the ordinate ALT (U/L) represents serum alanine aminotransferase. content, AST (U/L) represents serum aspartate aminotransferase content, ALP (U/L) represents serum alkaline phosphatase content.

Figure 11 is a heat map of Spearman's correlation analysis between gut microbiota at the genus level and metabolic-related parameters. +:P<0.05,*:P<0.01. Among them, picture A, picture B, picture C, picture D are respectively related to liver injury (ALT, AST and ALP indexes), lipid level (TC, LDL-C, TG and HDL-C content), oxidative stress level ( Correlation analysis between ROS, MDA, SOD and CAT levels) and inflammatory factors (IL-1β, TNF-α, MCP-1 and LBP levels) and gut microbiota.

Detailed ways

In the specific implementation process, the present invention uses red algae polysaccharide containing D-/L-galactose as a raw material, and carries out controllable degradation by physical method, chemical method, biological enzymatic method or any combination of the above methods to prepare and obtain different degrees of polymerization. The galacto-oligosaccharide and its derivatives contain D-galactose and L-galactose and their derivatives in their molecular backbone. The raw material of the product of the invention is derived from red algae polysaccharide, which has the advantages of abundant resources, simple preparation process, high safety, and easy industrialization. Non-alcoholic fatty liver disease caused by high-fat diet has broad application prospects in the fields of liver protection, lipid-lowering, weight loss and special medical food development.

Hereinafter, the technical solutions of the present invention will be further described with reference to specific embodiments.

Example 1: Containing 6-O-sulfuric acid-β-1,3-D-galactose (Gal6S) and α-1,4-L-3,6-lactone galactose (AnG) sulfur agar oligosaccharide ( Preparation of SAOs), oligosaccharide alcohols (SAOs-OH) and oligosaccharide acids (SAOs-OOH).

1 g of sulfur agar polysaccharide was prepared into a 10 mg/mL aqueous solution with dilute sulfuric acid with a molar concentration of 0.1 M, heated to 60 °C, stirred and degraded for 1.5 hours, neutralized with a molar concentration of 2 M NaOH aqueous solution after cooling, and the supernatant was collected by centrifugation. Add 3 times the volume of 95% medical ethanol (volume concentration) at 4 °C overnight, collect the precipitate by centrifugation, dissolve it with a small amount of water, and use a 200Da dialysis bag to dialysis and desalt. The inner solution is concentrated by rotary evaporation and then freeze-dried to obtain SAOs. Take 100 mg of SAOs, dissolve it in 10 mL of 100 mM NaBH ₄ aqueous solution (containing 100 mM NaOH) and react at 4 °C overnight, add acetic acid to adjust the pH to 7.0, desalt by dialysis, and freeze-dried to obtain oligosaccharide alcohol SAOs-OH . Then take 200 mg of SAOs, dissolve it in 5 mL of newly prepared Benedict reagent, heat at 55 °C until no brick-red precipitate is produced, centrifuge the supernatant, remove residual copper ions through cation exchange resin, and adjust the pH to neutral. , desalted by dialysis, and freeze-dried to obtain oligosaccharide acid SAOs-OOH.

The prepared SAOs series sulfur agar oligosaccharide alcohol, oligosaccharide acid and oligosaccharide have the following structural formulas:

In the formula, R=-SO ₃ Na; n=0-30;

Example 2: Laveran oligosaccharides (PYOs), oligosaccharide alcohols (PYOs) containing β-1,3-D-galactose (Gal) and 6-O-sulfate-α-1,4-galactose (Gal6S) -OH) and oligosaccharide acid (PYOs-OOH).

The seaweed was prepared into a 10 mg/mL aqueous solution with dilute sulfuric acid with a molar concentration of 0.1 M, heated to 80 °C, stirred and degraded for 2 hours, neutralized with a molar concentration of 2 M NaOH aqueous solution after cooling, and centrifuged to collect the supernatant. Add 4 times the volume 95% medical ethanol was kept at 4°C overnight, the precipitate was collected by centrifugation, dissolved in a small amount of water, desalted with a 200Da dialysis bag, concentrated by rotary evaporation, and freeze-dried to obtain PYOs (as shown in Figure 1A-D). Take 150 mg of PYOs oligosaccharide, dissolve it in 15 mL of 150 mM NaBH ₄ aqueous solution (containing 150 mM NaOH) and react at 4°C overnight, add acetic acid to adjust the pH to 7.0, desalinate by dialysis, and freeze-dry to obtain porphyrin Oligosaccharide alcohol PYOs-OH. Then take 100 mg of PYOs, dissolve it in 3 mL of newly prepared Benedict's reagent, heat and stir at 55 °C until no red precipitate is produced, centrifuge the supernatant, remove residual copper ions through cation exchange resin, and adjust the solution. pH to neutral, desalted by dialysis, freeze-dried to obtain porphyrin oligosaccharide acid PYOs-OOH.

The prepared Laveran PYOs oligosaccharide alcohol, oligosaccharide acid and its oligosaccharide have the following structural formulas:

In the formula, R=-H, or -SO ₃ Na; n=0-30;

Example 3: Agar oligosaccharides containing β-1,3-D-galactose (Gal) and α-1,4-L-3,6-lactogalactose (AnG) and their oligosaccharide alcohols and oligosaccharides Preparation of acid.

Dissolve the agarose in hot water, use dilute hydrochloric acid with a molar concentration of 0.1M to make a 10 mg/mL solution, stir at 80 °C for 0.5 hours, and neutralize it with an aqueous NaOH solution with a molar concentration of 2M after cooling, and collect the supernatant by centrifugation. Add 3.5 times the volume of 95% medical ethanol at 4 °C overnight, collect the precipitate by centrifugation, dissolve it in water, use a 200Da dialysis bag for dialysis desalination, rotate to concentrate and freeze-dry to obtain agar oligosaccharide AOs, and further reduce it with sodium borohydride to obtain agar oligosaccharide. Alcohol AOs-OH, or oxidized with Benedict's reagent to obtain agarose oligosaccharide acid AOs-OOH. The chemical structural formulas of agarose oligosaccharides, oligosaccharides and their oligosaccharides are as follows:

In the formula, n=0-30;

Example 4: New agarose oligosaccharides containing α-1,4-L-3,6-lactogalactose (AnG) and β-1,3-D-galactose (Gal) and their sugar alcohols and oligosaccharide acids preparation.

Dissolve the agarose with 60°C hot water to prepare a 10mg/mL aqueous solution, place it in a 35°C water bath and stir to cool down, add β-agarase, stir at a constant temperature for enzymatic hydrolysis for 3 hours, and immediately place it in a 95°C water bath to make the enzyme After denaturation for 10 minutes, it was cooled to room temperature, and the supernatant was collected by centrifugation. Then, 3 times the volume of 95% medical ethanol was added overnight at 4°C, and the precipitate was collected by centrifugation. New agar oligosaccharides NAOs are obtained, which are further reduced with sodium borohydride to obtain new agar oligosaccharides alcohol NAOs-OH, or oxidized with Benedict's reagent to obtain new agar oligosaccharides NAOs-OOH. The chemical structural formula of the prepared new agarose oligosaccharide alcohol, oligosaccharide acid and oligosaccharide is as follows:

In the formula, n=0-30;

In order to verify the sequence structure of the obtained oligosaccharide alcohol, the new agarose oligosaccharide obtained by enzymatic hydrolysis can be separated and purified with a Superdex 30 column to obtain pure new agarose tetraose (Figure 2A), which was prepared by the alkaline sodium borohydride reduction method. The high-resolution mass spectrometry (ESI-MS) analysis result of the obtained product of the new agarose alcohol is shown in Figure 2B. Similarly, the obtained new agartetraose was obtained by the Benedict directional oxidation method to obtain the new agartetraose acid product, and its high-resolution mass spectrometry (ESI-MS) analysis result is shown in FIG. 2C .

Example 5: Effects of PYOs on high-fat diet-induced colonic microbial disturbances in NAFLD mice.

In order to study the prebiotic effect of PYOs, 20-22 g male C57BL/6J mice were selected and fed with different diets after acclimation for one week. Among them, the Control group was fed with low-fat diet, and the other groups were fed with high-fat diet. After 6 months of feeding, the NAFLD model was successfully established. After that, the Metf group, PYOs-L and PYOs-H groups were given 225 mg/kg/dmetformin, 100 mg/kg/d PYOs and 300 mg/kg/d PYOs for one month, respectively, and the other groups were given The same volume of sterile normal saline in the stomach. At the end of the experiment, the colonic contents of mice in different treatment groups were taken for 16S rDNA sequencing and analysis. According to PCoA analysis (as shown in Figure 3A), compared with the Control group, the composition and structure of the colonic microbiota of the NAFLD mice were significantly changed, but the supplementation of PYOs significantly changed the structure of the colonic microbiota of the NAFLD mice and was more inclined to the Control group. Group. The colonic microbiota structures of the PYOs-L group and the PYOs-H group were completely separated, indicating that the regulation of colonic microbiota by PYOs was dose-dependent (as shown in Figure 3A).

The regulatory effect of PYOs on the colonic microbiota of NAFLD mice was evaluated at the phylum level (as shown in Figure 3B-F), and the relative abundance of Bacteroidetes and Firmicutes was lower in the colon of NAFLD mice compared with the Control group. , the Firmicutes/Bacteroidetes ratio was significantly increased (P<0.05), while supplementation with PYOs was able to significantly reverse this (P<0.05) (Figure 3B and C). Both PYOs groups significantly increased the abundance of Verrucomicrobia in NAFLD mice, while decreasing the relative abundance of Deferribacteres and Candidatus Saccharibacteria (P<0.05) (Fig. 3D-F), these changes have a positive effect on improving NAFLD in mice.

The regulatory effects of PYOs on the colonic microbiota of NAFLD mice at the genus level were investigated (as shown in Figure 4A-F), and PYOs significantly increased the relative abundance of Akkermansia spp. and Alloprevotella spp. in the colon of NAFLD mice (P<0.05). ), while reducing the relative abundances of Helicobacter spp., Mucispirillum spp. and Barnesiella spp. (P<0.05), returning them to relatively normal levels.

The above results show that PYOs can significantly improve the colonic flora disorder in NAFLD mice from the phylum and genus levels, can effectively reshape the colonic flora of NAFLD mice, and play a positive role in the anti-NAFLD of PYOs.

Example 6: Effects of PYOs on HFD-induced obesity and liver index in mice.

Obesity, especially abdominal obesity, is associated with the occurrence and development of NAFLD. It was observed that the waist circumference of the Model group was significantly larger than that of the Control group; PYOs significantly reduced the waist circumference of mice after six weeks of feeding (Fig. 5A), so PYOs could significantly reduce the obesity degree of mice. In addition, NAFLD mice had significantly higher body weight and body mass index (BMI) than Control mice (P<0.05) (Fig. 5B-D), and PYOs could significantly improve HFD-induced obesity in NAFLD mice (P<0.05). Furthermore, as shown in Figure 5E, the liver index of NAFLD mice was significantly increased (P<0.05) compared with the Control group, which was significantly reversed by PYOs treatment (P<0.05). The above results indicated that PYOs significantly inhibited obesity and increased liver index in NAFLD mice in a dose-dependent manner.

Example 7: Effects of PYOs on liver lipid accumulation in NAFLD mice.

Studies have shown that a significant histological feature of NAFLD is the presence of a large amount of vesicular fat in liver tissue accompanied by the migration of nuclei to the cell edge. The effects of PYOs on hepatic steatosis and cellular structure were investigated by liver histological analysis. As shown in Figure 6A and B, the livers of the mice in the Control group were bright reddish-brown, and the hepatic lobules were intact and clear; the livers of the mice in the Model group were pale, showing fat particles visible to the naked eye, and infiltrated by leukoplakia. There was a large area of necrosis in the center of the hepatic lobule, which was characterized by lysis, disappearance of nuclei, and cell rupture, proving that hepatocytes were damaged; after treatment with PYOs or Metf, the above symptoms were significantly reversed compared with the Model group, and tended to be closer to normal. The results of Oil Red O staining showed (as shown in Figure 6C) that compared with the Control group, the Model group had abnormal lipid accumulation in the liver, while PYOs or Metf treatment significantly alleviated the HFD-induced intrahepatic lipid accumulation. As shown in Figure 6D-F, compared with the Control group, NAFLD mice had significantly increased TG, FFA, and Cholesterol contents (P<0.05), which were significantly reversed by PYOs or Metf treatment (P<0.05). The above results indicated that NAFLD was successfully induced in HFD mice, and PYOs could significantly inhibit the accumulation of hepatic fat in NAFLD mice.

Example 8: Effects of PYOs on oxidative stress and inflammatory status in the liver of NAFLD mice.

Studies have shown that hepatic oxidative stress and inflammatory state play an important role in the occurrence of NAFLD. The oxidized DHE fluorescence intensity was positively correlated with the tissue ROS content. As shown in Figure 7A, compared with the Control group, the ROS content in the Model group was significantly increased, and PYOs or Metf treatment could significantly reduce the liver ROS level in NAFLD mice. Compared with the Control group, the activities of SOD, CAT and GSH in the liver of NAFLD mice were significantly decreased, while the content of MDA in the liver was significantly increased (P<0.05) (Fig. 7B-E), and these conditions were significantly improved after supplementation with PYOs (P<0.05), and in a dose-dependent manner, indicating that PYOs can alleviate hepatic oxidative stress in NAFLD.

Studies have shown that oxidative stress-mediated signal transduction mechanisms are involved in the occurrence and development of NAFLD inflammation. As shown in Figure 8A-C, compared with the Control group, liver IL-6, TNFα and MCP-1 levels were significantly increased in NAFLD mice (P<0.05), while PYOs significantly inhibited hepatic pro-inflammatory cells in a dose-dependent manner factor increased (P<0.05).

Example 9: Improvement of liver fibrosis in NAFLD mice by PYOs.

Studies have shown that oxidative stress in hepatocytes can lead to increased collagen synthesis, which leads to the occurrence and development of liver fibrosis. Liver fibrosis is an important biomarker of NAFLD progression, and portal fibrosis is considered an important clinical feature of severe NAFLD. The development of hepatocyte fibrosis in NAFLD mice was characterized by Masson's trichrome and α-SMA immunohistochemistry, and as shown in Figure 9A and B, PYOs effectively ameliorated liver fibrosis status in NAFLD mice. In addition, the levels of TGF-β and its related extracellular matrix proteins (such as FN, Col III, and Col IV) were significantly increased in the Model group compared with the Control group (P<0.05), while PYOs treatment significantly The levels of liver fibrosis-related proteins were decreased (P<0.05) (Fig. 9C-F). These results suggest that PYOs can significantly alleviate the occurrence and development of HFD-induced liver fibrosis in NAFLD mice.

Example 10: Protective effect of PYOs on liver injury in NAFLD mice.

Liver function enzymes (ALT, AST, and ALP) mainly exist in the liver, but can enter the blood in large quantities when the liver cell membrane is damaged. The activities of these related enzymes in serum can reflect the degree of liver damage. As shown in Figure 10A-C, the serum ALT, AST and ALP enzyme activities of the mice in the Model group were significantly higher than those in the Control group (P<0.05), which were significantly reversed by PYOs or Metf treatment (P<0.05). The above results indicated that 100 mg/kg/d and 300 mg/kg/d of PYOs had significant protective effects on the liver of NAFLD mice, which were better than those of 225 mg/kg/d of Metf.

Example 11: The correlation between the regulation of colonic flora by PYOs and the physiological indicators of NAFLD mice.

Using Spearman correlation analysis to elucidate the relationship between colonic flora and physiological indicators of NAFLD mice (as shown in Figure 11A-D), Veillonella spp. and Olsenella spp. were positively correlated with serum ALT and ALP levels; Clostridium XVIII spp. The relative abundance of , Roseburia spp., Enterorhabdus spp., Brachyspira spp., Anaeroplasma spp., Bifidobacterium spp., Parabacteroides spp., Desulfovibriospp., Clostridium XIVa spp. and Clostridium XIVb spp. were negatively correlated with the degree of liver injury; increased Pararevotella The relative abundance of spp. can significantly increase LDL-C content, while Alloprevotella spp., Parabacteroides spp., Enterorhabdus spp., Roseburia spp., Brachyspira spp., Bifidobacterium spp., Anaeroplasma spp., Clostridum XIVb spp. and Clostridum XIVaspp. The relative abundance of . was negatively correlated with TC, LDL-C and TG accumulation; the relative abundance of Paraprevotella spp., Clostridium XIspp. and Barnesiella spp. was negatively correlated with HDL-C content, while Eubacterium spp., Akkermansia spp. The relative abundances of , Alloprevotella spp. and Parabacteroides spp. were positively correlated with HDL-C content; the relative abundances of Anaeroplasm spp., Parabacteroides spp., Desulfovibrio spp., Brachyspira spp., Clostridum XIVa spp. and Clostridum XIVb spp. CAT and SOD were positively correlated, but negatively correlated with the contents of ROS, MDA, IL-1β, TNFα, MCP-1 and LBP. In conclusion, PYOs increased the relative abundance of probiotics while reducing the content of harmful microorganisms in NAFLD mice. Response and protection of liver damage are closely related. PYOs can improve HFD-induced NAFLD by regulating the relative abundance of the above-mentioned genera.

The anti-NAFLD effect of PYOs was evaluated using a high-fat diet-induced NAFLD mouse model. The results showed that PYOs modulated the phylum level of colonic microbiota in NAFLD mice (PYOs significantly increased the relative abundance of Bacteroidetes and Verrucomicrobia, and decreased the relative abundance of Firmicutes, Deferribacteres and Candidatus Saccharibacteria) and genus level (PYOs significantly increased the relative abundance of NAFLD mice. The relative abundance of Akkermansia spp. in the murine colon, while reducing the relative abundance of Helicobacter spp., the composition and abundance changes, alleviated the colonic microbiota disturbance in NAFLD mice, and then played a role in alleviating HFD-induced NAFLD. Spearman correlation analysis showed that PYOs increased the relative abundance of probiotics while reducing the abundance of harmful microorganisms in NAFLD mice. levels, inflammatory response, and protection from liver damage are closely related. It was further demonstrated that PYOs can improve HFD-induced NAFLD by regulating the relative abundance of the above-mentioned genera. The prebiotic effect of PYOs was preliminarily explained from the perspective of improving colonic flora, so that PYOs can significantly reduce HFD-induced obesity and liver index increase in NAFLD mice, improve liver oxidative stress and inflammation in NAFLD mice, and significantly reduce liver lipid accumulation . In addition, PYOs significantly alleviated oxidative stress-induced liver fibrosis, thereby ameliorating liver damage. In conclusion, PYOs significantly ameliorated HFD-induced NAFLD in mice through the above-mentioned protective mechanisms.

To sum up, the oligosaccharide of the present invention has the effect of resisting non-alcoholic fatty liver by improving the homeostasis of intestinal flora, and is suitable as a candidate drug for anti-fatty liver, liver protection, hypolipidemic or anti-metabolic syndrome drugs or health care. product or compound preparation application. The results of the examples show that the porphyrin oligosaccharides (PYOs) of the present invention can be used as prebiotics to regulate the disturbance of intestinal flora related to non-alcoholic fatty liver disease, thereby exerting the anti-non-alcoholic fatty liver effect, and can be used as the prevention and treatment of non-alcoholic fatty liver disease. Medicines or supplements for liver-related diseases. PYOs can significantly reduce lipid accumulation, alleviate hepatic oxidative stress, inflammation, fibrosis, and tissue apoptosis, thereby achieving liver protection and relieving non-alcoholic fatty liver disease. The product of the invention is derived from marine red algae oligosaccharide, and has the advantages of abundant resources, easy industrialization, safety and effectiveness, etc., and has broad development in the prevention and treatment of non-alcoholic fatty liver, liver protection, hyperlipidemia and anti-metabolic syndrome application prospects.

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still The recorded technical solutions are modified, or some technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims (8)

1. the application of a galactooligosaccharide and derivative thereof in the medicine for preventing and treating non-alcoholic fatty liver, it is characterized in that, the general structural formula of galactooligosaccharide and derivative thereof is as follows:

In the formula, R=-H or -SO ₃ Na, n=0～30;

2. according to the application of galacto-oligosaccharide according to claim 1 and derivative thereof in medicine for preventing and treating non-alcoholic fatty liver, it is characterized in that, to be rich in D-galactose/L-galactose and derivative thereof The red algae polysaccharide is used as raw material, and the oligosaccharide and its derivatives with different degrees of polymerization are prepared by one or more of physical degradation, chemical degradation and enzymatic degradation. -1,3-D-galactose (D-Gal) residue and α-1,4-L-galactose (L-Gal) residue, or both D-Gal and α-1,4-L- 3,6-lactone galactose (L-AnG) residue; the C6 hydroxyl group of D-Gal and L-Gal sugar residues contains different degrees of sulfate group (Gal6S); the non-reducing end of the prepared oligosaccharide Is Gal, Gal6S or AnG, the reducing end is Gal or sugar alcohol (Gal-OH) and sugar acid (Gal-OOH), or AnG sugar alcohol (AnG-OH), or Gal6S and its sugar alcohol (Gal6S-OH) and Sugar acid (Gal6S-OOH). 3. according to the application of galacto-oligosaccharide and derivative thereof in claim 2 as a medicine for preventing and treating nonalcoholic fatty liver, it is characterized in that, this galactooligosaccharide and derivative thereof adopts following preparation technique: Dissolve red algae-derived agarose (Agarose) in hot water at 60 °C, prepare a 10 mg/mL solution with buffer, place it in a 30 °C water bath, add β-agarase (CAS#37288-57-6) and Stir the enzymatic hydrolysis for 4 hours, centrifuge after cooling, collect the supernatant, add 3 times the volume of 95% medical ethanol at 4°C overnight, centrifuge, collect the precipitate and dissolve it with water, use a 200Da dialysis bag to dialysis and desalt, and spin the inner solution. Concentrate and freeze-dry to obtain a new agarose oligosaccharide mixture, which is further reduced with sodium borohydride to obtain a new agarose oligosaccharide alcohol, or oxidized with Benedict's reagent to obtain a new agarose oligosaccharide acid; Dissolve in hot water, use 0.1M dilute hydrochloric acid to make a 10mg/mL solution, stir and degrade at 80°C for 0.5 hours, neutralize with 2M NaOH aqueous solution after cooling, collect the supernatant by centrifugation, and then add 3 times the volume of 95 % medical ethanol at 4°C overnight, centrifuge to collect the precipitate, dissolve the precipitate with water, use a 200Da dialysis bag for dialysis desalination, rotary evaporation, concentration and freeze-drying to obtain an oligosaccharide mixture, which is further reduced with sodium borohydride to obtain agarose oligosaccharide alcohol, or Agaropectin was obtained by oxidation with Benedict's reagent; alternatively, sulfur agar (Agaropectin) was prepared into a 10 mg/mL aqueous solution with 0.1 M dilute sulfuric acid, heated to 60 °C, stirred and degraded for 1.5 hours, and cooled with molar Neutralize with 2M NaOH aqueous solution, collect the supernatant by centrifugation, then add 3 times the volume of 95% medical ethanol at 4°C overnight, collect the precipitate by centrifugation, dissolve the precipitate in water, use a 200Da dialysis bag for dialysis desalination, and then concentrate by rotary evaporation And freeze-dried to obtain sulfur agar oligosaccharide mixture, and then further reduced by sodium borohydride to obtain sulfur agar oligosaccharide alcohol, or oxidized with Benedict's reagent to obtain sulfur agar oligosaccharide acid; Molar concentration of 0.1M dilute sulfuric acid was made into a 10mg/mL aqueous solution, heated to 80 °C, stirred and degraded for 2 hours, cooled and neutralized with a molar concentration of 2M NaOH aqueous solution, centrifuged to collect the supernatant, and then added 3 times the volume of 95% medical ethanol At 4°C overnight, the precipitate was collected by centrifugation. After dissolving the precipitate in water, dialysis and desalting were carried out with a 200Da dialysis bag, followed by rotary evaporation, concentration and freeze drying to obtain an oligosaccharide mixture. Oxidation of Nitik's reagent yields laver oligosaccharide acid. 4. according to the application of the galacto-oligosaccharide and its derivatives described in one of claims 1 to 3 as a medicine for preventing and treating non-alcoholic fatty liver, it is characterized in that, the galac-oligosaccharides and derivatives are used as prebiotics and functional The factors play a role in preventing and treating non-alcoholic fatty liver. Galacto-oligosaccharides and their derivatives with these structural characteristics can effectively improve the intestinal disorders of patients with non-alcoholic fatty liver, and increase the proportion of beneficial bacteria and reduce the proportion of harmful bacteria by regulating the intestinal flora. It can reduce liver fat accumulation, anti-oxidation, anti-inflammatory, relieve liver fibrosis, and reduce liver cell damage, as a drug or health care product for the prevention and treatment of non-alcoholic fatty liver and related diseases. 5. according to the application of the galacto-oligosaccharide and its derivative in claim 4 as the medicine for preventing and treating non-alcoholic fatty liver, it is characterized in that, this galac-oligosaccharide and its derivative significantly increase the non-alcoholic fatty liver on the gate level. The relative abundance of beneficial bacteria Bacteroidete and Verrucomicrobia in the cecum of alcoholic fatty liver mice, while reducing the relative abundance of harmful bacteria Firmicutes, Deferribacteres and Candidatus saccharibacteria; at the genus level, the galactooligosaccharide and its derivatives significantly increased the NAFLD small Akkermansia, Parabacteroides, Alloprevotella and Clostridium XIVa in the murine cecum, and decreased relative abundances of Helicobacter, Mucispirillum and Barnesiella, returned the gut microbiota to normal levels. 6. The application of the galactooligosaccharide and its derivatives as claimed in claim 4 in the prevention and treatment of non-alcoholic fatty liver disease, wherein the galactooligosaccharides and derivatives thereof can significantly reduce oxidative stress, Inflammation, accumulation of liver lipids and fibrosis, it is used to prepare drugs against non-alcoholic fatty liver, liver protection, insulin resistance, anti-metabolic syndrome, anti-hyperlipidemia or hypolipidemic. 7. according to the application of galacto-oligosaccharide and derivatives thereof in claim 4 as prevention and treatment of non-alcoholic fatty liver, it is characterized in that, this galactooligosaccharide and its derivatives are used for anti-fatty liver, protection Liver or lipid-lowering health care products; or used in beverages, beer, dietary supplements, or in combination with other hepatoprotective drugs, or in combination with blood-lipid lowering drugs; or containing the galacto-oligosaccharide and its derivatives Compound preparation; or derivatives prepared by using the galacto-oligosaccharide and its derivatives as the core nucleus for anti-fatty liver, anti-insulin resistance, anti-metabolic syndrome, anti-hyperlipidemia drugs, functional foods or biological products middle. 8. according to the application of the galacto-oligosaccharide and derivative thereof in claim 4 as the medicine for preventing and treating non-alcoholic fatty liver, it is characterized in that, this galacto-oligosaccharide and derivative thereof and ursodeoxycholic acid, Vitamin E, pioglitazone, metformin or related clinical drugs form a compound preparation.

Images