CN111388488A

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

Info

Publication number: CN111388488A
Application number: CN202010277455.3A
Authority: CN
Inventors: 于广利; 王学良; 蒋昊; 蔡超
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2020-07-10

Abstract

The invention belongs to the field of marine drugs, and particularly relates to application of oligosaccharide containing D-galactose and L-galactose and derivatives thereof in preparing non-alcoholic fatty liver disease prevention and treatment drugs, wherein the D-/L-galactose-containing red algal polysaccharide is used as a raw material, and is controllably degraded by a physical method, a chemical method, a biological enzyme method or any combination of the methods to prepare the galacto-oligosaccharide and the derivatives thereof with different polymerization degrees, and the molecular skeleton of the galacto-oligosaccharide and the derivatives thereof contains D-galactose and L-galactose.

Description

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

Technical Field

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

Background

NAF L D refers to a clinical pathological syndrome characterized by abnormal accumulation of liver fat, excluding excessive drinking, viral infection, or other liver diseases NAF L D consists of a range of liver diseases, including simple hepatic steatosis, nonalcoholic steatohepatitis (NASH), and NASH-associated cirrhosis and hepatocellular carcinoma, NAF L D has an increasing incidence and has now become the most common chronic liver disease worldwide NAF L D is a liver disease associated with insulin resistance and genetic susceptibility, the exact mechanism of which is not yet well understood, Day et al propose a "secondary hit" theory that the first time is accumulation of Triglycerides (TG) in NAF, while the second time is oxidative stress-induced parenchymal inflammation, which ultimately leads to NASH. researchers also propose a "multifactorial co-hit" theory that says that NAF does not have a significant physical modification of NAF Triglycerides (TG) accumulation in NAF, or a variety of lipid stress-induced parenchymal inflammation, and that the secondary hit is not yet has been identified with the clinical stress-related dietary modification of NAF 152, NAF L D, NAF 8678, and NAF 865 has no clinical implications for the development of a variety of dietary modifications, including the mechanisms of dietary modifications that are suggested.

The intestinal flora is a group of commensal microbiota which act synergistically with the host. The intestinal flora comprises more than 1x10¹⁴The number of genes of more than 1000 different bacterial species in a cell, which is as many as 30 ten thousand genes, is 150 times the number of human genomes, and is called "human second genome". Enterobacteriaceae flora in humans is not constant and is easily affected by various factors such as genetics, diet, medicine, and sanitary environment. L iu et al, by administering different types of diet to SD ratsIt was found that the induction of NAF L D by a high-fat, high-sugar diet is not associated with caloric intake, but rather with changes in intestinal flora, suggesting that intestinal flora may play an important role in the pathogenesis of NAF L D, since the liver and intestine are connected by the portal vein, the liver is more easily exposed to translocating bacteria, bacterial products, lipopolysaccharides, and inflammatory mediators, under certain pathological conditions, disruption of the intestinal barrier can lead to translocation of bacteria and their metabolites and abnormal activation of the immune system, leading to liver inflammation and injury.

In models of mice and rodents, prognosis with dried strains of lactobacillus rhamnosus and lactobacillus casei, hepatic steatosis improves at both biochemical and histological levels, revealing the protective role of gut probiotics in diet-induced NAF L D intestinal flora may promote the onset of NAF L D by multiple mechanisms affecting energy metabolism, inducing endotoxemia, regulating bile acid metabolism, and so on, from which it is seen that intestinal flora plays an important role in NAF L D pathogenesis, it has been found that patients with NAF 567D may develop a phenomenon of small intestinal bacterial overgrowth and intestinal dysbiosis.

The research of the group shows that acidic polysaccharide and oligosaccharide (such as chondroitin sulfate and oligosaccharide thereof, keratan sulfate, fucosan sulfate and enteromorpha polysaccharide (publication No. CN108440681A) and the like) can be used as prebiotics to improve intestinal flora disorder, further play the roles of reducing blood sugar, reducing blood fat, resisting inflammation and improving metabolic syndrome, the research of the group also shows that agar oligosaccharide (publication No. CN105168232A) has the activity of reducing blood fat and the like, fucosan sulfate has the activity of inhibiting α -glycosidase (publication No. CN103288978A), algin oligosaccharide and derivatives thereof have the activity of improving insulin resistance and reducing blood sugar (publication No. CN101649004A, publication No. CN101691410A) and the like, but the research of agar containing galactan oligosaccharide derived from red algae and derivatives thereof to improve intestinal flora disorder of nonalcoholic fatty liver mice has not been found so far, the galactans derived from red algae mainly have three structural series, namely carrageenan series, agar series and carrageenan series, wherein the polysaccharide and oligosaccharide of carrageenan series are both composed of D-galactose and derivatives thereof (publication No. CN1513880A, publication No. CN101279991A, CN series, agar are different from agar polysaccharide and agar, CN-agar polysaccharide, CN-agar, NO, NO. III, NO. 7, NO. 5, NO. 7, NO. 5, NO. 7, NO. 5, NO. 7, NO..

Disclosure of Invention

The invention aims to provide the application of galacto-oligosaccharide and derivatives thereof in medicines for preventing and treating non-alcoholic fatty liver diseases, and the galacto-oligosaccharide and derivatives thereof are obtained from marine red algae polysaccharide, and proved to have the effects of improving intestinal microbial disturbance and have wide application prospects in prevention and treatment of related diseases such as non-alcoholic fatty liver diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of galacto-oligosaccharide and derivatives thereof in the preparation of medicaments for preventing and treating non-alcoholic fatty liver disease is disclosed, wherein the galacto-oligosaccharide and derivatives thereof have the following structural general formula:

wherein R is-H or-SO₃Na，n＝0～30；

The galacto-oligosaccharide and the derivative thereof are applied to the medicine for preventing and treating the non-alcoholic fatty liver, the red algal polysaccharide rich in D-galactose/L-galactose and the derivative thereof is used as a raw material, and the oligosaccharide and the derivative thereof with different polymerization degrees are prepared by one or the combination of more than two degradation methods of physical degradation, chemical degradation and enzymatic degradation, the prepared compound structure simultaneously contains β -1, 3-D-galactose (D-Gal) residues and α -1, 4-L-galactose (L-Gal) residues, or simultaneously contains D-Gal and α -1, 4-L-3, 6-diether galactose (L-AnG) residues, hydroxyl groups at C38 positions of the D-Gal and L-Gal sugar residues contain sulfate ester groups (Gal6S) with different degrees, the non-reducing end of the prepared oligosaccharide is Gal, Gal6S or AnG, the reducing end is Gal or sugar alcohol (Gal-OH) and saccharic acid (Gal-39 AnG), or Gal-OO 638) and the sugar alcohol (OO) and 638-6-OO) residues.

The galacto-oligosaccharide and the derivative thereof are applied to the medicine for preventing and treating the non-alcoholic fatty liver, and the galacto-oligosaccharide and the derivative thereof adopt the following preparation processes:

dissolving Agarose (agar) from red algae in 60 ℃ hot water, preparing 10mg/M L solution with buffer solution, placing the solution in a 30 ℃ water bath, adding β -agarase (CAS #37288-57-6) to stir and degrade for 4 hours, cooling and centrifuging, collecting supernatant, adding 95% medical ethanol with 3 times volume to 4 ℃ overnight, centrifuging, collecting precipitate, dissolving the precipitate with water, desalting with 200Da dialysis bag, concentrating the inner liquid by rotary evaporation and freeze drying to obtain new agar oligosaccharide mixture, further reducing with sodium borohydride to obtain new agar oligosaccharide, or oxidizing with Benedick reagent to obtain new agar oligosaccharide acid, or dissolving with 60 ℃ hot water, preparing 10mg/M L solution with 0.1M dilute hydrochloric acid, degrading with 80 ℃ stirring for 0.5 hours, cooling and neutralizing with 2M NaOH aqueous solution, collecting supernatant, adding 95% medical ethanol with 2 times volume to 4 ℃ precipitation, precipitating with NaOH, precipitating with 0.1M dilute NaOH solution, precipitating with NaOH, precipitating with 200.1% agar, further reducing agar with 200. mu. C, precipitating with NaOH, dissolving agar oligosaccharide in 200. mu. agar, precipitating with 200. mu. M, drying, concentrating with NaOH, concentrating with 200. mu. agar, precipitating with NaOH, concentrating, dialyzing with 200. mu. medium to obtain new agar oligosaccharide, concentrating, dissolving agar oligosaccharide, dissolving agar to obtain new agar oligosaccharide, dissolving agar to obtain new agar, dissolving agar oligosaccharide, dialyzing, dissolving agar to obtain new agar, dissolving agar oligosaccharide, dissolving agar to obtain new agar oligosaccharide, dissolving agar to obtain new agar oligosaccharide, dissolving agar to obtain new agar oligosaccharide, dissolving agar to obtain new agar, dissolving agar oligosaccharide, dissolving agar, dialyzing, dissolving agar to obtain new agar oligosaccharide, dissolving agar to obtain agar, dissolving agar to obtain new agar, dialyzing, dissolving agar to obtain new agar.

The galacto-oligosaccharide and the derivative thereof are applied to the medicine for preventing and treating the non-alcoholic fatty liver, the galacto-oligosaccharide and the derivative thereof are used as prebiotics and functional factors to play a role in preventing and treating the non-alcoholic fatty liver, the galacto-oligosaccharide and the derivative thereof with the structural characteristics effectively improve the intestinal disorder of a non-alcoholic fatty liver patient, the proportion of beneficial bacteria is increased by regulating the intestinal flora to reduce the proportion of harmful bacteria, the accumulation of liver fat is reduced, the oxidation resistance and the anti-inflammatory effect are realized, the hepatic fibrosis is relieved, the hepatic cell injury is reduced, and the galacto-oligosaccharide and the derivative thereof are used as the medicine or health care product.

The galacto-oligosaccharide and the derivatives thereof are applied to the medicine for preventing and treating the nonalcoholic fatty liver, the relative abundance of the caecum Bacteroides and Verrucomicrobia of the nonalcoholic fatty liver mice is remarkably increased on a phylum level, the relative abundance of Firmicutes, Deferribacteria and Candidatus is reduced, the relative abundance of the Akkermansia, Parabacterioides, Alloprewlla and Clostridium XIVa of the NAF L D mice is remarkably increased on a genus level, the relative abundance of the Helicobacter, Mucistillus and Barnesiella is reduced, and the intestinal flora is restored to a normal level.

The galacto-oligosaccharide and the derivative thereof can obviously reduce oxidative stress, inflammation, liver lipid accumulation and fibrosis, and are used for preparing medicines for resisting non-alcoholic fatty liver, protecting liver, resisting insulin resistance, resisting metabolic syndrome, resisting hyperlipidemia or reducing blood fat.

The galacto-oligosaccharide and the derivative thereof are applied to the medicine for preventing and treating the non-alcoholic fatty liver, and the galacto-oligosaccharide and the derivative thereof are used for health care products for preventing the fatty liver, protecting the liver or reducing the fat; or for beverages, beer, dietary supplements, or in combination with other liver-protecting drugs, or with lipid-lowering drugs; or a compound preparation containing the galacto-oligosaccharide and the derivative thereof; or the derivative prepared by taking the galactooligosaccharide and the derivative thereof as mother nucleus is used in drugs, functional foods or biological products for resisting fatty liver, insulin resistance, metabolic syndrome and hyperlipidemia.

The galacto-oligosaccharide and the derivative thereof are applied to the medicine for preventing and treating the non-alcoholic fatty liver, and the galacto-oligosaccharide and the derivative thereof form a compound preparation with ursodeoxycholic acid, vitamin E, pioglitazone, metformin or related clinical medicines.

The invention has the advantages and beneficial effects that:

1. the oligosaccharide containing D-and L-galactose residues and the derivative thereof can improve the disturbance of the intestinal flora of non-alcoholic fatty liver disease mice.

2. The oligosaccharide containing D-and L-galactose residues and the derivatives thereof have obvious effects of reducing liver fat accumulation, reducing oxidative stress and inflammation of the liver and liver fibrosis, have good protection effect on the liver, and can be used for preventing and treating fatty liver and hyperlipidemia.

3. The raw materials of the product are derived from marine red algae polysaccharide, and the product has the advantages of rich resources, simple preparation process, good product stability, easy industrialization, high safety, unique effect and the like, is used for improving the disorder of intestinal flora caused by the non-alcoholic fatty liver, and has wide development and application prospects in the development fields of new medicines and special medical foods for preventing and treating the non-alcoholic fatty liver, protecting the liver, reducing fat, metabolizing syndrome and the like.

4. According to the invention, the prepared series of galacto-oligosaccharides are subjected to NAF L D alleviation and other related functional evaluations by adopting a non-alcoholic fatty liver animal model constructed by high-fat diet induction, research results show that the oligosaccharides containing D-and L-galactose residues and derivatives thereof can remarkably improve the intestinal flora disorder of non-alcoholic fatty liver mice induced by high-fat diet, further remarkably reduce oxidative stress, inflammation, fibrosis and lipid accumulation of the liver, further play a good protection role on the liver, and have the effect of treating the non-alcoholic fatty liver and hyperlipidemia.

Drawings

FIG. 1A, B, C and D are high-resolution mass spectrograms and structural formulas of porphyran oligosaccharide (PYOs) trisaccharide, pentasaccharide, heptasaccharide and nonasaccharide, respectively. In the figure, the abscissa m/z represents the mass-to-charge ratio, and the ordinate Relative Absundance represents the Relative Abundance.

FIG. 2A, B and C are high-resolution mass spectrograms and structural formulas of neoagarotetraose, sugar alcohol and sugar acid thereof, respectively. In the figure, the abscissa m/z represents the mass-to-charge ratio, and the ordinate Relative Absundance represents the Relative Abundance.

FIG. 3 is a graph showing the effect of phylum PYOs on the structure of NAF L D mouse cecal bacteria group, PYOs represents porphyran oligosaccharide, Control represents a low-fat diet group, Model represents a high-fat diet group, Metf represents a high-fat diet plus metformin treatment group, PYOs-L represents a high-fat diet plus 100mg/kg/D PYOs treatment group, PYOs-H represents a 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 Os-L group, A is a PCoA graph of different groups of mouse intestinal bacteria group, B is a graph showing the overall change of phylum of mouse intestinal bacteria group, C, D, E, F graphs respectively show the change of mouse intestinal bacteria group in phylum, cecal bacteria group, filtration bacteria group, and filtration bacteria group.

FIG. 4 is a graph showing the effect of PYOs on the structure of NAF L D mouse cecal flora on the genus level, Control represents the group of low-fat diet, Model represents the group of high-fat diet, Metf represents the group of high-fat diet plus metformin, PYOs-L represents the group of high-fat diet plus 100mg/kg/D PYOs, PYOs-H represents the group of high-fat diet plus 300mg/kg/D PYOs. P <0.05, compared with Control group, # P <0.05, compared with Model group, & P <0.05, and PYOs-H group compared with PYOs-L, wherein A is a graph showing the overall change in the genus level of mouse intestinal flora in different treatment groups, B, C, D, E, and F are graphs showing the Relative change (%) of Helicobacter, Kermansmia, Allovocyte, Mucispiella, and Barnella, respectively, wherein the Relative scale coordinates of Ak represent the Relative graduation coordinates of the groups, and Akaban scale division represents the Relative coordinates of Ak groups.

FIG. 5 is a graph showing the effect of PYOs on weight loss in high fat diet-induced NAF L D 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<0.05, compared to Control group; # P<0.05, compared to the Model group. Wherein, A is a mouse body state diagram at the end of the test; b is a graph of the change in Body weight of mice, the abscissa represents different treatment groups, the ordinate Body weight (g) represents the Body weight of mice, the non-slashed group represents the Body weight before modeling, and the slashed group represents the Body weight at the end of the experiment; FIG. C is a graph showing the results of Weight gain of mice during the experiment, with the abscissa representing different treatment groups and the ordinate Weight gain (g) representing the amount of Weight gain of mice; graph D is body mass index of mice, with the abscissa representing different treatment groups and the ordinate BMI (kg/m)²) Representing the body mass index, E is a liver index map of each group, the abscissa represents different treatment groups, and the ordinate L driver index (mg/kg) represents the liver index.

FIG. 6 is a graph showing the effect of PYOs on inhibiting NAF L D mouse liver lipid accumulation, wherein Control represents a low-fat diet group, Model represents a high-fat diet group, Metf represents a high-fat diet plus metformin treatment group, PYOs-L represents a high-fat diet plus 100mg/kg/D PYOs treatment group, and PYOs-H represents a high-fat diet plus 300mg/kg/D Os treatment group, P <0.05, compared with the Control group, # P <0.05, compared with the Model group, & P <0.05, and compared with PYOs-L, wherein A is the appearance of mouse liver in different treatment groups, B is a graph showing the result of mouse liver H & E staining in different treatment groups, C is a graph showing the result of mouse liver Oil Red O staining in different treatment groups, D, E, F, and F are respectively showing the results of mouse liver triglyceride, fatty acid, Cholesterol content in different treatment groups, and Cholesterol content in Cholesterol (TG) in different treatment groups.

FIG. 7 is a graph showing the results of PYOs relieving the oxidative stress of the liver of NAF L D mice, Control represents a low-fat diet group, Model represents a high-fat diet group, Metf represents a high-fat diet plus metformin treatment group, PYOs-L represents a high-fat diet plus 100mg/kg/D PYOs treatment group, PYOs-H represents a high-fat diet plus 300mg/kg/D PY treatment group, P <0.05, compared with the Control group, # P <0.05, compared with the Model group, & P <0.05, PYOs-H group compared with the PYOs-L group, wherein A is the level of active oxygen (ROS) in the liver of different treatment groups, B, C, D, E are graphs of the contents of glutathione, malondialdehyde, catalase and catalase in the liver, respectively, and the abscissa represents the contents of glutathione (MDA), while GSH (nmol/mg) represents the content of glutathione, malondialdehyde (nmg/mg) in the liver, and CAT (CAT/mg) represents the content of superoxide dismutase (SOD).

FIG. 8 is a graph showing the results of relieving inflammation of liver in mice NAF L D with PYOs, wherein Control represents a low-fat diet group, Model represents a high-fat diet group, Metf represents a high-fat diet plus metformin treatment group, PYOs-L represents a high-fat diet plus 100mg/kg/dPYOs treatment group, PYOs-H represents a high-fat diet plus 300mg/kg/D Os treatment group,. P <0.05, compared with the Control group,. compared with the Model group,. P <0.05, compared with the PYOs-L group, and the A, B, C graphs show the contents of liver interleukin-6, TNF level α and monocyte chemotactic protein-1 in different treatment groups, respectively, and the abscissa shows the contents of different treatment groups, I L-6 (nmol/mg) shows the content of interleukin-6, TNF α (pg/m) shows the content of tumor necrosis factor L-861 (MCP-78/m) shows the content of monocyte chemotactic protein in different treatment groups.

FIG. 9 is a graph showing the results of PYOs on the improvement of the liver fibrosis of NAF L D mice, wherein Control represents a low-fat diet group, Model represents a high-fat diet group, Metf represents a high-fat diet plus metformin treatment group, PYOs-L represents a high-fat diet plus 100mg/kg/D PYOs treatment group, and PYOs-H represents a high-fat diet plus 300mg/kg/D PYOs treatment group, wherein A is a liver Masson trichrome staining result graph, wherein yellow arrows represent liver fibers, B is an immunohistochemical result graph of liver α -SMA, wherein yellow arrows represent α -SMA positivity, C, D, E and F are western collagen types of TGF- β, FN, ColIII and ColIV respectively, wherein TGF- β represents transforming growth factor- β represents fibronectin, ColIII represents III protein, and ColIV protein.

FIG. 10 is a graph showing the result of protecting liver cells of NAF L D mice with PYOs, wherein Control represents a low-fat diet group, Model represents a high-fat diet group, Metf represents a high-fat diet plus metformin treatment group, PYOs-L represents a high-fat diet plus 100mg/kg/D PYOs treatment group, PYOs-H represents a high-fat diet plus 300mg/kg/D PYOs treatment group. times P <0.05, compared with the Control group, # P <0.05, compared with the Model group, & P <0.05, and compared with PYOs-L group, wherein A, B, and C are serum glutamic pyruvic transaminase, glutamic oxalacetic transaminase, and alkaline phosphatase activity maps of the different treatment groups, respectively, the abscissa represents the different treatment groups, and the ordinate represents A L T (U/L) serum glutamic pyruvic transaminase, AST (U/382) serum 387, and the abscissa represents U L P.

FIG. 11 is a heat map of a Spearman's correlation analysis of gut flora between genus level and metabolic related parameters P <0.05 and P <0.01, wherein panels A, B, C, D are correlation analyses with liver injury (A L T, AST and A L P indicators), lipid levels (TC, L D L-C, TG and HD L-C content), oxidative stress levels (ROS, MDA, SOD and CAT levels), and inflammatory factors (I L-1 β, TNF- α, MCP-1 and L BP levels), respectively, and gut flora.

Detailed Description

In the specific implementation process, the invention takes red alga polysaccharide containing D-/L-galactose as a raw material, and prepares galacto-oligosaccharide and derivatives thereof with different polymerization degrees by controllable degradation through a physical method, a chemical method, a biological enzyme method or any combination of the methods, and the molecular skeleton of the galacto-oligosaccharide and the derivatives thereof contains D-galactose, L-galactose and the derivatives thereof.

The technical solution of the present invention will be further described with reference to specific examples.

Example 1 preparation of Sulfoagaro oligosaccharides (SAOs), oligosaccharide alcohols (SAOs-OH) and oligosaccharide acids (SAOs-OOH) comprising 6-O-sulfuric acid- β -1, 3-D-galactose (Gal6S) and α -1, 4-L-3, 6-lacto-galactose (AnG).

Preparing 1g of sulfur agar polysaccharide into 10mg/M L aqueous solution by using dilute sulfuric acid with the molar concentration of 0.1M, heating to 60 ℃, stirring and degrading for 1.5 hours, cooling, neutralizing by using NaOH aqueous solution with the molar concentration of 2M, centrifuging to collect supernatant, adding 3 times of 95% medical ethanol (volume concentration) in volume at 4 ℃ overnight, centrifuging to collect precipitate, dissolving by using a small amount of water, dialyzing and desalting by using a 200Da dialysis bag, carrying out rotary evaporation and concentration on the internal solution, freeze-drying to obtain SAOs, dissolving 100mg of SAOs in 10M L mM NaBH with the molar concentration of 100mM to obtain the SAOs, and dissolving the SAOs in 10M L₄Reacting water solution (containing NaOH with a molar concentration of 100 mM) at 4 ℃ overnight, adding acetic acid to adjust the pH value to 7.0, dialyzing, desalting, freeze-drying to obtain oligosaccharide alcohol SAOs-OH, taking 200mg SAOs, dissolving in newly prepared Benedick reagent of 5m L, heating at 55 ℃ to react until no brick red precipitate is generated, centrifuging to take supernatant, removing residual copper ions by cation exchange resin, adjusting the pH value to neutrality, dialyzing, and freeze-drying to obtain oligosaccharide acid SAOs-OOH.

The structural formulas of the prepared SAOs series sulfur agar oligo-sugar alcohol, oligosonic acid and oligosaccharide are as follows:

wherein R is-SO₃Na；n＝0-30；

Example 2 preparation of Porphyra gel oligosaccharides (PYOs), oligosaccharide alcohols (PYOs-OH) and oligosaccharide acids (PYOs-OOH) containing β -1, 3-D-galactose (Gal) and 6-O-sulfuric acid- α -1, 4-galactose (Gal 6S).

Preparing laver gel into 10mg/M L water solution with 0.1M dilute sulphuric acid, heating to 80 deg.C, stirring for degradation for 2 hr, cooling, neutralizing with 2M NaOH water solution, centrifuging to collect supernatant, adding 4 times volume of 95% medical ethanol at 4 deg.C overnight, centrifuging to collect precipitate, dissolving in small amount of water, desalting with 200Da dialysis bag, rotary steaming, concentrating, freeze drying to obtain laver gel oligosaccharide PYOs (shown in figures 1A-D), collecting PYOs oligosaccharide 150mg, dissolving in 15M L mM NaBH 150mM₄Reacting an aqueous solution (containing NaOH with the molar concentration of 150 mM) at 4 ℃ overnight, adding acetic acid to adjust the pH value to 7.0, dialyzing, desalting, freeze-drying to obtain the porphyran oligosaccharide alcohol PYOs-OH, dissolving 100mg of PYOs in a Benedict reagent newly prepared with 3m L, heating and stirring at 55 ℃ for reaction until no red-transfer precipitate is generated, centrifuging to obtain a supernatant, removing residual copper ions through cation exchange resin, adjusting the pH value to be neutral, dialyzing, desalting, and freeze-drying to obtain the porphyra oligosaccharide acid PYOs-OOH.

The structural formulas of the prepared porphyra gel PYOs oligo-sugar alcohol, oligosacchride acid and oligosacchride thereof are as follows:

wherein R is-H, or-SO₃Na；n＝0-30；

EXAMPLE 3 preparation of agar oligosaccharides containing β -1, 3-D-galactose (Gal) and α -1, 4-L-3, 6-lacto-galactose (AnG) and its oligosaccharide alcohols and oligosaccharide acids.

Dissolving agarose in hot water, preparing a 10mg/M L solution by using dilute hydrochloric acid with the molar concentration of 0.1M, stirring and degrading at 80 ℃ for 0.5 hour, cooling, neutralizing by using NaOH aqueous solution with the molar concentration of 2M, centrifuging, collecting supernatant, adding 3.5 times of medical ethanol with the volume of 95 percent at 4 ℃ overnight, centrifuging, collecting precipitate, dissolving in water, dialyzing and desalting by using a 200Da dialysis bag, carrying out rotary evaporation, concentration and freeze drying to obtain agar oligosaccharide AOs, further reducing by using sodium borohydride to obtain agar oligosaccharide AOs-OH, or oxidizing by using a Benedict reagent to obtain agar oligosaccharide acid AOs-OOH, wherein the chemical structural formulas of the agar oligosaccharide, the oligosaccharide acid and the oligosaccharide thereof are as follows:

wherein n is 0 to 30;

EXAMPLE 4 preparation of New agar oligosaccharide containing α -1, 4-L-3, 6-lacto galactose (AnG) and β -1,3-D galactose (Gal) and sugar alcohol and oligosaccharide acid thereof.

Dissolving agarose with hot water at 60 ℃ to prepare a 10mg/m L aqueous solution, placing the aqueous solution in a water bath kettle at 35 ℃ for stirring and cooling, adding β -agarase, stirring and carrying out enzymolysis at constant temperature for 3 hours, immediately placing the solution in a water bath kettle at 95 ℃ for enzyme denaturation for 10 minutes, cooling the solution to room temperature, centrifuging the solution to collect supernatant, then adding 3 times of medical ethanol with the volume of 95 percent at 4 ℃ for overnight, centrifuging the solution to collect precipitate, dissolving a small amount of the solution in water, carrying out dialysis and desalination by using a 200Da dialysis bag, carrying out rotary evaporation and concentration, freezing and drying the solution to obtain new agaro-oligosaccharide NAOs, and further reducing the new agaro-oligosaccharide alcohol NAOs-OH by using sodium borohydride, or oxidizing the new agaro-oligosaccharide acid NAOs-OOH by using Benedict reagent to obtain the new agaro oligosaccharide alcohol, oligosaccharide acid and oligosaccharide with the:

wherein n is 0 to 30;

in order to verify the sequence structure of the obtained oligosaccharide alcohol, the neoagaro-oligosaccharide obtained by enzymolysis can be separated and purified by a Superdex 30 column to obtain a neoagaro-tetrasaccharide pure product (figure 2A), and the neoagaro-tetrasaccharide alcohol is prepared by an alkaline sodium borohydride reduction method, and the analysis result of the obtained product by high resolution mass spectrometry (ESI-MS) is shown in figure 2B. Similarly, the neoagarotetraose obtained is subjected to Benedict directional oxidation to obtain a neoagarotetraenoic acid product, and the analysis result of high resolution mass spectrometry (ESI-MS) is shown in FIG. 2C.

Example 5 Effect of PYOs on high fat diet induced colonic microbial disturbance of NAF L D mice.

In order to study the prebiotic effect of PYOs, 20-22 g of male C57B L/6J mice were selected and adapted for one week and fed with different feeds, wherein the Control group was fed with low-fat feed, the other groups were fed with high-fat feed, the NAF L D model was successfully made after 6 months of feeding, then the Metf group, PYOs-L and PYOs-H groups were separately gavaged with 225mg/kg/dmetformin, 100mg/kg/D PYOs and 300mg/kg/D PYOs for one month, the remaining groups were gavaged with the same volume of sterile physiological saline, at the end of the experiment, the contents of mice of the different treatment groups were taken for 16S rDNA sequencing and analysis, as can be seen by the analysis of NAF Colon (as shown in FIG. 3A), the composition structure of NAF L D colon flora was significantly changed compared with the Control group, but the PY Os significantly changed the structure of colon flora L D mice and the colon was more completely changed as shown in the colon Control group of NAF-PY L.

PYOs were evaluated at the phylum level for their modulatory effects on NAF L D mouse colonic flora (as shown in figures 3B-F), and compared to Control group, NAF L D mouse colon was in lower relative abundance of bacteroides, Firmicutes was in higher relative abundance, and the Firmicutes/bacteroides ratio was significantly increased (P <0.05), whereas the supplement PYOs was able to significantly reverse this (P <0.05) (figures 3B and C). the PYOs group both significantly increased the abundance of verrucucomicubia in NAF L D mice, while decreasing the relative abundance of defericides and Candidatus saccharomyces (P <0.05) (figures 3D-F), which changes had a positive effect on improving mouse NAF L D.

The modulating effect of PYOs on NAF L D mouse colonic flora at the genus level was studied (as shown in figures 4A-F), and PYOs significantly increased the relative abundance of Akkermansia spp. and Alloprevotella spp. (P <0.05) in the colon of NAF L D mouse, while decreasing the relative abundance of Helicobacter spp., mucispillum spp. and Barnesiella spp. (P <0.05), restoring them to relatively normal levels.

The results show that the PYOs can obviously improve the colon flora disorder of NAF L D mice from phylum level and genus level, can effectively reshape the colon flora of NAF L D mice, and plays a positive role in resisting NAF L D for the PYOs.

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

Furthermore, NAF L D mice had significantly increased body weight and Body Mass Index (BMI) (P <0.05) over Control mice (FIG. 5B-D), PYOs significantly improved HFD-induced NAF L D mouse obesity (P < 0.05). furthermore, as shown in FIG. 5E, NAF L D mice had a significant increase in liver index (P <0.05) compared to Control mice (P <0.05), while PYOs treatment significantly reversed this (P < 0.05). the above results indicate that PYOs significantly inhibited NAF 539 2D mice in a dose-dependent manner.

Example 7 Effect of PYOs on liver lipid accumulation in NAF L D mice.

The study demonstrated that NAF L D has a significant histological feature of the presence of a significant amount of vesicular fat in liver tissue with cell nuclei migrating to the cell margins, and that PYOs was examined for its effects on hepatic steatosis and cell structure by liver histological analysis, as shown in fig. 6A and B, the liver of mice in the Control group was bright reddish brown with intact and well defined lobular structure, the liver of mice in the Model group was pale, showed macroscopic fat granules and was infiltrated with white spots, there was extensive necrosis in the center of the lobules characterized by lysis, nucleus loss and cell rupture, demonstrating damage to the hepatocytes, which was significantly reversed and tended to approach normal after treatment with PYOs or Metf, the results of oil red O staining, as shown in fig. 6C, showed abnormal liver reversal of lipid accumulation in the Model group, while treatment with osppy or Metf, significantly reduced HFD-induced intrahepatic lipid accumulation compared to the Control group, as shown in fig. 6D-F, NAF 56 and NAF 56, L g, showed significant inhibition of fat accumulation in mice (NAF) and no less fatty accumulation than NAF g 3 g).

Example 8 Effect of PYOs on hepatic oxidative stress and inflammatory status in NAF L D mice.

The studies showed that oxidative DHE fluorescence intensity was positively correlated with tissue ROS content, as shown in fig. 7A, Model group ROS content was significantly increased compared to Control group, PYOs or Metf treatment significantly reduced NAF L D mouse liver ROS levels, NAF L D mouse liver activity was significantly reduced with significant increase in liver content (P <0.05) compared to Control group, these conditions were significantly improved after PYOs supplementation (P <0.05) and exhibited certain dose dependence, indicating that os py could reduce NAF L D liver oxidative stress.

As shown in FIGS. 8A-C, NAF L D mice showed significantly higher liver I L-6, TNF α and MCP-1 levels (P <0.05) compared to the Control group, while PYOs significantly inhibited the rise of liver pro-inflammatory cytokines (P <0.05) in a dose-dependent manner.

Example 9 improvement of liver fibrosis by PYOs in NAF L D mice.

In addition, compared with Control mice, TGF- β and its associated extracellular matrix proteins (such as FN, Col III and Col IV) levels were significantly increased in Model mice (P <0.05), while PYOs treatment significantly decreased levels of liver fibrosis-associated proteins (P <0.05) (fig. 9C-F), these results indicate that PYOs can significantly alleviate the development and progression of liver fibrosis in NAF L D mice induced by HFD.

Example 10 protective effects of PYOs on liver injury in NAF L D mice.

As shown in FIGS. 10A-C, the activities of A L T, AST and A L P enzymes in the serum of Model group mice are significantly higher than that of Control group (P <0.05), while the treatment with PYOs or Metf significantly reversed the situation (P < 0.05). the results show that 100mg/kg/D and 300mg/kg/D PYOs have significant protective effect on the liver of NAF L D mice, and better than 225mg/kg/D Metf.

Example 11 PYOs modulate the association of colonic flora with the physiological indices of NAF L D mice.

The use of Spearman correlation analysis to elucidate the relationship between colon flora and NAF L D mouse physiological indicators (as shown in FIGS. 11A-D), Veillonella spp. and Olsenera spp. are positively correlated with serum A L T and A L P levels, the relative abundance of Clostridium XVIII spp., Rosebularia spp., Entherhabdus spp., Brachyspira spp., Anateplasma spp., Bifiorobacterium spp., Paraaberration spp., Desulfovivibrio spp., Clostridia spp., and Clostridia XIspp, the relative abundance of Paracoccus spp. can be correlated with the degree of liver injury, the relative abundance of Paragranulolla spp., Euonymus XIb.sp., Euonymus sp., Allotella sp., Anacetera, Paragranuloma sp., Euonymus sp.sp., Euonymus sp., Euonymus sp.sp.sp.sp., Euonymus sp.sp., Euonymus sp., Euonymus sp.sp.sp., Euonymus strain H.sp., Euonymus strain, Clostridium sp., Euonymus strain H.sp., Euonymus strain H7, and Microsporum strain related to induce a physiological indicators, such a positive effects that the relative abundance of accumulation of lipid accumulation of the strain, accumulation of the strain in vivo, accumulation of the strain, or the strain of the strain in mice, or the strain in vivo, or the strain of the strain, or strain of the strain, or the strain of the strain, or strain of the strain in mice, or the strain of the strain in mice, or the strain of the strain in the strain of.

The results show that PYOs can remarkably improve the relative abundance of NAF L D mouse colon bacillites and Verrucomicrobia, reduce the relative abundance of Firmicutes, Deferribacteria and Candidatus Saccharobacteria by regulating the phylum of NAF L D mouse colon flora (PYOs remarkably improves the relative abundance of NAF L D mouse colon bacillites and Verrucomicrobia), and the genus level (PYOs remarkably increases the relative abundance of NAF L D mouse colon Akkermannsp. while reducing the relative abundance of Helicobacter sp. NAF L D mouse), relieve the colonic microbiota disturbance of NAF L D mice, thus serving to relieve the effect of NAF L D induced by HFD, and demonstrate that NAF 36L D mice have a remarkably reduced relative abundance of NAF 3651D, NAF 364A reduction in the relative abundance of NAF, and a further improvement in the liver stress induced by the above mentioned oxidative stress induced by a Spearmand correlation analysis.

In conclusion, the oligosaccharide provided by the invention has the effect of resisting non-alcoholic fatty liver by improving the steady state of intestinal flora, and is suitable for being used as a candidate drug, a health product or a compound preparation of a drug for resisting fatty liver, protecting liver, reducing blood fat or resisting metabolic syndrome. The example results show that the porphyran oligosaccharide (PYOs) can be used as a prebiotic to regulate intestinal flora disturbance related to the non-alcoholic fatty liver, so as to play a role in resisting the non-alcoholic fatty liver, and can be used as a medicament or a health-care product for preventing and treating diseases related to the non-alcoholic fatty liver. The PYOs can obviously reduce lipid accumulation, relieve oxidative stress of the liver, inflammation, fibrosis and apoptosis of histiocyte, thereby realizing the functions of protecting the liver and relieving the non-alcoholic fatty liver. The product is derived from marine red algae oligosaccharide, has the advantages of rich resources, easy industrialization, safety, effectiveness and the like, and has wide development and application prospects in the aspects of preventing and treating non-alcoholic fatty liver, protecting liver, hyperlipemia, resisting metabolic syndrome and the like.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims

1. The application of galacto-oligosaccharide and derivatives thereof in the preparation of medicines for preventing and treating non-alcoholic fatty liver disease is characterized in that the galacto-oligosaccharide and derivatives thereof have the following structural general formula:

wherein R is-H or-SO₃Na，n＝0～30；

2. The use of galacto-oligosaccharides and their derivatives as drugs for the prevention and treatment of non-alcoholic fatty liver disease according to claim 1, wherein the compounds prepared from red algal polysaccharides rich in D-galactose/L-galactose and its derivatives by physical degradation, chemical degradation, enzymatic degradation or a combination of two or more degradation methods comprise β -1, 3-D-galactose (D-Gal) residues and α -1, 4-L-galactose (L-Gal) residues, or D-Gal and α -1, 4-L-3, 6-diether galactose (L-AnG) residues, and sulfate ester groups (Gal6S) with different degrees at the C38 hydroxyl groups of D-Gal and L-Gal residues, and the non-reducing end of the oligosaccharides prepared from Gal, Gal S or AnG, the reducing end of Gal-OH (Gal-OH) and sugar acids (OO-OH) and OO-6-OH (OO-OO) and its derivatives, and the non-reducing end of the compounds prepared from Gal-galactose (OO-OH) and its derivatives by physical degradation, chemical degradation, and enzymatic degradation.

3. The application of the galacto-oligosaccharide and the derivative thereof in the medicine for preventing and treating the non-alcoholic fatty liver disease according to claim 2 is characterized in that the galacto-oligosaccharide and the derivative thereof adopt the following preparation processes:

dissolving Agarose (agar) from red algae in 60 ℃ hot water, preparing 10mg/M L solution with buffer solution, placing the solution in a 30 ℃ water bath, adding β -agarase (CAS #37288-57-6) to stir and perform enzymolysis for 4 hours, cooling and then centrifuging, collecting supernatant, adding 3 times of 95% medical ethanol in volume to 4 ℃ overnight, centrifuging, collecting precipitate and dissolving the precipitate with water, performing dialysis desalination by using a 200Da dialysis bag, performing rotary evaporation, concentration and freeze drying on the inner liquid to obtain a new agar oligosaccharide mixture, further performing reduction by using sodium borohydride to obtain new agar oligosaccharide, or performing oxidation by using a Benedick reagent to obtain new agar oligosaccharide acid, or preparing 60 ℃ hot water to dissolve agar in 60 ℃ hot water, preparing 10mg/M L solution by using 0.1M dilute hydrochloric acid, performing rotary evaporation and degradation for 0.5 hours at 80 ℃, cooling and then neutralizing with 2M NaOH aqueous solution of 2M, collecting supernatant, then adding 3 times of 95% medical ethanol in 4 ℃ hot water, collecting precipitate, precipitating by using NaOH, precipitating by using NaOH 2.1M, performing centrifugal evaporation, drying after dissolving, dissolving agar in 200. mu. C, performing dialysis, dissolving agar oligosaccharide in 200. agar, precipitating, obtaining a 200. C by using Porphym agar, precipitating, obtaining a 5. agar, obtaining a new agar oligosaccharide mixture, performing dialysis, obtaining a new agar oligosaccharide mixture, further performing dialysis, obtaining a new agar oligosaccharide mixture, performing dialysis, performing centrifugation, obtaining a new agar oligosaccharide mixture, obtaining a new agar oligosaccharide by using Porphym oligosaccharide, obtaining a new agar by using NaOH solution, obtaining a new agar oligosaccharide, performing centrifugation, obtaining a new agar by using NaOH solution, performing centrifugation, performing dialysis, performing centrifugation, obtaining a new agar oligosaccharide by using NaOH solution, performing centrifugation, performing dialysis, obtaining a new agar oligosaccharide by using NaOH solution, obtaining a new agar oligosaccharide mixture, performing dialysis, adding a new agar oligosaccharide, performing centrifugation, obtaining a new agar oligosaccharide by using 10.

4. The use of galacto-oligosaccharides and derivatives thereof according to any one of claims 1 to 3 as a medicament for the prevention and treatment of non-alcoholic fatty liver disease, wherein the galacto-oligosaccharides and derivatives thereof function as prebiotics and functional factors for the prevention and treatment of non-alcoholic fatty liver disease, the galacto-oligosaccharides and derivatives thereof having these structural characteristics are effective in ameliorating intestinal disorders in patients with non-alcoholic fatty liver disease, and reducing the proportion of harmful bacteria by regulating the intestinal flora to increase the proportion of beneficial bacteria to reduce the proportion of harmful bacteria to achieve the effects of reducing liver fat accumulation, antioxidation, anti-inflammation, relieving liver fibrosis, and reducing liver cell damage, and are useful as a medicament or health product for the prevention and treatment of non-alcoholic fatty liver disease and related.

5. Use of galacto-oligosaccharides and derivatives thereof as agents for the control of non alcoholic fatty liver disease according to claim 4, characterized in that at the phylum level the galacto-oligosaccharides and derivatives thereof significantly increase the relative abundance of the beneficial bacteria Bacteroides and Verrucomicrobia in the caecum of non alcoholic fatty liver mice, while at the same time decreasing the relative abundance of the harmful bacteria Firmicutes, Deferribacteria and Candidatuscaria, and at the genus level the galacto-oligosaccharides and derivatives thereof significantly increase the relative abundance of NAF L D mouse caecum Akkermansia, Parabiades, Alloprevilla and Clostridium XIVa and decrease the relative abundance of Helicobacter, Mucispira and Barnesiella, restoring the normal intestinal flora.

6. The use of galacto-oligosaccharides and derivatives thereof according to claim 4 as a medicament for the prevention and treatment of non-alcoholic fatty liver disease, wherein the galacto-oligosaccharides and derivatives thereof significantly reduce oxidative stress, inflammation, liver lipid accumulation and fibrosis, and are useful for the preparation of medicaments against non-alcoholic fatty liver disease, hepatoprotection, insulin resistance, antimetabolite syndrome, antihyperlipidemia or hypolipidemic.

7. The use of galacto-oligosaccharides and derivatives thereof according to claim 4 as a medicament for the prevention and treatment of non-alcoholic fatty liver disease, characterized in that the galacto-oligosaccharides and derivatives thereof are used in health products for anti-fatty liver, liver protection or lipid lowering; or for beverages, beer, dietary supplements, or in combination with other liver-protecting drugs, or with lipid-lowering drugs; or a compound preparation containing the galacto-oligosaccharide and the derivative thereof; or the derivative prepared by taking the galactooligosaccharide and the derivative thereof as mother nucleus is used in drugs, functional foods or biological products for resisting fatty liver, insulin resistance, metabolic syndrome and hyperlipidemia.

8. The application of the galacto-oligosaccharide and the derivative thereof in the preparation of the drugs for preventing and treating the non-alcoholic fatty liver disease according to claim 4, characterized in that the galacto-oligosaccharide and the derivative thereof form a compound preparation with ursodeoxycholic acid, vitamin E, pioglitazone, metformin or related clinical drugs.