CN110812364A

CN110812364A - Application of galactooligosaccharide and derivatives thereof as SGLTs inhibitor

Info

Publication number: CN110812364A
Application number: CN201911004019.2A
Authority: CN
Inventors: 于广利; 王学良; 郝杰杰; 蔡超; 蒋昊; 李国云
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2020-02-21

Abstract

The invention belongs to the field of marine medicines, and particularly relates to application of D-galactose and L-galactose-containing oligosaccharide and derivatives thereof as SGLTs inhibitors. The preparation method comprises the steps of taking red algae polysaccharide containing D-/L-galactose as a raw material, and degrading by a physical method, a chemical method, a biological enzyme method or any combination of the methods to prepare galacto-oligosaccharides with different polymerization degrees and derivatives thereof, wherein the molecular skeleton of the galacto-oligosaccharides contains D-galactose, L-galactose and derivatives thereof. The raw materials of the product are derived from the red algae polysaccharide, the product has the advantages of rich resources, simple preparation process, high safety, definite target spot, easy industrialization and the like, is used as SGLT1 and 2 inhibitors, and has wide application prospect in the development of medicaments for resisting diseases such as diabetes, obesity, diabetic nephropathy, glycolipid metabolic disorder and the like and food for improving the function of nonalcoholic fatty liver.

Description

Application of galactooligosaccharide and derivatives thereof as SGLTs inhibitor

Technical Field

The invention belongs to the field of marine medicines, and particularly relates to application of D-galactose and L-galactose-containing oligosaccharide and derivatives thereof as SGLTs (SGLT1 and SGLT2) inhibitors.

Background

Diabetes (diabetes mellitis) is a metabolic disease which is characterized by chronic hyperglycemia and is generated by absolute or relative insufficiency of insulin secretion of pancreatic islet β cells, diabetes patients reach 4.25 hundred million in 2017 all over the world, 6.29 million is expected in 2045 years, Chinese diabetes patients are about 1.144 million and become countries with the largest number of patients, the morbidity of adults in China is 9.7 percent, the morbidity of the early stage of diabetes is higher by 15.5 percent, wherein more than 90 percent of the patients are type 2 diabetes, long-term chronic hyperglycemia causes complications, large, small and peripheral neuropathy causes damage to organs such as heart, brain, kidney and eyes, and the like, blood sugar control is a key for treating type 2 diabetes and delaying the progress of the complications.

Sodium-glucose cotransporter1 (SGLT 1) is a transporter with high affinity and low transport capacity, and is mainly distributed at the brush border of the small intestine and the distant S3 segment of the kidney from the proximal convoluted tubule to absorb glucose produced by food digestion and supply the glucose for life activities. Inhibiting the transporter can reduce glucose absorption in intestinal tract and lower postprandial blood sugar. The SGLT2 protein is a low affinity, high capacity transporter present in the kidney and capable of reabsorbing glucose from the glomerular filtrate back into the blood. When blood flows through the kidney and is filtered by the glomeruli, the resulting filtrate flows through the tubules, and glucose in the filtrate is transported back into the blood by SGLT2 located at the segments S1 and S2, preventing glucose from being excreted with urine. The human kidney filters about 180g of glucose per day, with more than 90% of the renal glucose reabsorption being accomplished by SGLT2, and the remainder by SGLT 1. SGLT2 inhibitors, by blocking this process, inhibit the transport of glucose by the SGLT2 protein, leaving the glucose in the filtered urine for eventual excretion with the urine, thereby lowering blood glucose.

In recent years, SGLT is considered to be one of the hot targets for developing hypoglycemic drugs, wherein SGLT1 and 2 inhibitors (SGLTi) play a role in lowering blood sugar by reducing the absorption of glucose by the small intestine and the reabsorption of glucose by the kidney.

As the earliest SGLTi studied, phlorizin (phloridzin) is a dual inhibitor of SGLT1 and SGLT2, which has poor selectivity for SGLT1 and 2 and is easily decomposed by the human body. On the basis of the development of SGLT2 selective inhibitors, empagliflozin (empagliflozin) which is currently on the market internationally is the most selective SGLT2i (>2500:1), followed by dapagliflozin (dapagliflozin, >1200:1) and canagliflozin (canagliflozin, >250: 1). SGLTi has good development and market prospects, receives more and more attention, and more developed medicines, but no SGLTi from marine sources is reported.

The research shows that seaweed polysaccharide and oligosaccharide have the functions of resisting oxidation, reducing blood sugar, reducing blood fat, resisting inflammation, enhancing immunity and the like, the research of the team makes a great deal of research work in the field, agar oligosaccharide (publication No. CN105168232A) has the activity of reducing blood fat and the like, fucosyl 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 oligosaccharide and derivatives thereof containing D-galactose and L-galactose have no report of inhibiting SGLT1 and SGLT2 in a targeted mode for preventing and treating diabetes related diseases, the galactan derived from red algae mainly comprises carrageenan series, agar series and agar series, and agar series, wherein the polysaccharide and agar series are both composed of D-galactose and sulfuric acid derivatives thereof (publication No. CN1513880A, publication No. CN 6348, CN101279991A are different from the polysaccharide and oligosaccharide of D-galactose and agar series and sulfuric acid derivatives thereof (publication No. CN-No. 11, CN-27. the preparation method is different from the polysaccharide and agar oligosaccharide, CN-No. 11, the polysaccharide and agar polysaccharide are prepared by a new agar oligosaccharide preparation method, a new agar oligosaccharide and a new agar oligosaccharide degrading oligosaccharide by a method, a new agar oligosaccharide preparation method of agar oligosaccharide and a new agar oligosaccharide (publication No. CN-agar oligosaccharide preparation method of agar oligosaccharide, CN-agar oligosaccharide preparation method, CN-agar oligosaccharide and agar oligosaccharide, CN-agar oligosaccharide preparation method of degrading oligosaccharide, CN-agar oligosaccharide preparation method, CN-agar oligosaccharide and agar oligosaccharide are different from which are different from a new agar oligosaccharide, CN-agar oligosaccharide and a new agar oligosaccharide preparation method for degrading oligosaccharide, CN-agar oligosaccharide preparation method for degrading oligosaccharide, CN-agar oligosaccharide preparation method for degrading oligosaccharide, CN-agar oligosaccharide, CN.

Disclosure of Invention

The invention aims to provide an application of galactooligosaccharide and a derivative thereof in preparation of SGLT1 and 2, obtains a series of galactooligosaccharide and a derivative thereof from marine red algae polysaccharide, and proves that the galactooligosaccharide and the derivative thereof have the functions of reducing blood sugar, improving glycolipid metabolism and preventing and treating diseases related to type 2 diabetes.

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

the application of galacto-oligosaccharide and derivatives thereof as SGLTs inhibitors is disclosed, wherein the oligosaccharide has the following structural general formula:

wherein R is-H or-SO₃Na，n＝0～30；

The galacto-oligosaccharide and the derivative thereof with the structural characteristics can be used for inhibiting the activity of SGLT1 and SGLT2 in a targeted manner, and can be used as medicines or health products for preventing and treating diabetes, diabetic nephropathy and cardiovascular diseases.

The galacto-oligosaccharide and the derivatives thereof can be used as SGLTs inhibitors, and can be combined with SGLTs proteins in a targeted mode to inhibit glucose transport activity and regulate glycolipid metabolism.

The galacto-oligosaccharide and the derivative thereof can obviously reduce blood sugar and are used for preparing medicines for reducing blood sugar, relieving insulin resistance, resisting type 2 diabetes, resisting metabolic syndrome, resisting non-alcoholic fatty liver, resisting hyperlipidemia, protecting liver and reducing blood fat.

The galacto-oligosaccharide and the derivatives thereof are applied to SGLTs inhibitors, and the SGLTs inhibitors are applied to medicines or health care products for resisting diabetes, diabetic nephropathy and cardiovascular diseases; or in beverages, beer, dietary supplements, or in combination with other antidiabetic agents, or hypolipidemic agents; or in combination with an agent that resists diabetic nephropathy; or a compound preparation containing the galacto-oligosaccharide and the derivative thereof; or in combination with anti-cardiovascular agents; or the derivatives prepared by taking the series of oligosaccharides as parent nucleus are used as preparations for SGLTs antidiabetics, antidiabetic nephropathy and cardiovascular disease resistant medicines or functional preparations.

The galacto-oligosaccharide and the derivative thereof are applied to being used as SGLTs inhibitors, the galacto-oligosaccharide and the derivative thereof and metformin, dapagliflozin, canagliflozin or acarbose related clinical hypoglycemic drugs form a compound preparation, and the galacto-oligosaccharide and the derivative thereof are applied to being used as SGLTs inhibitors to prevent and treat diabetes, diabetes and nephropathy and cardiovascular related diseases.

The galacto-oligosaccharide and the derivative thereof are applied to SGLTs inhibitors, and the preparation process of the galacto-oligosaccharide and the derivative thereof is as follows:

the method comprises the steps of preparing oligosaccharides with different polymerization degrees and derivatives thereof by taking red algal polysaccharides rich in D-/L-galactose and derivatives thereof as raw materials through one or a combination of more than two degradation methods of physical degradation, chemical degradation and enzymatic degradation, wherein 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 at C6 of the D-Gal and L-Gal sugar residues contains sulfuric acid esterification (Gal6S) modification with different degrees, the non-reducing end of the prepared oligosaccharides is Gal, Gal6S or AnG, the reducing end of the oligosaccharides is Gal or sugar alcohol (Gal-OH) and sugar acid (Gal-OOH) or AnG sugar alcohol (AnG-OH), or Gal6S and sugar alcohol (Gal-OH) and sugar alcohol (Gal-6S-6S).

The galacto-oligosaccharide and the derivatives thereof are applied to SGLTs inhibitors, and the galacto-oligosaccharide and the derivatives thereof adopt the following specific preparation processes:

dissolving agarose in 60 ℃ hot water, preparing 10mg/mL solution by using buffer solution, placing the solution in a 30 ℃ water bath, adding β -agarase (CAS #37288-57-6) into the water bath, stirring and degrading for 4 hours, cooling, centrifuging, collecting supernatant, adding 2 times of volume of 95% medical ethanol into the solution at 4 ℃, centrifuging, collecting supernatant, performing rotary evaporation to remove ethanol, performing dialysis and desalination by using a 200Da dialysis bag, performing rotary evaporation, concentration and freeze drying to obtain a new agaro-oligosaccharide mixture, further performing reduction by using sodium borohydride to obtain new agaro-oligosaccharide, or performing oxidation by using a Benedick reagent to obtain new agaro-oligosaccharide acid, or dissolving agarose in 80 ℃ hot water, preparing 10mg/mL solution by using 0.1M dilute hydrochloric acid, performing stirring and degradation at 80 ℃ for 0.5 hours, cooling, neutralizing by using 2M NaOH aqueous solution, performing centrifugal evaporation, collecting supernatant, then adding 2 times of volume of 95% medical ethanol into 4 ℃, collecting supernatant, performing rotary evaporation, removing ethanol, performing dialysis by using 200Da bags, performing dialysis, drying, further performing centrifugal evaporation to obtain a 2M aqueous solution, heating, removing the supernatant, performing reduction by using NaOH, drying, adding 2.1.1 mg of agar, performing dialysis to obtain a 200. agar, concentrating, performing dialysis to obtain a 2. agar, adding 5. agar, concentrating, performing dialysis to obtain a supernatant, performing dialysis to obtain a 2. agar, further performing dialysis, adding 2. agar-agar oligosaccharide mixture, performing centrifugation, removing agar-agar, drying, concentrating agar-agar, heating, performing centrifugation, drying, heating, removing agar, concentrating agar, removing agar, performing dialysis reagent, concentrating agar, heating, concentrating agar, performing dialysis, drying, concentrating agar, performing dialysis to obtain a 5-agar, performing dialysis solution, heating, drying, performing dialysis solution.

The invention has the design idea that the oligosaccharide compound which is high in safety and can play a role of reducing blood sugar in multiple targets and multiple ways is prepared, and the oligosaccharide and the derivatives thereof can directly inhibit the activity of SGLT1 in intestinal tracts and can enter the kidney through blood to inhibit the activity of SGLT 2.

The invention further carries out directional reduction and oxidation reaction on various prepared oligosaccharides on the basis of the existing degradation technology to obtain oligosaccharide derivatives with different structures and sequences and sugar alcohol or sugar acid structures at the reducing ends, and experiments prove that the oligosaccharides and the derivatives thereof have the activities of remarkably targeted inhibiting SGLT1 and SGLT2, and can be used for preparing medicines and functional products for preventing and treating diabetes, diabetes nephropathy and cardiovascular diseases.

The invention has the advantages and beneficial effects that:

1. the oligosaccharide containing D-and L-galactose residues and the derivatives thereof can target SGLT1 and 2, inhibit the glucose transport function of the oligosaccharide, and further relieve insulin resistance.

2. The oligosaccharide containing D-and L-galactose residues and the derivatives thereof have obvious effects of reducing blood fat and protecting liver, and can be used for preventing and treating hyperlipidemia and fatty liver.

3. The raw materials of the product are derived from marine polysaccharide, and the product has the advantages of rich resources, simple preparation process, good product stability, easy industrialization, safety, effectiveness and the like, is used for reducing blood sugar, and has wide development and application prospects in the development fields of new medicines for preventing and treating type 2 diabetes and special medical foods for reducing blood sugar, reducing blood fat, improving fatty liver and the like.

4. The oligosaccharide of the invention has the functions of inhibiting hyperglycemia, obesity, lipid accumulation, insulin resistance and non-alcoholic fatty liver caused by high-fat diet.

5. According to the invention, relevant function evaluation such as SGLT1 and 2 inhibitory activity and the like is carried out on the prepared series of oligosaccharides by adopting human SGLT1 and 2 stable transgenic cell strains and a type 2 diabetes animal model constructed by high fat diet induction. Research results show that the oligosaccharide containing D-and L-galactose residues and derivatives thereof can target and inhibit SGLT1 and 2 glucose transport activity, further has the effect of reducing blood sugar, and has the effects of obviously reducing body weight, hyperlipidemia and hyperglycemia, improving oxidative stress state and inflammation, and obviously increasing insulin sensitivity, thereby improving insulin resistance, and having the effect of treating diabetes, fatty liver, hyperlipidemia and cardiovascular and cerebrovascular diseases.

Drawings

FIG. 1 shows high resolution mass spectra and structural formulas of neoagarotetraose (a), sugar alcohol (b) and sugar acid (c). In the figure, the abscissa m/z represents the mass-to-charge ratio, and the ordinate RELATIVISUNDANCE represents the relative abundance.

FIG. 2 shows the construction and verification of human SGLT1 and 2 stably transfected cell lines, in which Control represents the untransfected blank Control group, EP (empty plasmid) represents the empty plasmid transfected group, SGLT1P represents the SGLT1 recombinant plasmid transfected group, SGLT1 represents the SGLT1 protein expression level, SGLT2P represents the SGLT2 recombinant plasmid transfected group, SGLT2 represents the SGLT2 protein expression level, β -actin represents β -actin, HEK293 represents the untransfected group, HEK293-SGLT1 represents the SGLT1 stably transfected cell group, HEK293-SGLT2 represents the SGLT2 stably transfected cell group, P <0.05 > is compared with the untransfected blank Control group, # P <0.05 > is compared with the empty plasmid transfected group, wherein (a) is a graph showing that SGLT1 stably transfected cell line protein expression n (protein) and the left stably transfected cell line (light) are shown in the SGLT1 and 2 stably transfected cell line), and the right stably transfected line 8653 (SGLT) is shown in the SGLT transverse chart, and the SGLT transverse chart shows the change of the SGLT 8458 and the horizontal change of the horizontal and vertical coordinate (SGLT) of the horizontal change of the SGLT 8458 and vertical coordinate (SGLT 849 and vertical coordinate of the horizontal change of the protein expression of the horizontal change of the protein of the SGLT1 and the horizontal change of the SGLT transverse coordinate of the stable cell line representing the SGLT1 and the horizontal change of the SGLT longitudinal change of the SGLT transverse graph (SGLT 849 and the stable cell line of the horizontal change of the cell line of the sample).

Fig. 3 shows that sulfoagarotriose targets SGLT1 and 2 to inhibit its glucose transport activity. GF3 represents sulfur agarotriose, SGLT1 IC50 represents the half inhibition concentration of sulfur agarotriose on SGLT1 activity, SGLT2 IC50 represents the half inhibition concentration of sulfur agarotriose on SGLT2 activity, and% inhibition represents the inhibition rate of different concentrations of GF3 on SGLT activity. Wherein (a) is a graph showing the results of the inhibition rate of GF3 on SGLT 1; (b) the graph shows the results of the inhibition rate of GF3 against SGLT 2.

FIG. 4 is a graph of the results of an Oral Glucose Tolerance Test (OGTT) with a series of galactooligosaccharides to lower postprandial blood glucose. Control represents blank Control group, SAOs represents sulfoagaro-oligosaccharide treatment group, AOs represents agaro-oligosaccharide treatment group, and POs represents porphyran-oligosaccharide treatment group. Wherein, the graph (a) is a result graph of Blood glucose levels at different time points of an OGTT experiment, the abscissa represents the testing time (min), and the ordinate represents the Blood glucose (mmol/L) represents the Blood glucose level; (b) the figure is a graph of the integration result of the Area under the curve of the OGTT experiment, the abscissa represents different treatment groups, and the ordinate represents the Area under the curve (mmol/L.min).

FIG. 5 is a graph of the results of SAOs decreasing blood glucose and increasing insulin sensitivity and urine glucose in type 2 diabetic (T2DM) mice. P <0.05, compared to normal group mice; # P <0.05, compared to group T2 DM. Control stands for low-fat diet treated group, Model stands for high-fat diet treated group, Metf stands for high-fat diet plus metformin treated group, Cana stands for high-fat diet plus canagliflozin treated group, SAOs-L stands for high-fat diet plus 100mg/kg/d sulfur agar oligosaccharide treated group, and SAOs-H stands for high-fat diet plus 300mg/kg/d sulfur agar oligosaccharide treated group. Wherein, the graph (a) is a graph of the results of oral glucose tolerance tests of different treatment groups, the abscissa represents the testing time (min), and the ordinate represents Blood glucose (mmol/L) represents the Blood glucose values at different time points; (b) the graph is a graph of the result of an insulin tolerance experiment of the abdominal cavity, wherein the abscissa represents the testing time (min), and the ordinate represents Blood glucose (mmol/L) represents the Blood glucose value at different time points; (c) the figure is a result graph of Fasting blood glucose determination after oligosaccharide feeding for six weeks, the abscissa represents different treatment groups, and the ordinate represents Fasting blood glucose (mmol/L); (d) the graph is a graph of the determination result of Urine glucose content, the abscissa represents different treatment groups, and the ordinate represents Urine glucose (mmol/L) represents the value of Urine glucose level.

FIG. 6 is a graph of SAOs decreasing T2DM mouse body weight. P <0.05, compared to normal group mice; # P <0.05, compared to group T2 DM. Control stands for low-fat diet treated group, Model stands for high-fat diet treated group, Metf stands for high-fat diet plus metformin treated group, Cana stands for high-fat diet plus canagliflozin treated group, SAOs-L stands for high-fat diet plus 100mg/kg/d sulfur agar oligosaccharide treated group, and SAOs-H stands for high-fat diet plus 300mg/kg/d sulfur agar oligosaccharide treated group. Wherein, the figure (a) is a mouse body state figure at the end of the test; (b) the figure is a graph of the weight change of mice, the abscissa represents different treatment groups, the ordinate Bodyweight (g) represents the weight of mice, the histogram without slash represents the weight before modeling, and the histogram with slash represents the weight at the end of the experiment; (c) the figure is a graph of the results of the Weight gain of mice during the experiment, with the abscissa representing the different treatment groups and the ordinate Weight gain (g) representing the Weight gain of the mice.

FIG. 7 is a graph of SAOs improving T2DM non-alcoholic fatty liver disease. Control stands for low-fat diet treated group, Model stands for high-fat diet treated group, Metf stands for high-fat diet plus metformin treated group, Cana stands for high-fat diet plus canagliflozin treated group, SAOs-L stands for high-fat diet plus 100mg/kg/d sulfur agar oligosaccharide treated group, and SAOs-H stands for high-fat diet plus 300mg/kg/d sulfur agar oligosaccharide treated group. Wherein, the picture (a) is the oil red O staining result of the frozen section of the liver tissue of different treatment groups; (b) the figure shows hematoxylin-eosin staining results of liver tissue sections of different treatment groups.

FIG. 8 is a graph of SAOs improving T2DM hyperlipidemia. Control stands for low-fat diet treated group, Model stands for high-fat diet treated group, Metf stands for high-fat diet plus metformin treated group, Cana stands for high-fat diet plus canagliflozin treated group, SAOs-L stands for high-fat diet plus 100mg/kg/d sulfur agar oligosaccharide treated group, and SAOs-H stands for high-fat diet plus 300mg/kg/d sulfur agar oligosaccharide treated group. Wherein, the graph (a) is a result graph of the Serum triglyceride content of different treatment groups, the abscissa represents different treatment groups, and the ordinate Serum TG represents the Serum triglyceride content (mmol/L); (b) the graph is a result graph of the total cholesterol content of the Serum of different treatment groups, the abscissa represents different treatment groups, and the ordinate Serum TC represents the total cholesterol content (mmol/L) of the Serum; (c) the figure is a result graph of the content of low density lipoprotein in serum of different treatment groups, the abscissa represents different treatment groups, and the ordinate SerumLDL-C represents the content (mmol/L) of low density lipoprotein in serum; (d) the figure shows the result of high-density lipoprotein content in Serum of different treatment groups, wherein the abscissa represents different treatment groups, and the ordinate represents Serum high-density lipoprotein content (mmol/L) in Serum HDL-C.

Detailed Description

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 1000mg of sulfur agar polysaccharide into 10mg/mL 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 medical ethanol (with the concentration of 95 wt%) with the volume of 95% at 4 ℃ for overnight, centrifuging to collect supernatant, carrying out reduced pressure rotary evaporation to remove ethanol, dialyzing and desalting by using a 200Da dialysis bag, and carrying out rotary evaporation concentration and freeze drying to obtain SAOs. 100mg of SAOs were dissolved in 10 ml of 100mM NaBH₄Reacting with water solution (containing 100mM NaOH) at 4 deg.C overnight, adding acetic acid to adjust pH to 7.0, dialyzing to desalt, and freeze drying to obtain oligosaccharide alcohol SAOs-OH. And dissolving 200mg of SAOs in 5 ml of newly prepared Benedict reagent, heating at 55 ℃ for reaction until no brick red precipitate is generated, centrifuging to obtain supernatant, removing residual copper ions through cation exchange resin, adjusting pH to be neutral, dialyzing for desalting, and freeze-drying to obtain the 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 gum oligosaccharides (POs), oligosaccharide alcohols (POs-OH) and oligosaccharide acids (POs-OOH) containing β -1, 3-D-galactose (Gal) and 6-O-sulfuric acid- α -1, 4-galactose (Gal 6S).

Preparing the laver glue into 10mg/mL aqueous solution by using dilute sulfuric acid with the molar concentration of 0.1M, heating to 80 ℃, stirring and degrading for 2.0 hours, cooling, neutralizing by using NaOH aqueous solution with the molar concentration of 2M, centrifuging, collecting supernatant, adding medical ethanol with the volume of 4 times 95% to the temperature of 4 ℃ overnight, centrifuging, collecting supernatant, carrying out reduced pressure rotary evaporation to remove the ethanol, desalting by using a 200Da dialysis bag, and carrying out rotary evaporation concentration and freeze drying to obtain the laver glue oligosaccharide POs. 150mg of POs oligosaccharide was dissolved in 15 ml of 150mM NaBH₄Reacting with water solution (containing 150mM NaOH) at 4 deg.C overnight, adding acetic acid to adjust pH to 7.0, dialyzing to desalt, and freeze drying to obtain agar gel oligosaccharide sugar alcohol POs-OH. And dissolving 100mg of POs in 3mL of newly prepared Benedick reagent, heating and stirring at 55 ℃ for reaction until no red-turning precipitate is generated, centrifuging to obtain a supernatant, removing residual copper ions through cation exchange resin, adjusting the pH value to be neutral, dialyzing for desalting, and freeze-drying to obtain the porphyra gum oligosaccharide acid POs-OOH.

The structural formulas of the prepared laver glue POs oligosaccharide alcohol, oligosaccharide acid and oligosaccharide 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/mL 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 a NaOH aqueous solution with the molar concentration of 2M, centrifuging, collecting a supernatant, adding 2.5 times of medical ethanol with the volume of 95% at 4 ℃ overnight, centrifuging, collecting a supernatant, performing rotary evaporation to remove the ethanol, dialyzing and desalting by using a 200Da dialysis bag, performing rotary evaporation, concentration and freeze drying to obtain agaro-oligosaccharide AOs, and further performing reduction by using sodium borohydride to obtain agaro-oligosaccharide AOs-OH, or oxidizing by using a Benedict reagent to obtain agaro-oligosaccharide AOs-OOH. The chemical structural formulas of the agar oligo-sugar alcohol, 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 10mg/mL aqueous solution, placing the aqueous solution in a water bath kettle at 30 ℃, adding β -agarase, stirring at constant temperature for enzymolysis for 4 hours, immediately placing the aqueous solution in a water bath kettle at 95 ℃ to denature the enzyme for 10 minutes, cooling to room temperature, centrifuging to collect supernatant, then adding medical ethanol with the volume of 3 times 95% to the temperature of 4 ℃ for overnight, centrifuging to collect supernatant, performing rotary evaporation to remove the ethanol, dialyzing and desalting the solution by using a 200Da dialysis bag, performing rotary evaporation, concentration and freeze drying to obtain new agaro-oligosaccharide NAOs, and further performing reduction by using sodium borohydride to obtain new agaro-oligosaccharide alcohol NAOs-OH, 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 chemical structural formulas as follows:

wherein n is 0 to 30;

in order to verify the sequence structure of the obtained oligosaccharide alcohol, the neoagaro-oligosaccharide obtained by enzymolysis is separated and purified by a Superdex 30 column to obtain a neoagarotetraose pure product (figure 1a), and the neoagarotetraitol is further prepared by an alkaline sodium borohydride reduction method, and the analysis result of high resolution mass spectrometry (ESI-MS) of the obtained product is shown in figure 1 b. 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. 1 c.

Example 5: construction of human SGLT1 and 2 Stable transgenic cell line

In order to study the inhibitory effect of the compound on human SGLT, a recombinant plasmid containing full-length human SGLT1 or SGLT2 was transferred into HEK293 cells by using Lipofectamine 2000, and G418 was used for screening to obtain a stable transfer cell strain capable of stably expressing SGLT or SGLT 2. The results of western blot prove that stable transgenic cell strains of SGLT1 (FIG. 2a) and SGLT2 (FIG. 2b) are successfully constructed. By using flow cytometry, it is further verified that the stably transfected cell line obtained by screening can significantly improve the activity of transporting glucose compared with the untransfected group (fig. 2c), and can be used for screening SGLT1 and SGLT2 inhibitors.

Example 6: thioagarotriose (GF3) targeted SGLT1 and 2 inhibits glucose transport activity thereof

To investigate the effect of GF3 on the inhibition of glucose transport activity in SGLT1 and 2, the inhibition of SGLT1 and 2-transporting fluorescent D-glucose homolog (2-NBDG) by series of GF3 concentrations was analyzed by flow cytometry. As shown in fig. 3, sulfoagarotriose targeted SGLT1 and 2 to inhibit their glucose transport activity curves. The experimental result shows that the inhibition rate of the GF3 on the glucose transport activity of SGLT1 and SGLT2 is obviously improved along with the increase of the concentration of the GF 3. The half inhibitory concentrations of GF3 on SGLT1 and SGLT2 glucose transport activity were calculated to be 1363nmol/L and 12.58nmol/L, respectively, using Graphpadprism software. GF3 is therefore a selective SGLT2 inhibitor, selective for SGLT1/SGLT2 ═ 108.34.

Example 7: oral Glucose Tolerance Test (OGTT) characterization series galactooligosaccharides reduce postprandial blood glucose effect

To confirm the effect of the series of galactooligosaccharides on inhibiting SGLT1 at the animal level, 18-22g male Kunming mice were taken and fed with 2g/kg body weight glucose solution after fasting for 12h, and blood glucose was measured at 0min, 30min, 60min, 90min and 120min and the area under the curve was calculated. As shown in fig. 4, a graph of the results of the Oral Glucose Tolerance Test (OGTT) of galactooligosaccharides lowering postprandial blood glucose. The experimental result shows that compared with the control group, SAOs, AOs and POs can obviously reduce the postprandial blood sugar of mice and improve the glucose tolerance, which shows that the series of galactooligosaccharides can obviously reduce the activity of SGLT1 and further has the effect of reducing the postprandial blood sugar.

Example 8: animal model characterization of type 2 diabetes (T2DM) SAOs hypoglycemic and insulin sensitivity increasing experiment

In order to further verify that series of galactooligosaccharides inhibit SGLT1 and SGLT2 at the animal level so as to further play a role in reducing hyperglycemia, 20-22 g of male Kunming mice are selected to be adaptive to different feeds for feeding after one week. Wherein Control group was fed with low fat diet and the other groups were fed with high fat diet. After 6 months of successful molding, the animals were fed with different compounds for one month, and then their oral glucose sensitivity, intraperitoneal insulin sensitivity and urine glucose level were measured. As shown in fig. 5, the results of SAOs decreased oral glucose tolerance, blood glucose, and increased insulin sensitivity and urine glucose in type 2 diabetes (T2DM) mice are plotted. The experimental results showed that SAOs significantly enhanced oral glucose tolerance (fig. 5a), insulin sensitivity (fig. 5b), decreased fasting glucose (fig. 5c) and increased urine glucose (fig. 5d) levels in T2DM mice compared to the Model group. It can be seen that SAOs inhibit SGLT1 and SGLT2 glucose transport activity, thereby lowering blood glucose and increasing urine glucose levels.

Example 9: SAOs weight reduction in T2DM mice

The weight loss effect of SAOs was further evaluated using the T2DM animal model created in example 8. The experimental results showed that mice were significantly reduced in size and waist circumference after SAOs gavage compared to the Model group (fig. 6a), and significantly reduced in weight gain (fig. 6b and 6 c). Therefore, the SAOs can obviously reduce the weight of the T2DM mouse, and has good weight-reducing effect.

Example 10: experiment for improving T2DM mouse non-alcoholic fatty liver disease by SAOs

The effect of SAOs on the alleviation of non-alcoholic fatty liver was further evaluated using the T2DM animal model created in example 8. As shown in fig. 7, the results of oil-red O staining (fig. 7a) and hematoxylin-eosin staining (fig. 7b) showed that the liver cells were uniformly distributed, morphologically well-defined, lipid droplet accumulation and cell degeneration were significantly reduced, and hepatic balloon-like changes and numbers of fatty vesicles were reduced in mice fed with SAOs compared to the Model group. The above results indicate that SAOs can regulate liver lipid metabolism and lipid accumulation of T2DM mice to alleviate non-alcoholic fatty liver disease.

Example 11: experiment for improving hyperlipidemia of T2DM mice by SAOs

The effect of SAOs in ameliorating hyperlipidemia was further evaluated using the T2DM animal model created in example 8. As shown in fig. 8, mice after SAOs feeding had significantly decreased serum triglyceride (fig. 8a), total cholesterol (fig. 8b), and low density lipoprotein levels (fig. 8c), significantly increased high density lipoprotein levels (fig. 8d) and exhibited some dose dependence compared to the Model group. The above results indicate that SAOs can alleviate hyperlipidemia of T2DM mice, thus having a certain protective effect on cardiovascular and cerebrovascular diseases.

The results of the above experiments show that galactooligosaccharides can target and inhibit the activity of SGLT1 and 2 and have certain SGLT2 selectivity. The experimental result of a T2 DM-making mouse model animal fed with high-fat feed shows that the series of galactooligosaccharides can obviously improve the hyperglycemia, the glucose tolerance and the insulin sensitivity of a T2DM mouse. Meanwhile, the health-care food has the efficacy of losing weight, and can remarkably improve non-alcoholic fatty liver, hyperlipidemia and cardiovascular and cerebrovascular diseases caused by high-fat diet.

In conclusion, the oligosaccharide can inhibit SGLT1 and 2 glucose transport activities in a targeted mode, has certain SGLT2 selectivity, has the effects of reducing blood sugar, relieving insulin resistance, resisting type 2 diabetes, resisting metabolic syndrome and hyperlipemia and relieving nonalcoholic fatty liver by inhibiting glucose transport and renal reabsorption, and is suitable for being used as candidate medicines or health products or compound preparations for relieving insulin resistance, preventing and treating fatty liver and reducing blood sugar, preventing and treating type 2 diabetes, preventing and treating metabolic syndrome and treating hyperlipemia. The results of the examples show that the series of galactooligosaccharide compounds have obvious targeted inhibition activity on SGLT1 and SGLT2, and further have obvious effects of relieving insulin resistance, protecting fatty liver and regulating glycolipid metabolism. The galacto-oligosaccharide and the derivative thereof can obviously enhance insulin sensitivity, relieve cell lipid accumulation and increase the metabolism function of cell glycolipid, thereby realizing the treatment effect on insulin resistance, type 2 diabetes, metabolic syndrome, fatty liver and hyperlipidemia. 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 insulin resistance, type 2 diabetes, metabolic syndrome, fatty liver, hyperlipidemia, hypertension 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 as SGLTs inhibitors is characterized in that the oligosaccharide has the following structural general formula:

wherein R is-H or-SO₃Na，n＝0～30；

The galacto-oligosaccharide and the derivatives thereof with the structural characteristics can be used as medicines or health products for preventing and treating diabetes, diabetic nephropathy and cardiovascular diseases by targeted inhibition of SGLT1 and SGLT2 activities.

2. Use of galacto-oligosaccharides and derivatives thereof as SGLTs inhibitors according to claim 1, characterized in that the galacto-oligosaccharides and derivatives thereof can target binding to SGLTs proteins inhibiting their glucose transport activity and regulating glycolipid metabolism.

3. The use of galacto-oligosaccharides and derivatives thereof as SGLTs inhibitors according to claim 1, wherein the galacto-oligosaccharides and derivatives thereof are capable of significantly lowering blood glucose and are used for the preparation of medicaments for lowering blood glucose, alleviating insulin resistance, resisting type 2 diabetes, resisting metabolic syndrome, resisting non-alcoholic fatty liver, resisting hyperlipidemia, protecting liver, and lowering blood lipid.

4. Use of galacto-oligosaccharides and derivatives thereof as SGLTs inhibitors according to claim 1, characterized in that the SGLTs inhibitors are used in drugs or health products for anti-diabetes, anti-diabetic nephropathy, anti-cardiovascular diseases; or in beverages, beer, dietary supplements, or in combination with other antidiabetic agents, or hypolipidemic agents; or in combination with an agent that resists diabetic nephropathy; or a compound preparation containing the galacto-oligosaccharide and the derivative thereof; or in combination with anti-cardiovascular agents; or the derivatives prepared by taking the series of oligosaccharides as parent nucleus are used as preparations for SGLTs antidiabetics, antidiabetic nephropathy and cardiovascular disease resistant medicines or functional preparations.

5. The use of galacto-oligosaccharides and derivatives thereof as SGLTs inhibitors according to claim 1, wherein the galacto-oligosaccharides and derivatives thereof form a complex formulation with metformin, dapagliflozin, canagliflozin or acarbose related clinical hypoglycemic agents, and are used as SGLTs inhibitors in drugs for the prevention and treatment of diabetes, diabetic nephropathy and cardiovascular related diseases.

6. Use of galacto-oligosaccharides and derivatives thereof as SGLTs inhibitors according to one of the claims 1 to 5, characterized in that the galacto-oligosaccharides and derivatives thereof are prepared as follows:

7. The use of galacto-oligosaccharides and derivatives thereof as SGLTs inhibitors according to claim 6, characterized in that the galacto-oligosaccharides and derivatives thereof are prepared by the following specific preparation process: