A chondroitin sulfate oligosaccharide derivative and its preparation method and application

Abstract

The present application belongs to the field of biomedicine technology, and specifically relates to a chondroitin sulfate oligosaccharide derivative and a preparation method and application thereof. The structure of the chondroitin sulfate oligosaccharide derivative is shown in Formula I, #imgabs0#Formula I, where R1 is SO3H, R2 is H or R1 is H, R2 is SO3H, and the polymerization degree of the chondroitin sulfate oligosaccharide derivative is equal to 8; the chondroitin sulfate oligosaccharide derivative has good promotion of chronic hepatitis repair and anti-inflammatory activity, and has a clear structure, and can be used for oral treatment of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and more specifically, to a chondroitin sulfate oligosaccharide derivative and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Glycosaminoglycans are a class of linear anionic polysaccharides composed of repeating disaccharide units. They are commonly found on the cell surface and in the extracellular matrix and play an important role in regulating biological and physiological processes. According to the different disaccharide units and the degree of sulfation, glycosaminoglycans can be divided into heparin (HP), heparan sulfate (HS), chondroitin sulfate (CS), etc. Among them, chondroitin sulfate has anti-inflammatory, antioxidant, anti-thrombotic, anticoagulant and immunomodulatory effects. However, due to the characteristics of chondroitin sulfate such as large molecular weight, high negative charge, hydrophilicity, and instability under acidic conditions, the oral bioavailability of chondroitin sulfate is low, which is not enough to provide the expected clinical treatment effect, and can only be administered parenterally such as intravenous injection. This is acceptable for short-term treatment, but for long-term treatment, patients need to endure pain, easy infection, inconvenience of outpatient treatment, etc., and often need a more convenient and more patient-friendly administration method. However, the inventors found that among many routes of administration, oral administration is the safest, most convenient, most economical, and most easily accepted by patients and clinicians. Therefore, it is of great significance to seek an effective strategy to improve the oral bioavailability of chondroitin sulfate.

[0004] Non-alcoholic fatty liver disease (NAFLD) refers to chronic liver damage characterized by excessive fat deposition in liver cells, excluding alcohol and other clear liver-damaging factors. It is one of the most common chronic liver diseases in the world, with a global prevalence of about 25% of the adult population, and the prevalence is still increasing year by year worldwide. Moreover, NAFLD can further develop into non-alcoholic steatohepatitis (NASH), cirrhosis and even liver cancer. In addition, NAFLD is closely related to metabolic syndrome, which not only affects liver health, but also increases the risk of a series of serious diseases such as type 2 diabetes, hypertension and dyslipidemia. Therefore, it is of great significance to seek an effective drug for the treatment of non-alcoholic fatty liver disease and a route of administration for long-term treatment. Summary of the invention

[0005] The present application is provided to solve the above defects in the prior art. A chondroitin sulfate oligosaccharide derivative and its preparation method and application are needed, a compound capable of improving the oral bioavailability of chondroitin sulfate is provided, and a compound having a therapeutic effect on non-alcoholic fatty liver disease is provided.

[0006] In a first aspect of the present application, a chondroitin sulfate oligosaccharide derivative is provided, wherein the structure of the chondroitin sulfate oligosaccharide derivative is as shown in Formula I,

[0007] Formula I, wherein R is SO3H or H, and the degree of polymerization of the chondroitin sulfate oligosaccharide derivative is equal to 8.

[0008] In a second aspect of the present application, a method for preparing the chondroitin sulfate oligosaccharide derivatives described in any embodiment of the present application is provided, wherein chondroitin sulfate is prepared into low molecular weight chondroitin sulfate by enzymatic hydrolysis; the low molecular weight chondroitin sulfate is separated by a P10 chromatographic column to obtain chondroitin sulfate oligosaccharides; the chondroitin sulfate oligosaccharides are subjected to a desulfurization reaction to remove part of N -6-sulfate group of acetylgalactosamine to obtain a chondroitin sulfate oligosaccharide desulfurization product; the chondroitin sulfate oligosaccharide desulfurization product is carboxyl activated and reacted with deoxycholeethylamine ligand to obtain a chondroitin sulfate oligosaccharide derivative.

[0009] The third aspect of the present application provides the use of the chondroitin sulfate oligosaccharide derivatives described in any embodiment of the present application in the preparation of promoting the repair of chronic hepatitis and / or anti-inflammation.

[0010] The fourth aspect of the present application provides the use of the chondroitin sulfate oligosaccharide derivative described in any embodiment of the present application in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0011] The chondroitin sulfate oligosaccharide derivatives and their preparation methods and applications provided in various embodiments of the present application, the chondroitin sulfate oligosaccharide derivatives involved can effectively promote the oral absorption of chondroitin sulfate oligosaccharides, and greatly improve the oral bioavailability of chondroitin sulfate oligosaccharide derivatives; the chondroitin sulfate oligosaccharide derivatives have good promotion of chronic hepatitis repair and anti-inflammatory activity, and have a clear structure, and can be used for oral treatment of non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar parts. The drawings generally illustrate various embodiments by way of example and not limitation. When appropriate, the same reference numerals are used throughout the drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

[0013] Figure 1 shows the P10 chromatogram of chondroitin sulfate oligosaccharide prepared according to the first embodiment of the present application;

[0014] Figure 2 The HSQC NMR spectrum of chondroitin sulfate oligosaccharide prepared according to the first embodiment of the present application is shown;

[0015] Figure 3 The preparation method of chondroitin sulfate oligosaccharide removal according to the second embodiment of the present application is shown. N -HSQC NMR spectrum of the 6-sulfate group of acetylgalactosamine;

[0016] Figure 4 shows a mass spectrum of deoxycholeethylamine prepared according to the third embodiment of the present application;

[0017] Figure 5 The chondroitin sulfate oligosaccharide derivative prepared according to the third embodiment of the present application is shown. 1 H NMR spectrum;

[0018] Figure 6 An Oil Red O dyeing diagram according to the fourth embodiment of the present application is shown. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0020] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements.

[0021] According to an embodiment of the present application, a chondroitin sulfate oligosaccharide derivative is provided, characterized in that the structure of the chondroitin sulfate oligosaccharide derivative is as shown in Formula I,

[0022] Formula I, wherein R is SO3H or H, and the degree of polymerization of the chondroitin sulfate oligosaccharide derivative is equal to 8.

[0023] The main structure of the non-reducing end of the chondroitin sulfate oligosaccharide derivative is glucuronic acid, and the reducing end is N -Acetylgalactosamine. Part N The sulfate group at position 6 of -acetylgalactosamine was removed by chemical reaction. The carboxyl groups of some uronic acids were coupled with deoxycholic acid by chemical derivatization.

[0024] The chondroitin sulfate oligosaccharide derivative not only has the advantage of small molecular weight of chondroitin sulfate oligosaccharide, but also inherits the anti-inflammatory effect of chondroitin sulfate. The derivative can be used to promote the repair of chronic hepatitis and anti-inflammatory activity, and has a clear structure and can be used for oral treatment of non-alcoholic fatty liver disease.

[0025] According to an embodiment of the present application, a chondroitin sulfate oligosaccharide is further provided, and the structure of the chondroitin sulfate oligosaccharide is shown in Formula II:

[0026]

[0027] Formula II, wherein R1 is SO3H, R2 is H or R1 is H, R2 is SO3H, and the polymerization degree of the chondroitin sulfate oligosaccharide is equal to 8.

[0028] The chondroitin sulfate oligosaccharide is a linear polysaccharide formed by connecting disaccharide units. Compared with chondroitin sulfate with a larger molecular weight, it can effectively promote the oral absorption effect and greatly improve the oral bioavailability of chondroitin sulfate oligosaccharide. For other utilization methods such as intravenous injection, it can also have a good absorption effect. The NHAc in the chondroitin sulfate oligosaccharide is an acetamide group. In this application, chondroitin sulfate oligosaccharide is also called chondroitin sulfate octose.

[0029] According to an embodiment of the present application, a method for preparing the chondroitin sulfate oligosaccharide described in any embodiment of the present application is also provided, the preparation method comprising: preparing low molecular weight chondroitin sulfate by enzymatic hydrolysis of chondroitin sulfate; and separating the low molecular weight chondroitin sulfate by a P10 chromatographic column to obtain chondroitin sulfate oligosaccharide.

[0030] According to the embodiments of the present application, a method for preparing the chondroitin sulfate oligosaccharide derivative described in any embodiment of the present application is also provided, wherein chondroitin sulfate is prepared into low molecular weight chondroitin sulfate by enzymatic hydrolysis; the low molecular weight chondroitin sulfate is separated by a P10 chromatographic column to obtain chondroitin sulfate oligosaccharides; the chondroitin sulfate oligosaccharides undergo a desulfurization reaction to remove the 6-position sulfate group to obtain a chondroitin sulfate oligosaccharide desulfurization product; the chondroitin sulfate oligosaccharide desulfurization product is carboxyl activated and reacted with a deoxycholeethylamine ligand to obtain a chondroitin sulfate oligosaccharide derivative.

[0031] In some embodiments, chondroitin sulfate is prepared by enzymatic hydrolysis to obtain low molecular weight chondroitin sulfate, including: dissolving chondroitin sulfate in a partial enzymatic hydrolysis buffer, adding chondroitin sulfate ABC enzyme, and performing an enzymatic hydrolysis reaction; after the reaction, inactivating the chondroitin sulfate ABC enzyme in the mixed solution; centrifuging the inactivated mixed solution, taking the supernatant, and freeze-drying to obtain low molecular weight osteoin sulfate; separating the low molecular weight osteoin sulfate through a P10 chromatographic column to obtain chondroitin sulfate oligosaccharides.

[0032] In some embodiments, the partial enzymatic hydrolysis buffer comprises sodium acetate, calcium acetate and water. The partial enzymatic hydrolysis buffer is prepared from sodium acetate solution and calcium acetate solution, the concentration of sodium acetate solution is 0.08-0.12 mol / L, the concentration of calcium acetate solution is 0.008-0.012 mol / L, and the volume ratio of sodium acetate solution to calcium acetate solution is 20:2-4. In some embodiments, acetic acid is used to adjust the sodium acetate solution to neutrality.

[0033] In some embodiments, the mass volume ratio of chondroitin sulfate to partial enzymatic hydrolysis buffer is 1 g:28-32 mL.

[0034] In some embodiments, the amount of chondroitin sulfate ABC enzyme corresponding to 1 g of chondroitin sulfate is 0.4-0.6 IU.

[0035] In some embodiments, the incubation reaction is performed at a temperature of 30-45° C. for a period of 6-10 h.

[0036] In some embodiments, the mobile phase for separation with a P10 column is a 0.2 M aqueous solution of ammonium bicarbonate.

[0037] In some embodiments, the P10 chromatographic column is also pretreated, specifically comprising: soaking the filler of the P10 chromatographic column with 20% ethanol, and then replacing the 20% ethanol with high-purity water; compacting the filler with water, and then compacting the filler with 0.2 M ammonium bicarbonate mobile phase to obtain a pretreated P10 chromatographic column.

[0038] The preparation method of chondroitin sulfate oligosaccharides of the present application is conducive to the enzymatic hydrolysis of chondroitin sulfate and obtaining chondroitin sulfate oligosaccharides with activity and high bioavailability.

[0039] In some embodiments, the chondroitin sulfate oligosaccharide undergoes a desulfurization reaction to remove part of N -6-sulfate group of acetylgalactosamine to obtain a chondroitin sulfate oligosaccharide desulfurization product, including: chondroitin sulfate oligosaccharide and pyridine to obtain chondroitin sulfate oligosaccharide pyridine salt; chondroitin sulfate oligosaccharide pyridine salt and N -methyl- N -Trimethylsilyl trifluoroacetamide reaction to obtain chondroitin sulfate oligosaccharide desulfurization product.

[0040] In some embodiments, the reaction of chondroitin sulfate oligosaccharide and pyridine to obtain chondroitin sulfate oligosaccharide pyridine salt comprises: the aqueous solution of the chondroitin sulfate oligosaccharide is subjected to H + After the ion exchange column, an eluate is obtained, and the eluate is mixed with pyridine to react to obtain chondroitin sulfate oligosaccharide pyridinium salt. The structural formula of chondroitin sulfate oligosaccharide pyridinium salt is shown in Formula III.

[0041]

[0042] Formula III, wherein R1 is SO3H, R2 is H or R1 is H, R2 is SO3H; R3 is pyridine.

[0043] In some embodiments, the concentration of the aqueous solution of chondroitin sulfate oligosaccharide is 0.2-0.3 g / mL.

[0044] In some embodiments, the solution after the eluent and pyridine are mixed is neutral. In some embodiments, the temperature for the reaction of the eluent and pyridine is room temperature and the time is 0.5-2 h.

[0045] In some embodiments, chondroitin sulfate oligosaccharide pyridinium salt and N -methyl- N -trimethylsilyl trifluoroacetamide reaction to obtain chondroitin sulfate oligosaccharide desulfurization products, including: chondroitin sulfate oligosaccharide pyridinium salt and N -methyl- N -trimethylsilyl trifluoroacetamide is mixed in anhydrous pyridine and heated for reaction to obtain a chondroitin sulfate oligosaccharide desulfurization product.

[0046] Chondroitin sulfate oligosaccharide pyridinium salt and N -methyl- N -trimethylsilyl trifluoroacetamide reaction, so that the 6-sulfate group in the pyridinium salt of chondroitin sulfate oligosaccharide is removed. The desulfurization product of chondroitin sulfate oligosaccharide is shown in Formula IV,

[0047] Formula IV, wherein R in Formula IV is SO3H or H;

[0048] Wherein, R is SO3 - or H.

[0049] In some embodiments, chondroitin sulfate oligosaccharide pyridinium salt and N -methyl- N -trimethylsilyl trifluoroacetamide in anhydrous pyridine has a mass volume ratio of 1 g:1 mL:5-7 mL. In some embodiments, chondroitin sulfate oligosaccharide pyridinium salt and N -methyl- N-trimethylsilyl trifluoroacetamide is mixed in anhydrous pyridine, and the temperature of the heating reaction is 90-110°C, and the reaction time is 10-15 hours. In some embodiments, the reaction is diluted with water, and the diluted solution is dialyzed and freeze-dried to obtain a chondroitin sulfate oligosaccharide desulfurization product. Further, the dialysis time is 20-26 hours.

[0050] In some embodiments, the desulfurized product of chondroitin sulfate oligosaccharide is carboxyl activated and reacted with a deoxycholethamine ligand, including: the desulfurized product of chondroitin sulfate oligosaccharide reacts with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to obtain a desulfurized product of chondroitin sulfate oligosaccharide having an active carboxylic acid; the chondroitin sulfate oligosaccharide having an active carboxylic acid reacts with deoxycholethamine at room temperature to obtain a chondroitin sulfate oligosaccharide derivative.

[0051] In some embodiments, the mass ratio of the chondroitin sulfate oligosaccharide desulfurization product to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is 100:10-15. In some embodiments, the chondroitin sulfate oligosaccharide desulfurization product and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride are dissolved in formamide for reaction, and the mass volume ratio of the chondroitin sulfate octasaccharide desulfurization product to formamide is: 100:1.5-2.5 mL.

[0052] In some embodiments, the reaction time of chondroitin sulfate oligosaccharide with active carboxylic acid and deoxycholethamine at room temperature is 10-15 hours. The product is precipitated in acetone and then freeze-dried to obtain chondroitin sulfate oligosaccharide derivatives.

[0053] In some embodiments, a method for preparing deoxycholethamine is also included, and the preparation method comprises: activating the carboxyl group of deoxycholic acid; reacting the activated deoxycholic acid with ethylenediamine to obtain deoxycholethamine.

[0054] In some embodiments, deoxycholic acid carboxyl activation comprises: deoxycholic acid, dicyclohexylcarbodiimide and N -hydroxysuccinimide in dimethylformamide to obtain activated deoxycholic acid. Further, deoxycholic acid, dicyclohexylcarbodiimide, N -hydroxysuccinimide and dimethylformamide mass volume ratio is 190-200 mg:160-170 mg:90-95 mg:13-17 mL. Further, deoxycholic acid, dicyclohexylcarbodiimide and N The temperature of mixing the -hydroxysuccinimide in dimethylformamide is room temperature, and the reaction time is 3-7 h. In some embodiments, water is added to the mixture after the reaction to precipitate unreacted dicyclohexylcarbodiimide, which is filtered out. The resulting solution is added to water to precipitate activated deoxycholic acid.

[0055] In some embodiments, the molar ratio of activated deoxycholic acid to ethylenediamine is 2-4:1; in some embodiments, the temperature for the reaction of activated deoxycholic acid and ethylenediamine is room temperature, and the reaction time is 3-7 h.

[0056] According to the embodiments of the present application, there is also provided the use of the chondroitin sulfate oligosaccharide described in any embodiment of the present application in the preparation of anti-inflammatory, antioxidant, antithrombotic, anticoagulant and immunomodulatory drugs.

[0057] According to the embodiments of the present application, there is also provided the use of the chondroitin sulfate oligosaccharide derivative described in any embodiment of the present application in the preparation of drugs for promoting the repair of chronic hepatitis and / or anti-inflammatory drugs.

[0058] According to the embodiments of the present application, there is also provided the use of the chondroitin sulfate oligosaccharide derivative described in any embodiment of the present application in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0059] According to an embodiment of the present application, a drug is also provided, which includes the chondroitin sulfate oligosaccharide described in any embodiment of the present application and / or the chondroitin sulfate oligosaccharide derivative described in any embodiment of the present application.

[0060] The drug also includes pharmaceutically necessary carriers, and of course, the pharmaceutically acceptable carrier dosage should be harmless to the subject, specifically including but not limited to buffers, antioxidants, preservatives, bactericides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, surfactants, salt-forming counterions and metal complexes. Moreover, according to the usual method, it can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0061] First Example: Preparation of chondroitin sulfate octasaccharide by enzymatic hydrolysis.

[0062] 1. Weigh 1 g of chondroitin sulfate, dissolve it in 30 mL of partial enzymatic hydrolysis buffer, add 0.5 IU of chondroitin sulfate ABC enzyme, and incubate at 37℃ for 8 h. After the enzymatic hydrolysis is completed, boil it in a 100℃ water bath to inactivate the chondroitin sulfate ABC enzyme, centrifuge it at 12000rpm for 10 min, take the supernatant, and lyophilize it to obtain low molecular weight chondroitin sulfate.

[0063] 2. Preparation of partial enzymatic buffer: 0.1 mol / L sodium acetate (adjust pH to 7.0 with acetic acid): 0.01 mol / L calcium acetate = 20:3 (V / V). Specific preparation method: 0.1 mol / L sodium acetate: weigh 0.1641 g sodium acetate and dissolve it in 20 mL water, and adjust pH to 7.0 with acetic acid; 0.01 mol / L calcium acetate: weigh 22.89 mg calcium acetate and dissolve it in 12.99 mL water.

[0064] 3. P10 column separation and characterization of chondroitin sulfate octasaccharide: (1) Mobile phase configuration: 0.2 M ammonium bicarbonate aqueous solution, ultrasonic dissolution, filtration to remove impurities, and ultrasonic removal of bubbles. (2) P10 column filling: Add filler to a beaker, soak it with 20% ethanol, and then replace the 20% ethanol with high-purity water. Filter the filler to remove bubbles and pour it into the column. First compact the filler with water, then compact the filler with 0.2 M ammonium bicarbonate mobile phase, and then load the sample. (3) Dissolve 100 mg of the obtained low molecular weight chondroitin sulfate in water and separate it by high performance liquid chromatography. The data spectrum is shown in the following figure. Figure 1 shown.

[0065] 4. NMR spectroscopy characterization of chondroitin sulfate octasaccharide: 30 mg of the obtained chondroitin sulfate octasaccharide was dissolved in 180 μL of deuterated water and characterized by HSQC NMR spectroscopy. The data spectrum is shown in Figure 2 shown.

[0066] Second Example: Preparation of chondroitin sulfate octasaccharide desulfurization product.

[0067] 1. Preparation of chondroitin sulfate octasaccharide pyridinium salt: Dissolve 5 g chondroitin sulfate octasaccharide in 20 mL deionized water and purify by HO. + Ion exchange column. Pyridine was added to the eluent until the mixture was neutralized to pH = 7. After stirring at room temperature for 1 h, the mixture was concentrated in vacuo and freeze-dried to obtain chondroitin sulfate octasaccharide pyridinium salt.

[0068] 2. Weigh 1 g of chondroitin sulfate octasaccharide pyridinium salt and dissolve it in 6 mL of anhydrous pyridine. Add 1 mL N -methyl- N -trimethylsilyl trifluoroacetamide (MTSTFA). After stirring at 100°C for 12 h, the solution was diluted with 5 mL of pure water. The solution was dialyzed in water for 24 h and freeze-dried to obtain the chondroitin sulfate octasaccharide desulfurization product.

[0069] 3. Characterization of chondroitin sulfate octasaccharide desulfurization products. 15 mg of the obtained chondroitin sulfate octasaccharide desulfurization product was dissolved in deuterium oxide and lyophilized three times to replace the exchangeable protons. The lyophilized sample was dissolved in 200 μL deuterium oxide, vortexed, centrifuged, and the supernatant was analyzed using NMR HSQC spectra. The results are shown in Figure 3 shown.

[0070] The third example: Preparation of chondroitin sulfate octasaccharide derivatives.

[0071] 1. Activation of deoxycholic acid carboxyl group: Deoxycholic acid (196 mg), dicyclohexylcarbodiimide (165 mg) and N -Hydroxysuccinimide (92 mg) was dissolved in 15 mL of dimethylformamide in a molar ratio of 1:1.6:1.6. The reaction was carried out under vacuum at room temperature for 5 h. 1 mL of distilled water was added to precipitate the unreacted dicyclohexylcarbodiimide and filtered off. The resulting solution was added to 15 mL of distilled water to precipitate the activated deoxycholic acid.

[0072] 2. Ligand preparation: The activated deoxycholic acid was mixed with ethylenediamine (molar ratio 3:1) in dimethylformamide and reacted at room temperature for 5 h to generate deoxycholeethylamine.

[0073] 3. Activation of chondroitin sulfate carboxyl group: Dissolve the above chondroitin sulfate octasaccharide desulfurization product (100 mg) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (11.49 mg) in 2 mL formamide to activate the carboxyl group of chondroitin sulfate octasaccharide.

[0074] 4. Preparation of derivatives: Deoxycholeethylamine and the active carboxylic acid of chondroitin sulfate octasaccharide are reacted at 25° C. for 12 h, and the product is precipitated in acetone and freeze-dried to obtain the low molecular weight chondroitin sulfate derivative.

[0075] 5. Characterization of the ligand deoxycholethiazine. 15 mg of the obtained ligand deoxycholethiazine was dissolved in 180 µL DMSO-d6, vortexed, centrifuged, and the supernatant was taken for characterization using mass spectrometry. The results are shown in Figure 4 shown.

[0076] 6. Characterization of chondroitin sulfate octasaccharide derivatives. 15 mg of the obtained chondroitin sulfate octasaccharide derivative was dissolved in 180 μL of a mixed solution of DMSO-d6 and deuterium oxide (v:v=2:1), vortexed, centrifuged, and the supernatant was collected. 1 The results are shown in Figure 2. Figure 5 shown.

[0077] Fourth embodiment: the chondroitin sulfate octasaccharide derivative prepared in the third embodiment is orally taken to treat non-alcoholic fatty liver disease.

[0078] Materials: BALB / c mice.

[0079] 1. Experimental methods

[0080] 1.1 Experimental grouping: The mice were randomly divided into a normal group, a model group, and an experimental group. The experimental groups included the CS-dp8-DOCA group and the atorvastatin group.

[0081] 1.2 Modeling method: The model group and the experimental group were fed with 60% high-fat diet (HFD) for 8 weeks to establish the non-alcoholic fatty liver model in mice; the normal group was fed with ordinary diet.

[0082] 1.3 Administration method:

[0083] The atorvastatin group of the experimental group was subcutaneously injected with atorvastatin (10 mg / kg). The CS-dp8-DOCA group of the experimental group was gavaged with chondroitin sulfate oligosaccharide derivatives (30 mg / kg). The normal group and the model group were subcutaneously injected with an equal amount of normal saline. The drug was administered once every 24 h, and the mice continued to be fed with a 60% high-fat diet (HFD). After 6 weeks of administration, the heart was perfused and the samples were dissected. The liver lipid content of the normal group, model group, atorvastatin group and CS-dp8-DOCA group was measured: the accumulation of lipid droplets in the liver was evaluated by histological methods such as Oil Red O staining.

[0084] 2. Experimental results

[0085] Figure 6 The Oil Red O staining results of four groups are normal group, model group, atorvastatin group, and CS-dp8-DOCA group. It can be seen from the Oil Red O staining results that the present application successfully established a non-alcoholic fatty liver model in mice by feeding with a 60% high-fat diet (HFD), and the liver lipid content in the model group was significantly higher than that in the normal group. Atorvastatin group. The chondroitin sulfate octasaccharide derivative provided by the present application can significantly reduce the liver lipid content of mice by administration through the digestive system, achieving a better therapeutic effect, which is close to the therapeutic effect of atorvastatin. The above results show that the chondroitin sulfate octasaccharide derivative has a significant preventive and therapeutic effect on non-alcoholic fatty liver disease.

[0086] In addition, although exemplary embodiments have been described herein, their scope includes any and all embodiments based on the present application with equivalent elements, modifications, omissions, combinations (e.g., various embodiments intersecting schemes), adaptations or changes. Therefore, this specification and examples are intended to be considered as examples only.

[0087] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more of them) may be used in combination with each other. For example, a person of ordinary skill in the art may use other embodiments when reading the above description. In addition, in the above specific embodiments, various features may be grouped together to simplify the present application.

[0088] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A method for preparing a chondroitin sulfate oligosaccharide derivative, characterized in that: The chondroitin sulfate is prepared into low molecular weight chondroitin sulfate by enzymatic hydrolysis; the low molecular weight chondroitin sulfate is separated by a P10 chromatographic column to obtain chondroitin sulfate oligosaccharide, wherein the chondroitin sulfate oligosaccharide is 8 sugars; the chondroitin sulfate oligosaccharide undergoes a desulfurization reaction to remove part of N -6-sulfate group of acetylgalactosamine to obtain a chondroitin sulfate oligosaccharide desulfurization product; the chondroitin sulfate oligosaccharide desulfurization product is carboxyl activated and reacted with deoxycholeethylamine ligand to obtain a chondroitin sulfate oligosaccharide derivative.

2. The preparation method according to claim 1, characterized in that: Chondroitin sulfate is prepared into low molecular weight chondroitin sulfate by enzymatic hydrolysis, including: dissolving chondroitin sulfate in a partial enzymatic hydrolysis buffer, adding chondroitin sulfate ABC enzyme, and performing enzymatic hydrolysis reaction; after the reaction, inactivating the chondroitin sulfate ABC enzyme in the mixed solution; centrifuging the inactivated mixed solution, and then taking the supernatant and freeze-drying it to obtain low molecular weight chondroitin sulfate.

3. The preparation method according to claim 2, characterized in that: The partial enzymatic hydrolysis buffer comprises sodium acetate, calcium acetate and water; the mass volume ratio of chondroitin sulfate to the partial enzymatic hydrolysis buffer is 1 g:28-32 mL.

4. The preparation method according to claim 2, characterized in that: The amount of chondroitin sulfate ABC enzyme corresponding to 1 g of chondroitin sulfate is 0.4-0.6 IU.

5. The preparation method according to claim 1, characterized in that: The chondroitin sulfate oligosaccharide undergoes a desulfurization reaction to remove part of N -6-sulfate group of acetylgalactosamine to obtain a desulfurized product of chondroitin sulfate oligosaccharide, comprising: subjecting an aqueous solution of the chondroitin sulfate oligosaccharide to H + After the ion exchange column, an eluent is obtained, and the eluent is mixed with pyridine to react to obtain chondroitin sulfate oligosaccharide pyridine salt; chondroitin sulfate oligosaccharide pyridine salt and N -methyl- N -Trimethylsilyl trifluoroacetamide reaction to obtain chondroitin sulfate oligosaccharide desulfurization product.

6. The preparation method according to claim 1, characterized in that: The desulfurized product of chondroitin sulfate oligosaccharide is carboxyl activated and reacted with a deoxycholethamine ligand, including: the desulfurized product of chondroitin sulfate oligosaccharide reacts with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a carboxyl-activated chondroitin sulfate oligosaccharide with active carboxylic acid; the chondroitin sulfate oligosaccharide with active carboxylic acid reacts with deoxycholethamine at room temperature to obtain a chondroitin sulfate oligosaccharide derivative.

7. A chondroitin sulfate oligosaccharide derivative prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the chondroitin sulfate oligosaccharide derivative according to claim 7 in the preparation of a medicament for treating non-alcoholic fatty liver disease.

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