A heparin oligosaccharide, heparin oligosaccharide composition, and methods of making and using the same

Abstract

The application discloses heparin oligosaccharide, a heparin oligosaccharide composition, a preparation method and application, and belongs to the technical field of biological medicines. The structure of the heparin oligosaccharide is as follows: heparin is enzymatically hydrolyzed into a low-molecular-weight heparin mixture; the low-molecular-weight heparin mixture is separated by a P10 chromatogram to obtain heparin decasaccharide; the heparin decasaccharide is separated by a strong anion exchange column to obtain salt-containing heparin oligosaccharide; and the salt-containing heparin oligosaccharide is desalted by a gel chromatogram column to obtain heparin oligosaccharide. The heparin oligosaccharide prepared by the application is beneficial to realizing quality control of a drug, improving the safety of the drug, and the heparin oligosaccharide is combined with SNAC, effectively promotes oral absorption of the heparin oligosaccharide, significantly improves the oral bioavailability of the heparin decasaccharide, has good anti-liver injury activity, and can be used for oral treatment of drug-induced acute liver injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a heparin oligosaccharide, a heparin oligosaccharide composition, and a preparation method and application. BACKGROUND

[0002] Drug-induced acute liver injury is a liver toxicity reaction caused by prescription drugs, herbal medicines or dietary supplements, accounting for 10-20% of acute liver failure cases. The global incidence is about 1-20 / 100,000. Common pathogenic drugs include antibiotics (such as isoniazid), non-steroidal anti-inflammatory drugs (such as acetaminophen), antitumor drugs and traditional herbal medicines (such as how to use the root of wu). The pathogenesis involves direct hepatotoxicity or idiosyncratic reactions, and the clinical manifestations are jaundice, fatigue, nausea, etc., and severe cases can progress to liver failure. Immediate withdrawal of suspected drugs, 90% of mild patients can recover spontaneously, and in clinical practice, N-acetylcysteine is the specific antidote for acetaminophen poisoning, and severe patients require artificial liver support (such as plasma exchange) or liver transplantation, and some liver-protecting drugs (such as ursodeoxycholic acid, silymarin) can also be used to assist in improving liver biochemical indicators. However, long-term use can reduce the effectiveness of drugs and produce side effects and new hepatotoxicity. Therefore, it is urgent to develop more effective liver protection and low-toxicity treatment methods.

[0003] In recent years, glycosaminoglycans have attracted widespread attention from academia and the pharmaceutical industry. Glycosaminoglycans are a class of linear anionic polysaccharides composed of repeating disaccharide units, which are widely present on the cell surface and in the extracellular matrix, and play an important role in regulating biological physiological processes. Due to its negative charge, glycosaminoglycans can interact with various proteins and exert a wide range of biological activities, including anti-inflammatory, anti-tumor and anti-viral effects. According to the composition of the disaccharide unit and the degree of sulfation, glycosaminoglycans can be divided into heparin, heparan sulfate and chondroitin sulfate. However, due to the characteristics of glycosaminoglycans such as large molecular weight, high negative charge, hydrophilicity and instability in acidic conditions, the oral bioavailability of glycosaminoglycans is low, which is insufficient to provide the expected clinical therapeutic effect. Therefore, it is of great significance to seek an effective strategy to improve the oral bioavailability of glycosaminoglycans. SUMMARY

[0004] In view of the problems in the prior art, the present application provides a heparin oligosaccharide, a heparin oligosaccharide composition, and a preparation method and application. The method of the present application has a simple process, a clear compound structure, and realizes the controllable preparation of oral heparin oligosaccharides with anti-liver injury activity, and can be applied to the field of liver disease treatment.

[0005] The present application is realized by the following technical solutions:

[0006] In a first aspect of the present application, a heparin oligosaccharide is provided, and the structure of the heparin oligosaccharide is shown in the following formula (I):

[0007] The heparin oligosaccharide shown in formula (I) is a linear polysaccharide connected by repeating disaccharide units of uronic acid and glucosamine, the degree of polymerization is 10, and the domain contains 4 consecutive [GlcNS6S-IdoA2S] disaccharide units.

[0008] In the second aspect of the present application, a preparation method of the heparin oligosaccharide is provided, which specifically comprises the following steps:

[0009] (1) enzymatically hydrolyzing heparin into a low molecular weight heparin mixture;

[0010] (2) separating the low molecular weight heparin mixture by P10 chromatography to obtain heparin decasaccharide;

[0011] (3) separating the heparin decasaccharide prepared in step (2) by a strong anion exchange column to obtain a salt-containing heparin oligosaccharide;

[0012] (4) desalting the salt-containing heparin oligosaccharide in step (3) by a gel chromatography column to obtain the heparin oligosaccharide with the structure of formula (I).

[0013] Further, the specific operation of step (1) is as follows: heparin is dissolved in a partial enzymatic hydrolysis buffer, heparinase I is added, and the mixture is incubated at 25-37℃ for 4-8 h for enzymatic hydrolysis; after the enzymatic hydrolysis is completed, the reaction system is inactivated at high temperature, and the supernatant is obtained after centrifugation to obtain the low molecular weight heparin mixture.

[0014] Further, the partial enzymatic hydrolysis buffer is a mixture of sodium acetate solution and calcium acetate solution, the molar concentrations of the sodium acetate solution and the calcium acetate solution are 0.05-0.15 mol / L and 0.005-0.015 mol / L respectively, and the volume ratio of the sodium acetate solution to the calcium acetate solution is 18-22:2-4; the dosage ratio of the heparin, the partial enzymatic hydrolysis buffer and heparinase I is 1 g:25-30 mL:0.5-0.75 IU; the high-temperature inactivation condition is heating at 100℃ in a water bath for 10 min; and the centrifugation condition is centrifugation at 12000 rpm for 10-15 min.

[0015] Further, in step (2), the P10 chromatography separation condition is as follows: the chromatography column is a P10 chromatography column, the mobile phase is 0.2 mol / L ammonium bicarbonate aqueous solution, the flow rate is 0.5 mL / min, and the retention time of chromatography separation is 410-450 min.

[0016] Further, in step (3), the strong anion exchange column is Thermo CarboPac PA1 SAX column, the mobile phase A is 0.2 mol / L sodium chloride aqueous solution, the mobile phase B is 2.0-3.0 mol / L sodium chloride aqueous solution, and the flow rate is 1 mL / min; gradient elution is performed, specifically: 0-5 min 90% mobile phase A, 5-170 min 90%-0% mobile phase A, 170-190 min 90% mobile phase A; the retention time of chromatographic separation is 95-110 min.

[0017] Further, in step (4), the desalting parameters of the gel chromatography column are as follows: the chromatography column is a G10 chromatography column, the mobile phase is water, the flow rate is 1 mL / min, and the retention time of chromatographic separation is 9.5-13.5 min.

[0018] In a third aspect, the present application discloses the heparin oligosaccharide composition, wherein the heparin oligosaccharide composition contains an active dose of heparin oligosaccharide.

[0019] The "active dose" can be a drug dose for the purpose of prevention, improvement, treatment or adjuvant treatment according to the general understanding in the art, and the dose belongs to the technical content that can be conventionally inferred by the general technology mastered by the skilled person in the art after the therapeutic activity of the above-mentioned components is determined.

[0020] Further, the heparin oligosaccharide composition contains the active dose of heparin oligosaccharide and further contains other auxiliary ingredients, and the other auxiliary ingredients can be any one or a combination of at least two of an absorption promoter, a carrier, a diluent, a binder, a wetting agent, a disintegrating agent, an emulsifying agent, a solubilizing agent, a permeation pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

[0021] Further, the other auxiliary ingredient is an absorption promoter, and the absorption promoter is N sodium (8-[2-hydroxybenzoyl]-amino)octanoate (SNAC), and the mass ratio of heparin oligosaccharide to SNAC is 3:1.

[0022] In a fourth aspect, the present application discloses the use of the heparin oligosaccharide or the heparin oligosaccharide composition in the preparation of a drug for treating acute liver injury.

[0023] The existing research shows that low molecular weight glycosaminoglycans can play an effective therapeutic effect on the disease model of paracetamol-induced acute liver injury. The low molecular weight glycosaminoglycans are a very complex mixture of homogeneous structure, and the present application provides a specific structure of heparin oligosaccharide. From the perspective of drug preparation development, the research results of the present application are more conducive to realizing the quality control of the drug, and the person skilled in the art can obtain the corresponding compound based on the preparation method provided in the present application, or can be prepared by chemical synthesis. On the other hand, drug-induced liver injury is an acute disease that requires a large amount of liver-protecting drugs, and the subsequent rehabilitation still needs to take drugs, so a safe oral preparation is a more suitable preparation form for patients. However, heparin is a macromolecular substance, and the bioavailability is low. In order to improve the oral bioavailability of the above-mentioned compound, the present application extracts heparin oligosaccharide (HP-dp10-1) from heparin, and further designs and selects components that can promote the absorption of HP-dp10-1 for combined administration. According to the research results of the present application, the drug combination of HP-dp10-1 and SNAC can effectively reduce the expression level of HMGB1 inflammatory factor in the tissue of the drug-induced acute liver injury disease model mouse by oral intake, and improve the liver injury symptoms.

[0024] According to the form of the drug applied to the affected area, the dosage form of the drug-induced acute liver injury treatment drug includes but is not limited to muscle injection, subcutaneous injection, intravenous injection, oral preparation or interventional preparation taken by surgical means, etc. In one embodiment verified by the present application, the above-mentioned drug composition adopts an oral form, specifically, an oral tablet.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] (1) The present application obtains a specific structure of heparin oligosaccharide (HP-dp10-1) by enzymolysis and separation of heparin, which is conducive to realizing the quality control of the drug and improving the safety of the drug;

[0027] (2) The heparin oligosaccharide provided in the present application is combined with SNAC, which effectively promotes the oral absorption of heparin oligosaccharide, significantly improves the oral bioavailability of heparin oligosaccharide, has good anti-liver injury activity, and can be used for oral treatment of drug-induced acute liver injury. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is the chromatogram in the chromatographic separation process of heparin oligosaccharide in Example 1;

[0029] Figure 2 It is the chromatogram in the separation process of salt-containing heparin oligosaccharide in Example 1;

[0030] Figure 3Cluster sequencing sequence analysis chart of heparin oligosaccharide prepared in Example 1; (a) is the information of complete enzymatic disaccharide unit composition, (b) is the information of nitrous acid degradation disaccharide unit composition;

[0031] Figure 4 Blood concentration curve of subcutaneous injection of HP-dp10-1 and oral administration of composition in Example 4;

[0032] Figure 5 HMGB1 immunohistochemical staining chart and statistical analysis chart of average optical density of Example 4; wherein, (A) is the normal group, (B) is the model group, (C) is the administration group 1, (D) is the administration group 2, (E) is the administration group 3, and (F) is the HMGB1 positive area chart. DETAILED DESCRIPTION

[0033] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples without specific conditions are generally carried out according to the conventional conditions or according to the conditions recommended by the manufacturers.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or materials used in the application can be purchased through conventional routes. Unless otherwise specified, the reagents or materials used in the application are used according to the conventional methods in the art or according to the product instructions. In addition, any method and material similar or equivalent to those described can be applied to the method of the application. The preferred implementation methods and materials described in the application are only for demonstration. The application will be further described according to the drawings and specific examples.

[0035] Example 1

[0036] In this embodiment, a preparation method of heparin oligosaccharide is provided, comprising the following steps:

[0037] (1) Enzymatic preparation of low molecular weight heparin mixture

[0038] 1 g of heparin was weighed and dissolved in 30 mL of partial enzymatic buffer solution, 0.5 IU of heparase I was added, and incubated at 37℃ for 6 h. After the enzymatic reaction was completed, the heparase I was inactivated by heating in a 100℃ water bath for 10 min. The supernatant was obtained after high-speed centrifugation at 12000 rpm for 10 min, and was freeze-dried to obtain a low molecular weight heparin mixture;

[0039] The above partial enzymatic buffer solution was prepared by mixing 0.1 mol / L sodium acetate solution (pH adjusted to 7.0 with acetic acid) and 0.01 mol / L calcium acetate solution at a volume ratio of 20:3.

[0040] (2) P10 chromatographic separation

[0041] Mobile phase: 0.2 mol / L ammonium bicarbonate aqueous solution, mixed and ultrasonic degassing;

[0042] Chromatographic column: P10 filler self-filling; the filling method is as follows: a beaker is added with the filler, which is soaked and cleaned with 20% ethanol, and then the 20% ethanol is replaced with high-purity water and vacuum degassed, and then the wall is poured into the empty column skin to compact the filler with the mobile phase, and then the sample is loaded;

[0043] Flow rate of mobile phase: 0.5 mL / min;

[0044] Chromatographic separation: 200 mg of the low molecular weight heparin mixture obtained in step (1) is dissolved in 1 mL of water, and after loading, separation (separation, collection of components with a retention time of 410-450 min) is performed to obtain heparin decasaccharide, and then freeze-drying treatment is performed, and the chromatogram in the P10 chromatographic separation process is as shown in Figure 1 .

[0045] (3) Strong anion exchange column separation

[0046] Mobile phase A: 0.2 mol / L sodium chloride aqueous solution, mixed and ultrasonic degassing;

[0047] Mobile phase B: 2.5 mol / L sodium chloride aqueous solution, mixed and ultrasonic degassing;

[0048] Chromatographic column: Thermo CarboPac PA1 SAX;

[0049] Chromatographic separation: 1 mg of heparin decasaccharide obtained in step (2) is dissolved in 100 μL of water, and separation is performed in a strong anion exchange column with gradient elution: 0~5 min 90% mobile phase A, 5~170 min 90%~0% mobile phase A, 170~190 min 90% mobile phase A; the flow rate is 1 mL / min, and the retention time of chromatographic separation is 95-110 min) to obtain salt-containing heparin oligosaccharide, and the chromatogram in the separation process is as shown in Figure 2 .

[0050] (4) Desalination treatment of gel chromatographic column

[0051] Mobile phase: high-purity water solution, ultrasonic degassing;

[0052] Chromatographic column: G10 chromatographic column;

[0053] Flow rate: 1 mL / min;

[0054] Chromatographic separation: the salt-containing heparin oligosaccharide obtained in step (3) was desalted by G10 chromatography, and the components with retention time of 9.5-13.5 min were collected, followed by lyophilization treatment to obtain heparin oligosaccharide (HP-dp10-1).

[0055] (5) Characterization

[0056] The above HP-dp10-1 was subjected to sequence analysis using cluster sequencing method, and the results are shown in Figure 3 , wherein (a) is the complete enzymatic disaccharide unit composition information, and (b) is the nitrous acid degradation disaccharide unit composition information; it is confirmed that the structural formula of HP-dp10-1 is shown in formula (1) below, and the heparin oligosaccharide shown in formula (I) is a linear polysaccharide composed of repeating disaccharide units of uronic acid and glucosamine, with a degree of polymerization of 10 and a sequence of:

[0057] ΔUA-GIcNS6S-IdoA2S-GIcNS6S-IdoA2S-GIcNS6S-IdoA2S-GIcNS6S-IdoA2S-GIcNS6S, which contains 4 consecutive [GlcNS6S-IdoA2S] disaccharide units in the structure:

[0058] .

[0059] Example 2

[0060] In this embodiment, another method for preparing heparin oligosaccharide is provided, which is different from example 1 in that:

[0061] (1) Enzymatic preparation of low molecular weight heparin mixture

[0062] 1 g of heparin was weighed and dissolved in 25 mL of partial enzymatic buffer, 0.75 IU of heparase I was added, and incubated at 37°C for 4 h. After the enzymatic reaction was completed, the heparase I was inactivated by heating in a water bath at 100°C for 10 min, and the supernatant was obtained after high-speed centrifugation at 12,000 rpm for 15 min, followed by lyophilization to obtain a low molecular weight heparin mixture;

[0063] The above partial enzymatic buffer was prepared by mixing 0.1 mol / L sodium acetate (pH adjusted to 7.0 with acetic acid) and 0.01 mol / L calcium acetate at a volume ratio of 18:2.

[0064] (2) P10 chromatographic separation, same as step (2) of example 1.

[0065] (3) Strong anion exchange column separation

[0066] Mobile phase A: 0.2 mol / L sodium chloride aqueous solution, mixed and ultrasonic degassing;

[0067] Mobile phase B: 2.0 mol / L sodium chloride aqueous solution, mixed and ultrasonic deaeration;

[0068] Column: Thermo CarboPac PA1 SAX.

[0069] Flow rate: 1 mL / min;

[0070] Chromatographic separation: 1 mg of heparin decasaccharide obtained in step (2) was dissolved in 100 μL of water, and separated by strong anion exchange column (flow rate: 1 mL / min; gradient elution: 0 min 90% mobile phase A, 5 min 90% mobile phase A, 5-170 min 90%-0% mobile phase A, 170-190 min 90% mobile phase A; retention time of chromatographic separation: 120-135 min) to obtain salt-containing heparin oligosaccharide.

[0071] (4) Desalting treatment as in step (4) of Example 1.

[0072] (5) Characterization

[0073] As in Example 1, heparin oligosaccharide as shown in formula (I) of Example 1 was obtained.

[0074] Example 3

[0075] In this example, another method for preparing heparin oligosaccharide is provided, which is different from Example 1 in that:

[0076] (1) Enzymatic preparation of low molecular weight heparin mixture

[0077] 1 g of heparin was weighed and dissolved in 25 mL of partial enzymatic hydrolysis buffer, 0.75 IU of heparinase I was added, and incubated at 25°C for 8 h. After the completion of enzymatic hydrolysis, the heparinase I was inactivated by heating in a 100°C water bath for 10 min, and the supernatant was obtained after high-speed centrifugation at 12,000 rpm for 15 min, followed by freeze-drying to obtain a low molecular weight heparin mixture;

[0078] The above partial enzymatic hydrolysis buffer was prepared by mixing 0.1 mol / L sodium acetate (pH adjusted to 7.0 with acetic acid) and 0.01 mol / L calcium acetate at a volume ratio of 22:4.

[0079] (2) P10 chromatographic separation, as in step (2) of Example 1.

[0080] (3) Strong anion exchange column separation

[0081] Mobile phase A: 0.2 mol / L sodium chloride aqueous solution, mixed and ultrasonic deaeration;

[0082] Mobile phase B: 3.0 mol / L sodium chloride aqueous solution, mixed and ultrasonic deaeration;

[0083] Chromatographic column: Thermo CarboPac PA1 SAX;

[0084] Flow rate: 1 mL / min;

[0085] Chromatographic separation: 1 mg of heparin decasaccharide obtained in step (2) was dissolved in water and separated by strong anion exchange column (flow rate: 1 mL / min; gradient elution: 0 min 90% mobile phase A, 5 min 90% mobile phase A, 5-170 min 90%-0% mobile phase A, 170-190 min 90% mobile phase A; retention time of chromatographic separation: 80-95 min) to obtain salt-containing heparin oligosaccharides.

[0086] (4) Desalination treatment as in Example 1 step (4).

[0087] (5) Characterization

[0088] As in Example 1, heparin oligosaccharides as shown in Formula (I) of Example 1 were obtained.

[0089] Example 4

[0090] In this example, a heparin oligosaccharide composition containing the above-mentioned HP-dp10-1 is provided, which includes HP-dp10-1 prepared in Example 1 and also includes an absorption enhancer SNAC, the mass ratio of HP-dp10-1 to SNAC being 3:1, and the preparation of the composition is as follows:

[0091] Take 3 mg of HP-dp10-1 and 1 mg of SNAC, and ultrasonically mix for 30 min until homogeneous and without stratification, to obtain the heparin oligosaccharide composition.

[0092] Example 5

[0093] In this example, an oral tablet is provided, and the preparation is as follows:

[0094] The heparin oligosaccharide composition in Example 4 is dried and crushed to a particle size of 20 mesh, and the dried powder is placed in a tablet press to prepare tablets.

[0095] Biological verification

[0096] I. Bioavailability verification

[0097] 1. Experimental materials: C57BL / 6J mice, 8 weeks old.

[0098] 2. Experimental method

[0099] Subcutaneous injection of HP-dp10-1 in Example 1 and oral gavage of heparin oligosaccharide composition in Example 4 (composition oral administration) were performed on 18 mice each. The doses of 1 mg / kg for subcutaneous injection and 10 mg / kg for oral administration were administered; the internal canthus blood was taken at 5 min, 10 min, 20 min, 30 min, 60 min and 120 min after administration and placed in an anticoagulant tube; the whole blood taken was centrifuged at 3000 rpm for 15 min at 4°C, and the supernatant was the plasma; the plasma was mixed with methanol at a volume ratio of 1:9, and the solution was allowed to stand at room temperature for 10 min for alcohol precipitation, and then centrifuged at 1000 g for 10 min, and the supernatant was discarded; the precipitate was digested with neutral protease at 40°C for 24 h, and after the digestion was completed, the protease was inactivated by boiling at 100°C for 10 min; centrifugation at 12000 rpm for 10 min was performed to obtain the supernatant, which was then purified by anion exchange column to further wash the impurities and obtain relatively pure HP-dp10-1; the eluted sugar was desalted using an ultrafiltration tube with a molecular weight cut-off of 2 kDa and freeze-dried; the desalted HP-dp10-1 was completely digested into disaccharides using heparinase I; after the disaccharides were labeled with AMAC, LC-MS / MS-MRM quantitative analysis was performed using Nexera LC-20A UPLC liquid system and SCIEX Triple Quad™ 5500+ mass spectrometry system to analyze the blood drug concentration.

[0100] 3. Experimental results

[0101] Figure 4 The blood drug concentration curves of subcutaneous injection of HP-dp10-1 and composition oral administration are shown in Table 1, and the relevant parameters and bioavailability of subcutaneous injection and composition oral administration are shown in Table 1, C max is the maximum value of the plasma drug concentration.

[0102] Table 1 Relevant parameters and bioavailability of subcutaneous injection and composition oral administration

[0103]

[0104] From the quantitative results, the bioavailability of subcutaneous injection was 92.87%, and the bioavailability of the orally administered composition reached 9.13%. Generally, the oral absorption rate of low molecular heparin drugs is not more than 1%. And the actual absorption of drugs into the blood (i.e. AUC value) is the effective concentration that actually exerts the drug effect, so the AUC value can be increased by increasing the oral dose. In this study, an oral dose 10 times the injection dose was used. At this dose, the drug concentration of the heparin oligosaccharide composition group was 21.84 µg / mL, which was about 90% of the drug concentration of the subcutaneous injection group, indicating that the prepared composition had good oral absorption effect after administration, significantly improved the oral absorption rate of heparin oligosaccharides, and had great application potential.

[0105] II. Verification of the therapeutic effect of drug-induced acute liver injury

[0106] 1. Experimental materials: C57BL / 6J mice, 8 weeks old.

[0107] 2. Experimental method

[0108] 2.1 Experimental grouping

[0109] The mice were randomly divided into 5 groups, namely: (A) normal group, (B) model group, (C) drug administration group 1, drug being heparin, (D) drug administration group 2, drug being HP-dp10-1 prepared in Example 1, (E) drug administration group 3, drug being the heparin oligosaccharide composition in Example 4.

[0110] 2.2 Modeling method

[0111] B, C, D, and E groups were intraperitoneally injected with acetaminophen at a dose of 400 mg / kg to establish a drug-induced acute liver injury model; group A was intraperitoneally injected with an equal amount of normal saline as a negative control.

[0112] 2.3 Drug administration method

[0113] After the model was successfully established, drug treatment was started. Groups C and D were administered heparin and HP-dp10-1 at 30 minutes and 3 hours after modeling, respectively. Group E was administered heparin oligosaccharide composition at 30 minutes and 3 hours after modeling. Groups A and B were both administered an equal amount of normal saline by gavage as controls. The administration method for groups C and D was as follows: the administration dose of heparin and HP-dp10-1 was both 10 mg / kg per time. The administration method for group E was as follows: the administration dose of HP-dp10-1 was 10 mg / kg per time, and the administration dose of SNAC was 3.3 mg / kg per time.

[0114] 3. Experimental results

[0115] After the modeling was completed, liver tissue sections were subjected to immunohistochemical staining of HMGB1 inflammatory factors, and the results were quantified using average optical density (AOD), and the results are shown in Figure 5 Figure 6, wherein (A) is the normal group, (B) is the model group, (C) is the administration group 1, (D) is the administration group 2, (E) is the administration group 3, and (F) is the HMGB1 positive area chart. From the results of statistical analysis, the number of HMGB1 positives in the model group was significantly higher than that in the normal group after acetaminophen treatment, and the average optical density value was significantly different, indicating that the drug-induced acute liver injury model was successfully established. After treatment with different groups, the number of HMGB1 positives in the administration group 2 was significantly lower than that in the administration group 1, indicating that HP-dp10-1 has better anti-liver injury activity than heparin. After treatment with the composition in Example 4, the number of HMGB1 positives in the administration group 3 was significantly lower than that in the administration group 2 and the administration group 1, indicating that SNAC effectively promotes the absorption of HP-dp10-1, and the composition has good anti-liver injury activity and can be used for the treatment of drug-induced acute liver injury.

[0116] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a heparin oligosaccharide or a heparin oligosaccharide composition for the manufacture of a medicament for the treatment of acute liver injury, characterized in that, The structure of the heparin oligosaccharide is shown in the following formula (I): ； The heparin oligosaccharide shown in formula (I) is a linear polysaccharide connected by repeating disaccharide units of glucosamine and uronic acid, with a degree of polymerization of 10, and a domain containing 4 consecutive [GlcNS6S-IdoA2S] disaccharide units.

2. Use according to claim 1, characterized in that, The heparin oligosaccharide composition can further comprise N - Sodium (8-[2-hydroxybenzoyl]-amino)octanoate.

3. A method for the preparation of heparin oligosaccharides, characterized in that, The heparin oligosaccharide has the structural formula of claim 1 formula (I); the preparation method of the heparin oligosaccharide comprises the following steps: (1) enzymatic hydrolysis of heparin into a low molecular weight heparin mixture; (2) separation of the low molecular weight heparin mixture by P10 chromatography to obtain heparin decasaccharide; (3) separation of the heparin decasaccharide prepared in step (2) by strong anion exchange column to obtain salt-containing heparin oligosaccharide; (4) desalting of the salt-containing heparin oligosaccharide in step (3) by gel chromatography column to obtain heparin oligosaccharide of formula (I).

4. The method of preparing heparin oligosaccharides according to claim 3, characterized in that, The specific operation of step (1) is: dissolve heparin in part of the enzymatic hydrolysis buffer, add heparinase I, incubate at 25-37℃ for 4-8 h for enzymatic hydrolysis, after the enzymatic hydrolysis is completed, inactivate the reaction system by high temperature, centrifuge and take the supernatant to obtain a low molecular weight heparin mixture.

5. The method of preparing heparin oligosaccharides according to claim 4, characterized in that, The part of the enzymatic hydrolysis buffer is a mixture of sodium acetate solution and calcium acetate solution, the molar concentrations of the sodium acetate solution and the calcium acetate solution are 0.05-0.15 mol / L and 0.005-0.015 mol / L respectively, and the volume ratio of the sodium acetate solution to the calcium acetate solution is 18-22:2-4; the dosage ratio of the heparin, the part of the enzymatic hydrolysis buffer and the heparinase I is 1 g:25-30 mL:0.5-0.75 IU; the high temperature inactivation condition is water bath heating at 100℃ for 10 min; the centrifugation condition is 12000 rpm for 10-15 min.

6. The method of preparing heparin oligosaccharides according to claim 3, characterized in that, In step (2), the P10 chromatography separation condition is: the chromatography column is P10 chromatography column, the mobile phase is 0.2 mol / L ammonium bicarbonate aqueous solution, the flow rate is 0.5 mL / min, and the retention time of chromatography separation is 410-450 min.

7. The method of preparing heparin oligosaccharides according to claim 3, wherein, In step (3), the strong anion exchange column is Thermo CarboPac PA1 SAX chromatography column, the mobile phase A is 0.2 mol / L sodium chloride aqueous solution, the mobile phase B is 2.0-3.0 mol / L sodium chloride aqueous solution, and the flow rate is 1 mL / min; gradient elution, specifically: 0-5 min 90% mobile phase A, 5-170 min 90%-0% mobile phase A, 170-190 min 90% mobile phase A; the retention time of chromatography separation is 80-135 min.

8. The method of preparing heparin oligosaccharides according to claim 3, wherein, In step (4), the desalting parameters of the gel chromatography column are as follows: the chromatography column is G10 chromatography column, the mobile phase is water, the flow rate is 1 mL / min, and the retention time of chromatography separation is 9.5-13.5 min.

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