Heparin oligosaccharide, heparin oligosaccharide composition, preparation method and application

Abstract

The invention discloses heparin oligosaccharide, a heparin oligosaccharide composition, a preparation method and application, and belongs to the technical field of biological medicine. The structure of the heparin oligosaccharide is as follows: heparin is subjected to enzymolysis to form a low-molecular-weight heparin mixture; carrying out P10 chromatographic separation on the low-molecular-weight heparin mixture to obtain heparin decaose; the heparin decaose is separated through a strong anion exchange column to obtain salt-containing heparin oligosaccharide, and the salt-containing heparin oligosaccharide is desalted through a gel chromatographic column to obtain the heparin oligosaccharide. The prepared heparin oligosaccharide is beneficial to realizing quality control of the medicine and improving the safety of the medicine, and the heparin oligosaccharide and SNAC are combined for use, so that oral absorption of the heparin oligosaccharide is effectively promoted, the oral bioavailability of heparin decane is remarkably improved, and the heparin oligosaccharide has good anti-liver injury activity and can be used for oral treatment of drug-induced acute liver injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a heparin oligosaccharide, a heparin oligosaccharide composition, its preparation method, and its application. Background Technology

[0002] Drug-induced acute liver injury (DILI) is a hepatotoxic reaction caused by prescription drugs, herbal remedies, or dietary supplements, accounting for 10%-20% of acute liver failure cases. The global incidence is approximately 1-20 per 100,000. Common causative drugs include antibiotics (such as isoniazid), nonsteroidal anti-inflammatory drugs (NSAIDs) (such as acetaminophen), antitumor drugs, and traditional herbal remedies (such as Polygonum multiflorum). The pathogenesis involves direct hepatotoxicity or idiosyncratic reactions, with clinical manifestations including jaundice, fatigue, and nausea; severe cases can progress to liver failure. Immediate discontinuation of the suspected drug is crucial; 90% of mild cases recover spontaneously. N-acetylcysteine ​​is a specific antidote for acetaminophen poisoning, while severe cases require artificial liver support (such as plasma exchange) or liver transplantation. Some hepatoprotective drugs (such as ursodeoxycholic acid and silymarin) can also help improve liver biochemical indicators. However, long-term use can reduce drug effectiveness and produce side effects and new hepatotoxicities. Therefore, there is an urgent need to develop more effective liver-protective and low-toxicity treatments.

[0003] In recent years, glycosaminoglycans have attracted widespread attention from the academic and pharmaceutical industries. Glycosaminoglycans are linear anionic glycans composed of repeating disaccharide units, widely distributed on cell surfaces and in the extracellular matrix, playing a crucial role in regulating biological and physiological processes. Due to their negatively charged nature, glycosaminoglycans can interact with various proteins, exerting a wide range of biological activities, including anti-inflammatory, antitumor, and antiviral effects. Based on the different disaccharide units they constitute and the degree of sulfation, glycosaminoglycans can be classified into heparin, heparan sulfate, and chondroitin sulfate, among others. However, due to the generally large molecular weight, high negative charge, hydrophilicity, and instability under acidic conditions, the oral bioavailability of glycosaminoglycans is low, insufficient to provide the expected clinical therapeutic effects. Therefore, seeking an effective strategy to improve the oral bioavailability of glycosaminoglycans is of great significance. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a heparin oligosaccharide, a heparin oligosaccharide composition, a preparation method, and applications. The method of this invention features a simple process, well-defined compound structures, and achieves the controllable preparation of oral heparin oligosaccharides with anti-hepatotoxic activity, which can be applied in the field of liver disease treatment.

[0005] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a heparin oligosaccharide, the structure of which is shown in formula (I): 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 domain containing four consecutive [GlcNS6S-IdoA2S] disaccharide units.

[0006] In a second aspect, the present invention provides a method for preparing the heparin oligosaccharide, specifically comprising the following steps: (1) Heparin is enzymatically hydrolyzed into a mixture of low molecular weight heparin; (2) The mixture of low molecular weight heparin was separated by P10 chromatography to obtain heparin decasaccharide; (3) The heparin decasaccharide prepared in step (2) is separated by a strong anion exchange column to obtain salt-containing heparin oligosaccharide; (4) The salt-containing heparin oligosaccharide in step (3) is desalted by gel chromatography to obtain heparin oligosaccharide with the structure of formula (I).

[0007] Further, the specific operation of step (1) is as follows: dissolve heparin in a portion of the enzymatic digestion buffer, add heparinase I and incubate at 25-37℃ for 4-8 h for enzymatic digestion. After the enzymatic digestion is completed, the reaction system is inactivated at high temperature, and the supernatant is taken after centrifugation to obtain a mixture of low molecular weight heparin.

[0008] Further, the partial enzymatic hydrolysis buffer is a mixture of sodium acetate solution and calcium acetate solution, with molar concentrations of 0.05-0.15 mol / L and 0.005-0.015 mol / L, respectively, and a mixing volume ratio of 18-22:2-4; the dosage ratio of heparin, 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 in a water bath at 100°C for 10 min; and the centrifugation condition is centrifugation at 12000 rpm for 10-15 min.

[0009] Further, in step (2), the conditions for P10 chromatographic separation are as follows: the chromatographic column is a P10 chromatographic column, the mobile phase is a 0.2 mol / L ammonium bicarbonate aqueous solution, the flow rate is 0.5 mL / min, and the retention time for chromatographic separation is 410-450 min.

[0010] Further, in step (3), the strong anion exchange column is a Thermo CarboPac PA1 SAX column, the mobile phase A is a 0.2 mol / L sodium chloride aqueous solution, the mobile phase B is a 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 for chromatographic separation is 95-110 min.

[0011] Furthermore, in step (4), the parameters for desalting the gel chromatography column are as follows: the column is a G10 column, the mobile phase is water, the flow rate is 1 mL / min, and the retention time for chromatographic separation is 9.5-13.5 min.

[0012] In a third aspect, the present invention discloses the heparin oligosaccharide composition, wherein the heparin oligosaccharide composition contains an active dose of heparin oligosaccharides.

[0013] The "active dose" mentioned above, according to the general understanding in the art, can be the dosage of a drug for the purpose of prevention, improvement, treatment or adjuvant treatment. After the therapeutic activity of the above-mentioned components is clarified, the dosage is a technical content that can be conventionally inferred based on the general technology mastered by those skilled in the art.

[0014] Furthermore, the heparin oligosaccharide composition, in addition to containing the active dose of heparin oligosaccharides, also includes other excipients, such as absorption enhancers, carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers, or any combination of at least two of these.

[0015] Furthermore, the other excipients are absorption enhancers, and the absorption enhancers are... N Sodium 8-(8-[2-hydroxybenzoyl]-amino)octanoate (SNAC), with a mass ratio of heparin oligosaccharide to SNAC of 3:1.

[0016] In a fourth aspect, the present invention discloses the use of the heparin oligosaccharide or heparin oligosaccharide composition in the preparation of a medicament for treating acute liver injury.

[0017] Existing research indicates that low molecular weight glycosaminoglycans (HMWSAs) can effectively treat acetaminophen-induced acute liver injury models. HMWSAs are homogeneous mixtures of polysaccharides with highly complex structures. This invention provides a heparin oligosaccharide with a specific structure. From a pharmaceutical development perspective, the findings of this invention are more conducive to drug quality control. Those skilled in the art can obtain the corresponding compound based on the preparation method provided by this invention, or it can be prepared through chemical synthesis. On the other hand, drug-induced liver injury is an acute disease requiring large doses of hepatoprotective drugs, and subsequent recovery still requires medication. Therefore, safe oral formulations are more suitable for patients. However, since heparin is a large molecule with low bioavailability, to improve the oral bioavailability of the above-mentioned compound, this invention 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 this invention, the combination of HP-dp10-1 and SNAC, when taken orally, can effectively reduce the expression level of the HMGB1 inflammatory factor in the tissues of mice with drug-induced acute liver injury, thus improving liver injury symptoms.

[0018] Depending on the method of administration to the affected area, the dosage forms of the drug for treating drug-induced acute liver injury include, but are not limited to, intramuscular injections, subcutaneous injections, intravenous injections, oral preparations, or interventional preparations administered via surgical means. In one embodiment verified by this invention, the above-mentioned drug composition is administered orally, specifically as an oral tablet.

[0019] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) The present invention obtains heparin oligosaccharide (HP-dp10-1) with a specific structure by enzymatic hydrolysis and separation of heparin, which is beneficial to the quality control of drugs and improves drug safety; (2) The heparin oligosaccharide provided by the present invention, when used in combination with SNAC, effectively promotes the oral absorption of heparin oligosaccharide, significantly improves the oral bioavailability of heparin decasaccharide, has good anti-hepatic injury activity, and can be used for oral treatment of drug-induced acute liver injury. Attached Figure Description

[0020] Figure 1 This is a chromatogram of the heparin-decaose chromatographic separation process in Example 1; Figure 2 This is a chromatogram of the separation process of salt-containing heparin oligosaccharides in Example 1; Figure 3 The image shows the cluster sequencing sequence of the heparin oligosaccharide prepared in Example 1; (a) shows the composition information of the completely enzymatically hydrolyzed disaccharide unit, and (b) shows the composition information of the nitrite-degraded disaccharide unit. Figure 4 The blood concentration curves for subcutaneous injection of HP-dp10-1 and oral administration of the composition in Example 4 are shown. Figure 5 The images show the HMGB1 immunohistochemical staining map and the average optical density statistical analysis map of Example 4; where (A) is the normal group, (B) is the model group, (C) is the treatment group 1, (D) is the treatment group 2, (E) is the treatment group 3, and (F) is the HMGB1 positive area map. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods or product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only. The invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0023] Example 1 In this embodiment, a method for preparing heparin oligosaccharides is provided, comprising the following steps: (1) Enzymatic hydrolysis to prepare a mixture of low molecular weight heparin Weigh 1 g of heparin, dissolve it in 30 mL of partial enzymatic digestion buffer, add 0.5 IU of heparinase I, incubate at 37 °C for 6 h, after enzymatic digestion, heat in a 100 °C water bath for 10 min to inactivate heparinase I, and centrifuge at 12000 rpm for 10 min to collect the supernatant, freeze dry to obtain a mixture of low molecular weight heparin. The above-mentioned enzymatic digestion buffer was prepared by mixing 0.1 mol / L sodium acetate solution (adjusted to pH 7.0 with acetic acid) and 0.01 mol / L calcium acetate solution at a volume ratio of 20:3. (2) P10 chromatographic separation Mobile phase: 0.2 mol / L ammonium bicarbonate aqueous solution, mixed and then ultrasonically degassed; Chromatographic column: P10 self-packed packing; packing method as follows: add packing to beaker, soak and wash with 20% ethanol, then replace 20% ethanol with high-purity water and degas under vacuum, then pour it into the empty column shell along the wall and compact the packing with the mobile phase, then the sample can be loaded. Mobile phase flow rate: 0.5 mL / min; Chromatographic separation: 200 mg of the low molecular weight heparin mixture obtained in step (1) was dissolved in 1 mL of water, and after loading, separation was performed (separation and collection of components with retention times of 410-450 min) to obtain heparin decasaccharide, which was then lyophilized. The chromatogram of the P10 chromatographic separation process is shown in the figure. Figure 1 As shown.

[0024] (3) Separation by strong anion exchange column Mobile phase A: 0.2 mol / L sodium chloride aqueous solution, mixed and then ultrasonically degassed; Mobile phase B: 2.5 mol / L sodium chloride aqueous solution, mixed and then ultrasonically degassed; Column: Thermo CarboPac PA1 SAX; Chromatographic separation: 1 mg of heparin decasaccharide obtained in step (2) was dissolved in 100 µL of water and separated on a strong anion exchange column using gradient elution: 0–5 min 90% mobile phase A, 5–170 min 90%–0% mobile phase A, 170–190 min 90% mobile phase A; flow rate was 1 mL / min, and retention time was 95–110 min. The separation yielded salt-containing heparin oligosaccharides. The chromatogram during the separation process is shown below. Figure 2 As shown.

[0025] (4) Desalting treatment of gel chromatography column Mobile phase: high-purity aqueous solution, ultrasonically degassed; Chromatographic column: G10 column; Flow rate: 1 mL / min; Chromatographic separation: The salt-containing heparin oligosaccharide obtained in step (3) was desalted by G10 chromatography, and the components with a retention time of 9.5-13.5 min were collected and then lyophilized to obtain heparin oligosaccharide (HP-dp10-1).

[0026] (5) Characterization The above HP-dp10-1 sequences were analyzed using cluster sequencing, and the results are as follows: Figure 3 As shown, (a) represents the composition information of the completely enzymatically hydrolyzed disaccharide unit, and (b) represents the composition information of the nitrite-degraded disaccharide unit; the structure of HP-dp10-1 has been confirmed as shown in formula (1). 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 the sequence is: The structure ΔUA-GIcNS6S-IdoA2S-GIcNS6S-IdoA2S-GIcNS6S-IdoA2S-GIcNS6S-IdoA2S-GIcNS6S contains four consecutive [GlcNS6S-IdoA2S] disaccharide units: .

[0027] Example 2 In this embodiment, another method for preparing heparin oligosaccharides is provided, which differs from Example 1 in that: (1) Enzymatic hydrolysis to prepare a mixture of low molecular weight heparin Weigh 1 g of heparin and dissolve it in 25 mL of partial enzymatic digestion buffer. Add 0.75 IU of heparinase I and incubate at 37 °C for 4 h. After enzymatic digestion, heat in a 100 °C water bath for 10 min to inactivate heparinase I. After centrifugation at 12,000 rpm for 15 min, collect the supernatant, freeze-dry, and obtain a mixture of low molecular weight heparin. The above-mentioned enzymatic digestion 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.

[0028] (2) P10 chromatographic separation, same as step (2) in Example 1.

[0029] (3) Separation by strong anion exchange column Mobile phase A: 0.2 mol / L sodium chloride aqueous solution, mixed and then ultrasonically degassed; Mobile phase B: 2.0 mol / L sodium chloride aqueous solution, mixed and then ultrasonically degassed; Chromatographic column: Thermo CarboPac PA1 SAX.

[0030] Flow rate: 1 mL / min; Chromatographic separation: 1 mg of heparin decasaccharide obtained in step (2) was dissolved in 100 µL of water and separated by passing it through a strong anion exchange column (flow rate of 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 for chromatographic separation was 120-135 min) to obtain salt-containing heparin oligosaccharide.

[0031] (4) The desalination process is the same as step (4) in Example 1.

[0032] (5) Characterization As in Example 1, heparin oligosaccharides as shown in Formula (I) of Example 1 were obtained.

[0033] Example 3 In this embodiment, another method for preparing heparin oligosaccharides is provided, which differs from Example 1 in that: (1) Enzymatic hydrolysis to prepare a mixture of low molecular weight heparin Weigh 1 g of heparin, dissolve it in 25 mL of partial enzymatic digestion buffer, add 0.75 IU of heparinase I, and incubate at 25 °C for 8 h. After enzymatic digestion, heat in a 100 °C water bath for 10 min to inactivate heparinase I, and centrifuge at 12,000 rpm for 15 min. Collect the supernatant, freeze-dry, and obtain a mixture of low molecular weight heparin. The above-mentioned enzymatic digestion buffer was prepared by mixing 0.1 mol / L sodium acetate (adjusted to pH 7.0 with acetic acid) and 0.01 mol / L calcium acetate at a volume ratio of 22:4.

[0034] (2) P10 chromatographic separation, same as step (2) in Example 1.

[0035] (3) Separation by strong anion exchange column Mobile phase A: 0.2 mol / L sodium chloride aqueous solution, mixed and then ultrasonically degassed; Mobile phase B: 3.0 mol / L sodium chloride aqueous solution, mixed and then ultrasonically degassed; Column: Thermo CarboPac PA1 SAX; Flow rate: 1 mL / min; Chromatographic separation: 1 mg of heparin decasaccharide obtained in step (2) was dissolved in water and separated by passing it through a strong anion exchange column (flow rate of 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 for chromatographic separation is 80-95 min) to obtain salt-containing heparin oligosaccharide.

[0036] (4) The desalination process is the same as step (4) in Example 1.

[0037] (5) Characterization As in Example 1, heparin oligosaccharides as shown in Formula (I) of Example 1 were obtained.

[0038] Example 4 In this embodiment, a heparin oligosaccharide composition comprising the above-mentioned HP-dp10-1 is provided. The composition includes HP-dp10-1 prepared in Example 1 and also includes the absorption enhancer SNAC. The mass ratio of HP-dp10-1 to SNAC is 3:1. The composition is prepared as follows: Take 3 mg HP-dp10-1 and 1 mg SNAC, and sonicate for 30 min until they are mixed evenly without layering to obtain the heparin oligosaccharide composition.

[0039] Example 5 In this embodiment, an oral tablet is provided, prepared as follows: The heparin oligosaccharide composition in Example 4 was dried and pulverized to a particle size of 20 mesh. The dried powder was then placed in a tablet press and compressed to obtain tablets.

[0040] Biological verification I. Bioavailability Verification 1. Experimental materials: C57BL / 6J mice, 8 weeks old.

[0041] 2. Experimental Methods Eighteen mice were administered HP-dp10-1 subcutaneously in Example 1 and heparin oligosaccharide composition (orally administered) in Example 4 via oral gavage. The drug was administered subcutaneously at a dose of 1 mg / kg orally at a dose of 10 mg / kg. Blood samples were collected from the inner canthus at time points of 5 min, 10 min, 20 min, 30 min, 60 min, and 120 min after administration and placed in anticoagulant tubes. The collected whole blood was centrifuged at 3000 rpm for 15 min at 4°C, and the supernatant was obtained as plasma. The plasma was mixed with methanol at a volume ratio of 1:9 and allowed to stand at room temperature for 10 min for alcohol precipitation. The solution was then centrifuged at 1000 g for 10 min, and the supernatant was discarded. The precipitate was enzymatically hydrolyzed with neutral protease at 40°C for 24 h. After hydrolysis, the solution was boiled at 100°C for 10 min to inactivate the protease. The supernatant was obtained by centrifugation at 12000 rpm for 10 min and then purified by anion exchange column to further wash away impurities, yielding relatively pure HP-dp10-1. The eluted sugars were then purified using a molecular weight cutoff of 2. The kDa ultrafiltration tube was desalted and freeze-dried; the desalted HP-dp10-1 was completely enzymatically hydrolyzed into disaccharide using heparinase I; the disaccharide was labeled with AMAC and then quantitatively analyzed by LC-MS / MS-MRM using a Nexera LC-20A UPLC liquid chromatography system and a SCIEX Triple Quad™ 5500+ mass spectrometry system to analyze the blood drug concentration.

[0042] 3. Experimental Results Figure 4 The blood concentration curves for subcutaneous injection of HP-dp10-1 and oral administration of the composition are shown in Table 1. Table 1 presents relevant parameters and bioavailability for subcutaneous injection and oral administration of the composition. max This represents the maximum plasma drug concentration.

[0043] Table 1. Relevant parameters and bioavailability for subcutaneous injection and oral administration of the composition. Quantitative results show that the bioavailability of the subcutaneous injection was 92.87%, while the bioavailability of the oral administration composition reached 9.13%. Generally, the oral absorption rate of low molecular weight heparin drugs does not exceed 1%. Furthermore, the actual drug concentration absorbed into the bloodstream (i.e., the AUC value) is the effective concentration at which the drug exerts its therapeutic effect. Therefore, the AUC value can be increased by increasing the oral dosage. 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, reaching approximately 90% of the drug concentration of the subcutaneous injection group. This indicates that the prepared composition has good oral absorption after administration, significantly improving the oral absorption rate of heparin oligosaccharides and possessing great application potential.

[0044] II. Validation of the therapeutic efficacy of drug-induced acute liver injury 1. Experimental materials: C57BL / 6J mice, 8 weeks old.

[0045] 2. Experimental Methods 2.1 Experimental Grouping Mice were randomly divided into 5 groups: (A) normal group, (B) model group, (C) drug group 1, the drug being heparin, (D) drug group 2, the drug being HP-dp10-1 prepared in Example 1, and (E) drug group 3, the drug being the heparin oligosaccharide composition in Example 4.

[0046] 2.2 Modeling Methods Groups B, C, D, and E were intraperitoneally injected with acetaminophen 400 mg / kg to establish a drug-induced acute liver injury model; Group A was intraperitoneally injected with an equal volume of physiological saline as a negative control.

[0047] 2.3 Administration method After successful model establishment, drug treatment was initiated. Groups C and D were administered heparin and HP-dp10-1 by gavage at 30 minutes and 3 hours after model establishment, respectively. Group E was administered a heparin oligosaccharide combination by gavage at 30 minutes and 3 hours after model establishment. Groups A and B were administered an equal volume of physiological saline by gavage as controls. The administration methods for C and D were: heparin and HP-dp10-1 at a dose of 10 mg / kg / time. The administration methods for Group E were: HP-dp10-1 at a dose of 10 mg / kg / time and SNAC at a dose of 3.3 mg / kg / time.

[0048] 3. Experimental Results After modeling was completed, immunohistochemical staining of liver tissue sections for HMGB1 inflammatory factors was performed, and the results were quantified using mean optical density (AOD). The results are as follows: Figure 5 As shown, (A) is the normal group, (B) is the model group, (C) is the treatment group 1, (D) is the treatment group 2, (E) is the treatment group 3, and (F) is the HMGB1 positive area map. Statistical analysis shows that after acetaminophen treatment, the number of HMGB1 positive cells in the model group was significantly higher than that in the normal group, and the average optical density value showed a statistically significant difference, indicating that the drug-induced acute liver injury model was successfully established. After treatment with different groups, the number of HMGB1 positive cells in treatment group 2 was significantly lower than that in treatment group 1, indicating that HP-dp10-1 has superior anti-hepatic injury activity compared to heparin. After treatment with the composition in Example 4, the number of HMGB1 positive cells in treatment group 3 was significantly lower than that in both treatment groups 2 and 1, indicating that SNAC effectively promoted the absorption of HP-dp10-1. This composition has good anti-hepatic injury activity and can be used to treat drug-induced acute liver injury.

[0049] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A heparin oligosaccharide, characterized in that, The structure of the heparin oligosaccharide is shown in formula (I) below: 。 2. A heparin oligosaccharide composition, characterized in that, The heparin oligosaccharide composition comprises the heparin oligosaccharide of claim 1.

3. The heparin oligosaccharide composition according to claim 2, characterized in that, The heparin oligosaccharide composition also includes N -(8-[2-hydroxybenzoyl]-amino)octanoic acid sodium salt.

4. A method for preparing heparin oligosaccharides according to claim 1, characterized in that, The following steps are involved: (1) Heparin is enzymatically hydrolyzed into a mixture of low molecular weight heparin; (2) The mixture of low molecular weight heparin was separated by P10 chromatography to obtain heparin decasaccharide; (3) The heparin decasaccharide prepared in step (2) is separated by a strong anion exchange column to obtain salt-containing heparin oligosaccharide; (4) The salt-containing heparin oligosaccharide in step (3) is desalted by gel chromatography to obtain heparin oligosaccharide with the structure of formula (I).

5. The method for preparing heparin oligosaccharides according to claim 4, characterized in that, The specific operation of step (1) is as follows: dissolve heparin in a portion of the enzymatic digestion buffer, add heparinase I and incubate at 25-37℃ for 4-8 h for enzymatic digestion. After the enzymatic digestion is completed, the reaction system is inactivated at high temperature, and the supernatant is taken after centrifugation to obtain a mixture of low molecular weight heparin.

6. The method for preparing heparin oligosaccharides according to claim 5, characterized in that, The partial enzymatic hydrolysis buffer is a mixture of sodium acetate solution and calcium acetate solution, with molar concentrations of 0.05-0.15 mol / L and 0.005-0.015 mol / L, respectively, and a volume ratio of 18-22:2-4. The dosage ratio of heparin, 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 in a water bath at 100°C for 10 min. The centrifugation condition is centrifugation at 12000 rpm for 10-15 min.

7. The method for preparing heparin oligosaccharides according to claim 4, characterized in that, In step (2), the P10 chromatographic separation conditions are as follows: the chromatographic column is a P10 chromatographic 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 for chromatographic separation is 410-450 min.

8. The method for preparing heparin oligosaccharides according to claim 4, characterized in that, In step (3), the strong anion exchange column is a Thermo CarboPac PA1 SAX column, mobile phase A is 0.2 mol / L sodium chloride aqueous solution, 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 as follows: 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 for chromatographic separation is 80-135 min.

9. The method for preparing heparin oligosaccharides according to claim 4, characterized in that, In step (4), the parameters for desalting the gel chromatography column are as follows: the column is a G10 column, the mobile phase is water, the flow rate is 1 mL / min, and the retention time for chromatographic separation is 9.5-13.5 min.

10. Use of a heparin oligosaccharide composition according to claim 1 or according to claim 2 or 3 in the preparation of a medicament for treating acute liver injury.

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