Oral polysaccharide for the treatment of inflammatory bowel disease and method of preparation thereof

Abstract

The application relates to a preparation method of refined heparin polysaccharides, which comprises the following steps: adopting a gel exclusion chromatography column method to separate heparin polysaccharide raw materials, and treating the collected separation components through freeze-drying, alcohol precipitation and desalination to obtain refined heparin polysaccharide products. The application also relates to an oligosaccharide with specific polysaccharide structure characteristics and anti-UC effectiveness, which can be used for the prevention and treatment of inflammatory bowel disease, and diseases similar to inflammatory bowel disease in terms of complications and pathogenesis. The application also relates to an oligosaccharide with specific polysaccharide structure characteristics and anti-inflammatory effectiveness.

Description

Technical Field

[0001] This invention relates to a method for preparing refined heparin polysaccharides, and their use in the prevention and / or treatment of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of complex, incurable chronic inflammatory bowel diseases, including ulcerative colitis (UC) and Crohn's disease (CD). It has been reported that the prevalence of IBD is as high as 0.8% in Western countries, while in Asia, both the incidence and prevalence of IBD are showing a continuous and rapid upward trend, becoming a disease of widespread global concern. It is projected that the prevalence in many countries will increase by more than 40% in the next decade. Therefore, research and treatment of IBD are becoming increasingly important. However, current pathological research and clinical treatment methods for IBD face significant bottlenecks. Taking UC as an example, the mainstream view in the research field considers four major factors—immunity, genetics, environment, and gut microbiota—to be important factors influencing the pathogenesis of UC, but its specific pathogenesis remains unclear, and there is no unified conclusion yet.

[0003] In clinical practice, mature treatment options for IBD patients are very limited, mainly falling into categories such as drug therapy and surgical treatment. Drug therapy primarily utilizes medications including aminosalicylic acid derivatives, corticosteroids, immunosuppressants, and biologics. However, these drugs are mostly broad-spectrum medications, with major drawbacks including significant side effects, easy development of drug tolerance, and low treatment efficacy. Therefore, clinical IBD medication has significant limitations, lacking specific treatment drugs, and urgently requiring the emergence of new drugs and treatment methods.

[0004] Heparin is a glycosaminoglycan with a molecular weight of 3000–30000 Da, exhibiting high heterogeneity and complexity. Currently, its only clinical use is for its anticoagulant function. Although current research indicates that heparin is a multi-target drug, interacting with hundreds of proteins in vivo, such as cell growth factors, chemokines, and adhesion molecules, thus possessing diverse biological functions and affecting human physiological functions to varying degrees, such as regulating inflammatory responses, inhibiting tumor metastasis, fighting viral infections, and inhibiting smooth muscle cell proliferation, these functions are often considered independent of its anticoagulant activity. However, it has not yet been applied clinically.

[0005] Currently, some preclinical studies and clinical trials have reported that heparin drugs have certain therapeutic potential for IBD. However, unfortunately, the therapeutic effects of different types of heparin drugs on IBD vary greatly, and inconsistent efficacy conclusions are often found in preclinical studies and clinical trials. It is also worth noting that subcutaneous injection of heparin drugs is often ineffective in treating IBD. The reasons for these problems are twofold: First, currently commercially available heparin drugs are all designed for anticoagulation-related indications in clinical practice. Therefore, during development, only their anticoagulant function was considered, and other non-anticoagulant activities of the heparin molecule were neglected in the drug production process, which is primarily focused on anticoagulant function, potentially damaging the corresponding effective structure. Furthermore, the clinical dosage of heparin drugs is strictly monitored, and dosages that exert their non-anticoagulant activity may induce massive bleeding. Second, the highly complex molecular structure of heparin drugs, the unknown structure-activity relationship, and the unclear understanding of the active functional polysaccharide fragments for treating IBD limit their clinical application in treating IBD and other related conditions.

[0006] Numerous studies have shown that the non-anticoagulant biological activity of heparin is closely related to its molecular weight. However, the high heterogeneity and structural complexity of heparin greatly limit the exploration of its non-anticoagulant biological activity. At the level of heterogeneity, heparin is a highly heterogeneous linear polysaccharide with a molecular weight ranging from 3000 to 30000 Da, not a pure substance, and with an extremely complex molecular composition. At the level of structural complexity, heparin possesses abundant sulfonic acid and acetyl groups, and the modification process is not controlled by the central dogma, exhibiting a high degree of randomness. This also leads to an exponential increase in the fine structural complexity of heparin. Due to these complex molecular structural characteristics, the complete glycan molecular structure of heparin has not yet been resolved. Therefore, the separation and purification of heparin-like polysaccharides is of great significance for clarifying the mechanism of action and structure-activity relationship of their non-anticoagulant biological activity.

[0007] Current research on the preparation and separation of heparin-like polysaccharides largely focuses on separating heparin based on its anticoagulant and non-anticoagulant activities. This method cannot distinguish polysaccharides of different molecular weights, and there is currently no precise separation and preparation process targeting the specific molecular weight of heparin. Although existing literature reports the use of gel size exclusion chromatography for quantitative analysis of heparin molecular weight, this method remains at the analytical level and suffers from limitations such as low polysaccharide analysis volume, unknown processes for precise separation and purification, and inability to directly scale up the process. Therefore, controllable separation and large-scale preparation of heparin-like polysaccharides of different molecular weights cannot be achieved. Consequently, there are currently no reports or processes in the research field that provide precise separation and preparation of heparin-like polysaccharides based on their molecular weight.

[0008] However, there is a lack of systematic structure-activity relationship studies on the anti-IBD activity of heparin drugs, and the functional polysaccharide fragments of heparin drugs for treating IBD are still unclear. In addition, existing studies often overlook the problems of the preparation process of anticoagulant heparin damaging its anti-IBD activity and corresponding fine structure. These issues need to be addressed in this field. Summary of the Invention

[0009] The purpose of this application is to provide a novel oral medication for treating inflammatory bowel disease, which, through the precise separation of heparin-like polysaccharides, exhibits significant preventive and / or therapeutic effects against inflammatory bowel disease. This application also provides a method for the precise separation and preparation of the polysaccharide drug.

[0010] Inflammatory bowel disease (IBD), a chronic nonspecific inflammatory bowel disease with extremely complex etiologies and pathogenesis, is prone to relapse, carries a high risk of cancer, and is currently incurable. Clinically, treatment options for this disease are very limited. Existing drugs mainly focus on broad-spectrum anti-inflammatory agents, with unsatisfactory therapeutic effects and generally suffering from poor specificity, strong side effects, and easy development of drug tolerance. Therefore, there is an urgent need to develop novel and highly effective drugs for the treatment of IBD.

[0011] Heparin is a multi-target drug that interacts with hundreds of proteins in vivo, such as cell growth factors, chemokines, and adhesion molecules, thus exhibiting a wide variety of non-anticoagulant biological activities beyond anticoagulation. While some literature reports the potential efficacy of heparin in treating IBD, current research suffers from inconsistencies in heparin treatment outcomes, significant differences in efficacy, a severe lack of understanding of the effective molecular structure of heparin for IBD, the structure-activity relationship of drugs, and structure-activity analysis techniques and methods. These research and technological bottlenecks greatly limit the exploration of the biological activities of heparin-based polysaccharides in treating IBD. Therefore, establishing efficient methods for preparing the fine structures of heparin-based polysaccharides, deeply exploring their structure-activity relationships, and identifying the optimal heparin-based oligosaccharide structures for anti-IBD purposes are key research priorities for developing novel non-anticoagulant drugs based on heparin-based polysaccharides.

[0012] Heparin polysaccharides are generally characterized by highly complex molecular structures and diverse functions. While some simple separation methods exist for the preparation of heparin polysaccharides (e.g., separation based on molecular weight using ultrafiltration membranes), their separation precision and volume are limited, making further refined separation and preparation of heparin polysaccharide drugs challenging. Although techniques such as gel size exclusion chromatography can achieve quantitative molecular weight analysis of heparin polysaccharides, they cannot meet the requirements for refined preparation; that is, they do not achieve the purification or large-scale preparation (mg to g level) of heparin polysaccharides.

[0013] The inventors of this application are dedicated to innovative research on heparin industry technology and have developed a set of refining processes for heparin polysaccharides. This process enables the preparation and separation of heparin polysaccharides with controllable molecular weight, overcoming the problem of high structural heterogeneity of heparin polysaccharides. It provides new technologies, methods and products for the effective exploration of their non-anticoagulant biological activities such as anti-inflammatory, anti-tumor and anti-fat accumulation, as well as their application in the treatment of inflammatory bowel disease.

[0014] Specifically, this invention relates to the following:

[0015] 1. A method for preparing refined heparin-like polysaccharides, comprising:

[0016] Heparin raw material is de-anticoagulated and enzymatically hydrolyzed to obtain heparin-like polysaccharide raw material;

[0017] The heparin polysaccharide raw material was separated using a gel size exclusion chromatography column, and the separated components were collected.

[0018] The collected and separated components were freeze-dried.

[0019] The freeze-dried products of each component were desalted by alcohol precipitation to obtain refined heparin polysaccharides.

[0020] 2. The preparation method according to item 1, wherein,

[0021] The de-anticoagulation treatment of raw heparin is performed using periodic acid oxidation to obtain a de-anticoagulated product.

[0022] The decoagulated product is enzymatically hydrolyzed using heparinase I to obtain the heparin polysaccharide raw material.

[0023] 3. The preparation method according to item 1 or 2, wherein,

[0024] Freeze-drying involves pre-freezing the collected and separated components at -80°C before placing them into a freeze dryer for freeze-drying.

[0025] 4. The preparation method according to any one of items 1 to 3, wherein,

[0026] When separating the heparin polysaccharide raw material using a size exclusion chromatography column, the size exclusion column is a size exclusion chromatography column, preferably a HiPrep 16 / 60 Sephacryl or TSKgel G2000SW column, and the mobile phase used is a 0.15-1.0M NaCl aqueous solution, preferably 0.15-0.6M, and more preferably 0.2M.

[0027] 5. The preparation method according to item 4, wherein,

[0028] The flow rate of the mobile phase in the gel size exclusion chromatography column is 0.1–1.0 mL / min, preferably 0.3–0.7 mL / min, and more preferably 0.5 mL / min.

[0029] 6. The preparation method according to item 4, wherein,

[0030] The pH value of the NaCl aqueous solution is 3 to 10, preferably 5.

[0031] 7. The preparation method according to item 4, wherein,

[0032] The gel size exclusion column is a HiPrep 16 / 60 Sephacryl column, and the column packing material is Sephacryl S-100 High Resolution, Sephacryl S-200 High Resolution, or Sephacryl S-300 High Resolution.

[0033] 8. The preparation method according to item 4, wherein,

[0034] The gel size exclusion column is a TSKgel G2000SW column, and the column packing material is TSKgel G2000.

[0035] 9. The preparation method according to any one of items 1 to 8, wherein when the lyophilized products of each component are desalted by alcohol precipitation,

[0036] The lyophilized products of each component were resuspended and concentrated in distilled water.

[0037] After adding the aqueous ethanol solution, allow it to stand for alcohol precipitation.

[0038] After centrifugation, the supernatant of the precipitate after alcohol precipitation was discarded; and

[0039] The purified heparin polysaccharide was then obtained by resuspending it in distilled water.

[0040] 10. The preparation method according to item 9, wherein,

[0041] Add 20-50% by volume of distilled water to each component of the freeze-dried product for resuspension and concentration, preferably 30%.

[0042] 11. The preparation method according to item 10, wherein,

[0043] The volume of the ethanol-water solution added is 2 to 6 times the volume of the concentrated liquid after resuspension, preferably 5 to 6 times; and,

[0044] The concentration of the ethanol-water solution is 75% to 100%.

[0045] 12. The preparation method according to item 9, wherein,

[0046] The time for standing alcohol precipitation is 5 to 60 minutes, preferably 10 to 30 minutes.

[0047] 13. The preparation method according to items 1 to 12, further comprising:

[0048] The refined heparin polysaccharide obtained by desalting through concentrated alcohol precipitation is then freeze-dried.

[0049] 14. An oligosaccharide, characterized in that the oligosaccharide has the following structure:

[0050] [a]-[b,c,d,e,f,g]-[h], where,

[0051] a represents the number of open-ring structures of the oligosaccharide molecule;

[0052] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0053] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0054] d represents the amount of glucosamine in the oligosaccharide molecule, and 1 ≤ d ≤ 10;

[0055] e represents the number of acetyl groups in the oligosaccharide molecule;

[0056] f is the number of sulfonic acid groups in the oligosaccharide molecule, and f ≥ 2.5d.

[0057] g represents the number of 1,6-dehydrated structures in the oligosaccharide molecule.

[0058] h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection.

[0059] 15. The oligosaccharide according to claim 14, characterized in that, in the structural formula of the oligosaccharide,

[0060] a≤0.3d, b=1, c=d-1, e≤1.0, g=0.

[0061] 16. The oligosaccharide according to claim 14, characterized in that the oligosaccharide has one of the following structures: [0]-[1,2,3,0,9,0]-[0], [0]-[1,2,3,0,8,0]-[0], [0]-[1,3,4,0,12,0]-[0], [2]-[1,3,4,1,10,0]-[1], [0]-[1,4,5,0,15,0]-[5], [1]-[1,4,5,0,14,0]-[1], [1]-[1] ,4,5,0,13,0]-[0],[2]-[1,4,5,1,13,0]-[4],[2]-[1,4,5,1,11,0]-[0],[0]-[1,5,6,0,18,0]-[6],[2]-[1,5,6,1,16,0]-[5],[2]-[1,5,6,1,15,0]-[3],[2]-[1,5,6,1,14,0]-[3] or [1]-[1,5,6,0,15,0]-[3].

[0062] 17. An oligosaccharide, characterized in that,

[0063] The oligosaccharide has a sugar chain length between 2 and 20 sugars, and the basic disaccharide unit is composed of repeated arrangements of [0]-[1,0,1,0,3,0]-[0] and / or [0]-[1,0,1,0,2,0]-[0], wherein,

[0064] [0]-[1,0,1,0,3,0]-[0] represents a disaccharide fragment with 0 open-ring structures, 1 unsaturated uronic acid, 0 saturated uronic acid, 1 glucosamine, 0 acetyl groups, 3 sulfonic acid groups, 0 dehydration structures, and 0 ammonium ions carried in mass spectrometry.

[0065] [0]-[1,0,1,0,2,0]-[0] represents a disaccharide structure fragment with 0 open ring structures, 1 unsaturated uronic acid, 0 saturated uronic acid, 1 glucosamine, 0 acetyl groups, 2 sulfonic acid groups, 0 dehydrated structures, and 0 ammonium ions carried in mass spectrometry.

[0066] 18. The oligosaccharide according to claim 17, characterized in that the average number of sulfonic acid groups contained in the basic disaccharide unit is greater than or equal to 2.5.

[0067] 19. The oligosaccharide according to any one of claims 17 or 18, characterized in that the average number of open-ring structures of the sugar ring in the basic disaccharide unit is less than or equal to 0.3.

[0068] 20. A heparin derivative containing oligosaccharides as described in items 14 to 19, characterized in that the content of oligosaccharides as described in items 14 to 19 in the heparin derivative is 17% or more, preferably 18% or more, preferably 19% or more, preferably 20% or more, preferably 21% or more, preferably 22% or more, preferably 23% or more.

[0069] 21. Use of the oligosaccharides described in items 14-19 and the heparin derivatives described in item 20 in the preparation of medicaments for the prevention or treatment of inflammatory bowel disease, and diseases with inflammatory bowel disease-related complications and similar pathogenesis.

[0070] 22. According to the use described in item 21, the inflammatory bowel disease-related complications and diseases with similar pathogenesis include: irritable bowel syndrome, arthritis and other extraintestinal complications including ankylosing spondylitis, pyoderma gangrenosa, erythema nodosum, iritis, uveitis, episcleritis, primary sclerosing cholangitis and rheumatoid arthritis.

[0071] 23. The use as described in any one of items 21 or 22, wherein the oligosaccharide is prepared using any one of items 1 to 13.

[0072] Invention Effects

[0073] The method for preparing refined heparin polysaccharides provided in this application achieves de-anticoagulant modification and precise separation with controllable molecular weight, yielding refined polysaccharide components with relatively concentrated molecular weight distribution and no anticoagulant activity. This eliminates the potential risk of massive bleeding induced by heparin polysaccharides and significantly optimizes the high structural heterogeneity of heparin polysaccharides. Through in vitro and in vivo drug screening and efficacy evaluation, the refined polysaccharide component with the best efficacy in significantly alleviating the characteristic clinical symptoms of ulcerative colitis (UC), a major clinical symptom of IBD, was obtained, showing superior therapeutic effects compared to first-line UC medications. Further oligosaccharide structure analysis identified oligosaccharide fragments exhibiting the main biological activities for UC treatment from the refined polysaccharide component with the best efficacy. This provides valuable data support for further research on its application in the treatment of inflammatory bowel disease and the development of new anti-UC polysaccharide drugs. It also provides new ideas and methods for studying the structure-activity relationship of non-anticoagulant biological activities such as anti-inflammatory, anti-tumor, and anti-fat accumulation of heparin polysaccharides. Attached Figure Description

[0074] Figure 1 ANOVA analysis plot of body weight curve for UC mice

[0075] Figure 2 Representative images of the colon of UC mice and results of colon length measurement.

[0076] Figure 3Representative images of the spleen of UC mice and a schematic diagram of the spleen weight index.

[0077] Figure 4 Image showing H&E staining to evaluate histological changes in the colonic epithelium of UC mice.

[0078] Figure 5 Schematic diagram for evaluating the in vitro anti-inflammatory activity indicators of heparin derivatives

[0079] Figure 6 Complete glycan chain analysis chromatograms of heparin derivatives and each isolated component.

[0080] Figure 7 This is a graph showing the relationship between oligosaccharide coverage and oligosaccharide enrichment threshold for heparin derivatives and each isolated component.

[0081] Figure 8 Venn diagram of enriched oligosaccharides of heparin derivatives and various isolated components.

[0082] Figure 9 Structural feature analysis diagram of effective and ineffective anti-UC oligosaccharides of heparin derivatives Detailed Implementation

[0083] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are intended to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0084] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0085] This application relates to a method for preparing refined heparin-like polysaccharides, which, in one specific embodiment, includes the following steps:

[0086] Heparin raw material is de-anticoagulated and enzymatically hydrolyzed to obtain heparin-like polysaccharide raw material;

[0087] The heparin polysaccharide raw material was separated using a gel size exclusion chromatography column, and the separated components were collected.

[0088] The collected and separated components were freeze-dried.

[0089] The freeze-dried products of each component were desalted by alcohol precipitation to obtain refined heparin polysaccharides.

[0090] The heparin polysaccharide raw material described in this application, namely the deanticoagulated heparin derivative, is a substance obtained by deanticoagulating heparin or (ultra) low molecular weight heparin. It is heparin or (ultra) low molecular weight heparin that has no anticoagulation activity or low anticoagulation activity, and its anti-Xa factor is less than or equal to 70 IU / mg, preferably less than or equal to 60 IU / mg, preferably less than or equal to 50 IU / mg, preferably less than or equal to 40 IU / mg, preferably less than or equal to 30 IU / mg, preferably less than or equal to 20 IU / mg, and preferably less than or equal to 10 IU / mg.

[0091] In one specific embodiment, the raw material heparin is first subjected to anticoagulation treatment, and then the product of the anticoagulation treatment is enzymatically hydrolyzed to obtain the heparin polysaccharide raw material.

[0092] In one specific embodiment of this application, the raw material heparin is de-anticoagulated using periodic acid oxidation to obtain a de-anticoagulated product.

[0093] Heparin derivatives with de-anticoagulant activity can be obtained by periodic acid oxidation, while other biological activities are largely preserved, and the degree and form of sulfation remain basically unchanged. Periodic acid can selectively oxidize the ortho-carbon atom containing unsubstituted hydroxyl or amino groups, causing the C(2)-C(3) bond of the unsulfated uronic acid to break, thus destroying the glucuronic acid in the antithrombin binding pentasaccharide within the heparin molecule and losing its anticoagulant activity; the polyaldehyde oxidized heparin obtained by periodic acid oxidation is stabilized by reduction with borohydride (Islam, T., et al., Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site. Carbohydrate Research, 2002, 337(21–23): p.2239-2243.).

[0094] In one specific embodiment, raw material heparin (e.g., sodium heparin) is dissolved in water, and a sodium periodate solution is added to initiate the reaction. After a period of reaction, ethylene glycol is added to neutralize excess sodium periodate, followed by the addition of sodium borohydride. After adjusting the pH, the sample is filtered and collected. It is then concentrated and desalted using a dialysis bag or similar method to finally obtain a de-anticoagulant heparin derivative with removed anticoagulant activity.

[0095] In one specific embodiment of this application, heparinase, such as heparinase I, is used to enzymatically hydrolyze the product after anticoagulation treatment to obtain the heparin polysaccharide raw material.

[0096] In the prior art, the EC number for heparinase I is EC4.2.2.7. Commercially available heparinase I can be used, such as heparinase I purchased from Sigma or IBEX. Heparinase can also be recombinant heparinase I constructed using molecular biology methods, or a fusion protein formed by heparinase I with any fusion partner, as long as it possesses heparinase I activity. Preferably, heparinase I is a fusion protein of heparinase I, especially a fusion protein of heparinase I containing MBP.

[0097] In one specific embodiment, the reaction of heparinase I with the de-anticoagulated product can be batch-wise, continuous, or semi-continuous, and those skilled in the art can choose appropriately according to production needs.

[0098] In one specific embodiment of this application, a size exclusion chromatography column is used to separate the heparin-based polysaccharide raw material. Preferably, an AKTA Prime purification system combined with a size exclusion chromatography column for preparative liquid chromatography forms the basic hardware setup.

[0099] Gel size exclusion chromatography, also known as size exclusion chromatography, is a chromatographic technique that separates samples based on their molecular size. It is primarily used for the analysis and separation of the relative molecular mass distribution of soluble polymers in organic solvents. The packing material for gel size exclusion chromatography columns is a gel, a surface-inert substance containing numerous pores or a three-dimensional network structure of varying sizes. The pores of the gel only allow component molecules with diameters smaller than the pore opening to enter. These pores are quite large for mobile phase molecules, allowing them to diffuse freely in and out. Depending on the gel packing material used, gel size exclusion columns can separate oil-soluble and water-soluble substances, with a molecular mass range from several million to below 100.

[0100] In one specific embodiment of this application, the selected gel size exclusion column is a HiPrep 16 / 60 Sephacryl series column, a high-resolution gel filtration packing material generally used for fine separation. Specifically, HiPrep 16 / 60 Sephacryl S-100 High Resolution, HiPrep 16 / 60 Sephacryl S-200 High Resolution, and HiPrep 16 / 60 Sephacryl S-300 High Resolution from the HiPrep 16 / 60 Sephacryl series can be selected for the separation of polysaccharide raw materials in this application. In one specific embodiment, the preferred column packing material is HiPrep 16 / 60 Sephacryl S-100 High Resolution, and the column pressure used does not exceed 0.15 MPa.

[0101] In another specific embodiment of this application, the selected gel size exclusion chromatography column is a TSKgel G2000SW column. The packing material of the TSKgel SW series columns is a rigid spherical silica gel matrix with covalently bonded hydrophilic groups on its surface, specifically designed for the separation of proteins and peptides using gel size exclusion chromatography (GFC). The SW series column packing material possesses the necessary properties for high-performance size exclusion chromatography, namely low adsorption and good pore size distribution. Its pH range is 2.5–7.5, and it can use organic solvents completely miscible with water, such as acetonitrile, acetone, methanol, or ethanol. In one specific embodiment, the preferred column packing material is the TSKgel G2000, with a column pressure not exceeding 2.00 MPa.

[0102] In one specific embodiment of this application, the mobile phase used when employing the gel size exclusion chromatography column is a 0.15–1.0 M NaCl aqueous solution, for example, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 M NaCl aqueous solution. Preferably, the mobile phase is a 0.15–0.6 M NaCl aqueous solution. More preferably, the mobile phase is a 0.2 M NaCl aqueous solution.

[0103] In the separation theory of gel size exclusion chromatography, the mobile phase needs to fill the high-energy sites in the gel size exclusion column packing material with a certain concentration of salt ions to ensure separation efficiency; on the other hand, the selection of the mobile phase needs to take into account both the requirements of polysaccharide separation and polysaccharide purification. Current mobile phases can meet both of these requirements.

[0104] In one specific embodiment of this application, the pH value of the mobile phase NaCl aqueous solution is 3 to 10, preferably 5.

[0105] The pH of the mobile phase affects separation efficiency; generally, heparin polysaccharides show the best separation efficiency at pH 5. Some existing literature suggests that there is no significant difference in separation efficiency between pH 5 and 7.

[0106] In one specific embodiment of this application, when using the gel size exclusion column, the flow rate of the mobile phase is 0.1–1.0 mL / min, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mL / min. Preferably, the flow rate of the mobile phase is 0.3–0.7 mL / min. More preferably, the flow rate of the mobile phase is 0.5 mL / min. In one specific embodiment, a person skilled in the art can set the flow rate according to the operable range of the column.

[0107] In one specific embodiment, the polysaccharide raw material is dissolved in the mobile phase at a concentration of 10–240 mg / mL. Preferably, the concentration is 10–120 mg / mL.

[0108] In one specific implementation, the sample loading volume for a single separation is 0.5–5 mL. This parameter can be set according to the specific operating range of the chromatographic column.

[0109] In one specific embodiment of this application, the heparin-based polysaccharide raw material is dissolved in the mobile phase, and different component collection times are set according to the different molecular weights of the samples. In one specific embodiment, one component can be collected every 3 to 30 minutes. According to the principle of gel size exclusion chromatography, components with different molecular weights will be separated sequentially. The first separated component is the high molecular weight polysaccharide, and the later separated component is the low molecular weight polysaccharide. Those skilled in the art can set any collection interval time that allows for effective separation of the samples, thereby achieving the enrichment and purification of polysaccharides of specific molecular weights to obtain a refined polysaccharide salt solution with a specific molecular weight distribution.

[0110] In one specific embodiment, the heparin-based polysaccharide raw material is dissolved in the mobile phase, and collection begins 70–100 minutes after injection. This parameter can be set according to the injection volume.

[0111] In one specific embodiment of this application, the separation components were collected starting 80 minutes after sample injection, and then one component was collected every 10 minutes thereafter, resulting in 11 separation components with different molecular weights.

[0112] Through the above separation and purification steps of heparin-based polysaccharide raw materials, a salt solution of purified polysaccharide with a specific molecular weight distribution is obtained. In order to increase the concentration of polysaccharide and salt in the solution, so as to facilitate alcohol precipitation to obtain more polysaccharide products, the above-mentioned purified polysaccharide salt solution can be freeze-dried (single-stage freeze-drying) to remove excess water.

[0113] In one specific embodiment, the freeze-drying conditions are as follows: the salt solution of the refined polysaccharide is pre-frozen at -80°C, and then freeze-dried in a freeze dryer for 2 days until the moisture is completely removed.

[0114] In one specific embodiment, the freeze-dried product is concentrated and precipitated with alcohol to obtain a desalted purified polysaccharide aqueous solution.

[0115] In one specific embodiment, during the concentration and alcohol precipitation process, the lyophilized product can be resuspended in distilled water to enrich the refined polysaccharides and increase the yield of alcohol precipitation. Specifically, distilled water can be added in an amount of 20% to 50% of the volume of the lyophilized product, preferably 30%. In the above process, the reduction in volume will also further concentrate NaCl, promoting the precipitation of polysaccharides.

[0116] In one specific embodiment, after the above-described resuspension and concentration step, an aqueous ethanol solution is added for sedimentation and alcohol precipitation. Specifically, an aqueous ethanol solution with a concentration of 75% to 100% can be added in an amount 2 to 6 times the volume of the concentrated liquid. Preferably, anhydrous ethanol is added in an amount 5 to 6 times the volume of the concentrated liquid. In the industrial production of heparin, solutions with different ethanol concentrations are often used in the ethanol precipitation step. The precipitation volume is related to the concentration of the polysaccharide; the higher the polysaccharide concentration, the smaller the precipitation volume, and the lower the polysaccharide concentration, the larger the precipitation volume.

[0117] In one specific embodiment, the alcohol precipitation settling time can be 5–60 min. Preferably, it is 10–30 min, and more preferably 10 min. The main purpose of settling is to fully flocculate the polysaccharide and ensure the yield.

[0118] In one specific embodiment, the precipitate after alcohol precipitation is centrifuged, all supernatant is discarded, and distilled water is added again for resuspension to obtain a desalted purified polysaccharide aqueous solution of each component.

[0119] In a preferred embodiment, the precipitated product is centrifuged at 8000g, 4°C, and 10min.

[0120] In one specific embodiment, 0.5–10 mL of distilled water is added for resuspension. Preferably, 2 mL of distilled water is added. This parameter is related to the polysaccharide concentration; the higher the polysaccharide concentration, the larger the volume of distilled water required, and the lower the polysaccharide concentration, the smaller the volume of distilled water required.

[0121] In one specific embodiment, the desalted purified heparin polysaccharide aqueous solution obtained after concentration and alcohol precipitation is subjected to freeze-drying again (secondary freeze-drying) to obtain a purified polysaccharide product that is easy to store.

[0122] In one specific implementation, the processing conditions for secondary freeze-drying are the same as those for primary freeze-drying.

[0123] The method for preparing refined heparin polysaccharides provided in this application enables the controlled separation of heparin polysaccharides by molecular weight, resulting in refined polysaccharide components with relatively concentrated molecular weight distributions. This significantly optimizes the high heterogeneity of polysaccharides and provides favorable conditions for further research on their non-anticoagulant biological activities such as anti-inflammatory, anti-tumor, and anti-fat accumulation, as well as their application in the treatment of ulcerative colitis (UC).

[0124] After verifying the anti-UC biological activity of the various refined polysaccharide components prepared using the above-described method, it was found that some isolated components exhibited superior anti-UC efficacy. Following complete glycan chain analysis and Venn diagram analysis of enriched oligosaccharides in the corresponding refined polysaccharide isolated components, a class of oligosaccharides with specific polysaccharide structural characteristics was found in the anti-UC effective isolated components, suggesting that these are likely anti-UC effective polysaccharide fragments.

[0125] Based on this, this application provides an oligosaccharide with a specific polysaccharide structure, which is mainly obtained from purified polysaccharides effective against UC. The oligosaccharide is characterized by having the following structure:

[0126] [a]-[b,c,d,e,f,g]-[h], where,

[0127] a represents the number of open-ring structures of the oligosaccharide molecule;

[0128] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0129] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0130] d represents the amount of glucosamine in the oligosaccharide molecule;

[0131] e represents the number of acetyl groups in the oligosaccharide molecule;

[0132] f represents the number of sulfonic acid groups in the oligosaccharide molecule;

[0133] g represents the number of 1,6-dehydrated structures in the oligosaccharide molecule;

[0134] h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection;

[0135] Furthermore, the number of glucosamine groups in the structure of the oligosaccharide is between 1 and 10, i.e., 1≤d≤10, and the number of sulfonic acid groups is more than 2.5 times the number of glucosamine groups, i.e., f≥2.5d. This indicates that the oligosaccharide has a highly sulfonated structure.

[0136] In one specific embodiment, the oligosaccharide molecule contains only one unsaturated uronic acid, i.e., b = 1. Since the total number of unsaturated and saturated uronic acids is equal to the number of glucosamines, the number of saturated uronic acids is the number of glucosamines minus 1, i.e., c = d - 1.

[0137] In one specific embodiment, the structure of the above oligosaccharide molecule basically does not contain or contains a small amount of open-ring structures, so the number of open-ring structures is not greater than 0.3 times the number of glucosamine, that is, a≤0.3d.

[0138] In one specific embodiment, the structure of the above oligosaccharide molecule does not contain any dehydration structure, i.e., g = 0.

[0139] In one specific embodiment, the number of acetyl groups in the structure of the above oligosaccharide molecule is no greater than 1, that is, e≤1.0.

[0140] In specific embodiments, the oligosaccharides provided in this application have one of the following structures:

[0141] [0]-[1,2,3,0,9,0]-[0],[0]-[1,2,3,0,8,0]-[0],[0]-[1,3,4,0,12,0]-[0],[2]-[1,3,4,1,10,0]-[1],[0]-[1,4,5,0,15,0]-[5],[1]-[1,4,5,0,14,0]-[1],[1]-[1,4,5,0,13,0]-[0],[ 2]-[1,4,5,1,13,0]-[4],[2]-[1,4,5,1,11,0]-[0],[0]-[1,5,6,0,18,0]-[6],[2]-[1,5,6,1,16,0]-[5],[2]-[1,5,6,1,15,0]-[3],[2]-[1,5,6,1,14,0]-[3] or [1]-[1,5,6,0,15,0]-[3],

[0142] If the above structural formula is represented by [a]-[b,c,d,e,f,g]-[h], then in which,

[0143] a represents the number of open-ring structures of the oligosaccharide molecule;

[0144] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0145] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0146] d represents the amount of glucosamine in the oligosaccharide molecule;

[0147] e represents the number of acetyl groups in the oligosaccharide molecule;

[0148] f represents the number of sulfonic acid groups in the oligosaccharide molecule;

[0149] g represents the number of 1,6-dehydrated structures in the oligosaccharide molecule;

[0150] h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection.

[0151] In one specific embodiment, the oligosaccharide with effective anti-UC properties provided in this application has the following structural features:

[0152] The oligosaccharide has a sugar chain length between 2 and 20 sugars, and the basic disaccharide unit is composed of repeated arrangements of [0]-[1,0,1,0,3,0]-[0] and / or [0]-[1,0,1,0,2,0]-[0], wherein,

[0153] [0]-[1,0,1,0,3,0]-[0] represents a disaccharide structure fragment with 0 open ring structures, 1 unsaturated uronic acid, 0 saturated uronic acid, 1 glucosamine, 0 acetyl groups, 3 sulfonic acid groups, 0 dehydrated structures, and 0 ammonia in the mass spectrum.

[0154] [0]-[1,0,1,0,2,0]-[0] represents a disaccharide structure fragment with 0 open ring structures, 1 unsaturated uronic acid, 0 saturated uronic acid, 1 glucosamine, 0 acetyl groups, 2 sulfonic acid groups, 0 dehydrated structures, and 0 ammonia in the mass spectrum.

[0155] Furthermore, in one specific embodiment, the oligosaccharide exhibits highly sulfonated characteristics, with the average number of sulfonic acid groups in its basic disaccharide unit being greater than or equal to 2.5. This means that the basic disaccharide structural unit is mainly composed of the structure shown in [0]-[1,0,1,0,3,0]-[0], while the content of the structure shown in [0]-[1,0,1,0,2,0]-[0] is relatively small.

[0156] Furthermore, in one specific embodiment, the oligosaccharide contains essentially no or only a small amount of open-ring structures, and the average number of open-ring structures in the basic disaccharide unit is less than or equal to 0.3. This application also provides a heparin derivative containing the oligosaccharide with anti-UC efficacy as described above, and the content of said oligosaccharide is 17% or more, preferably 18% or more, preferably 19% or more, preferably 20% or more, preferably 21% or more, preferably 22% or more, preferably 23% or more.

[0157] On the other hand, this application provides an oligosaccharide with a specific polysaccharide structure, which is mainly obtained from purified polysaccharides with anti-inflammatory effects, characterized in that the oligosaccharide has the following structure:

[0158] [a]-[b,c,d,e,f,g]-[h], where,

[0159] a represents the number of open-ring structures of the oligosaccharide molecule;

[0160] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0161] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0162] d represents the amount of glucosamine in the oligosaccharide molecule;

[0163] e represents the number of acetyl groups in the oligosaccharide molecule;

[0164] f represents the number of sulfonic acid groups in the oligosaccharide molecule;

[0165] g represents the number of 1,6-dehydrated structures in the oligosaccharide molecule;

[0166] h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection;

[0167] Furthermore, the number of glucosamines in the structure of the oligosaccharide is between 1 and 10, i.e., ≤d≤10. Since the number of glucosamines d is also equal to the number of disaccharide units, it means that the oligosaccharide is composed of d disaccharide units, and the length of the entire oligosaccharide sugar chain is 2d. The number of sulfonic acid groups contained in each disaccharide is less than or equal to 2.7, i.e., f≤2.7d.

[0168] In one specific embodiment, the oligosaccharide molecule contains only one unsaturated uronic acid, i.e., b = 1. Since the total number of unsaturated and saturated uronic acids is equal to the number of glucosamines, the number of saturated uronic acids is the number of glucosamines minus 1, i.e., c = d - 1.

[0169] In one specific embodiment, the number of open sugar rings in each disaccharide of the above oligosaccharide molecule is greater than or equal to 0.25, that is, a≥0.25d.

[0170] In one specific embodiment, the structure of the above oligosaccharide molecule does not contain any dehydration structure, i.e., g = 0.

[0171] In one specific embodiment, the number of acetyl groups in each disaccharide of the above oligosaccharide molecule is greater than or equal to 0.1, that is, ≥0.1d.

[0172] In specific embodiments, the oligosaccharides provided in this application have one of the following structures:

[0173] [4]-[1,9,10,1,26,0]-

[12] ,[4]-[1,9,10,1,25,0]-

[10] ,[3]-[1,8,9,1,23,0]-[9],[3]-[1,8,9,1,24,0]-

[10] ,[3]-[1,7,8,1,19,0]-[7],[1]-[1,7,8,3,19,0]-[8],[3]-[1,7,8,1,20,0]-[8],[2]-[1,6,7,1,18,0]-[7],[3]-[1,8 ,9,1,22,0]-[8],[3]-[1,8,9,2,13,0]-[4],[1]-[1,5,6,0,17,0]-[5],[1]-[1,6,7,0,19,0]-[7],[2]-[1,7,8,1,20,0]-[8],[2]-[1,6,7,1,17,0]-[7],[1]-[1,6,7,0,20,0]-[8],[0]-[1,0,1,0,3,0]-[0]or[3]-[1,4,5,1,8,0]-[7],

[0174] If the above structural formula is represented by [a]-[b,c,d,e,f,g]-[h], then in which,

[0175] a represents the number of open-ring structures of the oligosaccharide molecule;

[0176] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0177] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0178] d represents the amount of glucosamine in the oligosaccharide molecule;

[0179] e represents the number of acetyl groups in the oligosaccharide molecule;

[0180] f represents the number of sulfonic acid groups in the oligosaccharide molecule;

[0181] g represents the number of 1,6-dehydrated structures in the oligosaccharide molecule;

[0182] h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection.

[0183] This application also provides a heparin derivative containing the anti-inflammatory oligosaccharide as described above, wherein the content of the oligosaccharide is 52% or more, preferably 65% ​​or more, preferably 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 93% or more.

[0184] In this application, there are no specific limitations on the methods for analyzing the structure and determining the content of polysaccharide components; any method known to those skilled in the art can be used.

[0185] In one specific implementation, the polysaccharide components in the sample to be tested are first extracted using methods known to those skilled in the art. Subsequently, the polysaccharide components are analyzed using a full-chain glycan map by the National Institute of Metrology (NIM) of China, obtaining detection results that record the oligosaccharide content and distribution information of the polysaccharide sample. Based on these detection results, the number of open-ring structures of the sugar rings, the number of unsaturated uronic acids, the number of saturated uronic acids, the number of glucosamines, the number of acetyl groups, the number of sulfonic acid groups, the number of 1,6-dehydrated structures, and the number of ammonium ions carried by the oligosaccharide molecules in mass spectrometry detection can be confirmed.

[0186] Based on the oligosaccharide content and distribution information, the structure of the polysaccharide component can be reconstructed using methods known to those skilled in the art (specific methods can be found in the description of the reconstruction process in Experimental Example 3 of this invention) to confirm the basic disaccharide unit structure of the oligosaccharide, as well as the average number of sulfonic acid groups contained in the basic disaccharide unit and the average number of sugar ring open structures in the basic disaccharide unit.

[0187] Furthermore, based on the whole sugar chain mapping analysis method, the oligosaccharide content and distribution information of the sample can be obtained, and the content of oligosaccharides that conform to each structure can also be calculated.

[0188] This application also relates to the use of the oligosaccharides provided in this application and de-anticoagulated heparin derivatives containing said oligosaccharides in the treatment of inflammatory bowel disease, such as ulcerative colitis and Crohn's disease.

[0189] This application also relates to the use of the oligosaccharides provided in this application and de-anticoagulated heparin derivatives containing said oligosaccharides in the preparation of medicaments for treating inflammatory bowel disease and diseases with similar pathogenesis and complications of inflammatory bowel disease, wherein the diseases with similar pathogenesis and complications of inflammatory bowel disease include, but are not limited to, irritable bowel syndrome, arthritis and other extraintestinal complications including ankylosing spondylitis, pyoderma gangrenosa, erythema nodosum, iritis, uveitis, episcleritis, primary sclerosing cholangitis and rheumatoid arthritis.

[0190] In a specific embodiment of this application, the oligosaccharide with anti-UC efficacy is prepared by the method for preparing the refined heparin polysaccharide provided in this application.

[0191] Example

[0192] Example 1: Preparation of polysaccharide raw materials

[0193] 20g of high-quality heparin (purchased from Changshan Biochemical Pharmaceutical Co., Ltd., product name: sodium heparin) was dissolved in 0.6L of deionized water. An equal volume of 0.2M sodium periodate solution (freshly prepared) was added to the 0.6L high-quality heparin (33g / L), and the mixture was reacted at 300rpm and 4℃ in the dark for 22 hours. 80mL of ethylene glycol was added to neutralize the excess sodium periodate, and then 28g of sodium borohydride was added, and the mixture was reacted at 4℃ for 16 hours. The pH was adjusted to 7.0 with HCl. The sample was filtered through a 0.22μm filter membrane and collected. The sample was then desalted using a dialysis bag or through a Millipore ultrafiltration device with a 1K filter membrane until the filtrate showed no color change when tested with 0.1M AgNO3, indicating that desalting was complete. The sample was frozen at -80℃ and then lyophilized in a freeze dryer. It was then pulverized into powder using a mortar and pestle or a small grinder for storage to obtain the de-anticoagulated heparin derivative (named NAHP).

[0194] The de-anticoagulated heparin derivative (NAHP) obtained above was dissolved in reaction buffer. Heparinase I, prepared according to method ZL200410038098.6, was added to the solution every 0.5–1 hour, with 20 IU of heparinase I added each time. The absorbance (A231) at 231 nm was monitored using a quartz cuvette with a 1 cm path difference and a UV spectrophotometer (the instrument was calibrated and zeroed using a pH 7.4 buffer; for accuracy, when the UV spectrophotometer reading A231 was greater than 0.6, the solution was diluted a certain factor to bring the reading to 0.2–0.6 for measurement). The reaction was stopped when A231 reached 46, at which point the total heparinase I activity reached approximately 220–250 IU. The method for ending the reaction was as follows: the enzyme in the reaction solution was inactivated in a boiling water bath at 100℃ for 5-10 min. The reaction system was then removed and cooled to room temperature. Six volumes of anhydrous ethanol were added to the reaction solution, and the mixture was stirred at room temperature for 10 min. Then, it was centrifuged at 4000 r / min for 15 min at room temperature. The precipitate was collected, and two to three times its mass of deionized water was added to the precipitate to dissolve it completely. The solution was then filtered through a 0.22 μm pore size filter membrane. The permeate was collected and frozen into solid ice blocks at -80℃. The ice blocks were then freeze-dried using a freeze dryer (cold trap temperature -50℃) and then pulverized into powder using a mortar and pestle or a small grinder to obtain low molecular weight de-anticoagulant heparin (named: LNAHP).

[0195] The de-anticoagulated heparin derivative (NAHP) obtained above was dissolved in the reaction solution. Heparinase I, prepared according to method ZL200410038098.6, was added to the solution every 0.5–1 hour, with 20 IU of heparinase I added each time. The absorbance (A231) at 231 nm was monitored using a quartz cuvette with a 1 cm path difference and a UV spectrophotometer (the instrument was calibrated and zeroed using a pH 7.4 buffer solution; for accuracy, when the UV spectrophotometer reading A231 was greater than 0.6, the solution was diluted a certain factor to bring the reading to 0.2–0.6 for measurement). The reaction was stopped when A231 reached 10⁶. At this point, the total heparinase I activity reached approximately 340–380 IU. The method for ending the reaction was to inactivate the enzyme in the reaction solution in a boiling water bath at 100℃ for 5-10 min, then remove the reaction system and cool it to room temperature. Six volumes of anhydrous ethanol were added to the reaction solution, and the mixture was stirred at room temperature for 10 min. Then, it was centrifuged at 4000 r / min for 15 min at room temperature. The precipitate was collected, dissolved in 2-3 times the mass of deionized water, filtered through a 0.22 μm membrane, and the permeate was collected and frozen into solid ice blocks at -80℃. The ice blocks were then freeze-dried in a freeze dryer (cold trap temperature -50℃) and then pulverized into powder using a mortar and pestle or a small grinder to obtain ultra-low molecular weight de-anticoagulant heparin (also named: ULNAHP).

[0196] Example 2: Preparation of purified polysaccharide components

[0197] Step (1): Isolation of polysaccharides

[0198] Experimental equipment: An AKTA Prime purification system was used, along with a gel size exclusion column (HiPrep 16 / 60 Sephacryl S-100HR) for preparative liquid chromatography.

[0199] Process parameters: The mobile phase is 0.2M NaCl aqueous solution (pH=5.00), the flow rate is 0.5mL / min, the column volume is 120mL, and the maximum sample loading for one separation is 5mL.

[0200] Experimental Method: The polysaccharide raw material LNAHP obtained in Example 1 was dissolved in the mobile phase at a concentration of 120 mg / mL. Depending on the molecular weight of the sample, the separated fractions were collected starting 80 minutes after injection. Based on the principle of gel size exclusion chromatography, the high molecular weight polysaccharides were separated first, followed by the low molecular weight polysaccharides. One fraction was collected every 10 minutes during the separation process, ending at 180 minutes. Based on the different collection times, fractions with different molecular weights were obtained, thereby achieving the enrichment and purification of polysaccharides with specific molecular weights, resulting in a refined polysaccharide salt solution with a specific molecular weight distribution. The refined polysaccharide fraction collected at 110 minutes after raw material injection was named S4, and the refined polysaccharide fraction collected at 130 minutes was named S6.

[0201] Step (2): One-time freeze-drying of polysaccharide solution

[0202] The salt solutions of each component of refined polysaccharide obtained in step (1) are pre-frozen at -80°C and then freeze-dried in a freeze dryer until the water is completely removed, thus obtaining the first freeze-dried product of each component of polysaccharide solution.

[0203] Step (3): Concentration and alcohol precipitation

[0204] The lyophilized products of each component obtained in step (2) were resuspended in distilled water at 30% of their original volume to enrich the refined polysaccharides and increase the alcohol precipitation yield. Subsequently, the polysaccharide solution was precipitated with anhydrous ethanol, thoroughly mixed, allowed to stand, and then centrifuged. After centrifugation, all supernatant (a mixture of ethanol and NaCl containing a small amount of soluble polysaccharides) was carefully discarded. The precipitate was then resuspended in an appropriate amount of distilled water to obtain the desalted purified heparin-like polysaccharide aqueous solution.

[0205] Step (4): Secondary freeze-drying of polysaccharide solution

[0206] The desalted purified polysaccharide aqueous solution obtained in step (3) is pre-frozen at -80℃ and then freeze-dried in a freeze dryer until the water is completely removed, thus obtaining the purified polysaccharide freeze-dried product.

[0207] After preliminary verification of the anti-UC function and anti-inflammatory effects of the isolated components obtained by the above method, it was found that component S6 had a superior anti-UC therapeutic effect; at the same time, components S4 and S6 also had superior anti-inflammatory effects. In the following experiments of this application, the anti-inflammatory and anti-UC biological activities of S4 and S6 will be further verified and their structure-activity relationship will be analyzed. Specifically, the de-anticoagulated heparin derivative (NAHP), low molecular weight de-anticoagulated heparin (LNAHP), and ultra-low molecular weight de-anticoagulated heparin (ULNAHP) used in the following experimental examples were all obtained by the method of Example 1; the polysaccharide isolated components S4 and S6 were obtained by the method of Example 2; the heparin (HP) used was ungraded heparin purchased from Hebei Changshan Biochemical, and the weight-average molecular weight (Mw) of this heparin was 17223 Da; the 5-aminosalicylic acid preparation (5-Amino) used... Salicylic Acid (5-ASA) is a mesalazine sustained-release granule purchased from a pharmacy. Its indications are: ulcerative colitis, used for acute exacerbations of ulcerative colitis to prevent recurrence; Crohn's disease, used for patients with frequently recurring Crohn's disease to prevent acute exacerbations. The NAEno used is a de-anticoagulated enoxaparin derivative obtained from enoxaparin (purchased from Changshan Biochemical) using the same de-anticoagulation modification process as NAHP in Example 1. The NAI45 used is a low molecular weight de-anticoagulated heparin derivative obtained from heparin (purchased from Changshan Biochemical) by enzymatic hydrolysis according to the method in Comparative Example 5 of ZL201810100469.0, followed by the same de-anticoagulation modification process as NAHP in Example 1 of this application. It has been found to be ineffective in treating UC in previous preliminary in vivo functional verification experiments.

[0208] Experimental Example 1: Determination of the molecular weight and distribution of purified heparin polysaccharide components

[0209] The weight-average molecular weight (Mw) and distribution coefficient (P) of the polysaccharide raw material LNAHP obtained in Example 1 and the purified polysaccharide fraction S6 obtained in Example 2 were determined by gel size exclusion high-performance liquid chromatography (HPLC). A TSK-GEL G2000SWXL column (TOSOH, Japan) was used, with a flow rate of 0.5 mL / min, a column temperature of 35 °C, and an injection volume of 25 μL. A WATERS (1525, USA) chromatographic system was used, with a UV detector and a differential detector connected in series at the column outlet. The UV detector wavelength was 234 nm. The method for determining molecular weight and its distribution can be found in Wu, Jingjun et al., "Controllable production of low molecular weight heparins by combinations of heparinase I / II / III." Carbohydrate polymers 101 (2014):484-492. Specific results are shown in Table 1.

[0210] Table 1. Molecular weights of refined polysaccharide raw materials and the separated components obtained from the refining process.

[0211]

[0212] As shown in Table 1, the purified components S4 and S6 exhibit significantly different molecular weight distributions compared to the polysaccharide raw material. Specifically, this difference is reflected in polydispersity, specifically the distribution coefficient P. Polydispersity is the ratio of weight-average molecular weight to number-average molecular weight, and is a positive number always greater than 1. The closer this value is to 1, the closer the substance is to a single substance with a clearly defined molecular weight. In this embodiment, the polydispersity of the polysaccharide raw material is greater than 1.6. After purification and separation, the polydispersity of the separated components is even less than 1.1, indicating that the molecular weight distribution of the purified polysaccharide is significantly more concentrated and closer to that of a pure substance.

[0213] Experiment Example 2: Verification of the anti-UC and anti-inflammatory biological activities of purified polysaccharide isolated fractions

[0214] 1. The in vivo efficacy of the purified polysaccharide fraction obtained in Example 2 against UC was investigated using DSS-induced UC mouse models. Evaluation indicators mainly included changes in body weight, colon length, spleen weight index, serum inflammatory factor levels, and colonic histopathological evaluation, systematically assessing the efficacy of the purified polysaccharide in treating UC.

[0215] Experimental methods:

[0216] The animal experimental model was selected from 6-8 week old male C57BL / 6J mice. A healthy control group (WT group), a DSS modeling group, and a post-modeling drug treatment group were set up.

[0217] On Day 0, except for the WT group, the drinking water of the other groups was replaced with 3% DSS (sodium dextran sulfate, Mw: 36,000-50,000 Da, MP biomedicals, LLC) aqueous solution to induce the UC mouse model, and the weight of the mice was recorded daily.

[0218] On Day 3, UC mouse models in different drug treatment groups were treated by gavage at a drug dose of 30 mg / kg / mouse / day, which continued until Day 7.

[0219] Day 7, the experiment concluded. The mice were euthanized and dissected to obtain spleen and colon tissue. The spleen tissue was weighed, and the spleen weight index was calculated based on spleen weight and body weight to evaluate the in vivo anti-inflammatory and immunomodulatory effects of the refined polysaccharide isolate. Colon length was measured, and the colon tissue was then paraffin-embedded and histopathological sections were stained with H&E. The in vivo anti-UC efficacy of the refined polysaccharide isolate was evaluated based on changes in the colonic epithelial tissue structure of UC mice. Specific experimental results are as follows: Figures 1-4 As shown.

[0220] Experimental results:

[0221] (1) Weight loss is one of the important phenotypes of DSS-induced UC mouse model, and this indicator can characterize the severity of the disease. Figure 1 The ANOVA analysis plot shows the weight loss curves of mice. It can be seen that during the treatment period from day 3 to day 7, the trend of weight loss in mice treated with oral HP, NAI45, and NAEno was not significantly improved compared to the DSS group. On day 6, the trend of weight loss in mice treated with oral effective drugs (LNAHP, ULNAHP) and purified polysaccharide isolate S6 was significantly alleviated compared to the DSS group (p<0.05, p<0.01, p<0.05). On day 7, oral NAHP and first-line clinical drugs (5ASA) significantly alleviated the DSS-induced weight loss trend in mice (p<0.05, p<0.01), while oral LNAHP, ULNAHP, and S6 mice showed a more significant alleviation of the weight loss trend (p<0.0001, p<0.0001, p<0.0001). The results show that the isolated component S6 can effectively alleviate the weight loss trend in UC mice, and compared with the first-line clinical drug 5ASA, it has a more significant effect in alleviating the symptoms of weight loss in UC mice.

[0222] (2) Shortened colon length is one of the characteristic phenotypes of the UC mouse model and is also closely related to the severity of the disease. Figure 2 The results of colon length measurement in mice of each experimental group are shown. Compared with the DSS group, except for the oral HP, NAEno, and NAI45 treatment groups, oral administration of other heparin derivatives and 5ASA effectively alleviated the colon length shortening induced by DSS in UC mice. Among them, the S6 treatment group showed the best effect in alleviating colon length shortening, with the greatest statistical significance compared to the DSS group (p<0.0001). These results indicate that the purified polysaccharide fraction S6 is more effective in alleviating the symptoms of colon length shortening in UC mice, and its efficacy is more significant than that of the first-line clinical drug 5ASA.

[0223] (3) As an important immune organ, the spleen becomes enlarged and its mass increases when the systemic immune system is activated. Splenomegaly is also one of the clinical symptoms of UC patients, which is closely related to the chronic and persistent activation of the immune system. The spleen weight index can be used to characterize the activation status of an individual's immune system. Figure 3 This is a schematic diagram of the spleen weight index. It can be seen that the DSS model group mice showed significantly greater splenomegaly and an increased spleen weight index compared to the WT group mice. Compared to the DSS group, oral administration of NAHP, LNAHP, S6, and 5ASA significantly alleviated the symptoms of splenomegaly in UC mice and inhibited the increase in spleen weight index (p<0.01, p<0.01, p<0.01, p<0.01); while oral administration of NAEno and NAI45 did not effectively alleviate splenomegaly and the increase in spleen weight index. These results indicate that the purified polysaccharide fraction S6 can significantly alleviate splenomegaly in UC mice and inhibit the increase in spleen weight index, exhibiting significant immunosuppressive ability; its efficacy in inhibiting splenomegaly and the increase in spleen weight index is comparable to that of 5ASA.

[0224] (4) Histopathological evaluation of colonic mucosa is the gold standard for clinical diagnosis, treatment and efficacy evaluation of UC, and it is also an important indicator for evaluating the biological activity of decoagulated heparin derivatives and purified isolated components in the treatment of UC. Figure 4H&E staining was used to evaluate the histopathological changes of the colonic epithelium in UC mice. The WT group mice exhibited normal colonic epithelial structure, with intact epithelial cells, normal crypt structure, regular crypt arrangement, abundant goblet cells, and no neutrophil infiltration. The DSS group mice showed severe neutrophil and macrophage infiltration in the colonic epithelium. Compared to the WT group, there was significant shedding and necrosis of the original colonic epithelial cells, a large loss of goblet cells, complete disruption of the colonic epithelial barrier, and complete disappearance of crypt structure. Compared to the DSS group mice, oral administration of HP, NAEno, and NAI45 did not effectively alleviate the disease symptoms such as colonic epithelial structure destruction and epithelial cell shedding. Oral administration of NAHP, ULNAHP, and 5ASA partially improved the loss of colonic crypts and epithelial cell shedding and necrosis, but the integrity of the colonic epithelium and colonic crypts was poor, crypt morphology and arrangement were abnormal, significant inflammatory cell infiltration remained between crypts, and goblet cell loss was still observed. Oral administration of LNAHP and S6 significantly protected the colonic epithelial tissue of mice, maintaining its structural integrity, normal crypt shape and regular arrangement, and exhibiting a normal distribution of goblet cells and a small amount of inflammatory cell infiltration. Histopathological evaluation showed that the purified polysaccharide fraction S6 significantly alleviated the damage to the colonic epithelial tissue structure in UC mice, restored the colonic epithelial tissue structure to normal, and inhibited inflammatory responses in the peripheral blood and lamina propria. Compared with the first-line clinical drug 5ASA, it exhibited superior biological activity for the treatment of UC.

[0225] Based on the above comprehensive evaluation indicators, the purified polysaccharide fraction S6 exhibits excellent anti-UC biological activity, and its efficacy-related indicators are superior to those of the first-line clinical drug 5ASA.

[0226] 2. An in vitro inflammation model was constructed using the lipopolysaccharide-induced mouse macrophage line RAW 264.7 to investigate the anti-UC biological activity of the purified polysaccharide fractions S4 and S6 obtained in Example 2 above.

[0227] Experimental methods:

[0228] Mouse macrophages RAW 264.7 (purchased from ATCC) were seeded into 48-well plates at a concentration of 150,000 cells / mL and cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. After overnight culture, the old medium was discarded, and the cells were washed once with PBS. A healthy control (WT) group and a lipopolysaccharide (LPS) modeling group were set up, with the remainder being drug treatment groups. The WT group received only serum-free medium, while the other groups received serum-free medium containing 100 ng / mL LPS. Cells from each group were then incubated in a cell culture incubator for 15 min. After 15 min, heparin, heparin derivatives, and isolated fractions S4 and S6 were added to the drug treatment groups at a concentration of 1 mg / mL, and treatment lasted for 24 hours. After 24 hours, the supernatant was collected, and ELISA was used to detect inflammatory markers such as IL-6 and TNF-α secreted by RAW264.7 cells (group setup and detection results are shown in the table below). Figure 5 (As shown).

[0229] Experimental results:

[0230] Persistent chronic inflammation, a key clinical phenotype of ulcerative colitis (UC), is the primary target of UC treatment drugs. In the aforementioned DSS-induced UC mouse model experiment, S6 was found to possess good anti-UC biological activity. However, the mechanism of action of effective anti-UC heparin derivatives in inhibiting inflammation remains unclear. To confirm whether the effective anti-UC polysaccharide components possess significant anti-inflammatory activity, LPS-stimulated RAW 264.7 cells were constructed as an in vitro model to evaluate the in vitro anti-inflammatory effects of heparin derivatives and purified heparin polysaccharides. RAW 264.7 cells, as mouse macrophages, are an important component of innate immunity. Upon stimulation by exogenous antigens such as LPS, they secrete large amounts of cytokines such as IL-6 and TNF-α, triggering sustained activation of the immune system. IL-6 and TNF-α are highly expressed in UC patients and are also effective targets of UC treatment drugs. Therefore, examining the expression levels of cytokines such as IL-6 and TNF-α can evaluate the overall level of inflammatory response.

[0231] from Figure 5As shown in the schematic diagram of the in vitro anti-inflammatory activity evaluation of each group, LPS stimulation caused RAW 264.7 cells to secrete large amounts of IL-6 and TNF-α. In terms of anti-IL-6 activity, anticoagulant heparin (HP) could effectively alleviate the increase in IL-6 secretion in RAW264.7 cells (p<0.01). NAHP, LNAHP, S4, S6, NAEno, and NAI45, obtained after HP was de-anticoagulated, exhibited more significant anti-IL-6 secretion activity (p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.0001). Regarding anti-TNF-α activity, anticoagulated heparin (HP) failed to effectively alleviate the increase in TNF-α levels in RAW 264.7 cells. In contrast, LNAHP, S4, S6, and 5ASA exhibited significant inhibitory activity against TNF-α secretion (p<0.01, p<0.001, p<0.05, p<0.01). These results indicate that LNAHP, S4, and S6 exhibited the best in vitro anti-inflammatory activity. These results also confirm that HP, after decoagulation modification and enzymatic hydrolysis, can exhibit more significant anti-inflammatory activity. This suggests that the anti-inflammatory activity of heparin drugs may be related to the preparation process of decoagulated heparin derivatives; heparin derivatives prepared using a process of first decoagulation followed by enzymatic hydrolysis generally exhibited the best in vitro anti-inflammatory effect.

[0232] Experiment Example 3: Verification of the anti-UC efficacy and anti-inflammatory activity of purified polysaccharide fractions and structure-activity relationship analysis

[0233] Through the screening of anti-UC performance and in vitro anti-inflammatory activity in Experiment Example 2 above, de-anticoagulated heparin derivatives and refined polysaccharide fractions S4 and S6 with relatively excellent performance were discovered. In order to further analyze the anti-inflammatory activity and structure-activity relationship of the refined polysaccharide fractions in this application for the treatment of UC, the National Institute of Metrology of China was commissioned to conduct complete glycan mapping analysis on several different heparin derivatives and refined polysaccharide fractions S4 and S6 to explore the structural fragments that exert the anti-inflammatory and anti-UC properties of heparin. Complete glycan mapping analysis is an important means of characterizing the structure of low molecular weight de-anticoagulated heparin, and it is a glycan structure analysis method based on liquid chromatography-high resolution mass spectrometry (LC-MS).

[0234] The specific method is as follows: LNAHP, ULNAHP, S4, S6, and the anti-UC ineffective heparin derivative NAI45 were selected. The heparin derivatives were dissolved in water, and the samples were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument (instrument manufacturer: Thermo Scientific, instrument model: UHPLC-LTQ-Orbitrap, instrument serial number: SN04010B) for detection, and the mass spectrometry signals were collected. The LC detection parameters were: column, 3μm HILIC 150×2mm; detector: high-resolution mass spectrometry; column temperature: 22℃; flow rate: 0.15ml / min; injection volume: 3μL; run time: 100min; mobile phase: acetonitrile-water system. Mass spectrometry detection parameters were: sheath gas flow: 20arb; aux gas flow: 5arb; I spray voltage: 4.2kV; capillary temp: 275℃; S-Lens RF Level: 50%. The raw mass spectrometry data were extracted using Xcalibur software to obtain the precise mass-to-nucleus ratio of specific glycan structures (accurate to 4 decimal places, mass tolerance set to 5ppm), and then integrated to obtain the peak area, thereby obtaining the structural and abundance information of each oligosaccharide component.

[0235] Having obtained the structural and abundance information of the aforementioned oligosaccharide components, we will further analyze which oligosaccharide components are effective against UC. The specific method is as follows:

[0236] Using R Studio software (Version 1.2.1335) on the Windows 10 operating system, and employing R programming language combined with packages such as pheatmap for visualization, the content levels of different oligosaccharides in different samples were visualized using heatmaps, yielding the total sugar chain spectral analysis results for the aforementioned groups of substances, such as... Figure 6 As shown in the figure (each row represents an oligosaccharide component, and each column represents the oligosaccharide enrichment status of different samples. The data has been row normalized. Components with enriched oligosaccharides are darker in color, components without enrichment are lighter in color, and components without oligosaccharides are white).

[0237] 1. By Figure 6 It can be seen that the oligosaccharide composition of the effectively separated component S6 differs significantly from that of other heparin derivatives. Based on this, to further obtain the functional fragments from which the effective heparin derivatives exert their biological activity, the oligosaccharides in the complete glycan map analysis were sorted from highest to lowest content, and their contents were calculated, resulting in a graph showing the relationship between oligosaccharide coverage and oligosaccharide enrichment thresholds. Figure 7 ).Depend on Figure 7 It can be seen that the oligosaccharide enrichment threshold is between 0% and 40%, and the oligosaccharide coverage increases rapidly, indicating that the top 40% of oligosaccharide components have good representativeness. Therefore, in subsequent analyses, the top 40% of oligosaccharides were selected as the enriched oligosaccharides for each heparin derivative and isolated component, representing the main components of each heparin derivative and isolated component.

[0238] Based on this, using R Studio software (Version 1.2.1335) on the Windows 10 operating system, and employing R language programming and packages such as UpSetR, Venn diagram analysis was performed on the enriched oligosaccharides of various heparin derivatives and isolated components. Figure 8 It is known that a common class of polysaccharides exists among the heparin derivatives effective against UC. Therefore, a class of polysaccharides enriched in LNAHP and S6 but not in NAI45 is defined as the effective component, a class of polysaccharides enriched in NAI45 but not in LNAHP and S6 is defined as the ineffective component, and the remaining components are defined as other components.

[0239] Using R Studio software (Version 1.2.1335) on a Windows 10 operating system, and combining R language programming with packages such as ggplot2, stringr, pheatmap, ggsci, and UpSetR, we can... Figure 8 The corresponding oligosaccharide data were analyzed, and the structural feature information of the corresponding components was extracted. Figure 9 It can be seen that, compared with the ineffective and other components, the effective component has a higher number of sulfonic acid groups and a lower number of open sugar rings. Therefore, it can be inferred that the effective oligosaccharide for anti-UC is characterized by high sulfonation and the absence of open sugar ring structures. Based on... Figure 8 The classification results are summarized in Table 2, which shows the information on the effective oligosaccharide components obtained from the enriched oligosaccharide classification.

[0240] Table 2 Summary of oligosaccharide information of effective anti-UC components

[0241]

[0242] Note: The oligosaccharide structure representation means [polysaccharide ring-opening structure] - [unsaturated uronic acid - saturated uronic acid - glucosamine - acetyl group - sulfonic acid group - dehydration structure] - [amino group (i.e., the number of ammonium ions carried in mass spectrometry, not necessarily the total number of ions in the polysaccharide structure itself)].

[0243] Based on this, the contents of various oligosaccharides in heparin derivatives and isolated components were statistically analyzed, and the results are shown in Table 3. The effective components listed in Table 3 were highly enriched in the anti-UC effective heparin derivatives LNAHP, ULNAHP, and S6, while their contents were lower in other heparin derivatives, which is consistent with the in vivo anti-UC efficacy of heparin derivatives.

[0244] Table 3 Summary of the content of various oligosaccharides in heparin derivatives and isolated fractions

[0245]

[0246]

[0247] Based on this, the effective components in Table 2 were further analyzed, and the oligosaccharides of the effective components were reconstructed in the following manner in conjunction with the biosynthesis process of heparin derivatives:

[0248] (1) First, reconstruct the polysaccharide backbone: determine the length of the heparin sugar chain based on the number of unsaturated uronic acid, uronic acid and glucosamine, and construct the basic backbone of the effective components in the form of alternating arrangement of uronic acid and glucosamine.

[0249] (2) Next, the open-ring structure and acetyl group are reconstructed: Based on the synthesis mechanism of heparin polysaccharide, the open-ring structure is reconstructed onto uronic acid, and the acetyl group is reconstructed onto glucosamine. Depending on the subsequent reconstruction of the sulfonic acid group, the above reconstructed structure may need to be finely adjusted according to the number of sulfonic acid groups.

[0250] (3) Finally, the sulfonic acid group is reconstructed: This is mainly determined by the number of sulfonic acid groups, combined with the number of open-ring structures and acetyl groups, as well as probability theory. Priority is given to reconstructing the sulfonic acid groups of the disaccharide with the NS domain that is highly present in heparin, and then the reconstruction of sulfonic acid groups on the sugar ring containing open-ring structures and acetyl groups is considered.

[0251] The molecular structure characteristics of the effective anti-UC oligosaccharide component can be obtained through the above reconstruction. Table 4 shows the polysaccharide structure drug composition, molecular formula, and structural formula of the effective component.

[0252] Table 4. Structural composition and molecular formula of effective anti-UC oligosaccharides

[0253]

[0254]

[0255]

[0256] Note 1: [Ring-opening]-[△HexA,HexA,HexN,Ac,SO3,Dehydration]-[NH3] indicates [polysaccharide ring-opening structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure]-[amino group (the number of ammonium ions carried in the mass spectrometry detection, not all of the polysaccharide structure itself)]

[0257] Note 2: In the "Reconstruction of Major and Minor Structures" column, the major structure is the molecular structure most likely to appear based on the characteristics of the biosynthesis process of heparin and the preparation process of heparin derivatives; the minor structure is the molecular structure that exists in the biosynthesis process of heparin and the preparation process of heparin derivatives, but is not the molecular structure reached by the major reaction. It is estimated that its number is at least one order of magnitude smaller than that of the major structure.

[0258] Note 3: In the "Reconstruction of Primary and Secondary Structures" column, △UA represents unsaturated uronic acid, HexA represents uronic acid, GlcA represents glucuronic acid, IdoA represents idurouronic acid, and GlcNAc represents N-acetylglucosamine; Ω is the symbol for ring-opening modification of the sugar ring, and NS / 6S / 3S / 2S represent N-sulfonic acid, 6-O-sulfonic acid, 3-O-sulfonic acid, and 2-O-sulfonic acid modifications on uronic acid and glucosamine, respectively; symbols such as "()" and "[]" indicate that polysaccharide structures within the same symbol can be interchanged in sequence.

[0259] Based on the data in Tables 2, 3, and 4, it can be found that the structural characteristics of effective polysaccharides against UC are that they contain little or no open-ring structures (produced by heparin decoction) and are highly sulfated polysaccharide fragments. Enrichment of these polysaccharide fragments and polysaccharides composed of these polysaccharide fragments have therapeutic effects on UC.

[0260] Thus, we have discovered a class of oligosaccharide components with specific polysaccharide structures. These oligosaccharide components are mainly derived from heparin derivatives effective against UC and are highly enriched in LNAHP and S6. Based on this discovery, further inferences suggest that ideal oligosaccharide fragments for exerting anti-UC biological activity should possess the following structural characteristics:

[0261] (1) The sugar chain length of polysaccharides is between 2 and 20 sugars. The basic disaccharide structural unit is composed of repeated combinations of [0]-[1,0,1,0,3,0]-[0] and [0]-[1,0,1,0,2,0]-[0]. The above representation means [polysaccharide ring-opening structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydrated structure]-[amino group (the number of amino groups in the mass spectrum of polysaccharide, not all of the structure of the polysaccharide itself)].

[0262] (2) It has a high degree of sulfonation, requiring that the average content of sulfonic acid groups in each disaccharide is greater than or equal to 2.5; this means that the basic disaccharide structural unit is mainly composed of [0]-[1,0,1,0,3,0]-[0], and the content of [0]-[1,0,1,0,2,0]-[0] is relatively small.

[0263] (3) On this basis, it basically does not contain or contains a small amount of open-ring structure, and the average content of open-ring structure in each disaccharide is required to be less than or equal to 0.3.

[0264] Based on the above structural characteristics, and according to the features of heparin biosynthesis, the preparation process of heparin derivatives, and the results of intact glycan analysis, we can infer that highly sulfonated heparin derivative polysaccharides containing little or no open-ring structures have therapeutic effects on UC. The above results can be further summarized using formulas, and the derivation process is as follows:

[0265] For any class of oligosaccharides: [a]-[b,c,d,e,f,g]-[h], the above structural formula represents the number of each structure in the oligosaccharide: [polysaccharide ring-opening structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure]-[amino group (the number of amino groups in the mass spectrum of the polysaccharide, not all of the structure of the polysaccharide itself)].

[0266] For heparin-based oligosaccharides, there is exactly one unsaturated uronic acid, i.e., b = 1. Since the number of uronic acids and glucosamines is equal, we have b + c = d. The number of glucosamines, d, is also equal to the number of disaccharide units, meaning the oligosaccharide consists of d disaccharide units. Here, 1 <= d <= 10. The total length of the oligosaccharide chain is 2d. Since the effective heparin derivative does not contain any dehydrated structures during preparation, g = 0. h represents the number of amino groups in the polysaccharide in the mass spectrometer, which is independent of the polysaccharide's structure and is not limited here.

[0267] Based on the structural characteristics of effective anti-UC oligosaccharides, these oligosaccharides possess a highly sulfonated structure, requiring an average sulfonic acid group content greater than 2.5 per disaccharide, i.e., f>=2.5d. Simultaneously, these oligosaccharides contain virtually no or only a small amount of open-ring structures, with an average open-ring structure content less than 0.3 per disaccharide, i.e., a<=0.3d. Regarding the number of acetyl groups e, according to... Figure 9 As a result, considering the characteristics of heparin biosynthesis, e is limited to 1.0.

[0268] Based on the above information, the variables are further organized. The structural formula of the effective anti-UC oligosaccharide can be obtained:

[0269] [a]-[1,d-1,d,e,f,0]-[h]

[0270] Where: 1 <= d <= 10, a <= 0.3d, f >= 2.5d, e <= 1.0, and h is not limited.

[0271] Based on the above formula, the molecular formula of this type of oligosaccharide can be further obtained:

[0272] C (12d+2e) H (2a-2b+19d+2e+2) O (1-b+10d+e+3f) N (d) S (f)

[0273] The molecular weight can be calculated from the molecular formula.

[0274] 2. According to Figure 6 To further analyze the functional fragments responsible for the anti-inflammatory activity of the above-mentioned refined polysaccharide components, the contents of all oligosaccharide fragments measured in the whole-glycan chromatogram analysis were summed in each of the heparin derivatives (LNAHP, S4, S6, and NAI45) with significant anti-inflammatory activity. The summed values ​​were then ranked from highest to lowest, and the top 15% of oligosaccharide fragments were selected as representative oligosaccharide fragments. The data were compiled and summarized in Table 5. The contents of the representative oligosaccharide fragments in each heparin derivative were further summarized in Table 6.

[0275] Table 5. Representative oligosaccharide fragments of anti-inflammatory heparin derivatives

[0276]

[0277]

[0278] Note: The oligosaccharide structure representation means [polysaccharide ring-opening structure] - [unsaturated uronic acid - saturated uronic acid - glucosamine - acetyl group - sulfonic acid group - dehydration structure] - [amino group (i.e., the number of ammonium ions carried in mass spectrometry, not necessarily the total number of ions in the polysaccharide structure itself)].

[0279] Table 6 Summary of the content of anti-inflammatory effective oligosaccharides in various heparin derivatives and isolated fractions

[0280]

[0281] Table 7. Molecular formulas and molecular weight information of effective anti-inflammatory oligosaccharides.

[0282]

[0283]

[0284] Thus, we have discovered a class of well-defined oligosaccharide fragments, which account for only 15% of all oligosaccharide types, but are highly enriched in anti-inflammatory heparin derivatives, exceeding 45% in content, especially in S4 where the content exceeds 90%. It can be inferred that these oligosaccharides are the functional fragments from which heparin derivatives exert their anti-inflammatory biological activity.

[0285] To further summarize the structural characteristics of the oligosaccharides with anti-inflammatory properties, their structural formulas are derived using formulas, as follows:

[0286] For any class of oligosaccharides: [a]-[b,c,d,e,f,g]-[h], the above structural formula represents the number of each structure in the oligosaccharide, such as [polysaccharide ring-opening structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure]-[amino group (the number of amino groups in the mass spectrometer, not all of the structure of the polysaccharide itself)].

[0287] For heparin-based oligosaccharides, there is exactly one unsaturated uronic acid, i.e., b = 1. Since the number of uronic acids and glucosamines is equal, we have b + c = d. The number of glucosamines, d, is also equal to the number of disaccharide units, meaning the oligosaccharide consists of d disaccharide units. Here, 1 <= d <= 10. The total length of the oligosaccharide chain is 2d. Since the effective heparin derivative does not contain any dehydrated structures during preparation, g = 0. h represents the number of amino groups in the polysaccharide in the mass spectrometer, which is independent of the polysaccharide's structure and is not limited here.

[0288] Based on the structural characteristics of effective anti-inflammatory oligosaccharides, most anti-inflammatory oligosaccharides meet the following conditions in terms of molecular structure: the number of acetyl groups in each disaccharide must be greater than or equal to 0.1, i.e., e>=0.1d; the number of sulfonic acid groups in each disaccharide must be less than or equal to 2.7, i.e., f<=2.7d; and the number of open sugar rings in each disaccharide must be greater than or equal to 0.25, i.e., a>=0.25d.

[0289] Based on the above information, the variables are further organized. The structural formula of the effective anti-inflammatory oligosaccharide can be obtained:

[0290] [a]-[1,d-1,d,e,f,0]-[h]

[0291] Where: 1 <= d <= 10, a >= 0.25d, f <= 2.7d, e >= 0.1d, and h is not limited.

[0292] Based on the above formula, the molecular formula of this type of oligosaccharide can be further obtained:

[0293] C (12d+2e) H (2a-2b+19d+2e+2) O (1-b+10d+e+3f) N (d) S (f)

[0294] The molecular weight can be calculated from the molecular formula.

[0295] 3. Comparison of structural similarities and differences between anti-inflammatory and anti-UC effective oligosaccharides

[0296] Based on the above summary of the two oligosaccharide structures, their structural similarities and differences are compared, and the specific results are shown in Table 8.

[0297] Table 8. Comparison of structural similarities and differences between anti-inflammatory and anti-UC effective oligosaccharides.

[0298]

[0299] Table 8 shows that anti-UC oligosaccharides and anti-inflammatory oligosaccharides have only a small overlap in the number of acetyl groups, sulfonic acid groups, and open-ring structures of sugar rings. Overall, the group composition of the two oligosaccharides still differs significantly. They are two types of oligosaccharide components with different compositions.

[0300] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application, without departing from the scope of the technical solution of this application, shall still fall within the protection scope of this application.

Claims

1. A method for preparing refined heparin-like polysaccharides, comprising: Heparin raw material is de-anticoagulated and enzymatically hydrolyzed to obtain heparin-like polysaccharide raw material; The heparin polysaccharide raw material was separated using a gel size exclusion chromatography column, and the separated components were collected. The collected and separated components were freeze-dried. The lyophilized products of each component were desalted by alcohol precipitation to obtain purified heparin-like polysaccharides. When separating the heparin-based polysaccharide raw material using a size exclusion chromatography column, the size exclusion column is a HiPrep 16 / 60 Sephacryl, the mobile phase is a 0.15~0.6 M NaCl aqueous solution, and the flow rate of the mobile phase is 0.3~0.7 mL / min. The collection time is 130 minutes after sample injection.

2. The preparation method according to claim 1, wherein, The de-anticoagulation treatment of raw heparin is performed using periodic acid oxidation to obtain a de-anticoagulated product. The decoagulated product is enzymatically hydrolyzed using heparinase I to obtain the heparin polysaccharide raw material.

3. The preparation method according to claim 1, wherein, Freeze-drying involves pre-freezing the collected and separated components at -80°C before placing them into a freeze dryer for freeze-drying.

4. The preparation method according to claim 1, wherein, The mobile phase is a 0.2 M NaCl aqueous solution.

5. The preparation method according to claim 1, wherein, The flow rate of the mobile phase in the gel size exclusion column is 0.5 mL / min.

6. The preparation method according to claim 1, wherein, The pH value of the NaCl aqueous solution is 3~10.

7. The preparation method according to claim 1, wherein, The pH value of the NaCl aqueous solution is 5.

8. The preparation method according to claim 1, wherein, The packing material for the gel size exclusion chromatography column is Sephacryl S-100 High Resolution, Sephacryl S-200 High Resolution, or Sephacryl S-300 High Resolution.

9. The preparation method according to any one of claims 1 to 8, wherein, When the lyophilized products of each component are desalted by alcohol precipitation, The lyophilized products of each component were resuspended and concentrated in distilled water. After adding the aqueous ethanol solution, allow it to stand for alcohol precipitation. After centrifugation, the supernatant of the precipitate after alcohol precipitation is discarded. as well as The purified heparin polysaccharide was then obtained by resuspending it in distilled water.

10. The preparation method according to claim 9, wherein, Add 20-50% by volume of distilled water to each freeze-dried product for resuspension and concentration.

11. The preparation method according to claim 9, wherein, Each component of the freeze-dried product was resuspended and concentrated by adding 30% of its volume of distilled water.

12. The preparation method according to claim 9, wherein, The volume of the ethanol-water solution added is 2 to 6 times the volume of the concentrated liquid after resuspension; and, The concentration of the ethanol aqueous solution is 75%~100%.

13. The preparation method according to claim 12, wherein, The volume of the ethanol-water solution added is 5 to 6 times the volume of the concentrated liquid after resuspension.

14. The preparation method according to claim 9, wherein, The time for the alcohol precipitation to stand is 5-60 min.

15. The preparation method according to claim 9, wherein, The time for the alcohol precipitation to stand is 10-30 minutes.

16. The preparation method according to claim 1, further comprising: The refined heparin polysaccharide obtained by desalting through concentrated alcohol precipitation is then freeze-dried.

17. An oligosaccharide, said oligosaccharide being prepared by the preparation method according to any one of claims 1 to 16, and comprising the following structures [a]-[b,c,d,e,f,g]-[h]: [0]-[1,2,3,0,9,0]-[0], [0]-[1,2,3,0,8,0]-[0], [0]-[1,3,4,0,12,0]-[0], [2]-[1,3,4,1,10,0]-[1], [0]-[1,4,5,0,15,0]-[5], [1]-[1,4,5,0,1]-[h]. 4,0]-[1],[1]-[1,4,5,0,13,0]-[0],[2]-[1,4,5,1,13,0]-[4],[2]-[1,4,5,1,11,0]-[0],[0]-[1,5,6,0,18,0]-[6],[2]-[1,5,6,1,16,0]-[5],[2]-[1,5,6,1,15,0]-[3],[2]-[1,5,6,1,14,0]-[3] or [1]-[1,5,6,0,15,0]-[3],wherein, a represents the number of open-ring structures of the oligosaccharide molecule; b represents the number of unsaturated uronic acids in the oligosaccharide molecule; c represents the number of saturated uronic acids in the oligosaccharide molecule; d represents the amount of glucosamine in the oligosaccharide molecule, and 1 ≤ d ≤ 10; e represents the number of acetyl groups in the oligosaccharide molecule; f is the number of sulfonic acid groups in the oligosaccharide molecule, and f ≥ 2.5d. g represents the number of 1,6-dehydrated structures in the oligosaccharide molecule. h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection.

18. The oligosaccharide according to claim 17, characterized in that, In the structural formula of the oligosaccharide a≤0.3d, b=1, c=d-1, e≤1.0, g=0.

19. The oligosaccharide according to claim 17, characterized in that, The oligosaccharide has a sugar chain length between 2 and 20 sugars, and the basic disaccharide unit is composed of repeated arrangements of [0]-[1,0,1,0,3,0]-[0] and / or [0]-[1,0,1,0,2,0]-[0], wherein, [0]-[1,0,1,0,3,0]-[0] represents a disaccharide fragment with 0 open-ring structures, 1 unsaturated uronic acid, 0 saturated uronic acid, 1 glucosamine, 0 acetyl groups, 3 sulfonic acid groups, 0 dehydration structures, and 0 ammonium ions carried in mass spectrometry. [0]-[1,0,1,0,2,0]-[0] represents a disaccharide structure fragment with 0 open ring structures, 1 unsaturated uronic acid, 0 saturated uronic acid, 1 glucosamine, 0 acetyl groups, 2 sulfonic acid groups, 0 dehydrated structures, and 0 ammonium ions carried in mass spectrometry.

20. The oligosaccharide according to claim 19, characterized in that, The basic disaccharide unit contains an average number of sulfonic acid groups greater than or equal to 2.

5.

21. The oligosaccharide according to claim 19 or 20, characterized in that, The average number of open-ring structures of sugar rings in the basic disaccharide unit is less than or equal to 0.

3.

22. A heparin derivative containing the oligosaccharides as described in claims 17-21, characterized in that, In the heparin derivative, the content of the oligosaccharide as described in claims 17-21 is 17% or more.

23. The heparin derivative according to claim 22, characterized in that, In the heparin derivative, the content of the oligosaccharide as described in claims 17-21 is 18% or more.

24. The heparin derivative according to claim 22, characterized in that, In the heparin derivative, the content of the oligosaccharide as described in claims 17-21 is 19% or more.

25. The heparin derivative according to claim 22, characterized in that, In the heparin derivative, the content of oligosaccharides as described in claims 17-21 is 20% or more.

26. The heparin derivative according to claim 22, characterized in that, In the heparin derivative, the content of the oligosaccharide as described in claims 17-21 is 21% or more.

27. The heparin derivative according to claim 22, characterized in that, In the heparin derivative, the content of the oligosaccharide as described in claims 17-21 is 22% or more.

28. The heparin derivative according to claim 22, characterized in that, In the heparin derivative, the content of oligosaccharides as described in claims 17-21 is 23% or more.

29. Use of the oligosaccharide as described in any one of claims 17 to 21 and the heparin derivative as described in any one of claims 22 to 28 in the preparation of a medicament for the prevention or treatment of inflammatory bowel disease.

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