Oral polysaccharide for anti-inflammatory treatment and method for its preparation

Abstract

The application relates to a preparation method of refined heparin polysaccharides. Gel exclusion chromatography column method is used to separate heparin polysaccharide raw materials, and the collected separated components are treated through freeze-drying, alcohol precipitation and desalting to obtain refined heparin polysaccharide products. The application also relates to an oligosaccharide with specific polysaccharide structure characteristics and anti-inflammatory effectiveness.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of refined heparin polysaccharides, and its use in the preparation of anti-inflammatory drugs. BACKGROUND

[0002] Heparin is a kind of glycosaminoglycan with a molecular weight of 3000-30000 Da, which has high heterogeneity and complexity. At present, the only use of heparin in clinic is its anticoagulant function. In addition to the anticoagulant activity, heparin also has a large number of non-anticoagulant biological activities, including anti-inflammatory, anti-tumor, anti-virus and anti-fat accumulation, etc. Among them, the anti-inflammatory activity is one of the most extensive and in-depth functions of the non-anticoagulant biological activities of heparin. A large number of research reports have been reported and the anti-inflammatory activity of heparin has been found. However, the anti-inflammatory activity of heparin has not been applied in clinic, which is mainly related to the strong anticoagulant biological activity of heparin drugs which can induce the risk of massive hemorrhage in the whole body, and the lack of clear structure-activity relationship analysis of the anti-inflammatory activity of heparin. The inconsistent results of preclinical and clinical studies on the anti-inflammatory activity of heparin also limit the research and development of the anti-inflammatory activity of heparin.

[0003] A large number of studies have shown that the anti-inflammatory activity and other non-anticoagulant biological activities of heparin are closely related to the size of the molecular weight. However, the high heterogeneity and structural complexity of heparin greatly limit the exploration of its non-anticoagulant biological activities and the analysis of effective molecular structure. In terms of heterogeneity, heparin is a highly heterogeneous linear polysaccharide with a molecular weight of 3000-30000 Da, which is not a pure substance and has extremely complex molecular composition. In terms of structural complexity, heparin has a rich modification of sulfonic acid groups and acetyl groups, and the modification process is not controlled by the central dogma and has high randomness, which also makes the complexity of the fine structure of heparin increase exponentially. Due to the complex molecular structure characteristics of heparin, the complete sugar chain molecular structure has not been resolved so far. Therefore, it is of great significance to separate and refine heparin polysaccharides to clarify the mechanism of action and drug structure-activity relationship of the non-anticoagulant biological activities of heparin polysaccharides.

[0004] For the preparation and separation of heparin polysaccharides, most of the current research focuses on the separation of the anticoagulant and non-anticoagulant activities of heparin. This separation method cannot distinguish polysaccharides of different molecular weights, and there is no fine separation and preparation process for the molecular weight of heparin in the prior art. Although the existing literature reports the use of gel exclusion chromatography to quantitatively analyze the molecular weight of heparin, the above method only stays at the analysis level, and there are defects such as small amount of polysaccharide analysis, unknown process for fine separation and purification of polysaccharides, and inability to directly realize process amplification, so it cannot realize the controllable separation and large-scale preparation of heparin polysaccharides with different molecular weights. Therefore, there is no process and report on the fine separation and preparation of heparin polysaccharides with different molecular weights in the current research field.

[0005] However, the anti-inflammatory activity of heparin lacks systematic structure-activity relationship study, and the anti-inflammatory polysaccharide fragments are not clear. In addition, the preparation process of anticoagulant heparin often destroys its anti-inflammatory activity and corresponding fine structure, which are problems to be solved in the field. SUMMARY

[0006] The present application provides a fine separation and preparation method of heparin polysaccharide. Through fine separation of heparin polysaccharide, controllable separation and large-scale preparation of heparin polysaccharide with different molecular weights are realized, which has anti-inflammatory effect, such as inflammation in pneumonia caused by novel coronavirus, pulmonary and liver fibrosis, arthritis, rheumatoid arthritis, irritable bowel syndrome, gastritis, skin inflammation, and inflammatory bowel disease. The present application also provides a drug with excellent anti-inflammatory performance.

[0007] Due to the complexity of the structure of heparin and the application limitation of strong anticoagulant activity of heparin which easily induces systemic massive hemorrhage, the research on non-anticoagulant biological activity of heparin in this field generally lacks the stability of anti-inflammatory activity of heparin, self-contradictory treatment results, effective molecular structure characteristics of heparin anti-inflammatory, drug structure-activity relationship, and extremely lack of structure-activity analysis technology and method. The above research and technical bottlenecks greatly limit the research on anti-inflammatory biological activity of heparin polysaccharide and the development of related drugs. Therefore, establishing an efficient fine structure preparation method of heparin polysaccharide, deeply exploring its structure-activity relationship, and excavating the most suitable heparin oligosaccharide structure with anti-inflammatory effect are the research focus of developing new heparin polysaccharide anti-inflammatory drugs.

[0008] Heparin polysaccharide generally has the characteristics of high complexity of molecular structure and diversity of biological activity. In the preparation of heparin polysaccharide, although there are some simple separation methods (such as using ultrafiltration membrane to separate according to molecular weight), the separation precision and separation amount are limited, and there is still some difficulty in further fine separation and preparation of heparin polysaccharide drugs. Although the gel exclusion chromatography method can realize the quantitative analysis of the molecular weight of heparin polysaccharide, it cannot meet the requirements of fine preparation, that is, the purification of heparin polysaccharide is not realized, and the large-scale preparation (mg-g level) of heparin polysaccharide is not realized.

[0009] The inventors of the present application are committed to the innovative research of heparin industry technology, and have constructed a set of fine process flow for heparin polysaccharide, which can realize the preparation and separation of heparin polysaccharide with controllable molecular weight, overcome the problem of high non-uniformity of heparin polysaccharide structure, and provide new technology, new method and new product for the effective excavation of non-anticoagulant biological activity of heparin polysaccharide such as anti-inflammatory, anti-tumor, and anti-fat accumulation and the application in anti-inflammatory drugs.

[0010] Specifically, the present application relates to the following:

[0011] 1. A method for preparing a refined heparinoid polysaccharide, comprising:

[0012] subjecting a raw heparin to a de-anticoagulation treatment and enzymatic hydrolysis to obtain a heparinoid polysaccharide raw material;

[0013] separating the heparinoid polysaccharide raw material using a gel exclusion chromatography column and collecting the separated components;

[0014] subjecting the collected separated components to a freeze-drying treatment;

[0015] subjecting the freeze-dried products of the components to a desalting treatment by alcohol precipitation to obtain a refined heparinoid polysaccharide.

[0016] 2. The method according to item 1, wherein,

[0017] subjecting the raw heparin to a de-anticoagulation treatment is subjecting the raw heparin to a de-anticoagulation treatment using a periodate oxidation method to obtain a de-anticoagulation treated product, and

[0018] subjecting the de-anticoagulation treated product to enzymatic hydrolysis is subjecting the de-anticoagulation treated product to enzymatic hydrolysis using heparinase I to obtain the heparinoid polysaccharide raw material.

[0019] 3. The method according to item 1 or 2, wherein,

[0020] subjecting to a freeze-drying treatment is subjecting the collected separated components to a pre-freezing at -80°C and then to a freeze-drying treatment in a freeze-dryer.

[0021] 4. The method according to any one of items 1 to 3, wherein,

[0022] when separating the heparinoid polysaccharide raw material using a gel exclusion chromatography column, the gel exclusion chromatography column is a size-exclusion chromatography column, preferably a HiPrep 16 / 60 Sephacryl or a TSKgel G2000SW chromatography column, and the mobile phase used is a 0.15 to 1.0 M NaCl aqueous solution, preferably 0.15 to 0.6 M, more preferably 0.2 M.

[0023] 5. The method according to item 4, wherein,

[0024] the flow rate of the mobile phase in the gel exclusion chromatography column is 0.1 to 1.0 mL / min, preferably 0.3 to 0.7 mL / min, more preferably 0.5 mL / min.

[0025] 6. The method according to item 4, wherein,

[0026] The pH value of the NaCl aqueous solution is 3-10, preferably 5.

[0027] 7. The production method according to item 4, wherein,

[0028] The gel exclusion chromatography column is a HiPrep 16 / 60 Sephacryl column, and the column filler is Sephacryl S-100 High Resolution, Sephacryl S-200 High Resolution or Sephacryl S-300 High Resolution.

[0029] 8. The production method according to item 4, wherein,

[0030] The gel exclusion chromatography column is a TSKgel G2000SW column, and the column filler is TSKgel G2000.

[0031] 9. The production method according to any one of items 1-8, wherein, when the freeze-dried product of each component is subjected to desalting treatment by alcohol precipitation,

[0032] The freeze-dried product of each component is resuspended and concentrated by adding distilled water;

[0033] After adding the aqueous ethanol solution, the product is allowed to stand for alcohol precipitation;

[0034] The precipitated product after alcohol precipitation is centrifuged, and the supernatant is discarded; and

[0035] The purified heparin-like polysaccharide is obtained after resuspension by subsequently adding distilled water.

[0036] 10. The production method according to item 9, wherein,

[0037] The freeze-dried product of each component is resuspended and concentrated by adding distilled water in an amount of 20-50%, preferably 30%, of the volume.

[0038] 11. The production method according to item 10, wherein,

[0039] The aqueous ethanol solution is added in an amount of 2-6 times, preferably 5-6 times, the volume of the resuspended and concentrated liquid; and,

[0040] The concentration of the aqueous ethanol solution is 75-100%.

[0041] 12. The production method according to item 9, wherein,

[0042] The time for which the product is allowed to stand for alcohol precipitation is 5-60 min, preferably 10-30 min.

[0043] 13. The preparation method according to any one of items 1 to 12, further comprising,

[0044] The refined heparin-like polysaccharide obtained by desalting treatment through concentrated alcohol precipitation is subjected to freeze-drying treatment.

[0045] 14. An oligosaccharide, characterized in that the oligosaccharide has the following structure:

[0046] [a]-[b,c,d,e,f,g]-[h], wherein,

[0047] a is the number of open ring structures of sugar rings in the oligosaccharide molecule;

[0048] b is the number of unsaturated uronic acids in the oligosaccharide molecule;

[0049] c is the number of saturated uronic acids in the oligosaccharide molecule;

[0050] d is the number of glucosamine in the oligosaccharide molecule, and 1≤d≤10;

[0051] e is the number of acetyl groups in the oligosaccharide molecule;

[0052] f is the number of sulfonic acid groups in the oligosaccharide molecule, and f≤2.7d,

[0053] g is the number of 1,6-anhydro structures in the oligosaccharide molecule,

[0054] h is the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection.

[0055] 15. The oligosaccharide according to claim 14, characterized in that in the structural formula of the oligosaccharide, a≥0.25d, b=1, c=d-1, e≥0.1d, and g=0.

[0056] 16. The oligosaccharide according to item 14, wherein the oligosaccharide has one of the following structures: [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], [3]-[1,4,5,1,8,0]-[7].

[0057] 17. A heparin derivative comprising the oligosaccharide according to any one of items 14 to 16, wherein the content of the oligosaccharide according to any one of items 14 to 16 in the heparin derivative is 52% or more.

[0058] 18. Use of the oligosaccharide according to any one of items 14 to 16 and the heparin derivative according to item 17 in the manufacture of an anti-inflammatory drug, preferably the inflammation is pneumonia caused by a novel coronavirus, inflammation in the process of pulmonary and hepatic fibrosis, arthritis, rheumatoid arthritis, irritable bowel syndrome, gastritis, skin inflammation, and inflammatory bowel disease.

[0059] 19. The use according to item 18, wherein the oligosaccharide is prepared by the method according to any one of items 1 to 13.

[0060] Effects of the Invention

[0061] The preparation method of the refined heparin polysaccharide provided in the application realizes the anticoagulant modification and the fine preparation and separation of the heparin polysaccharide with controllable molecular weight, and each refined polysaccharide component with a relatively concentrated molecular weight distribution and no anticoagulant activity can be obtained, the potential drug risk of heparin polysaccharide inducing massive hemorrhage is removed, and the characteristics of the great structural heterogeneity of heparin polysaccharide are significantly optimized. Through in vivo and in vitro drug screening and pharmacodynamic evaluation verification, the complete removal of heparin anticoagulant activity is realized, and the systemic investigation of the in vitro anti-inflammatory activity of the refined heparin polysaccharide component is realized. Using the LPS-induced RAW264.7 in vitro anti-inflammatory model, the anticoagulant heparin derivatives LNAHP, S4, S6 and NAI45 with in vitro anti-inflammatory effect are successfully screened and obtained. Through whole sugar chain mapping analysis, the composition and content of the representative oligosaccharide in the effective anti-inflammatory heparin derivative are determined. The representative oligosaccharide is a functional fragment of the heparin derivative for exerting anti-inflammatory activity, and provides a new idea and method for studying the structure-activity relationship of heparin polysaccharide anti-inflammatory, anti-tumor and anti-fat accumulation biological activities. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 UC mouse weight curve ANOVA analysis diagram

[0063] Figure 2 UC mouse colon representative picture and colon length measurement result diagram

[0064] Figure 3 UC mouse spleen representative picture and spleen weight index diagram

[0065] Figure 4 H&E staining evaluation of UC mouse colon epithelial histological changes diagram

[0066] Figure 5 Heparin derivative in vitro anti-inflammatory activity index evaluation diagram

[0067] Figure 6 Heparin derivative and each isolated component complete sugar chain analysis diagram

[0068] Figure 7 Heparin derivative and each isolated component oligosaccharide coverage and oligosaccharide enrichment threshold relationship diagram

[0069] Figure 8 Heparin derivative and each isolated component enriched oligosaccharide wein diagram

[0070] Figure 9 Heparin derivative effective and ineffective anti-UC oligosaccharide structure characteristic analysis diagram DETAILED DESCRIPTION

[0071] The application will be described in further detail below with reference to the specific embodiments. The embodiments are given to introduce the scope of the application fully and to provide the skilled in the art with a complete understanding of the application.

[0072] It should be noted that some terms are used in the description and claims. The skilled in the art should understand that the same component can be referred to by different terms. The description and claims of the present application do not distinguish components by the difference in terms, but by the difference in function. "Include" or "comprise" mentioned throughout the description and claims are open terms, which should be interpreted as "including but not limited to". The subsequent description is a preferred embodiment for implementing the present application, but the description is for the purpose of illustrating the general principles of the description, and is not intended to limit the scope of the present application. The scope of protection of the present application is defined by the appended claims.

[0073] The present application relates to a method for preparing refined heparin polysaccharides, and in a specific embodiment, the method comprises the following steps:

[0074] The raw heparin is subjected to anti-coagulation removal and enzymatic hydrolysis to obtain a heparin polysaccharide raw material;

[0075] The heparin polysaccharide raw material is separated by gel exclusion chromatography column and the separated components are collected;

[0076] The collected separated components are subjected to freeze-drying treatment;

[0077] The freeze-dried products of the components are subjected to desalination treatment by alcohol precipitation to obtain refined heparin polysaccharides.

[0078] The heparin polysaccharide raw material in the present application, i.e. the anti-coagulation removed heparin derivative, is a substance obtained by anti-coagulation removal of heparin or (ultra) low molecular weight heparin, which is heparin or (ultra) low molecular weight heparin with no or low anti-coagulation activity, i.e. the 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.

[0079] In a specific embodiment, the raw heparin is subjected to anti-coagulation removal, and then the product of the anti-coagulation removal is subjected to enzymatic hydrolysis to obtain the heparin polysaccharide raw material.

[0080] In one embodiment of the present application, the raw heparin is subjected to anti-coagulation removal treatment by periodate oxidation to obtain the anti-coagulation removed product.

[0081] The anti-coagulation removed heparin derivative can be obtained by periodate oxidation, and other biological activities are largely retained, and the degree and form of sulfation are basically unchanged. Periodate can selectively oxidize the adjacent carbon atom containing unsubstituted hydroxyl or amino group, break the C(2)-C(3) bond of the unsulfated uronic acid, and destroy the antithrombin binding pentasaccharide in the heparin molecule, thereby losing the anti-coagulation activity. The polyaldehyde oxidized heparin obtained by periodate oxidation is reduced by borohydride to maintain stability (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.).

[0082] In one embodiment, the raw heparin (e.g., heparin sodium) is dissolved in water, and a sodium periodate solution is added for reaction. After a period of time, ethylene glycol is added to neutralize the excess sodium periodate, and sodium borohydride is added for reaction. After adjusting the pH value, the sample is filtered and collected. Concentration and desalination are performed using a dialysis bag, and the anti-coagulation removed heparin derivative is finally obtained.

[0083] In one embodiment of the present application, the anti-coagulation removed product is subjected to enzymatic hydrolysis by heparinase, such as heparinase I, to obtain the heparin polysaccharide raw material.

[0084] In the prior art, the E.C. number of heparinase I is E.C. 4.2.2.7. Commercially available heparinase I, such as that purchased from Sigma or IBEX, can be used. The heparinase can also be a recombinant heparinase I constructed by molecular biology methods or a fusion protein formed by heparinase I and any fusion partner, as long as it has the activity of heparinase I. Preferably, the heparinase I is a fusion protein of heparinase I, especially a fusion protein of heparinase I containing MBP.

[0085] In one embodiment, the reaction between heparinase I and the anti-coagulation removed product can be batch, continuous or semi-continuous, which can be appropriately selected by those skilled in the art according to the needs of production.

[0086] In one embodiment of the present application, the heparin polysaccharide raw material is separated by using a gel exclusion chromatography column. Preferably, an AKTA Prime purification system is used in combination with a gel exclusion chromatography column for preparative liquid chromatography to form the basic hardware facilities.

[0087] Gel exclusion chromatography, also known as size exclusion chromatography, is a chromatographic technique based on the size of the sample molecules, mainly used for the analysis and separation of the relative molecular mass distribution of high polymers soluble in organic solvents. The chromatographic column filler of gel exclusion chromatography is gel, which is a substance with inert surface and containing many different size pores or three-dimensional network structures. The pores of the gel only allow molecules with a diameter smaller than the pore opening to enter, and these pores are relatively large for the mobile phase molecules so that the mobile phase molecules can freely diffuse in and out. Depending on the gel filler used, gel exclusion chromatography columns can separate oil-soluble and water-soluble substances, and separate relative molecular mass ranging from several million to 100 or less.

[0088] In one embodiment of the present application, the gel exclusion chromatography column used is a HiPrep 16 / 60 Sephacryl series chromatography column, which is a high-resolution gel filtration filler, generally used for fine separation. Specifically, HiPrep 16 / 60 Sephacryl S-100 High Resolution, HiPrep 16 / 60 Sephacryl S-200 High Resolution, HiPrep 16 / 60 Sephacryl S-300 High Resolution, etc. under the HiPrep 16 / 60 Sephacryl series can be used for the separation of polysaccharide raw materials in the present application. In one embodiment, the preferred chromatographic column filler is HiPrep 16 / 60 Sephacryl S-100 High Resolution, and the column pressure is not more than 0.15 MPa.

[0089] In another embodiment of the present application, the gel exclusion chromatography column used is a TSKgel G2000SW chromatography column. The filler of the TSKgel SW series chromatography column is a rigid spherical silica gel as the matrix, and a hydrophilic group is covalently bonded to its surface, which is specifically used for GFC separation of proteins and polypeptides. The filler of the SW series chromatography column has the necessary performance for high-performance size exclusion chromatography, i.e. low adsorption and good pore size distribution, and its pH range is 2.5-7.5, and organic solvents completely miscible with water, such as acetonitrile, acetone, methanol or ethanol, etc. can be used. In one embodiment, the preferred chromatographic column filler is TSKgel G2000, and the column pressure is not more than 2.00 MPa.

[0090] In one embodiment of the application, the mobile phase used in the gel 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, 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.

[0091] In the separation theory of gel exclusion chromatography, the mobile phase needs to have a certain concentration of salt ions to fill the high-energy sites in the gel exclusion chromatography column filler to ensure the separation effect; on the other hand, the selection of the mobile phase needs to take into account the needs of both polysaccharide separation and polysaccharide purification. The current mobile phase can meet the above two requirements.

[0092] In one embodiment of the application, the pH value of the mobile phase NaCl aqueous solution is 3-10, preferably 5.

[0093] The pH value of the mobile phase affects the separation efficiency, and generally the separation efficiency of heparin polysaccharides is best at pH 5. Some existing literature states that there is no significant difference in separation efficiency between pH 5-7.

[0094] In one embodiment of the application, the flow rate of the mobile phase used in the gel exclusion chromatography column 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, 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 embodiment, the skilled person can set it according to the operable range of the chromatography column.

[0095] In one embodiment, the polysaccharide raw material is dissolved in the mobile phase at a concentration of 10-240 mg / mL. Preferably, the dissolution concentration is 10-120 mg / mL.

[0096] In one embodiment, the sample loading amount for one separation is 0.5-5 mL. This parameter can be set according to the specific operable range of the chromatography column.

[0097] In one embodiment of the application, the heparin polysaccharide raw material is dissolved in the mobile phase, and different component collection times are set according to the different molecular weights of the sample. In one embodiment, one component can be collected every 3-30 minutes. According to the principle of gel exclusion chromatography, components of different molecular weights are separated in turn. The first separated component is a polysaccharide of large molecular weight, and the last separated component is a polysaccharide of small molecular weight. A person skilled in the art can set any collection interval that can effectively separate the sample, so as to realize the enrichment and purification of polysaccharides of specific molecular weight, and obtain a refined polysaccharide salt solution with specific molecular weight distribution.

[0098] In one embodiment, the heparin polysaccharide raw material is dissolved in the mobile phase, and collection is started from the 70th to 100th minute after sample injection. This parameter can be set according to the size of the sample injection amount.

[0099] In one embodiment of the application, the separation components are collected from the 80th minute after sample injection, and then one component is collected every 10 minutes, and 11 separation components of different molecular weights are collected.

[0100] Through the above separation and purification steps of the heparin polysaccharide raw material, a refined polysaccharide salt solution with specific molecular weight distribution is obtained. In order to increase the concentration of polysaccharide and salt in the solution, so as to obtain more polysaccharide product by alcohol precipitation, the above refined polysaccharide salt solution can be subjected to freeze-drying treatment (primary freeze-drying) to remove excess water.

[0101] In one embodiment, the freeze-drying treatment conditions are as follows: the refined polysaccharide salt solution is pre-frozen at -80°C, and then placed in a freeze dryer for 2 days until the water is completely removed.

[0102] In one embodiment, the product subjected to freeze-drying treatment is subjected to concentration and alcohol precipitation to obtain a refined polysaccharide aqueous solution after desalination.

[0103] In one embodiment, during the concentration and alcohol precipitation process, distilled water can be added to the product subjected to freeze-drying treatment to enrich the refined polysaccharide and increase the yield of alcohol precipitation. Specifically, distilled water can be added in an amount of 20%-50% of the volume of the freeze-dried product, and preferably 30%. In the above process, the volume reduction also causes the NaCl to be further concentrated, which promotes the precipitation of polysaccharide.

[0104] In a specific embodiment, after the above-mentioned resuspension and concentration step, an alcohol precipitation is performed by adding an aqueous ethanol solution. Specifically, an aqueous ethanol solution with a concentration of 75% to 100% can be added in an amount of 2 to 6 times the volume of the concentrated solution. Preferably, anhydrous ethanol is added in an amount of 5 to 6 times the volume of the concentrated solution. In the industrial production of heparin, the alcohol precipitation step often uses solutions with different ethanol concentrations. The volume of the alcohol precipitation is related to the concentration of the polysaccharide. The higher the concentration of the polysaccharide, the smaller the volume of the alcohol precipitation. The lower the concentration of the polysaccharide, the larger the volume of the alcohol precipitation.

[0105] In a specific embodiment, the alcohol precipitation can be performed for 5 to 60 minutes. Preferably, it is performed for 10 to 30 minutes, and more preferably, it is performed for 10 minutes. The main purpose of the standing is to allow the polysaccharide to flocculate sufficiently, thereby ensuring a high yield.

[0106] In a specific embodiment, after the alcohol precipitation, the precipitated product is centrifuged, and all the supernatant is discarded. Distilled water is then added to resuspend the product, thereby obtaining a desalted refined polysaccharide solution.

[0107] In a preferred embodiment, the precipitated product is centrifuged at 8000g, 4°C, for 10 minutes.

[0108] In a specific embodiment, 0.5 to 10 mL of distilled water is added to resuspend the product. Preferably, 2 mL of distilled water is added. This parameter is related to the concentration of the polysaccharide. The higher the concentration of the polysaccharide, the larger the volume of distilled water required. The lower the concentration of the polysaccharide, the smaller the volume of distilled water required.

[0109] In a specific embodiment, the desalted refined heparin polysaccharide solution obtained after the above-mentioned concentration and alcohol precipitation is subjected to a second freeze-drying process (secondary freeze-drying), thereby obtaining a refined polysaccharide product that is easy to store.

[0110] In a specific embodiment, the processing conditions of the secondary freeze-drying are the same as those of the primary freeze-drying.

[0111] The preparation method of the refined heparin polysaccharide provided in the present application enables the preparation and separation of heparin polysaccharides with controllable molecular weight, thereby obtaining refined polysaccharide components with a relatively concentrated molecular weight distribution. This significantly optimizes the non-uniformity of the polysaccharide, thereby providing favorable conditions for further research on its non-anticoagulant biological activities, such as anti-inflammatory, anti-tumor, and anti-fat accumulation activities, and for its application in the treatment of UC.

[0112] After the anti-UC biological activity of each of the refined polysaccharide components prepared by the above-mentioned preparation method of refined heparin-like polysaccharides is verified, it is found that some isolated components have relatively excellent anti-UC performance. After complete sugar chain analysis and enriched oligosaccharide Venn diagram analysis of the corresponding refined polysaccharide isolated components, it is found that there is a kind of oligosaccharide with specific polysaccharide structure characteristics in the anti-UC effective isolated components, which is deduced to be very likely to be an anti-UC effective polysaccharide fragment.

[0113] On this basis, the application provides an oligosaccharide with a specific polysaccharide structure, which is mainly obtained based on the anti-UC effective refined polysaccharide, and the oligosaccharide has the following structure:

[0114] [a]-[b,c,d,e,f,g]-[h], wherein,

[0115] a represents the number of open ring structures of the sugar ring in the oligosaccharide molecule;

[0116] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0117] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0118] d represents the number of glucosamine in the oligosaccharide molecule;

[0119] e represents the number of acetyl groups in the oligosaccharide molecule;

[0120] f represents the number of sulfonic acid groups in the oligosaccharide molecule;

[0121] g represents the number of 1,6-dehydration structures in the oligosaccharide molecule;

[0122] h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometric detection;

[0123] And the number of glucosamine in the structure of the oligosaccharide is between 1 and 10, that is, 1≤d≤10, and the number of sulfonic acid groups is 2.5 times the number of glucosamine, that is, f≥2.5d, which indicates that the oligosaccharide has a high sulfonic acid structure.

[0124] In a specific embodiment, the structure of the above-mentioned oligosaccharide molecule has only one unsaturated uronic acid, that is, b=1. Since the total number of unsaturated uronic acid and saturated uronic acid is equal to the number of glucosamine, the number of saturated uronic acid is the number of glucosamine-1, that is, c=d-1.

[0125] In a specific embodiment, the structure of the above-mentioned oligosaccharide molecule has substantially no open ring structure or contains a small amount of open ring structure, so the number of open ring structure is not more than 0.3 times the number of glucosamine, that is, a≤0.3d.

[0126] In one embodiment, the oligosaccharide molecule has no dehydrated structure, i.e. g = 0.

[0127] In one embodiment, the oligosaccharide molecule has no more than one acetyl group, i.e. e ≤ 1.0.

[0128] In one embodiment, the oligosaccharide molecule has one of the following structures,

[0129] [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],

[0130] If the structure is represented as [a]-[b,c,d,e,f,g]-[h], then in the structure,

[0131] a represents the number of open ring structures in the oligosaccharide molecule;

[0132] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0133] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0134] d represents the number of glucosamines in the oligosaccharide molecule;

[0135] e represents the number of acetyl groups in the oligosaccharide molecule;

[0136] f represents the number of sulfonic acid groups in the oligosaccharide molecule;

[0137] g represents the number of 1,6-dehydrated structures in the oligosaccharide molecule;

[0138] h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection.

[0139] In one embodiment, the oligosaccharide having anti-UC activity provided by the present application has the following structural characteristics:

[0140] The oligosaccharide has a sugar chain length between 2 and 20 saccharides, and the basic disaccharide unit is composed of a repeating combination of [0]-[1,0,1,0,3,0]-[0] and / or [0]-[1,0,1,0,2,0]-[0], wherein,

[0141] [0]-[1,0,1,0,3,0]-[0] represents a disaccharide structural fragment with 0 ring-opening structure, 1 unsaturated uronic acid, 0 saturated uronic acid, 1 glucosamine, 0 acetyl group, 3 sulfonic acid group, 0 dehydrated structure, and 0 ammonia in the mass spectrum;

[0142] [0]-[1,0,1,0,2,0]-[0] represents a disaccharide structural fragment with 0 ring-opening structure, 1 unsaturated uronic acid, 0 saturated uronic acid, 1 glucosamine, 0 acetyl group, 2 sulfonic acid group, 0 dehydrated structure, and 0 ammonia in the mass spectrum.

[0143] Further, in one embodiment, the above oligosaccharide has a high sulfonic acid group content, and the average number of sulfonic acid groups contained in the basic disaccharide unit is greater than or equal to 2.5. This means that the basic disaccharide unit is mainly composed of the structure represented by [0]-[1,0,1,0,3,0]-[0], and the content of the structure represented by [0]-[1,0,1,0,2,0]-[0] is small.

[0144] Further, in one embodiment, the above oligosaccharide has a low ring-opening structure content, and the average number of ring-opening structures contained in the basic disaccharide unit is less than or equal to 0.3.

[0145] The present application also provides a heparin derivative containing the above oligosaccharide having anti-UC activity, and the content of the oligosaccharide is greater than or equal to 17%, preferably greater than or equal to 18%, preferably greater than or equal to 19%, preferably greater than or equal to 20%, preferably greater than or equal to 21%, preferably greater than or equal to 22%, and preferably greater than or equal to 23%.

[0146] On the other hand, the present application provides an oligosaccharide having a specific polysaccharide structure, which is mainly based on a refined polysaccharide having anti-inflammatory activity, and is characterized in that the oligosaccharide has the following structure:

[0147] [a]-[b,c,d,e,f,g]-[h], wherein,

[0148] a represents the number of ring-opening structures in the oligosaccharide molecule;

[0149] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0150] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0151] d represents the number of glucosamines in the oligosaccharide molecule;

[0152] e represents the number of acetyl groups in the oligosaccharide molecule;

[0153] f represents the number of sulfonic acid groups in the oligosaccharide molecule;

[0154] g represents the number of 1,6-anhydro structures in the oligosaccharide molecule;

[0155] h represents the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection;

[0156] In addition, the number of glucosamines in the structure of the oligosaccharide is between 1 and 10, i.e. 1≤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.

[0157] In a specific embodiment, the structure of the above oligosaccharide molecule contains only one unsaturated uronic acid, i.e. b=1. Since the total number of unsaturated uronic acids and saturated uronic acids is equal to the number of glucosamines, the number of saturated uronic acids is equal to the number of glucosamines minus one, i.e. c=d-1.

[0158] In a specific embodiment, the structure of the above oligosaccharide molecule contains more than or equal to 0.25 ring-opening per disaccharide, i.e. a≥0.25d.

[0159] In a specific embodiment, the structure of the above oligosaccharide molecule does not contain any anhydro structure, i.e. g=0.

[0160] In a specific embodiment, the structure of the above oligosaccharide molecule contains more than or equal to 0.1 acetyl group per disaccharide, i.e. ≥0.1d.

[0161] In specific embodiments, the oligosaccharide provided by the present application has one of the structures shown below,

[0162] [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],

[0163] In case the above formula is represented as [a]-[b,c,d,e,f,g]-[h], then in this formula,

[0164] a represents the number of open ring structures in the saccharide rings of the oligosaccharide molecule;

[0165] b represents the number of unsaturated uronic acids in the oligosaccharide molecule;

[0166] c represents the number of saturated uronic acids in the oligosaccharide molecule;

[0167] d represents the number of glucosamines in the oligosaccharide molecule;

[0168] e represents the number of acetyl groups in the oligosaccharide molecule;

[0169] f represents the number of sulfonic acid groups in the oligosaccharide molecule;

[0170] g represents the number of 1,6-anhydro structures in the oligosaccharide molecule;

[0171] h represents the number of ammonium ions carried by the oligosaccharide molecule in the mass spectrometric detection.

[0172] The present application also provides a heparin derivative, which contains an anti- inflammatory effective oligosaccharide as described above and the content of the oligosaccharide is above 52%, preferably above 65%, preferably above 70%, preferably above 75%, preferably above 80%, preferably above 85%, preferably above 90%, preferably above 93%.

[0173] In the present application, the method for analyzing the structure and determining the content of polysaccharide components is not specifically limited, and any method known to those skilled in the art can be used.

[0174] In a specific embodiment, the polysaccharide components in the sample to be tested can be first extracted by a method known to those skilled in the art. Then, the polysaccharide components are subjected to whole sugar chain mapping analysis by the National Institute of Metrology, China, and the detection results recording the oligosaccharide content and oligosaccharide distribution information of the polysaccharide sample are obtained. Based on the detection results, the number of ring-opening structures in the oligosaccharide molecules, the number of unsaturated uronic acids in the oligosaccharide molecules, the number of saturated uronic acids in the oligosaccharide molecules, the number of glucosamine in the oligosaccharide molecules, the number of acetyl groups in the oligosaccharide molecules, the number of sulfonic acid groups in the oligosaccharide molecules, the number of 1, 6-dehydration structures in the oligosaccharide molecules, and the number of ammonium ions carried by the oligosaccharide molecules in mass spectrometric detection can be confirmed.

[0175] Based on the oligosaccharide content and oligosaccharide distribution information, the structure of the polysaccharide components can be reconstructed by a method known to those skilled in the art (for specific methods, see the description of the reconstruction process in Experimental Example 3 of the present application) to confirm the basic disaccharide unit structure of the oligosaccharide, and the average number of sulfonic acid groups and the average number of ring-opening structures in the basic disaccharide unit.

[0176] In addition, based on the oligosaccharide content and oligosaccharide distribution information obtained by the whole sugar chain mapping analysis method, the content of oligosaccharides conforming to each structure can be calculated.

[0177] The present application also relates to the use of the oligosaccharides provided by the present application and the unfractionated heparin derivatives containing the oligosaccharides for preparing anti-inflammatory drugs, preferably the inflammation is caused by pneumonia caused by the novel coronavirus, inflammation in the process of pulmonary and liver fibrosis, arthritis, rheumatoid arthritis, irritable bowel syndrome, gastritis, skin inflammation, and inflammatory bowel disease, etc.

[0178] In a specific embodiment of the present application, the oligosaccharides having anti-inflammatory effectiveness are prepared by the method for preparing the refined heparin-like polysaccharides provided by the present application.

[0179] Example 1 Preparation of polysaccharide raw material

[0180] The 20 g of fine heparin (purchased from Changshan Biochemical Pharmaceutical Co., Ltd., product name: heparin sodium) was dissolved in 0.6 L of deionized water, and an equal volume of 0.2 M sodium periodate solution (freshly prepared) was added to 0.6 L of fine heparin (33 g / L), and the reaction was carried out at 300 rpm and 4°C in the dark for 22 hours. 80 mL of ethylene glycol was added to neutralize the excess sodium periodate, and then 28 g of sodium borohydride was added at 4°C for 16 hours. The pH was adjusted to 7.0 with HCl. The filtrate was collected by suction filtration through a 0.22 μm filter membrane. The sample was then desalted using a dialysis bag or concentrated and desalted by ultrafiltration through a Millipore ultrafiltration device with a 1K filter membrane until the filtrate showed no color change when tested with 0.1 M AgNO3. The sample was frozen at -80°C and then lyophilized in a freeze dryer. The powder was then pulverized with a mortar or a small crusher and stored to obtain the anticoagulant-free heparin derivative (named NAHP).

[0181] The anticoagulant-free heparin derivative (NAHP) obtained above was dissolved in a reaction buffer, and heparinase I prepared according to the method of ZL200410038098.6 was added to the solution every 0.5-1 h, with each addition of 20 IU of heparinase I. The optical absorption A231 of the solution at 231 nm was monitored using a quartz cuvette with an optical path difference of 1 cm and an ultraviolet spectrophotometer (the instrument was calibrated to zero using a buffer at pH 7.4, and in order to ensure the accuracy of the test results, when the ultraviolet spectrophotometer reading A231 was greater than 0.6, the solution was diluted by a certain factor to make the reading between 0.2 and 0.6). When A231 reached 46, the reaction was stopped, and the total enzyme activity of heparinase I added was about 220-250 IU. The method of stopping the reaction was to inactivate the enzyme in the reaction solution by boiling in a water bath at 100°C for 5-10 min, remove the reaction system and cool it to room temperature, add 6 times the volume of anhydrous ethanol to the reaction solution, stir at room temperature for 10 min, then centrifuge at 4000 r / min at room temperature for 15 min, collect the precipitate, add 2-3 times the mass of deionized water to the precipitate to dissolve it thoroughly, then filter the solution using a filter membrane with a pore size of 0.22 μm, collect the filtrate and freeze it into a solid ice block in a -80°C freezer, then lyophilize it using a freeze dryer (the cold trap temperature was -50°C), and finally pulverize the powder with a mortar or a small crusher to obtain the low molecular weight anticoagulant-free heparin (named: LNAHP).

[0182] The obtained anti-coagulant heparin derivative (NAHP) was dissolved in the reaction solution, and heparase I prepared according to the method of ZL200410038098.6 was added to the solution every 0.5-1 h, 20 IU of heparase I was added each time, and the light absorption A231 of the solution at 231 nm was monitored using a quartz cuvette with an optical path difference of 1 cm and an ultraviolet spectrophotometer (the instrument was calibrated to zero using a buffer solution at pH 7.4, and in order to ensure the accuracy of the test results, when the ultraviolet spectrophotometer reading A231 was greater than 0.6, the solution was diluted by a certain multiple so that the reading was within 0.2-0.6). When A231 reached 106, the reaction was stopped, and the total enzyme activity of heparase I added was about 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°C for 5-10 min, then remove the reaction system and cool it to room temperature, add 6 times the volume of anhydrous ethanol to the reaction solution, stir at room temperature for 10 min, then centrifuge at room temperature at a speed of 4000 r / min for 15 min, collect the precipitate, dissolve it in deionized water with a mass of 2-3 times that of the precipitate, filter it using a 0.22 μm membrane, collect the filtrate and place it in a -80°C freezer to freeze into a solid ice block, then send it to a freeze dryer (the cold trap temperature is -50°C) for freeze-drying, then crush it into powder using a mortar or a small crusher, and obtain the ultra-low molecular weight anti-coagulant heparin (also named: ULNAHP).

[0183] Example 2 Preparation of a purified polysaccharide component

[0184] Step (1): Isolation of polysaccharide

[0185] Experimental equipment: An AKTA Prime purification system was used in combination with a gel exclusion chromatography column (HiPrep 16 / 60 Sephacryl S-100HR) for preparative liquid chromatography.

[0186] Process parameters: The mobile phase was 0.2M NaCl aqueous solution (pH = 5.00), the flow rate was 0.5 mL / min, and the column volume was 120 mL; the sample loading amount for one separation was a maximum of 5 mL.

[0187] Experimental method: The polysaccharide raw material LNAHP obtained in Example 1 was dissolved in the mobile phase at a concentration of 120 mg / mL, and the separated components were collected from the 80th min after sample injection according to the different molecular weights of the sample. According to the principle of gel exclusion chromatography, the first separated component was a polysaccharide with a large molecular weight, and the last separated component was a polysaccharide with a small molecular weight. During the separation, one component was collected every 10 min until the end of the 180th min. According to the different collection times, each separated component with different molecular weights was obtained, thereby realizing the enrichment and purification of polysaccharides with specific molecular weights, and obtaining a refined polysaccharide salt solution with specific molecular weight distribution. Among them, the refined polysaccharide separated component collected at the 110th min after the sample injection was named S4, and the refined polysaccharide separated component collected at the 130th min was named S6.

[0188] Step (2): Primary freeze-drying of polysaccharide solution

[0189] The salt solution of each component refined polysaccharide obtained in step (1) was pre-frozen at -80°C, and then placed in a freeze dryer to freeze-dry until the water was completely removed, thereby obtaining the primary freeze-dried product of each component polysaccharide solution.

[0190] Step (3): Concentrated alcohol precipitation

[0191] Each component primary freeze-dried product obtained in step (2) was resuspended in distilled water at an amount of 30% of the original volume to enrich the refined polysaccharide and increase the alcohol precipitation yield. Subsequently, the polysaccharide solution was precipitated with anhydrous ethanol, and after thorough mixing and standing, it was centrifuged. After centrifugation, all the supernatant (a mixture of ethanol and NaCl containing a small amount of soluble polysaccharide) was carefully discarded. Then, the precipitate was resuspended with an appropriate amount of distilled water to obtain the desalted water solution of each component refined heparin polysaccharide.

[0192] Step (4): Secondary freeze-drying of polysaccharide solution

[0193] The desalted water solution of each component refined polysaccharide obtained in step (3) was pre-frozen at -80°C, and then placed in a freeze dryer to freeze-dry until the water was completely removed, thereby obtaining the freeze-dried product of the refined polysaccharide.

[0194] After the preliminary anti-UC function and anti-inflammatory effect verification of each isolated component obtained by the above method, it was found that the S6 component had a relatively excellent anti-UC treatment effect; at the same time, the S4 and S6 components also had a relatively excellent anti-inflammatory effect. In the following experiments of the present application, the anti-inflammatory and anti-UC biological activities of S4 and S6 will be further verified and their drug structure-activity relationship will be analyzed. Specifically, the anticoagulant heparin derivatives (NAHP), low molecular weight anticoagulant heparin (LNAHP), and ultra-low molecular weight anticoagulant heparin (ULNAHP) used in the following experimental examples were obtained by the method of Example 1; the polysaccharide isolated components S4 and S6 used were obtained by the method of Example 2; the heparin (HP) used was unfractionated heparin purchased from Changshan Biochemical, Hebei, and the weight average molecular weight Mw of the heparin was 17223 Da; the 5-amino salicylic acid preparation (5-Amino Salicylic Acid, 5-ASA) used was mesalazine sustained-release granules purchased from a pharmacy, and the indications were: ulcerative colitis, used for acute attack of ulcerative colitis, prevention of recurrence; Crohn's disease, used for frequent onset of Crohn's disease patients, prevention of acute attack; the NAEno used was an anticoagulant enoxaparin derivative obtained by the same anticoagulant modification process of NAHP in Example 1 using enoxaparin (purchased from Changshan Biochemical) as the raw material; the NAI45 used was a low molecular weight anticoagulant heparin derivative obtained by the same anticoagulant modification process of NAHP in Example 1 after enzymolysis according to the method of Comparative Example 5 in ZL201810100469.0 using heparin (purchased from Changshan Biochemical) as the raw material, which was found to have no effect on the treatment of UC in the previous preliminary in vivo function verification experiment.

[0195] Determination of molecular weight and distribution of refined heparin polysaccharide components

[0196] The weight average molecular weight (Mw) and distribution coefficient (P) of the polysaccharide raw material LNAHP obtained in Example 1 and the refined polysaccharide separation component S6 obtained in Example 2 were determined by gel exclusion high performance liquid chromatography. The chromatographic column was TSK-GEL G2000SWXL (TOSOH, Japan), the control flow rate was 0.5 mL / min, the column temperature was 35°C, and the injection volume was 25 μL. A WATERS (1525, USA) chromatographic system, an ultraviolet detector and a differential detector were connected in series at the outlet of the chromatographic column in the order of ultraviolet detector and differential detector, and the wavelength of the ultraviolet detector was 234 nm. The method for determining the molecular weight and its distribution can refer to the method described 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. The specific determination results are shown in Table 1.

[0197] Table 1 Molecular weight of refined polysaccharide raw material and refined separation component

[0198]

[0199] From the molecular weight information in Table 1, it can be seen that the separation components S4 and S6 obtained after refining have obviously different molecular weight distribution compared with the polysaccharide raw material. From the specific indicators, it can be reflected in the polydispersity, i.e. the distribution coefficient P (Polydispersity). The polydispersity is the ratio of the weight average molecular weight to the number average molecular weight, which is a positive number greater than 1. The closer the value is to 1, the closer the substance is to a single substance with a definite molecular weight. The polydispersity of the polysaccharide raw material used in this embodiment is greater than 1.6, and after refining and preparation, the polydispersity of the separation component is even less than 1.1, indicating that the molecular weight distribution of the refined polysaccharide is obviously more concentrated and closer to a pure substance.

[0200] Experimental Example 2 Anti-UC and Anti-inflammatory Biological Activity Verification of Refined Polysaccharide Separation Component

[0201] 1. The anti-UC biological activity of the refined polysaccharide separation component obtained in Example 2 was investigated in vivo by DSS-induced UC model mice. In the evaluation index, mainly using the body weight change, colon length, spleen weight index, serum inflammatory factor content, colon pathological histology evaluation and other indicators, the efficacy of the refined polysaccharide in treating UC was systematically evaluated.

[0202] Experimental method:

[0203] Animal experimental model selection 6-8 weeks of male C57BL / 6J mice. Set up healthy control group (WT group), DSS modeling group, and drug treatment group after modeling.

[0204] Day 0, except for the WT group, the rest of the groups will replace the drinking water with 3% DSS (sodium dextran sulfate, Mw: 36,000-50,000 Da, MP biomedicals, LLC) aqueous solution to induce UC mouse model, and record the body weight of mice every day.

[0205] Day 3, at a drug dose of 30 mg / kg / one / day, UC mouse models in different drug treatment groups were treated by gavage, which lasted until Day 7.

[0206] Day 7, the experiment ended, the experimental mice were euthanized, and the spleen and colon tissues were obtained. The spleen tissue was weighed, and the spleen weight index was calculated according to the spleen weight and body weight to evaluate the in vivo anti-inflammatory and immune regulation efficacy of the isolated components of refined polysaccharides. The colon length was measured, and then the colon tissue was paraffin-embedded and pathological tissue section H&E staining was performed, and according to the structural changes of the colon epithelial tissue of the UC mice, the in vivo anti-UC efficacy of the isolated components of refined polysaccharides was evaluated. The specific experimental results are shown in Figures 1-4 .

[0207] Experimental results:

[0208] (1) Body weight loss is one of the important phenotypes of DSS-induced UC mouse models, and this index can characterize the severity of the disease. Figure 1 The ANOVA analysis chart of the body weight change curve of the mice can be seen. During the treatment period from the 3rd day to the 7th day, the body weight loss trend of the mice in the oral HP, NAI45, and NAEno treatment groups was not significantly improved compared with the DSS group. On the 6th day, the body weight loss trend of the mice in the oral effective treatment drug (LNAHP, ULNAHP) and the isolated component S6 of refined polysaccharides was significantly relieved compared with the DSS group (p<0.05, p<0.01, p<0.05). On the 7th day, the mice in the oral NAHP and the first-line clinical drug (5ASA) could significantly relieve the trend of body weight loss induced by DSS (p<0.05, p<0.01), while the mice in the oral LNAHP, ULNAHP, and S6 had more significant relief of the trend of body weight curve (p<0.0001, p<0.0001, p<0.0001). The above results show that the isolated component S6 can effectively relieve the body weight loss trend of UC mice, and has more significant efficacy in relieving the body weight loss symptoms of UC mice compared with the first-line clinical drug 5ASA.

[0209] (2) Colon length shortening is one of the characteristic phenotypes of UC mouse model, and is closely related to the severity of the disease. Figure 2 The results of colon length measurement of mice in each experimental group are shown. Compared with DSS group mice, oral administration of the rest of heparin derivatives and 5ASA, except for HP, NAEno and NAI45 treatment groups, can effectively alleviate the colon length shortening phenomenon of UC mice induced by DSS, among which: the S6 treatment group of mice has the best effect of alleviating colon length shortening, and the most significant difference compared with DSS group mice (p<0.0001). The above results show that the refined polysaccharide separation component S6 achieves more effective therapeutic effect on alleviating the symptoms of colon length shortening in UC mice, and the therapeutic effect is more significant than that of 5ASA, the first-line clinical drug.

[0210] (3) As an important immune organ, the spleen will appear splenomegaly and weight increase when the whole body 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 activation of the immune system of an individual can be represented by the spleen weight index. Figure 3 The schematic diagram of spleen weight index is shown. It can be seen that the DSS model group mice have obvious splenomegaly and increased spleen weight index compared with the WT group mice. Compared with the DSS group, oral administration of NAHP, LNAHP, S6 and 5ASA can very significantly alleviate the symptoms of splenomegaly in UC mice and inhibit the increase of spleen weight index (p<0.01, p<0.01, p<0.01, p<0.01); while oral administration of NAEno and NAI45 cannot effectively alleviate splenomegaly and the increase of spleen weight index. The above results show that the refined polysaccharide separation component S6 can significantly alleviate splenomegaly and inhibit the increase of spleen weight index in UC mice, and has significant immunosuppressive ability; its effect on inhibiting splenomegaly and the increase of spleen weight index is comparable to that of 5ASA.

[0211] (4) Histopathological evaluation of colon mucosa is the gold standard for clinical diagnosis, treatment and efficacy evaluation of UC, and is also an important indicator for evaluating the biological activity of anticoagulant heparin derivatives and refined separation components for UC treatment. Figure 4Figure 1 shows the pathological histomorphological changes of the colon epithelium of the UC mice evaluated by H&E staining. The WT group mice had normal colon epithelial structure, with intact colon epithelial cells, normal crypt structure, regular crypt arrangement, a large number of goblet cells, and no neutrophil infiltration. The colon epithelium of the DSS group mice showed severe neutrophil and macrophage infiltration, compared with the WT group mice, the original colon epithelial cells were shed and necrotic, the goblet cells were largely absent, the colon epithelial barrier was completely destroyed, and the crypt structure was completely absent. Compared with the DSS group mice, oral administration of HP, NAEno, and NAI45 could not effectively alleviate the destruction of the colon epithelial structure and the shedding of epithelial cells and other disease symptoms. Oral administration of NAHP, ULNAHP, and 5ASA could partially improve the colon crypt loss and epithelial cell shedding and necrosis, but the integrity of the colon epithelium and colon crypt was poor, the crypt shape and arrangement were abnormal, there was still obvious inflammatory cell infiltration between the crypts, and there was a phenomenon of goblet cell loss. Oral administration of LNAHP and S6 could significantly protect the colon epithelial tissue of the mice, with intact colon epithelial structure, normal crypt shape, regular crypt arrangement, normally distributed goblet cells, and a small amount of inflammatory cell infiltration. The results of the histopathological evaluation showed that the refined polysaccharide separation component S6 could significantly alleviate the destruction of the colon epithelial tissue structure of the UC mice and restore the colon epithelial tissue structure to normal, and inhibit the inflammatory response in the peripheral blood and intestinal lamina propria. Compared with the first-line clinical drug 5ASA, S6 has superior biological activity for treating UC.

[0212] Through the above comprehensive index evaluation, the anti-UC biological activity of the refined polysaccharide separation component S6 is excellent, and the efficacy-related indicators are better than those of the first-line clinical drug 5ASA.

[0213] 2. The anti-UC biological activity of the refined polysaccharide separation components S4 and S6 obtained in Example 2 above was investigated in vitro in terms of drug efficacy by constructing an in vitro inflammation model using a mouse macrophage cell line RAW 264.7 induced by lipopolysaccharide.

[0214] Experimental method:

[0215] Mouse macrophage RAW 264.7 (purchased from ATCC) was used, and was inoculated into a 48-well plate at a concentration of 150,000 cells / mL, and was cultured in a high-sugar DMEM medium plus 10% fetal bovine serum at 37°C, 5% CO2. After overnight culture, the old culture 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, and the rest were drug treatment groups. Among them, the WT group was only added with a serum-free medium, and the rest groups were added with a serum-free medium containing 100 ng / mL LPS. Then, the cells in each group were placed in a cell incubator for 15 min. After 15 min, heparin, heparin derivatives, and isolated components S4 and S6 were added to the drug treatment groups at a concentration of 1 mg / mL, and were treated for 24 hrs. After 24 hrs, the culture medium supernatant was used to detect inflammatory factors such as IL-6 and TNF-α secreted by RAW 264.7 by ELISA (the groups were set up and the detection results are shown in Table 1). Figure 5

[0216] Experimental results:

[0217] The sustained chronic inflammatory response is one of the important clinical phenotypes of UC, and is the main action target of UC treatment drugs in clinical practice. In the above-mentioned UC mouse model experiment induced by DSS, it was found that S6 had good anti-UC biological activity. However, the mechanism of the effective anti-UC heparin derivative in inhibiting inflammation has not been elucidated. In order to confirm whether the polysaccharide component with effective anti-UC activity has significant anti-inflammatory activity, a LPS-stimulated RAW 264.7 cell was constructed as an in vitro model to evaluate the in vitro anti-inflammatory effect of heparin derivatives and refined heparin polysaccharides. RAW 264.7 is a mouse macrophage, which is an important component of innate immunity, and will secrete a large amount of cytokines such as IL-6 and TNF-α under the stimulation of exogenous antigens such as LPS, thereby triggering the sustained activation of the immune system. IL-6 and TNF-α are highly expressed in UC patients, and are also effective action targets of UC treatment drugs. Therefore, by investigating the expression content of cytokines such as IL-6 and TNF-α, the overall inflammatory response level can be evaluated.

[0218] From Figure 5 ​As 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.

[0219] Experiment Example 3: Verification of the anti-UC efficacy and anti-inflammatory activity of purified polysaccharide fractions and structure-activity relationship analysis

[0220] 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).

[0221] 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 x 2 mm; detector, high resolution mass spectrometer; column temperature, 22 °C; flow rate, 0.15 ml / min; injection volume, 3 μL; run time 100 min; mobile phase, acetonitrile-water system. The mass spectrometry detection parameters were as follows: sheath gas flow, 20 arb; aux gas flow, 5 arb; I spray voltage, 4.2 kV; capillary temp, 275 °C; S-Lens RF Level, 50%. The obtained mass spectrometry raw data were extracted for the accurate mass-to-charge ratio (accurate to the fourth decimal place, mass tolerance set to 5 ppm) of specific sugar chain structures using Xcalibur software, and then integrated to obtain the mass spectrometry peak area, thereby obtaining the structural information and abundance information of each oligosaccharide component.

[0222] After obtaining the structural information and abundance information of the above oligosaccharide components, we further analyzed which oligosaccharide component was effective for anti-UC by the following method:

[0223] Using R studio software (Version 1.2.1335) on a Windows 10 operating system, R language programming was used in combination with pheatmap and other program packages for visualization, and the content of different oligosaccharides in different samples was visualized using a heat map to obtain the whole sugar chain map analysis results of the above groups, as shown in Figure 6 . (Each row in the figure represents an oligosaccharide component, and each column represents the oligosaccharide enrichment of the corresponding sample, the data are row-normalized, the oligosaccharide component is colored dark, the oligosaccharide component is colored white, and the oligosaccharide component is white).

[0224] 1. From Figure 6 It can be seen that the oligosaccharide composition of the effective separation component S6 is significantly different from other heparin derivatives. On this basis, in order to further obtain the functional fragments of the effective heparin derivatives for biological activity, the oligosaccharides in the whole sugar chain map analysis were sorted according to the content from high to low, and the content was calculated to obtain the threshold relationship graph of oligosaccharide coverage and oligosaccharide enrichment Figure 7 . It can be seen from Figure 7 that the oligosaccharide enrichment threshold is between 0% and 40%, and the oligosaccharide coverage rises rapidly, indicating that the oligosaccharide components with a content of 40% have good representativeness. Therefore, in the subsequent analysis, the oligosaccharides with a content of 40% were selected as the enriched oligosaccharides of each heparin derivative and separation component, representing the main components of each heparin derivative and separation component.

[0225] On this basis, the enriched oligosaccharides of each heparin derivative and isolated component were analyzed by Venn diagram using R studio software (Version 1.2.1335) on Windows 10 operating system, R language programming and combined with UpSetR and other packages, and the results are shown in Table 1. Figure 8 It can be seen that among the heparin derivatives effective against UC, there is a common polysaccharide. Therefore, the polysaccharide enriched in LNAHP and S6 but not in NAI45 is defined as an effective component, the polysaccharide enriched in NAI45 but not in LNAHP and S6 is defined as an ineffective component, and the remaining components are defined as other components.

[0226] On Windows 10 operating system, R studio software (Version 1.2.1335) was used to analyze the corresponding oligosaccharide data by R language programming and combined with ggplot2, stringr, pheatmap, ggsci, UpSetR and other packages. Figure 8 Figure 9 It can be seen that the effective component has a higher number of sulfonic acid groups and a smaller number of ring-opening structures compared to the ineffective component and other components. It can be inferred that the oligosaccharide effective against UC has the characteristics of high sulfonation and no ring-opening structure. According to the classification results, Figure 8 The effective component oligosaccharide information classified by enriched oligosaccharide is summarized as shown in Table 2:

[0227] Table 2 Summary of oligosaccharide information of effective components against UC

[0228]

[0229] Note: The oligosaccharide structure represents [polysaccharide ring-opening structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure]-[amino (i.e. the number of ammonium ions carried in mass spectrometry, not the structure of the polysaccharide itself)]

[0230] On this basis, the content of each type of oligosaccharide in heparin derivatives and isolated components was calculated, and the results are shown in Table 3. The effective components listed in Table 3 are highly enriched in anti-UC effective heparin derivatives LNAHP, ULNAHP, S6, and have low content in other heparin derivatives, which is consistent with the in vivo anti-UC efficacy of heparin derivatives.

[0231] Table 3 Summary of the content of each type of oligosaccharide in heparin derivatives and isolated components ​

[0232]

[0233] On this basis, the effective components in Table 2 are further analyzed, and the oligosaccharide reconstruction of the effective components is carried out in the following manner in combination with the biosynthesis process of heparin derivatives:

[0234] (1) First, reconstruct the polysaccharide skeleton: according to the number of unsaturated uronic acid, uronic acid and glucosamine, determine the length of the heparin sugar chain, and construct the basic skeleton of the effective component in the form of alternating arrangement of uronic acid and glucosamine.

[0235] (2) Second, reconstruct the open ring structure and acetyl group: according to the synthesis mechanism of heparin polysaccharide, the open ring structure is reconstructed to the uronic acid, and the acetyl group is reconstructed to the glucosamine. According to the subsequent reconstruction of sulfonic acid group, the above reconstructed structure may also need to be adjusted slightly according to the number of sulfonic acid groups.

[0236] (3) Finally, reconstruct the sulfonic acid group: mainly according to the number of sulfonic acid groups, combined with the number and probability of open ring structure and acetyl group, and comprehensive consideration of probability theory. Preferably, the NS domain disaccharide highly contained in heparin is reconstructed with sulfonic acid group, and then the sulfonic acid group on the sugar ring containing open ring structure and acetyl group is reconstructed.

[0237] After the above reconstruction, the molecular structure characteristics of the effective anti-UC oligosaccharide component can be obtained. The polysaccharide structure drug composition, molecular formula and structural formula of the effective component are shown in Table 4.

[0238] Table 4 Structure composition and molecular formula of effective anti-UC oligosaccharide

[0239]

[0240]

[0241]

[0242]

[0243] Note 1: [open ring]-[△HexA, HexA, HexN, Ac, SO3, dehydration]-[NH3] represents [polysaccharide open ring structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure]-[amino (the number of ammonium ions carried in mass spectrometry, not all the structure of polysaccharide itself)]

[0244] Note 2: In the column of "Reconstruction of main structure and secondary structure", the main structure is the molecular structure which is most likely to appear according to the process of heparin biosynthesis and the preparation process of heparin derivatives; the secondary structure is the molecular structure which exists in the process of heparin biosynthesis and the preparation process of heparin derivatives, but is not the main reaction reached, and it is estimated that its amount is at least one order of magnitude less than the main structure.

[0245] Note 3: In the column of "Reconstruction of main structure and secondary structure", △UA represents unsaturated uronic acid, HexA represents uronic acid, GlcA represents glucuronic acid, IdoA represents iduronic acid, and GlcNAc represents N-acetylglucosamine; Ω is a symbol of ring-opening modification, NS / 6S / 3S / 2S represents N-sulfonic acid group, 6-O-sulfonic acid group, 3-O-sulfonic acid group, and 2-O-sulfonic acid group modification on uronic acid and glucosamine, respectively; and the symbols such as "()" and "[]" represent that the polysaccharide structures in the same symbol can be replaced with each other in sequence

[0246] According to the data in Tables 2, 3 and 4, it can be found that the structural characteristics of the anti-UC effective polysaccharides are that the polysaccharide fragments containing no or a small amount of ring-opening structure (produced by heparin anticoagulation) are highly sulfated, and the polysaccharides enriched with such polysaccharide fragments or composed of such polysaccharide fragments have the efficacy of treating UC.

[0247] So far, we have found a group of oligosaccharide components with specific polysaccharide structure composition, which are mainly based on anti-UC effective heparin derivatives and are highly enriched in LNAHP and S6. Based on this discovery, further reasoning can be made that the ideal oligosaccharide fragments exerting anti-UC biological activity should have the following structural characteristics:

[0248] (1) The length of the polysaccharide sugar chain is between 2 and 20 sugars, and the basic disaccharide structural unit is composed of [0]-[1,0,1,0,3,0]-[0] and [0]-[1,0,1,0,2,0]-[0] repeated arrangement and combination, wherein the above-mentioned representation means [polysaccharide ring-opening structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure]-[amino (the number of polysaccharide amino groups in mass spectrometry, not all the structure of the polysaccharide itself)]

[0249] (2) It has the characteristic of high sulfation, and the average content of sulfonic acid group per 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 less.

[0250] (3) On this basis, it basically contains no or a small amount of ring-opening structure, and the average content of ring-opening structure per disaccharide is less than or equal to 0.3.

[0251] According to the above structural features, we can infer that the heparin derivatives polysaccharides with high sulfation, no or small amount of open ring structure have the effect of treating UC according to the characteristics of heparin biosynthesis process, the preparation process of heparin derivatives and the complete sugar chain analysis results. The above results can be further summarized using the formula, and the derivation process is as follows:

[0252] For any oligosaccharide material: [a]-[b,c,d,e,f,g]-[h], the above structural formula represents: the number of each structure of [polysaccharide open ring structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydrated structure]-[amino (the number of polysaccharide with amino in mass spectrum, not all the structure of polysaccharide itself)] in oligosaccharide.

[0253] For heparin oligosaccharides, there is only one unsaturated uronic acid, i.e. b = 1. And because the number of uronic acid and glucosamine is equal, b + c = d. The number of glucosamine d is also equal to the number of disaccharide units, which means that the oligosaccharide is composed of d disaccharide units, here 1 <= d <= 10. The length of the entire oligosaccharide sugar chain is 2d. Because the effective heparin derivatives do not contain any dehydrated structure in the preparation process, g = 0. h is the number of polysaccharide with amino in mass spectrum, which is irrelevant to the structure of polysaccharide itself, and is not limited here.

[0254] According to the structural characteristics of effective anti-UC oligosaccharides, such oligosaccharides have high sulfation structure, which requires the average content of sulfonic acid groups per disaccharide to be greater than 2.5, i.e. f >= 2.5d. At the same time, such oligosaccharides basically do not contain or contain a small amount of open ring structure, and the average content of open ring structure per disaccharide is less than 0.3, i.e. a <= 0.3d. For the number of acetyl groups e, according to Figure 9 As a result, combined with the characteristics of heparin biosynthesis, e <= 1.0.

[0255] According to the above information, further arrange the variables. The structural formula of the effective anti-UC oligosaccharide can be obtained as follows:

[0256] [a]-[1,d-1,d,e,f,0]-[h]

[0257] Where: 1 <= d <= 10, a <= 0.3d, f >= 2.5d, e <= 1.0, h is not limited.

[0258] According to the above formula, the molecular formula of such oligosaccharide material can be further obtained as follows:

[0259] C (12d+2e) H (2a-2b+19d+2e+2) O (1-b+10d+e+3f) N (d) S (f)

[0260] According to the molecular formula, the molecular weight can be calculated.

[0261] 2. According to Figure 6 The contents of all oligosaccharide fragments detected in the whole oligosaccharide mapping analysis in each heparin derivative (LNAHP, S4, S6 and NAI45) with significant anti-inflammatory activity were added together, and the oligosaccharide fragments were ranked from high to low according to the added values, and the top 15% of oligosaccharide fragments were taken as representative oligosaccharide fragments. The data was collated and summarized as shown in Table 5. The contents of the representative oligosaccharide fragments in each heparin derivative were further summarized as shown in Table 6.

[0262] Table 5 Anti-inflammatory heparin derivative representative oligosaccharide fragments

[0263]

[0264] Note: The oligosaccharide structure represents the method as [polysaccharide ring structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure]-[amino (i.e. the number of ammonium ions carried in mass spectrometry, not all the structure of the polysaccharide itself)]

[0265] Table 6 Anti-inflammatory effective oligosaccharide content summary in each heparin derivative and isolated component

[0266]

[0267] Table 7 Molecular formula and molecular weight information of anti-inflammatory effective oligosaccharide

[0268]

[0269] So far, we have found a class of oligosaccharide fragments with clear composition, which accounts for only 15% of the total number of oligosaccharide species, but is highly enriched in anti-inflammatory heparin derivatives, with a content of more than 45%, especially in S4, with a content of more than 90%. It can be speculated that the above oligosaccharide is the functional fragment of heparin derivative for anti-inflammatory biological activity.

[0270] In order to further summarize the structural characteristics of the above anti-inflammatory oligosaccharides, the structural formula is further summarized by using the formula, and the derivation process is as follows:

[0271] For any type of oligosaccharide: [a]-[b,c,d,e,f,g]-[h], where the above structural formula represents: the number of each structure in the oligosaccharide, such as [polysaccharide ring structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure]-[amino (the number of polysaccharide with ammonia in mass spectrometry, not all the structure of the polysaccharide itself)].

[0272] For heparin oligosaccharides, there is only one unsaturated uronic acid, i.e. b = 1. And because the number of uronic acids and glucosamines are equal, b + c = d. Where the number of glucosamines d is also equal to the number of disaccharide units, meaning that the oligosaccharide is composed of d disaccharide units, here, 1 <= d <= 10. The length of the entire oligosaccharide sugar chain is 2d. Since the effective heparin derivative does not contain any dehydrated structures in the preparation process, g = 0. h is the number of amino groups in the mass spectrum of polysaccharide, which is irrelevant to the structure of the polysaccharide itself, and is not limited here.

[0273] According to the structural characteristics of the effective anti-inflammatory oligosaccharide, the molecular structure of most anti-inflammatory oligosaccharides meets the following conditions: the number of acetyl groups contained in each disaccharide is greater than or equal to 0.1, i.e. e >= 0.1d; the number of sulfonic acid groups contained in each disaccharide is less than or equal to 2.7, i.e. f <= 2.7d; the number of ring-opening sugar rings contained in each disaccharide is greater than or equal to 0.25, i.e. a >= 0.25d.

[0274] According to the above information, further arrange the variables. The structural formula of the effective anti-inflammatory oligosaccharide can be obtained:

[0275] [a]-[1,d-1,d,e,f,0]-[h]

[0276] Where: 1 <= d <= 10, a >= 0.25d, f <= 2.7d, e >= 0.1d, h is not limited.

[0277] According to the above formula, the molecular formula of this type of oligosaccharide can be further obtained:

[0278] C (12d+2e) H (2a-2b+19d+2e+2) O (1-b+10d+e+3f) N (d) S (f)

[0279] According to the molecular formula, the molecular weight can be calculated.

[0280] 3. Comparison of the structure of anti-inflammatory effective oligosaccharides and anti-UC effective oligosaccharides

[0281] By summarizing the structural formula of the two oligosaccharides above, the similarities and differences in their structures are compared, and the specific results are shown in Table 8.

[0282] Table 8 Comparison of the structure of anti-inflammatory effective oligosaccharides and anti-UC effective oligosaccharides

[0283]

[0284] As can be seen from Table 8, there is only a small intersection between the anti-UC oligosaccharides and the anti-inflammatory oligosaccharides in the number of acetyl groups, sulfonic acid groups and ring-opening structures of the sugar ring, and overall, the group composition of the two types of oligosaccharides still has significant differences. They are two types of different oligosaccharide components.

[0285] The above description is merely preferred embodiments of the present application, but not any form of limitation to the present application. Any modification, equivalent replacement, and improvement made by any person with the technical contents disclosed by the present application shall fall within the protection scope of the present application.

Claims

1. A method for preparing a refined heparin polysaccharide, comprising: subjecting a raw heparin to a de-anticoagulation treatment and an enzymatic hydrolysis to obtain a heparin polysaccharide raw material; subjecting the heparin polysaccharide raw material to separation by using a gel exclusion chromatography column and collecting the separated components; subjecting the collected separated components to a freeze-drying treatment; subjecting the freeze-dried products of the components to a desalting treatment by alcohol precipitation to obtain a refined heparin polysaccharide, wherein, when the heparin polysaccharide raw material is subjected to separation by using a gel exclusion chromatography column, the gel exclusion chromatography column is a HiPrep 16 / 60 Sephacryl, and a mobile phase used is a 0.15-0.6 M NaCl aqueous solution, and a flow rate of the mobile phase is 0.3-0.7 mL / min. 2.The method according to claim 1, wherein, the de-anticoagulation treatment of the raw heparin is a de-anticoagulation treatment of the raw heparin by using a high-iodic acid oxidation method to obtain a de-anticoagulation treated product, and the enzymatic hydrolysis of the de-anticoagulation treated product is an enzymatic hydrolysis of the de-anticoagulation treated product by using heparinase I to obtain the heparin polysaccharide raw material. 3.The method according to claim 1, wherein, the freeze-drying treatment is a pre-freezing of the collected separated components at -80 ℃, and then the pre-frozen components are put into a freeze-dryer for freeze-drying treatment.

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

5. The production method according to claim 1, wherein, the flow rate of the mobile phase in the gel exclusion chromatography column is 0.5 mL / min. 6.The method according to claim 1, wherein, the pH value of the NaCl aqueous solution is 3-10. 7.The method according to claim 1, wherein, the pH value of the NaCl aqueous solution is 5. 8.The method according to claim 1, wherein, the filler of the gel exclusion chromatography column is Sephacryl S-100 High Resolution, Sephacryl S-200 High Resolution or Sephacryl S-300 High Resolution.

9. The production method according to any one of claims 1 to 8, wherein when the freeze-dried products of the components are subjected to a desalting treatment by alcohol precipitation, the freeze-dried products of the components are resuspended by adding distilled water; after adding an ethanol aqueous solution, alcohol precipitation is allowed to stand; the precipitated products after alcohol precipitation are centrifuged, and the supernatant is discarded; and then, the refined heparin polysaccharide is obtained by resuspending after adding distilled water. 10.The method according to claim 9, wherein, the freeze-dried products of the components are resuspended by adding distilled water with a volume of 20-50% of the volume of the freeze-dried products. 11.The method according to claim 9, wherein, the freeze-dried products of the components are resuspended by adding distilled water with a volume of 30% of the volume of the freeze-dried products. 12.The method according to claim 9, wherein, the volume of the ethanol aqueous solution added is 2-6 times the volume of the resuspended and concentrated liquid; and the concentration of the ethanol aqueous solution is 75%-100%.

13. The method of making according to claim 12, wherein, the volume of the ethanol aqueous solution added is 5-6 times the volume of the resuspended and concentrated liquid. 14.The method according to claim 9, wherein, The time for standing the alcohol precipitation is 5-60 min.

15. The preparation method of claim 9, wherein, The time for standing the alcohol precipitation is 10-30 min.

16. The preparation method of claim 1, further comprising, The refined heparin polysaccharide obtained after desalination by concentrated alcohol precipitation is subjected to freeze-drying treatment.

17. An oligosaccharide prepared by the preparation method of any one of claims 1-16.

18. The oligosaccharide of claim 17, wherein, The oligosaccharide has the following structure: [a]-[b,c,d,e,f,g]-[h], wherein, a is the number of open-loop structures of sugar rings in the oligosaccharide molecule; b is the number of unsaturated uronic acids in the oligosaccharide molecule; c is the number of saturated uronic acids in the oligosaccharide molecule; d is the number of glucosamine in the oligosaccharide molecule, and 1≤d≤10; e is the number of acetyl groups in the oligosaccharide molecule; f is the number of sulfonic acid groups in the oligosaccharide molecule, and f≤2.7d, g is the number of 1,6-anhydro structures in the oligosaccharide molecule, h is the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometric detection.

19. The oligosaccharide of claim 18, wherein, In the structural formula of the oligosaccharide, a≥0.25d, b=1, c=d-1, e≥0.1d, g=0.

20. The oligosaccharide of claim 17, wherein, The oligosaccharide has one of the following structures: [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], [3]-[1,4,5,1,8,0]-[7].

21. A heparin derivative comprising an oligosaccharide according to any one of claims 18 to 20, wherein the oligosaccharide is attached to the heparin backbone via a 2-O-sulfate group. In the heparin derivative, the content of the oligosaccharide of any one of claims 18-20 is above 52%.

22. Use of the oligosaccharide of any one of claims 18-20 and the heparin derivative of claim 21 in the preparation of an anti-inflammatory drug.

23. The use of claim 22, wherein, The anti-inflammatory drug is used for preventing and treating inflammation in the process of pneumonia, lung and liver fibrosis caused by the novel coronavirus, arthritis, irritable bowel syndrome, gastritis, skin inflammation and inflammatory bowel disease.

24. The use according to claim 23, wherein, The arthritis includes rheumatoid arthritis.

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