WO2023097925A1

WO2023097925A1 - Oral polysaccharide for treating inflammatory bowel disease and preparation method therefor

Info

Publication number: WO2023097925A1
Application number: PCT/CN2022/080641
Authority: WO
Inventors: 邢新会; 王怡; 曾文; 张翀
Original assignee: 清华大学
Priority date: 2021-11-30
Filing date: 2022-03-14
Publication date: 2023-06-08
Also published as: CN116200437A

Abstract

The present application relates to a preparation method for a refined heparin polysaccharide. Raw materials of the heparin polysaccharide are separated by means of size exclusion chromatography, and the collected separated components are subjected to treatments such as freeze-drying, alcohol precipitation, and desalination so as to obtain a refined heparin polysaccharide product. The present application further relates to an oligosaccharide having specific polysaccharide structural features and anti-UC effectiveness, which can be used for prevention and treatment of inflammatory bowel disease, complications related to inflammatory bowel disease, and diseases having similar pathogenesis. The present application further relates to an oligosaccharide having specific polysaccharide structural features and anti-inflammatory effectiveness.

Description

Oral polysaccharide for treating inflammatory bowel disease and preparation method thereof

technical field

The invention relates to a preparation method of refined heparin polysaccharide and its use for preventing and/or treating inflammatory bowel disease.

Background technique

Inflammatory bowel disease (Inflammatory Bowel Disease, IBD) is a group of complex and incurable chronic intestinal inflammatory diseases, including ulcerative colitis (Ulcerative Colitis, UC) and Crohn's disease (Crohn's disease, CD). According to reports, the prevalence of IBD in Western countries is as high as 0.8%. In Asia, the incidence and prevalence of IBD both show a continuous and rapid growth rate, becoming a disease of widespread concern worldwide. Prevalence increases of more than 40% are projected in many countries over the next decade. Therefore, the research and treatment of IBD are becoming more and more important. However, there is a huge bottleneck in the current pathological research and clinical treatment of IBD. Taking UC as an example, in the field of research, the mainstream view believes that the four major factors of "immunity, genetics, environment, and intestinal flora" are important factors affecting the pathogenesis of UC, but the specific pathogenesis is still unclear, and there is no unified conclusion .

Clinically, the mature treatment methods for IBD patients are very limited, mainly divided into drug therapy, surgical treatment and other methods. The main drugs used in drug therapy include: aminosalicylic acid preparations, corticosteroids, immunosuppressants, biological agents, etc. However, most of the above-mentioned drugs are broad-spectrum drugs, and their main defects include: obvious side effects, easy drug tolerance, and low treatment efficiency. Therefore, clinical IBD medications have great limitations, lack of specific therapeutic drugs, and the emergence of new drugs and treatment methods is urgently needed.

Heparin is a kind of glycosaminoglycan with a molecular weight of 3000-30000Da, which has a high degree of heterogeneity and complexity. Currently, the only clinical use is its anticoagulant function. Although current studies have shown that heparin is a multi-target drug that can interact with hundreds of proteins in vivo, such as cell growth factors, chemokines, adhesion molecules, etc. To a certain extent, it affects the physiological functions of the human body, such as regulating the inflammatory response, anti-tumor metastasis, anti-viral infection, and inhibiting the proliferation of smooth muscle cells. These functions are often considered to be independent of anticoagulant activity. However, it has not been applied clinically.

At present, there are also some preclinical studies and clinical trials reporting that heparins have a certain therapeutic potential for IBD, but unfortunately, the therapeutic effects of different types of heparins on IBD vary greatly, and preclinical studies and clinical trials often There are problems such as inconsistent efficacy conclusions, and it is worth noting that heparin drugs in the form of subcutaneous injection are often ineffective in the treatment of IBD. The reason for the above problems is that: on the one hand, the current commercialized heparin drugs are all oriented to anticoagulant-related indications in clinical practice, so only the anticoagulant function was paid attention to during the development process, while other heparin molecules Non-anticoagulant activity is ignored in the production process of drugs dominated by anticoagulant function, and the corresponding effective structure may be destroyed. The clinical dosage of heparin drugs is strictly monitored, and the dosage of drugs that exert their non-anticoagulant activity may induce the risk of massive bleeding; on the other hand, the highly complex molecular structure and unknown structure-activity relationship of heparin drugs, and their therapeutic activity and function The lack of clear polysaccharide fragments and other issues limit its clinical application for the treatment of IBD and other related diseases.

A large number of studies have shown that the non-anticoagulant biological activity of heparin is closely related to its molecular weight. However, the high heterogeneity and structural complexity of heparin greatly limit the exploration of its non-anticoagulant biological activity. In terms of heterogeneity, heparin is a highly heterogeneous linear polysaccharide with a molecular weight ranging from 3,000 to 30,000 Da. The molecular composition is extremely complex, and it is not a pure substance. In terms of structural complexity, heparin is rich in sulfonic acid groups and acetyl group modifications, and its modification process is not controlled by the central dogma and has a high degree of randomness, which also increases the complexity of the fine structure of heparin exponentially. Due to the complex molecular structure of heparin, the analysis of its complete sugar chain molecular structure has not been realized so far. Therefore, the separation and purification of heparin-like polysaccharides is of great significance for clarifying the mechanism of action of the non-anticoagulant biological activity of heparin-like polysaccharides and the drug structure-activity relationship.

For the preparation and separation of heparin-like polysaccharides, most of the current research focuses on the separation of anticoagulant and non-anticoagulant active units of heparin. This separation method cannot distinguish polysaccharides with different molecular weights. fine separation preparation process. Although the existing literature reports the quantitative analysis of the molecular weight of heparin by using gel exclusion chromatography, the above method only stays at the analysis level, and the analysis amount of polysaccharides is small. And other defects, so the controllable separation and mass production of heparin-like polysaccharides with different molecular weights cannot be realized yet. Therefore, in the current research field, there is no process and report for the fine separation and preparation of heparin-like polysaccharides according to their molecular weight.

However, there is a lack of systematic structure-activity relationship research on the anti-IBD activity of heparin drugs, and the functional polysaccharide fragments of its therapeutic IBD activity are still unclear. In the existing studies, the impact of the preparation process of anticoagulant heparin on the anti-IBD activity and Corresponding to problems such as the destruction of fine structures, the above-mentioned problems are to be solved in this field.

Contents of the invention

The purpose of this application is to provide a new oral drug for treating inflammatory bowel disease, which has a significant effect of preventing and/or treating inflammatory bowel disease through the fine separation of heparin polysaccharides. The application also provides a method for the fine separation and preparation of polysaccharide drugs.

As a chronic non-specific intestinal inflammatory disease with extremely complex etiology and pathogenesis, inflammatory bowel disease is prone to recurrent attacks and has a high risk of canceration. It is currently incurable. The clinical treatment methods for this disease are very limited. The clinical treatment is mainly based on broad-spectrum anti-inflammation. Therefore, there is an urgent need to develop new and specific therapeutic drugs for inflammatory bowel disease.

Heparin is a multi-target drug that can interact with hundreds of proteins in vivo, such as cell growth factors, chemokines, adhesion molecules, etc., so it has a variety of non-anticoagulant biological activities besides anticoagulation . There are also some literatures reporting the potential efficacy of heparin in the treatment of IBD, but at present there are inconsistent conclusions on heparin treatment, huge differences in curative effect, effective anti-IBD heparin molecular structure characteristics, drug structure-activity relationship and structure-activity analysis techniques and methods are extremely lacking. Technical bottlenecks greatly limit the discovery of the biological activity of heparin-like polysaccharides in the treatment of IBD. Therefore, establishing an efficient fine structure preparation method of heparin-like polysaccharides, in-depth exploration of its structure-activity relationship, and excavating the most suitable structure of heparin-like oligosaccharides for anti-IBD are the research focus of developing new non-anticoagulant drugs of heparin-like polysaccharides.

Heparin polysaccharides generally have the characteristics of highly complex molecular structure and diverse functions. In the preparation of heparin-like polysaccharides, although there are some simple separation methods (such as: using ultrafiltration membranes to separate according to molecular weight), their separation accuracy and separation volume are limited. Further fine separation and preparation of heparin-like polysaccharides There are still some difficulties. Although the quantitative analysis of the molecular weight of heparin-like polysaccharides can be achieved by means of gel exclusion chromatography, it cannot meet the requirements of fine preparation, that is, the purification of heparin-like polysaccharides and the large-scale preparation of heparin-like polysaccharides (mg~ g level).

The inventors of this application devoted themselves to the innovative research of heparin industry technology, and constructed a set of refining process for heparin-like polysaccharides, which can realize the preparation and separation of heparin-like polysaccharides with a controllable molecular weight, and overcome the heparin-like polysaccharides. The problem of high uniformity provides new technologies, new methods and new products for the effective mining of its anti-inflammatory, anti-tumor, anti-fat accumulation and other non-anticoagulant biological activities and its application in the treatment of inflammatory bowel disease.

Specifically, the present invention relates to the following:

1. A preparation method of refined heparin polysaccharides, comprising:

De-anticoagulant treatment and enzymatic hydrolysis of raw heparin to obtain heparin-like polysaccharide raw materials;

Separating the heparin-like polysaccharide raw material by using a gel size exclusion chromatography column and collecting the separated components;

Freeze-drying the collected separated components;

The freeze-dried products of each component are desalted through alcohol precipitation to obtain refined heparin-like polysaccharides.

2. The preparation method according to item 1, wherein,

The anticoagulant treatment of the raw heparin is to use the periodate oxidation method to perform anticoagulation treatment on the raw heparin to obtain the anticoagulant treatment product, and

The enzymatic hydrolysis of the anticoagulant-treated product is to use heparanase I to enzymolyze the anti-anticoagulant-treated product to obtain the heparin-like polysaccharide raw material.

3. The preparation method according to

item

1 or 2, wherein,

The freeze-drying process is to pre-freeze the collected and separated components at -80°C, and then put them into a freeze dryer for freeze-drying.

4. The production method according to any one of items 1 to 3, wherein,

When using a gel exclusion chromatography column to separate the heparin polysaccharide raw material, the gel exclusion chromatography column is a size gel exclusion chromatography column, preferably HiPrep 16/60 Sephacryl or TSKgel G2000SW chromatography column, using The mobile phase is 0.15-1.0M NaCl aqueous solution, preferably 0.15-0.6M, more preferably 0.2M.

5. The preparation method according to item 4, wherein,

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

6. The preparation method according to item 4, wherein,

The pH of the NaCl aqueous solution is 3-10, preferably 5.

7. The preparation method according to item 4, wherein,

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

8. The preparation method according to item 4, wherein,

The gel size exclusion chromatographic column is a TSKgel G2000SW chromatographic column, and the chromatographic column filler is TSKgel G2000.

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

Add distilled water to the freeze-dried product of each component to resuspend and concentrate;

After adding ethanol aqueous solution, let it stand for alcohol precipitation;

Centrifuge the precipitated product after alcohol precipitation and discard the supernatant; and

Then distilled water is added for resuspension to obtain the refined heparin-like polysaccharide.

10. The preparation method according to item 9, wherein,

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

11. The preparation method according to item 10, wherein,

The volume of the ethanol aqueous solution added is 2 to 6 times, preferably 5 to 6 times, the volume of the liquid after resuspension and concentration; and,

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

12. The preparation method according to item 9, wherein,

The time for standing for alcohol precipitation is 5-60 minutes, preferably 10-30 minutes.

13. The preparation method according to items 1-12, further comprising,

The refined heparin-like polysaccharides obtained after desalting by concentration and alcohol precipitation are then subjected to freeze-drying treatment.

14. An oligosaccharide, characterized in that, the oligosaccharide has a structure as shown below:

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

a is the number of sugar ring opening structures in the oligosaccharide molecule;

B is the quantity of unsaturated uronic acid in described oligosaccharide molecule;

C is the quantity of saturated uronic acid in described 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≥2.5d,

g is the number of 1,6-anhydrous structures in the oligosaccharide molecule,

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

15. The oligosaccharide according to item 14, characterized in that, in the structural formula of the oligosaccharide,

a≤0.3d, b=1, c=d-1, e≤1.0, g=0.

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

17. An oligosaccharide characterized in that,

The sugar chain length of the oligosaccharide is between 2 and 20 sugars, and the basic disaccharide units are [0]-[1,0,1,0,3,0]-[0] and/or [0]-[ 1,0,1,0,2,0]-[0] are composed of repeated permutations and combinations, among which,

[0]-[1,0,1,0,3,0]-[0] means that the number of sugar ring opening structures is 0, the number of unsaturated uronic acid is 1, the number of saturated uronic acid is 0, and the number of glucose The number of amines is 1, the number of acetyl groups is 0, the number of sulfonic acid groups is 3, the number of dehydration structures is 0, and the number of ammonium ions carried in mass spectrometry is 0.

[0]-[1,0,1,0,2,0]-[0] is 0 for sugar ring opening structure, 1 for unsaturated uronic acid, 0 for saturated uronic acid, and 0 for glucose The number of amines is 1, the number of acetyl groups is 0, the number of sulfonic acid groups is 2, the number of dehydration structures is 0, and the number of ammonium ion carried by mass spectrometry is 0.

18. The oligosaccharide according to item 17, characterized in that the average number of sulfonic acid groups contained in the basic disaccharide units is greater than or equal to 2.5.

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

20. A heparin derivative containing the oligosaccharides as described in Items 14 to 19, characterized in that in the heparin derivatives, the content of the oligosaccharides as described in Items 14 to 19 is more than 17%, preferably More than 18%, preferably more than 19%, preferably more than 20%, preferably more than 21%, preferably more than 22%, preferably more than 23%.

21. Use of the oligosaccharides as described in items 14 to 19 and the heparin derivatives as described in item 20 in the preparation of drugs for preventing or treating inflammatory bowel disease, as well as related complications of inflammatory bowel disease and diseases with similar pathogenesis use.

22. The use according to item 21, wherein, inflammatory bowel disease-related complications and diseases with similar pathogenesis include: irritable bowel syndrome, arthritis and other extraintestinal complications including ankylosing spondylitis, gangrenous pus Dermatosis, erythema nodosum, iritis, uveitis, episcleritis, primary sclerosing cholangitis, and rheumatoid arthritis.

23. The use according to any one of items 21 or 22, wherein the oligosaccharide is prepared by the method according to any one of items 1-13.

Invention effect

By adopting the preparation method of refined heparin-like polysaccharides provided by this application, the de-anticoagulant modification of heparin-like polysaccharides and fine preparation and separation of molecular weight controllable are realized, and various refined polysaccharide groups with concentrated molecular weight distribution and no anticoagulant activity can be obtained. In this way, the potential medication risk of heparin-like polysaccharides that can induce massive bleeding is removed, and the characteristic of large structural heterogeneity of heparin-like polysaccharides is significantly optimized. Through in vivo and in vitro drug screening and drug efficacy evaluation verification, the refined polysaccharide component that can significantly relieve the characteristic clinical disease symptoms of UC, the main clinical syndrome of IBD, and has the best curative effect has been obtained, and its therapeutic effect is better than that of the first-line clinical medicine for UC. Further through the analysis of oligosaccharide structure, the oligosaccharide fragments that play the main biological activity of UC treatment were resolved from the refined polysaccharide fraction with the best curative effect, which provides a basis for further research on its application in the treatment of inflammatory bowel disease drugs and the development of anti-inflammatory bowel disease. The new drug of UC polysaccharide provides favorable data support, and also provides new ideas and new methods for studying the structure-activity relationship of non-anticoagulant biological activities of heparin polysaccharides such as anti-inflammatory, anti-tumor, and anti-fat accumulation.

Description of drawings

Figure 1 is the ANOVA analysis chart of the body weight curve of UC mice

Figure 2 is a representative picture of the colon of UC mice and the measurement results of colon length

Figure 3 is a representative picture of the spleen of UC mice and a schematic diagram of the spleen weight index

Figure 4 is the H&E staining evaluation of the histological changes in the colonic epithelium of UC mice

Figure 5 is a schematic diagram of in vitro anti-inflammatory activity index evaluation of heparin derivatives

Figure 6 is the analysis chart of the complete sugar chain of heparin derivatives and each separated component

Figure 7 is a graph showing the relationship between oligosaccharide coverage and oligosaccharide enrichment threshold of heparin derivatives and each separated fraction

Fig. 8 is the enriched oligosaccharide Venn diagram of heparin derivatives and each fraction

Figure 9 is an analysis diagram of the structural characteristics of effective and ineffective anti-UC oligosaccharides of heparin derivatives

Detailed ways

The present application will be further described in detail below in conjunction with specific embodiments, and the given examples are for a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art should understand that they may use different terms to refer to the same component. The specification and claims do not use differences in nouns as a way of distinguishing components, but use differences in functions of components as a criterion for distinguishing. As mentioned throughout the specification and claims, "comprising" or "including" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred implementation mode for implementing the application, but the description is for the purpose of the general principles of the specification, and is not intended to limit the scope of the application. The scope of protection of the present application should be defined by the appended claims.

The present application relates to a method for preparing refined heparin-like polysaccharides, in a specific embodiment, comprising the following steps:

Freeze-drying the collected separated components;

The heparin-like polysaccharide raw material described in this application, that is, deanticoagulant heparin derivatives, is a substance obtained after deanticoagulation treatment of heparin or (ultra) low molecular weight heparin, and has no anticoagulant activity or low anticoagulant activity. Heparin or (ultra) low molecular weight heparin, the anti-Xa factor is less than or equal to 70IU/mg, preferably the anti-Xa factor is less than or equal to 60IU/mg, preferably the anti-Xa factor is less than or equal to 50IU/mg, preferably the anti-Xa factor is less than or equal to 40IU/mg, preferably The anti-Xa factor is less than or equal to 30 IU/mg, preferably the anti-Xa factor is less than or equal to 20 IU/mg, preferably the anti-Xa factor is less than or equal to 10 IU/mg.

In a specific embodiment, the raw material heparin is deanticoagulated first, and then the deanticoagulated product is enzymatically hydrolyzed to obtain the heparin-like polysaccharide raw material.

In a specific embodiment of the present application, the raw material heparin is deanticoagulated by a periodate oxidation method to obtain a deanticoagulated product.

Heparin derivatives that remove anticoagulant activity can be obtained by periodic acid oxidation, and other biological activities can be largely retained, and the degree and form of sulfation remain basically unchanged. Periodic acid can selectively oxidize the adjacent carbon atoms containing unsubstituted hydroxyl or amino groups, so that the unsulfated uronic acid C(2)-C(3) bond is broken, and the antithrombin in the heparin molecule binds to five The glucuronic acid in the sugar is thus destroyed and loses its anticoagulant activity; the polyaldehyde oxidized heparin obtained by oxidation of periodate is stabilized by borohydride reduction (Islam, T., et al., Further evidence that period cleavage of heparin occurs primarily through the antithrombin binding site. Carbohydrate Research, 2002.337(21–23):p.2239-2243.).

In a specific embodiment, raw material heparin (for example, sodium heparin) is dissolved in water, and sodium periodate solution is added for reaction. After reacting for a period of time, ethylene glycol was added to neutralize excess sodium periodate, and then sodium borohydride was added for reaction. After pH adjustment, filtered samples were collected by filtration. Concentration and desalination are then carried out using dialysis bags, etc., to finally obtain deanticoagulated heparin derivatives with no anticoagulant activity.

In a specific embodiment of the present application, heparinase, such as heparanase I, is used to enzymatically hydrolyze the anticoagulant-treated product to obtain the heparin-like polysaccharide raw material.

In the prior art, the E.C. number of heparanase I is E.C.4.2.2.7. Purchased heparanase I can be used, for example, heparanase I purchased from Sigma or IBEX. The heparanase can also be a recombinant heparanase I constructed by molecular biology methods or a fusion protein formed between heparanase I and any fusion partner, as long as it has the activity of heparanase I. Preferably, the heparanase I is a fusion protein of heparanase I, especially a fusion protein of heparanase I comprising MBP.

In a specific embodiment, the manner of reacting heparanase I with the anticoagulant-treated product can be batch, continuous or semi-continuous, and those skilled in the art can appropriately select according to the needs of production. .

In a specific embodiment of the present application, a gel exclusion chromatography column is used to separate the heparin-like polysaccharide raw material. Preferably, a set of AKTA Prime purification system is used together with a gel exclusion chromatographic column for the preparation of liquid phase to form the basic hardware facilities.

Gel size exclusion chromatography, also known as space exclusion chromatography, is a chromatographic technique that separates molecules according to the size of the sample. It is mainly used for the analysis and separation of the relative molecular mass distribution of soluble polymers in organic solvents. The chromatographic column filler of gel exclusion chromatography is gel, which is a surface inert substance containing many holes or three-dimensional network structures of different sizes. The pores of the gel only allow entry of component molecules whose diameter is smaller than the opening of the pores, which are quite large for the mobile phase molecules so that the mobile phase molecules can freely diffuse in and out. According to the different gel fillers used, the gel exclusion chromatography column can separate oil-soluble and water-soluble substances, and the relative molecular mass of the separation ranges from several million to less than 100.

In a specific embodiment of the present application, the selected gel exclusion chromatographic column is HiPrep 16/60 Sephacryl series chromatographic column, which is a high-resolution gel filtration filler and is generally used for fine separation. Specifically, you can choose HiPrep 16/60 Sephacryl S-100 High Resolution, HiPrep 16/60 Sephacryl S-200 High Resolution, HiPrep 16/60 Sephacryl S-300 High Resolution under the HiPrep 16/60 Sephacryl series for this application Separation of polysaccharide raw materials. In a specific embodiment, the preferred chromatographic column filler is HiPrep 16/60 Sephacryl S-100 High Resolution, and the column pressure used does not exceed 0.15Mpa.

In another specific embodiment of the present application, the selected gel exclusion chromatographic column is a TSKgel G2000SW chromatographic column. The filler of TSKgel SW series chromatographic column is made of rigid spherical silica gel, and hydrophilic groups are covalently bonded on its surface, which is specially used for GFC separation of proteins and peptides. The packing material of SW series chromatographic column has the performance necessary for high-performance size exclusion chromatography, that is, low adsorption and good pore size distribution. , methanol or ethanol, etc. In a specific embodiment, the preferred chromatographic column filler is TSKgel G2000, and its column pressure does not exceed 2.00Mpa.

In a specific embodiment of the present application, the mobile phase used when using the gel exclusion chromatography column is 0.15-1.0M NaCl aqueous solution, for example, it can be 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 , 0.8, 0.9, 1.0M NaCl aqueous solution. Preferably, the mobile phase is 0.15-0.6 M NaCl aqueous solution. More preferably, the mobile phase is 0.2M NaCl aqueous solution.

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 packing to ensure the separation effect; on the other hand, the choice of mobile phase needs to take into account the separation of polysaccharides And polysaccharide purification two parts of the demand. The current mobile phase can meet the requirements of the above two aspects.

In a specific embodiment of the present application, the mobile phase NaCl aqueous solution has a pH value of 3-10, preferably a pH value of 5.

The pH value of the mobile phase will affect the separation efficiency. Generally, when the pH value is 5, the separation efficiency of heparin polysaccharides is the best. Some existing literature states that there will be no significant difference in separation efficiency between

pH

5 and 7.

In a specific embodiment of the present application, when the gel exclusion chromatography column is used, the flow rate of the mobile phase is 0.1-1.0 mL/min, for example, it can be 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 a specific embodiment, technicians can set according to the operable range of the chromatographic column.

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

In a specific embodiment, the sample loading volume for one separation is 0.5-5 mL. This parameter can be set according to the specific operable range of the column.

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

In a specific embodiment, the heparin-like polysaccharide raw material is dissolved in the mobile phase and collected from 70 to 100 minutes after sample injection. This parameter can be set according to the size of the injection volume.

In a specific embodiment of the present application, the separated components were collected from the 80th minute after sample injection, and then one component was collected every 10 minutes, and 11 separated components with different molecular weights were collected.

Through the above step of separating and refining the heparin-like polysaccharide raw material, a salt solution of refined polysaccharide 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 products by alcohol precipitation, the above-mentioned refined polysaccharide salt solution can be freeze-dried (one-time freeze-drying) to remove excess water.

In a specific embodiment, the conditions of the freeze-drying treatment are: pre-freeze the salt solution of the refined polysaccharide at -80°C, and then freeze-dry it in a freeze dryer for 2 days until the water is completely removed.

In a specific embodiment, the freeze-dried product is concentrated and alcohol-precipitated to obtain a refined polysaccharide aqueous solution after desalination.

In a specific embodiment, during the concentrated alcohol precipitation process, distilled water can be added to the freeze-dried product to resuspend to enrich the refined polysaccharide and increase the yield of alcohol precipitation. Specifically, distilled water may be added in an amount of 20% to 50% of the volume of the freeze-dried product, preferably 30%. In the above process, the reduction in volume will also further concentrate NaCl and promote the precipitation of polysaccharides.

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

In a specific embodiment, the resting time for alcohol precipitation may be 5-60 minutes. Preferably it is 10 to 30 minutes, more preferably 10 minutes. The main purpose of standing still is to fully flocculate the polysaccharide to ensure the yield.

In a specific embodiment, the precipitated product after alcohol precipitation is centrifuged, all the supernatant is discarded, and distilled water is added again for resuspension, so as to obtain the desalted refined polysaccharide aqueous solution of each component.

In a preferred embodiment, the precipitated product is centrifuged under the conditions of 8000 g, 4° C., and 10 min.

In a specific embodiment, 0.5-10 mL of distilled water is added for resuspension. Preferably, 2 mL of distilled water is added. This parameter is related to the concentration of polysaccharides. The higher the concentration of polysaccharides, the larger the volume of distilled water required, and the lower the concentration of polysaccharides, the smaller the volume of distilled water required.

In a specific embodiment, the above-mentioned desalted refined heparin-like polysaccharide aqueous solution obtained after concentration and alcohol precipitation is subjected to freeze-drying (secondary freeze-drying) to obtain a refined polysaccharide product that is convenient for storage.

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

Using the preparation method of refined heparin-like polysaccharides provided by this application, the preparation and separation of heparin-like polysaccharides with a controllable molecular weight can be achieved, and various refined polysaccharide components with a relatively concentrated molecular weight distribution can be obtained, which significantly optimizes the heterogeneity of polysaccharides It provides favorable conditions for further research on its non-anticoagulant biological activities such as anti-inflammation, anti-tumor, and anti-fat accumulation, as well as its application in the treatment of UC.

After the anti-UC biological activity of each refined polysaccharide component prepared by the above-mentioned method for preparing refined heparin-like polysaccharide was verified, it was found that some isolated components had relatively excellent anti-UC efficacy. After the complete sugar chain analysis and the enriched oligosaccharide Venn diagram analysis of the corresponding purified polysaccharide fractions, it was found that there was a class of oligosaccharides with specific polysaccharide structural characteristics in the fractions that were effective against UC, and it was inferred that they were very It may be an effective anti-UC polysaccharide fragment.

On this basis, the present application provides an oligosaccharide with a specific polysaccharide structure, which is mainly obtained based on refined polysaccharides effective against UC, characterized in that the oligosaccharide has the following structure:

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

a represents the number of sugar ring opening structures in the oligosaccharide molecule;

B represents the quantity of unsaturated uronic acid in described oligosaccharide molecule;

C represents the quantity of saturated uronic acid in described oligosaccharide molecule;

d represents the quantity of glucosamine in the oligosaccharide molecule;

e represents the number of acetyl groups in the oligosaccharide molecule;

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

g represents the number of 1,6-anhydrous structures in the oligosaccharide molecule;

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

Moreover, 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 more than 2.5 times the number of glucosamine, that is, f≥2.5d, This indicates that this oligosaccharide has a highly sulfonated structure.

In a specific embodiment, there is only one unsaturated uronic acid in the structure of the above-mentioned oligosaccharide molecule, that is, b=1. Since the total amount of unsaturated uronic acid and saturated uronic acid is equal to the amount of glucosamine, the amount of saturated uronic acid is the amount of glucosamine-1, ie c=d-1.

In a specific embodiment, the structure of the oligosaccharide molecule basically does not contain or contains a small amount of ring-opening structures, so the number of ring-opening structures is not more than 0.3 times the number of glucosamine, ie a≤0.3d.

In a specific embodiment, the structure of the above-mentioned oligosaccharide molecule does not contain any dehydration structure, that is, g=0.

In a specific embodiment, the number of acetyl groups in the structure of the above-mentioned oligosaccharide molecule is not more than 1, that is, e≤1.0.

In a specific embodiment, the oligosaccharide provided by the application has one of the following structures,

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

The above structural formula is expressed as [a]-[b,c,d,e,f,g]-[h], wherein,

d represents the quantity of glucosamine in the oligosaccharide molecule;

e represents the number of acetyl groups in the oligosaccharide molecule;

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

In a specific embodiment, the oligosaccharides with effective anti-UC properties provided by the application have the following structural characteristics:

[0]-[1,0,1,0,3,0]-[0] means that the sugar ring opening structure is 0, the number of unsaturated uronic acid is 1, the number of saturated uronic acid is 0, and the number of glucosamine A disaccharide structure fragment with a quantity of 1, a quantity of acetyl groups of 0, a quantity of sulfonic acid groups of 3, a quantity of dehydration structure of 0, and a quantity of ammonia in the mass spectrum of 0,

[0]-[1,0,1,0,2,0]-[0] means that the sugar ring opening structure is 0, the number of unsaturated uronic acid is 1, the number of saturated uronic acid is 0, glucosamine A disaccharide structure fragment with a quantity of 1, an acetyl group of 0, a sulfonic acid group of 2, an anhydrous structure of 0, and an ammonia-bearing quantity of 0 in the mass spectrum.

Further, in a specific embodiment, the above-mentioned oligosaccharides have a highly sulfonated property, and the average number of sulfonic acid groups contained in the basic disaccharide units is greater than or equal to 2.5. This means that the basic disaccharide structural unit is mainly composed of the structure shown in [0]-[1,0,1,0,3,0]-[0], while [0]-[1,0,1, The structure shown in 0,2,0]-[0] has less content.

Further, in a specific embodiment, the above-mentioned oligosaccharides basically do not contain or contain a small amount of ring-opening structures, and the average number of basic disaccharide units containing ring-opening structures is less than or equal to 0.3. The present application also provides a heparin derivative, which contains the above-mentioned oligosaccharide with anti-UC effectiveness, and the content of the oligosaccharide is more than 17%, preferably more than 18%, preferably more than 19%, preferably More than 20%, preferably more than 21%, preferably more than 22%, preferably more than 23%.

On the other hand, the present application provides an oligosaccharide with a specific polysaccharide structure, which is mainly obtained based on an effective anti-inflammatory refined polysaccharide, characterized in that the oligosaccharide has the following structure:

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

d represents the quantity of glucosamine in the oligosaccharide molecule;

e represents the number of acetyl groups in the oligosaccharide molecule;

Moreover, the number of glucosamine in the structure of the oligosaccharide is between 1 and 10, that is, ≤d≤10, since the number d of glucosamine is also equal to the number of disaccharide units, it means that the oligosaccharide is composed of d disaccharides The sugar chain length of the whole oligosaccharide is 2d. The number of sulfonic acid groups contained in each disaccharide is less than or equal to 2.7, that is, f≤2.7d.

In a specific embodiment, the number of sugar ring openings contained in each disaccharide in the structure of the oligosaccharide molecule is greater than or equal to 0.25, that is, a≥0.25d.

In a specific embodiment, the number of acetyl groups per disaccharide in the structure of the above-mentioned oligosaccharide molecule is greater than or equal to 0.1, that is, ≥0.1d.

[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],

The above structural formula is expressed as [a]-[b,c,d,e,f,g]-[h], wherein,

d represents the quantity of glucosamine in the oligosaccharide molecule;

e represents the number of acetyl groups in the oligosaccharide molecule;

The present application also provides a heparin derivative, which contains the above-mentioned effective anti-inflammatory oligosaccharide, and the content of the oligosaccharide is more than 52%, preferably more than 65%, preferably more than 70%, preferably more than 75% % or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 93% or more.

In the present application, there are no specific limitations on the methods for structural analysis and content determination of polysaccharide components, and any method known to those skilled in the art can be used.

In a specific embodiment, firstly, the polysaccharide components in the sample to be tested can be extracted by methods known to those skilled in the art. Subsequently, the polysaccharide components were analyzed by the National Institute of Metrology for the whole sugar chain map, and the detection results recorded the oligosaccharide content and oligosaccharide distribution information of the polysaccharide samples were obtained. Based on the detection results, the number of sugar ring opening structures in the oligosaccharide molecule, the number of unsaturated uronic acid in the oligosaccharide molecule, the number of saturated uronic acid in the oligosaccharide molecule, and the number of glucosamine in the oligosaccharide molecule can be confirmed , the number of acetyl groups in oligosaccharide molecules, the number of sulfonic acid groups in oligosaccharide molecules, the number of 1,6-anhydrous structures in oligosaccharide molecules, and the number of ammonium ions carried by oligosaccharide molecules in mass spectrometry detection.

Based on the oligosaccharide content and oligosaccharide distribution information, the structure of the polysaccharide component can also be reconstructed by methods known to those skilled in the art (for specific methods, please refer to the description of the reconstruction process in Experimental Example 3 of the present invention) to confirm The basic disaccharide unit structure of the oligosaccharide, as well as the average number of sulfonic acid groups contained in the basic disaccharide unit and the average number of sugar ring opening structures in the basic disaccharide unit.

In addition, the oligosaccharide content and oligosaccharide distribution information obtained based on the whole sugar chain map analysis method can also calculate the content of oligosaccharides conforming to each structure.

The present application also relates to the use of the oligosaccharides provided in the present application and the deanticoagulated heparin derivatives containing the oligosaccharides to treat inflammatory bowel disease, for example, they can be used to treat ulcerative colitis and Crohn's disease.

The present application also relates to the use of the oligosaccharides provided in the present application and deanticoagulant heparin derivatives containing the oligosaccharides in the preparation of drugs for treating inflammatory bowel disease, as well as inflammatory bowel disease-related complications and diseases with similar pathogenesis Uses, wherein, inflammatory bowel disease-related complications and diseases with similar pathogenesis include but are not limited to irritable bowel syndrome, arthritis and other extraintestinal complications including ankylosing spondylitis, pyoderma gangrenosum, nodular Erythema, iritis, uveitis, episcleritis, primary sclerosing cholangitis, and rheumatoid arthritis.

In a specific embodiment of the present application, the oligosaccharides having anti-UC effectiveness are prepared by the preparation method of the purified heparin-like polysaccharide provided in the present application.

Example

The preparation of embodiment 1 polysaccharide raw material

Dissolve 20g of high-quality heparin (purchased from Changshan Biochemical Pharmaceutical Co., Ltd., product name: heparin sodium) in 0.6L of deionized water, and add an equal volume of 0.2M sodium periodate to 0.6L of high-quality heparin (33g/L) Solution (prepared now), react at 300 rpm, 4°C and protect from light for 22 hours. Add 80 mL of ethylene glycol to neutralize excess sodium periodate, then add 28 g of sodium borohydride and react at 4°C for 16 hours. The pH was adjusted to 7.0 with HCl. Filter through a 0.22 μm filter membrane to collect filtered samples. Then use the dialysis bag to desalinate or use the Millipore ultrafiltration device to add 1K filter membrane for ultrafiltration concentration and desalination until the filtrate has no color change after the 0.1M AgNO ₃ test, then the desalination is considered complete. The samples were frozen at -80°C and placed in a lyophilizer to freeze-dry, and then pulverized into powder with a mortar or a small pulverizer for storage to obtain a deanticoagulant heparin derivative (named NAHP).

Dissolve the anticoagulant heparin derivative (NAHP) obtained above in the reaction buffer, add heparanase I prepared according to the method of ZL200410038098.6 to the solution every 0.5 to 1 h, add 20 IU heparinase I each time, use The optical path difference is the quartz cuvette of 1cm and the light absorption A231 of 231nm of monitoring solution by ultraviolet spectrophotometer (using the buffer solution of pH 7.4 to calibrate and zero the instrument, in order to detect the accuracy of the result, when the ultraviolet spectrophotometer reading A231 When it is greater than 0.6, dilute the solution by a certain number of times so that the reading is between 0.2 and 0.6 for determination). When it is detected that A231 reaches 46, the reaction is terminated, and the enzyme activity of the added total heparanase I reaches about 220-250 IU at this time. The end method is to inactivate the enzyme in the reaction solution in a boiling water bath at 100°C for 5-10 minutes, take out the reaction system and cool it to room temperature, add 6 times the volume of absolute ethanol to the reaction solution, stir at room temperature for 10 minutes, and then Centrifuge at a speed of 4000r/min for 15min, collect the precipitate, add 2 to 3 times the mass of deionized water to the precipitate to make it fully dissolved, then filter the solution with a filter membrane with a pore size of 0.22 μm, collect the permeate and place it Freeze into solid ice cubes in a low-temperature refrigerator at -80°C, then freeze-dry them with a freeze dryer (the temperature of the cold trap is -50°C), and then crush them into powder with a mortar or a small pulverizer to obtain low molecular weight deanticoagulant heparin (named: LNAHP).

Dissolve the anticoagulant heparin derivative (NAHP) obtained above in the reaction solution, add heparinase I prepared according to the method of ZL200410038098.6 to the solution every 0.5 to 1 h, add 20 IU heparinase I each time, and use light A quartz cuvette with a path difference of 1 cm and an ultraviolet spectrophotometer monitor the light absorption A231 of the solution at 231nm (the buffer solution of pH 7.4 is used to calibrate and zero the instrument. For the accuracy of the detection results, when the ultraviolet spectrophotometer reading A231 is greater than 0.6, dilute the solution by a certain number of times, so that the reading is measured at 0.2-0.6). When it is detected that A231 reaches 106, the reaction is terminated, and the enzyme activity of the added total heparanase I reaches about 340-380 IU at this time. The final method is to inactivate the enzyme in the reaction solution in a boiling water bath at 100°C for 5-10 minutes, then take out the reaction system and cool it to room temperature, add 6 times the volume of absolute ethanol to the reaction solution, stir at room temperature for 10 minutes, and then Centrifuge at a speed of 4000r/min for 15min at room temperature, collect the precipitate, add deionized water 2 to 3 times the mass of the precipitate to dissolve, use a 0.22μm membrane to filter, collect the permeate and place it in a low-temperature refrigerator at -80°C to freeze. The solid ice cubes are then sent to a lyophilizer (the temperature of the cold trap is -50°C) to freeze-dry, and then crushed into powder with a mortar or a small pulverizer to obtain ultra-low molecular weight deanticoagulant heparin (also named: ULNAHP) .

The preparation of embodiment 2 refined polysaccharide components

Step (1): Separation of polysaccharides

Experimental equipment: A set of AKTA Prime purification system is used with a gel exclusion chromatography column (HiPrep 16/60 Sephacryl S-100HR) used in the preparative liquid phase.

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

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 minute after injection according to the molecular weight of the sample. According to the principle of gel exclusion chromatography, the polysaccharides with large molecular weight are separated first, and the polysaccharides with small molecular weight are separated later. During the separation period, a fraction was collected every 10 minutes until the 180th minute. According to different collection times, separate components with different molecular weights are obtained, so as to realize the enrichment and purification of polysaccharides with specific molecular weights, and obtain refined polysaccharide salt solutions with specific molecular weight distributions. Among them, the refined polysaccharide fraction collected at 110 minutes after raw material injection was named S4, and the refined polysaccharide fraction collected at 130 minutes was named S6.

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

Pre-freeze the refined polysaccharide salt solution of each component obtained in step (1) at -80°C, and then freeze-dry it in a lyophilizer until the water is completely removed to obtain a primary freeze-dried product of the polysaccharide solution of each component .

Step (3): concentrated alcohol precipitation

The primary freeze-dried products of each component obtained in step (2) were resuspended in distilled water according to 30% of the original volume to enrich the refined polysaccharide and increase the alcohol precipitation yield. Subsequently, the polysaccharide solution was alcohol-precipitated with absolute ethanol, thoroughly mixed and allowed to stand, and then centrifuged. After centrifugation all supernatants (a mixture of ethanol and NaCl containing a small amount of soluble polysaccharides) were carefully discarded. Adding an appropriate amount of distilled water to the precipitate for resuspension can obtain the desalted refined heparin-like polysaccharide aqueous solution of each component.

Step (4): secondary freeze-drying of polysaccharide solution

Pre-freeze the desalted refined polysaccharide aqueous solution of each component obtained in step (3) at -80°C, and then freeze-dry in a freeze dryer until the water is completely removed to obtain a refined polysaccharide freeze-dried product.

After preliminary verification of the anti-UC function and anti-inflammatory effect of each isolated fraction obtained by the above method, it was found that the S6 fraction has a relatively excellent anti-UC therapeutic effect; at the same time, the S4 and S6 fractions also have a relatively excellent anti-inflammatory effect. Effect. In the following experiments of this application, the anti-inflammatory and anti-UC biological activities of S4 and S6 will be further verified and their drug structure-activity relationship will be analyzed. Specifically, the deanticoagulated heparin derivatives (NAHP), low molecular weight deanticoagulated heparin (LNAHP) and ultra-low molecular weight deanticoagulated heparin (ULNAHP) used in the following experimental examples were all obtained by the method of Example 1; The used polysaccharide separation components S4 and S6 were obtained by the method of Example 2; the used heparin (HP) was from the unfractionated heparin purchased by Hebei Changshan Biochemical, and the weight average molecular weight Mw of the heparin was 17223Da; the used 5- Aminosalicylic acid preparations (5-Amino Salicylic Acid, 5-ASA) come from the mesalazine sustained-release granules bought in pharmacies. The indications are: ulcerative colitis, used for the acute attack of ulcerative colitis, to prevent recurrence; Rohn's disease, for patients with frequent onset of Crohn's disease, to prevent acute attacks; the NAEno used is based on enoxaparin (purchased from Changshan Biochemical) as a raw material, and has been modified by the same anticoagulation as NAHP in Example 1 De-anticoagulated enoxaparin derivatives obtained by the process; the NAI45 used is based on heparin (purchased from Changshan Biochemical) as a raw material, and after enzymatic hydrolysis according to the method of Comparative Example 5 in ZL201810100469.0, then using Example 1 of this application A low-molecular-weight de-anticoagulant heparin derivative obtained by the same de-anticoagulation modification process in NAHP, which has been found to be ineffective for the treatment of UC in previous preliminary in vivo functional verification experiments.

Experimental example 1 Determination of molecular weight and distribution of purified heparin polysaccharide components

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 fraction 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 0.5mL/min, the column temperature was 35°C, and the injection volume was 25μL. A WATERS (1525, U.S.) chromatographic system was adopted, and an ultraviolet detector and a differential detector were connected in series at the outlet of the chromatographic column in sequence, and the wavelength of the ultraviolet detector was 234nm. Molecular weight and its distribution determination method can refer to the method recorded 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 measurement results are shown in Table 1.

Table 1 The molecular weight of the refined polysaccharide raw material and the separated components obtained by refining

From the molecular weight information in Table 1, it can be seen that the separated components S4 and S6 obtained after refining have significantly different molecular weight distributions compared with polysaccharide raw materials. From the perspective of specific indicators, it can be reflected in polydispersity, that is, the distribution coefficient P( Polydispersity). Polydispersity is the ratio of weight average molecular weight to number average molecular weight, which is a positive number always 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 example is greater than 1.6. After refined separation and preparation, the polydispersity of the separated components is even less than 1.1, indicating that the molecular weight distribution of the refined polysaccharide is obviously more concentrated and closer to pure substances.

Experimental example 2 Verification of anti-UC and anti-inflammatory biological activity of refined polysaccharide fraction

1. by DSS-induced UC model mice, the anti-UC biological activity of the refined polysaccharide fraction obtained in the above-mentioned embodiment 2 is investigated in vivo. In terms of evaluation indicators, body weight change, colon length, spleen weight index, serum inflammatory factor content, and colon histopathological evaluation were mainly used to systematically evaluate the efficacy of refined polysaccharides in treating UC.

experimental method:

As the animal experimental model, male C57BL/6J mice aged 6-8 weeks were selected. Set up a healthy control group (WT group), a DSS modeling group, and a drug treatment group after modeling.

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

On

Day

3, 30 mg/kg/mouse/day was administered to the UC mouse models of different drug treatment groups by intragastric administration until Day 7.

On Day 7, when the experiment was over, the experimental mice were euthanized and dissected to obtain spleen and colon tissues. The spleen tissue was weighed, and the spleen weight index was calculated based on the spleen weight and body weight to evaluate the in vivo anti-inflammatory and immune-regulating efficacy of the purified polysaccharide fraction. The length of the colon was measured, and then the colon tissues were paraffin-embedded and histopathological sections H&E stained. According to the changes in the colonic epithelial tissue structure of UC mice, the in vivo anti-UC efficacy of the purified polysaccharide fraction was evaluated. The specific experimental results are shown in Figure 1-Figure 4.

Experimental results:

(1) Weight loss is one of the important phenotypes of the DSS-induced UC mouse model, which can characterize the severity of the disease. Figure 1 is an ANOVA analysis chart of the body weight change curve of mice. It can be seen that during the treatment period from the 3rd day to the 7th day, the tendency of the mice in the oral HP, NAI45, NAEno treatment group to lose weight was not significantly improved compared with the DSS group mice. On the 6th day, the body weight loss trend of S6 mice with oral effective therapeutic drugs (LNAHP, ULNAHP) and purified polysaccharide fraction compared with DSS group mice has been significantly alleviated (p<0.05, p<0.01, p<0.05) . On the 7th day, the oral administration of NAHP and clinical first-line drug (5ASA) mice can significantly alleviate the tendency of body weight loss induced by DSS (p<0.05, p<0.01), while oral administration of LNAHP, ULNAHP, S6 mice obtained more Significant remission of weight curve downtrend (p<0.0001, p<0.0001,

p<0.0001). The above results show that the isolated fraction S6 can effectively alleviate the weight loss trend of UC mice, and has a more significant effect on alleviating the symptoms of weight loss in UC mice than the clinical first-line drug 5ASA.

(2) Shortened colon length is one of the characteristic phenotypes of the UC mouse model, and it is also closely related to the severity of the disease. Figure 2 shows the measurement results of the colon length of mice in each experimental group. Compared with the mice in the DSS group, in addition to oral administration of HP, NAEno and NAI45 treatment groups, oral administration of other heparin derivatives and 5ASA can effectively alleviate the shortening of the colon length of UC mice induced by DSS, among which: mice in the S6 treatment group relieved Colon length shortening had the best effect, with the most significant difference compared to DSS group mice (p<0.0001). The above results show that the refined polysaccharide fraction S6 can more effectively alleviate the symptoms of shortened colon length in UC mice, and the curative effect is more significant than that of the clinical first-line drug 5ASA.

(3) As an important immune organ, the spleen will become enlarged and increase in mass when the systemic immune system is activated. Splenomegaly is also one of the clinical symptoms of UC patients, which is closely related to the chronic and persistent activation of the immune system. The activation of the individual immune system can be characterized by the spleen weight index. Figure 3 is a schematic diagram of the spleen weight index. It can be seen that the mice in the DSS model group showed obvious splenomegaly and increased spleen weight index compared with the mice in the WT group. Compared with the DSS group, oral administration of NAHP, LNAHP, S6 and 5ASA can significantly relieve 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 could not effectively alleviate splenomegaly and the increase of spleen weight index. The above results indicated that the refined polysaccharide fraction S6 could significantly alleviate splenomegaly and inhibit the increase of spleen weight index in UC mice, and had significant immunosuppressive ability; its efficacy in inhibiting splenomegaly and increase of spleen weight index was comparable to that of 5ASA.

(4) Histopathological evaluation of colonic mucosa is the gold standard for clinical diagnosis, treatment and drug efficacy evaluation of UC, and is also an important indicator for evaluating the biological activity of deanticoagulated heparin derivatives and refined fractions for UC treatment. Figure 4 is a diagram of H&E staining to evaluate the pathological and morphological changes of the colonic epithelium of UC mice. Among them, mice in the WT group had normal colonic epithelial structure, complete colonic epithelial cells, normal crypt structure, regular crypt arrangement, a large number of goblet cells, and no neutrophil infiltration. The colonic epithelium of the mice in the DSS group showed severe neutrophil and macrophage infiltration. was completely destroyed, and the crypt structure completely disappeared. Compared with the mice in the DSS group, oral administration of HP, NAEno and NAI45 could not effectively alleviate the disease symptoms such as the destruction of the colonic epithelial structure and the shedding of epithelial cells. Oral administration of NAHP, ULNAHP, and 5ASA can partially improve the loss of colonic crypts and epithelial cell detachment and necrosis, but the integrity of the colonic epithelium and colonic crypts is poor, the shape and arrangement of the crypts are abnormal, and the crypts still exist Significant inflammatory cell infiltration and absence of goblet cells. Oral administration of LNAHP and S6 can significantly protect the colonic epithelial tissue of mice, with complete colonic epithelial structure, normal crypt shape, regular crypt arrangement, normal distribution of goblet cells and a small amount of inflammatory cell infiltration. The results of histopathological evaluation showed that the refined polysaccharide fraction S6 could significantly alleviate the destruction of the colonic epithelial tissue structure in UC mice and restore the colonic epithelial tissue structure to normal, and inhibit the inflammatory response of peripheral blood and intestinal lamina propria, which is consistent with the clinical first-line drug Compared with 5ASA, it has superior biological activity in UC treatment.

Through the evaluation of the above comprehensive indicators, the refined polysaccharide fraction S6 has excellent anti-UC biological activity, and its efficacy-related indicators are better than the clinical first-line drug 5ASA.

2. The mouse macrophage cell line RAW 264.7 induced by lipopolysaccharide was used to construct an in vitro inflammation model, and the anti-UC biological activity of the purified polysaccharide fractions S4 and S6 obtained in the above-mentioned Example 2 was investigated in vitro.

experimental method:

Use mouse macrophage RAW 264.7 (purchased from ATCC), inoculate into 48-well plate at a concentration of 150,000 cells/mL, use high-glucose DMEM medium plus 10% fetal bovine serum at 37°C, 5% CO ₂ To cultivate. After overnight culture, discard the old medium and wash the cells once with PBS. A healthy control (WT) group and a lipopolysaccharide (LPS) model group were set up, and the rest were drug treatment groups. Among them, only serum-free medium was added to WT group, and serum-free medium containing 100 ng/mL LPS was added to other groups. The cells of each group were then placed in a cell culture incubator for 15 min. After 15 minutes, heparin, heparin derivatives and separated fractions S4 and S6 were added to the drug treatment group at a concentration of 1 mg/mL, and the drugs were added for 24 hrs. After 24hrs, the supernatant of the culture medium was taken and detected by ELISA to detect the inflammatory factor indicators such as IL-6 and TNF-α secreted by RAW264.7 (the settings and detection results of each group are shown in Figure 5).

Experimental results:

Persistent chronic inflammatory response, as one of the important clinical phenotypes of UC, is the most important target of clinical UC therapeutic drugs. In the above DSS-induced UC mouse model experiment, it was found that S6 has good anti-UC biological activity. However, the mechanism by which effective anti-UC heparin derivatives inhibit inflammation has not been elucidated. In order to confirm whether the effective anti-UC polysaccharide components have significant anti-inflammatory activity, LPS-stimulated RAW 264.7 cells were constructed as an in vitro model to evaluate the in vitro anti-inflammatory effects of heparin derivatives and refined heparin polysaccharides. As a mouse macrophage, RAW 264.7 is an important part of innate immunity. Under the stimulation of exogenous antigens such as LPS, it will secrete a large amount of cytokines such as IL-6 and TNF-α, triggering the continuous activation of the immune system. IL-6 and TNF-α are highly expressed in UC patients, and they are also effective targets for UC therapeutic drugs. Therefore, the overall level of inflammatory response can be evaluated by examining the expression levels of cytokines such as IL-6 and TNF-α.

From the schematic diagram of the in vitro anti-inflammatory activity evaluation of each group in Figure 5, it can be seen that LPS stimulation caused RAW 264.7 to secrete a large amount of IL-6 and TNF-α. In terms of anti-IL-6 activity, anticoagulant heparin HP can effectively alleviate the increase of IL-6 secretion in RAW 264.7 cells (p<0.01), while NAHP, LNAHP, S4, S6, NAEno and NAI45 exhibited more significant anti-IL-6 secretion activity (p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.0001). In terms of anti-TNF-α activity, anticoagulant heparin HP could not effectively alleviate the increase of TNF-α content in RAW 264.7 cells, while LNAHP, S4, S6 and 5ASA showed significant inhibition of TNF-α secretion Activity (p<0.01, p<0.001, p<0.05, p<0.01). The above results indicated that LNAHP, S4 and S6 exhibited the best anti-inflammatory activity in vitro. The above results also confirmed that HP can exhibit more significant anti-inflammatory activity after anticoagulant modification and bioenzyme hydrolysis. This also shows that the anti-inflammatory activity of heparin drugs may be related to the preparation process of deanticoagulated heparin derivatives, and the heparin derivatives obtained by the preparation process of deanticoagulation first and then enzymatic hydrolysis generally exert the best anti-inflammatory effect in vitro.

Experimental Example 3 Anti-UC Efficacy and Anti-inflammatory Activity Verification and Structure-Activity Relationship Analysis of Refined Polysaccharide Fraction

Through the screening of the anti-UC performance and in vitro anti-inflammatory activity of the above experimental example 2, it was found that deanticoagulant heparin derivatives and refined polysaccharide fractions S4, S6, etc., performed relatively well. In order to further analyze the refined polysaccharide fractions in this application In order to explore the anti-inflammatory and anti-inflammatory effects of heparin, the China Institute of Metrology was commissioned to conduct a complete sugar chain map analysis on several different heparin derivatives and refined polysaccharide fractions S4 and S6. Structural fragments for anti-UC properties. Complete sugar chain mapping (Chain Mapping) is an important means to characterize the structure of low molecular weight deanticoagulated heparin, and it is a sugar chain structure analysis method based on liquid chromatography-high resolution mass spectrometry (LC-MS) technology.

The specific method is: select LNAHP, ULNAHP, S4, S6 and anti-UC ineffective heparin derivative NAI45, dissolve the heparin derivative in water, and inject the sample into a liquid mass spectrometry instrument (instrument manufacturer: Thermo Scientific, instrument model: UHPLC -LTQ-Orbitrap, instrument number: SN04010B) to detect and collect mass spectrometry signals. Wherein, the liquid chromatography detection parameter is: chromatographic column,

3μm HILIC

150×2mm; detector, high-resolution mass spectrometry; column temperature, 22°C; flow rate, 0.15ml/min; injection volume, 3μL; run time 100min; mobile phase, acetonitrile-water system. Mass spectrometry detection parameters are: sheath gas flow, 20arb; aux gas flow, 5arb; I spray voltage, 4.2kV; capillary temp, 275°C; S-Lens RF Level, 50%. The obtained mass spectrum raw data was extracted by Xcalibur software to extract the exact mass-to-nucleus ratio of the specific sugar chain structure (accurate to the 4th decimal place, mass tolerance is set to 5ppm) and then integrated to obtain the mass spectrum peak area, thus obtaining each oligosaccharide component structure and abundance information.

After obtaining the structural information and abundance information of the above-mentioned oligosaccharide components, we will further analyze which oligosaccharide components are effective anti-UC oligosaccharides as follows. The specific method is:

Using R studio software (Version 1.2.1335) on the Windows 10 operating system, using R language programming combined with pheatmap and other packages for visualization, the content of different oligosaccharides in different samples was visualized with a heat map, and the above-mentioned parameters were obtained. The results of the whole sugar chain map analysis of the group of substances are shown in Figure 6 (each row in the figure represents a kind of oligosaccharide component, and each column represents the enrichment situation of oligosaccharide corresponding to different samples, and the data are normalized by row, and the enrichment The color of the oligosaccharide-accumulating component is black, the color of the non-enriched oligosaccharide component is white, and the oligosaccharide-free component is white).

1. As can be seen from Figure 6, the oligosaccharide composition of fraction S6 is significantly different from other heparin derivatives. On this basis, in order to further obtain the functional fragments of effective heparin derivatives to exert biological activity, the oligosaccharides in the complete glycan map analysis were sorted from high to low content, and their contents were calculated to obtain the oligosaccharide coverage and Threshold relation diagram of oligosaccharide enrichment (Fig. 7). It can be seen from Figure 7 that the oligosaccharide enrichment threshold is between 0% and 40%, and the oligosaccharide coverage increases rapidly, indicating that the top 40% oligosaccharide components are well represented. Therefore, in the subsequent analysis, the oligosaccharides with the top 40% of the oligosaccharide content were selected as the enriched oligosaccharides of each heparin derivative and isolated fraction, representing the main components of each heparin derivative and isolated fraction.

On this basis, using R studio software (Version 1.2.1335) on the Windows 10 operating system, using R language programming and combining programs such as UpSetR, the enriched oligosaccharides of various heparin derivatives and separated fractions were subjected to Venn From the graph analysis, it can be known from Fig. 8 that there is a common polysaccharide among the heparin derivatives effective against UC. Therefore, a class of polysaccharides enriched in LNAHP and S6 but not enriched in NAI45 is defined as an effective component (Effective component), and a class of polysaccharides enriched in NAI45 but not enriched in LNAHP and S6 is defined as Ineffective component (Ineffective component), the remaining components are defined as other components (Other component).

Using R studio software (Version 1.2.1335) on the Windows 10 operating system, using R language programming and combining ggplot2, stringr, pheatmap, ggsci, UpSetR and other program packages to analyze the corresponding oligosaccharide data in Figure 8, by extracting the corresponding As for the structural feature information of the components, it can be seen from Figure 9 that the effective components have a higher number of sulfonic acid groups and a smaller number of sugar ring openings than the ineffective components and other components. Therefore, it can be speculated that the effective anti-UC oligosaccharides are highly sulfonated and do not contain the characteristics of sugar ring opening structure. According to the classification results in Figure 8, the information on the effective component oligosaccharides obtained from the classification of enriched oligosaccharides was summarized, as shown in Table 2:

Table 2 Summary of oligosaccharide information of anti-UC active components

Note: The expression method of oligosaccharide structure means [polysaccharide ring-opening structure] - [unsaturated uronic acid - saturated uronic acid - glucosamine - acetyl group - sulfonic acid group - dehydration structure] - [amino (i.e. mass spectrum The number of ammonium ions carried in the detection is not owned by the structure of the polysaccharide itself)]

On this basis, the contents of various oligosaccharides in the heparin derivatives and isolated fractions were counted, and the results are shown in Table 3. The effective components listed in Table 3 are highly enriched in the effective anti-UC heparin derivatives LNAHP, ULNAHP, and S6, and their contents are lower in other heparin derivatives, which is also consistent with the in vivo anti-UC efficacy of heparin derivatives.

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

On this basis, the active components in Table 2 were further analyzed, and combined with the biosynthesis process of heparin derivatives, the oligosaccharides were reconstructed in the following manner:

(1) Reconstruct the polysaccharide skeleton first: determine the length of the heparin sugar chain according to the number of unsaturated uronic acid, uronic acid and glucosamine, and construct the basic structure of the effective components in the form of alternating arrangement of uronic acid and glucosamine skeleton.

(2) Second, reconstruct the ring-opening structure and acetyl group: According to the synthesis mechanism of heparin polysaccharide, reconstruct the ring-opening structure onto uronic acid, and reconstruct the acetyl group onto glucosamine. According to the subsequent reconfiguration of sulfonic acid groups, the above restructured structure may need to be finely adjusted according to the number of sulfonic acid groups.

(3) Final reconstruction of sulfonic acid groups: mainly based on the number of sulfonic acid groups, combined with the number of ring-opening structures and acetyl groups, and the comprehensive consideration of probability theory. Prioritize the remodeling of the NS domain disaccharides that are highly contained in heparin for sulfonic acid group remodeling, followed by remodeling of the sulfonic acid group on sugar rings containing ring-opening structures and acetyl groups.

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

Table 4 Structural composition and molecular formula of effective anti-UC oligosaccharides

Note 1: [Open ring]-[△HexA, HexA, HexN, Ac, SO3, dehydration]-[NH3] means [polysaccharide ring-opening structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine- Acetyl group-sulfonic acid group-dehydration structure]-[amino group (the number of ammonium ions carried in the mass spectrometry detection is not owned by the structure of the polysaccharide itself)]

Note 2: In the column of "reconstructed main structure and secondary structure", the main structure is the most likely molecular structure according to the biosynthesis process of heparin and the preparation process characteristics of heparin derivatives; It exists in the biosynthesis process and the preparation process of heparin derivatives, but it is not the molecular structure achieved by the main reaction. It is estimated that its quantity is at least one order of magnitude smaller than the main structure.

Note 3: In the column of "reconstructed primary structure and secondary structure", △UA means unsaturated uronic acid, HexA means uronic acid, GlcA means glucuronic acid, IdoA means iduronic acid, GlcNAc means N -Acetylglucosamine; Ω is the sugar ring opening modification symbol, NS/6S/3S/2S represent the N-sulfonic acid group, 6-O-sulfonic acid group, 3-O on uronic acid and glucosamine respectively -Sulfonic acid group, 2-O-sulfonic acid group modification; "()", "[]" and other symbols indicate that the polysaccharide structures within the same symbol can be replaced in sequence

According to the data in Table 2, Table 3 and Table 4, it can be found that the structural characteristics of effective anti-UC polysaccharides are that they do not contain or contain a small amount of open-ring structure (produced by heparin anticoagulation) and highly sulfated polysaccharide fragments, enriching this Polysaccharide-like fragments and polysaccharides composed of such polysaccharide fragments have therapeutic effects on UC.

So far, we have discovered a class of oligosaccharide components with a specific polysaccharide structure composition, which are mainly based on anti-UC effective heparin derivatives, and are highly enriched in LNAHP and S6. On the basis of this discovery, further inferences can be considered that the ideal oligosaccharide fragments that exert anti-UC biological activity should have the following structural characteristics:

(1) The sugar chain length of polysaccharides is between 2 and 20 sugars, and the basic disaccharide structural units are [0]-[1,0,1,0,3,0]-[0] and [0]-[1 ,0,1,0,2,0]-[0] repeated permutations and combinations, wherein the above representation method means [polysaccharide ring-opening structure]-[unsaturated uronic acid-saturated uronic acid-glucosamine- Acetyl group-sulfonic acid group-dehydration structure]-[amino group (the amount of ammonia in the polysaccharide in the mass spectrum is not owned by the structure of the polysaccharide itself)]

(2) It has the characteristics of highly sulfonated, requiring the average content of sulfonic acid groups per disaccharide to be 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.

(3) On this basis, it basically does not contain or contains a small amount of ring-opening structures, and the average content of ring-opening structures per disaccharide is required to be less than or equal to 0.3.

Based on the above structural characteristics, we can deduce that highly sulfonated polysaccharides of heparin derivatives that contain no or a small amount of ring-opened structures have therapeutic The effect of UC. The above results can be further summarized by using the formula, and the derivation process is as follows:

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

For heparin-based oligosaccharides, there is only one unsaturated uronic acid, ie b=1. And since the amounts of uronic acid and glucosamine are equal, there can be b+c=d. The number d of glucosamine is also equal to the number of disaccharide units, which means that the oligosaccharide is composed of d disaccharide units, and here, 1<=d<=10. The sugar chain length of the entire oligosaccharide is 2d. Since the effective heparin derivative does not contain any dehydrated structure during the preparation process, g=0. h is the amount of ammonia in the polysaccharide in the mass spectrum, which has nothing to do with the structure of the polysaccharide itself, and is not limited here.

According to the structural characteristics of effective anti-UC oligosaccharides, this type of oligosaccharides has a highly sulfonated structure, requiring that the average content of sulfonic acid groups per disaccharide is greater than 2.5, that is, f>=2.5d. At the same time, this type of oligosaccharide basically does not contain or contains a small amount of ring-opening structures, and the average content of ring-opening structures per disaccharide is less than 0.3, that is, a<=0.3d. For the number e of acetyl groups, according to the results in FIG. 9 , combined with the characteristics of heparin biosynthesis, e<=1.0 is defined.

Based on the information above, further organize the variables. The structural formula of effective anti-UC oligosaccharides can be obtained:

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

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

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

C _(12d+2e) H _{(2a-2b+19d+2e+2)} O _{(1-b+10d+e+3f)} N _(d) S _(f)

According to the molecular formula, the molecular weight can be calculated.

2. According to the results of the sugar chain spectrum in Figure 6, in order to further analyze the functional fragments of the above-mentioned refined polysaccharide components that exert anti-inflammatory activity, all the oligosaccharide fragments measured in the analysis of the full sugar chain spectrum were analyzed in each heparin with significant anti-inflammatory activity. The contents in the derivatives (LNAHP, S4, S6 and NAI45) are summed, sorted according to the summed value from high to low, and the top 15% oligosaccharide fragments are taken as representative oligosaccharide fragments, and the data are arranged and summarized as follows Table 5 shows. The content of representative oligosaccharide fragments in each heparin derivative is further summarized, as shown in Table 6.

Table 5 Representative oligosaccharide fragments of anti-inflammatory heparin derivatives

Table 6 Summary of content of effective anti-inflammatory oligosaccharides in each heparin derivative and isolated fraction

Table 7 Molecular formula and molecular weight information of effective anti-inflammatory oligosaccharides

So far, we have discovered a class of oligosaccharide fragments with a well-defined composition, which only account for 15% of all oligosaccharide species, but are highly enriched in anti-inflammatory heparin derivatives, with a content exceeding 45%, especially in The content in S4 exceeds 90%. It can be speculated that the above-mentioned oligosaccharides are functional fragments of heparin derivatives exerting anti-inflammatory biological activity.

In order to further summarize the structural characteristics of the above-mentioned oligosaccharides with anti-inflammatory properties, use the formula to further summarize its structural formula, and the derivation process is as follows:

For any type of oligosaccharide substances: [a]-[b,c,d,e,f,g]-[h], wherein the above structural formula represents: [polysaccharide ring-opening structure]-[unsaturated sugar in oligosaccharides Aldonic acid - saturated uronic acid - glucosamine - acetyl group - sulfonic acid group - dehydration structure] - [amino group (the amount of ammonia in the polysaccharide in the mass spectrum, not the structure of the polysaccharide itself)] and other structures.

According to the structural characteristics of effective anti-inflammatory oligosaccharides, the number of molecular structures of most anti-inflammatory oligosaccharides meets the following conditions: the number of acetyl groups contained in each disaccharide is required to be greater than or equal to 0.1, that is, e>=0.1d; each disaccharide is required to contain The number of sulfonic acid groups is less than or equal to 2.7, that is, f<=2.7d; the number of sugar ring openings contained in each disaccharide is required to be greater than or equal to 0.25, that is, a>=0.25d.

Based on the information above, further organize the variables. The structural formula of effective anti-inflammatory oligosaccharides can be obtained:

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

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

C _(12d+2e) H _{(2a-2b+19d+2e+2)} O _{(1-b+10d+e+3f)} N _(d) S _(f)

According to the molecular formula, the molecular weight can be calculated.

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

Through the above-mentioned induction of the structural formulas of the two oligosaccharides, the similarities and differences in their structures were compared, and the specific results are shown in Table 8.

Table 8 Comparison of structural similarities and differences between effective anti-inflammatory oligosaccharides and effective anti-UC oligosaccharides

It can be seen from Table 8 that the anti-UC oligosaccharides and anti-inflammatory oligosaccharides have only a small amount of overlap in the number of acetyl groups, sulfonic acid groups and sugar ring opening structures, and there are still significant differences in the group composition of the two oligosaccharides overall . They are two types of oligosaccharide components with different compositions.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application in any way. Any person skilled in the art may make use of the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present application without departing from the content of the technical solution of the present application still belong to the protection scope of the technical solution of the present application.

Claims

A method for preparing refined heparin-like polysaccharides, comprising:

De-anticoagulant treatment and enzymatic hydrolysis of raw heparin to obtain heparin-like polysaccharide raw materials;

Separating the heparin-like polysaccharide raw material by using a gel size exclusion chromatography column and collecting the separated components;

Freeze-drying the collected separated components;

The freeze-dried products of each component are desalted through alcohol precipitation to obtain refined heparin-like polysaccharides.
The preparation method according to claim 1, wherein,

The anticoagulant treatment of the raw heparin is to use the periodate oxidation method to perform anticoagulation treatment on the raw heparin to obtain the anticoagulant treatment product, and

The enzymatic hydrolysis of the anticoagulant-treated product is to use heparanase I to enzymolyze the anti-anticoagulant-treated product to obtain the heparin-like polysaccharide raw material.
The preparation method according to claim 1 or 2, wherein,

The freeze-drying process is to pre-freeze the collected and separated components at -80°C, and then put them into a freeze dryer for freeze-drying.
The preparation method according to any one of claims 1 to 3, wherein,

When using a gel exclusion chromatography column to separate the heparin polysaccharide raw material, the gel exclusion chromatography column is a size gel exclusion chromatography column, preferably HiPrep 16/60 Sephacryl or TSKgel G2000SW chromatography column, using The mobile phase is 0.15-1.0M NaCl aqueous solution, preferably 0.15-0.6M, more preferably 0.2M.
The preparation method according to claim 4, wherein,

The flow rate of the mobile phase in the gel size exclusion chromatography column is 0.1-1.0 mL/min, preferably 0.3-0.7 mL/min, more preferably 0.5 mL/min.
The preparation method according to claim 4, wherein,

The pH of the NaCl aqueous solution is 3-10, preferably 5.
The preparation method according to claim 4, wherein,

The gel exclusion chromatographic column is a HiPrep 16/60 Sephacryl chromatographic column, and the chromatographic column filler is Sephacryl S-100 High Resolution, Sephacryl S-200 High Resolution or Sephacryl S-300 High Resolution.
The preparation method according to claim 4, wherein,

The gel size exclusion chromatographic column is a TSKgel G2000SW chromatographic column, and the chromatographic column filler is TSKgel G2000.
The preparation method according to any one of claims 1 to 8, wherein, when the lyophilized products of each component are subjected to desalination treatment by alcohol precipitation,

Add distilled water to the freeze-dried product of each component to resuspend and concentrate;

After adding ethanol aqueous solution, let it stand for alcohol precipitation;

Centrifuge the precipitated product after alcohol precipitation and discard the supernatant; and

Then distilled water is added for resuspension to obtain the refined heparin-like polysaccharide.
The preparation method according to claim 9, wherein,

Add 20-50% of the volume of distilled water to the freeze-dried product of each component for resuspension and concentration, preferably 30%.
The preparation method according to claim 10, wherein,

The volume of the ethanol aqueous solution added is 2 to 6 times, preferably 5 to 6 times, the volume of the liquid after resuspension and concentration; and,

The concentration of the ethanol aqueous solution is 75%-100%.
The preparation method according to claim 9, wherein,

The time for standing for alcohol precipitation is 5-60 minutes, preferably 10-30 minutes.
According to the preparation method described in claims 1-12, it also comprises,

The refined heparin-like polysaccharides obtained after desalting by concentration and alcohol precipitation are then subjected to freeze-drying treatment.
A kind of oligosaccharide, it is characterized in that, described oligosaccharide has the structure as shown below:

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

a is the number of sugar ring opening structures in the oligosaccharide molecule;

B is the quantity of unsaturated uronic acid in described oligosaccharide molecule;

C is the quantity of saturated uronic acid in described 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≥2.5d,

g is the number of 1,6-anhydrous structures in the oligosaccharide molecule,

h is the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection.
The oligosaccharide according to claim 14, characterized in that, in the structural formula of the oligosaccharide,

a≤0.3d, b=1, c=d-1, e≤1.0, g=0.
The oligosaccharide according to claim 14, wherein the oligosaccharide has one of the following structures: [0]-[1,2,3,0,9,0]-[0], [0 ]-[1,2,3,0,8,0]-[0],[0]-[1,3,4,0,12,0]-[0],[2]-[1,3 ,4,1,10,0]-[1],[0]-[1,4,5,0,15,0]-[5],[1]-[1,4,5,0,14 ,0]-[1], [1]-[1,4,5,0,13,0]-[0], [2]-[1,4,5,1,13,0]-[4 ],[2]-[1,4,5,1,11,0]-[0],[0]-[1,5,6,0,18,0]-[6],[2]- [1,5,6,1,16,0]-[5],[2]-[1,5,6,1,15,0]-[3],[2]-[1,5,6 ,1,14,0]-[3] or [1]-[1,5,6,0,15,0]-[3].
An oligosaccharide, characterized in that,

The sugar chain length of the oligosaccharide is between 2 and 20 sugars, and the basic disaccharide units are [0]-[1,0,1,0,3,0]-[0] and/or [0]-[ 1,0,1,0,2,0]-[0] are composed of repeated permutations and combinations, among which,

[0]-[1,0,1,0,3,0]-[0] means that the number of sugar ring opening structures is 0, the number of unsaturated uronic acid is 1, the number of saturated uronic acid is 0, and the number of glucose The number of amines is 1, the number of acetyl groups is 0, the number of sulfonic acid groups is 3, the number of dehydration structures is 0, and the number of ammonium ions carried in mass spectrometry is 0.

[0]-[1,0,1,0,2,0]-[0] is 0 for sugar ring opening structure, 1 for unsaturated uronic acid, 0 for saturated uronic acid, and 0 for glucose The number of amines is 1, the number of acetyl groups is 0, the number of sulfonic acid groups is 2, the number of dehydration structures is 0, and the number of ammonium ions carried by mass spectrometry is 0.
The oligosaccharide according to claim 17, wherein the average number of sulfonic acid groups contained in the basic disaccharide unit is greater than or equal to 2.5.
The oligosaccharide according to any one of claims 17 or 18, characterized in that the average number of sugar ring-opening structures in the basic disaccharide unit is less than or equal to 0.3.
A heparin derivative containing the oligosaccharide according to claims 14-19, characterized in that, in the heparin derivative, the content of the oligosaccharide according to claim 14-19 is more than 17%, preferably More than 18%, preferably more than 19%, preferably more than 20%, preferably more than 21%, preferably more than 22%, preferably more than 23%.
Use of the oligosaccharides as claimed in claims 14 to 19 and the heparin derivatives as claimed in claim 20 in the preparation of medicaments for preventing or treating inflammatory bowel disease, as well as inflammatory bowel disease-related complications and diseases with similar pathogenesis use.
The use according to claim 21, wherein inflammatory bowel disease-related complications and diseases with similar pathogenesis include: irritable bowel syndrome, arthritis and other extraintestinal complications including ankylosing spondylitis, gangrenous pyoderma disease, erythema nodosum, iritis, uveitis, episcleritis, primary sclerosing cholangitis, and rheumatoid arthritis.
The use according to any one of claims 21 or 22, wherein the oligosaccharide is prepared by the method according to any one of claims 1-13.