CN116199802A

CN116199802A - An oral polysaccharide for anti-inflammatory treatment and its preparation method

Info

Publication number: CN116199802A
Application number: CN202111445111.XA
Authority: CN
Inventors: 邢新会; 王怡; 曾文; 张翀
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2023-06-02

Abstract

The application relates to a preparation method of refined heparin polysaccharide, which adopts gel exclusion chromatography to separate heparin polysaccharide raw materials, and carries out freeze-drying, alcohol precipitation and desalination treatment on collected separation components to obtain refined heparin polysaccharide products. The present application also relates to an oligosaccharide having specific polysaccharide structural features and having anti-inflammatory efficacy.

Description

An oral polysaccharide for anti-inflammatory treatment and its preparation method

Technical Field

The invention relates to a preparation method of refined heparin polysaccharide and application thereof in preparing anti-inflammatory drugs.

Background

Heparin is a class of glycosaminoglycans with a molecular weight of 3000-30000 Da, has high heterogeneity and complexity, and is currently used clinically for its anticoagulant function. Heparin, in turn, also exists in large amounts, independent of non-anticoagulant biological activity other than anticoagulant activity, including: anti-inflammatory, anti-tumor, antiviral, anti-cellulite, etc. Among them, the anti-inflammatory activity is one of the most widely studied and in-depth functions among heparin non-anticoagulation biological activities. There are a number of studies currently reporting and finding the anti-inflammatory activity of heparin. However, the anti-inflammatory activity of heparin has not been applied clinically, which is mainly related to the risk that the strong anti-coagulation biological activity of heparin drugs can induce systemic hemorrhage and the lack of clear analysis of the structure-activity relationship of the anti-inflammatory activity, and the problems of inconsistent results and the like of the current preclinical study and clinical study of the anti-inflammatory activity of heparin limit the research and development of the anti-inflammatory activity of heparin.

Numerous studies have shown that the anti-inflammatory and other non-anticoagulant biological activities of heparin are closely related to their molecular weight size, however the high heterogeneity and structural complexity of heparin greatly limits the discovery of their non-anticoagulant biological activities and resolution of the effective molecular structure. In the non-uniformity level, heparin is a linear polysaccharide with high non-uniformity, the molecular weight is different from 3000 Da to 30000Da, and the heparin is not a pure substance and has extremely complex molecular composition. In the aspect of structural complexity, heparin has rich sulfonic acid groups and acetyl groups for modification, the modification process is not controlled by a central rule, and the heparin has high randomness, so that the fine structural complexity of heparin is exponentially increased. Due to the complex molecular structure characteristics of heparin, the analysis of the complete sugar chain molecular structure of heparin has not been realized so far. Therefore, the separation and refining of heparin polysaccharide has important significance for defining the action mechanism and the medicine structure-activity relationship of the heparin polysaccharide non-anticoagulation biological activity.

Aiming at the preparation and separation of heparin polysaccharide, most of researches focus on the separation of anticoagulant and non-anticoagulant activities of heparin, and the separation method can not distinguish polysaccharide with different molecular weights, and no fine separation and preparation process aiming at the molecular weight of heparin exists in the prior art. Although the prior art reports quantitative analysis of heparin molecular weight by using gel exclusion chromatography, the method only stays at an analysis level, has the defects of small polysaccharide analysis amount, unknown process for fine separation and purification of polysaccharide, incapability of directly realizing process amplification and the like, and therefore, can not realize controllable separation and mass preparation of heparin polysaccharide with different molecular weight. Therefore, no technology and report for carrying out fine separation preparation aiming at the molecular weight of heparin polysaccharide exist in the research field at present.

The research on the lack of systematic structure-activity relationship of the anti-inflammatory activity of heparin is not clear, and the problems of the anti-inflammatory activity, the damage of the corresponding fine structure and the like caused by the preparation process of the anti-coagulation heparin are ignored in the existing research, so that the problems are solved in the field.

Disclosure of Invention

The application provides a fine separation and preparation method of heparin polysaccharide, which realizes the controllable separation and mass preparation of heparin polysaccharide with different molecular weights through the fine separation of heparin polysaccharide, so that the heparin polysaccharide has the anti-inflammatory effect, such as the effects of pneumonia, inflammation in the pulmonary and hepatic fibrosis process, arthritis, rheumatoid arthritis, irritable bowel syndrome, gastritis, skin inflammation, inflammatory bowel disease and the like caused by novel coronavirus. The application also provides a medicament with excellent anti-inflammatory performance.

Due to the structural complexity of heparin and the application limitation that the strong anticoagulation activity of heparin is easy to induce systemic hemorrhage, the research on the non-anticoagulation biological activity of heparin in the field at present generally has unstable anti-inflammatory activity of heparin, contradiction of treatment results, extremely lack of anti-inflammatory effective molecular structural characteristics, medicine structure-activity relationship and structure-activity analysis technology and method, and the research and technical bottlenecks greatly limit the research on the anti-inflammatory biological activity of heparin polysaccharide and the development of related medicines. Therefore, the establishment of an efficient preparation method of the fine structure of the heparin polysaccharide, the deep exploration of the structure-activity relationship thereof, the excavation of the most suitable heparin oligosaccharide structure such as anti-inflammatory and the like are important points for the research of developing new anti-inflammatory drugs of the heparin polysaccharide.

Heparin polysaccharide has the characteristics of high complexity of molecular structure, various biological activities and the like. In the preparation of heparin polysaccharide, although some simple separation means (such as ultrafiltration membrane separation according to molecular weight) exist at present, the separation precision and the separation amount are limited, and a certain difficulty still exists in further fine separation preparation of heparin polysaccharide drugs. Although the molecular weight quantitative analysis of heparin polysaccharide can be realized by adopting gel exclusion chromatography and other means at present, the heparin polysaccharide can not meet the requirements of fine preparation, namely: the purification of heparin polysaccharide is not realized, and the mass preparation (mg-g grade) of heparin polysaccharide is not realized.

The inventor of the application is dedicated to innovative research of heparin industry technology, constructs a set of refining process flow aiming at heparin polysaccharide, can realize preparation and separation of heparin polysaccharide with controllable molecular weight, overcomes the problem of high structural non-uniformity of heparin polysaccharide, and provides a new technology, a new method and a new product for effectively excavating non-anticoagulation biological activities such as anti-inflammatory activity, anti-tumor activity, fat accumulation resistance and the like and application in anti-inflammatory drugs.

In particular, the invention relates to the following:

1. a method for preparing refined heparin polysaccharide, comprising:

performing anticoagulation treatment and enzymolysis on raw heparin to obtain heparin polysaccharide raw materials;

separating the heparin polysaccharide raw material by adopting a gel exclusion chromatographic column and collecting components obtained by separation;

lyophilizing the collected separated components;

desalting the freeze-dried product of each component by alcohol precipitation to obtain refined heparin polysaccharide.

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

the anticoagulation treatment of the raw heparin is carried out by utilizing a periodate oxidation method to obtain a product of the anticoagulation treatment, and

and the enzymolysis of the product subjected to anticoagulation treatment is that heparinase I is utilized to carry out enzymolysis on the product subjected to anticoagulation treatment so as to obtain the heparin polysaccharide raw material.

3. The production method according to

item

1 or 2, wherein,

the freeze-drying treatment is to pre-freeze the collected separated components at-80 ℃ and then put the components into a freeze dryer for freeze-drying treatment.

4. The production process according to any one of the above 1 to 3, wherein,

when the heparin polysaccharide raw material is separated by adopting a gel exclusion chromatographic column, the gel exclusion chromatographic column is a size gel exclusion chromatographic column, preferably a HiPrep 16/60Sephacryl or TSKgel G2000SW chromatographic column, and the mobile phase used is 0.15-1.0M NaCl aqueous solution, preferably 0.15-0.6M, more preferably 0.2M.

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

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

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

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

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

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

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

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

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

adding distilled water into the freeze-dried products of the components for resuspension and concentration;

adding an ethanol water solution, and standing for alcohol precipitation;

centrifuging the precipitated product after alcohol precipitation, and discarding the supernatant; and

and adding distilled water to resuspend, thus obtaining the refined heparin polysaccharide.

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

Distilled water accounting for 20 to 50 percent of the volume of the freeze-dried product of each component is added for resuspension concentration, preferably 30 percent.

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

the volume of the ethanol water solution is 2 to 6 times, preferably 5 to 6 times, of the volume of the liquid after the suspension concentration; the method comprises the steps of,

the concentration of the ethanol water solution is 75% -100%.

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

the time of standing and alcohol precipitation is 5-60 min, preferably 10-30 min.

13. The production method according to item 1 to 12, which further comprises,

desalting by concentrating and alcohol precipitation to obtain the refined heparin polysaccharide, and freeze-drying.

14. An oligosaccharide, wherein the oligosaccharide has the structure shown below:

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

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

b is the amount of unsaturated uronic acid in the oligosaccharide molecule;

c is the amount of saturated uronic acid in the oligosaccharide molecule;

d is the amount of glucosamine in the oligosaccharide molecule, and d is more than or equal to 1 and less than or equal to 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 is less than or equal to 2.7d,

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

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

15. The oligosaccharide of claim 14, wherein in the structural formula of the oligosaccharide, a is greater than or equal to 0.25d, b=1, c=d-1, e is greater than or equal to 0.1d, g=0.

16. The oligosaccharide of claim 14, wherein the oligosaccharide has one of the following structures: [4] - [1,9,10,1,26,0] - [12],[4] - [1,9,10,1,25,0] - [10],[3] - [1,8,9,1,23,0] - [9],[3] - [1,8,9,1,24,0] - [10],[3] - [1,7,8,1,19,0] - [7],[1] - [1,7,8,3,19,0] - [8],[3] - [1,7,8,1,20,0] - [8],[2] - [1,6,7,1,18,0] - [7],[3] - [1,8,9,1,22,0] - [8],[3] - [1,8,9,2,13,0] - [4],[1] - [1,5,6,0,17,0] - [5],[1] - [1,6,7,0,19,0] - [7],[2] - [1,7,8,1,20,0] - [8],[2] - [1,6,7,1,17,0] - [7],[1] - [1,6,7,0,20,0] - [8],[0] - [1,0,1,0,3,0] - [0],[3] - [1,4,5,1,8,0] - [7].

17. A heparin derivative containing the oligosaccharide according to any one of claims 14 to 16, wherein the oligosaccharide according to any one of claims 14 to 16 is contained in an amount of 52% or more.

18. The use of the oligosaccharide according to any one of items 14 to 16 and the heparin derivative according to item 17 for the preparation of an anti-inflammatory drug, preferably wherein the inflammation is pneumonia caused by a novel coronavirus, inflammation in the course of pulmonary and hepatic fibrosis, arthritis, rheumatoid arthritis, irritable bowel syndrome, gastritis, skin inflammation, inflammatory bowel disease, and the like.

19. The use according to claim 18, wherein the oligosaccharide is prepared by the method according to any one of claims 1 to 13.

Effects of the invention

By adopting the preparation method of the refined heparin polysaccharide, the anticoagulation modification and the molecular weight controllable fine preparation and separation of the heparin polysaccharide are realized, and each refined polysaccharide component without anticoagulation activity with relatively concentrated molecular weight distribution can be obtained, so that the potential medication risk that the heparin polysaccharide can induce massive hemorrhage is removed, and the characteristic of high structural non-uniformity of the heparin polysaccharide is obviously optimized. Through in-vitro and in-vivo drug screening and drug effect evaluation verification, the complete removal of heparin anticoagulation activity and the systematic investigation of in-vitro anti-inflammatory activity of heparin polysaccharide refined components are realized. The anti-anticoagulation heparin derivatives LNAHP, S4, S6 and NAI45 with in-vitro anti-inflammatory effect are successfully screened and obtained by adopting an in-vitro anti-inflammatory model of RAW 264.7 induced by LPS. The composition and content of representative oligosaccharides in the effective anti-inflammatory heparin derivatives are determined by total sugar chain profile analysis. The representative oligosaccharide is a functional fragment of heparin derivatives which can play anti-inflammatory activity, and provides a new thought and a new method for researching the structure-activity relationship of non-anticoagulation biological activities such as anti-inflammatory, anti-tumor, anti-fat accumulation and the like of heparin polysaccharide.

Drawings

FIG. 1 is an ANOVA analysis of UC mice body weight curve

FIG. 2 is a graph showing representative pictures of the colon and colon length measurements of UC mice

FIG. 3 is a diagram showing spleen representative pictures and spleen weight index of UC mice

FIG. 4 is a graph showing the histological changes of the colon epithelium of UC mice evaluated by H & E staining

FIG. 5 is a schematic diagram showing in vitro anti-inflammatory Activity index evaluation of heparin derivatives

FIG. 6 is a diagram showing analysis of complete sugar chains of heparin derivatives and isolated fractions

FIG. 7 is a graph showing the relationship between coverage of heparin derivative and oligosaccharide concentration threshold of each fraction isolated

FIG. 8 is a diagram of an enriched oligosaccharide wien for heparin derivatives and fractions isolated

FIG. 9 is a structural characterization of heparin derivatives effective and ineffective anti-UC oligosaccharides

Detailed Description

The following examples are set forth in order to provide a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth the preferred embodiment for carrying out the present application, but is not intended to limit the scope of the present application in general, as the description proceeds. The scope of the present application is defined by the appended claims.

The application relates to a preparation method of refined heparin polysaccharide, which comprises the following steps in a specific embodiment:

lyophilizing the collected separated components;

The heparin polysaccharide raw material, namely the anticoagulation heparin derivative, is a substance obtained by performing anticoagulation treatment on heparin or (ultra) low molecular weight heparin, is heparin or (ultra) low molecular weight heparin which does not have anticoagulation activity or has low anticoagulation activity, and has Xa factor resistance of less than or equal to 70IU/mg, preferably Xa factor resistance of less than or equal to 60IU/mg, preferably Xa factor resistance of less than or equal to 50IU/mg, preferably Xa factor resistance of less than or equal to 40IU/mg, preferably Xa factor resistance of less than or equal to 30IU/mg, preferably Xa factor resistance of less than or equal to 20IU/mg, and preferably Xa factor resistance of less than or equal to 10IU/mg.

In a specific embodiment, the heparin raw material is subjected to anticoagulation treatment, and then the product of the anticoagulation treatment is subjected to enzymolysis to obtain the heparin polysaccharide raw material.

In one embodiment of the present application, the raw heparin is subjected to anticoagulation treatment by a periodate oxidation method to obtain a anticoagulation treatment product.

Heparin derivatives with removed anticoagulant activity can be obtained by periodate oxidation, and other biological activities can be largely preserved, and the sulfation degree and the form are basically unchanged. Periodic acid can selectively oxidize ortho-carbon atoms containing unsubstituted hydroxyl or amino, so that the bond between non-sulfated uronic acid C (2) -C (3) is broken, and antithrombin in heparin molecules is combined with glucuronic acid in pentasaccharide to be destroyed, and anticoagulant activity is lost; the polyaldehyde oxidized heparin obtained by oxidation of periodate is stabilized by reduction of borohydride (Islam, T., et al, further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site. Carbohydrate Research,2002.337 (21-23): p.2239-2243.).

In a specific embodiment, the starting heparin (e.g., heparin sodium) is dissolved in water and reacted with a sodium periodate solution. After a period of reaction, ethylene glycol is added to neutralize the excess sodium periodate, and sodium borohydride is added to react. After adjusting the pH, the filtered sample was collected by filtration. And concentrating and desalting by using a dialysis bag and the like to finally obtain the anticoagulated heparin derivative with the anticoagulation activity removed.

In a specific embodiment of the present application, the product of the anticoagulation treatment is subjected to enzymatic hydrolysis with a heparinase, e.g. heparinase I, to obtain the heparin-like polysaccharide material.

In the prior art, heparanase I has an e.c. number of e.c.4.2.2.7. A commercially available heparanase I may be used, for example, one available from Sigma or IBEX. The heparanase may also be recombinant heparanase I constructed by molecular biology methods or a fusion protein of heparanase I with any fusion partner, provided that it has heparanase I activity. Preferably, heparinase I is a heparinase I fusion protein, in particular comprising MBP.

In a specific embodiment, the manner in which heparinase I is reacted with the desublimated product may be batchwise, continuous or semi-continuous, and may be suitably selected by one of ordinary skill in the art depending on the needs of the production.

In a specific embodiment of the present application, the heparin-type polysaccharide starting material is separated using a gel exclusion chromatography column. Preferably, a set of AKTA Prime purification system is adopted to be matched with a gel exclusion chromatographic column for preparing liquid phase.

Gel exclusion chromatography, also known as space exclusion chromatography, is a chromatographic technique for separation according to the size of sample molecules, and is mainly used for analysis and separation of the mass distribution of high polymers soluble in organic solvents relative to the molecules. The column packing of gel exclusion chromatography is a gel, which is a surface inert, substance containing many pores of different sizes or a three-dimensional network structure. The pores of the gel only allow the entry of constituent molecules having diameters smaller than the opening of the pores, which are so large for mobile phase molecules that the mobile phase molecules can diffuse freely in and out. Depending on the gel packing used, gel exclusion chromatography columns can separate oil-soluble and water-soluble materials, with separation relative molecular masses ranging from millions to less than 100.

In one specific embodiment of the present application, the gel exclusion chromatography column selected is a HiPrep16/60Sephacryl series chromatography column, which is a high resolution gel filtration packing, typically used for fine separation. Specifically, the separation of the polysaccharide raw material used in the present application, such as HiPrep16/60Sephacryl S-100High Resolution, hiPrep16/60Sephacryl S-200High Resolution, and HiPrep16/60Sephacryl S-300High Resolution, under the HiPrep16/60Sephacryl series, can be selected. In a specific embodiment, the chromatographic column packing is preferably HiPrep16/60Sephacryl S-100High Resolution using a column pressure of no more than 0.15MPa.

In another specific embodiment of the present application, the gel exclusion chromatography column selected is a TSKgel G2000SW column. The packing of TSKgel SW series chromatographic column is made up by using rigid spherical silica gel as matrix and covalent bonding hydrophilic group on its surface, specially used for GFC separation of protein and polypeptide. The packing of the SW series chromatographic column has the performance necessary for high performance size exclusion chromatography, namely low adsorptivity and good pore size distribution, and the pH application range is 2.5-7.5, and organic solvents which are completely miscible with water, such as acetonitrile, acetone, methanol or ethanol, etc. can be used. In a specific embodiment, the preferred column packing is TSKgel G2000, which uses column pressures of no more than 2.00MPa.

In a specific embodiment of the present application, the mobile phase used in the gel exclusion chromatography column is an aqueous NaCl solution of 0.15 to 1.0M, for example, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0M. Preferably, the mobile phase is an aqueous NaCl solution of 0.15 to 0.6M. More preferably, the mobile phase is a 0.2M aqueous NaCl 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 requires a compromise between the requirements of polysaccharide separation and polysaccharide purification. The current mobile phase can meet the requirements of the two aspects.

In a specific embodiment of the present application, the pH of the aqueous solution of NaCl in the mobile phase is 3 to 10, preferably pH 5.

The pH of the mobile phase affects the separation efficiency, and heparin polysaccharide separation efficiency is best when ph=5. Some prior art documents state that there is no significant difference in separation efficiency between

pH

5 and 7.

In a specific embodiment of the present application, the flow rate of the mobile phase when the gel exclusion chromatography column is used is 0.1 to 1.0mL/min, for example, may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mL/min. Preferably, the flow rate of the mobile phase is 0.3 to 0.7mL/min. More preferably, the flow rate of the mobile phase is 0.5mL/min. In a specific embodiment, the skilled artisan can set the settings based on the operable range of the chromatographic column.

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

In a specific embodiment, the sample loading for a single separation is between 0.5 and 5mL. This parameter may be set according to the specific operable range of the chromatographic column.

In a specific embodiment of the present application, the heparin-like polysaccharide material is dissolved in the mobile phase, and the collection time of the components is set to be different according to the molecular weight of the sample. In a specific embodiment, one component may be collected every 3 to 30 minutes. According to the principle of gel exclusion chromatography, the components with different molecular weights are separated out successively. The polysaccharide is separated into high molecular weight polysaccharide and low molecular weight polysaccharide. The skilled artisan can set any collection interval that will allow for efficient separation of the sample, thereby achieving enrichment and purification of the polysaccharide of a particular molecular weight, resulting in a salt solution of the purified polysaccharide having a particular molecular weight distribution.

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

In a specific embodiment of the present application, the separated components are collected from 80min after sample introduction, followed by collecting one component every 10min, and collecting 11 separated components having different molecular weights.

The salt solution of the refined polysaccharide with specific molecular weight distribution is obtained through the above steps of separating and refining the heparin polysaccharide raw material. In order to increase the concentration of polysaccharide and salt in the solution, so as to facilitate the alcohol precipitation to obtain more polysaccharide products, the refined polysaccharide salt solution may be subjected to freeze-drying treatment (primary freeze-drying) to remove excessive water.

In a specific embodiment, the lyophilization conditions are: pre-freezing the salt solution of refined polysaccharide at-80deg.C, and lyophilizing in a lyophilizer for 2 days until water is completely removed.

In a specific embodiment, the freeze-dried product is subjected to concentrated alcohol precipitation to obtain a desalted purified polysaccharide aqueous solution.

In a specific embodiment, during the concentration and alcohol precipitation process, distilled water can be added to the freeze-dried product to concentrate and refine polysaccharide, so as to increase the yield of alcohol precipitation. Specifically, distilled water may be added in an amount of 20 to 50% by volume of the lyophilized product, preferably 30%. In the above process, the reduction of the volume also causes the NaCl to be further concentrated, thereby promoting the precipitation of polysaccharide.

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

In a specific embodiment, the alcohol settling time may be from 5 to 60 minutes. Preferably 10 to 30 minutes, more preferably 10 minutes. The main purpose of the standing is to flocculate the polysaccharide sufficiently to ensure the yield.

In a specific embodiment, the precipitated product after the alcohol precipitation is subjected to centrifugation to discard all supernatants, and distilled water is added again to resuspension, so as to obtain a purified polysaccharide aqueous solution of each component after desalination.

In a preferred embodiment, the precipitated product is subjected to centrifugation at 8000g at 4℃for 10min.

In a specific embodiment, 0.5 to 10mL of distilled water is added for resuspension. Preferably, 2mL of distilled water is added. This parameter is related to the polysaccharide concentration, the higher the polysaccharide concentration, the greater the distilled water volume required, and the lower the polysaccharide concentration, the less distilled water volume required.

In a specific embodiment, the desalted purified heparin polysaccharide aqueous solution obtained after concentration and alcohol precipitation is subjected to freeze-drying treatment again (secondary freeze-drying), so that a purified polysaccharide product which is convenient to store can be obtained.

In a specific embodiment, the treatment conditions for the secondary lyophilization are the same as for the primary lyophilization.

By adopting the preparation method of the refined heparin polysaccharide, the preparation and separation of the heparin polysaccharide with controllable molecular weight are realized, each refined polysaccharide component with concentrated molecular weight distribution can be obtained, the characteristic of high polysaccharide non-uniformity is obviously optimized, and the preparation method provides favorable conditions for further researching the effective excavation of non-anticoagulation biological activities such as anti-inflammatory, anti-tumor, anti-fat accumulation and the like and the application for treating UC.

After functional verification of anti-UC biological activity of each refined polysaccharide component prepared by the preparation method of the refined heparin polysaccharide, certain separation components are found to have excellent anti-UC performance. After complete sugar chain analysis and enriched oligosaccharide Venn diagram analysis of the corresponding refined polysaccharide separation components, it was found that an oligosaccharide with specific polysaccharide structural characteristics exists in the anti-UC effective separation components, and it was deduced that the oligosaccharide is likely to be a polysaccharide fragment effective in anti-UC.

On the basis of this, the present application provides an oligosaccharide having a specific polysaccharide structure, which is mainly based on an anti-UC effective purified polysaccharide, characterized in that said oligosaccharide has the structure shown below:

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

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

b represents the amount of unsaturated uronic acid in the oligosaccharide molecule;

c represents the amount of saturated uronic acid in the oligosaccharide molecule;

d represents the amount 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-anhydro structures in the oligosaccharide molecule;

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

in addition, the number of glucosamine in the structure of the oligosaccharide is 1-10, namely, 1-10, and the number of sulfonic acid groups is more than 2.5 times of the number of glucosamine, namely, f is more than or equal to 2.5d, which indicates that the oligosaccharide has a high sulfonated structure.

In a specific embodiment, there are only 1 unsaturated uronic acid in the structure of the oligosaccharide molecule described above, i.e. 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 glucosamine-1, i.e. c=d-1.

In a specific embodiment, the oligosaccharide molecule has a structure that contains substantially no or a small amount of ring-opened structures, and thus the number of ring-opened structures is not more than 0.3 times the number of glucosamine, i.e., a.ltoreq.0.3 d.

In a specific embodiment, the oligosaccharide molecule does not have any dehydrated structure in its structure, i.e. g=0.

In a specific embodiment, the number of acetyl groups in the structure of the oligosaccharide molecule is not more than 1, i.e. e.ltoreq.1.0.

In a specific embodiment, the oligosaccharides provided herein have one of the structures shown below,

[0] - [1,2,3,0,9,0] - [0] - [1,2,3,0,8,0] - [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] - [1,4,5,0,13,0] - [0] - [2] - [1,4,5,1,13,0] - [4] - [2] - [1,4,5,1,11,0] - [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 structural formula is represented by [ a ] - [ b, c, d, e, f, g ] - [ h ], wherein,

d represents the amount 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 potent anti-UC properties provided herein have the following structural features:

the oligosaccharide has a sugar chain length of 2-20 sugar, and the basic disaccharide unit is formed by repeatedly arranging and combining [0] - [1,0,1,0,3,0] - [0] and/or [0] - [1,0,1,0,2,0] - [0],

[0] - [1,0,1,0,3,0] - [0] represents a disaccharide structure fragment having a sugar ring-opening structure of 0, an unsaturated uronic acid amount of 1, a saturated uronic acid amount of 0, a glucosamine amount of 1, an acetyl group amount of 0, a sulfonic acid group amount of 3, a dehydrated structure amount of 0, and an ammonia amount of 0 in mass spectrum,

[0] - [1,0,1,0,2,0] - [0] represents a disaccharide structure fragment having a sugar ring-opening structure of 0, an unsaturated uronic acid number of 1, a saturated uronic acid number of 0, a glucosamine number of 1, an acetyl group number of 0, a sulfonic acid group number of 2, a dehydrated structure number of 0, and an ammonia number of 0 in mass spectrum.

Further, in a specific embodiment, the above-mentioned oligosaccharide has a highly sulfonated property, and the average number of sulfonic acid groups contained in the basic disaccharide unit is 2.5 or more. This means that the basic disaccharide structural units are mainly composed of the structures shown in [0] to [1,0,1,0,3,0] to [0] with the structures shown in [0] to [1,0,1,0,2,0] to [0] being present in a smaller amount.

Further, in a specific embodiment, the oligosaccharide contains substantially no or a small amount of ring-opened structures, and the average number of ring-opened structures contained in the basic disaccharide unit is 0.3 or less.

The present application also provides a heparin derivative comprising the oligosaccharide having anti-UC effectiveness as described above, and the content of the oligosaccharide is 17% or more, preferably 18% or more, preferably 19% or more, preferably 20% or more, preferably 21% or more, preferably 22% or more, preferably 23% or more.

In another aspect, the present application provides an oligosaccharide having a specific polysaccharide structure, which oligosaccharide is mainly based on an anti-inflammatory effective purified polysaccharide, characterized in that the oligosaccharide has the structure shown below:

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

d represents the amount of glucosamine in the oligosaccharide molecule;

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

in addition, the number of glucosamine in the structure of the oligosaccharide is 1-10, namely 1-10, and the number d of glucosamine is equal to the number of disaccharide units, so that the oligosaccharide is composed of d disaccharide units, and 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, namely f is less than or equal to 2.7d.

In a specific embodiment, the oligosaccharide molecule has a structure in which the number of ring-opening sugar rings per disaccharide is 0.25 or more, that is, a.gtoreq.0.25 d.

In a specific embodiment, the oligosaccharide molecule has a structure in which the number of acetyl groups per disaccharide is 0.1 or more, i.e., 0.1d or more.

[4] 1,9,10,1,26,0, 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, 5,1,6,7,0,19,0, 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,

d represents the amount 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-described anti-inflammatory effective oligosaccharide, and the content of the oligosaccharide is 52% or more, preferably 65% or more, preferably 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 93% or more.

In the present application, the method for structural analysis and content measurement of the polysaccharide component is not particularly limited, and may be performed using any method known to those skilled in the art.

In a specific embodiment, the polysaccharide component of the sample to be tested may first be extracted by methods known to those skilled in the art. And then carrying out full sugar chain spectrum analysis on the polysaccharide component by depending on China national institute of metrology, and obtaining a detection result recording oligosaccharide content and oligosaccharide distribution information of the polysaccharide sample. Based on the detection result, the number of sugar ring-opened 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, the number of glucosamine in the oligosaccharide molecule, the number of acetyl groups in the oligosaccharide molecule, the number of sulfonic acid groups in the oligosaccharide molecule, the number of 1, 6-dehydrated structures in the oligosaccharide molecule, and the number of ammonium ions carried by the oligosaccharide molecule in mass spectrometry detection can be confirmed.

The structure of the polysaccharide component can also be reconstructed by methods known to those skilled in the art based on the oligosaccharide content and oligosaccharide distribution information (a specific method can be referred 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 ring-opened structures of sugar rings in the basic disaccharide unit.

In addition, the oligosaccharide content and oligosaccharide distribution information of the sample obtained based on the whole sugar chain map analysis method can also be used for calculating the oligosaccharide content conforming to each structure.

The application also relates to the application of the oligosaccharide and the anticoagulation heparin derivative containing the oligosaccharide in the preparation of anti-inflammatory medicaments, wherein the inflammation is preferably inflammation caused by novel coronavirus in the processes of pneumonia, pulmonary and hepatic fibrosis, arthritis, rheumatoid arthritis, irritable bowel syndrome, gastritis, skin inflammation, inflammatory bowel disease and the like.

In a specific embodiment of the present application, the oligosaccharides with anti-inflammatory activity are prepared by the method for preparing the refined heparin-like polysaccharide provided herein.

EXAMPLE 1 preparation of polysaccharide starting material

20g of refined heparin (product name: heparin sodium, purchased from Changshan Biochemical pharmaceutical Co., ltd.) was dissolved in 0.6L of deionized water, and 0.2M sodium periodate solution (now prepared) was added to 0.6L of refined heparin (33 g/L) in the same volume, and reacted at 300rpm at 4℃for 22 hours in the absence of light. 80mL of ethylene glycol was added to neutralize the excess sodium periodate, and 28g of sodium borohydride was added to react at 4℃for 16 hours. The pH was adjusted to 7.0 with HCl. Suction-filtering with 0.22 μm filter membrane, and collecting the filtered sampleThe product is obtained. Desalting with dialysis bag or ultrafiltration concentrating and desalting with 1K filter membrane by Millipore ultrafiltration device until the filtrate passes through 0.1M AgNO ₃ And (5) checking that no color change exists, and considering that the desalination is completed. The sample is frozen at-80 ℃ and then placed in a freeze dryer for freeze drying, and then crushed into powder by a mortar or a small crusher for preservation, thus obtaining the anticoagulated heparin derivative (named NAHP).

The anticoagulated heparin derivative (NAHP) obtained above was dissolved in a reaction buffer, heparinase I prepared according to ZL200410038098.6 was added to the solution every 0.5 to 1h, 20IU of heparinase I was added each time, the light absorption A231 of the solution 231nm was monitored using a quartz cuvette and an ultraviolet spectrophotometer with an optical path difference of 1cm (the instrument was calibrated and zeroed using a buffer solution with a pH of 7.4, and for the accuracy of the detection result, when the ultraviolet spectrophotometer reading A231 was more than 0.6, the solution was diluted by a certain factor to give a reading of 0.2 to 0.6. When A231 was detected to reach 46, the reaction was ended, at which time the total heparanase I added had an enzyme activity of about 220-250IU. The ending method comprises inactivating enzyme in the reaction solution in boiling water bath at 100 ℃ for 5-10 min, taking out the reaction system, cooling to room temperature, adding 6 times of absolute ethyl alcohol into the reaction solution, stirring for 10min at room temperature, centrifuging for 15min at 4000r/min at room temperature, collecting precipitate, adding deionized water with 2-3 times of the mass of the precipitate into the precipitate to fully dissolve the precipitate, filtering the solution by using a filter membrane with 0.22 mu m aperture, collecting the permeate, placing the permeate in a low-temperature refrigerator at-80 ℃ to freeze into solid ice cubes, freeze-drying by a freeze dryer (the cold trap temperature is-50 ℃), and crushing into powder by using a mortar or a small crusher to obtain low molecular weight anticoagulant heparin (named as LNAHP).

The anticoagulated heparin derivative (NAHP) obtained above was dissolved in the reaction solution, and heparinase I prepared by the method ZL200410038098.6 was added to the solution every 0.5 to 1h, and 20IU of heparinase I was added each time, and the light absorption A231 of the solution 231nm was monitored by using a quartz cuvette with an optical path difference of 1cm and an ultraviolet spectrophotometer (the apparatus was calibrated and zeroed by using a buffer solution with a pH of 7.4, and when the ultraviolet spectrophotometer reading A231 was more than 0.6, the solution was diluted by a certain factor to give a reading of 0.2 to 0.6 for the accuracy of the detection result. When A231 was detected to reach 106, the reaction was ended, at which time the enzyme activity of total heparanase I added reached about 340-380IU. The method is finished by inactivating enzyme in the reaction solution for 5-10 min in a boiling water bath at 100 ℃, taking out the reaction system, cooling to room temperature, adding 6 times of absolute ethyl alcohol into the reaction solution, stirring for 10min at room temperature, centrifuging for 15min at 4000r/min at room temperature, collecting precipitate, adding deionized water with the mass 2-3 times of that of the precipitate for dissolving, filtering by using a membrane with the mass of 0.22 mu m, collecting permeate, placing the permeate in a refrigerator at-80 ℃ for freezing into solid ice cubes, then sending the ice cubes into a freeze dryer (the cold trap temperature is-50 ℃) for freeze drying, and then crushing the ice cubes into powder by using a mortar or a small crusher to obtain ultra-low molecular weight anticoagulated heparin (also named as ULMAHP).

EXAMPLE 2 preparation of refined polysaccharide component

Step (1): separation of polysaccharides

Experimental facilities: a set of AKTA Prime purification system is adopted together with a gel exclusion chromatographic column (HiPrep 16/60Sephacryl S-100 HR) for preparing liquid phase.

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

The experimental method comprises the following steps: the polysaccharide starting material LNAHP obtained in example 1 was dissolved in the mobile phase at a concentration of 120mg/mL, and the separated fractions were collected from 80min after sample introduction, depending on the molecular weight of the sample. According to the principle of gel exclusion chromatography, the polysaccharide with large molecular weight is separated first, and the polysaccharide with small molecular weight is separated later. The separation period was completed with one fraction collected every 10min to 180 min. According to the difference of the collecting time, each separated component with different molecular weight is obtained, thereby realizing the enrichment and purification of polysaccharide with specific molecular weight and obtaining the refined polysaccharide salt solution with specific molecular weight distribution. Wherein, the refined polysaccharide separation component collected at 110min after the raw material sample injection is named as S4, and the refined polysaccharide separation component collected at 130min is named as S6.

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

Pre-freezing the salt solution of the refined polysaccharide of each component obtained in the step (1) at the temperature of-80 ℃, and then freeze-drying the salt solution in a freeze dryer until the water is completely removed, thus obtaining a primary freeze-dried product of the polysaccharide solution of each component.

Step (3): concentrating and precipitating with ethanol

Adding distilled water into the primary freeze-dried products of the components obtained in the step (2) according to the amount of 30% of the original volume for resuspension to enrich the refined polysaccharide, and increasing the alcohol precipitation yield. And then, carrying out alcohol precipitation on the polysaccharide solution by using absolute ethyl alcohol, fully mixing, standing and centrifuging. After centrifugation, all supernatants (ethanol and NaCl mixture with a small amount of soluble polysaccharide) were carefully discarded. And adding a proper amount of distilled water into the precipitate for resuspension, thus obtaining the refined heparin polysaccharide aqueous solution of each component after desalination.

Step (4): secondary lyophilization of polysaccharide solutions

Pre-freezing the desalted refined polysaccharide aqueous solution of each component obtained in the step (3) at the temperature of-80 ℃, and then freeze-drying the aqueous solution in a freeze dryer until the water is completely removed, thus obtaining the refined polysaccharide freeze-dried product.

After preliminary anti-UC function and anti-inflammatory effect verification is carried out on each separated component obtained by the method, the S6 component is found to have excellent anti-UC treatment effect; meanwhile, the S4 and S6 components also have excellent anti-inflammatory effect. The anti-inflammatory and anti-UC biological activity will be further validated against S4 and S6 and its drug structure-activity relationship will be resolved in the experiments described below in this application. Specifically, the desanti-coagulant heparin derivative (NAHP), low molecular weight desanti-coagulant heparin (LNAHP) and ultra low molecular weight desanti-coagulant heparin (ULNAHP) used in the following experimental examples were all obtained by the method of example 1; the polysaccharide separation components S4 and S6 used were obtained by the method of example 2; heparin (HP) used was from unfractionated heparin, biochemically purchased from Hebei dichroa, having a weight average molecular weight Mw of 17223Da; the 5-aminosalicylic acid preparation (5-Amino Salicylic Acid, 5-ASA) used was derived from mesalamine slow release granules purchased at pharmacies and was indicated for the following indications: ulcerative colitis, for the acute onset of ulcerative colitis, to prevent recurrence; crohn's disease, for patients with frequent incidences of Crohn's disease, preventing acute episodes; the NAEno used was a desoaked enoxaparin derivative obtained from enoxaparin (purchased from Changshan biochemistry) by the same desoaking modification process as that of NAHP in example 1; the NAI45 used was a low molecular weight desoaked heparin derivative obtained by the same desoaking modification process as that of NAHP in example 1 of the present application, after enzymatic hydrolysis according to comparative example 5 in ZL201810100469.0 using heparin (purchased from dichroa biochemical), which was found to be ineffective in the treatment of UC in a previous preliminary in vivo functional verification experiment.

Experimental example 1 determination of molecular weight and distribution of refined heparin polysaccharide component

The polysaccharide starting material LNAHP obtained in example 1 and the purified polysaccharide fraction S6 obtained in example 2 were measured for weight average molecular weight (Mw) and distribution coefficient (P) by gel exclusion high performance liquid chromatography. The column was TSK-GEL G2000SWXL (TOSOH, japan), the flow rate was controlled at 0.5mL/min, the column temperature was 35℃and the sample injection volume was 25. Mu.L. Using a water (1525, usa) chromatography system, an ultraviolet detector and a differential detector were connected in series in tandem at the outlet of the column, the ultraviolet detector wavelength being 234nm. The method for measuring the molecular weight and the distribution thereof can be described in Wu, jingjun et al, "Controllable production of low molecular weight heparins by combinations of heparinase I/II/III." Carbohydrate polymers 101 (2014): 484-492. The specific measurement results are shown in Table 1.

TABLE 1 molecular weight of purified polysaccharide starting material and isolated fraction obtained by purification

From the molecular weight information in table 1, it is clear that the separated components S4, S6 obtained after refining have a significantly different molecular weight distribution compared to the polysaccharide starting material, and from a specific index, can be reflected in polydispersity, i.e., distribution coefficient P (Polydispersity). The polydispersity is a positive number constantly greater than 1, the ratio of weight average molecular weight to number average molecular weight. The closer the value is to 1, the closer the material is to a single material of well-defined molecular weight. The polydispersity of the polysaccharide material employed in this example was greater than 1.6, and even less than 1.1 for the isolated component after preparation by refining separation, indicating that the molecular weight distribution of the refined polysaccharide was significantly more concentrated and closer to pure material.

Experimental example 2 validation of anti-UC and anti-inflammatory biological Activity of isolated fraction of purified polysaccharide

1. The anti-UC biological activity of the isolated fraction of purified polysaccharide obtained in example 2 above was examined in vivo by DSS-induced UC model mice. In the evaluation indexes, mainly adopting indexes such as weight change, colon length, spleen weight index, serum inflammatory factor content, colon pathology histology evaluation and the like to systematically evaluate the efficacy of the refined polysaccharide in treating UC.

The experimental method comprises the following steps:

the animal experimental model selects male C57BL/6J mice of 6-8 weeks. A healthy control group (WT group), a DSS building module, and a post-modeling drug treatment group were set.

Day 0, except for the WT group, the remaining groups replaced drinking water with 3% aqueous dss (dextran sodium sulfate, mw:36,000-50,000Da,MP biomedicals,LLC) to induce the UC mouse model, with daily recordings of mouse body weight.

Day 3, the intragastric treatment of UC mouse models of different drug treatment groups was performed at a drug dose of 30 mg/kg/Day, up to Day 7.

Day 7, end of the experiment, euthanized and dissected to obtain spleen and colon tissue. Spleen tissue is weighed and spleen weight index is calculated according to spleen weight and body weight to evaluate in vivo anti-inflammatory and immunity regulating effects of the refined polysaccharide isolated component. The colon length was measured, and then the colon tissue was paraffin-embedded and pathological tissue sections H & E stained to evaluate the in vivo anti-UC efficacy of the refined polysaccharide isolate according to the changes in the colonic epithelial tissue structure of UC mice. The specific experimental results are shown in fig. 1-4.

Experimental results:

(1) Weight loss is one of the important phenotypes of DSS-induced UC mouse models, an indicator that can characterize the severity of the disease. FIG. 1 is an ANOVA analysis of the body weight change curve of mice. It can be seen that the tendency of the oral HP, NAI45, NAEno treated mice to lose weight was not significantly improved over the DSS group mice during the treatment period from day 3 to day 7. On day 6, the trend of weight loss in orally active therapeutic drug (LNAHP, ULNAHP) and purified polysaccharide fraction S6 mice had been significantly reduced compared to DSS group mice (p <0.05, p <0.01, p < 0.05). On day 7, oral NAHP and clinical first line drug (5 ASA) mice could significantly alleviate the trend of weight loss induced by DSS (p <0.05, p < 0.01), while oral LNAHP, ULNAHP, S mice gave a more pronounced trend of weight loss alleviation curve (p <0.0001 ). The above results show that the isolated fraction S6 can effectively relieve the weight reduction trend of UC mice, and has more remarkable curative effect on relieving the weight reduction symptoms of UC mice compared with the clinical first-line administration of 5 ASA.

(2) The reduced colon length is one of the characteristic phenotypes of the UC mouse model and is also closely related to disease severity. Fig. 2 shows colon length measurements for each experimental group of mice. In comparison to DSS group mice, except for the oral HP, NAEno and NAI45 treatment groups, oral heparin derivatives and 5ASA were effective in alleviating the reduction in colon length in UC mice induced by DSS, wherein: s6 treated mice had the best effect of alleviating the shortening of colon length, with the greatest difference in significance compared to DSS mice (p < 0.0001). The results show that the refined polysaccharide separation component S6 achieves the curative effect of more effectively relieving the symptoms of the reduction of the colon length of the UC mice, and has more remarkable curative effect than the clinical first-line medicine 5 ASA.

(3) Spleen is an important immune organ, and spleen enlargement and mass increase occur when the whole immune system is activated. Splenomegaly is also one of the clinical symptoms of UC patients, which is closely related to chronic sustained activation of the immune system. Activation of the immune system of an individual can be characterized by spleen weight index. FIG. 3 is a graph showing spleen weight index. It can be seen that DSS model mice developed a phenotype of pronounced splenomegaly and exponentially increased splenomegaly compared to WT mice. Compared with DSS groups, oral administration of NAHP, LNAHP, S6 and 5ASA can very significantly alleviate symptoms of splenomegaly in UC mice, inhibit elevation of spleen weight index (p <0.01 ); whereas oral NAEno and NAI45 are not effective in alleviating splenomegaly and the increase in spleen weight index. The above results show that the refined polysaccharide separation component S6 can remarkably relieve splenomegaly of UC mice and inhibit the index rise of spleen weight, and has remarkable immunosuppressive ability; the efficacy of the composition in inhibiting splenomegaly and spleen weight index increase is equivalent to that of 5 ASA.

(4) The pathological histology evaluation of the colonic mucosa is a gold standard for clinical diagnosis, treatment and drug effect evaluation of UC, and is also an important index for evaluating the biological activity of the UC treatment of the anticoagulated heparin derivative and the refined separation component. FIG. 4 is a graph showing the morphological changes of the colon epithelium pathology of UC mice evaluated by H & E staining. Wherein, the WT group mice have normal colon epithelial structure, the colon epithelial cells are complete, the crypt structure is normal, the crypt arrangement is regular, a large amount of cup-shaped cells exist, and neutrophil infiltration does not exist. The colon epithelium of the DSS mice has serious neutrophil infiltration and macrophage infiltration, and compared with the WT mice, the colon epithelium has the advantages of shedding necrosis, massive goblet cell loss, complete destruction of the colon epithelium barrier and complete disappearance of the crypt structure. Compared with DSS mice, oral administration of HP, NAEno and NAI45 was not effective in alleviating disease symptoms such as disruption of colonic epithelial structures and shedding of epithelial cells. While oral NAHP, UNAHP and 5ASA can partially improve the phenomena of colonic crypt depletion and epithelial cell shedding necrosis, the colonic epithelium and colonic crypt have poor integrity, abnormal crypt morphology and arrangement, obvious inflammatory cell infiltration exists between crypts, and goblet cell depletion exists. Oral administration of LNAHP and S6 can significantly protect the colon epithelial tissue of mice, which has the conditions of complete colon epithelial structure, normal crypt shape, regular crypt arrangement, normal distribution of goblet cells and small inflammatory cell infiltration. The pathological histology evaluation result shows that the refined polysaccharide separation component S6 can obviously relieve the damage of the colon epithelial tissue structure of the UC mice, restore the colon epithelial tissue structure to be normal, inhibit inflammatory reaction of peripheral blood and intestinal lamina propria, and has more excellent UC treatment biological activity compared with the clinical first-line drug 5 ASA.

Through the comprehensive index evaluation, the UC-resistant biological activity of the refined polysaccharide separation component S6 is excellent, and the curative effect related indexes are all better than those of the clinical first-line medicine 5ASA.

2. An in vitro inflammation model was constructed using lipopolysaccharide-induced mouse macrophage cell line RAW264.7, and the anti-UC biological activities of the purified polysaccharide fractions S4 and S6 obtained in example 2 above were examined in an in vitro layer.

The experimental method comprises the following steps:

the cells were inoculated into 48-well plates using mouse macrophage RAW264.7 (available from ATCC) at a concentration of 150,000cells/mL, and 10% fetal bovine serum was added at 37℃with 5% CO using high-sugar DMEM medium ₂ Culturing under the condition. After overnight incubation, old media was discarded and cells were washed once with PBS. A healthy control (WT) group and one Lipopolysaccharide (LPS) building module were set up, the remainder being the drug treatment group. Of these, the WT group was fed with serum-free medium alone, and the remaining groups were fed with serum-free medium containing 100ng/mL of LPS. Each group of cells was then placed in a cell incubator for 15min incubation. Heparin, heparin derivatives and fractions S4 and S6 were added to the drug-treated group at a concentration of 1mg/mL after 15min, and the drug was administered for 24hrs. After 24hrs, culture supernatants were assayed for inflammatory factor indicators such as IL-6 and TNF- α secreted by RAW264.7 by ELISA (settings and assay results are shown in FIG. 5).

Experimental results:

persistent chronic inflammatory response is one of important clinical phenotypes of UC, and is the most important acting target of clinical UC therapeutic drugs. In the above-described experiment with DSS-induced UC mice model, S6 was found to possess good anti-UC biological activity. However, the mechanism of action of the effective anti-UC heparin derivatives in inhibiting inflammation has not been elucidated. In order to confirm whether the polysaccharide component of effective anti-UC has significant anti-inflammatory activity, the in vitro anti-inflammatory effects of heparin derivatives and refined heparin polysaccharide were evaluated by constructing LPS-stimulated RAW264.7 cells as an in vitro model. RAW264.7 is an important component of innate immunity as a mouse macrophage, and under the stimulation of exogenous antigens such as LPS, cytokines such as IL-6, TNF-alpha and the like can be secreted in a large quantity, so as to trigger the continuous activation of an immune system. IL-6 and TNF-alpha are highly expressed in UC patients, and are also effective acting targets of UC therapeutic drugs. Therefore, the level of inflammatory response in the whole can be evaluated by examining the expression levels of cytokines such as IL-6 and TNF- α.

From the in vitro anti-inflammatory activity evaluation scheme of each group in FIG. 5, it can be seen that LPS stimulation causes RAW264.7 to secrete IL-6 and TNF-alpha in a large amount. The anticoagulated heparin HP can effectively relieve the increase of the IL-6 secretion amount of RAW264.7 cells (p < 0.01) on the anti-IL-6 activity, and NAHP, LNAHP, S, S6, NAEno and NAI45 obtained by the anticoagulation modification of the HP show more remarkable anti-IL-6 secretion activity (p <0.0001 ). While anticoagulated heparin HP was not effective in alleviating the rise in TNF- α content in RAW264.7 cells at anti-TNF- α activity, LNAHP, S4, S6 and 5ASA showed significant TNF- α secretion inhibiting activity (p <0.01, p <0.001, p <0.05, p < 0.01). The above results indicate that LNAHP, S4 and S6 exhibit optimal anti-inflammatory activity in vitro. The above results also demonstrate that HP can exhibit more significant anti-inflammatory activity after anticoagulation modification and biological enzymolysis. It also shows that the anti-inflammatory activity of heparin medicine may be related to the preparation process of anticoagulated heparin derivative, and the heparin derivative obtained by adopting the preparation process of firstly anticoagulating and then enzymolysis generally exerts the optimal in vitro anti-inflammatory effect.

Experimental example 3 anti-UC efficacy and anti-inflammatory Activity verification and Structure-Activity relationship analysis of purified polysaccharide fraction

Through the screening of the anti-UC performance and the in vitro anti-inflammatory activity of the experimental example 2, the anti-anticoagulation heparin derivatives and the refined polysaccharide separation components S4 and S6 which are excellent in performance are found, and in order to further analyze the anti-inflammatory activity of the refined polysaccharide separation components and the medicine structure-activity relationship for treating UC in the application, the Chinese research institute of metering is entrusted with carrying out complete sugar chain pattern analysis on several different heparin derivatives and the refined polysaccharide separation components S4 and S6 so as to excavate structural fragments which exert the anti-inflammatory and anti-UC performance of heparin. Complete sugar Chain map analysis (Chain Mapping) is an important means for characterizing low molecular weight desanti-coagulation heparin structure, and is a sugar Chain structure analysis method based on liquid chromatography-high resolution mass spectrometry (LC-MS) technology.

The specific method comprises the following steps: LNAHP, ULNAHP, S4, S6 and UC-resistant invalid heparin derivative NAI45 are selected, the heparin derivative is dissolved in water, a sample is injected into a liquid chromatography-mass spectrometer (instrument manufacturer: thermo Scientific, instrument model: UHPLC-LTQ-Orbitrap, instrument number: SN 04010B) for detection, and a mass spectrum signal is collected. Wherein, the liquid chromatography detection parameters are as follows: a chromatographic column is provided with a plurality of chromatographic columns,

3μm HILIC/>

150X 2mm; a detector, high resolution mass spectrometry; column temperature, 22 ℃; a flow rate of 0.15ml/min; sample injection amount, 3 μl; running for 100min; mobile phase, acetonitrile-water system. The mass spectrum detection parameters are as follows: the flow of the shath gas is 20arb; aux gas flow,5arb; i spray voltage,4.2kV; the calilla temp,275 ℃; S-Lens RF Level,50%. The obtained mass spectrum raw data was integrated after extracting the exact mass-to-core ratio (4 th position after the decimal point is accurate, mass tolerance is set to 5 ppm) of the specific sugar chain structure by Xcalibur software to obtain mass spectrum peak areas, thereby obtaining structure information and abundance information of each oligosaccharide component.

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

the total sugar chain spectrum analysis results of the above-mentioned groups of substances are obtained by using R studio software (Version 1.2.1335) on Windows 10 operating system, utilizing R language programming and combining with packages such as phepatmap for visualization, and using heat maps for visualization representation of the content of different oligosaccharides in different samples, as shown in FIG. 6 (each row in the figure represents an oligosaccharide component, each column represents the oligosaccharide enrichment condition corresponding to different samples, the data is normalized, the color of the oligosaccharide enriched component is blackish, the color of the non-enriched oligosaccharide component is whiter, and the non-contained oligosaccharide component is white).

1. As can be seen from fig. 6, the oligosaccharide constituent of the effective separation fraction S6 has a significant difference from other heparin derivatives. On the basis, in order to further obtain a functional fragment of the effective heparin derivative which plays a role in biological activity, the oligosaccharides in the complete sugar chain map analysis are sequenced from high to low according to the content, and the content is calculated to obtain a threshold relation graph of oligosaccharide coverage and oligosaccharide enrichment (figure 7). As can be seen from FIG. 7, the oligosaccharide enrichment threshold reached between 0% and 40%, the oligosaccharide coverage increased rapidly, indicating that the oligosaccharide fraction 40% before the content was better represented. Thus, in subsequent analyses, oligosaccharides with a first 40% oligosaccharide content were selected as the enriched oligosaccharides of the respective heparin derivative and the separate component, representing the main constituent of the respective heparin derivative and separate component.

On this basis, R studio software (version 1.2.1335) was used on the Windows 10 operating system, and the various heparin derivatives and the isolated oligosaccharides were subjected to venn diagram analysis using R programming in the language in combination with program packages such as UpSetR, and as is apparent from fig. 8, among the heparin derivatives effective against UC, there was a common polysaccharide. Thus, a class of polysaccharides enriched in LNAHP and S6 and not in NAI45 is defined as the active component (Effective component), a class of polysaccharides enriched in NAI45 and not in LNAHP and S6 is defined as the inactive component (Ineffective component), and the remaining components are defined as the Other components (Other components).

The corresponding oligosaccharide data in fig. 8 is analyzed by using R studio software (Version 1.2.1335) on the Windows 10 operating system by programming with R language and combining with program packages such as ggplot2, stringr, phepatmap, ggsci, upSetR, etc., and by extracting structural feature information of the corresponding components, as can be seen from fig. 9, the effective components have a higher number of sulfonic acid groups and a smaller number of sugar ring open rings than the ineffective components and other components. From this, it is assumed that the anti-UC effective oligosaccharides have high sulfonation degree and do not contain sugar ring-opening structure. According to the classification result of fig. 8, the oligosaccharide information of the effective components obtained by the oligosaccharide enrichment classification is summarized as shown in table 2:

TABLE 2 summary of oligosaccharide information for anti-UC active Components

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

Based on this, the contents of various oligosaccharides in heparin derivatives and isolated fractions were counted, and the results are shown in Table 3. The active ingredients listed in table 3 are highly enriched in the anti-UC active heparin derivative LNAHP, ULNAHP, S, with lower levels in other heparin derivatives, which is also consistent with the in vivo anti-UC efficacy of the heparin derivatives.

TABLE 3 summary of the content of various oligosaccharides in heparin derivatives and isolated fractions

On this basis, the active ingredients in table 2 were further analyzed and oligosaccharide reconstitution was performed in the following manner in connection with the biosynthesis process of heparin derivatives:

(1) Firstly, reconstructing a polysaccharide skeleton: the length of heparin sugar chain is determined according to the number of unsaturated uronic acid, uronic acid and glucosamine, and the basic skeleton of the effective component is constructed in the form of alternating uronic acid and glucosamine.

(2) And reconstructing a ring-opening structure and an acetyl group: according to the mechanism of heparin polysaccharide synthesis, the ring-opened structure is reconstructed to uronic acid, and acetyl groups are reconstructed to glucosamine. Depending on the subsequent reconstruction of the sulfonic acid groups, the above reconstruction may also require fine adjustments depending on the number of sulfonic acid groups.

(3) Finally, the sulfonic acid group is reconstructed: mainly according to the number of sulfonic acid groups, and simultaneously combining the ring-opening structure with the number of acetyl groups, and comprehensively considering probability theory. The reconstruction of sulfonic acid groups on the NS domain disaccharide highly contained in heparin is preferably performed, and the reconstruction of sulfonic acid groups on the sugar ring containing the ring-opened structure and acetyl groups is also preferably performed.

The molecular structural characteristics of the effective anti-UC oligosaccharide component can be obtained through the reconstruction. The polysaccharide structural drug composition, molecular formula and structural formula of the active ingredient are shown in table 4.

TABLE 4 structural composition and formula of effective anti-UC oligosaccharides

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Note 1: [ Ring opening ] - [ delta HexA, hexA, hexN, ac, SO3, dehydration ] - [ NH3] represents [ polysaccharide ring opening structure ] - [ unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure ] - [ amino group (number of ammonium ion carried in mass spectrometry detection, not all of the structure of polysaccharide itself) ]

And (2) injection: in the column of the 'reconstructed main structure and secondary structure', the main structure is the molecular structure which is most likely to appear according to the biosynthesis process of heparin and the preparation process characteristics of heparin derivatives; the secondary structure is a molecular structure which exists in the biosynthesis process of heparin and the preparation process of heparin derivatives, but is not achieved by the main reaction, and the number of the secondary structure is calculated to be at least one order of magnitude smaller than that of the main structure.

And (3) injection: in the column of "primary and secondary structures of reconstruction", deltaUA represents unsaturated uronic acid, hexA represents uronic acid, glcA represents glucuronic acid, idoA represents iduronic acid, glcNAc represents N-acetylglucosamine; omega is a sugar ring opening modification symbol, and NS/6S/3S/2S respectively represents N-sulfonic acid group, 6-O-sulfonic acid group, 3-O-sulfonic acid group and 2-O-sulfonic acid group modification on uronic acid and glucosamine; symbols "()", "[ ]", etc. indicate that the polysaccharide structures within the same symbol can be replaced with each other in sequence

From the data in tables 2, 3 and 4, it can be seen that the structural features of the anti-UC effective polysaccharide are that the polysaccharide fragments which do not contain or contain a small amount of open-loop structures (resulting from heparin anticoagulation) and are highly sulfated, are enriched for this type of polysaccharide fragments, and that the polysaccharide having the composition of this type of polysaccharide fragments has therapeutic efficacy in treating UC.

To this end, we have found a class of oligosaccharide components with specific polysaccharide structural compositions, which are mainly based on anti-UC effective heparin derivatives, highly enriched in LNAHP and S6. Further conclusions based on this finding may be drawn that the ideal oligosaccharide fragment exhibiting anti-UC biological activity should have the following structural features:

(1) The polysaccharide has a sugar chain length of 2-20 sugar, and the basic disaccharide structural unit is formed by repeatedly arranging and combining [0] - [1,0,1,0,3,0] - [0] and [0] - [1,0,1,0,2,0] - [0], wherein the expression method means [ polysaccharide open-loop structure ] - [ unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure ] - [ amino group (the quantity of the polysaccharide with ammonia in mass spectrum is not the whole structure of the polysaccharide) ]

(2) Has high sulfonation property, and requires that the average content of sulfonic acid groups in each disaccharide is 2.5 or more; this means that the basic disaccharide structural units are mainly composed of [0] - [1,0,1,0,3,0] - [0], and the content of [0] - [1,0,1,0,2,0] - [0] is small.

(3) On this basis, the compound contains substantially no or a small amount of ring-opened structure, and the average content of ring-opened structure per disaccharide is required to be 0.3 or less.

According to the structural characteristics, the heparin derivative polysaccharide which is highly sulfonated and does not contain or contains a small amount of ring-opening structure can be deduced to have the effect of treating UC according to the characteristics of the biosynthesis process of heparin, the preparation process flow of the heparin derivative and the analysis result of complete sugar chains. The above results can be further summarized by using the formula, and the deduction process is as follows:

for any class of oligosaccharide material: [a] - [ b, c, d, e, f, g ] - [ h ], wherein the structural formula is represented by: the number of structures in the oligosaccharide [ polysaccharide ring-opened structure ] - [ unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydrated structure ] - [ amino group (the number of amino groups carried by polysaccharide in mass spectrum, not all the structures of polysaccharide itself) ].

For heparin-like oligosaccharides there are and only 1 unsaturated uronic acid, i.e. b=1. And since the numbers of uronic acid and glucosamine are equal, there may be b+c=d. Wherein the glucosamine number d is also equal to the disaccharide unit number, meaning that the oligosaccharide is composed of d disaccharide units, here 1< = d < = 10. The sugar chain length of the whole oligosaccharide is 2d. Since the effective heparin derivative does not contain any dehydrated structure during the preparation, g=0. h is the ammonia carrying amount of the polysaccharide in the mass spectrum, is irrelevant to the structure of the polysaccharide, and is not limited herein.

According to the structural characteristics of the effective anti-UC oligosaccharides, the oligosaccharides have a highly sulfonated structure, requiring an average content of sulfonic acid groups per disaccharide of greater than 2.5, i.e. f > =25d. Meanwhile, the oligosaccharides basically contain no or a small amount of open-loop structures, and the average content of the open-loop structures in each disaccharide is less than 0.3, namely a < = 0.3d. For the number of acetyl groups e, e < = 1.0 was defined according to the results of fig. 9, in combination with heparin biosynthesis characteristics.

And further sorting the variables according to the information. The structural formula of the effective anti-UC oligosaccharide can be obtained:

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

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

The molecular formula of the oligosaccharide substance can be further obtained according to the formula:

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

the molecular weight can be calculated according to the molecular formula.

2. According to the results of the sugar chain spectrum in fig. 6, in order to further analyze the functional fragments of the polysaccharide purified fraction exhibiting anti-inflammatory activity, the contents of all oligosaccharide fragments measured in the total sugar chain spectrum analysis in the respective heparin derivatives (LNAHP, S4, S6 and NAI 45) having remarkable anti-inflammatory activity were added, the order was made from high to low according to the addition and the number, the first 15% of the oligosaccharide fragments were taken as representative oligosaccharide fragments, and the data were collated, and summarized as shown in table 5. 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 the content of anti-inflammatory effective oligosaccharides in each heparin derivative and isolated fraction

TABLE 7 molecular formula and molecular weight information for anti-inflammatory effective oligosaccharides

To this end we have found a well-defined class of oligosaccharide fragments which account for only 15% of the total oligosaccharide species but are highly enriched in anti-inflammatory heparin derivatives in amounts exceeding 45%, in particular exceeding 90% in S4. It is presumed that the oligosaccharide is a functional fragment of the heparin derivative exhibiting anti-inflammatory biological activity.

In order to further generalize the structural characteristics of the oligosaccharide with anti-inflammatory property, the structural formula is further generalized by using the formula, and the deduction process is as follows:

for any class of oligosaccharide material: [a] - [ b, c, d, e, f, g ] - [ h ], wherein the structural formula is represented by: the number of structures in the oligosaccharide [ polysaccharide ring-opening structure ] - [ unsaturated uronic acid-saturated uronic acid-glucosamine-acetyl group-sulfonic acid group-dehydration structure ] - [ amino group (the number of polysaccharide with ammonia in mass spectrum, not all the structures of the polysaccharide itself) ].

According to the structural features of effective anti-inflammatory oligosaccharides, the number of molecular structures of most anti-inflammatory oligosaccharides satisfies the following conditions: the number of acetyl groups contained per disaccharide is required to be 0.1 or more, i.e., > e=0.1 d; the number of sulfonic acid groups contained per disaccharide is required to be 2.7 or less, i.e. f < = 2.7d; the number of ring-opened sugar rings per disaccharide is required to be 0.25 or more, i.e., a > =0.25 d.

And further sorting the variables according to the information. Can obtain the structural formula of effective anti-inflammatory oligosaccharide:

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

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

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

The molecular weight can be calculated according to the molecular formula.

3. Structural dissimilarity comparison of anti-inflammatory and anti-UC effective oligosaccharides

By generalizing the structural formulas of the two oligosaccharides, the structural differences are compared, and specific results are shown in Table 8.

TABLE 8 structural dissimilarity comparison of anti-inflammatory and anti-UC effective oligosaccharides

From table 8, it can be seen that the anti-UC oligosaccharides and the anti-inflammatory oligosaccharides have only a small number of intersections in the number of acetyl groups, sulfonic acid groups and sugar ring opening structures, and that there is still a significant difference in the group composition of the two oligosaccharides as a whole. Is two kinds of oligosaccharide components with different compositions.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application in any way. Any person skilled in the art may make variations or modifications to the equivalent embodiments using the teachings disclosed above. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. A method for preparing refined heparin polysaccharide, comprising:

lyophilizing the collected separated components;

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

3. The preparation method according to claim 1 or 2, wherein,

4. The production process according to any one of claim 1 to 3, wherein,

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

6. An oligosaccharide, wherein the oligosaccharide has the structure shown below:

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

b is the amount of unsaturated uronic acid in the oligosaccharide molecule;

c is the amount of saturated uronic acid in the oligosaccharide molecule;

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

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

7. The oligosaccharide as claimed in claim 6, wherein in the oligosaccharide has the formula,

a≥0.25d，b＝1，c＝d-1，e≥0.1d，g＝0。

8. a heparin derivative containing the oligosaccharide according to claims 6 to 7, wherein the oligosaccharide according to claims 6 to 7 is contained in the heparin derivative in an amount of 52% or more.

9. Use of the oligosaccharide according to claims 6-7 and the heparin derivative according to claim 8 for the preparation of anti-inflammatory medicaments, preferably wherein the inflammation is caused by novel coronaviruses and is caused by pneumonia, pulmonary and hepatic fibrosis, arthritis, rheumatoid arthritis, irritable bowel syndrome, gastritis, skin inflammation, inflammatory bowel disease and the like.

10. The use according to claim 9, wherein the oligosaccharide is prepared by the method of any one of claims 1 to 5.