Use of liquid sulfur dioxide in the preparation of chondroitin sulfate or chondroitin sulfate salts and methods of use

Abstract

The application belongs to the technical field of chondroitin sulfate preparation, and particularly relates to application and application method of liquid sulfur dioxide in preparation of chondroitin sulfate or chondroitin sulfate salt. In the application, liquid sulfur dioxide is used as a solvent to perform sulfonation modification treatment on unsulfated chondroitin, on the one hand, the use of traditional high-boiling organic reagents is avoided, the difficulty of three-waste treatment is reduced, and the process is more green and environmentally friendly; on the other hand, the post-treatment operation of the sulfonation reaction is simpler, and sulfur dioxide can be removed from the system by only temperature control or pressure adjustment, so that solvent residues possibly existing in the final product are avoided, and the product is safer. In addition, by adjusting the key conditions of the sulfonation reaction, the proportion of chondroitin sulfate with 6 independent sulfation in the disaccharide unit of the sulfonation product is 70% to 90%, the reaction is stable and reliable, and the sulfonation rate is high.

Description

Technical Field

[0001] This invention belongs to the field of chondroitin sulfate preparation technology, and specifically relates to the application and method of liquid sulfur dioxide in the preparation of chondroitin sulfate or chondroitin sulfate salt. Background Technology

[0002] Chondroitin sulfate (CS) is a naturally occurring glycosaminoglycan (GAG) that is covalently linked to proteins to form proteoglycans. It is formed by the sulfation modification of repeating disaccharide units (glucuronic acid and N-acetylgalactosamine) and is widely distributed in the extracellular matrix and cell surface of animal tissues.

[0003] Traditionally, chondroitin sulfate is primarily extracted from animal tissues via chemical and enzymatic hydrolysis. Terrestrial animals (such as cartilage from cattle, pigs, and chickens) and marine organisms (such as sharks, cod, squid, and sea cucumbers) are the main raw materials. Patents CN106397629A, CN101701043A, and CN103641935A all employ this method to directly extract chondroitin sulfate from animal tissues. However, animal extraction methods face challenges such as limited raw material supply, risks of pathogen contamination (e.g., mad cow disease), and ethical and religious issues, prompting researchers to explore more controllable alternative sources.

[0004] A non-sulfated chondroitin framework can be prepared by fermentation technology, followed by chemical sulfation and controlled depolymerization processes to obtain low molecular weight chondroitin sulfate with a molecular weight distribution of 1000-5000 Daltons. Compared with natural chondroitin sulfate, this modified product exhibits significantly improved bioavailability and superior safety characteristics while maintaining equivalent anti-inflammatory activity, making it an important alternative to naturally derived chondroitin sulfate.

[0005] Currently, there has been considerable research on the sulfation modification of chondroitin. For example, patent CN103582653A discloses a series of treatments, including adding a protecting group, rearranging, sulfation, and deprotection, to obtain chondroitin sulfate with a specific composition from unsulfated chondroitin polysaccharide. Although this method can obtain chondroitin sulfate with a specific composition, it involves numerous steps, complex operations, strict control of reaction conditions, and requires a large amount of reagents and organic solvents, resulting in high process costs. It is not suitable for large-scale industrial production and is also not environmentally friendly.

[0006] Unlike the sulfation process disclosed in patent CN103635491A, patents CN119823299A and CN112279936A omit the operation of protecting the hydroxyl group. Instead, they directly dissolve the unsulfated chondroitin ammonium salt and sodium salt in solvents such as N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), or formamide, and then use sulfur trioxide complexes for sulfation to prepare chondroitin sulfate. The result is mainly 6-sulfated shark-like chondroitin sulfate. The preparation process of chondroitin sulfate is greatly simplified and the cost is significantly reduced.

[0007] In addition, some documents (such as patent CN103298837A) attempt to refine the sulfation design of the chondroitin skeleton through methods such as protection-deprotection and multi-step organic synthesis to obtain chondroitin sulfate with specific disaccharide combinations. However, these methods involve numerous steps, complex reagents, and large amounts of organic solvents, making it difficult to achieve a comprehensive balance between economy, environmental protection, and consistent quality in industrial-scale production. In summary, existing technologies for the sulfation or sulfonation of fermentation-derived chondroitin to prepare chondroitin sulfate generally suffer from the following problems:

[0008] Firstly, the over-reliance on high-boiling-point nitrogen-containing organic solvents such as DMF, NMP, and formamide results in a heavy burden on solvent recovery and waste treatment, making it difficult for the process to meet current requirements for green manufacturing and environmental friendliness.

[0009] Secondly, high-boiling-point organic solvents are prone to leaving residues in the product, and post-processing operations are complex, requiring purification by means of ion exchange chromatography, gel chromatography, ultrafiltration dialysis, etc., resulting in high costs for product safety and batch-to-batch stability control.

[0010] Therefore, to address the problems faced by existing sulfonation processes for chondroitin, it is necessary to develop a novel sulfonation system that does not use high-boiling-point nitrogen-containing organic solvents such as DMF, NMP, and formamide. This system would enable efficient sulfation of unsulfated chondroitin under mild conditions, avoiding the cumbersome post-processing required in chondroitin sulfate preparation and reducing processing costs. Simultaneously, it would allow the resulting chondroitin sulfate to exhibit reproducible and scalable structural characteristics in terms of molecular weight, charge density, and unsulfated disaccharide content. This would maintain good bioactivity while also ensuring better safety and process scale-up adaptability, providing a new technical solution for large-scale commercial production. Summary of the Invention

[0011] To address the above technical problems, this invention proposes the application and method of liquid sulfur dioxide in the preparation of chondroitin sulfate or chondroitin sulfate salt.

[0012] The technical solution of this invention is:

[0013] The first aspect of the invention is the protection of the use of liquid sulfur dioxide in the preparation of chondroitin sulfate or chondroitin sulfate salt.

[0014] Also protected are the applications of the aforementioned liquid sulfur dioxide as the sole solvent in the preparation of chondroitin sulfate or chondroitin sulfate salt; and the applications of the aforementioned liquid sulfur dioxide as the sole solvent in the sulfonation process in the preparation of chondroitin sulfate or chondroitin sulfate salt.

[0015] In this invention, chondroitin or chondroitin salt is used as raw material, and liquid sulfur dioxide is used as the only organic solvent to disperse the raw material. Then, a sulfonation reaction is carried out under the action of a sulfonating agent. The entire sulfonation reaction process is carried out in the organic phase, and finally chondroitin sulfate or chondroitin sulfate salt is obtained.

[0016] Preferably, the sulfonating agent is selected from one or a combination of several of concentrated sulfuric acid, triethylamine sulfur trioxide, pyridine sulfur trioxide, DMF sulfur trioxide, and chlorosulfonic acid.

[0017] Preferably, the sulfonation reaction temperature is -30℃ to 50℃.

[0018] More preferably, the sulfonation reaction temperature is -20℃ to 40℃.

[0019] Preferably, the mass ratio of chondroitin or chondroitin salt to liquid sulfur dioxide is 1:3 to 60, and the molar ratio of chondroitin disaccharide unit to sulfonating agent in the chondroitin or chondroitin salt is 1:1 to 5.

[0020] This invention improves upon the conventional sulfonation process of chondroitin by using liquid sulfur dioxide instead of high-boiling-point nitrogen-containing organic reagents such as DMF, NMP, and formamide disclosed in existing technologies. After the reaction, the transformation of sulfur dioxide between gas and liquid states can be achieved by controlling the temperature or pressure, thus realizing the removal and recovery of liquid sulfur dioxide. Even if there is a small amount of residual sulfur dioxide, it will be converted into sodium sulfite and removed during the desalination process. The method of this invention greatly reduces environmental pressure, the process is simple, the product has no solvent residue, and it is more environmentally friendly and safe.

[0021] Furthermore, by controlling the ratio of liquid sulfur dioxide, sulfonating reagent, and sulfonation reaction temperature, this invention can achieve sulfonation substitution of hydroxyl groups at different positions. In this process, no specific protection operation is required for the hydroxyl groups, simplifying the complex process of traditional sulfonation reaction while obtaining a high content of 6-substituted chondroitin sulfate sodium salt. The results show that the prepared chondroitin sulfate sodium salt is composed of alternating disaccharide sequences of D-glucuronic acid (G1cA) and N-acetyl-D-galactosamine (GaINAc) with different sulfation residues linked by β (1->3). The sulfation disaccharide units account for 60%~99%, the unsulfation disaccharide units account for 1%~40% of the chondroitin sulfate content, and the 6-position independently sulfonated chondroitin sulfate accounts for 70%~90%.

[0022] A second aspect of the present invention is to provide a method for preparing chondroitin sulfate or chondroitin sulfate salt by sulfonation, comprising the following steps:

[0023] (1) Mix chondroitin or chondroitin salt with liquid sulfur dioxide to form a dispersion system of chondroitin or chondroitin salt in liquid sulfur dioxide, then add sulfonating agent to the dispersion system and carry out sulfonation reaction at a temperature of -30℃~50℃ to obtain the product of sulfonation reaction.

[0024] The mass ratio of chondroitin to liquid sulfur dioxide is 1 g: 3-60; the molar ratio of chondroitin disaccharide units to sulfonating reagent in the chondroitin or chondroitin salt is 1:1-5; the sulfonating reagent is selected from at least one of concentrated sulfuric acid, triethylamine sulfur trioxide, pyridine sulfur trioxide, DMF sulfur trioxide, and chlorosulfonic acid; the sulfonation reaction time is 0.5-36 h, preferably 3-10 h.

[0025] (2) The product of the sulfonation reaction in (1) is purified and dried to obtain chondroitin sulfate or chondroitin sulfate salt.

[0026] Preferably, in the sulfonation method, after the sulfonation reaction in (1) is completed, the system is heated to a temperature greater than the boiling point of sulfur dioxide, or the pressure is reduced to lower the boiling point of sulfur dioxide, so that the liquid sulfur dioxide is converted into gas and removed from the reaction system, thereby realizing the recovery of sulfur dioxide.

[0027] A third aspect of the present invention is the use of chondroitin sulfate or chondroitin sulfate salt prepared by the above method in the preparation of medicaments for the prevention and / or treatment of osteoarthritis or medicaments with lipid-lowering effects.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] This invention uses liquid sulfur dioxide as a solvent to sulfonate unsulfated chondroitin. On the one hand, it avoids the use of traditional high-boiling-point organic reagents, reduces the difficulty of waste treatment, and makes the process more environmentally friendly. On the other hand, the sulfonation reaction process is simple, does not require protection of specific groups, and the post-sulfonation processing is simpler. Sulfur dioxide can be removed from the system simply by adjusting the temperature or pressure, avoiding solvent residues that may exist in the final product, making the product safer.

[0030] Furthermore, this invention can achieve high sulfation of chondroitin simply by controlling the key conditions of the sulfonation reaction. The results show that the proportion of chondroitin sulfate with independent sulfonation at the 6-position in the disaccharide unit of the chondroitin sulfonation product prepared by the process of this invention is 70%~90%, the reaction is stable and reliable, and the sulfonation rate is high. Attached Figure Description

[0031] Figure 1 The graph shows the analysis results of the content of unsulfated chondroitin sodium disaccharide prepared in Example 1;

[0032] Figure 2 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 2;

[0033] Figure 3 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 3;

[0034] Figure 4 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 4;

[0035] Figure 5 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 5;

[0036] Figure 6 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 6;

[0037] Figure 7 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 7;

[0038] Figure 8 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 8;

[0039] Figure 9 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 9.

[0040] Figure 10 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 10;

[0041] Figure 11The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 11;

[0042] Figure 12 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 12;

[0043] Figure 13 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 13;

[0044] Figure 14 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 14;

[0045] Figure 15 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 15;

[0046] Figure 16 The graph shows the analysis results of the content of chondroitin sulfate sodium disaccharide prepared in Example 16. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions to be protected by this invention will be clearly and completely described below in conjunction with specific embodiments. The described embodiments are only some preferred solutions of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0048] Example 1

[0049] In this invention, chondroitin sodium salt is first prepared, and the specific preparation method is as follows:

[0050] Using *Escherichia coli* JCRG01 as the production strain, the culture was carried out in shake flasks and then transferred to a 10L seed tank. The seed tank culture medium included 20 g / L glucose, 10 g / L yeast extract, 5 g / L potassium dihydrogen phosphate, and 2 g / L ammonium sulfate. After 18 hours of culture, the culture was transferred to a 50L fermenter. The fermenter culture medium included 10 g / L glucose, 2 g / L ammonium sulfate, 5 g / L phosphate, 4 g / L magnesium sulfate, 5 g / L potassium sulfate, and 10 g / L sodium chloride. During the culture, 25% ammonia was added to maintain the pH at 7.0. Feed was added using glucose as the carbon source. Fermentation was carried out for 40-50 hours until the OD600 reached 120-150, at which point the fermenter was discharged. The fermentation broth contained 10-14 g / L of chondroitin. The fermentation broth was filtered through a 50 nm pore size ceramic membrane, and the filtrate was collected. The filtrate was then passed through an anion exchange resin and eluted with 1M sodium chloride. The eluent was collected and used for 5... Ultrafiltration was performed using a 5 kDa ultrafiltration membrane. The concentrated solution was collected and hydrolyzed with 0.1 M hydrochloric acid for 20 min. The hydrolysate was then ultrafiltered using a 5 kDa membrane. The concentrated solution was collected, and the pH was adjusted to 7.0 with 2 M sodium hydroxide solution. The solution was spray-dried to obtain chondroitin sodium salt powder. The content of chondroitin sodium salt was determined by HPLC. The results are as follows: Figure 1 As shown.

[0051] Example 2

[0052] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0053] (1) After the high pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 24 g of sulfur trioxide pyridine complex solid is added. The high pressure vessel is sealed and heated to 0°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0054] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried to obtain 24.1g of sodium chondroitin sulfate. The content of sodium chondroitin sulfate was detected by HPLC, and the results are as follows: Figure 2 As shown.

[0055] Example 3

[0056] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0057] (1) After the high pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 24 g of sulfur trioxide pyridine complex solid is added, the high pressure vessel is sealed, and the reaction is carried out at -20°C for 5 h.

[0058] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 20.5g. The content of chondroitin sulfate sodium salt was detected by HPLC. The results are as follows: Figure 3 As shown.

[0059] Example 4

[0060] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0061] (1) After the high pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 16 g of sulfur trioxide pyridine complex solid is added. The high pressure vessel is sealed and heated to 0°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0062] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 22.3g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 4 As shown.

[0063] Example 5

[0064] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0065] (1) After the high pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 16 g of sulfur trioxide pyridine complex solid is added. The high pressure vessel is sealed and heated to 20°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0066] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 22.9g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 5 As shown.

[0067] Example 6

[0068] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0069] (1) After the pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 18 g of sulfur trioxide triethylamine complex solid is added. The pressure vessel is sealed and heated to 0°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0070] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 19.9g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 6 As shown.

[0071] Example 7

[0072] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0073] (1) After the pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 27 g of sulfur trioxide triethylamine complex solid is added. The pressure vessel is sealed and heated to 0°C. The reaction is carried out for 5 hours. After the reaction is completed, the pressure is released by cooling to below -10°C.

[0074] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 23.1g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 7 As shown.

[0075] Example 8

[0076] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0077] (1) After the pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 143 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 27 g of sulfur trioxide triethylamine complex solid is added. The pressure vessel is sealed and heated to 10°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0078] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 23.5g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 8 As shown.

[0079] Example 9

[0080] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0081] (1) After the pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 143 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 27 g of sulfur trioxide triethylamine complex solid is added. The pressure vessel is sealed and heated to 10°C. The reaction is carried out for 24 h. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0082] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 23.8g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 9 As shown.

[0083] Example 10

[0084] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0085] (1) After the pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 86g of liquid sulfur dioxide and 20g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 27g of sulfur trioxide triethylamine complex solid is added. The pressure vessel is sealed and heated to 10°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0086] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 22.6g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 10 As shown.

[0087] Example 11

[0088] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0089] (1) After the reaction vessel is fully purged with nitrogen, the temperature is lowered to about -20°C. 286g of liquid sulfur dioxide and 20g of chondroitin sodium prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 12g of chlorosulfonic acid is slowly added dropwise at -20°C. After the addition is completed, the reaction is maintained at -20°C for 5 hours.

[0090] (2) Slowly raise the temperature to 20~25℃, recover liquid sulfur dioxide, add 200g of water to the autoclave to dissolve it, adjust the pH to neutral with liquid alkali, ultrafilter, and freeze dry to obtain 24.3g of chondroitin sulfate sodium. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows. Figure 11 As shown.

[0091] Example 12

[0092] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0093] (1) After the pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 57g of liquid sulfur dioxide and 20g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 16g of sulfur trioxide pyridine complex solid is added. The pressure vessel is sealed and heated to 0°C. The reaction is carried out for 5 hours. After the reaction is completed, the pressure is released by cooling to below -10°C.

[0094] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 21.7g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 12 As shown.

[0095] Example 13

[0096] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0097] (1) After the high pressure vessel is fully purged with nitrogen, the temperature is lowered to about -30°C. Take 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 and put them into the reaction vessel and stir them thoroughly. Add 16 g of sulfur trioxide pyridine complex solid, seal the high pressure vessel, control the temperature at -30°C, and react for 5 h.

[0098] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 19.1g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 13 As shown.

[0099] Example 14

[0100] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0101] (1) After the pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 16 g of sulfur trioxide pyridine complex solid is added. The pressure vessel is sealed and heated to 50°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0102] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 22.3g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 14 As shown.

[0103] Example 15

[0104] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0105] (1) After the high pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 8 g of sulfur trioxide pyridine complex solid is added. The high pressure vessel is sealed and heated to 0°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0106] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 21.5g. The content of chondroitin sulfate sodium salt was detected by HPLC. The results are as follows: Figure 15 As shown.

[0107] Example 16

[0108] The specific steps for preparing chondroitin sulfate sodium salt by sulfonation are as follows:

[0109] (1) After the high pressure vessel is fully purged with nitrogen, it is cooled to about -20°C. 286 g of liquid sulfur dioxide and 20 g of sodium chondroitin prepared in Example 1 are added to the reaction vessel and stirred thoroughly. 48 g of sulfur trioxide pyridine complex solid is added. The high pressure vessel is sealed and heated to 0°C. The reaction is carried out for 5 hours. After the reaction is completed, the temperature is lowered to below -10°C and the pressure is released.

[0110] (2) Then, the temperature was slowly raised to 20°C to recover sulfur dioxide. 200g of water was added to the autoclave to dissolve the sulfur dioxide. The pH was adjusted to 7.0 with 10% alkali solution. The solution was then desalted by ultrafiltration and freeze-dried. The mass of the obtained chondroitin sulfate sodium powder was 27.1g. The content of chondroitin sulfate sodium salt was detected by HPLC, and the results are as follows: Figure 16 As shown.

[0111] The structures of sodium chondroitin sulfate prepared in Examples 2-16 were determined and analyzed.

[0112] According to the analytical method on page 1595 of Part II of the Chinese Pharmacopoeia 2020, the unsulfated chondroitin sodium obtained in Example 1 and the chondroitin sodium prepared in Examples 2-9 were enzymatically hydrolyzed with chondroitin sulfate ABC enzyme. The chondroitin sulfate sodium was analyzed by HPLC using a Hypersil SAX column with a detection wavelength of 232 nm. The disaccharide units sulfonated at different positions eluted sequentially, which can be used to analyze the sulfonation status of chondroitin sulfate.

[0113] The analysis results are shown in Table 1 and Figures 1-16 Table 1 shows the disaccharide distribution of chondroitin sulfate sodium prepared in Examples 2-16. Figures 1-16 The disaccharide distribution of the products prepared in Examples 1-16 are shown respectively.

[0114] Table 1. Chondroitin sulfate sulfonation status (content / %) batch number CS-0S CS-6S CS-4S CS-2,6S CS-4,6S CS-2,4S Example 2 1.89 86.899 0.053 7.572 2.79 0.796 Example 3 27.004 68.46 0.563 2.527 0.85 0.596 Example 4 19.075 74.051 0.573 4.536 1.657 0.108 Example 5 14.722 76.328 0.944 5.681 2.246 0.079 Example 6 30.953 65.2 0.285 2.386 0.714 0.461 Example 7 15.059 79.076 0.163 3.651 1.221 0.83 Example 8 10.584 83.117 0.116 3.954 1.334 0.895 Example 9 1.257 81.261 0.044 12.043 4.857 0.539 Example 10 10.629 74.899 0.082 10.915 2.975 0.489 Example 11 2.378 56.238 0.847 23.02 5.238 4.488 Example 12 19.086 66.711 0.063 10.321 2.975 0.844 Example 13 40.628 55.652 0.462 2.146 0.665 0.447 Example 14 7.184 52.207 1.649 17.328 3.87 2.971 Example 15 47.864 49.404 0.725 1.453 0.492 0.081 Example 16 0.906 60.32 / 26.598 4.942 /

[0115] The results showed that the chondroitin sulfate prepared in Examples 2-16 could all be recognized and hydrolyzed by chondroitinase ABC. According to the test results, the chondroitin sulfate obtained by the sulfonation method of the present invention is mainly a 6-position independent sulfonation product, namely shark-like chondroitin sulfate. The 6-position independent sulfonation product can reach more than 65%, and can reach up to 86%. The degree of sulfation can be adjusted by controlling the reaction conditions. The sulfonation process provided by the present invention can obviously replace the traditional organic solvents to complete the sulfation modification of chondroitin. The final product does not undergo structural modification that affects the recognition and hydrolysis of chondroitinase ABC.

[0116] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. Application of liquid sulfur dioxide in the preparation of chondroitin sulfate or chondroitin sulfate salt.

2. Application of liquid sulfur dioxide as the sole solvent in the preparation of chondroitin sulfate or chondroitin sulfate salt.

3. Application of liquid sulfur dioxide as the sole solvent in the preparation of chondroitin sulfate or chondroitin sulfate salt during sulfonation.

4. A method for preparing chondroitin sulfate or chondroitin sulfate salt by applying liquid sulfur dioxide to any one of claims 1 to 3, characterized in that, Using chondroitin or chondroitin salt as raw material and liquid sulfur dioxide as the sole solvent, a sulfonation reaction is carried out under the action of a sulfonating agent to obtain chondroitin sulfate or chondroitin sulfate salt.

5. The method as described in claim 4, characterized in that, The sulfonating agent is selected from one or a combination of several of concentrated sulfuric acid, triethylamine sulfur trioxide, pyridine sulfur trioxide, DMF sulfur trioxide, and chlorosulfonic acid.

6. The method as described in claim 4, characterized in that, The sulfonation reaction is carried out at a temperature of -30℃ to 50℃.

7. The method as described in claim 4, characterized in that, The mass ratio of chondroitin or chondroitin salt to liquid sulfur dioxide is 1:3~60, and the molar ratio of chondroitin disaccharide unit to sulfonating agent in chondroitin or chondroitin salt is 1:1~5.

8. A method for preparing chondroitin sulfate or chondroitin sulfate salt by sulfonation, characterized in that, The steps include the following: (1) Mix chondroitin or chondroitin salt with liquid sulfur dioxide to form a dispersion system of chondroitin or chondroitin salt in liquid sulfur dioxide, then add sulfonating agent to the dispersion system and carry out sulfonation reaction at -30℃~50℃ to obtain the product of sulfonation reaction. The mass ratio of chondroitin to liquid sulfur dioxide is 1:3~60; the molar ratio of chondroitin disaccharide units to sulfonating reagent in the chondroitin or chondroitin salt is 1:1~5; the sulfonating reagent is selected from at least one of concentrated sulfuric acid, triethylamine sulfur trioxide, pyridine sulfur trioxide, DMF sulfur trioxide, and chlorosulfonic acid; the sulfonation reaction time is 0.5~36 h. (2) The product of the sulfonation reaction in (1) is purified and dried to obtain chondroitin sulfate or chondroitin sulfate salt.

9. The method as described in claim 8, characterized in that, After the sulfonation reaction in (1) is completed, the system is heated to a temperature greater than the boiling point of sulfur dioxide, or the pressure is reduced to lower the boiling point of sulfur dioxide, so that the liquid sulfur dioxide is converted into gas and removed from the reaction system, thereby realizing the recovery of sulfur dioxide.

10. The use of chondroitin sulfate or chondroitin sulfate salt prepared by the method of any one of claims 8 or 9 in the preparation of a medicament for the prevention and / or treatment of osteoarthritis or a medicament for the lowering of blood lipids.

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