Preparation method and application of sodium chondroitin sulfate

Abstract

The invention discloses a preparation method and application of chondroitin sulfate sodium, and belongs to the technical field of chondroitin sulfate. The preparation method of the sodium chondroitin sulfate comprises the following steps: dispersing sodium chondroitin which is not sulfated by using an organic solvent, adding a sulfonating reagent for sulfonation reaction, terminating the reaction, centrifuging, and collecting precipitate; and decoloring and drying the collected precipitate to obtain the chondroitin sulfate sodium. The average molecular weight of the final product obtained by the method is controllable, the 6-site overall sulfonation rate is up to 90% or above, the 6-site independent sulfonation rate is up to 60%-85%, the quality of products among batches is stable, and sodium chondroitin sulfate based on the characteristics is more suitable for human body absorption, high in bioavailability and higher in safety and effectiveness; moreover, the method can realize abundant sources, uniform structure, stable quality, mild reaction and reasonable and controllable production cycle without complex extraction and purification processes, and is suitable for large-scale commercial production and application.

Description

[0001] This application is a divisional application of the invention patent application with application date of March 18, 2025, application number 202510314994.2, and invention name “A preparation method of sodium chondroitin sulfate and its application”. Technical Field

[0002] The invention relates to a preparation method of sodium chondroitin sulfate and application thereof, belonging to the technical field of chondroitin sulfate. Background Art

[0003] Chondroitin sulfate (ChS / CS) is a naturally occurring bioactive macromolecule that is widely distributed in almost all invertebrates and vertebrates. Its sugar chain is composed of alternating disaccharide units of glucuronic acid (GlcA) or iduronic acid (IdoA) and N-acetylgalactosamine (GalNAc), and has sulfate groups in varying numbers and positions, such as positions 2 or 3 of GlcA or IdoA and positions 4 or 6 of GalNAc. It has been shown to have profound effects on many physiological processes, including inflammation, tumor progression and metastasis, vascular remodeling, and anti-oxidation.

[0004] Currently, commercial-grade, industrial production of chondroitin sulfate (CS) relies solely on animal tissues (e.g., cattle, pigs, chickens, cartilaginous fish, sharks, rays, and bony fish). Despite the plentiful raw material, this presents safety and quality challenges. In fact, the polysaccharide chains of CS from different sources may contain varying numbers of disaccharides and sulfate groups at different positions. Each CS is not entirely composed of a single ideal repeating disaccharide unit, but rather consists of multiple disaccharide structures with varying contents. Even known CS samples primarily comprise disaccharide units with varying percentages of N-acetylgalactosamine monosulfated at the 4th and 6th positions, disaccharides with varying percentages and positions of sulfate groups can be found within their polysaccharide chains. Consequently, CS from different sources exhibits a degree of heterogeneity in terms of relative molecular mass, charge density, and chemical properties. This not only complicates the study of the physicochemical properties and structural characterization of CS but also poses a disadvantage for quality control.

[0005] In addition, factors such as the source of raw materials and differences in structural characteristics (such as the composition, ratio, and position of the sulfate group of the disaccharide) can also affect the overall bioavailability and pharmacokinetic activity of chondroitin sulfate. Chondroitin sulfates of different structures have been shown to have different properties. For example, sulfated ChS at the 4th position can block the presentation of RANTES on the cell surface or bind to red blood cells infected with Plasmodium falciparum. Therefore, determining the source of chondroitin sulfate is crucial.

[0006] Chondroitin sulfate from sharks has been shown to exhibit anti-angiogenic and anti-tumor activities, as well as neurite outgrowth promoting activity in animals and humans. It has also shown significant efficacy in the treatment of osteoarthritis, rheumatoid arthritis, progressive systemic sclerosis, and neurovascular glaucoma, and is now widely used. Functional group identification has shown that the chondroitin isolated from shark cartilage is primarily C-type chondroitin sulfate, with the main component being monosulfated at the 6-position with GalNAc. This structural difference significantly distinguishes it from chondroitin sulfates from other sources. For example, ChS from bovine, porcine, chicken, and whale sources is primarily A-type chondroitin sulfate, with the main component being monosulfated at the 4-position with GalNAc. ChS from squid is primarily E-type chondroitin sulfate, with the main component being disulfated at the 4 and 6-positions with GalNAc. This may be the primary reason for its functional differences from other sources. However, shark resources are limited, and the supply of shark-derived chondroitin sulfate is far lower than market demand. Therefore, shark-like chondroitin with structural characteristics similar to natural shark chondroitin has become a current development hotspot.

[0007] European Patent Publication No. EP1304338B1 discloses a method for obtaining GalNAc-6-sulfated chondroitin sulfate using a microbial fermentation method. This method uses Escherichia coli K4 polysaccharide as a starting material, obtains an acidic polysaccharide after purification by acid treatment, and then protects the 4- and 6-positions of GalNAc and the 2- and 3-positions of GlcA by dibenzylidene and O-acetylation, respectively. The dibenzylidene group is separated by treatment with acetic acid, and the 4- and 6-positions of GalNAc are deprotected. The product is then sulfated to obtain chondroitin sulfate C sulfonated at the 6-position of GalNAc. This method requires a two-step protection and deprotection strategy, is cumbersome, and the introduction of more reagents increases the difficulty of subsequent purification, making it unsuitable for industrial large-scale production. Chinese patent CN103582653B also discloses a method for producing chondroitin sulfate sulfated at the 6-position with GalNAc. This method first converts chondroitin sodium salt into its free acid, pyridinium salt, or methyl ester, and then obtains a compound with repeating disaccharide units under acid catalysis. The hydroxyl groups at the 2' and 3' positions of the glucuronic acid unit are then protected, and the orthoester functional groups are rearranged to obtain an ester derivative. The ester derivative is then further sulfated to remove the O-acyl group present in the compound obtained in the previous step, thereby obtaining a sodium chondroitin sulfate in which all N-acetyl-D-galactosamine units in the same polysaccharide chain are randomly or monosulfated at the 4-position or 6-position. Although this method can also achieve effective control of a high proportion of a single component, the sulfonation process requires protection, deprotection, and orthoester functional group rearrangement strategies, resulting in a cumbersome process and unsuitable for large-scale industrial production. Summary of the Invention

[0008] In order to solve the above problems, the present invention provides a new method for preparing sodium chondroitin sulfate. The method uses unsulfated sodium chondroitin as the starting material, adopts a sulfonation strategy without a protective group, and realizes the sulfonation of chondroitin through a one-step sulfonation. The proportion of sodium chondroitin sulfate with independent sulfonation of GalNAc at the 6-position in the obtained sodium chondroitin sulfate can reach more than 60%.

[0009] The independent sulfonation of GalNAc 6-position in the present invention refers to the situation where sulfation occurs only at GalNAc 6-position of chondroitin sulfate sodium (ie, the disaccharide structure is 4GlcAβ1, 3GalNAc6Sβ1).

[0010] Specifically, the present invention provides a method for preparing sodium chondroitin sulfate, the preparation method comprising the following steps:

[0011] Step 1) Sulfonation: Disperse unsulfated sodium chondroitin in an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, terminate the reaction, centrifuge, and collect the precipitate;

[0012] Step 2) post-processing: decolorizing and drying the precipitate collected in step 1 to obtain sodium chondroitin sulfate;

[0013] The sulfonation reagent in step 1 is preferably one or more selected from concentrated sulfuric acid, sulfur trioxide trimethylamine complex (CAS: 3162-58-1), and sulfur trioxide pyridine complex (CAS: 26412-87-3).

[0014] Furthermore, the organic solvent in step 1 is selected from one or more of N,N-dimethylformamide or formamide.

[0015] Furthermore, the weight ratio of the unsulfated sodium chondroitin and the sulfonated reagent (ie, W 软骨素钠:W sulfonated reagent, g / g) ≤ 1:0.5; preferably 1:0.5-5 (such as 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7 :2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, or any non-integer ratio of the above ratios can be rounded up. In some preferred embodiments, the weight ratio (g / g) of the unsulfated sodium chondroitin and the sulfonated reagent in step 1 is about W 软骨素钠 :W 磺化试剂 =1:1-5.

[0016] It is understood that the sulfonation reagent in step 1 can be directly added to the organic solvent dispersion of unsulfated sodium chondroitin, or the sulfonation reagent can be dissolved in the same or different organic solvent as the above-mentioned dispersion of unsulfated sodium chondroitin and then added to the organic solvent dispersion of unsulfated sodium chondroitin.

[0017] In some specific embodiments, the sulfonation reagent is usually dissolved in an appropriate amount of an organic solvent different from the organic solvent used to disperse the unsulfated sodium chondroitin, and then added to the organic solvent dispersion of the unsulfated sodium chondroitin.

[0018] In some specific embodiments, the sulfonating reagent in step 1 is selected from one or more of concentrated sulfuric acid, sulfur trioxide trimethylamine complex, and sulfur trioxide pyridine complex, and the organic solvent in step 1 is N,N-dimethylformamide and formamide; in other specific embodiments, the sulfonating reagent in step 1 is preferably selected from one or more of concentrated sulfuric acid, sulfur trioxide trimethylamine complex, and sulfur trioxide pyridine complex, and the organic solvent in step 1 is N,N-dimethylformamide or formamide.

[0019] Furthermore, the weight-to-volume ratio (g / ml) of the unsulfated sodium chondroitin and the organic solvent in step 1 is about W 软骨素钠 :V 有机溶剂=1:4-50 (e.g. 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 9, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, or 1:50, or any non-integer ratio that can be rounded up to the above ratios).

[0020] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the unsulfated sodium chondroitin and the organic solvent in step 1 is about 软骨素钠 :V 有机溶剂 =1:8-20.

[0021] Further, the sulfonation reaction temperature in step 1 is about 20-50°C (such as 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or any two temperature ranges within the interval). Preferably, the sulfonation reaction temperature in step 1 is about 30-40°C.

[0022] Furthermore, the sulfonation reaction time in step 1 is about 0.5-6 h (such as 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, or any non-integer time rounded to the above time points); preferably, the sulfonation reaction time in step 1 is about 2-4 h.

[0023] Furthermore, the sulfonation reaction in step 1 is terminated by using anhydrous ethanol.

[0024] Furthermore, in the above-mentioned termination reaction, 3-10 times the volume (such as 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or any non-integer ratio of the above multiples) of anhydrous ethanol can be added to the entire sulfonation reaction system to terminate the reaction; preferably, in the above-mentioned termination reaction, 4-8 times the volume of anhydrous ethanol can be added to the entire sulfonation reaction system to terminate the reaction.

[0025] Furthermore, the centrifugal speed in step 1 is about 4000-8000 rpm (such as 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm); preferably, the centrifugal speed in step 1 is 5000-6000 rpm.

[0026] In order to better remove the solvent residue in the precipitate, in some specific embodiments, the precipitate collected by centrifugation is usually washed once or multiple times with anhydrous ethanol.

[0027] Furthermore, in step 2, activated carbon fiber membrane or hydrogen peroxide is used for decolorization.

[0028] In some preferred embodiments, step 2 uses activated carbon fiber membrane for decolorization; in other preferred embodiments, step 2 uses hydrogen peroxide for decolorization.

[0029] In some specific embodiments, before decolorization, an appropriate amount of purified water can be used to dissolve the precipitate collected in step 1.

[0030] Furthermore, the drying in step 2 is freeze drying or spray drying.

[0031] Furthermore, the unsulfated sodium chondroitin in step 1 is commercially available unsulfated sodium chondroitin.

[0032] Furthermore, the unsulfated sodium chondroitin in step 1 is unsulfated sodium chondroitin obtained through microbial fermentation.

[0033] In some specific embodiments, the unsulfated chondroitin sodium described in step 1 is obtained by extracting the fermentation broth obtained by fermenting Escherichia coli DH001; further, the extraction comprises necessary ultrafiltration, acid hydrolysis, purification, etc. The Escherichia coli DH001 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 26, 2024, with the deposit number: CGMCC No. 32081, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified as Escherichia coli.

[0034] In some specific embodiments, obtaining the unsulfated sodium chondroitin may comprise the following process:

[0035] Fermentation: The fermentation broth containing unsulfated sodium chondroitin is obtained by fermentation with Escherichia coli DH001;

[0036] Ultrafiltration: centrifuge the fermentation broth, collect the supernatant, and ultrafilter to obtain the filtrate;

[0037] Acid hydrolysis: add acid hydrolysis agent to the filtrate for acid hydrolysis, centrifuge and collect the supernatant;

[0038] Purification: The supernatant after acid hydrolysis is purified by ion exchange to obtain unsulfated chondroitin sodium.

[0039] It is understandable that the above-mentioned fermentation process also involves the use of a culture medium necessary for maintaining the growth, metabolism and fermentation of the strain. The culture medium contains a carbon source, a nitrogen source, inorganic salts and trace elements necessary for growth, etc., and can be any option that can be provided by the prior art and should not be regarded as a limitation of the present invention.

[0040] In some preferred embodiments, the carbon source involved in the above fermentation process is glucose.

[0041] In other preferred embodiments, the nitrogen source involved in the above fermentation process is an organic nitrogen source and / or an inorganic nitrogen source; preferably, the organic nitrogen source may be yeast extract powder, and / or the inorganic nitrogen source may be ammonium sulfate; more preferably, the nitrogen source involved in the above fermentation process is yeast extract powder and ammonium sulfate.

[0042] In other preferred embodiments, the fermentation process further involves the use of inorganic salts and trace elements necessary for strain growth, such as phosphorus, magnesium, iron, etc.; in some preferred embodiments, these inorganic salts and trace elements can be selected from one or more of potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and ferrous sulfate heptahydrate.

[0043] In some specific embodiments, the fermentation broth in the above fermentation process is obtained by fermenting Escherichia coli DH001 in a culture medium containing a carbon source, a nitrogen source, and inorganic salts and trace elements necessary for growth.

[0044] In some more specific embodiments, the fermentation broth in the above fermentation process is obtained by fermenting Escherichia coli DH001 in a culture medium containing glucose as a carbon source, yeast extract and ammonium sulfate as nitrogen sources, and inorganic salts and trace elements necessary for growth.

[0045] Furthermore, the centrifugal speed described in the above ultrafiltration process is about 6000-20000rpm (such as 6000rpm, 7000rpm, 8000rpm, 9000rpm, 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm, 15000rpm, 16000rpm, 17000rpm, 18000rpm, 19000rpm, 20000rpm); preferably, the centrifugal speed described in the above ultrafiltration process is 8000-10000rpm.

[0046] Furthermore, the pore size of the ultrafiltration membrane described in the above ultrafiltration process is 5-30kDa (such as 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 11kDa, 12kDa, 13kDa, 14kDa, 15kDa, 16kDa, 17kDa, 18kDa, 19kDa, 20kDa, 21kDa, 22kDa, 23kDa, 24kDa, 25kDa, 26kDa, 27kDa, 28kDa, 29kDa, 30kDa); preferably, the pore size of the ultrafiltration membrane described in the above ultrafiltration process is 10-20kDa.

[0047] Furthermore, the acid hydrolysis process refers to acid hydrolysis of the filtrate after ultrafiltration, and the acid hydrolysis agent can be an organic acid or an inorganic acid reagent.

[0048] Furthermore, the acidolysis agent in the above acidolysis process is selected from one or more of sulfuric acid, hydrochloric acid and acetic acid.

[0049] Furthermore, the acid hydrolysis pH of the above-mentioned acid hydrolysis process is about 1.0-5.0 (such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0); preferably, the acid hydrolysis pH of the above-mentioned acid hydrolysis process is about 1.0-2.0.

[0050] Furthermore, the acid hydrolysis time of the above acid hydrolysis process is about 2-10 hours (such as 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours).

[0051] Furthermore, the acid hydrolysis temperature of the above-mentioned acid hydrolysis process is about 50-90°C (such as 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, or any two temperature ranges within the interval).

[0052] Furthermore, the ion exchange in the above purification process is anion resin exchange; preferably D280 anion resin exchange or D980 anion resin exchange.

[0053] Furthermore, the ion exchange eluent involved in the above purification process is a 1-3 mol / L (such as 1 mol / L, 2 mol / L, 3 mol / L) sodium chloride solution; in some specific embodiments, the ion exchange eluent involved in the above purification process is a 2 M sodium chloride solution.

[0054] Furthermore, the above purification process may further include the steps of re-ultrafiltration, concentration, and drying, that is, the eluate obtained by ion exchange is re-ultrafiltered (such as the ultrafiltration membrane pore size is 2-30 kDa), and the collected filtrate is concentrated and dried to obtain unsulfated sodium chondroitin. These processes are only for better or more optimal extraction of unsulfated sodium chondroitin, and their number, sequence, process parameters, etc. should not be regarded as limiting the present invention.

[0055] Furthermore, the average molecular weight of the unsulfated sodium chondroitin obtained by fermentation with Escherichia coli DH001 provided by the present invention is about 5-30 kDa (such as 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, or any non-integer kDa value that can be rounded to the above integer kDa value, that is, the average molecular weight of the unsulfated sodium chondroitin obtained can be any integer or non-integer kDa value within the range).

[0056] In some preferred embodiments, the average molecular weight of sodium chondroitin sulfate obtained by the method provided by the present invention is 5-30 kDa (such as 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, or any non-integer kDa value that can be rounded to the above integer kDa value, that is, the average molecular weight of sodium chondroitin sulfate obtained by the method provided by the present invention can be any integer or non-integer kDa value within the range).

[0057] In some preferred embodiments, the proportion of sodium chondroitin sulfate obtained by the method provided by the present invention, wherein the proportion of sodium chondroitin sulfate independently sulfonated at the GalNAc6 position is higher than 60% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any non-integer ratio of the above ratios can be rounded off), that is, more than 60% (including 60%) of the sodium chondroitin sulfate obtained by the method provided by the present invention is sodium chondroitin sulfate with independent sulfonation at the 6-position of GalNAc; in some more preferred embodiments, the proportion of sodium chondroitin sulfate with independent sulfonation at the 6-position of GalNAc in the sodium chondroitin sulfate obtained by the method provided by the present invention is in the range of 60%-85%, that is, 60%-85% (including 60% and 85%) of the sodium chondroitin sulfate obtained by the method provided by the present invention is sodium chondroitin sulfate with independent sulfonation at the 6-position of GalNAc.

[0058] The present invention also provides a method for sulfating sodium chondroitin, wherein the sodium chondroitin is not sulfated and has an average molecular weight of about 5-30 kDa. The sulfation method comprises the sulfonation (i.e., step 1) and post-treatment (i.e., step 2) processes described above.

[0059] Specifically, the sulfation method of chondroitin sodium provided by the present invention comprises the following steps:

[0060] Step 1) Sulfonation: Disperse chondroitin sodium in an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, terminate the reaction, centrifuge, and collect the precipitate;

[0061] Step 2) post-processing: decolorizing and drying the precipitate collected in step 1 to obtain sodium chondroitin sulfate;

[0062] in:

[0063] The sodium chondroitin is not sulfated and has an average molecular weight of about 5-30 kDa;

[0064] The sulfonating agent is preferably one or more selected from concentrated sulfuric acid, sulfur trioxide trimethylamine complex (CAS: 3162-58-1), and sulfur trioxide pyridine complex (CAS: 26412-87-3).

[0065] The organic solvent is selected from one or more of N,N-dimethylformamide or formamide.

[0066] Furthermore, the unsulfated sodium chondroitin is commercially available unsulfated sodium chondroitin.

[0067] Furthermore, the unsulfated sodium chondroitin is unsulfated sodium chondroitin obtained through microbial fermentation.

[0068] In some specific embodiments, the unsulfated sodium chondroitin in step 1 is obtained by extracting the fermentation broth obtained by fermenting Escherichia coli DH001; further, the extraction comprises necessary ultrafiltration, acid hydrolysis, purification, etc. The Escherichia coli DH001 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 26, 2024, with the deposit number: CGMCC No. 32081, the deposit address is Beijing, China, and the classification name is Escherichia coli.

[0069] The present invention also relates to the use of any of the above-mentioned methods for preparing sodium chondroitin sulfate or any of the above-mentioned methods for sulfating sodium chondroitin in the preparation of sodium chondroitin sulfate sulfated at the GalNAc 6 position; preferably, the sodium chondroitin sulfate sulfate sulfated at the GalNAc 6 position refers to sodium chondroitin sulfate independently sulfonated at the GalNAc 6 position.

[0070] The present invention also relates to a method for preparing sodium chondroitin sulfate having an independent sulfonation ratio of GalNAc at position 6 greater than 60%, wherein the average molecular weight of the sodium chondroitin sulfate is 5-30 kDa, and the method comprises the sulfonation (i.e., step 1) and post-treatment (i.e., step 2) processes described in any one of the above items; more preferably, the present invention also relates to a method for preparing sodium chondroitin sulfate having an independent sulfonation ratio of GalNAc at position 6 of 60%-85%, wherein the average molecular weight of the sodium chondroitin sulfate is 5-30 kDa, and the method comprises the sulfonation (i.e., step 1) and post-treatment (i.e., step 2) processes described in any one of the above items.

[0071] The present invention also provides sodium chondroitin sulfate having an independent sulfonation ratio of GalNAc at position 6 greater than 60%, wherein the sodium chondroitin sulfate has an average molecular weight of 5-30 kDa, and the sodium chondroitin sulfate is obtained using any of the above-mentioned methods for preparing sodium chondroitin sulfate or methods for sulfating sodium chondroitin. More preferably, the present invention also provides sodium chondroitin sulfate having an independent sulfonation ratio of GalNAc at position 6 greater than 60%, wherein the sodium chondroitin sulfate has an average molecular weight of 5-30 kDa, and the sodium chondroitin sulfate is obtained using any of the above-mentioned methods for preparing sodium chondroitin sulfate or methods for sulfating sodium chondroitin, or methods for preparing sodium chondroitin sulfate having an independent sulfonation ratio of GalNAc at position 6 greater than 60%, or methods for preparing sodium chondroitin sulfate having an independent sulfonation ratio of GalNAc at position 6 of 60%-85%.

[0072] The present invention has the following technical advantages:

[0073] The preparation method of sodium chondroitin sulfate or the sulfation method of sodium chondroitin provided by the present invention uses unsulfated sodium chondroitin as a starting material and realizes the GalNAc 6-position directed sulfonation of chondroitin through a "one-step" chemical sulfonation strategy. In the sodium chondroitin sulfate obtained by the method provided by the present invention, the proportion of chondroitin sulfate independently sulfonated at the GalNAc 6-position is as high as more than 60% (60%-85%). Based on this characteristic, the sodium chondroitin sulfate obtained is more uniform in structure, has higher safety and effectiveness while ensuring more reliable quality; and its components are more suitable for human absorption and have high bioavailability.

[0074] The present invention has mild reaction conditions, does not require high temperature and high pressure, has simple operation steps, has low requirements for reaction equipment, is green and environmentally friendly, does not require a large amount of organic reagents, has a stable and reliable reaction, has a high sulfonation rate of sodium chondroitin sulfate (which can be as high as more than 70%), has a controllable product molecular weight (preferably 5-30 kDa), and has stable product quality between batches, making it more suitable for industrial promotion and use.

[0075] The method provided by the present invention uses unsulfated sodium chondroitin derived from microbial fermentation as raw material, which can effectively avoid cross contamination between organisms, and has a stable raw material source, uniform structure, and safe and stable quality.

[0076] In summary, the chemical sulfonation process involved in the preparation method and application provided by the present invention is simple, the reaction is mild, and no complex reaction steps (such as protection and deprotection strategies, functional group rearrangement strategies, etc.) are required to achieve directional position-specific sulfation of the final product. This can greatly reduce the dependence on the production site and specific equipment during the production process, which is beneficial to the control of production costs. In addition, the production process is simple and does not require the use of complex chemical reagents (the protecting group strategy requires about 15 organic reagents, while the present invention only requires 4 organic reagents). While reducing the reagent use cost and recovery cost, it also reduces the possibility of environmental pollution and reduces the chemical component residues that may exist in the final product, further ensuring the safety of clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is the sugar chain structure diagram of chondroitin sulfate mentioned in the background technology;

[0078] Figure 2 This is a graph showing the molecular weight analysis results of unsulfated sodium chondroitin obtained in Example 1;

[0079] Figure 3 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 2;

[0080] Figure 4 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 3;

[0081] Figure 5 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 4;

[0082] Figure 6 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 5;

[0083] Figure 7 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 6;

[0084] Figure 8 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 7;

[0085] Figure 9 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 8;

[0086] Figure 10 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 9;

[0087] Figure 11 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Comparative Example 1;

[0088] Figure 12 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Comparative Example 2;

[0089] Figure 13 This is a diagram showing the disaccharide content analysis results of the unsulfated sodium chondroitin obtained in Example 1;

[0090] Figure 14 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 2;

[0091] Figure 15 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 3;

[0092] Figure 16 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 4;

[0093] Figure 17 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 5;

[0094] Figure 18 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 6;

[0095] Figure 19 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 7;

[0096] Figure 20 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 8;

[0097] Figure 21 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 9;

[0098] Figure 22 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Comparative Example 1;

[0099] Figure 23 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Comparative Example 2;

[0100] Figure 24 This is the carbon NMR spectrum of the sodium chondroitin sulfate prepared in Example 2;

[0101] Figure 25 This is the carbon NMR spectrum of sodium chondroitin sulfate prepared in Example 3;

[0102] Figure 26 This is the carbon NMR spectrum of the sodium chondroitin sulfate prepared in Example 4. DETAILED DESCRIPTION

[0103] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0104] Example 1 Preparation of Chondroitin Sodium

[0105] (1) Fermentation: Escherichia coli DH001 was used as the production strain. After activation, it was inoculated at 1%-5% into a 30 L seed tank (containing medium 1, components of which are glucose 5-10 g / L; yeast extract 0.8-2 g / L; ammonium sulfate 0.5-1 g / L; potassium dihydrogen phosphate 10-12 g / L; magnesium sulfate heptahydrate 3-5 g / L; and defoamer 0.2-0.3 g / L) and cultured for 8-10 h until OD 600 When the pH reaches 2.5-5, the seeds are transferred into a 100L fermentation tank (containing medium 2, components of which are glucose 10-15g / L; yeast extract 2-5g / L; ammonium sulfate 1.5-2.5g / L; magnesium sulfate heptahydrate 4-8g / L; potassium dihydrogen phosphate 5-10g / L; ferrous sulfate heptahydrate 0.1-0.3g / L; sodium chloride 5-10g / L; defoamer 0.2-0.5g / L) at a transplant rate of 5-10% for fermentation, with initial stirring at 200-300rpm, ventilation 1-4vvm, dissolved oxygen correction 100%, and dissolved oxygen control at 15%-30% throughout the process. 25% (w / v) ammonia water is fed to control the pH at 7.0-8.0, and 40%-60% mass fraction of glucose is fed as the carbon source for feeding, and fermentation is carried out for 42-48h to OD 600 When the temperature reaches 110-135, the tank is put into the fermentation broth, and the content of sodium chondroitin (unsulfated) in the fermentation broth is 8-10 g / L;

[0106] (2) Ultrafiltration: The fermentation broth obtained by fermentation was centrifuged at 8000 rpm, the supernatant was collected, and ultrafiltration was performed using a 10 kDa ultrafiltration membrane until the conductivity of the concentrate was stable;

[0107] (3) Acid hydrolysis: The pH of the ultrafiltration concentrate was adjusted to 1.5 with hydrochloric acid, and the mixture was reacted at 80°C for 6 h for acid hydrolysis. The precipitate was removed by centrifugation at 8000 rpm to obtain the supernatant.

[0108] (4) Purification: D280 anion exchange resin was added to the obtained supernatant, stirred and adsorbed for 2 h, and then eluted with a 2 mol / L sodium chloride solution. The eluate was ultrafiltered using a 10 kDa ultrafiltration membrane until the conductivity of the concentrate was stable. The concentrate was concentrated and dried to obtain 410 g of sodium chondroitin (unsulfated sodium chondroitin).

[0109] Molecular weight analysis: According to the "Low Molecular Weight Heparin Molecular Weight Detection Method", the molecular weight of the chondroitin sodium obtained by the above method was determined using a TSKgel G3000SWXL column. The average molecular weight Mw of the chondroitin sodium was 11.1 kDa (see Figure 2 , retention time is 18.8min).

[0110] Example 2 Preparation of Sodium Chondroitin Sulfate 1

[0111] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of N,N-dimethylformamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 10g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:1, disperse with 50 mL of formamide, add to the above chondroitin sodium dispersion, stir in an oil bath at 40°C for 2 h, add 0.7 L of anhydrous ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L of anhydrous ethanol;

[0112] (2) Post-treatment: The washed precipitate was redissolved in 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and the mixture was stirred at 25°C for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 11.8 g of sodium chondroitin sulfate.

[0113] Molecular weight analysis: According to the "Low Molecular Weight Heparin Molecular Weight Detection Method", the molecular weight of the sodium chondroitin sulfate prepared in Example 2 was determined using a TSKgel G3000SWXL column. The average molecular weight Mw of the sodium chondroitin sulfate obtained in Example 2 was 13.4 kDa (see Figure 3 , retention time is 17.5min).

[0114] Example 3 Preparation of Sodium Chondroitin Sulfate 2

[0115] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of formamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 10g of sulfur trioxide pyridine complex, W 软骨素钠 :W磺化试剂 =1:1, disperse with 50 mL N,N-dimethylformamide, add to the above chondroitin sodium dispersion, stir in an oil bath at 40°C for 2 h, add 0.7 L anhydrous ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L anhydrous ethanol;

[0116] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and the mixture was stirred at 25°C for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 12.1 g of sodium chondroitin sulfate. The average molecular weight Mw of the sodium chondroitin sulfate obtained in Example 3 was determined to be 13.7 kDa (see Figure 4 , retention time is 17.6min).

[0117] Example 4 Preparation of Sodium Chondroitin Sulfate 3

[0118] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of formamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 10g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:1, disperse with 50 mL of formamide, add to the above chondroitin sodium dispersion, stir in an oil bath at 40°C for 2 h, add 0.7 L of anhydrous ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L of anhydrous ethanol;

[0119] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and the mixture was stirred at 25°C for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 11.2 g of sodium chondroitin sulfate. The average molecular weight Mw of the sodium chondroitin sulfate obtained in Example 4 was determined to be 12.1 kDa (see Figure 5 , retention time is 18.5min).

[0120] Example 5 Preparation of Sodium Chondroitin Sulfate

[0121] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of N,N-dimethylformamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 10g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂=1:1, disperse with 50 mL of N,N-dimethylformamide, add to the above chondroitin sodium dispersion, stir in an oil bath at 40°C for 2 h, add 0.7 L of anhydrous ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L of anhydrous ethanol;

[0122] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and the mixture was stirred at 25° C. for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 11.3 g of sodium chondroitin sulfate. The average molecular weight Mw of the sodium chondroitin sulfate obtained in Example 5 was determined to be 12.3 kDa (see Figure 6 , retention time is 18.3min).

[0123] Example 6 Preparation of Sodium Chondroitin Sulfate

[0124] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of formamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 50g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:5, disperse with 50 mL of formamide, add to the above chondroitin sodium dispersion, stir in an oil bath at 40°C for 2 h, add 0.7 L of anhydrous ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L of anhydrous ethanol;

[0125] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and the mixture was stirred at 25°C for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 11.5 g of sodium chondroitin sulfate. The average molecular weight Mw of the sodium chondroitin sulfate obtained in Example 6 was determined to be 13.2 kDa (see Figure 7 , retention time is 17.6min).

[0126] Example 7 Preparation of Sodium Chondroitin Sulfate

[0127] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of N,N-dimethylformamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 10g sulfur trioxide trimethylamine complex, W 软骨素钠 :W 磺化试剂=1:1, dispersed with 50 mL of formamide, added to the above chondroitin sodium dispersion, stirred in an oil bath at 50°C for 6 h, added 0.7 L of anhydrous ethanol to terminate the reaction, centrifuged at 6000 rpm, collected the precipitate, and washed with 0.7 L of anhydrous ethanol.

[0128] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water and decolorized by repeated filtration 5 times using 6 layers of activated carbon fiber membrane. The decolorized liquid was freeze-dried to obtain 11.6 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Example 7 was determined to be 12.8 kDa (see Figure 8 , retention time is 18.2min).

[0129] Example 8 Preparation of Sodium Chondroitin Sulfate

[0130] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of N,N-dimethylformamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 10g of concentrated sulfuric acid, W 软骨素钠 :W 磺化试剂 =1:1, dispersed with 50 mL of formamide, added to the above chondroitin sodium dispersion, stirred in an oil bath at 30°C for 4 h, added 0.7 L of anhydrous ethanol to terminate the reaction, centrifuged at 6000 rpm, collected the precipitate, and washed with 0.7 L of anhydrous ethanol.

[0131] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water and decolorized by repeated filtration 5 times using 6 layers of activated carbon fiber membrane. The decolorized liquid was freeze-dried to obtain 11.2 g of sodium chondroitin sulfate. The average molecular weight Mw of the sodium chondroitin sulfate obtained in Example 8 was determined to be 12.2 kDa (see Figure 9 , retention time is 17.9 min).

[0132] Example 9 Preparation of Sodium Chondroitin Sulfate 8

[0133] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of N,N-dimethylformamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 5g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:0.5, dispersed with 50 mL of formamide, added to the above chondroitin sodium dispersion, stirred in an oil bath at 40°C for 2 h, added 0.7 L of anhydrous ethanol to terminate the reaction, centrifuged at 6000 rpm, collected the precipitate, and washed with 0.7 L of anhydrous ethanol.

[0134] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water and decolorized by repeated filtration 5 times using 6 layers of activated carbon fiber membrane. The decolorized liquid was freeze-dried to obtain 10.9 g of sodium chondroitin sulfate. The average molecular weight Mw of the sodium chondroitin sulfate obtained in Example 9 was determined to be 11.9 kDa (see Figure 10 , retention time is 18.3min).

[0135] Comparative Example 1

[0136] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of N,N-dimethylformamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 10g of chlorosulfonic acid, W 软骨素钠 :W 磺化试剂 =1:1, disperse with 50 mL of formamide, add to the above chondroitin sodium dispersion, stir in an oil bath at 40°C for 2 h, add 0.7 L of anhydrous ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L of anhydrous ethanol;

[0137] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and the mixture was stirred at 25°C for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 11.4 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Comparative Example 1 was determined to be 12.5 kDa (see Figure 11 , retention time is 18.0 min).

[0138] Comparative Example 2

[0139] (1) Sulfonation: 10 g of sodium chondroitin prepared in Example 1 was dispersed in 50 mL of N,N-dimethylformamide. 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, preheat in an oil bath at 40°C; take 3g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:0.3, dispersed with 50 mL of formamide, added to the above chondroitin sodium dispersion, stirred in an oil bath at 40°C for 2 h, added 0.7 L of anhydrous ethanol to terminate the reaction, centrifuged at 6000 rpm, collected the precipitate, and washed with 0.7 L of anhydrous ethanol;

[0140] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and the mixture was stirred at 25°C for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 10.5 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Comparative Example 2 was 11.3 kDa (see Figure 12 , retention time is 18.7 min).

[0141] Result Analysis

[0142] 1. Analysis of the sodium chondroitin sulfate prepared in Examples 2 to 9

[0143] According to the analysis method on page 1595 of Part II of the Chinese Pharmacopoeia 2020 Edition, the unsulfated sodium chondroitin obtained in Example 1 and the sodium chondroitin sulfate prepared in Examples 2 to 9 and Comparative Examples 1 to 2 were enzymatically hydrolyzed with chondroitin sulfate ABC enzyme to hydrolyze the macromolecular chondroitin sulfate into disaccharide units (→GalA-GalNAc→). HPLC analysis was performed using a Hypersil SAX column with a detection wavelength of 232 nm. The sulfonated disaccharide units at different positions peaked in sequence, and the sulfonation of chondroitin sulfate could be analyzed.

[0144] The analysis results are shown in Table 1 and Figure 13-23 Table 1 shows the disaccharide distribution of sodium chondroitin sulfate prepared in Examples 2 to 9 and Comparative Examples 1 to 2. Figure 13 This is the disaccharide content analysis result of chondroitin sodium obtained in Example 1. Figure 14 This is the disaccharide distribution analysis result of the sodium chondroitin sulfate prepared in Example 2. Figure 15 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Example 3. Figure 16 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Example 4; Figure 17 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Example 5; Figure 18 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Example 6; Figure 19 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Example 7; Figure 20 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Example 8; Figure 21 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Example 9; Figure 22 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Comparative Example 1; Figure 23 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Comparative Example 2.

[0145] Table 1 Sulfonation of chondroitin sulfate

[0146] The result shows that the chondroitin sulfate obtained in Examples 2 to 9 can be recognized and hydrolyzed by chondroitinase ABC. From the perspective of the hydrolysis product, the sulfonation reaction of the method provided by the present invention is relatively thorough. In each preparation example, only no more than 30% of the chondroitin (i.e., OS-CS) is not sulfonated, and the sulfonation rate can be as high as more than 70%. In particular, in Examples 2-3, the sulfonation rate can be as high as more than 90% (such as the OS-CS% of Example 2 is only 6.94%, and the OS-CS% of Example 3 is only 3.44%), and the vast majority are sulfonated at the 6th position of GalNAc. In Examples 2 and 3, the vast majority of the sulfonated products are distributed in the 6th position of the independently sulfonated chondroitin sulfate (i.e., 6S-CS, the disaccharide structure is 4G lcAβ1,3GalNAc6Sβ1), 2,6-sulfonated chondroitin sulfate (i.e., 2,6S-CS, disaccharide structure is 4GlcAβ1,3GalNAc6Sβ1), 4,6-sulfonated chondroitin sulfate (i.e., 4,6S-CS, disaccharide structure is 4GlcAβ1,3GalNAc4,6Sβ1), and only less than 1% of 2,4-sulfonated chondroitin sulfate (i.e., 2,4S-CS, disaccharide structure is 4GlcA2Sβ1,3GalNAc4Sβ1); in Examples 4-9, all products were sulfonated at the 6-position of GalNAc.

[0147] The sulfonated products are evenly distributed, and the proportion of products independently sulfonated only at the GalNAc 6-position (i.e., 6S-CS) is as high as more than 60%. For example, the proportion of the 6-position independently sulfonated product in Example 2 is 80.57%, the proportion of the 6-position independently sulfonated product in Example 3 is 80.69%, the proportion of the 6-position independently sulfonated product in Example 4 is 65.41%, the proportion of the 6-position independently sulfonated product in Example 5 is 70.67%, the proportion of the 6-position independently sulfonated product in Example 6 is 73.15%, the proportion of the 6-position independently sulfonated product in Example 7 is 76.06%, the proportion of the 6-position independently sulfonated product in Example 8 is 70.47%, and the proportion of the 6-position independently sulfonated product in Example 9 is 62.95%. This shows that the preparation method provided by the present invention can achieve high-proportion directional 6-position sulfonation of chondroitin, and can stably and effectively control the proportion of the GalNAc 6-position independently sulfonated product in the final product to more than 60%, especially can well control the GalNAc in the final product. The proportion of the 6-position independent sulfonation product is between 60% and 85%. In actual production, the final product with the target proportion can be obtained according to product requirements.

[0148] In addition, the sulfonation process of the preparation method provided by the present invention is mild, and the final product does not undergo structural modification that may affect the recognition and enzymatic hydrolysis of natural enzymes.

[0149] As can be seen from Comparative Example 1, compared to Example 3, after changing the type of sulfonating agent, the proportion of independent sulfonation products at position 6 decreased to 23.50%, indicating that the type of sulfonating agent in the preparation method of the present invention can significantly determine the degree of directional sulfonation at position 6 of chondroitin sodium. As can be seen from Comparative Example 2, compared to Example 3, reducing the amount of sulfonating agent reduced the sulfonation rate to 50.52%, indicating that the addition ratio of the sulfonating agent in the preparation method of the present invention has a greater impact on the sulfonation rate. This also directly demonstrates that the preparation method provided by the present invention can control the proportion of independent sulfonation products at position 6 of GalNAc in the final product to more than 60%.

[0150] 2. C-NMR spectroscopy analysis of sodium chondroitin sulfate

[0151] C-NMR spectrometer model: AV500 (CAq08);

[0152] The detection conditions are as follows: magnetic field strength (FS): 125 MHz; number of scans (NS): 12288 times; temperature (TE): approximately 40°C (313.2 K); sampling time (AQ): 1.1 S; delay time (D1): 2.0 S; sampling center (O1P): 100.0 ppm; decoupling center (O2P): 4.0 ppm; spectral width (SW): 220-240 ppm; pulse (PUL): 30°; exponential linewidth window function (LB): 1.0 Hz; NMR tube diameter: 5 mm; acquisition method: D_GLP_E.glp.

[0153] The analysis results can be found in Figures 24-26 , Figures 24-26 The carbon NMR spectra of sodium chondroitin sulfate prepared in Examples 2 to 4 are respectively, the 49.5 ppm peak is the methanol positioning peak, and the carbon spectrum has the main characteristic peaks of CS-C, among which the 68.7 ppm peak is the characteristic signal peak of sulfonated carbon No. 6 in CS-C, and 176.2 ppm and 23.8 ppm are the characteristic signal peaks of carbon No. 7 and carbon No. 8 in GalNAc, respectively.

[0154] The results showed that the carbon chain structure of sodium chondroitin sulfate obtained in Example 2-4 was consistent with the carbon chain structure of CS-C, proving that chondroitin sulfate sulfonated at the 6-position of GalNAc was obtained using the method of the present invention.

[0155] In addition, it can be seen from Examples 2-9 (see Figure 3-10), the average molecular weights (Mw) of sodium chondroitin sulfate obtained by sulfonation of sodium chondroitin via the method provided by the present invention are similar, indicating that the sulfonation method provided by the present invention is mild and, despite undergoing a chemical reaction process, minimal damage to the disaccharide backbone structure. The average molecular weights (Mw) of the obtained sodium chondroitin sulfates indicate that the average molecular weight (Mw) distribution of sodium chondroitin sulfate obtained using the sulfonation method provided by the present invention is stable across batches, demonstrating that the sulfonation method provided by the present invention is operationally stable across batches and is not significantly dependent on the operating environment, operating methods, compound dosage, and ratio.

[0156] In summary, the present invention uses unsulfated sodium chondroitin as raw material and can achieve directional position-specific sulfation of the final product through a simple chemical sulfonation process, thereby obtaining a 6-position sulfonated product with stable molecular weight and high ratio.

[0157] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the order of operation. Obvious improvements made to the present invention by those skilled in the art in combination with existing common knowledge also fall within the scope of protection of the present invention.

Claims

1. A sodium chondroitin sulfate having an independent sulfonation ratio of 60% to 85% at position 6 of GalNAc, characterized in that: The sodium chondroitin sulfate is obtained by sulfation of unsulfated sodium chondroitin obtained by fermentation of Escherichia coli DH001. The Escherichia coli DH001 was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on September 28, 2024. The registration number of the collection center is: CGMCC No. 32081, and the collection address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The sodium chondroitin sulfate according to claim 1, characterized in that Described sulfation comprises the steps: Step 1) Sulfonation: Disperse unsulfated sodium chondroitin in an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, terminate the reaction, centrifuge, and collect the precipitate; Step 2) post-processing: decolorizing and drying the precipitate collected in step 1 to obtain sodium chondroitin sulfate; in: The sulfonating agent is selected from any one of concentrated sulfuric acid, sulfur trioxide trimethylamine complex, and sulfur trioxide pyridine complex; The organic solvent is selected from one or more of N,N-dimethylformamide or formamide; The sulfonation reaction temperature is 20-50°C and the reaction time is 0.5-6h; The weight ratio of the unsulfated sodium chondroitin and the sulfonated reagent is W 软骨素钠 :W 磺化试剂 =1:0.5-5.

3. The sodium chondroitin sulfate according to claim 2, characterized in that The weight-to-volume ratio of the unsulfated sodium chondroitin and the organic solvent in step 1 is W sodium chondroitin:V organic solvent=1:4-50, g / ml.

4. The sodium chondroitin sulfate according to claim 1, characterized in that The unsulfated sodium chondroitin obtained by fermentation of Escherichia coli DH001 has an average molecular weight of 5-30 kDa after sulfation.

5. The sodium chondroitin sulfate according to claim 1, characterized in that The average molecular weight of the sodium chondroitin sulfate is 5-30 kDa.

6. A method for preparing sodium chondroitin sulfate with an independent sulfonation ratio of GalNAc 6 at 60% to 85%, characterized in that: The method comprises the following steps: Step 1) Sulfonation: Disperse unsulfated sodium chondroitin in an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, terminate the reaction, centrifuge, and collect the precipitate; Step 2) post-processing: decolorizing and drying the precipitate collected in step 1 to obtain sodium chondroitin sulfate; in: The unsulfated chondroitin sodium is obtained by fermentation of Escherichia coli DH001. The Escherichia coli DH001 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 28, 2024. The registration number of the collection center is: CGMCC No. 32081. The storage address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The sulfonating agent is selected from any one of concentrated sulfuric acid, sulfur trioxide trimethylamine complex, and sulfur trioxide pyridine complex; The organic solvent is selected from one or more of N,N-dimethylformamide or formamide; The sulfonation reaction temperature is 20-50°C and the reaction time is 0.5-6h; The weight ratio of the unsulfated sodium chondroitin and the sulfonation reagent is W sodium chondroitin:W sulfonation reagent = 1:0.5-5.

7. The preparation method according to claim 6, characterized in that The weight-to-volume ratio of the unsulfated sodium chondroitin and the organic solvent in step 1 is W sodium chondroitin:V organic solvent=1:4-50, g / ml.

8. The preparation method according to claim 6, characterized in that The average molecular weight of the unsulfated sodium chondroitin after sulfation is 5-30 kDa.

9. The preparation method according to claim 6, characterized in that The average molecular weight of the sodium chondroitin sulfate is 5-30 kDa.

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