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%, and the quality of products among batches is stable. The sodium chondroitin sulfate based on the characteristics is more suitable for being absorbed by a human body, high in bioavailability and higher in safety and effectiveness; the method can be realized without a complex extraction and purification process, and is rich in source, uniform in structure, stable in quality, mild in reaction, reasonable and controllable in production cycle and suitable for large-scale commercial production and application.

Description

Technical Field

[0001] The invention relates to a preparation method of sodium chondroitin sulfate and application thereof, belonging to the technical field of chondroitin sulfate. Background Art

[0002] Chondroitin sulfate (ChS / CS) is a naturally derived 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 different numbers and positions, such as the 2nd or 3rd position of G1cA or IdoA and the 4th or 6th position of Gal1NAc. It has been confirmed that it has a profound effect on many physiological processes such as inflammation, tumor progression and metastasis, vascular remodeling, and anti-oxidation.

[0003] At present, the commercial-grade industrial production of chondroitin sulfate uses animal tissue as raw material (such as cattle, pigs, chickens, cartilaginous fish, sharks, rays, bony fish, etc.). Although the raw material source is sufficient, it also brings safety and quality problems. In fact, the polysaccharide chain of ChS from different sources may have different numbers of disaccharides and sulfate groups in different positions. Each ChS is not completely composed of an ideal disaccharide repeating unit, but is composed of a variety of disaccharide structures with different contents. Even if the known ChS sample is mainly composed of two different percentages of disaccharide units, monosulfated at the 4th position and monosulfated at the 6th position of N-acetylgalactosamine, disaccharides with different percentages of sulfate groups and positions can also be found in its polysaccharide chain. Therefore, chondroitin sulfate from different sources has certain heterogeneity in relative molecular mass, charge density, chemical properties, etc., which not only makes it difficult to study the physical and chemical properties and structural characterization of ChS, but also brings unfavorable factors to its quality control.

[0004] 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 disaccharides) will 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, it is crucial to determine the source of chondroitin sulfate.

[0005] Chondroitin sulfate from sharks has been shown to have anti-angiogenic and anti-tumor activities, neurite outgrowth promoting activities in animals and humans, and has significant effects in treating osteoarthritis, rheumatoid arthritis, progressive systemic sclerosis and neurovascular glaucoma, and has now been widely used. Functional group identification shows that the chondroitin isolated from shark cartilage is mainly C-type chondroitin sulfate, that is, its main component is GalNAc 6-monosulfated chondroitin, which makes its structural characteristics significantly different from chondroitin sulfate from other sources, such as ChS from cattle, pigs, chickens, whales, etc., which is mainly composed of A-type chondroitin sulfate, and its main component is GalNAc 4-monosulfated chondroitin; ChS from squid is mainly composed of E-type chondroitin sulfate, and its main component is GalNAc 4,6-disulfated chondroitin, which may be the main reason for its functional difference from chondroitin from other sources. However, shark resources are limited, and the supply of shark-derived chondroitin sulfate is far less than the market demand. Therefore, shark-like chondroitin with structural characteristics similar to natural shark chondroitin has become a current development hotspot.

[0006] European patent with publication number EP1304338B1 discloses a method for obtaining chondroitin sulfate sulfated at position 6 of GalNAc by using a microbial fermentation method. The method uses Escherichia coli K4 polysaccharide as a starting material, obtains an acidic polysaccharide after purification by acid treatment, and then protects positions 4 and 6 of GalNAc and positions 2 and 3 of GlcA by dibenzylidene and O-acetylation, respectively, separates the dibenzylidene group by treatment with acetic acid, deprotects positions 4 and 6 of GalNAc, and then sulfates to obtain chondroitin sulfate C sulfonated at position 6 of GalNAc. The method requires a two-step protection and deprotection strategy, the process is cumbersome, and the introduction of more reagents increases the difficulty of subsequent purification, which is not suitable for industrial large-scale production. Chinese patent CN103582653B also discloses a method for producing chondroitin sulfate sulfated at the 6th position of GalNAc, wherein the sodium salt of chondroitin is first converted into its free acid or pyridinium salt or methyl ester, and then a compound of repeating disaccharide units is obtained under acid catalysis, and then the hydroxyl groups at the 2' and 3' positions of the glucuronic acid unit are protected, and the orthoester functional groups are rearranged to obtain an ester derivative, which is then sulfated to remove the O-acyl group present in the compound obtained in the previous step, thereby obtaining a sodium salt of chondroitin sulfate in which all N-acetyl-D-galactosamine units in the same polysaccharide chain are randomly or monosulfated at the 4-position or the 6-position. Although this method can also achieve effective control of a high proportion of a single component, the sulfonation process of this method requires protection, deprotection and orthoester functional group rearrangement strategies, and the process is cumbersome and not suitable for industrial large-scale production. Summary of the invention

[0007] 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 a 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 position 6 in the obtained sodium chondroitin sulfate can reach more than 60%.

[0008] 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).

[0009] Specifically, the present invention provides a method for preparing sodium chondroitin sulfate, the preparation method comprising the following steps: Step 1) Sulfonation: Disperse the unsulfated sodium chondroitin with an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, terminate the reaction, centrifuge, and collect the precipitate; Step 2) post-treatment: decolorizing and drying the precipitate collected in step 1 to obtain sodium chondroitin sulfate; Wherein, the sulfonation reagent described in step 1 is preferably one or more selected from concentrated sulfuric acid, sulfur trioxide trimethylamine complex (Sulfurtrioxide trimethylamine complex, CAS: 3162-58-1), and sulfur trioxide pyridine complex (Pyridinesulfur trioxide, CAS: 26412-87-3).

[0010] Furthermore, the organic solvent described in step 1 is selected from one or more of N, N-dimethylformamide or formamide.

[0011] Further, the weight ratio of the unsulfated sodium chondroitin and the sulfonated reagent described in step 1 (i.e., W 软骨素钠 :W 磺化试剂，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 , 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 off). In some preferred embodiments, the weight ratio (g / g) of the unsulfated sodium chondroitin and the sulfonated reagent described in step 1 is about W 软骨素钠 :W 磺化试剂 =1:1-5.

[0012] It is understood that the sulfonated reagent in step 1 can be directly added to the organic solvent dispersion of unsulfated sodium chondroitin, or the sulfonated 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.

[0013] In some specific embodiments, the sulfonation reagent is usually dissolved in an appropriate amount of an organic solvent different from the above-mentioned dispersion of unsulfated sodium chondroitin, and then added to the organic solvent dispersion of unsulfated sodium chondroitin.

[0014] In some specific embodiments, the sulfonating agent described 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 described in step 1 is N, N-dimethylformamide and formamide; in other specific embodiments, the sulfonating agent described 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 described in step 1 is N, N-dimethylformamide or formamide.

[0015] Further, 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=1:4-50 (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, 1:2 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 thereof rounded off).

[0016] 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.

[0017] 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.

[0018] Further, the sulfonation reaction time in step 1 is about 0.5-6h (such as 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, or can be rounded to any non-integer time of the above time points); preferably, the sulfonation reaction time in step 1 is about 2-4h.

[0019] Furthermore, the sulfonation reaction in step 1 is terminated by using anhydrous ethanol.

[0020] Further, 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 rounded off) 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.

[0021] Furthermore, the centrifugal speed described in step 1 is about 4000-8000 rpm (such as 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm); preferably, the centrifugal speed described in step 1 is 5000-6000 rpm.

[0022] 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.

[0023] Furthermore, in step 2, activated carbon fiber membrane or hydrogen peroxide is used for decolorization.

[0024] In some preferred embodiments, step 2 uses activated carbon fiber membrane for decolorization; in other preferred embodiments, step 2 uses hydrogen peroxide for decolorization.

[0025] In some specific embodiments, before decolorization, an appropriate amount of purified water can be taken to dissolve the precipitate collected in step 1.

[0026] Furthermore, the drying in step 2 is freeze drying or spray drying.

[0027] Furthermore, the unsulfated sodium chondroitin in step 1 is commercially available unsulfated sodium chondroitin.

[0028] Furthermore, the unsulfated sodium chondroitin in step 1 is unsulfated sodium chondroitin obtained by microbial fermentation.

[0029] In some specific embodiments, the unsulfated sodium chondroitin 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 and other steps. The Escherichia coli DH001 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 26, 2024, with a deposit number of CGMCC No. 32081, a deposit address of Beijing, China, and a classification name of Escherichia coli.

[0030] In some specific embodiments, the unsulfated sodium chondroitin can be obtained by the following process: Fermentation: The fermentation liquid containing unsulfated sodium chondroitin is obtained by fermentation with Escherichia coli DH001; Ultrafiltration: centrifuge the fermentation broth, collect the supernatant, and ultrafilter to obtain the filtrate; Acid hydrolysis: add acid hydrolysis agent to the filtrate for acid hydrolysis, centrifuge and collect the supernatant; Purification: The supernatant after acid hydrolysis is purified by ion exchange to obtain unsulfated chondroitin sodium.

[0031] It is understandable that the above-mentioned fermentation process also involves the use of a necessary culture medium 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., which can be any option that can be provided by the prior art and should not be regarded as a limitation of the present invention.

[0032] In some preferred embodiments, the carbon source involved in the above fermentation process is glucose.

[0033] 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.

[0034] In other preferred embodiments, the fermentation process also involves the use of inorganic salts and trace elements necessary for the growth of the strain, 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.

[0035] 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.

[0036] 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.

[0037] Furthermore, the centrifugal speed described in the above ultrafiltration process is about 6000-20000 rpm (such as 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm, 15000 rpm, 16000 rpm, 17000 rpm, 18000 rpm, 19000 rpm, 20000 rpm); preferably, the centrifugal speed described in the above ultrafiltration process is 8000-10000 rpm.

[0038] Further, 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.

[0039] 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.

[0040] Furthermore, the acidolysis agent in the above acidolysis process is selected from one or more of sulfuric acid, hydrochloric acid and acetic acid.

[0041] Further, the acid hydrolysis pH of the above 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 acid hydrolysis process is about 1.0-2.0.

[0042] Furthermore, the acid hydrolysis time of the above acid hydrolysis process is about 2-10 h (such as 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h).

[0043] Furthermore, the acid hydrolysis temperature of the above 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, 70°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).

[0044] Furthermore, the ion exchange in the above purification process is anion resin exchange; preferably D280 anion resin exchange or D980 anion resin exchange.

[0045] 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 2M sodium chloride solution.

[0046] Furthermore, the above purification process may also include the steps of re-ultrafiltration, concentration, drying, etc., that is, after the eluate obtained by ion exchange is re-ultrafiltered (such as the ultrafiltration membrane pore size is 2-30 kDa), 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 the number, sequence, process parameters, etc. should not be regarded as limiting the present invention.

[0047] Further, the average molecular weight of the unsulfated sodium chondroitin obtained by fermentation of 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 interval).

[0048] 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 interval).

[0049] In some preferred embodiments, in the sodium chondroitin sulfate obtained by the method provided by the present invention, the proportion of the sodium chondroitin sulfate independently sulfonated at the GalNAc6 position is higher than 60% (such as 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%, 100%, or any non-integer ratio of the above ratios rounded off), that is, in the sodium chondroitin sulfate obtained by the method provided by the present invention, more than 60% (including 60%) of the components are sodium chondroitin sulfate independently sulfonated at the GalNAc6 position; in some more preferred embodiments, in the sodium chondroitin sulfate obtained by the method provided by the present invention, GalNAc The proportion of sodium chondroitin sulfate independently sulfonated at the 6th position ranges from 60% to 85%, that is, among the sodium chondroitin sulfate obtained by the method provided by the present invention, 60% to 85% (including 60% and 85%) of the components are sodium chondroitin sulfate independently sulfonated at the 6th position of GalNAc.

[0050] 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, and the sulfation method comprises the sulfonation (i.e., step 1) and post-treatment (i.e., step 2) processes described in any one of the above.

[0051] Specifically, the sulfation method of sodium chondroitin provided by the present invention comprises the following steps: Step 1) Sulfonation: Disperse sodium chondroitin with an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, terminate the reaction, centrifuge, and collect the precipitate; Step 2) post-treatment: decolorizing and drying the precipitate collected in step 1 to obtain sodium chondroitin sulfate; in: The sodium chondroitin is not sulfated and has an average molecular weight of about 5-30 kDa; The sulfonating agent is preferably one or more selected from concentrated sulfuric acid, sulfur trioxide trimethylamine complex (Sulfur trioxidetrimethylamine complex, CAS: 3162-58-1), and sulfur trioxide pyridine complex (Pyridine sulfurtrioxide, CAS: 26412-87-3); The organic solvent is selected from one or more of N, N-dimethylformamide or formamide.

[0052] Furthermore, the unsulfated sodium chondroitin is commercially available unsulfated sodium chondroitin.

[0053] Furthermore, the unsulfated sodium chondroitin is unsulfated sodium chondroitin obtained through microbial fermentation.

[0054] In some specific embodiments, the unsulfated sodium chondroitin 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 and other steps. The Escherichia coli DH001 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 26, 2024, with a deposit number of CGMCC No. 32081, a deposit address of Beijing, China, and a classification name of Escherichia coli.

[0055] 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 preparing sodium chondroitin sulfate sulfated at the 6-position of GalNAc; preferably, the sodium chondroitin sulfate sulfated at the 6-position of GalNAc refers to sodium chondroitin sulfate independently sulfonated at the 6-position of GalNAc.

[0056] The present invention also relates to a method for preparing sodium chondroitin sulfate with an independent sulfonation ratio of GalNAc at position 6 higher 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 with 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.

[0057] The present invention also provides a sodium chondroitin sulfate with an independent sulfonation ratio of GalNAc at position 6 higher than 60%, the average molecular weight of the sodium chondroitin sulfate is 5-30 kDa, and the sodium chondroitin sulfate is obtained by any of the above-mentioned methods for preparing sodium chondroitin sulfate or methods for sulfating sodium chondroitin; more preferably, the present invention also provides a sodium chondroitin sulfate with an independent sulfonation ratio of GalNAc at position 6 higher than 60%, the average molecular weight of the sodium chondroitin sulfate is 5-30 kDa, and the sodium chondroitin sulfate is obtained by any of the above-mentioned methods for preparing sodium chondroitin sulfate or methods for sulfating sodium chondroitin or methods for preparing sodium chondroitin sulfate with an independent sulfonation ratio of GalNAc at position 6 higher than 60%, or methods for preparing sodium chondroitin sulfate with an independent sulfonation ratio of GalNAc at position 6 of 60%-85%.

[0058] The present invention has the following technical advantages: The method for preparing sodium chondroitin sulfate or the method for sulfating sodium chondroitin provided by the present invention uses unsulfated sodium chondroitin as a starting material, and realizes the directional sulfonation of GalNAc 6-position 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 GalNAc 6-position is as high as more than 60% (60%-85%). Based on this characteristic, the sodium chondroitin sulfate obtained has a more uniform structure, and has higher safety and effectiveness while ensuring more reliable quality; its components are more suitable for human absorption and have high bioavailability.

[0059] The reaction conditions of the present invention are mild, high temperature and high pressure are not required, the operation steps are simple, the requirements for reaction equipment are low, it is green and environmentally friendly, a large amount of organic reagents are not required, the reaction is stable and reliable, the sulfonation rate of sodium chondroitin sulfate is high (up to 70% or more), the molecular weight of the product is controllable (preferably 5-30 kDa), the quality of the product between batches is stable, and it is more suitable for industrial promotion and use.

[0060] The method provided by the invention uses unsulfated sodium chondroitin derived from microbial fermentation as a raw material, which can effectively avoid cross contamination between organisms, and has a stable source of raw materials, uniform structure, and safe and stable quality.

[0061] 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, which can greatly reduce the dependence on the production site and specific equipment in the production process, which is beneficial to the control of production costs. The production process is simple and does not require the use of complex chemical reagents (the protecting group strategy requires about 15 organic reagents, and the present invention only requires 4 organic reagents). While reducing the cost of reagent use and recovery costs, it 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 applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The sugar chain structure result diagram of chondroitin sulfate mentioned in the background technology; Figure 2 This is a graph showing the molecular weight analysis results of unsulfated sodium chondroitin obtained in Example 1; Figure 3 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 2; Figure 4 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 3; Figure 5 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 4; Figure 6 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 5; Figure 7 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 6; Figure 8 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 7; Fig. 9 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 8; Fig.10 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Example 9; Fig.11 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Comparative Example 1; Fig.12 This is a graph showing the molecular weight analysis results of sodium chondroitin sulfate prepared in Comparative Example 2; Fig.13 This is a diagram showing the disaccharide content analysis results of the unsulfated sodium chondroitin obtained in Example 1; Fig.14 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 2; Fig.15This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 3; Fig.16 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 4; Fig.17 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 5; Fig.18 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 6; Fig.19 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 7; Fig. 20 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 8; Fig.21 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Example 9; Fig. 22 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Comparative Example 1; Fig.23 This is a diagram showing the disaccharide content analysis results of sodium chondroitin sulfate prepared in Comparative Example 2; Fig.24 The carbon NMR spectrum of sodium chondroitin sulfate prepared in Example 2; Fig.25 The carbon NMR spectrum of sodium chondroitin sulfate prepared in Example 3; Fig.26 This is the carbon nuclear magnetic resonance spectrum of the sodium chondroitin sulfate prepared in Example 4. DETAILED DESCRIPTION

[0063] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] Example 1 Preparation of Sodium Chondroitin (1) Fermentation: Escherichia coli DH001 was used as the production strain. After activation, it was inoculated at a rate of 1%-5% into a 30 L seed tank (containing medium 1, components of which are glucose 5-10 g / L; yeast extract powder 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) for fermentation and cultured for 8-10 h until OD 600When the pH reaches 2.5-5, the seeds are transferred into a 100 L fermentation tank (containing medium 2, components of which are glucose 10-15 g / L; yeast extract 2-5 g / L; ammonium sulfate 1.5-2.5 g / L; magnesium sulfate heptahydrate 4-8 g / L; potassium dihydrogen phosphate 5-10 g / L; ferrous sulfate heptahydrate 0.1-0.3 g / L; sodium chloride 5-10 g / L; defoamer 0.2-0.5 g / L) at a transfer rate of 5-10%, with initial stirring at 200-300 rpm, ventilation 1-4 vvm, dissolved oxygen correction 100%, and dissolved oxygen control at 15%-30% throughout the process. 25% (w / v) ammonia water is added to control the pH at 7.0-8.0, and 40%-60% mass fraction of glucose is added as the carbon source for feeding, and fermentation is carried out for 42-48 h until 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 8-10g / L; (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; (3) Acid hydrolysis: The pH of the ultrafiltration concentrate was adjusted to 1.5 with hydrochloric acid, and the reaction was carried out at 80°C for 6 h for acid hydrolysis. The precipitate was removed by centrifugation at 8000 rpm to obtain the supernatant; (4) Purification: Add D280 anion exchange resin to the obtained supernatant, stir and adsorb for 2 h, then elute with 2 mol / L sodium chloride solution, ultrafilter the eluate with a 10 kDa ultrafiltration membrane until the conductivity of the concentrate is stable, concentrate and dry the concentrate to obtain 410 g of sodium chondroitin (unsulfated sodium chondroitin).

[0065] Molecular weight analysis: According to the "Low Molecular Weight Heparin Molecular Weight Detection Method", the molecular weight of the sodium chondroitin obtained by the above method was determined using a TSKgel G3000SWXL column. The average molecular weight of the sodium chondroitin Mw was 11.1 kDa (see Figure 2 , retention time is 18.8min).

[0066] Example 2 Preparation of Sodium Chondroitin Sulfate 1 (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1 and disperse it 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 a 40 ℃ oil bath for later use; take 10g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂=1:1, dispersed with 50 mL of formamide, added to the above sodium chondroitin 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 the precipitate with 0.7 L of anhydrous ethanol; (2) Post-treatment: The washed precipitate was re-dissolved 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.

[0067] 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).

[0068] Example 3 Preparation of Sodium Chondroitin Sulfate 2 (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1 and disperse it 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 a 40 ℃ oil bath for later use; take 10g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:1, dispersed with 50 mL N, N-dimethylformamide, 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 the precipitate with 0.7 L of anhydrous ethanol; (2) Post-treatment: The washed precipitate was re-dissolved 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 12.1 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Example 3 was determined to be 13.7 kDa (see Figure 4 , retention time is 17.6min).

[0069] Example 4 Preparation of Sodium Chondroitin Sulfate 3 (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1 and disperse it 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 a 40 ℃ oil bath for later use; take 10g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂=1:1, dispersed with 50 mL of formamide, added to the above sodium chondroitin 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 the precipitate with 0.7 L of anhydrous ethanol; (2) Post-treatment: The washed precipitate was re-dissolved 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 11.2 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Example 4 was determined to be 12.1 kDa (see Figure 5 , retention time is 18.5min).

[0070] Example 5 Preparation of Sodium Chondroitin Sulfate (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1 and disperse it 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 a 40 ℃ oil bath for later use; take 10g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:1, dispersed with 50 mL N, N-dimethylformamide, 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 the precipitate with 0.7 L of anhydrous ethanol; (2) Post-treatment: The washed precipitate was re-dissolved 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 11.3 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Example 5 was determined to be 12.3 kDa (see Figure 6 , retention time is 18.3min).

[0071] Example 6 Preparation of Sodium Chondroitin Sulfate (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1 and disperse it 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 a 40 ℃ oil bath for later use; take 50g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:5, dispersed with 50 mL of formamide, added to the above sodium chondroitin 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 the precipitate with 0.7 L of anhydrous ethanol; (2) Post-treatment: The washed precipitate was re-dissolved 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 11.5 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Example 6 was determined to be 13.2 kDa (see Figure 7 , retention time is 17.6min).

[0072] Example 7 Preparation of Sodium Chondroitin Sulfate (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1 and disperse it 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 a 40 ℃ oil bath for later use; 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 a 50 ℃ oil bath for 6 h, added 0.7 L of anhydrous ethanol to terminate the reaction, centrifuged at 6000 rpm, collected the precipitate, and washed the precipitate with 0.7 L of anhydrous ethanol.

[0073] (2) Post-treatment: The washed precipitate was re-dissolved with 100 mL of purified water, and decolorized by repeated filtration with 6 layers of activated carbon fiber membrane for 5 times. 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).

[0074] Example 8 Preparation of Sodium Chondroitin Sulfate (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1 and disperse it 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 a 40 ℃ oil bath for later use; take 10g of concentrated sulfuric 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 30 °C for 4 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.

[0075] (2) Post-treatment: The washed precipitate was re-dissolved with 100 mL of purified water, and decolorized by repeated filtration with 6 layers of activated carbon fiber membrane for 5 times. The decolorized liquid was freeze-dried to obtain 11.2 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Example 8 was 12.2 kDa (see Fig. 9 , retention time is 17.9min).

[0076] Example 9 Preparation of Sodium Chondroitin Sulfate 8 (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1 and disperse it 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 a 40 ℃ oil bath for later use; 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 a 40 ℃ oil bath for 2 h, added 0.7 L of anhydrous ethanol to terminate the reaction, centrifuged at 6000 rpm, collected the precipitate, and washed the precipitate with 0.7 L of anhydrous ethanol.

[0077] (2) Post-treatment: The washed precipitate was re-dissolved with 100 mL of purified water, and decolorized by repeated filtration with 6 layers of activated carbon fiber membrane for 5 times. The decolorized liquid was freeze-dried to obtain 10.9 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Example 9 was determined to be 11.9 kDa (see Fig.10 , retention time is 18.3min).

[0078] Comparative Example 1 (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 a 40 ℃ oil bath for later use; take 10 g chlorosulfonic acid, W 软骨素钠 :W 磺化试剂 =1:1, dispersed with 50 mL of formamide, added to the above sodium chondroitin 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 the precipitate with 0.7 L of anhydrous ethanol; (2) Post-treatment: The washed precipitate was re-dissolved 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 11.4 g of sodium chondroitin sulfate. The average molecular weight Mw of sodium chondroitin sulfate obtained in Comparative Example 1 was 12.5 kDa (see Fig.11 , retention time is 18.0min).

[0079] Comparative Example 2 (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 a 40 ℃ oil bath for later use; 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 sodium chondroitin 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 the precipitate with 0.7 L of anhydrous ethanol; (2) Post-treatment: The washed precipitate was re-dissolved 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 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 Fig.12 , retention time is 18.7min).

[0080] Results Analysis 1. Determination and analysis of sodium chondroitin sulfate prepared in Examples 2 to 9 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→), and HPLC analysis was performed using a HypersilSAX 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.

[0081] 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. Fig.13 The disaccharide content analysis results of sodium chondroitin obtained in Example 1 are as follows: Fig.14 This is the disaccharide distribution analysis result of the sodium chondroitin sulfate prepared in Example 2. Fig.15 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Example 3. Fig.16 The disaccharide distribution analysis results of sodium chondroitin sulfate prepared in Example 4; Fig.17 The disaccharide distribution analysis results of sodium chondroitin sulfate prepared in Example 5; Fig.18 The disaccharide distribution analysis results of sodium chondroitin sulfate prepared in Example 6; Fig.19 The disaccharide distribution analysis results of sodium chondroitin sulfate prepared in Example 7; Fig. 20 The disaccharide distribution analysis results of sodium chondroitin sulfate prepared in Example 8; Fig.21 The disaccharide distribution analysis results of sodium chondroitin sulfate prepared in Example 9; Fig. 22 The disaccharide distribution analysis results of sodium chondroitin sulfate prepared in Comparative Example 1; Fig.23 This is the disaccharide distribution analysis result of sodium chondroitin sulfate prepared in Comparative Example 2.

[0082] Table 1 Sulfonation of chondroitin sulfate

[0083] The results show that the chondroitin sulfate obtained in Examples 2 to 9 can be recognized and hydrolyzed by chondroitinase ABC. From the perspective of the hydrolysis products, the sulfonation reaction of the method provided by the present invention is relatively thorough, and only no more than 30% of the chondroitin (i.e., OS-CS) in each preparation example is not sulfonated, and the sulfonation rate can be as high as more than 70%, especially 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 1.34%. % is only 3.44%), and most of them are sulfonated at the 6-position of GalNAc. For example, in Examples 2 and 3, most of the sulfonated products are distributed in chondroitin sulfate independently sulfonated at the 6-position (i.e., 6S-CS, disaccharide structure is 4GlcAβ1,3GalNAc6Sβ1), chondroitin sulfate sulfonated at the 2,6-position (i.e., 2,6S-CS, disaccharide structure is 4GlcA2Sβ1,3GalNAc6Sβ1), and chondroitin sulfate sulfonated at the 4,6-position (i.e., 4,6S-CS, disaccharide structure is 4GlcAβ1,3GalNAc4,6Sβ1), and only less than 1% of chondroitin sulfate sulfonated at the 2,4-position (i.e., 2,4S-CS, disaccharide structure is 4GlcA2Sβ1,3GalNAc4Sβ1); in Examples 4-9, all products are sulfonated at the 6-position of GalNAc.

[0084] The sulfonated products are evenly distributed, and the proportion of products that are independently sulfonated only at the 6th position of GalNAc (i.e., 6S-CS) is as high as more than 60%, such as the proportion of the 6th position independently sulfonated product of Example 2 is 80.57%, the proportion of the 6th position independently sulfonated product of Example 3 is 80.69%, the proportion of the 6th position independently sulfonated product of Example 4 is 65.41%, the proportion of the 6th position independently sulfonated product of Example 5 is 70.67%, the proportion of the 6th position independently sulfonated product of Example 6 is 73.15%, the proportion of the 6th position independently sulfonated product of Example 7 is 76.06%, the proportion of the 6th position independently sulfonated product of Example 8 is 70.47%, and the proportion of the 6th position independently sulfonated product of Example 9 is 62.95%, indicating 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 6th 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.

[0085] 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. As can be seen from Comparative Example 1, compared with Example 3, after changing the type of sulfonating agent, the proportion of independent sulfonated products at position 6 is reduced to 23.50%, which indicates 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 sodium chondroitin. As can be seen from Comparative Example 2, compared with Example 3, the amount of sulfonating agent is reduced, and the sulfonation rate is reduced to 50.52%, which indicates 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 illustrates that the preparation method provided by the present invention can control the proportion of independent sulfonated products at position 6 of GalNAc in the final product to more than 60%.

[0086] 2. Analysis of Sodium Chondroitin Sulfate by C NMR Spectroscopy NMR carbon spectrum detection instrument model: AV500 (CAq08); The detection conditions are as follows: magnetic field strength (FS): 125MHz; number of scans (NS): 12288 times; temperature (TE): about 40℃ (313.2K); sampling time (AQ): 1.1S; delay time (D1): 2.0S; sampling center (O1P): 100.0ppm; decoupling center (O2P): 4.0ppm; spectral width (SW): 220~240ppm; pulse (PUL): 30°; exponential line width window function (LB): 1.0Hz; NMR tube diameter: 5mm; acquisition method: D_GLP_E.glp.

[0087] The analysis results can be found in Figures 24 to 26 , Figures 24 to 26 The 49.5 ppm peak is the methanol localization peak. The carbon spectrum contains 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 the 176.2 ppm and 23.8 ppm peaks are the characteristic signal peaks of carbon No. 7 and carbon No. 8 in GalNAc, respectively.

[0088] 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.

[0089] In addition, it can be seen from Examples 2-9 (see Figure 3-10 ), the average molecular weight Mw of sodium chondroitin sulfate obtained by sulfonating sodium chondroitin via the method provided by the present invention is not much different, which shows that the sulfonation method provided by the present invention is mild in reaction. Although it undergoes a chemical reaction process, the process has minimal damage to the disaccharide skeleton structure. From the average molecular weight Mw of the obtained sodium chondroitin sulfate, it can be seen that the average molecular weight Mw distribution of sodium chondroitin sulfate obtained by the sulfonation method provided by the present invention is stable between batches, which shows that the sulfonation method provided by the present invention is stable in operation between batches and has little dependence on the operating environment, operating means, compound dosage and proportion, etc.

[0090] In summary, the present invention uses unsulfated sodium chondroitin as a 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 a stable molecular weight and a high ratio.

[0091] The above embodiments 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 method for preparing sodium chondroitin sulfate, characterized in that: The preparation method comprises the following steps: Step 1) Sulfonation: Disperse the unsulfated sodium chondroitin with an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, terminate the reaction, centrifuge, and collect the precipitate; Step 2) post-treatment: decolorizing and drying the precipitate collected in step 1 to obtain sodium chondroitin sulfate; in: The average molecular weight of the unsulfated sodium chondroitin described in step 1 is 5-30 kDa; The sulfonating agent in step 1 is selected from one or more of concentrated sulfuric acid, sulfur trioxide trimethylamine complex, and sulfur trioxide pyridine complex; The organic solvent in step 1 is selected from one or more of N, N-dimethylformamide or formamide; The sodium chondroitin sulfate obtained in step 2 has more than 60% of the sodium chondroitin sulfate independently sulfonated at the 6-position of GalNAc, and the average molecular weight of the sodium chondroitin sulfate obtained in step 2 is 5-30 kDa.

2. The preparation method according to claim 1, characterized in that: The weight ratio of the unsulfated sodium chondroitin and the sulfonated reagent in step 1 is W 软骨素钠 :W 磺化试剂 ≤1:0.

5.

3. The preparation method according to claim 1, 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 preparation method according to claim 1, characterized in that: The sulfonation reaction temperature in step 1 is 20-50° C.; the sulfonation reaction time is 0.5-6 h.

5. The preparation method according to claim 1, characterized in that: The unsulfated sodium chondroitin described in step 1 is unsulfated sodium chondroitin obtained through microbial fermentation.

6. The preparation method according to claim 1, characterized in that The unsulfated chondroitin sodium described in step 1 is obtained by fermentation of Escherichia coli DH001, and the Escherichia coli Escherichiacoli DH001 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on September 26, 2024, with a deposit number of CGMCC No.32081, a deposit address of Beijing, China, and a classification name of Escherichia coli Escherichiacoli.

7. A method for sulfating sodium chondroitin, characterized in that: The sodium chondroitin is not sulfated and has an average molecular weight of 5-30 kDa. The sulfation method comprises the sulfonation and post-treatment process according to any one of claims 1-6.

8. Use of the preparation method according to any one of claims 1 to 6 or the sulfation method of sodium chondroitin according to claim 7 in the preparation of sodium chondroitin sulfate sulfated at the 6-position of GalNAc, wherein the sodium chondroitin sulfate sulfated at the 6-position of GalNAc is sodium chondroitin sulfate independently sulfonated at the 6-position of GalNAc.

9. A method for preparing sodium chondroitin sulfate with an independent sulfonation ratio of GalNAc 6 higher than 60%, characterized in that: The average molecular weight of the sodium chondroitin sulfate is 5-30 kDa, and the method comprises the sulfonation and post-treatment process according to any one of claims 1-6.

10. A sodium chondroitin sulfate having an independent sulfonation ratio of GalNAc at position 6 higher than 60%, characterized in that: The average molecular weight of the sodium chondroitin sulfate is 5-30 kDa, and the sodium chondroitin sulfate is obtained by the preparation method described in claims 1-6 or the sulfation method of sodium chondroitin according to claim 7 or the preparation method of sodium chondroitin sulfate with an independent sulfonation ratio of GalNAc at position 6 higher than 60% according to claim 9.

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