A preparation method and application of sodium chondroitin sulfate

Abstract

The present invention discloses a preparation method and application of sodium chondroitin sulfate, belonging to the technical field of chondroitin sulfate. The preparation of sodium chondroitin sulfate disclosed in the present invention includes: dispersing unsulfated sodium chondroitin in an organic solvent, adding a sulfonating reagent for sulfonation reaction, terminating the reaction, centrifuging, and collecting the precipitate; then decolorizing and drying the collected precipitate to obtain sodium chondroitin sulfate. The average molecular weight of the final product obtained by this method is controllable, the overall sulfonation rate at the 6th position is as high as over 90%, the independent sulfonation rate at the 6th position is as high as 60% - 85%, and the product quality is stable among batches. Sodium chondroitin sulfate based on this characteristic is more suitable for human absorption, has high bioavailability, and has higher safety and effectiveness; moreover, this method can be achieved without going through a complex extraction and purification process, has rich sources, uniform structure, stable quality, mild reaction, and reasonable and controllable production cycle, and is suitable for large-scale commercial production and application.

Description

Technical Field

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

[0002] Chondroitin sulfate (ChS / CS) is a naturally occurring bioactive macromolecule that is almost widely distributed in 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 different numbers and positions of sulfate groups, such as the 2- or 3-position of G1cA or IdoA and the 4- or 6-position of Ga1NAc. It has now been confirmed that it has a profound impact on many physiological processes such as inflammation, tumor progression and metastasis, vascular remodeling, and antioxidant.

[0003] Currently, the commercial industrial production of chondroitin sulfate uses animal tissues as raw materials (such as cattle, pigs, chickens, chondrichthyans, sharks, rays, bony fish, etc.). Although the raw material sources are sufficient, it also brings safety and quality problems. In fact, the polysaccharide chains of ChS from different sources may have different numbers of disaccharides and different positions of sulfate groups. Each ChS is not completely composed of an ideal disaccharide repeating unit, but is composed of multiple disaccharide structures with different contents. Even if the known ChS samples are mainly composed of two different percentages of disaccharide units of 4-sulfated and 6-sulfated N-acetylgalactosamine, disaccharides with different percentages of the number and position of sulfate groups can also be found in its polysaccharide chain. Therefore, chondroitin sulfates from different sources have certain heterogeneity in terms of relative molecular mass, charge density, chemical properties, etc. This not only causes difficulties in the research of the physicochemical properties and structural characterization of ChS, but also brings adverse factors to its quality control.

[0004] In addition, factors such as raw material sources and structural feature differences (such as the composition, ratio, and position of sulfate groups of disaccharides, etc.) will also affect the overall bioavailability and pharmacokinetic activity of chondroitin sulfate. Chondroitin sulfates with different structures have been confirmed to have different properties. For example, 4-sulfated ChS 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 derived from sharks has been proven to have anti-angiogenic and anti-tumor activities, promote neurite outgrowth activity in animals and humans, and have significant effects in the treatment of osteoarthritis, rheumatoid arthritis, progressive systemic sclerosis, and neurovascular glaucoma. It has now been widely used. Functional group identification shows that chondroitin isolated from shark cartilage is mainly chondroitin sulfate C, that is, its main component is GalNAc 6-monosulfated chondroitin, which makes its structural characteristics significantly different from chondroitin sulfates from other sources. For example, ChS from sources such as cattle, pigs, chickens, and whales is mainly chondroitin sulfate A, and its main component is GalNAc 4-monosulfated chondroitin; ChS from squid is mainly chondroitin sulfate E, and its main component is GalNAc 4,6-disulfated chondroitin. This may be the main reason for the functional differences from chondroitin sulfates from other sources. However, shark resources are limited, and the supply of chondroitin sulfate from sharks is far lower than the market demand. Therefore, shark-like chondroitin with structural characteristics similar to natural shark chondroitin has become a current research and development hotspot.

[0006] European Patent EP1304338B1 discloses a method for obtaining GalNAc 6-sulfated chondroitin sulfate by microbial fermentation. This method uses Escherichia coli K4 polysaccharide as the starting material, obtains an acidic polysaccharide after acid treatment and purification, and then protects the 4- and 6-positions of GalNAc and the 2- and 3-positions of GlcA by dibenzylation and O-acetylation respectively. The dibenzyl group is separated by treatment with acetic acid, the 4- and 6-positions of GalNAc are deprotected, and then sulfated to obtain chondroitin sulfate C with sulfonic acid at the 6-position of GalNAc. This 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 large-scale industrial production. Chinese Patent CN103582653B also discloses a method for producing GalNAc 6-sulfated chondroitin sulfate. This method first converts chondroitin sodium salt into its free acid or pyridine salt or methyl ester, then obtains a compound of repeating disaccharide units under acid catalysis, and then protects the 2' and 3' hydroxyl groups of the glucuronic acid unit, and the orthoester functional group rearranges to obtain an ester derivative, and then sulfates, and removes the O-acyl groups present in the compound obtained in the previous step, thereby obtaining sodium chondroitin sulfate in which all N-acetyl-D-galactosamine units in the same polysaccharide chain are randomly or monosulfated at the 4- or 6-position. Although this method can also effectively control a high proportion of a single component, the sulfonation process of this method requires protection, deprotection, and orthoester functional group rearrangement strategies, the process is cumbersome, and it is not suitable for large-scale industrial production. Summary of the Invention

[0007] To solve the above problems, the present invention provides a novel method for preparing sodium chondroitin sulfate. This method uses unsulfated sodium chondroitin as the starting material and adopts a sulfonation strategy without protecting groups to achieve the sulfonation of chondroitin through one-step sulfonation. The proportion of sodium chondroitin sulfate with independent sulfonation at the 6-position of GalNAc in the obtained sodium chondroitin sulfate can reach more than 60%.

[0008] The independent sulfonation at the 6-position of GalNAc described in the present invention refers to the situation where sulfonation occurs only at the 6-position of GalNAc in sodium chondroitin sulfate (i.e., the disaccharide structure is 4GlcAβ1,3GalNAc6Sβ1).

[0009] Specifically, the present invention provides a method for preparing sodium chondroitin sulfate, and the preparation method includes the following steps:

[0010] Step 1) Sulfonation: Disperse unsulfated sodium chondroitin in an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, then terminate the reaction, centrifuge, and collect the precipitate;

[0011] Step 2) Post-treatment: Decolorize and dry the precipitate collected in Step 1 to obtain sodium chondroitin sulfate;

[0012] Among them, the sulfonation reagent described in Step 1 is preferably selected from one or more of concentrated sulfuric acid, sulfur trioxide trimethylamine complex (CAS: 3162 - 58 - 1), and pyridine sulfur trioxide complex (CAS: 26412 - 87 - 3).

[0013] Furthermore, the organic solvent described in Step 1 is selected from one or more of N, N - dimethylformamide or formamide.

[0014] Furthermore, the weight ratio of the unsulfated sodium chondroitin and the sulfonation reagent 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 that can be rounded to the above ratios). In some preferred embodiments, the weight ratio (g / g) of the unsulfated chondroitin sodium and the sulfonating reagent in step 1 is about W 软骨素钠 :W 磺化试剂 = 1:1 - 5.

[0015] It can be understood that the sulfonating reagent in step 1 can be directly added to the organic solvent dispersion of unsulfated chondroitin sodium, or the sulfonating reagent can be dissolved in the same or different organic solvents as those used to disperse the unsulfated chondroitin sodium, and then added to the organic solvent dispersion of unsulfated chondroitin sodium.

[0016] In some specific embodiments, the sulfonating reagent is usually dissolved in an appropriate amount of an organic solvent different from that used to disperse the unsulfated chondroitin sodium, and then added to the organic solvent dispersion of unsulfated chondroitin sodium.

[0017] In some specific embodiments, the sulfonating reagent in step 1 is selected from one or more of concentrated sulfuric acid, trimethylamine sulfur trioxide complex, and pyridine sulfur trioxide complex, and the organic solvent in step 1 is N, N-dimethylformamide and formamide; in some other specific embodiments, the sulfonating reagent in step 1 is preferably selected from one or more of concentrated sulfuric acid, trimethylamine sulfur trioxide complex, and pyridine sulfur trioxide complex, and the organic solvent in step 1 is N, N-dimethylformamide or formamide.

[0018] Furthermore, the weight-to-volume ratio (g / ml) of the unsulfated chondroitin sodium and the organic solvent in step 1 is about W 软骨素钠 :V 有机溶剂= 1:4 - 50 (such as 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, 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 to the above ratios).

[0019] In some preferred embodiments, the weight - volume ratio (g / ml) of the unsulfated sodium chondroitin and the organic solvent described in step 1 is about W 软骨素钠 :V 有机溶剂 = 1:8 - 20.

[0020] Furthermore, the sulfonation reaction temperature described 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.

[0021] Furthermore, the sulfonation reaction time described 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 that can be rounded to the above time points); preferably, the sulfonation reaction time described in step 1 is about 2 - 4 h.

[0022] Furthermore, the sulfonation reaction in step 1 is terminated with absolute ethanol.

[0023] Furthermore, in the above 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 that can be rounded to the above multiples) of absolute ethanol can be added to the entire sulfonation reaction system for termination reaction; preferably, in the above termination reaction, 4 - 8 times the volume of absolute ethanol can be added to the entire sulfonation reaction system for termination reaction.

[0024] Further, the centrifugation speed in step 1 is about 4000 - 8000 rpm (such as 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm); preferably, the centrifugation speed in step 1 is 5000 - 6000 rpm.

[0025] In order to better remove the solvent residue in the precipitate, in some specific embodiments, anhydrous ethanol is usually used to wash the precipitate collected by centrifugation one or more times.

[0026] Further, in step 2, decolorization is carried out using activated carbon fiber membrane or hydrogen peroxide.

[0027] In some preferred embodiments, in step 2, decolorization is carried out using an activated carbon fiber membrane; in some other preferred embodiments, in step 2, decolorization is carried out using hydrogen peroxide.

[0028] In some specific embodiments, before decolorization, an appropriate amount of pure water can be taken to dissolve the precipitate collected in step 1.

[0029] Further, the drying in step 2 is freeze-drying or spray-drying.

[0030] Further, the sodium chondroitin without sulfation described in step 1 is commercially available sodium chondroitin without sulfation.

[0031] Further, the sodium chondroitin without sulfation described in step 1 is sodium chondroitin without sulfation obtained by microbial fermentation.

[0032] In some specific embodiments, the sodium chondroitin without sulfation described in step 1 is obtained by extracting the fermentation broth obtained after fermentation by Escherichia coli DH001; further, the extraction includes necessary steps such as ultrafiltration, acid hydrolysis, purification, etc. The Escherichia coli DH001 was deposited on September 26, 2024 at the General Microbiology Center of the China Microbial Culture Collection Center, with the deposit number: CGMCC No. 32081, and the deposit address is Beijing, China, and the taxonomic name is Escherichia coli.

[0033] In some specific embodiments, the obtaining of the sodium chondroitin without sulfation may include the following process:

[0034] Fermentation: Ferment using Escherichia coli DH001 to obtain a fermentation broth containing sodium chondroitin without sulfation;

[0035] Ultrafiltration: Centrifuge the fermentation broth, collect the supernatant, and perform ultrafiltration to obtain the filtrate;

[0036] Acid hydrolysis: Add an acid hydrolysis agent to the filtrate for acid hydrolysis, centrifuge, and collect the supernatant;

[0037] Purification: Purify the acid-hydrolyzed supernatant by ion exchange to obtain sodium chondroitin without sulfation.

[0038] It can be understood that the above fermentation process also involves the use of a necessary culture medium for maintaining the growth, metabolism, and fermentation of the strain. The described culture medium contains a carbon source, a nitrogen source, inorganic salts and trace elements necessary for growth, etc., and can be any selection provided by the prior art, and should not be regarded as a limitation to the present invention.

[0039] In some preferred embodiments, the carbon source involved in the above fermentation process is glucose.

[0040] In some 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 can be yeast extract powder, and / or the inorganic nitrogen source can be ammonium sulfate; more preferably, the nitrogen source involved in the above fermentation process is yeast extract powder and ammonium sulfate.

[0041] In some other preferred embodiments, the above 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.

[0042] 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.

[0043] 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 the carbon source, yeast extract powder and ammonium sulfate as the nitrogen source, and inorganic salts and trace elements necessary for growth.

[0044] Further, the centrifugal speed 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 in the above ultrafiltration process is 8000 - 10000 rpm.

[0045] Further, the membrane pore size of the ultrafiltration in the above ultrafiltration process 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); preferably, the ultrafiltration membrane pore size in the above ultrafiltration process is 10 - 20 kDa.

[0046] Further, the above 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.

[0047] Furthermore, the acid hydrolysis agent in the above acid hydrolysis process is selected from one or more of sulfuric acid, hydrochloric acid, and acetic acid.

[0048] Further, the acid hydrolysis pH in 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 in the above acid hydrolysis process is about 1.0 - 2.0.

[0049] Further, the acid hydrolysis time in 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).

[0050] Further, the acid hydrolysis temperature in 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).

[0051] Further, the ion exchange in the above purification process is anion resin exchange; preferably D280 anion resin exchange or D980 anion resin exchange.

[0052] Furthermore, the eluent for the ion exchange involved in the above purification process is a sodium chloride solution with a concentration of 1 - 3 mol / L (such as 1 mol / L, 2 mol / L, 3 mol / L); in some specific embodiments, the eluent for the ion exchange involved in the above purification process is a 2M sodium chloride solution.

[0053] Furthermore, the above purification process may further include steps such as ultrafiltration, concentration, and drying, that is, after ultrafiltrating (such as the membrane pore size of ultrafiltration is 2 - 30 kDa) the eluent obtained through ion exchange, the collected filtrate is concentrated and dried to obtain chondroitin sulfate sodium without sulfation. These processes are only for better or more optimal realization of the extraction of chondroitin sulfate sodium without sulfation, and the number of times, sequence, process parameters, etc. should not be regarded as a limitation to the present invention.

[0054] Further, the average molecular weight of the chondroitin sulfate sodium without sulfation 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 chondroitin sulfate sodium without sulfation obtained can be any integer or non-integer kDa value within the interval).

[0055] In some preferred embodiments, the average molecular weight of sodium chondroitin sulfate obtained by using 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 values, that is, the average molecular weight of sodium chondroitin sulfate obtained by using the method provided by the present invention can be any integer or non-integer kDa value within the range).

[0056] In some preferred embodiments, in the sodium chondroitin sulfate obtained by using the method provided by the present invention, the proportion of sodium chondroitin sulfate with independent sulfonation at the 6-position of GalNAc 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 proportion that can be rounded to the above proportion), 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 with independent sulfonation at the 6-position of GalNAc; in some more preferred embodiments, in the sodium chondroitin sulfate obtained by using the method provided by the present invention, the proportion range of sodium chondroitin sulfate with independent sulfonation at the 6-position of GalNAc is 60% - 85%, that is, in the sodium chondroitin sulfate obtained by the method provided by the present invention, 60% - 85% of the components (including 60% and 85%) are sodium chondroitin sulfate with independent sulfonation at the 6-position of GalNAc.

[0057] The present invention also provides a sulfation method for sodium chondroitin, wherein the sodium chondroitin is unsulfated and its average molecular weight is about 5 - 30 kDa, and the sulfation method includes the sulfonation (i.e., step 1) and post-treatment (i.e., step 2) processes described in any one of the above.

[0058] Specifically, the sulfation method for sodium chondroitin provided by the present invention includes the following steps:

[0059] Step 1) Sulfonation: Disperse sodium chondroitin in an organic solvent, add a sulfonation reagent to carry out a sulfonation reaction, terminate the reaction, centrifuge, and collect the precipitate;

[0060] Step 2) Post-treatment: Decolorize and dry the precipitate collected in Step 1 to obtain sodium chondroitin sulfate;

[0061] Wherein:

[0062] The chondroitin sodium is unsulfated and has an average molecular weight of about 5 - 30 kDa;

[0063] The sulfonating reagent is preferably selected from one or more of concentrated sulfuric acid, sulfur trioxide trimethylamine complex (CAS: 3162 - 58 - 1), and pyridine sulfur trioxide complex (CAS: 26412 - 87 - 3);

[0064] The organic solvent is selected from one or more of N, N - dimethylformamide or formamide.

[0065] Furthermore, the unsulfated chondroitin sodium is commercially available unsulfated chondroitin sodium.

[0066] Furthermore, the unsulfated chondroitin sodium is unsulfated chondroitin sodium obtained by microbial fermentation.

[0067] In some specific embodiments, the unsulfated chondroitin sodium described in Step 1 is obtained by extracting the fermentation broth obtained after fermentation by Escherichia coli DH001; furthermore, the extraction includes necessary steps such as ultrafiltration, acid hydrolysis, and purification. The Escherichia coli DH001 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 26, 2024, with the deposit number: CGMCC No. 32081, the deposit address is Beijing, China, and the taxonomic name is Escherichia coli.

[0068] The present invention also relates to the use of the method for preparing sodium chondroitin sulfate described in any one of the above or the method for sulfating chondroitin sodium described in any one of the above in the preparation of sodium chondroitin sulfate with sulfation at the 6 - position of GalNAc; preferably, the sodium chondroitin sulfate with sulfation at the 6 - position of GalNAc refers to sodium chondroitin sulfate with independent sulfonation at the 6 - position of GalNAc.

[0069] The present invention also relates to a method for preparing sodium chondroitin sulfate with a GalNAc 6 - independent sulfonation ratio 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; more preferably, the present invention also relates to a method for preparing sodium chondroitin sulfate with a GalNAc 6 - independent sulfonation ratio 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.

[0070] The present invention also provides a sodium chondroitin sulfate with a GalNAc 6 - independent sulfonation ratio higher than 60%, wherein the average molecular weight of the sodium chondroitin sulfate is 5 - 30 kDa, and the sodium chondroitin sulfate is obtained by using the method for preparing sodium chondroitin sulfate or the sulfation method of chondroitin sodium described in any one of the above; more preferably, the present invention also provides a sodium chondroitin sulfate with a GalNAc 6 - independent sulfonation ratio of 60% - 85%, wherein the average molecular weight of the sodium chondroitin sulfate is 5 - 30 kDa, and the sodium chondroitin sulfate is obtained by using the method for preparing sodium chondroitin sulfate or the sulfation method of chondroitin sodium or the method for preparing sodium chondroitin sulfate with a GalNAc 6 - independent sulfonation ratio higher than 60% or the method for preparing sodium chondroitin sulfate with a GalNAc 6 - independent sulfonation ratio of 60% - 85% described in any one of the above.

[0071] The present invention has the following technical advantages:

[0072] The method for preparing sodium chondroitin sulfate or the sulfation method of chondroitin sodium provided by the present invention uses unsulfated chondroitin sodium as the 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 using the method provided by the present invention, the proportion of chondroitin sulfate with independent sulfonation at GalNAc 6 - position is as high as more than 60% (60% - 85%). Based on this characteristic, the structure of the obtained sodium chondroitin sulfate is more uniform, and it has higher safety and effectiveness on the premise of ensuring more reliable quality; its components are more suitable for human absorption and have high bioavailability.

[0073] The reaction conditions of the present invention are mild, without high temperature and high pressure, the operation steps are simple, the requirements for reaction equipment are low, it is green and environmentally friendly, without a large amount of organic reagents, the reaction is stable and reliable, the sulfonation rate of sodium chondroitin sulfate is high (up to more than 70%), 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.

[0074] The method provided by the present invention uses unsulfated sodium chondroitin derived from microbial fermentation as the raw material, which can effectively avoid cross-contamination between organisms, and the raw material source is stable, the structure is uniform, and the quality is safe and stable.

[0075] 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 the final product can be sulfonated in a targeted position-specific manner without complex reaction steps (such as protection and deprotection strategies, functional group rearrangement strategies, etc.). It can greatly reduce the dependence on the production area and specific equipment during the production process, is conducive to cost control of production, and the production process is simple and does not require the use of complex chemical reagents (about 15 organic reagents are required for the protection group strategy, and only 4 organic reagents are required in the present invention). While reducing the reagent usage cost and recovery cost, it reduces the possibility of environmental pollution and reduces the possible chemical composition residues in the final product, further ensuring the safety of clinical applications. Description of the Drawings

[0076] Figure 1 It is a diagram of the sugar chain structure of chondroitin sulfate mentioned in the background art;

[0077] Figure 2 It is a diagram of the molecular weight analysis result of unsulfated sodium chondroitin obtained in Example 1;

[0078] Figure 3 It is a diagram of the molecular weight analysis result of sodium chondroitin sulfate prepared in Example 2;

[0079] Figure 4 It is a diagram of the molecular weight analysis result of sodium chondroitin sulfate prepared in Example 3;

[0080] Figure 5 It is a diagram of the molecular weight analysis result of sodium chondroitin sulfate prepared in Example 4;

[0081] Figure 6 It is a diagram of the molecular weight analysis result of sodium chondroitin sulfate prepared in Example 5;

[0082] Figure 7 It is a diagram of the molecular weight analysis result of sodium chondroitin sulfate prepared in Example 6;

[0083] Figure 8 It is a diagram of the molecular weight analysis result of sodium chondroitin sulfate prepared in Example 7;

[0084] Figure 9 It is a diagram of the molecular weight analysis result of sodium chondroitin sulfate prepared in Example 8;

[0085] Figure 10 It is a diagram of the molecular weight analysis result of sodium chondroitin sulfate prepared in Example 9;

[0086] Figure 11 The molecular weight analysis result chart of sodium chondroitin sulfate prepared in Comparative Example 1;

[0087] Figure 12 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Comparative Example 2;

[0088] Figure 13 The disaccharide content analysis result chart of non-sulfated sodium chondroitin obtained in Example 1;

[0089] Figure 14 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Example 2;

[0090] Figure 15 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Example 3;

[0091] Figure 16 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Example 4;

[0092] Figure 17 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Example 5;

[0093] Figure 18 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Example 6;

[0094] Figure 19 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Example 7;

[0095] Figure 20 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Example 8;

[0096] Figure 21 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Example 9;

[0097] Figure 22 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Comparative Example 1;

[0098] Figure 23 The disaccharide content analysis result chart of sodium chondroitin sulfate prepared in Comparative Example 2;

[0099] Figure 24 The nuclear magnetic resonance carbon spectrum of sodium chondroitin sulfate prepared in Example 2;

[0100] Figure 25 The nuclear magnetic resonance carbon spectrum of sodium chondroitin sulfate prepared in Example 3;

[0101] Figure 26 The nuclear magnetic resonance carbon spectrum of sodium chondroitin sulfate prepared in Example 4. Detailed implementation mode

[0102] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present invention.

[0103] Example 1 Preparation of sodium chondroitin

[0104] (1) Fermentation: Using Escherichia coli DH001 as the production strain, after activation, inoculate it into a 30 L seed tank (containing Medium 1, with components: 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; antifoaming agent 0.2 - 0.3 g / L) at an inoculation amount of 1% - 5%, and ferment for 8 - 10 h until OD 600 reaches 2.5 - 5, and transfer the seeds into a 100 L fermenter (containing Medium 2, with components: glucose 10 - 15 g / L; yeast extract powder 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; antifoaming agent 0.2 - 0.5 g / L) at a transfer inoculation amount of 5% - 10%, with an initial stirring speed of 200 - 300 rpm, an aeration rate of 1 - 4 vvm, a dissolved oxygen correction of 100%, the whole process with dissolved oxygen controlled at 15% - 30%, adding 25% (w / v) ammonia water to control the pH at 7.0 - 8.0, and adding glucose as the carbon source for feeding at a mass fraction of 40% - 60%, ferment for 42 - 48 h until OD 600 reaches 110 - 135 and then discharge the tank to obtain the fermentation broth, and the content of (unsulfated) sodium chondroitin in the fermentation broth is 8 - 10 g / L;

[0105] (2) Ultrafiltration: Centrifuge the fermentation broth obtained by fermentation at 8000 rpm, collect the supernatant, and perform ultrafiltration with a 10 kDa ultrafiltration membrane until the conductivity of the concentrated solution is stable;

[0106] (3) Acid hydrolysis: Adjust the pH of the concentrated solution after ultrafiltration to 1.5 with hydrochloric acid, react at 80 °C for 6 h for acid hydrolysis, centrifuge at 8000 rpm to remove the precipitate, and obtain the supernatant;

[0107] (4) Purification: D280 anion exchange resin was added to the obtained supernatant, stirred and adsorbed for 2 h, then eluted with 2 mol / L sodium chloride solution, and the eluate was ultrafiltered with a 10 kDa ultrafiltration membrane until the conductivity of the concentrated solution was stable. The concentrated solution was concentrated and dried to obtain 410 g of sodium chondroitin (unsulfated sodium chondroitin).

[0108] Molecular weight analysis: According to the "Detection Method for Molecular Weight of Low Molecular Heparin", the molecular weight of the sodium chondroitin obtained by the above method was determined using a TSKgel G3000SWXL column. After determination, the average molecular weight Mw of the above sodium chondroitin was 11.1 kDa (see Figure 2 , and the retention time was 18.8 min).

[0109] Example 2 Preparation of Sodium Chondroitin Sulfate 1

[0110] (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1, disperse it with 50 mL of N, N-dimethylformamide, i.e., W 软骨素钠 :V 有机溶剂 =1:5, g:ml; form a sodium chondroitin dispersion, and preheat it in a 40 °C oil bath for standby; take 10 g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 =1:1, disperse it with 50 mL of formamide, then add it to the above sodium chondroitin dispersion, stir and react in a 40 °C oil bath for 2 h, add 0.7 L of absolute ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L of absolute ethanol;

[0111] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and stirring was carried out at 25 °C for 3 h to complete decolorization. The decolorized feed liquid was spray-dried to obtain 11.8 g of sodium chondroitin sulfate.

[0112] Molecular weight analysis: According to the "Detection Method for Molecular Weight of Low Molecular Heparin", the molecular weight of the sodium chondroitin sulfate prepared in Example 2 was determined using a TSKgel G3000SWXL column. After determination, the average molecular weight Mw of the sodium chondroitin sulfate obtained in Example 2 was 13.4 kDa (see Figure 3 , and the retention time was 17.5 min).

[0113] Example 3 Preparation of Sodium Chondroitin Sulfate 2

[0114] (1) Sulfonation: Take 10 g of sodium chondroitin prepared in Example 1, disperse it with 50 mL of formamide, i.e., W 软骨素钠 :V 有机溶剂= 1:5, g:ml; Prepare a chondroitin sodium dispersion, preheat it in an oil bath at 40 °C for standby; Take 10 g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 = 1:1. After dispersing with 50 mL of N, N-dimethylformamide, add it to the above chondroitin sodium dispersion. Stir and react in an oil bath at 40 °C for 2 h, add 0.7 L of absolute ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L of absolute ethanol;

[0115] (2) Post-treatment: Redissolve the washed precipitate with 100 mL of purified water, add 1.5 mL of hydrogen peroxide, stir at 25 °C for 3 h to complete decolorization. Spray-dry the decolorized material liquid to obtain 12.1 g of chondroitin sodium sulfate. It is measured that the average molecular weight Mw of the chondroitin sodium sulfate obtained in Example 3 is 13.7 kDa (see Figure 4 , and the retention time is 17.6 min).

[0116] Preparation of chondroitin sodium sulfate in Example 4 3

[0117] (1) Sulfonation: Take 10 g of chondroitin sodium prepared in Example 1, disperse it with 50 mL of formamide, i.e., W 软骨素钠 :V 有机溶剂 = 1:5, g:ml; Prepare a chondroitin sodium dispersion, preheat it in an oil bath at 40 °C for standby; Take 10 g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 = 1:1. After dispersing with 50 mL of formamide, add it to the above chondroitin sodium dispersion. Stir and react in an oil bath at 40 °C for 2 h, add 0.7 L of absolute ethanol to terminate the reaction, centrifuge at 6000 rpm, collect the precipitate, and wash the precipitate with 0.7 L of absolute ethanol;

[0118] (2) Post-treatment: Redissolve the washed precipitate with 100 mL of purified water, add 1.5 mL of hydrogen peroxide, stir at 25 °C for 3 h to complete decolorization. Spray-dry the decolorized material liquid to obtain 11.2 g of chondroitin sodium sulfate. It is measured that the average molecular weight Mw of the chondroitin sodium sulfate obtained in Example 4 is 12.1 kDa (see Figure 5 , and the retention time is 18.5 min).

[0119] Preparation of chondroitin sodium sulfate in Example 5 4

[0120] (1) Sulfonation: Take 10 g of chondroitin sodium prepared in Example 1, disperse it with 50 mL of N, N-dimethylformamide, i.e., W 软骨素钠 :V 有机溶剂 = 1:5, g:ml; Prepare a chondroitin sodium dispersion, preheat it in an oil bath at 40 °C for standby; Take 10 g of sulfur trioxide pyridine complex, W软骨素钠 :W 磺化试剂 = 1:1. Dispersed in 50 mL of N, N - dimethylformamide, then added to the above chondroitin sulfate sodium dispersion. Stirred and reacted in an oil bath at 40 °C for 2 h. Added 0.7 L of absolute ethanol to terminate the reaction, centrifuged at 6000 rpm, collected the precipitate, and washed the precipitate with 0.7 L of absolute ethanol;

[0121] (2) Post - treatment: The washed precipitate was redissolved in 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and stirred at 25 °C for 3 h to complete decolorization. The decolorized feed liquid was spray - dried to obtain 11.3 g of chondroitin sulfate sodium. It was determined that the average molecular weight Mw of the chondroitin sulfate sodium obtained in Example 5 was 12.3 kDa (see Figure 6 , and the retention time was 18.3 min).

[0122] Example 6 Preparation of chondroitin sulfate sodium 5

[0123] (1) Sulfonation: Take 10 g of chondroitin sulfate sodium prepared in Example 1, dispersed in 50 mL of formamide, i.e., W 软骨素钠 :V 有机溶剂 = 1:5, g:ml; formed a chondroitin sulfate sodium dispersion, pre - heated in an oil bath at 40 °C for standby; Take 50 g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂 = 1:5, dispersed in 50 mL of formamide, then added to the above chondroitin sulfate sodium dispersion, stirred and reacted in an oil bath at 40 °C for 2 h, added 0.7 L of absolute ethanol to terminate the reaction, centrifuged at 6000 rpm, collected the precipitate, and washed the precipitate with 0.7 L of absolute ethanol;

[0124] (2) Post - treatment: The washed precipitate was redissolved in 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and stirred at 25 °C for 3 h to complete decolorization. The decolorized feed liquid was spray - dried to obtain 11.5 g of chondroitin sulfate sodium. It was determined that the average molecular weight Mw of the chondroitin sulfate sodium obtained in Example 6 was 13.2 kDa (see Figure 7 , and the retention time was 17.6 min).

[0125] Example 7 Preparation of chondroitin sulfate sodium 6

[0126] (1) Sulfonation: Take 10 g of chondroitin sulfate sodium prepared in Example 1, dispersed in 50 mL of N, N - dimethylformamide, i.e., W 软骨素钠 :V 有机溶剂 = 1:5, g:ml; formed a chondroitin sulfate sodium dispersion, pre - heated in an oil bath at 40 °C for standby; Take 10 g of trimethylamine sulfur trioxide complex, W 软骨素钠 :W 磺化试剂= 1:1. After dispersing with 50 mL of formamide, it was added to the above chondroitin sulfate sodium dispersion solution. The reaction was stirred in an oil bath at 50 °C for 6 h. 0.7 L of absolute ethanol was added to terminate the reaction, and centrifuged at 6000 rpm to collect the precipitate, and the precipitate was washed with 0.7 L of absolute ethanol.

[0127] (2)Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, and decolorization was completed by repeating filtration 5 times with a 6-layer activated carbon fiber membrane. The decolorized feed liquid was freeze-dried to obtain 11.6 g of chondroitin sulfate sodium. It was determined that the average molecular weight Mw of the chondroitin sulfate sodium obtained in Example 7 was 12.8 kDa (see Figure 8 , and the retention time was 18.2 min).

[0128] Example 8 Preparation of Chondroitin Sulfate Sodium 7

[0129] (1)Sulfonation: Take 10 g of chondroitin sulfate sodium prepared in Example 1 and disperse it with 50 mL of N, N-dimethylformamide, i.e., W 软骨素钠 :V 有机溶剂 = 1:5, g:ml; form a chondroitin sulfate sodium dispersion solution, and preheat it in an oil bath at 40 °C for standby; take 10 g of concentrated sulfuric acid, W 软骨素钠 :W 磺化试剂 = 1:1. After dispersing with 50 mL of formamide, it was added to the above chondroitin sulfate sodium dispersion solution. The reaction was stirred in an oil bath at 30 °C for 4 h. 0.7 L of absolute ethanol was added to terminate the reaction, and centrifuged at 6000 rpm to collect the precipitate, and the precipitate was washed with 0.7 L of absolute ethanol.

[0130] (2)Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, and decolorization was completed by repeating filtration 5 times with a 6-layer activated carbon fiber membrane. The decolorized feed liquid was freeze-dried to obtain 11.2 g of chondroitin sulfate sodium. It was determined that the average molecular weight Mw of the chondroitin sulfate sodium obtained in Example 8 was 12.2 kDa (see Figure 9 , and the retention time was 17.9 min).

[0131] Example 9 Preparation of Chondroitin Sulfate Sodium 8

[0132] (1)Sulfonation: Take 10 g of chondroitin sulfate sodium prepared in Example 1 and disperse it with 50 mL of N, N-dimethylformamide, i.e., W 软骨素钠 :V 有机溶剂 = 1:5, g:ml; form a chondroitin sulfate sodium dispersion solution, and preheat it in an oil bath at 40 °C for standby; take 5 g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂= 1:0.5. After dispersing with 50 mL of formamide, it was added to the above chondroitin sulfate sodium dispersion solution. The reaction was stirred in an oil bath at 40 °C for 2 h. 0.7 L of absolute ethanol was added to terminate the reaction, and the mixture was centrifuged at 6000 rpm. The precipitate was collected and washed with 0.7 L of absolute ethanol.

[0133] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, and decolorization was completed by repeatedly filtering 5 times with a 6-layer activated carbon fiber membrane. The decolorized liquid was freeze-dried to obtain 10.9 g of chondroitin sulfate sodium. It was determined that the average molecular weight Mw of the chondroitin sulfate sodium obtained in Example 9 was 11.9 kDa (see Figure 10 , and the retention time was 18.3 min).

[0134] Comparative Example 1

[0135] (1) Sulfonation: Take 10 g of chondroitin sulfate sodium prepared in Example 1 and disperse it with 50 mL of N, N-dimethylformamide, i.e., W 软骨素钠 :V 有机溶剂 = 1:5, g:ml; to form a chondroitin sulfate sodium dispersion solution, which was preheated in an oil bath at 40 °C for standby; take 10 g of chlorosulfonic acid, W 软骨素钠 :W 磺化试剂 = 1:1. After dispersing with 50 mL of formamide, it was added to the above chondroitin sulfate sodium dispersion solution. The reaction was stirred in an oil bath at 40 °C for 2 h. 0.7 L of absolute ethanol was added to terminate the reaction, and the mixture was centrifuged at 6000 rpm. The precipitate was collected and washed with 0.7 L of absolute ethanol;

[0136] (2) Post-treatment: The washed precipitate was redissolved with 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and stirring was carried out at 25 °C for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 11.4 g of chondroitin sulfate sodium. It was determined that the average molecular weight Mw of the chondroitin sulfate sodium obtained in Comparative Example 1 was 12.5 kDa (see Figure 11 , and the retention time was 18.0 min).

[0137] Comparative Example 2

[0138] (1) Sulfonation: Take 10 g of chondroitin sulfate sodium prepared in Example 1 and disperse it with 50 mL of N, N-dimethylformamide, i.e., W 软骨素钠 :V 有机溶剂 = 1:5, g:ml; to form a chondroitin sulfate sodium dispersion solution, which was preheated in an oil bath at 40 °C for standby; take 3 g of sulfur trioxide pyridine complex, W 软骨素钠 :W 磺化试剂= 1:0.3. After dispersing in 50 mL of formamide, it was added to the above chondroitin sulfate sodium dispersion. The reaction was stirred in an oil bath at 40 °C for 2 h. 0.7 L of absolute ethanol was added to terminate the reaction. The precipitate was collected by centrifugation at 6000 rpm, and the precipitate was washed with 0.7 L of absolute ethanol;

[0139] (2) Post-treatment: The washed precipitate was redissolved in 100 mL of purified water, 1.5 mL of hydrogen peroxide was added, and stirring was carried out at 25 °C for 3 h to complete decolorization. The decolorized liquid was spray-dried to obtain 10.5 g of chondroitin sulfate sodium. After determination, the average molecular weight Mw of the chondroitin sulfate sodium obtained in Comparative Example 2 was 11.3 kDa (see Figure 12 , and the retention time was 18.7 min).

[0140] Result analysis

[0141] 1. The chondroitin sulfate sodium prepared in Examples 2-9 was determined and analyzed

[0142] According to the analysis method on page 1595 of Part II of the Chinese Pharmacopoeia 2020 Edition, chondroitin ABC enzyme was used to enzymatically hydrolyze the unsulfated chondroitin sulfate sodium obtained in Example 1 and the chondroitin sulfate sodium prepared in Examples 2-9 and Comparative Examples 1-2. The macromolecular chondroitin sulfate was enzymatically hydrolyzed into disaccharide units (→GalA-GalNAc→). HPLC analysis was carried out using a Hypersil SAX chromatographic column, and the detection wavelength was 232 nm. The disaccharide units sulfonated at different positions eluted in sequence, and thus the sulfonation situation of chondroitin sulfate could be analyzed.

[0143] The analysis results are shown in Table 1 and Figures 13 - 23 . Among them, Table 1 shows the disaccharide distribution of the chondroitin sulfate sodium prepared in Examples 2-9 and Comparative Examples 1-2, Figure 13 is the analysis result of the disaccharide content of the chondroitin sulfate sodium obtained in Example 1, Figure 14 is the analysis result of the disaccharide distribution of the chondroitin sulfate sodium prepared in Example 2, Figure 15 is the analysis result of the disaccharide distribution of the chondroitin sulfate sodium prepared in Example 3, Figure 16 is the analysis result of the disaccharide distribution of the chondroitin sulfate sodium prepared in Example 4; Figure 17 is the analysis result of the disaccharide distribution of the chondroitin sulfate sodium prepared in Example 5; Figure 18 is the analysis result of the disaccharide distribution of the chondroitin sulfate sodium prepared in Example 6; Figure 19 is the analysis result of the disaccharide distribution of the chondroitin sulfate sodium prepared in Example 7; Figure 20 is the analysis result of the disaccharide distribution of the chondroitin sulfate sodium prepared in Example 8; Figure 21 is the analysis result of the disaccharide distribution of the chondroitin sulfate sodium prepared in Example 9; Figure 22Analysis results of disaccharide distribution of sodium chondroitin sulfate prepared in Comparative Example 1; Figure 23 Analysis results of disaccharide distribution of sodium chondroitin sulfate prepared in Comparative Example 2.

[0144] Table 1 Sulfonation of chondroitin sulfate

[0145]

[0146] The results show that: The chondroitin sulfates obtained in Examples 2-9 can all be recognized and enzymatically hydrolyzed by chondroitinase ABC. From the situation of the enzymatic hydrolysis products, the sulfonation reaction of the method provided by the present invention is relatively complete. In each preparation example, no more than 30% of chondroitin (i.e., 0S-CS) is unsulfonated, 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% (for example, the 0S-CS % in Example 2 is only 6.94%, and the 0S-CS % in Example 3 is only 3.44%), and most of them are sulfonated at the 6th position of GalNAc. For example, in Examples 2 and 3, most of the sulfonated products are distributed in chondroitin sulfate sulfonated at the 6th position alone (i.e., 6S-CS, disaccharide structure: 4GlcAβ1,3GalNAc6Sβ1), chondroitin sulfate sulfonated at the 2nd and 6th positions (i.e., 2,6S-CS, disaccharide structure: 4GlcA2Sβ1,3GalNAc6Sβ1), chondroitin sulfate sulfonated at the 4th and 6th positions (i.e., 4,6S-CS, disaccharide structure: 4GlcAβ1,3GalNAc4,6Sβ1), and only less than 1% of chondroitin sulfate sulfonated at the 2nd and 4th positions (i.e., 2,4S-CS, disaccharide structure: 4GlcA2Sβ1,3GalNAc4Sβ1); in Examples 4-9, all products are sulfonated at the 6th position of GalNAc.

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

[0148] In addition, the sulfonation process of the preparation method provided by the present invention is mild, and no structural modification that can affect the recognition and enzymatic hydrolysis by natural enzymes occurs in the final product.

[0149] It can be seen from Comparative Example 1 that, compared with Example 3, after changing the type of sulfonating agent, the proportion of products with independent sulfonation at the 6-position decreased 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 6-sulfonation of sodium chondroitin. It can be seen from Comparative Example 2 that, compared with Example 3, when the amount of sulfonating agent is reduced, the sulfonation rate decreased to 50.52%, which indicates that the addition ratio of the sulfonating agent in the preparation method of the present invention has a great influence on the sulfonation rate. This also directly shows that the preparation method provided by the present invention can control the proportion of products with independent sulfonation at the 6-position of GalNAc in the final product to be over 60%.

[0150] 2. Nuclear magnetic resonance carbon spectrum analysis of sodium chondroitin sulfate

[0151] Model of the nuclear magnetic resonance carbon spectrum detection instrument: AV500 (CAq08);

[0152] The detection conditions are as follows: magnetic field strength (FS): 125 MHz; number of scans (NS): 12288 times; temperature (TE): about 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 line width window function (LB): 1.0 Hz; diameter of the nuclear magnetic tube: 5 mm; acquisition method: D_GLP_E.glp.

[0153] The analysis results are shown in Figures 24 - 26 , Figures 24 - 26 which are the carbon-13 NMR spectra of sodium chondroitin sulfate prepared in Examples 2 to 4, respectively. The peak at 49.5 ppm is the methanol positioning peak. There are main characteristic peaks in the carbon spectrum of CS-C. Among them, the peak at 68.7 ppm is the characteristic signal peak of sulfonated carbon 6 in CS-C, and the peaks at 176.2 ppm and 23.8 ppm are the characteristic signal peaks of carbon 7 and carbon 8 in GalNAc, respectively.

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

[0155] In addition, it can be seen from Examples 2 - 9 (see Figures 3 - 10 ), the average molecular weights Mw of sodium chondroitin sulfate obtained by sulfonating sodium chondroitin via the method provided by the present invention are not very different, which indicates that the sulfonation method provided by the present invention has a mild reaction. Although it undergoes a chemical reaction process, this process has minimal damage to the disaccharide backbone structure. From the average molecular weights Mw of the obtained sodium chondroitin sulfate, it can be seen that the distribution of the average molecular weights Mw between batches of sodium chondroitin sulfate obtained by using the sulfonation method provided by the present invention is stable, which indicates that the sulfonation method provided by the present invention has stable operation between batches and has little dependence on the operating environment, operating means, compound dosage and ratio, etc.

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

[0157] The above-provided examples are not intended to limit the scope covered by the present invention, and the described steps are not intended to limit the order of its operations. Obvious improvements made by those skilled in the art to the present invention in combination with the existing common general knowledge also fall within the protection scope 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; the sulfonation reaction temperature is 20-50°C, and the reaction time is 0.5-6h; The weight ratio of the unsulfated sodium chondroitin to the sulfonated reagent is W 软骨素钠 :W 磺化试剂= 1:0.5-5; 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 described in step 1 is selected from any one 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-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.

3. 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.

4. 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.

5. Use of the preparation method according to any one of claims 1 to 4 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.

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