Novel one-pot production method for polysulfated chondroitin sulfate

Abstract

The present invention provides a one-pot production method for polysulfated chondroitin sulfate having the following physicochemical properties: a) a sulfonic acid group content of 20.1-43.7%; and b) an intrinsic viscosity of 0.08-0.20, the production method comprising converting chondroitin sulfate into a water-insoluble alkylpyridinium complex in a solution of the chondroitin sulfate or a salt thereof.

Description

Novel one-pot manufacturing method for polysulfated chondroitin sulfate

[0001] The present invention relates to a novel one-pot (using the same reaction vessel without changing the reaction vessel) method for producing polysulfated chondroitin sulfate.

[0002] Polysulfated chondroitin sulfate exhibits excellent effects such as inhibiting blood coagulation, promoting peripheral blood circulation, inhibiting fibroblast proliferation, moisturizing the skin, and inhibiting hyaluronidase activity, and is used as a pharmaceutical or cosmetic.

[0003] Chondroitin sulfate, the raw material for polysulfated chondroitin sulfate, is a glycosaminoglycan, a linear polysaccharide with a repeating structure of amino sugar derivatives and hexose derivatives. Chondroitin sulfate is widely distributed in animal tissues as a proteoglycan bound to core proteins and plays an important role in animal development, differentiation, growth, and regeneration. For example, as a major component in cartilage, chondroitin sulfate contributes to hydration and elasticity, thus aiding in cartilage tissue formation. Chondroitin sulfate also exhibits binding affinity to various physiologically active molecules, playing a role in the storage, stabilization, or masking of these molecules, and cooperating with cell membrane receptors to regulate signal transduction mechanisms. Furthermore, in the nervous system, chondroitin sulfate promotes or inhibits the extension of nerve cell axons, and in immune system cells, it is located downstream, controlling the accumulation and release of immune substances. In infections caused by malaria parasites and viruses, chondroitin sulfate is known to act as a receptor and exert an inhibitory effect on infection. In many cases, chondroitin sulfates such as those in the D and E structures described below are known to exhibit particularly high physiological activity.

[0004] Patent Document 1 discloses a method for polysulfating chondroitin sulfate, specifically a method of sulfating chondroitin sulfate (C) sodium salt in concentrated sulfuric acid at low temperature. This method is advantageous because sulfation and demolecularization occur simultaneously in a single step. However, it is difficult to independently control sulfation and demolecularization with this method, making quality control difficult. Furthermore, the need to control the reaction temperature to a low temperature, and the requirement for a large amount of ether-based solvent, make it industrially hazardous and undesirable from an environmental protection standpoint.

[0005] Patent Document 2 discloses a method for sulfating all hydroxyl groups in a molecule of chondroitin sulfate using the tributylamine salt, and Patent Documents 3 and 4 disclose a method for polysulfating an organic amine salt of chondroitin sulfate with a sulfur trioxide complex or a sulfate-carbodiimide mixture. However, neither of these methods leads to a reduction in the molecular weight of chondroitin sulfate; therefore, to obtain polysulfated chondroitin sulfate of the desired molecular weight, chondroitin sulfate of a predetermined molecular weight must be used beforehand. Furthermore, chondroitin sulfate, the raw material, is usually available in the form of a sodium salt, but sodium salts are poorly soluble in organic solvents, and the reaction tends to be heterogeneous. Therefore, the sodium salt is converted to an organic amine salt that is easily soluble in organic solvents before polysulfation. The conversion of sodium salt to an organic amine salt can be done using ion exchange resins, but this is disadvantageous for industrial production because it is complicated and costly.

[0006] Patent Document 5 discloses a method for obtaining a polysulfated product by suspending sodium chondroitin sulfate, which is insoluble in aprotic solvents, directly in an organic amine (e.g., pyridine), adding a large amount of sulfur trioxide pyridine complex, and heating for a long time. However, dematuration due to prolonged high-temperature heating is unavoidable, and because sodium chondroitin sulfate is insoluble in aprotic solvents, the sulfation reaction efficiency is low. Although the document states that polysulfated chondroitin sulfate with a certain molecular weight can be obtained, in reality, dematuration of the raw material chondroitin sulfate is unavoidable, and the molecular weight cannot be controlled. Furthermore, it requires a large amount of sulfation reagent, making it costly and disadvantageous for industrial production.

[0007] Patent document 6 discloses a method for sulfating chondroitin sulfate using a mixture of sulfuric acid and chlorosulfonic acid. However, in this method, sulfation and demolecularization proceed simultaneously, making it difficult to independently control sulfation and demolecularization. Furthermore, the same document states that this method results in excessive demolecularization of chondroitin sulfate.

[0008] JP 47-30167, JP 11-166001, JP 62-27402, US Patent No. 6388060, JP 2005-344073, JP 59-133201

[0009] Chondroitin sulfate has a linear polysaccharide structure with a molecular weight of tens of thousands (20 to 400 monosaccharides). This structure is based on a repeating disaccharide structure in which glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) are alternately linked at β1-3 and β1-4. Chondroitin sulfate is a substance in which chondroitin has undergone various sulfate group modifications, such as those shown in Figure 1.

[0010] Specifically, chondroitin sulfate (abbreviated as CS in Figure 1) includes a variety of sulfated disaccharide units, such as the A structure (CSA) in which the 4th position of the GalNAc residue of the chondroitin (CH) disaccharide unit is sulfated (4S), the C structure (CSC) in which the 6th position of the GalNAc residue is sulfated (6S), the D structure (CSD) in which two positions of the disaccharide unit, the 2nd position of the GlcA residue and the 6th position of the GalNAc residue, are sulfated (2S, 6S), the E structure (CSE) in which two positions of the GalNAc residue, the 4th and 6th positions, are sulfated (4S, 6S), and the triS structure (CStriS or CtriS) in which a total of three positions, the 2nd position of the GlcA residue and the 4th and 6th positions of the GalNAc residue, are sulfated (2S, 4S, 6S). Chondroitin sulfate has an extremely complex polysaccharide structure, formed by the combination of these sulfate group modification structures. Therefore, depending on the source and manufacturer of chondroitin sulfate, it exhibits a wide range of physicochemical properties, making it unsuitable as a raw material for pharmaceuticals, cosmetics, and other products. Thus, when using chondroitin sulfate as a raw material for pharmaceuticals, cosmetics, and other products, there is a need for a method to produce polysulfated chondroitin sulfate with consistent physicochemical properties, i.e., with quality control in which the molecular weight and sulfate group content are within a certain range.

[0011] Through diligent research, the inventors have discovered that a one-pot manufacturing method, which involves converting chondroitin sulfate or its salt into a water-insoluble alkylpyridinium complex in a solution of chondroitin sulfate or its salt, can produce polysulfated chondroitin sulfate with consistent physical and chemical properties, i.e., with controlled molecular weight and sulfate group content within a certain range. This discovery has led to the completion of the present invention.

[0012] Accordingly, the present invention provides, in particular, the following: [1] A one-pot method for producing polysulfated chondroitin sulfate having the following physicochemical properties: a) sulfate group content: 20.1 to 43.7%, and b) intrinsic viscosity: 0.08 to 0.20, the method comprising converting chondroitin sulfate into a water-insoluble alkylpyridinium complex in a solution of chondroitin sulfate or a salt thereof. [2] The method for producing according to [1], wherein the alkylpyridinium is selected from the group consisting of cetylpyridinium fluoride, chloride, iodide, and bromide. [3] The method for producing according to [1], further comprising polysulfating the alkylpyridinium complex using a sulfur trioxide complex in an aprotic solvent. [4] The method for producing according to [3], wherein the sulfur trioxide complex is selected from the group consisting of sulfur trioxide pyridine complex, N,N-dimethylformamide complex, and trimethylamine complex. [5] The method for producing the product according to [3], wherein the aprotic solvent is selected from the group consisting of pyridine, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide. [6] The method for producing the product according to [1], wherein the polysulfated chondroitin sulfate is a chondroitin sulfate having an E structure in which two positions, 4 and 6, of the N-acetylgalactosamine of chondroitin are sulfated. [7] The method for producing the product according to [1] to [6], wherein the chondroitin sulfate is derived from a source of fish or mammal. [8] The method for producing the product according to [7], wherein the chondroitin sulfate is derived from a source selected from the group consisting of salmon, shark, ray, whale, cattle, and pig.

[0013] According to the present invention, it is possible to produce polysulfated chondroitin sulfate with consistent physical and chemical properties, that is, with quality control in which the molecular weight and sulfate group content are within a certain range. Furthermore, while conventional production methods require the use of purified, high-purity sodium chondroitin sulfate as a raw material, the method of the present invention allows for easy polysulfation even if the raw material sodium chondroitin sulfate is not a highly purified product.

[0014] This figure shows the structures of chondroitin sulfate that have undergone various sulfate group modifications. In the figure, CS represents chondroitin sulfate. This is an electrophoresis diagram of the raw material chondroitin sulfate and the polysulfated chondroitin sulfate obtained in Example 1 (Test Example 2). 1: Raw material chondroitin sulfate, 2: Polysulfated chondroitin sulfate obtained in Example 1. This is a graph comparing the polysulfated chondroitin sulfate obtained in Example 1 and the polysulfated chondroitin sulfate produced by the conventional manufacturing method with respect to anticoagulation activity (antithrombin activity) (Figure 3A) and growth inhibitory activity on the growth curve of cultured human fibroblast cells (Figure 3B) (Test Example 3). This is a graph showing the hyaluronidase activity inhibitory effect of the polysulfated chondroitin sulfate obtained in Example 1 and the polysulfated chondroitin sulfate obtained by the conventional manufacturing method (Test Example 4). This is the proton nuclear magnetic resonance spectrum (MSR) analysis of the insertion positions of sulfate groups in polysulfated chondroitin sulfate obtained in Example 1 and polysulfated chondroitin sulfate obtained by the conventional manufacturing method (Test Example 5). 1: Signal from the 4-position proton of N-acetylgalactosamine, where sulfate groups are simultaneously inserted into the hydroxyl groups at positions 4 and 6. 2: Signal from the N-acetyl group of N-acetylgalactosamine, where sulfate groups are simultaneously inserted into the hydroxyl groups at positions 4 and 6. These signals are not observed in polysulfated chondroitin sulfate obtained by the conventional manufacturing method. This is a comparison of the MRS proton nuclear magnetic resonance spectra of polysulfated chondroitin sulfate obtained in Example 1 and polysulfated chondroitin sulfate obtained in Example 2 (Test Example 6).

[0015] One embodiment of the present invention provides a one-pot method for producing polysulfated chondroitin sulfate having the following physicochemical properties: a) sulfate group content: 20.1 to 43.7%, and b) intrinsic viscosity: 0.08 to 0.20, the method comprising converting chondroitin sulfate into a water-insoluble alkylpyridinium complex in a solution of chondroitin sulfate or a salt thereof.

[0016] In the present invention, the "sulfate group content" of polysulfated chondroitin sulfate refers to the sulfate groups (-SO) contained in the structure of polysulfated chondroitin sulfate. ３H) represents the percentage (%w / w), and its value can be determined by hydrolyzing polysulfated chondroitin sulfate with a 1 mol / L hydrochloric acid solution for 3 hours, followed by measuring the sulfate group content by HPLC. The HPLC measurement conditions are as follows, for example: Standard: Potassium sulfate Column: TSKgel IC-ANION PW (Tosoh) Mobile phase: Borate buffer Flow rate: 1.0 mL / min Injection volume: 10 μL Column temperature: 40°C Detector: Electrical conductivity detector (Waters)

[0017] The "sulfate group content" of polysulfated chondroitin sulfate, that is, the sulfate groups (-SO) contained in the structure of polysulfated chondroitin sulfate. ３ The proportion of H) (% w / w) can be adjusted to the desired proportion by, for example, adjusting the amount of sulfur trioxide pyridine complex added when producing polysulfide chondroitin sulfate from sodium chondroitin sulfate.

[0018] "Intrinsic viscosity" is a common technical term in the polymer field. The intrinsic viscosity of polysulfated chondroitin sulfate can be measured using a capillary viscometer such as an Ubbelohde viscometer or an Ostwald viscometer, but it is preferable to measure it using an Ubbelohde viscometer. The calculation of intrinsic viscosity using an Ubbelohde viscometer can be performed, for example, as follows: Determine the reduced viscosity using an Ubbelohde viscometer, and then determine the intrinsic viscosity by extrapolation. The viscosity of the solution to be measured is η, and the viscosity of the solvent is η. ｓ In this case, the rate of increase in viscosity is (η - η ｓ ) / η ｓ This is the specific viscosity η ｓｐ This is defined as follows: The increase in viscosity per unit concentration c of the solution being measured is η ｓｐ / c becomes this, and this is the reduced viscosity η ｒｅｄ It is defined as follows. The intrinsic viscosity [η] can be determined by extrapolating the vertical axis (reduced viscosity) so that the concentration of the solution being measured (horizontal axis) becomes zero.

[0019] In this invention, polysulfated chondroitin sulfate is produced in the same reaction vessel without changing the vessel. The reaction vessel used is not particularly limited, as long as it is a container large enough to accommodate the raw materials and the target product, and is made of a material that does not adversely affect the synthesis reaction of polysulfated chondroitin sulfate. Examples of such reaction vessels include ceramic, porcelain, glass, and stainless steel containers. Among these, stainless steel containers are preferred for factory-scale production, and glass containers are preferred for laboratory-scale production.

[0020] The present invention relates to a manufacturing method comprising converting chondroitin sulfate into a water-insoluble alkylpyridinium complex in a solution of chondroitin sulfate or a salt thereof. The solvent used when preparing the solution of chondroitin sulfate or a salt thereof is not particularly limited as long as it is a liquid capable of carrying out the complex formation reaction between chondroitin sulfate and alkylpyridinium, but examples include N,N-dimethylformamide, dimethyl sulfoxide, and water, with water being preferred.

[0021] In one embodiment of the present invention, the alkylpyridinium is selected from the group consisting of cetylpyridinium fluoride, chloride, iodide, and bromide. Among these, cetylpyridinium chloride is preferred.

[0022] In one embodiment of the present invention, a desired polysulfated chondroitin sulfate can be obtained by polysulfating the alkylpyridinium complex with a sulfur trioxide complex in an aprotic solvent. The sulfur trioxide complex can be selected from the group consisting of a sulfur trioxide pyridine complex, an N,N-dimethylformamide complex, and a trimethylamine complex, with the sulfur trioxide pyridine complex being preferred. The aprotic solvent can be selected from the group consisting of pyridine, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide, with the mixed solvent of pyridine and N,N-dimethylformamide being preferred.

[0023] The polysulfated chondroitin sulfate produced in the present invention is preferably a chondroitin sulfate with an E structure in which two positions, 4 and 6, of the N-acetylgalactosamine of chondroitin are sulfated. The origin of the raw material chondroitin sulfate or its salt is not particularly limited, but for example, it may be derived from a fish or mammal source, preferably from a source selected from the group consisting of salmon, shark, ray, whale, cattle, and pig, and particularly preferably from a shark source. Examples of chondroitin sulfate salts include sodium salt, potassium salt, and calcium salt, and among these, sodium chondroitin sulfate sodium, which is a sodium salt, is preferred.

[0024] In a preferred embodiment of the present invention, for example, polysulfated chondroitin sulfate can be produced by the following method: Sodium chondroitin sulfate derived from sharks is dissolved in water, cetylpyridinium chloride is added and stirred at room temperature, the precipitate formed is collected, the aqueous layer is removed as much as possible, and an appropriate amount of 80% ethanol is added to remove excess cetylpyridinium chloride. Pyridine is added, and 10 mL of N,N-dimethylformamide (DMF) containing a sulfur trioxide pyridine complex is added and heated. After removing the solvent, water is added to dissolve the crude product, the pH is adjusted to 10 or higher with a sodium hydroxide solution, and then dialyze is performed against water. After adding NaCl to the obtained crude product aqueous solution, it is added dropwise to ethanol, the precipitate is filtered off to obtain the desired polysulfated chondroitin sulfate.

[0025] The present invention will be described in more detail below with reference to examples and test examples, but the present invention is not limited to these specific examples.

[0026] Example 1 A Pyrex glass test tube with a screw cap was used as the reaction vessel for one-pot production. 10 g of shark-derived chondroitin sulfate sodium (manufactured by Nippon Yakuhin Co., Ltd.; average number of sulfate groups per disaccharide unit: 1.06) was dissolved in 100 mL of water, and 5 g of cetylpyridinium chloride was added and the mixture was stirred at room temperature for 30 minutes. The precipitate was collected, the aqueous layer was removed as much as possible, and ethanol was added to remove excess cetylpyridinium chloride. 80 mL of pyridine was added, and 10 mL of DMF containing 10 g of sulfur trioxide pyridine complex was added and the mixture was heated for 3 hours. After removing the solvent, water was added to dissolve the crude product, the pH was adjusted to 10 or higher with a 30% sodium hydroxide solution, and then dialyzed against water. NaCl was added to the obtained crude product aqueous solution, and then it was added dropwise to ethanol. The precipitate was filtered off to obtain the desired polysulfated chondroitin sulfate (average number of sulfate groups per disaccharide unit: 2.4 ± 0.3). By changing the amount of sulfur trioxide pyridine complex added to 2g, 5g, 7.5g, and 15g respectively, polysulfated chondroitin sulfates with average sulfate groups per disaccharide unit of 1.2±0.2, 1.5±0.2, 1.8±0.2, and 3.7±0.3 were also produced. The average sulfate groups per disaccharide unit mentioned above are shown as the average sulfate group content per disaccharide unit and the degree of variation (±) obtained by measuring the sulfate group content of polysulfated chondroitin sulfate synthesized simultaneously using four test tubes with the method of Test Example 1. Test Example 1 The sulfate content of the shark-derived chondroitin sulfate sodium, the starting material of Example 1, and each polysulfated chondroitin sulfate with a different average sulfate group per disaccharide unit produced in Example 1 was measured by the following method. The starting material, shark-derived chondroitin sulfate sodium, or the polysulfated chondroitin sulfate obtained in Example 1, was hydrolyzed in a 1 mol / L hydrochloric acid solution for 3 hours, and the sulfate group content was measured by HPLC. Potassium sulfate (reagent grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a standard.Column used: TSKgel IC-ANION PW (Tosoh) Mobile phase: Borate buffer Flow rate: 1.0 mL / min Injection volume: 10 μL Column temperature: 40°C Detector: Electrical conductivity detector (Waters) <Preparation of borate buffer> 300 mg of potassium gluconate, 360 mg of boric acid, 500 mg of sodium tetraborate decahydrate, 5.00 g of glycerin, 30 mL of 1-butanol and 120 mL of acetonitrile were dissolved in water to make 1000 mL (pH 8.5). <Method for calculating the average number of sulfate groups per disaccharide unit> Sulfate group content = Quantitative value of sulfate groups (as SO3) ÷ (Quantitative value of sulfated disaccharide) Example) If there are 0 sulfate groups: 0 ÷ 379 × 100 = 0% If there is 1 sulfate group: 80 ÷ 459 × 100 = 17.4% If there are 2 sulfate groups: 160 ÷ 539 × 100 = 29.7% If there are 3 sulfate groups: 240 ÷ 619 × 100 = 38.8% If there are 4 sulfate groups: 320 ÷ 699 × 100 = 47.8%. <Results> The results are as follows:

[0027] Test Example 2 Polyacrylamide electrophoresis was performed using ATTO Corporation's Pagel NPG-520L type gel (5-20% gradient gel), with 25 mM Tris and 192 mM glycine buffer used as electrode solutions. Figure 2 shows the electrophoresis results of the raw material chondroitin sulfate sodium and the polysulfated chondroitin sulfate obtained in Example 1 (average number of sulfate groups per disaccharide unit: 2.6). As can be seen from Figure 2, the electrophoresis distance of the polysulfated chondroitin sulfate obtained in Example 1 is shorter than that of the raw material chondroitin sulfate sodium, due to the increase in molecular weight associated with the introduction of sulfate groups or adsorption to the gel due to the introduction of sulfate groups.

[0028] Test Example 3 The starting material of Example 1, shark-derived chondroitin sulfate sodium, and the polysulfated chondroitin sulfate obtained in Example 1 were compared with polysulfated chondroitin sulfate produced by a conventional manufacturing method (method described in Japanese Patent Publication No. 11-166001) for 1) anticoagulation activity (antithrombin activity) and 2) growth inhibitory activity on the growth curve of cultured human fibroblast cells. 1) Outline of the conventional manufacturing method (method described in Japanese Patent Publication No. 11-166001): Sodium chondroitin sulfate salt was passed through a column packed with cation exchange resin (Dowex X8 400mesh) to remove sodium, the eluted chondroitin sulfate (proton type) was converted to tributylamine salt, and then freeze-dried. The sample obtained by further drying this in a vacuum in the presence of phosphorus pentoxide for 16 hours was dissolved in N,N-dimethylformamide, and a 10-fold molar amount of sulfur trioxide pyridine complex was added relative to the hydroxyl groups of chondroitin sulfate. Sulfation was carried out at 40-50°C for 3 hours to obtain polysulfurized chondroitin sulfate (average number of sulfate groups per disaccharide unit: 2.7 ± 0.3). By changing the amount of sulfur trioxide pyridine complex added to 15-fold molar amount, polysulfated chondroitin sulfate with an average number of sulfate groups per disaccharide unit of 3.7 ± 0.3 was also produced. The average number of sulfate groups per disaccharide unit mentioned above is shown as the average number of sulfate groups per disaccharide unit and the degree of variation (±) obtained by measuring the sulfate group content of polysulfated chondroitin sulfate synthesized simultaneously using six test tubes with the method of Test Example 1. 2) Anticoagulation activity (antithrombin activity) The anticoagulation activity was evaluated for the shark-derived chondroitin sulfate sodium (average number of sulfate groups per disaccharide unit: 1.06) used as the starting material in Example 1, the polysulfated chondroitin sulfate obtained in Example 1 (average number of sulfate groups per disaccharide unit: 2.8, 3.8), and the polysulfated chondroitin sulfate produced by the conventional manufacturing method (average number of sulfate groups per disaccharide unit: 2.4, 4.0). 50 μL of polysulfated chondroitin sulfate, 30 μL of human serum, and 20 μL of human-derived thrombin (1.2 NIH units / mL) were mixed and incubated at 37°C for 30 seconds.50 μL (1.9 μmol / mL) of the chromogenic reagent Chromogenic TH (ethylmalonyl-Pro-Arg-p-nitroanelide hydrochloride) was added, and the absorbance at 405 nm was measured. The control was USP Heparin Reference Standard (K-3) (U.S. Pharmaceutical Convention, Rockville, MD). The results are shown in Figure 3A. The polysulfated chondroitin sulfate obtained in Example 1 and the polysulfated chondroitin sulfate produced by the conventional method showed almost equivalent results. 3) Proliferation inhibitory activity of polysulfated chondroitin sulfate on the proliferation curve of cultured human fibroblasts. A commercially available cell line (derived from adult humans, Takara Bio Inc.) was used as the human fibroblast cell line. From the polysulfated chondroitin sulfate obtained in Example 1, a sample with an average of 2.8 sulfate groups per disaccharide was added to fibroblast culture medium (Takara Bio Co., Ltd.) at the respective concentrations. The polysulfated chondroitin sulfate obtained by the conventional manufacturing method (method described in Japanese Patent Publication No. 11-166001; described above) also had an average of 2.8 sulfate groups per disaccharide. The results are shown in Figure 3B. The polysulfated chondroitin sulfate obtained in Example 1 and the polysulfated chondroitin sulfate produced by the conventional manufacturing method showed almost equivalent results, although the results are not shown.

[0029] Test Example 4 <Evaluation of Hyaluronidase Inhibitory Activity> To 0.1 mL of hyaluronidase (3 mg / mL), 0.1 mL of polysulfated chondroitin sulfate obtained by a conventional manufacturing method (method described in Japanese Patent Publication No. 11-166001; described above), having 2.4 sulfate groups per disaccharide, or polysulfated chondroitin sulfate produced in Example 1, having 2.6 sulfate groups per disaccharide, were added, and the mixture was incubated at 37°C for 10 minutes. Hyaluronidase isolated from bovine testes (Sigma-Aldrich) was used. To the incubated solution, 0.1 mL of 1.5 mol NaCl and 0.7 mL of bovine vitreous-derived hyaluronic acid (Sigma-Aldrich) were added as hyaluronidase activators, and the mixture was reacted at 37°C for 40 minutes. The hyaluronidase was then inactivated in a boiling water bath for 5 minutes. A solution of p-dimethylaminobenzaldehyde dissolved in concentrated sulfuric acid, diluted 10-fold, was added to a solution in which hyaluronidase had been inactivated. After incubation at 37°C for 20 minutes, the absorbance of the solution was measured at a wavelength of 585 nm, and the hyaluronidase activity inhibition rate was determined by the following formula. The results are shown in Figure 4. The hyaluronidase activity inhibitory effect of the polysulfated chondroitin sulfate obtained in Example 1 was significantly stronger than that of the polysulfated chondroitin sulfate obtained by the conventional manufacturing method (P < 0.05). Therefore, it is expected that the sulfate group is attached to the polysulfated chondroitin sulfate obtained in Example 1 in a different manner than that of the polysulfated chondroitin sulfate obtained by the conventional manufacturing method. In Figure 4, "hyaluronic acid" refers to the amount of hyaluronic acid (final concentration 10 mg / mL), which is the degradation substrate of hyaluronidase; "os existing method" refers to the amount of sample prepared by the existing polysulfation method; and "os CPC method" refers to the polysulfation method using CPC according to the present invention. The vertical axis of the graph shows the amount of reducing ends of oligosaccharides obtained with different amounts of polysulfated chondroitin sulfate added, relative to a sample containing only hyaluronic acid (the amount of reducing ends of oligosaccharides produced by enzymatic decomposition is set to 100%). (If hyaluronidase is inhibited, the amount will always be less than 100%). Test Example 5 To confirm the structural characteristics of polysulfated chondroitin sulfate obtained in Example 1, polysulfated chondroitin sulfate with approximately the same average number of sulfate groups was prepared using the manufacturing method of Example 1 (average number of sulfate groups per disaccharide: 2.1) or a conventional manufacturing method (the method described in Japanese Patent Publication No. 11-166001; described above) (average number of sulfate groups per disaccharide: 2.1), and the insertion positions of the sulfate groups were investigated by proton nuclear magnetic resonance spectroscopy. The results are shown in Figure 5. According to Figure 5, it was found that in the manufacturing method of Example 1, sulfate groups were inserted at the 4th and 6th positions of N-acetylgalactosamine from an early stage, which was difficult with the conventional manufacturing method.

[0030] Test Example 6 For the raw material shark-derived chondroitin sulfate sodium (average number of sulfate groups per disaccharide: 1.06), and the polysulfated chondroitin sulfate sample 1 (average number of sulfate groups per disaccharide: 2.4) and polysulfated chondroitin sulfate sample 2 (average number of sulfate groups per disaccharide: 3.8) produced by the method described in Example 1, 0.05 g / 100 mL, 0.10 g / 100 mL, and 0.2 g / mL of each sample at 30 °C were prepared, and the reduced viscosity was determined using an Ubbelohde viscometer (Shibata Scientific Viscometer Ubbelohde SU SU No. 1 026130-001), and the intrinsic viscosity was determined by the extrapolation method. When the viscosity of the measurement target solution is η and the viscosity of the solvent is η ｓ , the viscosity increase rate is (η - η ｓ ) / η ｓ , which is defined as the specific viscosity η ｓｐ . The increase in viscosity per unit concentration c of the measurement target solution is η ｓｐ / c, which is defined as the reduced viscosity η ｒｅｄ . The intrinsic viscosity [η] is obtained by extrapolating the vertical axis (reduced viscosity) so that the concentration of the measurement target solution (horizontal axis) becomes zero. As a result, viscosities of 0.20, 0.12, and 0.08 (Pa·s) were obtained from the raw material, sample 1, and sample 2, respectively.

[0031] Test Example 7 For 10 g of a sample (manufactured by Nippon Pharmaceutical Co., Ltd.) containing approximately 70% of shark-derived chondroitin sulfate sodium dissolved in 100 mL of water and approximately 10% of other type II collagen, 5 g of cetylpyridinium chloride was added and stirred at room temperature for 30 minutes. The resulting precipitate was collected, the aqueous layer was removed as much as possible, ethanol was added to remove excess cetylpyridinium chloride, 80 mL of pyridine was added, 10 mL of DMF containing 10 g of sulfur trioxide pyridine complex was added, and heated for 3 hours. After removing the solvent, water was added to the crude product to dissolve it, adjusted to pH 10 or higher with 30% sodium hydroxide solution, and then dialyzed against water. After adding NaCl to the aqueous solution of the crude product, it was dropped into ethanol. The precipitate was collected by filtration to obtain a polysulfated product. The same post-treatment was carried out 6 hours after the reaction to obtain polysulfated chondroitin sulfate.

[0032] In Test Example 8, the proton nuclear magnetic resonance spectra of the polysulfated chondroitin sulfate obtained in Example 1 (average number of sulfate groups per disaccharide: 2.6) and the polysulfated chondroitin sulfate obtained in Test Example 7 were compared (Figure 6). From Figure 6, it can be seen that even when prepared using approximately 70% chondroitin sulfate sodium with a low degree of purity as the raw material (Test Example 7), polysulfated chondroitin sulfate with the same average number of sulfate groups per disaccharide as when prepared using purified 100% chondroitin sulfate sodium (Example 1) was obtained. In the conventional manufacturing method (the method described in Japanese Patent Publication No. 11-166001; described above), it is essential to use purified, high-purity chondroitin sulfate sodium as the raw material. However, from the above results, it can be seen that in the method of the present invention, polysulfation can be easily carried out even if the raw material chondroitin sulfate sodium is not a highly purified product.

Claims

1. A one-pot method for producing polysulfated chondroitin sulfate having the following physicochemical properties: a) sulfate group content: 20.1 to 43.7%, and b) intrinsic viscosity: 0.08 to 0.20, the method comprising converting chondroitin sulfate into a water-insoluble alkylpyridinium complex in a solution of chondroitin sulfate or a salt thereof.

2. The manufacturing method according to claim 1, wherein the alkylpyridinium is selected from the group consisting of cetylpyridinium fluoride, chloride, iodide, and bromide.

3. The method for producing the alkylpyridinium complex according to claim 1, comprising polysulfating the alkylpyridinium complex in an aprotic solvent using a sulfur trioxide complex.

4. The manufacturing method according to claim 3, wherein the sulfur trioxide complex is selected from the group consisting of a pyridine complex of sulfur trioxide, an N,N-dimethylformamide complex, and a trimethylamine complex.

5. The production method according to claim 3, wherein the aprotic solvent is selected from the group consisting of pyridine, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

6. The production method according to claim 1, wherein the polysulfated chondroitin sulfate is a chondroitin sulfate having an E structure in which two positions, 4 and 6, of the N-acetylgalactosamine of chondroitin are sulfated.

7. The manufacturing method according to claims 1 to 6, wherein the chondroitin sulfate is derived from a fish or mammal.

8. The manufacturing method according to claim 7, wherein the chondroitin sulfate is derived from a source selected from the group consisting of salmon, shark, ray, whale, cattle, and pigs.

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