Chondroitin sulfate polysaccharides, semi-synthetic preparation method and uses thereof

Abstract

The present invention relates to the field of pharmaceutical technology, in particular to chondroitin sulfate polysaccharide, its semi-synthetic preparation method, and its use. The chondroitin sulfate polysaccharide metal salt provided by the present invention has anti-inflammatory effects and can be used to prepare anti-inflammatory disease drugs. Specifically, the chondroitin sulfate polysaccharide metal salt provided by the present invention has anti-inflammatory and bone-protecting effects and can be used to prepare anti-rheumatoid arthritis disease drugs and anti-osteoarthritis disease drugs. The present invention also provides a method for preparing the chondroitin sulfate polysaccharide metal salt, which can be obtained by semi-synthetic means with different sulfation degrees, and the method is simple and suitable for large-scale production.

Description

[Technical Field]

[0001] The present invention relates to the pharmaceutical field, specifically to chondroitin sulfate polysaccharide metal salts and their preparation methods and uses. [Background technology]

[0002] Chondroitin sulfate polysaccharide (CS) is a linear, sulfated glycosaminoglycan composed of alternating β(1→3) and β(1→4) glycosidic bonds between glucose and galactosamine. Due to varying degrees of sulfation at the 2nd and 3rd positions of glucuronic acid and the 4th and 6th positions of galactosamine, 11 natural subtypes are derived, with the specific structures shown below. [ka]

[0003] Chondroitin sulfate polysaccharides are widely distributed in animal bodies and are also endogenous substances in humans. They play important roles in physiological and pathological processes such as those in the nervous system, cancer, and inflammation by regulating the expression levels of multiple enzymes and factors.

[0004] All chondroitin sulfate medicines and supplements currently on the market are derived from cartilage tissue extracts of terrestrial animals and consist of type A with a small amount of type C. However, chondroitin sulfate polysaccharides derived from marine animals differ in that their main components are often subtypes such as type C (shark cartilage extract) and type E (giant squid cartilage extract). Research has shown that chondroitin sulfate polysaccharides with different compositions often exhibit significant differences in pharmacological activity, and there remains a significant gap in the development of highly pharmacologically active chondroitin sulfate polysaccharide drugs. Summary of the Invention

[0005] An object of the present invention is to provide a chondroitin sulfate polysaccharide metal salt, a method for preparing the same, and uses thereof. The chondroitin sulfate polysaccharide metal salt provided by the present invention has excellent pharmacological activity.

[0006] In order to solve the above problems, the present invention provides the following technical means.

[0007] A first aspect of the present invention provides a chondroitin sulfate polysaccharide metal salt (Formula I), which is electrically neutral as a whole and comprises a chondroitin sulfate polysaccharide anion and a metal cation. [ka]

[0008] The average molecular weight of the chondroitin sulfate polysaccharide anion is 1,000 to 15,000 Da.

[0009] -SO3 in the chondroitin sulfate polysaccharide anion - and -COO - The molar ratio of -SO3 in the chondroitin sulfate polysaccharide anion is preferably 1.46 to 2.73. - and -COO - The molar ratio is 1.9 to 2.5.

[0010] The range of the chondroitin sulfate polysaccharide anion n is 2≦n≦45.

[0011] Preferably, the average molecular weight of the chondroitin sulfate polysaccharide anion is 4,000 to 15,000 Da.

[0012] Preferably, the metal cations include sodium ions and / or calcium ions and / or potassium ions.

[0013] Preferably, the range of n for the chondroitin sulfate polysaccharide anion is 6≦n≦20.

[0014] In the present invention, the chondroitin sulfate polysaccharide corresponding to the chondroitin sulfate polysaccharide anion mainly contains chondroitin sulfate polysaccharide C, chondroitin sulfate polysaccharide E, and chondroitin sulfate polysaccharide A, with the remainder containing other subtypes of chondroitin sulfate polysaccharides. When the weight content of the chondroitin sulfate polysaccharide is taken as 100%, the total weight content of the chondroitin sulfate polysaccharide C, chondroitin sulfate polysaccharide E, and chondroitin sulfate polysaccharide A is preferably 60 to 92%; the weight content of the chondroitin sulfate polysaccharide C is preferably 0 to 30%, more preferably 10 to 25%; the weight content of the chondroitin sulfate polysaccharide E is preferably 0 to 80%, more preferably 10 to 60%; and the weight content of the chondroitin sulfate polysaccharide A is preferably 0 to 90%, more preferably 0 to 70%.

[0015] In the present invention, the -SO3 in the chondroitin sulfate polysaccharide anion - The weight content is preferably 14 to 27%.

[0016] In the present invention, the weight content of alduronic acid in the chondroitin sulfate polysaccharide anion is preferably 20 to 35%, and the weight content of hexosamine is preferably 22 to 32%.

[0017] In the present invention, the metal cations preferably include sodium ions and / or calcium ions and / or potassium ions, and more preferably sodium ions or calcium ions.

[0018] A second aspect of the present invention provides a method for preparing the chondroitin sulfate polysaccharide metal salt of the first aspect, comprising the steps of:

[0019] a step of mixing the polysaccharide chondroitin sulfate raw material, a sulfation reagent, and an organic solvent, and carrying out a sulfation reaction to obtain a sulfated product; The sulfated product system is subjected to a first precipitation treatment, a salt formation treatment, dialysis, a second precipitation treatment and column purification in order to obtain a chondroitin sulfate polysaccharide metal salt; the salt-forming reagent used in the salt formation treatment is an aqueous metal hydroxide solution.

[0020] The polysaccharide chondroitin sulfate raw material contains chondroitin sulfate polysaccharide A and chondroitin sulfate polysaccharide C, of ​​which the weight content of chondroitin sulfate polysaccharide A is 70-90%, and the weight content of chondroitin sulfate polysaccharide C is 10-30%. In the present invention, the polysaccharide chondroitin sulfate raw material used was purchased from Yantai Dongcheng Pharmaceutical Group Co., Ltd. The polysaccharide chondroitin sulfate raw material has an average molecular weight of 20,000-23,000 Da and -SO3 - and -COO - The molar ratio of chondroitin sulfate polysaccharide A is 70-90%, and the weight content of chondroitin sulfate polysaccharide C is 10-30%, which meets the relevant requirements of the 2015 edition of the "Chinese Pharmacopoeia".

[0021] The average molecular weight of the polysaccharide chondroitin sulfate raw material is 20,000 to 23,000 Da; In the raw material of the polysaccharide chondroitin sulfate, -SO3 - and -COO - The molar ratio is 0.9 to 1.1.

[0022] The sulfating reagent includes one or more of sulfur trioxide trimethylamine complex, sulfur trioxide pyridine complex, and sulfur trioxide triethylamine complex; The equivalent ratio of the sulfation reagent to the repeating disaccharide fragment in the polysaccharide chondroitin sulfate raw material is (1 to 10):1, more preferably (3 to 8):1.

[0023] The sulfation reaction is carried out at a temperature of 40 to 120°C for 2 to 36 hours, more preferably 6 to 24 hours. In the present invention, the sulfation reaction is preferably carried out under stirring conditions; the stirring speed is not particularly limited in the present invention, and may be any stirring speed well known to those skilled in the art.

[0024] The salt-forming reagent is an aqueous sodium hydroxide solution and / or an aqueous potassium hydroxide solution; the concentration of the salt-forming reagent is 1 to 4 mol / L.

[0025] The first precipitation reagent used in the first precipitation treatment is an aqueous ethanol solution with a volume fraction of 90 to 95%; The second precipitation reagent used in the second precipitation treatment is ethanol; The cut-off molecular weight of the dialysis bag used in the dialysis is 3,000 to 12,000 Da.

[0026] In the present invention, the addition method, addition rate, and amount of the first precipitation reagent are not particularly limited as long as the sulfated product in the sulfated product system is completely precipitated. In the present invention, the time for the first precipitation treatment is preferably 25 to 35 minutes, and the first precipitation treatment is preferably carried out at room temperature under stirring conditions.

[0027] After the first precipitation treatment is completed, the present invention preferably filters the resulting system, dissolves the resulting solid by adding water, and then mixes the resulting solution with a salt-forming reagent to perform a salt-forming treatment. In the present invention, the ratio of water to solid used in the dissolution is preferably (0.8-1.2) mL:1 g, more preferably 1 mL:1 g. In the present invention, the salt-forming reagent is preferably an aqueous sodium hydroxide solution and / or an aqueous potassium hydroxide solution, more preferably an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution. The concentration of the salt-forming reagent is preferably 1-4 mol / L, more preferably 2-3 mol / L. When the salt-forming reagent is an aqueous sodium hydroxide solution and / or an aqueous potassium hydroxide solution, the concentration of the salt-forming reagent is the sum of the concentrations of sodium hydroxide and potassium hydroxide. In the present invention, there is no particular limitation on the amount of salt-forming reagent added, but it is preferable that the pH value of the resulting system after mixing the dissolved substance and salt-forming reagent is neutral, ensuring the formation of the corresponding sodium salt and / or potassium salt.

[0028] After the salt formation treatment is completed, in the present invention, the obtained solution is subjected to dialysis, and the cut-off molecular weight of the dialysis bag used in the dialysis is preferably 3,000 to 12,000 Da, more preferably 8,000 to 12,000 Da. In the present invention, the dialysis time is preferably 2 to 3 days, more preferably 2 days; during the dialysis process, it is desirable to change the water every 12 hours.

[0029] After the dialysis is completed, the present invention preferably evaporates the resulting system, adds an aqueous sodium acetate solution (NaOAc solution) to the resulting residue to dissolve it, and then mixes the resulting dissolved substance with ethanol to perform a second precipitation treatment. In the present invention, the mass concentration of the NaOAc aqueous solution is preferably 2 to 5%, more preferably 2 to 3%. In the present invention, the reason for using the NaOAc aqueous solution to dissolve the residue is to ensure that the residue is more soluble in ethanol, facilitating the subsequent second precipitation treatment. In the present invention, the ratio of the amount of the residue, the aqueous NaOAc solution, and the amount of ethanol used is preferably 1 g:(8 to 12) mL:(3.5 to 4.5) mL, more preferably 1 g:10 mL:4 mL.

[0030] After the second precipitation treatment is completed, the resulting system is preferably centrifuged, and the resulting solid is a crude chondroitin sulfate polysaccharide metal salt product. To ensure that the final chondroitin sulfate polysaccharide metal salt has high purity, the present invention preferably repeats the first precipitation treatment, salt formation treatment, dialysis, and second precipitation treatment on the crude chondroitin sulfate polysaccharide metal salt product, and then performs subsequent column purification on the resulting crude product. In the present invention, the gel column used in the column purification is preferably a G25 gel column, and the elution solvent used in the column purification is preferably water.

[0031] After the column purification is completed, the eluate is preferably dried to obtain the chondroitin sulfate polysaccharide metal salt. In the present invention, the drying is preferably freeze-drying. In the present invention, the temperature and time of the freeze-drying are not particularly limited, as long as the substance can be sufficiently dried.

[0032] The third aspect of the present invention provides a pharmaceutical composition. The composition comprises the chondroitin sulfate polysaccharide metal salt of the first aspect and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared by methods known in the art. The compound of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or auxiliaries to form any dosage form suitable for human or animal use. The weight content of the compound of the present invention in the pharmaceutical composition is usually 0.1-95%.

[0033] The compound of the present invention or a pharmaceutical composition containing the same can be administered in the form of a unit dose, and the route of administration may be digestive or non-digestive, for example, orally, intravenously, intramuscularly, subcutaneously, via the nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0034] The dosage form may be a liquid, solid, or semi-solid preparation. Liquid preparations may include solutions (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil, and double emulsions), suspensions, injections (including liquid injections, powder injections, and drops), eye drops, nasal drops, lotions, and liniments. Solid preparations may include tablets (including inclusion tablets, enteric-coated tablets, troches, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, pills, suppositories, membranes, patches, aerosols (powders), and sprays. Semi-solid preparations may include ointments, gels, pastes, and the like.

[0035] The compound of the present invention may be formulated into a general formulation, or may be formulated into a sustained-release formulation, a controlled-release formulation, a targeting formulation, or various microparticle drug delivery systems.

[0036] In order to form the compound of the present invention into tablets, various excipients generally known in the art may be used, specifically, diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents may be water, ethanol, isopropanol, etc.; binders may be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic slurry, gelatin paste, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyglycol, etc.; disintegrants may be dry starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethylcellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfonate, etc.; lubricants and flow aids may be talc powder, silica, stearates, tartaric acid, liquid paraffin, polyglycol, etc.

[0037] Furthermore, tablets may be coated tablets, for example sugar-coated tablets, thin-coated tablets, enteric-coated tablets, or double-layered and multi-layered tablets.

[0038] To prepare a dosage unit as a capsule, the active ingredient compound of the present invention may be mixed with a diluent and a flow aid, and the mixture may be directly placed in a hard or soft capsule. Alternatively, the active ingredient compound of the present invention may first be prepared as granules or micro-pills with a diluent, binder, and disintegrant, and then placed in a hard or soft capsule. The types of diluents, binders, wetting agents, disintegrants, and flow aids used in preparing tablets of the present compound may also be applied to preparing capsules of the present compound.

[0039] To prepare the compound of the present invention as an injection, water, ethanol, isopropanol, propylene glycol, or a mixture thereof may be used as a solvent, and an appropriate amount of a solubilizer, dissolution aid, pH adjuster, or osmolality adjuster commonly used in the art may be added. The solubilizer or dissolution aid may be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmolality adjuster may be sodium chloride, mannitol, glucose, phosphate, acetate, etc. When preparing a freeze-dried powder injection, mannitol, glucose, etc. may be added as a proppant.

[0040] If necessary, coloring agents, preservatives, flavoring agents, taste-correcting agents or other additives may be added to the pharmaceutical preparation.

[0041] To achieve the intended purpose of administration and enhance the therapeutic effect, the drug or drug composition of the present invention may be administered by any known administration method.

[0042] The dosage of the compound pharmaceutical composition of the present invention may vary widely depending on the nature and progression of the disease to be prevented or treated, the individual condition of the patient or animal, the administration route and dosage form, etc. Generally, the preferred daily dosage range of the compound of the present invention may be 0.001-150 mg / kg body weight, preferably 0.1-100 mg / kg body weight, more preferably 1-60 mg / kg body weight, and most preferably 2-30 mg / kg body weight. The dosage may be administered in a single dosage unit or divided into multiple dosage units, and may be appropriately determined according to the doctor's clinical experience and the dosage plan including other treatment measures.

[0043] The compounds or compositions of the present invention can be administered alone or in combination with other therapeutic drugs or symptom-treating drugs. When the compounds of the present invention have a synergistic effect with other therapeutic drugs, the dosage can be adjusted according to the actual situation.

[0044] A fourth aspect of the present invention is the use of the chondroitin sulfate polysaccharide metal salt according to the first aspect of the present invention in the preparation of an anti-inflammatory drug for treating inflammatory diseases, including osteoarthritis and rheumatoid arthritis.

[0045] The present invention provides a chondroitin sulfate polysaccharide metal salt. The chondroitin sulfate polysaccharide metal salt provided by the present invention exhibits anti-inflammatory effects by inhibiting the NF-κB signaling pathway and simultaneously suppressing the expression and function of inflammation-related factors and enzymes. As can be seen from the results of the examples, the chondroitin sulfate polysaccharide metal salt provided by the present invention has anti-inflammatory effects and can be used to prepare anti-inflammatory drugs.

[0046] The present invention provides a chondroitin sulfate polysaccharide metal salt. The chondroitin sulfate polysaccharide metal salt provided by the present invention not only has anti-inflammatory activity, but also has the effect of increasing bone mineral density. As can be seen from the results of the examples, the chondroitin sulfate polysaccharide metal salt provided by the present invention has anti-inflammatory and bone-protecting effects and can be used to prepare anti-rheumatoid arthritis drugs.

[0047] The present invention provides a chondroitin sulfate polysaccharide metal salt. The chondroitin sulfate polysaccharide metal salt provided by the present invention can increase the water content of cartilage and has the effect of protecting cartilage. As can be seen from the results of the examples, the chondroitin sulfate polysaccharide metal salt provided by the present invention has anti-inflammatory and cartilage-protecting effects and can be used to prepare anti-osteoarthritis drugs.

[0048] Beneficial technical effects: When the sulfation degree of the compounds of the present invention is high (the ratio of sulfate ions to carboxylate ions is within a specific range), they have outstanding in vivo anti-inflammatory activity, exceeding that of naturally occurring CS-E. There is a clear structure-activity relationship between the sulfation degree of the compounds of the present invention and their in vivo anti-inflammatory activity. In an animal model of acute ulcerative proctitis, the compounds of the present invention also exhibit outstanding biological activity and have significant therapeutic effects. Compared with naturally occurring CS-A and CS-E, the compounds of the present invention exhibit outstanding biological activity and have significant therapeutic effects in both an animal model of anti-type I collagen-induced mouse toe swelling (RA) and a papain-induced rat arthritis (OA) model.

[0049] The present invention provides a method for preparing the chondroitin sulfate polysaccharide metal salt, which can be produced by semi-synthetic means to produce chondroitin sulfate polysaccharide metal salts with different sulfation degrees, and the method is simple to operate and suitable for large-scale production. [Brief explanation of the drawings]

[0050] [Figure 1] 1 is an HPLC spectrum of polysaccharide 1 prepared in Example 1; [Figure 2] 1 is an HPLC spectrum of polysaccharide 2 prepared in Example 2; [Figure 3] 1 is an HPLC spectrum of polysaccharide 3 prepared in Example 3; [Figure 4] 1 is an HPLC spectrum of polysaccharide 4 prepared in Example 4; [Figure 5] This is a comparison diagram of the weight changes of UC mice in different groups in Experimental Example 3. Here, ##p<0.01 vs. Con; *p<0.05 vs. Mod; [Figure 6] This is a comparison diagram of the colon length of UC mice in different groups in Experimental Example 3. Here, ##p<0.01 vs. Con; *p<0.05 vs. Mod; [Figure 7]This is a comparison diagram of DAI scores of mice in different groups in Experimental Example 3, where ##p<0.01 vs. Con; **p<0.01, *p<0.05 vs. Mod. [Figure 8] MRI images of rat knee joints from different groups in Test Example 5. Figure 8 shows that in the CON group, the joint space was normal, the articular cartilage surface was intact and undamaged, the subchondral bone was normal, the cartilage thickness was normal, there was a moderate amount of infrapatellar fat, and there was a synovial membrane and a small amount of synovial fluid in the joint; in the MOD group, the joint space was narrowed, the articular cartilage surface was damaged and missing, the cartilage margin was incomplete, the cartilage shape was unclear, and the cartilage thickness was reduced; in the 5H group, the joint shape was clear, the joint space had returned to normal, and there was a small amount of synovial fluid. [Figure 9] CT images of rat knee joints from different groups in Test Example 5. Figure 9 shows the shape of the rat knee joint in the CON group; in the MOD model group, bone destruction appeared in the bone beneath the knee joint cartilage, with significant erosion, obvious cavities formed in the joint, and deformation of the bone joint; in the POS-positive drug group and 5L group, bone destruction and erosion appeared to be relatively mild compared to the model group; and in the 5M and 5H groups, significant improvements in bone destruction and erosion were observed compared to the model group. DETAILED DESCRIPTION OF THE INVENTION

[0051] Example 1 (1) 26.8 g of commercialized polysaccharide chondroitin sulfate A (Yantai Dongcheng Pharmaceutical Group Co., Ltd., No. CSJ1170701) was dissolved in 360 mL of dimethyl sulfoxide, and 24 g (3 eq.) of sulfur trioxide trimethylamine complex was added. The mixture was stirred and reacted at 50°C for 6 hours;

[0052] (2) The resulting reaction system was cooled to room temperature, and 400 mL of 95% ethanol solution was added. After stirring for 30 minutes, the system was filtered. Water was added to the solid to dissolve it (the ratio of water to solid was 1 mL:1 g). The pH was adjusted to 7.0 with 2 mol / L NaOH solution. The resulting solution was placed in a dialysis bag (cutoff molecular weight: 12,000 Da). After dialysis for 2 days, the solid was obtained by evaporation.

[0053] (3) The obtained solid was dissolved in a 2% by mass aqueous solution of NaOAc, and then absolute ethanol was added to the solution to cause sufficient precipitation, followed by centrifugation to obtain a crude product; the ratio of the amount of the solid, the aqueous NaOAc solution, and the amount of absolute ethanol used was 1 g:10 mL:40 mL;

[0054] (4) The obtained crude product was repeatedly subjected to steps (2) and (3), and then the obtained substance was purified on a G25 gel column (the eluent used was HO). Finally, the obtained eluate was freeze-dried to obtain 21.3 g of chondroitin sulfate polysaccharide sodium salt (referred to as Polysaccharide 1).

[0055] Example 2 Chondroitin sulfate polysaccharide was prepared according to the process of Example 1. The difference was that in step (1), 6.7 g of commercial polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and 8 g (4 eq.) of sulfur trioxide trimethylamine complex was added; the mixture was stirred and reacted at 60°C for 24 hours. Finally, 4.4 g of chondroitin sulfate polysaccharide sodium salt (referred to as polysaccharide 2) was obtained.

[0056] Example 3 Chondroitin sulfate polysaccharide was prepared according to the process of Example 1. The difference is that in step (1), 6.7 g of commercial polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and 10 g (5 eq.) of sulfur trioxide trimethylamine complex was added; the mixture was stirred and reacted at 60°C for 24 hours, and the cut-off molecular weight of the dialysis bag was 7000 Da. Finally, 4.1 g of chondroitin sulfate polysaccharide sodium salt (referred to as polysaccharide 3) was obtained.

[0057] Example 4 Chondroitin sulfate polysaccharide was prepared according to the process of Example 1. The difference is that in step (1), 6.7 g of commercial polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and 12 g (6 eq.) of sulfur trioxide trimethylamine complex was added; the mixture was stirred and reacted at 70°C for 20 hours, and the cut-off molecular weight of the dialysis bag was 5000 Da. Finally, 3.9 g of chondroitin sulfate polysaccharide sodium salt (referred to as polysaccharide 4) was obtained.

[0058] Example 5 Chondroitin sulfate polysaccharide was prepared according to the process of Example 1. The difference is that in step (1), 1.0 g of commercial polysaccharide chondroitin sulfate A was dissolved in 13 mL of dimethyl sulfoxide, and 2.4 g (8 eq.) of sulfur trioxide trimethylamine complex was added; the mixture was stirred and reacted at 100°C for 36 hours, and the cut-off molecular weight of the dialysis bag was 3500 Da. Finally, 1.0 g of chondroitin sulfate polysaccharide sodium salt (referred to as polysaccharide 5) was obtained.

[0059] Pharmacological experiments Experimental Example 1 The compositions of polysaccharides 1 to 5 prepared in Examples 1 to 5 were analyzed. Specifically, the average molecular weight (Da), -SO3 - / -COO - (molar ratio value), -SO3 -The content, alduronic acid content, hexosamine content, anticoagulant capacity (IU), degradable polysaccharide content, and proportion of each subtype component (Polysaccharides 1 to 5 prepared in Examples 1 to 5 were degraded with chondroitin sulfate ABC enzyme, and the proportion of each subtype component contained therein, specifically, polysaccharide chondroitin sulfate A, polysaccharide chondroitin sulfate C, and polysaccharide chondroitin sulfate E, was measured using HPLC. Note that polysaccharides 1 to 5 also contain other subtypes of chondroitin sulfate polysaccharides, so no further limitations are given here; the HPLC spectra of polysaccharides 1 to 4 are shown in Figures 1 to 4), and the specific results are shown in Table 1.

[0060] [Table 1]

[0061] Experimental Example 2 The anti-inflammatory effects of polysaccharides 1 to 5 prepared in Examples 1 to 5 were evaluated, the details of which are as follows:

[0062] (1) Test method: Experiment to evaluate the pharmacodynamic activity of CS compounds in a mouse ear model of acute swelling and inflammation induced by rhododendron oil Animals: Balb / c mice, male (20-22 g); 8 mice / group.

[0063] Grouping: The rats were divided into a model group, an active drug indomethacin group (5 mg / kg), a natural extract CS-E group (200 mg / kg), and a polysaccharide group (200 mg / kg, 100 mg / kg, 50 mg / kg). Both the active drug and CS-based compounds were prepared in 0.5% sodium carboxymethylcellulose by mass and stored in a refrigerator at 4°C.

[0064] Administration and measurement: Administration was once daily for 3 days. After the final administration, the percentage of ear swelling induced by the compound and the percentage of ear swelling inhibition by the drug were calculated.

[0065] Statistical analysis: Experimental results are expressed as "mean ± standard deviation." Statistical differences between the two groups were analyzed by t-test. * indicates p<0.05, ** indicates p<0.01.

[0066] (2) The anti-inflammatory effects of polysaccharides are shown in Tables 2 to 4.

[0067] [Table 2]

[0068] [Table 3]

[0069] [Table 4] As can be seen from Tables 2 to 4, there is a structure-activity relationship between the degree of sulfation and anti-inflammatory activity of polysaccharides 1 to 5. Polysaccharides 2 to 5 exhibited excellent anti-inflammatory effects in a mouse ear swelling model, superior to the naturally occurring CS-E polysaccharide. In particular, polysaccharide 3 exhibited significant anti-inflammatory activity within the range of 50 to 200 mg / kg, with a clear dose-response relationship.

[0070] Experimental Example 3 Polysaccharide 3 (referred to as SEMI5) prepared in Example 3 was subjected to pharmacodynamic evaluation in an anti-DSS model, the details of which are as follows:

[0071] I. Materials and Methods 1. Experimental animals C57BL / 6J mice, male (18~20g), 6 / group. Purchased from Beijing Huafukang Biological Technology Co., Ltd.; Permit number: SCXK (King) 2014-0004.

[0072] 2. Experimental group assignment (1) Normal control group: 0.5% sodium carboxymethylcellulose was orally administered by force.

[0073] (2) UC (Ulcerative Colitis) model group: Carboxymethylcellulose sodium was administered orally at a mass concentration of 0.5%.

[0074] (3) SASP (positive drug salazosulfapyridine) group: Shanghai Xinyi Pharmaceutical Co., Ltd. (lot number: 036151102); 500 mg / kg, prepared with 0.5% carboxymethylcellulose sodium, stored at 4°C, and administered orally once daily.

[0075] (4) SEMI5-50 group: 50 mg / kg, prepared with distilled water, stored at 4°C, and administered orally once daily.

[0076] (5) SEMI5-150 group: 150 mg / kg, prepared with distilled water, stored at 4°C, and administered orally once daily.

[0077] 3. Experimental method C57BL / 6J mice were housed in an SPF animal house (Experimental Animal Use Permit Number: SYXK (Kyoto) 2014-0023) and maintained on an adapted diet for one week before being randomly assigned to five groups according to the protocol described above. The UC model and treatment groups were administered daily DSS (MP, CA9011-18-1, US) using a laboratory-established method for modeling ulcerative colonic inflammation. The normal control and UC model groups were administered 0.5% sodium carboxymethylcellulose by oral gavage once daily. The SASP, SEMI5-50, and SEMI5-150 groups were administered 0.5% sodium carboxymethylcellulose by oral gavage once daily according to the experimental protocol described in the "Experimental Grouping" section. Seven days after modeling, animals in the UC model group exhibited typical UC symptoms, including fatigue, decreased activity, weight loss, loose stools, and bloody stools. At the end of the experiment, the animals in each group were euthanized, and various parameters related to colon inflammation were measured (as shown in the experimental results section), and the anti-UC pharmacodynamic activity of each compound was comprehensively evaluated.

[0078] 2. Experimental results 1. Effect of various concentrations of SEMI5 on body weight in DSS-induced UC model mice (Table 5 and Figure 5).

[0079] [Table 5]

[0080] As can be seen from Table 5 and Figure 5, compared with the normal control group, the UC model mice had a significant weight loss, which was statistically significant, suggesting successful UC modeling. The SASP and SEMI5-150 groups were unable to effectively alleviate DSS-induced acute UC, resulting in weight loss in C57 BL / 6J mice. However, when comparing the SEMI5-50 group with the UC model group, weight loss was effectively alleviated, which was statistically significant.

[0081] 2. Effect of various concentrations of SEMI5 on colonic contracture in UC mice (Table 6 and Figure 6).

[0082] [Table 6]

[0083] As can be seen from Table 6 and Figure 6, in the DSS-induced C57BL / 6J mouse acute UC animal model, the colon contracture of the UC model mice was significantly shortened compared to the normal control group, with a statistically significant difference. The SASP group failed to effectively improve colon contracture in the model animals. However, when compared with the UC model group, the SEMI5-50 and SEMI5-150 groups effectively improved colon contracture, with a statistically significant difference.

[0084] 3. The effect of each concentration of SEMI5 on the disease index (DAI) score in UC mice (Table 7 and Figure 7).

[0085] [Table 7]

[0086] As can be seen from Table 7 and Figure 7, the disease comprehensive index (DAI) of the UC model animals was significantly increased compared to the normal control group, with a statistically significant difference, suggesting successful modeling. Compared to the UC model group, the SEMI5-50 and SEMI5-150 groups showed a significant decrease in the experimental animal disease comprehensive index (DAI) scores, with a statistically significant difference. The SASP group showed some improvement, but the difference was statistically significant. The DAI score was evaluated based on indicators such as the degree of animal weight loss, stool characteristics, and stool blood. A lower DAI score indicates an animal's physiological state is closer to normal. The DAI score criteria are shown in Table 8.

[0087] [Table 8]

[0088] Experimental Example 4 Polysaccharide 3 (referred to as SEMI5) prepared in Example 3 was evaluated in an anti-type I collagen-induced mouse toe swelling model, the details of which are as follows:

[0089] I. Materials and Methods 1 Animal: DBAI mice, male (20-22 g); 7 mice / group.

[0090] The animals were divided into two groups: a blank control group, a model group, an active drug indomethacin group (5 mg / kg), a CSA group (200 mg / kg), a CSE group (200 mg / kg), a CS-E-semi3 group (200 mg / kg), and a CS-Semi-5 group (200 mg / kg). The active drug and CS compounds were prepared in 0.5% sodium carboxymethylcellulose and stored in a refrigerator at 4°C.

[0091] 3 Dosage frequency: once daily for 3 days.

[0092] 4. Experimental Method: Following established laboratory procedures, mice were weighed weekly after the first immunization. After the second shock immunization on day 21, the degree of joint swelling in each group of mice was observed weekly and evaluated using a joint swelling index score.

[0093] 5. Statistical analysis: Experimental results were expressed as "mean ± standard deviation." Statistical differences between the two groups were analyzed using the t-test. * indicates p<0.05, ** indicates p<0.01.

[0094] 2. Experimental Results [Table 9]

[0095] [Table 10]

[0096] [Table 11]

[0097] As can be seen from the comparison of the mouse weight results at termination in Table 9, SEMI5 drugs have a clear restorative effect on mouse weight loss. Table 10 shows that mouse joint swelling generally appears at 4 weeks, progressing from the toes to the soles of the feet and even to the heel-ankle joints. The lesions peak at 5-6 weeks, with a modeling success rate of 100%. SEMI5 drugs have a clear ameliorating effect on joint swelling. Table 11 suggests that SEMI5 drugs have a clear improving effect on bone mineral density, which is superior to that of active compounds. These results suggest that semisynthetic SEMI5 drugs have anti-RA activity.

[0098] Experimental Example 5 Polysaccharide 3 (designated SEMI5) prepared in Example 3 was evaluated in the anti-papain-induced OA model, details of which are as follows:

[0099] Materials and Methods: 1 animal: Male SD rats (180-220 g); 6 rats / group; purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.; Permit number: SCXK(Kyoto)2012-0001.

[0100] 2 groups: (1) Normal control group (Con group): DDW oral gavage.

[0101] (2) Model group (Mod group): DDW oral gavage.

[0102] (3) Positive drug celecoxib group (Pos group): purchased from Pfizer (W47055); prepared in DDW, stored at 4°C, and administered orally by gavage once daily.

[0103] (4) Compound semi5 low dose group (5L group): 50 mg / kg, prepared in DDW, stored at 4°C, and administered orally by gavage once daily.

[0104] (5) Compound semi5 medium dose group (5M group): 100 mg / kg, prepared in DDW, stored at 4°C, and administered orally by force once daily.

[0105] (6) Compound semi5 high dose group (5H group): 200 mg / kg, prepared in DDW, stored at 4°C, and administered orally by gavage once daily.

[0106] 2. Experimental Results 1. Net width of knee joint The width of the knee joint, with the skin and fat removed, is measured with a vernier caliper, and the degree of swelling of the joint is assessed intuitively.

[0107] [Table 12]

[0108] 2. Nuclear magnetic resonance results The degree of damage to rat knee articular cartilage was assessed using nuclear magnetic resonance (see Figure 8). At the same time, the change in cartilage mineralization measured under corresponding conditions was calculated to reflect the water content of the articular cartilage in each group. The higher the water content, the closer the articular cartilage is to a normal physiological state (see Table 13).

[0109] [Table 13]

[0110] As can be seen from Table 13, the mineralization index of the model group (Mod) animals was statistically significantly different from that of the normal control group (Con), strongly suggesting successful modeling of cartilage destruction; the mineralization index of the high-dose group animals was statistically significantly different from that of the model group (Mod). These results suggest that cartilage in the model group was significantly destroyed, while the treatment group had a significant improvement effect. **p<0.01 vs. Con; ##p<0.01 vs. Mod.

[0111] 3 CT appraisal results CT can be used to identify the subchondral bone in rat knee joints and determine the state of subchondral bone remodeling.

[0112] 3. Experimental conclusions 1. Pharmacodynamic evaluation of anti-DSS-induced acute ulcerative colitis model 1) The UC model animals showed significant weight loss, bloody stools, diarrhea, colonic contracture, and increased DAI composite index score and colonic tissue pathology score, suggesting successful modeling of the DSS-induced mouse ulcerative colitis model, and demonstrating that it is a reliable system suitable for evaluating the activity of anti-UC compounds.

[0113] 2) In the experiment using the SASP group, although it was unable to effectively reduce weight loss in UC model animals, it was able to improve phenomena such as diarrhea, bloody stool, and colonic contracture in UC model animals, suggesting that SASP has a certain anti-UC activity.

[0114] 3) The SEMI5-50 group effectively reduced weight loss in the model animals, and also demonstrated a certain degree of anti-UC activity in terms of reducing diarrhea, bloody stools, and DAI scores in the UC model animals, with statistically significant differences.These results demonstrate that, under this measurement system, SEMI5-50 has a clear therapeutic effect on the DSS-induced UC animal model.

[0115] 4) The SEMI5-150 group failed to reduce weight loss in UC model animals, which was not statistically significant, but it did improve colonic contracture, which was statistically significant, and it also demonstrated anti-UC activity in terms of diarrhea, bloody stool, and reduction of DAI and tissue scores in UC model animals, which was statistically significant. These results demonstrate that, under this measurement system, SEMI5-150 has a certain therapeutic effect on DSS-induced UC animal models.

[0116] 2. Pharmacodynamic evaluation of anti-type I protein-induced mouse toe swelling model 1) As can be seen from the comparison of the mouse weights at the end of treatment, SEMI5 was found to have a clear restorative effect on the weight loss of arthritic mice.

[0117] 2) SEMI5 has a clear improving effect on joint swelling.

[0118] 3) SEMI5 showed a clear improvement in joint bone mineral density, and the improvement was superior to that of anti-RA drugs. These results suggest that semi-synthetic SEMI5 has anti-RA activity.

[0119] 3. Pharmacodynamic evaluation of antipapain-induced osteoarthritis in rats 1) The knee joint width of the model group (Mod) animals was significantly increased compared with the normal control group (Con), with a statistically significant difference, suggesting that obvious swelling appeared in the joints of the model group animals; there was no statistical difference between the positive drug group (Pos) and the model group; there was a statistical difference between the knee joint width of the animals in each treatment group and the model group (Mod), suggesting that SEMI5 has relatively excellent anti-inflammatory activity.

[0120] 2) In the CON group, the joint space was normal, the articular cartilage surface was intact and undamaged, the subchondral bone was normal, the cartilage thickness was normal, there was a moderate amount of infrapatellar fat, and there was a synovial membrane and a small amount of synovial fluid in the joint; in the MOD group, the joint space was narrowed, the articular cartilage surface was missing due to damage, the outer cartilage edge was incomplete, the cartilage shape was unclear, and the cartilage thickness was reduced; in the SEMI5H group, the joint shape was clear, the joint space had returned to normal, and there was a small amount of synovial fluid.

[0121] 3) The calcification index of the model group (Mod) animals was statistically significantly different from that of the normal control group (Con), suggesting that significant cartilage destruction was successfully modeled; the calcification index of the high-dose group of SEMI5 animals was statistically significantly different from that of the model group (Mod). From these results, while cartilage destruction was significant in the model group, a clear improvement was observed in the treatment group.

[0122] 4) Compared with the normal knee joint shape of rats in the CON group, in the MOD model group, bone destruction was observed in the bone below the knee articular cartilage, with significant erosion, obvious cavities formed in the joint, and deformation of the bone joint was observed; in the POS-positive drug group and SEMI5L group, bone destruction and erosion appeared relatively mild compared with the model group; in the SEMI5M group and SEMI5H group, there was a clear improvement in bone destruction and erosion compared with the model group.

[0123] These results suggest that semisynthetic SEMI5 has anti-OA activity.

[0124] The above is merely a preferred embodiment of the present invention, and it goes without saying that even if a person skilled in the art makes various improvements and modifications without departing from the principles of the present invention, these improvements and modifications should also be considered to be within the scope of protection of the present invention.

Claims

1. A chondroitin sulfate polysaccharide metal salt represented by the following general formula (I), having the following structural general formula: 【Chemistry 1】 R=H or SO ３ － (I) It is electrically neutral overall and contains chondroitin sulfate polysaccharide anions and metal cations. the average molecular weight of the chondroitin sulfate polysaccharide anion is 4,000 to 15,000 Da; In the chondroitin sulfate polysaccharide anion, -SO ３ － and-COO － The molar ratio of is 1.9 to 2.5, The chondroitin sulfate polysaccharide metal salt, wherein the chondroitin sulfate polysaccharide anion n is in the range of 2≦n≦45.

2. 2. The chondroitin sulfate polysaccharide metal salt according to claim 1, wherein the metal cations include sodium ions and / or calcium ions and / or potassium ions.

3. a step of mixing a polysaccharide chondroitin sulfate raw material, a sulfation reagent, and an organic solvent to carry out a sulfation reaction, thereby obtaining a sulfated product system; and a step of sequentially subjecting the sulfated product system to a first precipitation treatment, a salt formation treatment, dialysis, a second precipitation treatment and column purification to obtain a chondroitin sulfate polysaccharide metal salt, 3. The method for preparing a chondroitin sulfate polysaccharide metal salt according to claim 1, wherein the salt-forming reagent used in the salt-forming treatment is an aqueous solution of a metal hydroxide.

4. The polysaccharide chondroitin sulfate raw material includes chondroitin sulfate polysaccharide A and chondroitin sulfate polysaccharide C, in which the weight content of the chondroitin sulfate polysaccharide A is 70-90% and the weight content of the chondroitin sulfate polysaccharide C is 10-30%; The average molecular weight of the polysaccharide chondroitin sulfate raw material is 20,000 to 23,000 Da; In the polysaccharide chondroitin sulfate raw material, -SO ３ － and-COO － The preparation method according to claim 3, characterized in that the molar ratio of is 0.9 to 1.

1.

5. the sulfating reagent comprises one or more of sulfur trioxide trimethylamine complex, sulfur trioxide pyridine complex, and sulfur trioxide triethylamine complex; The preparation method according to claim 3 or 4, characterized in that the equivalent ratio of the sulfation reagent to the repeating disaccharide fragment in the polysaccharide chondroitin sulfate raw material is (1-10):

1.

6. The method of claim 5, wherein the sulfation reaction temperature is 40-120°C, the reaction time is 2-36 hours, and the equivalent ratio of the sulfation reagent to the repeating disaccharide fragment in the polysaccharide chondroitin sulfate raw material is (3-8):

1.

7. The preparation method according to claim 3, characterized in that the salt-forming reagent is an aqueous sodium hydroxide solution and / or an aqueous potassium hydroxide solution, and the concentration of the salt-forming reagent is 1-4 mol / L.

8. the first precipitation reagent used in the first precipitation treatment is an aqueous ethanol solution having a volume fraction of 90 to 95%; the second precipitation reagent used in the second precipitation treatment is ethanol; The preparation method according to claim 3, characterized in that the cut-off molecular weight of the dialysis bag used in the dialysis is 3,000 to 12,000 Da.

9. A pharmaceutical composition comprising the chondroitin sulfate polysaccharide metal salt of claim 1 or 2 and a pharmaceutically acceptable carrier or excipient.

10. 3. Use of the chondroitin sulfate polysaccharide metal salt according to claim 1 or 2 in the preparation of an anti-inflammatory disease drug.

11. 11. The use according to claim 10, characterized in that the inflammatory diseases include ulcerative colitis, osteoarthritis and rheumatoid arthritis.

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