RU2843181C9 - Low-molecular chondroitin sulphate, composition thereof, method of producing and using thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to low-molecular chondroitin sulphate and a method for production thereof. Disclosed is a low molecular weight chondroitin sulphate with an average molecular weight of less than 1000 Da, having a narrow range of molecular weight distribution, content of chondroitin sulphate disaccharide 43–60% and content of chondroitin sulphate tetrasaccharide 30–45%, total content of chondroitin sulphate disaccharide and chondroitin sulphate tetrasaccharide more than 87%. Said low-molecular chondroitin sulphate is obtained through depolymerisation of chondroitin sulphate lyase with macromolecular chondroitin sulphate as an initial substance. Invention also discloses a method for the preparation of said low molecular weight chondroitin sulphate, use thereof to reduce joint inflammation, relieve pain and/or relieve or treat osteoporosis, as well as a composition containing same and a method for reducing inflammation of the joint, relieving pain and/or relieving or treating osteoporosis using same.

EFFECT: compared to the usual market macromolecular chondroitin sulphate, the proposed product has a more marked restorative action at concentration of 50–100 mcg/ml on chondrocytes, damaged by 1 mM hydrogen peroxide, with a strong reducing ability and a degree of restoration of 14–23%.

25 cl, 12 dwg, 2 tbl, 24 ex

Description

PRIOR ART

Field of technology

[0001] The invention relates to the biochemical field of technology, in particular to low molecular weight chondroitin sulfate, its composition, method of production and use.

Description of the Prior Art

[0002] Chondroitin sulfate (CS, namely macromolecular chondroitin sulfate or chondroitin sulfate polysaccharide) is a class of linear polysaccharide-containing polyanion. D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) are linked by a β-1,3 glycosidic bond to form disaccharide units, which are linked to each other by a β-1,4 glycosidic bond, and sulfate groups are introduced at different positions in the subsequent biosynthetic process. Chondroitin sulfate is widely distributed in cartilage and connective tissue of various animals.

[0003] A large number of studies have shown that CS has blood lipid-lowering activity, anti-atherosclerosis activity, immune-enhancing activity, anti-viral hepatitis activity and anti-tumor activity, and has been clinically widely used in medicine and food field for orthopedic diseases, ophthalmology, cardiovascular diseases and oral diseases. For example, in the treatment guideline for knee osteoarthritis published by the European League of Rheumatism, CS is regarded as an effective drug for the treatment of knee osteoarthritis; in Japan, CS is prepared as an oral preparation to relieve joint pain or as an eye drop to replenish tears or protect the cornea; in the United States, CS is sold as a food supplement; in Australia, CS is mainly included in compound preparations and sold as food supplements; Chondroitin sulfate sodium salt (CS-Na), CS-Na tablets and CS-Na capsules are also collected in the 2015 edition of Chinese Pharmacopoeia, and they are classified as drugs for lowering blood lipids and treating bone and joint diseases. Therefore, CS, as a kind of macromolecular substance with diverse biological activities and wide applications, has great value for development and application. However, the traditional high molecular weight CS has high apparent viscosity, complex structure, and is difficult to penetrate the cell membrane. In clinical use, it mainly faces the problems of low bioavailability, poor oral absorption and unstable efficacy.

[0004] The relative molecular weight (Mr) of natural CS is usually 50-100 kDa, and the range of Mr of CS obtained by different methods and from different sources is usually 10-40 kDa. When the Mr is below 10 kDa, then it is called low molecular weight chondroitin sulfate (LMWCS) or chondroitin sulfate oligosaccharide (CSO).

[0005] LMWCS is generally produced by cleaving CS products, including acid hydrolysis methods, alkaline hydrolysis methods and enzymatic depolymerization methods. The reaction products of acid hydrolysis have many impurities and are difficult to remove. The sulfonic acid groups on chondroitin sulfate will also be cleaved to varying degrees during the reaction and cause environmental pollution. In contrast, the reaction conditions of enzymatic depolymerization are mild, and therefore the pollution is reduced and easy to control, and the enzymatic depolymerization will not cause the destruction of the sulfonic acid group, which facilitates industrial production.

[0006] According to the prior art of patent literature, CN 102676613B discloses that bovine testicular hyaluronidase has low specificity and low efficiency, and a mixture of chondroitin sulfate disaccharide, tetrasaccharide and hexasaccharide is obtained and then separated into three monomers having a molecular weight of 521 Da, 1024 Da and 1527 Da, respectively, which are different in composition and molecular weight from the low molecular weight chondroitin sulfate of the present invention. The molecular weight of the disaccharide in the present method is about 379 Da and about 459 Da, and the molecular weight of the tetrasaccharide is about 838 Da and about 918 Da. In addition, no pharmacodynamic studies of the obtained product have been conducted in the patent literature.

[0007] Patent application CN 108070627 A discloses the use of chondroitin sulfate AC enzyme with high cost for producing chondroitin sulfate D tetrasaccharide with a specific structure and molecular weight, but its molecular weight is 1078 Da, and it is different from the molecular weight of the present low molecular weight chondroitin sulfate tetrasaccharide of about 838 Da and about 918 Da. In addition, no pharmacodynamic studies of the obtained product were conducted in this patent application.

[0008] Patent CN 103602711B discloses the use of a fermentation liquid after fermentation from Bacteria sulfolifolia , the efficiency of which is low, 1000L of the fermentation liquid can catalyze only 400kg of products, resulting in chondroitin sulfate disaccharide having a molecular weight of 450-480 Da, and the content of the disaccharide is more than 97%. Unlike the low molecular weight chondroitin sulfate in composition and molecular weight of the present invention, the molecular weight of the disaccharide in the present method is about 379 Da and about 459 Da, and the content ratio of the disaccharide is 42-58%. In addition, this patent discloses that the products are obtained from chicken cartilage, pig cartilage and bovine cartilage, and they are used for the treatment of myocarditis.

[0009] The periodical literature "Preparation of Chondroitin Sulfate Oligosaccharides by Enzymatic Method and Its Antioxidant Activity" (Food Industry Science and Technology, 2017, 13, 48-52) discloses the use of chondroitin sulfate enzyme (molecular weight 76 kDa) after fermentation from the enzyme-producing strain Acinetobacter sp.C26 . The catalytic efficiency is low, the reaction concentration is reported as only 2%, and the proportion of oligosaccharides is not analyzed. Chondroitin sulfate disaccharides and tetrasaccharides are obtained by pyrolysis, m/z of disaccharides is 342 Da and 458 Da, m/z of tetrasaccharides is 939 Da, which is different from the molecular weight of disaccharide of about 379 Da and about 459 Da and the molecular weight of tetrasaccharides of about 838 Da and about 918 Da in the present invention. In addition, the product obtained in this literature was tested only for antioxidant activity, and no other pharmacodynamic studies were conducted.

[0010] Therefore, the research on the pharmacodynamics of low molecular weight chondroitin sulfate at home and abroad is insufficient, especially for chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide as the main components, which have the content controlled within a certain range and the average molecular weight stably controlled to less than 1000 Daltons, and a narrow molecular weight distribution. Further research and identification of low molecular weight chondroitin sulfate for the treatment of joint damage are still needed to complement the research in this technical field at home and abroad.

SUMMARY OF THE INVENTION

[0011] The invention aims to overcome the above-mentioned disadvantages of the prior art and to provide a new low molecular weight chondroitin sulfate, its composition, method of production and use.

[0012] In order to realize the above-mentioned objects, one of the objects of the present invention is to provide the following technical solution: a low molecular weight chondroitin sulfate with an average molecular weight of less than 1000 Daltons and a narrow molecular weight distribution, containing chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide as the main components, of which the content of chondroitin disaccharide is 43-60%, and the content of chondroitin sulfate tetrasaccharide is 30-45%, the sum of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is more than 87%; the general formula of the structure of the low molecular weight chondroitin sulfate is shown in the following formula I:

Formula I: n=0-5, and n is an integer, R ₁ , R ₂ , R ₃ = -H or -SO ₃ Na.

[0013] Further, the average molecular weight of the low molecular weight chondroitin sulfate is 590-830 Da and preferably 677-742 Da.

[0014] Furthermore, in the low molecular weight chondroitin sulfate, the content of chondroitin sulfate disaccharide is 48-55%, and the content of chondroitin sulfate tetrasaccharide is 35-40%.

[0015] The second object of the invention is to provide the following technical solution: a method for producing low molecular weight chondroitin sulfate, wherein macromolecular chondroitin sulfate as a starting material is depolymerized by chondroitin sulfate lyase to obtain a product which is low molecular weight chondroitin sulfate with an average molecular weight stably controlled to less than 1000 Daltons and a narrow molecular weight distribution, wherein the low molecular weight chondroitin sulfate contains chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide as the main components, of which the content of chondroitin sulfate disaccharide is 43-60%, and the content of chondroitin sulfate tetrasaccharide is 30-45%, the sum of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is more than 87%; The general formula of the structure of low molecular weight chondroitin sulfate is shown in the following formula I:

Formula I: n=0-5, and n is an integer, R ₁ , R ₂ , R ₃ = -H or -SO ₃ Na.

[0016] Next, chondroitin sulfate lyase is obtained by carrying out the following steps: screening and identifying soil, wastewater or sludge samples from coastal areas, river banks, farmers' markets, slaughterhouses and canteens, and optimally expressed by Escherichia coli or Bacillus subtilis .

[0017] Further, the technical method is to use commercially available macromolecular chondroitin sulfate as a raw material for production, and this raw material is obtained from the cartilage tissue of land and sea animals. The raw material also refers to one or more mixtures of chicken cartilage, pig cartilage, cattle cartilage or shark bone, preferably shark bone.

[0018] Further, the working conditions of the enzymatic depolymerization reaction are as follows: the added amount of chondroitin sulfate lyase relative to the fermentation broth per liter is 100-300 U/L, the concentration of macromolecular chondroitin sulfate as a starting material is 100-700 g/L, the enzymatic depolymerization time is 6-40 hours, the enzymatic depolymerization temperature is 25-35°C, the stirring speed is 100-700 rpm, and the pH of the enzymatic depolymerization is 6.5-8.5. Further, the protein is removed from the hydrolysate with mixed solvents after the enzymolysis reaction in which the volume ratio of the hydrolysate and the mixed solvents is 2-5:1, and the volume ratio of dichloromethane and isopropyl alcohol in the mixed solvents is 3-5:1; The mixture is stirred at 100-500 rpm for 10-40 min, centrifuged at 3000-5000 rpm for 10-30 min, and the upper layer of the reaction solution is taken.

[0019] Further, the protein can also be removed from the hydrolyzate after the enzymatic depolymerization reaction by ultrafiltration to obtain a reaction solution.

[0020] Next, the reaction liquid of the upper layer is filtered and sterilized through a 0.22 μm capsule filter after removing the protein, and the reaction solution is added to 8-12 times the volume of anhydrous ethanol for precipitation and dried in a vacuum.

[0021] Next, the reaction solution is filtered and sterilized through a 0.22 μm capsule filter after removing the protein, and then spray dried.

[0022] Furthermore, compared with the macromolecular chondroitin sulfate from shark bone, the low molecular weight chondroitin sulfate obtained by enzymatic depolymerization of one or more than one of shark bone and chicken cartilage has a more significant restorative effect at a concentration of 50-100 μg/mL on chondrocytes damaged by 1 mM hydrogen peroxide, and the recovery rate is 14%-23%.

[0023] Furthermore, low molecular weight chondroitin sulfate with a specific range of disaccharide and tetrasaccharide content can repair chondrocytes damaged by 1 mM hydrogen peroxide in the concentration range of 50-1600 μg/mL, and the recovery rate is 20%-62.4%.

[0024] Further, low molecular weight chondroitin sulfate with a specific range of disaccharide and tetrasaccharide content can repair chondrocytes damaged by 1 mM hydrogen peroxide in a concentration range of 50-1600 μg/ml, and the recovery rate is more than 20%, preferably more than 40%, preferably more than 50%, and even more preferably more than 60%.

[0025] Furthermore, low molecular weight chondroitin sulfate finds application in the fields of preparing pharmaceuticals, cosmetics, health care products and foods.

[0026] According to another aspect of the invention, there is provided a composition of hydrolyzed chondroitin sulfate for increasing bone density and relieving arthritis. The daily dose of the composition of hydrolytic chondroitin sulfate and glucosamine is lower than the daily dose of the currently known commercially available composition of chondroitin sulfate and glucosamine; for example, the daily dose of chondroitin sulfate and glucosamine in Move free is 1700 mg/day; the daily dose of chondroitin sulfate and glucosamine in BJ Tomson is 1160 mg/day. By reducing the dosage, patient compliance can be improved, but the effect of increasing bone density and relieving arthritis is not reduced.

[0027] In the following context, glucosamine is sometimes referred to simply as "ammonia sugar".

[0028] In this context, the term "hydrolytic chondroitin sulfate" has the same meaning as the term "chondroitin sulfate oligosaccharide (CSO)" and the term "low molecular weight chondroitin sulfate (nmCS)".

[0029] The term "hydrolytic chondroitin sulfate" as used herein refers to mixtures of hydrolytic chondroitin sulfate of varying molecular weights. For example, the molecular weight can be greater than about 300 Da and less than 10,000 Da, such as 379-10,000 Da, such as greater than about 350 Da, greater than about 400 Da, greater than about 500 Da, greater than about 600 Da, greater than about 700 Da, greater than about 800 Da, greater than about 900 Da, less than about 9,000 Da, less than about 8,000 Da, less than about 7,000 Da, less than about 6,000 Da, less than about 5,000 Da, less than about 4,000 Da, less than about 3,000 Da, less than about 2,000 Da, or less than about 1,000 Da, preferably less than about 1,000 Da of the low molecular weight chondroitin sulfate product, more preferably about 590 Da-830 Yes, the same as about 600 Da-750 Da and even more preferable 677-742 Da.

[0030] In one embodiment of the invention, a hydrolyzed chondroitin sulfate composition is provided, which accepts the following technical proposal: the invention relates to a composition containing hydrolyzed chondroitin sulfate, which is characterized in that it contains a daily dosage of 50 mg-800 mg of hydrolyzed chondroitin sulfate for a human. In some embodiments, compositions containing hydrolyzed chondroitin sulfate contain about 50 mg to 800 mg of hydrolyzed chondroitin sulfate. In another embodiment, the composition comprising hydrolyzed chondroitin sulfate consists of about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, approximately 740 mg, approximately 750 mg, approximately 760 mg, approximately 770 mg, approximately 780 mg, approximately 790 mg, approximately 800 mg hydrolyzed chondroitin sulfate.

[0031] A composition containing hydrolyzed chondroitin sulfate is characterized in that this composition may contain glucosamine.

[0032] The invention relates to a composition containing hydrolyzed chondroitin sulfate, which is characterized in that the glucosamine can be a mixture of one or both glucosamine hydrochloride and glucosamine sulfate.

[0033] A composition containing hydrolyzed chondroitin sulfate is characterized in that the hydrolyzed chondroitin sulfate is a mixture of hydrolyzed chondroitin sulfate with different molecular weights.

[0034] A composition containing hydrolyzed chondroitin sulfate is characterized in that the ratio of hydrolyzed chondroitin sulfate to glucosamine is about 1:3-1:10, such as about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, preferably about 1:6-1:4, more preferably about 1:5. The composition may contain a daily dose for humans of about 150 mg-8000 mg of glucosamine. In some embodiments, the composition may comprise about 7200 mg, about 6400 mg, about 5600 mg, about 4800 mg, about 4000 mg, about 3200 mg, about 2400 mg, about 1600 mg, about 1200 mg, about 800 mg, about 600 mg, about 450 mg, about 400 mg, about 350 mg, about 300 mg, about 250 mg, about 200 mg, about 150 mg of glucosamine. The composition more preferably comprises about 100 mg of hydrolyzed chondroitin sulfate and about 500 mg of glucosamine. The composition may also comprise about 200 mg of hydrolyzed chondroitin sulfate and about 1000 mg of glucosamine.

[0035] The invention relates to a composition of hydrolyzed chondroitin sulfate, wherein the hydrolyzed chondroitin sulfate is a mixture obtained by enzymatic depolymerization, purification, concentration and spray drying of chondroitin sulfate, and its molecular weight is 379-10000 Da.

[0036] The composition containing hydrolyzed chondroitin sulfate also contains pharmaceutically acceptable excipients that are selected from fillers, disintegrants, adhesives, flavoring agents, lubricants and film coating agents.

[0037] The invention relates to a hydrolyzed chondroitin sulfate composition and a method for producing the same, which is characterized in that the fillers include, but are not limited to, microcrystalline cellulose, starch, dextrin, mannitol, lactose, etc. Disintegrants include, but are not limited to, crospovidone, sodium croscarmellose, sodium carboxymethyl starch, hydroxypropyl starch, pregelatinized starch, low-substituted hydroxypropyl cellulose (L-hydroxypropyl cellulose), sodium bicarbonate, citric acid, tartaric acid, etc. Adhesives include, but are not limited to, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, etc. Lubricants include, but are not limited to, magnesium stearate, silicon dioxide, talc, hydrogenated vegetable oil, polyethylene glycol, stearic acid, etc. The film coating agent composition includes, but is not limited to, hydroxypropyl methylcellulose, polyethylene glycol, color lake, etc.

[0038] The invention relates to a composition of hydrolyzed chondroitin sulfate and a method for producing it, which is characterized in that the preparation includes, but is not limited to, a tablet, a granule, a capsule, and a pill.

[0039] The composition of the invention can be used to produce medical products or drugs for the purpose of alleviating arthritis and relieving pain. In particular, the composition can be used to produce medical products or drugs for alleviating osteoarthritis, increasing bone density, alleviating osteoporosis, or alleviating pain.

[0040] A composition of low molecular weight chondroitin sulfate or hydrolyzed chondroitin sulfate can reduce serum levels of inflammatory cytokines IL-6, IL-1β and TNF-α and/or serum levels of the C5b-9 component.

[0041] A low molecular weight chondroitin sulfate composition or a hydrolyzed chondroitin sulfate composition may increase the level of hydroxyproline in the femur.

[0042] Compared with the prior art, hydrolyzed chondroitin sulfate obtained by enzymatic depolymerization of chondroitin sulfate can be used to produce medical products for increasing bone density, relieving osteoporosis and alleviating arthritis, and the dosage is low and the gastrointestinal irritation is less. The daily dose for humans of hydrolyzed chondroitin sulfate and glucosamine, the components constituting the composition of the invention, is lower than the daily dose of currently known chondroitin sulfate and glucosamine available on the market. By reducing the dosage, patient compliance can be improved, but the effect of increasing bone density and relieving arthritis is not reduced. The results of the evaluation of the degree of arthritis pain in mice and the daily dose in humans showed that the daily dose of hydrolyzed chondroitin sulfate from 50 to 800 mg has an obvious alleviating and treating effect on osteoarthritis pain, and the addition of glucosamine to the composition can increase the alleviating and treating effect on osteoarthritis pain and osteoporosis pain.

[0043] Compared with the prior art, the invention has the following advantages:

1. Chondroitin sulfate lyase is obtained by screening and identifying the soil, wastewater or sludge samples from coastal areas, river banks, farmers' markets, slaughterhouses and canteens, and optimally expressed by Escherichia coli or Bacillus subtilis; chondroitin sulfate lyase is used for enzymatic depolymerization, and it has good specificity and higher enzymatic activity. Low molecular weight chondroitin sulfate with an average molecular weight of less than 1000 Dalton can be stably obtained, especially low molecular weight chondroitin sulfate with an average molecular weight of 590-830 Da, which has a narrow molecular weight distribution.

2. The protein is removed by a solvent method or ultrafiltration method, and the protein content is no more than 0.5%.

3. 100L of fermentation broth catalyzes more than 400kg of macromolecular chondroitin sulfate. It provides the advantages of short production cycle, high efficiency, and is suitable for industrial scale-up.

4. The proportion of oligosaccharides of different components is determined by LC/MS (liquid chromatography/mass spectrometry) analysis. The quality of the product is stable. The total content of oligosaccharides of low molecular weight chondroitin sulfate with disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is more than 97%, and chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide are the main components; the content of chondroitin sulfate disaccharide is 43-60%, and the content of chondroitin sulfate tetrasaccharide is 30-45%, the sum of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is more than 87%.

5. Compared with the macromolecular chondroitin sulfate from shark bone, the low molecular weight chondroitin sulfate obtained by enzymatic depolymerization of one or more of shark bone and chicken cartilage has a more significant repair effect at a concentration of 50-100 μg/ml on chondrocytes damaged by 1 mM hydrogen peroxide, and the recovery rate is 14%-23%, which can be used for the treatment of joint damage. The repair effect of the low molecular weight chondroitin sulfate obtained from shark bone is better than that obtained from chicken cartilage, pig cartilage, bovine cartilage and mixed bone. At a concentration of 50 μg/ml, low molecular weight chondroitin sulfate obtained from shark bone, chicken cartilage, porcine cartilage, bovine cartilage and mixed bone exerted a more significant restorative effect on chondrocytes damaged by 1 mM hydrogen peroxide than macromolecular chondroitin sulfate obtained from shark bone.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0044] Fig. 1 is a distribution spectrum of low molecular weight chondroitin sulfate oligosaccharides from shark bone in Example 9.

[0045] Fig. 2 is a distribution spectrum of low molecular weight chondroitin sulfate oligosaccharides from shark bone in Example 10.

[0046] Fig. 3 is a distribution spectrum of low molecular weight chondroitin sulfate oligosaccharides from shark bone in Example 11.

[0047] Fig. 4 is a distribution spectrum of the low molecular weight chondroitin sulfate oligosaccharides from shark bone in Example 12.

[0048] Fig. 5 shows the results of tests for the efficacy and activity of low molecular weight chondroitin sulfate from different sources in Example 14.

[0049] Fig. 6 shows the effect of the composition of the invention on the body weight of mice.

[0050] Fig. 7 shows a diagram of the effect of the composition of the invention on the support force of mice.

[0051] Fig. 8 shows the distribution spectrum of low molecular weight chondroitin sulfate oligosaccharides with a purity of 97.72% in control group 1.

[0052] Fig. 9 shows the restorative effect of the sample from Example 9 and control groups 1-2 on chondrocytes damaged by 1 mM hydrogen peroxide at different concentration levels.

[0053] Fig. 10A shows the effects of different dose groups on IL-6 (A), IL-1β (B), TNF-α (C), and C5b-9 (D) levels in mouse serum.

[0054] Fig. 10B shows the determination of hydroxyproline (HYP) levels in mouse femurs.

[0055] Fig. 10C shows safranin O-fast green (×100) staining of mouse knee joint.

[0056] Fig. 10D shows the pathological score after safranin O-fast green staining of the mouse knee joint.

[0057] Fig. 10E shows immunohistochemical staining of C5b-9 (×100) in the mouse knee joint.

[0058] Fig. 10F shows the number of C5b-9-positive cells in the mouse knee joint.

[0059] Fig. 11A shows changes in body weight in rats in a CSO dose study in a rat model of osteoarthritis induced by papain injection into the knee joint.

[0060] Fig. 11B shows the level of IL-6 in the serum of rats in a CSO dose study in a rat model of osteoarthritis induced by papain injection into the knee joint.

[0061] Fig. 11C shows the level of IL-1β in the serum of rats in a CSO dose study in a rat model of osteoarthritis induced by papain injection into the knee joint.

[0062] Fig. 11D shows the serum TNF-α level in rats in a CSO dose-response study in a rat model of osteoarthritis induced by papain injection into the knee joint.

[0063] Fig. 11E shows pathological staining (×100) of the rat knee joint in a CSO dose study in a rat model of osteoarthritis induced by papain injection into the knee joint.

[0064] Fig. 11F shows the pathological score in the rat knee joint in a CSO dose study in a rat model of osteoarthritis induced by papain injection into the knee joint.

[0065] Fig. 12A shows changes in body weight in rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0066] Fig. 12B shows changes in body weight in male rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0067] Fig. 12C shows the femur weight ratio of male rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular osteoporosis in male rats.

[0068] Fig. 12D shows the femur weight ratio of female rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular osteoporosis in male rats.

[0069] Fig. 12E shows the bone mineral density of female rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0070] Fig. 12F shows the bone mineral density of male rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0071] Fig. 12G shows the ash content of rat femurs in a study of the therapeutic effects of CSO on ovariectomy-induced osteoporosis in female rats and testicular osteoporosis in male rats.

[0072] Fig. 12H shows the bone phosphorus level in rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0073] Fig. 12I shows the serum alkaline phosphatase (ALP) level in rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0074] Fig. 12J shows the serum level of bone morphogenetic protein-4 (BMP-4) in rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0075] Fig. 12K shows the serum calcitonin (CT) level in rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0076] Fig. 12L shows the level of parathyroid hormone (PTH) in the serum of rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0077] Fig. 12M shows the area of femoral trabeculae in rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0078] Fig. 12N shows the percentage of femoral trabecular area in rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

[0079] Fig. 12O shows the number of femoral trabeculae in rats in a study of the therapeutic effect of CSO on ovariectomy-induced osteoporosis in female rats and testicular excision-induced osteoporosis in male rats.

DESCRIPTION OF EMBODIMENTS

[0080] In order to facilitate those skilled in the art in understanding the present invention, the technical solutions of the invention will be described further below with reference to examples, but the following should in no way limit the scope of the invention claimed in the appended claims.

[0081] The substances, reagents, etc. used in the following examples are commercially available unless otherwise specifically stated. Chondroitin sulfate lyase was obtained by screening and identifying soil, wastewater, or sludge samples from coastal areas, river banks, farmers' markets, slaughterhouses, and canteens, and was optimally expressed by Escherichia coli or Bacillus subtilis . The highest activity for the enzyme is 11976.5 U/L, and the enzyme contains 998 amino acids and has a molecular weight of 113 kDa. The amino acid sequence of chondroitin sulfate lyase is disclosed in another patent application for the invention of the same applicant, filed on April 3, 2019, application number 201910264385.5, published on June 21, 2019, and publication number CN 109913437 A. All relevant information from that patent application is incorporated into this patent application.

[0082] The formula for calculating the average molecular weight of low molecular weight chondroitin sulfate is as follows:

where r _t =r _U1 +r _U2 +r _U3 +r _U4 +r _U5 ,

where r _u1 : the peak response value of component 1 (hexasaccharide and octasaccharide) in the sample solution; M _w1 means the molecular weight of component 1 in the sample solution;

r _u2 : the peak response value of component 2 (tetrasaccharide) in the sample solution; M _w2 means the molecular weight of component 2 in the sample solution;

r _u3 : the peak response value of component 3 (tetrasaccharide) in the sample solution; M _w3 means the molecular weight of component 3 in the sample solution;

r _u4 : the peak response value of component 4 (disaccharide) in the sample solution; M _w4 means the molecular weight of component 4 in the sample solution;

r _u5 : the peak response value of component 5 (disaccharide) in the sample solution; M _w5 means the molecular weight of component 5 in the sample solution;

r _t : the sum of the peak response values of component 1, component 2, component 3, component 4 and component 5 in the sample solution.

[0083] The molecular weights of the disaccharide (n=0), tetrasaccharide (n=1), hexasaccharide (n=2), and octasaccharide (n=3) in the low molecular weight chondroitin sulfate obtained by enzymatic depolymerization of the macromolecular chondroitin sulfate from shark bone were different. The content of decosacharide (n=4) and the content of dodecosacharide (n=5) were very low, so the average molecular weight of the oligosaccharide compositions was ignored in the calculation. The molecular weights of the disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide measured by liquid chromatography/mass spectrometry in the samples obtained in Example 9 are shown in Table 1 below.

[0084] Example 1. Enzymatic depolymerization reaction

[0085] 2 L of purified water were added to a 5 L beaker, then 800 g of shark bone chondroitin sulfate was added while stirring at 400 rpm. After all the substance was dissolved, sodium hydroxide solution was used to adjust the pH to 7.0, and 200 U/L of chondroitin sulfate lyase was added. The system was stirred at 30°C for 6 h, and it was determined whether the average molecular weight was less than 1000 Da. If the reaction was not complete, the reaction time was increased for another 4 h and central monitoring was continued. The reaction was continued until the average molecular weight was less than 1000 Da.

[0086] Example 2. Enzymatic depolymerization reaction

[0087] 2 L of purified water were added to a 5 L beaker, then 400 g of shark bone chondroitin sulfate was added while stirring at 700 rpm. After all the substance was dissolved, sodium hydroxide solution was used to adjust the pH to 6.5, and 300 U/L of chondroitin sulfate lyase was added. The system was stirred at 35°C for 6 h, and it was determined whether the average molecular weight was less than 1000 Da. If the reaction was not complete, the reaction time was increased for another 4 h and central monitoring was continued. The reaction was continued until the average molecular weight was less than 1000 Da.

[0088] Example 3. Enzymatic depolymerization reaction

[0089] 2 L of purified water were added to a 5 L beaker, then 200 g of shark bone chondroitin sulfate was added while stirring at 100 rpm. After all the substance was dissolved, sodium hydroxide solution was used to adjust the pH to 8.0, and 100 U/L of chondroitin sulfate lyase was added. The system was stirred at 25°C for 6 h, and it was determined whether the average molecular weight was less than 1000 Da. If the reaction was not complete, the reaction time was increased for another 4 h and central monitoring was continued. The reaction was continued until the average molecular weight was less than 1000 Da.

[0090] Example 4. Enzymatic depolymerization reaction

[0091] 2 L of purified water were added to a 5 L beaker, the stirring speed was controlled to 500 rpm, and then 1400 g of shark bone chondroitin sulfate was added. After all the substance was dissolved, sodium hydroxide solution was used to adjust the pH to 8.5 and 280 U/L of chondroitin sulfate lyase was added. The system was stirred at 28°C for 6 h and it was determined whether the average molecular weight was below 1000 Da. If the reaction was not complete, the reaction time was increased for another 4 h and central monitoring was continued. The reaction was continued until the average molecular weight was less than 1000 Da.

[0092] Example 5. Protein Removal

[0093] 2 L of the hydrolysate after the reaction in Example 1 was taken and transferred to a centrifuge. The hydrolysate after the reaction was centrifuged at 4200 rpm for 15 min to remove the thallus, the supernatant was collected, 0.4 L of an organic solvent (volume ratio of dichloromethane to isopropyl alcohol=5:1) was added to remove the protein. After stirring at 100 rpm for 40 min and centrifuging at 4200 rpm for 15 min, the upper layer of the reaction solution was collected and poured out.

[0094] Example 6. Protein Removal

[0095] 2.1 L of the hydrolysate after the reaction in Example 2 was taken and transferred to a centrifuge. The hydrolysate after the reaction was centrifuged at 4200 rpm for 15 min to remove the thallus, the supernatant was collected, 0.7 L of an organic solvent (volume ratio of dichloromethane to isopropyl alcohol=4:1) was added to remove the protein. After stirring at 500 rpm for 10 min and centrifuging at 3000 rpm for 30 min, the upper layer of the reaction solution was collected and poured out.

[0096] Example 7. Protein Removal

[0097] 2 L of the hydrolysate after the reaction in Example 3 was taken and transferred to a centrifuge. The hydrolysate after the reaction was centrifuged at 4200 rpm for 15 min to remove the thallus, the supernatant was collected, 1 L of an organic solvent (volume ratio of dichloromethane to isopropyl alcohol=3:1) was added to remove the protein. After stirring at 300 rpm for 30 min and centrifuging at 5000 rpm for 10 min, the upper layer of the reaction solution was collected and poured out.

[0098] Example 8. Protein Removal

[0099] 2.3 L of the hydrolysate after the reaction in Example 4 was taken and subjected to ultrafiltration through an ultrafiltration system using a membrane bag with a molecular weight cutoff of 50,000 to 80,000 under low temperature conditions so that the protein was removed, to obtain an ultrafiltration reaction solution.

[00100] Example 9. Alcohol precipitation and drying

[00101] The 2-liter upper layer of the reaction solution obtained in Example 5 was filtered and sterilized into a clean area through a 0.22 μm capsule filter. Then, the resulting filtrate was added dropwise to 20 L of anhydrous ethanol, stirred for 0.5 h and left for 2 h. After the solid was completely precipitated, the supernatant was removed. The solid was collected by centrifugal filtration and then dried in a vacuum drying oven at 45°C for 24 h until the weight loss was not more than 10%. Then, 650 g of the product, which was low molecular weight chondroitin sulfate, was obtained with a yield of 81.3% (i.e., the ratio of 650 g of low molecular weight chondroitin sulfate to 800 g of the original shark bone chondroitin sulfate). The protein content determined by Coomassie blue staining was 0.3%. The molecular weight distribution was determined by liquid chromatography mass spectrometry, as shown in Fig. 1, the maximum retention time of component 1 was 5.879 min, and the content of component 1 was 9.51%, which was mainly composed of hexasaccharide and octasaccharide. Component 2 with a maximum retention time of 8.632 min was tetrasaccharide and the content was 33.83%. Component 3 with a maximum retention time of 8.966 min was also tetrasaccharide and the content was 5.07%. Component 4 with a maximum retention time of 9.846 min was disaccharide and the content was 47.08%. The component 5 with the maximum retention time of 10.786 min was also a disaccharide, the content of which was 2.03%. The component 6 with the maximum retention time of 12.506 min was a salt peak. Therefore, the total content of low molecular weight chondroitin sulfate including disaccharide, tetrasaccharide, hexasaccharide and octasaccharide was 97.52%, containing disaccharide and tetrasaccharide as the main components, wherein the total content of disaccharide and tetrasaccharide was 88.01%. The average molecular weight of the low molecular weight chondroitin sulfate obtained by enzymatic depolymerization of shark bone was 704.3-741.2 Da, and the specific calculation was as follows:

[00102] Let us assume that component 1 was all hexasaccharide, then

Let's assume that component 1 was all octasaccharide, then

[00103] The molecular weight was analyzed by liquid chromatography/mass spectrometry (LC/MS) under the following specific analytical conditions:

Chromatographic conditions:

Chromatography Column 1: TSK Guard Column SWXL 7 µm, 6 mm × 4 cm

Chromatography column 2: TSK G3000 SWXL 5 µm, 7.8 mm × 30 cm

Flow rate: 1 ml/min

Sample quantity: 10 µl

Column temperature: 30°C

Detection wavelength: 195 nm

Collection time: 25 min

Buffer: 0.38 g of ammonium formate was diluted with water to 2000 ml, mixed thoroughly, filtered and obtained.

Mobile phase: the volume ratio of buffer to methanol was 9:1.

Mass spectral conditions:

Ion registration mode: negative ion registration mode [M-H]-

Pulse voltage: 70 V

Mass to charge ratio range: 300-1000 m/z

Dry gas flow rate: 12 l/min

Spray Pressure: 35 PSI

Capacitor voltage: 3000 V.

[00104] The results of the molecular weight determination were as follows: the corresponding molecular weights of the disaccharide were 378.1 and 458.1, and the corresponding molecular weights of the tetrasaccharide were 837.2 and 917.1.

[00105] Example 10. Alcohol precipitation and drying

[00106] The 2-liter upper layer of the reaction solution obtained in Example 6 was filtered and sterilized into a clean area through a 0.22 μm capsule filter. Then, the resulting filtrate was added dropwise to 16 L of anhydrous ethanol, stirred for 0.5 h and left for 2 h. After all the solid was precipitated, the supernatant was removed. The solid was collected by centrifugal filtration and then dried in a vacuum drying oven at 50°C for 24 h until the weight loss was not more than 10%. Then, 320 g of the product, which was low molecular weight chondroitin sulfate, was obtained with a yield of 80.0% (i.e., the ratio of 320 g of low molecular weight chondroitin sulfate to 400 g of the original shark bone chondroitin sulfate). The protein content determined by Coomassie blue staining was 0.4%. The molecular weight distribution was determined by liquid chromatography mass spectrometry, as shown in Fig. 2, the maximum retention time of component 1 was 5.878 min, and the content of component 1 was 9.50%, and it was mainly composed of hexasaccharide and octasaccharide. Component 2 with a maximum retention time of 8.625 min was a tetrasaccharide and the content was 33.90%. Component 3 with a maximum retention time of 8.958 min was also a tetrasaccharide and the content was 5.08%. Component 4 with a maximum retention time of 9.838 min was a disaccharide and the content was 46.86%. Component 5 with the maximum retention time of 10.785 min was also a disaccharide, the content of which was 2.13%. Component 6 with the maximum retention time of 12.505 min was a salt peak. Therefore, the total content of low molecular weight chondroitin sulfate including disaccharide, tetrasaccharide, hexasaccharide and octasaccharide was 97.47%, mainly disaccharide and tetrasaccharide, and the content of disaccharide and tetrasaccharide in total was 87.97%. The average molecular weight of the low molecular weight chondroitin sulfate obtained by enzymatic depolymerization of shark bone was 704.7-741.6 Da, and the specific calculation method was as follows:

[00107] Let us assume that component 1 was all hexasaccharide, then

Let's assume that component 1 was all octasaccharide, then

[00108] Example 11. Alcohol precipitation and drying

[00109] The 2-liter upper layer of the reaction solution obtained in Example 7 was filtered and sterilized into a clean area through a 0.22 μm capsule filter. Then, the resulting filtrate was added dropwise to 24 L of anhydrous ethanol, stirred for 0.5 h and left for 2 h. After all the solid was precipitated, the supernatant was removed. The solid was collected by centrifugal filtration and then dried in a vacuum drying oven at 40°C for 24 h until the weight loss was not more than 10%. Then, 150 g of the product, which was low molecular weight chondroitin sulfate, was obtained in a yield of 75.0% (i.e., the ratio of 150 g of low molecular weight chondroitin sulfate to 200 g of the original shark bone chondroitin sulfate). The protein content determined by Coomassie blue staining was 0.4%. The molecular weight distribution was determined by liquid chromatography mass spectrometry, as shown in Fig. 3, the maximum retention time of component 1 was 5.879 min, and the content of component 1 was 8.62%, and it was mainly composed of hexasaccharide and octasaccharide. Component 2 with a maximum retention time of 8.632 min was a tetrasaccharide and the content was 30.79%. Component 3 with a maximum retention time of 8.966 min was also a tetrasaccharide and the content was 4.41%. Component 4 with a maximum retention time of 9.872 min was a disaccharide and the content was 52.49%. Component 5 with the maximum retention time of 10.786 min was also a disaccharide, the content of which was 2.02%. Component 6 with the maximum retention time of 12.512 min was a salt peak. Therefore, the total content of low molecular weight chondroitin sulfate including disaccharide, tetrasaccharide, hexasaccharide and octasaccharide was 98.32%, mainly disaccharide and tetrasaccharide, and the content of disaccharide and tetrasaccharide in total was 89.70%. The average molecular weight of the low molecular weight chondroitin sulfate obtained by enzymatic depolymerization of shark bone was 679.2-712.5 Da, and the specific calculation method was as follows:

[00110] Let us assume that component 1 was all hexasaccharide, then

Let's assume that component 1 was all octasaccharide, then

[00111] Example 12. Spray drying

[00112] The 2-liter upper layer of the reaction solution obtained in Example 8 was filtered and sterilized into a clean zone through a 0.22 μm capsule filter, and then spray dried. The spray drying parameters were as follows: the inlet air temperature was 120°C, the outlet air temperature was 60°C, and the flow rate was 100 rpm. Then, 1,200 g of a low molecular weight chondroitin sulfate product was obtained at a yield of 85.7% (that is, the ratio of 1,200 g of the low molecular weight chondroitin sulfate to 1,400 g of the original shark bone chondroitin sulfate). The protein content determined by the Coomassie bright blue staining method was 0.5%. The molecular weight distribution was determined by liquid chromatography mass spectrometry, as shown in Fig. 4, the maximum retention time of component 1 was 5.876 min, and the content of component 1 was 8.50%, and it was mainly composed of hexasaccharide and octasaccharide. Component 2 with the maximum retention time of 8.636 min was a tetrasaccharide, the content of which was 30.59%. Component 3 with the maximum retention time of 8.963 min was also a tetrasaccharide, the content of which was 4.43%. Component 4 with the maximum retention time of 9.870 min was a disaccharide, the content of which was 52.69%. Component 5 with the maximum retention time of 10.783 min was also a disaccharide, the content of which was 2.14%. Component 6 with the maximum retention time of 12.510 min was a salt peak. Therefore, the total content of low molecular weight chondroitin sulfate including disaccharide, tetrasaccharide, hexasaccharide and octasaccharide was 98.35%, mainly disaccharide and tetrasaccharide, and the content of disaccharide and tetrasaccharide in total was 89.85%. The average molecular weight of low molecular weight chondroitin sulfate obtained by enzymatic depolymerization of shark bone was 677.4-710.2 Da, and the specific calculation method was as follows:

[00113] Let us assume that component 1 was all hexasaccharide, then

Let's assume that component 1 was all octasaccharide, then

[00114] Example 13

[00115] According to the enzymatic depolymerization reaction of Example 1, the protein removal method of Example 5, and the alcohol precipitation and drying method of Example 9, four more types of low molecular weight chondroitin sulfate from different sources were obtained using bovine cartilage, pig cartilage, chicken cartilage, and mixed bone chondroitin sulfate from chicken cartilage and shark bone, respectively, with a content of 90% from Shandong Baolijiao Company.

[00116] Example 14. Test for potency and activity

[00117] (1) Sample preparation:

(1.1) The sample of Example 9 was obtained as a result of the enzymatic depolymerization reaction by the method described in Example 1.

(1.2) Control group sample 1: a mixture of 0.63 g of disaccharide with a purity of 97.72% and 0.41 g of oligomeric peptide from deep-sea fish (Ningxia Vanilla Biotechnology Co., Ltd., specification 98%). 61.56% of disaccharide and 40.18% of oligopeptides from deep-sea fish were calculated.

The method for preparing samples of 97.72% disaccharide was as follows:

Preparation of mobile phase:

A: 20 mM Tris, pH adjusted to 7.5 with HCl;

B: 20 mM Tris, 1 M NaCl, pH adjusted to 7.5 with HCl.

Sample processing:

The sample of Example 9 was dissolved in mobile phase A by weighing 20 g of sample powder and dissolving in 1 L of mobile phase A.

Steps: Before purification, the Q-Sepharose-FF packed column was washed with mobile phase B and then equilibrated with mobile phase A. The sample was loaded onto the Q-Sepharose-FF packed column and the penetration peaks were collected. The tetrasaccharide absorbed in the column was washed with mobile phase B and the eluted peaks were collected and re-passed through the column for chromatographic purification until a single disaccharide peak was detected.

Deionization: The detected single peak of disaccharide was pooled and collected and then deionized using a glucose gel column. Before loading, the sample was washed with purified water until the conductivity was below 0.1 mS/cm and the sample loading volume was 20%-30% of the column volume. Then, the peaks with conductivity below 1 mS/cm were collected by washing the column with purified water.

Lyophilization: After collecting the deionized peak and pre-freezing at -20°C, it was placed in a freeze dryer for freeze drying. After lyophilization to powder, the disaccharide with a purity of 97.72% was collected. The purity of the disaccharide was determined as follows: HPLC was used to determine the molecular weight distribution, as shown in Fig. 8. Component 1 with a peak retention time of 9.265 min was a tetrasaccharide with a content of 2.28%. Component 2 with a peak retention time of 10.070 min was a disaccharide with a content of 65.79%. Component 3 with a peak retention time of 10.937 min was a disaccharide with a content of 31.93%. The salt peak in Fig. 8 was not integrated.

(1.3) Control group sample 2: Using shark bone macromolecular chondroitin sulfate with a content of 90% from Shandong Baoliga Company, with an average molecular weight of about 70,000 Da, which contained almost no disaccharide and tetrasaccharide.

[00118] (2) Preparation of culture medium, solution and cells:

(2.1) Minimal medium: DMEM/F-12 medium.

(2.2) Growth medium: 20 ml of fetal bovine serum (FBS) was added to 180 ml of minimal medium and stored at 2°C-8°C.

(2.3) Complete medium: DMEM (Dulbecco's modified Eagle's medium) + fetal bovine serum Cell P5 (5th generation).

(2.4) 1 mM _H2O2 solution for stimulation: 30% (9790 mM) hydrogen peroxide _was filtered through a sterile 0.2 μm filter and then diluted to 1 mM with minimal medium for use.

(2.5) Stock solutions of samples 1 and 2: A certain amount of samples [bovine bone CSO oligosaccharide (from Example 13), pork bone CSO oligosaccharide (from Example 13), shark bone CSO oligosaccharide (from Example 9), chicken bone CSO oligosaccharide (from Example 13), chicken bone and shark bone CSO oligosaccharide (from Example 13), and shark bone CSO polysaccharide (Shandong Baolijia)] were weighed. DPBS (Dulbecco's phosphate buffered saline) was used as a diluent to prepare 10 mg/mL of stock solution of sample 1, and then filtered through a 0.2 μm sterile filter membrane for use.

Weighing a certain amount of samples (sample of Example 9, sample in control groups 1-2), DPBS was used as a diluent to prepare 10 mg/mL stock solution of sample 1, and then filtered through a 0.2 μm sterile filter membrane for use.

(2.6) Samples were detected in gradient solutions 1 and 2:

The sample 1 solution prepared above was further diluted in minimal medium to prepare two serial dilutions at concentrations of 50 and 100 μg/ml, respectively.

The sample solution 2 prepared above was further diluted in minimal medium to prepare seven serial dilutions in concentrations ranging from 50 to 3200 μg/mL, namely: 50, 100, 200, 400, 800, 1600 and 3200 μg/mL, respectively.

(2.7) Preparation of cell culture:

ATDC5 cells were revived with growth medium and incubated in a humidified incubator at 37±2°C and 5±1% carbon dioxide. Growth medium was used for subculture when approximately 70%-90% confluency of the cultures was observed using a microscope with appropriate magnification (such as 10-40×).

The cell medium was removed for cell passage. The cells were washed once with DPBS. Then, the cells were filtered with about 0.5 ml of a solution containing 0.25% trypsin for about 1 minute, and the cells became round and detached from the surface. Under the microscope, the morphology of the cells became round, some cells detached from the vessel wall, and they were directly added to the complete medium for final digestion. Use a pipette to suck out the medium, blow off the cells from the vessel wall, disperse the cells evenly in the medium, and transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 min. After centrifugation, the supernatant was discarded, the cells were resuspended in 2 ml of complete medium, and then the cells were transferred to 2 new T25 rectangular flasks respectively. Each rectangular flask was added with 5 ml of complete medium, mixed gently, and placed in a carbon dioxide-containing incubator (37°C, 5% _CO2 ) for culturing.

The cells were subcultured for 2-5 days using cell passages of 4-20 and confluence of 70%-90%. According to the cell culture preparation procedure, an appropriate volume of cell solution containing 2× ¹⁰⁶ /mL of living cells was prepared in the growth medium. A 96-well transparent cell culture plate was taken and 100 μL of cell solution was added to each well so that each well contained approximately 6000 cells. To prevent cell sedimentation and to ensure homogeneity in each well, the cell solution was mixed frequently before addition. The plates were incubated in a carbon dioxide incubator (37°C, 5% _CO2 ) overnight (24 h).

After culturing on the following day (24 h), the original medium was discarded and 50 μl _{of 1} mM _H2O2 was added to each well (see Step 4.2 for the method of preparing 1 mM _H2O2 solution for stimulation). Then, chondroitin sulfate gradient detection solutions from different sources and at different concentrations were added to each well (see Step 4.3 for the method of preparing gradient detection solutions) and then placed in an incubator containing carbon dioxide (37 °C, 5% _CO2 ) for additional culturing for 12 h.

After culturing, 10 μl of CCK-8 solution was added to each well for another 2 h on the next day (12 h). Immediately after the reaction, the absorbance value at 450 nm was detected using a multifunctional immunoassay plate reader. Each sample will be tested in duplicate, and the average absorbance value will be taken at the end.

[00119] (3) Comparison of cellular activity of low molecular weight chondroitin sulfate from different animal sources and macromolecular chondroitin sulfate:

CCK assay was used to investigate the reparative effect on damaged chondrocytes for low molecular weight chondroitin sulfate from different animal sources and macromolecular chondroitin sulfate. The results of the efficacy test shown in Fig. 5 showed that, compared with macromolecular chondroitin sulfate (chondroitin sulfate polysaccharide) from shark bone, low molecular weight chondroitin sulfate obtained by enzymatic hydrolysis of one or more shark bone and chicken bone had a more significant reparative effect at concentrations of 50-100 μg/mL on chondrocytes damaged by 1 mM hydrogen peroxide, and the reparative ability was 14% to 23%, which can be used for the treatment of joint damage.

[00120] (4) Comparison of cellular activity of low molecular weight shark bone chondroitin sulfate containing different components and macromolecular shark bone chondroitin sulfate:

In the control group, 1 0.63 g of disaccharide with a purity of 91.12% was mixed with 0.41 g of fish collagen oligopeptide. The disaccharide content was 61.56%, and the content of deep-sea fish collagen oligopeptide was 40.18% when calculated (Ningxia Vanilla Biotechnology Co., Ltd., specification 98%).

The results are shown in Table A and Fig. 9. The regenerative effect of the inventive product on chondrocytes damaged by 1 mM hydrogen peroxide was more significant than that of the control groups 1-2 in the concentration range of 50-3200 μg/ml. Among them, the inventive product had an improvement in the regenerative rate of chondrocytes in the concentration range of 50-1600 μg/ml, and the improvement was greater as the concentration increased. However, when the concentration reached 3200 μg/ml, an inhibition effect appeared because the concentration was too high, and the regenerative rate fluctuated from 20% to 62.4%, showing an obvious decreasing trend.

[00121] Example 15. Hydrolyzed chondroitin sulfate capsules

[00122] Recipe:

[00123] Manufacturing method:

(1) The hydrolyzed chondroitin sulfate from Example 9 and microcrystalline cellulose were separately passed through an 80 mesh sieve to prepare for use;

(2) After thoroughly mixing hydrolyzed chondroitin sulfate and microcrystalline cellulose, the prescribed amount of crospovidone, colloidal silicon dioxide and magnesium stearate were added sequentially and mixed for 15 min;

(3) This mixture was filled into the capsule shell using a capsule filling machine and capsules were formed.

[00124] Example 16. Hydrolyzed chondroitin sulfate tablets

[00125] Recipe:

[00126] Manufacturing method:

(1) The hydrolyzed chondroitin sulfate from Example 9 and microcrystalline cellulose were separately passed through an 80 mesh sieve to prepare for use;

(2) After thoroughly mixing hydrolyzed chondroitin sulfate and microcrystalline cellulose, the prescribed amount of sodium croscarmellose, colloidal silicon dioxide and magnesium stearate were added sequentially, and the mixture was mixed for 15 min;

(3) The mixture was pressed by a rotary tablet press and controlled to obtain a tablet hardness of 6-10 kg;

(4) A coating containing 10% solids was prepared using purified water;

(5) The coating was applied to the tablets using a high-efficiency coating machine. The inlet air temperature was set at 55°C, the spray pressure was 0.2 MPa, and the base sheet temperature was controlled at 40-45°C. After the coating was completed, the tablets were manufactured.

[00127] Example 17. Hydrolyzed chondroitin sulfate tablets

[00128] Recipe:

[00129] Method of manufacture:

(1) The hydrolyzed chondroitin sulfate from Example 9 and lactose were separately passed through an 80 mesh sieve to prepare for use;

(2) After thoroughly mixing hydrolyzed chondroitin sulfate and lactose, the prescribed amount of hydroxypropyl cellulose, L-hydroxypropyl cellulose, colloidal silicon dioxide and magnesium stearate were added sequentially, and the mixture was mixed for 15 min;

(3) The mixture was pressed by a rotary tablet press and controlled to obtain a tablet hardness of 6-10 kg;

(4) A coating containing 10% solids was prepared using purified water;

(5) The tablets were coated using a high-efficiency coating machine. The inlet air temperature was set at 55°C, the spray pressure was 0.2 MPa, and the base sheet temperature was controlled at 40-45°C. After the coating was completed, the tablets were manufactured.

[00130] Example 18. Hydrolyzed chondroitin sulfate tablets

[00131] Recipe:

[00132] Manufacturing method:

(1) The hydrolyzed chondroitin sulfate from Example 9, starch and dextrin were separately passed through an 80 mesh sieve to prepare for use;

(2) After thoroughly mixing hydrolyzed chondroitin sulfate, starch and dextrin, the prescribed amount of hydroxypropyl cellulose, sodium carboxymethyl cellulose, colloidal silicon dioxide and magnesium stearate were added sequentially, and the mixture was mixed for 15 min;

(3) The mixture was pressed by a rotary tablet press and controlled to obtain a tablet hardness of 6-10 kg;

(4) A coating containing 10% solids was prepared using purified water;

(5) The tablets were coated using a high-efficiency coating machine. The inlet air temperature was set at 55°C, the spray pressure was 0.2 MPa, and the base sheet temperature was controlled at 40-45°C. After the coating was completed, the tablets were manufactured.

[00133] Example 19. Hydrolyzed chondroitin sulfate capsules

[00134] Manufacturing method:

(1) The low molecular weight chondroitin sulfate from Example 9 and microcrystalline cellulose were separately passed through an 80 mesh sieve to prepare for use;

(2) After thoroughly mixing low molecular weight chondroitin sulfate and microcrystalline cellulose, the prescribed amount of sodium croscarmellose, colloidal silicon dioxide and magnesium stearate were added sequentially and mixed for 15 min;

(3) This mixture was filled into the capsule shell using a capsule filling machine and capsules were formed;

[00135] Example 20. Hydrolyzed chondroitin sulfate and glucosamine sulfate tablets

[00136] Recipe:

[00137] Manufacturing method:

(1) The hydrolyzed chondroitin sulfate from Example 9, glucosamine sulfate, microcrystalline cellulose and lactose were individually passed through an 80 mesh sieve to prepare for use;

(2) After thoroughly mixing hydrolyzed chondroitin sulfate, glucosamine sulfate, microcrystalline cellulose and lactose, the prescribed amount of sodium croscarmellose, colloidal silicon dioxide and magnesium stearate were added sequentially, and the mixture was mixed for 15 min;

(3) The mixture was pressed by a rotary tablet press and controlled to obtain a tablet hardness of 6-10 kg;

(4) A coating containing 10% solids was prepared using purified water;

(5) The tablets were coated using a high-efficiency coating machine. The inlet air temperature was set at 55°C, the spray pressure was 0.2 MPa, and the base sheet temperature was controlled at 40-45°C. After the coating was completed, the tablets were manufactured.

[00138] Example 21. Testing of the composition obtained according to the invention in a mouse model of medial meniscus instability (DMM)

[00139] 1. Materials

1.1 Test sample: The composition was prepared according to Examples 15-20 of the present invention, and the recommended daily dosage of the composition of Example 15, Example 16, Example 17, Example 18, Example 19 and Example 20 was 2 tablets (granules) per day, and 1 tablet (granules) in the morning and evening.

1.2 Preparation of test substance: The test sample to be administered was mixed into the feed. For Examples 15, 16, 17, 18, 19, and 20, 150 mg/day samples were administered to mice daily.

1.3 Route of administration to subjects: Samples of each Example were administered to animals in each group via the intragastric route.

[00140] 2. Creating a model

[00141] Mice were anesthetized with chloral hydrate and the hair on the knee joint of the right hind limb was shaved. After disinfection with iodine and alcohol, a longitudinal incision of approximately 1 cm was made on the internal skeleton of the mice to expose the knee joint. Microsurgical scissors were used to open the joint cavity and the medial meniscal ligament on the tibial plateau was transected. 6-0 absorbable suture was used to close the joint capsule. 6-0 suture was used to suture the joint skin and a small amount of penicillin was applied to the sutured skin to prevent infection. A control group (9 mice) was subject to the same procedure except that the medial meniscal ligament was not transected. On the second day after the DMM surgery, the mice were randomly divided into 7 groups with 13 rats in each group (1. Model control group; 2. Example 15; 3. Example 16; 4. Example 17; 5. Example 18; 6. Example 19; 7. Example 20).

[00142] 3. Experimental results

[00143] On the second day after the operation, the mice were administered intragastrically, and the blank group and the model group were administered the same volume of normal saline intragastrically. The animals were administered once a day for 12 weeks. The animals were weighed once a week, and the dose was adjusted according to body weight.

[00144] Conclusion: As shown in Fig. 6, from Day 0 of the experiment, the body weight of mice in each group showed a slight increasing trend, and there was no significant difference between the groups at each time point (P>0.05).

[00145] After intragastric administration to mice for 12 weeks, the YLS-11A mouse foot support force measuring device operating in the channel mode was used to detect the difference in the support force of the two hind paws of the mouse when standing, to evaluate the degree of osteoarthritic pain in the mice. The mice were driven into a single channel for a 60-degree slope climbing experiment. When the mouse started to stand on the slope, the difference in support force between the left and right hind paws was recorded. The greater the difference in support force, the more severe the degree of osteoarthritis.

[00146] The degree of osteoarthritic pain in mice was evaluated by determining the difference in the support force in mice. As shown in Fig. 7, the difference in the support force in the model group was the largest, indicating that the degree of osteoarthritic pain in the model group was the most severe. There was a significant difference compared with the sham-operated group, indicating that the model was successfully implemented. Compared with the model group, the compounds of Examples 15, 16, 19 and 20 could significantly reduce the difference in support force (P<0.01), and the compounds of Examples 17 and 18 also reduced the difference in support force (P<0.05). The daily dose for mice can be calculated by the formula: human dose=mouse dose*body weight/dose equivalent ratio. The daily dose for humans is calculated as follows:

Note: 1. The equivalent dose ratio for mice and humans was 9.1; 2. The dose of hydrolyzed chondroitin sulfate for mice = the dose of the composition for mice

* prescribed dose of hydrolyzed chondroitin sulfate/total tablet weight.

[00147] The results showed that a daily dose of hydrolyzed chondroitin sulfate from 50 to 800 mg had a significant relieving and treating effect on osteoarthritis pain, and the addition of glucosamine to the composition could enhance the relieving and treating effect on osteoarthritis pain.

[00148] Example 22. To evaluate the therapeutic effect of chondroitin sulfate oligosaccharide (CSO) and the combination of CSO with glucosamine (an amino sugar) on osteoarthritis caused by medial meniscus instability (DMM) in mice by pathological and immunohistochemical detection of inflammatory cytokines, bone acid hydroxyproline, and safranin O-fast green staining in serum

[00149] Male C57BL/6 mice aged 8-9 weeks, 100 mice, were given free access to water and food. The mice were anesthetized with chloral hydrate, and the hair of the right hind limb knee joint was shaved. The mice were anesthetized with chloral hydrate, and the hair of the right hind limb knee joint was shaved. After disinfection with iodine and alcohol, a longitudinal incision of approximately 1 cm was made on the internal skeleton of the mice to expose the knee joint. Microsurgical scissors were used to open the joint cavity, and the medial meniscal ligament on the tibial plateau was transected.

Absorbable 6-0 suture was used to close the joint capsule. 6-0 suture was used to close the joint skin, and a small amount of penicillin was applied to the sutured skin to prevent infection. The sham-operated group (9 mice) was subject to the same procedure, but the tibiofibular ligament of the medial meniscus was not cut.

[00150] On the second day after DMM surgery, mice were randomly divided into 7 groups with 13 rats in each group:

1) model control group;

2) chondroitin sulfate oligosaccharide group at the highest dose (60 mg/kg);

3) chondroitin sulfate oligosaccharide group in high dose (30 mg/kg);

4) chondroitin sulfate oligosaccharide group in an average dose (15 mg/kg);

5) chondroitin sulfate oligosaccharide group at a low dose (7.5 mg/kg);

6) chondroitin sulfate oligosaccharide group (15 mg/kg) + glucosamine hydrochloride (7.5 mg/kg).

[00151] On the second day after the operation, the mice were intragastrically administered a volume of 0.2 ml/20 g. The sham-operated group and the model group were administered the same volume of normal saline. High molecular weight chondroitin sulfate (CS) (80 mg/kg) + glucosamine (400 mg/kg) was suspended in 0.5% CMC-NA (sodium carboxymethyl cellulose). The animals were administered once a day for 12 weeks. The animals were weighed once a week, and the dose was adjusted according to body weight.

[00152] Blood samples were collected from the eyeballs of mice, and the serum was separated. TNF-α, IL-1β, IL-6, and C5b-9 were determined by ELISA. Mice were sacrificed, the right hind femur was collected, and bone hydroxyproline acid was determined according to the kit method. The knee joint of the right hind limb was collected and examined for pathology (staining with safranin O-fast green) and C5b-9 expression (immunohistochemistry). The evaluation indices are summarized as follows:

[00153] Effect on serum inflammatory cytokine levels

[00154] The severity of osteoarthritis in mice was assessed by measuring the levels of inflammatory cytokines IL-6, IL-1β, TNF-α and complement C5b-9 in the serum. As shown in Fig. 10A, the levels of inflammatory cytokines IL-6, IL-1β, TNF-α and complement C5b-9 in the model group were the highest, indicating that the model group had the most severe osteoarthritis. There was a significant difference compared with the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00155] As shown in Fig. 10A, compared with the model group, the levels of inflammatory cytokines IL-6, IL-1β, TNF-α and complement C5b-9 in the highest, high, middle and low dose of chondroitin sulfate oligosaccharide group were reduced to different degrees, indicating that different doses of chondroitin sulfate oligosaccharide had an attenuating and therapeutic effect on osteoarthritis.

[00156] Compared with the medium dose group, the serum levels of inflammatory cytokines IL-6, IL-1β, TNF-α and complement C5b-9 in the CSO+glucosamine group were reduced to varying degrees, indicating that the combination of chondroitin sulfate oligosaccharide and glucosamine can improve the attenuation and treatment of osteoarthritis.

[00157] Compared with the macromolecular chondroitin sulfate+glucosamine group, the levels of inflammatory cytokines IL-6, IL-1β, TNF-α and complement C5b-9 in the blood serum of the CSO+glucosamine group and the oligosaccharide group of the invention were significantly reduced, indicating that the therapeutic effect of chondroitin sulfate oligosaccharide on osteoarthritis was better than that of the macromolecular chondroitin sulfate+glucosamine group.

[00158] Effect on femoral hydroxyproline levels

[00159] The severity of osteoarthritis in mice was evaluated by measuring hydroxyproline in the femur of mice. As shown in Fig. 10B, the hydroxyproline level in the femur of the model group was the lowest, indicating that the osteoarthritis in the model group was the most severe. There was a significant difference compared with the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00160] Compared with the model group, the highest, high, medium and low dose chondroitin sulfate oligosaccharide groups and the chondroitin sulfate oligosaccharide+glucosamine group significantly increased the hydroxyproline level in the femur of mice (P<0.01).

[00161] Compared with the medium dose group, the hydroxyproline level in the CSO+glucosamine group was significantly increased, indicating that the combination of glucosamine and CSO can promote the treatment efficacy of osteoarthritis.

[00162] Compared with the macromolecular chondroitin sulfate+glucosamine group, the data of the CSO+glucosamine group showed that the effect of CSO was superior to that of CS.

[00163] Pathological examination of the knee joint

[00164] The severity of osteoarthritis in mice was evaluated by staining the knee joint with safranin O-fast green. The results are shown in Fig. 10C and Fig. 10D. The pathological score after staining with safranin O-fast green in the knee joint of mice in the model group was the highest, indicating that the model group had the most severe osteoarthritis. There was a significant difference compared with the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00165] Compared with the model group, the highest dose and high dose chondroitin sulfate oligosaccharide groups and the middle dose chondroitin sulfate oligosaccharide+glucosamine group could significantly decrease the pathological score after safranin O-fast green staining (P<0.05), indicating that chondroitin sulfate oligosaccharide has an alleviating effect on osteoarthritis.

[00166] Level C5b-9 on the cartilaginous surface of the knee joint

[00167] The severity of osteoarthritis in mice was evaluated by immunohistochemical staining of C5b-9 in the knee joint. The results are shown in Fig. 10E and Fig. 10F. The number of C5b-9-positive cells in the knee joint of mice in the model group was the highest, indicating that the osteoarthritis in the model group was the most severe. There was a significant difference compared with the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00168] Compared with the model group, the chondroitin sulfate oligosaccharide high dose and high dose group, the oligosaccharide medium dose+glucosamine group significantly decreased the number of C5b-9-positive cells in the knee joint of mice (P<0.01), indicating that chondroitin sulfate oligosaccharide can attenuate cartilage damage in osteoarthritis.

[00169] Example 23. Effect of Different Doses of CSO in a Rat Model of Osteoarthritis Induced by Papain Injection into the Knee Joint

[00170] Eighty male Wistar rats of 180-220 g were mixed with 4% papain and 0.03 mol/L L-cysteine at a ratio of 2:1 and left to stand for 30 min. On Day 0, 3 and 6 of the experiment, 0.3 ml of the mixed solution was injected into the knee joint cavity of the rats to induce an osteoarthritis model. The normal control group was injected with an equal volume of normal saline.

[00171] After the last injection of papain mixture, the model animals were randomly divided into 7 groups of 10 rats in each group:

1) model control group;

2) the highest chondroitin sulfate oligosaccharide group (30 mg/kg);

3) chondroitin sulfate oligosaccharide group in high dose (15 mg/kg);

4) chondroitin sulfate oligosaccharide group at an average dose (7.5 mg/kg);

5) chondroitin sulfate oligosaccharide group at a low dose (3.8 mg/kg);

6) chondroitin sulfate oligosaccharide group (7.5 mg/kg) + glucosamine hydrochloride (37.5 mg/kg);

7) group of macromolecular chondroitin sulfate (40 mg/kg) + glucosamine (200 mg/kg).

[00172] On the second day after the modeling, the rats were intragastrically administered a volume of 0.2 ml/100 g. The normal control group and the model group were administered the same volume of normal saline. Macromolecular chondroitin sulfate (40 mg/kg) + glucosamine (200 mg/kg) was suspended in 0.5% CMC-Na. The drug was administered once a day for 12 weeks. The animals were weighed every 3 days, and the dose was adjusted according to body weight.

[00173] The width of both knee joints was measured before modeling, before administration, and 1, 3, 6, 9, and 12 weeks after administration. The degree of joint swelling was calculated using the following formula: Degree of joint swelling (mm) = knee width after inflammation (after administration) - knee width before inflammation.

[00174] After 12 weeks of intragastric administration, a blood sample was collected from the abdominal aorta of the rats and the serum was separated. TNF-α, IL-1β and IL-6 were determined by ELISA. The femur of the rats was collected and the hydroxyproline acid in the bone was determined according to the kit method. The knee joint was taken and examined for pathology (HE (hematoxylin and eosin) staining). The evaluation indices are summarized as follows:

[00175] As shown in Fig. 11A, from Day 0 of the experiment, the body weight of the rats in each group showed an increasing trend and no significant difference between the groups at each time point. The results showed that chondroitin sulfate oligosaccharide did not have a significant effect on the body weight of the rats after 12 weeks.

[00176] The severity of osteoarthritis in rats was evaluated by measuring the levels of inflammatory cytokines IL-6, IL-1β and TNF-α in the serum of rats. The results are shown in Fig. 11B, Fig. 11C and Fig. 11D. The levels of inflammatory cytokines IL-6, IL-1β and TNF-α in the serum of the model group were the highest, indicating that the model group had the most severe osteoarthritis. There was a significant difference compared with the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00177] Compared with the medium dose group, the levels of inflammatory cytokines IL-6, IL-1β and TNF-α in the serum of rats in the medium dose oligosaccharide+glucosamine group were reduced to varying degrees, indicating that the combination of chondroitin sulfate oligosaccharide and glucosamine can enhance the effect of relieving and treating osteoarthritis.

[00178] Compared with the macromolecular chondroitin sulfate+glucosamine group, the levels of inflammatory cytokines IL-6, IL-1β and TNF-α in the serum of rats in the CSO+glucosamine group were significantly increased, indicating that the effect of chondroitin sulfate oligosaccharide on osteoarthritis was better than that of the macromolecular chondroitin sulfate+glucosamine group.

[00179] The severity of osteoarthritis in rats was evaluated by pathological examination of the knee joint. The results are shown in Fig. 11E and Fig. 11F. The pathological score of the knee joint in rats in the model group was the highest, indicating that the osteoarthritis in the model group was the most severe. There was a significant difference compared with the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00180] Compared with the model group, the highest dose and high dose chondroitin sulfate oligosaccharide group, the medium dose oligosaccharide+glucosamine group could significantly reduce the pathological score (P<0.01), indicating that chondroitin sulfate oligosaccharide has the effect of alleviating osteoarthritis.

[00181] Example 24. Therapeutic Effect of CSO on Osteoporosis in Ovariectomized Female Rats and Testicularly Removed Male Rats

[00182] Female rats:

[00183] Ninety female SD rats weighing 200-220 g were used. The rats were acclimated to the environment for 3 days. Before surgery, the rats were fasted but were given water freely for 24 hours. The rats were injected intraperitoneally with 3% chloral hydrate and anesthetized. Ten mice were used as a sham-operated group with longitudinal incisions made on the skin and muscle on both sides of the lumbar and dorsal spine without removing the ovaries. For the remaining animals, longitudinal incisions were made on both sides of the lumbar and dorsal spine to cut the skin and muscle, and both ovaries were removed and the wounds were sutured. All animals were given intramuscular injections of penicillin for 3 consecutive days after surgery. (Male rats were operated in the same manner with removal of the testicles.)

[00184] On the second day after surgery, ovariectomized rats were randomly divided into 8 groups with 8 rats in each group:

1) false-operated group;

2) model group;

3) calcium acetate group (158 mg/kg);

4) chondroitin sulfate oligosaccharide group at the highest dose (30 mg/kg) + calcium acetate (158 mg/kg);

5) chondroitin sulfate oligosaccharide group in high dose (15 mg/kg) + calcium acetate (158 mg/kg);

6) chondroitin sulfate oligosaccharide group in an average dose (7.5 mg/kg) + calcium acetate (158 mg/kg);

7) chondroitin sulfate oligosaccharide group at a low dose (3.8 mg/kg) + calcium acetate (158 mg/kg);

8) a group of chondroitin sulfate oligosaccharide (7.5 mg/kg) + glucosamine hydrochloride (37.5 mg/kg) + calcium acetate (158 mg/kg);

9) a group of macromolecular chondroitin sulfate (40 mg/kg) + glucosamine (200 mg/kg) + calcium acetate (158 mg/kg).

[00185] On the second day after the surgery, the rats were intragastrically administered a volume of 0.2 ml/100 g. The sham-operated group and the model group were administered the same volume of normal saline. After 12 weeks of continuous administration, the animals were weighed once a week, and the dose was adjusted according to body weight.

[00186] Blood samples were collected from the rats' orbits 24 h after the last administration. Serum alkaline phosphatase (ALP), bone morphogenetic protein (BGP), parathyroid hormone (PTH), and calcitonin (CT) were determined.

[00187] The rats were sacrificed and the femur was removed from both sides. The right femur was taken and the following parameters were measured: bone weight ratio (g/100 g body weight), bone density, ash content, bone calcium or bone phosphorus, bone hydroxyproline, HE pathology. The evaluation parameters are summarized as follows:

[00188] Male rats:

[00189] A total of 90 male SD rats weighing 200-220 g were used. The rats were acclimated to the environment for 3 days. Before surgery, the rats were fasted but had free access to water for 24 hours. A total of 90 male SD rats weighing 200-220 g were used. The rats were acclimated to the environment for 3 days. Before surgery, the rats were fasted but had free access to water for 24 hours. The rats were injected intraperitoneally with 3% chloral hydrate, anesthetized, and the scrotal skin was disinfected in a horizontal position with iodine and alcohol. Two longitudinal incisions were made on each side of the mediastinum at a distance of 1 cm. After cutting the proper testicular membrane of 10 of them, their bilateral testes were separated from the epididymis (without excision), and then placed back into the scrotum, and the incisions were sutured, and they were used as a sham-operated group. In the remaining 80 males with excised testes, the bilateral testes were found and excised in the same way. All animals were injected with penicillin for 3 days after the operation.

[00190] On the second day after surgery, ovariectomized rats were randomly divided into 8 groups of 8 rats each:

The remaining stages were the same as for female rats.

[00191] Experimental Results

[00192] Changes in body weight

[00193] As shown in Fig. 12A and Fig. 12B, from Day 0 of the experiment, the body weight of the rats in each group showed an increasing trend, and there was no significant difference between the groups at each time point. The results indicate that chondroitin sulfate oligosaccharide does not have a significant effect on the body weight of female and male rats after 12 weeks of administration.

[00194] Bone weight coefficient

[00195] As shown in Fig. 12C and Fig. 12D, compared with the sham-operated group, the bone weight ratio of the rats in the model group was significantly reduced (P<0.01), indicating successful modeling. Compared with the model group, the bone weight ratio of the female and male rats in the high-dose oligosaccharide group was significantly increased (P<0.05).

[00196] Bone mineral density

[00197] The severity of osteoporosis was evaluated by determining the bone mineral density of the rats. As shown in Fig. 12E and Fig. 12F, the bone mineral density of the rats in the model group was significantly reduced, indicating that the bone mineral density of the rats in the model group was the most severe, and there was a significant difference between the model group and the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00198] Compared with the model group, the BMD (bone mineral density) of the femoral body and epiphysis of the female osteoporotic rats was significantly increased in each treatment group (P<0.05, P<0.01), indicating that chondroitin sulfate oligosaccharide has a positive effect on osteoporosis.

[00199] Compared with the model group, the highest dose group, high dose group, medium dose group, low dose group, CSO+glucosamine group and macromolecular chondroitin sulfate+glucosamine group significantly increased the femoral epiphyseal BMD in male rats (P<0.05, P<0.01); the CSO+glucosamine group and macromolecular chondroitin sulfate+glucosamine group significantly increased the femoral corpus bone mineral density of male rats (P<0.05).

[00200] Ash level

[00201] The severity of osteoporosis in rats was evaluated by measuring the ash content of the femur in rats. As shown in Fig. 12G, the ash content of the bone in the model group was significantly reduced, indicating that the model group had the most severe osteoporosis, which was significantly different from that in the sham group (P<0.01, P<0.05), indicating that the model was successfully implemented.

[00202] Compared with the model group, the CSO+glucosamine group significantly increased the ash content of the femur in female rats (P<0.05), the other groups tended to increase, and there was no statistical difference (P>0.05). The high dose, medium dose and low dose CSO groups, the CSO+glucosamine group and the Ca group significantly increased the ash content of the femur in male rats (P<0.01, P<0.05). The results showed that chondroitin sulfate oligosaccharide has a positive effect on osteoporosis.

[00203] Bone phosphorus

[00204] The severity of osteoporosis was evaluated by measuring the bone phosphorus level in the rats. As shown in Fig. 12H, the bone phosphorus level in the rats in the model group was significantly reduced, indicating that the bone phosphorus level in the model group was the most severe and significantly different from the phosphorus level in the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00205] Compared with the model group, the bone phosphorus level of female rats in the high dose and medium dose CSO groups, the macromolecular chondroitin sulfate+glucosamine group and the CSO+glucosamine group significantly increased (P<0.05, P<0.01). In the low dose CSO group, the CSO+glucosamine group and the CA group, the bone phosphorus level of male rats significantly increased (P<0.05, P<0.01). The results showed that chondroitin sulfate oligosaccharide has a positive effect on osteoporosis.

[00206] Serum alkaline phosphatase

[00207] The alkaline phosphatase (ALP) level in the rat serum was measured to evaluate the severity of osteoporosis. As shown in Fig. 121, the alkaline phosphatase level in the rat serum in the model group was significantly reduced, indicating that the model group had the most severe osteoporosis, and this level was significantly different from that in the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00208] Compared with the model group, the serum alkaline phosphatase level of female osteoporotic rats in the treatment groups increased significantly (P<0.05, P<0.01). Except for the low-dose CSO group, the serum alkaline phosphatase level of male osteoporotic rats in the other treatment groups increased significantly (P<0.01).

[00209] Bone morphogenetic protein-4 serum level

[00210] The level of bone morphogenetic protein-4 (BMP-4) in the serum of rats was measured to evaluate the severity of osteoporosis. As shown in Fig. 12J, the level of bone morphogenetic protein-4 (BMP-4) in the serum of rats in the model group was significantly reduced, indicating that the model group had the most severe osteoporosis, and it was significantly different from the sham group (P<0.01), indicating that the model was successfully implemented.

[00211] Compared with the model group, the serum BMP-4 level of female osteoporotic rats in all groups increased significantly (P<0.05, P<0.01). Except for the low-dose CSO group, the serum BMP-4 level in the other treatment groups increased significantly (P<0.05, P<0.01).

[00212] Serum calcitonin

[00213] The level of calcitonin (CT) in the serum of rats was measured to evaluate the severity of osteoporosis. As shown in Fig. 12K, the level of calcitonin (CT) in the serum of rats in the model group was significantly reduced, indicating that the model group had the most severe osteoporosis, and it was significantly different from the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00214] Compared with the model group, the serum CT level of female osteoporotic rats in each administration group increased significantly (P<0.05, P<0.01). Except for the low-dose CSO group, the serum CT of male osteoporotic rats in the other administration groups increased significantly (P<0.05, P<0.01).

[00215] Serum parathyroid hormone

[00216] The level of parathyroid hormone (PTH) in the serum of rats was measured to evaluate the severity of osteoporosis. As shown in Fig. 12L, the level of PTH in the serum of rats in the model group was significantly reduced, indicating that the model group had the most severe osteoporosis, and this level was different from that in the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00217] Compared with the model group, except for the low dose CSO group and the CA group, the serum PTH level of female osteoporotic rats in the other administration groups was significantly decreased (P<0.05, P<0.01). Except for the macromolecular chondroitin sulfate+glucosamine group, the serum PTH level of male osteoporotic rats in the other administration groups was significantly decreased (P<0.05, P<0.01).

[00218] Femoral trabecular area, percentage of trabecular area, and number of trabeculae

[00219] To evaluate the severity of osteoporosis, the femoral trabecular area, the percentage of trabecular area, and the number of trabeculae in the rats were measured. The results were as shown in Fig. 12M, Fig. 12N, and Fig. 12O. The femoral trabecular area, the percentage of trabecular area, and the number of trabeculae in the rats in the model group were significantly reduced, indicating that the rats in the model group had the most severe osteoporosis. This was significantly different from the sham-operated group (P<0.01), indicating that the model was successfully implemented.

[00220] Compared with the model group, in the highest, high and medium dose groups and in the medium dose oligosaccharide+glucosamine group, the femoral trabecular area, the percentage of trabecular area and the number of trabeculae were significantly increased in male osteoporotic rats (P<0.05, P<0.01). Compared with the model group, in the treatment groups, the femoral trabecular area, the percentage of trabecular area and the number of trabeculae were significantly increased in female osteoporotic rats (P<0.05, P<0.01).

[00221] Compared with the model group, the number of femoral trabeculae in male osteoporotic rats was significantly increased in all administration groups except the Ca group (P<0.05, P<0.01). Compared with the model group, except the low dose CSO group and the Ca group, the number of femoral trabeculae in female osteoporotic rats was significantly increased in all treatment groups (P<0.05, P<0.01).

[00222] These results showed that chondroitin sulfate oligosaccharide had a positive effect on alleviating osteoporosis.

[00223] According to the available experimental data, the following conclusions can be drawn: different doses of chondroitin oligosaccharide sulfate have an effect on relieving and treating pain in osteoarthritis and osteoporosis. Chondroitin oligosaccharide sulfate in combination with glucosamine can enhance the effect on relieving and treating pain in osteoarthritis and osteoporosis. The effect of chondroitin oligosaccharide sulfate on relieving osteoarthritis and osteoporosis is better than the effect of macromolecular chondroitin sulfate.

[00224] Each of the references cited in the description of the application above is incorporated herein by reference. In the event of a conflict between the above description and the references, the description provided in this application will prevail.

Claims (39)

1. A low molecular weight chondroitin sulfate, wherein the average molecular weight of the low molecular weight chondroitin sulfate is less than 1000 Daltons, and the molecular weight distribution of the low molecular weight chondroitin sulfate is narrow, containing chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide as the main components, of which the content of chondroitin sulfate disaccharide is 43-60% and the content of chondroitin sulfate tetrasaccharide is 30-45%, the sum of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is more than 87%; wherein the content of chondroitin sulfate disaccharide and the content of chondroitin sulfate tetrasaccharide are determined by liquid chromatography mass spectrometry; and wherein the general formula of the structure of the low molecular weight chondroitin sulfate is shown in the following formula I: Formula I where n=0-5, and n is an integer, R ₁ , R ₂ , R ₃ = -H or -SO ₃ Na, wherein the low molecular weight chondroitin sulfate is obtained by the following method: macromolecular chondroitin sulfate as a starting material is depolymerized with chondroitin sulfate lyase to obtain a product representing low molecular weight chondroitin sulfate with an average molecular weight stably controlled to less than 1000 Daltons; Chondroitin sulfate lyase was obtained by screening and identifying soil, wastewater or sludge samples from coastal areas, river banks, farmers' markets, slaughterhouses and canteens, and expressed by Escherichia coli or Bacillus subtilis ; wherein the macromolecular chondroitin sulfate as a starting material is obtained from cartilaginous tissue of land and sea animals selected from one or more than one of chicken cartilage, pig cartilage, bovine cartilage or shark bone; and wherein the working conditions of the enzymatic depolymerization reaction are as follows: the added amount of chondroitin sulfate lyase per liter of fermentation broth is 100-300 U/L, the concentration of macromolecular chondroitin sulfate as a starting material is 100-700 g/L, the enzymatic depolymerization time is 6-40 h, the enzymatic depolymerization temperature is 25-35°C, the stirring speed is 100-700 rpm, and the pH of the enzymatic depolymerization is 6.5-8.5. 2. Low molecular weight chondroitin sulfate according to claim 1, wherein the average molecular weight of the low molecular weight chondroitin sulfate is 590-830 Da and preferably 677-742 Da. 3. The low molecular weight chondroitin sulfate according to claim 1, wherein, compared with the macromolecular chondroitin sulfate from shark bone, the low molecular weight chondroitin sulfate obtained as a result of enzymatic depolymerization from one or more than one of shark bone and chicken cartilage has a more significant restorative effect at a concentration of 50-100 μg/ml on chondrocytes damaged by 1 mM hydrogen peroxide, and the degree of restoration is 14-23%; or the low molecular weight chondroitin sulfate with a specific range of disaccharide and tetrasaccharide content specified in claim 1, in a concentration range of 50-1600 μg/ml can restore chondrocytes damaged by 1 mM hydrogen peroxide, and the degree of restoration is 20-62.4%. 4. A method for producing low molecular weight chondroitin sulfate according to any one of claims 1, 2, wherein macromolecular chondroitin sulfate as a starting material is depolymerized with chondroitin sulfate lyase to obtain a product that is low molecular weight chondroitin sulfate with an average molecular weight stably controlled to less than 1000 Daltons, Chondroitin sulfate lyase is obtained by screening and identifying soil, wastewater or sludge samples from coastal areas, river banks, farmers' markets, slaughterhouses and canteens, and expressed by Escherichia coli or Bacillus subtilis ; wherein the macromolecular chondroitin sulfate as a starting material is obtained from cartilaginous tissue of land and sea animals selected from one or more than one of chicken cartilage, pig cartilage, bovine cartilage or shark bone; and wherein the working conditions of the enzymatic depolymerization reaction are as follows: the added amount of chondroitin sulfate lyase per liter of fermentation broth is 100-300 U/L, the concentration of macromolecular chondroitin sulfate as a starting material is 100-700 g/L, the enzymatic depolymerization time is 6-40 h, the enzymatic depolymerization temperature is 25-35°C, the stirring speed is 100-700 rpm, and the pH of the enzymatic depolymerization is 6.5-8.5; and wherein the low molecular weight chondroitin sulfate contains chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide as the main components, of which the content of chondroitin sulfate disaccharide is 43-60%, and the content of chondroitin sulfate tetrasaccharide is 30-45%, the sum of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is more than 87%; and wherein the general formula of the structure of the low molecular weight chondroitin sulfate is shown in the following formula I: Formula I where n=0-5, and n is an integer, R ₁ , R ₂ , R ₃ = -H or -SO ₃ Na. 5. A method for producing low-molecular chondroitin sulfate according to claim 4, wherein the macromolecular chondroitin sulfate as a starting material is obtained from shark bone. 6. The method for producing low molecular weight chondroitin sulfate according to claim 4, wherein the protein is removed from the hydrolyzate with mixed solvents after an enzymolysis reaction in a reaction mixture in which the volume ratio of the hydrolyzate and mixed solvents is 2-5:1, and the volume ratio of dichloromethane and isopropyl alcohol in the mixed solvents is 3-5:1; and wherein the reaction mixture is stirred at 100-500 rpm for 10-40 min, centrifuged at 3000-5000 rpm for 10-30 min, and the upper layer of the reaction solution is taken. 7. A method for producing low molecular weight chondroitin sulfate according to claim 4, wherein the protein is removed from the hydrolyzate after the enzymolysis reaction by ultrafiltration to obtain a reaction solution. 8. The method for producing low molecular weight chondroitin sulfate according to claim 6, wherein the upper reaction solution is filtered and sterilized through a 0.22 μm capsule filter after removing the protein, and then the reaction solution is added to 8-12 times the volume of anhydrous ethanol for precipitation and dried in a vacuum. 9. The method for producing low molecular weight chondroitin sulfate according to claim 7, wherein the reaction solution is filtered and sterilized through a 0.22 μm capsule filter after removing the protein and then spray dried. 10. The use of low molecular weight chondroitin sulfate according to any one of claims 1, 2, in the fields of preparing pharmaceutical preparations for reducing joint inflammation, relieving pain and/or relieving or treating osteoporosis in a subject. 11. A composition containing low molecular weight chondroitin sulfate according to claim 1 or 2, which contains a daily dosage of 50-800 mg of low molecular weight chondroitin sulfate for humans, intended for use in pharmaceuticals, healthcare products and food products. 12. A composition containing low molecular weight chondroitin sulfate according to claim 11, which may contain glucosamine. 13. A composition containing low molecular weight chondroitin sulfate according to claim 12, wherein the glucosamine may be glucosamine hydrochloride, glucosamine sulfate, or a mixture thereof. 14. A composition containing low molecular weight chondroitin sulfate according to claim 11, wherein the low molecular weight chondroitin sulfate is a mixture of low molecular weight chondroitin sulfate with different molecular weights. 15. A composition containing low molecular weight chondroitin sulfate according to claim 14, wherein the average molecular weight of the low molecular weight chondroitin sulfate is 379-1000 Da. 16. A composition containing low molecular weight chondroitin sulfate according to claim 11, which contains pharmaceutically acceptable excipients selected from fillers, disintegrants, adhesives, fragrances, lubricants and film coating agents. 17. A composition comprising low molecular weight chondroitin sulfate according to claim 16, wherein the fillers include, but are not limited to, microcrystalline cellulose, starch, dextrin, mannitol, lactose; wherein the disintegrants include, but are not limited to, crospovidone, sodium croscarmellose, sodium carboxymethyl starch, hydroxypropyl starch, pregelatinized starch, low-substituted hydroxypropyl cellulose, sodium bicarbonate, citric acid, tartaric acid; wherein the adhesives include, but are not limited to, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone; wherein lubricants include, but are not limited to, magnesium stearate, colloidal silicon dioxide, talc, hydrogenated vegetable oil, polyethylene glycol, stearic acid; and wherein film coating agents include, but are not limited to, hydroxypropyl methylcellulose, polyethylene glycol and color lake. 18. A composition comprising low molecular weight chondroitin sulfate according to claim 11, which is in dosage forms including, but not limited to, tablets, granules, capsules and pills. 19. A method for reducing joint inflammation, relieving pain, and/or relieving or treating osteoporosis in a subject, comprising administering to the subject a composition comprising low molecular weight chondroitin sulfate according to any one of claims 11-18. 20. The method of claim 19, which is designed to reduce joint inflammation by reducing the level of inflammatory cytokines or complement in the blood serum or reducing the pathological score in safranin O-fast green staining, wherein the inflammatory cytokines are one of the following: IL-6, IL-1β or TNF-α; wherein the complement is C5b-9. 21. The method of claim 19, which is intended to reduce joint inflammation by improving the level of hydroxyproline acid in the femur. 22. The method of claim 19, which is intended to alleviate or treat osteoporosis by increasing the level of one of the following: bone mass index, bone density, ash level, bone phosphorus level, serum alkaline phosphatase, bone morphogenetic protein, calcitonin, femoral trabecular area, percentage of trabecular area, or number of trabeculae, or by decreasing the level of parathyroid hormone. 23. The method according to claim 19, wherein said composition contains a daily dose of 50-800 mg of low molecular weight chondroitin sulfate for humans. 24. The method according to claim 19, wherein said composition is administered in the dosage forms of tablets, granules, capsules or pills. 25. The method according to claim 19, wherein said composition is administered once a day or twice a day.