Cartilage polypeptide compositions and methods of making same

Abstract

The application provides a kind of cartilage polypeptide composition and its preparation method, the cartilage polypeptide 30-45%, auxiliary agent 10-25%, growth agent 3-8%, slow-release agent 2-9%, the balance is water, the application will cartilage polypeptide, auxiliary agent, growth agent and slow-release agent several ingredients scientific compatibility, preparation has the composition of protecting cartilage tissue, anti-inflammatory pain, effectively increase bone density effect, improve synovial tissue, there is no rebound ineffective condition after drug withdrawal.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a cartilage polypeptide composition and a preparation method thereof. Background Art

[0002] Chondroitin polypeptides are enzymatically hydrolyzed products made from animal cartilage. Rich in collagen peptides and chondroitin sulfate, they promote healthy joints, skin, and connective tissue. Cartilage contains many types of collagen, the most abundant of which is type II collagen. Type II collagen was discovered in the 1970s by French scholar Miller, who isolated it from chicken cartilage. It is composed of three identical α chains and is primarily produced by chondrocytes. Its characteristic amino acid, hydroxylysine, is significantly higher in content than type I or type III collagen. Chondroitin sulfate (CS) is an important natural acidic mucopolysaccharide, widely found in animal cartilage tissue, and is a representative of animal proteoglycans. Due to its pharmacological effects, including anti-inflammatory, anti-tumor, lipid-lowering, and anti-atherosclerotic plaque formation, its effectiveness is excellent and its side effects are minimal, making it an ideal choice for applications in medicine, food, biomaterials, and cosmetics.

[0003] Osteoarthritis is a disease characterized by degeneration of joint cartilage, pain that worsens with weight-bearing, wear and loss of articular cartilage, bone hyperplasia leading to joint deformity, creaking during movement, local tenderness, and mild swelling. Currently, both Traditional Chinese Medicine and Western Medicine are available for the treatment of osteoarthritis. However, oral administration of Traditional Chinese Medicine (TCM) is slow to produce immediate results, while Western medicine primarily relies on painkillers or injections. However, these medications have significant side effects, and injections are not effective long-term.

[0004] Osteoporosis is a systemic skeletal disease characterized by low bone mass and microstructural degeneration. This leads to increased bone brittleness and a heightened risk of fracture. Osteoporosis is a reduction in bone tissue mass, including a proportional decrease in both organic and inorganic matter. Early on, it manifests as thinning, fracture, and loss of cancellous trabeculae, a decrease in trabeculae, and increased load on the remaining trabeculae, leading to microfractures and structural damage. As the disease progresses, a small portion of the inner surface of the cortical bone gradually transforms into a structure similar to cancellous bone, thinning the cortical bone and significantly reducing its strength, including both elasticity and hardness. Fracture is the most important clinical complication of osteoporosis. The incidence and prevalence of osteoporotic fractures at all sites steadily increase with age. While current chondroitin medications can improve osteoporosis and repair articular cartilage to a certain extent, their effectiveness is less than ideal, their effects are limited, and their efficacy in enhancing bone density is limited. Summary of the Invention

[0005] In view of this, the present application provides a cartilage polypeptide composition and a preparation method thereof, which solve the above problems.

[0006] The technical scheme of the present application is implemented as follows: a cartilage polypeptide composition comprises the following raw materials in percentages: 30-45% of cartilage polypeptide, 10-25% of auxiliary agent, 3-8% of growth promoter, and 2-9% of sustained-release agent, and the rest is water.

[0007] Further, the composition comprises the following raw materials in percentages: 36% of cartilage polypeptide, 18% of auxiliary agent, 5% of growth promoter, and 6% of sustained-release agent, and the rest is water.

[0008] Further, the cartilage polypeptide is one or more of fish head cartilage, tail cartilage, and vertebral cartilage.

[0009] Further, the extraction method of the cartilage polypeptide comprises the following steps:

[0010] S1, cartilage pretreatment: the above fish spine or fish head bone is stripped of periosteum, washed clean, and crushed to an average particle size of 0.1-0.8 mm to obtain cartilage powder;

[0011] S2, defatting treatment: the cartilage powder is soaked in an 80-90 (wt) % ethanol solution for 5-8 h, wherein the mass ratio of the cartilage powder to the ethanol solution is 1-4:10, and the cartilage powder is shaken in a gas bath shaking box at 20-28 ℃ and a rotation speed of 180-250 rpm for 10-15 h, and then deionized water is used to remove ethanol, and the defatted cartilage powder is dried to a water content of 2-8%;

[0012] S3, enzymolysis: the defatted cartilage powder is hydrolyzed using a composite enzyme under the assistance of ultrasonic waves, and the mass ratio of the cartilage powder to the composite enzyme is 5-9:1, the composite enzyme is alkaline protease, trypsin, and β-glucosidase in a mass ratio of (1-3):(2-6):(0.3-1.5), the enzyme activity is 10-13 × 10 5 cfu / g, the enzymolysis temperature is 20-40 ℃, the pH is 7-9, the ultrasonic power is 400-600 W, the supernatant is obtained by centrifugation, and the concentrated solution is obtained by ultrafiltration and concentration;

[0013] S4, the concentrated solution is adjusted to a pH of 3-7, dehydrated by ultrafiltration, the ultrafiltration membrane used has a molecular weight cut-off of 5000-10000 Da, the effluent I is collected, and then pure water is used for elution, the effluent II is collected, the effluent I and the effluent II are combined, hydrogen peroxide is added, and the mixture is oxidized at a temperature of 20-40 ℃ for 40-80 min, the mixture is concentrated by ultrafiltration, the pH value is adjusted to 5.0-6.0, 90-97% ethanol is added for crystallization, the ethanol concentration is adjusted to 65-75%, and then the cartilage polypeptide is obtained by centrifugation, washing, and drying after crystallization.

[0014] Furthermore, the auxiliary agent includes the following raw materials in parts by weight: 3-9 parts of Achyranthes bidentata powder, 0.1-0.5 parts of antler glue, 2-4 parts of pearl powder, and 2-5 parts of pentapeptide-34 trifluoroacetate.

[0015] Furthermore, the growth-promoting agent comprises the following raw materials in parts by weight: 5-15 parts of water-soluble coenzyme Q10, 1-3 parts of protamine, 3-5 parts of betulinic acid, and 2-4 parts of glycerophospholipid.

[0016] Furthermore, the sustained-release agent is a mixture of sodium carboxymethyl cellulose, maleic acid, and mannitol in a mass ratio of (1-3): (0.3-0.8): (1.8-2.0).

[0017] Furthermore, a method for preparing a cartilage polypeptide composition comprises the following steps:

[0018] S1. Weigh cartilage polypeptide and auxiliary agent respectively, mix and dissolve in distilled water, heat to 70-80°C and keep stirring for 0.5-1h, and control the stirring speed at 400-600 rpm to obtain gelatinized liquid;

[0019] S2. Add the growth promoter to the gelatinized liquid and stir until it is completely dissolved to obtain solution A;

[0020] S3. Slowly add the sustained-release agent to solution A, heat to 60-70° C., and use a disperser to disperse and homogenize at high speed to obtain an emulsion, which is then spray-dried to obtain a cartilage polypeptide composition powder.

[0021] Furthermore, the cartilage polypeptide composition prepared by the method for preparing the cartilage polypeptide composition is in the form of capsules, tablets, granules or powders.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention scientifically combines several components, including cartilage polypeptide, adjuvant, growth promoter and sustained-release agent, to prepare a composition with the functions of protecting cartilage tissue, resisting inflammation and pain, and effectively increasing bone density. The present invention adopts the cartilage polypeptide and the adjuvant in combination, which can promote the production and replenishment of joint synovial fluid in large quantities, promote the regeneration of cartilage cells, lubricate the joint cartilage surface, reduce wear, increase the flexibility of the joint area, improve the synovial tissue, and prevent rebound after drug withdrawal; when used in combination with the growth promoter, the active ingredients in the growth promoter can assist the cartilage polypeptide and the adjuvant in combining with the cartilage polypeptide to protect connective tissue, promote blood circulation and metabolism, fully inhibit the activity of cartilage degrading enzymes, stimulate the regeneration of connective tissue cells, and thus accelerate the reconstruction and recovery of soft tissue, thereby playing a role in preventing and treating joint degeneration; the sustained-release agent can promote the cartilage polypeptide composition to slowly release the drug effect in the body and maintain steady state; the various components have a synergistic effect, improve bone metabolism, promote calcium absorption, better increase bone density, and delay osteoporosis.

[0024] The present invention can efficiently extract cartilage polypeptides. During the extraction process, air bath vibration is used for effective degreasing, and complex enzyme hydrolysis is performed. The complex enzyme is specifically proportioned, and its proportion and temperature are controlled. Ultrasonic vibration is also used to control the ultrasonic conditions to have a positive effect on the complex enzyme, thereby improving the efficiency of the enzymatic hydrolysis process, thereby significantly improving the extraction efficiency of the cartilage polypeptides.

[0025] The invention is prepared by compounding active ingredients that have health-care effects on bones and joints. The raw materials have little side effects and good effects, and have a broad market. DETAILED DESCRIPTION

[0026] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0027] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0028] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0029] Example 1

[0030] The cartilage polypeptide composition comprises the following raw materials in percentage: 30% cartilage polypeptide, 10% auxiliary agent, 3% growth promoter, 2% sustained-release agent, and the balance is water;

[0031] The cartilage polypeptide is tilapia head cartilage, and the extraction method comprises the following steps:

[0032] S1. Cartilage pretreatment: The periosteum of the tilapia head cartilage was removed, the tilapia head cartilage was rinsed clean, and then ground into an average particle size of 0.1 mm to obtain cartilage powder;

[0033] S2. Degreasing: Soak the cartilage powder in 80 wt% ethanol solution for 5 h, with the mass ratio of cartilage powder to ethanol solution being 1:10. Shake the powder in an air bath at 20°C and 180 rpm for 10 h, remove the alcohol with deionized water, and dry the powder to obtain a defatted cartilage powder with a moisture content of 2-8%.

[0034] S3. Enzymatic hydrolysis: Defatted cartilage powder was hydrolyzed with a composite enzyme under ultrasound assistance. The mass ratio of cartilage powder to composite enzyme was 5:1. The composite enzyme consisted of alkaline protease, trypsin, and β-glucosidase in a mass ratio of 1:2:0.3. The enzyme activity was 10×10 5 cfu / g, enzymatic hydrolysis temperature was 20℃, pH was 7 for 2 h, ultrasonic power was 400 W, supernatant was collected by centrifugation, and concentrated by ultrafiltration to obtain concentrate;

[0035] S4. The concentrated solution is adjusted to pH 3, and ultrafiltration is performed for dehydration. The molecular weight cutoff of the ultrafiltration membrane used is 5000Da. The effluent I is collected and eluted with pure water. The effluent II is collected. The effluent I and the effluent II are combined and added into hydrogen peroxide for oxidation at a temperature of 20°C for 40 minutes. The ultrafiltration is concentrated and the pH value is adjusted to 5.0. 90% ethanol is added for crystallization to make the ethanol concentration reach 65%. After crystallization, the cartilage polypeptide is obtained by centrifugation, washing, and drying.

[0036] The auxiliary agent comprises the following raw materials in parts by weight: 3 parts of Achyranthes bidentata powder, 0.1 parts of antler glue, 2 parts of pearl powder, and 2 parts of pentapeptide-34 trifluoroacetate;

[0037] The growth-promoting agent comprises the following raw materials in parts by weight: 5 parts of water-soluble coenzyme Q10, 1 part of protamine, 3-5 parts of betulinic acid, and 2 parts of glycerophospholipid;

[0038] The sustained-release agent is a mixture of sodium carboxymethyl cellulose, maleic acid, and mannitol in a mass ratio of 1:0.3:1.8.

[0039] Example 2

[0040] The cartilage polypeptide composition comprises the following raw materials in percentage: 45% of cartilage polypeptide, 25% of auxiliary agent, 8% of growth promoter, 9% of sustained-release agent, and the balance of water;

[0041] The cartilage polypeptide is fish head cartilage, and the extraction method comprises the following steps:

[0042] S1. Cartilage pretreatment: The periosteum of the fish head cartilage was removed, the fish head cartilage was rinsed clean, and then crushed to an average particle size of 0.8 mm to obtain cartilage powder;

[0043] S2. Degreasing: Soak the cartilage powder in a 90 wt% ethanol solution for 8 h, with the mass ratio of cartilage powder to ethanol solution being 4:10. Shake the solution in an air bath shaker at 28°C and 250 rpm for 15 h, remove the alcohol with deionized water, and dry the solution to obtain a defatted cartilage powder with a moisture content of 8%.

[0044] S3. Enzymatic hydrolysis: The defatted cartilage powder was hydrolyzed with a composite enzyme under ultrasound assistance. The mass ratio of cartilage powder to composite enzyme was 5-9:1. The composite enzyme was alkaline protease, trypsin, and β-glucosidase in a mass ratio of 3:6:1.5, and the enzyme activity was 10-13×10 5 cfu / g, enzymatic hydrolysis temperature was 40℃, pH was 9.0 for 5 h, ultrasonic power was 600 W, supernatant was collected by centrifugation, and concentrated by ultrafiltration to obtain concentrate;

[0045] S4, the concentrated solution is adjusted to pH 7, and is dehydrated by ultrafiltration, the molecular weight cut-off of the ultrafiltration membrane used is 10000 Da, the effluent I is collected, and then the effluent II is collected by elution with pure water, the effluent I and the effluent II are combined, and then are oxidized by hydrogen peroxide at a temperature of 40℃ for 80 min, and are concentrated by ultrafiltration, the pH value is adjusted to 6.0, and crystallization is performed by adding 97% ethanol, so that the ethanol concentration reaches 75%, and then the cartilage polypeptide is obtained by centrifugation, washing and drying after crystallization.

[0046] The auxiliary agent comprises the following raw materials in parts by weight: 9 parts of Radix Achyranthis Bidentatae powder, 0.5 parts of Cornu Cervi Degelatinatum, 4 parts of pearl powder, and 5 parts of penta-peptide-34 trifluoroacetate;

[0047] The growth promoter comprises the following raw materials in parts by weight: 15 parts of water-soluble coenzyme Q10, 3 parts of protamine, 5 parts of white birch acid, and 4 parts of glycerophospholipid.

[0048] The sustained-release agent is a mixture of sodium carboxymethyl cellulose, maleic acid and mannitol in a mass ratio of 3:0.8:2.0.

[0049] Example 3

[0050] The cartilage polypeptide composition comprises the following raw materials in percentages: 36% of cartilage polypeptide, 18% of auxiliary agent, 5% of growth promoter, 6% of sustained-release agent, and the balance of water.

[0051] The cartilage polypeptide is chicken breast cartilage, and the extraction method comprises the following steps:

[0052] S1, cartilage pretreatment: the chicken breast cartilage is cleaned and crushed to an average particle size of 0.5 mm after removing periosteum, to obtain cartilage powder;

[0053] S2, defatting treatment: the cartilage powder is soaked in an 85(wt)% ethanol solution for 6 h, the mass ratio of the cartilage powder to the ethanol solution is 2:10, and the defatting treatment is performed in a shaking incubator at 25℃ and a rotation speed of 220 rpm for 13 h, and then the ethanol is removed by deionized water, and the defatting cartilage powder is dried to a water content of 5%;

[0054] S3, enzymolysis: the defatting cartilage powder is hydrolyzed by using a composite enzyme under the assistance of ultrasonic waves, the mass ratio of the cartilage powder to the composite enzyme is 7:1, the composite enzyme comprises alkaline protease, trypsin and β-glucosidase in a mass ratio of 2:4:0.9, the enzyme activity is 12×10 5 cfu / g, the enzymolysis is performed at a temperature of 30℃ and a pH of 8 for 3 h, the ultrasonic power is 500 W, the supernatant is obtained by centrifugation, and the concentrated solution is obtained by ultrafiltration and concentration;

[0055] S4, the concentrated solution is adjusted to pH 5, and is dehydrated by ultrafiltration, the molecular weight cut-off of the ultrafiltration membrane used is 7000 Da, the effluent I is collected, and the effluent II is collected by elution with pure water, the effluent I and the effluent II are combined, and are oxidized with hydrogen peroxide at a temperature of 30℃ for 60 min, and are concentrated by ultrafiltration, the pH value is adjusted to 5.0, and crystallization is performed by adding 95% ethanol, the ethanol concentration is adjusted to 70%, and the cartilage polypeptide is obtained by centrifugation, washing, and drying after crystallization.

[0056] The auxiliary agent comprises the following raw materials in parts by weight: 6 parts of Radix Achyranthis Bidentatae powder, 0.3 parts of cornu cervi, 3 parts of pearl powder, and 4 parts of penta-peptide-34 trifluoroacetate;

[0057] The growth promoter comprises the following raw materials in parts by weight: 10 parts of water-soluble coenzyme Q10, 2 parts of protamine, 4 parts of white birch acid, and 3 parts of glycerophospholipid;

[0058] The sustained-release agent is a mixture of sodium carboxymethyl cellulose, maleic acid, and mannitol in a mass ratio of 2:05:1.9.

[0059] The cartilage polypeptide compositions in the above Examples 1-3 are prepared by the following method:

[0060] S1, the cartilage polypeptide and the auxiliary agent are weighed respectively, are mixed and dissolved in distilled water, are heated to 75℃ and are kept stirring for 0.8 h, the stirring rate is controlled at 500 rpm, and a gelatinized liquid is obtained;

[0061] S2, the gelatinized liquid is added to the growth promoter and is stirred until completely dissolved, and a solution A is obtained;

[0062] S3, the sustained-release agent is slowly added to the solution A, is heated to 65℃, and is uniformly dispersed by using a disperser at a high speed, an emulsion is obtained, and a cartilage polypeptide composition powder is obtained by spray drying.

[0063] Example 4

[0064] A cartilage polypeptide composition and a preparation method thereof: the composition comprises the following raw materials in percentage: cartilage polypeptide 36%, auxiliary agent 18%, growth promoter 5%, sustained-release agent 6%, and the balance is water;

[0065] The cartilage polypeptide is chicken breast cartilage, and the extraction method comprises the following steps:

[0066] S1, cartilage pretreatment: the chicken breast cartilage is removed from the periosteum, is washed clean, is crushed to an average particle size of 0.5 mm, and cartilage powder is obtained;

[0067] S2, defatting treatment: the cartilage powder is soaked in 85(wt)% ethanol solution for 6h, the mass ratio of cartilage powder to ethanol solution is 2:10, the solution is shaken in a gas bath shaking box at 25℃, 220rpm for 13h, deionized water is used to remove ethanol, and the defatted cartilage powder is dried to a moisture content of 5%;

[0068] S3, enzymatic hydrolysis: the defatted cartilage powder is hydrolyzed using a composite enzyme under ultrasonic assistance, the mass ratio of cartilage powder to composite enzyme is 7:1, the composite enzyme is alkaline protease, trypsin and β-glucosidase with a mass ratio of 2:4:0.9, the enzyme activity is 12x10 5 cfu / g, the enzymatic hydrolysis is carried out at 30℃, pH 8 for 3h, the ultrasonic power is 500W, the supernatant is obtained by centrifugation, and the concentrated solution is obtained by ultrafiltration concentration;

[0069] S4, the concentrated solution is adjusted to pH 5, dehydrated by ultrafiltration, the molecular weight cut-off of the ultrafiltration membrane used is 7000Da, the effluent I is collected, and then eluted with pure water, the effluent II is collected, the effluent I and the effluent II are combined, and then hydrogen peroxide is added to oxidize at 30℃ for 60min, the concentrated solution is obtained by ultrafiltration concentration, the pH value is adjusted to 5.0, 95% ethanol is added for crystallization, the ethanol concentration is adjusted to 70%, and the cartilage polypeptide is obtained by centrifugation, washing and drying after crystallization.

[0070] The auxiliary agent includes the following raw materials by weight: 6 parts of Radix Achyranthis Bidentatae powder, 0.3 parts of deerhorn glue, 3 parts of pearl powder, and 4 parts of pentapeptide-34 trifluoroacetate;

[0071] The growth promoter includes the following raw materials by weight: 10 parts of water-soluble coenzyme Q10, 2 parts of protamine, 4 parts of white birch acid, and 3 parts of glycerophospholipid;

[0072] The sustained-release agent is a mixture of carboxymethyl cellulose sodium, maleic acid and mannitol with a mass ratio of 2:05:1.9.

[0073] The above-mentioned cartilage polypeptide composition is prepared by the following method:

[0074] S1, the cartilage polypeptide and the auxiliary agent are weighed respectively, mixed and stirred to dissolve in distilled water, heated to 70℃ and kept stirring for 0.5h, and the stirring rate is controlled at 400rpm to obtain a gelatinized solution;

[0075] S2, the gelatinized solution is added to the growth promoter and stirred until completely dissolved to obtain solution A;

[0076] S3, the sustained-release agent is slowly added to solution A, heated to 60℃ and uniformly dispersed using a disperser at high speed to obtain an emulsion, and the cartilage polypeptide composition powder is obtained by spray drying.

[0077] Example 5

[0078] A cartilage polypeptide composition and a preparation method thereof: the composition comprises the following raw materials in percentage: 36% cartilage polypeptide, 18% auxiliary agent, 5% growth promoter, 6% sustained-release agent, and the balance is water;

[0079] The cartilage polypeptide is chicken sternal cartilage, and the extraction method comprises the following steps:

[0080] S1. Cartilage pretreatment: The periosteum of the chicken sternal cartilage was removed, the chicken sternal cartilage was rinsed clean, and then ground into an average particle size of 0.5 mm to obtain cartilage powder;

[0081] S2. Degreasing: Soak the cartilage powder in 85 wt% ethanol solution for 6 h, with the mass ratio of cartilage powder to ethanol solution being 2:10. Shake the solution in an air bath shaker at 25°C and 220 rpm for 13 h, remove the alcohol with deionized water, and dry the solution to obtain defatted cartilage powder with a moisture content of 5%.

[0082] S3. Enzymatic hydrolysis: Defatted cartilage powder was hydrolyzed with a composite enzyme under ultrasound assistance. The mass ratio of cartilage powder to composite enzyme was 7:1. The composite enzyme consisted of alkaline protease, trypsin, and β-glucosidase in a mass ratio of 2:4:0.9. The enzyme activity was 12×10 5 cfu / g, enzymatic hydrolysis temperature was 30℃, pH was 8 for 3 h, ultrasonic power was 500 W, supernatant was collected by centrifugation, and concentrated by ultrafiltration to obtain concentrate;

[0083] S4. The concentrated solution is adjusted to pH 5, and ultrafiltration is performed for dehydration. The molecular weight cutoff of the ultrafiltration membrane used is 7000 Da. The effluent I is collected and eluted with pure water. The effluent II is collected. The effluent I and the effluent II are combined and added into hydrogen peroxide for oxidation at a temperature of 30° C. for 60 minutes. The ultrafiltration is concentrated and the pH value is adjusted to 5.0. 95% ethanol is added for crystallization to make the ethanol concentration reach 70%. After crystallization, the cartilage polypeptide is obtained by centrifugation, washing, and drying.

[0084] The auxiliary agent comprises the following raw materials in parts by weight: 6 parts of Achyranthes bidentata powder, 0.3 parts of antler glue, 3 parts of pearl powder, and 4 parts of pentapeptide-34 trifluoroacetate;

[0085] The growth-promoting agent comprises the following raw materials in parts by weight: 10 parts of water-soluble coenzyme Q10, 2 parts of protamine, 4 parts of betulinic acid, and 3 parts of glycerophospholipid;

[0086] The sustained-release agent is a mixture of sodium carboxymethyl cellulose, maleic acid, and mannitol in a mass ratio of 2:05:1.9.

[0087] The above cartilage polypeptide composition is prepared by the following method:

[0088] S1. Weigh cartilage polypeptide and auxiliary agent respectively, mix and dissolve in distilled water, heat to 80°C and keep stirring for 1 hour, and control the stirring speed at 600 rpm to obtain gelatinized liquid;

[0089] S2. Add the growth promoter to the gelatinized liquid and stir until completely dissolved to obtain solution A;

[0090] S3. Slowly add the sustained-release agent to solution A, heat to 70° C., and use a disperser to disperse and homogenize at high speed to obtain an emulsion, which is then spray-dried to obtain a cartilage polypeptide composition powder.

[0091] Comparative Example 1

[0092] The difference between this comparative example and Example 3 is that the cartilage polypeptide composition comprises the following raw materials in the following percentages: 60% cartilage polypeptide, 8% auxiliary agent, 12% growth promoter, 11% sustained-release agent, and the balance is water.

[0093] Comparative Example 2

[0094] The difference between this comparative example and Example 3 is that the cartilage polypeptide composition does not contain an adjuvant, and the remaining raw materials and operations are the same, as follows:

[0095] The composition comprises the following raw materials in the following percentages: 36% cartilage polypeptide, 18% auxiliary agent, 5% growth promoter, 6% sustained-release agent, and the balance is water;

[0096] The cartilage polypeptide is chicken sternal cartilage, and the extraction method comprises the following steps:

[0097] S1. Cartilage pretreatment: The periosteum of the chicken sternal cartilage was removed, the chicken sternal cartilage was rinsed clean, and then ground into an average particle size of 0.5 mm to obtain cartilage powder;

[0098] S2. Degreasing: Soak the cartilage powder in 85 wt% ethanol solution for 6 h, with the mass ratio of cartilage powder to ethanol solution being 2:10. Shake the solution in an air bath shaker at 25°C and 220 rpm for 13 h, remove the alcohol with deionized water, and dry the solution to obtain defatted cartilage powder with a moisture content of 5%.

[0099] S3. Enzymatic hydrolysis: Defatted cartilage powder was hydrolyzed with a composite enzyme under ultrasound assistance. The mass ratio of cartilage powder to composite enzyme was 7:1. The composite enzyme consisted of alkaline protease, trypsin, and β-glucosidase in a mass ratio of 2:4:0.9. The enzyme activity was 12×10 5 cfu / g, enzymatic hydrolysis temperature was 30℃, pH was 8 for 3 h, ultrasonic power was 500 W, supernatant was collected by centrifugation, and concentrated by ultrafiltration to obtain concentrate;

[0100] S4. The concentrated solution is adjusted to pH 5, and ultrafiltration is performed for dehydration. The molecular weight cutoff of the ultrafiltration membrane used is 7000 Da. The effluent I is collected and eluted with pure water. The effluent II is collected. The effluent I and the effluent II are combined and added into hydrogen peroxide for oxidation at a temperature of 30° C. for 60 minutes. The ultrafiltration is concentrated and the pH value is adjusted to 5.0. 95% ethanol is added for crystallization to make the ethanol concentration reach 70%. After crystallization, the cartilage polypeptide is obtained by centrifugation, washing, and drying.

[0101] The growth-promoting agent comprises the following raw materials in parts by weight: 10 parts of water-soluble coenzyme Q10, 2 parts of protamine, 4 parts of betulinic acid, and 3 parts of glycerophospholipid;

[0102] The sustained-release agent is a mixture of sodium carboxymethyl cellulose, maleic acid, and mannitol in a mass ratio of 2:05:1.9;

[0103] The above cartilage polypeptide composition is prepared by the following method:

[0104] S1. Weigh cartilage polypeptides respectively and dissolve them in distilled water, heat to 80°C and stir for 1 hour at a stirring rate of 600 rpm to obtain a gelatinized solution;

[0105] S2. Add the growth promoter to the gelatinized liquid and stir until it is completely dissolved to obtain solution A;

[0106] S3. Slowly add the sustained-release agent to solution A, heat to 70° C., and use a disperser to disperse and homogenize at high speed to obtain an emulsion, which is then spray-dried to obtain a cartilage polypeptide composition powder.

[0107] Comparative Example 3

[0108] The difference between this comparative example and Example 3 is that the cartilage polypeptide composition does not contain a growth-promoting agent, and the remaining raw materials and operations are the same, as follows:

[0109] The cartilage polypeptide composition comprises the following raw materials in percentage: 36% of cartilage polypeptide, 18% of auxiliary agent, 6% of sustained-release agent, and the balance of water;

[0110] The cartilage polypeptide is chicken sternal cartilage, and the extraction method comprises the following steps:

[0111] S1. Cartilage pretreatment: The periosteum of the chicken sternal cartilage was removed, the chicken sternal cartilage was rinsed clean, and then ground into an average particle size of 0.5 mm to obtain cartilage powder;

[0112] S2. Degreasing: Soak the cartilage powder in 85 wt% ethanol solution for 6 h, with the mass ratio of cartilage powder to ethanol solution being 2:10. Shake the solution in an air bath shaker at 25°C and 220 rpm for 13 h, remove the alcohol with deionized water, and dry the solution to obtain defatted cartilage powder with a moisture content of 5%.

[0113] S3, enzymatic hydrolysis: defatted cartilage powder is hydrolyzed using a composite enzyme under ultrasonic assistance, the mass ratio of cartilage powder to composite enzyme is 7:1, the composite enzyme is alkaline protease, trypsin and β-glucosidase with a mass ratio of 2:4:0.9, and the enzyme activity is 12×10 5 cfu / g, the enzymatic hydrolysis is carried out at 30℃ and pH 8 for 3h, the ultrasonic power is 500W, the supernatant is obtained by centrifugation, and the concentrated liquid is obtained by ultrafiltration concentration;

[0114] S4, the concentrated liquid is adjusted to pH 5, dehydrated by ultrafiltration, the molecular weight cut-off of the ultrafiltration membrane used is 7000Da, the effluent I is collected, and then eluted with pure water, the effluent II is collected, the effluent I and the effluent II are combined, and then hydrogen peroxide is added to oxidize at 30℃ for 60min, concentrated by ultrafiltration, adjusted to pH 5.0, and crystallized by adding 95% ethanol to make the ethanol concentration reach 70%, and then centrifuged, washed and dried to obtain cartilage polypeptide.

[0115] The auxiliary agent includes the following raw materials by weight: 6 parts of Radix Achyranthis Bidentatae powder, 0.3 parts of Cornu Cervi Degelatinatum, 3 parts of pearl powder, and 4 parts of penta-peptide-34 trifluoroacetate;

[0116] The sustained-release agent is a mixture of carboxymethyl cellulose sodium, maleic acid and mannitol in a mass ratio of 2:05:1.9.

[0117] The above cartilage polypeptide composition is prepared by the following method:

[0118] S1, cartilage polypeptide and auxiliary agent are weighed and mixed respectively, dissolved in distilled water, heated to 75℃ and kept stirring for 0.8h, and the stirring rate is controlled at 500rpm to obtain a gelatinized liquid;

[0119] S2, the sustained-release agent is slowly added to the gelatinized liquid, heated to 65℃ and uniformly dispersed by high-speed dispersion to obtain an emulsion, and then spray dried to obtain cartilage polypeptide composition powder.

[0120] Comparative Example 4

[0121] The difference between this comparative example and Example 3 is that the cartilage polypeptide composition does not contain a sustained-release agent, and the rest of the raw materials and operations are the same, as follows:

[0122] The cartilage polypeptide composition includes the following raw materials by percentage: cartilage polypeptide 36%, auxiliary agent 18%, and growth promoter 5%, and the balance is water;

[0123] The cartilage polypeptide is chicken breast cartilage, and the extraction method includes the following steps:

[0124] S1, cartilage pretreatment: the above chicken breast cartilage is removed from the periosteum, washed and crushed to an average particle size of 0.5mm to obtain cartilage powder;

[0125] S2. Degreasing: Soak the cartilage powder in 85 wt% ethanol solution for 6 h, with the mass ratio of cartilage powder to ethanol solution being 2:10. Shake the solution in an air bath shaker at 25°C and 220 rpm for 13 h, remove the alcohol with deionized water, and dry the solution to obtain defatted cartilage powder with a moisture content of 5%.

[0126] S3. Enzymatic hydrolysis: Defatted cartilage powder was hydrolyzed with a composite enzyme under ultrasound assistance. The mass ratio of cartilage powder to composite enzyme was 7:1. The composite enzyme consisted of alkaline protease, trypsin, and β-glucosidase in a mass ratio of 2:4:0.9. The enzyme activity was 12×10 5 cfu / g, enzymatic hydrolysis temperature was 30℃, pH was 8 for 3 h, ultrasonic power was 500 W, supernatant was collected by centrifugation, and concentrated by ultrafiltration to obtain concentrate;

[0127] S4. The concentrated solution is adjusted to pH 5, and ultrafiltration is performed for dehydration. The molecular weight cutoff of the ultrafiltration membrane used is 7000 Da. The effluent I is collected and eluted with pure water. The effluent II is collected. The effluent I and the effluent II are combined and added into hydrogen peroxide for oxidation at a temperature of 30° C. for 60 minutes. The ultrafiltration is concentrated and the pH value is adjusted to 5.0. 95% ethanol is added for crystallization to make the ethanol concentration reach 70%. After crystallization, the cartilage polypeptide is obtained by centrifugation, washing, and drying.

[0128] The auxiliary agent comprises the following raw materials in parts by weight: 6 parts of Achyranthes bidentata powder, 0.3 parts of antler glue, 3 parts of pearl powder, and 4 parts of pentapeptide-34 trifluoroacetate;

[0129] The growth-promoting agent comprises the following raw materials in parts by weight: 10 parts of water-soluble coenzyme Q10, 2 parts of protamine, 4 parts of betulinic acid, and 3 parts of glycerophospholipid;

[0130] The above cartilage polypeptide composition is prepared by the following method:

[0131] S1. Weigh cartilage polypeptide and auxiliary agent respectively, mix and dissolve in distilled water, heat to 75°C and keep stirring for 0.8h, and control the stirring speed at 500rpm to obtain gelatinized liquid;

[0132] S2. Add the growth promoter to the gelatinized liquid and stir until it is completely dissolved to obtain solution A, which is spray-dried to obtain cartilage polypeptide composition powder.

[0133] 1. Efficacy Test

[0134] 1. Arthritis swelling measurement

[0135] Experimental animals: 100 rats, 3 months old, were selected as animal models. The experiment was divided into 10 groups, with 10 rats in each group.

[0136] Inflammatory Methods: Adjuvant arthritis was induced in rats by a single intradermal injection of 1 mL of a mixture consisting of heat-killed Mycobacterium casei in incomplete Freund's adjuvant at the base of the tail.

[0137] Experimental method: The mice in Group 1 were not given any medication. The mice in Groups 2-10 were injected with 1.5 mL of the compositions of Examples 1-5 and Comparative Examples 1-4 of the present invention per 1 kg rat. The mice were fed continuously for one week without any medication. From the second week, oral administration was started, and the medication was given once a day for three consecutive weeks. The medication was then stopped for one week. The circumference of the same part of the right hind ankle joint of the rats was measured by the wire-tying method every week. The swelling value = (circumference of the ankle joint after inflammation - circumference of the ankle joint before inflammation) / circumference of the ankle joint before inflammation.

[0138] The experimental results are shown in the following table:

[0139]

[0140] As can be seen from the results in the above table, compared with the blank group, the cartilage polypeptide composition of the present invention significantly reduced the swelling of the rat joints, effectively inhibited the swelling of the arthritis rat joints, improved the synovial tissue, and there was no rebound after drug withdrawal; compared with the example group and the comparative example 1, the scientific ratio of the raw materials of the cartilage polypeptide composition synergistically exerted its effect, entered the cartilage to protect the cartilage tissue, and was able to inhibit the edema of the arthritis rats induced by Freund's complete adjuvant; compared with the example group and the comparative example 2, the adjuvant added by the present invention, the components in the adjuvant have anti-inflammatory, analgesic, and immunomodulatory effects The invention discloses a method for the treatment of arthritis in rats with arthritis, wherein the ...

[0141] 2. Bone mineral density (BMD) measurement

[0142] The midpoint and distal end bones of the left femur of the rat were taken and fixed on a plexiglass box stand. The bone density (BMD) was scanned using a Piximus bone densitometer.

[0143]

[0144] As can be seen from the above table, the cartilage polypeptide composition of the present invention is scientifically formulated to increase bone density, resist inflammation and pain, and effectively increase bone density. The components have a synergistic effect, easily promote calcium absorption, better increase bone density, and delay osteoporosis.

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cartilage polypeptide composition, characterized in that: The composition is composed of the following raw materials in the following percentages: 30-45% cartilage polypeptide, 10-25% auxiliary agent, 3-8% growth promoter, 2-9% sustained-release agent, and the balance is water; The cartilage polypeptide is derived from chicken sternal cartilage or fish vertebral cartilage; The method for extracting the cartilage polypeptide comprises the following steps: S1. Cartilage pretreatment: Remove the periosteum from the chicken sternal cartilage or fish vertebral cartilage, rinse it, and then grind it into an average particle size of 0.1-0.8 mm to obtain cartilage powder; S2. Degreasing: Soak the cartilage powder in 80-90 wt% ethanol solution for 5-8 hours, wherein the mass ratio of cartilage powder to ethanol solution is 1-4:10, and shake in an air bath shaker at 20-28°C and 180-250 rpm for 10-15 hours. Remove the alcohol with deionized water and dry the powder to obtain defatted cartilage powder with a moisture content of 2-8%. S3. Enzymatic hydrolysis: Defatted cartilage powder was hydrolyzed with a composite enzyme under ultrasound assistance. The mass ratio of cartilage powder to composite enzyme was 5-9:

1. The composite enzyme consisted of alkaline protease, trypsin, and β-glucosidase in a mass ratio of (1-3):(2-6):(0.3-1.5), and the enzyme activity was 10-13×10 5 cfu / g, enzymatic hydrolysis temperature is 20-40℃, pH is 7.0-9.0, enzymatic hydrolysis is carried out for 2-5h, ultrasonic power is 400-600W, supernatant is collected by centrifugation, and concentrated by ultrafiltration to obtain a concentrate; S4. The concentrated solution is adjusted to a pH of 3-7, and ultrafiltration is performed to dehydrate the solution. The ultrafiltration membrane used has a molecular weight cutoff of 5000-10000 Da. Effluent I is collected and eluted with pure water. Effluent II is collected. Effluent I and effluent II are combined and then added into hydrogen peroxide for oxidation at a temperature of 20-40° C. for 40-80 min. The solution is concentrated by ultrafiltration and the pH is adjusted to 5.0-6.

0. 90-97% ethanol is added for crystallization to a concentration of 65-75% ethanol. After crystallization, the solution is centrifuged, washed, and dried to obtain the cartilage polypeptide. The auxiliary agent is composed of the following raw materials in parts by weight: 3-9 parts of Achyranthes bidentata powder, 0.1-0.5 parts of antler glue, 2-4 parts of pearl powder, and 2-5 parts of pentapeptide-34 trifluoroacetate; The growth-promoting agent is composed of the following raw materials in parts by weight: 5-15 parts of water-soluble coenzyme Q10, 1-3 parts of protamine, 3-5 parts of betulinic acid, and 2-4 parts of glycerophospholipid; The sustained-release agent is a mixture of sodium carboxymethyl cellulose, maleic acid, and mannitol in a mass ratio of (1-3): (0.3-0.8): (1.8-2.0); The method for preparing the cartilage polypeptide composition comprises the following steps: S1. Weigh cartilage polypeptide and auxiliary agent respectively, mix and dissolve in distilled water, heat to 70-80°C and keep stirring for 0.5-1h, and control the stirring speed at 400-600 rpm to obtain gelatinized liquid; S2. Add the growth promoter to the gelatinized liquid and stir until it is completely dissolved to obtain solution A; S3. Slowly add the sustained-release agent to solution A, heat to 60-70° C., and use a disperser to disperse and homogenize at high speed to obtain an emulsion, which is then spray-dried to obtain a cartilage polypeptide composition powder.

2. The cartilage polypeptide composition according to claim 1, wherein: The composition is composed of the following raw materials in the following percentages: 36% of cartilage polypeptide, 18% of auxiliary agent, 5% of growth promoter, 6% of sustained-release agent, and the balance of water.

3. The cartilage polypeptide composition according to claim 1, wherein the cartilage polypeptide composition prepared by the preparation method is in the form of capsules, tablets, granules or powders.

Images