Multifunctional bone peptide compound and application thereof in prevention and treatment of osteoporotic osteoarthritis

Abstract

The invention discloses a multifunctional bone peptide compound and application thereof in prevention and treatment of osteoporotic osteoarthritis, and relates to the technical field of bone peptides. The multifunctional bone peptide compound is prepared from the following raw materials: giant salamander bone meal, bovine bone articular cartilage powder, bovine bone meal, sturgeon bone meal, oyster bone meal, deer-horn glue powder, calcium chloride and fructo-oligosaccharide. According to the multifunctional bone peptide compound disclosed by the invention, the composite aggregate is subjected to enzymolysis step by step by using the compound enzyme, the bone peptide and calcium ions are compounded through a chelation reaction, the absorbability of calcium is improved, one part of the bone peptide in the composite bone peptide has a function of promoting osteoblast activity, and the other part of the bone peptide possibly has a function of inhibiting overactivity of osteoclasts; part of the bone peptide has an anti-inflammatory effect, can regulate bone metabolism, repair articular cartilage and relieve inflammatory response, and can improve the effect of the bone peptide product in the aspects of preventing and treating osteoporosis, osteoarthritis and other diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of osteopeptide, and in particular to a multifunctional osteopeptide complex and its application in preventing and treating osteoporosis and osteoarthritis. Background Art

[0002] Bone diseases such as osteoporosis and osteoarthritis have become a public health problem that seriously affects people's health and quality of life.

[0003] Osteoporosis is a systemic bone disease characterized by decreased bone mass, destruction of bone tissue microstructure, increased bone brittleness and susceptibility to fractures. According to statistics, about 200 million women and 120 million men worldwide suffer from osteoporosis. In my country, with the rapid growth of the elderly population, the number of osteoporosis patients has increased sharply, especially in postmenopausal women, whose incidence rate is higher. Traditional osteoporosis treatments mainly rely on calcium, vitamin D supplements and anti-osteoporosis drugs, such as bisphosphonates and calcitonins. However, these treatments have certain limitations. Although calcium and vitamin D can supplement basic nutrition, their use alone has limited effect on improving bone density; anti-osteoporosis drugs are often accompanied by adverse reactions such as gastrointestinal discomfort and renal damage, and patients have poor tolerance for long-term use.

[0004] Osteoarthritis is also a common chronic joint disease. Its main pathological features are degeneration and wear of articular cartilage, subchondral bone sclerosis, osteophyte formation, and synovial inflammation, which lead to joint pain, swelling, stiffness, and limited movement. Its incidence rate increases significantly with age. About half of people over 50 years old suffer from osteoarthritis to varying degrees. At present, the clinical treatment of osteoarthritis mostly uses non-steroidal anti-inflammatory drugs to relieve pain, intra-articular injection of hyaluronic acid to improve joint lubrication, and joint replacement surgery when the condition is severe. However, non-steroidal anti-inflammatory drugs are prone to cause complications such as gastrointestinal bleeding and ulcers. Joint replacement surgery is high-risk, expensive, and has a long postoperative recovery period, which brings great physical pain and economic burden to patients.

[0005] In the process of seeking more effective and safer treatment methods, natural biomaterials have gradually entered the field of vision of scientific researchers. As a type of bioactive peptide extracted from animal bones or bone tissue, bone peptides have many potential functions, such as promoting bone cell proliferation and differentiation, regulating calcium and phosphorus metabolism, and certain anti-inflammatory activity. For example, bovine bone powder peptides have been found to stimulate the activity of osteoblasts and increase the synthesis of bone matrix; some marine fish bone peptides have shown a protective effect on articular chondrocytes and can delay cartilage degeneration. However, bone peptides from a single source are often limited in function and it is difficult to meet the needs of comprehensive prevention and treatment of osteoporosis and osteoarthritis at the same time.

[0006] In recent years, with the in-depth study of various bone-derived bioactive components, the development of multifunctional complexes integrating multiple components with synergistic effects has become a trend. The bone peptide compositions in the prior art have poor effects and slow onset in the process of preventing and treating osteoporosis and osteoarthritis. Summary of the invention

[0007] The present invention provides a multifunctional bone peptide complex and its application in preventing and treating osteoporosis and osteoarthritis, which solves the technical defects of the prior art that the bone peptide complex has poor therapeutic effect and slow onset in the process of preventing and treating osteoporosis and osteoarthritis.

[0008] In order to solve the above-mentioned invention object, the technical solution provided by the present invention is as follows: A multifunctional bone peptide complex, whose raw materials, by weight, include 20-30 parts of giant salamander bone powder, 15-20 parts of bovine joint cartilage powder, 30-40 parts of bovine bone powder, 10-20 parts of sturgeon bone powder, 3-8 parts of oyster bone powder, 3-6 parts of deer antler glue powder, 7-11 parts of calcium chloride and 3-5 parts of oligofructose.

[0009] Preferably, the raw materials of the complex further include 0.8-1.2 parts of L-lactate calcium, 0.8-1.2 parts of vitamin D3 and 2-3 parts of β-cyclodextrin by mass.

[0010] Preferably, the preparation method of the multifunctional osteopeptide complex comprises the following steps: S1, mixing 3-8 parts of giant salamander bone powder, bovine bone articular cartilage powder, bovine bone powder, sturgeon bone powder and oyster bone powder and crushing them to obtain a mixed aggregate; S2, mixing the mixed aggregate with deionized water at a material-liquid ratio of 1 g:20-25 mL, placing in a constant temperature water bath at 50-55° C., adjusting the pH to 9.0-10, adding alkaline protease, performing a first enzymatic hydrolysis, and obtaining a first enzymatic hydrolysis solution; S3, adjusting the pH value of the first enzymatic hydrolysis solution to 7-7.5, adding a mixed enzyme, and performing a second enzymatic hydrolysis at 50-55° C., wherein the mixed enzyme includes a neutral protease, an elastase, and a papain, to obtain a second enzymatic hydrolysis solution; S4, heating the second enzymatic hydrolysis solution at 95-100° C. for 10-15 min to inactivate the enzyme, thereby obtaining an enzyme-inactivated solution; S5, centrifuging the enzyme-inactivated solution, taking the supernatant, and freeze-drying it to obtain a bone powder extract; S6, adding the bone powder extract to a calcium chloride aqueous solution, adjusting the pH value of the mixed solution to 7.5, performing a chelate reaction, and obtaining a chelate reaction solution; S7, adding ethanol to the chelation reaction solution (the added amount is 4 times the volume of the chelation reaction solution), taking the precipitate after centrifugation, and freeze-drying (-20°C, time is 24h) to obtain bone powder chelate; S8. Mixing the bone powder chelate with antler glue powder, oligofructose, L-lactate calcium, vitamin D3 and β-cyclodextrin to obtain the multifunctional bone peptide complex.

[0011] Preferably, the particle size of the mixed aggregate in S1 is less than 50 μm.

[0012] Preferably, the amount of alkaline protease added during the first enzymatic hydrolysis in S2 is 8000-8500 U / g, and the time of the first enzymatic hydrolysis is 3-4 h.

[0013] Preferably, the amount of the mixed enzyme added during the second enzymatic hydrolysis in S3 is 11000-13000 U / g, and the time of the second enzymatic hydrolysis is 3-4 h.

[0014] Preferably, the mass ratio of neutral protease, elastase and papain in the mixed enzyme in S3 is 2:0.5-1:0.5-1.

[0015] Preferably, the mass concentration of the calcium chloride aqueous solution in S6 is 5-10 mg / mL.

[0016] Preferably, the chelating reaction time in S6 is 3-5 hours.

[0017] A use of the multifunctional osteopeptide complex as described above in the preparation of a health product or medicine for preventing and / or treating osteoporosis and osteoarthritis.

[0018] Giant salamander bone powder bone peptide, bovine bone articular cartilage powder bone peptide, bovine bone powder bone peptide and sturgeon bone powder bone peptide can be extracted through enzymatic treatment.

[0019] Giant salamander bone meal is rich in collagen. The bone peptides obtained after enzymatic hydrolysis are rich in collagen peptides and some bioactive factors, such as calcium regulatory proteins and growth factors. The bone peptides of sturgeon bone meal and oyster bone meal are rich in collagen peptides, especially type I and type III collagen peptides. It may also contain some trace elements, such as zinc, magnesium and other element-binding peptides related to bone metabolism.

[0020] Collagen peptides can provide a structural framework for bone tissue, which is conducive to osteoblasts depositing calcium salts on it and promoting the formation of new bones. In the treatment of osteoporosis, it helps to increase bone density. For osteoarthritis, the collagen peptides in giant salamander bone powder peptides can supplement the collagen components in articular cartilage. Articular cartilage is mainly composed of collagen and proteoglycans. Supplementing collagen can enhance the toughness of cartilage, relieve joint pain and improve joint mobility. The nutrients such as amino acids can provide nutrition for bone cells and chondrocytes, promote cell metabolism, and help maintain the health of bones and cartilage. Peptides bound to elements such as zinc and magnesium may be involved in regulating the physiological functions of bone cells. For example, zinc plays an important role in the proliferation and differentiation of osteoblasts, and magnesium can affect the activity of calcium channels and regulate the distribution of calcium inside and outside cells. These trace element bound peptides can transport trace elements to bone cells and play their role in regulating bone metabolism. In addition, sturgeon bone powder peptides may contain some peptides with antioxidant activity. In the pathological process of osteoarthritis and osteoporosis, oxidative stress can damage bone cells and chondrocytes. These antioxidant peptides can scavenge free radicals, reduce the damage of oxidative stress to cells, and protect the health of bones and cartilage.

[0021] The bone peptides produced after enzymatic hydrolysis of bovine articular cartilage powder mainly include cartilage-specific collagen (such as type II collagen), chondroitin sulfate peptides and hyaluronic acid peptides. These components are important components of articular cartilage. At the same time, it also contains some small molecule peptides with biological activity. Type II collagen peptides can directly participate in the repair of articular cartilage matrix. Cartilage wear is a common problem in patients with osteoarthritis. These peptides can supplement the collagen components in the cartilage matrix and promote chondrocytes to synthesize new cartilage matrix, thereby repairing damaged cartilage. Chondroitin sulfate peptides and hyaluronic acid peptides can increase the viscoelasticity of synovial fluid. Synovial fluid plays a lubricating role in joint movement. These peptides can improve the quality of synovial fluid, reduce friction between joint surfaces, reduce joint wear, and relieve pain in patients with osteoarthritis.

[0022] The main component of bovine bone powder peptide is type I collagen peptide. It also contains mineral binding peptides such as calcium and phosphorus, as well as a variety of amino acids, which can promote the differentiation of osteoblasts and the absorption of calcium. Type I collagen is the most important type of collagen in bone tissue and constitutes the fiber framework of bones. After being absorbed, type I collagen peptide can build a solid fiber network for bone tissue, making the bone tissue more dense. In the prevention and treatment of osteoporosis, it works synergistically with minerals such as calcium to promote the deposition of calcium salts on collagen fibers, increase bone density, improve bone strength, and reduce the risk of fractures. The collagen peptides and amino acids in bovine bone powder peptide can provide raw materials for bone repair. They can stimulate the proliferation and differentiation of surrounding osteoblasts, accelerate the formation of callus, and promote the healing of bone tissue.

[0023] The composite bone peptide is chelated with calcium chloride to form a stable chelate. The speed and effect of the effective ingredients of the bone peptide are improved. After the composite bone peptide is chelated with calcium chloride, a relatively stable structure is formed. This structure can prevent calcium from reacting with other substances (such as oxalic acid, phytic acid, etc.) in the gastrointestinal tract to form a precipitate that is difficult to absorb, and at the same time it is beneficial for the intestinal cells to absorb calcium. In the process of intestinal absorption, there are two ways of active absorption and passive absorption. Chelated calcium may enter the cell by actively absorbing it by binding to special transport proteins on the surface of intestinal cells. This absorption method is not limited by the calcium concentration in the intestine, and the absorption rate is relatively fast. When the chelated calcium reaches the bone, under the guidance of the composite bone peptide, calcium can be more accurately deposited on the bone matrix, promote bone mineralization, and help prevent and treat bone diseases such as osteoporosis.

[0024] The combination of multiple bone peptides promotes the role of the multifunctional bone peptide complex in preventing and treating osteoporosis and osteoarthritis in all aspects. At the same time, deer antler glue powder, oligofructose, etc. are added to promote the absorption of the effective ingredients of the bone peptide composition by the small intestine. Deer antler glue powder is rich in amino acids and animal proteins, some of which can promote the absorption of bone calcium, effectively resist osteoporosis, stimulate the proliferation and differentiation of osteoblasts, increase the synthesis of bone matrix, and inhibit the excessive absorption of bone by osteoclasts, thereby helping to increase bone density. At the same time, oligofructose can improve the absorption of the effective ingredients of bone peptides by the small intestine.

[0025] Bone powder is hydrolyzed to obtain composite bone peptides. The enzymatic hydrolysis is carried out in a step-by-step manner, so that polypeptides of different lengths can be "split" to ensure that the polypeptides in the bone powder can be fully dissolved. First, alkaline protease is used to perform the first enzymatic hydrolysis on the defatted bone powder. For raw materials such as giant salamander bone powder, bovine bone articular cartilage powder, bovine bone powder and sturgeon bone powder, alkaline protease can decompose the proteins therein into smaller peptide segments. It mainly acts on the sites where the carboxyl side of the peptide bond is a hydrophobic amino acid (such as leucine, phenylalanine, etc.). The long-chain protein is gradually decomposed into short-chain peptides. Collagen has a complex triple helix structure, and alkaline protease can destroy this structure and decompose collagen into collagen peptides. These collagen peptides are important components of bone peptides and play an important role in promoting bone formation and improving the function of articular cartilage. For example, in the treatment of osteoarthritis, collagen peptides obtained from bone enzymatic hydrolysis can replenish the collagen components lost in articular cartilage and enhance the toughness of cartilage.

[0026] In the second enzymatic hydrolysis process, a mixed enzyme (neutral protease, elastase and papain) is used for secondary enzymatic hydrolysis. After the initial enzymatic hydrolysis by alkaline protease, the protein structure in the bone raw material is partially destroyed, but there may still be some complex protein aggregates or proteins with special structures. Neutral protease, elastase and papain can decompose these remaining complex structures from different angles. For example, neutral protease performs general peptide bond cleavage to further fragment the protein; elastase decomposes the elastin component to increase the diversity of peptide segments; papain uses its specific cleavage method to process those protein parts that are difficult to be decomposed by other enzymes. Through this multi-level decomposition method, the protein in the bone raw material can be more comprehensively converted into various useful bone peptides. The synergistic effect of these three enzymes can expose these hidden parts by gradually decomposing the protein. For example, some peptide segments that promote the proliferation and differentiation of bone cells may not work in the complete protein molecule, but after the synergistic enzymatic hydrolysis of the three enzymes, these peptide segments are released, thereby enhancing the efficacy of bone peptide products in promoting bone formation and repair. The peptides produced by neutral proteases are of varying lengths, elastase produces peptides with special structures targeting specific elastins, and the peptides produced by papain may be unique in amino acid sequence.

[0027] The technical solution of the present invention has at least the following beneficial effects compared with the prior art: The various bone peptide components in the composite bone peptide of the present invention, such as bone peptides from giant salamander bone powder, cattle bone powder, etc., are rich in collagen peptides, which can provide structural support for bone tissue, stimulate osteoblast proliferation and differentiation, promote new bone formation, and increase bone density.

[0028] The bovine bone articular cartilage powder peptides of the present invention contain type II collagen peptides, chondroitin sulfate peptides, etc., which can accurately repair articular cartilage damage, supplement cartilage matrix components, and enhance cartilage toughness. Chondroitin sulfate increases the viscoelasticity of synovial fluid, plays an excellent lubricating role, reduces friction on the joint surface, relieves pain, and is beneficial for the treatment of arthritis.

[0029] Composite bone peptides chelate with calcium chloride. Bone peptides build the bone matrix framework and guide calcium deposition. Calcium supplements stabilize the structure. The two complement each other and jointly promote bone tissue health. At the same time, bone peptides and vitamin D3 cooperate with each other to promote the absorption and utilization of bone peptides.

[0030] Composite bone peptides are obtained by enzymatic hydrolysis of bones in a step-by-step manner and by adding complex enzymes. Alkaline protease efficiently hydrolyzes collagen in a specific alkaline environment, laying the foundation for subsequent enzymatic hydrolysis and improving the solubility and biological activity of bone peptides. During the secondary enzymatic hydrolysis, neutral protease, elastase and papain work synergistically to fully decompose the complex protein structure, further optimizing the quality and functional diversity of bone peptides.

[0031] In the multifunctional osteopeptide complex of the present invention, a part of the osteopeptide has the function of promoting the activity of osteoblasts, another part of the osteopeptide may have the function of inhibiting the overactivity of osteoclasts, and another part of the osteopeptide has an anti-inflammatory effect. These osteopeptides with different functions work together to more comprehensively regulate bone metabolism, repair joint cartilage and reduce inflammatory reactions, thereby improving the quality and effect of osteopeptide products in preventing and treating diseases such as osteoporosis and osteoarthritis. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the embodiments will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Deer antler glue powder: Hubei Kangyuan Pharmaceutical Co., Ltd., approval number: National Medicine Standard Z42021903. Giant salamander bone powder comes from the fourth generation of artificially cultivated giant salamanders. Sturgeon comes from artificially cultivated Siberian sturgeon. Cattle bones come from artificially cultivated cattle.

[0034] The methods for obtaining giant salamander bone powder, cattle bone articular cartilage powder, cattle bone powder, sturgeon bone powder and oyster bone powder are as follows: fresh giant salamander bones, cattle bone articular cartilage, cattle bones, sturgeon bones or oyster shells are taken out, the minced meat and fascia on the surface are removed, and the bones are repeatedly washed with tap water, boiled in boiling water for 10 minutes to remove the surface fat, and the giant salamander bones, cattle bone articular cartilage, cattle bones, sturgeon bones and oyster shells are washed again. After being placed in a high-pressure steamer at 0.1MPa and boiled for 40 minutes, they are repeatedly washed in hot water to remove grease. Then, the giant salamander bones, cattle bone articular cartilage, cattle bones, sturgeon bones and oyster shells are placed in a constant temperature hot air drying oven at 80℃ for drying. The dried bones are put into a grinder in batches and crushed, and the corresponding pre-treated bone powder is obtained by passing through an 80-mesh sieve. The different pre-treated bone powders were mixed with 10% anhydrous ethanol at a solid-liquid ratio of 1:10 (w / v), and placed on a magnetic stirrer for defatting for 24 hours, with the organic solution replaced every 12 hours. After defatting, the defatted bone powder was repeatedly washed with distilled water, centrifuged at 8000 rpm for 15 minutes in a high-speed centrifuge, and then placed in an oven at 80°C for drying to obtain giant salamander bone powder / bovine bone articular cartilage powder / bovine bone powder / sturgeon bone powder / oyster bone powder.

[0035] The particle size of antler glue powder is less than 130 microns. Fructooligosaccharide: Shanghai Jining Biological Research Co., Ltd., item number BR-T1135. β-cyclodextrin: Jiangsu Lanyu Bioengineering Co., Ltd., item number 1441225. Alkaline protease: activity 200,000 U / g, item number S10154, Shanghai Yuanye Biotechnology. Neutral protease: activity 100U / mg, item number S10013, Shanghai Yuanye Biotechnology. Elastase: activity 10U / mg, item number S27996, Shanghai Yuanye Biotechnology. Papain: activity 800U / mg, item number S10011, Shanghai Yuanye Biotechnology.

[0036] Example 1 The present embodiment provides a multifunctional bone peptide complex, the raw materials of which include, by mass, 20 parts of giant salamander bone powder, 15 parts of bovine bone articular cartilage powder, 30 parts of bovine bone powder, 10 parts of sturgeon bone powder, 3 parts of oyster bone powder, 3 parts of deer antler glue powder, 7 parts of calcium chloride, 3 parts of oligofructose, 0.8 parts of L-lactate calcium, 0.8 parts of vitamin D3 and 2 parts of β-cyclodextrin.

[0037] The preparation method of the multifunctional osteopeptide complex comprises the following steps: S1, mixing giant salamander bone powder, bovine bone articular cartilage powder, bovine bone powder and sturgeon bone powder and crushing them to obtain a mixed aggregate with a particle size of less than 50 μm; S2, the mixed aggregate and deionized water were mixed at a material-liquid ratio of 1g:20mL, the mixture was placed in a constant temperature water bath at 50°C, the pH was adjusted to 9.0, alkaline protease was added in an amount of 8000U / g (8000U / g means that 8000U equivalent of alkaline protease was added to 1g of mixed aggregate, and the following embodiments were measured in the same manner as this embodiment), and the first enzymolysis was performed for 4h to obtain a first enzymolysis solution; S3, adjusting the pH value of the first enzymatic solution to 7, adding a mixed enzyme, the addition amount of the mixed enzyme is 11000U / g (11000U / g refers to the addition of 11000U equivalent of the mixed enzyme per 1g of mixed aggregate, and the embodiments hereinafter are the same as this embodiment), the mass ratio of neutral protease, elastase and papain in the mixed enzyme is 2:0.5:0.5, and performing a second enzymatic hydrolysis at 50°C, and obtaining a second enzymatic hydrolysis solution after 4h of enzymatic hydrolysis; S4, heating the second enzymatic hydrolysis solution at 95° C. for 10 min to inactivate the enzyme, thereby obtaining an enzyme-inactivated solution; S5, centrifuging the enzyme-inactivated solution, taking the supernatant, and freeze-drying it to obtain a bone powder extract; S6, preparing a calcium chloride aqueous solution with a mass concentration of 5 mg / mL with the calcium chloride in the amount recorded in the formula, adding the bone powder extract to the calcium chloride aqueous solution, adjusting the pH value of the mixed solution to 7.5, performing a chelation reaction, and obtaining a chelation reaction solution after reacting for 3 hours; S7, adding ethanol to the chelation reaction solution (the added amount is 4 times the volume of the chelation reaction solution), taking the precipitate after centrifugation, and freeze-drying (-20°C, time is 24h) to obtain bone powder chelate; S8. Mixing the bone powder chelate with antler glue powder, oligofructose, L-lactate calcium, vitamin D3 and β-cyclodextrin to obtain the multifunctional bone peptide complex.

[0038] Example 2 The present embodiment provides a multifunctional bone peptide complex, the raw materials of which include, by mass, 30 parts of giant salamander bone powder, 20 parts of bovine articular cartilage powder, 40 parts of bovine bone powder, 20 parts of sturgeon bone powder, 8 parts of oyster bone powder, 6 parts of deer antler glue powder, 11 parts of calcium chloride, 5 parts of oligofructose, 1.2 parts of calcium L-lactate, 1.2 parts of vitamin D3 and 3 parts of β-cyclodextrin.

[0039] The preparation method of the multifunctional osteopeptide complex comprises the following steps: S1, mixing giant salamander bone powder, bovine bone articular cartilage powder, bovine bone powder and sturgeon bone powder and crushing them to obtain a mixed aggregate with a particle size of less than 50 μm; S2, the mixed aggregate and deionized water were mixed at a material-liquid ratio of 1g:25mL, the pH was adjusted to 10 in a constant temperature water bath at 55°C, alkaline protease was added in an amount of 8500U / g, and the first enzymatic hydrolysis was performed for 4h to obtain a first enzymatic hydrolysis solution; S3, adjusting the pH value of the first enzymatic solution to 7.5, adding a mixed enzyme, wherein the amount of the mixed enzyme added is 13000 U / g, wherein the mass ratio of neutral protease, elastase and papain is 2:1:1, performing a second enzymatic hydrolysis at 55° C., and obtaining a second enzymatic hydrolysis solution after enzymatic hydrolysis for 3 hours; S4, heating the second enzymatic hydrolysis solution at 100° C. for 15 min to inactivate the enzyme, thereby obtaining an enzyme-inactivated solution; S5, centrifuging the enzyme-inactivated solution, taking the supernatant, and freeze-drying it to obtain a bone powder extract; S6, preparing a calcium chloride aqueous solution with a mass concentration of 10 mg / mL with the calcium chloride in the amount recorded in the formula, adding the bone powder extract to the calcium chloride aqueous solution, adjusting the pH value of the mixed solution to 7.5, performing a chelation reaction, and obtaining a chelation reaction solution after reacting for 5 hours; S7, adding ethanol to the chelation reaction solution (the added amount is 4 times the volume of the chelation reaction solution), taking the precipitate after centrifugation, and freeze-drying (-20°C, time is 24h) to obtain bone powder chelate; S8. Mixing the bone powder chelate with antler glue powder, oligofructose, L-lactate calcium, vitamin D3 and β-cyclodextrin to obtain the multifunctional bone peptide complex.

[0040] Example 3 The present embodiment provides a multifunctional bone peptide complex, the raw materials of which include, by mass, 25 parts of giant salamander bone powder, 18 parts of bovine articular cartilage powder, 35 parts of bovine bone powder, 16 parts of sturgeon bone powder, 5 parts of oyster bone powder, 4 parts of deer antler glue powder, 8 parts of calcium chloride, 4 parts of oligofructose, 1 part of L-lactate calcium, 1 part of vitamin D3 and 2.2 parts of β-cyclodextrin.

[0041] The preparation method of the multifunctional osteopeptide complex comprises the following steps: S1, mixing giant salamander bone powder, bovine bone articular cartilage powder, bovine bone powder and sturgeon bone powder and crushing them to obtain a mixed aggregate with a particle size of less than 50 μm; S2, the mixed aggregate and deionized water were mixed at a material-liquid ratio of 1g:23mL, the pH was adjusted to 9.5 in a constant temperature water bath at 53°C, alkaline protease was added in an amount of 8300U / g, and the first enzymatic hydrolysis was performed for 3.5h to obtain a first enzymatic hydrolysis solution; S3, adjusting the pH value of the first enzymatic solution to 7.3, adding a mixed enzyme, wherein the amount of the mixed enzyme added is 12000 U / g, wherein the mass ratio of neutral protease, elastase and papain is 2:0.7:0.6, performing a second enzymatic hydrolysis at 53° C., and obtaining a second enzymatic hydrolysis solution after 3.5 hours of enzymatic hydrolysis; S4, heating the second enzymatic hydrolysis solution at 96° C. for 12 min to inactivate the enzyme, thereby obtaining an enzyme-inactivated solution; S5, centrifuging the enzyme-inactivated solution, taking the supernatant, and freeze-drying it to obtain a bone powder extract; S6, preparing a calcium chloride aqueous solution with a mass concentration of 6 mg / mL with the calcium chloride in the amount recorded in the formula, adding the bone powder extract to the calcium chloride aqueous solution, adjusting the pH value of the mixed solution to 7.5, performing a chelation reaction, and obtaining a chelation reaction solution after reacting for 4 hours; S7, adding ethanol to the chelation reaction solution (the added amount is 4 times the volume of the chelation reaction solution), taking the precipitate after centrifugation, and freeze-drying (-20°C, time is 24h) to obtain bone powder chelate; S8. Mixing the bone powder chelate with antler glue powder, oligofructose, L-lactate calcium, vitamin D3 and β-cyclodextrin to obtain the multifunctional bone peptide complex.

[0042] Comparative Example 1 This comparative example is the same as Example 1, except that in this comparative example, the bovine articular cartilage powder is replaced with an equal amount of bovine bone powder.

[0043] Comparative Example 2 This comparative example is the same as Example 1, except that in this comparative example, an equal amount of elastase in S3 is replaced by a neutral protease.

[0044] Comparative Example 3 This comparative example is the same as Example 1, except that in this comparative example, an equal amount of papain in S3 is replaced by neutral protease.

[0045] Comparative Example 4 This comparative example is the same as Example 1, except that the mass ratio of neutral protease, elastase and papain in the mixed enzyme S3 in this comparative example is 2:0.3:1.5.

[0046] Comparative Example 5 This comparative example is the same as Example 1, except that the mass ratio of neutral protease, elastase and papain in the mixed enzyme S3 in this comparative example is 2:1.5:0.2.

[0047] test: Wild-type AB strain zebrafish were bred by natural pair mating. Zebrafish aged 3 days after fertilization (3dpf) were used. 3dpf normal wild-type AB strain zebrafish were placed in a six-well plate, 30 fish per well, and 3 ml of prednisone (prednisone concentration was 10μg / mL) was placed in each well for 3 days. The osteoporosis model was induced by treating zebrafish with prednisone to obtain osteoporotic zebrafish.

[0048] The model group consisted of zebrafish with osteoporosis, which were placed in an incubator at 28°C for 4 days, and then ten zebrafish were selected, stained with 0.2% calcein and photographed, and data were collected and statistically analyzed for the fluorescence intensity of the first three segments of the zebrafish vertebrae (S); Group 1: osteoporotic zebrafish were placed in a six-well plate, with 30 zebrafish in each well. 3 mL of a 1000 μg / mL suspension of the multifunctional osteopeptide complex in Example 1 was added to each well. After incubation at 28°C for 4 days, ten zebrafish were selected, stained with 0.2% calcein and photographed. Data were collected and the fluorescence intensity (S) of the first three segments of the zebrafish vertebrae was statistically analyzed. Group 2, osteoporotic zebrafish were placed in a six-well plate, with 30 zebrafish in each well, 3 mL of a 1000 μg / mL suspension of the multifunctional bone peptide complex in Example 2 was added to each well, and the plate was placed in a 28°C incubator for 4 days. Ten zebrafish were selected, stained with 0.2% calcein and photographed, and data were collected and statistically analyzed for the fluorescence intensity (S) of the first three segments of the zebrafish vertebrae; Group 3, osteoporotic zebrafish were placed in a six-well plate, with 30 zebrafish in each well, 3 mL of a 1000 μg / mL suspension of the multifunctional osteopeptide complex in Example 3 was added to each well, and the plate was placed in a 28°C incubator for 4 days. Ten zebrafish were selected, stained with 0.2% calcein and photographed, data were collected, and the fluorescence intensity (S) of the first three segments of the zebrafish vertebrae was statistically analyzed; Group 4, osteoporotic zebrafish were placed in a six-well plate, with 30 zebrafish in each well, 3 mL of a suspension of the multifunctional bone peptide complex in Comparative Example 1 with a concentration of 1000 μg / mL was added to each well, and the plate was placed in a 28°C incubator for 4 days. Ten zebrafish were selected, stained with 0.2% calcein and photographed, and data were collected and statistically analyzed for the fluorescence intensity (S) of the first three segments of the zebrafish vertebrae; Group 5, osteoporotic zebrafish were placed in a six-well plate, with 30 zebrafish in each well, 3 mL of a suspension of the multifunctional bone peptide complex in Comparative Example 2 at a concentration of 1000 μg / mL was added to each well, and the plate was placed in a 28°C incubator for 4 days. Ten zebrafish were selected, stained with 0.2% calcein and photographed, and data were collected and statistically analyzed for the fluorescence intensity (S) of the first three segments of the zebrafish vertebrae; Group 6, osteoporotic zebrafish were placed in a six-well plate, with 30 zebrafish in each well, 3 mL of a suspension of the multifunctional bone peptide complex in Comparative Example 3 at a concentration of 1000 μg / mL was added to each well, and the plate was placed in a 28°C incubator for 4 days. Ten zebrafish were selected, stained with 0.2% calcein and photographed, and data were collected and statistically analyzed for the fluorescence intensity (S) of the first three segments of the zebrafish vertebrae; Group 7, osteoporotic zebrafish were placed in a six-well plate, with 30 zebrafish in each well, 3 mL of a suspension of the multifunctional bone peptide complex in Comparative Example 4 with a concentration of 1000 μg / mL was added to each well, and the plate was placed in a 28°C incubator for 4 days. Ten zebrafish were selected, stained with 0.2% calcein and photographed, and data were collected and statistically analyzed for the fluorescence intensity (S) of the first three segments of the zebrafish vertebrae; Group 8, osteoporotic zebrafish were placed in a six-well plate, with 30 zebrafish in each well, and 3 mL of a suspension of the multifunctional bone peptide complex in Comparative Example 5 with a concentration of 1000 μg / mL was added to each well. After incubation in a 28°C incubator for 4 days, ten zebrafish were selected, stained with 0.2% calcein and photographed, and data were collected and statistically analyzed for the fluorescence intensity (S) of the first three segments of the zebrafish vertebrae.

[0049] The statistical results of the fluorescence intensity of the first three segments of the zebrafish vertebrae in each group are shown in Table 1.

[0050] Table 1 Statistical results of fluorescence intensity of the first three segments of zebrafish vertebrae Test Group Vertebral fluorescence intensity (pixels, mean ± SE) Model Group 31267 Example 1 76892 Example 2 75673 Example 3 77659 Comparative Example 1 75334 Comparative Example 2 60543 Comparative Example 3 58980 Comparative Example 4 66045 Comparative Example 5 64367

[0051] 45 experimental mice of similar size and mass were prepared. The mice were 6-7 week old female C57BL / 6 mice with a body mass of 20 g. They were divided into 9 groups, with five mice in each group. The average value was taken when calculating the relevant data.

[0052] Complete Freund's adjuvant (CFA) (Chondrex, USA, batch number: 210370) was used as an arthritis induction agent for C57BL / 6 mice. CFA was repeatedly aspirated using a 5 mL syringe until CFA was completely emulsified to form an oil-in-water state. On the 0th day of modeling, 0.01 mL of completely emulsified CFA was injected subcutaneously into the left and right hind limb foot pads of animals in the Model, 1, 2, 3, 4, 5, 6, 7, and 8 groups. The rheumatoid arthritis mouse model was established at the same time as the injection of CFA. The mice in the Model, 1, 2, 3, 4, 5, 6, 7, and 8 groups were subcutaneously injected with 0.1 mg / kg of epinephrine hydrochloride injection into the back of the mice, and 2 hours later, they were placed in 4°C cold water to swim for 5 minutes, once a day, for 7 consecutive days.

[0053] Drug intervention treatment for group 1, group 2, group 3, group 4, group 5, group 6, group 7, and group 8. The multifunctional bone peptide complexes in Examples 1-3 and Comparative Examples 1-5 were made into 0.20 mL capsules and fed to groups 1, 2, 3, 4, 5, 6, 7, and 8 of mice, twice a day, for a total of 14 days. The Model group was not fed with drugs and was raised normally for 14 days.

[0054] Mouse arthritis index scoring: The effect of the multifunctional bone peptide complex was evaluated by scoring the swelling of the mouse hind foot joints. The degree of swelling of the mouse paws was evaluated on the 14th day of treatment, and the severity of the disease was evaluated from the aspects of erythema and edema. Referring to the arthritis index scoring evaluation method of SEEUWS et al., the severity of the disease was quantified by 0 to 4 points. 0 points: no signs of swelling; 1 point: mild redness and swelling of the limbs; 2 points: moderate redness and swelling of the limbs; 3 points: severe ankle and tarsometatarsal joint involvement or mild twisting of the limbs; 4 points: severe redness, swelling or twisting of the ankle and the entire limb. The arthritis index score was the average of the sum of the scores of the left and right joints of each mouse's hind foot. The statistics of the arthritis index scores of mice in each group are shown in Table 2.

[0055] Table 2 Arthritis index scores of mice Test Group Arthritis index score Model 7.21 Example 1 4.96 Example 2 4.78 Example 3 4.89 Comparative Example 1 6.34 Comparative Example 2 5.87 Comparative Example 3 5.44 Comparative Example 4 5.23 Comparative Example 5 5.17

[0056] From the data in Table 1 and Table 2, it can be seen that the addition of bovine bone articular cartilage powder has little effect on the effect of the composite of the present invention in treating osteoporosis, but has a great effect on the effect of treating arthritis. It can also be seen that the use of the composite enzyme in the second enzymatic hydrolysis process is conducive to improving the effectiveness of the composite of the present invention in treating arthritis and osteoporosis.

[0057] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. The protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A multifunctional bone peptide complex, characterized in that: The raw materials include 20-30 parts of giant salamander bone powder, 15-20 parts of bovine joint cartilage powder, 30-40 parts of bovine bone powder, 10-20 parts of sturgeon bone powder, 3-8 parts of oyster bone powder, 3-6 parts of deer antler glue powder, 7-11 parts of calcium chloride and 3-5 parts of oligofructose by mass.

2. A multifunctional bone peptide complex according to claim 1, characterized in that: The raw materials of the complex further include 0.8-1.2 parts of L-lactate calcium, 0.8-1.2 parts of vitamin D3 and 2-3 parts of beta-cyclodextrin by weight.

3. A multifunctional bone peptide complex according to claim 2, characterized in that: The preparation method of the multifunctional osteopeptide complex comprises the following steps: S1, mixing giant salamander bone powder, cattle bone articular cartilage powder, cattle bone powder, sturgeon bone powder and oyster bone powder and crushing them to obtain mixed aggregate; S2, mixing the mixed aggregate with deionized water at a material-liquid ratio of 1 g:20-25 mL, placing in a constant temperature water bath at 50-55° C., adjusting the pH to 9.0-10, adding alkaline protease, performing a first enzymatic hydrolysis, and obtaining a first enzymatic hydrolysis solution; S3, adjusting the pH value of the first enzymatic hydrolysis solution to 7-7.5, adding a mixed enzyme, and performing a second enzymatic hydrolysis at 50-55° C., wherein the mixed enzyme includes a neutral protease, an elastase, and a papain, to obtain a second enzymatic hydrolysis solution; S4, heating the second enzymatic hydrolysis solution at 95-100° C. for 10-15 min to inactivate the enzyme, thereby obtaining an enzyme-inactivated solution; S5, centrifuging the enzyme-inactivated solution, taking the supernatant, and freeze-drying it to obtain a bone powder extract; S6, adding the bone powder extract to a calcium chloride aqueous solution, adjusting the pH value of the mixed solution to 7.5, performing a chelate reaction, and obtaining a chelate reaction solution; S7, adding ethanol to the chelation reaction solution, taking out the precipitate after centrifugation, and freeze-drying to obtain the bone powder chelate; S8. Mixing the bone powder chelate with antler glue powder, oligofructose, L-lactate calcium, vitamin D3 and β-cyclodextrin to obtain the multifunctional bone peptide complex.

4. A multifunctional bone peptide complex according to claim 3, characterized in that: The particle size of the mixed aggregate in S1 is less than 50 μm.

5. The multifunctional bone peptide complex according to claim 3, characterized in that: In the first enzymatic hydrolysis process described in S2, the amount of alkaline protease added is 8000-8500 U / g, and the time of the first enzymatic hydrolysis is 3-4 hours.

6. The multifunctional bone peptide complex according to claim 3, characterized in that: In the second enzymatic hydrolysis process described in S3, the amount of the mixed enzyme added is 11000-13000 U / g, and the time of the second enzymatic hydrolysis is 3-4 hours.

7. The multifunctional bone peptide complex according to claim 3, characterized in that: The mass ratio of neutral protease, elastase and papain in the mixed enzyme in S3 is 2:0.5-1:0.5-1.

8. The multifunctional bone peptide complex according to claim 3, characterized in that: The mass concentration of the calcium chloride aqueous solution in S6 is 5-10 mg / mL.

9. A multifunctional bone peptide complex according to claim 3s, characterized in that: The chelating reaction time in S6 is 3-5h.

10. Use of the multifunctional osteopeptide complex according to any one of claims 1 to 9 in the preparation of health products or medicines for preventing and / or treating osteoporosis and osteoarthritis.