A bone repair composition and method of making the same

Abstract

The present application relates to a kind of bone repair compositions and its preparation method, belong to biological medicine technical field, pharmaceutical composition includes acid anhydride bovine beta-lactoglobulin peptide, casein phosphopeptide, yak bone peptide, loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid and cyperus rotundus extract;Acid anhydride bovine beta-lactoglobulin peptide is prepared using special enzymolysis method.Acid anhydride bovine beta-lactoglobulin peptide, casein phosphopeptide and yak bone peptide are loaded using carboxymethyl chitosan-hydroxyapatite composite microspheres, then using sodium alginate-sodium carboxymethyl cellulose containing loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid and cyperus rotundus extract one coating, then using sodium alginate-sodium carboxymethyl cellulose secondary coating.The present application develops high-efficiency pharmaceutical combination component, using polymer material to coat drug to form sustained-release system, improve bioavailability, using granular carrier to increase the stability and solubility of drug, promote intestinal absorption, improve bone repair process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a bone repair composition and a preparation method thereof. BACKGROUND

[0002] Bone injury and disease, such as delayed fracture healing, osteoporotic bone defect, postoperative bone loss of bone tumor, etc., seriously affect the quality of life and physical health of patients. In addition to traditional bone repair methods such as surgical implantation of artificial bone or autologous bone transplantation, drug repair treatment is also needed. Among them, bone repair oral drugs, with the advantages of non-invasiveness and high patient compliance, have become an important research direction in the field of bone repair.

[0003] From a biological point of view, bone is in a dynamic metabolic process, and the balance between osteoblasts and osteoclasts maintains the health of bone tissue. Oral drugs aim to regulate cell activity, intervene in metabolic pathways, and promote bone tissue regeneration and repair. For example, for bone loss caused by osteoporosis, oral drugs can inhibit the bone resorption activity of osteoclasts, while activating the function of osteoblasts to restore bone remodeling balance. For traumatic bone defects, drugs need to regulate growth factor signaling pathways to promote the differentiation of mesenchymal stem cells into osteoblasts and accelerate bone tissue regeneration.

[0004] In terms of drug component development, early-stage basic nutritional supplements such as vitamin D and calcium preparations were mainly used. These drugs can supplement the basic substances needed for bone growth, maintain bone mineralization balance, and have certain effect on preventing and treating bone diseases caused by nutritional deficiencies. With further research, biologically active molecules such as parathyroid hormone analogs (PTH) and bisphosphonate drugs have gradually become mainstream. Parathyroid hormone analogs can promote osteoblast proliferation and differentiation through intermittent stimulation; bisphosphonates can be adsorbed on the bone surface to inhibit osteoclast activity and reduce bone resorption. In addition, natural plant extracts such as icariin have also become a new hotspot for the development of oral drugs due to their potential to promote osteogenesis and inhibit osteoclasts.

[0005] However, oral administration faces technical bottlenecks such as complex gastrointestinal environment and first-pass effect. Stomach acid and digestive enzymes can destroy the structure of the drug and reduce its bioavailability; liver metabolism can inactivate some drugs before they enter the blood circulation, reducing drug efficacy. In addition, the components of bone repair oral drugs also need to be further developed to improve efficiency. With the cross-fusion of biotechnology, material science and pharmacy, bone repair oral drugs need to break through the limitations of existing technology to provide safer and more effective treatment options for patients with bone diseases. SUMMARY

[0006] In view of the problems of low drug efficacy, first-pass effect, easy inactivation and reduced bioavailability of existing oral bone repair drug components, the present application provides a bone repair composition and a preparation method thereof, and develops a high-efficiency drug combination component including acid anhydride bovine beta-lactoglobulin peptide, casein phosphopeptide, yak bone peptide, loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid and cyperus rotundus extract, uses high molecular material to coat the drug to form a sustained-release system, controls the release of the drug in the gastrointestinal tract, reduces the degradation of the drug, improves the bioavailability, uses a granular carrier to increase the stability and solubility of the drug, improves the particle size and surface properties of the drug to promote intestinal absorption, and comprehensively improves the bone repair process.The specific technical scheme is as follows:

[0007] A bone repair composition is prepared from the following components by mass fraction: acid anhydride bovine beta-lactoglobulin peptide 6-8 parts, casein phosphopeptide 4-6 parts, yak bone peptide 6-8 parts, loquat leaf extract 1.5-2.5 parts, pterostilbene 2-3 parts, honeysuckle chlorogenic acid 0.2-0.5 parts and cyperus rotundus extract 2-3 parts; the acid anhydride bovine beta-lactoglobulin peptide, casein phosphopeptide and yak bone peptide are loaded on carboxymethyl chitosan-hydroxyapatite composite microspheres, and then coated with sodium alginate-carboxymethyl cellulose sodium containing loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid and cyperus rotundus extract once, and then coated with sodium alginate-carboxymethyl cellulose sodium twice.

[0008] In the above composition, the preparation method of the carboxymethyl chitosan-hydroxyapatite composite microspheres includes: according to the mass ratio, deionized water: carboxymethyl chitosan: hydroxyapatite = (90-100):(3-5):(6-8), carboxymethyl chitosan is dissolved in deionized water to prepare a carboxymethyl chitosan solution, the pH is adjusted to 5-6, hydroxyapatite is added, and ultrasonic dispersion is performed to obtain a mixed solution; the mixed solution is dropped into liquid paraffin, stirred and emulsified, then tannic acid aqueous solution is dropped, stirred and cross-linked, the microspheres are collected by centrifugation, washed with phosphate buffer, and freeze-dried to obtain carboxymethyl chitosan-hydroxyapatite composite microspheres.

[0009] Further, the amount of liquid paraffin is 2-3 times the volume of the mixed solution; the liquid paraffin contains 2wt%-3wt% Tween 80; the stirring and emulsification is 300r / min-400r / min stirring and emulsification for 30min-40min.

[0010] Further, the tannic acid aqueous solution contains 10wt%-15wt% tannic acid; the amount of tannic acid solution is 1-1.2 times the mass of carboxymethyl chitosan; the stirring and cross-linking reaction is stirring and cross-linking reaction at 55℃-60℃ and 300r / min-400r / min for 4h-5h.

[0011] Further, the phosphate buffer is washed 4-6 times, and the pH value of the phosphate buffer is 7.0-7.4.

[0012] In the composition, the preparation method of the anhydride-modified bovine beta-lactoglobulin peptide comprises the following steps: dispersing the anhydride-modified bovine beta-lactoglobulin in 6-8 times the mass of a phosphate buffer with a pH value of 7.0-7.4, adding 0.4-0.8% of the mass of the anhydride-modified bovine beta-lactoglobulin of natto kinase, and 0.6-1.0% of the mass of the anhydride-modified bovine beta-lactoglobulin of serratia peptidase, enzymatically hydrolyzing at 45-50°C for 60-90 min, inactivating the enzyme at 85-90°C for 10-15 min, performing ultrafiltration using a 10,000 Da ultrafiltration membrane, taking the product below 10,000 Da, and freeze-drying to obtain the anhydride-modified bovine beta-lactoglobulin peptide.

[0013] The preparation method of the bone repair composition comprises the following steps:

[0014] S1: adding 6-8 parts of the anhydride-modified bovine beta-lactoglobulin peptide, 4-6 parts of the casein phosphopeptide, and 6-8 parts of the yak bone peptide to deionized water to prepare a peptide solution; adding 40-50 parts of the carboxymethyl chitosan-hydroxyapatite composite microspheres to the peptide solution, stirring and adsorbing, centrifuging to take the precipitate, and freeze-drying to obtain peptide-loaded microspheres;

[0015] S2: preparing a coating liquid I by adding 400-450 parts of deionized water, 1.5-2.5 parts of loquat leaf extract, 2-3 parts of pterostilbene, 0.2-0.5 parts of honeysuckle chlorogenic acid, 2-3 parts of cyperi rhizoma extract, 10-15 parts of sodium alginate, and 8-10 parts of carboxymethyl cellulose sodium; then adding the peptide-loaded microspheres, intermittently oscillating and soaking and dispersing, freeze-drying, and breaking up to obtain the first coated peptide-loaded microspheres;

[0016] S3: preparing a coating liquid II by adding 200-250 parts of deionized water, 10-15 parts of sodium alginate, and 4-6 parts of carboxymethyl cellulose sodium; then adding the first coated peptide-loaded microspheres, stirring and soaking and dispersing, freeze-drying, and breaking up to obtain the composition.

[0017] In S1 of the preparation method, the amount of the deionized water is 30-35 times the total mass of the anhydride-modified bovine beta-lactoglobulin peptide, the casein phosphopeptide, and the yak bone peptide; the stirring and adsorbing is stirring and adsorbing at 4-8°C and 100-200 r / min for 6-8 h; and the centrifuging is centrifuging at 8,000-8,500 r / min for 10-15 min.

[0018] In S2 of the preparation method, the parameters of the intermittent oscillation soaking and dispersing are as follows: oscillation amplitude 1cm-2cm, oscillation frequency 10 times / min-20 times / min, duration of each oscillation 3min-5min, interval time of stopping oscillation 20min-30min, and total soaking time 60min-90min.

[0019] In S3 of the preparation method, the stirring soaking and dispersing is 20r / min-30r / min for 3min-5min.

[0020] The bone repair composition and the preparation method thereof have the following beneficial effects.

[0021] I. After the acid anhydride-converted bovine beta-lactoglobulin peptide is subjected to specific enzymatic treatment, it becomes a small-molecule peptide fragment with high activity. Compared with macromolecular proteins, the transmembrane transport efficiency is significantly improved, and it is more easily absorbed through the gastrointestinal mucosa into the blood circulation. These peptide fragments can specifically bind to the receptors on the surface of osteoblasts, activate the intracellular signaling pathways related to proliferation and differentiation, and promote the proliferation and differentiation of osteoblasts into mature bone cells. Casein phosphopeptide can tightly bind to calcium to form a soluble complex, preventing calcium from combining with other anions in the gastrointestinal tract to form insoluble precipitates, thereby maintaining the solubility of calcium in the intestinal tract and greatly improving the absorption efficiency of calcium. In addition, casein phosphopeptide can also promote the active transport of calcium in the intestine, allowing more calcium to be absorbed into the blood, providing sufficient calcium source for bone mineralization, and playing a key role in maintaining the balance of bone mineralization. Yak bone peptide is rich in various amino acids and active ingredients, which can provide rich nutrients for the growth and metabolism of osteoblasts; at the same time, it can regulate the balance between osteoblasts and osteoclasts, inhibit the excessive activity of osteoclasts, reduce bone resorption, and promote the synthesis and repair of bone tissue.

[0022] II. The carboxymethyl chitosan-hydroxyapatite composite microspheres can adsorb a large amount of peptide substances and achieve efficient loading; the composite microspheres loaded with peptides can improve the stability of the peptides and prevent them from being rapidly degraded in the gastrointestinal tract.

[0023] Three, sodium alginate-sodium carboxymethyl cellulose, in aqueous solution, form a certain viscosity and plasticity of the gel. When coated, they form a tight gel layer of drug ingredients, forming a physical barrier to prevent the stomach and digestive enzymes in the gastrointestinal tract and drug direct contact, thereby reducing the degradation of the drug. At the same time, the gel layer according to the environment in the gastrointestinal tract (including pH, ionic strength, etc.) to control the release rate and location of the drug, to achieve the directional release of the drug, improve the efficiency of drug use. Sodium alginate-sodium carboxymethyl cellulose secondary coating, on the basis of the first coating, secondary coating further strengthen the protection of the drug, forming a multi-layer slow-release structure. Multi-layer structure to delay the drug and gastrointestinal environment, making the drug in the gastrointestinal tract is more slow and lasting, which helps to maintain the effective concentration of the drug in the blood and bone tissue, to avoid the dramatic fluctuations in drug concentration.

[0024] Four, strong anti-inflammatory and antioxidant capacity of pterostilbene and green tea of honeysuckle chlorogenic acid angiogenesis, mutual cooperation, the formation of a comprehensive regulation of bone repair microenvironment. When bone injury or disease occurs, local tissue will produce inflammation and oxidative stress, which will not only hinder the repair of bone tissue, but also lead to further damage to bone tissue. Pterostilbene by eliminating reactive oxygen species and inhibit the production of inflammatory mediators, reduce inflammation and oxidative stress on bone cells, protect osteoblasts and other bone tissue cells normal function; and green tea of honeysuckle chlorogenic acid to promote angiogenesis, to bring a wealth of oxygen and nutrients to the damaged bone tissue, while accelerating the excretion of metabolic waste. Both synergistic effect in a certain proportion, on the one hand, to improve the survival environment of bone tissue, for the growth and differentiation of bone cells to provide good conditions; on the other hand, to promote the transport and exchange of the material required in the process of bone tissue repair, accelerate the formation of callus and bone tissue regeneration.

[0025] V. Loquat leaf extract and Cyperus rotundus extract cooperate with each other in regulating osteocyte metabolism and maintaining bone microenvironment homeostasis. Loquat leaf extract reduces the damage of inflammation to osteocytes through anti-inflammatory effect, creating conditions for normal metabolism of osteocytes; Cyperus rotundus extract affects the function of osteocytes by regulating hormone levels and cytokine secretion in bone tissue. The synergistic effect of the two in a certain proportion can more comprehensively regulate the metabolic activity of osteocytes, including the proliferation, differentiation of osteoblasts, and synthesis of bone matrix, as well as the regulation of osteoclast activity. Excessive activity of osteoclasts can lead to increased bone resorption, affecting bone repair. Loquat leaf extract and Cyperus rotundus extract inhibit the generation and activity of osteoclasts through different mechanisms. The active ingredients in loquat leaf extract interfere with the differentiation signaling of osteoclast precursor cells, reducing the generation of osteoclasts; Cyperus rotundus extract affects the function and survival of osteoclasts by regulating related hormones and cytokines. The combined action of the two can more effectively inhibit the excessive activity of osteoclasts, reduce bone resorption, and achieve a better balance between bone resorption and bone formation, which is conducive to the reconstruction and restoration of normal bone structure and function during bone repair.

[0026] In summary, a variety of peptide components (anhydrous bovine beta-lactoglobulin peptide, casein phosphopeptide, and yak bone peptide) and plant extracts (loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid, and Cyperus rotundus extract) together constitute a synergistic regulation network for cell activity. Peptide substances such as anhydrous bovine beta-lactoglobulin peptide, casein phosphopeptide, and yak bone peptide can act on osteoblasts to promote their proliferation, differentiation, and function; plant active ingredients such as loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid, and Cyperus rotundus extract create a good microenvironment for bone repair through anti-inflammatory, antioxidant, bone metabolism regulation, and angiogenesis promotion. These components synergize with each other, and compared to single components, they can more comprehensively and efficiently promote the regeneration and repair of bone tissue. The loading and coating of high molecular materials (carboxymethyl chitosan-hydroxyapatite composite microspheres, sodium alginate-carboxymethyl cellulose sodium) provide a good protection and delivery system for other components. It not only protects the drug components from the damage of the gastrointestinal environment, but also improves the stability, solubility, and absorption performance of the drug. By forming a sustained-release system, the drug can be released at the right time and location, ensuring that each component can continuously function in different stages of bone repair. This mutual cooperation and synergistic effect among components significantly improves the overall efficacy of the drug, providing a more effective solution for the treatment of skeletal injuries and diseases. DETAILED DESCRIPTION

[0027] The application will be further described in conjunction with specific implementation examples, but the application is not limited to these examples.

[0028] Example 1

[0029] A bone repair composition is prepared from the following raw materials in mass parts: 6 parts of anhydridized bovine beta-lactoglobulin peptide, 4 parts of casein phosphopeptide, 6 parts of yak bone peptide, 1.5 parts of loquat leaf extract, 2 parts of pterostilbene, 0.2 parts of green acid from honeysuckle flower, and 2 parts of rhizoma cyperi extract; the anhydridized bovine beta-lactoglobulin peptide, the casein phosphopeptide, and the yak bone peptide are loaded on carboxymethyl chitosan-hydroxyapatite composite microspheres, and then coated once with sodium alginate-carboxymethyl cellulose sodium containing the loquat leaf extract, the pterostilbene, the green acid from honeysuckle flower, and the rhizoma cyperi extract, and then coated twice with sodium alginate-carboxymethyl cellulose sodium.

[0030] The preparation method of the carboxymethyl chitosan-hydroxyapatite composite microspheres comprises the following steps: according to the mass ratio of deionized water: carboxymethyl chitosan: hydroxyapatite = 90:3:6, the carboxymethyl chitosan is dissolved in deionized water to prepare a carboxymethyl chitosan solution, the pH is adjusted to 5, the hydroxyapatite is added, and ultrasonic dispersion is performed to obtain a mixed solution; the mixed solution is dropped into liquid paraffin with 2wt% Tween 80, and stirred and emulsified at 300r / min for 30min, then 10wt% tannic acid aqueous solution with a mass of 1 times that of the carboxymethyl chitosan is dropped, and crosslinking reaction is performed at 55℃ and 300r / min for 4h, the microspheres are collected by centrifugation, washed 4 times with pH 7.0 phosphate buffer, and freeze-dried to obtain the carboxymethyl chitosan-hydroxyapatite composite microspheres.

[0031] The preparation method of the anhydridized bovine beta-lactoglobulin peptide comprises the following steps: the anhydridized bovine beta-lactoglobulin is dispersed in 6 times the mass of pH 7.0 phosphate buffer, 0.4% nattokinase and 0.6% serralysin are added, and enzymolysis is performed at 45℃ for 60min, and the enzyme is inactivated at 85℃ for 15min, then ultrafiltration is performed with a 10000Da ultrafiltration membrane, the product below 10000Da is taken, and freeze-drying is performed to obtain the anhydridized bovine beta-lactoglobulin peptide.

[0032] The preparation method of the above-mentioned bone repair composition comprises the following steps:

[0033] S1: according to mass parts, 6 parts of anhydridized bovine beta-lactoglobulin peptide, 4 parts of casein phosphopeptide, and 6 parts of yak bone peptide are added to deionized water with 30 times the mass of the total peptide to prepare a peptide solution; 40 parts of carboxymethyl chitosan-hydroxyapatite composite microspheres are added to the peptide solution, and stirring adsorption is performed at 4℃ and 100r / min for 6h, and the precipitate is collected by centrifugation at 8000r / min for 15min, and freeze-drying is performed to obtain peptide-loaded microspheres.

[0034] S2: By mass fractions, 400 parts deionized water, 1.5 parts loquat leaf extract, 2 parts pterostilbene, 0.2 parts honeysuckle chlorogenic acid, 2 parts cyperus extract, 10 parts sodium alginate, and 8 parts sodium carboxymethyl cellulose were used to prepare coating solution I. Then, peptide-loaded microspheres were added, and the mixture was intermittently shaken and soaked to disperse the microspheres. The parameters were: shaking amplitude 1 cm, shaking frequency 10 times / min, duration of each shaking 3 min, and interval between shaking stops 20 min, for a total soaking time of 60 min. The microspheres were then freeze-dried and dispersed to obtain one-time coated peptide-loaded microspheres.

[0035] S3: Prepare coating solution II by mass fractions of 200 parts deionized water, 10 parts sodium alginate, and 4 parts sodium carboxymethyl cellulose. Then add the coated peptide-loaded microspheres, stir and soak at 20 r / min for 3 min, freeze dry, and break up to obtain the composition.

[0036] Example 2

[0037] A bone repair composition is made from the following raw materials in parts by weight: 7 parts of anhydride-modified bovine β-lactoglobulin peptide, 5 parts of casein phosphopeptide, 7 parts of yak bone peptide, 2 parts of loquat leaf extract, 2.5 parts of pterostilbene, 0.35 parts of honeysuckle chlorogenic acid, and 2.5 parts of cyperus extract. The anhydride-modified bovine β-lactoglobulin peptide, casein phosphopeptide, and yak bone peptide are loaded onto carboxymethyl chitosan-hydroxyapatite composite microspheres, then coated once with sodium alginate-sodium carboxymethyl cellulose containing loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid, and cyperus extract, and then coated twice with sodium alginate-sodium carboxymethyl cellulose.

[0038] The preparation method of carboxymethyl chitosan-hydroxyapatite composite microspheres includes: dissolving carboxymethyl chitosan in deionized water at a mass ratio of 95:4:7 to prepare a carboxymethyl chitosan solution, adjusting the pH to 5.5, adding hydroxyapatite, and ultrasonically dispersing to obtain a mixed solution; adding the mixed solution dropwise to 2.5 times the volume of liquid paraffin containing 2.5 wt% Tween 80, stirring and emulsifying at 350 r / min for 35 min, then adding 1.1 times the mass of carboxymethyl chitosan in a 12 wt% tannic acid aqueous solution, stirring and crosslinking at 58℃ and 350 r / min for 4.5 h, collecting the microspheres by centrifugation, washing five times with pH 7.2 phosphate buffer, and lyophilizing to obtain carboxymethyl chitosan-hydroxyapatite composite microspheres.

[0039] The preparation method of anhydride-modified bovine β-lactoglobulin peptide includes: dispersing anhydride-modified bovine β-lactoglobulin in 7 times its mass of pH 7.2 phosphate buffer, adding 0.6% of the anhydride-modified bovine β-lactoglobulin protein with nattokinase and 0.8% of the anhydride-modified bovine β-lactoglobulin protein with sarapeptidase, enzymatically hydrolyzing at 48℃ for 80 min, inactivating the enzyme at 88℃ for 12 min, ultrafiltration using a 10000 Da ultrafiltration membrane, taking the product with a content below 10000 Da, and lyophilizing to obtain anhydride-modified bovine β-lactoglobulin peptide.

[0040] The method for preparing the above-mentioned bone repair composition includes the following steps:

[0041] S1: According to the mass fractions, add 7 parts of anhydride-modified bovine β-lactoglobulin peptide, 5 parts of casein phosphopeptide and 7 parts of yak bone peptide to 32 times the total peptide mass of deionized water to prepare peptide solution; add 45 parts of carboxymethyl chitosan-hydroxyapatite composite microspheres to peptide solution, stir and adsorb at 6℃ and 150r / min for 7h, centrifuge at 8200r / min for 12min, take the precipitate, freeze dry to obtain peptide-loaded microspheres.

[0042] S2: By mass fractions, 430 parts deionized water, 2 parts loquat leaf extract, 2.5 parts pterostilbene, 0.35 parts honeysuckle chlorogenic acid, 2.5 parts cyperus extract, 13 parts sodium alginate, and 9 parts sodium carboxymethyl cellulose were used to prepare coating solution I. Then, peptide-loaded microspheres were added, and the mixture was intermittently shaken and soaked to disperse the microspheres. The parameters were: shaking amplitude 1.5 cm, shaking frequency 15 times / min, duration of each shaking 4 min, and interval between shaking stops 25 min, for a total soaking time of 80 min. The microspheres were then freeze-dried and dispersed to obtain one-time coated peptide-loaded microspheres.

[0043] S3: Prepare coating solution II by mass fractions of 230 parts deionized water, 12 parts sodium alginate, and 5 parts sodium carboxymethyl cellulose. Then add the coated peptide-loaded microspheres, stir and soak at 25 r / min for 4 min, freeze dry, and break up to obtain the composition.

[0044] Example 3

[0045] A bone repair composition is made from the following raw materials in parts by weight: 8 parts of anhydride-modified bovine β-lactoglobulin peptide, 6 parts of casein phosphopeptide, 8 parts of yak bone peptide, 2.5 parts of loquat leaf extract, 3 parts of pterostilbene, 0.5 parts of honeysuckle chlorogenic acid, and 3 parts of cyperus extract; the anhydride-modified bovine β-lactoglobulin peptide, casein phosphopeptide, and yak bone peptide are loaded onto carboxymethyl chitosan-hydroxyapatite composite microspheres, then coated once with sodium alginate-sodium carboxymethyl cellulose containing loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid, and cyperus extract, and then coated twice with sodium alginate-sodium carboxymethyl cellulose.

[0046] The preparation method of the carboxymethyl chitosan-hydroxyapatite composite microspheres comprises the following steps: carboxymethyl chitosan is dissolved in deionized water to prepare a carboxymethyl chitosan solution, the pH value is adjusted to 6, hydroxyapatite is added, and ultrasonic dispersion is performed to obtain a mixed solution; the mixed solution is dropped into liquid paraffin with 3 wt% Tween 80, and emulsification is performed at 400 r / min for 40 min; then, 15 wt% tannic acid aqueous solution with a mass of 1.2 times that of the carboxymethyl chitosan is dropped, and crosslinking reaction is performed at 60°C and 400 r / min for 5 h; the microspheres are collected by centrifugation, washed with pH 7.4 phosphate buffer solution for 6 times, and freeze-dried to obtain the carboxymethyl chitosan-hydroxyapatite composite microspheres.

[0047] The preparation method of the anhydride-modified bovine beta-lactoglobulin peptide comprises the following steps: the anhydride-modified bovine beta-lactoglobulin is dispersed in 8 times the mass of pH 7.4 phosphate buffer solution, 0.8% natto kinase and 1.0% serratia peptidase are added, and enzymolysis is performed at 50°C for 90 min; the enzyme is inactivated at 90°C for 10 min; 10000 Da ultrafiltration membrane is used for ultrafiltration; the product below 10000 Da is taken; and freeze-drying is performed to obtain the anhydride-modified bovine beta-lactoglobulin peptide.

[0048] The preparation method of the above-mentioned bone repair composition comprises the following steps:

[0049] S1: 8 parts of anhydride-modified bovine beta-lactoglobulin peptide, 6 parts of casein phosphopeptide and 8 parts of yak bone peptide are added to 35 times the mass of deionized water of the total mass of the peptides to prepare a peptide solution; 50 parts of carboxymethyl chitosan-hydroxyapatite composite microspheres are added to the peptide solution, stirring is performed at 8°C and 200 r / min for 8 h, centrifugation is performed at 8500 r / min for 10 min, the precipitate is taken, and freeze-drying is performed to obtain peptide-loaded microspheres.

[0050] S2: 450 parts of deionized water, 2.5 parts of loquat leaf extract, 3 parts of pterostilbene, 0.5 parts of honeysuckle chlorogenic acid, 3 parts of cyperus rotundus extract, 15 parts of sodium alginate and 10 parts of carboxymethyl cellulose sodium are prepared to prepare coating liquid I, and then the peptide-loaded microspheres are added; intermittent shock soaking and dispersion are performed, and the parameters are as follows: shock amplitude 2 cm, shock frequency 20 times / min, duration of each shock 5 min, interval time of stopping shock 30 min, and the total soaking time is 90 min; freeze-drying is performed, and the peptide-loaded microspheres are dispersed to obtain primary coated peptide-loaded microspheres.

[0051] S3: 250 parts of deionized water, 15 parts of sodium alginate and 6 parts of carboxymethyl cellulose sodium are prepared to prepare coating liquid II, and then the primary coated peptide-loaded microspheres are added; stirring soaking and dispersion are performed at 30 r / min for 5 min; freeze-drying is performed, and the peptide-loaded microspheres are dispersed to obtain the composition.

[0052] The raw material sources in the above embodiments: anhydridized bovine beta-lactoglobulin was from Xi'an Yahuabio Technology Co., Ltd., with a purity of 98%. Casein phosphopeptide was from Shaanxi Hengruikang Health Industry Co., Ltd., with a product number of HRK029417. Yak bone peptide was from Xi'an Yahuabio Technology Co., Ltd., with a model number of YH-HNGT. Loquat leaf extract was from Shaanxi Lvsygen Biological Products Manufacturing Co., Ltd., with a product number of LSYSW-PPYTQ W. Pterostilbene was from Xi'an Jianglin Biological Technology Co., Ltd., with a purity of 98%. Green original acid of honeysuckle was from Xi'an Meihewo Biological Technology Co., Ltd., with a purity of 98%. Rhizoma cyperi extract was from Xi'an Zeyuankang Biological Technology Co., Ltd., with a product number of ZYK415. Carboxymethyl chitosan was from Xi'an Aoptical Biological Technology Co., Ltd., with a product number of AT-7404. Hydroxyapatite was from Shaanxi Fengxiwu Biological Technology Co., Ltd., with a food-grade hydroxyapatite of 20-80 μm spherical. Liquid paraffin was from Xi'an Tianzheng Pharmaceutical Auxiliary Co., Ltd., with a pharmaceutical grade. Tween 80 was from Guangzhou Jincheng Chemical Co., Ltd., with a food grade. Tannic acid was from Henan Anrui Biological Technology Co., Ltd., with a food grade. Natto kinase was from Shaanxi Feimi Biological Technology Co., Ltd., with an enzyme activity of 20,000 U / g. Serratiopeptase was from Shenzhen Hengsheng Biological Technology Co., Ltd., with an enzyme activity of 200,000 U / g. Sodium alginate was from Guangdong Ousman Biological Technology Co., Ltd., with a food grade. Sodium carboxymethyl cellulose was from Henan Puhui Biological Co., Ltd., with a food grade.

[0053] Comparative Example 1

[0054] In the composition, anhydridized bovine beta-lactoglobulin peptide was replaced by anhydridized bovine beta-lactoglobulin; other parameters and methods were the same as in Example 1.

[0055] Comparative Example 2

[0056] In the composition, anhydridized bovine beta-lactoglobulin peptide was 2 parts; casein phosphopeptide was 8 parts; other parameters and methods were the same as in Example 1.

[0057] Comparative Example 3

[0058] In the composition, pterostilbene and green original acid of honeysuckle were not added; other parameters and methods were the same as in Example 1.

[0059] Comparative Example 4

[0060] In the composition, pterostilbene was 1 part, and green original acid of honeysuckle was 1.2 parts; other parameters and methods were the same as in Example 1.

[0061] Comparative Example 5

[0062] In the composition, loquat leaf extract was 3 parts, and rhizoma cyperi extract was 0.5 parts; other parameters and methods were the same as in Example 1.

[0063] Comparative Example 6

[0064] In the preparation method of the anhydridized bovine β-lactoglobulin peptide, the serrapeptase is replaced by papain (enzyme activity 200,000 U / g); other parameters and methods are the same as in Example 1.

[0065] Comparative Example 7

[0066] In the preparation method of the anhydridized bovine β-lactoglobulin peptide, the nattokinase is replaced by bromelain (enzyme activity 100,000 U / g); other parameters and methods are the same as in Example 1.

[0067] Comparative Example 8

[0068] In the preparation method of the composition, the secondary coating of sodium alginate-sodium carboxymethyl cellulose is not performed; other parameters and methods are the same as in Example 1.

[0069] Comparative Example 9

[0070] In the preparation method of the composition, no loading coating treatment is performed; the anhydridized bovine β-lactoglobulin peptide, casein phosphopeptide, yak bone peptide, loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid, and cyperus rotundus extract are directly mixed with 40 parts of carboxymethyl chitosan-hydroxyapatite composite microspheres dry powder; other parameters and methods are the same as in Example 1.

[0071] I. Toxicity test

[0072] 1. Acute toxicity test: healthy adult Kunming mice weighing 18-22 g were selected and randomly divided into 12 groups, 6 in each group (half male and half female). They are Example 1-Example 3 groups, Comparative Example 1-Comparative Example 9 groups. Gavage 50 mg / kg body weight. After administration, observe and detect the mice for 14 days to see if they have symptoms of poisoning and death. The results are all normal.

[0073] 2. Long-term toxicity test: healthy SD rats weighing 180-220 g were selected and randomly divided into 13 groups, 6 in each group (half male and half female). They are Example 1-Example 3 groups, Comparative Example 1-Comparative Example 9 groups, and blank control group (no administration). Gavage 50 mg / kg body weight, continuous administration for 90 days. During the administration period, observe whether the rats have symptoms of poisoning and death, and detect pathological changes after the experiment. The results are all normal.

[0074] II. In vivo bone repair effect detection

[0075] Animal model establishment and grouping: 60 healthy SD rats (body weight 180-220 g, male) were selected, and anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg). Under sterile conditions, the right forelimb skin of the rat was incised, the radius was exposed, a 4 mm long bone defect model (non-fracture) was prepared in the middle of the radius using a micro bone saw, and then the muscle and skin were sutured layer by layer after marking. Postoperative intramuscular injection of penicillin sodium for 3 consecutive days to prevent infection. The rats were randomly divided into 15 groups (Examples 1-3, Comparative Examples 1-9, blank control group, yak bone peptide group, icariin group), 4 rats in each group.

[0076] The composition was diluted with water to 20 mg / mL, and each experimental group of rats was given 4 mL / kg body weight of the composition by gavage every day (2 mL / kg body weight in the morning and 2 mL / kg body weight in the evening). The blank control group was given the same amount of normal saline without the composition by gavage, the yak bone peptide group was given the same amount of yak bone peptide (diluted to 20 mg / mL) by gavage, and the icariin group was given the same amount of icariin (diluted to 20 mg / mL) by gavage. Continuous gavage was performed for 56 days. After 2 hours after the second administration on the 28th day and after 2 hours after the second administration on the 56th day, the rats were placed under an animal X-ray machine for X-ray examination to detect and analyze the area ratio of the callus at the bone defect site (callus area / original bone defect area x 100%), and the average value was taken to evaluate the bone repair progress.

[0077] Table 1 In vivo bone repair results

[0078]

[0079] From the above results, it can be seen that the active ingredients in the compositions of Examples 1 to 3 are less than 50% of the yak bone peptide group and the icariin group, and still have a relatively high repair effect, indicating that the composition has good bioavailability, and the use of each component can well promote bone repair.

[0080] Comparative Example 1 uses anhydrous bovine beta-lactoglobulin instead of anhydrous bovine beta-lactoglobulin peptide. Anhydrous bovine beta-lactoglobulin is a macromolecular protein, and its transmembrane transport efficiency is low compared to small molecule peptides, making it difficult to be absorbed into the blood circulation through the gastrointestinal mucosa. At the same time, in the complex environment of the gastrointestinal tract, macromolecular proteins are more easily degraded by gastric acid and proteases, making it difficult to reach the action site intact. Anhydrous bovine beta-lactoglobulin peptide is a small molecule fragment after proteolysis, has better bioavailability, and participates in cell metabolism and signal transduction. Therefore, after using anhydrous bovine beta-lactoglobulin instead, the components effective for bone repair are reduced, resulting in a decrease in bone repair effect.

[0081] The acid anhydride bovine beta-lactoglobulin peptide in the present application plays a key role in promoting the proliferation and differentiation of osteoblasts; the casein phosphopeptide helps the absorption and utilization of calcium, but the function of a single component is relatively limited. When the acid anhydride bovine beta-lactoglobulin peptide is reduced and the casein phosphopeptide is increased, the promotion of osteoblast proliferation and differentiation is weakened, the synergistic balance between the original components is destroyed, the key signal transduction and cell activity required in the bone tissue regeneration process are inhibited, and thus the bone repair effect is reduced.

[0082] The counterexample 3 does not add pterostilbene and honeysuckle chlorogenic acid. Pterostilbene has anti-inflammatory and antioxidant properties, can regulate oxidative stress and inflammatory response in the bone metabolism process, and creates a good microenvironment for bone repair; honeysuckle chlorogenic acid promotes angiogenesis and provides sufficient nutrients and oxygen for bone tissue regeneration. After the absence of the two, the bone repair process lacks the corresponding microenvironment regulation and nutritional support, affecting the activity of osteoblasts and the synthesis of bone matrix, and making the callus formation slow.

[0083] The counterexample 4 changes the ratio of pterostilbene and honeysuckle chlorogenic acid. Pterostilbene and honeysuckle chlorogenic acid synergistically act through different mechanisms in bone repair. When the ratio is changed, the regulation of the two on the bone metabolism-related pathways is unbalanced. The activation of pterostilbene on some key transcription factors and the promotion of honeysuckle chlorogenic acid on vascular endothelial growth factor cannot achieve the best coordination, resulting in weakened anti-inflammatory and pro-angiogenic effects, and thus affecting the bone repair process.

[0084] The counterexample 5 changes the ratio of loquat leaf extract and cyperus rotundus extract. Loquat leaf extract and cyperus rotundus extract synergistically regulate bone cell metabolism and maintain bone microenvironment homeostasis. After the ratio is changed, the regulation ability of the two on hormone levels and cytokine secretion in bone tissue changes. The synergistic effect on the apoptosis and function inhibition of osteoclasts is weakened, leading to an imbalance between bone resorption and bone formation, and a poor bone repair effect.

[0085] The counterexample 6 uses papain instead of serrapeptase. Different proteases have different enzyme cutting sites and products for acid anhydride bovine beta-lactoglobulin. Serrapeptase can specifically hydrolyze acid anhydride bovine beta-lactoglobulin into highly active peptide fragments, which can effectively participate in the bone repair process and promote the expression of osteoblast differentiation-related genes. The peptide fragments produced by papain have different structures and activities, and cannot effectively play the role of promoting bone repair like the hydrolysis products of serrapeptase, thus leading to a decrease in callus formation ability.

[0086] Comparative Example 7 replaces nattokinase with bromelain. Nattokinase produces bioactive peptide sequences in the acid anhydride bovine beta-lactoglobulin peptide preparation process through specific enzymatic action. These sequences bind to bone cell surface receptors and activate bone repair-related signaling pathways. Bromelain has different enzymatic properties, and the peptide sequences produced cannot efficiently activate the corresponding signaling pathways, efficiently promote osteoblast differentiation, and reduce bone repair effectiveness.

[0087] Comparative Example 8 does not perform secondary coating with sodium alginate-sodium carboxymethyl cellulose. The multi-layer sustained-release structure formed by secondary coating can further protect the drug, allowing it to be slowly released at specific sites in the gastrointestinal tract. Without secondary coating, the drug coated once is more susceptible to attack by gastric acid and digestive enzymes in the gastrointestinal tract, leading to premature release and degradation of the drug, which cannot provide effective ingredients continuously during the key stage of bone repair. At the same time, lacking the protection of secondary coating, the stability of the drug in the gastrointestinal tract is reduced, thereby affecting the effectiveness of bone repair.

[0088] Comparative Example 9 does not perform loading coating treatment and directly mixes dry powder. Without loading coating treatment, the drug components are directly exposed to the gastrointestinal environment, making them extremely susceptible to rapid degradation by gastric acid and digestive enzymes, resulting in a significant reduction in bioavailability. At the same time, the drug without forming a sustained-release system is rapidly released in the gastrointestinal tract, making it difficult to maintain an effective drug concentration and continuously act on the bone repair process, thereby making it difficult to efficiently promote callus formation.

Claims

1. A bone repair composition, characterized in that, The composition is made from the following raw materials in parts by weight: 6 to 8 parts of acid-anhydride bovine β-lactoglobulin peptide, 4 to 6 parts of casein phosphopeptide, 6 to 8 parts of yak bone peptide, 1.5 to 2.5 parts of loquat leaf extract, 2 to 3 parts of pterostilbene, 0.2 to 0.5 parts of honeysuckle chlorogenic acid, and 2 to 3 parts of cyperus extract; The acid-anhydride bovine β-lactoglobulin peptide, casein phosphopeptide, and yak bone peptide were loaded onto carboxymethyl chitosan-hydroxyapatite composite microspheres, and then coated once with sodium alginate-sodium carboxymethyl cellulose containing loquat leaf extract, pterostilbene, honeysuckle chlorogenic acid, and cyperus extract, and then coated a second time with sodium alginate-sodium carboxymethyl cellulose. The preparation method of the anhydride-modified bovine β-lactoglobulin peptide includes: dispersing anhydride-modified bovine β-lactoglobulin in 6 to 8 times its mass of pH 7.0 to 7.4 phosphate buffer, adding 0.4% to 0.8% of the anhydride-modified bovine β-lactoglobulin protein mass of nattokinase and 0.6% to 1.0% of the anhydride-modified bovine β-lactoglobulin protein mass of sarapeptidase, enzymatically hydrolyzing at 45℃ to 50℃ for 60 to 90 min, inactivating the enzyme at 85℃ to 90℃ for 10 to 15 min, ultrafiltration using a 10000 Da ultrafiltration membrane, taking the product with a mass below 10000 Da, and lyophilizing to obtain the anhydride-modified bovine β-lactoglobulin peptide.

2. The bone repair composition according to claim 1, characterized in that, The preparation method of the carboxymethyl chitosan-hydroxyapatite composite microspheres includes: dissolving carboxymethyl chitosan in deionized water at a mass ratio of (90-100): (3-5): (6-8) to prepare a carboxymethyl chitosan solution, adjusting the pH to 5-6, adding hydroxyapatite, and ultrasonically dispersing to obtain a mixed solution; adding the mixed solution dropwise into liquid paraffin, stirring to emulsify, then adding tannic acid aqueous solution, stirring to crosslink the reaction, collecting the microspheres by centrifugation, washing with phosphate buffer, and lyophilizing to obtain carboxymethyl chitosan-hydroxyapatite composite microspheres.

3. The bone repair composition according to claim 2, characterized in that, The amount of liquid paraffin used is 2 to 3 times the volume of the mixed liquid; the liquid paraffin contains 2 wt% to 3 wt% Tween 80; the stirring and emulsification is carried out at 300 r / min to 400 r / min for 30 min to 40 min.

4. The bone repair composition according to claim 2, characterized in that, The tannic acid aqueous solution contains 10wt% to 15wt% tannic acid; the amount of tannic acid solution used is 1 to 1.2 times the mass of carboxymethyl chitosan; the stirring crosslinking reaction is carried out at 55℃ to 60℃ and 300r / min to 400r / min for 4h to 5h.

5. The bone repair composition according to claim 2, characterized in that, The phosphate buffer solution is washed 4 to 6 times; the pH value of the phosphate buffer solution is 7.0 to 7.

4.

6. A method for preparing a bone repair composition according to claim 1, characterized in that, Includes the following steps: S1: According to the mass fractions, add 6 to 8 parts of anhydride-modified bovine β-lactoglobulin peptide, 4 to 6 parts of casein phosphopeptide and 6 to 8 parts of yak bone peptide to deionized water to prepare peptide solution; add 40 to 50 parts of carboxymethyl chitosan-hydroxyapatite composite microspheres to peptide solution, stir to adsorb, centrifuge to collect the precipitate, freeze dry to obtain peptide-loaded microspheres. S2: Prepare coating solution I by mass fractions of 400-450 parts deionized water, 1.5-2.5 parts loquat leaf extract, 2-3 parts pterostilbene, 0.2-0.5 parts honeysuckle chlorogenic acid, 2-3 parts cyperus extract, 10-15 parts sodium alginate, and 8-10 parts sodium carboxymethyl cellulose. Then add peptide-loaded microspheres, disperse by intermittent shaking and soaking, freeze-dry, and break up to obtain one-time coated peptide-loaded microspheres. S3: Prepare coating solution II by mass fractions of 200-250 parts deionized water, 10-15 parts sodium alginate, and 4-6 parts sodium carboxymethyl cellulose. Then add the coated peptide-loaded microspheres, stir, soak, disperse, freeze-dry, and break up to obtain the composition.

7. The method for preparing a bone repair composition according to claim 6, characterized in that, In S1, the amount of deionized water used is 30 to 35 times the total mass of acid-anhydrated bovine β-lactoglobulin peptide, casein phosphopeptide, and yak bone peptide; the stirring adsorption is carried out at 4℃ to 8℃ and 100r / min to 200r / min for 6 to 8 hours; the centrifugation is carried out at 8000r / min to 8500r / min for 10 to 15 minutes.

8. The method for preparing a bone repair composition according to claim 6, characterized in that, In S2, the parameters for the intermittent oscillation soaking dispersion are: oscillation amplitude 1cm~2cm, oscillation frequency 10 times / min~20 times / min, duration of each oscillation 3min~5min, interval between stopping oscillation 20min~30min, and total soaking time 60min~90min.

9. The method for preparing a bone repair composition according to claim 6, characterized in that, In S3, the stirring, soaking, and dispersing is carried out at a speed of 20 r / min to 30 r / min for 3 min to 5 min.

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