Multi-effect synergistic pharmaceutical composition for bone repair and preparation method thereof

Abstract

The invention relates to a multi-effect synergistic pharmaceutical composition for bone repair and a preparation method thereof, and belongs to the technical field of biological medicine, the pharmaceutical composition comprises five-black chicken yolk peptide, tegillarca granosa peptide, epimedium extract and rhizoma drynariae extract. The five-black chicken egg yolk peptide is obtained by carrying out enzymolysis on five-black chicken egg yolk freeze-dried powder by lipase and trypsin, then carrying out enzymolysis by ficin and bromelain, and then sequentially purifying by a macroporous adsorption resin column and a phenyl agarose gel column. The tegillarca granosa peptide is obtained by fermenting tegillarca granosa meat through bacillus marinus; the herba epimedii extract is obtained by purifying an upper phase extracted from herba epimedii powder through an aqueous two-phase system through a sephadex chromatographic column; the rhizoma drynariae extract is obtained by performing enzymolysis on rhizoma drynariae powder with cellulase, extracting with a sodium dioctyl sulfosuccinate-isooctane reverse micelle solution, taking an upper organic phase, breaking micelles, taking a lower water phase and performing ultrafiltration. The four components are matched according to a specific proportion to realize multi-effect regulation of promoting formation, inhibiting bone resorption and regulating hormone.

Description

TECHNICAL FIELD

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

[0002] Osteoporosis is a systemic bone disease characterized by bone loss, bone microstructure destruction, and increased bone fragility. Its incidence is increasing year by year and has become a serious public health problem. According to statistics, an osteoporotic fracture occurs every three seconds worldwide, and fractures not only significantly reduce the quality of life of patients, but also increase the risk of disability and death. Traditional calcium and vitamin D supplements are the basic treatment, but they can only relieve symptoms and cannot effectively reverse bone loss.

[0003] Traditional anti-osteoporosis drugs are mainly divided into two categories: bone resorption inhibitors and bone formation promoters. Bone resorption inhibitors (such as bisphosphonates and estrogen receptor modulators) reduce bone loss by inhibiting osteoclast activity, and are widely used in clinical practice. However, long-term use may cause jaw necrosis, atypical fractures, and other side effects, with poor safety. Bone formation promoters (such as teriparatide) can stimulate osteoblast activity and increase bone density, but their mechanism is single, the treatment cycle is short, and there is a risk of hypercalcemia, with poor safety.

[0004] In terms of administration, anti-osteoporosis drugs are mainly divided into oral and injectable types. Oral drugs (such as alendronate sodium and raloxifene) have the advantages of convenient use and low cost, and are suitable for long-term maintenance therapy. However, due to low oral absorption efficiency, long-term gastrointestinal irritation and patient compliance issues need to be addressed. Injectable drugs (such as denosumab and romosozumab) have low administration frequency (such as once every six months), high absorption efficiency compared to oral drugs, and are particularly suitable for patients with severe osteoporosis, high fracture risk, or unable to tolerate oral drugs. However, there are problems such as high cost and potential side effects (such as injection site reactions).

[0005] In recent years, with the deepening understanding of bone metabolism mechanisms, new drug technologies focus on precise regulation of bone metabolism balance and multi-target synergistic effects. For example, monoclonal antibodies targeting the RANKL pathway significantly reduce the risk of fractures by specifically blocking osteoclast differentiation and activation. Small molecule drugs inhibit sclerostin (SOST) and activate the Wnt signaling pathway, achieving the dual effects of promoting bone formation and inhibiting bone resorption. In addition, the development of nanotechnology and drug delivery systems has further improved the targeting and safety of drugs. For example, nanoparticle carriers can achieve sustained-release drug delivery, reducing systemic adverse reactions. Bone-targeting modification technology can precisely enrich drugs in bone tissue. Gene therapy and stem cell therapy also show potential application value by regulating bone metabolism-related gene expression and promoting osteogenic differentiation.

[0006] Although the anti-osteoporosis drug research and development has made significant progress, but still face many challenges, in the preparation of cost economy, composition safety, high efficiency and so on still need continuous innovation and improvement, in order to better adapt to the needs of patients. SUMMARY

[0007] In view of the existing composition safety, poor single effect, high efficiency and other aspects of the existing bone repair drug need to be further improved, the present application provides a kind of bone repair multi-effect synergistic drug composition and preparation method thereof, five black chicken egg yolk peptide, mud clam peptide is prepared by special enzymolysis and purification method, special extraction method is used to prepare Epimedium extract and rhizoma drynaria extract, four are matched according to specific proportion, through five black chicken egg yolk peptide and mud clam peptide promote bone formation, rhizoma drynaria extract inhibits bone resorption, Epimedium extract regulates hormone, multi-component synergy, realizes the multi-effect regulation of "promoting formation-inhibiting osteoclast activation-antioxidation", avoids the mechanism limitation of single target drug, and improves the bone repair property. Its specific technical scheme is as follows:

[0008] A kind of bone repair multi-effect synergistic drug composition, including five black chicken egg yolk peptide, mud clam peptide, Epimedium extract and rhizoma drynaria extract, the mass ratio of five black chicken egg yolk peptide:mud clam peptide:Epimedium extract:rhizoma drynaria extract=(8-10):(3-5):(0.5-1):(1-1.5);Wherein: the five black chicken egg yolk peptide is five black chicken egg yolk freeze-dried powder, which is enzymolyzed by lipase and trypsin, and then enzymolyzed by ficin and bromelain to obtain an enzymolysis solution, which is filtered by microfiltration membrane, and then purified by LX-68 macroporous adsorption resin column and phenyl sepharose column in turn, and ultrafiltration is carried out to obtain a freeze-dried product with a molecular weight of 500Da-5kDa;Mud clam peptide is mud clam meat fermented by marine bacillus fermentation to obtain fermentation broth, which is filtered after ultrasonic treatment, and the filtrate is treated by electrodialysis to remove heavy metal ions and sodium ions, and ultrafiltration is carried out to obtain a freeze-dried product with a molecular weight of 1kDa-3kDa;Epimedium extract is the upper phase extracted from Epimedium powder by hydroxypropyl methyl cellulose-potassium citrate trihydrate aqueous two-phase system, which is purified by dextran gel LH-20 chromatographic column, dried, and the obtained product is obtained;Rhizoma drynaria extract is obtained by enzymolysis of rhizoma drynaria powder with cellulase, extraction by dioctyl sodium sulfosuccinate-isooctane reverse micelle solution, centrifugation, standing and layering, taking the upper organic phase, adding sodium citrate aqueous solution, stirring to break the micelles, standing, centrifugation, standing and layering, taking the lower aqueous phase, and ultrafiltration to obtain a freeze-dried product with a molecular weight of 500Da-5kDa.

[0009] The preparation method of the Wuhe black chicken egg yolk peptide comprises the following steps: diluting egg yolk freeze-dried powder with normal saline to prepare an egg yolk diluent, adding lipase and trypsin, adjusting pH to 7.5-8.0, and performing enzymolysis at 36-38℃ for 60-90 min, then increasing the temperature to 50-55℃, adjusting pH to 6.5-7, adding ficin and bromelain, and performing enzymolysis for 60-80 min, increasing the temperature to inactivate the enzyme, centrifuging, taking the supernatant, loading the supernatant onto an LX-68 macroporous adsorption resin column, eluting with 5%-6% (by volume) ethanol water solution for 0-1.5 BV, eluting with 20%-25% (by volume) ethanol water solution for 1.5-2.5 BV, collecting the eluent for 1.5-2.5 BV, evaporating to obtain a concentrated solution, loading the concentrated solution onto a phenyl sepharose column, eluting with 1.4-1.6 M (NH4)2SO4 water solution for 0-0.5 BV, eluting with 0.8-1 M (NH4)2SO4 water solution for 0.5-1.5 BV, eluting with 0.3-0.5 M (NH4)2SO4 water solution for 1.5-3 BV, collecting the eluent for 1.5-3 BV, performing ultrafiltration with an ultrafiltration membrane, taking the component between 500 Da and 5 kDa, and freeze-drying to obtain the Wuhe black chicken egg yolk peptide.

[0010] In the preparation method of the Wuhe black chicken egg yolk peptide, the egg yolk freeze-dried powder is prepared from a Wuhe green skin chicken egg; the amount of the normal saline is 8-10 times the mass of the egg yolk freeze-dried powder; the amount of the added lipase is 0.5%-1% of the mass of the egg yolk freeze-dried powder; the amount of the added trypsin is 1%-1.5% of the mass of the egg yolk freeze-dried powder; the amounts of the added ficin and bromelain are both 0.5%-0.8% of the mass of the egg yolk freeze-dried powder; the stirring rotation speed of the enzymolysis is 100-150 rpm; the temperature for inactivating the enzyme is 75-80℃ for 10-15 min; the centrifugation is performed at 5000-6000 rpm for 10-15 min; the flow rate of the supernatant is 1.5-2 BV / h; the evaporation is performed at 35-40℃ under vacuum to a volume of 20%-30%; the flow rate of the concentrated solution is 1-1.5 BV / h; the height-diameter ratio of the LX-68 macroporous adsorption resin column is (4-5):1; and the height-diameter ratio of the phenyl sepharose column is (3-4):1.

[0011] The preparation method of the above composition of mud clam peptide includes: adding mud clam meat to 1 to 2 times its mass of sterile water, crushing and homogenizing to obtain clam meat homogenate, adding 3 to 4 times its mass of sterile water to dilute, adjusting the pH to 7.2 to 7.5, inoculating with marine Bacillus for fermentation to obtain fermentation broth; ultrasonically treating the fermentation broth to dissolve active peptides; then filtering through a nylon mesh and a ceramic membrane to collect the filtrate; transferring the filtrate to an electrodialysis device for electrodialysis treatment to remove heavy metal ions and sodium ions; ultrafiltration of the electrodialysis solution through an ultrafiltration membrane to collect the fraction between 1 kDa and 3 kDa, freeze-drying to obtain mud clam peptide.

[0012] In the above-mentioned method for preparing mud clam peptides, the mud clam meat is freshly washed mud clam meat; the crushing and homogenization is carried out at 6000rpm-8000rpm for 5min-8min; the inoculation amount of marine Bacillus is 3%-5% of the mass of the mud clam meat; the fermentation is carried out at 30℃-35℃ and 100rpm-120rpm with stirring for 48h-52h; the ultrasonic treatment is carried out at 30℃-35℃ and 200rpm-300rpm with stirring, 200W-250W, and 30kHz-40kHz for 30min-50min; the nylon mesh is 200-250 mesh; the ceramic membrane has a pore size of 0.45μm; the electrodialysis device is equipped with anion and cation exchange membranes; the electrodialysis parameters are set as follows: operating voltage is 12V-15V, and current density is 5mA / cm². 2 ~10mA / cm 2 The processing time is 1.5h to 2h.

[0013] The preparation method of Epimedium extract in the above composition includes: mixing hydroxypropyl methylcellulose: tripotassium citrate: water in a mass ratio of (6-8): (12-15): (85-90), stirring evenly to construct an aqueous two-phase system; adding Epimedium powder to the aqueous two-phase system in a mass ratio of 1: (5-6), extracting, refrigerating and allowing to stand to separate the two phases, and taking the upper phase; loading the sample through a dextran gel LH-20 chromatography column, eluting 0-3 BV with 30%-35% volume concentration ethanol aqueous solution, collecting 1.5-2.5 BV of eluent, evaporating and concentrating, and lyophilizing to obtain Epimedium extract.

[0014] In the above-mentioned method for preparing Epimedium extract, the Epimedium powder is passed through an 80-100 mesh sieve; the extraction is carried out by stirring at 55-60℃ and 80-100 rpm for 1-1.5 hours; the refrigeration and standing temperature is 4-6℃; the evaporation and concentration is carried out by vacuum rotary evaporation at 35-40℃; and the height-to-diameter ratio of the dextran gel LH-20 chromatography column is (4-5):1.

[0015] The preparation method of the Rhizoma Drynariae extract in the composition comprises the following steps: adding Rhizoma Drynariae powder into deionized water, adding cellulase, enzymolysis, heating and deactivating the enzyme, and evaporating to remove water to obtain an enzymolysis product; dissolving sodium dioctyl sulfosuccinate in isooctane to prepare a reverse micelle solution with a concentration of 0.08 mol / L-0.1 mol / L; adding the enzymolysis product with a material-liquid mass ratio of 1: (5-6) into the reverse micelle solution, extracting, centrifuging, and standing to separate layers, and collecting the organic phase in the upper layer; adding a sodium citrate aqueous solution into the organic phase, homogenizing the reverse micelles at 7500 rpm-8500 rpm for 8 min-12 min, standing, centrifuging, and standing to separate layers, and collecting the water phase in the lower layer, and then the water phase is subjected to ultrafiltration with an ultrafiltration membrane, and the components with a molecular weight of 500 Da-5 kDa are collected, and then the components are freeze-dried to obtain the Rhizoma Drynariae extract.

[0016] In the preparation method of the Rhizoma Drynariae extract, the Rhizoma Drynariae powder is sieved through a 100 mesh-120 mesh screen; the deionized water is used in an amount of 4-6 times the mass of the Rhizoma Drynariae powder; the cellulase is added in an amount of 1%-3% of the mass of the Rhizoma Drynariae powder; the enzymolysis is performed at pH 4.5-5, 50°C-55°C, and 100 rpm-150 rpm for 3 h-4 h; the heating and deactivating the enzyme is performed at 85°C-90°C for 10 min-15 min; the evaporation is performed by vacuum rotary evaporation at 35°C-40°C; the extraction is performed by oscillating extraction at 30°C-35°C and a rotation speed of 120 rpm-150 rpm for 2 h-2.5 h; the sodium citrate aqueous solution has a concentration of 0.1 mol / L-0.12 mol / L; the sodium citrate aqueous solution is added in an amount of 10%-12% of the volume of the organic phase; the centrifuging is all performed at 4000 rpm-5000 rpm for 8 min-12 min; and the standing is all performed for 10 min-15 min.

[0017] The preparation method of the multi-effect synergistic pharmaceutical composition for bone repair comprises the following steps: mixing and preparing five black chicken egg yolk peptides, mud clam peptides, Epimedium extract and Rhizoma Drynariae extract according to a mass ratio to obtain the multi-effect synergistic pharmaceutical composition.

[0018] The multi-effect synergistic pharmaceutical composition for bone repair and the preparation method thereof have the following beneficial effects: I. The five black chicken egg yolk peptides are purified through an LX-68 macroporous adsorption resin column and a phenyl sepharose column, and the anti-nutritional factors (including high-phosphorus proteins) and un-enzymolysis protein aggregates in the egg yolk can be effectively removed. If the impurities are left, the metal ions (including calcium ions) necessary for bone cells are chelated, the alkaline phosphatase (ALP) activity is inhibited, the immune response is triggered, or the cell membrane structure is damaged. The mud clam peptides are treated by electrodialysis to remove heavy metal ions and sodium ions, so that the damage of the metal ions to the red blood cell membrane is avoided.

[0019] II. Both Wujia chicken egg yolk peptide and Naihe peptide in the composition are small molecular peptide segments obtained by enzymatic hydrolysis of natural proteins, with concentrated molecular weight and low antigenicity, which is not easy to cause allergic reactions. The extracts of Herba Epimedii and Drynariae Radix are purified by aqueous two-phase system and reverse micelle extraction, reducing the residue of organic solvents and the damage of heat-sensitive components.

[0020] III. The bone formation promoting effect of Wujia chicken egg yolk peptide: The active peptide segments (including adhesion peptides) produced by specific enzymatic hydrolysis of Wujia chicken egg yolk specifically bind to integrin receptors on the surface of osteoblasts, activate the signaling pathway, up-regulate the expression of osteogenic transcription factors, promote cell cycle progression, and accelerate osteoblast proliferation and protein synthesis.

[0021] IV. The extract of Herba Epimedii activates the signaling pathway, promotes osteoblast differentiation and gene transcription of osteocalcin. At the same time, its flavonoid components scavenge reactive oxygen species (ROS) in cells, reduce the damage of oxidative stress to osteoblasts, and maintain the cell proliferation microenvironment.

[0022] V. Synergistic induction effect of Drynariae Radix extract: The components such as naringin and protocatechuic acid in Drynariae Radix extract promote the differentiation of mesenchymal stem cells into osteoblasts by activating the signaling pathway. In addition, the active ingredients retained by the reverse micelle extraction process enhance the calcium and phosphorus uptake capacity of osteoblasts and promote bone matrix mineralization.

[0023] VI. The synergistic effect network of multiple components: Wujia chicken egg yolk peptide activates osteoblast proliferation through the signaling pathway, and Naihe peptide provides peptides containing glycine and proline. When the two are compounded in a specific ratio, the small molecular active peptides in Naihe peptide act as cofactors for egg yolk peptides, enhancing their binding efficiency to cell receptors and avoiding the competitive inhibition of receptors caused by excessive single components.

[0024] VII. Synergy of antioxidant and signal regulation: The flavonoid components of Herba Epimedii extract and the protocatechuic acid of Drynariae Radix extract form an antioxidant system, scavenge ROS produced during the differentiation of osteoclasts, and reduce DNA damage to osteoblasts. Specific proportioning can improve efficacy, and Drynariae Radix extract inhibits RANKL-induced osteoclast activation, forming a "inhibit osteoclast activation-promote formation" two-way regulation with icariin.

[0025] VIII. Precise release of active peptides by stepwise enzymatic hydrolysis: Wujia chicken egg yolk peptide uses lipase and trypsin to pretreat and hydrolyze egg yolk lipoprotein, and then uses ficin and bromelain for secondary enzymatic hydrolysis to specifically cut the triple helix structure of collagen, releasing peptide segments with angiogenic and cell adhesion functions.

[0026] Nine, purification technology to improve the specificity of ingredients: phenyl sepharose column (6FF high resolution) by hydrophobic interaction separation of active peptides, excluding macromolecular impurities (including endotoxin) and small molecule toxins; dextran gel LH-20 chromatographic column purification of Epimedium extract, using molecular sieve effect to retain icariin, remove polysaccharide and other hydrophilic impurities, avoid impurities and active ingredients competing for cell receptors.

[0027] Ten, multi-target regulation of bone metabolism balance: traditional anti-osteoporosis drugs (such as bisphosphonates) only inhibit bone resorption, teriparatide only promotes bone formation, while the composition realizes the multi-effect regulation of "promoting formation-inhibiting osteoclast activation-antioxidation" through the multi-component synergy of five black chicken egg yolk peptide and mud clam peptide (promoting formation), Rhizoma Drynaria extract (inhibiting osteoclast activation) and Epimedium extract (hormone regulation), avoiding the mechanism limitations of single-target drugs.

[0028] Eleven, reduce the safety risk of long-term medication: traditional oral drugs (such as alendronate sodium) have gastrointestinal irritation, and injection drugs (such as denosumab) have injection site reactions, while the composition reduces the toxicity and side effects of chemical synthesis drugs through natural ingredient purification and precise proportioning. The phospholipid peptide component of five black chicken egg yolk peptide can stabilize the cell membrane, and the electrodialysis impurity removal process of mud clam peptide reduces the toxicity of metal ions. DETAILED DESCRIPTION

[0029] The application will be further described below in conjunction with specific implementation examples, but the application is not limited to these examples.

[0030] Glossary: BV is the volume of the resin column.

[0031] Example 1 A multi-effect synergistic drug composition for bone repair, comprising five black chicken egg yolk peptide, mud clam peptide, Epimedium extract and Rhizoma Drynaria extract, with a mass ratio of five black chicken egg yolk peptide:mud clam peptide:Epimedium extract:Rhizoma Drynaria extract = 8:3:0.5:1.

[0032] The preparation method of the Wuzihe egg yolk peptide comprises the following steps: diluting egg yolk freeze-dried powder prepared from Wuzihe green-shelled eggs with 8 times the mass of normal saline to obtain an egg yolk diluent, adding lipase with a mass of 0.5% of the egg yolk freeze-dried powder and trypsin with a mass of 1% of the egg yolk freeze-dried powder, adjusting the pH to 7.5, and performing enzymatic hydrolysis at 36℃ and 100 rpm for 60 min, then increasing the temperature to 50℃, adjusting the pH to 6.5, adding ficin and bromelain, both with a mass of 0.5% of the egg yolk freeze-dried powder, and performing enzymatic hydrolysis at 100 rpm for 60 min, increasing the temperature to 75℃ to inactivate the enzymes for 10 min, and centrifuging at 5000 rpm for 10 min, and then taking the supernatant, loading the supernatant onto an LX-68 macroporous adsorption resin column with a column height-diameter ratio of 4:1 at a flow rate of 1.5 BV / h, eluting with 5% by volume of an ethanol aqueous solution for 0-1.5 BV, eluting with 20% by volume of an ethanol aqueous solution for 1.5-2.5 BV, collecting the eluent for 1.5-2.5 BV, and performing vacuum rotary evaporation at 35℃ until the volume is 20% to obtain a concentrated solution; loading the concentrated solution onto a phenyl sepharose column with a column height-diameter ratio of 3:1 at a flow rate of 1 BV / h, eluting with 1.4M (NH4)2SO4 for 0-0.5 BV, eluting with 0.8M (NH4)2SO4 for 0.5-1.5 BV, eluting with 0.3M (NH4)2SO4 for 1.5-3 BV, collecting the eluent for 1.5-3 BV, performing ultrafiltration, taking components with a molecular weight of 500 Da-5 kDa, and freeze-drying to obtain the Wuzihe egg yolk peptide.

[0033] The preparation method of the mud clam peptide comprises the following steps: adding fresh and washed mud clam meat into 1 times the mass of sterile water, crushing and homogenizing the mud clam meat at 6000 rpm for 5 min to obtain a homogenate, diluting the homogenate with 3 times the mass of sterile water, adjusting the pH to 7.2, inoculating 3% of the mass of the mud clam meat with Bacillus marinus, and performing aerobic fermentation at 26℃ and 100 rpm for 48 h to obtain a fermentation liquor; performing ultrasonic treatment on the fermentation liquor at 35℃ and 200 rpm, 200W and 30 kHz for 30 min to dissolve active peptides; then filtering the fermentation liquor through a 200-mesh nylon screen and a 0.45-μm ceramic membrane to obtain a filtrate; transferring the filtrate into an electrodialysis device, which is equipped with anion and cation exchange membranes, setting the operating voltage to 12V, the current density to 5 mA / cm2, and the treatment time to 1.5 h to remove heavy metal ions and sodium ions; performing ultrafiltration on the solution after the electrodialysis through an ultrafiltration membrane, collecting components with a molecular weight of 1 kDa-3 kDa, and freeze-drying to obtain the mud clam peptide. 2

[0034] ​The preparation method of the Epimedium extract includes the following steps: mixing hydroxypropyl methylcellulose, potassium citrate and water according to a mass ratio of 6:12:85, stirring uniformly, and constructing a two-water-phase system; adding Epimedium powder under 80 mesh to the two-water-phase system according to a solid-liquid mass ratio of 1:5, stirring extraction at 55 DEG C and 80 rpm for 1 h, separating the two phases by 4 DEG C cold storage and standing, and taking the upper phase; loading the upper phase through a dextran gel LH-20 chromatographic column with a column height-diameter ratio of 4:1, eluting with 30% volume concentration ethanol aqueous solution, collecting 1.5-2.5 BV eluent, concentrating by vacuum rotary evaporation at 35 DEG C, and freeze-drying to obtain the Epimedium extract.

[0035] The preparation method of the Rhizoma Drynariae extract includes the following steps: adding Rhizoma Drynariae powder under 100 mesh to 4 times of deionized water, adding cellulase with a mass ratio of 1% of the Rhizoma Drynariae powder, stirring enzyme hydrolysis at pH 4.5, 50 DEG C and 100 rpm for 3 h, heating to 85 DEG C for 10 min to inactivate the enzyme, and removing water by vacuum rotary evaporation at 35 DEG C to obtain an enzyme hydrolysate; dissolving sodium dioctyl sulfosuccinate in isooctane to prepare a reverse micelle solution with a concentration of 0.08 mol / L; adding the enzyme hydrolysate to the reverse micelle solution according to a solid-liquid mass ratio of 1:5, oscillating extraction at 30 DEG C and 120 rpm for 2 h, centrifuging at 4000 rpm for 8 min, standing for 10 min to separate the layers, collecting the organic phase in the upper layer, adding 10% of the organic phase to a 0.1 mol / L sodium citrate aqueous solution, homogenizing the micelles at 7500 rpm for 8 min, standing for 10 min, centrifuging at 4000 rpm for 8 min, standing for 10 min to separate the layers, and collecting the water phase in the lower layer; ultrafiltrating the water phase by using an ultrafiltration membrane, collecting components with a molecular weight of 500 Da-5 kDa, and freeze-drying to obtain the Rhizoma Drynariae extract.

[0036] The preparation method of the multi-effect synergistic pharmaceutical composition for bone repair includes the following steps: mixing Wuhai chicken egg yolk peptide, mud clam peptide, Epimedium extract and Rhizoma Drynariae extract according to a mass ratio to obtain the multi-effect synergistic pharmaceutical composition.

[0037] Example 2 The multi-effect synergistic pharmaceutical composition for bone repair includes Wuhai chicken egg yolk peptide, mud clam peptide, Epimedium extract and Rhizoma Drynariae extract, and the mass ratio of the Wuhai chicken egg yolk peptide, the mud clam peptide, the Epimedium extract and the Rhizoma Drynariae extract is 9:4:0.7:1.3.

[0038] The preparation method of the Wuzihe egg yolk peptide comprises the following steps: diluting egg yolk freeze-dried powder prepared from Wuzihe green-shelled eggs with 9 times the mass of normal saline to obtain an egg yolk diluent, adding lipase with a mass of 0.8% of the egg yolk freeze-dried powder and trypsin with a mass of 1.2% of the egg yolk freeze-dried powder, adjusting the pH to 7.8, and performing enzymatic hydrolysis at 37℃ and 120 rpm for 80 min, then increasing the temperature to 52℃, adjusting the pH to 6.8, adding ficin and bromelain, both with a mass of 0.6% of the egg yolk freeze-dried powder, and performing enzymatic hydrolysis at 120 rpm for 70 min, increasing the temperature to 78℃ to inactivate the enzymes for 12 min, and centrifuging at 5500 rpm for 12 min, and then taking the supernatant, loading the supernatant onto an LX-68 macroporous adsorption resin column with a column height-diameter ratio of 4.5:1 at a flow rate of 1.8 BV / h, eluting with 5.5% volume concentration of an ethanol aqueous solution for 0-1.5 BV, eluting with 22% volume concentration of an ethanol aqueous solution for 1.5-2.5 BV, collecting the eluent for 1.5-2.5 BV, and performing vacuum rotary evaporation at 38℃ until the volume is 25% to obtain a concentrated solution; loading the concentrated solution onto a phenyl sepharose column with a column height-diameter ratio of 3.5:1 at a flow rate of 1.2 BV / h, eluting with 1.5M (NH4)2SO4 aqueous solution for 0-0.5 BV, eluting with 0.9M (NH4)2SO4 aqueous solution for 0.5-1.5 BV, eluting with 0.4M (NH4)2SO4 aqueous solution for 1.5-3 BV, collecting the eluent for 1.5-3 BV, performing ultrafiltration with an ultrafiltration membrane, taking components with a molecular weight of 500Da-5kDa, and freeze-drying to obtain the Wuzihe egg yolk peptide.

[0039] The preparation method of the mud clam peptide comprises the following steps: adding fresh and washed mud clam meat into 1.5 times the mass of sterile water, crushing and homogenizing the mud clam meat at 7000 rpm for 6 min to obtain a homogenate, diluting the homogenate with 3.5 times the mass of sterile water, adjusting the pH to 7.4, inoculating 4% of the mass of the mud clam meat with Bacillus marinus, and performing aerobic fermentation at 27℃ and 110 rpm for 50 h to obtain a fermentation liquor; performing ultrasonic treatment on the fermentation liquor at 32℃ and 250 rpm for 40 min under the conditions of 200W and 35kHz to dissolve active peptides; then filtering the fermentation liquor through a 200-mesh nylon screen and a 0.45-μm ceramic membrane to obtain a filtrate; transferring the filtrate into an electrodialysis device, which is equipped with cation and anion exchange membranes, setting the operating voltage to 13V, the current density to 8mA / cm 2 , and the treatment time to 1.5h, removing heavy metal ions and sodium ions; performing ultrafiltration on the solution after the electrodialysis through an ultrafiltration membrane, collecting components with a molecular weight of 1kDa-3kDa, and freeze-drying to obtain the mud clam peptide.

[0040] The preparation method of the Epimedium extract includes the following steps: mixing hydroxypropyl methylcellulose, potassium citrate and water according to a mass ratio of 7:13:88, stirring uniformly, and constructing a two-water-phase system; adding Epimedium powder under 80 mesh to the two-water-phase system according to a material-liquid mass ratio of 1:5.5, stirring extraction at 58 ℃ and 90 rpm for 1 h, and separating the two phases by 5 ℃ cold storage and standing; loading the upper phase through a dextran gel LH-20 chromatographic column with a column height-diameter ratio of 4.5:1, eluting 1.5-2.5 BV of eluent with 32% volume concentration ethanol aqueous solution, concentrating by vacuum rotary evaporation at 38 ℃, and freeze-drying to obtain the Epimedium extract.

[0041] The preparation method of the Rhizoma Drynariae extract includes the following steps: adding Rhizoma Drynariae powder under 100 mesh to 5 times of deionized water, adding cellulase with a mass ratio of 2% of the Rhizoma Drynariae powder, stirring enzyme hydrolysis at pH 4.8, 52 ℃ and 120 rpm for 3.5 h, heating to 88 ℃ for 12 min to inactivate the enzyme, and removing water by vacuum rotary evaporation at 38 ℃ to obtain an enzyme hydrolysate; dissolving sodium dioctyl sulfosuccinate in isooctane to prepare a reverse micelle solution with a concentration of 0.09 mol / L; adding the enzyme hydrolysate to the reverse micelle solution according to a material-liquid mass ratio of 1:5.5, oscillating extraction at 32 ℃ and 130 rpm for 2 h, centrifuging at 4500 rpm for 10 min, standing for 12 min to separate the layers, collecting the organic phase in the upper layer, adding sodium citrate aqueous solution with a concentration of 0.11 mol / L and a volume of 11% of the organic phase, homogenizing the micelles at 8000 rpm for 10 min, standing for 12 min, centrifuging at 4500 rpm for 10 min, standing for 12 min to separate the layers, and collecting the water phase in the lower layer; ultrafiltration is performed on the water phase, and components with a molecular weight of 500 Da-5 kDa are collected and freeze-dried to obtain the Rhizoma Drynariae extract.

[0042] The preparation method of the multi-effect synergistic pharmaceutical composition for bone repair includes the following steps: mixing Wuhai chicken egg yolk peptide, mud clam peptide, Epimedium extract and Rhizoma Drynariae extract according to a mass ratio to obtain the multi-effect synergistic pharmaceutical composition.

[0043] Example 3 A multi-effect synergistic pharmaceutical composition for bone repair includes Wuhai chicken egg yolk peptide, mud clam peptide, Epimedium extract and Rhizoma Drynariae extract, and the mass ratio of the Wuhai chicken egg yolk peptide, the mud clam peptide, the Epimedium extract and the Rhizoma Drynariae extract is 10:5:1:1.5.

[0044] The preparation method of the Wuzi black chicken egg yolk peptide comprises the following steps: diluting egg yolk freeze-dried powder prepared from Wuzi black chicken green-shelled eggs with 10 times the mass of normal saline to prepare an egg yolk diluent, adding lipase in an amount of 1% of the mass of the egg yolk freeze-dried powder and trypsin in an amount of 1.5% of the mass of the egg yolk freeze-dried powder, adjusting the pH to 8.0, and performing enzymatic hydrolysis at 38℃ and 150 rpm for 90 min, then increasing the temperature to 55℃, adjusting the pH to 7, adding ficin and bromelain, both in an amount of 0.8% of the mass of the egg yolk freeze-dried powder, and performing enzymatic hydrolysis at 150 rpm for 80 min, increasing the temperature to 80℃ to inactivate the enzymes for 15 min, and centrifuging at 6000 rpm for 15 min, and taking the supernatant, and loading the supernatant onto an LX-68 macroporous adsorption resin column with a column height-diameter ratio of 5:1 at a flow rate of 2 BV / h, eluting with 6% by volume of an ethanol aqueous solution for 0-1.5 BV, eluting with 25% by volume of an ethanol aqueous solution for 1.5-2.5 BV, collecting the eluate for 1.5-2.5 BV, and vacuum rotary evaporating at 40℃ to a volume of 30% to obtain a concentrated solution; loading the concentrated solution onto a phenyl sepharose column with a column height-diameter ratio of 4:1 at a flow rate of 1.5 BV / h, eluting with 1.6M (NH4)2SO4 aqueous solution for 0-0.5 BV, eluting with 1M (NH4)2SO4 aqueous solution for 0.5-1.5 BV, eluting with 0.5M (NH4)2SO4 aqueous solution for 1.5-3 BV, collecting the eluate for 1.5-3 BV, performing ultrafiltration on the eluate, collecting components with a molecular weight of 500 Da-5 kDa, and freeze-drying to obtain the Wuzi black chicken egg yolk peptide.

[0045] The preparation method of the mud clam peptide comprises the following steps: adding fresh and washed mud clam meat into 2 times the mass of sterile water, crushing and homogenizing the mud clam meat at 8000 rpm for 8 min to obtain a homogenate, diluting the homogenate with 4 times the mass of sterile water, adjusting the pH to 7.5, inoculating 5% of the mass of the mud clam meat with Bacillus marinus, and performing aerobic fermentation at 28℃ and 120 rpm for 52 h to obtain a fermentation liquor; performing ultrasonic treatment on the fermentation liquor at 30℃ and 300 rpm for 50 min under the conditions of 250 W and 40 kHz to dissolve active peptides; then filtering the fermentation liquor through a 250-mesh nylon screen and a 0.45-μm ceramic membrane to obtain a filtrate; transferring the filtrate into an electrodialysis device, and setting the operating voltage to 15 V, the current density to 10 mA / cm 2 , and the treatment time to 2 h to remove heavy metal ions and sodium ions; performing ultrafiltration on the solution after the electrodialysis, collecting components with a molecular weight of 1 kDa-3 kDa, and freeze-drying to obtain the mud clam peptide.

[0046] The preparation method of the Epimedium extract includes the following steps: mixing hydroxypropyl methylcellulose, potassium citrate and water according to a mass ratio of 8:15:90, stirring uniformly, and constructing a two-water-phase system; adding Epimedium powder under 100 mesh to the two-water-phase system according to a solid-liquid mass ratio of 1:6, stirring extraction at 60 DEG C and 100 rpm for 1.5 h, separating the two phases by 6 DEG C cold storage and standing, and taking the upper phase; loading the upper phase through a dextran gel LH-20 chromatographic column with a column height-diameter ratio of 5:1, eluting 0-3 BV of 35% volume concentration ethanol aqueous solution, collecting 1.5-2.5 BV of eluent, concentrating by vacuum rotary evaporation at 40 DEG C, and freeze-drying to obtain the Epimedium extract.

[0047] The preparation method of the Rhizoma Drynariae extract includes the following steps: adding Rhizoma Drynariae powder under 120 mesh to 6 times of deionized water, adding cellulase with a mass ratio of 3% of the Rhizoma Drynariae powder, stirring enzyme hydrolysis at pH 5, 55 DEG C and 150 rpm for 4 h, heating to 90 DEG C for 15 min to inactivate the enzyme, and removing water by vacuum rotary evaporation at 40 DEG C to obtain an enzyme hydrolysate; dissolving sodium dioctyl sulfosuccinate in isooctane to prepare a reverse micelle solution with a concentration of 0.1 mol / L; adding the enzyme hydrolysate to the reverse micelle solution according to a solid-liquid mass ratio of 1:6, oscillating extraction at 35 DEG C and 150 rpm for 2.5 h, centrifuging at 5000 rpm for 12 min, standing for 15 min to separate the layers, collecting the organic phase in the upper layer, adding 12% of the organic phase to a 0.12 mol / L sodium citrate aqueous solution, homogenizing the micelles at 8500 rpm for 12 min, standing for 15 min, centrifuging at 5000 rpm for 12 min, standing for 15 min to separate the layers, collecting the water phase in the lower layer, ultrafiltering by using an ultrafiltration membrane, collecting components with a molecular weight of 500 Da-5 kDa, and freeze-drying to obtain the Rhizoma Drynariae extract.

[0048] The preparation method of the multi-effect synergistic pharmaceutical composition for bone repair includes the following steps: mixing Wuhai chicken egg yolk peptide, Lutraria peptide, Epimedium extract and Rhizoma Drynariae extract according to a mass ratio to obtain the multi-effect synergistic pharmaceutical composition.

[0049] In the above embodiments, the lipase is from Henan Anrui Biological Technology Co., Ltd., with an enzyme activity of 100,000 U / g. The trypsin is from Ningxia Xiasheng Industry Group Co., Ltd., model number FDG-2281, with an enzyme activity of 200,000 U / g. The ficin is from Nanjing Songguan Biological Technology Co., Ltd., with an enzyme activity of 100,000 U / g. The bromelain is from Peizuo Biological Technology (Xi'an) Co., Ltd., with an enzyme activity of 100,000 U / g. The LX-68 macroporous adsorption resin is from Mabio (Shanghai) Biochemical Technology Co., Ltd. The phenyl sepharose is from Shanghai Huicheng Biological Technology Co., Ltd., with a high resolution of 6FF. The marine bacillus is from Shanghai Guyan Industry Co., Ltd., with a product number GOY-4887, and is activated before being added. The hydroxypropyl methyl cellulose is from Shanghai Yuanye Biological Technology Co., Ltd., with a model number S14174. The tripotassium citrate is from Zhengzhou Yuhao Food Additives Co., Ltd. The dextran gel LH-20 is from Shanghai Yuanye Biological Technology Co., Ltd., with a product number S14037. The cellulase is from Shanghai Yuanchi Biological Technology Co., Ltd., with an enzyme activity of 100,000 U / g. The dioctyl sodium sulfosuccinate is from Wuhan Huaxiang Kejebio Technology Co., Ltd. The isooctane is from Shaanxi Liedu Pharmaceutical Chemical Co., Ltd.

[0050] Comparative Example 1 The difference from Example 1 is that the Wujia chicken egg yolk peptide is changed to 3 parts, and the Naiqie peptide is changed to 8 parts.

[0051] Comparative Example 2 The difference from Example 1 is that the Yiyanghao extract is changed to 1.2 parts, and the Gushu bu extract is changed to 0.3 parts.

[0052] Comparative Example 3 The difference from Example 1 is that the Wujia chicken egg yolk peptide is changed to 3 parts, the Naiqie peptide is changed to 8 parts, the Yiyanghao extract is changed to 1.2 parts, and the Gushu bu extract is changed to 0.3 parts.

[0053] Comparative Example 4 The difference from Example 1 is that in the preparation method of the Wujia chicken egg yolk peptide, the trypsin is changed to alkaline protease.

[0054] Comparative Example 5 The difference from Example 1 is that in the preparation method of the Wujia chicken egg yolk peptide, the ficin is changed to papain.

[0055] Comparative Example 6 The difference from Example 1 is that in the preparation method of the Wujia chicken egg yolk peptide, the ficin is not added.

[0056] Comparative Example 7 The difference from Example 1 is that in the preparation method of the Wujia chicken egg yolk peptide, the bromelain is changed to neutral protease.

[0057] Comparative Example 8 The difference from Example 1 is that the LX-68 macroporous adsorption resin column is replaced by AB-8 macroporous adsorption resin in the preparation method of Wuzhe chicken egg yolk peptide.

[0058] Comparative Example 9 The difference from Example 1 is that the LX-68 macroporous adsorption resin column is replaced by D101 macroporous adsorption resin in the preparation method of Wuzhe chicken egg yolk peptide.

[0059] Comparative Example 10 The difference from Example 1 is that the phenyl sepharose gel column (6FF high resolution) is replaced by phenyl sepharose gel column (CL-4B) in the preparation method of Wuzhe chicken egg yolk peptide.

[0060] Comparative Example 11 The difference from Example 1 is that the marine Bacillus is replaced by Bacillus licheniformis in the preparation method of Naihe peptide.

[0061] Comparative Example 12 The difference from Example 1 is that the dextran gel LH-20 is replaced by dextran gel G-25 in the preparation method of Herba Epimedii extract.

[0062] Comparative Example 13 The difference from Example 1 is that the Herba Epimedii powder is boiled in 6 times the volume of water for 30 min, filtered, and the water extract is loaded into the dextran gel LH-20 column in the preparation method of Herba Epimedii extract.

[0063] Comparative Example 14 The difference from Example 1 is that the Herba Epimedii powder is percolated with 6 times the volume of ethanol to obtain a percolation extract, which is evaporated at 50°C under reduced pressure for 8 h to obtain Herba Epimedii extract in the preparation method of Herba Epimedii extract.

[0064] Comparative Example 15 The difference from Example 1 is that the Wuzhe chicken egg yolk freeze-dried powder is replaced by ordinary farmed white chicken egg yolk freeze-dried powder.

[0065] The replacement raw material sources involved in the above comparative examples: alkaline protease was from Ningxia Xiasen Industrial Group Co., Ltd., model FDG-2202, enzyme activity 200,000 U / g. Papain was from Ningxia Xiasen Industrial Group Co., Ltd., model FDG-2203, enzyme activity 100,000 U / g. Neutral protease (serratiopeptidase) was from Zhengzhou Weifeng Biological Technology Co., Ltd., enzyme activity 100,000 U / g. AB-8 macroporous adsorption resin was from Shanghai Yuanye Biological Technology Co., Ltd. D101 macroporous adsorption resin was from Shanghai Yuanye Biological Technology Co., Ltd. Phenyl sepharose gel column (CL-4B) was from Shanghai Huicheng Biological Technology Co., Ltd. Bacillus licheniformis was from Shanghai Guyan Industrial Co., Ltd., model GOY-4853. Dextran gel G-25 was from Shanghai Yuanye Biological Technology Co., Ltd.

[0066] I. Preparation of sample dilution: 0.1 g of the pharmaceutical composition of each example and comparative example was weighed, 10 mL of sterile PBS buffer was added, and vortexed for 10 min to fully dissolve, to obtain a mother liquor of 10 mg / mL. The mother liquor was diluted by 10 times and 100 times in turn with DMEM medium containing 10% fetal bovine serum to obtain a working solution of 1 mg / mL and 0.1 mg / mL, which was used for various tests.

[0067] II. Project testing: 1. Hemolysis rate test: to evaluate the damage of the drug to red blood cells, the lower the hemolysis rate, the higher the safety of the drug in the blood system.

[0068] Peripheral anticoagulated blood (EDTA anticoagulation) of healthy people was taken, washed with normal saline for 3 times, 2000 rpm centrifugation for 10 min each time, and the supernatant was discarded to obtain a red blood cell suspension. The red blood cell suspension was diluted with normal saline to 2% (v / v). 1 mg / mL of the working solution of the sample was mixed with an equal volume of the red blood cell suspension (500 μL / well each), added to a 24-well plate, 3 replicate wells were set for each sample, and a negative control group (normal saline + red blood cell suspension) and a positive control group (distilled water + red blood cell suspension) were also set. After incubation at 37°C with constant temperature and 100 rpm shaking for 1 h, centrifugation was performed at 3000 rpm for 10 min, and the supernatant was taken. The absorbance value A was measured at 540 nm wavelength by using a microplate reader, and the hemolysis rate was calculated: hemolysis rate (%) = (A experimental group - A negative control group) / (A positive control group - A negative control group) x 100%.

[0069] 2. Osteoblast proliferation test: CCK-8 method was used to determine the promoting effect of the pharmaceutical composition on the proliferation of hFOB 1.19 cells, and the higher the cell proliferation rate, the stronger the ability to promote cell proliferation.

[0070] hFOB 1.19 cells were seeded in a 96-well plate at a density of 3 x 10 3Cells were seeded per well in a 96-well plate and cultured for 24 h. The original culture medium was discarded, and 100 μL of 1 mg / mL sample working solution was added per well, with 5 replicates per sample. A blank control group (cell-free culture medium) and a negative control group (cells + culture medium, no drug) were also set up. After culturing at 37℃ and 5% CO2 for 72 h, the medium was replaced with fresh serum-free medium, i.e., 100 μL of fresh serum-free medium containing 10% CCK-8 was added to each well, and incubated for 2 h. The absorbance value A was measured at 450 nm using a microplate reader, and the cell proliferation rate was calculated as follows: Cell proliferation rate (%) = (A experimental group - A blank control group) / (A negative control group - A blank control group) × 100%.

[0071] 3. Alkaline phosphatase (ALP) activity assay: ALP is a key marker of osteoblast differentiation. By detecting its activity, we can assess the ability of drugs to promote osteoblast differentiation. The higher the activity, the better the differentiation promotion effect.

[0072] hFOB 1.19 cells were cultured at a concentration of 1×10⁻⁶. 5 Cells were seeded per well in a 24-well plate and cultured for 24 hours. The original culture medium was discarded, and 500 μL of 0.1 mg / mL sample working solution was added per well. Three replicates were set up for each sample. A blank control group (cell-free culture medium) and a negative control group (cells + culture medium, no drug) were also included. After 7 days of culture, the culture medium was discarded, and the cells were washed twice with PBS. 100 μL of cell lysis buffer (pH 7.4 PBS buffer containing 1% Triton X-100) was added to each well, and the cells were lysed on ice for 30 minutes. The cells were then centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected. Follow the instructions for the ALP detection kit (disodium p-nitrophenyl phosphate method). Measure the absorbance at 405 nm using an ELISA reader. Determine the total protein concentration (mg / mL) of the supernatant using the BCA method. Calculate the ALP concentration (U / L) based on the ALP standard curve. Calculate ALP activity: ALP activity (U / mg protein) = ALP concentration (U / L) / [protein concentration (mg / mL) × 1000]; 1000 is the unit conversion factor.

[0073] 4. Osteocalcin (OCN) secretion detection: OCN is an important non-collagenous protein in bone tissue. Its secretion is detected by enzyme-linked immunosorbent assay (ELISA). The higher the relative secretion of OCN, the better the effect of the drug in promoting bone formation.

[0074] hFOB 1.19 cells were cultured at a concentration of 1×10⁻⁶. 5Inoculate 1 cell / hole into 24-well plates and culture for 24 h. Discard the original culture medium and add 0.1 mg / mL sample working solution 500 μL / hole. Set 3 replicate holes for each sample, and set a negative control group (cells + culture medium without drugs). After 14 days of culture, collect the cell culture supernatant, centrifuge at 12,000 rpm for 10 min, and take the supernatant. According to the operation manual of the ELISA detection kit, measure the absorbance value at 450 nm wavelength on an enzyme label instrument, and obtain the absolute amount of OCN (ng / mL) from the standard curve (experimental group and negative control group). Calculate the relative secretion amount of OCN: relative secretion amount of OCN (%) = absolute amount of OCN in the experimental group (ng / mL) / absolute amount of OCN in the negative control group (ng / mL) x 100%.

[0075] Table 1. Detection data (interval value between parallel samples)

[0076] Note: Hemolysis rate: 0% hemolysis for the negative control (normal saline) and 100% hemolysis for the positive control (distilled water).

[0077] From the above results, it can be seen that the preparation method of each component of Comparative Examples 1 to 3 can obtain more effective and safe components, and the design of the component ratio can improve the synergistic effect, and each index is excellent.

[0078] Comparative Example 1: Adjustment of the ratio of five black chicken egg yolk peptide and mud clam peptide. Decreased osteogenesis activity: five black chicken egg yolk peptide is rich in active peptide segments that promote the proliferation and differentiation of osteoblasts, activate signal pathways, up-regulate the expression of transcription factors, promote cell cycle progression, and accelerate bone matrix synthesis. When the ratio of five black chicken egg yolk peptide decreases from 8 parts to 3 parts, the key active ingredients are insufficient, and the proliferation and differentiation of osteoblasts are weakened. Imbalance of synergistic effect: although mud clam peptide can provide amino acids and small molecule active substances to assist bone repair, excessive mud clam peptide can break the synergistic balance with egg yolk peptide. Some components in high-concentration mud clam peptide (including free cysteine) can compete with cell surface receptors for active sites of egg yolk peptide, inhibiting the bone formation effect of egg yolk peptide. In addition, metal ion chelating peptides contained in mud clam peptide can chelate trace elements (including zinc and copper) necessary for cell growth when in excess, leading to cell metabolic disorder. Reduced safety: five black chicken egg yolk peptide has a certain cell membrane protection effect, and the phospholipid peptide component contained therein stabilizes the cell membrane structure. After the ratio is reduced, the drug's ability to protect the red blood cell membrane is weakened, and the residual protease or incompletely purified protein fragments in mud clam peptide attack the red blood cell membrane.

[0079] Example 1: The proportion of Epimedium extract and Drynaria extract is adjusted. Signal pathway imbalance: the main active ingredients of Epimedium extract (including icariin) activate the estrogen receptor by mimicking the action of estrogen, upregulate the signal pathway, and promote osteoblast differentiation. However, excessive activation leads to excessive oxidative stress in cells, producing excessive reactive oxygen species (ROS), damaging cell DNA and mitochondrial function, and thus inhibiting cell proliferation. At the same time, the proportion of Drynaria extract is reduced, and the content of effective components (including naringin and protocatechuic acid) is reduced, leading to a decrease in the efficiency of osteogenic induction. Component interaction changes: icariin and the effective components in Drynaria extract (including flavonoids) have a synergistic antioxidant effect. After the proportion is imbalanced, the antioxidant capacity is weakened, and the accumulation of free radicals in cells affects the expression of cell cycle proteins. In addition, icariin has poor water solubility and is prone to form microcrystals when taken in excess, increasing mechanical damage to the red blood cell membrane.

[0080] Example 3: The proportions of multiple components are simultaneously adjusted. Core active component loss: the proportion of Wujhei egg yolk peptide and Drynaria extract is greatly reduced, directly reducing the key signal molecules that promote bone formation, leading to the inability of osteoblasts to effectively start the proliferation and differentiation program, and a significant decrease in cell proliferation rate and ALP activity. Metabolic disorder and increased toxicity: the overuse of Naidiao peptide and Epimedium extract, the accumulation of the amino acid metabolites (including ammonia) of the former exceeds the cell's metabolic capacity, causing an imbalance in the intracellular environment pH; the increase in icariin crystals and impurities in the latter increases drug toxicity. Synergistic network collapse: the multi-effect synergy of the drug composition depends on the proportional contribution of each component. Proportional chaos makes it impossible for various signal pathways to be activated synergistically, and the secretion of osteocalcin (OCN) drops sharply.

[0081] Example 4: In the preparation of Wujhei egg yolk peptide, trypsin is replaced by alkaline protease. Active peptide segment structure changes: trypsin specifically cleaves lysine and arginine carboxyl-terminal peptide bonds, etc., and can accurately release the bone formation-promoting active peptides (including adhesion peptides) in Wujhei egg yolk. The alkaline protease has a wide range of action sites, and the sequence integrity and conformation of the active peptide segments in the enzymatic products are destroyed, leading to a decrease in the binding ability of the active peptides to the integrin receptors of osteoblasts, a decrease in cell proliferation rate, and a decrease in OCN secretion. Accumulation of byproducts: alkaline protease over-enzymolysis produces a large amount of short peptide impurities. These byproducts compete with active peptides for cell transport proteins, reducing the effective uptake of active peptides. In addition, short peptide byproducts interfere with normal metabolic pathways in cells.

[0082] Comparative Example 5: In the preparation of Wuhai chicken egg yolk peptides, fig protease was replaced with papain. Insufficient extraction of specific active ingredients: Fig protease specifically degrades anti-nutritional factors in egg yolk and releases encapsulated angiogenic peptides. Although papain is also a cysteine ​​protease, its degradation efficiency for egg yolk high-phosphoprotein is low, leading to residual anti-nutritional factors. These substances bind to receptors on the surface of osteoblasts, competitively inhibiting the action of active peptides, thus reducing cell proliferation rate and ALP activity. Increased immunogenicity: Papain has poor specificity, generating new antigenic epitopes during enzymatic hydrolysis, triggering an immune response. These antigenic peptides bind to proteins on the erythrocyte membrane, forming antigen-antibody complexes, activating the complement system, and causing erythrocyte lysis. Simultaneously, the immune response consumes cellular energy, indirectly affecting osteoblast function.

[0083] Comparative Example 6: No fig protease was added during the preparation of Wuhai chicken egg yolk peptides. Anti-nutritional factor inhibition: Egg yolk high-phosphorus protein has a strong metal ion chelating ability. When not degraded, it chelates into calcium and magnesium ions essential for osteocytes, inhibiting the metal ion-assisted catalysis of the alkaline phosphatase (ALP) active site, leading to a decrease in ALP activity. Furthermore, egg yolk high-phosphorus protein binds to anion channels on the cell membrane, altering the membrane potential, affecting the cell's absorption of nutrients, and inhibiting cell proliferation.

[0084] Comparative Example 7: Bromelain was replaced with neutral protease in the preparation of Wuhai chicken egg yolk peptides. Reduced bioavailability of active peptides: Bromelain contains various thiol proteases that can specifically hydrolyze the triple helix structure in egg yolk collagen, releasing peptides with pro-angiogenic and cell adhesion functions. Neutral protease has poor substrate specificity, resulting in reduced yields of these functional peptides in the enzymatic hydrolysis products. This leads to insufficient vascular endothelial growth factor (VEGF)-like activity in the osteoblast microenvironment, affecting cell proliferation and differentiation. Peptide toxicity: Neutral protease produces certain hydrophobic peptides that readily insert into the cell membrane phospholipid bilayer, disrupting membrane fluidity and integrity, leading to increased erythrocyte membrane permeability.

[0085] Comparative Example 8: In the preparation of Wuhai chicken egg yolk peptides, the LX-68 macroporous adsorption resin column was replaced with AB-8 macroporous adsorption resin. Loss of active peptides: LX-68 resin is a polar adsorption resin with high selectivity for hydrophilic active peptides containing hydroxyl and carboxyl groups. AB-8 resin is a weakly polar resin with strong adsorption capacity for hydrophobic impurities, leading to the loss of hydrophilic active peptides during elution. These active peptides are key substances for activating osteoblast signaling pathways; their reduction directly leads to a decrease in cell proliferation rate and OCN secretion. Impact of residual impurities: After AB-8 resin adsorbs impurities, some impurities (including lipid-soluble pigments and incompletely enzymatically hydrolyzed protein fragments) are co-eluted with the active peptides. Although these impurities have low toxicity to erythrocytes, they can interfere with intracellular signal transduction in osteoblasts, affecting cell cycle regulation.

[0086] Comparative Example 9: In the preparation of Wuzi Yuzhi Peptide, the LX-68 macroporous adsorption resin column was replaced by D101 macroporous adsorption resin. Selective adsorption difference: D101 resin is a non-polar macroporous adsorption resin, and has weak adsorption force for the active ingredients in Wuzi Yuzhi Peptide, and is easy to adsorb fat-soluble macromolecular impurities. A large amount of active peptide segments are lost during adsorption-elution process, resulting in reduced bone formation activity. Membrane damage mechanism: Fat-soluble impurities (including long-chain fatty acid esters) remaining in the D101 resin eluate insert into the red blood cell membrane, resulting in increased hemolysis rate.

[0087] Comparative Example 10: In the preparation of Wuzi Yuzhi Peptide, the phenyl sepharose gel column (6FF high resolution) was replaced by phenyl sepharose gel column (CL-4B). Separation precision is reduced: the pore size of CL-4B is larger, and the separation precision is insufficient, resulting in co-elution of active peptide segments and impurities (including endotoxins and macromolecular protein aggregates), which affects the reduction of ALP activity. It reduces the affinity of osteoblast receptors, and further affects the secretion of OCN.

[0088] Comparative Example 11: In the preparation of Naihe Peptide, the marine Bacillus was replaced by Bacillus licheniformis. Enzyme system difference: The protease secreted by marine Bacillus is adapted to high-salt environment and specifically degrades myosin in Naihe meat to generate bone formation peptides, which can promote extracellular matrix mineralization. The alkaline protease secreted by Bacillus licheniformis has poor specificity in degrading myosin, producing a large amount of inactive or low-activity peptide segments, resulting in insufficient matrix components required for osteoblast proliferation and differentiation. Metabolic product toxicity: Lipopeptide substances (including surfactin) are produced during the fermentation of Bacillus licheniformis. These substances have strong cell membrane solubility, although they do not cause a significant increase in hemolysis rate, but they can destroy the lipid raft structure on the membrane of osteoblasts, affecting the aggregation of integrin receptors and signal transduction, and indirectly inhibiting the secretion of osteocalcin (OCN).

[0089] Comparative Example 12: In the preparation of Herba Epimedii extract, the dextran gel LH-20 was replaced by dextran gel G-25. Separation range mismatch: The separation range of dextran gel LH-20 is suitable for the purification of icariin; the separation range of G-25 has weak retention capacity for icariin, resulting in increased impurity content in the eluate. These impurities (including low-molecular-weight flavonoid aglycone and polysaccharide fragments) can compete with icariin for estrogen receptors on osteoblasts, reducing the efficiency of icariin-activated signaling pathways, and reducing cell proliferation rate and ALP activity. Active ingredient loss: Due to the poor separation effect of G-25 gel on icariin, part of icariin will be eluted early with impurities, resulting in insufficient content of active ingredients in the final extract, and the loss of active ingredients still affects the overall effect of promoting osteoblast proliferation of the drug.

[0090] Comparative Example 13: The extract of Epimedium was extracted by water boiling method. Active ingredient destruction: The glycosidic bond of icariin was hydrolyzed under high temperature (100°C) in the water boiling method, generating aglycone with lower activity. At the same time, high temperature also destroyed other heat-sensitive active ingredients (including flavonoid carbon glycosides) retained in the aqueous two-phase system extraction, resulting in a significant decrease in overall bone formation activity, cell proliferation rate, and ALP activity. Impurity introduction and toxicity increase: The water extraction method cannot effectively remove hydrophilic impurities (including polysaccharides, proteins), which will increase the osmotic pressure of the solution, leading to osmotic hemolysis of red blood cells.

[0091] Comparative Example 14: The extract of Drynaria was extracted by ethanol percolation method. Active ingredient extraction bias: The bone formation active ingredients of Drynaria (including naringin, protocatechuic acid) are water-soluble substances, while the ethanol percolation method mainly extracts fat-soluble components (including flavonoids, volatile oils). This method will result in a large loss of key water-soluble active ingredients, which cannot effectively activate the signal pathway, thereby inhibiting osteoblast differentiation, ALP activity, and OCN relative secretion. Organic solvent toxicity: High-concentration ethanol (70-80°C, still possible to remain after evaporation under reduced pressure) has direct toxicity to cells, which can damage the lipid structure of the cell membrane, leading to the rupture of the red blood cell membrane. At the same time, ethanol can interfere with the active center conformation of alkaline phosphatase in osteoblasts, reducing its catalytic efficiency; it can also inhibit the transcription process of osteocalcin genes in cells, further weakening the drug's promotion of osteoblast proliferation.

[0092] Comparative Example 15: Different types of chicken egg yolk. The protein quality is different, including content, type, peptide chain structure, and activity, resulting in some differences in product ingredients and effects.

Claims

1. A multi-effect synergistic pharmaceutical composition for bone repair, characterized in that, It includes five black chicken egg yolk peptides, mud clam peptides, epimedium extract, and drynaria extract, with a mass ratio of five black chicken egg yolk peptides: mud clam peptides: epimedium extract: drynaria extract = (8-10): (3-5): (0.5-1): (1-1.5). The Wuhai chicken egg yolk peptide is obtained by enzymatic hydrolysis of Wuhai chicken egg yolk freeze-dried powder with lipase and trypsin, followed by enzymatic hydrolysis with fig protease and bromelain to obtain the enzymatic hydrolysate, which is then filtered through a microfiltration membrane and purified sequentially through an LX-68 macroporous adsorption resin column and a phenyl agarose gel column, and ultrafiltration to obtain a freeze-dried product with a value between 500 Da and 5 kDa. The mud clam peptide is obtained by fermenting mud clam meat with marine Bacillus, followed by ultrasonic treatment and filtration. The filtrate is then subjected to electrodialysis to remove heavy metal ions and sodium ions, and ultrafiltration to obtain a freeze-dried product with a value between 1 kDa and 3 kDa. The Epimedium extract is the upper phase obtained by extracting Epimedium powder using a hydroxypropyl methylcellulose-tripotassium citrate aqueous two-phase system, and then purifying it using a dextran gel LH-20 column. The extract of Drynaria fortunei is obtained by enzymatic hydrolysis of Drynaria fortunei powder with cellulase, followed by extraction with sodium dioctyl succinate sulfonate-isooctane reverse micelle solution. The upper organic phase is taken, and sodium citrate aqueous solution is added and stirred to break the micelles. The lower aqueous phase is then taken and ultrafiltered to obtain a lyophilized product with a value between 500 Da and 5 kDa.

2. The multi-effect synergistic pharmaceutical composition for bone repair according to claim 1, characterized in that, The preparation method of the Wuhai chicken egg yolk peptide includes: diluting egg yolk freeze-dried powder with physiological saline to prepare egg yolk dilution, adding lipase and trypsin, adjusting the pH to 7.5-8.0, enzymatically hydrolyzing at 36℃-38℃ for 60-90 min, then raising the temperature to 50℃-55℃, adjusting the pH to 6.5-7, adding fig protease and bromelain, enzymatically hydrolyzing for 60-80 min, inactivating the enzymes by raising the temperature, centrifuging, collecting the supernatant, and loading it onto an LX-68 macroporous adsorption resin column. 0-1.5 BV is eluted with 5%-6% (v / v) ethanol aqueous solution, and 1.5-2.5 BV is eluted with 20%-25% (v / v) ethanol aqueous solution. The 1.5-2.5 BV eluent is collected, evaporated, and a concentrated solution is obtained. The concentrated solution is loaded onto a phenyl agarose gel column, and 0-0.5 BV is eluted with 1.4M-1.6M... Elute with (NH4)2SO4 aqueous solution. Elute 0.5–1.5 BV with 0.8 M–1 M (NH4)2SO4 aqueous solution. Elute 1.5–3 BV with 0.3 M–0.5 M (NH4)2SO4 aqueous solution. Collect the eluent of 1.5–3 BV, ultrafilter using an ultrafiltration membrane, and take the fraction between 500 Da and 5 kDa. Freeze-dry to obtain Wuhai chicken egg yolk peptide.

3. The multi-effect synergistic pharmaceutical composition for bone repair according to claim 2, characterized in that, The egg yolk freeze-dried powder is prepared from the egg yolk of Wuhai chickens with green shells; the amount of physiological saline is 8 to 10 times the mass of the egg yolk freeze-dried powder; the amount of lipase added is 0.5% to 1% of the mass of the egg yolk freeze-dried powder; the amount of trypsin added is 1% to 1.5% of the mass of the egg yolk freeze-dried powder; the amounts of figase and bromelain added are both 0.5% to 0.8% of the mass of the egg yolk freeze-dried powder; the stirring speed for enzymatic hydrolysis is 100 rpm to 150 rpm; the enzyme inactivation is carried out at 75℃ to 80℃ for 10 to 15 minutes; the centrifugation is carried out at 5000 rpm to 6000 rpm for 10 to 15 minutes; the supernatant is loaded at a flow rate of 1.5 to 2 BV / h; the evaporation is carried out at 35℃ to 40℃ under vacuum rotary evaporation to 20% to 30% of the volume; the concentrate is prepared at 1 to 1.5... The sample is loaded at a flow rate of BV / h; the height-to-diameter ratio of the LX-68 macroporous adsorption resin column is (4-5):1; the height-to-diameter ratio of the phenyl agarose gel column is (3-4):

1.

4. The multi-effect synergistic pharmaceutical composition for bone repair according to claim 1, characterized in that, The preparation method of the mud clam peptide includes: adding mud clam meat to 1 to 2 times its weight of sterile water, crushing and homogenizing to obtain a clam meat homogenate, adding 3 to 4 times its weight of sterile water to dilute, adjusting the pH to 7.2 to 7.5, inoculating with marine Bacillus for fermentation to obtain a fermentation broth; ultrasonically treating the fermentation broth to dissolve the active peptides; then filtering through a nylon mesh and a ceramic membrane to collect the filtrate; transferring the filtrate to an electrodialysis device for electrodialysis treatment to remove heavy metal ions and sodium ions; ultrafiltration of the electrodialysis solution through an ultrafiltration membrane to collect the components between 1 kDa and 3 kDa, freeze-drying to obtain the mud clam peptide.

5. The multi-effect synergistic pharmaceutical composition for bone repair according to claim 4, characterized in that, The mud clam meat is freshly washed mud clam meat; the crushing and homogenization is carried out at 6000rpm-8000rpm for 5-8 minutes; the inoculation amount of marine Bacillus is 3%-5% of the mud clam meat mass; the fermentation is carried out at 30℃-35℃ and 100rpm-120rpm with stirring for 48-52 hours; the ultrasonic treatment is carried out at 30℃-35℃ and 200rpm-300rpm with stirring, 200W-250W, and 30kHz-40kHz for 30-50 minutes; the nylon mesh is 200-250 mesh; the ceramic membrane has a pore size of 0.45μm; the electrodialysis device is equipped with anion and cation exchange membranes; the electrodialysis parameters are set as follows: operating voltage of 12V-15V and current density of 5mA / cm³. 2 ~10mA / cm 2 The processing time is 1.5h to 2h.

6. The multi-effect synergistic pharmaceutical composition for bone repair according to claim 1, characterized in that, The preparation method of the Epimedium extract includes: mixing hydroxypropyl methylcellulose:tripotassium citrate:water in a mass ratio of (6-8):(12-15):(85-90), stirring evenly to construct an aqueous two-phase system; adding Epimedium powder to the aqueous two-phase system in a material-to-liquid mass ratio of 1:(5-6), extracting, refrigerating and allowing to stand to separate the two phases, and taking the upper phase; loading the sample through a dextran gel LH-20 chromatography column, eluting 0-3 BV with 30%-35% volume concentration ethanol aqueous solution, collecting 1.5-2.5 BV of eluent, evaporating and concentrating, and lyophilizing to obtain the Epimedium extract.

7. The multi-effect synergistic pharmaceutical composition for bone repair according to claim 6, characterized in that, The Epimedium powder is passed through an 80-100 mesh sieve; the extraction is carried out at 55-60℃ and 80-100 rpm with stirring for 1-1.5 hours; the refrigeration and standing temperature is 4-6℃; the evaporation and concentration are carried out at 35-40℃ under vacuum rotary evaporation; the height-to-diameter ratio of the dextran gel LH-20 chromatography column is (4-5):

1.

8. The multi-effect synergistic pharmaceutical composition for bone repair according to claim 1, characterized in that, The preparation method of the *Drynaria fortunei* extract includes: adding *Drynaria fortunei* powder to deionized water, adding cellulase, enzymatic hydrolysis, heating to inactivate the enzyme, evaporating to remove water to obtain the enzymatic hydrolysate; dissolving sodium dioctyl succinate sulfonate in isooctane to prepare a reverse micelle solution with a concentration of 0.08 mol / L to 0.1 mol / L; adding the enzymatic hydrolysate to the reverse micelle solution at a material-to-liquid mass ratio of 1:(5-6), extracting, centrifuging, allowing to stand and separate into layers, and collecting the upper organic phase; adding sodium citrate aqueous solution to the organic phase, homogenizing at 7500 rpm to 8500 rpm for 8 min to 12 min to break the micelles, allowing to stand, centrifuging, allowing to stand and separate into layers, collecting the lower aqueous phase, ultrafiltration using an ultrafiltration membrane, taking the fraction between 500 Da and 5 kDa, lyophilizing to obtain the *Drynaria fortunei* extract.

9. A multi-effect synergistic pharmaceutical composition for bone repair according to claim 8, characterized in that, The *Drynaria fortunei* powder is sieved through a 100-120 mesh sieve; the amount of deionized water used is 4-6 times the mass of the *Drynaria fortunei* powder; the amount of cellulase added is 1%-3% of the mass of the *Drynaria fortunei* powder; the enzymatic hydrolysis is carried out at pH 4.5-5, 50-55℃, and 100-150 rpm for 3-4 hours with stirring; the enzyme inactivation is carried out at 85-90℃ for 10-15 minutes; the evaporation is carried out under vacuum at 35-40℃. The extraction process involved evaporation; extraction was performed at 30℃~35℃ and 120rpm~150rpm with shaking for 2h~2.5h; the concentration of the sodium citrate aqueous solution was 0.1mol / L~0.12mol / L; the amount of sodium citrate aqueous solution added was 10%~12% of the organic phase volume; centrifugation was performed at 4000rpm~5000rpm for 8min~12min; and settling was performed for 10min~15min.

10. A method for preparing a multi-effect synergistic pharmaceutical composition for bone repair as described in claim 1, characterized in that, The process includes the following steps: mixing five-black chicken egg yolk peptide, mud clam peptide, epimedium extract and drynaria extract in a certain mass ratio to obtain a multi-effect synergistic drug composition.

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