Composition for bone repair and growth and preparation method thereof

Abstract

The invention relates to a composition for bone repair and growth and a preparation method thereof, and belongs to the technical field of medicines, the composition comprises a glue layer extracted peptide in sandjellyfish and a tabasheer-grifola frondosa composite bacterial peptide; the extract peptide of the glue layer in the sand jellyfish contains a product between 500Da and 2000Da, wherein the product is obtained by sequentially carrying out reaction on the glue layer in the sand jellyfish through lumbrukinase, serrapeptase and glutamine transaminase; the tabasheer-grifola frondosa composite bacterial peptide contains a product between 500Da and 2000Da, which is obtained by performing common enzymolysis on tabasheer mushrooms and grifola frondosa mushrooms through cellulase and chitinase, then performing common enzymolysis on the tabasheer mushrooms and the grifola frondosa mushrooms through nattokinase and serrapeptase and finally performing common reaction on aminopeptidase and glutamine transaminase. According to the invention, a special method is adopted to prepare the glue layer extracted peptide and the tabasheer-grifola frondosa compound bacterium peptide in the sandfish, and the glue layer extracted peptide and the tabasheer-grifola frondosa compound bacterium peptide are matched for use to achieve good biological activity, stability and absorption and utilization rate, so that the defects of the existing skeleton repair and growth medicines are overcome, and the repair and growth of skeletons are effectively promoted.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a composition for bone repair and growth and a preparation method thereof. Background Art

[0002] Bone health is crucial for the human body, not only supporting the body structure but also participating in various physiological functions. However, affected by various factors, bone injuries and diseases are very common, such as fractures, osteoporosis, bone tumors, etc. These problems seriously affect the quality of life of patients and even endanger their lives. Therefore, the research and development of technologies and products for promoting bone repair and growth have always been the focus and hotspot in the fields of medicine and biomaterials.

[0003] Fracture is a common bone injury, and its healing is a complex physiological process, which is divided into the hematoma inflammation organization stage, the primary callus formation stage, and the bone plate formation and shaping stage. However, the self-repair ability of bones is limited and slow, often resulting in delayed healing or even non-healing of fractures in patients, leading to nonunion, which brings great pain to the physical and mental health of patients. Especially for elderly fracture patients, due to the decline in bone repair ability, it is very difficult to quickly reduce swelling, relieve pain and form callus at the fracture site only by early rehabilitation exercise after surgery. The swelling pain and long-term non-healing of callus in rehabilitation treatment make it difficult for many patients to adhere to rehabilitation treatment, further delaying the healing speed of the fracture site. Osteoporosis is a common disease affecting national health, and the repair of bone defects caused by its concurrent fractures, tumors, trauma, inflammation, etc. faces huge challenges. This disease will damage the osteogenic / angiogenic and bone remodeling abilities in the body, resulting in limited efficacy of traditional bone repair materials.

[0004] Currently, there are various products and methods for improving bone health in the market, such as calcium supplements, vitamin D, and specific drug therapies. However, these methods have limitations. Long-term use may produce side effects. For example, calcium supplements may cause gastrointestinal discomfort and an increased risk of stones; excessive vitamin D will cause poisoning; some drugs may affect liver and kidney functions after long-term use. Moreover, the absorption rate of these components is low, and the absorption effects vary greatly among different individuals, with limited applicability to specific populations. For example, people with certain diseases or special constitutions may not be able to use them or may have poor effects. To solve these problems, some studies have used tissue engineering technology to composite mesenchymal stem cells with scaffold materials and then perform osteogenic induction and differentiation. However, mesenchymal stem cells are easily regulated by the local microenvironment, and inflammatory cytokines will inhibit their differentiation into osteoblasts, affecting bone regeneration and repair, and there is a risk of tumor formation due to stem cell proliferation. There is also a method of percutaneous injection of bone growth factors. Although it has less trauma and a wide range of indications, the extraction and purification process of bone growth factors is complex, and they are easily inactivated during the purification, preservation, and use processes, with a short effective time, and continuous injection is required to take effect. With the in-depth study of the mechanism of bone repair and growth, people have found that some bioactive substances and materials have a positive effect on promoting bone repair and growth. Such as milk calcium, colostrum basic protein, etc., but they are also affected by absorption and preparation costs, and the utilization rate is not high.

[0005] In summary, there are many deficiencies in the existing methods and products for promoting bone repair and growth. Therefore, it is still necessary to continuously develop bone repair and growth drugs that are safe, efficient, and have a wide range of applications. Summary of the Invention

[0006] In view of the defects of existing bone repair and growth drugs, such as poor safety, absorption utilization rate, and stability, which affect the bone repair and growth effect, the present invention provides a composition for bone repair and growth and its preparation method. By using a special method to prepare the peptide extracted from the mesoglea of Stomolophus meleagris and the peptide of the compound bacteria of Daldinia concentrica and Grifola frondosa, and using them in combination, good biological activity, stability, and absorption utilization rate are achieved, overcoming the defects of existing bone repair and growth drugs and effectively promoting bone repair and growth. The specific technical solutions are as follows: A composition for bone repair and growth, the composition includes the peptide extracted from the mesoglea of Stomolophus meleagris and the peptide of the compound bacteria of Daldinia concentrica and Grifola frondosa; the peptide extracted from the mesoglea of Stomolophus meleagris contains the product between 500Da and 2000Da obtained by the sequential reaction of the mesoglea of Stomolophus meleagris with lumbrokinase, serrapeptase, and transglutaminase; the peptide of the compound bacteria of Daldinia concentrica and Grifola frondosa contains the product between 500Da and 2000Da obtained by first enzymatically hydrolyzing Daldinia concentrica and Grifola frondosa with cellulase and chitinase, then enzymatically hydrolyzing with nattokinase and serrapeptase, and finally reacting with aminopeptidase and transglutaminase.

[0007] Further, the preparation method of the peptide extracted from the mesoglea of Stomolophus meleagris includes the following steps: Take the dehydrated mesoglea of Stomolophus meleagris and crush it, add a phosphate buffer solution according to the mass ratio of the material to the liquid of 1:(10 - 15), and perform ultrasonic treatment to obtain an extract; add lumbrokinase to the extract, enzymatically hydrolyze at 37°C - 40°C for 2h - 2.5h, inactivate the enzyme by heating, cool down to 45°C - 50°C and keep it at a constant temperature, add serrapeptase, enzymatically hydrolyze for 1.5h - 2h, inactivate the enzyme by heating, cool down to 37°C - 40°C and keep it at a constant temperature, add transglutaminase, react for 1h - 1.5h, and inactivate the enzyme by heating; filter through a sieve, ultrafilter the filtrate through an ultrafiltration membrane, collect the components between 500Da and 2000Da, and freeze-dry to obtain the peptide extracted from the mesoglea of Stomolophus meleagris.

[0008] In the preparation method of the peptide extracted from the mesoglea of Stomolophus meleagris, the pH value of the phosphate buffer solution is 7.0 - 7.4; the power of the ultrasonic treatment is 200W - 300W, the temperature of the ultrasonic treatment is 30°C - 35°C, and the time of the ultrasonic treatment is 30min - 45min.

[0009] In the preparation method of the peptide extracted from the mesoglea of Stomolophus meleagris, the addition amount of lumbrokinase is 0.5% - 1.0% of the mass of the mesoglea of Stomolophus meleagris; the addition amount of serrapeptase is 0.8% - 1.5% of the mass of the mesoglea of Stomolophus meleagris; the addition amount of transglutaminase is 1.0% - 1.5% of the mass of the mesoglea of Stomolophus meleagris.

[0010] In the preparation method of the peptide extracted from the mesoglea of Rhopilema esculentum, the temperature for heat inactivation of enzymes is 85°C to 90°C, and the time for heat inactivation of enzymes is 10 min to 15 min; the mesh number of the sieve is 250 mesh to 325 mesh.

[0011] Furthermore, the preparation method of the Shiraia - Grifola frondosa compound peptide comprises the following steps: Mix and pulverize Shiraia bambusicola mushroom and Grifola frondosa mushroom according to the mass ratio of (3 - 4):(1 - 2) to obtain mushroom powder; Add an acetic acid - sodium acetate buffer solution according to the solid - liquid mass ratio of 1:(10 - 15), add cellulase and chitinase, perform ultrasonic enzymolysis, heat inactivate the enzymes, cool down to 45°C to 50°C for constant temperature, adjust the pH value to 7.0 - 7.5, add nattokinase and serrapeptase, enzymolyze for 2 h to 3 h, heat inactivate the enzymes, cool down to 40°C to 45°C, adjust the pH value to 7.5 - 8.0, add aminopeptidase and transglutaminase, react for 1.5 h to 2 h, heat inactivate the enzymes, filter through a sieve, ultrafilter the filtrate through an ultrafiltration membrane, collect the components between 500 Da and 2000 Da, and freeze - dry to obtain the Shiraia - Grifola frondosa compound peptide.

[0012] In the preparation method of the Shiraia - Grifola frondosa compound peptide, the pH value of the acetic acid - sodium acetate buffer solution is 5.0 - 5.5; the addition amount of cellulase is 1.0% - 1.5% of the mass of the mushroom powder; the addition amount of chitinase is 0.3% - 0.6% of the mass of the mushroom powder.

[0013] In the preparation method of the Shiraia - Grifola frondosa compound peptide, the ultrasonic power of ultrasonic enzymolysis is 250 W - 350 W, the temperature is 40°C - 45°C, and the time is 3 h - 4 h.

[0014] In the preparation method of the Shiraia - Grifola frondosa compound peptide, the addition amounts of both nattokinase and serrapeptase are 0.5% - 1.0% of the mass of the mushroom powder; the addition amount of aminopeptidase is 0.5% - 0.8% of the mass of the mushroom powder; the addition amount of transglutaminase is 0.5% - 1.0% of the mass of the mushroom powder.

[0015] In the preparation method of the Shiraia - Grifola frondosa compound peptide, the temperature for heat inactivation of enzymes is 85°C to 90°C, and the time for heat inactivation of enzymes is 10 min to 15 min; the mesh number of the sieve is 250 mesh to 325 mesh.

[0016] The preparation method of the above - mentioned composition for bone repair and growth comprises the following steps: Mix according to the mass ratio, peptide extracted from the mesoglea of Rhopilema esculentum: Shiraia - Grifola frondosa compound peptide=(15 - 20):(8 - 10) to obtain the composition.

[0017] The above composition for bone repair and growth is mixed with drugs and / or pharmaceutical excipients, sterilized, and then prepared into oral preparations or injections for bone repair and growth.

[0018] A composition for bone repair and growth provided by the present invention and its preparation method have the following beneficial effects: I. The mesoglea of Rhopilema esculentum is rich in collagen, mucopolysaccharides and bioactive peptides. Its three-dimensional fiber network structure is similar to the bone matrix, providing a bionic structural basis for bone repair. The present invention designs a stepwise enzymatic hydrolysis strategy: pretreatment with lumbrokinase opens the collagen fiber bundle through fibrinolytic action and specifically cuts it to expose the internal active sites; secondary enzymatic hydrolysis with serrapeptase specifically cuts the carboxyl terminus of hydrophobic amino acids (including leucine and phenylalanine) to release oligopeptide fragments with chemotactic effects; transglutaminase modification catalyzes the cross-linking of γ-glutamyl groups and lysine ε-amino groups to form a stable three-dimensional network structure and prolong the half-life of active peptides. Peptide segments with a molecular weight of 500 Da - 2000 Da have the best transmembrane transport efficiency and can effectively activate signal pathways to promote osteoblast differentiation.

[0019] II. The synergistic effect of the compound peptide of Daldinia concentrica - Grifola frondosa. Cellulase and chitinase are combined to break the cell wall of the mycelium synergistically, improving the release rate of intracellular active substances. The combination of nattokinase and serrapeptase, the former specifically cuts the carboxyl terminus of lysine and arginine, and the latter acts on hydrophobic sites to produce short peptides with pro-angiogenic activity. Aminopeptidase modification removes the N-terminal hydrophobic amino acid residues to improve water solubility and bioavailability. Transglutaminase modification catalyzes the cross-linking of γ-glutamyl groups and lysine ε-amino groups to form a stable three-dimensional network structure and prolong the half-life of active peptides. The obtained product can effectively regulate the balance between osteoclasts and osteoblasts, promote the absorption of components such as calcium ions, and improve the activation of osteoblasts driven by calcium influx; effectively regulate cell proliferation, differentiation and extracellular matrix synthesis, and promote fracture healing.

[0020] III. The synergistic mechanism of the reasonably proportioned composition: Rhopilema esculentum peptide activates pathways such as FAK / PI3K / Akt through integrin receptors, while mushroom peptide activates pathways such as cAMP / PKA through GPCR. Different pathways synergistically promote the expression of the Runx2 transcription factor. Some peptides in the mushroom peptide reverse the inhibitory effect of the inflammatory microenvironment on the osteogenic differentiation of mesenchymal stem cells. Rhopilema esculentum peptide provides sites (chelating through the carboxyl group of Glu / Gln residues with Ca² + ), and the components in the mushroom peptide inhibit the acidic secretion of osteoclasts, immunomodulate, have anti-inflammatory effects, and promote the absorption of components such as calcium ions. Through the synergistic effect of each component, good biological activity, stability and absorption utilization rate are achieved, overcoming the defects of existing drugs for bone repair and growth, and promoting bone repair and growth. Detailed implementation manners

[0021] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments.

[0022] Example 1: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the mesoglea of Rhopilema esculentum: the compound peptide of Shiraia bambusicola-Grifola frondosa = 15:8.

[0023] The preparation of the peptide extracted from the mesoglea of Rhopilema esculentum includes: taking the dehydrated mesoglea of Rhopilema esculentum and pulverizing it, adding a phosphate buffer solution with a pH value of 7.0 according to the mass ratio of material to liquid of 1:10, performing ultrasonic treatment at an ultrasonic power of 200 W and a temperature of 30 °C for 30 min to obtain an extract; adding 0.5% of the mass of the mesoglea of Rhopilema esculentum of earthworm kinase to the extract, enzymolyzing at 37 °C for 2 h, heat-inactivating the enzyme at 85 °C for 10 min, cooling to 45 °C and keeping it constant, adding 0.8% of the mass of the mesoglea of Rhopilema esculentum of serrapeptase, enzymolyzing for 1.5 h, heat-inactivating the enzyme at 85 °C for 10 min, cooling to 37 °C and keeping it constant, adding 1.0% of the mass of the mesoglea of Rhopilema esculentum of transglutaminase, reacting for 1 h, heat-inactivating the enzyme at 85 °C for 10 min; filtering through a 250-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze-drying.

[0024] The preparation of the compound peptide of Shiraia bambusicola-Grifola frondosa includes: mixing and pulverizing Shiraia bambusicola and Grifola frondosa according to the mass ratio of 3:1 to obtain a bacterial powder; adding an acetic acid-sodium acetate buffer solution with a pH value of 5.0 according to the mass ratio of material to liquid of 1:10, adding 1.0% of the mass of the bacterial powder of cellulase and 0.3% of the mass of the bacterial powder of chitinase, performing ultrasonic enzymolysis at an ultrasonic power of 200 W and a temperature of 40 °C for 3 h, heat-inactivating the enzyme at 85 °C for 10 min, cooling to 45 °C and keeping it constant, adjusting the pH value to 7.0, adding 0.5% of the mass of the bacterial powder of nattokinase and 0.5% of the mass of the bacterial powder of serrapeptase, enzymolyzing for 2 h, heat-inactivating the enzyme at 85 °C for 10 min, cooling to 40 °C, adding 0.5% of the mass of the bacterial powder of aminopeptidase and 0.5% of the mass of the bacterial powder of transglutaminase, reacting for 1.5 h, heat-inactivating the enzyme at 85 °C for 10 min, filtering through a 250-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze-drying.

[0025] Example 2: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the mesoglea of Rhopilema esculentum: the compound peptide of Shiraia bambusicola-Grifola frondosa = 15:10.

[0026] The preparation of peptides extracted from the mesoglea of Rhopilema esculentum includes: taking the dehydrated mesoglea of Rhopilema esculentum, crushing it, adding a phosphate buffer solution with a pH value of 7.4 according to the mass ratio of material to liquid of 1:10, performing ultrasonic treatment at an ultrasonic power of 200 W and a temperature of 35 °C for 30 min to obtain an extract; adding 1.0% of the mass of the mesoglea of Rhopilema esculentum of earthworm kinase to the extract, enzymatically hydrolyzing at 37 °C for 2.5 h, heat-inactivating the enzyme at 85 °C for 15 min, cooling to 45 °C and keeping it constant, adding 1.5% of the mass of the mesoglea of Rhopilema esculentum of serrapeptase, enzymatically hydrolyzing for 1.5 h, heat-inactivating the enzyme at 90 °C for 10 min, cooling to 40 °C and keeping it constant, adding 1.0% of the mass of the mesoglea of Rhopilema esculentum of transglutaminase, reacting for 1.5 h, heat-inactivating the enzyme at 85 °C for 15 min; filtering through a 250-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze-drying.

[0027] The preparation of the compound peptide of Daldinia concentrica-Grifola frondosa includes: mixing and crushing Daldinia concentrica and Grifola frondosa according to the mass ratio of 4:1 of Daldinia concentrica mushroom to Grifola frondosa mushroom to obtain a bacterial powder; adding an acetic acid-sodium acetate buffer solution with a pH value of 5.0 according to the mass ratio of material to liquid of 1:15, adding 1.5% of the mass of the bacterial powder of cellulase and 0.3% of the mass of the bacterial powder of chitinase, performing ultrasonic enzymatic hydrolysis at an ultrasonic power of 250 W and a temperature of 40 °C for 4 h, heat-inactivating the enzyme at 85 °C for 15 min, cooling to 45 °C and keeping it constant, adjusting the pH value to 7.5, adding 0.5% of the mass of the bacterial powder of nattokinase and 1.0% of the mass of the bacterial powder of serrapeptase, enzymatically hydrolyzing for 2 h, heat-inactivating the enzyme at 90 °C for 10 min, cooling to 45 °C, adding 0.5% of the mass of the bacterial powder of aminopeptidase and 1.0% of the mass of the bacterial powder of transglutaminase, reacting for 1.5 h, heat-inactivating the enzyme at 90 °C for 10 min, filtering through a 325-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze-drying.

[0028] Example 3: The mixing mass ratio of the composition for bone repair and growth is as follows: peptides extracted from the mesoglea of Rhopilema esculentum: compound peptide of Daldinia concentrica-Grifola frondosa = 17.5:9.

[0029] The preparation of peptides extracted from the mesoglea of Rhopilema esculentum includes: taking the dehydrated mesoglea of Rhopilema esculentum, crushing it, adding a phosphate buffer solution with a pH value of 7.2 according to the mass ratio of material to liquid of 1:12, performing ultrasonic treatment at an ultrasonic power of 250 W and a temperature of 32 °C for 40 min to obtain an extract; adding 0.8% of the mass of the mesoglea of Rhopilema esculentum of earthworm kinase to the extract, enzymatically hydrolyzing at 38 °C for 2 h, heat-inactivating the enzyme at 88 °C for 12 min, cooling to 48 °C and keeping it constant, adding 1.2% of the mass of the mesoglea of Rhopilema esculentum of serrapeptase, enzymatically hydrolyzing for 1.5 h, heat-inactivating the enzyme at 88 °C for 12 min, cooling to 38 °C and keeping it constant, adding 1.2% of the mass of the mesoglea of Rhopilema esculentum of transglutaminase, reacting for 1 h, heat-inactivating the enzyme at 88 °C for 12 min; filtering through a 325-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze-drying.

[0030] The preparation of Shiraia bambusicola - Grifola frondosa compound bacteriopeptide includes: mixing and pulverizing Shiraia bambusicola mushroom and Grifola frondosa mushroom according to the mass ratio of 3.5:1.5 to obtain a bacterial powder; adding an acetic acid - sodium acetate buffer solution with a pH value of 5.2 according to the mass ratio of material to liquid of 1:12, adding 1.2% of the mass of the bacterial powder of cellulase and 0.5% of the mass of the bacterial powder of chitinase, performing ultrasonic enzymatic hydrolysis at an ultrasonic power of 220 W and a temperature of 42°C for 3.5 h, inactivating the enzyme at 88°C for 12 min, cooling to a constant temperature of 48°C, adjusting the pH value to 7.2, adding 0.8% of the mass of the bacterial powder of nattokinase and 0.8% of the mass of the bacterial powder of serrapeptase, enzymatically hydrolyzing for 2.5 h, inactivating the enzyme at 88°C for 12 min, cooling to 43°C, adding 0.6% of the mass of the bacterial powder of aminopeptidase and 0.7% of the mass of the bacterial powder of glutamine transaminase, reacting for 1.5 h, inactivating the enzyme at 88°C for 12 min, filtering through a 325 - mesh sieve, subjecting the filtrate to ultrafiltration through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze - drying.

[0031] Example 4: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the mesoglea of Rhopilema esculentum: Shiraia bambusicola - Grifola frondosa compound bacteriopeptide = 20:9.

[0032] The preparation of the peptide extracted from the mesoglea of Rhopilema esculentum includes: taking the dehydrated mesoglea of Rhopilema esculentum for pulverization, adding a phosphate buffer solution with a pH value of 7.4 according to the mass ratio of material to liquid of 1:15, performing ultrasonic treatment at an ultrasonic power of 300 W and a temperature of 35°C for 45 min to obtain an extract; adding 1.0% of the mass of the mesoglea of Rhopilema esculentum of lumbrokinase to the extract, enzymatically hydrolyzing at 40°C for 2.5 h, inactivating the enzyme at 90°C for 15 min, cooling to a constant temperature of 50°C, adding 1.5% of the mass of the mesoglea of Rhopilema esculentum of serrapeptase, enzymatically hydrolyzing for 2 h, inactivating the enzyme at 90°C for 15 min, cooling to a constant temperature of 40°C, adding 1.5% of the mass of the mesoglea of Rhopilema esculentum of glutamine transaminase, reacting for 1.5 h, inactivating the enzyme at 90°C for 15 min; filtering through a 325 - mesh sieve, subjecting the filtrate to ultrafiltration through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze - drying.

[0033] The preparation of Shiraia bambusicola - Grifola frondosa compound bacteriopeptide includes: mixing and pulverizing Shiraia bambusicola mushroom and Grifola frondosa mushroom according to the mass ratio of 4:1.5 to obtain a bacterial powder; adding an acetic acid - sodium acetate buffer solution with a pH value of 5.5 according to the mass ratio of material to liquid of 1:15, adding 1.5% of cellulase based on the mass of the bacterial powder and 0.6% of chitinase based on the mass of the bacterial powder, performing ultrasonic enzymatic hydrolysis at an ultrasonic power of 250 W and a temperature of 45 °C for 4 h, inactivating the enzyme at 90 °C for 15 min, cooling to 50 °C and keeping it constant, adjusting the pH value to 7.5, adding 1.0% of nattokinase based on the mass of the bacterial powder and 1.0% of serrapeptase based on the mass of the bacterial powder, performing enzymatic hydrolysis for 3 h, inactivating the enzyme at 90 °C for 15 min, cooling to 45 °C, adding 0.8% of aminopeptidase based on the mass of the bacterial powder and 1.0% of transglutaminase based on the mass of the bacterial powder, reacting for 2 h, inactivating the enzyme at 90 °C for 15 min, filtering through a 325 - mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze - drying.

[0034] Example 5: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the mesoglea of Rhopilema esculentum: Shiraia bambusicola - Grifola frondosa compound bacteriopeptide = 20:8.

[0035] The preparation of the peptide extracted from the mesoglea of Rhopilema esculentum includes: pulverizing the dehydrated mesoglea of Rhopilema esculentum, adding a phosphate buffer solution with a pH value of 7.0 according to the mass ratio of material to liquid of 1:15, performing ultrasonic treatment at an ultrasonic power of 300 W and a temperature of 30 °C for 45 min to obtain an extract; adding 0.5% of lumbrokinase based on the mass of the mesoglea of Rhopilema esculentum to the extract, performing enzymatic hydrolysis at 40 °C for 2 h, inactivating the enzyme at 90 °C for 10 min, cooling to 50 °C and keeping it constant, adding 0.8% of serrapeptase based on the mass of the mesoglea of Rhopilema esculentum, performing enzymatic hydrolysis for 2 h, inactivating the enzyme at 85 °C for 15 min, cooling to 37 °C and keeping it constant, adding 1.5% of transglutaminase based on the mass of the mesoglea of Rhopilema esculentum, reacting for 1 h, inactivating the enzyme at 90 °C for 10 min; filtering through a 325 - mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze - drying.

[0036] The preparation of Shiraia bambusicola - Grifola frondosa compound bacteriopeptide includes: mixing and pulverizing Shiraia bambusicola mushroom and Grifola frondosa mushroom according to the mass ratio of 3:2 to obtain bacterial powder; adding an acetic acid - sodium acetate buffer solution with a pH value of 5.5 according to the mass ratio of material to liquid of 1:10, adding 1.0% of cellulase based on the mass of the bacterial powder and 0.6% of chitinase based on the mass of the bacterial powder, performing ultrasonic enzymolysis at an ultrasonic power of 200 W and a temperature of 45 °C for 3 h, inactivating the enzyme at 90 °C for 10 min, cooling to 50 °C for constant temperature, adjusting the pH value to 7.0, adding 1.0% of nattokinase based on the mass of the bacterial powder and 0.5% of serrapeptase based on the mass of the bacterial powder, enzymolyzing for 3 h, inactivating the enzyme at 85 °C for 15 min, cooling to 40 °C, adding 0.8% of aminopeptidase based on the mass of the bacterial powder and 0.5% of transglutaminase based on the mass of the bacterial powder, reacting for 2 h, inactivating the enzyme at 85 °C for 15 min, filtering through a 250 - mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting the components between 500 Da and 2000 Da, and freeze - drying.

[0037] The composition for bone repair and growth prepared in each of the above - mentioned examples is mixed with a drug and / or pharmaceutical excipient, sterilized, and then an oral preparation or an injection is prepared for bone repair and growth.

[0038] In each of the above - mentioned examples, the enzyme activity of lumbrokinase is 20,000 U / g, which is sourced from Xi'an Muguo Biotechnology Co., Ltd. The enzyme activity of serrapeptase is 200,000 U / g, which is sourced from Shenzhen Hengsheng Biotechnology Co., Ltd. The enzyme activity of transglutaminase is 100,000 U / g, which is sourced from Shenzhen Hengsheng Biotechnology Co., Ltd. The enzyme activity of cellulase is 100,000 U / g, which is sourced from Shandong Yaqiu Biotechnology Co., Ltd. The enzyme activity of chitinase is 100,000 U / g, which is sourced from Guangdong Yuanfeng Chemical Reagent Co., Ltd. The enzyme activity of nattokinase is 40,000 U / g, which is sourced from Shaanxi Yunhe Biotechnology Co., Ltd. The enzyme activity of aminopeptidase is 20,000 U / g, which is sourced from Shandong Xinxiong Biotechnology Co., Ltd.

[0039] Comparative Example 1 The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the mesoglea of Rhopilema esculentum: Shiraia bambusicola - Grifola frondosa compound bacteriopeptide = 8:15. Other methods and parameters are the same as those in Example 1.

[0040] Comparative Example 2 The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the mesoglea of Rhopilema esculentum: Shiraia bambusicola - Grifola frondosa compound bacteriopeptide = 15:4. Other methods and parameters are the same as those in Example 1.

[0041] Comparative Example 3 The mesoglea of Rhopilema esculentum is replaced with the mesoglea of Stomolophus meleagris; other methods and parameters are the same as those in Example 1.

[0042] Comparative Example 4 In the preparation of Shiraia bambusicola - Grifola frondosa compound bacteriopeptide: mixing and pulverizing Shiraia bambusicola mushroom and Grifola frondosa mushroom according to the mass ratio of 1:3; other methods and parameters are the same as those in Example 1.

[0043] Comparative Example 5 In the preparation of peptides extracted from the mesoglea of Rhopilema esculentum: nattokinase was used to replace lumbrokinase; other methods and parameters were the same as in Example 1.

[0044] Comparative Example 6 In the preparation of peptides extracted from the mesoglea of Rhopilema esculentum: serrapeptase was used to replace nattokinase; other methods and parameters were the same as in Example 1.

[0045] Comparative Example 7 In the preparation of peptides extracted from the mesoglea of Rhopilema esculentum: no transglutaminase was added for the reaction; other methods and parameters were the same as in Example 1.

[0046] Comparative Example 8 In the preparation of the peptide of Shiraia bambusicola-Grifola frondosa complex: no transglutaminase was added for the reaction; other methods and parameters were the same as in Example 1.

[0047] Comparative Example 9 In the preparation of the peptide of Shiraia bambusicola-Grifola frondosa complex: no aminopeptidase was added for the reaction; other methods and parameters were the same as in Example 1.

[0048] Comparative Example 10 In the preparation of the peptide of Shiraia bambusicola-Grifola frondosa complex: neither aminopeptidase nor transglutaminase was used for the reaction; other methods and parameters were the same as in Example 1.

[0049] Comparative Example 11 In the preparation of the peptide of Shiraia bambusicola-Grifola frondosa complex: both nattokinase and serrapeptase were replaced by bromelain (the enzyme activity of bromelain was 200,000 U / g); other methods and parameters were the same as in Example 1.

[0050] I. Safety Detection: 1. Acute toxicity test: Animal model: ICR mice (5 males and 5 females, 10 mice in each group); Dose design: single intraperitoneal injection at 5 mg / kg, 10 mg / kg, 20 mg / kg; Blank control group: injected with an equal volume of normal saline. Observe and detect poisoning phenomena. 2. Subacute toxicity test: Animal model: SD rats (5 males and 5 females, 10 rats in each group); Daily intramuscular injection: 4 mg / kg for 28 consecutive days; Observe and detect poisoning phenomena. 3. Allergic reaction test (guinea pig maximization test): Guinea pigs (male, 6 in each group) were subcutaneously injected with the composition (5 mg / kg) on days 0 and 7; On day 21, the composition (1 mg / kg) was injected into the ear vein, and allergic phenomena were observed and detected. 4. Genotoxicity test (Ames test): Strains: TA97, TA98, TA100, TA1535 (+ / - S9 metabolic activation); Dose: 1.0 mg / plate; Judgment criterion: The number of revertant colonies ≥ 2 times that of the negative control is positive. The compositions of each example and each comparative example were non-toxic and had no allergic effect after the above safety tests, and the Ames test was negative (no mutagenicity).

[0051] II. Mechanism research: 1. Signal pathway activation experiment: 1.1 Detection of FAK / PI3K / Akt pathway (effect of jellyfish peptide): Detection method: Western Blot.

[0052] Steps: Cell model: mouse mesenchymal stem cells (C3H10T1 / 2).

[0053] Grouping and treatment: Control group: serum-free medium; Jellyfish peptide group (150 μg / mL); Combined group (jellyfish peptide 150 μg / mL + mushroom peptide 80 μg / mL); Inhibitor control group: jellyfish peptide 150 μg / mL + PI3K inhibitor LY294002 (10 μM).

[0054] Treatment time: 60 min.

[0055] Protein extraction: Total protein was extracted with RIPA lysis buffer.

[0056] Detection targets: Phosphorylated FAK (p-FAK Tyr397).

[0057] Phosphorylated PI3K p85 (Tyr458).

[0058] Phosphorylated Akt (Ser473).

[0059] Antibody dilution ratio: 1:1000 (phosphorylated antibody), 1:2000 (total protein antibody).

[0060] 1.2 Detection of cAMP / PKA pathway (effect of mushroom peptide): Detection method: ELISA + Western Blot.

[0061] Steps: Cell model: Mouse mesenchymal stem cells (C3H10T1 / 2).

[0062] Grouping and treatment: Control group: Serum-free medium; Mushroom peptide group (80 μg / mL); Combined group (150 μg / mL of jellyfish peptide + 80 μg / mL of mushroom peptide); Inhibitor control group: 80 μg / mL of mushroom peptide + PKA inhibitor H89 (10 μM); cAMP detection: After 30 min of treatment, the intracellular cAMP level was detected using an ELISA kit.

[0063] PKA detection: Phosphorylated PKA substrate (CREB Ser133) was detected by Western Blot.

[0064] 2 Runx2 transcriptional regulation experiment: 2.1 Expression of Runx2 protein and mRNA: Detection method: qRT-PCR + Western Blot.

[0065] Steps: Cells and treatment: Mouse mesenchymal stem cells (C3H10T1 / 2), treated for 48 h.

[0066] Grouping and treatment: Control group: Serum-free medium; Jellyfish peptide group (150 μg / mL); Mushroom peptide group (80 μg / mL); Combined group (150 μg / mL of jellyfish peptide + 80 μg / mL of mushroom peptide); qRT-PCR: Primer: Runx2 F: 5'-CCGCACGACAACCGCACCAT-3', R: 5'-CGCTCCGGCCCACAAATCTC-3'.

[0067] Internal reference: GAPDH.

[0068] Western Blot: Anti-Runx2 antibody (1:1000).

[0069] 2.2 Runx2 transcriptional activity: Detection method: Dual-luciferase reporter system.

[0070] Steps: Plasmid transfection: Cells were transfected with a luciferase reporter plasmid driven by the Runx2 response element (OSE2).

[0071] Treatment: The grouping was the same as before, and the treatment lasted for 24 h.

[0072] Detection: Luciferase activity (Firefly / Renilla ratio).

[0073] 3 Regulation of the inflammatory microenvironment (effect of mushroom peptide): 3.1 Inhibition of inflammatory factor secretion: Detection method: LPS-induced inflammation model + ELISA.

[0074] Steps: Cell model: Macrophages (RAW264.7) were co-cultured with hBMSCs.

[0075] Inflammation induction: Macrophages were stimulated with LPS (1 μg / mL) for 24 h.

[0076] Treatment groups: LPS group; LPS + mushroom peptide group (80 μg / mL); LPS + combined peptide group (150 μg / mL of jellyfish peptide + 80 μg / mL of mushroom peptide).

[0077] Detection: ELISA was used to detect the levels of TNF-α and IL-6 in the supernatant.

[0078] 3.2 Osteogenic differentiation rescue experiment: Detection method: ALP staining + qRT-PCR.

[0079] Steps: Co-culture system: LPS-activated macrophages were co-cultured with hBMSCs in Transwell.

[0080] Treatment: A peptide combination (150 μg / mL of jellyfish peptide + 80 μg / mL of mushroom peptide) was added to the co-culture system, and osteogenic induction was performed for 7 days.

[0081] Detection: ALP activity (BCIP / NBT staining).

[0082] mRNA expression of osteogenic genes (ALP, OCN).

[0083] 4 Calcium metabolism regulation: 4.1 Calcium ion chelating ability (effect of jellyfish peptide): Detection method: Pyrene red colorimetry (Calcium Binding Assay).

[0084] Steps: Sample: Jellyfish peptide solution (1 mg / mL).

[0085] Control: BSA control group (1 mg / mL).

[0086] Reaction: Add CaCl2 (final concentration 2 mM) and incubate at 37 °C for 30 min.

[0087] Detection: Add pyrene red (50 μM) and measure the absorbance at OD540 nm.

[0088] 4.2 Promotion of mineralized nodule formation: Detection method: Alizarin red staining.

[0089] Steps: Cells and treatment: hBMSCs were osteogenically induced for 21 days and grouped as follows: BSA control group (1 mg / mL), jellyfish peptide group (1 mg / mL), combined group (peptide combination 15:8; 1 mg / mL).

[0090] Staining: Fix with 4% PFA and stain with 2% alizarin red (pH 4.2) for 30 min.

[0091] Quantification: Dissolve the dye with 10% CPC and measure OD562 nm.

[0092] Table 1 Detection results (5 parallel samples)

[0093] Explanation of key results: 1. Pathway synergy: FAK / PI3K / Akt pathway: The expression of p-Akt in the combined group was 4 times that of the control group (3 times for jellyfish peptide alone), and LY294002 completely inhibited the activation. cAMP / PKA pathway: The cAMP level in the combined group was 3.8 times that of the control group (3 times for mushroom peptide alone), and p-CREB was upregulated synchronously.

[0094] 2. Runx2 cascade amplification: The combined treatment increased the transcriptional activity (luciferase) of Runx2 to 4 times and the mRNA to more than 3 times, significantly higher than that of the single peptide group (p < 0.05).

[0095] 3. Inflammatory reversal ability: Mushroom peptide reduced the secretion of TNF-α induced by LPS by 60% (*p < 0.01), and under combined treatment, the osteogenic differentiation markers (ALP, OCN) were restored to 80% of the normal level.

[0096] 4. Dual pathways of calcium metabolism: The Ca 2+ chelation rate of jellyfish peptide reached 65% (*p < 0.01 vs BSA); the amount of mineralized nodules formed in the combined group increased by 250%, confirming the synergistic effect on bone mineralization.

[0097] Note: 1. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using one-way ANOVA and Tukey's test (*p < 0.05, **p < 0.01, ***p < 0.001); 2. NS: no significant difference; ↑ / ↓: fold increase or percentage decrease compared to the control group; 3. The inhibitor control group was used to verify pathway specificity (e.g., p-Akt did not increase after inhibition of PI3K by LY294002). 4. In the above detections, the combined group was always the jellyfish peptide and mushroom peptide at a mass ratio of 15:8; the drug components involved above were all obtained by the preparation method of Example 1.

[0098] III. Animal experiments for bone repair and growth detection: Experimental animals: 102 healthy SD rats at 8 weeks of age (half male and half female), with a body weight of 200 g - 260 g. After 1 week of adaptive feeding, the experiment was carried out.

[0099] Fracture model construction: The rats were anesthetized by intraperitoneal injection of 3 wt% sodium pentobarbital at 30 mg / kg, and a closed fracture model was created at the right forelimb humerus.

[0100] Drug administration: The rats were randomly divided into 17 groups (1 blank control group, 5 experimental groups of examples, and 11 experimental groups of comparative examples), with 6 rats in each group (half male and half female). The rats in the experimental groups were intramuscularly injected with the corresponding composition solution (5 mg / mL) around the fracture site, and the injection dose was 0.2 mL / time. The blank control group was injected with an equal volume of PBS buffer. Then, the drug was injected once every 2 days. The rats were continuously fed, and vitamin C, zinc gluconate and other nutritional components and trace elements were normally supplemented in the feed.

[0101] Experimental detection: On the 0th, 14th, 28th, and 42nd days of feeding, a bone densitometer was used to measure the bone density of the right forelimb humerus fracture site of the rats. On the 15th, 18th, 21st, 24th, 27th, 30th, 33rd, 36th, 39th, and 42nd days of feeding, a transmission examination was performed on the right forelimb of the rats to judge the fracture healing situation. When the fracture line was blurred and continuous callus passed through the fracture line, it was determined that the fracture had healed, and the fracture healing time was recorded. The results are shown in Table 2 below.

[0102] Table 2 Bone repair and growth detection results (mean values)

[0103] From the above results, it can be seen that the combinations of Examples 1 to 5 have a better effect on promoting bone repair and growth, and the fracture healing time is effectively shortened.

[0104] In Comparative Example 1, the relative content of the peptide extracted from the mesoglea of Rhopilema esculentum is relatively low, and it cannot form the best synergistic effect with the peptide of the compound bacteria of Daldinia concentrica and Grifola frondosa. Bone repair is a complex process involving multiple cellular activities and signaling pathways. The two types of bioactive peptides act on different links respectively (including the peptide extracted from the mesoglea of Rhopilema esculentum promoting the proliferation of osteoblasts, and the peptide of the compound bacteria of Daldinia concentrica and Grifola frondosa regulating the synthesis of the extracellular matrix of bone cells). When the peptide extracted from the mesoglea of Rhopilema esculentum is insufficient, the proliferation rate of osteoblasts is limited, affecting the formation of new bone tissue, resulting in slow bone density growth and prolonged fracture healing time.

[0105] In Comparative Example 2, there is too little peptide of Daldinia concentrica and Grifola frondosa, and the anti-inflammatory peptide it provides is insufficient, and it cannot effectively inhibit the interference of the local inflammatory microenvironment on osteogenic differentiation. The synthesis of the extracellular matrix of bone cells is inhibited, and it cannot provide a good attachment and growth environment for osteoblasts, affecting the osteogenic process. At the same time, it cannot efficiently regulate the cytokine balance in the local microenvironment, which is not conducive to the subsidence of the inflammatory response and tissue repair during the fracture healing process, resulting in insignificant bone density increase and delayed fracture healing.

[0106] In Comparative Example 3, the collagen type in the mesoglea of Stomolophus meleagris is different, and the content of the peptide promoting callus formation generated after enzymatic hydrolysis is significantly reduced. There are differences in the chemical composition between the mesoglea of Stomolophus meleagris and the mesoglea of Rhopilema esculentum, and these differences will lead to differences in the amino acid composition, sequence and spatial structure of the extracted peptides. And the structure of the peptide determines its function, including the binding ability to osteogenic-related receptors. The peptide extracted from the mesoglea of Stomolophus meleagris is difficult to effectively activate the relevant signaling pathways in osteoblasts (including the mitogen-activated protein kinase (MAPK) signaling pathway, and the peptide of Rhopilema esculentum activates the FAK / PI3K / Akt pathway through the integrin α2β1 receptor) like the peptide extracted from the mesoglea of Rhopilema esculentum, and these pathways are crucial for the proliferation, differentiation and survival of osteoblasts. It affects the increase of bone density and the fracture healing rate.

[0107] In Comparative Example 4, the mass ratio of Daldinia concentrica to Grifola frondosa changes to 1:3, changing the component composition of the compound bacteria peptide. Peptides produced by different mushrooms have different biological activities and play different roles in functions beneficial to bone repair such as promoting angiogenesis, regulating immune response, and antioxidant. For example, the peptide extracted from Daldinia concentrica has a significant effect on promoting the expression of vascular endothelial growth factor (VEGF) and activates the cAMP / PKA pathway through GPCR; while the peptide extracted from Grifola frondosa is more critical for the regulation of immune cells. When the ratio changes, the expression of VEGF and the expression of the Runx2 transcription factor are insufficient, the angiogenesis at the fracture site decreases, the supply of nutrients and oxygen is limited, the metabolism and function of osteoblasts are affected, and finally the bone density increases slowly and the fracture healing time is prolonged.

[0108] In Comparative Example 5, the earthworm kinase was replaced with nattokinase. Nattokinase has poor cleavage site specificity for collagen, resulting in a deviation in the molecular weight distribution of the generated peptides and changes in the peptide amino acid sequence, which affects its binding efficiency with integrin receptors. During the preparation of peptides extracted from the mesoglea of Rhopilema esculentum, earthworm kinase can specifically cleave certain peptide bonds to produce peptide segments with specific structures and activities. After being replaced by nattokinase, the structures and sequences of the enzymatically cleaved peptide segments change. These new peptide segments cannot effectively activate the osteogenesis-related signaling pathway, thereby affecting bone density growth and fracture healing.

[0109] In Comparative Example 6, serrapeptase was replaced with nattokinase. Serrapeptase has a unique three-dimensional substrate recognition ability and can release specific anti-inflammatory peptides; nattokinase cannot replace this function, resulting in insufficient local inflammation inhibition. Serrapeptase participates in the modification and generation of peptides at specific steps. It acts on specific amino acid residues of peptides extracted from the mesoglea of Rhopilema esculentum, affecting the final structure and function of the peptides. After being replaced by nattokinase, the unique modification function of serrapeptase cannot be completed, reducing the affinity of the extracted peptides for the receptors on the surface of osteoblasts and blocking intracellular signal transduction, which is not conducive to the differentiation of osteoblasts and the synthesis of bone matrix, resulting in poor bone density improvement and delayed fracture healing.

[0110] In Comparative Example 7, during the preparation of peptides extracted from the mesoglea of Rhopilema esculentum, transglutaminase was absent. The peptide cross-linking reaction mediated by transglutaminase was absent, resulting in a decrease in peptide chain stability, a shortened half-life, a decrease in hydrophilicity, a decrease in absorption and utilization rate, and a reduction in the effective action time. Transglutaminase catalyzes the cross-linking reaction between glutamine residues and lysine residues during peptide preparation to form stable isopeptide bonds. The absence of it causes the extracted peptides to be unable to form specific higher-order structures, affecting their biological activities. For example, it cannot effectively bind and activate insulin-like growth factor (IGF), which has an important promoting effect on osteoblast proliferation and bone matrix synthesis. After the activity of IGF is affected, the metabolic activities of osteoblasts slow down, bone density growth slows down, and the fracture healing time is prolonged.

[0111] In Comparative Example 8, during the preparation of the bamboo fungus-grifola frondosa complex bacterium peptide, transglutaminase was absent. Transglutaminase also participates in the formation of peptide segments with stable structures and functions. The lack of it makes the complex bacterium peptide structure unstable and unable to effectively regulate the balance between osteoclasts and osteoblasts. The functions of osteoblasts are restricted and cannot replenish new bone in time, affecting bone density improvement and fracture healing.

[0112] In the preparation of the Shiraia bambusicola - Grifola frondosa compound bacteriopeptide of Comparative Example 9, the absence of aminopeptidase led to the retention of N - terminal hydrophobic amino acids (including Leu, Phe), inhibited the interaction between the peptide and calcium ion channels (including Cav1.2), and reduced the activation of osteoblasts driven by calcium influx. After the absence of aminopeptidase, the amino acid sequence of the compound bacteriopeptide could not be correctly modified, affecting its recognition and binding to cell - surface receptors. This included the inability to activate the fibroblast growth factor (FGF) signaling pathway, resulting in a decrease in bone repair ability, slow growth of bone density, and delayed fracture healing.

[0113] In the preparation of the Shiraia bambusicola - Grifola frondosa compound bacteriopeptide of Comparative Example 10, the simultaneous absence of aminopeptidase and transglutaminase reactions led to a situation where the peptide chain could neither form a stable structure nor undergo correct amino acid modification, leaving a residual inhibitory N - terminal, which synergistically weakened the pro - bone - regeneration signaling pathway; during the fracture - healing process, it was unable to effectively regulate cell proliferation, differentiation, and extracellular matrix synthesis, and there was almost no significant increase in bone density, and the fracture - healing time was greatly prolonged.

[0114] Bromelain preferentially cleaved basic amino acids (including Lys, Arg), destroying the functional peptide segments, and the effect became worse. The enzymatic cleavage specificity and mechanism of action of bromelain were different from those of nattokinase and serrapeptase. Using bromelain produced a mixture of peptide segments different from the normal preparation process. These peptide segments had different charge distributions, spatial conformations, and biological activities. They were difficult to activate key intracellular signaling pathways related to bone repair, including the bone morphogenetic protein (BMP) signaling pathway; they could not efficiently promote the differentiation of bone cells and the reconstruction of bone tissue, resulting in slow growth of bone density and prolonged fracture - healing time.

Claims

1. A composition for bone repair and growth, characterized in that, The composition comprises a peptide extracted from the mesoglea of *Rhopilema esculentum* and a compound peptide of *Dendrobium catenatum* and *Grifola frondosa*; the peptide extracted from the mesoglea of *Rhopilema esculentum* contains products between 500 Da and 2000 Da obtained by successively reacting the mesoglea of *Rhopilema esculentum* with earthworm kinase, serrapeptase, and transglutaminase; the compound peptide of *Dendrobium catenatum* and *Grifola frondosa* contains products between 500 Da and 2000 Da obtained by first co-enzymatically hydrolyzing *Dendrobium catenatum* and *Grifola frondosa* with cellulase and chitinase, then co-enzymatically hydrolyzing with nattokinase and serrapeptase, and finally reacting with aminopeptidase and transglutaminase.

2. The composition for bone repair and growth according to claim 1, wherein The preparation method of the peptide extracted from the mesoglea of *Rhopilema esculentum* comprises the following steps: Take the dehydrated mesoglea of *Rhopilema esculentum*, crush it, add a phosphate buffer solution according to the mass ratio of material to liquid of 1:(10 - 15), perform ultrasonic treatment to obtain an extract; add earthworm kinase to the extract, enzymatically hydrolyze at 37°C - 40°C for 2 h - 2.5 h, heat inactivate the enzyme, cool to 45°C - 50°C for constant temperature, add serrapeptase, enzymatically hydrolyze for 1.5 h - 2 h, heat inactivate the enzyme, cool to 37°C - 40°C for constant temperature, add transglutaminase, react for 1 h - 1.5 h, heat inactivate the enzyme; filter through a sieve, ultrafilter the filtrate through an ultrafiltration membrane, collect the components between 500 Da and 2000 Da, and freeze-dry to obtain the peptide extracted from the mesoglea of *Rhopilema esculentum*.

3. The composition for bone repair and growth according to claim 2, characterized in that, The pH value of the phosphate buffer solution is 7.0 - 7.4; the power of the ultrasonic treatment is 200 W - 300 W, the temperature of the ultrasonic treatment is 30°C - 35°C, and the time of the ultrasonic treatment is 30 min - 45 min.

4. The composition for bone repair and growth according to claim 2, wherein The addition amount of the earthworm kinase is 0.5% - 1.0% of the mass of the mesoglea of *Rhopilema esculentum*; the addition amount of the serrapeptase is 0.8% - 1.5% of the mass of the mesoglea of *Rhopilema esculentum*; the addition amount of the transglutaminase is 1.0% - 1.5% of the mass of the mesoglea of *Rhopilema esculentum*.

5. The composition for bone repair and growth according to claim 1, characterized in that, The preparation method of the compound peptide of *Dendrobium catenatum* and *Grifola frondosa* comprises the following steps: Mix and crush *Dendrobium catenatum* and *Grifola frondosa* according to the mass ratio of (3 - 4):(1 - 2) to obtain a bacterial powder; add an acetic acid - sodium acetate buffer solution according to the mass ratio of material to liquid of 1:(10 - 15), add cellulase and chitinase, perform ultrasonic enzymatic hydrolysis, heat inactivate the enzyme, cool to 45°C - 50°C for constant temperature, adjust the pH value to 7.0 - 7.5, add nattokinase and serrapeptase, enzymatically hydrolyze for 2 h - 3 h, heat inactivate the enzyme, cool to 40°C - 45°C, add aminopeptidase and transglutaminase, react for 1.5 h - 2 h, heat inactivate the enzyme, filter through a sieve, ultrafilter the filtrate through an ultrafiltration membrane, collect the components between 500 Da and 2000 Da, and freeze-dry to obtain the compound peptide of *Dendrobium catenatum* and *Grifola frondosa*.

6. The composition for bone repair and growth according to claim 5, wherein The pH value of the acetic acid - sodium acetate buffer solution is 5.0 - 5.5; the addition amount of the cellulase is 1.0% - 1.5% of the mass of the bacterial powder; the addition amount of the chitinase is 0.3% - 0.6% of the mass of the bacterial powder.

7. A composition for bone repair and growth according to claim 5, characterized in that, The ultrasonic power of the ultrasonic enzymatic hydrolysis is 250 W - 350 W, the temperature is 40°C - 45°C, and the time is 3 h - 4 h.

8. A composition for bone repair and growth according to claim 5, characterized in that, The addition amounts of nattokinase and serrapeptase are both 0.5% - 1.0% of the mass of the bacterial powder; the addition amount of aminopeptidase is 0.5% - 0.8% of the mass of the bacterial powder; the addition amount of transglutaminase is 0.5% - 1.0% of the mass of the bacterial powder.

9. A composition for bone repair and growth according to claim 2 or 5, characterized in that, The temperature for heat inactivation of enzymes is 85°C - 90°C, and the time for heat inactivation of enzymes is 10 min - 15 min; the mesh number of the sieve is 250 mesh - 325 mesh.

10. A method for preparing the composition for bone repair and growth according to claim 1, characterized in that, It includes the following steps: According to the mass ratio, the peptide extracted from the mesoglea of Rhopilema esculentum: the peptide of the complex bacteria of Daldinia concentrica - Grifola frondosa = (15 - 20):(8 - 10), and they are mixed to obtain a composition.

Images