A compound type bone health composition containing astragalus polysaccharide and polypeptide, and a preparation method and application thereof

Abstract

The application provides a compound bone health composition containing astragalus polysaccharide and polypeptide and a preparation method and application thereof, and belongs to the technical field of biological medicine. The composition is prepared by compounding astragalus polysaccharide and collagen polypeptide, the astragalus polysaccharide is prepared by high-pressure pulse electric field combined with low eutectic solvent ultrasonic extraction, and the collagen polypeptide is prepared by compound microbial fermentation, step-by-step enzymolysis and purification. The astragalus polysaccharide and the collagen polypeptide have a significant synergistic effect, the astragalus polysaccharide has anti-inflammatory and antioxidant effects and optimizes the repair microenvironment, and the collagen polypeptide provides repair raw materials and promotes tissue regeneration. The composition can efficiently promote the repair of joint movement injury, can improve the chondrocyte activity, can remove active oxygen and can inhibit the secretion of inflammatory factors. Cell experiments and zebra fish animal experiments show that the composition has excellent repair effect, the bone health product prepared based on the composition also has good repair performance, and has important clinical value and market prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a composite bone-strengthening composition containing astragalus polysaccharides and polypeptides, its preparation method, and its application. Background Technology

[0002] Joints are key structural and functional units of the human musculoskeletal system, and their health directly affects an individual's athletic ability and quality of life. During sports and daily physical activities, joints, especially weight-bearing or frequently used joints such as the knees, ankles, shoulders, and elbows, are highly susceptible to sports injuries due to acute trauma or chronic strain. These injuries not only include structural damage such as cartilage wear, subchondral bone microfractures, and ligament tears, but are also often accompanied by complex inflammatory responses, oxidative stress, and metabolic imbalances within the joint cavity. If repair is not timely or appropriate, it can lead to decreased joint stability, chronic pain, functional impairment, and a significantly increased risk of developing osteoarthritis.

[0003] Currently, clinical interventions for joint sports injuries mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), physical therapy, intra-articular injections, and surgical repair. While these methods can alleviate symptoms to some extent, they have significant limitations: long-term use of medications can cause serious side effects; injection therapy may damage cartilage; and surgical treatment is highly invasive and time-consuming. Therefore, developing safe products that can promote active tissue repair and regulate the intra-articular environment is an urgent need in this field.

[0004] Bioactive ingredients have become a research hotspot due to their multi-target effects and high safety. Astragalus polysaccharides, extracted from the rhizome of Astragalus membranaceus, have effects such as regulating blood pressure, protecting the liver, and enhancing immunity; collagen peptides have effects such as improving skin elasticity, accelerating wound healing, and improving hair quality.

[0005] However, there are currently no technical solutions for combining astragalus polysaccharides with collagen peptides for bone health, nor are there efficient preparation processes for such compositions. Therefore, developing a safe, efficient, and multi-target synergistic compound bone health composition containing astragalus polysaccharides and collagen peptides has significant clinical value and market potential. Summary of the Invention

[0006] The purpose of this invention is to provide a composite bone-strengthening composition containing astragalus polysaccharides and polypeptides, its preparation method, and its application. The composite bone-strengthening composition provided by this invention has anti-inflammatory and antioxidant properties and effectively promotes cartilage repair and regeneration.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a composite bone-strengthening composition containing astragalus polysaccharide and polypeptide, comprising the following raw materials in parts by weight: 1-3 parts of astragalus polysaccharide and 1-3 parts of collagen peptide.

[0009] Preferably, the extraction method of Astragalus polysaccharide is as follows: Astragalus is mixed with water and extracted by high voltage pulse electric field. After the reaction system is concentrated, a eutectic solvent is added for ultrasonic extraction. After filtration, anhydrous ethanol is added to the filtrate, the mixture is allowed to stand, centrifuged, washed, and dried to obtain Astragalus polysaccharide.

[0010] More preferably, the material-to-liquid ratio extracted by the high-voltage pulsed electric field is 1:5-10 g / mL, the electric field strength is 15-20 kV / cm, the pulse width is 15-25 μs, the pulse frequency is 200-300 Hz, and the total number of pulses is 200-300.

[0011] More preferably, the eutectic solvent is composed of betaine and glycerol in a molar ratio of 1:1.5-2.5.

[0012] Preferably, the preparation method of the collagen peptide includes: using fresh animal tissue rich in collagen as raw material, fermenting it in liquid with compound microorganisms, then sequentially hydrolyzing it with flavor protease and trypsinase, and purifying it to obtain collagen peptide.

[0013] More preferably, the composite microorganism consists of Lactobacillus plantarum CICC 24988 and Pediococcus spp. CICC 6282.

[0014] The present invention also provides an application of the above-described composite bone-strengthening composition in the preparation of bone-strengthening products.

[0015] Preferably, the composite bone-strengthening composition can promote the repair of joint movement injuries.

[0016] The present invention also provides a bone-strengthening product based on the above-mentioned composite bone-strengthening composition, comprising: 30-40 parts of the composite bone-strengthening composition, 0.002-0.003 parts of vitamin D3, 0.008-0.012 parts of vitamin K2, 5-10 parts of calcium citrate, and 20-30 parts of vegetable oil.

[0017] The present invention also provides a method for preparing the above-mentioned bone-strengthening product, comprising: mixing vitamin D3, vitamin K2 and vegetable oil to obtain an oil phase; mixing calcium citrate and a compound bone-strengthening composition to obtain a powder mixture; adding the powder mixture to the oil phase, stirring, homogenizing, and degassing to obtain the bone-strengthening product.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention provides a composite bone-strengthening composition containing astragalus polysaccharide and polypeptides, comprising: 1-3 parts of astragalus polysaccharide and 1-3 parts of collagen peptides. The composite bone-strengthening composition contains the following raw materials in parts by weight: 1-3 parts of astragalus polysaccharide and 1-3 parts of collagen peptides; the astragalus polysaccharide is obtained by ultrasonic extraction using a high-voltage pulsed electric field combined with a eutectic solvent, and the collagen peptides are prepared by combined fermentation of *Lactobacillus plantarum* CICC 24988 and *Pediococcus* CICC 6282, stepwise enzymatic hydrolysis, and purification.

[0020] This invention demonstrates a synergistic effect between Astragalus polysaccharides and collagen peptides: Astragalus polysaccharides alleviate joint inflammation and oxidative stress by inhibiting the release of pro-inflammatory factors and scavenging reactive oxygen species, creating a favorable microenvironment for repair; collagen peptides, as small molecule raw materials, target and accumulate at the injury site, promoting the regeneration of cartilage matrix and collagen fibers, and inhibiting cartilage degradation. Simultaneously, Astragalus polysaccharides enhance the absorption and utilization rate and targeted aggregation ability of collagen peptides, jointly strengthening the mechanical properties of repaired tissues. Cellular experiments show that the composition of this invention significantly promotes chondrocyte proliferation, inhibits oxidative damage, and suppresses inflammatory responses, making it suitable for soft tissue repair. This composition provides a safe and efficient new solution for joint motion injury repair, possessing significant clinical value and market potential. Detailed Implementation

[0021] This invention provides a composite bone-strengthening composition containing astragalus polysaccharide and polypeptide, comprising the following raw materials in parts by weight: 1-3 parts of astragalus polysaccharide and 1-3 parts of collagen peptide.

[0022] The extraction method of Astragalus polysaccharide according to the present invention includes: mixing Astragalus with water, extracting under a high-voltage pulsed electric field, concentrating the reaction system, adding an equal volume of eutectic solvent for ultrasonic extraction, filtering, adding anhydrous ethanol to the filtrate, allowing to stand, centrifuging, washing, and drying to obtain Astragalus polysaccharide; the preferred material-to-liquid ratio of the high-voltage pulsed electric field extraction is 1:5-10 g / mL, more preferably 1:8 g / mL, the preferred electric field strength is 15-20 kV / cm, more preferably 18 kV / cm, the preferred pulse width is 15-25 μs, more preferably 20 μs, the preferred pulse frequency is 200-300 Hz, more preferably 250 Hz, and the preferred total number of pulses is 200-300, more preferably 250; the preferred eutectic solvent is composed of betaine and glycerol in a molar ratio of 1:1.5-2.5, more preferably 1:2.

[0023] This invention utilizes an electric field to disrupt the cell wall of Astragalus membranaceus, efficiently releasing polysaccharide components and improving the extraction rate; ultrasonic extraction with an equal volume of eutectic solvent can specifically dissolve polysaccharides, reduce impurity dissolution, and enhance purification effect; adding anhydrous ethanol and centrifuging allows polysaccharides to precipitate through alcohol precipitation.

[0024] The Astragalus polysaccharide prepared by this invention can promote the repair of joint sports injuries. It can inhibit the release of pro-inflammatory factors at the injury site, reduce inflammation and edema of the synovial membrane, and activate the anti-inflammatory phenotype of macrophages, creating a microenvironment conducive to repair. It can also enhance the proliferation activity of fibroblasts, accelerate collagen synthesis in damaged tendons, ligaments, and other connective tissues, improve the mechanical strength of repaired tissues, and shorten the healing cycle, thus playing a positive role in inflammation control and tissue regeneration after joint sports injuries.

[0025] The preferred method for preparing collagen peptides according to the present invention includes: using fresh animal tissue rich in collagen as raw material, subjecting it to anaerobic fermentation in liquid form by compound microorganisms, followed by enzymatic hydrolysis with flavor protease and trypsinase, and purification to obtain collagen peptides; the fresh animal tissue rich in collagen is preferably animal skin, animal bones, animal tendons, and animal scales, more preferably cowhide, cow bones, pig tendons, and fish scales; the compound microorganisms are preferably composed of *Lactobacillus plantarum* CICC 24988 and *Pediococcus* CICC 6282, and the number of *Lactobacillus plantarum* CICC 24988 in the compound microorganisms is preferably (5-9) × 10⁻⁶. 8 7 × 10⁻⁶ cells / g, more preferably 7 × 10⁻⁶ cells / g. 8 The optimal bacterial count for *Pediococcus* spp. CICC 6282 is (3-8) × 10⁻⁶ / g. 8 5 × 10⁻⁶ cells / g, more preferably 5 × 10⁻⁶ cells / g. 8The anaerobic fermentation temperature is preferably 35-40℃, more preferably 37℃, the pH value is preferably 6-6.5, more preferably 6.3, and the time is preferably 54-72h, more preferably 62h; the enzymatic hydrolysis temperature of the flavor protease is preferably 50-55℃, more preferably 53℃, the pH value is preferably 6-7, more preferably 6.5, and the time is preferably 80-120min, more preferably 100min; the enzymatic hydrolysis temperature of the pancreatic peptidase is preferably 45-50℃, more preferably 48℃, the pH value is preferably 6.5-7.5, more preferably 7, and the time is preferably 60-80min, more preferably 70min. The purification process preferably includes: centrifuging the enzymatic hydrolysate at 8000-10000 rpm for 20-30 min, collecting the supernatant, filtering it through a 1000-3000 Da ultrafiltration membrane at a pressure of 0.2-0.3 MPa, a temperature of 25-35℃, and a flow rate of 10-15 mL / min, and collecting the permeate; adding 0.5%-1.0% activated carbon by mass of the permeate, stirring at 30-40℃ and a stirring speed of 150-200 rpm for 30-60 min, filtering through a plate and frame filter, and collecting the clarified liquid; concentrating the clarified liquid to 1 / 5-1 / 8 of its volume using 200-500 Da nanofiltration at a pressure of 0.4-0.6 MPa and a temperature of 30-40℃, collecting the concentrated liquid, and freeze-drying it under vacuum to obtain collagen peptides. The purification process more preferably includes: centrifuging the enzymatic hydrolysate at 9000 rpm for 25 min, collecting the supernatant, filtering it through a 2000 Da ultrafiltration membrane at a pressure of 0.25 MPa, a temperature of 30 °C, and a flow rate of 12 mL / min, and collecting the permeate; adding 0.8% activated carbon by mass of the permeate, stirring at 35 °C and a stirring speed of 180 rpm for 40 min, filtering through a plate and frame filter, and collecting the clarified liquid; concentrating the clarified liquid to 1 / 6 of its volume using a 300 Da nanofiltration membrane at a pressure of 0.5 MPa and a temperature of 35 °C, collecting the concentrated liquid, and freeze-drying it under vacuum to obtain collagen peptides.

[0026] This invention utilizes anaerobic fermentation by compound microorganisms with the help of specific strains to initially degrade animal tissue collagen into small molecule peptide precursors. Flavor protease and trypsinase further hydrolyze the precursors into small molecule peptides through stepwise enzymatic hydrolysis. Centrifugation removes residual bacterial cells, ultrafiltration membrane filtration removes large molecular impurities while retaining target peptides, activated carbon adsorption decolorizes and deodorizes to improve sensory qualities, and nanofiltration improves the purity of collagen peptides.

[0027] The collagen peptides described in this invention can directly participate in the repair of joint movement injuries. Their small molecular structure makes them easily absorbed and allows them to target and accumulate at the injury site, providing raw materials for chondrocytes and fibroblasts to synthesize collagen. This promotes the regeneration and remodeling of cartilage matrix and tendon collagen fibers, repairing damaged cartilage surfaces and tendon fiber bundles. It can also enhance chondrocyte activity, inhibit the degradation of cartilage matrix by matrix metalloproteinases, maintain joint tissue homeostasis, and improve the movement function of injured joints.

[0028] This invention demonstrates a synergistic effect between Astragalus polysaccharide and collagen peptides: Astragalus polysaccharide creates a favorable microenvironment for collagen peptides to function through anti-inflammatory and immunomodulatory effects, reducing the inhibition of collagen regeneration by inflammation; collagen peptides provide structural raw materials for tissue repair, accelerating the regeneration of damaged tissues; simultaneously, Astragalus polysaccharide enhances the absorption and utilization rate of collagen peptides, promoting their targeted aggregation to the damaged site, jointly improving the collagen content and mechanical properties of the repaired tissue. Compared with single components, it is more effective in relieving joint pain, improving joint mobility, and significantly improving the repair efficiency and quality of joint sports injuries.

[0029] The present invention also provides an application of the above-mentioned composite bone-strengthening composition in the preparation of bone-strengthening products, wherein the composite bone-strengthening composition can promote the repair of joint movement injuries.

[0030] The present invention also provides a bone-strengthening product based on the above-mentioned composite bone-strengthening composition, comprising: 30-40 parts of the composite bone-strengthening composition, 0.002-0.003 parts of vitamin D3, 0.008-0.012 parts of vitamin K2, 5-10 parts of calcium citrate, and 20-30 parts of vegetable oil.

[0031] In this invention, the vegetable oil used is an edible vegetable oil, including but not limited to at least one of olive oil, soybean oil, peanut oil, rapeseed oil, and camellia oil.

[0032] The present invention also provides a method for preparing the above-mentioned bone-strengthening product, comprising: mixing vitamin D3, vitamin K2 and vegetable oil to obtain an oil phase; mixing calcium citrate and a compound bone-strengthening composition to obtain a powder mixture; adding the powder mixture to the oil phase, stirring, homogenizing, and degassing to obtain the bone-strengthening product.

[0033] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the following embodiments are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Flavor protease (also known as flavor enzyme), with an enzyme activity of 100,000 U / g, is sourced from Nanning Pangbo Bioengineering Co., Ltd.; Pancreatic peptidase (also known as elastase), with an enzyme activity of 100,000 U / g, is sourced from Guangzhou Wenbosen Biomedical Co., Ltd.

[0037] Lactiplantibacillus plantarum CICC 24988, Pediococcus sp. CICC 6282, Brevibacterium casei CICC 23944, and Lactococcus sp. CICC 6291 are all derived from the China Industrial Microbial Culture Collection Center.

[0038] Example 1: Preparation of a composite bone-strengthening composition

[0039] (1) Extraction of Astragalus polysaccharides

[0040] Astragalus membranaceus was pulverized and passed through a 200-mesh sieve. Deionized water was added at a material-to-liquid ratio of 1:8 g / mL. High-voltage pulsed electric field extraction was performed under an electric field strength of 18 kV / cm, a pulse width of 20 μs, a pulse frequency of 250 Hz, and a total of 250 pulses. After extraction, the reaction system was concentrated to 1 / 4 of its original volume. An equal volume of eutectic solvent (betaine to glycerol molar ratio of 1:2) was added, and ultrasonic extraction was performed for 30 min. The mixture was filtered, and 3 times the volume of anhydrous ethanol was added to the filtrate. The mixture was allowed to stand at 4℃ for 12 h, centrifuged at 8000 rpm for 15 min, and the precipitate was washed 3 times with anhydrous ethanol. The precipitate was then freeze-dried under vacuum to obtain Astragalus membranaceus polysaccharide.

[0041] (2) Preparation of collagen peptides

[0042] Fresh tilapia scales were crushed and pulverized, then mixed with an equal mass of glucose to obtain a mixture. This mixture was then mixed with water at a ratio of 1:10 g / mL, sterilized, and inoculated with 5% (by weight) of a compound microbial culture (containing 7 × 10⁻⁶ Lactobacillus plantarum CICC 24988). 8 CFU / g, Pediococcus spp. CICC 6282 bacterial count 5×10⁻⁶ 8The collagen peptides were anaerobic fermented at 37℃ and pH 6.3 for 62 h, sterilized, and filtered. The fermentation broth was then subjected to enzymatic hydrolysis with flavor protease (53℃, pH 6.5, 100 min) and trypsinase (48℃, pH 7, 70 min) in sequence. The enzymes were inactivated and filtered. The hydrolysate was centrifuged at 9000 rpm for 25 min. The supernatant was filtered through a 2000 Da ultrafiltration membrane at 0.25 MPa, 30℃, and a flow rate of 12 mL / min. 0.8% activated carbon by weight of the permeate was added to the permeate. The mixture was stirred at 35℃ and 180 rpm for 40 min and then filtered through a plate and frame filter. The clarified liquid was concentrated to 1 / 6 of its original volume by nanofiltration at 0.5 MPa and 35℃ using a 300 Da nanofiltration membrane. The resulting product was then freeze-dried under vacuum to obtain collagen peptides.

[0043] (3) Preparation of composite bone-strengthening composition

[0044] Weigh out 2 parts of Astragalus polysaccharide and 2 parts of collagen peptide by weight, mix them, and obtain a compound bone-strengthening composition.

[0045] Example 2: Preparation of a composite bone-strengthening composition

[0046] (1) Extraction of Astragalus polysaccharides

[0047] Astragalus membranaceus was pulverized and passed through a 150-mesh sieve. Deionized water was added at a material-to-liquid ratio of 1:5 g / mL. High-voltage pulsed electric field extraction was performed under an electric field strength of 15 kV / cm, a pulse width of 15 μs, a pulse frequency of 200 Hz, and a total number of 200 pulses. After extraction, the reaction system was concentrated to 1 / 3 of its original volume. An equal volume of eutectic solvent (betaine to glycerol molar ratio of 1:1.5) was added, and ultrasonic extraction was performed for 35 min. The mixture was filtered, and 2.5 times the volume of anhydrous ethanol was added to the filtrate. The mixture was allowed to stand at 4℃ for 12 h, centrifuged at 8000 rpm for 15 min, and the precipitate was washed three times with anhydrous ethanol and dried under vacuum at 60℃ to obtain Astragalus membranaceus polysaccharide.

[0048] (2) Preparation of collagen peptides

[0049] Fresh yak bones were crushed and pulverized, then mixed with an equal mass of glucose to obtain a mixture. This mixture was then mixed with water at a ratio of 1:8 g / mL, sterilized, and inoculated with 4% (by weight) of a compound microbial culture (containing 5 × 10⁶ *Lactobacillus plantarum* CICC 24988). 8 CFU / g, Pediococcus spp. CICC 6282 bacterial count 3×10⁻⁶ 8The collagen peptides were fermented at 35℃ and pH 6 for 72 h, sterilized, and filtered. The fermentation broth was then sequentially hydrolyzed with flavor protease (50℃, pH 6, 80 min) and trypsinase (45℃, pH 6.5, 60 min), the enzymes were inactivated, and the broth was filtered. The hydrolysate was centrifuged at 8000 rpm for 30 min. The supernatant was ultrafiltered at 0.2 MPa, 25℃, and 10 mL / min using 1000 Da ultrafiltration. 0.5% activated carbon was added to the permeate, and the mixture was stirred at 30℃ and 150 rpm for 60 min before plate and frame filtration. The clarified liquid was concentrated to 1 / 5 of its original volume by nanofiltration at 0.4 MPa and 30℃ using 200 Da nanofiltration. The resulting product was then freeze-dried under vacuum to obtain collagen peptides.

[0050] (3) Preparation of composite bone-strengthening composition

[0051] Weigh out 1 part of Astragalus polysaccharide and 3 parts of collagen peptide by weight, mix them, and obtain a compound bone-strengthening composition.

[0052] Example 3: Preparation of a composite bone-strengthening composition

[0053] (1) Extraction of Astragalus polysaccharides

[0054] Astragalus membranaceus was pulverized and passed through a 250-mesh sieve. Deionized water was added at a material-to-liquid ratio of 1:10 g / mL. High-voltage pulsed electric field extraction was performed under an electric field strength of 20 kV / cm, a pulse width of 25 μs, a pulse frequency of 300 Hz, and a total of 300 pulses. After extraction, the reaction system was concentrated to 1 / 5 of its original volume, and an equal volume of eutectic solvent (betaine to glycerol molar ratio of 1:2.5) was added. The mixture was ultrasonically extracted for 25 min, filtered, and 3 times the volume of anhydrous ethanol was added to the filtrate. The mixture was allowed to stand at 4℃ for 12 h, centrifuged at 8000 rpm for 15 min, and the precipitate was washed three times with anhydrous ethanol and dried under vacuum at 60℃ to obtain Astragalus membranaceus polysaccharide.

[0055] (2) Preparation of collagen peptides

[0056] Fresh yak hides were crushed and pulverized, then mixed with an equal mass of glucose to obtain a mixture. This mixture was then mixed with water at a ratio of 1:12 g / mL, sterilized, and inoculated with 7% of the mixture's mass of a compound microbial culture (containing 9 × 10⁶ *Lactobacillus plantarum* CICC 24988). 8 CFU / g, Pediococcus spp. CICC 6282 count 8 × 10⁻⁶ 8The collagen peptides were anaerobic fermented at 37℃ and pH 6.3 for 54 h, sterilized, and filtered. The fermentation broth was then subjected to enzymatic hydrolysis with flavor protease (55℃, pH 7, 120 min) and trypsinase (50℃, pH 7.5, 80 min) in sequence. The enzymes were inactivated and filtered. The hydrolysate was centrifuged at 10,000 rpm for 20 min. The supernatant was filtered through a 3000 Da ultrafiltration membrane at 0.3 MPa, 35℃, and 15 mL / min. 1% activated carbon by weight of the permeate was added to the permeate. The mixture was stirred at 40℃ and 200 rpm for 30 min and then filtered through a plate and frame filter. The clarified liquid was concentrated to 1 / 8 of its original volume by nanofiltration at 0.6 MPa and 40℃ using a 500 Da nanofiltration membrane. The resulting product was then freeze-dried under vacuum to obtain collagen peptides.

[0057] (3) Preparation of composite bone-strengthening composition

[0058] Weigh out 3 parts by weight of Astragalus polysaccharide and 1 part by weight of collagen peptide, mix them, and obtain a compound bone-strengthening composition.

[0059] Comparative Example 1: Only one component, Astragalus polysaccharide

[0060] The difference between this comparative example and Example 1 is that only Astragalus polysaccharide is used, and collagen peptides are omitted, and step (2) is omitted accordingly.

[0061] Comparative Example 2: Component consisting only of collagen peptides

[0062] The difference between this comparative example and Example 1 is that only collagen peptides are used, and Astragalus polysaccharide is omitted, and step (1) is omitted accordingly.

[0063] Comparative Example 3: Preparation of Astragalus Polysaccharides by Hot Water Extraction

[0064] The difference between this comparative example and Example 1 is that the preparation method of Astragalus polysaccharide in step (1) is as follows: Astragalus is crushed, passed through a 200-mesh sieve, and deionized water is added at a material-to-liquid ratio of 1:8 g / mL. It is extracted twice at 90℃ for 2 hours each time, filtered, and the filtrates are combined. Three times the volume of anhydrous ethanol is added to the filtrate, and it is allowed to stand at 4℃ for 12 hours. It is then centrifuged at 8000 rpm for 15 minutes. The precipitate is washed three times with anhydrous ethanol and then freeze-dried under vacuum to obtain Astragalus polysaccharide.

[0065] Comparative Example 4: Preparation of Collagen Peptides by Changing the Types of Complex Microorganisms

[0066] The difference between this comparative example and Example 1 is that the composite microorganism in step (2) consists of *Brevibacterium casei* CICC23944 and *Lactococcus* CICC 6291, with the number of *Brevibacterium casei* CICC 23944 in the composite microorganism being 7 × 10⁻⁶. 8 Lactococcus spp. CICC 6291 count: 5 × 10⁶ cells / g 8pcs / g, other conditions remain unchanged.

[0067] Comparative Example 5: Preparation of collagen peptides by changing the order of enzymatic hydrolysis

[0068] The difference between this comparative example and Example 1 is that in step (2), the fermentation broth is first hydrolyzed with pancreatic peptidase (48℃, pH 7, hydrolysis for 70 min), and then hydrolyzed with flavor protease (53℃, pH 6.5, hydrolysis for 100 min).

[0069] Experimental Example 1: Cell Experiment

[0070] The effects of the composite bone-strengthening compositions of Examples 1-3 and Comparative Examples 1-5 on the viability of chondrocyte C28 / I2 cells, as well as on the cell viability, ROS content, and inflammatory factors of H2O2-induced chondrocyte C28 / I2 cells, were detected respectively.

[0071] All cell experiments were independently repeated 3 times (n=3).

[0072] (1) CCK-8 assay to detect the effect of the compound bone-strengthening composition on the cell viability of C28 / I2 cells

[0073] Preparation of sample culture medium: The compound bone-strengthening compositions of Examples 1-3 and Comparative Examples 1-5 were respectively mixed with a culture medium containing 10% fetal bovine serum and 100 U·mL⁻¹. -1 Penicillin and 100 μg·mL -1 Streptomycin was mixed with RPMI-1640 medium, dissolved by sonication, and filtered through a microporous membrane to obtain composite bone-strengthening composition sample culture media with concentrations of 6.25, 12.5, 25, 50, and 100 mg / mL.

[0074] Grouping:

[0075] Drug intervention group: Sample culture medium obtained from Examples 1-3 and Comparative Examples 1-5 + C28 / I2 chondrocytes + CCK-8;

[0076] Blank control group: containing 10% fetal bovine serum, 100 U / mL -1 Penicillin and 100 μg·mL -1 Streptomycin-containing RPMI-1640 medium + C28 / I2 chondrocytes + CCK-8;

[0077] Negative control group: Contains only 10% fetal bovine serum, 100 U / mL -1 Penicillin and 100 μg·mL -1 Streptomycin in RPMI-1640 medium + CCK-8.

[0078] C28 / I2 chondrocytes in the logarithmic growth phase were adjusted to a concentration of 8 × 10⁻⁶ using sample medium / RPMI-1640 medium. 3 Seeds were administered at a rate of 100 μL per well in a 96-well cell culture plate and incubated at 37°C with 5% CO2 for 24 h. After incubation, 10 μL of CCK-8 solution was added to each well, and the plate was gently shaken to mix. The plate was then returned to the incubator for another h of incubation.

[0079] The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. The viability of Examples 1-3 and Comparative Examples 1-5 at different concentrations against C28 / I2 chondrocytes was calculated.

[0080] Cell viability (%) = (OD value of drug intervention group - OD value of negative control group) / (OD value of blank control group - OD value of negative control group) × 100%.

[0081] The cell viability results of C28 / I2 chondrocytes at different concentrations in each group are shown in Table 1.

[0082] Table 1. Cell viability (%) of chondrocytes at different concentrations in each group

[0083] Grouping 6.25 mg / mL 12.5 mg / mL 25mg / mL 50mg / mL 100mg / mL Example 1 102.35 105.81 108.58 112.25 115.34 Example 2 101.60 104.48 107.86 110.57 113.72 Example 3 102.18 104.73 108.15 111.73 114.51 Comparative Example 1 100.34 102.16 104.29 107.18 109.06 Comparative Example 2 100.19 101.30 103.40 105.62 108.15 Comparative Example 3 100.21 101.97 103.74 106.26 108.60 Comparative Example 4 100.53 102.72 104.91 107.89 109.27 Comparative Example 5 100.82 102.69 105.67 108.64 110.43

[0084] Note: Cell viability >100% indicates that the sample promotes the proliferation of C28 / I2 chondrocytes.

[0085] Table 1 shows that the composite bone-strengthening compositions of Examples 1-3 of this invention have a mild yet significant proliferative effect on C28 / I2 chondrocytes. Cell viability increases in a gradient with increasing concentration, with Example 1 showing the highest cell viability, significantly higher than the comparative examples. The single-component groups (Comparative Examples 1 and 2) showed limited improvement in cell viability; the groups with insufficient process optimization (Comparative Examples 3-5) also showed improvement. These data illustrate the synergistic effect of Astragalus polysaccharide and collagen peptides: Astragalus polysaccharide creates a suitable proliferation environment through immune regulation, while collagen peptides provide nutrients for cell growth; the two synergistically promote chondrocyte proliferation. Simultaneously, the optimized high-voltage pulsed electric field extraction, specific composite microbial fermentation, and stepwise enzymatic hydrolysis processes of this invention maximize the preservation of the bioactivity of the two active ingredients, further enhancing the synergistic effect and providing a sufficient cellular basis for the repair of joint movement injuries.

[0086] (2) Effects of the compound bone-strengthening composition on H2O2-induced chondrocytes

[0087] Preparation of sample culture medium: The compound bone-strengthening compositions of Examples 1-3 and Comparative Examples 1-5 were respectively mixed with a culture medium containing 10% fetal bovine serum and 100 U·mL⁻¹. -1 Penicillin and 100 μg·mL-1 Streptomycin was mixed with RPMI-1640 medium, dissolved by sonication, and filtered through a microporous membrane to obtain sample culture media of the compound bone-strengthening composition with concentrations of 50 and 100 mg / mL.

[0088] Grouping:

[0089] Drug intervention group: Sample culture medium of Examples 1-3 and Comparative Examples 1-5 + C28 / I2 chondrocytes + H2O2 + CCK-8;

[0090] Model group: containing 10% fetal bovine serum, 100 U·mL -1 Penicillin and 100 μg·mL -1 Streptomycin in RPMI-1640 medium + C28 / I2 chondrocytes + H2O2 + CCK-8;

[0091] Negative control group: containing 10% fetal bovine serum, 100 U / mL -1 Penicillin and 100 μg·mL -1 Streptomycin in RPMI-1640 medium + H2O2 + CCK-8;

[0092] Blank control group: containing 10% fetal bovine serum, 100 U / mL -1 Penicillin and 100 μg·mL -1 Streptomycin in RPMI-1640 medium + C28 / I2 chondrocytes + CCK-8.

[0093] C28 / I2 chondrocytes in the logarithmic growth phase were adjusted to a concentration of 8 × 10⁻⁶ using sample medium / RPMI-1640 medium. 3 Seeds were generated at a volume of 100 μL per well in a 96-well cell culture plate. 200 μM H2O2 was added, and the plate was incubated at 37°C and 5% CO2 for 24 h.

[0094] 1) Detection of cell viability

[0095] After incubation, add 10 μL of CCK-8 solution to each well and gently shake the culture plate to mix. Return the culture plate to the incubator and continue incubation for 1 h.

[0096] The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. The viability of H2O2-induced chondrocytes in Examples 1-3 and Comparative Examples 1-5 at different concentrations was calculated.

[0097] Cell viability (%) = (OD value of drug intervention group or model group - OD value of negative control group) / (OD value of blank control group - OD value of negative control group) × 100%.

[0098] The cell viability results of chondrocytes induced by H2O2 at different concentrations in each group are shown in Table 2.

[0099] Table 2. Cell viability (%) of chondrocytes induced by H2O2 at different concentrations in each group.

[0100]

[0101] Table 2 shows that the viability of chondrocytes in the model group was significantly lower than that in the blank control group after H2O2 induction, indicating that the cell model was successfully constructed. The compositions in Examples 1-3 significantly improved the survival ability of damaged cells, showing a significant improvement compared to the model group, and the effect was far superior to the comparative examples. The single-component groups (Comparative Examples 1 and 2) had weaker cell protection effects due to a lack of synergistic effects; the groups with insufficient process optimization (Comparative Examples 3-5) had limited anti-damage capabilities due to insufficient purity of active ingredients. These results indicate that the anti-inflammatory and antioxidant effects of Astragalus polysaccharides and the cellular nutrient supply of collagen peptides form a powerful synergy. Astragalus polysaccharides clear oxidative stress products and reduce cell damage, while collagen peptides repair damaged cell structure and maintain cell function. The two work synergistically to resist H2O2-induced cell damage, and the optimized process enhances the synergistic efficiency of the active ingredients, providing crucial cellular protection for tissue repair after joint injury.

[0102] 2) Detection of MDA content

[0103] After incubation, the MDA content of each group of compound bone-strengthening compositions induced by H2O2 was detected using a malondialdehyde (MDA) kit. The specific results are shown in Table 3.

[0104] Table 3. MDA content (nM / mg protein) of H2O2-induced chondrocytes in each group

[0105]

[0106] Table 3 shows that the MDA content in the model group was significantly higher than that in the blank control group, indicating severe oxidative damage. The compositions in Examples 1-3 reduced MDA levels in a concentration-dependent manner, significantly lower than the model group and superior to the control groups. As a marker of lipid peroxidation, the reduction in MDA content reflects the antioxidant capacity of the composition, an effect attributed to the synergistic effect of Astragalus polysaccharide and collagen peptides: Astragalus polysaccharide reduces lipid peroxidation by scavenging free radicals and inhibiting oxidation reactions; collagen peptides repair oxidatively damaged cell structures and enhance cellular antioxidant capacity, with both synergistically alleviating oxidative damage to chondrocytes. The optimized preparation process of this invention improves the purity and activity of the two components, further enhancing the synergistic antioxidant effect, effectively improving the oxidative stress microenvironment at joint injury sites, and creating favorable conditions for tissue repair.

[0107] 3) Detection of reactive oxygen species (ROS) content

[0108] After incubation, cells were collected, and flow cytometry using the DCFH-DA reactive oxygen species probe was used to detect the ROS levels of H2O2-induced chondrocytes at various concentrations of the composite bone-strengthening composition, and the relative ROS levels were calculated. Specific results are shown in Table 4.

[0109] Relative ROS (%) = (ROS fluorescence intensity of drug intervention group or model group / ROS fluorescence intensity of blank control group) × 100%.

[0110] Table 4. Relative ROS levels (%) of H2O2-induced chondrocytes in each group

[0111]

[0112] Table 4 shows that the ROS level in the model group was significantly higher than that in the blank control group, indicating that oxidative stress damage in chondrocytes was successfully induced. Compared with the model group, the compositions of Examples 1-3 significantly downregulated ROS content and were superior to the comparative proportions. Excessive ROS accumulation can aggravate joint inflammation and tissue damage. The ROS scavenging effect of the compositions is due to the synergistic effect of Astragalus polysaccharide and collagen peptides: Astragalus polysaccharide inhibits ROS generation through anti-inflammatory pathways, while collagen peptides reduce ROS damage to cells through cell protection mechanisms. The two work synergistically to scavenge reactive oxygen species from the dual dimensions of source inhibition and damage repair. At the same time, the optimized process ensures the high activity and high purity of the two components, allowing the synergistic effect to be fully exerted, effectively reducing the damage of oxidative stress to chondrocytes, and creating a low-oxidative stress microenvironment for joint damage repair, demonstrating the technical innovation of this invention.

[0113] 4) Expression of inflammatory factor proteins using ELISA kits

[0114] After incubation, cell supernatant was collected, and the effects of a series of concentrations of the compound bone-strengthening composition on the expression of inflammatory factors IL-1β, TNF-α, and IL-6 proteins in H2O2-induced chondrocytes were detected using an ELISA kit. The specific results are shown in Table 5.

[0115] Table 5. Effects of each group on H2O2-induced inflammatory factors in chondrocytes

[0116]

[0117] Table 5 shows that the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in the model group were significantly upregulated compared to the blank control group, indicating that the chondrocyte inflammatory injury model was successfully constructed. The compositions in Examples 1-3 effectively inhibited their secretion in a concentration-dependent manner, significantly superior to the comparative examples. The single-component groups (Comparative Examples 1 and 2) showed limited anti-inflammatory effects due to a lack of synergy; the groups with insufficient process optimization (Comparative Examples 3-5) had low synergistic efficiency of active ingredients, making it difficult to effectively regulate the inflammatory response. These results confirm that Astragalus polysaccharide and collagen peptides have a strong synergistic effect in anti-inflammation: Astragalus polysaccharide inhibits the release of pro-inflammatory factors by regulating inflammatory pathways, while collagen peptides repair damaged cells and reduce the production of inflammatory mediators; the two synergistically improve the inflammatory microenvironment at the joint injury site. Optimized processes further enhance the synergistic anti-inflammatory efficiency of the two components, effectively reducing synovial inflammation and cartilage damage, providing an important guarantee for the repair of joint movement injuries.

[0118] Example 4: Bone-strengthening products

[0119] (1) Weighing

[0120] Accurately weigh the following components according to their weight proportions: 35 parts of the compound bone-strengthening composition of Example 1, 0.0025 parts of vitamin D3, 0.01 parts of vitamin K2, 8 parts of calcium citrate, and 25 parts of olive oil.

[0121] (2) Preparation

[0122] Under light-protected conditions, vitamin D3 and vitamin K2 were added to olive oil and stirred at 400 rpm for 18 min to obtain the oil phase; calcium citrate and the compound bone-strengthening composition were stirred at 150 rpm for 12 min to obtain the powder mixture; the powder mixture was added to the oil phase and stirred at 35°C and 2500 rpm for 12 min, then homogenized at 40°C and 200 bar for 2 cycles, degassed, and the bone-strengthening product was obtained.

[0123] Example 5: Bone-strengthening products

[0124] (1) Weighing

[0125] Accurately weigh the following components according to their weight proportions: 30 parts of the compound bone-strengthening composition of Example 2, 0.003 parts of vitamin D3, 0.008 parts of vitamin K2, 10 parts of calcium citrate, and 20 parts of soybean oil.

[0126] (2) Preparation

[0127] Under light-protected conditions, vitamin D3 and vitamin K2 were added to soybean oil and stirred at 300 rpm for 20 min to obtain the oil phase; calcium citrate and the compound bone-strengthening composition were stirred at 100 rpm for 15 min to obtain the powder mixture; the powder mixture was added to the oil phase and stirred at 30℃ and 2000 rpm for 15 min, then homogenized at 35℃ and 150 bar for 3 cycles, degassed, and the bone-strengthening product was obtained.

[0128] Example 6: Bone-strengthening products

[0129] (1) Weighing

[0130] Accurately weigh the following components according to their weight proportions: 40 parts of the compound bone-strengthening composition of Example 3, 0.002 parts of vitamin D3, 0.012 parts of vitamin K2, 5 parts of calcium citrate, and 30 parts of peanut oil.

[0131] (2) Preparation

[0132] Under light-protected conditions, vitamin D3 and vitamin K2 were added to peanut oil and stirred at 500 rpm for 15 min to obtain the oil phase; calcium citrate and the compound bone-strengthening composition were stirred at 200 rpm for 10 min to obtain the powder mixture; the powder mixture was added to the oil phase and stirred at 40℃ and 3000 rpm for 10 min, then homogenized at 45℃ and 250 bar for 2 cycles, degassed, and the bone-strengthening product was obtained.

[0133] Comparative Example 6

[0134] The difference between this comparative example and Example 4 is that "Composite Bone Strengthening Composition of Example 1" is replaced with the composite bone strengthening composition prepared in Composite Example 1.

[0135] Comparative Example 7

[0136] The difference between this comparative example and Example 4 is that "Composite Bone Strengthening Composition of Example 1" is replaced with the composite bone strengthening composition prepared in Composite Example 2.

[0137] Comparative Example 8

[0138] The difference between this comparative example and Example 4 is that "Composite Bone Strengthening Composition of Example 1" is replaced with the composite bone strengthening composition prepared in Composite Example 3.

[0139] Comparative Example 9

[0140] The difference between this comparative example and Example 4 is that "Composite Bone Strengthening Composition of Example 1" is replaced with the composite bone strengthening composition prepared in Composite Example 4.

[0141] Comparative Example 10

[0142] The difference between this comparative example and Example 4 is that "Composite Bone Strengthening Composition of Example 1" is replaced with the composite bone strengthening composition prepared in Composite Example 5.

[0143] Experiment Example 2: Zebrafish Animal Experiment

[0144] (1) Group design

[0145] Three days after fertilization, transgenic zebrafish with cartilage fluorescence were divided into nine groups (blank group, model group, positive group, and experimental group (Example 4, Comparative Example 6, Comparative Example 7, Comparative Example 8, Comparative Example 9, and Comparative Example 10)), with 30 fish in each group.

[0146] The standard culture medium has a temperature of 28℃±0.5℃, a pH of 7.0-7.5, dissolved oxygen ≥6mg / L, and a DMSO volume fraction of 0.1%.

[0147] The bone-strengthening products of Example 4 and Comparative Examples 6-10 were diluted with standard aquaculture solution to a concentration of 0.5 mg / mL.

[0148] The positive group used chondroitin sulfate, which was diluted with standard culture medium to a working solution with a concentration of 1 mg / mL.

[0149] The specific grouping arrangements are as follows:

[0150] Control group: cultured in standard culture medium;

[0151] Model group: 12 μM dexamethasone induction + standard culture medium culture;

[0152] Positive group: 12 μM dexamethasone induction + 1 mg / mL working solution culture;

[0153] Experimental group: 12 μM dexamethasone induction + 10 mg / mL sample solution culture.

[0154] (2) Model building and intervention

[0155] Zebrafish in the model group, positive group and experimental group were placed in a sample solution / working solution containing 100 μM dexamethasone and the corresponding concentration, and continuously immersed and cultured at 28℃±0.5℃ for 72h. During this period, oxygen was continuously supplied, and the culture medium was changed every 24h (in accordance with the group design in (1)). The blank control group was cultured in a standard culture medium containing 0.1% DMSO.

[0156] After soaking and culturing for 72 hours, the zebrafish were rinsed three times with PBS solution, anesthetized with MS-222, and observed and photographed using a fluorescence microscope.

[0157] The average fluorescence intensity (FI) of zebrafish cartilage tissue in each group was measured using an image analysis system. Ten zebrafish were randomly selected for each sample, and three different fields of view were measured for each fish. The average value was taken as the fluorescence intensity value of the group.

[0158] The cartilage regeneration promotion rate of each group was calculated based on the average fluorescence intensity of the model group.

[0159] The specific results of fluorescence intensity and cartilage regeneration promotion rate for each group are shown in Table 6.

[0160] The cartilage regeneration promotion rate (%) = (mean FI of the positive group or experimental group - mean FI of the model group) / mean FI of the model group × 100%.

[0161] Table 6. Cartilage tissue regeneration promotion rate (%) in each group of zebrafish

[0162] Group Cartilage fluorescence intensity (pixels) Cartilage regeneration promotion rate (%) control group 1074396 — Model group 637248 — positive group 996592 56.39 Example 4 976073 53.17 Comparative Example 6 911520 43.04 Comparative Example 7 895461 40.52 Comparative Example 8 909225 42.68 Comparative Example 9 938411 47.26 Comparative Example 10 945994 48.45

[0163] Table 6 shows that after dexamethasone induction, the pixel value of cartilage fluorescence intensity in the model group was significantly lower than that in the blank group, indicating that the cartilage injury model was successfully constructed. The cartilage fluorescence intensity in the Example 4 group was significantly higher than that in the model group and significantly better than that in the comparative examples. The regeneration promotion rates of Comparative Examples 6 (Astragalus polysaccharide only) and 7 (collagen peptide only) were much lower than those in Example 4, indicating a synergistic effect between the two components, and that their combination can significantly improve repair efficiency. The regeneration promotion rates of Comparative Examples 8-10 were lower than those in Example 4 due to changes in the extraction process of Astragalus polysaccharide or the preparation process of collagen peptide, indicating that the optimized high-voltage pulsed electric field extraction, specific compound microbial fermentation, and stepwise enzymatic hydrolysis processes of this invention can maximize the preservation of the bioactivity of the two components and enhance the synergistic effect. Therefore, the synergistic effect of Astragalus polysaccharide and collagen peptide is the core of promoting cartilage regeneration, and the optimized preparation process provides a guarantee for the full realization of this synergistic effect, enabling the bone-strengthening product containing both to exhibit excellent cartilage injury repair potential.

[0164] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composite bone-strengthening composition containing astragalus polysaccharides and polypeptides, characterized in that, It is composed of the following raw materials in parts by weight: 1-3 parts of Astragalus polysaccharide and 1-3 parts of collagen peptide; The extraction method of Astragalus polysaccharide is as follows: Astragalus is mixed with water and extracted under a high-voltage pulsed electric field. After the reaction system is concentrated, a eutectic solvent is added for ultrasonic extraction. The mixture is filtered, anhydrous ethanol is added to the filtrate, and the mixture is allowed to stand, centrifuged, washed, and dried to obtain Astragalus polysaccharide. The high-voltage pulsed electric field extraction has a material-to-liquid ratio of 1:5-10 g / mL, an electric field strength of 15-20 kV / cm, a pulse width of 15-25 μs, a pulse frequency of 200-300 Hz, and a total number of pulses of 200-300. The eutectic solvent is composed of betaine and glycerol in a molar ratio of 1:1.5-2.

5. The preparation method of the collagen peptides includes: using fresh animal tissue rich in collagen as raw material, after anaerobic fermentation by compound microorganisms, followed by enzymatic hydrolysis by flavor protease and trypsinase, and purification to obtain collagen peptides. The collagen-rich fresh animal tissue is one of the following: cowhide, cow bone, pig tendon, or fish scale; The composite microorganism consists of *Lactobacillus plantarum* CICC 24988 and *Pediococcus* CICC 6282, with the *Lactobacillus plantarum* CICC 24988 count being (5-9) × 10⁻⁶. 8 The number of *Pediococcus* spp. CICC 6282 cells / g is (3-8)×10⁻⁶. 8 pcs / g; The anaerobic fermentation was carried out at a temperature of 35-40℃, a pH of 6-6.5, and a time of 54-72 hours. The enzymatic hydrolysis temperature of the flavor protease is 50-55℃, the pH value is 6-7, and the time is 80-120 min; The pancreatic peptidase hydrolysis temperature is 45-50℃, the pH value is 6.5-7.5, and the time is 60-80 min; The purification process includes: centrifuging the enzymatic hydrolysate at 8000-10000 rpm for 20-30 min, collecting the supernatant, filtering it through a 1000-3000 Da ultrafiltration membrane at a pressure of 0.2-0.3 MPa, a temperature of 25-35℃, and a flow rate of 10-15 mL / min, and collecting the permeate; adding 0.5%-1.0% activated carbon by weight of the permeate, stirring at 30-40℃ and a stirring speed of 150-200 rpm for 30-60 min, filtering through a plate and frame filter, and collecting the clarified liquid; concentrating the clarified liquid to 1 / 5-1 / 8 of its volume using 200-500 Da nanofiltration at a pressure of 0.4-0.6 MPa and a temperature of 30-40℃, collecting the concentrated liquid, and freeze-drying it under vacuum to obtain collagen peptides.

2. The application of the composite bone-strengthening composition according to claim 1 in the preparation of bone-strengthening products.

3. The application according to claim 2, characterized in that, The composite bone-strengthening composition can promote the repair of joint movement injuries.

4. A bone-strengthening product comprising the composite bone-strengthening composition of claim 1, characterized in that, include: The compound bone-strengthening composition contains 30-40 parts of vitamin D3, 0.002-0.003 parts of vitamin K2, 0.008-0.012 parts of calcium citrate, and 20-30 parts of vegetable oil.

5. A method for preparing the bone-strengthening product according to claim 4, characterized in that, include: Vitamin D3, vitamin K2, and vegetable oil are mixed to obtain the oil phase; Calcium citrate and a compound bone-strengthening composition are mixed to obtain a powder mixture; The powder mixture is added to the oil phase and stirred, homogenized, and degassed to obtain the bone-strengthening product.

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