A pharmaceutical composition for improving bone density and a preparation method thereof

Abstract

The application provides a kind of drug composition for improving bone density and preparation method thereof, belongs to the biological medicine technical field, and the drug composition includes milk slurry fungus extraction peptide, hair potato skin extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2 and vitamin D3.Milk slurry fungus extraction peptide is sequentially zymolyzed by bromelain and subtilisin, and is extracted by LH-60 dextran gel column purification of zymolyzate.Hair potato skin extract is the epidermis of hair potato tuber extracted by ethanol aqueous solution, and the extract is extracted by AB-8 macroporous adsorption resin column purification.Herring trout extract is sequentially zymolyzed by trypsin and earthworm kinase by fish head, fish skin and fish scale of herring trout, and the product below 5kDa is obtained.Each component is compounded according to a specific ratio, and the core effect of improving bone density is realized by "two-way regulation of bone metabolism + optimization of calcium utilization + improvement of safety".

Description

Technical Field

[0001] This invention pertains to the field of biomedical technology, specifically relating to a pharmaceutical composition for improving bone density and its preparation method. Background Technology

[0002] Bone mineral density (BMD) is a key indicator of bone health, and its decrease significantly increases the risk of osteoporosis and other bone diseases. Physiologically, bone tissue is constantly undergoing metabolism. Osteoblasts are responsible for bone formation, while osteoclasts participate in bone resorption. Under normal circumstances, these two cells maintain a dynamic balance, keeping bone density stable. However, with age, hormonal changes, malnutrition, lack of exercise, and unhealthy lifestyle habits, this balance is disrupted. Osteoclast activity relatively increases, while osteoblast function gradually weakens, leading to bone loss and decreased bone density.

[0003] Traditional medical interventions for decreased bone density have many limitations. Early approaches relied primarily on calcium and vitamin D supplementation to improve bone density, but simply supplementing these nutrients often fails to address the root cause of bone metabolic imbalances, and the results are less than ideal for individuals with already significant bone density decline. Later, anti-osteoporosis drugs emerged, such as bisphosphonates. These drugs inhibit osteoclast activity and reduce bone resorption, but long-term use may cause side effects such as gastrointestinal discomfort, and their effect on promoting bone formation is relatively weak. Furthermore, estrogen replacement therapy was once used to prevent and treat osteoporosis in postmenopausal women, but estrogen use increases the risk of breast cancer, endometrial cancer, and other diseases, limiting its widespread application.

[0004] Therefore, from the perspective of patients' needs, there is a strong desire for safer, more effective, and comprehensive methods to regulate bone metabolism and improve bone density. Patients want drugs that can both inhibit bone resorption and promote bone formation, while also having minimal side effects and being suitable for long-term use. Against this backdrop, the development of a novel drug composition that effectively improves bone density is particularly urgent. Such a drug composition needs to integrate multiple mechanisms of action to synergistically regulate bone metabolism, thereby achieving a better effect in improving bone density and meeting people's needs for maintaining bone health. Summary of the Invention

[0005] Existing bone density-enhancing drugs fail to adequately meet the needs for safe, effective, and comprehensive health maintenance. This invention provides a pharmaceutical composition for enhancing bone density and its preparation method. The pharmaceutical composition includes *Lactobacillus subtilis* extract peptides, *Dioscorea opposita* peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2, and vitamin D3. It achieves the core effect of enhancing bone density through "bidirectional regulation of bone metabolism + optimized calcium utilization + improved safety." The specific technical solution is as follows:

[0006] A pharmaceutical composition for improving bone density comprises *Dactylogyrus rubra* extract peptide, yam peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2, and vitamin D3 in a mass ratio of (3-5):(1-2):(5-8):(3-5):(0.05-0.1):(0.01-0.03). The *Dactylogyrus rubra* extract peptide is obtained by sequentially enzymatically hydrolyzing *Dactylogyrus rubra* with bromelain and *Bacillus subtilis* protease, followed by purification and extraction of the hydrolysate using an LH-60 dextran gel column. The yam peel extract is obtained by extracting the epidermis of yam tubers with an ethanol-water solution, followed by purification and extraction using an AB-8 macroporous adsorption resin column. The rainbow trout extract is obtained by sequentially enzymatically hydrolyzing the head, skin, and scales of rainbow trout with trypsin and lumbrokinase to obtain a product with a mass of less than 5 kDa. The calcium citrate-glycine chelate has a mass ratio of calcium citrate to glycine of (2.2-2.6):1.

[0007] The preparation of the *Dactylogyrus pulveratum* extract peptide in the above-mentioned pharmaceutical composition includes: pulverizing *Dactylogyrus pulveratum* into a slurry, inactivating endogenous enzymes, adding deionized water at a material-to-liquid mass ratio of 1:(12-15), adjusting the pH to 6.0-7.5, adding bromelain, and enzymatically hydrolyzing at 50℃-55℃ for 1.5-2 hours. Then, adjusting the pH to 7.5-8.0, adding subtilisin, and enzymatically hydrolyzing at 50℃-55℃ for 1-1.5 hours, inactivating the enzyme, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading onto an LH-60 dextran gel column, rinsing with deionized water for 2-3 BV to remove impurities, eluting with 50%-70% (v / v) ethanol aqueous solution for 3-4 BV, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the *Dactylogyrus pulveratum* extract peptide.

[0008] In the preparation of the above-mentioned *Dactylogyrus mollissima* extract peptides, the *Dactylogyrus mollissima* is a fresh fruiting body; the endogenous enzyme is inactivated by incubation at 90℃~95℃ for 10min~15min; the amount of bromelain added is 0.8%~1.5% of the mass of the bacterial pulp; the amount of Bacillus subtilis protease added is 0.8%~1.5% of the mass of the bacterial pulp; the enzyme is inactivated at 90℃~95℃ for 10min~15min; and the centrifugation is performed at 8000rpm~8500rpm for 10min~15min.

[0009] The preparation of the yam peel extract in the above-mentioned pharmaceutical composition includes: taking the epidermis of fresh yam tubers, grinding and pulverizing it, adding 10 to 12 times its mass of 70% to 75% (v / v) ethanol aqueous solution, refluxing and extracting to obtain the extract, concentrating under reduced pressure, centrifuging, collecting the supernatant, loading it onto an AB-8 macroporous adsorption resin column, rinsing with deionized water for 2 to 3 BV to remove impurities, rinsing with 10% to 20% (v / v) ethanol aqueous solution for 2 to 3 BV to remove impurities, eluting with 60% to 70% (v / v) ethanol aqueous solution for 4 to 5 BV, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the yam peel extract.

[0010] In the preparation of the above-mentioned yam peel extract, the reflux extraction is performed by reflux extraction at 75℃~80℃ 2 to 3 times, each time for 1h~1.5h; the centrifugation is performed by centrifugation at 4000rpm~5000rpm for 10min~15min.

[0011] The preparation of the rainbow trout extract in the above-mentioned pharmaceutical composition includes: taking the head, skin and scales of fresh rainbow trout, drying and pulverizing them to obtain powder; adding deionized water at a material-to-liquid mass ratio of 1:(8-10), adjusting the pH to 8.0-8.5, adding trypsin, and enzymatically hydrolyzing at 35℃-40℃ for 1.5-2 hours, then adjusting the pH to 7.5-8.0, adding lumbrokinase, and enzymatically hydrolyzing at 50℃-55℃ for 1-1.5 hours, inactivating the enzyme, centrifuging, taking the supernatant, ultrafiltration using a 5kDa ultrafiltration membrane, taking the fraction with less than 5kDa, and freeze-drying to obtain the rainbow trout extract.

[0012] In the preparation of the rainbow trout extract, the fish head is a fish head with gills removed but gill covers retained; the drying temperature is 60℃~65℃; the particle size of the powder is sieved through an 80-100 mesh sieve; the amount of trypsin added is 0.8%~1.5% of the powder mass; the amount of lumbrokinase added is 0.8%~1.5% of the powder mass; the enzyme inactivation is performed at 90℃~95℃ for 10-15 minutes; and the centrifugation is performed at 8000rpm~8500rpm for 10-15 minutes.

[0013] The preparation method of the above-mentioned pharmaceutical composition for improving bone density includes the following steps: mixing milk vetch extract peptide, yam peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2 and vitamin D3 in a mass ratio to obtain the pharmaceutical composition.

[0014] The above-mentioned pharmaceutical composition is mixed with pharmaceutically acceptable excipients to prepare an oral dosage form.

[0015] The use of the above-mentioned pharmaceutical composition in the preparation of a medicament for improving bone density.

[0016] This invention provides a pharmaceutical composition for improving bone density and a method for preparing the same, with the following beneficial effects:

[0017] I. The pharmaceutical composition of this invention achieves its core effect of improving bone density through "bidirectional regulation of bone metabolism + optimized calcium utilization + enhanced safety". It can activate osteoblast proliferation and differentiation, promote collagen matrix secretion and mineralized nodule formation through active ingredients (milk worm extract peptides and rainbow trout extract), while simultaneously inhibiting osteoclast differentiation and reducing bone resorption, breaking the pathological state of "osteoclast activity > osteogenic activity" and fundamentally maintaining stable bone mass. The calcium citrate-glycine chelate avoids the formation of insoluble precipitates of calcium in the gastrointestinal tract through its chelation structure, allowing it to directly enter the body through amino acid absorption channels. This also reduces the irritation of free calcium to the gastrointestinal tract, solving the problems of "poor absorption and significant side effects" associated with traditional calcium supplements. All components undergo purification processes (LH-60 dextran gel, AB-8 macroporous adsorption resin, ultrafiltration) to remove impurities, resulting in low in vitro hemolysis and no significant toxicity, making it suitable for long-term use and meeting clinical requirements for drug safety.

[0018] II. Peptides extracted from *Millettia dielsiana*: Contains specific bioactive peptides that can activate osteoblast signaling pathways (promoting ALP synthesis and enhancing cell proliferation), thereby increasing osteoblast density. *Millettia dielsiana* fruiting bodies undergo stepwise enzymatic hydrolysis with bromelain and subtilisin to directionally cleave proteins into small-molecule bioactive peptides. LH-60 dextran gel column purification enriches the bioactive peptides, removes interfering impurities, and improves osteogenic efficiency.

[0019] III. Sweet Potato Peel Extract: Contains components that help regulate the bone metabolism microenvironment and synergistically enhance osteogenic activity. Fresh sweet potato peel is extracted by reflux with an ethanol-water solution for efficient extraction of active ingredients. Purification using an AB-8 macroporous adsorption resin column (10%–20% ethanol for impurity removal + 60%–70% ethanol for elution) removes harmful impurities, preventing them from stimulating erythrocytes or inhibiting osteoblast function, while simultaneously enriching flavonoid components to enhance synergistic osteogenic activity.

[0020] IV. Rainbow trout extract: Contains <5kDa active protein peptides, which can promote osteoblast migration and mineralization while inhibiting osteoclast signaling pathway activation. Using fish head (gills removed, gill cover retained), fish skin, and fish scales as raw materials, the protein is degraded into active small molecule peptides through stepwise enzymatic hydrolysis by trypsin and lumbrokinase, enhancing the "promote mineralization-inhibit osteoclast" effect.

[0021] V. Calcium citrate-glycine chelate: As a calcium supplement, it improves calcium absorption efficiency and reduces gastrointestinal irritation. Calcium citrate is chelated with glycine to prevent calcium from decomposing in the acidic environment of the stomach / weakly alkaline environment of the intestine. The hydrophilicity of glycine increases solubility and reduces the irritation of free calcium to the gastrointestinal mucosa.

[0022] VI. Synergistic regulation of osteogenic and osteoclast-reactive processes: The combination of lactobacillus extract peptides (which activate osteogenic signals) and rainbow trout extract (which inhibits osteoclast-reactive signals) forms a bidirectional regulatory mechanism that promotes osteogenic processes and inhibits osteoclast-reactive processes. This avoids the limitations of single ingredients that "only promote osteogenic processes but cannot prevent bone loss" or "only inhibit osteoclast-reactive processes but cannot increase bone mass," and significantly improves the balance of bone metabolism.

[0023] VII. Synergistic effect of calcium absorption and deposition: Calcium citrate-glycine chelate (enhances calcium absorption), vitamin D3 (promotes calcium absorption), and vitamin K2 (guides calcium deposition) work together to form a complete calcium utilization chain of "absorption-transport-deposition", solving the problems of "poor absorption and abnormal deposition" of traditional calcium supplements and ensuring that calcium can be effectively used for bone formation;

[0024] 8. Synergistic Effect of Active Ingredients and Purification Processes: The purification processes of each component (LH-60 dextran gel, AB-8 macroporous adsorption resin, ultrafiltration) remove impurities and prevent them from interfering with the action of active ingredients (such as tannins in yam peel extract and macromolecular peptides in rainbow trout extract). At the same time, each component is mixed in a specific ratio to ensure that the concentration of active ingredients is within the saturation range of cell receptors (such as the limited proportion of peptides extracted from milk vetch to avoid excessive absorption). This maximizes the synergistic effect and ultimately achieves the overall advantages of "high safety and significant bone density improvement". Detailed Implementation

[0025] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0026] Example 1

[0027] A pharmaceutical composition for improving bone density comprises, in a mass ratio of 4:1.5:7:4:0.08:0.02, *Dactylogyrus pulveratum* extract peptide, *Potamogeton crispus* peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2, and vitamin D3.

[0028] The preparation of *Millettia speciosa* extract peptides includes: taking fresh *Millettia speciosa* fruiting bodies, crushing them into a slurry, incubating at 92℃ for 12 min to inactivate endogenous enzymes, adding deionized water at a material-to-liquid mass ratio of 1:13, adjusting the pH to 6.8, adding 1% (by weight) of bromelain from the slurry, and enzymatically hydrolyzing at 52℃ for 1.5 h, then adjusting the pH to 7.8, adding 1% (by weight) of Bacillus subtilis protease from the slurry, and enzymatically hydrolyzing at 52℃ for 1 h, inactivating the enzyme at 92℃ for 12 min, centrifuging at 8200 rpm for 12 min, collecting the supernatant, concentrating under reduced pressure at 48℃ to 25% of its volume, loading the sample onto an LH-60 dextran gel column, rinsing with deionized water for 2.5 BV to remove impurities, eluting with 60% (by weight) ethanol aqueous solution for 3.5 BV, collecting the eluent, concentrating under reduced pressure at 48℃ to remove ethanol, and freeze-drying to obtain *Millettia speciosa* extract peptides.

[0029] The preparation of the yam peel extract includes: taking the epidermis of fresh yam tubers, grinding and pulverizing it, adding 11 times the mass of 73% (v / v) ethanol aqueous solution, reflux extraction twice at 78℃ for 1 hour each time, combining the extracts, concentrating under reduced pressure at 48℃ to remove ethanol, centrifuging at 4500 rpm for 12 minutes, collecting the supernatant, loading it onto an AB-8 macroporous adsorption resin column, rinsing with deionized water for 2.5 BV to remove impurities, rinsing with 15% (v / v) ethanol aqueous solution for 2.5 BV to remove impurities, eluting with 65% (v / v) ethanol aqueous solution for 4.5 BV, collecting the eluent, concentrating under reduced pressure at 48℃ to remove ethanol, and freeze-drying to obtain the yam peel extract.

[0030] The preparation of rainbow trout extract includes: taking the head (removing gills, retaining gill covers), skin, and scales of fresh rainbow trout, removing the fishy smell (soaking in 5wt% light salt water for 30 min, washing with deionized water), drying at 62℃, pulverizing, and passing through an 80-mesh sieve to obtain powder; adding deionized water at a material-to-liquid mass ratio of 1:9, adjusting the pH to 8.2, adding 1.2% trypsin by weight of the powder, enzymatically hydrolyzing at 38℃ for 1.5 h, then adjusting the pH to 7.8, adding 1.2% lumbrokinase by weight of the powder, enzymatically hydrolyzing at 52℃ for 1.5 h, inactivating the enzyme at 92℃ for 12 min, centrifuging at 8200 rpm for 12 min, collecting the supernatant, ultrafiltration using a 5 kDa ultrafiltration membrane, collecting the fraction below 5 kDa, freeze-drying to obtain rainbow trout extract.

[0031] The preparation of calcium citrate-glycine chelate includes: weighing raw materials according to a mass ratio of calcium citrate to glycine of 2.4:1; dissolving glycine in 9 times its mass of deionized water, adding calcium citrate under stirring at 55℃ and 350 rpm, and stirring continuously until homogeneous to obtain a mixture; adjusting the pH of the mixture to 8.0 with 1.5 wt% sodium hydroxide aqueous solution, heating to 75℃, stirring at 350 rpm for 2 hours, filtering, taking the filtrate, adding 3.5 times its volume of 92% volume concentration ethanol aqueous solution under stirring at 250 rpm, letting it stand at 5℃ for 3 hours, taking the lower precipitate, washing it 3 times with anhydrous ethanol, vacuum drying at 55℃ for 7 hours, breaking it up, and passing it through a 100-mesh sieve to obtain calcium citrate-glycine chelate.

[0032] Example 2

[0033] A pharmaceutical composition for improving bone density comprises, in a mass ratio of 5:2:8:5:0.1:0.03, a lactucalysin extract peptide, a taro peel extract, a rainbow trout extract, a calcium citrate-glycine chelate, vitamin K2, and vitamin D3.

[0034] The preparation of *Millettia speciosa* extract peptides includes: taking fresh *Millettia speciosa* fruiting bodies, crushing them into a slurry, incubating at 95℃ for 15 min to inactivate endogenous enzymes, adding deionized water at a material-to-liquid mass ratio of 1:15, adjusting the pH to 7.5, adding 1.5% (by weight) of bromelain (by weight) of the slurry, and enzymatically hydrolyzing at 50℃ for 2 h, then adjusting the pH to 8.0, adding 1.5% (by weight) of Bacillus subtilis protease (by weight) of the slurry, and enzymatically hydrolyzing at 55℃ for 1.5 h, inactivating the enzyme at 95℃ for 15 min, centrifuging at 8500 rpm for 15 min, collecting the supernatant, concentrating under reduced pressure at 50℃ to 30% of the volume, loading the sample onto an LH-60 dextran gel column, rinsing with deionized water for 3 BV to remove impurities, eluting with 70% (by weight) ethanol aqueous solution for 4 BV, collecting the eluent, concentrating under reduced pressure at 50℃ to remove ethanol, and freeze-drying to obtain *Millettia speciosa* extract peptides.

[0035] The preparation of the yam peel extract includes: taking the epidermis of fresh yam tubers, grinding and pulverizing it, adding 12 times the mass of 75% (v / v) ethanol aqueous solution, refluxing at 80℃ for 3 times, 1.5 h each time, combining the extracts, concentrating under reduced pressure at 50℃ to remove ethanol, centrifuging at 5000 rpm for 15 min, collecting the supernatant, loading it onto an AB-8 macroporous adsorption resin column, rinsing with deionized water for 3 BV to remove impurities, rinsing with 20% (v / v) ethanol aqueous solution for 3 BV to remove impurities, eluting with 70% (v / v) ethanol aqueous solution for 5 BV, collecting the eluent, concentrating under reduced pressure at 50℃ to remove ethanol, and freeze-drying to obtain the yam peel extract.

[0036] The preparation of rainbow trout extract includes: taking fresh rainbow trout heads (removing gills, retaining gill covers), skin, and scales, removing the fishy smell (soaking in 5wt% light salt water for 35 min, washing with deionized water), drying at 65℃, pulverizing, and passing through a 100-mesh sieve to obtain powder; adding deionized water at a material-to-liquid mass ratio of 1:10, adjusting the pH to 8.5, adding 1.5% trypsin by weight of the powder, enzymatically hydrolyzing at 40℃ for 2 h, then adjusting the pH to 7.5, adding 1.5% lumbrokinase by weight of the powder, enzymatically hydrolyzing at 55℃ for 1.5 h, inactivating the enzyme at 95℃ for 15 min, centrifuging at 8500 rpm for 15 min, taking the supernatant, ultrafiltration using a 5 kDa ultrafiltration membrane, taking the fraction below 5 kDa, freeze-drying to obtain rainbow trout extract.

[0037] The preparation of calcium citrate-glycine chelate includes: weighing raw materials according to a mass ratio of calcium citrate to glycine of 2.6:1; dissolving glycine in 10 times its mass of deionized water, adding calcium citrate under stirring at 60℃ and 400 rpm, and stirring continuously until homogeneous to obtain a mixture; adjusting the pH of the mixture to 8.5 with 2wt% sodium hydroxide aqueous solution, heating to 80℃, stirring at 400 rpm for 2.5 h, filtering, taking the filtrate, adding 4 times its volume of 95% volume concentration ethanol aqueous solution under stirring at 300 rpm, letting it stand at 6℃ for 4 h, taking the lower precipitate, washing 3 times with anhydrous ethanol, vacuum drying at 60℃ for 8 h, breaking it up, and passing it through a 120-mesh sieve to obtain calcium citrate-glycine chelate.

[0038] Example 3

[0039] A pharmaceutical composition for improving bone density comprises, in a mass ratio of 3:1:5:3:0.05:0.01, a lactucalysin extract peptide, a taro peel extract, a rainbow trout extract, a calcium citrate-glycine chelate, vitamin K2, and vitamin D3.

[0040] The preparation of *Millettia speciosa* extract peptides includes: taking fresh *Millettia speciosa* fruiting bodies, crushing them into a slurry, incubating at 90℃ for 10 min to inactivate endogenous enzymes, adding deionized water at a material-to-liquid mass ratio of 1:12, adjusting the pH to 6.0, adding 0.8% (by weight) of bromelain (by weight) of the slurry, and enzymatically hydrolyzing at 55℃ for 1.5 h, then adjusting the pH to 7.5, adding 0.8% (by weight) of Bacillus subtilis protease (by weight) of the slurry, and enzymatically hydrolyzing at 50℃ for 1 h, inactivating the enzyme at 90℃ for 10 min, centrifuging at 8000 rpm for 10 min, collecting the supernatant, concentrating under reduced pressure at 45℃ to 20% of the volume, loading the sample onto an LH-60 dextran gel column, rinsing with deionized water for 2 BV to remove impurities, eluting with 50% (by weight) ethanol aqueous solution for 3 BV, collecting the eluent, concentrating under reduced pressure at 45℃ to remove ethanol, and freeze-drying to obtain *Millettia speciosa* extract peptides.

[0041] The preparation of the yam peel extract includes: taking the epidermis of fresh yam tubers, grinding and pulverizing it, adding 10 times the mass of 70% (v / v) ethanol aqueous solution, refluxing and extracting twice at 75℃ for 1 hour each time, combining the extracts, concentrating under reduced pressure at 45℃ to remove ethanol, centrifuging at 4000 rpm for 10 min, collecting the supernatant, loading it onto an AB-8 macroporous adsorption resin column, rinsing with deionized water for 2 BV to remove impurities, rinsing with 10% (v / v) ethanol aqueous solution for 2 BV to remove impurities, eluting with 60% (v / v) ethanol aqueous solution for 4 BV, collecting the eluent, concentrating under reduced pressure at 45℃ to remove ethanol, and freeze-drying to obtain the yam peel extract.

[0042] The preparation of rainbow trout extract includes: taking fresh rainbow trout heads (removing gills, retaining gill covers), skin, and scales, removing the fishy smell (soaking in 5wt% light salt water for 30 min, washing with deionized water), drying at 60℃, pulverizing, and passing through an 80-mesh sieve to obtain powder; adding deionized water at a material-to-liquid mass ratio of 1:8, adjusting the pH to 8.0, adding 0.8% of the powder mass of trypsin, enzymatically hydrolyzing at 35℃ for 1.5 h, then adjusting the pH to 8.0, adding 0.8% of the powder mass of lumbrokinase, enzymatically hydrolyzing at 50℃ for 1 h, inactivating the enzyme at 90℃ for 10 min, centrifuging at 8000 rpm for 10 min, taking the supernatant, ultrafiltration using a 5 kDa ultrafiltration membrane, taking the fraction below 5 kDa, freeze-drying to obtain rainbow trout extract.

[0043] The preparation of calcium citrate-glycine chelate includes: weighing raw materials according to a mass ratio of calcium citrate to glycine of 2.2:1; dissolving glycine in 8 times its mass of deionized water, adding calcium citrate under stirring at 50℃ and 300 rpm, and stirring continuously until homogeneous to obtain a mixture; adjusting the pH of the mixture to 7.5 with 1 wt% sodium hydroxide aqueous solution, heating to 70℃, stirring at 300 rpm for 1.5 h, filtering, taking the filtrate, adding 3 times its volume of 90% volume concentration ethanol aqueous solution under stirring at 200 rpm, letting it stand at 4℃ for 2 h, taking the lower precipitate, washing twice with anhydrous ethanol, vacuum drying at 50℃ for 6 h, breaking it up, and passing it through a 100-mesh sieve to obtain calcium citrate-glycine chelate.

[0044] Example 4

[0045] A pharmaceutical composition for improving bone density comprises, by mass ratio of *Dactylogyrus rubra* extract peptide, *Potamogeton crispus* peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2, and vitamin D3, in the form of *Dactylogyrus rubra* extract peptide, *Potamogeton crispus* peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2, and vitamin D3.

[0046] The preparation of *Millettia speciosa* extract peptides includes: taking fresh *Millettia speciosa* fruiting bodies, crushing them into a slurry, incubating at 90℃ for 15 min to inactivate endogenous enzymes, adding deionized water at a material-to-liquid mass ratio of 1:12, adjusting the pH to 7.5, adding 0.8% (by weight) bromelain from the slurry, and enzymatically hydrolyzing at 53℃ for 1.5 h, then adjusting the pH to 8.0, adding 0.8% (by weight) Bacillus subtilis protease from the slurry, and enzymatically hydrolyzing at 55℃ for 1 h, inactivating the enzyme at 95℃ for 10 min, centrifuging at 8500 rpm for 10 min, collecting the supernatant, concentrating under reduced pressure at 50℃ to 20% of the volume, loading the sample onto an LH-60 dextran gel column, rinsing with deionized water for 3 BV to remove impurities, eluting with 50% (by volume) ethanol aqueous solution for 4 BV, collecting the eluent, concentrating under reduced pressure at 45℃ to remove ethanol, and freeze-drying to obtain *Millettia speciosa* extract peptides.

[0047] The preparation of the yam peel extract includes: taking the epidermis of fresh yam tubers, grinding and pulverizing it, adding 12 times the mass of 70% volume concentration ethanol aqueous solution, refluxing and extracting twice at 80℃ for 1.5 hours each time, combining the extracts, concentrating under reduced pressure at 45℃ to remove ethanol, centrifuging at 5000 rpm for 10 minutes, collecting the supernatant, loading it onto an AB-8 macroporous adsorption resin column, rinsing with deionized water for 3 BV to remove impurities, rinsing with 10% volume concentration ethanol aqueous solution for 3 BV to remove impurities, eluting with 60% volume concentration ethanol aqueous solution for 5 BV, collecting the eluent, concentrating under reduced pressure at 45℃ to remove ethanol, and freeze-drying to obtain the yam peel extract.

[0048] The preparation of rainbow trout extract includes: taking fresh rainbow trout heads (removing gills, retaining gill covers), skin, and scales, removing the fishy smell (soaking in 5wt% light salt water for 40 min, washing with deionized water), drying at 65℃, pulverizing, and passing through an 80-mesh sieve to obtain powder; adding deionized water at a material-to-liquid mass ratio of 1:10, adjusting the pH to 8.0, adding 1.5% trypsin by weight of the powder, enzymatically hydrolyzing at 35℃ for 2 h, then adjusting the pH to 7.6, adding 1.5% lumbrokinase by weight of the powder, enzymatically hydrolyzing at 50℃ for 1.5 h, inactivating the enzyme at 90℃ for 15 min, centrifuging at 8000 rpm for 15 min, taking the supernatant, ultrafiltration using a 5 kDa ultrafiltration membrane, taking the fraction below 5 kDa, freeze-drying to obtain rainbow trout extract.

[0049] The preparation of calcium citrate-glycine chelate includes: weighing raw materials according to a mass ratio of calcium citrate to glycine of 2.2:1; dissolving glycine in 10 times its mass of deionized water, adding calcium citrate under stirring at 50℃ and 400 rpm, and stirring continuously until homogeneous to obtain a mixture; adjusting the pH of the mixture to 8.5 with 1 wt% sodium hydroxide aqueous solution, heating to 70℃, stirring at 400 rpm for 1.5 h, filtering, taking the filtrate, adding 3 times the volume of 95% volume concentration ethanol aqueous solution under stirring at 300 rpm, letting it stand at 4℃ for 4 h, taking the lower precipitate, washing twice with anhydrous ethanol, vacuum drying at 60℃ for 6 h, breaking it up, and passing it through a 120-mesh sieve to obtain calcium citrate-glycine chelate.

[0050] A method for preparing a pharmaceutical composition for improving bone density according to the above embodiments includes the following steps: mixing *Dactylogyrus rubra* extract peptide, *Potamogeton crispus* peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2, and vitamin D3 in a mass ratio to obtain the pharmaceutical composition. The pharmaceutical composition is then mixed with pharmaceutically acceptable excipients to prepare an oral dosage form. The use of the pharmaceutical composition in the preparation of a medicament for improving bone density is also described.

[0051] Comparative Example 1

[0052] The difference from Example 1 is that the mass ratio of Mycorrhiza uralensis extract peptide, yam peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2 and vitamin D3 is 1:4.5:7:4:0.08:0.02.

[0053] Comparative Example 2

[0054] The difference from Example 1 is that the mass ratio of Mycorrhiza uralensis extract peptide, yam peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2 and vitamin D3 is 9:1.5:2:4:0.08:0.02.

[0055] Comparative Example 3

[0056] The difference from Example 1 is that the mass ratio of Mycorrhiza uralensis extract peptide, yam peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2 and vitamin D3 is 4:6.5:2:4:0.08:0.02.

[0057] Comparative Example 4

[0058] The difference from Example 1 is that in the preparation of the milk vetch extract peptide, bromelain is replaced by papain.

[0059] Comparative Example 5

[0060] The difference from Example 1 is that in the preparation of the milk vetch extract peptide, the subtilis protease is replaced by serrepeptidase (enzymatic hydrolysis conditions 48°C, pH 7.0).

[0061] Comparative Example 6

[0062] The difference from Example 1 is that in the preparation of the milk vetch extract peptide, LH-60 dextran gel is replaced with dextran gel G-75.

[0063] Comparative Example 7

[0064] The difference from Example 1 is that in the preparation of the yam peel extract, the AB-8 macroporous adsorption resin is replaced by macroporous adsorption resin D101.

[0065] Comparative Example 8

[0066] The difference from Example 1 is that trypsin is not used for enzymatic hydrolysis in the preparation of rainbow trout extract.

[0067] Comparative Example 9

[0068] The difference from Example 1 is that papain was used instead of lumbrokinase in the preparation of rainbow trout extract.

[0069] The raw materials used in the above embodiments and comparative examples are as follows: Vitamin K2 is from Zhengzhou Rongyuan Chemical Products Co., Ltd.; Vitamin D3 is from Shaanxi Mingcheng Pharmaceutical Co., Ltd.; Bromelain is from Peptide Biotechnology (Xi'an) Co., Ltd., with an enzyme activity of 100,000 U / g; Bacillus subtilis protease is from Nanjing Bermuda Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g; LH-60 dextran gel is from Nanjing Dulai Biotechnology Co., Ltd.; AB-8 macroporous adsorption resin is from Nanjing Dulai Biotechnology Co., Ltd.; Trypsin is from Qingdao Haiweisen Biotechnology Co., Ltd., with an enzyme activity of 4000 U / g; Lumbrokinase is from Hubei Xinkang Pharmaceutical Chemical Co., Ltd., model XK3368; Calcium citrate is from Nanjing Jiayixinwei Chemical Co., Ltd., with a purity of 99%; Glycine is from Henan Jiuxu Biotechnology Co., Ltd.; Papain is from Xi'an Changhe Pharmaceutical Co., Ltd., with an enzyme activity of 100,000 U / g; Serapeptidase is from Zhengzhou Huafeng Food Technology Co., Ltd., with an enzyme activity of 100,000 U / g; and Dextran gel G-75 is from Nanjing Dulai Biotechnology Co., Ltd. The macroporous adsorption resin D101 is sourced from Nanjing Dulai Biotechnology Co., Ltd.

[0070] I. Hemolytic test:

[0071] Sample preparation: The drug was diluted with physiological saline to prepare a test solution of 1 mg / mL, and then filtered through a 0.22 μm microfiltration membrane for sterilization.

[0072] The detection method included: Rabbit anticoagulated whole blood (10% sodium citrate anticoagulation, anticoagulant to whole blood volume ratio 1:9), centrifuged at 1500 rpm for 10 min, discarded the supernatant, and washed red blood cells three times with physiological saline, centrifuged at 1500 rpm for 10 min after each wash, finally preparing a 2% (v / v) red blood cell suspension (diluted with physiological saline). Identical test tubes were used for grouping, with the following added: test sample group (1 mL test sample solution + 1 mL red blood cell suspension), negative control group (1 mL physiological saline + 1 mL red blood cell suspension), and positive control group (1 mL distilled water + 1 mL red blood cell suspension); each group had 5 replicates. After gentle mixing, the mixture was incubated in a 37℃ constant temperature water bath for 3 h. After incubation, centrifuged at 1500 rpm for 10 min. The supernatant was collected, and the OD value was measured at a wavelength of 545 nm using a UV spectrophotometer, zeroed with physiological saline before measurement. Hemolysis rate (%) = (OD test sample - OD negative control) / (OD positive control - OD negative control) × 100%.

[0073] II. Detection of osteoblast proliferation promotion:

[0074] Sample preparation: The drug was diluted with α-MEM complete medium (containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, pH 7.4) to a solution of 250 μg / mL and then filtered through a 0.22 μm microfiltration membrane for sterilization.

[0075] The detection method includes: MC3T3-E1 cells in logarithmic growth phase are subjected to a 5×10⁻⁶ thiocyanate incubation. 3 Cells were seeded at a density of 100 μL per well in 96-well plates. After incubation at 37°C and 5% CO2 for 24 h, the original culture medium was discarded, and 100 μL of the test solution was added to each well, with 5 replicates. An equal volume of α-MEM complete culture medium was added to the negative control group. After culturing for another 72 h, 10 μL of CCK-8 reagent was added directly to each well (avoiding the generation of air bubbles). After culturing for another 4 h, the OD value was measured at 450 nm using a microplate reader, with a reference wavelength set to 650 nm. The relative cell proliferation rate (%) = (OD of test solution / OD of negative control) × 100%.

[0076] III. Detection of osteoblast differentiation promotion (alkaline phosphatase (ALP) activity assay):

[0077] Sample preparation: The drug was diluted to 250 μg / mL with osteogenic induction medium (α-MEM complete medium containing 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate sodium, and 100 nM dexamethasone, pH 7.4) and sterilized by filtration through a 0.22 μm microfiltration membrane.

[0078] The detection method includes: MC3T3-E1 cells at a concentration of 2×10⁻⁶ 4Cells were seeded at a density of 500 μL of α-MEM complete medium per well in 24-well plates. The plates were incubated at 37°C in a 5% CO2 incubator until confluence reached 80%, then the medium was replaced with the test solution. A blank control group (α-MEM complete medium) and a negative control group (osteogenic induction medium) were also included, with 5 replicates per group. The medium was changed every 2 days for 7 days. The medium was discarded, and the cells were washed twice with PBS (pH 7.4). 200 μL of 0.1% Triton X-100 lysis buffer was added to each well, and the cells were incubated on ice for 30 min, gently shaking every 10 min. The lysis buffer was collected and centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. 50 μL of the supernatant was mixed with 100 μL of pNPP substrate solution (1 mg / mL, dissolved in 0.1 M carbonate buffer at pH 10.5, containing 2 mM MgCl2), and incubated at 37°C in the dark for 30 min. The reaction was terminated by adding 50 μL of 0.1 M NaOH, and the OD value was measured at 405 nm. The protein concentration of the same supernatant was determined using a BCA protein quantification kit. ALP activity (U / mg protein) was calculated. Relative ALP activity (%) = (ALP activity of test sample / ALP activity of negative control) × 100%.

[0079] IV. Detection of Promotion of Mineralized Nodule Formation:

[0080] Sample preparation: The drug was diluted to a 250 μg / mL test solution using mineralization induction medium (α-MEM complete medium containing 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate sodium, pH 7.4) and then sterilized by filtration through a 0.22 μm microfiltration membrane.

[0081] The detection method includes: MC3T3-E1 cells at a concentration of 2×10⁻⁶ 4Cells were seeded at a density of 500 μL per well in 24-well plates. After incubation at 37°C and 5% CO2 for 24 h, the solution was replaced with the test solution. A negative control (mineralization induction medium) was also included, with 5 replicates per group. The medium was changed every 3 days for 28 consecutive days (obvious mineralization nodules appeared in the negative control group). After incubation, the medium was discarded, and the plate was washed twice with PBS (pH 7.4). 500 μL of 4% (w / v) paraformaldehyde fixative (prepared with PBS, pH 7.4) was added for fixation for 30 min. The fixative was discarded, and the plate was washed twice with deionized water. 1 mL of 0.2% (w / v) Alizarin Red S solution (pH 4.2) was added, and the plate was stained at room temperature for 30 min, gently agitating the plate every 10 min. The plate was then washed thoroughly with deionized water four times until no free dye remained. After drying, add 500 μL of 10% (w / v) hexadecylpyridine ammonium chloride solution (dissolved in 10 mM sodium phosphate buffer, pH 7.0) to each well and shake at room temperature for 30 min to dissolve the bound dye. Transfer 200 μL of the solution to a 96-well plate and measure the OD value at 562 nm. Mineralization promotion rate (%) = [(OD test sample - OD negative control) / OD negative control] × 100%.

[0082] V. Osteoclast Differentiation Inhibition Detection:

[0083] Sample preparation: The drug was diluted to 500 μg / mL of test sample stock solution with osteoclast induction medium (α-MEM complete medium containing 30 ng / mL RANKL and 25 ng / mL M-CSF, pH 7.4) and sterilized by filtration through a 0.22 μm microfiltration membrane.

[0084] The detection method included: bone marrow mononuclear cells (BMMs) were collected from the femur of 6-week-old SPF-grade C57BL / 6 male mice, pre-cultured in α-MEM complete medium containing 25 ng / mL M-CSF for 48 h, adherent stromal cells were removed, and non-adherent cells were collected and cultured at 1.5 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of 1:1 / well in 24-well plates. Each well was initially incubated with 500 μL of α-MEM complete medium containing 25 ng / mL M-CSF and cultured at 37°C for 24 h in a 5% CO2 incubator. The old medium was then discarded. New medium was added to the following groups: blank group (500 μL medium containing 25 ng / mL M-CSF), model control group (500 μL osteoclastogenesis induction medium containing 30 ng / mL RANKL and 25 ng / mL M-CSF), and test sample group (250 μL osteoclastogenesis induction medium + 250 μL test sample stock solution, i.e., a final drug concentration of 250 μg / mL). Each group had 5 replicates. Continue culturing in a 37℃, 5% CO2 incubator, changing the medium every 2 days for 7 days. Discard the medium, wash twice with PBS (pH 7.4), add 500 μL of 4% (w / v) paraformaldehyde fixative (prepared with PBS, pH 7.4) for 30 min, discard the fixative, wash twice with PBS (pH 7.4), add 500 μL of TRAP staining solution, and incubate at 37℃ in the dark for 1 h. Rinse slowly with tap water 3 times and air dry. Under an optical microscope (magnification 200×), randomly select 5 fields of view from each well and count the number of TRAP-positive osteoclasts with ≥3 nuclei in each field of view. Osteoclast differentiation inhibition rate (%) = [1 - (average number of osteoclasts in the test group / average number of osteoclasts in the model control group)] × 100%.

[0085] Table 1 Test Results

[0086]

[0087] The above results are averages.

[0088] The results above indicate that in Examples 1 to 3, each raw material was prepared using standard processes: the *Dactylogyrus* extract peptide was purified using LH-60 dextran gel electrophoresis to remove impurities; the *Potamogeton crispus* peel extract was enriched with active ingredients using AB-8 resin; and the rainbow trout extract underwent stepwise enzymatic hydrolysis and ultrafiltration screening to prevent impurities from stimulating erythrocytes. The components, in a reasonable ratio, worked synergistically to enhance cell proliferation, promote cell differentiation, aid in the formation of mineralized nodules, moderately inhibit osteoclastosis, chelate calcium to prevent the body from initiating osteoclast compensation, and maintain bone metabolic balance.

[0089] In Comparative Example 1, the proportion of peptides extracted from *Lactobacillus thuringiensis* decreased, while the proportion extracted from *Polygonum cuspidatum* peel increased. *Lactobacillus thuringiensis* extract contains specific bioactive peptides that promote cell proliferation and ALP synthesis by activating osteoblast signaling pathways; the osteogenic effect of *Polygonum cuspidatum* peel extract is weaker than that of the bioactive peptides. When the proportion of *Lactobacillus thuringiensis* extract peptides decreased and the proportion of *Polygonum cuspidatum* peel extract increased, the bioactive signals required for osteoblast activation were insufficient, leading to weakened cell proliferation and ALP activity. Simultaneously, the small amount of tannins introduced by the excessive amount in *Polygonum cuspidatum* peel extract slightly inhibited osteoclast apoptosis, weakening the osteoclast-inhibiting effect. Furthermore, impurities in the *Polygonum cuspidatum* peel extract slightly irritated erythrocytes, resulting in a slight increase in hemolysis rate. Weakened osteoblast function directly affects collagen matrix secretion and calcium deposition, ultimately reducing the mineralization promotion rate.

[0090] In Comparative Example 2, the proportion of rainbow trout extract decreased, while the proportion of *Lactobacillus thuringiensis* extract peptides increased. Collagen peptides in rainbow trout extract promote osteoblast migration and mineralization through signaling pathways and inhibit osteoclast signaling pathway activation. When the proportion of rainbow trout extract decreased, insufficient collagen peptide supply led to weakened osteoblast migration and mineral matrix synthesis, thus reducing the inhibition of osteoclast activity. Although the proportion of *Lactobacillus thuringiensis* extract peptides increased, it exceeded the saturation concentration of cell receptors, resulting in limited absorption and therefore limited efficacy. Excessive amounts of these components also produced mild toxicity, interfering with normal osteoblast metabolism and reducing cell proliferation and ALP activity. High levels of incompletely purified small molecule impurities also slightly damaged erythrocyte membranes, leading to increased hemolysis.

[0091] In Comparative Example 3, the proportion of yam peel extract increased while the proportion of rainbow trout extract decreased. Diosgenin from yam peel extract needs to work synergistically with collagen peptides from rainbow trout extract to help regulate the bone metabolic microenvironment. When the proportion of yam peel extract increased and the proportion of rainbow trout extract decreased, their synergistic effect was disrupted: insufficient collagen peptides led to reduced extracellular matrix synthesis in osteoblasts, resulting in a lack of structural support for the formation of mineralized nodules; excessive polysaccharides in yam peel extract competitively bound to receptors on the surface of osteoblasts, inhibiting active signal transduction and further weakening cell proliferation and ALP activity. Simultaneously, the reduced proportion of rainbow trout extract weakened the inhibition of RANKL-induced osteoclast activation, decreasing the osteoclast-inhibiting effect; excessive accumulation of impurities in yam peel extract also slightly irritated erythrocytes, leading to increased hemolysis.

[0092] In Comparative Example 4, bromelain was replaced with papain. Bromelain specifically cleaves peptide bonds in the fruiting body proteins of *Millettia dielsiana*, generating osteogenic small peptides; papain's cleavage sites are concentrated on basic amino acids, and its enzymatic products are mainly inactive large peptides. After the enzyme replacement, the content of active peptides in *Millettia dielsiana* extract was significantly reduced, resulting in insufficient activation of osteoblast signaling pathways, leading to weakened cell proliferation and ALP activity. The lack of active peptides also reduces the secretion of osteoblast mineralization-related proteins, lowers the mineralization promotion rate, and weakens the apoptosis-inducing effect on osteoclasts, thus reducing the osteoclast inhibition rate.

[0093] In Comparative Example 5, subtilisin was replaced with serratide peptide. Subtilisin can further degrade the enzymatic hydrolysis products of bromelain, generating more active small molecule peptides; serratide peptide does not have a synergistic effect with the original process, resulting in insufficiently degraded peptides with different sequences, reducing penetration of osteoblast membranes and causing insufficient activation of intracellular signaling pathways in osteoblasts, leading to decreased cell proliferation and ALP activity; the expression of mineralization-related genes is reduced, further decreasing the mineralization promotion rate. At the same time, insufficient concentration of active peptides cannot effectively block the osteoclast NF-κB signaling pathway, weakening the osteoclast inhibition effect.

[0094] In Comparative Example 6, the LH-60 dextran gel was replaced with G-75. G-75 and LH-60 have different separation ranges; replacement with G-75 resulted in the loss of some effective bioactive peptides and the introduction of other harmful impurities. The decreased bioactive peptide content led to insufficient osteoblast activation signals, weakened cell proliferation and ALP activity; simultaneously, insufficient bioactive peptides failed to effectively inhibit the expression of osteoclast differentiation-related genes, resulting in a decreased osteoclast inhibition rate.

[0095] In Comparative Example 7, the AB-8 macroporous adsorption resin was replaced with D101. After purification with D101 resin, the purity of the active ingredients decreased; insufficient flavonoids weakened the role of assisting osteoblast activation, failing to synergistically promote ALP gene transcription, leading to decreased cell proliferation and ALP activity; simultaneously, it affected collagen fiber cross-linking maturation, reducing the mineralization promotion rate. Furthermore, flavonoids inhibit the generation of reactive oxygen species in osteoclasts, and increased impurities slightly promoted osteoclast activation, thus decreasing the osteoclast inhibition rate; the tannins adsorbed by D101 resin had a slight irritant effect on erythrocytes, resulting in a slight increase in hemolysis rate.

[0096] In Comparative Example 8, trypsin hydrolysis was omitted. Collagen in rainbow trout heads, skin, and scales requires initial enzymatic hydrolysis with trypsin to break it down into effective small peptides, followed by further degradation with lumbrokinase into bioactive peptides <5kDa. Omitting trypsin reduces the efficiency of lumbrokinase's direct action on large collagen molecules, resulting in decreased bioactive peptide production and the introduction of impurity peptides. Insufficient bioactive peptides lead to inadequate activation of osteoblast proliferation pathways and a decreased cell proliferation rate. Simultaneously, the lack of bioactive peptides weakens osteoblast differentiation signals, reduces ALP activity, and decreases mineralized matrix synthesis. Furthermore, the synergistic inhibitory effect of bioactive peptides on osteoclasts is weakened, resulting in a decreased osteoclast inhibition rate; the introduction of impurity peptides also compromises safety.

[0097] In Comparative Example 9, lumbrokinase was replaced with papain. Lumbrokinase can specifically degrade protein peptides after trypsin digestion, generating specific active peptides that promote osteoblast adhesion and proliferation. Papain has a significantly different cleavage site than lumbrokinase, and cannot effectively cleave to form specific sequence active peptides. The different peptide structures result in different efficacy directions and active components, leading to differences in drug efficacy.

Claims

1. A pharmaceutical composition for improving bone density, characterized in that, The pharmaceutical composition consists of milkweed extract peptide, yam peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2 and vitamin D3 in a mass ratio of (3-5):(1-2):(5-8):(3-5):(0.05-0.1):(0.01-0.03); The preparation of the yam peel extract includes: taking the epidermis of fresh yam tubers, grinding and pulverizing it, adding 10 to 12 times the mass of 70% to 75% volume concentration ethanol aqueous solution, refluxing and extracting to obtain the extract, concentrating under reduced pressure, centrifuging, collecting the supernatant, loading it onto an AB-8 macroporous adsorption resin column, rinsing with deionized water for 2 to 3 BV to remove impurities, rinsing with 10% to 20% volume concentration ethanol aqueous solution for 2 to 3 BV to remove impurities, eluting with 60% to 70% volume concentration ethanol aqueous solution for 4 to 5 BV, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the yam peel extract; The calcium citrate to glycine chelate chelate has a chelate mass ratio of (2.2-2.6):

1. The preparation of the *Millettia speciosa* extract peptides includes: crushing *Millettia speciosa* fruiting bodies into a slurry, inactivating endogenous enzymes, adding deionized water at a material-to-liquid mass ratio of 1:(12-15), adjusting the pH to 6.0-7.5, adding 0.8%-1.5% bromelain by weight of the slurry, and enzymatically hydrolyzing at 50℃-55℃ for 1.5-2 hours, then adjusting the pH to 7.5-8.0, adding 0.8%-1.5% Bacillus subtilis protease by weight of the slurry, and enzymatically hydrolyzing at 50℃-55℃ for 1-1.5 hours, inactivating the enzymes, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading the sample onto an LH-60 dextran gel column, rinsing with deionized water for 2-3 BV to remove impurities, eluting with 50%-70% ethanol aqueous solution for 3-4 BV, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain *Millettia speciosa* extract peptides; The preparation of the rainbow trout extract includes: taking the head, skin, and scales of fresh rainbow trout, drying them, pulverizing them to obtain powder; adding deionized water at a material-to-liquid mass ratio of 1:(8-10), adjusting the pH to 8.0-8.5, adding 0.8%-1.5% trypsin by weight of the powder, and enzymatically hydrolyzing at 35℃-40℃ for 1.5-2 hours; then adjusting the pH to 7.5-8.0, adding 0.8%-1.5% lumbrokinase by weight of the powder, and enzymatically hydrolyzing at 50℃-55℃ for 1-1.5 hours; inactivating the enzyme; centrifuging; taking the supernatant; ultrafiltration using a 5kDa ultrafiltration membrane; taking the fraction with a mass below 5kDa; and freeze-drying to obtain the rainbow trout extract.

2. The pharmaceutical composition for improving bone density according to claim 1, characterized in that, In the preparation of peptides extracted from *Dactylogyrus latifolia*, the endogenous enzyme is inactivated by incubation at 90℃~95℃ for 10min~15min; the enzyme is inactivated at 90℃~95℃ for 10min~15min; and the centrifugation is performed at 8000rpm~8500rpm for 10min~15min.

3. The pharmaceutical composition for improving bone density according to claim 1, characterized in that, In the preparation of the yam peel extract, the reflux extraction is performed by reflux extraction at 75℃~80℃ 2 to 3 times, each time for 1h~1.5h; the centrifugation is performed by centrifugation at 4000rpm~5000rpm for 10min~15min.

4. The pharmaceutical composition for improving bone density according to claim 1, characterized in that, In the preparation of rainbow trout extract, the fish head is a fish head with gills removed but gill covers retained; the drying temperature is 60℃~65℃; the particle size of the powder is sieved through an 80-100 mesh sieve; the enzyme inactivation is performed at 90℃~95℃ for 10-15 minutes; and the centrifugation is performed at 8000rpm~8500rpm for 10-15 minutes.

5. A method for preparing a pharmaceutical composition for improving bone density according to claim 1, characterized in that, The process includes the following steps: mixing milk vetch extract peptides, yam peel extract, rainbow trout extract, calcium citrate-glycine chelate, vitamin K2 and vitamin D3 in a specific mass ratio to obtain a pharmaceutical composition.

6. The method for preparing a pharmaceutical composition for improving bone density according to claim 5, characterized in that, The pharmaceutical composition is mixed with pharmaceutically acceptable excipients to prepare an oral dosage form.

7. Use of the pharmaceutical composition for increasing bone density as described in claim 1 in the preparation of a medicament for increasing bone density.

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