A compound preparation for promoting fracture regeneration and healing and its preparation method

Abstract

This invention provides a compound preparation for promoting fracture regeneration and healing, and its preparation method. The compound preparation contains bioactive peptides, active ingredients from traditional Chinese medicine, angiogenesis-promoting components, bone morphogenetic proteins, chondroitin sulfate, vitamin D3, and alginate, among other active ingredients. The active ingredients from traditional Chinese medicine include various raw materials such as puerarin. The bioactive peptides and angiogenesis-promoting components are specifically formulated. During preparation, the bioactive peptides and active ingredients from traditional Chinese medicine are microencapsulated, the angiogenesis-promoting components are made into a nanoemulsion, and the bone morphogenetic proteins are freeze-dried, ensuring the stability and bioavailability of these active ingredients, thereby further enhancing the efficacy of the preparation. This compound preparation not only effectively promotes angiogenesis and bone tissue repair at the fracture site but also accelerates the fracture healing process and reduces patient pain. It demonstrates significant advantages in promoting fracture regeneration and healing, providing patients with a more efficient and safer treatment option.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, and in particular to a compound preparation for promoting fracture regeneration and healing, and its preparation method. Background Technology

[0002] Fractures are a common clinical trauma. The regeneration and healing process after a fracture involves complex physiological mechanisms, requiring not only the bone tissue's own repair capabilities but also factors such as good blood supply, cytokine regulation, and a suitable microenvironment. Timely and effective treatment is crucial for restoring bone function and reducing complications.

[0003] Currently, there are numerous methods and medications available in clinical practice to promote fracture healing, but all have certain limitations. Traditional treatment methods mainly include surgical reduction and fixation, and adjuvant drug therapy. While surgical reduction and fixation can restore the anatomical structure of the fracture site, the postoperative recovery process is lengthy and carries risks such as infection and secondary injury.

[0004] In terms of drug treatment, existing drugs for promoting fracture healing have many shortcomings. Common calcium and vitamin D supplements mainly function to replenish the basic building blocks of bone, but their effects on cell regulation and angiogenesis during the fracture healing process are limited. Bisphosphonates can inhibit bone resorption, but their effect on promoting new bone formation is not ideal. Parathyroid hormone analogs can promote osteoblast activity, but long-term use may lead to some adverse reactions.

[0005] Traditional Chinese medicine preparations have certain advantages in promoting fracture healing and can regulate the body's functions as a whole. However, their ingredients are complex, making quality control difficult, and the efficacy of different batches of products may vary.

[0006] Biologics such as bone growth factor drugs and platelet-rich plasma can directly promote bone formation and angiogenesis, but they have problems such as poor stability and harsh storage and use conditions, which limit their widespread application.

[0007] Furthermore, most existing drugs for promoting fracture healing only focus on a single aspect, such as bone formation or angiogenesis, lacking comprehensive regulation of the complex process of fracture healing. Moreover, the dosage forms and administration methods of these drugs are also inadequate, making it difficult to accurately deliver the drugs to the fracture site and affecting the full effectiveness of their therapeutic effects.

[0008] Therefore, developing a compound preparation that can comprehensively regulate multiple aspects of fracture healing, has good stability, safety, and efficacy, as well as a matching preparation method, is of great clinical significance for improving the treatment effect of fractures. Summary of the Invention

[0009] In view of this, the present invention proposes a compound preparation for promoting fracture regeneration and healing and its preparation method, thereby solving the above problems.

[0010] The technical solution of this invention is achieved as follows: a compound preparation for promoting fracture regeneration and healing, comprising the following active ingredients by weight percentage: 5-15% bioactive peptides, 5-10% traditional Chinese medicine active ingredients, 10-20% angiogenesis-promoting ingredients, 2-6% bone morphogenetic protein, 1-3% chondroitin sulfate, 0.5-4% vitamin D3, 2-3% alginate, with the remainder being a pharmaceutical carrier; the traditional Chinese medicine active ingredients comprise the following raw materials by weight: 13-20 parts puerarin, 12-15 parts berberine, 5-10 parts sanguisorba officinalis polysaccharide, 5-12 parts tanshinone IIA, 5-8 parts datura stramonium glycoside, 3-8 parts datura saponin VI, 2-7 parts tanshinone B, 3-5 parts matrine, 4-9 parts astragaloside A, and 1-3 parts oridonine A.

[0011] Furthermore, it contains the following active ingredients by weight percentage: 10% bioactive peptides, 8% traditional Chinese medicine active ingredients, 15% angiogenesis-promoting ingredients, 4% bone morphogenetic protein, 2% chondroitin sulfate, 2.2% vitamin D3, 2.5% alginate, with the remainder being a pharmaceutical carrier.

[0012] Furthermore, the bioactive peptides are deer antler polypeptide powder, yak bone collagen peptides and oyster peptides in a mass ratio of (3.5-5.5):(1.3-2.5):(6.5-8.1), which promote bone cell proliferation.

[0013] Furthermore, the angiogenic components are allicin, ginkgolide, and naringin in a mass ratio of (10-15):(5-8):(2-4).

[0014] Furthermore, the bone morphogenetic protein is any one or a combination of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14.

[0015] Furthermore, the pharmaceutical carrier includes, but is not limited to, fillers, disintegrants, lubricants, and binders; wherein the filler is microcrystalline cellulose, the disintegrant is sodium carboxymethyl starch, the lubricant is magnesium stearate, and the binder is hydroxypropyl methylcellulose.

[0016] Furthermore, the dosage form of this compound preparation is any one of tablets, capsules, granules, injections, gels, or sprays.

[0017] Furthermore, a method for preparing a compound preparation for promoting fracture regeneration and healing includes the following steps:

[0018] S1. Mix the bioactive peptides and active ingredients of traditional Chinese medicine evenly, then mix them with the wall material solution, and prepare microcapsules by spray drying. The microcapsule particle size is controlled at 10-50μm.

[0019] S2. Add the angiogenesis-promoting ingredients to Tween-80 and Span-80 as emulsifiers, then slowly add water, and emulsify in a high-speed homogenizer at a speed of 10,000-15,000 rpm to form a nanoemulsion with an average particle size of 50-200 nm. Spray dry the nanoemulsion to obtain nanoscale angiogenesis-promoting ingredient powder for later use.

[0020] S3. Dissolve bone morphogenetic protein in a buffer solution containing a protective agent and freeze-dry to obtain dried bone morphogenetic protein powder.

[0021] S4. The microcapsules of S1, the angiogenesis-promoting component powder of S2, the bone morphogenetic protein powder, chondroitin sulfate, vitamin D3 and alginate are ultrasonically mixed at 10-30 rpm for 30-120 minutes to ensure that all components are fully mixed and homogeneous. The pharmaceutical carrier is then added according to the dosage form, which can be any one of tablets, capsules, granules, injections, gels or sprays.

[0022] Furthermore, the wall material solution of S1 is a mixture of chitosan and 0.5-1% v / v acetic acid solution, with a concentration of 1-3% w / v.

[0023] Furthermore, the amounts of Tween-80 and Span-80 added in S2 are 2-5% and 1-3% of the mass of the angiogenesis-promoting components, respectively, and the amount of water added is 2-4 times the mass of the angiogenesis-promoting components.

[0024] Furthermore, the ultrasonic frequency of S4 is 20-40kHz, and the power is 100-300W.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] The compound preparation for promoting fracture regeneration and healing, and its preparation method, present in this invention, exhibits several significant advantages. The formulation contains a precise and synergistic ratio of various active ingredients, including bioactive peptides, active components of traditional Chinese medicine, angiogenesis-promoting components, and bone morphogenetic proteins. This comprehensive approach accelerates the fracture healing process across multiple stages, from promoting cell proliferation and differentiation, regulating bone metabolism, improving blood circulation, to inducing bone formation, overcoming the limitations of traditional drugs that focus on only a single stage. The well-defined composition not only ensures stable and controllable quality, reducing the instability in efficacy caused by the complexity of components and batch variations in traditional Chinese medicine preparations, but also facilitates in-depth research into the mechanism of action. The unique preparation process, employing spray drying to prepare microcapsules, nanoemulsion technology, and freeze-drying, enhances drug stability and bioavailability, ensuring the effectiveness of each component. Furthermore, this compound preparation can be formulated into various dosage forms to meet the needs of different clinical scenarios and patients. Combined with pharmaceutically acceptable carriers, it ensures the formation, disintegration, flow, and stability of the preparation, comprehensively improving its quality and efficacy. Detailed Implementation

[0027] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0028] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Example 1

[0030] A compound preparation for promoting fracture regeneration and healing contains the following active ingredients by weight percentage: 5% bioactive peptides, 5% active ingredients from traditional Chinese medicine, 10% pro-angiogenic ingredients, 2% bone morphogenetic protein, 1% chondroitin sulfate, 0.5% vitamin D3, 2% alginate, and the remainder being a pharmaceutical carrier.

[0031] The bioactive peptides are deer antler polypeptide powder, yak bone collagen peptides and oyster peptides in a mass ratio of 3.5:1.3:6.5.

[0032] The active ingredients of the traditional Chinese medicine include the following raw materials in parts by weight: 13 parts puerarin, 12 parts berberine, 5 parts sanguisorbin, 5 parts tanshinone IIA, 5 parts datura stramonium glycoside, 3 parts chuanxiong saponin VI, 2 parts tanshinone B, 3 parts matrine, 4 parts astragaloside A, and 1 part oridonin A.

[0033] The angiogenic components are allicin, ginkgolide, and naringin in a mass ratio of 10:5:2.

[0034] The bone morphogenetic protein mentioned is bone morphogenetic protein-2. Example 2

[0035] A compound preparation for promoting fracture regeneration and healing contains the following active ingredients by weight percentage: 15% bioactive peptides, 10% traditional Chinese medicine active ingredients, 20% angiogenesis-promoting ingredients, 6% bone morphogenetic protein, 3% chondroitin sulfate, 4% vitamin D3, 3% alginate, and the remainder being a pharmaceutical carrier.

[0036] The bioactive peptides are deer antler polypeptide powder, yak bone collagen peptides and oyster peptides in a mass ratio of 5.5:2.5:8.1.

[0037] The active ingredients of the traditional Chinese medicine include the following raw materials in parts by weight: 20 parts of puerarin, 15 parts of berberine, 10 parts of sanguisorba officinalis polysaccharide, 12 parts of tanshinone IIA, 8 parts of datura stramonium glycoside, 8 parts of datura saponin VI, 7 parts of salvianolic acid B, 5 parts of matrine, 9 parts of astragaloside A, and 3 parts of oridonine A.

[0038] The angiogenic components are allicin, ginkgolide, and naringin in a mass ratio of 15:8:4.

[0039] The bone morphogenetic protein mentioned is bone morphogenetic protein-7. Example 3

[0040] A compound preparation for promoting fracture regeneration and healing contains the following active ingredients by weight percentage: 10% bioactive peptides, 8% active ingredients from traditional Chinese medicine, 15% pro-angiogenic ingredients, 4% bone morphogenetic protein, 2% chondroitin sulfate, 2.2% vitamin D3, 2.5% alginate, with the remainder being a pharmaceutical carrier.

[0041] The bioactive peptides are deer antler polypeptide powder, yak bone collagen peptides and oyster peptides in a mass ratio of 4.5:1.8:7.

[0042] The active ingredients of the traditional Chinese medicine include the following raw materials in parts by weight: 18 parts of puerarin, 14 parts of berberine, 8 parts of sanguisorba officinalis polysaccharide, 8 parts of tanshinone IIA, 7 parts of datura stramonium glycoside, 5 parts of chuanxiong saponin VI, 5 parts of salvianolic acid B, 4 parts of matrine, 6 parts of astragaloside A, and 2 parts of oridonine A.

[0043] The angiogenic components are allicin, ginkgolide, and naringin in a mass ratio of 13:7:3.

[0044] The bone morphogenetic protein is bone morphogenetic protein-14.

[0045] The above Examples 1-3 were prepared using the following methods:

[0046] S1. Mix the bioactive peptides and active ingredients of traditional Chinese medicine evenly, and then mix them with the wall material solution. The wall material solution is a mixture of chitosan and 0.7% v / v acetic acid solution with a concentration of 2% w / v. Microcapsules are prepared by spray drying, and the microcapsule particle size is controlled at 130 μm.

[0047] S2. Add Tween-80 and Span-80 as emulsifiers to the angiogenesis-promoting components at 4% and 2% of the mass of the angiogenesis-promoting components, respectively. Then slowly add water at 3 times the mass of the angiogenesis-promoting components and emulsify in a high-speed homogenizer at 13,000 rpm to form a nanoemulsion with an average particle size of 150 nm. Spray dry the nanoemulsion to obtain nano-sized angiogenesis-promoting component powder for later use.

[0048] S3. Dissolve bone morphogenetic protein in a buffer solution containing a protective agent and freeze-dry it to obtain dried bone morphogenetic protein powder.

[0049] S4. The microcapsules of S1, the angiogenesis-promoting component powder of S2, the bone morphogenetic protein powder, chondroitin sulfate, vitamin D3 and alginate are ultrasonically mixed at a speed of 10-30 rpm for 80 minutes. The ultrasonic frequency is 30 kHz and the power is 200 W to ensure that all components are fully mixed and homogeneous, thus preparing a spray.

[0050] Comparative Example 1

[0051] The difference between this comparative example and Example 3 is that the compound preparation does not contain bioactive peptides.

[0052] Comparative Example 2

[0053] The difference between this comparative example and Example 3 is that the compound preparation does not contain any active ingredients of traditional Chinese medicine.

[0054] Comparative Example 3

[0055] The difference between this comparative example and Example 3 is that the compound preparation does not contain any angiogenesis-promoting ingredients.

[0056] I. In vitro cell experiments

[0057] 1. Osteoblast proliferation experiment

[0058] 1.1 Cell Culture: Osteoblast cell line (MC3T3-E1 cells) was cultured in α-MEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator.

[0059] 1.2 Grouping and Drug Administration: Cells were seeded in 96-well plates. After cell attachment, they were divided into a blank control group (containing only culture medium), a positive control group (teriparatide), and an experimental group containing the compound preparation of this invention. Each group received the corresponding treatment, with multiple replicates for each group.

[0060] 1.3 Cell proliferation assay: Cell proliferation was detected using the MTT assay. At different time points after drug administration (days 1, 3, 5, and 7), MTT solution (5 mg / mL) was added to 96-well plates, and after incubation for 24 hours, the absorbance was measured using a microplate reader. The cell proliferation rate was calculated based on the absorbance values ​​to evaluate the effect of the compound preparation on osteoblast proliferation.

[0061] 2. Cell differentiation detection

[0062] 2.1 Alkaline phosphatase (ALP) activity assay: Cells were collected at a specific time point (7 days) after drug administration, and cells were lysed with cell lysis buffer. The ALP activity in the cell lysis buffer was measured according to the ALP assay kit instructions to reflect the early differentiation of osteoblasts.

[0063] 2.2 Osteocalcin (OCN) secretion detection: The OCN content in the culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). At different time points after drug administration (14 days), cell culture supernatant was collected, and the OCN secretion level was detected according to the steps of the OCN ELISA kit to assess the late differentiation of osteoblasts.

[0064] 2.3 Calcium nodule formation detection: Calcium nodule formation was observed 21 days after drug administration using alizarin red staining. The culture medium was discarded, cells were washed with PBS, fixed with 4% paraformaldehyde, and then stained with alizarin red. The number and area of ​​calcium nodules were observed and counted under a microscope to assess the effect of the compound preparation on osteoblast mineralization capacity.

[0065] Test Results

[0066] Table 1: Results of osteoblast proliferation assay

[0067]

[0068] Table 2: Results of tests on osteoblast differentiation-related indicators

[0069]

[0070] This suggests that these compound preparations have good potential to promote bone regeneration and healing, possibly by regulating osteoblast growth and differentiation signaling pathways, increasing the number and activity of osteoblasts, thereby accelerating bone tissue formation and repair.

[0071] II. Fracture Model Experiment

[0072] 1. Model Establishment: Healthy adult SD rats, weighing 250-300g, half male and half female, were purchased from a reputable experimental animal center. The animals were acclimatized for one week in an environment with a temperature of (22±2)℃ and a relative humidity of (50±10)%, with free access to food and water. A fracture model was created surgically. Under anesthesia, the femur or tibia of the animal was exposed, and a fracture was induced using a bone saw or bone forceps. The fracture site was then fixed with Kirschner wires or plates to ensure the stability and consistency of the fracture model.

[0073] 2. Grouping and Administration: Rats were randomly divided into 8 groups of 10 rats each: a blank control group (administered 0.5 mL of physiological saline), a positive control group (administered bone-setting tablets), and an experimental group containing the compound preparation of this invention. Administration began post-surgery via injection of 0.5 mL once daily until the end of the experiment, and the healing time was calculated.

[0074] 3. Imaging examinations

[0075] X-ray examination: X-ray images of the fracture sites in rats were taken at 2 and 8 weeks post-surgery. The fracture healing status was observed, including callus formation and the degree of blurring of the fracture line. The X-ray callus scoring scale (1-10 points, where 1 point indicates a clear fracture line and no callus formation; 10 points indicates the fracture line has disappeared and the callus is well-formed) was used for scoring.

[0076] CT scan: Twelve weeks post-surgery, the fracture sites in rats were scanned using a CT scanner to measure changes in bone mineral density (BMD). BMD values ​​in the fracture areas were analyzed using software and compared with pre-operative normal BMD values.

[0077] 4. Test Results

[0078]

[0079] The results, considering fracture healing time, X-ray callus score, and CT bone mineral density changes, clearly demonstrate that the compound preparations in Examples 1-3 exhibit superior performance in promoting fracture healing. They not only shorten healing time but also significantly promote callus growth and increase bone mineral density. Example 3 showed the most outstanding effect, with all indicators superior to the other groups. The positive control drug also showed some effect in promoting fracture healing, but the compound preparations in the example groups were significantly more effective.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound preparation for promoting fracture regeneration and healing, characterized in that: It is made from the following active ingredients by weight percentage: 5-15% bioactive peptides, 5-10% traditional Chinese medicine active ingredients, 10-20% angiogenesis-promoting ingredients, 2-6% bone morphogenetic protein, 1-3% chondroitin sulfate, 0.5-4% vitamin D3, 2-3% alginate, and the remainder being a pharmaceutical carrier; the traditional Chinese medicine active ingredients include the following raw materials by weight: 13-20 parts puerarin, 12-15 parts berberine, 5-10 parts Sanguisorba officinalis polysaccharide, 5-12 parts tanshinone IIA, 5-8 parts datura stramonium glycoside, 3-8 parts datura saponin VI, 2-7 parts salvianolic acid B2, 3-5 parts matrine, 4-9 parts astragaloside A, and 1-3 parts oridonine A; the bioactive peptides are deer antler polypeptide powder, yak bone collagen peptides, and oyster peptides in a mass ratio of (3.5-5.5):(1.3-2.5):(6.5-8.1); The preparation method of the compound preparation for promoting fracture regeneration and healing includes the following steps: S1. Mix bioactive peptides and active ingredients of traditional Chinese medicine evenly, then mix with wall material solution, and prepare microcapsules by spray drying method, with microcapsule particle size controlled at 130μm; S2. Add the angiogenesis-promoting ingredients to Tween-80 and Span-80 as emulsifiers, then slowly add water, and emulsify in a high-speed homogenizer at a speed of 10,000-15,000 rpm to form a nanoemulsion with an average particle size of 50-200 nm. Spray dry the nanoemulsion to obtain nanoscale angiogenesis-promoting ingredient powder for later use. S3. Dissolve bone morphogenetic protein in a buffer solution containing a protective agent and freeze-dry it to obtain dried bone morphogenetic protein powder. S4. The microcapsules of S1, the angiogenesis-promoting component powder of S2, the bone morphogenetic protein powder, chondroitin sulfate, vitamin D3 and alginate are ultrasonically mixed at 10-30 rpm for 30-120 minutes to ensure that all components are fully mixed and homogeneous. The pharmaceutical carrier is then added according to the dosage form, which can be any one of tablets, capsules, granules, injections, gels or sprays.

2. The compound preparation for promoting fracture regeneration and healing as described in claim 1, characterized in that: It is made from the following active ingredients by weight percentage: 10% bioactive peptides, 8% active ingredients from traditional Chinese medicine, 15% pro-angiogenic ingredients, 4% bone morphogenetic protein, 2% chondroitin sulfate, 2.2% vitamin D3, 2.5% alginate, and the remainder is a pharmaceutical carrier.

3. The compound preparation for promoting fracture regeneration and healing as described in claim 1, characterized in that: The angiogenic components are allicin, ginkgolide, and naringin in a mass ratio of (10-15):(5-8):(2-4).

4. The compound preparation for promoting fracture regeneration and healing as described in claim 1, characterized in that: The bone morphogenetic protein is any one or a combination of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14.

5. The compound preparation for promoting fracture regeneration and healing as described in claim 1, characterized in that: The pharmaceutical carrier includes, but is not limited to, fillers, disintegrants, lubricants, and binders; wherein the filler is microcrystalline cellulose, the disintegrant is sodium carboxymethyl starch, the lubricant is magnesium stearate, and the binder is hydroxypropyl methylcellulose.

6. The compound preparation for promoting fracture regeneration and healing as described in claim 1, characterized in that: The wall material solution of S1 is a mixture of chitosan and 0.5-1% v / v acetic acid solution, with a concentration of 1-3% w / v.

7. The compound preparation for promoting fracture regeneration and healing as described in claim 1, characterized in that: The amounts of Tween-80 and Span-80 added in S2 are 2-5% and 1-3% of the mass of the angiogenesis-promoting components, respectively, and the amount of water added is 2-4 times the mass of the angiogenesis-promoting components.

8. The compound preparation for promoting fracture regeneration and healing as described in claim 1, characterized in that: The ultrasonic frequency of S4 is 20-40kHz, and the power is 100-300W.

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