Compound preparation for promoting fracture regeneration and healing and preparation method thereof

Abstract

The invention provides a compound preparation for promoting fracture regeneration and healing and a preparation method thereof.The compound preparation comprises bioactive peptide, traditional Chinese medicine active ingredients, angiogenesis promoting ingredients, bone morphogenetic protein, chondroitin sulfate, vitamin D3, alginate and other active ingredients, and the traditional Chinese medicine active ingredients cover puerarin and other raw materials; in the preparation process, the bioactive peptide and the traditional Chinese medicine active ingredients are subjected to microencapsulation treatment, the blood vessel growth promoting ingredients are prepared into nano-emulsion, and the bone morphogenetic protein is freeze-dried, so that the stability and bioavailability of the active ingredients are ensured, and the curative effect of the preparation is further enhanced. The compound preparation disclosed by the invention not only can effectively promote the revascularization and bone tissue repair of a fracture part, but also can accelerate the fracture healing process and relieve the pain of a patient. The compound has remarkable advantages in the aspects of promoting fracture regeneration and healing, and provides a more efficient and safer treatment choice for patients.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a compound preparation for promoting fracture regeneration and healing and a preparation method thereof. Background Art

[0002] Fractures are common clinical injuries. The regeneration and healing process after fracture involves complex physiological mechanisms, which not only requires the repair ability of bone tissue itself, but also depends on a variety of factors such as good blood supply, cytokine regulation, and a suitable microenvironment. Timely and effective treatment is crucial to restore bone function and reduce complications.

[0003] At present, there are many methods and drugs to promote fracture healing in clinical practice, but they all have certain limitations. Traditional treatment methods mainly include surgical reduction and fixation and drug-assisted treatment. Although surgical reduction and fixation can restore the anatomical structure of the fracture site, the postoperative recovery process is long and there are risks such as infection and secondary injury.

[0004] In terms of drug treatment, existing drugs that promote fracture healing have many shortcomings. Common calcium and vitamin D drugs mainly supplement the basic raw materials for bone construction, but have limited effects on cell regulation and angiogenesis during fracture healing. Although bisphosphonates can inhibit bone resorption, their effect on promoting new bone formation is not ideal. Although parathyroid hormone analogs can promote osteoblast activity, long-term use may cause some adverse reactions.

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

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

[0007] In addition, most existing drugs that promote fracture healing only focus on a single link, such as bone formation or angiogenesis, and lack comprehensive regulation of the complex process of fracture healing. Moreover, the dosage form and administration method of the drug are also insufficient, making it difficult to accurately deliver the drug to the fracture site, affecting the full effectiveness of the drug.

[0008] Therefore, developing a compound preparation that can comprehensively regulate multiple links of fracture healing and has good stability, safety and efficacy, as well as a matching preparation method, is of great clinical significance for improving the therapeutic 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 a preparation method thereof to solve the above problems.

[0010] The technical solution of the present invention is achieved as follows: a compound preparation for promoting fracture regeneration and healing, comprising the following active ingredients in percentage by weight: 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 in parts by weight: 13-20 parts of puerarin, 12-15 parts of berberine, 5-10 parts of sanguisorba officinalis polysaccharide, 5-12 parts of tanshinone ⅡA, 5-8 parts of galangal glycosides, 3-8 parts of chuanxiong saponin VI, 2-7 parts of salvianolic acid B, 3-5 parts of matrine, 4-9 parts of astragaloside IV, and 1-3 parts of oridonin A.

[0011] Furthermore, the active ingredients are contained in the following weight percentages: 10% bioactive peptides, 8% active ingredients of traditional Chinese medicine, 15% angiogenesis promoting ingredients, 4% bone morphogenetic protein, 2% chondroitin sulfate, 2.2% vitamin D3, 2.5% alginate, and the remainder is a pharmaceutical carrier.

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

[0013] Furthermore, the angiogenesis-promoting 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 of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, or a combination of several of them.

[0015] Furthermore, the pharmaceutical carrier includes but is not limited to a filler, a disintegrant, a lubricant, and a binder; 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 the 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 comprises the following steps:

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

[0019] S2. Add Tween-80 and Span-80 as emulsifiers to the angiogenesis-promoting component, then slowly add water, and emulsify in a high-speed homogenizer at a rotation 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 nano-scale angiogenesis-promoting component powder for later use.

[0020] S3. Dissolving the bone morphogenetic protein in a buffer solution containing a protective agent, and freeze-drying the solution to obtain dry bone morphogenetic protein powder.

[0021] S4. Ultrasonic mixing of the microcapsules of S1, the angiogenesis promoting component powder of S2, the bone morphogenetic protein powder, chondroitin sulfate, vitamin D3 and alginate at a speed of 10-30 rpm for 30-120 min to fully mix the components, and adding a pharmaceutical carrier according to the dosage form, which is 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 added amounts of Tween-80 and Span-80 in the S2 are 2-5% and 1-3% of the mass of the angiogenesis-promoting component, respectively, and the added amount of water is 2-4 times the mass of the angiogenesis-promoting component.

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

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The compound preparation for promoting fracture regeneration and healing of the present invention and its preparation method show significant advantages in many aspects. The proportions of various active ingredients such as bioactive peptides, active ingredients of traditional Chinese medicine, angiogenesis promoting ingredients, bone morphogenetic proteins, etc. in the preparation are accurate and mutually coordinated, accelerating the fracture healing process in all aspects from promoting cell proliferation and differentiation, regulating bone metabolism, improving blood circulation to inducing bone formation, etc., overcoming the deficiency of traditional drugs that only focus on a single link. The clear composition of ingredients not only achieves stable and controllable quality, but also reduces the problem of unstable efficacy caused by complex ingredients and batch differences of similar traditional Chinese medicine preparations, and is also convenient for in-depth study of the mechanism of action. The unique preparation process is processed by spray drying to prepare microcapsules, nanoemulsion technology and freeze drying, which improves the stability and bioavailability of the drug and ensures the effectiveness of each ingredient. In addition, the compound preparation can be made into a variety of dosage forms to meet different clinical scenarios and patient needs, and is matched with a pharmaceutical carrier that meets pharmaceutical standards to ensure the molding, disintegration, flow and stability of the preparation, and comprehensively improve the quality and efficacy of the preparation. DETAILED DESCRIPTION

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

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

[0029] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources. Example 1

[0030] A compound preparation for promoting fracture regeneration and healing, comprising the following active ingredients in percentage by weight: 5% bioactive peptide, 5% traditional Chinese medicine active ingredient, 10% angiogenesis promoting ingredient, 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 velvet antler polypeptide powder, yak bone collagen peptide and oyster peptide in a mass ratio of 3.5:1.3:6.5.

[0032] The active ingredients of traditional Chinese medicine include the following raw materials in parts by weight: 13 parts of puerarin, 12 parts of berberine, 5 parts of Sanguisorba officinalis polysaccharide, 5 parts of tanshinone ⅡA, 5 parts of galangal glycosides, 3 parts of dipsaponin VI, 2 parts of salvianolic acid B, 3 parts of matrine, 4 parts of astragaloside IV, and 1 part of oridonin.

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

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

[0035] A compound preparation for promoting fracture regeneration and healing, comprising the following active ingredients in percentage by weight: 15% bioactive peptides, 10% traditional Chinese medicine active ingredients, 20% angiogenesis promoting ingredients, 6% bone morphogenetic protein, 3% chondroitin sulfate, 34% vitamin D, 3% alginate, and the remainder being a pharmaceutical carrier.

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

[0037] The active ingredients of 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 galangal glycosides, 8 parts of dipsaponin VI, 7 parts of salvianolic acid B, 5 parts of matrine, 9 parts of astragaloside IV, and 3 parts of oridonin A.

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

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

[0040] A compound preparation for promoting fracture regeneration and healing, comprising the following active ingredients in percentage by weight: 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, and the remainder being a pharmaceutical carrier.

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

[0042] The active ingredients of 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 galangal glycosides, 5 parts of dipsaponin VI, 5 parts of salvianolic acid B, 4 parts of matrine, 6 parts of astragaloside IV, and 2 parts of oridonin A.

[0043] The angiogenesis-promoting 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 adopt the following preparation method:

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

[0047] S2, adding Tween-80 and Span-80 as emulsifiers to the angiogenesis-promoting component, the added amounts being 4% and 2% of the mass of the angiogenesis-promoting component respectively, and then slowly adding water 3 times the mass of the angiogenesis-promoting component, emulsifying in a high-speed homogenizer at a speed of 13000 rpm to form a nanoemulsion with an average particle size of 150 nm, spray-drying the nanoemulsion to obtain a nano-scale angiogenesis-promoting component powder for later use;

[0048] S3, dissolving the bone morphogenetic protein in a buffer solution containing a protective agent, and freeze-drying to obtain a dry bone morphogenetic protein powder;

[0049] S4, ultrasonically mix the microcapsules of S1, the angiogenesis promoting component powder of S2, the bone morphogenetic protein powder, chondroitin sulfate, vitamin D3 and alginate at a speed of 10-30 rpm for 80 minutes, with an ultrasonic frequency of 30 kHz and a power of 200 W to fully mix the components and prepare 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 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 angiogenesis promoting components.

[0056] 1. In vitro cell experiments

[0057] 1. Osteoblast Proliferation Assay

[0058] 1.1 Cell culture: Osteoblastic cell line (MC3T3-E1 cells) were 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 administration: The cells were inoculated in a 96-well plate, and after the cells adhered to the wall, they were divided into a blank control group (containing only culture medium), a positive control group (teriparatide) and an experimental group of the compound preparation of the present invention. The corresponding treatments were given respectively, and multiple replicates were set for each group.

[0060] 1.3 Cell proliferation detection: The MTT method was used to detect cell proliferation. At different time points (1, 3, 5, and 7 days) after administration, MTT solution (5 mg / mL) reagent was added to the 96-well plate. After incubation for 24 hours, the absorbance value was measured with an enzyme marker, and the cell proliferation rate was calculated based on the absorbance value to evaluate the effect of the compound preparation on osteoblast proliferation.

[0061] 2. Cell differentiation detection

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

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

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

[0065] Test Results

[0066] Table 1: Osteoblast proliferation test results

[0067]

[0068] Table 2: Test results of osteoblast differentiation related indicators

[0069]

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

[0071] 2. Fracture Model Experiment

[0072] 1. Model establishment: Healthy adult SD rats weighing 250-300g, half male and half female, were purchased from a regular experimental animal center. The experimental animals were adaptively raised in an environment with a temperature of (22±2)℃ and a relative humidity of (50±10)% for 1 week, with free access to food and water. A fracture model was created by surgery. Under anesthesia, the femur or tibia of the animal was exposed, and a bone saw or bone rongeur was used to cause a fracture, and then the fracture site was fixed with a Kirschner wire or a steel plate to ensure the stability and consistency of the fracture model.

[0073] 2. Grouping and administration: Rats were randomly divided into 8 groups, 10 rats in each group, and divided into a blank control group (administered 0.5 mL of physiological saline), a positive control group (Jieguqili tablets) and an experimental group of the compound preparation of the present invention. Administration began after surgery, with the administration route being injection of 0.5 mL, once a day, and continued until the end of the experiment, and the healing time was calculated.

[0074] 3. Imaging examination

[0075] X-ray examination: The fracture sites of rats were photographed with an X-ray machine at 2 weeks and 8 weeks after surgery. Fracture healing, including callus formation and fracture line blur, was observed and scored using the X-ray callus scoring standard (1-10 points, 1 point for clear fracture line and no callus formation; 10 points for the disappearance of fracture line and good callus shaping).

[0076] CT examination: 12 weeks after surgery, the fracture site of the rats was scanned using a CT scanner to measure the changes in bone density at the fracture site. The bone density value of the fracture area was analyzed by software and compared with the normal bone density value before surgery.

[0077] 4. Test Results

[0078]

[0079] The results of the three aspects of comprehensive fracture healing time, X-ray callus score and CT bone density change can clearly show that the compound preparations of Examples 1-3 are excellent in promoting fracture healing, not only shortening the fracture healing time, but also significantly promoting callus growth and improving bone density. Among them, the effect of Example 3 is the most prominent, and all indicators are better than other groups. The positive control drug also shows a certain effect of promoting fracture healing, but in comparison, the compound preparation of the embodiment group is 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound preparation for promoting fracture regeneration and healing, characterized in that: The invention comprises the following active ingredients in percentage by weight: 5-15% bioactive peptides, 5-10% active ingredients of traditional Chinese medicine, 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 is a pharmaceutical carrier; the active ingredients of traditional Chinese medicine include the following raw materials in parts by weight: 13-20 parts of puerarin, 12-15 parts of berberine, 5-10 parts of sanguisorba officinalis polysaccharide, 5-12 parts of tanshinone ⅡA, 5-8 parts of galangal glycosides, 3-8 parts of dipsaponin VI, 2-7 parts of salvianolic acid B, 3-5 parts of matrine, 4-9 parts of astragaloside IV, and 1-3 parts of oridonin A.

2. A compound preparation for promoting fracture regeneration and healing as claimed in claim 1, characterized in that: The invention comprises the following active ingredients in percentage by weight: 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, and the remainder is a pharmaceutical carrier.

3. A compound preparation for promoting fracture regeneration and healing as claimed in claim 1, characterized in that: The bioactive peptides are antler polypeptide powder, yak bone collagen peptide and oyster peptide in a mass ratio of (3.5-5.5):(1.3-2.5):(6.5-8.1).

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

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

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

7. The method for preparing a composite preparation for promoting fracture regeneration and healing according to claim 1, characterized in that: The following steps are involved: S1, mixing the bioactive peptides and the active ingredients of traditional Chinese medicine evenly, and then mixing them with the wall material solution, preparing microcapsules by spray drying, and controlling the particle size of the microcapsules to be 10-50 μm; S2, adding Tween-80 and Span-80 as emulsifiers to the angiogenesis-promoting component, and then slowly adding water, emulsifying in a high-speed homogenizer at a rotation speed of 10000-15000 rpm to form a nanoemulsion with an average particle size of 50-200 nm, spray-drying the nanoemulsion to obtain a nano-scale angiogenesis-promoting component powder for standby use; S3, dissolving the bone morphogenetic protein in a buffer solution containing a protective agent, and freeze-drying to obtain a dry bone morphogenetic protein powder; S4. Ultrasonic mixing of the microcapsules of S1, the angiogenesis promoting component powder of S2, the bone morphogenetic protein powder, chondroitin sulfate, vitamin D3 and alginate at a speed of 10-30 rpm for 30-120 min to fully mix the components, and adding a pharmaceutical carrier according to the dosage form, which is any one of tablets, capsules, granules, injections, gels or sprays.

8. The method for preparing a composite preparation for promoting fracture regeneration and healing according to claim 7, 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.

9. The method for preparing a composite preparation for promoting fracture regeneration and healing according to claim 7, characterized in that: The added amounts of Tween-80 and Span-80 in the S2 are 2-5% and 1-3% of the mass of the angiogenesis-promoting component respectively, and the added amount of water is 2-4 times of the mass of the angiogenesis-promoting component.

10. The method for preparing a composite preparation for promoting fracture regeneration and healing according to claim 7, characterized in that: The ultrasonic frequency of the S4 is 20-40kHz, and the power is 100-300W.

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