A pharmaceutical composition for promoting fracture healing and a preparation method and application thereof

Abstract

This invention provides a pharmaceutical composition for promoting fracture healing, its preparation method, and its application. The composition, by weight, comprises 25-50 parts of an active core component, 8-25 parts of an osteogenic synergistic bioactive component, 3-15 parts of a bone-targeting transdermal penetration enhancer, and 5-15 parts of a moisturizing dispersant. The active core component is based on bone-healing traditional Chinese medicines such as calcined natural copper nanoparticles and Eupolyphaga sinensis polypeptides. The osteogenic synergistic component is based on collagen peptides of specific molecular weights and graded sodium hyaluronate. The bone-targeting transdermal penetration enhancer is a compound of eugenol and limonene. This invention utilizes a three-dimensional synergistic formulation structure and a phase-separated low-temperature preparation process, overcoming the shortcomings of traditional topical medications, such as poor transdermal targeting, weak efficacy, easy inactivation, and low safety. It can achieve targeted enrichment at fracture ends, synergistically promoting fracture healing throughout its entire life cycle, and simultaneously repairing bone and soft tissue. It is suitable for promoting the repair of various fractures and possesses extremely high clinical value and promising prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of topical pharmaceutical preparations, and in particular to a pharmaceutical composition for promoting fracture healing, its preparation method, and its application. Background Technology

[0002] Fractures are one of the most common traumatic diseases in clinical orthopedics. Their healing is a complex biological process involving multiple stages, such as hematoma organization, fibrous callus formation, bony callus formation, and callus remodeling. Approximately 5%-10% of fractures will result in complications such as delayed healing and nonunion. In particular, osteoporotic fractures, old fractures, and high-energy injury fractures in the elderly are difficult to heal, have long treatment cycles, seriously affect the patient's quality of life, and bring a heavy medical burden.

[0003] Currently, clinical fracture treatment is mainly divided into two categories: surgical internal fixation and conservative external fixation. Adjunctive treatments to promote fracture healing primarily involve oral medications, including traditional Chinese medicine bone-healing preparations, calcium supplements, and bone metabolism regulators. However, oral medications have significant clinical limitations: firstly, after absorption through the gastrointestinal tract, they are distributed throughout the body, resulting in low local drug concentrations at the fracture site and a slow onset of action; secondly, long-term use can easily cause adverse reactions such as gastrointestinal irritation and burden on liver and kidney function, making them particularly unsuitable for elderly patients and patients with impaired liver or kidney function.

[0004] Topical drug delivery is an important research direction in the adjuvant treatment of fractures, as it can act directly on the skin of the fracture site, avoiding the side effects of systemic administration. However, existing topical drugs for promoting fracture healing still have many technical limitations: First, the drug composition is complex, the effective ingredients are unclear, and the quality is difficult to control; second, traditional plasters have low transdermal absorption efficiency, making it difficult to achieve effective local blood drug concentrations; third, most formulations only focus on promoting blood circulation, removing blood stasis, reducing swelling and relieving pain, and are not targeted at the core links of bone regeneration such as osteoblast activation and collagen synthesis, thus having limited effectiveness in complex cases such as osteoporotic fractures and poor bone integration after internal fixation; in addition, traditional dosage forms such as black plasters have problems such as discomfort during application, poor breathability, and easy allergies, while some modern gels have defects such as low drug loading, poor adhesion, and unsatisfactory sustained-release effect. Summary of the Invention

[0005] In view of this, the present invention proposes a pharmaceutical composition for promoting fracture healing, its preparation method and application, thereby solving the above problems.

[0006] The technical solution of the present invention is achieved as follows: a pharmaceutical composition for promoting fracture healing, comprising the following raw materials in parts by weight: 25-64 parts of active core component, 8-23 parts of osteogenic synergistic bioactive component, 3-15 parts of bone-targeting transdermal penetration enhancer, 5-15 parts of moisturizing dispersant, 0.5-2 parts of borneol, and 0.5-2 parts of menthol.

[0007] Furthermore, the active core components include the following components: 3-8 parts of vinegar-calcined nano-natural copper powder, 4-9 parts of Drynaria fortunei extract, 3-8 parts of Dipsacus asper extract, 3-7 parts of Eupolyphaga sinensis polypeptide extract, 2-6 parts of Eucommia ulmoides extract, 2-6 parts of Angelica sinensis extract, 2-5 parts of Carthamus tinctorius extract, 2-4 parts of Daemonorops draco extract, 1-4 parts of Boswellia carterii resin extract, 2-4 parts of Commiphora myrrha resin extract, and 1-3 parts of Curcumin extract.

[0008] Preferably, pure natural copper is taken, calcined at high heat until dark red according to the calcination and quenching method of the Chinese Pharmacopoeia, quenched with rice vinegar, and repeatedly operated until the texture is brittle. After drying, it is ultra-finely pulverized to the nanoscale to obtain vinegar-calcined nano natural copper powder.

[0009] Preferably, the following herbs were taken: Drynaria fortunei, Dipsacus asper, Eucommia ulmoides, Angelica sinensis, and Carthamus tinctorius. After cleaning and pulverizing, they were extracted using low-temperature ultrasonic extraction at 35℃ with 70% v / v ethanol as the extraction solvent and a solid-liquid ratio of 1g:10mL. The extraction was performed three times, each time for 30 minutes. The extracts were combined, concentrated under reduced pressure, and purified by macroporous resin to obtain the corresponding standardized extracts.

[0010] Preferably, the extraction method of the *Eupolyphaga sinensis* polypeptide extract includes the following steps:

[0011] (1) Pretreatment: Crush the dried ground beetle and pass it through a 40-60 mesh sieve to obtain ground beetle coarse powder;

[0012] (2) Compound enzymatic hydrolysis: Mix the crude powder of ground beetle with 5-10 times its weight of phosphate buffer solution with pH 7.0-8.0, add 0.8%-1.5% of the weight of the crude powder of ground beetle compound protease, and enzymatically hydrolyze at 45-55℃ for 2-4 hours. The compound protease is composed of neutral protease and trypsin in a mass ratio of 1:1-2.

[0013] (3) Inactivation and separation: Heat the enzyme hydrolysate to 85-95℃ and maintain for 10-15 minutes to inactivate enzyme activity. After cooling, centrifuge and take the supernatant.

[0014] (4) Purification: The supernatant is sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa for fractionation and separation, and peptide components with molecular weights between 3 and 10 kDa are collected.

[0015] (5) Drying: The refined polypeptide components are freeze-dried or spray-dried to obtain the Eupolyphaga polypeptide extract.

[0016] Preferably, cleaned dragon's blood, frankincense, and myrrh are taken separately, pulverized and passed through a 30-mesh sieve, and extracted with ethyl acetate as the solvent at a solid-liquid ratio of 1g:12mL. The mixture is placed in a low-temperature environment of 30℃ and ultrasonically extracted with an ultrasonic power of 200W and an ultrasonic frequency of 40kHz for 30 minutes each time. The extracts are combined and filtered through a 0.45μm organic filter membrane to remove impurities. The filtrate is concentrated under reduced pressure at 40℃ and a vacuum degree of -0.08MPa until there is no ethyl acetate odor, and a resin extract is obtained. The resin extract is vacuum dried at 35℃ and a vacuum degree of -0.09MPa to constant weight, pulverized and passed through a 100-mesh sieve to obtain dragon's blood resin extract, frankincense resin extract, and myrrh resin extract, which are then stored in a light-proof and sealed container for later use.

[0017] Preferably, the cleaned turmeric material is pulverized and passed through a 40-mesh sieve. Using 95% v / v pharmaceutical ethanol as the extraction solvent, a solid-liquid ratio of 1g:12mL is added. The mixture is placed at 35℃ and ultrasonically extracted twice, for 30 minutes each time, using 300W of ultrasonic power and 40kHz of ultrasonic frequency. The extracts are combined and filtered through a 0.45μm organic filter membrane to remove impurities. The filtrate is concentrated under reduced pressure at 40℃ and a vacuum of -0.08MPa to obtain a turmeric extract with a relative density of 1.12 (measured at 40℃). The turmeric extract is then further processed with 95% v / v pharmaceutical ethanol. The alcohol was dissolved by stirring at 35°C to prepare a saturated solution. While hot, the solution was filtered through a 0.22μm organic filter membrane. Purified water at 4°C was added to the filtrate at a volume ratio of 1:4 at a uniform rate. After stirring evenly, the solution was cooled to 5°C at a rate of 0.5°C / min. The solution was allowed to stand in the dark for 12-18 hours to grow crystals. The solution was then filtered. The filter cake was washed three times with 50% v / v ethanol at 4°C. The solution was recrystallized twice to obtain curcumin wet crystals. The wet crystals were then vacuum dried at 35°C and a vacuum degree of -0.09MPa to a constant weight. The solution was then pulverized to obtain high-purity curcumin, which was stored in a dark and sealed container for later use.

[0018] All the above active components are mixed evenly through a 100-mesh sieve and set aside for later use.

[0019] Furthermore, the osteogenic enhancing bioactive component comprises the following components: 3-8 parts of deer bone collagen peptide, 2-6 parts of humanized type I recombinant collagen peptide, 2-4 parts of oyster oligopeptides, and 1-5 parts of graded molecular weight sodium hyaluronate, wherein the molecular weight distribution of the deer bone collagen peptide is 1000-3000 Da; and the graded molecular weight sodium hyaluronate is composed of high molecular weight sodium hyaluronate with a molecular weight of 1000-1500 kDa and low molecular weight sodium hyaluronate with a molecular weight of 10-30 kDa in a mass ratio of 2:1.

[0020] Furthermore, the bone-targeting transdermal penetration enhancer is a mixture of eugenol and limonene in a mass ratio of 1:1-3.

[0021] Further, the composition is a topical patch, gel, or poultice, the matrix of which contains at least one of carbomer, polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA), and the matrix accounts for 60-90% of the composition by mass.

[0022] The present invention also provides a method for preparing the above-mentioned topical pharmaceutical composition for promoting fracture healing, comprising the following steps:

[0023] S1. Pre-dispersion of active components: Take the following ingredients according to the formula: vinegar-calcined nano natural copper powder, Drynaria fortunei extract, Dipsacus asper extract, Eupolyphaga sinensis polypeptide extract, Eucommia ulmoides extract, Angelica sinensis extract, Carthamus tinctorius extract, Daemonorops draco extract, Boswellia carterii resin extract, Commiphora myrrha resin extract, and Curcumin extract. Add them to a moisturizing dispersant and stir at 40-55℃ until completely dissolved and the powder is evenly dispersed to obtain a pre-dispersion of active components. Keep it warm for later use.

[0024] S2. Preparation of osteogenic enhancement bioactive solution: Take deer bone collagen peptide, humanized type I recombinant collagen peptide, oyster oligopeptide, and graded molecular weight sodium hyaluronate according to the formula, add them to sterile phosphate buffer solution with pH 5.5-6.5, stir at low speed at 20-30℃ in the dark until completely dissolved, and obtain bioactive solution. Keep it at low temperature in the dark for later use.

[0025] S3. Preparation of blank matrix: Take the raw material of the external application matrix according to the formula, add the remaining deionized water, swell at room temperature for 12-24 hours, stir until a uniform gel matrix system without lumps is formed, and then degas under vacuum for later use.

[0026] S4. Premixed bone-targeted transdermal permeation enhancement system: Take eugenol and limonene according to the formula, add them to anhydrous ethanol, stir at 20-25℃ in the dark until completely miscible, and obtain a homogeneous transdermal permeation enhancement solution. Seal and protect from light for later use.

[0027] S5. Final preparation of the composition: Under light-protected conditions at 20-30℃, first add the pre-dispersion of the active component obtained in S1 to the blank matrix in S3, and stir at medium speed until completely mixed; then slowly add the bioactive liquid obtained in S2, and stir at low speed until mixed; finally add the transdermal penetration enhancer obtained in S4, borneol, and menthol, and stir at low speed until the system is homogeneous, adjust the pH to 5.5-7.0, degas under vacuum, and fill / coat to form the topical drug composition.

[0028] Further, in step S1, the particle size D90 of the calcined natural copper nanoparticles is ≤100nm; the moisturizing and dispersing agent is one or more of glycerol, propylene glycol, and 1,3-butanediol; the stirring speed is 300-500r / min, and the stirring time is 20-40min.

[0029] Further, in step S2, the low-speed stirring speed is 100-200 r / min, and the stirring time is 15-30 min; in step S4, the amount of anhydrous ethanol added is 2-5 times the total mass of the transdermal permeation-enhancing solution; in step S5, the medium-speed stirring speed is 250-350 r / min, the low-speed stirring speed is 80-150 r / min, and the system temperature does not exceed 30℃ throughout the stirring process.

[0030] The present invention also provides the above-mentioned topical pharmaceutical composition for promoting fracture healing, and its use in the preparation of topical pharmaceutical formulations for promoting fracture end repair.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] This invention combines traditional active core components for promoting blood circulation, removing blood stasis, and healing bones with modern bioactive substances that have a clear function of promoting bone growth and replenishing bone matrix, and is supplemented with a highly efficient bone-targeting transdermal penetration enhancer. The three components work synergistically to improve local microcirculation, eliminate hematoma and inflammation, directly provide raw materials for bone repair, and stimulate osteoblast activity, thus promoting fracture healing from multiple pathological aspects, with particularly significant effects on complex fracture types with delayed healing.

[0033] The active core components utilize acetic acid-calcined nano-sized natural copper powder, increasing the bioavailability of copper ions. Its nano-size facilitates transdermal absorption and participation in collagen cross-linking. The *Eupolyphaga sinensis* polypeptide extract, obtained through a specific method, exhibits enhanced angiogenesis and cell migration-promoting activity. In the osteogenic enhancing components, the combination of deer bone collagen peptides of specific molecular weights and humanized type I recombinant collagen peptides provides the most favorable collagen signals and structures for osteoblast recognition, adhesion, and growth; the combination of high- and low-molecular-weight sodium hyaluronate provides both physical lubrication and support, as well as biological signal regulation.

[0034] A specific ratio of eugenol and limonene was used as a penetration enhancer. The two not only have good transdermal penetration enhancement effects, but the study also found that their combination has a certain affinity for bone tissue, which can guide the active ingredients of the drug to accumulate more effectively in the subcutaneous bone tissue, thereby improving the drug's targeting and utilization rate.

[0035] The preparation method employs a stepwise pre-dispersion and pre-mixing strategy. First, the heat-sensitive active ingredients of the traditional Chinese medicine are uniformly dispersed with a humectant at low temperature. Then, the bioactive peptides are dissolved under mild conditions, and finally, they are mixed with the matrix at low temperature. This process minimizes the damage to the activity of proteins, peptides, and volatile components caused by high temperatures and prolonged shearing, ensuring the bioavailability of all components in the final product.

[0036] The composition can be formulated as a plaster, gel, or poultice, exhibiting good skin adhesion, breathability, and moisturizing properties. Gels and poultices, in particular, have a high drug loading capacity and strong plasticity, allowing them to adhere closely to the skin surface of fractures at different locations. They are suitable for uneven areas such as joints, improving patient compliance and comfort. Detailed Implementation

[0037] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0038] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0040] Example 1

[0041] Pharmaceutical composition formulation:

[0042] Active core components 25g: calcined nano natural copper powder 3g, Drynaria fortunei extract 4g, Dipsacus asper extract 3g, Eupolyphaga sinensis polypeptide extract 3g, Eucommia ulmoides extract 2g, Angelica sinensis extract 2g, Carthamus tinctorius extract 2g, Daemonorops draco extract 2g, Boswellia carterii resin extract 1g, Commiphora myrrha resin extract 2g, Curcuma longa extract 1g.

[0043] Osteogenic and enhancing bioactive components (8g): 3g deer bone collagen peptide, 2g humanized type I recombinant collagen peptide, 2g oyster oligopeptide, and 1g graded molecular weight sodium hyaluronate. The deer bone collagen peptide has a molecular weight distribution of 1000 Da. The graded molecular weight sodium hyaluronate is composed of high molecular weight sodium hyaluronate with a molecular weight of 1000 kDa and low molecular weight sodium hyaluronate with a molecular weight of 10 kDa in a mass ratio of 2:1.

[0044] Bone-targeted transdermal penetration enhancer 3g: Eugenol and limonene in a mass ratio of 1:1;

[0045] 5g of moisturizing dispersant: glycerin;

[0046] Borneol 0.5g;

[0047] Menthol 0.5g;

[0048] External application matrix: Carbomer 940 20g and PVP K30 30g, with the balance being deionized water 50g, the total matrix accounting for 60% of the composition mass;

[0049] The extraction method for the Eupolyphaga sinensis polypeptide extract is as follows:

[0050] (1) Pretreatment: The dried ground beetle is crushed and passed through a 50-mesh sieve to obtain ground beetle coarse powder;

[0051] (2) Compound enzymatic hydrolysis: Mix the crude powder of ground beetle with 8 times its weight of phosphate buffer at pH 7.5, add 1.2% of the weight of the crude powder of ground beetle compound protease, and hydrolyze at 50℃ for 3 hours. The compound protease is composed of neutral protease and trypsin in a mass ratio of 1:1.5.

[0052] (3) Inactivation and separation: Heat the enzyme hydrolysate to 90°C and maintain for 12 min to inactivate the enzyme. After cooling to room temperature, centrifuge at 5000 r / min for 18 min and take the supernatant.

[0053] (4) Purification: The supernatant is sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa for fractionation, and the polypeptide fractions of 3-10 kDa are collected.

[0054] (5) Drying: The purified polypeptide liquid is freeze-dried under vacuum to obtain the Eupolyphaga polypeptide extract.

[0055] Example 2

[0056] Pharmaceutical composition formulation:

[0057] Active core components 64g: calcined nano natural copper powder 8g, Drynaria fortunei extract 9g, Dipsacus asper extract 8g, Eupolyphaga sinensis polypeptide extract 7g, Eucommia ulmoides extract 6g, Angelica sinensis extract 6g, Carthamus tinctorius extract 5g, Daemonorops draco extract 4g, Boswellia carterii resin extract 4g, Commiphora myrrha resin extract 4g, Curcuma longa extract 3g.

[0058] Osteogenic and enhancing bioactive components 23g: 8g deer bone collagen peptide, 6g humanized type I recombinant collagen peptide, 4g oyster oligopeptide, and 5g graded molecular weight sodium hyaluronate, wherein the deer bone collagen peptide has a molecular weight distribution of 3000 Da; the graded molecular weight sodium hyaluronate is composed of high molecular weight sodium hyaluronate with a molecular weight of 1500 kDa and low molecular weight sodium hyaluronate with a molecular weight of 30 kDa in a mass ratio of 2:1.

[0059] Bone-targeted transdermal penetration enhancer 15g: Eugenol and limonene in a mass ratio of 1:2;

[0060] Moisturizing dispersant 5-15g: propylene glycol;

[0061] 2g of borneol;

[0062] 2g of menthol;

[0063] External application matrix: Carbomer 940 20g and PVP K30 30g, with the balance being deionized water 50g, the total matrix accounting for 90% of the composition mass;

[0064] The extraction method of the Eupolyphaga sinensis polypeptide extract is the same as in Example 1.

[0065] Example 3

[0066] Pharmaceutical composition formulation:

[0067] Active core components 44g: calcined nano natural copper powder 5g, Drynaria fortunei extract 7g, Dipsacus asper extract 5g, Eupolyphaga sinensis polypeptide extract 5g, Eucommia ulmoides extract 4g, Angelica sinensis extract 4g, Carthamus tinctorius extract 3g, Daemonorops draco extract 3g, Boswellia carterii resin extract 3g, Commiphora myrrha resin extract 3g, Curcumin extract 2g.

[0068] Osteogenic and enhancing bioactive components 15g: 5g deer bone collagen peptide, 4g humanized type I recombinant collagen peptide, 3g oyster oligopeptide, and 3g graded molecular weight sodium hyaluronate, wherein the deer bone collagen peptide has a molecular weight distribution of 2000 Da; the graded molecular weight sodium hyaluronate is composed of high molecular weight sodium hyaluronate with a molecular weight of 1200 kDa and low molecular weight sodium hyaluronate with a molecular weight of 20 kDa in a mass ratio of 2:1.

[0069] Bone-targeted transdermal penetration enhancer 9g: Eugenol and limonene in a mass ratio of 1:2;

[0070] 10g of moisturizing dispersant: in 1,3-butanediol;

[0071] 1g of borneol;

[0072] 1g of menthol;

[0073] External application matrix: Carbomer 940 20g and PVP K30 30g, with the balance being deionized water 50g, the total matrix accounting for 70% of the composition mass;

[0074] The extraction method of the Eupolyphaga sinensis polypeptide extract is the same as in Example 1.

[0075] Hydrogel matrix that promotes fracture healing

[0076] The pharmaceutical compositions were prepared using the formulations of Examples 1-3 above.

[0077] S1. Pre-dispersion of active components: Take the following ingredients according to the formula: calcined natural copper powder, drynaria extract, dipsacus extract, Eupolyphaga extract, eucommia extract, angelica extract, safflower extract, dragon's blood extract, frankincense resin extract, myrrh resin extract, and curcumin extract. Add them to the moisturizing and dispersing agent and stir at 5°C until completely dissolved and the powder is evenly dispersed. The stirring speed is 400 r / min and the stirring time is 30 min to obtain the pre-dispersion of active components. Keep it warm for later use.

[0078] S2. Preparation of osteogenic enhancement bioactive solution: Take deer bone collagen peptide, humanized type I recombinant collagen peptide, oyster oligopeptide, and graded molecular weight sodium hyaluronate according to the formula, add them to sterile phosphate buffer at pH 6.0, stir at low speed of 150 r / min for 25 min at 25℃ in the dark until completely dissolved, and obtain the bioactive solution. Keep it at low temperature in the dark for later use.

[0079] S3. Preparation of blank matrix: Take the raw material of the external application matrix according to the formula, add the remaining deionized water, swell at room temperature for 24 hours, stir until a uniform gel matrix system without lumps is formed, and then degas under vacuum for later use.

[0080] S4. Premixed bone-targeted transdermal permeation enhancement system: Take eugenol and limonene according to the formula, add them to anhydrous ethanol at 3 times the total mass of the transdermal permeation enhancement solution, stir at 20-25℃ in the dark until completely miscible, and obtain a homogeneous transdermal permeation enhancement solution. Seal and protect from light for later use.

[0081] S5. Final preparation of the composition: Under light-protected conditions at 25°C, first add the pre-dispersion of the active component obtained in S1 to the blank matrix of S3, and stir at a medium speed of 300 r / min until completely mixed; then slowly add the bioactive liquid obtained in S2, and stir at a low speed of 100 r / min until mixed; finally add the transdermal penetration enhancer obtained in S4, borneol, and menthol, and stir at a low speed of 100 r / min until the system is homogeneous, adjust the pH to 6.0, degas under vacuum, and fill into containers to obtain the hydrogel.

[0082] Example 4: A poultice to promote fracture healing

[0083] The composition formulation of this embodiment is the same as that of Example 3, except that the external application matrix is ​​a poultice matrix. The formulation is: 50g PVA, 30g PVP K90, 20g gelatin, 100g glycerin, 20g kaolin, and the remainder deionized water. The total proportion of the matrix is ​​70% of the composition mass.

[0084] The preparation method step S3 is adjusted as follows: PVA and gelatin are added to deionized water according to the formula, heated to 90°C to dissolve until homogeneous, cooled to 40°C, PVP K90, glycerin and kaolin are added, stirred until completely dispersed, swollen at room temperature for 2 hours, and degassed under vacuum to obtain the blank matrix of the poultice; the remaining steps are the same as in Example 3, and finally coated on the non-woven fabric backing layer, covered with an anti-adhesive layer, and cut to obtain the poultice.

[0085] Example 5: A plaster to promote fracture healing

[0086] The composition formulation of this embodiment is the same as that of Example 3, except that the external application matrix is ​​a hot melt adhesive plaster matrix. The formulation is: 100g thermoplastic elastomer SIS, 80g hydrogenated rosin glyceryl ester, 20g lanolin, and 50g liquid paraffin. The total matrix accounts for 70% of the composition mass.

[0087] The preparation method has been adjusted as follows:

[0088] S1. Pre-dispersion of active components: Same as in Example 3;

[0089] S2. Preparation of osteogenic and bioactive liquid: Same as in Example 3;

[0090] S3. Preparation of blank matrix: Take SIS and hydrogenated rosin glycerol ester according to the formula, heat to 120℃ to melt and mix, cool to 60℃, add lanolin and liquid paraffin, stir until uniform, and obtain blank hot melt adhesive matrix. Cool to 35℃ and keep warm for later use.

[0091] S4, Premixed bone-targeted transdermal penetration enhancement system: Same as in Example 3;

[0092] S5. Final preparation of the composition: Under light-protected conditions at 30°C, add the materials of S1, S2, and S4, along with borneol and menthol, to the blank matrix in sequence, stir at low speed until completely mixed, adjust the pH to 6.0, coat it onto the non-woven fabric backing layer, cover it with an anti-adhesive layer, and cut it to obtain the plaster.

[0093] Comparative Example 1

[0094] The only difference between this comparative example and Example 3 is that no bone-targeting transdermal penetration enhancer (eugenol, limonene) is added to the formulation; the rest of the formulation and preparation method are completely the same.

[0095] Comparative Example 2

[0096] The only difference between this comparative example and Example 3 is that no osteogenic synergistic bioactive components are added to the formulation; the rest of the formulation and preparation method are completely the same.

[0097] Comparative Example 3

[0098] The only difference between this comparative example and Example 1 is that the traditional water extract of Eupolyphaga sinensis is used instead of the polypeptide extract of Eupolyphaga sinensis. The preparation method of the water extract is as follows: Eupolyphaga sinensis crude powder is boiled twice with 10 times the amount of water, 1 hour each time. The decoctions are combined, filtered, concentrated, and freeze-dried to obtain the final product. The rest of the formula and preparation method are completely the same.

[0099] Comparative Example 4

[0100] This comparative example is a commercially available external preparation of Jiegu Qili San (Heilongjiang Shenge Pharmaceutical Co., Ltd.), and its formulation conforms to the standards of the Chinese Pharmacopoeia.

[0101] Comparative Example 5

[0102] The formulation of this comparative example is completely identical to that of Example 3, except that the preparation method adopts the traditional one-pot method: all raw materials, matrix and deionized water are added at one time, stirred and mixed evenly at 50°C, pH is adjusted, defoaming and filling are performed to obtain the product.

[0103] Experimental Example 1: In vitro transdermal absorption and bone-targeted enrichment assay.

[0104] 1. In vitro transdermal rate test

[0105] A modified Franz diffusion cell was used, with isolated abdominal skin of SD rats as the transdermal barrier. The receiving solution was a mixture of physiological saline and ethanol (7:3) at pH 7.4, and the effective diffusion area was 3.14 cm². 2 The receiving pool volume was 15 mL, the water bath temperature was 37℃, and the stirring speed was 300 r / min. Samples from Examples 1-5 and Comparative Examples 1-5 were taken and evenly applied to the stratum corneum of the skin. The receiving liquid was collected at 2h, 4h, 8h, 12h, and 24h. The cumulative permeation amount of the active indicator component (naringin, derived from Drynaria fortunei extract) was detected by high performance liquid chromatography, and the cumulative transdermal rate over 24h was calculated. The results are shown in Table 1.

[0106]

[0107] As shown in Table 1, the cumulative transdermal permeability of Examples 1-5 of the present invention is significantly higher than that of the comparative examples. In particular, the transdermal permeability of Example 3 is higher than that of Comparative Examples 1 and 4, which proves that the bone-targeted transdermal permeation enhancement system of the present invention can significantly improve the transdermal absorption efficiency of active ingredients. Meanwhile, the transdermal permeability of Comparative Example 5 is significantly lower than that of Example 3, which proves that the phase separation preparation process of the present invention can effectively improve the dispersibility and transdermal permeability of active ingredients.

[0108] 2. In vivo bone-targeted enrichment assay

[0109] Sixty SPF-grade SD rats, half male and half female, weighing 220±20g, were used to create a closed fracture model of the right femur. They were randomly divided into 6 groups of 10 rats each: Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Blank Control Group.

[0110] Drug administration began 24 hours after modeling. The corresponding sample was applied topically to the right fracture site of the rats, with a dosage of 1 g per rat, administered once every 24 hours for 7 consecutive days. 24 hours after the last administration, the rats were anesthetized and euthanized. Bone tissue from the fracture site, surrounding muscle tissue, and skin tissue were collected. The concentration of the indicator components in the tissues was detected using liquid chromatography-mass spectrometry (LC-MS), and the drug concentration ratio (target enrichment coefficient) in bone / muscle tissue was calculated. The results are shown in Table 2.

[0111]

[0112] As shown in Table 2, the drug concentration in the bone tissue of the fractured bone ends of rats in Example 1 of this invention was significantly higher than that in all comparative examples, with an enrichment coefficient of 3.97, which was much higher than that of Comparative Example 1 (0.76). This demonstrates that the eugenol-limonene composition has excellent bone-targeting enrichment ability, which can guide the active ingredients to specifically accumulate in the bone tissue of the fractured bone ends, thus solving the core problem of traditional topical drugs being retained in soft tissue and having low concentration in bone tissue.

[0113] Experimental Example 2: Pharmacodynamic Test of a Rat Fracture Model

[0114] 1. Seventy-two SPF-grade SD rats, half male and half female, weighing 220±20g, were randomly divided into nine groups of eight rats each: Example 3 group, Example 4 group, Comparative Examples 1-5 group, Model Control Group (blank matrix applied externally), and Sham Operation Group (only incision without modeling).

[0115] 2. Except for the sham-operated group, a closed fracture model of the midshaft of the right femur was established in all other groups of rats. The drug was administered 24 hours after modeling, and the corresponding sample was applied externally to the fracture site at a dose of 1 g / rat. The drug was changed every 24 hours and administered continuously for 6 weeks.

[0116] 3. Six weeks after administration, rats were anesthetized and euthanized. The right femur was harvested to detect bone mineral density and biomechanical parameters (maximum load and bending strength). Serum samples were collected to detect osteogenic related parameters: osteocalcin (BGP) and alkaline phosphatase (ALP). The results are shown in Tables 3 and 4.

[0117]

[0118]

[0119] As shown in Tables 3 and 4, the bone mineral density, biomechanical strength, and serum osteogenic indicators BGP and ALP levels in Examples 3 and 4 of this invention were significantly higher than those in all comparative examples and the model control group (P<0.01), demonstrating that the composition of this invention can significantly promote osteoblast activity, accelerate callus growth and mineralization, improve bone biomechanical properties after fracture healing, and significantly shorten the fracture healing period. Meanwhile, all indicators in comparative examples 1-5 were significantly lower than those in Example 3, demonstrating that the three-dimensional synergistic formulation, targeted penetration-enhancing system, and proprietary preparation process of this invention are all core keys to improving efficacy and are indispensable.

[0120] Test Example 3: Skin Irritation Test

[0121] Twelve healthy rabbits, half male and half female, weighing 2.5 ± 0.5 kg, were randomly divided into three groups of four rabbits each: intact skin group, damaged skin group, and blank control group.

[0122] 24 hours before drug administration, hair was removed from both sides of the spine on the back of rabbits, covering an area of ​​approximately 3cm × 3cm. For the damaged skin group, a "#" mark was made in the hair removal area with a needle until bleeding occurred. 1g / rabbit of the sample from Example 1 was applied topically to both the intact skin group and the damaged skin group, while a blank control group was treated with a blank matrix. The dressings were fixed in place. 24 hours after drug administration, residual drug was removed with warm water. Skin reactions were observed at 1 hour, 24 hours, 48 ​​hours, and 72 hours after drug removal, and skin irritation was scored according to the "Cosmetic Safety Technical Specifications".

[0123] The results showed that no skin reactions such as erythema, edema, or rash appeared in any rabbits in the intact skin group and the damaged skin group at any time point, and the skin irritation score was 0, which was not different from the blank control group. After 7 days of continuous administration, no skin irritation reaction was observed, which proves that the composition of the present invention is non-irritating to the skin and has excellent safety for external use.

[0124] 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 pharmaceutical composition for promoting fracture healing, characterized in that, It includes the following raw materials in parts by weight: 25-64 parts of active core component, 8-23 parts of osteogenic and synergistic bioactive component, 3-15 parts of bone-targeting transdermal penetration enhancer, 5-15 parts of moisturizing dispersant, 0.5-2 parts of borneol, and 0.5-2 parts of menthol.

2. The pharmaceutical composition according to claim 1, characterized in that, The active core components include the following: 3-8 parts of vinegar-calcined nano-natural copper powder, 4-9 parts of Drynaria fortunei extract, 3-8 parts of Dipsacus asper extract, 3-7 parts of Eupolyphaga sinensis polypeptide extract, 2-6 parts of Eucommia ulmoides extract, 2-6 parts of Angelica sinensis extract, 2-5 parts of Carthamus tinctorius extract, 2-4 parts of Daemonorops draco extract, 1-4 parts of Boswellia carterii resin extract, 2-4 parts of Commiphora myrrha resin extract, and 1-3 parts of Curcumin extract.

3. The pharmaceutical composition according to claim 2, characterized in that, The extraction method of the Eupolyphaga sinensis polypeptide extract includes the following steps: (1) Pretreatment: Crush the dried ground beetle and pass it through a 40-60 mesh sieve to obtain ground beetle coarse powder; (2) Compound enzymatic hydrolysis: Mix the crude powder of ground beetle with 5-10 times its weight of phosphate buffer solution with pH 7.0-8.0, add 0.8%-1.5% of the weight of the crude powder of ground beetle compound protease, and enzymatically hydrolyze at 45-55℃ for 2-4 hours. The compound protease is composed of neutral protease and trypsin in a mass ratio of 1:1-2. (3) Inactivation and separation: Heat the enzyme hydrolysate to 85-95℃ and maintain for 10-15 minutes to inactivate enzyme activity. After cooling, centrifuge and take the supernatant. (4) Purification: The supernatant is sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa for fractionation and separation, and peptide components with molecular weights between 3 and 10 kDa are collected. (5) Drying: The refined polypeptide components are freeze-dried or spray-dried to obtain the Eupolyphaga polypeptide extract.

4. The pharmaceutical composition according to claim 1, characterized in that, The osteogenic enhancing bioactive component consists of the following components: 3-8 parts of deer bone collagen peptide, 2-6 parts of humanized type I recombinant collagen peptide, 2-4 parts of oyster oligopeptides, and 1-5 parts of graded molecular weight sodium hyaluronate. The molecular weight distribution of the deer bone collagen peptide is 1000-3000 Da. The graded molecular weight sodium hyaluronate is composed of high molecular weight sodium hyaluronate with a molecular weight of 1000-1500 kDa and low molecular weight sodium hyaluronate with a molecular weight of 10-30 kDa in a mass ratio of 2:

1.

5. The pharmaceutical composition according to claim 1, characterized in that, The bone-targeting transdermal penetration enhancer is a mixture of eugenol and limonene in a mass ratio of 1:1-3.

6. The pharmaceutical composition according to claim 1, characterized in that, The composition is a plaster, gel, or poultice, and the matrix contains at least one of carbomer, polyvinylpyrrolidone, and polyvinyl alcohol, wherein the matrix accounts for 60-90% of the composition by mass.

7. The method for preparing the topical pharmaceutical composition for promoting fracture healing according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Pre-dispersion of active components: Take the following ingredients according to the formula: vinegar-calcined nano natural copper powder, Drynaria fortunei extract, Dipsacus asper extract, Eupolyphaga sinensis polypeptide extract, Eucommia ulmoides extract, Angelica sinensis extract, Carthamus tinctorius extract, Daemonorops draco extract, Boswellia carterii resin extract, Commiphora myrrha resin extract, and Curcumin extract. Add them to a moisturizing dispersant and stir at 40-55℃ until completely dissolved and the powder is evenly dispersed to obtain a pre-dispersion of active components. Keep it warm for later use. S2. Preparation of osteogenic enhancement bioactive solution: Take deer bone collagen peptide, humanized type I recombinant collagen peptide, oyster oligopeptide, and graded molecular weight sodium hyaluronate according to the formula, add them to sterile phosphate buffer solution with pH 5.5-6.5, stir at low speed at 20-30℃ in the dark until completely dissolved, and obtain bioactive solution. Keep it at low temperature in the dark for later use. S3. Preparation of blank matrix: Take the raw material of the external application matrix according to the formula, add the remaining deionized water, swell at room temperature for 12-24 hours, stir until a uniform gel matrix system without lumps is formed, and then degas under vacuum for later use. S4. Premixed bone-targeted transdermal permeation enhancement system: Take eugenol and limonene according to the formula, add them to anhydrous ethanol, stir at 20-25℃ in the dark until completely miscible, and obtain a homogeneous transdermal permeation enhancement solution. Seal and protect from light for later use. S5. Final preparation of the composition: Under light-protected conditions at 20-30℃, first add the pre-dispersion of the active component obtained in S1 to the blank matrix in S3, and stir at medium speed until completely mixed; then slowly add the bioactive liquid obtained in S2, and stir at low speed until mixed; finally add the transdermal penetration enhancer obtained in S4, borneol, and menthol, and stir at low speed until the system is homogeneous, adjust the pH to 5.5-7.0, degas under vacuum, and fill / coat to form the topical drug composition.

8. The preparation method according to claim 7, characterized in that, In step S1, the particle size D90 of the calcined natural copper nanoparticles is ≤100nm; the moisturizing and dispersing agent is one or more of glycerol, propylene glycol, and 1,3-butanediol; the stirring speed is 300-500r / min, and the stirring time is 20-40min.

9. The preparation method according to claim 7, characterized in that, In step S2, the low-speed stirring speed is 100-200 r / min, and the stirring time is 15-30 min; in step S4, the amount of anhydrous ethanol added is 2-5 times the total mass of the transdermal permeation-enhancing solution; in step S5, the medium-speed stirring speed is 250-350 r / min, the low-speed stirring speed is 80-150 r / min, and the system temperature does not exceed 30℃ throughout the stirring process.

10. The topical pharmaceutical composition for promoting fracture healing according to any one of claims 1-6, or the topical pharmaceutical composition prepared by the preparation method according to any one of claims 7-8, in the preparation of a topical pharmaceutical formulation for promoting fracture end repair.

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