Pharmaceutical composition for osteoporotic fracture as well as preparation method and application of pharmaceutical composition

Abstract

The invention provides a pharmaceutical composition for osteoporotic fracture as well as a preparation method and application of the pharmaceutical composition. The composition comprises active ingredients such as a traditional Chinese medicine active extract, cod bone polypeptide freeze-dried powder, a calcium alginate chelate and a nano-hydroxyapatite / chitosan-fibroin compound, the traditional Chinese medicine extract is prepared from various Chinese herbal medicines according to a specific proportion and has the effects of tonifying kidney, strengthening bone, promoting blood circulation to remove blood stasis and the like; the cod bone polypeptide freeze-dried powder can induce osteogenesis, the calcium alginate chelate can promote calcium absorption, and the nano compound is used as a bone targeting carrier; the preparation method comprises the steps of traditional Chinese medicine extraction, liposome suspension formation, double-layer microcapsule encapsulation, mixing and the like, and various dosage forms can be prepared. The pharmaceutical composition promotes fracture healing through multi-way and multi-target effects, integrates multiple functions of osteogenesis induction, calcium absorption promotion, bone targeted repair and the like, and is expected to effectively promote osteoporotic fracture healing.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine compositions, and in particular to a medicine composition for osteoporotic fractures, and a preparation method and application thereof. Background Art

[0002] As the global population is aging, osteoporosis has become a major factor endangering public health. Osteoporosis is characterized by decreased bone mass and degeneration of bone tissue microstructure, which significantly increases the brittleness of bones and greatly increases the risk of fractures. Osteoporotic fractures, one of the most serious complications of osteoporosis, not only cause severe physical pain to patients, but also seriously reduce their quality of life, while also bringing a heavy medical burden to society and families.

[0003] At present, the clinical treatment of osteoporotic fractures mainly includes surgical treatment and drug treatment. The core purpose of surgical treatment is to restore the anatomical structure of the fracture site and ensure its stability. However, the healing process of postoperative fractures is often interfered by many factors, such as the patient's age, basic health status, and the severity of osteoporosis. In terms of drug treatment, there are many types of anti-osteoporosis drugs, including calcium, vitamin D and its derivatives, bisphosphonates, calcitonins, etc. Although these drugs can increase bone density and reduce the risk of fractures to a certain extent, they have obvious limitations in promoting the healing of osteoporotic fractures. For example, calcium and vitamin D are mainly used to supplement the calcium nutrition required by the human body, and their direct promotion effect on fracture healing is relatively limited; bisphosphonates mainly reduce bone resorption by inhibiting the activity of osteoclasts, but this may have an adverse effect on the bone remodeling process at the fracture site; although calcitonins can effectively relieve pain symptoms after fractures, the overall effect on fracture healing is unsatisfactory.

[0004] In addition, traditional drug treatments often fail to effectively intervene in the key links of the fracture healing process. The healing of osteoporotic fractures is an extremely complex biological process, involving multiple key links such as osteoblast proliferation and differentiation, calcium absorption and utilization, bone matrix synthesis and mineralization, and targeted repair of bone tissue. Existing drugs are difficult to take all these links into account at the same time, which leads to prolonged fracture healing time, difficulty in ensuring healing quality, and even possible serious complications such as non-union of fractures. Summary of the invention

[0005] In view of this, the present invention proposes a pharmaceutical composition for osteoporotic fractures and a preparation method and application thereof to solve the above problems.

[0006] The technical scheme of the present invention is achieved as follows: a pharmaceutical composition for osteoporotic fractures comprises the following active ingredients in parts by weight: 30-50 parts of traditional Chinese medicine active extracts, 10-15 parts of cod bone polypeptide freeze-dried powder, 8-12 parts of calcium alginate chelate, and 2-10 parts of nano-hydroxyapatite / chitosan-fibroin complex; the traditional Chinese medicine active extracts comprise the following raw materials in parts by weight: 2-4 parts of drynariae, 3-5 parts of epimedium, 4-6 parts of prepared rehmannia, 1-3 parts of antler glue, 3-5 parts of dipsaccharum, 1-3 parts of natural copper, 2-4 parts of earthworm, 3-5 parts of eucommia, 3-5 parts of achyranthes, 5-7 parts of astragalus, 1-3 parts of cynomorium songaricum, 2-6 parts of cynomorium truncatum, and 3-6 parts of liquorice.

[0007] Furthermore, the active extract of traditional Chinese medicine includes the following raw materials in parts by weight: 3 parts of Drynaria fortunei, 4 parts of Epimedium, 5 parts of Rehmannia glutinosa, 2 parts of antler glue, 4 parts of Dipsacus asper, 2 parts of natural copper, 3 parts of Eucommia bark, 4 parts of Eucommia ulmoides, 4 parts of Achyranthes bidentata, 6 parts of Astragalus, 2 parts of Cynomorium songaricum, 4 parts of Cynomorium fortunei, and 5 parts of Licorice.

[0008] Further, the preparation method of the active extract of traditional Chinese medicine is as follows: firstly, natural copper is calcined to red, quenched with vinegar, and the earthworm is roasted with wine and then dried at low temperature, all the raw materials of traditional Chinese medicine are mixed and crushed to 40-60 meshes according to proportion, and supercritical carbon dioxide is used for extraction, the extraction pressure is set to 20-30MPa, the extraction temperature is controlled at 35-45°C, the carbon dioxide flow rate is 15-25L / h, and the extraction time is 2-3 hours, and the obtained extract is subjected to alcohol extraction, and 8-10 times the volume fraction of 70%-80% ethanol solution is added, and reflux extraction is performed at 60-70°C for 2-3 times, each time for 1-2 hours. The alcohol extract is combined, and it is concentrated under reduced pressure to a clear paste with a relative density of 1.10-1.20 (60°C), and then water precipitation is performed, and 4-6 times the amount of purified water is added to the clear paste, after stirring evenly, it is allowed to stand for precipitation at 3-5°C for 12-24 hours, and the supernatant is taken, and it is concentrated under reduced pressure and dried to obtain the active extract of traditional Chinese medicine.

[0009] Furthermore, the codfish bone polypeptide freeze-dried powder is prepared by adding the washed and crushed deep-sea codfish bones to a buffer solution with a pH value of 7.5-8.0, adding 500-800U of neutral protease and 300-500U of alkaline protease per gram of codfish bones, performing enzymolysis at 37-40°C for 4-6 hours, removing insoluble impurities by centrifugation and filtration after the enzymolysis, collecting the supernatant, concentrating the enzymolysis product supernatant obtained above, and then adding activated polyethylene glycol reagent at a ratio of active peptide to polyethylene glycol (PEG) molecule molar ratio of 1:(3-5), reacting at 25-30°C in the dark for 8-12 hours, removing unreacted polyethylene glycol and other impurities by dialysis and ultrafiltration after the reaction, and freeze-drying to obtain the codfish bone polypeptide freeze-dried powder.

[0010] Further, the calcium alginate chelate is prepared by selecting fresh seaweed raw materials of kelp or sargassum, cleaning and crushing them into particles of 2-5 mm, mixing the seaweed raw materials with deionized water in a mass ratio of 1:(10-15), soaking them at 80-90° C. for 2-3 hours to dissolve the alginic acid preliminarily; then cooling the soaking liquid to 35-37° C., inoculating activated plant lactobacillus with an inoculation amount of 3%-5% (v / v), and fermenting them in a constant temperature shaker at a speed of 150-180 r / min for 2 hours. 4-36 hours; after the fermentation is completed, the fermentation liquid is centrifuged at a speed of 4000-5000r / min for 15-20 minutes, and the supernatant is collected; a calcium chloride solution with a mass concentration of 10%-15% is slowly added to the supernatant, and stirring is performed while adding until no precipitation is generated, at which time the alginate and the calcium ions are fully chelated; the obtained precipitate is washed with deionized water and anhydrous ethanol for 3-5 times in turn to remove impurities, and finally vacuum dried at 50-60°C for 8-12 hours to obtain calcium alginate chelate.

[0011] Further, the nano-hydroxyapatite / chitosan-fibroin complex is prepared by mixing and stirring a chitosan solution with a mass concentration of 1-3% and a fibroin solution with a mass concentration of 5-8% until they are completely dissolved, with a weight ratio of fibroin to chitosan being 1:1-3, to obtain a blended solution; then, calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in deionized water, mixed in a ratio of calcium to phosphorus molar ratio of 1.67-1.75:1, and slowly added dropwise to the blended solution under magnetic stirring, while adjusting the pH value to 9-10 with ammonia water; stirring and reacting in a constant temperature water bath at 50-60°C for 3-5 hours; after the reaction is completed, the product is centrifuged, repeatedly washed with deionized water and anhydrous ethanol for 1-3 times, and finally vacuum dried at 60-80°C for 12-24 hours, crushed to a particle size of 20-80nm, to obtain a nano-hydroxyapatite / chitosan-fibroin complex.

[0012] Furthermore, a method for preparing a pharmaceutical composition for osteoporotic fractures comprises the following steps:

[0013] S1. Weigh soybean lecithin and cholesterol in a mass ratio of 3-5:1, dissolve in chloroform-methanol in a volume ratio of 2-3:1, and after rotary evaporation to form a film, add a phosphate buffer solution containing cod bone polypeptide freeze-dried powder and calcium alginate chelate, the pH value is 6.5-7.4, and after hydration, high pressure homogenize to a particle size of ≤200nm to form a liposome suspension;

[0014] S2, mixing the liposome suspension with β-cyclodextrin in a mass ratio of 1:4-6, spray drying to form double-layer microcapsules with an encapsulation efficiency of ≥85%;

[0015] S3. Add active extracts of traditional Chinese medicine, double-layer microcapsules and nano-hydroxyapatite / chitosan-fibroin complexes into the gel solution, place them in a three-dimensional motion mixer, and mix them at a speed of 30-50 r / min for 30-60 minutes at a low temperature below 20°C and a relative humidity below 30% to obtain a pharmaceutical composition. The mixed pharmaceutical composition can be made into different dosage forms as needed.

[0016] Furthermore, the mass volume ratio of the cod bone polypeptide freeze-dried powder of S1 to the calcium alginate chelate and the phosphate buffer solution is (10-15):(8-12):100 g / mL.

[0017] Furthermore, the dosage form includes tablets, capsules or granules.

[0018] Furthermore, the pharmaceutical composition is used in the preparation of a drug for treating osteoporotic fractures.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The various active ingredients of this pharmaceutical composition play a synergistic role. Among the active extracts of traditional Chinese medicine, the combination of various traditional Chinese medicines such as Drynaria fortunei and Epimedium can nourish the kidney and strengthen bones, promote blood circulation and remove blood stasis, regulate the body's qi and blood and viscera functions as a whole, and provide a good internal environment for fracture healing. As an osteogenic induction active ingredient, cod bone polypeptide freeze-dried powder can directly act on osteogenic precursor cells, induce them to differentiate into osteoblasts, and accelerate new bone formation. Calcium alginate chelate effectively promotes calcium absorption, provides sufficient calcium source for bone repair, and ensures the smooth progress of bone mineralization. As a bone-targeted carrier system, nanohydroxyapatite / chitosan-fibroin complex can accurately deliver drugs to the fracture site, increase the local concentration of drugs, and enhance the therapeutic effect.

[0021] The preparation process of active extracts of traditional Chinese medicine using supercritical carbon dioxide extraction combined with alcohol extraction and water precipitation can effectively extract the active ingredients in traditional Chinese medicine, remove impurities, improve the purity of the medicine, and ensure the stability of the efficacy. The enzymatic hydrolysis, modification and separation and purification process of cod bone polypeptide freeze-dried powder can retain and enhance the activity of active peptides. Calcium alginate chelate is prepared through fermentation, precipitation and other steps to improve its bioavailability. When preparing the pharmaceutical composition, a double-layer microcapsule is first made to improve the encapsulation rate of cod bone polypeptide freeze-dried powder and calcium alginate chelate, so as to protect them from the influence of the external environment, achieve slow and stable release, and prolong the drug action time. DETAILED DESCRIPTION

[0022] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0023] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0024] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0025] Example 1

[0026] A pharmaceutical composition for osteoporotic fractures, comprising the following active ingredients in parts by weight: 30 parts of traditional Chinese medicine active extract, 10 parts of cod bone polypeptide freeze-dried powder, 8 parts of calcium alginate chelate, and 2 parts of nano-hydroxyapatite / chitosan-fibroin complex; the traditional Chinese medicine active extract comprises the following raw materials in parts by weight: 2 parts of drynariae, 3 parts of epimedium, 4 parts of prepared rehmannia, 1 part of antler glue, 3 parts of dipsacus, 1 part of natural copper, 2 parts of earthworm, 3 parts of eucommia, 3 parts of achyranthes, 5 parts of astragalus, 1 part of cynomorium songaricum, 2 parts of cynomorium truncatum, and 3 parts of liquorice;

[0027] The preparation method of the active extract of traditional Chinese medicine is as follows: first, natural copper oxide is calcined to red, quenched with vinegar, and the earthworm is roasted with wine and then dried at low temperature. All the raw materials of traditional Chinese medicine are mixed and crushed to 40 meshes according to proportion, and supercritical carbon dioxide is used for extraction. The extraction pressure is set to 20MPa, the extraction temperature is controlled at 35°C, the carbon dioxide flow rate is 15L / h, and the extraction time is 2 hours. The obtained extract is subjected to alcohol extraction, and 8 times the volume fraction of 70% ethanol solution is added, and reflux extraction is performed at 60°C for 2 times, each time for 1 hour. The alcohol extracts are combined, concentrated under reduced pressure to a clear paste with a relative density of 1.10 (60°C), and then water precipitation is performed. 4-6 times the amount of purified water is added to the clear paste, and after stirring evenly, it is allowed to stand for precipitation at 4°C for 12 hours, and the supernatant is taken, concentrated under reduced pressure and dried to obtain the active extract of traditional Chinese medicine;

[0028] The codfish bone polypeptide freeze-dried powder is prepared by adding washed and crushed deep-sea codfish bones to a buffer solution with a pH value of 7.5, adding 500U of neutral protease and 300U of alkaline protease per gram of codfish bones, performing enzymolysis at 37°C for 4 hours, removing insoluble impurities by centrifugation and filtration after the enzymolysis, collecting the supernatant, concentrating the enzymolysis product supernatant obtained above, and then adding an activated polyethylene glycol reagent at a molar ratio of active peptide to polyethylene glycol (PEG) molecules of 1:3, reacting at 25°C under light-proof conditions for 8 hours, removing unreacted polyethylene glycol and other impurities by dialysis and ultrafiltration after the reaction, and freeze-drying to obtain the codfish bone polypeptide freeze-dried powder;

[0029] The calcium alginate chelate selects fresh kelp as the seaweed raw material, cleans it and crushes it to a particle size of 2 mm, mixes the seaweed raw material with deionized water in a mass ratio of 1:10, and soaks it at 80° C. for 2 hours to dissolve the alginic acid preliminarily; then cools the soaking liquid to 35° C., inoculates activated plant lactobacillus with an inoculation amount of 3% (v / v), and ferments it in a constant temperature shaker at a speed of 150 r / min for 24 hours; after the fermentation is completed, centrifuges the fermentation liquid at a speed of 4000 r / min for 15 minutes, and collects the supernatant; slowly adds a calcium chloride solution with a mass concentration of 10% to the supernatant, stirring while adding, until no precipitation is generated; washes the obtained precipitate with deionized water and anhydrous ethanol three times in sequence to remove impurities, and finally vacuum dries it at 50° C. for 8 hours to obtain the calcium alginate chelate;

[0030] The nano-hydroxyapatite / chitosan-fibroin complex is prepared by mixing and stirring a chitosan solution with a mass concentration of 1-3% and a silk fibroin solution with a mass concentration of 5% until they are completely dissolved, wherein the weight ratio of the silk fibroin to the chitosan is 1:1-3, to obtain a blended solution; then, calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in deionized water, mixed in a ratio of calcium to phosphorus molar ratio of 1.67:1, and slowly added dropwise to the blended solution under magnetic stirring, and the pH value is adjusted to 9 with ammonia water; the reaction is continuously stirred in a constant temperature water bath at 50°C for 3 hours; after the reaction is completed, the product is centrifuged, repeatedly washed once with deionized water and anhydrous ethanol, and finally vacuum dried at 60°C for 12 hours, crushed to a particle size of 20nm, to obtain the nano-hydroxyapatite / chitosan-fibroin complex.

[0031] Example 2

[0032] A pharmaceutical composition for osteoporotic fractures, comprising the following active ingredients in parts by weight: 50 parts of traditional Chinese medicine active extract, 15 parts of cod bone polypeptide freeze-dried powder, 12 parts of calcium alginate chelate, and 10 parts of nano-hydroxyapatite / chitosan-fibroin complex; the traditional Chinese medicine active extract comprises the following raw materials in parts by weight: 4 parts of drynariae, 5 parts of epimedium, 6 parts of prepared rehmannia, 3 parts of antler glue, 5 parts of dipsaccharum officinale, 3 parts of natural copper, 4 parts of earthworm, 5 parts of eucommia, 5 parts of achyranthes bidentata, 7 parts of astragalus, 3 parts of cynomorium songaricum, 6 parts of cynomorium truncatum, and 6 parts of liquorice;

[0033] The preparation method of the active extract of traditional Chinese medicine is as follows: first, natural copper oxide is calcined to red, quenched with vinegar, and the earthworm is roasted with wine and then dried at low temperature. All the raw materials of traditional Chinese medicine are mixed and crushed to 60 meshes according to proportion, and supercritical carbon dioxide is used for extraction. The extraction pressure is set to 30MPa, the extraction temperature is controlled at 45°C, the carbon dioxide flow rate is 25L / h, and the extraction time is 3 hours. The obtained extract is subjected to alcohol extraction, and 10 times the volume fraction of 80% ethanol solution is added, and reflux extraction is performed at 70°C for 3 times, each time for 2 hours. The alcohol extracts are combined, concentrated under reduced pressure to a clear paste with a relative density of 1.20 (60°C), and then water precipitation is performed. 6 times the amount of purified water is added to the clear paste, and after stirring evenly, it is allowed to stand for precipitation at 4°C for 24 hours, and the supernatant is taken, concentrated under reduced pressure and dried to obtain the active extract of traditional Chinese medicine;

[0034] The codfish bone polypeptide freeze-dried powder is prepared by adding washed and crushed deep-sea codfish bones to a buffer solution with a pH value of 8.0, adding 800U of neutral protease and 500U of alkaline protease per gram of codfish bones, performing enzymolysis at 40°C for 6 hours, removing insoluble impurities by centrifugation and filtration after the enzymolysis, collecting the supernatant, concentrating the enzymolysis product supernatant obtained above, and then adding an activated polyethylene glycol reagent at a molar ratio of active peptide to polyethylene glycol (PEG) molecules of 1:5, reacting at 30°C in a dark environment for 12 hours, removing unreacted polyethylene glycol and other impurities by dialysis and ultrafiltration after the reaction, and freeze-drying to obtain the codfish bone polypeptide freeze-dried powder;

[0035] The calcium alginate chelate comprises fresh Sargassum as the seaweed raw material, which is cleaned and crushed to a particle size of 5 mm, mixed with the seaweed raw material and deionized water in a mass ratio of 1:15, and soaked at 90° C. for 3 hours to dissolve the alginic acid preliminarily; then the soaking liquid is cooled to 37° C., and activated plant lactobacillus is inoculated with an inoculation amount of 5% (v / v), and fermented in a constant temperature shaker at a speed of 180 r / min for 36 hours; after the fermentation is completed, the fermentation liquid is centrifuged at a speed of 5000 r / min for 20 minutes, and the supernatant is collected; a calcium chloride solution with a mass concentration of 15% is slowly added to the supernatant, and stirred while adding until no precipitation is generated; the obtained precipitate is washed with deionized water and anhydrous ethanol for 5 times in sequence to remove impurities, and finally vacuum dried at 60° C. for 12 hours, and the crushed particle size is 80 nm, so as to obtain the calcium alginate chelate;

[0036] The nano-hydroxyapatite / chitosan-fibroin complex is prepared by mixing and stirring a chitosan solution with a mass concentration of 3% and a fibroin solution with a mass concentration of 8% until they are completely dissolved, wherein the weight ratio of fibroin to chitosan is 1:3, to obtain a blended solution; then, calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in deionized water, mixed in a ratio of calcium to phosphorus molar ratio of 1.75:1, and slowly added dropwise to the blended solution under magnetic stirring, and the pH value is adjusted to 10 with ammonia water; the reaction is continuously stirred in a constant temperature water bath at 60°C for 5 hours; after the reaction is completed, the product is centrifuged, repeatedly washed with deionized water and anhydrous ethanol for 3 times, and finally vacuum dried at 80°C for 24 hours to obtain the nano-hydroxyapatite / chitosan-fibroin complex.

[0037] Example 3

[0038] A pharmaceutical composition for osteoporotic fractures comprises the following active ingredients in parts by weight: 40 parts of traditional Chinese medicine active extract, 12 parts of cod bone polypeptide freeze-dried powder, 10 parts of calcium alginate chelate, and 8 parts of nano-hydroxyapatite / chitosan-fibroin complex; the traditional Chinese medicine active extract comprises the following raw materials in parts by weight: 3 parts of drynariae, 4 parts of epimedium, 5 parts of prepared rehmannia, 2 parts of antler glue, 4 parts of dipsaccharum officinale, 2 parts of natural copper, 3 parts of earthworm, 4 parts of eucommia, 4 parts of achyranthes bidentata, 6 parts of astragalus, 2 parts of cynomorium songaricum, 4 parts of cynomorium truncatum, and 5 parts of liquorice;

[0039] The preparation method of the active extract of traditional Chinese medicine is as follows: first, natural copper oxide is calcined until it is red, quenched with vinegar, and the earthworm is roasted with wine and then dried at low temperature. All the raw materials of traditional Chinese medicine are mixed and crushed to 50 meshes according to proportion, and supercritical carbon dioxide is used for extraction. The extraction pressure is set to 25MPa, the extraction temperature is controlled at 40°C, the carbon dioxide flow rate is 20L / h, and the extraction time is 2 hours. The obtained extract is subjected to alcohol extraction, and 9 times the volume fraction of 75% ethanol solution is added. Reflux extraction is performed at 65°C for 3 times, each time for 2 hours. The alcohol extracts are combined, concentrated under reduced pressure to a clear paste with a relative density of 1.20 (60°C), and then water precipitation is performed. 5 times the amount of purified water is added to the clear paste, and after stirring evenly, it is allowed to stand for precipitation at 4°C for 20 hours. The supernatant is taken, concentrated under reduced pressure and dried to obtain the active extract of traditional Chinese medicine;

[0040] The codfish bone polypeptide freeze-dried powder is prepared by adding washed and crushed deep-sea codfish bones to a buffer solution with a pH value of 7.8, adding 700U of neutral protease and 400U of alkaline protease per gram of codfish bones, performing enzymolysis at 38°C for 5 hours, removing insoluble impurities by centrifugation and filtration after the enzymolysis, collecting the supernatant, concentrating the enzymolysis product supernatant obtained above, and then adding an activated polyethylene glycol reagent according to a molar ratio of active peptide to polyethylene glycol (PEG) molecules of 1:4, reacting at 28°C under light-proof conditions for 10 hours, removing unreacted polyethylene glycol and other impurities by dialysis and ultrafiltration after the reaction, and freeze-drying to obtain the codfish bone polypeptide freeze-dried powder;

[0041] The calcium alginate chelate is prepared by selecting fresh seaweed raw materials of kelp or sargassum, cleaning and crushing them to a particle size of 3 mm, mixing the seaweed raw materials with deionized water in a mass ratio of 1:12, and soaking them at 85° C. for 3 hours to dissolve the alginic acid preliminarily; then cooling the soaking liquid to 36° C., inoculating activated plant lactobacillus with an inoculation amount of 4% (v / v), and fermenting them at a speed of 170 r / min for 30 hours in a constant temperature shaker; after the fermentation is completed, centrifuging the fermented liquid at a speed of 4500 r / min for 18 minutes, and collecting the supernatant; slowly adding a calcium chloride solution with a mass concentration of 13% to the supernatant, stirring while adding, until no precipitation is generated; washing the obtained precipitate with deionized water and anhydrous ethanol for 4 times in sequence to remove impurities, and finally vacuum drying at 55° C. for 10 hours to obtain the calcium alginate chelate;

[0042] The nano-hydroxyapatite / chitosan-fibroin complex is prepared by mixing and stirring a chitosan solution with a mass concentration of 2% and a fibroin solution with a mass concentration of 7% until they are completely dissolved, wherein the weight ratio of fibroin to chitosan is 1:2, to obtain a blended solution; then, calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in deionized water, mixed in a ratio of calcium to phosphorus molar ratio of 1.67:1, and slowly added dropwise to the blended solution under magnetic stirring, and the pH value is adjusted to 10 with ammonia water; the mixture is continuously stirred in a constant temperature water bath at 55° C. for 4 hours for reaction; after the reaction is completed, the product is centrifuged, repeatedly washed twice with deionized water and anhydrous ethanol, and finally vacuum dried at 70° C. for 20 hours, and crushed to a particle size of 50 nm to obtain a nano-hydroxyapatite / chitosan-fibroin complex;

[0043] The above examples 1-3 adopt the following preparation method:

[0044] S1. Weigh soybean lecithin and cholesterol in a mass ratio of 4:1, dissolve in chloroform-methanol in a volume ratio of 3:1, and after rotary evaporation to form a film, add a phosphate buffer solution containing cod skeletal polypeptide lyophilized powder and calcium alginate chelate, the pH is 7.0, and the mass volume ratio of cod skeletal polypeptide lyophilized powder and calcium alginate chelate to phosphate buffer solution is 12:10:100 g / mL. After hydration, high pressure homogenization is performed to a particle size of ≤200 nm to form a liposome suspension;

[0045] S2, mixing the liposome suspension with β-cyclodextrin in a mass ratio of 1:5, spray drying to form double-layer microcapsules with an encapsulation efficiency of ≥85%;

[0046] S3. Add active extracts of traditional Chinese medicine, double-layer microcapsules and nano-hydroxyapatite / chitosan-fibroin complexes into the gel solution, place them in a three-dimensional motion mixer, and mix them at a speed of 40 r / min for 50 minutes at a low temperature below 20°C and a relative humidity below 30% to obtain a pharmaceutical composition. The mixed pharmaceutical composition can be made into different dosage forms as needed.

[0047] Comparative Example 1

[0048] The difference between this comparative example and Example 3 is that Cynomorium songaricum and Cynomorium iron oxide in the active Chinese medicine extract are removed, and the remaining ingredients and contents are the same as those in the example, that is, the active Chinese medicine extract consists of 3 parts of Drynaria fortunei, 4 parts of Epimedium, 5 parts of Rehmannia glutinosa, 2 parts of antler glue, 4 parts of Dipsacus asper, 2 parts of natural copper, 3 parts of Eucommia ulmoides, 4 parts of Eucommia ulmoides, 4 parts of Achyranthes bidentata, 6 parts of Astragalus membranaceus, and 5 parts of Licorice.

[0049] Comparative Example 2

[0050] The difference between this comparative example and Example 3 is that the pharmaceutical composition does not contain cod bone polypeptide freeze-dried powder.

[0051] Comparative Example 3

[0052] The difference between this comparative example and Example 3 is that the pharmaceutical composition does not contain calcium alginate chelate.

[0053] 1. Pharmacodynamic Verification

[0054] Determination of drug effects on osteoblast differentiation

[0055] 1. Cell culture: Osteogenic MC3T3-E1 cells were cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO. 2 Culture the cells in a cell culture incubator and perform subculture or experiments when the cell confluence reaches 80%.

[0056] 2. Grouping and dosing: Set up the example drug group, each comparative drug group and blank control group (only culture medium). Cells were cultured at a density of (5×10 4 The cells were inoculated into a 24-well plate and cultured for 24 hours until the cells adhered to the wall. Then, culture media containing different drugs were added. The drug concentration was determined according to the appropriate gradient of preliminary experiments. 3-5 replicate wells were set for each group.

[0057] 3. Detection indicators and methods:

[0058] (1) Alkaline phosphatase (ALP) activity detection: On the 7th day after administration, the culture medium was discarded, the cells were washed three times with PBS, and cell lysis solution was added and lysed on ice for 30 minutes. The cells were centrifuged at 4°C and 12,000 r / min for 15 minutes, and the supernatant was taken. The ALP activity was determined using the ALP detection kit according to the instructions. Disodium p-nitrophenyl phosphate was used as the substrate, and the absorbance was measured at a wavelength of 405 nm. The higher the absorbance value, the stronger the ALP activity and the higher the degree of osteoblast differentiation.

[0059] (2) Detection of osteocalcin (OC) expression: At the late stage of osteogenic differentiation (day 14), the OC content in the cell culture supernatant or cell lysate was detected by ELISA.

[0060] (3) Detection of cell mineralized nodules: On the 14th day after administration, the culture medium was discarded, the cells were washed three times with PBS, and fixed with 4% paraformaldehyde for 30 minutes. Then, the cells were stained with 0.1% Alizarin Red staining solution (pH 4.2) for 30 minutes, and the excess staining solution was gently rinsed to remove it. The cells were observed and photographed under a microscope, and the area and number of mineralized nodules were analyzed by ImageJ software to evaluate the mineralization ability of osteoblasts. The more mineralized nodules, the more mature the osteoblast differentiation.

[0061] 4. Test results:

[0062]

[0063] From the above results, we can see that:

[0064] From the perspective of ALP activity, Example 3 has the highest ALP activity. Comparative Example 1 has a lower ALP activity of 0.60±0.03 due to the removal of Cynomorium songaricum and Cynomorium truncatum in the active extract of traditional Chinese medicine. This indicates that Cynomorium songaricum and Cynomorium truncatum play an important role in promoting the early differentiation of osteoblasts, and affect ALP synthesis and secretion by regulating intracellular signaling pathways. Comparative Example 2 shows that cod bone polypeptide freeze-dried powder can directly act on osteoblast precursor cells, induce their differentiation into osteoblasts and increase ALP activity. Comparative Example 3 shows that although calcium alginate chelate mainly promotes calcium absorption, it also has a positive regulatory effect on the early differentiation of osteoblasts, and may indirectly affect ALP activity by affecting intracellular calcium homeostasis.

[0065] In terms of OC content, the OC content of Example 3 is 20.0±1.8ng / mL, which is significantly higher than that of other groups. Compared with Comparative Example 1, it can be seen that Cynomorium songaricum and Cynomorium truncatum can promote the synthesis and secretion of OC by osteoblasts, and the reduction of their content will affect the mineralization and maturation of bone matrix. Compared with Comparative Example 2, it shows that cod bone polypeptide freeze-dried powder is critical to the maturation and function of osteoblasts, and can regulate OC gene expression to promote OC synthesis and secretion. Compared with Comparative Example 3, it shows that calcium alginate chelate can actively maintain the normal synthesis level of OC, and provide sufficient calcium source for OC synthesis and bone mineralization by promoting calcium absorption.

[0066] Regarding the area and number of mineralized nodules, Example 3 is compared with Comparative Example 1, which shows that these two Chinese medicines can regulate the mineralization of the extracellular matrix, promote calcium salt deposition, and help the growth of mineralized nodules. Compared with Comparative Example 2, it shows that cod bone polypeptide freeze-dried powder can activate the mineralization signaling pathway, promote matrix protein secretion and calcium salt deposition. Compared with Comparative Example 3, calcium alginate chelate can enhance the cell's calcium uptake and utilization, and provide calcium for the formation of mineralized nodules.

[0067] Comparative Example 4

[0068] The difference between this comparative example and Example 3 is that the nano-hydroxyapatite / chitosan-fibroin complex in the pharmaceutical composition is replaced by an equal amount of nano-hydroxyapatite-chitosan complex; specifically, calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in deionized water, mixed in a calcium-phosphorus molar ratio of 1.67:1, and slowly added dropwise to a chitosan solution with a mass concentration of 2% under magnetic stirring, and the pH value is adjusted to 10 with ammonia water; the reaction is continuously stirred in a constant temperature water bath at 55°C for 4 hours; after the reaction, the product is centrifuged, repeatedly washed twice with deionized water and anhydrous ethanol, and finally vacuum dried at 70°C for 20 hours, crushed to a particle size of 50 nm, and a nano-hydroxyapatite-chitosan complex is obtained.

[0069] 2. In vitro targeting experiments

[0070] 1. Experimental materials:

[0071] (1) Cell line: Human osteoblast cell line (MG-63 cells) was selected as a cell model for studying bone targeting;

[0072] (2) Drug samples: the drug composition of Example 3 and the drug composition of Comparative Example 4

[0073] 2. Experimental Procedure

[0074] (1) Drug labeling: Take the drugs of Example 3 and Comparative Example 4, add FITC solution (dissolved in DMSO, 0.1 mg / mL), shake at room temperature in the dark for 2-4 hours, dialyze the labeled drugs with PBS for 24 hours, change the solution 3-4 times, collect the drugs by centrifugation, resuspend them in culture medium, and adjust the concentration to 1 mg / mL.

[0075] (2) Cell treatment: MG-63 cells were collected at 5×10 5 6-well plates were inoculated with 100 μL / well, 2 mL of culture medium was added, and the cells were cultured for 24 hours to adhere to the wall. The original culture medium was discarded, and the cells were divided into 3 groups of Example and 4 groups of Comparative Example, with 3 replicates in each group. Both groups were added with equal volumes (200 μL) of labeled drugs and cultured for 2, 4, and 6 hours.

[0076] (3) Intake detection:

[0077] Fluorescence microscopy: at each time point, discard the drug-containing culture medium, wash three times with PBS, fix with 4% paraformaldehyde for 20 minutes, wash three more times with PBS, and observe and take pictures under the microscope.

[0078] Flow cytometry: Digest the cells with trypsin, discard the supernatant by centrifugation, resuspend in PBS, measure the fluorescence intensity, and analyze the uptake rate.

[0079] 3. Experimental results

[0080]

[0081] The above results show that the cell uptake rate and average fluorescence intensity of Example 3 at each time point are higher than those of Comparative Example 4, indicating that the nano-hydroxyapatite / chitosan-fibroin complex has better targeting performance in vitro.

[0082] Comparative Example 5

[0083] The difference between this comparative example and Example 3 is that double-layer microcapsules are not prepared; cod bone polypeptide freeze-dried powder and calcium alginate chelate are directly added to a gel solution containing active extracts of traditional Chinese medicine and nano-hydroxyapatite-chitosan complex, placed in a three-dimensional motion mixer, and mixed at a speed of 40 r / min for 50 minutes under a low temperature of below 20°C and a relative humidity of below 30% to obtain a pharmaceutical composition, and the subsequent steps for preparing different dosage forms are the same as those in the example.

[0084] 3. In vitro release experiment

[0085] 1. Experimental Procedure

[0086] (1) Sample preparation: Weigh the pharmaceutical compositions of Example 3 and Comparative Example 5 to a concentration of 1 μg / mL, respectively put them into treated dialysis bags, tie the bag mouths tightly, and set 3 parallel samples for each group.

[0087] (2) Simulated gastric fluid release: Add 50 mL of simulated gastric fluid to a 100 mL conical flask, place the dialysis bag containing the drug, and shake at 37°C and 100 r / min. At 0.5 h, 2 h, 4 h, and 8 h, remove the dialysis bag, blot dry with filter paper, and place in a new conical flask containing 50 mL of simulated gastric fluid and continue shaking. At the same time, take 1 mL of the release solution, centrifuge at 10,000 r / min for 10 minutes, and filter the supernatant through a 0.45 μm filter membrane for testing.

[0088] (3) Simulated intestinal fluid release: After the simulated gastric fluid release is completed, the dialysis bag is rinsed three times with PBS and placed in a conical flask with 50 mL of simulated intestinal fluid, and the above oscillation, sampling, and processing steps are repeated.

[0089] 2.HPLC analysis

[0090] (1) Chromatographic conditions: C18 column, mobile phase (methanol-water or acetonitrile-water gradient elution), detection wavelength determined by component, flow rate 1.0 mL / min, column temperature 30 °C.

[0091] (2) Standard curve: Inject reference substance solutions of different concentrations into HPLC to measure the peak area, and draw a standard curve with concentration as the horizontal axis and peak area as the vertical axis.

[0092] (3) Sample determination: The treated sample filtrate was injected into HPLC to measure the peak area, and the concentration of each component was calculated according to the standard curve.

[0093] 3. Data processing

[0094] The cumulative release rate of each component at different time points is calculated = (the sum of the concentrations of the components in the release medium at a certain time point / the sum of the initial concentrations of the components in the pharmaceutical composition) × 100%.

[0095] 4. Experimental Results

[0096]

[0097] From the data, it can be seen that the release rate of the three active ingredients in the comparative example 5 in the simulated gastric juice and the simulated intestinal juice is faster than that in the example 3, and a higher cumulative release rate is achieved in the early stage, lacking a sustained release effect. The presence of the double-layer microcapsules in the example 3 significantly affects the release characteristics of the drug components, and it has a sustained release effect on naringin, icariin and antler collagen, which helps to maintain the stable release of the drug in the body and ensure the continuous exertion of the drug effect, which has a positive significance for the treatment of osteoporotic fractures.

[0098] 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 pharmaceutical composition for osteoporotic fractures, characterized in that: The invention comprises the following active ingredients in parts by weight: 30-50 parts of traditional Chinese medicine active extract, 10-15 parts of cod bone polypeptide freeze-dried powder, 8-12 parts of calcium alginate chelate, and 2-10 parts of nano-hydroxyapatite / chitosan-fibroin complex; the traditional Chinese medicine active extract comprises the following raw materials in parts by weight: 2-4 parts of drynariae, 3-5 parts of epimedium, 4-6 parts of prepared rehmannia, 1-3 parts of antler glue, 3-5 parts of dipsaccharum officinale, 1-3 parts of natural copper, 2-4 parts of earthworm, 3-5 parts of eucommia, 3-5 parts of achyranthes bidentata, 5-7 parts of astragalus, 1-3 parts of cynomorium songaricum, 2-6 parts of cynomorium truncatum, and 3-6 parts of liquorice.

2. A pharmaceutical composition for osteoporotic fractures according to claim 1, characterized in that: The active extract of traditional Chinese medicine comprises the following raw materials in parts by weight: 3 parts of Drynaria fortunei, 4 parts of Epimedium, 5 parts of Rehmannia glutinosa, 2 parts of antler glue, 4 parts of Dipsacus asper, 2 parts of natural copper, 3 parts of Eucommia bark, 4 parts of Eucommia ulmoides, 4 parts of Achyranthes bidentata, 6 parts of Astragalus, 2 parts of Cynomorium songaricum, 4 parts of Cynomorium fortunei, and 5 parts of Licorice.

3. A pharmaceutical composition for osteoporotic fractures according to claim 1 or 2, characterized in that: The preparation method of the active extract of traditional Chinese medicine comprises the following steps: firstly calcining natural copper oxide until it is red, quenching with vinegar, roasting earthworm with wine and then drying at low temperature, taking all the raw materials of traditional Chinese medicine in proportion, mixing and crushing to 40-60 mesh, extracting with supercritical carbon dioxide, setting the extraction pressure to 20-30MPa, controlling the extraction temperature to 35-45°C, the carbon dioxide flow rate to 15-25L / h, and the extraction time to 2-3 hours, performing alcohol extraction on the obtained extract, adding 8-10 times the volume fraction of 70%-80% ethanol solution, refluxing extraction at 60-70°C for 2-3 times, each time for 1-2 hours, combining the alcohol extracts, concentrating under reduced pressure to a clear paste with a relative density of 1.10-1.20, then performing water precipitation treatment, adding 4-6 times the volume of purified water to the clear paste, stirring evenly, standing and precipitating at 3-5°C for 12-24 hours, taking the supernatant, concentrating under reduced pressure and drying, and obtaining the active extract of traditional Chinese medicine.

4. A pharmaceutical composition for osteoporotic fractures according to claim 1, characterized in that: The codfish bone polypeptide freeze-dried powder is prepared by adding washed and crushed deep-sea codfish bones to a buffer solution with a pH value of 7.5-8.0, adding 500-800U of neutral protease and 300-500U of alkaline protease per gram of codfish bones, performing enzymolysis at 37-40°C for 4-6 hours, removing insoluble impurities by centrifugation and filtration after the enzymolysis, collecting the supernatant, concentrating the enzymolysis product supernatant obtained above, and then adding an activated polyethylene glycol reagent at a molar ratio of active peptide to polyethylene glycol molecules of 1:(3-5), reacting at 25-30°C in the dark for 8-12 hours, removing unreacted polyethylene glycol and other impurities by dialysis and ultrafiltration after the reaction, and freeze-drying to obtain the codfish bone polypeptide freeze-dried powder.

5. The pharmaceutical composition for osteoporotic fracture according to claim 1, characterized in that: The calcium alginate chelate is prepared by selecting fresh seaweed raw materials of kelp or sargassum, cleaning and crushing them into particles with a diameter of 2-5 mm, mixing the seaweed raw materials with deionized water in a mass ratio of 1:(10-15), soaking them at 80-90° C. for 2-3 hours to preliminarily dissolve the alginate; then cooling the soaking liquid to 35-37° C., inoculating activated plant lactobacillus with an inoculation amount of 3%-5% (v / v), and shaking them in a constant temperature shaker at a rotation speed of 150-180 r / min. The method comprises the steps of: rapidly fermenting the mixture for 24-36 hours; after the fermentation is completed, centrifuging the fermentation liquid at a speed of 4000-5000 r / min for 15-20 minutes, and collecting the supernatant; slowly adding a calcium chloride solution with a mass concentration of 10%-15% to the supernatant, stirring while adding, until no more precipitation is produced; washing the obtained precipitate with deionized water and anhydrous ethanol for 3-5 times in turn to remove impurities, and finally vacuum drying the mixture at 50-60° C. for 8-12 hours to obtain calcium alginate chelate.

6. A pharmaceutical composition for osteoporotic fractures according to claim 1, characterized in that: The nano-hydroxyapatite / chitosan-fibroin complex is prepared by mixing and stirring a chitosan solution with a mass concentration of 1-3% and a fibroin solution with a mass concentration of 5-8% until they are completely dissolved, wherein the weight ratio of fibroin to chitosan is 1:1-3, to obtain a blended solution; then, calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in deionized water, mixed in a ratio of calcium to phosphorus molar ratio of 1.67-1.75:1, and slowly added dropwise to the blended solution under magnetic stirring, and the pH value is adjusted to 9-10 with ammonia water; the reaction is continuously stirred in a constant temperature water bath at 50-60°C for 3-5 hours; after the reaction is completed, the product is centrifuged, repeatedly washed with deionized water and anhydrous ethanol for 1-3 times, and finally vacuum dried at 60-80°C for 12-24 hours, and crushed to a particle size of 20-80nm, to obtain the nano-hydroxyapatite / chitosan-fibroin complex.

7. The method for preparing a pharmaceutical composition for osteoporotic fractures according to claim 1, characterized in that: The following steps are involved: S1. Weigh soybean lecithin and cholesterol in a mass ratio of 3-5:1, dissolve in chloroform-methanol in a volume ratio of 2-3:1, and after rotary evaporation to form a film, add a phosphate buffer solution containing cod bone polypeptide freeze-dried powder and calcium alginate chelate, the pH value is 6.5-7.4, and after hydration, high pressure homogenize to a particle size of ≤200nm to form a liposome suspension; S2, mixing the liposome suspension with β-cyclodextrin in a mass ratio of 1:4-6, spray drying to form double-layer microcapsules with an encapsulation efficiency of ≥85%; S3. Add active extracts of traditional Chinese medicine, double-layer microcapsules and nano-hydroxyapatite / chitosan-fibroin complexes into the gel solution, place them in a three-dimensional motion mixer, mix them at a speed of 30-50 r / min for 30-60 minutes under an environment below 20°C and a relative humidity below 30% to obtain a pharmaceutical composition, and prepare the mixed pharmaceutical composition into different dosage forms.

8. The method for preparing a pharmaceutical composition for osteoporotic fractures according to claim 7, characterized in that: The mass volume ratio of the cod bone polypeptide freeze-dried powder of S1 to the calcium alginate chelate and the phosphate buffer solution is (10-15):(8-12):100 g / mL.

9. The method for preparing a pharmaceutical composition for osteoporotic fractures according to claim 7, characterized in that: The dosage forms include tablets, capsules or granules.

10. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a medicament for treating osteoporotic fractures.

Images