Preparation method of bone melon extract powder injection and pharmaceutical composition
By deep degreasing and decalcification, specific enzymatic hydrolysis and ultrafiltration purification, and macroporous adsorption resin purification, combined with composite stabilizers and lyophilization protectants, the problem of component fluctuation in bone melon extract powder injection was solved, achieving uniformity and long-term stability of active ingredients, and improving the consistency of drug quality and efficacy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEILONGJIANG DILONG PHARM CO LTD
- Filing Date
- 2026-03-30
- Publication Date
- 2026-07-14
AI Technical Summary
In the preparation of bone melon extract powder injection, the extraction processes of bone polypeptide and melon seed polypeptide are independent and the conditions are poorly controlled, resulting in fluctuations in the composition and ratio of effective components between different batches, which affects the consistency of drug quality and the stability of efficacy.
The molecular weight distribution of bone peptides was controlled by a combination of deep defatting and decalcification treatment with enzymatic hydrolysis and two-step ultrafiltration purification under specific conditions. The raw material of melon seeds was pretreated by defatting and extracted with alcohol-water solution under specific pH conditions, followed by selective purification with macroporous adsorption resin. A composite stabilizer of specific amino acids and antioxidants was introduced to optimize the freeze-drying protectant system.
It improves the uniformity of active ingredients and batch-to-batch consistency of bone polypeptide and melon seed polypeptide, enhances the purity and physicochemical stability of powder injection, and ensures the stability of therapeutic efficacy under long-term storage.
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing a bone melon extract powder injection and a pharmaceutical composition. Background Technology
[0002] The bone melon extract is a compound preparation made from fresh or frozen pig limb bones and dried mature seeds of melon (a plant in the Cucurbitaceae family). The extract is prepared as a sterile aqueous solution or a sterile freeze-dried product after extraction from these materials. The bone melon extract contains a variety of active polypeptides and free amino acids, which can regulate bone metabolism, stimulate osteoblast proliferation, promote new bone formation, regulate calcium and phosphorus metabolism, increase bone calcium deposition, and prevent osteoporosis.
[0003] Currently, in the preparation of bone melon extract powder injection, the extraction processes of bone polypeptide and melon seed polypeptide are relatively independent and the conditions are poorly controlled, making it difficult to accurately control the molecular weight distribution range of the two polypeptide active ingredients. This results in fluctuations in the composition and ratio of effective ingredients between different batches, affecting the consistency of the final drug quality and the stability of its efficacy.
[0004] Therefore, a method for preparing bone melon extract powder for injection and a pharmaceutical composition are proposed to solve the above problems. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing bone melon extract powder for injection and a pharmaceutical composition, which solves the problem mentioned in the background art where the composition and proportion of effective components fluctuate between different batches, affecting the consistency of the final drug quality and the stability of its efficacy.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a bone melon extract powder injection and a pharmaceutical composition, comprising the following steps: Step 1: Pretreatment of bone raw materials. Select long bones from the limbs of healthy pigs or cattle, wash and crush them, and then perform deep degreasing and decalcification treatments in sequence to obtain bone matrix raw materials. Step 2: Bone polypeptide extraction. The pretreated bone matrix raw material is subjected to enzymatic hydrolysis. After the reaction is completed, the enzyme is inactivated and the primary extract of bone polypeptide is obtained. Then, it is purified by ultrafiltration, concentrated and dried to obtain bone polypeptide extract. Step 3: Pre-treatment of melon seed raw materials. Select mature melon seeds, wash and dry them, then crush and defatted them to obtain defatted melon seed powder. Step 4: Extraction of melon seed polypeptides. Defatted melon seed powder is extracted using an alcohol-water mixture. The extract is filtered, concentrated, purified, eluted, concentrated, and dried using macroporous adsorption resin to obtain melon seed polypeptide extract. Step 5: Preparation of stabilizer composition: Specific amino acids and antioxidants are mixed in proportion and co-ground to obtain a composite stabilizer; Step Six: Preparation of freeze-dried formulation. The bone polypeptide extract obtained in Step Two, the melon seed polypeptide extract obtained in Step Four, the stabilizer composition obtained in Step Five, as well as the freeze-drying protectant and excipients, are dissolved, mixed, filtered and sterilized under specific conditions, then dispensed and freeze-dried to obtain bone melon extract powder for injection.
[0007] Preferably, the deep degreasing and decalcification treatment in step one includes the following steps: immersing the crushed bone particles in a degreasing solution containing organic solvent, and ultrasonically degreasing at 45-60℃ for 1-3 hours. After degreasing, washing with purified water until neutral, draining, immersing the degreased bone particles in a decalcification solution, stirring and reacting at room temperature for 4-12 hours, washing with purified water until neutral, and freeze-drying at -50℃ to -40℃ for 24-48 hours to obtain a porous, sponge-like clean bone matrix raw material; The degreasing solution is a mixed solution of chloroform and methanol in a volume ratio of 2:1, with a material-to-liquid ratio of 1g:8-12mL. The decalcification solution is a 0.5 mol / L to 1.5 mol / L hydrochloric acid solution or a disodium ethylenediaminetetraacetate solution, with a material-to-liquid ratio of 1 g: 10-15 mL.
[0008] Preferably, the frequency of the ultrasonic-assisted treatment in the deep degreasing process is 28-40 kHz, the temperature of the degreasing process is 50-55℃, and the degreasing process time is 1.5-2 hours.
[0009] Preferably, the bone polypeptide extraction in step two includes the following specific steps: Enzymatic hydrolysis: Mix the pretreated bone matrix raw material with purified water at a material-to-liquid ratio of 1:8-1:15, adjust the pH to 6.5-7.5 with acid or alkali, add 0.5%-2.0% of the weight of the bone matrix raw material with compound protease, and enzymatically hydrolyze at 50-60℃ for 4-8 hours. Enzyme inactivation and separation: Heat the enzyme hydrolysate to 85-95℃ and maintain for 10-20 minutes to inactivate the enzyme. Then, first filter it through a 100-200 mesh filter, and then centrifuge it at 4℃ and 8000-12000 r / min for 15-25 minutes. Collect the supernatant to obtain the primary extract of bone polypeptide. Purification and concentration: The primary extract of bone peptides was subjected to two-step ultrafiltration by passing it through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The permeate with molecular weights less than 10 kDa and greater than 3 kDa was collected. The permeate was concentrated under reduced pressure to 1 / 10 to 1 / 15 of its original volume under conditions of a temperature not exceeding 50°C and a vacuum degree not lower than -0.09 MPa to obtain concentrated bone peptides. Drying: The concentrated bone polypeptide solution is spray-dried with the inlet temperature controlled at 160-180℃ and the outlet temperature controlled at 75-85℃ to obtain a white to off-white bone polypeptide extract.
[0010] Preferably, the complex protease used in the enzymatic hydrolysis reaction is composed of alkaline protease, neutral protease and trypsin, and the enzyme activity ratio of the three is 1-2:1-3:0.5-1.5.
[0011] Preferably, the pretreatment of melon seed raw materials in step three includes the following steps: washing the melon seeds with purified water, drying them in a forced-air dryer at 40-50℃ until the moisture content is less than 8%, then crushing them with a pulverizer and passing them through a 40-60 mesh sieve to obtain coarse melon seed powder; mixing the coarse melon seed powder with petroleum ether at a material-to-liquid ratio of 1:4-1:6, refluxing and defatting at 50-60℃ for 2-4 hours, repeating the defatting process 1-3 times until the filtrate is colorless, and vacuum drying the defatted melon seed powder at 40-50℃ until no solvent residue remains to obtain defatted melon seed powder.
[0012] Preferably, the extraction of melon seed polypeptides in step four includes the following specific steps: Extraction: Mix defatted melon seed powder with an ethanol aqueous solution with a volume concentration of 50%-70% at a material-to-liquid ratio of 1:10-1:20, adjust the pH to 8.0-9.5 with acid or alkali, and extract by stirring at 60-80℃ for 2-4 hours. Repeat the extraction 1-2 times and combine the extracts. Preliminary concentration and impurity removal: The combined extracts are concentrated under reduced pressure at a temperature not exceeding 60℃ until they are alcohol-free and reduced to 1 / 5-1 / 8 of their original volume. Then, they are allowed to stand at 4℃ for 12-24 hours, centrifuged to remove insoluble impurities, and the supernatant is collected. Resin purification: The supernatant is loaded onto a pretreated macroporous adsorption resin column at a flow rate of 1-3 column volumes / hour. The macroporous adsorption resin is at least one of AB-8, D101, or HPD-100. After loading, impurities are removed by elution with 3-5 BV of purified water at a flow rate of 2-4 BV / hour. Then, elution is performed with 3-6 BV of 30%-50% ethanol aqueous solution at a flow rate of 1-3 BV / hour. This eluent is collected. Concentration and drying: The collected eluent was concentrated under reduced pressure at a temperature not exceeding 50°C to a paste-like consistency, and then freeze-dried or spray-dried to obtain a pale yellow melon seed polypeptide extract.
[0013] Preferably, the specific amino acid and antioxidant in step five include L-arginine, glycine, and glutathione. The preparation of the stabilizer composition includes the following steps: weighing 3-8 parts of L-arginine, 2-5 parts of glycine, and 0.5-2 parts of glutathione by weight, placing the three in a nano-grinding mill, and grinding them together at a speed of 300-500 r / min for 30-60 minutes under inert gas protection until the average particle size D90 of the mixture is less than 10 micrometers, thereby obtaining the composite stabilizer.
[0014] Preferably, the preparation of the lyophilized formulation in step six includes the following specific steps: Preparation of solution: Pour 70%-80% of the total volume of water for injection into the mixing tank, cool to 2-8℃, and add 10-30 parts of bone polypeptide extract, 5-15 parts of melon seed polypeptide extract, 60-85 parts of freeze-drying protectant and excipient, and 1-5 parts of stabilizer composition in sequence. Stir until completely dissolved, and adjust the pH of the solution to 5.5-7.0 with acid or alkali during the process. The freeze-drying protectant and excipient are composed of the following components in parts by weight: 40-60 parts mannitol, 10-20 parts sucrose, and 5-10 parts dextran. Volume adjustment and filtration: Add water for injection to the total volume, continue stirring at 2-8℃ for 15-30 minutes to mix evenly, and then filter through 0.45-micron and 0.22-micron microporous membranes for sterilization to obtain sterile drug solution; Dispensing and Freeze-drying: The sterile drug solution was dispensed into vials under aseptic conditions, partially capped, and then transferred to a freeze dryer for freeze-drying. The freeze-drying process was as follows: First, the plate temperature was lowered to below -45°C and maintained for 2-4 hours to completely freeze the drug solution. Then, a first drying was performed, with the plate temperature raised to -25°C at a rate of 0.5-1°C / min and maintained at this temperature for 20-30 hours, with the vacuum degree controlled at 10-30 Pa. Next, a second drying was performed, with the plate temperature raised to 25-30°C at a rate of 0.3-0.5°C / min and maintained at this temperature for 8-12 hours, with the vacuum degree controlled at 5-15 Pa. After freeze-drying, the vials were fully capped under vacuum or nitrogen purging conditions to obtain the bone marrow extract powder for injection.
[0015] Preferably, the composition is made from raw materials comprising the following parts by weight: 10-30 parts of bone polypeptide extract, 5-15 parts of melon seed polypeptide extract, 60-85 parts of freeze-drying protectant and excipient, and 1-5 parts of stabilizer composition; wherein the weight ratio of polypeptides in the bone polypeptide extract to the melon seed polypeptide extract is 1:1 to 6:1.
[0016] Compared with the prior art, the present invention provides a method for preparing bone melon extract powder for injection and a pharmaceutical composition, which has the following beneficial effects: 1. In this invention, by subjecting bone raw materials to deep degreasing and decalcification, combined with enzymatic hydrolysis under specific conditions and a two-step ultrafiltration purification process, non-target impurities in the bone matrix can be removed, and the molecular weight distribution range of bone polypeptides can be controlled, thereby improving the uniformity and batch-to-batch consistency of the active ingredients in the bone polypeptides. Simultaneously, the complex protease hydrolysis strategy used in bone polypeptide extraction can synergistically act on bone proteins, increasing polypeptide yield and laying the foundation for stable control of the proportion of active ingredients in the final pharmaceutical composition.
[0017] 2. In this invention, by pre-treating the raw material of melon seeds with defatting and extracting it with an alcohol-water solution under specific pH conditions, combined with selective purification using macroporous adsorption resin, the target polypeptide components in melon seeds can be enriched while removing sugars and pigment impurities, thereby improving the purity of the melon seed polypeptide extract and providing a guarantee for the preparation of high-purity powder injections with low related substances.
[0018] 3. In this invention, by introducing a composite stabilizer made by co-grinding specific amino acids and antioxidants, the composition is well compatible with bone polypeptides and melon seed polypeptides, and works synergistically with an optimized freeze-drying protectant system. During freeze-drying and long-term storage, it can protect the active structure of bone polypeptides and melon seed polypeptides, inhibit their degradation, aggregation and oxidation, thereby improving the physicochemical stability, reconstitution properties and therapeutic stability of the powder injection under long-term storage. Detailed Implementation
[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0020] Example 1: A method for preparing a bone marrow extract powder injection and a pharmaceutical composition, comprising the following steps: Step 1: Pretreatment of bone raw materials. Select long bones from the limbs of healthy pigs, wash and crush them, and then perform deep degreasing and decalcification treatments in sequence to obtain bone matrix raw materials. Step 2: Bone polypeptide extraction. The pretreated bone matrix raw material is subjected to enzymatic hydrolysis. After the reaction is completed, the enzyme is inactivated and the primary extract of bone polypeptide is obtained. Then, it is purified by ultrafiltration, concentrated and dried to obtain bone polypeptide extract. Step 3: Pre-treatment of melon seed raw materials. Select mature melon seeds, wash and dry them, then crush and defatted them to obtain defatted melon seed powder. Step 4: Extraction of melon seed polypeptides. Defatted melon seed powder is extracted using an alcohol-water mixture. The extract is filtered, concentrated, purified, eluted, concentrated, and dried using macroporous adsorption resin to obtain melon seed polypeptide extract. Step 5: Preparation of stabilizer composition: Specific amino acids and antioxidants are mixed in proportion and co-ground to obtain a composite stabilizer; Step Six: Preparation of freeze-dried formulation. The bone polypeptide extract obtained in Step Two, the melon seed polypeptide extract obtained in Step Four, the stabilizer composition obtained in Step Five, as well as the freeze-drying protectant and excipients, are dissolved, mixed, filtered and sterilized under specific conditions, then dispensed and freeze-dried to obtain bone melon extract powder for injection.
[0021] The deep degreasing and decalcification process in step one includes the following steps: the crushed bone particles are soaked in a degreasing solution containing organic solvent and ultrasonically degreased at 45°C for 1 hour. After degreasing, the particles are washed with purified water until neutral and drained. The degreased bone particles are then soaked in a decalcification solution and stirred at room temperature for 4 hours. After the reaction is complete, the particles are washed with purified water until neutral and freeze-dried at -50°C for 24 hours to obtain a porous, sponge-like clean bone matrix raw material. The degreasing solution is a mixed solution of chloroform and methanol in a volume ratio of 2:1, with a material-to-liquid ratio of 1g:8mL. The decalcification solution is a 0.5 mol / L hydrochloric acid solution, and the material-to-liquid ratio is 1 g: 10 mL.
[0022] In the deep degreasing process, the frequency of the ultrasonic-assisted treatment was 28 kHz, the temperature of the degreasing treatment was 50 ℃, and the degreasing treatment time was 1.5 hours.
[0023] Step two, bone polypeptide extraction, includes the following specific steps: Enzymatic hydrolysis: The pretreated bone matrix raw material was mixed with purified water at a material-to-liquid ratio of 1:8, the pH was adjusted to 6.5 with acid, and 0.5% of the weight of the bone matrix raw material was added with compound protease. The mixture was then enzymatically hydrolyzed at 50°C for 4 hours. Enzyme inactivation and separation: The enzyme hydrolysate was heated to 85°C and held for 10 minutes to inactivate the enzyme. Then, it was first coarsely filtered through a 100-mesh filter and then centrifuged at 4°C and 8000 r / min for 15 minutes. The supernatant was collected to obtain the primary extract of bone polypeptide. Purification and concentration: The primary extract of bone peptides was subjected to two-step ultrafiltration by passing it through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The permeate with molecular weights less than 10 kDa and greater than 3 kDa was collected. The permeate was then concentrated under reduced pressure to 1 / 10 of its original volume at a temperature not higher than 50°C and a vacuum degree not lower than -0.09 MPa to obtain concentrated bone peptides. Drying: The concentrated bone polypeptide solution was spray-dried with the inlet temperature controlled at 160℃ and the outlet temperature controlled at 75℃ to obtain a white to off-white bone polypeptide extract.
[0024] The complex protease used in the enzymatic hydrolysis reaction consists of alkaline protease, neutral protease, and trypsin, with an enzyme activity ratio of 1:1:0.5.
[0025] Step 3, the pretreatment of melon seed raw materials, includes the following steps: After washing the melon seeds with purified water, dry them at 40°C with forced air until the moisture content is less than 8%, then crush them with a pulverizer and pass them through a 40-mesh sieve to obtain coarse melon seed powder; mix the coarse melon seed powder with petroleum ether at a material-to-liquid ratio of 1:4, reflux and defatt it at 50°C for 2 hours, repeat the defatting once, until the filtrate is colorless, and vacuum dry the defatted melon seed powder at 40°C until there is no solvent residue to obtain defatted melon seed powder.
[0026] Step four, the extraction of polypeptides from melon seeds, includes the following specific steps: Extraction: Defatted melon seed powder was mixed with 50% ethanol aqueous solution at a material-to-liquid ratio of 1:10. The pH was adjusted to 8.0 with acid, and the mixture was stirred and extracted at 60°C for 2 hours. The extraction was repeated once, and the extracts were combined. Preliminary concentration and impurity removal: The combined extracts were concentrated under reduced pressure at a temperature not exceeding 60°C until they were alcohol-free and concentrated to 1 / 5 of their original volume. Then, they were allowed to stand at 4°C for 12 hours, centrifuged to remove insoluble impurities, and the supernatant was collected. Resin purification: The supernatant was loaded onto a pretreated macroporous adsorption resin column at a flow rate of 1 column volume / hour. The macroporous adsorption resin was AB-8 type. After loading, impurities were removed by elution with 3 BV of purified water at a flow rate of 2 BV / hour, and then eluted with 3 BV of 30% ethanol aqueous solution at a flow rate of 1 BV / hour. This eluent was collected. Concentration and drying: The collected eluent was concentrated under reduced pressure at a temperature not exceeding 50°C to a paste-like consistency, and then freeze-dried to obtain a pale yellow melon seed polypeptide extract.
[0027] The specific amino acids and antioxidants in step five include L-arginine, glycine, and glutathione. The preparation of the stabilizer composition includes the following steps: Weigh 3 parts of L-arginine, 2 parts of glycine, and 0.5 parts of glutathione by weight, place the three in a nano-grinding mill, and grind them together for 30 minutes at a speed of 300 r / min under inert gas protection until the average particle size D90 of the mixture is less than 10 micrometers, to obtain the composite stabilizer.
[0028] Step six, the preparation of the lyophilized formulation, includes the following specific steps: Preparation of solution: Pour 70% of the total volume of water for injection into the mixing tank, cool to 2°C, and then add 10 parts of bone polypeptide extract, 5 parts of melon seed polypeptide extract, 60 parts of freeze-drying protectant and excipient, and 1 part of stabilizer composition in sequence. Stir until completely dissolved, and adjust the pH of the solution to 5.5 with acid during the process. The freeze-drying protectant and excipients consist of the following components in parts by weight: 40 parts mannitol, 10 parts sucrose, and 5 parts dextran. Volume adjustment and filtration: Add water for injection to the total volume, continue stirring at 2°C for 15 minutes to mix evenly, and then filter through 0.45-micron and 0.22-micron microporous membranes for sterilization to obtain sterile drug solution; Dispensing and Freeze-drying: The sterile drug solution was dispensed into vials under aseptic conditions, partially capped, and then transferred to a freeze dryer for freeze-drying. The freeze-drying process was as follows: First, the plate temperature was lowered to below -45°C and maintained for 2 hours to completely freeze the drug solution. Then, a first drying was performed, with the plate temperature raised to -25°C at a rate of 0.5°C / min and maintained at this temperature for 20 hours, with the vacuum degree controlled at 10 Pa. A second drying was then performed, with the plate temperature raised to 25°C at a rate of 0.3°C / min and maintained at this temperature for 8 hours, with the vacuum degree controlled at 5 Pa. After freeze-drying, the vials were fully capped under vacuum to obtain the bone marrow extract powder for injection.
[0029] The composition is made from the following raw materials in parts by weight: 10 parts bone polypeptide extract, 5 parts melon seed polypeptide extract, 60 parts freeze-drying protectant and excipient, and 1 part stabilizer composition; wherein the weight ratio of polypeptides in the bone polypeptide extract to those in the melon seed polypeptide extract is 1:1.
[0030] Example 2: A method for preparing a bone melon extract powder injection and a pharmaceutical composition, comprising the following steps: Step 1: Pretreatment of bone raw materials. Select long bones from the limbs of healthy pigs, wash and crush them, and then perform deep degreasing and decalcification treatments in sequence to obtain bone matrix raw materials. Step 2: Bone polypeptide extraction. The pretreated bone matrix raw material is subjected to enzymatic hydrolysis. After the reaction is completed, the enzyme is inactivated and the primary extract of bone polypeptide is obtained. Then, it is purified by ultrafiltration, concentrated and dried to obtain bone polypeptide extract. Step 3: Pre-treatment of melon seed raw materials. Select mature melon seeds, wash and dry them, then crush and defatted them to obtain defatted melon seed powder. Step 4: Extraction of melon seed polypeptides. Defatted melon seed powder is extracted using an alcohol-water mixture. The extract is filtered, concentrated, purified, eluted, concentrated, and dried using macroporous adsorption resin to obtain melon seed polypeptide extract. Step 5: Preparation of stabilizer composition: Specific amino acids and antioxidants are mixed in proportion and co-ground to obtain a composite stabilizer; Step Six: Preparation of freeze-dried formulation. The bone polypeptide extract obtained in Step Two, the melon seed polypeptide extract obtained in Step Four, the stabilizer composition obtained in Step Five, as well as the freeze-drying protectant and excipients, are dissolved, mixed, filtered and sterilized under specific conditions, then dispensed and freeze-dried to obtain bone melon extract powder for injection.
[0031] The deep degreasing and decalcification process in step one includes the following steps: the crushed bone particles are soaked in a degreasing solution containing organic solvent and ultrasonically degreased at 50°C for 2 hours. After degreasing, the particles are washed with purified water until neutral and drained. The degreased bone particles are then soaked in a decalcification solution and stirred at room temperature for 8 hours. After the reaction is complete, the particles are washed with purified water until neutral and freeze-dried at -45°C for 36 hours to obtain a porous, sponge-like clean bone matrix raw material. The degreasing solution is a mixed solution of chloroform and methanol in a volume ratio of 2:1, with a material-to-liquid ratio of 1g:10mL. The decalcification solution is a 1 mol / L hydrochloric acid solution, and the material-to-liquid ratio is 1 g: 12 mL.
[0032] In the deep degreasing process, the frequency of the ultrasonic-assisted treatment was 35 kHz, the temperature of the degreasing treatment was 53 ℃, and the degreasing treatment time was 1.7 hours.
[0033] Step two, bone polypeptide extraction, includes the following specific steps: Enzymatic hydrolysis: The pretreated bone matrix raw material was mixed with purified water at a material-to-liquid ratio of 1:12, the pH was adjusted to 7 with acid, 1% of the weight of the bone matrix raw material was added with compound protease, and enzymatic hydrolysis was carried out at 55℃ for 6 hours. Enzyme inactivation and separation: The enzyme hydrolysate was heated to 90℃ and kept at 15 minutes to inactivate the enzyme. Then, it was first coarsely filtered through a 150-mesh filter and then centrifuged at 4℃ and 10000r / min for 20 minutes. The supernatant was collected to obtain the primary extract of bone polypeptide. Purification and concentration: The primary extract of bone peptides was subjected to two-step ultrafiltration by passing it through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The permeate with molecular weights less than 10 kDa and greater than 3 kDa was collected. The permeate was concentrated under reduced pressure to 1 / 12 of its original volume at a temperature not higher than 50°C and a vacuum degree not lower than -0.09 MPa to obtain concentrated bone peptides. Drying: The concentrated bone polypeptide solution was spray-dried with the inlet temperature controlled at 170℃ and the outlet temperature controlled at 80℃ to obtain a white to off-white bone polypeptide extract.
[0034] The complex protease used in the enzymatic hydrolysis reaction consists of alkaline protease, neutral protease, and trypsin, with an enzyme activity ratio of 1.5:2:1.
[0035] Step 3, the pretreatment of melon seed raw materials, includes the following steps: After washing the melon seeds with purified water, dry them at 45°C with forced air until the moisture content is less than 8%, then crush them with a pulverizer and pass them through a 50-mesh sieve to obtain coarse melon seed powder; mix the coarse melon seed powder with petroleum ether at a material-to-liquid ratio of 1:5, reflux and defatt it at 55°C for 3 hours, repeat the defatting process twice until the filtrate is colorless, and vacuum dry the defatted melon seed powder at 45°C until there is no solvent residue to obtain defatted melon seed powder.
[0036] Step four, the extraction of polypeptides from melon seeds, includes the following specific steps: Extraction: Defatted melon seed powder was mixed with 60% ethanol aqueous solution at a material-to-liquid ratio of 1:15. The pH was adjusted to 8.5 with acid, and the mixture was stirred and extracted at 70°C for 3 hours. The extraction was repeated twice, and the extracts were combined. Preliminary concentration and impurity removal: The combined extracts were concentrated under reduced pressure at a temperature not exceeding 60°C until they were alcohol-free and reduced to 1 / 6 of their original volume. Then, they were allowed to stand at 4°C for 18 hours, centrifuged to remove insoluble impurities, and the supernatant was collected. Resin purification: The supernatant was loaded onto a pretreated macroporous adsorption resin column at a flow rate of 2 column volumes / hour. The macroporous adsorption resin was AB-8 type. After loading, impurities were removed by elution with 4 BV purified water at a flow rate of 3 BV / hour. Then, 5 BV of 40% ethanol aqueous solution was used for elution at a flow rate of 2 BV / hour. This eluent was collected. Concentration and drying: The collected eluent was concentrated under reduced pressure at a temperature not exceeding 50°C to a paste-like consistency, and then freeze-dried to obtain a pale yellow melon seed polypeptide extract.
[0037] The specific amino acids and antioxidants in step five include L-arginine, glycine, and glutathione. The preparation of the stabilizer composition includes the following steps: weigh 5 parts of L-arginine, 3 parts of glycine, and 1 part of glutathione by weight, place the three in a nano-grinding mill, and grind them together at a speed of 400 r / min for 45 minutes under inert gas protection until the average particle size D90 of the mixture is less than 10 micrometers, thus obtaining the composite stabilizer.
[0038] Step six, the preparation of the lyophilized formulation, includes the following specific steps: Preparation of solution: Pour 75% of the total volume of water for injection into the mixing tank, cool to 6°C, and then add 20 parts of bone polypeptide extract, 10 parts of melon seed polypeptide extract, 70 parts of freeze-drying protectant and excipient, and 3 parts of stabilizer composition in sequence. Stir until completely dissolved, and adjust the pH of the solution to 6.5 with acid during the process. The freeze-drying protectant and excipients are composed of the following components in parts by weight: 50 parts mannitol, 15 parts sucrose, and 7 parts dextran. Volume adjustment and filtration: Add water for injection to the total volume, continue stirring at 5°C for 25 minutes to mix evenly, and then filter through 0.45-micron and 0.22-micron microporous membranes for sterilization to obtain sterile drug solution; Dispensing and Lyophilization: The sterile drug solution was dispensed into vials under aseptic conditions, partially capped, and then transferred to a lyophilizer for lyophilization. The lyophilization process was as follows: First, the plate temperature was lowered to below -45°C and maintained for 3 hours to completely freeze the drug solution. Then, a first drying was performed, with the plate temperature raised to -25°C at a rate of 0.7°C / min and maintained at this temperature for 25 hours, with the vacuum degree controlled at 20 Pa. Next, a second drying was performed, with the plate temperature raised to 30°C at a rate of 0.4°C / min and maintained at this temperature for 10 hours, with the vacuum degree controlled at 10 Pa. After lyophilization, the vials were fully capped under vacuum to obtain the bone marrow extract powder for injection.
[0039] The composition is made from the following raw materials in parts by weight: 20 parts bone polypeptide extract, 10 parts melon seed polypeptide extract, 70 parts freeze-drying protectant and excipient, and 3 parts stabilizer composition; wherein the weight ratio of polypeptides in the bone polypeptide extract to those in the melon seed polypeptide extract is 3:1.
[0040] Example 3: A method for preparing a bone melon extract powder injection and a pharmaceutical composition, comprising the following steps: Step 1: Pretreatment of bone raw materials. Select long bones from the limbs of healthy pigs, wash and crush them, and then perform deep degreasing and decalcification treatments in sequence to obtain bone matrix raw materials. Step 2: Bone polypeptide extraction. The pretreated bone matrix raw material is subjected to enzymatic hydrolysis. After the reaction is completed, the enzyme is inactivated and the primary extract of bone polypeptide is obtained. Then, it is purified by ultrafiltration, concentrated and dried to obtain bone polypeptide extract. Step 3: Pre-treatment of melon seed raw materials. Select mature melon seeds, wash and dry them, then crush and defatted them to obtain defatted melon seed powder. Step 4: Extraction of melon seed polypeptides. Defatted melon seed powder is extracted using an alcohol-water mixture. The extract is filtered, concentrated, purified, eluted, concentrated, and dried using macroporous adsorption resin to obtain melon seed polypeptide extract. Step 5: Preparation of stabilizer composition: Specific amino acids and antioxidants are mixed in proportion and co-ground to obtain a composite stabilizer; Step Six: Preparation of freeze-dried formulation. The bone polypeptide extract obtained in Step Two, the melon seed polypeptide extract obtained in Step Four, the stabilizer composition obtained in Step Five, as well as the freeze-drying protectant and excipients, are dissolved, mixed, filtered and sterilized under specific conditions, then dispensed and freeze-dried to obtain bone melon extract powder for injection.
[0041] The deep degreasing and decalcification process in step one includes the following steps: the crushed bone particles are soaked in a degreasing solution containing organic solvent and ultrasonically degreased at 60°C for 3 hours. After degreasing, the particles are washed with purified water until neutral and drained. The degreased bone particles are then soaked in a decalcification solution and stirred at room temperature for 12 hours. After the reaction is complete, the particles are washed with purified water until neutral and freeze-dried at -40°C for 48 hours to obtain a porous, sponge-like clean bone matrix raw material. The degreasing solution is a mixed solution of chloroform and methanol in a volume ratio of 2:1, with a material-to-liquid ratio of 1g:12mL. The decalcification solution is a 1.5 mol / L hydrochloric acid solution, and the material-to-liquid ratio is 1 g: 15 mL.
[0042] In the deep degreasing process, the frequency of the ultrasonic-assisted treatment was 40kHz, the temperature of the degreasing treatment was 55℃, and the degreasing treatment time was 2 hours.
[0043] Step two, bone polypeptide extraction, includes the following specific steps: Enzymatic hydrolysis: The pretreated bone matrix raw material was mixed with purified water at a material-to-liquid ratio of 1:15, the pH was adjusted to 7.5 with acid, and 2.0% of the weight of the bone matrix raw material was added with compound protease. The mixture was then enzymatically hydrolyzed at 60°C for 8 hours. Enzyme inactivation and separation: The enzyme hydrolysate was heated to 95℃ and kept at 20 minutes to inactivate the enzyme. Then, it was first coarsely filtered through a 200-mesh filter and then centrifuged at 4℃ and 12000r / min for 25 minutes. The supernatant was collected to obtain the primary extract of bone polypeptide. Purification and concentration: The primary extract of bone peptides was subjected to two-step ultrafiltration by passing it through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The permeate with molecular weights less than 10 kDa and greater than 3 kDa was collected. The permeate was then concentrated under reduced pressure to 1 / 15 of its original volume at a temperature not higher than 50°C and a vacuum degree not lower than -0.09 MPa to obtain concentrated bone peptides. Drying: The concentrated bone polypeptide solution was spray-dried with the inlet temperature controlled at 180℃ and the outlet temperature controlled at 85℃ to obtain a white to off-white bone polypeptide extract.
[0044] The complex protease used in the enzymatic hydrolysis reaction consists of alkaline protease, neutral protease, and trypsin, with an enzyme activity ratio of 2:3:1.5.
[0045] Step 3, the pretreatment of melon seed raw materials, includes the following steps: After washing the melon seeds with purified water, dry them at 50°C with forced air until the moisture content is less than 8%, then crush them with a pulverizer and pass them through a 60-mesh sieve to obtain coarse melon seed powder; mix the coarse melon seed powder with petroleum ether at a material-to-liquid ratio of 1:6, reflux and defatt it at 60°C for 4 hours, repeat the defatting process 3 times until the filtrate is colorless, and vacuum dry the defatted melon seed powder at 50°C until there is no solvent residue to obtain defatted melon seed powder.
[0046] Step four, the extraction of polypeptides from melon seeds, includes the following specific steps: Extraction: Defatted melon seed powder was mixed with 70% ethanol aqueous solution at a material-to-liquid ratio of 1:20. The pH was adjusted to 9.5 with acid, and the mixture was stirred and extracted at 80°C for 4 hours. The extraction was repeated twice, and the extracts were combined. Preliminary concentration and impurity removal: The combined extracts were concentrated under reduced pressure at a temperature not exceeding 60°C until they were alcohol-free and reduced to 1 / 8 of their original volume. Then, they were allowed to stand at 4°C for 24 hours, centrifuged to remove insoluble impurities, and the supernatant was collected. Resin purification: The supernatant was loaded onto a pretreated macroporous adsorption resin column at a flow rate of 3 column volumes / hour. The macroporous adsorption resin was AB-8 type. After loading, impurities were removed by elution with 5 BV purified water at a flow rate of 4 BV / hour. Then, 6 BV of 50% ethanol aqueous solution was used for elution at a flow rate of 3 BV / hour. This eluent was collected. Concentration and drying: The collected eluent was concentrated under reduced pressure at a temperature not exceeding 50°C to a paste-like consistency, and then freeze-dried to obtain a pale yellow melon seed polypeptide extract.
[0047] The specific amino acids and antioxidants in step five include L-arginine, glycine, and glutathione. The preparation of the stabilizer composition includes the following steps: weigh 8 parts of L-arginine, 5 parts of glycine, and 2 parts of glutathione by weight, place the three in a nano-grinding mill, and grind them together at a speed of 500 r / min for 60 minutes under inert gas protection until the average particle size D90 of the mixture is less than 10 micrometers, thus obtaining the composite stabilizer.
[0048] Step six, the preparation of the lyophilized formulation, includes the following specific steps: Preparation of solution: Pour 80% of the total volume of water for injection into the mixing tank, cool to 8°C, and then add 30 parts of bone polypeptide extract, 15 parts of melon seed polypeptide extract, 85 parts of freeze-drying protectant and excipient, and 5 parts of stabilizer composition in sequence. Stir until completely dissolved, and adjust the pH of the solution to 7.0 with acid during the process. The freeze-drying protectant and excipients consist of the following components in parts by weight: 60 parts mannitol, 20 parts sucrose, and 10 parts dextran. Volume adjustment and filtration: Add water for injection to the total volume, continue stirring at 8°C for 30 minutes to mix evenly, and then filter through 0.45-micron and 0.22-micron microporous membranes for sterilization to obtain sterile drug solution; Dispensing and Freeze-drying: The sterile drug solution was dispensed into vials under aseptic conditions, partially capped, and then transferred to a freeze dryer for freeze-drying. The freeze-drying process was as follows: First, the plate temperature was lowered to below -45°C and maintained for 4 hours to completely freeze the drug solution. Then, a first drying was performed, in which the plate temperature was raised to -25°C at a rate of 1°C / min and maintained at this temperature for 30 hours, with the vacuum degree controlled at 30 Pa. Next, a second drying was performed, in which the plate temperature was raised to 30°C at a rate of 0.5°C / min and maintained at this temperature for 12 hours, with the vacuum degree controlled at 15 Pa. After freeze-drying, the vials were fully capped under vacuum to obtain the bone marrow extract powder for injection.
[0049] The composition is made from the following raw materials in parts by weight: 30 parts bone polypeptide extract, 15 parts melon seed polypeptide extract, 85 parts freeze-drying protectant and excipient, and 5 parts stabilizer composition; wherein the weight ratio of polypeptides in the bone polypeptide extract to those in the melon seed polypeptide extract is 6:1.
[0050] Comparative Example 1: The difference between this comparative example and Example 1 is that the defatted bone particles were not subjected to ultrasound-assisted treatment during the preparation of the bone polypeptide extract in this comparative example.
[0051] Comparative Example 2 differs from Example 1 in that the enzyme used in the enzymatic hydrolysis reaction during the preparation of the bone polypeptide extract in this comparative example is a single neutral protease.
[0052] Comparative Example 3 differs from Example 1 in that macroporous adsorption resin was not used for purification when preparing the melon seed polypeptide extract in this comparative example.
[0053] Comparative Example 4 differs from Example 1 in that no nanoscale composite stabilizer was added during the preparation of the lyophilized formulation in this comparative example.
[0054] The performance of the bone melon extract powder for injection prepared in Examples 1-3 and Comparative Examples 1-4 was tested. The test items and test methods are as follows: Degreasing and decalcification rate tests: After pretreatment of bone raw materials, Soxhlet extraction was performed using ether as solvent for 6 hours. The amount of residual fat and the degreasing rate were calculated. The calcium ion content was determined by atomic absorption spectrometry at the characteristic wavelength of calcium, and the decalcification rate was calculated. Polypeptide molecular weight distribution test: High performance liquid chromatography-evaporative light scattering detection method was used to determine the molecular weight distribution of bone polypeptide extract and melon seed polypeptide extract using polypeptide standards with known molecular weight as a control, and the percentage of the main molecular weight range was calculated. Detection of related substances: High performance liquid chromatography was used with octadecylsilane-bonded silica gel as the packing material and acetonitrile-water as the mobile phase for gradient elution. The detection wavelength was 220 nm. The purity of the main peptide component and the content of related substances in the powder injection were determined. Accelerated stability test: The powder injection was placed under accelerated stability test conditions of 40±2°C and 75±5% relative humidity, and samples were taken at 0, 1, 2, 3 and 6 months to determine the peptide content, related substances and reconstitution time.
[0055] The bone marrow extract powder for injection prepared using the processes in Examples 1-3 showed advantages in key quality attributes compared to the powder for injection prepared using the processes in Comparative Examples 1-4. This indicates that by performing deep defatting and decalcification treatment on bone raw materials, combined with enzymatic hydrolysis under specific conditions and a two-step ultrafiltration purification process, non-target impurities in the bone matrix can be removed, and the molecular weight distribution range of bone polypeptides can be controlled, thereby improving the uniformity and batch-to-batch consistency of the active ingredients of bone polypeptides. Simultaneously, the complex protease hydrolysis strategy used in bone polypeptide extraction can synergistically act on bone proteins, increasing polypeptide yield and laying the foundation for stable control of the proportion of active ingredients in the final drug composition. By pre-treating the melon seed raw materials with defatting and extracting with an alcohol-water solution under specific pH conditions, combined with selective purification using macroporous adsorption resin, the target polypeptide components in the melon seeds can be enriched, while removing sugars and pigment impurities, thereby improving the purity of the melon seed polypeptide extract and ensuring the preparation of high-purity powder for injection with low related substances. By introducing a composite stabilizer made by co-grinding specific amino acids and antioxidants, this composition is well compatible with bone peptides and melon seed peptides. In synergy with an optimized freeze-drying protectant system, it can protect the active structures of bone peptides and melon seed peptides during freeze-drying and long-term storage, inhibiting their degradation, aggregation and oxidation, thereby improving the physicochemical stability, reconstitution properties and therapeutic stability of the powder injection under long-term storage.
[0056] The bone marrow extract powder injection prepared by the preparation process provided by this invention exhibits consistent superiority in key indicators such as raw material pretreatment efficiency, active ingredient quality control, product purity, and long-term stability, demonstrating excellent comprehensive performance.
[0057] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.
[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a bone melon extract powder injection, characterized in that: Includes the following steps: Step 1: Pretreatment of bone raw materials. Select long bones from the limbs of healthy pigs or cattle, wash and crush them, and then perform deep degreasing and decalcification treatments in sequence to obtain bone matrix raw materials. Step 2: Bone polypeptide extraction. The pretreated bone matrix raw material is subjected to enzymatic hydrolysis. After the reaction is completed, the enzyme is inactivated and the primary extract of bone polypeptide is obtained. Then, it is purified by ultrafiltration, concentrated and dried to obtain bone polypeptide extract. Step 3: Pre-treatment of melon seed raw materials. Select mature melon seeds, wash and dry them, then crush and defatted them to obtain defatted melon seed powder. Step 4: Extraction of melon seed polypeptides. Defatted melon seed powder is extracted using an alcohol-water mixture. The extract is filtered, concentrated, purified, eluted, concentrated, and dried using macroporous adsorption resin to obtain melon seed polypeptide extract. Step 5: Preparation of stabilizer composition: Specific amino acids and antioxidants are mixed in proportion and co-ground to obtain a composite stabilizer; Step Six: Preparation of freeze-dried formulation. The bone polypeptide extract obtained in Step Two, the melon seed polypeptide extract obtained in Step Four, the stabilizer composition obtained in Step Five, as well as the freeze-drying protectant and excipients, are dissolved, mixed, filtered and sterilized under specific conditions, then dispensed and freeze-dried to obtain bone melon extract powder for injection.
2. The method for preparing a bone marrow extract powder injection according to claim 1, characterized in that: The deep degreasing and decalcification process in step one includes the following steps: immersing the crushed bone particles in a degreasing solution containing organic solvent, and ultrasonically degreasing them at 45-60℃ for 1-3 hours. After degreasing, washing with purified water until neutral, draining, immersing the degreased bone particles in a decalcification solution, stirring and reacting at room temperature for 4-12 hours, washing with purified water until neutral, and freeze-drying at -50℃ to -40℃ for 24-48 hours to obtain a porous, sponge-like clean bone matrix raw material; The degreasing solution is a mixed solution of chloroform and methanol in a volume ratio of 2:1, with a material-to-liquid ratio of 1g:8-12mL. The decalcification solution is a 0.5 mol / L to 1.5 mol / L hydrochloric acid solution or a disodium ethylenediaminetetraacetate solution, with a material-to-liquid ratio of 1 g: 10-15 mL.
3. The method for preparing a bone marrow extract powder injection according to claim 2, characterized in that: The ultrasonic-assisted treatment in the deep degreasing process has a frequency of 28-40kHz, a temperature of 50-55℃, and a duration of 1.5-2 hours.
4. The method for preparing a bone marrow extract powder injection according to claim 1, characterized in that: Step two, the extraction of bone polypeptides, includes the following specific steps: Enzymatic hydrolysis: Mix the pretreated bone matrix raw material with purified water at a material-to-liquid ratio of 1:8-1:15, adjust the pH to 6.5-7.5 with acid or alkali, add 0.5%-2.0% of the weight of the bone matrix raw material with compound protease, and enzymatically hydrolyze at 50-60℃ for 4-8 hours. Enzyme inactivation and separation: Heat the enzyme hydrolysate to 85-95℃ and maintain for 10-20 minutes to inactivate the enzyme. Then, first filter it through a 100-200 mesh filter, and then centrifuge it at 4℃ and 8000-12000 r / min for 15-25 minutes. Collect the supernatant to obtain the primary extract of bone polypeptide. Purification and concentration: The primary extract of bone peptides was subjected to two-step ultrafiltration by passing it through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The permeate with molecular weights less than 10 kDa and greater than 3 kDa was collected. The permeate was concentrated under reduced pressure to 1 / 10 to 1 / 15 of its original volume under conditions of a temperature not exceeding 50°C and a vacuum degree not lower than -0.09 MPa to obtain concentrated bone peptides. Drying: The concentrated bone polypeptide solution is spray-dried with the inlet temperature controlled at 160-180℃ and the outlet temperature controlled at 75-85℃ to obtain a white to off-white bone polypeptide extract.
5. The method for preparing a bone melon extract powder injection according to claim 4, characterized in that: The complex protease used in the enzymatic hydrolysis reaction consists of alkaline protease, neutral protease and trypsin, with an enzyme activity ratio of 1-2:1-3:0.5-1.
5.
6. The method for preparing a bone melon extract powder injection according to claim 1, characterized in that: The pretreatment of melon seed raw materials in step three includes the following steps: after washing the melon seeds with purified water, they are dried in a forced-air dryer at 40-50℃ until the moisture content is less than 8%, and then crushed by a pulverizer and passed through a 40-60 mesh sieve to obtain coarse melon seed powder; the coarse melon seed powder is mixed with petroleum ether at a material-liquid ratio of 1:4-1:6, and refluxed at 50-60℃ for 2-4 hours, and the degreasing is repeated 1-3 times until the filtrate is colorless, and the degreased melon seed powder is vacuum dried at 40-50℃ until there is no solvent residue to obtain defatted melon seed powder.
7. The method for preparing a bone melon extract powder injection according to claim 1, characterized in that: Step four, the extraction of melon seed polypeptides, includes the following specific steps: Extraction: Mix defatted melon seed powder with an ethanol aqueous solution with a volume concentration of 50%-70% at a material-to-liquid ratio of 1:10-1:20, adjust the pH to 8.0-9.5 with acid or alkali, and extract by stirring at 60-80℃ for 2-4 hours. Repeat the extraction 1-2 times and combine the extracts. Preliminary concentration and impurity removal: The combined extracts are concentrated under reduced pressure at a temperature not exceeding 60℃ until they are alcohol-free and reduced to 1 / 5-1 / 8 of their original volume. Then, they are allowed to stand at 4℃ for 12-24 hours, centrifuged to remove insoluble impurities, and the supernatant is collected. Resin purification: The supernatant is loaded onto a pretreated macroporous adsorption resin column at a flow rate of 1-3 column volumes / hour. The macroporous adsorption resin is at least one of AB-8, D101, or HPD-100. After loading, impurities are removed by elution with 3-5 BV of purified water at a flow rate of 2-4 BV / hour. Then, elution is performed with 3-6 BV of 30%-50% ethanol aqueous solution at a flow rate of 1-3 BV / hour. This eluent is collected. Concentration and drying: The collected eluent was concentrated under reduced pressure at a temperature not exceeding 50°C to a paste-like consistency, and then freeze-dried or spray-dried to obtain a pale yellow melon seed polypeptide extract.
8. The method for preparing a bone marrow extract powder injection according to claim 1, characterized in that: The specific amino acids and antioxidants in step five include L-arginine, glycine, and glutathione. The preparation of the stabilizer composition includes the following steps: Weigh out 3-8 parts by weight of L-arginine, 2-5 parts by weight of glycine and 0.5-2 parts by weight of glutathione, place the three components in a nano-grinding mill, and grind them together for 30-60 minutes at a speed of 300-500 r / min under inert gas protection until the average particle size D90 of the mixture is less than 10 micrometers, to obtain the composite stabilizer.
9. The method for preparing a bone marrow extract powder injection according to claim 1, characterized in that: Step six, the preparation of the lyophilized formulation, includes the following specific steps: Preparation of solution: Pour 70%-80% of the total volume of water for injection into the mixing tank, cool to 2-8℃, and add 10-30 parts of bone polypeptide extract, 5-15 parts of melon seed polypeptide extract, 60-85 parts of freeze-drying protectant and excipient, and 1-5 parts of stabilizer composition in sequence. Stir until completely dissolved, and adjust the pH of the solution to 5.5-7.0 with acid or alkali during the process. The freeze-drying protectant and excipient are composed of the following components in parts by weight: 40-60 parts mannitol, 10-20 parts sucrose, and 5-10 parts dextran. Volume adjustment and filtration: Add water for injection to the total volume, continue stirring at 2-8℃ for 15-30 minutes to mix evenly, and then filter through 0.45-micron and 0.22-micron microporous membranes for sterilization to obtain sterile drug solution; Dispensing and Freeze-drying: The sterile drug solution was dispensed into vials under aseptic conditions, partially capped, and then transferred to a freeze dryer for freeze-drying. The freeze-drying process was as follows: First, the plate temperature was lowered to below -45°C and maintained for 2-4 hours to completely freeze the drug solution. Then, a first drying was performed, with the plate temperature raised to -25°C at a rate of 0.5-1°C / min and maintained at this temperature for 20-30 hours, with the vacuum degree controlled at 10-30 Pa. Next, a second drying was performed, with the plate temperature raised to 25-30°C at a rate of 0.3-0.5°C / min and maintained at this temperature for 8-12 hours, with the vacuum degree controlled at 5-15 Pa. After freeze-drying, the vials were fully capped under vacuum or nitrogen purging conditions to obtain the bone marrow extract powder for injection.
10. A medicinal composition for injection of *Calamus esculenta* extract powder, prepared by any one of the methods for preparing *Calamus esculenta* extract powder for injection according to claims 1-9, characterized in that: The composition is made from the following raw materials in parts by weight: 10-30 parts of bone polypeptide extract, 5-15 parts of melon seed polypeptide extract, 60-85 parts of freeze-drying protectant and excipient, and 1-5 parts of stabilizer composition; wherein the weight ratio of polypeptides in the bone polypeptide extract to those in the melon seed polypeptide extract is 1:1 to 6:1.