Active bone marrow peptide for increasing bone mineral density as well as preparation method and application of active bone marrow peptide

Abstract

The invention discloses an active bone marrow peptide for increasing bone mineral density and a preparation method and application thereof, and belongs to the technical field of bioengineering.The preparation method comprises the steps that carboxylated Fe3O4 nanoparticles, chitosan, an EDC cross-linking agent and bone marrow peptide are covalently coupled to form a magnetic composite material, and bone marrow peptide powder is obtained after the magnetic composite material is subjected to magnetic field adsorption separation, active peptide elution and freeze-drying. The KH-560 is used for enhancing the interface bonding strength, the PEG molecular chain is used for reducing the non-specific adsorption, and the Tween-20 is used for promoting the dissociation of the active peptide, so that the adsorption efficiency, the selectivity and the reusability of the magnetic composite material are remarkably improved. The sturgeon active bone marrow peptide is efficiently extracted, the bone mineral density can be effectively increased, and technical support is provided for development of bone mineral density functional products.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and more specifically, to an active bone marrow peptide for increasing bone density, its preparation method and application. Background Art

[0002] Bone marrow peptide is a small molecule active peptide extracted from animal bone marrow, usually obtained by enzymolysis or biotechnological decomposition of bone marrow protein. Such peptides are rich in components such as collagen, amino acids, minerals and growth factors, and are considered to have various biological activities, such as enhancing immunity, antioxidant and anti-aging, blood replenishment and hematopoiesis, anti-fatigue, and neuroprotection.

[0003] In the prior art, the preparation of bone marrow peptide mostly adopts enzymolysis method, acid / alkali hydrolysis method, physical assisted extraction method, fermentation method or ultrafiltration chromatography purification method. However, the above methods have the following technical problems: low enzymolysis efficiency: the structure of bone marrow protein is complex (such as high cross-linking degree of collagen), and conventional enzymolysis is difficult to completely hydrolyze, and composite enzymes need to act synergistically, but the cost is high and process optimization is difficult; low yield of active peptide: the molecular weight distribution of the target peptide segment is wide, and the existing separation techniques (such as ultrafiltration) may lose small molecule active peptides or cannot effectively remove bitter peptides; poor product stability: the extracted peptide is easily degraded by temperature and pH, and the endogenous enzymes (such as lipase) contained in bone marrow may cause a decrease in activity during storage and problems such as inability to mass produce.

[0004] Currently, magnetic adsorption technology is mostly used for the separation and purification of proteins and polypeptides, and there is also an application of magnetic adsorption technology in the preparation of active peptides. For example, the invention patent with the publication number CN 106011206 A discloses a method for preparing active peptides by immobilizing double enzymes with a composite carrier magnetic nanoparticle. However, in this prior art, the magnetic carrier aims at enzyme immobilization, the active sites on the material surface are easily occupied by enzymes, the loading efficiency is low, and the enzyme immobilized material is prone to poor reusability due to enzyme inactivation or shedding, resulting in poor separation and preparation effect.

[0005] Therefore, how to provide a reusable magnetic adsorption material and how to provide the acquisition rate of active peptides are technical problems that those skilled in the art urgently need to solve. Summary of the Invention

[0006] In view of this, the present invention provides an active bone marrow peptide for increasing bone density, its preparation method and application. The active bone marrow peptide is prepared by using a magnetic composite material, which not only simplifies the preparation process of the active bone marrow peptide, but also the magnetic composite material can be reused. Moreover, by using this magnetic composite material, an active bone marrow peptide with the efficacy of increasing bone density can be successfully prepared, providing technical support for the development of bone density functional products.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme: A preparation method of active bone marrow peptide for increasing bone density, comprising the following steps: S1: Prepare a magnetic composite material according to the composition and ratio of 50 - 70 parts of carboxylated Fe 3 O 4 nanoparticles, 15 - 25 parts of chitosan, 10 - 20 parts of bone marrow peptide and 3 - 7 parts of EDC crosslinking agent, and set aside; S2: Mix the magnetic composite material obtained in S1 with bone marrow homogenate, add 0.1 - 0.3% of sodium citrate based on the volume of the homogenate, and stir at 20 - 30 °C for 20 - 40 min; S3: Place the product obtained after S2 treatment in a 0.2 - 0.4 T magnetic field, and magnetically separate to obtain a magnetic composite material - biomolecule complex; S4: Use glycine - HCl buffer solution to oscillate, dissociate and elute the magnetic composite material - biomolecule complex, concentrate with an ultrafiltration membrane and freeze - dry to prepare the active bone marrow peptide.

[0008] As a preferred technical solution, the specific preparation method of the magnetic composite material described in S1 is as follows: (1) Carboxyl activation: Disperse carboxylated Fe 3 O 4 nanoparticles in MES buffer solution, then add EDC and NHS crosslinking agents, and oscillate at room temperature for 15 - 45 min to obtain a suspension; (2) Coupling reaction: Add chitosan to the suspension obtained in step (1), adjust the pH to 5.5 - 6.5, and react at room temperature for 2 - 4 h; then add bone marrow peptide, adjust the pH to 7.0 - 7.4, and continue to react for 6 - 12 h; (3) Termination and washing: Add glycine to the suspension in step (2) to block unreacted groups, magnetically separate and wash to prepare the magnetic composite material.

[0009] As a preferred technical solution, the concentration of the carboxylated Fe 3 O 4 nanoparticles is 0.5 - 2 mg / ml; the pH of the MES buffer solution is 5.5 - 6.5; the concentration of the EDC is 8 - 12 mM; the concentration of the NHS is 4 - 6 mM; the molar ratio of the EDC and NHS crosslinking agents is 2:1; the concentration of the glycine is 0.05 - 0.2 M, and the pH is 7.5 - 8.5.

[0010] As a preferred technical solution, the particle size of the carboxylated Fe 3 O 4 nanoparticles is 15 - 25 nm, and its surface is modified with silane coupling agent KH - 560; the degree of deacetylation of the chitosan ≥ 85%, and its surface is modified with PEG; the molecular weight of the bone marrow peptide < 1.5 kDa.

[0011] As a preferred technical solution, the magnetic composite material and the bone marrow homogenate in S2 are mixed at a mass-to-volume ratio of 1:5–1:20; the pH of the bone marrow homogenate is 5.5-6.5; the magnetic composite material can be reused 3-7 times; As a preferred technical solution, the time for magnetic separation in S3 is 1-3 min; As a preferred technical solution, the pH of the glycine-HCl buffer solution in S4 is 2.0-3.0, containing 0.01-0.05% (volume percentage) of Tween-20; the dosage of the glycine-HCl buffer solution is 1.5-2.5 times the volume of the magnetic composite material-biological molecule complex; the time for oscillatory dissociation and elution is 8-12 min; the cut-off molecular weight of the ultrafiltration membrane is 2-5 kDa.

[0012] The beneficial effect of the above operation is that Tween-20, as a surfactant, promotes the dissociation of the bioactive peptide from the surface of the magnetic composite material by reducing the surface tension of the solution, and at the same time reduces the loss of the bioactive peptide during the dissociation process.

[0013] Another object of the present invention is to provide: the bioactive bone marrow peptide prepared by the above method.

[0014] Another object of the present invention is to provide: the application of the above bioactive bone marrow peptide in the preparation of products for improving bone density.

[0015] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects: The method of the present invention first covalently couples carboxylated Fe 3 O 4 nanoparticles, chitosan, an EDC cross-linking agent with sturgeon bone marrow peptide to form a magnetic composite material. Chitosan (amino group) and carboxylated Fe 3 O 4 (carboxyl group) are cross-linked through EDC to form a stable network, binding to the amino / carboxyl group of the bone marrow peptide; a step-by-step coupling process: first couple chitosan, and then introduce the bone marrow peptide to avoid competitive binding. Among them, the surface of the carboxylated Fe 3 O 4 nanoparticles is modified with KH-560, and KH-560 binds to the surface of the Fe 3 O 4 nanoparticles through a siloxane bond, enhancing the interfacial binding strength, ensuring that the magnetic composite material still maintains a high adsorption efficiency after multiple uses, and at the same time improving the mechanical stability and anti-degradation performance of the magnetic composite material.

[0016] The chitosan molecular chain is cross-linked with carboxylated Fe 3 O 4The carboxyl groups on the surface of the nanoparticles undergo an esterification reaction to form covalent bond linkages. The chitosan molecular chains form a three-dimensional network structure through hydrogen bonding, and this network structure can effectively load sturgeon bone marrow peptides and enhance their binding ability. The bone marrow peptides undergo an amidation reaction with the amino groups on the surface of chitosan through an EDC cross-linking agent to form stable covalent bond linkages, improving the loading efficiency and selectivity of the bone marrow peptides on the magnetic composite material.

[0017] The PEG molecular chains in the surface modification layer of the magnetic composite material reduce the non-specific adsorption of impurities such as proteins through steric hindrance effects, enhance the hydrophilicity of the material, improve the selectivity and purity in the separation process of active peptides, delay the performance degradation of the magnetic composite material, and ensure that it still maintains a high adsorption efficiency after multiple uses. At the same time, it improves the dispersibility and stability of the magnetic composite material in the aqueous phase system.

[0018] During the separation process, after the magnetic composite material is mixed with sturgeon bone marrow homogenate, the sturgeon bone marrow peptides are adsorbed onto the surface of the magnetic composite material through covalent bonds and hydrogen bonding. After separating the magnetic composite material by magnetic field adsorption, glycine-HCl buffer solution is used for elution. The eluted active peptides are concentrated by an ultrafiltration membrane and then freeze-dried to obtain the final product.

[0019] In summary, the innovation of the technical route of the present invention lies in directly separating natural active bone marrow peptides from bone marrow homogenate using a magnetic composite material from enzymatic hydrolysis to direct separation, omitting the enzymatic hydrolysis step, avoiding enzyme activity loss and side reactions, significantly improving the product purity, shortening the process flow (reducing 2 - 3 steps), reducing energy consumption, and being more suitable for the efficient preparation of natural active peptides. In addition, the bone marrow peptides separated by magnetic separation in the present invention are directly applied to bone density products for the first time, opening up a new usage path for bone marrow peptides. Detailed implementation mode

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] The EDC cross-linking agent used in the embodiments of the present invention is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; The silane coupling agent KH-560 used is γ-glycidoxypropyltrimethoxysilane; The PEG used is epoxy-PEG, Shanghai Aladdin Biochemical Technology Co., Ltd., product number E163664; The NHS cross-linking agent used is N-hydroxysuccinimide ester.

[0022] The present invention adopts the following embodiments: A magnetic composite material comprising the following components in parts by weight: 50 - 70 parts of carboxylated Fe 3 O 4 nanoparticles, 15 - 25 parts of chitosan, 10 - 20 parts of sturgeon bone marrow peptide, and 3 - 7 parts of EDC crosslinking agent; The above-mentioned carboxylated Fe 3 O 4 nanoparticles are prepared by the following method: (1) Carboxylation: Disperse Fe 3 O 4 in 0.1 M citric acid solution, reflux at 80 °C for 6 hours, magnetically separate and wash until neutral to prepare carboxylated Fe 3 O 4 nanoparticles (particle size 15 - 25 nm); (2) Surface modification with silane coupling agent KH-560: A: Preparation of hydrolysis solution: Dissolve KH-560 in ethanol / water mixed solution (volume ratio 9:1), with a final concentration of 2 - 5% (w / v), adjust the pH to 4 - 5 with dilute hydrochloric acid, and stir magnetically for 30 min to fully hydrolyze KH-560 to form silanol; B: Coupling reaction: Disperse Fe 3 O 4 nanoparticles in ethanol to prepare a suspension of Fe 3 O 4 nanoparticles, then add it dropwise to the hydrolysis solution, ultrasonically disperse for 10 min, and under nitrogen protection, stir and react at a constant temperature of 70 °C for 6 - 12 hours; C: Cleaning: After the reaction, magnetically separate the particles, wash them 3 times each with ethanol and deionized water to remove unreacted KH-560 and by-products, and dry them under vacuum at 40 °C for 12 h to prepare carboxylated Fe 3 O 4 nanoparticles with KH-560 surface modification.

[0023] The above-mentioned chitosan is prepared by the following method: (1) Preparation of chitosan solution: Dissolve chitosan (degree of deacetylation ≥ 85%) in 1% acetic acid solution (pH 5.0) with a concentration of 1 - 2% (w / v); (2) Configuration of reaction system: Mix the chitosan solution and borate buffer solution in a volume ratio of 1:1, adjust the pH to 9.0 - 10.0 (alkaline conditions promote the ring-opening of epoxy groups, too low leads to slow reaction, too high may damage the chitosan structure temperature), add epoxy-PEG (molar ratio 3 - 5 times that of chitosan amino groups), and stir until completely dissolved; (3) Coupling reaction: Stir and react for 6 - 8 hours in a constant temperature water bath at 60 °C (to accelerate the reaction and avoid degradation of chitosan caused by high temperature); (4) Termination and purification: After cooling to room temperature, adjust the pH to neutral with 0.1 M HCl, and dialyze (cut-off molecular weight 3.5 kDa) to remove unreacted PEG (for 3 days, changing water 3 times a day), then freeze-dry to obtain chitosan with PEG modified on its surface.

[0024] The above sturgeon bone marrow peptide is prepared by the following method Bone marrow homogenate: Take sturgeon bone marrow, add PBS at a ratio of 1:5 (w / v), homogenize and then centrifuge (10,000×g, 20 minutes, 4 °C); Enzymatic hydrolysis: Use trypsin or alkaline protease for compounding (enzyme / substrate ratio 1:50, pH 8.0, 37 °C) to hydrolyze for 4 hours, and inactivate with boiling water; Purification: Ultrafiltration (cut-off molecular weight 10 kDa) combined with HPLC (C18 column, gradient elution with acetonitrile / water).

[0025] Preparation method of a magnetic composite material The carboxylated Fe 3 O 4 nanoparticles, chitosan and sturgeon bone marrow peptide are added with an EDC cross-linking agent for preparation: (1) Carboxyl activation: Disperse the carboxylated Fe 3 O 4 nanoparticles (1 mg / mL) in MES buffer (pH 6.0), then add EDC (10 mM) and NHS (5 mM), and shake at room temperature for 30 min; (2) Coupling reaction: Add chitosan to the suspension obtained in step (1), adjust the pH to 5.5 - 6.5, and react at room temperature for 2 - 4 h; then add sturgeon bone marrow peptide, adjust the pH to 7.0 - 7.4, and react at room temperature for 6 - 12 h; (3) Termination and washing: Add 0.1 M glycine (pH 8.0) to the suspension in step (2) to block unreacted groups, perform magnetic separation, and wash 3 times with PBS to prepare the magnetic composite material.

[0026] Example 1;

[0027] Preparation method of an active bone marrow peptide for increasing bone density, comprising the following steps: S1: According to 60 parts of carboxylated Fe 3 O 4A magnetic composite material was prepared for later use according to the following ratio: 60 parts of carboxylated Fe S2: The magnetic composite material obtained in S1 was mixed with bone marrow homogenate at pH 6.0 at a mass-to-volume ratio of 1:12, and 0.2% sodium citrate was added as a buffer, followed by stirring at 25 °C for 30 min; S3: The product obtained after the treatment in S2 was placed in a 0.3 T magnetic field for magnetic separation for 2 min to obtain a magnetic composite material-biological molecule complex; S4: The magnetic composite material-biological molecule complex was eluted and dissociated by shaking with a glycine-HCl buffer solution at pH 2.5 (containing 0.03% Tween-20 by volume percentage) for 10 min. The amount of the glycine-HCl buffer solution was twice the volume of the magnetic composite material-biological molecule complex to obtain active bone marrow peptide, which was concentrated by a 3 kDa ultrafiltration membrane and freeze-dried to prepare active bone marrow peptide for increasing bone density; The bone marrow homogenate was prepared by the following method: Homogenization: Bone marrow and lysis buffer (containing protease inhibitor) were mixed at a ratio of 1:3 and treated with an ultrasonic crusher (200 W, 5 s pulse / 10 s interval, ice bath) for 10 min; Centrifugation: Centrifuge at 12,000×g for 30 minutes at 4 °C, take the supernatant, and filter through a 0.22 μm filter membrane.

[0028] Example 2;

[0029] A preparation method of active bone marrow peptide for increasing bone density, comprising the following steps: S1: According to the ratio of 60 parts of carboxylated Fe 3 O 4 nanoparticles (particle size 25 nm), 20 parts of chitosan (degree of deacetylation ≥ 85%), 15 parts of sturgeon bone marrow peptide (molecular weight < 1.5 kDa) and 5 parts of EDC crosslinking agent, a magnetic composite material was prepared for later use with reference to the above preparation method; S2: The magnetic composite material obtained in S1 was mixed with bone marrow homogenate at pH 6.0 at a mass-to-volume ratio of 1:5, and 0.2% sodium citrate was added as a buffer, followed by stirring at 25 °C for 30 min; S3: The product obtained after the treatment in S2 was placed in a 0.3 T magnetic field for magnetic separation for 2 min to obtain a magnetic composite material-biological molecule complex; S4: Use glycine-HCl buffer solution with pH 2.5 (containing 0.03% Tween-20 by volume) to oscillate, dissociate and elute the magnetic composite-biomolecule complex for 10 min. The dosage of glycine-HCl buffer solution is 2 times the volume of the magnetic composite-biomolecule complex to obtain active bone marrow peptides. Concentrate with a 3 kDa ultrafiltration membrane and lyophilize to prepare active bone marrow peptides for increasing bone density. The preparation of bone marrow homogenate is the same as that in Example 1.

[0030] Example 3;

[0031] A preparation method of active bone marrow peptides for increasing bone density includes the following steps: S1: According to the ratio of 70 parts of carboxylated Fe 3 O 4 nanoparticles (particle size 20 nm), 25 parts of chitosan (degree of deacetylation ≥ 85%), 20 parts of sturgeon bone marrow peptides (molecular weight < 1.5 kDa) and 7 parts of EDC crosslinking agent, refer to the above preparation method to prepare magnetic composites for standby; S2: Mix the magnetic composite obtained in S1 with bone marrow homogenate with pH 6.5 at a mass-to-volume ratio of 1:20, add 0.3% sodium citrate as a buffer, and stir at 30 °C for 40 min; S3: Place the product obtained after S2 treatment in a 0.4 T magnetic field for magnetic separation for 3 min to obtain a magnetic composite-biomolecule complex; S4: Use glycine-HCl buffer solution with pH 3 (containing 0.05% Tween-20 by volume) to oscillate, dissociate and elute the magnetic composite-biomolecule complex for 12 min. The dosage of glycine-HCl buffer solution is 2.5 times the volume of the magnetic composite-biomolecule complex to obtain active bone marrow peptides. Concentrate with a 5 kDa ultrafiltration membrane and lyophilize to prepare active bone marrow peptides for increasing bone density. The preparation of bone marrow homogenate is the same as that in Example 1.

[0032] Example 4;

[0033] A preparation method of active bone marrow peptides for increasing bone density includes the following steps: S1: According to the ratio of 50 parts of carboxylated Fe 3 O 4 nanoparticles (particle size 25 nm), 15 parts of chitosan (degree of deacetylation ≥ 85%), 10 parts of sturgeon bone marrow peptides (molecular weight < 1.5 kDa) and 3 parts of EDC crosslinking agent, refer to the above preparation method to prepare magnetic composites for standby; S2: Mix the magnetic composite material obtained in S1 with the bone marrow homogenate at pH 5.5 at a mass-to-volume ratio of 1:20, add 0.1% sodium citrate as a buffer, and stir at 20 °C for 20 min; S3: Place the product obtained after the treatment in S2 in a 0.2 T magnetic field and perform magnetic separation for 2 min to obtain a magnetic composite material-biological molecule complex; S4: Use a glycine-HCl buffer solution at pH 2.0 (containing 0.01% Tween-20 by volume percentage) to oscillate, dissociate, and elute the magnetic composite material-biological molecule complex for 8 min. The amount of glycine-HCl buffer solution is 1.5 times the volume of the magnetic composite material-biological molecule complex to obtain active bone marrow peptides. Concentrate and lyophilize with a 2 kDa ultrafiltration membrane to prepare active bone marrow peptides for increasing bone density; The preparation of the bone marrow homogenate is the same as in Example 1.

[0034] Example 5;

[0035] A preparation method of active bone marrow peptides for increasing bone density, comprising the following steps: S1: According to the ratio of 55 parts of carboxylated Fe 3 O 4 nanoparticles (particle size 18 nm), 22 parts of chitosan (degree of deacetylation ≥ 85%), 18 parts of sturgeon bone marrow peptides (molecular weight < 1.5 kDa), and 6 parts of EDC crosslinking agent, refer to the above preparation method to prepare a magnetic composite material for standby; S2: Mix the magnetic composite material obtained in S1 with the bone marrow homogenate at pH 6.2 at a mass-to-volume ratio of 1:10, add 0.25% sodium citrate as a buffer, and stir at 28 °C for 35 min; S3: Place the product obtained after the treatment in S2 in a 0.3 T magnetic field and perform magnetic separation for 2.5 min to obtain a magnetic composite material-biological molecule complex; S4: Use a glycine-HCl buffer solution at pH 2.8 (containing 0.04% Tween-20 by volume percentage) to oscillate, dissociate, and elute the magnetic composite material-biological molecule complex for 11 min. The amount of glycine-HCl buffer solution is 2.2 times the volume of the magnetic composite material-biological molecule complex to obtain active bone marrow peptides. Concentrate and lyophilize with a 5 kDa ultrafiltration membrane to prepare active bone marrow peptides for increasing bone density; The preparation of the bone marrow homogenate is the same as in Example 1.

[0036] Example 6;

[0037] A preparation method of active bone marrow peptides for increasing bone density, comprising the following steps: S1: According to the ratio of 65 parts of carboxylated Fe 3 O 4Nanoparticles (particle size 22 nm), 23 parts of chitosan (degree of deacetylation ≥ 85%), 17 parts of sturgeon bone marrow peptide (molecular weight < 1.5 kDa) and 5 parts of EDC crosslinking agent were mixed according to the above preparation method to prepare a magnetic composite material for later use; S2: The magnetic composite material obtained in S1 was mixed with bone marrow homogenate at pH 6.3 at a mass-to-volume ratio of 1:15, and 0.2% sodium citrate was added as a buffer, and stirred at 27 °C for 32 min; S3: The product obtained after the treatment in S2 was placed in a 0.3 T magnetic field and magnetically separated for 2.0 min to obtain a magnetic composite material-biological molecule complex; S4: The magnetic composite material-biological molecule complex was shaken, dissociated and eluted with glycine-HCl buffer solution at pH 2.7 (containing 0.03% Tween-20 by volume percentage) for 10 min. The amount of glycine-HCl buffer solution was 2.0 times the volume of the magnetic composite material-biological molecule complex to obtain active bone marrow peptide, which was concentrated by a 5 kDa ultrafiltration membrane and freeze-dried to prepare active bone marrow peptide for increasing bone density; The preparation of bone marrow homogenate was the same as that in Example 1.

[0038] Comparative Example 1 In Comparative Example 1, based on Example 1, the use of the magnetic composite material was omitted, and the active peptide was directly eluted with glycine-HCl buffer solution.

[0039] Comparative Example 2 In Comparative Example 2, based on Example 1, the bone marrow homogenate was not pretreated with pH and was directly mixed with the magnetic composite material.

[0040] Comparative Example 3 In Comparative Example 3, based on Example 1, Tween-20 was not added, and the active peptide was directly eluted with glycine-HCl buffer solution.

[0041] Comparative Example 4 In Comparative Example 4, based on Example 1, carboxylated Fe 3 O 4 nanoparticles modified with KH-560 were not used.

[0042] Effect verification of the preparation method of active bone marrow peptide for increasing bone density In order to verify the effects of the preparation methods of different groups and the effects of the products, the following experiments were carried out: (I) Test of active peptide recovery rate Test method: The active peptide recovery rate of the bone marrow peptide powder prepared in Examples 1-6 and Comparative Examples 1-4 of the present invention was detected by high performance liquid chromatography (HPLC).

[0043] The test conditions are as follows: Chromatographic column: C18 reverse-phase chromatographic column (4.6 mm × 250 mm, 5 μm); Mobile phase: 0.1% trifluoroacetic acid aqueous solution (phase A) and acetonitrile (phase B); Gradient elution program: 0 - 10 min, 5% - 30% B; 10 - 20 min, 30% - 60% B; Flow rate: 1.0 mL / min; Detection wavelength: 214 nm (absorption of peptide bond).

[0044] It was determined that the recovery rates of active bone marrow peptides in different groups are shown in Table 1.

[0045] Table 1 Recovery rates of active bone marrow peptides in different groups

[0046] Result analysis: The recovery rates of active peptides in Examples 1 - 6 are all above 92%, indicating that the preparation method of the present invention can efficiently recover active peptides; the recovery rates of active peptides in Comparative Examples 1 - 4 are significantly lower than those in the example group, especially in Comparative Example 1 (without using magnetic composite material) and Comparative Example 2 (without pH pretreatment), and their recovery rates are only 68.9% and 72.1% respectively.

[0047] (II) Bone density increase experiment Experimental subjects: 66 female mice at 6 weeks old, with a body weight of 18 - 22 g; Experimental grouping: The mice were divided into 11 groups, with 6 mice in each group; Blank control group: Administered physiological saline by gavage.

[0048] Example 1 group: Administered the bone marrow peptide powder prepared in Example 1 by gavage.

[0049] Example 2 group: Administered the bone marrow peptide powder prepared in Example 2 by gavage.

[0050] Example 3 group: Administered the bone marrow peptide powder prepared in Example 3 by gavage.

[0051] Example 4 group: Administered the bone marrow peptide powder prepared in Example 4 by gavage.

[0052] Example 5 group: Administered the bone marrow peptide powder prepared in Example 5 by gavage.

[0053] Example 6 group: Administered the bone marrow peptide powder prepared in Example 6 by gavage.

[0054] Comparative Example 1 group: Administered the bone marrow peptide powder prepared in Comparative Example 1 by gavage.

[0055] Comparative Example 2 group: Administered the bone marrow peptide powder prepared in Comparative Example 2 by gavage.

[0056] Control group 3: intragastric administration of the bone marrow peptide powder prepared in Control Example 3.

[0057] Control group 4: intragastric administration of the bone marrow peptide powder prepared in Control Example 4.

[0058] Feeding method: The mice were intragastrically administered with the corresponding substance at 0.5 mL / 20 g body weight every day for 4 consecutive weeks.

[0059] Test method: Dual-energy X-ray absorptiometry (DXA) was used to measure the bone density of the mice, which was measured before the start of the experiment and at the end of the experiment respectively. The experimental results are shown in Table 2.

[0060] Table 2 Increase in bone density of active bone marrow peptides in different groups

[0061] Result analysis: Compared with Control Examples 1-4, the active bone marrow peptides prepared in Examples 1-6 of the present invention showed a significant increase in bone density. Among them, Example 1 had the best weight gain effect, indicating that the method of the present invention can effectively improve the quality of the product and the effect of increasing bone density.

[0062] In order to further verify the performance of the magnetic composite material in Example 1, the following experiment was carried out to verify its reusability: First use: Adsorb and elute the active bone marrow peptide under the conditions of Example 1, and measure the adsorption efficiency; Recycling use: The eluted magnetic composite material was reused for adsorption, repeated 3-7 times; the experimental results are shown in Table 3.

[0063] Table 3 Reusability of the magnetic composite material prepared in Example 1

[0064] Result analysis: It can be seen from the content of Table 3 that the magnetic composite material prepared by the present invention can be reused, and the performance reduction of the adsorption efficiency, elution rate and material recovery rate is not obvious, indicating that the magnetic composite material of the present invention can be reused 1-7 times.

[0065] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other.

[0066] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an active bone marrow peptide for increasing bone density, characterized in that: The steps include: S1: preparing a magnetic composite material according to the composition and ratio of 50-70 parts of carboxylated Fe3O4 nanoparticles, 15-25 parts of chitosan, 10-20 parts of myeloid peptide and 3-7 parts of EDC cross-linking agent, and setting aside; S2: mixing the magnetic composite material obtained in S1 with the bone marrow homogenate, adding 0.1-0.3% sodium citrate by volume of the homogenate, and stirring at 20-30°C for 20-40 minutes; S3: placing the product obtained after treatment in S2 in a 0.2-0.4T magnetic field, and magnetically separating to obtain a magnetic composite material-biomolecule complex; S4: The magnetic composite material-biomolecule complex is shaken, dissociated and eluted using glycine-HCl buffer, concentrated using an ultrafiltration membrane, and freeze-dried to prepare active bone marrow peptide.

2. The method for preparing an active bone marrow peptide for increasing bone density according to claim 1, characterized in that: The specific preparation method of the magnetic composite material described in S1 is as follows: Carboxyl activation: disperse the carboxylated Fe3O4 nanoparticles in MES buffer, then add EDC and NHS cross-linking agents, shake at room temperature for 15-45 min to obtain a suspension; Coupling reaction: add chitosan to the suspension obtained in step (1), adjust the pH to 5.5-6.5, and react at room temperature for 2-4 hours; then add bone marrow peptide, adjust the pH to 7.0-7.4, and continue the reaction for 6-12 hours; Termination and washing: adding glycine to the suspension of step (2) to block the unreacted groups, performing magnetic separation and washing to prepare a magnetic composite material.

3. The method for preparing an active bone marrow peptide for increasing bone density according to claim 2, characterized in that: The concentration of the carboxylated Fe3O4 nanoparticles is 0.5-2 mg / ml; the pH of the MES buffer solution is 5.5-6.5; the concentration of the EDC is 8-12 mM; the concentration of the NHS is 4-6 mM; the molar ratio of the EDC and NHS crosslinking agent is 2:1; the concentration of the glycine is 0.05-0.2 M, and the pH is 7.5-8.

5.

4. The method for preparing an active bone marrow peptide for increasing bone density according to claim 2, characterized in that: Said The particle size of the carboxylated Fe3O4 nanoparticles is 15-25nm, and the surface of the nanoparticles is modified with a silane coupling agent KH-560; the deacetylation degree of the chitosan is ≥85%, and the surface of the nanoparticles is modified with PEG; and the molecular weight of the bone marrow peptide is <1.5kDa.

5. The method for preparing an active bone marrow peptide for increasing bone density according to claim 1, characterized in that: S2 The magnetic composite material and the bone marrow homogenate are mixed in a mass volume ratio of 1:5-1:20; the pH of the bone marrow homogenate is 5.5-6.5; the magnetic composite material can be reused 3-7 times; S3 The magnetic separation time is 1-3min; S4 The pH of the glycine-HCl buffer is 2.0-3.0, containing 0.01-0.05% Tween-20 by volume; the amount of the glycine-HCl buffer is 1.5-2.5 times the volume of the magnetic composite material-biomolecule complex; the time of the oscillation dissociation elution is 8-12 minutes; the molecular weight cutoff of the ultrafiltration membrane is 2-5kDa.

6. The active bone marrow peptide for increasing bone density prepared by the method according to any one of claims 1 to 5.

7. Use of the active bone marrow peptide for increasing bone density according to claim 6 in the preparation of products for increasing bone density.

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