Bovine collagen peptide for increasing bone density and method for preparing the same

Abstract

The application discloses a kind of bovine collagen peptide for increasing bone density and a preparation method thereof, and relates to the technical field of enzyme processing preparation of protein peptide.The application relates to the application of bovine collagen peptide in the field of composition.The composition containing bovine collagen peptide is prepared from the following components in mass fraction:bovine collagen peptide 25-45 parts, calcium citrate 12-28 parts, vitamin D 0.0002-0.0004 parts, extract of rhizoma drynariae 6-14 parts, extract of astragalus 4-9 parts, auxiliary materials 12-38 parts, and zoledronate derivative 0.01-0.05 parts.The application breaks through the limitation of single component and poor synergistic effect in the prior art, and forms a multi-dimensional action system of "inhibiting bone resorption, promoting bone formation and supplementing matrix" by matching bovine collagen peptide, calcium citrate, traditional Chinese medicine extract and modified zoledronate derivative, so that the effect of improving bone density is improved.

Description

Technical Field

[0001] This invention relates to the field of enzyme processing for the preparation of protein peptides, specifically to a bovine collagen peptide that increases bone density and its preparation method. Background Technology

[0002] With the aging population and the influence of unhealthy lifestyle habits, bone health problems such as osteoporosis and decreased bone density are becoming increasingly common, affecting not only the quality of life of middle-aged and elderly people but also gradually spreading to younger and middle-aged populations. Decreased bone density easily leads to symptoms such as fractures and joint pain, seriously threatening human health. Therefore, developing safe and effective products to increase bone density has become a hot research topic in the industry.

[0003] Currently, bone health products on the market are mainly divided into three categories: single-ingredient calcium supplements, vitamin D supplements, and traditional Chinese medicine extracts. Single-ingredient calcium supplements, such as calcium carbonate and calcium phosphate, suffer from low absorption efficiency and lack the ability to repair the bone matrix, making it difficult to fundamentally improve bone density. While vitamin D products can promote calcium absorption, their effect when used alone is limited and cannot meet the complex needs of bone health. Traditional Chinese medicine extracts, such as Drynaria fortunei and Astragalus membranaceus, have certain bone-strengthening effects, but the utilization rate of active ingredients is low, and their synergistic effects with chemical components have not been fully explored.

[0004] Furthermore, while existing chemical-based bone density-improving drugs such as zoledronic acid can inhibit osteoclast activity, long-term use can easily cause side effects such as gastrointestinal irritation and kidney damage, raising concerns about their safety. Additionally, traditional products often lack targeted supplementation of collagen, a crucial component of bones; its loss leads to decreased bone resilience and hinders bone density improvement.

[0005] In summary, existing products suffer from drawbacks such as single-ingredient composition, poor synergistic effects, low absorption efficiency, and insufficient safety, making it difficult to achieve the goal of efficiently and safely increasing bone density. Therefore, developing a composition that combines natural extracts, nutrients, and modified chemical derivatives, and features synergistic effects and low side effects, has become a key need for addressing current bone health issues. Summary of the Invention

[0006] The purpose of this invention is to address the problems of existing technologies, such as single ingredients, poor synergistic effect, low absorption efficiency, insufficient safety, and lack of targeted collagen supplementation. This invention provides a bovine collagen peptide for increasing bone density, containing bovine collagen peptide, calcium citrate, vitamin D, extracts of Drynaria fortunei and Astragalus membranaceus, and a modified zoledronic acid derivative. This peptide exhibits synergistic effects in increasing bone density, is safe with low side effects, and has a feasible preparation method.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a method for preparing bovine collagen peptides that increase bone density, comprising the following steps:

[0008] S1: Fresh beef bones are washed, crushed into bone paste, added to water and subjected to repeated freeze-thaw cycles, followed by defatting and decalcification to obtain pretreated bone matrix;

[0009] S2: Place the pretreated bone matrix in a sealed reaction vessel, add distilled water, and while performing high-temperature and high-pressure cooking, apply intermittent ultrasonic treatment at a frequency of 20-40kHz to adjust the pH and temperature of the reaction system. First, add alkaline protease for primary hydrolysis, and then add a compound enzyme of flavor protease and trypsin for secondary deep hydrolysis to inactivate the enzyme. After the enzymatic hydrolysis is completed, raise the temperature to inactivate the enzyme.

[0010] S3: Centrifuge the enzymatic hydrolysate to remove the precipitate, and pass the supernatant sequentially through a microfiltration membrane and an ultrafiltration membrane with a molecular weight cutoff of 1000-3000 Da. Collect the permeate with a molecular weight cutoff of less than 1000 Da, add a calcium source, and carry out a chelation reaction at 40-50℃. After the reaction solution is concentrated and freeze-dried, the bovine collagen peptide that increases bone density is obtained.

[0011] Furthermore, the specific enzymatic hydrolysis process in S2 is as follows: adjust the pH of the feed solution to 8.5-9.5, the temperature to 50-55℃, add alkaline protease at 0.5%-1.0% of the substrate mass, and hydrolyze for 1.5-2 hours; then adjust the pH back to 7.0-7.5, the temperature to 45-50℃, add a mixture of flavor protease and trypsin at 0.3%-0.8% of the substrate mass, and continue hydrolysis for 2-3 hours; during the secondary hydrolysis process, ultrasonic assistance is activated every 30 minutes, with an ultrasonic power of 300-500W, for a duration of 5-10 minutes;

[0012] The mass ratio of the flavor protease to trypsin mixture is 2:1 to 3:1.

[0013] Furthermore, the calcium source in S3 is calcium chloride or calcium lactate, and the mass ratio of its addition to collagen peptides is 1:2 to 1:4; the pH value is controlled at 7.5-8.5 during the chelation reaction, and the reaction time is 40-60 minutes; before freeze drying, the step of adding 0.1%-0.5% by mass of vitamin D3 microcapsule powder to the chelation solution for compounding is also included.

[0014] Applications of the bovine collagen peptide composition prepared by the above-described method for increasing bone density.

[0015] A composition containing bovine collagen peptides, wherein, by weight, the raw materials of the composition are composed of the following components: 25-45 parts bovine collagen peptides, 12-28 parts calcium citrate, 0.0002-0.0004 parts vitamin D, 6-14 parts Drynaria fortunei extract, 4-9 parts Astragalus membranaceus extract, 12-38 parts excipients, and 0.01-0.05 parts zoledronic acid derivative;

[0016] The bovine collagen peptides were prepared according to the above preparation method;

[0017] The zoledronic acid derivative has the structure shown in Formula 1:

[0018] Formula 1;

[0019] R1 in Formula 1 is any one of halogen, aryl, ethyl ester, or alkoxy with 1-5 carbon atoms;

[0020] The zoledronic acid derivatives can be prepared into pharmaceutically acceptable salts or solvates.

[0021] Furthermore, the preparation method of the Drynaria fortunei extract is as follows: Drynaria fortunei is pulverized to 20-40 mesh, 10 times its weight of 75% ethanol solution is added, and the mixture is refluxed and extracted twice, 1.5 h each time. The extracts are combined and concentrated under reduced pressure at a vacuum of 0.08-0.09 MPa and a temperature of 60-70℃ until there is no alcohol odor. Then, it is vacuum dried until the moisture content is ≤3%, pulverized, and passed through a 100-mesh sieve to obtain the Drynaria fortunei extract.

[0022] The preparation method of the Astragalus extract is as follows: slice Astragalus into pieces with a thickness of 2-3 mm, add 10 times the mass of 75% ethanol solution, reflux extract twice, 1.5 h each time, combine the extracts, concentrate under reduced pressure at a vacuum degree of 0.08-0.09 MPa and a temperature of 60-70℃ until there is no alcohol odor, then vacuum dry until the moisture content is ≤3%, pulverize, and pass through a 100-mesh sieve to obtain the Astragalus extract.

[0023] Furthermore, the excipients are composed of fillers, binders, disintegrants and lubricants in a mass ratio of 5:2:1:0.5.

[0024] Furthermore, the filler is a mixture of microcrystalline cellulose and lactose in a mass ratio of 3:2;

[0025] The adhesive is hydroxypropyl methylcellulose;

[0026] The disintegrant is crospovidone;

[0027] The lubricant is a mixture of magnesium stearate and micronized silica gel in a mass ratio of 2:1.

[0028] Furthermore, the zoledronic acid derivative is any one of the compounds shown in the following structures:

[0029] zoledronic acid derivative 1;

[0030] 2. Zoledronic acid derivatives;

[0031] 3. Zoledronic acid derivatives;

[0032] 4. Zoledronic acid derivatives.

[0033] A method for preparing a composition containing bovine collagen peptides includes the following steps:

[0034] (1) Raw material pretreatment: The bovine collagen peptide and calcium citrate are passed through a 90-110 mesh sieve, and the bone drynaria extract and astragalus extract are passed through a 100 mesh sieve.

[0035] (2) Mixing: Add the bovine collagen peptides and vitamin D to a double cone mixer and mix at 150-250 rpm for 12-18 min. Then add the calcium citrate, Drynaria fortunei extract, Astragalus membranaceus extract and zoledronic acid derivative and continue mixing for 25-35 min to obtain the premix.

[0036] (3) Granulation: Add 6-9% of the adhesive aqueous solution by mass of the premix to the premix, stir until a uniform soft material is formed, and then granulate using a swing granulator to obtain wet granules;

[0037] (4) Drying and granulation: The wet granules are placed in a vacuum drying oven and dried at 55-65℃ for 2.5-3.5h. After drying, they are granulated by passing them through an 18-35 mesh sieve to obtain dry granules.

[0038] (5) Preparation of finished product: The filler, disintegrant and lubricant are added to the dry granules and mixed for 18-25 minutes using a three-dimensional mixer. The mixture is then compressed into tablets or capsules to obtain a composition containing bovine collagen peptides.

[0039] Furthermore, the mass concentration of the adhesive aqueous solution is 8-12%.

[0040] This invention addresses the core problems of existing technologies—namely, single-component composition, poor synergy, low absorption efficiency, insufficient safety, and lack of targeted collagen supplementation—by utilizing the mass proportions of each component to form a synergistic system. Bovine collagen peptides supplement key bone matrix components, compensating for the neglect of collagen loss in traditional products, enhancing bone toughness, and laying the structural foundation for increased bone density. Calcium citrate provides a highly efficient calcium source, combined with trace amounts of vitamin D, leveraging vitamin D's promoting effect on calcium absorption to overcome the low absorption efficiency of single-component calcium supplements, ensuring effective calcium deposition in bones. The mass proportion ratio of *Drynaria fortunei* extract and *Astragalus membranaceus* extract ensures the effective concentration of active ingredients in traditional Chinese medicine and enhances bone-strengthening effects through their synergistic effect, while also working in conjunction with bovine collagen peptides and calcium citrate. Calcium citrate and other ingredients complement each other in terms of nutritional supplementation and natural conditioning, solving the problems of low utilization rate of active ingredients and insufficient synergy with chemical components in traditional Chinese medicine extracts. The addition of trace amounts of zoledronic acid derivatives inhibits osteoclast activity, forming a two-way synergy of "inhibiting osteoclast activity and promoting bone growth" with calcium and collagen supplements, breaking through the limitations of single-component effects. Excipients are combined with fillers, binders, etc. in specific proportions to ensure the stability of the formulation and the uniform release of active ingredients, further improving absorption efficiency. Finally, through the scientific proportion of each component by weight, a comprehensive synergy of natural extracts, nutrients, and modified chemical derivatives is achieved, which not only solves the single defects of traditional products, but also achieves the goal of increasing bone density efficiently and safely through a multi-dimensional mechanism of action.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] 1. Synergistic effect of multiple components for better bone density improvement: Overcoming the limitations of existing technologies with single components and poor synergistic effect, this product combines bovine collagen peptides, calcium citrate, traditional Chinese medicine extracts and modified zoledronic acid derivatives to form a multi-dimensional action system of "inhibiting osteoclasts + promoting osteoclasts + replenishing matrix", thereby improving the effect of bone density improvement.

[0043] 2. Optimized ingredients and structure for enhanced safety: Addressing the issue of significant side effects associated with traditional chemical bone density drugs, a modified zoledronic acid derivative is used, combined with natural Chinese herbal extracts and nutrients, to reduce the risk of side effects and improve product safety.

[0044] 3. Balancing matrix replenishment and nutrient absorption for more comprehensive bone health improvement: It overcomes the shortcomings of traditional products that neglect collagen replenishment, using bovine collagen peptides to replenish key bone matrix, combined with vitamin D to promote calcium absorption, and relying on traditional Chinese medicine extracts to achieve dual improvement in bone toughness and calcium deposition.

[0045] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0047] Figure 1 This is the NMR spectrum of compound 3 described in this invention.

[0048] Figure 2 This is the NMR spectrum of the zoledronic acid derivative 1 described in this invention. Detailed Implementation

[0049] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0051] Preparation Example 1

[0052] Preparation of zoledronic acid derivative 1:

[0053] ;

[0054] Compound 1: 4-chloro-1-ethynyl-2-fluorobenzene;

[0055] Compound 2: (4-amino-1-hydroxybutane-1,1-diyl)bisphosphonic acid;

[0056] Compound 3: (4-((4-ethynyl-3-fluorophenyl)amino)-1-hydroxybutane-1,1-diyl)bisphosphonic acid;

[0057] Under a nitrogen atmosphere, 5.00 g of compound 1, 8.06 g of compound 2, and 70 mL of toluene were added to the reaction system and mixed thoroughly. Then, under a flowing nitrogen atmosphere, 0.57 g of 1,3-bis(diphenylphosphine)propane palladium(II) chloride and 6.55 g of triethylamine were added sequentially. The mixture was refluxed at 110 °C for 12 hours under a flowing nitrogen atmosphere. The mixture was diluted with 150 mL of water, stirred thoroughly, and the pH was adjusted to 6.5 with 0.1 M HCl. The aqueous phase was washed with ethyl acetate (3 × 50 mL), the organic phase was discarded, and the aqueous phase was retained. The aqueous phase was decolorized with activated carbon, filtered, and filtered through a 0.45 μm filter membrane. The aqueous phase was then loaded onto a preparative C18 HPLC system with the following mobile phases: A: water + 0.1% formic acid, B: acetonitrile gradient elution. The main peak fraction was collected, freeze-dried, and 6.58 g of compound 3 was obtained.

[0058] ;

[0059] Compound 3: (4-((4-ethynyl-3-fluorophenyl)amino)-1-hydroxybutane-1,1-diyl)bisphosphonic acid;

[0060] Compound 4: (2-(4,7-dibromo-1H-benzo[d]imidazol-1-yl)-1-hydroxyethane-1,1-diyl)bisphosphonic acid;

[0061] Zoledronic acid derivative 1: {4-[(4-{[7-bromo-1-(2-hydroxy-2,2-bisphosphonoethyl)benzo[d]imidazol-4-yl]ethynyl}-3-fluorophenyl)amino]-1-hydroxy-1-phosphonobutyl}phosphonic acid;

[0062] Under a nitrogen atmosphere, 6.58 g of compound 3, 8.60 g of compound 4, and 90 mL of a mixed solvent of dioxane and diisopropylamine (60 mL / 30 mL) were added to the reaction system. After stirring until homogeneous, 0.48 g of bis(1,2-bis(diphenylphosphine)ethane)palladium and 0.1 g of CuI were added sequentially. The mixture was stirred until homogeneous, heated to 90 °C, and refluxed for 6 h. The mixture was then diluted with 150 mL of water, stirred until homogeneous, and the pH was adjusted to 6.5 with 0.1 M HCl. The aqueous phase was washed with ethyl acetate (3 × 50 mL), and the organic phase (containing impurities) was discarded. The aqueous phase (containing the product) was retained and filtered through a 0.45 μm filter membrane. The aqueous phase was then loaded onto a preparative C18 HPLC system. The mobile phase consisted of a gradient elution of A: water + 0.1% formic acid and B: acetonitrile. The main peak fraction was collected, freeze-dried, and 5.67 g of zoledronic acid derivative 1 was obtained as a white solid.

[0063] Compound structure identification data:

[0064] Compound structure identification can rely on 1H NMR spectroscopy (NMR spectroscopy). 1¹H NMR, mass spectrometry (MS), and other methods are commonly used core identification methods in this field. For those skilled in the art, given a clear understanding of the raw material composition, reaction conditions, and raw material ratios, combining any one of the above-mentioned compound structure identification data is sufficient to form a clear and accurate judgment on the specific chemical structure of the final product.

[0065] Mass spectrum of compound 3: 368 [M+1];

[0066] Mass spectrum of zoledronic acid derivative 1: 765 [M+1];

[0067] The NMR spectra of compound 3 are shown in [reference needed]. Figure 1 ;

[0068] The NMR data for zoledronic acid derivative 1 are shown below. Figure 2 .

[0069] Preparation Examples 2-4

[0070] In Preparation Examples 2-4, zoledronic acid derivative 2-zoledronic acid derivative 4 were prepared sequentially, following the preparation method of Preparation Example 1, except that compound 1 was replaced. All other aspects, such as feed ratio and post-treatment, were kept the same as in Preparation Example 1. For details, please refer to Table 1.

[0071] Table 1

[0072]

[0073] Preparation Example 5

[0074] Preparation of a bovine collagen peptide that increases bone density:

[0075] 1. Raw materials: fresh beef bones (with fascia, muscle and other impurities removed), distilled water, alkaline protease (enzyme activity ≥ 200,000 U / g), flavor protease (enzyme activity ≥ 100,000 U / g), trypsin (enzyme activity ≥ 150,000 U / g), calcium source (calcium chloride or calcium lactate), vitamin D3 microcapsule powder (vitamin D3 content ≥ 5,000 IU / g).

[0076] 2. Preparation steps:

[0077] S1: Select fresh bovine bones, rinse the surface with clean water to remove impurities and attached fascia and muscle tissue, then crush them into bone blocks with a particle size of 5-10mm in a crusher, and then grind them into uniform bone paste using a colloid mill; mix the bone paste with distilled water at a mass ratio of 1:3, freeze in a -20℃ freezer for 4 hours, remove and thaw naturally at room temperature of 25℃, repeat the freeze-thaw operation 3 times to destroy the bone matrix cell structure; heat the frozen and thawed bone paste suspension to 60℃, add 3% of the bone paste mass of food-grade defatting agent, stir and react for 2 hours, stirring for 5 minutes every 20 minutes, then centrifuge (3000rpm, 15 minutes) to remove the upper oil layer and collect the lower precipitate; add 5 times the volume of 0.5mol / L hydrochloric acid solution to the precipitate, stir and react at 30℃ for 3 hours, and monitor the pH value of the system in real time. When the pH stabilizes at 2.0-2.5, stop the reaction, wash the precipitate repeatedly with distilled water until the pH of the washing liquid is neutral, centrifuge and collect the precipitate to obtain the pretreated bone matrix;

[0078] S2: Mix the pretreated bone matrix with distilled water at a mass ratio of 1:8, place the mixture in a sealed high-temperature and high-pressure reactor, turn on the reactor heating device and ultrasonic generator, set the ultrasonic frequency to 30kHz, and adopt an intermittent working mode (working for 10 minutes, pausing for 20 minutes); introduce high-pressure steam into the reactor to raise the temperature to 120℃ and maintain the pressure at 0.15MPa, hold for 30 minutes, then cool down to 55℃; adjust the pH of the reaction system to 9.0, add alkaline protease at 0.8% of the substrate (pretreated bone matrix) mass, and maintain the reaction at 52℃. Hydrolysis was performed for 1.8 hours under intermittent ultrasonic assisted by 30kHz. The reaction system temperature was then raised to 90℃ and held for 20 minutes to inactivate the alkaline protease. The system temperature was then lowered to 48℃, the pH was adjusted to 7.2, and a complex enzyme of flavor protease and trypsin (mass ratio 2.5:1) was added at 0.5% of the substrate mass. Hydrolysis continued for 2.5 hours under intermittent ultrasonic assisted by 30kHz (ultrasound was turned on every 30 minutes, power 400W, for 8 minutes). The reaction system temperature was then raised to 95℃ and held for 25 minutes to completely inactivate the complex enzyme, yielding the enzymatic hydrolysate.

[0079] S3: Cool the enzymatic hydrolysate to room temperature, place it in a centrifuge, and centrifuge at 4000 rpm for 20 minutes to remove insoluble precipitates and collect the supernatant. Filter the supernatant through a 0.22 μm microfiltration membrane to remove large molecular impurities and incompletely hydrolyzed protein particles. Pass the microfiltration permeate into an ultrafiltration device and perform fractional filtration using ultrafiltration membranes with molecular weight cutoffs of 3000 Da and 1000 Da, collecting the permeate with a molecular weight cutoff of less than 1000 Da, which is the crude bovine collagen peptide extract. Add a calcium source (calcium chloride) to the crude bovine collagen peptide extract. The mass ratio of calcium source to collagen peptides was 1:3. The pH of the system was adjusted to 8.0, and the chelation reaction was carried out at 45℃ with stirring for 50 minutes. Vitamin D3 powder with a mass fraction of 0.3% was added to the chelation reaction solution, stirred evenly, and the reaction was continued at the temperature for 10 minutes. The compounded reaction solution was concentrated to 1 / 5 of the original volume through a vacuum concentration device (vacuum degree 0.085MPa, temperature 60℃), and then placed in a freeze dryer for freeze drying at -50℃ and vacuum degree 10Pa for 12 hours. After pulverization, it was passed through a 100-mesh sieve to obtain the bovine collagen peptide product.

[0080] Example 1

[0081] Preparation of a composition containing bovine collagen peptides:

[0082] 1. Raw material quality allocation method:

[0083] 1.1 Bovine collagen peptides: 35 parts, bovine collagen peptides prepared in Preparation Example 5;

[0084] 1.2 Calcium citrate: 20 parts;

[0085] 1.3 Vitamin D: 0.0003 parts;

[0086] 1.4 Drynaria fortunei extract: 10 parts;

[0087] 1.5 Astragalus extract: 6.5 parts;

[0088] 1.6 Auxiliary materials: 25 parts;

[0089] 1.6.1 Filler: 14.7 parts (8.82 parts microcrystalline cellulose, 5.88 parts lactose, in a mass ratio of 3:2);

[0090] 1.6.2 Adhesive (hydroxypropyl methylcellulose): 5.88 parts;

[0091] 1.6.3 Disintegrant (crospovidone): 2.94 parts;

[0092] 1.6.4 Lubricant: 1.47 parts (0.98 parts magnesium stearate, 0.49 parts micronized silica gel, in a mass ratio of 2:1).

[0093] 1.7 Zoledronic acid derivative (zoledronic acid derivative 1 prepared in Preparation Example 1): 0.03 parts;

[0094] Preparation of Drynaria fortunei extract: Drynaria fortunei was pulverized and passed through a 30-mesh sieve, and 10 times its weight of 75% ethanol solution was added. The mixture was refluxed and extracted twice, 1.5 h each time. The extracts were combined and concentrated under reduced pressure at a vacuum of 0.08-0.09 MPa and a temperature of 70°C until no alcohol odor was detected. The mixture was then vacuum dried until the moisture content was ≤3%, pulverized, and passed through a 100-mesh sieve to obtain Drynaria fortunei extract.

[0095] Preparation of Astragalus extract: Slice Astragalus into pieces 2-3 mm thick, add 10 times the mass of 75% ethanol solution, reflux extract twice, 1.5 h each time, combine the extracts, concentrate under reduced pressure at 0.08-0.09 MPa and 70℃ until no alcohol odor remains, then vacuum dry until the moisture content is ≤3%, pulverize, and pass through a 100-mesh sieve to obtain Astragalus extract.

[0096] 2. Preparation method:

[0097] (1) Raw material pretreatment: Bovine collagen peptides and calcium citrate were passed through a 100-mesh sieve, and Drynaria fortunei extract and Astragalus membranaceus extract were passed through a 100-mesh sieve to ensure that the raw materials had uniform particle size and no obvious clumping.

[0098] (2) Mixing: Add the sieved bovine collagen peptides and vitamin D to a double cone mixer and mix at 200 rpm for 15 min. Then add calcium citrate, Drynaria fortunei extract, Astragalus membranaceus extract and zoledronic acid derivative and continue mixing at the same speed for 30 min to obtain a uniform premix.

[0099] (3) Granulation: Prepare a 10% hydroxypropyl methylcellulose aqueous solution, add 7% of the adhesive aqueous solution to the premix, stir until a uniform soft material that can be formed by hand and easily dispersed by light touch is formed, and granulate using a swing granulator (18 mesh screen) to obtain wet granules.

[0100] (4) Drying and granulation: The wet granules are placed in a vacuum drying oven and dried at 60°C and 0.085MPa for 3 hours. After drying, they are granulated through a 24-mesh sieve to remove excessive lumps and fine powder, and dry granules are obtained.

[0101] (5) Preparation of finished product: Add filler, disintegrant and lubricant in a preset ratio to dry granules, mix in a three-dimensional mixer for 20 minutes, and after mixing evenly, compress into tablets with a diameter of 8 mm and a tablet weight of 0.5 g using a tablet press to obtain the finished product of the bovine collagen peptide composition that increases bone density.

[0102] Examples 2-5

[0103] The preparation of a composition containing bovine collagen peptides is carried out according to the preparation method of Example 1, except that zoledronic acid derivative 1 is replaced sequentially with zoledronic acid derivative 2-zoledronic acid derivative 4, and the rest is the same as in Example 1.

[0104] Comparative Example 1

[0105] Preparation of a composition containing bovine collagen peptides, referring to the preparation method of Example 1, except that zoledronic acid derivative 1 is replaced with zoledronic acid ( The rest remains the same as in Example 1.

[0106] Comparative Example 2

[0107] The preparation of a composition containing bovine collagen peptides is carried out according to the preparation method of Example 1, except that the zoledronic acid derivative 1 is replaced with risedronic acid, and the rest is the same as in Example 1.

[0108] Comparative Example 3

[0109] The preparation of a composition containing bovine collagen peptides is carried out according to the preparation method of Example 1, except that the zoledronic acid derivative 1 is replaced with ibandronic acid, and the rest is the same as in Example 1.

[0110] Comparative Example 4

[0111] The preparation of a composition containing bovine collagen peptides was carried out according to the preparation method of Example 1, except that zoledronic acid derivative 1 was not added, and the rest remained the same as in Example 1.

[0112] Comparative Example 5

[0113] The preparation of a composition containing bovine collagen peptides was carried out according to the preparation method of Example 1, except that the Astragalus extract was not added, and the rest remained the same as in Example 1.

[0114] Comparative Example 6

[0115] The preparation of a composition containing bovine collagen peptides was carried out according to the preparation method of Example 1, except that the Drynaria fortunei extract was not added, and the rest remained the same as in Example 1.

[0116] Comparative Example 7

[0117] The preparation of a composition containing bovine collagen peptides is carried out according to the preparation method of Example 1, except that the bovine collagen peptides are not added, and the rest is the same as in Example 1.

[0118] Performance testing:

[0119] Female ICR mice or C57BL / 6 mice, aged 6-8 weeks and weighing 20-25g, were selected. After one week of acclimatization, they were randomly divided into four groups: a blank control group, a model control group, an example group, and a comparative group. Except for the blank control group, the mice in the other groups underwent ovariectomy to establish a postmenopausal osteoporosis model. Surgical procedure: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg / kg). After depilation and disinfection of the abdomen, the skin and muscle layers were incised, and both ovaries were removed. The incision was sutured layer by layer. Postoperatively, penicillin was injected to prevent infection, and the mice were fed routinely. In the blank control group, only abdominal incision and suturing were performed, without removal of the ovaries. Drug administration began one week after surgery. Mice in each group were treated as follows and administered the drug continuously for 12 weeks: Blank control group and model control group: The mice were given an equal volume of physiological saline by gavage. Example group and comparative group: The corresponding composition was dissolved in physiological saline at a dose of 50mg / kg of mouse body weight to prepare a suspension for gavage administration once daily.

[0120] 1. Skeletal biomechanical performance testing: After separating the right femur of the mouse and removing the soft tissue, a three-point bending test was performed using a universal testing machine. The span was set to 10 mm, the loading speed to 2 mm / min, and the maximum load was recorded. The data are shown in Table 2.

[0121] 2. Serum biochemical index detection: Blood was collected from the orbital cavity of mice, and serum was separated by centrifugation. The levels of osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) in the serum were detected by enzyme-linked immunosorbent assay (ELISA). The data are shown in Table 2.

[0122] Table 2

[0123]

[0124] Compared to the blank control group, the model control group showed a decreasing trend in bone mechanical properties, a decrease in osteocalcin levels, and an increase in anti-tartrate acid phosphatase levels, indicating the successful establishment of the osteoporosis model. Compared to the model control group, the example group showed a significant recovery in bone mechanical properties, a marked recovery in osteocalcin levels, and a decrease in anti-tartrate acid phosphatase levels, with an overall trend similar to the blank control group, demonstrating the improving effect of the composition on bone density. The trends of relevant indicators among the example groups were similar, indicating that zoledronic acid derivatives with different structures could exert good effects. Compared to the example groups, the comparative groups (regardless of whether they were replaced with traditional phosphonates or lacked zoledronic acid derivatives, Astragalus extract, or Drynaria extract) showed a weaker recovery in bone mechanical properties and a weaker recovery in osteocalcin levels, with no significant decrease in anti-tartrate acid phosphatase levels. The comparative group lacking key components showed even worse improvement effects, highlighting the synergistic effect of the modified zoledronic acid derivatives and bovine collagen peptides, traditional Chinese medicine extracts, and other components in this invention. 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 composition containing bovine collagen peptides, characterized in that, The raw materials of the composition, by weight, consist of the following components: 25-45 parts bovine collagen peptide, 12-28 parts calcium citrate, 0.0002-0.0004 parts vitamin D, 6-14 parts Drynaria fortunei extract, 4-9 parts Astragalus membranaceus extract, 12-38 parts excipients, and 0.01-0.05 parts zoledronic acid derivative. The zoledronic acid derivative is ; The preparation method of the Drynaria fortunei extract is as follows: Drynaria fortunei is pulverized to 20-40 mesh, 10 times the mass of 75% ethanol solution is added, and the mixture is refluxed and extracted twice, 1.5 h each time. The extracts are combined and concentrated under reduced pressure at a vacuum of 0.08-0.09 MPa and a temperature of 60-70℃ until there is no alcohol odor. Then, it is vacuum dried until the moisture content is ≤3%, pulverized, and passed through a 100-mesh sieve to obtain the Drynaria fortunei extract. The preparation method of the Astragalus extract is as follows: slice Astragalus into pieces with a thickness of 2-3 mm, add 10 times the mass of 75% ethanol solution, reflux extract twice, 1.5 h each time, combine the extracts, concentrate under reduced pressure at a vacuum degree of 0.08-0.09 MPa and a temperature of 60-70℃ until there is no alcohol odor, then vacuum dry until the moisture content is ≤3%, pulverize, and pass through a 100-mesh sieve to obtain the Astragalus extract; The preparation method of the bovine collagen peptide includes the following steps: S1: Fresh beef bones are washed, crushed into bone paste, added to water and subjected to repeated freeze-thaw cycles, followed by defatting and decalcification to obtain pretreated bone matrix; S2: Place the pretreated bone matrix in a sealed reaction vessel, add distilled water, and while performing high-temperature and high-pressure cooking, apply intermittent ultrasonic treatment at a frequency of 20-40kHz to adjust the pH and temperature of the reaction system. First, add alkaline protease for primary hydrolysis, and then add a compound enzyme of flavor protease and trypsin for secondary deep hydrolysis to inactivate the enzyme. After the enzymatic hydrolysis is completed, raise the temperature to inactivate the enzyme. S3: Centrifuge the enzymatic hydrolysate to remove the precipitate, and pass the supernatant sequentially through a microfiltration membrane and an ultrafiltration membrane with a molecular weight cutoff of 1000-3000 Da. Collect the permeate with a molecular weight cutoff of less than 1000 Da, add a calcium source, and carry out a chelation reaction at 40-50℃. After the reaction solution is concentrated and freeze-dried, the bovine collagen peptide is obtained.

2. The composition containing bovine collagen peptides according to claim 1, characterized in that, The specific enzymatic hydrolysis process in S2 is as follows: adjust the pH of the feed solution to 8.5-9.5, the temperature to 50-55℃, add alkaline protease at 0.5%-1.0% of the substrate weight, and hydrolyze for 1.5-2 hours; then adjust the pH back to 7.0-7.5, the temperature to 45-50℃, add a mixture of flavor protease and trypsin at 0.3%-0.8% of the substrate weight, and continue hydrolysis for 2-3 hours; during the secondary hydrolysis process, ultrasonic assistance is activated every 30 minutes, with an ultrasonic power of 300-500W, for a duration of 5-10 minutes; The mass ratio of the flavor protease to trypsin mixture is 2:1 to 3:

1.

3. The composition containing bovine collagen peptides according to claim 1, characterized in that, The calcium source in S3 is calcium chloride or calcium lactate, and the mass ratio of its addition to collagen peptides is 1:2 to 1:

4. During the chelation reaction, the pH value is controlled at 7.5-8.5, and the reaction time is 40-60 minutes. Before freeze-drying, the process also includes a step of adding 0.1%-0.5% by mass of vitamin D3 microcapsule powder to the chelation solution for compounding.

4. The composition containing bovine collagen peptides according to claim 1, characterized in that, The excipients consist of fillers, binders, disintegrants and lubricants in a mass ratio of 5:2:1:0.

5.

5. The composition containing bovine collagen peptides according to claim 4, characterized in that, The filler is a mixture of microcrystalline cellulose and lactose in a mass ratio of 3:2; The adhesive is hydroxypropyl methylcellulose; The disintegrant is crospovidone; The lubricant is a mixture of magnesium stearate and micronized silica gel in a mass ratio of 2:

1.

6. A method for preparing a composition containing bovine collagen peptides as described in claim 1, characterized in that, Includes the following steps: (1) Raw material pretreatment: The bovine collagen peptide and calcium citrate are passed through a 90-110 mesh sieve, and the bone drynaria extract and astragalus extract are passed through a 100 mesh sieve. (2) Mixing: Add the bovine collagen peptides and vitamin D to a double cone mixer and mix at 150-250 rpm for 12-18 min. Then add the calcium citrate, Drynaria fortunei extract, Astragalus membranaceus extract and zoledronic acid derivative and continue mixing for 25-35 min to obtain the premix. (3) Granulation: Add 6-9% of the binder aqueous solution by mass of the premix to the premix, stir until a uniform soft material is formed, and then granulate using a swing granulator to obtain wet granules; (4) Drying and granulation: The wet granules are placed in a vacuum drying oven and dried at 55-65℃ for 2.5-3.5h. After drying, they are granulated by passing them through an 18-35 mesh sieve to obtain dry granules. (5) Preparation of finished product: Add filler, disintegrant and lubricant to dry granules, mix for 18-25 min using a three-dimensional mixer, and then fill into tablets or capsules to obtain a composition containing bovine collagen peptides.

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