Hugu active peptide complex and its application in bone health care

Abstract

This invention discloses a Hu sheep active peptide complex and its application in bone health care. Using healthy Hu sheep bones as raw material, collagen is extracted after defatting and decalcification. This collagen is then enzymatically hydrolyzed with trypsin and alkaline protease, followed by ultrafiltration, desalting, and freeze-drying to obtain Hu sheep bone collagen peptides. These collagen peptides are mixed with pharmaceutical excipients, calcium source compounds, chondroprotective agents, and fat-soluble vitamins. An ethanol-water binder solution containing palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside is added, and the mixture is granulated, dried, sized, and pressed to obtain the active peptide complex. This complex, through the synergistic effect of its components, can significantly promote osteoblast proliferation and inhibit apoptosis, effectively increasing bone density in osteoporosis model animals. It also exhibits excellent biocompatibility and no cytotoxicity, solving the problems of limited function, insufficient preparation efficiency, and instability of existing single active peptides. This makes it suitable for the development and application of bone health-related nutritional supplements.

Description

Technical Field

[0001] This invention belongs to the field of nutritional health products, specifically relating to a lake sheep active peptide complex and its application in bone health care. Background Technology

[0002] Osteoporosis is a chronic disease caused by multiple factors. Its core pathological features are decreased bone density and destruction of bone tissue microstructure, mainly due to the imbalance between the bone-forming capacity of osteoblasts and the bone-resorbing capacity of osteoclasts.

[0003] While traditional treatments can improve bone density in the short term, their long-term use has many limitations. For example, estrogen replacement therapy, although it can inhibit bone resorption, may increase the risk of breast cancer, endometrial cancer, and thromboembolic diseases, limiting its use in certain populations; bisphosphonates are prone to causing adverse reactions such as gastrointestinal irritation and kidney damage, and precise dosage control is difficult; fluoride drugs, while promoting bone mineralization, may increase bone fragility, thus increasing the risk of fractures.

[0004] With the deepening of research on functional foods, food-derived bioactive peptides have become a research hotspot in the field of bone health due to their high safety and good biocompatibility. In existing technologies, gelatin hydrolysates, soybean protein hydrolysates, and egg yolk peptides have been proven to promote osteoblast proliferation, inhibit apoptosis, or promote bone growth, with their mechanisms often related to regulating the JNK signaling pathway, EGFR signaling pathway, or enhancing the expression of bone metabolism markers. However, bioactive peptides from a single source have limited functions, and when combined, their combined effects are often insufficient, resulting in limited bone health benefits. Furthermore, issues such as hydrolysis efficiency, retention rate of active ingredients, and complex stability in the current preparation process of bioactive peptides have not been effectively resolved, limiting their industrial application.

[0005] Huzhou sheep, a unique and superior meat sheep breed in my country, is characterized by its rapid growth, strong reproductive performance, and tender meat. Its bones, muscles, and blood are rich in high-quality protein, which, after enzymatic hydrolysis, can generate small molecule peptides with various biological activities, providing a high-quality and novel raw material source for the development of food-derived bioactive peptides. Currently, there are no reports, either domestically or internationally, on the preparation of bioactive peptide complexes using Huzhou sheep as raw material and their application in the field of bone health. Existing technologies for the preparation of bioactive peptides mostly rely on conventional enzymatic hydrolysis processes and lack in-depth research on the synergistic effects of niche and complex organic compounds with bioactive peptides. This results in the incomplete utilization of the functional activity of bioactive peptides, and the stability and application effects of the complexes need further improvement. Therefore, developing a bioactive peptide complex using Huzhou sheep as raw material, optimizing the enzymatic hydrolysis process and component compatibility, and possessing a highly efficient synergistic bone-protective effect would not only fill the current technological gap but also provide a safer and more effective new product for the prevention and health maintenance of osteoporosis, possessing significant academic value and industrialization prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a Hu sheep active peptide complex with optimized preparation process and highly efficient components, using Hu sheep bone as raw material, and its application in bone health care. This invention solves the problems of existing active peptides having single functions, insufficient synergistic effects, and poor preparation efficiency and stability, and achieves the effects of promoting osteoblast proliferation, inhibiting osteoclast activity, and increasing bone density.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: A Hu sheep active peptide complex includes Hu sheep active peptides, composite adhesives, bone functional excipients, and pharmaceutical excipients.

[0008] Preferably, the active peptides from Hu sheep include Hu sheep bone collagen peptides.

[0009] Lake sheep bone collagen peptides are the core active ingredient prepared from the bones of healthy lake sheep. As an important nutrient substrate required for bone formation, it provides a key material basis for osteoblast proliferation and mineralization. By participating in the regulation of bone metabolism-related pathways, it further enhances osteoblast proliferation signal transduction, inhibits osteoblast apoptosis, and balances osteoclast bone resorption, ultimately effectively improving bone density in osteoporosis model animals. Moreover, it has excellent biocompatibility and no cytotoxicity, providing safe and core functional support for bone health.

[0010] Preferably, the composite binder comprises palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside.

[0011] Preferably, the mass ratio of sheep bone collagen peptide to palmitoyl aspartate diethyl ester is 0.5-2:0.1-0.3.

[0012] Preferably, the mass ratio of Hu sheep bone collagen peptide to hesperidin-3-O-glucoside is 0.5-2:0.04-0.12. Diethyl palmitoyl aspartate and hesperidin-3-O-glucoside form a highly efficient synergistic system. Together with sheep bone collagen peptides, they promote the transport and absorption of active peptides in cells, regulate osteoblast proliferation-related signaling pathways, and participate in bone metabolism regulation. This not only significantly enhances osteoblast proliferation activity and inhibits osteoblast apoptosis, but also promotes the efficient absorption and utilization of active ingredients in vivo. Together, they strengthen the bone formation process and optimize bone metabolism balance, ultimately effectively increasing bone density in osteoporosis model animals and fully leveraging the synergistic effect of bone health care.

[0013] Preferably, the functional bone excipient includes a calcium-derived compound.

[0014] Preferably, the calcium source compound includes one or more of calcium lactate, calcium carbonate, and calcium hydrogen phosphate.

[0015] Preferably, the mass ratio of sheep bone collagen peptides to calcium source compounds is 1:1-3.

[0016] Preferably, the functional bone excipient includes a chondroprotectant.

[0017] Preferably, the chondroprotective agent includes chondroitin sulfate and / or hyaluronic acid.

[0018] Preferably, the mass ratio of sheep bone collagen peptide to chondroprotectant is 1:0.2-1.

[0019] Preferably, the bone functional excipient includes fat-soluble vitamins.

[0020] Preferably, the fat-soluble vitamins include vitamin D3 and vitamin K2.

[0021] Preferably, the mass ratio of sheep bone collagen peptides to fat-soluble vitamins is 1:0.001-0.004.

[0022] Preferably, the formulation excipients include fillers.

[0023] Preferably, the filler includes one or more of microcrystalline cellulose, mannitol, lactose, and starch.

[0024] Preferably, the mass ratio of sheep bone collagen peptides to fillers is 1:0.7-2.3.

[0025] Preferably, the formulation excipients include lubricants.

[0026] Preferably, the lubricant includes one or more of magnesium stearate, calcium stearate, and talc.

[0027] Preferably, the mass ratio of sheep bone collagen peptide to lubricant is 1:0.15-0.25.

[0028] A method for preparing a lake sheep active peptide complex, comprising, Collagen extract was obtained from the bones of Hu sheep through defatting, decalcification and purification. Trypsin and alkaline protease were added to the collagen extract. After enzymatic hydrolysis, enzyme inactivation, centrifugation, ultrafiltration, desalting through ion exchange resin, and freeze drying were performed to obtain Hu sheep bone collagen peptides. The collagen peptides from sheep bones were uniformly mixed with pharmaceutical excipients, calcium source compounds were added and mixed uniformly, chondroprotective agents were added and mixed uniformly, and binder solution was added. The active peptide complex was obtained by granulation, drying, granulation and compression.

[0029] Preferably, defatting is performed using Soxhlet extraction with an organic solvent.

[0030] Preferably, the organic solvent includes n-hexane and / or petroleum ether.

[0031] Preferably, decalcification is performed using a chelating agent solution.

[0032] Preferably, the chelating agent includes disodium EDTA and / or tetrasodium EDTA.

[0033] Preferably, the enzymatic hydrolysis includes an enzymatic hydrolysis aid.

[0034] Preferably, the enzymatic hydrolysis aid includes potassium phenethyl thiogluconate.

[0035] Preferably, the mass-to-volume ratio of potassium phenethyl thiogluconate to collagen extract is 0.2-0.5 mg:1 mL.

[0036] Potassium phenethyl thiogluconate optimizes the enzymatic hydrolysis efficiency of trypsin and alkaline protease. By improving the enzymatic reaction environment, it promotes the full hydrolysis of collagen, allowing the prepared sheep bone collagen peptides to retain more highly active functional fragments. These high-quality active peptides can not only provide nutritional substrates for osteoblast proliferation and mineralization more efficiently, but also more precisely regulate bone metabolism-related pathways. They can also form synergistic effects with composite adhesives and functional bone excipients, further amplifying the osteoblast proliferation promotion effect and the bone density improvement effect in osteoporosis model animals. This provides important process optimization support for improving the bone health benefits of active peptide complexes.

[0037] Preferably, the mass-to-volume ratio of alkaline protease to collagen extract is 1 mg: 0.5-5 mL.

[0038] Preferably, the mass-to-volume ratio of the complex protease to the collagen extract is 1 mg: 0.5-5 mL.

[0039] More preferably, the chondroprotective agent includes 5,7-dihydroxy-6,8-dimethoxyflavone, and the mass ratio of sheep bone collagen peptide to 5,7-dihydroxy-6,8-dimethoxyflavone is 1:0.05-0.15.

[0040] The additional introduction of 5,7-dihydroxy-6,8-dimethoxyflavonoids as a chondroprotective agent can specifically activate osteoblast proliferation and mineralization-related signaling pathways, while enhancing the inhibitory effect on osteoclast bone resorption. It works synergistically with the transport-promoting effect of palmitoyl aspartate diethyl ester and the apoptosis-inhibiting and osteogenic mineralization-promoting effects of hesperidin-3-O-glucoside to comprehensively regulate the balance between bone formation and bone resorption, further enhance osteoblast proliferation activity, and significantly increase bone mineral density in osteoporosis model animals. This makes the bone health care effect of the active peptide complex more comprehensive and efficient, providing an important functional supplement for the upgrade of the complex's function.

[0041] This invention also provides a method for preparing a collagen extract, comprising: Remove the surface muscles, fascia, and adipose tissue from the limb and trunk bones of healthy Hu sheep. Wash with deionized water 3-5 times, drain, and pulverize at 20-30℃ and 500-1500 rpm to obtain Hu sheep bone powder. Add the Hu sheep bone powder to n-hexane and extract with Soxhlet at 60-64℃ for 1-3 hours, repeating the extraction 2-3 times. Place in a ventilated area for 2-5 hours, and dry at 40-50℃ for 5-7 hours to obtain defatted bone powder. Mix the defatted bone powder with 0.4-0.6 mol / L disodium EDTA solution, adjust the pH to 7.2-7.6, and dry at 32-38℃ with 100... React at -300 rpm for 4-6 hours, wash with deionized water until the pH of the washing solution is 6.9-7.1, centrifuge at 3000-4000 rpm for 5-15 minutes, and dry at 35-45℃ for 4-12 hours to obtain decalcified bone powder; add the decalcified bone powder to deionized water, adjust the pH to 6.5-6.9, and react and extract at 60-64℃ for 10-18 hours, stirring every 1-3 hours at a speed of 100-300 rpm, filter, centrifuge at 8000-10000 rpm for 15-25 minutes, and collect the supernatant to obtain collagen extract.

[0042] Preferably, the mass-to-volume ratio of sheep bone powder to hexane is 1g:5-20mL.

[0043] Preferably, the mass-to-volume ratio of defatted bone meal to 0.4-0.6 mol / L EDTA disodium solution is 1 g: 5-15 mL.

[0044] Preferably, the mass-to-volume ratio of decalcified bone meal to deionized water is 1g:10-30mL.

[0045] This invention also provides a method for preparing Hu sheep bone collagen peptides, comprising: The collagen extract was subjected to enzymatic hydrolysis at 48-52℃ and pH 8.4-8.6 with the addition of alkaline protease and complex protease. The mixture was stirred at 44-46℃, pH 7.4-7.6, and 100-300 rpm for 3-5 hours to obtain the hydrolysate. The hydrolysate was then heated to 88-92℃ and maintained at this temperature for 10-20 minutes. After cooling to room temperature, the mixture was centrifuged at 7000-9000 rpm for 15-25 minutes, and the supernatant was collected. After filtration, a crude extract was obtained. The crude extract was purified by ultrafiltration at 0.08-0.12 MPa and 24-26℃, and the permeate was collected. The permeate was desalted by ion exchange resin and eluted with deionized water to obtain the eluent. The eluent was pre-frozen at -45℃ to -35℃ for 2-6 h and freeze-dried at 0.06-0.1 MPa and -25℃ to -15℃ for 12-18 h to obtain Hu sheep bone collagen peptides.

[0046] Preferably, the mass-to-volume ratio of alkaline protease to collagen extract is 1 mg: 0.5-5 mL.

[0047] Preferably, the mass-to-volume ratio of the complex protease to the collagen extract is 1 mg: 0.5-5 mL.

[0048] More preferably, an enzymatic hydrolysis aid, including potassium phenethyl thiogluconate, is added to the collagen extract.

[0049] More preferably, the mass-to-volume ratio of potassium phenethyl thiogluconate to collagen extract is 0.2-0.5 mg:1 mL.

[0050] Preferably, the molecular weight cutoff of the ultrafiltration membrane is 4-5 kDa.

[0051] Preferably, the ion exchange resin includes type 732 cation exchange resin and type 717 anion exchange resin.

[0052] Preferably, the volume ratio of type 732 cation exchange resin to type 717 anion exchange resin is 1:0.6-1.4.

[0053] Preferably, the sample loading flow rate is 0.5-2 BV / h.

[0054] This invention also provides a method for preparing an active peptide complex, comprising: Ethanol and deionized water were mixed, and palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside were added and mixed evenly to obtain an adhesive solution. Lake sheep bone collagen peptides, microcrystalline cellulose, and mannitol were mixed evenly and stirred at 200-400 rpm for 10-20 min. A calcium source compound and a chondroprotectant were added and stirred at 200-400 rpm for 10-20 min. Fat-soluble vitamins and a lubricant were added and stirred at 200-400 rpm for 10-20 min. The adhesive solution was added and stirred to obtain a soft material. This material was granulated through a 14-18 mesh sieve, dried at 42-48℃ for 1-3 h, granulated through a 16-20 mesh sieve, and pressed to obtain an active peptide complex.

[0055] Preferably, in the adhesive solution, the volume ratio of ethanol to deionized water is 1:0.5-1.5.

[0056] Preferably, in the adhesive solution, the mass-to-volume ratio of palmitoyl aspartate diethyl ester to ethanol is 0.5-1.5 g: 100 mL.

[0057] Preferably, in the adhesive solution, the mass-to-volume ratio of hesperidin-3-O-glucoside to ethanol is 0.2-0.6 g: 100 mL.

[0058] Preferably, the filler includes one or more of microcrystalline cellulose, mannitol, lactose, and starch.

[0059] Preferably, the mass ratio of sheep bone collagen peptides to fillers is 1:0.7-2.3.

[0060] Preferably, the calcium source compound includes one or more of calcium lactate, calcium carbonate, and calcium hydrogen phosphate.

[0061] Preferably, the mass ratio of sheep bone collagen peptides to calcium source compounds is 1:1-3.

[0062] Preferably, the chondroprotective agent includes chondroitin sulfate and / or hyaluronic acid.

[0063] Preferably, the mass ratio of sheep bone collagen peptide to chondroitin sulfate is 1:0.1-0.5.

[0064] Preferably, the mass ratio of sheep bone collagen peptide to hyaluronic acid is 1:0.1-0.5.

[0065] Preferably, the fat-soluble vitamins include vitamin D3 and vitamin K2.

[0066] Preferably, the mass ratio of sheep bone collagen peptides to vitamin D3 is 1:0.0005-0.002.

[0067] Preferably, the mass ratio of sheep bone collagen peptides to vitamin K2 is 1:0.0005-0.002.

[0068] Preferably, the lubricant includes one or more of magnesium stearate, calcium stearate, and talc.

[0069] Preferably, the mass ratio of sheep bone collagen peptide to lubricant is 1g:0.015-0.025.

[0070] Preferably, the volume of the adhesive solution is measured by the volume of ethanol therein, and the mass-to-volume ratio of sheep bone collagen peptide to ethanol is 0.5-2g:20mL.

[0071] Preferably, the weight of the compressed tablet is 0.4-0.6g / tablet.

[0072] Preferably, the pressing pressure is 6-10 MPa.

[0073] More preferably, the chondroprotective agent includes 5,7-dihydroxy-6,8-dimethoxyflavone.

[0074] More preferably, the mass ratio of sheep bone collagen peptide to 5,7-dihydroxy-6,8-dimethoxyflavone is 1:0.05-0.15.

[0075] This invention utilizes healthy Hu sheep bones as raw material, preparing highly active Hu sheep bone collagen peptides through defatting, decalcification, targeted enzymatic hydrolysis, ultrafiltration purification, and freeze-drying. A composite binder composed of palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside is introduced, along with bone functional excipients and formulation excipients, to prepare a Hu sheep active peptide complex. Therefore, it exhibits the following beneficial effects: the Hu sheep active peptide complex significantly promotes osteoblast proliferation and inhibits their apoptosis, effectively inhibits osteoclast bone resorption, and substantially increases bone density in osteoporosis model animals; preparation efficiency and product activity are simultaneously improved; the complex demonstrates excellent stability, superior biocompatibility, and no cytotoxicity. Therefore, this invention represents a highly efficient, safe, and effective Hu sheep active peptide complex suitable for the development and application of bone health-related nutritional supplements, and its applications. Attached Figure Description

[0076] Figure 1 This is a schematic diagram of the test results for the activity of the active peptide complex in promoting osteocyte proliferation.

[0077] Figure 2 This is a schematic diagram showing the results of the test on the increase in bone mineral density of osteoporosis model mice by the active peptide complex.

[0078] Figure 3 This is a schematic diagram of the biosafety test results for the active peptide complex. Detailed Implementation

[0079] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0080] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0081] Example 1: Preparation of collagen extract: The surface muscles, fascia, and adipose tissue of the limb and trunk bones of healthy Hu sheep were removed. The bones were washed four times with deionized water, drained, and then pulverized at 25℃ and 1000 rpm to obtain Hu sheep bone powder. The Hu sheep bone powder was added to n-hexane and extracted using a Soxhlet extractor at 62℃ for 2 hours. This extraction was repeated twice. The bones were then placed in a ventilated area for 4 hours and dried at 45℃ for 6 hours to obtain defatted bone powder. The defatted bone powder was mixed with a 0.5 mol / L disodium EDTA solution, and the pH was adjusted to... 7.4. The mixture was reacted at 35℃ and 200 rpm for 5 h. It was washed with deionized water until the pH of the washing solution reached 7.0. The solution was centrifuged at 3500 rpm for 10 min and dried at 40℃ for 8 h to obtain decalcified bone powder. The decalcified bone powder was added to deionized water, the pH was adjusted to 6.7, and the mixture was reacted and extracted at 62℃ for 14 h, stirring every 2 h at 200 rpm. After filtration, the mixture was centrifuged at 9000 rpm for 20 min, and the supernatant was collected to obtain collagen extract. The mass-to-volume ratio of sheep bone powder to hexane was 1 g:10 mL, the mass-to-volume ratio of defatted bone powder to 0.5 mol / L disodium EDTA solution was 1 g:10 mL, and the mass-to-volume ratio of decalcified bone powder to deionized water was 1 g:20 mL.

[0082] Preparation of Hu sheep bone collagen peptides: Collagen extract was subjected to enzymatic hydrolysis at 50℃ and pH 8.5 with the addition of alkaline protease and complex protease. The mixture was stirred at 45℃, pH 7.5, and 200 rpm for 4 hours to obtain the hydrolysate. The hydrolysate was heated to 90℃ and held at that temperature for 15 minutes. After cooling to room temperature, the mixture was centrifuged at 8000 rpm for 20 minutes. The supernatant was collected and filtered to obtain the crude extract. The crude extract was purified by ultrafiltration at 0.1 MPa and 25℃, and the permeate was collected. The permeate was desalted using an ion exchange resin and eluted with deionized water to obtain the eluent. The eluent was pre-frozen at -40℃ for 4 hours and then freeze-dried at 0.08 MPa and -20℃ for 15 hours to obtain Hu sheep bone collagen peptides. The mass-to-volume ratio of alkaline protease to collagen extract was 1 mg:1 mL, and the mass-to-volume ratio of complex protease to collagen extract was 1 mg:1 mL; the molecular weight cutoff of the ultrafiltration membrane was 3 kDa; the ion exchange resin included type 732 cation exchange resin and type 717 anion exchange resin, with a volume ratio of type 732 cation exchange resin to type 717 anion exchange resin of 1:1, and the loading flow rate was 1 BV / h.

[0083] Preparation of the active peptide complex: Ethanol and deionized water were mixed, and palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside were added and mixed evenly to obtain a binder solution; Hu sheep bone collagen peptides, microcrystalline cellulose and mannitol were mixed evenly and stirred at 300 rpm for 15 min; calcium lactate, chondroitin sulfate and hyaluronic acid were added and stirred at 300 rpm for 15 min; vitamin D3, vitamin K2 and magnesium stearate were added and stirred at 300 rpm for 5 min; the binder solution was added and stirred to obtain a soft material; the material was granulated through a 16-mesh sieve, dried at 45℃ for 2 h, granulated through an 18-mesh sieve, and pressed to obtain the active peptide complex. In the adhesive solution, the volume ratio of ethanol to deionized water is 1:1, the mass-to-volume ratio of palmitoyl aspartate diethyl ester to ethanol is 1 g:100 mL, and the mass-to-volume ratio of hesperidin-3-O-glucoside to ethanol is 0.2 g:100 mL; the mass ratio of Hu sheep bone collagen peptides to microcrystalline cellulose is 1:1, the mass ratio of Hu sheep bone collagen peptides to mannitol is 1:0.5, the mass ratio of Hu sheep bone collagen peptides to calcium lactate is 1:2, and the mass ratio of Hu sheep bone collagen peptides to chondroitin sulfate is 1:0. 2. The mass ratio of Hu sheep bone collagen peptide to hyaluronic acid is 1:0.2, the mass ratio of Hu sheep bone collagen peptide to vitamin D3 is 1:0.001, the mass ratio of Hu sheep bone collagen peptide to vitamin K2 is 1:0.001, and the mass ratio of Hu sheep bone collagen peptide to magnesium stearate is 1:0.02; the volume of the binder solution is measured by the volume of ethanol in it, and the mass-volume ratio of Hu sheep bone collagen peptide to ethanol is 1g:20mL; the weight of the compressed tablet is 0.5g / tablet, and the compression pressure is 8MPa.

[0084] Example 2: The only difference between this example and Example 1 is the preparation of the active peptide complex.

[0085] Preparation of the active peptide complex: Ethanol and deionized water were mixed, and palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside were added and mixed evenly to obtain a binder solution; Hu sheep bone collagen peptides, microcrystalline cellulose and mannitol were mixed evenly and stirred at 300 rpm for 15 min; calcium lactate, chondroitin sulfate and hyaluronic acid were added and stirred at 300 rpm for 15 min; vitamin D3, vitamin K2 and magnesium stearate were added and stirred at 300 rpm for 5 min; the binder solution was added and stirred to obtain a soft material; the material was granulated through a 16-mesh sieve, dried at 45℃ for 2 h, granulated through an 18-mesh sieve, and pressed to obtain the active peptide complex. In the adhesive solution, the volume ratio of ethanol to deionized water is 1:1, the mass-to-volume ratio of palmitoyl aspartate diethyl ester to ethanol is 1 g:100 mL, and the mass-to-volume ratio of hesperidin-3-O-glucoside to ethanol is 0.4 g:100 mL; the mass ratio of Hu sheep bone collagen peptides to microcrystalline cellulose is 1:1, the mass ratio of Hu sheep bone collagen peptides to mannitol is 1:0.5, the mass ratio of Hu sheep bone collagen peptides to calcium lactate is 1:2, and the mass ratio of Hu sheep bone collagen peptides to chondroitin sulfate is 1:0. 2. The mass ratio of Hu sheep bone collagen peptide to hyaluronic acid is 1:0.2, the mass ratio of Hu sheep bone collagen peptide to vitamin D3 is 1:0.001, the mass ratio of Hu sheep bone collagen peptide to vitamin K2 is 1:0.001, and the mass ratio of Hu sheep bone collagen peptide to magnesium stearate is 1:0.02; the volume of the binder solution is measured by the volume of ethanol in it, and the mass-volume ratio of Hu sheep bone collagen peptide to ethanol is 1g:20mL; the weight of the compressed tablet is 0.5g / tablet, and the compression pressure is 8MPa.

[0086] Example 3: The only difference between this example and Example 2 is the preparation of the lake sheep bone collagen peptide.

[0087] Preparation of Hu sheep bone collagen peptides: Collagen extract was subjected to enzymatic hydrolysis at 50℃ and pH 8.5 with the addition of alkaline protease, complex protease, and potassium phenethyl thiogluconate. The mixture was stirred at 45℃, pH 7.5, and 200 rpm for 4 hours to obtain the hydrolysate. The hydrolysate was heated to 90℃ and held at that temperature for 15 minutes. After cooling to room temperature, it was centrifuged at 8000 rpm for 20 minutes. The supernatant was collected and filtered to obtain the crude extract. The crude extract was purified by ultrafiltration at 0.1 MPa and 25℃, and the permeate was collected. The permeate was desalted by ion exchange resin and eluted with deionized water to obtain the eluent. The eluent was pre-frozen at -40℃ for 4 hours and then freeze-dried at 0.08 MPa and -20℃ for 15 hours to obtain Hu sheep bone collagen peptides. The mass-to-volume ratio of alkaline protease to collagen extract was 1 mg:1 mL, the mass-to-volume ratio of complex protease to collagen extract was 1 mg:1 mL, and the mass-to-volume ratio of potassium phenethyl thiogluconate to collagen extract was 0.3 mg:1 mL. The ultrafiltration membrane had a molecular weight cutoff of 3 kDa. The ion exchange resins included type 732 cation exchange resin and type 717 anion exchange resin, with a volume ratio of type 732 cation exchange resin to type 717 anion exchange resin of 1:1. The loading flow rate was 1 BV / h.

[0088] Example 4: The only difference between this example and Example 3 is the preparation of the active peptide complex.

[0089] Preparation of the active peptide complex: Ethanol and deionized water were mixed, and palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside were added and mixed evenly to obtain a binder solution; Hu sheep bone collagen peptides, microcrystalline cellulose and mannitol were mixed evenly and stirred at 300 rpm for 15 min; calcium lactate, chondroitin sulfate, hyaluronic acid and 5,7-dihydroxy-6,8-dimethoxyflavone were added and stirred at 300 rpm for 15 min; vitamin D3, vitamin K2 and magnesium stearate were added and stirred at 300 rpm for 5 min; the binder solution was added and stirred to obtain a soft material; the material was granulated through a 16-mesh sieve, dried at 45℃ for 2 h, granulated through an 18-mesh sieve, and pressed to obtain the active peptide complex. In the adhesive solution, the volume ratio of ethanol to deionized water is 1:1; the mass-to-volume ratio of palmitoyl aspartate diethyl ester to ethanol is 1 g:100 mL; and the mass-to-volume ratio of hesperidin-3-O-glucoside to ethanol is 0.2 g:100 mL. The mass ratio of Hu sheep bone collagen peptides to microcrystalline cellulose is 1:1; the mass ratio of Hu sheep bone collagen peptides to mannitol is 1:0.5; the mass ratio of Hu sheep bone collagen peptides to calcium lactate is 1:2; the mass ratio of Hu sheep bone collagen peptides to chondroitin sulfate is 1:0.2; and the mass ratio of Hu sheep bone collagen peptides to hyaluronic acid is... The mass ratios of the following components are as follows: 1:0.2; 1:0.1; 1:0.001; 1:0.001; 1:0.001; 1:0.02; and 1:0.02. The volume of the binder solution is measured by the volume of ethanol contained within it, and the mass-to-volume ratio of the sheep bone collagen peptide to ethanol is 1 g:20 mL. The weight of each compressed tablet is 0.5 g, and the compression pressure is 8 MPa.

[0090] Comparative Example 1: The only difference between this comparative example and Example 1 is the preparation of the active peptide complex.

[0091] Preparation of the active peptide complex: Ethanol and deionized water were mixed, and palmitoyl aspartate diethyl ester was added and mixed evenly to obtain a binder solution; sheep bone collagen peptides, microcrystalline cellulose and mannitol were mixed evenly and stirred at 300 rpm for 15 min; calcium lactate, chondroitin sulfate and hyaluronic acid were added and stirred at 300 rpm for 15 min; vitamin D3, vitamin K2 and magnesium stearate were added and stirred at 300 rpm for 5 min; the binder solution was added and stirred to obtain a soft material; the material was granulated through a 16-mesh sieve, dried at 45℃ for 2 h, granulated through an 18-mesh sieve, and pressed to obtain the active peptide complex. In the adhesive solution, the volume ratio of ethanol to deionized water is 1:1, and the mass-to-volume ratio of palmitoyl aspartate diethyl ester to ethanol is 1 g:100 mL; the mass ratio of Huzhou sheep bone collagen peptide to microcrystalline cellulose is 1:1, the mass ratio of Huzhou sheep bone collagen peptide to mannitol is 1:0.5, the mass ratio of Huzhou sheep bone collagen peptide to calcium lactate is 1:2, the mass ratio of Huzhou sheep bone collagen peptide to chondroitin sulfate is 1:0.2, the mass ratio of Huzhou sheep bone collagen peptide to hyaluronic acid is 1:0.2, the mass ratio of Huzhou sheep bone collagen peptide to vitamin D3 is 1:0.001, the mass ratio of Huzhou sheep bone collagen peptide to vitamin K2 is 1:0.001, and the mass ratio of Huzhou sheep bone collagen peptide to magnesium stearate is 1:0.02; the volume of the adhesive solution is measured by the volume of ethanol in it, and the mass-to-volume ratio of Huzhou sheep bone collagen peptide to ethanol is 1 g:20 mL; the weight of the compressed tablet is 0.5 g / tablet, and the compression pressure is 8 MPa.

[0092] Comparative Example 2: The only difference between this comparative example and Example 1 is the preparation of the active peptide complex.

[0093] Preparation of the active peptide complex: Ethanol and deionized water were mixed, and palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside were added and mixed evenly to obtain a binder solution; Hu sheep bone collagen peptides, microcrystalline cellulose and mannitol were mixed evenly and stirred at 300 rpm for 15 min; calcium lactate, chondroitin sulfate and hyaluronic acid were added and stirred at 300 rpm for 15 min; vitamin D3, vitamin K2 and magnesium stearate were added and stirred at 300 rpm for 5 min; the binder solution was added and stirred to obtain a soft material; the material was granulated through a 16-mesh sieve, dried at 45℃ for 2 h, granulated through an 18-mesh sieve, and pressed to obtain the active peptide complex. In the adhesive solution, the volume ratio of ethanol to deionized water is 1:1, the mass-to-volume ratio of palmitoyl aspartate diethyl ester to ethanol is 1 g:100 mL, and the mass-to-volume ratio of hesperidin-3-O-glucoside to ethanol is 0.8 g:100 mL; the mass ratio of Hu sheep bone collagen peptides to microcrystalline cellulose is 1:1, the mass ratio of Hu sheep bone collagen peptides to mannitol is 1:0.5, the mass ratio of Hu sheep bone collagen peptides to calcium lactate is 1:2, and the mass ratio of Hu sheep bone collagen peptides to chondroitin sulfate is 1:0. 2. The mass ratio of Hu sheep bone collagen peptide to hyaluronic acid is 1:0.2, the mass ratio of Hu sheep bone collagen peptide to vitamin D3 is 1:0.001, the mass ratio of Hu sheep bone collagen peptide to vitamin K2 is 1:0.001, and the mass ratio of Hu sheep bone collagen peptide to magnesium stearate is 1:0.02; the volume of the binder solution is measured by the volume of ethanol in it, and the mass-volume ratio of Hu sheep bone collagen peptide to ethanol is 1g:20mL; the weight of the compressed tablet is 0.5g / tablet, and the compression pressure is 8MPa.

[0094] Comparative Example 3: The only difference between this comparative example and Example 1 is the preparation of the active peptide complex.

[0095] Preparation of the active peptide complex: Ethanol and deionized water were mixed, and povidone K30 was added and mixed evenly to obtain an adhesive solution; Hu sheep bone collagen peptides, microcrystalline cellulose and mannitol were mixed evenly and stirred at 300 rpm for 15 min; calcium lactate, chondroitin sulfate and hyaluronic acid were added and stirred at 300 rpm for 15 min; vitamin D3, vitamin K2 and magnesium stearate were added and stirred at 300 rpm for 5 min; the adhesive solution was added and stirred to obtain a soft material; the material was granulated through a 16-mesh sieve, dried at 45℃ for 2 h, granulated through an 18-mesh sieve, and pressed to obtain the active peptide complex. In the adhesive solution, the volume ratio of ethanol to deionized water is 1:1, and the mass-to-volume ratio of povidone K30 to ethanol is 1 g:100 mL; the mass ratio of Huzhou sheep bone collagen peptide to microcrystalline cellulose is 1:1, the mass ratio of Huzhou sheep bone collagen peptide to mannitol is 1:0.5, the mass ratio of Huzhou sheep bone collagen peptide to calcium lactate is 1:2, the mass ratio of Huzhou sheep bone collagen peptide to chondroitin sulfate is 1:0.2, the mass ratio of Huzhou sheep bone collagen peptide to hyaluronic acid is 1:0.2, the mass ratio of Huzhou sheep bone collagen peptide to vitamin D3 is 1:0.001, the mass ratio of Huzhou sheep bone collagen peptide to vitamin K2 is 1:0.001, and the mass ratio of Huzhou sheep bone collagen peptide to magnesium stearate is 1:0.02; the volume of the adhesive solution is measured by the volume of ethanol in it, and the mass-to-volume ratio of Huzhou sheep bone collagen peptide to ethanol is 1 g:20 mL; the weight of the compressed tablet is 0.5 g / tablet, and the compression pressure is 8 MPa.

[0096] Comparative Example 4: The only difference between this comparative example and Example 2 is the preparation of the sheep bone collagen peptide.

[0097] Preparation of Hu sheep bone collagen peptides: Collagen extract was subjected to enzymatic hydrolysis at 50℃ and pH 8.5 with the addition of alkaline protease, complex protease, and rhamnine. The mixture was stirred at 45℃, pH 7.5, and 200 rpm for 4 hours to obtain the hydrolysate. The hydrolysate was heated to 90℃ and held at that temperature for 15 minutes. After cooling to room temperature, the mixture was centrifuged at 8000 rpm for 20 minutes. The supernatant was collected and filtered to obtain the crude extract. The crude extract was purified by ultrafiltration at 0.1 MPa and 25℃, and the permeate was collected. The permeate was desalted using an ion exchange resin and eluted with deionized water to obtain the eluent. The eluent was pre-frozen at -40℃ for 4 hours and then freeze-dried at 0.08 MPa and -20℃ for 15 hours to obtain Hu sheep bone collagen peptides. The mass-to-volume ratio of alkaline protease to collagen extract was 1 mg:1 mL, the mass-to-volume ratio of complex protease to collagen extract was 1 mg:1 mL, and the mass-to-volume ratio of rhamnine to collagen extract was 0.3 mg:1 mL. The molecular weight cutoff of the ultrafiltration membrane was 3 kDa. The ion exchange resin included type 732 cation exchange resin and type 717 anion exchange resin, with a volume ratio of type 732 cation exchange resin to type 717 anion exchange resin of 1:1. The loading flow rate was 1 BV / h.

[0098] Experimental Example 1: Assay on the osteocyte proliferation activity of bioactive peptide complex.

[0099] Test samples: Active peptide complexes prepared in each example and comparative example.

[0100] Test method: MC3T3-E1 osteoblasts were seeded into 96-well plates, and α-MEM medium containing 10% fetal bovine serum was added to each well. The plates were incubated at 37℃ and 5% CO2 for 24 h to allow cell adhesion. The original medium was discarded, and 100 μL of active peptide complex solution diluted to 100 μg / mL with sterile PBS buffer was added to each well. The blank control group was added with 100 μL of sterile PBS buffer. After culturing for another 48 h, 20 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated in the dark for 4 h. After discarding the liquid in the wells, 150 μL of DMSO was added to each well, and the plates were shaken for 10 min to dissolve the crystals. The absorbance of each well was measured at 490 nm using a microplate reader. The relative proliferation rate of osteoblasts was calculated based on the absorbance value. The relative proliferation rate of osteoblasts (%) = (absorbance value of experimental group / absorbance value of blank control group) × 100%.

[0101] The results of the osteocyte proliferation activity test of the active peptide complex prepared in this invention are as follows: Figure 1As shown, Example 1 uses a composite binder composed of palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside. Palmitoyl aspartate diethyl ester can promote the transport and absorption of active peptides in cells, while hesperidin-3-O-glucoside can help inhibit osteoblast apoptosis and enhance proliferation signal transduction, thus jointly enhancing osteoblast proliferation activity. Therefore, the relative proliferation rate shows a significant advantage, which is significantly higher than that of the blank control group. In Example 2, the amount of hesperidin-3-O-glucoside was increased. Within an appropriate dosage range, the increased amount of this component further enhanced its synergistic effect with palmitoyl aspartate diethyl ester and Hu sheep bone collagen peptides, resulting in a more significant signal regulation effect and a better osteoblast apoptosis inhibition effect. Therefore, the proliferation rate increased compared to Example 1. In Example 3, potassium phenethyl thiogluconate was added during the enzymatic hydrolysis process. This component optimized the enzymatic hydrolysis efficiency of trypsin and alkaline protease, allowing the prepared Hu sheep bone collagen peptides to retain more highly active functional fragments, providing a higher quality nutrient substrate for osteoblast proliferation. Meanwhile, the synergistic effect of the composite adhesive system still exists, and the combination of high-quality active peptides and the synergistic system further amplifies the proliferation-promoting effect. Therefore, the proliferation rate of Example 3 is significantly higher than that of Examples 1 and 2. Example 4 additionally added 5,7-dihydroxy-6,8-dimethoxyflavonoids, which can further activate osteoblast proliferation-related signaling pathways. It forms a quadruple synergistic system with the transport-promoting effect of palmitoyl aspartate diethyl ester, the apoptosis-inhibiting effect of hesperidin-3-O-glucoside, and the highly active bone collagen peptides optimized by phenethyl ester thiogluconate potassium, which comprehensively enhances osteoblast proliferation activity. Therefore, the relative proliferation rate of Example 4 is the highest among all groups. Comparative Example 1, without the addition of hesperidin-3-O-glucoside, had an incomplete synergistic system, resulting in a lower relative osteoblast proliferation rate than in Example 1. Comparative Example 2, with excessive hesperidin-3-O-glucoside, led to decreased compatibility of the complex system and slightly inhibited osteoblast proliferation signaling pathways, resulting in a lower relative proliferation rate than the blank control group and no promoting effect. This verified that functional components must be used within a reasonable dosage range to exert a synergistic effect. Comparative Example 3 used conventional povidone K30 as a binder. This component only acts as a binder and cannot form any synergistic proliferation effect with the sheep bone collagen peptides. Relying solely on the nutritional supplementation effect of the sheep bone collagen peptides themselves, the effect on promoting osteoblast proliferation was weak. Furthermore, povidone K30 had a slight effect on cells, resulting in the lowest relative proliferation rate among all groups, significantly lower than the examples using the composite binder. Comparative Example 4 used rhamnine instead of potassium phenethyl thiogluconate during enzymatic hydrolysis. Rhamnine's effect on optimizing enzymatic hydrolysis efficiency and its effect on preserving active fragments of collagen peptides are significantly weaker than those of potassium phenethyl thiogluconate. The prepared sheep bone collagen peptides have insufficient bioactivity, and therefore the relative proliferation rate is lower than that in Example 3.

[0102] Experimental Example 2: Test on the increase in bone mineral density of osteoporosis model mice by active peptide complex.

[0103] Test samples: Active peptide complexes prepared in each example and comparative example.

[0104] Test methods: The active peptide complexes prepared in each example and comparative example were ground into fine powder using a grinder and passed through a 100-mesh sieve to remove coarse particles, thus obtaining active peptide complex powder. 10 mg of active peptide complex powder was added to 1 mL of sterile physiological saline and stirred at room temperature for 30 min to obtain an active peptide complex suspension. Female ICR mice aged 6-8 weeks were randomly divided into 10 groups, corresponding to each example and comparative example, including a sham-operated group and a model control group. After 1 week of adaptive feeding, an osteoporosis model was established by bilateral ovariectomy. In the sham-operated group, only the adipose tissue around the ovary was removed, and the mice recovered for 2 weeks after surgery. Mice in each experimental group were given the corresponding active peptide complex suspension by gavage daily at a dose of 10 mL / kg. The sham-operated group and the model control group were given an equal volume of sterile PBS by gavage for 8 consecutive weeks. After the administration, the mice were fasted for 12 h and anesthetized with 10% chloral hydrate. The bone mineral density of the L1-L4 lumbar vertebrae of the mice was detected by dual-energy X-ray absorptiometry.

[0105] The results of the test on bone mineral density improvement in osteoporosis model mice by the active peptide complex prepared in this invention are as follows: Figure 2As shown in Example 1, sheep bone collagen peptides provide core nutritional substrates for bone formation. The composite binder composed of palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside can promote the absorption and transport of active ingredients in vivo, inhibit osteoclast activity, and promote osteoblast mineralization. Calcium lactate supplements the calcium required for bone mineralization, chondroitin sulfate and hyaluronic acid protect cartilage tissue, and vitamin D3 and K2 synergistically promote calcium absorption and bone calcium deposition. Under the combined action of multiple components, bone formation and bone resorption are effectively balanced, thus the bone density is significantly higher than that of the model control group; Example 2 provides... Within an appropriate dosage range, increasing the dosage of hesperidin-3-O-glucoside further enhanced the inhibitory effect on osteoclast activity and significantly promoted osteoblast mineralization. In Example 3, potassium phenethyl thiogluconate was added during enzymatic hydrolysis. This component optimized the hydrolysis efficiency of trypsin and alkaline protease, allowing the prepared sheep bone collagen peptides to retain more highly active functional fragments, further amplifying the bone density-enhancing effect, thus further improving bone density. In Example 4, 5,7-dihydroxy-6,8-dimethyl... Oxyflavonoids further activated osteoblast mineralization-related signaling pathways and enhanced the inhibitory effect on osteoclast bone resorption, maximizing bone formation and inhibiting bone resorption, resulting in the highest bone density among all experimental groups. Comparative Example 1, without the addition of hesperidin-3-O-glucoside, showed limited bone metabolism regulation, with bone density lower than in Example 1. In Comparative Example 2, the amount of hesperidin-3-O-glucoside was significantly increased. Excessive hesperidin-3-O-glucoside led to decreased compatibility of the complex system, weakening its inhibitory effect on osteoclasts and potentially affecting… Osteoblast mineralization was slightly disrupted, and no significant increase in bone density was observed. Comparative Example 3 used conventional povidone K30 as a binder, which only served as a binder in the formulation. Relying solely on the nutritional supplementation effect of the sheep bone collagen peptides themselves and the basic functions of calcium lactate and vitamins, the effect on improving bone density in osteoporosis model mice was weak. Comparative Example 4 used rhamnine in the enzymatic hydrolysis process. Rhamnine's effect on optimizing enzymatic hydrolysis efficiency and preserving active fragments of bone collagen peptides was significantly weaker than that of potassium phenethyl thiogluconate, therefore the bone density was not higher than that of Example 3. This invention, through the scientific design of composite binder components and dosage, optimization of enzymatic hydrolysis process, and rational addition of functional auxiliary components, constructed a multi-dimensional synergistic active peptide complex system, which significantly improved the bone density of osteoporosis model mice. In contrast, component deficiency, improper dosage, substitution of conventional raw materials, or insufficient process optimization all led to a decrease in the effect on improving bone density, fully demonstrating the scientific nature and superiority of the formulation design and process optimization of this invention, and providing reliable experimental evidence for the development of bone health nutritional supplements.

[0106] Experimental Example 3: Biosafety test of bioactive peptide complex.

[0107] Test samples: Active peptide complexes prepared in each example and comparative example.

[0108] Test method: The active peptide complexes prepared in each example and comparative example were ground into fine powder using a grinder, and the coarse particles were removed by passing through a 100-mesh sieve to obtain active peptide complex powder; 20 mg of active peptide complex powder was added to 1 mL of sterile physiological saline, stirred at room temperature for 30 min, and filtered through a 0.22 μm filter membrane for sterilization to obtain an active peptide complex suspension; L929 fibroblasts were seeded in DMEM medium containing 10% fetal bovine serum and cultured in a 37℃, 5% CO2 incubator until the logarithmic growth phase, and then seeded at 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL / well in 96-well plates, and 100 μL of cell suspension was added to each well. The cells were cultured for 24 h to allow them to adhere. The original culture medium was discarded, and 100 μL of each active peptide complex suspension was added to the experimental group, while 100 μL of sterile physiological saline was added to the negative control group. After culturing for another 24 h, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated in the dark for 4 h. After discarding the liquid in the wells, 150 μL of LDMSO was added to each well, and the cells were shaken for 10 min to dissolve the crystals. The absorbance of each well was measured at 490 nm using a microplate reader, and the relative cell viability was calculated.

[0109] The biosafety test results of the active peptide complex prepared in this invention are as follows: Figure 3 As shown, the relative cell viability of all tested samples was higher than 93%. The raw materials used in Examples 1-4, such as sheep bone collagen peptide, palmitoyl aspartate diethyl ester, hesperidin-3-O-glucoside, phenethyl ester thiogluconate potassium, and 5,7-dihydroxy-6,8-dimethoxyflavone, are all biocompatible components, and the dosage of each component is controlled within a safe range. No toxic effects were produced after compounding, thus maintaining a high cell viability. The raw materials used in Comparative Examples 1-4 were also food-grade or pharmaceutical-grade safe components. Even if some common components were missing or the type of binder was changed, no toxic or harmful substances were introduced, thus resulting in a high relative cell viability. These test results indicate that the active peptide complex prepared in this invention and all comparative samples have good biocompatibility, no cytotoxicity, and can meet the food safety requirements for bone health nutritional supplements.

[0110] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0111] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A lake sheep active peptide complex, characterized in that: The Hu sheep active peptide complex comprises Hu sheep active peptides, a composite binder, bone functional excipients, and pharmaceutical excipients; the Hu sheep active peptides include Hu sheep bone collagen peptides, the composite binder comprises palmitoyl aspartate diethyl ester and hesperidin-3-O-glucoside, the mass ratio of Hu sheep bone collagen peptides to palmitoyl aspartate diethyl ester is 0.5-2:0.1-0.3, and the mass ratio of Hu sheep bone collagen peptides to hesperidin-3-O-glucoside is 0.5-2:0.04-0.

12.

2. The lake sheep active peptide complex according to claim 1, characterized in that: The bone functional excipient includes a calcium source compound, which includes one or more of calcium lactate, calcium carbonate and calcium hydrogen phosphate, and the mass ratio of the sheep bone collagen peptide to the calcium source compound is 1:1-3.

3. The lake sheep active peptide complex according to claim 1, characterized in that: The functional bone excipient includes a chondroitin sulfate and / or hyaluronic acid, and the mass ratio of the sheep bone collagen peptide to the chondroitin sulfate is 1:0.2-1.

4. The lake sheep active peptide complex according to claim 1, characterized in that: The bone functional excipient includes fat-soluble vitamins, including vitamin D3 and vitamin K2, and the mass ratio of the sheep bone collagen peptide to the fat-soluble vitamins is 1:0.001-0.

004.

5. The lake sheep active peptide complex according to claim 1, characterized in that: The formulation excipients include a filler, which includes one or more of microcrystalline cellulose, mannitol, lactose and starch, and the mass ratio of the sheep bone collagen peptide to the filler is 1:0.7-2.

3.

6. The lake sheep active peptide complex according to claim 1, characterized in that: The formulation excipients include a lubricant, which includes one or more of magnesium stearate, calcium stearate, and talc. The mass ratio of the sheep bone collagen peptide to the lubricant is 1:0.15-0.

25.

7. A method for preparing a lake sheep active peptide complex according to any one of claims 1-6, characterized in that: include, Collagen extract was obtained from the bones of Hu sheep through defatting, decalcification and purification. Alkaline protease and complex protease were added to the collagen extract. After enzymatic hydrolysis, the extract was inactivated, centrifuged, ultrafiltered, desalted through ion exchange resin, and freeze-dried to obtain Hu sheep bone collagen peptides. The collagen peptides from sheep bones were uniformly mixed with pharmaceutical excipients, calcium source compounds were added and mixed uniformly, chondroprotective agents were added and mixed uniformly, and binder solution was added. The active peptide complex was obtained by granulation, drying, granulation and compression.

8. The method for preparing a lake sheep active peptide complex according to claim 7, characterized in that: The defatting is performed using Soxhlet extraction with an organic solvent, the organic solvent including n-hexane and / or petroleum ether, and the decalcification is performed using a chelating agent solution, the chelating agent including disodium EDTA and / or tetrasodium EDTA.

9. The method for preparing a lake sheep active peptide complex according to claim 7, characterized in that: The enzymatic hydrolysis includes an enzymatic hydrolysis aid, which includes potassium phenethyl thiogluconate, and the mass-to-volume ratio of potassium phenethyl thiogluconate to collagen extract is 0.2-0.5 mg:1 mL.

10. The method for preparing a lake sheep active peptide complex according to claim 7, characterized in that: The mass-to-volume ratio of the alkaline protease to the collagen extract is 1 mg: 0.5-5 mL, and the mass-to-volume ratio of the complex protease to the collagen extract is 1 mg: 0.5-5 mL.

Images