Food-derived bone health peptide and its use in the preparation of bone density improving products

Abstract

This application discloses a food-derived bone health peptide and its application in the preparation of products that improve bone density. The amino acid sequence of the bone health peptide is NDFYPK. This application focuses on OPG, a key target of bone metabolism, and screens novel hexapeptide NDFYPK from bovine colostrum, a product of in vitro gastrointestinal digestion simulating the human body. It verifies NDFYPK's core characteristic of direct release via in vitro gastrointestinal digestion of bovine colostrum. Efficacy is validated at the in vitro cellular level and in vivo in zebrafish model, and its mechanism of action in improving osteoporosis is elucidated through molecular biology experiments, providing a scientific basis for further development of bovine colostrum and its bioactive peptides.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a hexapeptide NDFYPK that is naturally present in bovine colostrum, can be directly and completely released after oral administration through the physiological digestion of the human gastrointestinal tract, and has the function of improving osteoporosis, and its application. Background Technology

[0002] The core pathology of osteoporosis lies in the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, ultimately leading to decreased bone density, bone microstructure degeneration, and a significantly increased risk of fracture. Current clinical treatments for osteoporosis primarily utilize bisphosphonates, estrogen replacement therapy, and RANKL monoclonal antibodies. While these can slow bone loss to some extent, long-term use can cause severe gastrointestinal reactions, atypical fractures, mandibular osteonecrosis, and rapid bone rebound after discontinuation. Furthermore, existing treatments largely rely on inhibiting bone resorption as their core mechanism, exhibiting significant limitations in improving mineral absorption and promoting osteoblast activation and new bone formation.

[0003] The OPG / RANKL / RANK signaling axis is a core pathway regulating bone metabolism homeostasis. Osteoporosis progenitor (OPG), a key endogenous regulator of this pathway, competitively binds to RANKL, blocking its binding to RANK receptors on the surface of osteoclast precursor cells. This inhibits osteoclast differentiation, maturation, and activation, reducing bone resorption. Simultaneously, it positively regulates osteoblast activity, making OPG a core target for maintaining bone metabolic homeostasis and preventing osteoporosis. Upregulating OPG expression levels and enhancing its biological activity and stability can fundamentally restore the dynamic balance between bone formation and bone resorption, representing a significant research direction in the field of osteoporosis prevention and treatment.

[0004] Food-derived bioactive peptides are derived from the enzymatic hydrolysis or fermentation of everyday food proteins. They naturally possess high safety, low immunogenicity, and are easily absorbed due to their small molecular weight. In recent years, their potential in the field of bone metabolism regulation has gradually attracted attention. Bovine colostrum, the milk secreted by cows within three days after natural calving, is rich in various bioactive components such as immunoglobulins, lactoferrin, lysozyme, lactoperoxidase, antimicrobial peptides, and growth factors, making it one of the important sources of food-derived bioactive peptides. However, existing research mainly focuses on the in vitro targeted enzymatic hydrolysis to prepare bovine colostrum complex peptides or single peptides. While confirming that bovine colostrum peptides obtained through in vitro enzymatic hydrolysis have osteoprotective activity, it has not identified the core active molecules that can be directly released from natural bovine colostrum after physiological digestion in the human gastrointestinal tract. Therefore, it cannot be confirmed that daily consumption of bovine colostrum can directly improve osteoporosis. In addition, most of the identified single-sequence active peptides use RANKL as a single target, which has weak specificity and low affinity for OPG targets. They cannot achieve bidirectional regulation of bone metabolism by upregulating OPG expression and activity, and have problems such as insufficient osteogenic activity, narrow effective concentration range, and cytotoxicity at low concentrations, making it difficult to achieve industrialization. Summary of the Invention

[0005] This application focuses on OPG, a key target of bone metabolism, and screens a novel hexapeptide NDFYPK from bovine colostrum, a product of in vitro gastrointestinal digestion simulating the human body. It verifies NDFYPK's core characteristic of direct release via in vitro gastrointestinal digestion of bovine colostrum and its gastrointestinal digestibility. Then, it is prepared using a mature solid-phase synthesis process. The efficacy is systematically verified at the cellular level and in vivo in zebrafish models. Furthermore, molecular biology experiments elucidate its mechanism of action in improving osteoporosis, providing a scientific basis for further development of bovine colostrum and its bioactive peptides.

[0006] Based on this, this application provides a food-derived bone health peptide and its application in the preparation of products that improve bone density.

[0007] A dietary bone health peptide, a hexapeptide derived from bovine colostrum, wherein the amino acid sequence of the hexapeptide is NDFYPK.

[0008] Verification has shown that the hexapeptide NDFYPK possesses three core characteristics: First, this peptide naturally exists in the bovine colostrum protein sequence and can be directly and completely released by endogenous enzymes in the digestive tract through a process that simulates human gastrointestinal digestion; second, this peptide can also be efficiently released from bovine colostrum through a food-grade active peptide-directed enzymatic hydrolysis process; third, the hexapeptide NDFYPK released through enzymatic hydrolysis can still exist stably in its complete active form in a simulated human gastrointestinal digestive environment, exhibiting excellent gastrointestinal digestive stability.

[0009] This application also provides the application of the aforementioned food-derived bone health peptide in the preparation of health foods or functional foods for improving bone density.

[0010] This application also provides a health food or functional food for improving bone density, including the aforementioned food-derived bone health peptide.

[0011] Optionally, food-grade carriers or excipients may also be included.

[0012] When used in the preparation of health foods or functional foods, the hexapeptide can be prepared with food-acceptable excipients according to conventional food preparation methods. These excipients include solvents, binders, fillers, sugars, and sweeteners, which can be selected according to different needs. The types of health foods or functional foods can be any of the following: tablets, capsules, oral liquids, compressed candies, solid beverages, pills, granules, decoctions, pastes, syrups, drop pills, or tea bags.

[0013] This application also provides the use of the dietary bone health peptide in the preparation of a medicament for the prevention or treatment of osteoporosis.

[0014] This application also provides a medicament for the prevention or treatment of osteoporosis, comprising a therapeutically effective amount of the dietary bone health peptide.

[0015] Therapeutic effective dose refers to the range of doses in which a drug, in clinical application, can produce a clear therapeutic effect on the target disease while keeping adverse reactions within an acceptable range. This range can vary depending on the severity of the disease and the physical condition, age, weight, and sex of the subject to be treated.

[0016] Optionally, pharmaceutically acceptable carriers or excipients may also be included.

[0017] The therapeutically effective hexapeptide can be incorporated with pharmaceutically acceptable carriers or excipients, such as diluents, binders, disintegrants, lubricants, flavoring agents, and fragrances. Dosage forms of the drug include granules, tablets, capsules, powders, oral liquids, drops, microcapsules, or injections.

[0018] Optionally, the improvement of bone density or prevention or treatment of osteoporosis includes: promoting the mRNA expression of osteogenic genes OPG, OCN, and ALP; and inhibiting the mRNA expression of osteoclast-related genes RANKL and mMP9.

[0019] This application also provides the use of the dietary bone health peptide in the preparation of a formulation for promoting the proliferation, differentiation and mineralization of osteoblasts MC3T3-E1.

[0020] This application also provides an agent for promoting the proliferation, differentiation and mineralization of osteoblasts MC3T3-E1, comprising a therapeutically effective amount of the dietary bone health peptide.

[0021] This application also provides a bovine colostrum enzymatic hydrolysate containing the aforementioned dietary bone health peptide. This hydrolysate is prepared by targeted enzymatic hydrolysis of bovine colostrum freeze-dried powder using a food-grade complex protease; the food-grade complex protease is composed of a complex protease consisting of neutral protease, papain, and trypsin in a mass ratio of 1~2:2~3:6.

[0022] The complete hexapeptide NDFYPK was also identified in the digestion products of the bovine colostrum enzymatic hydrolysis product after simulated digestion, and it has good gastrointestinal digestibility.

[0023] Compared with the prior art, this application has at least one of the following beneficial effects: (1) The active peptides in this application are derived from natural food raw materials, which naturally have high safety, low immunogenicity, small molecular weight and easy absorption.

[0024] (2) The hexapeptide of this application can be obtained by screening bovine colostrum from human in vitro gastrointestinal digestion products, which verifies its core characteristic that it can be directly released through in vitro gastrointestinal digestion of bovine colostrum.

[0025] (3) The hexapeptide of this application can also be prepared by food-grade compound protease hydrolysis of bovine colostrum freeze-dried powder. It has been verified that the hexapeptide NDFYPK released by this hydrolysis process can still exist stably in an intact active form in a simulated human gastrointestinal digestive environment, and has excellent gastrointestinal digestive stability.

[0026] (4) The hexapeptide of this application can significantly promote the proliferation, differentiation and mineralization of osteoblasts MC3T3-E1.

[0027] (5) The hexapeptide of this application can significantly promote the mRNA expression of osteogenic genes OPG, OCN and ALP; and inhibit the mRNA expression of osteoclast-related genes RANKL and mMP9.

[0028] (6) The hexapeptide of this application has strong specificity and high affinity for OPG target binding. It can achieve bidirectional regulation of bone metabolism by upregulating OPG expression and activity, and has no cytotoxicity within the range of 0~200 μg / mL. Attached Figure Description

[0029] Figure 1 This is the total ion chromatogram from Example 1; Figure 2 This is a diagram showing the molecular docking of the hexapeptide NDFYPK with the OPG protein. Figure 3 This is a secondary mass spectrum of the hexapeptide NDFYPK; Figure 4 This is a primary mass spectrum of the hexapeptide NDFYPK. Figure 5 Figure showing the effect of hexapeptide NDFYPK on cell viability of MC3T3-E1 osteoblasts; Figure 6 Figure showing the effect of tetrapeptide EYIV on cell viability of MC3T3-E1 osteoblasts; Figure 7 The figure shows the effect of hexapeptide NDFYPK on alkaline phosphatase (ALP) activity in MC3T3-E1 osteoblasts. Figure 8A This is a chromogenic image of the hexapeptide NDFYPK on the formation of mineralized nodules in osteoblasts. Figure 8B This is a graph showing the quantitative results of the hexapeptide NDFYPK on the formation of mineralized nodules in osteoblasts; Figure 9A Figure showing the effect of hexapeptide NDFYPK on bone mineralization area in zebrafish, an osteoporosis model. Figure 9B The figure shows the effect of hexapeptide NDFYPK on the fluorescence intensity of zebrafish, an osteoporosis model. Figure 10AThe figure shows the effect of hexapeptide NDFYPK on the expression of osteogenic-related gene OPG mRNA in zebrafish. Figure 10B The figure shows the effect of hexapeptide NDFYPK on the expression of osteogenic-related gene OCN mRNA in zebrafish. Figure 10C Figure showing the effect of hexapeptide NDFYPK on the expression of osteogenic-related gene ALP mRNA in zebrafish. Figure 11A Figure showing the effect of hexapeptide NDFYPK on the expression of RANKL mRNA, a gene related to osteoclastosis in zebrafish. Figure 11B The figure shows the effect of hexapeptide NDFYPK on the expression of the osteoclast-related gene MMP9 mRNA in zebrafish. Detailed Implementation

[0030] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0031] 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 belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0032] The bovine colostrum powder used in the following examples is commercially available lyophilized bovine colostrum powder, which is readily available. Preferably, 24-hour lyophilized bovine colostrum powder can be used.

[0033] Example 1 Validation of gastrointestinal release and stability of hexapeptide NDFYPK 1.1 Preparation of simulated digestive fluid Based on the physiological electrolyte composition of the human gastrointestinal tract, simulated saliva (SSF), simulated gastric juice (SGF), and simulated intestinal juice (SIF) electrolyte stock solutions with a concentration of 1.25× were prepared. Specific formulations are shown in Table 1. All stock solutions were sterilized by filtration through a 0.22 μm filter membrane and stored in a sealed, light-protected container at -20 ℃. Before use, they were preheated in a 37 ℃ water bath. CaCl2(H2O)2 should be added immediately before the start of each digestion stage to avoid precipitation caused by premature addition.

[0034] Table 1. Formula for simulated digestive fluid electrolyte reserve solution (preparation volume 400 mL)

[0035] 1.2 Gastrointestinal Digestion of Bovine Colostrum This experiment constructed an in vitro digestive system simulating the entire human oral cavity-stomach-intestinal tract to verify that bovine colostrum can directly release the complete hexapeptide NDFYPK through the normal physiological digestive process of the human gastrointestinal tract. The specific experimental steps are as follows: 1.2.1 Oral Digestion Stage Take 5 g of bovine colostrum powder sample and mix it thoroughly with 5 g of 1.25×SSF stock solution at a ratio of 1:1 (wt / wt). Add CaCl2(H2O)2 to the final system concentration of 1.5 mM and adjust the pH of the system to 7.0. Since bovine colostrum does not contain starch, salivary α-amylase is not added at this stage. Place the mixture in a 37 ℃ constant temperature shaking incubator and incubate for 2 min to complete oral digestion and obtain oral chyme.

[0036] 1.2.2 Gastric Digestion Stage Take 10 mL of the above oral chyme and mix it thoroughly with 10 mL of 1.25×SGF stock solution at a ratio of 1:1 (vol / vol). Add CaCl2(H2O)2 to a final system concentration of 0.15 mM. Add pepsin to a final system enzyme activity of 2000 U / mL and add pepsin from rabbit stomach extract to a final system enzyme activity of 60 U / mL. Simultaneously subtract the pepsin activity contained in RGE to ensure that the total pepsin activity of the system meets the set requirements. Precisely adjust the pH of the system to 3.0 using 1M HCl and incubate in a 37 ℃ constant temperature shaking incubator for 2 h to complete gastric digestion and obtain gastric digestion products.

[0037] 1.2.3 Intestinal Digestion Stage Take 20 mL of the above gastric digestion products and mix them thoroughly with 20 mL of 1.25×SIF stock solution at a ratio of 1:1 (vol / vol). Add CaCl2(H2O)2 to a final system concentration of 0.6 mM. Add bovine bile extract to a final system bile salt concentration of 10 mM. Add trypsin to a final system trypsin activity of 100 U / mL. Adjust the pH of the system to 7.0 precisely using 1 M NaOH. Incubate the system in a 37 ℃ constant temperature shaking incubator for 2 h to complete the digestion of the entire gastrointestinal tract and obtain the intestinal digestion end products.

[0038] 1.2.4 Reaction Termination and Sample Pretreatment Immediately after digestion, 5 mM MPefablocSC (serine protease inhibitor) and 1 μM pepstatin A (aspartic protease inhibitor) were added to the end product of intestinal digestion to terminate the enzyme reaction. The digestive enzymes were then completely inactivated by boiling in a water bath for 10 min. The inactivated sample was centrifuged at 4 ℃ and 12000 r / min for 15 min. The supernatant was desalted and enriched by C18 solid-phase extraction column, concentrated by nitrogen blowing, and then reconstituted with the initial mobile phase. The solution was then filtered through a 0.22 μm organic filter membrane to obtain the in vitro digested bovine colostrum sample.

[0039] 1.3 Preparation of active peptides from bovine colostrum by enzymatic hydrolysis Bovine colostrum powder was mixed with pure water at a ratio of 1:10 to obtain bovine colostrum base material. Food-grade complex protease (a complex protease of neutral protease, papain, and trypsin in a mass ratio of 1.5:2.5:6) was added, and the mixture was hydrolyzed at 55 ℃ with shaking for 4 h. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 min, and the supernatant was collected by centrifugation at 4 ℃ and 8000 r / min for 15 min. Large molecular weight proteins were removed by ultrafiltration through a 3 kDa membrane, and the permeate was collected and freeze-dried to obtain bovine colostrum active peptide powder, i.e., the enzymatic hydrolysis product.

[0040] 1.4 Gastrointestinal Digestion of Bovine Colostrum Enzymatic Hydrolysates Take 5 g of the above-mentioned 10 mg / mL bovine colostrum active peptide solution (prepared by mixing the bovine colostrum active peptide powder obtained in 1.3 with pure water), and process it strictly according to the simulated human whole gastrointestinal digestion process in 1.3.1-1.3.4 of this example; simultaneously set up an enzyme blank control group (no peptide powder sample, only digestive liquid and corresponding digestive enzyme added) to eliminate the interference of enzyme self-degradation products on the detection results; and obtain the sample after digestion of bovine colostrum enzymatic hydrolysate.

[0041] 1.5 Virtual screening and structural confirmation of active peptides via molecular docking 1.5.1 Mass Spectrometry Analysis Three different bovine colostrum peptide samples prepared in steps 1.2, 1.3, and 1.4 were dissolved in pure water at 2 mg each, centrifuged (12000 rpm, 10 min) using a 10 kDa ultrafiltration tube, washed, and the peptide fractions with a molecular weight less than 10 kDa were collected and their concentrations determined using Nanodrop. 100 μg of the above peptides were taken and dithiothreitol (DTT) was added to a final concentration of 10 mMol / L, and the mixture was reduced in a 56℃ water bath for 1 h. Iodoacetamide (IAM) was then added to a final concentration of 20 mMol / L, and the mixture was reacted at room temperature in the dark for 40 min. Unreacted IAM was neutralized again with DTT (10 mMol / L). The treated samples were desalted using a C18 stage-tip and dried under vacuum at 45℃ for later use.

[0042] The processed samples were analyzed by LC-MS / MS: Capillary liquid chromatography conditions: (1) Pre-column: 150 μm·d. × 50 mM, packing material is Reprosil-Pur120C18-AQ 3 μm; Analytical column: 150 μm·d. × 170 mM, packing material is Reprosil-Pur120C18-AQ 1.9 μm; (2) Mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 80% acetonitrile solution containing 0.1% formic acid; (3) Flow rate: 600 nL / min; (4) Analysis time for each component: 66 min; (5) Mobile phase gradient elution method: 0 min, 4% B; 2 min, 8% B; 35 min, 28% B; 55 min, 40% B; 56 min, 95% B; 66 min, 95% B.

[0043] Mass spectrometry parameters: The acquisition mode is data-dependent acquisition (DDA) mode, and the specific parameter settings are as follows: (1) Primary mass spectrometry parameters: Resolution: 70000; AGCtarget: 3e6; MaximumIT: 100 ms; Scanrange: 100~1500 m / z; (2) Secondary mass spectrometry parameters: Resolution: 17500; AGCtarget: 1e5; MaximumIT: 50 ms; TopN: 20; NCE / steppedNCE: 28.

[0044] After LC-MS / MS analysis, the raw mass spectrometry file was used for peptide sequence resolution using the PEAKSDenovo method. The search parameters were as follows: Fixed modifications: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (Peptide N-term); Enzyme: Nonspecific; Peptide Mass Tolerance (PMS): 20 ppm; Fragment Mass Tolerance (FMS): 0.02 Da.

[0045] 1.5.2 Molecular docking virtual screening Based on the sequencing results in Section 1.5.1, approximately 13,000 peptide mass spectrometry data points were accumulated in the preliminary stage. Among them, the total ion spectrum of the bovine colostrum in vitro digestion sample in section 1.2 is shown below. Figure 1As shown. Using OPG, a key target of bone metabolism, as the research object, the crystal structure of OPG was downloaded from the PDB database. Water molecules and proligands were removed from the structure using PyMOL software. Preprocessing was performed using AutoDockTools software, including hydrogenation, Gasteiger charge calculation, active pocket definition, and redundant side chain removal. The result was saved in pdbqt format for subsequent docking. Simultaneously, MSA (multiple sequence alignment) information of the OPG target protein was downloaded from the AlphaFoldDB database to provide basic data for subsequent structure prediction.

[0046] A virtual peptide library of hexapeptides with 20 random combinations of natural amino acids (a total of 64,000,000 combinations) was constructed. AlphaFold3 was used to predict the three-dimensional structure of all hexapeptides and the three-dimensional structure of the complexes of hexapeptides with OPG target proteins. Each peptide-target combination generated an independent predicted structure model. Candidate peptides with high reliability in complex interface prediction were screened with an ipTM (interface prediction TM-score) > 0.75 as the initial screening threshold. After optimization by AMBER force field, all were saved in pdbqt format.

[0047] AutoDockVina software was used to perform molecular docking between the candidate active peptides after initial screening and the OPG target protein. The initial screening threshold was set at binding energy ≤ -7.0 kcal / mol. Multiple indicators were used to evaluate the positive peptides after initial screening. The evaluation indicators included interface specificity score, binding energy efficiency, ligand interface contribution, shape complementarity, Rosetta interface binding energy, etc. Finally, the hexapeptide NDFYPK (Asn-Asp-Phe-Tyr-Pro-Lys) with the best binding activity to the OPG target was selected from the active library.

[0048] The final screening results are shown in Table 2. The optimal binding energy of the hexapeptide NDFYPK to OPG protein is as low as -9.2 kcal / mol, the interface prediction score (ipTM) reaches 0.91, the interface specificity score is 0.1872, it can form 6 stable hydrogen bonds with key amino acid residues of OPG protein, and the shape complementarity is 0.426. All indicators confirm that it has extremely strong binding affinity and targeting specificity to the OPG target.

[0049] Table 2. Molecular docking results of hexapeptide NDFYPK and control tetrapeptide EYIV

[0050] Molecular docking diagram of hexapeptide NDFYPK and OPG protein is shown below. Figure 2 As shown; the secondary mass spectrum of the hexapeptide NDFYPK is as follows. Figure 3 As shown.

[0051] 1.6 Stability verification of hexapeptide NDFYPK monomer in a simulated human gastrointestinal digestive system The samples of 1.2 bovine colostrum digest, 1.3 directional enzymatic hydrolysis product, and 1.4 bovine colostrum hydrolysis product after digestion were subjected to hexapeptide NDFYPK mass spectrometry detection. The results are shown in Table 3.

[0052] Table 3. Mass spectrometry detection results of hexapeptide NDFYPK in samples from different sources.

[0053] The results show that the hexapeptide NDFYPK obtained in this application has three core characteristics: First, the peptide can be directly and completely released by endogenous enzymes in the digestive tract through a simulated human gastrointestinal digestion process; second, the peptide can be efficiently released from bovine colostrum through a food-grade active peptide-directed enzymatic hydrolysis process; and third, the hexapeptide NDFYPK released through the enzymatic hydrolysis process can still exist stably in its complete active form in a simulated human gastrointestinal digestion environment, exhibiting excellent gastrointestinal digestive stability.

[0054] Example 2 2.1 Solid-phase synthesis of hexapeptide NDFYPK The hexapeptide NDFYPK was prepared using the Fmoc manual solid-phase synthesis strategy and was commissioned to Shanghai Jier Biochemical Co., Ltd. The specific raw materials and preparation steps are as follows: 2.1.1 Core Raw Materials and Reagents: (1) Protect amino acid raw materials: Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH; (2) Condensation reagents: Pybop, DIEA, pyridine; (3) Solvents: DMF, DCM, methanol; (4) Solid support: Fmoc-Lys(Boc)-Wang resin; (5) Deprotection reagent: 20% piperidine / DMF solution; (6) Pyrolysis buffer: TFA 94.5%, water 2.5%, EDT 2.5%, TIS 1%; (7) Others: nitrogen, anhydrous ether, ninhydrin test reagent.

[0055] 2.1.2 Specific preparation steps (1) Resin swelling: Fmoc-Lys(Boc)-Wang resin was placed in a solid-phase synthesis reaction tube, and dichloromethane (DCM) was added at a ratio of 15 mL / g resin. The mixture was shaken at room temperature for 30 min to allow the resin to fully swell. The solvent was then removed by filtration through a sand core. (2) First amino acid coupling: Add 3 times molar excess of Fmoc-Pro-OH amino acid and 3 times molar excess of condensing agent Pybop to the reaction tube, then add 3 times molar excess of N,N-diisopropylethylamine DIEA, add DMF to dissolve, and shake at room temperature for 30 min. (3) Deprotection: Remove the reaction solution, add 20% piperidine / DMF solution at a ratio of 15 mL / g resin, react at room temperature for 5 min, remove the solution, add the same 20% piperidine / DMF solution again, react at room temperature for 15 min to complete the removal of Fmoc protecting group; (4) Ninhydrin detection: Remove the piperidine solution, take more than ten resin grains, add one drop each of ninhydrin, KCN and phenol solution in sequence, heat at 105 ℃-110 ℃ for 5 min, the resin turns dark blue, indicating that the deprotection is complete; (5) Washing: Wash twice with DMF (10 mL / g resin), twice with DCM (10 mL / g resin), and twice with DMF (10 mL / g resin) in sequence; (6) Amino acid condensation: Take the corresponding Fmoc protected amino acid in the order from C-terminus to N-terminus of the peptide chain, add 3 times the molar excess of Pybop, dissolve it with a small amount of DMF and add it to the reaction tube, immediately add 3 times the molar excess of DIEA, and shake at room temperature for 30 min; after the reaction is completed, take the resin for ninhydrin detection. If the resin is transparent white, the condensation reaction is complete. (7) Repeat washing: Wash twice with DMF (10 mL / g resin), twice with DCM (10 mL / g resin), and twice with DMF (10 mL / g resin); (8) Full sequence synthesis: Repeat steps (3)-(7), and connect Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Asn(Trt)-OH in sequence from C-terminus to N-terminus of the peptide chain to complete the coupling of the full sequence of the hexapeptide; after the last amino acid Fmoc-Asn(Trt)-OH is connected, repeat step (3) to complete the N-terminal deprotection. (9) Resin shrinkage: Wash twice with DMF (10 mL / g resin), twice with DCM (10 mL / g resin), and twice with methanol (10 mL / g resin) in sequence, and then vacuum dry for 10 min. (10) Peptide cleavage: Prepare a lysis buffer at a ratio of 10 mL / g resin with a volume ratio of TFA:water:EDT:TIS=94.5:2.5:2.5:1. Add the lysis buffer to the dried resin and cleave at room temperature with shaking for 120 min. (11) Drying and washing: After lysis, the filtrate is collected by sand core filtration. The lysate is dried as much as possible by nitrogen. Pre-cooled anhydrous ether is added to precipitate the peptide. After standing, centrifugation is performed to discard the supernatant. The precipitate is washed 6 times with anhydrous ether and the ether is evaporated at room temperature to obtain crude hexapeptide. (12) Crude product detection: Take a small amount of crude product, dissolve it in methanol, and then use Shimadzu LCMS for mass spectrometry detection. After confirming the target molecular weight, proceed with subsequent purification. (13) Purification and freeze-drying: The crude product was prepared and purified using an LC3000 high performance liquid chromatograph. The mobile phase was 0.1% TFA aqueous solution-0.1% TFA acetonitrile solution, with linear gradient elution. The target peak fraction was collected, and after removing the organic phase by rotary evaporation, it was freeze-dried to obtain pure hexapeptide NDFYPK.

[0056] 2.2 Solid-phase synthesis of control tetrapeptide EYIV The tetrapeptide EYIV was prepared using the Fmoc manual solid-phase synthesis strategy. The core reagents, resin swelling, deprotection, ninhydrin detection, washing, lysis, purification and lyophilization steps were the same as those for the preparation of the hexapeptide NDFYPK in 2.2. The only differences were: (1) the starting solid-phase synthesis resin was replaced with Fmoc-Val-Wang resin; (2) the types and order of the amino acids coupled in step (8) were replaced with Fmoc-Tyr (tBu)-OH and Fmoc-Glu (OtBu)-OH in the order from C-terminus to N-terminus of the peptide chain; (3) after lysis, the anhydrous diethyl ether was used to wash the precipitate 6 times, which was consistent with the hexapeptide preparation process.

[0057] 2.3 Structural confirmation of the synthesized peptide 2.3.1 High Performance Liquid Chromatography (HPLC) Purity Detection The purity of the hexapeptide NDFYPK was determined by high performance liquid chromatography (HPLC). The detection parameters were as follows: column: 4.6 × 250 mM, Sinochrom ODS-BP 5 μm; detection wavelength: 220 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: 0.1% TFA aqueous solution (phase A) and 0.1% TFA acetonitrile solution (phase B). Gradient elution results showed that the retention time of the characteristic peak of the target hexapeptide was 10.865 min, and the product purity calculated by peak area normalization was ≥98%, which meets the purity requirements for subsequent cell and in vivo experiments.

[0058] 2.3.2 Mass Spectrometry (MS) Molecular Mass Confirmation The molecular weight of the synthesized hexapeptide NDFYPK was confirmed by electrospray ionization mass spectrometry. NDFYPK: Results of full-scan mass spectrometry (Level 1 mass spectrometry) Figure 4 The [M+H]+ molecular ion peak mass-to-charge ratio of the target peptide was 783.84 m / z, which is highly consistent with the theoretical relative molecular mass of the hexapeptide NDFYPK of 782.84 Da, confirming that the product obtained by solid-phase synthesis is the target hexapeptide.

[0059] Example 3 In vitro osteogenic cell assays of hexapeptide NDFYPK: This embodiment uses MC3T3-E1 mouse embryonic osteogenic progenitor cells as a model to systematically verify the in vitro osteogenic activity of the hexapeptide NDFYPK and to verify the performance advantages of the hexapeptide. The experiment was divided into a blank control group (without peptide) and NDFYPK administration groups at different concentrations (5, 10, 20, 50, 100, 200 μg / mL), with 6 replicates in each group.

[0060] 3.1 Effect of peptides on osteoblast proliferation activity detected by CCK-8 assay MC3T3-E1 cells were loaded at 5×10 3 Cells were seeded at a concentration of [insert concentration here] per well in 96-well plates and incubated at 37 °C, 5% CO2 for 24 h until complete cell adhesion. The original culture medium was discarded, and the cells were washed twice with PBS. α-MEM complete culture medium containing different concentrations of peptides was added, and incubation continued for 72 h. After incubation, 10 μL of LCK-8 reagent was added to each well, and the cells were incubated in the dark for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The formula for calculating cell viability is: ×100% The blank group was a culture medium group without cells or samples, and the control group was a normal culture medium group without peptides.

[0061] The results are as follows Figure 5 As shown, the hexapeptide NDFYPK significantly promoted the proliferation of MC3T3-E1 cells in the concentration range of 5-50 μg / mL, with 50 μg / mL being the optimal concentration, which was significantly different from the blank control group (P<0.05). When the concentration was increased to 200 μg / mL, there was no statistically significant difference in cell viability compared with the blank control group (P>0.05), and no cytotoxicity was observed.

[0062] Using tetrapeptide EYIV as a comparative example, the results of the comparative example are as follows: Figure 6As shown, the tetrapeptide EYIV exhibited significant dose-dependent toxicity to MC3T3-E1 osteoblasts at test concentrations ranging from 0 to 200 μg / mL, with a concentration of 20 μg / mL significantly inhibiting cell viability. The comparison between the two confirms that the hexapeptide NDFYPK possesses superior osteoblast safety and proliferative activity.

[0063] 3.2 Effects of hexapeptide NDFYPK on osteoblast alkaline phosphatase (ALP) activity Sample preparation: MC3T3-E1 cells were prepared at a concentration of 2×10⁻⁶. 4 Cells were seeded per well in 6-well plates and, after adhesion, were added to osteogenic induction medium (containing 10 mmol / L sodium β-glycerophosphate + 50 μg / mL ascorbic acid + 102 nmol / L dexamethasone) at concentrations of 0, 25, and 50 μg / mL NDFYPK and cultured for 7 days. After discarding the culture medium, the cells were washed twice with pre-cooled PBS. 150 μL of RIPA lysis buffer without phosphatase inhibitors was added to each well, and the cells were lysed on ice for 5 min. The cell suspension was collected and centrifuged at 4 °C and 1200 r / min for 10 min. The supernatant was used as the protein sample for analysis. The total protein concentration of the sample was determined using the BCA method.

[0064] ALP activity assay: Prepare 0.01 mg / mL phenol standard working solution with double-distilled water. Prepare buffer, matrix solution, and chromogenic reagent according to the kit instructions. Set up blank wells (30 μL double-distilled water), standard wells (30 μL 0.01 mg / mL phenol standard working solution), and assay wells (30 μL protein sample). Add 50 μL buffer and 50 μL matrix solution to each well sequentially, mix well, and incubate at 37 ℃ for 15 min. Immediately add 150 μL chromogenic reagent to stop the reaction and develop color. Measure the absorbance (OD) at 520 nm using a microplate reader.

[0065] Result Calculation: ALP activity is calculated using the following formula: ×c(standard) ÷c(protein) In the formula, c(standard) is the concentration of the phenol standard solution (0.01 mg / mL), and c(protein) is the total protein concentration of the sample to be tested (g / mL).

[0066] The results are as follows Figure 7 As shown, the hexapeptide NDFYPK can increase ALP activity in MC3T3-E1 cells in a concentration-dependent manner. Specifically, 50 μg / mL NDFYPK significantly increased ALP activity, reaching 147.62±4.58% of the control group, with a highly statistically significant difference between groups (P<0.01). This result confirms that the hexapeptide NDFYPK can significantly promote early differentiation and maturation of osteoblasts.

[0067] 3.3 Effects of hexapeptide NDFYPK on osteoblast mineralization nodule formation MC3T3-E1 cells were loaded at 2×10 4 Cells were seeded at a concentration of 50 μg / mL NDFYPK in 6-well plates. After cell adhesion, the medium was replaced with osteogenic induction medium containing 50 μg / mL NDFYPK (same as 3.2.1). A blank control group was also set up. The medium was changed every 3 days and cultured until day 21.

[0068] After culture, the culture medium was discarded, the cells were rinsed twice with PBS, and fixed with 95% ethanol at room temperature for 30 min. The fixative was discarded, the cells were rinsed twice again with PBS, and 0.1% Alizarin Red S staining solution (pH=4.2) was added. The cells were stained at room temperature in the dark for 30 min. After staining, the cells were thoroughly washed with distilled water to remove non-specific staining. The formation of mineralized nodules was observed under an inverted microscope and photographed. The proportion of mineralized area was quantitatively analyzed using ImageJ software.

[0069] The staining phenotype results are shown in Figure 8A. In the blank control group, only a few scattered mineralized nodules were formed, and there was almost no obvious positive staining under the microscope. In the 50 μg / mL NDFYPK drug group, the number of orange-red positive mineralized nodules increased significantly, and they were distributed in dense patches, indicating a significant improvement in the degree of mineralization.

[0070] Quantitative results such as Figure 8B As shown, the mineralization area in the 50 μg / mL NDFYPK group was 11.53±1.83%, which was significantly higher than that in the blank control group (7.56±0.72%), and the difference between the groups was statistically significant (P<0.05). This result confirms that hexapeptide NDFYPK can significantly promote terminal differentiation and mineralization maturation of osteoblasts and has excellent in vitro osteogenic activity.

[0071] Example 4 Validation of the in vivo improvement effect of hexapeptide NDFYPK on a zebrafish osteoporosis model 4.1 Construction of a zebrafish osteoporosis model Adult zebrafish were reared at a constant temperature of 28 ℃ under a 14 h / 10 h light-dark cycle. Fertilized eggs were obtained through natural mating at a female-to-male ratio of 2:2 and incubated at a constant temperature of 28 ℃. Normal juveniles that reached 3 dpf were randomly assigned to 6-well plates (15 fish / well) and set up a blank control group, a model group, and different doses of NDFYPK (1, 5, 10, 15 μg / mL).

[0072] The solution settings for each group are as follows: Blank control group: zebrafish system water containing 0.2% (m / v) dimethyl sulfoxide (DMSO); Model group: system water containing 0.2% dimethyl sulfoxide + dexamethasone (20 μM); NDFYPK dosage groups: system water containing 0.2% dimethyl sulfoxide + dexamethasone (20 μM) + NDFYPK (final concentrations of 1, 5, 10, and 15 μg / mL).

[0073] The culture medium was changed every other day for each group, and the intervention was continued until 7 dpf, after which the juvenile fish were collected for bone staining.

[0074] 4.2 Alizarin Red Staining and Result Analysis After zebrafish juveniles reached 7 days post-flop (dpf), the system water containing the test substance was aspirated from the 6-well plates. The plates were washed once with system water, and the juveniles were euthanized with 5X anesthetic. After washing again with system water and aspirating all liquid from the wells, the entire staining process was performed sequentially: First, 3 mL of 4% paraformaldehyde was added to each well and fixed at 4 °C for 24 h; after discarding the solution, 3 mL of 50% ethanol was added to each well and rinsed on a shaker at room temperature for 10 min; after discarding the solution, 3 mL of freshly prepared 3% H₂O₂ + 0.5% KOH bleaching solution was added to each well and decolorized on a shaker at room temperature for approximately 20 min until the fish bodies turned white and the eyes turned pale yellow; after discarding the solution, 3 mL of 25% glycerol + 0.1% KOH was added to each well and rinsed 2-3 times, 10 min each time; after discarding the solution, 3 mL of 0.01% alizarin red staining solution was added to each well and stained in the dark for 2 h; after discarding the staining solution, 3 mL of 50% ethanol was added to each well. Rinse the zebrafish 2-3 times with 0.1% KOH for 10 minutes each time. Finally, transfer the treated juvenile fish to a transparent glass plate for photography. If photography cannot be completed on the same day, transfer the juvenile fish to 100% KOH for preservation. Images of the zebrafish skull were acquired using a stereomicroscope, and the bone mineralization area and alizarin red staining fluorescence intensity were quantitatively analyzed using ImageJ software.

[0075] Quantitative results such as Figure 9A and Figure 9B As shown, where Figure 9A This is a map showing the results of bone mineralization area. Figure 9B The fluorescence intensity results show that, compared with the blank control group, the mineralized area of ​​the zebrafish skull in the model group decreased to 9.75±0.25 mm. 2 The alizarin red fluorescence intensity decreased to 1562±298.51, and the difference between groups was statistically significant (P<0.01), indicating that the dexamethasone-induced zebrafish osteoporosis model was successfully established. After intervention with hexapeptide NDFYPK, the mineralization level of zebrafish bones showed a significant dose-dependent recovery, with the mineralized area in the 10 μg / mL treatment group recovering to 11.71±0.52 mm. 2The difference compared to the model group was statistically significant (P<0.05); the mineralized area in the 15 μg / mL drug administration group recovered to 11.43±0.12 mm. 2 The fluorescence intensity recovered to 1793.42±41.23, showing a highly statistically significant difference compared to the model group (P<0.01). The 10 μg / mL and 15 μg / mL administration groups restored bone mineralization indicators to normal levels, with no statistically significant difference compared to the blank control group (P>0.05). These results confirm that the hexapeptide NDFYPK prepared in this application has clear in vivo osteoproliferative activity, can effectively improve dexamethasone-induced bone mineralization deficiency, and reverse osteoporosis pathological damage.

[0076] Example 5 qPCR experimental verification of the molecular mechanism by which hexapeptide NDFYPK improves osteoporosis: In this embodiment, qPCR technology was used to detect the mRNA expression levels of osteogenic and osteoclast-related genes in zebrafish after intervention with hexapeptide NDFYPK, elucidating the molecular mechanism by which it improves osteoporosis.

[0077] Sample processing and RNA extraction: The zebrafish rearing, grouping, and intervention procedures were the same as in Example 4, except that the intervention group was the NDFYPK group: system water containing 0.2% dimethyl sulfoxide + dexamethasone (20 μM) + NDFYPK (10 μg / mL). After intervention for 7 dpf, juvenile fish (10 fish / group) were collected from each group, washed three times with pre-cooled PBS, and excess liquid was aspirated. Trizol reagent was added, and the mixture was thoroughly ground and lysed. Total RNA was extracted using the Trizol method. The RNA concentration and purity were determined using Nanodrop 2000. RNA with OD260 / OD280 between 1.8 and 2.0 was used for subsequent reverse transcription experiments.

[0078] Reverse transcription and qPCR amplification: Total RNA was reverse transcribed into cDNA using a reverse transcription kit. Using cDNA as a template, qPCR amplification was performed using a SYBR Green quantitative PCR kit. β-actin was used as an internal reference gene to detect the mRNA expression levels of osteogenic genes (OPG, OCN, ALP) and osteoclast-related genes (RANKL, mMP9). Primer sequences are shown in Table 4.

[0079] Table 4. Primer sequences for qPCR amplification

[0080] qPCR reaction program: 95 ℃ pre-denaturation for 30 s; 95 ℃ denaturation for 5 s, 60 ℃ annealing for 30 s, 40 cycles; melting curve analysis: 95 ℃ for 15 s, 60 ℃ for 1 min, 95 ℃ for 15 s. Each sample was set up in 3 replicates.

[0081] 5.3 Experimental Results Osteogenesis-related gene expression results as follows Figures 10A-10C As shown, compared with the blank control group, the model composition contained bone-related gene OPG ( Figure 10A ), OCN ( Figure 10B ), ALP Figure 10C The mRNA expression levels of the osteogenic genes were all significantly downregulated (P<0.01). After intervention with 10 μg / mL hexapeptide NDFYPK, the expression levels of each osteogenic gene significantly rebounded. The relative expression levels of OPG were upregulated to 0.84±0.09, OCN to 0.88±0.08, and ALP to 9.1±0.02. The differences compared with the model group were all statistically significant (P<0.01) and were basically close to the levels of the blank group.

[0082] The results of osteoclast-related gene expression are as follows: Figure 11A and Figure 11B As shown, compared with the blank control group, the osteoclast-related gene RANKL ( Figure 11A ) and mMP9 ( Figure 11B The mRNA expression levels of the osteoclasts were all significantly upregulated (P<0.01); after intervention with 10 μg / mL hexapeptide NDFYPK, the expression levels of each osteoclast gene were significantly downregulated, with the relative expression level of RANKL downregulated to 1.52±0.14 and mMP9 downregulated to 1.68±0.29, and the differences were statistically significant compared with the model group (P<0.01).

[0083] The above results confirm that hexapeptide NDFYPK can promote bone formation by upregulating the expression of key osteogenic genes and inhibit bone resorption by downregulating the expression of key osteoclast genes, thereby bidirectionally regulating bone metabolism balance at the transcriptional level and playing a core role in improving osteoporosis.

[0084] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A food-derived bone health peptide, characterized in that, The hexapeptide is derived from bovine colostrum, and the amino acid sequence of the hexapeptide is NDFYPK.

2. The application of the food-derived bone health peptide as described in claim 1 in the preparation of health foods or functional foods for improving bone density.

3. A health food or functional food for improving bone density, characterized in that, Includes the food-derived bone health peptides as described in claim 1.

4. The health food or functional food according to claim 3, characterized in that, It also includes food science-acceptable carriers or additives.

5. The use of the dietary bone health peptide as described in claim 1 in the preparation of a medicament for the prevention or treatment of osteoporosis.

6. A drug for the prevention or treatment of osteoporosis, characterized in that, Includes a therapeutically effective amount of the dietary bone health peptide as described in claim 1.

7. The drug according to claim 6, characterized in that, It also includes pharmaceutically acceptable carriers or excipients.

8. The use of the dietary bone health peptide as described in claim 1 in the preparation of a formulation for promoting the proliferation, differentiation and mineralization of osteoblasts MC3T3-E1.

9. A formulation for promoting the proliferation, differentiation, and mineralization of osteoblasts MC3T3-E1, characterized in that, Includes a therapeutically effective amount of the dietary bone health peptide as described in claim 1.

10. A bovine colostrum enzymatic hydrolysis product, characterized in that, It contains the food-derived bone health peptide as described in claim 1.

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