A bovine colostrum-derived active peptide and its use in the preparation of a product for improving osteoporosis

Abstract

The application discloses a bovine colostrum-derived active peptide and application thereof in preparing an osteoporosis-improving product. The amino acid sequence of the bovine colostrum-derived active peptide is SLLP. The application takes RANKL as a core docking target, and a novel tetrapeptide SLLP is screened from a product of bovine colostrum simulated human whole gastrointestinal tract digestion. The natural release characteristics and digestion stability of the tetrapeptide SLLP are verified by using a standardized in-vitro digestion system. Target binding activity verification, large-scale synthesis and in-vivo evaluation are completed, and the mechanism of the tetrapeptide SLLP in improving osteoporosis is systematically clarified. The application provides a new candidate molecule and complete theoretical support for the development of drugs for preventing and treating osteoporosis and health-care food for improving bone density.

Description

Technical Field

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

[0002] The core pathology of osteoporosis lies in the disruption of the dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, accompanied by impaired absorption of minerals such as calcium, magnesium, and phosphorus in the intestines, ultimately leading to decreased bone density, degeneration of bone microstructure, and a significantly increased risk of fracture.

[0003] The RANKL / RANK signaling pathway is a core pathway for regulating bone metabolism balance. RANKL binds to the RANK receptor on the surface of osteoclast precursor cells, which can drive osteoclast differentiation and activation and promote bone resorption. Blocking the binding of the two can effectively inhibit bone resorption and restore bone metabolism balance, and is currently the core target for the prevention and treatment of osteoporosis.

[0004] Currently, the main clinical treatments for osteoporosis are bisphosphonates, estrogen replacement therapy, and RANKL monoclonal antibodies. Although these can slow down bone loss to some extent, long-term use can lead to serious gastrointestinal reactions, atypical fractures, mandibular osteonecrosis, and rapid rebound of bone mass after discontinuation. Moreover, most of these treatments focus on inhibiting bone resorption as their core mechanism, which has significant limitations in improving mineral absorption, promoting osteoblast activation, and new bone formation.

[0005] Therefore, the discovery of safe, non-toxic, and highly active osteoprotective peptides that can target and bind to RANKL from natural food matrices has become a research hotspot in this field. Food-derived bioactive peptides have advantages such as small molecular weight, good water solubility, easy oral absorption, strong targeting, no immunogenicity, and a wide safety window, showing great development potential in the prevention and treatment of osteoporosis; bovine colostrum is rich in milk proteins and immune-active substances, and its protein sequence contains a large number of functional bioactive peptides, making it a high-quality natural source of osteoprotective peptides.

[0006] Currently, a variety of osteogenic peptides have been reported, but there are still many technical bottlenecks that cannot be overcome: (1) Most studies use complex peptide mixtures, and the core active amino acid sequence is not clearly defined, resulting in poor product quality control; (2) Most of the identified single-sequence peptides are obtained by artificial directional enzymatic hydrolysis or chemical synthesis, and are not endogenous peptides that are naturally present in food matrix and can be directly released by human gastrointestinal digestion, resulting in insufficient safety and natural affinity for human body; (3) Existing short peptides generally have poor gastrointestinal stability. After oral administration, they are easily degraded by various proteases such as pepsin and trypsin in the digestive tract, and cannot reach the target in the complete active form to exert their effects. The oral bioavailability is extremely low, and there is a lack of standardized gastrointestinal stability verification system, and the experimental results have no physiological relevance; (4) At the same time, existing active peptides generally have weak RANKL target binding force, insufficient osteogenic activity, and narrow effective concentration range. They mainly inhibit bone resorption in a single direction and lack synergistic improvement on bone formation and mineral absorption, and cannot fundamentally restore the balance of bone metabolism. Summary of the Invention

[0007] Given the current state of existing technologies, the challenge lies in identifying novel osteoactive peptides that are derived from natural bovine colostrum, have well-defined sequences, can be directly and completely released through human gastrointestinal tract digestion, have proven excellent gastrointestinal digestibility, exhibit strong RANKL target binding specificity, demonstrate superior osteogenic activity, possess high biocompatibility, and possess bidirectional regulatory functions for both mineral absorption and bone metabolism.

[0008] This application uses RANKL as the core docking target and screens the product of bovine colostrum digested in a simulated human gastrointestinal tract to obtain a novel tetrapeptide SLLP. A standardized in vitro digestion system is used to verify its natural release characteristics and digestive stability. Target binding activity verification, large-scale synthesis and in vivo evaluation are completed, and its mechanism of action in improving osteoporosis is systematically elucidated.

[0009] Based on this, this application provides an active peptide derived from bovine colostrum and its application in the preparation of products that improve osteoporosis.

[0010] An active peptide derived from bovine colostrum, with the amino acid sequence SLLP (SEQ ID NO: 1).

[0011] This application also provides the use of the bioactive peptide derived from bovine colostrum in the preparation of a medicament for the prevention or treatment of osteoporosis.

[0012] Optionally, the prevention or treatment of osteoporosis includes at least one of the following: (a) Reversing inhibition of longitudinal bone growth; (b) Reversing osteoporotic bone microstructure damage; (c) Promotes mineral deposition in bones; (d) Regulate bone metabolism balance.

[0013] Optionally, the reversal of longitudinal bone growth inhibition includes promoting tibial and femoral length growth.

[0014] Optionally, the reversal of osteoporotic bone microstructure damage includes increasing bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and reducing trabecular bone separation (Tb.Sp).

[0015] Optionally, the minerals include calcium (Ca), magnesium (Mg), phosphorus (P), zinc (Zn), and manganese (Mn) found in the femur.

[0016] Optionally, the regulation of bone metabolism balance includes: promoting osteoblast differentiation and new bone formation, inhibiting osteoclast activation and bone resorption, and improving the body's calcium and phosphorus metabolism levels. Further, this is manifested in a highly significant increase in serum PINP, TGF-β1, and IGF-1 levels (P<0.01), and a decrease in CTX-I levels (P<0.01); by targeting and regulating the RANKL pathway, on the one hand, it upregulates the expression of key osteoblast differentiation genes such as Runx2, OCN, and ALP, promoting osteoblast differentiation and new bone formation; on the other hand, it downregulates the expression of key osteoclast differentiation genes such as RANKL and MMP9, inhibiting osteoclast activation and bone resorption.

[0017] This application also provides a medicament for the prevention or treatment of osteoporosis, comprising a therapeutically effective amount of the said bovine colostrum-derived active peptide.

[0018] 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.

[0019] Optionally, pharmaceutically acceptable carriers or excipients may also be included.

[0020] The therapeutically effective tetrapeptide can be incorporated with pharmaceutically acceptable carriers or excipients, such as diluents, binders, disintegrants, lubricants, flavoring agents, and aromatics. Dosage forms of the drug include granules, tablets, capsules, powders, oral liquids, drops, microcapsules, or injections.

[0021] This application also provides the use of the bioactive peptide derived from bovine colostrum in the preparation of health foods for improving bone density.

[0022] This application also provides a health food for improving bone density, comprising active peptides derived from bovine colostrum.

[0023] Optionally, food-grade carriers or excipients may also be included.

[0024] When used in the preparation of health food products, the tetrapeptide 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 type of health food product can be any of the following: tablets, capsules, oral liquids, compressed candies, solid beverages, pills, granules, decoctions, pastes, syrups, drop pills, or tea bags.

[0025] Compared with the prior art, this application has at least one of the following beneficial effects: (1) The active peptides in this application are food-derived bioactive peptides, which have advantages such as small molecular weight, good water solubility, easy oral absorption, strong targeting, no immunogenicity, and wide safety window.

[0026] (2) The active peptides obtained by screening natural bovine colostrum have clear sequences, can be directly and completely released through human gastrointestinal physiological digestion, have been verified to have excellent gastrointestinal digestion stability, strong RANKL target binding specificity, excellent osteogenic activity, high biosafety, and have the dual function of promoting mineral absorption and bone metabolism.

[0027] (3) The active peptides of this application can also be screened by enzymatic hydrolysis, and the active peptides obtained by enzymatic hydrolysis and targeted screening have been verified to have excellent gastrointestinal digestive stability.

[0028] (4) The active peptides of this application have good activity in reversing the inhibition of bone longitudinal growth, reversing osteoporosis-induced bone microstructure damage, promoting mineral deposition in bone, and regulating bone metabolism balance. Attached Figure Description

[0029] Figure 1 The total ion mass spectrum of Example 1; Figure 2 This is a diagram showing the docking of the tetrapeptide SLLP with the target RANKL. Figure 3 This is the secondary mass spectrum of the tetrapeptide SLLP; Figure 4 This is the primary mass spectrum of the tetrapeptide SLLP; Figure 5 The results show the effects of tetrapeptides SLLP and DIFP on bone length in mice (where A represents tibia length and B represents femur length). Figure 6 The figure shows the effect of tetrapeptide SLLP on bone mineral density and bone microstructure parameters of mouse femur (where A is bone mineral density, B is bone volume fraction, C is the number of trabeculae, D is the trabeculae thickness, and E is the trabeculae separation). 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 of ordinary skill 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 Gastrointestinal release, peptide screening and identification, and stability verification of tetrapeptide SLLP: 1.1 Preparation of simulated digestive fluid Based on the physiological electrolyte composition of the human gastrointestinal tract, 1.25× concentration simulated saliva (SSF), simulated gastric juice (SGF), and simulated intestinal juice (SIF) electrolyte stock solutions were prepared, with specific formulas shown in Table 1. All stock solutions were sterilized by filtration through a 0.22 μm filter membrane and then stored sealed and protected from light at -20 ℃. Before use, they were preheated in a 37 ℃ water bath. Calcium chloride dihydrate (CaCl2) was included. 2H2O should be added immediately before 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 Experiment of Bovine Colostrum 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. Add 2H2O 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 components, salivary α-amylase is not added at this stage. Place the mixed system 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, mix it thoroughly with 10 mL of 1.25×SGF stock solution at a ratio of 1:1 (vol / vol), and add CaCl2. 2H2O was added to a final system concentration of 0.15 mM; pepsin was added to a final system enzyme activity of 2000 U / mL, and pepsin from rabbit gastric extract (RGE) was added to a final system enzyme activity of 60 U / mL. The pepsin activity inherent in RGE was simultaneously subtracted to ensure that the total pepsin activity of the system met the set requirements; the pH of the system was precisely adjusted to 3.0 using 1 M HCl, and the system was incubated in a 37℃ constant temperature shaking incubator for 2 h to complete gastric digestion and obtain the gastric digestion product.

[0037] 1.2.3 Intestinal Digestion Stage Take 20 mL of the above gastric digestion products, mix thoroughly with 20 mL of 1.25×SIF stock solution at a ratio of 1:1 (vol / vol), and add CaCl2. 2H2O was added to a final system concentration of 0.6 mM; bovine bile extract was added to a final system bile salt concentration of 10 mM; trypsin was added to a final system trypsin activity of 100 U / mL; the pH of the system was precisely adjusted to 7.0 using 1 M NaOH, and the system was incubated 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 product.

[0038] 1.2.4 Reaction Termination and Sample Pretreatment Immediately after digestion, 5 mM PefablocSC (serine protease inhibitor) and 1 μM pepstatin A (aspartic protease inhibitor) were added to the end product of intestinal digestion to terminate the enzymatic 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 digestion sample of bovine colostrum.

[0039] 1.3 Enzymatic hydrolysis process for preparing bioactive peptides from bovine colostrum Bovine colostrum powder was mixed with purified water at a ratio of 1:10 to prepare bovine colostrum base. Food-grade complex protease (a complex of neutral protease, papain, and trypsin in a mass ratio of 1.5:2.5:6) was added, with the total enzyme content equal to 2% of the substrate protein mass. The mixture was then enzymatically hydrolyzed at 55 ℃ with shaking for 4 h. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 min. 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. 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 Experiment of Bovine Colostrum Enzymatic Hydrolysates Take 5 g of the above-mentioned 10 mg / mL bovine colostrum active peptide solution (the preparation of bovine colostrum active peptide powder obtained in 1.3 and pure water), and process it strictly according to the simulated human whole gastrointestinal digestion process described in 1.3.1-1.3.4 of this example; at the same time, set up an enzyme blank control group (without peptide powder sample, only digestive liquid and corresponding digestive enzyme are added) to eliminate the interference of enzyme self-degradation products on the detection results, and finally obtain the sample to be tested after digestion of bovine colostrum enzymatic hydrolysate.

[0041] 1.5 Mass Spectrometry Detection and Result Analysis 1.5.1 Mass Spectrometry Detection Three different bovine colostrum samples prepared in Examples 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 reduced in a 56 °C water bath for 1 h. Iodoacetamide (IAM) was then added to a final concentration of 20 mMol / L, and 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 °C for later use.

[0042] The processed samples were analyzed by LC-MS / MS: Capillary liquid chromatography conditions: (1) Pre-column: 150 μm·d.×50mM, packing material was Reprosil-Pur120C18-AQ3 μm; Analytical column: 150 μm·d.×170mM, packing material was Reprosil-Pur120C18-AQ1.9 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] Among them, the total ion chromatogram of the bovine colostrum in vitro digestion sample is as follows: Figure 1 As shown.

[0046] 1.5.2 Molecular docking virtual screening Based on the sequencing results in Section 1.5.1, approximately 13,000 peptide mass spectrometry data were accumulated in the preliminary stage. Taking RANKL, a key target of bone metabolism, as the research object, the crystal structure of RANKL was downloaded from the PDB database. Water molecules and proligands in the structure were removed using PyMOL software. Preprocessing was performed using AutoDockTools software, including hydrogenation, Gasteiger charge calculation, active pocket definition, and removal of redundant side chains. The data was saved in pdbqt format for subsequent docking. At the same time, the MSA (multiple sequence alignment) information of the RANKL target protein was downloaded from the AlphaFoldDB database to provide basic data for subsequent structure prediction.

[0047] A virtual peptide library of tetrapeptides 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 tetrapeptides and the three-dimensional structure of the complexes of tetrapeptides with RANKL target proteins. Each peptide-target combination generated an independent predicted structure model. Candidate peptides with high reliability of 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.

[0048] AutoDockVina software was used to perform molecular docking between the candidate tetrapeptides after initial screening and the RANKL 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 tetrapeptide SLLP with the best binding activity to the RANKL target was obtained from the tetrapeptide library.

[0049] The final screening results are shown in Table 2. The optimal binding energy of the tetrapeptide SLLP to RANKL protein was as low as -7.5 kcal / mol. It can form three stable hydrogen bonds with key amino acid residues of the target protein. The interface prediction score was 0.82, the interface specificity score was 0.1526, and the shape complementarity was 0.387. All indicators meet the screening criteria for highly active binding peptides. The above results confirm that the tetrapeptide SLLP has extremely strong binding affinity and target specificity to RANKL, a core target of bone metabolism. It can block downstream signaling pathways by stably binding to RANKL, and has excellent potential activity in improving osteoporosis.

[0050] Table 2 Molecular docking results of tetrapeptide SLLP and tetrapeptide DIFP

[0051] The docking results of the tetrapeptide SLLP with the RANKL protein are as follows: Figure 2 As shown, the secondary mass spectra of the tetrapeptide SLLP are as follows: Figure 3 As shown.

[0052] 1.6 Stability verification results of active tetrapeptide SLLP 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 tetrapeptide SLLP mass spectrometry detection. The results are shown in Table 3.

[0053] Table 3. Mass spectrometry detection results of tetrapeptide SLLP in samples from different sources.

[0054] The results showed that after simulated human oral administration and complete physiological digestion, the intact tetrapeptide SLLP could be directly detected in bovine colostrum, with a molecular weight of 428.2635, which highly matched the theoretical molecular weight. This directly confirms that routine oral administration of bovine colostrum, without any additional processing, can directly release the intact active tetrapeptide SLLP through the physiological digestion of endogenous proteases in the human digestive tract. Simultaneously, the tetrapeptide SLLP could also be obtained through in vitro targeted enzymatic hydrolysis of bovine colostrum proteins, further corroborating the sequence accuracy of this peptide and its bovine colostrum origin.

[0055] Example 2: Synthesis and structural confirmation of the tetrapeptide SLLP 2.1 Solid-phase synthesis of tetrapeptide SLLP The tetrapeptide SLLP was prepared using the Fmoc manual solid-phase synthesis strategy and synthesized by Shanghai Jier Biochemical Co., Ltd. The specific raw materials and preparation steps are as follows: After swelling with H-Pro-2-Chlorotrityl resin, amino acids were sequentially coupled with a 3-fold excess of Fmoc protectant in the order from C-terminus to N-terminus using Pybop / DIEA as the condensing agent. Deprotection was performed with 20% piperidine / DMF, and the endpoint was monitored using the ninhydrin method. After the complete sequence synthesis, the crude peptide was obtained by TFA / water / EDT / TIS (94.5:2.5:2.5:1) cleavage and anhydrous diethyl ether precipitation. The crude peptide was purified by HPLC gradient elution and freeze-dried to obtain pure SLLP. The peptide was identified by ESI-MS. Figure 4 The [M+H]+ ion peak m / z was 429.31, which is highly consistent with the theoretical molecular mass (428.52 Da), confirming that the product is the target tetrapeptide.

[0056] 2.2 Solid-phase synthesis of control tetrapeptide DIFP The tetrapeptide DIFP was prepared using the Fmoc manual solid-phase synthesis strategy. The steps were the same as those for the preparation of the tetrapeptide SLLP in 2.1, except that the starting solid-phase synthesis resin was replaced with Fmoc-Pro-Wang resin; and the types and order of the coupled amino acids were changed to Fmoc-Ile-OH and Fmoc-Asp(OtBu)-OH sequentially from the C-terminus to the N-terminus of the peptide chain.

[0057] Example 3 Validation of the in vivo improvement effect of tetrapeptide SLLP on a mouse osteoporosis model: This embodiment uses an ovariectomized (OVX) induced mouse osteoporosis model to systematically verify the in vivo osteoprotective activity of the tetrapeptide SLLP. A sham-operated group was set up as a blank control. Combined with bone phenotype, serum biochemistry, and gene expression detection, the efficacy and mechanism of action of SLLP in improving osteoporosis were comprehensively analyzed.

[0058] 3.1 Animal model construction and grouping for drug administration Seventy-two 6-week-old female C57BL / 6J mice were housed in an SPF-grade environment at a temperature of 22±2 ℃ and a humidity of 50±5%, with a 12 h / 12 ​​h light-dark cycle. They were allowed free access to food and water and underwent formal experiments after one week of acclimatization.

[0059] The mice were randomly divided into 6 groups of 12 mice each, as follows: Sham surgery group: Only open surgery was performed without removing the ovaries, and the same volume of normal saline was administered by gavage daily after the operation; Model group (OVX): underwent bilateral oophorectomy and were given an equal volume of normal saline by gavage daily after surgery; Low-dose SLLP group (SLLP-L): Bilateral oophorectomy was performed, and SLLP was administered by gavage daily postoperatively at a dose of 1 mg / (kg). d); High-dose SLLP group (SLLP-H): Bilateral oophorectomy was performed, and SLLP was administered by gavage daily postoperatively at a dose of 10 mg / (kg). d).

[0060] Low-dose DIFP group (DIFP-L): Patients underwent bilateral oophorectomy and were given DIFP by gavage daily postoperatively at a dose of 1 mg / (kg). d); High-dose DIFP group (DIFP-H): Underwent bilateral oophorectomy, and received DIFP by gavage daily postoperatively at a dose of 10 mg / (kg). d).

[0061] All mice were routinely fed post-surgery and subjected to continuous gavage intervention for 12 weeks. For the last 12 hours of the experiment, mice were fasted but allowed free water. They were anesthetized with sodium pentobarbital, and euthanized by cervical dislocation after blood was collected via the eyeball. The femurs and tibias were dissected bilaterally, and surrounding soft tissues were removed. The right femur was flash-frozen in liquid nitrogen and stored at -80 °C for subsequent gene expression detection; the left femur and tibia were used for bone mineral density, bone microstructure, and bone mineral content detection. Blood samples were allowed to stand at room temperature for 2 hours, then centrifuged at 3000 r / min for 15 min to separate serum, which was then stored at -80 °C for subsequent biochemical assays.

[0062] 3.2 Detection of bone phenotype and bone mineral content 3.2.1 Bone length measurement The results of femur and tibia length measurements in each group of mice are as follows: Figure 5As shown in the figure, A represents tibia length and B represents femur length. The results showed that after ovariectomy, the total length of the femur and tibia in the OVX model group was significantly shortened by 10.99% and 7.32% respectively compared to the sham operation group (P<0.05). After SLLP intervention, the bone length of the mice recovered in a dose-dependent manner. Specifically, the high-dose SLLP group (SLLP-H) showed an increase of 8.36% and 7.04% in femur and tibia length respectively compared to the OVX model group, with the tibia length essentially returning to normal levels (P<0.01). However, all DIFP dose groups showed no significant improvement in bone length (all increases were <4%, P>0.05). These results indicate that SLLP can effectively reverse the inhibition of longitudinal bone growth in osteoporotic model mice and possesses clear in vivo osteoprotective activity.

[0063] 3.2.2 Bone mineral density and bone microstructure detection The left femur of mice was scanned using micro-CT with the following parameters: voltage 50 kV, current 200 μA, resolution 10 μm, and rotation step size 0.5°. After scanning, the distal femur cancellous bone region was selected as the region of interest using the accompanying analysis software to reconstruct the three-dimensional structure and quantitatively analyze bone microstructural parameters such as bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp).

[0064] The results are as follows Figure 6 As shown, A is bone mineral density (BMD), B is bone volume fraction (BV / TV), C is the number of trabeculae (Tb.N), D is the trabecular thickness (Tb.Th), and E is the trabecular separation (Tb.Sp). The results showed that, compared with the sham group, the femoral bone mineral density (BMD), bone volume fraction (BV / TV), number of trabeculae (Tb.N), and trabecular thickness (Tb.Th) of mice in the OVX model group were all significantly reduced (P<0.01), while the trabecular separation (Tb.Sp) was significantly increased (P<0.01), exhibiting a typical osteoporotic bone microstructure degeneration phenotype, and the model was successfully established. Compared with the OVX model group, the SLLP dose group could improve bone microstructure parameters in a dose-dependent manner. Among them, the high-dose group could significantly increase bone mineral density (BMD), bone volume fraction (BV / TV), number of trabeculae (Tb.N), and trabecular thickness (Tb.Th), and reduce trabecular separation (Tb.Sp) (P<0.01), reversing osteoporotic bone microstructure damage.

[0065] 3.2.3 Determination of mineral content in bone tissue The left femur of mice was dried in an oven at 105 ℃ to constant weight, ground in a mortar and pestle, and then digested by wet digestion. After digestion, the femur was diluted to a final volume with deionized water, filtered through a 0.22 μm filter membrane, diluted by the appropriate factor, and the contents of calcium (Ca), magnesium (Mg), phosphorus (P), zinc (Zn), and manganese (Mn) in the femur were determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0066] The results are shown in Table 4. Compared with the Sham group, the contents of Ca, Mg, P, Zn, and Mn in the femur of mice in the OVX model group were significantly decreased (P<0.01). Compared with the OVX model group, the SLLP dosage group increased the contents of multiple minerals in the bone in a dose-dependent manner. Among them, the contents of Ca, Mg, P, Zn, and Mn in the high-dose group increased by 18.76%, 12.43%, 16.89%, 22.57%, and 58.32% respectively compared with the model group, with extremely significant differences (P<0.01), which were basically close to those in the Sham group. These results confirm that SLLP can effectively promote the deposition of minerals in bone and improve the level of bone mineralization.

[0067] Table 4 Effect of tetrapeptide SLLP on mineral content in mouse femur (μg / g, Mean±SD, n=8)

[0068] 3.3 Serum biochemical index detection Using the corresponding enzyme-linked immunosorbent assay (ELISA) kits and strictly following the instructions, bone metabolism-related indicators in mouse serum were detected: bone formation marker type I procollagen N-terminal peptide (PINP), bone resorption marker type I collagen C-terminal peptide (CTX-I), transforming growth factor-β1 (TGF-β1), insulin-like growth factor-1 (IGF-1), and calcium and phosphorus metabolism indicators serum calcium (Ca) and serum phosphorus (P).

[0069] The results are shown in Table 5. Compared with the Sham group (sham surgery), the OVX model group mice showed significantly decreased serum PINP, TGF-β1, and IGF-1 levels (P<0.01) and significantly increased CTX-I levels (P<0.01), exhibiting a bone metabolism imbalance phenotype of bone formation inhibition and bone resorption hyperactivity. Compared with the OVX model group, the high-dose SLLP group significantly increased serum PINP, TGF-β1, and IGF-1 levels (P<0.01), decreased CTX-I levels (P<0.01), and significantly increased serum calcium and phosphorus levels (P<0.05), essentially approaching the levels of the Sham group. These results confirm that the tetrapeptide SLLP can bidirectionally regulate bone metabolism balance by promoting bone formation and inhibiting bone resorption, while simultaneously improving the body's calcium and phosphorus metabolism levels.

[0070] Table 5 Effects of SLLP on serum biochemical parameters in mice (Mean±SD, n=12)

[0071] 3.4 Detection of bone tissue-related gene expression We used real-time quantitative PCR (qPCR) to detect the mRNA expression levels of osteogenic and osteoclast-related genes in mouse femoral tissue, elucidating the molecular mechanism by which the tetrapeptide SLLP improves osteoporosis.

[0072] 3.4.1 Total RNA extraction and reverse transcription Mouse femoral tissue stored at -80℃ was ground with liquid nitrogen, and Trizol reagent was added for complete lysis. Total RNA was extracted using the Trizol method. RNA concentration and purity were determined using Nanodrop 2000. RNA with an OD260 / OD280 ratio between 1.8 and 2.0 was used for subsequent reverse transcription experiments. Total RNA was reverse transcribed into cDNA using a reverse transcription kit and stored at -20℃ for later use.

[0073] 3.4.2 qPCR amplification qPCR amplification was performed using the SYBR Green quantitative PCR kit, with β-actin as the internal reference gene. The mRNA expression levels of osteogenic genes (Runx2, OCN, ALP) and osteoclast-related genes (RANKL, MMP9) were detected. The primer sequences are shown in Table 6.

[0074] Table 6. Primer sequences for qPCR amplification

[0075] 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. Three technical replicates were set up for each sample, and the experiment was independently repeated three times. (-ΔΔCt) The relative expression levels of genes in each group were calculated using this method.

[0076] 3. Experimental Results The results of osteogenic gene expression are shown in Table 7. Compared with the Sham group, the mRNA expression levels of Runx2, OCN, and ALP in the bone tissue of mice in the OVX model group were significantly downregulated (P<0.05). Compared with the OVX model group, the SLLP dose group upregulated the expression of the above-mentioned key osteogenic genes in a dose-dependent manner. Among them, the expression levels of each gene in the high-dose group were significantly higher than those in the model group (P<0.05), which was basically close to that of the Sham group.

[0077] The results of osteoclast-related gene expression are shown in Table 7. Compared with the Sham group, the mRNA expression levels of RANKL and MMP9 in the bone tissue of mice in the OVX model group were significantly upregulated (P<0.05). Compared with the OVX model group, the SLLP dose group downregulated the expression of the above osteoclast-related genes in a dose-dependent manner. Among them, the expression levels of RANKL and MMP9 in the high-dose group were only 22.43% and 19.67% of those in the model group, respectively, which was significantly different (P<0.05) and basically close to that of the Sham group.

[0078] The above results confirm that the tetrapeptide SLLP can regulate the RANKL pathway by targeting and regulating the expression of key genes for osteogenic differentiation such as Runx2, OCN, and ALP, thereby promoting osteoblast differentiation and new bone formation. On the other hand, it can downregulate the expression of key genes for osteoclast differentiation such as RANKL and MMP9, thereby inhibiting osteoclast activation and bone resorption. By regulating bone metabolism balance at the transcriptional level, it can play a core role in improving osteoporosis.

[0079] Table 7. Effects of SLLP on the relative expression levels of mRNAs of genes related to mouse bone tissue (Mean±SD, n=8)

[0080] 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. Application of bovine colostrum-derived bioactive peptide SLLP in the preparation of drugs for the prevention or treatment of osteoporosis.

2. The application according to claim 1, characterized in that, The prevention or treatment of osteoporosis includes at least one of the following: (a) Reversing inhibition of longitudinal bone growth; (b) Reversing osteoporotic bone microstructure damage; (c) Promotes mineral deposition in bones; (d) Regulate bone metabolism balance.

3. The application according to claim 2, characterized in that, The reversal of longitudinal bone growth inhibition includes: promoting tibial and femoral length growth; The reversal of osteoporotic bone microstructure damage includes: increasing bone mineral density, bone volume fraction, number of trabeculae, trabeculae thickness, and reducing trabeculae separation. The regulation of bone metabolism balance includes: promoting osteoblast differentiation and new bone formation, and inhibiting osteoclast activation and bone resorption.

4. A drug for the prevention or treatment of osteoporosis, characterized in that, It includes a therapeutically effective amount of bioactive peptides derived from bovine colostrum, wherein the amino acid sequence of the bioactive peptides is SLLP.

5. The drug according to claim 4, characterized in that, It also includes pharmaceutically acceptable carriers or excipients.

6. Application of SLLP, an active peptide derived from bovine colostrum, in the preparation of health foods for improving bone density.

7. A health food product for improving bone density, characterized in that, It contains bioactive peptides derived from bovine colostrum, wherein the amino acid sequence of the bioactive peptides is SLLP.

8. The health food according to claim 7, characterized in that, It also includes food science-acceptable carriers or additives.

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