A milk-derived polypeptide arhphlsf for promoting osteoblast mineralization and application thereof

Abstract

The application discloses a milk-derived polypeptide ARHPHPHLSF for promoting osteoblast mineralization and application thereof, and belongs to the field of bioactive peptides. The milk-derived bioactive polypeptide ARHPHPHLSF has a significant effect of promoting osteoblast proliferation, can significantly promote osteoblast mineralization, and is expected to be used for preparing medicines for promoting osteoblast proliferation and preventing osteoporosis. The milk-derived bioactive polypeptide ARHPHPHLSF is convenient to synthesize, can be produced industrially, and has a good application prospect in the fields of medicines, cosmetics and the like.

Description

Technical Field

[0001] This invention relates to a milk-derived polypeptide ARHPHPHLSF that promotes osteoblast mineralization and its applications, belonging to the field of bioactive peptides. Background Technology

[0002] Osteoporosis is a systemic metabolic bone disease characterized by a decrease in overall bone mass and damage to the microstructure of bone tissue. In healthy individuals, bone metabolism is maintained through a balance between bone resorption and bone formation. However, endocrine disorders, poor calcium absorption, nutritional deficiencies, hormonal imbalances, aging, and numerous pathological processes disrupt this balance, ultimately leading to osteoporosis. Currently, clinical treatment for osteoporosis primarily relies on chemical drugs, but these often carry certain risks, with long-term use potentially causing diseases such as breast cancer and cardiovascular disease. Therefore, the search for safer, food-sourced, natural alternatives that can promote bone formation and reverse bone structural damage is receiving increasing attention.

[0003] Peptides are protein degradation products, and various bioactive peptides have been reported to have functions such as regulating immunity, preventing cardiovascular disease, and promoting bone health. These peptides can be obtained through microbial fermentation, digestive enzymatic hydrolysis, artificial synthesis via genetic engineering, and chemical synthesis. Most bioactive peptides consist of 2-20 amino acid residues and are easily digested and absorbed by the human body. In 2007, Huttunen et al. reported that the milk tripeptide IPP can promote osteoblast proliferation and mineralization; however, compared to the abundant milk-derived peptides, research reports are still relatively limited. Therefore, exploring milk-derived bioactive peptides that promote bone health and prevent osteoporosis is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a new application of bioactive peptides in promoting bone mineralization and / or preventing osteoporosis.

[0005] This invention provides the use of the bioactive peptide ARHPHPHLSF (amino acid sequence Ala-Arg-His-Pro-His-Pro-His-Leu-Ser-Phe) in the preparation of drugs that promote bone mineralization and / or prevent osteoporosis.

[0006] In one embodiment, the bioactive polypeptide is a milk-derived polypeptide derived from amino acid residues at positions 96-105 of β-casein; the amino acid sequence of the Kappa-casein is shown in SEQ ID NO:2.

[0007] In one embodiment, the drug further comprises a pharmaceutically acceptable carrier.

[0008] In one embodiment, the application includes, but is not limited to, promoting osteoblast proliferation and promoting osteoblast mineralization.

[0009] This invention also provides the application of the bioactive peptide ARHPHPHLSF in the preparation of health products that help improve bone density.

[0010] In one embodiment, the health product includes, but is not limited to, functional fermented dairy products. This invention also provides the application of the bioactive peptide ARHPHPHLSF in the preparation of biomimetic mineralization or bone repair materials.

[0011] This invention also provides a product that promotes bone cell mineralization, comprising the bioactive peptide ARHPHPHLSF or a derivative thereof; the derivative thereof refers to a peptide derivative obtained by modifying the amino acid side chain groups, amino terminus, or carbonyl terminus of the bioactive peptide ARHPHPHLSF with hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification, or glycosylation.

[0012] The beneficial effects are as follows: The milk-derived bioactive polypeptide ARHPHPHLSF of the present invention has a significant effect on promoting osteoblast proliferation and can significantly promote osteoblast mineralization, and is expected to be used in the preparation of drugs that promote osteoblast proliferation and prevent osteoporosis. The milk-derived bioactive polypeptide ARHPHPHLSF of the present invention is easy to synthesize and can be industrially produced, and has good application prospects in the fields of medicine and cosmetics. Attached Figure Description

[0013] Figure 1 HPLC identification results of the bioactive peptide ARHPHPHLSF.

[0014] Figure 2 LC-MS identification results of the bioactive peptide ARHPHPHLSF.

[0015] Figure 3 The effect of bioactive peptide ARHPHPHLSF on osteoblast proliferation. The effects of bioactive peptides IPP and ARHPHPHLSF at concentrations of 0.02, 0.2, and 2 µM on osteoblast proliferation were tested; CCK-8 assays were quantified by measuring absorbance at 450 nm using a microplate reader. n=3, data are expressed as mean ± SEM, and analysis was performed by one-dimensional ANOVA followed by Tukey multiple comparison assays. p <0.05.

[0016] Figure 4Effects of bioactive peptides IPP and ARHPHPHLSF on osteoblast mineralization: (a) Cells were cultured with ascorbic acid and sodium β-glycerophosphate and treated with different concentrations (0.02, 0.2, and 2 µM) of bioactive peptides IPP and ARHPHPHLSF. Cells were then stained with Alizarin Red and images were taken; (b) Absorbance at 562 nm was measured after destaining cells with cetylpyridinium chloride. Data are the average of three independent experiments ± SEM. p <0.05. Detailed Implementation

[0017] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0018] Example 1: Artificial Synthesis of Milk-Derived Bioactive Peptide ARHPHPHLSF 1. Resin activation: Starting from the first amino acid at the C-terminus, select 0.5 mmol / g of Fmoc-An-Resin to protect the amino acid and add it to a 150 mL solid-phase reactor. Then, add 50 mL of dichloromethane (DCM) to swell the resin for 30 min.

[0019] 2. Deprotection: After soaking the resin for 30 minutes, remove the solution and wash three times with nitrogen-dimethylamide (DMF). Add a 20% (v / v) hexahydropyridine DMF solution and react for 5 minutes. Add another 20% (v / v) hexahydropyridine DMF solution and react for 10 minutes, washing once with DMF in between. After the reaction is complete, remove the solution and wash three times with DMF. Then, perform a deprotection reaction with piperidine for 30 minutes.

[0020] 3. Deprotection: After deprotection for 30 minutes, remove piperidine and wash 6 times with DMF. After washing 6 times, use ninhydrin to detect the deprotection color. If there is color, it means that deprotection is successful.

[0021] 5. Feeding: After washing and deprotection, and after testing, add the weighed Ala and 1-timo-benzotriazole (HOBT) in the order of ARHPHPHLSF peptides, then add 20 ml of DMF to dissolve them, and then add 3 ml of N,N-diisopropylcarbodiimide (DIC). Mix well and add to the reactor to react for 30 minutes.

[0022] 6. Detection: After reacting for 30 minutes, use a certain amount of acetic anhydride to cap the reaction mixture (acetic anhydride: DIE A: DCM = 1:1:2, v:v:v) for 30 minutes. Then remove the reaction solution, wash with DMF 3 times, and test to check whether the reaction is complete.

[0023] 7. After confirming successful ligation, repeat steps 2-6, sequentially ligating Arg-His-Pro-His-Pro-His-Leu-Ser-Phe. If the test fails, soak the amino acid in DCM, then continue ligating the amino acid from the previous step without deprotection until successful ligation. After ligating the last amino acid, deprotect the amino acid, wash four times with DMF, and then dry the resin with methanol. Slowly add the prepared lysis buffer (TFA: anisole sulfide: phenol: triisopropylsilane: water = 82.5: 7.5: 5: 3: 2) at 0°C with slow stirring. React at low temperature for 0.5 hours, then at room temperature for 2 hours. Filter to obtain the lysis buffer, slowly add the lysis buffer to anhydrous ice-cold ether while stirring, filter to separate the crude peptide, wash three times with ice-cold ether to obtain the crude peptide.

[0024] 8. Purification: Take a small amount of sample and dissolve it by sonication. After dissolution, take 20 μL of sample. Analyze using an analytical high-performance liquid chromatograph (HPLC) with a gradient of 10-100 and a time of 0-25 minutes. Pump A uses 100% acetonitrile plus 0.1% TFA, and pump B uses 100% water plus 0.1% TFA. Inject the sample and analyze. After finding the target peak, confirm the mass spectrometry to ensure it is correct. After confirming the target peak, provide the corresponding gradient for preparation. The mobile phase and analysis time of the preparative HPLC should be the same. After the target peak appears, confirm the mass spectrometry. After confirming the mass spectrometry, provide the corresponding gradient for analysis. After the analysis is qualified, freeze-dry the sample.

[0025] The synthesized bioactive peptides were identified using UPLC and LC-MS.

[0026] (1) UPLC identification The UPLC conditions are as follows: Instrument: Wasters ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer; Column: Gemini-NX 5μ C18 110A, 4.6 250 mm; Flow rate: 1.0 mL / min; Temperature: 50℃; Ultraviolet detection wavelength: 220 nm; Injection volume: 20 μL; Gradient conditions: Solution A: Acetonitrile containing 0.1% formic acid (v / v); Solution B: Water containing 0.1% formic acid (v / v);

[0027] (2) Liquid chromatography-mass spectrometry The LC-MS conditions are as follows: Instrument: Agilent liquid chromatography-mass spectrometry system; Detector: ESI; Atomizer flow rate: 1.5 L / min; CDL: -20.0v; CDL temperature: 250℃; Block temperature: 200℃; Probe voltage: +4.5 kV; Detector voltage: 1.5 kV; Mobile phase: 50% water / 50% acetonitrile; Flow rate: 0.2 mL / min; Based on the above analytical methods, the bioactive peptide ARHPHPHLSF was analyzed by chromatographic and mass spectrometric analysis using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-MS / MS) and liquid chromatography-mass spectrometry (LC-MS / MS). The mass chromatogram is shown below. Figure 1 As shown, the retention time is as follows Figure 2 As shown, the peptide mass-to-charge ratio of this peak is 1198.7 Da, and the retention time is 11.791 min.

[0028] Example 2: Effects of milk-derived bioactive peptides on osteoblast proliferation Mouse embryonic osteoblast precursor cells MC3T3-E1 were seeded in 96-well plates at a density of 2 × 10⁶ cells / well. 4 Cells / well. After incubation at 37°C for 24 h, α-MEM complete medium containing 0, 0.02, 0.2, and 2 μmol / L bioactive peptides IPP and ARHPHPHLSF were added respectively. After incubation at 37°C for 24 h, 10 μL of CCK-8 was added to each well under aseptic conditions, and the cells were incubated for 30 min. The cells were then shaken well and the absorbance was measured at 450 nm using a microplate reader.

[0029] like Figure 3 As shown, milk-derived peptides IPP and ARHPHPHLSF can promote osteoblast proliferation, and the effect increases with increasing dosage. However, the effect of ARHPHPHLSF is significantly higher than that of IPP. Furthermore, milk-derived peptides ARHPHPHLSF at concentrations of 0.02, 0.2, and 2 μmol / L increased the proliferation of MC3T3-E1 cells by 10%, 23%, and 27%, respectively.

[0030] Example 3: Effect of bioactive peptide ARHPHPHLSF on osteoblast mineralization Calcium deposition reflects the level of osteogenic differentiation in cells; the greater the calcium deposition, the higher the level of osteogenic differentiation. Alizarin red chelates with calcium ions to form a deep red or purplish-red complex, which is commonly used to observe mineralized calcium nodules in osteoblasts. The alizarin red dye solution and the calcium ion complex can be dissolved in 10% hexadecylpyridine chloride for quantitative analysis of calcium nodules. The absorbance values ​​of stained calcium nodules and extracted samples are proportional.

[0031] MC3T3-E1 cells were divided into 1×10 5 Cells were seeded in 12-well plates, with 1 mL of α-MEM complete medium added to each well. The next day, the medium was replaced with α-MEM (10% FBS, 50 μg / mL L-ascorbic acid, and 10 mM sodium β-glycerophosphate) induction medium containing different concentrations (0, 0.02, 0.2, 2 μmol / L) of bioactive peptides IPP and ARHPHPHLSF. After 21 days, cells were fixed with 4% paraformaldehyde, stained with 1% Alizarin Red S, and incubated at 37°C for 30 min. After incubation, cells were rinsed and photographed under a microscope. 1 mL of 10% hexadecyl chloropyridine was added to each well, and after incubation at room temperature for 1 hour, the OD value at 562 nm was measured.

[0032] like Figure 4 As shown, milk-derived peptides IPP and ARHPHPHLSF can promote the formation of mineralized nodules in osteoblasts, and the effect increases with increasing dosage. However, the effect of ARHPHPHLSF is significantly higher than that of IPP. Furthermore, the effects of milk-derived peptide ARHPHPHLSF at concentrations of 0.02, 0.2, and 2 μmol / L increased by 15%, 46%, and 63%, respectively.

[0033] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Application of bioactive peptide ARHPHPHLSF in the preparation of drugs for the prevention of osteoporosis.

2. The application according to claim 1, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

3. Application of bioactive peptide ARHPHPHLSF in the preparation of health products that help improve bone density.

4. The application according to claim 3, characterized in that, The health products mentioned include functional fermented dairy products.

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