Milk-derived polypeptide for promoting proliferation and differentiation of osteoblasts and application of milk-derived polypeptide
By preparing the milk-source bioactive peptides WLKPDPS and NE(pS)PEQTDDL, the risk of chemotherapy for osteoporosis and insufficient research on milk-source peptides has been solved, and safe and effective osteoblast proliferation and mineralization have been achieved, with wide application prospects.
Patent Information
- Application Number
- CN202510470637.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-07-25
AI Technical Summary
In the prior art, chemical drugs have certain risks in the treatment of osteoporosis, and there are few researches on promoting bone health in milk-derived polypeptides, and there is a lack of safe and effective natural alternatives.
Develop milk-source bioactive peptides WLKPDPS and NE(pS)PEQTDDL, which are prepared by solid phase synthesis, enzymatic lysis or microbial expression method, and are used to prepare food, medicine, health products or nutritional products to promote osteoblast proliferation and differentiation.
Milk-derived bioactive peptides significantly promote osteoblast proliferation and mineralization, have significant biosafety, and are suitable for the preparation of drugs for preventing osteoporosis, with wide application prospects.
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Figure CN120365404A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a milk-derived polypeptide that promotes the proliferation and differentiation of osteoblasts and its application, belonging to the technical field of bioactive peptides. Background Art
[0002] Bone mineral density mainly refers to the density of bone mineral content in unit volume of bone, which mainly reflects the toughness of bone. Improving bone mineral density is an important measure to maintain bone health, especially playing a significant role in preventing osteoporosis. Clinically, the treatment of osteoporosis still mainly relies on chemical drugs, but they often have certain risks, and long-term use may cause diseases such as gastrointestinal discomfort and cardiovascular diseases. Therefore, searching for safer and food-derived natural substitutes that can promote bone formation and reverse bone structure damage is receiving increasing attention.
[0003] Polypeptides are protein degradation products. It has been reported that a variety of bioactive peptides have functions such as regulating immunity, preventing cardiovascular diseases, and promoting bone health. These polypeptides can be obtained through various ways such as microbial fermentation, digestive enzyme hydrolysis, artificial synthesis by genetic engineering, and chemical synthesis. Most bioactive peptides are composed of 2 - 20 amino acid residues and are easily digested and absorbed by the human body. There is a rich variety of milk-derived polypeptides, but the research related to bone health is less. In 2007, Huttunen et al. reported that the milk tripeptide IPP has the function of promoting the proliferation, differentiation, and signal transduction of osteoblasts. However, compared with the rich milk-derived polypeptides, the research reports are still relatively limited.
[0004] Therefore, it is of great significance to explore milk-derived bioactive peptides that promote bone health and prevent osteoporosis. Summary of the Invention
[0005] To solve the above problems, the present invention provides a milk-derived bioactive peptide WLKPDPS and NE(pS)PEQTDDL, which has extremely high biological safety and the function of promoting the proliferation and differentiation of osteoblasts.
[0006] The first object of the present invention is to provide a milk-derived bioactive peptide, and the amino acid sequence of the milk-derived bioactive peptide is WLKPDPS or NESPEQTDDL.
[0007] In one embodiment, the serine in the milk-derived bioactive peptide NESPEQTDDL can also be phosphorylated serine.
[0008] In one embodiment, the milk-derived bioactive peptide is WLKPDPS or NE(pS)PEQTDDL.
[0009] In one embodiment, the preparation method of the milk-derived bioactive peptide includes solid-phase synthesis method, enzymatic hydrolysis method, and microbial expression method.
[0010] The second object of the present invention is to provide a food, medicine, health product or nutritional product, which contains an effective dose of any of the above-mentioned milk-derived bioactive peptides;
[0011] Optionally, the food, medicine, health product or nutritional product may also contain any of the above-mentioned milk-derived bioactive peptides; the derivatives of the milk-derived bioactive peptides refer to the milk-derived bioactive peptide derivatives obtained by hydroxylating, carbonylating, carboxylating, methylating, acetylating, phosphorylating, esterifying or glycosylating the amino acid side chain groups, amino terminus or carbonyl terminus of the milk-derived bioactive peptides.
[0012] In one embodiment, the medicine further contains pharmaceutically acceptable pharmaceutical excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.
[0013] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid esters and fatty acid glycerides; coloring agents such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc powder and polyethylene glycol; coating materials such as acrylic resin, hypromellose, polyvinylpyrrolidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents may be added to the composition;
[0014] Optionally, the dosage forms of the medicine include but are not limited to oral dosage forms, injection dosage forms, inhalation dosage forms;
[0015] Optionally, the oral dosage forms include but are not limited to tablets, capsules, granules, oral liquids, oral suspensions;
[0016] Optionally, the injection dosage forms include but are not limited to injection solutions, injection powder for injection;
[0017] Optionally, the inhalation dosage forms include but are not limited to aerosols, powder aerosols.
[0018] The third object of the present invention is to provide the use of any of the above-mentioned milk-derived bioactive peptides in the preparation of a food, medicine, health product or nutritional product, which is used to promote calcium absorption in the food, medicine, health product or nutritional product.
[0019] In one embodiment, the milk-derived bioactive peptides can also be used to prepare products for antioxidant, anti-inflammatory, cartilage repair and tissue regeneration (such as promoting bone matrix mineralization, enhancing bone density, accelerating bone tissue growth, shortening the fracture healing cycle), regulating metabolic and immune functions (such as regulating enzyme activity, enhancing immunity), preventing dental caries, and improving digestive function.
[0020] In one embodiment, the food includes, but is not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, beverages;
[0021] The food also includes special dietary foods;
[0022] The health products also contain acceptable excipients.
[0023] In one embodiment, the food, medicine, health product or nutritional product is used to promote calcium absorption.
[0024] In one embodiment, the medicine also contains pharmaceutically acceptable medicinal excipients; the medicinal excipients refer to conventional drug carriers in the pharmaceutical field.
[0025] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid esters and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc powder and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, polyvinylpyrrolidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can be added to the composition;
[0026] Optionally, the dosage form of the drug includes, but is not limited to, oral dosage forms, injection dosage forms, inhalation dosage forms;
[0027] Optionally, the oral dosage forms include, but are not limited to, tablets, capsules, granules, oral liquids, oral suspensions;
[0028] Optionally, the injection dosage forms include, but are not limited to, injection solutions, injection powder for injection;
[0029] Optionally, the inhalation dosage forms include, but are not limited to, aerosols, powder aerosols;
[0030] Optionally, the food includes, but is not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, beverages; the food also includes special dietary foods; the health products also contain acceptable excipients.
[0031] Advantages of the present invention
[0032] The milk-derived bioactive polypeptides WLKPDPS and NE(pS)PEQTDDL of the present invention have a significant effect on promoting the proliferation of osteoblasts and can significantly promote osteoblast mineralization, and are expected to be used in the preparation of drugs for promoting osteoblast proliferation and preventing osteoporosis.
[0033] The milk-derived bioactive polypeptides WLKPDPS and NE(pS)PEQTDDL of the present invention are convenient to synthesize and can be industrially produced, and have good application prospects in the fields of food, medicine, cosmetics, etc. Description of the drawings
[0034] Figure 1 It is the HPLC detection result of the milk-derived bioactive peptide WLKPDPS;
[0035] Figure 2 It is the LC-MS detection result of the milk-derived bioactive peptide WLKPDPS;
[0036] Figure 3 It is the HPLC detection result of the milk-derived bioactive peptide NE(pS)PEQTDDL;
[0037] Figure 4 It is the LC-MS detection result of the milk-derived bioactive peptide NE(pS)PEQTDDL;
[0038] Figure 5 It is the influence of the milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL on the proliferation of osteoblasts;
[0039] Figure 6 It is the influence of the milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL on the differentiation of osteoblasts;
[0040] Figure 7 It is the influence of the milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL on the related pathways of osteoblasts. Detailed implementation manners
[0041] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.
[0042] Raw materials used in the embodiments:
[0043] LPN was isolated from fresh milk, and the specific method was referred to the literature (Ma Ping, Sun Jie, Liu Ning. Isolation, purification and identification of osteopontin in milk [J]. Food and Fermentation Industries, 2008, (06): 135-139.);
[0044] Pepsin, pancreatin and bile salts were purchased from Sigma-Aldrich Company in the United States, and potassium chloride, sodium chloride and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd.
[0045] The milk-derived bioactive peptides used in the examples were all synthesized by Shanghai Kopeptide Biotechnology Co., Ltd.
[0046] Test method:
[0047] 1. HPLC method
[0048] High performance liquid chromatography (HPLC) was used. A Kromasil 100-5-C18 (4.6×250mm×5μm) chromatographic column was used, and 0.1% trifluoroacetic acid aqueous solution (A) and 0.1% trifluoroacetic acid acetonitrile solution (B) were used as the mobile phase. The total flow rate was 1 mL / min, and the detection wavelength was set at 214 nm. The sample was dissolved in ultrapure water, and the injection volume was 7 μL. The gradient elution program was as follows:
[0049] Time (min) Phase A (%) Phase B (%) 0.01 90 10 30 60 40 33 0 100 38 0 100 40 0 10 50 End
[0050] 2. LC-MS method
[0051] An electrospray ionization (ESI) interface was used. The detector voltage was set at -0.2 kV, the CDL temperature was 250 °C, the CDL voltage was 0 V, the block temperature was 200 °C, the atomizing gas flow rate was 1.5 L / min, the preset bias was +4.5 kV, and the T-Flow was 0.2 ml / min. The sample dissolution method was a mixed solvent of 15% acetonitrile (ACN) and 85% water (H2O), and the injection volume was 0.2 μL.
[0052] Example 1: Preparation of milk-derived bioactive peptides
[0053] To prepare milk-derived bioactive peptides, an in vitro simulated digestion was carried out using an infant model, and the steps were as follows:
[0054] (1) Gastric digestion stage:
[0055] Add 10 mg / mL LPN solution to the enzyme reactor for preheating, adjust the pH to 5.3, and then mix it with simulated gastric juice (pH 5.3, 724.3 U pepsin / mL, 13 mM potassium chloride, 94 mM sodium chloride) at a ratio of 63:37 (v / v); react at 37 °C and pH 5.3 for 60 min, and adjust the pH to 7.0 with 2 M sodium hydroxide to terminate the digestion at this stage to obtain the product of the gastric digestion stage;
[0056] (2) Intestinal digestion stage:
[0057] Adjust the pH of the product of the gastric digestion stage to 6.6, and then mix it with simulated intestinal fluid (pH 6.6, 42.1 U trypsin / mL, 8.2 mM bile salt, 10 mM potassium chloride, 249 mM sodium chloride) at a ratio of 62:38 (v / v); digest at 37 °C and pH 6.6 for 60 min, and terminate the intestinal digestion at 95 °C for 10 min to obtain the final digestion product.
[0058] (3) Polypeptide composition identification:
[0059] Analyze the final digestion product by LC-MS / MS equipped with an online nanoelectrospray ion source. The whole system is an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) in series with EASY-nanoLC 1200. A total of 5 μL of the sample is loaded (analysis column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample is separated with a 60-min gradient, the column flow rate is controlled at 300 nL / min, the column temperature is 40 °C, the electrospray voltage is 2 kV, the gradient starts from 4% of phase B, and is increased to 50% in a non-linear gradient within 53 minutes and 40 seconds, increased to 95% in 40 seconds, and maintained for 5 minutes and 40 seconds. The mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisitions.
[0060] The mass spectrometry parameters are set as follows: (1) MS: scanning range (m / z): 100 - 1500; resolution: 120,000; Normalized AGC target: 200%; maximum injection time: 100 ms; (2) HCD-MS / MS: resolution: 50,000; Normalized AGC target: 200%; maximum injection time: 86 ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30 s. The tandem mass spectrometry maps were analyzed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database was Uniprot-Bos taurus (version 2024, 26635 entries). The database search parameters were as follows: fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm, maximum missed cleavage: 2, fixed modification: Carbamidomethylation 57.02, variable modifications: Oxidation(M) 15.99, Deamidation(NQ) 0.98, Acetylation(Protein N-term) 42.01, Phosphorylation(STY) 79.97. The protein false discovery rate (FDR) cutoff was 1%, with at least 1 unique peptide; the peptide FDR cutoff was 1%.
[0061] A total of 242 peptides derived from LPN were identified. Molecular docking of the polypeptides was performed with BMPR1A as the target, and potential milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL were screened according to the binding energy.
[0062] Shanghai Kopeptide Biotech Co., Ltd. was commissioned to synthesize the milk-derived bioactive peptides WLKPDP, WLKPDPS, and NE(pS)PEQTDDL.
[0063] Example 2: Detection of milk-derived bioactive peptides
[0064] The milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL prepared in Example 1 were taken, and the detection results are as follows:
[0065] (1) HPLC detection results
[0066] The HPLC detection results are as Figure 1 (WLKPDPS) and Figure 3 (NE(pS)PEQTDDL) shown.
[0067] (2) LC-MS detection results
[0068] The LC-MS detection results are as follows Figure 2 (WLKPDPS) and Figure 4 (NE(pS)PEQTDDL) as shown.
[0069] (3) Promoting effect on osteoblasts
[0070] To detect the effects of milk-derived bioactive peptides VFTP (VP4), WLKPDP (WP6), WLKPDPS (WS7), and NE(pS)PEQTDDL (NL10) on the proliferation of osteoblasts, the steps are as follows:
[0071] MC3T3-E1 Subclone14 cells were seeded in 96-well plates at a density of 5000 cells / well. After incubation for 24 h, the samples were dissolved in α-MEM medium, filtered and sterilized with a 0.22 μm membrane, and the media containing 100 μg / mL IPP, 1, 10, 100 μg / mL VP4, WP6, WS7, NL10 were added to the wells respectively. After incubation for 24 h, the cell number was detected using the CCK-8 reagent.
[0072] The results are as follows Figure 5 shown. The results indicate that each sample has no significant effect on cell viability, suggesting that 100 μg / mL IPP, 1, 10, 100 μg / mL VP4, WP6, WS7, NL10 have no cytotoxicity to MC3T3-E1 Subclone14 cells.
[0073] (4) Effects on osteoblast differentiation
[0074] To detect the effect of milk-derived bioactive peptide WLKPDPS on osteoblast differentiation, the steps are as follows:
[0075] 1×10 5 cells / well were seeded in 12-well plates, and 1 mL of medium was added to each well. After 24 h, the medium was replaced with induction medium containing 100 μg / mL IPP, 100 μg / mL VP4, WP6, WS7, NL10 respectively, and the medium was changed every two days. After 3 days and 7 days of intervention, the cell culture supernatant was taken, and the ALP activity in the supernatant was measured using an ALP kit.
[0076] The results are as follows Figure 6As shown, the results indicate that after 3 days (left figure) and 7 days (right figure) of induced differentiation, compared with the control group and the IPP group, the activity of alkaline phosphatase in the cell culture supernatant of WLKPDPS increased significantly (different lowercase letters indicate significant differences between groups, p < 0.05); on the 3rd day, the ALP activity of the WS7 group was 803.06 ± 22.19 U / mL, and that of the NL10 group was 802.43 ± 53.39 U / mL, which were increased by 39.7% and 39.6% respectively compared with IPP. Similar results were also presented on the 7th day.
[0077] (5) Effects on osteoblast-related pathways
[0078] To detect the effects of the milk-derived bioactive peptide WLKPDPS on osteoblast differentiation, the steps are as follows:
[0079] Inoculate 1×10 5 cells / well into a 12-well plate, and add 1 mL of medium to each well. After 24 h, replace the medium with the induction medium containing 100 μg / mL WLKPDPS, and change the medium every two days. After 3 days of intervention, extract the cell RNA using an animal RNA extraction kit, reverse transcribe it into cDNA, and then measure the expression levels of related genes on the BMP / Smad pathway by QPCR.
[0080] The results are as Figure 7 shown. The results indicate that compared with the control group, WLKPDPS significantly up-regulated the mRNA expression levels of related Bmpr, Smad1, Smad5, and Runx2 by 558.9%, 58.2%, 30.1%, and 81.4% respectively, and NE(pS)PEQTDDL significantly up-regulated the mRNA expression levels of related Bmpr, Smad1, Smad5, and Runx2 by 533.9%, 32.3%, 10.6%, and 151.8% respectively.
[0081] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims
1. A milk source bioactive peptide, characterized in that, The amino acid sequence of the milk-derived bioactive peptide is WLKPDPS or NESPEQTDDL.
2. The milk source bioactive peptide according to claim 1, wherein The serine in the milk-derived bioactive peptide NESPEQTDDL can also be phosphorylated serine.
3. The milk source bioactive peptide according to any one of claims 1 to 2, characterized in that, The preparation methods of the milk-derived bioactive peptide include solid-phase synthesis, enzymatic hydrolysis, and microbial expression.
4. A food, drug, health product or nutritional product, characterized in that, The food, drug, health product, or nutritional product contains an effective dose of the milk-derived bioactive peptide according to any one of claims 1 to 3; Optionally, the food, drug, health product, or nutritional product may also contain the milk-derived bioactive peptide according to any one of claims 1 to 3; the derivatives of the milk-derived bioactive peptide refer to the milk-derived bioactive peptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification, or glycosylation modification on the amino acid side chain groups, amino terminus, or carbonyl terminus of the milk-derived bioactive peptide.
5. The food, medicine, health product or nutritional product according to claim 4, characterized in that, The drug also contains pharmaceutically acceptable pharmaceutical excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.
6. The food, drug, health product or nutritional product according to claim 5, characterized in that, The excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid esters, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hypromellose, polyvinylpyrrolidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition; Optionally, the dosage form of the drug includes, but is not limited to, oral dosage forms, injection dosage forms, and inhalation dosage forms; Optionally, the oral dosage forms include, but are not limited to, tablets, capsules, granules, oral liquids, and oral suspensions; Optionally, the injection dosage forms include, but are not limited to, injection solutions and injection powder injections; Optionally, the inhalation dosage forms include, but are not limited to, aerosols and powder aerosols.
7. The food, drug, health product or nutritional product according to claim 4, characterized in that, The food includes, but is not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soybean products, and beverages; The food also includes special dietary foods; The health product also contains acceptable excipients.
8. Use of the milk source bioactive peptide according to any one of claims 1 to 3 in the preparation of food, medicine, health care product or nutritional product, characterized in that, The food, drug, health product, or nutritional product is used to promote calcium absorption.
9. The application according to claim 8, characterized in that, The drug also contains pharmaceutically acceptable pharmaceutical excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.
10. The application according to claim 9, characterized in that, The excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dried starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbates, sorbitan fatty acid esters, and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resins, hypromellose, polyvinylpyrrolidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can be added to the composition. Optionally, the dosage form of the drug includes, but is not limited to, oral dosage forms, injection dosage forms, inhalation dosage forms. Optionally, the oral dosage forms include, but are not limited to, tablets, capsules, granules, oral liquids, oral suspensions. Optionally, the injection dosage forms include, but are not limited to, injection solutions, injection powder for injection. Optionally, the inhalation dosage forms include, but are not limited to, aerosols, powder inhalations. Optionally, the foods include, but are not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soybean products, beverages; the foods also include special dietary foods; the health products also contain acceptable excipients.