Milk-derived polypeptide sequence for promoting proliferation of skeletal muscle cells and use thereof

Abstract

The application discloses a milk-derived polypeptide sequence for promoting proliferation of skeletal muscle cells and application thereof, and belongs to the field of bioactive peptides. The milk-derived bioactive polypeptide RHPHPHLSF provided by the application has the effect of promoting proliferation of muscle cells, and is expected to be used for preparing medicines for promoting proliferation of muscle cells and preventing sarcopenia. The milk-derived bioactive polypeptide RHPHPHLSF is convenient to synthesize, can be produced industrially, and has a good application prospect in the fields of food, medicines and cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to a milk-derived polypeptide sequence for promoting proliferation of skeletal muscle cells and application thereof, and belongs to the field of bioactive peptides. BACKGROUND

[0002] Sarcopenia, also known as muscle loss, is a progressive, systemic skeletal muscle disease associated with aging, mainly manifested as reduced muscle mass, decreased muscle strength and decreased muscle physiological function.

[0003] Sarcopenia is characterized by loss of muscle mass and impaired muscle function, including reduced muscle cross-sectional area and muscle strength due to aging, while the body is not suffering from disease. At the same time, sarcopenia is usually accompanied by metabolic abnormalities, including impaired insulin sensitivity, impaired oxidative defense, and decreased mitochondrial function. In order to slow down muscle loss, various treatment methods have emerged. The usual treatment for sarcopenia is to regulate hormone levels. It has been proven that administration of growth hormone and / or testosterone can alleviate muscle loss by improving certain muscle functions in elderly men. However, testosterone administration is associated with a greater risk of polycythemia, venous thromboembolism, prostate cancer and cardiovascular events. Therefore, there is increasing interest in finding safer, food-derived natural alternatives that can promote muscle proliferation and alleviate muscle damage.

[0004] In recent years, bioactive peptides and proteins are expected to make further progress in the treatment of muscle abnormalities. For example, Kitakaze et al. revealed that lactoferrin promotes C2C12 proliferation and differentiation, and myotube hypertrophy. And Iwasa et al. determined that a milk casein hydrolysis-derived peptide promotes glucose uptake in skeletal muscle cells and optimizes human metabolism. However, compared to the rich milk-derived polypeptides, the research reports are still limited. Therefore, it is meaningful to explore milk-derived bioactive peptides that promote skeletal muscle health and prevent muscle loss. SUMMARY

[0005] The purpose of the present application is to provide a new use of bioactive peptides in promoting proliferation of skeletal muscle cells and / or preventing muscle loss.

[0006] The present application provides a bioactive peptide: RHPHPHLSF (9 peptides, the amino acid sequence is Arg-His-Pro-His-Pro-His-Leu-Ser-Phe), ARHPHPHLSF (10 peptides, the amino acid sequence is Ala-Arg-His-Pro-His-Pro-His-Leu-Ser-Phe).

[0007] In one embodiment of the present application, the bioactive polypeptide is a milk-derived polypeptide, ARHPHPHLSF (10 peptides, SEQ ID NO. 2) and RHPHPHLSF (9 peptides, SEQ ID NO. 3) are derived from kappa-casein, located at 96-105 and 97-105, respectively.

[0008] The amino acid sequence of the kappa-casein is shown in SEQ ID NO. 1.

[0009] Kappa-casein (SEQ ID NO. 1)

[0010] MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTM ARHPHPHLSF MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV.

[0011] In one embodiment of the present application, the bioactive polypeptide is used as a food, a drug or a health product; it is used to promote skeletal muscle cell proliferation and muscle cell growth.

[0012] Preferably, the food, drug, health product or nutritional product can also contain a derivative of the polypeptide; the derivative of the polypeptide refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, the amino terminal or the carbonyl terminal of the polypeptide;

[0013] The drug also contains a pharmaceutically acceptable pharmaceutical excipient; the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field;

[0014] Preferably, the excipient includes one or more of the following: a binder such as a cellulose derivative, an alginate, a gelatin and a polyvinylpyrrolidone; a diluent such as starch, pre-gelatinized starch, dextrin, sucrose, lactose, mannitol; a filler such as starch, sucrose; a humectant such as glycerol; a disintegrant such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; an absorption promoter such as a quaternary ammonium compound; a surfactant such as polysorbate, sorbitan fatty acid and fatty acid glyceride; a colorant such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red and the like; a lubricant such as hydrogenated vegetable oil, talc and polyethylene glycol; a coating material such as an acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate phthalate; in addition, other auxiliary agents such as a flavoring agent, a sweetener can also be added in the composition;

[0015] Preferably, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, inhalation dosage form;

[0016] Preferably, the oral dosage form includes, but is not limited to, tablet, capsule, granule, oral liquid, oral suspension;

[0017] Preferably, the injection dosage form includes, but is not limited to, injection liquid, injection powder;

[0018] Preferably, the inhalation dosage form includes, but is not limited to, aerosol, powder aerosol.

[0019] Preferably, the food includes, but is not limited to, cereal product, vegetable product, fruit product, meat product, seafood product, egg product, dairy product, bean product, beverage; the food also includes special dietary food;

[0020] The health product also contains acceptable adjuvant.

[0021] The present application also provides an expression vector or recombinant microorganism, which contains the above-mentioned ARHPHPHLSF peptide and / or RHPHPHLSF peptide.

[0022] Preferably, the vector is selected from DNA vector, RNA vector, plasmid, transposon vector, CRISPR / Cas9 vector, or viral vector;

[0023] Preferably, the recombinant microorganism is bacteria or fungi.

[0024] The present application also provides a food, drug, health product or nutritional product, which contains an effective dose of at least one of the above-mentioned polypeptides;

[0025] Preferably, the food, drug, health product or nutritional product can also contain a derivative of the polypeptide; the derivative of the polypeptide refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino terminal or carbonyl terminal of the polypeptide.

[0026] In an embodiment of the present application, the drug also contains pharmaceutically acceptable pharmaceutical adjuvant; the pharmaceutical adjuvant refers to conventional drug carrier in the field of pharmacy;

[0027] Preferably, the adjuvants 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; humectants such as glycerin; 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 and fatty acid glyceride; colorants such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red and the like; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; other adjuvants such as flavoring agents, sweeteners can also be added to the composition;

[0028] Preferably, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, inhalation dosage form;

[0029] Preferably, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, oral suspensions;

[0030] Preferably, the injection dosage form includes, but is not limited to, injection liquid, injection powder;

[0031] Preferably, the inhalation dosage form includes, but is not limited to, aerosol, powder aerosol.

[0032] In an embodiment of the present application, the food includes, but is not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, bean products, beverages; the food also includes special dietary foods;

[0033] The health products also contain acceptable adjuvants.

[0034] The present application also provides the use of the above-mentioned ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of food, drugs, health products or nutritional products; the food, drugs, health products or nutritional products are used to promote skeletal muscle cell proliferation and promote muscle cell growth.

[0035] In an embodiment of the present application, the drug also contains a pharmaceutically acceptable carrier.

[0036] In an embodiment of the present application, the use includes, but is not limited to, promoting muscle cell proliferation.

[0037] The drug also contains a pharmaceutically acceptable pharmaceutical adjuvant; the pharmaceutical adjuvant refers to the conventional drug carrier in the pharmaceutical field;

[0038] Preferably, the adjuvants 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; humectants such as glycerin; 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 and fatty acid glyceride; colorants such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red and the like; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; other adjuvants such as flavoring agents, sweeteners can also be added to the composition;

[0039] Preferably, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, inhalation dosage form;

[0040] Preferably, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, oral suspensions;

[0041] Preferably, the injection dosage form includes, but is not limited to, injection liquid, injection powder;

[0042] Preferably, the inhalation dosage form includes, but is not limited to, aerosol, powder aerosol.

[0043] In an embodiment of the present application, the food includes, but is not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, bean products, beverages; the food also includes special dietary foods;

[0044] The health care product also contains acceptable adjuvants.

[0045] The present application also provides the use of the bioactive peptide RHPHPHLSF and / or ARHPHPHLSF in the preparation of a health care product for improving muscle mass.

[0046] In an embodiment of the present application, the health care product includes, but is not limited to, functional fermented dairy products.

[0047] The present application also provides the use of the bioactive peptide RHPHPHLSF and / or ARHPHPHLSF in the preparation of a drug for promoting skeletal muscle cell proliferation and / or preventing sarcopenia.

[0048] In an embodiment of the present application, the drug also contains a pharmaceutically acceptable carrier.

[0049] In an embodiment of the present application, the use includes, but is not limited to, promoting muscle cell proliferation.

[0050] The medicine also contains pharmaceutically acceptable pharmaceutical adjuvants; the pharmaceutical adjuvants refer to conventional pharmaceutical carriers in the pharmaceutical field;

[0051] Preferably, the adjuvants include one or more of the following: binders such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; humectants 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 and fatty acid glyceride; colorants such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red and the like; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; other adjuvants such as flavoring agents, sweeteners can also be added to the composition;

[0052] Preferably, the dosage form of the medicine includes, but is not limited to, oral dosage form, injection dosage form, inhalation dosage form;

[0053] Preferably, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, oral suspensions;

[0054] Preferably, the injection dosage form includes, but is not limited to, injection liquid, injection powder;

[0055] Preferably, the inhalation dosage form includes, but is not limited to, aerosol, powder aerosol.

[0056] The present application also provides a product for promoting muscle cell proliferation, which includes the bioactive peptide RHPHPHLSF or ARHPHPHLSF peptide or a derivative of the bioactive polypeptide RHPHPHLSF or ARHPHPHLSF peptide;

[0057] In an embodiment of the present application, the product for promoting muscle cell proliferation includes a health product for promoting muscle mass increase and a medicine for promoting muscle cell proliferation; the derivative of the bioactive polypeptide RHPHPHLSF or ARHPHPHLSF peptide refers to a polypeptide derivative obtained by modifying the amino acid side chain group, the amino terminal or the carbonyl terminal of the bioactive polypeptide RHPHPHLSF or ARHPHPHLSF peptide by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation.

[0058] Beneficial effects

[0059] The milk-derived bioactive polypeptide RHPHPHLSF of the application has a significant effect of promoting proliferation of skeletal muscle cells, and is expected to be used for preparing a drug or functional food for promoting proliferation of skeletal muscle cells and preventing sarcopenia. The RHPHPHLSF is convenient to synthesize and can be produced industrially, and has a good application prospect in the fields of food, medicine and cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 : Schematic diagram of screening whey peptide and FGFR1 receptor molecule docking. Pymol 1.5 and LigPlot were used to show the specific binding site of the polypeptide and FGFR1. The interaction force includes hydrogen bond combination and hydrophobic interaction force.

[0061] Figure 2 : Effect of polypeptide on proliferation of skeletal muscle cells. The effect of bioactive peptide RHPHPHLSF at a concentration of 0.1 μM on the proliferation of skeletal muscle cells was tested; the absorbance at 450 nm was measured by a microplate reader to quantify the CCK-8 assay. n = 3, data are expressed as mean ± SEM, and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p < 0.05).

[0062] Figure 3 : EdU proliferation staining results of polypeptide. (a) Cells were treated with a modified version of DMEM medium and different concentrations of 0.1 μM bioactive peptide RHPHPHLSF. Then, the cells were stained with EdU staining solution, and images were taken with an inverted fluorescence microscope; (b) The number of positive cells was counted using image software, and the data were the mean ± SEM from 9 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the control group.

[0063] Figure 4 : Effect of polypeptide on muscle cell cycle. The effect of bioactive peptide RHPHPHLSF at a concentration of 0.1 μM on the cell cycle of skeletal muscle cells was tested; the number of cells at each stage was collected and analyzed by flow cytometry, and plotted using Modefit5, and the data were analyzed. n = 3, data are expressed as mean ± SEM, and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p < 0.05).

[0064] Figure 5Effect of polypeptide on muscle cell ROS content. The effect of bioactive peptide RHPHPHLSF at a concentration of 0.1 μM on skeletal muscle cell oxidative damage was tested; the relative content of ROS in the cells was quantified by measuring the absorbance at 532 nm with a microplate reader. n = 3, data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p < 0.05). **** p < 0.0001 compared with the control group.

[0065] Figure 6 Effect of polypeptide on muscle cell MDA content. The effect of bioactive peptide RHPHPHLSF at a concentration of 0.1 μM on skeletal muscle cell oxidative damage was tested; the MDA content in the cells was quantified by measuring the absorbance at 532 nm with a microplate reader and normalized with the BCA protein content. n = 3, data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p < 0.05). * p < 0.05, ** p < 0.01 compared with the control group. DETAILED DESCRIPTION

[0066] The EdU working solution and cell cycle detection kit used in the following examples were purchased from Wuhan Savi Biological Technology Co., Ltd. and Shanghai Biyun Tian Biological Technology Co., Ltd., respectively.

[0067] The LC-MS / MS analysis method used in the following examples is as follows:

[0068] LC-MS / MS analysis was performed using an EASY nLC1200 system equipped with an Acclaim PepMap™ RSLC column (50 μm x 15 cm, 2 μm, 100 μm). Mobile phase A included a solution of water containing 0.1% (v / v) formic acid and 2% (v / v) acetonitrile, and mobile phase B was a solution of water containing 0.1% (v / v) formic acid and 90% (v / v) acetonitrile. The gradient elution program was: 6% to 20% B, 0-40 min; 20% to 32% B, 40-52 min; 32% to 80% B, 52-56 min, at a flow rate of 0.2 μL / min. The MS spectrum acquisition range was 150-2000 m / z, and the resolution was 60000. The MS / MS data collection range was 180-2000 m / z, and the scan time was 0.1 s.

[0069] The active peptide sequence identification method used in the following examples is as follows:

[0070] Peptide identification was performed using bovine proteins from the UniProt database (https: / / www.uniprot.org / ) as a reference database. Raw data were searched using Maxquant (v.1.5.2.8) in the non-specific enzyme cleavage mode with a mass tolerance of 0.02 Da, a tolerance of 10 ppm for in silico synthesis, and a tolerance of 7 ppm for isotope removal in MS / MS. The protein and peptide segment matching false discovery rate (FDR) threshold was set to 1%. In order to find bioactive peptides, three repeated analyses were performed for each sample, and if a peptide appeared at least twice, it was considered to be significant.

[0071] The method for screening the ability of whey peptides to dock with FGFR1 receptor molecules involved in the following examples:

[0072] For virtual screening and molecular docking, the molecular structure of FGFR1 receptor (PDB ID: 1EVT) was downloaded from the RCSB protein database (https: / / www.rcsb.org / ). Water molecules and irrelevant atoms were removed before docking. Peptide structures were constructed and energy minimized using Discovery Studio 2019. Virtual screening was performed using AutoDock Vina 1.2. The docking method was referred to, the center position of the box was set to -10.083, 2.000, -12.444, the size was 120x120x120, and other parameters were kept at default values. The conformation with the highest affinity was selected as the result. Under the same conditions, the binding mode and direction of FGFR1 receptor were analyzed and selected. The first three conformations of each peptide docking with FGFR1 were generated, and the best conformation was selected based on the docking results for further analysis. Discovery Studio (DS) 2019, Pymol 1.5 and LigPlot were used.

[0073] The culture medium involved in the following examples is as follows:

[0074] DMEM complete medium: purchased from Thermo Fisher Scientific (China) Co., Ltd. in Shanghai, China.

[0075] Example 1: Preparation of bioactive polypeptides

[0076] 1. Isolation and preparation of polypeptides

[0077] (1) Preparation of fermented whey

[0078] The purified strain CCFM1263 (described in the Chinese invention patent application document with publication number CN117305158A) was subcultured on a first generation MRS solid plate and two generations of MRS liquid tube, centrifuged at 6000 x g for 5 min, resuspended with 11% (w / w) skim milk, inoculated into 11% (w / w) skim milk at an inoculum of 2% (v / v), shaken uniformly, and then cultured in an anaerobic incubator at 37°C for 18 h. Then, the same method was used for the skim milk subculture, and after being cultured in an anaerobic incubator at 37°C for 18 h, the protein was washed with Tris acid, and the viable cell count was adjusted to 1 x 10 6 CFU / mL, inoculated into 11% (w / w) skim milk at an inoculum of 2% (v / v), and fermented in a constant temperature incubator at 37°C for 48 h.

[0079] Different fermented milk samples were taken, 1 mol / L sodium hydroxide solution was added to adjust the sample pH to 4.6, and then pasteurized in a high-pressure steam sterilization pot at 95°C for 10 min. The sterilized fermented milk sample was centrifuged at 10000 x g at 4°C for 10 min, and the supernatant was taken and filtered with a 0.45 μm organic filter membrane to obtain the Lactobacillus helveticus fermented milk whey sample. The collected Lactobacillus helveticus fermented milk whey was freeze-dried into powder and stored in a freeze-dried bottle at 4°C for standby use.

[0080] (2) The C18 solid phase extraction column was used for desalting and purification of the fermented whey. The C18 solid phase extraction desalting column was used for desalting and purification of the sample, and the loading amount was 3 mg. It was divided into five steps of wetting, leveling, loading, desalting, and washing, and a constant pressure pump was used to control the flow rate.

[0081] (3) After purification, the peptide solution was dried using a cold centrifugal vacuum concentrator, and reconstituted in an aqueous solution consisting of 0.1% (v / v) formic acid and 2% (v / v) acetonitrile to achieve a peptide concentration of 0.5 mg / mL. After stirring the solution at 12000g and 4°C for 15 min, the whey was collected and stored in a sample vial, and then subjected to LC-MS / MS analysis.

[0082] The sequence of the peptide in the mixed solution was analyzed.

[0083] 2. Identification of active peptide sequences, screening of whey peptides for docking ability with FGFR1 receptor molecules

[0084] The sequence of the peptide obtained in step 1 was identified, and the docking ability of the whey peptide with the FGFR1 receptor molecule was screened.

[0085] The results are shown in Table 1

[0086] Table 1: Results of molecular docking

[0087]

[0088]

[0089] The results show that: the lower the binding energy, the better the effect of binding with the receptor. Ultimately, two groups of polypeptides with better virtual binding effect with the receptor are obtained, ARHPHPHLSF and RHPHPHLSF. The schematic diagram of the docking of the two groups of polypeptides with FGFR1 molecules is shown in Figure 1 .

[0090] Example 2: Effect of bioactive polypeptides on muscle cells

[0091] 1. Effect on muscle cell proliferation

[0092] The specific method is as follows:

[0093] (1) Chemically synthesized by Shanghai Gil Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder, respectively.

[0094] (2) ARHPHPHLSF and RHPHPHLSF polypeptide powders obtained in step (1) are added to DMEM complete medium respectively, so that the final concentration of polypeptides in the medium is 0.001 μmol / L, 0.01 μmol / L and 0.1 μmol / L respectively. Finally, DMEM complete medium containing 0.001 μmol / L, 0.01 μmol / L and 0.1 μmol / L bioactive peptide ARHPHPHLSF is prepared;

[0095] DMEM complete medium containing 0.001 μmol / L, 0.01 μmol / L and 0.1 μmol / L bioactive peptide RHPHPHLSF.

[0096] (3) Mouse myoblast C2C12 cells are inoculated in a 96-well plate at a seeding amount of 2×10 4 cells / well. After 24 h of culture at 37°C, DMEM complete medium containing 0 μmol / L, 0.001 μmol / L, 0.01 μmol / L and 0.1 μmol / L bioactive peptide ARHPHPHLSF (named ARH10) and RHPHPHLSF (named RH9) is added, respectively. After 24 h of culture at 37°C, 10 μL of CCK-8 solution is added to each well under sterile conditions, and the incubator (37°C constant temperature) is cultured for 30 min. Shake well, and measure the absorbance value at 450 nm with a microplate reader.

[0097] The results are shown in Table 2 and Figure 2 .

[0098] Table 2: Effect of different concentrations of polypeptides on muscle cell proliferation

[0099]

[0100]

[0101] Results show that milk-derived peptide RHPHPHLSF can promote the proliferation of myoblast cells, and increase with the increase of dose, and the proliferation rate reaches 127% when the concentration of milk-derived peptide RHPHPHLSF is 0.1 μmol / L. ARHPHPHLSF can also promote the proliferation of myoblast cells, and increase with the increase of dose.

[0102] 2. Effect on EdU proliferation staining of muscle cells

[0103] The specific method is as follows:

[0104] (1) Chemically synthesized by Shanghai Gilmore Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder respectively.

[0105] (2) Add ARHPHPHLSF polypeptide and RHPHPHLSF polypeptide powder obtained in step (1) to DMEM complete medium respectively, so that the final concentration of polypeptide in the medium is 0.1 μmol / L. Prepare DMEM complete medium containing ARHPHPHLSF polypeptide with a final concentration of 0.1 μmol / L and DMEM complete medium containing RHPHPHLSF polypeptide with a final concentration of 0.1 μmol / L.

[0106] (3) Seed mouse myoblast C2C12 cells in a 96-well plate at a seeding amount of 2×10 4 cells / well, and incubate at 37°C for 24 h. Then add DMEM complete medium containing 0 μmol / L and 0.1 μmol / L of bioactive peptide ARHPHPHLSF (named ARH10) and RHPHPHLSF (named RH9) respectively, and incubate at 37°C for 24 h.

[0107] (4) Add 500 μL of 37°C preheated EdU working solution to the intervened cells and incubate for 2 h. Discard the EdU working solution, add 1 mL of fixing solution, and fix at room temperature for 15 min. Discard the fixing solution and wash with PBS containing 5% BSA for 3 times. Add 1 mL of 0.3% Triton X-100 per well and incubate at room temperature for 15 min. Discard Triton X-100 and wash with washing solution for 3 times. Add 500 μL of Click reaction solution and incubate at room temperature for 30 min in the dark. Discard the Click reaction solution and wash with washing solution for 3 times. Add 1 mL of Hoechsty solution and incubate at room temperature for 10 min in the dark. Discard the Hoechsty solution and wash with washing solution for 3 times. Collect images under fluorescence microscope.

[0108] Results are shown in Table 3 and Figure 3

[0109] Table 3: Effect of different concentrations of polypeptides on the proportion of positive cells in muscle cells

[0110]

[0111] Results are shown in Table 3 and Figure 3 RHPHPHLSF can significantly increase the proportion of positive cells in muscle cells, and the proliferation effect is significantly higher than that of ARHPHPHLSF on C2C12. When the concentration of RHPHPHLSF is 0.1 μmol / L, the proportion of positive cells is increased by 20.11% compared with the normal group.

[0112] 3. Effect on the growth cycle of muscle cells

[0113] The specific method is as follows:

[0114] (1) Chemically synthesized by Shanghai Gilmore Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder, respectively.

[0115] (2) ARHPHPHLSF polypeptide and RHPHPHLSF polypeptide powder obtained in step (1) are added to DMEM complete medium respectively, so that the final concentration of polypeptide in the medium is 0.1 μmol / L. DMEM complete medium containing ARHPHPHLSF polypeptide with a final concentration of 0.1 μmol / L and DMEM complete medium containing RHPHPHLSF polypeptide with a final concentration of 0.1 μmol / L are prepared.

[0116] (3) Mouse myoblast C2C12 cells are inoculated in a 12-well plate at a seeding amount of 1 x 10 5 After 24 h of culture at 37℃, 0 μmol / L, 0.1 μmol / L of bioactive peptide ARHPHPHLSF (named ARH10) and RHPHPHLSF (named RH9) are added respectively, and cultured at 37℃ for 24 h.

[0117] ​(4) Discard the culture medium in the culture plate, and after lysing the cells with the lysis solution, collect the cells into a 1.5 mL centrifuge tube, discard the supernatant after centrifugation, add pre-cooled 0.5 mL PBS buffer solution to dissolve the precipitate, discard the supernatant after centrifugation, and repeat the washing with PBS solution for 2-3 times. Then add propidium iodide (PI) staining solution to disperse the cells, and then stain overnight at 4°C in the dark (more than 24 h). Finally, analyze the fluorescence of the cells using a flow cytometer. The DNA signal represented by the red fluorescence staining intensity after PI staining is collected by computer, and the forward scatter (FSC) and side scatter (SSC) data are real-time collected. All data are analyzed using Modefit5.

[0118] The results are shown in Table 4 and Figure 4

[0119] Table 4: Effect of different polypeptides on muscle cell cycle

[0120]

[0121] The results are shown in Table 4 and Figure 4 As shown in Table 4, the milk-derived RHPHPHLSF can accelerate the progress of muscle cell cycle. When the concentration of the milk-derived peptide RHPHPHLSF is 0.1 μmol / L, the number of G0 / G1 phase cells is significantly reduced by 5.6% compared with the normal group, and the number of S phase cells is significantly increased by 5.71%, indicating that RHPHPHLSF treatment accelerates the progress of cell cycle and promotes cell growth.

[0122] 4. Determination of ROS content in muscle cells

[0123] The specific method is as follows:

[0124] (1) Chemically synthesized by Shanghai Gil Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder, respectively.

[0125] (2) Add ARHPHPHLSF and RHPHPHLSF polypeptide powder obtained in step (1) to DMEM complete medium to make the final concentration of polypeptide in the medium 0.1 μmol / L. At the same time, dissolve TNF-α powder synthesized by Shanghai Genechem Co., Ltd. in DMEM medium to make the final concentration of TNF-α in the medium 20 ng / mL. Finally, prepare DMEM complete medium containing 0.1 μmol / L bioactive peptide ARHPHPHLSF, DMEM complete medium containing 0.1 μmol / L bioactive peptide RHPHPHLSF, and DMEM complete medium containing 20 ng / mL TNF-α.

[0126] ​(3) Mouse myoblast C2C12 cells were seeded into six-well plates at 1 x 10 5 cells per well, and cultured for 24 h before grouping intervention. 0 μmol / L, 0.1 μmol / L bioactive peptide ARHPHPHLSF (named: ARH10), RHPHPHLSF (named: RH9) containing DMEM complete medium and 20 ng / mL TNF-α containing DMEM complete medium were added respectively, and cultured at 37°C for 24 h.

[0127] (4) The old culture medium was discarded, 500 μL of DCFH-DA solution with a final concentration of 10 μmol / L was added, and incubated at 37°C for 20-40 min. Serum-free medium (purchased from Thermo Fisher Scientific) was washed 3 times to remove the DCFH-DA that did not enter the cells, and finally the absorbance value was measured at 532 nm wavelength.

[0128] Results are shown in Table 5 and Figure 5

[0129] Table 5: Effect of different concentrations of polypeptides on the content of ROS in muscle cells

[0130]

[0131] Results are shown in Table 5 and Figure 5 The content of ROS in the milk peptide RHPHPHLSF treatment group was reduced by 1.79 times compared with the modeling group.

[0132] 5. Determination of MDA content in muscle cells

[0133] The specific method is as follows:

[0134] (1) Chemically synthesized by Shanghai Gil Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder respectively.

[0135] (2) ARHPHPHLSF and RHPHPHLSF polypeptide powders obtained in step (1) were added to DMEM complete medium to make the final concentration of polypeptide in the medium 0.1 μmol / L, and Shanghai Shenguo chemically synthesized TNF-α powder was dissolved in DMEM medium to make the final concentration of TNF-α in the medium 20 ng / mL. Finally, DMEM complete medium containing 0.1 μmol / L bioactive peptide ARHPHPHLSF, DMEM complete medium containing 0.1 μmol / L bioactive peptide RHPHPHLSF and DMEM complete medium containing 20 ng / mL TNF-α were prepared

[0136] ​(3) Mouse myoblast C2C12 cells were inoculated into a six-well plate at 1x10 5 cells per well, and after 24h of culture, the cells were subjected to grouping intervention. DMEM complete medium containing 0 μmol / L, 0.1 μmol / L bioactive peptide ARHPHPHLSF (named: ARH10), RHPHPHLSF (named: RH9) and 20 ng / mL TNF-α DMEM complete medium were added, respectively, and the cells were further cultured at 37°C for 24h.

[0137] (4) After treatment, the cells were lysed using a lysis solution, and the supernatant was collected in a 1.5 mL centrifuge tube after centrifugation at 12000g for 10 min. 0.1 ml of homogenate solution and lysis solution were added to a new centrifuge tube as a blank control, 0.1 ml of different concentrations of standard samples were added for making a standard curve, and 0.1 ml of sample was added for determination; then 0.2 ml of MDA detection working solution was added. After mixing, 100°C or boiling water bath heating was performed for 15 min. The water bath was cooled to room temperature, and centrifugation was performed at 1000g for 10 min at room temperature. 200 μL of supernatant was taken and added to a 96-well plate, and then the absorbance was measured at 532 nm using an enzyme-labeled instrument.

[0138] Results are shown in Table 6 and Figure 6

[0139] Table 6: Effect of different concentrations of polypeptides on the content of MDA in muscle cells

[0140]

[0141] Results are shown in Table 6 and Figure 6 As shown in the results, compared with the modeling group, the content of MDA in the milk peptide RHPHPHLSF treatment group was reduced by 1.15 times.

[0142] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.​

Claims

1. Use of ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of food; the food is used to promote skeletal muscle cell proliferation and muscle cell growth.

2. Use according to claim 1, characterized in that, The food is cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, bean products, beverages.

3. Use according to claim 1, characterized in that, The food is special dietary food.

4. Use of ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of health products; the health products are used to promote skeletal muscle cell proliferation and muscle cell growth.

5. Use according to claim 4, characterized in that, The health products also contain acceptable adjuvants.

6. Use of ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of nutritional products; the nutritional products are used to promote skeletal muscle cell proliferation and muscle cell growth.

7. Use of ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of drugs for preventing sarcopenia.

8. Use according to claim 7, characterized in that, The drugs also contain pharmaceutically acceptable pharmaceutical adjuvants; the pharmaceutical adjuvants refer to conventional drug carriers in the pharmaceutical field.

9. Use according to claim 8, characterized in that, The adjuvants include one or more of the following: binding agents: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: starch, dextrin, sucrose, lactose, mannitol; fillers: starch, sucrose; wetting agents: glycerol; disintegrating agents: sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; absorption promoters: quaternary ammonium compounds; surfactants: polysorbate, sorbitan fatty acid and fatty acid glyceride; coloring agents: titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red; lubricants: hydrogenated vegetable oil, talc and polyethylene glycol; coating materials: acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; adjuvants: flavoring agents, sweeteners. The dosage forms of the drugs are oral dosage forms, injection dosage forms, inhalation dosage forms. The oral dosage forms are tablets, capsules, granules, oral liquids.

10. Use according to claim 8, characterized in that, The oral dosage forms are oral suspensions.

11. Use according to claim 10, characterized in that, The injection dosage forms are injection solutions, injection powders.

12. The use according to claim 10, characterized in that, The inhalation dosage forms are aerosols, powder aerosols.

13. The use according to claim 10, characterized in that, 15. Use of ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of health products for improving muscle mass.

14. The use according to claim 10, characterized in that, ​ ​

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