CN115349629A - Composition for activating muscle satellite cells and method of use thereof - Google Patents
Composition for activating muscle satellite cells and method of use thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
Compositions for activating myosatellite cells and methods of using the same are provided. The composition of the present invention comprises 0.1 to 9 parts by weight of ginseng powder or fermented ginseng powder, 10 to 60 parts by weight of soybean peptide, and 1 to 15 parts by weight of D-ribose. The composition activates skeletal muscle satellite cells, promotes the proliferation of the skeletal muscle satellite cells by activating an mTOR signal path, and can be applied to products for repairing and increasing muscles after exercise and preventing muscular atrophy.
Description
Technical Field
The invention relates to the field of health foods, in particular to a composition for activating muscle satellite cells and a using method thereof.
Background
The muscle satellite cell is a skeletal muscle stem cell, is mainly distributed below a basal layer around muscle fibers and plays a vital role in the process of muscle repair after muscle growth and sports injury. The number and function of myosatellite cells is affected by normal aging and by some diseases, for example in sarcopenia the proliferative capacity of myosatellite cells is affected, resulting in a reduction in the number of myosatellite cells. Reduction in the number of muscle satellite cells and functional decline can lead to inefficient recovery or muscle atrophy after injury. Accordingly, methods and compositions are sought for activating muscle satellite cells, promoting proliferation of muscle satellite cells, and for beneficial effects in the repair of muscle damage following exercise, stimulation of muscle growth, and prevention of muscle atrophy in the elderly.
The activation, proliferation and differentiation of the muscle satellite cells are regulated by an mTOR signaling pathway, and the activated mTOR signaling pathway promotes the proliferation and differentiation of the muscle satellite cells, so that the growth of muscles and the repair of muscle injuries can be accelerated. When skeletal muscle is damaged, a human body can activate the muscle satellite cells to a certain degree in order to repair the damaged muscle, the division of the muscle satellite cells is started, the muscle satellite cells of the damaged muscle are converted into myoblasts after being activated, and the myoblasts are divided and differentiated into muscle cells to repair and fill the damaged part. However, when the body is aged or affected by some diseases, the growth rate and repair ability of the muscle are weakened. Thus, activation of the mTOR pathway in muscle satellite cells contributes to proliferation and differentiation of muscle satellite cells.
Many patents and literature currently utilize peptides or carbohydrates to support the repair of damaged muscle and the growth of muscle. The invention patent with Chinese patent application number CN201710726385.3 discloses a composition for resisting exercise-induced fatigue and promoting recovery after exercise and a preparation method thereof, wherein the composition consists of 5-25 parts of separated whey protein, 1-10 parts of chondrocollagen, 1-10 parts of collagen peptide, 1-5 parts of olive fruit powder, 1-5 parts of Chinese date powder, 1-5 parts of yam powder, 1-5 parts of glucose, 1-5 parts of isomaltulose and 0.1-0.5 part of sucralose, and experiments prove that the composition has the effect of promoting recovery after exercise. The composition can supplement protein and energy requirements in the process after sports injury repair and eliminate free radicals generated in sports, but the mechanism for promoting the recovery after sports is not deeply researched and researched. Patent application No. AU2017213796A1 provides a method for inducing, enhancing or increasing proliferation of muscle satellite cells, which includes using a composition of one or more of a kinase inhibitor, a G protein-coupled receptor (GPCR) modulator, a histone deacetylase modulator, an epigenetic modulator, a neuropeptide, a dopamine receptor modulator, a 5-hydroxytryptamine receptor modulator, a histamine receptor modulator, an adenosine receptor modulator, an ionophore, an ion channel modulator, and the like, and animal experiments prove that these compounds can enhance proliferation of mouse primary satellite cells, but these drugs have relatively significant toxic side effects and are relatively expensive to use, and thus have limited use scenarios. Chinese patent CN 113368123A uses rhamnosyl icariside II as active ingredient of stem and leaf extract of herba Epimedii in promoting repair and regeneration of injured muscle, but the extraction process for obtaining icariside II monomer is complicated and has low purity (CN 102824394A).
There is a need in the art for compositions that promote repair and regeneration of damaged muscle by activating proliferation and differentiation of skeletal muscle satellite cells.
Disclosure of Invention
The present invention is based in part on the following findings of the inventors: the composition can synergistically activate the proliferation and differentiation of skeletal muscle satellite cells and can be applied to products for repairing muscle injury after exercise, increasing muscles and preventing muscular atrophy. The composition has simple preparation process and definite functional components, and can promote the repair and regeneration of damaged muscles by activating the proliferation and differentiation of skeletal muscle satellite cells.
In one aspect, the present invention provides a composition comprising 0.1 to 9 parts by weight of ginseng powder or fermented ginseng powder, 10 to 60 parts by weight of soybean peptide, and 1 to 15 parts by weight of D-ribose.
In one embodiment, the composition of the present invention is a composition for repairing muscle damage, augmenting muscle, and/or preventing muscle atrophy after exercise. In one embodiment, the composition of the present invention is a composition that activates proliferation and differentiation of skeletal muscle satellite cells and/or promotes repair and regeneration of damaged muscle.
In one embodiment, the composition comprises 3-9 parts by weight of ginseng powder or fermented ginseng powder.
In one embodiment, the composition comprises 5 to 15 parts by weight of D-ribose. In one embodiment, the composition comprises 10 to 15 parts by weight of D-ribose.
In one embodiment, the composition comprises 14 to 60 parts by weight of a soy peptide. In one embodiment, the composition comprises 14 to 30 parts by weight of a soybean peptide.
In one embodiment, the ginseng powder or fermented ginseng powder has a total ginsenoside content of 1wt% to 10wt%. In one embodiment, the peptide content in the soybean peptide is 70wt% to 99wt%.
In one embodiment, the fermented ginseng powder is a fermented ginseng powder obtained by yeast fermentation and lactic acid bacteria fermentation of a ginseng powder raw material.
In one embodiment, the composition comprises carnosine. In one embodiment, the composition does not comprise carnosine.
In one embodiment, the composition comprises an adjuvant or additive in a pharmaceutical or food product. In one embodiment, the adjuvant or additive is one or more of a sweetener, an acid modulator, a filler, a flavoring agent, a coloring agent, an antioxidant, a thickener, a stabilizer, an emulsifier, an anticaking agent, a glidant, a lubricant.
In one embodiment, the composition is an oral formulation, preferably, the oral formulation is a powder, capsule, tablet, or emulsion.
In one aspect, the invention provides a method of activating the mTOR signaling pathway in a myosatellite cell or activating proliferation and differentiation of a myosatellite cell in vitro comprising contacting the myosatellite cell with a composition of the invention. In one embodiment, the myosatellite cell differentiates into a myoblast or muscle cell.
In another aspect, the invention provides methods of activating muscle satellite cells in a subject, methods of promoting muscle repair in a subject, methods of promoting recovery after exercise, or methods of stimulating muscle growth or muscle gain. These methods may be non-therapeutic methods. The methods may comprise administering to a subject a composition of the invention.
The advantages of the invention include:
(1) The present invention provides a novel composition which activates proliferation and differentiation of myosatellite cells, increases the number of myosatellite cells, enhances the function of myosatellite cells, has a wide range of applications, and can be applied not only to products for promoting recovery after exercise but also to products for stimulating muscle development and preventing muscular atrophy.
(2) The composition of the invention can contain fermented ginseng powder which is ginseng powder fermented by food-grade yeast and lactic acid bacteria, so that the dryness of ginseng is reduced, more rare ginsenoside is obtained, and the problem of absorption and utilization degree of ginsenoside is improved.
(3) Compared with micromolecular drugs such as kinase inhibitors in the prior art (AU 2017213796A 1), the composition provided by the invention comprises food-grade ginseng powder or fermented ginseng powder, soybean peptide and D-ribose, is safer to use, has no toxic or side effect, is closer to the use requirement of daily life, and can be applied to sports nutritional foods or common foods.
(4) Compared with patent CN201710726385.3, the invention has more definite and deeper action mechanism; compared with the traditional Chinese medicine icariin for promoting muscle injury repair used in patent CN 113368123A, the preparation process of the composition is simple, the complex process flow of extracting high-purity icariin monomers and the organic solvent introduced in the extraction process are avoided, and the safety is improved.
Drawings
FIG. 1 shows a fluorescent micrograph of the proliferation of satellite cells detected by the Edu method.
FIG. 2 shows a bar graph of the number of cells treated in different groups as determined by the EdU method. Note: the letters a and b in the histogram indicate the comparison results of different groups, and the absence of the same letter between any two groups indicates that there is a significant difference between the two groups (p < 0.05), and the presence of a duplicate letter between the two groups indicates that there is no significant difference between the two groups.
Figure 3 shows the effect of different groups of treatments on the muscle satellite cell cycle. Note: letters a, b and c on the histogram indicate comparison results of different groups, no same letter appears between any two groups to indicate that the two groups have significant difference, and repeated letters between the two groups to indicate that the two groups have no significant difference
Figure 4 shows immunoblots of different groups of treatments and the corresponding quantification results.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention provides compositions for activating myosatellite cells. The composition can be used for activating proliferation and/or differentiation of skeletal muscle satellite cells; increasing the number of myosatellite cells; strengthening the function of the muscle satellite cells; repairing muscle damage after exercise; muscle augmentation; and/or to help prevent muscle atrophy.
The composition of the present invention may comprise ginseng powder, soybean peptide and D-ribose.
The fermented ginseng powder is prepared by dissolving ginseng powder raw materials, sterilizing, fermenting with yeast, fermenting with lactobacillus, filtering, spraying powder, and sieving. The fermented ginseng powder is prepared by dissolving ginseng powder raw materials, sterilizing, fermenting with yeast, fermenting with lactobacillus, filtering, spraying powder, and sieving. The composition of the present invention may comprise 0.1 to 9 parts by weight of ginseng powder or fermented ginseng powder. In particular, the composition of the present invention may comprise 3 to 9 parts by weight of ginseng powder or fermented ginseng powder. For example, the composition of the present invention may comprise 0.2, 0.5, 0.8, 1, 2, 3, 4, 5, 6, 7 or 8 parts by weight of ginseng powder or fermented ginseng powder. The ginseng powder or fermented ginseng powder used in the present invention may have a content of total saponins of ginseng of 1wt% or more, for example, 1wt% to 10wt%, and may be commercially available. For example, the total ginsenoside content in the ginseng powder or the fermented ginseng powder can be 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, or 9wt%. The ginseng powder used in the present invention may comply with national or local standards or industry standards (such as DBS 22/033) for ginseng powder. The fermented ginseng powder generally contains more than 1wt% of total ginsenosides. In addition, the fermented ginseng powder contains more rare ginsenosides than ginseng powder.
The soybean peptide is prepared by performing enzymolysis on soybean protein. The composition of the present invention may comprise 10 to 60 parts by weight of soybean peptide. In particular, the composition may comprise 14 to 60 parts by weight of soy peptide, preferably 14 to 30 parts by weight of soy peptide. For example, the composition of the present invention may comprise 15, 20, 25, 30, 35, 40, 45, 50, or 55 parts by weight of soybean peptide. The peptide content of the soybean peptide used in the present invention may be 70wt% to 99wt%, and is commercially available. For example, the peptide content in the soybean peptide used in the present invention may be 71wt%, 72wt%, 73wt%, 74wt%, 75wt%, 76wt%, 77wt%, 78wt%, 79wt%, 80wt%, 81wt%, 82wt%, 83wt%, 84wt%, 85wt%, 86wt%, 87wt%, 88wt%, 89wt%, 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, 96wt%, 97wt%, or 98wt%. The soybean peptide used in the present invention may meet national or local standards or industrial standards for ginseng powder (QB/T2653-2004).
The composition of the present invention may further comprise 5 to 15 parts by weight of D-ribose. In particular, the composition of the present invention may contain 10 to 15 parts by weight of D-ribose. For example, the composition of the present invention may comprise 6, 7, 8, 9, 10, 11, 12, 13 or 14 parts by weight of D-ribose.
The compositions of the invention may also contain other active ingredients, as desired. For example, the compositions of the invention may also comprise carnosine. The compositions of the invention may also contain no carnosine.
"carnosine" is a natural small molecule dipeptide that is present in relatively high concentrations in muscle and brain tissue in mammals and humans. Carnosine has antioxidant and anti-glycosylation properties and is thought to improve muscle function, allowing muscle recovery from fatigue. Herein, the composition may or may not have carnosine added. The inventors found that the addition of carnosine did not enhance the effect of the composition of the invention.
In order to prepare the composition of the present invention into a consumable product, the composition may further comprise adjuvants or additives in pharmaceutical products or foods. For example, the adjuvant or additive is one or more of sweetener, acid regulator, filler, flavoring agent, colorant, antioxidant, thickener, stabilizer, emulsifier, anticaking agent, glidant, and lubricant. The composition of the present invention may be prepared into oral preparations such as powders, capsules, tablets, or emulsions.
The compositions of the invention can be used in methods for activating a muscle satellite cell in vitro, and also for non-therapeutic purposes of activating a muscle satellite cell, promoting muscle repair, stimulating muscle growth, or augmenting muscle in a subject.
Examples
The objects, technical features and advantageous effects of the present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Examples 1 to 5: preparation of composition for activating muscle satellite cell
Ginseng powder, soybean peptide and D-ribose were all purchased commercially, the ginseng powder used in the examples contained 6% total saponins of ginseng, and the peptide content of the soybean peptide used in the examples was not less than 85% (on a dry basis).
The compositions of examples 1-5 and the compositions of comparative examples 1-3 were prepared by mixing the components at the components and contents shown in table 1.
TABLE 1 weight parts composition of compositions for activating myosatellite cells
Example 6: edu method for detecting proliferation of rat skeletal muscle satellite cells
Rat skeletal muscle satellite cells (purchased from Wuhan Punuoise Life technologies, inc., trade name CP-R223, culture medium being DMEM medium containing FBS, growth additives, penicillin, streptomycin) were seeded in 96-well plates and placed at 37 ℃ with 5% CO 2 After 24 hours of incubation in the incubator, rat skeletal muscle satellite cells were treated with each of the examples and comparative examples (each sample was completely dissolved in DMEM medium and used after filtration through 0.45. Mu.M filter) for 24 hours (37 ℃ C.5％CO 2 ) Each set of samples was set up in 3 replicates. After 24 hours of treatment of each group, the effect of cell proliferation was examined using an EdU (5-bromo-2-deoxyuracil) kit (purchased from Kyoto Biotechnology Ltd., KGA 331-100). EdU (5-bromo-2-deoxyuracil) is a pyrimidine analog that intercalates into DNA double strands during DNA synthesis and allows detection of cells in a proliferative state labeled with EdU using fluorescence imaging techniques. And after the EdU kit dyes the cells, observing the cells by using a fluorescence microscope, photographing and recording the cells, and analyzing the number of the positive cells marked by fluorescence of each processing group by using Image J Image processing software. The larger the number of cells fluorescently labeled in the same size field, the larger the number of cells in the proliferation phase, and the better the growth state of the cells. The experimental results are shown in fig. 1 and 2, and the statistical data are summarized in table 2; wherein blank refers to the experimental group without the addition of example or comparative sample.
As can be seen from table 2, the average number of positive cells in example 3 was 219, which is increased by 68 relative to the blank; and the comparative examples 1, 2 and 3 are respectively increased by 7, 7 and 23 and the sum of the three is 37 compared with the blank control, and the increased number of the EdU staining positive cells of the example 3 compared with the blank control is larger than the sum of the fluorescence intensity of each of the comparative examples 1 to 3 compared with the blank control, which shows that the composition can synergistically promote the proliferation of the rat skeletal muscle satellite cells.
Table 2: effect of different groups of treatments on cell proliferation
| Treatment group | Number of positive cells |
| Blank control | 151±16 |
| Example 1 | 208±10 |
| Example 2 | 201±4 |
| Example 3 | 219±6 |
| Example 4 | 208±2 |
| Example 5 | 198±5 |
| Comparative example 1 | 158±19 |
| Comparative example 2 | 158±15 |
| Comparative example 3 | 174±10 |
Example 7: flow cytometry detection of the cell cycle of satellite cells
Rat skeletal muscle satellite cells (purchased from GmbH of Wuhan Protech & ltd., CP-R223, culture medium DMEM containing FBS, growth additives, penicillin, streptomycin) were inoculated into 6-well plates, placed at 37 ℃ and 5% CO 2 After 24 hours of incubation in the incubator, rat skeletal muscle satellite cells were treated with each of the examples and comparative samples (each sample was completely dissolved in DMEM medium, used after filtration through 0.45. Mu.M filter) for 24 hours (37 ℃, 5% CO) 2 ). After 24 hours, the cells were digested with trypsin and collected, and the number of cells was 2X 10 5 ～2×10 6 Centrifuging at 2000rpm/min for 3 min, and discarding the pancreasThe enzyme medium was washed 1 time with PBS, centrifuged at 2000rpm/min for 3 minutes, and the PBS was discarded. Cells were stained with PI (propidium iodide, available from 70-CCS012, union, hangzhou Co., ltd.), 600. Mu.L of PI staining solution (1% v/v) was added to each cell group, vortexed for 5 to 10 seconds, incubated at room temperature for 30 minutes, and then the cell cycle of rat skeletal muscle satellite cells was examined using a flow cytometer (Pcy 5.5 channel for cell collection). The results of the experiment are shown in fig. 2 and table 3.
The effect of each treatment group on the cell cycle distribution of rat skeletal muscle satellite cells was examined by flow cytometry. Cells in the G0 phase are in a silent or quiescent state, and thus G0 phase cells are also referred to as quiescent phase cells. Cells in the G1 phase are usually in the growth substance preparation phase, cells in the S phase undergo DNA replication, and the G2/M phase is the mitotic phase of the cells. Therefore, the more diploid cells in the G0/G1 phase, the less cells in the cell growth/division phase. Conversely, the smaller the number of cells in the G0/G1 phase, the larger the number of cells in the S + G2/M phase, and the more the cells in the cell growth/division phase. From the statistical results in fig. 3 and table 3, it can be seen that the cell ratio in G0/G1 phase is significantly decreased and the cell ratio in S + G2/M phase is increased after the treatment of the groups of examples 1, 2, 3, 4 and 5 for 24 hours compared to the blank control and the comparative groups, which indicates that the composition formulated in a specific ratio can activate the rat skeletal muscle satellite cells in a silent or quiescent state, and accelerate the cells to enter the cell cycle process of proliferation and division.
As can be seen from Table 3, the proportion of cells in the S + G2/M phase in example 3 was 38.10%, which was increased by 8.92% relative to the blank; comparative examples 1, 2 and 3 were increased by 1.22%, 0.18%, 4.99%, and 6.39% in total, respectively, relative to the blank, and example 3 was increased to S + G2/M phase cells more than the sum of the respective increases of comparative examples 1-3 to S + G2/M phase cells, indicating that the compositions of the present invention synergistically accelerate cell cycle progression into proliferation and division.
Table 3: effect of different groups of treatments on satellite cell cycle distribution
Example 8: immunoblotting method for detecting activity of mTOR (mammalian target of rapamycin) signaling pathway of satellite cells
Rat skeletal muscle satellite cells (purchased from GmbH of Wuhan Protech & ltd., CP-R223, culture medium DMEM containing FBS, growth additives, penicillin, streptomycin) were inoculated into 12-well plates, placed at 37 ℃ and 5% CO 2 After 24 hours of incubation in the incubator, the samples of each example and comparative example (each sample was completely dissolved in DMEM medium, used after filtration through 0.45. Mu.M filter) were treated (37 ℃ C., 5% CO) 2 ) Rat skeletal muscle satellite cells for 24 hours.
(1) Preparation of cell protein sample
1) After the cells are treated for 24 hours by using the samples of each example and comparative example, the culture medium of a 12-well plate is discarded, the precooled PBS is washed once and then the PBS is discarded, 90 mu L of precooled NP-40 cell lysate is added into each well, and the mixture is placed on ice for standing and lysis for 10 minutes;
2) Scraping the lysed cells by using a cell scraper, transferring a lysate into a centrifuge tube, placing the centrifuge tube on a vortex oscillator to oscillate for 5 seconds, then inserting the centrifuge tube on ice to stand for 5 minutes, repeating the step for 3 times to completely lyse the cells, then placing the centrifuge tube in a low-temperature centrifuge which is precooled to 4 ℃ in advance, centrifuging for 10 minutes at 12000g, and sucking 2 mu L of supernatant for measuring the protein concentration;
3) The BCA method is used for measuring the protein concentration, the absorption amount of protein lysate is calculated according to the measurement result of the protein concentration, the volume of each sample is filled with NP-40 lysate, 5 times of sample loading buffer solution with the corresponding amount is added to enable the sample loading buffer solution to be 1 times of working concentration, the sample is evenly mixed after being oscillated for 5 seconds on a vortex oscillation instrument, the mixture is placed in a protein sample heater to be boiled for 5 minutes, 10000g of the mixture is centrifuged for 1 minute in a normal-temperature centrifuge, and the prepared protein sample is used for subsequent glue running or is placed in a refrigerator at the temperature of-20 ℃ to be stored.
(2) Detection of protein expression by immunoblotting
1) Electrophoresis: preparing SDS-polyacrylamide gel, adding the protein sample prepared by the method into a sample loading hole of the gel, installing an electrophoresis apparatus, inserting a wire and a power supply, performing electrophoresis at a constant voltage of 90V, and stopping electrophoresis when a target strip runs to a proper position;
2) Film transferring: taking down the gel, transferring the gel into a film transferring clamp, inserting the gel into a film transferring device, placing the film transferring device in a film transferring groove, pouring cold 1 Xelectric transferring buffer solution, inserting ice bricks, inserting electric wires and a power supply, and performing constant-voltage film transferring for 1 hour under the voltage of 90V;
3) And (3) sealing: taking out the membrane after the membrane is transferred, placing the membrane in 5% of skimmed milk, and placing the membrane on a shaking table to seal for 1 hour at room temperature;
4) Incubating the primary antibody: cutting the target band, and inoculating with p-mTOR (obtained from CST, phospho-mTOR (Ser 2448) (D9C 2) XP
Rabbitmab # 5536) and mTOR (purchased from CST, mTOR (7C 10) rabbitmab # 2983) were expressed as 1: diluting the mixture in TBST solution containing 1% skimmed milk at a ratio of 1000, placing the strip in an antibody incubation tank with proper size, adding corresponding antibody diluent, and incubating overnight in a shaking table in a refrigerator at 4 deg.C; recovering primary antibody the next day, washing the membrane with 1 × TBST on a shaker with a high rotation speed, changing the solution once in 10 minutes, and washing 3 times in total;
5) Incubation of secondary antibody: according to the following steps: 10000 of rabbit secondary antigen solution, adding the secondary antigen dilution solution to a membrane, placing the membrane on a slow shaking table, incubating for 1 hour at room temperature, then removing the secondary antibody, washing the membrane on the shaking table with 1 xTBST at a higher rotating speed, changing the solution once in 10 minutes, and washing for 4 times in total;
6) And (3) developing: and mixing ECL solution A and ECL solution B according to the ratio of 1:1, uniformly dripping the mixture on a film, transferring the film to clean cellophane in a developing clamp, and immediately developing in a dark room to obtain a result strip;
7) Image J Image processing software is used for counting the gray levels of p-mTOR and mTOR, the ratio of p-mTOR to mTOR is calculated, data are summarized in a table 4, and the experimental result is shown in a figure 4.
Table 4: effect of different group treatment on p-mTOR/mTOR ratio
| Treatment group | p-mTOR/mTOR |
| Blank control | 0.162±0.006 |
| Example 1 | 0.37±0.008 |
| Example 2 | 0.425±0.021 |
| Example 3 | 0.453±0.002 |
| Example 4 | 0.336±0.003 |
| Comparative example 1 | 0.213±0.011 |
| Comparative example 2 | 0.136±0.004 |
| Comparative example 3 | 0.176±0.005 |
Activation and proliferation of muscle satellite cells is regulated by the mTOR signaling pathway. Therefore, the inventors used immunoblotting to detect the activity of mTOR pathway in rat skeletal muscle satellite cells after different groups of treatments. The results of the experiment are shown in FIG. 4. The groups of example 1, example 2, example 3 and example 4 significantly improve the expression level of p-mTOR in rat skeletal muscle satellite cells, while the groups of comparative example 1, comparative example 2 and comparative example 3 have insignificant improvement of the expression level of p-mTOR, and the improvement of the p-mTOR/mTOR ratio of each example group is significantly better than that of each comparative example. As can be seen from Table 4, the mean value of p-mTOR/mTOR in example 3 was 0.453, which is an increase of 0.291 relative to the blank control; compared with the blank control, the comparative examples 1, 2 and 3 respectively increase 0.051, -0.026 and 0.014, and the sum of the three is 0.039, and the activation effect of the example 3 on the mTOR pathway is better than the sum of the activation effects of the comparative examples 1 to 3 on the mTOR pathway, so that the composition compounded according to the specific proportion can activate the mTOR signaling pathway and has a synergistic effect. In combination with the above experimental results, it was found that the composition promotes the proliferative activity of skeletal muscle satellite cells by activating mTOR signaling pathway in skeletal muscle satellite cells.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A composition comprising 0.1-9 parts by weight of ginseng powder or fermented ginseng powder, 10-60 parts by weight of soybean peptide and 1-15 parts by weight of D-ribose.
2. The composition of claim 1, wherein the composition comprises 3-9 parts by weight of ginseng powder or fermented ginseng.
3. The composition according to claim 1 or 2, wherein the composition comprises 5-15 parts by weight of D-ribose, preferably 10-15 parts by weight of D-ribose.
4. The composition according to any one of claims 1-3, wherein the composition comprises 14-60 parts by weight of soybean peptide, preferably 14-30 parts by weight of soybean peptide.
5. The composition according to any one of claims 1-4, wherein the ginseng powder or fermented ginseng powder has a total ginsenoside content of more than 1wt%, such as 1wt% to 10wt%, and/or a peptide content in the soy peptide is 70wt% to 99wt%.
6. The composition of any one of claims 1-5, comprising carnosine or not.
7. The composition according to any one of claims 1 to 6, comprising an adjuvant or additive in a pharmaceutical or food product; preferably wherein the adjuvant or additive is one or more of a sweetener, an acid regulator, a filler, a flavoring agent, a coloring agent, an antioxidant, a thickening agent, a stabilizer, an emulsifier, an anticaking agent, a glidant, a lubricant.
8. The composition of any one of claims 1-7, which is an oral formulation, preferably the oral formulation is a powder, capsule, tablet, or emulsion.
9. A method of activating the mTOR signaling pathway in a muscle satellite cell or activating proliferation and/or differentiation of a muscle satellite cell in vitro comprising contacting a muscle satellite cell with the composition of any one of claims 1-8.
10. A method for the non-therapeutic purpose of activating muscle satellite cells, promoting muscle repair, stimulating muscle growth or augmentation, or promoting recovery after exercise in a subject, comprising administering to the subject a composition according to any one of claims 1-8.
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