Differentiation promoter for skeletal muscle stem cells

Abstract

To discover a highly safe material derived from natural products that acts on skeletal muscle stem cells and promotes their differentiation, thereby enabling easy and efficient regeneration of skeletal muscle, and to provide the discovered material as a differentiation promoter for skeletal muscle stem cells and a regeneration promoter for skeletal muscle.SOLUTION: A differentiation promoter for skeletal muscle stem cells comprises an extract of Rosa roxburghii as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an agent for promoting the differentiation of skeletal muscle stem cells.

Background Art

[0002] Skeletal muscles in the living body atrophy with aging and may develop locomotive syndromes including sarcopenia. Recent studies have shown that the aging of skeletal muscles is a risk factor for heart failure and that the maintenance of healthy skeletal muscles is important in the treatment and prevention of various diseases. Although there are individual differences in the age-related changes of skeletal muscles, various factors are considered to be involved, such as chronic inflammation, oxidative stress, stem cells, various hormones, apoptosis, insulin resistance, etc. In particular, skeletal muscle stem cells present in skeletal muscle tissue are considered to be important. Skeletal muscle stem cells, also called satellite cells, are scattered while maintaining a quiescent state around muscle fibers. When they receive a signal of muscle injury, their proliferation is promoted, followed by differentiation into myoblasts, and finally they are incorporated as a part of muscle fibers. After muscle regeneration is completed, some stem cells maintain a quiescent state again around muscle fibers to prepare for the next muscle injury (Non-Patent Document 1). In recent years, it has also been found that with aging, the number of satellite cells in muscle tissue decreases, and the regenerative ability of individual satellite cells also decreases (Non-Patent Document 2).

[0003] In addition, the facial muscles that move the cheeks, eyes, nose, mouth, etc. of the face also deteriorate with aging. For example, the orbicularis oculi muscle is one of the facial muscles around the eyes and is responsible for opening and closing the eyes. However, with aging, the muscle becomes thinner, and it is known that the extrusion of adipose tissue causes eye bags around the eyes. Therefore, exploring and identifying functional substances that control the differentiation of skeletal muscle stem cells and promote skeletal muscle regeneration is considered an important approach not only for the medical utility against age-related muscle atrophy but also for aesthetics. Several means of promoting muscle regeneration have been studied so far. For example, a muscle production promoter containing a compound having histamine H3 receptor agonist activity as an active ingredient (Patent Document 1), a skeletal muscle regeneration promoter containing chondroitin sulfate-degrading enzyme as an active ingredient (Patent Document 2), etc. have been reported, but they do not act on skeletal muscle stem cells (muscle satellite cells) and are not derived from natural products.

[0004] Rosa roxburghii (scientific name: Rosa roxburghii) is a plant of the genus Rosa in the family Rosaceae (Rosaceae) and has been reported to have effects such as preventing or improving spots (Patent Document 3) and promoting the production of bleomycin hydrolase (Patent Document 4), etc., but nothing is known about its effect of promoting the differentiation of skeletal muscle stem cells.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] In view of the above circumstances, an object of the present invention is to find a highly safe natural product-derived material that acts on skeletal muscle stem cells to promote their differentiation and can simply and efficiently regenerate skeletal muscle, and to provide it as an agent for promoting the differentiation of skeletal muscle stem cells.

Means for Solving the Problems

[0008] As a result of intensive research to solve the above problems, the present inventors have found that an extract of Rosa multiflora var. cathayensis has an excellent effect of promoting the differentiation of skeletal muscle stem cells, and have completed the present invention.

[0009] That is, the present invention includes the following. (1) An agent for promoting the differentiation of skeletal muscle stem cells, containing a solvent extract of Rosa multiflora var. cathayensis as an active ingredient. (2) An agent for promoting the differentiation of skeletal muscle stem cells, containing a solvent extract of the filtration residue of the aqueous extract of Rosa multiflora var. cathayensis as an active ingredient. (3) The agent for promoting the differentiation of skeletal muscle stem cells according to (2), wherein the temperature of the aqueous extraction is 20°C or lower, and the solvent extract of the filtration residue is an extract extracted at a temperature 20°C or higher than the temperature of the aqueous extraction. (4) The differentiation promoter for skeletal muscle stem cells according to (1) or (2), wherein the solvent is one or more selected from the group consisting of water, lower alcohols, and liquid polyhydric alcohols. (5) A composition for skeletal muscle regeneration, comprising the differentiation promoter for skeletal muscle stem cells according to (1) or (2). (6) A composition for improving the impairment of muscle regeneration by reactive oxygen species, comprising the differentiation promoter for skeletal muscle stem cells according to (1) or (2). (7) A method for promoting the differentiation of skeletal muscle stem cells, comprising the step of culturing skeletal muscle stem cells in a medium containing an extract of Rosa multiflora var. cathayensis.

Advantages of the Invention

[0010] According to the present invention, there is provided a differentiation promoter for skeletal muscle stem cells that can promote the differentiation of skeletal muscle stem cells and simply and efficiently regenerate skeletal muscle. Therefore, the differentiation promoter for skeletal muscle stem cells of the present invention is effective in improving and preventing sarcopenia, muscle weakness, wrinkles, and flaccidity caused by the decline of skeletal muscle in the body and face associated with aging, and can greatly contribute to the fields of anti-aging medicine and cosmetic medicine.

Modes for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described in detail. The differentiation promoter for skeletal muscle stem cells according to the present invention (hereinafter, also referred to as "the agent of the present invention") contains an extract of Rosa multiflora var. cathayensis obtained by solvent extraction as an active ingredient.

[0012] In the present invention, the "skeletal muscle stem cell" refers to a mononuclear stem cell called a satellite cell that exists between the cell membrane and the basement membrane of muscle fibers. When stimulated, such as when muscle fibers are damaged, skeletal muscle stem cells are activated to become myoblasts. After myoblasts proliferate through several rounds of cell division, they differentiate into muscle cells, and multiple muscle cells fuse with each other to form multinucleated muscle tubes. Therefore, by promoting the proliferation and differentiation of skeletal muscle stem cells, skeletal muscle regeneration is promoted. In the present invention, the origin of skeletal muscle stem cells is not limited, and it can exert effects on skeletal muscle stem cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

[0013] "Skeletal muscle" mainly refers to the muscles that attach to the movable parts of the skeleton and function in maintaining posture and movement. The tissue of skeletal muscle is composed of multinucleated giant cells called muscle fibers, and several to dozens of satellite cells, which are skeletal muscle stem cells, adhere to each muscle fiber and are regenerated by the function of these satellite cells. The cells of skeletal muscle are characterized by the gene expression and protein expression of skeletal muscle-specific markers such as MYOG (Myogenin), DES (Desmin), CKM (Creatine Kinase Muscle), MHC3 (Myosin heavy chain 3), MLC1 (Myosin light chain 1), Skeletal Muscle Troponin-I, Mb (Myoglobin), FABP (Fatty acid-binding protein), etc., morphology such as multinucleation and formation of muscle fibers, and functions such as contractility.

[0014] Rosa roxburghii used in the present invention is a deciduous shrub of the genus Rosa in the family Rosaceae. It is a variety of rose native to China named after the moon on the 16th night because some of its petals are not completely round but are missing. In the present invention, in addition to the original species, varieties, hybrids, etc. of Rosa roxburghii can also be used.

[0015] In the present invention, the part of Rosa multiflora Thunb. var. carnea Thory used for extraction may be any part of the plant body such as flowers, fruits, pericarp, stems, leaves, branches, roots, seeds, etc., or the whole plant body (whole herb), or a mixture thereof, but fruits are preferred. Also, for extraction, the plant body may be used as it is, or may be subjected to treatments such as drying, pulverization, or cutting into small pieces.

[0016] In addition, as the fruit of the above Rosa multiflora Thunb. var. carnea Thory, those having a major axis length in the range of 1 cm to 10 cm are preferably used, and those having a major axis length in the range of 1 cm to 4 cm are more preferably used. Also, the fruits to be used may be raw, or dried products such as natural dried products or sun-dried products may be used.

[0017] In a preferred embodiment of the present invention, the filtration residue of the aqueous extract of Rosa multiflora Thunb. var. carnea Thory is used as the extraction material. The water used for the aqueous extraction is not particularly limited, and examples include tap water, distilled water, deionized water, purified water, etc. Also, the water used for the aqueous extraction may be water with pH adjusted by adding an acid or an alkali. There is no particular limitation on the amount of water used for the aqueous extraction. For example, it is 3 times or more, preferably 5 times or more, more preferably 10 times or more based on the extraction raw material (dry weight). However, in the present invention, it is preferably 100 times or less for the convenience of the operation of filtering the aqueous extract of Rosa multiflora Thunb. var. carnea Thory and collecting the residue. The filtration is not particularly limited, and can be performed using filter paper, mesh, sieve, etc. The temperature and time of the aqueous extraction can be appropriately selected depending on the pressure during extraction, etc. The temperature is, for example, 20°C or lower, preferably 2 to 15°C, more preferably 5 to 10°C, and the time is, for example, 10 to 30 hours, preferably 15 to 25 hours. In the present invention, the operation of the above aqueous extraction of Rosa multiflora Thunb. var. carnea Thory, filtering the obtained aqueous extract to separate it into a filtrate and a residue, and collecting the residue is sometimes referred to as "pretreatment".

[0018] In the present invention, the extraction material may be untreated Rosa multiflora Thunb. var. carnea Thory, or Rosa multiflora Thunb. var. carnea Thory subjected to the above pretreatment. The Rosa multiflora Thunb. var. carnea Thory subjected to pretreatment (filtration residue of the aqueous extract of Rosa multiflora Thunb. var. carnea Thory) may be dried before being subjected to extraction, or may be used for extraction as it is without drying.

[0019] As the extraction solvent for obtaining an extract from the above extraction material, water or hot water, or a mixed solvent of water and an organic solvent can be used. As the organic solvent, lower alcohols (such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), and liquid polyhydric alcohols (such as 1,3-butylene glycol, propylene glycol, glycerin, etc.) can be used, and one or more of these can be mixed and used. Among them, a water-ethanol-based mixed polar solvent (an ethanol aqueous solution of 30 to 70 v / v%) or ethanol is preferable, and an ethanol aqueous solution of 50 v / v% is more preferable. Also, an acid or an alkali can be added to the above extraction solvent to use a solvent with adjusted pH.

[0020] The extraction temperature is a temperature below the boiling point of the solvent used for extraction, and is not particularly limited. For example, it may be heat extraction or normal temperature extraction. The extraction time is 1 to 2 weeks in the case of normal temperature extraction, and 30 minutes to 24 hours, preferably 1 to 10 hours in the case of heat extraction, but it can be appropriately adjusted according to conditions such as the type of extraction solvent and extraction temperature.

[0021] In the present invention, when using the filtration residue of the aqueous extract of Rosa multiflora Thunb. var. cathayensis Rehd. as the extraction material, the extraction temperature is preferably 20°C or higher than the temperature of the aqueous extraction. Among them, when using water as the extraction solvent, it is preferably 70°C or higher, more preferably 80°C or higher, and most preferably 90°C or higher than the temperature of the aqueous extraction.

[0022] The extract may be used as the extracted solution as it is, but if necessary, within the range that does not affect its effect, it may be used after performing treatments such as concentration (concentration by an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0023] The solvent extract of Rosa multiflora Thunb. var. carnea Thory obtained in this way, and the solvent extract of the filtration residue of the aqueous extract of Rosa multiflora Thunb. var. carnea Thory (hereinafter, these are collectively referred to as the "extract of Rosa multiflora Thunb. var. carnea Thory") have the effect of promoting the differentiation from skeletal muscle stem cells to skeletal muscle both at the in vivo level (in the living body) and at the in vitro level (in vitro), so they can be used as the active ingredient of a differentiation promoter for skeletal muscle stem cells.

[0024] The content of the extract of Rosa multiflora Thunb. var. carnea Thory in the agent of the present invention is not particularly limited. For example, it is preferably 0.00001 to 10% by weight in terms of dry matter based on the total amount of the agent, and more preferably 0.0001 to 1% by weight.

[0025] In vitro, the agent of the present invention can be used as an additive for cell culture media, a research reagent, and a medical reagent for promoting the differentiation from skeletal muscle stem cells to skeletal muscle.

[0026] When the agent of the present invention is used in vivo, it can be used as it is, but within a range that does not impair the effects of the present invention, it can be formulated with appropriate additives into various compositions such as cosmetics, pharmaceuticals, quasi-drugs, foods and drinks, etc., and provided as a composition for promoting skeletal muscle regeneration. It should be noted that the pharmaceuticals of the present invention include drugs used for animals, that is, veterinary drugs.

[0027] When the agent of the present invention is used, for example, for the purpose of improving wrinkles, sagging, depressions, etc. caused by the decline of facial muscles (orbicularis oris muscle, zygomatic major and minor muscles, orbicularis oculi muscle, mentalis muscle, etc.), it is preferably in the form of cosmetics or quasi-drugs, or in the form of foods and drinks such as beauty drinks. Also, when the agent of the present invention is used, for example, for the purpose of treating and / or preventing diseases (such as sarcopenia, etc.) caused by damage or functional decline of skeletal muscle, it is preferably used in the form of pharmaceuticals or supplements.

[0028] When formulating the agent of the present invention into cosmetics or quasi-drugs, a topical skin composition is preferred, and any dosage form such as an aqueous solution system, solubilization system, emulsion system, powder system, powder dispersion system, oil solution system, gel system, ointment system, aerosol system, water-oil two-layer system, or water-oil-powder three-layer system may be used. Further, the cosmetics or quasi-drugs may be selected and appropriately formulated with various components, additives, bases, etc. commonly used in topical skin compositions together with the above-mentioned Rosa multiflora var. cathayensis extract according to their types, and can be manufactured according to methods known in the art. The form may be any of liquid, emulsion, cream, gel, paste, spray, etc. Examples of the components to be formulated in the topical skin composition include fats and oils (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (isopropyl myristate, isopropyl palmitate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidonecarboxylic acid, etc.), saccharides (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins (β-carotene (vitamin A), vitamin C, vitamin E, etc.), plant and animal extract components, various surfactants, humectants, ultraviolet absorbers, pH adjusters, stabilizers, preservatives, bactericides, fragrances, etc.

[0029] Examples of the types of cosmetics and quasi-drugs include, but are not limited to, lotion, emulsion, gel, essence, general cream, sunscreen cream, pack, mask, facial wash, cosmetic soap, foundation, powder, bath agent, body lotion, body shampoo, etc.

[0030] When the agent of the present invention is formulated into a pharmaceutical product, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various pharmaceutical preparations in a dosage form suitable for application to the affected area. As pharmacologically and pharmaceutically acceptable additives, depending on the dosage form and use, appropriately selected pharmaceutical bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegrant aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffering agents, solubilizing agents or solubilizing aids, isotonic agents, pH adjusters, propellants, coloring agents, sweetening agents, flavoring agents, fragrances, etc. can be appropriately added, and it can be prepared into various dosage forms that can be administered orally or parenterally, systemically or locally by various known methods. When the pharmaceutical product of the present invention is provided in the above-mentioned various forms, it can be manufactured by manufacturing methods commonly used by those skilled in the art, such as the manufacturing methods shown in each article of the General Rules of Preparations of the Japanese Pharmacopoeia [2].

[0031] For oral dosage forms, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegrant aids such as carboxymethyl cellulose, carboxymethyl cellulose calcium, starch, or hydroxypropyl cellulose; binders such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropyl methylcellulose, sucrose, polyethylene glycol, or titanium oxide; bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat, etc. can be used, but are not limited thereto.

[0032] For parenteral dosage forms, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum solution, vegetable oil, etc.; isotonic agents such as glucose, sodium chloride, D-mannitol, etc.; pH adjusters such as inorganic acids, organic acids, inorganic bases or organic bases, etc. can be used, but are not limited thereto.

[0033] The form of the pharmaceutical of the present invention is not particularly limited. For example, oral preparations such as tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspensions, elixirs, etc., injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drip infusions, suppositories, transdermal absorbents, transmucosal absorbents, patches and other parenteral preparations can be mentioned. Further, it may be a dry product that is redissolved when used, and in the case of an injectable preparation, it is provided in the state of a unit dose ampoule or a multi-dose container.

[0034] Since the active ingredient of the pharmaceutical of the present invention is derived from a natural product and has high safety and no side effects, when used as a pharmaceutical for preventing and / or treating diseases caused by skeletal muscle damage or functional decline, it can be orally or parenterally administered to mammals such as humans, mice, rats, rabbits, dogs, cats, etc. in a wide range of dosages.

[0035] The composition for promoting skeletal muscle regeneration of the present invention is expected to have effects such as promoting muscle differentiation, increasing muscle mass, and muscle hypertrophy because the extract of Rosa multiflora var. cathayensis contained therein has the effect of promoting the differentiation of skeletal muscle stem cells into skeletal muscle, and is effective for treating, improving, and preventing pathological conditions or diseases caused by skeletal muscle damage or functional decline. Examples of pathological conditions or diseases caused by skeletal muscle damage or functional decline include, in the case of facial skeletal muscle, wrinkles, eye bags, nasolabial folds, marionette lines, eyelid depressions, eye bags (under-eye bags and tear troughs), loss of firmness and elasticity, etc. on the cheeks, forehead, and eyelids due to the decline of facial expression muscles (orbicularis oris muscle, zygomatic major and minor muscles, orbicularis oculi muscle, mentalis muscle, etc.) associated with aging. In the case of skeletal muscle of the body, sarcopenia, locomotive syndrome, physical frailty, muscle atrophy and muscle damage caused by oxidative stress, trauma, and muscle diseases (muscular dystrophy, amyotrophic lateral sclerosis (ALS), spinal progressive muscular atrophy (SPMA), etc.) can be mentioned. Muscle atrophy includes myogenic atrophy caused by the muscle itself, neurogenic atrophy caused by motor neurons, and disuse atrophy caused by long-term inactivity of muscles. The above-mentioned oxidative stress can be caused by ultraviolet rays, air pollution, smoking, drugs, excessive exercise, physical and mental stress, etc., regardless of the cause. Since the extract of Rosa multiflora var. cathayensis has been shown to improve the suppression of differentiation of skeletal muscle stem cells by hydrogen peroxide as shown in the following examples, it is particularly effective as a composition for improving the impairment of muscle regeneration by reactive oxygen species.

[0036] The dosage or administration amount of the cosmetics, quasi-drugs, and pharmaceuticals of the present invention can be appropriately determined according to their types and forms, the age, sex, body weight, degree of symptoms of the use or administration target, etc. For example, when orally administered to adults, it is in the range of 0.1 to 1000 mg / day, preferably 1 to 500 mg / day, more preferably 5 to 300 mg / day as the extract of Rosa multiflora var. cathayensis, and is administered once to several times a day respectively. In some cases, an amount less than the above administration range may be sufficient, and in other cases, it may be necessary to administer in excess of the range.

[0037] When the extract of Rosa multiflora var. cathayensis is incorporated into cosmetics, quasi-drugs, or pharmaceuticals, its content is not particularly limited, but in terms of the dry solid content of the extract of Rosa multiflora var. cathayensis, 0.001 to 30% by weight (w / w) is preferable, and 0.01 to 10% by weight (w / w) is more preferable, based on the total weight of the formulation (composition). If it is less than 0.001% by weight (w / w), the effect is low, and even if it exceeds 30% by weight (w / w), no significant enhancement of the effect is likely to be observed. Also, regarding the method of adding the active ingredient in formulation, it may be added in advance or during the manufacturing process, and it may be appropriately selected considering workability.

[0038] In addition, the agent of the present invention can also be incorporated into foods and drinks. By providing it in the form of foods and drinks, it becomes easy to ingest the active ingredient of the present invention daily or continuously. In the present invention, foods and drinks include, in addition to general foods and drinks, foods that can be ingested for the purpose of maintaining or promoting health other than pharmaceuticals, for example, health foods, functional foods, foods with health claims, or foods for special purposes. Health foods include foods provided under names such as dietary supplements, health supplements, and supplements. Foods with health claims are defined by the Food Sanitation Law or the Food Promotion Law and include foods for specified health uses and foods with nutritional claims that can display specific health effects, functions of nutritional components, reduction of disease risks, etc. The form of foods and drinks may be any form suitable for consumption, for example, solid, liquid, granular, powdery, capsule, cream, or paste.

[0039] Examples of the types of foods and drinks include breads, noodles, confectioneries, dairy products, processed fishery and livestock products, fats and oils and processed fat and oil products, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, milk drinks, etc.) and concentrated stock solutions and powder for adjustment of such beverages, but are not limited thereto.

[0040] The food or drink of the present invention may be appropriately blended with additives commonly used according to its type. Any additive that is acceptable in food hygiene can be used. For example, sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavors, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics can be mentioned.

[0041] The blending amount of the extract of Rosa multiflora var. cathayensis in the food or drink of the present invention may be any amount that can exert the action of promoting skeletal muscle regeneration, but it may be appropriately set in consideration of the general intake amount of the target food or drink, the form of the food or drink, efficacy and effect, taste, palatability, and cost.

[0042] The present invention also relates to a method for promoting the differentiation of skeletal muscle stem cells into skeletal muscle, which includes a step of culturing skeletal muscle stem cells in a medium containing an extract of Rosa multiflora var. cathayensis. The extract of Rosa multiflora var. cathayensis used here may be an untreated extract of Rosa multiflora var. cathayensis or an extract of Rosa multiflora var. cathayensis subjected to the above-mentioned pretreatment.

[0043] In the method according to the present invention, a medium generally used for the proliferation, differentiation, and maturation of mesenchymal stem cells may be used. Also, the conditions and operations of the culture method can be carried out according to conventional conditions and operations in the technical field. For example, a basal medium containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), specifically, Dulbecco’s Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), Hank’s balanced salt solution, etc. can be mentioned. When inducing differentiation, one or more of insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) are added as myocyte differentiation-inducing factors. Also, if necessary, epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, biotin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, etc. may be added to the medium, and antibiotics (penicillin, streptomycin, dexamethasone, amphotericin B, etc.) may also be added. Each component of the medium is sterilized by a suitable method and then used. It is also possible to use a commercially available medium in which the above components are appropriately added to the basal medium.

[0044] In addition, other than the above, it is preferable that the medium contains serum at a content rate of 1 to 20%. However, since the components of serum vary depending on the lot and there are variations in its effects, it is preferable to use it after performing a lot check.

[0045] As a dedicated maintenance medium for commercially available skeletal muscle stem cells, StemLife Sk Comp kit manufactured by KURABO INDUSTRIES LTD. can be used, and as a dedicated differentiation medium, MyoLife Complete Myogenesis Dif. Med manufactured by KURABO INDUSTRIES LTD. can be used.

[0046] Examples of the incubators used for cell culture include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, and the like.

[0047] The incubator may be non-cell adhesive or cell adhesive, and is appropriately selected according to the purpose. For the purpose of improving the adhesiveness to cells, an incubator with cell adhesiveness may be treated with a cell support substrate such as an extracellular matrix. Examples of the extracellular matrix include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, and the like.

[0048] The addition concentration of the extract of Rosa multiflora var. cathayensis to the medium used for cell culture is, for example, 0.1 to 1000 μg / mL, preferably 1 to 100 μg / mL. Further, during the cell culture period, the extract of Rosa multiflora var. cathayensis may be added to the medium periodically.

[0049] The cell culture conditions may follow normal conditions and do not require special control. For example, the culture temperature is not particularly limited, but is about 30 to 40°C, preferably 36 to 37°C. The CO2 gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. It is preferable to change the medium once every 2 to 3 days, and more preferably every day. The above culture conditions can also be set by appropriately varying within the range where the cells can survive and proliferate.

[0050] The differentiation of skeletal muscle stem cells into skeletal muscle can be confirmed, for example, by determining whether the expression levels of MYOG (Myogenin) and DES (Desmin), which are markers of skeletal muscle, are significantly increased at the mRNA level or protein level compared to the expression levels at the start of culture in cells cultured in the presence of an extract of Rosa multiflora var. cathayensis compared to cells cultured in the absence of the extract of Rosa multiflora var. cathayensis. At the mRNA level, methods of confirmation include RT-PCR, quantitative PCR, and Northern blotting using primers and probes specific to, for example, the MYOG gene or the DES gene. Also, at the protein level, immunological methods such as ELISA, flow cytometry, and Western blotting using antibodies specific to the proteins encoded by the MYOG gene or the DES gene can be mentioned.

Example

[0051] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to these examples.

[0052] [Example 1] Preparation of extract of Rosa multiflora var. cathayensis (Production Example 1) Preparation of hot water extract of Rosa multiflora var. cathayensis without pretreatment To 10 g of the dried product of the fruit of Rosa multiflora var. cathayensis, 20 times the amount of water was added, and extraction was performed at 95°C for 2 hours. The obtained extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 1.2 g of a hot water extract of Rosa multiflora var. cathayensis (without pretreatment / hot water extract of Rosa multiflora var. cathayensis fruit).

[0053] (Production Example 2) Preparation of 50% ethanol extract of Rosa multiflora var. cathayensis without pretreatment To 10 g of the dried product of the fruit of Rosa multiflora var. cathayensis, it was immersed in 20 times the amount of a 50% ethanol aqueous solution at 25°C for 7 days for extraction. The obtained extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 1.7 g of a 50% ethanol extract of Rosa multiflora var. cathayensis (without pretreatment / 50% ethanol extract of Rosa multiflora var. cathayensis fruit).

[0054] (Production Example 3) Preparation of Ethanol Extract of Rosa multiflora var. cathayensis without Pretreatment 20 g of dried fruit of Rosa multiflora var. cathayensis was immersed in 20-fold volume of ethanol at 25°C for 7 days for extraction. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.3 g of an ethanol extract of Rosa multiflora var. cathayensis (without pretreatment / ethanol extract of Rosa multiflora var. cathayensis fruit).

[0055] (Production Example 4) Preparation of Hot Water Extract of Rosa multiflora var. cathayensis with Pretreatment 10-fold volume of water was added to 10 g of dried fruit of Rosa multiflora var. cathayensis, and extraction was carried out at 5°C for 24 hours (the increase in the solid content concentration of the extract stopped within 6 hours and no further extraction occurred). After the obtained extract was filtered through filter paper, the residue was dried, 20-fold volume of water was added to the obtained dried residue, and extraction was carried out at 95°C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.3 g of a hot water extract of Rosa multiflora var. cathayensis (with pretreatment / hot water extract of Rosa multiflora var. cathayensis fruit).

[0056] (Production Example 5) Preparation of 50% Ethanol Extract of Rosa multiflora var. cathayensis with Pretreatment 10-fold volume of water was added to 10 g of dried fruit of Rosa multiflora var. cathayensis, and extraction was carried out at 5°C for 24 hours (the increase in the solid content concentration of the extract stopped within 6 hours and no further extraction occurred). After the obtained extract was filtered through filter paper, the residue was dried, and the obtained dried residue was immersed in 20-fold volume of 50% ethanol aqueous solution at 25°C for 7 days for extraction. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.4 g of a 50% ethanol extract of Rosa multiflora var. cathayensis (with pretreatment / 50% ethanol extract of Rosa multiflora var. cathayensis fruit).

[0057] (Production Example 6) Preparation of Ethanol Extract of Rosa multiflora var. cathayensis with Pretreatment To 20 g of the dried fruit of Rosa multiflora Thunb., 10 times the amount of water was added, and extraction was carried out at 5°C for 24 hours (the increase in the solid content concentration of the extract stopped within 6 hours, and no further extraction occurred). After filtering the obtained extract with filter paper, the residue was dried, and the obtained dried residue was immersed in 20 times the amount of ethanol at 25°C for 7 days for extraction. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.2 g of the ethanol extract of Rosa multiflora Thunb. (with pretreatment / ethanol extract of Rosa multiflora Thunb. fruit).

[0058] [Example 2] Evaluation of the effect of promoting the differentiation of the extract of Rosa multiflora Thunb. on skeletal muscle stem cells A commercially available human normal skeletal muscle stem cell (alias: normal human skeletal muscle satellite cell) (manufactured by KURABO) was treated with the extract of Rosa multiflora Thunb. to evaluate how the extract of Rosa multiflora Thunb. is involved in promoting the differentiation of skeletal muscle stem cells. Skeletal muscle stem cells cultured in a dedicated maintenance medium StemLife Sk Comp kit (manufactured by KURABO) were seeded at 1×10 5 cells per well in a 24-well culture plate and cultured for 2 days until confluent. When the cells became confluent, the medium was replaced with a dedicated differentiation medium MyoLife Complete Myogenesis Dif. Med (manufactured by KURABO), and at the same time, the test substances (the extracts of Rosa multiflora Thunb. from Production Examples 1 to 6) were added so that the final concentration was 10 μg / ml and cultured. The cells 2 days after the addition of the extract of Rosa multiflora Thunb. were collected, and the gene expression levels of MYOG (Myogenin) and DES (Desmin), which are skeletal muscle differentiation markers, were analyzed.

[0059] The gene expression analysis of MYOG and DES was performed as follows. After the cells were washed twice with PBS(-) after the addition of the extract of Rosa multiflora var. cathayensis, RNA was extracted from the cells using Trizol Reagent (manufactured by Invitrogen). As a control, cells before induction of differentiation were also collected and gene expression analysis was performed in the same manner. Using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), the extracted RNA was reverse-transcribed into cDNA, and then real-time PCR (95°C: 15 seconds, 60°C: 30 seconds, 40 cycles) was performed using the following primer sets with ABI7300 (manufactured by Applied Biosystems) to measure the gene expression levels of MYOG and DES. Other operations were carried out according to the defined methods.

[0060] Primer set for MYOG (skeletal muscle differentiation marker): 5'-CAGTGAATATTGGGAACCTTCCAG-3' (SEQ ID NO: 1) 5'-ATGGCAGCTTTACAAACAACAC-3' (SEQ ID NO: 2) Primer set for DES (skeletal muscle differentiation marker): 5'-CAGCCAACAAGAACAACGAC-3' (SEQ ID NO: 3) 5'-GGAATCGTTAGTGCCCTTCAG-3' (SEQ ID NO: 4) Primer set for 18srRNA (internal standard): 5'-CCGAGCCGCCTGGATAC-3' (SEQ ID NO: 5) 5'-CAGTTCCGAAAACCAACAAAATAGA-3' (SEQ ID NO: 6)

[0061] The effect of promoting the differentiation of skeletal muscle stem cells was calculated as the ratio of the expression levels of the mRNAs of MYOG and DES, which are differentiation markers in skeletal muscle stem cells before the start of differentiation induction, to the expression level of 18s ribosomal RNA (18srRNA) as an internal standard (relative gene expression level of differentiation marker gene / expression level of 18srRNA gene), with the value being set to 1. In contrast, the values of the relative gene expression levels of MYOG and DES in the cells after 2 days of culture were calculated and evaluated. The results of these tests are shown in Table 1 below.

[0062]

Table 1

[0063] As shown in Table 1, it was shown that the extract of Rosa multiflora Thunb. var. cathayensis Rehd. induces and promotes the differentiation of skeletal muscle stem cells. When 50% ethanol was used as the extraction solvent, the effect of promoting differentiation was high, and particularly for the extract of Rosa multiflora Thunb. var. cathayensis Rehd. with pretreatment, a higher effect of promoting differentiation was obtained.

[0064] [Example 3] Efficacy of the extract of Rosa multiflora Thunb. var. cathayensis Rehd. in the case of impaired differentiation of skeletal muscle stem cells by hydrogen peroxide It has been found that when skeletal muscle tissue is damaged by intense exercise or the like, reactive oxygen species accumulate in the tissue. The effect of reactive oxygen species on the induction of differentiation of skeletal muscle stem cells was analyzed, and the efficacy when the extract of Rosa multiflora Thunb. var. cathayensis Rehd. was allowed to act was evaluated. Skeletal muscle stem cells cultured in a dedicated maintenance medium StemLife Sk Comp kit (manufactured by KURABO Industries Ltd.) were seeded at 1×10 5Cells were seeded one by one and cultured until they became confluent. When the cells became confluent, the medium was replaced with a dedicated differentiation medium, MyoLife Complete Myogenesis Dif. Med (manufactured by KURABO), and at the same time, hydrogen peroxide (H2O2) was added to a final concentration of 100 μM. In addition, the test substances (extracts of Rosa multiflora var. cathayensis from Production Examples 1 to 6) were added simultaneously to a final concentration of 10 μg / ml and cultured. The cells after 3 days were collected, and the gene expression levels of MYOG (Myogenin) and DES (Desmin), which are skeletal muscle differentiation markers, were analyzed as indicators.

[0065] The gene expression analysis of MYOG and DES was performed as follows. After the cells after the addition of the extract of Rosa multiflora var. cathayensis were washed twice with PBS(-), RNA was extracted from the cells using Trizol Reagent (manufactured by Invitrogen). As a control, cells before differentiation induction were also collected and gene expression analysis was performed in the same manner. After reverse-transcribing the extracted RNA into cDNA using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), real-time PCR (95°C: 15 seconds, 60°C: 30 seconds, 40 cycles) was performed using the primer set described in Example 2 with ABI7300 (manufactured by Applied Biosystems) to measure the gene expression levels of MYOG and DES. Other operations were carried out according to the defined methods.

[0066] The effect of promoting the differentiation of skeletal muscle stem cells was calculated as the ratio of the expression levels of the mRNAs of MYOG and DES, which are differentiation markers in skeletal muscle stem cells before the start of differentiation induction, to the expression level of 18s ribosomal RNA (18srRNA) as an internal standard (gene relative expression level = differentiation marker gene expression level / 18srRNA gene expression level), with the value of 1 for the gene relative expression level of MYOG and DES in the cells before differentiation induction. In contrast, the values of the gene relative expression levels of MYOG and DES in the cells after 3 days of culture were calculated and evaluated. The results of these tests are shown in Table 2 below.

[0067]

Table 2

[0068] As shown in Table 2, it was shown that hydrogen peroxide inhibits the differentiation of skeletal muscle stem cells. When the extract of Rosa multiflora was added, it was shown that the inhibition of differentiation of skeletal muscle stem cells by hydrogen peroxide was improved in all cases. However, when 50% ethanol was used as the extraction solvent, the effect of improving the inhibition of differentiation was high. In particular, in the extract of Rosa multiflora subjected to pretreatment, a higher effect of improving the inhibition of differentiation was obtained.

Industrial Applicability

[0069] The skeletal muscle stem cell differentiation promoter of the present invention can promote the differentiation of skeletal muscle stem cells and regenerate skeletal muscle in vivo or in vitro. Therefore, the present invention can be used in the field of manufacturing cosmetics and pharmaceuticals for anti-aging.

Claims

1. A differentiation promoter for skeletal muscle stem cells, containing an extract obtained by solvent extraction of Rosa multiflora var. cathayensis as an active ingredient.

2. A differentiation promoter for skeletal muscle stem cells, containing an extract obtained by solvent extraction of the filtration residue of an aqueous extract of Rosa multiflora var. cathayensis as an active ingredient.

3. The differentiation promoter for skeletal muscle stem cells according to Claim 2, wherein the temperature of the aqueous extraction is 20°C or lower, and the extract obtained by solvent extraction of the filtration residue is an extract extracted at a temperature 20°C or higher than the temperature of the aqueous extraction.

4. The differentiation promoter for skeletal muscle stem cells according to Claim 1 or 2, wherein the solvent is one or more selected from the group consisting of water, lower alcohols, and liquid polyhydric alcohols.

5. A composition for skeletal muscle regeneration, characterized by containing the differentiation promoter for skeletal muscle stem cells according to Claim 1 or 2.

6. A composition for improving the impairment of muscle regeneration by reactive oxygen species, characterized by containing the differentiation promoter for skeletal muscle stem cells according to Claim 1 or 2.

7. A method for promoting the differentiation of skeletal muscle stem cells, comprising the step of culturing skeletal muscle stem cells in a medium containing an extract of Rosa multiflora var. cathayensis.

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