Differentiation promoter for skeletal muscle stem cells

Abstract

To discover a highly safe material derived from natural sources that acts on skeletal muscle stem cells to promote their differentiation and enables simple 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 Althaea officinalis 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 of a living body atrophy with aging, and there is a possibility of developing a locomotive syndrome including sarcopenia. Recent studies have revealed 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 these skeletal muscles, various factors are considered to be involved. For example, chronic inflammation, oxidative stress, stem cells, various hormones, apoptosis, insulin resistance, etc. are mentioned, but in particular, skeletal muscle stem cells existing 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, and then they differentiate into myoblasts and finally engraft as 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 aging causes a decrease in the number of satellite cells in muscle tissue and a decrease in the regenerative ability of individual satellite cells (Non-Patent Document 2).

[0003] In addition, facial muscles that move the cheeks, eyes, nose, mouth, etc. also deteriorate with aging. For example, the orbicularis oculi muscle is one of the facial muscles around the eyes and closes and opens the eyes. It has been found that with aging, the muscle becomes thinner and the extrusion of adipose tissue causes eye bags around the eyes. Therefore, searching for 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 usefulness against age-related muscle atrophy but also for aesthetics. Some means for promoting muscle regeneration have been studied so far. For example, histamine H 3Although a muscle production promoter containing a compound having 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, they do not act on skeletal muscle stem cells (muscle satellite cells) and are not derived from natural products.

[0004] Althaea (scientific name: Althaea officinalis) is a perennial plant belonging to the genus Althaea in the family Malvaceae. It has been reported to have effects such as a whitening effect (Patent Document 3) and an anti-inflammatory effect (Patent Document 4).

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 studies to solve the above problems, the present inventors have found that an extract of Althaea 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 an extract of Althaea as an active ingredient. (2) An agent for promoting the differentiation of skeletal muscle stem cells, containing an extract of Althaea cultivated by irradiation with a combination of two types of artificial light having different wavelength ranges as an active ingredient. (3) The agent for promoting the differentiation of skeletal muscle stem cells according to (2), wherein the two types of artificial light having different wavelength ranges are blue light having a wavelength range of 400 to 515 nm and red light having a wavelength range of 570 to 730 nm. (4) The agent for promoting the differentiation of skeletal muscle stem cells according to (3), wherein the ratio of the photosynthetic photon flux density (PPFD) of the blue light to the red light is 4:1 to 2:1. (5) A composition for skeletal muscle regeneration, characterized by containing the agent for promoting the differentiation of skeletal muscle stem cells according to (1) or (2). (6) A composition for improving the impairment of muscle regeneration by reactive oxygen species, characterized by containing the agent for promoting the differentiation of skeletal muscle stem cells according to (1) or (2). (7) A method for promoting the differentiation of skeletal muscle stem cells, including the step of culturing skeletal muscle stem cells in a medium containing an extract of Althaea.

Effects of the Invention

[0010] According to the present invention, there is provided a myoblast differentiation promoter that can promote the differentiation of skeletal muscle stem cells and simply and efficiently regenerate skeletal muscle. Therefore, the myoblast differentiation promoter of the present invention is effective in improving and preventing sarcopenia, muscle strength decline, wrinkles, sagging, etc. 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.

Mode for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described in detail. The myoblast differentiation promoter according to the present invention (hereinafter, also referred to as "the agent of the present invention") contains an extract of Althaea as an active ingredient.

[0012] In the present invention, the "skeletal muscle stem cell" refers to a mononuclear stem cell called a muscle satellite cell that exists between the cell membrane and the basement membrane of muscle fibers. When stimulated such as muscle fiber damage, skeletal muscle stem cells are activated to become myoblasts, and myoblasts proliferate by several cell divisions and then 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 an effect 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] The "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., as well as morphology such as multinucleation and formation of muscle fibers, and functions such as contractility.

[0014] Althaea (scientific name: Althaea officinalis) used in the present invention is a perennial plant belonging to the genus Althaea in the Malvaceae family, and is also called by other names such as Marshmallow. It has cold tolerance and is widely distributed across Europe, North Africa, and West Asia. The leaves are green, ovate-heart-shaped with serrations, and have stellate hairs.

[0015] In the present invention, the extract of Althaea refers to the extract of a part of the plant body such as the flowers, seeds, leaves, stems, roots of Althaea or the whole plant body (whole herb), or a mixture thereof. However, the parts used as extraction raw materials in the present invention are preferably the roots and leaves. Also, for extraction, the plant body can be used as it is, or treatments such as drying, pulverization, and cutting into small pieces can be performed.

[0016] In a preferred embodiment of the present invention, as the extraction material of hollyhock, hollyhock cultivated by irradiating a combination of two types of artificial light with different wavelength ranges is used. As a cultivation method of hollyhock, it can be carried out by cultivation using soil or hydroponics. When carried out by hydroponics, after sowing the seeds, in the state of having taken root, it can be subjected to hydroponics. The cultivation is preferably carried out in a facility where the temperature, light, and carbon dioxide concentration are controlled. The cultivation temperature is 15 to 30 °C, preferably 20 to 25 °C. The cultivation period varies depending on the conditions of irradiating the above artificial light, but it can generally be harvested in 10 to 30 days. It is also possible to cultivate for a longer period.

[0017] The light source for irradiating artificial light may be any light source that can selectively irradiate a specific wavelength range, and examples include optical semiconductor elements such as light-emitting diodes (LEDs) and laser diodes, and LEDs are preferred.

[0018] As the two types of artificial light with different wavelength ranges irradiated on hollyhock, blue light with a wavelength range of 400 to 515 nm and red light with a wavelength range of 570 to 730 nm are preferred, and blue light with a wavelength range of 430 to 460 nm and red light with a wavelength range of 630 to 680 nm are more preferred. These lights are most preferably irradiated simultaneously. The wavelength at this time refers to the maximum wavelength (peak wavelength) of the irradiation spectrum. As long as it is a light source having such a wavelength peak, it can be used either a self-made one or a commercially available one. In addition to the above two types of artificial light, light sources such as sunlight and fluorescent lamps can also be used. When using light sources such as sunlight and fluorescent lamps, an optical filter may be used so that the above wavelengths can be selectively irradiated.

[0019] The amount of light irradiated is expressed as the photosynthetically active photon flux density (PPFD), and when irradiating by combining two types of light emitters, it means the total amount of light. The amount of light is preferably 10 to 300 μmol·m -2 s -1 and preferably 50 to 200 μmol·m -2 s -1is more preferable. In the case of light amounts outside this range, growth disorders or poor growth may occur. Irradiation is preferably performed from a position 10 to 50 cm above the top of the althaea. The irradiation time can be appropriately changed according to the characteristics and purposes of the plants, but it is preferably 6 hours or more per day, and more preferably 12 to 24 hours.

[0020] The ratio of the photosynthetic photon flux density (PPFD) of blue light to red light can be selected according to purposes such as yield and effectiveness, but the ratio of blue light:red light is preferably 4:1 to 2:1, and more preferably 2:1.

[0021] In the present invention, the extraction material for obtaining the extract of althaea may be althaea cultivated under natural light, or althaea cultivated by irradiating a combination of two types of artificial light having different wavelength ranges. The extraction method for obtaining the extract from althaea is not particularly limited, and for example, a heat extraction method may be used, or a normal temperature or cold temperature extraction method may also be used. As the extraction solvent, water or hot water, or a mixed solvent of water and an organic solvent can be used. As the organic solvent, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) and the like can be used, but water-soluble organic solvents such as ethanol, methanol, acetone, n-propanol, t-butanol, propylene glycol, and 1,3-butylene glycol are preferable, and one or more of these may be mixed and used. Among them, water or hot water, a water-ethanol-based mixed polar solvent (30 to 70 v / v% ethanol aqueous solution) is more preferable, and hot water is even more preferable. In addition, an acid or an alkali can be added to the above extraction solvent to use a solvent with adjusted pH.

[0022] The amount of the solvent used is not particularly limited. For example, it may be 10 times or more, preferably 20 times or more, based on the extraction raw material (dry weight), but it is preferably 100 times or less for the convenience of operations when performing concentration or isolation after extraction. Also, the extraction temperature and time depend on the type of solvent used. For example, it can be exemplified as 10 to 100 °C, preferably 30 to 90 °C, and 30 minutes to 24 hours, preferably 1 to 10 hours. More specifically, for example, by adding water to the roots and leaves of Althaea and performing hot water extraction at 95 to 100 °C, an extract of Althaea can be obtained. Alternatively, by adding a lower alcohol (such as ethanol, etc.) to the roots and leaves of Althaea and performing extraction at room temperature (for example, 5 to 35 °C), an extract of Althaea can be obtained.

[0023] The extract may be used as the extracted solution as it is, but if necessary, within a range that does not affect its effect, treatments such as concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc., deodorization, and ethanol precipitation may be performed before use. 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.

[0024] The extract of Althaea obtained in this way has the effect of promoting the differentiation from skeletal muscle stem cells to skeletal muscle both at the biological level (in vivo) and at the culture level (in vitro), so it can be used as an active ingredient of a differentiation promoter for skeletal muscle stem cells.

[0025] The content of the extract of Althaea in the agent of the present invention is not particularly limited, but for example, in terms of the dry matter, it is preferably 0.00001 to 10% by weight, more preferably 0.0001 to 1% by weight, based on the total amount of the agent.

[0026] 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 in vitro.

[0027] When the agent of the present invention is used in vivo, it can be used as it is, but it can be formulated into various compositions such as cosmetics, pharmaceuticals, quasi-drugs, foods and drinks, together with appropriate additives within the range that does not impair the effects of the present invention, and can be 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.

[0028] When the agent of the present invention is used for the purpose of improving wrinkles, sagging, depressions, etc. caused by the decline of facial muscles (such as orbicularis oris muscle, zygomatic major / minor muscle, 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. Further, when the agent of the present invention is used 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.

[0029] When the agent of the present invention is incorporated into cosmetics or quasi-drugs, a topical skin composition is preferred, and it may be in any dosage form such as an aqueous solution system, solubilized 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. Further, the cosmetics or quasi-drugs can be selected and appropriately formulated with various components, additives, bases, etc. commonly used in topical skin compositions together with the above-mentioned extract of Althaea 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 incorporated into the topical skin composition include fats and oils (such as olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (such as lanolin, beeswax, carnauba wax, etc.), hydrocarbons (such as liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (such as myristyl alcohol, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (such as isopropyl myristate, isopropyl palmitate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (such as citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidonecarboxylic acid, etc.), saccharides (such as maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and hydrolysates of proteins, amino acids and their salts, vitamins (such as 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.

[0030] Examples of the types of cosmetics and quasi-drugs include, but are not limited to, lotions, emulsions, gels, beauty essences, general creams, sunscreen creams, packs, masks, facial cleansers, cosmetic soaps, foundations, face powders, bath agents, body lotions, body shampoos, etc.

[0031] 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, buffers, solubilizers or solubilizing aids, isotonic agents, pH adjusters, propellants, coloring agents, sweeteners, 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 Pharmaceutical Preparations of the Japanese Pharmacopoeia [2].

[0032] 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.

[0033] 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.

[0034] The form of the pharmaceutical of the present invention is not particularly limited. For example, oral preparations such as tablets, dragees, 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 absorption agents, transmucosal absorption agents, patches, and other parenteral preparations can be mentioned. Further, it may be a dry product that is redissolved at the time of use. In the case of an injectable preparation, it is provided in the state of a unit dose ampoule or a multi-dose container.

[0035] 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 doses.

[0036] 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 Althaea officinalis L. blended therein has the effect of promoting the differentiation from 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 muscles, 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 eye corners 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 muscles 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 non-use of muscles. The above-mentioned oxidative stress may be caused by ultraviolet rays, air pollution, smoking, drugs, excessive exercise, physical and mental stress, etc., regardless of the cause. Since the extract of Althaea officinalis L. 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.

[0037] The usage amount or dosage 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, etc. of the usage or administration target. For example, when orally administered to adults, the extract of Althaea officinalis L. is in the range of 0.1 to 1000 mg / day, preferably 1 to 500 mg / day, more preferably 5 to 300 mg / day, and is administered once to several times a day respectively. In some cases, an amount less than the above dosage range may be sufficient, and in other cases, it may be necessary to administer beyond the range.

[0038] When the extract of the althaea 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 the althaea, 0.001 to 30% by weight (w / w) is preferred, and 0.01 to 10% by weight (w / w) is more preferred, 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.

[0039] 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, health functional foods, or special-purpose foods. Health foods include foods provided under names such as dietary supplements, health supplements, and supplements. Health functional foods are defined by the Food Sanitation Law or the Food Promotion Law and include foods for specified health uses and foods with nutritional functions 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, pellet, powder, capsule, cream, or paste.

[0040] 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.

[0041] 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.

[0042] The blending amount of the extract of Althaea in the food or drink of the present invention may be any amount that can exert the effect 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 effects, taste, palatability, and cost.

[0043] 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 Althaea. The extract of Althaea used here may be an extract of Althaea cultivated under normal natural light, or may be an extract of Althaea cultivated by irradiating a combination of two types of artificial light with different wavelength ranges described above.

[0044] 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 art. 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 used. It is also possible to use a commercially available medium in which the above components are appropriately added to the basal medium.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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 with 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.

[0049] The addition concentration of the extract of Arteia to the medium used for cell culture is, for example, 0.1 to 1000 μg / mL, preferably 1 to 100 μg / mL. Also, during the cell culture period, the extract of Arteia may be periodically added to the medium.

[0050] The cell culture conditions may follow normal conditions, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40 °C, preferably 36 to 37 °C. CO 2 The 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 appropriately varied and set within the range where the cells can survive and proliferate.

[0051] 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 Althaea officinalis L. compared to cells cultured in the absence of the extract of Althaea officinalis L. At the mRNA level, methods for confirmation include RT-PCR, quantitative PCR, and Northern blotting using primers and probes specific for, for example, the MYOG gene or DES gene. At the protein level, immunological methods such as ELISA, flow cytometry, and Western blotting using antibodies specific for the proteins encoded by the MYOG gene or DES gene can be mentioned.

Example

[0052] 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.

[0053] [Example 1] (1) Experimental materials and growth conditions Seeds of Althaea officinalis L. were sown in vermiculite containing moisture, germinated in the dark at a temperature of 22 to 25°C, cultivated under fluorescent lights at 22 to 26°C, and seedlings were grown. Thereafter, the young shoots were wrapped in a sponge, and using a hydroponic cultivation device, at room temperature of 21 to 25°C for 24 hours, from a position 30 cm directly above the plant, blue LEDs (peak wavelength 450 nm) and red LEDs (peak wavelength 660 nm) were simultaneously irradiated, and cultivation was performed such that the total photosynthetically active photon flux density of the blue LED and the red LED was 100 μmol·m -2 s -1 −2, and the light quantity ratio of blue light to red light was set to 4:1, 3:1, or 2:1. During cultivation, the light quantity ratio was not changed. Cultivation was also performed under sunlight. After culturing for 4 weeks in all cases, the plants were harvested and dried in warm air at approximately 60°C to obtain a dried product of Althaea officinalis L.

[0054] (2) Production example of an extract of Althaea officinalis L. (1) The hollyhock cultivated under sunlight obtained in (1) was designated as "hollyhock (sunlight cultivation)", and the hollyhock cultivated with the light quantity ratio of blue light to red light being 4:1, 3:1, and 2:1 was designated as "hollyhock (blue:red = 4:1 cultivation)", "hollyhock (blue:red = 3:1 cultivation)", and "hollyhock (blue:red = 2:1 cultivation)", respectively. Using the roots and leaves of the hollyhock cultivated under each of these conditions as extraction materials, the extracts of hollyhock were produced as follows.

[0055] (Production Example 1) Preparation of hot water extract of hollyhock (sunlight cultivation) To 10 g of the dried product of hollyhock (sunlight cultivation), 200 mL of water was added, and extraction was carried out at 95 - 100 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.0 g of the hot water extract of hollyhock (sunlight cultivation).

[0056] (Production Example 2) Preparation of 50% ethanol extract of hollyhock (sunlight cultivation) 10 g of the dried product of hollyhock (sunlight cultivation) was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. After the obtained extract was filtered, it was concentrated to dryness by an evaporator to obtain 1.5 g of the 50% ethanol extract of hollyhock (sunlight cultivation).

[0057] (Production Example 3) Preparation of ethanol extract of hollyhock (sunlight cultivation) 10 g of the dried product of hollyhock (sunlight cultivation) was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After the obtained extract was filtered, it was concentrated to dryness by an evaporator to obtain 0.5 g of the ethanol extract of hollyhock (sunlight cultivation).

[0058] (Production Example 4) Preparation of hot water extract of hollyhock (blue:red = 4:1 cultivation) To 10 g of the dried product of hollyhock (blue:red = 4:1 cultivation), 200 mL of water was added, and extraction was carried out at 95 - 100 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.6 g of the hot water extract of hollyhock (blue:red = 4:1 cultivation).

[0059] (Production Example 5) Preparation of 50% Ethanol Extract of Althaea (cultivated with blue:red = 4:1) 10 g of the dried product of Althaea (cultivated with blue:red = 4:1) was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 1.2 g of 50% ethanol extract of Althaea (cultivated with blue:red = 4:1).

[0060] (Production Example 6) Preparation of Ethanol Extract of Althaea (cultivated with blue:red = 4:1) 10 g of the dried product of Althaea (cultivated with blue:red = 4:1) was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 0.4 g of ethanol extract of Althaea (cultivated with blue:red = 4:1).

[0061] (Production Example 7) Preparation of Hot Water Extract of Althaea (cultivated with blue:red = 3:1) 200 mL of water was added to 10 g of the dried product of Althaea (cultivated with blue:red = 3:1), and extraction was carried out at 95 - 100 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.2 g of hot water extract of Althaea (cultivated with blue:red = 3:1).

[0062] (Production Example 8) Preparation of 50% Ethanol Extract of Althaea (cultivated with blue:red = 3:1) 10 g of the dried product of Althaea (cultivated with blue:red = 3:1) was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 1.0 g of 50% ethanol extract of Althaea (cultivated with blue:red = 3:1).

[0063] (Production Example 9) Preparation of Ethanol Extract of Althaea (cultivated with blue:red = 3:1) 10 g of the dried product of Althaea (cultivated with blue:red = 3:1) was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 0.4 g of ethanol extract of Althaea (cultivated with blue:red = 3:1).

[0064] (Production Example 10) Preparation of Hot Water Extract of Althaea (Cultivated with Blue:Red = 2:1) To 10 g of the dried product of Althaea (cultivated with blue:red = 2:1), 200 mL of water was added, and extraction was carried out at 95 - 100 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.3 g of the hot water extract of Althaea (cultivated with blue:red = 2:1).

[0065] (Production Example 11) Preparation of 50% Ethanol Extract of Althaea (Cultivated with Blue:Red = 2:1) 10 g of the dried product of Althaea (cultivated with blue:red = 2:1) was immersed in 200 mL of a 50% ethanol aqueous solution at room temperature for 7 days for extraction. After the obtained extract was filtered, it was concentrated to dryness with an evaporator to obtain 1.2 g of the 50% ethanol extract of Althaea (cultivated with blue:red = 2:1).

[0066] (Production Example 12) Preparation of Ethanol Extract of Althaea (Cultivated with Blue:Red = 2:1) 10 g of the dried product of Althaea (cultivated with blue:red = 2:1) was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After the obtained extract was filtered, it was concentrated to dryness with an evaporator to obtain 0.3 g of the ethanol extract of Althaea (cultivated with blue:red = 2:1).

[0067] [Example 2] Evaluation of the Effect of Althaea Extract on the Differentiation Promotion of 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 Althaea extract to evaluate how the Althaea extract is involved in the promotion of 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 5Cells were seeded one by one and cultured for 2 days 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, the test substances (extracts of Arteia in Production Examples 1 to 12) were added at a final concentration of 10 μg / ml and cultured. Cells were collected 2 days after the addition of the Arteia extract, and the gene expression levels of MYOG (Myogenin) and DES (Desmin), which are skeletal muscle differentiation markers, were analyzed.

[0068] The gene expression analysis of MYOG and DES was performed as follows. After the cells after the addition of the Arteia extract 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 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.

[0069] 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)

[0070] 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), which is an internal standard (differentiation marker gene expression level / 18srRNA gene expression level). The value was 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.

[0071] [Table 1]

[0072] As shown in Table 1, it was shown that the extract of Althaea induces and promotes the differentiation of skeletal muscle stem cells. The hot water extract showed a high differentiation-promoting effect under each condition. In addition, the differentiation-promoting effect of the extract of Althaea irradiated with a combination of artificial light of blue light with a wavelength of 450 nm and red light with a wavelength of 660 nm was high, and the extract with a light intensity ratio of blue light to red light of 2:1 had the highest differentiation-promoting effect.

[0073] [Example 3] Efficacy of the extract of Althaea in the impairment of 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 Althaea was allowed to act was evaluated. 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 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 reactive oxygen species H 2O 2 was added to a final concentration of 100 μM. Also, the test substances (extracts of Arteia in Production Examples 1 to 12) were simultaneously added to a final concentration of 10 μg / ml and cultured. After 3 days, the cells were collected, and the gene expression levels of MYOG (Myogenin) and DES (Desmin), which are differentiation markers of skeletal muscle, were analyzed as indicators.

[0074] The gene expression analysis of MYOG and DES was performed as follows. After the cells after the addition of the Arteia extract were washed twice with PBS(-), 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. 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 performed according to the defined methods.

[0075] The promoting effect on 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, and the gene relative expression level (differentiation marker gene expression level / 18srRNA gene expression level) with a value of 1. In contrast, 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.

[0076]

Table 2

[0077] As shown in Table 2, it was shown that hydrogen peroxide suppresses the differentiation of skeletal muscle stem cells. When the extract of Althaea was added, it was shown that the suppression of the differentiation of skeletal muscle stem cells by hydrogen peroxide was improved in all cases, and the hot water extract showed a high improvement effect. In addition, the improvement effect of the extract of Althaea irradiated with a combination of artificial light of blue light with a wavelength of 450 nm and red light with a wavelength of 660 nm was high, and the one with a light quantity ratio of blue light to red light of 2:1 had the highest improvement effect.

Industrial Applicability

[0078] 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 promoter for the differentiation of skeletal muscle stem cells, containing an extract of Althaea as an active ingredient.

2. A promoter for the differentiation of skeletal muscle stem cells, containing an extract of Althaea cultivated by irradiation with a combination of two types of artificial light having different wavelength ranges as an active ingredient.

3. The promoter for the differentiation of skeletal muscle stem cells according to Claim 2, wherein the two types of artificial light having different wavelength ranges are blue light having a wavelength range of 400 to 515 nm and red light having a wavelength range of 570 to 730 nm.

4. The promoter for the differentiation of skeletal muscle stem cells according to Claim 3, wherein the ratio of the photosynthetic photon flux density (PPFD) of the blue light to the red light is 4:1 to 2:

1.

5. A composition for skeletal muscle regeneration, characterized by containing the promoter for the differentiation of 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 promoter for the differentiation of 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 Althaea.