Muscle strengthening agent

Abstract

The purpose of the present invention is to provide a relatively safe muscle enhancer that, by activating muscle satellite cells, is capable of effectively increasing or maintaining muscle mass and muscle strength, and capable of preventing or suppressing any loss in muscle mass or muscle strength. The muscle enhancer according to the present invention is characterized by containing a specific ellagitannin as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a muscle enhancer that can increase or maintain muscle mass and muscle strength, or prevent or suppress a decrease in muscle mass and muscle strength.

Background Art

[0002] In recent years, with the progress of lack of exercise due to the development of transportation means and the aging of the population, a decrease in muscle mass and muscle strength (muscle atrophy) has become a major problem. Generally, it is said that muscle mass and muscle strength decrease from around 40 years old, and many elderly people are said to be in a state of sarcopenia (age-related muscle loss). Sarcopenia mainly refers to a decrease in muscle mass and muscle strength due to aging, and is an important factor in locomotive syndrome (a syndrome caused by disorders of locomotor organs such as muscles, bones, joints, cartilage, and intervertebral discs resulting in a decrease in mobility function) and frailty (a state in which muscle strength and physical and mental vitality are decreased), and it has been pointed out that it causes the need for care. Thus, a decrease in muscle mass and muscle strength is closely related not only to a decrease in the quality of life (QOL), but also to a decrease in activities of daily living (ADL), the occurrence of complications, and an increase in the care burden.

[0003] It is said that both appropriate nutrition intake and exercise are necessary to enhance muscles, that is, to increase and maintain muscle mass and muscle strength, and further to prevent or suppress a decrease in muscle mass and muscle strength. However, especially in the elderly, in addition to a decrease in food intake due to a decrease in appetite, the biological function efficiency of converting ingested proteins and amino acids into muscle proteins is also decreased. Furthermore, in the elderly with decreased physical strength and physical function, it is difficult to continuously perform exercise, and the efficiency of increasing muscle mass by exercise is also decreased. Therefore, for example, when the activity level of the elderly decreases due to injury or illness, the muscle mass and muscle strength further decrease accordingly, and they may fall into a negative cycle of further decrease in activity level and worsening of muscle atrophy. Thus, in the elderly, muscle enhancement by nutrition intake and exercise is insufficient, and more effective muscle enhancement means are desired.

[0004] In addition, for the purposes of maintaining health, improving exercise ability, preventing or improving obesity and metabolic syndrome, losing weight, etc., and also for the purposes of promoting growth, increasing muscle mass, improving meat quality, etc. in animals other than humans, enhancing muscles is desired.

[0005] In order to enhance muscles, it is necessary to enlarge or maintain the muscle fibers that make up the muscles. Muscle fibers are multinucleated cells, and the number of their nuclei controls the cell size. Therefore, in order to enlarge or maintain muscle fibers, it is necessary to increase the number of nuclei. However, since the nuclei incorporated into muscle fibers (muscle nuclei) do not proliferate, it is essential to supply new nuclei to muscle fibers from outside the cells. The supply of nuclei to muscle fibers is carried out by muscle satellite cells (also called satellite cells) that exist between the basement membrane and the cell membrane of muscle fibers. Usually, muscle satellite cells are in a "resting state" with a stopped cell cycle and are in an undifferentiated state, but they are activated during exercise load, muscle damage, and growth, and shift to an "activated state" in which the cell cycle progresses. Activated muscle satellite cells supply new nuclei to muscle fibers through a series of processes such as proliferation, differentiation, and fusion with muscle fibers, and as a result, the muscle fibers hypertrophy. Therefore, activating muscle satellite cells contributes to muscle enhancement.

[0006] Growth hormone is known as a component for enhancing muscles. However, growth hormone has problems such as doping issues, side effects such as high blood pressure, carcinogenicity, liver damage, testicular atrophy, amenorrhea, delusions, and paranoia, and it is not preferable for daily and long-term continuous intake.

[0007] Therefore, substances with muscle-strengthening effects are being explored from safe ingredients. For example, Patent Document 1 discloses a muscle function decline inhibitor containing catechins included in tea beverages as an active ingredient. Patent Document 2 describes a muscle-strengthening agent containing a plant of the genus Saraca of the Caesalpiniaceae family that grows wild in India, Sri Lanka, and Southeast Asian countries or an extract thereof. Further, Non-Patent Document 1 reports the effect of leucine, which is a branched-chain amino acid, on muscle protein synthesis. Moreover, Patent Document 3 describes the activation of mitochondria in skeletal muscle-derived cells by extracts of pomegranate, punicalagin and ellagic acid contained therein, and urolithin, which are metabolites thereof, and the increase in muscle mass by ingesting punicalagin, ellagic acid, and urolithin. Although Patent Documents 1 to 3 each describe the action on muscles, none of them activate satellite cells. Further, Patent Document 4 discloses a satellite cell differentiation promoter containing Rafflesia, which is a plant of the family Rafflesiaceae, or an extract thereof as an active ingredient, but it only shows an effect of inducing differentiation on skeletal muscle-derived cells and does not activate satellite cells. In contrast, Patent Document 5 describes that an extract of Citrus limetta shows an action of activating satellite cells.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] As described above, in recent years, with the advent of a super-aged society, the decline in muscle mass and muscle strength has become a serious problem, and countermeasures are being sought. Although research has been conducted to increase and maintain muscle mass and muscle strength, appropriate nutrition intake and exercise are only recommended as actual countermeasures. Although drugs for preventing or suppressing the decline in muscle mass and muscle strength are on the market, drugs that are safe and show a sufficient muscle strengthening effect have not yet been on the market. Therefore, an object of the present invention is to provide a relatively safe muscle strengthening agent that can effectively increase or maintain muscle mass and muscle strength and prevent or suppress the decline in muscle mass and muscle strength by activating satellite cells.

Means for Solving the Problems

[0011] The present inventors have conducted intensive research to solve the above problems. As a result, they have found that a specific ellagitannin, which is a kind of polyphenol, activates satellite cells that play an important role in increasing and maintaining muscle mass and muscle strength, and thus completed the present invention. The present invention is shown below.

[0012] [1] A muscle strengthening agent characterized by containing an ellagitannin represented by the following formula (I) as an active ingredient.

Chemical formula

[10] The use according to [8] above, wherein the ellagitannin represented by the above formula (I) is stachyurin.

[11] The use according to any one of [8] to

[10] above, for increasing and / or maintaining muscle mass and / or muscle strength, or for preventing and / or suppressing a decrease in muscle mass and / or muscle strength.

[12] The use according to any one of [8] to

[11] above, for administering ellagitannin represented by the above formula (I) to a human.

[13] The use according to any one of [8] to

[11] above, for administering ellagitannin represented by the above formula (I) to a human having or having a possibility of having one or more diseases selected from the group consisting of sarcopenia, locomotive syndrome, and frailty, or to an elderly person.

[14] The use according to any one of [8] to

[13] above, for administering ellagitannin represented by the above formula (I) at 0.1 mg / kg body weight / day or more.

[15] The use according to any one of [8] to

[14] above, for administering ellagitannin represented by the above formula (I) as a component of a composition for food or drink.

[16] A muscle strengthening method, comprising the step of administering ellagitannin represented by the above formula (I) as an active ingredient to a subject.

[17] The muscle strengthening method according to

[16] above, wherein the ellagitannin represented by the above formula (I) is casuarinin.

[18] The muscle strengthening method according to

[16] above, wherein the ellagitannin represented by the above formula (I) is stachyurin.

[19] The muscle strengthening method according to any one of

[16] to

[18] above, which increases and / or maintains muscle mass and / or muscle strength, or prevents and / or suppresses a decrease in muscle mass and / or muscle strength.

[20] The muscle strengthening method according to any one of

[16] to

[19] above, wherein the subject is a human.

[21] The muscle strengthening method according to any one of

[16] to

[19] above, wherein the subject is a human having or having a possibility of having one or more diseases selected from the group consisting of sarcopenia, locomotive syndrome, and frailty, or an elderly person.

[22] The muscle strengthening method according to any one of

[16] to

[21] above, wherein the ellagitannin represented by the above formula (I) is administered at 0.1 mg / kg body weight / day or more.

[23] The muscle strengthening method according to any one of

[16] to

[22] above, wherein the ellagitannin represented by the above formula (I) is administered as a component of a food or drink composition.

[24] A muscle strengthening method characterized by including a step of administering the muscle strengthening agent according to any one of [1] to [7] above.

[25] The muscle strengthening method according to

[24] above, wherein the administration target is a person having or having a possibility of having one or more diseases selected from the group consisting of sarcopenia, locomotive syndrome, and frailty, or an elderly person.

[26] A myosatellite cell activator characterized by containing the ellagitannin represented by the above formula (I) as an active ingredient.

[27] The myosatellite cell activator according to

[26] above, wherein the ellagitannin represented by the above formula (I) is casuarinin.

[28] The myosatellite cell activator according to

[26] above, wherein the ellagitannin represented by the above formula (I) is stachyurin.

[29] The satellite cell activator according to any one of

[26] to

[28] above, which is for increasing and / or maintaining muscle mass and / or muscle strength, or for preventing or suppressing a decrease in muscle mass and / or muscle strength.

[30] The satellite cell activator according to any one of

[26] to

[29] above, which is for administering ellagitannin represented by the above formula (I) at 0.1 mg / kg body weight / day or more.

[31] The satellite cell activator according to any one of

[26] to

[30] above, which is a composition for food and drink.

[32] The satellite cell activator according to

[31] above, which is a food with function claims.

[33] A method for activating satellite cells, comprising a step of administering ellagitannin represented by the above formula (I) as an active ingredient to a subject.

[34] The method for activating satellite cells according to

[33] above, wherein the ellagitannin represented by the above formula (I) is casuarinin.

[35] The method for activating satellite cells according to

[33] above, wherein the ellagitannin represented by the above formula (I) is stachyurin.

[36] The method for activating satellite cells according to any one of

[33] to

[35] above, which is for increasing and / or maintaining muscle mass and / or muscle strength, or for preventing and / or suppressing a decrease in muscle mass and / or muscle strength.

[37] The method for activating satellite cells according to any one of

[33] to

[36] above, wherein the subject is a human.

[38] The method for activating satellite cells according to any one of

[33] to

[37] above, wherein the subject is a human having or having a possibility of having one or more diseases selected from the group consisting of sarcopenia, locomotive syndrome, and frailty, or an elderly person.

[39] The method for activating satellite cells according to any one of

[33] to

[38] above, which is for administering ellagitannin represented by the above formula (I) at 0.1 mg / kg body weight / day or more.

[40] The method for activating satellite cells according to any one of

[33] to

[39] above, which is for administering ellagitannin represented by the above formula (I) as a component of a composition for food and drink. [Effect of the Invention]

[0013] The muscle enhancer according to the present invention contains, as an active ingredient, a specific ellagitannin which is a kind of polyphenol, and thus is relatively safe. It exhibits an action of activating satellite cells in the same manner as exercise, and can be used for increasing or maintaining muscle mass and muscle strength, and further for preventing or suppressing a decrease in muscle mass and muscle strength. [Brief Description of the Drawings]

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

[0015] The muscle enhancer according to the present invention contains, as an active ingredient, an ellagitannin represented by the formula (I) (hereinafter referred to as "ellagitannin (I)"). The muscle enhancer according to the present invention has a muscle enhancing action.

[0016] In the present invention, "muscle strengthening" means increasing or maintaining muscle mass and muscle strength, and in addition, preventing or suppressing a decrease in muscle mass and muscle strength. Therefore, the "muscle strengthening agent" of the present invention can be used to increase or maintain muscle mass and muscle strength, and further prevent or suppress a decrease in muscle mass and muscle strength. "Muscle mass" refers to the amount of muscle, and "muscle strength" refers to the force exerted by the muscle. Further, since the muscle strengthening agent of the present invention has an action of activating satellite cells, satellite cells that have proliferated and differentiated by ingestion of the muscle strengthening agent of the present invention fuse with muscle fibers and become new muscle nuclei, thereby increasing muscle mass. That is, it is also a muscle strengthening agent and a satellite cell activator due to an increase in the number of satellite cells. The muscle strengthening agent of the present invention can be used to promote muscle strengthening in healthy individuals and athletes who require muscle strengthening, children and young people who require physical development such as weight gain. Furthermore, the muscle strengthening agent of the present invention can also be used for the prevention and improvement of muscle mass and muscle strength decline due to sarcopenia caused by aging, inactivity due to lack of exercise, long-term bed rest, gibbs fixation, microgravity, etc., secondary sarcopenia due to malnutrition, invasion, cachexia, etc., myogenic diseases (muscular dystrophy, congenital myopathy, etc.), neurodegenerative diseases (amyotrophic lateral sclerosis, spinal muscular atrophy, etc.). It can also be used for the prevention and improvement of conditions and diseases caused by a decrease in muscle mass and muscle strength such as locomotive syndrome and frailty. That is, the muscle strengthening agent of the present invention can also be used to maintain or enhance muscle mass and muscle strength in those with decreased muscle mass and muscle strength, for example, the elderly. Here, the elderly are, for example, those 60 years old or older, more preferably 70 years old or older. In addition, the muscle strengthening agent of the present invention can also be used for animals such as livestock for which growth promotion, increased meat mass, and improved meat quality are desired. Incidentally, since the muscle strengthening agent of the present invention can strengthen muscles, it can also be used for the purpose of preventing or improving obesity and metabolic syndrome, and losing weight.

[0017] A satellite cell is an undifferentiated muscle stem cell existing between the basement membrane and the cell membrane of a muscle fiber. "Activation of satellite cells" in the present invention means that quiescent satellite cells transition to the proliferation phase, and through differentiation and fusion with muscle fibers, supply nuclei to the muscle fibers. Activation of satellite cells increases the number of nuclei that control the size of muscle fibers, leading to muscle fiber hypertrophy and, thus, muscle strengthening. Activation of satellite cells can be evaluated by measuring the number of cells, the amount of nucleic acid, and the uptake amount of DNA analogs such as bromodeoxyuridine.

[0018] Ellagitannin is a type of polyphenol widely present in the plant kingdom such as the bark of oaks, and is a general term for a group of compounds having a structure in which a polyhydric alcohol such as glucose and a polyhydric phenolic acid such as hexahydroxydiphenic acid and gallic acid are ester-bonded. The hexahydroxydiphenic acid structural part in the molecule of ellagitannin is called a hexahydroxydiphenoyl group, and the gallic acid structural part is called a galloyl group. Ellagitannin is a highly stable compound, but is hydrolyzed by acids, alkalis, and enzymes. The hexahydroxydiphenic acid released from the parent skeleton by hydrolysis rapidly undergoes intramolecular dehydration condensation as shown in the following formula to produce stable ellagic acid. Many plants containing ellagitannin are used as crude drugs, and ellagitannin having physiological activities such as antioxidant action and anticancer activity is known.

[0019]

Chemical formula

[0020] Ellagitannin (I) is a compound in which two hexahydroxydiphenoyl groups and one galloyl group are ester-bonded to glucose. The glucose in ellagitannin (I) has an open-chain structure by ring-opening, and two hexahydroxydiphenoyl groups are ester-bonded to the hydroxyl groups at the 2- and 3-positions and the 4- and 6-positions of glucose, respectively, and a galloyl group is ester-bonded to the hydroxyl group at the 5-position. Furthermore, the carbon atom (anomeric carbon atom) at the 1-position of glucose forms a C-glycosidic bond with the hexahydroxydiphenoyl group bonded to the 2- and 3-positions. Examples of ellagitannin (I) include casuarinin and stachyurin. Casuarinin and stachyurin differ in the configuration at the 1-position of glucose, with casuarinin having an α-glucose structure and stachyurin having a β-glucose structure.

[0021] [Chemical formula]

[0022] The method for obtaining ellagitannin (I) is not particularly limited and can be chemically synthesized from known compounds. Alternatively, ellagitannin (I) may be purified from plants. For example, casuarinin and / or stachyurin are contained in Casuarina stricta of the genus Casuarina in the family Casuarinaceae; Stachyurus praecox of the genus Stachyurus in the family Stachyuraceae; Psidium guajava and Psidium cattleianum of the genus Psidium in the family Myrtaceae; Syzygium jambos, Syzygium samarangense, and Syzygium aqueum of the genus Syzygium in the family Myrtaceae; Feijoa sellowiana of the genus Feijoa in the family Myrtaceae; Eucalyptus viminalis of the genus Eucalyptus in the family Myrtaceae; and Pimenta dioica of the genus Pimenta in the family Myrtaceae. In addition, the present inventors have uniquely found that casuarinin and stachyurin are also contained in Backhousia citriodora of the genus Backhousia in the family Myrtaceae.

[0023] The method for purifying ellagitannin (I) from plants is not particularly limited. Since ellagitannin (I) is water-soluble due to having a large number of hydroxyl groups, it can be extracted from parts such as leaves, stems, roots, branches, and flowers of plants containing ellagitannin (I) using aqueous solvents such as water, water-miscible organic solvents, and mixed solvents thereof. The obtained extract may be used as it is, or dried by vacuum drying, freeze drying, drum drying, spray drying, etc., and then the target ellagitannin (I) may be purified by chromatography or the like.

[0024] Ellagitannin (I) may form a salt in a muscle enhancer, and the technical scope of the present invention includes muscle enhancers containing a salt of ellagitannin (I). Such salts include, for example, sodium salts and potassium salts.

[0025] The dosage form of the muscle-strengthening agent according to the present invention is not particularly limited. For example, it may be ellagitannin (I) itself, a composition combined with other components, or a solution or suspension thereof. In addition, plants containing ellagitannin (I) and fractions extracted and purified from plants can also be used as the active ingredient of the present invention. The dosage form of the muscle-strengthening agent is not particularly limited, and examples thereof include tablets, powders, capsules, dragees, granules, liquids, external preparations, and the like. For the muscle-strengthening agent according to the present invention, pharmaceutically acceptable additives may be used according to the dosage form. Examples of such additives include excipients, disintegrants, lubricants, binders, antioxidants, coloring agents, sweeteners, anti-aggregation agents, solubilizing agents for active ingredients, stabilizers, and the like. The above excipients are not particularly limited, and examples thereof include sucrose, lactose, glucose, corn starch, dextrin, mannitol, crystalline cellulose, calcium phosphate, calcium sulfate, magnesium sulfate, and the like. The above disintegrants are not particularly limited, and examples thereof include starch, agar, calcium citrate, calcium carbonate, sodium hydrogen carbonate, dextrin, crystalline cellulose, carboxymethyl cellulose, tragacanth, and the like. The above lubricants are not particularly limited, and examples thereof include talc, magnesium stearate, polyethylene glycol, silica, hydrogenated vegetable oil, and the like. The above binders are not particularly limited, and examples thereof include ethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, tragacanth, shellac, gelatin, gum arabic, polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, sorbitol, and the like. The above antioxidants are not particularly limited, and examples thereof include ascorbic acid, tocopherol, sodium bisulfite, sodium thiosulfate, sodium pyrosulfite, citric acid, and the like. The above coloring agents are not particularly limited, and examples thereof include those permitted to be added to pharmaceuticals and foods. The above sweeteners are not particularly limited, and examples thereof include those permitted to be added to pharmaceuticals and foods.The anti-aggregation agent is not particularly limited, and examples thereof include stearic acid, talc, light anhydrous silicic acid, hydrous silicon dioxide, and the like. The solubilizing agent for the active ingredient is not particularly limited, and examples thereof include organic acids such as fumaric acid, succinic acid, and malic acid. The stabilizer is not particularly limited, and examples thereof include benzoic acid, sodium benzoate, ethyl paraoxybenzoate, propylene glycol, and the like.

[0026] The muscle strengthening agent according to the present invention can also be used, for example, as a composition for food and drink, a pharmaceutical product, a quasi-drug, a feed, pet food, or an animal medicine.

[0027] The muscle strengthening agent according to the present invention may be used or ingested as it is as a composition for food and drink such as a food for specified health use or a food with functional claims that exhibits a muscle strengthening effect, a pharmaceutical product, a quasi-drug, a supplement, a feed, pet food, or an animal medicine, or it may be used by being contained in a composition for food and drink, a pharmaceutical product, a quasi-drug, a feed, pet food, an animal medicine, etc. Further, the muscle strengthening agent of the present invention may be in the form of a preparation for oral ingestion. Examples of the preparation for oral ingestion include forms that can be orally ingested such as capsules (hard capsules, microcapsules, soft capsules), tablets, powders, chewable preparations, syrups, and liquids. The capsule base material in the case of a capsule agent is not particularly limited, and includes gelatin derived from bovine bone, cowhide, pigskin, fish skin, etc., and other base materials, for example, seaweed-derived products such as carrageenan and alginic acid that can be used as food additives, plant seed-derived products such as locust bean gum and guar gum, microorganism-derived products such as pullulan and curdlan, and production agents containing celluloses can also be used.

[0028] In addition, the muscle strengthening agent according to the present invention can also be added to general foods and drinks. The foods and drinks to which the muscle strengthening agent according to the present invention is added are not particularly limited. For example, beverages such as milk beverages, soft drinks, sports drinks, nutritional drinks, beauty drinks, and liquid nutritional agents; confectioneries such as chewing gum, chocolate, candies, jelly, cakes, biscuits, and crackers; frozen confections such as ice cream and iced confections; noodles such as udon, Chinese noodles, spaghetti, and instant noodles; processed products such as fish cakes, fish sausage, and fish slices; seasonings such as dressings, mayonnaise, and sauces; bread, ham, congee, rice, soup, various retort foods, various frozen foods, etc. can be appropriately added and used. The foods and drinks containing the muscle strengthening agent according to the present invention can be used for applications such as so-called health foods, supplements, functional foods, foods with functional claims, dietary supplements, foods for specified health uses, foods with nutritional functions, nursing foods, smile care foods, chewing and swallowing assist foods, thick liquid foods, foods for patients, etc. Needless to say, it can also be used in other food forms. Furthermore, it can also be used for pet foods and livestock feeds.

[0029] The muscle strengthening agent of the present invention may be in the form of a parenteral preparation. For example, it can also be in the form of being applied to the skin. In this case, the dosage form is not particularly limited. For example, the muscle strengthening agent according to the present invention is dissolved or mixed and dispersed in a base to form a cream, paste, jelly, gel, emulsion, or liquid form (such as ointments, liniments, lotions, sprays, etc.), a preparation in which ellagitannin (I) is dissolved or mixed and dispersed in a base and spread on a support (such as patches, etc.), a preparation in which the above composition is dissolved or mixed and dispersed in an adhesive and spread on a support (such as plasters, tapes, etc.).

[0030] When the muscle strengthening agent of the present invention is used as a quasi-drug, the quasi-drug refers to a quasi-drug defined in the Pharmaceutical Affairs Law, and examples include oral preparations (liquid preparations such as extracts, elixirs, syrups, tinctures, and lemonades, and solid preparations such as capsules, granules, pills, powders, and tablets).

[0031] The muscle strengthening agent of the present invention contains ellagitannin represented by the formula (I) as an active ingredient. The proportion of the ellagitannin in the muscle strengthening agent depends on the dosage form and the like, but for example, it can be 0.0001% by mass or more. As the proportion, 0.001% by mass or more or 0.003% by mass or more is preferable, 0.01% by mass or more or 0.03% by mass or more is more preferable, and 0.1% by mass or more or 0.3% by mass or more is even more preferable. Regarding the upper limit of the proportion, it may be 100% by mass or less, but 50% by mass or less or 40% by mass or less is preferable, 20% by mass or less or 10% by mass or less is more preferable, and 7% by mass or less or 5% by mass or less is even more preferable.

[0032] The muscle strengthening agent according to the present invention exhibits excellent muscle strengthening effects such as an activating effect on satellite cells. Such an activating effect on satellite cells can be evaluated, for example, by the following in vitro test. Specifically, after adding a test substance to the culture solution of satellite cells isolated from an animal and culturing for 24 hours, bromodeoxyuridine (BrdU) is added so that the final concentration becomes 10 μM, and the mixture is incubated for 2 hours. Next, the satellite cells are fixed at 4°C for 10 minutes using ice-cold methanol added with 0.1% H2O2, and further subjected to DNA denaturation treatment at 37°C for 1 hour using 2N hydrochloric acid. Then, an anti-BrdU antibody is used as the primary antibody and an HRP-conjugated anti-mouse IgG antibody is used as the secondary antibody, and color development is carried out with diaminobenzidine (DAB) to detect BrdU-positive cells. The proportion of BrdU-positive cells in the total number of cells can be determined and used as the satellite cell activation rate. Regarding the satellite cell activation rate of the muscle strengthening agent according to the present invention, if the satellite cell activation rate increases due to the treatment with the test substance as compared with the solvent control, the satellite cells are considered to be activated. As the increase rate compared with the solvent control, 2% or more is preferable, 3% or more is more preferable, and 5% or more is even more preferable.

[0033] The muscle strengthening method according to the present invention includes the step of administering the muscle strengthening agent according to the present invention. Examples of patients to whom the muscle strengthening agent according to the present invention should be administered include, for example, patients who should enhance muscle mass and muscle strength, and patients who should prevent a decrease in muscle mass and muscle strength. More specifically, as the administration target, there are mentioned those who have or are likely to have one or more diseases selected from the group consisting of sarcopenia, locomotive syndrome, and frailty, or the elderly.

[0034] The muscle strengthening agent according to the present invention can be administered not only to humans but also to animals other than humans. That is, the present invention also relates to a muscle strengthening method characterized by including the step of administering the muscle strengthening agent according to the present invention to an animal. Examples of the animal to be administered include, for example, farm animals, pet animals, and sports animals. The farm animals are not particularly limited, but examples include livestock such as horses, cows, pigs, sheep, goats, camels, and llamas, experimental animals such as mice, rats, guinea pigs, and rabbits, poultry such as chickens, ducks, turkeys, and ostriches, fish, crustaceans, or shellfish. The pet animals are not particularly limited, but examples include dogs and cats. The sports animals are not particularly limited, but examples include racehorses.

[0035] The administration frequency and dosage of the muscle strengthening agent according to the present invention may be appropriately adjusted according to the administration target, age, gender, condition, etc., and an amount capable of exerting a muscle strengthening effect is administered to the administration target. For example, the lower limit of the dosage for humans is not particularly limited, but the dosage of ellagitannin (I) per day is preferably 0.1 mg / kg body weight or more, or 0.5 mg / kg body weight or more, more preferably 1 mg / kg body weight or more, or 2 mg / kg body weight or more, and even more preferably 5 mg / kg body weight or more. Also, for example, the upper limit of the dosage for humans is not particularly limited, but the dosage of ellagitannin (I) per day is preferably 1000 mg / kg body weight or less, more preferably 500 mg / kg body weight or less, and even more preferably 100 mg / kg body weight or less. Also, the number of administrations per day is not particularly limited, and within the desired administration range, it may be administered once or divided into several times.

[0036] The muscle strengthening agent according to the present invention can be appropriately used in combination with exercises such as resistance training, physical therapy, rehabilitation, etc. for the purpose of improving the effects of increasing or maintaining muscle mass and muscle strength. Further, the muscle strengthening agent according to the present invention can also be used in combination with other pharmaceuticals, foods having a muscle strengthening effect, etc. for the purpose of improving the effects of increasing or maintaining muscle mass and muscle strength. The food having the above muscle strengthening effect is not particularly limited, but examples include amino acids such as whey protein, whey peptide, casein, casein peptide, soy protein, soy peptide, wheat protein, wheat peptide, valine, leucine, isoleucine, arginine, citrulline, ornithine, creatine, β-hydroxy-β-methylbutyric acid, and the like.

[0037] This application claims the benefit of priority based on Japanese Patent Application No. 2019-67321 filed on March 29, 2019. The entire contents of the specification of Japanese Patent Application No. 2019-67321 filed on March 29, 2019 are incorporated herein by reference for reference purposes.

Examples

[0038] Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, and it is of course possible to appropriately modify and implement within the range that can conform to the gist of the foregoing and following, and all of them are included in the technical scope of the present invention.

[0039] Example 1: Preparation of casuarinine Dried leaves (100 g) of lemon myrtle (Backhousia citriodora), which is a plant of the Rutaceae family, were immersed in water (500 mL), and extraction was carried out while stirring at 50 °C for 2 hours. The solid content was separated by filtration to obtain an extract. The obtained extract was concentrated under reduced pressure and freeze-dried to remove the solvent, and a lemon myrtle extract (16.1 g) was obtained. The obtained extract (120 mg) was dissolved in water (1 mL) and fractionated by high performance liquid chromatography (HPLC) under the following conditions. By repeating the fractionation, casuarinine (40 mg) was obtained. The structure of the compound was confirmed by NMR analysis and mass spectrometry. [HPLC Conditions] Column: 「YMC-Pack ODS-A」 manufactured by YMC, φ10 mm × 150 mm Eluent: Gradient of a mixed solution of acetonitrile - methanol and 0.1% aqueous formic acid solution

[0040] Example 2: Preparation of Stakurine In the same manner as in Example 1, stakurine was prepared from lemon martle.

[0041] Example 3: Evaluation of Myosatellite Cell Activation Ability (1) Isolation of Myosatellite Cells Muscle tissues were excised from the thigh, hip, and back of 6-month-old male SD rats (Japan SLC), and after removing and mincing fat and connective tissues, etc., they were treated with PBS containing 1.25 mg / mL protease (Sigma) at 37°C for 1 hour. After removing muscle fiber fragments etc. by centrifugation, they were seeded on plates coated with polylysine (Sigma) and fibronectin (Sigma), and after pre-culturing at 37°C for 24 hours in a 5% CO2 atmosphere, myosatellite cells were isolated by washing with PBS. As the medium, DMEM (「Dulbecco’s Modified Eagle Medium」, Thermo Fisher Scientific) supplemented with 10% horse serum (Thermo Fisher Scientific) was used. Hereinafter, this medium will be abbreviated as 「10% HS-DMEM」.

[0042] (2) Treatment with Casuarinine or Stakurine After dissolving casuarinin prepared in Example 1 or stakurinin prepared in Example 2 in water, it was added to 10% HS-DMEM to prepare 10% HS-DMEM containing casuarinin or stakurinin. The medium of isolated muscle satellite cells was replaced with 10% HS-DMEM containing casuarinin or stakurinin and cultured for 24 hours. The treatment concentration of casuarinin was 0.01 - 1.1 μM (13 - 1000 ng / mL), the treatment concentration of stakurinin was 0.09 - 0.4 μM (83 - 333 ng / mL), and 10% HS-DMEM was used as a solvent control.

[0043] (3) Evaluation of muscle satellite cell activation ability Two hours before the end of the culture, bromodeoxyuridine (BrdU, Sigma) was added to the medium to a final concentration of 10 μM. After the end of the culture, the muscle satellite cells treated with the test substance were fixed at 4°C for 10 minutes using ice-cold methanol containing 0.1% H2O2. Further, after performing DNA denaturation treatment at 37°C for 1 hour using 2N hydrochloric acid, an anti-BrdU antibody (Sigma) was used as the primary antibody and an HRP-conjugated anti-mouse IgG antibody (Sigma) was used as the secondary antibody, and color development was performed with diaminobenzidine (DAB, Sigma) to detect BrdU-positive cells. The ratio of BrdU-positive cells to the total number of cells was determined and used as the muscle satellite cell activation rate. The experiment was performed 12 times for the solvent control and 3 times for each concentration in the case of casuarinin or stakurinin, and the mean value and standard deviation were calculated. The results are shown in Table 1 and Figures 1 and 2. In Figures 1 and 2, "*" indicates a significant difference with p < 0.05 compared to the solvent control in the Dunnett's test.

[0044]

Table 1

[0045] As shown in the results of Table 1 and Figures 1 and 2, significant muscle satellite cell activation effects were observed for casuarinin and stakurinin.

[0046] Comparative Example 1: Evaluation of the muscle satellite cell activation ability of polyphenols To confirm whether the effects of casuarinin and stachyurin on skeletal muscle satellite cell activation are specific to these compounds, the polyphenols other than ellagitannin, catechin (manufactured by Wako Pure Chemical Industries, Ltd.), myricitrin (manufactured by Adooq Bioscience), hyperin (manufactured by EXTRASYNTHESE), and quercitrin (manufactured by EXTRASYNTHESE), were evaluated for their ability to activate skeletal muscle satellite cells in the same manner as in Example 3. The treatment concentration of each compound was 100 ng / mL. The results are shown in Table 2 and Figure 3.

[0047]

Chemical formula

[0048]

Table 2

[0049] As shown in the results of Table 2 and Figure 3, the polyphenols other than the above ellagitannin did not show a clear skeletal muscle satellite cell activation effect compared to the solvent control.

[0050] Comparative Example 2: Evaluation of the ability of other ellagitannins and partial structures to activate skeletal muscle satellite cells To confirm whether the same skeletal muscle satellite cell activation effects as those of casuarinin and stachyurin are also observed in other ellagitannins and partial structures, casuarictin (manufactured by Nagara Science Co., Ltd.) having a structure in which the glucose moiety of casuarinin is cyclized, castalagin (manufactured by Sigma) having a structure in which a 2,3-hexahydroxydiphenoyl group and a 5-galloyl group are bonded in casuarinin, gallic acid (manufactured by Wako Pure Chemical Industries, Ltd.) which is a partial structure of casuarinin and stachyurin, and ellagic acid (manufactured by Wako Pure Chemical Industries, Ltd.) in which the hexahydroxydiphenoyl groups of casuarinin and stachyurin are intramolecularly dehydrated and condensed were evaluated for their ability to activate skeletal muscle satellite cells in the same manner as in Example 3. The treatment concentration of each compound was 100 ng / mL. The results are shown in Table 3 and Figure 4.

[0051]

Chemical formula

[0052]

Table 3

[0053] As shown in the results of Table 3 and Figure 4, ellagitannins other than casuarinin and stachyurin, and partial structures of casuarinin and stachyurin did not show a clear myoblast activation effect compared to the solvent control.

[0054] From the above results, it was revealed that the excellent myoblast activation effect is specific to casuarinin and stachyurin.

Claims

1. A satellite cell activator characterized by containing, as an active ingredient, one or more ellagitannins selected from casuarinin and stachyurin.

2. The satellite cell activator according to claim 1, which is for increasing and / or maintaining muscle mass and / or muscle strength, or preventing or suppressing a decrease in muscle mass and / or muscle strength.

3. The satellite cell activator according to claim 1 or 2, which contains 0.0000013% by mass or more of the above ellagitannin.

4. The satellite cell activator according to any one of claims 1 to 3, which contains 5% by mass or less of the above ellagitannin.

5. The satellite cell activator according to any one of claims 1 to 4, which is for administering 0.1 mg / kg body weight / day or more of the above ellagitannin.

6. The satellite cell activator according to any one of claims 1 to 5, which is a composition for food and drink.

7. The satellite cell activator according to claim 8, which is a food with function claims.

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