Composition for promoting expression of fast-twitch muscle fibers and composition for promoting fast-twitching of muscle

Abstract

To provide a novel application of cacao water extract.SOLUTION: Cacao water extract is employed as an active constituent in a composition used to promote the expression of fast-twitch muscle fibers (composition for promoting expression of fast-twitch muscle fibers), and a composition used to promote the fast-twitching of muscle (composition for promoting fast-twitching of muscle).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for promoting fast-twitch muscle formation, which has the effect of promoting the expression of fast-twitch muscle fibers and is used to promote the formation of fast-twitch muscle fibers. [Background technology]

[0002] Skeletal muscle is a multifunctional tissue involved in various physiological processes. For example, skeletal muscle plays an important role in energy metabolism, the uptake of glucose, and other nutrients into the body, as well as in daily movements such as walking and maintaining posture, and in physical activities such as exercise. Skeletal muscle is formed as follows: satellite cells first differentiate into myoblasts through cell division, and then the myoblasts differentiate into myotubes. These myotubes then fuse to form muscle fibers. Connective cells fill the spaces between these cells to form muscle bundles, which then finally form skeletal muscle. Therefore, efficient differentiation of satellite cells and myoblasts is crucial for the formation of muscle fibers, the basic units of skeletal muscle.

[0003] Human skeletal muscle is composed of various types of muscle fibers. For example, based on their contractile and metabolic properties, muscle fiber types are classified as type I (slow-twitch, slow oxidative) and type II (fast-twitch), and type II is further subdivided into type IIa (fast-twitch, fast oxidative), type IIx (fast-twitch, intermediate), and type IIb (fast-twitch, fast glycolytic). Slow-twitch fibers are characterized by slow contraction and low force, but are rich in mitochondria and myoglobin, allowing for prolonged contraction and high endurance (fatigue resistance). Therefore, they are important muscle fibers for aerobic endurance exercise. On the other hand, fast-twitch fibers are low in mitochondria and myoglobin and have poorer endurance (fatigue resistance), but are fast contraction and capable of generating high power. Therefore, they are important muscle fibers for exercise requiring explosive power.

[0004] Muscle fibers are dynamic structures whose phenotypes change variably from fast-twitch to slow-twitch or from slow-twitch to fast-twitch depending on various conditions, such as increases or decreases in neuromuscular activity, increases or decreases in mechanical load, changes in hormone secretion (especially thyroid hormone), and aging. As mentioned above, fast-twitch muscle fibers can exert powerful force instantaneously. Therefore, efficient formation and conversion of skeletal muscle fibers to fast-twitch muscle fibers is important not only for promoting physical health but also for improving athletic performance, particularly in sports requiring explosive power. Furthermore, muscle atrophy (thinning) due to aging results in a decrease in fast-twitch muscle fibers, which leads to a decline in the ability to exert powerful force instantaneously. One reason why elderly people are prone to falls is that they are unable to quickly react with their other foot when they trip. Therefore, there is a great need to strengthen and increase muscles, especially fast-twitch muscle fibers, in order to prevent the decline in muscle capacity associated with aging and to live longer, healthier lives.

[0005] It has been known that green tea extract, the main component of which is catechin, improves lipid metabolism and thereby enhances endurance (Non-Patent Document 1), and that high molecular weight polyphenols extracted from fermented tea have the effect of promoting muscle slowing (Patent Document 1). However, these studies both increase slow-twitch muscle fibers, and there is no description of muscle strengthening by promoting differentiation into fast-twitch muscle fibers. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-37323 [Non-patent literature]

[0007] [Non-Patent Document 1] Murase T et al., Green tea extract improves endurance capacity and increase muscle lipid oxidation in mice. Am J Physiol Regul Integr Comp Physiol 2005; 288; R708-715 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a composition used to promote the expression of fast-twitch muscle fibers (a composition for promoting the expression of fast-twitch muscle fibers) and a composition for promoting the formation of fast-twitch muscle fibers (a composition for promoting the formation of fast-twitch muscle fibers). [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above-mentioned problems, and as shown in the experimental examples described below, they have found that when a water extract of cacao nibs, a raw material for chocolate (aqueous cacao extract), is added to mouse myoblasts (C2C12 cells), expression of the fast-twitch muscle fiber gene (MyhcIIb) is significantly induced. Based on this finding, the present inventors have confirmed that the aqueous cacao extract can promote the expression of fast-twitch muscle fibers, thereby promoting the development of fast-twitch muscle (enhancing fast-twitch muscle), and that the aqueous cacao extract is effective as an active ingredient in a composition for promoting the expression of fast-twitch muscle fibers and a composition for promoting the development of fast-twitch muscle fibers.

[0010] The present invention was completed based on these findings and includes the following embodiments. (1) A composition for promoting the expression of fast-twitch muscle fibers, comprising a water extract of cocoa as an active ingredient. (2) A composition for promoting fast muscle growth, comprising a cacao water extract as an active ingredient. (3) The composition according to (1) or (2), wherein the cacao water extract is a water extract of whole cacao beans or a portion of the cacao beans containing at least the endosperm. (4) The composition according to any one of (1) to (3), which is an oral composition. [Effects of the Invention]

[0011] The present invention provides a new use for the aqueous cocoa extract. Specifically, the aqueous cocoa extract can be used as an active ingredient in a composition for promoting the expression of fast-twitch muscle fibers and a composition for promoting fast-twitch muscle formation. In other words, a composition for promoting the expression of fast-twitch muscle fibers and a composition for promoting fast-twitch muscle formation containing the aqueous cocoa extract as an active ingredient can be provided.

[0012] The composition for promoting the expression of fast-twitch muscle fibers or the composition for promoting fast-twitch muscle formation of the present invention can promote the expression of fast-twitch muscle fibers and promote the transformation of muscles into fast-twitch muscle fibers (transition / conversion to fast-twitch muscle fibers) based on the action of the aqueous cocoa extract. As described above, fast-twitch muscle fibers are important for exerting explosive power, so the composition for promoting the expression of fast-twitch muscle fibers or the composition for promoting fast-twitch muscle formation of the present invention can be effectively used to strengthen the muscles of sprint athletes or to compensate for the muscle loss that occurs with aging. [Brief explanation of the drawings]

[0013] [Figure 1] In the experimental example, (A) shows the results of measuring the mRNA expression level of MyhcI, a slow-twitch muscle fiber marker, and (B) shows the results of measuring the mRNA expression level of MyhcIIb, a fast-twitch muscle fiber marker. In the figure, "Cacao" refers to cacao water extract, and "Dex" refers to dexamethasone. (Data are means ± SE, n = 3, *P< 0.05, ***P< 0.001 vs. Dex+) DETAILED DESCRIPTION OF THE INVENTION

[0014] (1) Cacao water extract Each of the compositions of the present invention contains an aqueous cacao extract as an active ingredient. The cacao plant or processed product thereof from which the aqueous cacao extract is obtained is a part of the cacao bean containing at least the endosperm, and examples thereof include whole cacao beans, various parts of the cacao bean (the part from which the outer skin has been removed, the germ, the endosperm, etc.), and processed cacao beans such as cacao nibs, cacao mass, defatted cacao mass, cocoa powder, etc. Here, the term "cacao beans" refers to raw beans harvested from the cacao plant that have been fermented and then dried. Cocoa nibs are the endosperm obtained by roughly crushing cocoa beans and removing the shell (seed coat) and germ. Cocoa mass is obtained by grinding cocoa beans, particularly cocoa nibs, and defatted cocoa mass can be obtained by removing fats and oils from the cocoa mass. The method for removing fats and oils is not particularly limited and can be carried out by known methods such as pressing. Cocoa powder can be obtained by grinding the defatted cocoa mass. Furthermore, when extracting using a cocoa plant or a processed product thereof as a raw material, it is preferable to use cocoa nibs, cocoa mass, or cocoa powder that have been subjected to a micronization process such as grinding or pulverization, from the viewpoint of extraction efficiency. Note that these processed cocoa bean products may also be roasted during any manufacturing process.

[0015] The origin (country of origin) of the cacao plant is not particularly limited as long as it can provide the aqueous cacao extract of the present invention. For example, Venezuela, the country of origin of the cacao nibs (cacao beans) used in the experimental examples described below, can be exemplified.

[0016] The preparation of a cacao water extract from a cacao plant or a processed product thereof can be carried out by a known extraction method using water as an extraction solvent or a known steam distillation method, preferably a method of extraction with high-temperature water at about 90 to 100°C under atmospheric pressure, or a method of steam distillation at 100°C under atmospheric pressure. As described in the experimental examples below, the aqueous cocoa extract used in the present invention may be substantially free of polyphenols. Here, "substantially free" means that the total polyphenol content in the aqueous cocoa extract is 0.1% by mass (0.1 g / 100 g) or less, preferably 0.10% by mass (0.10 g / 100 g) or less. The polyphenol content in the aqueous cocoa extract can be measured by the Folin-Ciocalteu method. The Folin-Ciocalteu method is a measurement method based on the oxidation-reduction of phenols and can be measured according to the method established by the Chocolate Industry Fair Trade Commission. During measurement, the amount of polyphenols can be calculated using commercially available epicatechin as a standard substance. Therefore, the method for preparing the aqueous cocoa extract is preferably a method for preparing a cocoa extract that is substantially free of polyphenols.

[0017] The cacao water extract may be in any form, and may be in the form of a water-soluble extract liquid, or may be in the form of a concentrate (sol or paste) obtained by concentrating the extract liquid, or may be in the form of a dried product (powder, granules, or particulates) prepared by subjecting the extract liquid to a drying process such as freeze-drying or spray-drying.

[0018] (2) Effects of cacao water extract As shown in the experimental examples described below, the aqueous cocoa extract promotes the expression of MyhcIIb, a fast-twitch muscle fiber marker (see Figure 1(B)). This suggests that the aqueous cocoa extract promotes the expression of fast-twitch muscle fibers and the transition or conversion of formed muscle fibers to fast-twitch muscle fibers, thereby promoting the development of fast-twitch muscle. Therefore, administration (ingestion) of the aqueous cocoa extract exerts this effect in vivo, making it possible to promote the development of fast-twitch muscle. In other words, the aqueous cocoa extract can be incorporated into oral compositions for the purposes of promoting the development of fast-twitch muscle fibers and / or promoting the development of fast-twitch muscle (skeletal muscle). Furthermore, as shown in the experimental examples, it has been confirmed that this effect also works in the presence of a muscle atrophy inducer. Therefore, the aqueous cocoa extract exerts this effect and can promote the development of fast-twitch muscle, even in situations where muscle atrophy is progressing, such as with aging.

[0019] (3) Cocoa water extract-containing composition The present invention provides a composition (hereinafter referred to as "the composition") containing an aqueous cacao extract having the aforementioned effects. The composition can be an oral composition or a parenteral composition (e.g., an intramuscular composition), but is preferably an oral composition.

[0020] The present composition exerts the aforementioned effects when administered to animals, including humans, and the content of the aqueous cocoa extract is not particularly limited, so long as this is the case. For example, the composition may be prepared and provided as one containing 0.01% or more of the aqueous cocoa extract by weight, calculated on a solid basis. The lower limit of the content is preferably 0.1% or more by weight, more preferably 0.5% or more by weight, and even more preferably 1% or more by weight. There is no upper limit to the content, but examples include 99% or less by weight, 50% or less by weight, 30% or less by weight, and 10% or less by weight.

[0021] The term "solids equivalent" refers to the content of the cocoa water extract in the composition converted into the solid content of the cocoa water extract. The term "solids content of the cocoa water extract" refers to the solid content of the cocoa water extract after removing water from the cocoa water extract. The solids content of the cocoa water extract can be determined by the atmospheric pressure heat drying method (sand-mixing method). Specifically, after a weighing dish containing sea sand and a glass rod is weighed to a constant weight, the test sample (cocoa water extract) and distilled water are added and the dish is dried in a dryer at 99°C for 4 hours. The moisture content of the test sample is then determined from the difference in mass between before and after drying. The mass of the solids contained in the test sample (before drying) can then be calculated from the moisture content thus measured.

[0022] The cacao aqueous extract used in the present invention preferably contains aroma components (cacao-derived aroma components) in a total amount of 0.001 to 1% by mass based on 100% by mass of the cacao aqueous extract. The content of cacao-derived aroma components in the cacao aqueous extract is more preferably 0.01 to 1% by mass. These cacao-derived aroma components include, but are not limited to, acetic acid, isovaleric acid, tetramethylpyrazine, 2,3-butanediol-2, 2,3-butanediol monoacetate-2, 2-methylbutyric acid, 2-phenylethanol, phenylacetaldehyde, 2,3-butanediol-1, and acetoin. A method for measuring the cacao aroma components in the cacao aqueous extract will be described in the Examples. The present composition preferably contains the cacao-derived aroma component in an amount of 0.0001 to 0.1%, and more preferably 0.001 to 0.1% by mass, based on 100% by mass of the present composition.

[0023] The present composition can be used as a composition for promoting the expression of fast-twitch muscle fibers based on the effect of promoting the expression of fast-twitch muscle fibers exhibited by the aqueous cocoa extract, which is an active ingredient of the composition. Furthermore, the present composition can be used as a composition for promoting the formation of fast-twitch muscle fibers based on the effect. The effect of these compositions in promoting fast muscle fiber expression can be evaluated using the mRNA expression level of MyhcIIb, a fast muscle fiber marker, as an indicator. Details of this can be obtained according to the method described in the Experimental Examples below.

[0024] (4) Use in food, beverages, medicines, quasi-drugs, feed, etc. The present composition is preferably in an oral administration (oral ingestion) form. The type of oral administration form (foods and beverages [including health functional foods and supplements such as foods for specified health uses, foods with functional claims, and nutritional functional foods], pharmaceuticals, and quasi-drugs) is not particularly limited. The present composition may also be applied to feed or pet food for animals other than humans (including livestock, poultry, and pets). Furthermore, the present composition may also be an additive added to foods and beverages, pharmaceuticals, quasi-drugs, feed, or pet food.

[0025] The oral dosage form is not limited, and examples thereof include liquids (including extracts and syrups), jellies, powders, fine granules, or granules; capsules (hard capsules, soft capsules) in which liquids, powders, or granules are filled into capsules; or tablets obtained by further compressing powders or granules (solid preparations). Compositions having such dosage forms can be prepared by blending the aqueous cacao extract with conventionally known edible carriers, excipients, etc. that are acceptable for pharmaceutical use or as food or feed, and molding the mixture into various dosage forms (oral dosage forms).

[0026] When the present composition is in the form of a liquid preparation, a wide variety of carriers conventionally known in the art can be used. Examples of suitable additives for preparing liquid preparations and syrups include water, ethanol, sucrose, invert sugar, glucose, maltose, and reduced starch syrup. When the present composition is in the form of a solid preparation, such as a tablet, a wide variety of carriers conventionally known in the art can be used. Examples of such carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, and silicic acid; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, crystalline cellulose, hydroxypropylcellulose, hypromellose, and sodium alginate; and binders such as dry starch, powdered agar, powdered laminaran, sodium bicarbonate, and polyisoprene. Disintegrants such as ethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, crospovidone, povidone, and low-substituted hydroxypropyl cellulose can be used. Disintegration inhibitors such as stearin, cocoa butter, and hydrogenated oils can be used. Absorption promoters such as quaternary ammonium salts and sodium lauryl sulfate can be used. Wettable tablets can be coated with conventional coatings, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, and film-coated tablets, or can be double- or multi-layered tablets. Furthermore, capsules containing the active ingredient can be prepared by filling conventional capsules made from materials such as gelatin, pullulan, starch, gum arabic, and hydroxypropylmethylcellulose (HPMC).

[0027] In addition to the above, additives such as surfactants, absorption enhancers, adsorbents, fillers, preservatives, stabilizers, emulsifiers, solubilizers, etc. may be appropriately selected and used depending on the form of the preparation.

[0028] All of these forms can be prepared by conventional methods in the art, for example, tablets can be obtained by adding the above-mentioned active ingredient and other excipients necessary for obtaining tablets as appropriate, mixing and dispersing them well, and then compressing them into tablets, while powders can be obtained by adding the above-mentioned active ingredient and other excipients necessary for obtaining powders as appropriate, mixing them by a suitable method, and pulverizing them.

[0029] The composition may be in the form of a formulation as described above, or may be in the form of a conventional food or beverage. Such a food or beverage can be produced by adding the aqueous cacao extract described above to various foods or beverages. The food or beverage may be in any form that can be orally ingested, such as a solution, suspension, emulsion, jelly (gel), sol, powder, or solid molding, and is not particularly limited. Specific examples include beverages such as soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, cocoa drinks, tea drinks, powdered drinks, concentrated drinks, nutritional drinks, and alcoholic beverages; flour products such as bread, pasta, noodles, cake mix, fried chicken powder, and breadcrumbs; confectioneries such as candy, caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, and dessert sweets; and instant foods such as instant noodles, retort pouch foods, canned foods, microwaveable foods, instant soups and miso soups, and freeze-dried foods. Examples include: condiments such as sauces, processed tomato seasonings, flavor seasonings, cooking mixes, sauces, dressings, soups, and curry and stew bases; dairy products such as milk drinks, yogurts, cheese, fermented milk, lactic acid bacteria drinks, ice cream, and cream; processed egg products such as pudding and mayonnaise; processed seafood products such as fish ham and sausage, and fish paste products; processed livestock products such as meat ham and sausage; processed agricultural products such as canned agricultural products, jams and marmalades, pickles, boiled beans, and cereals; frozen foods, nutritional foods, etc.

[0030] Such foods and beverages (including foods in the form of formulations) may be labeled so that the effects of the composition are apparent to consumers, such as foods for specified health uses, foods with functional claims, etc. Examples of such labeling include, but are not limited to, "supports muscle-building power," "increases muscle strength," "increases explosive power," "suppresses muscle and strength decline," "maintains muscle mass and strength," "macho," "slim and muscular," "suppresses, maintains, improves, or enhances the decline of walking ability," and "prevents falls."

[0031] The composition may also be in the form of a conventional feed or pet food, which can also be produced by adding the aqueous cocoa extract described above to various feeds or pet foods.

[0032] The content of the aqueous cocoa extract in the composition can be set as appropriate depending on the form and type (food, drink, pharmaceutical, quasi-drug, feed, pet food, etc.) described above, with an upper limit of 100% by mass. The dosage (intake) of the composition can be varied as appropriate depending on the type of human or animal, the sex and age of the subject, and the condition and severity of symptoms (pathological condition) of the subject. For example, the daily dosage (intake) for a human adult (body weight 50 kg) is typically about 0.25 to 2.5 mg, calculated as the amount of solids of the aqueous cocoa extract contained in the composition, without limitation.

[0033] The subject of administration (ingestion) of the present composition may be any person who needs to strengthen their muscles, preferably those who need to strengthen fast-twitch muscles that exert explosive power. Examples of subjects include, but are not limited to, those who are concerned about the decline in leg and lower back muscle strength due to aging, those with weakened legs and lower back (e.g., elderly people), athletes and sports players (e.g., sprinters, baseball players, rugby players, soccer players, jumpers, karate players, etc.), sports enthusiasts, etc.

[0034] (5) How to use the cacao water extract The present invention also provides methods of using the aqueous cocoa extract. One of the methods of use is to use the aqueous cocoa extract to impart the effect of promoting the expression of fast-twitch muscle fibers and / or the effect of promoting the formation of fast-twitch muscle fibers to oral compositions (including foods, beverages, pharmaceuticals, quasi-drugs, feed, pet food, etc.). This method can be carried out by incorporating an effective amount of the aqueous cocoa extract into an oral composition to promote the expression of fast-twitch muscle fibers and / or the formation of fast-twitch muscle fibers. The type of aqueous cocoa extract to be incorporated, the method for evaluating the effect, and the type of oral composition to be incorporated are as described above, and the descriptions therein are incorporated by reference herein.

[0035] As described above, in this specification, the terms "comprise" and "contain" encompass the meanings of "consist of" and "consist essentially of." [Example]

[0036] The present invention will be explained below using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. Unless otherwise specified, "%" and "parts" in the following descriptions mean "% by mass" and "parts by mass," respectively.

[0037] The materials used in the following experiments and their sources are as follows: Cocoa nibs: Fermented and dried cocoa beans (from Venezuela) are roasted, dehusked, and then roughly crushed (including the germ and endosperm). C2C12 cells: mouse myoblasts (derived from mouse striated muscle) (obtained from ATCC). FBS-containing DMEM medium: Prepared using FBS (Sigma-Aldrich) and DMEM (Fujifilm Wako Pure Chemical Industries, Ltd.). Total RNA extraction reagent: TRI Reagent (Cosmo Bio). A mixture of guanidine thiocyanate and phenol in a single-phase solution, used to isolate DNA, RNA, and proteins from biological samples. Dexamethasone: obtained from Sigma-Aldrich. Prime Script RT reagent kit: manufactured by Takara Bio Inc. SsoAdvanced Universal SYBR Green Supermix: Manufactured by Bio-Rad Laboratories, Inc. Custom DNA primers: Invitrogen. Internal standard substance: decane

[0038] Experimental Method 1. Preparation of aqueous cocoa extract 100 kg of cacao nibs frozen with liquid nitrogen were placed in a steam distiller and distilled at 100°C under atmospheric pressure. The resulting steam was cooled and designated aroma A. 100 kg of water was added to the residue, and the mixture was stirred and extracted at 40°C under atmospheric pressure for 1 hour. The extract was then filtered through a 100 μm pore filter, and the filtrate was designated extract B. The mixture of aroma A and extract B was designated as the aqueous cacao extract (total volume 100 mL) and used in the following experiments. The polyphenol content per 100% by mass of the prepared cacao water extract was 0.1% (measured by the Folin-Ciocalteu method).

[0039] The total amount of aroma components (cacao-derived aroma components) contained in 100% by mass of the prepared cacao water extract was 0.0144% by mass. The content of aroma components in the cacao water extract was measured by the following method.

[0040] (Measurement of aroma component content) 90g of each sample (cocoa water extract) was mixed with 1% internal standard (IS) and subjected to liquid-liquid extraction with 2 volumes of diethyl ether. The extract was transferred to a separatory funnel, and the separated diethyl ether phase was evaporated in an evaporator (evaporation temperature: 40°C) to obtain an aroma concentrate. The resulting aroma concentrate was subjected to GC-MS / FID measurement under the following conditions.

[0041] [GC-MS / FID] Apparatus: Agilent 5975 (manufactured by Agilent Technologies) <GC Conditions> Carrier gas: Helium Oven temperature: 40 °C Oven hold time: 30 minutes Ionization method: EI (Electron Impact) Ionization energy: 70 eV Scan range: m / z 30 to 600 <FID Conditions> Hydrogen flow: 30 mL / min Air flow: 300 mL / min Gas supply: Methane Fuel gas flow: 1 mL / min

[0042] By this measurement, it was confirmed that the cacao-derived aroma components contain at least acetic acid, isovaleric acid, tetramethylpyrazine, 2,3-butanediol 2, 2,3-butanediol monoacetate 2, 2-methylbutyric acid, 2-phenylethanol, phenylacetaldehyde, 2,3-butanediol 1, and acetoin.

[0043] 2. Evaluation using C2C12 cells C2C12 cells were seeded into the wells of a 12-well plate at a density of 5×10 4 cells / mL and pre-cultured at 37 °C for 24 hours in DMEM medium containing 10% FBS. Then, the medium was changed to a differentiation medium (DMEM medium containing 0.5% FBS, the same below), and the cells were further cultured at 37 °C for 96 hours. Thereafter, the medium was changed to a differentiation medium containing a cacao water extract (total amount of cacao aroma components 5 ppm), and the cells were cultured at the same temperature for 24 hours. Next, the medium was further changed to a differentiation medium containing a cacao water extract (total amount of cacao aroma components 5 ppm) and 1 μM dexamethasone (Dex), and the cells were cultured at the same temperature for 24 hours. Next, cells were recovered from the culture, the recovered cells were lysed with a total RNA extraction reagent, RNA was recovered, and cDNA was synthesized using a Prime Script RT reagent kit according to its manual. Using the obtained cDNA as a template, primers (custom DNA primers) for each of the MyhcI and MyhcIIb genes and SsoAdvanced Universal SYBR Green Supermix, quantitative real-time PCR was performed according to its manual to measure the mRNA expression levels of MyhcI and MyhcIIb. As an endogenous control, primers for Actb (actin β) were used to measure the mRNA expression level of Actb.

[0044] Note that MyhcI and MyhcIIb are genes of myosin heavy chain (MyHC) molecular species that constitute skeletal muscle. The molecular species mainly observed in human skeletal muscle include MyHCI, MyHCIIA, MyHCIIX / D, and MyHCIIB. There is a relationship of MyHCI < MyHCIIA < MyHCX / D < MyHCIIB in the maximum shortening velocity of single muscle fibers containing these myosin heavy chain molecular species. From this, MyhcI, which is the gene of MyHCI, is useful as a marker gene for slow muscle fibers, and the mRNA expression level of MyhcI serves as an indicator of muscle slow muscle transformation (induction or promotion). On the other hand, MyhcIIb, which is the gene of MyHCIIB, is useful as a marker gene for fast muscle fibers, and the mRNA expression level of MyhcIIb serves as an indicator of the promotion of fast muscle fiber expression and the induction or promotion of muscle fast muscle transformation.

[0045] Glucocorticoids are important mediators of muscle breakdown, inducing ubiquitin-proteasome pathway-dependent proteolysis in skeletal muscle. Furthermore, dexamethasone, a synthetic glucocorticoid, has been shown to increase MuRF1 and atrogin-1 expression in skeletal muscle. Because the metabolic changes induced by dexamethasone are similar to those observed in age-related muscle atrophy in animals and humans, dexamethasone has been used as a muscle atrophy inducer in many studies. The test system used in this study (assessing MyhcI or MyhcIIb expression in the presence of dexamethasone) takes aging into consideration, specifically evaluating the effects of cacao aqueous extract on muscle atrophy associated with aging. However, it goes without saying that the subjects in this study who experience muscle atrophy with age are not limited to elderly individuals.

[0046] Experimental results The results of measuring the mRNA expression levels of MyhcI and MyhcIIb, which are muscle fiber markers, are shown in Figure 1(A) and Figure 1(B), respectively. As shown in Figure 1(A), the aqueous cocoa extract did not affect the mRNA expression level of MyhcI, a slow-twitch muscle fiber marker, in the presence of dexamethasone. In contrast, as shown in Figure 1(B), the mRNA expression level of MyhcIIb, a fast-twitch muscle fiber marker, was significantly increased by treatment with the aqueous cocoa extract, even in the presence of dexamethasone. These results demonstrate that the aqueous cocoa extract acts on myoblasts to promote the expression of fast-twitch muscle fibers and the transition of muscle fibers to fast-twitch muscle fibers, thereby promoting the development of fast-twitch muscle, even in cases where muscle atrophy progresses with age. The aqueous cocoa extract used in this experiment had a polyphenol content of 0.1% and was added to the medium at a ratio such that the total amount of cocoa aroma components was 5 ppm. Therefore, the aforementioned effects of the aqueous cocoa extract in promoting the expression of fast-twitch muscle fibers and promoting muscle fast-twitch muscle formation are not thought to be due solely to the polyphenols in the aqueous cocoa extract.

Claims

1. A composition for promoting the expression of fast-twitch muscle fibers, comprising an aqueous cocoa extract as an active ingredient.

2. A composition for promoting fast-twitch muscle growth, comprising an aqueous cacao extract as an active ingredient.

3. 3. The composition according to claim 1, wherein the aqueous cocoa extract is an aqueous extract of whole cocoa beans or a portion of cocoa beans containing at least the endosperm.

4. 3. The composition of claim 1 or 2, which is an oral composition.

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