Composition for inhibiting muscle atrophy

Abstract

To provide a composition capable of inhibiting muscle atrophy, suppressing expression of a muscle atrophy responsible gene, or promoting increase in a protein of a muscle cell.SOLUTION: A composition for inhibiting muscle atrophy, a composition for suppressing expression of a muscle atrophy responsible gene, or a composition for promoting increase in a protein of a muscle cell comprises: i) a water-soluble turmeric extract and leucine; (ii) bisacurone and leucine; or (iii) turmeronol B.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition or kit for inhibiting muscle atrophy. The present invention relates to a composition or kit for suppressing the expression of genes causing muscle atrophy. The present invention relates to a composition or kit for promoting an increase in muscle cell proteins.

Background Art

[0002] In Japan where the aging society is progressing, it is considered that the number of patients with sarcopenia, which is muscle atrophy due to aging, and disuse muscle atrophy, which is muscle atrophy due to being bedridden, will increase. Such muscle atrophy may require hospitalization, which also causes problems such as an increase in medical costs and the occupancy rate of hospital beds.

[0003] The treatment of sarcopenia and disuse muscle atrophy is difficult, and the development of preventive and treatment methods is being studied. On the other hand, exercise is known as an effective means for suppressing muscle atrophy, but it is difficult for the elderly and bedridden patients to exercise daily. Therefore, an approach from food has attracted attention as a countermeasure against skeletal muscle atrophy. Since food is easy to incorporate into daily life, there is great expectation for food having an inhibitory effect on skeletal muscle atrophy.

[0004] Inventions related to foods useful for countermeasures against skeletal muscle atrophy are disclosed in, for example, the following documents. Patent Document 1 describes a composition for inhibiting muscle atrophy containing a water-soluble turmeric extract as an active ingredient, and a composition for inhibiting muscle atrophy containing bisacron as an active ingredient.

[0005] Patent Document 2 describes that muscle atrophy can be recovered by activating myoblasts and strengthening muscles with an agent containing a turmeric extract as an active ingredient. It is also described that the turmeric extract contains sesquiterpenes.

[0006] Patent Document 3 describes a method for improving a biomarker selected from ATP, nitric oxide, e.Nos, MAO, ACHE, muscle protein, and myogenin, and enhancing muscle cell proliferation, using a composition comprising turmeric extract and one other component.

[0007] Non-patent document 1 describes how lower limb skeletal muscle atrophy in streptozotocin (STZ)-treated diabetic model mice (T1DM) was reduced by curcumin administration, and how curcumin administration significantly suppressed atrophy in the rectus femoris and gastrocnemius muscles. Furthermore, non-patent document 1 describes how the mRNA expression of MuRF1 and Atrogin-1 / MAFbx, which was increased in the STZ group, was significantly decreased by curcumin administration. Non-patent document 2 describes how leucine enhances protein synthesis and inhibits protein degradation. Non-patent document 3 describes the possibility that a combination of aerobic exercise and leucine supplementation may help maintain skeletal muscle protein synthesis and mitigate age-related muscle weakness. Patent Document 4 describes an oral amino acid-containing composition for preventing or improving skeletal muscle mass loss in the elderly, which contains L-leucine in a molar composition ratio of 35-66% of the total essential amino acids. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2018-188428 [Patent Document 2] Japanese Patent Publication No. 2008-156294 [Patent Document 3] Special Publication No. 2016-505615 [Patent Document 4] Japanese Patent Publication No. 2014-65740 [Non-patent literature]

[0009] [Non-Patent Document 1] Taisuke Ono, "Curcumin suppresses increased protein degradation and reduces atrophy of lower limb skeletal muscles associated with diabetes," Doctoral dissertation, Hokkaido University, No. 9809, degree awarded March 24, 2011. [Non-Patent Document 2] Chemistry and Biology, Vol. 45, No. 3, pp. 203-210, 2007 [Non-Patent Document 3] Food Funct.,2017 Dec 13;8(12):4528-4538 [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention aims to provide a composition or kit capable of inhibiting muscle atrophy, suppressing the expression of genes causing muscle atrophy, or promoting protein increase in muscle cells. [Means for solving the problem]

[0011] This invention encompasses the following inventions. (1)(i)Water-soluble turmeric extract and leucine, (ii) Bisacron and leucine, or (iii) Turmeronol B A composition for inhibiting muscle atrophy, including the following: (2) The composition described in (1), which is a food or beverage. (3)(i)Water-soluble turmeric extract and leucine, (ii) Bisacron and leucine, or (iii) Turmeronol B A composition for suppressing the expression of muscle atrophy-causing genes, including the above. (4) The composition described in (3), which is a food or beverage. (5)(i) Water-soluble turmeric extract and leucine, (ii) Bisacron and leucine, or (iii) Turmeronol B A composition for promoting the increase of protein in muscle cells, including the following: (6) The composition described in (5), which is a food or beverage. (7) A kit for inhibiting muscle atrophy, comprising water-soluble turmeric extract and leucine in a separated form. (8) A kit for suppressing the expression of muscle atrophy-causing genes, comprising water-soluble turmeric extract and leucine in a separated form. (9) A kit for promoting an increase in muscle cell protein, comprising water-soluble turmeric extract and leucine in a separated form. (10) A kit for inhibiting muscle atrophy, comprising bisacron and leucine in a separated form. (11) A kit for suppressing the expression of muscle atrophy-causing genes, comprising bisacron and leucine in a separated form. (12) A kit for promoting an increase in muscle cell protein, comprising bisacron and leucine in a separated form.

Advantages of the Invention

[0012] The composition or kit for inhibiting muscle atrophy according to the present invention has a high effect of inhibiting muscle atrophy. The composition or kit for suppressing the expression of muscle atrophy-causing genes according to the present invention has a high effect of suppressing the expression of muscle atrophy-causing genes. The composition or kit for promoting an increase in muscle cell protein according to the present invention has a high effect of promoting an increase in muscle cell protein.

Brief Description of the Drawings

[0013] [Figure 1] Figure 1 shows the results of Example 1. The left side of Figure 1 shows the mRNA expression level of the Mafbx gene in muscle cells under each addition condition of dexamethasone (DEX), water-soluble turmeric extract (AE), and leucine (Leu), normalized as a relative value to the mRNA expression level of the internal standard gene Ppia, and further shown as a relative value to the normalized mRNA expression level of the Mafbx gene in the control (DEX: -, AE: 0, Leu: 0) group. The right side of Figure 1 shows the mRNA expression level of the Murf1 gene in muscle cells under each addition condition of DEX, AE, and Leu in the same relative value. [Figure 2]Figure 2 shows the results of Example 2. Figure 2 left shows the measured total protein content of muscle cells cultured under conditions in which 100 μg / mL of water-soluble turmeric extract (AE) and 10 μM leucine (Leu) were added individually or in combination. Figure 2 right shows the measured total protein content of muscle cells cultured under conditions in which 100 μg / mL of water-soluble turmeric extract (AE) and 5 μM leucine (Leu) were added individually or in combination. [Figure 3] Figure 3 shows the results of Example 3. It shows the measured total protein content of muscle cells cultured under conditions in which 2.5 μM bisacron (Bis) and 10 μM leucine (Leu) were added, either individually or in combination. [Figure 4] Figure 4 shows the results of Example 4. Figure 4 left shows the mRNA expression levels of the Mafbx gene in muscle cells under the conditions of dexamethasone (DEX) and turmeronol B (TB) addition, normalized as a relative value to the mRNA expression level of the internal standard gene Ppia, and further shown as a relative value to the normalized mRNA expression level of the Mafbx gene in the control group (DEX:-, TB:0). Figure 4 right similarly shows the mRNA expression levels of the Murf1 gene in muscle cells under the conditions of DEX and TB addition as relative values. [Modes for carrying out the invention]

[0014] Turmeric In this invention, "turmeric" refers to *Curcuma longa*, a plant belonging to the genus *Curcuma* in the family Zingiberaceae. *Curcuma longa* is sometimes also called "autumn turmeric."

[0015] <Water-soluble turmeric extract> Water-soluble turmeric extract refers to an extract (turmeric extract) of the turmeric plant raw material using a hydrophilic extraction solvent as described later, and may be further obtained by heating and / or reducing the pressure to volatilize the extraction solvent and drying as needed. These heating, pressure reduction, and drying methods are not particularly limited, and conventionally known methods can be used, for example.

[0016] Turmeric plant raw materials include turmeric rhizomes. Turmeric rhizomes may be used as is after being collected from the soil and washed, or appropriate parts of the rhizome may be used in their original form, cut to appropriate dimensions or shapes, or in the form of crushed material. The turmeric plant raw material may be dried.

[0017] The method for extracting turmeric extract from plant materials is not particularly limited, and conventionally known methods can be used.

[0018] The hydrophilic extraction solvent is preferably at least one selected from the group consisting of water and hydrophilic organic solvents, more preferably water, a hydrophilic organic solvent, or a mixed solvent of water and a hydrophilic organic solvent, and particularly preferably water or a mixed solvent of water and a hydrophilic organic solvent. The hydrophilic organic solvent may be a mixed solvent of multiple types of hydrophilic organic solvents. "Water" also includes hot water. Examples of hydrophilic organic solvents include at least one type of alcohol (may be a mixed solvent of multiple types of alcohols), and while the alcohol is not particularly limited, ethanol is preferred.

[0019] When using a mixed solvent of a hydrophilic organic solvent and water as the hydrophilic extraction solvent, the mixing ratio of the two is not particularly limited, but for example, the weight ratio of hydrophilic organic solvent to water is preferably in the range of 10:90 to 90:10, and more preferably in the range of 20:80 to 50:50. The temperature during extraction is not particularly limited.

[0020] Water-soluble turmeric extract extracted using a hydrophilic extraction solvent contains more water-soluble compounds compared to water-insoluble turmeric extract extracted using a hydrophobic organic solvent such as ethyl acetate. Therefore, the components contained in water-soluble and water-insoluble turmeric extracts are different from each other.

[0021] In the present invention, as the water-soluble turmeric extract, the extract obtained from turmeric rhizomes using a hydrophilic extraction solvent as described above can be used as is, or the extract obtained from turmeric rhizomes using a hydrophilic extraction solvent can be further treated by dilution, concentration, drying, etc. Conventional known methods can be used for dilution, concentration, drying, etc.

[0022] The water-soluble turmeric extract used in the present invention is preferably characterized by containing bisacron. Bisacron is preferably contained in an amount of 0.1% by weight or more, more preferably 0.15% by weight or more, and particularly preferably 0.2% by weight or more, relative to the total amount of the water-soluble turmeric extract. The amount of bisacron in the water-soluble turmeric extract can be determined by mixing the water-soluble turmeric extract with ethyl acetate, centrifuging the supernatant to obtain a solution from which the ethyl acetate has been removed by vacuum distillation, and then dissolving the solution in acetonitrile. This solution is then subjected to high-performance liquid chromatography (HPLC) as an analytical sample.

[0023] The water-soluble turmeric extract used in the present invention preferably does not contain curcumin, or if it does contain curcumin, it contains 0.5% by weight or less. More preferably, the water-soluble turmeric extract used in the present invention does not contain curcuminoids (a general term for curcumin and its related substances), or if it does contain curcuminoids, the total amount is 0.5% by weight or less.

[0024] The amounts of curcumin and curcuminoids can be measured by dissolving a water-soluble turmeric extract in 50% acetonitrile, centrifuging the supernatant, and then measuring it using high-performance liquid chromatography (Agilent 1100).

[0025] <Bisacron> Bisacrons are compounds classified as bisaborane-type sesquiterpenes and include compounds or salts thereof having the following planar structural formula. Bisacrons have an asymmetric carbon at the position indicated by the * in the planar structural formula, and therefore several optical isomers exist, but in this specification, bisacron is a concept that encompasses all of these optical isomers.

[0026] [ka]

[0027] Bisacron may be in the form of a water-soluble turmeric extract containing bisacron, or in a purified form. The origin of bisacron is not particularly limited; artificially synthesized bisacron or bisacron extracted and purified from plants of the genus Curcuma (not limited to autumn turmeric) of the family Zingiberaceae can be used.

[0028] <Turmeronol B> Turmeronol B is a compound having the following planar structure.

[0029] [ka]

[0030] In the natural product isolated from turmeric, turmeronol B has an S-isomer configuration at the 6th carbon in the 2-methyl-2-hepten-4-one substructure. However, in the present invention, turmeronol B only needs to have the above-mentioned planar structure, and the aforementioned configuration may be the S-isomer, the R-isomer, or a mixture of the S-isomer and the R-isomer.

[0031] Turmeronol B may be in the form of a water-soluble turmeric extract containing turmeronol B, or in a purified form. The origin of turmeronol B is not particularly limited; artificially synthesized turmeronol B or turmeronol B extracted and purified from plants of the genus Curcuma (not limited to autumn turmeric) of the family Zingiberaceae can be used.

[0032] <Leucine> In this invention, leucine preferably refers to L-leucine. Leucine may also be in the form of a salt acceptable for use in food, beverages, or pharmaceuticals.

[0033] <Compositions for inhibiting muscle atrophy, compositions for suppressing the expression of genes causing muscle atrophy, or compositions for promoting the increase of protein in muscle cells> A first embodiment of the present invention relates to a composition for inhibiting muscle atrophy, comprising (i) a water-soluble turmeric extract and leucine, (ii) bisacroline and leucine, or (iii) turmeronol B. A second embodiment of the present invention relates to a composition for suppressing the expression of muscle atrophy-causing genes, comprising (i), (ii), or (iii) above. A third embodiment of the present invention relates to a composition for promoting the increase of muscle cell protein, comprising (i), (ii), or (iii) above. The compositions according to each embodiment of the present invention are compositions intended for humans or non-human animals, preferably humans. The compositions according to the first, second, or third embodiment may contain (i), (ii), or (iii) as an active ingredient, and may consist only of (i), (ii), or (iii), or may consist of (i), (ii), or (iii) and at least one other component. The shape of the compositions according to the first, second, or third embodiment is not particularly limited and may be any shape, such as liquid, fluid, gel, semi-solid, or solid.

[0034] The aforementioned at least one other component is not particularly limited, but preferably includes components that are permissible in the final form of food, beverages, pharmaceuticals, etc., and that can be taken orally.

[0035] Other such ingredients include, for example, sweeteners, acidulants, vitamins, minerals, thickeners, emulsifiers, antioxidants, and water. Additionally, if necessary, colorants, flavorings, preservatives, antifungal agents, and other physiologically active substances may be added.

[0036] Examples of sweeteners include monosaccharides and disaccharides such as glucose, fructose, sucrose, lactose, maltose, palatinose, trehalose, and xylose; isomerized sugars (glucose-fructose syrup, fructose-glucose syrup, sugar-mixed isomerized sugar, etc.); sugar alcohols (erythritol, xylitol, lactitol, palatinose, sorbitol, reduced starch syrup, etc.); honey; and high-intensity sweeteners (sucralose, acesulfame potassium, thaumatin, stevia, aspartame, etc.).

[0037] Acidulants include citric acid, malic acid, gluconic acid, tartaric acid, lactic acid, phosphoric acid, or salts thereof, and one or more of these can be used.

[0038] Examples of vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin E, niacin, and inositol. Minerals include calcium, magnesium, zinc, and iron.

[0039] Examples of thickening agents include carrageenan, gellan gum, xanthan gum, gum arabic, tamarind gum, guar gum, locust bean gum, karaya gum, agar, gelatin, pectin, soybean polysaccharides, and carboxymethylcellulose (CMC).

[0040] Examples of emulsifiers include glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, lecithin, plant sterols, and saponins.

[0041] Antioxidants include vitamin C, tocopherol (vitamin E), enzyme-treated rutin, and catechins.

[0042] The aforementioned other components may be appropriately blended in amounts that are typically used by those skilled in the art in compositions of food, beverages, pharmaceuticals, etc.

[0043] The composition obtained by formulating (i), (ii), or (iii) above with at least one other component by appropriate means may be in the form of a solid composition such as a capsule, a tablet (including coated tablets such as sugar-coated tablets or multilayer tablets, orally disintegrating agents, chewable tablets, etc.), a powder, or granules, or it may be in the form of a liquid composition.

[0044] The compositions according to the first, second, or third embodiments of the present invention are preferably, and more preferably, food or beverage products themselves. Here, "food or beverage product" includes food additives and food or beverage ingredients used in combination with other food materials to manufacture food or beverage products. When a composition containing (i), (ii), or (iii) as an active ingredient is a food or beverage product, or a food or beverage ingredient used in combination with other food materials to manufacture food or beverage products, "food or beverage product" is preferably a functional food, a food for specified health uses, a supplement for nutritional supplementation, etc., and its form is not particularly limited, but examples include confectionery (e.g., gum, candy, caramel, chocolate, cookies, snacks, jelly, gummies, tablets, etc.), noodles (e.g., soba, udon, ramen, etc.), dairy products (e.g., milk, ice cream, yogurt, etc.), seasonings (e.g., miso, soy sauce, etc.), soups, beverages (e.g., juice, coffee, black tea, tea, carbonated drinks, sports drinks, etc.), and is preferably a beverage.

[0045] In this specification, "inhibiting muscle atrophy" means preventing, reducing, or treating muscle atrophy such as sarcopenia and disuse muscle atrophy in humans or non-human animals, preferably humans. The composition for inhibiting muscle atrophy according to the first embodiment of the present invention can be taken to maintain or improve muscles that decline with age, or to maintain or improve walking ability that declines with age.

[0046] In this specification, "muscle atrophy-causing genes" typically refer to one or more of the Mafbx and Murf1 genes in humans. These muscle atrophy-causing genes are known to be upexpressed in patients with muscle atrophy and to cause muscle atrophy. "Suppressing the expression of muscle atrophy-causing genes" means preventing the upexpression of muscle atrophy-causing genes in subjects such as humans for whom prevention of muscle atrophy is required, and reducing the upexpression of muscle atrophy-causing genes in subjects such as humans who have symptoms of muscle atrophy. The composition for suppressing the expression of muscle atrophy-causing genes according to the second embodiment of the present invention can also be taken to maintain or improve muscles that decline with age, or to maintain or improve walking ability that declines with age. The suppression of the expression of muscle atrophy-causing genes can be confirmed by collecting cells, preferably muscle cells (skeletal muscle cells, smooth muscle cells, cardiomyocytes, etc.) from a subject that has ingested the composition according to the second embodiment of the present invention, preparing cDNA from RNA in the cells, and detecting the amount of amplified product by PCR using a primer set capable of amplifying muscle atrophy-causing genes with the cDNA as a template. An example of a primer set for amplifying the Mafbx gene is a set of primers containing the nucleotide sequence shown in SEQ ID NO: 1 and primers containing the nucleotide sequence shown in SEQ ID NO: 2. An example of a primer set for amplifying the Murf1 gene is a set of primers containing the nucleotide sequence shown in SEQ ID NO: 3 and primers containing the nucleotide sequence shown in SEQ ID NO: 4.

[0047] The suppression of the expression of muscle atrophy-causing genes can also be confirmed by collecting cells, preferably muscle cells (skeletal muscle cells, smooth muscle cells, cardiomyocytes, etc.), from a subject who has ingested the composition according to the second embodiment of the present invention, and detecting proteins derived from muscle atrophy-causing genes in the cells. For example, if the amount of proteins derived from muscle atrophy-causing genes, such as MAFbx, a protein derived from the Mafbx gene, and MuRF1, a protein derived from the Murf1 gene, is reduced in the cells, it can be confirmed that the expression of muscle atrophy-causing genes is suppressed.

[0048] A composition for promoting the increase of muscle cell protein, according to a third embodiment of the present invention, can also be ingested to maintain or improve age-related muscle weakness, or to maintain or improve age-related walking ability. The promotion of muscle cell protein increase can be confirmed by collecting muscle cells (skeletal muscle cells, smooth muscle cells, cardiomyocytes, etc.) from a subject who has ingested the composition according to the third embodiment of the present invention and measuring the total protein content in the muscle cells. The total protein content can be measured by the bisiconic acid method.

[0049] A composition for promoting the increase of muscle cell protein according to a third embodiment of the present invention can be used to inhibit muscle atrophy.

[0050] When the composition according to the first embodiment of the present invention contains (i) water-soluble turmeric extract and leucine as active ingredients, the content of water-soluble turmeric extract and leucine can be appropriately set so that they can synergistically inhibit muscle atrophy. The composition contains water-soluble turmeric and leucine in an amount of preferably 3 to 1200 parts by weight, more preferably 300 to 1200 parts by weight, of water-soluble turmeric extract per 1 part by weight. The content of water-soluble turmeric extract in the composition according to the first embodiment of the present invention is preferably 10 mg to 1000 mg, and the content of leucine is preferably 0.1 g to 10 g.

[0051] When the composition according to the first embodiment of the present invention contains (ii) bisacron and leucine as active ingredients, the amounts of bisacron and leucine can be appropriately set so that they can synergistically inhibit muscle atrophy. Bisacron and leucine are included in such a way that leucine is preferably in an amount of 500 to 5000 parts by weight, more preferably 1000 to 3000 parts by weight, per 1 part by weight of bisacron. The amount of bisacron in the composition according to the first embodiment of the present invention is preferably 80 μg to 4000 μg, and the amount of leucine is preferably 0.1 g to 10 g.

[0052] When the composition according to the first embodiment of the present invention contains (iii) turmeronol B as an active ingredient, the amount of turmeronol B can be appropriately set to exert an effect of inhibiting muscle atrophy. The amount of turmeronol B in the composition according to the first embodiment of the present invention is preferably 5 μg to 250 μg.

[0053] When the composition according to the second embodiment of the present invention contains (i) water-soluble turmeric extract and leucine as active ingredients, the content of water-soluble turmeric extract and leucine can be appropriately set so that they can synergistically suppress the expression of muscle atrophy-causing genes. The composition contains water-soluble turmeric and leucine in an amount of preferably 3 to 1200 parts by weight, more preferably 300 to 1200 parts by weight, of water-soluble turmeric extract per 1 part by weight. The content of water-soluble turmeric extract in the composition according to the second embodiment of the present invention is preferably 10 mg to 1000 mg, and the content of leucine is preferably 0.1 g to 10 g.

[0054] When the composition according to the second embodiment of the present invention contains (ii) bisacron and leucine as active ingredients, the amounts of bisacron and leucine can be appropriately set so that they synergistically suppress the expression of muscle atrophy-causing genes. Bisacron and leucine are included in an amount of preferably 500 to 5000 parts by weight, more preferably 1000 to 3000 parts by weight, of 1 part by weight of bisacron. The amount of bisacron in the composition according to the second embodiment of the present invention is preferably 80 μg to 4000 μg, and the amount of leucine is preferably 0.1 g to 10 g.

[0055] When the composition according to the second embodiment of the present invention contains (iii) turmeronol B as an active ingredient, the amount of turmeronol B can be appropriately set to exert an effect of suppressing the expression of muscle atrophy-causing genes. The amount of turmeronol B in the composition according to the second embodiment of the present invention is preferably 5 μg to 250 μg.

[0056] When the composition according to the third embodiment of the present invention contains (i) water-soluble turmeric extract and leucine as active ingredients, the content of water-soluble turmeric extract and leucine can be appropriately set so that they can synergistically promote the increase of muscle cell protein. The composition contains water-soluble turmeric and leucine in an amount of preferably 3 to 1200 parts by weight, more preferably 3 to 20 parts by weight, of water-soluble turmeric extract per 1 part by weight. The content of water-soluble turmeric extract in the composition according to the third embodiment of the present invention is preferably 10 mg to 1000 mg, and the content of leucine is preferably 0.1 g to 10 g.

[0057] When the composition according to the third embodiment of the present invention contains (ii) bisacron and leucine as active ingredients, the content of bisacron and leucine can be appropriately set so that they can synergistically promote the increase of muscle cell protein. Bisacron and leucine are included in such a way that leucine is preferably in an amount of 500 to 5000 parts by weight, more preferably 1000 to 3000 parts by weight, per 1 part by weight of bisacron. The content of bisacron in the composition according to the third embodiment of the present invention is preferably 80 μg to 4000 μg, and the content of leucine is preferably 0.1 g to 10 g.

[0058] If the composition according to the third embodiment of the present invention contains (iii) turmeronol B as an active ingredient, the amount of turmeronol B can be appropriately set to promote the increase of muscle cell protein. The amount of turmeronol B in the composition according to the third embodiment of the present invention is preferably 5 μg to 250 μg.

[0059] <Kits for inhibiting muscle atrophy, kits for suppressing the expression of genes causing muscle atrophy, or kits for promoting the increase of muscle cell protein> A fourth embodiment of the present invention relates to a kit for inhibiting muscle atrophy, comprising a water-soluble turmeric extract and leucine in separate forms. A fifth embodiment of the present invention relates to a kit for suppressing the expression of muscle atrophy-causing genes, comprising a water-soluble turmeric extract and leucine in separate forms. A sixth embodiment of the present invention relates to a kit for promoting the increase of muscle cell protein, comprising a water-soluble turmeric extract and leucine in separate forms. A seventh embodiment of the present invention relates to a kit for inhibiting muscle atrophy, comprising bisacron and leucine in separate forms. An eighth embodiment of the present invention relates to a kit for suppressing the expression of muscle atrophy-causing genes, comprising bisacron and leucine in separate forms. A ninth embodiment of the present invention relates to a kit for promoting the increase of muscle cell proteins, comprising bisacron and leucine in separate forms.

[0060] A kit for inhibiting muscle atrophy according to the fourth or seventh embodiment of the present invention can be used for the same purposes as the composition for inhibiting muscle atrophy according to the first embodiment.

[0061] A kit for suppressing the expression of muscle atrophy-causing genes according to the fifth or eighth embodiment of the present invention can be used for the same purposes as the composition for suppressing the expression of muscle atrophy-causing genes according to the second embodiment.

[0062] A kit for promoting the increase of muscle cell protein according to the sixth or ninth embodiment of the present invention can be used in the same applications as the composition for promoting the increase of muscle cell protein according to the third embodiment.

[0063] A kit according to the fourth, fifth, or sixth embodiment of the present invention contains a water-soluble turmeric extract and leucine in separate forms. The water-soluble turmeric extract may consist of water-soluble turmeric extract alone, or it may be a composition containing water-soluble turmeric extract and at least one other component, but preferably it is a composition containing water-soluble turmeric extract and at least one other component. On the other hand, the leucine may consist of leucine alone, or it may be a composition containing leucine and at least one other component, but preferably it is a composition containing leucine and at least one other component. The form of the composition containing water-soluble turmeric extract and at least one other component and the composition containing leucine and at least one other component are not particularly limited and may be any form such as liquid, fluid, gel, semi-solid, or solid, and it is preferably, more preferably, a food or beverage or pharmaceutical in itself. Specific examples of at least one other component and food or beverage are as described with respect to the composition according to the first, second, or third embodiment. The amounts of water-soluble turmeric extract and leucine in the kit according to the fourth, fifth, or sixth embodiment of the present invention are as described with respect to the case where the active ingredient is (i) in the composition according to the first, second, or third embodiment of the present invention.

[0064] A kit according to the seventh, eighth, or ninth embodiment of the present invention contains bisacron and leucine in separate forms. Bisacron may consist of bisacron alone, or it may be a composition containing bisacron and at least one other component, but preferably it is a composition containing bisacron and at least one other component. On the other hand, leucine may consist of leucine alone, or it may be a composition containing leucine and at least one other component, but preferably it is a composition containing leucine and at least one other component. The form of the composition containing bisacron and at least one other component and the composition containing leucine and at least one other component are not particularly limited and may be any form such as liquid, fluid, gel, semi-solid, or solid, and it is preferably, more preferably, a food or beverage or pharmaceutical product. Specific examples of at least one other component and food or beverage are as described with respect to the composition according to the first, second, or third embodiment. The amounts of bisacron and leucine in the kit according to the seventh, eighth, or ninth embodiment of the present invention are as described with respect to the case where the active ingredient is (ii) in the composition according to the first, second, or third embodiment of the present invention. [Examples]

[0065] <Sample> Water-soluble turmeric extract (autumn turmeric extract) Water-soluble turmeric extract refers to an extract obtained by extracting the rhizome of *Curcuma longa*, commonly known as autumn turmeric, using a hydrophilic extraction solvent. The rhizome portion of the turmeric was extracted using an extraction solvent such as water, and the solvent was evaporated by heating and / or reducing the pressure. The water-soluble turmeric extract used in the experiment contained 0.15% by weight or more of bisacron and 0.1% by weight or less of curcuminoids.

[0066] Bisacron As bisacron, purified (≧99% (HPLC)) bisacron reagent was obtained from Nagara Science Co., Ltd. and used.

[0067] Leucine As leucine, we obtained and used L-leucine reagent from Fujifilm Wako Pure Chemical Industries, Ltd.

[0068] Turmeronol B For the turmenol B reagent, we obtained purified (≧99% (HPLC)) turmenol B from Nagara Science Co., Ltd. and used it.

[0069] <Experimental Method 1: Confirmation of the effect on the expression of muscle atrophy-causing genes> Mouse skeletal muscle-derived cell line C2C12 was divided into 1.75 × 10⁻¹⁴ cells. 4 The cells were suspended in DMEM supplemented with 10% fetal bovine serum to achieve a cell / well ratio, seeded in a collagen-coated 24-well plate, and then cultured at 37°C under 5% CO2 conditions.

[0070] On the third day of culture, when the cells reached 90% confluence, they were washed with PBS, and then differentiated by adding 4% horse serum-supplemented DMEM and culturing at 37°C under 5% CO2 conditions. The culture medium was changed every two days.

[0071] On day 7 of differentiation induction, after washing with PBS, serum-free DMEM was added and the cells were cultured for 10 hours at 37°C under 5% CO2 conditions (culture under starvation conditions). Next, the solution was replaced with serum-free DMEM containing (or not containing) 10 μM dexamethasone (Fujifilm Wako Pure Chemical Industries, Ltd.), and a predetermined concentration of water-soluble turmeric extract and bisacron (or not containing bisacron), and the cells were incubated at 37°C under 5% CO2 conditions for 14 hours.

[0072] Next, the solution was replaced with serum-free DMEM containing (or not containing) a predetermined concentration of leucine, and the cells were incubated at 37°C under 5% CO2 conditions for 1 hour.

[0073] After culturing, the cells were washed with PBS, harvested, and RNA was extracted from them. The RNeasy Mini Kit (QIAGEN) was used for cell harvesting and RNA extraction, following the instructions provided.

[0074] The obtained RNA was used to measure the expression levels of Mafbx and Murf1, as well as Ppia as an internal standard gene, by real-time PCR. The procedure was performed using the One Step TB Green PrimeScript RT-PCR Kit II (Takara Bio) according to the included protocol. The primer sequences are shown below.

[0075] [Table 1]

[0076] <Experimental Method 2. Experiment to Confirm Total Protein Amount> Using the same conditions and procedures as in Experimental Method 1, mouse skeletal muscle-derived cell line C2C12 was cultured in DMEM supplemented with 10% fetal bovine serum. Differentiation induction culture was performed in DMEM supplemented with 4% horse serum from day 3 of culture, and from day 7 of differentiation induction, the cells were cultured under starvation conditions in serum-free DMEM for 10 hours.

[0077] Next, the culture medium was replaced with serum-free DMEM containing (or not containing) a predetermined concentration of water-soluble turmeric extract, bisacron, and leucine, and the cells were incubated at 37°C under 5% CO2 conditions for 15 hours.

[0078] After washing with PBS, RIPA buffer (10 mM Tris (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM DTT, 1.0% Nonidet P-40) was added, and the cells were collected into 1.5 mL tubes using a cell scraper.

[0079] The mixture was left to stand on ice for 1 hour with periodic vortexing, then centrifuged at 12000 rpm at 4°C for 20 minutes. The supernatant was collected as the total protein extract and stored at -20°C.

[0080] After the stored supernatant was returned to room temperature, its total protein content was measured using the bisiconate method. The Micro BCA® Protein Assay Kit (Thermo Scientific) was used for the measurement, and the procedure was followed according to the included instructions.

[0081] <Example 1: Suppression of muscle atrophy-causing gene expression by combined use of water-soluble turmeric extract and leucine> The inhibitory effect of either or both of water-soluble turmeric extract and leucine on the expression of muscle atrophy-causing genes (Mafbx gene, Murf1 gene) in the presence of dexamethasone was confirmed using the method described in Experimental Method 1. As a control, the mRNA expression levels of muscle atrophy-causing genes were measured under conditions without dexamethasone, water-soluble turmeric extract, or leucine. The mRNA expression levels of muscle atrophy-causing genes were normalized as relative values ​​to the mRNA expression levels of the internal standard gene Ppia. Furthermore, the normalized mRNA expression levels of muscle atrophy-causing genes under each condition were expressed as relative values ​​to the normalized mRNA expression levels of muscle atrophy-causing genes under the control conditions.

[0082] The results are shown in Figure 1. In Figure 1, DEX represents dexamethasone, AE represents water-soluble turmeric extract, and Leu represents leucine. Figure 1 shows the mean and standard deviation of the results from the three trials. The significance probability (##: less than 1%, student's t-test) in the comparative test between the (DEX:-,AE:0,Leu:0) group and the (DEX:+,AE:0,Leu:0) group is shown. The significance probability (*: 1% or more and less than 5%, $: 5% or more and less than 10%, Dunnett's test) in the comparative test between the (DEX:+,AE:0,Leu:0) group and the AE and / or Leu-added group is shown.

[0083] The addition of dexamethasone (DEX) increased the mRNA expression levels of the Mafbx and Murf1 genes in cells, suggesting that DEX induces muscle atrophy. Furthermore, when both 2.5 μg / mL of water-soluble turmeric extract and 10 mM leucine were added simultaneously, the expression levels of each gene were significantly lower, indicating a strong inhibitory effect. This result suggests that the combined use of water-soluble turmeric extract and leucine synergistically inhibits muscle atrophy.

[0084] <Example 2: Total protein increase effect by combining water-soluble turmeric extract and leucine> The method described in Experimental Method 2 confirmed the effect of either or both of water-soluble turmeric extract and leucine on increasing total protein in muscle cells.

[0085] The results are shown in Figure 2. In Figure 2, AE represents water-soluble turmeric extract, and Leu represents leucine. Figure 2 shows the mean and standard deviation of the results from the three trials. The significance probability ($: 5% to less than 10%, Dunnett's test) in the comparative test with the control group (AE: 0, Leu: 0) is shown.

[0086] The combined effect of adding water-soluble turmeric extract (100 μg / mL) and leucine (5 mM or 10 mM) was more pronounced in increasing total muscle protein than adding either one alone, suggesting a synergistic effect of combining both substances on muscle growth.

[0087] <Example 3: Muscle protein increase effect of combined use of bisacron and leucine> The method described in Experimental Method 2 confirmed the effect of bisacron, leucine, or both on increasing the total protein content of muscle cells.

[0088] The results are shown in Figure 3. In Figure 3, Bis represents bisacron and Leu represents leucine. Figure 3 shows the mean and standard deviation of the results from the three trials. The significance probability (*: 1% to less than 5%, Dunnett's test) in the comparative test with the control group (Bis: 0, Leu: 0) is shown.

[0089] The combined addition of bisacron (2.5 μM) and leucine (10 mM) resulted in a stronger increase in total muscle protein than the addition of bisacron alone or leucine alone, suggesting a synergistic effect of muscle growth when both are used together.

[0090] <Example 4: Suppressive effect of turmeronol B on the expression of muscle atrophy-causing gene expression> The inhibitory effect of turmeronol B on the expression of muscle atrophy-causing genes (Mafbx gene, Murf1 gene) in the presence of dexamethasone was confirmed using the method described in Experimental Method 1. As a control, the mRNA expression levels of muscle atrophy-causing genes were measured under conditions without dexamethasone or turmeronol B. The mRNA expression levels of muscle atrophy-causing genes were normalized as relative values ​​to the mRNA expression level of the internal standard gene Ppia. Furthermore, the normalized mRNA expression levels of muscle atrophy-causing genes under each condition were expressed as relative values ​​to the normalized mRNA expression levels of muscle atrophy-causing genes under control.

[0091] The results are shown in Figure 4. In Figure 4, DEX represents dexamethasone and TB represents turmeronol B. Figure 4 shows the mean and standard deviation of the results from the three trials. The significance probability (##: less than 1%, student's t-test) in the comparative test between the (DEX:-,TB:-) group and the (DEX:+,TB:-) group is shown. The significance probability (**: less than 1%, *: 1% or more and less than 5%, Dunnett's test) in the comparative test between the (DEX:+,TB:0) group and the groups with TB 25μM, 50μM, or 75μM added is shown.

[0092] Turmeronol B (25 μM, 50 μM, 75 μM) was found to suppress the increase in mRNA expression levels of the Mafbx and Murf1 genes caused by dexamethasone (DEX), suggesting that turmeronol B has an inhibitory effect on muscle atrophy. [Industrial applicability]

[0093] The compositions according to the present invention are useful in the fields of food and beverages and pharmaceuticals.

Claims

1. (i) 10 mg to 1000 mg of water-soluble turmeric extract and 0.1 g to 10 g of leucine, (ii) 80 μg to 4000 μg of bisacron and 0.1 g to 10 g of leucine, or (iii) 5 μg to 250 μg of turmeronol B A composition for inhibiting muscle atrophy, including the following:

2. The composition according to claim 1, which is a food or beverage.

3. (i) 10 mg to 1000 mg of water-soluble turmeric extract and 0.1 g to 10 g of leucine, (ii) 80 μg to 4000 μg of bisacron and 0.1 g to 10 g of leucine, or (iii) 5 μg to 250 μg of turmeronol B A composition for suppressing the expression of muscle atrophy-causing genes, including the above.

4. The composition according to claim 3, which is a food or beverage.

5. (i) 10 mg to 1000 mg of water-soluble turmeric extract and 0.1 g to 10 g of leucine, (ii) 80 μg to 4000 μg of bisacron and 0.1 g to 10 g of leucine, or (iii) 5 μg to 250 μg of turmeronol B A composition for promoting the increase of protein in muscle cells, including the following:

6. The composition according to claim 5, which is a food or beverage.

7. A kit for inhibiting muscle atrophy, comprising 10 mg to 1000 mg of water-soluble turmeric extract and 0.1 g to 10 g of leucine in separate forms.

8. A kit for suppressing the expression of muscle atrophy-causing genes, comprising 10 mg to 1000 mg of water-soluble turmeric extract and 0.1 g to 10 g of leucine in separate forms.

9. A kit for promoting the increase of muscle cell protein, comprising 10 mg to 1000 mg of water-soluble turmeric extract and 0.1 g to 10 g of leucine in separate forms.

10. A kit for inhibiting muscle atrophy, comprising 80 μg to 4000 μg of bisacron and 0.1 g to 10 g of leucine in separate forms.

11. A kit for suppressing the expression of a gene causing muscle atrophy, comprising 80 μg to 4000 μg of bisacron and 0.1 g to 10 g of leucine in separate forms.

12. A kit for promoting the increase of muscle cell protein, comprising 80 μg to 4000 μg of bisacron and 0.1 g to 10 g of leucine in separate forms.

Images