Inhibition of muscle atrophy

Abstract

To provide a composition for suppressing muscular atrophy.SOLUTION: A composition for inhibiting muscle atrophy comprises, as an active ingredient, Agrimol B and / or a concentrated Agrimonia extract containing 0.3% or more of Agrimol B.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to the inhibition of muscle atrophy. [Background technology]

[0002] Elderly people are prone to muscle atrophy and muscle weakness due to aging, making them more susceptible to injuries such as muscle damage and fractures. When these people are placed under activity restrictions such as bed rest or cast immobilization for treatment and recuperation, muscle strength further declines. In addition, muscle atrophy, which generally occurs in the elderly and causes a decrease in muscle mass and strength, can be attributed to disuse muscle atrophy and sarcopenia. When muscle atrophy occurs, a further decline in muscle function is observed. Elderly people are prone to muscle atrophy due to a decline in muscle strength caused by lack of exercise, which can lead to a vicious cycle as described above, and in the worst cases, they can become bedridden. On the other hand, it is known that a certain amount of forced exercise can prevent disuse muscle atrophy and decline in muscle function in order to maintain daily living functions and quality of life (QOL).

[0003] Until now, attempts to prevent muscle atrophy and decline in muscle function have been limited to continuing moderate exercise while healthy or physical therapy during rehabilitation, and more effective methods of suppressing muscle atrophy are desired. In recent years, in addition to exercise and physical therapy, researchers have been searching for compounds that can prevent muscle atrophy and the resulting decline in muscle function, ultimately leading to bedriddenness. Genetic research is also being conducted. Non-Patent Document 1 identifies the gene responsible for muscle atrophy as atrogin and clarifies the molecular mechanism of disuse muscle atrophy. In particular, it describes that disuse muscle atrophy can be suppressed by suppressing the expression of a gene called Atrogin-1. Therefore, it is believed that if the expression of the Atrogin-1 gene can be suppressed, it will be possible to prevent and improve muscle atrophy that occurs with the activation of Atrogin-1 gene expression, such as sarcopenia. Patent Document 1 describes an Atrogin-1 gene expression inhibitor containing ε-viniferin as an active ingredient. Patent Document 2 describes an Atrogin-1 gene expression inhibitor containing gnetin C as an active ingredient. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-71549 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-140314 [Non-patent literature]

[0005] [Non-Patent Document 1] Takeshi Futagawa, "Ubiquitin ligase as a therapeutic target for disuse muscle atrophy," Biochemistry, Vol. 81, No. 7, Japanese Biochemical Society, 2009, pp. 614-618 Summary of the Invention [Problem to be solved by the invention]

[0006] The problem to be solved by the present invention is to provide a composition for inhibiting muscle atrophy. [Means for solving the problem]

[0007] As a result of intensive research to solve the above-mentioned problems, the inventors of the present invention found that agrimony extract and Agrimol B, a component contained in agrimony extract, suppress TNFα-induced increase in Atrogin-1 expression, thereby finding that the above-mentioned problems can be solved, and have completed the present invention. That is, the present invention is as follows. (1) A composition for inhibiting muscle atrophy containing Agrimol B as an active ingredient. (2) A composition for inhibiting muscle atrophy, containing concentrated agrimony containing 0.3% or more of Agrimol B as an active ingredient. [Effects of the Invention]

[0008] The present invention provides a composition for inhibiting muscle atrophy that suppresses TNFα-induced increase in Atrogin-1 expression. The composition for inhibiting muscle atrophy that suppresses TNFα-induced increase in Atrogin-1 expression of the present invention contains Agrimol B and / or concentrated Agrimony extract as active ingredients, and by suppressing TNFα-induced increase in Atrogin-1 expression, it ultimately suppresses the progression of disuse muscle atrophy or sarcopenia. As a result, it suppresses the decline in muscle function. Therefore, it can be used as a medicine or health food for preventing bedriddenness. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a graph showing the inhibitory effect of an extract of Agrimony on TNF-α-induced Atrogin-1 expression. [Figure 2] FIG. 1 shows the inhibitory effect of concentrated agrimony extract on TNF-α-induced Atrogin-1 expression. [Figure 3] FIG. 1 shows the inhibitory effect of Agrimol B on TNF-α-induced Atrogin-1 expression. DETAILED DESCRIPTION OF THE INVENTION

[0010] In the present invention, "muscle atrophy" refers to a decrease in muscle mass due to a decrease or shrinkage of muscle cells, and includes those caused by long-term bed rest, immobilization in a plaster cast due to a fracture, or exposure to microgravity (disuse muscle atrophy), and those caused by aging (sarcopenia). Therefore, inhibiting muscle atrophy means inhibiting the decrease in muscle mass that accompanies inactivity and aging. The muscle atrophy-inhibiting composition of the present invention contains Agrimol B and / or concentrated agrimony extract as active ingredients and is a composition for inhibiting TNFα-induced increase in Atrogin-1 expression. Activation of the TNFα-inducible Atrogin-1 gene promotes the breakdown of skeletal muscle proteins, leading to muscle atrophy. Meanwhile, Agrimol B and / or concentrated agrimony extract inhibits the TNFα-induced increase in Atrogin-1 expression, resulting in muscle growth. Therefore, Agrimol B and / or concentrated agrimony extract can be used as agents for preventing and improving muscle atrophy and sarcopenia.

[0011] Agrimol B has the following formula (1): [ka] In the present invention, it can be obtained by extraction from plants or chemical synthesis. Commercially available products may also be used. It is preferably obtained by extraction from plants, and particularly preferably obtained from an extract of agrimony.

[0012] Agrimonia pilosa (scientific name: Agrimonia pilosa) is a perennial herb of the Rosaceae family, belonging to the genus Agrimonia. It is found in forest edges, fields, and roadsides in Honshu, Shikoku, Kyushu, and elsewhere, and is readily available. It is also known as dragon's tooth plant or sedge plant. In the present invention, dried Agrimonia pilosa, commercially available as an ingredient in traditional Chinese medicine, folk remedies, and health foods (herbal tea), can be used. Wild or cultivated Agrimonia pilosa, or the whole plant can be harvested and naturally or heat-dried. The Agrimonia pilosa is shredded and immersed in approximately 10 times its volume of water or 0-80% (v / v) ethanol for 3-5 days for extraction at room temperature, or extracted at 50-80°C for 5-24 hours using a reflux condenser, followed by filtration to recover the Agrimonia extract. The water and ethanol are removed from the extract using a reduced-pressure vacuum dryer such as a rotary evaporator or a freeze-drying device to obtain the Agrimonia extract.

[0013] In a composition for inhibiting muscle atrophy containing a concentrated agrimony extract as an active ingredient, the concentration of Agrimol B in the concentrated agrimony extract is preferably, for example, 0.30% by mass or more.

[0014] A composition for inhibiting muscle atrophy containing Agrimol B and / or concentrated agrimony extract as active ingredients may contain Agrimol B and / or concentrated agrimony extract as is, or may be administered to animals and humans as a pharmaceutical composition with a conventional pharmaceutical carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be selected appropriately as needed. Examples include oral preparations such as tablets, capsules, granules, fine granules, and powders, and parenteral preparations such as injections and suppositories. The appropriate dosage for an adult is typically 10 to 1,000 mg of the extract taken in divided doses several times a day.

[0015] In the present invention, oral preparations such as tablets, capsules, granules, fine granules, and powders are prepared according to conventional methods using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, cornstarch, and inorganic salts. The amount of Agrimol B and / or concentrated agrimony extract in these preparations is not particularly limited and can be appropriately determined. In addition to the composition of the present invention, these preparations may also contain suitable additives such as binders, disintegrants, surfactants, lubricants, flow enhancers, flavoring agents, colorants, and fragrances.

[0016] Among the ingredients other than these active ingredients, examples of binders include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, methylcellulose sodium, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, and macrogol. Examples of disintegrants include starch, hydroxypropyl starch, carboxymethylcellulose sodium, carboxymethylcellulose calcium, carboxymethylcellulose, and low-substituted hydroxypropyl cellulose. Examples of surfactants include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid esters, and polyoxyethylene sorbitan fatty acid esters. Examples of lubricants include talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, and polyethylene glycol. Examples of flow enhancers include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate. The present invention will be described in more detail below with reference to examples. [Example]

[0017] [Suppression of Atrogin-1 expression by Agrimony in mouse skeletal muscle-derived myotube cells (C2C12)] <Preparation of Agrimony Extract and Concentrated Agrimony Extract> 100 g of agrimony (Senkakuso, Fukuda Ryu Co., Ltd.) was added to 10 volumes of 90% ethyl alcohol (1 kg), and reflux extraction was repeated twice. After completion, solid-liquid separation was performed to obtain an extract. The obtained extract was treated with activated carbon and filtered through diatomaceous earth in the usual manner, then concentrated and freeze-dried to obtain a crude extract (solid amount 9.28 g, yield 9.28% (relative to raw material)). This crude extract was used in this test as "Agrimony Extract." 8.51 g of the dried extract of Cinmizuhiki was dissolved and suspended (solid content: 10%) in a 20% ethanol solution heated to 60°C, and then filtered through diatomaceous earth by a conventional method to recover the residue. The residue was dissolved again in 90% ethanol, filtered through diatomaceous earth by a conventional method, and then concentrated and freeze-dried again to obtain a concentrate (solid content: 0.59 g, yield: 6.93% (relative to the Cinmizuhiki extract)). This was designated as the concentrated Cinmizuhiki extract. This concentrated Cinmizuhiki extract had the fat-soluble components containing Agrimol B concentrated by removing the 20% ethanol-soluble components from the Cinmizuhiki extract.

[0018] <Measurement of the Content of Agrimol B in Cinmizuhiki Extract and Concentrated Cinmizuhiki Extract> For the test samples used in this example, the content of Agrimol B was measured by a conventional method. It was contained at 0.036% by mass in the Cinmizuhiki extract and 0.447% by mass in the concentrated Cinmizuhiki extract. The content of Agrimol B in the Cinmizuhiki extract and the concentrated Cinmizuhiki extract was measured by HPLC under the following conditions. (Measurement Conditions by HPLC) · Column: Wakosil-ll5C18 AR 4.6 mm × 150 mm · Mobile Phase: Distilled Water: Acetonitrile: Phosphoric Acid = 100:900:1 · Flow Rate: 1 mL / min · Detection: UV288 nm · Injection Volume: 10 μL

[0019] <Culture and Differentiation Induction of C2C12 Cells> C2C12 was cultured in DMEM / F-12 (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Hyclone laboratories) and 1% penicillin-streptomycin (Sigma Aldrich). The pre-cultured C2C12 was at 2.5×10 5Suspended to a concentration of cells / mL, seeded 400 μL each into a Collagen Coated 48 well Plate (CORNING), and cultured at 37 °C under 5% CO₂. The next day, the entire culture medium was replaced with DMEM / F-12 (differentiation induction medium) containing 2% horse serum (HS, Gibco) and 1% penicillin-streptomycin to initiate differentiation induction. The culture medium was replaced by half on the 2nd and 4th days of differentiation.

[0020] <Samples and TNF-α treatment for muscle tube cells> On the 5th day of differentiation, after removing the culture supernatant, 400 μL of a new medium in which the samples were dissolved was added. The dissolved concentrations were adjusted so that the concentrations of the Kinhimizuhiki extract were 1 μg / mL, 3 μg / mL, 10 μg / mL, and the concentrations of the concentrated Kinhimizuhiki extract were 1 μg / mL, 3 μg / mL, 10 μg / mL, respectively. After culturing for 24 hours, the culture supernatant was removed, replaced with a new medium in which TNF-α (final concentration 12 ng / mL) was dissolved, and further cultured. After 3 hours, the culture supernatant was removed, and the samples for RNA were collected using RLT Lysis buffer (QUIAGEN).

[0021] <Analysis of Atrogin-1 expression> RNA was prepared using the RNeasy Mini kit (QIAGEN) according to the attached instructions. Using 100 ng of the prepared RNA, cDNA was synthesized using the PrimeScript RT reagent kit (Takara) according to the attached instructions. The expression level of Atrogin-1 was measured using Quanti Studio (Applied Biosystems) under the reaction conditions of 95°C for 20 seconds, (95°C for 1 second → 60°C for 20 seconds) × 45 cycles by mixing cDNA, mouse Atrogin-1 taq man probe (TaqMan Gene expression assays: Applied Biosystems) and Premix Ex Taq Perfect Real Time (TaKaRa). The expression level of GAPDH was measured in the same reaction as above using the mouse GAPDH taq man probe (TaqMan Gene expression assays: Applied Biosystems) as an internal standard. The relative gene expression levels of each sample were calculated by the ΔΔCt method using GAPDH as an internal standard from the Ct values obtained by the measurement. The primers used were as follows. Atrogin-1 (alias: Fbxo32 F-box protein 32): Assay ID: Mm00499523_m1 Gapdh (Glyceraldehyde-3-phosphate dehydrogenase): Assay ID: Mm99999915_g1

[0022] The results are shown in Figures 1 and 2. The extract of Kinmizuhiki shown in Figure 1 did not suppress the TNF-α-induced increase in Astrogin-1 expression. However, it was found that the concentrated extract of Kinmizuhiki shown in Figure 2 suppressed the TNF-α-induced increase in Astrogin-1 expression.

[0023] [Atrogin-1 Expression Inhibition Test of Agrimol B on Mouse Skeletal Muscle-Derived Myotube Cells (C2C12)] [Culture and Differentiation Induction of C2C12 Cells] C2C12 was cultured in DMEM (High glucose) containing 10% FBS and 1% penicillin-streptomycin. The pre-cultured C2C12 was suspended to a concentration of 4×10 5 cells / mL and seeded at 100 μl per well in a 96-well plate (CORNING), and cultured at 37 °C under 5% CO2. The next day, the entire medium was replaced (200 μl) with DMEM / F-12 containing 2% HS and 1% penicillin-streptomycin to initiate differentiation induction. Half of the medium was replaced on the 2nd and 4th days of differentiation.

[0024] <Samples and TNF-α treatment for myotubes> Six days after the induction of differentiation, the culture supernatant was removed, and 100 μl of fresh differentiation induction medium was added. Agrimol B (Chem Faces) was treated here to a final concentration of 2.5 μM, 5 μM, or 10 μM and cultured for 24 hours. After culturing in the presence of Agrimol B, the culture supernatant was removed, and 100 μl of fresh differentiation induction medium was added. TNF-α was added here to a final concentration of 12 ng / mL. After 3 hours, the culture supernatant was removed, and samples for RNA were collected using RLT Lysis buffer (QUIAGEN).

[0025] <Analysis of Atrogin-1 expression> RNA was prepared using the RNeasy mini plus kit (QIAGEN) according to the attached instructions. 200 ng of the prepared RNA was used to prepare cDNA using the PrimeScript RT reagent kit (Takara) according to the attached instructions. Atrogin-1 expression levels were measured by mixing cDNA, a mouse Atrogin-1 TaqMan probe (TaqMan Gene expression assays: Applied Biosystems), and Premix Ex Taq Perfect Real Time (TaKaRa) in a Quanti Studio (Applied Biosystems) reaction mixture at 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second, followed by 60°C for 20 seconds. GAPDH expression levels were measured using a mouse GAPDH TaqMan probe (TaqMan Gene expression assays: Applied Biosystems) as an internal standard in the same reaction. The relative gene expression levels of each sample were calculated from the Ct values obtained by the assay using the ΔΔCt method with GAPDH as the internal standard.

[0026] The results are shown in Figure 3. As shown in Figure 3, Agrimol B was found to suppress the TNF-α-induced increase in Astrogin-1 expression.

Claims

1. A composition for inhibiting muscle atrophy, containing Agrimol B as an active ingredient.

2. A composition for inhibiting muscle atrophy, comprising concentrated agrimony containing 0.3% or more of Agrimol B as an active ingredient.

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