Muscle atrophy inhibition

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 suppression of muscle atrophy.

Background Art

[0002] In the elderly, muscle atrophy and muscle strength decline due to aging are observed, and injuries such as muscle damage and increased fracture susceptibility are likely to occur. For the treatment and convalescence, when placed under activity restrictions such as rest or plaster fixation, muscle strength further declines. In general, muscle atrophy in the elderly, in which muscle mass and muscle strength decrease, includes disuse muscle atrophy and sarcopenia. When muscle atrophy occurs, a further decline in muscle function is observed. The elderly tend to develop muscle atrophy from a decline in muscle strength due to lack of exercise, and are likely to fall into the above-mentioned vicious cycle from muscle atrophy. In the worst case, they become bedridden. On the other hand, it is known that to some extent forced exercise can suppress disuse muscle atrophy and decline in muscle function in order to maintain life functions and quality of life (QOL).

[0003] Hitherto, attempts to prevent muscle atrophy and decline in muscle function have been limited to continuing appropriate exercise during normal health or physical therapy for rehabilitation, and a more effective method for suppressing muscle atrophy is desired. In recent years, not only exercise and physical therapy but also the search for components that can prevent muscle atrophy and the accompanying decline in muscle function, and ultimately prevent bedriddenness, has been carried out. Furthermore, genetic research has also been conducted. Non-Patent Document 1 identifies that the gene causing muscle atrophy is atrogins, and the molecular mechanism of disuse muscle atrophy has been clarified. In particular, it is described that disuse muscle atrophy can be suppressed by suppressing the expression of a gene called Atrogin-1. Therefore, it is said that if the expression of the Atrogin-1 gene can be suppressed, muscle atrophy that occurs with the activation of the expression of the Atrogin-1 gene such as sarcopenia can be prevented and improved. 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

Patent Document 2

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that the extract of Agrimonia pilosa Ledeb. and Agrimol B, which is a component contained in the extract of Agrimonia pilosa Ledeb., suppresses the increase in TNFα-induced Atrogin-1 expression, and thus have found that the above problems can be solved, leading to the completion of the present invention. That is, the present invention is as follows. (1) A composition for suppressing muscle atrophy containing Agrimol B as an active ingredient. (2) A composition for suppressing muscle atrophy containing concentrated Agrimonia pilosa Ledeb. containing 0.3% or more of Agrimol B as an active ingredient.

Advantages of the Invention

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

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out 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, which is caused by long-term bed rest, plaster fixation due to fractures, or exposure to microgravity (referred to as disuse muscle atrophy), or is associated with aging (referred to as sarcopenia). Therefore, suppressing muscle atrophy means suppressing the decrease in muscle mass associated with inactivity or aging. The composition for suppressing muscle atrophy of the present invention is a composition for suppressing muscle atrophy that contains Agrimol B and / or concentrated Agrimonia pilosa extract as an active ingredient and suppresses the increase in TNFα-induced Atrogin-1 expression. When the TNFα-induced Atrogin-1 gene is activated, the degradation of skeletal muscle proteins is promoted and muscle atrophy progresses. On the other hand, Agrimol B and / or concentrated Agrimonia pilosa extract suppress the increase in TNFα-induced Atrogin-1 expression and strengthen the muscles. Therefore, Agrimol B and / or concentrated Agrimonia pilosa extract can be used as a preventive and ameliorating agent for muscle atrophy and sarcopenia.

[0011] Agrimol B is a compound represented by the following formula (1):

Chemical formula

[0012] Agrimonia pilosa (scientific name: Agrimonia pilosa) is a perennial herb of the genus Agrimonia in the Rosaceae family. It grows on the edges of forests, fields, and roadsides in Honshu, Shikoku, Kyushu, etc., and is easily available. It is also known as Longyacao and Xianhecao. In the present invention, as Agrimonia pilosa, a dried product commercially available as a crude Chinese medicine, a folk remedy, or a raw material for health foods (herbal tea) can be used. It may be wild or cultivated, and the whole plant can be collected and naturally dried or heat-dried. This is cut into small pieces and immersed in about 10 times the amount of water or ethanol with a water content concentration of 0 to 80% (v / v) for 3 to 5 days and extracted at room temperature, or extracted with a reflux condenser at 50 to 80°C for 5 to 24 hours, and filtered to recover the Agrimonia pilosa extract. This extract is made into an Agrimonia pilosa extract by removing water and ethanol using a vacuum drying device such as a rotary evaporator or a freeze-drying device.

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

[0014] The composition for suppressing muscle atrophy containing Agrimol B and / or the extract of concentrated Agrimol B as an active ingredient may use Agrimol B and / or the extract of concentrated Agrimol B as it is, and can be administered to animals and humans as a pharmaceutical composition together with a conventional pharmaceutical carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as needed. For example, oral preparations such as tablets, capsules, granules, fine granules, powders, etc., and parenteral preparations such as injections, suppositories, etc. can be mentioned. The dosage is usually appropriate for adults to take 10 to 1000 mg of the extract by weight several times a day.

[0015] In the present invention, oral preparations such as tablets, capsules, granules, fine granules, powders, etc. are produced according to a conventional method using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, etc. The blending amount of Agrimol B and / or the extract of concentrated Agrimol B in these preparations is not particularly limited and can be appropriately designed. In addition to the composition of the present invention, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, etc. can be appropriately used in this type of preparation.

[0016] Among the formulation components other than these active ingredients, examples of the binder include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, sodium methylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol, and the like. Examples of the disintegrant include starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, low-substituted hydroxypropylcellulose, and the like. Examples of the surfactant include sodium lauryl sulfate, soy lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester, and the like. Examples of the lubricant include talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol, and the like. Examples of the fluidity promoter include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate, and the like. Hereinafter, the present invention will be described in more detail with reference to examples.

Examples

[0017] [Test for inhibiting Atrogin-1 expression of Kinnmizu Hiki on mouse skeletal muscle-derived myotube cells (C2C12)] [Preparation of Kinnmizu Hiki extract and concentrated Kinnmizu Hiki extract] To 100 g of Kinnmizu Hiki (Agrimonia pilosa Ledeb., Fukuda Ryuzo Co., Ltd.), 10 times the amount (1 kg) of 90% ethyl alcohol was added, 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 by a conventional method, and then concentrated and freeze-dried to obtain a crude extract (solid content 9.28 g, yield 9.28% (relative to the raw material)). This crude extract was used in this test as the "Kinnmizu Hiki extract". 8.51 g of the dried extract of *Kinmizuhiki* 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 *Kinmizuhiki* extract)). This was designated as the concentrated *Kinmizuhiki* extract. By removing the 20% ethanol-soluble components from the *Kinmizuhiki* extract, the fat-soluble components including Agrimol B were concentrated in this concentrated *Kinmizuhiki* extract.

[0018] <Measurement of the Content of Agrimol B in the *Kinmizuhiki* Extract and the Concentrated *Kinmizuhiki* Extract> Regarding 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 *Kinmizuhiki* extract and 0.447% by mass in the concentrated *Kinmizuhiki* extract. The content of Agrimol B in the *Kinmizuhiki* extract and the concentrated *Kinmizuhiki* extract was measured by HPLC under the following conditions. (Measurement Conditions by HPLC) · Column: Wakosil-II 5C18 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 medium was replaced with DMEM / F-12 (differentiation induction medium) containing 2% horse serum (HS, Gibco) and 1% penicillin-streptomycin to initiate differentiation induction. Half of the medium was replaced 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 fresh medium for lysing the samples 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 fresh medium containing TNF-α (final concentration 12 ng / mL), and further cultured. After 3 hours, the culture supernatant was removed, and the samples for RNA were collected using RLT Lysis buffer (QUIAGEN).

[0021] <Atrogin-1 expression analysis> 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) with 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 Figure 1 and Figure 2. The Kinhimizuki extract shown in Figure 1 did not suppress the TNF-α-induced increase in Astrogin-1 expression. However, it was found that the concentrated Kinhimizuki extract 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. On the next day, the entire culture medium was replaced (200 μl) with DMEM / F-12 containing 2% HS and 1% penicillin-streptomycin to initiate differentiation induction. Half of the culture medium was replaced on the second and fourth days of differentiation.

[0024] <Samples and TNF-α treatment for myotube cells> 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. Using 200 ng of the prepared RNA, cDNA was prepared using the PrimeScript RT reagent kit (Takara) according to the attached instructions. The expression level of Atrogin-1 was measured by mixing cDNA, the mouse Atrogin-1 taq man probe (TaqMan Gene expression assays: Applied Biosystems) and Premix Ex Taq Perfect Real Time (TaKaRa), and 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) × 40 cycles. 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.

[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

**Claim 1** A composition for suppressing the increase in TNFα-induced Atrogin-1 expression, having Agrimol B as an active ingredient. **Claim 2** A composition for suppressing the increase in TNFα-induced Atrogin-1 expression, having a concentrated extract of Agrimonia pilosa Ledeb. containing 0.3% by mass or more of Agrimol B as an active ingredient.

Images