JP2015140314A - Atrogin-1 inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical or food and drink which inhibit myofunction decrease and muscle atrophy, are useful for preventing a bedridden state, and have a high level of safety.SOLUTION: An atrogin-1 gene expression inhibitor comprises gnetin C as an active ingredient. The expression of the atrogin-1 gene is inhibited, thereby preventing and improving disuse muscle atrophy, sarcopenia and the like, in order to prevent and improve muscle strength decrease.

Description

  The present invention relates to an atrosin-1 inhibitor and a muscular atrophy prevention or muscular strength prevention / amelioration agent containing the same.

In elderly people, muscle atrophy and muscle strength decrease due to aging are recognized, and injuries such as muscular damage and fracture tend to occur. The muscle strength is further reduced when the subject is placed in a resting state for such treatment or medical treatment or under the restriction of activities such as cast fixation.
In general, muscle atrophy in which the muscle mass and muscle strength of an elderly person decrease include disused muscle atrophy and sarcopenia. When muscle atrophy occurs, the muscle function further decreases.
Elderly people become muscular atrophy due to a decrease in muscular strength, and are more likely to fall into the vicious circle as described above from muscular atrophy, and in the worst case, become bedridden. For this reason, in order to improve life function and maintain quality of life (QOL), it is said that disuse muscular atrophy and deterioration of muscle function can be suppressed by a certain amount of forced exercise. (Non-patent document 1: Ministry of Health and Welfare, 1991, regarding the spread of 10 articles to bedridden zero).

Until now, attempts to prevent muscle atrophy and deterioration of muscle function have been limited to normal continuation of exercise or physical therapy for rehabilitation, and a more effective method for suppressing muscle atrophy is desired.
In addition to exercise and physical therapy, search has already been made for ingredients that can prevent muscle atrophy and the accompanying decline in muscle function and eventually bedridden.
Inhibition of muscle atrophy by fruit polyphenol (Patent Document 1: JP 2001-89387 A), suppression of muscle protein degradation by lycopene (Patent Document 2: JP 2004-59518 A), reduction of muscle oxidative stress by superoxide dismutase ( Patent Document 3: Japanese Patent Laid-Open No. 2006-62976), Muscle Function Decrease Inhibitor (Patent Document 4: Japanese Patent Laid-Open No. 2008-13473) containing catechins as an active ingredient, Myostatin antagonist by myostatin peptide (Patent Document 5: JP-T-2008-530004), a muscle strength enhancer (Patent Document 6: WO2008-123417 International Publication), and L-leucine in a molar ratio of 35 to 66% in total essential amino acids. For preventing skeletal muscle loss (Japanese Patent Application Laid-Open No. 2012-13181) JP) and the like.

  On the other hand, genetic studies have also been conducted, and a muscle atrophy-causing gene (atrogines) has been identified and the molecular mechanism of disuse muscle atrophy has been clarified. In particular, it has been clarified that by suppressing the expression of a gene called atrodin-1, it is possible to suppress disuse muscle atrophy (Non-patent Document 2: Biochemical Vol. 81, No. 7, pages 614-618, 2009). Year). Therefore, if it is possible to suppress the expression of the atrodin-1 gene, it is said that the muscular atrophy that occurs along with the activation of the expression of the atrodin-1 gene such as sarcopenia can be prevented and improved.

JP 2001-89387 A JP 2004-59518 A JP 2006-62976 A JP 2008-13473 A Special table 2008-530004 gazette WO2008-123417 International Publication JP 2012-131819 A

About spread of "10 articles to bedridden zero": March 7, 1991 Genko 18th (announced by the Ministry of Health and Welfare) Journal “Biochemistry” Vol. 81, No. 7, pp. 614-618, 2009, published by the Japanese Biochemical Society

  An object of the present invention is to provide a pharmaceutical or a food or drink useful for bedridden prevention by suppressing a decrease in muscle function or muscle atrophy.

  Then, when this inventor examined the component derived from a natural product, he found the inhibitory action of the atrogin-1 gene expression strong against gnethin C which is a plant-derived natural component.

The present invention has the following configuration.
(1) An agent that suppresses the expression of attronidine-1 gene containing gnetin C as an active ingredient.
(2) A muscular atrophy inhibitor comprising the atrodin-1 gene expression inhibitor according to (1).
(3) A food or drink containing the muscle atrophy inhibitor according to (2).
(4) A sarcopenia preventive / ameliorating agent comprising the muscle atrophy inhibitor according to (2).

  According to the present invention, an inhibitor of atrodin-1 gene expression is provided. The atrodin-1 gene expression inhibitor of the present invention contains gnetin C as an active ingredient, and finally suppresses the progression of disuse muscle atrophy or sarcopenia by suppressing the gene expression of atrodin-1. As a result, the decrease in muscle function is suppressed. Therefore, it can be used as a medicine or health food for bedridden prevention.

It is a graph which shows the measurement result by the relative quantification method ((DELTA) (DELTA) Ct method) which confirmed that gnetin C suppresses the expression of the atrodin-1 gene in the myoblast derived from a striated muscle (a mouse | mouth ACTB gene is made into a reference gene) If you have). It is a graph which shows the measurement result by the relative quantification method ((DELTA) (DELTA) Ct method) which confirmed that gnetin C suppresses the expression of the atrogin-1 gene in the myoblast derived from a striated muscle (a mouse | mouth GAPD gene is referred to as a reference gene) If you have).

Hereinafter, the present invention will be described in detail.
In the present invention, “muscle atrophy” refers to a decrease in muscle mass due to a decrease or reduction in muscle cells, such as cast fixation by prolonged bed rest or fracture, or exposure to microgravity (disused muscles). Atrophy), and those associated with aging (called sarcopenia). Therefore, suppression of muscular atrophy means suppressing a decrease in muscle mass associated with inactivity or aging.
The present invention is an inhibitor of atrodin-1 containing gnetin C as an active ingredient. When the atrodin-1 gene is activated, the degradation of skeletal muscle protein is promoted and muscle atrophy is promoted. On the other hand, when the muscle is enlarged by IGF-1, the expression of the atrodin-1 gene is suppressed and the muscle is strengthened. Therefore, gnetin C can be used as a preventive / ameliorating agent for muscle atrophy and sarcopenia.

  Gnetin C used in the present invention is a kind of resveratrol classified into a group of polyphenols called stilbenoids (stilbene derivatives). The system name is (2R, 3R) -2- (4-hydroxyphenyl) -3- (3,5-dihydroxyphenyl) -6-[(E) -4-hydroxystyryl] -2,3-dihydrobenzofuran- 4-ol. The structural formula of gnetin C is shown in the following chemical formula 1.

Gnetin C is a stilbene compound contained in seeds and fruits of Gnetum family plants such as Gnemon (scientific name: Ginum gnemon, also known as Melinjo) distributed in Southeast Asia. It is also known as a kind of grape phytoalexin. Gnetin C can be extracted from Gnetsum seeds (also known as merillo) using various extraction solvents, preferably methanol or methanol. The extracted gnetin C can be separated and purified by separation and purification means such as preparative high performance liquid chromatography.
Moreover, the high purity product synthesize | combined using organic chemistry or microorganisms can be used for the gnetin C of this invention.
Extraction, for example, is known to contain a lot of gnetin C in the seeds of merillo, so after drying, it is immersed in an extraction solvent for a certain period of time or brought into contact with an extraction solvent that is heated to reflux, It can then be filtered, concentrated and further collected by preparative chromatography as described above. As the extraction solvent, any solvent that is usually used for extraction can be used. For example, water, methanol, ethanol, propylene glycol, 1,3-butylene glycol, glycerol and other alcohols, chloroform, dichloroethane, four Organic solvents such as carbon chloride, acetone and ethyl acetate can be used alone or in combination.
The extract obtained by extraction with the above solvent is used as it is, or concentrated extracts are used, or these extracts are adsorbed, for example, impurities are removed using an ion exchange resin, porous polymers (for example, Amberlite XAD- After adsorbing on the column of 2), elution with methanol or ethanol and concentration can also be used.

  The atrogin-1 inhibitor of the present invention can be administered to humans and animals as a pharmaceutical preparation, and can also be ingested even if blended with various foods and drinks, feeds (eg pet food).

The pharmaceutical preparation is appropriately used orally or parenterally (intravenous administration, intraperitoneal administration, etc.).
The dosage form is also arbitrary, for example, oral solid preparations such as tablets, granules, powders and capsules, oral liquid preparations such as internal liquids and syrups, and parenteral liquid preparations such as injections. These forms can also be appropriately prepared by known methods.
For these pharmaceutical preparations, commonly used binders, disintegrants, thickeners, dispersants, reabsorption accelerators, corrigents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonicity Excipients such as agents, stabilizers and pH adjusters may be used as appropriate.

  In the pharmaceutical preparation of the present invention, the dosage of gnetin C, which is an active ingredient, varies depending on its purity, dosage form, patient age, body weight, indication symptoms, etc. For example, in the case of oral administration, it may be an adult. For example, it is administered once to several times a day, and about 1 mg to 200 mg, preferably about 3 mg to 20 mg / person is administered once a day.

  As a form of food / beverage products, it can shape | mold arbitrarily, such as a granular form, a granular form, a paste form, a gel form, solid form, or a liquid form, for example. These include various known substances that are recognized to be contained in foods, such as binders, disintegrants, thickeners, dispersants, reabsorption accelerators, flavoring agents, buffers, surfactants, Excipients such as solubilizers, preservatives, emulsifiers, isotonic agents, stabilizers and pH adjusters can be contained as appropriate.

  The content of gnetin C, which is an active ingredient contained in the food and drink of the present invention, can be appropriately determined according to the type, purpose, form, usage method, and the like, for example, about 1 to 10% by mass. can do. In particular, when used as a health food or drink, it is preferable to contain the active ingredient of the present invention in such an amount that a predetermined effect is sufficiently exhibited. Therefore, in such a case, the food / beverage product of the present invention contains gnetin C, and the food / beverage product with an indication that it is used for the prevention or improvement of various diseases that become difficult to act due to muscle atrophy. It can be.

  Hereinafter, the present invention will be described in more detail based on examples and reference examples. In addition, this invention is not limited to a following example. Moreover, all compounding quantities are shown by the mass%.

Example <Suppression test of atrodin-1 gene in striated myoblasts>
The C2C12 cell line, which is a mouse striated myoblast cell line, has been used for studies on muscle protein synthesis and degradation systems and on muscle differentiation, and is suitable for the purpose of observing gene expression in muscles.

1. Test sample A high purity reagent (manufactured by Wako Pure Chemical Industries, Ltd.) of gnetin C purified from commercially available meringo was used as a test sample. For comparison purposes, Piceatannol derived from red wine, which is a compound belonging to resveratrol, was used.
Piceatannol is a stilbenoid compound similar to gnetin C having the following structural formula, and is classified as resveratrol.

2. Cell Line and Culture C2C12 cells, a mouse striated myoblast cell line, were used for real-time PCR (Polymerase Chain Reaction) studies. C2C12 cells are used for studies on muscle protein synthesis and degradation systems and on muscle differentiation. On the other hand, in skeletal muscles of mice, increased expression of the atrodin-1 gene has been reported along with muscle atrophy, and in skeletal muscles of rats and human skeletal muscles, an increase in the atrosin-1 gene has been reported with aging. Using the mRNA expression of C2C12 cells as an index, suppression of the expression of the atrodin-1 gene was examined.
C2C12 cells (DS Pharma Biomedical Co., Ltd.) was purchased in the vial (> 10 ⁶ cells), which was defined as the F ₀ generation. While the subculture was performed, the generation number was not changed, but the cells having the number of passages of 10 or less were subjected to the experiment (F ₀ cells were frozen and stored in a liquid nitrogen liquid phase. when used in the experiment was remelted, the cells were the F ₁ generation). C2C12 cells were subcultured at a ratio of 1:20 after reaching submaximal confluent, and cells that reached about 30% confluent after 24 hr were used. Since C2C12 cells are adherent cells, acutase (Innovative Cell Technologies, Inc. San Diego, CA, USA) was used for cell detachment. Cell culture medium is low glucose (1,000 mg / L, D-glucose) DMEM (Dulbecco's modified Eagle medium) basic liquid medium (500 mL, 12320-032, 20 mM HEPES [4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid], 110 mM sodium pyruvate, Life Technologies Japan, Hatchobori, Tokyo), 10% HyClone (registered trademark) fetal bovine serum (FBS [Fetal Bovine Serum]: Australian, Lot No .: 14301101, Thermo Scientific, Tauranga, New Zealand) and antibacterial-antimycotic (Antibiotic-Antimycotic, 10 mg / L, Life Technologies Japan, Hatchobori, Tokyo [penicillin G; 100 mg / mL, streptomycin sulfate; 100 mg / mL, amphotericin B; 0.25 mg / mL]) was added. At 0 hr, as a food-derived component, a final concentration of 10, 30 mM piceatannol (Lot No .: WEG2634, Wako Pure Chemical Industries, Ltd., Doshumachi, Osaka) and a final concentration of 10, 30 mM gnetin C (Lot No.: TLG0417, Wako Pure Chemical Industries, Ltd., Doshumachi, Osaka) was added, and ethanol (EtOH, 99.5 v / v%, Wako Pure Chemical Industries, Ltd.), the solvent of the above components, was added to the culture supernatant of the control group. , Doshumachi, Osaka) was added in the same amount (10 mL) as the ingredients were added. After 24 hours and 48 hours after the start of the test, the culture supernatant was aspirated and removed to obtain an mRNA recovery sample.

3. Measurement of suppression of Atrodin-1 gene expression Collected cells (10 × 10 ⁶ cells / sample) were obtained from DPBS (Dulbecco's Phosphate Buffered Saline, (-) CaCl ₂ (-) MgCl ₂ , Life Technologies Japan, Hatchobori, Tokyo) After washing, total RNA was extracted from C2C12 cells using High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany). Next, Transcriptor Universal cDNA Master (4 mL; Reverse Tanscriptase, 1 mL; Transcriptor Reaction Buffer containing random hexamer primers, 1 mL; Template RNA, 14 mL; PCR grade water: final volume 20 mL / tube) Roche Diagnostics, Mannheim, Germany) A reverse transcription reaction was performed to prepare cDNA. Using 2 mL of this cDNA, real-time PCR was performed using a LightCycler (registered trademark) 480 (Roche Diagnostics, Mannheim, Germany) in a final volume of 20 mL (Forward primer; 0.4 mL, Reverse primer; 0. 4 mL, Probe 0.4 mL, PCR grade water; 6.8 mL, 480 Master Mix; 10 mL: final volume 20 mL / well). The target primer was synthesized by Life Technologies Japan, and the reference primer and other reagents (Probe, PCR grade water, 480 Master Mix) were from Roche Diagonisitics.

4). Analysis Method Data processing was performed using analysis software in the LightCycler (registered trademark) 480 system. Since the PCR reaction was performed by the probe method, the target gene expression level was calculated by the relative quantification method by comparison with the reference gene expression level: ΔΔCt method. Mouse ACTB (b-actin) and mouse GAPD (glyceraldehyde-3-phosphate dehydrogenase) were used as reference genes.
The primer sequence of mouse atrodin-1, which is the target gene, is as follows.
m Atrosin-1 forward: 5'-AGTGAGGACCGGCTACTGTG-3 '
m Atrosin-1 reverse: 5'-GATCAAACGCTTGCGAATCT-3 '

5. Results The measurement and analysis results are shown in FIGS.
10 μM and 30 μM piceatannol did not suppress the expression of the atrodin-1 gene in striated myoblasts, whereas gnetin C suppressed the expression of the atrodin-1 gene. Piceatanol also promoted the expression of the atrodin-1 gene.
It can be said that the suppression of the expression of the atrodin-1 gene is an action specific to gnetin C.
From the above results, it was found that gnetin C can suppress muscle atrophy caused by the expression of the atrodin-1 gene.

  Supplement tablets, soft capsules, gummi, soft drinks, tablet confectionery, and candy can be produced according to conventional methods according to the formulations shown in Tables 1 to 6 below.

Claims (4)

  An agent that inhibits the expression of attronidine-1 gene comprising gnetin C as an active ingredient.   A muscle atrophy inhibitor comprising the atrodin-1 gene expression inhibitor according to claim 1.   Food-drinks containing the muscle atrophy inhibitor of Claim 2.   A sarcopenia preventive / ameliorating agent comprising the muscle atrophy inhibitor according to claim 2.