JP2015071549A - Atrogin-1 inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide highly safe pharmaceuticals or foods and drink which suppress reduced muscle function or muscle atrophy and are useful for prevention of bedridden.SOLUTION: The invention relates to an atrogin-1 gene silencing agent containing ε-viniferin as an active ingredient.

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 Strength Function Reduction 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), muscle strengthening agent containing Tedokoro extract as an active ingredient (Patent Document 6: WO2008 / 123417 International Publication), L-leucine in a total essential amino acid in a molar ratio of 35 to 66%. For preventing skeletal muscle mass loss (Patent Document 7: JP2012-1318A) 9 No.), and the like.

  On the other hand, genetic studies have also been conducted, and a muscle atrophy-causing gene (atrogins) 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 the spread of "10 articles to bedridden zero": March 18, 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 muscle function decline or muscle atrophy.

  Then, when this inventor examined the component derived from a natural product, he found the inhibitory action of atrodin-1 gene expression strong against (epsilon) -viniferin which is a plant-derived natural component.

The present invention has the following configuration.
(1) An atrosin-1 gene expression inhibitor containing ε-viniferin 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 comprises ε-viniferin as an active ingredient, and finally suppresses the disuse of muscle atrophy or the progression of 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 preventing bedridden.

It is a graph which shows that (epsilon) -viniferin has suppressed the expression of the atrodin-1 gene in the myoblast derived from a striated muscle.

  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 ε-viniferin 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, ε-viniferin can be used as a preventive / ameliorating agent for muscle atrophy and sarcopenia.

  The ε-viniferin used in the present invention is a stilbene compound and is known as a kind of grape phytoalexin. Resveratrol in the present invention refers to a polyphenol monomer having a stilbene skeleton as a basic structure. ε-viniferin is this dimer. Ε-viniferin can be extracted from a plant body containing a stilbene compound, preferably from grape side branches, using various extraction solvents, preferably 70% methanol. The extracted ε-viniferin can be separated and purified by separation and purification means such as preparative high performance liquid chromatography.

  Although ε-viniferin is collectively referred to as resveratrol, it is used separately in the present invention. In the present invention, as described above, resveratrol is a kind of polyphenol present in grape leaves, shoots, pericarp and the like, and refers to a monomer of a stilbene compound, and trans-resveratrol is a typical compound.

  As ε-viniferin, it is preferable to use a high-purity product synthesized using organic chemistry or microorganisms. However, it cannot be supplied in large quantities yet. Moreover, it can extract and use from plant bodies, such as the above-mentioned grape, In that case, you may use an extract or resveratrol obtained from the extract as ε-viniferin high concentration content You may use as it is. The natural product may be any as long as the content of ε-viniferin is controlled, and examples thereof include grapes, peanuts, and teraceae plants such as Japanese knotweed and clover.

Grape is particularly preferred as a raw material for extraction from natural products. The type of grape is not particularly limited, but Delaware, Kyoho, Koshu, Pione, Muscat, Chenin Blanc, Grenache, Mataro, Muller Thurgau, Trebbiano, Berry A, Cabernet Sauvignon, Merlot, Pinot Noir, Cabernet Franc, Schiller, Chardonnay, Sauvignon Blanc, Semillon, Schiller, Gamay, Riesling, Alligote and the like are preferred.
A grape extract refers to an extract from a product derived from grape berries, grape leaves or grapes. The thing derived from grape means grape juice, wine, the residue at the time of wine manufacture, wine concentrate, etc.
Extraction, for example, resveratrols are known to be abundant in grape skins, shoots, leaves, vines, so after drying them as necessary, soak in a solvent for a certain period of time, Alternatively, it can be brought into contact with a heating refluxing extraction solvent, then filtered, concentrated, and further collected by the above-described preparative chromatography. 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 removed by using an adsorption method, for example, ion exchange resin, or a porous polymer (for example, Amberlite XAD- After adsorbing on the column of 2), elution with methanol or ethanol and concentration can also be used. Further, a partitioning method, for example, an extract extracted with water / ethyl acetate can be used. Moreover, since it is contained also in wine, the concentrate of wine, or the solid substance concentrated and dried, they can also be used. The grape residue generated during wine production can be used as it is, or can be extracted in the same manner as the above extraction method. As the organic solvent, alcohol such as ethanol is preferable, and ethanol is particularly preferable.

  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 dose of ε-viniferin, which is an active ingredient, varies depending on its purity, its dosage form, patient age, body weight, indication symptoms, etc. For example, in the case of oral administration, adult 1 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-drinks, it can shape | mold arbitrarily, for example in the shape of a granule, a granular form, a paste form, a gel form, solid form, or liquid. 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 ε-viniferin, 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, 1 to 10% by mass. Can be about. 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 ε-viniferin and is labeled as being used for the prevention or improvement of various diseases that become difficult to act due to muscle atrophy. It can be a food and drink.

  Hereinafter, the present invention will be described in more detail based on reference examples and 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. Cell lines and cultures C2C12 cells (DS Pharma Biomedical Co., Ltd.) are purchased in vials (> 10 ⁶ cells), cultured until reaching confluence, and further subcultured at a ratio of 1:20. After 24 hours, cells that reached approximately 30% confluent were used for the test. The culture was performed under conditions of 37 ° C., 5%, CO ₂ .
For cell detachment, actase (Innovative Cell Technologies, Inc.) was used.
As a medium for cell culture, low glucose (1,000 mg / l, D-glucose) DMEM (Dulbecco's modified Eagle medium) is selected as a basic liquid medium, 10% HyClone fetal calf serum (Thermo Scientific) and Antibacterial-antifungal agent was added at 10 mg / l. The antibacterial-antifungal agent is a mixture of penicillin G; 100 mg / ml, streptomycin sulfate; 100 mg / ml, amphotericin B; 0.25 mg / ml.

2. Measurement of Atrosin-1 Gene Expression Suppression Immediately before culturing, trans resveratrol (Wako Pure Chemical Industries, Ltd.) to a final concentration of 10 and 30 mM, and ε-viniferin (Japanese Kojun Pharmaceutical Co., Ltd.) was added. In addition, 10 ml of ethanol which is the solvent used for the preparation of the above components was added to the culture supernatant of the control group. After 24 hours from the start of the test, the culture supernatant was removed by aspiration, and the cells were recovered and used as an mRNA recovery sample.
The collected cells (10 × 10 ⁶ cells / sample) were washed with DPBS (Dulbecco's Phosphate Buffered Saline, (−) CaCl 2 (−) MgCl 2, Life Technologies), and then High High RNA Isolation Kit (Roche) Total RNA was extracted from the cells. Next, reverse transcription reaction was performed using Transscriptor Universal cDNA Master (Roche Diagnostics) to prepare cDNA. Using this cDNA, real-time PCR was performed using LightCycler 480 (Roche Diagnostics). The target gene primer used was synthesized by Life Technologies Japan, and the reference primer (mouse ACTB) and other reagents designated by Roche Diagnostics were used.
The primer sequence of mouse Atrogin-1, which is the target gene, is as follows.
mAtrogin-1 forward: 5′-AGTGAGGACCGGCTACTGTG-3 ′
mAtrogin-1 reverse: 5′-GATCAAACGCTTGCGAATCT-3 ′

3. Analysis Method Data processing was performed using analysis software in the LightCycler® 480 system. Since the PCR reaction was performed by the probe method, the target gene expression level was calculated by the relative quantification method: ΔCt method by comparison with the reference gene expression level.

4). Results Measurement and analysis results are shown in FIG. 10 μM resveratrol, 30 μM resveratrol and 10 μM ε-viniferin did not suppress the expression of the atrogin-1 gene of striated myoblasts, whereas 30 μM ε-viniferin did not suppress the atrosin-1 gene. Was suppressed. 30 μM resveratrol 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 unique to ε-viniferin among resveratrols.
From the above results, ε-viniferin can suppress muscle atrophy caused by the expression of the atrodin-1 gene.

Reference example 1
Example of preparation of ε-viniferin from natural products ε-viniferin was extracted from about 5 kg of grape branch (variety Delaware) with 10 l of 70% methanol, and separated and purified by preparative HPLC to obtain about 200 mg of ε-viniferin. Obtained.
Acetonitrile / water (7:13) is used as a mobile phase during HPLC fractionation, and it is detected after about 8 minutes at a flow rate of 1 ml / min. Detection was performed with an ultraviolet absorption of 288 nm.
Moreover, the residue obtained by distilling off the solvent after methanol extraction was used as the vinyliferin in the following formulation examples as crude purified ε-viniferin.

Formulation Example 1: Supplement (Compounding ingredient) (mass%)
Lactose 54.0
Crystalline cellulose 35.0
Starch degradation product 10.0
ε-viniferin 1.0

Formulation Example 2: Soft capsule (formulation component) (mass%)
Edible soybean oil 50.0
ε-viniferin 1.0
Glycerin fatty acid ester 12.0
Beeswax 5.0
Gelatin 12.0
Water 20.0

Formulation Example 3: Gummy (Compounding ingredient) (mass%)
Reduced water tank 44.0
Granulated sugar 20.0
Glucose 20.0
Gelatin 4.7
Water 9.6
ε-viniferin 1.0
Flavor 0.6
Dye 0.1

Formulation Example 4: Soft drink (compounding ingredient) (mass%)
Fructose dextrose liquid sugar 30.0
Grape juice 20.0
Emulsifier 0.5
ε-viniferin 1.0
Perfume Appropriate amount of purified water

Formulation Example 5: Tablet confectionery (compounding ingredients) (mass%)
76.0 sugar
Glucose 19.0
ε-viniferin 1.0
Sucrose fatty acid ester 0.2
Purified water 3.8

Formulation Example 6: Candy (formulation component) (mass%)
Sugar 50.0
Minamata 33.0
Water 14.0
Organic acid 2.0
ε-viniferin 0.5
Fragrance 0.5

Claims (4)

  Atrodin-1 gene expression inhibitor comprising ε-viniferin 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.