JP6856280B2 - Muscle atrophy inhibitors and foods and medicines containing them - Google Patents

Description

The present invention relates to a muscular atrophy inhibitor and foods and pharmaceuticals containing the same.

Skeletal muscle is the tissue that occupies the largest volume in the human body, and plays an important role in physical activities such as uptake of sugars, lipids, and amino acids into the body, energy metabolism, and exercise. The quantity and quality of skeletal muscle are important for the expression of its function, and the quantity and quality of protein contained in skeletal muscle are strictly controlled by synthesis and decomposition, and homeostasis is maintained. Muscle mass is also regulated by the degree of physical activity, hormones, growth factors, stress and nutritional status.

During exercise, the decrease in skeletal muscle mass and skeletal muscle strength reduces the performance during exercise (that is, the amount of exercise) and the fatigue resistance after exercise. In addition, syndromes characterized by progressive and systemic skeletal muscle mass and skeletal muscle weakness represented by sarcopenia often occur especially in the elderly, and it is said that the estimated number of patients is 4.5 million. Patients suffering from such syndromes suffer from physical weakness such as falls, decreased oral function, cognitive decline, urinary incontinence, sleeplessness due to muscle weakness, gait disorders and joint disorders due to decreased skeletal muscle mass and strength. QOL (Quality of Life) deteriorates due to the restriction of various activities. Preventing muscular atrophy is considered to be significant in improving performance during exercise and improving QOL in the elderly. In order to solve these problems, it was necessary to develop a material that can inhibit muscular atrophy by ingesting it easily.

Recent studies have shown that glucocorticoids are involved in muscular atrophy. Glucocorticoids are nuclear receptor-type ligand-gated transcription factors secreted from the adrenal cortex under the control of the hypothalamic-pituitary system and regulate target gene expression through binding to glucocorticoid receptors (GRs). .. Glucocorticoids are known to cause protein assimilation suppression and catabolism in skeletal muscle, leading to muscular atrophy.

Atrogin-1, which is one of the GR target genes in skeletal muscle, is expressed in skeletal muscle and myocardium, and mice in which this gene is knocked out showed resistance to muscular atrophy. ) (See Non-Patent Document 1). Atrogin-1 is upregulated by at least 13 types of muscular atrophy such as glucocorticoid treatment of muscle, muscle non-use and oxidative stress, and is considered to be a marker of muscular atrophy phenomenon (see Non-Patent Document 2). ).

Similarly, Myostatin, one of the GR target genes in skeletal muscle, is a 26 kDa glycoprotein belonging to the TGF-β superfamily and has a function as a negative regulator of muscle growth (non-patent literature). 3). Myostatin is synthesized mainly in skeletal muscle and suppresses the growth of skeletal muscle. It has been reported that a decrease in Myostatin expression level leads to an increase in muscle mass and a decrease in body fat, and an increase in Myostatin expression level leads to a decrease in muscle mass and exhaustion.

In addition to glucocorticoids, factors affecting muscular atrophy include increased levels of inflammatory cytokines. For example, interleukin (IL) -1β is a kind of inflammatory cytokine and is considered to be associated with muscular atrophy by having a catabolic function (see Non-Patent Document 4). Since IL-1β causes skeletal muscle atrophy in vitro, IL-1β-treated myotube cells are an evaluation system used to investigate the mechanism of muscle atrophy. Tumor necrosis factor (TNF) -α and the like are also known as inflammatory cytokines.

The blood concentration of inflammatory cytokines such as IL-1β and TNF-α may increase with aging, may increase with rapid exercise, and the like. These inflammatory cytokines are thought to directly increase the expression of genes that cause muscular atrophy and cause muscular atrophy (muscle catabolism). Experimentally, it has been reported that the expression of muscular atrophy-causing genes is increased when inflammatory cytokines are allowed to act on muscle cells (see Non-Patent Document 5).

On the other hand, with respect to the components having a muscular atrophy inhibitory action, little has been clarified except that some polyphenols have been reported to be effective. Regarding the action of polyphenols on skeletal muscle muscular atrophy, it is recommended that fruit polyphenols (specifically, apple polyphenols) be contained as an active ingredient for the purpose of suppressing disuse muscle atrophy, which has been suggested to be involved in oxidative stress. A characteristic disuse muscular atrophy inhibitory composition has been reported (see Patent Document 1). The main components of apple polyphenols are procyanidins and proanthocyanidins. In addition, a muscle atrophy inhibitor containing proanthocyanidin as an active ingredient (see Patent Document 2), and a muscle fiber type migration inhibitor that suppresses the migration of muscle fiber type during disuse muscle atrophy containing fruit-derived polyphenol as an active ingredient. (Refer to Patent Document 3), an agent for suppressing muscle atrophy containing catechins as an active ingredient (see Patent Document 4) is also known.

However, although these documents describe the effect of polyphenols and the like on suppressing muscular atrophy, there is no description that polyphenols and the like act on genes that cause muscular atrophy, and therefore their effects on humans are limited. In addition, since the content of polyphenols in plants is low, it may be difficult to obtain a large amount of extracts. Further, since many polyphenols are insoluble in water, there is no choice but to use an organic solvent for extraction, and there is a problem that it is difficult to use for foods and pharmaceuticals mainly composed of water. Furthermore, the effect of polyphenols on inhibiting muscle atrophy was low, and there was still room for improvement.

By the way, the present inventors have been studying agar so far. Agar is dehydrated and dried mucilage extracted from red algae seaweed such as gelidiaceae and Gracilaria. Agar is a type of polysaccharide and is composed of agarose and agaropectin. Agarobiose, which is a repeating unit of agarose, has a structure in which β-D-galactopyranose bound at the 1st and 3rd positions and 3,6 anhydro-α-L-galactopyranose bound at the 1st and 4th positions are alternately bound. There is. Agaropectin is a general term for ionic polysaccharides other than agarose in agar, and its structure has the same binding mode as agarose, but it is partially rich in sulfate ester, methoxyl group, pyruvate group, and carboxyl group. Includes. The weight average molecular weight (Mw) of agar is generally 200,000 to 400,000. By decomposing this agar with an acid or an enzyme, agarooligosaccharides (agarobiose, agarotetraose, agarohexaose, agarooctaose, etc.) can be produced. Here, the agarooligosaccharide consists of repeating agarobiose (disaccharide) which is a disaccharide, and is composed of agarotetraose (4 sugar), agarohexaose (6 sugar), agarooctaose (8 sugar), and agarodecaose. It is a general term for (10 sugars) and the like.

Conventionally, agarooligosaccharides are preventive agents for various diseases such as systemic lupus erythematosus, immune-mediated glomerulonephritis, multiple sclerosis, collagen disease, autoimmune disease, and rheumatism, and further, antioxidants that suppress active oxygen production. It is known that it is an agent (see Patent Document 5). It is also known that agarooligosaccharides prevent aging of skin and bone by suppressing metalloprotease production (see Patent Document 6) and have a blood glucose lowering effect (see Patent Document 7). However, it has not been known so far that agarooligosaccharide has a muscular atrophy inhibitory effect.

JP 2001-89387 (Claim 1 and the like) JP-A-2002-338464 (Claim 1 and the like) Japanese Unexamined Patent Publication No. 2006-328031 (Claim 1 and the like) Japanese Unexamined Patent Publication No. 2008-13473 (Claim 1 and the like) Japanese Patent No. 4007760 (Claim 1, Example 6, etc.) Japanese Unexamined Patent Publication No. 2011-21370 (Claim 1 and the like) JP-A-2004-149471 (Claim 1 and the like)

"Science", 2001, vol. 294, p. 1704 "Int. J. Biochem. Cell Biol.", 2003, vol. 35, p. 698-705 "J. Cachexia Sarcopenia Muscle 2", 2011, p. 143-151 "Am. J. Physiol. Cell Physiol.", 2009, vol. 293 (3), C706-C714 "The FASEB Journal", 2005, vol. 19 (3), p. 362-370

The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel muscular atrophy inhibitor having a high muscular atrophy inhibitory effect. Another object of the present invention is to provide foods and pharmaceuticals containing such muscular atrophy inhibitors.

The present inventors investigated the effect of agarooligosaccharides on the expression of muscle atrophy-causing genes in the skeletal muscle atrophy system caused by the inflammatory cytokine IL-1β, and found that agarooligosaccharides may inhibit muscle atrophy. I found it. Furthermore, the present inventors conducted a human test by oral administration of agarooligosaccharide, confirmed the muscular atrophy inhibitory activity, and completed the present invention.

That is, the present invention is a muscular atrophy inhibitor characterized in that at least one agarooligosaccharide selected from the group consisting of agarobiose, agarotetraose, agarohexaose and agarooctaose is used as an active ingredient. ..

In this case, the muscular atrophy inhibitor has an action of inhibiting muscular atrophy by suppressing the expression of the muscular atrophy-causing gene, and further, the muscular atrophy-causing gene is one or more selected from Atrogin-1 and Myostatin. It is preferably the gene of. Alternatively, as the muscular atrophy inhibitor, the agarooligosaccharide is preferably at least one selected from the group consisting of agarobiose and agarotetraose.

The present invention also relates to a food containing the above-mentioned muscular atrophy inhibitor. Alternatively, the present invention relates to a pharmaceutical product containing the above-mentioned muscular atrophy inhibitor. In these cases, it is preferable to contain 1 mg or more of the muscular atrophy inhibitor.

According to the present invention, it is possible to provide a novel muscular atrophy inhibitor having a high muscular atrophy inhibitory effect. Further, according to the present invention, it is possible to provide foods and pharmaceuticals having a high muscular atrophy inhibitory effect.

It is a graph which shows the muscular atrophy inhibitory effect of an Example. It is a graph which shows the muscular atrophy inhibitory effect of an Example. It is a graph which shows the muscular atrophy inhibitory effect of an Example. It is a graph which shows the muscular atrophy inhibitory effect of an Example.

1. 1. Muscle Atrophy Inhibitor The muscle atrophy inhibitor of the present invention will be described below. The muscular atrophy inhibitor of the present invention (hereinafter, may be simply referred to as “muscle atrophy inhibitor”) is at least one selected from the group consisting of agarobiose, agarotetraose, agarohexaose and agarooctaose. The active ingredient is agarooligosaccharide.

The muscular atrophy inhibitor may contain only one of these agarooligosaccharides, or may contain two or more of these agarooligosaccharides.

In particular, the agarooligosaccharide contained in the muscular atrophy inhibitor is preferably at least one selected from the group consisting of agarobiose and agarotetraose. In this case, agarobiose and agarotetraose may be used alone or may be a composition containing both of these two types of agarooligosaccharides. In this case, the contents of agarobiose and agarotetraose are preferably in the range of 1 to 100% by mass, more preferably in the range of 10 to 100% by mass, and 50 to 100% by mass. It is particularly preferable that it is within the range.

In addition, the muscular atrophy inhibitor may contain agarodecaose, which is a kind of agarooligosaccharide. The content of agarodecaose is preferably in the range of 1 to 20% by mass, and particularly preferably in the range of 5 to 15% by mass. The composition of the agarooligosaccharide contained in the muscular atrophy inhibitor can be measured by the same method as the high performance liquid chromatography in the examples described later.

The agarooligosaccharide contained in the muscular atrophy inhibitor can be produced by hydrolyzing agar. As a specific method thereof, a method of acid decomposition can be mentioned. Examples of the acid decomposition method include the use of the solid acid described in Japanese Patent No. 4796697, the use of mineral acids such as sulfuric acid and hydrochloric acid, and the use of organic acids such as acetic acid and citric acid. However, either method may be used. The agarooligosaccharide produced by hydrolyzing with an acid is generally called "agarooligosaccharide".

On the other hand, those produced by enzymatic decomposition with β-agarase are called "neo-agarooligosaccharides" because of the difference in the cleavage location of the agar molecule main chain. The sugar compound produced by enzymatically decomposing agar with α-agarase becomes agarooligosaccharide as in the case of hydrolyzing with acid. Both agarooligosaccharides and neoagalooligosaccharides have an inhibitory effect on muscle atrophy. Therefore, the "agalo-oligosaccharide" in the present invention includes both "agaro-oligosaccharide" by acid decomposition and "neo-agaro-oligosaccharide" by enzymatic decomposition.

As a procedure for producing agarooligosaccharide by acid decomposition, first, an agar solution is prepared, and an acid is added to the agar solution for acid decomposition. Water can be mentioned as a solvent of the agar solution. The content of agar contained in the agar solution is not particularly limited, but is usually in the range of 0.1 to 50% by mass, preferably in the range of 1 to 10% by mass. The acid decomposition temperature is not particularly limited, but is usually in the range of 10 to 150 ° C, preferably in the range of 50 to 100 ° C. The acid decomposition may be carried out in a stationary state or under stirring.

Next, unnecessary components are removed from the obtained acid decomposition product. Filter paper, activated carbon, or the like can be used to remove unnecessary components. If necessary, the pH may be adjusted with an alkali such as sodium hydroxide. The agarooligosaccharide solution thus obtained can be stored in powder form by vacuum freeze-drying or the like.

The agarooligosaccharide can be produced by using a solution obtained by hot water extraction of not only agar but also red algae such as Gelidiaceae, Gracilaria, and Igis, which are raw materials for agar. Examples of red algae of the family Gelidiaceae include Maxa, Onigusa, Oobusa, Hiluxa, Obakusa, Yuikiri and the like. In addition, examples of red algae of the family Gracilaria include Gracilaria and Gracilaria. Further, examples of red algae of the family Igis include Igis, Egonori and the like. As a raw material for agarooligosaccharide, one type or a combination of two or more of these red algae can be used.

The muscular atrophy inhibitor has the effect of inhibiting muscular atrophy in tissues such as skeletal muscle. Skeletal muscle is atrophied due to long-term administration of glucocorticoid, diseases such as cancer, diabetes, AIDS, aging, lack of nutrition, or when skeletal muscle is not used for a long time due to cast fixation or bedridden, and skeletal muscle function declines. Is known, resulting in a decrease in QOL.

The muscular atrophy inhibitor has an action of suppressing the expression of the muscular atrophy-causing gene. Examples of the muscular atrophy-causing gene include Atrogin-1 and Myostatin. These genes are GR target genes, are induced in the early stages of muscle atrophy, and are involved in various muscle atrophy. Therefore, the muscular atrophy inhibitor of the present invention can also be referred to as an Atrogin-1 expression inhibitor or a Myostatin expression inhibitor.

The muscular atrophy inhibitory effect of the muscular atrophy inhibitor can be evaluated by measuring the expression level of the muscular atrophy-causing gene. The expression level of the muscle atrophy-causing gene can be evaluated not only in humans but also in mice. Examples of this evaluation method include a method of quantitatively measuring mRNA complementary to these muscular atrophy-causing genes by real-time PCR (RT-PCR) or the like.

By ingesting a muscular atrophy inhibitor, a muscular atrophy inhibitory effect can be obtained. As a result, exercise can be sustained due to health promotion, motor function improvement, muscular strength improvement, QOL improvement, etc., and fatigue resistance improvement effect, and as a result, health promotion, motor function improvement, muscular strength improvement, and QOL improvement are realized. be able to. Examples of the method of ingesting the muscular atrophy inhibitor include various methods such as oral ingestion and subcutaneous injection.

Therefore, as an invention of a method using a muscular atrophy inhibitor, a method for inhibiting muscular atrophy in humans or mammals (other than humans), which comprises agarobiose, agarotetraose, agarohexaose and agarooctaose. It can be said that it is a method for inhibiting muscular atrophy, which comprises ingesting at least one agarooligosaccharide more selected as an active ingredient.

2. Foods / Pharmaceuticals Muscle atrophy inhibitors are preferably contained in foods or pharmaceuticals and ingested. Examples of foods include general foods, dietary supplements, functional foods, and beverages. The general food is not particularly limited, but for example, confectionery such as candy, jelly, cake, chocolate, meat products such as hamburger, kamaboko, hanpen, fried fish cake, dressing, seasoning such as mayonnaise, soft drink, coffee, etc. Examples include sports drinks and beverages such as beer. The pharmaceutical product is not particularly limited, and examples thereof include tablets, capsules, pills, powders, granules, fine granules, jellies, liquids, ointments, creams, suspensions, emulsions, lotions and the like. be able to.

The content of the muscular atrophy inhibitor contained in foods or pharmaceuticals is not particularly limited, but is usually 1 mg or more, preferably in the range of 10 to 10000 mg, and particularly preferably in the range of 100 to 1000 mg.

Hereinafter, the present invention will be specifically described based on examples, but these do not limit the object of the present invention.

(1) Preparation of agarooligosaccharides Three types of agarooligosaccharides 1 to 3 were prepared. The preparation method of each agarooligosaccharide and the composition ratio of the constituent sugars are as follows.
(A) Agalo-oligosaccharide 1
(Adjustment method)
A commercially available agarooligosaccharide (agaoligo: manufactured by Takara Bio Inc.) was purchased.
(Constituent sugar)
The composition ratio of the oligosaccharide contained in the agarooligosaccharide 1 was measured by high performance liquid chromatography. Measurement conditions Column TOSOH TSK-GEL ALPHA-2500 series two coupling, solvent _H 2 O, flow rate 0.3 ml / min, eluting with a temperature of 60 ° C., detection was RI (differential refractometer). The results were as follows (numerical values are mass%, the same applies hereinafter).
Disaccharide (Agarobiose): 26.2
Tetrasaccharide (Agarotetraose): 27.7
6 sugar (agarohexaose): 23.1
8 sugar (agaro octaose): 18.9
10 sugar (agaro de chaos): 4.1

(B) Agalo-oligosaccharide 2
(Adjustment method)
After 50 g of agar (Ultra Agar AX-30: manufactured by Ina Food Industry Co., Ltd.) was heated and dissolved in 1000 g of purified water, 2 g of concentrated sulfuric acid was added and the mixture was stirred at 90 ° C. for 3 hours. After adjusting the pH to 3.5 with sodium hydroxide, it was treated with activated carbon, filtered through a filter paper, and further filtered through a 0.1 μm filter to obtain a solution. This solution was pulverized by vacuum freeze-drying.
(Constituent sugar)
When the composition ratio of the oligosaccharide contained in the agarooligosaccharide 2 was measured by the same method as that of the agarooligosaccharide 1, the results were as follows.
Disaccharide (Agarobiose): 31.5
Tetrasaccharide (Agarotetraose): 30.1
6 sugar (agarohexaose): 21.2
8 sugar (agaro octaose): 11.6
10 sugar (agaro de chaos): 5.6

(C) Agalo-oligosaccharide 3
(Adjustment method)
Neo-agaro oligosaccharides were produced by the production method described in Example 1 of JP-A-2-65789.
(Constituent sugar)
When the composition ratio of the oligosaccharide contained in the agarooligosaccharide 3 was measured by the same method as that of the agarooligosaccharide 1, the results were as follows.
Disaccharide (Agarobiose): 1.5
Tetrasaccharide (Agarotetraose): 37.5
6 sugar (agarohexaose): 41.3
8 sugar (agaro octaose): 6.3
10 sugar (agaro de chaos): 13.4

(2) Effect of agarooligosaccharide on expression suppression of Atrogin-1 and Myostatin Mouse skeletal muscle-derived C2C12 cells were used. The density of C2C12 cells was adjusted and cultured under the conditions of _{37 ° C. and 5% CO 2.} DMEM containing 10% bovine serum was used as the medium. Upon reaching 10 ⁵ / cm ^2, medium was replaced with DMEM containing 2% bovine serum, the agarooligosaccharides 1 final concentration 1 [mu] g / ml (Example 1), 10 [mu] g / ml (Example 2), 50 [mu] g / A system added to the medium so as to be ml (Example 3) and 100 μg / ml (Example 4) was prepared. For comparison, a system without agarooligosaccharide was also prepared (Comparative Example 1). After the addition, it was confirmed that the cells differentiated into myotube cells 5 days after culturing under the conditions of 37 ° C. and 5% CO _2.

After that, IL-1β was added to induce muscular atrophy, and 24 hours later, myotube cells were collected with a cell scraper. Total RNA of the recovered cells was isolated using TRIzol (guanidine thiosianate, phenol, chloroform), chloroform, and isopropanol. Next, cDNA was prepared using reverse transcriptase, and the expression levels of each mRNA of Atrogin-1 and Myostatin were measured by real-time PCR using it as a template. The results are shown in FIGS. 1 (a) and 1 (b). The vertical axis of the figure shows the expression level of cDNA of each gene. In addition, "IL-1β" on the horizontal axis of the figure indicates that "+" is added, "-" indicates no addition, and "AOS" indicates the amount of agarooligosaccharide 1 added.

As shown in this figure, in Atrogin-1, 50 μg / ml or more of agarooligosaccharide 1 was added (in the figure) as compared with the case where agarooligosaccharide was not added (“AOS” is “0” in the figure). It was found that the gene expression level decreased when "AOS" was "50" and "100"). Further, in Myostatin, compared with the case where no agarooligosaccharide was added (“AOS” is “0” in the figure), when 1 μg / ml or more of agarooligosaccharide 1 was added (“AOS” is “1” in the figure). , "10", "50", "100"), it was found that the expression level of the gene decreased. Therefore, it was found that the activity of the gene that promotes muscular atrophy is suppressed by adding agarooligosaccharide to skeletal muscle-derived cells.

In particular, when the concentration of agarooligosaccharide was 50 μg / ml or more, the expression level of all genes was decreased, and thus it was found that the addition amount was more preferable as compared with the case where the concentration was less than 50 μg / ml. It was. In addition, a comparison between Atrogin-1 and Myostatin revealed that agarooligosaccharides were particularly effective in suppressing the expression of Myostatin.

(3) Comparison between agarooligosaccharides 1 to 3 and polyphenols Similar to the above "(2) Effect of agarooligosaccharide on suppressing expression of Atrogin-1 and Myostatin", mouse skeletal muscle-derived C2C12 cells were used. The density of C2C12 cells was adjusted and cultured under the conditions of _{37 ° C. and 5% CO 2.} DMEM containing 10% bovine serum was used as the medium. When the cell density reached ¹⁰ 5 / cm ^2, medium was replaced with DMEM containing 2% bovine serum, agarooligosaccharide 1 to 3 (respectively Examples 5-8) to a final concentration of 50 [mu] g / ml A system added to the medium was prepared. For comparison, a system in which no additives (Comparative Example 1), proanthocyanidins (Comparative Example 2) and epigallocatechin (Comparative Example 3) were added to the medium to a final concentration of 50 μg / ml was also prepared. After the addition, it was confirmed that the cells differentiated into myotube cells 5 days after culturing under the conditions of 37 ° C. and 5% CO _2.

After that, IL-1β was added to induce muscular atrophy, and 24 hours later, myotube cells were collected with a cell scraper. Total RNA of the recovered cells was isolated using TRIzol (guanidine thiosianate, phenol, chloroform), chloroform, and isopropanol. Next, cDNA was prepared using reverse transcriptase, and the expression levels of each mRNA of Atrogin-1 and Myostatin were measured by real-time PCR using it as a template. The results are shown in FIGS. 2 (a) and 2 (b). The vertical axis of the figure shows the expression level of cDNA of each gene. In addition, "IL-1β" on the horizontal axis of the figure indicates that "+" is added, "-" indicates no addition, and "additive substance" indicates that "no" is not added. It indicates that the substance was added.

As shown in this figure, in both Atrogin-1 and Myostatin, the addition of agarooligosaccharides 1 to 3 suppresses the activity of genes that promote muscular atrophy, and moreover than proanthocyanidins epigallocatechin. It turned out that the effect was high. In addition, a comparison between Atrogin-1 and Myostatin revealed that agarooligosaccharides were particularly effective in suppressing the expression of Myostatin. Furthermore, when the agarooligosaccharides 1 to 3 were compared, it was found that the agarooligosaccharide 2 having a low expression level in any of the genes was particularly preferable.

(4) Isolation of agarobiose, agarotetraose, agarohexaose and agarooctaose in agarooligosaccharide and verification of the effect of each component 0.024 g of agarooligosaccharide 1 was used in the column TOSOH TSK-GEL ALPHA-2500 ( was introduced in series two coupling), solvent _H 2 O, flow rate 0.3 ml / min, eluting with a temperature of 60 ° C., the eluate was collected in fraction collector by 0.5 ml. Among the recovered fractions, fraction 11 contained agarobiose, 12 and 13 contained agarotetraose, 14 and 15 contained agarohexaose, and 18 and 19 contained agarooctaose. Each fraction was dried to obtain 0.0049 g of agarobiose, 0.0049 g of agarotetraose, 0.0017 g of agarohexaose and 030,000 g of agarooctaose.

Mouse skeletal muscle-derived C2C12 cells were used in the same manner as in the above-mentioned "(2) Effect of agarooligosaccharide on suppressing expression of Atrogin-1 and Myostatin". The density of C2C12 cells was adjusted and cultured under the conditions of _{37 ° C. and 5% CO 2.} DMEM containing 10% bovine serum was used as the medium. When the cell density reached 10 5 ^/ cm ^{2, 2%} bovine serum medium was replaced with DMEM containing, agarobiose, agarotetraose, agarohexaose and A Gallo octa ose each final concentration of 10 and 50μM A system added to the medium was prepared so as to be (Examples 9 to 17). For comparison, an additive-free system was also prepared (Comparative Example 1). After the addition, it was confirmed that the cells differentiated into myotube cells 5 days after culturing under the conditions of 37 ° C. and 5% CO _2.

After that, IL-1β was added to induce muscular atrophy, and 24 hours later, myotube cells were collected with a cell scraper. Total RNA of the recovered cells was isolated using TRIzol (guanidine thiosianate, phenol, chloroform), chloroform, and isopropanol. Next, cDNA was prepared using reverse transcriptase, and the expression levels of each mRNA of Atrogin-1 and Myostatin were measured by real-time PCR using it as a template. The results are shown in FIGS. 3 (a) and 3 (b). The vertical axis of the figure shows the expression level of cDNA of each gene. In addition, "IL-1β" on the horizontal axis of the figure indicates that "+" is added, "-" indicates no addition, and "additive substance" indicates that "no" is not added. It indicates that the substance was added.

As shown in this figure, in both Atrogin-1 and Myostatin, the activity of genes that promote muscular atrophy is suppressed by the addition of agarobiose, agarotetraose, agarohexaose, and agarooctaose. Furthermore, it was found that the effect was higher than that of Agarohexaose and Agarooctaose.

(6) Verification of effect when the composition ratio of agarooligosaccharide was changed As shown in Table 1, agarooligosaccharides 4 to 10 in which the mixing ratios of agarobiose, agarotetraose, agarohexaose and agarooctaose were changed were prepared. (Examples 18 to 24).

Mouse skeletal muscle-derived C2C12 cells were used in the same manner as in the above-mentioned "(2) Effect of agarooligosaccharide on suppressing expression of Atrogin-1 and Myostatin". The density of C2C12 cells was adjusted and cultured under the conditions of _{37 ° C. and 5% CO 2.} DMEM containing 10% bovine serum was used as the medium. When the cell density reached ¹⁰ 5 / cm ^2, medium was replaced with DMEM containing 2% bovine serum were prepared and the system was added to the medium to the agarooligosaccharides 4-10 respectively to a final concentration of 50 [mu] M. For comparison, an additive-free system was also prepared (Comparative Example 1). After the addition, it was confirmed that the cells differentiated into myotube cells 5 days after culturing under the conditions of 37 ° C. and 5% CO _2.

After that, IL-1β was added to induce muscular atrophy, and 24 hours later, myotube cells were collected with a cell scraper. Total RNA of the recovered cells was isolated using TRIzol (guanidine thiosianate, phenol, chloroform), chloroform, and isopropanol. Next, cDNA was prepared using reverse transcriptase, and the expression levels of each mRNA of Atrogin-1 and Myostatin were measured by real-time PCR using it as a template. The results are shown in FIGS. 4 (a) and 4 (b). The vertical axis of the figure shows the expression level of cDNA of each gene. In addition, "IL-1β" on the horizontal axis of the figure indicates that "+" is added, "-" indicates no addition, and "additive substance" indicates that "no" is not added. It indicates that the substance was added.

As shown in this figure, it was found that the addition of agarooligosaccharides 4 to 10 suppresses the activity of genes that promote muscle atrophy in both Atrogin-1 and Myostatin. In particular, it was found that the addition of agarooligosaccharide having a high proportion of agarobiose or agarotetraose has a high effect of suppressing the activity of a gene that promotes muscular atrophy.

(6) Preparation of foods and pharmaceuticals (a) Example 25 (beverage)
A beverage intended to inhibit muscular atrophy was prepared with the following formulation.
Agalo-oligosaccharide 1 2.5g
Sucrose 50.0g
Citric acid 5.0g
Orange juice (5-fold concentrated) 200.0 g
Appropriate amount of orange fragrance (b) Example 26 (pharmaceutical product)
A tablet (1 tablet 0.3 g) for the purpose of inhibiting muscular atrophy was prepared with the following composition.
Agalo-oligosaccharide 1 50.0%
Lactose 30.0%
Crystalline cellulose 19.5%
Magnesium stearate 0.5%
(C) Example 27 (pharmaceutical product)
A powder was prepared for the purpose of inhibiting muscular atrophy with the following formulation.
Agalo-oligosaccharide 1 50.0%
Lactose 40.0%
Cornstarch 9.7%
Magnesium stearate 0.3%

(7) Muscle fatigue reduction test A muscle fatigue reduction test was conducted on 9 healthy adults. Specifically, nine people who are running every day were selected, and the beverages and medicines of Examples 25 to 27 were used as beverages so that the intake of agarooligosaccharide was 500 mg per subject for each subject group shown below. I had them take medicine and run 10km on a treadmill (manufactured by Nakao Health Co., Ltd.). Immediately after the end of running, we conducted an awareness survey on muscle fatigue one day and two days later. Nine subjects were asked to run for 10 km without ingesting the product of the present invention 10 days before the test, and responded by comparison with that time.

(Intake items)
Subjects 1-3: Example 25
Subjects 4-6: Example 26
Subjects 7-9: Example 27
(result)
Less muscle fatigue than when not ingested 7/9 people Same as when not ingested 2/9 people More muscle fatigue than when not ingested 0/9

As described above, most of the subjects had the impression that they had less muscular fatigue. As described above, it was found that the foods and pharmaceuticals containing agarooligosaccharide have a muscular atrophy inhibitory effect.

Claims (7)

An agent for suppressing the expression of a muscular atrophy-causing gene, which comprises at least one agarooligosaccharide selected from the group consisting of agarobiose, agarotetraose, agarohexaose and agarooctaose as an active ingredient. The agent for suppressing the expression of a muscular atrophy-causing gene according to claim 1, wherein the muscular atrophy-causing gene is one or more genes selected from Atrogin-1 and Myostatin. The muscular atrophy-causing gene expression inhibitor according to claim 1 or 2, wherein the agarooligosaccharide is at least one selected from the group consisting of agarobiose and agarotetraose. A food for suppressing muscular atrophy-causing gene expression, which comprises the muscular atrophy-causing gene expression-suppressing agent according to any one of claims 1 to 3. The food for suppressing the expression of a muscular atrophy-causing gene according to claim 4, which contains 1 mg or more of the muscular atrophy-causing gene expression-suppressing agent. Muscle atrophy causes gene silencing drugs characterized by containing muscle atrophy causes gene expression inhibitor according to claims 1-3. Muscle atrophy causes gene silencing drugs according to claim 6, characterized in that it contains the muscle atrophy causative gene expression inhibitor or 1 mg.

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