JP2020169126A - Muscle enhancer - Google Patents

Abstract

To provide a novel muscle enhancer useful for enhancing muscle.SOLUTION: A muscle enhancer contains phloretic acid as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to muscle builders.

Muscle is formed by the differentiation of muscle cells. In the differentiation of myocytes, myoblasts, which are progenitor cells, proliferate to a certain number of cells, move to the planned muscle region, and fuse with each other to become differentiated polygonal myotube cells. Then, in myotube cells, genes representing muscle-specific traits such as muscle contraction proteins and specific enzymes are expressed, and a muscle contraction device is constructed.

Mitochondria in muscle cells are also closely involved in muscle function. The main function of mitochondria is energy (ATP) production in the TCA cycle. In order to improve the metabolic function of muscle, it is important to increase the amount of mitochondria or increase its activity.

Furthermore, it is known that the amount of sugar (glucose) taken up by muscle cells is involved in the anti-fatigue effect. That is, in order to suppress the decrease of muscle glycogen and enhance the anti-fatigue effect of muscle, it is important to increase the amount of glucose taken up by muscle cells.

Since strengthening muscles and enhancing their functions are important for maintaining and promoting motor function and maintaining physical health, various muscle-building means have been studied. For example, Patent Document 1 discloses a muscle differentiation-inducing promoter containing rice bran and / or a fermented extract of rice germ as an active ingredient. Further, Patent Document 2 discloses a method of activating mitochondria during exercise using glutathione. Further, Patent Document 3 describes that a tripeptide having a pyroglutamic acid residue at the N-terminal has an effect of promoting glucose uptake into muscle cells or muscle tissues.

Japanese Unexamined Patent Publication No. 2017-193947 JP-A-2018-27983 JP-A-2018-115131

An object of the present invention is to provide a novel muscle-building agent useful for building muscle.

The present inventors have found by in vitro tests that phloretic acid has a muscle-building effect.

The first aspect of the present invention provides a muscle-building agent containing phloretic acid as an active ingredient.

A second aspect of the present invention provides a myotube cell differentiation promoter containing phloretic acid as an active ingredient.

A third aspect of the present invention provides a mitochondrial activator containing phloretic acid as an active ingredient.

A third aspect of the present invention provides an agent for promoting sugar uptake into myotube cells, which contains phloretic acid as an active ingredient.

According to the present invention, it is possible to provide a novel muscle-building agent useful for building muscle.

Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments.

The muscle-building agent of the present invention has a muscle-building effect. The muscle-building action herein includes promoting myoblast-to-myoblast differentiation, promoting myoblast differentiation, activating mitochondria in muscle, activating mitochondria, and activating myotube cells. Includes a sugar uptake promoting effect on myoblasts, which increases the amount of sugar uptake. That is, it can be said that the present invention provides a myotube cell differentiation promoter, a mitochondrial activator, or a sugar uptake promoter into myotube cells.

Mitochondrial activation action includes the action of increasing the amount of mitochondria present in cells (muscle cells) in muscle (the amount of mitochondria per cell), and the activity of mitochondria present in muscle cells (mitochondrial activity per cell). ) Is included. That is, the present invention can also be said to provide a mitochondrial bulking agent in muscle cells or a mitochondrial activator in muscle cells.

The action of promoting sugar uptake into myotube cells may be an action of increasing the amount of sugar taken up into myotube cells after myoblasts have differentiated into myotube cells. The sugar is mainly glucose. The sugar uptake promoter for myotube cells according to the present invention promotes sugar uptake into myotube cells in at least one of the presence of insulin and the absence of insulin. Incorporation of sugar into myotube cells enhances muscle endurance, anti-fatigue, and fatigue recovery. Therefore, the present invention can also be said to provide an anti-fatigue agent by incorporating sugar into myotube cells.

The muscle-building agent according to one embodiment contains phloretic acid as an active ingredient.

Phloretic acid is a type of aromatic hydroxy acid and is also a compound also called 3- (4-hydroxyphenyl) propionic acid. Phloretic acid may be produced by hydrogenation of the 2-propene acid side chain of p-coumaric acid or hydrolysis of phloretin by phloretin hydrolase. The phloretic acid may be a commercially available one, or p-coumaric acid may be reduced by a microorganism. When phloretic acid is obtained from p-coumaric acid, the raw material p-coumaric acid may be commercially available or separated from the lignin decomposition product. At this time, the lignin decomposition product may be derived from a gramineous plant, or may be derived from sugar cane or bagasse.

The muscle-building agent may consist only of the active ingredient phloretic acid, and may further contain a food composition, a quasi-drug, or a material that can be used in a pharmaceutical product. The ingredients that can be used in food compositions, non-pharmaceutical products, or pharmaceuticals are not particularly limited, but for example, amino acids, proteins, carbohydrates, fats and oils, sweeteners, minerals, vitamins, flavors, excipients, etc. Examples thereof include binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents and the like.

Examples of the protein include milk casein, whey, soybean protein, wheat protein, egg white and the like. Examples of carbohydrates include cornstarch, cellulose, pregelatinized starch, wheat starch, rice starch, potato starch and the like. Examples of fats and oils include salad oil, corn oil, soybean oil, safflower oil, olive oil, palm oil and the like. Examples of the sweetener include sugars such as glucose, sucrose, fructose, high fructose corn syrup, high fructose corn syrup, sugar alcohols such as xylitol, erythritol, and martitol, and artificial sweeteners such as sucralose, aspartame, saccharin, and acesulfam K. Examples include fructose and stevia sweeteners. Examples of minerals include calcium, potassium, phosphorus, sodium, manganese, iron, zinc, magnesium and the like, and salts thereof. Examples of vitamins include vitamin E, vitamin C, vitamin A, vitamin D, B vitamins, biotin, niacin and the like. Examples of excipients include dextrin, starch, lactose, crystalline cellulose and the like. Examples of the binder include polyvinyl alcohol, gelatin, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc and the like. Examples of the disintegrant include crystalline cellulose, agar, gelatin, calcium carbonate, sodium hydrogen carbonate, dextrin and the like. Examples of the emulsifier or surfactant include sucrose fatty acid ester, citric acid, lactic acid, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, lecithin and the like. Examples of the base include cetostearyl alcohol, lanolin, polyethylene glycol and the like. Examples of the dissolution aid include polyethylene glycol, propylene glycol, sodium carbonate, sodium citrate and the like. Examples of the suspending agent include glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxymethyl cellulose, sodium alginate and the like. These may be used alone or in combination of two or more.

When the muscle-building agent contains other materials, the content of phloretic acid, which is an active ingredient, may be appropriately set according to the form, purpose of use, etc. of the muscle-building agent described later, but the muscle-building effect is further enhanced. From the viewpoint of effective exertion, the solid content is preferably 0.5% by mass or more, more preferably 1% by mass or more, further preferably 3% by mass or more, and preferably 50% by mass or less. It is more preferably 40% by mass or less, and further preferably 30% by mass or less.

The muscle-building agent can be used as a food composition, a drug or a quasi-drug. The food composition for muscle building may be provided in the form of, for example, a health food, a food for specified health use, a functional food, a nutritionally functional food, a supplement or the like. That is, according to the present invention, it can be said that a muscle-building food composition, a muscle-building drug, or a muscle-building quasi-drug is provided.

The shape of the muscle-building agent is not limited and may be any shape such as solid (powder, granule, etc.), liquid (solution, suspension, etc.), paste, etc., powder, pill, granule, tablet, etc. It may be in any dosage form such as capsules, lozenges, liquids and suspensions.

The muscle-building agent may be orally administered, or may be administered parenterally, such as intravenously.

When the muscle-building agent is orally administered, for example, the dose of phloretic acid is preferably 100 μg / kg (body weight) or more, and 150 μg / kg (body weight) or more. It is more preferable to administer the drug so as to be 200 μg / kg (body weight) or more. Further, phloretic acid is preferably administered so as to be 300 μg / kg (body weight) or more per day, more preferably 450 μg / kg (body weight) or more, and 500 μg / kg (body weight). It is more preferable to administer the above. Further, phloretic acid is preferably administered so as to be 1000 mg / kg (body weight) or less, more preferably 800 mg / kg (body weight) or less, and 600 mg / kg (body weight) or less. ) It is more preferable to administer the following. Further, phloretic acid is preferably administered so as to be 3000 mg / kg (body weight) or less per day, more preferably 2000 mg / kg (body weight) or less, and 1000 mg / kg (body weight) or less. It is more preferable to administer as follows. Within this range, a sufficient blood concentration can be achieved, and a muscle-building effect can be more effectively expressed.

When the muscle-building agent is administered parenterally, for example, the dose of phloretic acid is preferably 100 μg / kg (body weight) or more, and 200 μg / kg (body weight) or more. It is more preferable to administer the drug so as to be 300 μg / kg (body weight) or more. Further, phloretic acid is preferably administered so as to be 150 μg / kg (body weight) or more per day, more preferably 300 μg / kg (body weight) or more, and 500 μg / kg (body weight). It is more preferable to administer as described above. Further, phloretic acid is preferably administered so as to be 2000 mg / kg (body weight) or less, and more preferably 1000 mg / kg (body weight) or less. It is more preferable that the dose is less than or equal to the body weight. Further, phloretic acid is preferably administered so as to be 4000 mg / kg (body weight) or less per day, more preferably 3000 mg / kg (body weight) or less, and 2000 mg / kg (body weight) or less. It is more preferable to administer as follows. Within this range, a sufficient blood concentration can be achieved, and a muscle-building effect can be more effectively expressed.

The muscle-building agent of the present embodiment can be used for humans or animals. When a muscle-building agent is used for animals, it can be used as a feed or a feed additive. Examples of the feed include companion animal feed such as dog food and cat food, livestock feed, poultry feed, and farmed fish and shellfish feed. "Feed" includes anything that an animal ingests orally for nutritional purposes. More specifically, when classified in terms of nutrient content, all of roughage, concentrated feed, inorganic feed, and special feed are included, and when classified in terms of official standards, all of compound feed, mixed feed, and simple feed. Including. In addition, when classified in terms of feeding method, it includes all feeds that are directly fed, feeds that are mixed with other feeds, or feeds that are added to drinking water to supplement nutrients.

The muscle-building agent according to the present embodiment may be used to promote muscle formation, to promote recovery of damaged muscle, or to activate the function of the formed muscle.

The specific embodiment of the mitochondrial activator, the myotube cell differentiation promoting agent, or the sugar uptake promoting agent into myotube cells according to the embodiment may be the same as that of the muscle strengthening agent described above. That is, in the mitochondrial activator or myotube cell differentiation promoter according to the embodiment, in the above description of the muscle strengthening agent, "muscle strengthening agent" is referred to as "mitochondria activator", "myotube cell differentiation promoting agent". Alternatively, it may be read as "sugar uptake promoter for myotube cells".

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.

<Test>
[material]
In the following test examples, the materials shown below were used.
(cell)
Mouse myoblasts C2C12 cells (ATCC, CRL-1772)

(Culture medium)
Growth medium composition: DMEM medium, 10% FBS, antibiotics added Differentiation medium composition: DMEM medium, 0.5% FBS, antibiotics added

(Test reagent)
Dulbecco Modified Eagle's Medium (DMEM Medium, Nacalai Tesque Co., Ltd.)
Fetal Bovine Serum (FBS) (Cell Culture Bioscience)
Penicillin-streptomycin mixed solution (Nacalai Tesque, Inc.)
0.25% trypsin / EDTA mixed solution (Nacalai Tesque, Inc.)
Dulbecco PBS (-) (Nissui Pharmaceutical Co., Ltd.)
Gelatin (A type, MP Biomedicals)
Hoechst (Nuclear staining reagent, Hoechst 33342 solution, Dojin Chemical Laboratory Co., Ltd.)
MitoTracker Mitochondrion-Selective Probes, Invitrogen
Rhodamine (VectaCell Rhodamine 123, Funakoshi Co., Ltd.)
4% -paraformaldehyde / phosphate buffer (Nacalai Tesque, Inc.)
Primary antibody (Anti-Myosin Heavy Chain Purified clone: MF20, eBioscience)
Secondary antibody (Alexa Fluor 555 F (ab) 2 fragment of goat anti-mouse IgG (H + L), Life Technologies Japan Co., Ltd.)
Insulin (Insulin, Human Recombinant Zinc solution, Gibco)

[Pre-cell culture]
The cells used in the following test examples were pre-cultured.
C2C12 cells were awakened in a T-75 (75 cm ² U-shaped cantoneck cell culture flask, Corning) flask using growth medium. C2C12 cells were cultured in a T-75 flask placed in a CO ₂ incubator (5% CO ₂ , 37 ° C., wet). Medium exchange was performed every other day and cells were harvested when 80% confluence was reached and used in the test. In the following test, a plate coated with gelatin by the coating method shown below was used as the well plate when culturing C2C12 cells.
(1) A 0.75% aqueous gelatin solution was sterilized in an autoclave.
(2) 100 μL of 0.75% gelatin aqueous solution was added to each well in the plate, and this well was allowed to stand in a CO ₂ incubator (5% CO ₂ , 37 ° C.) for 2 hours.
(3) The 0.75% aqueous gelatin solution was removed, and the obtained plate was used.

[Test Example 1: Mitochondrial activation test (evaluation of activity per cell)]
Pre-cultured cells were seeded on a 96-well plate for fluorescence observation (optical bottom plate, Nunc) at 4 × 10 ⁴ cells / 0.1 mL / well using growth medium. This well plate was cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.) for 2 days. Then, the medium was replaced with a differentiation medium, and the cells were cultured for 4 days to form myotube cells. After forming myotube cells, a differentiation medium containing floretic acid and 1% (v / v) ethanol (Examples 1-1 to 1-3) and a differentiation medium containing no floretoic acid and 1% ethanol (Comparative Example). 1-1) or a differentiation medium containing 50 μM resveratrol and 1% ethanol (positive control 1) was replaced and cultured respectively. The culture was terminated 48 hours after the start of the culture. These were used as test samples of Examples 1-1 to 1-3, Comparative Example 1-1, or Positive Control 1. In Examples 1-1 to 1-3, the final concentration of phloretic acid in the differentiation medium is shown in Table 1.

After removing the culture supernatant, a differentiation medium containing 5 μg / mL Hoechst (nuclear staining reagent) or 10 μg / mL rhodamine (mitochondrial active staining reagent) was added to the well plate and cultured at 37 ° C. for 30 minutes. .. Then, the fluorescence intensity was measured with a fluorescence plate reader. The mitochondrial activity per cell was calculated by dividing the fluorescence intensity of the differentiation medium containing rhodamine by the fluorescence intensity of the differentiation medium containing Hoechst. The relative value (%) in the test sample of Examples 1-1 to 1-3 was determined when the mitochondrial activity in the test sample of Comparative Example 1-1 was 100%. The test was carried out 5 times each, and the average value is shown in Table 1.

As shown in Table 1, the mitochondrial activity per cell was higher in the test samples of Examples 1-1 to 1-3 to which phloretic acid was added, as compared with the test sample of Comparative Example 1-1 to which phloretic acid was not added. Increased. When a significant difference test with Comparative Example 1-1 (two-sided test by Student's T-test) was performed, the test samples of Examples 1-1 to 1-3 were compared with the test samples of Comparative Example 1-1. The mitochondrial activity per cell was significantly increased. That is, the results showed that Example 1-1 had a significant difference at p <0.001, and Examples 1-2 and 1-3 had a significant difference at p <0.05. Since the mitochondrial activity was also increased in the positive control 1, it can be said that the test was carried out without any problem.

Subsequently, the mitochondrial activity of the test sample after culturing for 72 hours was evaluated by the same method as described above. The results are shown in Table 1. As shown in Table 1, even after culturing for 72 hours, the test samples of Examples 1-1 to 1-3 to which phloretic acid was added were compared with the test samples of Comparative Example 1-1 to which phloretic acid was not added. , Increased mitochondrial activity per cell.

[Test Example 2: Myotube cell differentiation promotion test]
Pre-cultured cells were seeded on a 96-well plate for fluorescence observation at 4 × 10 ⁴ cells / 0.1 mL / well using growth medium. This well plate was cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.) for 2 days. Then, a differentiation medium containing phloretic acid and 1% (v / v) ethanol (Example 2-1), and a differentiation medium not containing phloretic acid and containing 1% (v / v) ethanol (Comparative Example 2-1). , Phloretic acid-free, 0.1% (v / v) DMSO-containing differentiation medium (Comparative Example 2-2), or 0.5 μM LDN-193189 (4- (6- (4- (piperazin-1-)). It was replaced with a differentiation medium (positive control 2) containing (il) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline) and 0.1% (v / v) DMSO. After exchanging the medium, the cells were cultured for 3 days, and after fixation, immunostaining with an anti-myosin heavy chain (MHC) antibody was performed. In Example 2-1 the final concentration of phloretic acid in the differentiation medium is shown in Table 2.
After completion of the culture, 4% -paraformaldehyde / phosphate buffer was added at 100 μL / well, and the cells were allowed to stand at 4 ° C. for 15 minutes. After standing, the cells were washed 3 times with Dulbecco PBS (DPBS), and then blocked with DPBS containing 0.1% Triton-X and 3% bovine serum albumin (BSA) for 1 hour at room temperature. Subsequently, a 3% BSA / DBPS mixed solution containing a primary antibody (anti-myosin heavy chain antibody: diluted 300-fold) was added, and the reaction was carried out at 4 ° C. overnight. After the reaction, the mixture was washed 3 times with a 3% BSA / DBPS mixed solution. Next, a 3% BSA / DBPS mixed solution containing a secondary antibody (500-fold dilution) and Hoechst (nuclear staining reagent, 1000-fold dilution) was added, reacted at room temperature in the dark for 2 hours, and washed with DPBS after the reaction. did. These were used as test samples of Example 2-1 and Comparative Examples 2-1 to 2-2 and Positive Control 2.

The data for 1 well was set to n = 1, and the analysis was performed with a total of 5 wells (n = 5). The total number of Hoechst-positive nuclei and the number of MHC-positive nuclei were counted, and the Fusion index (% of MHC + nuclei) was calculated by the following formula. The average value (n = 5) of Fusion index is shown in Table 2.
Fusion audit (% of MHC + nuclei) = MHC-positive number of nuclei / total number of nuclei x 100

As shown in Table 2, in the test sample of Example 2-1 to which phloretic acid was added, Fusion index was increased as compared with the test sample of Comparative Example 2-1 to which phloretic acid was not added. The Fusion index in the positive control 2 was 63.7 ± 0.88, and the Fusion index increased in the positive control as compared with Comparative Example 2-2. Therefore, it can be said that the test was carried out without any problem.

[Test Example 3: Sugar uptake test]
Pre-cultured cells were seeded on a 96-well plate for fluorescence observation at 4 × 10 ⁴ cells / 0.1 mL / well using growth medium. This well plate was cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.) for 2 days. Then, the medium was replaced with a differentiation medium, and the cells were cultured for 4 days to form myotube cells. After forming myotube cells, a differentiation medium containing phloretic acid and 1% (v / v) ethanol (Examples 3-1 to 2-3), or a differentiation medium containing no phloretic acid and containing 1% ethanol (Examples 3-1 to 2-3). It was replaced with Comparative Example 3-1) and cultured for 24 hours each. Then, the medium was replaced with a desensitized medium (differentiation medium without FBS added) and cultured for 18 hours. After culturing in desensitized medium, cells were treated with a differentiation medium containing insulin and a test substance (phloretic acid or solvent) for 1 hour. These were used as test samples of Examples 3-1 to 2-3 and Comparative Example 3-1. In Examples 3-1 to 2-3, the final concentration of phloretic acid in the differentiation medium is shown in Table 3. Glucose uptake into cells in the test sample was evaluated by measuring the luminescence intensity using the Glucose Uptake-Glo Assay kit (Promega). The test was carried out 5 times each, and the average value of the emission intensity (n = 5) was calculated. The results are shown in Table 3.

As shown in Table 3, the emission intensity was increased in the test samples of Examples 3-1 to 3-2 to which phloretic acid was added, as compared with the test sample of Comparative Example 3-1 to which phloretic acid was not added. It was found that the amount of sugar uptake was increased. When a significant difference test (two-sided test by Student's T-test) was performed, the luminescence intensity of the test sample of Example 3-1 was significantly increased as compared with the test sample of Comparative Example 3-1 (p <0). .01).

＊）有意な増加（ｐ＜０．０１）

*) Significant increase (p <0.01)

Claims (4)

A muscle-building agent containing phloretic acid as an active ingredient. A myotube cell differentiation promoter containing phloretic acid as an active ingredient. A mitochondrial activator containing phloretic acid as an active ingredient. An agent that promotes sugar uptake into myotube cells, which contains phloretic acid as an active ingredient.