Skeletal muscle differentiation promoter

Abstract

To provide a skeletal muscle differentiation promoter having excellent activity and derived from safe food materials.SOLUTION: The present invention provides a skeletal muscle differentiation promoter comprising, as an active ingredient, 4'-demethylnobiletin and / or 4'-demethyltangeretin. 4'-demethylnobiletin and 4'-demethyltangeretin are producible by conversion from nobiletin and tangeretin, citrus peel constituents, through koji mold fermentation, so that the pharmaceuticals or functional foods and beverages of the present disclosure are highly safe and free from the risk of side effects.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a skeletal muscle differentiation promoter containing 4'-demethylnobiletin and / or 4'-demethyltangeretin as an active ingredient, more specifically to a composition containing 4'-demethylnobiletin and / or 4'-demethyltangeretin as an active ingredient and having the effect of inducing differentiation from skeletal muscle-derived myoblasts to myotubes. [Background technology]

[0002] In the process of skeletal muscle tissue formation, an important step is the differentiation of myoblasts, which are produced from satellite cells, stem cells present in skeletal muscle tissue, into muscle cells, which fuse with each other and differentiate into multinucleated myotube cells to form myotubes (see, for example, Non-Patent Document 1). During this process, MyoD, a muscle differentiation control factor, acts from satellite cell activation to the early stage of myotube differentiation. As muscle differentiation progresses further, expression of myogenin is induced. As a result, myosin heavy chain (MHC) is expressed in myotubes, which are mature muscle fibers from the late stage of muscle differentiation (see, for example, Non-Patent Document 2). Therefore, compounds that promote the differentiation marker MHC from myoblasts to myotubes can promote skeletal muscle differentiation and suppress loss of skeletal muscle mass.

[0003] Previous attempts to enhance muscle differentiation have included the use of muscle differentiation promoters using isolated and purified biotin (see Patent Document 1), oligo-DNA (see Patent Document 2), and technologies that utilize components containing polyphenols (see Patent Document 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Charge, SBP et al., Physiol Rev. 84(1);209-238, 2004 [Non-patent document 2] Daiki Seko et al., Basic Aging Research 40(1); 19-25, 2016 [Patent documents]

[0005] [Patent Document 3] Patent No. 6664956 specification [Patent Document 4] Patent No. 7386507 specification [Patent Document 5] Patent No. 6859336 specification Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure aims to provide an excellent skeletal muscle differentiation promoter from among safe food materials. [Means for solving the problem]

[0007] Nobiletin, a polymethoxyflavonoid, is a flavonoid unique to citrus fruits, and in recent years has become known for its various physiological effects, including cancer prevention, anti-aging, and anti-arteriosclerosis. The present inventors have demonstrated that by using citrus peels containing large amounts of nobiletin and tangeretin and fermenting them with a specific type of koji mold, the main components nobiletin and tangeretin are converted to 4'-demethylnobiletin (formula (I) below) and 4'-demethyltangeretin (formula (II) below). The present inventors have also discovered that 4'-demethylnobiletin and 4'-demethyltangeretin have excellent memory-improving effects (see Japanese Patent No. 5667561). Furthermore, the present inventors have also discovered that 4'-demethyltangeretin has excellent wrinkle-preventing or improving effects (see Japanese Patent No. 7262760).

[0008] [ka]

[0009] [ka]

[0010] The present inventors further investigated the functionality of 4'-demethylnobiletin and 4'-demethyltangeretin and found that they have the effect of promoting myotube formation and skeletal muscle formation by promoting MHC expression and strongly inducing the differentiation of skeletal muscle-derived myoblasts into myotubes. Based on these findings, the present inventors have completed the present disclosure.

[0011] That is, the present disclosure relates to a skeletal muscle differentiation promoter containing 4'-demethylnobiletin and / or 4'-demethyltangeretin as an active ingredient. [Effects of the Invention]

[0012] The present disclosure makes it possible to provide an excellent agent for promoting skeletal muscle differentiation by containing 4'-demethylnobiletin and / or 4'-demethyltangeretin as an active ingredient.

[0013] Since 4'-demethylnobiletin and 4'-demethyltangeretin are food-derived components contained in a koji mold fermentation product of citrus peel components, the skeletal muscle differentiation promoter of the present disclosure is highly safe and has no risk of side effects. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is an electrophoretic photograph showing the effect of adding 4′-demethylnobiletin (DN) to promote the expression of myotube differentiation marker, myosin heavy chain (MHC). [Figure 2] FIG. 1 shows the effect of adding 4′-demethylnobiletin (4′-DemNob) on promoting differentiation of C2C12 cells from myoblasts to myotubes. [Figure 3] FIG. 1 shows the effect of adding 4′-demethyltangeretin (4′-DemTan) to promote differentiation of C2C12 cells from myoblasts to myotubes. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present disclosure will be described in detail. The present embodiment relates to a skeletal muscle differentiation promoter containing 4'-demethylnobiletin and / or 4'-demethyltangeretin as an active ingredient.

[0016] [4'-demethylnobiletin, 4'-demethyltangeretin] Although 4'-demethylnobiletin and 4'-demethyltangeretin (see Formula (I) and Formula (II) above) are not commercially available, pure 4'-demethylnobiletin and 4'-demethyltangeretin (isolates) or compositions containing 4'-demethylnobiletin and 4'-demethyltangeretin obtained by koji mold fermentation using citrus fruits containing nobiletin and tangeretin, particularly the peel, using synthetic products, or the previously reported methods of Japanese Patent Nos. 5,667,561 and 7,262,760, can be used. However, the methods of Japanese Patent Nos. 5,667,561 and 7,262,760, which utilize bioconversion of nobiletin and tangeretin by koji mold fermentation, are preferred because they are simpler and less expensive.

[0017] 4'-demethylnobiletin and 4'-demethyltangeretin are also among the metabolic products produced after nobiletin and tangeretin are absorbed in the body. Citrus peel is used as an ingredient in sweets such as marmalade and candied fruits, and mandarin oranges (Citrus reticulata), which have a high content of nobiletin and tangeretin, have a long history of being eaten as tangerine, and 4'-demethylnobiletin, 4'-demethyltangeretin, and compositions containing them are extremely safe.

[0018] The methods for producing 4'-demethylnobiletin, 4'-demethyltangeretin, and compositions containing them, which are described in Japanese Patent Nos. 5,667,561 and 7,262,760, are described below.

[0019] [Fermentation ingredients] The raw material for koji fermentation is citrus 'fruit' (the entire fruit including peel, juice, pulp, seeds, etc.), which contains the polymethoxyflavonoids nobiletin and tangeretin; however, it is particularly desirable to use 'fruit peel' from the standpoint of the nobiletin and tangeretin content and the effective use of waste materials. Furthermore, any variety or lineage of citrus fruit (for example, ponkan, shikuwasha, tangerine, tachibana, etc.) can be used as long as it contains nobiletin and tangeretin. The fermentation raw material may contain other parts of citrus plants (for example, leaves, buds, stems, flowers, etc.), but it is desirable that the fermentation raw material does not contain these parts in terms of the content of nobiletin and tangeretin.

[0020] The above citrus fruits are preferably harvested and collected fresh, or washed, but dried, frozen, or long-term stored fruits can also be used. Citrus fruits may be used in their original form, but it is preferable to chop, crush or grind them. This process includes a wide range of actions, such as roughly chopping the citrus fruits into several pieces, shredding into small pieces, crushing, grinding, powdering, etc. Preferably, this process can be carried out by chopping the citrus fruits into roughly one to several centimeters in size.

[0021] Furthermore, extracts (extracts, dried products) obtained by extracting nobiletin and tangeretin in advance from these fermentation raw materials, and pure nobiletin and tangeretin isolated therefrom can also be used. It is preferable to heat-treat these fermentation raw materials to sterilize any unwanted bacteria in the raw materials before carrying out the koji mold fermentation described below.

[0022] [Koji mold fermentation] Examples of koji molds that can be used to ferment the fermentation raw materials include Aspergillus kawachii, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus saitoi, Aspergillus usamii, and Rhizopus filamentous fungi (also known as Rhizopus), and mixtures of these may also be used. Among the koji molds, Aspergillus kawachii, Aspergillus awamori, Aspergillus oryzae, and Aspergillus niger are preferably used, as these can produce 4'-demethylnobiletin and 4'-demethyltangeretin at high contents. Two or more of these koji molds can also be used in combination.

[0023] The method of inoculating the koji mold into the fermentation raw material can be to sprinkle koji mold spores directly onto the fermentation raw material to attach them to the fermentation raw material. Alternatively, the inoculum source can be a medium in which the koji mold has been pre-fermented by liquid culture, and this can be inoculated so that the spores are distributed throughout the fermentation raw material. When the koji mold is inoculated into the fermentation raw material, the microbial fermentation is preferably carried out under aerobic conditions, and therefore a suitable container is, for example, a cylindrical container with a wide bottom and a shallow depth. It is advisable to spread the fermentation raw materials evenly on the bottom of such a container so that the contact area with the air becomes large.

[0024] The fermentation temperature is not particularly limited as long as it is a suitable condition for the growth of the koji mold, and can be preferably 10 to 40°C, more preferably 20 to 40°C, and even more preferably 25 to 32°C. In addition, as a suitable condition for the growth of the koji mold, fermentation is preferably carried out in a dark place. Furthermore, it is preferable that the raw materials contain sufficient moisture. The fermentation period for microbial fermentation should be such that 4'-demethylnobiletin and 4'-demethyltangeretin can be obtained in large amounts, and is preferably 2 to 21 days, more preferably 3 to 14 days, and even more preferably 4 to 12 days. If the fermentation period is less than two days, the microbial fermentation by the koji mold hardly progresses, and sufficient 4'-demethylnobiletin and 4'-demethyltangeretin are not obtained. Conversely, if the fermentation period exceeds 21 days, decomposition of the 4'-demethylnobiletin and 4'-demethyltangeretin produced by microbial conversion progresses, and the desirable citrus-derived aroma is lost.

[0025] Furthermore, in the koji mold fermentation, nobiletin and tangeretin are demethylated by enzymes secreted from the koji mold, converting them to 4'-demethylnobiletin and 4'-demethyltangeretin. Therefore, instead of performing koji mold fermentation, it is also possible to obtain 4'-demethylnobiletin and 4'-demethyltangeretin by performing solution extraction from the koji mold or a fermentation product obtained after fermentation to obtain an enzyme solution containing an enzyme that demethylates nobiletin and tangeretin, and then performing an enzymatic reaction with the raw material using the enzyme to obtain a reaction product.

[0026] By carrying out the above-mentioned koji mold fermentation, all of the polymethoxyflavonoids nobiletin and tangeretin contained in the citrus raw material are converted to 4'-demethylnobiletin and 4'-demethyltangeretin. Specifically, by fermenting the citrus raw material with koji mold, it is possible to obtain a koji mold fermentation product with a high content of 4'-demethylnobiletin of about 0.5 to 1.5% by mass (specifically, about 1% by mass) per dry weight and 4'-demethyltangeretin of about 0.3 to 0.9% by mass (specifically, about 0.6% by mass) per dry weight. Therefore, the koji mold fermentation product obtained here can be used as an active ingredient of the skeletal muscle differentiation promoter of this embodiment, either in the form in which it is obtained or after processing (e.g., by shredding, crushing, powdering, drying, etc.).

[0027] [Solution extraction] In consideration of purity, in the production of the skeletal muscle differentiation promoter of this embodiment, it is desirable to obtain an extract by performing solution extraction from the fermentation product obtained after the koji mold fermentation. The solution extraction step can be performed directly on the koji mold fermentation product, but is preferably performed on the koji mold fermentation product after further processing such as shredding, crushing, grinding, or powdering.

[0028] The solvent used in the solution extraction step may be water, a buffer solution, an organic solvent, or a water-containing solvent thereof. Examples of the organic solvent include lower aliphatic alcohols such as ethanol, methanol, isopropanol, and butanol, as well as acetone, ethyl acetate, and chloroform. Among these solvents, water, ethanol, or aqueous ethanol is particularly preferred in terms of extraction efficiency, ease of handling, and safety.

[0029] In particular, aqueous ethanol is preferred, and performing extraction using aqueous ethanol at a final concentration of more preferably 55% or more, even more preferably 60% or more, and particularly preferably 80% or more (all v / v) is preferred because this can suppress the elution of polysaccharide impurities and improve the extraction efficiency of 4'-demethylnobiletin and 4'-demethyltangeretin. The extraction conditions are as follows: the raw material (preferably the crushed material) is added with 1 to 50 times, preferably 2 to 15 times (both by mass) the amount of the solvent; and the raw material is soaked or shaken at a temperature of 0°C to the boiling point of the solvent, preferably room temperature to a temperature below the boiling point of the solvent, for 5 minutes to 1 month, preferably 20 minutes to 1 week.

[0030] The obtained extract can be concentrated to dryness by freeze-drying or drying using an evaporator or the like. Furthermore, the solution extraction step can be performed multiple times using multiple different solvents. In particular, if the first extraction is performed with water or a low-concentration aqueous alcohol, the extraction efficiency of 4'-demethylnobiletin and 4'-demethyltangeretin can be improved by subsequently performing an extraction using aqueous ethanol of the specific concentration or higher. The extract obtained as described above (the extract liquid or concentrated dried product) has excellent differentiation-inducing activity from skeletal muscle-derived myoblasts to myotube cells, and can therefore be used as is as the active ingredient of the skeletal muscle differentiation promoter of this embodiment.

[0031] 〔purification〕 Furthermore, by subjecting these to a purification step, the contents of 4'-demethylnobiletin and 4'-demethyltangeretin can be further increased. As a purification step, a high-content composition can be obtained by liquid-liquid separation extraction or column purification using silica gel, chemically modified silica gel, activated carbon, synthetic adsorption resin carrier, etc. An example of suitable purification conditions is shown below.

[0032] (Purification of 4'-demethylnobiletin) First, the extraction solvent (specifically, ethanol) is removed from the extract, and the resulting removed solution is applied to a column of porous synthetic adsorption resin (specifically, Diaion HP-20 [manufactured by Mitsubishi Chemical Corporation]) equilibrated with water. Then, after removing components eluted with water from the column, components eluted with 30 to 35% (v / v) ethanol are further removed. Next, components eluted with 44 to 46% (v / v) ethanol are collected from the column, thereby obtaining a composition with a high content of 4'-demethylnobiletin (specifically, a purity of 15% (w / w) or more). Furthermore, the 4'-demethylnobiletin-rich composition obtained as described above can be further subjected to ODS column chromatography (specifically, 45% [v / v] methanol elution), thin layer chromatography (TLC) (specifically, hexane / ethanol [7:3]), or ODS-HPLC (specifically, 37% [v / v] acetonitrile-water mixed solvent), and the target peak can be collected to isolate pure 4'-demethylnobiletin. The 4'-demethylnobiletin obtained as described above is a monodemethyl form of nobiletin in which the 4'-position has been demethylated. 4'-demethylnobiletin becomes more polar due to demethylation, and has superior solubility in alcohol and water compared to nobiletin.

[0033] (Purification of 4'-demethyltangeretin) First, an extract obtained by extraction with aqueous ethanol (specifically, an ethanol concentration of 35 to 40% [v / v]) is applied to a column of porous synthetic adsorption resin (specifically, Diaion HP20 [manufactured by Mitsubishi Chemical Corporation]) equilibrated with aqueous ethanol having the same ethanol concentration as the extraction solvent. Then, components eluted with 39 to 41% (v / v) ethanol (specifically, 40% (v / v) ethanol) are removed from the column. Next, components eluted with 42 to 44% (v / v) ethanol (specifically, 43% (v / v) ethanol) are collected from the column, thereby selectively isolating 4'-demethyltangeretin and obtaining a composition rich in 4'-demethyltangeretin (specifically, a purity of 30% (w / w) or more). Furthermore, the 4'-demethyltangeretin-rich composition obtained as described above can be further subjected to ODS column chromatography (specifically, elution with 40% [v / v] methanol), thin-layer chromatography (TLC) (specifically, hexane / ethanol [7:3]), or ODS-HPLC (specifically, elution with a 33% [v / v] acetonitrile / water mixed solvent), and pure 4'-demethyltangeretin can be isolated by collecting the target peak. The 4'-demethyltangeretin obtained by the above process is a monodemethylated form of tangeretin in which the 4'-position has been demethylated. 4'-Demethyltangeretin becomes more polar due to demethylation and has better solubility in alcohol and water than tangeretin.

[0034] [Medicines and functional foods and beverages] The present embodiment relates to a skeletal muscle differentiation promoter containing 4'-demethylnobiletin and / or 4'-demethyltangeretin as an active ingredient. The "skeletal muscle differentiation promoter" according to this embodiment encompasses drugs and functional foods and drinks.

[0035] In the present embodiment, 4'-demethylnobiletin and 4'-demethyltangeretin can be used as active ingredients in pharmaceuticals or functional foods and beverages by mixing them with various raw materials as the compositions obtained in the above steps ('composition directly containing the fermentation product,' 'solution extract,' 'purified product,' etc.) or as 'isolates'.

[0036] In this embodiment, the term "drug" includes pharmaceuticals and quasi-drugs. The pharmaceuticals and quasi-drugs may be in the form of either an oral preparation or an external preparation. Furthermore, the term "functional food and drink" in this embodiment includes functional foods and functional drinks.

[0037] When orally taken as an internal medicine or functional food or drink, the effective intake amount of 4'-demethylnobiletin and 4'-demethyltangeretin is 1 mg or more, preferably 5 mg or more, per day for an adult weighing 60 kg. By orally taking these amounts, excellent effects can be obtained in inducing the differentiation of skeletal muscle-derived myoblasts into myotube cells and promoting myotube formation and skeletal muscle formation, as well as excellent effects in improving the decrease in skeletal muscle mass caused by aging.

[0038] The content of 4'-demethylnobiletin and 4'-demethyltangeretin in the drug or functional food or drink according to this embodiment may be any amount that ensures the necessary intake amount, and specifically, the content can be 0.001% by mass or more, preferably 0.01% by mass or more, and more preferably 0.1% by mass or more. The upper limit can be 20% by mass or less.

[0039] Therefore, it is expected that the above-mentioned pharmacological effects can be obtained by ingesting the drug or functional food or drink of this embodiment in a form and by an ingestion method (frequency, amount) that can ensure this required amount. However, it is desirable to appropriately determine the intake amount depending on the subject's age, weight, symptoms, ingestion schedule, formulation form, etc.

[0040] The form of the drug according to this embodiment can be, for example, powder, fine granules, granules, toothpaste, etc. when taken, and in addition to being filled into capsules, it can also be in the form of a solution dispersed in water, a cream, or a tablet obtained by mixing with excipients, etc. The drug according to this embodiment may contain, in addition to 4'-demethylnobiletin, 4'-demethyltangeretin, or a composition containing them, various carriers, additives, other medicinal ingredients, and the like, as long as the effects of the present disclosure are achieved.

[0041] With regard to the food and beverage composition according to this embodiment, 4'-demethylnobiletin, 4'-demethyltangeretin, or a composition containing thereof can be mixed with various food ingredients and additives to form the food and beverage composition, for example, in the form of biscuits, snacks, gum, chewable tablets, soft drinks, drinks, soups, jellies, candies, etc. [Example]

[0042] The present disclosure will be described below with reference to examples, but the scope of the present disclosure is not limited to these examples.

[0043] Example 1 Preparation of a 4'-demethylnobiletin-containing composition In accordance with the method described in Japanese Patent No. 5667561, a nobiletin conversion product, 4'-demethylnobiletin, was prepared by fermenting Ponkan peel with koji mold.

[0044] Specifically, 10 kg of Ponkan peel was chopped into small pieces and sterilized by steaming. The resulting Ponkan peel was inoculated with Aspergillus awamori (manufactured by Bioc Co., Ltd.) so that it was distributed throughout the peel. Fermentation (Koji mold fermentation) was then carried out aerobically in a thermostatic chamber at 30°C for 5 days to obtain a Koji mold fermentation product. 60 L of water was added to 5 kg of the resulting Koji mold fermentation product, and hot water extraction was carried out at 100°C for 1 hour to obtain an extract.

[0045] The obtained extract was applied to a Diaion HP20 (porous synthetic adsorption resin column) that had been equilibrated with water in advance, and after removing non-adsorbed components with 3 L of water, the components eluted with 2 L of 35% (v / v) ethanol were further removed. Next, the components eluted with 2 L of 45% (v / v) ethanol were collected, and the collected matter was concentrated to dryness using a rotary evaporator to obtain a 4'-demethylnobiletin-containing composition (4'-demethylnobiletin content 19.1%).

[0046] Example 2: Preparation of 4'-demethylnobiletin isolate 2 g of the 4'-demethylnobiletin-containing composition obtained in Example 1 was dissolved in 20% (v / v) methanol and subjected to ODS column chromatography (a column having an inner diameter of 20 mm and a length of 30 cm was packed with 30 g of Wako Gel 50C18). Components eluted with 40% (v / v) methanol were removed, and components eluted with 60% (v / v) methanol were collected.

[0047] The recovered components were then subjected to preparative TLC chromatography (silica gel 70PF) using a developing solvent of hexane / ethanol 7:3. 254 A Wako plate (film thickness 0.75 mm, manufactured by Wako Pure Chemical Industries) was used, and the fractions containing 4'-demethylnobiletin were collected while checking using a UV lamp.

[0048] The obtained fraction was then applied to a preparative HPLC column (TSK GEL ODS, manufactured by Tosoh Corporation, 4.6 mm×25 cm), and 20 mg of pure 4′-demethylnobiletin was obtained using a mobile phase of 37% (v / v) acetonitrile.

[0049] Example 3 Preparation of a 4'-demethyltangeretin-containing composition According to the method described in Japanese Patent No. 7262760, 4'-demethyltangeretin, a tangeretin conversion product, was prepared by fermenting Ponkan peel with Aspergillus oryzae.

[0050] Specifically, 10 kg of Ponkan peel was chopped into small pieces and sterilized by steaming. The resulting Ponkan peel was inoculated with Aspergillus awamori (manufactured by Bioc Co., Ltd.) so that it was distributed throughout the peel. Fermentation (Koji mold fermentation) was then carried out aerobically in a thermostatic chamber at 30°C for two days to obtain a Koji mold fermentation product.

[0051] 75 L of 40% (v / v) ethanol was added to 6 kg of the resulting koji mold fermentation product, and the extract was extracted at 100°C for 1 hour to obtain an extract. The extract was applied to a Diaion HP20 (porous synthetic adsorption resin column) previously equilibrated with 40% (v / v) ethanol, and non-adsorbed components were removed with 3 L of 40% (v / v) ethanol. After that, the components eluted with 10 L of 43% (v / v) ethanol were recovered. The recovered material was concentrated to dryness using a rotary evaporator to obtain a 4'-demethyltangeretin-containing composition (4'-demethyltangeretin content: 32%).

[0052] Example 4: Preparation of 4'-demethyltangeretin isolate Two grams of the 4'-demethyltangeretin-containing composition obtained in Example 3 was dissolved in 20% (v / v) methanol and subjected to ODS column chromatography (a column with an inner diameter of 20 mm and a length of 30 cm packed with 30 g of Wako Gel 50C18). The component eluted with 30% (v / v) methanol was removed, and the component eluted with 40% (v / v) methanol was obtained.

[0053] The resulting components were then subjected to preparative TLC chromatography (silica gel 70PF) using a developing solvent of hexane / ethanol 7:3. 254 The fraction containing 4'-demethyltangeretin was collected using a Wako plate (film thickness 0.75 mm, manufactured by Wako Pure Chemical Industries, Ltd.) while checking with a UV lamp.

[0054] The obtained fraction was then applied to a preparative HPLC column (TSK GEL ODS, manufactured by Tosoh Corporation, 4.6 mm×25 cm), and 30 mg of pure 4′-demethyltangeretin was obtained using a mobile phase of 33% (v / v) acetonitrile.

[0055] <Example 5> Examination of the effects of 4'-demethylnobiletin and 4'-demethyltangeretin on skeletal muscle differentiation Using the 4'-demethylnobiletin and 4'-demethyltangeretin isolates obtained in Examples 2 and 4, the effects of these substances on myosin heavy chain (MHC) as a myotube cell differentiation marker and on the differentiation of myoblasts into myotubes (myotube formation) as an indicator of skeletal muscle formation were evaluated at the cellular level.

[0056] The cells used were C2C12 cells, a myoblast cell line derived from mouse skeletal muscle satellite cells. The cells were cultured in a growth medium (DMEM medium (Thermo Fisher Scientific, Gibco® #11966-025) containing 10% fetal bovine serum (BioWest, #S1400-500)) in a 37°C incubator under 5% CO2, and subcultured every 2–3 days by 0.25% trypsin treatment. The morphological changes of the cells were observed by phase contrast observation using an all-in-one fluorescence microscope (Keyence (registered trademark), BZ-X710).

[0057] For biochemical analysis and cell morphology observation, C2C12 cells were seeded (6 × 10) onto a 35 mm plastic dish (Falcon® #353001). 4 After overnight culture in a 100% PBS culture medium (100 cells / dish), the medium was replaced with the growth medium supplemented with each isolate of 4'-demethylnobiletin and 4'-demethyltangeretin (products of Examples 2 and 4), and cultured for two days. Thereafter, the medium was replaced with DMEM medium (differentiation medium) supplemented with 2% horse serum (Gibco (registered trademark) #16050-122), and cultured for a predetermined period. The medium was replaced every 1 to 2 days. In addition, as a (comparison) control group, the cells were cultured for two days in the growth medium supplemented with 0.1% DMSO (Fujifilm Wako Pure Chemical Industries) instead of the growth medium supplemented with each isolate of 4'-demethylnobiletin and 4'-demethyltangeretin, and then the medium was replaced with the differentiation-inducing medium as described above.

[0058] For preparation of cell extracts and biochemical analysis, C2C12 cells were washed with cold DPBS(-) and then lysed in lysis buffer (2% SDS, 1 mM EDTA, 50 mM Tris-HCl, pH 7.5) supplemented with a protease inhibitor cocktail (Nacalai Tesque®, #25995-24) and a phosphatase inhibitor cocktail (PhosSTOP®, Roche®, #04906845001). After 5 minutes of sonication, the cells were centrifuged at 10,000 × g for 10 minutes, and the resulting supernatant was used as the cell extract. The prepared cell extract was aliquoted and stored in a deep freezer at -80°C until use in experiments. Total protein was quantified using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific, #23225).

[0059] The cell extract was boiled in Laemmli's sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for 5 minutes, and the protein sample was separated on a 4-20% SDS-PAGE gel. Precision Plus Protein Dual Color Standards (Bio-Rad®, #161-0374) were used as protein molecular weight markers. After electrophoresis, the sample was blotted onto a PVDF membrane. The blot was then immersed in 5% skim milk / TBST (TBST: 150 mM NaCl, 20 mM Tris-HCl (pH 7.5) buffer containing 0.5% (w / v) Tween-20) and blocked with shaking at room temperature for 1 hour. The membrane was then incubated overnight at 4°C with a primary antibody solution (mouse anti-MHC monoclonal antibody (R&D Systems, #MF20) diluted 1:1,000 in 5% skim milk / TBST). After washing with TBST at room temperature, the blot membrane was incubated with a secondary antibody solution (horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG antibody (Cell Signaling Technology®; CST, #7076; diluted 2,000-fold with 5% skim milk / TBST) at room temperature for 1 hour. After washing with TBST, the blot membrane was incubated with a chemiluminescent reagent (SuperSignal® West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific, #34580) at room temperature for 5 minutes, and then the MHC signal was detected using an image analyzer (Invitrogen®, iBright® FL1500 Imaging System).

[0060] The blot membrane was then stripped and blocked again for further reprobing with anti-GAPDH antibody, and then reacted with anti-GAPDH antibody solution (rabbit monoclonal anti-GAPDH antibody (CST, #2118) diluted 1,000-fold with 5% skim milk / TBST) overnight at 4°C. After washing at room temperature, the blot membrane was reacted with HRP-labeled goat anti-rabbit IgG antibody solution (CST, #7074; diluted 2,000-fold with 5% skim milk / TBST) for 1 hour at room temperature. After washing, the blot membrane was reacted with the above-mentioned chemiluminescent reagent to detect the GAPDH bands using ChemiDoc. TM Detection was performed using a Touch Imaging System (Bio-Rad®).

[0061] To evaluate the ability to induce differentiation of skeletal myogenic myoblasts into myotubes (myotube formation), C2C12 cells were cultured in growth medium containing 0.1% DMSO for two days in the control group, then switched to a differentiation-inducing medium and cultured for six days to induce differentiation. In the 4'-demethylnobiletin and 4'-demethyltangeretin-added groups, cells were cultured for two days in growth medium containing 0.1% DMSO supplemented with 4'-demethylnobiletin or 4'-demethyltangeretin to a final concentration of 20 μM, then switched to a differentiation-inducing medium and cultured for six days, as in the control group. After six days, photographs of the morphology of differentiated cells were taken and recorded for the control group and each of the 4'-demethylnobiletin and 4'-demethyltangeretin-added groups. Results were assessed by comparing the morphology with that of the control group and confirming the appearance of myotubes.

[0062] The results of SDS-PAGE are shown in Figure 1, and the results of cell morphology observation are shown in Figures 2 and 3, respectively. Figure 1 is an electrophoretic photograph showing the effect of adding 4'-demethylnobiletin to promote the expression of the myotube differentiation marker MHC. "C1" and "C2" indicate the control groups, "DN1" and "DN2" indicate the 4'-demethylnobiletin-added groups, and "2 days" and "6 days" indicate the number of days of culture in the differentiation-inducing medium, respectively. "M" is a protein molecular weight marker (Precision Plus Protein Dual Color Standard, Bio-Rad (registered trademark)). Figure 1 shows that in the 4'-demethylnobiletin-added groups (DN1, DN2), the band for the myotube differentiation marker MHC was darker both 2 days and 6 days after addition compared to the control groups (C1, C2), indicating differentiation into myotubes.

[0063] 2 and 3 are micrographs showing the effect of adding 4'-demethylnobiletin (4'-DemNob) or 4'-demethyltangeretin (4'-DemTan), respectively, to promote the differentiation of C2C12 cells from myoblasts to myotubes (myotube formation). In each figure, A shows the cell morphology of the control group, and B shows the 4'-DemNob or 4'-DemTan addition group. In FIG. 2, with the addition of 4'-demethylnobiletin (final concentration 20 μM), many myotubes were observed within the range of the photograph (FIG. 2B), but there were clearly fewer myotubes in the control group without 4'-demethylnobiletin (FIG. 2A). Furthermore, in Figure 3, the addition of 4'-demethyltangeretin (final concentration 20 μM) resulted in a large number of myotubes within the photographed area (Figure 3B), whereas the control group without 4'-demethyltangeretin had significantly fewer myotubes (Figure 3A).

[0064] From the above, it was demonstrated that 4'-demethylnobiletin and 4'-demethyltangeretin have a very strong effect of promoting the differentiation of myoblasts into myotubes.

[0065] The above has described in detail the embodiments and examples of the present disclosure with reference to the drawings, but the specific configurations are not limited to these, and design changes that do not deviate from the gist of the present disclosure are included in the present disclosure.

[0066] For example, in the above examples, the skeletal muscle differentiation promoting effect of 4'-demethylnobiletin and 4'-demethyltangeretin extracted and purified from Ponkan peel or a composition containing these has been described; however, the present disclosure is not limited to this, and the present disclosure can also include skeletal muscle differentiation promoters and the like that contain 4'-demethylnobiletin and 4'-demethyltangeretin as active ingredients, extracted from any citrus fruit containing nobiletin or tangeretin instead of Ponkan. [Industrial Applicability]

[0067] The pharmaceutical agents or functional foods and beverages disclosed herein containing 4'-demethylnobiletin or 4'-demethyltangeretin as active ingredients have the effect of inducing differentiation of skeletal muscle-derived myoblasts into myotubes, and are therefore expected to be useful for, for example, treating a decrease in skeletal muscle mass that accompanies aging, and are expected to have an effect of improving QOL.

Claims

[Claim 1] A skeletal muscle differentiation promoter comprising 4'-demethylnobiletin and / or 4'-demethyltangeretin as active ingredients.

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