Composition for promoting sugar uptake

Abstract

To provide a composition that promotes sugar uptake into a muscle cell.SOLUTION: A sugar uptake promoting composition contains at least one plant material selected from Pueraria hirsuta, chili pepper, pine bark, and barley young leaves.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition that promotes sugar uptake into muscle cells. [Background technology]

[0002] Muscles are large organs that have the function of taking in glucose, and the uptake of glucose into muscles has a significant impact on blood sugar control. Normally, when blood sugar levels rise after a meal, additional insulin is secreted, and this insulin causes glucose to be taken up into muscles, but it is known that in obese people, for example, the uptake of glucose into muscles by insulin is reduced.

[0003] In order to suppress such a decrease in sugar uptake into muscles, a sugar uptake promoter has been proposed which contains as an active ingredient one or more extracts selected from the group consisting of beet, chaga, quince, wolfberry, bracken, Cassia japonica, ginseng, hawthorn, shell ginger leaves, sweet tea, eucommia tea, silver vine, pine cone, and purple brown rice (see Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2015-98438 A Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a composition that promotes sugar uptake into muscle cells. [Means for solving the problem]

[0006] In the course of studying the functions of plant-derived materials, the present inventors discovered that kudzu flowers, chili peppers, pine bark, and young barley leaves have the effect of promoting sugar uptake into muscle cells, leading to the completion of the present invention. In particular, they discovered that young barley leaves promote sugar uptake into muscle cells by activating insulin signaling.

[0007] That is, the present invention is as follows. [1] A composition for promoting sugar uptake, comprising at least one plant material selected from kudzu flowers, chili peppers, pine bark, and young barley leaves. [2] A composition for promoting sugar uptake according to [1], which is for oral use. Effect of the Invention

[0008] The composition of the present invention can promote glucose uptake into muscle cells. [Brief description of the drawings]

[0009] [Figure 1] 1 is a graph showing the amount of glucose uptake when the composition of the present invention (kudzu flower, chili pepper, pine bark, or young barley leaves) was applied. [Diagram 2] 1 is a graph showing the change in glucose uptake due to the addition of Wortmannin, a PI3K inhibitor, and Compound C, an AMPK inhibitor, when the composition of the present invention (young barley leaves) was applied. [Diagram 3] 1 is a graph showing the change in p-Akt expression level (phosphorylation rate of Akt) relative to the total expression level of Akt by the addition of wortmannin, a PI3K inhibitor, when the composition of the present invention (young barley leaves) was applied. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The composition of the present invention is not particularly limited as long as it contains at least one plant material selected from kudzu flowers, chili peppers, pine bark, and young barley leaves, and may contain two or more types.

[0011] The composition of the present invention can promote the uptake of sugar into muscle cells and effectively utilize carbohydrates, and therefore can provide effects such as lowering blood sugar levels, preventing and improving diabetes, dieting (preventing, preventing and / or eliminating obesity), and promoting metabolism, as well as improving muscle function by utilizing carbohydrates in muscles.

[0012] Furthermore, since the composition of the present invention containing young barley leaves activates insulin signaling, in addition to the effects described above, it is possible to obtain various effects brought about by insulin signaling activation (e.g., lowering blood neutral fat levels, increasing muscle mass, etc.). Furthermore, it is possible to prevent or improve various symptoms caused by insulin, such as poor insulin secretion (e.g., diabetes, hyperlipidemia, hypertension, osteoporosis, etc.).

[0013] Each plant material contained in the composition of the present invention will be described below. (Kuzuka) Kudzu is a creeping perennial plant belonging to the genus Pueraria in the family Fabaceae. Kudzu flowers may be harvested at any stage from bud to fully opened flower, or may be mixed and used. There is no particular restriction on the type of kudzu, but examples include Pueraria thomsonii, Pueraria lobata, and Pueraria thunbergiana, and Pueraria thomsonii is preferred because it has excellent sugar uptake properties.

[0014] As the kudzu flower, a processed product of the collected kudzu flower can be used. Examples of the processed form include dried products, fermented products, crushed products, squeezed products, extracts, etc. Examples of the solvent used for extraction include organic solvents such as water, methanol, ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, diethyl ether, and hexane, acidic aqueous solutions such as acetic acid, citric acid, and aqueous solutions thereof, and mixed solvents thereof. The temperature of the extraction solvent can be appropriately set within the range of room temperature to the boiling point or lower depending on the solvent used. Examples of the processed form include (dried) powder, finely chopped products, granules, paste, liquid, etc. As the kudzu flower of the present invention, a crushed product, a water (warm water, hot water) extract, an ethanol extract, or a hydroethanol extract is preferred, with a water (warm water, hot water) extract, an ethanol extract, or a hydroethanol extract being more preferred, and a hot water extract being the most preferred since it provides an excellent sugar uptake effect.

[0015] (Chili) The chili pepper is not particularly limited as long as it contains capsaicin or a capsaicinoid-like substance, and the variety, the place of origin, etc. are not particularly limited. Specifically, chili pepper varieties CH-19 Sweet, Fushimi Kannaga, Shishito, Yamashina, Manganji, Takanotsume, Kagawa Hontaka, Aomori Takanotsume, Sapporo Daicho, California Wonder, Cherry Bomb, etc. can be used, and Fushimi Kannaga is preferred because it provides excellent sugar uptake. Various chili peppers may be used alone or in combination of two or more.

[0016] Any part of the chili pepper may be used so long as it contains capsaicin or capsaicinoid-like substances. However, since capsaicin or capsaicinoid-like substances are found in large amounts in the placenta, it is preferable to use the placenta or a fruit containing the placenta.

[0017] As the chili pepper, a processed product of the harvested chili pepper can be used. Examples of the processed form include dried products, fermented products, crushed products, juice products, extracts, etc. Examples of the solvent used for extraction include organic solvents such as water, methanol, ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, diethyl ether, and hexane, acidic aqueous solutions such as acetic acid, citric acid, and aqueous solutions thereof, and mixed solvents thereof. The temperature of the extraction solvent can be appropriately set within a range from room temperature to the boiling point or lower depending on the solvent used. Examples of the processed form include (dried) powder, finely chopped products, granules, paste, liquid, etc. As the chili pepper of the present invention, fermented products, crushed products, water (warm water, hot water) extracts, ethanol extracts, and aqueous ethanol extracts are preferred, with fermented products, water (warm water, hot water) extracts, ethanol extracts, and aqueous ethanol extracts being more preferred, and fermented products being the most preferred as they provide excellent sugar uptake activity.

[0018] The fermentation process decomposes capsaicinoid-like substances contained in chili peppers, for example. The fermentation process is carried out by contacting microorganisms capable of producing organic acids and assimilating fatty acids produced by the decomposition of capsaicinoid-like substances, etc. The capsaicinoid-like substances, etc. are decomposed by changes in pH due to the organic acids, etc. produced by the microorganisms.

[0019] Fermentation includes lactic acid fermentation, citric acid fermentation, alcohol fermentation, acetic acid fermentation, and fermentation by a combination of these. Depending on the type of fermentation, lactic acid bacteria, yeast bacteria, acetic acid bacteria, etc. are contacted with the chili pepper. These bacteria may be used alone for fermentation, or multiple bacteria may be added simultaneously for fermentation, or different bacteria may be added stepwise for fermentation. Among these, lactic acid fermentation is preferred. As the chili pepper of the present invention, lactic acid fermentation is the most preferred because it provides excellent sugar uptake.

[0020] Examples of lactic acid bacteria include Leuconostoc mesentroides, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus casei, Streptococcus thermophilus, Streptococcus faecalis, and Bifidobacterium longum, which may be used alone or in combination. For example, when used alone, Lactobacillus plantarum is preferred in terms of its acid resistance, growth temperature, and growth rate.

[0021] (pine bark) Pine is an evergreen coniferous tree of the genus Pinus in the family Pinaceae. Examples of raw materials for pine bark include French maritime pine (Pinus Martima), larch, black pine, red pine, Japanese red pine, Japanese white pine, Korean pine, Japanese stone pine, Ryukyu pine, Japanese white pine, and white pine. Among these, French maritime pine is preferred because it has an excellent sugar uptake effect.

[0022] Pine bark contains proanthocyanidins as one of its main components. Proanthocyanidins are a group of compounds consisting of condensation polymers with a degree of polymerization of 2 or more, whose constituent units are flavan-3-ol and / or flavan-3,4-diol.

[0023] As the pine bark, a processed product of harvested pine bark can be used. Examples of the processed form include dried products, fermented products, crushed products, squeezed products, extracts, etc. Examples of the solvent used for extraction include organic solvents such as water, methanol, ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, diethyl ether, and hexane, acidic aqueous solutions such as acetic acid, citric acid, and aqueous solutions thereof, and mixed solvents thereof. The temperature of the extraction solvent can be appropriately set within a range from room temperature to the boiling point or lower depending on the solvent used. Examples of the processed form include (dried) powder, finely chopped products, granules, paste, liquid, etc. The pine bark of the present invention is preferably a crushed product, a water (warm water, hot water) extract, an ethanol extract, or a hydroethanol extract, more preferably a water (warm water, hot water) extract, an ethanol extract, or a hydroethanol extract, and most preferably a hydroethanol extract since it has an excellent sugar uptake effect. In addition, a commercially available product can be used as the pine bark, and for example, a hydroethanol extract of pine bark (Flavangenol (registered trademark)) manufactured by Toyo Shinyaku Co., Ltd. can be used.

[0024] In addition, the pine bark preferably contains a condensation polymer having a degree of polymerization of 2 or more as proanthocyanidin. In particular, proanthocyanidin containing a large amount of condensation polymers having a low degree of polymerization is preferable. The condensation polymers having a low degree of polymerization are, for example, condensation polymers (dimers to tridemers) having a degree of polymerization of 2 to 30, preferably condensation polymers (dimers to decamers) having a degree of polymerization of 2 to 10, and more preferably condensation polymers (dimers to tetramers) having a degree of polymerization of 2 to 4. In this specification, polymers having a degree of polymerization of 2 to 4 are called OPC (oligomeric proanthocyanidin). The pine bark in the composition of the present invention preferably contains 10% by mass or more of OPC, more preferably 20% by mass or more, and even more preferably 30% by mass or more.

[0025] (Young barley leaves) Barley (Hordeum vulgare L.) is an annual or biennial herb that is said to be native to Central Asia and belongs to the family Poaceae, and is roughly classified into two-row barley, six-row barley, etc., depending on the ear shape. In the present invention, either two-row barley or six-row barley may be used. In addition, it may contain stems together with young leaves.

[0026] As the young barley leaves, a processed product obtained by processing the young barley leaves harvested can be used. Examples of the processed form include dried products, fermented products, crushed products, juices, extracts, etc. Examples of the crushing method include a method in which the leaves and / or stems of barley are dried, coarsely crushed, heated at 110°C or higher, and further finely crushed (see JP 2003-033151 A), and a method in which the leaves and / or stems of barley are blanched, dried, and then crushed (see JP 2002-065204 A). Examples of the solvent used for extraction include organic solvents such as water, methanol, ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, diethyl ether, and hexane, acidic aqueous solutions such as acetic acid, citric acid, or aqueous solutions thereof, and mixed solvents thereof. The temperature of the extraction solvent can be appropriately set within a range from room temperature to the boiling point or lower depending on the solvent used. The processed material may be in the form of a (dry) powder, a finely chopped material, a granule, a paste, a liquid, or the like. As the young barley leaves of the present invention, crushed material, squeezed material, water (warm water, hot water) extract, ethanol extract, and aqueous ethanol extract are preferred, with squeezed material, water (warm water, hot water) extract, ethanol extract, and aqueous ethanol extract being more preferred, and squeezed material being the most preferred as it provides an excellent sugar uptake effect.

[0027] The composition for promoting sugar uptake of the present invention can be used for oral or parenteral administration, but is preferably used for oral administration.

[0028] Examples of parenteral compositions include injections, drops, nasal drops, ear drops, eye drops, and the like.

[0029] Oral compositions include, for example, so-called health foods, such as functional foods whose efficacy has been approved by a designated organization, such as foods for specified health uses, foods with nutritional functions, and foods with functional claims, and pharmaceuticals (including quasi-drugs).

[0030] That is, the oral composition for promoting sugar uptake (oral agent for promoting sugar uptake) of the present invention contains at least one plant material (component of the present invention) selected from kudzu flowers, chili pepper, pine bark, and young barley leaves, and is not particularly limited as long as it can be distinguished from other products as a product in terms of its use for promoting sugar uptake. For example, the scope of the present invention includes a product that displays a sugar uptake promoting function or a carbohydrate utilization function on the main body, packaging, instruction manual, or advertising material of the product of the present invention. In addition, the scope of the present invention also includes a product that displays a blood glucose level lowering function, which is a direct effect of the promotion of sugar uptake. Furthermore, for a composition containing young barley leaves, a product that displays an insulin signal activating ability is also included in the scope of the present invention. Note that the oral composition for promoting sugar uptake of the present invention is not limited to a product that displays the component of the present invention as an active ingredient for promoting sugar uptake, etc. on the packaging of the product. For example, the active ingredient may not be specified, and a specific other ingredient may be displayed as the active ingredient.

[0031] Examples of the form of the oral composition of the present invention include tablets, capsules, powders, granules, liquids, granules, rods, plates, blocks, solids, rounds, pastes, creams, caplets, gels, chewables, and sticks. Among these, tablets, capsules, powders, granules, and liquids are particularly preferred. Specific examples include supplements, containerized beverages filled in PET bottles, cans, bottles, etc., instant beverages (powdered beverages) to be dissolved in water (hot water), milk, fruit juice, green juice, etc., and food additives. These are preferred because they are easy to drink during meals and can increase palatability.

[0032] The content of the component of the present invention in the oral composition of the present invention may be appropriately contained within the range in which the effect is exerted.Depending on the form, for example, the component of the present invention is preferably 0.01% or more of the whole composition of the present invention in terms of dry mass, more preferably 1% or more, and even more preferably 5% or more.In order to more effectively exert the effect of the present invention, the component of the present invention is preferably 70% or more of the whole composition of the present invention in terms of dry mass, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 100%.

[0033] The intake amount of the oral composition of the present invention is not particularly limited, but from the viewpoint of more clearly exerting the effect of the present invention, the intake amount of the component of the present invention per day for an adult is preferably 10 mg / day or more, more preferably 50 mg / day or more, and even more preferably 100 mg / day or more, calculated as dry mass. The upper limit is, for example, 10,000 mg / day, preferably 5,000 mg / day, and more preferably 3,000 mg / day. The oral composition of the present invention can be stored as a daily amount in one container or divided into, for example, 2 to 3 multiple containers so that the daily intake amount is the above-mentioned intake amount.

[0034] The composition of the present invention can be produced by a known method by adding other components other than the components of the present invention as necessary. Examples of other components other than the components of the present invention include vitamins such as water-soluble vitamins (vitamin B1, B2, B3, B5, B6, B12, B13, B15, B17, biotin, choline, folic acid, inositol, PABA, vitamin C, vitamin P) and oil-soluble vitamins (vitamins A, D, E, K); minerals such as calcium, magnesium, phosphorus, and iron; sulfur-containing compounds contained in taurine and garlic; flavanoids or flavonoids such as hesperidin and quercetin; proteins such as collagen; peptides; amino acids; animal fats and oils; vegetable fats and oils; crushed products or extracts of animals and plants, etc. EXAMPLES

[0035] The present invention will be described below with reference to examples. [Example 1] (raw materials) The kudzu flower used was kudzu flower hot water extract powder (manufactured by Toyo Shinyaku Co., Ltd.), which was made by extracting kudzu flowers (Pueraria thomsonii) with hot water and drying the extract. The chili pepper used was Fushimi Kancho lactic acid bacteria fermentation powder (manufactured by Toyo Shinyaku Co., Ltd.). As the pine bark, "Flavangenol (registered trademark)" (manufactured by Toyo Shinyaku Co., Ltd.), a hydrous ethanol extract powder of French maritime pine bark, was used. As the young barley leaves, we used barley leaf juice powder (manufactured by Toyo Shinyaku Co., Ltd.), which is made by squeezing the juice from young barley leaves and drying them.

[0036] (Preparation of test substances) Test substances for kudzu flower hot water extract powder, Fushimi Kancho lactic acid bacteria fermentation powder, and young barley leaf juice powder were prepared as follows. 1) The raw material was adjusted to 60 mg / mL with ultrapure water and stirred for 1 hour using a rotary mixer. 2) Centrifuge at 15,000 rpm for 5 minutes and use the supernatant. 3) Dilute 300-fold with dilution buffer (200 μg / mL).

[0037] A test substance was prepared as follows for the aqueous ethanol extract powder of French maritime pine bark. 1) The raw material was adjusted to 60 mg / mL with DMSO and stirred on a rotary mixer for 1 hour. 2) Centrifuge at 15,000 rpm for 5 minutes and use the supernatant. 3) Dilute 300-fold with KRPH buffer (200 μg / mL).

[0038] The KRPH buffer and dilution buffer used in the preparation of the above test substances are as follows. In the following procedures, the KRPH buffer and dilution buffer are the same.

[0039] <KRPH Buffer> Final concentration is 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 7H 2 O, 1.3 mM CaCl 2 The KRPH buffer was prepared by dissolving the solution in ultrapure water to a concentration of 118 mM NaCl, 5 mM KCl, and 30 mM Hepes (pH 7.5).

[0040] <Dilution Buffer> DMSO was diluted 300-fold with KRPH buffer to prepare a dilution buffer.

[0041] (Preparation of measurement samples) 1) 37°C, 5% CO 2 In the incubator, 75 cm 2 A mouse myoblast cell line (C2C12) was cultured in a flask in a normal medium. 2) The cells were suspended by trypsinization and then transferred to a 75 cm 2 The cells were seeded from the flask into each well of a 96-well plate at a cell density of 10,000 cells / well. 3) 37°C, 5% CO 2 The cells were precultured in an incubator until they became confluent. 4) Replace with differentiation induction medium and incubate at 37℃, 5% CO 2 The cells were cultured in an incubator and induced to differentiate into myotube cells. 5) Replace with 2% BSA-DMEM and store at 37°C, 5% CO overnight. 2 The cells were cultured in an incubator. 6) The plate was washed once with KRPH buffer, and 100 μL / well of KRPH buffer was added. 7) Add the test substance (100 μL / well) at twice the final concentration and incubate at 37°C, 5% CO 2 The cells were cultured in an incubator for 3 hours. As a control, a dilution buffer was added instead of the test substance.

[0042] 8) Wash three times with KRPH buffer that had been pre-warmed to 37°C. 9) Add 1 mM 2DG-KRPH and incubate at 37°C, 5% CO 2 The plate was incubated in an incubator for 30 minutes. 10) The cells were quickly washed three times with ice-cold PBS, and 0.1 N NaOH was added at 25 μL / well for cell disruption (lysis), followed by freezing in a -80°C freezer to further disrupt the cells. 11) After thawing, 25 μL / well of 0.1 N HCl was added (neutralized). 12) Heat at 85℃ for 45 minutes. 13) Cool to room temperature and add 25 μL / well of 3x buffer to prepare the measurement sample.

[0043] The normal medium, differentiation-inducing medium, and 3x buffer used in the preparation of the above measurement samples are as follows. In the following procedures, the normal medium, differentiation-inducing medium, and 3x buffer are the same.

[0044] <Normal medium> The normal medium was prepared by adding 1% p / s (penicillin / streptomycin) to DMEM to make 10% FBS.

[0045] <Differentiation Induction Medium> The differentiation induction medium was prepared by adding 1% p / s to DMEM, 2% horse serum (HS), and 0.1 μg / mL insulin.

[0046] 3 x Buffer Final concentration is 150mM 2,2',2''-Nitrilotriethanol (TEA) (pH8.1), 150mM KCl, 1.5mM MgCl 2 , 0.06% BSA, 0.36 μM NADP + ATP was adjusted to 8.0 mM with ultrapure water to prepare a 3× buffer.

[0047] (Glucose uptake measurement) The amount of glucose uptake was measured using a glucose uptake measurement kit (Cosmo Bio Co., Ltd.). 1) 25 μL of the calibration curve or measurement sample was transferred to a 96-well black plate. 2) A glucose uptake measurement solution was prepared and added at 50 μL / well. 3) After addition, the mixture was incubated at 37°C for 3 hours in the dark. 4) Fluorescence was measured (excitation wavelength: 540 nm / fluorescence wavelength: 590 nm). 5) A calibration curve was prepared and the concentration of incorporated 2DG (2-Deoxy-D-glucose) was calculated.

[0048] (Protein quantification) The protein amount was measured using a BCA protein measurement kit (Thermo Fisher Scientific). 1) 5 μL of the calibration curve or sample and 100 μL of the color reagent were added. 2) After incubation at 37°C for 1 hour, the wavelength was measured at 562 nm.

[0049] (Calculation of results) 1) 2DG concentration / protein amount was calculated and compared.

[0050] (result) The results are shown in Figure 1. As shown in Figure 1, it was revealed that the addition of kudzu flower hot water extract powder, Fushimi Kancho lactic acid bacteria fermentation powder, pine bark hydroethanol extract powder, and barley leaf juice powder increased the amount of sugar uptake by C2C12 cells induced to differentiate into myotubes compared to the control.

[0051] [Example 2] (raw materials) As the young barley leaves, we used barley leaf juice powder (manufactured by Toyo Shinyaku Co., Ltd.), which is made by squeezing the juice from young barley leaves and drying them.

[0052] (Preparation of test substances) 1) Barley leaf juice powder was adjusted to 60 mg / mL with ultrapure water and stirred in a rotary mixer for 1 hour. 2) Centrifuge at 15,000 rpm for 5 minutes and use the supernatant. 3) The following test substance solutions were prepared using the supernatant: Barley leaf juice powder 100 μg / mL, Barley leaf juice powder 100 μg / mL + Compound C 12.5 μM, Barley leaf juice powder 100 μg / mL + Wortmannin 10 μM. The solvent composition for each test substance was 0.1% DMSO / 2% BSA-KRPH buffer.

[0053] (Preparation of measurement samples) 1) 37°C, 5% CO 2 In the incubator, 75 cm 2 A mouse myoblast cell line (C2C12) was cultured in a flask in a normal medium. 2) The cells were suspended by trypsinization and then transferred to a 75 cm 2 The cells were seeded from the flask into each well of a 96-well plate at a cell density of 10,000 cells / well. 3) 37°C, 5% CO 2 The cells were precultured in an incubator until they became confluent. 4) Replace with differentiation induction medium and incubate at 37℃, 5% CO 2 The cells were cultured in an incubator and induced to differentiate into myotube cells. 5) Replace with 1 mM AICAR-2% BSA-DMEM and incubate overnight at 37°C in 5% CO 2 The cells were cultured in an incubator. 6) Wash three times with DMEM (100 μL / well). 7) Replace with 2% BSA-DMEM and incubate at 37°C, 5% CO 2 The cells were cultured in an incubator for 1 hour. 8) Wash three times with KRPH buffer. 9) Add the test substance (100 μL / well) and incubate at 37°C, 5% CO 2 The cells were cultured in an incubator for 3 hours. Note that a control (0.1% DMSO / 2% BSA-KRPH) was used without adding the test substance.

[0054] 10) Wash three times with KRPH buffer that had been pre-warmed to 37°C. 11) Add 1 mM 2DG-KRPH and incubate at 37°C, 5% CO 2 The plate was incubated in an incubator for 30 minutes. 12) The cells were quickly washed three times with ice-cold PBS, and 0.1 N NaOH was added at 25 μL / well to disrupt (dissolve) the cells. The cells were then frozen in a -80°C freezer to further disrupt the cells. 13) After thawing, 25 μL / well of 0.1 N HCl was added (neutralized). 14) Heat at 85℃ for 45 minutes. 15) Cool to room temperature and add 25 μL / well of 3x buffer to prepare the measurement sample.

[0055] (Glucose uptake measurement and protein quantification) As in Example 1, glucose uptake measurement and protein quantification were carried out.

[0056] (Calculation of results) 1) 2DG concentration / protein amount was calculated and compared.

[0057] (result) The results are shown in Figure 2. As shown in Figure 2, compared to the control, the group to which young barley leaf juice powder was added showed an increase in the amount of glucose uptake into C2C12 cells that had been induced to differentiate into myotube cells. Furthermore, compared to the group to which young barley leaf juice powder was added, the group to which young barley leaf juice powder and Compound C were added showed no decrease in the amount of glucose uptake, but the group to which young barley leaf juice powder and wortmannin were added showed a decrease in the amount of glucose uptake. There are two pathways for glucose uptake into muscle cells: the insulin-mediated pathway and the AMPK-mediated pathway. Glucose uptake was inhibited by the addition of wortmannin, an inhibitor of PI3K downstream of insulin signaling, but not by the addition of Compound C, an AMPK inhibitor, suggesting that the sugar uptake-promoting effect of young barley leaves is mediated by activation of insulin signaling.

[0058] [Example 3] (raw materials) As in Example 2, barley leaves were squeezed and dried to prepare barley leaf juice powder, which was used as a raw material. (Preparation of test substances) 1) The same treatment as in Example 2 was carried out, and the following test substance solutions were prepared using the supernatant: barley young leaf juice powder 100 μg / mL, barley young leaf juice powder 100 μg / mL + wortmannin 10 μM. The solvent composition for each test substance was 0.1% DMSO / 2% BSA-KRPH buffer.

[0059] (Preparation of measurement samples) 1) 37°C, 5% CO 2 In the incubator, 75 cm 2 A mouse myoblast cell line (C2C12) was cultured in a flask in a normal medium. 2) Collagen solution (manufactured by Toyobo Co., Ltd.) was added to a 24-well plate at 300 μL / well and coated for 1 hour. 3) After collecting the collagen solution, it was washed twice with PBS. 4) The cells were suspended by trypsinization and then transferred to a 75 cm 2 The cells were seeded from the flask into each well of a 24-well plate (3) at a cell density of 60,000 cells / well. 5) 37°C, 5% CO 2 The cells were precultured in an incubator until they became confluent. 6) Replace with differentiation induction medium and incubate at 37℃, 5% CO 2 The cells were cultured in an incubator and induced to differentiate into myotube cells.

[0060] 7) The differentiation medium was replaced with 1 mM AICAR-containing differentiation medium and incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 24 hours. 8) Wash three times with DMEM. 9) Replace with 2% BSA-DMEM and incubate at 37°C, 5% CO 2 The cells were cultured in an incubator for 3 hours. 10) Remove the medium and add the test substance (100 μL / well). Incubate at 37°C, 5% CO 2The cells were cultured in an incubator for 30 minutes. Note that the control was a medium containing no test substance (0.1% DMSO / 2% BSA-DMEM).

[0061] 11) Wash once with ice-cold PBS and add 50 μL of cell lysis solution. 12) To further disrupt the cells, they were frozen in a -80°C freezer. 13) The solution was quickly dissolved at room temperature and then placed on ice. 14) The cells were scraped off with a bent tip, transferred to a 1.5 mL tube, and frozen again in a -80°C freezer to further disrupt the cells. 15) The solution was quickly dissolved at room temperature and centrifuged at 15,000 rpm at 4°C for 10 minutes. 16) The supernatant was collected in a new 1.5 mL tube and used as the measurement sample.

[0062] The cell lysis solutions used in the preparation of the above test substances are as follows:

[0063] <Cell lysate> A solution prepared by diluting 100× protease inhibitor and 100× phosphatase inhibitor 100-fold with RIPA buffer (Thermo Fisher Scientific Co., Ltd.) was used as a cell lysate.

[0064] (Protein quantification) In the same manner as in Example 1, protein quantification in the measurement sample was carried out.

[0065] (Western Blotting) 1) The protein amount of the measurement sample was adjusted to 5 μg / well. 2) The prepared sample dilutions were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels. 3) After electrophoresis, the proteins were transferred to a PVDF membrane. 4) The expression levels of p-Akt or Akt were measured on the membrane blocked with 5% BSA-TBST using rabbit anti-p-Akt (Thr308) antibody or rabbit anti-Akt antibody (Cell Signaling Technology) as the primary antibody and HRP-anti-rabbit IgG antibody (Cell Signaling Technology) as the secondary antibody. Quantification was performed using a Chemi Doc XRS+ system (Bio-Rad). 5) The signal intensity was quantified, and p-Akt / Akt was calculated and compared with the control, which was set at 1.

[0066] (result) The results of p-Akt expression are shown in Figure 3. As shown in Figure 3, p-Akt expression increased in the group to which young barley leaf juice powder was added, and decreased with the addition of wortmannin. In other words, the addition of young barley leaf juice powder increased p-Akt expression, and this increase was reduced by the addition of wortmannin, suggesting that the sugar uptake-promoting effect of young barley leaves is due to activation of insulin signaling, and that the site of action is upstream of PI3K.

[0067] From the above results, it was found that the amount of sugar uptake into myoblasts was increased by using any of kudzu flowers, chili peppers, pine bark, and young barley leaves. In particular, it was suggested that the composition for promoting sugar uptake of the present invention containing young barley leaves can promote sugar uptake into muscle cells by activating insulin signaling. [Industrial Applicability]

[0068] The sugar uptake-promoting composition of the present invention can be used as so-called health foods, etc., and is therefore industrially useful.

Claims

1. A composition for promoting sugar uptake into muscles, comprising at least one plant material selected from young barley leaves and lactic acid fermentation products of chili peppers.

2. 2. The composition for promoting sugar uptake into muscles according to claim 1, which is for oral administration.

Images