Composition for improving endurance, and method for improving endurance using the same

Abstract

To provide a novel composition for improving endurance more efficiently than before.SOLUTION: A composition for improving endurance contains γ-aminobutyric acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to endurance-enhancing compositions and methods for improving endurance using the same. [Background technology]

[0002] Improving endurance is essential for building a body that is less susceptible to fatigue and for speeding up recovery from fatigue, not only in sports competitions that require long periods of exercise, but also in everyday activities such as housework and walking to work, and all physical activities such as leisure hobbies. Therefore, it is desirable to develop muscle capillaries and increase the amount of blood flowing through the muscles (muscle blood flow) through continuous daily exercise, thereby generating more energy and improving cardiopulmonary function, thereby enabling a long-term energy supply.

[0003] On the other hand, in modern society, it is widely known that regular exercise is desirable for improving endurance and maintaining health. However, the automation of housework and work, and the development of transportation have led to a decline in physical activity, and the percentage of people who actually exercise regularly is low. In addition to the decline in physical activity, changes in dietary habits have also contributed to the recent increase in lifestyle-related diseases. Furthermore, improving endurance through exercise does not produce sufficient results in the short term; repeated exercise is required over a long period of time. This risk of injury and difficulty in feeling the training effects associated with long-term exercise are also factors that prevent people from continuing to exercise.

[0004] Therefore, improving endurance efficiently through training is important not only for athletes who require endurance, but also for everyone, allowing many people to continue exercising without straining themselves. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made in view of the above circumstances, and has as its object to provide a novel composition for improving endurance more efficiently than conventional compositions. [Means for solving the problem]

[0006] As a result of extensive research to solve the above-mentioned problems, the inventors have confirmed that gamma-aminobutyric acid (GABA) enhances the endurance-improving effect, and by conducting further research based on this finding, have completed the present invention.

[0007] Therefore, according to a main aspect of the present disclosure, the following invention is provided. (A1) A composition for improving endurance containing γ-aminobutyric acid. (A2) The composition described in the above item, wherein the content of γ-aminobutyric acid is about 10 mg or more. (A3) The composition according to any one of the preceding items, which is formulated or packaged so as to contain about 10 to about 2000 mg of the γ-aminobutyric acid per day. (A4) A composition according to any one of the preceding items, which is taken orally. (A5) The composition according to any one of the above items for ingestion during training. (A6) The composition according to item (A5), which is ingested before training, during training, or after training. (A7) The composition according to item (A5) or (A6) for enhancing the effect of training on improving endurance. (A8) The composition according to any one of the preceding items, which is a composition for improving muscle endurance. (A9) A composition described in any one of the above items for increasing glycogen levels in skeletal muscles. (A10) A composition described in any one of the above items for promoting glycogen loading in skeletal muscle. (A11) A composition according to any one of the preceding items for reducing plasma insulin levels. (A12) The composition according to any one of the preceding items for inhibiting phosphorylation of protein kinase B (Akt) in skeletal muscle. (A13) The composition according to any one of the preceding items for increasing the expression level of PGC-1α protein in skeletal muscle. (A14) A composition according to any one of the preceding items for increasing the expression level of AMPK protein in skeletal muscle. (A15) A composition according to any one of the preceding items for increasing the expression level of myosin protein in skeletal muscle. (A16) A composition according to any one of the preceding items for increasing the expression level of myosin heavy chain (MHC) 2a. (A17) A composition according to any one of the above items for increasing slow muscle mass in skeletal muscles. (A18) A composition according to any one of the preceding items for enhancing type I muscle fibers in skeletal muscles. (A19) A composition according to any one of the preceding items for enhancing type IIa muscle fibers in skeletal muscles. (A20) The composition according to any one of the above items, which is a food or drink, a food additive, a quasi-drug, or a pharmaceutical.

[0008] According to another main aspect of the present disclosure, the following inventions are also provided. (B1) A non-therapeutic method for improving endurance, comprising the step of ingesting a composition described in any one of the preceding items. (B2) The method according to item (B1), wherein the content of γ-aminobutyric acid is about 10 mg or more. (B3) The method according to item (B1) or (B2), wherein the γ-aminobutyric acid is ingested in an amount of about 10 to about 2000 mg per day. (B4) The method according to any one of items (B1) to (B4), wherein the composition is taken orally. (B5) The method according to any one of items (B1) to (B5), wherein the composition is ingested during training. (B6) The method according to item (B5), wherein the composition is ingested before training begins, during training, or after training ends. (B7) The method according to item (B5) or (B6), wherein the composition is ingested to enhance the endurance-improving effect of training.

[0009] According to another main aspect of the present disclosure, the following inventions are also provided. (C1) A method for improving endurance, comprising the step of ingesting a composition described in any one of the preceding items. (C2) The method according to item (C1), wherein the content of γ-aminobutyric acid is about 10 mg or more. (C3) The method according to item (C1) or (C2), wherein the γ-aminobutyric acid is ingested at about 10 to about 2000 mg per day. (C4) The method according to any one of items (C1) to (C4), wherein the composition is taken orally. (C5) The method according to any one of items (C1) to (C5), wherein the composition is ingested during training. (C6) The method according to item (C5), wherein the composition is ingested before training begins, during training, or after training ends. (C7) The method according to item (C5) or (C6), wherein the composition is ingested to enhance the endurance-improving effect of training.

[0010] In addition, other features and significant actions and effects of the present disclosure will become apparent to those skilled in the art by referring to the following description of the preferred embodiments of the present invention and the drawings. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram illustrating a training and driving test schedule according to one embodiment of the present disclosure. [Figure 2] FIG. 2 is a graph showing measurement results of limit travel time according to one embodiment of the present disclosure. [Figure 3]FIG. 3 is a graph showing the measurement results of glycogen amount in skeletal muscle according to one embodiment of the present disclosure. [Figure 4] FIG. 4 is a graph showing the results of measuring the amount of insulin in plasma according to one embodiment of the present disclosure. [Figure 5] FIG. 5 is a graph showing the measurement results of the relative expression level of PGC-1α protein in skeletal muscle according to one embodiment of the present disclosure. [Figure 6] FIG. 6 is a graph showing the measurement results of the relative expression level of AMPK protein according to one embodiment of the present disclosure. [Figure 7] FIG. 7 is a graph showing the ratio of phosphorylated AKT / AKT in skeletal muscle according to one embodiment of the present disclosure. [Figure 8] FIG. 8 is a graph showing the results of measuring the amount of mitochondrial DNA in skeletal muscle according to one embodiment of the present disclosure. [Figure 9] FIG. 9 is a graph showing the measurement results of the relative expression level of myosin 2a protein in skeletal muscle (soleus muscle) according to one embodiment of the present disclosure. BEST MODE FOR CARRYING OUT THE INVENTION

[0012] Hereinafter, an embodiment and examples according to the present disclosure will be described with reference to the drawings. As described above, one embodiment of the present disclosure is an endurance-enhancing composition containing γ-aminobutyric acid.

[0013] In recent years, gamma-aminobutyric acid (GABA, 4-aminobutyric acid) has attracted attention as an amino acid widely distributed in nature that can be added to foods without impairing their flavor. GABA is an inhibitory neurotransmitter found in large quantities in the mammalian central nervous system. It is known to suppress the excessive secretion of excitatory neurotransmitters, calm nerve excitement, and exert relaxation and anti-stress effects. It is also known to have a wide range of physiological activities, including lowering blood pressure, lowering cholesterol, and suppressing immune system decline. Because GABA is found in vegetables, grains, and the human body, it is easy to add to foods, and GABA-containing chocolate and numerous supplements are available on the market.

[0014] Regarding the effects of GABA intake during training, it has been reported that taking GABA in addition to whey protein, which stimulates skeletal muscle hypertrophy, stimulates muscle hypertrophy and increases lean muscle mass more than whey protein alone (J Clin Med Res. 2019;11(6):428-434). On the other hand, it is also known that taking steroids with muscle hypertrophy effects does not improve endurance performance (e.g., Eur.J. Appl. Physiol. Nov 2006, Vol. 98, Issue 4, pp. 329-340). Muscle hypertrophy and improved endurance are phenomena with different properties and mechanisms.

[0015] GABA is an amino acid widely distributed in nature, such as in vegetables and grains. Therefore, in one embodiment of the present disclosure, the origin of GABA is not particularly limited as long as it can be used in foods and beverages. For example, GABA-containing plant extracts or purified products may be used, or GABA can be prepared from a fermented product obtained by adding glutamic acid decarboxylase or a microorganism containing the enzyme, such as lactic acid bacteria, to a glutamic acid-containing raw material. GABA-containing products and commercially available GABA can also be used as raw materials for the compositions of the present disclosure, as long as they do not impair the effects of the compositions of the present disclosure.

[0016] In one embodiment of the present disclosure, the GABA-containing endurance-enhancing composition can be taken during training. In this case, it can be taken before, after, or during training. In another embodiment of the present disclosure, the composition can be taken with meals or repeatedly taken daily. The training is not particularly limited as long as it applies a certain level of load over a certain period of time. Examples of training include whole-body endurance training such as interval training, circuit training, walking, jogging, running, LSD (long, slow, distance), swimming, aqua aerobics, cycling, stationary bike, aerobics, cross-country skiing, step exercise, and hypoxic training; strength training including bodyweight training, free weight training, tube training, machine training, slow training, and core training; explosive power training such as plyometric training and ballistics training; agility training such as sprint training and quickness training; balance training such as stabilization; and flexibility training such as stretching and PNF stretching. In other embodiments, training can include not only so-called exercise activities, including sports and aerobic and anaerobic exercise, but also muscle labor in daily life and physical activities that involve a certain level of load.

[0017] In one embodiment of the present disclosure, the composition of the present disclosure can be ingested during the above-described training to enhance the endurance-improving effect of training. The composition of the present disclosure is characterized by containing GABA, which improves endurance through the action of GABA and preferably further enhances the endurance-improving effect of training. Continuous training develops capillaries around muscle fibers. As capillaries develop, the amount of blood flowing into muscle fibers increases, and the amount of oxygen transported increases in proportion to the increase in blood volume, thereby increasing ATP production by mitochondria and improving endurance. In one embodiment of the present disclosure, the composition of the present disclosure can further enhance the normal endurance-improving effect of such training through the action of GABA.

[0018] In one embodiment of the present disclosure, the training intensity at which the endurance-improving effect of the composition of the present disclosure is enhanced may be low to moderate intensity or higher, such as walking or jogging, and may preferably be about 2 METs or higher, about 3 METs or higher, about 4 METs or higher, about 5 METs or higher, about 6 METs or higher, about 7 METs or higher, about 8 METs or higher, about 9 METs or higher, about 10 METs or higher, about 11 METs or higher, about 12 METs or higher, about 13 METs or higher, about 14 METs or higher, or about 15 METs or higher. METs (METs) is an index of training intensity expressed as the oxygen intake per kg of body weight per unit time. 1 MET is defined as the oxygen intake at rest (3.5 mL / kg / min), with 3 to 6 METs being moderate-intensity exercise and 7 METs or higher being high-intensity exercise.

[0019] For example, walking at 5.6 to 6.4 km / hour for exercise purposes is equivalent to 4.8 METs (National Institute of Health and Nutrition, Revised Edition, Physical Activity METs Table), and walking at this rate for one hour is equivalent to 4.8 METs. Therefore, in one embodiment of the present disclosure, the composition of the present disclosure can enhance the endurance-improving effect of such walking.

[0020] In one embodiment of the present disclosure, the training intensity at which the endurance-improving effect of the composition of the present disclosure is enhanced can be a total of about 5 METs or more, about 10 METs or more, about 15 METs or more, about 20 METs or more, about 25 METs or more, about 30 METs or more, about 35 METs or more, about 40 METs or more, about 45 METs or more, or about 50 METs or more per week. For example, training at 30 METs per week requires 6 METs of training (e.g., running at 6.4 km / h) (see Table 1) for 5 hours per week. This can be done for 5 days, or for 2.5 hours twice per week. Alternatively, a total of 30 METs can be achieved by running at 8.4 km / h for 1 hour (9 METs) (see Table 1) twice per week and running at 6.4 km / h for 1 hour (6 METs) (see Table 1) twice per week. In one embodiment of the present disclosure, the composition of the present disclosure can enhance the endurance-improving effect of training that results in a predetermined total number of METs or more.

[0021] Furthermore, in one embodiment of the present disclosure, the training intensity at which the endurance-improving effect of the composition of the present disclosure is enhanced may be such that the value obtained by (heart rate / maximum heart rate)×100 is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or about 100% or more. For example, if a person with a maximum heart rate of 190 attempts to train at 80% intensity, they should perform training so that their heart rate reaches 152 (=0.8×190). In other embodiments, the training intensity can also be calculated by {(heart rate−resting heart rate) / (maximum heart rate−resting heart rate)}×100. In one embodiment of the present disclosure, the composition of the present disclosure can enhance the endurance-improving effect of training at or above such a predetermined training intensity.

[0022] In one embodiment of the present disclosure, the training intensity at which the endurance-improving effect of the composition of the present disclosure is enhanced may be, in the case of strength training, a low load of about 12 to about 30 RM or more, a moderate load of about 4 to about 12 RM, or a high load of about 1 to about 3 RM. RM is an index of training intensity expressed as the maximum lifted weight, and a load that can be lifted only once is referred to as 1 RM. In one embodiment of the present disclosure, the composition of the present disclosure can enhance the endurance-improving effect of training at or above such a predetermined training intensity.

[0023] As used herein, "improved endurance" refers to extending the duration of a certain exercise or training, or improving performance within a certain period of time. For example, in one embodiment of the present disclosure, the endurance-improving effect of the composition of the present disclosure refers to extending the duration of a 6.4 km / hour run (6 METs) as described above, or enabling a person to run, for example, 8.4 km / hour (9 METs) within the same period of time, when compared before and after ingestion of the composition of the present disclosure.

[0024] In a preferred embodiment, the composition of the present disclosure can enhance training-induced improvements in endurance. That is, compared to improvements in endurance achieved by continuing a certain exercise or training for a certain period of time, the degree of improvement in endurance achieved by continuing a certain exercise or training for a certain period of time while ingesting the composition of the present disclosure can be greater. For example, compared to a case in which a person continues walking at 5.6 to 6.4 km / h (4.8 METs·h) and, as a result of the training effect, is able to run at 6.4 km / h (6 METs·h) in the same period of time, the composition of the present disclosure can enable a person to run at 8.4 km / h (9 METs·h).

[0025] Alternatively, in one embodiment of the present disclosure, the endurance-improving effect of the composition of the present disclosure can be, for example, an increase in the number of repetitions that can be lifted or an increase in the weight equivalent to 5RM when strength training with a load of 5RM is performed before and after taking the composition of the present disclosure. In another embodiment, for example, if the weight equivalent to 5RM is 50 kg, and the weight equivalent to 5RM increases to 60 kg as a result of continued 5RM strength training, the composition of the present disclosure can enable training with a weight equivalent to 5RM of 70 kg.

[0026] As used herein, "endurance" refers to the ability to sustain the above-mentioned exercise or training for a long period of time, and includes sustained muscle contraction activity or muscle anti-fatigue action. Endurance includes muscular endurance, which is the ability to sustain local muscle exercise for a long period of time, and whole-body endurance, which is the ability to sustain whole-body exercise for a long period of time.

[0027] As used herein, "muscle endurance" refers to the endurance of skeletal muscles, i.e., the ability of skeletal muscles to repeatedly contract over a long period of time, or the ability to sustain exercise using a portion of skeletal muscles for a long period of time. For example, improved muscle endurance enables one to continue running for a long period of time, or suppresses the decline in exercise performance even when performing repeated short, intermittent exercises. Furthermore, as used herein, "whole-body endurance" refers to the ability to sustain whole-body exercise using the muscles of the entire body, including cardiopulmonary functions such as the myocardium, for a long period of time.

[0028] In one embodiment of the present disclosure, the composition of the present disclosure may be used to increase glycogen levels in skeletal muscles. In another embodiment, the composition of the present disclosure may be used to promote glycogen loading in skeletal muscles. Among athletes and sportspeople, glycogen loading (carbohydrate loading), a method of storing carbohydrates, an energy source, in the muscles and liver before a game or competition, is known in the hope of improving endurance. The glycogen loading method utilizes the activation of glycogen synthase in the muscles and liver, which are depleted of energy sources, by reducing glycogen levels in the muscles and liver through prior training and then switching to a high-carbohydrate diet. Since an increase in glycogen levels in muscles is known to improve endurance, the composition of the present disclosure can improve endurance by increasing glycogen levels in skeletal muscles or promoting glycogen loading.

[0029] When an athlete's endurance is to be efficiently improved by an orally ingested substance, doping must be prevented at the same time. Therefore, it is desirable to efficiently improve endurance by ingesting nutrients that are commonly used in foods. From this perspective, the GABA of the present invention is preferable as an orally ingested supplement for improving endurance (especially endurance improvement through training).

[0030] Furthermore, when it comes to improving endurance in daily life, because people may be in poor physical condition or already taking medicines, it is desirable to efficiently improve endurance by ingesting nutrients that are widely distributed in nature and easy to use, rather than using unnatural chemically synthesized products. Furthermore, when supplementing with nutrients, especially during exercise or before and after exercise, those that leave an unpleasant aftertaste should be avoided. From this perspective, the GABA of the present invention is preferable as an oral supplement for improving endurance (especially for improving endurance through training).

[0031] In one embodiment of the present disclosure, the composition of the present disclosure can be used to reduce the amount of insulin in plasma.When the amount of insulin released into plasma by the composition of the present disclosure is reduced, insulin sensitivity is increased and insulin resistance is improved, thereby promoting the uptake of blood glucose into muscle cells and increasing the amount of glycogen.Therefore, the composition of the present disclosure can reduce the amount of insulin in plasma and increase the amount of glycogen in skeletal muscles, thereby improving endurance.

[0032] In one embodiment of the present disclosure, the composition of the present disclosure may be for inhibiting the phosphorylation of protein kinase B (AKT) in skeletal muscle. It is known that AKT is phosphorylated and activated when insulin is secreted. The composition of the present disclosure can improve endurance by reducing the amount of insulin in plasma and increasing the amount of glycogen in skeletal muscle, and therefore the composition of the present disclosure can also inhibit the phosphorylation of AKT.

[0033] In one embodiment of the present disclosure, the composition of the present disclosure may be used to increase the expression level of AMPK protein in skeletal muscle. AMPK (AMP-activated protein kinase) is known to play an important role in regulating energy metabolism. During repeated exercise, skeletal muscle contractions increase the intracellular AMP / ATP ratio, which is thought to reflect the intracellular energy, oxygen, or stress state. Sensing this, AMPK is phosphorylated and activated, promoting glucose and lipid metabolism. It is believed that exercise activates AMPK in skeletal muscle. AMPK activation promotes mitochondrial proliferation, increases glucose transporter 4 (GLUT4), increases uncoupling protein type 3 (UCP3), promotes PGC-1α phosphorylation, and deacetylates PGC-1α, thereby promoting glucose and lipid metabolism. The composition of the present disclosure promotes the phosphorylation or activation of AMPK in skeletal muscle, thereby promoting glucose and lipid metabolism, enabling efficient energy supply and improving endurance.

[0034] In one embodiment of the present disclosure, the composition of the present disclosure may be for increasing the expression level of PGC-1α protein in skeletal muscle. PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) regulates energy metabolism in vivo. PGC-1α is a transcriptional coactivator that regulates the production of ATP, and is known to translocate from the cytoplasm to the cell nucleus upon activation of AMPK, where it interacts with various transcription factors to promote mitochondrial biogenesis and increase the expression of the glucose transporter GLUT4. Expression of PGC-1α increases in skeletal muscle during exercise, and it is involved in the increased expression of genes related to mitochondrial biogenesis and energy metabolism. Based on this regulatory effect on energy production, PGC-1α is known to be involved in improving endurance. The composition of the present disclosure can improve endurance by increasing the expression of PGC-1α protein in skeletal muscle.

[0035] PGC-1α protein is known to be involved in the slow-twitching of skeletal muscles (Nature Aug 2002, 418, 797-801), and an increase in the expression level of PGC-1α protein enhances the mass of slow-twitch muscles. This can lead to increased energy production. Therefore, in one embodiment of the present disclosure, the composition of the present disclosure can be used to increase the mass of slow-twitch muscle in skeletal muscle. "Increasing slow-twitch muscle mass" refers to an increase in the cross-sectional area, number, or proportion of slow-twitch muscle fibers in skeletal muscle, or an increase in the number of capillaries in slow-twitch muscle fibers in skeletal muscle, resulting in the production of more energy. Slow-twitch muscle fibers contract at a slow rate and can continuously exert small forces for a long period of time. Therefore, the composition of the present disclosure can improve endurance by increasing the mass of slow-twitch muscle. Furthermore, as used herein, "enhancing muscle fibers" refers to an increase in the cross-sectional area, number, or proportion of type I muscle fibers or type IIa muscle fibers in skeletal muscle, or an increase in the number of capillaries in type I muscle fibers or type IIa muscle fibers in skeletal muscle, resulting in the production of more energy.

[0036] Muscle fibers are histochemically classified into slow-twitch and fast-twitch muscle fibers, and further divided into four types based on differences in myosin heavy chain (MHC), which is the most abundant muscle protein: slow-twitch type I muscle fibers (Type 1, (MHC1)) and fast-twitch type II muscle fibers (Type 2 (MHC2a, MHC2b, MHC2x)).In many skeletal muscles, these muscle fibers with different metabolic profiles exist in a mosaic pattern.

[0037] In one embodiment of the present disclosure, the composition of the present disclosure may be used to increase the expression level of myosin protein, preferably MHC2a, in skeletal muscle. Myosin is a myofibrillar protein present in skeletal muscle, and together with actin, accounts for approximately 50 to 60% of muscle protein. Myosin is a hexameric protein consisting of two myosin heavy chains (MHC) and four myosin light chain subunits. Because the region responsible for the ATPase activity of myosin is present in the MHC, the MHC is considered to be an important part of myosin's motor protein.

[0038] MHC1 is known to have slower muscle contraction speed and weaker isometric tension than MHC2, but its high mitochondrial content and high ATP synthesis capacity make it less susceptible to fatigue. On the other hand, MHC2 has faster muscle contraction speed and stronger isometric tension than MHC1, but lower ATP resynthesis capacity. The three MHC2 isoforms (MHC2a, MHC2x, and MHC2b) are known to have characteristics similar to slow-twitch muscle fibers, with the order 2a > 2x > 2b. 2a in particular is strongly associated with athletic performance. While the ratio of fiber types in the general human population is 2a:2b + 2x, or 2:1, elite athletes are dominated by 2a regardless of the type of sport they engage in, and it is known that 2b and 2x types are rarely present in the fibers of marathon runners and competitive swimmers in particular. From this, it is believed that increased expression of myosin proteins, particularly MHC2a, leads to the acquisition of endurance ability, and the composition of the present disclosure can improve endurance by increasing the expression level of myosin proteins, preferably myosin heavy chain (MHC) 2a, in skeletal muscles.

[0039] In one embodiment of the present disclosure, the composition of the present disclosure can be used to increase the amount of mitochondrial DNA in skeletal muscle. Increasing the amount of mitochondrial DNA or activating mitochondria is thought to have the same effect as training, improving endurance. For example, during aerobic exercise, ATP produced using oxygen via the TCA cycle and subsequent electron transport chain in mitochondria is supplied as an energy source, while during anaerobic exercise, ATP produced from anaerobic systems such as the ATP-CP system and the lactate system is mainly utilized. Therefore, the composition of the present disclosure can improve endurance by increasing the amount of mitochondrial DNA in skeletal muscle.

[0040] In one embodiment of the present disclosure, the content of GABA in the composition of the present disclosure is not particularly limited as long as the concentration or weight is such that the effects of the composition of the present disclosure can be obtained, depending on the administration method, etc. The content of GABA in the composition of the present disclosure is preferably about 10 to about 2000 mg, and the lower limit of the content may be, for example, about 10 mg or more, about 30 mg or more, about 50 mg or more, about 70 mg or more, about 100 mg or more, about 150 mg or more, about 200 mg or more, about 300 mg or more, about 400 mg or more, about 500 mg or more, or about 700 mg or more, and the upper limit of the content may be, for example, about 2000 mg or less, about 1800 mg or less, about 1500 mg or less, about 1300 mg or less, about 1000 mg or less, about 800 mg or less, about 700 mg or less, about 600 mg or less, or about 500 mg or less, and the content may be any numerical range between these lower and upper limits.

[0041] In one embodiment of the present disclosure, the composition of the present disclosure may contain, in addition to GABA, any additives or any components usable in ingestible compositions, depending on the form. Examples of these additives and components include, but are not limited to, vitamins including vitamin E and vitamin C, minerals, nutritional components, physiologically active ingredients such as flavorings, excipients added during formulation, binders, emulsifiers, tonicity agents (isotonicity agents), buffers, solubilizers, preservatives, stabilizers, antioxidants, colorants, coagulants, or coating agents.

[0042] In one embodiment of the present disclosure, the form of the composition of the present disclosure is not particularly limited as long as it contains GABA, and can be, for example, a pharmaceutical composition, a food or beverage (including functional foods such as foods for specified health uses, foods with nutrient functions, and foods with functional claims, health supplements, health foods, and supplements), a food additive, a quasi-drug, or a pharmaceutical.

[0043] In one embodiment of the present disclosure, the composition of the present disclosure can be taken orally, and when used as an oral preparation, its form can be, for example, a tablet (including coated tablets), capsule, powder, granule, powder, liquid, suspension, emulsion, granule, powder, pill, paste, cream, caplet, gel, chewable, stick, etc. Furthermore, it can be prepared into a form that is easy to incorporate, such as a powder or granule, and then used as a raw material for other pharmaceuticals.

[0044] In one embodiment of the present disclosure, the composition of the present disclosure can be used for non-therapeutic purposes, and ingesting the composition of the present disclosure can also be used as a non-therapeutic method for improving endurance. Such methods do not include therapeutic or medical procedures, and are not particularly limited to specific subjects. For example, athletes who train regularly and healthy individuals who do not usually exercise much can be provided with the endurance-improving effect by ingesting the composition of the present disclosure. Furthermore, when used as a pharmaceutical or quasi-drug, efficacy such as improved endurance can be promoted.

[0045] In one embodiment of the present disclosure, the daily intake amount of GABA contained in the composition of the present disclosure can be appropriately determined depending on the form of the composition, the method of intake, the purpose of use, and the age, weight, symptoms, etc. of the subject. For example, from the viewpoint of more significantly exerting the effects of the GABA contained in the composition of the present disclosure, it is preferable to ingest the GABA contained in the composition of the present disclosure so that the daily intake amount is about 10 mg / day or more, more preferably about 25 mg / day or more, about 50 mg / day or more, about 60 mg / day or more, about 80 mg / day or more, about 100 mg / day or more, about 120 mg / day or more, about 150 mg / day or more, about 180 mg / day or more, about 200 mg / day or more, about 300 mg / day or more, about 400 mg / day or more, about 500 mg / day or more, about 800 mg / day or more, about 1000 mg / day or more, about 1200 mg / day or more, about 1500 mg / day or more, about 1800 mg / day or more, or about 2000 mg / day or more. There is no particular upper limit to the daily intake amount of GABA contained in the composition of the present disclosure, as long as it is within a range that allows the composition of the present disclosure to exert its endurance-improving effect.

[0046] In one embodiment of the present disclosure, the frequency of application of the method of the present disclosure or the frequency of ingestion of the composition of the present disclosure may be once or multiple times a day within the desired intake range, and the ingestion period may also be appropriately set within a range in which the endurance-improving effect of the composition of the present disclosure can be exerted.

[0047] As used herein, "about" means ±10% of the preceding numerical value. [Example]

[0048] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to these examples.

[0049] The experimental methods and materials used in the present disclosure are described below. Note that although the following experimental methods are used in the present embodiment, similar results can be obtained by using other experimental methods. Example 1

[0050] Examination of the endurance-improving effect of GABA Repeated high-intensity training improves physical function. Therefore, the following test was conducted to confirm the effect of GABA intake on the improvement of endurance through training.

[0051] (Running test mice group) Mice used to measure limit running time on a treadmill were divided into four groups as shown in Table 1 below. [Table 1] Specifically, 10-week-old male ICR mice were classified into three groups (9 mice per group): a control group with free access to normal food and no training; a GABA group with free access to normal food supplemented with 0.5% GABA (1.8 g in human equivalent) and no training; a training group with free access to normal food and training; and a GABA training group with free access to normal food and training.

[0052] (Treadmill training) During the test period, mice classified as described above were given free access to either a normal diet or a GABA-containing diet at approximately 2.7 to 5.0 g / day per individual. The training and GABA training groups were forced to run for 60 minutes three times a week (every 1 or 2 days apart). This training continued until 3 days after the third running test, described below. For training, mice were forced to run on a small animal treadmill (Melquest) at a 0-degree gradient at a speed of 25 m / min. Since mice run at speeds of 6 to 24 m / min without load, a running speed of 25 m / min is slightly faster than normal, i.e., a low-to-moderate intensity.

[0053] (Measurement of maximum running time on a treadmill) Four groups of mice prepared as described above were used to measure their maximum running time on a small animal treadmill (Melquest). For the running test, mice were run on a 5-degree gradient treadmill, and the time until exhaustion and no longer running was measured. The running speed was initially 15 m / min for 5 min, then increased by 2 m / min every 2 min until reaching 30 m / min. After reaching 30 m / min, the running speed was fixed and the running test continued. Running tests were conducted three times, at the start of the test, two weeks later, and four weeks later, according to the schedule shown in Figure 1. Mice were considered unable to run if they collided with the rear wall of the treadmill five times in a row without running for more than three seconds. The running time until exhaustion at the start of the test (first test), two weeks later (second test), and four weeks later (third test) are shown in Figure 2.

[0054] In both the GABA training group and the non-training group, the running time until exhaustion in the running test increased with continued training, indicating that training improves endurance. The increase in running time due to training was significantly greater or tended to increase in the GABA training group compared to the non-training group (P=0.04 at 2 weeks and P=0.14 at 4 weeks). These results demonstrate that GABA enhances the endurance-improving effects of training. Example 2

[0055] (Measurement of glycogen content in skeletal muscle) Two days after the final training, the gastrocnemius muscles (skeletal muscles) of the mice in each group were collected and glycogen levels were measured using a Glycogen Colorimetric / Fluorometric Assay Kit (BioVision) as follows.

[0056] At least 10 mg of mouse gastrocnemius muscle was placed in a 1.5 mL plastic tube, and 20 volumes of distilled water were added and homogenized. The mixture was boiled in hot water for 10 minutes and then centrifuged at 18,000 g for 10 minutes. 50 μL of each sample was placed in a 96-well plate, and 2 μL of hydrolase mix was added and vortexed. The solution was left to stand at room temperature (20-25°C) for 30 minutes. A reaction mixture was prepared in the following proportions, and 50 μL of this reaction mixture was added to each well.

[0057] Reaction mixture (50 μL) Development buffer 46 μL Development enzyme mix 2 μL OxiRed probe 2 μL

[0058] The solution containing the reaction mixture was left standing at room temperature for 30 minutes in the dark. The absorbance was measured (OD 570 nm) and the amount of glycogen in the skeletal muscle was calculated.

[0059] The results of measuring glycogen levels are shown in Figure 3. Compared to the control group, glycogen levels in the gastrocnemius muscles of the GABA group tended to increase (P = 0.09). Furthermore, glycogen levels in the gastrocnemius muscles of the GABA training group were significantly higher than those of the training group (P = 0.04). These results demonstrate that GABA increases glycogen levels in skeletal muscle, and that even when training is performed, taking GABA in conjunction with training also increases glycogen levels in skeletal muscle. Example 3

[0060] (Measurement of plasma insulin levels) Two days after the final training, blood samples were taken from the mice in the group and plasma insulin levels were measured using Levis® Insulin-Mouse T (Fujifilm Wako Shibayagi Co., Ltd.) as follows.

[0061] An antibody-coated 96-well plate was prepared and washed four times with washing buffer. 100 μL of biotin-conjugated anti-insulin antibody was added and mixed. 10 μL of serum samples from each group of mice or standard insulin solution was added and mixed, and the mixture was allowed to stand at room temperature (20-25°C) for 2 hours. The 96-well plate was washed four times with washing buffer, and 100 μL of peroxidase-avidin conjugate was added and mixed. The mixture was allowed to stand at room temperature (20-25°C) for 30 minutes. 100 μL of tetramethylbenzidine solution was added, and the 96-well plate was washed four times with washing buffer. 100 μL of 1M sulfuric acid solution was added to each well to stop the reaction. The absorbance was measured (OD 450 nm) and the amount of insulin in the plasma was calculated.

[0062] The results of measuring insulin levels as described above are shown in Figure 4. Compared to the control group, insulin levels were reduced in the GABA group, training group, and GABA training group. Furthermore, when the training group was compared with the GABA training group, insulin levels were reduced in the GABA training group (P=0.07). These results indicate that GABA or training reduces plasma insulin levels, and that GABA in particular further enhances the insulin-reducing effect of training. Example 4

[0063] (Measurement of relative expression levels of PGC1α and AMPK proteins in skeletal muscle) [Western blotting] (Tissue sample preparation) Two days after the final training, gastrocnemius (skeletal muscle) was collected from each group of mice. Gastrocnemius muscle tissue fragments were placed in 1.5 mL plastic tubes and weighed. Three times the tissue fragment weight of RIPA Buffer (Nacalai Tesque) was added, homogenized, and centrifuged at 18,000 g × 20. Protein content was quantified using bovine serum albumin as a standard protein according to the protocol of the TaKaRa BCA Protein Assay Kit (Takara Bio Inc.). Protein concentration was adjusted to 50 μg / mL with RIPA Buffer. 2x4X SDS sample buffer (150 mM Tris-HCl, pH 7.0, 12% sodium dodecyl sulfate, 25% glycerol, 0.02% bromophenol blue, 5% 2-mercaptoethanol) was added to one-quarter the volume of the extraction solution and heated at 98°C for 3 minutes to prepare the sample for electrophoresis.

[0064] (SDS-polyacrylamide gel electrophoresis (SDS-PAGE)) SDS-PAGE was performed according to the method of Laemmli. The separating gel was 5-20% and the stacking gel was 5%. The electrophoresis buffer was 25 mM Tris, 192 mM glycine, and 0.1% SDS. Molecular weight markers were Precision Plus Protein I. TMA two-color standard (Biorad) was used. A current of 20 mA was applied per wide gel during electrophoresis.

[0065] (Western blotting) After SDS-PAGE, the membrane was infiltrated with 20% ethanol for 5 minutes by shaking. Protein transfer was performed using the iBlot dry blotting system (Thermo Fisher). After transfer, the PVDF membrane was blocked for 1 hour with PBS-T (Fujifilm Wako Pure Chemical Industries) containing 0.1% BSA. Primary antibodies (rabbit anti-PGC1α antibody (Novus Biologicals), rabbit anti-AMPK (Cell Signaling Technology)) diluted in PBS-T were used. The antigen-antibody reaction was carried out overnight in a 4°C incubator. The membrane was then washed four times with PBS-T for 5 minutes each. HRP-conjugated secondary antibodies that bind to each primary antibody were diluted in PBS-T and incubated for 1 hour. The PVDF membrane was then washed four times with PBS-T for 5 minutes each, and developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare). Detection was performed using a WSE-6100 LuminoGraph I (ATTO). The PVDF membrane was stripped with Western blot stripping buffer and diluted with the primary antibody (GAPDH, monoclonal antibody, peroxidase-conjugated). The antigen-antibody reaction was carried out for 2 hours. The PVDF membrane was then washed three times with TBS(-) for 3 minutes each, developed using ECL Prime Western Blotting Detection Reagent, and detected using a WSE-6100 LuminoGraph I. Image analysis was performed using ImageJ (Nat Methods Jul 2012 9, 671-675) to measure the relative expression levels of GAPDH and target proteins.

[0066] [result] The results of measuring the relative expression levels of PGC-1α protein and the relative expression levels of AMPK protein are shown in Figure 5 and Figure 6, respectively. Compared with the control group, PGC-1α protein expression in skeletal muscle in the GABA group tended to increase. Furthermore, PGC-1α protein expression in skeletal muscle in the GABA training group was significantly higher than in the training group (P = 0.01). These results demonstrate that GABA increases PGC-1α protein expression in skeletal muscle. Furthermore, even in the presence of training, GABA supplementation increased PGC-1α protein expression in skeletal muscle. These results also suggest that GABA supplementation promotes slow-twitch muscle contraction.

[0067] Furthermore, compared to the control group, AMPK protein expression levels were increased in the training group and the GABA training group (P=0.008 and P=0.0008, respectively). Furthermore, AMPK protein expression was higher in the GABA training group than in the training group (P=0.02). These results indicate that GABA intake in conjunction with training further enhances the training-induced increase in AMPK protein expression. Example 5

[0068] (Skeletal muscle phosphorylated AKT / AKT ratio) Three days after the third running test, gastrocnemius muscles (skeletal muscles) were collected from mice in each group, and the expression levels of phosphorylated AKT and AKT in the skeletal muscles were measured in the same membrane using a method similar to the Western blot method described above, and the expression ratio between phosphorylated AKT and AKT was calculated.

[0069] The results of measuring the levels of phosphorylated AKT and AKT insulin as described above are shown in Figure 7. Compared to the control group, the phosphorylated AKT / total AKT ratio in skeletal muscle of the GABA group tended to decrease. Furthermore, the phosphorylated AKT / total AKT ratio in skeletal muscle of the GABA training group was significantly lower than that of the training group (P = 0.0001). These results demonstrate that GABA reduces the amount of phosphorylated AKT in skeletal muscle, and that even when training is performed, taking GABA in conjunction with training also reduces the amount of phosphorylated AKT in skeletal muscle. Example 6

[0070] (Measurement of mitochondrial DNA content) 20–50 mg of tissue sample was placed in a 2 mL plastic tube and purified using NucleoSpin® DNA RapidLyse (Takara Bio Inc.) according to the genomic DNA purification protocol. The relative expression level of mitochondria to mouse nuclear DNA was quantified by real-time PCR using a Thermal Cycler Dice® Real Time System II (Takara Bio Inc.), and the mitochondrial DNA content was determined.

[0071] The results of measuring the amount of mitochondrial DNA in skeletal muscle as described above are shown in Figure 8. Compared to the control group, GABA group, and training group, the amount of mitochondrial DNA in skeletal muscle of the GABA training group tended to be higher. These results demonstrate that taking GABA in conjunction with training increases the amount of mitochondrial DNA in skeletal muscle. Example 7

[0072] (Measurement of relative expression level of myosin 2a protein in skeletal muscle (soleus muscle)) [Myosin Western blotting] (SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for myosin measurement) Two days after the final training, mouse skeletal muscle (soleus muscle) tissue was mashed on ice using a biomasher. The mashed soleus muscle tissue was dissolved in 1 ml of RIPA buffer to prepare a protein extract. The protein concentration of the protein extract was measured using a protein assay BCA kit, and the protein extract was diluted with RIPA buffer to a protein concentration of 4 mg / ml. The protein extract was then diluted with 4x sample buffer and RIPA buffer to a final concentration of 1 mg / ml and a final concentration of 5% mercaptoethanol. This diluted solution was heated in a block heater at 60°C for 10 minutes to prepare the measurement sample.

[0073] Next, 5 mL of the electrophoresis gel prepared as shown in the table below was placed on two overlapping glass plates (10 cm x 12 cm). [Table 2] Approximately 1 ml of MilliQ water was added and the gel was left to solidify.

[0074] Subsequently, a stacking gel was prepared as shown in the table below. [Table 4] After discarding the water and wiping off the moisture with a Kimwipe, 2 mL of concentrated gel was added, a comb was inserted, and the gel was left to harden.

[0075] Next, the electrophoresis buffer was prepared as shown in the table below. [Table 3] The electrophoresis buffer 1 was placed in the anode side of the electrophoresis tank, the gel was set, and bubbles at the bottom of the gel were removed. The electrophoresis buffer 2 was placed in the cathode side of the electrophoresis tank, and the comb was removed. The glycerol in the sample application wells was removed by pipetting, and 2 μL of a molecular weight marker (Biorad) and 3 μL of the sample were applied to the wells. Electrophoresis was performed at a constant voltage of 70 V for 24 hours at 4°C.

[0076] During electrophoresis, a blocking solution (1% BSA / PBS) was prepared, the gel was cut out, and rocked in 20% EtOH for 5 minutes. Using iBlot2, the gel was set according to the iBlot protocol and transferred to a PVDF membrane. The standard protocol was P0.

[0077] After transfer, the membrane was rocked in a blocking solution (22°C (room temperature) for 1 hour), and then rocked in PBS / 0.05% Tween 20 (PBS-T) for 5 minutes. The blocking solution was diluted 10-fold with PBS-T to prepare a primary antibody dilution, and the primary antibody (anti-mouse myosin IIa antibody (RandD Systems, Inc.) was diluted (see the antibody data sheet for the dilution). The membrane was placed in a nylon bag, heat-sealed in three places, and filled with approximately 8 mL of the primary antibody dilution. Bubbles were removed from the nylon bag, and the fourth place was sealed. The primary antibody reaction was carried out at 22°C (room temperature) for 1 hour or at 4°C overnight. The membrane was then removed from the bag, placed in an appropriate container, and washed four times with PBS-T for 5 minutes. Next, for the secondary antibody reaction, a secondary antibody dilution (approximately 1 / 3000) was prepared in PBS-T and reacted at 22°C (room temperature) for 1 hour. After the reaction, the membrane was washed five times with PBS-T for 5 minutes, and the color-developing substrate was allowed to return to room temperature. After returning to room temperature, the substrate solution was prepared.

[0078] The membrane was placed on a plastic wrap, excess PBS-T was removed with Kimwipes, and substrate solution was applied (approximately 1 mL). Excess substrate solution was removed with Kimwipes, and the membrane was sandwiched between nylon sheets and heat-sealed, and then placed in the detection device.

[0079] Figure 9 shows the results of measuring the relative expression level of myosin 2a protein in skeletal muscle (soleus muscle) using the method described above. Compared to the control group and the training group, the GABA group showed a tendency for an increased expression level of myosin 2a protein. In addition, the GABA training group showed a significantly increased expression level of myosin 2a protein compared to the other three groups, demonstrating a synergistic effect of GABA intake and training. These results demonstrate that GABA intake in conjunction with training increases the relative expression level of myosin 2a protein in skeletal muscle (soleus muscle).

[0080] Although the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure should be interpreted solely by the claims. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2020-28755, filed on February 21, 2020, with the Japan Patent Office, the contents of which are incorporated by reference in their entirety as if they constitute the contents of this application.

Claims

[Claim 1] The invention described in this specification.

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