Oral composition

Abstract

To provide an oral composition containing amino acids that exhibits excellent muscle-building effects. [Solution] An oral composition containing leucine and threonine, wherein the molar composition ratio of threonine to leucine is 0.05 or less.

Description

Technical Field

[0001] The present invention relates to an oral composition containing leucine and threonine. Background Art

[0002] In recent years, various health foods have been developed against the background of increasing public interest in health, for example, health foods containing amino acids have been developed.

[0003] As health foods containing such amino acids, for example, health foods containing amino acids such as alanine, arginine, and leucine, which are used for enhancing learning and memory ability, have been proposed (see, for example, Patent Document 1). Prior Art Documents Patent Documents

[0004] Patent Document 1 Japanese Unexamined Patent Application Publication No. 2013-184956 Summary of the Invention Problems to be Solved by the Invention

[0005] An object of the present invention is to provide an oral composition containing amino acids and exhibiting an excellent muscle strengthening effect. Means for Solving the Problems

[0006] As a result of intensive studies conducted by the present inventors to solve the above problems, they have found that an oral composition containing leucine and threonine in a specific ratio exhibits an excellent muscle strengthening effect, and thus completed the present invention.

[0007] That is, the present invention includes the following aspects. [1] An oral composition containing leucine and threonine, wherein the molar composition ratio of said threonine to said leucine is 0.05 or less. [2] The oral composition according to [1] above, wherein the molar composition ratio of threonine to leucine is 0.02 or more and 0.04 or less. [3] The oral composition according to [1] or [2] above, further comprising phenylalanine. [4] The oral composition according to [3] above, wherein the molar composition ratio of phenylalanine to threonine is 0.5 or more and 2.0 or less.

[0008] [5] The oral composition according to [4], wherein the molar composition ratio of phenylalanine to threonine is 0.7 or more and 1.8 or less. [6] The oral composition according to any one of [1] to [5] above, wherein the molar composition ratio of the leucine content to the total content of essential amino acids is 0.7 or more. [7] The oral composition according to any one of [1] to [6] above, further comprising lysine, valine and isoleucine. [8] The oral composition according to any one of [1] to [7] above, further comprising methionine, histidine, and tryptophan. [9] An oral composition according to any of the above [1] to [8], for muscle building. [Effects of the Invention]

[0009] The oral composition of the present invention exhibits excellent muscle-building effects. [Modes for carrying out the invention]

[0010] The present invention relates to an oral composition containing leucine and threonine, characterized in that the molar composition ratio of threonine to leucine is 0.05 or less.

[0011] The oral composition of the present invention contains leucine and threonine in specific proportions, thereby promoting protein synthesis in the body and exhibiting excellent muscle-building effects. For example, a small amount can provide muscle-building effects comparable to those of conventional whey protein. The oral composition of the present invention can be used for muscle building in athletes and healthy individuals, muscle recovery after illness, and muscle maintenance in the elderly. Furthermore, when the oral composition of the present invention contains leucine and threonine in specific proportions, the flavor is improved, making it easier to consume without resistance.

[0012] The following describes each component contained in the oral composition of the present invention. [Leucine] Leucine is a type of amino acid, a branched-chain amino acid, and one of the essential amino acids in humans, and is abbreviated as Leu or L. Leucine exists in isomers such as L-form, D-form, and DL-form, and any of them can be used in the oral composition of the present invention. The leucine of the present invention refers to leucine that exists as a free amino acid, and does not refer to leucine that exists as a component of proteins. Free amino acids (amino acids that exist individually and independently away from proteins) have different properties as compounds from amino acids that exist as components of proteins, and the two are clearly distinguished. Note that the amino acids such as threonine, which will be described later, also refer to free amino acids.

[0013] The leucine content relative to the total essential amino acid content in the oral composition of the present invention (the molar composition ratio of leucine content when the total essential amino acid content is set to 1) is not particularly limited, but from the viewpoint of more significantly exhibiting the effects of the present invention and providing an oral composition with excellent taste, it is preferably 0.7 or more, more preferably 0.7 to 0.99 (essential amino acid:leucine = 1:0.7 to 0.99), even more preferably 0.72 to 0.9, and particularly preferably 0.75 to 0.85. In the oral composition of the present invention, the effects of the present invention are more effectively exhibited when the proportion of leucine is high, but from the viewpoint of taste, it is better not to have too much leucine, so the above range is preferred.

[0014] [Threonine] Threonine is an amino acid that has a hydroxyethyl group in its side chain, and is also called threonine. It is one of the essential amino acids in humans and is abbreviated as Thr or T. Because threonine has two optically active sites, there are four isomers: L-threonine, D-threonine, L-alrosreonine, and D-alrosreonine. Any of these can be used in the oral composition of the present invention.

[0015] As described above, the molar ratio of leucine to threonine in the oral composition of the present invention is 0.05 or less in terms of the molar composition ratio of threonine to leucine. However, from the viewpoint of enjoying the effects of the present invention, it is preferable that it be 0.005 or more and 0.05 or less (leucine:threonine = 1:0.005~0.05), more preferably 0.01 or more and 0.045 or less, even more preferably 0.02 or more and 0.025 or more and 0.04 or less.

[0016] Leucine and threonine of the present invention can be used not only in free form but also in the form of salts. Examples of the salt forms include acid addition salts and salts with bases, and it is preferable to select a pharmacologically acceptable salt. Specific examples of the salts include salts with inorganic bases, organic bases, inorganic acids, and organic acids. In the present invention, from the viewpoint that the effects of the present invention can be more fully obtained, free form or organic acid salts are preferable, free form or hydrochloride is more preferable, and free form is particularly preferable. Commercially available products that are generally available as food raw materials can be used as the leucine and threonine of the present invention.

[0017] <Other amino acids> The oral composition of the present invention preferably further contains other essential amino acids other than leucine and threonine. This allows intake of essential amino acids in a well-balanced manner, and enables more effective muscle enhancement. Specific examples of the essential amino acids include phenylalanine, lysine, valine, isoleucine, methionine, histidine, and tryptophan.

[0018] Preferable examples of essential amino acids used together with leucine and threonine in the oral composition of the present invention include phenylalanine alone, a combination of lysine, valine and isoleucine, and a combination of methionine, histidine and tryptophan, and it is more preferable to use these in combination. Further, it is most preferable that the composition contains all essential amino acids.

[0019] [Phenylalanine] Phenylalanine is a type of amino acid, which is an aromatic amino acid having a benzyl group in its side chain. It is one of the essential amino acids in humans, and is represented by the abbreviation Phe or F. Phenylalanine has isomers such as L-form, D-form and DL-form, and any of these can be used in the oral composition of the present invention.

[0020] When the oral composition containing leucine and threonine of the present invention contains phenylalanine, the mixing ratio (molar composition ratio) of phenylalanine to threonine is preferably 0.5 or more and 2.0 or less, more preferably 0.7 or more and 1.8 or less, and still more preferably 0.8 or more and 1.5 or less, in terms of the molar composition ratio of phenylalanine to threonine, from the viewpoint that the effects of the present invention can be more fully obtained.

[0021] [Lysine] Lysine is a type of amino acid and is a basic amino acid. It is one of the essential amino acids for humans, is also called lysine, and is represented by Lys or K as an abbreviation. Lysine has isomers such as L-form, D-form and DL-form, any of which can be used in the oral composition of the present invention.

[0022] As for the content of lysine contained in the oral composition of the present invention, from the viewpoint that the effects of the present invention can be more fully obtained, the molar composition ratio relative to the total content of essential amino acids is preferably 0.02 or more, more preferably 0.02 or more and 0.08 or less, and still more preferably 0.03 or more and 0.08 or less.

[0023] [Valine] Valine is a type of amino acid and is a branched-chain amino acid. It is one of the essential amino acids for humans, is also called 2-aminoisovaleric acid, and is represented by Val or V as an abbreviation. Valine has isomers such as L-form, D-form and DL-form, any of which can be used in the oral composition of the present invention.

[0024] As for the content of valine contained in the oral composition of the present invention, from the viewpoint that the effects of the present invention can be more fully obtained, the molar composition ratio relative to the total content of essential amino acids is preferably 0.02 or more, more preferably 0.02 or more and 0.08 or less, and still more preferably 0.03 or more and 0.08 or less.

[0025] [Isoleucine] Isoleucine is a branched-chain amino acid. It is one of the essential amino acids in humans and is also called 2-amino-3-methylpentanoic acid (2-amino-3-methylvaleric acid), and is abbreviated as Ile or I. Isoleucine exists in isomers such as L-, D-, and DL-, and any of them can be used in the oral composition of the present invention.

[0026] The isoleucine content in the oral composition of the present invention is preferably 0.02 or more in molar composition ratio to the total content of essential amino acids, more preferably 0.02 or more and 0.06 or less, and even more preferably 0.03 or more and 0.06 or less, from the viewpoint of enjoying the effects of the present invention.

[0027] Furthermore, when the oral composition of the present invention contains lysine, valine, and isoleucine, the total molar composition ratio of the lysine, valine, and isoleucine content to the total essential amino acid content is preferably 0.08 or higher, more preferably 0.08 to 0.22, and even more preferably 0.11 to 0.22, in order to better enjoy the effects of the present invention.

[0028] [Methionine] Methionine is a type of amino acid, a hydrophobic amino acid containing sulfur in its side chain. It is one of the essential amino acids in humans and is abbreviated as Met or M. Methionine exists in isomers such as L-, D-, and DL-, and any of these can be used in the oral composition of the present invention.

[0029] Regarding the methionine content in the oral composition of the present invention, from the viewpoint of enjoying the effects of the present invention, it is preferable that the molar composition ratio of the total essential amino acid content be 0.01 or more, more preferably 0.01 to 0.04, and even more preferably 0.015 to 0.04.

[0030] [Histidine] Histidine is an aromatic amino acid with a heteroaromatic ring in its side chain. It is one of the essential amino acids in humans and is abbreviated as His or H. Histidine exists in isomers such as L-, D-, and DL-, and any of these can be used in the oral composition of the present invention.

[0031] Regarding the histidine content in the oral composition of the present invention, from the viewpoint of enjoying the effects of the present invention, it is preferable that the molar composition ratio of the total essential amino acid content be 0.008 or more, more preferably 0.008 or more and 0.03 or less, and even more preferably 0.01 or more and 0.03 or less.

[0032] [Tryptophan] Tryptophan is an aromatic amino acid with an indole ring in its side chain. It is one of the essential amino acids in humans, and its systematic name is 2-amino-3-(indolyl)propionic acid, abbreviated as Trp or W. Tryptophan exists in isomers such as L-, D-, and DL-, and any of these can be used in the oral composition of the present invention.

[0033] Regarding the tryptophan content in the oral composition of the present invention, from the viewpoint of enjoying the effects of the present invention, it is preferable that the molar composition ratio of the tryptophan content to the total content of essential amino acids be 0.002 or more, more preferably 0.002 or more and still preferable 0.003 or more and 0.02 or less.

[0034] The other essential amino acids of the present invention, like leucine and threonine, can be used not only in free form but also in salt form, and commercially available products that are commonly available as food ingredients can be used.

[0035] The oral composition of the present invention may contain other commonly used substances in addition to the above-mentioned leucine, threonine, and other essential amino acids. Examples of other substances include various excipients, binders, glazing agents, lubricants, stabilizers, diluents, fillers, thickeners, emulsifiers, antioxidants, pH adjusters, colorants, flavorings, and additives.

[0036] There are no particular restrictions on the amount of oral composition of the present invention to be taken, but in order to better enjoy the effects of the present invention, the daily intake of leucine may be 200 mg / day or more, preferably 1000 mg / day or more, and more preferably 1500 mg / day or more. There are no particular restrictions on the upper limit of intake, but in order to better enjoy the effects of the present invention, the daily intake of leucine may be 15000 mg / day or less, preferably 5000 mg / day or less, and more preferably 2500 mg / day or less.

[0037] The oral composition of the present invention is not particularly limited as long as it is in a form for oral intake, and may be any of the following: pharmaceuticals (including quasi-drugs), so-called health foods, foods for specified health uses, or foods with functional claims.

[0038] Examples of the oral composition of the present invention include tablets, capsules, powders, granules, liquids, rods, plates, blocks, solids, rounds, pastes, creams, caplets, gels, chewables, sticks, and the like, with tablets, capsules, powders, granules, and liquids being preferred.

[0039] The oral composition of the present invention can be used as an oral composition for muscle building. Such an oral composition for muscle building is not particularly limited as long as it contains leucine and threonine and can be distinguished from other products in that it is used for muscle building. For example, any product that displays that it has a muscle-building function on the product itself, packaging, instructions, or promotional materials is included within the scope of the present invention. Furthermore, in the case of foods with functional claims, products that use muscle building as the scientific basis for their functionality are also included.

[0040] Specifically, examples include products that claim to have functions such as suppressing, maintaining, or increasing the decline of muscle mass (muscle weight, muscle cross-sectional area, muscle density), suppressing, maintaining, or improving the decline of muscle quality, and suppressing, maintaining, or increasing the decline of muscle strength. More specifically, examples include products that claim to "enhance muscle strength," "support the ability to build muscle," "suppress muscle breakdown," "support muscle training," "support body building," "muscular," "lean and muscular," and "maintain muscle mass and strength."

[0041] Furthermore, the oral muscle-enhancing composition of the present invention is not limited to products in which the combination of components (leucine and threonine) of the present invention is indicated as the active ingredient for muscle enhancement on the product packaging, etc. For example, it may be a product in which the active ingredient is not specified. Also, even general food products that are manufactured and sold with a suggested function or use are included in the scope of the oral muscle-enhancing composition of the present invention. For example, this includes products that are sold with testimonials from people who have consumed them, mentioning the function or use as personal impressions, posted on websites, etc.

[0042] The oral composition of the present invention is not particularly limited to individuals who require muscle strengthening, but is preferably used by people who aim to lose weight, athletes, and elderly people with weakened legs and hips. [Examples]

[0043] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0044] <Test 1: Evaluation of cell-activating activity> The cell-activating activity of the mouse myoblast cell line (C2C12) was confirmed using the oral composition of the present invention. C2C12 is widely used as a model for evaluating effects such as muscle mass increase, and by confirming its cell-activating activity, the muscle-building effect can be confirmed.

[0045] [Test substance] The following substances were used as test materials. • Leucine: Reagents of L-leucine • Lysine: Reagent for L-lysine monohydrochloride • Valine: L-valine reagent • Isoleucine: Reagent of L-isoleucine • Threonine: L-threonine reagent • Phenylalanine: L-phenylalanine reagent • Methionine: L-methionine reagent • Histidine: L-histidine reagent • Tryptophan: L-tryptophan reagent

[0046] (Preparation of test substance) Each test substance was suspended in 10% FBS-amino acid-free DMEM medium to a concentration of 20 mM. The suspension was sterilized by passing the suspension through a 0.2 μm filter, and then diluted to a concentration of 10 μM in the same medium to obtain the test substance preparation solution. The test substance-containing media were prepared by mixing each test substance preparation solution to achieve the molar composition ratios shown in Tables 1-3. For the "other essential amino acids," a mixture of lysine, valine, isoleucine, methionine, histidine, and tryptophan was used.

[0047] The specific experimental procedure is as follows: 75cm in a 37℃, 5V %CO2 incubator 2C2C12 cells were cultured in a flask using 10% FBS-DMEM. Then, cells suspended by trypsin were placed in each well of a collagen-coated 96-well plate in groups of 8 × 10⁶. 3 Cells were seeded at a cell density of cells / well and pre-cultured for 24 hours in a 37°C, 5V %CO2 incubator.

[0048] After removing the culture medium from each well, wash once with PBS, add 100 μL / well of culture medium containing the test substance, and incubate for 3 hours in a 37°C, 5V %CO2 incubator. 10% FBS-amino acid-free DMEM was used as the culture medium, and the total molar concentration of the test substance in the medium was set to 10 μM. In addition, a 10% FBS-amino acid-free DMEM medium, which did not contain the test substance, was added as a control. This was designated as Comparative Example 1.

[0049] After 3 hours of incubation, the culture medium was removed, and 150 μL of CellCounting Kit-8 (Dojin Chemical Co., Ltd.), diluted 20-fold with amino acid-free DMEM, was added to each well. After standing in a 37°C, 5V %CO2 incubator to allow for appropriate color development, the absorbance of the formazan product at 450 nm, which is proportional to the number of viable cells in each well, was measured.

[0050] The chromogenic reagent WST-8 in CellCounting Kit-8 is reduced to formazan by the action of dehydrogenase via electron transport molecules. The more living cells there are, the higher the dehydrogenase activity and the greater the formazan production. Since formazan has a maximum absorption around 450 nm, its production was measured based on this absorbance to confirm C2C12 proliferation. A higher absorbance based on formazan indicates that C2C12 has proliferated, and therefore this absorbance serves as an indicator of myoblast activation activity. Additionally, as a blank, the absorbance of CellCounting Kit-8 diluted 20-fold with amino acid-free DMEM was measured.

[0051] The difference between the absorbance of each test substance well and the absorbance of the blank was calculated as the cell-activating activity in each test substance well. The results of evaluating this activity relative to the cell-activating activity of the control well (Comparative Example 1) are shown in Tables 1-3, as shown by the following formula.

[0052] Cell activation activity = (Absorbance of each test substance well - Absorbance of the blank well) × 100 / (Absorbance of the control well - Absorbance of the blank well)

[0053] [Table 1]

[0054] [Table 2]

[0055] [Table 3]

[0056] As shown in Table 1, the compositions of Examples 1 to 3 of the present invention, in which threonine was blended in a specific ratio to leucine, exhibited cell-activating activity, as is evident from the comparison with the control (Comparative Example 1) which did not contain leucine or threonine. In particular, higher cell-activating activity was observed in the composition of Example 2, in which the ratio of threonine to leucine was approximately 0.03. Furthermore, the compositions of Examples 1 to 3 of the present invention showed improved cell-activating activity compared to the compositions of Comparative Examples 2 and 3, which contained leucine and threonine individually. That is, by blending leucine and threonine (Comparative Examples 2 and 3), which do not show cell-activating activity individually, in specific ratios, cell-activating activity was improved. In addition, the compositions of Examples 1 to 3 of the present invention showed higher cell-activating activity compared to the compositions of Comparative Examples 4 and 5, which had a higher ratio of threonine to leucine.

[0057] Furthermore, as shown in Table 2, compositions of Examples 4-6, which contained threonine in a specific ratio to leucine, along with other amino acids, exhibited cell-activating activity, as is evident from a comparison with the control (Comparative Example 1) which did not contain leucine or threonine. In particular, compositions of Examples 5 and 6 showed high cell-activating activity.

[0058] As shown in Tables 2 and 3, high cell-activating activity was observed in the compositions of Examples 5 and 6 and Reference Examples 5 and 6, in which phenylalanine was blended in a specific ratio to threonine.

[0059] As described above, the composition of the present invention, by incorporating threonine to leucine in a specific ratio, allows for the balanced administration of essential amino acids effective for protein synthesis in the body, thereby improving the cell-activating activity of C2C12. Therefore, the composition of the present invention can promote muscle strengthening.

[0060] <Test 2: Sensory Test> The flavor of the oral composition of the present invention was evaluated.

[0061] [Preparation of the test substance (beverage)] The test substances shown in Table 4 were dissolved in pure water to a concentration of 7.5 mg / mL to obtain the liquid beverages of Comparative Example 10 and Example 7. For the "other essential amino acids," a mixture of lysine, valine, isoleucine, methionine, histidine, and tryptophan was used.

[0062] [Evaluation Method] A sensory evaluation test was conducted on five subjects with knowledge and experience in sensory testing, who consumed a liquid beverage. The evaluation items were "leucine-specific bitterness," "drinkability," and "aftertaste." For each item of the sensory evaluation, we assigned scores as follows: Leucine's characteristic bitterness: Strong bitterness 1 ← 3 → 5 Weak bitterness Ease of drinking: Difficult to drink 1 ← 3 → 5 Easy to drink Aftertaste: Bad aftertaste 1 ← 3 → 5 Good aftertaste

[0063] [Test Results] Table 4 shows the average score for each item.

[0064] [Table 4]

[0065] As shown in Table 4, the oral composition of the present invention was improved in all aspects, including the characteristic bitterness of leucine, ease of drinking, and aftertaste, compared to a beverage containing leucine alone (comparative example). Mixing leucine and threonine improved the flavor and resulted in an oral composition that is easier to consume.

[0066] <Test 3: Animal Test> Animal experiments were conducted in which the oral composition of the present invention was administered to mice, and the amount of puromycin-binding protein was evaluated (SunSET method). Since puromycin is incorporated into newly synthesized peptide chains, an increase in the amount of puromycin incorporated into muscle protein over a certain period of time serves as an indicator of muscle-building effect.

[0067] (Preparation of the administration solution) The test substances shown in Table 5 were suspended in pure water to a concentration of 40 mg / mL to prepare the administration solution for Example 8. Comparative Example 11 used only pure water without the test substances as the administration solution, and Comparative Example 12 used WPC80 (whey protein content: 75% by mass or more) as the test substance to prepare an administration solution with a concentration of 354.7 mg / mL so that the leucine dosage (amount of leucine constituting the protein) was the same as in Example 8. For the "other essential amino acids," a mixture of lysine, valine, isoleucine, methionine, histidine, and tryptophan was used.

[0068] [Table 5]

[0069] The specific evaluation method is as follows: Seven-week-old male C57BL / 6J mice were acclimated for one week and then fasted overnight. The prepared test substance solution was orally administered at a dose of 10 mL / kg. Fifteen minutes later, puromycin (0.04 mmol / kg) was administered intraperitoneally. Thirty minutes later (45 minutes after administration of the test substance), hind limb muscle (gastrocnemius muscle) was collected, and puromycin-labeled peptide was analyzed by Western blotting as described below.

[0070] (Western blotting) The hind limb muscles obtained above were freeze-milled and further pulverized in RIPA buffer (Thermo) supplemented with protease inhibitor and phosphatase inhibitor to obtain a protein extract. The protein extract was centrifuged at 10,000 × g for 10 minutes, and the supernatant was collected. The protein concentration was measured using a BCA protein assay kit (Thermo). The protein extract, RIPA buffer, and 4x Laemmli Sample Buffer were mixed and heated at 95°C for 5 minutes to prepare electrophoresis samples at a concentration of 10 μg / well.

[0071] Electrophoresis was performed at 100V using a 10% precast polyacrylamide gel (Bio-Rad). After electrophoresis, the protein was transferred from the gel to a PVDF membrane. Blocking was performed by shaking at room temperature for 1 hour using Block Ace (KAC). The sample was immersed in TBST Buffer containing the primary antibody (anti-puromycin antibody, Merck) and shaken at room temperature for 1 hour, then washed with TBST for 5 minutes x 3 times. Subsequently, the sample was shaken at room temperature for 1 hour with the secondary antibody (Anti-mouse IgG, HRP-linked Antibody; Cell Signaling Technology). After washing with TBST for 5 minutes x 3 times, chemiluminescence measurement was performed and the amount of puromycin-labeled protein in the protein extract was analyzed using the Chemi Doc XRS+ system (Bio-Rad). CBB staining was also performed to correct for the amount of puromycin-labeled peptide, and the results when the amount of puromycin-containing protein in Comparative Example 11 was set to 1 are shown in Table 6.

[0072] [Table 6]

[0073] As shown in Table 6, the composition of Example 8 of the present invention showed an increased relative amount of puromycin-containing protein compared to pure water (Comparative Example 11), confirming an improvement in the rate of muscle protein synthesis. Furthermore, compared to the case using whey protein in Comparative Example 12, although the dose was small and the amount of leucine administered was the same (however, Example 8 uses leucine as a free amino acid, while Comparative Example 12 uses leucine as an amino acid that constitutes protein), an improvement in the rate of muscle protein synthesis was confirmed in Example 8. Therefore, it was confirmed in animal studies that the oral composition of the present invention exhibits excellent muscle-building effects. [Industrial applicability]

[0074] The oral composition of the present invention is industrially useful because it can be used as a health food or the like.

Claims

[Claim 1] The invention described in the specification.

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