Oral composition

Abstract

To provide an oral composition containing an amino acid and exhibiting excellent muscle-enhancing effect.SOLUTION: The oral composition contains leucine and threonine, wherein the molar composition ratio of threonine to leucine is ≤ 0.05.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to oral compositions containing leucine and threonine. [Background technology]

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

[0003] Health foods containing such amino acids have been proposed, for example, for enhancing learning and memory abilities, containing amino acids such as alanine, arginine, and leucine (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-184956 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an oral composition that contains amino acids and exhibits excellent muscle-building effects. [Means for solving the problem]

[0006] As a result of intensive research to solve the above problems, the inventors discovered that an oral composition containing leucine and threonine in a specific ratio exhibits excellent muscle-building effects, and thus completed the present invention.

[0007] That is, the present invention includes the following aspects. [1] An oral composition containing leucine and threonine, An oral composition, 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 the threonine to the 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 the phenylalanine to the threonine is 0.5 or more and 2.0 or less.

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

[0009] The oral composition of the present invention exhibits excellent muscle-building effects. DETAILED DESCRIPTION OF THE INVENTION

[0010] The oral composition of the present invention is 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 a specific ratio, which promotes protein production in the body and exhibits excellent muscle-building effects. For example, even with a small amount of intake, muscle-building effects comparable to those of conventional whey protein can be achieved. The oral composition of the present invention can be used for muscle building in athletes and healthy individuals, muscle recovery after illness, muscle maintenance in the elderly, etc. Furthermore, when the oral composition of the present invention contains leucine and threonine in a specific ratio, the flavor is improved and the composition can be ingested without resistance.

[0012] Each component contained in the oral composition of the present invention will be described below. [Leucine] Leucine is a type of amino acid, a branched-chain amino acid that is one of the essential amino acids for humans, and is abbreviated as Leu or L. Leucine exists in isomers such as L-, D-, and DL-isomers, and all of these 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 include leucine that exists as a component of protein. Free amino acids (amino acids that exist independently and separately from proteins) have different chemical properties from amino acids that exist as components of proteins, and the two are clearly distinguished. Note that amino acids such as threonine, which will be described later, are also referred to as free amino acids.

[0013] The content of leucine relative to the total content of essential amino acids in the oral composition of the present invention (the molar composition ratio of leucine content when the total content of essential amino acids is taken as 1) is not particularly limited, but in order to more significantly exhibit the effects of the present invention and provide an oral composition with excellent taste, it is preferably 0.7 or more, more preferably 0.7 to 0.99 (essential amino acids:leucine=1:0.7-0.99), 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 leucine content is high, but from the viewpoint of taste, it is better not to have too high a leucine content, so the above range is preferred.

[0014] [Threonine] Threonine, a type of amino acid with a hydroxyethyl group in the side chain, is also called threonine. It is one of the essential amino acids in humans and is abbreviated as Thr or T. Threonine has two optically active centers, and therefore exists as four isomers: L-threonine, D-threonine, L-allothreonine, and D-allothreonine. All of these isomers can be used in the oral composition of the present invention.

[0015] As described above, the blending ratio (molar composition ratio) of leucine and 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, but in order to better enjoy the effects of the present invention, it is preferably 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 0.04 or less, and particularly preferably 0.025 or more and 0.04 or less.

[0016] The leucine and threonine of the present invention can be used not only in the free form but also in the form of a salt. Examples of salts include acid addition salts and salts with bases, and it is preferable to select a pharmacologically acceptable salt. Specific examples of salts include salts with inorganic bases, organic bases, inorganic acids, and organic acids. In the present invention, in order to further enjoy the effects of the present invention, the free form or organic acid salts are preferred, the free form or hydrochloride salts are more preferred, and the free form is particularly preferred. The leucine and threonine of the present invention can be commercially available products that are usually available as food ingredients.

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

[0018] The essential amino acids used together with leucine and threonine in the oral composition of the present invention include, for example, 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.Moreover, it is most preferable to include all essential amino acids.

[0019] [Phenylalanine] Phenylanine is an aromatic amino acid with a benzyl group in the side chain. It is an essential amino acid in humans and is abbreviated as Phe or F. Phenylanine exists as L-, D-, and DL-isomers, all of which 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 blending 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 even more preferably 0.8 or more and 1.5 or less, in order to better enjoy the effects of the present invention.

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

[0022] In order to obtain the effects of the present invention more effectively, the content of lysine in the oral composition of the present invention is preferably such that the molar composition ratio relative to the total content of essential amino acids is 0.02 or more, more preferably 0.02 or more and 0.08 or less, and even more preferably 0.03 or more and 0.08 or less.

[0023] [Ballin] Valine is a branched-chain amino acid. It is one of the essential amino acids for humans and is also known as 2-aminoisovaleric acid, abbreviated as Val or V. Valine exists as an isomer, such as L-, D-, or DL-isomer, and any of these can be used in the oral composition of the present invention.

[0024] In order to better enjoy the effects of the present invention, the content of valine contained in the oral composition of the present invention is preferably such that the molar composition ratio relative to the total content of essential amino acids is 0.02 or more, more preferably 0.02 or more and 0.08 or less, and even 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 for humans and is also known as 2-amino-3-methylpentanoic acid (2-amino-3-methylvaleric acid), abbreviated as Ile or I. Isoleucine exists in the form of L-, D-, and DL-isomers, all of which can be used in the oral composition of the present invention.

[0026] In order to further enjoy the effects of the present invention, the content of isoleucine contained in the oral composition of the present invention is preferably a molar composition ratio of 0.02 or more 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.

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

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

[0029] In order to obtain the effects of the present invention more effectively, the molar composition ratio of methionine contained in the oral composition of the present invention to the total content of essential amino acids is preferably 0.01 or more, more preferably 0.01 or more and 0.04 or less, and even more preferably 0.015 or more and 0.04 or less.

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

[0031] In order to obtain the effects of the present invention more effectively, the content of histidine in the oral composition of the present invention is preferably such that the molar composition ratio relative to the total content of essential amino acids is 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 an essential amino acid in humans and has the systematic name 2-amino-3-(indolyl)propionic acid, abbreviated as Trp or W. Tryptophan exists in the form of L-, D-, and DL-isomers, all of which can be used in the oral composition of the present invention.

[0033] In order to better enjoy the effects of the present invention, the content of tryptophan contained in the oral composition of the present invention is preferably such that the molar composition ratio relative to the total content of essential amino acids is 0.002 or more, more preferably 0.002 or more and 0.02 or less, and even more preferably 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 the free form but also in the form of a salt, and commercially available products that are usually available as food ingredients can also be used.

[0035] In addition to the above-mentioned leucine, threonine, and other essential amino acids, the oral composition of the present invention may contain other commonly used substances, such as various excipients, binders, glossing agents, lubricants, stabilizers, diluents, bulking agents, thickeners, emulsifiers, antioxidants, pH adjusters, colorants, flavors, and additives.

[0036] The intake amount of the oral composition of the present invention is not particularly limited, but from the viewpoint of further enjoying 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. The upper limit of the intake amount is not particularly limited, but from the viewpoint of further enjoying 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 that can be taken orally, and may be any of pharmaceuticals (including quasi-drugs), so-called health foods, foods for specified health uses, and foods with functional claims.

[0038] Examples of the form of the oral composition of the present invention include tablets, capsules, powders, granules, liquids, rods, plates, blocks, solids, rounds, pastes, creams, caplets, gels, chewable tablets, and sticks, 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, the scope of the present invention includes products that display the muscle building function on the product itself, packaging, instructions, or promotional materials. Functional food claims also include those that claim muscle building as a scientific basis for their functionality.

[0040] Specifically, examples include products that claim to have functions such as inhibiting, maintaining, or increasing muscle mass (muscle weight, muscle cross-sectional area, muscle density), inhibiting, maintaining, or improving muscle quality, inhibiting, maintaining, or increasing muscle strength, etc. More specifically, examples include products that claim to "increase muscle strength," "support muscle-building ability," "suppress muscle breakdown," "support muscle training," "support body building," "macho," "slim-macho," "maintain muscle mass and strength," etc.

[0041] The oral composition for muscle building of the present invention is not limited to those labeled on the product packaging, etc., with the combined components of the present invention (leucine and threonine) as the active ingredient for muscle building. For example, it may be a product in which the active ingredient is not specified. Furthermore, even general foods that are manufactured and sold with suggested functions and uses are included in the scope of the oral composition for muscle building of the present invention. For example, there are products sold with personal testimonials from people who have taken the product posted on their websites, etc., mentioning its functions and uses.

[0042] The subjects who can take the oral composition of the present invention are not particularly limited as long as they are people who need to strengthen their muscles, but preferred examples include people who want to get in shape, athletes, and elderly people with weak legs and hips. [Example]

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

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

[0045] [Test substance] The following substances were used as test substances. Leucine: Reagent for L-leucine Lysine: L-Lysine monohydrochloride reagent Valine: Reagent for L-valine Isoleucine: Reagent for L-isoleucine Threonine: L-threonine reagent Phenylalanine: Reagent for L-phenylalanine 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 through a 0.2 μm filter and diluted with the same medium to a concentration of 10 μM to obtain a test substance preparation solution. Test substance-containing media were prepared by mixing each test substance preparation solution to achieve the molar composition ratios listed in Tables 1 to 3. Note that a mixture of lysine, valine, isoleucine, methionine, histidine, and tryptophan was used as the "other essential amino acids."

[0047] The specific experimental procedures are as follows. In a 37°C, 5% CO2 incubator, 75 cm 2C2C12 cells were cultured in a flask with 10% FBS-DMEM. Then, the cells were suspended by trypsinization and placed in each well of a collagen-coated 96-well plate at 8 × 10 3 The cells were seeded at a density of 100 cells / well and pre-cultured for 24 hours in a 5% by volume CO2 incubator at 37°C.

[0048] After removing the medium from each well, the wells were washed once with PBS, and 100 μL / well of medium containing the test substance was added, followed by culturing for 3 hours in a 37° C., 5% by volume CO 2 incubator. The medium used was 10% FBS-amino acid-free DMEM, and the total molar concentration of the test substance in the medium was 10 μM. As a control, 10% FBS-amino acid-free DMEM medium containing no test substance was added. This was designated Comparative Example 1.

[0049] After 3 hours of incubation, the medium was removed, and 150 μL of Cell Counting Kit-8 (Dojindo Laboratories) diluted 20-fold with amino acid-free DMEM was added to each well. The plates were placed in a 37°C, 5% CO2 incubator to allow for adequate color development, and the absorbance at 450 nm of the formazan product, which is proportional to the number of viable cells in each well, was measured.

[0050] The WST-8 color reagent in the Cell Counting Kit-8 is reduced to formazan by the action of dehydrogenase via electron transfer substances. The more viable cells there are, the higher the dehydrogenase activity and the greater the amount of formazan produced. Since formazan has a maximum absorption around 450 nm, the amount of formazan produced was measured based on this absorbance to confirm C2C12 proliferation. Higher formazan absorbance indicates greater C2C12 proliferation, so this absorbance serves as an indicator of myoblast-stimulating activity. As a blank, the absorbance of Cell Counting Kit-8 alone 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 of each test substance well, and the results were evaluated relative to the cell-activating activity of the control well, Comparative Example 1, using the formula below. Tables 1 to 3 show the results.

[0052] Cell stimulating activity = (absorbance of each test substance well - absorbance of blank) x 100 / (absorbance of control well - absorbance of blank)

[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, which contain a specific ratio of threonine to leucine, exhibited cell-activating activity, as evident from a comparison with the control (Comparative Example 1) containing no leucine or threonine. In particular, the composition of Example 2, which contains a threonine to leucine ratio of approximately 0.03, exhibited higher cell-activating activity. Furthermore, the compositions of Examples 1 to 3 of the present invention exhibited improved cell-activating activity compared with the compositions of Comparative Examples 2 and 3, which contain leucine and threonine alone. In other words, the cell-activating activity was improved by incorporating leucine and threonine (Comparative Examples 2 and 3) at a specific ratio, which do not exhibit cell-activating activity alone. Furthermore, the compositions of Examples 1 to 3 of the present invention exhibited higher cell-activating activity compared with the compositions of Comparative Examples 4 and 5, which contain a high ratio of threonine to leucine.

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

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

[0059] As described above, the composition of the present invention contains a specific ratio of leucine to threonine, allowing for a 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 building.

[0060] <Test 2: Sensory Test> The oral compositions of the present invention were evaluated for flavor.

[0061] [Preparation of test substance (drink)] 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. As 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 by having five subjects with knowledge and experience in sensory testing ingest the liquid beverage. The evaluation items were the "bitterness specific to leucine," "ease of drinking," and "aftertaste." Each item in the sensory evaluation was scored as follows: Leucine's characteristic bitterness: Strong bitterness 1 ← 3 → 5 Weak bitterness Drinkability: Difficult to drink 1 ← 3 → 5 Easy to drink Aftertaste: Bad aftertaste 1 ← 3 → 5 Good aftertaste

[0063] [Test Results] The average scores for each item are shown in Table 4.

[0064] [Table 4]

[0065] As shown in Table 4, the oral composition of the present invention was improved in all respects, including the bitterness specific to leucine, ease of drinking, and aftertaste, compared to the reference beverage containing leucine alone (comparison example). By mixing leucine and threonine, the flavor was improved, resulting in an oral composition that was easier to ingest.

[0066] <Test 3: Animal Test> In an animal experiment, mice were administered the oral composition of the present invention, and the amount of puromycin-binding protein was evaluated (SunSET method). Because 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 is an indicator of muscle-building effects.

[0067] (Preparation of administration solution) The test substance shown in Table 5 was suspended in pure water to a concentration of 40 mg / mL to prepare the administration solution of Example 8. In Comparative Example 11, pure water alone containing no test substance was used as the administration solution, and in Comparative Example 12, WPC80 (whey protein content: 75% by mass or more) was used as the test substance to prepare an administration solution with a concentration of 354.7 mg / mL so that the dosage of leucine (amount of leucine constituting the protein) was the same as in Example 8. Note that a mixture of lysine, valine, isoleucine, methionine, histidine, and tryptophan was used as the "other essential amino acids."

[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. 15 minutes later, puromycin (0.04 mmol / kg) was administered intraperitoneally. Thirty minutes later (45 minutes after test substance administration), hind leg muscles (gastrocnemius muscles) were harvested, and puromycin-labeled peptides were analyzed by Western blotting as described below.

[0070] (Western blotting) The hindlimb muscle obtained above was freeze-pulverized and further pulverized in RIPA buffer (Thermo) supplemented with protease inhibitors and phosphatase inhibitors 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 a sample for electrophoresis at 10 μg / well.

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

[0072] [Table 6]

[0073] As shown in Table 6, the composition of Example 8 of the present invention had an increased relative amount of puromycin-containing protein compared to pure water (Comparative Example 11), confirming an improvement in the muscle protein synthesis rate. Furthermore, compared to the whey protein used in Comparative Example 12, a smaller amount was administered, and the amount of leucine administered was the same (however, Example 8 differs in that leucine is a free amino acid, while Comparative Example 12 uses leucine as a protein-constituting amino acid), but an improvement in the muscle protein synthesis rate was confirmed in Example 8. Therefore, animal tests also confirmed that the oral composition of the present invention exhibits excellent muscle-building effects. [Industrial Applicability]

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

Claims

1. 1. An oral composition comprising leucine and threonine, An oral composition, wherein the molar composition ratio of said threonine to said leucine is 0.05 or less.

2. The oral composition of claim 1 , further comprising phenylalanine.

3. The oral composition according to claim 2, wherein the molar composition ratio of said phenylalanine to said threonine is 0.5 or more and 2.0 or less.

4. The oral composition according to claim 1, wherein the molar composition ratio of the leucine content to the total content of essential amino acids is 0.7 or more.

5. The oral composition according to any one of claims 1 to 4, which is for muscle building.

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