Muscle damage repair promoter, muscle damage treatment drug, muscle damage repair-promoting composition, muscle damage repair-promoting food composition, animal feed, and method for treating muscle-damaged non-ruminant livestock or pet animal

Abstract

To provide a muscle injury repair-promoting agent, a muscle injury-treating agent, a muscle injury repair-promoting composition and a muscle injury repair-promoting food composition each containing oleic acid, to provide an animal feed containing the muscle injury repair-promoting agent, and to provide a method for treating muscle-injured non-ruminant domestic animals and pet animals.SOLUTION: A muscle injury repair promoter containing oleic acid as an active ingredient is preferably orally ingested 230mg / day to 480mg / day, and an ingestion target may be a human, a non-ruminant livestock, or a pet.SELECTED DRAWING: Figure 2A

Description

[Technical Field]

[0001] The present invention relates to a muscle damage repair promoter containing oleic acid, a muscle damage therapeutic agent, a composition for promoting muscle damage repair, and a food composition for promoting muscle damage repair, as well as animal feed containing the muscle damage repair promoter and a method for treating muscle-damaged non-ruminant livestock and companion animals. [Background technology]

[0002] Skeletal muscle tissue is the largest energy-consuming organ in the body and is an essential locomotive organ for our activities. Furthermore, when muscles are damaged, they are rapidly repaired and regenerated, and in most cases, recovery is achieved without dysfunction.

[0003] Satellite cells, which are muscle stem cells, contribute to this high regenerative capacity of muscle. When muscle is damaged, satellite cells, which are pooled in a dormant state on the basement membrane of muscle fibers, are activated, migrate to the damaged area, proliferate there, and differentiate into myoblasts. The myoblasts then fuse with each other to form multinucleated myotubes. These myotubes then fuse to fill the damaged area, and the muscle eventually becomes mature myofibers, recovering to a level comparable to the original tissue.

[0004] Regarding tissue regeneration, research is underway into new treatments using cytokines and angiogenic factors. However, the growth factors and other treatments currently used in research and treatment are extracted or recombinant proteins consisting of several hundred amino acids, and the problems of infection and side effects remain unresolved. The inventors focused on oleic acid, a component found in foods consumed on a daily basis and whose safety has been established.

[0005] Oleic acid is a type of fatty acid found in food fats and oils, and is classified as an n-9 unsaturated fatty acid. Regarding oleic acid and skeletal muscle, research using muscle cells has shown that oleic acid increases the mRNA expression level of genes that promote lipid metabolism (Non-Patent Document 1), decreases the expression level of glucose transporters (Non-Patent Document 2), and improves palmitic acid-induced muscle cell differentiation disorders (Non-Patent Document 3).

[0006] It is also known that oral administration of oleic acid increases the mRNA expression level of genes involved in fatty acid oxidation (Non-Patent Document 4).The inventors have also found that oleic acid increases lipid-burning muscle fibers in animal tests using muscle cells and mice (Non-Patent Documents 5 and 6). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Tumova J. et al., Protective Effect of Unsaturated Fatty Acids on Palmitic Acid-Induced Toxicity in Skeletal Muscle Cells is not Mediated by PPARδ Activation, Lipids, 50, 955-964 (2015). [Non-patent document 2] Poletto AC et al., Oleic and linoleic fatty acids downregulate Slc2a4 / GLUT4 expression via NFKB and SREBP1 in skeletal muscle cells, Molecular and Cellular Endocrinology, 401, 65-72 (2015). [Non-patent document 3] Xue M. et al., Oleate Ameliorates Palmitate-Induced Impairment of Differential Capacity in C2C12 Myoblast Cells, Stem Cells and Development, 30, 289-300 (2021). [Non-patent document 4] Lim JH et al., Oleic Acid Stimulates Complete Oxidation of Fatty Acids through Protein Kinase A-dependent Activation of SIRT1-PGC1α Complex, The Journal of Biological chemistry, 288, 10, 7117-7126 (2013). [Non-Patent Document 5] Watanabe N. et al., Oleic acid up-regulates myosin heavy chain (MyHC) 1 expression and increases mitochondrial mass and maximum respiration in C2C12 myoblasts, Biochemical and Biophysical Research Communications, 525, 406-411 (2020). [Non-patent document 6] Komiya Y. et al., Dietary oleic acid intake increases the proportion of type 1 and 2X muscle fibers in mice, Scientific Reports, 14, 755 (2024). Summary of the Invention [Problem to be solved by the invention]

[0008] As described above, various studies have been conducted on the relationship between oleic acid and skeletal muscle, but there have been no reports on the muscle regeneration-promoting effect of oleic acid intake. The present invention aims to provide a muscle damage repair promoter, a muscle damage therapeutic agent, a composition for promoting muscle damage repair, and a food composition for promoting muscle damage repair that can promote muscle damage repair when a subject (human, non-ruminant livestock, or pet) suffers muscle damage. Another aim of the present invention is to provide an animal feed that can promote muscle damage repair. Furthermore, the present invention aims to provide a method for treating muscle-damaged non-ruminant livestock and pets. [Means for solving the problem]

[0009] The present invention includes the following aspects. (1) A muscle damage repair promoter containing oleic acid as an active ingredient. (2) The muscle damage repair promoter according to (1) above, in which oleic acid is orally ingested at 230 mg / day to 480 mg / day. (3) The muscle damage repair promoter according to (2) above, which is intended for consumption by humans, non-ruminant livestock, or pets. (4) A muscle damage treatment drug containing oleic acid as an active ingredient. (5) The muscle damage treatment drug according to (4) above, in which oleic acid is orally ingested at 230 mg / day to 480 mg / day. (6) The muscle damage therapeutic agent according to (5) above, which is intended for administration to humans, non-ruminant livestock, or pets. (7) A composition for promoting muscle damage repair, comprising oleic acid as an active ingredient and a pharmaceutically and / or food-acceptable carrier. (8) The composition for promoting muscle damage repair described in (7) above, which is an oral preparation. (9) The composition for promoting muscle damage repair described in (8) above, which is in the form of a tablet, capsule, or granule. (10) A composition for promoting muscle damage repair according to any one of (7) to (9) above, in which oleic acid is ingested at 230 mg / day to 480 mg / day. (11) The composition for promoting muscle damage repair according to (10) above, wherein the subject of administration is a human, a non-ruminant livestock animal, or a pet animal. (12) A food composition for promoting muscle damage repair, containing oleic acid as an active ingredient and further containing a carrier acceptable for food. (13) A food composition for promoting muscle damage repair described in (12) above, which is a supplement. (14) The food composition for promoting muscle damage repair according to (12) or (13) above, wherein oleic acid is ingested at 230 mg / day to 480 mg / day. (15) The food composition for promoting muscle damage repair according to (14) above, which is intended for consumption by humans, non-ruminant livestock, or pets. (16) An animal feed containing the muscle damage repair promoter described in (1) or (2) above. (17) A method for treating muscle damage, comprising administering the muscle damage repair promoter described in (1) or (2) above to non-ruminant livestock or pets. [Effects of the Invention]

[0010] The present invention provides a muscle damage repair promoter, a muscle damage therapeutic agent, a composition for promoting muscle damage repair, and a food composition for promoting muscle damage repair, all of which are capable of promoting muscle damage repair. Daily ingestion of this agent, this drug, this composition, or this food composition after muscle damage can repair damaged skeletal muscle in a shorter period than the period required for natural healing of muscle damage caused by overload exercise or trauma. It also shifts muscle fiber type toward slow-twitch muscle after injury. Furthermore, the present invention provides animal feed capable of promoting muscle damage repair, and also provides a method for treating muscle-damaged non-ruminant livestock and companion animals. [Brief explanation of the drawings]

[0011] [Figure 1A] FIG. 1 is a diagram illustrating Example 1, showing an HE stained image of the tibialis anterior muscle after injury. [Figure 1B] FIG. 1 is a diagram illustrating Example 1, showing an observation image of cells in regenerated skeletal muscle tissue. [Figure 1C] FIG. 1 is a diagram illustrating Example 1, and is a graph showing the results of measuring the number of central nuclei in regenerated muscle cells after injury. [Figure 2A]FIG. 10 is a diagram illustrating Example 2, showing quadruple immunohistochemical staining images obtained by staining muscle sections from the oleic acid intake group 14 days after injury with four MyHC antibodies. [Figure 2B] FIG. 10 is a diagram illustrating Example 2, and is a table showing the muscle fiber type staining composition ratios, where the number of each positive cell in the section is expressed as a ratio. [Figure 2C] 2C is a graph based on the table of FIG. 2B. [Figure 2D] FIG. 10 is a diagram illustrating Example 2, and is a graph showing the mRNA expression levels of each Myh gene 7 days after injury. DETAILED DESCRIPTION OF THE INVENTION

[0012] A muscle damage repair promoter and a muscle damage therapeutic agent according to one embodiment of the present invention contain oleic acid as an active ingredient. A composition for promoting muscle damage repair according to one embodiment of the present invention contains oleic acid as an active ingredient and further contains a pharmaceutically and / or food-acceptable carrier. A food composition for promoting muscle damage repair according to one embodiment of the present invention contains oleic acid as an active ingredient and further contains a food-acceptable carrier.

[0013] Oleic acid is an 18-carbon omega-9 monounsaturated fatty acid. It is found in olive oil and animal fats in the form of triacylglycerol.

[0014] The present invention is based on the discovery that oral ingestion of oleic acid promotes repair of muscle damage. The present inventors have found that oral ingestion of oleic acid promotes repair of muscle damage.

[0015] Furthermore, it was also revealed that the proportion of slow-twitch muscle fiber types, which are excellent at burning fat and are fatigue-resistant, was increased among the regenerated skeletal muscle fiber types.

[0016] The muscle damage repair promoter, muscle damage therapeutic agent, composition for promoting muscle damage repair, and food composition for promoting muscle damage repair according to this embodiment preferably reach peripheral (skeletal muscle) tissue in the form of free fatty acids. Whether oleic acid is ingested in the form of triacylglycerol or free fatty acids, it is transported to skeletal muscle in the form of free fatty acids, so the intake form is not particularly limited. Specific examples of intake forms include oils and fats rich in oleic acid (olive oil) and oral medications and oral supplements in the form of free fatty acids.

[0017] As used herein, "a pharmaceutically and / or food-acceptable carrier" refers to a pharmaceutically acceptable carrier and / or a food-acceptable carrier. Examples of pharmaceutically acceptable carriers include conventionally known excipients, binders, lubricants, isotonicity agents, stabilizers, preservatives, flavoring agents, solubilizers, emulsifiers, etc., while examples of food-acceptable carriers include conventionally known food additives used in food manufacturing or for the processing and preservation of food, such as preservatives, sweeteners, coloring agents, and flavoring agents.

[0018] One embodiment of the present invention includes an animal feed containing the muscle damage repair promoter. Animals include humans, non-ruminant livestock such as pigs and horses, and pets such as dogs and cats. The muscle damage repair promoter, muscle damage therapeutic agent, muscle damage repair promoting composition, and muscle damage repair promoting food composition according to this embodiment can be primarily intended for consumption by humans, non-ruminant livestock, and pets, not limited to animal feed. Therefore, one embodiment of the present invention is a method for treating muscle damage by administering the muscle damage repair promoter according to this embodiment, or the animal feed, muscle damage therapeutic agent, muscle damage repair promoting composition, or muscle damage repair promoting food composition containing the same, to non-ruminant livestock or pets with muscle damage, thereby promoting muscle damage repair in these animals.

[0019] The muscle damage repair promoter, muscle damage therapeutic agent, muscle damage repair promoting composition, and muscle damage repair promoting food composition according to this embodiment can be produced by incorporating oleic acid as an active ingredient. The amount of oleic acid as an active ingredient in these muscle damage repair promoters, muscle damage therapeutic agent, muscle damage repair promoting composition, and muscle damage repair promoting food composition is not particularly limited, but the concentration is preferably 230 mg / day to 480 mg / day for humans, non-ruminant livestock, and pets, and is preferably taken continuously for at least two weeks after injury.

[0020] The forms of the muscle damage repair promoter, muscle damage therapeutic agent, muscle damage repair promoting composition, and muscle damage repair promoting food composition according to the present embodiment are not particularly limited, and may be, for example, oral forms such as tablets, capsules, granules, powders, oral liquids, syrups, oils, or oral jellies. When the muscle damage repair promoting food composition is a supplement, it may be in the form of a general health food capsule (e.g., one derived from gelatin or dietary fiber). By using oleic acid when producing the muscle damage repair promoter, muscle damage therapeutic agent, muscle damage repair promoting composition, and muscle damage repair promoting food composition according to the present embodiment, an agent or composition having the effect of promoting muscle damage repair can be obtained. The form of oleic acid in this case is not limited, and it may be in the form of an oil (triacylglycerol) such as olive oil, or in the form of a free fatty acid.

[0021] The present invention will be described below with reference to examples. The following examples are given to illustrate the present invention, but are not intended to limit the present invention. [Example]

[0022] Example 1 Eight-week-old male C57BL / 6J mice were intramuscularly injected with 100 μL of 10 μM cardiotoxin, a muscle injury-inducing agent. After injury, the mice were fed a powdered diet containing 10% oleic acid ad libitum for two weeks. A control group received a diet containing 10% palmitic acid (n=4).

[0023] Three, seven, and 14 days after muscle injury, the tibialis anterior muscles were excised, immediately embedded in OCT compound, and rapidly frozen in isopentane cooled with liquid nitrogen to prepare frozen blocks. 10 μm sections were cut from the blocks using a cryostat, stained with hematoxylin and eosin, and examined under a microscope for morphological analysis.

[0024] Figure 1A shows HE-stained images of tibialis anterior muscle at various days after injury. Figure 1B shows cells with a central nucleus, an indicator of skeletal muscle tissue regeneration from injury. In Figure 1B, the dark area at the tip of the arrow indicates the central nucleus. Generally, skeletal muscle cells have nuclei at the cell periphery, but in regenerating muscle cells, the nucleus is located in the center of the cytoplasm. Therefore, it is possible to estimate the number of regenerating muscle cells by counting the number of cells with nuclei located in the center of the cytoplasm in an observation image of skeletal muscle tissue.

[0025] Figure 1C is a graph showing the results of measuring the number of cells with central nuclei (centronuclear count) in images of skeletal muscle tissue 7 and 14 days after injury. As shown in Figure 1C, there was no significant difference in the number of central nuclei between the oleic acid intake group and the control group 7 days after injury, but 14 days after injury, the number of central nuclei was significantly reduced in the oleic acid intake group compared to the control group (P<0.05). This result indicates that the injured muscles of the oleic acid intake group recovered more quickly than those of the control group.

[0026] Furthermore, as shown in Example 2 below, when the muscle fiber type of the regenerated skeletal muscle was evaluated, it was confirmed that there was an increase in slow-twitch muscle fibers, which are excellent at burning fat and have anti-fatigue properties (P<0.05).

[0027] Based on these experimental results, we concluded that oleic acid intake after muscle damage promotes muscle repair. Furthermore, when the amount of oleic acid consumed by mice was calculated based on their daily food intake, it was found to be 230-480 mg / day, and we concluded that this range was the effective amount.

[0028] Example 2 Quadruple immunohistochemical staining was performed on muscle sections from the oleic acid-fed group 14 days after injury, as prepared in Example 1, using monoclonal antibodies specific for four myosin heavy chain (MyHC) isoforms, which serve as muscle fiber type markers: 4B51E8 (α-MyHC1-Alexa Fluor 647), 8F72C8 (α-MyHC2A-Alexa Fluor 350), 6F12H3 (α-MyHC2X-Fluorescein), and 2G72F10 (α-MyHC2B-AnaTag™ HiLyte™ Fluor 594 Fluorophores).

[0029] The quadruple immunohistochemical staining image is shown in Figure 2A. In Figure 2A, white indicates MyHC1 (slow-twitch muscle type), light gray indicates MyHC2A (intermediate-twitch muscle type), gray indicates MyHC2X (intermediate-twitch muscle type), and dark gray indicates MyHC2B (fast-twitch muscle type).

[0030] Similar images to those shown in Figure 2A were also obtained from muscle sections 14 days after injury in the control group. The number of positive cells in sections stained with each of the four MyHC antibodies was counted from these images. Duplicate staining was counted as hybrid fibers. The results are presented as a table in Figure 2B, and a graph of Figure 2B is shown in Figure 2C.

[0031] As shown in Figure 2B and Figure 2C, oleic acid intake significantly increased the proportion of MyHC1, 2A, and 2A+2X-positive cells in muscle tissue 14 days after injury, and significantly decreased the number of MyHC2B-positive cells (*, P<0.05).

[0032] Figure 2D is a graph showing the mRNA expression levels of each Myh gene 7 days after injury. Total RNA was extracted from muscle tissue from the oleic acid-fed and control groups 7 days after injury, cDNA was prepared from the mRNA, and mRNA expression levels of the Myh gene encoding MyHC were analyzed using qPCR. As a result, the mRNA expression levels of Myh7, a slow-twitch muscle fiber, and Myh2, an intermediate-twitch muscle fiber, were significantly increased 7 days after injury in the oleic acid-fed group. This result tended to be consistent with the immunostaining results. In other words, Example 2 suggests that oleic acid intake favors the slow-twitch muscle fiber type after regeneration.

[0033] The above-described embodiments and examples have been described to facilitate understanding of the present invention, and are not intended to limit the present invention. Therefore, each element disclosed in the above embodiments is intended to include all design modifications and equivalents that fall within the technical scope of the present invention. [Industrial Applicability]

[0034] The present invention makes it possible to provide a muscle damage repair promoter, a muscle damage therapeutic agent, a composition for promoting muscle damage repair, and a food composition for promoting muscle damage, each containing oleic acid. Furthermore, the present invention makes it possible to provide animal feed containing the muscle damage repair promoter and a method for treating muscle-damaged non-ruminant livestock and pet animals.

Claims

1. A muscle damage repair promoter containing oleic acid as an active ingredient.

2. 2. The muscle damage repair promoter according to claim 1, wherein oleic acid is orally ingested at 230 mg / day to 480 mg / day.

3. The muscle damage repair promoter according to claim 2, wherein the subject of administration is a human, a non-ruminant livestock animal, or a pet animal.

4. A muscle damage treatment drug containing oleic acid as an active ingredient.

5. The muscle damage therapeutic agent according to claim 4, wherein oleic acid is orally ingested at 230 mg / day to 480 mg / day.

6. The muscle damage therapeutic agent according to claim 5, wherein the subject of administration is a human, a non-ruminant livestock animal, or a pet animal.

7. A composition for promoting muscle damage repair, comprising oleic acid as an active ingredient and further comprising a pharmaceutically and / or food-acceptable carrier.

8. The composition for promoting muscle damage repair according to claim 7, which is an oral agent.

9. The composition for promoting muscle damage repair according to claim 8, which is in the form of a tablet, capsule, or granule.

10. The composition for promoting muscle damage repair according to any one of claims 7 to 9, wherein oleic acid is ingested at 230 mg / day to 480 mg / day.

11. The composition for promoting muscle damage repair according to claim 10, wherein the subject of administration is a human, a non-ruminant livestock animal, or a pet animal.

12. A food composition for promoting muscle damage repair, which contains oleic acid as an active ingredient and further contains a carrier acceptable for food.

13. The food composition for promoting muscle damage repair according to claim 12, which is a supplement.

14. The food composition for promoting muscle damage repair according to claim 12 or 13, wherein oleic acid is ingested at 230 mg / day to 480 mg / day.

15. The food composition for promoting muscle damage repair according to claim 14, wherein the subject of intake is a human, a non-ruminant livestock animal, or a pet animal.

16. An animal feed containing the muscle damage repair promoter according to claim 1 or 2.

17. A method for treating muscle damage, comprising administering the muscle damage repair promoter according to claim 1 or 2 to non-ruminant livestock or pet animals.

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