Peptides having muscle loss inhibitory and muscle mass enhancing activities, and uses thereof

Abstract

The present application relates to a peptide having muscle loss inhibitory and muscle mass increasing activities, and uses thereof, a peptide consisting of the amino acid sequence of SEQ ID NO: 1, a pharmaceutical composition for preventing or treating muscle diseases containing the peptide as an active ingredient, and a food composition for preventing or improving muscle diseases containing the peptide as an active ingredient are provided.

Description

[Technical Field]

[0001] The present invention relates to a peptide having muscle loss-inhibiting and muscle mass-increasing activity, and uses thereof.

[0002] This application claims priority to Korean Patent Application No. 10-2022-0071033, filed on June 10, 2022, the disclosure of which is incorporated herein by reference. [Background technology]

[0003] The aging population is rapidly increasing worldwide, and the United Nations predicts that by 2050, the population aged 60 and over will exceed 2 billion. Skeletal muscle is the largest organ, accounting for 50% of total body weight. Muscle loss and weakening are among the major physical changes that occur with aging. After the age of 30, skeletal muscle tends to decrease by approximately 1% per year, and after the age of 65, the decline accelerates. This rapid muscle loss significantly impacts quality of life, reducing physical ability and increasing the risk of falls and fractures. Reflecting this concern, the World Health Organization (WHO) officially recognized sarcopenia as a disease in 2017.

[0004] Sarcopenia, induced by the degeneration of spinal nerves, motor nerves, or skeletal muscle fibers associated with muscle diseases, is one of the typical intractable diseases whose cause has not yet been elucidated. According to the research so far, the motor nerves that induce the contraction of skeletal muscles degenerate, the contraction of skeletal muscles does not progress, or in skeletal muscles, the expression of proteins involved in muscle contraction is reduced, or the said proteins are deformed, and the contraction of normal skeletal muscles does not progress. In the long term, it is known that the aforementioned motor nerves or skeletal muscles are deformed into fibrous tissues. Since the fundamental cause of such sarcopenia has not yet been elucidated and no method has been developed to prevent or recover the degeneration of motor nerves and skeletal muscles, currently, research is actively underway to develop a method to blunt the progression of the said sarcopenia. Currently, exercise, protein, and calorie supplementation are known to be helpful for sarcopenia, but they are not very helpful for the elderly, who account for most of the sarcopenia patients, and there is an urgent need for a therapeutic agent for sarcopenia.

[0005] Note that the size or muscle mass of muscles is regulated by the intracellular signal transduction process that induces anabolic and catabolic actions occurring within the muscles. Specifically, when the signal transduction reaction that induces the synthesis of muscle proteins is dominant compared to the degradation of muscle proteins, muscle protein synthesis increases, which is shown as muscle size increase (hypertrophy) or muscle fiber number increase (hyperplasia). Also, the differentiation and formation of muscle cells are regulated by various muscle regulatory factors. MyoD initiates the expression of muscle-specific genes, induces muscle satellite cells to differentiate into myoblasts, and the induction of myogenin expression by the activity of MyoD is involved in the formation of myotubes as the most important factor in the fusion of myoblasts. The muscle fibers formed through such a process form bundles and ultimately form muscles.

[0006] Under such a technical background, extensive research has been conducted to inhibit muscle loss caused by external or internal factors and promote muscle protein synthesis (Korean Registered Patent No. 10-2064387), but the current situation is still not sufficient.

Summary of the Invention

Problems to be Solved by the Invention

[0007] One aspect is to provide a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0008] Another aspect is to provide a muscle loss inhibitory composition and a muscle mass enhancing composition containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0009] Other objects and advantages of the present application will become more apparent from the following detailed description together with the appended claims and the drawings. Contents not described in this specification can be fully recognized and analogized by those skilled in the art within the technical field of this application or a similar technical field, so the description thereof is omitted.

Means for Solving the Problems

[0010] Each description and embodiment disclosed in the present application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. Also, it cannot be considered that the scope of the present application is limited by the specific descriptions described below.

[0011] One aspect provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0012] As used herein, the term "peptide" may mean a linear molecule formed by the binding of amino acid residues to each other by peptide bonds. The peptide can be produced by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques or liquid-phase synthesis techniques (U.S. Patent No. 5,516,891). As a result of intensive efforts to develop peptides having biologically effective activities, the present inventors have investigated the peptide consisting of the amino acid sequence of SEQ ID NO: 1. Here, the biologically effective activity is selected from any one or more of the following characteristics: (a) enhancement of the expression of MyoG (myogenin) and Myf5 (myogenic factor 5), which are early differentiation markers of myoblasts; (b) enhancement of the expression of MyHC (Myosin heavy chain), which is a late differentiation marker of myoblasts or a muscle fiber constituent protein, and α-actinin; and (c) reduction of the expression of α-SMA (α-smooth muscle actin) and IL-6 (interleukin-6), which are inflammatory proteins. Therefore, the peptide can be utilized for the prevention, improvement, or treatment of muscle diseases.

[0013] The peptide may have a protecting group attached to its N-terminus or C-terminus in order to acquire chemical stability, enhanced pharmacological properties (such as half-life, water absorbency, titer, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activities), and reduced antigenicity. In one specific example, the N-terminus of the peptide is bound to any one protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG: polyethylene glycol), and / or the C-terminus of the peptide may be bound to any one protecting group selected from the group consisting of an amino group (-NH2), a tertiary alkyl group, and an azide (-NHNH2). Further, the peptide may optionally further contain a targeting sequence, a tag, a labeled residue, an amino acid sequence produced for a specific purpose to increase the half-life or peptide stability.

[0014] The peptide is artificially synthesized or non-naturally occurring or engineered, where "non-naturally occurring or engineered" means a state that is not in its natural state of existence but is produced with artificial modification. Here, the artificial modification includes mimicking a plurality of amino acid structures and artificially synthesizing an amino acid sequence, or being engineered as described above to acquire chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0015] As used herein, the term "stability" may mean not only in vivo stability that protects the peptide from the attack of proteolytic enzymes in vivo, but also storage stability (for example, storage stability at room temperature).

[0016] Another aspect provides a pharmaceutical composition for preventing or treating muscle diseases, comprising as an active ingredient a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0017] Among the terms or elements mentioned in the description related to the peptide, those that are the same as those already mentioned are as described above.

[0018] As used herein, the term "prevention" means all acts of suppressing or delaying the onset of a disease by administration of the composition.

[0019] As used herein, the term "treatment" means any form of treatment that provides an effect including improvement of the state (for example, one or more symptoms) of an individual suffering from a disease or at risk of developing a disease, delay of disease progression, delay of symptom occurrence, or attenuation of symptom progression. Accordingly, the above-mentioned "treatment" and "prevention" are not intended to mean cure or complete removal of symptoms.

[0020] As used herein, the term "muscle disease" is a muscle disease, disorder or condition associated with reduced proliferation or differentiation of myoblasts, and also generally refers to muscle diseases due to, for example, reduced muscle function, muscle wasting or muscle degeneration. The muscle diseases include, but are not limited to, for example, sarcopenia, atony, muscular dystrophy, muscular atrophy, muscle degeneration, muscle stiffness, amyotrophic lateral sclerosis, muscle weakness and cachexia.

[0021] In one specific example, a pharmaceutical composition according to an embodiment can enhance muscle mass or muscle strength or improve muscle function by promoting the proliferation and differentiation of myoblasts.

[0022] Conventional functional peptides, despite having effective biological activities, have drawbacks in that due to the size of the peptide itself, they cannot effectively penetrate into target tissues or target cells, or have a short half-life and disappear in the body in a short period. On the other hand, a pharmaceutical composition according to an embodiment contains a peptide consisting of about 10 or fewer amino acids as an active ingredient, whereby the cell penetration rate of the active ingredient is very excellent. For example, when administered locally, a therapeutic effect on effective muscle diseases can be obtained.

[0023] According to one embodiment, the peptide of the present application can significantly increase the expression of early differentiation markers (such as MyoG and Myf5) and late differentiation markers of myoblasts, or muscle fiber constituent proteins (such as myosin heavy chain, α-actinin), and promote the differentiation into muscle cells or muscle fibers. In addition, the peptide of the present application not only improves muscle mass recovery / regeneration during muscle injury caused by external factors, but also infiltrates immune cells into the damaged muscle tissue, reduces the accumulation of collagen tissue, alleviates the symptoms of muscle diseases, and inhibits the progression of the disease. The peptide can be utilized as an active ingredient of a pharmaceutical composition for the treatment of muscle diseases.

[0024] The pharmaceutical composition also includes a pharmaceutically effective amount of the peptide and / or a pharmaceutically acceptable carrier, but is not limited thereto.

[0025] As used herein, the term "pharmaceutically effective amount" may mean an amount sufficient to achieve the therapeutic efficacy of the pharmaceutical composition for bone diseases.

[0026] The weight ratio of the peptide to the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500. As an example, the weight ratio may be 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2, but is not limited thereto.

[0027] The pharmaceutically acceptable carrier is one generally used in the manufacture of pharmaceuticals and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0028] The pharmaceutical composition may also contain, in addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives, etc., but is not limited thereto.

[0029] The pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, it may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0030] The dosage of the pharmaceutical composition is 0.0001 to 1,000 μg, 0.001 to 1,000 μg, 0.01 to 1,000 μg, 0.1 to 1,000 μg, or 1.0 to 1,000 μg per day, but is not limited thereto, and can be administered in various ways depending on factors such as the formulation method, administration method, age, weight, sex, medical condition, food, administration time, administration route, excretion rate, and reactivity of the patient.

[0031] The pharmaceutical composition is formulated into a unit dosage form or can be manufactured by being incorporated into a multi-dose container by using a pharmaceutically acceptable carrier and / or excipient by a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the invention pertains.

[0032] The dosage form is a solution, suspension, or emulsion in an oil or aqueous medium, or is in the form of an ointment, cream, gel, transdermal absorbent, cataplasm, patch, paste, extract, powder, granule, tablet, or capsule, and may also contain a dispersant and / or stabilizer added thereto.

[0033] The peptide can be contained in a nanosome or nanoparticle, for example, to further improve cell penetration problems or stability problems. For example, the nanosome uses lecithin as a raw material and is manufactured by a microfluidizer and can be contained, for example, within lecithin particles. Any method can be used as long as the method for manufacturing the nanosome is known. The size of the nanosome particles is also 10 to 200 nm, for example, 10 to 180 nm, 10 to 160 nm, 10 to 140 nm, 10 to 120 nm, 10 to 100 nm, 10 to 80 nm, 10 to 60 nm, 10 to 40 nm, 10 to 20 nm, 50 to 200 nm, 50 to 180 nm, 50 to 160 nm, 50 to 140 nm, 50 to 120 nm, 50 to 100 nm, 50 to 80 nm, or 50 to 60 nm.

[0034] The pharmaceutical composition may also contain an ingredient beneficial for the prevention or treatment of muscle diseases, and the ingredient may be, for example, any one selected from vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin H, vitamin PP, provitamin B5, vitamin A, vitamin D, vitamin E, vitamin K1 or carotene, or a mixture thereof, but is not limited thereto.

[0035] Another aspect provides a food composition for preventing or improving muscle diseases, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0036] Among the terms or elements mentioned in the description related to the foregoing peptide and pharmaceutical composition, those that are the same as those already mentioned are as described above.

[0037] As used herein, the term "improvement" may mean all acts that at least reduce a parameter related to the alleviation or treatment of a condition, such as the degree of symptoms.

[0038] In one specific example, the food composition according to an embodiment can increase muscle mass or muscle strength or improve muscle function through promoting the proliferation and differentiation of myoblasts.

[0039] The food composition can add the peptide as it is or use it together with other foods or food ingredients, and can be appropriately used by ordinary methods. The food composition may also contain a food auxillary additive acceptable in food science in addition to the active ingredient, and the mixing amount of the active ingredient can be appropriately determined according to the purpose of use (preventive, health or therapeutic treatment).

[0040] The food composition may also contain components beneficial for the prevention or improvement of muscle diseases, and the components include, for example, any one selected from vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin H, vitamin PP, provitamin B5, vitamin A, vitamin D, vitamin E, vitamin K1 or carotene, or a mixture thereof, but is not limited thereto.

[0041] In this specification, the term "food supplement" means a component that can be added to food supplementarily, and is added for manufacturing health functional foods of each dosage form, and can be appropriately selected and used by those skilled in the art. Examples of the food supplement include various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of the food supplement are not limited by the foregoing examples.

[0042] The food composition may also include health functional foods. In this specification, the term "health functional food" refers to a food manufactured and processed into forms such as tablets, capsules, powders, granules, liquids and pills using raw materials and components having useful functions for the human body. Here, "functionality" means adjusting nutrients or obtaining effects useful for health purposes such as physiological effects on the structure and function of the human body. The health functional food can be manufactured by methods generally used in the normal technical field, and during the foregoing manufacturing, raw materials and components generally added in the normal technical field can be added for manufacturing. Also, the dosage form of the health functional food can be manufactured without limitation as long as it is a dosage form recognized as a health functional food. The food composition is manufactured into various forms of dosage forms, and different from general drugs, it has the advantages of using food as a raw material and having no side effects that may occur during long-term taking of drugs, excellent portability, and a health functional food according to one embodiment can be ingested as an adjuvant for enhancing the therapeutic effect of muscle diseases.

[0043] In addition, there is no limitation on the types of health foods to which the composition according to an embodiment can be used / applied. The food composition containing the peptide as an active ingredient can be produced by mixing appropriate other auxiliary components that can be contained in health functional foods and known additives according to the selection of those skilled in the art. Examples of foods to which addition is possible include meats, sausages, bread, chocolate, candies, snacks, confectioneries, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, drinking water, tea, drink agents, alcoholic beverages, and vitamin complexes. In addition to these, it can also be produced by adding to juices, tea, jelly, and juices, etc.

[0044] Still another aspect provides a method for preventing or treating a muscle disease, which includes the step of administering to an individual a composition containing, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0045] Among the terms or elements mentioned in the descriptions related to the aforementioned peptide, pharmaceutical composition, food composition, etc., those that are the same as those already mentioned are as described above.

[0046] In this specification, the terms "administer" or "apply" are used interchangeably, and they can mean providing a predetermined substance to an individual or patient by any appropriate method. Further, the terms can mean causing at least partial localization of the composition according to an embodiment to a desired site, or disposing the composition according to an embodiment within an individual by an administration route.

[0047] In this specification, the term "individual" means a subject in need of improving skin condition, and more specifically, means a human or a non-human primate, and mammals such as mouse, dog, cat, horse, and cow.

[0048] The composition is, for example, also a pharmaceutical composition or a food composition containing, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0049] The content of the peptide as an active ingredient contained in the composition can be appropriately and non - restrictively selected according to the form of food / drug, desired use, etc. For example, it can be added at 0.01 to 15% by weight of the total composition weight. Also, for example, in a health drink composition, it can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml, but is not limited thereto.

Advantages of the Invention

[0050] According to the peptide according to one aspect, it can improve the expression of factors involved in the differentiation of myoblasts and promote the differentiation into muscle cells or muscle fibers.

[0051] According to the peptide according to one aspect, when there is muscle damage caused by external factors, it not only improves muscle mass recovery / regeneration, but also infiltrates immune cells into the damaged muscle tissue, reduces the accumulation of collagen tissue, alleviates the symptoms of muscle diseases, and inhibits the progression of the disease.

[0052] Therefore, the peptide according to one aspect can be utilized as an active ingredient of a composition for preventing, improving or treating muscle diseases.

Brief Description of the Drawings

[0053]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0054] Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

[0055] Example 1. Synthesis of Peptide Using an automatic peptide synthesizer (Milligen 9050 (Millipore (USA))), a peptide having the amino acid sequence of SEQ ID NO: 1 described in Table 1 below was synthesized, and C18 reverse phase high performance liquid chromatography (HPLC) (Waters Associates (USA)) was used to purify the synthesized peptides. The column used was ACQUITY UPLC BEH300 C18 (2.1 mm X 100 mm, 1.7 μm (Waters Co (USA))).

[0056]

Table 1

[0057] Example 2. Confirmation of the effect of promoting the differentiation of myoblasts in the initial differentiation stage In this example, by evaluating the change in the expression of the initial differentiation marker protein of C2C12 cells, which are mouse myoblasts, the effect of the peptide according to one example on the differentiation of myoblasts into muscle cells / muscle fibers in the initial differentiation stage was confirmed.

[0058] 2-1. Evaluation through protein expression analysis After culturing C2C12 cells in DMEM medium containing 1% P / S and 2% BCS, when the cell confluency reached about 70 - 80%, they were seeded into a 6-well culture plate. Then, when the cell confluency of the cells in the 6-well culture plate reached 100%, while replacing the culture medium with a differentiation medium (1% P / S, 2% horse serum), 5, 50, or 100 μg / ml of the peptide of SEQ ID NO: 1 was added thereto. Thereafter, the same amount of the peptide of SEQ ID NO: 1 as the differentiation medium was added alternately once every two days. Three days after the first medium replacement, lysis buffer was added to the culture to lyse the culture, and then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain the protein, which was quantified using a BCA kit. SDS-PAGE was performed on the sample containing the protein to separate each protein, and then it was electrotransferred to a membrane. The membrane to which the protein was attached was treated with 5% non-fat dry milk for blocking, and then reacted with the primary antibody at 4°C overnight. After washing with PBS-T, it was reacted with the secondary antibody at room temperature for 1 hour, and further washed with PBS-T, and then detected via Gel Doc (Bio-Rad (Hercules (CA, USA))) and Western detection reagent (Elpis Biotech (Daejeon (Korea))). As the primary antibody, anti-Myo G (Santa Cruz (USA)) was used, anti-Myf5 (Abcam (UK)) and anti-GAPDH (Santa Cruz (USA)) were used, as the control group, the non-treated group (Non) was used, and as the comparison group, the group treated with only the differentiation medium (CM) was used.

[0059] As a result, as shown in FIGS. 1 and 2, it was confirmed that the expression of Myo G (myogenin) and Myf5, which are early differentiation markers of myoblasts, was increased by the treatment with the peptide of SEQ ID NO: 1.

[0060] 2 - 2. Evaluation via immunofluorescence method C2C12 cells were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached 70-80%, they were seeded into 6-well culture plates. When cell confluency reached 100%, the culture medium was replaced with differentiation medium (1% P / S, 2% horse serum) and 100 μg / ml of SEQ ID NO: 1 peptide was added. Subsequently, differentiation medium and 100 μg / ml of SEQ ID NO: 1 peptide were alternately added every two days. Three days after the initial medium change, the cultured cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes and permeabilized with 0.1% Triton X-100 for 5 minutes. The fixed cells were then blocked with PBS, 10% FBS, and 0.1% Triton X-100 at room temperature for 1 hour, followed by treatment with the primary antibody Myo G (Santa Cruz, USA) and incubation at room temperature for 1 hour. The cells were then washed and further incubated with the secondary antibody (goat anti-mouse IgG-TR (Santa Cruz, USA)) at room temperature for 30 minutes. Cell nuclei were then stained with DAPI (Santa Cruz, USA) and observed under a LEICA fluorescence microscope (TCSSP8 (LEICA, Germany)).

[0061] As a result, as shown in Figures 3 and 4, it was confirmed that treatment with the peptide of Sequence No. 1 not only increased the expression of Myo G (myogenin), an early differentiation marker of myoblasts, but also increased its expression in the nuclear region of myoblasts.

[0062] Taking the above experimental results together, it was found that the peptide according to one embodiment contributes to promoting the early differentiation of myoblasts into muscle cells.

[0063] Example 3. Confirmation of the effect of promoting differentiation of myoblasts at the late differentiation stage In this example, by evaluating the expression changes of the initial differentiation marker proteins of C2C12 cells, which are mouse myoblasts, the effect of the peptide according to one example on the differentiation of myoblasts into muscle cells / muscle fibers in the late differentiation stage was confirmed.

[0064] 3-1. Evaluation via protein expression analysis Except for the time point when the lysis buffer was added (7 days after the first medium change), the procedure was carried out in the same manner as in Example 2-1, and the expression level of MyHC (Myosin heavy chain) protein, which is a late differentiation marker of myoblasts, was confirmed. As the primary antibodies, anti-MyHC (Santa Cruz, USA) and anti-GAPDH (Santa Cruz, USA) were used, and as the control group, the non-treated group (Non) was used, and as the comparison group, the group treated with only the differentiation medium (CM) was used.

[0065] As a result, as shown in FIGS. 5 and 6, it was confirmed that the expression of MyHC (Myosin heavy chain), which is a late differentiation marker of myoblasts, was increased by the treatment with the peptide of SEQ ID NO: 1.

[0066] 3-2. Evaluation via immunofluorescence method Except for the fixation time point (7 days after the first medium change), the procedure was carried out in the same manner as in Example 3-1, and the expression level of MyHC (Myosin heavy chain) protein, which is a late differentiation marker of myoblasts, was confirmed. As the primary antibody, anti-α-actinin (Santa Cruz, USA) was used, and as the secondary antibody, goat anti-mouse IgG-TR (Santa Cruz, USA) was used, and as the comparison group, the group treated with only the differentiation medium (CM) was used.

[0067] As a result, as shown in FIGS. 7 and 8, it was confirmed that the expression of α-actinin, which is a late differentiation marker of myoblasts, was increased by the treatment with the peptide of SEQ ID NO: 1.

[0068] Based on the comprehensive experimental results, it was found that the peptide according to one embodiment contributes to promoting the late differentiation of myoblasts into muscle cells.

[0069] Example 4. Confirmation of muscle loss inhibitory effect using an animal model In this example, for an animal model in which muscle injury was induced by BaCl2, by evaluating the change in muscle mass, the infiltration area of immune cells in muscle tissue, the level of accumulated collagen, and the expression levels of muscle fiber constituent proteins and inflammatory proteins, the effect of the peptide according to one embodiment on inhibiting muscle loss was confirmed.

[0070] 4-1. Muscle mass evaluation A 1.2% (w / v) 50 μl aqueous BaCl2 solution was injected into the entire tibialis anterior (TA) muscle of 8-week-old male C57BL / 6 mice to induce muscle injury in the mice for 1 day. Thereafter, 20 mg of the peptide of SEQ ID NO: 1 was orally administered to the mice with induced muscle injury at a concentration of 0.1 mg / μl at intervals of 1 day for a total of 6 times. For the positive control group, 0.5 μg of IGF-I was intramuscularly injected at a concentration of 0.01 μg / μl. On the 7th day from the day when muscle injury was induced, the mice were sacrificed, and the change in the entire tibialis anterior (TA) muscle was visually confirmed and its weight was measured. As the control group, a non-treated group (Non) was used, as the negative control group, a group (N.C) in which only muscle injury by BaCl2 was induced was used, and as the positive control group, a group (P.C) in which IGF-I was treated in the group in which muscle injury by BaCl2 was induced was used.

[0071] As a result, as shown in FIGS. 9 and 10, it was confirmed that the muscle mass of the entire tibialis anterior (TA) muscle of mice with muscle injury induced by BaCl2 was increased by treatment with the peptide of SEQ ID NO: 1, and thereby it was found that the regeneration of the damaged muscle could be promoted.

[0072] 4-2. Evaluation via muscle tissue staining The muscle tissue obtained from the mouse with induced muscle injury in Example 4-1 was washed with PBS at room temperature and then fixed with 4% paraformaldehyde (PFA). Subsequently, the fixed muscle tissue was washed three times with PBS and dehydrated with a gradient ethanol series (from 70% to 100%). The muscle tissue sample was sectioned to a size of 4 μm. The tissue slide containing the sectioned specimen was dewaxed and hydrated through a gradient ethanol series, stained with hematoxylin solution for 1 minute, and then stained with eosin solution for 10 seconds. After the staining was completed, the tissue slide was immersed successively in 90% and 100% ethanol, and finally immersed in xylene twice for 5 minutes each, and then mounting of the tissue slide was carried out (H&E staining).

[0073] Also, the tissue slide containing the aforementioned sectioned specimen was dewaxed and hydrated as described above, stained with Picro-Sirius Red Solution for 60 minutes, immersed twice in 1% acetic acid solution for 2 minutes each, and washed with D.W. Subsequently, the tissue slide was immersed in ethanol and finally immersed in xylene twice for 5 minutes each, and then mounting of the tissue slide was carried out (Sirius red staining). As a result, as shown in FIGS. 11 and 12, it was confirmed that the infiltration area of immune cells in the tissue with muscle injury induced by BaCl2 was reduced by the treatment with the peptide of SEQ ID NO: 1. Also, as shown in FIGS. 13 and 14, it was confirmed that the accumulation of collagen in the tissue with muscle injury induced by BaCl2 was reduced by the treatment with the peptide of SEQ ID NO: 1.

[0074] 4-3. Evaluation through Protein Expression Analysis in Muscle Tissue The muscle tissue obtained from the mouse in which muscle injury was induced in Example 4-1 was disrupted using a homogenizer, and a lysis buffer was added thereto to dissolve the disrupted matter. Then, centrifugation was performed at 4°C and 12,000 rpm for 30 minutes to obtain proteins, and they were quantified using a BCA kit. After SDS-PAGE was performed on the sample containing the proteins to separate each protein, it was electrotransferred onto a membrane. After the membrane to which the proteins adhered was treated with 5% non-fat dried milk for blocking, a primary antibody was reacted overnight at 4°C. After washing with PBS-T, a secondary antibody was reacted at room temperature for 1 hour and then washed with PBS-T, and then it was detected via GelDoc (Bio-Rad, Hercules, CA (USA)) and a Western detection reagent (ElpisBiotech, Daejeon, Korea). On the other hand, as the primary antibody, anti-MyHC (Santa Cruz (USA)) was used, and anti-α-actinin (Santa Cruz (USA)), anti-α-SMA (Abcam, UK), anti-IL-6 (Santa Cruz (USA)), and anti-α-tubulin (Santa Cruz (USA)) were used. As a result, as shown in FIGS. 15 and 16, it was confirmed that the expression of MyHC and α-actinin, which are intramuscular fiber constituent proteins in the tissue in which muscle injury was induced by BaCl2, was increased by the treatment with the peptide of SEQ ID NO: 1. Further, as shown in FIGS. 17 and 18, it was confirmed that the expression of α-SMA and IL-6, which are inflammatory proteins in the tissue in which muscle injury was induced by BaCl2, was reduced by the treatment with the peptide of SEQ ID NO: 1.

[0075] From the above experimental results, it can be known that the peptide according to one embodiment can not only cause the regeneration or recovery of damaged muscle, but also contribute to suppressing the progression of muscle injury, such as an inflammatory reaction that causes muscle injury.

[0076] Formulation Example 1. Production of Peptide Nanosomes 50 mg of the peptide of Example 1 was dissolved by sufficient stirring with 500 ml of distilled water. After mixing the formulation solution with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and some oiliness, the volume was adjusted with distilled water to a total volume of 1 L, and then emulsified using a microfluidizer under high pressure to produce peptide nanosomes of about 100 nm in size.

[0077] Formulation Example 2. Pharmaceutical Preparation 2-1. Preparation of Powder The following components are mixed and filled into an airtight package to produce a powder. 20 mg of the peptide of the present invention 100 mg of lactose 10 mg of talc

[0078] 2-2. Preparation of Tablet After mixing the following components, they are tabletted by a usual tablet preparation method to produce tablets. 10 mg of the peptide of the present invention 100 mg of corn starch 100 mg of lactose 2 mg of magnesium stearate

[0079] 2-3. Preparation of Capsule The following components are mixed by a usual capsule preparation method and filled into gelatin capsules to produce capsules. 10 mg of the peptide of the present invention 3 mg of crystalline cellulose 14.8 mg of lactose 0.2 mg of magnesium stearate

[0080] 2-4. Preparation of Injectable It is prepared by a usual injectable preparation method with the following component contents per ampoule (2 ml). 10 mg of the peptide of the present invention 180 mg of mannitol 2,974 mg of sterile distilled water for injection 26 mg of Na2HPO4·2H2O 2-5. Production of Liquid Agent

[0081] By the usual method for producing a liquid agent, each component is added to purified water and dissolved, and after mixing the following components, purified water is added, the whole is adjusted to 100 ml, and then filled into a brown bottle and sterilized to produce a liquid agent. 10 mg of the peptide of the present invention 10 g of isomerized sugar 5 g of mannitol Purified water (appropriate amount)

[0082] The above description of the present invention is for illustrative purposes, and those with ordinary knowledge in the technical field to which the present invention pertains will be able to understand that it can be easily deformed into other specific forms without changing the technical idea and essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all aspects and not restrictive.

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, wherein the N-terminus of the peptide is linked to any one protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG).

3. The C-terminus of the peptide is bonded to any one protecting group selected from the group consisting of an amino group (-NH ２ ), a tertiary alkyl group, and an azide (-NHNH ２ ). The peptide according to claim 1.

4. The peptide according to claim 1, wherein the peptide exhibits any one or more of the following characteristics: (a) Enhancement of the expression of myogenin and myogenic factor 5, (b) Enhancement of the expression of myosin heavy chain and α-actinin, and (c) Reduction of the expression of α-smooth muscle actin and interleukin-6.

5. A pharmaceutical composition for preventing or treating muscle diseases, comprising the peptide according to any one of claims 1 to 4 as an active ingredient.

6. The pharmaceutical composition according to claim 5, wherein the muscle disease is a muscle disease caused by muscle function decline, muscle wasting, or muscle degeneration.

7. The pharmaceutical composition according to claim 6, wherein the muscle disease is any one of sarcopenia, atony, muscular dystrophy, muscular atrophy, muscle degeneration, muscle stiffness, amyotrophic lateral sclerosis, myasthenia gravis, and cachexia.

8. A food composition for preventing or improving muscle diseases, comprising the peptide according to any one of claims 1 to 4 as an active ingredient.

9. The food composition according to claim 8, wherein the muscle disease is a muscle disease caused by muscle function decline, muscle wasting, or muscle degeneration.

10. The food composition according to claim 8, further comprising any one component selected from vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin H, vitamin PP, provitamin B5, vitamin A, vitamin D, vitamin E, vitamin K1 or carotene, or a mixture thereof.

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