Novel peptide having activities of inhibiting muscle loss and promoting muscle growth, and uses thereof

Abstract

The present invention relates to a peptide having the activities of inhibiting sarcopenia, promoting muscle growth, and inhibiting fatty liver. The peptide according to the present invention can promote the proliferation of muscle cells and the differentiation thereof into muscle fibers, has a muscle regeneration effect even under conditions of muscular atrophy, and can significantly inhibit the expression of fat synthesis-related genes / proteins in hepatocytes. Thus, the peptide according to the present invention is expected to be effectively used for inhibiting sarcopenia, promoting muscle growth, and inhibiting fatty liver.

Description

Novel peptides having muscle loss inhibition and muscle mass increase activities and their uses

[0001] [Cross-citation with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2024-0005392, filed January 12, 2024, the entire contents of which are incorporated herein by reference.

[0003] [Technical Field]

[0004] The present invention relates to a novel peptide having muscle loss inhibition and muscle mass increase activity, and its use. Furthermore, the present invention relates to the use of the novel peptide for the inhibition of fatty liver disease.

[0005] With the aging population rapidly increasing worldwide, a United Nations report projects that the number of people aged 60 and older will exceed 2 billion by 2050. Skeletal muscle is the largest organ in the body, accounting for 50% of total body weight, and muscle loss and weakening are among the major physical changes that occur with aging. Skeletal muscle mass tends to decrease by approximately 1% per year after age 30, and then rapidly declines after age 65. This rapid loss of muscle mass can significantly impact quality of life, including diminished physical ability and an increased risk of falls and fractures. Reflecting these concerns, the World Health Organization (WHO) officially recognized sarcopenia as a disease in 2017.

[0006] Sarcopenia, caused by degeneration of spinal nerves, motor nerves, or skeletal muscle fibers associated with muscle disease, is a representative intractable disease with unknown causes. Previous research has shown that the motor nerves that drive skeletal muscle contraction degenerate, preventing skeletal muscle contraction. Alternatively, the expression of proteins involved in muscle contraction within skeletal muscle is reduced or altered, preventing normal skeletal muscle contraction. Over the long term, the motor nerves or skeletal muscle are known to transform into fibrous tissue. Because the fundamental cause of sarcopenia remains unknown, and methods to prevent or reverse the degeneration of motor nerves or skeletal muscles have yet to be developed, active research is currently underway to develop methods to slow the progression of sarcopenia. While exercise, protein, and calorie supplementation are known to be helpful for sarcopenia, these treatments are not particularly effective in the elderly, who account for the majority of patients. Therefore, there is a pressing need for treatments for sarcopenia.

[0007] Meanwhile, muscle size or mass is regulated by intracellular signaling processes that induce anabolic or catabolic reactions within the muscle. Specifically, when signaling responses that induce muscle protein synthesis are dominant over those that induce muscle protein degradation, muscle protein synthesis increases, resulting in increased muscle size (hypertrophy) or muscle fiber number (hyperplasia). Furthermore, muscle cell differentiation and muscle formation are regulated by various muscle regulatory factors. MyoD initiates the expression of muscle-specific genes and induces the differentiation of muscle satellite cells into myoblasts. The induction of myogenin expression by MyoD activation is a crucial factor in the fusion of myoblasts, contributing to the formation of myotubes. Muscle fibers formed through this process form bundles, ultimately forming muscles.

[0008] Under this technological background, various studies are being conducted to inhibit muscle loss caused by external or internal factors and to promote muscle protein synthesis (Korean Patent No. 10-2064387), but the results are still insufficient.

[0009] [Prior Art Literature]

[0010] [Patent Document]

[0011] Republic of Korea Patent No. 10-2064387

[0012] The present inventors have conducted research to develop an effective active substance that can be used to prevent, improve, or treat muscle diseases by suppressing sarcopenia and promoting muscle growth. As a result, the inventors experimentally confirmed that the novel peptide they synthesized exhibits excellent sarcopenia suppression and muscle growth promotion activities, as well as excellent fatty liver inhibition activity, thereby completing the present invention.

[0013] Accordingly, the purpose of the present invention is to provide a novel peptide having various physiological activities such as inhibition of muscle loss, increase in muscle mass, and inhibition of fatty liver, and a pharmaceutical composition or health functional food containing the same.

[0014] In order to achieve the purpose of the present invention described above,

[0015] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0016] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising the peptide as an active ingredient.

[0017] Another aspect of the present invention provides a food composition for preventing or improving muscle disease, comprising the peptide as an active ingredient.

[0018] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating fatty liver comprising the peptide as an active ingredient.

[0019] Another aspect of the present invention provides a food composition for preventing or improving fatty liver, comprising the peptide as an active ingredient.

[0020] The present invention is described in detail below.

[0021] 1. Peptides and their activities

[0022] According to one aspect of the present invention, a peptide comprising an amino acid sequence of SEQ ID NO: 1 is provided.

[0023] [Amino acid sequence of sequence number 1]

[0024] DTVEN

[0025] The term "peptide" as used herein means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.

[0026] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention can be used without modification, but a variant or fragment of amino acids having a different sequence by deletion, insertion, substitution, or a combination thereof of amino acid residues can be used within a range that does not affect the original activity of the peptide, such as muscle loss inhibition, muscle mass increase, and fatty liver inhibition activity.

[0027] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., within a range that does not change its activity.

[0028] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to a peptide comprising an amino acid sequence of SEQ ID NO: 1, and a variant or active fragment thereof. The substantially identical amino acid sequence refers to an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or 98% or more, with the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0029] The peptide of the present invention may be modified at the N-terminus and / or C-terminus to select a portion of the amino acid sequence and increase its activity. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptide of the present invention, for example, increasing the half-life of the peptide when administered in vivo. The term "stability" encompasses not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0030] The above N-terminal modification may be a modification in which a 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, and polyethylene glycol (PEG) is bonded to the N-terminus of the peptide. The above C-terminal modification may be a modification in which a hydroxyl group (-OH), an amino group (-NH2), a hydrazine group (-NHNH2), or the like is bonded to the C-terminus of the peptide, but is not limited thereto.

[0031] The peptide of the present invention can be manufactured using various methods widely known in the art. For example, the peptide of the present invention can be manufactured using chemical synthesis methods known in the art, particularly solid-phase synthesis techniques or liquid-phase synthesis techniques.

[0032] In addition, the peptide of the present invention is artificially synthesized, or non-naturally occurring or engineered, wherein "non-naturally occurring or engineered" means a state that is not a state that occurs in nature, but is created by artificial modification. Here, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking a plurality of amino acid structures, or engineering to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0033] The peptide of the present invention has muscle loss inhibition and muscle mass increase activity.

[0034] In addition, the peptide of the present invention has fatty liver inhibitory activity.

[0035] Therefore, the peptide of the present invention can be used for the prevention, improvement or treatment of muscle diseases and for the prevention, improvement or treatment of fatty liver.

[0036] In one embodiment, the peptides of the present invention have one or more of the following activities:

[0037] (i) Activity that promotes proliferation and differentiation of myoblasts;

[0038] (ii) activity that increases the expression levels of pPI3K and p70S6K proteins, which are myogenic markers;

[0039] (iii) Activity that increases the expression levels of mRNA and protein of muscle fiber differentiation markers MyoG, Myf4, Myf5, Myf6 and α-actinin;

[0040] (iv) Activity in increasing the expression levels of mRNA and protein of SIRT1, AKT, mTOR, p70S6K and IGF-1, which are muscle protein synthesis markers;

[0041] (v) activity that increases the myotube synthesis capacity of myoblasts; and

[0042] (vi) Activity that reduces the expression levels of mRNA and protein of SREBP1 and FAS, which are fat synthesis markers.

[0043] 2. Composition for preventing, improving or treating muscle diseases

[0044] pharmaceutical composition

[0045] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating muscle disease is provided, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0046] The peptide containing the amino acid sequence of the above sequence number 1 is identical to the peptide described in the above section “1. Peptide and its activity”, and a detailed description thereof is cited and not described repeatedly.

[0047] The peptide of the present invention having the above-described activity exhibits excellent efficacy in suppressing muscle loss and increasing muscle mass, and can be used for the prevention or treatment of muscle diseases.

[0048] Meanwhile, conventional functional peptides, despite their effective biological activity, have shown the disadvantage of not being able to effectively enter target tissues or cells due to the size of the peptide itself, or being eliminated from the body in a short period of time due to a short half-life. On the other hand, the pharmaceutical composition for preventing or treating muscle diseases of the present invention comprises a peptide composed of about 10 amino acids or less as an active ingredient, and therefore has excellent cell penetration rate of the active ingredient, and for example, when administered topically, it can obtain an effective preventive or therapeutic effect for muscle diseases.

[0049] As used herein, the term "muscle disease" may collectively refer to a muscle disease, illness or condition associated with a decrease in the proliferation or differentiation of myoblasts, for example, muscle diseases resulting from muscle dysfunction, muscle wasting or muscle degeneration. The muscle disease may be, but is not limited to, one or more selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle rigidity, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration and cachexia.

[0050] In one embodiment, the peptide of the present invention can promote the proliferation and differentiation of myogenic cells, thereby inhibiting muscle loss and increasing muscle mass. Specifically, the peptide of the present invention can promote the proliferation of myogenic cells and significantly increase the expression of early differentiation markers, mid-differentiation markers, and late differentiation markers of myogenic cells, thereby promoting differentiation into muscle cells or muscle fibers. In addition, the peptide of the present invention can significantly increase the expression of myogenic markers and muscle protein synthesis markers, and can also increase the myotube synthesis capacity of myogenic cells, and can enhance muscle mass recovery / regeneration when myogenic cells are damaged by exogenous factors.

[0051] Therefore, the peptide of the present invention can be effectively utilized as an active ingredient of a pharmaceutical composition for preventing or treating muscle diseases, and the pharmaceutical composition of the present invention can be effectively utilized for preventing or treating muscle diseases.

[0052] As used herein, the term “prevention” means any act of inhibiting or delaying the onset of a disease by administering the composition.

[0053] As used herein, the term "treatment" refers to any form of treatment that provides a benefit to a subject suffering from a disease or at risk of developing a disease, including improving the condition of the subject (e.g., one or more symptoms), delaying the progression of the disease, delaying the onset of symptoms, or slowing the progression of symptoms. Accordingly, the terms "treatment" and "prevention" are not intended to imply a cure or complete elimination of symptoms.

[0054] As used herein, the term "administration" means introducing a given substance into an individual by any appropriate method, and the pharmaceutical composition of the present invention may be administered via any general route capable of reaching an in vivo target. For example, the route of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be administered orally or parenterally, and in the case of parenteral administration, may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0055] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 to 1000 μg, 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg per day, and may be administered in various amounts depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. In addition, the frequency of administration of the pharmaceutical composition may be 1 to 4 times, 2 to 3 times, or 2 times per day, and the administration period may be, but is not limited to, 4 weeks or more, 8 weeks or more, 4 to 12 weeks, or 8 to 12 weeks.

[0056] The pharmaceutical composition of the present invention may additionally comprise a therapeutically effective amount of the above-described peptide and / or an appropriate carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

[0057] The term "therapeutically effective amount" above means an amount sufficient for the peptide, which is an active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, for example, an amount sufficient to achieve the efficacy of preventing or treating muscle disease.

[0058] Examples of carriers, excipients or diluents usable in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0059] The pharmaceutical composition of the present invention can be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, and can be manufactured in the form of unit doses or by being placed in multi-dose containers.

[0060] When formulating, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0061] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium, styrax, and talc can also be used.

[0062] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0063] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include withepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0064] Preparations for transdermal administration may include dusting powders, emulsions, suspensions, oils, sprays, ointments, cream pastes, gels, foams, or solutions. The pharmaceutical compositions of the present invention may be an anhydrous ointment, containing paraffin, especially low viscosity paraffin, which is suitable for topical use and is liquid at body temperature, or may contain natural or partially synthetic fats, for example coconut fatty acid triglycerides, hydrogenated oils, for example hydrogenated peanut oil or castor oil, partial fatty acid esters of glycerol, for example glycerol monostearate and distearate, silicones, for example polymethylsiloxanes, such as hexamethyldisiloxane or octamethyltrisiloxane, fatty alcohols, for example those associated with aqueous creams and which increase the water absorption capacity, and sterols, wool wax, other emulsifiers and / or other additives.

[0065] The dosage of the peptide comprising the amino acid sequence of SEQ ID NO: 1 contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and period of administration, and may be appropriately selected depending on the case. For example, the peptide comprising the amino acid sequence may be administered at a dosage of 0.0001 to 1000 mg / kg per day, specifically 0.1 to 1000 mg / kg, and the application may be administered once a day or divided into several times. The dosage of the peptide comprising the peptide comprising the amino acid sequence of the present invention may be increased or decreased depending on the route of administration, the severity of the disease, gender, weight, age, etc. Therefore, the dosage does not limit the scope of the present invention in any way.

[0066] The pharmaceutical composition of the present invention may contain a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention at a concentration of 0.01 μM to 1000 μM, specifically, the peptide is present at a concentration of 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; It may be included at a concentration of, but is not limited to, 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1 μM to 200 μM; 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM.

[0067] The weight ratio between the peptide and the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500, and 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 It can be, but is not limited to, 1:4, or 2:1 or 1:2.

[0068] Additionally, the peptide may be incorporated into nanosomes or nanoparticles, for example, to further improve cell penetration or stability. For example, the nanosomes may be manufactured using a microfluidizer using lecithin as a raw material, and may be incorporated into lecithin particles, for example. Any known method for manufacturing the nanosomes may be used. The size of the nanosome particles may be from 10 to 200 nm, for example, from 10 to 180 nm, from 10 to 160 nm, from 10 to 140 nm, from 10 to 120 nm, from 10 to 100 nm, from 10 to 80 nm, from 10 to 60 nm, from 10 to 40 nm, from 10 to 20 nm, from 50 to 200 nm, from 50 to 180 nm, from 50 to 160 nm, from 50 to 140 nm, from 50 to 120 nm, from 50 to 100 nm, from 50 to 80 nm, or from 50 to 60 nm.

[0069] Meanwhile, the pharmaceutical composition of the present invention may additionally contain one or more ingredients that exhibit improvement, alleviation, treatment, or prevention of muscle disease in addition to the peptide comprising the amino acid sequence of SEQ ID NO: 1. The ingredient may be, for example, any one ingredient 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 mixtures thereof, but is not limited thereto.

[0070] Another aspect of the present invention provides a method for preventing or treating muscle disease, comprising the step of administering to a subject a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a pharmaceutical composition comprising the peptide as an active ingredient.

[0071] In one embodiment, the subject comprises a subject in need of prevention or treatment of a muscle disorder.

[0072] The term "subject" above refers to a human or non-human animal, such as humans, primates, mammals and vertebrates.

[0073] In addition, another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a pharmaceutical composition comprising the peptide as an active ingredient for preventing or treating muscle disease.

[0074] Food composition

[0075] According to another aspect of the present invention, a food composition for preventing or improving muscle disease is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0076] Among the terms or elements mentioned in the description of the above peptide and pharmaceutical composition, those already mentioned are as described above.

[0077] In one embodiment, the peptide of the present invention can promote the proliferation and differentiation of myogenic cells, thereby inhibiting muscle loss and increasing muscle mass. Therefore, the peptide of the present invention can be effectively utilized as an active ingredient in a food composition for preventing or ameliorating muscle diseases, and the food composition of the present invention can be effectively utilized for the prevention or amelioration of muscle diseases.

[0078] As used herein, the term "improvement" may mean any action that at least reduces the severity of a symptom, for example, a parameter associated with alleviating or treating a condition.

[0079] The food composition described above can be used by adding the peptide as is or in combination with other foods or food ingredients, and can be used appropriately according to a conventional method. For example, it can contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. Examples of the natural carbohydrates described above include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. In addition to the flavoring agents described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrates described above can be appropriately determined by a person skilled in the art.

[0080] In addition to the above, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), 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. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0081] In addition, the food composition of the present invention may additionally contain one or more ingredients that exhibit improvement, alleviation, treatment, or prevention of muscle disease, in addition to the peptide comprising the amino acid sequence of SEQ ID NO: 1. The ingredient may be, for example, any one ingredient 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 mixtures thereof, but is not limited thereto.

[0082] Meanwhile, the food composition may include a health functional food. As used herein, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients that have functionality useful to the human body. Here, "functionality" means obtaining a beneficial effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food may be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art may be added. In addition, the formulation of the health functional food may be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition may be manufactured in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material, and is highly portable. According to one embodiment, the health functional food can be consumed as a supplement to enhance the therapeutic effect of muscle disease.

[0083] In addition, there is no limitation on the type of health functional food to which the food composition according to one embodiment can be used / applied. The food composition containing the peptide as an active ingredient can be prepared by mixing other appropriate auxiliary ingredients that can be included in health functional foods and known additives according to the selection of a person skilled in the art. Examples of foods to which the peptide can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and in addition, it can be prepared by adding it to juice, tea, jelly, and juice.

[0084] 3. Composition for preventing or treating fatty liver

[0085] pharmaceutical composition

[0086] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating fatty liver is provided, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0087] The peptide containing the amino acid sequence of the above sequence number 1 is identical to the peptide described in the above "1. Peptide and its activity" section, and the terms or elements mentioned in the description of the pharmaceutical composition and food composition are identical to those already mentioned in the above "2. Composition for preventing, improving or treating muscle disease" section, and the specific description is based on this and is not described repeatedly.

[0088] The peptide of the present invention having the above-described activity exhibits excellent efficacy in inhibiting fat synthesis and can be used for the prevention or treatment of fatty liver.

[0089] In one embodiment, the peptide of the present invention can significantly reduce the expression of factors involved in fat synthesis in hepatocytes. Therefore, the peptide of the present invention can be effectively utilized as an active ingredient in a pharmaceutical composition for the prevention or treatment of fatty liver, and the pharmaceutical composition of the present invention can be effectively utilized for the prevention or treatment of fatty liver.

[0090] Meanwhile, the pharmaceutical composition of the present invention may additionally contain one or more ingredients that exhibit improvement, alleviation, treatment or prevention of fatty liver, in addition to the peptide comprising the amino acid sequence of sequence number 1.

[0091] Another aspect of the present invention provides a method for preventing or treating fatty liver, comprising the step of administering to a subject a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a pharmaceutical composition comprising the peptide as an active ingredient.

[0092] In one embodiment, the subject comprises a subject in need of prevention or treatment of fatty liver.

[0093] In addition, another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a pharmaceutical composition comprising the peptide as an active ingredient for preventing or treating fatty liver.

[0094] Food composition

[0095] According to another aspect of the present invention, a food composition for preventing or improving fatty liver is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0096] Among the terms or elements mentioned in the description of the above peptide and pharmaceutical composition, those already mentioned are as described above.

[0097] In one embodiment, the peptide of the present invention can significantly reduce the expression of factors involved in fat synthesis in hepatocytes. Therefore, the peptide of the present invention can be effectively utilized as an active ingredient in a food composition for preventing or improving fatty liver disease, and the food composition of the present invention can be effectively utilized for the prevention or improvement of fatty liver disease.

[0098] Meanwhile, the food composition of the present invention may additionally contain one or more ingredients that exhibit improvement, alleviation, treatment or prevention of fatty liver, in addition to the peptide comprising the amino acid sequence of sequence number 1.

[0099] The peptide of the present invention has sarcopenia inhibition, muscle growth promotion, and fatty liver inhibition activities. Specifically, the peptide of the present invention can promote muscle cell proliferation and differentiation into muscle fibers, has a muscle regeneration effect even under muscle atrophy conditions, and can significantly suppress the expression of fat synthesis-related genes / proteins in hepatocytes. Therefore, the peptide of the present invention is expected to be effectively utilized for sarcopenia inhibition, muscle growth promotion, and fatty liver inhibition.

[0100] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0101] Figure 1 shows that the peptide of the present invention promotes cell proliferation of C2C12 myoblasts.

[0102] Figure 2 shows that the peptide of the present invention increases the protein expression levels of pPI3K and p70S6K, which are myogenesis-related proteins, in C2C12 cells.

[0103] Figures 3a and 3b show that the peptide of the present invention increases the mRNA expression levels of MyoG, Myf6, Myf5 and Myf4 genes, which are early / mid-stage differentiation-related genes, in C2C12 cells.

[0104] Figures 4a and 4b show that the peptide of the present invention increases the protein expression levels of Myf5, MyoG and α-actinin, which are early / middle / late differentiation-related proteins, in C2C12 cells.

[0105] Figures 5a and 5b show that the peptide of the present invention increases the mRNA expression levels of SIRT1, mTOR, AKT, P70S6K, and IGF-1 genes, which are genes related to muscle protein synthesis, in C2C12 cells.

[0106] Figures 6a and 6b show that the peptide of the present invention increases the protein expression levels of SIRT1, IGF-1, pmTOR, and p70S6K, which are proteins related to muscle protein synthesis, in C2C12 cells.

[0107] Figure 7 shows that the peptide of the present invention increases myotube synthesis ability in C2C12 cells.

[0108] Figure 8 shows that the peptide of the present invention restores the protein expression levels of MyoD and α-actinin, which are differentiation-related proteins, in C2C12 cells in which an atrophy environment is induced.

[0109] Figure 9 shows that the peptide of the present invention inhibits the mRNA expression levels of SREBP1 and FAS genes, which are fat synthesis-related genes, in HepG2 cells.

[0110] Figure 10 shows that the peptide of the present invention inhibits the protein expression level of SREBP1, a fat synthesis-related protein, in HepG2 cells.

[0111] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0112] Manufacturing Example 1: Manufacturing of peptides

[0113] A peptide having the amino acid sequence of SEQ ID NO: 1 shown in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was purified using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm Х 100 mm, 1.7 μm, Waters Co, USA).

[0114] Sequence number Amino acid sequence (N-terminal → C-terminal) 1DTVEN

[0115] The efficacy of the peptide of sequence number 1 manufactured above was evaluated through the following experiment.

[0116] Experimental Example 1: Analysis of peptides' myogenic cell proliferation activity

[0117] To analyze the effect of the peptide manufactured in Manufacturing Example 1 on the proliferation of myoblasts, a cell viability / cytotoxicity assay was performed.

[0118] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at a density of 5 x 10 3Cells were seeded in 96-well culture plates at a density of 200 μl per well. After 1 day, the medium was replaced with serum-free DMEM medium (1% P / S), and 4 hours later, the medium was replaced with DMEM containing 0.2% FBS, and the peptide of SEQ ID NO: 1 was added at a concentration of 5 μM, 50 μM, or 100 μM. After 48 and 72 hours of addition, the reaction was performed using EZ-cytox (DoGenBio Co., Ltd, Seoul, Korea) until the absorbance of Non reached 1.0, and the absorbance at 450 nm was measured using a spectrophotometer (SPECTRAMAX M2e).

[0119] Experimental results showed that the level of cell proliferation in C2C12 myoblasts was increased in a concentration-dependent manner by the peptide of sequence number 1. Specifically, when treated with the peptide of sequence number 1 for 48 hours, the proliferation level of C2C12 myoblasts was confirmed to increase by approximately 17% at a peptide concentration of 50 μM, and when treated with the peptide of sequence number 1 for 72 hours, the proliferation level of C2C12 myoblasts was confirmed to increase by approximately 42% at a peptide concentration of 100 μM (Fig. 1).

[0120] Experimental Example 2: Analysis of protein expression related to myogenesis

[0121] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on myogenesis, the expression level of myogenesis-related protein markers in myogenic cells was evaluated through Western blot analysis.

[0122] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at a density of 5 x 10 5Cells were seeded in 6-well culture plates at a density of 2 ml per well. After 1 day, the medium was replaced with serum-free DMEM medium (1% P / S), and 4 hours later, the peptide of SEQ ID NO: 1 was added at a concentration of 5 μM or 50 μM. After 1 hour, the solution was dissolved by adding lysis buffer, and the protein was obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes, which was quantified using a BCA kit. The protein was subjected to SDS-PAGE and electrotransferred to a membrane. After blocking the protein-electrophoresed membrane with 5% skim milk, the membrane was incubated overnight at 4°C with anti-pPI3K primary antibodies (Cell signaling Technology, USA) and anti-p70S6K primary antibodies (Cell signaling Technology, USA) and, as a loading control, anti-β-actin primary antibodies (Santa Cruz, USA). The membrane was washed with PBS-T and incubated with peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) and peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) for 1 hour at room temperature. After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0123] Experimental results showed that the expression of myogenesis-related protein markers pPI3K and p70S6K was increased in a concentration-dependent manner by the peptide of sequence number 1 in C2C12 myoblasts. Specifically, pPI3K was confirmed to increase by approximately 39% at a peptide concentration of 50 μM, and p70S6K was confirmed to increase significantly by approximately 135% at a peptide concentration of 50 μM (Fig. 2).

[0124] Experimental Example 3: Analysis of gene expression related to early / mid-stage differentiation

[0125] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the differentiation of myoblasts into muscle cells / muscle fibers, the expression levels of early / mid-stage differentiation-related gene markers in myoblasts were evaluated through RT-PCR analysis.

[0126] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded in 6-well culture plates at a density of 2 ml per well. When the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and the peptide of SEQ ID NO: 1 was added at a concentration of 5 μM or 50 μM. Thereafter, the differentiation medium and peptide of SEQ ID NO: 1 were replaced every other day, and 3 days after the first replacement, the cells were harvested, and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was used to obtain complementary DNA using TOPScript™ RT DryMIX (enzynomics, Korea), and then polymerase chain reaction (PCR) was performed on early / mid-stage differentiation marker genes using TOPsimple™ DryMIX-nTaq (enzynomics, Korea). The results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 2.

[0127] Primer sequence (5'-> 3')SEQ ID NO:MyoG Forward(5') ACC AGG AGC CCC ACT TCT AT (3')2MyoG Reverse(5') ACG ATG GAC GTA AGG GAG TG (3')3Myf6 Forward(5') ATC AGC TAC ATT GAG CGT CTA CA (3')4Myf6 Reverse(5') CCT GGA ATG ATC CGA AAC ACT TG (3')5Myf5 Forward(5') TAT GAA GGC TCC TGT CC (3')6Myf5 Reverse(5') ACG TGC TCC TCA TCG TCT G (3')7Myf4 Forward(5') ATC AGC TAC ATT GAG CGT CTA CA (3')8Myf4 Reverse(5') CCT GGA ATG ATC CGA AAC ACT TG (3')9GAPDH Forward(5') GTG ATG GCA TGG ACT GTG GT (3')10GAPDH Reverse(5') GGA GCC AAA AGG GTC ATC AT (3')11

[0128] As a result of the experiment, it was confirmed that the mRNA expression levels of the genes MyoG, Myf4, Myf5, and Myf6, which are gene markers related to early / mid-stage differentiation of myoblasts, were significantly increased in a peptide concentration-dependent manner by the peptide of sequence number 1 in C2C12 myoblasts (Fig. 3a). In particular, the mRNA expression levels of Myf4 were significantly increased by approximately 100-fold or more at a peptide concentration of 50 μM, and the mRNA expression levels of Myf6 were significantly increased by approximately 48-fold or more at a peptide concentration of 50 μM (Fig. 3b).

[0129] Experimental Example 4: Analysis of protein expression related to early, mid, and late differentiation

[0130] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the differentiation of myoblasts into muscle cells / muscle fibers, the expression levels of early / middle / late differentiation-related protein markers in myoblasts were evaluated through Western blot analysis.

[0131] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded in 6-well culture plates at a density of 2 ml per well. When the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and the peptide of SEQ ID NO: 1 was added at a concentration of 5 μM or 50 μM. Thereafter, the differentiation medium and the peptide of SEQ ID NO: 1 were replaced every other day, and 3 days after the first replacement, the solution was dissolved by adding lysis buffer, and the protein was obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes, which was quantified using a BCA kit. The protein was subjected to SDS-PAGE and electrotransferred to a membrane. After blocking the electrophoretic membrane with 5% skim milk, the membrane was incubated overnight at 4°C with anti-Myf5 primary antibody (Abcam, UK), anti-MyoG primary antibody (Santa Cruz, USA), and anti-α-actinin primary antibody (Santa Cruz, USA), and anti-β-actin primary antibody as a loading control (Santa Cruz, USA). The membrane was washed with PBS-T and incubated with peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch, USA) and peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) for 1 hour at room temperature. After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0132] Experimental results confirmed that the expression of Myf5, MyoG, and α-actinin, which are protein markers related to early / middle / late differentiation of myoblasts, was increased in a concentration-dependent manner by the peptide of sequence number 1 in C2C12 myoblasts (Fig. 4a). In particular, the protein expression level of Myf5 was found to be significantly increased by approximately 37-fold or more at a peptide concentration of 50 μM (Fig. 4b).

[0133] Experimental Example 5: Analysis of gene expression related to muscle protein synthesis

[0134] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to muscle protein synthesis, the expression level of muscle protein synthesis-related signaling gene markers in myogenic cells was evaluated through RT-PCR analysis.

[0135] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded in 6-well culture plates at a density of 2 ml per well. When the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and the peptide of SEQ ID NO: 1 was added at a concentration of 5 μM or 50 μM. Thereafter, the differentiation medium and peptide of SEQ ID NO: 1 were replaced every other day, and 3 days after the first replacement, the cells were harvested, and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was used to obtain complementary DNA using TOPScript™ RT DryMIX (enzynomics, Korea), and then polymerase chain reaction (PCR) was performed on signal transduction genes related to muscle protein synthesis using TOPsimple™ DryMIX-nTaq (enzynomics, Korea). The results were run on a 1.5% agarose gel and the mRNA expression levels of the markers were compared under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 3.

[0136] Primer sequence (5'-> 3')SEQ ID NO:SIRT1 Forward(5') GAT CCT TTG GAT TCC TGC AA (3')12SIRT1 Reverse(5') AGT TCC AGC CGT CTC TGT GT (3')13mTOR Forward(5') TTG AGG TCG CTA TGA CCA GAG AGA A (3')14mTOR Reverse(5') TTA CCA GAA GGG ACA CCA GCC AAT G (3')15AKT Forward(5') GGT AGC GAT AAT GGA GGT CT (3')16AKT Reverse(5') GCT TCT GTC CTC TTC TCC TT (3')17P70S6K Forward(5') GGA GCC TGG GAG CCC TGA TGT (3')18P70S6K Reverse(5') GAA GCC CTC TTT GAT GCT GTC C (3')19IGF-1 Forward(5') TGC TCT CAA CAT CTC CCA TC (3')20IGF-1 Reverse(5') GCC TCC TTA GAT CAC AGC TCC (3')21GAPDH Forward(5') GTG ATG GCA TGG ACT GTG GT (3')10GAPDH Reverse(5') GGA GCC AAA AGG GTC ATC AT (3')11

[0137] As a result of the experiment, it was confirmed that the mRNA expression levels of SIRT1, AKT, mTOR, P70S6K, and IGF-1 genes, which are gene markers related to muscle protein synthesis, were significantly increased in a peptide concentration-dependent manner by the peptide of sequence number 1 in C2C12 myogenic cells (Fig. 5a and Fig. 5b).

[0138] Experimental Example 6: Analysis of protein expression related to muscle protein synthesis

[0139] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of proteins related to muscle protein synthesis, the expression level of muscle protein synthesis-related signaling protein markers in myoblasts was evaluated through Western blot analysis.

[0140] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded in 6-well culture plates at a density of 2 ml per well. When the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and the peptide of SEQ ID NO: 1 was added at a concentration of 5 μM or 50 μM. Thereafter, the differentiation medium and the peptide of SEQ ID NO: 1 were replaced every other day, and 3 days after the first replacement, the solution was dissolved by adding lysis buffer, and the protein was obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes, which was quantified using a BCA kit. The protein was subjected to SDS-PAGE and electrotransferred to a membrane. After blocking the electrophoretic membrane with 5% skim milk, the membrane was incubated overnight at 4°C with anti-SIRT1 primary antibody (Cell signaling Technology, USA), anti-IGF-1 primary antibody (Cell signaling Technology, USA), anti-pmTOR primary antibody (Cell signaling Technology, USA), and anti-p70S6K primary antibody (Cell signaling Technology, USA), and anti-β-actin primary antibody (Santa Cruz, USA) as a loading control. The membrane was washed with PBS-T and incubated with peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch, USA) and peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) for 1 hour at room temperature. After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0141] As a result of the experiment, it was confirmed that the protein expression levels of SIRT1, IGF-1, pmTOR, and p70S6K, which are protein markers related to muscle protein synthesis, were significantly increased in a peptide concentration-dependent manner by the peptide of sequence number 1 in C2C12 myogenic cells (Fig. 6a and Fig. 6b).

[0142] Experimental Example 7: Analysis of myotube synthesis ability

[0143] To analyze the effect of the peptide manufactured in Manufacturing Example 1 on the myotube synthesis ability in myoblasts, differentiation-induced myoblasts were observed under a microscope and the thickness of the myotubes was compared.

[0144] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 80%, 5 x 10 5 The cells were seeded in 6-well culture plates at a density of 2 ml per well. When the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and the peptide of SEQ ID NO: 1 was added at a concentration of 5 μM or 50 μM. Thereafter, the differentiation medium and the peptide of SEQ ID NO: 1 were replaced every other day, and 3 days after differentiation induction, images were taken with an optical microscope (FLEXACAM C1, Leica, Germany) to compare the thickness of the myotubes.

[0145] Experimental results showed that myotubes formed by the peptide of sequence number 1 in C2C12 myotubes fused with surrounding myotubes to form thicker myotubes, and this occurred in a peptide concentration-dependent manner (Fig. 7).

[0146] Experimental Example 8: Analysis of Differentiation-Related Protein Expression in Muscle Atrophy-Induced Myogenic Cells

[0147] Western blot analysis was used to evaluate whether the peptide manufactured in Manufacturing Example 1 could restore the expression levels of differentiation-related proteins whose expression was reduced in myogenic cells that induced muscle atrophy.

[0148] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded in 6-well culture plates at a density of 2 ml per well. When the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum) to induce differentiation. The differentiation batch was then replaced every other day, and on the fourth day of differentiation induction, the peptide of SEQ ID NO: 1 was pretreated for 30 minutes at a concentration of 5 μM or 50 μM. Next, the cells were treated with soluble dexamethasone (Sigma, USA) and cultured for 24 hours to induce an atrophic environment. After incubation, the cells were lysed with lysis buffer and centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins, which were then quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to membranes. After blocking the protein-electrophoresed membrane with 5% skim milk, the membrane was incubated overnight at 4°C with anti-MyoD primary antibodies (Santa Cruz, USA) and anti-α-actinin primary antibodies (Santa Cruz, USA), and anti-α-tubulin primary antibodies (Santa Cruz, USA) as a loading control. The membrane was washed with PBS-T and incubated with peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) and peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) for 1 hour at room temperature. After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0149] As a result of the experiment, when C2C12 myoblasts were treated with dexamethasone, the expression levels of the differentiation-related proteins MyoD and α-actinin were significantly reduced to less than half, but it was confirmed that the reduced protein expression levels were restored in a peptide concentration-dependent manner by the peptide of sequence number 1 (Fig. 8).

[0150] Experimental Example 9: Analysis of Gene Expression Related to Fat Synthesis

[0151] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to fat synthesis, the expression level of fat synthesis-related gene markers in hepatocytes was evaluated through RT-PCR analysis.

[0152] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at a density of 1 x 10 5Cells were seeded into 12-well culture plates at a density of 1 ml per well. When the cell confluency reached approximately 70–80% in the 12-well culture plates, the medium was replaced with serum-free DMEM. Four hours later, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, USA), and the peptide of SEQ ID NO: 1 was treated at a concentration of 5 μM or 50 μM, followed by incubation for 48 hours. After incubation, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was used to obtain complementary DNA using TOPScript™ RT DryMIX (enzynomics, Korea), and then polymerase chain reaction (PCR) was performed on genes related to fat synthesis using TOPsimple™ DryMIX-nTaq (enzynomics, Korea). The results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 4.

[0153] Primer sequence (5'-> 3') SEQ ID NO: SREBP1 Forward(5') GAC CGA CAT CGA AGG TGA AG (3')22SREBP1 Reverse(5') AAG AGA GGA GCT CAA TGT GGC (3')23FAS Forward(5') CGG AAA CTG CAG GAG CTG TC (3')24FAS Reverse(5') CAC GGA GTT GAG CCG CAT (3')25GAPDH Forward(5') GAG TCA ACG GAT TTG GTC GT (3')26GAPDH Reverse(5') GAC AAG CTT CCC GTT CTC AG (3')27

[0154] As a result of the experiment, it was confirmed that when oleic acid was treated in HepG2 hepatocytes, the mRNA expression levels of SREBP1 and FAS genes, which are fat synthesis-related genes, significantly increased, but the mRNA expression levels of genes increased by the peptide of sequence number 1 decreased in a peptide concentration-dependent manner (Fig. 9).

[0155] Experimental Example 10: Analysis of Protein Expression Related to Fat Synthesis

[0156] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of proteins related to fat synthesis, the expression level of protein markers related to fat synthesis in hepatocytes was evaluated through Western blot analysis.

[0157] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at a density of 1 x 10 5Cells were seeded in 12-well culture plates at a density of 1 ml per well. When the cell confluency in the 12-well culture plates reached approximately 70-80%, the medium was replaced with serum-free DMEM. After 4 hours, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, USA), and the peptide of SEQ ID NO: 1 was treated at a concentration of 5 μM or 50 μM, followed by incubation for 48 hours. After incubation, the solution was dissolved by adding lysis buffer, and the protein was obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes, which was quantified using a BCA kit. The protein was subjected to SDS-PAGE and electrotransferred to a membrane. The membrane to which the proteins were transferred was blocked with 5% skim milk and then incubated overnight at 4°C with anti-SREBP1 primary antibody (Abcam, UK) and anti-β-actin primary antibody (Santa Cruz, USA) as a loading control. The membrane was washed with PBS-T and incubated with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) at room temperature for 1 h. The membrane was washed again with PBS-T and visualized using a Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0158] As a result of the experiment, it was confirmed that when oleic acid was treated in HepG2 hepatocytes, the protein expression level of SREBP1, a protein related to fat synthesis, was significantly increased, but the expression level of the protein increased by the peptide of sequence number 1 was decreased (Fig. 10).

[0159] Through the above experiments, it was confirmed that the peptide of SEQ ID NO. 1 of the present application can promote the proliferation of muscle cells and differentiation into muscle fibers, and has a muscle regeneration effect even under muscle atrophy conditions. Furthermore, it was confirmed that the peptide of SEQ ID NO. 1 of the present application can significantly suppress the expression of genes / proteins related to fat synthesis in hepatocytes. Therefore, the peptide of SEQ ID NO. 1 of the present application is expected to be effectively utilized for the purpose of suppressing sarcopenia, promoting muscle growth, and suppressing fatty liver.

[0160] Although representative embodiments of the present application have been described above as examples, the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the relevant field will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A peptide comprising the amino acid sequence of sequence number 1.

2. In claim 1, The above peptide is a peptide having muscle loss inhibition and muscle mass increasing activity.

3. In claim 1, The above peptide is a peptide having fatty liver inhibitory activity.

4. In claim 1, The above peptide has one or more of the following activities: (i) Activity that promotes proliferation and differentiation of myoblasts; (ii) activity that increases the expression levels of pPI3K and p70S6K proteins, which are myogenic markers; (iii) activity in increasing the expression levels of mRNA and protein of myofiber differentiation markers MyoG, Myf4, Myf5, Myf6 and α-actinin; (iv) activity in increasing the expression levels of mRNA and protein of SIRT1, AKT, mTOR, p70S6K and IGF-1, which are muscle protein synthesis markers; (v) activity that increases the myotube synthesis capacity of myoblasts; and (vi) Activity in reducing the expression levels of mRNA and protein of SREBP1 and FAS, which are markers of fat synthesis.

5. A pharmaceutical composition for preventing or treating muscle disease, comprising the peptide of claim 1 as an active ingredient.

6. In claim 5, A pharmaceutical composition for preventing or treating a muscle disease, wherein the muscle disease is at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle stiffness, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration and cachexia.

7. A food composition for preventing or improving muscle disease, comprising the peptide of claim 1 as an active ingredient.

8. In claim 7, A food composition for preventing or improving muscle disease, wherein the muscle disease is at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle stiffness, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration and cachexia.

9. A pharmaceutical composition for preventing or treating fatty liver, comprising the peptide of claim 1 as an active ingredient.

10. A food composition for preventing or improving fatty liver, comprising the peptide of claim 1 as an effective ingredient.

11. A method for preventing or treating muscle disease, comprising the step of administering to a subject the peptide of claim 1 or a pharmaceutical composition comprising the peptide as an active ingredient.

12. In claim 11, A method for preventing or treating a muscle disease, wherein the muscle disease is at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle stiffness, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration, and cachexia.

13. A method for preventing or treating fatty liver, comprising the step of administering to a subject the peptide of claim 1 or a pharmaceutical composition comprising the peptide as an active ingredient.

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