Composition for preventing, alleviating, or treating muscle diseases, comprising mackerel protein hydrolysate

Abstract

The present invention relates to a composition for preventing, alleviating, or treating muscle diseases, comprising mackerel protein hydrolysate, wherein the mackerel protein hydrolysate has an excellent protective effect against muscular atrophy, and thus can be used as a food, functional food, and pharmaceutical material to effectively prevent, alleviate, or treat various muscle diseases related to sarcopenia and muscular atrophy.

Description

Composition for the prevention, improvement, or treatment of muscle diseases containing mackerel protein hydrolysate

[0001] The present invention relates to a composition for preventing, improving, or treating muscle diseases, and more specifically, to a composition for preventing, improving, or treating muscle diseases containing mackerel protein hydrolysate as an active ingredient.

[0002] Muscle is an important component of the human body and is a tissue derived from mesenchymal stem cells. Muscles account for approximately 40% of our body weight; they are supported by bones and tendons and consist of bundles of muscle fibers that move with one another and change cell size to induce contraction. Muscles are classified into skeletal, cardiac, and visceral muscles; each generates force and induces movement at its respective location, while also playing a role in protecting bodily organs such as bones, joints, and internal organs. Furthermore, muscles possess regenerative capabilities; if damaged, they can be regenerated into muscles with their original contractile and relaxation abilities after undergoing degeneration through satellite cells and the surrounding environment.

[0003] Muscle diseases are caused by congenital genetic or environmental factors, and diseases related to muscle loss are on the rise in recent years due to the trend of an aging society and increased life expectancy. Human muscle mass decreases by more than 1% annually starting from age 40, and by age 80, it has declined to 50% of its maximum mass; consequently, muscle loss in old age is recognized as the most significant cause of a decline in overall physical function. These muscle diseases are on the rise globally compared to the past.

[0004] Among muscle diseases, sarcopenia is a degenerative disease characterized by a decrease in muscle mass and strength. Unlike the muscle loss that occurs during the normal aging process, it refers to a condition in which muscle mass decreases abnormally and rapidly. Age-related sarcopenia is reported to increase the risk of falls, fractures, frailty, metabolic diseases, and death in the elderly due to limitations in physical performance. Although this sarcopenia tends to gradually increase as the body ages, no clear cause or distinct treatment method for age-related sarcopenia has yet been identified.

[0005] Muscular atrophy is a condition resulting from the weakening of muscles and refers to the loss of muscle tissue. The most common cause is a lack of exercise, but it is known that reduced physical activity due to injury or disease, nutritional deficiencies, and genetic factors can also cause muscle atrophy. Additionally, a decrease in protein synthesis in the body due to aging can lead to muscle atrophy, and nutritional deficiencies caused by dieting are also a cause.

[0006] Muscle loss and muscle atrophy are closely correlated. Looking at the current state of the treatment industry for sarcopenia and muscle atrophy, the decline in physical function and muscle strength among the elderly—caused by spinal diseases, cancer, aging, or internal and surgical issues—is a major problem in the aging society. Preventing age-related decline in muscle function—that is, stopping muscle atrophy and maintaining contractile function and muscle elasticity—is a crucial factor for healthy physical activity and independent daily living in old age.

[0007] Therefore, in accordance with the global trend of an aging society, efforts are needed to maintain the musculoskeletal function of the elderly to increase their quality of life and healthy life expectancy. Consequently, there is a need to develop new materials that improve muscle strength or reduce muscle atrophy without causing other side effects, while demonstrating excellent efficacy in improving muscle atrophy.

[0008] The objective of the present invention is to provide a composition for the prevention, improvement, or treatment of muscle diseases comprising a naturally derived hydrolysate having an excellent protective effect against muscle atrophy.

[0009] Another objective of the present invention is to provide a method for producing the above-mentioned natural product-derived hydrolysate.

[0010] To achieve the above objective, the present invention provides a food composition for preventing or improving muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

[0011] The present invention provides a health functional food composition for preventing or improving muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

[0012] The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

[0013] In addition, the present invention provides a method for preparing a mackerel protein hydrolysate, comprising the steps of: adding mackerel powder to distilled water to prepare a substrate solution and heat-treating it to inactivate the autoenzyme; adding a protein hydrolyzing enzyme to the substrate solution in which the autoenzyme has been inactivated to hydrolyze it; heat-treating the hydrolyzed solution to inactivate the enzyme and centrifuging it; and filtering the supernatant obtained by centrifugation and freeze-drying it.

[0014] The present invention relates to a novel natural material having an excellent protective effect against muscle atrophy. By utilizing the mackerel protein hydrolysate according to the present invention as a material for food, functional food, and pharmaceuticals, various muscle diseases related to muscle loss and muscle atrophy can be effectively prevented, improved, or treated.

[0015] Figure 1 compares the protective effect against muscle atrophy through the results of measuring the diameter of C2C12 cells according to treatment with mackerel protein hydrolysate by enzyme.

[0016] Figure 2 confirms the cytotoxicity of mackerel alkalase protein hydrolysate (MHA).

[0017] Figure 3 shows microscopic images of a C2C12 cell muscle atrophy model according to treatment with different MHA concentrations.

[0018] Figure 4 is the result of measuring the diameter of the C2C12 cells in Figure 3.

[0019] Figure 5 shows the change in myosin heavy chain (MYH) protein expression following MHA treatment.

[0020] Figure 6 confirms the effect of MHA treatment on the expression of muscle atrophy-related proteins.

[0021] The present invention will be described in detail below.

[0022]

[0023] The inventors completed the present invention by confirming that a mackerel protein hydrolysate prepared by treating mackerel with various protein hydrolyzing enzymes has an excellent protective effect against muscle atrophy in a muscle atrophy model using C2C12 cells.

[0024]

[0025] The present invention provides a food composition for preventing or improving muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

[0026] The above mackerel protein hydrolysate may be prepared by treating a substrate solution to which mackerel powder has been added with a protein hydrolyzing enzyme selected from alcalase, bromelain, flavourzyme, neutrase, papain, or a combination thereof, and preferably by treating with alcalase, but is not limited thereto.

[0027] In this specification, "hydrolysis" refers to a chemical reaction in which a large molecule, originally a single molecule, breaks down into several ions or molecules upon reacting with water. Protein hydrolysis is a chemical reaction that hydrolyzes the peptide bonds of proteins to produce amino acids or peptides; it is physiologically catalyzed by enzymes and chemically catalyzed by acids or alkalis, and the enzymes are mostly proteolytic enzymes.

[0028] Among the above protein hydrolyzing enzymes, alcalase is a protein hydrolyzing enzyme produced by bacteria or fungi, and can exhibit strong protein degradation ability under conditions around pH 7-9.

[0029] The above bromelain is a protein hydrolyzing enzyme found in pineapple stems, and can exhibit strong protein degradation ability under conditions around pH 6-9.

[0030] The above-mentioned flavourzyme is a protein hydrolyzing enzyme obtainable from fungi, and can exhibit strong protein degradation ability under conditions around pH 6-7.

[0031] The above neutrase is a protein hydrolyzing enzyme obtainable from Bacillus bacteria, and can exhibit protein degradation ability by hydrolyzing peptide bonds within proteins under conditions of pH 5.5-7.5.

[0032] The above papain is a protein hydrolyzing enzyme found in papaya (Carica papaya) and can exhibit strong protein degradation ability under conditions around H 5-7.5.

[0033]

[0034] The composition containing the above-mentioned mackerel protein hydrolysate as an active ingredient has an excellent protective effect against muscle atrophy. In addition, the composition can increase the expression of myosin heavy chain (MYH), a protein that makes up muscle cells, and decrease the expression of MAFbx (muscle atrophy F-box) or MuRF1 (muscle RING-finger protein-1), which are major factors inducing muscle atrophy, thereby enabling the prevention or improvement of muscle diseases.

[0035]

[0036] The above muscle disease may include muscle diseases resulting from decreased muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration. Specifically, the above muscle disease may be one or more selected from the group consisting of sarcopenia, muscular atrophy, muscular dystrophy, myasthenia gravis, myosophagosis, and muscle degeneration, but is not limited thereto.

[0037]

[0038] The above food composition may include all forms such as functional food, nutritional supplement, health food, or food additives.

[0039] The food composition according to the present invention may be prepared as a powder, granule, tablet, capsule, syrup, or beverage, etc., for the purpose of preventing or improving muscle diseases, and there are no restrictions on the form in which the food may take place, and it may include all foods in the conventional sense. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and its processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.), etc. are possible, and it may include all functional foods in the conventional sense. In addition, it may also include foods used as animal feed.

[0040] The above food composition may be prepared by further including food-grade acceptable food additives and appropriate other auxiliary ingredients commonly used in the industry. Unless otherwise stipulated, suitability as a food additive may be determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety. Items listed in the above 'Food Additives Codex' include, for example, chemically synthesized products such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0041] The above other auxiliary ingredients may additionally contain, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents, etc. In particular, the above natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol, and as sweeteners, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.

[0042] The effective dose of mackerel protein hydrolysate contained in the above food composition can be appropriately adjusted according to the purpose of use, such as the prevention or improvement of muscle diseases. Since the above composition is made from natural products and food ingredients, it has the advantage of not having side effects that may occur with the long-term use of general medicines, and due to its excellent portability, it can be consumed as an adjuvant for the prevention or improvement of muscle diseases.

[0043]

[0044] The present invention provides a health functional food composition for preventing or improving muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

[0045] In this specification, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body, and is a food with high medical or medical effects that is processed to efficiently exhibit biological regulatory functions in addition to nutritional supply; it may be used interchangeably with terms known in the art, such as functional food.

[0046] Corresponding features can be substituted in the aforementioned section.

[0047]

[0048] The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

[0049] Corresponding features can be substituted in the aforementioned section.

[0050]

[0051] In this specification, the term "pharmaceutical composition" means a composition administered for the purpose of preventing or treating a specific disease, and for the purposes of the present invention, means administered for the treatment of a muscle disease or a disease or complication caused by it.

[0052] The above pharmaceutical composition may be prepared according to conventional methods in the pharmaceutical field. Depending on the formulation, the above pharmaceutical composition may be combined with a suitable pharmaceutically acceptable carrier and, if necessary, may be prepared by further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. The above suitable carrier, etc., may be selected differently depending on the administration form and formulation, as it does not impair the activity and properties of the mackerel protein hydrolysate according to the present invention.

[0053] Examples of carriers, excipients, diluents, etc. that may be included in the above pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc.

[0054] The above pharmaceutical composition can be applied in any dosage form, and more specifically, can be formulated and used in oral dosage forms, topical preparations, suppositories, and parenteral dosage forms of sterile injectable solutions according to conventional methods, but is not limited thereto.

[0055] Among the above oral formulations, solid formulations may be in the form of tablets, pills, powders, granules, capsules, etc., and may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc., and may also include lubricants such as magnesium stearate and talc in addition to simple excipients. Furthermore, in the case of capsule formulations, in addition to the substances mentioned above, liquid carriers such as fatty oils may be further included. Among the above oral formulations, liquid formulations may include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0056] The parenteral formulations described above may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc., may be used. However, they are not limited thereto, and any suitable formulation known in the art may be used.

[0057]

[0058] The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.

[0059] In this specification, "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects.

[0060] The effective dose level of the above pharmaceutical composition may be determined differently depending on factors including the purpose of use, the patient's age, gender, weight and health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. For example, although not constant, it may generally be administered at a dose of 0.001 to 1000 mg / kg, preferably 0.01 to 100 mg / kg, once or several times daily. The above dosage does not limit the scope of the present invention in any way.

[0061] The above pharmaceutical composition may be administered to any animal capable of developing a muscle disease, and said animal may include, for example, humans and primates, as well as livestock such as cattle, pigs, horses, and dogs.

[0062] The above pharmaceutical composition may be administered via a suitable route of administration depending on the formulation form, and may be administered via various oral or parenteral routes as long as it can reach the target tissue. The method of administration may be administered by conventional methods, such as oral, rectal or intravenous, intramuscular, topical application, respiratory inhalation, intrauterine dura mater, or intracerebroventricular injection, without needing to be particularly limited.

[0063] The above pharmaceutical composition may be used alone for the prevention or treatment of muscle diseases, or may be used in combination with surgery or other drug treatments.

[0064]

[0065] In addition, the present invention provides a method for preparing a mackerel protein hydrolysate, comprising the steps of: adding mackerel powder to distilled water to prepare a substrate solution and heat-treating it to inactivate the autoenzyme; adding a protein hydrolyzing enzyme to the substrate solution in which the autoenzyme has been inactivated to hydrolyze it; heat-treating the hydrolyzed solution to inactivate the enzyme and centrifuging it; and filtering the supernatant obtained by centrifugation and freeze-drying it.

[0066]

[0067] In the manufacturing method according to the present invention, the step of preparing a substrate solution by adding the mackerel powder to distilled water and heat-treating to inactivate the autoenzyme can be performed by preparing a substrate solution by adding mackerel powder in an amount of 1 to 10% (w / v), preferably 3 to 5% (w / v), relative to distilled water, and heat-treating at 90 to 100°C for 10 minutes to 1 hour, preferably at 95°C for 10 to 30 minutes.

[0068] The step of hydrolyzing by adding a proteolytic enzyme to the substrate solution in which the self-enzyme has been inactivated can be carried out by adding 0.5 to 2% (v / w), preferably 1% (v / w), of the proteolytic enzyme relative to the substrate solution in which the self-enzyme has been inactivated, and reacting at 50 to 60°C for 1 to 24 hours, preferably at 55°C for 4 to 12 hours. The proteolytic enzyme may be selected from alcalase, bromelain, flavourzyme, neutrase, papain, or a combination thereof, and preferably may be alcalase.

[0069] The step of heat-treating the hydrolyzed solution to inactivate the enzyme and centrifuging can be performed by heat-treating at 90 to 100°C for 10 minutes to 1 hour, preferably at 95°C for 10 to 30 minutes to inactivate the enzyme, and after inactivation, centrifuging at 10,000 to 20,000 ×g for 10 to 30 minutes, preferably at 13,000 ×g for 20 minutes.

[0070] The step of filtering and freeze-drying the supernatant obtained by centrifugation above can be performed by filtering the supernatant obtained by centrifugation under reduced pressure using a 0.2 to 0.3 μm filter and freeze-drying for 60 to 80 hours to obtain a mackerel protein hydrolysate. Additionally, a hydrolysate containing low molecular weight peptides of 3 kDa to 1 kDa or less can be obtained through ultrafiltration.

[0071]

[0072] The mackerel protein hydrolysate produced by the above manufacturing method can be used as a composition for improving muscle function, preventing, improving, or treating muscle diseases.

[0073] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0074]

[0075] <Example 1> Preparation of Mackerel Protein Hydrolysate

[0076] A substrate solution was prepared by adding distilled water to mackerel powder at a ratio of 4% (w / v), and the autoenzymes were inactivated at 95°C for 20 minutes. Next, proteolytic enzymes, including alcalase, bromelain, flavourzyme, neutrase, and papain, were added at 1% (v / w) relative to the substrate, and hydrolysis was carried out at 55°C for 12 hours. Subsequently, the final reaction mixture was heated at 95°C for 20 minutes to inactivate the enzymes, and the supernatant was obtained by centrifugation at 13,000 ×g for 20 minutes. The supernatant was then filtered under reduced pressure using a 0.22 μm filter and freeze-dried for 72 hours to obtain the mackerel protein hydrolysate.

[0077]

[0078] Table 1 below shows the yield of freeze-dried mackerel protein hydrolysates by enzyme.

[0079] Hydrolysate yield (%) by enzyme 1) No enzyme 12.25±0.01 d2) Alcalase47.18±0.00 a Bromelain 39.67±0.00 b Flavourzyme 36.34±0.10 bc Neutrase29.26±0.01 c Papain 19.43±0.01 d

[0080] * 1) Yield (%) = [Total solid content of protein hydrolysate - Total solid content of blank (no substrate)] / Total substrate content × 100

[0081] 2) Each value is mean ± SD (n = 3), and various superscripts (ae) showed significant differences at P < 0.05 by Duncan's multiple range test.

[0082]

[0083] Referring to Table 1 above, the alcalase-treated mackerel protein hydrolysate showed the highest yield at 47.18%, followed by the bromelain-treated group at 39.67%, the flavozyme-treated group at 36.34%, the nutase-treated group at 29.26%, the papain-treated group at 19.43%, and the enzyme-untreated group at 12.25%.

[0084]

[0085] <Experimental Example 1> Confirmation of the protective effect of mackerel protein hydrolysate against muscle atrophy by enzyme

[0086] The C2C12 cells used in the experiment were obtained from ATCC (Manassas, VA, USA). DMEM (Dulbeccos modified Eagles medium high glucose) was prepared by adding 10% fetal bovine serum (FBS) and 1% antibiotics, penicillin and streptomycin, to the medium, and the cells were cultured in a 5% CO2 incubator at 37°C. Once the cells reached a confluent state, differentiation was induced for 48 hours by adding DMEM supplemented with 2% FBS or horse serum. Subsequently, the DMEM supplemented with 2% FBS or horse serum was replaced every 2 days to induce myotube formation.

[0087] Cells with differentiated and formed myoducts were treated with each enzyme-specific mackerel protein hydrolysate at a concentration of 100 μg / mL for 1 hour, then treated with TNF-α 20 ng / mL (Recombinant Murine TNF-α, PEPROTECH, USA) or H2O2 2 mM for 24 hours to induce muscle atrophy, and the myoduct diameter of C2C12 was measured using the i solution program.

[0088] As a result, as shown in Figure 1, it was confirmed that the cells treated with mackerel alkalase protein hydrolysate exhibited the highest tube diameter compared to other enzyme-treated groups and showed the most excellent protective effect on the tube cells.

[0089] Accordingly, subsequent experiments were conducted using mackerel alkalase protein hydrolysate.

[0090]

[0091] <Experimental Example 2> Confirmation of Cytotoxicity of Mackerel Hydrolysate Alcalase (MHA)

[0092] The effect of mackerel alkalase protein hydrolysate (MHA) on the viability of mouse myoblast cell line C2C12 cells was confirmed by the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay. C2C12 cells at 3.5 × 10⁶ 4 Differentiated cells were seeded into 24 wells at a cell / well density and treated with MHA at different concentrations for 1 hour, followed by treatment with TNF-α 20 ng / mL or H2O2 2 mM for 24 hours. Subsequently, 20 μL of MTT reagent at a concentration of 2.5 mg / mL was added to each well and incubated for 4 hours. After removing the supernatant and adding 100 μL of dimethyl sulfoxide (DMSO) to each well to dissolve the formed formazan crystals, the absorbance was measured at 550 nm using a Biotek microplate spectrophotometer, and cytotoxicity was calculated as the absorbance of the sample treatment group relative to the absorbance of the control group.

[0093] As a result of confirming the cytotoxicity of MHA, as shown in Figure 2, it was confirmed that there was no cytotoxicity or a protective effect at concentrations of 200 μg / mL or lower.

[0094]

[0095] <Experimental Example 3> Confirmation of the protective effect against muscle atrophy of mackerel alkalase protein hydrolysate (MHA)

[0096] After differentiating C2C12 cells, muscle atrophy was induced by treating them with MHA at different concentrations for 1 hour, followed by treatment with TNF-α 20 ng / mL or H2O2 2 mM for 24 hours. Subsequently, the cells were imaged at 100x magnification using an inverse phase-contrast microscope (Leica Microsystems, Wetzlar, Germany), and the canal diameter of C2C12 was measured using the i solution program.

[0097] As a result, as shown in Figures 3 and 4, treatment with MHA in two muscle atrophy models was confirmed to protect against muscle atrophy, showing significant activity particularly at 50-200 μg / mL.

[0098]

[0099] <Experimental Example 4> Confirmation of the effect of MHA treatment on MYH protein expression

[0100] Immunofluorescence was performed to measure the expression of muscle atrophy-related proteins in C2C12 cells induced by MHA treatment. A 12 mm glass coverslip was placed on a 24-well plate at 3.5 × 10⁻⁶ 4Cells were seeded at a cell / well density, and differentiation of C2C12 cells was induced as in Experimental Example 1 above. Subsequently, C2C12 cells were pretreated with MHA at various concentrations for 1 hour. TNF-α 20 ng / mL or H2O2 2 mM was added to the samples for 24 hours to induce muscle atrophy. Coverslips were washed with PBS, and cells were fixed with 4% paraformaldehyde (in PBS) for 20 minutes. Then, the cells were washed with PBS, and blocking was performed for 1 hour with a blocking buffer containing 0.3% Triton X-100 and 5% BSA. After incubating the MYH antibody (Santa Cruz) at a 1:50 ratio overnight at 4°C, a secondary antibody conjugated with FITC (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488), abcam) was reacted at a 1:200 ratio at 4°C for 2 hours. After washing three times with PBS, the sample was enclosed, and the expression pattern of MYH was analyzed using a fluorescence microscope (Zeiss Axioskop 50 fluorescence microscope, Carl Zeiss, Oberkochen, Germany) equipped with a fluorescence camera (Jenoptik ProgRes C5 cool, Jena, Germany).

[0101] As a result, as shown in Figure 5, it was confirmed that the expression level of MYH and the diameter of the canal increased in C2C12, in which muscle atrophy was induced by treatment with TNF-α or H2O2, compared to the control group in which only muscle atrophy was induced.

[0102]

[0103] <Experimental Example 5> Confirmation of the effect of MHA on the expression of muscle atrophy-related proteins

[0104] Western blot analysis was performed to measure the expression of muscle atrophy-related proteins in C2C12 cells induced by MHA treatment. Differentiated C2C12 cells were pretreated with MHA at various concentrations for 1 hour. TNF-α 20 ng / mL or H2O2 2 mM was added to the samples for 24 hours to induce muscle atrophy. Cell proteins were separated in amounts ranging from 5 to 30 μg using 8% and 10% SDS-polyacrylamide gels via electrophoresis at 100 V. The separated proteins were transferred to a PVDF membrane at 100 V for 60 minutes, followed by blocking with 5% skim milk solution for 1 hour. Subsequently, the primary antibody was diluted 1:1,000 in 5% BSA solution and incubated overnight at 4°C. Afterward, the membrane was washed three times with phosphate buffered saline with tween 20 (PBST), and a secondary antibody conjugated with horseradish peroxidase (HRP) was diluted 1:3,000 in a 5% skim milk solution and reacted at room temperature for 2 hours, followed by washing three times again with PBST. For protein development, a solution of chromogenic reagents A and B from the ECL detection kit (Bio-Rad. USA) mixed in a 1:1 ratio was applied to the membrane, and the protein band was observed by developing it on an X-ray film (CP-G plus, Agfa Healthcare Ltd, New Orleans, LA, USA).

[0105] As a result, referring to Figure 6, it was confirmed that MHA (50-200 μg / mL) increased the expression of myosin heavy chain (MYH), a protein that makes up muscle cells, in C2C12 cells where muscle atrophy was induced by treatment with TNF-α 20 ng / mL or H2O2 2 mM compared to the control group. In addition, it was confirmed that the expression of MAFbx and MuRF1, ubiquitin E3 ligases which are major factors inducing muscle atrophy, decreased upon treatment with MHA (50-200 μg / mL). Through this, it was confirmed that MHA is an effective substance for improving muscle atrophy.

[0106]

[0107] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A food composition for preventing or improving muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

2. In Paragraph 1, The above mackerel protein hydrolysate is, A food composition characterized by being prepared by treating a substrate solution to which mackerel powder has been added with a protein hydrolyzing enzyme selected from alcalase, bromelain, flavorzyme, neutrase, papain, or a combination thereof.

3. In Paragraph 1, The above composition is, A food composition characterized by having a protective effect against muscle atrophy.

4. In Paragraph 1, The above composition is, A food composition characterized by increasing the expression of myosin heavy chain (MYH), a protein that makes up muscle cells, and decreasing the expression of MAFbx or MuRF1, which are major factors that induce muscle atrophy.

5. In Paragraph 1, The above muscle disease is, A food composition characterized by having one or more selected from the group consisting of sarcopenia, muscular atrophy, muscular dystrophy, myasthenia gravis, myasthenia rigidity, and muscle degeneration.

6. A health functional food composition for preventing or improving muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

7. A pharmaceutical composition for the prevention or treatment of muscle diseases, containing mackerel protein hydrolysate as an active ingredient.

8. A step of preparing a substrate solution by adding mackerel powder to distilled water and heat-treating it to inactivate the autoenzyme; A step of hydrolyzing by adding a protein hydrolyzing enzyme to a substrate solution in which the above-mentioned autoenzyme has been inactivated; A step of heat-treating the hydrolyzed solution to inactivate the enzyme and centrifuging; and A method for preparing mackerel protein hydrolysate, comprising the step of filtering the supernatant obtained by centrifugation and freeze-drying it.

9. In Paragraph 8, The heat treatment for the above enzyme inactivation is, A manufacturing method characterized by being performed at 90 to 100℃ for 10 minutes to 1 hour.

10. In Paragraph 8, The above-mentioned protein hydrolyzing enzyme is, A method of preparation characterized by being selected from alcalase, bromelain, flavourzyme, neutrase, papain, or a combination thereof.

11. In Paragraph 8, The above hydrolysis step is, A method of preparation characterized by adding 0.5 to 2% (v / w) of a protein hydrolyzing enzyme relative to a substrate solution in which the above-mentioned autoenzyme is inactivated, and performing the process at 50 to 60°C for 1 to 24 hours.

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