Composition for promoting muscle cell differentiation or preventing or treating muscle diseases comprising amentoflavone as active ingredient

Abstract

The present invention relates to a composition for promoting muscle cell differentiation, or preventing or treating muscle diseases, the composition comprising amentoflavone as an active ingredient. It was confirmed that amentoflavone promotes the differentiation of muscle cells by regulating the expression of muscle-specific markers and promoting creatine kinase activity, and therefore the composition can be effectively used as a composition for promoting muscle cell differentiation, producing cultured meat, or preventing, treating or ameliorating muscle diseases.

Description

A composition for promoting muscle cell differentiation or for preventing or treating muscle diseases containing amentoflavone as an active ingredient

[0001] The present invention relates to a composition for promoting muscle cell differentiation or for preventing or treating muscle diseases, comprising amentoflavone as an active ingredient.

[0002] According to the Food and Agriculture Organization of the United Nations (FAO), as the global population continues to grow, meat consumption is projected to increase by 1.3% annually from 3.04 million tons in 2018 to reach 4.55 million tons by 2050. The FAO has announced that more than 200 million tons of additional meat must be produced annually to meet this demand. Food shortages resulting from future population growth entail various issues, including environmental and ethical concerns; therefore, the development of alternative meats capable of addressing all these areas is necessary. These "alternative livestock products," which can replace conventional meat, generally require lower resource input and greenhouse gas emissions compared to existing meat, and can reduce the generation of environmental pollutants such as odors.

[0003] Representative alternative meats attracting attention include plant-based meat substitutes, edible insects, and cultured meat. Among these, "cultured meat" refers to edible meat produced by growing cells in a laboratory rather than raising and slaughtering livestock directly. Various methods are being devised for the technology of manufacturing and processing cultured meat. Basically, cultured meat is produced by differentiating animal muscle cells, culturing them into three-dimensional tissues, and processing them.

[0004] Serum, essential for animal cell culture for cultured meat production, is isolated from animals such as calves, horses, sheep, pigs, dogs, and goats, with Fetal Bovine Serum (FBS) being the most commonly used. However, the process of isolating serum has disadvantages, including being unethical, environmentally unfriendly, and costly. Consequently, serum-free media have recently garnered attention as an alternative to FBS. Serum-free media are cell culture media that eliminate the need for serum by producing and adding various hormones and growth factors essential for cell culture using recombinant protein synthesis technology to synthetic media. However, the types of cell lines that can be cultured in serum-free media are limited, the cell line adaptation process is cumbersome, and high costs persist because the addition of recombinant proteins is required to replace FBS. Therefore, active research is currently underway to produce cultured meat more quickly and at a lower cost than existing methods.

[0005] Meanwhile, muscles are important tissues that perform various roles, such as bodily movement, maintaining posture, and metabolic activity. Muscle diseases that cause damage to or decline in the function of these muscles can significantly reduce the quality of life. In particular, as global aging progresses rapidly, sarcopenia—a condition characterized by a continuous annual decrease in muscle mass among the elderly—is increasing sharply. The World Health Organization (WHO) recognizes sarcopenia as a disease and treats it as a problem that threatens the quality of life of the elderly population and public health. Consequently, research aimed at preventing and treating sarcopenia is actively underway.

[0006] The object of the present invention is to provide a medium composition for promoting muscle cell differentiation comprising amentoflavone as an active ingredient.

[0007] Another objective of the present invention is to provide an animal feed composition for promoting muscle cell differentiation comprising amentoflavone as an active ingredient.

[0008] Another objective of the present invention is to provide a culture medium composition for producing cultured meat comprising amentoflavone as an active ingredient.

[0009] Another objective of the present invention is to provide a method for producing cultured meat, comprising the step of treating muscle cells of an individual other than a human with amentoflavone.

[0010] Another objective of the present invention is to provide a composition for the prevention, treatment, or improvement of muscle diseases comprising amentoflavone as an active ingredient.

[0011] To achieve the above objective, the present invention provides a culture medium composition for promoting muscle cell differentiation comprising amentoflavone as an active ingredient.

[0012] In addition, the present invention provides an animal feed composition for promoting muscle cell differentiation comprising amentoflavone as an active ingredient.

[0013] In addition, the present invention provides a culture medium composition for producing cultured meat comprising amentoflavone as an active ingredient.

[0014] In addition, the present invention provides a method for producing cultured meat, comprising the step of treating muscle cells of an individual other than a human with amentoflavone.

[0015] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising amentoflavone as an active ingredient.

[0016] In addition, the present invention provides a health functional food composition for preventing or improving muscle diseases, comprising amentoflavone as an active ingredient.

[0017] According to the present invention, by confirming that amentoflavone promotes the differentiation of muscle cells through the regulation of muscle-specific marker expression and the promotion of creatine kinase activity, it can be usefully utilized as a composition for promoting muscle cell differentiation, for producing cultured meat, or for preventing, treating, or improving muscle diseases.

[0018] Figure 1 shows the results of analyzing the intermolecular binding of amentoflavone (hereinafter referred to as the sample) and myostatin (hereinafter referred to as MSTN). Specifically, the results show the analysis of possible binding sites of the sample and MSTN through in silico molecular modeling.

[0019] Figure 2 shows the results of analyzing the intermolecular binding stability of the sample and MSTN.

[0020] Figure 3 shows the results of analyzing the effects of the samples on bovine muscle stem cells. Specifically, Figure 3A shows changes in intracellular creatine kinase activity; Figure 3B shows changes in intracellular myosin heavy chain (hereinafter referred to as MYH); Figure 3C shows changes in muscle canal width; Figure 3D shows changes in gene and protein expression of muscle-specific markers; and Figure 3E shows the results of analyzing changes in reactive oxygen species (hereinafter referred to as ROS) activity. *p<0.05, **p<0.01, ***p<0.001.

[0021] Figure 4 shows the results of analyzing the effects of the samples on porcine muscle stem cells. Specifically, Figure 4A shows changes in intracellular creatine kinase activity; Figure 4B shows changes in intracellular MYH; Figure 4C shows changes in muscle canal width; Figure 4D shows changes in gene and protein expression of muscle-specific markers; and Figure 4E shows the results of analyzing changes in ROS activity. **p<0.01, ***p<0.001.

[0022] Figure 5 shows the results of analyzing the effects of the samples on porcine muscle stem cells. Specifically, Figure 5A shows changes in intracellular creatine kinase activity; Figure 5B shows changes in intracellular MYH; Figure 5C shows changes in muscle canal width; Figure 5D shows changes in gene and protein expression of muscle-specific markers; and Figure 5E shows the results of analyzing changes in ROS activity. *p<0.05, **p<0.01, ***p<0.001.

[0023] Figure 6 shows the results of analyzing the effects of the samples on human muscle stem cells. Specifically, Figure 6A shows changes in intracellular creatine kinase activity; Figure 6B shows changes in intracellular MYH; Figure 6C shows changes in muscle canal width; Figure 6D shows changes in gene and protein expression of muscle-specific markers; and Figure 6E shows the results of analyzing changes in ROS activity. *p<0.05, **p<0.01, ***p<0.001.

[0024] Figure 7 shows the results of animal experiments conducted to confirm the effect of the sample on improving muscle diseases. Specifically, Figure 7A shows changes in mouse body weight; Figure 7B shows changes in mouse muscle weight; Figure 7C shows changes in protein expression of muscle-specific markers in mice; Figure 7D shows changes in creatine kinase activity in mouse muscles; Figure 7E shows changes in mouse muscle strength and endurance; Figure 7F shows changes in mouse muscle canal width; and Figure 7F shows the results of the analysis of changes in MYH within mouse muscles. *p<0.05, **p<0.01, ***p<0.001.

[0025] The present invention will be described in more detail below.

[0026]

[0027] The present invention provides a culture medium composition for promoting muscle cell differentiation comprising amentoflavone as an active ingredient.

[0028] The above amentoflavone is a compound represented by the following chemical formula 1, where the chemical formula is C 30 H 18 0 10 and the molecular weight is 538.46 g / mol.

[0029] [Chemical Formula 1]

[0030]

[0031] The above amentoflavone can promote creatine kinase activity.

[0032] In addition, the above amentoflavone can inhibit reactive oxygen species (ROS) activity.

[0033] In addition, the amentoflavone may promote the expression of one or more selected from the group consisting of myodi, myogenin, and myosin heavy chain, but is not limited thereto.

[0034] In addition, the above amentoflavone can inhibit myostatin expression.

[0035] The above muscle cells may be derived from one or more animals selected from the group consisting of humans, chickens, cattle, pigs, and mice, but are not limited thereto.

[0036] The above culture medium composition may additionally include components for muscle cell growth and differentiation in addition to amentoflavone. The amentoflavone may be included in the culture medium composition and may be used alone as a culture medium adjuvant.

[0037]

[0038] In addition, the present invention provides an animal feed composition for promoting muscle cell differentiation comprising amentoflavone as an active ingredient.

[0039]

[0040] In addition, the present invention provides a culture medium composition for producing cultured meat comprising amentoflavone as an active ingredient.

[0041] The cultured meat may be derived from one or more animals selected from the group consisting of chickens, cattle, pigs, and mice, but is not limited thereto.

[0042] "Cultured meat" refers to an alternative meat produced by multiplying cells harvested from living animals. It primarily utilizes stem cells to culture animal tissues and has recently garnered attention as a meat source that can be obtained without raising or slaughtering livestock. Since cultured meat is based on tissue culture and eliminates livestock farming, it has the advantage of minimizing issues regarding animal welfare, ethics, and the environment. However, it currently has disadvantages, such as high production costs per kilogram and long cultivation times.

[0043]

[0044] In addition, the present invention provides a method for producing cultured meat, comprising the step of treating muscle cells of an individual other than a human with amentoflavone.

[0045]

[0046] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising amentoflavone as an active ingredient.

[0047] The above muscle diseases may be one or more selected from the group consisting of muscular atrophy, muscular dystrophy, sarcopenia, myopathy, myasthenia, and muscular injury, but are not limited thereto.

[0048] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0049] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0050] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0051] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms, but is not limited thereto.

[0052] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically affinities for the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0053] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0054] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0055] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration, route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0056]

[0057] In addition, the present invention provides a health functional food composition for preventing or improving muscle diseases, comprising amentoflavone as an active ingredient.

[0058] The present invention can be generally used as a commonly used food.

[0059] The food composition of the present invention may be used as a health functional food. 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 in accordance with the Health Functional Foods Act, and the term “functional properties” refers to consuming the food for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0060] The above-mentioned health functional food composition may include ordinary food additives, and unless otherwise specified, suitability as a “food additive” shall be determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0061] Examples of items listed in the above “Food Additives Codex” include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon 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.

[0062] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a conventional hard capsule with the composition according to the present invention and additives such as excipients, and soft capsules can be manufactured by mixing the composition according to the present invention with additives such as excipients and filling it into a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0063] The definitions of terms regarding the above excipients, binders, disintegrants, lubricants, flavoring agents, etc., are those described in literature known in the art and include those with identical or similar functions. There are no special restrictions on the types of food mentioned above, and they include all health functional foods in the conventional sense.

[0064] In the present invention, the term “prevention” refers to any act of suppressing or delaying muscle disease through the administration of a composition according to the present invention.

[0065] In this invention, the term “treatment” refers to any act of improving or beneficially altering the symptoms of a muscle disease through the administration of a composition according to this invention.

[0066] In this invention, the term “improvement” refers to any act of improving a poor condition of a muscle disease through the administration of a composition according to this invention.

[0067] 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.

[0068]

[0069] [Experimental Example]

[0070] 1. Computer Analysis (in silico)

[0071] Components constituting Ginkgo biloba were collected through a literature search, and the structures of these components were collected from the PubChem (https: / pubchem.ncbi.nlm.nih.gov / ) compound database. To verify MSTN and the binding potential of the collected components, Discovery Studio and Autodock intermolecular binding analysis programs were used.

[0072]

[0073] 2. Isolation and Culture of Muscle Stem Cells

[0074] To extract muscle stem cells from cattle, pigs, and chickens, muscle tissue was collected from the rump of a cow, the foreleg of a pig, and the leg of a chicken embryo, finely minced, placed in a 0.1% pronase enzyme solution, and stirred at 37°C for 2 hours. Afterward, centrifugation was performed at 1,000g for 3 minutes to remove the supernatant, and the tissue was suspended in DMEM medium containing 1% penicillin / streptomycin (hereinafter referred to as P / S) and 10% fetal bovine serum (hereinafter referred to as FBS), and then the degraded cells from the muscle tissue were separated through a 100μm filter. The isolated cells were centrifuged at 1,000g for 5 minutes to remove the supernatant, suspended in Ham's F10 medium containing 1% P / S, 20% FBS, and 5 ng / mL fibroblast growth factor 2 (FGF2), then transferred to a cell culture dish and cultured at 37°C and 5% CO2. The beef rump and pork foreleg were purchased from Green Meat Distribution (1F, Building N, 17 Josan-ro, Aplyang-eup, Gyeongsan-si, Gyeongbuk), and the chicken embryos were purchased from Egg Han Kkureomi (308 Wonhyo-ro, Gyeongsan-si, Gyeongbuk).

[0075] Human skeletal muscle cells (purchased from ATCC) were cultured in proliferation medium [Ham's F10 medium containing 20% ​​FBS, 1% P / S and 5 ng / mL fibroblast growth factor 2 (hereinafter referred to as FGF2)].

[0076]

[0077] 3. Muscle Differentiation Analysis

[0078] To determine the effect of the sample (Amentoflavone; Ametoflavone) (sigma Aldrich, CAS No: 1617-53-4) on muscle differentiation, when the muscle stem cells cultured in Experimental Example 2 proliferated to cover more than 90% of the surface of the culture dish, the medium was replaced with differentiation medium (DMEM medium containing 1% P / S and 2% FBS), and the sample was diluted to different concentrations and treated. After 4 days of differentiation culture, all the medium was removed and washed with physiological saline (phosphate buffer saline; hereinafter referred to as PBS). Then, 200 μL of fresh PBS was added, and the cells in the culture dish were recovered. The cell membranes of the recovered cells were disrupted by sonication, and the supernatant was separated by centrifugation.

[0079] For the creatine kinase activity assay, a muscle-specific enzyme, an assay mixture (100 μL buffer, 10 μL substrate, and 1 μL enzyme) of the creatine enzyme activity assay kit was prepared, and 10 μL of the supernatant obtained from cell lysis was mixed with 100 μL of the assay mixture and reacted. Subsequently, the absorbance at 340 nm was measured using a microplate reader at reaction times of 20 minutes and 25 minutes, respectively. The measured creatine enzyme activity was calculated using the following Equation 1.

[0080] [Mathematical Formula 1]

[0081] Creatine kinase activity (U / L) = (25-minute OD value - 20-minute OD value) / (calibrator OD value - distilled water OD value) × 600

[0082]

[0083] 4. Analysis of gene expression of muscle-specific markers

[0084] RT-PCT analysis was performed to determine the effect of the sample on the gene expression of muscle-specific markers. First, to extract RNA, muscle stem cells treated with the sample were differentiated for 4 days. Subsequently, all cell culture media were removed, and 1 mL of Trizol reagent was added to lyse the cells. 200 μL of chloroform was added to the lysed cells, and centrifugation was performed at 12,000 rpm for 10 minutes. Afterward, the clear liquid layer of the supernatant was transferred to a new tube, and 500 μL of isopropanol was added. The mixture was then stirred thoroughly, and centrifugation was performed at 12,000 rpm for 10 minutes. Subsequently, all supernatant excluding the RNA precipitate was removed, and 1 mL of 70% ethanol was added for washing. Afterward, all ethanol was removed, and the RNA precipitate was dissolved in distilled water containing DEPC (Diethyl pyrocarbonate). The acquired RNA was quantified using a spectrophotometer. 2 μg of RNA was reacted with reverse transcriptase to synthesize cDNA. Gene expression was confirmed via RT-PCR analysis. The synthesized cDNA was mixed with primers designed using the primer design tool provided by the National Center for Biotechnology Information (NCBI) (Table 1) and a reagent containing SYBR green fluorescent dye. RT-PCR analysis was performed using the Applied Biosystems 7500 real-time PCR.

[0085] Gene Species Size (bp) Temperature (°C) Forward primer (5' → 3') Reverse primer (5' → 3') GAPDH 20859 GGG TCA TCT CTG CAC CT (Sequence No. 1) ACA GTC TTC TGG GTG GCA GT (Sequence No. 2) MYOD 22959 GAT GAC CCG TGT TTC GAC TC (Sequence No. 3) TAG TCG TCT TGC GTT TGC AC (Sequence No. 4) MYOG 19759 TGG GCG TGT AAG GTG TGT AA (Sequence No. 5) TGC AGG CGC TCT ATG TAC TG (Sequence No. 6) MYH 23557 GGA GAT GCG AGA TGA AAA GC (Sequence No. 7) CAT GTT GGC CAT TTC CTT CT (Sequence No. 8) MSTN 24059 TGC CCA CGG AGT CTG ATC TT (Sequence No. 9) CAG TGC CTG GGT TCA TGT CA (Sequence No. 10) GAPDH Pig 14760 TCG GAG TGA ACG GAT TTG GC (Sequence No. 11) TGC CGT GGG TGG AAT CAT AC (Sequence No. 12) MYOD Pig 19660 GCA CTA CAG CGG TGA CTC AG (Sequence No. 13) CAC GAT GCT GGA CAG ACA GT (Sequence No. 14) MYOG Pig 16458 CAG TGA ATG CAG TTC CCA CA (Sequence No. 15) CCA CAT CCT CCA CTG TGA TG (Sequence No. 16) MYH Pig 16258 TTC CTT CCA AAC CGT CTC TG (Sequence No. 17) TTA CAC CTC AGC TGG TGC AG (Sequence No. 18) MSTN Pig 24259 CAT GCC TAC AGA GTC TGA TCT T (Sequence No. 19) CAG TGC CTG GGT TCA TGT CA (Sequence No. 20) GAPDH Chicken 15759 CAA CAT CAA ATG GGC AGA TG (Sequence No. 21) GAC ACC CCA TCA CAA ACA TGG (Sequence No. 22) MYOD Chicken 16059 AGC TCT CGC AGG AGA AAC AG (Sequence No. 23) CTG GAG GCA GTATGG GAC AT (Sequence No. 24) MYOG Chicken 20559 CGC CAT CAG TAC AAT CGA G (Sequence No. 25) ATC GCT CAG GAG GTG ATC TG (Sequence No. 26) MYH Chicken 12859 CTG CCG ATG AAA AAG TGG CTA (Sequence No. 27) AGC CTT GTC TGC AAC TTC TG (Sequence No. 28) MSTN Chicken 19759 AGT AGC GAT GGC TCT TTG GA (Sequence No. 29) GGT TTT TGG ACT TGC CTC AA (Sequence No. 30)

[0086]

[0087] 5. Analysis of Protein Expression of Muscle-Specific Markers

[0088] Western blot analysis was performed to determine the effect of the sample on the protein expression of muscle-specific markers. Muscle stem cells treated with the sample were differentiated for 4 days. Afterward, all cell culture media were removed, and the cells were washed with PBS. Subsequently, a mixture containing RIPA (Radioimmunoprecipitation) buffer and 1% protease inhibitor was added to the cells, the cells were scraped, centrifuged at 12,000 rpm for 10 minutes, the supernatant was collected, and the protein concentration was measured using a spectrophotometer. 60 μg of the extracted protein was electrophoresed on a 10% polyacrylamide gel and transferred to a PVDF (Polyvinylidene fluoride) membrane. Then, the membrane was blocked for 1 hour in a TBS (tris-buffered saline) solution containing 3% skim milk and 1% surfactant, after which the primary antibody was added. The sample was reacted at 4°C for 16 hours, washed three times for 10 minutes each with TBS (tris-buffered saline) buffer containing 1% surfactant, and then reacted with the secondary antibody at room temperature for 1 hour. Afterward, it was washed three times for 10 minutes each with TBS buffer containing 1% surfactant, and then developed after adding a chemical chromogen (Super Signal West Pico Chemiluminescent Substrate).

[0089]

[0090] 6. ROS Activation Analysis

[0091] To determine the effect of the sample on ROS (reactive oxygen species) activity, muscle stem cells treated with the sample were differentiated for 4 days. Afterward, all cell culture medium was removed, and 10 μM of 2',7'-dichlorofluorescein (Sigma-Aldrich, St. Louis) was added and incubated at 37°C for 2 hours. Subsequently, the cells were washed twice with PBS, and fluorescence was measured using a spectrophotometer (excitation 498 nm / emission 522 nm).

[0092]

[0093] 7. Analysis of the Effect of Improving Muscle Disorders

[0094] To confirm the effect of the sample on improving muscle disease, animal experiments were conducted. These animal experiments were performed under the approval of the Animal Ethics Committee of NDIC Co., Ltd. in accordance with the Animal Protection Act (Act No. 4379 enacted on May 31, 1991, and Act No. 19880 partially amended on January 2, 2024) (Approval No.: P251001). Muscle atrophy was induced in male C57BL / 6N mice (purchased from Coatec) by administering dexamethasone (hereinafter referred to as DEX) intraperitoneally at a dose of 25 mg / kg once daily for a total of 10 days, and simultaneously with the administration of dexamethasone, amentoflavone was orally administered at a dose of 1 mg / kg once daily for a total of 10 days. During the administration period, the body weight of the mice was measured at two-day intervals, and on the 10th day after administration of amentoflavone, the weight of the gastrocnemius muscle and quadriceps femoris muscle was measured and proteins were extracted through dissection. To measure the muscle strength and endurance of the mice, the grip strength test and the rotarod test were performed, respectively.

[0095]

[0096] 8. Statistical Analysis

[0097] The Tukey test was used to analyze the differences between the means of gene expression. The software used was SAS version 9.0, and one-way ANOVA was performed using PROC GLM. A p-value of ≤ 0.05 was considered to indicate statistical significance.

[0098]

[0099] [Example]

[0100] 1. Analysis of MSTN and intermolecular binding

[0101] As a result of analyzing the intermolecular binding of the ginkgo constituents identified in Experimental Example 1 and MSTN, it was predicted that the sample (amentoflavone; AMF) exhibits excellent intermolecular binding with MSTN. As shown in Figure 1, it was predicted that the sample and MSTN could interact at a binding free energy level of -8.20 kcal / mol. Additionally, as shown in Table 2, it was predicted that the sample could interact with the amino acids constituting MSTN (Phe2, Val50, Phe51, Leu52, Gln53, Lys54, Tyr55, Pro56, His57, Thr58, and His59). Furthermore, as shown in Table 3, the small intestinal absorption rate of the sample was predicted to be 84.356%, and negative results were obtained regarding carcinogenicity, hepatotoxicity, and skin irritation, confirming that it is safe when applied to the human body.

[0102]

[0103]

[0104]

[0105] 2. Analysis of Intermolecular Binding Stability of Samples and MSTN

[0106] To verify the binding stability of the sample and MSTN, a molecular dynamics simulation study was performed. As a result, as shown in Fig. 2, when the stability of the sample and MSTN complex was evaluated based on hydrogen bonding, the graph showed low variability, confirming that it exhibited good binding stability (Fig. 2A). In addition, although a maximum of five hydrogen bonds were predicted for the complex, it was confirmed that only three hydrogen bonds were ultimately maintained stably (Fig. 2B).

[0107]

[0108] 3. Analysis of Effects on Bovine Muscle Stem Cells

[0109] According to Experimental Example 3 above, the effect of the sample on muscle differentiation in bovine muscle stem cells was analyzed, and as shown in Fig. 3A, creatine kinase activity significantly increased starting from a treatment concentration of 1 nM of the sample. In addition, when the myosin heavy chain (MYH) protein constituting muscle fibers was identified through fluorescence staining, as shown in Fig. 3B, the MYH area significantly increased in the sample-treated group (AMF, 1 nM) compared to the control group (untreated group; Con), and as shown in Fig. 3C, the canal width significantly increased in the sample-treated group compared to the control group (an increase of approximately 8.92 μm).

[0110] In addition, according to Experimental Examples 4 and 5 above, the effect of the sample on muscle-specific marker expression in bovine muscle stem cells was analyzed, and as shown in Fig. 3D, compared to the control group (untreated group; Con), the gene and protein expression of MYOD (hereinafter referred to as MYOD), Myogenin (hereinafter referred to as MYOG), and MYH were all significantly increased in the sample-treated group (AMF, 1 nM), and the gene and protein expression of MSTN were significantly decreased.

[0111] In addition, according to Experimental Example 6 above, the effect of the sample on ROS activity in bovine muscle stem cells was analyzed, and as shown in Fig. 3E, ROS activity was significantly reduced in the sample-treated group (AMF, 1 nM) compared to the control group (untreated group; Con) (reduced by about 20%).

[0112]

[0113] 4. Analysis of the effects on porcine muscle stem cells

[0114] According to Experimental Example 3 above, the effect of the sample on muscle differentiation in porcine muscle stem cells was analyzed, and as shown in Fig. 4A, creatine kinase activity significantly increased starting from a treatment concentration of 10 nM of the sample. In addition, when MYH (myosin heavy chain) protein was confirmed by fluorescence staining, as shown in Fig. 4B, the MYH area significantly increased in the sample-treated group (AMF, 10 nM) compared to the control group (untreated group; Con), and as shown in Fig. 4C, the root canal width significantly increased in the sample-treated group compared to the control group (an increase of approximately 6.51 μm).

[0115] In addition, according to Experimental Examples 4 and 5 above, the effect of the sample on muscle-specific marker expression in porcine muscle stem cells was analyzed, and as shown in Figure 4D, the gene and protein expression of MYOD, MYOG, and MYH were all significantly increased in the sample-treated group (AMF, 10 nM) compared to the control group (untreated group; Con), and the gene and protein expression of MSTN were significantly decreased.

[0116] In addition, according to Experimental Example 6 above, the effect of the sample on ROS activity in porcine muscle stem cells was analyzed, and as shown in Fig. 4E, ROS activity was significantly reduced (about 20% reduction) in the sample-treated group (AMF, 10 nM) compared to the control group (untreated group; Con).

[0117]

[0118] 5. Analysis of the effects on chicken muscle stem cells

[0119] According to Experimental Example 3 above, the effect of the sample on muscle differentiation in chicken muscle stem cells was analyzed, and as shown in Fig. 5A, creatine kinase activity significantly increased starting from a treatment concentration of 1 nM of the sample. In addition, when MYH (myosin heavy chain) protein was confirmed by fluorescence staining, as shown in Fig. 5B, the MYH area significantly increased in the sample-treated group (AMF, 10 nM) compared to the control group (untreated group; Con), and as shown in Fig. 5C, the root canal width significantly increased in the sample-treated group compared to the control group (an increase of approximately 7.47 μm).

[0120] In addition, according to Experimental Examples 4 and 5 above, the effect of the sample on muscle-specific marker expression in chicken muscle stem cells was analyzed, and as shown in Fig. 5D, the gene and protein expression of MYOD, MYOG, and MYH were all significantly increased in the sample-treated group (AMF, 10 nM) compared to the control group (untreated group; Con), and the gene and protein expression of MSTN were significantly decreased.

[0121] In addition, according to Experimental Example 6 above, the effect of the sample on ROS activity in chicken muscle stem cells was analyzed, and as shown in Fig. 5E, ROS activity was significantly reduced (about 20% reduction) in the sample-treated group (AMF, 10 nM) compared to the control group (untreated group; Con).

[0122]

[0123] 6. Analysis of effects on human muscle stem cells

[0124] According to Experimental Example 3 above, the effect of the sample on muscle differentiation in human muscle stem cells was analyzed, and as shown in Fig. 6A, creatine kinase activity significantly increased starting from a treatment concentration of 100 nM of the sample. In addition, when MYH (myosin heavy chain) protein was confirmed by fluorescence staining, as shown in Fig. 6B, the MYH area significantly increased in the sample-treated group (AMF, 100 nM) compared to the control group (untreated group; Con), and as shown in Fig. 6C, the root canal width significantly increased in the sample-treated group compared to the control group (an increase of approximately 5.96 μm).

[0125] In addition, according to Experimental Examples 4 and 5 above, the effect of the sample on muscle-specific marker expression in human muscle stem cells was analyzed, and as shown in Fig. 6D, the gene and protein expression of MYOG and MYH were both significantly increased in the sample-treated group (AMF, 100 nM) compared to the control group (untreated group; Con), and the gene and protein expression of MSTN were significantly decreased.

[0126] In addition, according to Experimental Example 6 above, the effect of the sample on ROS activity in human muscle stem cells was analyzed, and as shown in Fig. 6E, ROS activity was significantly reduced in the sample-treated group (AMF, 100 nM) compared to the control group (untreated group; Con) (reduced by more than 20%).

[0127]

[0128] 7. Analysis of the Effect of Improving Muscle Disorders

[0129] According to Experimental Example 7 above, the effect of the sample on improving muscle disease was analyzed. As shown in Fig. 7A, body weight was significantly reduced in the DEX-only treatment group (DEX) compared to the control group, whereas body weight was significantly increased in the sample treatment group (DEX+AMF) compared to the DEX-only treatment group. Additionally, as shown in Fig. 7B, muscle weight was significantly reduced in the DEX-only treatment group, while muscle weight was significantly increased in the sample treatment group compared to the DEX-only treatment group.

[0130] In addition, according to Experimental Example 5, the effect of the sample on the protein expression of muscle-specific markers in mice (Experimental Example 7) was analyzed, and as shown in Fig. 7C, the protein expression of MYOD, MYOG, and MYH was significantly increased and the protein expression of MSTN was significantly decreased in the sample-treated group compared to the group treated with DEX alone.

[0131] In addition, according to Experimental Example 3, the effect of the sample on muscle differentiation in mice (Experimental Example 7) was analyzed, and as shown in Fig. 7D, creatine kinase activity was significantly reduced in the group treated with DEX alone compared to the control group in the gastrocnemius (GAS) and quadriceps (Quad) muscles, whereas creatine kinase activity was significantly increased in the sample treatment group compared to the group treated with DEX alone.

[0132] In addition, according to Experimental Example 7, a grip strength test was performed to evaluate the muscle strength of mice and a rota-rod test was performed to evaluate endurance. As shown in Figure 7E, muscle strength and endurance were significantly reduced in the DEX-only treatment group compared to the control group, whereas muscle strength and endurance were significantly increased in the sample treatment group compared to the DEX-only treatment group.

[0133] In addition, the area of ​​the root canal was measured by H&E (Hematoxylin & Eosin staining) staining of the gastrocnemius and quadriceps muscles of mice, and as shown in Fig. 7F, it was confirmed that the area of ​​the root canal was restored in the sample treatment group. Furthermore, as shown in Fig. 7G, it was confirmed that the MYH (myosin heavy chain) protein had more MYH expression sites in the sample treatment group compared to the DEX-only treatment group when examined by fluorescent staining.

[0134]

[0135] 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 culture medium composition for promoting muscle cell differentiation containing amentoflavone as an active ingredient.

2. A culture medium composition according to claim 1, characterized in that the amentoflavone promotes creatine kinase activity.

3. A culture medium composition according to claim 1, characterized in that the amentoflavone inhibits reactive oxygen species (ROS) activity.

4. A culture medium composition according to claim 1, characterized in that the amentoflavone promotes the expression of one or more selected from the group consisting of myodi, myogenin, and myosin heavy chain.

5. A culture medium composition according to claim 1, characterized in that the amentoflavone inhibits myostatin expression.

6. A culture medium composition according to claim 1, characterized in that the muscle cells are derived from one or more animals selected from the group consisting of humans, chickens, cattle, pigs, and mice.

7. An animal feed composition for promoting muscle cell differentiation containing amentoflavone as an active ingredient.

8. A culture medium composition for producing cultured meat containing amentoflavone as an active ingredient.

9. A culture medium composition according to claim 8, characterized in that the cultured meat is derived from one or more animals selected from the group consisting of chickens, cattle, pigs, and mice.

10. A method for producing cultured meat comprising the step of treating muscle cells of an organism other than a human with amentoflavone.

11. A pharmaceutical composition for the prevention or treatment of muscle diseases containing amentoflavone as an active ingredient.

12. A pharmaceutical composition according to claim 11, characterized in that the muscle disease is one or more selected from the group consisting of muscular atrophy, muscular dystrophy, sarcopenia, myopathy, myasthenia, and muscular injury.

13. A health functional food composition for the prevention or improvement of muscle diseases containing amentoflavone as an active ingredient.

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