Composition for preventing or ameliorating muscle diseases, comprising hemp seed extract

Abstract

The present invention relates to the use of a hemp seed extract for preventing or ameliorating muscle diseases by promoting myoblast differentiation or muscle regeneration. In the present invention, the hemp seed extract may exhibit preventive or therapeutic effects on various muscle diseases by increasing muscle mass, strengthening muscle power, improving muscle function, and enhancing exercise performance, and also has an effect of improving muscle function in healthy individuals in addition to treating muscle diseases.

Description

Composition for preventing or improving muscle diseases containing hemp seed extract

[0001] The present invention relates to the use of hemp seed extract for the prevention or improvement of muscle diseases, and more specifically, includes the use of improving muscle function and preventing or treating related diseases through the effect of the hemp seed extract on the proliferation and differentiation of root cells.

[0002]

[0003] Muscles play a crucial role in bodily functions such as energy metabolism and exercise capacity, and can be damaged or weakened by various factors including sarcopenia due to aging, muscular atrophy caused by nutritional imbalances or lack of exercise, other diseases such as cancer, and aging.

[0004] Sarcopenia, a major disease that damages muscles, is a condition in which muscle strength declines as skeletal muscle mass decreases due to aging. The most significant characteristic of sarcopenia is the decrease in muscle mass, and the types of muscle fibers may also change. While type 1 and type 2 muscle fibers decrease at similar rates with aging, the thickness of type 1 muscle fibers decreases more noticeably in patients with sarcopenia. It is reported that this sarcopenia causes muscle weakness and functional impairment among the elderly (Roubenoff R., Can. J. Appl. Physiol. 26, 78-89, 2001).

[0005] In addition, muscle atrophy, another major muscle disease, can occur due to nutritional deficiencies or prolonged disuse of muscles; it can develop as the balance between normal protein synthesis and breakdown is disrupted, leading to the decomposition of proteins within the muscles.

[0006] Various treatment methods are currently under development to fundamentally treat these muscle diseases. In particular, therapeutic approaches utilizing mechanisms that strengthen muscles by promoting the proliferation of muscle stem cells or enhancing the differentiation of muscle cells from them, or by regenerating and increasing muscle mass, are being proposed. Since these methods enable a fundamental cure for muscle diseases, research into various therapeutic substances that can make this possible is currently necessary.

[0007] Accordingly, the inventors discovered that an extract of hemp seeds promotes the differentiation of root cells and, through this, improves muscle function, thereby having a preventive or therapeutic effect against muscle diseases, and completed the present invention regarding a composition utilizing hemp seed extract.

[0008] Therefore, the object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of muscle diseases comprising hemp seed extract.

[0009] Another objective of the present invention is to provide a health functional food composition for the prevention or improvement of muscle diseases comprising hemp seed extract.

[0010] Another objective of the present invention is to provide a composition for increasing muscle mass comprising hemp seed extract.

[0011] Another objective of the present invention is to provide a composition for strengthening muscle or improving muscle function comprising hemp seed extract.

[0012]

[0013] To achieve the above objectives, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a hemp seed extract.

[0014] To achieve another objective of the present invention, the present invention provides a health functional food composition for the prevention or improvement of muscle diseases comprising hemp seed extract.

[0015] To achieve another objective of the present invention, the present invention provides a composition for increasing muscle mass comprising a hemp seed extract.

[0016] To achieve another objective of the present invention, the present invention provides a composition for strengthening muscle strength or improving muscle function comprising a hemp seed extract.

[0017]

[0018] The present invention will be described in detail below.

[0019] The present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a hemp seed extract.

[0020] In the present invention, the cannabis seeds are the seeds of the cannabis (Cannabis sativa) plant. In particular, cannabis seed oil, which is extracted from cannabis seeds, contains a large amount of an antioxidant component called cannabinoid and is also utilized in the treatment of stress, depression, and cancer. Cannabis seed oil is known to contain approximately 20 types of amino acids, is rich in fiber that helps improve digestive function and immunity, and contains a large amount of gamma-linolenic acid that helps balance female hormones. In particular, it contains unsaturated fatty acids, such as omega-6 and omega-3, in appropriate proportions, providing effects such as anti-inflammatory and inhibition of blood clot formation.

[0021] In one embodiment of the present invention, the hemp seed extract comprises a preparation obtained by extracting a natural product through a suitable solvent or by evaporating and concentrating the extract, but is not limited thereto. It may be an extract obtained by extraction treatment, a diluted or concentrated extract, a dried product obtained by drying the extract, a modified or purified product thereof. The hemp seed extract may be prepared using general extraction, separation, and purification methods known in the art.

[0022] In the present invention, the hemp seed extract may be extracted according to various known extraction methods and may particularly include an unsaponifiable extract. The extraction method may use, for example, supercritical CO₂ extraction, solvent extraction, cold pressing, microwave extraction, or ultrasonic extraction, but is not limited to such methods.

[0023] In particular, the hemp seed extract of the present invention may include an unsaponifiable extract of hemp seeds, and the unsaponifiable extract, i.e., 'unsaponifiable matter,' comprises all fatty components that are not well soluble or insoluble in water after saponification in a strong alkaline medium, and are soluble in organic solvents, such as ethyl ether, aromatic hydrocarbons, and chlorinated solvents. The hemp seed unsaponifiable extract of the present invention refers to an extract containing components that are not saponified with alkali from hemp seeds.

[0024] In one aspect of the present invention, the hemp seed extract of the present invention has the effect of promoting the differentiation of muscle stem cells or root cells, thereby enhancing muscle regeneration and increasing muscle mass, and furthermore, may have a preventive or therapeutic effect against muscle diseases caused by decreased muscle function, muscle loss, muscle atrophy, muscle wasting, or muscle degeneration.

[0025] The hemp seed extract of the present invention has an effect of “promoting myoblast differentiation,” and “myoblast differentiation” refers to the process in which mononuclear myoblasts form multinuclear myotubes through fusion, and refers to inducing this process. Cells in the differentiation stage that form myotubes are identified through the expression of related markers such as Myh3 and MyoG. When myoblast differentiation is promoted, muscle cells increase, and from an individual perspective, this can ultimately lead to an increase in muscle mass.

[0026] That is, the hemp seed extract of the present invention has a “muscle regeneration” effect. The “muscle regeneration” refers to the ability of damaged muscles to recover to a normal state. The muscle regeneration may be an effect resulting from the promotion of differentiation of root cells, which increases the absolute amount of muscle cells, or from an increase in the diameter of individual muscle tubes.

[0027] Furthermore, the hemp seed extract of the present invention has the effect of "increasing muscle mass" or "strengthening muscle strength." "Increasing muscle mass" may refer to a state in which the amount of muscle cells is increased through the differentiation of root cells, or a state in which the formation of myotubes is increased and the diameter of myotubes is increased. "Strengthening muscle strength" refers to the effects of enhancing physical performance, enhancing maximum endurance, increasing muscle mass, enhancing muscle recovery, reducing muscle fatigue, improving the energy balance, or a combination thereof. As previously explained, the extract of the present invention can increase total muscle mass by increasing muscle mass through the ability to differentiate root cells into muscle cells, and by promoting muscle regeneration, it can enhance maximum endurance, thereby enhancing physical performance and reducing muscle fatigue. Additionally, because muscle cells can be rapidly replaced, muscle damage can be healed quickly.

[0028] In addition, the hemp seed extract of the present invention can improve exercise performance through muscle strength enhancement, wherein "exercise performance" refers to the ability to perform exercise using muscle strength. The muscle strength can be improved by increasing muscle mass, muscle endurance, oxidative muscle mass, muscle recovery capacity, and the improvement of the energy balance within the muscle, and can also be enhanced by reducing fatigue substances within the muscle, but the hemp seed extract of the present invention has the effect of enhancing muscle strength particularly through effects such as promoting myoblast differentiation, increasing muscle mass, and regenerating muscle.

[0029] The hemp seed extract of the present invention has an effect of improving muscle function, which refers to the ability to exert force through muscle contraction and includes muscle strength, which is the ability of a muscle to exert maximum contractile force to overcome resistance; muscle endurance, which is the ability of a muscle to repeat contraction and relaxation for a given weight for a long time or many times; and explosiveness, which is the ability to exert strong force in a short period of time. Such muscle function is proportional to muscle mass, and "improvement of muscle function" means enhancing muscle function for the better. Furthermore, since the hemp seed extract of the present invention promotes mitochondrial activity within the muscle, it consequently improves the energy balance in the muscle, thereby having an effect of improving muscle function.

[0030] The composition containing the hemp seed extract of the present invention can be prepared in the form of a food composition, a food additive, or a health food composition for promoting differentiation of root cells, muscle regeneration, increasing muscle mass, strengthening muscle power, improving exercise performance, and improving muscle function. In particular, the muscle-strengthening composition of the present invention has effects of muscle generation, increasing muscle mass, and strengthening muscle power not only for the prevention or treatment of muscle diseases caused by aging or disease, but also for general individuals with normal muscle function. Therefore, it can be utilized in the form of a functional food or an adjuvant thereof.

[0031] In another aspect, the present invention relates to a composition for the prevention, improvement, or treatment of muscle diseases comprising a hemp seed extract or a pharmaceutically acceptable salt thereof. That is, the hemp seed extract of the present invention has a preventive, improvement, or therapeutic effect for muscle diseases. The term "muscle disease" refers to a muscle disease that occurs when muscle strength is weakened due to damage or loss of muscle caused by aging or disease, and this may be caused by various factors such as: aging-related diseases such as genetic predisposition, hypertension, impaired glucose tolerance, diabetes, obesity, dyslipidemia, atherosclerosis, or cardiovascular disease; chronic diseases such as cancer, autoimmune diseases, infectious diseases, AIDS, chronic inflammatory diseases, arthritis, malnutrition, kidney disease, chronic obstructive pulmonary disease, emphysema, rickets, chronic lower back pain, peripheral nerve damage, central nerve damage, and chemical damage; loss of movement due to causes such as fractures or trauma or long-term bed rest; and aging. In other words, the aforementioned muscle disease refers to one caused by aging, decline in muscle function, muscle wasting, muscle degeneration, disuse, or muscle damage.

[0032] The above muscle disease may be one or more muscle diseases selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, and sarcopenia, and more specifically, may include muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, sarcopenia, and muscle loss caused by aging or diseases such as cancer.

[0033] In this specification, the term "prevention" refers to any act that can suppress muscle disease or delay its onset by administering the pharmaceutical composition according to the present invention.

[0034] In this specification, the term "treatment" refers to any act in which symptoms are improved or benefited by the administration of the pharmaceutical composition according to the present invention.

[0035] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods, and may additionally include carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients, and diluents that may additionally be included in the active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0036] For example, solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, or gelatin, with the extract or compound. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration 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.

[0037] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, 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, cocoa oil, laurin oil, glycerogelatin, etc. may be used.

[0038] The pharmaceutical composition of the present invention may be administered orally or parenterally (intravenous injection, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time, and may be selected in an appropriate form by those skilled in the art.

[0039] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceuticalally effective amount" refers to a reasonable amount applicable to medical treatment, meaning an amount sufficient to treat a disease, and the criteria may be determined based on the patient's disease, severity, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitant components, and other factors. The pharmaceutical composition of the present invention may be administered in combination with an individual therapeutic agent or other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, the dosage may be determined to a level that minimizes side effects, and this can be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, gender, etc., and generally, an amount of 0.001 to 150 mg, more preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, 1 to 3 times a day. However, this is for illustrative purposes only, and the above dosage may be set differently as needed.

[0040] In addition, the composition of the present invention may be a food or a health functional food, and in particular, 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 pursuant to Article 6727 of the Health Functional Foods Act, and the term "functionality" means consuming for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological action on the structure and function of the human body.

[0041] The food or health functional food of the present invention can be manufactured and processed into pharmaceutical administration forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, etc., or into health functional foods such as tea bags, infusions, beverages, candies, jellies, and gums for the purpose of preventing and improving muscle diseases.

[0042] The food or health functional food composition of the present invention may be used as a food additive and may be manufactured into a product either alone or in combination with other ingredients. Additionally, it may include nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents and promoters, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The above ingredients may be used alone or in combination, and may be combined and used in appropriate amounts.

[0043] As another embodiment of the present invention, the present invention relates to a feed or feed additive composition comprising a hemp seed extract.

[0044] In the present invention, "feed" refers to a substance that supplies organic or inorganic nutrients necessary to sustain the life of an animal. The feed includes nutrients such as energy, protein, lipids, vitamins, and minerals required by animals such as livestock, and may be plant-based feed such as grains, root fruits, food processing by-products, algae, fibers, oils, starches, meal, grain by-products, or animal-based feed such as proteins, inorganic substances, oils, minerals, oils, and single-cell proteins, but is not limited thereto.

[0045] In the present invention, the term "feed additive" refers to a substance added to feed to improve the productivity or health of animals, and is not limited thereto, but may further include amino acid preparations, vitamin preparations, enzyme preparations, flavoring agents, silicate preparations, buffering agents, extractants, oligosaccharides, etc. for promoting growth, preventing disease, etc. The feed or feed additive composition of the present invention may have the effect of promoting differentiation of animal muscle cells, muscle regeneration, or muscle strength enhancement, and furthermore may have the effect of preventing, treating, or improving muscle-related diseases of animals.

[0046] In other words, the composition containing the hemp seed extract of the present invention can be utilized for various purposes, such as pharmaceuticals, health functional foods, functional foods, animal feed, and cell culture medium compositions. It promotes the differentiation of root cells, thereby providing effects of muscle regeneration, increased muscle mass, improved muscle function, enhanced muscle strength, and increased exercise capacity. Furthermore, it has effects of preventing, improving, or treating muscle diseases.

[0047] The present invention relates to a composition comprising an unsaponifiable extract of hemp seeds that has a muscle differentiation-promoting or muscle differentiation effect. The composition regenerates muscle through the enhancement of muscle differentiation, and thereby can have preventive or therapeutic effects on various muscle diseases due to increased muscle mass, strengthened muscle power, improved muscle function, and increased exercise performance. Since it is also effective for improving muscle function in the general population, it can be used for various purposes, such as pharmaceutical compositions, functional foods, and additives.

[0048]

[0049] Figure 1 is a graph showing cell viability by concentration after treating mouse C2C12 cell lines with hemp seed extract at concentrations of 0, 1, 10, 100, 1,000, and 10,000 ng / ml, respectively, to analyze the cytotoxicity of hemp seed extract by concentration, and then performing an MTT assay.

[0050] Figure 2 shows the results of measuring luciferase activity against PGC-1α after treating C2C12 cells with hemp seed extract at concentrations of 0.1 and 1 μg / mL, respectively. Statistics are expressed as p<0.0001 through a one-way ANOVA test.

[0051] Figures 3 to 6 show the results of confirming the differentiation-enhancing effect of C2C12 cells.

[0052] Figure 3 shows the results of MHC immunofluorescence staining performed to analyze the effect of hemp seed extract on muscle cell differentiation in C2C12 cells when treated at different concentrations (0.1, 1 μg / mL). Figure 3A is an image of the MHC staining results, and Figure 3B is a graph analyzing the diameter of the root canal. (Scale bar: 100 μm) Statistics are expressed as *p<0.05 through a one-way ANOVA test.

[0053] Figure 4 shows the results of enhancing mRNA expression of genes involved in muscle differentiation (Myh3, Myogenin-1) when C2C12 cells were treated with hemp seed extract at different concentrations (0.01, 0.1, 1 μg / mL). Statistics are expressed as *p<0.05, ***p<0.001, and ****p<0.0001 through a one-way ANOVA test.

[0054] Figure 5 shows the results of an immunoblock analysis on the protein expression of muscle differentiation signaling markers PGC1α, p-AKT, AKT, p-p70, and p70 when C2C12 was treated with hemp seed extract.

[0055] Figure 6 shows the results (n=3) of qRT-PCR analysis on factors related to mitochondrial activity in muscle after treating root cells (C2C12) with hemp seed extract at different concentrations (0.01, 0.1, 1 μg / mL). Statistics are expressed as **p<0.01 and ****p<0.0001 through a one-way ANOVA test.

[0056] Figure 7 shows the results of MHC immunofluorescence staining performed after treatment with dexamethasone to analyze the inhibitory effect of hemp seed extract on muscular dystrophy. Figure 7A is an image of the MHC staining results, and Figure 7B is a graph analyzing the diameter of the root canal. (Scale bar: 100μm). (scale bar: 100μm) Statistics are expressed as *p<0.05 through an unpaired t-test.

[0057] Figure 8 shows the results of analyzing the difference in expression of muscle atrophy-related markers (Atrogin-1, MuRF-1) after treatment with dexamethasone to analyze the inhibitory effect of hemp seed extract on muscular dystrophy using real-time polymerase chain reaction. Statistics are expressed as *p<0.05 and ***p<0.001 through an unpaired t-test.

[0058]

[0059] Hereinafter, embodiments are described in detail to specifically explain the present specification. However, the embodiments according to the present specification may be modified in various different forms, and the scope of the present specification is not to be interpreted as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those with average knowledge in the art.

[0060]

[0061] Example 1. Preparation of hemp seed extract

[0062] To prepare the hemp seed unsaponifiable extract, the hemp seed unsaponifiable extract was obtained using the saponification extraction method. Specifically, for saponification, approximately 4.0 g of the hemp seed sample was added to a saponification reaction vessel, 20 mL of 6% Pyrogallol and 8 mL of 6% KOH were added, and the container was filled with nitrogen. The mixture was reacted in a water bath at 75°C for 50 minutes, after which it was cooled in an ice box. 30 mL of 2% NaCl and 20 mL of extraction solvent (0.01% BHT hexane:ethyl acetate = 85:15) were added. After shaking and subsequent phase separation, the supernatant was extracted, concentrated under reduced pressure to remove the solvent, and then freeze-dried to obtain the hemp seed unsaponifiable extract of the present invention. Additionally, the obtained extract was stored at -20°C and used as a sample for this experiment.

[0063]

[0064] Example 2. Culture and Differentiation Induction of Root Cell Line C2C12

[0065] C2Cl2 is a myoblastic cell line obtained from live mice of the C3H species and is widely used in myoblastic differentiation research. The above C2C12 cells were cultured in general cell culture medium and differentiation medium, respectively. DMEM supplemented with 15% fetal bovine serum was used as the normal cell culture medium (GM, growth media), and DMEM containing 2% horse serum was used as the differentiation medium (DM, differentiation media).

[0066] Cells were seeded into cell culture medium (GM) and cultured for 24 hours, then DMSO and cannabis seed extract (0.1 and 1 μM / mL) were added to differentiation medium (DM) at different concentrations and differentiation was induced for 3 days.

[0067] Example 3. mRNA expression analysis via qPC-PCR

[0068] C2C12 cells proliferated or differentiated in Example 2 above were lysed using the Easy-spin Total RNA Extraction Kit, chloroform was added, and centrifugation was performed. After separating the supernatant, an equal volume of isopropanol was added and binding was carried out. After centrifugation, the mixture was washed with 70% EtOH mixed with DEPC water, and 20–50 μl of DEPC solution was added to quantify the RNA concentration. The RNA was reverse transcribed using the PrimeScript cDNA synthesis kit to synthesize cDNA. Gene expression was analyzed using qRT-PCR with a Real-Time PCR system utilizing SYBR Premix EX Taq. The fold change in gene expression was normalized relative to the expression of ribosomal gene 18S rRNA.

[0069] Example 4. Immunoblot

[0070] C2C12 cells differentiated in Example 2 above were obtained and centrifuged at 13,000 rpm. The cells were lysed in Lysis buffer (ATTA) containing a Protease Inhibitor and a Phosphatase Inhibitor (ATTA). After centrifugation, the lysed cell samples were quantified, and an equal amount of protein was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature and then washed with TBST (RAON). Primary antibodies PGC1α, p-AKT, AKT, p-p70, p70, β-tubulin, and Total-OxPhos were diluted 1:500 and added to TBST containing 5% BSA, followed by overnight incubation at 4°C. The next day, a secondary antibody was diluted 1:5000 and added to TBST containing skim milk, reacted at room temperature, washed with TBST, and then ECL (Enhanced Chemiluminescent solution, Thermo Fisher Scienfix) was added. The membrane was exposed to a chemiluminescent machine (e-Blot touch imager) to determine the amount of protein.

[0071]

[0072] Experimental Example 1. Evaluation of cell viability against hemp seed extract-induced toxicity

[0073] C2C12 myocells were treated with cannabis seed extracts at concentrations of 0, 1, 10, 100, 1,000, and 10,000 ng / ml for 24 hours, and cell viability was confirmed by performing an MTT assay. Myocells (3.5 x 10⁶) on cell culture medium 5Cells (96-well) were reacted with 0.2 mg / ml of MTT for 4 hours, and the eluted product was then measured at 595 nm absorbance. As a result, cell viability was nearly similar to the control group up to a concentration of ~1000 nM of hemp seed extract, and it was confirmed that cell viability significantly increased at a concentration of 1 ng / ml. (Fig. 1)

[0074]

[0075] Experimental Example 2. PGC1-α reporter assay

[0076] 1.5 x 10 per well in a 96-well culture plate 4 Cells were seeded into each well, and immediately transfected by treating with PGC-1α promoter luciferase (DNA) and Mirus (transfection reagent) at a ratio of [150 ng / 10 μl] + [0.3 μl / 10 μl] per well, respectively. After 24 hours, differentiated hemp seed extracts were treated at 0.1 μg and 1 μg / ml, and differentiation was induced for 24 hours. 24 hours after drug treatment, cells were lysed in 30 μl of 1x cell culture lysis reagent (Promega) per well for 30 minutes. The lysates were then transferred to a plate for luminometer reading, and luciferin was added to induce an enzymatic reaction, after which the luminescence intensity was measured. The expression level of luciferase in PGC-1α reporter cell lines depends on the stimulation received by the PGC-1α promoter. In other words, if the compound is a PGC-1α expression inducer, it stimulates the PGC-1α promoter to increase luciferase expression, and if it is a PGC-1α expression inhibitor, it decreases luciferase expression. The degree of expression was assessed by measuring the luminescence produced upon the addition of a luciferase substrate to determine the activity or inhibition of PGC-1α transcription by the hemp seed extract.

[0077] As a result, as shown in Figure 2, it was confirmed that luciferase activity against PGC-1α increased in the group treated with hemp seed extract.

[0078]

[0079]

[0080] Experimental Example 3. Confirmation of effect promoting myocell differentiation

[0081] MHC immunofluorescence staining was performed to analyze the effect of hemp seed extract on the differentiation of myocells into muscle cells (Fig. 3). C2C12 cell lines were treated with control DMSO hemp seed extract at concentrations of 0.1 μg / ml and 1 μg / ml, respectively, and muscle cell differentiation was induced as in Example 2. The degree of myotube formation was compared and analyzed by performing immunofluorescence staining with MHC antibodies on the third day after differentiation induction. As a result, the hemp seed extract promoted muscle cell differentiation compared to the control group (Fig. 3A). It was confirmed that the formation of multinucleated myotubes increased starting from a concentration of 0.1 μg / ml, and the diameter of the myotubes increased as the concentration increased (Fig. 3B).

[0082] In addition, the C2Cl2 cell line of Example 2 was inoculated into a cell culture medium and cultured in DMEM medium for 24 hours. Then, hemp seed extracts (0 (DMSO), 0.01, 0.1, and 1 μg / mL) were added to the differentiation medium at different concentrations, and differentiation was induced for 3 days while replacing the medium every other day. After differentiation, mRNA expression levels were analyzed using Myogenin-1 and Myh3 primers, which are myogenic cell differentiation markers, using the method of Example 3, or protein expression levels of p-AKT and p-p70, which are muscle differentiation signaling markers, were confirmed using the method of Example 4.

[0083] As a result, as shown in Figure 4, the effect of enhancing mRNA expression of Myh3 and Myogenin-1 (MyoG), genes involved in muscle differentiation, was confirmed in myocells treated with hemp seed extract. Additionally, as shown in Figure 5, it was confirmed that the protein expression of PGC1α, p-ATK, and p-p70 increased in myocells treated with hemp seed extract. From the above results, it was confirmed that the hemp seed extract promotes the differentiation of myocells by maximally increasing the mRNA or protein expression of Myh3, PGC1α, p-ATKT, and p-p70.

[0084]

[0085] Experimental Example 4. Confirmation of mitochondrial activity enhancement effect

[0086] To induce differentiation of C2Cl2 myocells in the same manner as in Example 2 and to analyze the regulation of muscle activity, the expression of mitochondrial-related genes was analyzed using the method of Example 3 (Fig. 6). C2C12 myocells were treated with DMSO or hemp seed extract at concentrations of 1 μg / ml (0 μg / mL, 0.01, 0.1, and 1 μg / mL), respectively, and muscle cell differentiation was induced for 3 days. Subsequently, the mRNA expression levels of mitochondrial-related genes ATP5a, Sdhb, MTCO1, and NdufB8 were analyzed by performing real-time polymerase chain reaction. As a result, it was confirmed that mitochondrial activity, which is important for the regulation of muscle activity, generally increased upon treatment with hemp seed extract.

[0087]

[0088] Experimental Example 5. Confirmation of the effect of improving DEX (dexamethasone)-induced muscular dystrophy

[0089] To analyze the inhibitory effect of hemp seed extract on muscular dystrophy, dexamethasone (DEX) was administered and MHC immunofluorescence staining was performed. C2C12 myocells were differentiated in DM (differentiation medium) for 4 days, then treated with 50 μM DEX while simultaneously being treated with 1 μg / mL of hemp seed extract, and cultured in DM for an additional 24 hours.

[0090] As a result, the diameter of the DEX-treated C2C12 root canals was thinned compared to the control group, and it was observed that the canals were cut in places. However, upon treatment with hemp seed extract, it was confirmed that the diameter of the root canals thinned by dexamethasone was restored / improved to a level equivalent to the control group (DMSO-treated group) (Figs. 7a, 7b).

[0091] In addition, the mRNA expression levels of Atrogin-1 and MuRF-1, which are mediators of muscle atrophy, were analyzed using the method of Example 3, and it was confirmed that the mRNA expression of MuRF-1 and Atrogin-1, which was increased by dexamethasone, decreased upon treatment with hemp seed extract (Fig. 8). That is, from the above results, it was determined that hemp seed extract has the efficacy to prevent or improve muscle atrophy by inhibiting the expression of MuRF-1 and Atrogin-1, which are skeletal muscle-specific ubiquitin ligases, and by restoring and upregulating the diameter of the root canal induced by dexamethasone.

[0092]

[0093] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

[0094]

[0095] In one embodiment, the present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a hemp seed extract.

[0096] In another aspect, the present invention relates to a health functional food composition for the prevention or improvement of muscle diseases comprising hemp seed extract.

[0097] In one embodiment, the muscle disease is selected from the group comprising atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, muscle wasting, and sarcopenia.

[0098] In one embodiment, the muscle disease is caused by aging, decreased muscle function, muscle wasting, muscle degeneration, disused or damaged muscle.

[0099] In another aspect, the present invention relates to a composition for increasing muscle mass comprising hemp seed extract.

[0100] In another aspect, the present invention relates to a composition for strengthening muscle or improving muscle function comprising a hemp seed extract.

[0101] In one embodiment, the hemp seed extract has the effect of promoting root cell differentiation or muscle regeneration.

[0102] In one embodiment, the hemp seed extract is an unsaponifiable extract,

[0103] 1) A step of adding pyrogallol and potassium hydroxide to hemp seeds and filling with nitrogen;

[0104] 2) A step of reacting in a water bath at a temperature of 70 to 80°C and then cooling;

[0105] 3) Sodium chloride (NaCl); and dibutylhydroxytoluene to the reactants after cooling is complete

[0106] (BHT) A mixed extraction solvent of hexane and ethyl acetate is added and shaken to obtain the supernatant; and

[0107] 4) It is prepared by an extraction method comprising the steps of removing the solvent from the obtained supernatant and freeze-drying.

[0108] In another aspect, the present invention relates to the use of hemp seed unsaponifiable extract for the prevention or treatment of muscle diseases through the effect of promoting muscle stem cell differentiation or muscle regeneration; improvement of muscle function; or increase of muscle mass.

Claims

1. A pharmaceutical composition for the prevention or treatment of muscle diseases comprising hemp seed extract.

2. A health functional food composition for the prevention or improvement of muscle diseases comprising hemp seed extract.

3. In either Paragraph 1 or Paragraph 2, A composition in which the above muscle disease is selected from the group comprising atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, muscle wasting, and sarcopenia.

4. In Paragraph 3, A composition characterized in that the above-mentioned muscle disease is caused by aging, decreased muscle function, muscle wasting, muscle degeneration, disused, or muscle damage.

5. A composition for increasing muscle mass comprising hemp seed extract.

6. A composition for strengthening muscles or improving muscle function comprising hemp seed extract.

7. In any one of Paragraph 1, Paragraph 2, Paragraph 5, or Paragraph 6, A composition characterized by having a muscle cell differentiation promoting or muscle regeneration effect.

8. In any one of Paragraph 1, Paragraph 2, Paragraph 5, or Paragraph 6, A composition in which the above-mentioned cannabis seed extract is an unsaponifiable extract.

9. In Paragraph 8, The above unsaponifiable extract is, 1) A step of adding pyrogallol and potassium hydroxide to hemp seeds and filling with nitrogen; 2) A step of reacting in a water bath at a temperature of 70 to 80°C and then cooling; 3) Sodium chloride (NaCl); and dibutylhydroxytoluene to the reactants after cooling is complete (BHT) A mixed extraction solvent of hexane and ethyl acetate is added and shaken to obtain the supernatant; and 4) A composition prepared by an extraction method comprising the steps of removing the solvent from the obtained supernatant and freeze-drying.

10. Use for the prevention or treatment of muscle diseases through the effect of promoting muscle stem cell differentiation or muscle regeneration of hemp seed unsaponifiable extract; improvement of muscle function; or increase in muscle mass.

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