Pharmaceutical composition containing supercritical cannabis extract for preventing or treating muscle diseases

Abstract

The present invention relates to a pharmaceutical composition comprising a supercritical cannabis extract for preventing or treating muscle diseases, and, more specifically, to a pharmaceutical composition comprising a supercritical cannabis extract, thereby exhibiting effects of inhibiting muscle cell death, protecting muscle cells, promoting muscle cell differentiation and alleviating muscle inflammation.

Description

Pharmaceutical composition for the prevention or treatment of muscle diseases containing cannabis supercritical extract

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a cannabis supercritical extract.

[0002]

[0003] Sarcopenia is a condition characterized by an excessive decrease in muscle mass that restricts movements in daily life, and it can be caused by various factors such as aging, chronic diseases, lack of physical activity, and nutritional deficiencies. Since muscles play a crucial role in energy consumption and physical function within the human body, functional impairment and quantitative loss of muscle mass reduce a patient's quality of life. Furthermore, sarcopenia is associated with the vulnerable health status of the elderly and can influence the development and progression of various other diseases.

[0004] Sarcopenia is on the rise alongside the increase in the elderly population; as of 2000, there were 50 million people with sarcopenia worldwide, and it is projected that this number will exceed 200 million by 2040 (Cruz-Jentoft). Consequently, social and economic burdens caused by sarcopenia are also expected to arise. To date, most treatments and prevention efforts for sarcopenia have focused on exercise and nutritional supplementation, but in most cases, complete prevention or effective treatment is difficult with these methods alone.

[0005] Muscle cells are a core component of muscle tissue, and failure to maintain their health can lead to problems in muscle performance and quality. The death of these muscle cells is directly linked to their aging and functional decline; furthermore, frequent occurrences of cell damage, inflammation, and imbalances in cell cycle regulation can negatively impact muscle structure and function. Therefore, protecting myoblasts and inhibiting their death are considered crucial strategies for the prevention and treatment of sarcopenia. To achieve this, it is essential to understand the biological processes of muscle cells and consider the factors influencing them. Consequently, there is a pressing need to develop new agents capable of protecting these muscle cells and preventing sarcopenia.

[0006]

[0007] Against this background, while researching treatments for muscle diseases, the inventors completed the present invention by developing a pharmaceutical composition for the prevention or treatment of muscle diseases that includes a supercritical cannabis extract and has the effects of inhibiting muscle cell death, protecting muscle cells, promoting muscle cell differentiation, and alleviating muscle inflammation.

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

[0009] Another objective of the present invention is to provide a functional food composition for improving muscle function comprising a hemp supercritical extract.

[0010]

[0011] To solve the above-mentioned problem, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a cannabis supercritical extract.

[0012] In the above pharmaceutical composition, the cannabis may be any one selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.

[0013] In the above pharmaceutical composition, the cannabis supercritical extract may be extracted from one or more selected from the group consisting of cannabis flower buds, whole plant, roots, stems, leaves, bark, fruits, and seeds.

[0014] In the above pharmaceutical composition, the cannabis supercritical extract may be included at a concentration of 0.1 to 5 μg / ml.

[0015] In the above pharmaceutical composition, the cannabis supercritical extract may be extracted using supercritical carbon dioxide as a solvent and supercritical methanol or supercritical ethanol as a co-solvent.

[0016] In the above pharmaceutical composition, supercritical carbon dioxide and an auxiliary solvent may be used in a ratio of 6:1 (v / v) to 10:1 (v / v).

[0017] In the above pharmaceutical composition, the cannabis supercritical extract may be prepared by a method comprising the steps of: (a) obtaining a cannabis sample by drying cannabis flower buds at 30 to 70 ℃ for 24 to 120 hours; (b) decarbonizing the cannabis sample at 100 to 200 ℃ for 15 to 60 minutes; and (c) supercritical extracting the decarbonized cannabis sample at a pressure of 200 to 400 bar and a temperature of 30 to 80 ℃.

[0018] In the above pharmaceutical composition, the muscle disease may be any one selected from the group consisting of sarcopenia, muscular dystrophy, myotonia, hypotonia, amyotrophic lateral sclerosis, and myasthenia.

[0019] In addition, the present invention provides a functional food composition for improving muscle function comprising a hemp supercritical extract.

[0020]

[0021] The pharmaceutical composition of the present invention exhibits effects of inhibiting muscle cell apoptosis and protecting muscle cells, can promote the differentiation of muscle cells, and can alleviate muscle inflammation. In addition, the pharmaceutical composition of the present invention can be utilized for the prevention or treatment of muscle diseases.

[0022] The food composition of the present invention exhibits effects of inhibiting muscle cell death and protecting muscle cells, can promote the differentiation of muscle cells, and can alleviate muscle inflammation, thereby improving overall muscle function.

[0023]

[0024] Figure 1 shows the results of confirming cell viability when C2C12 cells were treated with cannabis supercritical extracts at concentrations of 0.15, 0.3, 0.6, 1.25, or 2.5 μg / ml after dexamethasone treatment.

[0025] Figure 2 is a fluorescence microscope image of Total MHC in myotube cells after immunofluorescence staining and nuclei after staining with DAPI, showing the results of analyzing the effects of cannabis supercritical extracts at concentrations of 0.25 and 0.5 μg / ml on myotube cell differentiation.

[0026] Figure 3 shows the quantification of the length of myotube cells using the Image J program, confirming the effect of cannabis supercritical extracts at concentrations of 0.25 and 0.5 μg / ml on the length of myotube cells shortened by dexamethasone.

[0027] Figure 4 shows the results of quantifying the area of ​​myotube cells using the Image J program and confirming the effect of cannabis supercritical extracts at concentrations of 0.25 and 0.5 μg / ml on the area of ​​myotube cells reduced by dexamethasone.

[0028] Figure 5 is a graph quantifying the concentration of the inflammatory factor NO by measuring it with ELISA, and is the result of confirming whether the amount of NO, which was significantly increased by dexamethasone treatment, decreased by treatment with cannabis supercritical extract.

[0029] Figure 6 is a graph quantifying the concentration of the inflammatory factor IL6 by measuring it with ELISA, and is the result of confirming whether the amount of IL6, which was significantly increased by dexamethasone treatment, decreased by treatment with cannabis supercritical extract.

[0030] Figure 7 is a graph quantifying the expression level of the inflammatory factor IL1 mRNA by measuring it with RT-qPCR, and is the result of confirming whether the expression level of the IL1 gene, which was significantly increased by dexamethasone treatment, was significantly reduced by treatment with cannabis supercritical extract.

[0031] Figure 8 is a graph quantifying the expression level of the inflammatory factor IL4 mRNA by measuring it with RT-qPCR, and is the result of confirming whether the expression level of the IL4 gene, which was significantly increased by dexamethasone treatment, was significantly reduced by treatment with cannabis supercritical extract.

[0032] Figure 9 is a graph quantifying the expression level of the inflammatory factor MCP1 mRNA by measuring it with RT-qPCR, and is the result of confirming whether the expression level of the MCP1 gene, which was significantly increased by dexamethasone treatment, was significantly reduced by treatment with cannabis supercritical extract.

[0033] Figure 10 is a process flow diagram of cannabis supercritical extraction.

[0034]

[0035] The present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases and a functional food composition for improving muscle function, comprising a cannabis supercritical extract.

[0036] The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a cannabis supercritical extract.

[0037] Cannabis is a type of plant belonging to the genus Cannabis, and there are various varieties, the major varieties of which include Cannabis sativa, Cannabis indica, and Cannabis ruderalis. Cannabis contains pharmacological components such as cannabidiol (CBD) and tetrahydrocannabinol (THC).

[0038] Supercritical extraction refers to a method of extracting active ingredients using supercritical fluids. A supercritical fluid is defined as a substance existing under conditions above the critical point pressure and temperature, where the distinction between liquid and gas is difficult. Supercritical fluids possess unique characteristics distinct from ordinary liquids or gases; while their density is similar to that of a liquid, their viscosity is similar to that of a gas. Due to their liquid-like density, they have high solubility, and due to their gas-like low viscosity, their diffusion and penetration rates are rapid. Supercritical extraction is an extraction method that utilizes these properties of supercritical fluids.

[0039] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis may be any one selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis. For example, it may be Cannabis sativa.

[0040] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted from one or more selected from the group consisting of flower buds, whole plant, roots, stems, leaves, bark, fruits, and seeds of cannabis. For example, it may be extracted from flower buds.

[0041] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be included at a concentration of 0.1 to 5 μg / ml, 0.1 to 4 μg / ml, 0.1 to 3 μg / ml, 0.1 to 2 μg / ml, 0.1 to 1.5 μg / ml, or 0.25 to 0.5 μg / ml.

[0042] A pharmaceutical composition according to one embodiment of the present invention may treat muscle diseases by inhibiting the death of muscle cells, protecting muscle cells, promoting the differentiation of muscle cells, and alleviating muscle inflammation.

[0043] In a pharmaceutical composition according to one embodiment of the present invention, the muscle disease may be any one selected from the group consisting of sarcopenia, muscular dystrophy, myotonia, hypotonia, amyotrophic lateral sclerosis, and myasthenia. For example, it may be sarcopenia.

[0044] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted using one or more supercritical fluids selected from the group consisting of supercritical carbon dioxide, supercritical methanol, or supercritical ethanol as a solvent. For example, it may be extracted using supercritical carbon dioxide as a solvent.

[0045] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted using supercritical carbon dioxide as a solvent and supercritical methanol or supercritical ethanol as a co-solvent.

[0046] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted using supercritical carbon dioxide and an auxiliary solvent in a ratio of 6:1 (v / v) to 10:1 (v / v). For example, it may be extracted using supercritical carbon dioxide and an auxiliary solvent in a ratio of 8:1 (v / v).

[0047] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted at a pressure of 200 to 400 bar, 250 to 350 bar, or 280 to 320 bar. For example, it may be extracted at a pressure of 300 bar.

[0048] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted at a temperature of 30 to 80 ℃, 40 to 70 ℃, or 40 to 60 ℃. For example, it may be extracted at a temperature of 50 ℃.

[0049] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted using supercritical carbon dioxide as a solvent at a pressure of 300 bar and a temperature of 50 ℃.

[0050] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be prepared by a method comprising the following steps:

[0051] (a) A step of obtaining a hemp sample by drying hemp flower buds at 30 to 70 ℃ for 24 to 120 hours,

[0052] (b) a step of decarbonizing the hemp sample at 100 to 200 ℃ for 15 to 60 minutes, and

[0053] (c) A step of supercritical extraction of the above decarboxylated hemp sample at a pressure of 200 to 400 bar and a temperature of 30 to 80 ℃.

[0054] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be prepared by a method comprising the following steps:

[0055] (a) A step of obtaining a hemp sample by drying hemp flower buds at 55°C for 72 hours,

[0056] (b) a step of decarbonizing the hemp sample at 140°C for 15 to 60 minutes, and

[0057] (c) A step of supercritical extraction of the above decarboxylated hemp sample at a pressure of 300 bar and a temperature of 50 ℃ for 10 to 1 hour.

[0058] In step (c) above, supercritical extraction may be performed using supercritical carbon dioxide as a solvent and supercritical methanol or supercritical ethanol as a co-solvent. Preferably, extraction may be performed using supercritical carbon dioxide and a co-solvent in a ratio of 6:1 (v / v) to 10:1 (v / v). For example, extraction may be performed using supercritical carbon dioxide and a co-solvent in a ratio of 8:1 (v / v).

[0059] FIG. 10 is a process flow diagram of a cannabis supercritical extraction for producing a cannabis supercritical extract of the present invention, and is composed of a CO₂ cylinder (1), an auxiliary solvent cylinder (1'), a meter (2), a pre-cooler (3), a CO₂ pump (4), an auxiliary solvent pump (4'), a preheater (5), an extraction vessel (6), an internal basket loading section (7), a pressure gauge (8), a back pressure regulator (9), a product collection valve (10), a cooling medium circulator (11), a heating medium circulator (12), and a data logger (13).

[0060] A pharmaceutical composition according to one embodiment of the present invention may further include a suitable carrier, excipient, and diluent, etc., that are commonly used in the manufacture of pharmaceutical compositions. The carrier may be used without limitation as long as it is known in the art, such as a buffer, preservative, analgesic, solubilizing agent, isotonic agent, stabilizer, base, lubricant, etc.

[0061] A pharmaceutical composition according to one embodiment 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. Furthermore, it may be used in the form of external skin preparations such as ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or gels. When formulating, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, humectants, disintegrants, and surfactants.

[0062] The dosage of a pharmaceutical composition according to one embodiment of the present invention can be appropriately adjusted according to the form and purpose of use, the patient's condition, the type and severity of symptoms, etc.

[0063] The present invention provides a method for preventing or treating muscle disease, comprising the step of administering a cannabis supercritical extract according to one embodiment of the present invention to a subject requiring treatment.

[0064] A preventive or therapeutic method according to one embodiment of the present invention may be to administer a cannabis supercritical extract in a therapeutically effective amount.

[0065] The present invention provides a method for preventing or treating muscle disease, comprising the step of administering a pharmaceutical composition according to one embodiment of the present invention to a subject in need.

[0066] A method for prevention or treatment according to one embodiment of the present invention may be to administer a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0067] In this specification, "subject requiring treatment" may mean mammals, including humans, and "administration" means providing a specific substance to the subject by any appropriate method. "Therapeutic effective dose" 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, and this may be determined by a person skilled in the art based on factors including the patient's sex, age, weight, health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration, release rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. It is desirable to apply a specific therapeutic effective dose for a specific patient differently based on various factors and similar factors well known in the medical field, including the specific composition, including the type and degree of response to be achieved and whether other preparations are used in some cases, the patient's age, weight, general health status, sex and diet, time of administration, route of administration and release rate of the composition, duration of treatment, and drugs used with or concurrently with the specific composition.

[0068] The present invention provides a use of a cannabis supercritical extract according to one embodiment of the present invention for improving muscle function.

[0069] The present invention provides a use of a cannabis supercritical extract according to one embodiment of the present invention for the prevention or treatment of muscle diseases.

[0070] The present invention provides a use of a cannabis supercritical extract according to one embodiment of the present invention in the manufacture of a drug for the prevention or treatment of muscle diseases.

[0071] The present invention provides a use of a pharmaceutical composition according to one embodiment of the present invention for the prevention or treatment of muscle diseases.

[0072] The present invention provides a use of a pharmaceutical composition according to one embodiment of the present invention in the manufacture of a drug for the prevention or treatment of muscle diseases.

[0073] The present invention provides a functional food composition for improving muscle function comprising a hemp supercritical extract.

[0074] In a food composition according to one embodiment of the present invention, the same applies to the pharmaceutical composition of the present invention, unless it contradicts the foregoing matters.

[0075] In a food composition according to one embodiment of the present invention, the cannabis supercritical extract may be included at a concentration of 0.1 to 5 μg / ml, 0.1 to 4 μg / ml, 0.1 to 3 μg / ml, 0.1 to 2 μg / ml, 0.1 to 1.5 μg / ml, or 0.25 to 0.5 μg / ml.

[0076] A food composition according to one embodiment of the present invention may include conventional food additives, and unless otherwise specified, suitability as a food additive is 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. In addition, a food composition according to one embodiment of the present invention may include a suitable carrier commonly used in the manufacture of food compositions.

[0077] A food composition formulation according to one embodiment of the present invention is prepared according to a conventional method and can be encapsulated after drying with a carrier, or formulated into the form of powder, granules, pills, tablets, capsules, candy, syrup, and beverage.

[0078] A food composition according to one embodiment of the present invention can be prepared as any form of food. For example, the food may be any one selected from the group consisting of drinks, meat, sausage, bread, candies, snacks, noodles, ice cream, dairy products, soup, isotonic drinks, beverages, alcoholic drinks, chewing gum, tea, and vitamin complexes.

[0079] The present invention provides a use of a food composition according to one embodiment of the present invention for improving muscle function.

[0080] In the present invention, the matters mentioned regarding the cannabis supercritical extract, pharmaceutical composition, food composition, preventive or therapeutic method, and use are applied equally unless they contradict each other.

[0081] Hereinafter, to specifically explain the present invention, manufacturing examples and experimental examples will be described in detail.

[0082]

[0083] Preparation Example

[0084] Preparation Example 1. Pretreatment of hemp raw material and preparation of dried hemp sample

[0085] The cannabis (Cannabis Sativa L., Purple 52 variety) used in this study was cultivated at N.Thing, a legally licensed farm in Korea. The cannabis was cultivated and harvested under specific conditions, and the harvested flower buds were dried to remove moisture. The flower buds were dried using a dryer at 55°C for 72 hours. This process ensured the production of a uniform sample, making it suitable for storage and use. The dried sample was finely ground using a small grinder (RT-08, Hanil Electric, Seoul, South Korea) and stored in a sealed glass container under refrigerated conditions at 4°C.

[0086]

[0087] Preparation Example 2. Decarbonation Process

[0088] Decarboxylation of cannabis samples was performed as a pretreatment process to remove carboxyl groups in order to activate cannabinoids. This process was carried out using an oven maintained at 140°C. Depending on the desired level of activation, the samples were treated for a duration between 15 and 60 minutes. The samples were heat-treated at 140°C for the set duration, and after decarboxylation was completed, they were collected for a subsequent extraction process.

[0089]

[0090] Preparation Example 3. Particle Size Analysis

[0091] Particle size distribution was evaluated using a sieve separator (CISA BA 200 N, CISA Cedaceria Industrial, Barcelona, ​​Spain) and seven types of standard sieves (45 μm, 100 μm, 150 μm, 180 μm, 250 μm, 300 μm, 500 μm; crystal sieve, Gunpo, South Korea). 8 g of decarboxylated sample was placed in the sieve separator and operated at a vibration amplitude of 2 mm. This process was repeated at 10-minute intervals until the particle size distribution became uniform. The weight of each fraction was measured using a precision balance (OHAUS Adventurer - Model AR2140), and the particle size distribution was evaluated based on these measurements.

[0092]

[0093] Preparation Example 4. Preparation of hemp supercritical extract through supercritical carbon dioxide extraction

[0094] Supercritical CO₂ extraction was used to extract cannabinoids from cannabis. In addition, to establish optimal extraction conditions, extraction efficiency was evaluated by adjusting various conditions, such as extraction time and the use of auxiliary solvents. The extraction conditions were set to a pressure of 300 bar, a temperature of 50 ℃, and an extraction time of 10 minutes to 1 hour. The solvent used was either pure supercritical CO₂ or a mixture of supercritical CO₂ and ethanol or methanol as auxiliary solvents. For extraction, 8 g of cannabis sample was loaded into a cylindrical inner basket measuring 187 mm in length and 13 mm in diameter. One end of the basket was constructed with a connecting joint, while the other end was filled with 0.2 g of glass fiber to prevent sample loss due to gravity and to facilitate the flow of supercritical CO₂. The cylindrical basket was mounted concentrically inside a cylindrical extraction vessel measuring 465 mm in length and 50 mm in diameter. One end of the extraction vessel was connected to a metering pump, and the other end was connected to an outlet and a back pressure regulator (BPR). The temperature of the extraction vessel was maintained at 50°C using a circulating water bath. The CO₂ flow rate was set to 8 mL / min using a high-pressure metering pump. CO₂ with a purity of 99.5% was used, and the pressure was raised to 300 bar using a high-pressure pump and then regulated via the BPR. The extract obtained during extraction passed through the BPR and flowed into a separator, where the pressure inside the separator was maintained at 30 bar via another BPR. After the extraction was completed, the extract remaining in the piping was collected by washing with an ethanol or methanol auxiliary solvent using a pump. When an auxiliary solvent was used, it was mixed with supercritical CO₂ at an 8:1 (v / v) ratio and injected in liquid form via a high-pressure pump.

[0095]

[0096] Preparation Example 5. Preparation of cannabis Soxhlet extract as a control

[0097] Cannabis Soxhlet Extract (CSE) was prepared by the following process. First, a condenser was attached to the top of the Soxhlet extraction apparatus, and a round-bottom flask containing 300 ml of 99.5 wt% ethanol was connected to the bottom. 8 g of finely ground cannabis sample was placed in a cellulose extraction thimble (CET Cellulose Extraction Thimbles, CHMLAB GROUP, Barcelona, ​​Spain), and this extraction thimble was inserted into the Soxhlet extraction apparatus. The round-bottom flask at the bottom of the Soxhlet extraction apparatus was set to be positioned on an Extraction Multi Heating Mantle (Misung Scientific Co., Ltd., Seoul, South Korea), and a recirculating chiller (Jeio Tech, Daejeon, South Korea) was operated to allow cooling water to circulate through the condenser. The heating mantle was set to the maximum temperature, and extraction was carried out for 3 hours at a solvent temperature of 78.4 °C. After the extraction was completed, the power to the heating mantle was turned off, and the round-bottom flask was separated to obtain the extract. Subsequently, the obtained extract was subjected to decarboxylation at 140 °C for 1 hour to finally obtain the hemp syllet extract.

[0098]

[0099] Preparation Example 6. Preparation of room-temperature cannabis extract as a control

[0100] Cannabis room temperature extract (CRE) was prepared by the following process. 8 g of finely ground cannabis sample was placed in a batch extractor and extracted three times at room temperature. The first extraction was performed in 150 mL of 99.5 wt% EtOH for 24 hours, the second extraction in 150 mL of 99.5 wt% EtOH for 24 hours, and the third extraction in 100 mL of 99.5 wt% EtOH for 24 hours.

[0101]

[0102] Experimental Example

[0103] Experimental Example 1. Muscle cell protective and apoptosis inhibitory effects of Cannabis Supercritical CO₂ Extract (CSCE)

[0104] We investigated whether the supercritical extract of cannabis possessed cytoprotective and apoptotic inhibitory effects in a cell model. First, mouse-derived muscle cell line C2C12 cells were seeded into a 96-well microplate and cultured for 48 hours. Subsequently, the medium was changed to differentiation media, and the myoblasts were differentiated into myotubes for 24 hours. After the myotubes were formed, muscle atrophy was induced using the synthetic glucocorticoid dexamethasone. Simultaneously, cannabis supercritical extracts were mixed into the medium at concentrations of 0.15, 0.3, 0.6, 1.25, and 2.5 μg / ml, respectively, and treated for 24 hours. After the culture was complete, 20 μl of MTT labeling reagent (final concentration 0.5 mg / ml) was added to each well, and the 96-well microplate was cultured in an incubator (above 37°C, 5–6.5% CO2) for 4 hours. 100 μl of DMSO solution was added to each well to confirm the complete dissolution of the purple formazan crystals inside the 96-well microplate, and the absorbance of the sample was measured at a wavelength of 575 nm using a microplate reader.

[0105] As a result, it was confirmed that treatment with cannabis supercritical extracts at concentrations of 0.6, 1.25, and 2.5 μM could significantly increase cell viability, which was significantly reduced by dexamethasone treatment (Fig. 1) (p<0.001).

[0106]

[0107] Experimental Example 2. Muscle differentiation-promoting efficacy of Cannabis Supercritical CO₂ Extract (CSCE)

[0108] The efficacy of cannabis supercritical extract in promoting muscle differentiation was confirmed. First, mouse-derived muscle cell line C2C12 cells were seeded into 6-well microplates and cultured for 48 hours. When the cells grew to a density of over 90%, the differentiation medium was replaced to differentiate the myoblasts into myotubes. Muscle atrophy was induced by treating the differentiation medium with dexamethasone, and at the same time, cannabis supercritical extract (concentrations: 0.15, 0.3, 0.6, 1.25, and 2.5 μg / ml, respectively) was applied for a total of 96 hours, changing the medium every 24 hours.

[0109] After treatment, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 minutes. After fixation, the cells were washed again three times with PBS and treated with 0.1% Triton X-100 for 5 minutes to increase cell membrane permeability. After treatment with Triton X-100, the cells were washed three times with PBS, followed by treatment with 3% BSA solution for 1 hour to block non-specific antibody binding. Subsequently, the anti-MHC (Myosin Heavy Chain) primary antibody was applied at an appropriate dilution ratio and incubated at 4°C for 16 hours.

[0110] After the primary antibody reaction was completed, the cells were washed three times with PBS, and a secondary antibody (Alexa Fluor 488) conjugated with a fluorescent dye was incubated at room temperature for one hour. The cells were washed three more times with PBS, the nuclei were stained using DAPI staining, and the stained myotube cells were observed using a fluorescence microscope. The degree of muscle differentiation was confirmed through MHC staining, and the effects of the cannabis supercritical extract on myotube cell differentiation were compared and analyzed.

[0111] As a result, compared to the group treated with dexamethasone alone, an increase in myotube formation and Total MHC levels was observed in the groups treated with 0.25 and 0.5 μg / ml of cannabis supercritical extract (Fig. 2). Regarding the length and area of ​​myotube cells, it was confirmed that the length of myotube cells, which had been significantly shortened by dexamethasone, increased significantly with treatment with cannabis supercritical extract at concentrations of 0.25 and 0.5 μg / ml (Fig. 3). In addition, regarding the area of ​​myotube cells, which had been significantly reduced by dexamethasone, it was confirmed that the area of ​​myotube cells increased significantly with treatment with cannabis supercritical extract at concentrations of 0.25 and 0.5 μg / ml (Fig. 4).

[0112]

[0113] Experimental Example 3. Efficacy of Cannabis Supercritical CO₂ Extract (CSCE) in Alleviating Muscle Inflammation

[0114] The efficacy of cannabis supercritical fluid extract in alleviating muscle inflammation was confirmed. First, mouse-derived muscle cell line C2C12 cells were seeded into 6-well microplates and cultured for 48 hours. Once the cells reached a density of over 90%, the medium was replaced with differentiation medium to differentiate myoblasts into myotubes. Muscle atrophy was induced by mixing dexamethasone into the differentiation medium and simultaneously treating the cells with cannabis supercritical fluid extract (concentrations: 0.05, 0.1, 0.25, 0.5, 1 μg / ml), changing the medium every 24 hours for a total of 96 hours. After 96 hours, the medium was collected, and an ELISA kit was used to measure the expression levels of NO (Nitric Oxide) and IL6 (Interleukin-6) in each sample. The collected medium was dispensed into each well of the ELISA plate, and the expression levels were measured by comparing them to standard samples of NO and IL6 according to the manufacturer's protocol. To measure NO, nitrate (NO3-) is converted into nitride (Nitrite, NO2-) through the Griess reaction. - After reduction to ), the concentration was measured, and in the case of IL6, the expression level was evaluated through antibody-based ELISA analysis.

[0115] After all cultures and reactions were completed, the absorbance in each well of the ELISA plate was measured using a microplate reader (NO was measured at 540 nm, IL6 at 450 nm). From this, the changes in the expression levels of NO and IL-6 following treatment with cannabis supercritical extract were quantitatively evaluated, and the effect of the extract on regulating inflammatory responses was analyzed.

[0116] RNA was extracted from cells cultured on plates after collecting the culture medium. Transcriptome RNA was purified using an RNA extraction kit, and the concentration and purity of the purified RNA were measured using NanoDrop. Reverse transcription was performed for subsequent cDNA synthesis. RT-qPCR was conducted using the reverse-transcribed cDNA to evaluate the expression of inflammation-related genes IL1, IL4, and MCP1. The PCR reaction was carried out using commercial SYBR Green Master Mix and primer sets specific to each gene. Reaction conditions were set according to standard RT-qPCR conditions, and amplification curves were monitored in real-time using a Cycler. The results were analyzed using the ΔΔCt method, and the relative expression levels of IL1, IL4, and MCP1 mRNA in each sample were quantified. From this, the effect of cannabis supercritical extract on the expression of inflammatory factors was quantitatively evaluated, and the patterns of change in inflammatory factor expression were analyzed according to treatment concentration.

[0117] As a result, it was confirmed that the amount of NO, which had significantly increased due to dexamethasone treatment, was significantly reduced by treatment with cannabis supercritical extracts at concentrations of 0.5 and 1 μg / ml (Fig. 5). IL6, which had significantly increased due to dexamethasone treatment, was also significantly reduced by treatment with cannabis supercritical extracts at concentrations of 0.05, 0.1, and 1 μg / ml (Fig. 6).

[0118] Likewise, the expression levels of IL1 mRNA, IL4 mRNA, and MCP1 mRNA, which were increased by dexamethasone treatment, were also significantly reduced by cannabis supercritical extracts at concentrations of 0.25 and 0.5 μg / ml (Figs. 7 to 9). From these results, it can be seen that the cannabis supercritical extract of the present invention can alleviate inflammation in myotube cells.

[0119]

[0120] [National R&D projects that supported this invention]

[0121] [Project ID] 1545027852

[0122] [Assignment No.] 122036032HD030

[0123] [Ministry Name] Ministry of Agriculture, Food and Rural Affairs

[0124] [Project Management (Specialized) Agency Name] Korea Institute of Planning and Evaluation for Food, Agriculture and Forestry Technology

[0125] [Research Project Name] Technology Commercialization Support

[0126] [Research Project Title] Development of Production Technology for Cannabis for Food and Pharmaceuticals through Vertical Integration of Cultivation, Extraction, and Separation / Purification

[0127] [Name of Project Performing Organization] Bobsnu Co., Ltd.

[0128] [Research Period] April 1, 2022 ~ December 31, 2024

[0129]

[0130] [Explanation of the symbol]

[0131] 1: CO₂ cylinder 1': Auxiliary solvent cylinder

[0132] 2: Meter 3. Pre-cooler

[0133] 4. CO₂ Pump 4'. Auxiliary Solvent Pump

[0134] 5. Preheater 6. Extraction vessel

[0135] 7. Internal basket loading section 8. Pressure gauge

[0136] 9. Back pressure regulator 10. Product collection valve

[0137] 11. Cooling medium circulator 12. Heating medium circulator

[0138] 13. Data Logger

Claims

1. A pharmaceutical composition for the prevention or treatment of muscle diseases comprising a cannabis supercritical extract.

2. A pharmaceutical composition according to claim 1, wherein the cannabis is any one selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.

3. A pharmaceutical composition according to claim 1, wherein the cannabis supercritical extract is extracted from one or more selected from the group consisting of cannabis flower buds, whole plant, root, stem, leaf, bark, fruit, and seeds.

4. A pharmaceutical composition according to claim 1, wherein the cannabis supercritical extract is included at a concentration of 0.1 to 5 μg / ml.

5. A pharmaceutical composition according to claim 1, wherein the cannabis supercritical extract is extracted using supercritical carbon dioxide as a solvent and supercritical methanol or supercritical ethanol as a co-solvent.

6. A pharmaceutical composition according to claim 5, wherein the supercritical carbon dioxide and the auxiliary solvent are used in a ratio of 6:1 (v / v) to 10:1 (v / v).

7. In Claim 1, the cannabis supercritical extract is (a) A step of obtaining a hemp sample by drying hemp flower buds at 30 to 70 ℃ for 24 to 120 hours, (b) a step of decarbonizing the hemp sample at 100 to 200 ℃ for 15 to 60 minutes, and (c) A pharmaceutical composition prepared by a method comprising the step of supercritical extraction of the above decarboxylated cannabis sample at a pressure of 200 to 400 bar and a temperature of 30 to 80 ℃.

8. A pharmaceutical composition according to claim 1, wherein the muscle disease is any one selected from the group consisting of sarcopenia, muscular dystrophy, myotonia, hypotonia, amyotrophic lateral sclerosis, and myasthenia.

9. A functional food composition for improving muscle function comprising a hemp supercritical extract.

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