Pharmaceutical composition for preventing or treating muscle diseases comprising Ribes fasciculatum var. chinense Maxim. extract as an active ingredient

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases comprising an extract of *Ribesfasciculatum var. chinenseMaxim.* as an active ingredient. By confirming that the extract promotes the expression of muscle formation-related factors and inhibits the expression of muscle degradation-related factors in a cell model in which myotube differentiation inhibition or muscle atrophy is induced, the composition can be usefully utilized as a composition for the prevention, treatment, or improvement of muscle diseases.

Description

Technology Field

[0001] The present invention relates to the crowberry ( Ribes fasciculatum var. chinense This invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases comprising an extract of Maxim.) as an active ingredient. Background Technology

[0002] Sarcopenia, caused by the degeneration of spinal nerves, motor nerves, or skeletal muscle fibers, is one of the representative intractable diseases whose etiology has not yet been identified. According to research conducted so far, motor nerves that induce skeletal muscle contraction degenerate, preventing skeletal muscle contraction, or the expression of proteins involved in muscle contraction within skeletal muscle decreases (sarcopenia), or the said proteins are modified, preventing normal skeletal muscle contraction, and it is known that in the long term, said motor nerves or skeletal muscles are deformed into fibrous tissue.

[0003] Among the various forms of sarcopenia, degenerative sarcopenia is defined as an age-related decrease in muscle mass and is increasingly recognized for its significant impact on the quality of life in old age. While degenerative sarcopenia affects 13–24% of individuals under the age of 70, it occurs in over 50% of those over 80. Muscle weakness associated with muscle loss is known to be linked to fatigue, reduced work performance, and an increased risk of fractures, such as femoral fractures. Patients with sarcopenia have skeletal muscle markers below baseline levels and experience difficulties in daily life, such as decreased grip strength or walking speed. In terms of body composition, there is a distinct loss of muscle mass, while body fat tends to increase.

[0004] Nutritional approaches and exercise training methods are being studied as treatments for sarcopenia. Treatments using appetite stimulants and protein compounds such as testosterone have been proposed, but their therapeutic effects are not satisfactory. Exercise training is not considered a suitable alternative, as it is often difficult for elderly individuals with progressing sarcopenia to even attempt exercise. Pharmacologically, methods to slow the progression of sarcopenia primarily involve inhibiting muscular atrophy, which is caused by the degeneration or progressive mutation of muscle cells—a type of sarcopenia.

[0005] Meanwhile, the crowberry ( Ribes fasciculatum var. chinense (Raspberry) is a deciduous shrub belonging to the Saxifragaceae family of the Rosales order of dicotyledonous plants, and it grows naturally under trees in valleys of mountainous regions in Korea. While the crowberry has been reported to possess biological activities such as anti-allergic and anti-obesity properties, there have been no reports to date regarding its use in improving muscle diseases. Prior art literature

[0006] 1. Republic of Korea Published Patent KR 10-2023-0161339 (Published Nov. 27, 2023) The problem to be solved

[0007] The objective of the present invention is to provide a composition for the prevention, treatment, or improvement of muscle diseases. means of solving the problem

[0008] To achieve the above objective, the present invention relates to a crowberry ( Ribes fasciculatum var. chinense The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising an extract of Maxim.) as an active ingredient.

[0009] In addition, the present invention provides a health functional food composition for preventing or improving muscle diseases, comprising the above extract as an active ingredient. Effects of the invention

[0010] According to the present invention, crowberry ( Ribes fasciculatum var. chinense By confirming that the extract of Maxim.) promotes the expression of muscle formation-related factors and inhibits the expression of muscle degradation-related factors in cell models in which myotube differentiation inhibition or muscle atrophy is induced, it can be usefully utilized as a composition for the prevention, treatment, or improvement of muscle diseases. Brief explanation of the drawing

[0011] Figure 1A is a schematic diagram showing the process of preparing a conditioned medium (Experimental Example 2-1); Figure 1B is a schematic diagram showing the process of preparing a cell model that inhibits myotube differentiation (Experimental Example 2-2); and Figure 1C is a schematic diagram showing the process of preparing a cell model that inhibits muscle atrophy (Experimental Example 2-3). Figures 2 and 3 show the results of analyzing the effect of crowberry extract (hereinafter referred to as the sample) on the expression of muscle-related factors. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. Control Group 2 (refer to Experimental Example 2-2); ####p<0.0001 vs. Negative Control Group (NC) (refer to Experimental Example 2-2). RFE; Ribes fasciculatum var. chinense Maxim. fruit extract. Specific details for implementing the invention

[0012] The present invention will be described in more detail below.

[0014] The present invention relates to the crowberry ( Ribes fasciculatum var. chinense The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising an extract of Maxim.) as an active ingredient.

[0015] The above extract may be obtained by extraction with water, (C1~C4) alcohol, or a mixed solvent thereof.

[0016] In addition, the extract may be derived from one or more parts selected from the group consisting of flowers, leaves, stems, roots, fruits, and branches of the crowberry, but is not limited thereto.

[0017] In addition, the above extract may promote the expression of one or more selected from the group consisting of MyHC (Myosin Heavy Chain), MyoD (myoblast determination protein 1), MyoG (Myogenin), and MRF4 (myogenic regulatory factor 4), or inhibit the expression of MuRF1 (Muscle RING-finger protein-1) or MaBbx (Muscle Atrophy F-box), but is not limited thereto.

[0018] The above muscle disease may be one or more selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, amyotrophic lateral sclerosis, and inflammatory myopathy, but is not limited thereto.

[0019] The above muscle disease may be caused by inflammation.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0029] In addition, the present invention relates to a crowberry ( Ribes fasciculatum var. chinense The present invention provides a health functional food composition for the prevention or improvement of muscle diseases comprising an extract of Maxim.) as an active ingredient.

[0030] The present invention can be generally used as a commonly used food.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0040] 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.

[0042] [ Experimental Example 1] Experiment Preparation

[0043] 1-1. Sample

[0044] The crowberry used as a sample in this experiment ( Ribes fasciculatum var. chinenseTo prepare the Maxim.) extract, crowberry leaves were purchased from the Natural Product Central Bank (Jeollanam-do) and used (the above leaves were collected in Gochang-gun, Jeollabuk-do, South Korea in 2016, and the specimen (KRIB 0084079) is stored at the Herbarium of the Korea Research Institute of Biotechnology). 100g of crowberry leaves were dried in the shade and powdered, and 1L of 99.9% methyl alcohol (HPLC grade) was added. Then, the extract was extracted 30 times at room temperature using an ultrasonic extractor (SDN-900H, SD-ULTRASONIC CO., LTD) (40KHz, 1500W, ultrasonic treatment for 15 minutes, and standing for 120 minutes per cycle). Afterwards, the sample (47.1g) was obtained by filtering with a filter (Qualitative Filter No.100, HYUNDAI MICRO CO., LTD) and drying under reduced pressure.

[0046] 1-2. Cell Model

[0047] The mouse muscle progenitor cells C2C12 used as the cell model for this experiment were purchased from ATCC (Mannassas, VA, USA), and the mouse macrophages Raw264.7 were purchased from the Korean Cell Line Bank (Seoul, Korea). The two types of cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% (v / v) FBS (fetal bovine serum) (Gibco, CA, USA), 100 μg / mL penicillin, and 100 μg / mL streptomycin, under conditions of 37°C and 5% CO2.

[0049] [ Experimental Example 2] root canal Inhibition of differentiation; or muscle atrophy Cell model production

[0050] 2-1. Conditional badge manufacturing

[0051] To inhibit myotube differentiation of C2C12 cells or to induce muscle atrophy, a conditioned medium (CM) using Raw264.7 cells was first prepared. As shown in Fig. 1A, Raw264.7 cells were placed in a 6-well plate (1×10⁶ 5 Cells were seeded into wells (cells / well) and cultured at 37°C and 5% CO2. Cells were attached overnight, and LPS (1 μg / mL) and / or samples (50, 100, or 200 μg / mL) were treated for 24 hours. Subsequently, cells were washed twice with PBS (phosphate-buffered saline) and cultured in serum-free DMEM medium for 24 hours. The conditioned medium obtained from the above process was centrifuged at 3000 rpm for 20 minutes and filtered through a 0.2 μM filter. The conditioned medium was stored at -80°C until use.

[0053] 2-2. root canal Production of a differentiation inhibition cell model

[0054] As shown in Fig. 1B, when the confluence of C2C12 cells reached 90%, the culture medium was replaced with a medium mixed in a 1:1 weight ratio of DMEM differentiation medium (DM) supplemented with 2% horse serum and conditioned medium (Experimental Example 2-1). Differentiation into myotubes was induced through the differentiation medium, while inhibition of differentiation into myotubes was induced through the conditioned medium. The medium was replaced every 2 days. The experimental groups were set as follows. The negative control (NC) was treated with serum-free DMEM medium instead of the conditioned medium.

[0055] 1) Normal group (Negative control; NC): Differentiation medium (serum-free DMEM)

[0056] 2) Control Group 1: Conditioned medium without LPS and sample treatment + Differentiation medium (DMEM containing horse serum) treatment

[0057] 3) Control Group 2: LPS (1 μg / mL) treated, and untreated sample treated in conditioned medium + differentiation medium (DMEM containing horse serum).

[0058] 4) Sample Treatment Group 1: Conditioning medium treated with LPS (1 μg / mL) and sample (50 μg / mL) + differentiation medium (DMEM containing horse serum)

[0059] 5) Sample Treatment Group 2: Conditioning medium treated with LPS (1 μg / mL) and sample (100 μg / mL) + Differentiation medium (DMEM containing horse serum)

[0060] 6) Sample Treatment Group 3: Conditioned medium treated with LPS (1 μg / mL) and sample (200 μg / mL) + Differentiation medium treatment (DMEM containing horse serum)

[0062] 2-3. muscle atrophy Cell model production

[0063] As shown in Fig. 1C, when the confluence of C2C12 cells reached 90%, the culture medium was replaced with a differentiating medium (DMEM) supplemented with 2% horse serum to induce differentiation of the cells into myotubes. The medium was replaced every 2 days. On the 5th day of the experiment, the medium was replaced with a mixture of the differentiation medium and the conditioning medium (Experimental Example 2-1) in a 1:1 weight ratio and cultured for 48 hours. Muscle atrophy was induced using the mixed medium (conditioning medium). The experimental group was set up in the same way as in Experimental Example 2-2.

[0065] [ Experimental Example 3] Giemsa dyeing

[0066] C2C12 cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes. Subsequently, the cells were stained with a 10% Giemsa solution in distilled water (DW) for 40 minutes. The Giemsa solution was filtered using a 0.2 μM filter before use. Afterward, the Giemsa solution was removed, the cells were washed three times with PBS, and cell images were captured using a Leica microscope.

[0068] [ Experimental Example 4] Western Blot analyze

[0069] Western blot analysis was performed to determine the effect of the samples on the protein expression of muscle-related factors. C2C12 cells cultured in 6-well plates were lysed using PRO-PREP™ (iNtRON Biotechnology, Seoul, Korea). Protein concentration was measured using Bradford solution with bovine serum albumin as the standard. After adding 5X SDS-loading buffer and boiling at 100°C for 10 minutes, 30 μg of protein was obtained and used in the experiment. Protein samples were separated via SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane. Subsequently, the membranes were blocked from non-specific proteins for 1 hour using 5% skim milk dissolved in TBS-T (containing 150 mM NaCl, 10 mM Tris of pH 8.0, and 0.05% Tween 20). Afterward, each membrane was incubated overnight at 4°C with the primary antibody. Subsequently, the membranes were washed three times with TBS-T for 10 minutes each, and incubated at room temperature for 1 hour with secondary anti-rabbit IgG or anti-mosue IgG antibodies conjugated with HRP (horseradish peroxidase). The membranes were washed three times again with TBS-T for 10 minutes each, and proteins were detected using ECL Western blot detection reagent (GE, Healthcare, Buckinghamshire, UK). Proteins were quantified using ImageJ software.

[0071] [ Examples 1] Muscles Related factors Expression analysis

[0072] 1-1. Muscles Related factors Expression Analysis 1

[0073] According to Experimental Example 4 above, the effect of the sample on the expression of muscle formation-related factors was analyzed for the cell model of Experimental Example 2-2 above. As shown in Figure 2, compared to the normal group, the expression of MyHC (Myosin Heavy Chain) (Invitrogen, Carlsbad, CA), MyoD (myoblast determination protein 1) (Cell Signaling Technology, Beverly, MA), MyoG (Myogenin) (abcam, Cambridge, MA, USA), and MRF4 (myogenic regulatory factor 4) (abcam, Cambridge, MA, USA) was significantly decreased in the control group 2, while the expression of the four muscle-related factors was significantly increased in the sample treatment group compared to the control group 2 in a concentration-dependent manner.

[0075] 1-2. Muscles Related factors Expression Analysis 2

[0076] According to Experimental Examples 3 and 4 above, the effect of the sample on the expression of muscle-related factors was analyzed for the cell models of Experimental Examples 2-3. As shown in Figure 3, compared to the normal group, MyHC (Myosin Heavy Chain) expression was significantly decreased and MuRF1 (Muscle RING-finger protein-1) (Cell Signaling Technology, Beverly, MA) and MaBbx (Muscle Atrophy F-box) (Cell Signaling Technology, Beverly, MA) expression was significantly increased in the control group 2, whereas compared to the control group 2, MyHC expression was significantly increased and MuRF1 and MaBbx expression were significantly decreased in a concentration-dependent manner.

[0078] 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

Claim 1 Crow's-food tree Ribes fasciculatum var. chinense A pharmaceutical composition for the prevention or treatment of muscle diseases comprising an extract of Maxim.) as an active ingredient, wherein the muscle disease is caused by inflammation, and the muscle disease is one or more selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, amyotrophic lateral sclerosis, and inflammatory myopathy. Claim 2 A pharmaceutical composition according to claim 1, characterized in that the extract is obtained by extraction with water, (C1-C4) alcohol, or a mixed solvent thereof. Claim 3 A pharmaceutical composition according to claim 1, wherein the extract is derived from one or more parts selected from the group consisting of flowers, leaves, stems, roots, fruits, and branches of the crowberry. Claim 4 A pharmaceutical composition according to claim 1, characterized in that the extract promotes the expression of one or more selected from the group consisting of MyHC (Myosin Heavy Chain), MyoD (myoblast determination protein 1), MyoG (Myogenin), and MRF4 (myogenic regulatory factor 4). Claim 5 A pharmaceutical composition according to claim 1, characterized in that the extract inhibits the expression of MuRF1 (Muscle RING-finger protein-1) or MaBbx (Muscle Atrophy F-box). Claim 6 delete Claim 7 delete Claim 8 Crow's-food tree Ribes fasciculatum var. chinense A health functional food composition for preventing or improving muscle diseases comprising an extract of Maxim.) as an active ingredient, wherein the muscle disease is caused by inflammation, and the muscle disease is one or more selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, amyotrophic lateral sclerosis, and inflammatory myopathy.

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