WO2022177236A1

WO2022177236A1 - Pharmaceutical composition for preventing or treating muscular atrophy or cachexia, comprising gintonin

Info

Publication number: WO2022177236A1
Application number: PCT/KR2022/002120
Authority: WO
Inventors: 권혁영; 나승열; 토니 위자야요셉; 세티아완타니아
Original assignee: 순천향대학교 산학협력단; 건국대학교 산학협력단
Priority date: 2021-02-16
Filing date: 2022-02-11
Publication date: 2022-08-25
Also published as: KR20220117115A

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscular atrophy or cachexia, comprising an extract containing ginseng-derived gintonin, a fraction thereof, or gintonin, as an active ingredient.

Description

Pharmaceutical composition for preventing or treating muscular atrophy or cachexia containing gintonin

The present invention relates to a ginseng-derived gintonin-containing extract, a fraction thereof, or a pharmaceutical composition for preventing or treating muscular atrophy or cachexia comprising gintonin as an active ingredient.

Skeletal muscle mass accounts for about 40-50% of human body weight and accounts for the largest amount of tissue in the human body and is a major protein store. Maintaining sustained muscle mass is important for slowing the risk of metabolic syndrome and age-related muscular dystrophy. A balance of myofibrillar protein synthesis and breakdown is inevitably important for maintaining muscle mass. Several signal transduction pathways, such as AKT/mTOR and the ubiquitin-proteasome system (UPS), are known to be involved in muscle hypertrophy and atrophy. Hypertrophic symptoms are associated with an increase in muscle mass, due to an increase in pre-existing skeletal muscle fibers by increased protein synthesis without an apparent change in the number of muscle fibers. Conversely, atrophy is due to changes in protein synthesis and increased degradation of muscle proteins. Muscle atrophy such as sarcopenia and cachexia, which causes weakness and atrophy of the body, is associated with many diseases such as aging, cancer, diabetes and metabolic syndrome.

Sarcopenia is a syndrome occurring mainly in the elderly with multifactorial causes such as sedentary lifestyle, motor unit number and satellite cell number/function, mitochondrial dysfunction, altered protein homeostasis and inflammation. Cachexia, on the other hand, is defined as a syndrome related to inflammation from the altered metabolic processes caused by the disease. In most clinical manifestations, sarcopenia is associated with low muscle mass and weak muscle function associated with strength or physical performance. During aging, increased fat mass and pro-inflammatory cytokines synergized with each other to accelerate the loss of muscle mass and function in sarcopenia patients. In the cachexia condition, the patient presented with additional symptoms such as decreased strength, fatigue, anorexia, insulin resistance, increased muscle proteolysis and inflammation. Particularly in cancer, muscle atrophy occurs in most cancer patients and is known as cancer-related cachexia. Since such muscular atrophy reduces the quality of life and longevity of patients, it is essential to find a therapeutic strategy to delay or prevent skeletal muscular atrophy.

Cachexia is a symptom of skeletal muscle loss known as cancer-induced cachexia that occurs in the majority of patients with advanced cancer, particularly lung cancer, and is also observed in several diseases such as acquired immunodeficiency syndrome (AIDS), chronic obstructive pulmonary disease (COPD), diabetes and hormone deficiency. In cancer cachexia, skeletal muscle loss in cancer patients is the most obvious symptom as it progresses rapidly. 60% of cancer patients develop cancer cachexia, resulting in significant weight loss, resulting in poor quality of life and poor prognosis and outcome. Cancer cachexia is also thought to contribute to an increase in mortality in 20-30% of cancer patients.

Cachexia is driven by a variety of conditions, such as altered energy balance, increased production of pro-cachexia cytokines and factors, and decreased adipose tissue. The best known cachexia-inducing factors are myostatin, activin, growth differentiation factor 15 (GDF15), tumor necrosis factor-like weak inducer of apoptosis (TWEAK), and interferon γ (IFNγ), tumor necrosis factor α (TNFα) , interleukin 1α and β (IL-1α and IL-1β), and interleukin (IL)-6. These factors induce catabolism by inducing proteolysis through activation of the ubiquitin-proteasome system (UPS) and autophagy-lysosome system (ALS). During physiological conditions, serine/threonine-protein kinase (AKT) phosphorylates FoxO3, causing cytoplasmic localization. In cachexia conditions, AKT activity is inhibited by the influence of inflammatory cytokines or reduced levels of insulin-like growth factor 1 (IGF1). Reduced AKT activity causes transnuclear migration after dephosphorylation of FoxO3a protein, enabling transcription of Murf-1 and Atrogin-1. A general convergent step in regulating inflammatory cytokines is nuclear factor kappa B (NF-κB) and nuclear factor kappa B (NF-κB), a common transcription factor that mediates cellular responses to various stimuli, such as lipopolysaccharides, reactive oxygen species (ROS), and several cytokines. are related It has been demonstrated that TNF-α up-regulates inflammatory cytokines through NF-κB, acting as an upstream component of the general pathway to produce catabolic cytokines.

Glucocorticoids (GC) are endocrine hormones involved in the response to stressful conditions and are known to have anti-inflammatory and immunosuppressive properties. Therefore, dexamethasone (DEX), a synthetic glucocorticoid, is widely used as an immunosuppressant and anti-inflammatory agent. Interestingly, DEX has also recently been shown to be an effective treatment for COVID-19. Despite this widely used therapeutic agent, it has been reported that GCs, including DEX, induce muscle atrophy. This symptom results from inhibition of protein synthesis as well as upregulation of protein degradation. Proteolysis is known to be induced through activation of two ubiquitin E3 ligases, the muscular atrophy F-box (atrogin-1) and the muscle ring finger (MuRF1), as well as proteasome degradation. The mammalian target of the mTOR (mammalian target of rapamycin) pathway is a major pathway in protein synthesis and is thought to be inhibited by GCs causing muscle atrophy. This causes DEX-induced atrogin-1 and MuRF1 to degrade MyoD (myogenic differentiation antigen) causing muscle atrophy. Therefore, it is important to demonstrate a strategy to mitigate protein degradation caused by DEX and increase protein synthesis.

Ginseng, the root of ginseng (Panax ginseng Meyer), has been used as a traditional herbal medicine in many Eastern countries, particularly Korea, Japan and China. 'Panax' means 'all treatments' and represents the traditional belief that ginseng has the ability to heal all parts of the human body. Ginseng root and root extract have been traditionally used in Korea as a drug to energize the body and mind, increase muscle and physical strength, prevent aging, and increase energy (Korean Patent No. 10-1584722).

Accordingly, the inventors of the present invention screened a ginseng-derived component and found that gintonin (GT), a ginseng-derived lysophosphatidic acid receptor (LPAR) ligand, increases myotube cell diameter and fusion of both mouse C2C12 and human HSkM cells, and , The present invention was completed by confirming that dexamethasone (DEX)-induced muscle atrophy and cachexia were prevented and treated in vivo.

Accordingly, the present invention provides a ginseng-derived gintonin-containing extract, a fraction thereof, or a pharmaceutical composition and functional health food for the prevention or treatment of muscular atrophy or cachexia comprising gintonin as an active ingredient, and prevention or treatment of muscular atrophy or cachexia using the same to provide a way

In order to achieve the object of the present invention as described above, the present invention provides a ginseng-derived gintonin-containing extract, a fraction thereof, or a pharmaceutical composition for preventing or treating muscular atrophy or cachexia comprising gintonin as an active ingredient.

In addition, the present invention provides a functional health food for preventing or improving muscular atrophy or cachexia comprising an extract containing ginseng-derived gintonin, a fraction thereof, or gintonin as an active ingredient.

In addition, the present invention provides a method for preventing or treating muscular atrophy or cachexia, comprising administering to a subject the pharmaceutical composition for the prevention or treatment of muscular atrophy or cachexia according to the present invention.

The present invention is a natural ingredient derived from ginseng-derived gintonin-containing extract, its fraction, or gintonin, which is biocompatible and has no side effects, applied to the prevention or treatment of muscular atrophy or cachexia, and acts as a lysophosphatidic acid receptor (LPAR) ligand Therefore, it can be used as a promising candidate to treat skeletal muscular atrophy and cachexia under physiological and pathological conditions.

1a to 1c show the results of staining with myosin light chain (MHC) after treating various ginseng-derived components in differentiated C2C12 myotube cells. It shows that ginseng-derived components induce hypertrophy and fusion of C2C12 myotube cells: (a) Representative images; (b) Mean myotube cell diameter (data presented as mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001)). (c) Number of nuclei per myotube cell (data presented as mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001)).

2A to 2E show GT-induced hypertrophy and fusion of C2C12 myotube cells. (a, b) Cell viability of C2C12 myotube cells treated with various concentrations of GT in the presence and absence of DEX. (c-e) C2C12 myotube cells were treated with different concentrations of GT and stained with MHC. (c) A representative image is shown. (d) Mean myotube cell diameter, and data are presented as the mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (e) Shows the number of nuclei per myotube cell, and data are presented as the mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

3a to 3b show that GT protects C2C12 myotubes against dexamethasone (DEX)-induced atrophy. (a-c) C2C12 myotube cells were treated with GT with DEX (25 μM) and stained with MHC: (a) Representative image. (b) Mean myotube cell diameter, and data are presented as the mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (c) The number of nuclei per myotube cell, and the data are presented as the mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

4A to 4H show that GT induces hypertrophy of C2C12 myotube cells through LPAR. (a) Results of quantification of LPAR expression by RT-qPCR after GT treatment in the presence or absence of DEX in C2C12 myotube cells. Data are presented as the mean ± SEM of two independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (b-d) C2C12 myotube cells were treated with GT, DEX, ki16425, or LPA and then stained with MHC: (b) Representative image; (c) Mean myotube cell diameter, and data are presented as mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (d) The number of nuclei per myotube cell, and the data are presented as the mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (e-h) C2C12 myotube cells were treated with GT, DEX, Ki16425, or LPA, and the cell lysate was isolated and immunoblotted. (e, g) A representative image is shown. (f, h) The images measured with ImageJ software are shown, and the data are presented as the mean ± SEM of three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

5A-5E show that GT-enriched fraction (GEF) prevents DEX-induced muscle dysfunction in vivo. Mice were injected intraperitoneally with Dex (5 mg/kg body weight/day) or PBS once a day for 7 days as an excipient control. Then, the mice were orally administered with GEF (25 and 50 mg/kg body weight/day) or PBS once a day for 7 days as an excipient control. Control (n=4), DEX (n=9), DEX+GEF 25 mg/kg (n=6) and DEX+GEF 50 mg/kg (n=6). (a) Body weight, organ (heart, liver, spleen) weight, and excised skeletal muscle (Gast, splenic muscle, EDL, and TA) weight are shown. Data are presented as mean ± SEM (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (b) It shows the grip strength measured at intervals of 2-3 days. Data are presented as mean ± SEM (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (c-e) HE staining of TA muscle. (c) A representative image is shown. (d) The cross-sectional area of the TA muscle fiber is shown. The graph was prepared based on the frequency. (e) Mean CSA of TA muscle fibers. Data are presented as mean ± SEM (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

6A-6C show that GT protects against atrophy of primary normal human skeletal myoblasts (HSkM). (a-c) HSkM myotube cells were treated with GT without or with DEX for 2 days and then stained with MHC. (a) A representative image of myotube cell culture was taken with a phase contrast microscope set at ×100 magnification. (b) Mean myotube cell diameter of a total of 60 myotube cell diameters in 10 random fields. (c) Shows the number of nuclei per myotube cell. Data are presented as the mean ± SEM of two independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

7a to 7c show the results of staining with anti-MHC Ab after treating various ginseng-derived components in differentiated C2C12 myotube cells, showing that ginseng-derived components induce hypertrophy and fusion of C2C12 myotube cells: ( a) representative images; (b) Mean myotube cell diameter (data presented as mean±SEM of three independent experiments). (c) Number of nuclei per myotube cell (data presented as mean ± SEM of three independent experiments (*, P ≤ 0.05; ***, P ≤ 0.0001)).

8a is a graph showing the results of the MTT assay confirming the cell viability of C2C12 myotube cells treated with GT. Data are presented as mean ± SEM. ns, not significant. Figure 8b shows a representative image of GT-treated C2C12 myotube cells stained with anti-MHC Ab. 8c is a graph showing the average diameter of C2C12 myotube cells treated with GT. 8D is a graph showing the results of quantifying the number of nuclei per myotube cell. Data are presented as the mean ± SEM of more than 280 myotubes from 10 randomly selected fields (*P ≤ 0.05; ***P ≤ 0.0001). 8e shows representative images of C2C12 myotube cells treated with GT and TNFα and IFNγ stained with anti-MHC Ab. 8f is a graph showing the average diameter of C2C12 myotube cells treated with GT, TNFα and IFNγ. 8g is a graph showing the results of quantifying the number of nuclei per C2C12 myotube cells treated with GT, TNFα and IFNγ. Data are presented as mean ± SEM of more than 450 myotubes from 10 randomly selected fields (*P ≤ 0.05; ***P ≤ 0.0001).

Figure 9a shows the results of RT-PCR quantification of LPARs expression in C2C12 myotube cells treated with GT (*P ≤ 0.05). 9B shows representative images of C2C12 myotube cells treated with GT, TNFα and IFNγ , or Ki16425 stained with anti-MHC Ab. 9c is a graph showing the average diameter of C2C12 myotube cells treated with GT, TNFα and IFNγ , or Ki16425. 9D is a graph showing the number of nuclei per C2C12 myotube cells treated with GT, TNFα and IFNγ , or Ki16425 (*P≤0.05; ***P≤0.0001). 9E shows the results of RT-PCR measuring the mRNA level of Gαi2 in C2C12 cells transfected with control (non-target) (siCtrl) or Gαi2 siRNA (***P ≤ 0.0001). Figure 9f is a representative image treated with GT (100 ng/mL) after the transfected cells were differentiated into myotube cells. 9G shows the average diameter of more than 600 myotube cells from 10 random fields (***P ≤ 0.001; ns, not significant).

Figure 10a shows the results of the FACS profile measuring the ROS level of C2C12 myoblasts treated with TNFα, GT, or N-acetyl cysteine (NAC, negative control). Figure 10b is a graph showing the ROS level of C2C12 myoblasts treated with TNFα, GT, or N-acetyl cysteine (NAC, negative control) (*P ≤ 0.05; ***P ≤ 0.0001). Figure 10c is a FACS profile showing the mitochondrial membrane potential (ΔΨm) of C2C12 myoblasts treated with TNFα or GT. Figure 10d shows the mitochondrial membrane potential (ΔΨm) of C2C12 myoblasts treated with TNFα or GT (*P ≤ 0.05). 10e shows the results of analyzing the effect of GT on the scavenging of hydroxyl radicals (*P ≤ 0.05; ***P ≤ 0.0001). FIG. 10f shows IL-6 levels in C2C12 myotube cells treated with TI (TNFα at 20 ng/mL and IFNγ at 100 u/mL) or GT (100 ng/mL). 10g shows the Nox-2 level of C2C12 myotube cells treated with TI (TNFα at 20 ng/mL and IFNγ at 100 u/mL) or GT (100 ng/mL) (*, P ≤ 0.05).

11A shows the schedule of the LLC1-induced cancer cachexia mouse experiment. 11B shows the tumor volume of LLC1-induced cancer cachexia mice. 11C shows the body weight of LLC1-induced cancer cachexia mice. 11D shows images of skeletal muscles (GA, SOL, TA, and EDL) of LLC1-induced cancer cachexia mice. 11E shows the weight of skeletal muscle of LLC1-induced cancer cachexia mice. 11F shows organ weights of LLC1-induced cancer cachexia mice (*P ≤ 0.05; ***P ≤ 0.0001). 11f is a result of evaluating the grip strength of LLC1-induced cancer cachexia mice. 11H shows the results of hanging experiments in LLC1-induced cancer cachexia mice (*P ≤ 0.05; ***P ≤ 0.0001). 11I is an HE-stained cannon image of GA muscle. 11J is a graph showing the cross-section (CSA) mean of GA muscle fibers. 11K shows the results of plotting the cross section (CSA) of GA muscle fibers according to frequency (*P ≤ 0.05; ***P ≤ 0.0001). 11L is a Western blot showing the protein levels of muscle atrophy-related genes in GA tissue lysates. 11M is a graph analyzing the Western blot results showing the protein levels of muscle atrophy-related genes (n=6; *P ≤ 0.05).

Figure 12a shows an image of HSkM myoblasts differentiated into myotubes, treated with GT in the presence or absence of TNFα, and stained with anti-MHC Ab (100x magnification). 12B shows the average diameter of myotube cells. 12C shows the number of nuclei per myotube cell (*P ≤ 0.05; ***P ≤ 0.0001).

13 shows a molecular pathway model of GT for cancer cachexia.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known techniques known to those skilled in the art may be omitted. In addition, in describing the present invention, if it is determined that a detailed description of a related well-known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terms used in this specification are terms used to properly express a preferred embodiment of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present invention relates to a ginseng-derived gintonin-containing extract, a fraction thereof, or a pharmaceutical composition for the prevention or treatment of muscle atrophy or cachexia and a health functional food comprising gintonin as an active ingredient.

The ginseng-derived gintonin-containing extract is obtained by extracting ginseng with an appropriate solvent, and any suitable solvent for the extraction may be any pharmaceutically acceptable organic solvent, and water or an organic solvent may be used. For example, as an extraction solvent, purified water, methanol, ethanol, propanol, isopropanol, butanol, etc., containing 1-4 carbon atoms anhydrous or hydrous lower alcohol, propylene glycol, etc. , butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, ether, benzene, methylene chloride, and various solvents such as cyclohexane can be used alone or in combination, For example, it may be an alcohol extract or an ethanol extract, but is not limited thereto.

As the extraction method, any one of methods such as hot water extraction, cold extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method. There is no limitation on the method for preparing the gintonin-containing extract of the present invention, and any known method may be used.

The fraction of the gintonin-containing extract derived from ginseng can be fractionated using a polar solvent such as water, methanol, ethanol, or a non-polar solvent such as hexane and ethyl acetate, for example, a butanol fraction may, but is not limited thereto.

The gintonin-containing extract, fraction thereof, or gintonin activates lysophosphatidic acid receptor (LPAR), for example, LPAR1 and LPAR3 to induce hypertrophy and fusion in both myotube cells, but this not limited 13 shows a molecular pathway model of gintonin, which binds to the LPA receptor and activates Gαi2. Gαi2 activation inhibits TNFα-induced atrophy, thereby suppressing atrophy and inflammation-related genes. In addition, gintonin reduces oxidative stress by scavenging ROS and inhibits mitochondrial membrane potential loss.

In the present invention, the muscle atrophy is insoluble muscle atrophy (disuse atrophy of muscles), absence of mechanical stimulation and denervation of skeletal muscle, cachexia, drug induced muscle atrophy, malnutrition muscle It may be selected from the group consisting of atrophy (Malnutrition induced muscle atrophy), muscular dystrophy (muscular dystrophy) and sarcopenia, but is not limited thereto. The drug-induced muscular atrophy may be induced by glucocorticoids or dexamethasone, but is not limited thereto.

In the present invention, the term "treatment" means, unless otherwise stated, a disease or condition to which the term applies, or one or more symptoms of the disease or disorder to reverse, alleviate, inhibit the progression, or prevent. and, as used herein, the term treatment refers to the act of treating when “treating” is defined as above. Thus, "treatment" or "therapeutic therapy" of muscular atrophy or cachexia in a mammal may include one or more of the following:

(1) arrest the development of muscle atrophy or cachexia;

(2) prevent muscle atrophy or the spread of cachexia;

(3) relieve muscle atrophy or cachexia;

(4) preventing recurrence of muscle atrophy or cachexia; and

(5) alleviating symptoms of muscle atrophy or cachexia

The ginseng-derived gintonin-containing extract, a fraction thereof, or the composition of the present invention comprising gintonin as an active ingredient may be used as a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, A lubricant or flavoring agent may be used.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For formulation in the form of, for example, tablets or capsules, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or required, suitable binders, lubricants, disintegrants and color-developers may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by an appropriate method in the art.

The ginseng-derived gintonin-containing extract of the present invention, a fraction thereof, or gintonin may be included in an amount of 0.001 to 20% by weight based on the total weight of the composition.

The composition of the present invention may also be a food composition, and the food composition of the present invention may contain various flavoring agents or natural ingredients, such as a conventional food composition, in addition to containing an active ingredient, ginseng-derived gintonin-containing extract, a fraction thereof, or gintonin. Carbohydrates and the like may be contained as additional ingredients.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-mentioned flavoring agents can advantageously use natural flavoring agents (Taumatine), stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements. There is this.

In addition, the food composition contains various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and natural flavoring agents, coloring agents, and thickening agents (cheese, chocolate, etc.) ), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages.

The present invention also provides a health functional food for preventing or improving muscle atrophy or cachexia comprising an extract containing ginseng-derived gintonin, a fraction thereof, or gintonin as an active ingredient.

The health functional food of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of improving muscle atrophy or cachexia.

In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with Act No. 6727 of the Health Functional Food Act, and provides nutrients for the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes, such as controlling or physiological effects.

The health functional food of the present invention may contain normal food additives, and unless otherwise specified, whether it is suitable as a food additive is related to the item according to the general rules and general test method of food additives approved by the Food and Drug Administration. It is judged according to the standards and standards.

The items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodles-added alkalis, preservatives, and tar dye preparations.

For example, the health functional food in tablet form is granulated by a conventional method by mixing the active ingredient of the present invention, ginseng-derived gintonin-containing extract, a fraction thereof, or a mixture of gintonin with excipients, binders, disintegrants and other additives. Then, a lubricant or the like may be added for compression molding, or the mixture may be directly compression molded. In addition, the health functional food in the form of tablets may contain a corrosive agent or the like, if necessary.

Among health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with an extract containing ginseng-derived gintonin, which is the active ingredient of the present invention, a fraction thereof, or a mixture of gintonin with additives such as excipients, Soft capsules can be prepared by filling a capsule base such as gelatin with an extract containing ginseng-derived gintonin or a mixture of gintonin with additives such as excipients. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the form of a ring can be prepared by molding the active ingredient of the present invention, ginseng-derived gintonin-containing extract, a fraction thereof, or a mixture of gintonin and excipients, binders, and disintegrants by a known method. , if necessary, it can be coated with sucrose or other skinning agent, or the surface can be coated with a material such as starch or talc.

The health functional food in the form of granules can be prepared in a granular form by a known method by mixing the active ingredient, ginseng-derived gintonin-containing extract, a fraction thereof, or a mixture of gintonin and excipients, binders, and disintegrants of the present invention. , a flavoring agent, a flavoring agent, etc. may be contained as needed.

The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.

In addition, the present invention provides a method for preventing or treating muscular atrophy or cachexia, comprising administering to an individual a ginseng-derived gintonin-containing extract, a fraction thereof, or a pharmaceutical composition containing gintonin as an active ingredient.

The subject may be a mammal, for example, a human, but is not limited thereto.

As used herein, the term “therapeutically effective amount” refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as conceived by a researcher, veterinarian, physician or other clinician, which an amount that induces amelioration of the symptoms of the disease or disorder being treated. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other components contained in the composition, the type of formulation, and the age, weight, and general health of the patient It can be adjusted according to various factors including condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. In the treatment method of the present invention, in the case of adults, it is preferable to administer the serrated jasmine extract of the present invention once to several times a day, at a dose of 0.01 mg/kg to 100 mg/kg.

In the treatment method of the present invention, the ginseng-derived gintonin-containing extract of the present invention, a fraction thereof, or a composition containing gintonin as an active ingredient can be administered orally, rectal, intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, Administration may be in a conventional manner via topical, intraocular or intradermal routes.

Hereinafter, the present invention will be described in more detail by way of Examples. The following examples are merely for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

<실시예><Example>

실시예 1Example 1

GT 및 GEF의 제조Manufacture of GT and GEF

GT (gintonin) and GEF were prepared as previously described. 1 kg of 4-year-old ginseng was ground into small pieces (>3 mm) and refluxed 8 times with 70% ethanol (EtOH) at 80°C for 8 hours. The EtOH extract was concentrated (350 g), dissolved in cold distilled water at a ratio of 1-10, and stored in a cold chamber at 4° C. for 24 hours. After centrifugation, the precipitate was lyophilized. The obtained fraction was named GEF.

세포 배양cell culture

The mouse myoblast line, C2C12, was obtained from the American Type Culture Collection (Manassas, VA, USA) at 5% CO ₂ , 37° C. at 10% fetal bovine serum [FBS, Corning Life Sciences] and 1% penicillin-streptomycin [Corning Life Sciences] supplemented growth medium (GM, Dulbecco's modified Eagle's medium [DMEM, Corning Life Sciences, Oneonta NY, USA]). For differentiation, 1×10 ⁵ cells were inoculated into each well of a 24-well plate and another Cultured until confluency unless otherwise noted Confluent C2C12 cells were myoblasts in differentiation medium (DM, DMEM containing 2 % heat-inactivated horse serum [Sigma] and 1 % penicillin-streptomycin) for up to 4 days. Culture for differentiation.Differentiation medium was replaced with fresh differentiation medium every 2 days.For Dex-induced atrophy study, differentiated C2C12 cells were treated with 25 uM Dex (Sigma) and then incubated in DM for 2 days. Human skeletal myoblasts (HSkM) were obtained from Thermo Fisher Scientific.For differentiation, 1×10 ⁵ cells were seeded in each well of a 24-well plate and cultured until confluency. After differentiation in DMEM [Gibco] containing 2% heat-inactivated horse serum [Sigma] and 1% penicillin-streptomycin) for up to 7 days, and after treatment with GT for 2 days, the diameter and fusion index of myotube cells were checked.

웨스턴 블롯 분석Western blot analysis

Cell lysates were harvested for 30 minutes using normal cell lysis buffer on ice and centrifuged at 13,000 rpm for 5 minutes at 4°C. The protein concentration of the supernatant was confirmed by Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., USA). An equal amount of each protein extract (30 μg) was dissolved using 10% polyacrylamide gel and 0.45 μm hybridization nitrocellulose filter (HATF) membrane (Millipore) using Trans-blot turbo (Bio-Rad Laboratories, Inc., USA). , USA). Anti-atrogin-1 (sc-166806, Santa Cruz Biotechnology, CA, USA), anti-p-protein kinase B (AKT) (#9271, Cell Signaling Technology, MA, USA), anti-AKT (#9272) as an antibody , Cell Signaling Technology, MA, USA) and anti-actin (ab1801, Abcam, MA, USA) were used.

세포 증식 검정 (MTT 검정)Cell proliferation assay (MTT assay)

MTT assay was performed to confirm cell proliferation after GT treatment. Assays were performed using Cell Proliferation Kit I (Roche, Netherlands). Cells (1×10 ⁴ ) were seeded in 96-well plates and incubated for 4 days to differentiate them. Then, myotube cells were treated with various concentrations of GT without or with DEX for 24 hours. After incubation, 10 μl MTT solution was added to the cells for 4 hours, followed by the addition of 100 μl solubilizing solution overnight. Absorbance was measured at 575 nm using a microplate reader.

RNA 추출 및 RT-qPCRRNA extraction and RT-qPCR

RNA was isolated using Hybrid R (Gene All), and the same amount of RNA was converted to cDNA using ReverTra Ace® qPCR Kit (Toyobo). In order to confirm the gene expression level, PCR was performed with the qPCR Master Mix Kit (Toyobo) using the primer sequences shown in Table 1. Results were normalized to GAPDH levels.

동물 실험animal testing

C57BL/6 wild-type mice (12-16 weeks old, 26 ± 1.5 g, male) were purchased from Orient (Korea). All animals were maintained in a sterile environment with free access to food and water and a 12 hour light/dark cycle. The procedures for handling and experimenting with mice were approved by the Animal Ethics Committee of Soonchunhyang University. All mice used were in normal health and had not previously been used in any experiments. Mice were divided into 4 groups: control, DEX alone, and combined GT and DEX treatment at 25 and 50 mg/kg body weight. Mice were intraperitoneally injected with DEX (5 mg/kg body weight/day) or PBS as a control once a day for 7 days. After 7 days of DEX treatment, GEF (25 and 50 mg/kg body weight/day) or PBS as an excipient control was orally administered to mice once a day for 7 days. At the end of the experiment, the tibialis anterior, gastrocnemius, EDL, and soleus muscle (splenic muscle) were obtained and their weight was measured. The ratio of each skeletal muscle to body weight was calculated.

헤마톡실린 및 에오신 염색 및 면역형광 염색Hematoxylin and eosin staining and immunofluorescence staining

Immunofluorescence staining and cryosection of cultured cells were performed with a slight modification of the conventional method. Cryosections (10 μm thick) of TA muscle were stained with hematoxylin (Sigma-Aldrich) for 10 minutes and eosin (Sigma-Aldrich) for 3 minutes, dehydrated with ethanol, and washed with Xylene solution (Daejung). Images were taken using a Nikon digital SLR camera (DS-i2) equipped with a Nikon Eclipse Ti-U inverted microscope. Samples were fixed with 4% formaldehyde in 1x PBS for 15 minutes, washed and blocked with 1x PBS with 5% normal Donkey serum for 1 hour. Sacometric myosin heavy chain (MyHC) (MF20, 0.3 μg/mL; Developmental Studies Hybridoma Bank) was used. Primary antibodies were incubated overnight at 4°C. A secondary anti-mouse IgG antibody conjugated to Cy3 (1:800 dilution; Jackson Immuno-Research Laboratories, West Grove, PA, USA) was used. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen, Waltham, MA, USA). Cell images were taken using a Nikon digital SLR camera (DS-i2) equipped with a Nikon eclipse Ti-U inverted microscope. Counting and measurement were performed using ImageJ software.

통계 분석statistical analysis

All in vitro and in vivo experiments were repeated three times, and the results were expressed as mean±SEM (standard error mean). The sample size for each experiment is indicated in the figure description. Experimental groups were compared using the two-tailed Student's t-test. Graphs and bar diagrams were prepared using GraphPad Prism software. P-value *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, and a P-value of <0.05 was considered statistically significant.

실시예 2 Example 2

인삼-유래 성분은 C2C12 근관세포의 비대 및 융합을 유도한다Ginseng-derived ingredients induce hypertrophy and fusion of C2C12 myotube cells

It has been reported that ginseng induces muscle strength and reduces muscle damage and inflammatory responses in mice. In addition, it has been reported that ginsenosides such as ginsenoside Rg1 have a protective effect on muscles. Therefore, we hypothesized that ginseng-derived ingredients are effective for muscle atrophy. To substantiate this hypothesis, various ginseng-derived components (arginine-fructose-glucose (AFG), GT, non-saponin extracts, and whole saponin extracts at 100 ng/ml; ginsenosides Rb1, Rb2, Rc at 100 nM) , Rd, Re, Rf, Rg1, Rg2, Rg3R, and Rg3 isomers such as Rg3R, and Rg3S) were screened to confirm the protective effect on C2C12 myotube cells. After differentiation of C2C12 myoblasts into myotubes, ginseng-derived components were treated for 2 days. Cell hypertrophy and fusion index were confirmed by measuring the cell diameter and counting the number of nuclei per cell, respectively. Several ginsenosides, such as ginsenosides Rd, Rg2, Rg3R, and Rg3S, induced cell hypertrophy and fusion ( FIG. 1 ). This is consistent with previous studies that ginsenosides Rb1 and Rg3 induce muscle hypertrophy and myoblast differentiation. Interestingly, among the tested ginseng components, GT (100 ng/ml) showed the highest level of C2C12 myotube cell hypertrophy and fusion ( FIG. 1 ).

C2C12 근관세포의 GT-유도 비대 및 융합GT-induced hypertrophy and fusion of C2C12 myotube cells

Since it showed a better hypertrophic effect of myotube cells compared to other ginsenosides, it was confirmed whether GT had an effect of preventing muscle wasting. First, the effect of GT on C2C12 cell viability was confirmed. GT treatment showed no cytotoxicity to C2C12 myotube cells with or without DEX ( FIGS. 2a and 2b ). To confirm the dose-dependent hypertrophic effect of GT on myotube cells, C2C12 myotube cells were treated with GT in the range of 10 ng/mL to 10,000 ng/mL and stained with myosin heavy chain (MHC), a marker of myogenic differentiation. To measure myotube cell diameter, MHC-positive myotube cells with more than 10 nuclei were counted at 10 different locations and the average diameter was calculated. GT treatment increased myotube cell diameter by about 2.5 times compared to the control ( FIGS. 2c and 2d ). To confirm cell fusion induced by GT, the fusion index was calculated by counting the number of nuclei per MHC-positive myocyte (single nuclei, 2 to 5 nuclei, and 6 or more nuclei per myotube cell). GT significantly increased the frequency of myotubes containing multiple nuclei (2-5 nuclei and more than 6 nuclei) compared to the control, whereas the frequency of mononuclear myocytes was significantly decreased upon GT treatment (Fig. 2e), which indicates that GT suggests the formation of larger myotube cells that induce muscle cell fusion.

GT는 DEX-유도 위축에 대해 C2C12 근관세포를 보호한다GT Protects C2C12 Myotubes Against DEX-Induced Atrophy

As GT increases muscle cell size and fusion index, it was investigated whether GT protects muscle cells from drug- or stress-induced muscle atrophy. To this end, the protective effect of GT against muscle cell atrophy was confirmed by treating differentiated C2C12 cells with DEX alone or with DEX and GT. GT treatment consistently increased muscle cell size even in the presence of DEX (Figs. 3a, 3b). In particular, co-treatment with gintonin (100 ng/ml) and DEX increased myotube cell diameter by 2.7-fold compared to treatment with DEX alone ( FIGS. 3a and 3b ). In addition, the frequency of multinucleated myotubes was significantly increased in the co-treatment of GT and DEX compared to DEX alone ( FIG. 3c ). These results indicate that GT protects C2C12 myotubes against DEX-induced myocyte atrophy.

GT는 리소포스파티드산 수용체 (LPAR)를 통해 C2C12 근관세포의 비대를 유도한다GT induces hypertrophy of C2C12 myotube cells via lysophosphatidic acid receptor (LPAR)

It has been reported that GT activates the lysophosphatidic acid receptor (LPAR) and inhibits heat stress-induced inflammation through LPAR. Therefore, it was confirmed whether the effect of GT in muscle cells was related to LPAR. RT-qPCR was used to determine if it was affected by GT. Expression of both LPAR1 and LPAR3 was significantly induced by GT treatment, but LPAR2 expression was not changed (Fig. 4a). To further confirm these results, it was confirmed that the LPAR1/3 antagonist Ki16425 was functionally associated with GT. Differentiated C2C12 myotube cells were pre-treated with Ki16425 for 30 minutes, then GT-treated, and the diameter and fusion of myotube cells were confirmed. The diameter and fusion index induced by GT were significantly decreased by the dual treatment of GT and Ki16425 ( FIGS. 4b-4d ), suggesting that GT induces myocyte hypertrophy through LPAR. It was confirmed whether LPA could affect muscle cells. As expected, LPA increased both cell diameter and fusion index of C2C12 myotubes, but the effect of LPA was not as good as that of GT ( FIGS. 4b-4d ). Then, the effects of gintonin on the two pathway protein synthesis (PI3K-mTOR) and degradation signal pathway (Atrogin1), which are important determinants in muscle atrophy, were confirmed by Western blot. As expected, DEX reduced the level of the protein synthesis axis by reduced AKT phosphorylation, which was significantly reduced by GT treatment. In addition, GT significantly abrogated the DEX-induced increase in Atrogin-1 expression ( FIGS. 4E-4H ). In addition, the effect of GT on this pathway was abrogated with the LPA antagonist Ki16425 ( FIGS. 4E-4H ). These results indicate that GT induces protein synthesis signaling that protects DEX-induced muscular atrophy through LPAR.

GT가 풍부한 분획 (GEF)이 인 비보에서 DEX-유도 근육 기능이상을 보호한다GT-Rich Fraction (GEF) Protects DEX-Induced Muscle Dysfunction In Vivo

To further confirm the in vivo effect of GT on muscle atrophy, a DEX-induced muscle atrophy mouse model was used. For the in vivo study, the gintonin-rich fraction (GEF) mass-produced from ginseng using ethanol and water was used. To determine the effect of GEF on muscle wasting, mice were injected with DEX (5 mg/kg of body weight) 7 times daily, followed by oral gavage of GEF (25 and 50 mg/kg) daily for 7 days. did. First, the body weight of the control, DEX or GEF+DEX co-treated mice, the weight of various organs and the hindlimb muscle size were checked. Body weights and weights of organs including liver and spleen did not differ significantly between controls, DEX and GEF and DEX co-treatment ( FIG. 5A ). Interestingly, however, cardiac weight was slightly increased by DEX but decreased significantly by co-administration of GEF and DEX ( FIG. 5A ). There was no significant difference in the weight of muscles such as spleen (SOL), extensor longus (EDL), gastrocnemius (GAS), and tibialis anterior (TA) between the experimental groups (Fig. 5a).

Grip strength was assessed on different days each time during GEF treatment. The muscle strength decreased by DEX was completely restored by GEF treatment started from day 2, which was comparable to the control ( FIG. 5b ). To understand the molecular-level protective effect of GEF on muscular atrophy, hematoxylin and eosin (H&E) staining was performed. DEX treatment reduced the cross-sectional area (CSA) of TA muscle fibers compared to PBS (Fig. 5c). Consistent with the grip strength results, GEF treatment significantly increased CSA in DEX-treated mice ( FIGS. 5C-5E ). At 25 and 50 mg/kg of GEF treatment, mean CSA was approximately 1.7 and 2 fold higher, respectively, compared to DEX alone treatment ( FIG. 5e ). These results suggest that GEF can prevent DEX-induced muscular atrophy in vivo.

GT는 일차 정상 인간 골격 근원세포 (HSkM)의 위축을 보호한다GT Protects Atrophy of Primary Normal Human Skeletal Myoblasts (HSkM)

Based on the results that GT protects muscle cell atrophy in both in vitro and in vivo mouse systems, it was confirmed whether the protective effect of GT was also applied to human cells. To this end, we analyzed the effect of GT on human muscle cell atrophy using primary normal human skeletal myoblasts HSkM. HSkM cells were differentiated in low-glucose DMEM with 2% horse serum condition for 7 days, and after GT treatment for 2 days, the diameter and fusion index of myotube cells were confirmed (FIG. 6a). GT-treated HSkM myotubes showed significantly larger diameters compared to the control ( FIG. 6b ). In addition, the frequency of cells containing 5 or more nuclei was significantly higher in the GT-treated group compared to the control group ( FIG. 6c ). Consistently, in the dexamethasone-induced atrophy model, we found that GT increased both cell diameter and cell frequency with 5 or more nuclei ( FIG. 6 ). These results suggest that it protects atrophy of both mouse and human muscle cells and thus may be a promising candidate for atrophy-related muscle diseases.

결론conclusion

Muscle atrophy is found not only in prolonged inactivity, but also in various diseases such as aging, cancer, diabetes and renal failure. Despite many efforts to develop therapeutic agents, there are still few effective treatments for the treatment of muscular atrophy. In the present invention, it was confirmed that GT, a ginseng-derived ingredient, protects and alleviates DEX-mediated muscle atrophy in vitro and in vivo. GT induced hypertrophy and fusion of both mouse C2C12 and human HSkM myotubes in the presence and absence of DEX. This indicates that GT can be applied to muscle atrophy caused by both GC-related and GC-unrelated conditions. The activity of GT is dependent on the LPA receptor, as demonstrated by the LPAR antagonist Ki16425 abrogating the effect of GT on C2C12 myotubes. It is widely known that LPA receptors are highly expressed in many cell types and are responsible for proliferation, survival, cytoskeletal changes, and calcium influx. The LPA receptor is a G protein-coupled receptor (GPCR) that can bind to heterotrimeric G proteins (Gi, Gq, G12/13 alpha subunits) and exhibit multiple cellular responses by LPA stimulation. The biological activity of LPA is mostly mediated through the activation of six receptors, LPA1 to LPA6. We also found that GT treatment induced expression of both LPAR1 and LPAR3, but not LPAR2. Thus, the present invention suggests that GT can prevent muscle atrophy through activation of LPARs, particularly LPAR1 and LPAR3.

It has been reported to increase the size of cardiomyocytes and induce cardiac hypertrophy, which is associated with signs of heart failure. It was consistently confirmed that in the present invention, the DEX-treated group exhibited greater cardiac mass. Interestingly, we found that cardiac mass increased by DEX was slightly but significantly decreased by GEF treatment. Recently, it was reported that the ginsenoside Rg1 reduces cardiac mass increased by DEX and has a protective effect on cardiac hypertrophy induced by pressure overload. Both ginsenosides Rg1 and GT are thought to have the potential to reduce DEX-induced cardiac hypertrophy. Therefore, further studies are needed to study the mechanisms and correlations between GT and cardiac hypertrophy that can alleviate heart failure or related symptoms.

DEX, a glucocorticoid drug, is used to modulate physiological processes and is commonly applied to treat various diseases such as anti-inflammatory. However, one of the side effects of DEX is that it causes muscle atrophy when taken for a long time. This phenomenon was caused by an increased FOXO3a pathway due to increased activity of the ubiquitin-proteasome pathway. Activation of FOXO3 alone is sufficient to trigger proteolysis through the ubiquitin-proteasome system (UPS). Conversely, overproduction of IGF1 or AKT in mice, either through transgenic expression into muscle or by electrophoresis, is sufficient to reduce muscle weight loss by induction of muscle systemic hypertrophy and higher muscle strength. Various novel anticancer agents recently used or in clinical trials can inhibit PI3K-AKT-mTOR signaling and thus inhibit muscle growth and promote atrophy. Thus, novel approaches to stimulate these kinases may be important in preventing muscle wasting, particularly during recovery and recovery. The present invention confirmed grip strength and CSA of muscle fibers in a mouse model of muscular atrophy and demonstrated that oral administration of GEF prevented or alleviated DEX-induced muscular atrophy. We also found that the level of pAKT was higher in GEF-treated muscle compared to DEX, whereas the level of Atrogin-1 was significantly reduced in GEF-treated mice. Thus, AKT and Atrogin-1 are thought to be important pathways for GT to exhibit its protective activity against DEX-induced muscular atrophy. The inventors of the present invention demonstrated in the previous literature that GEF administration exhibited a potentially significant effect on cognition, thus demonstrating that it is safe and tolerated in the cognitively impaired elderly. Since GEF has been reported to be relatively safe in small clinical trials, it is suggested that GEF be tested clinically to improve muscle wasting.

In the present invention, it was found that gintonin, a novel ginseng component, induces hypertrophy and fusion in both mouse and human myotube cells. In addition, gintonin prevented or alleviated DEX-induced muscle wasting in a mouse model. Therefore, the present invention suggests that gintonin may be a promising candidate for treating skeletal muscular atrophy under physiological and pathological conditions.

실시예 3Example 3

GT 및 GEF의 제조Manufacture of GT and GEF

GT and GEF were prepared according to previously published methods. Briefly, to prepare GT from ginseng root, 8 kg of 4-year-old ginseng was ground into small pieces (>3 mm) and refluxed 8 times at 80°C for 8 hours with 80% ethanol (EtOH). The MeOH extract (1.3 kg) was concentrated in vacuo and partitioned between n-butanol (n-BuOH) and water. The n-BuOH fraction of ginseng (fr., 300 g) was dissolved in PBS (pH 7.2) and loaded onto a column filled with DEAE sepharose CL-6B (GE Healthcare) and equilibrated with PBS (pH 7.2). Unbound material was eluted with the same buffer and bound material was eluted with a linear gradient of 0 to 1 M NaCl in PBS (pH 7.2). The eluted fraction was further diluted with 1000-fold excess distilled water (DW) using a Spectra/Por dialysis membrane (molecular weight cut off 6,000-8,000; Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) at 4°C for 8 hours. to remove small molecule components and other components such as ginsenosides in a yield of 0.2% (Pyo, 2011 #134).

세포 배양cell culture

The mouse myoblast line, C2C12, was obtained from the American Type Culture Collection (Manassas, VA, USA) at 5% CO ₂ , 37° C. at 10% fetal bovine serum [FBS, Corning Life Sciences] and 1% penicillin-streptomycin [Corning Life Sciences] supplemented growth medium (GM, Dulbecco's modified Eagle's medium [DMEM, Corning Life Sciences, Oneonta NY, USA]). For differentiation, 1×10 ⁵ cells were inoculated into each well of a 24-well plate and another Cultured until confluency unless otherwise noted Confluent C2C12 cells were myoblasts in differentiation medium (DM, DMEM containing 2 % heat-inactivated horse serum [Sigma] and 1 % penicillin-streptomycin) for up to 4 days. Culture for differentiation.Differentiation medium was replaced with fresh differentiation medium every 2 days.For Dex-induced atrophy study, differentiated C2C12 cells were treated with 25 uM Dex (Sigma) and then incubated in DM for 2 days. Human skeletal myoblasts (HSkM) were obtained from Thermo Fisher Scientific.For differentiation, 1×10 ⁵ cells were seeded in each well of a 24-well plate and cultured until confluency. After differentiation up to 7 days in DMEM [Gibco] containing 2% heat-inactivated horse serum [Sigma] and 1% penicillin-streptomycin), GT treatment for 3 days, the diameter and fusion index of myotube cells were checked. Cain was purchased from Peprotech (USA).

웨스턴 블롯 분석Western blot analysis

The tissue lysate was homogenized and lysed using a normal cell lysis buffer, followed by centrifugation at 13,000 rpm at 4°C for 20 minutes. The protein concentration of the supernatant was confirmed by Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., USA). An equal amount of each protein extract (30 μg) was dissolved using 10% polyacrylamide gel and 0.45 μm hybridization nitrocellulose filter (HATF) membrane (Millipore) using Trans-blot turbo (Bio-Rad Laboratories, Inc., USA). , USA). Anti-atrogin-1 (sc-166806, Santa Cruz Biotechnology, CA, USA), anti-MuRF-1 (ab172479, Abcam, MA, USA), anti-GDF8/myostatin (sc-398333, Santa Cruz Biotechnology) as antibodies , CA, USA), and anti-GAPDH (#9272, Cell Signaling Technology, MA, USA) were used.

세포 증식 검정 (MTT 검정)Cell proliferation assay (MTT assay)

MTT assay was performed to confirm cell proliferation after GT treatment. Assays were performed using Cell Proliferation Kit I (Roche, Netherlands). Cells (1×10 ⁴ ) were seeded in 96-well plates and differentiated by incubation for 24 hours. Then, myoblasts were treated with various concentrations of GT for 96 hours. After incubation, 10 μl MTT solution was added to the cells for 4 hours, followed by the addition of 100 μl solubilizing solution overnight. After absorbance was measured at 575 nm using a microplate reader, background noise measured at 650 nm was removed using a microplate reader Multiskan GO spectrophotometer (Thermo Fisher Scientific, USA).

RNA 추출 및 RT-qPCRRNA extraction and RT-qPCR

총 ROS 생산의 측정Measurement of total ROS production

C2C12 myoblasts were pre-incubated with GT (100 ng/mL) in the presence or absence of TNFα (20 ng/mL) for 4 hours. Then, the cells were incubated with CellRox Deep Red reagent (Life Technologies, USA) for 30 min at 37 °C under dark charge. Analysis was carried out on a FACS Canto or FACS Aria III (BD Biosciences, USA) and data were analyzed with FlowJo software (Tree Star Inc., USA).

히드록실 라디칼 소거 검정Hydroxyl radical scavenging assay

ROS clearance assays were performed as previously published. Briefly, an iron(II)-dependent thiobarbital acid (TBA) reactive substance was used to evaluate hydroxyl radical-mediated damage to deoxyribose. The reaction system contained 20 μl iron ammonium sulfate (10 mM), 50 μl H2O2 (10 mM), 25 μl EDTA (10 mM), 25 μl 2-deoxyribose, and GT in 150 μl PBS, pH 7.4. did. After the mixture was incubated at 37° C. for 4 hours, 250 μl of 2.8% trichloroacetic acid (TCA) was added to the mixture along with 250 μl of 1% TBA. Then, the mixture was boiled at 100° C. for 15 minutes and cooled, and chromogen was quantified at 535 nm with a Multiskan GO spectrophotometer (Thermo Fisher Scientific, USA). All chemicals were purchased from Sigma-Aldrich (USA).

미토콘드리아 막 전위 (ΔΨm) 검정 Mitochondrial Membrane Potential (ΔΨm) Assay

Mitochondrial membrane potential was confirmed using MitoProbe™ DiIC1(5) Assay Kit (Molecular Probe, USA). C2C12 myoblasts were pre-incubated with GT (100 ng/mL) in the presence or absence of TNFα (20 ng/mL) for 24 hours. Then, the cells were stained with 5 μl of 10 μM DiIC1(5) at 37° C. for 15 minutes and washed once with PBS buffer. Cells were pelleted by centrifugation at 1000 rpm for 5 minutes and resuspended in 500 μl of PBS. Fluorescence was analyzed using a FACS Canto and data were analyzed with FlowJo software (Tree Star Inc., USA). The excitation and emission peaks of DiIC1(5) were 638 nm and 658 nm, respectively.

Gαi2 siRNA 트랜스펙션Gαi2 siRNA transfection

C2C12 myoblasts were transfected with 5 pM of Gαi2 siRNA (Bioneer, pre-designed siRNA14678-1) and control non-target siRNA (Bioneer, SN-1003) using Lipofectamine RNAiMAX (Invitrogen). After the cells were 80% confluent, differentiation was initiated by replacing them with DMEM supplemented with 2% horse serum. Cells were differentiated for 4 days and then treated with GT (100 ng/ml) and vehicle (DMSO) for 2 days. Then, the diameter of the myotube cells was measured.

동물 실험animal testing

C57BL/6 wild-type mice (10-weeks-old, 25 ± 1.0 g, male) were purchased from Orient (Korea). All animals were maintained in a sterile environment with free access to food and water and a 12 hour light/dark cycle. The procedures for handling and experimenting with mice were approved by the Animal Ethics Committee of Soonchunhyang University. All mice used were in normal health and had not previously been used in any experiments. To evaluate the anti-cancer cachexia effect of GT, LLC1 cells (2x10 ⁶ cells) were injected subcutaneously in the right flank. When tumors were detected on day 6, mice were randomly divided into three groups: healthy control group, PBS treated LLC1, and GEF treated LLC1. Mice were orally administered GEF (50 mg/kg body weight) or PBS as an excipient control daily until day 21. At the end of the experiment, the tibialis, gastrocnemius, EDL, and splenic muscles were collected and weighed.

근육 강도 평가muscle strength assessment

Muscle behavior was assessed in two independent experiments. First, the grip force test was recorded using a grip force meter in mice (Model 47200, Ugo-Basile, Varese, Italy). To measure the grip strength of the mouse's forepaws, the mice were held by a metal bar by gently grabbing their tails. As soon as the mouse grasped the metal bar, it was grabbed by the mouse's tail and pulled back vertically until the metal bar was released. Experiments were performed 5 times at an interval of 2 minutes for each mouse. The three maximum values on each experimental day were divided by body weight and normalized. The hanging experiment was conducted using Kondziela's inverted screen test. After each mouse was placed in the center of a wire mesh screen (40x40 cm), the screen was rotated slowly, turned upside down, and kept 40 cm above the floor. The time the mouse fell was measured, and the maximum time was 8 minutes. The experiment was performed three times at least 30 minutes apart.

Immunofluorescence staining and cryosection of cultured cells were performed with a slight modification of the conventional method. Cryosections (10 μm thick) of TA muscle were stained with hematoxylin (Sigma-Aldrich) for 10 minutes and eosin (Sigma-Aldrich) for 3 minutes, dehydrated with ethanol, and washed with Xylene solution (Daejung). Images were taken using a Nikon digital SLR camera (DS-i2) equipped with a Nikon Eclipse Ti-U inverted microscope. Samples were fixed with 4% formaldehyde in 1x PBS for 15 minutes, washed and blocked with 1x PBS with 5% normal Donkey serum for 1 hour. Sacometric myosin heavy chain (MyHC) (MF20, 0.3 μg/mL; Developmental Studies Hybridoma Bank) was used. Primary antibodies were incubated overnight at 4°C. A secondary anti-mouse IgG antibody conjugated to Cy3 (1:800 dilution; Jackson Immuno-Research Laboratories, West Grove, PA, USA) was used. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen, Waltham, MA, USA). Cell images were taken using a Nikon digital SLR camera (DS-i2) equipped with a Nikon eclipse Ti-U inverted microscope. Counting and measurement were performed using ImageJ software. The myotube cell diameter was determined as the average diameter of at least 250 myotube cells from 10 random fields selected in each condition.

통계분석statistical analysis

All in vitro and in vivo experiments were repeated three times, and the results were expressed as mean±SEM (standard error mean). The sample size for each experiment is indicated in the figure description. Experimental groups were compared using two-tailed Student's t-test and analysis of variance (ANOVA). Graphs and bar diagrams were prepared using GraphPad Prism software. A P-value of <0.05 was considered statistically significant (*P ≤ 0.05 and ***P ≤ 0.0001).

실시예 4Example 4

To hypothesize that ginseng-derived ingredients have an effect on muscle atrophy and to substantiate this hypothesis, various ginseng-derived ingredients (arginine-fructose-glucose (AFG), GT, non-saponin extract, and 100 ng/ml total Saponin extract; 100 nM ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, Rg3R, and Rg3 isomers such as Rg3R, and Rg3S) were screened to confirm the protective effect on C2C12 myotube cells. . After differentiation of C2C12 myoblasts into myotubes, ginseng-derived components were treated for 2 days. Cell hypertrophy and fusion index were respectively confirmed by measuring the cell diameter and counting the number of nuclei per cell. To determine the diameter of myotubes, MHC-positive myotubes with more than 10 nuclei were counted at 10 different locations and the average diameter was calculated. To quantify cell fusion induced by the ginseng component, the fusion index was calculated by counting the number of nuclei per MHC-positive myocyte (monocytes, 2 to 5 nuclei, and 6 or more nuclei per myotube cell). Several ginsenosides, such as ginsenosides Rd, Rg2, Rg3R, and Rg3S, induced cell hypertrophy and fusion ( FIGS. 7A-7C ). This is consistent with previous studies that ginsenosides Rb1 and Rg3 induce muscle hypertrophy and myoblast differentiation. Interestingly, among the tested ginseng components, GT (100 ng/ml) showed the highest level of C2C12 myotube cell hypertrophy and fusion ( FIGS. 7A to 7C ).

GT는 C2C12 근관세포의 비대 및 융합을 유도한다GT induces hypertrophy and fusion of C2C12 myotube cells

Since GT showed a better hypertrophic effect of myotube cells compared to other ginsenosides, it was confirmed whether GT had the effect of preventing muscle wasting. First, the effect of GT on C2C12 cell viability was confirmed. GT treatment showed no cytotoxicity to C2C12 myotube cells with or without DEX ( FIG. 8a ). To confirm the dose-dependent hypertrophic effect of GT on myotube cells, C2C12 myotube cells were treated with GT in the range of 10 ng/mL to 10,000 ng/mL and stained with myosin heavy chain (MHC), a marker of myogenic differentiation. To measure myotube cell diameter, MHC-positive myotube cells with more than 10 nuclei were counted at 10 different locations and the average diameter was calculated. GT treatment increased myotube cell diameter by about 2.5 times compared to the control ( FIGS. 8b and 8c ). GT significantly increased the frequency of myotube cells containing multiple nuclei (2-5 nuclei and 6 or more nuclei) compared to the control ( FIGS. 8b and 8d ). On the other hand, the frequency of mononuclear myocytes was significantly decreased upon GT treatment ( FIGS. 8b and 8d ), suggesting the formation of larger myotubes in which GT induces muscle cell fusion.

GT는 TNFα/IFNγ- 유도 근세포 위축으로부터 C2C12 근관세포를 보호한다GT protects C2C12 myotubes from TNFα/IFNγ-induced myoblast atrophy.

As GT increases muscle cell size and fusion index, we investigated whether GT could protect muscle cells in cancer cachexia condition. To mimic cancer cachexia in vitro, differentiated C2C12 cells were treated with TNFα/IFNγ (TI), and the effect of GT on cellular atrophy was confirmed. The myotube cell diameter reduced by TI was significantly increased by GT treatment in a dose-dependent manner ( FIGS. 8e , 8f ). Moreover, the frequency of myotube cells with multiple nuclei was significantly increased in the GT + TI treatment group compared to the TI alone treatment group ( FIGS. 8g and 8g ). Taken together, these results indicate that GT protects C2C12 myotubes from TI-induced myocyte atrophy.

GT는 리소포스파티드산 수용체 (LPAR)를 통해 세포성 위축을 방지한다GT prevents cellular atrophy through the lysophosphatidic acid receptor (LPAR)

It has been reported that GT activates the lysophosphatidic acid receptor (LPAR) and inhibits heat stress-induced inflammation through LPAR. Therefore, it was confirmed whether the effect of GT in muscle cells was related to LPAR. First, it was confirmed by RT-qPCR whether LPAR (LPAR1, LPAR2, and LPAR3) expression in C2C12 cells was affected by GT. The expression of both LPAR1 and LPAR3 was significantly induced by GT treatment, but LPAR2 was not induced ( FIG. 9A ). In addition, it was confirmed whether LPAR was functionally related to GT using the LPAR antagonist Ki16425. Differentiated C2C12 myotube cells were pretreated with Ki16425 for 30 minutes, and then treated with GT. Then, the diameter and fusion index of myotube cells were confirmed. Diameter and fusion indices induced by GT were significantly reduced with dual treatment of GT and Ki16425 ( FIGS. 9b-9d ), suggesting that GT induces muscle cell hypertrophy through LPAR. It has been demonstrated that Gai2, a signaling protein downstream of LPA/LPAR, stimulates PI3K-AKT to induce cell growth and promotes muscle hypertrophy by inhibiting proteolysis through reduction of E3 ubiquitin ligase atrogin-1. Using Gαi2 siRNA, it was tested whether the hypertrophic effect of GT was related to Gαi2. As a result, hypertrophy of C2C12 myotube cells inhibited by Gαi2 siRNA was induced by GT ( FIGS. 9E-9G ). These results suggest that GT prevents cell atrophy through the LPAR/Gαi2 pathway.

GT는 C2C12 세포에서 산화 스트레스를 방지한다GT Prevents Oxidative Stress in C2C12 Cells

As it has been demonstrated that GT is a common mechanism of cancer cachexia and reduces the inflammatory response, we hypothesized that GT reduces oxidative stress or inflammation, thereby protecting C2C12 myoblasts from oxidative damage caused by TNFα. To substantiate this hypothesis, total ROS levels in C2C12 cells were measured using 2',7'-dichlorofluorescein diacetate (DCFDA) in the presence and absence of TNFα. As expected, TNFα induced a higher level of ROS compared to the excipient ( FIGS. 10a and 10b ). Interestingly, the accumulated ROS levels by TNFα were significantly reduced by GT or the known ROS scavenger, N-acetylcysteine (NAC) ( FIGS. 10a , 10b ). Since it is known that high levels of ROS reduce mitochondrial membrane potential (ΔΨm), it was confirmed using the MitoProbe DilC ₁ (5) assay kit whether GT can rescue mitochondrial membrane potential. TNFα was observed to induce ΔΨm disruption in C2C12 myoblasts; However, this phenomenon was restored by the addition of GT (Figs. 10c, 10d). Therefore, to identify the mechanism by which GT reduces ROS levels, two independent methods were used: a radical scavenging assay and inflammation-related gene regulation by GT. First, the radical scavenging ability of GT was confirmed by a hydroxyl radical scavenging assay. The hydroxyl radicals generated from H ₂ O ₂ were dose-dependently decreased by the addition of GT. Interestingly, 1 ng/ml GT showed a significant scavenging effect compared to ascorbic acid (125 ng/ml), a well-known scavenger (Fig. 10e). Second, inflammation-related genes such as NOX2 and IL-6 induced by TNFα are known to be involved in ROS production. TNFα induced the expression of IL-6 and NOX-2, which was significantly reduced by GT ( FIGS. 10f , 10g ). These results suggest that GT prevents cell atrophy through ROS scavenging and reduction of inflammation-related genes.

GEF(GT-enriched fraction)는 인 비보에서 루이스 폐 암종(LLC1)-유도 암 악액질을 방지한다GT-enriched fraction (GEF) prevents Lewis lung carcinoma (LLC1)-induced cancer cachexia in vivo

To confirm the effect of GT in cancer cachexia in vivo, an LLC1-induced cachexia mouse model was used. In the in vivo study, GEF mass-produced from ginseng using ethanol and water was used. To confirm the effect of GEF on muscle wasting, LLC1 cells were injected subcutaneously into the flank of C57BL6 mice. Then, from the 6th day after cancer transplantation, GEF was orally administered daily for 15 days. Grip strength was measured every 2 days during the treatment period ( FIG. 11A ). The LLC1-induced cachexia model was well established to show a significant loss of muscle mass and an apparent reduction in tumor-free body weight compared to wild-type control mice (Figs. 11b-11e). GEF-treated mice and PBS-treated mice were found to have similar tumor sizes, so GEF did not show an anti-tumor effect (Fig. 11b). However, GEF clearly improved several characteristics of cancer cachexia, such as tumor-free body weight, muscle mass, grip strength, and hang time ( FIGS. 11C-11H ). PBS-treated mice (LLC1+PBS) showed a tumor-free weight loss of 9.51% compared to healthy mice control, whereas tumor-free body weights of GEF-treated mice (LLC+GEF) recovered significantly ( FIG. 11C ). The weights of the splenic muscle (SOL), extensor longus (EDL), gastrocnemius (GAS), and tibialis anterior (TA) were decreased in PBS-treated mice compared to healthy control mice, but their weights were restored in GEF-treated mice (Fig. 11d, 11e). Organ weights such as heart, lung, and spleen were not affected by GEF treatment ( FIG. 11F ). The reduced grip strength during the treatment period (days 9-21) was partially restored by GEF treatment compared to healthy control mice ( FIG. 11G ). Consistently, the low levels in the hanging experiments induced by LLC1 were restored to similar levels in healthy mice by GEF treatment ( FIG. 11H ). To understand the protective effect of GEF on muscle atrophy at the molecular level, hematoxylin and eosin (H&E) staining was performed. The mean of the cross-sectional area (CSA) of the GEF-treated group was 1.8 times higher than that of the PBS-treated group ( FIGS. 11i and 11j ). The PBS-treated group had a smaller distribution of CSA, but the GEF-treated group showed a larger CSA, which is similar to that observed in healthy mice. To further study the possible anti-cachexia mechanism of GEF, the levels of several muscle atrophy-related signaling pathways such as myostatin, MuRF-1 and atrozin-1 in GA muscle were confirmed by Western blot. The levels of myostatin, MuRF-1 and atrozin-1 were significantly increased in PBS-treated mice compared to healthy mice, but this was partially restored by GEF treatment ( FIGS. 11L , 11M ). These results suggest that GEF prevents LLC1-induced muscle atrophy in vivo.

As GT protects muscle cell atrophy in mouse systems both in vitro and in vivo, to confirm that GT also has a clear protective effect in human cells, we used HSkM to analyze the effect of GT on human muscle cell atrophy. did. HSkM cells were differentiated for 7 days in low glucose DMEM with 2% horse serum and treated with GT for 3 days in the presence or absence of TNFα. Then, the diameter and fusion index of myotube cells were confirmed ( FIGS. 12a and 12b ). GT-treated HSkM myotubes showed significantly larger diameters than control myotubes ( FIGS. 12a and 12b ). In addition, the frequency of cells with 5 or more nuclei was much higher in the GT-treated group compared to the control group (Fig. 12cC). Consistently, even in the TNFα-induced atrophy model, it was observed that GT increased both cell diameter and the frequency of cells with 5 or more nuclei ( FIGS. 12A-12C ). These results suggest that GT may prevent atrophy in both mouse and human muscle cells, making it a promising candidate treatment for cancer cachexia.

결론conclusion

In the present invention, it was found that GT, a component derived from ginseng, has an anti-atrophic effect on skeletal muscle both in vitro and in vivo. GT prevented TNFα/IFNγ-induced muscle atrophy in both mouse C2C12 and human HSkM myotubes in vitro. Furthermore, in a cancer cachexia mouse model, GT maintained tumor-free body weight and grip strength, and increased muscle mass including GA, SOL, TA, and EDL. However, GT did not show a clear anti-tumor effect at the doses used, suggesting that the anti-cachexia effect of GT is associated with skeletal muscle anti-atrophy but does not reduce tumor size.

GT treatment induced expression of both LPAR1 and LPAR3. The activity of GT is dependent on the LPAR/Gαi2 signaling pathway, as evidenced by the abolition of the effect of GT in C2C12 myotubes, both LPAR antagonists Ki16425 and siGαi2. Consistently, the anti-atrophic effect of Gαi2 has been reported, indicating that Gαi2 acts as a parallelogram to murf-1 and atrogine1-mediated atrophy, thereby inhibiting the TNFα-induced atrophic effect. Therefore, in the present invention, it was proposed to prevent muscle atrophy through activation of the GT LPAR/Gαi2 signaling pathway.

In the present invention, it was confirmed that GT exhibits antioxidant effects by direct elimination of ROS and reduction of inflammation-related genes such as IL6 and NOX2. Thus, GT has been shown to reduce the inflammatory response in many types of tissue, including muscle and nervous system. Previously, the inventors of the present invention confirmed that GEF administration was stable and tolerated by the elderly with cognitive impairment, and showed a possible effective effect on participants' ingestion in a small clinical trial. Therefore, the present invention suggested that GEF is a good therapeutic candidate for preventing or alleviating cancer cachexia-related signaling and inflammatory response, and can be clinically tested to improve muscle wasting.

In summary, the present invention found that GT, a novel ginseng component, induces hypertrophy and fusion in mouse and human myotube cells. GT can also protect and alleviate LLC1-induced muscle wasting through LPAR and Gαi2 in a mouse model ( FIG. 13 ). Therefore, the present invention proposes that GT may be a promising therapeutic candidate for treating skeletal muscle atrophy in both physiological and pathological conditions.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims

A pharmaceutical composition for the prevention or treatment of muscular atrophy or cachexia, comprising an extract containing ginseng-derived gintonin, a fraction thereof, or gintonin as an active ingredient.
The method of claim 1,

The muscle atrophy is insoluble muscle atrophy (disuse atrophy of muscles), lack of mechanical stimulation and denervation of skeletal muscle, cachexia, drug induced muscle atrophy, malnutrition Induced muscle atrophy), muscular dystrophy (muscular dystrophy) and sarcopenia (Sarcopenia) will be selected from the group consisting of, the prevention or treatment of muscle atrophy or cachexia pharmaceutical composition.
The method of claim 1,

The ginseng-derived gintonin-containing extract is a pharmaceutical composition for the prevention or treatment of muscular atrophy or cachexia, which is extracted by water or an organic solvent.
The method of claim 1,

The fraction is a ginseng-derived gintonin-containing alcohol extract obtained by fractionation using water, alcohol, chloroform, ethyl acetate, hexane or a combination thereof, a pharmaceutical composition for the prevention or treatment of muscular atrophy or cachexia.
3. The method of claim 2,

The drug-induced muscular atrophy is induced by glucocorticoids or dexamethasone, a pharmaceutical composition for the prevention or treatment of muscular atrophy or cachexia.
The method of claim 1,

The gintonin-containing extract, its fraction or gintonin is to activate the lysophosphatidic acid receptor (LPAR), a pharmaceutical composition for the prevention or treatment of muscular atrophy or cachexia.
A health functional food for preventing or improving muscular atrophy or cachexia, comprising an extract containing ginseng-derived gintonin, a fraction thereof, or gintonin as an active ingredient.
A method for preventing or treating muscular atrophy or cachexia comprising administering the composition of any one of claims 1 to 6 to a subject other than a human.