WO2022211396A1 - Pharmaceutical composition including salvia plebeia r. br. extract or compound derived therefrom as active ingredient for prevention or treatment of muscular dystrophy - Google Patents

Abstract

The present invention relates to a composition including a Salvia plebeia R. Br. extract or its derived compound rosmarinic acid and/or hispidulin as an active ingredient for prevention, palliation, or treatment of muscular dystrophy. The extract or the compound derived therefrom inhibits the expression of a muscular dystrophy-related protein and increases the activity of a protein involved in the synthesis of muscular proteins, thus preventing muscle fibers from decreasing in thickness. Since the extract or the compounds are derived from a natural product, they have almost no side effects and are highly safe, thus finding advantageous applications in preventing or treating muscular dystrophy.

Description

A pharmaceutical composition for preventing or treating muscular atrophy comprising Baeam chazugi extract or a compound derived therefrom as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating muscular atrophy comprising an extract of Salvia plebeia R. Br. or a compound derived therefrom, rosmarinic acid and/or hispidulin as an active ingredient. it's about

There are about 460 muscles in our body, and the muscles play an important role in body functions such as maintaining posture and balance or moving. In addition, muscles are the largest organs, accounting for 45-55% of body weight, and play an important role in energy metabolism and thermogenesis. When these muscle masses decrease due to various causes, a vicious cycle of musculoskeletal degeneration begins based on the weakening of muscle strength for physical activity. A decrease in walking speed and weakness in grip strength are the main symptoms and indicators of muscle mass loss, which may additionally lead to falls, fractures, joint damage, metabolic disorders, and cardiovascular diseases.

Decreased muscle mass can occur naturally according to the aging of the body, and is caused by muscle non-use or lack of exercise, or other pathological conditions (cachexia, sepsis, starvation, chemotherapy, excessive exposure to stress hormones, etc.) ) may be a secondary cause. This can be bundled into muscle cell atrophy due to anti-anabolic and catabolic action of type 1 and type 2 muscle fibers.

Therefore, as one treatment for muscle loss that can be caused by aging, uselessness, or disease, a natural product-derived biological material that can help maintain muscle mass even in a state that induces a decrease in muscle fiber proteins can be used.

In most cases, stress hormones (Cortisol, Glucocorticoids, etc.) in our body cause molecular and biological changes in muscle fibers and are directly or indirectly involved in anti-anabolism and catabolism. Glucocorticoids act to inhibit the PI3K/Akt/mTOR pathway as an anti-anabolic action, which inhibits the activities of downstream effectors 4E-BP1 and p70S6K, such as eIF4G (Eukaryotic translation initiation factor 4 G) and eIF4E (Eukaryotic translation initiation factor 4 E). This inhibits the mRNA translation process for protein synthesis, resulting in inhibition of muscle fiber synthesis and muscle fiber atrophy due to protein degradation.

Glucocorticoids not only inhibit muscle synthesis, but also break down proteins to induce muscle atrophy. According to the mechanism leading to 'PI3K/Akt-induced FOXO (Forkhead box O transcription factor) activation and GSK3 (Glycogen synthase kinase 3) inactivation', the gene that induces muscle atrophy is Atrogene (Atrogin-1/MAFbx). , MuRF-1, etc.), the gene induces degradation of proteins represented by the ubiquitin/proteasome system.

Salvia plebeia R. Br.) is a biennial plant of the Lamiaceae family. It is called baeba cabbage because its leaves are wrinkled and look like gombo cabbage and snake scales. It is recorded as Yeochicho (枝草) because it is a grass with fragrant branches on the bonchogangmok (本草綱目). Leaves are 3-8 cm long, clustered at the root, spread over the ground, opposite each other, and have dull sawtooths on the edge of the leaf, and sometimes there are fine hairs on the front and back, so the petiole has a long petiole. When they are young, they are crumpled and flat, attached to the ground, and overwinter. The whole grass is medicinal. It has the effects of blood volume, dihydration, detoxification, and insecticide, so it is used to treat blood loss, hematemesis, hematuria, sore throat, tooth ulcers, etc.

As a prior art related to Baeam chazugi, Korean Patent Registration No. 10-1794567 discloses allergic asthma inhibitory activity of baeam chazugi extract or fraction, and Korean Patent No. 10-1702120 discloses gout prevention or Although the therapeutic effect is disclosed, the preventive or therapeutic effect of baeam chazugi extract or a compound derived therefrom is not known.

Accordingly, the present inventors have intensively studied ingredients derived from natural products that can alleviate skeletal muscle reduction, and as a result, the extract of Baeam chazugi and its derived compounds rosmarinic acid and / or hispidulin ), it was confirmed that the preventive or therapeutic effect of the composition containing the muscle atrophy is excellent, and completed the present invention.

The technical problem to be achieved by the present invention is to provide a pharmaceutical composition for preventing or treating muscle atrophy comprising an extract of baeam chazugi as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or improving muscle loss comprising the extract of Baeam chazugi as an active ingredient.

Another object of the present invention is to provide a method for preparing the baeam chazugi extract.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating muscle atrophy comprising an extract of baeam chazugi as an active ingredient.

In addition, the present invention provides a method for preventing or treating muscle atrophy comprising administering the baeam chazugi extract to an individual.

In addition, the present invention provides a use of the baeam chazugi extract for the preparation of a medicament for the prevention or treatment of muscle atrophy.

As an embodiment of the present invention, the baeam chazugi extract is water (H ₂ O), C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, It may be extracted with one or more solvents selected from the group consisting of methylene chloride, and a mixed solvent thereof, and preferably 50% ethanol as a solvent.

In another embodiment of the present invention, the composition may have a preventive and/or ameliorating effect on the reduction of muscle fiber bundles, inflammatory cell infiltration and local fibrosis of the muscle.

As another embodiment of the present invention, the muscle atrophy is sarcopenia, insoluble muscle atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, drug-induced muscular atrophy (Drug induced atrophy), nutritional deficiency muscle atrophy (Malnutritional atrophy), and may be at least one selected from the group consisting of muscular dystrophy (Muscular dystrophy).

In another embodiment of the present invention, the composition may reduce the expression of one or more selected from the group consisting of MuRF-1 and Atrogin-1/MAFbx in myotube cells.

As another embodiment of the present invention, the composition may increase phosphorylation of p70S6K in myotube cells.

As another embodiment of the present invention, the baeam chazugi extract may include rosmarinic acid represented by the following [Formula 1] as an active ingredient.

[Formula 1]

As another embodiment of the present invention, the baeam chazugi extract may include hispidulin represented by the following [Formula 2] as an active ingredient.

[Formula 2]

In addition, the present invention provides a health functional food composition for preventing or improving muscle loss comprising a baeam chazugi extract as an active ingredient.

As an embodiment of the present invention, the baeam chazugi extract is water (H ₂ O), C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, It may be extracted with one or more solvents selected from the group consisting of methylene chloride, and a mixed solvent thereof, and preferably 50% ethanol as a solvent.

In another embodiment of the present invention, the muscle loss may mean including a qualitative loss due to a decrease in the diameter of myotube cells, and may include both a quantitative loss due to a decrease in the number of myotube cells.

In another embodiment of the present invention, the composition may reduce the expression of one or more selected from the group consisting of MuRF-1 and Atrogin-1/MAFbx in myotube cells.

In another embodiment of the present invention, the composition may increase the phosphorylation of Akt in myotube cells.

As another embodiment of the present invention, the baeam chazugi extract may include rosmarinic acid represented by the above [Formula 1] as an active ingredient.

As another embodiment of the present invention, the baeam chazugi extract may include hispidulin represented by the above [Formula 2] as an active ingredient.

In addition, the present invention provides a method for preparing a composition for preventing, improving or treating muscular atrophy, comprising the following steps.

(a) pulverizing the dried baeam chazugi to prepare baeam chazugi powder; and

(b) water (H2O), C1 to C4 lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixtures thereof Extracting by mixing with one or more solvents selected from the group consisting of solvents.

As an embodiment of the present invention, after the step (b), it may further include a step of lyophilizing after concentrating the extracted extract.

In another embodiment of the present invention, the muscle atrophy is sarcopenia, insoluble muscle atrophy, muscle atrophy caused by the absence of mechanical stimulation, denervational atrophy, drug-induced muscular atrophy ( Drug induced atrophy), nutritional deficiency muscle atrophy (Malnutritional atrophy), and may be at least one selected from the group consisting of muscular dystrophy (Muscular dystrophy).

In addition, the present invention provides a pharmaceutical composition for preventing or treating muscular atrophy comprising rosmarinic acid represented by Formula 1 and/or hispidulin represented by Formula 2 as active ingredients.

In addition, the present invention provides a method for preventing or treating muscular atrophy comprising administering to a subject rosmarinic acid represented by Formula 1 and/or hispidulin represented by Formula 2 above.

In addition, the present invention provides the use of rosmarinic acid represented by the above formula (1) and/or hispidulin represented by the above formula (2) for the manufacture of a medicament for the prevention or treatment of muscular atrophy.

On the other hand, in the prevention or treatment method of the present invention, the subject may have a genetic predisposition to muscular dystrophy or a patient with amyotrophic disease.

In addition, as an embodiment of the present invention, the prophylactic or therapeutic method may be to additionally perform one or more of the following steps (1) to (6) of the subject before and/or after the administration:

(1) Measurement of the diameter or number of myotube cells of an individual

(2) measurement of the intramuscular level of inflammation in the subject

(3) Observation of muscle cell fibrosis in individuals

(4) Measurement of the expression level of MuRF-1 and/or Atrogin-1/MAFbx in myotube cells of an individual

(5) Measurement of Akt phosphorylation level in myotube cells of individuals

(6) Measurement of the individual's athletic ability and/or muscle strength.

The present invention relates to a composition for preventing, improving or treating muscle atrophy, which includes an extract of Salvia plebeia R. Br. or a compound derived therefrom, rosmarinic acid and/or hispidulin as an active ingredient. As a result, the extract or a compound derived therefrom inhibits the expression of a muscle fiber atrophy-related protein, increases the activity of a protein involved in muscle protein synthesis to prevent a decrease in muscle fiber thickness, and has almost no side effects and high safety because it is derived from a natural product, It can be usefully used to prevent or treat muscle atrophy.

1 shows the HPLC chromatography results of the baeam chazugi extract of the present invention and the structures of rosmarinic acid and hispidulin, which are derived compounds thereof.

Figure 2 is (A) Cytotoxicity of myotube cells C2C12 cells in the control group of the present invention, a negative control group treated with dexamethasone to induce muscle atrophy, a Baeam chazugi extract treated group, and a dexamethasone and Baeam chazugi extract treated group. , (B) staining of myosin heavy chain (MHC) (C) area of myosin heavy chain, (D) a change in cell thickness and (E) a change in Fusin index is compared.

Figure 3 is a control group of the present invention, negative control group, baeam chazugi extract treatment group, dexamethasone and baeam chazugi treated group (A) western blot results, (B) myosin heavy chain expression level, (C) Atrogin-1 / MAFbx, (D) MuRF-1 and (E) shows the phosphorylation degree of Akt.

4 is a comparison of the thickness change of myotube C2C12 cells in the control group of the present invention, a negative control group treated with dexamethasone to induce muscle atrophy, a dexamethasone and rosmarinic acid treatment group, and a dexamethasone and hispidulin treatment group.

The present inventors have intensively researched on Salvia plebeia R. Br. extract or a compound derived thereof, rosmarinic acid and / or hispidulin, as a result of intensive study, the hypertrophy-related protein of the extract or compound The present invention was completed by confirming the alleviation effect of increasing expression and decreasing skeletal muscle.

More specifically, after inducing muscle atrophy by treatment with dexamethasone in myotube cells C2C12, the diameter of myotube increases as a result of treatment with the extract or compound, and phosphorylation and/or expression of a protein that promotes muscle protein synthesis It was confirmed that the degree of recovery was restored.

From the above results, the present invention provides a pharmaceutical composition for preventing or treating muscular atrophy comprising the baeam chazugi extract or a compound derived therefrom as an active ingredient.

As used herein, the term "extract" refers to a liquid component obtained by immersing a target substance in various solvents and then extracting it for a certain period of time at room temperature or heated state, a solid obtained by removing the solvent from the liquid component, etc. means the result of In addition, in addition to the result, it can be comprehensively interpreted as including all of the dilutions of the results, their concentrates, their preparations, and their purified products.

In the present invention, the extract can be extracted from various organs of natural, hybrid, and variegated plants as Salvia plebeia R. Br. extract, for example, roots, above-ground parts, stems, leaves, petals, flowers. It can be extracted from buds, fruit body, fruit skin as well as plant tissue culture.

The baeam chazugi extract of the present invention can be extracted according to a conventional method known in the art for obtaining an extract from a natural product, that is, using a conventional solvent under conditions of conventional temperature and pressure. For example, the baeam chazugi extract is selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof. It can be extracted using one or more solvents, but is not particularly limited as long as an extract having a preventive or therapeutic effect on muscular atrophy can be obtained. However, preferably, the extraction may be performed using a mixed solvent of water and an alcohol having 1 to 4 carbon atoms. In this case, water and alcohol may be mixed in a volume ratio of 1:5 to 1:1, but more preferably It can be extracted using 50% ethanol. Depending on the mixing ratio, the extraction efficiency of the effective substance having the effect of improving the muscular atrophy of Bangui may vary.

The method for obtaining the extract is also cold-chilled extraction method for extracting at room temperature of 10 to 25°C, hot water extraction method for extraction by heating to 40 to 100°C, ultrasound, as long as an extract having a preventive or therapeutic effect of muscle atrophy can be obtained It may be an ultrasonic extraction method of extracting by adding a reflux extraction method using a reflux condenser, but is not particularly limited thereto.

The prepared extract may then be filtered or concentrated or dried to remove the solvent, and both filtration, concentration and drying may be performed, and the filtration, concentration or drying process may be performed several times. For example, the filtration may be performed using a filter paper or a reduced pressure filter, the concentration may be performed by a vacuum concentrator, and the drying may be performed by a spray drying method, a freeze drying method, and the like, but is not particularly limited thereto.

In addition, the extract extracted with the solvent may then be further subjected to a fractionation process with a solvent selected from the group consisting of butanol, n-hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, or mixtures thereof. , but not limited thereto.

Accordingly, the present invention is a baeam chazugi grinding step of (a) grinding the dried baeam chazugi to prepare baeam chazugi powder; (b) extracting the pulverized baeam chazugi powder with a solvent of 50% ethanol; and (c) concentrating the extract extracted therefrom and then freeze-drying.

As used herein, the term “muscle atrophy” refers to a state in which muscle mass is reduced due to atrophy of muscle or a decrease in the number or cross-sectional area of muscle fibers. In general, muscle atrophy is accompanied by physiological, histochemical, and biochemical changes due to a decrease in muscle protein, and may cause intrinsic dysfunction of skeletal muscle. Fidulin may be used for the purpose of maintaining and preserving the intrinsic function of skeletal muscle resulting from muscle atrophy from the function of increasing the activity of the muscle fiber and protecting the muscle fiber.

The muscle atrophy is a non-limiting example, and the muscle atrophy is sarcopenia, insoluble muscle atrophy (Disuse atrophy), muscle atrophy due to absence of mechanical stimulation, denervational atrophy, drug-induced muscle It includes atrophy (Drug induced atrophy), malnutritional atrophy (Malnutritional atrophy), muscular dystrophy (Muscular dystrophy) and the like.

The insoluble muscle atrophy includes a decrease in the thickness of the muscle due to activity restrictions due to aging or disease, long bed life, cast or plaster fixation (Gips), etc., peripheral nerve damage, cancer, sepsis, etc. It is accompanied by long-term bed rest and aging processes as well as clinical diseases. The absence of mechanical stimulation and loss of innervation muscle atrophy cause a quantitative decrease and dysfunction of mitochondria, and increase mitochondrial reactive oxygen species (ROS) production, resulting in apoptosis of muscle cells by oxidative damage ( muscle atrophy due to apoptosis). The muscular dystrophy is characterized by muscle atrophy and muscle weakness caused by the absence of dystrophin-glycoprotein complex formation, which is a component of the striated muscle plasma membrane due to gene mutation.

In addition, cortisol (cortisol) is a substance that can be secreted in the body due to various acute stresses, and serves to supply energy against stress, but also causes muscle atrophy of the body according to its mechanism. In addition, synthetic corticosteroids may cause steroid induced atrophy, muscle parenchymal damage, etc. as an adverse reaction.

The composition for preventing, improving or treating muscle atrophy comprising the baeam chazugi extract of the present invention or a compound derived therefrom increases the thickness or mass of muscle fibers beyond indirectly preventing muscle loss by reducing the expression of inflammatory cytokines, By inhibiting the induction of muscle atrophy by protein degradation, it is possible to directly prevent, improve or treat the aforementioned muscular atrophy.

As used herein, the term “prevention” refers to any action that inhibits or delays the onset of muscle atrophy by administration of the composition according to the present invention, and “treatment” refers to suspected and onset of muscular atrophy by administration of the pharmaceutical composition. It refers to any action in which the symptoms of an individual are improved or changed to a beneficial effect.

In the present invention, the term "pharmaceutical composition" means one prepared for the purpose of preventing or treating a disease, and each may be formulated in various forms according to a conventional method and used. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated in the form of external preparations, suppositories, and sterile injection solutions.

In the present invention, "included as an active ingredient" means that the ingredient is included in an amount necessary or sufficient to realize a desired biological effect. The amount can be determined taking into account other non-toxic factors and may vary depending on various factors, such as, for example, the disease or condition being treated, the form of the composition being administered, the size of the subject, or the severity of the disease or condition. A person of ordinary skill in the art to which the present invention pertains can empirically determine the effective amount of an individual composition without undue experimentation.

In addition, the composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above according to each formulation. The pharmaceutically acceptable carrier may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, if necessary, antioxidants, buffers, bacteriostats It may further include other conventional additives, such as. 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 may be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA).

The composition of the present invention may be administered orally or parenterally in a pharmaceutically effective amount according to a desired method, and the term "pharmaceutically effective amount" of the present invention means a disease with a reasonable benefit/risk ratio applicable to medical treatment. It means an amount sufficient to treat the drug and does not cause side effects, and the effective dose level includes the patient's health condition, severity, drug activity, drug sensitivity, administration method, administration time, administration route, and excretion rate; It may be determined according to factors including the duration of treatment, combination or concurrent drugs, and other factors well known in the medical field.

As used herein, the term "individual" may be a mammal, such as a rat, livestock, mouse, or human, preferably a human.

The pharmaceutical composition of the present invention may be formulated in various forms for administration to a subject, and a representative formulation for parenteral administration is an injection formulation, preferably an isotonic aqueous solution or suspension. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be dissolved in saline or buffer to be formulated for injection. In addition, formulations for oral administration include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups and wafers. , sucrose, mannitol, sorbitol, cellulose and/or glycine) and glidants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). The tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid or its It may further include disintegrating agents such as sodium salts, absorbents, coloring agents, flavoring agents and/or sweetening agents. The formulation may be prepared by conventional mixing, granulating or coating methods.

In addition, the pharmaceutical composition of the present invention is a binder, lubricant, disintegrant, colorant, preservative, sweetening agent, flavoring agent, stabilizer, diluent, preservative, wetting agent, emulsification accelerator, auxiliaries such as salts or buffers for regulating osmotic pressure and others It may further include a therapeutically useful substance, and may be formulated according to a conventional method.

The pharmaceutical composition according to the present invention may be administered through several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient may vary depending on the route of administration, age, sex, weight, and severity of the patient. It may be appropriately selected according to several factors. In addition, the composition of the present invention may be administered in parallel with a known compound capable of enhancing the desired effect.

The pharmaceutical composition according to the present invention may be administered orally or parenterally, such as intravenously, subcutaneously, intranasally or intraperitoneally, to humans and animals. Oral administration also includes sublingual application. Parenteral administration includes injection methods such as subcutaneous injection, intramuscular injection and intravenous injection and drip method.

On the other hand, the present invention can be used as a health functional food composition for preventing or improving muscle atrophy and/or muscle loss comprising the baeam chazugi extract or a compound derived therefrom, rosmarinic acid and/or hispidulin as active ingredients.

As used herein, the term "health functional food composition" means that the baeam tea extract or a compound derived therefrom is added to food materials such as beverages, teas, spices, gum, confectionery, etc., or food prepared by encapsulation, powdering, suspension, etc. As such, it means that it brings a specific health effect when ingested, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long period of time using food as a raw material.

The health functional food composition is selected from meat, grains, caffeinated beverages, general drinks, chocolate, breads, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic beverages, alcohol, vitamin complexes and other health supplements. may be any one of

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Example 1. Preparation of Baeam Chazugi Extract

After purchasing the dried baeam tea, ground part was crushed to 0.30 to 0.50 mm based on the average particle diameter through a grinder, and extraction was performed for 1 to 4 hours with a 50:50 mixed solvent of ethanol and water. After concentrating the extract through a rotary vacuum evaporator, it was freeze-dried using Heto Power Dry LL3000 to obtain 14.9 g of extract from 500 g of Baeam Chazugi and 279 g of raw material. In order to confirm the constituents of the extract of baeam chazugi, the results of confirming the baeam chazugi extract, rosmarinic acid standard, and hispidulin standard through HPLC are shown in FIG. 1 . At this time, HPLC conditions are shown in Table 1 below.

Column Shiseido Capcell Pak UG120 (4.6 x 250 mm, 5 μm) Solvent condition acetonitrile with 0.1% formic acid (A), water with 0.1% formic acid (B), 0 min, 95% B; 10 min, 80% B; 55 min, 60% B. flow rate 1 ml/min UV 254 nm

As a result of the HPLC, it was confirmed that the baeam chazugi extract of the present invention contains rosmarinic acid and hispidulin (FIG. 1).

Example 2. in vitro Confirmation of the prevention, improvement or treatment effect of baeam chazugi extract in

2-1. C2C12 cell culture and differentiation induction

The C2C12 mouse myoblast cells used in this experiment were provided by the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center, and Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. was used as a growth medium and incubated under 5% CO ₂ , 37 °C conditions. C2C12 myoblasts were dispensed in a 12 well plate and cultured. When the cell density reached 80-85%, use a differentiation medium (DMEM containing 2% horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin) every two days. They were replaced with fresh medium and differentiated for 6 days.

2-2. Induction of muscle atrophy with dexamethasone and treatment of baeam chazugi extract

Dexamethasone induces muscle atrophy by inhibiting muscle synthesis and causing protein degradation. In order to confirm the effect of preventing, improving, or treating baeam chazugi extract, the C2C12 cells induced to differentiate in Example 2-1 were treated with dexamethasone to induce muscle atrophy, and the muscle atrophy induced cells were treated with embryo cancer. The tea extract was treated. Specific experimental conditions are as follows.

(1) A control group was prepared by treating C2C12 myotube cells that had completed differentiation in a differentiation medium supplemented with only 1% Penicillin for 24 hours.

(2) As a negative control (negative control), the dexamethasone-treated group was prepared by preparing a differentiation medium supplemented with 1% Penicillin to have a 10 μM Dexamethasone composition, and then treating the differentiated C2C12 myotube cells for 24 hours.

(3) Dexamethasone and Baeam chazugi extract treated group were prepared by additionally treating Baeam chazugi extract with a composition of 1, 5, and 10 μg/mL for 24 hours under the same conditions as the negative control of (2).

2-3. Assessment of viability of C2C12 myotubes

In order to confirm whether the baeam chazugi extract recovers the cell viability reduction effect caused by dexamethasone, C2C12 myotube cells were simultaneously treated with the baeam chazugi extract at concentrations of 1, 5, and 10 μg/ml and 10 μM dexamethasone. After 24 hours, cell viability was measured using the CCK-8 kit.

Group Cell viability (%) control 100 Dexamethasone 81.19 ^a Dexamethasone + Baeam Chazugi 1 μg/mL 84.64 Dexamethasone + Baeam Chazugi 5 μg/mL 83.80 Dexamethasone + Baeam Chazugi 10 μg/mL 86.56 ^b

^a : Significance compared to control group (p<0.005), ^b Significance compared to Dex treatment group (p<0.05)

As a result of the above experiment, it was confirmed that the C2C12 myotube cell viability decreased to 81.19% by dexamethasone treatment, but increased by 6.6% when 10 μg/mL of Bae cancer tea was treated (Table 2 and FIG. 2A).

2-4. Analysis of MyHC positive myotube cells, measurement of C2C12 myotube diameter, and analysis of polynuclear myotube formation index (Fusion index)

In Example 2-2, each control and experimental group treated with the sample for 24 hours were immunofluorescently stained with myosin heavy chain (MyHC), and the results were visualized in green, and the cell nuclei labeled with Hoechst33342 were visualized in blue. This was photographed using JuLiomated, an automated cell imaging system, and analyzed using ImageJ. The thickness of each cell after imaging was measured by 50 per cell group and the average was obtained. After analyzing the number of multinucleated myotube cells, the total number of cells was applied to determine the relative multinucleated- The number of MHC-positive myotubes (MHC-positive myotubes) was calculated. Each experiment was repeated three times.

Group MyHC positive area Diameter Fusion index control 1.00 1.00 1.00 Dexamethasone 0.87 ^a 0.73 ^b 0.72 ^b Dexamethasone + Baeam Chazugi 1 μg/mL 1.00 ^c 0.88 ^c 0.92 ^d Dexamethasone + Baeam Chazugi 5 μg/mL 1.03 ^c 0.95 ^c 0.97 ^d Dexamethasone + Baeam Chazugi 10 μg/mL 1.01 ^c 0.94 ^c 0.92 ^d

^a significant compared to control group (p<0.01), ^b significant compared to control group (p<0.005), ^c significant compared to Dex treatment group (p<0.005), ^d significant compared to Dex treatment group (p<0.01)

As a result of the above experiment, the myosin heavy chain (MyHC) positive area, the cell diameter, and the fusion index of C2C12 myotube were decreased by dexamethasone treatment, but it was improved by the Baeam chazugi extract (Table 3, Figure 2 (B)) - Fig. 2(E)).

More specifically, (1) the area of the myosin heavy chain stained in C2C12 myotube cells in the dexamethasone 10 μM administration group was reduced by 13% (p<0.01), and the thickness was 27% (p<0.005) compared to the control group, which is a group administered with the basic differentiation medium. , the fusion index decreased by 28% (p<0.005). (2) Compared to the negative control group administered with dexamethasone in the basal differentiation medium, the area of the myosin heavy chain of C2C12 myotube cells was 18.4% (p<0.005), and the thickness was 30.1% ( p<0.005), the fusion index increased by 34.7% (p<0.005). Therefore, it was confirmed that the baeam chazugi extract of the present invention showed a significant recovery rate in the dexamethasone-induced muscle atrophy model.

2-5. Identification of related proteins

In Example 2-2, MyHC, Atrogin-1/MAFbx, MuRF1, Akt, and p-Akt proteins acting on the pathways of muscle atrophy/synthesis in each of the control and experimental groups treated for 24 hours were subjected to Western blotting (Western blotting). blotting) was used.

The expression levels of myosin heavy chain (MyHC), Atrogin-1/MAFbx, MuRF1, Akt, and p-Akt in the control group and the experimental group are shown in Table 4 below.

As a result of the experiment, it was confirmed that the myosin heavy chain (MyHC) protein was decreased in C2C12 myotubes by dexamethasone treatment, the Atrogin-1/MAFbx and MuRF-1 proteins were increased, and the phosphorylation degree of Akt was decreased. It was confirmed that MyHC decreased by the treatment with baeam chazugi extract increased, the increased Atrogin-1/MAFbx and MuRF-1 proteins decreased, and the phosphorylation degree of Akt was improved. (Table 4 and Figure 3).

Group MyHC Atrogin-1/MAFbx MuRF1 p-Akt/t-Akt control 1.00 1.00 1.00 1.00 Dexamethasone 0.62 ^a 1.16 ^a 1.69 ^a 0.66 ^a Dexamethasone + Baeam Chazugi 1 μg/mL 0.90b 1.04 1.54 0.91b Dexamethasone + Baeam Chazugi 5 μg/mL 0.81 ^b 0.90 1.32b 0.73 Dexamethasone + Baeam Chazugi 10 μg/mL 0.82 ^b 0.64 ^b 0.95 ^b 0.88 ^b

^a Significance compared to the control group (p<0.05), ^b Significance compared to the Dex treatment group (p<0.05)

More specifically, (1) in the dexamethasone 10 1 meta administration group compared to the control group administered with the basic differentiation medium, myosin heavy chain (MyHC) decreased by 38%, Atrogin-1/MAFbx, and MuRF1 increased by 16% and 69%, respectively, The phosphorylation level of Akt (p-Akt/t-Akt) was reduced by 38%. (2) Compared to the dexamethasone-administered group, the myosin heavy chain (MyHC) increased by 32.2% in the group administered with 10 μg/ml of Baeam Chazugi, Atrogin-1/MAFbx decreased by 44.8 times, and MuRF1 decreased by 43.8 times. In addition, the phosphorylation degree of Akt (p-Akt/t-Akt) was increased by 33.3 times by the treatment of Baeam chazugi extract. Therefore, it was confirmed that the baeam chazugi extract of the present invention improved the atrophy of muscle cells caused by dexamethasone and inhibited the activation of the muscle atrophy factor.

Example 3. in vitro Confirmation of the prevention, improvement, or therapeutic effect of a compound derived from Baeam chazugi in

3-1. Induction of muscle atrophy with dexamethasone and treatment of compounds derived from embryonic cancer

In order to check the effect of preventing, improving, or treating the muscle atrophy of the compound derived from the embryonic cancer, dexamethasone was treated to the C2C12 cells induced in the differentiation in Example 2-1 to induce muscle atrophy, and the muscle atrophy-induced cells were treated with dexamethasone. It was treated with rosmarinic acid or hispidulin, a compound derived from chazugi extract. Specific experimental conditions are as follows.

(1) A control group was prepared by treating C2C12 myotube cells that had completed differentiation in a differentiation medium supplemented with only 1% Penicillin for 48 hours.

(2) As a negative control (negative control), the dexamethasone-treated group was prepared by preparing a differentiation medium supplemented with 1% Penicillin to have a 1 μM Dexamethasone composition, and then treating the differentiation medium with C2C12 for 48 hours.

(3) Dexmethasone and rosmarinic acid treatment group were prepared by additionally treating for 48 hours with a composition of 30 μM rosmarinic acid under the same conditions as the negative control group of (2).

(4) Dexmethasone and hispidulin treated group were prepared by additionally treating with a composition of 30 μM rosmarinic acid for 48 hours under the same conditions as the negative control group in (2).

3-2. Measurement of C2C12 myotube diameter

In Example 3-1, each of the control and experimental groups treated with the sample for 48 hours was photographed using the JuLi ^TM Stage, an automated cell imaging system, and the thickness of each cell that was photographed was measured using ImageJ, a cell processing program. 50 measurements were taken per group, the mean and standard error were calculated, and the data were organized (Table 5). Each experiment was repeated three times.

control Dex treatment group Dex + rosmarinic acid Dex + Hispidulin Relative thickness of C2C12 myotube cells (% of control) 100% 77.4% 100.6% 104.3%

(Dex: dexamethasone (1 μM), rosmarinic acid, hispidulin (30 μM))

As a result of the above experiment, it was confirmed that the thickness of C2C12 myotube was decreased by treatment with dexamethasone, and an improvement effect appeared by treatment with rosmarinic acid or hispidulin, a compound derived from Baeam chazugi (Table 5 and FIG. 5).

More specifically, (1) the thickness of C2C12 myotube cells was reduced by 22.6% in the dexamethasone 1 μM administration group compared to the control group, which is the basal differentiation medium administration group. (2) The thickness of C2C12 myotube cells increased by 23.2% in the group additionally administered with 30 μM of rosmarinic acid compared to the negative control group administered with dexamethasone in the basal differentiation medium. (3) The thickness of C2C12 myotube cells increased by 26.9% in the group additionally administered hispidulin 30 μM compared to the negative control group administered with dexamethasone in the basal differentiation medium.

Therefore, it was confirmed that the thickness of myotube cells increased in the muscle atrophy model induced by dexamethasone, respectively, when rosmarinic acid and hispidulin derived from the baeam chazugi extract of the present invention were increased, thereby preventing, improving or treating muscle atrophy. .

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention confirmed the effect of reducing the expression of muscle atrophy-related proteins of baeam chazugi extract and its derived rosmarinic acid and hispidulin, and increasing the activity of muscle protein synthesis-related proteins, prevention or treatment of natural-derived muscular atrophy diseases It is expected to be usefully used in pharmaceuticals, quasi-drugs, cosmetic materials, and functional food materials.

Claims (19)

A pharmaceutical composition for preventing or treating muscle atrophy comprising an extract of Salvia plebeia R. Br. as an active ingredient. According to claim 1, The Baeam chazugi extract is water (H ₂ O), C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof A pharmaceutical composition, characterized in that extracted with one or more solvents selected from the group consisting of. 3. The method of claim 2, The Baeam Chazugi extract is a pharmaceutical composition, characterized in that 50% ethanol is extracted as a solvent. According to claim 1, The muscle atrophy is sarcopenia, insoluble muscle atrophy (Disuse atrophy), muscle atrophy due to the absence of mechanical stimulation, denervational atrophy, drug induced atrophy, nutritional deficiency Muscle atrophy (Malnutritional atrophy), and muscular dystrophy (Muscular dystrophy) characterized in that at least one muscle atrophy selected from the group consisting of, a pharmaceutical composition. According to claim 1, The composition is characterized in that it reduces the expression of one or more selected from the group consisting of MuRF-1 and Atrogin-1 / MAFbx in myotube cells, a pharmaceutical composition. According to claim 1, The composition is characterized in that to increase the phosphorylation of Akt in myotube cells, a pharmaceutical composition. According to claim 1, The baeam chazugi extract is a pharmaceutical composition for preventing or treating muscle atrophy, comprising rosmarinic acid represented by the following [Formula 1] as an active ingredient. [Formula 1]

According to claim 1, The baeam chazugi extract is a pharmaceutical composition for preventing or treating muscle atrophy, comprising hispidulin represented by the following [Formula 2] as an active ingredient. [Formula 2]

A health functional food composition for preventing or improving muscle loss comprising an extract of Salvia plebeia R. Br. as an active ingredient. 10. The method of claim 9, The Baeam chazugi extract is water (H ₂ O), C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof A health functional food composition, characterized in that extracted with one or more solvents selected from the group consisting of. 10. The method of claim 9, The muscle loss is a health functional food composition, characterized in that it includes a qualitative loss due to a decrease in the diameter of myotube cells and a quantitative loss due to a decrease in the number of myotube cells. 10. The method of claim 9, The composition is a health functional food composition, characterized in that it reduces the expression of one or more selected from the group consisting of MuRF-1 and Atrogin-1 / MAFbx in myotube cells. 10. The method of claim 9, The composition is characterized in that to increase the phosphorylation of Akt in myotube cells, health functional food composition. 10. The method of claim 9, The Baeam tea extract is a health functional food composition comprising rosmarinic acid represented by the following [Formula 1] as an active ingredient. [Formula 1]

10. The method of claim 9, The baeam chazugi extract is a health functional food composition comprising hispidulin represented by the following [Formula 2] as an active ingredient. [Formula 2]

A method for preparing a composition for preventing, improving or treating muscular atrophy, comprising the steps of: (a) pulverizing the dried baeam chazugi to prepare baeam chazugi powder; and (b) water (H ₂ O), C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, And extracting by mixing with one or more solvents selected from the group consisting of these solvents. 17. The method of claim 16, After the step (b), the method further comprising the step of lyophilizing after concentrating the extracted extract. A method of preventing or treating muscle atrophy, comprising administering an extract of Salvia plebeia R. Br. to a subject. Use of Salvia plebeia R. Br. extract for the manufacture of a medicament for the prevention or treatment of muscular atrophy.

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