KR102605187B1 - A pharmaceutical composition for preventing or treating muscle atrophy comprising extract of Salvia plebeia R. Br. or compound derived from the same as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating muscle atrophy containing Salvia plebeia R. Br. extract or its derived compounds, rosmarinic acid and/or hispidulin, as active ingredients. Regarding this, the extract or its derived compound inhibits the expression of proteins related to muscle fiber atrophy and prevents a decrease in muscle fiber thickness by increasing the activity of proteins involved in muscle protein synthesis. Since it is derived from a natural product, it has few side effects and is highly safe, It can be useful in preventing or treating muscle atrophy.

Description

A pharmaceutical composition for preventing or treating muscle atrophy comprising extract of Salvia plebeia R. Br. or compound derived from the same as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating muscular atrophy containing Salvia plebeia R. Br. extract or its derived compounds, rosmarinic acid and/or hispidulin, as active ingredients. It's about.

There are about 460 muscles in our body, and muscles play an important role in bodily functions such as maintaining our body's posture and balance and moving. Additionally, muscles are the largest organ, accounting for 45-55% of body weight, and play an important role in energy metabolism and heat generation. When these muscle mass decreases due to various causes, a vicious cycle of musculoskeletal degeneration begins, based on a weakening of muscle strength for physical activity. Reduced walking speed and weakened grip strength are the main symptoms and indicators of decreased muscle mass, which can additionally lead to falls, fractures, joint damage, metabolic disorders, and cardiovascular disease.

Decreased muscle mass can occur naturally as the body ages, and can occur due to muscle unuse or lack of exercise, or due to other pathological conditions (cachexia, sepsis, starvation, chemotherapy, excessive exposure to stress hormones). ) may occur secondaryly. This can be lumped into muscle cell atrophy due to the anti-anabolic and catabolic effects of type 1 and type 2 muscle fibers.

Therefore, as a treatment for muscle loss that may be caused by aging, disuse, or disease, natural biological materials derived from natural products that can help maintain muscle mass even in conditions that induce a decrease in muscle fiber proteins can be used.

In most cases, our body's stress hormones (Cortisol, Glucocorticoids, etc.) cause molecular biological changes in muscle fibers and are directly or indirectly involved in anti-anabolism and catabolism. Glucocorticoids have an anti-anabolic action and play a role in inhibiting the PI3K/Akt/mTOR Pathway, which inhibits the activity of downstream effectors such as 4E-BP1 and p70S6K, leading to eIF4G (Eukaryotic translation initiation). It blocks the operation of factor 4 G) and eukaryotic translation initiation factor 4 E (eIF4E). This inhibits the mRNA translation process for protein synthesis, which results 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. This is because the gene that induces muscle atrophy according to the mechanism leading to ‘FOXO (Forkhead box O transcription factor) activation and GSK3 (Glycogen synthase kinase 3) inactivation by PI3K/Akt’ is Atrogene (Atrogin-1/MAFbx). , MuRF-1, etc.), which induces the degradation of proteins represented by the Ubiquitin/Proteasome system.

Salvia plebeia R. Br. is a biennial plant of the Lamiaceae family. It is called morel cabbage because its leaves are wrinkled, and snake cabbage because they look like snake scales. It is recorded as Yeochicho (枝草) in the Materia Medica class because it is a herb with fragrant branches. The leaves are 3 to 8 cm long, grow in clumps from the roots and spread over the ground, grow two at a time, have dull sawtooth edges, and sometimes have fine hairs on the front and back, so the petioles are long. When young, they spend the winter crinkled and flat attached to the ground, and as they grow, the leaves become less wrinkled and grow gently square and straight. The whole plant is used for medicinal purposes and has the effect of reducing blood flow, clearing water, detoxifying, and killing insects. It is used in the treatment of inflammatory bowel disease, dental ulcers, etc.

As prior art related to Baeam Chazugi, Republic of Korea Patent No. 10-1794567 discloses the allergic asthma suppressing activity of Baeam Chazugi extract or fraction, and Republic of Korea Patent No. 10-1702120 discloses that the Baeam Chazugi extract or fraction prevents or prevents gout. Although the therapeutic effect has been disclosed, the preventive or therapeutic effect of muscle atrophy of Baeamchazugi extract or its derived compounds is not known.

Accordingly, the present inventors conducted intensive research on ingredients derived from natural products that can alleviate skeletal muscle decline, and as a result, they found extracts of Baeam Chazugi and their derived compounds, rosmarinic acid and/or hispidulin. ), and the present invention was completed after confirming that the composition containing was excellent in preventing or treating muscle atrophy.

KR 10-1794567

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

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

Another object of the present invention is to provide a method for producing the Baeam Chazugi extract.

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

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

Additionally, the present invention provides a method for preventing or treating muscle atrophy comprising administering the extract of Baeamchazugi to an individual.

In addition, the present invention provides the use of the extract of Baeam Chazugi for the production of a drug for preventing or treating muscular atrophy.

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

As another embodiment of the present invention, the composition may have a prevention and/or improvement effect on reduction of muscle fiber bundles, inflammatory cell infiltration of muscles, and local fibrosis.

As another embodiment of the present invention, the muscle atrophy includes sarcopenia, disuse atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, and drug-induced muscle atrophy. It may be one or more selected from the group consisting of drug induced atrophy, malnutritional atrophy, and muscular dystrophy.

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

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

As another embodiment of the present invention, the extract of Baeam Chazugi may contain rosmarinic acid represented by the following [Chemical Formula 1] as an active ingredient.

[Formula 1]

.

As another embodiment of the present invention, the extract of Baeam Chazugi may contain 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, containing pear extract as an active ingredient.

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

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

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

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

As another embodiment of the present invention, the extract of Baeam Chazugi may contain rosmarinic acid represented by the following [Chemical Formula 1] as an active ingredient.

[Formula 1]

.

As another embodiment of the present invention, the extract of Baeam Chazugi may contain hispidulin represented by the following [Formula 2] as an active ingredient.

[Formula 2]

.

Additionally, the present invention provides a method for producing a composition for preventing, improving, or treating muscle atrophy, comprising the following steps.

(a) a pulverizing step of pulverizing the dried baeam chazuki to produce baeam chazugi powder; and

(b) The pulverized Baeamchazugi powder was mixed with water (H2O), C1 to C4 lower alcohol, n-hexane, ethyl acetate, acetone, butylacetate, 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 step (b), the step of concentrating the extracted extract and then freeze-drying may be additionally included.

In another embodiment of the present invention, the muscle atrophy includes sarcopenia, disuse atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, drug-induced muscle atrophy ( It may be one or more selected from the group consisting of drug induced atrophy, malnutritional atrophy, and muscular dystrophy.

The present invention is directed to a composition for preventing, improving or treating muscle atrophy containing Salvia plebeia R. Br. extract or its derived compounds, rosmarinic acid and/or hispidulin, as active ingredients. Regarding this, the extract or its derived compound inhibits the expression of proteins related to muscle fiber atrophy, prevents a decrease in muscle fiber thickness by increasing the activity of proteins involved in muscle protein synthesis, and has almost no side effects and is highly safe as it is derived from a natural product, It can be useful in preventing or treating muscle atrophy.

Figure 1 shows the HPLC chromatography results of the Baeamchazugi extract of the present invention and the structures of its derived compounds, rosmarinic acid and hispidulin.
Figure 2 compares changes in the thickness of myotube cells C2C12 cells in the control group of the present invention, the negative control group treated with dexamethasone to induce muscle atrophy, the Baeamchazugi extract treatment group, and the dexamethasone and Baeamchazugi extract treatment group. will be.
Figure 3 shows the mRNA expression levels of MuRF-1 and Atrogin-1/MAFbx in the control group, negative control group, Baeam chazgi extract treatment group, and dexamethasone and Baeam chazgi treatment group of the present invention.
Figure 4 shows the degree of p70S6K phosphorylation in myotube C2C12 cells after dexamethasone treatment and treatment with Baeam Chazgi extract by western blot.
Figure 5 compares changes in the thickness of myotube cells C2C12 cells in the control group of the present invention, the negative control group treated with dexamethasone to induce muscle atrophy, the group treated with dexamethasone and rosmarinic acid, and the group treated with dexamethasone and hispidulin.

The present inventors conducted intensive studies on Salvia plebeia R. Br. extract or its derived compounds, rosmarinic acid and/or hispidulin, and found that the extract or compound contained proteins related to muscle hypertrophy. The present invention was completed by confirming the effect of increasing expression and alleviating skeletal muscle decline.

More specifically, after inducing muscle atrophy by treating myotube cells C2C12 with dexamethasone, treatment with the extract or compound resulted in an increase in the diameter of myotubes and phosphorylation and/or expression of proteins that promote muscle protein synthesis. It was confirmed that the degree was recovered.

From the above results, the present invention provides a pharmaceutical composition for preventing or treating muscle atrophy containing an extract of Baeam Chazugi or a compound derived therefrom as an active ingredient.

In the present invention, the term "extract" refers to a liquid component obtained by immersing the target material in various solvents and then extracting it at room temperature or at a heated state for a certain period of time, a solid component obtained by removing the solvent from the liquid component, etc. means the result of In addition, in addition to the above results, it can be comprehensively interpreted to include all dilutions of the results, concentrates thereof, crude preparations, purifications, etc. of the above results.

In the present invention, the extract is an extract of Salvia plebeia R. Br. and can be extracted from various organs of natural, hybrid, and variant plants, such as roots, aerial parts, stems, leaves, petals, and flowers. It can be extracted from buds, fruit bodies, and fruit peels, as well as plant tissue cultures.

The Baeam chazugi extract of the present invention can be extracted according to a conventional method known in the art for obtaining extracts from natural products, that is, using a conventional solvent under conventional conditions of temperature and pressure. For example, the Baeam Chazugi extract is selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butylacetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof. Extraction can be performed using one or more solvents, but there is no particular limitation as long as an extract having an effect for preventing or treating muscle atrophy can be obtained. However, extraction may preferably be done using a mixed solvent of water and 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 effective substances that improve muscle atrophy may vary.

Methods for obtaining the extract include cold needle extraction at room temperature of 10 to 25°C, hot water extraction by heating to 40 to 100°C, and ultrasonic waves, as long as it is possible to obtain an extract that has the effect of preventing or treating muscle atrophy. It may be an ultrasonic extraction method, a reflux extraction method using a reflux condenser, etc., but is not particularly limited thereto.

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

In addition, the extract extracted with the above solvent may 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 is not limited to this.

In addition, in the present invention, when extracting the Baeamchazugi, an acid for drinking purposes can be added to the solvent for extraction, preferably acetic acid, citric acid, or malic acid. One or more acids selected from the group consisting of acid, succinic acid, fumaric acid, tartaric acid, amino acid, and lactic acid can be added to the solvent. .

Accordingly, the present invention includes (a) pulverizing dried Baeam Chazugi to produce Baeam Chazugi powder; (b) extracting the pulverized Baeamchazugi powder with a solvent of 50% ethanol; and (c) concentrating the extract extracted from the above and freeze-drying it.

In the present invention, the term “muscular atrophy” refers to a state in which muscles are atrophied or muscle mass is reduced due to 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 can result in intrinsic dysfunction of skeletal muscle. Pydulin can be used for the purpose of maintaining and preserving the intrinsic function of skeletal muscles resulting from muscle atrophy due to its function of increasing the activity of muscle fibers and protecting muscle fibers.

The muscle atrophy is a non-limiting example, and the muscle atrophy includes sarcopenia, disuse atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, and drug-induced muscle atrophy. Includes drug induced atrophy, malnutritional atrophy, muscular dystrophy, etc.

The insoluble muscle atrophy includes a decrease in muscle thickness due to activity restrictions due to aging, disease, etc., long periods of bed rest, and the use of splints (casts) or plasters (Gips), etc., and can be caused by various causes such as peripheral nerve damage, cancer, and sepsis. It is accompanied not only by clinical disease but also by long-term bed rest and the aging process. The absence of mechanical stimulation and loss of innervation result in muscular atrophy, which results in a quantitative decrease and dysfunction of mitochondria, increases the production of reactive oxygen species (ROS) in mitochondria, and causes apoptosis of muscle cells due to oxidative damage ( It causes muscle atrophy due to apoptosis. The main symptoms of muscular dystrophy are muscle atrophy and muscle weakness caused by the absence of dystrophin-glycoprotein complex, a component of the rhabdoid plasma membrane, due to a genetic mutation.

In addition, cortisol is a substance that can be secreted from the body due to various acute stresses. It plays a role in supplying energy to combat stress, but depending on its mechanism, it also causes muscle atrophy in the body. Additionally, synthetic corticosteroids may cause adverse reactions such as steroid induced atrophy and damage to muscle parenchyma.

The composition for preventing, improving or treating muscle atrophy containing the Baeamchazugi extract or a compound derived therefrom of the present invention not only indirectly prevents muscle loss by reducing the expression of inflammatory cytokines, but also increases the thickness or mass of muscle fibers, By suppressing the induction of muscle atrophy by protein degradation, the above-mentioned muscle atrophy can be directly prevented, improved, or treated.

In the present invention, 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 the suspicion and onset of muscle atrophy by administration of the pharmaceutical composition. It refers to any action that improves or changes an entity's symptoms in a beneficial way.

In the present invention, the term “pharmaceutical composition” refers to a product prepared for the purpose of preventing or treating disease, and can be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be formulated and used 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 the desired biological effect. In actual application, determination of the amount included as an active ingredient is necessary to treat the target disease. The amount may be determined taking into account the fact that it does not cause other toxicities and may vary depending on various factors such as, for example, the disease or condition being treated, the type of composition administered, the size of the subject, or the severity of the disease or condition. A person skilled in the art 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, depending on each formulation. The pharmaceutically acceptable carrier may be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these ingredients, and may include antioxidants, buffers, and bacteriostatic agents as necessary. Other common additives may also be included. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Furthermore, it may be preferably formulated according to each disease or ingredient 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 can be administered orally or parenterally in a pharmaceutically effective amount according to the desired method, and the term “pharmaceutically effective amount” in the present invention refers to a disease with a reasonable benefit/risk ratio applicable to medical treatment. It refers to an amount that is sufficient to treat This may depend on factors including the duration of treatment, drugs combined or used simultaneously, and other factors well known in the medical field.

In the present invention, the term “individual” may be a mammal such as a rat, livestock, mouse, or human, and preferably may be a human.

The pharmaceutical composition of the present invention can be formulated in various forms for administration to an individual, and a representative formulation for parenteral administration is an injectable formulation, and an isotonic aqueous solution or suspension is preferred. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline or buffer solution and formulated for injection. In addition, dosage forms for oral administration include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. These dosage forms contain diluents (e.g. lactose, dextrose) in addition to the active ingredient. , sucrose, mannitol, sorbitol, cellulose and/or glycine) and lubricants (e.g., 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 in some cases starch, agar, alginic acid or the like. It may further include disintegrants such as sodium salts, absorbents, colorants, flavoring and/or sweetening agents. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, the pharmaceutical composition of the present invention contains adjuvants such as binders, lubricants, disintegrants, colorants, preservatives, sweeteners, flavoring agents, stabilizers, diluents, preservatives, wetting agents, emulsification accelerators, salts or buffers for adjusting osmotic pressure, and other It may additionally contain therapeutically useful substances and may be formulated according to conventional methods.

The pharmaceutical composition according to the present invention can be administered through various routes, including orally, transdermally, subcutaneously, intravenously, or intramuscularly, and the dosage of the active ingredient depends on the route of administration, the patient's age, gender, weight, and severity of the patient. It can be appropriately selected depending on several factors. Additionally, the composition of the present invention can be administered in combination with known compounds that can enhance the desired effect.

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

Meanwhile, the present invention can be used as a health functional food composition for preventing or improving muscle atrophy and/or muscle loss, containing the extract of Baeamchazugi or its derived compounds, rosmarinic acid and/or hispidulin, as active ingredients.

In the present invention, the term “health functional food composition” refers to foods manufactured by adding the extract of Baeam Chazugi or its derived compounds to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspending them. This means that when consumed, it brings about specific health effects. However, unlike general drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food.

The health functional food composition may be selected from meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, pizza, jelly, noodles, gum, ice cream, alcoholic beverages, alcohol, vitamin complexes, and other health supplements. It can be any one of them.

Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated in the drawings and explained in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

Example 1. Preparation of Baeamchazugi extract

The dried aerial part of Baeamchazugi was purchased, ground to an average particle size of 0.30 to 0.50 mm through a grinder, and extracted with a 50:50 mixed solvent of ethanol and water for 1 to 4 hours. The extract was concentrated using a rotary vacuum evaporator and then 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 components 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 Figure 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 conditions 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 1ml/min UV 254nm

As a result of the HPLC, it was confirmed that the Baeam Chazugi extract of the present invention contains rosmarinic acid and hispidulin (Figure 1).

Example 2. In vitro Confirm the effect of Baeam Chazugi extract on preventing, improving or treating muscle atrophy

2-1. Culture and differentiation induction of C2C12 cells

C2C12 mouse myoblast cells used in this experiment were provided by the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center, and were stored in 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 cultured under 5% CO ₂ and 37°C conditions. C2C12 myoblasts were distributed and cultured in a 12-well plate, and when the cell density reached 80-85%, differentiation medium (DMEM containing 2% horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin) was used every two days. Replaced with new medium and differentiated for 6 days.

2-2. Induction of muscle atrophy with dexamethasone and treatment with Baeam Chazugi extract

Dexamethasone induces muscle atrophy by inhibiting muscle synthesis and causing protein breakdown. In order to confirm the effect of the Baeamchazugi extract on preventing, improving, or treating muscle atrophy, the C2C12 cells induced in Example 2-1 were treated with dexamethasone to induce muscle atrophy, and the cells in which muscle atrophy was induced were treated with embryonic cancer. Chazugi extract was treated. The specific experimental conditions are as follows.

(1) The control group was prepared by treating differentiated C2C12 myotube cells with differentiation medium containing only 1% Penicillin for 48 hours.

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

(3) The Baeam Chazugi extract treatment group was prepared under the same conditions as the control group in (1) above and additionally treated with a composition of 30 μg/mL of Baeam Chazugi extract for 48 hours.

(4) The group treated with dexamethasone and pear extract was prepared under the same conditions as the negative control group in (2) above by additionally treating the group with 30 μg/mL of pear extract extract for 48 hours.

2-3. Measurement of C2C12 myotube diameter

In Example 2-2, each control and experimental group treated with samples for 48 hours was photographed using JuLi™Stage, an automated cell imaging system, and the thickness of each photographed cell was measured using ImageJ, a cell processing program. Data were organized by measuring 50 items per group and calculating the mean and standard error (Table 2). Each experiment was repeated three times.

control group Dex treatment group Baeam Chazugi Dex + Baeamchazugi C2C12 Relative thickness of myotube cells
(% of control) 100% 71.3% ^a 103.9% 98.8 ^b

(Dex: Dexamethasone (1 μM), Baeam Chazugi: Baeam Chazugi extract (30 μg/ml))

^a : Significance compared to the control group (p< 0.05), ^b Significance compared to the Dex treatment group (negative control) (p<0.01)

As a result of the above experiment, it was confirmed that the thickness of C2C12 myotubes was reduced by dexamethasone treatment, and that the improvement effect was observed by treatment with Baeam Chazugi extract (Table 2 and Figure 2).

More specifically, (1) the thickness of C2C12 myotube cells decreased by 28.7% in the group administered 1 μM dexamethasone compared to the control group administered basic differentiation medium (p<0.05). (2) Compared to the control group, which was administered basic differentiation medium, the thickness of C2C12 myotube cells increased by 3.9% in the group administered 30 μg/ml of embryonic chazgi, but the range was not statistically significant. (3) Compared to the negative control group administered dexamethasone to the basic differentiation medium, the thickness of C2C12 myotube cells increased by 38.6% in the group additionally administered 30 μg/ml of embryonic chazgi, which was very statistically significant (p<0.01). It was a shame.

Therefore, it was confirmed that the Baeamchazugi extract of the present invention showed a significant (38.6%) recovery rate in the dexamethasone-induced muscle atrophy model.

2-4. Confirmation of muscle atrophy-related mRNA expression level and protein phosphorylation level

In Example 2-2, the expression levels of mRNA of atrogin-1/MAFbx and MuRF-1, which act on the muscle atrophy inducing pathway, were measured in each control and experimental group treated for 48 hours by real time-PCR, and Western The degree of phosphorylation of p70S6K, which acts on the muscle synthesis pathway, was confirmed using Western blotting.

The mRNA expression levels of Atrogin-1/MAFbx and MuRF-1 in the control and experimental groups are shown in Table 3 below. As a result of the experiment, it was confirmed that the mRNA expression levels of Atrogin-1/MAFbx and MuRF-1 increased in C2C12 myotubes by treatment with dexamethasone, and that the expression levels of each mRNA decreased by treatment with Baeam Chazugi extract (Table 3 and Figure 3 ).

control group Dex treatment group Baeam Chazugi Dex + Baeamchazugi Atrogin-1 mRNA One 3.37 0.53 1.87 MuRF-1 mRNA One 2.28 0.86 0.73

More specifically, (1) compared to the control group administered with basic differentiation medium, the mRNA expression levels of atrogin-1/MAFbx and MuRF-1 increased by 3.37-fold and 2.28-fold, respectively, in the group administered 1 μM dexamethasone. (2) Compared to the control group, the mRNA expression level of atrogin-1/MAF decreased by 0.53-fold and the mRNA expression level of MuRF-1 decreased by 0.86-fold in the group administered 30 μg/ml Baeamchazgi. (3) Compared to the control group, in the group administered 1 μM of dexamethasone and 30 μg/ml of Baeam Chazgi, the mRNA expression level of atrogin-1/MAFbx was 1.87 times that of MuRF-1, and the mRNA expression level of MuRF-1 was 0.73 times that of ( 4) When compared to the negative control group administered dexamethasone to the basic differentiation medium, the expression level of atrogin-1/MAFbx mRNA increased by dexamethasone treatment was decreased by 44.5% by administering Baeamchazugi extract, and the mRNA of MuRF-1 The expression level decreased by 68.0%.

Therefore, it was confirmed that the Baeamchazugi extract of the present invention inhibits the activation of atrophy factors in muscle cells by dexamethasone.

In addition, the degree of phosphorylation of p70S6K, a downstream mechanism of the Akt/mTOR pathway, was confirmed through Western blotting. In the negative control group treated with dexamethasone, the phosphorylation of p70S6K protein was reduced by 61% compared to the control group, and additionally, in the negative control group treated with dexamethasone, the phosphorylation of p70S6K protein was decreased by 61% compared to the control group. It was confirmed that protein synthesis was promoted in the group by recovering the reduced degree of phosphorylation by 58.3% compared to the negative control group (Figure 4).

Example 3. In vitro Confirmation of muscle atrophy prevention, improvement, or treatment effects of compounds derived from Baeamchazugi

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

In order to confirm the effectiveness of the compound derived from Baeamchazgi in preventing, improving, or treating muscle atrophy, the C2C12 cells induced in Example 2-1 were treated with dexamethasone to induce muscle atrophy, and the cells in which muscle atrophy was induced were treated with embryonic cancer. It was treated with rosmarinic acid or hispidulin, a compound derived from Chazugi extract. The specific experimental conditions are as follows.

(1) The control group was prepared by treating differentiated C2C12 myotube cells with differentiation medium containing only 1% Penicillin for 48 hours.

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

(3) The dexmethasone and rosmarinic acid treatment group was prepared by additionally treating the group with 30 μM rosmarinic acid for 48 hours under the same conditions as the negative control group in (2) above.

(4) The dexmethasone and hispidulin treated group was prepared by additionally treating the group with 30 μM rosmarinic acid for 48 hours under the same conditions as the negative control group in (2) above.

3-2. Measurement of C2C12 myotube diameter

In Example 3-1, each control group and experimental group treated for 48 hours were photographed using JuLi™Stage, an automated cell imaging system, and the thickness of each photographed cell was measured using ImageJ, a cell processing program. Data were organized by measuring 50 items per group, calculating the mean and standard error (Table 4). Each experiment was repeated three times.

control group Dex treatment group Dex + rosmarinic acid Dex + hispidulin C2C12 Relative thickness of 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 the C2C12 myotube was reduced by treatment with dexamethasone, and that an improvement effect was observed by treatment with rosmarinic acid or hispidulin, a compound derived from Baeamchazugi (Table 4 and Figure 5).

More specifically, (1) the thickness of C2C12 myotube cells decreased by 22.6% in the group administered 1 μM dexamethasone compared to the control group administered basic differentiation medium. (2) Compared to the negative control group administered dexamethasone to the basic differentiation medium, the thickness of C2C12 myotube cells increased by 23.2% in the group additionally administered 30 μM rosmarinic acid. (3) Compared to the negative control group administered dexamethasone to the basic differentiation medium, the thickness of C2C12 myotube cells increased by 26.9% in the group additionally administered 30 μM of hispidulin.

Therefore, it was confirmed that when treated with rosmarinic acid and hispidulin derived from the extract of Baeamchazugi of the present invention, the thickness of myotube cells increased in a muscle atrophy model induced by dexamethasone, showing an effect in preventing, improving, or treating muscle atrophy. .

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

A pharmaceutical composition for preventing or treating muscle atrophy containing Salvia plebeia R. Br. extract as an active ingredient.
According to paragraph 1,
The Baeam Chazugi extract is water (H ₂ O), C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butylacetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof. A pharmaceutical composition, characterized in that it is extracted with one or more solvents selected from the group consisting of.
According to paragraph 2,
A pharmaceutical composition, characterized in that the Baeam Chazugi extract is extracted with 50% ethanol as a solvent.
According to paragraph 1,
The muscular atrophy includes sarcopenia, disuse atrophy, muscular atrophy due to absence of mechanical stimulation, denervational atrophy, drug induced atrophy, and nutritional deficiency. A pharmaceutical composition, characterized in that it is at least one muscular atrophy selected from the group consisting of malnutritional atrophy and muscular dystrophy.
According to paragraph 1,
A pharmaceutical composition, characterized in that the composition reduces the expression of one or more mRNAs selected from the group consisting of MuRF-1 and Atrogin-1/MAFbx in myotube cells.
According to paragraph 1,
A pharmaceutical composition, characterized in that the composition increases phosphorylation of p70S6K in myotube cells.
According to paragraph 1,
A pharmaceutical composition for preventing or treating muscular atrophy, wherein the Baeamchazugi extract contains rosmarinic acid represented by the following [Formula 1] as an active ingredient.
[Formula 1]
.
According to paragraph 1,
A pharmaceutical composition for preventing or treating muscular atrophy, wherein the Baeamchazugi extract contains hispidulin represented by the following [Formula 2] as an active ingredient.
[Formula 2]
.
A health functional food composition for preventing or improving muscle loss containing Salvia plebeia R. Br. extract as an active ingredient.
According to clause 9,
The Baeam Chazugi extract is water (H ₂ O), C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butylacetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof. A health functional food composition, characterized in that it is extracted with one or more solvents selected from the group consisting of.
According to clause 9,
A health functional food composition, wherein the muscle loss includes qualitative loss due to a decrease in the diameter of myotube cells and quantitative loss due to a decrease in the number of myotube cells.
According to clause 9,
The composition is a health functional food composition, characterized in that it reduces the expression of one or more mRNAs selected from the group consisting of MuRF-1 and Atrogin-1/MAFbx in myotube cells.
According to clause 9,
A health functional food composition characterized in that the composition increases phosphorylation of p70S6K in myotube cells.
According to clause 9,
A health functional food composition wherein the pear amchazugi extract contains rosmarinic acid represented by the following [Formula 1] as an active ingredient.
[Formula 1]
.
According to clause 9,
A health functional food composition wherein the Baeamchazugi extract contains hispidulin represented by the following [Formula 2] as an active ingredient.
[Formula 2]
.
Method for producing a composition for preventing, improving or treating muscle atrophy comprising the following steps:
(a) a pulverizing step of pulverizing the dried baeam chazuki to produce baeam chazugi powder; and
(b) The pulverized Baeamchazugi powder was mixed with water (H ₂ O), C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butylacetate, 1,3-butylene glycol, methylene chloride, and extraction by mixing with one or more solvents selected from the group consisting of mixed solvents thereof.
According to clause 16,
After step (b), the manufacturing method additionally includes the step of concentrating the extracted extract and then freeze-drying it.