KR102186564B1 - Composition for preventing or treating muscle weakness diseases comprising alverine, 4-hydroxy alverine or pharmaceutically acceptable salts thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diseases related to muscle weakness, comprising alverine, 4-hydroxy alverine, or a pharmaceutically acceptable salt thereof, and a composition for promoting differentiation of myoblasts in vitro. When myoblast are treated with alverine, 4-hydroxy alverine, or the pharmaceutically acceptable salt thereof, which is an active component of the present invention, differentiation into root canal cells is promoted. In addition, when a mouse is treated with the alverine, 4-hydroxy alverine, or the pharmaceutically acceptable salt thereof, it is possible to increase muscle mass and improve muscle strength. Therefore, the pharmaceutical composition according to the present invention can be usefully used for promoting differentiation of myoblasts and preventing or treating diseases related to muscle weakness.

Description

A pharmaceutical composition for preventing or treating muscle weakness-related diseases comprising alberine, 4-hydroxy alberine or a pharmaceutically acceptable salt thereof TECHNICAL FIELD [COMPOSITION FOR PREVENTING OR TREATING MUSCLE WEAKNESS DISEASES COMPRISING ALVERINE, 4-HYDROXY ALVERINE OR PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF}

The present invention relates to a pharmaceutical composition for preventing or treating diseases related to muscle weakness, including alberine, 4-hydroxy alberine, and a pharmaceutically acceptable salt thereof, and a composition for promoting differentiation of myoblasts in vitro.

Diseases that weaken muscle strength include sarcopenia that progresses with aging and muscle dystrophy, a degenerative myopathy in which muscle fibers are necrotized by genetic mutations.

Sarcopenia is a symptom in which muscle strength decreases due to loss of muscle mass during aging. In sarcopenia, not only muscle mass decreases, but also the types of muscle fibers change. This sarcopenia causes senility and dysfunction in the elderly. In addition, muscular dystrophy can lead to disability or death through necrosis and degeneration of muscle fibers due to a lack or mutation of a muscle-related gene. In addition, cachexia is a muscle weakness disease that frequently occurs in cancer patients. In cancer patients, due to the increase in metabolism, the metabolic reaction of skeletal muscle decomposition occurs excessively, resulting in decreased muscle mass and decreased muscle strength. This metabolic imbalance eventually reaches a stage in which recovery is not possible even with food intake, so active treatment such as drug therapy is required.

Methods of treating muscle loss include exercise therapy and medication. Exercise increases protein synthesis in skeletal muscle in the short term, and increases muscle strength and individual mobility. However, it is extremely limited to apply exercise therapy to patients. Testosterone or anabolic steroids are sometimes prescribed as drug therapy. However, in the case of such drug therapy, it shows a rapid effect, but induces masculinization in women, and causes side effects such as prostate symptoms in men. In addition, drugs that prescribe DHEA (dehydroepiandrosterone) and growth hormone, and SARMs (Selective Androgen Receptor Modulators), which are improved drugs that minimize the side effects of male hormones such as Androgen, and preserve anabolic efficacy, are also used (DD Thompson, J. Musculoskelet Neuronal Interact 7, 344-345, 2007). To date, there are no treatments for sarcopenia approved by the US FDA (Sung Sup Park, Young-Kyo Seo and Ki-Sun Kwon, BMB Reports 2019; 52(1): 64-69).

Recently, a stem cell treatment method that separates satellite cells and differentiates them from the body and then re-introduces them into the body and a method that promotes myogenesis by directly activating satellite cells in the body to maintain or strengthen muscle It is emerging as a method of treating muscle weakness-related diseases (Shihuan Kuang, and Michael A. Rudnicki, Trends in Molecular Medicine 14, 82-31, 2008).

That is, as a fundamental treatment method for diseases related to muscle weakness without side effects, a method of differentiating myoblasts is required, and accordingly, development of a substance capable of promoting the differentiation of myoblasts is required.

D.D. Thompson, J. Musculoskelet Neuronal Interact 7, 344-345, 2007 Sung Sup Park, Young-Kyo Seo and Ki-Sun Kwon, BMB Reports 2019; 52(1): 64-69 Shihuan Kuang, and Michael A. Rudnicki, Trends in Molecular Medicine 14, 82-31, 2008

Accordingly, the present inventors have tried to develop a substance capable of increasing muscle mass and effectively recovering muscle function by promoting differentiation of myoblasts. As a result, the present invention was completed by confirming that alberine, 4-hydroxy alberine or a pharmaceutically acceptable salt thereof promotes differentiation of myoblasts and restores muscle strength.

In order to solve the above problems, an aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle weakness-related diseases comprising alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a composition for promoting differentiation of myoblasts in vitro, comprising alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a method for promoting differentiation of myoblasts, comprising treating the myoblasts in vitro with alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a method for producing a myotube cell comprising the step of differentiating myoblasts by treating the myoblasts in vitro with alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof. do.

Another aspect of the present invention provides a food composition for preventing or improving muscle weakness-related diseases, including alberine, 4-hydroxy alberine, or a food pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a composition for strengthening muscle strength, comprising alberine, 4-hydroxy alberine, a pharmaceutically acceptable salt thereof, or a food pharmaceutically acceptable salt thereof.

When the myoblasts are treated with alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof, which are active ingredients of the pharmaceutical composition of the present invention, differentiation into root canal cells is promoted. In addition, when a mouse is treated with the alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof, it is possible to increase muscle mass and improve muscle strength. Accordingly, the pharmaceutical composition according to the present invention can be usefully used for promoting differentiation of myoblasts and preventing or treating diseases related to muscle weakness.

1 is a photograph taken by fluorescent staining of myotube cells differentiated by treatment with DMSO, insulin or alberine citrate on myoblasts.
2 is a graph showing the eMyHC fluorescence staining area of myotube cells differentiated by treatment with DMSO, insulin or alberine citrate on myoblasts.
3 is a diagram schematically illustrating an experiment schedule using animals to confirm the effect of alberine citrate on improving muscle strength.
Figure 4 is a graph showing the measurement value of the grip strength of the mouse according to the administration concentration of alberine citrate.
Figure 5 is a diagram showing the running time of mice in the normal group and the experimental group.
6 is a graph showing the time that the mice in the normal group and the experimental group were hung on the Rotor-rod.
7 is a graph showing the muscle mass of the spleen muscle (GA) and tibialis anterior muscle (TA) obtained from mice in the normal and experimental groups.
FIG. 8 is a diagram schematically illustrating an experiment schedule using sarcopenia-causing animals to confirm the muscle strength enhancing effect of alberine citrate.
9 is a graph showing the running time of mice in the normal group, the control group and the experimental group.
10 is a graph showing the muscle mass of the spleen muscle (GA) and tibialis anterior muscle (TA) obtained from mice in the normal group, the control group, and the experimental group.
11 is a photograph taken by fluorescence staining of myotubes differentiated by treatment with DMSO, insulin, alberine citrate or 4-hydroxy alverine (4HA) on myoblasts.
12 is a graph showing the eMyHC fluorescence staining area of myotubes differentiated by treatment with DMSO, insulin, alberine citrate or 4-hydroxy alberine (4HA) on myoblasts.
13 is a photograph taken by fluorescence staining of myotube cells induced muscle atrophy with a cancer cell culture solution (CM) with alberine citrate (AC) or 4-hydroxy alberine (4HA).
14 is a graph showing the diameter of myotube cells inducing muscle atrophy with a cancer cell culture solution (CM) using alberine citrate (AC) or 4-hydroxy alberine (4HA).

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases related to muscle weakness, comprising alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof.

The term "Alverine" used in the present invention, the name of IUPAC is N-Ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine, the chemical formula is C ₂₀ H ₂₇ N, It refers to a compound having a molecular weight of 281.44 g/mol. Alberine is a drug generally used for gastrointestinal disorders, and is known as a compound used to relax smooth muscle, which is an involuntary muscle involved in contraction of internal organs and uterus, but no association with the differentiation of myoblasts is known. The structure of alberine is shown in Formula 1 below.

[Formula 1]

The 4-hydroxy alberine is a biological metabolite of alberine, and the structure of 4-hydroxy alberine is shown in Formula 2 below.

[Formula 2]

The term "pharmaceutically acceptable salt" used in the present invention means a salt prepared according to a conventional method in the art, and such a preparation method is known to those skilled in the art. Specifically, the pharmaceutically acceptable salt includes, but is not limited to, pharmacologically or physiologically acceptable salts derived from inorganic acids and organic acids and bases below. Examples of suitable acids are hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid. , Benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, or potassium, alkaline earth metals such as magnesium. In one embodiment of the present invention, citrate was used as a pharmaceutically acceptable salt.

The disease related to muscle weakness refers to all diseases that can occur due to muscle weakness, and is a disease in which the differentiation ability of myoblasts is reduced or weakened due to decreased muscle cells in the body or decreased satellite cell activity. It refers to a disease that can be expected to improve or treat. Specifically, the disease related to muscle weakness may be sarcopenia, muscle atrophy, muscle dystrophy, or cachexia.

The term "myopenia" as used in the present invention refers to a symptom of a decrease in muscle strength due to a decrease in muscle mass during aging. It is thought that the decrease in the activity of satellite cells is an important cause of the reduction in muscle mass in sarcopenia. Satellite cells are activated by stimulation such as exercise or injury and proliferate into myoblasts, and when differentiation proceeds, they fuse with other cells to form multinuclear muscle fibers.

The term "muscular dystrophy" as used in the present invention means a symptom in which the muscles of the limb are gradually atrophy in an almost symmetrical direction. It can cause progressive degeneration of motor nerve fibers and cells in the spinal cord, leading to amyotrophic lateral sclerosis (ALS) and spinal progressive muscular atrophy (SPMA).

The term "muscular dystrophy" as used in the present invention refers to a degenerative myopathy characterized by necrosis of muscle fibers regardless of the central and peripheral nervous systems. Muscular dystrophy has slight differences between muscular atrophy and clinical symptoms. Muscular dystrophy occurs mainly in childhood, and muscular dystrophy occurs in adolescence. In addition, muscular dystrophy occurs in the proximal muscles and muscular dystrophy occurs in the distal muscles. Muscular stiffness symptoms are not found in muscular dystrophy, but in muscular atrophy. Muscular dystrophy has a certain hereditary nature, but muscular atrophy has a rare genetic tendency.

The term "cachexia" used in the present invention means a high degree of systemic weakness that can be seen at the end of cancer, tuberculosis, and hemophilia. In addition, cachexia is considered a type of poisoning caused by various organ disorders throughout the body. Symptoms of cachexia include muscle weakness, rapid shedding, anemia, lethargy, and yellowing of the skin. The causative diseases of cachexia include malignant tumors, basedo disease, and hypoplasia of the pituitary gland. Bioactive substances such as tumor necrosis factor (TNF) produced by macrophages have also been found to be factors that enhance cachexia.

The concentration of alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof in the pharmaceutical composition may be 10 μg/ml to 180 μg/ml. Specifically, the concentration of alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof is 20 μg/ml to 160 μg/ml, 40 μg/ml to 140 μg/ml or 60 μg/ml to 120 It may be μg/ml.

In addition, the dosage of the pharmaceutical composition may be administered based on the dosage of the active ingredients alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof. Specifically, the dosage of alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof may be administered from 60 mg to 240 mg per day, and if the dosage is large, administration over 1 to 3 times a day can do. Preferably, the dosage of alberine, 4-hydroxy alberine or a pharmaceutically acceptable salt thereof is 60 mg to 120 mg, and may be administered once to twice a day. In addition, the use of alberine is recommended for adults and children over 12 years of age.

In one embodiment of the present invention, alberine citrate, a pharmaceutically acceptable salt of alberine, was orally administered at a dose of 17 mg/kg/day, 50 mg/kg/day, or 150 mg/kg/day. Alberine citrate at concentrations of 1.7 mg/ml, 5 mg/ml, and 15 mg/ml were administered orally in 200 µl each to mice having an average body weight of 20 g.

The pharmaceutical composition may additionally include a pharmaceutically acceptable carrier. The carrier is commonly used in the manufacture of drugs, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrroly Money, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive selected from the group consisting of lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and combinations thereof.

The pharmaceutical composition may be appropriately administered to a subject according to a conventional method, route of administration, and dosage used in the art. Specifically, the pharmaceutical composition may be selected according to a method known in the art, and an appropriate dosage and number of administrations may be selected. It can be appropriately determined by various factors such as route, sex, health status, diet, age and weight of the individual, and the severity of the disease.

Another aspect of the present invention provides a composition for promoting differentiation of myoblasts in vitro, comprising alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof.

The alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof is the same as described above in the pharmaceutical composition.

The myoblast refers to a muscle cell in an undifferentiated state. The myotubes form multinuclear myotubes through fusion of myoblasts, which are mononuclear cells, during differentiation. In the late stages of differentiation, the expression of myosin heavy chain (MyHC) increases.

The composition for promoting differentiation may be a medium for differentiation of DMEM containing serum, but any medium or composition capable of promoting differentiation of myoblasts may be included without limitation. In addition, the composition may further contain additional substances required for cell culture or differentiation.

The concentration of alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof in the composition for promoting differentiation may be 0.01 μM to 100 μM. Specifically, the concentration of alberine, 4-hydroxy alberine or a pharmaceutically acceptable salt thereof is 0.1 μM to 50 μM, 0.1 μM to 25 μM, 0.5 μM to 25 μM, 0.1 μM to 10 μM, 0.5 μM To 10 μM, 1 μM to 10 μM, 0.1 μM to 5 μM, 0.5 μM to 5 μM, 1 μM to 5 μM, 0.1 μM to 2 μM, 0.5 μM to 2 μM, or 1 μM to 2 M. In one embodiment of the present invention, alberine or 4-hydroxy alberine at concentrations of 0.01 μM, 0.1 μM, 1 μM, and 10 μM were treated on C2C12 cells, which are mouse-derived myoblasts.

Another aspect of the present invention provides a method for promoting differentiation of myoblasts, comprising treating the myoblasts in vitro with alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof. The method of promoting differentiation of myoblasts may be performed in vitro or ex vivo .

Another aspect of the present invention provides a method for producing a myotube cell comprising the step of differentiating myoblasts by treating the myoblasts in vitro with alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof. do. The method for producing the myotube cells may be performed in vitro or ex vivo .

Another aspect of the present invention provides a food composition for preventing or improving muscle weakness-related diseases, including alberine, 4-hydroxy alberine, or a food pharmaceutically acceptable salt thereof. When the food composition is used as a food additive, it may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food composition may be used before or after the onset stage of muscle weakness-related disease in order to prevent or improve muscle weakness-related disease, and simultaneously or separately with a drug for treating the disease. Specifically, the food composition is characterized by promoting differentiation of myoblasts.

Diseases related to muscle weakness are the same as described above in the pharmaceutical composition.

Another aspect of the present invention provides a composition for strengthening muscle strength comprising alberine, 4-hydroxy alberine, a pharmaceutically acceptable salt thereof, or a food pharmaceutically acceptable salt thereof. The composition for strengthening muscle strength may be used as a pharmaceutical composition or a food composition.

The strengthening of muscle strength means increasing muscle mass, strengthening muscle recovery, and reducing muscle fatigue. The composition for strengthening muscle strength may increase total muscle mass by increasing muscle mass through the ability to differentiate myoblasts into muscle cells, thereby reducing muscle fatigue. In addition, because muscle cells can be replaced quickly, they can heal quickly against muscle damage. The composition for strengthening muscle strength of the present invention may be used as feed or feed additive.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Confirmation of the effect of alberine citrate to promote differentiation of myoblasts

Example 1.1. Of myoblast main C2Cl2 culture

C2Cl2 (American Type Culture Collection, CRL-1772™) cells are myoblasts obtained from C3H mice and are used for differentiation studies of myoblasts. The C2C12 cells were cultured using a culture medium, and then cultured using a differentiation medium when inducing differentiation. At this time, DMEM added with 10% fetal bovine serum was used as the growth media (GM), and 5% horse serum (HS) was used as the differentiation media (DM). This added DMEM was used.

Example 1.2. Promote differentiation of myoblast line

To confirm the effect of promoting differentiation of myoblasts of alberine citrate, C2C12 cells were dispensed into the culture medium of Example 1.1 and cultured for 24 hours, and then DMSO, insulin, or alberine citrate, respectively, to C2C12 cells. (Alverine citrate) was treated and differentiation was induced for 3 days. Thereafter, differentiation into myotubes and changes in thickness and diameter of myotubes were observed through fluorescence staining using an antibody against myosin H chain (hereinafter MyHC). At this time, DMSO, insulin and alberine citrate used in the experiment were all purchased from Sigma-Aldrich. The group treated with insulin was used as a positive control.

Specifically, 5 × 10 ⁵ C2C12 cells per plate were dispensed into a 6-well-plate treated with the culture medium of Example 1.1, and replaced with DMEM medium added with 5% horse serum after 24 hours. Differentiation was induced. Then, DMSO, insulin (1.72 μM) or alberine citrate (0.01 μM, 0.1 μM, 1 μM or 10 μM) were treated, respectively. After 3 days, the medium was removed, washed with a phosphate buffer solution (1X PBS), and then treated with paraformaldehyde (4%) to fix the cells at room temperature for 15 minutes. Thereafter, after washing three times with a phosphate buffer solution (1X PBS), PBS containing 0.3% triton X-100 was treated as a permeabilization buffer, and then reacted at room temperature for 10 minutes.

After washing three times with phosphate buffer solution (1X PBS), PBST (blocking buffer, PBS containing 0.5% Tween 20) containing 2% bovine serum albumin was treated and reacted for 30 minutes to make non-specific The binding of the antibody was inhibited. After washing three times with a phosphate buffer solution (1X PBS), 100 µl of a primary antibody (SC-20641, Santa Cruz Biotechnology) against MYH3 diluted in a 1:500 ratio was added and reacted at room temperature for 1 hour. Then, washed three times with a phosphate buffer solution (1X PBS), and added 100 µl of a secondary antibody (Goat anti-Rabbit IgG-HRP) diluted in a 1:5,000 ratio, and reacted at room temperature for 1 hour. After 1 hour, in order to stain the cell nuclei, the cells were washed 3 times with a phosphate buffer solution (1X PBS), treated with a DAPI staining reagent diluted in the blocking buffer, and then reacted at room temperature for 10 minutes. Thereafter, the phosphate buffer solution (1X PBS) was washed three times, and the washed cover glass was subjected to comparative analysis by measuring absorbance at 450 nm wavelength and taking a fluorescence photograph using a fluorescence microscope. The staining area of the anti-MyHC antibody was analyzed using imageJ (Java-based image processing program).

As a result, it was confirmed that myotubes treated with insulin and alberine citrate were more differentiated into myotubes than myoblasts treated with DMSO alone. In particular, as the treatment concentration of alberine citrate increased, the diameter of myotube cells also increased (FIGS. 1 and 2 ).

Example 2. Confirmation of the muscle strength improvement effect of alberine citrate using normal mice

In order to confirm the muscle strength improvement effect of alberine citrate, after administration of alberine citrate to mice, exercise capacity such as grip strength, running, and balance coordination were measured. Muscle mass was measured after measuring exercise capacity. Specifically, a 10-week-old C57BL/6 male mouse was given alberine citrate once a day for 4 weeks at 17 mg/kg/day (AC-L), 50 mg/kg/day (AC-M) or 150 mg/day. 200 µl per mouse was orally administered at a concentration of kg/day (AC-H), and this was set as an experimental group (Fig. 3). C57BL/6 male mice, 10 weeks old, were purchased from Daehan Biolink.

Example 2.1. Grip measurement

The grip strength was measured using a grip strength meter for mice (bioseb, USA). Specifically, the mouse was placed on the wire mesh connected to the instrument panel to observe the strength of the grip force, and the strength of the mouse gripping the wire mesh was measured while pulling it down by grabbing the tail. At this time, the measurement was repeated five times in succession, and the average value of the five measurement values was calculated.

As a result, the grip strength of the mice in the experimental group was all measured higher than that of the mice in the normal group. Through this, it was confirmed that alberine citrate is effective in improving muscle strength (FIG. 4).

Example 2.2. Measuring running ability

Running ability was measured using a self-produced Treadmill. Specifically, first, an electrical stimulation flowed at the starting point, so that a feeling of rejection was felt. It was acclimated for 10 minutes at a speed of 8 m/min before measurement. After each group of mice was placed in an isolated lane and allowed to run, the time until the mice were tired was measured. The determination of whether the mouse was exhausted was determined that the mouse was exhausted and the time was recorded when the time to stay outside the lane without running for more than 10 seconds elapsed. This experiment could not be repeated experiments on the same mouse. The mouse was placed on a trade mill and started at a speed of 8 rpm, accelerated by 2 rpm every 10 minutes, and run at a maximum of 20 rpm. In addition, the inclination was increased by 5 degrees after 30 minutes, starting with no inclination.

As a result, the running time of the mice in the experimental group increased more than that of the mice in the normal group, and in particular, the running time in the experimental group treated with alberine citrate at a concentration of 150 mg/kg/day significantly increased (FIG. 5).

Example 2.3. Measurement of balance control ability

The balance control ability was measured through the Rota-Rod Test. The rotor rod device consists of four test zones with a shaft diameter of 3 cm and a lane width of 9 cm, and consists of five rotatable circular partitions with a diameter of 60 cm and a circular rod. The measurement was started with a rotation speed of 10 rpm and accelerated until reaching a speed of up to 40 rpm for 5 minutes, and the time remaining in the rota rod device without the mouse hanging from the circular rod was measured. After 15 minutes of rest, the process of measuring again was repeated three times to calculate the average value of the measured values.

As a result, the time that the mice in the experimental group were hung on the circular rods increased compared to the time in which the mice in the normal group were hung on the circular rods. In particular, as the concentration of alberine citrate treatment increased, the experimental mice were suspended from the circular rods. The time there was also increased proportionally (Fig. 6).

Example 2.4. Muscle mass measurement

In order to measure muscle mass, the gastrocnemius (GA) and tibialis anterior (TA) muscles of the mouse hind legs of each group were excised and the weight was measured. As a result, the muscle mass of the spleen and tibialis anterior muscle of the mice in the experimental group was increased more than that of the normal mice, and in particular, the muscle mass of the spleen and tibialis anterior muscle of the mice in the experimental group increased as the concentration of alberine citrate treatment increased (Fig. 7). .

Example 3. Confirmation of muscle strength improvement effect of alberine citrate using sarcopenia-induced mice

In order to confirm the muscle strength improvement effect of alberine citrate, after administration of alberine citrate to mice inducing sarcopenia, the running ability was measured. After measuring running ability, muscle mass was measured. Specifically, the hind legs of the 10-week-old C57BL/6 male mice were fixed using a surgical stapler (Autosuture Royal 35W stapler). After 5 days, the stapler was removed and a 3-day recovery period was taken. Then, the running ability was measured. Alberine was orally administered at a concentration of 17 mg/kg/day (AC-L), 50 mg/kg/day (AC-M) or 150 mg/kg/day (AC-H) 200 µl per mouse, This was set as an experimental group. A sarcopenia-induced mouse that was not treated with anything was set as a control group (FIG. 8). C57BL/6 male mice, 10 weeks old, were purchased from Daehan Biolink.

Example 3.1. Measuring running ability

Running ability was measured in the same manner as in Example 2.2. As a result, in the case of the control mice, the running time was reduced by about 10 minutes compared to the normal mice. On the other hand, the running time of the mice in the experimental group increased in proportion to the treatment concentration of alberine citrate, and in particular, the running time of the experimental group in which alberine at a dose of 150 mg/kg/day (AC-H) was administered orally compared to the normal mice. This increased (Fig. 9).

Example 3.2. Muscle mass measurement

In order to measure muscle mass, the spleen muscle (GA) and tibialis anterior muscle (TA) of the hind limbs of each group of mice were excised and the weight was measured. As a result, in the control group, the weight of the splenic muscle and tibialis anterior muscle significantly decreased compared to the normal group. However, in the case of the experimental group, as the concentration of the treatment with alberine citrate increased, the muscle mass of the spleen and tibialis anterior muscles of the mice in the experimental group also increased (FIG. 10).

Example 4. Confirmation of the effect of promoting differentiation of myoblasts of 4-hydroxy alberine

To confirm the differentiation promoting effect of 4-hydroxy alberine in myoblasts, DMSO, insulin (1.72 μM), alberine citrate (0.01 μM, 0.1 μM, 1 μM or 10 μM) in the same manner as in Example 1. , 4-hydroxy alberine (0.01 μM, 0.1 μM, 1 μM, or 10 μM) was treated, respectively, and then differentiation into myotubes and changes in thickness and diameter of myotubes were observed. At this time, the alberine and 4-hydroxy alberine are shown in Table 1 below.

[Table 1]

As a result, it was confirmed that myotubes treated with alberine citrate or 4-hydroxy alberine were more differentiated into myotubes than those treated with DMSO or insulin (FIGS. 11 and 12 ). 4-hydroxy alberine was superior to alberine in promoting differentiation at the same concentration (Fig. 12).

Example 5. Confirmation of cachexia treatment effect of alberine citrate and 4-hydroxy alberine

In order to confirm the effect of alberine citrate and 4-hydroxy alberine on cachexia, an in vitro approach was performed with a cancer cell conditioned medium (CM) to mimic the environment in which the tumor was created. First, a cancer cell culture solution was prepared using a C26 cancer cell line. At this time, RPMI1640 medium to which 10% fetal bovine serum was added was used as a growth media (GM) for cancer cell culture. The C26 cancer cell line was cultured using a 100 mm cell culture plate and the cancer cell culture medium. At this time, the medium was removed when the cell confluency reached 90%. Then, after washing with a phosphate buffer solution (1X PBS), the differentiation medium prepared in the same manner as in Example 1 was treated. After 24 hours, CM (Conditioned medium), which is a C26 cancer cell line culture solution, was obtained, and was used after filtering with Bottle Top Filters (Thermo, PES, 1L).

5 × 10 ⁵ C2C12 cells per plate were dispensed into a 6-well-plate treated with the culture medium of Example 1.1, and after 24 hours, 5% horse serum was added to the DMEM medium to induce differentiation. I did. After 4 days, the differentiated myotube cells were treated with a differentiation medium containing 33% CM, and myotube cell atrophy was induced for 3 days. At this time, the experimental group was treated with alberine citrate (AC) and 4-hydroxy alverine (4HA) at a concentration of 1 μM to evaluate the effect of inhibiting atrophy of root canal cells. DMSO was used as a negative control, and ursolic acid (UA) was used as a positive control. In the same manner as in Example 1.2, the expression level of the myosin heavy chain was measured through fluorescence staining using an antibody against the myosin H chain (hereinafter, MyHC).

As a result, it was confirmed that the root canal cells of the negative control group were atrophy by CM. On the other hand, it was confirmed that treatment with alberine citrate or 4-hydroxy alberine inhibited atrophy of root canal cells by CM (FIGS. 13 and 14 ).

Claims (10)

As a pharmaceutical composition for preventing or treating diseases related to muscle weakness, comprising alberine, 4-hydroxy alverine, or a pharmaceutically acceptable salt thereof,
The muscular weakness-related disease is sarcopenia, muscular atrophy, muscular dystrophy, or cachexia. A pharmaceutical composition for preventing or treating muscular weakness-related diseases. delete The method of claim 1,
The pharmaceutical composition for preventing or treating diseases related to muscle weakness, wherein the pharmaceutically acceptable salt is citrate. A composition for promoting differentiation of myoblasts in vitro comprising alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof. The method of claim 4,
The alberine, 4-hydroxy alberine or a pharmaceutically acceptable salt thereof has a concentration of 0.01 μM to 100 μM, a composition for promoting differentiation of myoblasts Alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof, a method for promoting differentiation of myoblasts comprising the step of treating myoblasts in vitro. Alberine, 4-hydroxy alberine, or a pharmaceutically acceptable salt thereof is treated with myoblasts in vitro to differentiate myoblasts. As a food composition for preventing or improving muscle weakness-related diseases comprising alberine, 4-hydroxy alberine or a food pharmaceutically acceptable salt thereof,
The muscle weakness-related disease is sarcopenia, muscular atrophy, muscular dystrophy, or cachexia. delete Alberine, 4-hydroxy alberine, a pharmaceutically acceptable salt thereof or a composition for strengthening muscle strength comprising a food acceptable salt.

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