KR101725327B1 - Pharmaceutical compositions for Preventing or Treating Muscle Atrophy Diseases Comprising Bakuchiol - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing and treating muscle atrophy, comprising as an active ingredient bakuchiol derived from bovine bone. The pharmaceutical composition is effective for promoting muscle cell regeneration and inducing differentiation into muscle cells.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating muscle atrophy,

The present invention relates to a pharmaceutical composition for the prevention or treatment of Muscle Atrophy. More specifically, the present invention relates to a composition for promoting the differentiation of MyoD-mediated myoblasts and activating muscle regeneration ability through activation of the p38 MAPK signaling pathway The present invention relates to a pharmaceutical composition containing Bakuchiol as an active ingredient for treating muscle atrophy.

The present invention also relates to a functional food for the prevention or amelioration of Muscle Atrophy, and more particularly to a method for promoting the differentiation of MyoD-mediated myoblasts through activation of the p38 MAPK signaling pathway, Functional food containing Bakuchiol as an active ingredient to treat muscle atrophy.

Diseases associated with muscle disorders are muscle loss and related diseases such as muscle hypoxia, cachexia, muscle damage, muscular dystrophy and muscle fatigue. Treatment of muscle disorders includes maintenance of muscle performance, strength and muscle function.

Muscle loss is characterized by gradual loss of muscle mass, especially weakness and degeneration of the skeletal muscle or the vasculature and cardiac muscle. The process by which atrophy and hyperplasia occur occurs in all mammalian species. Various studies have demonstrated that the same molecular mechanisms, cellular and physiological processes are identical in atrophy of both rodents and humans.

Muscle loss is caused by a variety of causes and is associated with various pathologies, diseases and diseases. These include muscular dystrophy caused by gene abnormalities, such as Duchenne Muscular Dystrophy (DMD), progressive muscular dystrophy, Becker muscular dystrophy, Dejerine-Landouzy, But are not limited to, muscular dystrophy, Erb's muscular dystrophy, spinal muscular atrophy and infantile nerve axonal atrophy.

Muscle loss can also be caused by a variety of chronic diseases and aging processes. As the body ages, some of the newly generated skeletal muscle is replaced with fibrous tissue. Thus, normal human aging is associated with decreased progression of skeletal muscle mass and intensity, a condition called muscle hypoxia, which causes attenuation and degeneration.

In addition, age-related diseases such as hypertension, impaired glucose tolerance and diabetes, obesity, dyslipidemia, atherosclerosis and cardiovascular disease are also associated with loss of muscle mass.

In addition, the present invention also relates to a pharmaceutical composition for the treatment of cancer, autoimmune disease, infectious disease, HIV infection, AIDS, chronic inflammation, arthritis, malnutrition, kidney disease, COPD, emphysema, rickets, chronic lower back pain, , Chemical damage, and central nervous system (CNS) damage can also be associated with muscle loss or muscle loss. Finally, conditions that cause muscle loss can result from lung conditions such as organ fixation due to disease, or from inability such as long-term wheelchair use, long-term bed rest, fracture or trauma. Post-operative bed care is estimated to cause a loss of skeletal muscle mass of about 10% per week.

Untreated muscle loss can lead to serious health problems. Changes that occur at the time of muscle loss can cause a weakened physical condition, resulting in incompetent physical performance and harmful health conditions.

Thus, myopathy can seriously limit the return of the patient to society. Muscle loss due to chronic disease can lead to premature loss of motility and increase the risk of disease-related mortality. Muscle loss due to lungs is a particularly serious problem in older people who may already have age related deficits in muscle function and muscle mass, leading to permanent disability and premature death as well as increased fracture rates. Despite the clinical significance of the condition, there are few therapies to prevent or reverse the condition, and a solution is needed.

KR Publication No. 2013-0125411 (published by Unigen, Inc., Nov. 18, 2013) JP Publication No. 2005-32512 (SINPHAR PHARMACEUTICAL CO LTD, Nov. 24, 2005)

Accordingly, the present inventors have developed a pharmaceutical composition for the prevention or treatment of Muscle Atrophy. More specifically, the inventors of the present invention have identified the phenomenon that Bakuchiol promotes the differentiation of MyoD-mediated source cells through activation of the p38 MAPK signaling pathway Thus completing the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising Bakuchiol or a pharmaceutically acceptable salt thereof as an active ingredient for enhancing muscle regeneration ability to prevent or treat muscular atrophy.

It is another object of the present invention to provide a pharmaceutical composition or functional food for preventing or treating muscular atrophy.

It is still another object of the present invention to provide a therapeutic agent for muscle regeneration for damaged muscles.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating muscular atrophy, which comprises as an active ingredient bakuchiol or a pharmaceutically acceptable salt thereof.

The muscular atrophy may also include aging-related muscular hypoxia, degenerative muscle hypoxia, and diabetic myopathy.

The muscular atrophy may also include, for example, Parkinson's disease, Becker's muscular dystrophy, Ducement's muscular dystrophy, myotonic muscular dystrophy, Emery-Dreypuss's muscular dystrophy, Wynne's muscular dystrophy, facial diplopia, and congenital muscular dystrophy.

The present invention provides a pharmaceutical composition for inducing muscle cell differentiation or regeneration, which comprises bakuchiol or a pharmaceutically acceptable salt thereof as an active ingredient for solving the above problems.

The muscle cell differentiation and regeneration induction may be characterized by controlling the activity of myoD, a muscle transcription factor, to differentiate root canal cells into root canals or fibroblasts into myo cells.

The present invention provides a health functional food for prevention or amelioration of muscle atrophy containing bakuchiol or a pharmaceutically acceptable salt thereof.

The bakuchiol may be characterized in that it is obtained from a bovine seed extract.

The extract may be characterized in that it is obtained by using water, a C ₁ -C ₄ lower alcohol or a mixed solvent thereof.

The pharmaceutical composition or functional food for preventing or ameliorating muscle atrophy comprising as an active ingredient the bovine origin derived from the bovine bone of the present invention is derived from a bovine seed extract and is characterized in that the myoin, myoginin and myosin heavy chain heavy chain, MHC), promotes differentiation of muscle cells into muscle cells by regulating muscle differentiation activity, transforms embryonic fibroblasts into source cells, transforms-differentiates Promotes muscle cell regeneration, and promotes multinucleated muscle fiber formation without cell death, thereby treating, preventing, or ameliorating muscle atrophy and myopathy.

FIG. 1 shows the activity of inducing the expression of muscle specific marker protein (MHC) by the ethanol extract of bovine bone and its ethyl acetate fraction.
Fig. 2 is a chemical structural formula of Bakuchiol derived from bovine (Fig. 2-A), and the expression of muscle-specific proteins including Cdo, MHC, MyoD and myogenin in the bakuchiol treated C2C12 cells 2-B). The result of immunostaining with anti-MHC antibody and DAPI (Fig. 2-C) to confirm the degree of promoting the formation of myotube in C2C12 cells, The cells are reduced and the result is the increase of 6 or more polynuclear canaliculus cells (Fig. 2-D).
FIG. 3 is a graph showing the increase in the phosphorylation level of p38 MAPK (p-p38) (FIG. 3-A) in bakuchiol treatment and the SB203580 or Baku (Fig. 3-B) in which the expression of MHC, MyoD, and myogenin, the muscle-specific proteins, was decreased during the treatment of cholin.
FIG. 4 shows the recovery of muscle differentiation in Cdo-deficient C2C12 cells. C2C12 cells transformed with pSuper and Cdo shRNA (shCdo) expression vectors were treated with Bakuchiol and DMSO, respectively, and anti-MHC antibody (Fig. 4-A), the result of incompletely formed endodontic fibers recovered by treatment with Bakuchiol in C2C12 / shCdo cells (Fig. 4-B), and the result of Bakuchiol (Fig. 4-C), indicating that the level of phosphorylation of p38 MAPK was restored when treated with Bakuchiol in C2C12 / shCdo cells compared with the group treated with DMSO 4-D, Fig. 4-E).
FIG. 5 is a graph showing the effect of Bakuchiol on dysplasia and increased transcriptional activity of MyoD in the presence of DMSO or Baku in C2C12 cells (Figure 5-A) and MyoD-transformed 293T cells (Figure 5-B) (Fig. 5-C) as a result of western blotting with MyoD antibody after immune precipitation with E2A antibody after treatment with cholinesterase, and with increasing levels of myogenin and Baf60C in Bakuchiol-treated C2C12 cells as a ChIP assay .
FIG. 6 is a view for examining whether MyoD-transformed 10T1 / 2 fibroblasts are transfected into myoblasts. MyoD-transformed 10T1 / 2 cells were treated with Bakuchiol and then treated with MHC, MyoD, (Fig. 6-A) showing the expression of pan-Cadherin protein and quantifying the results of the experiment of Fig. 6-A (Fig. 6-B).
(Fig. 6-C) showing the degree of production of p-p38 MAPK (pp38) which is a phosphorylation form of p38 MAPK according to whether or not Bakuchiol treatment is performed.
FIG. 7 is a graph showing the effect of Bakuchiol on the regeneration of MyoD-transformed osteosarcoma (Rhabdomyosarcoma: RD) cells into myoblasts. MHC, MyoD, myogenin, p-p38, p38, p21, cyclin D1, (Fig. 7-A) and the result of immunostaining with anti-MHC antibody and DAPI (Fig. 7-B). FIG. 7-C is a Cdo-mediated p38 MAPK activation model induced through differentiation bakchiol treatment during transduction of source cells.

Source cell differentiation is a finely coordinated multistage process involving proliferation of myoblast, reduction of cell cycle, expression of muscle-specific transcriptional activity, cell proliferation and fusion to root canal. This process relies on the myogenic base helix-loop-helix (bHLH) enzyme system, which includes MyoD, myogenin and MEF2.

In particular, MyoD can convert non-myogenic cell types into bone muscle cells and act as a major regulator in tissue-specific transcription. MyoD forms a heterodimer with the E proteins (E12 and E47), binds to the common DNA sequence, and is present in the regulatory region of the muscle gene. MyoD works in conjunction with myocyte enhancer factor 2 (MEF2) to activate muscle-specific gene expression.

p38 mitogen-activated protein kinase (p38MAPK) regulates MyoD function through phosphorylation of several proteins including myoD heterodimeric partner E47 and chromosomal aberrant SWI / SNF subunit Baf60c in muscle-specific gene expression. It regulates myogenic differentiation. The p38MAPK pathway plays an important role in muscle differentiation, especially in cell-to-cell mediated muscle differentiation.

In order to identify potential activators for myocyte differentiation, natural substances for MyoD-reactive reporter activity and expression of the myosin heavy chain (MHC) in the source cells were screened from various medicinal plants, Bakuchiol, a phenolic mono terpene that was prenylated from Psoralea corylifolia seeds, was selected.

The present inventors evaluated the increase of MHC expression in the bovine ethanol extract and the ethyl acetate fraction in order to induce MyoD activity and anticipate the differentiation effect of muscle stem cells (see FIG. 1) Bakuchiol enhanced the expression of myogenic markers, MHC, on myoblast differentiation.

Bakuchiol is known to have antibacterial, anti-inflammatory, anti-cancer, anti-aging, liver protection and immunosuppressive properties. Bakuchiol has the effect of lowering the levels of blood glucose and blood triglycerides and exerts excellent effects in improving osteoblast differentiation.

The present inventors have studied the effects of bakchiol and molecular mechanisms on myoblast differentiation through experiments. As a result, Bakuchiol improves MyoD-mediated muscle differentiation, and can restore the inhibition of Cdo deficiency-induced differentiation through activation of p38MAPK. MyoD-transformed embryonic fibroblasts (10T1 / 2) Muscle differentiation and regeneration of osteosarcoma cells (Rhabdomyosarcoma (RD)). Finally, Bakuchiol can be used to regenerate damaged muscle or to treat muscle atrophy by positively controlling MyoD-mediated source cell differentiation.

Bakuchiol, a natural substance, enhances p38 MAPK-induced MyoD activity while promoting differentiation of source cells and inducing transfection (differentiation) of embryonic fibroblasts into source cells.

Muscle differentiation presents a good model for understanding mechanisms of cell fate identification and differentiation to improve bone muscle regeneration.

MyoD, a major regulator of osteoclast differentiation, coordinates the process of specificiation, differentiation and activation of muscle differentiation in cooperation with the MEF2 family, a transcription factor to activate the expression of muscle-specific genes. In fact, MyoD can form dysplasia with ubiquitous E proteins (E47) and regulate the activity of muscle bHLH transcription factors.

Bakuchiol enhances MyoD expression and promotes heterodimerization of MyoD and E proteins. MyoD, which can form a dysplasia with E protein, activates muscle specific gene expression and at the same time activates the chromosome-degenerating enzyme SWI / SNF subunit Baf60c. And suppresses the expression of non-muscle genes. Bakuchiol increases the transcriptional activity of MyoD, thereby regulating myogenin and Baf60c positively. Bakuchiol-induced MyoD activity is dependent on the activity of p38MAPK. MyoD interacts directly with the BAF60c subunit of the SWI / SNF complex. BAF60 phosphorylation of p38MAPK appears to induce complementation of the Brg1-SWI / SNF complex with subsequent chromatin remodeling, and binds to a regulatory factor that initiates differentiation. This allows Bakuchiol to strongly regulate the transcriptional activity of MyoD, which is responsible for efficient cell differentiation and maintenance of differentiated stages of cells.

p38MAPK positively regulates the activity of MyoD through phosphorylation of the E-protein associated with the partner and promotes heterodimerization of the MyoD and E proteins. Bakuchiol activates p38 MAPK (Fig. 3-A) but does not restore the effect of the p38 MAPK inhibitor (Fig. 3-B). Cdo in the cytoplasm is formed by multiple scaffold proteins, including JLP and Bnip-2, for p38 MAPK, leading to cell contact-mediated signaling from the cell surface to the muscle differentiation control network. In Cdo-deficient C2C12 cells, Bakuchiol restores the phosphorylation level of p38 MAPK (Fig. 4-D) and restores expression of muscle specific proteins including MyoD. Bakuchiol induces Cdo expression and restores Cdo expression in Cdo-deficient cells (Fig. 2-B and Fig. 4-C). Muscle Differentiation Transcription Enzymes result in a positive feedback network that automatically and cross-activates yet another enzyme, resulting in amplification of the muscle differentiation signaling pathway for differentiation. And maintains the muscle differentiation phenotype. Bakuchiol activates the upstream signaling kinase of p38MAPK and activates downstream of the Cdo signaling complex.

MyoD converts various cell types into muscle cells. Bakuchiol can stimulate differentiation of embryonic fibroblasts into myoblasts, which can be activated by up-regulation of p38 MAPK. MyoD-dependent muscle differentiation of fibroblasts requires activation of p38 MAPK

Bakuchiol also induces regeneration of osteosarcoma (RD) cells into canaliculus cells. Osteosarcoma (RD) cells express muscle bHLH factors such as MyoD, myogenin and MEF2. However, these myogenic regulatory factors (MRFs) can not induce differentiation into bone muscle cells. MyoD can bind DNA to the same DNA binding site of MyoD as a heterodimer with E protein, but can not be effectively transcribed in RD cells. Activity inhibition of MyoD occurs through binding with bHLH inhibitory protein. The inhibitory effect is reversible due to increased heterozygosity of MyoD and E protein, which can convert RD cells into myoblast differentiation stage. Experiments show that when transduced from RD cells to MyoD, bakuchiol can enhance the expression of muscle-specific proteins and promote the formation of MHC-positive canaliculus cells. Besides. In Puri et al, p38MAPK can restore RD cell differentiation in response to MKK6, and inhibits anti-muscle differentiation function of Pax7, thereby promoting differentiation of myoblast cells. Bakuchiol upregulates the activity of p38 MAPK or its upstream to promote differentiation of source cells and induces trans-differentiation of embryonic fibroblasts (Figs. 3, 6, 7).

Replacing damaged muscle tissue with healthy muscle cells provides a potentially effective treatment for a variety of degenerative muscle disorders, including muscle atrophy. And direct conversion of fibroblasts into muscle cells by MyoD transduction can support muscle regeneration. In this respect, bakchiol has the potential as a useful pharmaceutical candidate to reduce tumorigenesis and convert it into myoblast differentiation.

Muscular atrophy may include aggressive muscle weakness, degenerative muscle weakness, and diabetic muscle weakness.

Myocardial atrophy is also known to be associated with myocardial infarction, including Becker Muscular Dystrophy (BMD), Duchenne Muscular Dystrophy (DMD), Myotonic Muscular Dystrophy, Emery-Dreifuss May include Muscular Dystrophy (EDMD), Distal Muscular Dystrophy (DD), Facioscapulohumeral Muscular Dystrophy (FSHD), and Congenital Muscular Dystrophy (CMD).

According to the present invention, a pharmaceutical composition for promoting differentiation or regeneration of muscle cells containing bakchiol derived from a bovine bone as an active ingredient can be prepared.

The muscle cell differentiation may be differentiation into a source cell and a muscle cell such as a source cell or a fibroblast differentiable into a muscle cell.

Promoting differentiation into the muscle cells may be by modulating the activity of the MyoD transcription factor.

The muscle cell regeneration may be regeneration of damaged muscle cells. Such damage may be due to genetic factors such as surgery, cancer, inflammation, infection, and environmental factors.

According to an embodiment of the present invention, the composition for treating or preventing muscle atrophy may be prepared from a pharmaceutical composition. While it is possible to administer the present invention becuchiol itself for use in therapy and prophylaxis, it is preferred that the abovementioned bakuciol is included as the active ingredient of the pharmaceutical composition.

The pharmaceutical composition may contain a pharmaceutically acceptable salt of bakuchiol as an active ingredient.

The bakuciol according to the present invention can be prepared as a corresponding salt by a known method. Such salts are preferably water-soluble without toxicity. Preferred salts include salts of alkali metals such as potassium, sodium and the like; Salts of alkaline earth metals such as calcium, magnesium and the like; There may be mentioned amine compounds such as choline, diethanolamine, ethanolamine, ethyldiamine, meglumine, tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, di Salts of pharmaceutically acceptable amines such as ethanolamine, tris (hydroxymethyl) amine, lysine, arginine, N-methyl-D-glucamine and the like.

Further, the pharmaceutical composition contains the above-mentioned vanutiol as an active ingredient, and may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed.

It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants.

Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.

The method of administering the pharmaceutical composition of the present invention is not particularly limited, but it may be parenterally or orally administered intravenously, subcutaneously, intraperitoneally, by inhalation, skin application or topical application according to the intended method.

The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

The daily dose refers to the amount of the therapeutic substance of the present invention which is sufficient to treat a relieved disease state by being administered to an individual in need of treatment. The effective amount of the therapeutic substance will depend on the particular compound, the disease state and its severity, the individual in need of treatment, which can be routinely determined by one of ordinary skill in the art. As a non-limiting example, the dosage for the human body of the composition according to the present invention may vary depending on the age, weight, sex, dosage form, health condition and disease severity of the patient and is based on adult patients weighing 70 kg , It is generally 0.01 to 1000 mg / day, preferably 1 to 500 mg / day, and may be dividedly administered once to several times a day at predetermined time intervals.

According to one embodiment of the present invention, the composition for preventing or ameliorating atrophy of muscles may be manufactured from a food composition. When the composition of the present invention is prepared with a food composition, it contains not only the above-mentioned bakuciol as an active ingredient but also a component that is ordinarily added during the production of a food. Examples thereof include proteins, carbohydrates, fats, And flavoring agents. For example, when the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, etc. in addition to the bakuchiol of the present invention.

The bakuciol may be characterized in that it is obtained from a bovine extract. The extract may be obtained using water, a C ₁ -C ₄ lower alcohol, or a mixed solvent thereof.

The bogolium extract is not only contained in the leached liquid obtained by leaching and transferring from the bogolin but also the concentrate obtained by partially or entirely concentrating the leachate or the stagnant, premix, periodic, fluid extract and bogolin prepared by drying the concentrate again It includes both the chemical itself as well as the chemical itself, which is the main effect. In the present invention, extracts from all parts of mulberry trees such as stem, root, leaf, flower, and fruit can be used, but more preferably, seed extract can be used.

A known method can be used as a method for producing the extract of the present invention.

For example, the bogie is dried by any method such as natural drying or forced drying, and then finely pulverized. Thereafter, a polar solvent such as water, ethanol, butanol or acetone; Non-polar solvents such as ether, hexane, benzene, chloroform, and ethyl acetate; A mixed solvent of the non-polar solvent and the polar solvent; Alkaline water or vegetable oil such as soybean oil or sesame oil and the like and leached by any method such as cold water treatment, percolation, warming, etc. to obtain an extract containing an effective ingredient.

The extract may be suspended in water and sequentially fractionated using n-hexane, ethyl acetate, chloroform, and butanol to obtain each fraction, and then the extract containing the bakuchiol can be isolated.

Hereinafter, embodiments are described to help understand the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

1. Example

(1) Separation of Bakuchiol

540g of bovine bone, purchased from Kyungdong market, was extracted with 3 times for 2 hours while refluxing with ethanol, and then concentrated under reduced pressure to obtain an alcoholic extract (130g). This was dispersed in water and then extracted with hexane to separate the hexane-soluble fraction (28 g), followed by extraction with ethyl acetate to obtain an ethyl acetate-soluble fraction (53 g). The ethyl acetate fractions were separated into ten fractions with a n-hexane / ethyl acetate gradient elution system (50: 1 - > 51: 1). The third fraction was further subjected to silica gel column chromatography gradient elution with hexane / acetone (50: 1 to 3: 1) to separate 2.1 g of pure bakchiol.

(2) Cell culture

The source cells C2C12, embryonic fibroblast 10T1 / 2 cell line and embryonic kidney 293T cells were cultured in a conventional manner. C2C12 origin cells containing 2% HS (differentiation medium, DM ) in DMEM containing the cell line near the fusion point to induce the differentiation 37, 15% FBS (growth medium , GM) in a CO ₂ incubator (myoblast) , And myotube differentiation was observed for 48 to 72 hours. The efficiency of myotube formation was quantified using a transient differentiation assay.

To generate C2C12 cells transiently overexpressing shRNA corresponding to Cdo, the cells were each transformed with the indicated expression vector and lipofectamine 2000, and the culture medium was selected from the puromycin containing medium. In experiments involving p38MAPK inhibitors, C2C12 cells were treated with 500 nM Bakuchiol in a fresh culture medium with 2.5 μM SB203580 for 30 min and the elderly after challenge. For trans-differentiation studies, 10T1 / 2 cell lines and RD cells were cultured in DMEM containing 10% FBS in a 100 mm culture dish and transformed with 5 mu g MyoD or a control vector (pBp). After 24-48 hr of incubation, 10T1 / 2 cell lines and RD cells were treated with 500 nM Bakuchiol at 2% HS for 2 days.

(3) Western blot and immunoprecipitation

Cell lines were treated with extraction buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl ₂ , 1 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na ₃ VO ₄ , complete protease inhibitor cocktail) And SDS PAGE was performed. The primary antibodies include anti-CDO, anti-pan-cadherin, anti-p38, anti-p-p38 (active phosphorylated form of p38MAPK), anti-MHC, anti-myogeinin, troponin- T) and anti-MyD were used.

For immunoprecipitation analysis, C2C12 or 293T cells were transformed into MyoD for 36 hours. After 24 hours of treatment with Bakuchiol, the whole cell extract was incubated with 4 Overnite with anti-E2A and protein G agarose beads (Roche Diagnostics). The beads were washed three times with extraction buffer and resuspended in extraction buffer. The samples were analyzed by western blotting.

(4) Immunocytochemistry and microscopy

The C2C12 cell line was fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton X-100 in phosphate buffered saline, sealed and stained with anti-MHC. And then treated with Alexa Fluor 568-conjugated secondary antibody (Molecular Probes). Images were taken and processed with a Nikon ECLIPSE TE-2000U microscope, NIS-Elements F software (Nikon), and Photoshop CS5 (Adobe).

(5) Chromatin immunoprecipitation ChiP assa y

Chromatin immunoprecipitation assays were performed on c2c12 cells transfected with the pSuper, GST-tagged PRK2, and PRK2 shRNA vectors using the EZ-ChIP kit according to the manufacturer's instructions. Quantitative PCR analysis was performed on immunoprecipitated DNA and normalized to whole chromatin input or whole histone H3. The following primers were used.

Miyogenerin: 5'-GAATCACATGTAATCCACTGGA-3 ',

After amplification, the PCR products were separated on 1% agarose gel and visualized by bromide etidium.

2. Experimental Example

(1) Bakuchiol to promote myoblast differentiation

To identify potential activators of MyoD-mediated muscle regeneration, we screened natural substances from various medicinal plants and performed Western blot and MyoD-reporter assays using anti-MHC antibodies on C2C12 cells

Bakuchiol isolated from P. corylifoliae among candidate materials showed consistent results from both screening experiments (Fig. 2-A).

The ethanol extract of bakuchiol isolated from the bogolium increased the expression of MHC in a dose - dependent manner (up to 1000ng / ml), and the content of bakuciol in the extract was 17.2%. In the ethyl acetate fraction, MHC expression increased from 1 to 10 ng / mL, but decreased at concentrations above 100 ng / mL. At this time, the content of bakuchiol in the fraction was 61.6%. (Fig. 1)

Since myofibroblasts, osteoblasts and adipocytes were derived from the mesodermal system in embryonic development, the inventors studied the mechanism of bakchiol in myoD-mediated muscle differentiation of mesodermal-derived source cells. The present inventors first ascertained whether or not bakuciol stimulates the differentiation of myofibers by increasing the expression of muscle-specific proteins. Bakuchiol (10-500 nM) at various concentrations was treated and induced differentiation of C2C12 myocytes for 2 days. As a result of Annexin-V / PI staining, Bakuchiol did not induce severe cytotoxic effects in both growth and differentiation conditions of C2C12 myocytes. Bakuchiol treatment on C2C12 cells increased the expression of muscle-specific proteins including MHC, MyoD, and myogenin in a dose-related manner, and Bakuchiol treatment increased the expression of promyogenic receptors in C2C12 cells The level was increased sharply.

In order to investigate the effect of bakuchiol in the induction of root canal cell formation, C2C12 cells treated with DMSO and Bakuchiol were induced to differentiate for 2 days. Immunohistochemical staining for anti-MHC antibody and DAPI staining Lt; / RTI > In FIG. 2-C, C2C12 cells treated with Bakuchiol had relatively more nuclei and longer canaliculus cells than C2C12 cells treated with DMSO. MHC-positive cells were counted as cells with 2 to 5 nuclei, cells with 6 or more nuclei, or mononuclear cells. Finally, Bakuchiol treatment increased the specific gravity of canaliculus cells with more than 6 nuclei, and decreased the specific gravity of mononuclear cells, depending on the dose.

(2) Bakuchiol to activate p38MAPK in myoblast differentiation

The p38 MAPK signaling pathway plays an important role in osteogenic differentiation. To investigate the effect of bakuchiol on the promyogenic signaling pathway, in particular p38MARK, C2C12 cells were treated with various amounts of bakchiol and the level of phosphorylation of p38MARK was determined by Western blot analysis. Bakuchiol treatment dramatically increased the level of phosphorylated p38 MAPK depending on the dose and the level of expression of the whole protein remained relatively constant (Fig. 3-A). The present inventors examined whether bakuchiol-mediated muscle differentiation is necessary for p38 MAPK activation. For this, C2C12 cells were treated with the pharmaceutical inhibitor SB205380 for 30 min before treatment with Bakuchiol, and differentiated for 48 h. This was analyzed by Western blot. SB203580 treatment reduced the degree of phosphorylation of p38MAPK (Fig. 3-A) and inhibited the expression of muscle specific proteins MHC, MyoD and myogenin (Fig. 3-B). In addition, Bakuchiol treatment did not restore the activation state of p38 MAPK and did not restore the expression of muscle specific protein in p38 MAPK-deficient C2C12 cells induced by treatment with SB203580. These results indicate that the promyogenic activity of Bakuchiol stimulates the differentiation of myoblasts and is dependent on the activity of p38MAPK.

(3) In Cdo-deficient C2C12 cells Bakuchiol to restore myoblast differentiation

Cdo partially regulates cell-to-cell adhesion mediated muscle differentiation signaling through p38 MAPK activation. Bakuchiol increases the expression of Cdo and promotes differentiation of myocytes through activation of p38MAPK. Therefore, the present inventors have studied whether the incomplete differentiation can be restored by Bakuchiol due to Cdo-deficient source cells. To test this, C2C12 cells were transiently transfected with pSuper and Cdo shRNA (shCdo) expression vectors. C2C12 / pSuper and C2C12 / shCdo cells were treated with DMSO or Bakuchiol and induced differentiation for 2 days. Followed by immunostaining with anti-MHC antibody and DAPI. As a result, bakuchiol consistently increased the production of C2C12 / pSuper cells and canaliculus cells and increased the specific gravity of macrocystocytes with more than 6 nuclei when compared to DMSO-treated C2C12 / pSuper cells (Fig. 4-A And 4-B).

DMSO treatment of C2C12 / shCdo cells resulted in incomplete root canal production, but was restored to similar level to that of control cells due to the treatment with Bakuchiol in C2C12 / shCdo cells. Western blot analysis showed that the expression of muscle specific protein was potentially reduced in C2C12 / shCdo cells. As with the MHC staining results, Bakuchiol treatment in C2C12 / shCdo cells restored the expression levels of Cdo and muscle specific proteins when compared to control cells (Fig. 4-C).

Furthermore, the present inventors have studied whether bakuchiol treatment can restore p38 MAPK activity in C2C12 / shCdo cells. As seen in Figures 4-D and 4-E, DMSO treatment of C2C12 / shCdo reduces the phosphorylation level of p38MAPK and is restored to a similar level by bakchiol treatment in C2C12 / shCdo cells. These results indicate that Bakuchiol affects Cdo expression, promotes muscle differentiation, and is dependent on the Cdo-mediated p38 MAPK signaling pathway.

(4) Bakuchiol, which upregulates the transcription of the source gene of interest through MyoD

Cdo activates p38 MAPK in muscle differentiation, resulting in increased heterodimerization of MyoD with E protein. In order to investigate whether Bakuchiol treatment promotes heterozygoticization of E protein and MyoD in myoblast differentiation, C2C12 cells were treated with Bakuchiol to induce differentiation and harvested 2 days later. The cells were then immunoprecipitated with E47 antibody and immunoblotted with MyoD.

As seen in Figure 5-A, Bakuchiol treatment increased the binding of endogenous E47 protein and MyoD gradually as compared to control cells. The level of E47 protein remained constant in the disruption solution and the level of MyoD expression increased in C2C12 cells treated with bakuchiol. Control rabbit IgG was not detected under both conditions. Next, the present inventors analyzed whether exogenous overexpression of MyoD and E47 protein interact with each other in bakuchiol-treated 293T cells. Like endogenous interactions, Bakuchiol treatment increased MyoD binding to the E47 protein (Figure 5-B). This data suggests that becuchiol increases the heterodimerization of MyoD and E proteins through activation of the Cdo-mediated p38 MAPK pathway.

In order to examine whether MycoD transcription activity of Bakuchiol was regulated, we performed Chromatin immunoprecipitation (ChIP) analysis. As shown in FIG. 5-C, C2C12 cells treated with Bakuchiol increased the transcription levels of myogenin and Baf60C, indicating that Bakuchiol positively regulates the transcriptional activity of MyoD.

(5) Bakuchiol induces trans-differentiation of embryonic fibroblasts and induces regeneration of RD cells into myoblasts

 Using 10T1 / 2 fetal embryonic fibroblasts, the present inventors have studied whether treatment with Bakuchiol can promote 10T1 / 2 fibroblasts to MyoD-mediated trans-differentiation into myoblasts. 10T1 / 2 cells were transfected with pBp (pBabe-puro) and pBp / MyoD expression vectors and differentiated for 2 days after treatment with DMSO or Bakuchiol. And Western blot analysis. As shown in Fig. 6-A, regardless of the treatment with Bakuchiol, no expression of muscle-specific protein was observed in pBp-transformed 10T1 / 2 cells. Conversely, pBp / MyoD-transduced 10T1 / 2 cells expressed muscle specific proteins such as MyoD, myogenin and MHC. When treated with Bakuchiol in pBp / MyoD-transformed 10T1 / 2 cells, the expression levels of MHC and myogenin were significantly increased and the expression of MyoD was gradually increased (Fig. 6- A and Fig. 6-B).

Next, the present inventors examined whether p38 MAPK activation is involved in this activity. Transo-differentiation of 10T1 / 2 cells increased the level of p-p38 due to MyoD transformation, and treatment with Bakuchiol of pBp / MyoD-transformed 10T1 / 2 cells further increased the level of p-p38 C and 6-D). These results indicate that Bakuchiol increases the activity of MyoD through activation of p38 MAPK, and as a result, Bakuchiol induces trans-differentiation in which fibroblasts of the embryo become source cells.

Rhabdomyosarcoma (RMS) arises from precursor cells that fail to differentiate into normal muscle. RD cells were transiently transfected with pBp (pBabe-puro) and pBp / MyoD expression vectors to determine if bakuchiol treatment could convert RD cells into specialized musculoskeletal cells. And then treated with DMSO or Bakuchiol and subjected to Western blot analysis. The expression of MHC (hallmark of differentiated cell) was rapidly stimulated when treated with Bakuchiol in pBp / MyoD-transformed RD cells, and the expression of MyoD was increased in comparison with DMSO-treated cells (Fig. 7-A) Respectively. As with these results, polynuclear MHC-positive cells were increased in the paclitaxel-treated pBp / MyoD-transformed RD.

Claims (9)

A pharmaceutical composition for prevention and treatment of muscle atrophy, which comprises as an active ingredient bakuchiol.
The method according to claim 1, wherein the muscular atrophy is selected from the group consisting of aging-related muscular dysgenesis, degenerative muscular dysgenesis, diabetic muscular dysgenesis, geckomandibular dystrophy, Becker Muscular Dystrophy (BMD), Duchenne Muscular Dystrophy Myotonic Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy (EDMD), Distal Muscular Dystrophy (DD), Facioscapulohumeral Muscular Dystrophy (FSHD) or Congenital Muscular Dystrophy Muscular Dystrophy, CMD) for the treatment and prevention of muscle atrophy.
delete The pharmaceutical composition for prevention and treatment of muscle atrophy according to claim 1, wherein the composition for inducing differentiation of root cells into root canal cells or inducing differentiation of fibroblasts into root cells, which regulates the activity of MyoD, A pharmaceutical composition for preventing and treating fibrosis.
delete The pharmaceutical composition according to claim 1, wherein the bakuciol is obtained from a bovine extract obtained by using water, a C ₁ -C ₄ lower alcohol or a mixed solvent thereof.
A health functional food for prevention and improvement of muscle atrophy, which comprises as an active ingredient bakuchiol or a pharmaceutically acceptable salt thereof.
delete The health functional food for preventing and improving muscular atrophy according to claim 7, wherein the bakuchiol is obtained from a bovine extract obtained by using water, a C ₁ -C ₄ lower alcohol or a mixed solvent thereof.

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