KR20180048416A - Composition for Improving, Preventing or Treating Muscular Diseases Comprising Natural Extract - Google Patents

Abstract

The present invention provides a composition for alleviating, preventing, or treating muscle wasting and inflammation, which contains a Kadsura japonica fruit extract, a Fatoua japonica (Thunb.) Blume extract, a mixture thereof, or isobavachalcone as effective component. The isobavachalcone of the present invention exhibits effects of suppressing oxidative stress and reducing muscle wasting due to anti-inflammatory reaction.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for improving, preventing or treating muscular diseases, including natural product extracts. [0002] Composition for Improving, Preventing or Treating Muscular Diseases [

The present invention relates to a composition for improving, preventing or treating a muscular disease including a natural product extract.

Skeletal muscle is the largest organ in the human body, accounting for 40-50% of total body weight and plays an important role in many metabolic functions in the body including energy homeostasis and heat production. The muscle of a person decreases by 1% or more every year since the age of 40. At 80 years old, 50% of the maximum amount of muscle is decreased, and the decrease of the old age is recognized as the most important factor which decreases the overall body function. As the aging progresses, the body shape changes such as muscle and fat content and skeletal distortion are recognized. The prevalence of obesity due to the decrease of old age muscle is continuously increasing at more than 30% level worldwide. If insulin secretion is not enough to supply the energy to the cell can cause muscle development disorder, diabetes mellitus is increased in people with diabetes. In addition, the decrease in muscle causes arthritis, back pain, chronic pain, abdominal obesity incontinence, and fracture injury can lead to depression in old age, Is a major cause of poor quality of life in connection with various diseases. It is known that myopenia is closely related to chronic diseases such as osteoporosis, insulin resistance and arthritis, and it is possible to prevent the reduction of physical activity due to aging by preventing or improving myopenia. The world's progressive ataxia and weakness treatment market is estimated at about $ 14 billion in 2011 and is expected to grow at an annual CAGR of 9.4% and reach about $ 23.5 billion by 2017. Increased mitochondrial production, suppression of muscle protein degradation, and anti - inflammatory drugs have been proposed as treatments for muscle weakness, but there are no clear remedies. In addition, it is suggested that 25-30 g of high quality protein should be consumed per meal in the diet that is proposed to prevent the myopenia of the elderly. This means that 4-5 eggs or about 120 g of chicken breast should be consumed for every meal, making it difficult for the general public to practice everyday life. Therefore, although many people choose protein supplements as an alternative in recent years, protein supplements are likely to cause adverse effects by causing excessive protein intake. Moreover, in the case of kidney disease, high protein diet can not be done and the kidney function is decreased by aging. Therefore, other alternative to high protein intake is needed to prevent myopenia.

Currently, natural substances which are effective in improving muscle diseases include camphorafabi flora (registered patent No. 10-1528023), red ginseng (patent application No. 2014-0089793), scarlet (registered patent No. 10-1759779) (Japanese Patent No. 10-1725327), and the like are known. In addition, anti-mycobacterial activity, antiparasitic activity, estrogenic activity and antioxidative activity are known in association with isobaricarcone, and Ginkgo biloba is known as a natural product having activity to reduce muscle decline.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop natural products and their active substances for improvement of muscle diseases. As a result, it was confirmed that the extracts of Namamisia and Pomonopsis sieboldii had differentiation inducing effects on the myoflagic cells, and it was confirmed that isobavacalcone, which is commonly contained in the above Namomizae and Pomonopsis thaliana, .

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating muscle diseases.

Another object of the present invention is to provide a health functional food for increasing muscle mass or for promoting differentiation from muscle stem cells to muscle cells.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating inflammation.

Another object of the present invention is to provide a health functional food for antioxidation.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a muscle disorder, comprising Kamesura japonica extract, Fatoua Villosa extract or a mixture thereof as an active ingredient.

As used herein, the term "muscle disorder" refers to an external factor such as contusion, laceration, crush, internal factors such as sudden or severe falls or muscle strain during sports activities, Muscle loss, and / or loss of muscle function resulting from possible blood supply interruption, which may be due to genetic and / or environmental factors.

It is to be understood that the above-mentioned "muscle disorder" includes at least all muscle diseases related directly or indirectly with the expression of MAFbx or MuRF1 protein described later.

According to one embodiment of the present invention, the muscle disease is selected from the group consisting of muscular atrophy, sacopenia, muscular dystrophy, ataxia, atony, muscular dystrophy, ≪ / RTI > or more. Specifically, the muscle disease is at least one selected from the group consisting of muscular dystrophy, muscular dystrophy, muscular dystrophy, ataxia, or tense depression.

The muscular atrophy of the limbs is accompanied by progressive denaturation of motor nerve fibers and cells in the spinal cord, resulting in amyotrophic lateral sclerosis (ALS) and spinal progressive muscular dystrophy muscular atrophy, SPMA).

According to another embodiment of the present invention, the muscular atrophy is aggravated muscular dystrophy or obesity muscular dystrophy.

The 'aging muscular dystrophy' is characterized by the gradual loss of muscle mass and muscle strength due to aging, and the increase of visceral fat and accompanying chronic low inflammation (inflammation-induced cytokine) -grade inflammation leads to muscular atrophy.

Obesity is an obesity-related phenomenon that obesity increases the muscle decline. Obesity results in fat accumulation in the muscles resulting in decreased muscle mass, increased mast cell-derived inflammatory factors, and increased intramuscular mitochondria The function of the muscles may be further weakened due to the decrease of the function of the insulin secretion. If the insulin secretion is abnormally caused by obesity, the insufficient supply of energy to the cells may cause the developmental disorder of the muscles.

The term 'myopenia (muscular dystrophy)' means a gradual decrease in skeletal muscle amount due to aging, which directly leads to a decrease in muscle strength, resulting in a decrease in various bodily functions and a disorder.

The above-mentioned 'muscle degeneration atrophy' is a disease in which gradual muscular atrophy and muscle weakness appear, which means degenerative myopathy characterized by necrosis of muscle fibers pathologically.

The 'heart atrophy' is a condition in which the heart is contracted by an external or internal factor, such as starvation, consuming disease, and aging, which causes the myocardial fiber to become thinner and thinner, leading to a decrease in adipose tissue.

The pharmaceutical composition for preventing or treating a muscle disorder of the present invention comprises an extract of Namio mizuna, an extract of Momomori mori, or a mixture thereof as an active ingredient.

The extract of Namio mizutana or Momomori mori extract may be a crude extract obtained by extracting Namomi or Momomori mollusk with at least one solvent selected from the group consisting of water and linear or branched alcohols having 1 to 4 carbon atoms.

(V / v), more than 20% and more than 100% (v / v) when a mixture of water and alcohol is used in a solvent used for preparing a crude extract of Namio mizuna extract (V / v), less than 100% (v / v), less than 100% (v / v), less than 70% to less than 100% (v / v), less than 10% to less than 90% (v / v), less than 20% (v / v), more than 40% to less than 90% (v / v), more than 50% to less than 90% for example, an aqueous solution of a linear or branched alcohol having 1 to 4 carbon atoms of 1 to 4% (v / v) such as 80% (v / v) of a linear or branched alcohol having 1 to 4 carbon atoms.

The alcohol aqueous solution may be at least one selected from the group consisting of an aqueous methanol solution, an aqueous ethanol solution, an aqueous propanol solution, and an aqueous butanol solution, and may be, for example, an aqueous ethanol solution, but is not limited thereto.

The extract of Namio mizuna or Momomori mofusi according to the present invention may be a solvent fraction obtained by fractionating a solvent crude extract with an additional solvent. For example, the solvent extract may contain one kind selected from the group consisting of ethyl ether, ethyl acetate, and butanol Or more of the solvent fraction.

For example, a solvent crude extract obtained by extracting the above Namomisuga or Mongolian mollusk with at least one solvent selected from the group consisting of water and a straight chain or branched alcohol having 1 to 4 carbon atoms is selected from the group consisting of ethyl ether, ethyl acetate, and butanol May be a solvent fraction using one or more solvents.

The extracts of Namamisja extract or Mabunomycorrhizae extract include solvent extracts and solvent fractions of Namio mizuna or Mabunomycorrhizae sp.

The extract of Namio mizutana or Momomori mori extract may be prepared by mixing at least one species selected from the group consisting of flowers, leaves, stems and roots of Namamisawa or Momomori mulberry with at least one selected from the group consisting of water and linear or branched alcohols having 1 to 4 carbon atoms And may be a crude extract obtained by extracting with a solvent.

(V / v), less than 100% (v / v), more than 30% and less than 100% (v / v), less than 50% to less than 100% (v / v), less than 60% to less than 100% (v / (v / v), 20% or more to less than 90% (v / v), 30% or more to less than 90% A linear or branched aqueous alcohol solution of from 1 to 4 carbon atoms of less than 90% (v / v), from 60% to less than 90% (v / v) or from 60% to less than 80% (v / v) Or a linear or branched alcohol aqueous solution of 1 to 70% (v / v) carbon atoms.

The process for preparing the extract of Namio mizuna or Momomori mofusi according to the present invention will be described in detail as follows: After cutting or removing the stomata by cutting and washing with water, the Momomisu mugwort or Momomori mori extract is added to about 10 To 20 times by weight of an extraction solvent. After extraction, the filtrate is collected by filtration. The extraction temperature is not particularly limited, but is preferably from 15 to 110, and more preferably from 20 to 90. [

The extraction process may be repeated once or several times. In a preferred example of the present invention, a method of re-extraction after the first extraction may be adopted. In the case of mass production of herbal extracts, The loss is generated due to the high water content, so that the extraction efficiency is lowered only by the first extraction. In addition, the extraction efficiency of each step was verified, and it was found that about 80 to 90% of the total extraction amount was extracted by the second extraction.

In one example of the present invention, when the extraction step is repeated twice, the obtained residue is subjected to reflux extraction again with an extraction solvent, about 5 to 15 times by volume, preferably 8 to 12 times by volume. After the extraction, the filtrate is combined with the previously obtained filtrate and concentrated under reduced pressure to prepare a Namyomisan extract or a mulberry Momollipule extract. The extraction efficiency can be increased by mixing the extraction solution obtained after the second extraction and the filtrate obtained after each extraction, but the extract of the present invention is not limited to the extraction number.

If the amount of the solvent used in the preparation of the extract is too low, the stirring becomes difficult and the solubility of the extract is lowered and the extraction efficiency becomes poor. When the amount of the solvent used in the next purification step is large Therefore, the amount of the solvent used is preferably within the above range.

The thus-obtained filtered extract is used in an amount of about 10 to 30 times by weight, preferably 15 to 25 times by weight, more preferably about 15 times by weight, The mixture is concentrated to an azeotropic ratio of 1 to 5 times, preferably 2 to 3 times, by 20 times by weight of water. The same amount of water is added again to homogeneously suspend the suspension, followed by lyophilization to obtain a powdery Namomi extract or a mulberry extract.

The extraction method used in the present invention may be any conventionally used method such as, but not limited to, cold-water extraction, hot water extraction, ultrasonic extraction, or reflux-cooling extraction.

As used herein, the term " extract " includes a solvent crude extract, a specific solvent soluble extract (solvent fraction) and a solvent fraction of a solvent crude extract, and the Namio wisteria extract or mulberry wool extract contains all the solutions, concentrates or powders do.

The pharmaceutical composition for preventing or treating the above-mentioned muscle diseases promotes the differentiation of muscle stem cells into muscle cells.

The stem cells may be satellite cells or myoblasts, for example, myoblasts, but are not limited thereto.

The muscle cells may be, but are not limited to, muscle cells or root canal cells, for example, canalicular cells.

The pharmaceutical composition for preventing or treating muscle diseases according to claim 1, wherein the composition comprises at least one selected from the group consisting of 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 10 to 100, 10 to 90, But are not limited to, the following ranges: 50-80, 10-70, 10-60, 20-100, 20-90, 20-80, 20-70, or 20-60 ug / ml.

The pharmaceutical composition of the present invention can be used as a pharmaceutical composition comprising a pharmaceutically effective amount of a safflower extract, a mulberry root extract or a mixture thereof and / or a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the aforementioned Namio wisdom extract, mulberry wool extract or a mixture thereof.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical compositions according to the present invention can be administered to mammals, including humans, in a variety of routes. The mode of administration may be any conventional manner and may be administered, for example, by oral, skin, intravenous, intramuscular, subcutaneous, and the like routes, preferably orally.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-1000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets, capsules or gels (e.g., hydrogels), and may additionally contain dispersing or stabilizing agents .

According to another aspect of the present invention, there is provided a health functional food for increasing muscle mass or promoting differentiation of muscle cells into muscle cells, which comprises an extract of Namio mizuna, a extract of Momomori mori, or a mixture thereof.

The above-mentioned health functional food for increasing the muscle mass or promoting differentiation from muscle stem cells to muscle cells promotes differentiation from muscle stem cells into muscle cells.

The stem cells may be satellite cells or myoblasts, for example, myoblasts, but are not limited thereto.

The muscle cells may be, but are not limited to, muscle cells or root canal cells, for example, canalicular cells.

The health functional food for increasing muscle mass or for promoting differentiation from muscle stem cells to muscle cells according to the present invention includes components that are ordinarily added in food production and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings do. For example, when it is made of a drink, as an active ingredient, a flavoring agent or a natural carbohydrate may be added as an additional ingredient in addition to a Namibia extract, a Phellinus linteus extract or a mixture thereof. For example, natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharides (e.g., maltose, sucrose, etc.); oligosaccharide; Polysaccharides (e.g., dextrin, cyclodextrin and the like); And sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). Natural flavoring agents (e.g., tau marin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) may be used as flavorings.

Since the health functional food for promoting differentiation of muscular cells from the muscle mass-increasing or muscle stem cell of the present invention is the same as the active ingredient and the effect of the pharmaceutical composition for the prevention or treatment of the muscle disorder, In order to avoid the excessive complexity of the present description, the description thereof is omitted.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating inflammation comprising an extract of Namio mizuna as an active ingredient.

As used herein, the term "inflammation " refers to a protective response involving immune cells, blood vessels, and molecular biological intermediates as one of the biological responses to harmful stimulation sources of biological tissue, such as pathogens, damaged cells, and stimulus sources.

According to one embodiment of the present invention, the inflammation is TNF-α mediated inflammation.

Since the pharmaceutical composition for preventing or treating inflammation according to the present invention has the same effectiveness as the pharmaceutical composition for the prevention or treatment of the muscle disorder, the common content between the two is to prevent the excessive complexity of the present invention. It is omitted.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a muscle disorder, comprising a compound of the formula (1) as an active ingredient.

[Chemical Formula 1]

The isobaricarcone which is the active ingredient of the present invention has the empirical formula C ₂₀ H ₂₀ O ₄ , the molecular weight is 324.27, and (2E) -1- [2,4-dihydroxy-3- ] -3 - (4-hydroxyphenyl) -2-propen-1-one, 2 ', 4,4'-trihydroxy- It is synonymous with corylifolinin.

According to one embodiment of the present invention, the composition induces myogenesis.

As demonstrated in the following examples, it was confirmed that when the compound of Chemical Formula 1 was treated with myofibroblast, which induced myotube reduction by treating TNF-a, the reduction of root canal was inhibited in a concentration-dependent manner.

According to one embodiment of the present invention, the composition inhibits expression of a protein associated with muscle decline induction. According to another embodiment of the present invention, the protein associated with muscle decline induction is MAFbx (muscle atrophy F-box, Atrogin 1) and MuRF1 (muscle RING-finger 1).

As demonstrated in the following example, in the myofibroblasts that induced root canal reduction by treating TNF-a, the expression of the muscle-decreasing induction related proteins decreased in a concentration-dependent manner when the compound of formula 1 was treated.

According to one embodiment of the present invention, the composition inhibits the degradation of muscle protein.

As demonstrated in the following examples, in the myofibroblasts that induced root canal reduction by treating TNF- [alpha], the expression of atrogin or mufr1 mediates the degradation of muscle protein when treated with the compound of formula 1 Smad phosphorylation to inhibit muscle protein degradation and improve muscle tone.

According to another aspect of the present invention, there is provided a health functional food for increasing muscle mass or for promoting differentiation of muscle cells into muscle cells, which comprises the compound of Chemical Formula 1 as an active ingredient.

The pharmaceutical composition for preventing or treating muscle diseases comprising the compound of Chemical Formula 1 as an active ingredient of the present invention and the health functional food for increasing muscle mass or for promoting differentiation from muscle stem cells into muscle cells are the same as the above- Since the pharmaceutical composition for preventing or treating muscle diseases comprising the extract of Mulberry Mossypii or a mixture thereof as an active ingredient and the use of the composition for enhancing muscle mass or the health functional food for promoting differentiation from muscle stem cells to muscle cells are the same, Are omitted in order to avoid the excessive complexity of the present specification.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating inflammation comprising the compound of the formula (1) as an active ingredient.

[Chemical Formula 1]

According to one embodiment of the invention, the composition inhibits phosphorylation of inflammatory mediators. According to another embodiment of the present invention, the inflammatory mediator protein is p65 (nuclear factor NF-kappa-B p65 subunit) and p38 (p38 mitogen-activated protein kinases).

As demonstrated in the following examples, in the myofibroblasts that induced TNF-a to induce root canal reduction, inhibition of the phosphorylation of inflammatory mediators p65 and p38 when treated with the compound of formula 1 above.

According to still another aspect of the present invention, there is provided an antioxidant health functional food comprising the compound of the formula (1) as an active ingredient.

[Chemical Formula 1]

According to one embodiment of the present invention, the composition inhibits oxidative stress.

As demonstrated in the following examples, in the myofibroblasts that have induced root canal reduction by treating TNF- [alpha], translocation into the nucleus of the transcription factor NRF1 (Nuclear respiratory factor 1) To induce the expression of antioxidant genes. The expression of the antioxidant protein HO-1 (heme oxygenase (decycling) 1, HMOX1) was increased in a concentration-dependent manner by NRF1.

The description of the pharmaceutical composition, the food composition and the health functional food composition is common to that described above, and the description thereof is omitted in order to avoid the excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for improving, preventing or treating myopenia and inflammation, which comprises an extract of Namio mizuna, an extract of Momomori mori, a mixture thereof or isobaricarone as an active ingredient.

(b) Isobarakalcone, an active ingredient of the present invention, exhibits an effect of improving muscle decline due to oxidative stress and inflammatory reaction.

(c) The active ingredient of the present invention is a natural or natural material-derived material, which can be easily supplied and received, and has an economical advantage.

Figs. 1A and 1B show the effect of improving the muscle atrophy of the isobaricarone-containing natural product, Namibia. FIG. 1A shows the result of treatment with 25 μg / ml or 50 μg / ml of the extract of Namomisai extract or Mongolian mushroom extract against myoblasts (C1C12, myoblast) induced by root canal reduction by TNF-α treatment . IBC is an experimental group treated with 10 μM isobavachalcone. FIG. 1B shows the results of analysis of expression of total major histocompatibility complex (MHC) and major histocompatibility complex class II integral membrane beta chain (MHC2B) by treating Namibia extract with 1, 5 and 10 ug / ml, respectively.
FIGS. 2A to 2E are graphs showing changes in cell proliferation and expression of muscle-associated proteins by treatment with isobarachalcone (IBC) on myoblasts (C1C12, myoblast) induced by root canal reduction by TNF-α treatment . FIG. 2A shows the results of myoblast proliferation of experimental groups treated with isobaricacone 0.1, 1 and 10 uM together with a negative control (CON) and a positive control (TNF-α) treated with TNF-α and TNF-α . Fig. 2b shows the root canal diameters of the experimental group treated with isobaricarcon 0.1, 1 and 10 uM and the control group. FIG. 2c shows the degree of protein expression of MyHC and Myogenin in the experimental group treated with 0.1, 1 and 10 uM isobaricarboxylate on the myoblasts. Figure 2d shows the degree of expression of MyHC protein in the experimental group treated with isobaricarcon 0.1, 1 and 10 uM in the myoblasts. FIG. 2E shows the degree of expression of MyoG protein in the experimental group treated with isobaricarcon 0.1, 1 and 10 uM in the myoblasts.
3A to 3E show results of analysis of expression of muscle contraction inducing proteins by isobaricolone treatment on myotobia induced root canal reduction. FIG. 3A shows the results of examining changes in expression of MAFbx and MuRF1 proteins by treating isobarbacon 0.1, 1 and 10 uM with the myofibroblasts. FIGS. 3B and 3C show the relative changes in expression of MAFbx (FIG. 3B) and MuRF1 (FIG. 3C) protein by treating isobarbacon 0.1, 1 and 10 uM with the myofibroblast, respectively. FIGS. 3d and 3e show the relative changes in expression of MAFbx (FIG. 3D) and MuRF1 (FIG. 3E) mRNA by treatment with isobaricarcon 0.1, 1 and 10 uM on the myoblasts, respectively.
FIGS. 4A to 4D show results of nuclear translocation and phosphorylation analysis of p65, an inflammatory mediator protein, by isobaricolone treatment on myoblasts induced by reduction of root canal by TNF-α treatment. FIG. 4A shows the results of confirming changes in nucleus and cytoplasmic p-p65, p65, and Lamin B protein expression by treating isobarbacon 0.1, 1 and 10 uM on the myoblasts. FIG. 4B shows the result of relatively observing changes in the expression of p-p65 in the nucleus by treatment of the myoblasts with isobaricarcon 0.1, 1 and 10 uM. FIG. 4C shows the result of relatively observing changes in the expression of p-p65 in the nucleus by treating the myoblasts with isobaricarcon 0.1, 1 and 10 uM. FIG. 4D shows the result of relatively observing changes in the expression of p-IkBα protein by treatment of the myoblasts with isobaricarcon 0.1, 1 and 10 uM.
FIGS. 5A to 5D show the results of phosphorylation analysis of inflammation-mediated protein p38 and muscle protein degradation-mediated protein smad3 by isobaricolone treatment on myofibroblasts induced to decrease by TNF-α treatment. FIG. 5A shows the results of confirming the changes in protein expression of p-p38, p38, p-smad3 and smad3 by treating isobarbacon 0.1, 1 and 10 uM with the myofibroblast. FIG. 5B shows the results of relative expression of p-p38 / p38 expression in the myoblasts treated with isobaricarcon 0.1, 1 and 10 uM. FIG. 5c shows the results of relative expression of p-smad3 / smad3 expression in the myoblasts treated with isobaricarcon 0.1, 1 and 10 uM. FIG. 5D shows the results of relative expression of p-FOXO1 / FOXO1 expression by treating isobarbacon 0.1, 1 and 10 uM with the myofibroblasts.
FIGS. 6A to 6D show the results of analysis of the oxidative stress defense factor Nrf2 and the oxidative stress defense factor HO-1 by isobaricolone treatment on root canal induced root cancellation. FIG. 6A shows the results of confirming changes in protein expression of HO-1 and nuclear and cytoplasmic Nfr2 by treating isobarbacon 0.1, 1 and 10 uM on the myoblasts. FIG. 6 (b) shows the result of relatively observing changes in protein expression of HO-1 by treating isobarbacon 0.1, 1 and 10 uM with the myofibroblast. FIG. 6C shows the result of relatively observing changes in protein expression of Nrf2 in the nucleus by treating the myoblasts with isobaricarcon 0.1, 1 and 10 uM. FIG. 6D shows a result of relatively confirming the change in the protein expression of cytoplasmic Nrf2.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Improvement of muscle atrophy of natural products containing isobaricarboxyl

Experimental Method

1. Preparation of extract

The dried Kamesura japonica outpost and Fatoua Villosa outpost were each precipitated in 80% ethanol, ultrasonicated and extracted at room temperature for more than 2 hours, and ethanol was removed by vacuum distillation and lyophilized.

2. Cell culture and induction of differentiation into canaliculus cells

C2C12 mouse myoblast cells were purchased from ATCC (Manassas, Va., USA) and cultured in DMEM supplemented with 5% FBS and 1% antibiotic-antimycotic And cultured in a 5% CO ₂ incubator. For induction of differentiation into myoblasts, cultures were treated with DMEM medium containing 2% horse serum every 2 days. After 4-6 days, formation of myotube was observed under a microscope and treated with TNF-α 10 ng / ml to induce differentiation inhibition.

3. Treatment of Extracts

The extracts of Namimizae and Mulberry Momopsis were dissolved in 0.1% DMSO and diluted to prepare 1 mM stock. For induction of differentiation into myoblasts, the cells were diluted with DMEM medium containing 2% horse serum to a final concentration of 0.1, 1, and 10 μM, and cultured for 2 days. After 6 days, myotubes were observed under a microscope. To confirm the effect of inhibiting muscle growth, differentiation was induced by treatment of differentiated myofibroblasts with TNF-α 10 ng / ml for 24 hours, Extracts or extracts of Mulberry Momolipule (24 hours).

result

The inhibitory effect of atrophy induced by atrophy induced by TNF-α of moxa and moxibustion with isobaricarcone was examined by TNF-α when treated with moxa extract or myomi extract. (Fig. 1A).

As a result of confirming the muscle building effect of the Namibia containing isovarbacalcone, it was confirmed that MHC protein as a muscle growth marker was increased in a concentration-dependent manner and MHC2B was also increased (Fig. 1B). The above results indicate that the extract of Namibia extract has muscle hypertrophy

Example 2: Improvement of muscle atrophy of isobavachalcone (IBC)

(Isobavachalcone: An overview, Chin J integ Med 2012, Jyl; 18 (7): 543-547), which is a compound commonly contained in Namamisja and Pomonopsis lupus of Example 1,

Experimental Method

1. Cell culture and induction of myotube differentiation

C2C12 mouse myoblast cells were purchased from ATCC (Manassas, Va., USA) and cultured in DMEM supplemented with 5% FBS and 1% antibiotic-antimycotic And cultured in a 5% CO ₂ incubator. For induction of differentiation into myoblasts, cultures were treated with DMEM medium containing 2% horse serum every 2 days. After 4-6 days, formation of myotube was observed under a microscope and treated with TNF-α 10 ng / ml to induce differentiation inhibition.

2. Measurement of root canal diameter

The canals were observed with a 200x microscope (Olympus CKX41) and at least 100 cells were randomly assigned to each group and diameters were measured and expressed as ratios.

3. Measurement of atrophy marker mRNA expression level

C2C12 mouse myoblasts were inoculated into 6-well plates and then differentiated into DMEM medium supplemented with 2% horse serum and treated with TNF-α (10 ng / ml) for 1 hour. One hour later, the cells were recovered and total RNA was isolated. The cDNA was synthesized with iSCRIPT ™ cDNA synthesis kit and qPCR was performed with iQ SYBR green. The primers used were atrogin-1 / MAFbx, F5'-AAG GCT GTT GGA GCT GAT AGC-3 ', R 5'-CAC CCA CAT GTT AAT GTT GCC-3'; 3 ', R ' -ATG GTG AAG GTC GGT GTG A-3 ', R ' 5'-AAT CTC CAC TTT GCC ACT GC-3 'and the expression level of the target mRNA was expressed as a ratio after normalization with GAPDH.

4. Measurement of muscle relaxation markers and related signaling protein expression

C2C12 myofibroblasts were inoculated into 6-well plates and differentiated for 6 days in canals and treated with TNF-α (10 ng / ml) to induce a decrease. After 24 hours, the cells were recovered and their protein expression was confirmed by MyHC (Myosin heavy chain), Myogenin (MyoG), MAFbx (Muscle Atrophy F-Box Protein), β-actin and MuRF1 (Muscle RING finger 1) P-p65, p-p38, p-38, p-smad3, smad3 and Lamin B were detected by western blot analysis after 30 minutes of treatment.

5. Suppression of oxidative stress

The causes of muscle loss are known to vary, but the most common cause is muscle cell death due to increased inflammatory response. To confirm whether the inhibitory factor of isobaricarboxylic acid is due to the increase of antioxidant factors, the amount of protein expression of NRF2 and H0-1, which are representative antioxidative factors, after isobaricolone treatment was confirmed by Western blot analysis. In particular, Nrf2 migrates into the nucleus during oxidative stress formation and acts as a transcription factor in the nucleus, thus expressing antioxidant factors such as HO-1. In this experiment, Nrf2 was divided into cytoplasm and nucleus, and the expression level of each site was confirmed.

6. Treatment of isobaricarcone

The isobaricarcone was dissolved in 0.1% DMSO and diluted to prepare a 1 mM stock concentration. For induction of differentiation into myoblasts, the cells were diluted with DMEM medium containing 2% horse serum to a final concentration of 0.1, 1, and 10 μM, and cultured for 2 days. After two days of treatment, the degree of differentiation and molecular biology experiments were performed after the last 6 days. In order to observe myotube formation under microscope and to examine the effect of inhibition of muscle relaxation, differentiated myofibroblasts were treated with TNF-α 10 ng / ml for 24 hours to induce differentiation inhibition, (24 hours) to confirm the effect of inhibiting muscle relaxation.

Experiment result

1. Establishment of TNF-α induction reduction model and evaluation of efficacy of isobaricarcone

After culturing C2C12 myofibroblasts in differentiation medium for 6 days, differentiation was induced and TNF-α 10 ng / ml was treated to observe differentiation cell death (differentiation inhibition). As a result, diameter of root canal Was decreased by TNF- [alpha], which tended to increase in a concentration-dependent manner in the group treated with isobaricarcone by concentration, and increased significantly at 10 uM of isobaricarcone (Figs. 2a and 2b).

In addition, the amount of protein required for root canal production such as MyHC and Myogenin was confirmed by western blot, which was also found to be significantly increased depending on the concentration of isobaricolone (Figs. 2C to 2E). This suggests that isobaricarone has an inhibitory effect on muscle differentiation by increasing the amount of protein expression of MyHC and Myogenin.

2. Isobarbacalcone inhibiting effect of marker expression inhibition

MAFbx (atrogin) and MuRF1 are representative muscle loss markers and are activated upon muscle loss. In this study, expression levels of MAFbx and MuRF1 were confirmed by qPCR and Western blotting. As a result, both the protein and mRNA of MAFbx and MuRF1 which were increased by TNF-α were decreased in a concentration-dependent manner in isobaricolone concentration, and particularly showed a significant decrease at 10 ug / ml (FIGS.

3. Control of anti-inflammatory and antioxidant mechanisms of isobaricarcone

Oxidative stress (ROS) by TNF-α is known to regulate the translocation of NFkB into the nucleus. In this study, C2C12 cells were treated with TNF-α and isobaricarcone in concentrations of C2C12 cells and then fractionated into nuclear fraction and cytoplasmic fraction to determine whether C2C12 cells regulate the intracellular migration of NFkB increased by TNF- . As a result, p-p65, which was increased in TNF-α, was significantly decreased in 10 μg / ml of isobaricolone, and p-IkB was also decreased in 10 μg / (Fig. 4 (a) to 4 (d)). This regulated NFkB is thought to modulate muscle loss markers such as Murf1 and atrogin.

4. Isobaricarcone modulates muscle loss transcription factor

Smad and Forkhead box protein O (FOXO-1) are important transcription factors for muscle loss, and p38 is known to be an important regulator of muscle catabolism. In this study, we used western blot to determine the effect of isobaricarcone on the transcription factor and kinase regulation of muscle loss. As a result, activation of p38, which was increased in TNF-α, was confirmed to be reduced by isobaricolone, decreased Smad3 phosphorylation, and increased phosphorylation of FOXO-1, which was reduced by TNF-α, Regulating the expression of regulated kinase (Fig. 5 (a) to (d)).

5. Antioxidative effects of isobaricarcone

In addition, NRF2 is a transcription factor that transcribes an antioxidant-related gene, and has been reported to regulate the expression level of antioxidant enzyme in the loss of muscle cells caused by an increase in oxidative stress due to aging. In this study, it was confirmed that the expression level of NRF2 was significantly increased in a concentration-dependent manner in the group treated with isobaricarcone alone, and thus, the expression of HO-1 (Heme oxygenase 1) was also increased (FIGS. 6A to 6D).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (14)

A pharmaceutical composition for preventing or treating a muscle disorder, comprising Kamesura japonica extract, Fatoua Villosa extract or a mixture thereof as an active ingredient.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the Namio worm extract and mulberry wool extract are anhydrous or hydroalcoholic extracts having 1-4 carbon atoms.
The method of claim 1, wherein the muscle disorder is selected from the group consisting of muscular atrophy, sacopenia, muscular dystrophy, ataxia, atony, muscular dystrophy, muscle degeneration, Wherein the pharmaceutical composition is at least one selected from the group consisting of:
A functional food for increasing muscle mass or promoting differentiation of muscle cells from muscle stem cells comprising Kamesura japonica extract, Fatoua Villosa extract or a mixture thereof as an active ingredient.
[Claim 5] The health functional food according to claim 4, wherein the muscle stem cells are satellite cells or myoblasts, for promoting the differentiation of muscle cells from muscle stem cells or muscle stem cells.
5. The health functional food according to claim 4, wherein the muscle cell is myocyte or myotube, for promoting the differentiation of muscle cells from muscle stem cells or muscle stem cells.
A pharmaceutical composition for preventing or treating inflammation comprising an extract of Kamesura japonica as an active ingredient.
A pharmaceutical composition for preventing or treating a muscle disorder, comprising a compound of the formula (1) as an active ingredient.
[Chemical Formula 1]

10. The method of claim 8, wherein the muscle disorder is selected from the group consisting of muscular atrophy, sacopenia, muscular dystrophy, ataxia, atony, muscular dystrophy, muscle degeneration, Wherein the pharmaceutical composition is at least one selected from the group consisting of:
A health functional food for increasing muscle mass or promoting differentiation from muscle stem cells into muscle cells comprising the compound of formula (1) of claim 8 as an active ingredient.
[Claim 11] The health functional food according to claim 10, wherein the stem cell is a satellite cell or myoblast, for promoting the differentiation of muscle cells from muscle stem cells or muscle stem cells.
11. The health functional food according to claim 10, wherein the muscle cell is myocyte or myotube, for enhancing muscle mass or for promoting differentiation from muscle stem cells to muscle cells.
1. A pharmaceutical composition for preventing or treating inflammation comprising a compound of formula (1) as an active ingredient.
[Chemical Formula 1]

A health functional food for antioxidation comprising a compound of the following formula (1) as an active ingredient.
[Chemical Formula 1]

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