KR101924880B1 - Composition for promoting differentiation of muscle cells containing Terminalia chebula extract as effective component - Google Patents

Abstract

The present invention relates to a composition for promoting the muscle differentiation containing a Terminalia chebula extract as an effective component, wherein the Terminalia chebula extract, which is an effective component of the present invention, promotes the cell survival rate of myoblast cells, can induce and promote the differentiation of myoblast cells into muscle cells, and has an effect of regulating the expression amount of the muscle cell differentiation-related protein. Therefore, the Terminalia chebula extract of the preset invention can be usefully used for a muscle differentiation accelerator, medicines for muscular diseases, or health functional food for promoting muscle growth or preventing or treating senile muscle loss.

Description

[Technical Field] The present invention relates to a composition for accelerating muscle differentiation,

The present invention relates to a terminal chebula extract as an active ingredient.

Our body's muscles are attached to the bones to protect the bones and keep the body shape properly. Muscle also promotes calcium influx and increases bone density. However, as the body ages, changes in the constituents cause redistribution of body fat and body protein. At about age 50, muscle protein synthesis rate is slower than the rate of degradation, resulting in rapid muscle regeneration. Can be exposed.

Muscle hypofunction, which is one of the muscle weakness diseases, refers to a condition in which approximately 13 to 24% of the body weight is reduced. Muscle hypoxia significantly reduces the behavior of the body, thereby deteriorating the mental health and reducing the satisfaction of life. It can easily get injured and lead to serious serious injury. One of the causes of myopenia is a gradual decline in the amount and quality of skeletal muscle that occurs as the aging progresses, and weight loss, including fat and body fat content due to inadequate dietary energy intake.

Myoblasts, which are formed through cleavage and crystallization of precursor cells, differentiate into myocytes and fuse with adjacent cells to differentiate into myotubes. Differentiated canaliculus cells form muscles through the process of forming myofibrils (myofibrils). Some of the source cells are stationary as stem cells, and when the muscle tissue is exposed to a disease such as trauma or muscular dystrophy, the muscle stem cells, known as satellite cells, Healing and regeneration. In diseases such as muscle atrophy, these cells are particularly important for muscle stability and healing.

On the other hand, Gaza dried up the ripe fruit of Terminalia chebula Retzius. Known chemical components include gallic acid, tannin, and ellagic acid. It has been reported that Gaza contains a relatively large amount of tannins and has convergence and antidiarrheal activity. Gaza extract has antimicrobial activity and antifungal activity. In recent years, the effects of chebulagic acid isolated from Gadja extract on arthritis or inflammation have been studied.

Korean Patent Registration No. 0520995 discloses such a ghazu extract-related technology and a cosmetic product containing the Ghazu extract having an anti-aging effect on cells. Korean Patent Publication No. 2016-0080425 discloses a solid fermentation extract A composition for preventing skin aging is disclosed. However, a composition for promoting muscle differentiation comprising an extract of the present invention as an active ingredient has not been disclosed.

The present invention provides a composition for promoting muscle differentiation comprising an extract of Ghazia as an active ingredient. The present invention provides a composition for promoting muscle differentiation, comprising an extract of Ghazi, an effective ingredient, having excellent antioxidative activity, The present inventors have accomplished the present invention by not only significantly increasing the expression of the myeloid differentiation-related protein but also differentiation of myoblasts into muscle cells and the expression amount of myeloid-related protein.

In order to achieve the above object, the present invention provides an agent for promoting the differentiation of myoblasts, which comprises Gadjat extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating muscle diseases containing the muscle cell differentiation promoting agent as an active ingredient.

The present invention also provides a health functional food composition containing the muscle cell differentiation promoting agent as an active ingredient for preventing or improving muscular increase or age-related muscle loss.

The present invention relates to a composition for muscle differentiation comprising an extract of Ghazia as an active ingredient, wherein the extract of Ghazira, which is an active ingredient of the present invention, not only promotes the cell survival rate of the source cells but also induces and promotes differentiation into muscle cells And has an effect of regulating the amount of expression of myocyte differentiation-related proteins.

FIG. 1 shows the ABTS radical scavenging activity of the GAZA extract according to the present invention.
FIG. 2 shows the results of cell viability obtained by treating the Ghazapa extract according to the present invention by concentration.
FIG. 3 is a result of confirming the proliferation and differentiation of myofiber cells after 24 hours or 48 hours after treatment of Gadja extract according to the present invention.
FIG. 4 is a result of examining the expression amount of a protein involved in promoting muscle differentiation by immunoblot analysis after treating Gadja extract according to the present invention to a source cell.

The present invention relates to an agent for promoting the differentiation of myoblasts containing an extract of Gadjahal as an active ingredient.

The Gadja extract may be prepared by a method including, but not limited to, the following steps:

(1) extracting the extract with an extraction solvent;

(2) filtering the extract of step (1); And

(3) concentrating the filtered extract of step (2) and drying to produce an extract.

In the step (1), the extraction solvent is preferably selected from water, a C ₁ -C ₄ lower alcohol or a mixture thereof, more preferably water, but it is not limited thereto. In the above production method, any conventional method known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used. The extraction solvent is preferably added by 1 to 20 times the weight of the potato, more preferably 5 to 15 times, and further preferably 10 times. The extraction temperature is preferably 60 to 100 DEG C, but is not limited thereto. The extraction time is preferably 1 to 48 hours, more preferably 1 to 24 hours, most preferably 3 hours. In the above method, the concentration of the step (3) is preferably, but not limited to, using a vacuum rotary condenser or a vacuum rotary evaporator. In addition, drying is preferably carried out under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, more preferably freeze drying or the like.

The present invention also relates to a pharmaceutical composition for preventing or treating muscle diseases containing the muscle cell differentiation-promoting agent as an active ingredient.

The pharmacologically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one that is usually used at the time of formulation and is a salt solution such as saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, The present invention also relates to a method for producing a microcrystalline cellulose composition comprising the steps of dissolving a microcrystalline cellulose, a cross, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Tartrate, magnesium stearate, and mineral oil, and the like. A buffer, a bacteriostatic agent, a diluent, a surfactant, a binder, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent or a preservative agent in addition to the above components. A suitable dose of the pharmaceutical composition of the present invention may be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient . The route of administration of the pharmaceutical composition for preventing or treating muscle disorders of the present invention may be administered through any route generally accepted as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered orally or parenterally by intramuscular administration, topical administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, Rectal administration, and the like, and can be administered through a route of intramuscular administration in detail.

The present invention also relates to a health functional food composition containing the muscle cell differentiation promoting agent as an active ingredient for preventing or ameliorating muscle growth or senile muscle loss.

The health functional food composition may be any one selected from powder, granule, ring, tablet, capsule, candy, syrup and beverage, but is not limited thereto. The health functional food composition of the present invention can be prepared by adding the Ghazu extract as it is or mixing it with another food or food ingredient, and can be suitably prepared according to a conventional method. Examples of the food to which the Ghatti Extract can be added include dairy products including caramel, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, , A drink, an alcoholic beverage, and a vitamin complex, and includes all health functional foods in a conventional sense. That is, there is no particular limitation on the kind of the food. The health functional food composition may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, , pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices and vegetable drinks. The above components can be used independently or in combination. In addition, the health functional food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, and the natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, Polysaccharides such as dextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Although the ratio of the natural carbohydrate is not critical, it is preferably 0.01 to 0.04 g, more preferably 0.02 to 0.03 g, per 100 g of the composition of the present invention, but is not limited thereto. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

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 merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  1. Preparation of Gadja Extract

The terminal used in the present invention chebula ) were purchased from Oriental Pharmaceutical Company (Jeongseong Pharmacy, Daejeon, Korea).

1000 g of distilled water was added to 100 g of pulverized green potato, and the mixture was boiled for 90 minutes to obtain an extract. The extract was centrifuged at 150 × g for 15 minutes and the supernatant was dried using a freeze dryer. After lyophilization, the yield of hot water extract of Gaza was 11.58% (w / w).

Example  2. Cell culture

The cell line used in one embodiment of the present invention was obtained by isolating muscle tissue from the hind leg of mouse 3 days old with mouse primary myoblast. The cell line was supplemented with 10% cosmic calf serum, 10 ng / ml basic fibroblast growth factor and 1% antibiotic in Ham's F-10 nutrient mixture (F-10) Cultured in a culture dish coated with poly-L-ornithin and collagen at 37 ° C and 5% CO ₂ , which is a standard cell culture method.

Example  3. Evaluation of antioxidant efficacy

The antioxidant activity of the Gadja extract was measured using ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) assay.

Potassium persulfate (2.4 mM) and ABTS (7 mM) were mixed in a volume ratio of 1: 1 and reacted for 24 hours at room temperature in a shaded state to produce ABTS free radicals. The ABTS free radical was then diluted with distilled water and the ABTS reaction solution was prepared so that the absorbance at 650 nm was around 0.7. Each well of a 96-well plate was mixed with 80 μl of ABTS reaction solution and 20 μl of sample or distilled water, and reacted in a shaded state for 4 minutes. Absorbance was measured at 650 nm with a microplate reader.

The antioxidant activity was calculated using the following formula (1).

Equation (1)

ABTS radical scavenging activity (%) = {1 - [(absorbance of sample - absorbance of sample itself) / absorbance of control group]} × 100

As a result, the ABTS assay system was well established by confirming the antioxidative effect according to the increase of the concentration of the positive control group, resveratrol, and the GABA extract of the present invention abolished the ABTS radicals in a concentration-dependent manner, (Fig. 1).

Example  4. Analysis of cell viability and nutrient identification on cell differentiation

(3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -methoxycarbonylpiperidine (MTS) to investigate the effect of Gadja extract on cell viability of myoblasts. 2H-tetrazolium, inner salt) assay.

In 96 well plates, myoblasts were incubated with 2 x 10 < RTI ID = ^0.0 > After cells were divided into cells / wells and cultured for 24 hours, each well was replaced with a medium treated with the concentration of each sample. After 24 hours, the MTS reagent was added and absorbance was measured at 490 nm using a microplate reader. Relative cytotoxicity was measured by expressing the survival rate of the cells treated with the sample as a percentage compared to the control not treated with the sample.

Equation (2)

Cytotoxicity (%) = [(absorbance of sample-added group - absorbance of sample itself) / absorbance of control group] × 100

The cell viability was gradually increased by treatment with the Ghatti extract, and it was 137.54% higher when 200 μg / ㎖ of Ghatti extract was treated. Cell viability (%) was confirmed (Fig. 2).

In addition, we examined the effect of Gadja extract on the proliferation and differentiation of myoblast cells. As a result of treatment with 100 μg / ㎖ of Gadja extract, the shape of myoblasts changed from a circular shape to an elliptical shape, (Fig. 3). These results are similar to the morphological changes observed in the myocyte differentiation.

Example  5. Immunoblot  analysis( Immunoblot  analysis)

Myoblasts were washed with PBS and then lysed with lysis buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100) (lysate) was extracted. The same amount of protein lysate was mixed in a loading buffer (Laemmi sample buffer), and SDS-PAGE was used to separate the proteolytic fragments by size. Then, anti-pax3 / 7, anti-myh3 and anti-GAPDH To perform immunoblotting.

As a result, as shown in Fig. 4, it was confirmed that the expression of pax3 / 7 protein decreased and the expression of myh3 protein increased by GAZa extract. Based on these results, it was concluded that Gadjat extract induces differentiation of myoblasts.

Claims (5)

delete delete (Atony), muscular atrophy, muscular dystrophy, muscle regression, rhabdomyosarcoma, rhabdomyosarcoma, amyotrophic rhinosclerosis, myasthenia, cachexia and myopathy (sarcopenia). < RTI ID = 0.0 > 8. < / RTI > 4. The method of claim 3, wherein let the extract extraction solvents include water, C ₁ ~ C ₄ lower alcohol, and the prevention or treatment a pharmaceutical composition of the muscle disease of any one selected from a mixture thereof characterized in that a. A health-functional food composition for preventing or ameliorating a muscle increase or a senile muscle loss containing an extract of Ghazale as an active ingredient.

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