KR20170142658A - Composition for improving Myogenesis containing extraction of Atractylodes macrocephala Koidzumi - Google Patents

Abstract

The present invention relates to a composition containing a white atractylis extract as an effective component for promoting the differentiation and development of muscle. According to the present invention, as the white atractylis extract increases the expression of MyoD, which is a myogenic regulatory factor (MRF), and promotes the generation of a myosin heavy chain (MHC), which is muscle structure protein, an effect for the promotion of muscle differentiation is confirmed, and thus, the composition for the differentiation and development of muscle is able to be used as healthy functional food.

Description

TECHNICAL FIELD The present invention relates to a composition for promoting the differentiation and development of muscles containing an extract from Aspergillus oryzae as an active ingredient,

The present invention relates to a composition for promoting the differentiation and development of muscles containing an extract of Lachlan as an active ingredient.

Recently, skeletal muscle has been important in the study of obesity and related metabolic diseases. Skeletal muscle is the main organ for controlling insulin resistance as a major cause of insulin resistance, and at the same time is a key organ for regulation of living body energy through the action of energy production and heat production. In addition, as the aging progresses, sarcopenic obesity caused by sacropenia and fat accumulation due to increase in obesity inhibiting protein metabolism of muscles is recently treated as important. have. Myopenia or muscular dysfunction Obesity increases the risk of metabolic syndrome as well as physical dysfunction.

Therefore, it is necessary to develop a drug capable of promoting the differentiation of myofibers in order to treat diseases associated with weakness of the muscles.

Skeletal muscle cells undergo myogenesis process, and their morphology changes rapidly as they mature from myoblasts to myotubes. In the proliferative phase, mononuclear cells grow in size from a circular shape to gradually fusiform. In the differentiation phase, the cells gradually grow longer, and several cells around them are fused with each other to differentiate into tubular myotubes. And after the end of differentiation, myotube increases the diameter and length and makes the muscles grow bigger. Muscle differentiation is regulated by myogenic regulatory factors (MRFs) such as MyoD and MRF 5, and the expression of myosin heavy chain (MHC) is known to be increased in the late phase of differentiation. Thus, MRFs and MHC can be used as markers for muscle differentiation and their expression levels can be measured to determine muscle differentiation. After the differentiation, the size of the muscle is increased through the muscle hypertrophy process, in which the diameter of the canal becomes larger and the length becomes longer in the canaliculus cells fused through the protein synthesis pathway.

On the other hand, baekchul (Atractylodes macrocphala Koidzumi, Atractylodis Rhizoma Alba, ARA, Asteraceae) are used in Oriental medicine for the treatment of gastrointestinal diseases, abdominal pain and obesity. It has also been shown that the above extract has an anti-inflammatory, anti-ulcer, and antitumor effect (C.-Q. Li, L.-C. He, and J.-Q. Jin, Phytotherapy Research, vol. 4, pp. 347353, 2007).

The inventors of the present invention have investigated the skeletal muscle regulating action of the extract of Guanxi province, and confirmed that the muscles are differentiated by the treatment of the extract of Guanxi province, thus completing the present invention.

Accordingly, an object of the present invention is to provide a composition for promoting the differentiation and development of muscles containing an extract of Leukemia as an active ingredient.

Another object of the present invention is to provide a health food composition for promoting differentiation and development of muscles, comprising an extract of Lycopersicum as an active ingredient.

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

In order to accomplish the object of the present invention as described above, the present invention provides a composition for promoting the differentiation and development of muscles comprising an extract of Leukemia as an active ingredient.

In one embodiment of the present invention, the dried extract is prepared by mixing water, C1 to C4 lower alcohols, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, And is extracted with at least one solvent selected from the group consisting of.

In another embodiment of the present invention, the extract is extracted from the root of the extract.

In another embodiment of the present invention, the composition is characterized by an increase in the expression of myogenic regulatory factors (MRF).

In another embodiment of the present invention, the muscle differentiation regulator is MyoD.

In another embodiment of the present invention, the composition is characterized in that the expression of myosin heavy chain (MHC), which is a muscle structural protein, is increased.

The present invention also provides a health functional food composition for accelerating the differentiation and development of muscles comprising an extract of Lycopersicum as an active ingredient.

The present invention also provides a method for promoting differentiation and development of muscles, comprising the step of administering the extract to a subject.

In addition, the present invention provides the use of the extract of Leukemia for differentiation and development of muscles.

It has been confirmed that the extract obtained from the extract according to the present invention increases the expression of myogenic regulatory factors (MRF) and myosin heavy chain (MHC) It can be used for promoting development. It can also be used for the prevention and treatment of diseases that are closely related to muscle weakness and muscle loss including obesity and diabetes.

FIG. 1 shows a protocol of an embodiment of the present invention (DMEM: Dulbecco's modified Eagle's medium, FBS: fetal bovine serum, ARA: Atractylodis Rhizoma Alba, HS: myogenic regulatory factor, .
FIG. 2 shows morphological changes of C2C12 cells treated with the extract of the present invention in a proliferative phase.
FIG. 3A shows the results of morphological changes in C2C12 cells treated with the extract of the present invention.
FIG. 3b shows the results of confirming the changes of MyoD and MHC in the cell line of C2C12 cells treated with the extract of the present invention.
FIG. 4a shows the result of confirming the change of the Myotube length according to the concentration in the later stage of differentiation of C2C12 cells treated with the extract of the present invention.
FIG. 4B shows the result of confirming the change of MHC according to the concentration at the later stage of differentiation of C2C12 cells treated with the extract of the present invention.

The present invention provides a composition for accelerating the differentiation and development of muscles comprising a dried extract as an active ingredient.

The present invention also provides a health functional food composition for accelerating the differentiation and development of muscles comprising an extract of Lycopersicum as an active ingredient.

Hereinafter, the present invention will be described in detail.

The extract of the extract according to the present invention showed that the expression of myosin heavy chain (MHC), which is myogenic regulatory factors (MRF) and the muscle structural protein, was inhibited by extracting mouse muscle cells (C2C12) And it was confirmed that the extract had an effect of promoting differentiation and development of muscles.

Therefore, the extract of the present invention can be used variously as medicines useful for differentiation and development of muscles and as health functional foods for promoting differentiation and development of muscles.

The extract of the present invention can be prepared using conventional solvents known in the art, that is, under the conditions of ordinary temperature and pressure, using conventional solvents. Examples of the extraction solvent that can be used in the production of the extract of the present invention include water, C1 to C4 lower alcohols, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, And a mixed solvent thereof may be used alone or in combination. The solvent is preferably water, but is not limited thereto. The suitable amount of the extraction solvent is about 1 to 10 times the dry weight of the extract. Examples of the extraction method include a heat extraction method, a cold extraction method, a reflux cooling extraction method, an ultrasonic extraction method and the like. have. The extraction temperature is not particularly limited as long as the effective activity of the extractable component is not removed, but is preferably extracted by immersion at room temperature.

The composition of the present invention may contain one or more known active ingredients having an effect of promoting differentiation and development of muscles together with a dried extract.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient according to a suitable method in the art.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the patient's weight, age, , Diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the extract is preferably about 0.0001-500 mg / kg, preferably about 0.001-300 mg / kg, and is preferably administered once or several times a day.

The composition of the present invention can be used alone or in combination with other methods for the differentiation and development of muscles or using surgery, hormone therapy, drug therapy and biological response modifiers.

The composition of the present invention can be added to health functional foods for the purpose of preventing or improving metabolic diseases and promoting metabolism. When the dried extract of the present invention is used as a food additive, the dried extract may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the extract of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01-0.20 g, preferably about 0.04-0.10 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a method for promoting differentiation and development of muscles, comprising the step of administering the extract to a subject.

The subject is a mammal, including a human or non-human, and non-human mammals include, but are not limited to, mice, rats, dogs, cats, horses, cows, sheep, goats, pigs, rabbits and the like.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of 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.

Example  1: Preparation and method of experiment

1.1. Boiling Atractylodes macrocephala Koidzumi ) Preparation of extract

Pyridoxine was purchased from Kwangmyung Dang Pharmaceutical Co., Ltd. (Ulsan, Korea) and used as a sample after discrimination in Dongguk University Oriental Medical College. The resulting extract was filtered with a filter paper (Whatman No. 1; Sigma-Aldrich, St. Louis, MO, USA), and the filtrate was concentrated under reduced pressure using a rotary evaporator And then lyophilized to prepare a water extract. The yield was 26%.

1.2. Cell culture

The mouse-derived C2C12 cell line was purchased from the American Type Culture Collection (ATCC, CRL-1772; Manassas, VA, USA) and cultured at 37 ° C in 5% CO2. FBS was incubated with 2% horse serum (HS) for induction of differentiation, and Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS) medium containing 1% penicillin / streptomycin Respectively. The timing and frequency of drug treatment is based on the experimental method of Montesano et al. (Montesano A, Luzi L, Senesi P, Mazzocchi N, and Terruzzi I. Resveratrol promoter myogenesis and hypertrophy in murine myoblasts. J Transl Med 2013; (Fig. 1).

1.3. Shape observation

Cell morphology was observed using an optical microscope (Olympus CKX41; Olympus) equipped with a digital camera system (Olympus C7070; Olympus, Tokyo, Japan). The length and diameter of myotube were measured by Yeh et al. (Yeh TS, Hsu CC, Yang SC, Hsu MC, and Liu JF. Angelica Sinensis promotes myotube hypertrophy through the PI3K / Akt / mTOR pathway. ). Each of the wells was divided into 9 squares, and the linear length and diameter of 10 myotubes per zone were measured using a Leica application suite version 4.2 (Leica Microsystems, Heerbrugg, Swizerland, ) And the average value was calculated.

1.4. Real time polymerase chain reaction ( PCR ) analysis

Reverse transcription-PCR was performed to measure mRNA expression levels of MyoD and MHC. Each cell was harvested, centrifuged at 5,000 rpm for 5 minutes, and total RNA was isolated using TRIzol reagent. CDNA was synthesized from mRNA at 25 ° C for 10 minutes, at 42 ° C for 60 minutes, and at 70 ° C for 15 minutes by adding oligo- (dT) primer and Improm-IITM reverse transcriptase to the separated RNA. To perform PCR, 1 μg of cDNA synthesized from mRNA was added to mouse MyoD primers [sense; 5'-CAA CGC CAT CCG CTA CA-3 ', antisense; 5'-GTC TGG GTT CCC TGT TCT GT-3 '], mouse MHC primers [sense; 5'-TGA ACT GGA GGG TGA GGT AG-3 ', antisense; 5'-TTC GGT CTT CTT CTG TCT GG 3 '] and mouse GDPAH primers [sense; 5'-ATT CAA CGG CAC ACT CAA GG-3 ', antisense; (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 [mu] M dNTP, 1 U Tag polymerase, After mixing, 30 cycles were performed at 94 ° C for 30 seconds for denaturation, 30 seconds at 55 ° C to 60 ° C for annealing, and 60 seconds at 70 ° C for extension. PCR products were electrophoresed using 1% agarose gel containing EtBr and then confirmed by ultraviolet lamp. Expression rate of glyceraldehyde-3-phosphate dehydrogenase was determined using Image-J program (public domain) .

1.5. Immunocytochemistry Immunocytochemistry )

The C2Cl2 cells were differentiated in 0.1% gelatin-coated coverslip and treated with concentration by concentration. After 24 hours, the cells were washed with 1 × PBS, fixed with 3.7% paraformaldehyde at room temperature for 15 minutes, washed three times with 1 × PBS, permeabilized with permeabilization buffer The reaction was allowed to proceed at room temperature for 15 minutes. After washing 3 times with 1X PBS, the cells were reacted with PBST containing 1% BSA (blocking buffer, PBS containing 0.5% Tween 20) for 30 minutes to inhibit unspecific antibody binding. The primary antibody (SC-20641, Santa Cruz Biotechnology) for MHC was diluted 1: 500 in blocking buffer and reacted at room temperature for 1 hour. After washing three times with 1 × PBS, secondary antibody (Goat anti-Rabbit IgG-HRP) diluted 1: 5000 in blocking buffer was added and reacted at room temperature for 1 hour. 3 times. The cover glass was placed on a slide glass and analyzed by fluorescence microscopy (Leica Microsystems, Heerbrugg, Swizerland).

1.6. Statistical analysis

StatPad Prism (GraphPad Software Inc., San Diego, Calif., USA). Results of two replicates were expressed as mean ± standard errors of mean and analyzed using analysis of variance (Tukey's test). Statistical significance was accepted when the P-value was less than 0.05.

Example  2: Results

2.1. Observation of morphological changes of the proliferative phase

The morphological changes were observed after 40 hours of confluence treatment for 24 hours. As a result, it could be confirmed that the higher concentration of myocyte was observed in the control group than in the round-shaped cell shape in the myoblast state in the control group (FIG. 2).

2.2. Morphological changes and differentiation Marking factor  Confirm increase

After changing to a differentiation medium containing 2% HS in a 70% confluence state, the leaves were treated for 96 hours at intervals of 24 hours. In morphological changes, it was confirmed that the morphology of myotubes in the late stage of the differentiation was more evident in the 1.0 mg / ml aliquot (Fig. 3a). In addition, mRNA expression of MyoD and MHC among MRFs was analyzed by differentiation marker. The expression level of MyoD mRNA was increased at 1.0 mg / ml of extract, compared with the control, and the degree of canalicular cell formation was more specific The amount of mRNA expression of MHC was also significantly increased in 1.0 mg / ml of the extract compared to the control (P <0.05; FIG. 3b).

2.3. Morphological changes of late stage and confirmation of increase of differentiation marker

After 72 hours in 70% confluence, the differentiation medium was replaced and the drug was treated for 24 hours, and the efficacy of the extract on the root canal enlargement was evaluated by morphological observation. Both the length and diameter of canaliculus cells were significantly increased at 1.0 mg / ml of liquor compared to the control (P <0.01; Fig. 4a).

In addition, immunocytochemistry was used to evaluate the expression of muscle structural protein MHC, which is a typical differentiation marker of the late stage of differentiation. It was confirmed that the expression of MHC was very high at 1.0 mg / ml of extract (Fig. 4B).

2.4. Check the result

In the present invention, morphological changes were observed in the pancreas, the tuber, and the differentiation stage. As a result, it was confirmed that the pancreas caused a noticeable morphological change as compared with the control. In the proliferative phase, proliferation was more rapid in the spindle form than in the control group at 24 hours after the administration of the drug, and cell fusion and myotube formation were more prominent in the differentiation stage. When the morphology of late myotube was compared with that of the control group at the low concentration of 0.2 mg / ml, it was significantly increased at 1.0 mg / ml.

In addition, in the present invention, the degree of differentiation was evaluated by measuring the amount of expression using MyoD and MHC as the differentiation marker, and significant results were obtained. MyoD, myogenin, MRF 4 and MRF 5, which play a common role in promoting the fusion of myoblasts. The expression level of MyoD mRNA was measured at the differentiation stage, and the expression level was increased as the concentration of extract was higher.

In addition, the amount of expression of MHC, the main structural protein of myotube, was measured. MHC is more specific than MyoD in that it can evaluate the late stage of differentiation. The mRNA expression level of MHC in the differentiation stage was also increased as the concentration was increased and it was significantly increased at 1.0 mg / ml of extract. In addition, the immunocytochemical staining of the latter stage of the differentiation showed that the expression level of MHC was significantly increased at 1.0 mg / ml.

Therefore, it has been confirmed that the composition of the present invention has an effect of developing muscles by differentiating muscles.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation example  1: Preparation of pharmaceutical preparations

1. Manufacturing of powder

200 mg of crude extract

Lactose 100 mg

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

200 mg of crude extract

100 mg of corn starch

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of capsules

200 mg of crude extract

100 mg of corn starch

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

200 mg of crude extract

100 mg mannitol

Na ₂ HPO _{4 12} H ₂ O 2 mg

Sterile sterilized water for injection

Injection was prepared by mixing the above components per ampoule (2 mL) according to the usual injection preparation method.

Formulation example  2: Manufacturing of food

Foods containing the extract of the present invention were prepared as follows.

1. Preparation of cooking seasoning

A cooked sauce for health promotion was prepared from 20 to 95% by weight of the extract.

2. Manufacture of tomato ketchup and sauce

0.2-1.0 wt.% Of the extract was added to tomato ketchup or sauces to produce healthy tomato ketchup or sauce.

3. Manufacture of flour food

0.5 to 5.0% by weight of the extract was added to wheat flour, and bread, cake, cookies, crackers and noodles were prepared by using this mixture to prepare a food for health promotion.

4. Manufacture of soups and gravies

0.1-5.0% by weight of the extract was added to the soup and the juice to prepare health promotion meat product, noodle soup and juice.

5. Manufacture of ground beef

A ground beef for health promotion was prepared by adding 10% by weight of the extract from the ground beef.

6. Manufacture of dairy products

5-10% by weight of the extract was added to milk, and the milk was used to produce various dairy products such as butter and ice cream.

Formulation example  3: Manufacturing of beverages

1. Manufacture of carbonated beverages

The syrup was prepared by mixing 10-15% of the extract, 10-15% of sugar, 0.05-0.3% of citric acid, 0.005-0.02% of caramel and 0.1-1% of vitamin C and 75-80% of purified water . The syrup was sterilized at 85-98 ° C for 20-180 seconds, mixed with cooling water at a ratio of 1: 4, and 0.5-0.82% carbon dioxide gas was injected to prepare a carbonated drink containing the extract.

2. Manufacture of health drinks

(70%, 0.12%), vitamin C (0.02%), calcium pantothenate (0.02%), licorice extract (Solid content: 65%, 0.01%) were uniformly blended, instant sterilized, and packaged in small containers such as glass bottles and plastic bottles to produce health drinks.

3. Manufacture of vegetable juice

Healthy vegetable juice was prepared by adding 0.5 g of the extract of Lycopersicon esculentum to 1000 ml of tomato or carrot juice.

4. Manufacture of fruit juice

Healthy fruit juice was prepared by adding 0.1 g of dried extract to 1,000 ml of apple or grape juice.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (9)

A composition for accelerating the differentiation and development of muscles comprising an extract of Aspergillus as an active ingredient.
The method according to claim 1,
Wherein the extract is at least one selected from the group consisting of water, C1 to C4 lower alcohols, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, A composition for promoting muscle differentiation and development, characterized by being extracted with a solvent.
The method according to claim 1,
The composition for promoting muscle differentiation and development is characterized in that the extract is extracted from the roots of the extract.
The method according to claim 1,
Wherein said composition enhances the expression of myogenic regulatory factors (MRF).
5. The method of claim 4,
Wherein the muscle differentiation regulator is MyoD.
The method according to claim 1,
A composition for promoting muscle differentiation and development, characterized in that the composition increases the expression of myosin heavy chain (MHC), a muscle structural protein.
A health functional food composition for accelerating the differentiation and development of muscles containing an extract of Lolium japonica as an active ingredient.
8. The method of claim 7,
Wherein the extract is at least one selected from the group consisting of water, C1 to C4 lower alcohols, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, Wherein the composition is extracted with a solvent.
8. The method of claim 7,
Wherein said extract is extracted from the roots of the extract. &Lt; RTI ID = 0.0 &gt; 20. &lt; / RTI &gt;

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