KR20190100834A - Composition for strengthening of muscle, preventing and treating Sarcopenia comprising Liquidambar styraciflua L. - Google Patents

Abstract

The present invention relates to a composition for muscle strengthening or preventing and treating sarcopenia containing a Liquidambar styraciflua fruit extract. The Liquidambar styraciflua fruit extract of the present invention has no cytotoxicity and is excellent in anti-myostatin activity, thereby being able to be usefully used as a pharmaceutical composition for muscle strengthening or preventing or treating sarcopenia.

Description

Composition for strengthening of muscle, preventing and treating Sarcopenia comprising Liquidambar styraciflua L.}

The present invention relates to a composition for the prevention and treatment of muscle strengthening or myotropia comprising the extract of the American maple.

In the 20th century, the number of populations has doubled, but in the 21st century, it is difficult to double the growth of the population due to lower birth rates and longer life expectancy. According to the 2015 United Nations World population prospects: the 2015 revision, the population over 60, from 910 million in 2015, will grow to about 1.4 billion by 2030, to 2.1 billion by 2050. It is estimated. In addition, the elderly aged 80 and older will reach about 438 million by 2050, and this increase is expected to continue for decades to come. In Korea, the population aged 65 and over reached 7.2% of the total population in 2000 and entered the aging society.In 2018, the aged population reached 20.8%. It is expected that the National Statistical Office will enter a post-aged society. In addition, it is pointed out as a big problem that Korea is approaching a very fast time to reach an aging society or an old age society compared to other countries. This problem is thought to be caused by the recent rapidly lower fertility rate, resulting in a sharp increase in the number of elderly population.

Aging is a phenomenon in which the body's physical function degenerates over time, and in humans, at 80, hearing is reduced by 30%, blood output by 45%, lung capacity by 60%, and muscle mass by 50%. have. Various physiological activities are not reduced by aging and some enzyme activity or hormone secretion function is increased. Therefore, the physiological change due to aging is a defect of the target functional enhancement or control mechanism, so it can be said that the physiological change is not a decrease in physiological activity but a decrease in adaptation ability.

Representative physiological changes with age are a decrease in muscle mass and strength. It is known that the decrease in muscle mass occurs after about 40 years, and about 8% decrease occurs in 10 years until the 70s. After that, a sharp decrease occurs and it is known to occur up to 15% in 10 years. Sarcopenia in the elderly causes a direct decrease in muscle strength, which increases the risk of death due to decreased physical function and impairment, as well as reduced metabolism and reduced immunity, such as high blood pressure, diabetes, arthritis, obesity, and cancer. It is also a cause of increasing the prevalence of metabolic diseases.

In connection with the reduction of muscle mass, studies of myostatin that inhibits the growth of skeletal muscle have been made, and antibodies for inhibiting the function of myostatin have been prepared. However, side effects have appeared, and antimyostatin materials using natural products Although research is ongoing, most studies at the cellular level have not been made and no studies have been reached.

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of muscle strengthening or myotropia comprising the American Prunus fruit extract.

Another object of the present invention is to provide a dietary supplement for the prevention and improvement of muscle strengthening or myotropia containing the American Prunus fruit extract.

In addition, another object of the present invention is the step of extracting alcohol by adding alcohol to the dried American Prunus fruit; Concentrating the extract under reduced pressure to remove the solvent; It is to provide a method for producing a composition for preventing and treating muscle strengthening and muscular dystrophy comprising the step of recovering the supernatant by centrifuging the obtained US maple fruit extract.

In addition, another object of the present invention is to provide a feed additive comprising a US maple fruit extract.

The inventors of the present invention, while conducting various studies to develop a natural substance for preventing and treating muscle weakness due to muscle strengthening or aging, confirmed that the American maple fruit extract is active against antimyostatin. The agent for strengthening or reducing muscle strength of tree fruit extracts is not known at all until now, and can be said to have great significance in that it is first developed by the present inventors.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention and treatment of muscle strengthening or myotropia, including the American Prunus fruit extract.

In one embodiment of the present invention, the composition is characterized by having antimyostatin activity, improving muscle strength and muscle density, and having an effect of increasing muscle mass.

In one embodiment of the present invention, the muscular dystrophy may be muscular dystrophy, myasthenia gravis, muscular dystrophy, myopathy, myotonia, muscle weakness, muscular dystrophy, muscular dystrophy or myasthenia gravis.

In one embodiment of the present invention, the US Prunus fruit extract is an organic solvent extract, preferably water or alcohol extract, more preferably ethanol extract, but is not limited thereto.

As another aspect, the present invention provides a functional food for preventing and alleviating myopathy, including the American maple fruit extract as an active ingredient.

In still another aspect, the present invention provides a health functional food for strengthening muscles, comprising the extract of American Prunus as an active ingredient.

In another aspect, the present invention comprises the steps of extracting by applying hot water to the dried American Prunus fruit; Concentrating the extract under reduced pressure to remove the solvent; Provided is a method for producing a composition for preventing and treating myotropia or strengthening muscle strength, comprising the step of recovering the supernatant by centrifuging the obtained US Prunus fruit extract.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating myotropia or a food for preventing and alleviating myotropia, comprising the extract of the American Prunus fruit produced by the above method.

In still another aspect, the present invention provides a feed additive comprising the US Prunus fruit extract as an active ingredient.

The American Prunus vulgaris extract of the present invention is not cytotoxic and has an excellent effect on the activity of antimyostatin, and as a result can be used for the prevention, alleviation and treatment of senile muscle diseases such as myotropia.

Figure 1 is a schematic diagram showing the process of producing a US Prunus fruit extract.
Figure 2 is a graph showing the results of cytotoxic and antioxidant activity according to the concentration of US Prunus extract:
A: Cytotoxicity results according to concentrations of 1000, 100, 10 ug / ml of the American maple tree hydrothermal extract (LSWE),
B: Antioxidant results according to concentrations of 200, 100, 10, 1 ug / ml and 50 ug / ml AC (ascorbic acid) of American maple fruit hydrothermal extract (LSWE).
Figure 3 is a graph and film showing the results of the analysis of the anti myostatin activity, ActivinA and GDF11 inhibitory activity and myostatin signaling of the American maple fruit extract:
A: antimyostatin results according to the concentration of 10, 1, 0.1 ug / ml of the American maple fruit hot water extract (LSWE),
B: ActivinA inhibitory activity results according to the concentration of 10, 1.0, 0.1 ug / ml of the American maple fruit hydrothermal extract (LSWE),
C: results of GDF11 inhibitory activity according to the concentration of 10, 1.0, 0.1 ug / ml of American maple fruit hot water extract (LSWE),
D: Lane 1 is a control (no treatment), Lane 2 is a myostatin only, Lane 3 is a positive control and inhibits the phosphorylation of myostatin and SB431542 (myostatin-binding receptors) small molecules simultaneously, lanes 4 and 5 simultaneously treat myostatin and American maple tree hydrothermal extract (LSWE).
Figure 4 is a graph confirming the change in muscle strength according to the number of days treatment of American maple tree hot water extract (LSWE) in the animal model:
A: weight, B: strength, C: muscle density.
FIG. 5 is a graph showing triglycerides, glucose content and muscle fatigue in an animal model treated with American Maple Fruit Hot Water Extract (LSWE):
A: triglycerides, B: glucose, C: LDH activity.
FIG. 6 is a graph showing the antimyostatin activity of American Prunus vulgaris extract (hot water / ethanol):
A: antimyostatin results according to the concentration of 10, 1, 0.1 ug / ml of the American maple fruit hot water extract (LSWE),
B: Antimyostatin results according to concentrations of 10, 1, 0.5, 0.1 ug / ml of American Maple Fruit Ethanol Extract (LSEE).

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for the prevention or treatment of myotropia, including the extract of the American maple tree for preventing and treating myotropia due to muscle strength or aging.

The American tree, Liquidambar styraciflua L. , is a deciduous broad-leaved arboreous tree native to North America. It is a large tree that grows up to 20m in height and is a plant that is mainly used as a roadside tree, landscape tree, or park tree in Korea because of the autumn leaves in various colors such as yellow, orange, purple, and red in autumn. In addition, it is resistant to pests and grows rapidly, and is suitable for the restoration of forests and natural greens damaged by fire or flood. English is called Sweetgum, which is derived from the resin from the tree. These resins can be used to make chewing gum or as a source of perfume, while Native Americans can use it as a seasoning spice. Seeds blown away after the American fruit has matured have been pointed out as a winter problem because the shells are so hard that they can be dangerous if they fall on roads with many pedestrians or around school attended by young students.

In the present invention, the term "strength strengthening" means that muscle strength and muscle density increase.

As used herein, the term "myotropenia" refers to a disease in which muscle volume and strength gradually decline.

In the present invention, the type of the extraction solvent used for extracting the American Prunus fruit is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent may include water, alcohols having 1 to 4 carbon atoms or mixed solvents thereof, and these may be used alone or in combination of one or more thereof. Specifically, water or ethanol may be used, but is not limited thereto. The ethanol may be 1 to 100%, but is not limited thereto. In the present invention, the extract may be a hot water extract or an ethanol extract, but is not limited thereto.

The extract has anti myostatin activity.

That is, the present invention relates to a composition for preventing and treating myopathy, which comprises the extract of the American Prunus as an active ingredient, and the composition includes a pharmaceutical composition and a food composition. Sarcopenia is a disease caused by a decrease in skeletal muscle mass, which is known to directly cause a decrease in muscle strength, resulting in various physical functions and disorders, and increase the risk of death. It is a disease that is a result of gradual skeletal muscle loss associated with aging (Park, Sung-Won, 2007, Muscle Reduction of the Elderly, Korean Journal of Endocrinology, Vol. 22, No. 1). In particular, the United States Prunus fruit extract of the present invention specifically inhibits myostatin activity, which is a factor that inhibits the increase in muscle mass, and has a therapeutic and prophylactic effect for muscle reduction.

In addition, the present invention is directed to a composition for strengthening muscles comprising the extract of the US Prunus as an active ingredient, the composition includes a health food composition or a food composition. "Increased muscle strength" refers to the phenomenon in which the total muscle strength increases due to an increase in skeletal muscle mass or an improvement in muscle function, and does not mean a limited effect on a patient group of a specific disease.

As used herein, the term "myostatin (MSTN)" is a protein that regulates muscle growth, belongs to the transforming growth factor-β TGF-β family and grows and differentiation factor-8 (GDF-8). )to be.

It is known that myostatin binds directly to activin receptor type 2 and also affects Smad signaling mechanisms. Inhibiting the activity of myostatin is known to promote muscle differentiation and play an important role in the improvement of various metabolic diseases. The present invention provides a pharmaceutical composition and a food composition, including the extract of the American Maple Fruit, which can improve the symptoms of muscle diseases such as myotropin by adjusting the myostatin activity and enhance muscle strength.

The composition of the present invention may be prepared as a pharmaceutical composition. The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration. Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used.

On the other hand, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and rate of release, duration of treatment, factors including the drug being used in combination or co-administration, and other factors well known in the medical art can be determined. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient of the patient. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng to about 100 mg / kg, specifically 1 ng to about 10 mg / kg, per adult, and the frequency of administration of the composition of the present invention is particularly limited thereto. However, it may be administered once a day or several times in divided doses. However, since the dosage may vary depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

In another aspect, the present invention provides a health functional food for the prevention and improvement of muscle strength or myotropia, including the American Prunus fruit extract.

The food composition of the present invention includes compositions for all types of foods, such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, the health food can be prepared in the form of gum, vitamin complex, tea, juice and drink containing the US maple extract, granulating, encapsulating or powdering a food composition comprising the US maple extract as an active ingredient. It can be consumed by becoming angry. Functional food compositions of the present invention may include ingredients that are commonly added in the manufacture of food, and may include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured with a drink, in addition to the US Pung tree extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, licorice extract may be further included.

The food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids , Protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages and the like. In addition it may contain extracts for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination.

In addition, the present invention provides a feed composition comprising the US Prunus fruit extract as an active ingredient. The American maple fruit extract may be prepared in the same manner as the pharmaceutical composition or the food composition.

The American Prunus fruit extract of the present invention exhibits an overall increase in muscle strength due to an increase in skeletal muscle mass or an improvement in muscle function, and thus may be included in a feed composition as a growth promoter for animals or livestock.

Specifically, the feed comprising the US Prunus fruit extract according to the present invention as an active ingredient can be prepared in various forms of feed known in the art, and preferably, rich feed, research feed or special feed.

The rich feed includes seed fruits and grains containing grains such as wheat, oats and corn, and bran, soybeans, fluids, sesame seeds, linseed and coco palms including rice bran, wheat bran and barley bran. Concentrated fresh liquids obtained from fish oil, fish tails, and fish residues such as by-products such as oil products, sweet potatoes, potatoes, etc. Animal feed, yeast such as dried whey, which is the balance of the production of phosphorus fish soluble, meat meal, blood meal, milk powder, skim milk powder, milk from cheese, and casein from skim milk , Chlorella and algae.

Forage, raw vegetables such as grasses, grasses, and green grass, fodder turnips, fodder beets, and root vegetables such as Lutherbearger, a type of turnip, raw grass, green grass crops, and grains Silage (silage), grasses, grasses, and dried hay, straws of breeder crops, and leaves of legumes.

Special feeds contain mineral feeds such as shells and rock salts, urea feeds such as urea and its derivatives such as diureide isobutane, and natural feed ingredients. Feed additives, dietary supplements, and the like that are added to the.

The feed composition according to the present invention may comprise various feed additives.

In the present invention, the term "feed additive" refers to a substance added to a feed for various effects such as supplementation of nutrients and prevention of weight loss, enhancement of digestive utility of fiber in feed, improvement of oil quality, prevention of reproduction disorder and improvement of conception, and prevention of high temperature stress in summer. Say. The feed additive of the present invention corresponds to a supplementary feed under the Feed Control Act, and minerals such as sodium bicarbonate, bentonite, magnesium oxide, and composite minerals, and trace minerals such as zinc, copper, cobalt, and selenium. Preparations, vitamins such as keratin, vitamin E, vitamins A, D, E, nicotinic acid, and vitamin B complexes, protective amino acids such as methionine and lyric acid, protective fatty acids such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast culture Probiotics such as water, mold fermentation products, yeasts and the like may be further included.

The feed composition according to the present invention is not particularly limited as long as it is an individual for the purpose of increasing the total muscle strength and promoting growth due to an increase in the amount of skeletal muscle or an improvement in muscle function, and any one can be applied. Such subjects include animals such as non-primates (eg, cattle, pigs, horses, cats, dogs, rats and mice) and primates (eg, monkeys, such as cynomolgous monkeys) and Mammals, including chimpanzees). In another embodiment, the subject is a livestock animal (eg horse, cow, pig, etc.) or a pet animal (eg dog or cat). In addition, fish, for example, flounder, flounder, eel, freshwater eel, eel, rockfish.

The dosage of the feed composition according to the invention will depend on a number of factors such as the species, size, weight, age of the animal. In principle, typical dosages may range from 0.001 to 10 g per animal / day. However, the present invention is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of American Prunus Fruit Extract

The American Prunus fruit was sufficiently dried and extracted with hot water at a concentration of 20 mg / ml. Hot water extraction was extracted for 20 minutes at 90 ℃ using distilled water, it was repeatedly extracted three times. The extracted sample was removed from the water using a rotary vacuum concentrator and centrifuged at 4000 rpm for 20 minutes to recover only the supernatant. The supernatant was measured for the concentration of the sample using a centrifugal concentrator. The extraction was repeated three times, and the extraction yield of 7.1% was confirmed (FIG. 1).

<Example 2> Cytotoxicity and Antioxidant Activity of the American Prunus Fruit Extract

To determine the cytotoxicity of L. stryracifluca hot water extracts (LSWE), Cytotoxicity experiments were incubated in 5% CO ₂ incubator by seeding Hek293 cells per well in 96 well plate 1 × 10 ⁴ cells / 100 ul (in DMEM with 10% FBS and 1% penicillin / streptomycin) cells. After 24 hours, American maple tree hydrothermal extract (LSWE) was treated by concentration (0.5, 1, 10, 100 ug / ml) and incubated for 12 hours in a 5% CO ₂ incubator. Thereafter, 10 ul of WST reagent (DaeilLab, Korea) was added and the reaction was carried out for 1 hour, and then the absorbance was measured at 415 nm to determine cytotoxicity. Only cells treated without extracts were treated as positive controls, and H ₂ O ₂ treated groups as negative controls, and the results for cytotoxicity were compared using the following equation.

As a result, the American maple tree hydrothermal extract (LSWE) did not show cytotoxicity at the concentration of 100 ug / ml in Hek293 cells, it was confirmed that it has less than 5% toxicity at other concentrations (Fig. 2A).

In addition, in order to confirm the antioxidant activity of the US maple fruit extract, 100 ul of DPPH reagent and 100 ul of American maple fruit hydrothermal extract (LSWE) were treated to a final concentration of 10, 1, 0.1 ug / ml at room temperature and darkroom. After reacting for 30 minutes at, the absorbance at 562 nm was measured.

The control lane is a non-extractable lane, and AC (10) is a positive control, a lane treated with 10 g / ml of vitamin C (ascorbic acid), a representative antioxidant.

As a result, 10 ug / ml treatment of the US maple fruit hot water extract (LSWE) confirmed the antioxidant activity similar to that of vitamin C at a concentration of 10 ug / ml (Fig. 2B).

Example 3 Antimyostatin Activity Analysis of American Prunus Fruit Extract

In order to confirm the activity of the antimyostatin of the American Prunus fruit extract was confirmed through a luminase enzyme assay (luciferase assay) system.

Specifically, Hek293 cells were seeded with 2.0 × 10 ⁴ cells per well in 96 well plates in DMEM medium (10% FBS, 1% penicillin / streptomycine, 1% geneticine) and cultured in a 5% CO ₂ incubator. After 24 hours, the medium was replaced with DMEM without FBS, and cultured in a CO ₂ incubator treated with 1 nM of recombinant myostatin (R & D system, USA) and American maple fruit hydrothermal extract (LSWE). After 24 hours, the medium was removed and treated with 65 ul of DMEM medium and 65 ul of reagent (Bright-Glo luciferase assay system, USA) to measure luminescence on a micro plate luminometer. The measured values were expressed in% of antimyostatin activity using the values of positive control and negative control. The measured values were expressed by the following equation using the values of the positive control (myostatin only, MSTN only) and negative control (no myostatin, No MSTN). The antimyostatin activity was expressed in%. Myostatin Inhibitory Activity (%) = (Luminescence Value of 1 nM Myostatin Treatment-Luminous Value of American Maple Fruit Extract Treatment) x 100 / (Luminescence Value of 1 nM Myostatin Treatment- Luminescence Levels in Experimental Zones Without Myostatin Treatment)

As a result, it was confirmed that the activity inhibitory effect on myostatin increased in a concentration-dependent manner when treated with American Prunus extract, and a trendline formula of y = 7.1808x + 24.467 was obtained. The IC 50 value for 1 nM myostatin was found to be 3.56 ug / ml (FIG. 3A).

Example 4 Analysis of ActivinA and GDF11 Inhibitory Activities of American Prunus Fruit Extract

In order to confirm the ActivinA and GDF11 inhibitory activity of the extracts of the American maple tree, it was confirmed through a luciferase assay system.

Specifically, the inhibitory activity of ActivinA and GDF11 belonging to the superfamily, such as myostatin, was measured against the extract of American Prunus japonica. Hek293 cells were seeded with 2.0 × 10 ⁴ cells per well in 96 well plates in DMEM medium (10% FBS, 1% penicillin / streptomycine, 1% geneticine) and cultured in a 5% CO ₂ incubator. After 24 hours, the medium was replaced with DMEM without FBS, the ActivinA treatment group treated with ActivinA 0.5 nM, the GDF11 treated group treated with GDF11 1 nM per well, and each treated group was treated with American maple tree hydrothermal water. Extracts (LSWE) were treated by concentration and incubated in a CO ₂ incubator. After 24 hours, the medium was removed and treated with 65 ul of DMEM medium and 65 ul of reagent (Bright-Glo luciferase assay system, USA) to measure luminescence on a micro plate luminometer. The measured values were expressed in% of antimyostatin activity using the values of positive control and negative control. The measured values were for positive control (positive control, activinA or GDF11 only, activinA only or GDF11 only) and negative control (negative control, activinA or GDF11 not treated, No activinA or No GDF11). By using the following formula was expressed as antiactivinA or anti-GDF11 activity in%. activinA or GDF11 inhibitory activity (%) = (luminescence level of 0.5 nM activinA or 1 nM GDF11 only-Luminous value of American maple fruit extract) x 100 / (0.5 nM activinA or 1 nM GDF11 only) Luminescence value of spheres-Luminescence values of experimental groups not treated with 0.5 nM activinA or 1 nM GDF11)

As a result, signal transduction inhibition of American maple tree hydrothermal extract against 0.5 nM ActivinA was inhibited by about 60% at 1000 ug / ml, and 20% inhibition at 100 ug / ml. No inhibition occurred at / ml. From these results, a trendline formula of y = 11.654ln (x) -25.091 was obtained and the IC50 value for activinA of 0.5 nM was found to be 628.52 ug / ml (FIG. 3B).

In addition, the signal transduction inhibition of the American maple tree hydrothermal extract against 1.0 nM GDF11 was about 96% inhibited at 10 ug / ml of the American maple tree hydrothermal extract and 20% inhibition at 1.0 ug / ml. From these results, a trendline formula of y = 8.7544x + 8.8995 was obtained, and it was confirmed that the IC50 value for GDF11 of 1 nM was 4.69 ug / ml. Summarizing these results, it was confirmed that the American Prunus hydrothermal extract was 1.32 times higher than activinA and 176.54 times higher than GDF11 based on the IC50 value of myostatin (FIG. 3C).

Example 5 Myostatin Signal Transduction Analysis of American Prunus Fruit Extract

To determine the effect of LSWE on the myostatin signaling pathway, we examined the phosphorylation of smad 2 transcription factor through western blot.

Specifically, the smad transcription factor is a receptor for signal induction of the TGF-β receptor to which myostatin belongs, and plays an important role in cell growth and division. For Western blot, HepG2 cells were seeded at 2.0 × 10 ⁵ cells per well in 6 well plates in DMEM medium (10% FBS, 1% penicillin / streptomycine) and cultured in a 5% CO ₂ incubator. After 24 hours, the medium was replaced with DMEM without FBS, and after 4 hours, 10 nM of recombinant myostatin and American Prunus hydrothermal extracts at IC100 (105 ug / ml) and IC10 (7 ug / ml) concentrations ( LSWE) was treated for 30 minutes.

Cells were washed twice with PBS buffer and then RIPA buffer (20 mM tris-HCl, pH7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxychloate, 2.5 mM sodium pyrophosphate). , 1 mM β-glycerophosphate, 1 mM NA3VO4, 1 ug / ml leupeptin, cell signaling, USA), protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche, USA) were treated.

Cells were disrupted using a sonicator and centrifuged at 4 ° C. and 12,000 rpm for 20 minutes. Supernatants were quantified by BCA assay and electrophoresed on 10% polyacrylamide gel. After electrophoresis, transfer was made to PVDF membrane and blocked for 2 hours at room temperature using 5% BSA or 5% skim milk. Membrane was washed three times for 10 minutes at room temperature using TBS-T buffer and the primary antibody (Samd2, P-Smad2, Cell signaling, USA) was reacted for 2 hours at room temperature. After washing three times for 10 minutes at room temperature using TBS-T buffer and the secondary antibody (a nt i-Rabbit IgG (Cell signaling, USA)) was reacted for 2 hours at room temperature. Membrane was washed three times for 10 minutes at room temperature using TBS-T buffer, and was exposed to x-ray film using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, USA).

As a result, it was confirmed that the American maple tree hydrothermal extract (LSWE) inhibited myostatin signal transmission in a concentration-dependent manner through the western blot (phosphorylation of Smad transcription factor) by interfering with myostatin signal transduction. Myostatin (10 nM), SB431542 (small molecule that inhibits phosphorylation of receptors to which myostatin binds), and American Maple Fruit Hot Water Extract (LSWE) were singly or mixed to confirm phosphorylation of Smad2. When all of myostatin, SB431542, and American maple fruit hot water extract (LSWE) were not treated with the cells, myostatin signal was not transmitted to the receptor, so no Smad2 phosphorylation occurred. In cells treated with myostatin alone, myostatin signal was transferred into the cell, resulting in phosphorylation of Smad2. In cells treated with myostatin and SB431542, Smad2 phosphorylation did not occur by preventing SB431542 from transmitting myostatin signals intracellularly. For cells treated with myostatin and American maple fruit hydrothermal extract (LSWE) (IC100 (105 ug / ml), IC10 (7 ug / ml)), US maple fruit hydrothermal extract (LSWE) Phosphorylation of Smad2 did not occur concentration-dependently by preventing the signal from being delivered intracellularly (FIG. 3D). Therefore, it was confirmed that the extract of the American Prunus hydrothermal extract inhibits the signaling of myostatin and interferes with the phosphorylation of Smad2, thereby inhibiting the signal transduction into the nucleus.

Example 6 Analysis of the Effect on Muscle of the American Prunus Fruit Extract in Animal Model

To determine the effect of American maple tree hydrothermal extract (LSWE) on muscle at the in vivo level, ICR mice were orally administered to confirm the change in muscle strength.

Specifically, the mice used in the experiment were 4 week old male ICR mice, which were orally administered once a day for 14 days. The injection amount was orally administered 15 times to the IC50 value for 1 nM myostatin confirmed by luciferase assay experiment. Weight, strength test, and endurance test were conducted on days 0, 7, and 14, and muscle density was calculated by measuring the weight and volume of forelimb and hind limb muscles after the experiment.

In addition, blood samples were collected and serum triglycerides, glucose and LDH activities were measured. Triglycerides and glucose were measured using Lipidocare (SD biosense, Korea), and the content was measured using LDH activity and Biovision kit (Biovision, USA).

As a result, the mouse body weight did not show a significant difference compared to the control group through oral administration of the American Prunus hydrothermal extract (LSWE) for 14 days (FIG. 4A).

Muscle strength, however, increased significantly by 6.5% compared to the control group at 14 days (p <0.001) (FIG. 4B).

After 14 days of oral administration, the muscle density, which was calculated based on the mass and volume of the forelimbs and hind limbs, was significantly increased by 11.4% in the hind limbs compared to the control group (p <0.05). (FIG. 4C).

In addition, compared to the control group, triglycerides in the animal model treated with the American maple fruit hot water extract (LSWE) were significantly decreased by 37% (p <0.01) (FIG. 5A), and the glucose content was 16.8% significantly compared to the control group. Decreased (p <0.05) (FIG. 5B). LDH activity, an indicator of muscle fatigue, was significantly increased by 23.6% compared to the control group (p <0.05) (FIG. 5C).

Example 7 Analysis of Antimyostatin Activity of Extracts of American Prunus Fruits Extracted by Solvents

In order to confirm the antimyostatin activity of the extract of the American Prunus fruit according to the extraction solvent was confirmed through a luciferase assay system.

Specifically, Hek293 cells were seeded with 2.0 × 10 ⁴ cells per well in 96 well plates in DMEM medium (10% FBS, 1% penicillin / streptomycine, 1% geneticine) and cultured in a 5% CO ₂ incubator. After 24 hours, the medium was replaced with DMEM without FBS, and the concentration of 1 nM recombinant myostatin (R & D system, USA), American maple fruit hydrothermal extract (LSWE) and American maple fruit ethanol extract (LSEE) Each treatment was incubated in a CO ₂ incubator. After 24 hours, the medium was removed and treated with 65 ul of DMEM medium and 65 ul of reagent (Bright-Glo luciferase assay system, USA) to measure luminescence on a micro plate luminometer. The measured values were expressed in% of antimyostatin activity using the values of positive control and negative control. The measured values were expressed by the following equation using the values of the positive control (myostatin only, MSTN only) and negative control (no myostatin, No MSTN). The antimyostatin activity was expressed in%. Myostatin Inhibitory Activity (%) = (Luminescence Value of 1 NM Myostatin Treatment-Luminous Value of American Maple Fruit Extract Treatment (Heat / Ethanol)) x 100 / (1 nM Myostatin Treatment) Luminescence Values of the Experimental Zone-Luminescence Values of the Experimental Zone without Myostatin Treatment)

As a result, the activity inhibitory effect on myostatin showed that the American P. sulcus hydrothermal extract (LSWE) had an IC 50 value of 3.56 ug / ml for 1 nM myostatin (FIG. 6A). Extract (LSWE) was confirmed that the IC 50 value is 0.33 ug / ml (Fig. 6B).

Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (17)

Pharmaceutical composition for the prevention and treatment of muscle strengthening or myotropia comprising the extract of Liquidambar styraciflua L. fruit. The method of claim 1,
The composition is that having a myostatin (anti Myostatin) activity, pharmaceutical composition. The method of claim 1,
The composition is to improve muscle strength and muscle density, pharmaceutical composition. The method of claim 1,
The composition will have a muscle mass increase effect, pharmaceutical composition. The pharmaceutical composition of claim 1, wherein the extract is a hydrothermal extract or an ethanol extract. The pharmaceutical composition of claim 1, wherein the myopathy is muscular dystrophy, myasthenia gravis, muscular dystrophy, myopathy, myasthenia gravis, muscular weakness, muscular dystrophy, muscular dystrophy or myasthenia gravis. Health functional food for the prevention and alleviation of myopathy, which contains American maple fruit extract as an active ingredient. A health functional food for strengthening muscle strength, which contains the extract of American Maple Fruit as an active ingredient. (a) extracting by adding a solvent to the dried American Prune fruit;
(b) concentrating the extract under reduced pressure to remove the solvent; And,
(C) a method for producing a composition for preventing and treating myotropia, comprising recovering the supernatant by centrifuging the US Prunus fruit extract obtained in (b). The method of claim 9, wherein the solvent of step (a) is water or ethanol. The method of claim 9, wherein the extraction of step (a) is performed for 1 to 24 hours, and repeated for 1 to 3 times. 10. The method of claim 9, wherein in the step (a), the American Prunus fruit is prepared in a composition for preventing and treating myotropia, characterized in that it comprises a concentration of 10 to 30mg / ml. A pharmaceutical composition for preventing and treating myopathy, comprising the extract of the American Maple Fruit extracted by the method of any one of claims 9 to 12. A food for preventing and alleviating myopathy, comprising the extract of the American Prunus fruit extracted by the method of any one of claims 9 to 12. A muscle strengthening food comprising the extract of the American Maple fruit extracted by the method of any one of claims 9 to 12. Feed additives containing American maple fruit extract as an active ingredient. The feed additive of claim 16 wherein the extract is a hydrothermal extract or an ethanol extract.

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