KR101997060B1 - Composition for prevention or treatment of muscular disorder, or improvement of muscular functions comprising fermented deer antler - Google Patents

Abstract

The present invention relates to a composition for preventing or treating muscular disorder, or improving muscular functions comprising fermented Cornu cervi pantotrichum as an effective component. The composition comprising fermented Cornu cervi pantotrichum as an effective component increases protein expression of p-mTOR, p-p70S6K and p-4EPB-1 involved in synthesis of muscular protein and inhibits mRNA expression of MuRF1 and atrogin-1 involved in decomposition of muscular protein, thereby increasing muscle mass. In addition, the composition can be safely used without side effects by being derived from natural materials, thereby being usefully used for medicines or foods.

Description

TECHNICAL FIELD The present invention relates to a composition for prevention or treatment of muscular diseases or a composition for improving muscular function comprising fermented antler as an active ingredient,

The present invention relates to a composition for preventing or treating muscular diseases or a composition for improving muscle function, and more particularly, to a composition for preventing or treating muscular diseases or a composition for improving muscular function comprising Cornu cervi pantotrichum as an active ingredient .

Over the past 50 to 100 years, the decline in human physical activity has been associated with increased incidence of metabolic diseases such as type 2 diabetes, obesity, and cardiovascular disease. Lack of physical activity is the fourth leading cause of deaths reported by the World Health Organization. Because of these events, organizations such as the World Health Organization, the American Heart Association, and the Heart Foundation recommend at least 30 minutes of aerobic exercise for at least five days a week. Indeed, exercise has reduced the incidence of diabetes, obesity, breast cancer and colorectal cancer, and has shown good therapeutic effects in depression (British Journal of Pharmacology, 170: 1153-1166, 2013; American Journal of Cardiology, 110: 2012).

Muscle atrophy is caused by a gradual decrease in muscle mass and refers to muscle weakness and degeneration (Cell, 119 (7): 907-910, 2004). Atrophy is promoted by inactivity, oxidative stress, chronic inflammation and weakens muscle function and athletic performance (Clinical Nutrition, 26 (5): 524-534, 2007). The most important factor determining muscle function is muscle mass, which is maintained by a balance of protein synthesis and degradation. Muscular atrophy occurs when proteolysis occurs more than synthesis (The International Journal of Biochemistry and Cell Biology, 37 (10): 1985-1996, 2005).

Muscle size is regulated by intracellular signaling pathways that lead to anabolism or catabolism in the muscle. If the signaling pathway leads to synthesis rather than to muscle protein degradation This results in increased muscle mass (hypertrophy) and increased muscle fiber (hyperplasia) as muscle protein increases (The Journal of Sports Science, 20 (3): 1551-1561, 2011).

Factors involved in myoprotein synthesis induce protein synthesis by phosphorylating downstream proteins from the stimulation of phosphatidylinositol-3 kinase (PI3K) / Akt pathway in myocytes. The activity of the mammalian target of rapamycin (mTOR) by PI3K / Akt signaling is recognized as a central growth signaling factor that integrates various growth signals in the cell. mTOR induces muscle protein synthesis by activating two factors that initiate mRNA translation, 4E-binding protein (4EBP-1) and phosphorylated 70-kDa ribosomal S6 kinase (p70S6K) (The Journal of Sports Science, 20 (3): 1551-1561, 2011; The International Journal of Biochemistry and Cell Biology, 43 (9): 1267-1276, 2011). Conversely, when the transcription factor forhead box (FoxO) migrates from the cytoplasm to the nucleus, it increases the expression of the E3 ubiquitin ligase, atrogin-1 and MuRF1, involved in proteolysis (Disease Models and Mechanisms, 6: 25-39 , 2013). Increased expression levels of these proteins promotes protein degradation in muscles, resulting in reduced muscle mass. Thus, activation of mTOR and inhibition of atrogin-1 and MuRF1 expression increase muscle mass and increase muscle mass.

Muscle cell differentiation and muscle formation are regulated by a variety of muscle regulatory factors. Among them, MyoD induces the expression of muscle specific genes and induces the differentiation of muscle satellite cells into myoblasts. Induction of myogenin expression by the activity of MyoD is the most important factor in the fusion of myofibroblasts and is involved in the formation of myotubes. The muscle fibers formed through this process are bundled to finally form muscles (Cellular and Molecular Life Sciences, 70: 4117-4130, 2013).

On the other hand, Cornu cervi pantotrichum (antler) refers to the cartilage tissue regenerated annually by young horns which are dense and non-ossified in the hairs of plum, marrow and relative animals belonging to the deer family, It has been widely used in Korea and other eastern countries such as Japan and China as the best blood medicine tonic for a long time. Its properties and efficacy have been recorded in the literature of the main herb Gangmok and Dongbokgam.

Until now, research on antler has been restricted to the degree of clarifying the clinical effect in Oriental medicine and the physiological mechanism of antler in both sides. In recent years, active research has been carried out on physiologically active ingredients of antler antler, and it has been reported that antler antler has a wide variety of effects at the level of extract, such as immunity, anti-fatigue, anti-thrombosis, anti-aging, hypotensive effect and anti-inflammatory effect.

As described above, various methods have been studied to take antler with various effects. When the antler is used as a premix and powdered, since the absorption rate of antler is low, various ingredients of antler can not be absorbed into the body, . In addition, since the extracting method for extracting the antler extracts mainly focuses on the extraction of some active ingredients, there is a problem that it is difficult to extract all the various components contained in the antler extract.

In addition, since antler is accompanied by side effects such as diarrhea, it is used in combination with a component having astringent action in one room. In this process, the efficacy of antler may be attenuated. In the process of soaking in alcohol for use in oriental medicine, There is a problem that a large amount of the effective component is lost. In addition, the conventional method of hydrolysis, which is a conventional method for treating antler, takes a long time, and the composition of the extracted active ingredient is not constant, which makes it difficult to control the quality of the product. The extraction method using organic solvent extracts only the lipid component , It is difficult to extract the aqueous active ingredient and it is harmful to the human body due to the residual organic solvent which can not be removed.

Accordingly, various methods for enhancing the utility of the antler have been studied, and in particular, methods for improving the absorption rate and efficacy by fermenting antler have been developed.

The inventors of the present invention have searched for a natural substance which has excellent muscle-controlling activity and can be safely applied. As a result, the fermented antler fermented by using Bacillus subtilis strain exhibits superior muscular function improving effect alone .

Accordingly, the present invention has been completed for a long time by studying the fermented antler using the Bacillus subtilis strain for preventing or treating muscle diseases or improving muscle function.

KR 10-1448496 B

It is an object of the present invention to provide a food composition for preventing muscle disorders or improving muscular function comprising fermented antler as an active ingredient.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating muscle diseases comprising fermented antler as an active ingredient.

In order to achieve the above object, the present invention provides a food composition for preventing muscle disorders or improving muscular function comprising fermented antler as an active ingredient.

According to one embodiment of the present invention, the fermented antler can be obtained by fermenting antler antler with Bacillus subtilis .

According to one embodiment of the present invention, the composition may prevent or ameliorate one or more diseases selected from the group consisting of muscular atrophy, muscular dystrophy, muscle degeneration and sarcopenia.

According to an embodiment of the present invention, the antler may be an antler powder or an antler extract.

According to an embodiment of the present invention, the antler extract may be one obtained by extracting antler antler with at least one solvent selected from the group consisting of water, organic solvents having 1 to 6 carbon atoms, subcritical fluid and supercritical fluid.

The present invention also provides a pharmaceutical composition for preventing or treating muscle diseases, which comprises fermented antler as an active ingredient.

The composition comprising the fermented antler according to the present invention as an active ingredient increases protein expression of p-mTOR, p-p70S6K and p-4EPB-1 involved in muscle protein synthesis and inhibits MuRF1 and atrogin-1 Lt; RTI ID = 0.0 > mRNA < / RTI > expression.

Further, since the present invention is a composition derived from a natural product, it can be safely used without side effects and can be usefully used in medicines or foods.

FIG. 1 shows the results of measuring the amount of protein expression of p-mTOR in L6 muscle cells according to the fermented antler treatment of the present invention.
FIG. 2 shows the results of measuring the amount of protein expression of p-mTOR, p-p70S6K, and p-4EBP-1 according to the treatment of fermented product extracted with a barnyard subtilis KCCM 11402P in a L6 muscle cell .
FIG. 3 shows the results of measurement of the mRNA expression levels of atrogin-1 and MuRF1 according to the treatment of the fermented product extracted with the antler of Bacillus subtilis KCCM 11402P in L6 muscle cells.
FIG. 4 shows the results of measuring the amount of protein expression of p-mTOR, p-p70S6K, and p-4EBP-1 according to the treatment of the fermented product extracted with the barnyard subtilis KCTC 13022 in the L6 muscle cells .
FIG. 5 shows the results of measuring the expression levels of atrogin-1 and MuRF1 mRNA in the L6 muscle cells according to the treatment of Bacillus subtilis KCTC 13022 fermented with fermented extract.

Hereinafter, the present invention will be described in detail.

The present invention provides a food composition for preventing muscle disorders or improving muscular function comprising fermented antler as an active ingredient.

In the present specification, 'fermented antler' may be obtained by fermenting antler antler with Bacillus subtilis . Preferably, antler antler can be fermented with Bacillus subtilis SST-9960 [Deposit No. KCCM 11402P].

The deer antler may be a deer antler powder or a deer antler extract.

The antler extract may be obtained by extracting antler antler with at least one solvent selected from the group consisting of water, organic solvents having 1 to 6 carbon atoms, subcritical fluids and supercritical fluids. Among the above solvents, the organic solvent having 1 to 6 carbon atoms is preferably an alcohol having 1 to 6 carbon atoms, more preferably an alcohol having 1 to 3 carbon atoms.

The 'fermented antler' can be used without limitation as long as it is fermented by conventional fermentation methods known in the art.

However, in the present invention, the 'fermented deer antler' is used to (1) put the antler powder or the antler extract in a fermentation tray and add 100 parts by weight of antler powder or antler extract to bacillus subtilis (2) primary fermentation at 20 to 40 캜, preferably 25 to 35 캜, for 2 to 4 days, preferably 2.5 to 3.5 days, and (3) ) 20 to 80 parts by weight of the culture broth is added to 100 parts by weight of the first fermented green tea, and then the second fermentation is carried out under the same conditions as the first fermentation.

In particular, (4) after the secondary fermentation, the fermentation is carried out at 2 to 10 ° C for 5 to 24 hours at a low temperature, (5) ventilation drying is performed at 25 to 35 ° C, preferably 28 to 33 ° C, Is further preferable in terms of the effect when it is produced by further performing the step. According to one embodiment of the present invention, it has been confirmed specifically that the effect of improving muscle function is further enhanced when the fermented product of antler granules or antler extract is subjected to low-temperature aging.

The 'fermented antler' produced by the above method is more effective than the fermented antler prepared by another method or the fermented antler fermented and produced by another strain other than Bacillus subtilis, And further enhancement was confirmed.

In addition, according to one embodiment of the present invention, it is confirmed that the fermented antler obtained by fermenting the extract of antler extract is more effective than the fermented antler obtained by fermenting the antler granule.

The 'fermented deer antler' may be used after being dried and powdered, or may be extracted and used in the form of an extract.

In the present specification, the 'fermented product extracted from a barnyard extract' may be obtained by fermenting a barnyard extract with Bacillus subtilis .

In the present specification, 'muscle' refers to the tendons, muscles, and tendons comprehensively, and 'muscular function' refers to the ability to exert its force by contraction of muscles, and muscles exhibit maximum contraction force Muscular endurance which is the ability to indicate how long or how many times the muscle can repeat contraction and relaxation on a given weight, and the ability to exert a strong force in a short period of time. These muscle functions are hosted by the liver and are proportional to muscle mass. The term " muscle function improvement " refers to better improvement of muscle function. Also, "improvement of muscle function" refers to increasing muscle or inhibiting muscle loss.

The composition for prevention and treatment of muscular diseases or the composition for improving muscle function of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the fermented antler. For example, it may contain known muscle function improving ingredients. The addition of an additional ingredient may further enhance the muscle function-improving effect of the composition of the present invention. When the above ingredients are added, skin safety, easiness of formulation, and stability of effective ingredients can be considered according to the combined use. In one embodiment of the invention, the composition comprises muscle-improving ingredients known in the art as camphorafabi flora extract, Piper retrofractum Vahl .. fruit extract, myristate , cucumber beetaine extract , Pandurata extract, grape root extract, camel root extract, and red ginseng extract may be further included. The additional component may be contained in an amount of 0.0001 wt% to 10 wt% based on the total weight of the composition. For example, from 0.0001 wt% to 1 wt%, from 0.0001 wt% to 0.1 wt%, from 0.0001 wt% to 0.001 wt%, from 0.001 wt% to 10 wt%, from 0.001 wt% 0.001 wt% or more to 0.1 wt% or less, 0.01 wt% or more to 10 wt% or less, or 0.01 wt% or more to 1 wt% or less. The above content range may be adjusted according to requirements such as skin safety, easiness in formulation of the composition, and the like.

The composition for preventing muscle disorders and improving muscular function of the present invention may be a food composition. When the composition for preventing muscle disorders and improving muscular function of the present invention is a food composition, it can be used for prevention or improvement of muscle diseases due to muscle wasting or degeneration. Muscle exhaustion and degeneration are caused by genetic factors, acquired factors, aging, etc. Muscle exhaustion is characterized by gradual loss of muscle mass, weakness and degeneration of muscles, especially skeletal muscle or vascular and cardiac muscles. Examples of such diseases include atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and sarcopenia.

The food composition of the present invention is characterized in that the food composition includes all forms of functional food, nutritional supplement, health food, food additives and feed, And other animals.

Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art. Common foods include but are not limited to beverages (including alcoholic beverages), fruits and processed foods (eg, canned fruits, jam, maamalade, etc.), fish, meat and processed foods (Eg butter, chewing), edible vegetable oil, margarine (such as corn oil, etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), juice, various drinks, cookies, , Vegetable protein, retort food, frozen food, various kinds of seasoning (for example, soybean paste, soy sauce, sauce, etc.). The nutritional supplement may be prepared by adding the fermented deer antler to capsules, tablets, rings and the like. For example, the health functional food may be prepared by liquefying, granulating, encapsulating and pulverizing the fermented starch so that the fermented starch itself can be prepared in the form of tea, juice, and drink to be used for drinking (health drink) . In addition, in order to use the fermented antler as a food additive, it may be prepared into a powder or may be prepared into a concentrated form after being extracted. In addition, it can be prepared in the form of a composition by mixing with the fermented antler and a known active ingredient known to have an effect of improving muscle function.

When the composition for preventing muscular diseases and improving muscular function of the present invention is used as a health beverage composition, the health beverage composition may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

Fermented antler can be contained as an effective ingredient of a food composition for preventing muscle diseases and improving muscle function. The amount of the fermented antler is not particularly limited, but is preferably 0.01 to 100 parts by weight, By weight. The food composition of the present invention may be prepared by mixing with fermented antler and other active ingredients known to be effective in preventing muscle diseases and improving muscular function.

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjusting agent, Glycerin, an alcohol or a carbonating agent, and the like. In addition, the health food of the present invention may contain natural fruit juice, fruit juice drink, or pulp for the production of 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 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also provides a pharmaceutical composition for preventing or treating muscle diseases, which comprises fermented antler as an active ingredient.

The fermented deer antler may be obtained by fermenting antler antler with Bacillus subtilis.

The pharmaceutical composition for preventing or treating muscle diseases of the present invention can be used for preventing or treating muscle diseases due to muscle wasting or degeneration. Such muscle wasting or degeneration is caused by genetic factors, acquired factors, aging, etc., and muscle wasting is characterized by gradual loss of muscle mass, weakening and degeneration of muscles, especially skeletal muscle or veterinary muscle and heart muscle.

Specific examples of muscle diseases include atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and sarcopenia. The composition of the present invention has an effect of increasing muscle mass, and the kind of muscle is not limited.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable salt of a fermented antler. The term " pharmaceutically acceptable " as used herein refers to those that are physiologically acceptable and do not normally cause an allergic reaction or a similar reaction when administered to humans, wherein the salt includes a pharmaceutically acceptable free acid acid is preferred.

The pharmaceutically acceptable salt of the fermented antler may be an acid addition salt formed using an organic acid or an inorganic acid, and examples of the organic acid include formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, There may be mentioned acetic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, , p-toluenesulfonic acid or methanesulfonic acid. The inorganic acid includes, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt may preferably be in the form of a hydrochloride or an acetate, more preferably in the form of a hydrochloride.

The above-mentioned acid addition salt may be prepared by directly mixing an extract of fermented antler and an acid, or b) dissolving and mixing one of them in a solvent or a water solvent, or c) mixing the extract of the fermented antler with a solvent or an underwater solvent ≪ / RTI > and then mixing them.

Separately, additionally saltable forms include, but are not limited to, the salts of gabapentin, gabapentin, pregabalin, nicotinate, adipate, hemimarate, cysteine, acetylcysteine, methionine, arginine, lysine, Aspartate and the like.

In addition, the pharmaceutical composition for preventing and treating muscle diseases of the present invention may further comprise a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration.

Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like.

The carrier for parenteral administration may also contain water, suitable oils, saline, aqueous glucose and glycols and the like. In addition, stabilizers and preservatives may be further included. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, , Intranasal, enteral, topical, sublingual or rectal administration.

The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above. When formulated, one or more buffers (e.g., saline or PBS), antioxidants, bacteriostats, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants Side), suspending agents, thickening agents, disintegrating agents or surfactants, diluents or excipients.

Formulations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, etc. These solid preparations may contain at least one excipient, , Starch (including corn starch, wheat starch, rice starch and potato starch), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, , Methyl cellulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or tablets may be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and processing the mixture into granules.

In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions or syrups. In addition to water or liquid paraffin, which is a simple diluent commonly used, various excipients such as wetting agents, sweeteners, fragrances or preservatives may be included .

In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant, and may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent .

For parenteral administration, the pharmaceutical compositions of the present invention may be formulated in accordance with methods known in the art in the form of injectable, transdermal and nasal inhalers, together with suitable non-oral carriers. In the case of such injections, they must be sterilized and protected against contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injectables include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils . More preferably, suitable carriers include, but are not limited to, isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. may be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like may be further included. In addition, the injections may in most cases additionally include isotonic agents, such as sugars or sodium chloride.

Examples of transdermal dosage forms include ointments, creams, lotions, gels, solutions for external use, pastes, liniments, and air lozenges. By " transdermal administration " as used herein, it is meant that the pharmaceutical composition is locally administered to the skin, whereby an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

In the case of an inhalation dosage form, a propellant suitable for the composition according to the invention, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases, It can be conveniently delivered in aerosol spray form. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, commonly known in all pharmaceutical chemistries.

The pharmaceutical composition of the present invention can provide a desirable muscle function improving effect when it contains an effective amount of fermented antler. As used herein, the term " effective amount " refers to an amount that exhibits a further reaction than the negative control, and preferably refers to an amount sufficient to improve muscle function. The pharmaceutical composition of the present invention may contain 0.01 to 99.99% of fermented antler, and the remaining amount may be occupied by a pharmaceutically acceptable carrier. The effective amount of the fermented deer antler contained in the pharmaceutical composition of the present invention will vary depending on the form and the like of the composition.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses . The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the degree of the disease. In the case of parenteral administration, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per 1 kg of body weight per day on the basis of the fermented deer antler, and in the case of oral administration, May be administered one to several times in an amount of preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg per kg of body weight. However, the dosage of the fermented deer antler is determined based on various factors such as age, body weight, health condition, sex, severity of disease, diet and excretion rate of the patient as well as the administration route and the frequency of treatment of the pharmaceutical composition, It will be understood by one of ordinary skill in the art that the effective dose of fermented antler according to the particular use for improving muscle function may be determined. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The pharmaceutical composition for preventing and treating muscle diseases of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers.

The pharmaceutical composition for the prevention and treatment of muscle diseases of the present invention can also be provided as a formulation of an external preparation containing fermented antler as an active ingredient.

When the pharmaceutical composition for the prevention and treatment of muscle diseases according to the present invention is used as an external preparation for skin, it may further contain a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, Surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic active agents, lipophilic active agents Or any other ingredient conventionally used in skin topical agents such as lipid vesicles. The components can also be introduced in amounts commonly used in the field of dermatology.

When the pharmaceutical composition for preventing or treating muscle disorders of the present invention is provided as an external preparation for skin, it may be a formulation such as ointments, patches, gels, creams or sprays.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent, however, to those skilled in the art that these embodiments are for further explanation of the present invention and that the scope of the present invention is not limited thereby.

Example

Example 1. Bacillus subtilis ( Bacillus subtilis ) SST-9960 [Accession No .: KCCM 11402P] Production of fermented antler by strain

Example 1-1. Culture of fermentative bacteria and preparation of strain for inoculation

Bacillus subtilis SST-9960 [Accession No .: KCCM 11402P] was inoculated into LB liquid medium, followed by incubation at 30 ° C for 2 days. The culture was centrifuged at 3000 rpm for 10 minutes to obtain a supernatant . Then, 0.75% physiological saline was added to suspend the strain of Bacillus, and the saline was removed by centrifugation at 3000 rpm for 10 minutes. The step of adding the physiological saline solution was repeated three times to completely remove the medium components, and finally 0.75% physiological saline was added to prepare an inoculum strain such that the viable cell count was 10 ⁹ cfu / mL.

Examples 1-2. Fermentation of antler

The inoculum and glucose prepared in Example 1-1 were added to distilled water to be 2% (v / v), respectively, to prepare a strain culture.

500 g of antler grains were placed in a fermentation tray and 500 ml of the culture solution was added to immerse the antler in the fermentation broth. The fermentation was carried out at 30 캜 for 3 days under aerobic conditions at a stirring speed of 100 rpm. Then, 250 mL of the above culture broth was added again, and then the second fermentation was carried out under the same conditions.

Examples 1-3. Preparation of fermented antler extract

The fermented antler was refluxed for 3 hours at 100 ° C, filtered through a Kimble-filtering flask, lyophilized and stored frozen at 20 ° C.

Example 2. Preparation of fermented antler with low temperature aging

Proceeding as in Example 1-1 and Example 1-2, the antler was fermented for the second time.

The second fermented deer antler was aged at 5 캜 for 12 hours at a low temperature, followed by ventilation drying at 30 캜 for 6 hours.

A fermented antler extract was prepared in the same manner as in Example 1-3, using the prepared fermented deer antler.

Example 3. Bacillus subtilis ( Bacillus subtilis ) Preparation of fermented antler by KCCM 11496 strain

The fermented antler extract was prepared in the same manner as in Example 1. At this time, the zymogen mainly Bacillus subtilis (Bacillus subtilis) SST-9960 [Accession No: KCCM 11402P] was used instead of Bacillus subtilis (B. subtilis) strain KCCM 11496.

Example 4 Bacillus subtilis ( Bacillus subtilis ) Preparation of fermented antler by KCCM 11730 strain

The fermented antler extract was prepared in the same manner as in Example 1. At this time, the zymogen mainly Bacillus subtilis (Bacillus subtilis) SST-9960 [Accession No: KCCM 11402P] was used instead of Bacillus subtilis (B. subtilis) strain KCCM 11730.

Example 5 Bacillus subtilis ( Bacillus subtilis ) Preparation of fermented antler by KCCM 11402P strain

The dried deer antler was ground with a mixer, and 100 g of the ground antler sample was added to 1 L of water and extracted with stirring at 80 ° C for 2 hours. The extracts were filtered under reduced pressure with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent components and obtain a hot water extract of green tea.

2% (v / v) of the inoculum prepared in Example 1 was added to the hot water extract for deer, and the mixture was incubated at 30 ° C for 3 days. Thereafter, the extracted sample was filtered under reduced pressure with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components, and a fermented product of the antler extract was obtained.

Example 6. Bacillus subtilis ( Bacillus subtilis Production of fermented antler by KCTC 13022 strain

The fermented antler extract was prepared in the same manner as in Example 1. At this time, the zymogen mainly Bacillus subtilis (Bacillus subtilis) SST-9960 [Accession No: KCCM 11402P] was used instead of Bacillus subtilis (B. subtilis) strain KCCM 13022.

Example 7. Bacillus subtilis ( Bacillus subtilis ) Preparation of low temperature aged fermented antler by KCTC 13022 strain

The procedure of Example 2 was followed to prepare a low-temperature aged fermented antler extract. At this time, the zymogen mainly Bacillus subtilis (Bacillus subtilis) SST-9960 [Accession No: KCCM 11402P] was used instead of Bacillus subtilis (B. subtilis) strain KCCM 13022.

Example 8. Bacillus subtilis ( Bacillus subtilis ) Preparation of fermented antler by KCTC13022 strain

The fermented product of the antler extract was prepared in the same manner as in Example 5. At this time, the zymogen mainly Bacillus subtilis (Bacillus subtilis) SST-9960 [Accession No: KCCM 11402P] was used instead of Bacillus subtilis (B. subtilis) strain KCCM 13022.

Example 9. Bacillus subtilis ( Bacillus subtilis ) Preparation of fermented antler by KCCM 11402P strain

The dried deer antler was ground with a mixer, and 100 g of the ground antler sample was added to 1 L of 100% ethanol and extracted with stirring at 80 ° C for 2 hours. The extracted sample was filtered under reduced pressure with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent components, and then the antler seed extract was obtained. Thereafter, a fermented product of the antler extract was prepared in the same manner as in Example 5.

Example 10. Bacillus subtilis of a supernatant fluid extract of green Bacillus subtilis ) Preparation of fermented product by strain KCCM 11402P

The dried deer antler was pulverized with a mixer, and 10 g of the crushed green sample was charged into a sample cartridge and extracted using a supercritical fluid extraction device (SFX 3560, Isco Inc., Lincoln, NE, USA). Supercritical fluid extraction conditions were: extraction pressure 40 MPa, extraction temperature 50 ℃, supercritical carbon dioxide flow rate 60 mL / min, extraction time 60 min. Upon completion of the supercritical fluid extraction, supercritical fluid extraction was obtained by releasing the supercritical fluid state by lowering the pressure of the extraction device. Thereafter, a fermented product of the supernatant fluid extract of green tea supernatant was prepared in the same manner as in Example 5

Comparative Example. Production of hot water extract of antler

The dried deer antler was ground with a mixer, and 100 g of the ground antler sample was added to 1 L of water and extracted with stirring at 80 ° C for 2 hours. The extracts were filtered under reduced pressure with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent components and obtain a hot water extract of green tea.

Experimental Example

EXPERIMENTAL EXAMPLE 1: Increased expression of the major gene p-mTOR protein involved in myofunction

Lysine (L6) myoblast (American Type Culture Collection, Manassas, Va., USA) was dissolved in 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 100 U / mL penicillin, 100 ug / mL streptomycin (Gibco, Grand Island, NY, USA) in Dulbecco's modified Eagle's media (DMEM; Hyclone). L6 cells were treated with the samples prepared according to Examples 1 to 4 and Comparative Examples. After 24 hours, L6 cells were lysed with NP-40 buffer (ELPIS-Biotech, Daejeon, Korea) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo., USA) . The cells dissolved in the buffer solution were transferred to a 1.5-mL tube and centrifuged at 13,000 rpm for 10 minutes to take only the supernatant. The supernatant was quantified using Bradford (Bio-Rad Laboratories Inc., Hercules, CA, USA) method. The quantified protein was boiled for 5 minutes, separated by electrophoresis on 10% SDS-PAGE, and the separated proteins were transferred to the nitrocellulose membrane. Cellular signaling technology (Beverly, MA, USA) was diluted 1: 1000 in 2.5% bovine serum albumin (BSA) and reacted with proteins transferred to the nitrocellulose membrane at room temperature for 20 hours . The primary antibody was reacted and the nitrocellulose membrane was washed three times for 10 minutes using Tris-buffered saline Tween 20 (TBST). After washing, anti-rabbit secondary antibody (Bethyl Laboratories, Inc., Montgomery, TA, USA) with horseradish peroxidase conjugated to primary antibody was diluted to 1: 5000 in 2.5% BSA, The reaction was carried out at room temperature for 3 hours, and then washed three times for 10 minutes using TBST. Protein bands were developed using ECL western blotting detection reagents (Amersham, Tokyo, Japan) and protein expression of p-mTOR was confirmed using G; BOX EF imaging system (Syngene, Cambridge, UK) The amount of protein loading was constant with α-tubulin.

As a result, as shown in Fig. 1, protein expression of the major gene p-mTOR involved in improvement of myofunction by treating a sample fermented with antler by Bacillus subtilis SST-9960 [Accession No .: KCCM 11402P] .

Experimental Example 2: Bacillus subtilis ( Bacillus subtilis Effect of KCCM 11402P on the Expression of Muscle Protein Biomarkers in Fermented Extracts

(Gibco, USA) and 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 100 U / mL penicillin and 100 μg / mL streptomycin Grand Island, NY, USA) with DMEM (Hyclone) at a concentration of 0.3 × 10 ⁵ cells / well in a 6-well plate. When the cell density reached about 80 ~ 85%, the medium in the wells was removed, and myotube differentiation was induced by treating the cells with DMEM (Hyclone) containing 2% horse serum (Hyclone). Replacement with a new medium was performed once every two days, and the differentiation was carried out for a total of 6 days. L6 cells were treated with the samples prepared in Example 5 and Comparative Example. After 12 hours, L6 cells were lysed with NP-40 buffer (ELPIS-Biotech, Daejeon, Korea) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo., USA) . The cells dissolved in the buffer solution were transferred to a 1.5-mL tube and centrifuged at 13,000 rpm for 10 minutes to take only the supernatant. The supernatant was quantified using Bradford (Bio-Rad Laboratories Inc., Hercules, CA, USA) method. The quantified proteins were boiled for 5 minutes, separated by electrophoresis on 10% SDS-PAGE, and the separated proteins were transferred to the nitrocellulose membrane. Cell signaling technology (Beverly, MA, USA) was diluted in a ratio of 1: 1000 to 2.5% bovine serum albumin (BSA) And reacted with the protein transferred to the cellulose membrane at room temperature for 20 hours. The primary antibody was reacted and the nitrocellulose membrane was washed three times for 10 minutes using Tris-buffered saline Tween 20 (TBST). After washing, anti-rabbit secondary antibody (Bethyl Laboratories, Inc., Montgomery, TA, USA) with horseradish peroxidase conjugated to primary antibody was diluted to 1: 5000 in 2.5% BSA, The reaction was carried out at room temperature for 3 hours, and then washed three times for 10 minutes using TBST. Protein bands were developed using ECL western blotting detection reagents (Amersham, Tokyo, Japan) and p-mTOR, p-70S6K, p-4EBP-1 And the amount of protein loaded by α-tubulin was constant.

As a result, as shown in Fig. 2, the samples obtained by fermenting the antler extract with Bacillus subtilis KCCM 11402P strain showed that the major genes p-mTOR, p-70S6K, p- 4EBP-1 protein was significantly increased. This means that the fermented product obtained by the Bacillus subtilis KCCM 11402P of the present invention is superior to the antler extract in the ability to increase muscle protein synthesis in muscle cells.

Experimental Example 3: Bacillus subtilis ( Bacillus subtilis Inhibitory Effect of KCCM 11402P on the Expression of Biomarkers Induced by Protein Degradation in Fermented Extracts

After muscle cells were differentiated as in Experimental Example 2, DMEM (Hyclone) containing 50 ng / mL tumor necrosis factor alph (TNF-α; PeproTech, Rocky Hills, NJ, USA) The prepared samples were treated with cells. After 12 hours, total RNA was isolated using TRIzol solution (Takara, Tokyo, Japan). The isolated total RNA was quantitated using NanoDrop 1000 (Thermo Fisher Scientific Inc., Waltham, Mass., USA). The quantified 16 μL of RNA was amplified by PCR using Reverse Transcriptase Premix (ELPIS-Biotech) and PCR machine (Gene Amp PCR System 2700; Applied Biosystems, Foster City, CA, USA) at 42 ° C for 55 minutes and 70 ° C for 15 minutes cDNA. 4 μL of the cDNA generated in 16 μL was amplified by PCR using the following specific primers (Bioneer; Bioneer, Daejeon, Korea) and PCR premix (ELPIS-Biotech) for 30 seconds at 95 ° C., 1 minute at 60 ° C., Min for 30 cycles.

Atrogin-1

Forward primer: 5'-CCCTGAGTGGCATCGCCCAA-3 '(SEQ ID NO: 1)

Reverse primer: 5'-AGGTCCCGCCCATCGCTCA-3 '(SEQ ID NO: 2)

MuRF1

Forward primer: 5'-GAAATGCTATGCAGAACCTG-3 '(SEQ ID NO: 3)

Reverse primer: 5'-ATTCCTGCTTGTAGATGTCG-3 '(SEQ ID NO: 4)

β-Actin:

Forward primer: 5'-AGCCATGTACGTAGCCATCC-3 '(SEQ ID NO: 5)

Reverse primer: 5'-CTCTCAGCTGTGGTGCTGAA-3 '(SEQ ID NO: 6)

The amplified cDNA was separated by electrophoresis on 1.5% agarose gel, and the cDNA band was confirmed using G; BOX EF imaging system (Syngene). The results are shown in Fig.

As a result, as shown in Fig. 3, the samples fermented with the antler extract as a strain of Bacillus subtilis KCCM 11402P showed significantly higher mRNA expression levels of the major genes MuRF1 and atrogin-1, which are involved in the muscle protein degradation, compared to the antler extract Respectively. This means that the fermented product obtained by the fermentation of Bacillus subtilis KCCM 11402P of the present invention is superior to the antler extract in inhibiting muscle protein degradation in muscle cells.

Experimental Example 4 Bacillus subtilis ( Bacillus subtilis Effect of KCTC 13022 on the Expression of Muscle Protein Biomarkers in Fermented Extracts

The samples prepared in Example 8 and Comparative Example were treated with cells and the experiment was carried out in the same manner as in Experimental Example 2 above.

As a result, as shown in FIG. 4, the samples obtained by fermenting the antler extract with Bacillus subtilis KCCM 13022 strain showed that the major genes p-mTOR, p-70S6K, p- 4EBP-1 protein was significantly increased. This means that the fermented product of the extract of Bacillus subtilis KCCM 13022 of the present invention is superior to the extract of the antler in the ability to increase muscle protein synthesis in muscle cells.

Experimental Example 5 Bacillus subtilis ( Bacillus subtilis Inhibitory Effect of KCCM 13022 on the Expression of Biomarkers Induced by Protein Degradation in Fermented Extracts

Cells were treated with the samples prepared in Example 8 and Comparative Example, and the experiment was carried out in the same manner as Experimental Example 3.

As a result, as shown in FIG. 5, the sample fermented with the antler extract as a strain of Bacillus subtilis KCCM 13022 showed significantly higher mRNA expression levels of the major genes MuRF1 and atrogin-1, which are involved in the muscle protein degradation compared to the antler extract Respectively. This means that the fermented product obtained by the fermentation of Bacillus subtilis KCCM 13022 according to the present invention is superior to the antler extract in inhibiting muscle protein degradation in muscle cells.

Experimental Example 6. Confirmation of effect of increasing muscle mass in animal model

Five-week-old Wistar rats were adapted for 1 week and induced TNF-α 100 ng / g for 2 weeks to induce atrophy. Then, groups were randomly divided into 8 groups based on body weight Respectively. As the experimental group, the hot water extract of green tea prepared in Example 1 and the fermented antler extract prepared in the Example were respectively suspended in water at a concentration of 1,000 mg / kg body weight and administered once a day for 8 weeks at a constant time. At this time, TNF-α was used as a control group.

After 8 weeks of administration, the muscles under the right calf were excised and weighed with microbalance (Mettler PE160, USA). As a result, as shown in the following Table 1, the muscle water content of the antler extract of Comparative Example was not significantly increased, whereas the muscle weight of the fermented antler was 16.33% (p <0.05) and 21.32% (p < 0.05), 24.56% (p <0.01), 20.11% (p <0.05), 18.09% (p <0.05), 28.07% (p <0.01), 23.56% (p <0.01), 26.41% Respectively. These results indicate that the fermented antler according to the present invention is effective for increasing the muscle mass.

division Mean calf muscle weight (mg) Control group 410.8 ± 25.9 Example 1 477.9 ± 22.9 * Example 2 498.4 ± 28.6 * Example 5  511.7 ± 19.0 ** Example 6 493.4 ± 30.8 * Example 7 485.1 ± 26.9 * Example 8 526.1 ± 20.6 ** Example 9 507.6 ± 15.4 ** Example 10 519.3 ± 18.5 ** Comparative Example 428.0 ± 29.1

* p <0.05; ** p < 0.01

Hereinafter, a preparation example of a food or a medicament containing the sample of the above embodiment according to the present invention as an active ingredient will be described, but the present invention is not intended to be limited thereto but is specifically explained. The food or pharmaceutical composition of Preparation Examples 1 to 2 was prepared according to the conventional method according to the following composition components and composition ratio with the fermented deer antler having the effect of preventing or treating muscle diseases or improving muscular function.

Manufacturing example

Production Example 1. Preparation of Food

Production Example 1-1. Manufacture of Health Functional Foods

1,000 mg of the fermented deer extract of Example 1

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed , Granules may be prepared and used in the manufacture of a health functional food composition according to a conventional method.

Production Example 1-2. Manufacture of functional beverages

1,000 mg of the fermented deer extract of Example 1

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to the flask to obtain a total of 900 mL

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the functional beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Production Example 1-3. candy

Candy was prepared by mixing 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of flavor and 0.1% by weight of the fermented deer antler extract of Example 1.

Manufacturing Example 2. Preparation of Medicine

Production Example 2-1. Powder

20 mg of the fermented deer extract of Example 1

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Production Example 2-2. refine

10 mg of the fermented deer extract of Example 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Production Example 2-3. Capsule

10 mg of the fermented deer extract of Example 1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Production Example 2-4. Liquid

20 mg of the fermented deer extract of Example 1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the conventional liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water and adjusted to 100 as a whole. To prepare a liquid agent.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. It is natural.

Claims (10)

Muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and hypoxia, which are fermented deer antler obtained by fermenting antler granules or antler extract with Bacillus subtilis as an active ingredient, (sarcopenia). &lt; / RTI &gt; The method according to claim 1,
Wherein the composition enhances the expression of p-mTOR, p-p70S6K, and p-4EPB-1 protein. The method according to claim 1,
Wherein the composition reduces the expression of MuRF1 and atrogin-1. The method according to claim 1,
Wherein the antler extract is obtained by extracting antler antler with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid, . Muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and hypoxia, which are fermented deer antler obtained by fermenting antler granules or antler extract with Bacillus subtilis as an active ingredient, and sarcopenia. &lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; 6. The method of claim 5,
Wherein said composition increases the expression of p-mTOR, p-p70S6K, p-4EPB-1 protein. 6. The method of claim 5,
Wherein said composition reduces the expression of MuRF1 and atrogin-1. 6. The method of claim 5,
Wherein the antler extract is obtained by extracting antler antler with at least one solvent selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid, and a pharmaceutical composition for preventing or treating muscular disease . A food composition for enhancing muscular function by increasing muscle mass, which comprises fermented antler grains obtained by fermenting antler grains or antler extracts with Bacillus subtilis. delete

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