KR102354289B1 - Composition for preventing, ameliorating or treating of muscle disease containing Dioscorea nipponica Makino extract as effective component - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of muscle diseases, comprising an extract of fenugreek as an active ingredient. As an active ingredient of the present invention, the extract of fenugreek not only improves cell viability, but also can induce and promote differentiation into muscle cells, has the effect of increasing the expression level of myocyte differentiation-related genes, and promotes muscle regeneration and growth. It works. Therefore, the composition of the present invention can be usefully used as a health functional food for promoting muscle growth or preventing or improving muscle loss, and as a pharmaceutical for preventing or treating muscle disease.

Description

A composition for preventing, ameliorating or treating of muscle disease containing Dioscorea nipponica Makino extract as effective component;

The present invention relates to a composition for the prevention, improvement or treatment of muscle diseases, comprising an extract of fenugreek as an active ingredient.

The muscles of our body attach to the bones and perform various functions, such as protecting the bones and maintaining the correct body shape. In addition, the muscle promotes calcium influx and increases bone density. However, as the body ages, the redistribution of body fat and body protein occurs due to the change in composition, and when about 50 years old, the rate of protein synthesis in muscle cells becomes slower than the rate of decomposition, leading to rapid muscle degeneration, and may be exposed.

Sarcopenia, one of the diseases of muscle loss, refers to a state in which approximately 13 to 24% of one's body mass has decreased. Injuries are easy and can lead to serious injuries. One of the causes of sarcopenia is the gradual decrease in the quantity and quality of skeletal muscle that occurs with aging, and weight loss including fat and body fat components due to inadequate dietary energy intake.

Myoblasts formed through division and crystallization of precursor cells differentiate into myocytes, fuse with adjacent cells, and differentiate into myotubes. Differentiated myotube cells form muscle through the process of forming myofibril (myofibril). Some of myoblasts are in a stationary state as stem satellite cells, and when muscle tissue is exposed to a disease such as trauma or muscular dystrophy, muscle stem cells known as satellite cells are released from the damaged area of the muscle. heal and regenerate In diseases such as muscular dystrophy, these cells are particularly important for muscle stability and healing.

On the other hand, sagebrush is a vine-like perennial plant in the family Liliaceae, a monocotyledonous plant, and grows in mountains or fields. The rhizome extends laterally and is hard. The leaves are alternate phyllotaxis, the stem is long, and the leaves are divided into three and are egg-shaped. The middle-branched piece is lengthened and the side-branched piece is again divided into 3-5 pieces. The leaf axil protrudes from the back and has fine hairs. The flowers are unisexual and bloom in yellow-green in August, but do not open completely and form a water inflorescence. Male spikelets stand upright or obliquely in a stellate inflorescence, and female spikelets droop downwards. There are 6 pericarp fragments and 6 stamens. The fruit is a capsule (殼果), has three wings, and runs upward from the drooping stem. It is distributed in Korea, Japan, and China.

On the other hand, Korea Patent No. 1687271 discloses a composition for preventing or treating menopausal disorders comprising a palm cactus extract and a fermented horseradish extract, and U.S. Patent Publication No. 2018-0305399 of skeletal muscle of a prostan-3ol derivative Although the effect of promoting hypertrophy or treating atrophy of skeletal muscle has been disclosed, there is no disclosure of a composition for preventing, improving, or treating muscle disease comprising the extract of the present invention as an active ingredient.

The present invention has been derived from the above needs, and the present invention provides a composition for preventing, improving, or treating muscle diseases, comprising an extract of fenugreek as an active ingredient, and the extract of fenugreek of the present invention promotes cell viability The present invention was completed by confirming that it can induce and promote differentiation into muscle cells, increase the expression level of myocyte differentiation-related genes, and promote muscle regeneration and growth.

In order to achieve the above object, the present invention provides a health functional food composition for promoting muscle growth or preventing or improving muscle loss containing the extract of fenugreek as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases containing the extract of fenugreek as an active ingredient.

The present invention relates to a composition for the prevention, improvement or treatment of muscle diseases, comprising an extract of fenugreek as an active ingredient. As an active ingredient of the present invention, the extract of fenugreek not only improves cell viability, but also can induce and promote differentiation into muscle cells, has the effect of increasing the expression level of myocyte differentiation-related genes, and promotes muscle regeneration and growth. It works.

1 is a result of confirming the cell viability of the extract of the present invention. (A) is the viability of myoblasts, and (B) is the cell viability of myotubes. * and ** indicate that the cell viability of the group treated with the extract of B. Hemp compared to the group that was not treated with the A. Hemp extract was statistically significantly increased, * indicates p<0.05, and ** indicates p<0.01.
Figure 2 shows the time-dependent growth degree of myoblasts according to the treatment of the extract of the present invention. *** indicates that the cell growth rate of the group treated with the A. Hemp extract was statistically significantly increased compared to the vehicle that was not treated with the Hemp extract, p<0.001.
Figure 3 is the result of confirming the change in the expression level of the Cyclin gene according to the treatment of the extract of the present invention.
4 is a result confirming that myotube growth is promoted by the differentiation of myoblasts in the muscle cells treated with the extract of the present invention. ** and *** indicate that RNA levels of MyoD and MyoG were significantly increased compared to myoblasts, ** indicates p<0.01, and *** indicates p <0.001. ### indicates that the RNA levels of MyoD and MyoG were significantly increased in the group treated with the fan horses compared to the vehicle not treated with the fan horses, p<0.001.
5 is an H&E staining result confirming the muscle regeneration and growth promoting effect according to the administration of the extract of the present invention using an animal model of muscle damage. Control is the control group that did not consume the extract, and the control group is the experimental group fed the extract. Day 0 is just before the muscle injury, Day 4 is the 4th day after the muscle injury, and Day 8 is the 8th day after the muscle injury.
6 is a result of analyzing the muscle fiber area on the 8th day after muscle injury by section in the H&E staining results confirming the muscle regeneration and growth promoting effect according to the administration of the extract of the present invention using an animal model of muscle damage. Control is the control group that did not consume the extract, and the control group is the experimental group fed the extract.

The present invention is a fan ( Dioscorea) nipponica Makino ) relates to a health functional food composition for promoting muscle growth or preventing or improving muscle loss containing the extract as an active ingredient.

The fenugreek extract may be prepared by a method comprising the following steps, but is not limited thereto:

(1) extracting by adding an extraction solvent to the fan horsetail;

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

(3) Concentrating and drying the filtered extract of step (2) to prepare an extract.

In step (1), the extraction solvent _{is preferably selected from water, C 1} to C ₄ lower alcohols or mixtures thereof, more preferably ethanol, and still more preferably 70% (v/v) ethanol. do not limit In the above preparation method, the extraction method may use all conventional methods known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The extraction solvent is preferably added by adding 1 to 20 times the weight of the fan horseradish, more preferably 5 to 15 times, and even more preferably 10 times. The extraction temperature is preferably 40 ~ 100 ℃, but is not limited thereto. In addition, the extraction time is preferably 1 to 48 hours, more preferably 1 to 24 hours, and most preferably 3 hours, but is not limited thereto. In the above method, the concentration in step (3) is preferably using a vacuum rotary concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying, more preferably freeze drying, but not limited thereto.

The health functional food composition is preferably prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto. The health functional food composition of the present invention may be prepared by adding the extract of fenugreek as it is or by mixing it with other foods or food ingredients, and may be appropriately prepared according to a conventional method. Examples of foods to which the fenugreek extract can be added include caramel, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, It may be in any one form selected from tea, drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in a conventional sense. That is, there is no particular limitation on the type of the food. The health functional food composition includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavorants, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like. It may also contain pulp for the production of natural fruit juices and vegetable beverages. The above components may be used independently or in combination. In addition, the health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, and the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, dextrin, and cyclo polysaccharides such as dextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The proportion of the natural carbohydrate is not very important, but with respect to 100 g of the composition of the present invention, it is preferably 0.01 to 0.04 g, more preferably 0.02 to 0.03 g, but is not limited thereto. As the sweetener, natural sweeteners such as taumatine and stevia extract, and synthetic sweeteners such as saccharin and aspartame may be used.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases containing the extract of fenugreek as an active ingredient.

The muscle disease is dystonia (atony), muscular atrophy (muscular atrophy), muscle degeneration, muscle stiffness, muscular dystrophy (muscular dystrophy), amyotrophic axonal sclerosis, myasthenia gravis, cachexia (cachexia) and any selected from sarcopenia (sarcopenia) It is preferable that there is one, but is not limited thereto. It may further include a carrier, an excipient, or a diluent in addition to the extract of fenugreek.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, and water. Cross, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate ate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, antioxidants, buffers, bacteriostats, diluents, surfactants, binders, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents or preservatives may be further included. A suitable dosage of the pharmaceutical composition of the present invention may be prescribed variously depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient. can The route of administration of the pharmaceutical composition for the prevention or treatment of muscle disease of the present invention may be administered through any generally accepted route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but depending on the purpose, intramuscular administration, eye drop administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, It may be administered through a route such as rectal administration, and specifically, it may be administered through a route of intramuscular administration.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Example One. fan Preparation of Ethanol Extract

After drying 500 g of fan horsetail at 50° C. for 7 days with hot air, 10 times of 70% (v/v) ethanol was added, followed by extraction under reflux cooling twice for 3 hours each and filtration. After that, the extract was concentrated under reduced pressure at 45° C. to remove ethanol, and then freeze-dried at -80° C. for 7 days to obtain an extract of Fanima extract.

Example 2. Analysis of cell viability according to the treatment of the extract of F. horseradish

For cell viability analysis, 1×10 ⁴ cells/ml of C2C12 cells were spread in a 96-well plate, cultured for 24 hours, and treated with 1, 10, and 100 μg/ml F. E. extracts in each well for 48 hours. incubated during Thereafter, the cell viability was analyzed by measuring the absorbance at 490 nm, and the experiment was repeated 3 times and expressed as the mean ± standard deviation.

As a result, as shown in FIG. 1 , it was found that the Fiona extract of the present invention has almost no cytotoxicity. has grown From this, it was judged that the extract of fenugreek was effective for muscle regeneration and growth by regulating the growth of myoblasts.

In addition, as shown in FIG. 2 , when treated with the extract of F. horseradish, the growth rate of cells was statistically significantly increased.

Example 3. fan according to the treatment of the extract of myoblasts Confirmation of growth promotion effect

After culturing 2×10 ⁵ cells/ml C2C12 cells in a 6-well plate for 24 hours, the extracts were treated with fenugreek extract and cultured for 48 hours.

Then, RNA was extracted using QIAGEN's RNA mini extraction kit, and cell cycle-related cyclin D1, cycline D3, cyclin E1 and cyclin E2 cDNAs were synthesized using this, followed by real-time PCR analysis. The experiment was repeated 3 times and expressed as the mean ± standard deviation. As a result, as shown in FIG. 3 , it was confirmed that the expression levels of cyclin D1 and cyclin D3 were significantly increased by the treatment of the extract of F. E. horseradish. Therefore, it was determined that the proliferation of muscle cells was promoted by activating the cyclin D gene, which regulates the G1 phase of the cell cycle.

Example 4. fan according to the treatment of the extract of myotubes Confirmation of growth promotion effect

After culturing 2×10 ⁶ cells/ml C2C12 cells in a 6-well plate for 24 hours, they were treated with a horse chestnut extract and cultured in a differentiation medium containing 1% horse serum for 48 hours.

Then, RNA was extracted using QIAGEN's RNA mini extraction kit, and myoD and myoG were synthesized using this, followed by real-time PCR analysis. The experiment was repeated 3 times and expressed as the mean ± standard deviation. As a result, as shown in FIG. 4 , it was confirmed that the growth of myotube cells was significantly increased in the group treated with F. horseradish extract of the present invention.

Example 5. Using the muscle injury animal model of the present invention fan Confirmation of muscle regeneration and growth promotion effect according to the administration of the extract

After wearing 6-week-old BALB/c mice and undergoing an adaptation period for 2 weeks, they were divided into control and experimental groups. In order to induce muscle damage after 13 weeks of F. hemp extract diet, 1.2% BaCl₂ 50 μl of the solution was injected into the gastrocnemius muscle.

After sacrificing 5 mice per group on day 0 (just before muscle injury), day 4 and day 8, calf muscles were isolated and fixed with 4% paraformaldehyde (PFA).

Thereafter, for basic morphological analysis of muscles and IHC (immunohistochemistry) experiments, H&E staining using paraffin-embedded slides was performed, and observation was performed at a 400-fold ratio using an optical microscope. were compared.

As a result, as shown in FIG. 5 , on day 0, 1.2% BaCl2, a muscle damage chemical, Before 50 μl of the solution was injected, there was no significant difference in muscle tissue (cells). On the 4th day after muscle injury, both the control group and the experimental group were centered on the location of the nucleus of the muscle fibers, and it was confirmed that the size of the muscle fiber bundle was reduced compared to the 0th day before the muscle injury. The shift in the position of the nucleus in the muscle fiber to the center and the decrease in the area of the muscle fiber are pathological evidence indicating muscle damage. Although the area of muscle fibers in the experimental group was slightly larger than that of the control group, the difference was not evident.

However, on the 8th day after muscle injury, it was confirmed that the muscle in the experimental group compared to the control group was remarkably recovered. First, it was confirmed that the position of some nuclei was moved to a normal position, and the cross-sectional area of the muscle fiber was wider than that of the control group.

In other words, it was found that the experimental group fed with the extract of fenugreek showed a remarkable effect on promoting muscle growth compared to the control group.

In addition, in order to quantitatively compare the area of muscle fibers on the 8th day, the cross-sectional size of 220 muscle fibers per group (control group and experimental group) was compared. ^{The size of the cross-sectional area was measured by section [0~50μm 2} , 50~100μm ² , 101~200μm ² , 201~300μm ² , 301~400μm ² , 401~500μm ² of the muscle fiber cross-sectional area using the Image J program distributed free of charge by NIH. It was analyzed ^{501 ~ 600μm 2, 601 ~ 700μm} 2, 701 ~ 800μm 2, 801 ~ 900μm 2] distribution.

As a result, as shown in FIG. 6 , the number of muscle fibers in the section with a wide cross-sectional area was found to be more distributed in the experimental group than in the control group. That is, the control group is 0 to 50 μm ² , 50 to 100 μm ^{2 ,} and 101 to 200 μm ² . It was mainly distributed in the section, but it was found that the experimental group in which the fangs were fed relatively was distributed over a wider area.

Claims (6)

A health functional food composition for promoting muscle regeneration containing the extract of fenugreek as an active ingredient. [Claim 2] The health functional food composition for promoting muscle regeneration according to claim 1, wherein the extraction solvent for the extract of F. amaranth is water, C ₁ ~ C _{4 lower alcohol, or a mixture thereof.} The health functional food composition for promoting muscle regeneration according to claim 1, wherein the fenugreek extract is prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup and beverages. Of any one muscle disease selected from atony, muscular atrophy, muscle degeneration, muscular dystrophy, amyotrophic axonal sclerosis and sarcopenia containing the extract of fenugreek as an active ingredient A pharmaceutical composition for prophylaxis or treatment. delete [Claim 5] The pharmaceutical composition for the prevention or treatment of muscle disease according to claim 4, further comprising a carrier, excipient or diluent in addition to the extract of Fiona horseradish.

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