KR102346511B1 - Composition for Preventing or Treating Muscular disease containing Artemisia dracunculus L. extract - Google Patents

Abstract

The present invention relates to a composition for preventing or treating muscle diseases including sarcopenia, muscular atrophy, muscular dystrophy, muscle degeneration, etc., containing the tarragon extract, and more specifically, the tarragon extract of the present invention expresses the expression of a gene for producing muscle protein Since it increases the amount and reduces the amount of gene expression that destroys muscle proteins, as well as exhibits antioxidant and anti-inflammatory effects, the tarragon extract of the present invention can be usefully used as a therapeutic agent for muscle diseases derived from natural products without side effects. .

Description

Tarragon (Tarragon, Little Dragon, Mugwort, Estragon) containing an extract for preventing or treating a muscle disease composition for preventing or treating Muscular disease containing Artemisia dracunculus L. extract}

The present invention relates to a composition for preventing or treating muscle diseases, including sarcopenia, muscular atrophy, muscular dystrophy, muscle degeneration, etc. containing tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) extract. .

Muscle is responsible for about 40% of the human body, and in order to maintain the functional ability of the human body and prevent metabolic diseases, it is essential to secure an appropriate muscle mass. It is largely divided into smooth muscle, cardiac muscle, and skeletal muscle, and skeletal muscle occupies a significant portion of the entire body and promotes skeletal movement.

Muscular disease progresses through a process in which independent living becomes impossible due to the weakening of skeletal muscles, which gradually impairs walking and mobility functions and makes activities of daily living (ADL) difficult. In addition, since it causes cardiopulmonary dysfunction and other complications, it is important to correctly understand and approach the characteristics of each muscle disease.

In sarcopenia, the motor nerve that induces skeletal muscle contraction degenerates, so that skeletal muscle contraction does not proceed, or normal skeletal muscle contraction does not proceed because the expression of a protein involved in muscle contraction in skeletal muscle is reduced or altered. In the long term, it is known that the motor nerve or skeletal muscle is transformed into a fibrous tissue. It is also known to be caused by various causes such as aging, hormonal abnormalities, malnutrition, lack of physical activity, and inflammation and degenerative diseases.

It is known that exercise, protein, and calorie supplementation are helpful for sarcopenia, but it is not very helpful in the elderly, who account for most of sarcopenia patients, so a treatment for sarcopenia is urgently required. However, the drugs currently used for the treatment of sarcopenia are at the stage of clinical trials so far, and there is no drug that has finally received FDA approval.

Inflammatory myopathy, it is known that inflammation occurs in muscles and surrounding tissues, and the inflammation destroys the muscle tissue, which weakens the muscles and causes muscle pain and loss of strength, and in the long term, muscle mass decreases and muscle atrophy may appear. Because the muscle becomes inflamed, it is also called myositis and is largely classified into polymyositis and dermatomyositis. It is an autoimmune disease caused by abnormalities in the body's immune system and is known to be caused by viruses or some drugs (penicillamine, alpha interferon, cimetidine, phenytoin, hepatitis B vaccine, etc.).

Steroids are mainly used for the treatment of polymyositis or dermatomyositis, and the response is improved in 70 to 80% of patients. Used as a high-dose oral therapy or injection, muscle strength can be significantly improved, but side effects (weight gain, osteoporosis, exacerbation of diabetes, heartburn, indigestion, exacerbation of gastric ulcer, etc.) etc) are used together. In addition, when the therapeutic effect of various immunosuppressive agents is low or cannot be used due to side effects, intravenous immunoglobulin is sometimes used. On the other hand, although recently developed immunosuppressive agents are known to have some effects on polymyositis, there is a steady demand for the development of therapeutic agents without side effects.

In addition, muscular atrophy (muscular atrophy) is a disease in which the muscles of the extremities are atrophied, and the expression of MuRF-1 and Atrogin-1 is increased to decompose the myosin heavy chain in the muscle, resulting in muscle atrophy in which the size of the muscle is reduced. known to do

On the other hand, as a related prior art, Korean Patent No. 1,921,085 relates to a pharmaceutical composition for treating sarcopenia or muscular atrophy comprising a glucagon-like peptide-1 (GLP-1), a GLP-1 derived peptide, or a GLP-1 degradation inhibitor, Disclosed is a composition exhibiting an effect selected from the group consisting of weight gain, skeletal muscle weight gain, increased expression of a gene producing a muscle protein, inhibition of expression of a gene that disrupts muscle protein, and combinations thereof.

In addition, Korea Patent No. 1,809,156 relates to a composition for preventing, improving or treating or improving muscle function containing fucosterol, and for improving muscle function, fucosterol, mother and child, mother and child crushed product, mother and child extract, hibiscus extract, and turmeric powder. Alternatively, the extract of hibiscus inhibited the increase in the protein expression of p-mTOR, a major gene involved in muscle protein synthesis, suppression of the mRNA expression of MuRF-1 and Atrogin-1 involved in muscle protein degradation, and the mRNA expression of MyoD and Myogenin involved in muscle differentiation. As it increases, it has an excellent effect of enhancing muscle function, and since it is a natural product, it is disclosed that it can be safely used without side effects.

However, the above documents only disclose genes that generate and degrade muscle protein, and the antioxidant and anti-inflammatory effects of tarragon extract, increase in the expression levels of MyoD and Myogenin genes that generate muscle protein, and Atrogin-1 and Atrogin-1 that destroy muscle protein The effect of reducing the expression level of the MuRF-1 gene is not disclosed.

Accordingly, the inventors of the present invention have made efforts to develop natural products having an improving effect on muscle loss, and as a result, tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) extract has antioxidant and anti-inflammatory effects, It was newly found that it was possible to increase the expression levels of MyoD and Myogenin genes that generate muscle proteins and decrease the expression levels of Atrogin-1 and MuRF-1 genes that destroy muscle proteins, and the tarragon extract was used to treat muscle diseases. By revealing that it can be used in, the present invention was completed.

Korean Patent No. 1,921,085 Korean Patent No. 1,809,156

The present invention is to provide a pharmaceutical composition for preventing or treating muscle disease, and a health food, containing an extract of tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention and treatment of muscle diseases containing an extract of tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) as an active ingredient.

In addition, the present invention provides a health food for preventing or improving muscle disease containing tarragon extract as an active ingredient.

Tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) extract according to the present invention has antioxidant and anti-inflammatory effects, as well as an increase in the expression level of a gene for generating muscle protein, Since it reduces the gene expression level, it can be usefully used for the treatment of various muscle diseases.

1 is a schematic diagram of a tarragon extract manufacturing process according to a solvent.
Figure 2 is a diagram confirming the cytotoxicity of the tarragon extract.
3 is a view showing the measurement result of ROS production of tarragon extract.
4 is a view showing the measurement result of IL-1β production amount of tarragon ethanol extract.
5 is a diagram showing the measurement result of IL-6 production of tarragon extract.
6 is a diagram confirming the cytotoxicity of tarragon extract to C2C12 cells.
7 is a diagram showing the measurement result of MyoD gene expression of tarragon hot water extract.
8 is a diagram showing the measurement result of Myogenin gene expression level of tarragon extract.
9 is a diagram showing the results of measuring the Atrogin-1 gene expression level of the tarragon ethanol extract.
10 is a diagram showing the measurement result of MuRF-1 gene expression level of tarragon ethanol extract.
11 is a view showing a photograph of a change in the root canal width of tarragon hot water extract taken with an optical microscope.
12 is a view showing the measurement results of changes in root canal width of tarragon hot water extract.

Hereinafter, the present invention will be described in more detail.

Tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) of the present invention provides a pharmaceutical composition for preventing or treating muscle disease containing the extract as an active ingredient.

The tarragon extract is preferably prepared by a manufacturing method comprising the following steps, but is not limited thereto:

1) extracting by adding an extraction solvent to tarragon;

2) filtering the extract of step 1); and

3) drying the filtered extract of step 2) after concentrating under reduced pressure.

In the method, the extraction solvent of step 1) _{is preferably characterized in that the extraction is performed using water, C 1} to C ₂ lower alcohol or a mixture thereof as a solvent, but is not limited thereto.

In the method, the lower alcohol in step 1) is preferably ethanol or methanol, but is not limited thereto.

In the above method, it is preferable to use a vacuum vacuum concentrator or a vacuum rotary evaporator for the vacuum concentration in step 3), but is not limited thereto. In addition, drying under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying is preferable, but is not limited thereto.

The tarragon extract has antioxidant and anti-inflammatory effects, and it is preferable to have a preventive or therapeutic effect on muscle disease through the effect of increasing the expression level of the gene for generating muscle protein and reducing the expression level of the gene that destroys the muscle protein, but limited thereto doesn't happen

The muscle diseases include sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscle weakness, and muscular dystrophy. (muscular dystrophy), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis) and inflammatory myopathy (inflammatory myopathy) is preferably at least one disease selected from the group consisting of.

In a specific example of the present invention, the present inventors prepared tarragon hot water or ethanol extract (see FIG. 1).

In addition, in order to confirm the cytotoxicity of the tarragon extract, the extract was treated with RAW 264.7 cells and absorbance was measured at 450 nm, it was confirmed that the tarragon extract did not show cytotoxicity (Fig. 2, Table 5 and Table 6). Reference).

In addition, in order to confirm the antioxidant effect of the tarragon extract, the extract was treated with RAW 264.7 cells and the change according to the intensity of fluorescence intensity was measured with a flow cytometer. As a result, the tarragon extract reduced the amount of reactive oxygen species (ROS) production. Therefore, it was confirmed that there is an antioxidant effect (see Fig. 3, Table 7).

In addition, in order to confirm the anti-inflammatory effect of the tarragon extract, the extract was treated with RAW 264.7 cells and measured with Luminex. As a result, the tarragon extract reduced the production of IL-1β and IL-6, so it was confirmed that there was an anti-inflammatory effect. (see FIGS. 4, 5, 8 and 9).

In addition, in order to confirm the therapeutic effect of the tarragon extract on muscle disease, the extract was treated with C2C12 cells and absorbance was measured at 450 nm. As a result, it was confirmed that the tarragon extract was not toxic to muscle cells (Fig. 6, Table 10). and Table 11).

In addition, in order to confirm the muscle disease treatment effect of the tarragon extract, the C2C12 cells were treated with the extract to check the effect of gene expression involved in muscle protein synthesis or degradation. As a result, the tarragon extract is MyoD gene and Myogenin gene involved in muscle synthesis. By increasing the expression and reducing the expression of the Atrogin-1 gene and MuRF-1 gene involved in muscle degradation, it was confirmed that there is an effect of treating muscle diseases (Fig. 7, Fig. 8, Fig. 9, Fig. 10, Table 13, Table 14, see Table 15 and Table 16).

In addition, in order to confirm the therapeutic effect of the tarragon extract for muscle disease, the extract was treated with C2C12 cells to measure the width of the root canal. As a result, the tarragon extract increased the width of the root canal, and thus it was confirmed that there was an effect on the treatment of muscle diseases (FIG. 11). , see Figure 12 and Table 17).

Therefore, the tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) extract of the present invention exhibits an antioxidant or anti-inflammatory effect, and a gene involved in the increase and degradation of gene expression involved in muscle protein synthesis By showing the effect of reducing the expression and increasing the width of the root canal, the tarragon extract can be usefully used in a pharmaceutical composition for preventing or treating muscle diseases.

The composition containing the tarragon extract of the present invention may contain one or more active ingredients having the same or similar function in addition to the above ingredients.

The composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, and lactose. , mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate , sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

That is, the composition of the present invention may be administered in various oral and parenteral formulations during actual clinical administration. When formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants It can be prepared using Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the tarragon extract, for example, starch, calcium carbonate, sucrose ), lactose or gelatin may be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, and preservatives in addition to commonly used simple diluents such as water and liquid paraffin may be included. . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The composition of the present invention can be administered orally or parenterally according to a desired method, and when administered parenterally, external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection method It is preferable to select The dosage varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and in general, 0.1 mg to 100 mg per kg body weight, preferably 0.5 mg to 10 mg per kg body weight, is administered daily or every other day Or it can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

In addition, it provides a health food for preventing or improving muscle disease containing the tarragon extract of the present invention as an active ingredient.

The tarragon extract preferably has an activity for preventing or improving muscle disease, but the extract is not limited thereto.

Since the tarragon extract of the present invention significantly prevents or treats muscle disease, the tarragon extract can be usefully used as a health food composition for preventing or improving muscle disease.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include various foods such as sweets, breads and noodles, drinks such as water, soft drinks and fruit drinks, gum, tea, vitamin complexes, seasonings, health functional foods, etc., It includes all health functional foods in the ordinary sense.

The tarragon extract of the present invention may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, the amount of the compound in the health food can be added in an amount of 0.01 to 15% by weight, preferably 0.1 to 5% by weight of the total food weight, and 0.01 to 5.0 g, preferably 0.01 to 5.0 g based on 100, in the health beverage composition. It can be added in a proportion of 1.0 g. However, in the case of long-term intake for the purpose of health control, the above amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health functional beverage composition of the present invention is not particularly limited in other ingredients except for containing the compound as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; conventional sugars such as disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, erythritol and the like. As flavoring agents other than those described above, natural flavoring agents (taumakin, stevia extract, etc.), and synthetic flavoring agents (saccharin, aspartan, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 0.1 to 2.0 g, preferably about 0.1 to 1.0 g per 100 compositions of the present invention.

The health functional food of the present invention can be easily obtained by adding the above-described extract of the present invention during the food manufacturing process or by adding the above-described extract of the present invention after the food is manufactured. At this time, if necessary, a taste and odor correcting agent may be added.

In addition to the above, the tarragon extract of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and It may contain salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the tarragon extract of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not so important, but is generally selected in the range of about 20 parts by weight per 100 parts by weight of the tarragon extract of the present invention.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples specifically illustrate the present invention, and the content of the present invention is not limited by the Examples and Experimental Examples.

<Example 1> Preparation of tarragon extract

<1-1> Preparation of tarragon ethanol extract

Tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) was extracted using 16 times the amount of 70% ethanol at 45 to 75 ° C. for 8 to 14 hours, and filtered under reduced pressure (Whatman filter) Paper #2) was concentrated and then stored at -20℃.

In addition, the extraction yield according to the extraction temperature and extraction time is shown in [Table 1] and [Table 2].

On the other hand, in the following experimental example, an experiment was performed using an ethanol extract of tarragon obtained by extracting tarragon using 70% ethanol at 75° C. for 12 hours.

Samples transference number(%) 8 hours 10 hours 12 hours 14 hours Tarragon Ethanol Extract 20.1 24.5 28.9 29.0

Samples transference number(%) 45℃ 55℃ 65℃ 75℃ Tarragon Ethanol Extract 18.3 21.5 24.3 28.9

<1-2> Preparation of tarragon hot water extract

Using distilled water 16 times the amount of the tarragon sample, extraction was performed at 65 to 95° C. for 8 to 14 hours, cooled to room temperature, filtered and concentrated to prepare a tarragon hot water extract.

In addition, the extraction yield according to the extraction temperature and extraction time is shown in [Table 3] and [Table 4] below.

On the other hand, in the following experimental example, an experiment was performed using a tarragon hot water extract obtained by extracting tarragon at 95° C. for 12 hours using distilled water.

Samples transference number(%) 8 hours 10 hours 12 hours 14 hours Tarragon Hot Water Extract 20.1 24.5 28.9 29.0

Samples transference number(%) 45℃ 55℃ 65℃ 75℃ Tarragon Hot Water Extract 12.5 15.3 17.7 18.9

<Experimental Example 1> Confirmation of cytotoxicity

<1-1> RAW 264.7 cell passage

Frozen RAW 264.7 cells were transferred to a 50 ml tube, and 9 ml of PBS was added to float the cells, followed by centrifugation at 1,200 rpm for 5 minutes to remove the supernatant. Cells were suspended in 1 ml of DMEM (Dulbecco's modified Eagle's medium) medium composed of 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and cultured in a cell incubator (37° C., 5% CO ₂ ). The number of passages was 5 or more, and the samples were acclimatized for 24 hours before processing.

<1-2> Cytotoxicity measurement

In order to measure the cytotoxicity of the tarragon extract, RAW 264.7 cells cultured in Experimental Example <1-1> were used. RAW 264.7 cells were seeded in a 96-well plate at 1.5×10 ⁵ cells/well and cultured in a cell incubator (37° C., 5% CO ₂ ) for 24 hours. After culturing, the culture medium was replaced with a fresh medium, and the tarragon extract was treated at concentrations of 25, 50 and 100 μg/ml, and then cultured in a cell culture medium for 24 hours again. After incubation, 10 μl of WST solution was added and reacted for 30 minutes in _{a cell incubator (37° C., 5% CO 2 ).} After the reaction, the absorbance was measured at 450 nm to express the cell viability as a percentage for the control group that was not treated with the tarragon extract.

As a result, as shown in [FIG. 2], [Table 5] and [Table 6], the result of measuring cytotoxicity through RAW 264.7 cells was 100.0±1.0% of the control group, all treatment concentrations of tarragon extract showed a survival rate of 90% or more compared to the control group, confirming that it was safe (FIG. 2).

Unit : % Sample Name Control 25 μg/ml 50 μg/ml 100 μg/ml Tarragon Ethanol Extract 100.0±1.0 105.7±9.9 101.4±7.3 93.0±4.1

Unit : % Sample Name Control 25 μg/ml 50 μg/ml 100 μg/ml Tarragon Hot Water Extract 100.0±1.0 103.5±5.1 107.5±4.0 101.0±4.8

The following experiment was carried out by selecting a concentration of 50 μg/ml from among the concentrations in which cytotoxicity was not confirmed.

<Experimental Example 2> Confirmation of antioxidant effect

In order to confirm the antioxidant effect of the tarragon extract, the amount of reactive oxygen species (ROS) production was measured using RAW 264.7 cells prepared in Experimental Example <1-1>.

RAW 264.7 cells were seeded in a 12 well plate at 2×10 ⁵ cells/well and cultured in a cell incubator (37° C., 5% CO ₂ ) for 24 hours. After culturing, it was replaced with a new culture medium, and LPS was treated at a concentration of 50 μg/ml of tarragon extract at a concentration of 1 μg/ml, and then incubated in a cell incubator for 24 hours again. After incubation, the cells collected by centrifugation at 4°C and 1,200 rpm for 5 minutes were washed twice with PBS, and DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) was added to a concentration of 20 µM and stained in a cell incubator for 15 minutes. . After staining, centrifugation was performed again at 4°C and 1,200 rpm for 5 minutes, then the supernatant was removed, 400 μl of PBS was suspended again, and the change according to the intensity of fluorescence intensity was measured using a flow cytometer. is expressed as a percentage.

As a result, as shown in [Fig. 3] and [Table 7], when the ROS production amount of the control treated only with LPS was 100.0±1.0%, it was confirmed that the tarragon extract significantly reduced ROS production ( 3).

Unit : pg/ml Sample
Name Nor Con 50 μg/ml Sample
Name Nor Con 50 μg/ml tarragon
ethanol
extract 25.9
±4.3 100.0
±1.0 61.5
±1.0 tarragon
hot water
extract 19.0
±4.4 100.0
±1.0 61.9
±1.3

<Experimental Example 3> Confirmation of anti-inflammatory effect

In order to confirm the anti-inflammatory effect of the tarragon extract, the amount of Cytokine production was measured using RAW 264.7 cells prepared in Experimental Example <1-1>.

RAW 264.7 cells were seeded in a 12 well plate at 2×10 ⁵ cells/well and cultured in a cell incubator (37° C., 5% CO ₂ ) for 24 hours. After culturing, it was replaced with a new culture medium, and LPS was treated at a concentration of 50 μg/ml of tarragon extract at a concentration of 1 μg/ml, and then cultured again in a cell culture medium for 24 hours. After incubation, the supernatant was obtained by centrifugation at 1,200 rpm for 5 minutes and measured as follows using the article composed of the Milliplex map mouse cytokine/chemokine magnetic bead panel-immunology multiplex assay kit. 25 μl of the supernatant was dispensed into a 96 well plate, 25 μl of each of assay buffer, matrix buffer, and antibody-immobilized beads were added and mixed, followed by reaction at room temperature for 2 hours and washed twice with washing buffer. After washing, 25 μl of detection antibody was added, reacted in the dark at room temperature for 1 hour, and 25 μl of Streptavidin-Phycoerythrin was added to react at room temperature for 30 minutes, followed by washing twice using washing buffer solution. After washing, 150 μl of PBS was added, and after shaking for 5 minutes, measurements were made using Luminex.

As a result, as shown in [Fig. 4] and [Table 8], when the IL-1β production amount of the control treated only with LPS was 123.3±3.3 pg/ml, the tarragon ethanol extract of the present invention was the IL-1β It was confirmed that the production was significantly reduced (Fig. 4).

Unit : pg/ml Sample Name Nor Con 50 μg/ml Tarragon Ethanol Extract 46.0±0.8 123.3±3.3 93.9±8.3

In addition, as shown in [Fig. 5] and [Table 9], when the IL-6 production amount of the control treated only with LPS was 151839.8±2593.1 pg/ml and 145561.7±18836.0 pg/ml, respectively, the tarragon of the present invention The extract showed 111151.5±13370.4 and 100273.6±12658.0, respectively, confirming that IL-6 production was significantly reduced ( FIG. 5 ).

Unit : pg/ml Sample
Name Nor Con 50 μg/ml Sample
Name Nor Con 50 μg/ml tarragon
ethanol
extract 116.8
±27.3 151839.8
±2593.1 111151.5
±13370.4 tarragon
hot water
extract 118.3
±12.7 145561.7
±18836.0 100273.6
±12658.0

Therefore, it was confirmed that the tarragon extract of the present invention reduces the production of IL-1β and IL-6, which are inflammatory cytokines, and thus exhibits a significant anti-inflammatory effect.

<Experimental Example 4> Confirmation of the treatment effect of tarragon extract for muscle disease

<4-1> Reagent

Reagents were Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, USA), fetal bovine serum (FBS; Gibco BRL, USA), trypsin-EDTA (Thermofisher, USA), trypan blue (Sigma Co., USA), EZ-Cytox ( Daeilab, Korea), dulbecco's phosphate buffered saline (Welgene, Korea), RIPA buffer (Thermo Co., USA), sample loading buffer (BIO-RAD Co., Japan), 30% Acrylamide/Bis Solution 29:1 (3.3%) C) (BIO-RAD Co., Japan), Skim Milk (BD Co., France), Pierce BCA Proein Assay Kit (Thermo Co., USA), TEMED (BIO-RAD Co., Japan), phosphorylation-AKT ( p-AKT), AKT, phosphorylation-mTOR (p-mTOR), mTOR antibody (Danvers., USA), Protease inhibitor cocktail, phosphatase inhibitor cocktail (Sigma Co., USA), Total RNA prep kit (Intronbio, Korea), AccuPower CycleScript RT PreMix (Bioneer, Korea), SYBR Green (Qiagen, Germany), etc. were used.

<4-2> Device

The instrument is a centrifuge (Sigma Co., USA), deep-freezer (Sanyo Co., Japan), vortex mixer (Vision scientific Co., Korea), plate shaker (Lab-Line Co., USA), ELISA reader (Molecular Devices) Co., USA), optical microscope (Carl ZEISS Co., Germany), Light Microscope (Carl Zeiss, Co., Germany), Mini-Protean tetra Cell (BIO-RAD Co., Japan), Nanodrop (Thermo fisher, USA) ), Alpha Cycler 1 PCRmax (PCRmax, UK) real time PCR (Qiagen, Germany), etc. were used.

<4-3> C2C12 cell passage

C2C12 cells were purchased from American Type Culture Collection (ATCC, USA), and cultured in a cell incubator (37° C., 5% CO ₂ ) using DMEM medium supplemented with 10% FBS.

<4-4> Cytotoxicity measurement

In order to measure the cytotoxicity of the tarragon extract to muscle cells, the C2C12 cells cultured in Experimental Example <4-3> were used. C2C12 cells were seeded in a 96-well plate at 1×10 ⁴ cells/well and cultured for 24 hours. It was replaced with a fresh culture medium, and the tarragon extract was treated at concentrations of 25, 50 and 100 μg/ml and incubated for another 48 hours. After incubation, 10 μl of EZ-Cytox solution was added and reacted for 30 minutes in a cell incubator. After the reaction, the change in absorbance at 450 nm was measured, and the cell viability was expressed as a percentage for the control group that was not treated with the tarragon extract.

As a result, as shown in [Fig. 6], [Table 10] and [Table 11], the results of measuring cytotoxicity through C2C12 cells were 100.00±2.25% of the control group, at all treatment concentrations of the tarragon extract. A survival rate of 90% or more compared to the control group was confirmed to be safe (FIG. 6).

Unit : % Sample Name Control 25 μg/ml 50 μg/ml 100 μg/ml Tarragon Ethanol Extract 100.0±2.25 114.37±4.92 116.46±4.92 136.48±2.16

Unit : % Sample Name Control 25 μg/ml 50 μg/ml 100 μg/ml Tarragon Hot Water Extract 100.0±2.29 115.41±12.59 111.71±7.14 93.10±2.52

The following experiment was carried out by selecting a concentration of 50 μg/ml from among the concentrations in which cytotoxicity was not confirmed.

<4-5> Confirmation of the effect of gene expression involved in muscle protein synthesis or degradation

In order to check the effect of gene expression involved in muscle protein synthesis or degradation of the tarragon extract, the C2C12 cells prepared in Experimental Example <4-3> were used.

^{C2C12 cells were aliquoted at 6×10 5} cells/well in a 6 well plate and cultured for 24 hours. After 24 hours, the differentiation medium was replaced for cell differentiation, and the medium was exchanged every other day for 6 days. After 6 days, 50 μg/ml + TNF-α was treated and the reaction was performed again for 48 hours. 1 ml of cell lysis buffer was added and mixed, and then 200 μl of chloroform was added and mixed again. After centrifugation at 13,000 rpm for 10 minutes, 400 μl of the supernatant was recovered, 400 μl of binding buffer was added, mixed, injected into the column, and centrifuged. 700 μl of washing buffer A was added to the column and centrifuged, and 700 μl of washing buffer B was added and centrifuged. After centrifugation, 50 μl of elution buffer was added, followed by centrifugation to extract RNA.

For the reverse transcription reaction, 1 μg of total RNA was added to the RT premix (reverse transcriptase, reaction buffer, dNTPs, dT20 primer, stabilizer), and distilled water treated with DEPC (diethyl pyrocarbonate) was added so that the final volume was 20 μl. . After mixing the mixture, centrifugal sedimentation was performed at 2,000 rpm for 5 seconds and reacted for 60 minutes in a 45°C heating block to synthesize first-strand cDNA. This was again reacted at 95° C. for 5 minutes to inactivate M-MLV RT, and then the synthesized cDNA was used for PCR.

To amplify the synthesized cDNA, real-time PCR was performed, and 1 μl of cDNA, 2 μl of each primer, 10 μl of SYBR Green, and 5 μl of DEPC-DW were put into a real-time dedicated tube and proceeded as follows. The reaction was carried out at 94°C for 5 minutes, followed by 15 seconds at 94°C, 30 seconds at the annealing temperature of each primer, and 30 seconds at 72°C, followed by reaction at 94°C for 1 minute and then at 55°C for 1 minute. Finally, in the annealing of the primer, the final process was performed in which SYBR Green, a fluorescent conjugate material, was generated by raising the temperature by 0.5°C to 94°C until it reached 94°C. [Table 12] shows the primer sequence and annealing temperature used.

<Primer sequence of real-time PCR>
F : forward, R : reverse Primer F/R Sequences Size
(bp) Annealing
Temp. (℃) MyoD F ATGATGACCCGTGTTTCGACT (SEQ ID NO: 1) 119 65 R CACCGCAGTAGGGAAGTGT (SEQ ID NO: 2) Myogenin F GCAGGCTCAAGAAAGTGAATGA (SEQ ID NO: 3) 122 65 R TAGGCGCTCAATGTACTGGAT (SEQ ID NO: 4) Atrogin-1 F TCAAAGGCCTCACGCGATCACC (SEQ ID NO: 5) 215 65 R CCTCAATGACGTATCCCCCG (SEQ ID NO: 6) MuRF-1 F GAGGGGCTACCTTCCTCTCA (SEQ ID NO: 7) 210 65 R TTTACCCTCTGTGGTCACGC (SEQ ID NO: 8) GAPDH F TGCTGCATACGAGCATCCAT (SEQ ID NO: 9) 167 59 R TGTCCTCAAAGTGAACCGCA (SEQ ID NO: 10)

As a result, as shown in [Fig. 7] and [Table 13], when the MyoD gene expression level of the TNF-α-treated control group was 1.00 ± 0.00%, the tarragon hot water extract of the present invention suppressed MyoD gene expression. It was confirmed that there was a significant increase (FIG. 7).

Unit : % Sample Name Nor Con 50 μg/ml Tarragon Hot Water Extract 3.20±0.49 1.00±0.00 5.10±0.56

In addition, as shown in [Fig. 8] and [Table 14], when the Myogenin gene expression level of the control group treated only with TNF-α was 1.00 ± 0.00%, the tarragon extract of the present invention significantly increased Myogenin gene expression was confirmed (FIG. 8).

Unit : % Sample
Name Nor Con 50 μg/ml Sample
Name Nor Con 50 μg/ml tarragon
ethanol
extract 0.90
±1.0 1.00
±0.00 5.20
±0.92 tarragon
hot water
extract 0.90
±1.0 1.00
±0.00 1.60
±0.12

In addition, as shown in [Fig. 9] and [Table 15], when the Atrogin-1 gene expression level of the control group treated only with TNF-α was 1.00 ± 0.00%, the tarragon ethanol extract of the present invention was Atrogin-1 It was confirmed that the gene expression was significantly reduced (FIG. 9).

Unit : % Sample Name Nor Con 50 μg/ml Tarragon Ethanol Extract 0.80±0.03 1.00±0.00 0.50±0.08

In addition, as shown in [Fig. 10] and [Table 16], when the MuRF-1 gene expression level of the control group treated with TNF-α was 1.00±0.00%, the tarragon ethanol extract of the present invention was the MuRF-1 gene. It was confirmed that the expression was significantly reduced (FIG. 10).

Unit : % Sample Name Nor Con 50 μg/ml Tarragon Ethanol Extract 0.40±0.12 1.00±0.00 0.10±0.03

Therefore, the tarragon extract of the present invention increases the expression of the MyoD gene and Myogenin gene involved in muscle synthesis and decreases the Atrogin-1 gene and MuRF-1 gene expression involved in muscle degradation, so the tarragon extract can be used to treat sarcopenia was confirmed.

<4-6> Root canal width measurement

In order to confirm the effect of increasing the width of the root canal of the tarragon extract, the C2C12 cells prepared in Experimental Example <4-3> were used.

^{C2C12 cells were aliquoted at 6×10 5} cells/well in a 6 well plate and cultured for 24 hours. After 24 hours, it was replaced with a differentiation medium for cell differentiation, and the medium was exchanged every other day for 6 days. After 6 days, 50 μg/ml + TNF-α was treated and the reaction was performed again for 48 hours. After 48 hours of reaction, the width of the cell root canal was checked with an optical microscope equipped with a digital camera.

As a result, as shown in [Fig. 11], [Fig. 12], and [Table 17], in the control group treated with TNF-α only, the root canal width was 13.8±1.38 cm, and in the group treated with tarragon hot water extract, 22.20±2.47 cm , and it was confirmed that the area increased significantly ( FIGS. 11 and 12 ).

Unit : cm Sample Name Nor Con 50 μg/ml Tarragon Hot Water Extract 31.2±2.06 13.8±1.38 22.20±2.47

<110> Chong Kun Dang Healthcare Corp. <120> Composition for Preventing or Treating Muscular disease containing Artemisia dracunculus L. extract <130> PB2020-232 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MyoD primer F <400> 1 atgatgaccc gtgtttcgac t 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MyoD primer R <400> 2 caccgcagta gggaagtgt 19 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Myogenin primer F <400> 3 gcaggctcaa gaaagtgaat ga 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Myogenin primer R <400> 4 taggcgctca atgtactgga t 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Atrogin-1 primer F <400> 5 tcaaaggcct cacgcgatca cc 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Atrogin-1 primer R <400> 6 cctcaatgac gtatcccccg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MuRF-1 primer F <400> 7 gaggggctac cttcctctca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MuRF-1 primer R <400> 8 tttaccctct gtggtcacgc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer F <400> 9 tgctgcatac gagcatccat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer R <400> 10 tgtcctcaaa gtgaaccgca 20

Claims (3)

Tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) As a pharmaceutical composition for preventing or treating inflammatory myopathy containing hot water extract as an active ingredient,
The inflammatory myopathy is polymyositis or dermatomyositis,
The composition will inhibit IL-6 production, the composition.
delete Tarragon (English: Tarragon, Little Dragon, Mugwort, Estragon, scientific name: Artemisia dracunculus L.) As a health food for preventing or improving inflammatory myopathy containing hot water extract as an active ingredient,
The inflammatory myopathy is polymyositis or dermatomyositis,
The health food is to inhibit the production of IL-6, health food.

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