KR20220169668A - Composition comprising extract of Morindae radix for preventing, treating or improving muscular disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscle diseases, or a food composition for preventing or alleviating muscle diseases, containing an extract of Morindae radix as an active ingredient, wherein the extract of Morindae radix contains monotropane as a physiologically active substance to inhibit the expression of various factors that decompose muscle protein in muscle cells, so that it is possible to prevent muscle damage and loss and strengthen muscle function. Therefore, it is expected to prevent, alleviate, or treat muscular diseases through a food or pharmaceutical composition containing the extract of Morindae radix as the active ingredient.

Description

Composition comprising extract of Morindae radix for preventing, treating or improving muscular disease}

The present invention relates to a pharmaceutical composition for preventing or treating muscle diseases, or a food composition for preventing or improving muscle diseases, comprising an extract of Pogeukcheon as an active ingredient.

Metabolic syndrome, including abdominal obesity, hypertension, hyperglycemia and hyperlipidemia, seriously affects insulin control in glucose, fat and protein metabolism, and ultimately leads to the onset and development of type 2 diabetes mellitus (T2DM). promote The incidence of type 2 diabetes mellitus (T2DM), which is characterized by insulin resistance and defects in pancreatic islet cell function and insulin secretion, increases with age. volume and function gradually decline. Therefore, T2DM is an important factor deteriorating the quality of life of elderly people with many complications.

Skeletal muscle accounts for 80% of the body's glucose uptake. Decreased muscle mass results in decreased muscle capacity, insulin sensitivity and ability to manage peripheral glucose, increasing the risk of diabetes. Thus, skeletal muscle loss has been implicated in both cause and effect of diabetes, so a bidirectional relationship exists between muscular dystrophy and T2DM.

T2DM is primarily administered through diet and oral hypoglycemic drugs (eg, metformin and other antidiabetic drugs). Unfortunately, there are currently no effective treatments to stop the progression of diabetes. Over the past decade, the benefits of many traditionally used herbal medicines for diabetes have been demonstrated through in vitro and in vivo studies and clinical trials. In addition, Traditional Chinese medicine (TCM) and Traditional Korean medicine (TKM) are increasing in popularity because they are effective in treating many diseases with multiple targeting abilities and low side effects.

Recently, an attempt has been made to develop a composition for preventing or treating muscle diseases such as muscular dystrophy using herbal medicines such as hawthorn, rhodiola, and double evening primrose. Research is needed.

Republic of Korea Patent Registration No. 10-2208665 Republic of Korea Patent Registration No. 10-2208665 Republic of Korea Patent Registration No. 10-2208665

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of muscle diseases, which contains an extract of Paigeukcheon as an active ingredient.

In addition, an object of the present invention is to provide a food composition for preventing or ameliorating muscle diseases, comprising an extract of ragweed as an active ingredient.

One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a burdock root extract as an active ingredient.

Morindae radix is the dried root of Morinda officinalis HOW, an evergreen vine belonging to the Rubiaceae family, which is dried by removing the core and beard roots and flattening them. Roots are flat, cylindrical, 0.5 to 2 cm in diameter, usually curved and irregular in length. The characteristic of pageukcheon is that it has no odor and has a warm property with a sweet and spicy taste. It is known to strengthen muscles and bones, strengthen kidneys, and enhance immune function to act on the liver and kidneys. These pore springs contain various physiologically active substances such as monotropein, and are mainly prescribed for the treatment of kidney deficiency, menstrual irregularities, infertility, rheumatism, joint pain, and the like. However, there has been no study on the preventive or therapeutic effect of muscle disease by burpee cloth yet.

The "porous cloth extract" used in the present invention refers to an extract obtained by extraction treatment by using a cloth alone or in combination, a diluted or concentrated liquid of an extract, a dried product obtained by drying an extract, a refined product or a purified product of an extract, or a mixture thereof. Etc., the extract itself and extracts of all formulations that can be formed using the extract.

The ragweed extract may be obtained from nature or commercially cultivated and sold ragweed, but is not limited thereto.

The ragweed extract may be obtained using an extraction method known in the art without limitation. The extraction method may include, for example, cold extraction, ultrasonic extraction, reflux cooling extraction, hot water extraction, pressure extraction, solvent extraction, and the like.

In addition, the ragweed extract may be prepared in a powder state by an additional process such as concentration under reduced pressure and freeze-drying or spray-drying.

In addition, the ragweed extract includes not only an extract obtained by the above-described extraction method but also an extract subjected to a conventional purification process. For example, separation using an ultrafiltration membrane with a certain molecular weight cut-off value, separation by various chromatography (designed for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods may also be included in the extract of the present invention.

According to one embodiment of the present invention, the foam cloth extract may be obtained by reflux extraction of foam cloth.

More specifically, reflux extraction can be performed for 1 to 12 hours, preferably 2 to 6 hours, using 1 to 50 times, preferably 5 to 20 times, water compared to burst cloth, and sufficiently extracts the physiologically active substances of the break cloth. It can be extracted more than once to make it possible. This extract may be filtered one or more times to remove insoluble matter and impurities, and the filtrate may be concentrated under reduced pressure and then freeze-dried.

According to one embodiment of the present invention, the ragweed extract may contain monotropein as a physiologically active substance.

Monotropein belongs to an organic compound known as iridoid o-glycoside, and as a physiologically active substance in ragweed, it is known to be mainly effective in anti-inflammatory, but the effect of monotropein on preventing or treating muscle diseases was not known

According to one embodiment of the present invention, the ragweed extract may promote the differentiation of muscle cells and inhibit degradation.

According to one embodiment of the present invention, when the fibrous extract is administered to diabetic mice exhibiting muscle loss due to diabetes, the space between the widened muscle fiber bundles of skeletal muscle is maintained at a level similar to that of normal mice, and myogenesis occurs. And, it was confirmed that the biogenesis-related factors MyoD, Myogenin, MHC, and PGC-1α were significantly increased, and the muscular atrophy-related factor Atrogin1 was significantly decreased, and expressed at levels similar to those of normal mice.

In addition, according to one embodiment of the present invention, in myoblasts, which are progenitor cells differentiating into muscle fibers, the myoblast extract stimulates the expression of myogenic and biogenic factors to promote differentiation into myotube cells, and induces muscle atrophy. In cells, it was confirmed that monotropane increased the expression of the myogenesis-related factor Myogenin, while decreasing the expression of the myostatin-related factors Myostatin, MuRF1, and Atrogin-1.

By containing monotropane as a physiologically active substance, the ragweed extract according to the present invention can degrade muscle protein in muscle cells and inhibit the expression of various molecules that cause muscular atrophy, thereby improving muscle damage, muscle loss, and muscle dysfunction.

According to one embodiment of the present invention, the muscle disease is sarcopenia, muscular atrophy, muscular dystrophy, myotonia, hypotonia, amyotrophic lateral sclerosis sclerosis) and myasthenia.

A pharmaceutical composition containing the burdock chinensis extract as an active ingredient may further include pharmaceutically acceptable additives. The additives are starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, hydroxycalcium phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opa Dry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol and talc, but not limited thereto.

According to one embodiment of the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier. The carriers are commonly used in the manufacture of pharmaceuticals, and include lactose, dextrose, sucrose, sorbitol, mannitol, trehalose, hyaluronic acid, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto.

According to one embodiment of the present invention, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (22th ed., 2013).

The pharmaceutical composition of the present invention can be administered in various oral and parenteral formulations during actual clinical administration. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, can be prepared using Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one or more excipients, such as starch and calcium, added to the herbal composition in which the fat-soluble polyphenol component of the present invention is increased. It may be prepared by mixing carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally depending on the desired method, and in the case of parenteral administration, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method can be selected. can The dosage varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

The pharmaceutical composition of 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 with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, activity of the drug, drug It may be determined according to factors including sensitivity to, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The composition according to one embodiment 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 single or multiple times. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 mg to 1,000 mg, 0.01 mg to 100 mg, or 0.1 mg to 10 mg per 1 kg of body weight. It can be administered daily or every other day or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

Another aspect of the present invention provides a food composition for preventing or ameliorating muscle diseases comprising a burdock cloth extract as an active ingredient.

Since the description of the ragweed extract is the same as described above, it is omitted to avoid redundant description. A food composition for preventing or ameliorating muscle diseases containing such a ragweed extract can be expected to prevent muscle loss or improve muscle function by promoting differentiation and suppressing degradation of muscle cells.

"Food composition" as used in the present invention means a natural product or processed product containing one or more nutrients, preferably means a state that can be eaten directly through a certain degree of processing, and usually means As such, it includes all health functional foods, functional foods, beverages, food additives and beverage additives.

The food composition may be, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, and the like. In addition, in the present invention, the food includes special nutritional food (eg, formula milk, infant food, baby food, etc.), processed meat product, fish meat product, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplement food, seasoning food ( For example, soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (eg, snacks), dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.), drinks ( Examples include, but are not limited to, fruit and vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

The food, functional food, health functional food, beverage, food additive and beverage additive may be prepared by conventional manufacturing methods.

The burdock cloth extract according to the present invention is a physiologically active substance that inhibits the expression of various factors that degrade muscle protein in muscle cells, including monotropein, to prevent muscle damage and muscle loss and to enhance muscle function. It is expected that muscle diseases can be prevented, improved, or treated through a food or pharmaceutical composition containing them as ingredients.

Figure 1 shows the effect of burdock extract on the serological changes of HFD/STZ-induced diabetic mice.
Figure 2 shows the effect of the burdock extract on histological changes in skeletal muscle tissue of HFD/STZ-induced diabetic mice. The cross-sectional area of the calf muscle tissue was stained with H&E staining, and under a microscope, the nucleus was observed in blue and the cytoplasm in red. (A) is a stained representative histological photograph (magnification 200x) of each group, showing the organization of myofibrils (MF) within the bundle and the space between the myofibrils and the cross-sectional area of myofibrils. (B) is the quantification of the muscle fiber cross-section area.
Figure 3 shows the effect of the burdock extract on the muscle tissues of HFD/STZ-induced diabetic mice. The expression level of each protein was measured by Western blotting and compared with the expression level of β-actin, an internal control.
Figure 4 shows the effect of the burdock extract on the expression of myogenic protein in C2C12 myotube cells. Cells were treated with various concentrations of burdock extract (MORE) or metroformin (Met) for 24 hours. (A) the expression of Myogenin and MyoD, (B) the expression of MHC was measured by Western blot and compared with the expression level of β-actin, an internal control. (C) is an image (200x magnification) of cells stained with an anti-MHC antibody and DAPI and observed under a fluorescence microscope. MHC-positive cells were observed in green and DAPI-positive cells in blue.
Figure 5 shows the effect of the burdock extract on the expression of biogenesis regulators in C2C12 myotubes. The expression level of each protein was measured by Western blotting and compared with the expression level of β-actin, an internal control.
Figure 6 shows the effect of monotropane on the muscular atrophy of C2C12 myotubes. The expression level of each protein was measured by Western blotting and compared with the expression level of β-actin, an internal control.

Hereinafter, the present invention will be described in more detail. However, these descriptions are merely presented as examples to aid understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.

1. Materials and Methods

1-1. Preparation of Paigeukcheon Extract

The dried root of Morinda officinalis ( Morinda officinalis ) was purchased as a standard product from a herbal medicine sales company (Kwangmyeongdang Pharmaceutical, Korea). About 200 g of small pieces of ragweed were boiled in 2,000 mL of distilled water in a round-bottom flask under reflux and extracted twice for 3 hours. The crude extract was filtered through Whatman® Filter Paper (GE Healthcare UK Limited, UK), concentrated with a vacuum rotary evaporator (Eyela. Co. Ltd, Japan) at 60°C, and frozen at -80°C at 5 mTorr. It was rapidly freeze-dried with a dryer (IlShin Lab Co., Korea). The powder (yield = 58%) of Pogeukcheon extract (MORE) was stored at -20°C until used for in vivo and in vitro studies.

1-2. Production of disease animal models

Male C57BL/6N mice (Coretech, Korea) were used to construct the diabetic animal model. Laboratory animal care protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee of Dongguk University (IACU-2018-11). Mice were acclimated to the feeding protocol one week prior to the experiment. The mice were housed in a cage at 22±3° C. and 60±5% relative humidity, and the light/dark cycle was 12 h/12 h. Food and water were provided ad libitum. Mice were fed a standard diet (calorie 3.10 kcal/g, fat 18% kcal, carbohydrate 58% kcal, protein 24% kcal; En-vigo, Cat. 2018S, USA) and a high-fat diet for 8 weeks. (HFD) (calories 5.24 kcal/g, fat 60% kcal, carbohydrate 20% kcal, protein 20% kcal; Research diets, D12492, USA) was randomly assigned to a control group. After feeding, control mice were intraperitoneally (i.p.) injected with streptozotocin (STZ) (120 mg/kg body weight) dissolved in citrate buffer (pH 4.5). To confirm whether diabetes was induced, fasting blood glucose (FBG) levels were measured in whole blood of the tail using an Accu-Chek Inform II glucose meter (Roche, Switzerland). Mice with a blood glucose concentration of 300 mg/dL or more were used. All mice were normal group (Nor), HFD/STZ-induced diabetic control group (Control), MORE 100 mg/kg body weight group (MORE100) and MORE 200 mg/kg body weight group (MORE200) and metformin as a reference group. (metformin) 250 mg/kg body weight group (Met) were randomly assigned to 5 groups (n = 5 per group). The normal group was fed standard feed, and the rest group was fed HFD for 4 weeks while the drug was administered.

1-3. Measurement of physiological parameters

At the end of the experiment, the body weight and food intake of the mice were measured. Calculation of calorie intake is as follows:

Calorie intake of mice fed standard diet = feed intake x 3.1 kcal/g

Calorie intake of mice fed HFD diet = food intake x 5.24 kcal/g

1-4. Measurement of serological parameters

Mice were sacrificed using 5% isoflurane in the presence of anesthetic gas (75% O ₂ and 25% N ₂ O) after a 24-hour fast. For serological analysis, whole blood was collected from the abdominal aorta using a syringe. Blood samples were taken, and serum was separated by centrifugation twice for 10 minutes at room temperature at 3,000 rpm. Glucose (GLU), aspartate transaminase (AST), alanine aminotransferase (ALT), total Serological marker levels such as total cholesterol (TCHO), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) were measured. Insulin levels were assayed using an insulin ELISA kit (cat Crystal Chem, Inc., USA, Cat. 90082) according to the manufacturer's protocol. Insulin concentrations in serum samples were determined using a standard curve. Homeostasis model assessment of insulin resistance (HOMA-IR) level was calculated by the following equation: HOMA-IR = [fasting insulin (μU/ml) x fasting serum glucose (mM)] / 22.5.

1-5. histological observation

For histological observation and protein expression analysis, gastrocnemius tissues were obtained from mouse hind limbs. Calf muscle tissue was fixed with 4% paraformaldehyde for 24 hours and then embedded in paraffin. Tissue blocks were cut into 5 μm sections and stained with hematoxylin and eosin (H&E). Changes in muscle tissue were observed under a microscope (original magnification x 200). The size of the fiber was measured by image using ImageJ software (https://imagej.nih.gov/ij/), and the average cross-sectional area of the muscle fiber was shown (Rusbana, T.B., et al. Nutrients 2020, 12, 2409).

1-6. Cell culture and processing

C2C12 cells (ATCC, USA) are mouse myoblasts, ₁₀ % fetal bovine serum (FBS) (Merck Millipore, USA), penicillin/streptomycin (Corning ) containing DMEM (Corning, USA) and supplemented with DMEM containing 2% horse serum (Thermo Fisher Scientific, USA) for 4 days. Then, various concentrations of MORE (0.5, 1, 2 mg/mL) or monotropein (25, 50, 100 umol/mL), or Met 2.5 mM/mL were treated for 24 hours. In the MTT assay, no cytotoxicity was observed for MORE up to 2 mg/mL and monotropane up to 100 umol/mL.

Monotropein (Cat. Y0002220) and dexamethasone (Dexamethasone, Cat. D4902) were purchased from Merck (Sigma-Aldrich Inc., USA), and were dissolved in appropriate solvents according to the manufacturer's instructions.

1-7. immunochemical staining

C2C12 myoblasts were seeded onto Thermanox plastic coverslips (Nunc™, Thermo Fisher Scientific) and differentiated in 2% horse serum for 4 days. After treatment with MORE or metformin for 24 hours, C2C12 myotubes were washed three times with 1x PBS, fixed with 4% paraformaldehyde, placed on ice for 20 minutes, and incubated in 0.5% Triton X-100 (Sigma Sigma) for 5 minutes at room temperature. -Aldrich, USA). Then, the cells were washed with 1x PBS, blocked with 0.5% BSA for 2 hours, and incubated overnight at 4°C with an anti-MHC antibody (Santa Cruz Biotechnology, Inc., USA, Cat. Sc-376157). Myotube cells were washed three times with 1x PBS and incubated with goat anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, Cat. A11001). Nuclei were counterstained with DAPI. Cells were visualized using a fluorescence microscope (Leica DM2500, Leica Microsystems, Germany). MHC-positive cells were observed as green fluorescence in blue multinucleated myotubes stained with DAPI.

1-8. Western blot analysis

To isolate total protein, skeletal muscle tissue and C2C12 myotube cells were homogenized in a protein lysis buffer containing protease inhibitors and phosphatase inhibitors, and homogenized using a homogenizer (T10 basic, IKA®, Staufen im Breis T10 basic, IKA®, Staufen im Breisgau, Germany) was used to homogenize and centrifuged at 4° C. at 14,000 rpm for 20 minutes. After measuring the total protein concentration of each sample, the same amount of protein (30 μg) was separated with 8-10% acrylamide gel, and SDS-PAGE was performed. The gel was transferred to a nylon membrane and blocked with 5% skim milk for 1 hour at room temperature. The membrane is MyoD (Cat. sc-377460, Santa Cruz Biotechnology, USA), MHC (Cat. sc-376157), Myogenin (Cat. sc-52903), PGC-1α (Cat. NBP1-04676, Novus Biologicals, USA), SIRT1 (Cat. bs-0921R, Thermo Fisher Scientific., USA), NRF1 (Cat. 69432s, Cell Signaling Technology, USA), Atrogin1 (Cat. PA5-91959, Thermo Fisher Scientific) Myostatin (Cat. MA5-15486, Thermo Fisher Scientific), MuRF1 (Cat. 4305, Cell Signaling Technology) or TFAM (Cat. PA5-68789, Thermo Fisher Scientific) as primary antibodies and incubated overnight at 4°C. Then, the membrane was washed three times for 15 minutes with 1x TBST (tris-buffered saline (pH 7.4) containing 0.1% tween-20) buffer. The membrane was incubated with secondary antibody HRP-labeled anti-mouse or anti-rabbit IgG (Bio-Rad, USA) for 2 hours at room temperature and washed three times with 1x TBST buffer. Protein signals were detected using ECL substrate Western blotting detection reagent (Bio-Rad). The ChemiDoc MP Imaging System (Bio-Rad) was used to capture densitometrically quantified images using Image J programming software (Image J, NIH, USA). Expression of each target protein was normalized with β-actin (Sigma-Aldrich) as an internal control.

1-9. statistical analysis

All data are expressed as the mean ± standard deviation (in vivo n = 5, in vitro n = 3). Statistical analysis was performed using ANOVA, followed by Dunnetts test between the normal group and the control group or between the control group and the drug-treated group. A p-value < 0.05 was considered significant: # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the normal group, and *p < 0.05, ** p < 0.01, and *** p < versus the control group. is 0.001.

All statistical analyzes were performed in GraphPad Prism software Version 5.0 (GraphPad Software, USA).

2. Results

2-1. Effects of Pineapple Extract on Physiological Changes in Diabetic Animal Models

Changes in physiological and serological markers of diabetic mice induced by a high-fat diet and STZ administration (HFD/STZ) were measured to investigate the effect of sagebrush extract on diabetic symptoms. The results are shown in Table 1 below.

group weight
(g) calorie intake
(kcal) glucose in serum
(mg/dL) insulin in serum
(μU/mL) HOMA-IR
score Normal group 28.00±1.00 74.40±3.10 94.60±16.89 4.78±0.42 0.96±0.09 control group 32.33±2.08 ^### 99.56±5.24 ^### 516.20±33.10 ^### 3.19±0.38 ^## 3.31±0.31 ^### MORE100 28.67±2.08 ^** 85.59 ± 3.02 ^*** 461.60±35.23 ^* 4.11±0.61 2.61±0.47 ^* MORE200 28.33±2.51 ^** 90.83±8.01 ^* 403.40 ± 29.05 ^*** 4.45 ± 1.05 ^* 2.60±0.73 ^* Met 26.33 ± 1.52 ^*** 76.85 ± 8.00 ^*** 286.00 ± 37.58 ^*** 4.68±1.08 ^** 2.43±0.67 ^** Each data is presented as mean±SD. n = 5 per group

Referring to Table 1, the body weight, caloric intake, and glucose level of the diabetic mice (control group) were significantly increased, and the insulin level was significantly decreased compared to the normal group. The group treated with sagebrush extract (MORE100 and MORE200) and the group treated with metformin (Met) showed a significant weight loss compared to the control group. Caloric intake was significantly lower in both the MORE100 and MORE200 and metformin-administered groups of the sagebrush extract-administered groups compared to the control group.

Serum glucose levels were significantly decreased in MORE100, MORE200, and Met. Insulin levels were significantly higher in MORE200 and Met compared to controls. On the other hand, there was no significant difference between the control group and MORE100.

In the HOMA-IR score, the control group significantly increased compared to the normal group, and the insulin resistance scores of MORE100, MORE200, and Met significantly decreased compared to the control group.

2-2. Effects of Pogeukcheon Extract on Lipid Metabolite Changes in Diabetic Animal Models

Serum levels of lipid metabolites were measured in HFD/STZ-induced diabetic mice. The results are shown in Figure 1.

Referring to FIG. 1 , total cholesterol (TCO) and LDL-cholesterol (LDL-C) levels were higher in the control group, respectively. Total cholesterol (TCO) and LDL-cholesterol (LDL-C) levels were significantly reduced in MORE100 and MORE200 administered with the diabetic mouse extract. Total cholesterol (TCO) and LDL-cholesterol (LDL-C) levels were significantly reduced in Met. HDL-cholesterol (HDL-C) levels were lower in the control group than in the normal group and increased in MORE or Met.

AST and ALT levels were measured as indicators of liver damage. AST and ALT levels were significantly increased in the control group and significantly decreased in MORE100, MORE200, and Met.

2-3. Effects of Pineapple Extract on Histological Changes in Muscle Tissues of Diabetic Animal Models

Morphological changes were observed in the muscle tissue of HFD/STZ-induced diabetic mice, and the effect of the burdock extract on muscle damage was investigated. The results are shown in Figure 2.

The major histological features of skeletal muscle atrophy are reductions in muscle fiber (MF) diameter and areas with loosely arranged muscle fibers (see Ma, J.N., et al. J Ethnopharmacol 2020, 259, 112926).

Referring to FIG. 2 , it was confirmed that the gastrocnemius muscle fibers of the normal group were not degraded or necrosis through H&E staining.

In the control group, each bundle of muscle fibers was farther from other bundles, and the space (P) between muscle bundles was significant. Muscle fibers often go from rounded to angular in shape. In MORE100, MORE200, and Met, fibers increased significantly and had normal structures.

2-4. Effects of burdock extract on muscle tissue in HFD/STZ-induced diabetic animal models

The expression of various regulatory proteins including myogenic factors (MyoD, Myogenin and MHC), biogenesis factors (PGC-1α) and atrophy factors (Atrogin1) in HFD/STZ-induced diabetic mice was measured and investigated. The results are shown in FIG. 3 .

Referring to Figure 3, compared to the control group, the expression of MHC, MyoD, Myogenin, and PGC-1α in skeletal muscle tissue was significantly decreased, and the expression of Atrogin1 was significantly increased. In the diabetic mice MORE100 and MORE200 to which the extract was administered, the expression of MHC, MyoD, Myogenin, and PGC-1 in muscle tissue was significantly increased, but the expression of Atrogin1 was significantly decreased.

2-5. Effect of Paigeukcheon Extract on the Differentiation of C2C12 Myoblasts

Expressions of MHC, Myogenin, and MyoD were measured to investigate the effect of the parcole extract on the differentiation of C2C12 myogenic cells. The results are shown in FIG. 4 .

Referring to Figure 4, the expression of Myogenin and MyoD was significantly increased when the MORE 2 mg / mL treatment. It was observed that the myoblast extract (MORE) induces the myoblasts to differentiate into myotube cells having a long and wide cylinder shape and multinucleated cells. In addition, when myoblasts were treated with 0.5 mg/mL or 2 mg/mL of MORE, MHC expression was significantly increased. Expressions of Myogenin, MyoD, and MHC were markedly increased in metformin-treated C2C12 cells.

2-6. Effects of burdock extract on the expression of biogenic factors in C2C12 myotube cells

In order to investigate the effect of the papillae extract on the biogenesis of muscle cells, the expression of biogenetic factors PGC-1α, NRF1, SIRT1 and TFAM was measured in C2C12 myotube cells. The results are shown in FIG. 5 .

Referring to Figure 5, MORE treatment significantly increased the expression of PGC-1α, NRF1, SIRT1 and TFAM in a concentration-dependent manner.

2-7. Effects of monotropane on muscular atrophy of C2C12 myotubes

In order to investigate the effect of monotropane, a physiologically active substance in burdock cloth, on muscle atrophy, various factors such as myogenic factors (Myogenin) and myolytic factors (Myostatin, MuRF1, Atrogin-1) were regulated in C2C12 myotube cells where muscle atrophy occurred. Expression of the protein was measured. The results are shown in FIG. 6 .

Referring to FIG. 6 , in C2C12 myotube cells (DEX100) in which muscular atrophy was caused by treatment with dexamethasone, Myogenin was significantly decreased, and Myostatin, MuRF1, and Atrogin-1 were markedly increased, compared to normal cells (Nor). However, monotropane treatment increased the expression of Myogenin in a concentration-dependent manner and decreased the expression of Myostatin, MuRF1 and Atrogin-1. In particular, 50 umol/mL or 100 umol/mL of monotropane significantly reduced the expression of muscle degradation factors compared to DEX100.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (6)

A pharmaceutical composition for the prevention or treatment of muscle diseases comprising an extract of Paigeukcheon as an active ingredient. The method of claim 1,
The pharmaceutical composition obtained by reflux extraction of the foam cloth extract. The method of claim 1,
The Paigeukcheon extract is a pharmaceutical composition comprising monotropane as a physiologically active substance. The method of claim 1,
The pharmaceutical composition for promoting the differentiation of muscle cells and inhibiting the disassembly of the Paigeukcheon extract. The method of claim 1,
The muscle disease consists of sarcopenia, muscular atrophy, muscular dystrophy, myotonia, hypotonia, amyotrophic lateral sclerosis and myasthenia. A pharmaceutical composition that is one or more selected from the group. A food composition for the prevention or improvement of muscle disease, comprising an extract of Paigeukcheon as an active ingredient.

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