Composition for preventing obesity and preventing and treating muscle loss, comprising Rhododendron karamurasaki extract as an active ingredient

Abstract

The present invention provides an extract having excellent anti-obesity and muscle cell loss prevention effects, and a single compound obtained therefrom. A pharmaceutical composition for preventing and treating muscle loss and anti-obesity, comprising an extract of Rhododendron mucronulatum as an active ingredient, is provided.

Description

[Technical field]

[0001] The present invention relates to a composition for anti-obesity and preventing and treating muscle loss, which comprises an extract of Acanthus cerasifera as an active ingredient. [Background technology]

[0002] Lipid metabolism is necessary for the conservation and distribution of energy in the human body, the regulation of glucose metabolism, and the maintenance of energy homeostasis. Abnormalities in lipid metabolism can lead to symptoms such as obesity, diabetes, and hyperlipidemia. This lipid metabolism occurs mainly in the liver and adipose tissue, and in adipose tissue it is regulated by adipocytes that make up the tissue. Adipocytes are one of the important organs in the body's metabolism, and are not simply energy storage organs but also endocrine organs that secrete various hormones and play an active role in the metabolic process.

[0003] Adipocytes induce obesity when the amount of triglyceride in the adipocyte increases or the number of adipocytes increases. Therefore, in order to prevent and treat obesity, it is necessary to find a way to reduce fat accumulation and the number of adipocytes. In addition, since adipocytes are produced by the differentiation of preadipocytes, research into the mechanism of adipogenesis is also very important in understanding the role of adipose tissue. Recently, molecular biology research on the differentiation and regulatory organs of adipocytes that make up adipose tissue has been extensively conducted. However, there is a lack of research that clarifies the clear efficacy at the level of a single compound.

[0004] On the other hand, muscles can be divided into skeletal muscles, smooth muscles, and cardiac muscles in terms of structure and function. Double skeletal muscles are located just under the skin of the hands, feet, chest, abdomen, etc. and are about 600 voluntary muscles attached to bones through bones and tendons throughout the body. They are adapted to move or support bones through contraction. Contraction occurs and is regulated by nerve signals. They account for 40-50% of body weight and perform functions such as maintaining body temperature and generating energy. They are made up of fine muscle fibrils of actin and myosin in a regular arrangement, and horizontal stripes can be observed under a microscope (Lieber RL, 2002; Edwards RH, 1981).

[0005] Skeletal muscle fibers are biochemically classified into three types, Type I, Type IIa, and Type IIb, according to the mitochondrial content. Type I is a postural muscle made of red proximal fibers that maintains posture by maintaining a weak force for a long time. This muscle has a high mitochondrial content and is suitable for aerobic long-distance exercise. Type IIa is a fast-twitch muscle fiber that has the characteristics of fast-twitch fibers. When making movements, muscles made of white fast-twitch fibers are used, which are called active muscles and are classified as Type IIb. These muscles have a low mitochondrial content and are suitable for anaerobic exercise such as short-distance running. These skeletal muscle fibers are distributed in different proportions in different parts of the body (Tortora et al., 2008).

[0006] Muscle atrophy is the anti-anabolic and catabolic effect of imbalanced muscle fibers. Here, muscle atrophy refers to the loss of size and mass of muscle cells and muscle tissue when muscles are not used due to aging, diseased states (excessive exposure to stress hormones, cancer, sepsis, starvation, etc.), and reduced activity due to illness. When muscle atrophy occurs, muscle strength for physical activity weakens, starting a vicious cycle of musculoskeletal degeneration. Decreased walking speed and weakened grip strength are the main symptoms of muscle mass loss, which can lead to injuries, fractures, joint damage, metabolic disorders, and cardiovascular diseases.

[0007] Glucocorticoids in the human body cause molecular biological changes in muscle fibers and are directly and indirectly involved in anti-anabolic and catabolic actions. Dexamethasone, a glucocorticoid compound, acts as an anti-anabolic action by inhibiting the PI3K / Akt / mTOR pathway, which inhibits the activity of downstream effectors such as 4E-BP1 and S6K1, preventing the activation of eIF4G (Eukaryotic translation initiation factor 4G) and eIF4E (Eukaryotic translation initiation factor 4E). This suppresses the mRNA translation process for protein synthesis, which manifests as inhibition of muscle fiber synthesis and muscle fiber atrophy due to protein degradation (Shackman et al., 2013).

[0008] Dexamethasone can also induce muscle atrophy by inhibiting muscle synthesis and causing protein degradation. This is due to the mechanism of PI3K / Akt → FOXO activation and GSK3 inactivation, which leads to the expression of atrogenes (Atrogin-1, MuRF-1) that induce muscle atrophy, but these genes induce protein degradation represented by the ubiquitin-proteasome system.

[0009] Therefore, there is a need to develop a substance for preventing obesity and muscle loss that has the effect of simultaneously breaking down sarcopenia, a skeletal muscle loss disease, and fat. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, the problem to be solved by the present invention is to provide an extract having excellent anti-obesity and anti-loss of muscle cells at the same time, and a single compound obtained therefrom. [Means for solving the problem]

[0011] To achieve the above object, the present invention provides a pharmaceutical composition for anti-obesity and preventing and treating muscle loss, comprising an extract of Rhododendron mucronulatum as an active ingredient.

[0012] In one embodiment of the present invention, the extract of Rhododendron mucronulatum contains taxyporin glycosides or taxyporin non-glycosides.

[0013] In one embodiment of the present invention, the Rhododendron mucronulatum extract is obtained by supercritical extraction of the roots of Rhododendron mucronulatum.

[0014] In one embodiment of the present invention, the taxiporin glycoside comprises a compound of formula (1):

[0015] [ka]

[0016] In one embodiment of the present invention, the taxiporin glycoside comprises a compound of formula (2):

[0017] [ka]

[0018] In one embodiment of the present invention, there is provided a pharmaceutical composition for anti-obesity and prevention and treatment of muscle loss, comprising at least one compound selected from the group consisting of compounds of the following formulae and formula (2):

[0019] [ka]

[0020] [ka]

[0021] In one embodiment of the invention, the compound is extracted from the roots of Rhododendron calamum.

[0022] The present invention also provides a food composition for preventing obesity and muscle loss, comprising an extract of Rhododendron mucronulatum as an active ingredient.

[0023] In one embodiment of the present invention, the Rhododendron mucronulatum extract contains taxyporin glycoside or taxyporin non-glycosylated form, and the Rhododendron mucronulatum extract is obtained by supercritical extraction of the roots of Rhododendron mucronulatum.

[0024] In one embodiment of the present invention, the taxiporin glycoside comprises a compound of formula (1):

[0025] [ka]

[0026] In one embodiment of the present invention, the taxiporin glycoside comprises a compound of formula (2):

[0027] [ka]

[0028] The present invention provides a food composition for preventing obesity and muscle loss, comprising at least one compound selected from the group consisting of the compounds of the following formulae and (2):

[0029] [ka]

[0030] [ka]

[0031] The compound is extracted from the roots of Rhododendron karamurasakii.

[0032] In the present invention, the food composition for anti-obesity and preventing muscle loss is an additive for animal feed. Effect of the Invention

[0033] The anti-obesity composition according to the present invention is based on the taxyporin glycoside and taxyporin non-glycosylated form of the Rhododendron karamurasaki extract, and has the ability to inhibit adipocyte differentiation in a concentration-dependent manner. In particular, at the highest experimental concentration of 20ug / ml, the effect of inhibiting adipocyte differentiation by 40% or more was confirmed. Furthermore, it also has the effect of inhibiting muscle loss caused by hydrogen peroxide and dexamethasone. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 is a diagram illustrating an extraction process according to one embodiment of the present invention. [Diagram 2] FIG. 2 shows the results of TLC analysis of the Rhododendron karamurasakiense-derived high-content extract (RMRF). [Diagram 3] FIG. 3 shows the results of an analysis of the total phenol content of the Rhododendron ramosa-rich extract (RMRF). [Figure 4] FIG. 4 shows the results of RMRF analysis of taxyporin non-glycosylated forms against RMRF and spirit extract (RM).

[0035] [Diagram 5] FIG. 5 shows the results of RMRF analysis of taxyporin glycosides in RMRF and alcohol extract (RM). [Figure 6]FIG. 6 shows photographs of cells observed when treated with the compound "Taxifolin-3-O-arabinopyranoside," which has been shown to be an indicator substance and an effective substance for plants of the genus Rhododendron. [Figure 7] Figure 7 shows a photograph of cells observed when treated with a non-glycosylated compound, i.e., "Taxifolin-aglycone," produced through enzymatic hydrolysis from a glycoside compound, "Taxifolin-3-O-arabinopyranoside," well known as an indicator and effective substance derived from plants of the genus Rhododendron. [Figure 8] 8 and 9 show the results of analyzing cell viability in response to taxyporin glycoside (RM) and taxyporin non-glycosylated form (RMRF), respectively. [Figure 9] 8 and 9 show the results of analyzing cell viability in response to taxyporin glycoside (RM) and taxyporin non-glycosylated form (RMRF), respectively. [Figure 10] 10 and 11 show the results of analyzing cell viability when taxyporin glycoside (RM) and taxyporin non-glycosylated (RMRF) were used against H2O2 (hydrogen peroxide), respectively. [Figure 11] 10 and 11 show the results of analyzing cell viability when taxyporin glycoside (RM) and taxyporin non-glycosylated (RMRF) were used against H2O2 (hydrogen peroxide), respectively. [Figure 12] 12 and 13 show the results of analyzing cell viability when taxyporin glycoside (RM) and taxyporin non-glycosylated (RMRF) are used in comparison with dexamethasone, respectively. [Figure 13] 12 and 13 show the results of analyzing cell viability when taxyporin glycoside (RM) and taxyporin non-glycosylated (RMRF) are used in comparison with dexamethasone, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Hereinafter, specific embodiments of the present invention will be described with reference to the drawings. However, these are merely examples, and the present invention is not limited thereto. In describing the present invention, if it is determined that a detailed description of a known technology related to the present invention may unnecessarily obscure the gist of the present invention, the detailed description will be omitted. The terms described below are defined in consideration of the functions in the present invention, and may vary depending on the intention or practice of a user or operator. Therefore, the definitions should be based on the entire contents of this specification. The technical idea of ​​the present invention is determined by the claims, and the following examples are merely a means for efficiently explaining the technical idea of ​​the present invention to those skilled in the art to which the present invention belongs. The present invention provides a composition for preventing or treating hair loss, comprising an extract of a plant of the genus Rhododendron as an active ingredient. The present invention provides lipolysis inhibition of taxyporin glycosides and taxyporin non-glycosides derived from Rhododendron calamus native to Korea, as described below, and an anti-obesity composition based thereon. The anti-obesity composition according to the present invention includes both a pharmaceutical composition for anti-obesity and a food composition for prevention or improvement. In addition, the taxyporin glycoside and taxyporin non-glycosylated form derived from Rhododendron karamarasakiensis according to the present invention also have the effect of preventing and treating muscle loss by inhibiting muscle loss, which can be applied to both pharmaceutical compositions and food compositions for prevention or improvement.

[0037] FIG. 1 is a diagram illustrating an extraction process according to one embodiment of the present invention. Referring to FIG. 1, in one embodiment of the present invention, the roots (12 kg) of Rhododendron mucronulatum were extracted with 60% alcohol at room temperature for 7 days, filtered through filter paper, and the extract was concentrated under reduced pressure to recover an extract (440.54 g). The extract was then dissolved in distilled water and filtered through filter paper, and a Silicagel column (40 μm, YAMAZEN, Osaka, Japan) was used, and the solvent was prepared in a ratio of chloroform:methanol:water at a ratio of 70:30:4, and the mixture was run in an isocratic system, and the spots were confirmed by TLC. In order to improve the purity of the target compound, separation and purification using MPLC (YAMAZEN, Osaka, Japan) was performed. The solvent was repeatedly run through a gradient system (0%→50% MeOH / 20→80% MeOH) using an ODS column (50 μm, YAMAZEN, Osaka, Japan) with water and methanol as the solvent. Finally, two compounds were isolated and purified from the Rhododendron karamurasaki root extract, namely the glycoside taxifolin-3-O-arabinopyranoside and the non-glycosylated taxifolin, which will be described in more detail below.

[0038] Examples supercritical extraction Supercritical fluid extraction research equipment (ISA-SEFE-0500-0700-080, IlsinAutoclave, Daejeon, Korea) was used for supercritical extraction of Rhododendron karamarasaki root. To explain in detail, the sample was washed to remove foreign matter, dried in the shade, and used as the experimental material. The dried sample was crushed into 100g units to pass through a 200mesh crushing net, and the temperature of the Rhododendron karamarasaki root sample was adjusted to 40~60℃ and maintained. After that, when the temperature was stabilized, the Rhododendron karamarasaki root sample was placed in the extraction tank and CO2 gas was maintained at constant pressure, and then the high-pressure pump was used to inject CO2 gas through the line by adjusting the control valve until it reached the experimental pressure condition of 400~600bar. After the set pressure was reached, edible alcohol was added to the bottom of the extraction tank at 5mL or 10mL per minute for 60 or 240 minutes to proceed with the extraction. In order to remove the residual ethanol remaining in the sample, CO2 was pumped in using a high-pressure pump for 30 minutes at the set pressure and temperature to complete the extraction and produce the extract (RM).

[0039] Solvent fractionation The Rhododendron karam-rasakii root supercritical extract foil obtained after the above-mentioned supercritical extraction method was collected and extracted with edible alcohol (30-100%) for 3 days at room temperature, then filtered through filter paper, concentrated under reduced pressure, and freeze-dried to obtain the final Rhododendron karam-rasakii root supercritical extract foil alcohol extract (RMSCFR). The obtained RMSCFR was dissolved in distilled water (1st or 3rd distilled water) and filtered through filter paper, and the ethyl acetate (EtOAC) layer and water layer were separated using a separatory funnel. The obtained EtOAC extract was the Rhododendron karam-rasakii root high content extract (RMRF).

[0040] Experimental Example 1 TLC analysis In this experimental example, TLC analysis was carried out on the Rhododendron karamarasakiense-derived high-content extract (RMRF) produced by the above-mentioned method.

[0041] Figure 2 shows the results of TLC analysis of the Rhododendron karamarasakii extract (RMRF). As shown in Figure 2, it can be confirmed that the RMRF fraction contains taxifolin aglycone and taxifolin glycoside. That is, the results of chromatography of the non-glycosylated taxifolin (taxifolin aglycone), the glycoside taxifolin-3-O-arabinopyranoside, and RMRF I show that the taxifolin aglycone and taxifolin-3-O-arabinopyranoside (glycoside) are all present in the Rhododendron karamarasakiii extract (RMRF) according to the present invention.

[0042] Total phenol content analysis FIG. 3 shows the results of an analysis of the total phenol content of the Rhododendron ramosa-rich extract (RMRF). 3, the total phenol content (methyl gallate, ethyl gallate, gallic acid) was analyzed, and it was found that the total phenol content of RMRF, which was extracted and fractionated with a solvent, was more than three times higher than that of the RM extract. These results suggest that RMRF, which contains both taxyporin and taxyporin-3-O-arabinopyranoside, obtained by the supercritical extraction of Rhododendron karamara roots according to the present invention, can be expected to have strong biological activity.

[0043] HPLC analysis results For HPLC analysis, a Waters 2695 Separation module and a 2487 Dual λ Absorbance Detector were used. The columns used were a SkyPak C18 analytical column (5 μm) and a Phenomenex KJ0-4282 guard column. The mobile phase consisted of 1% Formic acid (A) and ACN (B) (Gradient program: 10% B 0 min, 60% B 0-40 min, 100% B 40-45 min, 10% B 45-50 min, 10% B 50-60 min).

[0044] Figure 4 shows the RMRF analysis results for taxyporin non-glycosylated forms in RMRF and alcohol extract (RM). Referring to Figure 4, taxyporin non-glycosylated forms were confirmed to be 1.2352ug / ml in the 60% alcohol extract (RM) of Rhododendron karamarasaki root, and 4.1530ug / ml in the RMRF extract. Therefore, it can be confirmed that the taxyporin non-glycosylated form content is increased by more than 336.22% compared to the existing 60% alcohol extract.

[0045] FIG. 5 shows the results of RMRF analysis of taxyporin glycosides in RMRF and alcohol extract (RM). Referring to FIG. 5, the taxyporin glycoside content in the Rhododendron karamarasakii-derived high-content taxyporin glycoside extract (RMRF) was analyzed and found to be increased by 684.17% compared to the 60% alcohol extract.

[0046] Structural identification The final single compounds were isolated and purified from the taxiporin non-glycosylated RM and RMRF through repeated purification such as MPLC column chromatography, and NMR and LC / MS data were measured to identify the structure of the isolated and purified compounds. The results are as follows:

[0047] White yellow amorphous powder Negative LC-MS: m / z 303.0 [MH]-1H-NMR (300 MHz, DMSO-d6): δ 11.92 (1H, s, 5-OH), 6.75~6.88 (3H in total, m, H-2', H-5' and H-6'), 5.92 (1H, d, J=2.1 Hz, H-8), 5.87 (1H, d, J=2.1 Hz, H-6), 5.00 (1H, d, J=11.1 Hz, H-2), 4.52 (1H, d, J=11.1 Hz, H-3).

[0048] 13C-NMR (75 MHz, DMSO-d6): 197.2 (C-4), 167.1 (C-7), 163.5 (C-5), 162.8 (C-9), 146.0 (C-4'), 145.1 (C-3'), 128.2 (C-1'), 119.6 (C-6'), 115.5 (C-5'), 115.2 (C-2'), 100.6 (C-10), 96.1 (C-6), 95.1 (C-8), 83.1 (C-2), 71.6 (C-3)

[0049] The structural formula of the resulting taxyporin non-glycosylated form is shown in Chemical Formula 1 below.

[0050] [ka]

[0051] Taxiporin glycoside The final single compound was isolated and purified from the extract of Korean wild Rhododendron moniliforme through repeated purification such as MPLC column chromatography, and NMR and LC / MS data were measured to identify the structure of the isolated and purified compound.

[0052] White yellow amorphous powder, LC-MS, (positive-ion mode) m / z 437.1109 [M+H]+; 1H-NMR, (700 MHz, MeOH-d4) δ : 3.38 (1H, dd, J = 11.2, 3.5 Hz, H-5''), 3.55 (1H, m, H-3''), 3.58 (1H, m, H-2''), 3.80 (1H, m, H-4''), 3.82 (1H, d, J = 3.5 Hz, H-1''), 3.91 (1H, dd, J=11.2, 7.0 Hz, H-5''), 4.79 (1H, d, J=10.5 Hz, H-3), 5.12 (1H, d, J=10.5 Hz, H-2), 5.90 (1H, d, J=2.1Hz, H-8), 5.92 (1H, d, J=2.1 Hz, H-6), 6.79 (1H, d, J=8.4 Hz, H-5'), 6.84 (1H, dd, J=8.4, 2.1 Hz, H-6'), 6.965 (1H, d, J=2.1 Hz, H-2'); 13 C-NMR, (175 MHz, MeOH-d4) δ : 196.17 (C-4), 169.04 (C-7), 165.74 (C-5), 164.28 (C-9), 147.18 (C-4'), 146.58 (C-3'), 128.98 (C-1'), 120.79 (C-6'), 116.30 (C-5'), 116.00 (C-2'), 102.40 (C-1"), 101.40 (C-10), 97.41 (C-6), 96.42 (C-8), 83.83 (C-2), 76.67 (C-3), 73.23 (C-2"), 71.12 (C-3"), 66.79 (C-4"), 63.36 (C-5").

[0053] The molecular weight confirmed through the final LC-MS was confirmed and compared with existing references, and it was confirmed that the final glycoside form of taxifolin was taxifolin-3-O-α-L-arabinopyranoside represented by the following formula 2.

[0054] [ka]

[0055] Experimental Example 2 The taxyporin glycoside or non-glycosylated form derived from Rhododendron karamarasakii according to the present invention has an anti-obesity effect of suppressing adipocyte differentiation. In this experimental example, the efficacy of natural product extracts (RM, RMRF) derived from Rhododendron karamarasakii, which is native to Korea, in suppressing adipocyte differentiation was evaluated. For this purpose, a 60% alcohol extract (RM) of the roots of Rhododendron karamarasakii, a plant native to Korea, and the taxyporin glycoside and non-glycosylated form, which are indicator and effective substances from RM, were used as test substances.

[0056] cell culture 3T3-L1 cells, which are mouse-derived adipose precursor cells, were purchased from the Korea Cell Line Bank. 3T3-L1 cells were cultured in a complete DMEM culture medium (Welgene) supplemented with 10% bovine calf serum (BCF), 100 units / mL penicillin, and 100 μg / mL streptomycin in a humidified CO2 incubator (5% CO2 / 95% air) at 37°C. When the cells filled about 80% of the culture dish, the cell monolayer was washed off with phosphate buffered saline (PBS, pH 7.4), and trypsin-2.65 mM EDTA was added to remove the cells and subculture them. The medium was changed every 2 days.

[0057] Measurement of cell viability 3 x 10 3T3-L1 cells 4The cells were then divided into 24-well plates at 100 cells / well and cultured for 24 hours. After culturing the cells for 24 hours, the cell culture medium was replaced with one containing the test substance and the cells were cultured for 72 hours. After culturing the cells for 72 hours, the MTT assay (Denizot F and Lang RJ Immunological Method 89:271-277,1986) was performed to measure the number of live cells. The MTT assay method is based on the principle that mitochondrial dehydrogenase reduces MTT (Amresco) to produce formazan, a blue substance. In this test, formazan was dissolved in isopropanol and the absorbance was measured at a wavelength of 570 nm.

[0058] Differentiation induction and treatment with test substances 1 x 10 3T3-L1 cells 5 The cells were dispensed into a 24-well plate at a concentration of 10 ...

[0059] Adipocyte differentiation (fat scale) measurement (Oil red-O staining) After inducing differentiation of 3T3-L1 cells and treating them with the test substances, the cells were washed with DPBS (Welgene) and fixed at room temperature for 1 hour with 4% paraformaldehyde (PFA, Biosesang). After fixing the cells, they were stained at room temperature for 1-2 hours with oil red O (Sigma-Aldrich) solution. After visually observing the degree of staining of the adipocytes, the cells were washed with distilled water and observed under a microscope. Figure 6 shows a cell photograph observed when treated with the compound 'Taxifolin-3-O-arabinopyranoside', which has been shown to be an indicator and effective substance of the plants of the genus Taxifolin. Referring to Figure 6, it is possible to significantly confirm the ability to inhibit adipocyte differentiation, which increases concentration-dependently depending on the treatment concentration. In particular, it was confirmed that the ability to inhibit adipocyte differentiation was inhibited by more than 40% at the highest concentration of 20ug / ml in the experiment. FIG. 7 is a photograph of cells observed when treated with a non-glycosylated compound, i.e., "Taxifolin-aglycone" compound, produced by enzymatic hydrolysis from a glycoside compound, i.e., "Taxifolin-3-O-arabinopyranoside," well known as an indicator substance and effective substance derived from plants of the genus Rhododendron. Referring to FIG. 7, it is possible to significantly confirm the effect of inhibiting adipocyte differentiation, which increases concentration-dependently depending on the treatment concentration. In particular, at the highest experimental concentration of 20ug / ml, it was confirmed that the effect of inhibiting adipocyte differentiation was inhibited by 40% or more. The above results suggest that the taxiporin glycoside or non-glycosylated form (RM, RMRF) derived from Rhododendron karamarasensis according to the present invention can be used as an idle ingredient in an anti-obesity therapeutic agent or functional food.

[0060] Experimental Example 3 Taxyporin glycoside or non-glycosylated form derived from Rhododendron karamarasakii according to the present invention has not only the fat-rotting ability but also the ability to inhibit muscle loss. In this experimental example, the muscle loss improving efficacy of natural product extracts (RM, RMRF) derived from Rhododendron karamarasakii, which is native to Korea, in preventing skeletal loss was evaluated. In particular, the protective effect against H2O2 and dexamethasone-induced muscle cell damage was tested in an in vitro system. For this purpose, a 60% alcohol extract (RM) from the root of Rhododendron karamarasakii, a plant native to Korea, and a high-content extract (RMRF) from RM, which is an indicator substance and has a high content of taxyporin glycoside and non-glycosylated form, a single compound, were used as test substances.

[0061] cell culture C2C12 cells, which are myoblasts derived from mouse skeletal muscle, were purchased from American Type Culture Collection (ATCC). C2C12 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin in a humidified CO2 incubator (5% CO2 / 95% air) at 37°C. When the cells filled approximately 80% of the culture dish, the cell monolayer was washed off with phosphate buffered saline (PBS, pH 7.4), and then trypsin-2.65 mM EDTA was added to remove the cells and subculture them. The medium was changed every 2 days.

[0062] Measurement of cell viability The cell viability of C2C12 cells was measured by the MTT assay (Denizot F and Lang RJ Immunological Method 89:271-277, 1986). C2C12 cells were incubated at 2.5 × 10 4The cells were dispensed into a 24-well plate at 100 cells / well and cultured for 24 hours. After culturing the cells for 24 hours, the cell culture medium was replaced with cell culture medium treated with various concentrations of test substances (0, 10, 50, 100, 150, 200μg / mL) and cultured for 24 hours. After culturing for 24 hours with the test substances, the cell culture medium was replaced with 1mg / mL MTT (Amresco) solution and the cells were further cultured for 2 hours, after which the formazan formed in living cells was eluted with isopropanol and the absorbance was measured at 570nm.

[0063] H 2 O 2 Protective effect against induced muscle cell damage 2.5 × 10 C2C12 cells 4 The cells were plated at 100μM H2O2 / well and cultured for 24 hours. After culturing C2C12 cells for 24 hours, 100μM H2O2 was treated to induce muscle cell damage, and to investigate the protective effect of each test substance against muscle cell damage, the cells were treated with various concentrations of test substances together with 100μM H2O2 and cultured for 24 hours. After culturing the cells for 24 hours, the MTT assay was performed in the same manner as above to measure cell viability.

[0064] Measurement of protective effect against dexamethasone-induced muscle cell injury 2.5 × 10 C2C12 cells 4 The cells were plated at 1000 cells / well in a 24-well plate and cultured for 24 hours. After culturing C2C12 cells for 24 hours, they were treated with 500μM dexamethasone to induce muscle cell damage, and to investigate the protective effect of each test substance against muscle cell damage, the cells were treated with various concentrations of test substances together with 500μM dexamethasone and cultured for 24 hours. After culturing the cells for 24 hours, the MTT assay was performed in the same manner as above to measure cell viability.

[0065] Statistical Processing All analytical values ​​are expressed as mean ± SEM. Collected results were analyzed using the GraphPad Prism 5.0 (GraphPad software, San Diego, CA, USA) program. Student's t-test and one-way analysis of variance (ANOVA) were used to compare the differences between the control group and the test substance-treated group. Only p<0.05 or higher was considered to be statistically significant.

[0066] result Effect of natural product extracts (RM, RMRF) on cell viability 8 and 9 show the results of analyzing cell viability for taxyporin glycoside (RM) and taxyporin non-glycosylated form (RMRF), respectively. Referring to FIGS. 8 and 9, in order to investigate the cytotoxicity of two kinds of natural product extracts (RM, RMRF) in C2C12 cells, various concentrations (0, 10, 50, 100, 150, 200 μg / mL) of natural product extracts were treated in cell culture medium, and the cells were cultured for 24 hours, and then MTT assay was performed. The cell viability of C2C12 cells was increased by treatment with RM and RMRF at various concentrations (10, 50, 100, 150, 200 μg / mL). The cell viability increased with increasing RM treatment concentration, and when treated with concentrations of 50 to 200 μg / mL, the cell viability increased by 11.3%, 24.8%, and 25.3%, respectively, compared to the control group (0 μg / mL). Similarly, cell viability increased with increasing RMRF treatment concentration, with treatment at concentrations of 50-200μg / mL increasing cell viability by 10.2%, 18.5%, 22.6%, and 27.4% respectively compared to the control group (0μg / mL).Based on these results, the extract of Rhododendron keta, native to Korea, which contains taxyporin glycosides and non-glycosylated taxyporin, is not toxic to normal muscle cells and is expected to reduce muscle loss by increasing muscle cells.

[0067] H 2 O 2 (Hydrogen peroxide) Analysis results 10 and 1 show the results of analyzing cell viability when taxyporin glycoside (RM) and taxyporin non-glycosylated (RMRF) were used against H2O2 (hydrogen peroxide), respectively.

[0068] H2O2 (hydrogen peroxide) is a strong oxidant that induces oxidative stress in vitro. To investigate the effects of RM and RMRF on muscle cell damage caused by oxidative stress, 100μM H2O2 was treated in the cell culture medium of C2C12 cells to induce oxidative stress, and then RM and RMRF were treated and cultured, and the cell viability of C2C12 cells was measured. Referring to Figures 10 and 11, when H2O2 was treated, the cell viability was significantly decreased compared to the control group [H2O2(-) / (-)] that was not treated with H2O2. When RM was treated at various concentrations (10, 50, 100, 200μg / mL), the cell viability was significantly increased compared to the control group [H2O2(+) / (-)] that was treated only with H2O2. When RM was treated at a concentration of 200μg / mL, the cell viability increased by 44% compared to the control group [H2O2(+) / (-)] that was treated only with H2O2. When RMRF was treated at concentrations of 10, 50, 100, and 200 μg / mL, cell viability increased significantly compared to the control group treated with H2O2 alone [H2O2(+) / (-)]. In particular, the cell viability increased statistically significantly by 53.4% ​​and 55.7% in the 100-200 μg / mL treatment group compared to the control group treated with H2O2 alone [H2O2(+) / (-)]. These results indicate that the extract of Rhododendron kalamarasakii, which is native to Korea and contains taxyporin glycosides and non-taxyporin glycosides, inhibits muscle loss caused by cell death induced by oxidative stress.

[0069] Dexamethasone analysis results 12 and 13 show the results of cell viability analysis when taxyporin glycoside (RM) and taxyporin non-glycosylated (RMRF) are used against dexamethasone, respectively. Dexamethasone is one of the representative glucocorticoids, and when misused in clinical practice, it causes the degradation of skeletal muscle, and is therefore widely used to induce muscle cell damage in an in vitro system. In order to investigate the effect of RM and RMRF according to the present invention on muscle cell damage caused by glucocorticoids, 500 μM dexamethasone was treated in the cell culture medium of C2C12 cells to induce muscle cell damage, and the cells were treated with RM and RMRF and then cultured, and the cell viability of C2C12 cells was measured. Referring to FIG. 12 and FIG. 13, when dexamethasone was treated, the cell viability was significantly reduced compared to the control group [DEX(-) / (-)] that was not treated with dexamethasone. RMRF at concentrations of 10-200μg / mL was confirmed to significantly increase the decrease in cell viability induced by dexamethasone. That is, as the concentration increased from 10 to 200μg / mL, the protective effect against cell damage increased statistically significantly, 39.5%, 49.4%, 53.6%, and 52.8%. In addition, when RMRF was treated at concentrations of 10, 50, and 100μg / mL, cell viability was significantly increased compared to the control group [DEX(+) / (-)] treated with dexamethasone only. RMRF at various concentrations (10, 50, and 100μg / mL) was confirmed to have a significant effect on the decrease in cell viability induced by dexamethasone. That is, as the concentration increased, cell viability increased by 39.8%, 44.5%, and 45.3%. Based on the above results, it can be seen that the extract of Rhododendron kalambara grown in Korea, which contains taxyporin glycoside and taxyporin non-glycosylated form, inhibits muscle loss caused by the breakdown of skeletal muscle and the death of muscle fiber cells, which is one of the side effects of glucocorticoid drugs. [Industrial Applicability]

[0070] The composition has industrial applicability as a composition for preventing and treating obesity and muscle slowing.

Claims

1. A pharmaceutical composition for preventing obesity and preventing and treating muscle loss, comprising an extract of Rhododendron mucronulatum as an active ingredient.

2. 2. The pharmaceutical composition for anti-obesity and prevention and treatment of muscle loss according to claim 1, wherein the extract of Rhododendron mucronulatum contains taxyporin glycoside or taxyporin non-glycosylated form.

3. 3. The pharmaceutical composition for anti-obesity and preventing and treating muscle loss according to claim 2, wherein the Rhododendron mucronulatum extract is obtained by supercritical extraction of the roots of Rhododendron mucronulatum.

4. The pharmaceutical composition for anti-obesity and prevention and treatment of muscle loss according to claim 2, characterized in that the taxiporin glycoside comprises a compound of the following formula (1): 【Chemistry 1】

5. The pharmaceutical composition for anti-obesity and prevention and treatment of muscle loss according to claim 2, characterized in that the taxiporin glycoside comprises a compound of the following formula (2): 【Chemistry 2】

6. A pharmaceutical composition for anti-obesity and prevention and treatment of muscle loss, comprising at least one compound selected from the group consisting of compounds of the following formulae and (2): 【Chemistry 3】 【Chemistry 4】

7. 7. The pharmaceutical composition for anti-obesity and prevention and treatment of muscle loss according to claim 6, wherein the compound is extracted from the roots of Rhododendron karamurosan.

8. A food composition for preventing obesity and muscle loss, comprising an extract of Rhododendron mucronulatum as an active ingredient.

9. 9. The food composition for anti-obesity and preventing muscle loss according to claim 8, wherein the Rhododendron mucronulatum extract contains taxyporin glycoside or taxyporin non-glycosylated form.

10. 10. The food composition for preventing obesity and muscle loss according to claim 9, wherein the Rhododendron mucronulatum extract is obtained by supercritical extraction of the roots of Rhododendron mucronulatum.

11. The food composition for anti-obesity and prevention of muscle loss according to claim 9, characterized in that the taxiporin glycoside comprises a compound of the following formula (1): 【Chemistry 5】

12. The food composition for anti-obesity and prevention of muscle loss according to claim 9, characterized in that the taxiporin glycoside comprises a compound of the following formula (2): 【Chemistry 6】

13. A food composition for preventing obesity and muscle loss, comprising at least one compound selected from the group consisting of compounds represented by the following formulae and (2): 【Chemistry 7】 【Chemistry 8】

14. The food composition for preventing obesity and muscle loss according to claim 13, characterized in that the compound is extracted from the roots of Rhododendron karamarasakii.

15. The food composition for anti-obesity and preventing muscle loss according to claim 13 or 14, which is an additive for animal feed.

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