Composition for the prevention and treatment of obesity and muscle loss, containing Rhododendron kaempferi 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 preventing and treating obesity and sarcopenia, containing an extract of Rhododendron kaempferi as an active ingredient.

Background Art

[0002] Lipid metabolism is necessary for the storage and distribution of human energy, the regulation of glucose metabolism, and the maintenance of energy homeostasis. If abnormalities occur in lipid metabolism, it can cause symptoms such as obesity, diabetes, and hyperlipidemia. Such lipid metabolism mainly occurs 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. They are not just simple energy storage organs but also endocrine organs that secrete various hormones and play an active role in the metabolic process.

[0003] Adipocytes will induce obesity when the amount of triglyceride in adipocytes increases or the number of adipocytes increases. Therefore, in preventing and treating obesity, it is necessary to find a solution to reduce fat accumulation and the number of adipocytes. Also, since adipocytes are formed by the differentiation of preadipocytes, studying the mechanism of adipogenesis is also very important for understanding the role of adipose tissue. Recently, extensive molecular biological research has been carried out on the differentiation of adipocytes that make up adipose tissue and the regulatory organs. However, the research on clarifying the clear efficacy at the single compound level is insufficient.

[0004] Muscles, on the other hand, can be divided into skeletal muscle, smooth muscle, and cardiac muscle in terms of structure and function. Skeletal muscle consists of over 600 voluntary muscles located just beneath the skin of the hands, feet, chest, and abdomen, attached to bones throughout the body via bones and tendons. It is suitable for moving or supporting bones through contraction. Contraction is caused and regulated by nerve signals. It accounts for 40-50% of body weight and performs functions such as maintaining body temperature and energy production. It is composed of actin and myosin, which are micromuscular fibrils, arranged in a regular pattern, and transverse stripes can be observed under a microscope (Lieber RL, 2002; Edwards RH, 1981).

[0005] Skeletal muscle fibers are classified into three types—Type I, Type IIa, and Type IIb—based on their mitochondrial content. Type I muscles, consisting of red proximal fibers, are postural muscles that maintain posture by sustaining weak force over extended periods. These muscles have a high mitochondrial content and are suitable for aerobic long-distance running. Type IIa muscles are fast-twitch fibers that also possess characteristics of proximal fibers. When creating movement, muscles composed of white fast-twitch fibers are used; these are called active muscles and are classified as Type IIb. These muscles have a low mitochondrial content and are suitable for anaerobic short-distance running. These skeletal muscle fibers are distributed in different proportions across different parts of the body (Tortora et al, 2008).

[0006] Muscle atrophy is described as an imbalance in the anti-anabolic and catabolic processes of 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 conditions (excessive exposure to stress hormones, cancer, sepsis, starvation, etc.), and reduced activity in a diseased lifestyle. When muscle atrophy occurs, muscle strength for physical activity weakens, initiating a vicious cycle of musculoskeletal degeneration. Decreased walking speed and weakened grip strength are the main symptom indicators of muscle mass loss, which can lead to injuries, fractures, joint damage, metabolic disorders, and cardiovascular diseases.

[0007] Human glucocorticoids induce molecular biological changes in muscle fibers, directly and indirectly participating in antianabolic and catabolic processes. Dexamethasone, a glucocorticoid compound, plays a role in inhibiting the PI3K / Akt / mTOR pathway as an anti-anabolic action. This inhibits the activity of downstream effectors such as 4E-BP1 and S6K1, preventing the action of eIF4G (Eukaryotic translation initiation factor 4G) and eIF4E (Eukaryotic translation initiation factor 4E). This suppresses the mRNA translation process for protein synthesis, manifesting 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 because the mechanism involves the activation of FOXO and inactivation of GSK3, leading to the expression of atrogenes (Atrogin-1, MuRF-1) that induce muscle atrophy. These genes, in turn, induce protein degradation, exemplified by the ubiquitin-proteasome system.

[0009] Therefore, there is a need to develop a substance that has the efficacy to simultaneously break down both muscular dystrophy (a disease characterized by a decrease in skeletal muscle) and fat, thus promoting anti-obesity and suppressing muscle loss. [Overview of the project] [Problems that the invention aims to solve]

[0010] Therefore, the problem that the present invention aims to solve is to provide an extract that simultaneously possesses excellent anti-obesity and anti-muscle cell reduction efficacy, and a single compound obtained therefrom. [Means for solving the problem]

[0011] To solve the aforementioned problems, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity and muscle loss, comprising an extract of Rhododendron mucronulatum as an active ingredient.

[0012] In one embodiment of the present invention, the Rhododendron mucronulatum extract comprises a taxiporine glycoside or a taxiporine nonglycoside.

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

[0014] In one embodiment of the present invention, the taxiporine glycoside comprises a compound of the following formula (1).

[0015] [ka]

[0016] In one embodiment of the present invention, the taxiporine glycoside comprises a compound of the following formula (2).

[0017] [ka]

[0018] In one embodiment of the present invention, a pharmaceutical composition for the prevention and treatment of obesity and muscle loss is provided, comprising one or more compounds selected from the group consisting of the following formulas and formula (2).

[0019] [ka]

[0020] [Chemical formula]

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

[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 example of the present invention, the extract of Rhododendron mucronulatum contains taxiphyllin glycoside or taxiphyllin aglycone, and the extract of Rhododendron mucronulatum is obtained by supercritical extraction of the roots of Rhododendron mucronulatum.

[0024] In one example of the present invention, the taxiphyllin glycoside contains a compound of the following formula (1).

[0025] [Chemical formula]

[0026] In one example of the present invention, the taxiphyllin glycoside contains a compound of the following formula (2).

[0027] [Chemical formula]

[0028] Provided is a food composition for preventing obesity and muscle loss, comprising any one or more selected from the group consisting of the compounds of the following formula and formula (2).

[0029] [Chemical formula]

[0030] [ka]

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

[0032] The present invention also relates to the anti-obesity and muscle loss prevention food composition, which is an animal feed additive. [Effects of the Invention]

[0033] The anti-obesity composition according to the present invention is based on taxiporine glycosides and non-taxiporine glycosides contained in Rhododendron kohlrabi extract, and has concentration-dependent inhibitory activity on adipocyte differentiation. In particular, at the highest experimental concentration of 20 ug / ml, an effect of suppressing adipocyte differentiation by more than 40% was confirmed. Furthermore, it also has an inhibitory effect on muscle loss in response to hydrogen peroxide and dexamethasone. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1 is a diagram illustrating an extraction process according to one embodiment of the present invention. [Figure 2] Figure 2 shows the TLC analysis results for high-content extract (RMRF) derived from Rhododendron parvifolium. [Figure 3] Figure 3 shows the results of the total phenol content analysis of the high-concentration extract (RMRF) derived from Rhododendron parvifolium. [Figure 4] Figure 4 shows the results of RMRF analysis against non-glycosaccharides of taxiporine in relation to alcohol extract (RM).

[0035] [Figure 5] Figure 5 shows the results of RMRF analysis of RM and taxiporine glycosides against alcohol extract (RM). [Figure 6]Figure 6 shows cell images observed after treatment with the compound "Taxifolin-3-O-arabinopyranoside," which has been shown to be both an indicator substance and an effective substance for plants of the genus Rhododendron. [Figure 7] Figure 7 shows cell images observed when treated with the non-glycosyl compound "Taxifolin-aglycone," which is produced by enzymatic hydrolysis of a glycoside compound, namely "Taxifolin-3-O-arabinopyranoside," a well-known indicator and active substance derived from plants of the genus Rhododendron. [Figure 8] Figures 8 and 9 show the results of the analysis of cell viability for taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF), respectively. [Figure 9] Figures 8 and 9 show the results of the analysis of cell viability for taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF), respectively. [Figure 10] Figures 10 and 11 show the results of analyzing cell viability when using taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF) in relation to H2O2 (hydrogen peroxide), respectively. [Figure 11] Figures 10 and 11 show the results of analyzing cell viability when using taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF) in relation to H2O2 (hydrogen peroxide), respectively. [Figure 12] Figures 12 and 13 show the results of analyzing cell viability when using taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF) in response to dexamethasone, respectively. [Figure 13] Figures 12 and 13 show the results of analyzing cell viability when using taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF) in response to dexamethasone, respectively. [Modes for carrying out the invention]

[0036] Specific embodiments of the present invention will be described below with reference to the drawings. However, these are merely illustrative, and the present invention is not limited thereto. In describing the present invention, if it is determined that a specific description of prior art related to the present invention may unnecessarily obscure the gist of the invention, such detailed description will be omitted. Furthermore, the terms described later are defined considering the function in the present invention, and these may change depending on the intent or convention of the user or operator. Therefore, their definitions should be based on the content throughout this specification. The technical idea of ​​the present invention is determined by the claims, and the following embodiments are merely one means of efficiently explaining the technical idea of ​​the present invention to a person with ordinary skill in the art to which the present invention belongs. The present invention provides a composition for the prevention or treatment of hair loss containing an extract of the genus Rhododendron as an active ingredient. The present invention provides a lipolysis inhibitor of taxiporine glycosides and non-taxiporine glycosides derived from the Korean native Rhododendron, as described below, and an anti-obesity composition based thereon. The anti-obesity composition according to the present invention includes all pharmaceutical compositions for anti-obesity purposes and food compositions for prevention or improvement. Furthermore, the taxiporine glycosides and non-taxiporine glycosides derived from Rhododendron kaempferi according to the present invention also possess muscle loss prevention and treatment effects, and these can be applied to both pharmaceutical compositions and food compositions for prevention or improvement.

[0037] Figure 1 is a diagram illustrating an extraction process according to one embodiment of the present invention. Referring to Figure 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 the extract (440.54 g). The extract was then dissolved in distilled water and filtered through filter paper. Using a Silicagel column (40 μm, YAMAZEN, Osaka, Japan), the solvent was prepared in a ratio of Chloroform:Methanol:Water of 70:30:4 and the process proceeded in an isocratic system, and spots were confirmed by TLC. To improve the purity of the target compound, MPLC (YAMAZEN, Osaka, Japan) separation and purification were performed. Using an ODS column (50 μm, YAMAZEN, Osaka, Japan), the solvent was water and methanol, and the process proceeded repeatedly in a gradient system (0% → 50% MeOH / 20 → 80% MeOH). Ultimately, two compounds were separated and purified from the root alcohol extract of Rhododendron parvifolium: the glycoside taxifolin-3-O-arabinopyranoside and the non-glycosyl taxifolin, which will be explained in more detail below.

[0038] Examples, etc. supercritical extraction For the supercritical extraction of Rhododendron kaempferi roots, we used supercritical fluid extraction research equipment (ISA-SEFE-0500-0700-080, IlsinAutoclave, Daejeon, Korea). Specifically, after removing foreign matter from the sample and washing it, we air-dried it and used it as experimental material. The dried sample was ground into 100g units so that it could pass through a 200-mesh grinding mesh, and the temperature of the Rhododendron kaempferi root sample was adjusted to 40-60°C and maintained. After the temperature stabilized, the Rhododendron kaempferi root sample was added, and the CO2 gas was maintained at an equal pressure. Then, using a high-pressure pump, the fluid was injected through the line by adjusting the control valve until the experimental pressure condition of 400-600 bar was reached. After reaching the set pressure, edible alcohol was added to the bottom of the extraction tank at a rate of 5 mL or 10 mL per minute for 60 minutes or 240 minutes to proceed with the extraction. To remove any remaining ethanol from the sample, CO2 was flowed through the sample 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 After supercritical extraction using the method described above, the supercritical extraction foil of Rhododendron kaempferi roots was collected and extracted with edible alcohol (30-100%) at room temperature for 3 days. The final alcoholic extract of Rhododendron kaempferi roots (RMSCFR) was obtained by vacuum concentration and freeze-drying through filter paper filtration. The obtained RMSCFR was dissolved in distilled water (primary or tertiary distilled water), filtered through filter paper, and then separated into an ethyl acetate (EtOAC) layer and an aqueous layer using a fractionation funnel. The EtOAC extract obtained at this time was identified as the high-content extract (RMRF) derived from Rhododendron kaempferi.

[0040] Experimental Example 1 TLC analysis In this experimental example, TLC analysis was performed on the high-content extract (RMRF) derived from Rhododendron parvifolium, which was produced by the method described above.

[0041] Figure 2 shows the TLC analysis results for the high-content extract (RMRF) derived from Rhododendron kaempferi. As shown in Figure 2, it can be confirmed that both non-glycoside and glycoside taxifolin are present in the RMRF fraction. Specifically, the chromatographic results for RMRF I show that both non-glycoside taxifolin (taxifolin aglycone) and glycoside taxifolin-3-O-arabinopyranoside (Taxifolin-3-O-arabinopyranoside, glycoside) are present in the high-content extract (RMRF) derived from Rhododendron kaempferi according to the present invention.

[0042] Total phenol content analysis Figure 3 shows the results of the total phenol content analysis of the high-concentration extract (RMRF) derived from Rhododendron parvifolium. Referring to Figure 3, analysis of the total phenol (Methyl gallate, Ethyl gallate, Gallic acid) content revealed that the total phenol content of RMRF, which was solvent-fractionated after extraction from the RM extract, was more than three times higher. These results indicate that RMRF containing both taxiporine and taxiporine-3-O-arabinopyranoside, obtained through supercritical root extraction of Rhododendron kaempferi according to the present invention, can be expected to exhibit potent physiological activity.

[0043] HPLC analysis results For HPLC analysis, a Waters 2695 Separation module and 2487 Dual λ Absorbance Detector were used. The columns used were SkyPak C18 analytical column (5 μm) and Phenomenex KJ0-4282 guard column. 1% Formic acid (A) and ACN (B) were used as mobile phases (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 results of RMRF analysis for non-taxyporine in RM and alcohol extract (RM). As shown in Figure 4, non-taxyporine was found at 1.2352 ug / ml in the 60% alcohol extract (RM) of Rhododendron parvifolium roots, and at 4.1530 ug / ml in the RMRF extract. Therefore, it can be confirmed that the non-taxyporine content increased by more than 336.22% compared to the existing 60% alcohol extract.

[0045] Figure 5 shows the results of RMRF analysis of RM and taxiporine glycosides against alcohol extract (RM). Referring to Figure 5, analysis of the taxyporine glycoside content in the high-content taxyporine glycoside extract (RMRF) derived from Rhododendron parvifolium showed a 684.17% increase compared to the 60% alcohol extract.

[0046] Structural identification The final single compound was separated and purified from the non-glycosate taxiporine RM and RMRF through repeated purification methods such as MPLC column chromatography. NMR and LC / MS data were then measured to perform structural identification of the separated and purified compound. 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 non-taxyporine obtained from this is shown in Chemical Formula 1 below.

[0050] [ka]

[0051] Taxiporine glycosides From an extract of the native Korean Rhododendron 'Calli', the final single compound was separated and purified through repeated purification methods such as MPLC column chromatography. NMR and LC / MS data were then measured to perform structural identification of the separated 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] By confirming the molecular weight through the final LC-MS and comparing it with existing references, it can be confirmed that the final glycoside form of taxifolin is taxifolin-3-O-α-L-arabinopyranoside, represented by chemical formula 2 below.

[0054] [ka]

[0055] Experimental Example 2 The taxiporine glycoside or non-glycosyl derivative derived from Rhododendron kaempferi according to the present invention has an anti-obesity effect that suppresses adipocyte differentiation. In this experiment, the efficacy of natural product extracts (RM, RMRF) derived from Rhododendron kaempferi native to Korea on the ability to suppress adipocyte differentiation was evaluated. For this purpose, a 60% alcohol extract (RM) of the root of Rhododendron kaempferi, a plant native to Korea, and taxiporine glycosides and non-glycosyl derivatives, which are indicator substances and active substances, were used as test substances.

[0056] cell culture 3T3-L1 cells, a type of adipocyte progenitor cell derived from mice, were purchased from the Korea Cell Line Bank. The 3T3-L1 cells were cultured in a humid CO2 incubator (5% CO2 / 95% air) at 37°C using a cell culture medium (complete DMEM culture medium) consisting of DMEM medium (Welgene) supplemented with 10% bovine calf serum (BCF), 100 units / mL penicillin, and 100 μg / mL streptomycin. When the cells filled approximately 80% of the culture dish, the cell monolayer was washed off with phosphate buffer saline (PBS, pH 7.4), and then the cells were removed by adding trypsin-2.65 mM EDTA and subcultured. The culture medium was changed every two days.

[0057] Measurement of cell viability 3T3-L1 cells, 3 × 10 4Cells were dispensed into 24-well plates to a concentration of cells / well and cultured for 24 hours. After 24 hours of culture, the cells were replaced with a cell culture medium containing the test substance and cultured for another 72 hours. After 72 hours of culture, the number of viable cells was measured using the MTT assay (Denizot F and Lang RJ Immunological Method 89:271-277, 1986). 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 of test substances 3T3-L1 cells 1 × 10 5 Cells were dispensed into 24-well plates at a concentration of cells / well. After the cells reached a state of confluence, the cell culture medium was sequentially changed with three different differentiation induction media (DMs) to induce differentiation into adipocytes. Specifically, the cell culture medium was changed to a differentiation induction media containing 10% FBS in DMEM medium supplemented with DMI (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 μg / mL insulin), and differentiation was stimulated for 2 days. After 2 days, the cell culture medium was changed to a fresh differentiation induction media containing 10% FBS in DMEM medium supplemented with 5 μg / mL insulin, and differentiation was stimulated again for 2 days. After stimulating differentiation for a total of 4 days, the cells were maintained in DMEM medium containing 10% FBS for 2 days to induce differentiation into adipocytes. To investigate the effect of test substances on adipocyte differentiation, the test substances were added to the differentiation induction media and the cells were treated.

[0059] Adipocyte differentiation (fat scale) measurement (Oil red-O staining) After inducing differentiation in 3T3-L1 cells and treating them with the test substance, the cells were washed with DPBS (Welgene) and then fixed at room temperature for 1 hour with 4% paraformaldehyde (PFA, Biosesang). After cell fixation, the cells were stained with oil red O (Sigma-Aldrich) solution at room temperature for 1-2 hours. After visually observing the degree of staining of adipocytes, the cells were washed with distilled water and observed under a microscope. Figure 6 shows cell images observed when treated with the compound "Taxifolin-3-O-arabinopyranoside," which has been shown to be an indicator substance and effective substance for plants of the genus Rhododendron. Referring to Figure 6, a significant concentration-dependent increase in the inhibitory ability of adipocyte differentiation can be confirmed depending on the treatment concentration. In particular, at the highest experimental concentration of 20 ug / ml, an effect of suppressing adipocyte differentiation by more than 40% was confirmed. Figure 7 shows cell images observed when cells were treated with the non-glycosyl compound, namely "Taxifolin-aglycone," produced by enzymatic hydrolysis of "Taxifolin-3-O-arabinopyranoside," a glycoside compound well known as an indicator substance and active ingredient derived from Rhododendron species. Referring to Figure 7, a significant concentration-dependent increase in the inhibitory ability of adipocyte differentiation can be confirmed depending on the treatment concentration. In particular, at the highest experimental concentration of 20 ug / ml, an effect of suppressing adipocyte differentiation by more than 40% was observed. These results suggest that the Rhododendron-derived taxipolin glycoside or non-glycosyl compound (RM, RMRF) according to the present invention can be used as an anti-obesity treatment agent or as a idle component in functional foods.

[0060] Experimental Example 3 The taxiporine glycosides or non-glycosides derived from Rhododendron kaempferi according to the present invention possess not only the ability to damage fat but also the ability to suppress muscle loss. In this experiment, the efficacy of natural product extracts (RM, RMRF) derived from Rhododendron kaempferi native to Korea in preventing skeletal muscle loss was evaluated. In particular, the protective effect against H2O2 and dexamethasone-induced muscle cell damage was experimentally examined in vitro. For this purpose, a 60% alcohol extract (RM) of the root of Rhododendron kaempferi, a plant native to Japan, and a high-content extract (RMRF) obtained by increasing the content of taxiporine glycosides and non-glycosides, which are indicator substances and single compounds derived from RM, were used as test substances.

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

[0062] Measurement of cell viability The cell viability of C2C12 cells was measured using the MTT assay method (Denizot F and Lang RJ Immunological Method 89:271-277, 1986). 2.5 × 10⁶ C2C12 cells were measured. 4Cells were dispensed into 24-well plates at a rate of cells / well and cultured for 24 hours. After 24 hours of cell culture, the cell culture medium was replaced with cell culture media treated with the test substance at various concentrations (0, 10, 50, 100, 150, 200 μg / mL), and the cells were cultured for another 24 hours. After 24 hours of culture with the test substance, the cell culture medium was replaced with 1 mg / mL MTT (Amresco) solution, and the cells were cultured for an additional 2 hours. Formazan formed in living cells was then eluted with isopropanol, and the absorbance was measured at 570 nm.

[0063] H 2 O 2 Measurement of protective effect against induced muscle cell damage 2.5 × 10⁻¹ C2C12 cells 4 Cells were dispensed into 24-well plates at a rate of cells / well and cultured for 24 hours. After culturing C2C12 cells for 24 hours, they were treated with 100 μM H2O2 to induce muscle cell damage, and then treated with various concentrations of each test substance along with 100 μM H2O2 to investigate the protective effect of each test substance on muscle cell damage, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, cell viability was measured by performing the MTT assay in the same manner as described above.

[0064] Measurement of the protective effect of dexamethasone-induced myocyte damage. 2.5 × 10⁻¹ C2C12 cells 4 Cells were dispensed into 24-well plates at a rate of cells / well and cultured for 24 hours. After culturing C2C12 cells for 24 hours, they were treated with 500 μM dexamethasone to induce muscle cell damage. To investigate the muscle cell damage protective effect of each test substance, cells were treated with various concentrations of the test substance along with 500 μM dexamethasone and cultured for 24 hours. After culturing the cells for 24 hours, cell viability was measured by performing the MTT assay in the same manner as described above.

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

[0066] result The effect of natural product extracts (RM, RMRF) on cell viability Figures 8 and 9 show the results of cell viability analysis for taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF), respectively. Referring to Figures 8 and 9, to investigate the cytotoxicity of two natural product extracts (RM and RMRF) in C2C12 cells, various concentrations (0, 10, 50, 100, 150, and 200 μg / mL) of the natural product extracts were treated in cell culture medium and cultured for 24 hours, after which an MTT assay was performed. The cell viability of C2C12 cells increased with treatment of various concentrations (10, 50, 100, 150, and 200 μg / mL) of RM and RMRF. As the treatment concentration of RM increased, cell viability increased, and when treated at concentrations of 50-200 μg / mL, 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 cell viability increasing by 10.2%, 18.5%, 22.6%, and 27.4% respectively when treated with concentrations of 50-200 μg / mL compared to the control group (0 μg / mL). These results suggest that the extract of native Korean Rhododendron kaempferi, containing both taxiporine glycosides and non-taxiporine glycosides, is non-toxic to normal muscle cells and can be expected to reduce muscle mass by increasing muscle cell count.

[0067] H 2 O 2 (Hydrogen peroxide) analysis results Figures 10 and 1 show the results of analyzing cell viability when using taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF), respectively, in relation to H2O2 (hydrogen peroxide).

[0068] H2O2 (hydrogen peroxide) is a strong oxidizing agent that induces oxidative stress in an in vitro system. To investigate the effects of RM and RMRF on muscle cell damage caused by oxidative stress, oxidative stress was induced by treating the cell culture medium of C2C12 cells with 100 μM H2O2. After treating with RM and RMRF and culturing the cells, the cell viability of the C2C12 cells was measured. Referring to Figures 10 and 11, cell viability decreased significantly when treated with H2O2 compared to the control group [H2O2(-) / (-)]. When treated with RM at various concentrations (10, 50, 100, 200 μg / mL), cell viability significantly increased compared to the control group [H2O2(+) / (-)] treated with H2O2 alone. When treated with RM at a concentration of 200 μg / mL, cell viability increased by 44% compared to the control group [H2O2(+) / (-)] treated with H2O2 alone. When RMRF was treated at concentrations of 10, 50, 100, and 200 μg / mL, cell viability was significantly increased compared to the control group treated with H2O2 alone [H2O2(+) / (-)]. In particular, in the 100-200 μg / mL treatment groups, a statistically significant increase in cell viability was observed, at 53.4% ​​and 55.7% respectively, compared to the control group treated with H2O2 alone [H2O2(+) / (-)]. Through these results, it was found that domestically grown Rhododendron kaempferi extract containing taxiporin glycosides and non-taxiporin glycosides suppresses muscle loss caused by oxidative stress-induced cell death in muscle cells.

[0069] Dexamethasone analysis results Figures 12 and 13 show the results of analyzing cell viability when using taxiporine glycosides (RM) and non-taxiporine glycosides (RMRF) in response to dexamethasone, respectively. Dexamethasone is one of the representative glucocorticoids and, when misused clinically, causes skeletal muscle degradation, and is therefore widely used in in vitro systems to induce muscle cell damage. To investigate the effects of RM and RMRF according to the present invention on glucocorticoid-induced muscle cell damage, muscle cell damage was induced by treating the cell culture medium of C2C12 cells with 500 μM dexamethasone, and then the cell viability of the C2C12 cells was measured after treating with RM and RMRF and culturing the cells. Referring to Figures 12 and 13, when treated with dexamethasone, the cell viability was significantly reduced compared to the control group [DEX(-) / (-)] that was not treated with dexamethasone. RM at concentrations of 10–200 μg / mL significantly increased the reduction in cell viability induced by dexamethasone. Specifically, as the concentration increased from 10–200 μg / mL, the protective effect against cell damage increased statistically significantly, at 39.5%, 49.4%, 53.6%, and 52.8%. Furthermore, treatment with RMRF at concentrations of 10, 50, and 100 μg / mL resulted in a significant increase in cell viability compared to the control group treated with dexamethasone alone [DEX(+) / (-)]. RMRF at various concentrations (10, 50, and 100 μg / mL) showed a significant effect on the reduction in cell viability induced by dexamethasone. Specifically, as the concentration increased, cell viability increased to 39.8%, 44.5%, and 45.3%. Based on these results, it can be seen that domestically grown Rhododendron kaempferi extract containing taxiporine glycosides and non-taxiporine glycosides suppresses muscle loss due to skeletal muscle breakdown and muscle fiber cell death, which are among the side effects of glucocorticoid-type drugs. [Industrial applicability]

[0070] It has industrial potential as a composition for preventing and treating obesity and muscle deceleration. <Note> The aspects of this disclosure include the following: <Section 1> A pharmaceutical composition for the prevention and treatment of obesity and muscle loss, containing an extract of Rhododendron mucronulatum as an active ingredient. <Section 2> The pharmaceutical composition for the prevention and treatment of obesity and muscle loss according to item 1, characterized in that the Rhododendron mucronulatum extract contains a taxiporine glycoside or a taxiporine non-glycoside. <Section 3> The aforementioned Rhododendron mucronulatum extract is obtained by supercritical extraction of the roots of Rhododendron mucronulatum, and is characterized by the pharmaceutical composition for the prevention and treatment of obesity and muscle loss as described in item 2. <Section 4> The pharmaceutical composition for the prevention and treatment of obesity and muscle loss according to item 2, characterized in that the taxiporine glycoside contains a compound of the following formula (1). [ka] <Section 5> The pharmaceutical composition for the prevention and treatment of obesity and muscle loss according to item 2, characterized in that the taxiporine glycoside contains a compound of the following formula (2). [ka] <Section 6> A pharmaceutical composition for the prevention and treatment of obesity and muscle loss, comprising one or more compounds selected from the group consisting of the compounds of formula (1) and formula (2) below. [ka] [ka] <Section 7> The pharmaceutical composition for the prevention and treatment of obesity and muscle loss according to item 6, characterized in that the compound is extracted from the roots of Rhododendron kaempferi. <Section 8> A food composition for preventing obesity and muscle loss, containing Rhododendron mucronulatum extract as an active ingredient. <Section 9> The food composition for preventing obesity and muscle loss according to item 8, characterized in that the Rhododendron mucronulatum extract contains a taxiporine glycoside or a non-taxiporine glycoside. <Section 10> The aforementioned Rhododendron mucronulatum extract is obtained by supercritical extraction of the roots of Rhododendron mucronulatum, as described in item 9, for the anti-obesity and muscle loss prevention food composition. <Section 11> The taxiporine glycoside is characterized by containing a compound of the following formula (1), as described in item 9, for anti-obesity and muscle loss prevention food composition. [ka] <Section 12> The taxiporine glycoside is characterized by containing a compound of the following formula (2) as described in item 9, making it an anti-obesity and anti-muscle loss food composition.

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Claims

[Claim 1] A pharmaceutical composition for preventing or treating obesity and muscle loss, comprising a compound of the following formula (1) or formula (2) as an active ingredient. 【Chemistry 1】 【Chemistry 2】

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