Application of composition in preparation of anti-obesity drugs for prevention and treatment

Abstract

The present invention relates to an application of a composition for the preparation of an anti-obesity drug for prevention and treatment of obesity, the composition comprising an extract of Rhododendron mucronatum as an active ingredient.

Description

[0001] This application is a divisional application of the invention patent application with the application date of May 10, 2022, application number 202280088465.X, and invention name "Composition for the prevention and treatment of obesity and muscle loss containing azalea extract as an active ingredient". Technical Field

[0002] The present invention relates to a composition for preventing and treating obesity and muscle loss, comprising a rhododendron extract as an effective ingredient. Background Art

[0003] Lipid metabolism is essential for maintaining energy storage and distribution, regulating glucose metabolism, and maintaining energy homeostasis. Abnormal lipid metabolism can lead to conditions such as obesity, diabetes, and hyperlipidemia. This lipid metabolism primarily occurs in the liver and adipose tissue, where it is regulated by the fat cells that make up the tissue. Fat cells are crucial organs in the body's metabolism, not simply serving as energy storage organs but also as endocrine organs that secrete a variety of hormones, playing an active role in metabolic processes.

[0004] Fat cells induce obesity due to an increase in the amount of neutral fat (triglyceride) in the fat cells or an increase in the number of fat cells. Therefore, in terms of preventing and treating obesity, it is necessary to find a solution that reduces fat accumulation and the number of fat cells. In addition, fat cells are made by differentiation of preadipocytes (preadipocytes). Therefore, the mechanism of adipogenesis is also very important for understanding the effect of adipose tissue. Recently, molecular biology research on the differentiation and regulation of the fat cells that constitute adipose tissue has been widely carried out. However, the actual situation is that it is still insufficient to disclose the research on the clear and definite efficacy on the single compound level.

[0005] Muscles, on the other hand, can be categorized structurally and functionally into skeletal, smooth, and cardiac muscles. Skeletal muscle is comprised of the more than 600 voluntary muscles located just beneath the skin in the hands, feet, chest, and abdomen, attached to bones throughout the body by bones or tendons. It is adapted to move or support bones through contraction. Contraction is initiated and regulated by nerve signals. It accounts for 40-50% of body weight and plays a role in maintaining body temperature and generating energy. The fine muscle fibrils, composed of actin and myosin, are arranged in a regular pattern, with striations visible under a microscope (Lieber RL, 2002; Edwards RH, 1981).

[0006] Skeletal muscle fibers are biochemically classified according to their mitochondrial content: Type I, Type IIa, and Type IIb. Type I muscles, composed of red slow-twitch fibers, maintain a weak force for extended periods and are therefore suitable for maintaining posture. Their high mitochondrial content makes them well-suited for aerobic exercise, such as long-distance running. Among fast-twitch fibers, those with slow-twitch characteristics are also called Type IIa. Muscles composed of white fast-twitch fibers are used for movement and are considered motor muscles, classified as Type IIb. Their low mitochondrial content makes them well-suited for anaerobic exercise, such as short-distance running. Skeletal muscle fibers are distributed in varying proportions throughout the body (Tortora et al., 2008).

[0007] Muscle atrophy is caused by the anti-anabolic and catabolic effects of unbalanced 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 reduced activity such as aging, disease states (overexposure to stress hormones, cancer, sepsis, hunger, etc.) and bedridden life. If muscle atrophy occurs, the muscle strength required for physical activity is weakened, thus starting a vicious cycle of musculoskeletal system degeneration. A decrease in walking speed and weakened gait are the main symptoms and indicators of muscle loss, which may lead to falls, fractures, joint injuries, metabolic disorders and cardiovascular diseases.

[0008] Glucocorticoids in our bodies induce molecular changes in muscle fibers, directly or indirectly contributing to their anti-anabolic and anti-catabolic effects. Dexamethasone, a glucocorticoid, inhibits the PI3K / Akt / mTOR signaling pathway through its anti-anabolic action. This action inhibits the activity of downstream effectors such as 4E-BP1 and S6K1, thereby preventing the operation of eukaryotic translation initiation factor 4G (eIF4G) and eukaryotic translation initiation factor 4E (eIF4E). This inhibition of mRNA translation, which is involved in protein synthesis, has been shown to contribute to muscle fiber atrophy through the blockage of myofiber synthesis and protein degradation (Shackman et al., 2013).

[0009] Dexamethasone inhibits muscle synthesis and protein degradation, thereby inducing muscle atrophy. This is due to the "PI3K / Akt → FOXO activation and GSK3 inactivation" mechanism, which leads to the expression of the muscle atrophy-inducing gene atrogene (Atrogin-1, MuRF-1). These genes induce protein degradation, typified by the ubiquitin-proteasome system.

[0010] Therefore, there is a need for the development of a substance for suppressing obesity and muscle loss that has the effect of simultaneously degrading sarcopenia, which is a disease of skeletal muscle loss, and fat. Summary of the Invention

[0011] Technical issues

[0012] Therefore, the problem to be solved by the present invention is to provide an extract and a single compound obtained therefrom that have excellent anti-obesity and anti-muscle cell reduction effects.

[0013] Technical Solution

[0014] In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing and treating obesity and muscle loss, comprising a Rhododendron mucronulatum extract as an active ingredient.

[0015] In one embodiment of the present invention, the Rhododendron mucronulatum extract contains taxifolin glycoside or taxifolin aglycone.

[0016] In one embodiment of the present invention, the Rhododendron mucronulatum extract is obtained by supercritical extraction of Rhododendron mucronulatum roots.

[0017] In one embodiment of the present invention, the taxifolin glycoside comprises a compound of the following formula (1).

[0018]

[0019] In one embodiment of the present invention, the taxifolin glycoside comprises a compound of the following formula (2).

[0020]

[0021] In one embodiment of the present invention, a pharmaceutical composition for preventing and treating obesity and muscle loss is provided, wherein the pharmaceutical composition comprises one or more compounds selected from the group consisting of the following formula (1) and formula (2).

[0022]

[0023] In one embodiment of the present invention, the compound is extracted from the root of Rhododendron odoriferum.

[0024] The present invention also provides a food composition for preventing obesity and muscle loss, comprising a Rhododendron mucronulatum extract as an effective ingredient.

[0025] In one embodiment of the present invention, the Rhododendron mucronulatum extract includes taxifolin glycoside or taxifolin aglycone, and the Rhododendron mucronulatum extract is obtained by supercritical extraction of Rhododendron mucronulatum roots.

[0026] In one embodiment of the present invention, the taxifolin glycoside comprises a compound of the following formula (1).

[0027]

[0028] In one embodiment of the present invention, the taxifolin glycoside comprises a compound of the following formula (2).

[0029]

[0030] The present invention provides a food composition for preventing obesity and muscle loss, wherein the food composition comprises one or more compounds selected from the group consisting of the following formula (1) and formula (2):

[0031]

[0032] The above compounds are extracted from the roots of Rhododendron.

[0033] In the present invention, the above-mentioned food composition for preventing obesity and muscle loss is also a feed additive for animals.

[0034] Beneficial effects

[0035] The anti-obesity composition of the present invention, based on the taxifolin glycosides and taxifolin aglycones contained in azalea extract, exhibits the ability to inhibit adipocyte differentiation in a concentration-dependent manner. In particular, at a concentration of 20 μg / ml, the highest experimental concentration, it was confirmed that adipocyte differentiation was inhibited by more than 40%. Furthermore, the composition also exhibits an inhibitory effect against muscle loss induced by hydrogen peroxide or dexamethasone. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1This is a diagram illustrating an extraction process according to one embodiment of the present invention.

[0037] Figure 2 The results of TLC analysis of a high-content extract (RMRF) from Rhododendron odoriferum.

[0038] Figure 3 The total phenolic content of the high-quality extract (RMRF) from Rhododendron odoriferum is analyzed.

[0039] Figure 4 The results of RMRF analysis of taxifolin aglycone in RMRF and alcohol extract (RM) are shown.

[0040] Figure 5 The results of RMRF analysis of taxifolin glycosides in RMRF and alcohol extract (RM) are shown.

[0041] Figure 6 This is a photograph of cells observed when treated with the compound "Taxifolin-3-O-arabinopyranoside," which is both an indicator substance for Rhododendron plants and revealed to be an effective substance.

[0042] Figure 7 The images show cells observed when an indicator substance derived from azalea plants and a glycoside compound known as an active substance (i.e., a compound in aglycone form produced from "Taxifolin-3-O-arabinopyranoside" by enzymatic hydrolysis, i.e., a "Taxifolin-aglycone" compound) were treated.

[0043] Figure 8 and Figure 9 These are the analysis results of the cellular activities of taxifolin glycoside (RM) and taxifolin aglycone (RMRF), respectively.

[0044] Figure 10 and Figure 11 The results of cell viability analysis were obtained when H2O2 (hydrogen peroxide) was used with taxifolin glycoside (RM) and taxifolin aglycone (RMRF).

[0045] Figure 12 and Figure 13 The results are analysis results of cell viability when dexamethasone was administered with taxifolin glycoside (RM) and taxifolin aglycone (RMRF). DETAILED DESCRIPTION

[0046] Hereinafter, specific embodiments of the present invention will be described with reference to the accompanying drawings. However, this is only an example and the present invention is not limited thereto.

[0047] During the description of the present invention, if a detailed description of a known technology related to the present invention is determined to be likely to cause unnecessary confusion regarding the main purpose of the present invention, such detailed description will be omitted. Furthermore, the terms described below are defined based on their functions within the present invention and may vary depending on the intentions or practices of users or operators. Therefore, their definitions should be based on the entire content of this specification.

[0048] The technical concept of the present invention is defined by the claims. The following examples are merely a means of effectively illustrating the technical concept of the present invention to persons skilled in the art. 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.

[0049] The present invention provides the following anti-lipolysis inhibitory effects of taxifolin glycosides and taxifolin aglycones derived from Korean native azaleas, and anti-obesity compositions based thereon. The anti-obesity compositions of the present invention include pharmaceutical compositions for the purpose of combating obesity and food compositions for preventing or ameliorating obesity. Furthermore, the taxifolin glycosides and taxifolin aglycones derived from azaleas of the present invention also have the ability to inhibit muscle loss, thereby preventing and treating muscle loss. These can be used in pharmaceutical compositions and food compositions for preventing or ameliorating obesity.

[0050] Figure 1 This is a diagram illustrating an extraction process according to one embodiment of the present invention.

[0051] like Figure 1As shown, in one embodiment of the present invention, 12 kg of Rhododendron mucronulatum roots were extracted with 60% alcohol at room temperature for 7 days. After filtration through filter paper, the extract was concentrated under reduced pressure to recover 440.54 g of the extract. The extract was then dissolved in distilled water and filtered through filter paper. The extract was then purified using a 40 μm silica gel column (YAMAZEN, Osaka, Japan) with a solvent ratio of 70:30:4 chloroform:methanol:water. The isocratic system was used, and a spot was confirmed by thin-layer chromatography (TLC). To improve the purity of the target compound, medium-pressure preparative chromatography (MPLC) (YAMAZEN, Osaka, Japan) was performed. Using an ODS column (50 μm, YAMAZEN, Osaka, Japan), water and methanol were used as solvents and a gradient system (0% → 50% methanol (MeOH) / 20 → 80% methanol (MeOH)) was repeated. Ultimately, two compounds, taxifolin-3-O-arabinopyranoside as a glycoside and taxifolin as an aglycone, were finally isolated and purified from the alcohol extract of Rhododendron odoriferum root. This will be described in more detail below.

[0052] Example

[0053] Supercritical extraction

[0054] For supercritical extraction of azalea roots, a supercritical fluid extraction research device (ISA-SEFE-0500-0700-080, Ilsin Autoclave, Daejeon, Korea) was used. The samples were carefully observed, free of foreign matter, cleaned, and air-dried before use as experimental materials.

[0055] The dried sample was crushed into 100g pieces, passing through a 200-mesh crushing screen. The temperature of the azalea root sample extraction tank was adjusted to 40-60°C and maintained. Once the temperature stabilized, the azalea root sample was added. After maintaining the pressure at an isobaric level, CO2 gas was introduced using a high-pressure pump through a line control valve until the experimental pressure reached 400-600 bar.

[0056] After reaching the set pressure, alcohol as total edible ethanol is added to the lower part of the extraction tank and extracted at a rate of 5 mL or 10 mL per minute for 60 minutes or 240 minutes. In order to remove residual ethanol remaining in the sample, CO2 is flowed for 30 minutes at the set pressure and temperature using a high-pressure pump to complete the extraction and produce an extract (RM).

[0057] Solvent fractionation

[0058] The Rhododendron root supercritical extract foil obtained after supercritical extraction according to the above method is recovered and extracted with edible ethanol (30-100%) at room temperature for three days. The extract is then filtered through filter paper, concentrated under reduced pressure, and freeze-dried to obtain the Rhododendron root supercritical extract foil alcohol extract (RMSCFR). The RMSCFR is dissolved in distilled water (single or triple distilled), filtered through filter paper, and separated into an ethyl acetate (EtOAC) layer and an aqueous layer using a fractionating funnel. The resulting EtOAC extract is the Rhododendron root high-content extract (RMRF).

[0059] Experimental Example 1

[0060] TLC analysis

[0061] In this experimental example, TLC analysis was performed on the Rhododendron odoriferum high-content extract (RMRF) prepared by the above method.

[0062] Figure 2 The results of TLC analysis of a high-content extract (RMRF) from Rhododendron odoriferum.

[0063] Reference Figure 2 In the RMRF fraction, it was confirmed that both taxifolin aglycone and taxifolin glycosides were present. Specifically, ① taxifolin aglycone as the aglycone, ② taxifolin-3-O-arabinoside as the glycoside, and ③ taxifolin-3-O-arabinoside as the glycoside. The RMRF I chromatography results indicate that both ① taxifolin aglycone and ② taxifolin-3-O-arabinopyranoside (glycoside) are present in the high-content azalea extract (RMRF) of the present invention.

[0064] Total phenol content analysis

[0065] Figure 3 The total phenolic content of the high-quality extract (RMRF) from Rhododendron odoriferum is analyzed.

[0066] Reference Figure 3 Analysis of total phenolic content (methyl gallate, ethyl gallate, and gallic acid) confirmed that the total phenolic content of RMRF, obtained through solvent fractionation after extraction, was more than three times higher than that of the RM extract. These results demonstrate that the present invention's supercritical extraction of azalea roots can be expected to yield potent physiological activities in RMRF, which contains taxifolin and taxifolin-3-O-arabinoside.

[0067] HPLC analysis results

[0068] For analytical HPLC, a Waters 2695 separation module and a 2487 Dualλ Absorbance Detector were used. The chromatographic column used was a SkyPak C18 analytical column (5 μm) and a Phenomenex KJ0-4282 guard column. The mobile phase used was 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).

[0069] Figure 4 The results of RMRF analysis of taxifolin aglycone in RMRF and alcohol extract (RM) are shown.

[0070] like Figure 4 As shown, the 60% alcohol extract (RM) of azalea root contained 1.2352 μg / ml of taxifolin aglycone, while the RMRF extract contained 4.1530 μg / ml. Therefore, the content of taxifolin aglycone increased by more than 336.22% compared to the conventional 60% alcohol extract.

[0071] Figure 5 The results of RMRF analysis of taxifolin glycosides in RMRF and alcohol extract (RM) are shown.

[0072] like Figure 5 As shown, analysis of the taxifolin glycoside content in the high-content taxifolin glycoside extract (RMRF) derived from Rhododendron odoriferum revealed an increase of 684.17% compared to the 60% alcohol extract.

[0073] Structural identification

[0074] Taxifolin aglycone

[0075] After repeated purification using MPLC column chromatography, a single compound was isolated from RM and RMRF. To identify the structure of the isolated compound, NMR and LC / MS data were measured. The results are shown below.

[0076] White yellow amorphous powder Negative LC-MS: m / z 303.0[MH]-1H-NMR (300MHz, DMSO-d6): δ11.92 (1H, s, 5-OH), 6.75~6.88 (3Hin total, m, H-2', H-5'and H-6'), 5.92 (1H, d, J=2.1Hz, H-8), 5.87 (1H, d, J=2.1Hz, H-6), 5.00 (1H, d, J=11.1Hz, H-2), 4.52 (1H, d, J=11.1Hz, H-3).

[0077] 13 C-NMR (75MHz, 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).

[0078] The structural formula of the taxifolin aglycone thus obtained is shown in Chemical Formula 1 below.

[0079] [Chemical Formula 1]

[0080]

[0081] Taxifolin glycosides

[0082] Through repeated extraction using MPLC column chromatography, a single compound was finally isolated from a Korean azalea extract. NMR and LC / MS data were then measured to identify the structure of the isolated compound.

[0083] White yellow amorphous powder, LC-MS, (positive-ion mode)m / z437.1109[M+H]+; 1H-NMR, (700MHz, MeOH-d4) δ: 3.38 (1H, dd, J=11.2, 3.5Hz, 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 = 3.5Hz, H-1”), 3.91 (1H, dd, J = 11.2, 7.0Hz, H- 5"), 4.79 (1H, d, J = 10.5Hz, H-3), 5.12 (1H, d, J = 10.5Hz, H-2), 5.90 (1H, d, J = 2.1Hz, H-8), 5.92 (1H, d, J = 2 .1Hz, H-6), 6.79 (1H, d, J=8.4Hz, H-5'), 6.84 (1H, dd, J=8.4, 2.1Hz, H-6'), 6.965 (1H, d, J=2.1Hz, H-2');

[0084] 13C-NMR, (175MHz, MeOH-d4) δ: 196.17(C-4), 169.04(C-7), 165.74(C-5), 164.2 8(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").

[0085] The molecular weight was finally confirmed by LC-MS and compared with existing references. It was finally confirmed to be taxifolin-3-O-α-L-arabinoside (arabinopyranoside) of the following chemical formula 2, which is a glycoside form of taxifolin.

[0086] [Chemical Formula 2]

[0087]

[0088] Experimental Example 2

[0089] The present invention's taxifolin glycosides and aglycones derived from azaleas have anti-obesity effects by inhibiting adipocyte differentiation. In this experimental example, the efficacy of natural extracts (RM, RMRF) derived from azaleas grown in Korea was evaluated for their ability to inhibit adipocyte differentiation. To this end, a 60% alcohol extract (RM) of azalea roots, a native Korean plant, and taxifolin glycosides and aglycones, which served as indicator and active substances in the RM, were used as test substances.

[0090] Cell culture

[0091] 3T3-L1 cells, derived from mouse preadipocytes, were purchased from the Korean Cell Line Bank and used. 3T3-L1 cells were cultured in a 37°C humidified CO2 incubator (5% CO2 / 95% air) using complete DMEM (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 with phosphate-buffered saline (PBS, pH 7.4), then the cells were removed by adding trypsin-2.65 mM EDTA and subcultured. The culture medium was replaced every two days.

[0092] Determination of cell viability

[0093] 3T3-L1 cells were cultured at 3×10 4 Cells / well were seeded into 24-well plates and cultured for 24 hours. After 24 hours of cell culture, cells were replaced with cell culture fluid containing test substance to culture cells for 72 hours. After 72 hours of cell culture, cell proliferation test (MTT assay) (Denizot F and Lang R.J Immunological Method 89:271-277, 1986) was performed to measure the number of surviving cells. The MTT assa method is based on the principle that mitochondrial dehydrogenase (dehydrogenase) reduces MTT (Amresco) and makes formazan (formazan) as a blue substance. In this test, absorbance was measured at a wavelength of 570nm after formazan was dissolved in isopropanol (isopropanol).

[0094] Induction of differentiation and treatment with test substances

[0095] 3T3-L1 cells were cultured at a rate of 1 × 10 5 The cells were seeded into a 24-well plate at a concentration of cells / well. After the cells reached a confluence state, the cell culture medium was replaced with three differentiation induction mediums (DM) in sequence to culture the cells, thereby inducing differentiation into adipocytes. That is, the cell culture medium was replaced with an induction differentiation medium in which DMI (1μM dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine (IBMX), 5μg / mL insulin) was added to a DMEM medium containing 10% FBS, and differentiation was stimulated for 2 days. After 2 days, the DMEM medium containing 10% fetal bovine serum (FBS) was replaced with a new induction differentiation medium supplemented with 5μg / mL insulin, and differentiation was stimulated again for 2 days. After stimulating differentiation for a total of 4 days, the cells were kept in a DMEM medium containing 10% FBS for 2 days to induce differentiation into adipocytes.

[0096] In order to investigate the effects of test substances on adipocyte differentiation, the test substances were added to the differentiation-inducing culture medium and the cells were treated.

[0097] Adipocyte differentiation (fat accumulation) assay (Oil red-O staining)

[0098] 3T3-L1 cells were induced to differentiate and treated with the test substances. The cells were then rinsed with DPBS (Welgene) and fixed with 4% paraformaldehyde (PFA) (Biosesang) for 1 hour at room temperature. Following fixation, the cells were treated with Oil Red O (Sigma-Aldrich) solution and stained for 1-2 hours at room temperature. The extent of adipocyte staining was visually observed, followed by rinsing with distilled water and microscopic observation of the adipocytes.

[0099] Figure 6 This is a photograph of cells observed when treated with the compound "Taxifolin-3-O-arabinopyranoside," which is both an indicator substance for Rhododendron plants and revealed to be an effective substance.

[0100] Reference Figure 6 The ability to inhibit adipocyte differentiation was significantly increased in a concentration-dependent manner depending on the treatment concentration. In particular, at the highest concentration tested, 20 μg / ml, the ability to inhibit adipocyte differentiation was inhibited by more than 40%.

[0101] Figure 7The images show cells observed when an indicator substance derived from azalea plants and a glycoside compound known as an active substance (i.e., a compound in aglycone form produced from "Taxifolin-3-O-arabinopyranoside" by enzymatic hydrolysis, i.e., a "Taxifolin-aglycone" compound) were treated.

[0102] Reference Figure 7 The ability to inhibit adipocyte differentiation was significantly increased in a concentration-dependent manner depending on the treatment concentration. In particular, at the highest concentration tested, 20 μg / ml, the ability to inhibit adipocyte differentiation was inhibited by more than 40%.

[0103] The above results indicate that the taxifolin glycosides or aglycones (RM, RMRF) derived from Rhododendron odoriferum of the present invention can be used as an effective ingredient of an anti-obesity therapeutic agent or functional food.

[0104] Experimental Example 3

[0105] The taxifolin glycosides or aglycones derived from azalea of ​​the present invention not only have this lipolysis ability, but also have a muscle loss inhibitory ability to inhibit muscle loss. In this experimental example, the muscle loss improvement effect of natural extracts (RM, RMRF) derived from azaleas grown in South Korea was evaluated for the prevention of skeletal muscle loss. In particular, in an in vitro (invitro) system, the protective effect on myocyte damage induced by H2O2 and dexamethasone was tested. For this purpose, a 60% alcohol extract (RM) of azalea roots, which is a native plant in South Korea, and a high-content extract (RMRF) in which the content of taxifolin glycosides and aglycones as an indicator substance and a single compound in RM was increased were used as test substances.

[0106] Cell culture

[0107] C2C12 cells, myoblasts derived from mouse skeletal muscle, were purchased from the American Type Culture Collection (ATCC) and used. 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 at 37°C in a humidified CO2 incubator (5% CO2 / 95% air). When the cells filled approximately 80% of the culture dish, the cell monolayer was washed with phosphate buffered saline (PBS, pH 7.4), and then trypsin-2.65 mM EDTA was added to remove the cells and subcultured. The medium was replaced every two days.

[0108] Determination of cell viability

[0109] The cell viability of C2C12 cells was determined by MTT assay (Denizot F and Lang R. J Immunological Method 89: 271-277, 1986). 4 Cells / well were seeded into a 24-well plate and cultured for 24 hours. After 24 hours of cell culture, the cell culture medium was replaced with cell culture medium containing the test substance at various concentrations (0, 10, 50, 100, 150, and 200 μg / mL) and cultured for another 24 hours. After treatment with the test substance and 24 hours of culture, the cell culture medium was replaced with a 1 mg / mL MTT solution (Amresco). After the cells were cultured for an additional 2 hours, the formazan formed in the surviving cells was dissolved with isopropanol, and the absorbance was measured at 570 nm.

[0110] Determination of the protective effect against H2O2-induced myocyte damage

[0111] C2C12 cells were cultured at 2.5×10 4Cells were seeded into 24-well plates at 100 μM H₂O₂ and cultured for 24 hours. After culturing for 24 hours, C2C12 cells were treated with 100 μM H₂O₂ to induce myocyte damage. To investigate the protective effects of each test substance against myocyte damage, the cells were treated with various concentrations of the test substance along with 100 μM H₂O₂ and cultured for 24 hours. After culturing for 24 hours, cell viability was measured using the same MTT assay as above.

[0112] Determination of the protective effect against dexamethasone-induced myocyte damage

[0113] C2C12 cells were cultured at 2.5×10 4 Cells were seeded into 24-well plates at 100 cells / well and cultured for 24 hours. After 24 hours of culture, C2C12 cells were treated with 500 μM dexamethasone to induce myocyte damage. To investigate the protective effects of each test substance against myocyte damage, the cells were treated with various concentrations of the test substance along with 500 μM dexamethasone and cultured for 24 hours. After 24 hours of culture, cell viability was measured using the same method as above using MTT assay.

[0114] Statistical processing

[0115] All values ​​are expressed as mean ± SEM. The collected data were analyzed using GraphPad Prism 5.0 (GraphPad software, San Diego, CA, USA). Student's t-test and one-way analysis of variance (ANOVA) were used to compare differences between the control group and the test substance-treated group. Statistical significance was considered only when p < 0.05 or higher.

[0116] result

[0117] Effects of natural product extracts (RM, RMRF) on cell viability

[0118] Figure 8 and Figure 9 These are the analysis results of the cellular activities of taxifolin glycoside (RM) and taxifolin aglycone (RMRF), respectively.

[0119] Reference Figure 8 and Figure 9To investigate the cytotoxicity of two natural extracts (RM and RMRF) in C2C12 cells, the cells were treated with various concentrations of the natural extracts (0, 10, 50, 100, 150, and 200 μg / mL) in the cell culture medium. After 24 hours of incubation, an MTT assay was performed. The cell viability of C2C12 cells increased with treatment with various concentrations of RM and RMRF (10, 50, 100, 150, and 200 μg / mL). The higher the treatment concentration of RM, the greater the increase in cell viability. When treated with concentrations of 50 to 200 μg / mL, cell viability increased by 11.3%, 24.8%, and 25.3%, respectively, compared to the control group (0 μg / mL).

[0120] Similarly, increasing the RMRF treatment concentration increased cell viability, with treatment at concentrations of 50 to 200 μg / mL increasing cell viability by 10.2%, 18.5%, 22.6%, and 27.4%, respectively, compared to the control group (0 μg / mL). These results suggest that the Korean native azalea extract containing taxifolin glycosides and taxifolin aglycones is non-toxic to normal muscle cells and can be expected to reduce muscle loss by increasing muscle cell numbers.

[0121] H2O2 (hydrogen peroxide) analysis results

[0122] Figure 10 and Figure 11 These are the results of analyzing cell activity when taxifolin glycoside (RM) and taxifolin aglycone (RMRF) were used for H 2 O 2 (hydrogen peroxide).

[0123] H2O2 (hydrogen peroxide), a strong oxidant, induces oxidative stress in vitro. To investigate the effects of RM and RMRF on muscle cell damage caused by oxidative stress, C2C12 cell culture medium was treated with 100 μM H2O2 to induce oxidative stress. After treatment with RM and RMRF and culture, the cell viability of the C2C12 cells was measured.

[0124] Reference Figure 10 and Figure 11 Compared with the control group [H2O2(-) / (-)] not treated with H2O2, the activity of cells treated with H2O2 was significantly reduced.

[0125] Compared to the control group treated with H2O2 alone [H2O2(+) / (-)], cell viability significantly increased when RM was treated at various concentrations (10, 50, 100, and 200 μg / mL). In particular, when RM was treated at a concentration of 200 μg / mL, cell viability increased to 44% compared to the control group treated with H2O2 alone [H2O2(+) / (-)]. When RMRF was treated at concentrations of 10, 50, 100, and 200 μg / mL, cell viability significantly increased compared to the control group treated with H2O2 alone [H2O2(+) / (-)]. In particular, in the groups treated with concentrations of 100 to 200 μg / mL, cell viability was confirmed to be 53.4% ​​and 55.7% compared to the control group treated with H2O2 alone [H2O2(+) / (-)], indicating a statistically significant increase. The above results indicate that the Korean native azalea extract containing taxifolin glycosides and taxifolin aglycone inhibits muscle loss caused by apoptosis of muscle cells induced by oxidative stress.

[0126] Dexamethasone analysis results

[0127] Figure 12 and Figure 13 The results are analysis results of cell viability when dexamethasone was administered with taxifolin glycoside (RM) and taxifolin aglycone (RMRF).

[0128] Dexamethasone is a typical glucocorticoid. Its clinical abuse leads to the breakdown of skeletal muscle and, based on this, is widely used to induce myocyte damage in vitro. To investigate the effects of RM and RMRF according to the present invention on glucocorticoid-induced myocyte damage, C2C12 cell culture medium was treated with 500 μM dexamethasone to induce myocyte damage. Following treatment with RM and RMRF and subsequent incubation, the cell viability of the C2C12 cells was measured.

[0129] Reference Figure 12 and Figure 13 Compared with the control group [DEX(-) / (-)] not treated with dexamethasone, the cell viability treated with dexamethasone was significantly reduced.

[0130] RM concentrations of 10 to 200 μg / mL were found to significantly enhance the reduction in cell viability induced by dexamethasone. Specifically, the protective effect against cell damage increased statistically significantly with increasing concentrations from 10 to 200 μg / mL, reaching 39.5%, 49.4%, 53.6%, and 52.8%.

[0131] Furthermore, when RMRF was treated at concentrations of 10, 50, and 100 μg / mL, cell viability significantly increased compared to the control group treated with dexamethasone alone (DEX(+) / (-)). RMRF at various concentrations (10, 50, and 100 μg / mL) was confirmed to have a significant effect on the decrease in cell viability induced by dexamethasone. Specifically, cell viability was confirmed to increase by 39.8%, 44.5%, and 45.3% with increasing concentration. These results indicate that the Korean native azalea extract containing taxifolin glycosides and taxifolin aglycones inhibits skeletal muscle breakdown and muscle loss caused by myofibroblast apoptosis, which are side effects of glucocorticoid drugs.

[0132] Industrial applicability

[0133] The present invention has industrial applicability as a composition for preventing and treating obesity and muscle loss.

Claims

1. A composition for use in preparing a drug for preventing and treating obesity, wherein the composition comprises azalea extract as an active ingredient.

2. The use according to claim 1, characterized in that The azalea extract contains taxifolin glycoside or taxifolin aglycone.

3. The use according to claim 2, characterized in that The azalea extract is obtained by supercritical extraction of azalea roots.

4. The use according to claim 2, characterized in that The above-mentioned taxifolin glycosides include compounds of the following formula (1):

5. The use according to claim 2, characterized in that The above-mentioned taxifolin glycosides include compounds of the following formula (2):

6. Use of a composition for preparing a drug for preventing and treating obesity, the composition comprising: One or more selected from the group consisting of compounds of the following formula (1) and formula (2):

7. The use according to claim 6, characterized in that The above compounds are extracted from the roots of Rhododendron.

8. Use of a composition for preparing a preventive food for anti-obesity, wherein the composition comprises azalea extract as an active ingredient.

9. The use according to claim 8, characterized in that The azalea extract contains taxifolin glycoside or taxifolin aglycone.

10. The use according to claim 9, characterized in that The azalea extract is obtained by supercritical extraction of azalea roots.

11. The use according to claim 9, characterized in that The above-mentioned taxifolin glycosides include compounds of the following formula (1):

12. The use according to claim 9, characterized in that The above-mentioned taxifolin glycosides include compounds of the following formula (2):

13. Use of a composition for preparing a preventive food for preventing obesity, the composition comprising: One or more selected from the group consisting of compounds of the following formula (1) and formula (2):

14. The use according to claim 13, characterized in that The above compounds are extracted from the roots of Rhododendron.

15. The use according to any one of claims 13 to 14, characterized in that The above composition is a feed additive for animals.

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