Pharmaceutical composition and health food for preventing and treating sarcopenia and periodontal diseases, containing both red poplar and elm extracts as active ingredients

Abstract

The present invention provides a pharmaceutical composition for preventing and treating muscle loss and periodontal diseases, which contains both a red poplar extract and an elm extract as active ingredients, and the extract of the present invention contains both a red poplar extract and an elm extract as active ingredients, and has a prevention and treatment effect of reducing muscle and periodontal diseases.

Description

Technical Field

[0001] This invention relates to a treatment agent and health food containing a compound extract of alder and elm as active ingredients for muscle reduction and periodontal disease, and more specifically, to a treatment agent and health food containing a compound extract of alder and elm as active ingredients that has excellent inhibitory and preventive effects on muscle reduction and periodontal disease. Background Technology

[0002] Muscles can be classified into skeletal muscles, smooth muscles, and cardiac muscles based on their structure or function. Skeletal muscles, located directly beneath the skin in areas such as the hands, feet, chest, and abdomen, consist of over 600 voluntary muscles attached to bones via the skeleton or tendons throughout the body. They are adapted for movement through contraction or for supporting the skeleton. Contraction is initiated and regulated by nerve signals. They constitute 40-50% of body weight and play functions such as maintaining body temperature and generating energy. Actin and myosin, which form microfibrils, are arranged in a regular pattern, and striations can be observed under a microscope (Lieber RL, 2002; Edwards RH, 1981).

[0003] Skeletal muscle fibers are classified into three biochemical types based on mitochondrial content: Type I, Type IIa, and Type IIb. Type I fibers are composed of red slow-twitch muscle fibers and are used to maintain posture with low force for extended periods. Due to their high mitochondrial content, they are suitable for aerobic exercises such as long-distance running. Type IIa fibers are fast-twitch muscle fibers that exhibit characteristics of slow-twitch fibers. During exercise, muscles composed of white fast-twitch muscle fibers are used; these are called active muscles and are classified as Type IIb. Due to their low mitochondrial content, they are suitable for anaerobic exercises such as sprinting. These skeletal muscle fibers are distributed in different proportions throughout the body (Tortora et al., 2008).

[0004] Muscle atrophy is caused by the uneven anti-anabolic and catabolic processes of muscle fibers. Muscle atrophy refers to the loss of size and mass of muscle cells and tissue due to reduced activity levels caused by aging, disease states (excessive exposure to stress hormones, cancer, sepsis, starvation, etc.), and bedridden living. If muscle atrophy occurs, the strength of muscles used for physical activity weakens, thus initiating a vicious cycle of musculoskeletal degeneration. Decreased walking speed and weakened grip strength are key symptoms and indicators of muscle loss, potentially leading to falls, fractures, joint injuries, metabolic disorders, and cardiovascular diseases.

[0005] Glucocorticoids in our bodies cause molecular biological changes in muscle fibers, directly or indirectly participating in anti-anabolic and anti-catabolic actions. Dexamethasone, a glucocorticoid compound, acts as an anti-anabolic agent by inhibiting the PI3K / Akt / mTOR pathway. This inhibits the activity of downstream effectors such as 4E-BP1 and S6K1, thereby blocking the operation of eukaryotic translation initiation factor 4G (eIF4G) and eukaryotic translation initiation factor 4E (eIF4E). This inhibits the mRNA translation process used for protein synthesis, manifesting as impaired muscle fiber synthesis and muscle fiber atrophy due to protein breakdown (Shackman et al., 2013).

[0006] Dexamethasone also inhibits muscle synthesis and protein degradation, thereby inducing muscle atrophy. This is based on the mechanism of "PI3K / Akt→FOXO activation and GSK3 inactivation" leading to the expression of atrogene genes that induce muscle atrophy (muscle atrophy F-box protein (Atrogin-1) and muscle-specific ring finger protein 1 (MuRF-1)). These genes induce protein degradation, represented by the ubiquitin-proteasome system. Therefore, it is necessary to develop substances for the prevention and treatment of sarcopenia, a disease of skeletal muscle loss.

[0007] Furthermore, periodontal disease refers to all diseases occurring in the periodontal tissues, and is classified into gingivitis and periodontitis according to the severity of the disease. Gingivitis refers to a milder, faster-recovering form of periodontal disease, where the inflammation is limited to the gums (i.e., soft tissue). Periodontitis, on the other hand, refers to a condition where the inflammation has spread to the gums and the surrounding gingival bone. Such periodontal diseases are not only a major cause of tooth loss after adulthood, but are also reported to have a strong association with systemic diseases such as dementia, arteriosclerosis, myocardial infarction, and stroke. In modern society, which is entering an aging phase, attention is increasingly focused on the prevention and treatment of periodontal disease.

[0008] Additionally, the genus *Ulmus* belongs to the family Ulmaceae in the order Rosales. These are deciduous or semi-deciduous trees found in the Northern Hemisphere, from Siberia to Indonesia, Mexico, and Japan. The genus *Ulmus* contains approximately 30-40 species, with seven species inhabiting Korea: *Ulmus spp.*, *Ulmus nigra*, *Ulmus parvifolia*, *Ulmus hainanensis*, *Ulmus spp.*, and *Ulmus macrocarpa*. The tree, root bark, leaves, flowers, fruits, and seeds of *Ulmus spp.* are all used medicinally. Representative medicinal uses include the milky white bark of the stems and roots of *Ulmus parvifolia*, *Ulmus spp.*, and *Ulmus macrocarpa*, as well as the root bark, which has diuretic, anti-inflammatory, insomnia-relieving, constipation-preventing, digestive-promoting, and antioxidant effects.

[0009] However, to date, there have been no specific reports on the preventive and therapeutic properties of elm plant extracts and their active ingredients for periodontal diseases such as gingivitis. Summary of the Invention

[0010] Technical issues Therefore, the technical problem to be solved by the present invention is to provide a muscle reduction and periodontal disease treatment agent containing plant-based ingredients as active ingredients, as well as a substance for the prevention, treatment or improvement of periodontal disease in health foods.

[0011] Technical solution To address the aforementioned technical problems, the present invention provides a pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease, which contains extracts of both alder and elm as active ingredients.

[0012] In one embodiment of the present invention, the elm extract includes catechin 7-O-β-D-apiofuranoside.

[0013] In one embodiment of the present invention, the alder extract contains oregonin.

[0014] In one embodiment of the present invention, the weight ratio of the alder and elm extracts is 3:7 to 7:3.

[0015] The present invention also provides a health food for the prevention and improvement of periodontal disease, which contains extracts of alder and elm as active ingredients.

[0016] In one embodiment of the present invention, the elm extract includes catechin 7-O-β-D-apiofuranoside.

[0017] In one embodiment of the present invention, the alder extract contains oregonin.

[0018] In one embodiment of the present invention, the weight ratio of the alder and elm extracts is 3:7 to 7:3.

[0019] The present invention also provides a feed containing extracts of alder and elm as active ingredients.

[0020] In one embodiment of the present invention, the elm extract includes catechin 7-O-β-D-apiofuranoside.

[0021] In one embodiment of the present invention, the alder extract contains oregonin.

[0022] The present invention also provides a toothpaste or oral cleanser that contains the above-mentioned alder and elm extracts as active ingredients.

[0023] Beneficial effects The extract of this invention contains both alder and elm extracts as active ingredients, and has preventive and therapeutic effects on muscle loss and periodontal disease. Attached Figure Description

[0024] Figure 1 This is a step diagram of a method for obtaining a compound extract of large-fruited elm and alder according to an embodiment of the present invention.

[0025] Figure 2 The results were obtained to analyze the content of oreganoin (formula 1) in each extract.

[0026] Figure 3 The results were used to analyze the content of catechin glycosides of chemical formula 2 in each extract.

[0027] Figure 4 The results of the analysis of the inhibitory effect of the extract of the present invention on TNF-α expression were compared with the main pharmaceutical components of existing commercial periodontal disease treatment agents (Igatan) lysozyme and vitamin C as control groups.

[0028] Figure 5 The results of the TNF-α expression inhibition analysis of the compound extract of the present invention are compared with those of using alder and elm alone.

[0029] Figure 6 The results of the IL-6 expression analysis of the extract of the present invention were compared with the main pharmaceutical components of existing commercial periodontal disease treatment agents (Igatan), namely lysozyme and vitamin C, as control groups.

[0030] Figure 7The results show the IL-6 expression levels of the compound extract of the present invention compared to the use of alder and elm trees alone.

[0031] Figure 8 The results of the IL-1β expression analysis of the extract of the present invention were obtained by using lysozyme and vitamin C, the main pharmaceutical components of existing commercial periodontal disease treatment agents (Igatan), as control groups.

[0032] Figure 9 The results show the IL-1β expression levels of the compound extract of the present invention compared to the use of alder tree alone and elm tree alone.

[0033] Figures 10 to 14 This is the result of analyzing the cell viability of TM1 to TM5.

[0034] Figures 15 to 19 The results are from the separate analysis of the effects of TM1 to TM5 on cell death.

[0035] Figure 20 The results are from measurements of the diameter of muscle cells in dexamethasone-induced muscle atrophy.

[0036] Best practice Hereinafter, specific embodiments of the present invention will be described with reference to the accompanying drawings. However, these are merely examples, and the present disclosure is not limited thereto.

[0037] In describing this invention, detailed descriptions of well-known technologies related to this invention will be omitted where it is determined that such detailed descriptions may unnecessarily obscure the subject matter of the invention. Furthermore, the terminology used thereafter is defined in consideration of the functionality within this invention and may vary depending on the intent or convention of the user, operator, etc. Therefore, its definition should be based on the entire contents of this specification.

[0038] The technical concept of this invention is defined by the claims. The following embodiments are merely a means to effectively illustrate the technical concept of this invention to those skilled in the art.

[0039] To solve the above-mentioned technical problems, the present invention provides a pharmaceutical composition for the prevention and treatment of periodontal disease containing elm plant extract as an active ingredient, and a health food for improving periodontal disease containing the same, the preparation method of which is as follows. Detailed Implementation

[0040] Example 1 Figure 1 This is a step diagram of a method for obtaining a composite extract of elm UBC and alder AJ according to an embodiment of the present invention. Figure 1 An example is shown where the weight ratio is 1:1.

[0041] Reference Figure 1 In one embodiment of the present invention, in order to obtain a composite extract of *Ulmus pumila* and *Alnus davidii*, *Ulmus pumila* UBC and *Alnus davidii* AJ were mixed in ratios of 3:7, 5:5, and 7:3, respectively. Figure 1 In the original material, the weight ratio of large-fruited elm and alder is 5:5, and the weight ratio of large-fruited elm and alder is made different in 300kg of raw material.

[0042] 300 kg of the prepared raw material was mixed with 3,000 kg of 50% ethanol (1:10, w / w) and extracted at 75 ± 5°C for 6 hours. After extraction, the mixture was cooled to room temperature, filtered through a 0.2 μm filter, and concentrated under reduced pressure to 60 Brix (55 ± 5°C, 60 bar) to obtain a compound concentrate of *Ulmus pumila* and *Alnus davidii* (E50) (Lot. No. DJTH-06466). After concentration, dextrin and purified water were mixed with the concentrate and freeze-dried. After freeze-drying, 30 kg of the *Ulmus pumila* and *Alnus davidii* extract powder (E50) (Lot. No. DJTH-06465) was recovered.

[0043] Here, the extract was analyzed by TLC and NMR and found to contain oregonin as an active ingredient.

[0044] [Chemical Formula 1]

[0045] In addition, the elm extract contains catechin glycoside represented by the following chemical formula 2, namely catechin 7-O-β-D-apiofuranoside.

[0046] [Chemical Formula 2]

[0047] Therefore, HPLC quantitative analysis was performed on the extracts of alder and elm in the above ratio, and the elm extract powder was used to confirm the content of oreganoin, an indicator substance of elm in different samples.

[0048] Figure 2 The results were obtained to analyze the content of oreganoin (formula 1) in each extract.

[0049] Reference Figure 2 It is known that the extracts in one embodiment of the present invention all contain oreganoin.

[0050] Figure 3 The results were used to analyze the content of catechin glycosides of chemical formula 2 in each extract.

[0051] Reference Figure 3 It is known that the extracts according to an embodiment of the present invention all contain catechin glycosides of chemical formula 2.

[0052] Experimental Example 1 Improvement effect of periodontal disease In this experimental case, in order to confirm the treatment and improvement effect of periodontal disease, an inflammatory environment composition model for periodontal disease was first constructed according to the following method.

[0053] 1) Analysis of intracellular inflammatory cytokine expression levels - Model of the composition of the endogenous inflammatory environment ① Gingival fibroblasts were cultured in a 12-well plate at a density of 1 x 10⁻⁶ cells / well. 5 The density of the / well was used for dispensing, and the culture was carried out for 12 hours.

[0054] ② After treating with 10 ng / ml TNF-α and the test substance at the specified concentration, further incubation was performed.

[0055] ③ Use a total RNA extraction kit to extract RNA from cultured cells.

[0056] ④ Synthesize cDNA using a reverse transcription kit.

[0057] ⑤ Quantitative RT-PCR was performed using the SYBR reagent. At this time, the cycle threshold (Ct) was derived using specific primers for three pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, etc.).

[0058] ⑥ The relative expression level of pro-inflammatory cytokine was analyzed using the △△Ct method.

[0059] - Model of the composition of the exogenous inflammatory environment ① Gingival fibroblasts were placed in a 12-well plate at a density of 1 x 10⁻⁶ cm⁻¹. 5 / well density is aliquoted and cultured for 12 hours.

[0060] ② After processing LPS (1 ug / ml) or extracellular vesicles from oral bacteria (such as Porphyromonas gingivalis) and a predetermined concentration of the test substance, further culture was performed.

[0061] ③ Use a total RNA extraction kit to extract RNA from cultured cells.

[0062] ④ Synthesize cDNA using a reverse transcription kit.

[0063] ⑤ Perform quantitative RT-PCR using SYBR reagent. At this time, use the specific primers for three pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, etc.) to derive the Ct (cycle threshold) value.

[0064] ⑥ The relative expression level of pro-inflammatory cytokine was analyzed using the △△Ct method.

[0065] 2) Evaluation of the molecular mechanisms of intracellular inflammatory signaling - Model of the composition of the endogenous inflammatory environment ① Gingival fibroblasts were placed in a 12-well plate at a density of 1 x 10⁻⁶ cm⁻¹. 5 / well density is aliquoted and cultured for 12 hours.

[0066] ② After treating with 10 ng / ml TNF-α and the test substance at the specified concentration, further incubation was performed.

[0067] ③ After lysing the cells with Laemmli sample buffer, the cells were sonicated and heated at 95°C for 5 minutes, and total protein was extracted from the cultured cells.

[0068] ④ After performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to a polyvinylidene fluoride membrane and the membrane was immersed in 5% skimmilk for 30 minutes for blocking.

[0069] ⑤ After binding specific antibodies to phosphorylated NFκB, phosphorylated STAT3, total NFκB, and total STAT3, the chemiluminescence signal was detected and quantified.

[0070] - Model of the composition of the exogenous inflammatory environment ① Gingival fibroblasts were placed in a 12-well plate at a density of 1 x 10⁻⁶ cm⁻¹. 5 / well density is aliquoted and cultured for 12 hours.

[0071] ② After treating oral bacteria (Porphyromonas gingivalis, etc.) with LPS (1 ug / ml) or extracellular vesicles and a predetermined concentration of the test substance, further culture was performed.

[0072] ③ After disrupting the cells using laemmli sample buffer, the cells were sonicated and heated at 95°C for 5 minutes to extract total protein from the cultured cells.

[0073] ④ After performing SDS-PAGE, the protein is transferred to a PVDF membrane, and the membrane is soaked in 5% skim milk for 30 minutes for blocking.

[0074] ⑤ After binding with specific antibodies against phospho NFκB, phospho STAT3, total NFκB, and total STAT3, the chemiluminescence signal was detected and quantified.

[0075] This experimental example uses three natural extracts: UBC60E, UBRF3, and UM / A 50E (refer to...). Figure 1 and Figure 2 The effects of concentrations ranging from 1 to 100 on the improvement of inflammation in fibroblasts were analyzed as follows.

[0076] Anti-inflammatory effects Analysis of intracellular inflammatory cytokine expression levels (model of endogenous inflammatory environment composition) Based on the above results, a comparative experiment was conducted to evaluate the anti-inflammatory efficacy of the extract of the present invention. For this purpose, the natural extract was first pretreated with TNF-α or LPS for 1 hour prior to treatment, and RNA was extracted and its expression level was measured 4 hours after TNF-α or LPS treatment. As a control group, lysozyme and vitamin C, the main pharmaceutical components of the periodontal disease treatment agent (Igatan), were used.

[0077] Figure 4 The results of the analysis of the inhibitory effect of the extract of the present invention on TNF-α expression were compared with the main pharmaceutical components of existing commercial periodontal disease treatment agents (Igatan) lysozyme and vitamin C as control groups.

[0078] Reference Figure 4 It is known that, in the case of the compound extract of one embodiment of the present invention, it has a significantly higher TNF-α expression inhibition effect compared with lysozyme and vitamin C, which are the main components of existing periodontal disease treatment agents.

[0079] Figure 5 The results of the TNF-α expression inhibition analysis of the compound extract of the present invention are compared with those of using alder and elm alone.

[0080] Reference Figure 5 It can be seen that, in the case of compound extract (alder:elm weight ratio = 7:3), it has a significantly higher TNF-α expression inhibition effect compared with alder alone and elm alone.

[0081] These results indicate that when both catechin 7-O-β-D-apiofuranoside (from elm glycosides) and oregonin (from alder glycosides) are present, a significantly enhanced inhibitory effect on TNF-α expression is confirmed.

[0082] Figure 6 The results of the IL-6 expression analysis of the extract of the present invention were compared with the main pharmaceutical components of existing commercial periodontal disease treatment agents (Igatan), namely lysozyme and vitamin C, as control groups.

[0083] Reference Figure 6 It is evident that, compared to lysozyme and vitamin C, which are the main components of existing periodontal disease treatments, the compound extract of one embodiment of the present invention exhibits a higher IL-6 expression inhibition effect. In particular, as shown in the chart on the right side of the box (alder:elm = 7:3), it is evident that it has a significant and exceptionally high IL-6 expression inhibition capacity.

[0084] Figure 7 The results show the IL-6 expression levels of the compound extract of the present invention compared to the use of alder and elm trees alone.

[0085] Reference Figure 7 It can be seen that, in the case of compound extract (alder:elm weight ratio = 7:3), it has a significantly higher IL-6 expression inhibition effect compared with alder alone and elm alone.

[0086] Figure 8 The results of the IL-1β expression analysis of the extract of the present invention were obtained by using lysozyme and vitamin C, the main pharmaceutical components of existing commercial periodontal disease treatment agents (Igatan), as control groups.

[0087] Reference Figure 8 It is known that, in the case of the compound extract of one embodiment of the present invention, it has a significantly higher IL-1β expression inhibition effect compared with lysozyme and vitamin C, which are the main components of existing periodontal disease treatment agents.

[0088] Figure 9 The results show the IL-1β expression levels of the compound extract of the present invention compared to the use of alder and elm trees alone.

[0089] Reference Figure 9 It can be confirmed that in the composite sample containing both Catechin 7-O-β-Dapiofuranoside, a glycoside derived from elm tree, and oregonin, a glycoside derived from alder tree, IL-1β expression was statistically significantly and very strongly inhibited.

[0090] This invention also provides a muscle-reducing therapeutic agent and health food based on an extract containing both alder and elm extracts. This will be described in more detail below.

[0091] Experiment Example 2 Material test substance The following extracts provided by the applicant were used as test materials: large-fruited elm extract (U, TM1), alder extract-01 (A, TM2), large-fruited elm compound extract-02 (elm:alder weight ratio = 7:3, TM3), large-fruited elm compound extract-03 (elm:alder weight ratio = 5:5, TM4), and large-fruited elm compound extract-04 (elm:alder weight ratio = 3:7, TM5). The preparation method of the compound extracts was the same as in the examples.

[0092] method Cell culture C2C12 cells, derived from mouse skeletal muscle myoblasts, were purchased from the 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). When the cells reached approximately 80% confluence, the cell monolayer was washed away with phosphate-buffered saline (PBS, pH 7.4), and then trypsin-2.65 mM EDTA was added to isolate the cells for passage. The medium was changed every two days. To induce C2C12 cells to differentiate into myotubes, the cells were cultured in DMEM medium supplemented with 2% horse serum (HS), and the myotube differentiation medium was changed every two days.

[0093] Determination of protective effect against H2O2-induced myoblast damage C2C12 cells were loaded at 5 × 10 4 Cells / wells were aliquoted into 24-well plates and cultured for 24 hours. After 24 hours of C2C12 cell culture, the cells were treated with 100 μM H2O2 to induce myocyte damage. To investigate the protective effect of the test substances against myocyte damage, five test substances were treated at different concentrations along with 100 μM H2O2, and the cells were cultured for 48 hours. After 48 hours of cell culture, cell viability was determined using the same MTT assay as described above.

[0094] Evaluation of H2O2-induced apoptosis in myocytes To assess the effect of the test substance on H2O2-induced myocyte apoptosis, C2C12 cells were subjected to a 5 × 10⁻⁶ thiocyanate incubation. 4Cells / wells were aliquoted into 24-well plates and cultured for 24 hours. After 24 hours of C2C12 cell culture, the cells were treated with 100 μM H2O2 to induce myocyte damage. To investigate the protective effect of the test substances against myocyte damage, five test substances were treated with different concentrations along with 100 μM H2O2, and the cells were cultured for 48 hours. The degree of myocyte apoptosis was determined according to the manufacturer's method using the Cellular DNA Fragmentation ELISA kit (Sigma-Aldrich) for the detection of 5-bromo-2'-deoxyuridine ((BrdU)-labeled DNA).

[0095] Determination of the protective effect against dexamethasone-induced myotube cell damage C2C12 cells were loaded at 5 × 10 4 Cells / wells were aliquoted into 24-well plates and cultured for 24 hours. Then, to induce C2C12 cell differentiation into myotubes, the cell culture medium was exchanged with myotube differentiation culture medium for 4 days. Subsequently, to induce myotube atrophy, cells were treated with 5 μM dexamethasone. To examine the protective effect of the test substances against myotube damage, five test substances were treated at various concentrations along with 5 μM dexamethasone, and the cells were cultured for 24 hours. After 24 hours of culture, cell viability was determined using the same MTT assay as described above.

[0096] Determination of the protective effect against dexamethasone-induced myotube cell atrophy C2C12 cells were cultured in a 24-well plate containing a cover glass for 24 hours until they reached a cell count of 5 × 10⁻⁶. 4Cells / well. To induce C2C12 cells to differentiate into myotubes, the cell culture medium was exchanged with myotube differentiation medium, and differentiation was induced for 4 days. Subsequently, to induce myotube atrophy, cells were treated with 5 μM dexamethasone. To investigate the protective effect of the test substances against myotube damage, five test substances were treated at various concentrations along with 5 μM dexamethasone, and the cells were cultured for 24 hours. The culture medium was removed, and the cells were washed with PBS, then fixed with 4% paraformaldehyde and 0.1% Triton X-100. After blocking in 5% BSA / TBST, a primary antibody step (MYH7, Santa Cruz) was performed. Subsequently, after staining the tissues with a secondary antibody (Anti-mouse IgG-Alexa-594, ThermoFisher Scientifice), protein expression was examined using an optical microscope (Carl Zeiss) with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) as a control.

[0097] Statistical processing All analytical data are expressed as mean ± SEM. The collected results were analyzed using GraphPad Prism 5.0 (GraphPad software, San Diego, CA, USA). To compare the differences between the treatment group and the control group, Student's t-test and one-way analysis of variance (ANOVA) were used. Statistical significance was considered only when p < 0.05.

[0098] result Effects of H2O2-induced myoblast damage H2O2 (hydrogen peroxide) is a strong oxidant that induces oxidative stress in in vitro systems. To investigate the effects of five test substances on H2O2-induced myocyte damage, C2C12 cell culture medium was treated with 100 μM H2O2 to induce oxidative stress. After treating with different concentrations of the five test substances and culturing for 48 h, the cell viability of C2C12 cells was measured.

[0099] Figures 10 to 14 This is the result of analyzing the cell viability of TM1 to TM5.

[0100] Reference Figures 10 to 14 Compared with the control group [H2O2(-) / (-)], the cell survival rate of the H2O2-treated group [H2O2(+) / (-)] was significantly reduced.

[0101] like Figure 10 As shown, compared with the H2O2 treatment group [H2O2(+) / (-)], the treatment with TM1 (5, 10, 50 μg / mL) significantly increased cell viability at a treatment concentration of 50 μg / mL.

[0102] like Figure 11 and Figure 12 As shown, compared with the H2O2 treatment group [H2O2(+) / (-)], the cell viability of TM2 and TM3 (5, 10, 50 μg / mL) treatments increased significantly starting from a treatment concentration of 10 μg / mL.

[0103] like Figure 13 As shown, compared with the H2O2 treatment group [H2O2(+) / (-)], the TM4 treatment (5, 10, 50 μg / mL) significantly increased cell viability at a treatment concentration of 50 μg / mL.

[0104] like Figure 14 As shown, compared with the H2O2 treatment group [H2O2(+) / (-)], the cell viability of TM5 (5, 10, 50 μg / mL) treatment was significantly increased starting from the 10 μg / mL treatment concentration.

[0105] Furthermore, when each test substance was treated at a concentration of 50 μg / mL, cell viability increased by 14.0% (TM1), 17.3% (TM2), 24.2% (TM3), 21.9% (TM4), and 23.3% (TM5) respectively compared with the H2O2 treatment group [H2O2(+) / (-)].

[0106] 3.3. Effects on H2O2-induced myoblast apoptosis It is well known that oxidative stress induces cell death by causing DNA damage. This cell death induced by oxidative stress such as H2O2 occurs through an apoptosis process of programmed cell death. Therefore, this study used the Cellular DNA Fragmentation ELISA kit to quantify fragmented DNA to evaluate the effects of five test substances on oxidative stress-induced cell death.

[0107] Figures 15 to 19 The results are from the separate analysis of the effects of TM1 to TM5 on cell death.

[0108] Reference Figures 15 to 19 Compared with the control group [H2O2(-) / (-)], the H2O2-treated group [H2O2(+) / (-)] showed a significant increase in cell death.

[0109] Compared to the H2O2 treatment group [H2O2(+) / (-)], TM1 treatment at the highest treatment concentration of 50 μg / mL reduced apoptosis by 14.6% ( Figure 15 Compared to the H2O2 treatment group [H2O2(+) / (-)], TM2 treatment significantly reduced cell death starting from a concentration of 10 μg / mL, and reduced cell death by 18.3% at the highest treatment concentration of 50 μg / mL. Figure 16 Compared to the H2O2 treatment group [H2O2(+) / (-)], TM3 treatment significantly reduced cell death starting from a treatment concentration of 10 μg / mL, and reduced cell death by 34.8% at the highest treatment concentration of 50 μg / mL. Figure 17 Compared to the H2O2 treatment group [H2O2(+) / (-)], TM4 treatment at the highest treatment concentration of 50 μg / mL reduced apoptosis by 26.3% ( ). Figure 18 Compared to the H2O2 treatment group [H2O2(+) / (-)], TM5 treatment significantly reduced cell death starting from a treatment concentration of 10 μg / mL, and at the highest treatment concentration of 50 μg / mL, cell death was reduced by 29.5% ( ). Figure 19 ).

[0110] 3.4. Effects on Dexamethasone-induced myotube cell damage Dexamethasone is a representative glucoseoid. Misuse and abuse in clinical practice can lead to skeletal muscle breakdown, and it is widely used to induce myocyte atrophy in in vitro systems. To investigate the effects of five test substances on dexamethasone-induced myotube atrophy, myotube diameter was measured after treating each test substance and culturing the cells. Specifically, C2C12 cells were cultured in myocyte differentiation medium for 4 days to induce myocyte differentiation (myotubes), then treated with 5 μM dexamethasone to induce myocyte atrophy. The cells were treated with the five test substances and cultured for 24 hours. To observe and quantify the myotube diameter, fluorescent staining with MYH antibody was performed, followed by observation and quantification of the myocyte diameter. Compared to the untreated dexamethasone control group [DEX(-) / (-)], treatment with 5 μM dexamethasone induced myocyte atrophy, and a significant decrease in myocyte diameter was observed.

[0111] Therefore, the diameter was significantly increased compared to the DEX-treated group [DEX(+) / (-)] after treatment with the five test substances.

[0112] Figure 20 The results are from measurements of the diameter of muscle cells in dexamethasone-induced muscle atrophy.

[0113] Reference Figure 20 The results of diameter measurements showed that, compared with the untreated dexamethasone control group [DEX(-) / (-)] (diameter 0.41–0.45 μm), the diameter of the DEX-treated group [DEX(+) / (-)] was 0.12–0.14 μm, indicating a significant decrease in muscle cell diameter. Therefore, all five test substances significantly increased muscle cell diameter. At the highest treatment concentration of 50 μg / mL (the highest concentration of the five test substances), the muscle cell diameter increased by 2.00-fold (TM1), 1.92-fold (TM2), 2.07-fold (TM3), 1.71-fold (TM4), and 2.33-fold (TM5) compared to the DEX-treated group [DEX(+) / (-)].

[0114] In summary, the compound extract of the present invention significantly increased the cell survival rate of C2C12 myoblasts, which was significantly reduced by H2O2 treatment. Furthermore, TM5, as the compound extract, increased the diameter of muscle cells reduced by dexamethasone treatment by a full 2.33 times, demonstrating a superior effect in protecting the myoblast atrophy-preventing properties of the compound extract of the present invention compared to individual extracts. Therefore, the possibility of developing it into a functional pharmaceutical material for the treatment and improvement of muscle loss (atrophy) can be confirmed in the future.

[0115] In this specification, the term "comprising...as an active ingredient" means that the extract of the present invention contains a sufficient amount to achieve the effect of preventing and treating muscle loss.

[0116] In this specification, the term "periodontal disease" refers to all diseases occurring in the periodontal tissues. The term "prevention" as used in this invention refers to all actions that inhibit or delay the onset of periodontal disease by administering the pharmaceutical composition of this invention. Furthermore, the term "treatment" as used in this invention refers to all actions that improve or become beneficial to the symptoms of periodontal disease by administering the pharmaceutical composition of this invention. The term "improvement" as used in this invention refers to all actions that at least reduce parameters related to the treatment state (e.g., symptom severity). Therefore, the functional food composition, in order to prevent or improve periodontal disease, can be used simultaneously or alone with a therapeutic agent before or after the onset of the relevant disease. The pharmaceutical compositions of the present invention for the prevention and treatment of periodontal disease may also contain pharmaceutically acceptable carriers, excipients or diluents.

[0117] In the compositions of the present invention, pharmaceutically acceptable carriers are commonly used in formulations, including but not limited to lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In addition to the above-mentioned components, the pharmaceutical compositions of the present invention may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0118] The pharmaceutical composition of the present invention can be administered orally or non-orally. In the case of non-oral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0119] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate and responsiveness. Generally, a skilled physician can easily determine and prescribe the dosage that is effective for the desired treatment or prevention.

[0120] The pharmaceutical composition of the present invention for the prevention and treatment of periodontal disease may contain other pharmaceutically active ingredients as active ingredients in addition to elm plant extracts, or may be used in combination with pharmaceutical compositions containing other active ingredients.

[0121] The food or health food compositions of the present invention may also contain food science-acceptable food additives. Food science-acceptable food additives that can be used in the present invention include, but are not limited to, sugars such as glucose, fructose, maltose, sucrose, dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol; natural flavoring agents such as sematriol and stevia extract; synthetic flavoring agents such as saccharin and aspartic acid; coloring agents; pectic acid or its salts; alginic acid or its salts; organic acids; protective colloid thickeners; pH adjusters; stabilizers; preservatives; glycerin; alcohols; and carbonating agents. The food compositions of the present invention can be in the form selected from the group consisting of powders, granules, tablets, capsules, candies, chewing gums, jellies, and beverages. The content of the elm extract in the above-mentioned food compositions can be appropriately selected considering the form, flavor, and taste of the food, for example, it can be in the range of 0.01 to 30% by weight relative to the total weight of the food. It will be apparent to those skilled in the art that the form, composition, and preparation method of the food composition according to the present invention can be suitably selected from conventional techniques known in the art.

[0122] Therefore, the composition of the present invention containing elm plant extract as an active ingredient can be used not only as a pharmaceutical composition or food composition, but also as a component of toothpaste and oral cleansers for humans and animals, and further as animal feed for pets and other animals.

[0123] In addition, the extract of the present invention can also be used as an active ingredient in toothpaste and oral hygiene products.

[0124] Industrial availability This invention relates to pharmaceutical compositions and health foods containing extracts of alder and elm as active ingredients for the prevention and treatment of sarcopenia and periodontal disease, and is industrially viable.

Claims

1. A pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease, wherein, It also contains alder and elm extracts as active ingredients.

2. The pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease according to claim 1, characterized in that, The elm extract mentioned above contains catechin 7-O-β-D-apiofuranoside.

3. The pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease according to claim 1, characterized in that, The alder extract contains oregonin.

4. The pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease according to claim 1, characterized in that, The weight ratio of the alder and elm extracts is 3:7 to 7:

3.

5. A health food product for the prevention and improvement of periodontal disease, wherein, It also contains extracts of alder and elm as active ingredients.

6. The health food for preventing and improving periodontal disease according to claim 5, characterized in that, The elm extract contains catechin 7-O-β-D-apiofuranoside.

7. The health food for preventing and improving periodontal disease according to claim 5, characterized in that, The alder extract contains oregonin.

8. The health food for preventing and improving periodontal disease according to claim 5, characterized in that, The weight ratio of the alder and elm extracts is 3:7 to 7:

3.

9. A feed containing extracts of both alder and elm as active ingredients.

10. A toothpaste that contains extracts of both alder and elm as active ingredients.

11. An oral cleanser containing both alder and elm extracts as active ingredients.

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