Pharmaceutical composition and health functional food including alnus japonica and ulmus davidiana var. japonica extracts together as active ingredients for preventing and treating sarcopenia and periodontal diseases

Abstract

Provided is a pharmaceutical composition including extracts of Alnus japonica and Ulmus davidiana var. japonica together as active ingredients for preventing and treating sarcopenia and periodontal diseases. The extracts according to the present invention include extracts of Alnus japonica and Ulmus davidiana var. japonica together as active ingredients, and have an effect of preventing and treating sarcopenia and periodontal diseases.

Description

Pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease and health functional food containing alder and elm extracts simultaneously as active ingredients

[0001] The present invention relates to a treatment for sarcopenia and periodontal disease and a health functional food containing a complex extract of alder and elm as an active ingredient, and more specifically, to a treatment for sarcopenia and periodontal disease and a health functional food containing a complex extract of alder and elm as an active ingredient having excellent inhibitory and preventive effects against sarcopenia and periodontal disease.

[0002] Muscles can be classified into skeletal muscle, smooth muscle, and cardiac muscle in terms of structure and function. Among these, skeletal muscle consists of approximately 600 voluntary muscles located just beneath the skin in areas such as the hands, feet, chest, and abdomen, attached to bones throughout the body via tendons. They are well-suited for moving or supporting bones through contraction. Contraction occurs and is regulated by nerve signals. They account for 40–50% of body weight and perform functions such as maintaining body temperature and generating energy. They are composed of microfilaments called actin and myosin arranged in a regular pattern, allowing transverse striations to be observed under a microscope (Lieber RL, 2002; Edwards RH, 1981).

[0003] Skeletal muscle fibers are biochemically classified into three types—Type I, Type IIa, and Type IIb—based on their mitochondrial content. Type I refers to postural muscles composed of red, slow-twitch fibers that maintain posture by sustaining weak force for extended periods. These muscles have a high mitochondrial content and are suitable for aerobic exercises such as long-distance running. Type IIa refers to fast-twitch fibers that possess the characteristics of slow-twitch fibers. Muscles composed of white, fast-twitch fibers are used to generate movement; these are called active muscles and are classified as Type IIb. These muscles have a low mitochondrial content and are suitable for anaerobic exercises such as short-distance running. These skeletal muscle fibers are distributed in different proportions across various parts of the body (Tortora et al, 2008).

[0004] Muscle atrophy is defined as the unbalanced anti-anabolic and catabolic processes of muscle fibers. In this context, muscle atrophy refers to the loss of muscle cells and tissue size and mass resulting from a lack of muscle use due to reduced activity caused by aging, disease conditions (such as excessive exposure to stress hormones, cancer, sepsis, and starvation), and bedridden living. When muscle atrophy occurs, muscle strength required for physical activity weakens, initiating a vicious cycle of musculoskeletal degeneration. Decrease in walking speed and weakened grip strength are the primary symptoms and indicators of muscle mass loss, which can lead to falls, fractures, joint injuries, metabolic disorders, and cardiovascular diseases.

[0005] Glucocorticoids in the body induce molecular biological changes in muscle fibers and are directly or indirectly involved in anabolic and catabolic processes. 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 function of eIF4G (Eukaryotic translation initiation factor 4G) and eIF4E (Eukaryotic translation initiation factor 4E). This suppresses the mRNA translation process for protein synthesis, resulting in inhibition of muscle fiber synthesis and muscle fiber atrophy due to protein degradation (Shackman et al., 2013).

[0006] Dexamethasone can induce muscle atrophy by inhibiting muscle synthesis and causing protein degradation. This leads to the expression of atrogen genes (Atrogin-1, MuRF-1) that induce muscle atrophy, following a mechanism involving 'PI3K / Akt → FOXO activation and GSK3 inactivation.' These genes induce protein degradation represented by the ubiquitin-proteasome system. Therefore, there is a need to develop substances for the prevention and treatment of sarcopenia, a disease characterized by the decline of skeletal muscle.

[0007] Furthermore, periodontal disease refers to any disease occurring in the periodontal tissues, and is classified into gingivitis and periodontitis depending on the severity of the disease. Among these, gingivitis is a relatively mild and fast-recovering form of periodontal disease where inflammation is confined only to the gums, or soft tissues, whereas periodontitis refers to cases where inflammation has progressed to the gums and surrounding bone. These periodontal diseases are not only a major cause of tooth loss after middle age but have also been reported to have a high correlation with systemic diseases such as dementia, arteriosclerosis, myocardial infarction, and stroke; consequently, interest in the prevention and treatment of periodontal disease is increasing in modern society, which has entered an aging society.

[0008] Meanwhile, the genus Ulmus belongs to the family Ulmaceae within the order Rosales and consists of deciduous or semi-deciduous trees found in the Northern Hemisphere, extending from Siberia to Indonesia, Mexico, and Japan. The genus includes approximately 30 to 40 species, and seven species—Ulmus parvifolia, Ulmus hirsuta, Ulmus davidiana, Ulmus parvifolia, Ulmus parvifolia var. spontanea and Ulmus parvifolia var. spontanea—inhabit Korea. All parts of the elm tree, including the tree trunk, root bark, leaves, flowers, fruits, and seeds, are used medicinally. Notably, the stem and root bark (*yubaekpi* and *yugeunpi*) of Ulmus parvifolia, Ulmus parvifolia, and Ulmus parvifolia var. spontanea are known for their diuretic, anti-inflammatory, insomnia-relieving, constipation-preventing, digestive-promoting, and antioxidant effects.

[0009] However, there have been no specific reports to date regarding the preventive and therapeutic properties of elm plant extracts and their active substances for periodontal diseases, such as gum inflammation.

[0010] Therefore, the problem that the present invention aims to solve is to provide a substance that prevents, treats, or improves periodontal disease, a therapeutic agent for muscle loss and periodontal disease containing a plant-based ingredient as an active ingredient, and a health functional food.

[0011] To solve the above problem, the present invention provides a pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease comprising alder and elm extracts simultaneously as active ingredients.

[0012] In one embodiment of the present invention, the elm extract comprises 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 functional food for the prevention and improvement of periodontal disease comprising alder and elm extracts simultaneously as active ingredients.

[0016] In one embodiment of the present invention, the elm extract comprises 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 alder and elm extracts simultaneously as active ingredients.

[0020] In one embodiment of the present invention, the elm extract comprises 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 mouthwash comprising the aforementioned alder and elm extracts simultaneously as active ingredients.

[0023] The extract according to the present invention contains alder and elm extracts simultaneously as active ingredients and has the effect of preventing and treating muscle loss and periodontal disease.

[0024] FIG. 1 is a step diagram of a method for obtaining a complex extract of king elm and alder according to one embodiment of the present invention.

[0025] Figure 2 shows the results of analyzing the content of oreganin of Chemical Formula 1 for each extract.

[0026] Figure 3 shows the results of analyzing the content of catechin glycoside of Chemical Formula 2 for each extract.

[0027] Figure 4 shows the results of analyzing the TNF-α expression inhibitory effect of the extract according to the present invention using lysozyme and vitamin C, which are the main pharmaceutical components of a commercially available periodontal disease treatment (Igatan), as a control group.

[0028] Figure 5 shows the results of the analysis of TNF-α expression inhibition using the complex extract according to the present invention compared to alder alone and elm alone.

[0029] Figure 6 shows the results of analyzing the IL-6 expression level of the extract according to the present invention using lysozyme and vitamin C, which are the main pharmaceutical components of a commercially available periodontal disease treatment (Igatan), as a control group.

[0030] Figure 7 shows the results of the analysis of IL-6 expression levels in the complex extract according to the present invention compared to alder alone and elm alone.

[0031] Figure 8 shows the results of analyzing the IL-1β expression level of the extract according to the present invention using lysozyme and vitamin C, which are the main pharmaceutical components of a commercially available periodontal disease treatment (Igatan), as a control group.

[0032] Figure 9 shows the results of the analysis of IL-1β expression levels in the complex extract according to the present invention compared to alder alone and elm alone.

[0033] Figures 10 to 14 show the results of analyzing the cell viability of TM1 to TM5.

[0034] Figures 15 to 19 show the results of analyzing the effects of TM1 to TM5 on apoptosis, respectively.

[0035] Figure 20 shows the results of measuring muscle cell diameter for Dexamethasone-induced muscle atrophy.

[0036] Hereinafter, specific embodiments of the present invention will be described with reference to the drawings. However, this is merely an example and the present invention is not limited thereto.

[0037] In describing the present invention, detailed descriptions of known technologies related to the invention are omitted if it is determined that such descriptions may unnecessarily obscure the essence of the invention. Furthermore, the terms described below are defined considering their functions in the present invention, and these definitions may vary depending on the intentions or practices of the user or operator. Therefore, their definitions should be based on the content throughout this specification.

[0038] The technical concept of the present invention is determined by the claims, and the following embodiments are merely a means to efficiently explain the technical concept of the present invention to those skilled in the art to which the present invention belongs.

[0039] To solve the aforementioned problems, the present invention provides a pharmaceutical composition for the prevention and treatment of periodontal disease comprising an extract of a plant of the genus Ulmus as an active ingredient, and a health functional food for improving periodontal disease comprising the same, wherein the manufacturing method is as follows.

[0040] Example 1

[0041] FIG. 1 is a flowchart of a method for obtaining a complex extract of elm (UBC) and alder (AJ) according to one embodiment of the present invention. FIG. 1 illustrates a case where the weight ratio is 1:1.

[0042] Referring to FIG. 1, in one embodiment of the present invention, Ulmus davidiana (UBC) and Alnus japonica (AJ) were mixed in ratios of 3:7, 5:5, and 7:3, respectively, for Ulmus davidiana-alnus complex extract. FIG. 1 shows the case where the weight ratio is 5:5, and the weight ratio of Ulmus davidiana and Alnus japonica is configured differently in 300 kg of raw material.

[0043] 300 kg of prepared raw material was mixed with 3,000 kg of 50% ethanol (1:10, w / w) and extracted for 6 hours at 75 ± 5℃. 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℃, 60 bar) to obtain a complex concentrate of Ulmus davidiana and Alnus japonica (E50) (Lot. No. DJTH-06466). After concentration was completed, dextrin and purified water were mixed based on the concentrate, and freeze-drying was performed. After freeze-drying, 30 kg of Ulmus davidiana and Alnus japonica extract powder (E50) (Lot. No. DJTH-06465) was recovered.

[0044] As a result of analysis using TLC and NMR, the above extract contains oregonin of the following chemical formula as an active ingredient.

[0045] [Chemical Formula 1]

[0046]

[0047]

[0048] In addition, the elm extract contains a catechin glycoside represented by the following chemical formula 2, 7-O-β-D-apiofuranoside.

[0049] [Chemical Formula 2]

[0050]

[0051]

[0052] To this end, HPLC quantitative analysis was performed on the extract powders and alder extract powder according to the ratio of alder to elm with respect to the above extract, and the content of oregonin, an indicator substance of alder, was confirmed for each sample.

[0053] Figure 2 shows the results of analyzing the content of oreganin of Chemical Formula 1 for each extract.

[0054] Referring to FIG. 2, it can be seen that all extracts according to one embodiment of the present invention contain oregonin.

[0055] Figure 3 shows the results of analyzing the content of catechin glycoside of Chemical Formula 2 for each extract.

[0056] Referring to FIG. 3, it can be seen that all extracts according to one embodiment of the present invention contain catechin glycoside of Formula 2.

[0057]

[0058] Experimental Example 1

[0059] Improvement effect on periodontal disease

[0060] In this experimental example, to verify the therapeutic and improvement effects of periodontal disease, an inflammatory environment creation model for periodontal disease was first constructed according to the following method.

[0061]

[0062] 1) Analysis of intracellular inflammatory cytokine expression levels

[0063] - Endogenous Inflammatory Environment Creation Model

[0064] ① 1 x 10 gingival fibroblasts in a 12-well plate 5 Dispense at a density of / well and incubate for 12 hours.

[0065] ② Additional culture after treatment with 10 ng / ml TNF-α and a specified concentration of the test substance.

[0066] ③ Extract RNA from cultured cells using a total RNA extraction kit.

[0067] ④ Synthesize cDNA using a reverse transcription kit.

[0068] ⑤ Perform quantitative RT-PCR using SYBR reagent, and derive the Ct (cycle threshold) value using specific primers for three types of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, etc.).

[0069] ⑥ Analyzed the relative expression levels of pro-inflammatory cytokines using the △△Ct method.

[0070] - Model for creating an exogenous inflammatory environment

[0071] ① 1 x 10 gingival fibroblasts in a 12-well plate 5 Dispense at a density of / well and incubate for 12 hours.

[0072] ② Additional culture after treatment with LPS (1 ug / ml) derived from oral bacteria (Porphyromonas gingivalis, etc.) or extracellular vesicles and a specified concentration of the test substance.

[0073] ③ Extract RNA from cultured cells using a total RNA extraction kit.

[0074] ④ Synthesize cDNA using a reverse transcription kit.

[0075] ⑤ Perform quantitative RT-PCR using SYBR reagent, and derive the Ct (cycle threshold) value using specific primers for three types of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, etc.).

[0076] ⑥ Analyzed the relative expression levels of pro-inflammatory cytokines using the △△Ct method.

[0077]

[0078] 2) Evaluation of intracellular inflammatory signaling molecular mechanisms

[0079] - Endogenous inflammatory environment creation model

[0080] ① 1 x 10 gingival fibroblasts in a 12-well plate 5 Dispense at a density of / well and incubate for 12 hours.

[0081] ② Additional culture after treatment with 10 ng / ml TNF-α and a specified concentration of the test substance.

[0082] ③ Total protein was extracted from cells cultured by sonication and heating at 95°C for 5 minutes after cell lysis with laemmli sample buffer.

[0083] ④ After performing SDS-PAGE, transfer the protein to a PVDF membrane and perform blocking by immersing the membrane in 5% skim milk for 30 minutes.

[0084] ⑤ After conjugating antibodies specific to phospho NFκB, phospho STAT3, total NFκB, and total STAT3, quantify by detecting the chemiluminescence signal

[0085]

[0086] - Model for Creating an Exogenous Inflammatory Environment

[0087] ① 1 x 10 gingival fibroblasts in a 12-well plate 5 Dispense at a density of / well and incubate for 12 hours.

[0088] ② Additional culture after treatment with LPS (1 ug / ml) derived from oral bacteria (Porphyromonas gingivalis, etc.) or extracellular vesicles and a specified concentration of the test substance.

[0089] ③ Total protein was extracted from cells cultured by sonication and heating at 95°C for 5 minutes after cell lysis with laemmli sample buffer.

[0090] ④ After performing SDS-PAGE, transfer the protein to a PVDF membrane and perform blocking by immersing the membrane in 5% skim milk for 30 minutes.

[0091] ⑤ After conjugating antibodies specific to phospho NFκB, phospho STAT3, total NFκB, and total STAT3, quantify by detecting the chemiluminescence signal

[0092]

[0093] In this experiment, the anti-inflammatory effects of three types of natural extracts—UBC60E, UBRF3, and UM / A 50E (see Figures 1 and 2)—were analyzed at concentrations of 1 to 100 on fibroblasts as follows.

[0094]

[0095] Anti-inflammatory effects

[0096] Analysis of intracellular inflammatory cytokine expression levels (endogenous inflammatory environment formation model)

[0097] Based on the above results, the anti-inflammatory efficacy of the extract according to the present invention was measured through comparative experiments. To this end, the natural extract was pretreated 1 hour before treatment with TNF-α or LPS, and RNA was extracted 4 hours after treatment with TNF-α or LPS to measure the expression level. At this time, lysozyme and vitamin C, which are the main pharmaceutical components of a periodontal disease treatment (Igatan), were used as control groups.

[0098] Figure 4 shows the results of analyzing the TNF-α expression inhibitory effect of the extract according to the present invention using lysozyme and vitamin C, which are the main pharmaceutical components of a commercially available periodontal disease treatment (Igatan), as a control group.

[0099] Referring to FIG. 4, it can be seen that the complex extract according to one embodiment of the present invention has a significantly higher TNF-α expression inhibitory effect compared to lysozyme and vitamin C, which are the main components of existing periodontal disease treatments.

[0100] Figure 5 shows the results of the analysis of TNF-α expression inhibition using the complex extract according to the present invention compared to alder alone and elm alone.

[0101] Referring to Figure 5, it can be seen that the complex extract (alder:elm weight ratio = 7:3) has a significantly higher TNF-α expression inhibitory effect compared to alder alone and elm alone.

[0102] This result confirms a superior synergistic effect of inhibiting TNF-α expression when Catechin 7-O-β-D apiofuranoside, a glycoside compound derived from elm, and oregonin, a glycoside compound derived from alder, are contained simultaneously.

[0103] Figure 6 shows the results of analyzing the IL-6 expression level of the extract according to the present invention using lysozyme and vitamin C, which are the main pharmaceutical components of a commercially available periodontal disease treatment (Igatan), as a control group.

[0104] Referring to FIG. 6, it can be seen that the complex extract according to one embodiment of the present invention has a high IL-6 expression inhibitory effect compared to lysozyme and vitamin C, which are the main components of existing periodontal disease treatments. In particular, in the case of the graph on the right within the box (Ora tree:Ulmus parvifolia = 7:3), it can be seen that it has a remarkably and uniquely high IL-6 expression inhibitory ability.

[0105] Figure 7 shows the results of the analysis of IL-6 expression levels in the complex extract according to the present invention compared to alder alone and elm alone.

[0106] Referring to Figure 7, it can be seen that the complex extract (alder:elm weight ratio = 7:3) has a significantly higher IL-6 expression inhibitory effect compared to alder alone and elm alone.

[0107] Figure 8 shows the results of analyzing the IL-1β expression level of the extract according to the present invention using lysozyme and vitamin C, which are the main pharmaceutical components of a commercially available periodontal disease treatment (Igatan), as a control group.

[0108] Referring to FIG. 8, it can be seen that the complex extract according to one embodiment of the present invention has a significantly higher IL-1β expression inhibitory effect compared to lysozyme and vitamin C, which are the main components of existing periodontal disease treatments.

[0109] Figure 9 shows the results of the analysis of IL-1β expression levels in the complex extract according to the present invention compared to alder alone and elm alone.

[0110] Referring to Figure 9, it can be confirmed that a composite sample containing both Catechin 7-O-β-D apiofuranoside, a glycoside compound derived from elm, and oregonin, a glycoside compound derived from alder, statistically significantly and very strongly inhibits IL-1β expression.

[0111]

[0112] The present invention also provides a sarcopenia treatment and a health functional food based on an extract containing both alder and elm extracts. This is described in more detail below.

[0113]

[0114] Experimental Example 2

[0115] ingredient

[0116] test substance

[0117] The king elm extract (U, TM1), alder extract-01 (A, TM2), king elm complex extract-02 (elm:alder weight ratio 7:3, TM3), king elm complex extract-03 (elm:alder weight ratio 5:5, TM4), and king elm complex extract-04 (elm:alder weight ratio 3:7, TM5) provided by the applicant were used as test substances, and the method of preparing the complex extracts is the same as in the examples.

[0118]

[0119] method

[0120] cell culture

[0121] C2C12 cells, myoblasts derived from mouse skeletal muscle, were purchased from the American Type Culture Collection (ATCC). C2C12 cells were cultured in a humid CO2 incubator (5% CO2 / 95% air) at 37°C using a cell culture medium prepared by adding 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin to Dulbecco's Modified Eagle Medium (DMEM). When the cells filled approximately 80% of the culture dish, the cell monolayer was washed with phosphate buffer saline (PBS, pH 7.4), and cells were detached and subcultured using trypsin-2.65 mM EDTA. The medium was changed every two days. To induce differentiation of C2C12 cells into myotubes, cells were cultured by replacing DMEM medium with a myotube differentiation medium containing 2% horse serum (HS), and the myotube differentiation medium was replaced every 2 days.

[0122]

[0123] Measurement of protective effect against H2O2-induced myoblast injury

[0124] 5 × 10 C2C12 cells4 Cells were seeded into a 24-well plate at cells / well and cultured for 24 hours. After culturing C2C12 cells for 24 hours, 100 μM H2O2 was added to induce myocyte damage. To investigate the protective effect of the test substances on myocyte damage, five types of test substances were added at various concentrations along with 100 μM H2O2, and the cells were cultured for 48 hours. After culturing the cells for 48 hours, the cell viability was measured by performing an MTT assay in the same manner as above.

[0125]

[0126] Evaluation of H2O2-induced apoptosis in myocells

[0127] To evaluate the effect of the test substance on H2O2-induced myocyte apoptosis, C2C12

[0128] 5 × 10 cells 4 Cells were seeded into 24-well plates at cells / well and cultured for 24 hours. After culturing C2C12 cells for 24 hours, 100 μM H2O2 was added to induce myocyte damage, and to investigate the protective effect of the test substances on myocyte damage, five types of test substances were added at various concentrations along with 100 μM H2O2, and the cells were cultured for 48 hours. The degree of myocyte apoptosis was measured using a Cellular DNA Fragmentation ELISA kit (Sigma-Aldrich) that detects 5'-Bromo-2'-deoxy-uridine (BrdU)-labeled DNA according to the method provided by the manufacturer.

[0129]

[0130] Measurement of protective effect against dexamethasone-induced myotube damage

[0131] 5 × 10 C2C12 cells 4Cells were seeded into a 24-well plate at cells / well and cultured for 24 hours. Subsequently, to induce differentiation of C2C12 cells into myotubes, the cell culture medium was replaced with myotube differentiation medium, and differentiation was induced for 4 days. Afterward, 5 μM dexamethasone was added to induce myotube atrophy, and to investigate the protective effect of the test substances on myotube damage, five types of test substances were added along with 5 μM dexamethasone at various concentrations and the cells were cultured for 24 hours. After culturing the cells for 24 hours, cell viability was measured by performing an MTT assay in the same manner as above.

[0132]

[0133] Measurement of protective effect against dexamethasone-induced myotube atrophy

[0134] 5 × 10 C2C12 cells 4Cells were cultured for 24 hours in 24-well plates containing cover glass to ensure a cell / well ratio. To induce differentiation of C2C12 cells into myotubes, the cell culture medium was replaced with myotube differentiation medium, and differentiation was induced for 4 days. Subsequently, 5 μM dexamethasone was added to induce myotube atrophy, and to investigate the protective effect of the test substances on myotube damage, cells were cultured for 24 hours with 5 μM dexamethasone and five test substances at various concentrations. After removing the medium and washing with PBS, the cells were fixed by treating with 4% paraformaldehyde and 0.1% Triton X-100. After blocking with 5% BSA / TBST, the primary antibody step (MYH7, Santa Cruz) was performed. Afterward, the tissue was stained with a secondary antibody (Anti-mouse IgG-Alexa-594, ThermoFisher Scientifice), counterstained with 4'-6-Diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and protein expression was examined using a light microscope (Carl Zeiss).

[0135]

[0136] Statistical processing

[0137] All analysis values ​​were expressed as mean ± SEM. The collected results were analyzed using GraphPad Prism 5.0 (GraphPad software, San Diego, CA, USA). Student's t-test and one-way analysis variance (ANOVA) were used to compare the differences between the test substance treatment group and the control group. Statistical significance was determined only when p < 0.05.

[0138]

[0139] result

[0140] Effects on H2O2-induced myoblast damage

[0141] H2O2 (hydrogen peroxide) is a strong oxidizing agent that induces oxidative stress in an in vitro system. To investigate the effects of five test substances on H2O2-induced muscle cell damage, oxidative stress was induced in the cell culture medium of C2C12 cells by treating them with 100 μM H2O2, and after treating them with the five test substances at various concentrations and culturing for 48 hours, the cell viability of C2C12 cells was measured.

[0142] Figures 10 to 14 show the results of analyzing the cell viability of TM1 to TM5.

[0143] Referring to Figures 10 to 14, the cell viability of the H2O2-treated group [H2O2(+) / (-)] was significantly reduced compared to the control group [H2O2(-) / (-)] that was not treated with H2O2.

[0144] As shown in Figure 10, treatment with TM1 (5, 10, 50 μg / mL) significantly increased cell viability at a treatment concentration of 50 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)].

[0145] As shown in Figures 11 and 12, treatment with TM2 and TM3 (5, 10, 50 μg / mL) significantly increased cell viability starting from a treatment concentration of 10 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)].

[0146] As shown in Figure 13, treatment with TM4 (5, 10, 50 μg / mL) significantly increased cell viability at a treatment concentration of 50 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)].

[0147] As shown in Figure 14, treatment with TM5 (5, 10, 50 μg / mL) significantly increased cell viability starting from a treatment concentration of 10 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)].

[0148] In addition, 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 to the H2O2 treatment group [H2O2(+) / (-)].

[0149]

[0150] 3.3. Effects on H2O2-induced myoocyte apoptosis

[0151] Oxidative stress is well known to induce cell death by causing DNA damage, and cell death caused by such oxidative stress, such as H2O2, occurs through the process of apoptosis, a type of programmed cell death. Therefore, in this study, fragmented DNA was quantified using a Cellular DNA Fragmentation ELISA kit to evaluate the effects of five test substances on oxidative stress-induced cell death.

[0152] Figures 15 to 19 show the results of analyzing the effects of TM1 to TM5 on apoptosis, respectively.

[0153] Referring to Figures 15 to 19, compared to the control group [H2O2(-) / (-)] that was not treated with H2O2, the H2O2-treated group [H2O2(+) / (-)] showed a significant increase in apoptosis.

[0154] Treatment with TM1 reduced apoptosis by 14.6% at the highest treatment concentration of 50 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)] (Fig. 15). Treatment with TM2 significantly reduced apoptosis starting from a concentration of 10 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)], resulting in an 18.3% reduction at the highest treatment concentration of 50 μg / mL (Fig. 16). Treatment with TM3 significantly reduced apoptosis starting from a treatment concentration of 10 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)], resulting in a 34.8% reduction at the highest treatment concentration of 50 μg / mL (Fig. 17). Treatment with TM4 reduced apoptosis by 26.3% at the highest treatment concentration of 50 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)] (Fig. 18). Treatment with TM5 significantly reduced apoptosis starting from a treatment concentration of 10 μg / mL compared to the H2O2 treatment group [H2O2(+) / (-)], resulting in a 29.5% reduction in apoptosis at the highest treatment concentration of 50 μg / mL (Fig. 19).

[0155]

[0156] 3.4. Effects on Dexamethasone-Induced Myotube Injury

[0157] Dexamethasone is one of the representative glucocorticoids that causes skeletal muscle degradation upon misuse in clinical practice; based on this, it is widely used to induce myotube atrophy in in vitro systems. To investigate the effects of five test substances on dexamethasone-induced myotube atrophy, cells were cultured after treatment with each test substance, and the diameter of the myotubes was measured. Specifically, C2C12 cells were cultured in myotube differentiation medium for 4 days to induce myotube differentiation, then treated with 5 μM dexamethasone to induce myotube atrophy, and finally treated with the five test substances and cultured for 24 hours. Fluorescent staining was performed using MYH antibodies to observe and quantify the diameter of the myotubes, after which the diameter of the myotubes was observed and quantified. Treatment with 5 μM dexamethasone induced muscle cell atrophy, and a significant decrease in muscle cell diameter was observed compared to the control group [DEX (-) / (-)] that was not treated with dexamethasone.

[0158] Accordingly, after treatment with 5 types of test substances, the diameter increased significantly compared to the DEX treatment group [DEX (+) / (-)].

[0159] Figure 20 shows the results of measuring muscle cell diameter for Dexamethasone-induced muscle atrophy.

[0160] Referring to Figure 20, the diameter was measured and compared to the control group [DEX (-) / (-)] which was not treated with dexamethasone, the diameter of the DEX-treated group [DEX (+) / (-)] was 0.12 to 0.14 μm, indicating a significant decrease in the diameter of the muscle cells. Accordingly, treatment with all five test substances significantly increased the diameter of the muscle cells. At the highest treatment concentration of the five test substances, 50 μg / mL, the diameter of the muscle cells increased by 2.00 times (TM1), 1.92 times (TM2), 2.07 times (TM3), 1.71 times (TM4), and 2.33 times (TM5) compared to the DEX-treated group [DEX (+) / (-)].

[0161] To summarize the above, the complex extract according to the present invention significantly increased the cell viability of C2C12 cells (myoblasts) that were significantly reduced by H2O2 treatment. In addition, the complex extract TM5 increased the diameter of muscle cells reduced by dexamethasone treatment by as much as 2.33 times, which demonstrates the superior protective effect against muscle cell atrophy possessed by the complex extract according to the present invention compared to each individual extract. Therefore, the potential for future development as a functional pharmaceutical material for the treatment and improvement of sarcopenia (atrophy) can be confirmed.

[0162] In this specification, the term “containing as an active ingredient” means that the extract of the present invention contains an amount sufficient to achieve efficacy in preventing and treating muscle loss.

[0163] In this specification, the term “periodontal disease” refers to any disease occurring in periodontal tissues, and the term “prevention” used in this invention refers to any act of suppressing periodontal disease or delaying its onset by administering a pharmaceutical composition according to this invention. Furthermore, the term “treatment” used in this invention refers to any act of improving or beneficially altering the symptoms of periodontal disease by administering a pharmaceutical composition according to this invention. The term “improvement” used in this invention refers to any act of at least reducing parameters related to the treated state, such as the severity of symptoms. In this case, the functional food composition may be used for the prevention or improvement of periodontal disease, either simultaneously with or separately from a therapeutic agent, either before or after the onset of the disease.

[0164] The pharmaceutical composition for the prevention and treatment of periodontal disease of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent.

[0165] Pharmaceutically acceptable carriers in the composition of the present invention are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include 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).

[0166] The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0167] The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion, and response sensitivity, and a physician who is normally skilled can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0168] The pharmaceutical composition for the prevention and treatment of periodontal disease according to the present invention may include other pharmaceutical active ingredients in addition to the elm plant extract as an active ingredient, or may be used mixed with a pharmaceutical composition containing other active ingredients.

[0169] The food or health functional food composition of the present invention may further include food-grade acceptable food additives. Food-grade acceptable food additives that may be used in the present invention include, but are not limited to, sugars such as glucose, fructose, maltose, sucrose, dextrin, and cyclodextrin, natural carbohydrates such as sugar alcohols such as xylitol, sorbitol, and erythritol, natural flavoring agents such as taumatin and stevia extract, synthetic flavoring agents such as saccharin and aspartamic acid, coloring agents, pectic acid or its salt, alginic acid or its salt, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents, etc. The food composition of the present invention may be in a form selected from the group consisting of powder, granules, tablets, capsules, candy, chewing gum, jelly, and beverage. The content of the elm plant extract in the above food composition can be appropriately selected considering the form, flavor, taste, etc. of the food, and, for example, may be in the range of 0.01 to 30% by weight relative to the total weight of the food. It is obvious to a person skilled in the art that the form, composition, and manufacturing method of the food composition according to the present invention can be appropriately selected from the ordinary technology known in the technical field.

[0170] Therefore, the composition containing an extract of a plant of the genus Ulmus according to the present invention as an active ingredient can be utilized as a component of toothpaste or mouthwash for humans and animals, in addition to pharmaceutical compositions or food compositions, and furthermore, can be utilized as animal feed for animals such as companion animals.

[0171] In addition, the extract according to the present invention can also be used as an active ingredient in toothpaste and mouthwash.

[0172] The present invention relates to a pharmaceutical composition and a health functional food for the prevention and treatment of sarcopenia and periodontal disease, comprising alder and elm extracts simultaneously as active ingredients, and is industrially applicable.

Claims

1. A pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease containing alder and elm extracts simultaneously as active ingredients.

2. In Paragraph 1, A pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease, characterized in that the above elm extract contains catechin 7-O-β-D-apiofuranoside.

3. In Paragraph 1, A pharmaceutical composition for the prevention and treatment of sarcopenia and periodontal disease, characterized in that the above alder extract contains oregonin.

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

3.

5. A health functional food for the prevention and improvement of periodontal disease containing alder and elm extracts as active ingredients.

6. In Paragraph 5, A health functional food for the prevention and improvement of periodontal disease, characterized in that the above elm extract contains catechin 7-O-β-D-apiofuranoside.

7. In Paragraph 5, A health functional food for the prevention and improvement of periodontal disease, characterized in that the above alder extract contains oregonin.

8. In Paragraph 5, A health functional food for preventing and improving periodontal disease, characterized in that the weight ratio of the above alder and elm extracts is 3:7 to 7:

3.

9. Feed containing alder and elm extracts simultaneously as active ingredients.

10. Toothpaste containing both alder and elm extracts as active ingredients.

11. A mouthwash containing alder and elm extracts simultaneously as active ingredients.

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