Composition for preventing or treating aging and aging-related diseases comprising sambucus spp. extract or compound isolated therefrom as active ingredient

Abstract

The present invention relates to a composition for preventing or treating aging and aging-related diseases, comprising a Sambucus spp. extract or a compound isolated therefrom as an active ingredient. It has been confirmed that the Sambucus spp. extract or the compound isolated therefrom exhibits effects of suppressing the expression of p16, which is a cellular aging biomarker, and promoting autophagy. Accordingly, the Sambucus spp. extract or the compound isolated therefrom of the present invention is expected to be applicable to the development of therapeutic agents, functional health foods for amelioration, veterinary medicines, and animal feed compositions for aging or aging-related diseases such as idiopathic pulmonary fibrosis, cardiovascular diseases, diabetes, neurodegenerative diseases, sarcopenia, and osteoarthritis-related diseases.

Description

Composition for preventing or treating aging and aging-related diseases, comprising an extract of a plant of the genus Elderberry or a compound isolated therefrom as an active ingredient

[0001] The present invention relates to a composition for preventing or treating aging and aging-related diseases, which contains as an effective ingredient an extract of a tree belonging to the genus Sambucus (Sambucus spp.), such as a Japanese maple, a Japanese maple, a Chinese ash tree, or a compound isolated therefrom.

[0002] Due to the ongoing global aging phenomenon, Korea is projected to enter a super-aged society by 2025, with those aged 65 and older accounting for over 20% of the total population. Consequently, interest in the prevention and treatment of aging and age-related diseases is also increasing. Aging is the gradual decline in biochemical and physiological functions over time. At the cellular level, it refers to the end of cell division. As we age, senescent cells accumulate in the body, which has a profound impact on age-related diseases such as cancer, cardiovascular disease, diabetes, and neurodegenerative diseases, thereby limiting human lifespan.

[0003] As cellular aging has been revealed to be closely related to age-related diseases, it is important to establish appropriate aging biomarkers that can be used to diagnose aging and establish disease treatment goals, as there can be a large difference between an individual's chronological age and biological age depending on genetic factors and environmental factors such as activity level, nutrition, and health status. According to the American Federation of Aging Research (AFAR), among various aging biomarker candidates such as inflammatory biomarkers and interleukins, p16 (p16) is particularly important. INK4A(also known as CDKN2A) is a cyclin-dependent kinase inhibitor (CDKI) that inhibits cell cycle progression and thus can be used as an effective aging biomarker because it is involved in cellular senescence. That is, since the progression of aging in most mammalian species is proportional to the increase in p16 expression, measuring p16 expression is considered a quantitative method for measuring biological age (Muss. et al., 2020).

[0004] Idiopathic pulmonary fibrosis (IPF), a geriatric disease known to occur frequently in the elderly, has been observed to have a particularly increased expression of p16 in lung cells. Idiopathic pulmonary fibrosis is a disease in which lung parenchyma becomes fibrotic due to abnormal tissue healing processes after damage to alveolar epithelial cells for unknown reasons. Symptoms include fibroblast activation, excessive extracellular matrix production, collagen accumulation, and alveolar wall thickening. It is a disease with a very poor prognosis with an average survival time of approximately 2-3 years after diagnosis, the third shortest survival time after pancreatic cancer and lung cancer. The incidence of idiopathic pulmonary fibrosis is 2-30 cases per 100,000 people worldwide, and the number of patients in Korea also more than doubled from 2012 to 2018, reaching 16.9 cases per 100,000 people as of 2021. Currently, FDA-approved drugs nintedanib and pirfenidone are mainly used for the treatment of IPF, but the therapeutic effect is insufficient, so there is an urgent need to develop new, more effective drug candidates, and there is a need for treatment methods or preventive methods that slow the progression of IPF.

[0005] p16 in IPF-derived alveolar epithelial and fibroblast cells INK4A, an increase in senescence markers such as p21CIP1 and senescence-associated beta-galactosidase was observed, indicating that aging is closely related to the initiation of IPF disease (Gulati, S., 2019). It was reported that a mixture of dasatinib and quercetin, which are senolytic inhibitors that selectively remove senescent cells, was effective in alleviating fibrosis by selectively removing senescent alveolar epithelial cells in both a bleomycin-treated damaged cell model that causes pulmonary fibrosis and a irradiated mouse model of lung fibrosis. These findings suggest that senescence of alveolar epithelial cells contributes to the development of pulmonary fibrosis, and that drugs that remove senescent cells or reduce the possibility of senescence can be used for the prevention and treatment of IPF. However, since senolytic inhibitors can also act to some extent on normal cells, they may not affect normal cells but instead target p16 in senescent cells. INK4A There is a need to develop substances that inhibit the expression of p16 in normal cells. INK4A Since it acts as a CDKI that inhibits cell cycle progression, development of compounds with fewer side effects is necessary.

[0006] Meanwhile, autophagy, a mechanism that regenerates energy and removes damaged substances by degrading aging or damaged materials and organelles within cells, plays a crucial role in maintaining normal cellular function. Various studies have reported that autophagy plays a crucial role in preventing aging by regulating age-related factors such as oxidative stress, DNA damage, telomere attrition, and inflammation. Furthermore, the use of autophagy-inducing agents, such as rapamycin, has been demonstrated to have potential effects in the prevention and treatment of aging and age-related diseases. These agents inhibit the transcription of p16, a biomarker of aging, and activate intracellular autophagy, which can be used to prevent or treat idiopathic pulmonary fibrosis (IPF), cardiovascular disease, diabetes, and neurodegenerative diseases. In particular, autophagy has been observed to be involved in the pathogenesis of IPF by regulating apoptosis and senescence in fibroblasts and alveolar epithelial cells. Therefore, modulating autophagy activity is emerging as a novel therapeutic target for pulmonary fibrosis. Conversely, inhibition of autophagy by TGF-β1 is known to induce differentiation of lung fibroblasts into myofibroblasts, inhibit collagen removal, and induce epithelial-mesenchymal transition (EMT). Furthermore, activation of the PI3 / Akt / mTOR signaling pathway and the JAK2 / STAT3 signaling pathway, both of which are involved in the pathogenesis of pulmonary fibrosis, is known to promote the progression of fibrosis by inducing inhibition of autophagy.

[0007] Among the various approaches to developing new drugs, the possibility of discovering new active ingredients from natural products used in traditional medicine is significantly higher than that of experimentally modifying existing drugs. Furthermore, because these active ingredients have been used for a long time, they have the advantage of reducing concerns about drug toxicity. In particular, for the prevention and treatment of idiopathic pulmonary fibrosis, which has a high incidence in the elderly and limited therapeutic options, the safety of therapeutic drugs is particularly important. In this regard, the exploration of natural products offers significant advantages.

[0008] Sambucus williamsii, also known as the elderberry, is a deciduous broadleaf shrub or small tree in the Adoxaceae family, native to Korea, China, and Japan. Since ancient times, the elderberry has been used in Korea as a remedy for muscular and bone sprains, and as a diuretic in folk medicine for kidney disease and dropsy. Furthermore, the leaves, stems, roots, and flowers have been used in traditional medicine to treat various conditions. The leaves, known for their blood circulation-boosting properties, have been used to improve circulation, relieve pain, and treat rheumatism and musculoskeletal pain. The flowers have been used for their diuretic and diaphoretic properties, and the roots have been used to treat gout, phlegm, fever, and jaundice. In the West, several plants in the genus Elder are called elder, and their fruit, elderberry, is one of the herbal dietary supplements frequently used for strengthening the immune system, preventing and treating colds, and maintaining skin health.

[0009] Native plants of the genus Sambucus (Sambucus spp.) in Korea include Sambucus williamsii, Sambucus racemosa H. hara, Sambucus sieboldiana, and Sambucus racemosa f. velutina. Reported chemical components of native elder plants in Korea include lignan glycosides in Sambucus williamsii (Machida et al., 2002), iriodoid glycosides in Sambucus sieboldiana (Suh et al., 2017), lignans, and sterols (Shen., et al., 2023). In addition, physiological activities such as the anti-inflammatory and analgesic action of the elderberry (Cho, SK, et al., 1994) and the antioxidant activity of the elderberry (Chae, JW, et al., 2012) have been reported, and in addition, it is known to have anti-aging, antiviral, antifungal, analgesic, and diuretic effects (Hwang, B., et al., 2010). However, there has been little research at the compound level as a substance for preventing and treating aging and aging-related diseases, and in particular, research on indigenous Korean elderberry plants has been even less conducted.

[0010] As a prior art, Korean Patent No. 0047290 describes the inhibitory effect of a composition containing an extract of the genus Amurense on cathepsin K enzyme activity, and Korean Patent Publication No. 0079546 describes the antioxidant effect of a composition containing an extract of the genus Amurense. However, the therapeutic effect of the extract of the plant of the genus Amurense and the compound isolated therefrom on aging or / and aging-related diseases is not described or implied at all.

[0011] The present invention provides a composition for preventing or treating aging and aging-related diseases, which comprises an extract of a plant of the genus Sambucus (Sambucus spp.) or a compound isolated therefrom as an active ingredient.

[0012] In addition, it is an object of the present invention to provide a compound isolated from an extract of a plant of the genus Atractylodes and a method for isolating the same.

[0013] The present invention provides a composition for preventing or treating aging and age-related diseases, comprising an extract of a plant of the genus Sambucus (Sambucus spp.) or a compound isolated therefrom as an active ingredient, a health functional food composition for improving aging and age-related diseases, an animal drug for preventing or treating aging and age-related diseases, or a composition for animal feed for improving aging and / or age-related diseases.

[0014] The above-mentioned elder plant may be at least one selected from the group consisting of elder (Sambucus. williamsii), Japanese elder (Sambucus racemosa H. hara), horse urine tree (Sambucus sieboldiana), and earthworm tree (Sambucus racemosaf. velutina).

[0015] The above extract of the plant of the genus Elder may be an extract obtained by extracting the plant of the genus Elder with one or more solvents selected from the group consisting of water, C1-C4 lower alcohol, C1-C4 acetic ester, acetone, and methylethylketone.

[0016] As the C1 to C4 lower alcohol, methanol, ethanol, ethanol, propanol, isopropanol, butanol, etc. can be used, and in addition, the C1 to C4 acetic acid ester is classified as a low-toxicity solvent in the “Residual Solvent Standard Guidelines for Pharmaceuticals” of the Ministry of Food and Drug Safety, and methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, etc. can be used. Preferably, it is at least one selected from the group consisting of water and C1 to C4 lower alcohol, more preferably ethanol, and most preferably 70% [v / v] ethanol.

[0017] The above solvent can be added in an amount of 200 to 500 times the weight of the plant of the genus Amurense.

[0018] The above Sambucus spp. plant extract may be an extract obtained by extracting a Sambucus spp. plant with at least one solvent selected from the group consisting of water, C1-C4 lower alcohol, C1-C4 acetic acid ester, acetone, and methyl ethyl ketone.

[0019] In addition, the Sambucus spp. plant extract may be a fraction produced by extracting and concentrating a Sambucus spp. plant with water, a lower alcohol of C1 to C4, or a mixed solvent thereof, and then sequentially fractionating the extract with n-hexane, ethyl acetate, n-butanol, 70% ethanol, and water after adding distilled water. Preferably, it is a hexane, ethyl acetate, or 70% ethanol aqueous solution, and more preferably, an ethyl acetate fraction.

[0020] The above-mentioned extract of the plant of the genus Coptis may include at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the following [chemical formula 1].

[0021] [Chemical Formula 1]

[0022]

[0023] The present invention also comprises a first step of obtaining an extract of a plant of the genus Atractylodes by extracting a plant of the genus Atractylodes using at least one solvent selected from the group consisting of water, a lower alcohol of C1 to C4, an acetic acid ester of C1 to C4, acetone, and methyl ethyl ketone; a second step of fractionating the extract of the plant of the genus Atractylodes of the first step with an organic solvent to secure fractions; A method for separating compounds 1, 4, 5 and 7 of the above chemical formula 1 is provided, comprising: a third step of separating at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5) and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the following chemical formula 1 by applying the fractions of the above two steps to column chromatography;

[0024] Compounds 1, 4, 5 and 7 of chemical formula 1 separated from the extract of the genus Elder can be obtained by fractionating the extract of the genus Elder and other plants by chromatography, and the chromatography is normal phase column chromatography, reverse phase column chromatography, Diaion HP-20 column chromatography, silica gel column chromatography, reverse phase silica gel column chromatography, RP-18 column chromatography, LH-20 column chromatography, preparative reversed-phase high performance chromatography, medium pressure liquid chromatography, high-performance liquid chromatography, It can be selected and used from HPLC, etc.

[0025] Meanwhile, the compound of the present invention can be synthesized according to a method conventional in the art and can be prepared as a pharmaceutically acceptable salt.

[0026] The above-mentioned plant of the genus Sambucus may be at least one species selected from the group consisting of Sambucus williamsii, Sambucus racemosa H. hara, Sambucus sieboldiana, and Sambucus racemosaf. velutina. The above-mentioned plant is at least one species selected from the group consisting of leaves, branches, stems, and roots.

[0027] The above aging may be aging of cells or tissues, and as cells or tissues age, the risk of developing cancer, cardiovascular disease, diabetes, neurodegenerative disorders, etc. increases. In particular, the above aging-related disease may be any one selected from the group consisting of idiopathic pulmonary fibrosis, cardiovascular disease, diabetes, neurodegenerative disease, sarcopenia, and osteoarthritis.

[0028] The above composition may be a pharmaceutical composition for preventing or treating aging and / or aging-related diseases.

[0029] The pharmaceutical composition may comprise an extract of a plant of the genus Atractylodes containing at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 of the above chemical formula 1, or at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 and a pharmaceutically acceptable carrier.

[0030] The above pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients, and diluents that can be included in the above pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one compound selected from the group consisting of compounds 1, 4, 5, and 7 of the above chemical formula 1 of the present invention with an extract of a plant of the genus Elderberry containing at least one compound selected from the group consisting of compounds 1, 4, 5, and 7, or at least one compound selected from the group consisting of compounds 1, 4, 5, and 7, and at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.Suppository bases that can be used include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0031] The dosage of the pharmaceutical composition will vary depending on the age, sex, and body weight of the subject being treated, the specific disease or pathological condition being treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. Determining the dosage based on these factors is within the skill of those skilled in the art, and the dosage is typically in the range of 1 to 1,000 mg / kg / day. A more preferred dosage is 1 to 500 mg / kg / day. Administration may be administered once daily or in multiple divided doses. The above dosage does not limit the scope of the present invention in any way.

[0032] The above pharmaceutical composition can be administered to mammals such as rats, livestock, companion animals, and humans via various routes. All modes of administration are conceivable, including oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebrovascular injection.

[0033] The above pharmaceutical composition can be used to prevent or treat idiopathic pulmonary fibrosis, cardiovascular disease, diabetes, neurodegenerative disease, etc. by inhibiting transcription of p16, an aging biomarker, and activating intracellular autophagy.

[0034] In addition, the composition may be a cosmetic or health functional food composition for improving aging or / and aging-related diseases.

[0035] The above health functional food composition may include an extract of a plant of the genus Atractylodes containing at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 of the above chemical formula 1, or at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 and a food-related acceptable food additive.

[0036] An extract of a plant of the genus Atractylodes containing at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 of the above chemical formula 1 or at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 may be included in an amount of 0.01 to 100 wt% based on the total weight of the health functional food composition of the present invention.

[0037] The health functional food composition of the present invention includes a form such as a tablet, capsule, pill or liquid, and the food to which the extract of a plant of the genus Atractylodes containing at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 of the chemical formula 1 of the present invention or at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 can be added includes, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, etc.

[0038] Another aspect of the present invention provides an animal drug for treating aging and aging-related diseases, comprising an extract of a plant of the genus Elderberry containing at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 of the chemical formula 1 of the present invention or a composition comprising at least one compound selected from the group consisting of compounds 1, 4, 5 and 7.

[0039] Another aspect of the present invention provides an animal feed composition for improving aging and aging-related diseases, comprising an extract of a plant of the genus Elderberry containing at least one compound selected from the group consisting of compounds 1, 4, 5 and 7 of the chemical formula 1 of the present invention or a composition containing at least one compound selected from the group consisting of compounds 1, 4, 5 and 7.

[0040] The above animal feed composition may include a feed additive. The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act.

[0041] The type of the above animal feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant feed such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal feed such as proteins, inorganic substances, oils, minerals, oils, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0042] The present invention also relates to a method for separating compounds 1, 4, 5 and 7 of the above chemical formula 1, comprising: a first step of obtaining an extract of a plant of the genus Elder by extracting the plant of the genus Elder with at least one solvent selected from the group consisting of water, a lower alcohol of C1 to C4, an acetic acid ester of C1 to C4, acetone and methyl ethyl ketone; a second step of fractionating the extract of the plant of the genus Elder of the step 1 with an organic solvent to secure fractions; A manufacturing method characterized by comprising a third step of applying the fractions of the above two steps to column chromatography to separate at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the above chemical formula 1.

[0043] The organic solvent of the above two steps may be a C1 to C4 lower alcohol, ethyl acetate, hexane, acetone, etc. The C1 to C4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, etc. Preferably, it is hexane or ethyl acetate, and more preferably, it is ethyl acetate.

[0044] The above two-step fraction is most preferably an ethyl acetate fraction obtained by sequentially adding hexane and ethyl acetate to the extract of the above first step after suspending it in distilled water.

[0045] The above three-step column chromatography can be selected from normal phase column chromatography, reverse phase column chromatography, Diaion HP-20 column chromatography, silica gel column chromatography, reverse phase silica gel column chromatography, RP-18 column chromatography, LH-20 column chromatography, preparative reverse phase-high performance liquid chromatography, medium pressure liquid chromatography, high performance liquid chromatography, etc.

[0046] The present invention provides a composition for preventing or treating aging and aging-related diseases, a health functional food composition for improving aging and aging-related diseases, an animal drug for preventing or treating aging and aging-related diseases, or a composition for animal feed for improving aging and aging-related diseases, characterized in that it comprises at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the above [chemical formula 1].

[0047] The present invention provides a novel compound 3-O-trans-caffeoylformolic acid (compound 1) of the following [chemical formula 2] isolated from a plant of the genus Amurense.

[0048] [Chemical Formula 2]

[0049]

[0050] The present invention provides a method for preventing or treating age-related diseases.

[0051] The method for preventing or treating an aging-related disease of the present invention comprises administering to a subject in need thereof a composition comprising at least one selected from the group consisting of an extract of a plant of the genus Elder; a fraction thereof; or a compound isolated therefrom.

[0052] The above age-related disease is any one selected from the group consisting of idiopathic pulmonary fibrosis, cardiovascular disease, diabetes, neurodegenerative disease, sarcopenia, and osteoarthritis.

[0053] The above Sambucus spp. plant extract is an extract obtained by extracting a Sambucus spp. plant using at least one solvent selected from the group consisting of water, C1-C4 lower alcohol, C1-C4 acetic acid ester, acetone, and methyl ethyl ketone.

[0054] The above fraction of the plant extract of the genus Elder is a fractional extract produced by extracting and concentrating the plant of the genus Elder with a solvent selected from water and a lower alcohol of C1 to C4, then adding distilled water and sequentially fractionating with n-hexane, ethyl acetate, n-butanol, 70% ethanol and water, and is preferably an ethanol fraction or an ethyl acetate fraction, and more preferably an ethyl acetate fraction.

[0055] The compound isolated from the above extract of the plant of the genus Amurense is at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the above [chemical formula 1].

[0056] The above-mentioned plant of the genus Sambucus is any one selected from the group consisting of Sambucus williamsii, Sambucus racemosa H. hara, Sambucus sieboldiana, and Sambucus racemosaf. velutina.

[0057] The above-mentioned extract of the plant of the genus Elderberry; a fraction thereof; or a compound isolated therefrom can inhibit cell aging or prevent or treat aging-related diseases by inhibiting the expression of p16, a aging-related protein, and increasing autophagy activity.

[0058] The present invention relates to a composition for preventing or treating aging and aging-related diseases, comprising an extract of a plant of the genus Elder or a compound isolated therefrom as an active ingredient, and it was confirmed that the extract of a plant of the genus Elder or a compound isolated therefrom has a transcriptional suppression effect of p16, an aging biomarker, and activation of autophagy.

[0059] Through this, it is expected that the extract of the plant of the genus Amurense of the present invention or a compound isolated therefrom can be used to develop a treatment agent for various geriatric diseases, a health functional food for improvement, an animal drug, and a composition for animal feed.

[0060] Figure 1 is a schematic diagram illustrating the bioactivity-guided isolation process for isolating autophagy-active fractions and compounds from extract fractions of Sambucus williamsii. (Activity was shown in the 70% EtOH extract (TE) and ethyl acetate (SW-EA) of Sambucus williamsii, fraction SW-EA3, and subfractions SW-EA3.4 and SW-EA3.4.3, and green fluorescent protein (GFP)-tagged LC3 spot confirmed in the HEK293A stable cell line.)

[0061] Figure 2 shows the molecular structures of compounds 1 to 7 obtained from the aerial part of Sambucus williamsii according to the present invention.

[0062] Figure 3 is a fluorescence micrograph showing the effect of compounds 1, 4, 5 and 7 isolated from the elderberry according to the present invention on autophagy activity.

[0063] FIG. 4 is a diagram showing the effect of novel compound 1 isolated from the elderberry according to the present invention on increasing autophagy in HEK293A cells infected with mCherry-GFP-LC3 adenovirus (quantification of autophagy flux was performed by measuring the relative fluorescence intensity of mCherry (red) and GFP (green) signals representing autophagosomes and autolysosomes, respectively).

[0064] Figure 5 is a drawing measuring the effect of novel compound 1 isolated from the elderberry according to the present invention on the expression of autophagy-related proteins in MCF-7 cells. After treating cells with compound 1 (5 and 1 μM), chloroquine (20 μM), an autophagy inhibitor, and rapamycin (0.25 μM), an autophagy inducer, (A) the expression levels of LC3-II, LC3-I, and p62 were analyzed by protein immunoblot analysis, (B) the relative expression of LC3-II / I normalized to β-actin, and (C) the relative expression of p62 normalized to β-actin.

[0065] Figure 6 shows a novel compound 1 isolated from the elderberry according to the present invention, p16 INK4a The results of measuring the effect on the inhibition of promoter activity. p16 for 5 hours INK4a A549 cells transfected with the Firefly luciferase reporter pGL3 plasmid encoding the promoter and the internal standard pSV-β-galactosidase plasmid were treated with compound 1 (10, 5, 1 μM) and Navitoclax (0.5 μM) for 24 h, and the relative luciferase activity was normalized to the control.

[0066] Figure 7 is a schematic diagram of a practical plan to confirm the effect of compound 1 (CPD1) in an animal model of pulmonary fibrosis.

[0067] Figure 8 is a graph showing the results of a Western blot confirming the effect of compound 1 on the expression of p16, p65, and LC3B in an animal model of pulmonary fibrosis, and its quantification.

[0068] Figure 9 shows the results of immunohistochemical staining for collagen (Masson's trichrome (MT) staining), p16, α-SMA, β-gal, LC3B, LAMP1, and p62 in lung tissue after treatment with compound 1 or nintedanib (NTD) in an animal model of pulmonary fibrosis.

[0069] Figure 10 is a graph quantifying the staining for collagen, p16, α-SMA, β-gal, LC3B, LAMP1, and p62 in Figure 9.

[0070] Figure 11 is a graph showing the correlation between p16 and a-SMA, p16 and LC3B, and a-SMA and LC3B when treated with compound 1, etc. in an animal model of pulmonary fibrosis.

[0071] Figure 12 shows the results of immunofluorescence staining confirming the effect of compound 1 on the expression of p16, α-SMA, and LC3B in an animal model of pulmonary fibrosis.

[0072] The present inventors used a library of traditional Korean medicinal plant extracts to develop p16 INK4A The compound was isolated by using a method to trace the activity against Sambucus williamsii, p16 INK4A It was confirmed that it exhibits inhibitory and autophagy-regulating activity. Autophagy activity and p16 INK4A The present invention was completed by isolating and confirming a compound exhibiting the above activity through the combined use of an activity tracking method.

[0073] Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The contents introduced herein are provided to sufficiently convey the spirit of the present invention.

[0074] <Example 1. Preparation of extracts of elderberry, fractions, and isolation of compounds using an activity tracking method>

[0075] Example 1-1. Preparation of elderberry extract

[0076] S. williamsii, which grows wild in the Seoul National University Herb Garden located in Ilsan-gu, Goyang-si, Gyeonggi-do, Korea, was used. 4.2 kg of the aerial parts of S. williamsii, including leaves and stems, were crushed, and 4 L of 70% [v / v] ethanol was added. Ultrasonic extraction was performed three times over two days, and the extract was concentrated under reduced pressure to obtain 105.0 g of a 70% ethanol extract.

[0077] Example 1-2. Fraction separation from the extract of the elderberry tree using a bioactivity-guided isolation method.

[0078] The 70% ethanol extract concentrate obtained in the above Example 1-1 was suspended in 1.5 L of distilled water to make a suspension, and sequential solvent fractionation was performed with n-hexane, ethyl acetate, n-butanol, 70% ethanol, and water to obtain four fractions of the elderberry, namely ethyl acetate (SW-EA), n-butanol (SW-Bu), 70% ethanol (SW-TE), and water (SW-W).

[0079] Autophagy plays a crucial role in cellular homeostasis and normal function by degrading intracellular waste and dysfunctional organelles. A decline in autophagy can lead to the accumulation of senescent cells, which can contribute to aging and various age-related diseases. It has been reported that senescent cells exhibit reduced autophagy activity. Therefore, inducing autophagy activation may be an effective way to manage various symptoms associated with reduced autophagy in age-related diseases such as idiopathic pulmonary fibrosis (IPF), including extracellular matrix (ECM) accumulation, epithelial-to-stromal transition (EMT), and collagen depletion failure. Therefore, the effects of each fraction of the elderberry extract on autophagy were investigated.

[0080] First, human fetal kidney cell line HEK-293A, engineered with LC3-GFP autophagy reporter, was seeded at low density (3 × 10 4 Cells were divided into 10 cells / mL and cultured for 24 h. Afterwards, 70% ethanol (SW-TE), ethyl acetate (SW-EA), n-butanol (SW-Bu), and water (SW-W) fractions were treated to a final concentration of 10 μg / mL and cultured for 6 h to induce autophagy. Rapamycin (0.25 μM) was treated as a positive control for autophagy regulation. Cells were then fixed with 4% (w / v) paraformaldehyde and mounted with DAPI mounting solution (ProLong) as a blue DNA stain. ™ Gold Antifade Mountant, Cell Signaling Technology, USA) and LC3 puncta were photographed using a fluorescence microscope (THUNDER Imager 3D Assay microscope, Leica, Germany). The results are shown in Figure 1.

[0081] As shown in Fig. 1, compared to the control group, autophagy activity increased in the 70% ethanol (SW-TE) and ethyl acetate (SW-EA) fractions among the 70% ethanol (SW-TE), ethyl acetate (SW-EA), n-butanol (SW-Bu) and water (SW-W) fractions, and among these, the ethyl acetate (SW-EA) fraction showed particularly high autophagy activity.

[0082] Accordingly, in order to isolate the compound with high autophagy activity of the extract of the above-mentioned elderberry, the ethyl acetate (SW-EA) fraction was fractionated stepwise by chromatography to obtain a fraction with high autophagy activity.

[0083] The extract obtained in Example 1-1 was suspended in 1.5 L of distilled water to make a suspension, and sequential solvent fractionation was performed with n-hexane and ethyl acetate to obtain 36.1 g of an ethyl acetate fraction. Thereafter, the ethyl acetate fraction (36.1 g) was subjected to normal phase column chromatography (particle size: 63-200 ㎛) under the gradient elution condition of n-hexane:ethyl acetate:methanol (5:1:0.1 → 0:1:1 [v:v]), and five fractions were obtained according to the thin-layer chromatography (TLC) profile (SW-EA1 to SW-EA5).

[0084] Among these, 4.5 g of SW-EA3 with high autophagy activity was subjected to reverse-phase column chromatography (particle size: 75 μm) under a concentration gradient elution condition of methanol:distilled water (1:1 → 1:0 [v:v]), thereby obtaining six subfractions (SW-EA3.1 to SW-EA3.6). The effect of each subfraction on autophagy was confirmed, and among these, the F.3.4 subfraction with high autophagy activity was selected.

[0085] The SW-EA3.4 fraction was subjected to Sephadex LH-20 column chromatography under 100% [v / v] methanol isocratic conditions to obtain four fractions (SW-EA3.4.1 to SW-EA3.4.4), and the SW-EA3.4.3 fraction was eluted with a concentration gradient of n-hexane:isopropanol (20:1→4:1 [v:v]) to obtain four fractions (SW-EA3.4.3.1 to SW-EA3.4.3.4), and the effect of these fractions on autophagy was confirmed. The fraction separation process according to the above activity tracking method (bioactivity-guided isolation) is shown in Fig. 1.

[0086] Example 1-3. Isolation of compounds from extracts of the elderberry tree using a bioactivity-guided isolation method.

[0087] The SW-EA3.4.3.2 fraction obtained from the SW-EA3.4.3 fraction above was purified by HPLC (column: Optima Pak C) with a gradient elution of methanol: distilled water (87:23→90:10 [v:v]) at a flow rate of 2 ml / min. 18 , 10×250 mm) was performed to separate compounds 6 (12.0 mg) and compound 7 (8.2 mg).

[0088] The SW-EA3.4.3.4 fraction was purified by HPLC (column: Optima Pak C) with isocratic elution of methanol: distilled water (85:25 [v:v]) at a flow rate of 2 ml / min. 18 , 10×250 mm) was performed to isolate compound 4 (10.6 mg).

[0089] The SW-EA3.4.3 fraction was eluted with methanol: distilled water (85:25 [v:v]) isocratic elution, 2 ㎖ / flow rate, HPLC (column: Optima Pak C) 18 , 10×250 mm) was performed to isolate compound 1 (8.9 mg).

[0090] The SW-EA.3.5 fraction was subjected to Sephadex LH-20 column chromatography under 100% [v / v] ethanol isocratic conditions to obtain two fractions (SW-EA.3.5.1 to SW-EA.3.5.2), and the SW-EA3.5.2 fraction was subjected to normal phase column chromatography (particle size: 63-200 μm) under gradient elution conditions of n-hexane:ethyl acetate (7:3→ 5:5 [v:v]) to isolate compound 5 (21 mg).

[0091] The SW-EA3.6 fraction was subjected to normal phase column chromatography (particle size: 63-200 μm) with a gradient elution condition of n-hexane:ethyl acetate (2:1→1:1 [v:v]) to obtain three fractions (SW-EA3.6.2 to SW-EA3.6.4), and then subjected to HPLC (column: Optima Pak C) with a gradient elution condition of methanol:distilled water (85:25→90:10 [v:v]) at a flow rate of 2 ml / min. 18 , 10×250 mm) was performed to separate compound 3 (7.5 mg) and compound 2 (6.0 mg).

[0092] <Example 2. Confirmation of the physicochemical structure of the compound>

[0093] Example 2-1. Compound isolated from extract of elderberry

[0094] The molecular structures of compounds 1 to 7 separated in Example 1 were determined by nuclear magnetic resonance (NMR) method, and the results are shown in Fig. 2.

[0095] 3-O-trans-caffeoylformolic acid (compound 1)

[0096] 3-O-trans-caffeoylpomolic acid;

[0097] white solid;

[0098] [α] +131°(c 0.50, methanol);

[0099] IR (KBr) Vmax: 3566, 2924, 2855, 1686, 1603, 1512, 1454, 1167,

[0100] 935 cm -1 ;

[0101] 1 H NMR and 13 C NMR data are shown in Tables 1 and 2 below;

[0102] HRESIMSm / z633.3812 [MH] - (calcd for C 39 H 54 O7, 633.3791).

[0103] 3-O-trans-caffeoyloleanolic acid (compound 2)

[0104] 3-O-trans-caffeoyloleanolic acid;

[0105] white solid;

[0106] [α] +77.5°(c 0.50, methanol);

[0107] IR (KBr) Vmax: 3325, 2946, 2877, 1698, 1604, 1518, 1449, 1365,

[0108] 1271, 1184, 1119, 1013, 977, 856, 813 cm -1 ;

[0109] HRESIMS m / z 617.3848 [MH] - ,(calcd for C 38 H 53 O 6, 617.3829).

[0110] ursolic acid (compound 3)

[0111] Ursolic acid;

[0112] white crystals;

[0113] [α] +62.7° (c 0.5, CHCl3);

[0114] IR (KBr) Vmax: 3389, 2965, 2927, 2868, 1689, 1456, 1383, 1314, 1278, 1186, 1089, 1035, 995, 827, 770 cm -1 ;

[0115] HRESIMSm / z455.3531 [MH] - (calcd for C 30 H 47 O4455.3526).

[0116] Formolic acid (compound 4)

[0117] Pomolic acid;

[0118] white crystals;

[0119] [α] +17.8°(c 0.5, CHCl3);

[0120] IR (KBr) Vmax: 3431, 1691, 1637, 1046, 1030 cm -1 ;

[0121] 1 H NMR and 13 C NMR data are shown in Tables 1 and 2 below;

[0122] HRESIMSm / z471.3482 [MH] - (calcd for C 30 H 47 O4471.3474).

[0123] Oleanolic acid (compound 5)

[0124] Oleanolic acid;

[0125] white crystals;

[0126] [α] +83°(c 0.5, CHCl3);

[0127] IR (KBr) Vmax: 3435, 3424, 2850, 2859, 1694, 1452, 1387, 1378, 1347, 1331, 1322, 1137, 1124, 1086, 1072 cm -1 ;

[0128] 1 H NMR and 13 C NMR data are shown in Tables 1 and 2 below;

[0129] HRESIMSm / z455.3507 [MH] - (calcd for C 30 H 47 O3455.3525).

[0130] Formonic acid (compound 6)

[0131] Pomonic acid;

[0132] white crystals;

[0133] [α] +50°(c 0.5, CHCl3);

[0134] IR (KBr) Vmax: 3575, 2969, 2935, 2877, 1690, 1460, 1383, 1269,

[0135] 1234, 1159, 1110, 1036, 938, 866, 769 cm -1 ;

[0136] HRESIMSm / z471.3480 [MH] - (calcd for C 30 H 47 O4471.3486).

[0137] 2-Hydroxy-3-oxoolean-12-en-28-oic acid (Compound 7)

[0138] 2-Hydroxy-3-oxoolean-12-en-28-oic acid;

[0139] white crystals;

[0140] [α] +25°(c 0.50, CHCl3);

[0141] IR (KBr) Vmax : 3435, 2850, 2859, 1694, 1452, 1387, 1347, 1322,

[0142] 1137, 1124, 1086, 1072 ㎝ -1 ;

[0143] 1H-NMR 및13C NMR 데이터는 하기 표 1 및 표 2 참조;

[0144] HRESIMS 469.3320 [M-H] - (calcd for C 30 H 45 O4469.3318).

[0145] 1H-NMR Spectroscopic Data (MeOD-d4, 400MHz)No.화합물 1화합물 4화합물 5화합물 711.73, m1.64, m1.65, m1.00, m1.62, m0.98, m2.30, m1.17, m22.00, m1.69, m1.73, m1.57, m4.58(dd, 12.7, 6.4)34.57(dd, 11.5, 4.5)3.15(dd, 11.2, 4.8)3.15(dd, 11.2, 4.8)-4----50.92, s0.76, m0.76, m1.18, m61.59, m1.49, m1.42, m1.55, m1.52, m1.42, m1.64, m1.54, m71.60, m1.35, m1.31, m1.56, m1.52, m1.36, m1.55, m8----91.75, m1.69, m1.58, m1.70, m10----111.69, m1.36, s1.97, m1.90, m2.06, m1.96, m125.29, m5.28 (t, 3.4)5.24 (t, 3.7)5.26 (t, 3.6)13----14----151.81, m1.02, m1.80(td, 13.5, 4.7)1.02, m1.76, m1.05, m1.80, m1.10, m162.59(td, 13.1, 4.4)1.54, m2.57(td, 13.2, 4.5)1.51, m2.02(td, 13.7, 3.9)1.58, m2.03, m1.61, m17----182.51, s2.50, s2.85(dd, 13.7, 3.5)2.86(dd, 13.9, 4.3)19--1.70, m1.11, m1.69, m1.14, m200.93 (d, 6.9)1.35, m--211.75, m1.24, m1.24, m1.20, m1.39, m1.22, m221.69, m1.11, m1.77, m1.72, m1.76, m1.56, m230.92, s0.98, s0.97, s1.12, s240.98, s0.78, s0.78, s1.11, s251.01, s0.95, s0.95, s1.30, s260.82, s0.80, s0.82, s0.88, s271.36, s1.33, s1.16, s1.16, s28----291.20, s1.19, s0.91, s0.91, s300.94, m0.93(d, 6.7)0.94, s0.94, s1'----2'7.03, m---3'----4'----5'6.77, m---6'6.94(dd, 8.3, 2.1)---7'7.52(d, 15.8)---8'6.25(d, 15.8)---9'----.

[0146] 13 C-NMR Spectroscopic Data (MeOD-d4, 100MHz) No. Compound 1 a Compound 4 Compound 5 Compound 7 139.0 39.8 39.85 0.5 224.7 27.9 27.9 40.5 70.4 38 2.4 79.8 79.72 17.04 39.0 39.8 39.8 49.35 56.8 56.7 56.7 58.8 6 19.5 19.6 19.5 20.37 34.1 34.2 34.0 33.8 8 2.4 4 1.0 40.5 40.69 39.0 48.6 49.1 48.9 10 56.8 38.1 38.2 38.9 11 24.7 24.724.524.712129.3129.5123.6123.213140.0140.0145.2145.51442.642.642.943.01529.629.628.828.81626.626.624.024.01749.048.647.647.61855.155.142.742.71973.6 73.647.247.22043.143.131.631.62127.327.334.934.92239.439.033.833.72328.728.728.725.42417.416.316.322.12515.915.915.916.42617.517.517.717.92724.824.826.4 26.428182.3182.3181.9181.82927.127.133.633.53016.616.624.024.01'127.7---2'115.1---3'146.8---4'149.6---5'116.5---6'122.9---7'146.7---8'115.6---9'169.2---

[0147] Among the above compounds, 3-O-trans-caffeoylpomolic acid (compound 1) was a novel compound isolated and identified from an extract of the elderberry tree, which had not been reported before.

[0148] Example 2-2. Confirmation of autophagy-inducing activity of compounds isolated from elderberry extract.

[0149] Compounds 1 to 7 isolated in Example 2-1 and the positive control rapamycin were tested for autophagy induction activity in the same manner as in Example 1-2. Human fetal kidney cell line HEK-293A, engineered with an LC3-GFP autophagy reporter, was seeded at a low density (3 × 10) on round coverslips in a 24-well plate. 4 cells / mL) and cultured for 24 hours. After that, compounds 1 to 7 isolated in Example 2-1 were treated to a final concentration of 10 μM and cultured for 6 hours to induce autophagy. Rapamycin (0.25 μM) was treated as a positive control for autophagy regulation. After that, cells were fixed with 4% (w / v) paraformaldehyde and mounted with DAPI mounting solution (ProLong), a blue DNA stain. ™ The cells were mounted on a gold antifade mountant (Cell Signaling Technology, USA) and stained, and LC3 puncta were observed using a fluorescence microscope (THUNDER Imager 3D Assay microscope, Leica, Germany). The autophagy-inducing effects of compounds 1, 4, 5, and 7 were remarkable. This is shown in Fig. 3.

[0150] <Example 3. Confirmation of the anti-aging effect of a compound isolated from the extract of the elderberry>

[0151] Example 3-1. Measurement of autophagy activation using mCherry-GFP-LC3 adenovirus vector

[0152] Using the LC3-GFP autophagy reporter used in Example 2-2 above, autophagy activation cannot be directly observed; only the presence or absence of autophagy activity can be observed. Therefore, additional experiments were conducted to confirm autophagy activation.

[0153] GFP-LC3 is a suitable marker for detecting autophagosomes, but it is unstable at acidic pH, making it difficult to detect during the autophagy process and when lysosomes fuse with autophagosomes to form autolysosomes with a pKa of 6.0. On the other hand, m-Cherry protein has considerable stability under acidic conditions, making it a suitable marker for detecting autolysosomes, which are essential for observing autophagy activation. In the dual fluorescently tagged LC3 system, the red signal of mCherry and the green signal of GFP-LC3 exist simultaneously at neutral pH, resulting in a final signal that is yellow or orange. However, mCherry, which is not degraded under acidic conditions, only detects a red signal. Therefore, when autophagy is activated, the ratio of autolysosomes to autophagosomes, represented by the ratio of yellow to red signals, increases, allowing us to directly measure autophagy activation within cells.

[0154] To detect the red fluorescent signal associated with the formation of autolysosomes during the autophagy phase, HEK-293A cells were cultured at high density (10 × 10 4Cells were seeded onto round coverslips in 24-well plates at a density of 100 cells / mL and cultured for 24 hours. After infection with mCherry-GFP-LC3 adenovirus vector for 1 hour, the cells were allowed to proliferate for 48 hours. After treatment with 0.25 μM rapamycin as a positive control for inducing autophagy, 20 μM chloroquine as a positive control for inhibiting autophagy, or novel compound 1 derived from the extract of elderberry according to the present invention at final concentrations of 1, 5, and 10 μM, the cells were cultured for 24 hours. Afterwards, cells were fixed with 4% (w / v) paraformaldehyde (Bioworld), treated with DAPI mounting solution (Cell Signaling Technology), a blue DNA stain, and photographed using a fluorescence microscope (THUNDER Imager 3D Assay microscope, Leica, Germany). The relative fluorescence intensities of mCherry (red) and GFP (green), which represent autophagosomes and autolysosomes, respectively, were measured to quantify the degree of autophagy activation, which was then standardized to the control group (Control), and the results are shown in Fig. 4.

[0155] As shown in Figure 4, compound 1 showed a marked increase in mCherry-LC3 punctate formation in a concentration-dependent manner, which resulted in an increase in red fluorescence signal in the merged image. This suggests that compound 1 can induce autophagy activity in a concentration-dependent manner.

[0156] Example 3-2. Measurement of autophagy activation using Western blot

[0157] LC3 and p62 (sequestosome 1) are used as markers for autophagy flux, indicating autophagy activation, because their transcription increases with autophagy induction. LC3 is converted from LC3-I to LC3-II by binding to phosphatidylethanolamine (PE) along with the formation of autophagosomes. Therefore, autophagy activity can be determined through the ratio of LC3-II / LC3-I. Furthermore, upregulation of p62 transcription has been studied as a mechanism by which it induces autophagy through inhibition of the ubiquitin-proteasome system (UPS), thereby contributing to the degradation of ubiquitinated transporters through autophagy (WJ Liu et al., 2016). On the other hand, p62 protein itself is known to be used as a substrate for autolysosomes, and its amount is known to decrease as autophagy becomes more active. Accordingly, the autophagy activation effect of compound 1 was confirmed by confirming the expression of LC3 and p62 at the mRNA and protein levels.

[0158] MCF-7 anticancer drug-resistant cells were treated with compound 1, chloroquinone, or rapamycin in each well and cultured. Compound 1 was treated at 1 and 5 μM, respectively, chloroquinone, used as an autophagy inhibitor, was treated at 20 μM, and rapamycin was treated at 250 nM. Protein levels were confirmed by Western blot analysis (Fig. 5A), and the expression of LC3-II / LC3-I and p62 was normalized to β-actin and shown in Fig. 5B and Fig. 5C.

[0159] As shown in Figure 5B, compound 1 significantly increased the expression of LC3-II / LC3-I in a pattern similar to rapamycin, a known autophagy-activating compound, while decreasing the expression of p62 in a concentration-dependent manner. Furthermore, compared to chloroquine, an autophagy inhibitor that blocks lysosomal fusion, it exhibited a higher LC3-II / LC3-I ratio, but it was confirmed that p62 degradation did not occur, inhibiting autolysosome formation.

[0160] Example 3-3. Measurement of inhibition ability of p16 expression increased in senescent cells.

[0161] Aging is a progressive process of change in cells and tissues and is considered a major risk factor for many diseases, including cancer, cardiovascular disease, diabetes, and neurodegenerative disorders. Among candidate biomarkers of aging, such as inflammatory markers and interleukins, the American Federation on Aging Research (AFAR) has identified p16 as a key feature associated with cellular aging. p16, a cyclin-dependent kinase inhibitor, is an effective biomarker of aging, and because p16 expression significantly increases with age in most mammalian species, measuring p16 expression is considered a quantitative method for assessing biological age (Muss, HB, et al., 2020).

[0162] To measure the activity of p16, experiments were performed using the A549 cell line, which is known to lack p16. A549 lung cancer cells were seeded in 96-well plates and cultured for 24 hours in DMEM medium containing 10% FBS. The cells were washed with cold PBS (phosphate buffer saline), treated with the plasmid mixture and Lipofectamine 2000, and cultured for 3 hours in OptiMEM medium. At this time, the plasmid mixture was human p16. INK4a pGL3_p 16 encoding the promoter INK4a plasmid 0.07 μg of the plasmid was prepared to contain firefly luciferase and 0.03 μg of RSV-β-galactosidase as an internal standard. Afterwards, the medium was replaced with RPMI 1640 medium containing 10% FBS and cultured overnight. The novel compound 1 isolated in Example 1-2 or navitoclax used as a positive control was treated to each well and cultured for 12 hours, and then the cells were lysed by treating with lysis buffer. At this time, the novel compound 1 of Example 1-2 was treated to be 1, 5, and 10 μM, respectively, and navitoclax was treated to be 0.5 μM. Luciferase activity was measured using a firefly luciferase assay kit (Promega, USA) and a luminescence plate reader, and normalized to β-galactosidase activity. The results are shown in Fig. 6.

[0163] As shown in Figure 6, p16 in A549 lung cancer cells INK4a As a result of measuring the level of luciferase expression by promoter activity, it was confirmed that novel compound 1, like the positive control navitoclax, concentration-dependently reduces p16 at the promoter activity level. Through this, it can be seen that novel compound 1 isolated from the elder extract of the present invention is effective in preventing and treating aging and aging-related diseases by suppressing the expression of p16, an aging marker.

[0164] <Example 4. Comparison of relative chemical compound contents of heterologous plants in the genus Elderberry>

[0165] Example 4-1. Securing plants of the genus Sambucus spp.

[0166] In order to confirm whether the compounds isolated from the elderberry (S. williamsii) of the above examples 1-2 are also contained in other plants of the genus Elderberry, which are of the same genus, plants of the genus Elderberry native to Korea were used.

[0167] As shown in Table 3 below, the elderberry (S. williamsii) was collected from the Seoul National University Medicinal Herb Garden located in Ilsan-gu, Goyang-si, Gyeonggi-do, Korea, and the Japanese maple (Sambucus racemosa H. hara), the horse urine tree (Sambucus sieboldiana), and the earthworm tree (Sambucus racemosaf. velutina) were collected from the Southern Academic Forest Estimation Experiment Station of the College of Agriculture and Life Sciences, Seoul National University.

[0168] Species Scientific name Part 1 Part 2 Part 3 Sambucus racemosa H. hara Roots and leaves - Elder tree Sambucus williamsii Roots and leaves Stem Horse urine tree Sambucus sieboldiana Roots and leaves Stem Earthworm tree Sambucus racemosa f. velutina Roots and leaves -

[0169] Example 4-2. Comparative analysis of relative compound contents using UPLC-DAD analysis.

[0170] For UPLC-DAD (ultra-performance liquid chromatography method with diode array detection) analysis, the plants of the elderberry, Chinese angelica tree, horse urn tree, and earthworm tree were extracted with a 70% ethanol solution and concentrated in the same manner as in Example 1. The extracts of each plant of the genus Elderberry were suspended in distilled water and fractionated using ethyl acetate. The fractionated ethyl acetate fractions were dried to a concentration of 2 mg / ml and subjected to UPLC-DAD analysis.

[0171] For UPLC-DAD analysis, UPLC was performed on a Thermo Scientific UltiMate 3000 UHPLC (Thermo Fisher Scientific Inc, USA), the column was OptimaPak-C18 (4.6 x 250 mm), and the detector (diode array detector; DAD) was a Thermo Scientific Dionex UltiMate 3000 Rapid Separation DAD (Thermo Fisher Scientific Inc, USA). The analytical conditions were as follows: the mobile phase of liquid chromatography was distilled water (mobile phase A) containing 0.1% [v / v] formic acid and acetonitrile (mobile phase B), flowing at a flow rate of 0.8 mL / min. The analysis was performed at a column temperature of 40 °C and a sample temperature of 25 °C. 10 μL of each sample was injected at a concentration of 2 mg / mL. After saturation with 50% mobile phase B for 10 minutes before analysis, the solution was flowed with a 50-100% mobile phase B concentration gradient for 0-40 minutes, and the compound peak was detected at a wavelength of 330 nm.

[0172] The presence or absence of substances included in the ethyl acetate fraction of each sample was compared through the retention time and UV-visible light absorption pattern of the compounds separated in the above Examples 1-2, and the relative contents were compared through comparison of peak intensities in the chromatogram, and the results are shown in Table 4 below.

[0173] Abbreviations for each part were indicated as leaf (L), stem (S), and root (R). The maximum content of each compound was confirmed in all samples, and the content of each part was standardized as a relative value by setting this to 100. If a compound was not identified, it was indicated as “-”.

[0174] Elderberry Plant Compounds 1457 S. racemosa H.hara R-96.6 1.0 13.2 L32.0 14.16.6 10.4 S. williamsii R7.9 88.7 1.886.1 L70.6-27.0 S11.5 76.2 49.5 17.1 S. sieboldiana R100.0 100.01.1100 L24.8 28.6 10.112.4 S. racemosa f. velutina R5 1.7 89.9 100.048.4 L44.5-22.4 S4.5 77.338.0- * Highest content was normalized to 100.

[0175] As shown in Table 4, the contents of compounds 1, 4, 5, and 7 of the present invention present in the ethyl acetate fractions of each part of the plants of the genus Elder obtained in Example 3-1 were compared, and it was confirmed that the compounds and contents present differed depending on the species and plant parts of the plants of the genus Elder, but most of them were present in the plants of the genus Elder.

[0176] <Example 5. Effect of Compound 1 in an animal model of pulmonary fibrosis>

[0177] Example 5-1. Creation and Experiment of an Animal Model of Pulmonary Fibrosis

[0178] To create a pulmonary fibrosis model, 10-week-old mice (C57BL6 / J males, 26 g, control and comparison groups) were administered 30 ㎕ of bleomycin (2.0 mg / kg) dissolved in saline (0%) into the airways. Compound 1 was administered orally (IP) once daily starting the day after bleomycin administration to induce fibrosis. For the compound 1 solvent, DMAc (saline (0.9%) N,N-Dimethylacetamide, Tween 80 in a 1:1:8 ratio) was used due to its low side effects and high yield. Table 5 shows the number of animals used in the experiment for each group, and Fig. 7 shows a schematic of the experimental schedule. Control: normal group; compound 1 (20 mg / kg) alone treatment group; BLM: bleomycin alone treatment group; BLM+CPD1(10); Bleomycin and compound 1 (10 mg / kg) combination treatment group; BLM+CPD1(20): Bleomycin and compound 1 (20 mg / kg) combination treatment group; BLM+NTD(60): Bleomycin and the positive control group, nintedanib (60 mg / kg) combination treatment group;

[0179] Control group Experimental group Control CPD1 (20) NTD (60) Bleomycin BLM + CPD1 (10) BLM + CPD1 (20) BLM + NTD (60) Number of animals 66613131313

[0180] Compound 1 was administered repeatedly daily after bleomycin administration. On the 21st day, the mice were sacrificed, and lung tissue was extracted and used for further experiments. (A) is a schematic diagram of the experimental schedule.

[0181] Example 5-2. Effect of compound 1 on p16, p65, and LC3B expression in an animal model of pulmonary fibrosis.

[0182] The effect of the compound 1 of the present invention confirmed above on the inhibition of bleomycin-induced senescence and autophagy flux was confirmed. In order to perform Western blotting, the lung tissue obtained in Example 5-1 was placed in Protein lysis buffer (100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM EDTA (pH 8.0), 0.5% NP-40, 0.5% Sodium deoxycholate, 50 mM β-glycerophosphate, 0.1 mM Na3VO4, 50 mM NaF) and ground using a homogenizer. Afterwards, centrifugation was performed, and only the supernatant was taken, and 0, 2, 4, 6, and 8 μL of 1% BSA solution was added as a standard using the Bradford assay, and 2 μL of the cell supernatant was mixed. After dispensing 200 μL each into a 96-well plate, the absorbance was measured and the same amount of protein was quantified to prepare samples. The samples were boiled in water for 5 minutes, and the same amount of protein was analyzed using SDS-PAGE. The expression levels of p16, p62, LC3B-I / II, and β-actin were confirmed, and the results are shown in Figure 8. The primary antibodies used for Western blotting were p16 (Invitrogen, PA5-20379), p62 (Santacruz, sc-48402), LC3B (Cell signaling, CST-2775), and β-actin (Santacruz, sc-47778). The secondary antibodies used were Goat anti rabbit and Goat anti mouse (Gendepot, #SA002-500 and #SA001-500).

[0183] As shown in Figure 8, the bleomycin alone group significantly increased the expression of p16, p62, LC3B, and β-actin, and when compound 1 according to the present invention was used in combination, the expression of the above proteins was suppressed in a concentration-dependent manner.

[0184] Example 5-3. Histological examination of an animal model of pulmonary fibrosis

[0185] The mouse lung tissues from each group obtained in Example 5-1 above were subjected to Masson's trichrome staining, immunohistochemical analysis, and immunofluorescence staining. The obtained tissues were fixed in a 10% formalin solution, solidified in a paraffin solution, and then sectioned at 4 μm, attached to slides, and deparaffinized in a xylene solution. Afterwards, Masson's trichrome (MT) staining was performed according to the manufacturer's manual using Masson's Trichrome Stain Kit (Polyscience, Inc.) to observe collagen, and immunohistochemical staining was performed using p16 (Santacruz, sc-1661), β-gal (Santacruz, sc-377257), α-SMA (Abcam, ab5694), LC3B (Novus Biologicals, NB100-2220), LAMP1 (Santacruz, sc-20011), and p62 (Santacruz, sc-48402) antibodies, and the results are shown in Fig. 9. In addition, the densities of collagen, p16, α-SMA, β-gal, LC3B, LAMP1, and p62 were analyzed using ImageJ software (NIH, Bethesda, MD, USA), and the results are shown in Fig. 10.

[0186] As shown in Figures 9 and 10, it was confirmed that treatment with compound 1 (20 mg / kg) reduced collagen deposition by up to 52% in bleomycin-induced fibrosis mice. In addition, immunohistochemical staining using p16, β-gal, α-SMA, LC3B, LAMP1, and p62 antibodies showed that BLM treatment significantly increased protein expression of p16, α-SMA, β-gal, LC3B, LAMP1, and p62 in lung tissue, and treatment with compound 1 according to the present invention inhibited the expression of these proteins in lung tissue.

[0187] Through this, it was confirmed that compound 1 according to the present invention suppressed increased collagen deposition and aging and autophagy marker expression due to BLM treatment, thereby effectively suppressing pulmonary fibrosis and facilitating the progression of autophagy flux.

[0188] Based on the above immunohistochemical analysis, the correlation between p16 and a-SMA, p16 and LC3B, and a-SMA and LC3B was confirmed, and the results are shown in Figure 11. As seen in Figure 11, the bleomycin alone group showed a tendency to converge toward the upper right, and the group treated with bleomycin plus compound 1 or nintedanib showed a very high correlation, converging toward the lower left.

[0189] In addition, the results of immunofluorescence analysis to determine whether p16 and LC3B are co-expressed in fibrotic tissue using p16 (Invitrogen, PA5-20379), α-SMA (Novus Biologicals, NB300-978), and LC3B (Novus Biologicals, NB100-2220) antibodies are shown in Fig. 12. As shown in Fig. 12, the bleomycin alone group showed increased expression of p16, α-SMA, and LC3B compared to the control group, and showed a tendency to decrease when compound 1 was co-treated. And the tissue parts that co-express these three proteins also showed a similar tendency.

[0190]

[0191] <Example 1. Pharmaceutical preparation>

[0192] Preparation Example 1-1. Preparation of tablets

[0193] 20 g of the extract of the elderberry or 7200 mg of the compound of Example 1-1 above were mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silica, respectively. A 10% gelatin solution was added to this mixture, which was then ground and passed through a 14-mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc, and 5 g of magnesium stearate were added thereto, resulting in a mixture that was made into tablets.

[0194] Preparation Example 1-2. Preparation of Injectable Solution

[0195] 0.3 g of the compound isolated in Example 1-2, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20°C for 30 minutes.

[0196] <Example 2. Manufacturing of health functional foods>

[0197] Preparation Example 2-1. Manufacturing of health functional foods

[0198] 20 g of the elderberry extract of Example 1-1 above, an appropriate amount of vitamin mixture, vitamin A acetate 70 μg, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 μg, vitamin C 10 mg, biotin 10 μg, nicotinamide 1.7 mg, folic acid 50 μg, calcium pantothenate 0.5 mg, an appropriate amount of mineral mixture, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, monobasic potassium phosphate 15 mg, dibasic calcium phosphate 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, and magnesium chloride 24.8 mg were mixed to manufacture granules, but various types are available depending on the intended use. It can be manufactured by modifying the formulation. In addition, the composition ratio of the above vitamin and mineral mixture may be modified arbitrarily, and the above ingredients can be mixed and manufactured according to a conventional health functional food manufacturing method.

[0199] Preparation Example 2-2. Manufacturing of health functional beverages

[0200] 1 g of the extract of the elderberry of Example 1-1, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water were mixed and stirred, heated, filtered, sterilized, and refrigerated according to a conventional beverage manufacturing method to produce a beverage.

Claims

A pharmaceutical composition for preventing or treating aging and aging-related diseases, characterized in that it comprises an extract of a plant of the genus Sambucus (Sambucus spp.), A pharmaceutical composition for preventing or treating aging and aging-related diseases, characterized in that the above-mentioned Sambucus spp. plant extract is an extract obtained by extracting a Sambucus spp. plant with at least one solvent selected from the group consisting of water, C1-C4 lower alcohol, C1-C4 acetic acid ester, acetone, and methyl ethyl ketone. In the first paragraph, A pharmaceutical composition for preventing or treating aging and aging-related diseases, characterized in that the above Sambucus spp. plant extract is a fraction produced by extracting and concentrating a Sambucus spp. plant with water, a lower alcohol of C1 to C4, or a mixed solvent thereof, adding distilled water, and then sequentially fractionating with n-hexane, ethyl acetate, n-butanol, 70% ethanol, and water. In the first paragraph, A pharmaceutical composition for preventing or treating aging and aging-related diseases, characterized in that the above-mentioned elderberry plant extract contains as an active ingredient at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the following [chemical formula 1]. [Chemical Formula 1] In the first paragraph, A pharmaceutical composition for preventing or treating aging and aging-related diseases, characterized in that the above-mentioned elder plant is at least one selected from the group consisting of elder (Sambucus. williamsii), Japanese yam (Sambucus racemosa H. hara), horse urine tree (Sambucus sieboldiana), and earthworm tree (Sambucus racemosaf. velutina). A pharmaceutical composition for preventing or treating aging and aging-related diseases, characterized in that it comprises at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the following [chemical formula 1]. [Chemical Formula 1] In any one of the first to fifth paragraphs, A pharmaceutical composition for preventing or treating aging and aging-related diseases, characterized in that the aging-related disease is any one selected from the group consisting of idiopathic pulmonary fibrosis, cardiovascular disease, diabetes, neurodegenerative disease, sarcopenia, and osteoarthritis. In paragraph 6, The pharmaceutical composition above is a pharmaceutical composition for preventing or treating aging and aging-related diseases, characterized by a p16 transcription inhibition effect, which is an aging biomarker, and an intracellular autophagy activation effect. A health functional food composition for improving aging and aging-related diseases, characterized in that it comprises an extract of a plant of the genus Sambucus (Sambucus spp.), A health functional food composition for improving aging and aging-related diseases, characterized in that the above Sambucus spp. plant extract is an extract obtained by extracting the Sambucus spp. plant with at least one solvent selected from the group consisting of water, C1-C4 lower alcohol, C1-C4 acetic acid ester, acetone, and methyl ethyl ketone. In paragraph 8, The above-mentioned Sambucus spp. plant extract is a health functional food composition for improving aging and aging-related diseases, characterized in that it is a fraction extract produced by extracting and concentrating a Sambucus spp. plant with water, a lower alcohol of C1 to C4, or a mixed solvent thereof, adding distilled water, and then sequentially fractionating with n-hexane, ethyl acetate, n-butanol, 70% ethanol, and water. In paragraph 8, A health functional food composition for improving aging and aging-related diseases, characterized in that the above-mentioned elderberry plant extract contains as an active ingredient at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the following [chemical formula 1]. [Chemical Formula 1] In paragraph 8, A health functional food composition for improving aging and aging-related diseases, characterized in that the above-mentioned elder plant is at least one selected from the group consisting of elder (Sambucus. williamsii), Japanese yam (Sambucus racemosa H. hara), horse urine tree (Sambucus sieboldiana), and earthworm tree (Sambucus racemosaf. velutina). In any one of the 8th to 11th clauses, A health functional food composition for improving aging and aging-related diseases, characterized in that the aging-related disease is any one selected from the group consisting of idiopathic pulmonary fibrosis, idiopathic pulmonary fibrosis, cardiovascular disease, diabetes, neurodegenerative disease, sarcopenia, and osteoarthritis. In an animal medicine for preventing or treating aging and aging-related diseases, characterized in that it contains an extract of a plant of the genus Sambucus spp., The above-mentioned Sambucus spp. plant extract is an animal drug for preventing or treating aging and aging-related diseases, characterized in that it is an extract obtained by extracting a Sambucus spp. plant with at least one solvent selected from the group consisting of water, C1-C4 lower alcohol, C1-C4 acetic acid ester, acetone, and methyl ethyl ketone. In Article 13, The above Sambucus spp. plant extract is characterized by being a fraction produced by extracting and concentrating a Sambucus spp. plant with water, a lower alcohol of C1 to C4, or a mixed solvent thereof, adding distilled water, and then sequentially fractionating with n-hexane, ethyl acetate, n-butanol, 70% ethanol, and water, and is an animal drug for preventing or treating aging and aging-related diseases. In Article 13, An animal drug for preventing or treating aging and aging-related diseases, characterized in that the above-mentioned elderberry plant extract contains as an active ingredient at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the following [chemical formula 1]. [Chemical Formula 1] In Article 13, An animal drug for preventing or treating aging and aging-related diseases, characterized in that the above-mentioned elder plant is at least one selected from the group consisting of elder (Sambucus. williamsii), Japanese yam (Sambucus racemosa H. hara), horse urine tree (Sambucus sieboldiana), and earthworm tree (Sambucus racemosaf. velutina). In any one of Articles 13 to 16, An animal drug for preventing or treating aging and aging-related diseases, characterized in that the aging-related disease is any one selected from the group consisting of idiopathic pulmonary fibrosis, idiopathic pulmonary fibrosis, cardiovascular disease, diabetes, neurodegenerative disease, sarcopenia, and osteoarthritis. A composition for animal feed for improving aging and aging-related diseases, characterized in that it comprises an extract of a plant of the genus Sambucus (Sambucus spp.), A composition for animal feed for improving aging and aging-related diseases, characterized in that the above Sambucus spp. plant extract is an extract obtained by extracting the Sambucus spp. plant with at least one solvent selected from the group consisting of water, C1-C4 lower alcohol, C1-C4 acetic acid ester, acetone, and methyl ethyl ketone. In Article 18, The above-mentioned Sambucus spp. plant extract is characterized in that it is a fraction produced by extracting and concentrating a Sambucus spp. plant with water, a lower alcohol of C1 to C4, or a mixed solvent thereof, adding distilled water, and then sequentially fractionating with n-hexane, ethyl acetate, n-butanol, 70% ethanol, and water, and a composition for animal feed for improving aging and aging-related diseases. In Article 18, A composition for animal feed for improving aging and aging-related diseases, characterized in that the above-mentioned Elderberry plant extract comprises as an active ingredient at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the following [chemical formula 1]. [Chemical Formula 1] In Article 18, A composition for animal feed for improving aging and aging-related diseases, characterized in that the above-mentioned elder plant is at least one selected from the group consisting of elder (Sambucus. williamsii), Japanese yam (Sambucus racemosa H. hara), horse urine tree (Sambucus sieboldiana), and earthworm tree (Sambucus racemosaf. velutina). In any one of Articles 18 to 21, A composition for animal feed for improving aging and aging-related diseases, characterized in that the aging-related disease is any one selected from the group consisting of idiopathic pulmonary fibrosis, idiopathic pulmonary fibrosis, cardiovascular disease, diabetes, neurodegenerative disease, sarcopenia, and osteoarthritis. A method for producing compounds 1, 4, 5, and 7 from plants of the genus Sambucus (Sambucus spp.), Step 1 of obtaining an extract of a Sambucus spp. plant by extracting the Sambucus spp. plant using at least one solvent selected from the group consisting of water, C1-4 lower alcohol, C1-4 acetic acid ester, acetone, and methyl ethyl ketone; Step 2: fractionating the extract of the plant of the genus Amurense obtained in Step 1 with an organic solvent to secure a fraction; and Step 3 of applying the fractions of the above step 2 to column chromatography to separate at least one compound selected from the group consisting of 3-O-trans-caffeoylformolic acid (compound 1), formolic acid (compound 4), oleanolic acid (compound 5), and 2-hydroxy-3-oxoolean-12-en-28-oic acid (compound 7) of the following [chemical formula 1]; A method for producing compound 1, 4, 5 or 7, characterized in that it consists of . [Chemical Formula 1] A novel compound, 3-O-trans-caffeoylformolic acid, having the following [chemical formula 2], isolated from a plant of the genus Sambucus (Sambucus spp.) having the following structural formula. [Chemical Formula 2]

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