Anti-aging composition derived from polygonum cuspidatum having cellular senescence and metabolic function recovery effects

Abstract

The present invention relates to an anti-aging composition comprising a Polygonum cuspidatum extract, or polydatin and 2-methoxystypandrone compounds, having cellular senescence and metabolic function recovery effects. The extract or compounds restore mitochondrial function in senescent cells, improve energy metabolism, activate the autophagy system, and reduce inflammatory factors while improving wrinkles and enhancing skin elasticity factors, thereby restoring senescent cells to a young cellular state. The extract or compounds can be applied as a senomorphic composition and can be readily used as a multifunctional cosmetic composition, pharmaceutical composition, or nutraceutical for anti-aging purposes.

Description

Anti-aging composition derived from Hojanggeun with cell aging and metabolic function recovery effects

[0001] The present invention relates to an anti-aging composition derived from Polygonum cuspidatum having the effect of preventing cell aging and restoring metabolic function.

[0002]

[0003] Aging is caused by functional changes in each organ and tissue, which in turn are driven by changes in the function of the cells that constitute the body's constituent units. For example, the loss of subcutaneous fat cells leads to decreased skin elasticity, and hair turns gray as hair follicle melanocytes lose their ability to produce melanin. Aging involves changes at the cellular level.

[0004] Cellular and individual aging share significant similarities not only physiologically but also molecularly. For example, neutral β-galactosidase (NAB) is known to be active only in cells that have entered a senescent state, and is used as a representative indicator of cellular aging, known as senescence-associated β-galactosidase (SA-β-gal). However, the activity of SA-β-gal has been observed only in aged human skin.

[0005] Therefore, cellular aging is considered to be an essential cause of individual aging, and research is ongoing to explore substances that regulate cellular aging through research at the cellular level, and to utilize them to understand individual aging and prevent and treat related diseases.

[0006] Cell senescence refers to the process of cell cycle arrest, characterized by the deterioration of cell characteristics and functions, followed by cell death or proliferation arrest. Cell senescence occurs as a result of stress or stimuli such as telomere shortening, oxidative stress, DNA damage, or the abnormal activation of oncogenes.

[0007] Cellular senescence can accelerate individual aging through two major mechanisms. First, senescent cells undergo cell cycle arrest, leading to decreased cell division and proliferation, ultimately resulting in decreased tissue regeneration capacity and impaired organismal function. Second, senescent cells secrete the senescence-associated secretory phenotype (SASP) to induce chronic inflammation. SASP is a key characteristic of senescent cells and encompasses cytokines, chemokines, ECM-degrading proteases, and growth factors secreted by senescent cells. These secreted SASPs can induce chronic inflammatory diseases in surrounding cells and tissues, or worsen and accelerate symptoms, ultimately leading to individual aging. In this regard, the concept of "inflammasome aging" has emerged, and the gradual increase in inflammation and the decline in immune function in response to inflammatory factors accelerate aging.

[0008] One strategy for treating aging is senomorphics, drugs that inhibit signaling pathways associated with SASP. Suppressing the activation of SASP factors such as IL-1α, IL-6, and IL-8 can halt the progression of aging.

[0009] Meanwhile, the expression of CXC motif chemokine ligand 12 (CXCL12) and slit guidance ligand 2 (SLIT2), known as anti-inflammatory chemokines, decreases in aged cells. CXCL12 plays an important role in maintaining skin homeostasis, promoting skin whitening, cell migration, and regeneration, while SLIT2 maintains the structural integrity of skin tissue and regulates cell-to-cell interactions, thereby maintaining skin elasticity and recovery. A recent study reported that when recombinant human SLIT2 protein (rhSLIT2) was treated with middle-aged fibroblasts, the cell morphology reverted to a small, spindle-shaped form like that of young cells, and proliferation ability was restored. Therefore, CXCL12 and SLIT2 can be used as functional identifiers of young cells.

[0010] Among the theories of aging, the 'mitochondrial theory of aging' states that mitochondria have their own genetic information and are particularly vulnerable to damage to genetic information due to the fact that they exist in places where a lot of oxidative stress is generated, and their repair system for damage is incomplete compared to the genetic information in the nucleus, so mitochondrial dysfunction causes aging and shortened lifespan.

[0011] Mitochondria are the energy powerhouses of cells, surrounded by a double membrane, with an outer and inner wall containing the electron transport chain. As electrons are transferred from Complex I to Complex V, hydrogen ions are released, and the resulting membrane potential difference is utilized to convert adenosine diphosphate (ADP) into adenosine triphosphate (ATP), a process known as oxidative phosphorylation (OXPHOS). Crucial energy metabolism occurs in the mitochondrial matrix, where sugar and lipid metabolism is essential for obtaining and storing energy during development, growth, and aging. Mitochondrial dysfunction contributes to the pathogenesis of age-related diseases. Cells with mitochondrial dysfunction exhibit a marked decline in division and proliferation compared to normal, young cells.

[0012] Mitochondria are also involved in maintaining the balance of calcium concentration, which acts as a coenzyme very important for intracellular metabolism and survival, and is involved in cell death.

[0013] Accordingly, research on improving mitochondrial function and delaying cellular aging has been increasing recently. For example, one study showed that oxazole derivatives stimulated the synthesis of ATP synthase, a mitochondrial inner membrane protein. It has been suggested that promoting ATP synthase activity helps restore mitochondrial inner membrane function and thus improve aging. However, while numerous studies have examined candidate compounds for improving mitochondrial function using synthetic single compounds, research on candidate compounds derived from natural products is lacking.

[0014] Polygonum cuspidatum, synonyms: Reynoutria japonica, Fallopia japonica) or Polygonum sachalinensis, synonyms: Reynoutria sachalinensis, Fallopia sachalinensis, giant knotweed or Sakhalin knotweed, is a dried medicinal herb. Traditionally, it has been used to treat rheumatic pain, dropsy, pregnancy turbidity, irregular menstruation, postpartum lochia, hepatitis, jaundice, osteomyelitis, dysentery, boils, and carcinoma due to its wind-clearing, diuretic, blood-stagnant, and swelling-reducing effects. Meanwhile, the anti-aging effects and detailed mechanisms of Polygonum cuspidatum have not yet been elucidated.

[0015]

[0016] [Prior Art Literature]

[0017] [Patent Document]

[0018] (Patent Document 1) KR 2564080 B1 (Title of the invention: Cosmetic composition having anti-aging effect and method for producing the same, Applicant: Bong-Sang Jo, Registration date: May 15, 2023)

[0019] (Patent Document 2) KR 2009-0125726 A (Title of the invention: Fermented extract of Hojanggeun and anti-aging herbal cosmetic composition containing the same, Applicant: Shinwha Pharmaceutical Co., Ltd., Publication date: October 22, 2009)

[0020]

[0021] The purpose of the present invention is to provide an anti-aging composition derived from Polygonum cuspidatum having the effect of preventing cell aging and restoring metabolic function.

[0022]

[0023] The present invention relates to an anti-aging composition characterized by containing an extract of Polygonum cuspidatum having anti-aging and metabolic function recovery effects.

[0024] The above extract may restore the function of aging cells to that of young cells.

[0025] The above extract may contain at least one of polydadtin and 2-methoxystypandrone as an active ingredient. At this time, the extract may contain 0.001 to 5.000 μg / ml of polydadtin or 2-methoxystypandrone, and, if necessary, more preferably, 0.1 to 5.0 μg / ml.

[0026] The above extract has minimal cytotoxicity toward senescent cells and has cell proliferation efficacy.

[0027] In addition, the above extract increases the membrane potential of mitochondria in aging cells and increases the oxygen consumption rate and ATP production rate, thereby improving the function of mitochondria in aging cells and energy metabolism.

[0028] The above extract activates the autophagy system of aged cells.

[0029] Additionally, the extract has the effect of reducing inflammatory factors, improving wrinkles, and enhancing elasticity factors.

[0030] The above extract has the effect of enhancing cell protection and moisturizing factors.

[0031] The present invention may be a cosmetic composition for anti-aging comprising the above composition.

[0032] In addition, a health functional food for anti-aging containing the above composition is provided.

[0033] The present invention also relates to a pharmaceutical composition for anti-aging comprising the composition.

[0034] In another aspect, the present invention relates to an anti-aging composition comprising one or more compounds of polydatin and 2-methoxystipandrone, wherein each compound may be included in the composition in an amount of 0.1 to 5.0 μg / ml.

[0035] Hereinafter, the present invention will be described in detail.

[0036] The compound of the present invention may be isolated from or synthesized from the plant Polygonum cuspidatum or Polygonum sp.

[0037] The above treatment with 2.5 ㎍ / ㎖ of the extract of Hojanggeun increases the proliferation of senescent cells by approximately 1.10 to 1.15 times, the ATP production rate by approximately 1.18 to 1.25 times, and the mitochondrial membrane potential (MMP) by 1.10 to 1.15 times.

[0038] At the same concentration, the extract of Hojanggeun reduces SA-β-gal-positive cells by 20-40%, has the effect of reducing lipofuscin accumulation by 40-50%, reducing lysosomal weight by 30-35%, and increasing autophagosome levels by 1.45-1.75 times. In addition, treatment with 2.5 ㎍ / ㎖ of Hojanggeun extract reduces p21 expression by 20-30%, and reduces IL-1β and IL-6 expression by 80-95%, restoring it to the level of young cells, and increases C-X-C motif chemokine ligand 12 (CXCL12) expression by 1.20-1.35 times. Treatment with 2.5 μg / ml of Hojanggeun extract reduced the expression of NLR family pyrin domain-containing protein 3 (NLRP3) protein by approximately 25-30%, restored the expression of IL-8 to the level of the untreated group with an inflammatory agent, and increased the expression of β-defensin 2 by 3.0-3.2 times compared to the group treated with an inflammatory agent, indicating an anti-inflammatory effect. In addition, protein carbonylation and the expression of calpain-1 were restored to the state before skin damage or skin aging. In addition, the expression of collagen type 1 alpha 1 (COL1A1) increased by 1.3-1.4 times, and the expression of collagen type 3 alpha 1 (COL3A1) increased by 1.1-1.3 times.

[0039] In addition, at least one compound selected from among polydatin and 2-methoxystipandrone, which are effective compounds contained in the extract of Hojanggeun, has an effect similar to that of the extract of Hojanggeun.

[0040] Preferably, treatment with 3.12 μg / ml (8 μM) of polydatin increases the proliferation of senescent cells by about 1.10 to 1.15 times, and increases the ATP production rate by about 1.5 to 1.7 times. At the same concentration, polydatin reduces lysosomal weight by 15 to 25% and increases autophagosome levels by 1.10 to 1.15 times. In addition, treatment with 3.12 μg / ml of polydatin reduces p21 expression by 35 to 40%, reduces IL-1β expression by 80 to 90%, and increases CXCL12 expression by 20 to 30%. Treatment with 3.12 μg / ml of polydatin reduces NLRP3 protein expression by about 30 to 40%, reduces SA-β-gal positive cells by 30 to 40%, and reduces p16 protein expression by 30 to 40%. Additionally, at the same concentration, polydatin restores protein carbonylation to a state prior to skin damage or skin aging.

[0041] Similarly, treatment with 0.5 μg / mL of 2-methoxystipandrone reduced SA-β-gal-positive cells by 40–50% and suppressed p16 protein expression by 35–45%. In addition, treatment with 0.5 μg / mL of 2-methoxystipandrone increased CXCL12 expression by 1.60–1.70-fold.

[0042] The extract derived from the root of the present invention can be extracted from a raw material sample plant for extraction using water, C1~C4 alcohol, 1,3-butylene glycol or a mixed solution thereof as a solvent, and the C1~C4 alcohol can be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol. The extract can also be a 20~90(v / v)% alcohol aqueous solution extract, and preferably a 50~80(v / v)% ethanol aqueous solution extract.

[0043] The water, C1~C4 alcohol, 1,3-butylene glycol or a mixed solution thereof used in the production of the above extract may be used in an amount 1~40 times the volume (1~40 ℓ per 1 kg) or 1~40 times the weight based on the initial weight of the raw material for extraction, and preferably 5~40 times the volume or 5~40 times the weight may be used. The extraction conditions of the above extract may be 20~100℃, or 20~80℃, or 20~50℃, for 1 minute~10 days, 1 minute~5 days, or 1 minute~2 days. The above process may be repeated 1~4 times.

[0044] After the above extraction, pre-filtration can be performed using non-woven fabric, paper, cloth, etc., or filtered using filters having various pore sizes. For example, filtering can be performed using filters having pore sizes of 10, 5, 2, 1, 0.6, 0.45, and 0.2 μm. Other filters can be used, added, or omitted as needed.

[0045] In addition, the above extract can be extracted by adding a buffer solution such as saline solution, Tri-HCl, saline solution, PBS (Phosphate-buffered saline), HBSS (Hank's balanced salt solution), etc. to the raw material sample; a buffer solution containing one or more of arginine, vitamin C, Tris-HCl, glycine, diphosphate, phosphate, potassium diphosphate, and potassium phosphate; and a buffer solution containing ionizable components such as calcium, iodine, iron, magnesium, selenium, zinc, sodium, phosphorus, sulfur, chlorine, copper, manganese, iron, and guanidine.

[0046] The extract or compound may be extracted using a conventional extraction device, an ultrasonic grinding extractor, or a fractionator. The extract or compound thus prepared may be subjected to hot air drying, reduced pressure drying, or freeze drying to remove the solvent. In addition, the extract or compound may be purified using column chromatography and then used.

[0047] The above extract or compound can be fractionated or purified and used by using a method known in the art for separating and extracting plant components, such as extraction using an organic solvent (alcohol, ether, acetone, etc.), distribution of hexane and water, or column chromatography, either singly or in an appropriate combination, according to the commercial law.

[0048] The above chromatography may be selected from silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography, thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

[0049] In addition, the present invention provides a pharmaceutical composition for anti-aging containing the extract, compound, and pharmaceutical excipient. The extract or compound may be added to the pharmaceutical composition of the present invention in an amount of 0.001 to 30 wt%.

[0050] 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 formulated, 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, and capsules, and these solid preparations are prepared by mixing the extract of the present invention with 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, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0051] The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and body weight of the subject to be treated, the specific disease or pathological condition to be 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 typically ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferred dosage is 1 mg / kg / day to 500 mg / kg / day. Administration may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0052] The pharmaceutical composition of the present invention can be administered to mammals, including rats, livestock, and humans, via various routes. All modes of administration are conceivable, including oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebrovascular injection. The composition of the present invention exhibits minimal toxicity and side effects, making it a safe drug for long-term use for preventive purposes.

[0053] In addition, the present invention provides a health functional food for anti-aging comprising an extract, a compound, and a food additive acceptable from a food science perspective. The extract and the compound may be added to the health functional food of the present invention in an amount of 0.001 to 30 wt%. The health functional food of the present invention includes forms such as tablets, capsules, pills, or liquids, and foods to which the extract of the present invention may be added include, for example, various drinks, meat, sausage, bread, candy, snacks, noodles, ice cream, dairy products, soups, sports drinks, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

[0054]

[0055] The present invention relates to an anti-aging composition characterized by containing an extract of Polygonum cuspidatum or a compound of polydatin or 2-methoxystypandrone having the effect of restoring cell aging and metabolic function, wherein the extract or compound restores the function of mitochondria in aging cells, improves energy metabolism, activates the autophagy system, reduces inflammatory factors, improves wrinkles and enhances elasticity factors, and enhances cell protection and moisturizing factors, thereby restoring aging cells to a young cell state, and can be applied as a cenomorphic composition, and can be easily used as a multifunctional cosmetic composition, pharmaceutical composition or health functional food that inhibits aging.

[0056]

[0057] Figure 1 shows the results of HPLC spectrum comparison of the extract of Hojanggeun and standard compounds.

[0058] Figure 2 is a graph confirming the effect of Hojanggeun extract on the proliferation of aged cells.

[0059] Figure 3 is a graph showing the ROS scavenging ability of aged cells treated with the extract of Hojanggeun.

[0060] Figure 4 is a graph showing the cytotoxicity results of the extract of Hojanggeun.

[0061] Figure 5 is a graph showing changes in the oxygen consumption rate (OCR), ATP production rate, and mitochondrial membrane potential (MMP) levels of senescent cells treated with the extract of Hojanggeun.

[0062] Figure 6 shows the results of the percentage of SA-β-gal staining positive cells accumulated in senescent cells treated with the extract of Hojanggeun, the accumulation rate of lipofuscin in the cells, the weight of lysosomes, and the level of autophagosomes.

[0063] Figure 7 is a graph showing the results of comparing the gene expression of p21, IL-1β, and IL-6, which are aging-related phenotypic factors, in aging cells treated with Hojanggeun extract with that of young cells.

[0064] Figure 8 is a graph showing the results of confirming the protein expression of NLRP3, an inflammatory factor, and the gene expression of IL-8 and β-defensin 2 in cells treated with the extract of Hojanggeun.

[0065] Figure 9 shows the results of confirming protein carbonylation and protein expression of Calpain-1 in senescent cells treated with the extract of Hojanggeun.

[0066] Figure 10 shows the results of confirming the gene expression of COL1A1 and COL3A1 in senescent cells treated with the extract of Hojanggeun.

[0067] Figure 11 is a graph showing the results of confirming the cell viability / cytotoxicity of polydatin on aged cells.

[0068] Figure 12 shows the results of confirming the effect of polydatin on the proliferation of aged cells.

[0069] Figure 13 is a graph showing the oxygen consumption rate (OCR) and ATP production rate of aged cells treated with polydatin.

[0070] Figure 14 shows the results of measuring the lipofuscin accumulation rate, lysosome weight, and autophagosome level in senescent cells treated with polydatin.

[0071] Figure 15 shows the results of confirming the gene expression of p21 and IL-1β, which are senescence-related phenotypic factors, in senescent cells treated with polydatin.

[0072] Figure 16 shows the results of confirming the protein expression of NLRP3, an inflammatory factor, in cells treated with polydatin.

[0073] Figure 17 shows the results of confirming protein carbonylation in senescent cells treated with polydatin.

[0074] Figure 18 shows the results of confirming the percentage of SA-β-gal staining positive cells and p16 protein expression in senescent cells treated with polydatin.

[0075] Figure 19 shows the results of confirming the percentage of SA-β-gal staining positive cells and p16 protein expression in senescent cells treated with 2-methoxystipandrone.

[0076] Figure 20 shows the results of confirming the gene expression of CXCL12 in senescent cells treated with extracts of Hojanggeun, Poladatin, and 2-methoxystipandrone.

[0077] Figure 21 shows the results of confirming the efficacy of Hojanggeun extract and polydatin in improving muscle loss in aged muscle cells.

[0078]

[0079] 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. Rather, the contents introduced herein are provided to ensure thoroughness and completeness, and to sufficiently convey the spirit of the present invention to those skilled in the art.

[0080] In this specification, cellular reverse aging refers to restoring the decreased mitochondrial function and energy metabolic efficiency of senescent cells to their previous state so that senescent cells that are unable to proliferate can proliferate and function like young cells again. This is different in concept from anti-aging, which simply treats young cells with a specific substance to delay the aging process of young cells (inhibit aging progression). What distinguishes cellular reverse aging from anti-aging is that in anti-aging, only a decrease in senescence markers is observed, while an increase in cell regeneration markers is not observed, whereas in cellular reverse aging, an increase in cell regeneration markers is observed along with a decrease in senescence markers. For example, in this specification, cellular reverse aging refers to decreasing the expression of senescence markers such as SA-β-gal and lipofuscin, while simultaneously increasing the expression of cell regeneration markers such as SLIT2 and CXCL12.

[0081]

[0082] <Example 1. Preparation of Polygonum cuspidatum extract and preparation of compounds>

[0083] The extract of Hojanggeun was prepared by the following method.

[0084] 100g of the root of Polygonum cuspidatum was mixed with 1000g of purified water and heated at 60℃ for 5 hours. The extract was filtered using a 5㎛ filter, then filtered through a 0.5㎛ filter to obtain a liquid, which was then concentrated to powder.

[0085] Polydatin (Sigma-Aldrich, 15721) was purchased from Sigma-Aldrich, and 2-methoxystypandrone was obtained as follows.

[0086] Add 2.4 L of 70% ethanol to 600 g of dried Korean ginseng root and extract for 24 hours with stirring at 40℃. Repeat this twice. Filter the extract through a 5 μm filter and then a 0.5 μm filter. Concentrate the filtrate under reduced pressure to remove as much ethanol as possible, then add 400 ml of purified water and 450 ml of ethyl acetate and fractionate. Repeat this twice. Concentrate only the ethyl acetate layer to obtain a powder. Dissolve the powder in methanol and then coat silica gel. The coating amount is 1.2 to 1.5 times the amount of sample to be loaded. After that, the powder is loaded onto a silica column and slowly eluted with hexane and ethyl acetate at a ratio of about 8:2 to 6:4, and the fraction containing 2-methoxystipandrone is collected and concentrated. Finally, 12.5 mg of 2-methoxystipandrone was obtained using reversed-phase (ODS) preparative high-performance liquid chromatography with methanol and purified water eluting conditions of 5:5 to 7.5:2.5. The physicochemical information of the obtained compound is as follows.

[0087] 2-Methoxystypandrone, Dark orange powder: mp 195 ℃; EI-MS 260.0692 (M+) (calcd. for C 14 H 12 O5260.0685); UV(MeOH) λmax; 216.0, 290.6 nm;

[0088] 1 H-NMR (400 MHz, CDCl3) δ12.50 (s, OH, 1H), 7.50 (s, 1H), 6.09 (s, 1H), 3.91 (s, 3H), 2.57 (s, 3H), 2.34 (s, 3H) ppm;

[0089] 13 C-NMR (100 MHz, CDCl3) δ 202.8, 190.2, 179.0, 160.9, 158.0, 143.4, 136.6, 130.4, 121.6, 112.3, 109.6, 56.8, 31.9, 20.0 ppm.

[0090]

[0091] <Example 2. Analysis of the extract of Hojanggeun>

[0092] Analysis of the polydatin and 2-methoxystipandrone contents of the extract of Hojanggeun in Example 1 was performed using HPLC (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA) and an HPLC column (Capcell Pak C 18 A 4.6 × 250 mm (Shiseido, Osaka, Japan) was used. The concentrated powder of Hojanggeun extract was dissolved in methanol (99.9%), filtered through a 0.45 μm filter, and analyzed. To prepare a standard solution of each compound, 7.6 mg of polydatin was dissolved in 50 ml of methanol, and 3.4 mg of 2-methoxystipandrone was dissolved in 25 ml of methanol.

[0093] The HPLC results comparing the extract of Hojanggeun and the standard compound are shown in Figure 1.

[0094] As a result of the analysis, polydatin and 2-methoxystipandrone in the extract of Hojanggeun were analyzed to be 3.05 wt% and 0.15 wt%, respectively. In addition, through repeated experiments, Hojanggeun extract containing 1.00 to 5.00 wt% of polydatin and 0.05 to 0.50 wt% of 2-methoxystipandrone was obtained.

[0095]

[0096] <Example 3. Culturing of young and senescent cells>

[0097] In the present invention, cells with a cell doubling time of 2 days or less (passage number 19-22) were used as young cells, and cells with a doubling time of 14 days or more through 1:2 subculture were used as aged cells (passage number 49-52).

[0098] Cultivation of each cell was basically performed in a 37°C, 5% CO2 incubator.

[0099] Additionally, depending on the cell type, an appropriate concentration of fetal bovine serum was mixed with Iscove's Modified Dulbecco's Medium (IMDM, Welgene, Korea), EpiLife (Gibco, USA), and Dulbecco's modified Eagle's Medium (DMEM, Welgene, Korea).

[0100]

[0101] <Experimental Example 1. Confirmation of the cell proliferation-inducing efficacy of Hojanggeun extract>

[0102] Human dermal fibroblasts (HDF; PCS-201-010; ATCC, Manassas, VA, USA) used in this experiment were cultured in DMEM containing 10% fetal bovine serum (FBS; Gibco, USA).

[0103] The cell proliferation induction efficacy was confirmed using the extract of the root of the plant in Example 1.

[0104] For this purpose, aged dermal fibroblasts were seeded at 1 × 10 per well in a 96-well plate. 3 The cells were cultured at a cell density of 10 cells / well. The extract of Hojanggeun was added to each well at a final concentration of 1.25–10.0 μg / ml every 4 days when the medium was replaced, and the degree of cell proliferation was counted using a DNA content-based method 12 days after sample treatment. Specifically, on the 12th day, the cells were washed twice with phosphate-buffered saline (PBS; AM9624; Invitrogen, USA), and 50 μl of 0.2% sodium lauryl sulfate (SLS; Sodium Lauryl sulfate, L3771, Sigma) was added to each well and incubated at 37°C for 1 hour. Afterwards, 150 μl of SYBR Green Ⅰ nucleic acid gel stain (Excitation / Emission: 485 nm / 535 nm, S-7567, Molecular Probes, USA) was added to each well, and the fluorescence intensity was measured using a VICTOR Multilabel Plate Reader (2030-0050, PerkinElmer, USA).

[0105] The results are shown in Fig. 2, and the results confirmed that the extract of Hojanggeun had cell proliferation induction efficacy at 1.25 ㎍ / ㎖ and 2.5 ㎍ / ㎖.

[0106]

[0107] <Experimental Example 2. Confirmation of the inhibitory effect of reactive oxygen species (ROS) production and cytotoxicity of the extract of Hojanggeun in aging cells>

[0108] The inhibitory effect of the extract of the root of the plant in Example 1 on the production of active oxygen in aged cells was confirmed.

[0109] Senescent fibroblasts were cultured in DMEM containing 10% fetal bovine serum (FBS; Gibco, USA). The prepared aged fibroblasts were treated with Hojanggeun extract at a final concentration of 1.25–10 μg / ml, with media replacement every 4 days for 12 days. To quantify reactive oxygen species, cells were treated with 30 μM DHR123 (10056-1; Biotium, Fremont, CA, USA) for 30 min at 37°C and subjected to flow cytometry analysis.

[0110] As a result, it can be confirmed that the inhibition effect of active oxygen production in aged cells is excellent at a concentration of 2.5 μg / ml, as shown in the results of Fig. 3.

[0111] Afterwards, the cell viability was confirmed for 2.5 ㎍ / ㎖ of the extract using Cedex HiRes Analyzer (05650216001, Roche, Switzerland) every 4 days for 12 days in the aged fibrotic cells, and as shown in the results of Fig. 4, no cytotoxicity was observed.

[0112] Meanwhile, optical microscopy revealed that the morphology of senescent fibroblasts, which were flat before extract treatment, changed to elongated and thinner, i.e., younger-looking, after treatment with the extract. This confirmed that extract treatment reversed cellular aging, and this was experimentally verified.

[0113]

[0114] Experimental Example 3. Confirmation of the efficacy of Hojanggeun extract in restoring mitochondrial function and improving cellular energy metabolism.

[0115] Restoring mitochondrial function is a prerequisite for recovery from aging. The mitochondrial electron transport chain not only transfers electrons but also pumps protons from the matrix into the mitochondrial intermembrane space, enabling oxidative phosphorylation.

[0116] In this example, the extract of the root of the Korean ginseng of Example 1 was used to confirm the efficacy of restoring mitochondrial function and improving cellular energy metabolism in aged fibroblasts.

[0117] For each experiment, aged fibroblasts were treated with the extract of Hojanggeun at a concentration of 2.5 μg / ml for a total of 12 days, with the medium replaced every 4 days. In subsequent experiments, with the exception of Experimental Examples 6 to 7, Experimental Examples 13 to 14, and 18, Hojanggeun extract was treated to the aged cells at the same concentration for the same period, regardless of cell type.

[0118] Using these cells, the efficiency of the mitochondrial electron transport chain was indirectly measured by measuring the efficiency of oxidative phosphorylation, and the oxygen consumption rate (OCR) and ATP production rate of the cells were measured as indicators of this oxidative phosphorylation efficiency.

[0119] To measure oxygen consumption rate, Seahorse XF Cell Mito stress test kit (103015-100; Aglient Technology, USA) was used, to measure ATP production rate, Seahorse XF Real-Time ATP Rate assay kit (103592-100, Aglient Technology, USA) was used, and for analysis, Seahorse XFe24 analyzer (Aglient Technology, USA) was used according to the manufacturer's manual.

[0120] Mitochondrial membrane potential (MMP) was measured using JC staining using cells treated under the same conditions. Cells were treated with medium containing 0.6 μg / ml of JC-10 (ENZ-52305, Enzo Life Science, USA), stained at 37°C for 30 minutes, and each measurement value was confirmed using flow cytometry (FACS analysis).

[0121] The results are shown in Figure 5. Referring to Figure 5, it can be seen that the extract of Hojanggeun showed a significantly higher oxygen consumption rate in aged cells compared to the control group, thereby increasing electron transport chain efficiency, ATP production, and mitochondrial membrane potential (MMP). Therefore, it can be seen that the extract of Hojanggeun is a composition that restores mitochondrial function in aged cells and increases cellular energy metabolism efficiency.

[0122]

[0123] <Experimental Example 4. Confirmation of the efficacy of Hojanggeun extract in restoring lysosomal / autophagy system function>

[0124] Removing dysfunctional organelles is a crucial process within cells. Traditionally, cells remove dysfunctional organelles through the autophagy system. However, as cells age, problems arise in lysosomes and the autophagy system, disrupting their normal functions. For example, a decrease in lysosomal activity leads to an increase in lysosomal weight to compensate for the reduced activity. This accumulation of dysfunctional organelles accelerates cellular aging.

[0125] Lipofuscin is a polymeric substance within lysosomes, mainly composed of cross-linked protein residues formed by iron-catalyzed oxidation. Since it is not degraded, it accumulates within cells during aging, so an improvement in lipofuscin accumulation can be seen as an indicator of improved aging. SA-β-gal also accumulates within cells, and SA-β-gal staining has traditionally been widely used as an indicator of improved aging.

[0126] Here, the efficacy of the extract of the Korean ginseng root of Example 1 in restoring the autophagy system function of aged fibroblasts was confirmed. SA-β-gal positive cells were identified using a staining kit (Cell Signaling, 9860), and the degree of lipofuscin accumulation was confirmed by flow cytometry without separate staining. The lysosome weight was confirmed by culturing aged fibroblasts in a medium containing 100 nM LysoTracker™ RED at 37°C for 30 minutes, staining them, and then analyzing them using a flow cytometer. The autophagosome level was determined by CYTO-ID in aged fibroblasts. ® After staining using (ENZ-51031-0050; Enzo Life Science, USA), the cells were analyzed using a flow cytometer.

[0127] The results are shown in Fig. 6, and the extract of the root of Hojanggeun of Example 1 decreased the percentage of SA-β-gal staining-positive cells and the accumulation of lipofuscin, which are indicators of accumulation in aged cells. This result was confirmed to result in a decrease in lysosomal weight, and it appears that the level of autophagosomes is increased.

[0128] This means that treatment with the extract of Hojanggeun has an excellent effect in improving the autophagy system by reducing non-functional lysosomes in aged fibroblasts.

[0129]

[0130] <Experimental Example 5. Confirmation of changes in aging-related phenotypic factors in extracts of Hojanggeun>

[0131] Using the extract of the root of Example 1, it was confirmed that increased aging-related phenotypic factors were reduced in aged fibroblasts.

[0132] To this end, we examined the expression patterns of the p21 gene, one of the pathways controlling cell cycle arrest, and gene expression changes in the senescence-associated secretory phenotype (SASP). Because SASP secretion significantly upregulates inflammatory responses and activates the IL-6, IL-8, IL-1β, and TNF-α pathways, we also examined gene expression changes in these factors.

[0133] Senescent cells treated with the extract of Hojanggeun were harvested, and total RNA was isolated using an RNase Mini Kit (74104; QIAGEN, Hilden, Germany). The isolated total RNA was reverse transcribed using a DiaStar™ RT Kit (DR22-R10k; SolGent, Seoul, Korea) to obtain cDNA.

[0134] qPCR was performed using Solg™ 2× Real Time PCR smart mix (SRH83-M40h; Solgent) in a CFX Connect™ Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA). qPCR was performed with denaturation at 95°C for 4 min, followed by 40 cycles of 94°C for 30 s, 57°C for 30 s, and 70°C for 10 s. All gene expression experiments were then performed under the same conditions.

[0135] Each result is shown in Fig. 7, and as a result, p21, which is related to cell cycle arrest, was expressed more in senescent cells than in young cells, but was decreased when treated with the Korean knotweed extract. This shows that the Korean knotweed extract restores irreversible cell cycle arrest. In addition, the expression of IL-1β and IL-6, which are inflammatory aging factors, also showed the same expression pattern as p21. This shows that the Korean knotweed extract has excellent efficacy in reducing infectious cell aging and restoring skin immunity by suppressing the inflammatory response in the skin of senescent cells and restoring the skin to a young cell state with high skin regeneration ability. This efficacy means that the Korean knotweed extract can be applied as a cenomorphic composition.

[0136]

[0137] <Experimental Example 6. Confirmation of the anti-inflammatory and skin defense-enhancing effects of Hojanggeun extract>

[0138] To further confirm the anti-inflammatory efficacy of the extract of Hojanggeun, the protein expression of NLRP3, an inflammatory factor, was confirmed by Western blot, and the gene expression levels of IL-8, an inflammatory cytokine, and β-defensin-2, an antimicrobial peptide, were confirmed using real-time PCR.

[0139] For this purpose, human epithelial keratinocytes (HaCaT, immortalized human epithelial keratinocytes) were used, and the cells were pretreated with LPS+ATP (NLRP3 confirmed), IFN-γ+TNF-α (IL-8 confirmed), and IL-4+IFN-γ+TNF-α (β-defensin-2 confirmed) to induce inflammation due to aging.

[0140] Each result is shown in Figure 8.

[0141] As a result, the extract of Korean knotweed suppressed the increased NLRP3 protein expression and IL-8 gene expression caused by pretreatment with inflammatory substances. The NLRP3 inflammasome is a protein complex that mediates and is activated by inflammatory responses, and causes acute and chronic inflammatory diseases in response to various exogenous pathogens and endogenous tissue damage. Activation of the NLRP3 inflammasome produces proinflammatory cytokines such as IL-1β and IL-18, which are known to be involved in aging-related inflammation. Recently, it has been reported that inhibiting abnormal NLRP3 inflammasome attenuates aging-related diseases and degenerative changes, thereby extending the lifespan of mice. Based on this, it can be confirmed that the extract of Korean knotweed has an aging-improving and recovery effect by reducing chronic inflammation through suppressing abnormal NLRP3 expression in aging cells.

[0142] Meanwhile, β-defensin-2, an antimicrobial peptide, is maintained at a relatively low level in normal skin, but when infection or wound occurs, its expression increases rapidly, thereby enhancing the skin's antimicrobial ability. Many studies have reported that the expression of antimicrobial peptides is reduced in the skin of patients with atopic dermatitis, and there have been several reports that antimicrobial peptides are reduced by Th2 cytokines such as IL-4, IL-10, and IL-13, which play an important role in the pathogenesis of atopic dermatitis. While β-defensin-2 gene expression in skin cells increases due to pretreatment with inflammatory inducers (IFN-γ, TNF-α) and then decreases due to IL-4, it was found that the extract of Hojanggeun increases its gene expression, thereby enhancing the skin cells' own protective ability.

[0143]

[0144] Experimental Example 7. Confirmation of the efficacy of Hojanggeun extract in alleviating cell damage and improving the skin barrier.

[0145] When cells age and are damaged, protein carbonylation, an irreversible oxidative protein modification, increases, damaging dermal fibers and causing skin aging such as wrinkles, sagging skin, and dullness. To confirm the efficacy of the extract of the Korean ginseng root on this, human epidermal keratinocytes (HEK; human epidermal keratinocytes, neonatal, C0015C; Invitrogen, Waltham, MA, USA) were used and cultured in EpiLife (Gibco, USA) medium containing 1% fetal bovine serum. The HEK cells were pretreated with 2500 μM of H2O as a cause of skin aging / damage, and then treated with 2.5 μg / ml of the Korean ginseng root extract. To measure protein carbonylation, cells were fixed by treating them with methanol for 5 minutes, stained with 20 μM fluorescein-5-thiosemicarbazide (FTSC, 46985, Sigma), and analyzed using a microplate reader (Tecan, Switzerland) and a fluorescence microscope (Leica, Germany).

[0146] In addition, we confirmed the protein expression of calpain-1, a transcript of a natural moisturizing factor and a key enzyme in the production of filaggrin, a component of the skin barrier, in HEK cells treated with the extract of Hojanggeun. To induce skin aging / damage, cells were pretreated with 200 ng / ㎖ IL-17A (200-17, Peprotech, USA), and then treated with 2.5 ㎍ / ㎖ of Hojanggeun extract, followed by culture for 3 days. Afterwards, proteins were extracted from the cells, and calpain-1 protein expression was confirmed by Western blotting.

[0147] As a result, as shown in Fig. 9, it can be seen that the extract of the root of the Korean rhizome of Example 1 is a composition that protects the skin by reducing protein carbonylation increased by cell damage / aging and improves the skin barrier by increasing the expression of calpain-1.

[0148]

[0149] <Experimental Example 8. Confirmation of the Elasticity-Improving Effect of Hojanggeun Extract>

[0150] To identify COA1A1 and COL3A1, which are factors related to collagen biosynthesis, aged fibroblasts were treated with 2.5 μg / ml of Hojanggeun extract and cultured for 12 days to collect RNA, synthesize cDNA, and perform real-time PCR.

[0151] Real-time PCR was performed by treating cultured cells with QIAzol Lysis Reagent (QIAGEN) and extracting RNA using the method provided by the manufacturer, and the isolated RNA was analyzed using Qubit™ After quantification using a fluorometer with RNA BR Assay kit, cDNA was synthesized and real-time PCR was performed. cDNA synthesis was performed using the qPCRBIO cDNA Synthesis Kit and the experiment was performed according to the kit's instructions. Real-time PCR was performed using 2x qPCRBIO SyGreen Blue mix Lo-ROX to amplify each gene and then quantitatively analyze the amplification products.

[0152] The results are shown in Figure 10, and it can be confirmed that the extract of Hojanggeun has the effect of improving skin elasticity and wrinkles by increasing the expression of COL1A1 and COL3A1, which are reduced in production during cell aging in fibroblasts.

[0153]

[0154] <Experimental Example 9. Confirmation of Cytotoxicity / Viability and Cell Proliferation Efficacy of Polydatin>

[0155] The cytotoxicity and cell proliferation efficacy were confirmed using the polydatin compound, an effective ingredient of the extract of Hojanggeun, using the methods disclosed in Experimental Examples 1 and 2. Under each experimental condition, the effective concentration and time of the compound were 3.12 μg / ml and 12 days, and the experimental conditions thereafter were the same as in Experimental Examples 1 and 2.

[0156] The results are shown in Figures 11 and 12, and it can be confirmed that the polydatin compound has no cytotoxicity at a concentration of 3.12 μg / ml (Figure 11) and has cell proliferation inducing efficacy (Figure 12).

[0157]

[0158] Experimental Example 10. Polydatin's Effect on Improving Cellular Energy Metabolism

[0159] The efficacy of restoring mitochondrial function was confirmed using the polydatin compound in the same manner as in Experimental Example 3.

[0160] The results are shown in Figure 13, and it was confirmed that the polydatin compound improves the efficiency of oxidative phosphorylation by increasing the oxygen consumption rate (OCR) and increases ATP production, thereby restoring mitochondrial function and inducing changes in cellular metabolism.

[0161] Major cellular bioenergetic pathways include glycolysis, which occurs in the cytoplasm, and oxidative phosphorylation, which occurs in the mitochondria. Glycolysis produces two ATP molecules per glucose molecule, while oxidative phosphorylation produces 32-28 ATP molecules per glucose molecule. Therefore, oxidative phosphorylation, which operates within the mitochondria, is a much more efficient energy synthesis process. Notably, oxidative phosphorylation consumes oxygen, whereas glycolysis does not. Therefore, an increase in oxygen consumption indicates that cells are relying on the more efficient oxidative phosphorylation process for energy production. This suggests that polydatin has the effect of reducing reliance on glycolysis.

[0162]

[0163] Experimental Example 11. Confirmation of the efficacy of polydatin in activating the autophagy system.

[0164] Using a polydatin compound, the accumulation of lipofuscin, lysosome weight, and autophagosome level were confirmed in the same manner as in Experimental Example 4.

[0165] The results are shown in Figure 14, and it can be confirmed that polydatin reduces lipofuscin accumulation and lysosomal weight, increases autophagosome levels, and restores the autophagy system that can remove damaged mitochondria, etc.

[0166]

[0167] Experimental Example 12. Confirmation of the efficacy of polydatin in improving aging-related phenotypic factors.

[0168] Using a polydatin compound, the expression of p21 and IL-1β, which are aging-related phenotypic factors, was confirmed in the same manner as in Experimental Example 5, and the results are shown in Figure 15.

[0169] As a result, it was found that polydatin is a substance that shows an effect of improving cell aging by reducing the expression of p21 and IL-1β, and increases the recovery ability for skin regeneration.

[0170]

[0171] Experimental Example 13. Confirmation of the Anti-Inflammatory Effect of Polydatin

[0172] The anti-inflammatory effect was confirmed by confirming the expression of NLRP3 protein using the polydatin compound in the same manner as in Experimental Example 6.

[0173] Referring to the results of Figure 16, it can be seen that the polydatin compound has an effect of improving skin immunity and inflammatory aging by reducing the expression of NLRP3, a major factor in the NLRP3 inflammasome complex that induces an inflammatory response in the skin.

[0174]

[0175] Experimental Example 14. Confirmation of the Cell Damage Recovery Efficacy of Polydatin

[0176] Protein carbonylation was confirmed using the method of Experimental Example 7 using a polydatin compound.

[0177] As a result, as shown in Fig. 17, it can be seen that the increased protein carbonylation, which is the cause of skin aging / damage, is restored to the control level by polydatin treatment, and it can also be confirmed that it is a substance that restores cell damage caused by aging.

[0178]

[0179] Experimental Example 15. Confirmation of the Anti-Aging Effect of Polydatin

[0180] The proportion of SA-β-gal positive cells was confirmed using the method of Experimental Example 4 using a polydatin compound. In addition, to confirm the protein expression of p16, the sample was treated and cultured in aged fibroblasts for 24 hours, and the protein was extracted from the cells and confirmed using the Western blot method.

[0181] SA-β-gal and p16 are representative markers of cellular senescence, and the proportion of senescent cells and the pattern of protein expression regulation were examined to determine the effect of polydatin on recovery from cellular senescence.

[0182] As a result, as shown in Fig. 18, polydatin was confirmed to have an anti-aging effect on aged cells by reducing the proportion of SA-β-gal positive cells by 30-40% and suppressing the protein expression of p16 by 30-40%.

[0183]

[0184] Experimental Example 16. Anti-aging efficacy of 2-methoxystipandrone.

[0185] As an effective compound in the extract of Hojanggeun, the 2-methoxystipandrone compound was used to confirm the SA-β-gal positive cell ratio and p16 protein expression using the method of Experimental Example 15.

[0186] As shown in Figure 19, 2-methoxystipandrone suppressed the proportion of SA-β-gal staining positive cells, a representative aging marker whose expression increases during cell aging, by 40-50% at 0.5 ㎍ / ㎖, and reduced p16 expression by 30-40%, confirming that it has an anti-aging effect on aging cells.

[0187]

[0188] <Experimental Example 17. Confirmation of the efficacy of extracts of Hojanggeun, polydatin, and 2-methoxystipandrone compounds in restoring young cell marker proteins>

[0189] The recovery effect on young cell marker proteins of aged fibroblasts was confirmed using the extract of Hojanggeun and the effective compound, polydatin and 2-methoxystipandrone.

[0190] To this end, the gene expression of the corresponding marker was confirmed.

[0191] Senescent cells treated with extracts of Hojanggeun, polydatin, and 2-methoxystipandrone were harvested, and total RNA was isolated using an RNase Mini Kit (74104; QIAGEN, Hilden, Germany). The isolated total RNA was reverse transcribed using a DiaStar™ RT Kit (DR22-R10k; SolGent, Seoul, Korea) to obtain cDNA.

[0192] qPCR was performed using Solg™ 2× Real Time PCR smart mix (SRH83-M40h; Solgent) in a CFX Connect™ Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA). qPCR was performed with denaturation at 95°C for 4 min, followed by 40 cycles of 94°C for 30 s, 57°C for 30 s, and 70°C for 10 s. All gene expression experiments were then performed under the same conditions.

[0193] The results are shown in Figure 20, and the results in Figure 20 indicate that the extract of Hojanggeun, polydatin, and 2-methoxystipandrone have excellent efficacy in increasing the expression of CXCL12, which is decreased in aged cells, thereby restoring them to the level of young cells with high skin regeneration ability.

[0194]

[0195] Experimental Example 18. Confirmation of the Muscle Loss-Improving Efficacy of Hojanggeun Extract and Polydatin Compounds

[0196] As we age, muscle tissue gradually deteriorates, resulting in a decline in muscle size and strength—a condition known as sarcopenia. This age-related muscle loss continues at a rate of approximately 1-2% per year until age 50. Furthermore, age-related changes in mitochondrial metabolism have been reported to lead to a decrease in maximal oxygen consumption, and this decrease in maximal oxygen consumption is a primary driver of sarcopenia.

[0197] Accordingly, the muscle loss improvement effect of Hojanggeun extract and polydatin was confirmed.

[0198] C2C12, a mouse-derived progenitor cell line used in the experiment (CRL-1772TM; ATCC, Manassas, VA, USA), was cultured in Dulbecco's Modified Eagle's Medium (DMEM, HyClone, USA) containing 10% fetal bovine serum.

[0199] C2C12 cells were seeded at 1 × 10 per well in a 12-well culture plate. 5After dividing the cells into cells according to their density, the medium was replaced and cultured until the cells formed a sufficient density. Afterwards, differentiation of C2C12 cells was induced by replacing the medium, and the medium was replaced and cultured for an additional 4 to 5 days until the cells formed a complete myotube morphology. After differentiation was complete, the culture medium was removed and the samples were pretreated for 2 hours. To induce aging by oxidative stress, H2O2 (Sigma-Aldrich, USA) was added to a final concentration of 50 μM and further cultured for 24 hours at 37°C under 5% CO2 conditions. The cells were washed with DPBS, dehydrated and fixed in methanol for 10 minutes, and then dried. Afterwards, myotubes were stained using Jenner's stain (Sigma-Aldrich, USA) and Giemsa Stain Modified Solution (Sigma-Aldrich, USA) and the morphology was observed using an optical microscope. Myotube diameter was measured using the Image J program (NIH, USA), and the fusion index was calculated using [Formula A] below.

[0200] [Formula A]

[0201] Fusion index (%) = (Number of nuclei forming a multinucleated structure / Total number of nuclei observed in the image) x 100

[0202] The results for this are shown in Fig. 21.

[0203] Figure 21a shows the results of microscopic examination of myotube morphology. When differentiated myotubes are treated with H2O2, the length, number, and diameter of myotubes decrease compared to the untreated control group (Non). On the other hand, when treated with Hojanggeun extract and polydatin, the length, number, and diameter of myotubes increase compared to the control group (control), confirming the efficacy of muscle loss recovery.

[0204] Figure 21b shows the results of measuring myotube diameter, and the diameter decreased by H2O2 treatment was increased by 1.6 times and 1.5 times, respectively, when treated with Hojanggeun extract and polydatin, confirming the efficacy of muscle loss recovery.

[0205] Table 1 shows the results of calculating the fusion index. Fusion index is an indicator of the degree to which myocytes fuse per unit area, indicating the degree to which muscle cells form muscle tissue. Treatment with Hojanggeun extract and polydatin increased the fusion index by 2-fold and 1.9-fold, respectively, confirming its efficacy in restoring muscle lost due to aging.

[0206] Test groupMyotube diameter (μm)Fusion index (%)H2O2Untreated control (Non)29.5 ± 1.4100 ± 0H2O2treated control (Con)9.9 ± 0.639.3 ± 5.9H2O2+ Hojanggeun extract 2.5 ㎍ / ㎖16.2 ± 0.978.5 ± 10.2H2O2+ Polydatin 0.15 ㎍ / ㎖15.3 ± 0.573.4 ± 10.6

[0207] Through the above experiments, we suggest that the extract of Hojanggeun and polydatin have anti-aging effects not only in skin cells but also in aged muscle cells.

[0208] Note: In the present invention, gene expression was confirmed using each primer disclosed in Table 2 below.

[0209] TargetPrimerSequence (5’-3’)Size (bp)36B4FWDCAGCAAGTGGGAAGGTGTAATCC23REVCCCATTCTATCATCAACGGGTACAA25GAPDHFWDCAATGACCCCTTCATTGACC20REVAAATGAGCCCCAGCCTTCT19β-actinFWDGGCACCCAGCACAATGAAG19REVCCGATCCACACGGAGTACTTG21COL1A1FWDAGCAAGAACCCCAAGGACAA20REVCGAACTGGAATCCATCGGTC20COL3A1FWDCTGATGGGGTCAAATGAAGGTG22REVCGTGCAACCATCCTCCAGAAC21p21FWDAGGTGGACCTGGAGACTCTCAG22REVTCCTCTTGGAGAAGATCAGCCG22IL-1βFWDCCACAGACCTTCCAGGAGAATG22REVGTGCAGTTCAGTGATCGTACAGG23IL-6FWDAGACAGCCACTCACCTCTTCAG22REVTTCTGCCAGTGCCTCTTTGCTG22IL-8FWDGAGAGTGATTGAGAGTGGACCAC23REVCACAACCCTCTGCACCCAGTTT22CXCL12FWDTCAGCCTGAGCTACAGATGC20REVCTTTAGCTTCGGGTCAATGC20

Claims

1. An anti-aging composition characterized by containing an extract of Polygonum cuspidatum, which has the effect of preventing cell aging and restoring metabolic function.

2. In paragraph 1, The above extract is an anti-aging composition characterized in that it restores the function of aging cells to young cells.

3. In paragraph 1, The above extract is an anti-aging composition characterized in that it contains at least one compound selected from the group consisting of polydatin and 2-methoxystypandrone as an active ingredient.

4. In paragraph 1, The above extract is an anti-aging composition characterized by minimal cytotoxicity in aging cells and cell proliferation efficacy.

5. In paragraph 1, The above extract is an anti-aging composition characterized in that it has the effect of restoring the function of mitochondria in aging cells and improving energy metabolism.

6. In paragraph 1, An anti-aging composition characterized in that the extract activates the autophagy system of aged cells.

7. In paragraph 1, An anti-aging composition characterized in that the extract reduces inflammatory factors of aging cells and enhances elasticity factors.

8. A cosmetic composition for anti-aging, characterized by comprising the composition of paragraph 1.

9. A health functional food for anti-aging characterized by containing the composition of paragraph 1.

10. A pharmaceutical composition for anti-aging, characterized by comprising the composition of paragraph 1.

11. An anti-aging composition characterized in that it contains at least one compound selected from the group consisting of polydatin and 2-methoxystypandrone as an active ingredient.

12. In paragraph 11, An anti-aging composition characterized in that the compound has the effect of restoring cell aging and metabolic function.

13. In paragraph 11, An anti-aging composition characterized in that the compound restores the function of aging cells to that of young cells.

14. In paragraph 11, An anti-aging composition characterized in that the compound has minimal cytotoxicity in aging cells and has cell proliferation efficacy.

15. In paragraph 11, An anti-aging composition characterized in that the compound has the effect of restoring the function of mitochondria in aging cells and improving energy metabolism.

16. In paragraph 11, An anti-aging composition characterized in that the compound activates the autophagy system of aged cells.

17. In paragraph 11, An anti-aging composition characterized in that the compound reduces inflammatory factors of aging cells and enhances elasticity factors.

18. A cosmetic composition for anti-aging, characterized by comprising the composition of Article 11.

19. A health functional food for anti-aging characterized by comprising the composition of Article 11.

20. A pharmaceutical composition for anti-aging, characterized by comprising the composition of claim 11.

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