Anti-aging composition derived from machilus thunbergii, having effect of restoring cellular senescence and metabolic function

Abstract

The present invention relates to an anti-aging composition containing a Magnolia officinalis extract or magnolol, honokiol and 4-O-methlyhonokiol compounds. The extract or compound restores mitochondrial functions of senescent cells, reduces damaged DNA in cells, and restores senescent cells to a youthful cell state, and thus can be effectively used as a multifunctional cosmetic composition, a pharmaceutical composition, or a health functional food for inhibiting aging.

Description

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

[0001] The present invention relates to an anti-aging composition derived from Magnolia officinalis having cell aging and metabolic function recovery effects.

[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 senescence-associated secretory phenotype factors (SASPs), inducing chronic inflammation. This has led to the emergence of the concept of "inflammasome aging," which proposes that the gradual increase in inflammation and the decline in immune function in response to inflammatory factors accelerate aging.

[0008] 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.

[0009] 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.

[0010] Mitochondria are the energy powerhouses of cells, surrounded by a double membrane with an outer and inner wall. The inner wall contains an electron transport chain, which releases hydrogen ions during the process of transferring electrons from complex I to complex V. This generates a membrane potential difference that converts ADP into ATP, forming oxidative phosphorylation (OXPHOS). A very important energy metabolism process takes place in the mitochondrial matrix, and this metabolism of sugars and lipids is an essential part of the process of obtaining or storing energy related to the development, growth, and aging of the body. Mitochondrial dysfunction contributes to the pathogenesis of age-related diseases. Cells that have begun to age due to mitochondrial dysfunction show a marked decline in energy metabolism function compared to young cells with normal division and proliferation functions.

[0011] 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.

[0012] 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.

[0013] Magnolia officinalis (Magnolia officinalis) is an evergreen tree native to East Asia, primarily Korea, Japan, and China. Magnolia officinalis primarily inhabits wetlands such as forests and swamps. Its bark, leaves, twigs, and roots have been used medicinally, as tea, and as oil. Several studies have demonstrated the pharmacological efficacy of Magnolia officinalis. One study demonstrated that Magnolia officinalis extract was nontoxic to MDA-MB-231 and HT-29 cells and had antidiabetic effects. However, the anti-aging effects and detailed mechanisms of Magnolia officinalis remain unknown.

[0014] [Prior Art Literature]

[0015] [Patent Document]

[0016] KR 0773863 B1 (Title of invention: Cosmetic composition for preventing skin aging and method for producing the same, Applicant: Byun Young-hoon, Registration date: April 11, 2006)

[0017] KR 0550934 B1 (Title of invention: Extract of Magnolia chinensis, Extract of Magnolia chinensis, Magnolia chinensis isolated therefrom, and Pharmaceutical composition and functional food containing them as active ingredients for preventing or treating diabetic complications, Applicant: Korea Institute of Oriental Medicine, Registration date: June 17, 2003)

[0018]

[0019] The purpose of the present invention is to provide an anti-aging composition derived from Magnolia officinalis having cell aging and metabolic function recovery effects.

[0020]

[0021] The present invention relates to an anti-aging composition characterized by containing an extract of Magnolia officinalis having anti-aging and metabolic function recovery effects.

[0022] The above extract may restore the function of aging cells to that of young cells.

[0023] The above extract may be extracted from the stem bark of the cinnamon tree.

[0024] The above extract may contain magnolol, honokiol and 4-O-methylhonokiol as active ingredients.

[0025] The above extract has minimal cytotoxicity toward senescent cells and has cell proliferation efficacy.

[0026] In addition, the above extract increases the membrane potential and reduces the weight of mitochondria in aging cells, thereby improving the function recovery and energy metabolism of mitochondria in aging cells.

[0027] The above extract activates the autophagy system of aged cells and reduces damaged DNA within cells. It also has the effect of reducing inflammatory factors, improving wrinkles, and enhancing elasticity.

[0028] The present invention may also relate to an anti-aging composition comprising a complex of magnolol, honokiol, and 4-O-methylhonokiol as active ingredients. Preferably, the present invention may relate to an anti-aging composition comprising 150 to 300 parts by weight of honokiol and 200 to 300 parts by weight of 4-O-methylhonokiol as active ingredients, based on 100 parts by weight of magnolol. In this case, magnolol may be included at a level of 0.0175 μg / ml.

[0029] The present invention may be a cosmetic composition for anti-aging comprising the above composition.

[0030] In addition, a health functional food for anti-aging containing the above composition is provided.

[0031] The present invention also relates to a pharmaceutical composition for anti-aging comprising the composition.

[0032] Hereinafter, the present invention will be described in detail.

[0033]

[0034] The compound of the present invention may be isolated or synthesized from Magnolia officinalis or Magnolia sp., or other Magnoliaceae plants.

[0035]

[0036] Treatment with 0.625 μg / ml of the above-mentioned extract of Magnolia chinensis increases cell proliferation by approximately 1.10-1.15 times, increases mitochondrial membrane potential (MMP) by 1.15-1.25 times, and reduces mitochondrial weight by 13-18%. At the same concentration, the extract of Magnolia chinensis has the effect of reducing basal glycolysis level, compensatory glycolysis, and post-2-DG acidification by 25-35%, and confirms the effect of suppressing gene damage by reducing DNA tail length by 10-15% through the Comet assay. The extract of Magnolia chinensis has the effect of increasing autophagosome level by 165-175%, reducing SA-β-gal (senescence associated β-galactosidase) positive cells by 20-30%, and reducing lipofuscin accumulation by 20-30%. In addition, 0.625 ㎍ / ㎖ of the extract of Magnolia chinensis increases the expression of slit guidance ligand 2 (SLIT2) by 3-4 times, the expression of CXC motif chemokine ligand 12 (CXCL12) increases by 7-8 times, the expression of protease-activated receptor 2 (PAR-2) decreases by 20-30%, the expression of collagen type 1 alpha 1 (COL1A1) increases by 2.8-3.2 times, and the expression of matrix metalloproteases 1 (MMP1) decreases by 85-95% compared to the control group and returns to the level of the normal group. Filaggrin expression increases 2-3 times, CC motif chemokines ligand 2 (CCL2) expression decreases by 40-45%, CCL5 expression decreases by 36-40%, Interleukin 6 (IL-6) expression decreases by 87-95%, and IL-8 expression decreases by 92-95%, all of which are restored to the normal group level.

[0037] In addition, in the present invention, when treating with the extract of Mackerel or a complex compound, based on 100 parts by weight of magnolol, 150 to 300 parts by weight of honokiol and 200 to 300 parts by weight of 4-O-methylhonokiol are included as effective ingredients, and at this time, when magnolol is 0.0175 ㎍ / ㎖, SLIT2 expression increases 5 to 7 times and CXCL12 expression increases 3 to 5 times. In particular, when the honokiol is included in an amount less than 150 parts by weight or more than 300 parts by weight based on 100 parts by weight of magnolol, the various anti-aging effects of the Mackerel extract and the compounds in the present invention are shown to be significantly reduced, and even when the minimum content of 4-O-methylhonokiol is less than 200 parts by weight, the anti-aging effect may not be well exhibited.

[0038] The extract derived from the bark 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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%.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050]

[0051] The present invention relates to an anti-aging composition characterized by containing an extract of Magnolia officinalis or compounds of magnolol, honokiol and 4-O-methylhonokiol having cell aging and metabolic function recovery effects, wherein the extract or compound restores the function of mitochondria in aging cells, improves energy metabolism, activates the autophagy system, reduces damaged DNA in cells, and enhances inflammatory factors, wrinkle improvement and elasticity 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.

[0052]

[0053] Figures 1a to 1d show the HPLC spectrum results of the extract of the bark and standard compounds.

[0054] Figure 2a is a graph showing the effect of the extract of Phellodendron amurense on the proliferation of aged cells, and Figure 2b is a photograph showing changes in cell morphology after treatment of aged cells with the extract of Phellodendron amurense.

[0055] Figure 3 is a graph showing the changes in mitochondrial membrane potential (MMP) and weight of aged cells treated with the extract of Hobak.

[0056] Figure 4a is a graph showing the change in the extracellular acidification rate (ECAR) of senescent cells treated with the extract of Magnolia chinensis, and Figure 4b shows the results confirming that the extract of Magnolia chinensis regulates the glycolytic proton efflux rate (GlycoPER) and acidification of 2-deoxyglucose (2-DG) of senescent cells.

[0057] Figure 5 shows the results of a Comet assay conducted using senescent cells treated with extracts of the bark of the genus Magnolia.

[0058] Figure 6 shows the results of the senescence associated β-galactosidase (SA-β-gal) staining positive cell ratio and lipofuscin accumulation in senescent cells treated with the extract of Magnolia chinensis.

[0059] Figure 7 shows the results of confirming the gene expression of slit guidance ligand 2 (SLIT2) and CXC motif chemokine ligand 12 (CXCL12) in senescent cells treated with the extract of Magnolia chinensis.

[0060] Figure 8a shows the results of confirming the gene expression of PAR-2 ​​(protease-activated receptor 2), collagen type 1 alpha 1 (COL1A1), and collagen type alpha 3 (COL3A1) in senescent cells treated with the extract of Magnolia chinensis, Figure 8b shows the results of confirming the matrix metalloproteases 1 (MMP1) gene, and Figure 8c shows the results of confirming the protein expression of filaggrin.

[0061] Figure 9 shows the results of confirming the gene expression of CC motif chemokines ligand 2 (CCL2), CCL5, interleukin-6 (IL-6), and IL-8 in senescent cells treated with the extract of Magnolia chinensis.

[0062] Figure 10 shows the results of confirming the effect of honokiol on the proliferation of aged cells.

[0063] Figure 11 is a graph showing the changes in mitochondrial membrane potential (MMP) and weight of aged cells treated with honokiol.

[0064] Figure 12a is a graph showing the change in the extracellular acidification rate (ECAR) of senescent cells treated with honokiol, and Figure 12b shows the results confirming that honokiol regulates the acidification of glycoPER (glycolytic proton efflux rate) and 2-DG (2-deoxyglucose) of senescent cells, and additionally, the results confirming the ratio of mitochondrial oxygen consumption (mitoOCR), which is an indicator that can confirm the main energy usage method of cells, and glycoPER.

[0065] Figure 13 shows the results of lysosomal weight, SA-β-gal positive cells, and lipofuscin accumulation in senescent cells treated with honokiol.

[0066] Figure 14 shows the results of confirming the gene expression of SLIT2 and CXCL12 in senescent cells treated with honokiol.

[0067] Figure 15a shows the results of confirming the MMP1 gene expression in senescent cells treated with honokiol, and Figure 15b shows the results of confirming the gene expression of inflammatory factors CCL2, CCL5, IL-6, and IL-8.

[0068] Figure 16a shows the results of confirming the expression of p16 protein and the percentage of SA-β-gal staining positive cells in senescent cells treated with 4-O-methylhonokiol, and Figure 16b shows the results of confirming the gene expression of MMP1 in the senescent cells.

[0069] Figure 17a shows the results of confirming the expression of MMP1 and IL-8 genes in senescent cells treated with a mixture of three compounds, 4-O-methylhonokiol, honokiol, and magnolol, and Figure 17b shows the results of confirming the expression of SLIT2 and CXCL12 genes in the senescent cells.

[0070] Figure 18 shows the results of confirming the efficacy of the extract of the bark and its effective compounds in improving muscle loss in aged muscle cells.

[0071] Figures 19 and 20 show the results of confirming each anti-aging factor in aged cells and young cells. (The sample in Figure 19 is a magnolia extract, and the sample in Figure 20 is a honokiol compound.

[0072]

[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. 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.

[0074] 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 rate of the 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 slit guidance ligand 2 (SLIT2) and CXC motif chemokine ligand 12 (CXCL12).

[0075]

[0076] <Example 1. Preparation of Magnolia officinalis extract and preparation of compounds>

[0077] The extract of the bark was prepared by the following method.

[0078] 100 g of the stem bark of Magnolia officinalis was mixed with 800 g of 70 (v / v)% aqueous ethanol solution and heated at 60°C for 3 hours. The extract was filtered using a 5 μm filter and then filtered using a 0.5 μm filter to obtain a liquid phase, which was then concentrated and powdered.

[0079] Magnolol (sigma-aldrich, M3445) and honokiol (sigma-aldrich, H4914) were purchased from Sigma-Aldrich.

[0080] 4-O-Methylhonokiol is obtained as follows. 2 g of the extract of Magnolia chinensis is suspended in 20 mL of distilled water, dissolved in 5 mL of hexane, and fractionally extracted. This process is repeated twice to collect the soluble portion in the hexane layer, and the hexane layer is concentrated under reduced pressure to obtain the hexane extract of Magnolia chinensis. This hexane extract is dissolved in methanol, adsorbed on a C18 column, and then mixed with methanol and water in a ratio of about 10:1 to 4:1 to obtain fractions, adsorbed on silica gel, and then silica gel column chromatography is performed 2 to 5 times to separate the active fraction, and finally 4-O-methylhonokiol is obtained using high-performance liquid chromatography. The physicochemical information of the obtained compound is as follows.

[0081] 1 H-NMR (400MHz, CDCl3): δppm 3.45(2H, d, J=6.0Hz, H-7'), 3.45(2H, d, J=6.0Hz, H-7), 3.90(3H, s, OMe), 5.10(4H, m, H-9 and H-9'), 6.01(2H, m, H-8) and H-8'), 6.95(1H, dd, J=8, 1.5Hz, H-3'), 6.97(1H, dd, J=8.0, 1.5Hz, H-3), 7.05(2H, m, H-2 and H-6'), 7.23(1H, dd, J=8.5, 1.6Hz, H-6), 7.30(1H, dd, J=8.5, 1.6Hz, H-4');

[0082] 13 C-NMR (100MHz, CDCl3): δppm 129.09(C-1), 130.67(C-2), 128.67(C-3), 156.94(C-4), 115.53(C-5), 127.86(C-6), 34.22(C-7), 136.48(C-8), 115.47(C-9), 127.83(C-1'), 150.79(C-2'), 110.88(C-3'), 129.64(C4'), 132.09(C-5'), 130.46(C-6'), 39.36(C-7'), 137.77(C-8'), 115.77(C-9'), 55.47(OMe).

[0083]

[0084] <Example 2. Analysis of the extract of Magnolia chinensis>

[0085] Analysis of the contents of honokiol, magnolol, and 4-O-methylhonokiol in the extract of the bark of 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. 23.3 mg of concentrated extract powder of Honokiol was dissolved in methanol (99.9%), filtered through a 0.45 μm filter, and analyzed. To prepare standard solutions of each compound, 4.0 mg of honokiol, 3.5 mg of magnolol, and 15.9 mg of 4-O-methylhonokiol were each dissolved in 50 ml of methanol (99.9%).

[0086] The HPLC results comparing the extract of the bark with the standard compound are shown in Figure 1.

[0087] The analysis results showed that the extract of Magnolia chinensis contained 3.86 wt% magnolol, 8.73 wt% honokiol, and 5.88 wt% 4-O-methylhonokiol. In addition, through repeated experiments, a Magnolia chinensis extract containing 3.5-4.0 wt% magnolol, 7.5-9.5 wt% honokiol, and 5.0-6.5 wt% 4-O-methylhonokiol was obtained.

[0088]

[0089] <Example 3. Culturing of young and senescent cells>

[0090] In the present invention, cells with a cell doubling time of 2 days or less (passage number 19-22) were designated as young cells, and these were cultured in a 1:2 subculture to obtain aged cells with a doubling time of 14 days or more (passage number 49-52), which were then cultured and used. All cell cultures were basically performed in a 37°C, 5% CO2 incubator.

[0091] 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).

[0092]

[0093] <Comparative Examples 1 to 10> Preparation of extracts from plants of the genus Magnolia or Maculfera

[0094] Each extract was prepared using the conditions in Table 1 below and the method of Example 1. However, analysis of the effective ingredients in the extracts revealed that most contained less than 1 wt% of magnolol and less than 3 wt% of honokiol, and in particular, 4-O-methylhonokiol was found to be absent or present in extremely small amounts in most extracts. Therefore, these extracts were not used as samples for subsequent analysis.

[0095] Extract Conditions Raw Material Part Part Comparison Example 1 Magnolia officinalis stem bark water Comparison Example 2 Magnolia officinalis root 70(v / v)% ethanol aqueous solution Comparison Example 3 Magnolia officinalis flower water Comparison Example 4 Magnolia officinalis flower 70(v / v)% ethanol aqueous solution Comparison Example 5 Magnolia officinalis root water Comparison Example 6 Korean Magnolia (Machilus thunbergii) stem bark 70(v / v)% ethanol aqueous solution Comparison Example 7 Japanese Magnolia (Magnolia obovata) stem bark 70(v / v)% ethanol aqueous solution Comparison Example 8 Magnolia liliflora stem bark 70(v / v)% ethanol aqueous solution Comparison Example 9 Magnolia (Magnolia kobus) Stem bark 70(v / v)% ethanol aqueous solution Comparative example 10 Magnolia rostrate stem bark 70(v / v)% ethanol aqueous solution

[0096]

[0097] <Experimental Example 1. Confirmation of the cell proliferation-inducing efficacy of the extract of Magnolia chinensis>

[0098] Human dermal fibroblasts (HDF; PCS-201-010; ATCC, Manassas, VA, USA) used in this experiment were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Welgene, Korea) containing 10% fetal bovine serum (FBS; Gibco, USA).

[0099] The cell proliferation induction efficacy was confirmed using the extract of the bark of Example 1.

[0100] 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 100 cells / well. The extract of the bark was added to each well at a final concentration of 0.625–10 μg / ml every 4 days during medium replacement, and after 12 days of drug treatment, the degree of cell proliferation was confirmed using the MTT assay.

[0101] The results for this are shown in Fig. 2a.

[0102] As a result, as shown in Fig. 2a, it was confirmed that the extract of the bark of the deciduous tree was not cytotoxic up to 10 μg / ml, and in particular, it was shown to exhibit cell proliferation efficacy at a concentration of 0.625 μg / ml, and thus the experiment was conducted using this as the effective concentration.

[0103]

[0104] <Experimental Example 2. Confirmation of the Cell Morphology Restoration Efficacy of Magnolia chinensis Extract>

[0105] Aged cells exhibit morphological changes, such as enlargement and flattening. Therefore, changes in cell morphology are considered important indicators of cell aging. Therefore, the efficacy of the extract of the bark of the Chinese juniper tree in restoring cell morphology was confirmed using Example 1.

[0106] For this purpose, 46-year-old adult (ATCC, CRL-2106, CCD-1090Sk) fibroblasts were used as aged fibroblasts. These cells were cultured in Minimum Essential Medium Eagle (MEM, Welgene, Korea) containing 10% fetal bovine serum (FBS; Gibco, USA).

[0107] First, the morphology of aged fibroblasts was confirmed through an optical microscope, and the cells were treated with 0.625 μg / ml of the extract of the bark, replaced every two days, and the cell images after 6 days are shown in Figure 2b.

[0108] Referring to Figure 2b, it is confirmed that the shape of large, flat senescent cells changes into an elongated, thin, i.e., young cell shape due to treatment with the extract of the bark of the perilla plant.

[0109] Accordingly, it can be seen that the extract of the present invention can reverse cell aging.

[0110]

[0111] Experimental Example 3. Confirmation of the mitochondrial function recovery effect of the extract of Magnolia chinensis

[0112] The efficacy of the extract of Magnolia chinensis in restoring mitochondrial function in aged fibroblasts was examined using the extract of Magnolia chinensis in Example 1. To this end, aged fibroblasts were treated with the extract at a concentration of 0.625 μg / ml, with media replacement every four days, for a total of 12 days. In subsequent experiments, with the exception of Experimental Examples 8 and 18, the extract of Magnolia chinensis was applied to aged cells at the same concentration for the same period, regardless of cell type.

[0113] Mitochondrial dysfunction in aged cells leads to significant changes, including increased intracellular reactive oxygen species and decreased energy production necessary for maintaining cellular function. Mitochondrial dysfunction can be identified by decreased mitochondrial membrane potential (MMP) and increased mitochondrial weight.

[0114] Aged fibroblasts treated with the extract of Hobak were stained with 50 nM MitoTracker green (M7514; Invitrogen) at 37°C for 30 minutes, and after cell fixation, mitochondrial weight was measured through flow cytometry (FACS) analysis.

[0115] Additionally, aged fibroblasts treated with the extract of Hobak were stained with JC-10 reagent containing 0.6 ㎍ / ml for 30 minutes at 37°C, and after cell fixation, mitochondrial membrane potential (MMP) was measured through flow cytometry (FACS) analysis.

[0116] The results for this are shown in Fig. 3.

[0117] The results in Figure 3 confirmed that the extract of Poria cocos increased mitochondrial membrane potential (MMP) and reduced weight in aged cells, thereby restoring mitochondrial function.

[0118]

[0119] <Experimental Example 4. Confirmation of the efficacy of the extract of Magnolia chinensis to improve cellular energy metabolism>

[0120] The efficacy of the extract of Magnolia chinensis in Example 1 to improve cellular energy metabolism in aged cells was confirmed. Cells synthesize energy through two processes: glycolysis and oxidative phosphorylation. These processes occur in the mitochondria. Compared to glycolysis, which breaks down one glucose molecule to produce two ATP molecules, oxidative phosphorylation, which generates energy using oxygen, is a much more efficient energy synthesis process, producing 32-38 ATP molecules. In other words, by assessing the indices of glycolysis and oxidative phosphorylation, the primary energy use method of a cell can be identified and the efficiency of energy metabolism can be determined.

[0121] To this end, we identified that as cell aging increases its dependence on this process, more protons are generated and released as pyruvate is converted to lactate, and through this, we confirmed the efficacy of reducing 2-deoxyglucose acidification.

[0122] To confirm the process, the Seahorse XF Glycolytic Rate Assay Kit (103344-100; Aglient Technolongy) as an extracellular acidification rate (ECAR) kit and the Seahorse XFe96 analyzer (Aglient Technolongy, Santa Clara, CA, USA) as an analyzer were used according to the manufacturer's manual.

[0123] The results of each experimental analysis are shown in Figures 4a and 4b.

[0124] Looking at Figure 4a, the effect of the extract of Magnolia chinensis on the extracellular acidification rate (ECAR) of aged fibroblasts was confirmed. When examined over time, both the basal glycolysis level before Rot / AA injection, an inhibitor of the mitochondrial electron transport chain, and the compensatory glycolysis results after Rot / AA injection were reduced.

[0125] That is, it can be seen that the extract of the bark improves the efficiency of cellular energy metabolism by reducing the dependence on this process.

[0126] In addition, as shown in Fig. 4b, it can be confirmed that the extract of Magnolia chinensis reduces glycoPER in aging cells, thereby inhibiting lactic acid production, an inefficient energy metabolism process, and reducing 2-DG acidification, one of the indicators of such inefficient energy metabolism.

[0127] Therefore, this again confirms that the extract of the bark effectively improves cellular energy metabolism.

[0128]

[0129] Experimental Example 5. Confirmation of DNA Damage Recovery Efficacy of Mackerel Extract

[0130] DNA plays a central role in life processes through replication and transcription. Damage to DNA can lead to genetic imbalances and accelerate aging. Damaged DNA exists in the nucleus in the form of small fragments.

[0131] The DNA damage repair efficacy of aged fibroblasts was confirmed using the extract of the bark of Example 1. To this end, a Comet assay was performed using the Comet Assay Single Cell Gel Electrophoresis Assay kit, and the DNA tail length was confirmed using Image J.

[0132] The results for this are shown in Fig. 5.

[0133] The results showed that the extract of Poria cocos reduces the increased DNA tail length in aging cells, thereby inhibiting DNA damage and DNA fragmentation.

[0134]

[0135] <Experimental Example 6. Confirmation of the Autophagy System Activation Efficacy of Magnolia chinensis Extract>

[0136] 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 function. The accumulation of dysfunctional organelles accelerates cellular aging.

[0137] In addition, lipofuscin is a polymeric substance within lysosomes, mainly composed of cross-linked protein residues formed by iron-catalyzed oxidation processes, and since it is not decomposed, it accumulates within cells during aging, so improvement in lipofuscin accumulation can be seen as an indicator of improvement in aging.

[0138] SA-β-gal also accumulates inside cells, and traditionally, SA-β-gal staining has been widely used as an indicator to confirm improvement in aging.

[0139] Here, the efficacy of the extract of the bark of Example 1 to restore autophagy system activation in aged fibroblasts was confirmed. SA-β-gal-positive cells were stained using a staining kit (Cell Signaling, 9860), and the degree of lipofuscin accumulation was confirmed by flow cytometry without separate staining. The results are shown in Figure 6.

[0140] The results in Fig. 6 confirm that the extract of Poria cocos improves the autophagy system by reducing the percentage of SA-β-gal staining positive cells and the accumulation of lipofuscin, which are indicators of accumulation in aged cells.

[0141]

[0142] <Experimental Example 7. Confirmation of the efficacy of extracts from the bark of the Chinese juniper berry on the recovery of young cell marker proteins>

[0143] The recovery effect of the extract of the bark of Example 1 on young cell marker proteins of aged fibroblasts was confirmed.

[0144] To this end, the gene expression of each marker was confirmed.

[0145] Senescent cells treated with the extract of Magnolia chinensis 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.

[0146] 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.

[0147] Each result is shown in Figure 7, and the results in Figure 7 indicate that the extract of the bark of the Chinese juniper berry has an excellent effect in restoring skin to a state similar to that of young cells with high skin regeneration ability by increasing the expression of SLIT2 and CXCL12, which are decreased in aged cells.

[0148]

[0149] <Experimental Example 8. Confirmation of the Skin Improvement Effect of Mackerel Extract>

[0150] In order to confirm the skin improvement effect using the extract of the bark of Example 1, the expression of factors related to skin tone-up, i.e., pigmentation inhibition effect, collagen biosynthesis, and skin barrier improvement was confirmed.

[0151] The cells used in each experiment were HaCaT cells, fibroblasts, and HEK cells. Human epithelial keratinocytes (HaCaT, human epithelial keratinocyte, immortalized; PCS-200-011; ATCC) were cultured in Dulbecco's modified Eagle's Medium (DMEM, Welgene, Korea) containing 10% fetal bovine serum (FBS; Gibco, USA), and fibroblasts (HDF; PCS-201-010; ATCC, Manassas, VA, USA) were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Welgene, Korea) containing 10% fetal bovine serum. Neonatal human epidermal keratinocytes (HEK; human epidermal keratinocytes, neonatal, C0015C; Invitrogen, Waltham, MA, USA) were cultured in EpiLife (Gibco, USA) medium containing 1% fetal bovine serum, and all cultures were performed at 37°C in a 5% CO2 incubator.

[0152] First, to confirm the expression of protease-activated receptor 2 (PAR-2), a tone-up-related factor, HaCaT cells were irradiated with 30 mJ of UVB, then treated with 0.625 μg / ml of the extract of Magnolia chinensis, cultured for 1 day, total RNA was collected, cDNA was synthesized, and real-time PCR was performed.

[0153] To identify COA1A1 and COL3A1, which are factors related to collagen biosynthesis, and MMP1, a collagen-degrading enzyme, aged fibroblasts were treated with 0.625 μg / ml of the extract of Magnolia chinensis and cultured for 12 days to collect RNA, synthesize cDNA, and perform real-time PCR.

[0154] Real-time PCR was performed by washing cells with PBS (phosphate buffered saline), then treating the cultured cells with QIAzol Lysis Reagent (QIAGEN) and extracting RNA using the method provided by the manufacturer. The isolated RNA was then analyzed by 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.

[0155] To confirm the expression of filaggrin, HEK cells were treated with 0.625 μg / ml of the extract of Magnolia chinensis, cultured for 3 days, analyzed by immunocytochemistry (ICC), and then quantified. The immunofluorescence analysis was performed as follows. The protein expression level of filaggrin, a skin barrier factor, was directly confirmed within the cells. To this end, HEK cells were fixed with a 4% paraformaldehyde solution, permeabilized with a 0.2% Triton X-100 solution, and then immunostained sequentially with anti-Filaggrin antibody and secondary fluorescent antibody, and images were obtained using a fluorescence microscope.

[0156] The results are shown in Figures 8a, 8b, and 8c.

[0157] The results in Fig. 8a show that the extract of Poria cocos has the effect of improving skin tone darkened by aging by reducing PAR-2 ​​expression in human epidermal keratinocytes (HaCaT) and inhibiting melanosome transfer and restoring the skin barrier. In addition, it can be seen that the extract of Poria cocos has the effect of improving skin elasticity and wrinkles by increasing the expression of COL1A1 and COL3A1, whose production decreases with cell aging, in fibroblasts.

[0158] In addition, the results in Figure 8b show that the extract of the bark of the magnolia tree has an effect of improving skin elasticity and wrinkles by reducing the increased expression of MMP1 in aged fibroblasts.

[0159] Figure 8c suggests that the expression of filaggrin, which plays an important role in maintaining the skin barrier and moisture, is increased in human epidermal keratinocytes (HEK), thereby also having a skin barrier and moisture strengthening effect.

[0160]

[0161] <Experimental Example 9. Confirmation of the anti-inflammatory effect of the extract of Magnolia chinensis>

[0162] In aged tissues, the inflammatory response is significantly upregulated by secretion of senescence-associated secretory phenotype factors (SASP), and when the IL-6 and TNF-α pathways are activated, the expression of related factors such as IL-6, IL-8, CCL2, CCL5, MIP1α, and MIP3α increases.

[0163] The efficacy of the extract of the bark of Example 1 in improving the expression of inflammation-related genes was confirmed using real-time PCR, and this is shown in Figure 9.

[0164] Referring to Figure 9, it can be seen that the extract of Magnolia chinensis significantly reduces the expression of CC motif chemokines ligand 2 (CCL2), CCL5, IL-6 (interleukin-6), and IL-8, which are inflammatory factors that accelerate skin aging, in aged fibroblasts, thereby reducing the inflammatory response in the skin and restoring skin immunity.

[0165]

[0166] <Experimental Example 10. Confirmation of the Cell Proliferation-Inducing Efficacy of Honokiol>

[0167] The cell proliferation induction efficacy was confirmed using the honokiol compound, an effective ingredient of the extract of Magnolia chinensis, using the same method as Experimental Example 1. The effective concentration and time of the compound were 1 μM and 12 days, and the experimental conditions thereafter were the same.

[0168] The results are shown in Fig. 10, and it can be confirmed that the honokiol compound also significantly increases cell proliferation and has an anti-aging effect.

[0169]

[0170] Experimental Example 11. Confirmation of Honokiol's Efficacy in Restoring Mitochondrial Function

[0171] The efficacy of honokiol compound in restoring mitochondrial function was confirmed using the same method as in Experimental Example 3.

[0172] The results for this are shown in Fig. 11.

[0173] Our results show that honokiol compounds also increase mitochondrial membrane potential (MMP) and reduce weight in aged cells, thereby restoring mitochondrial function.

[0174]

[0175] Experimental Example 12. Confirmation of the efficacy of honokiol in improving cellular energy metabolism.

[0176] Using the honokiol compound, the efficacy of improving cell energy metabolism, the efficacy of inhibiting lactic acid production, the efficacy of reducing 2-DG acidification, and the mitoOCR / glycoPER ratio were confirmed using the same method as in Experimental Example 4.

[0177] The results for this are shown in Figures 12a and 12b.

[0178] As shown in Figure 12a, honokiol improves cellular energy metabolism efficiency by decreasing the total, basal, compensatory extracellular acidification rate (ECAR) of senescent cells, thereby reducing their dependence on this process.

[0179] In addition, as explained through the results of Figure 12b, which show that as cell aging increases its dependence on this process, more protons are generated and released as pyruvate is converted to lactate, it can be confirmed that honokiol increases energy metabolic efficiency by reducing the glycolytic proton efflux rate (GlycoPER) and inhibiting lactate production, which is an inefficient energy metabolic process.

[0180] Honokiol also maintains efficient cellular energy metabolism by reducing 2-DG acidification, an indicator of inefficient energy metabolism.

[0181] These results also demonstrate that honokiol significantly increases the ratio of mitoOCR and glycoPER, which are important indicators of cellular metabolism that evaluate the activity of oxidative phosphorylation and glycolysis, thereby restoring mitochondrial function and inducing metabolic changes, thereby reducing dependence on glycolysis.

[0182]

[0183] Experimental Example 13. Confirmation of the efficacy of honokiol in activating the autophagy system.

[0184] Using the honokiol compound, the efficacy of activating the autophagy system was confirmed by applying the SA-β-gal positive cell staining and lipofuscin accumulation confirmation methods among the experimental methods of Experimental Example 6, and 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.

[0185] The results for this are shown in Fig. 13.

[0186] Looking at Figure 13, it can be confirmed that honokiol significantly reduces lysosomal weight, SA-β-gal positive cells, and lipofuscin accumulation, thereby restoring the autophagy system.

[0187]

[0188] Experimental Example 14. Confirmation of Honokiol's Effect on the Recovery of Young Cell Marker Proteins

[0189] Using the honokiol compound, the recovery effect on young cell marker genes was confirmed using the same method as in Experimental Example 7.

[0190] The results are shown in Fig. 14. As a result, it was found that honokiol also restores skin regeneration ability similar to that of young cells by increasing the expression of slit guidance ligand 2 (SLIT2) and CXC motif chemokine ligand 12 (CXCL12), whose expression is reduced in aged cells.

[0191]

[0192] Experimental Example 15. Confirmation of the Anti-Aging Effect of Honokiol

[0193] Using the honokiol compound, the expression of MMP1 and various aging-related inflammatory factors was confirmed using the method of Experimental Example 8, and the aging recovery effect was confirmed, and the results are shown in Fig. 15.

[0194] As a result, honokiol can be seen to have the effect of improving skin elasticity and wrinkles by reducing the increased expression of MMP1 in aging cells, as shown in Fig. 15a, and to significantly reduce the expression of CC motif chemokines ligand 2 (CCL2), CCL5, IL-6, and IL-8, inflammatory factors that accelerate skin aging, as shown in Fig. 15b, thereby reducing the inflammatory response in the skin and restoring skin immunity.

[0195]

[0196] Experimental Example 16. Confirmation of the Anti-Aging Effect of 4-O-Methylhonokiol

[0197] The anti-aging efficacy of the 4-O-methylhonokiol compound, an effective ingredient of the extract of Magnolia chinensis, was confirmed by the methods of Experimental Examples 6 and 8. As related factors, the expression of p16 and the proportion of SA-β-gal positive cells were confirmed, and the anti-aging efficacy was confirmed by confirming the expression of MMP1. The expression of p16 was confirmed by treating and culturing aged fibroblasts with the sample for 24 hours, extracting proteins from the cells, and then confirming the expression of p16 protein using the Western blot method.

[0198] The results for each experiment are shown in Fig. 16.

[0199] As a result, as shown in Fig. 16a, 4-O-methylhonokiol has an anti-aging effect on aged cells by reducing the expression of p16, a representative aging marker whose expression increases during cell aging, and the proportion of SA-β-gal staining positive cells, and as shown in Fig. 16b, it can be confirmed that it has an effect of improving skin elasticity and wrinkles by reducing the expression of MMP-1, which increases in aged cells.

[0200]

[0201] <Experimental Example 17. Confirmation of the Anti-Aging Effects of Magnolol, Honokiol, and 4-O-Methylhonokiol>

[0202] The anti-aging recovery effect was confirmed using the mixture of magnolol, honokiol and 4-O-methylhonokiol compounds using the methods of Experimental Examples 7 to 9.

[0203] Among the notations for each compound mixture sample, 1 indicates that the compound is contained at 0.0175 μg / mL, 2 indicates that it is contained at 0.0350 μg / mL, and 3 indicates that it is contained at 0.0525 μg / mL. That is, the 1:2:3 sample contains magnolol, honokiol, and 4-O-methylhonokiol at the following concentrations: 0.0175 μg / mL: 0.0350 μg / mL: 0.0525 μg / mL.

[0204] The results of each experiment are shown in Fig. 17.

[0205] Looking at Figure 17a, the mixing ratio of magnolol, honokiol, and 4-O-methylhonokiol, which are the main components of the extract of Magnolia chinensis, was changed to test the expression of MMP1 and IL-8 among senescence-associated secretory phenotypes (SASPs). The effect was higher when the content of honokiol and 4-O-methylhonokiol was higher than that of magnolol.

[0206] This suggests that honokiol and 4-O-methylhonokiol further contribute to the recovery of senescent cell phenotype.

[0207] Looking at Figure 17b, when evaluating the expression of SLIT2 and CXCL12, whose expression is reduced in aged cells, it can be seen that the expression of these genes increases further when the content of honokiol and 4-O-methylhonokiol is higher than that of magnolol. Furthermore, when the content of 4-O-methylhonokiol is the highest (1:2:3), the expression of SLIT2 and CXCL12 increases the most.

[0208] Therefore, it was confirmed that honokiol and 4-O-methylhonokiol further contribute to the anti-aging effect of senescent cells.

[0209]

[0210] Experimental Example 18. Confirmation of the Muscle Loss-Improving Efficacy of Mackerel Extract, Honokiol, and 4-O-Methylhonokiol

[0211] 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.

[0212] Accordingly, the muscle loss improvement efficacy of extracts of Honokiol, Honokiol, and 4-O-methylhonokiol was confirmed.

[0213] 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.

[0214] 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. After replacing the medium, differentiation of C2C12 cells was induced, and the medium was replaced and cultured for an additional 4 to 5 days until complete myotube morphology was formed. 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 and 5% CO2. 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.

[0215] [Formula A]

[0216] Fusion index (%) = (Number of nuclei forming a multinucleated structure / Total number of nuclei observed in the image) x 100

[0217] The results for this are shown in Fig. 18.

[0218] Figure 18a shows the results of microscopic examination of myotube morphology. When differentiated C2C12 myotubes were treated with H2O2, the length, number, and diameter of myotubes decreased compared to the untreated control group (Non). On the other hand, when treated with extracts of Magnolia chinensis, honokiol, and 4-O-methylhonokiol, the length, number, and diameter of myotubes increased compared to the control group, confirming the efficacy of muscle loss recovery.

[0219] Figure 18b shows the results of measuring myotube diameter, and the diameter decreased due to H2O2 treatment was increased by 2.6 times, 1.5 times, and 1.6 times, respectively, when treated with the extract of Phellodendron amurense, honokiol, and 4-O-methylhonokiol, confirming the efficacy of muscle loss recovery.

[0220] Table 2 shows the results of calculating the fusion index. The fusion index is an indicator of the degree to which myocytes have fused per unit area, indicating the degree to which muscle cells form muscle tissue. When treated with the extract of Magnolia chinensis, honokiol, and 4-O-methylhonokiol, the fusion index increased by 2.5-fold, 1.9-fold, and 2.1-fold, respectively, confirming the efficacy of restoring muscle lost due to aging.

[0221] Test groupFusion index (%)H2O2Untreated control group (Non)100 ± 0H2O2treated control group (Con)39.3 ± 5.9H2O2+ Mackerel extract 0.625 ㎍ / ㎖98.7 ± 0.3H2O2+ Honokiol 0.05 ㎍ / ㎖76.7 ± 2.8H2O2+ Methylhonokiol 0.03 ㎍ / ㎖80.7 ± 1.0

[0222] In addition, muscle recovery efficacy was also observed in experiments in which two or three compounds were combined, and this followed the trend of compound mixing ratio as shown in Figure 17b.

[0223] The above experiments suggest that extracts of Magnolia chinensis, Magnolia magnolol, Honokiol, and 4-O-methylhonokiol have anti-aging effects not only in skin cells but also in aged muscle cells.

[0224]

[0225] <Experimental Example 19. Comparison with the Young Cell Control Group>

[0226] Among the experimental results so far, for the group treated with the extract of the bark and the honokiol compound, the mitochondrial weight, the degree of lipofuscin accumulation, SA-β activity, and the inhibition rate of gene expression of IL-6 and MMP1 were presented, representatively, in the experiment on senescent cells compared to young cells.

[0227] Figure 19 shows the experimental results of the extract of the bark of Magnolia chinensis, and Figure 20 shows the experimental results of the honokiol compound. It can be confirmed that the aged cell treatment groups treated with the extract of Magnolia chinensis and the honokiol compound, respectively, were restored to a state similar to that of young cells, proving that these samples are anti-aging compositions.

[0228]

[0229] Note: In the present invention, gene expression was confirmed using each primer disclosed in Table 3 below.

[0230] TargetPrimerSequence (5'-3')Size (bp)36B4FWDCAGCAAGTGGGAAGGTGTAATCC23REVCCCATTCTATCATCAACGGGTACAA25GAPDHFWDCAATGACCCCTTCATTGACC20REVAAATGAGCCCCAGCCTTCT19β-actinFWDGGCACCCAGCACAATGAAG19REVCCGATCCACACGGAGTACTTG21PAR-2FWDCCACCTATCACTTCAGATCAACAAA25REVTTCCTGTAGGCCAGTGTCAAAAT23SLIT2FWDCAGAGCTTCAGCAACATGACCC22REVGAAAGCACCTTCAGGCACAACAG23CXCL12FWDTCAGCCTGAGCTACAGATGC20REVCTTTAGCTTCGGGTCAATGC20COL1A1FWDAGCAAGAACCCCAAGGACAA20REVCGAACTGGAATCCATCGGTC20COL3A1FWDCTGATGGGGTCAAATGAAGGTG22REVCGTGCAACCATCCTCCAGAAC21MMP1FWDATGAAGCAGCCCAGATGTGGAG22REVTGGTCCACATCTGCTCTTGGCA22CCL2FWDAGAATCACCAGCAGCAAGTGTCC23REVTCCTGAACCCACTTCTGCTTGG22CCL5FWDCCTGCTGCTTTGCCTACATTGC22REVACACACTTGGCGGTTCTTTCGG22IL-6FWDAGACAGCCACTCACCTCTTCAG22REVTTCTGCCAGTGCCTCTTTGCTG22IL-8FWDGAGAGTGATTGAGAGTGGACCAC23REVCACAACCCTCTGCACCCAGTTT22

Claims

1. An anti-aging composition characterized by containing an extract of Magnolia officinalis having 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 magnolol, honokiol and 4-O-methylhonokiol as active ingredients.

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, The above extract is an anti-aging composition characterized in that it reduces damaged DNA of aging cells, reduces inflammatory factors, 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 by containing a complex of magnolol, honokiol, and 4-O-methylhonokiol as an active ingredient.

12. In paragraph 11, The above complex is an anti-aging composition characterized in that it restores the function of aging cells to young cells.

13. In paragraph 11, The above complex is an anti-aging composition characterized by minimal cytotoxicity in aging cells and cell proliferation efficacy.

14. In paragraph 11, The above complex is an anti-aging composition characterized in that it has the effect of restoring the function of mitochondria and improving energy metabolism in aging cells.

15. In paragraph 11, The above complex is an anti-aging composition characterized in that it activates the autophagy system of aged cells.

16. In paragraph 11, The above complex is an anti-aging composition characterized in that it reduces damaged DNA of aging cells, reduces inflammatory factors, and enhances elasticity factors.

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

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

19. A pharmaceutical composition for anti-aging, characterized by comprising the composition of Article 11.

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