Composition for alleviating skin damage caused by particulate matter, comprising myricetin as active ingredient

Abstract

The present invention relates to a composition for alleviating skin damage caused by particulate matter (PM), the composition comprising myricetin as an active ingredient. Myricetin, which is an active ingredient of the composition for alleviating skin damage according to the present invention, can effectively reduce oxidative stress caused by PM2.5, endoplasmic reticulum stress and mitochondrial dysfunction, and apoptosis caused by cell exposure to PM2.5. Therefore, the composition comprising myricetin as an active ingredient according to the present invention shows great potential as a therapeutic candidate for treating skin damage caused by PM2.5.

Description

Composition for improving skin damage caused by ultrafine dust containing myricetin as an active ingredient

[0001] The present invention relates to a composition for improving skin damage caused by particulate matter (PM) containing myricetin as an active ingredient, and more specifically, to a PM composition characterized by containing myricetin as an active ingredient. 2.5 The present invention relates to a pharmaceutical, cosmetic and functional food composition for improving skin damage by inhibiting oxidative stress, endoplasmic reticulum stress and mitochondrial damage caused by .

[0002] The skin, a body part directly exposed to the external environment, not only acts as a protective barrier to protect vital organs, but also regulates moisture evaporation and protects against external infections. However, even though skin is capable of blocking viral invasion, external stressors like excessive UV rays and polluted environments can cause skin irritation, ultimately leading to skin aging.

[0003] Modern society is particularly vulnerable to the harmful effects of environmental pollution, such as air pollution. Among hazardous air pollutants, particulate matter (PM) is a serious problem generated from sources such as vehicles, factories, and power plants. Depending on particle size, PM is classified into coarse particles (diameter < 10 μm) and fine particles (diameter < 2.5 μm). Fine dust refers to dust with a diameter of 10 μm or less, and is commonly referred to as PM (particulate matter)-10 (PM) in environmental laws. 10 ) is called PM 10 It penetrates deep into the human alveoli and is a direct cause of various respiratory diseases. In particular, since it is generated by combustion, it is composed of harmful substances such as ionic components such as sulfate, nitrate, and ammonia, as well as metal compounds and carbon compounds. For this reason, it is regulated more strictly than general dust, and recently in developed countries, PM with a diameter of 2.5 ㎛ or less is regulated.2.5 A plan to manage it separately is also being considered.

[0004] PM is ultrafine dust with a diameter of 2.5 ㎛ or less contained in the atmosphere. 2.5 It is also called PM and serves as an important indicator for evaluating and monitoring the environmental quality of air pollution (Lee et al., 2020). 2.5 is composed of a mixture of various elements, metals, biological pollutants, organic and inorganic compounds. Recent studies have shown that PM 2.5 Associations have been identified between exposure and various disorders, including cardiovascular disease, respiratory problems, and skin diseases (Hayes et al., 2020; Wang et al., 2021; Park et al., 2022).

[0005] PM has the ability to penetrate human skin through hair follicles and damage the skin's protective barrier. This penetration allows PM to reach the deeper dermal layers, causing damage and worsening skin conditions (Neo et al., 2022). 2.5 Continuous exposure to PM can have a significant negative impact on the skin, promoting premature skin aging, pigmentation, acne problems, psoriasis exacerbation, and other skin problems. This occurs because the skin acts as a major barrier to external factors that can damage tissues (Delgado-Wicke et al., 2020). In a recent study, HaCaT cells were exposed to PM 2.5 Exposure to PM has been shown to increase intracellular reactive oxygen species (ROS) (Herath et al., 2024; Kang et al., 2024). ROS-induced endoplasmic reticulum (ER) stress, mitochondrial damage, and increased autophagy ultimately lead to cell death (Piao et al., 2018). These findings suggest that PM 2.5 It elucidates the complex mechanisms that cause skin damage and degeneration, suggesting specific targets for preventing and treating these harms.

[0006] PM 2.5 Numerous strategies have been devised to mitigate the adverse effects of oxidative stress on the skin. These approaches primarily focus on utilizing natural compounds with antioxidant properties to mitigate oxidative stress.

[0007] These fine dust particles cause stress within the skin, which is generated by reactive oxygen species. Reactive oxygen species act as intracellular signaling molecules that play a crucial role in maintaining normal cellular functions, such as electron transport within mitochondria and activation of white blood cells. However, reactive oxygen species are unstable and have high oxidizing power, so they easily react with biological substances. If they are not eliminated from the body, they cause oxidative stress (Jeong EJ, et al., NaturalProductSciences 2008, 14, 156-160; Parfenova H., et al., Am J.Physiol. Cell.Physiol. 2006, 290, 1399-1410).

[0008] Much research has been conducted on antioxidants that can prevent oxidative stress, and many antioxidants have been developed and used. Vitamins C and E, in particular, are widely used. Vitamin C is a water-soluble antioxidant, and vitamin E is a fat-soluble antioxidant. While these antioxidants exhibit powerful antioxidant effects, their instability leads to their rapid deterioration when exposed to air, which limits their use.

[0009] The skin is the largest organ in the human body and is exposed to most outdoor pollutants. Numerous epidemiological studies have shown that particulate matter 2.5 (PM) 2.5 ) highlights the link between exposure and skin aging, inflammation, and skin homeostasis disturbances.

[0010] Accordingly, in the present invention, PM 2.5The protective mechanism of myricetin against skin damage induced by .

[0011] This invention was supported by a national research and development project, and has the following information.

[0012] Assignment ID: 1345374806

[0013] Assignment Number: RS-2023-00270936

[0014] Ministry Name: Ministry of Education

[0015] Project Management (Professional) Institution Name: National Research Foundation of Korea

[0016] Research Project Name: University Key Research Institute Support Project

[0017] Research Project Name: Jeju Natural Medicine Research Center

[0018]

[0019] The technical problem to be solved in the present invention is particulate matter 2.5 (PM 2.5 ) to improve skin damage by inhibiting oxidative stress, endoplasmic reticulum stress and mitochondrial damage.

[0020] In order to solve the above technical problem, the present invention provides PM characterized by including myricetin as an active ingredient. 2.5 A composition for improving skin damage caused by .

[0021] The above composition for improving skin damage is characterized by being a composition for inhibiting oxidative stress, endoplasmic reticulum stress, and mitochondrial damage.

[0022] The above skin damage improvement is characterized by antioxidant, anti-aging or anti-inflammatory effects.

[0023] Preferably, the composition containing the above-mentioned myricetin as an active ingredient may be a cosmetic composition, a pharmaceutical composition or a functional food composition.

[0024] In this way, myricetin, an effective ingredient of the composition for improving skin damage of the present invention, is PM 2.5 Oxidative stress, endoplasmic reticulum stress and mitochondrial dysfunction due to PM 2.5 It can effectively reduce cell death caused by cell exposure to PM. Therefore, the composition of the present invention comprising myricetin as an active ingredient 2.5 This suggests that it has great potential as a therapeutic candidate for treating skin damage caused by .

[0025] The following drawings attached to this specification illustrate preferred embodiments of the present invention and, together with the contents of the invention described above, serve to further understand the technical idea of ​​the present invention, and therefore, the present invention should not be interpreted as being limited to matters described in such drawings.

[0026] Figure 1 shows the free radical scavenging activity of myricetin.

[0027] Figure 2 shows the ROS scavenging ability of myricetin.

[0028] Figure 3 shows the cytotoxic effect of myricetin on human HaCaT keratinocytes.

[0029] Figure 4 shows the superoxide anion removal effect of myricetin.

[0030] Figure 5 shows the hydroxyl radical removal effect of myricetin.

[0031] Figure 6 shows the results of flow cytometry analysis measuring the ROS scavenging ability of myricetin using H2DCFDA fluorescent dye.

[0032] Figure 7 is a confocal imaging photograph measuring the ROS scavenging ability of myricetin using H2DCFDA fluorescent dye.

[0033] Fig. 8 is PM 2.5Western blot analysis results showing the protective effect of myricetin against DNA damage induced by .

[0034] Fig. 9 is PM 2.5 The results observed the effect of myricetin on protein carbonylation levels due to oxidative stress induced by .

[0035] Fig. 10 is PM 2.5 The results observed the effects of myricetin on lipid peroxidation and cell membrane damage levels induced by .

[0036] Fig. 11 PM 2.5 This shows the effect of increasing cell survival by myricetin in cells treated with .

[0037] Figure 12 shows the results of a trypan blue analysis of the effect of myricetin on increasing cell viability.

[0038] Figure 13 shows mitochondrial Ca 2+ These are the results of flow cytometry analysis to evaluate the level.

[0039] Fig. 14 PM 2.5 This shows the effect of myricetin on mitochondrial ROS levels induced by .

[0040] Fig. 15 is PM 2.5 This is the result of a Western blot analysis that confirmed the expression level of proteins related to mitochondrial damage induced by .

[0041] Fig. 16 is PM 2.5 JC-1 staining results investigating the protective role of myricetin against mitochondrial damage.

[0042] Fig. 17 is PM 2.5 This study showed the depolarization-improving effect of myricetin on mitochondrial damage.

[0043] Fig. 18 is PM 2.5 The recovery effect of myricetin on the cellular ATP level decreased by .

[0044] Figure 19 shows the effect of myricetin on the release of mitochondrial cytochrome c into the cytoplasm.

[0045] Figure 20 shows the inhibitory effect of myricetin on the cleavage of caspases and PARP.

[0046] Figure 21 PM of myricetin 2.5 It shows the effect of reducing cell death caused by .

[0047] Figure 22 PM of myricetin 2.5 It shows the recovery effect of apoptotic bodies generated by .

[0048] The present invention will be described in more detail below.

[0049] The present invention provides a composition for improving skin damage caused by fine dust (PM) 2.5, characterized in that it contains myricetin as an active ingredient.

[0050] Myricetin (3,3′,4′,5,5′,7-hexahydroxylflavone) used in the present invention is a natural flavonoid present in various fruits, vegetables, and herbs, and exhibits various pharmacological effects including anti-inflammatory, anti-cancer, and antioxidant functions.

[0051] According to one embodiment of the present invention, the composition is characterized as a composition for inhibiting or improving oxidative stress, endoplasmic reticulum stress, and mitochondrial damage.

[0052] The above skin damage improvement is characterized by antioxidant, anti-aging or anti-inflammatory effects.

[0053] According to one embodiment of the present invention, myricetin is PM 2.5 increases the production of reactive oxygen species (ROS), which can induce oxidative stress, resulting in DNA damage, lipid peroxidation, protein carbonylation, and ultimately cell death. In particular, myricetin effectively counteracts these detrimental effects.

[0054] According to one embodiment of the present invention, myricetin is PM 2.5 Mitochondrial Ca induced 2+ Reduces mitochondrial ROS levels, mitochondrial membrane depolarization, and PM 2.5 It can ultimately improve cell death by alleviating cytochrome c release into the cytoplasm and caspase activation induced by PM. In addition, myricetin can also improve cell death by alleviating cytochrome c release into the cytoplasm and caspase activation induced by PM. 2.5 Cytoplasmic Ca induced 2+ It can significantly reduce the level.

[0055] In the present invention, the “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

[0056] The composition of the present invention may be a pharmaceutical composition, cosmetic composition, or functional food composition having a skin damage improvement effect.

[0057] The composition of the present invention may additionally include suitable carriers, excipients, or diluents commonly used in addition to the active ingredient. In this case, the amount of myricetin included in the composition is not particularly limited, but may be 0.0001 to 100 wt%, preferably 0.01 to 50 wt%, based on the total weight of the composition.

[0058] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to myricetin. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one commonly used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0059] The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered parenterally, more preferably by topical application.

[0060] The appropriate dosage of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. The dosage of the pharmaceutical composition of the present invention is generally 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, and more preferably 0.1 to 100 mg / kg for adults. In addition, in the case of external preparation, it is preferable to apply 1.0 to 3.0 ml once to 5 times a day for adults and continue for more than 1 month. However, the above dosage does not limit the scope of the present invention.

[0061] The pharmaceutical composition of the present invention can be manufactured in unit dosage form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person of ordinary skill in the art. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or stabilizer.

[0062] In the present invention, "cosmetic composition" means a product used on the human body to cleanse and beautify the human body, add attractiveness, brighten the appearance, or maintain or improve the health of the skin or hair, and which has a mild effect on the human body.

[0063] In addition to the active ingredient, the cosmetic composition of the present invention may further contain components commonly added to cosmetic compositions, such as conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0064] The cosmetic composition of the present invention can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it can be manufactured into the formulation of an ampoule, a nourishing cream, an astringent toner, an emollient toner, a lotion, an essence, a nourishing gel, or a massage cream.

[0065] When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0066] When the formulation of the present invention is a powder or spray, toluene, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0067] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0068] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth gum, etc. can be used as a carrier component.

[0069] When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0070] The cosmetic composition of the present invention may further contain adjuvants commonly used in the fields of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, film-forming agents, water, ionic or nonionic emulsifiers, fillers, metal ion sequestering and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in cosmetics. In addition, the above ingredients may be introduced in amounts commonly used in the field of dermatology.

[0071] The cosmetic composition of the present invention may be applied alone or in combination, or may be applied in combination with other cosmetic compositions other than those of the present invention. Furthermore, the cosmetic composition of the present invention may be used according to conventional methods, and the frequency of application may vary depending on the user's skin condition or preference.

[0072] The term "health functional food" used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients that have functionality useful to the human body. Here, functionality means obtaining a useful effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material, and can be highly portable.

[0073] The food composition according to the present invention may additionally include not only myricetin as an active ingredient, but also ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents.

[0074] Examples of the carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents, natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

[0075] For example, when the food composition of the present invention is manufactured as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia extract, jujube extract, licorice extract, etc. may be additionally included in addition to the myricetin of the present invention.

[0076] In this way, myricetin, an effective ingredient of the composition for improving skin damage of the present invention, is PM 2.5 Oxidative stress and mitochondrial dysfunction due to PM 2.5 It can effectively reduce cell death caused by cell exposure to PM. Therefore, the composition of the present invention comprising myricetin as an active ingredient 2.5 This suggests that it has great potential as a therapeutic candidate for treating skin damage caused by .

[0077]

[0078] Hereinafter, the present invention will be described in more detail through examples. These examples are intended merely to illustrate the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

[0079]

[0080] 1. Materials and Methods

[0081] (1) Reagents and antibodies

[0082] The following materials were purchased from Sigma-Aldrich (St. Louis, MO, USA): myricetin, diesel particulate matter NIST 1650b (PM 2.5), p38 inhibitor (SB203580), N-acetyl cysteine ​​(NAC), trypan blue solution, thiazolyl blue tetrazolium bromide (MTT), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), 2,2-diphenyl-1-picrylhydrazyl (DPPH). SP600125 (JNK inhibitor) was purchased from Tocris Bioscience (Minneapolis, MN, USA). Hoechst 33342 was purchased from Immuno Chemistry Technologies (Davis, CA, USA). Fluo-4 acetoxymethyl ester (Fluo-4 AM), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and Rhod-2 acetoxymethyl ester (Rhod-2 AM) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Diphenyl-1-pyrenylphosphine (DPPP) was purchased from Molecular Probes (Eugene, OR, USA). U0126 (MEK inhibitor) was purchased from Calbiochem (San Diego, CA, USA). Primary antibodies against phospho-H2A.X, myeloid leukemia-1 (Mcl-1), caspase-3, extracellular signal-regulated kinase (ERK), phospho-ERK, C / EBP-homologous protein (CHOP), inositol-requiring enzyme 1 (IRE1), Jun N-terminal kinase (JNK), and phospho-JNK were purchased from Cell Signaling Technology (Danvers, MA, USA).Primary antibodies against actin, cytochrome c, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Bcl-2-like protein 11 (Bim), caspase-9, p38, phospho-p38, cytochrome c oxidase IV (COX4), poly[ADP-ribose] polymerase (PARP), and protein kinase-like endoplasmic reticulum kinase (PERK), phospho-PERK, eukaryotic initiation factor 2 (eIF2), phospho-eIF2, X-box binding protein 1 (XBP-1), activating transcription factor 6 (ATF6), and glucose-regulated protein 78 kDa (GRP78) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Phospho-IRE1 was purchased from (Abcam, Cambridge, MA, USA). Anti-IgG secondary antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals and reagents were of analytical grade.

[0083]

[0084] (2) PM 2.5 preparation

[0085] PM 2.5 A stock solution was prepared by dissolving PM in dimethyl sulfoxide (DMSO). 2.5 The stock solution was sonicated for 30 minutes to disperse the particles and prevent cluster formation.

[0086]

[0087] (3) Cell culture

[0088] Human keratinocytes (HaCaT) (Cell Lines Service GmbH, Eppelheim, Germany) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotic-antimycotic solution and maintained at 37°C in an incubator with 5% CO2 and 100% humidity.

[0089]

[0090] (4) DPPH radical detection

[0091] Myricetin (1, 2.5, 5, 10, 20, and 40 μM) and NAC (1 mM) were added to a 96-well plate containing 0.15 mM DPPH. The mixture was shaken vigorously in the dark for 3 h, and the remaining DPPH amount was measured at a wavelength of 520 nm.

[0092]

[0093] (5) ROS measurement

[0094] 1.0 × 10 cells 5 Cells were plated at a density of 10 cells / mL. After a 16-h incubation period, cells were treated with myricetin (1, 2.5, 5, 10, 20, and 40 μM) or 1 mM NAC. After a 30-min incubation period, cells were exposed to H2O2 (1 mM). After 30 min, cells were stained with H2DCFDA (25 μM) and fluorescence emission was measured using a fluorescence spectrofluorometer (PerkinElmer, Waltham, MA, USA) for 20 min. Otherwise, cells were incubated with myricetin (10 μM) or 1 mM NAC for 30 min and then exposed to PM. 2.5 (50 μg / mL) for an additional 1 h. After 20 min of incubation with H2DCFDA staining, intracellular ROS levels were detected using an FV1200 laser scanning confocal microscope (Olympus, Tokyo, Japan) and a flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Mitochondrial ROS levels were measured using a mitochondrial ROS detection assay kit purchased from Cayman Chemical (Ann Arbor, Michigan, USA) according to the manufacturer's instructions.

[0095]

[0096] (6) Cell viability evaluation

[0097] 1 × 10 cells 5Cells were seeded in 24-well plates at a density of 10 cells / mL. After 16 h of culture, the cells were treated with taurorsodeoxycholic acid (TUDCA), an ER stress inhibitor, or chemical inhibitors targeting the MAPK pathway (U0126; SB203580; and SP600125), or NAC with or without myricetin for 30 min, and then incubated with PM for 24 h at 37°C. 2.5 (50 μg / mL). MTT solution was added to each well, and the formazan crystals in each well were dissolved with DMSO. The absorbance at 540 nm was read using a VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). In addition, the harvested cells were stained with 0.1% trypan blue solution and incubated at 20°C for 5 minutes. Viable cells that did not show staining were counted under an optical microscope at 20x magnification.

[0098]

[0099] (7) Superoxide anion detection evaluation

[0100] Superoxide anions were generated using xanthine and a xanthine oxidase system and then reacted with DMPO. The resulting DMPO / OOH adduct was identified by JES-FA electron spin resonance (ESR) spectroscopy (JEOL; Tokyo, Japan) as previously reported (Fernando et al., 2020).

[0101]

[0102] (8) Hydroxyl radical detection

[0103] Hydroxyl radicals were generated via the Fenton reaction (H2O2 + FeSO4) and then reacted with DMPO. The resulting DMPO / OH adduct was detected using ESR spectroscopy (JEOL) as previously reported (Piao et al., 2023).

[0104]

[0105] (9) Western blot

[0106] Extracted cell lysates were electrophoresed, transferred, and incubated with primary antibody (1:1,000), followed by secondary antibody. The presence of protein bands was detected using Amersham ECL Western Blot Detection Reagent (GE Healthcare Life Sciences, Buckinghamshire, UK).

[0107]

[0108] (10) Protein carbonylation

[0109] Quantification of protein carbonylation was performed using the OxiSelect™ Protein Carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA) according to the instructions provided by the manufacturer.

[0110]

[0111] (11) Lipid peroxidation analysis

[0112] Cells were treated with myricetin (10 μM) and / or PM 2.5 (50 μg / mL) for 24 h and stained with 5 μM of the fluorescent probe DPPP. Lipid adducts of DPPP oxide were detected using confocal microscopy.

[0113]

[0114] (12) Ca 2+ Numerical quantification

[0115] Myricetin and PM 2.5 Cells treated with were stained with Fluo-4 AM or Rhod-2 AM dye for 30 min. Then, intracellular and mitochondrial Ca 2+ The values ​​were examined using a confocal microscope (Olympus) and a flow cytometer (Becton Dickinson).

[0116]

[0117] (13) Measurement of mitochondrial membrane potential (Δψm)

[0118] Mitochondrial Δψm was analyzed by staining treated cells with the lipophilic cationic fluorescent dye JC-1 and then using a confocal microscope (Olympus) and flow cytometer (Becton Dickinson).

[0119]

[0120] (14) Determination of ATP levels

[0121] The amount of ATP in the treated cells was measured using an ATP detection kit (Invitrogen, Carlsbad, CA, USA) according to the instructions provided by the manufacturer.

[0122]

[0123] (15) Detection of apoptotic sub-G1 hypodiploid cells

[0124] After fixation in 70% ethanol, cells were washed twice with PBS + 2 mM ethylenediaminetetraacetic acid (EDTA). Cells were then stained with propidium iodide (PI) solution (PI 100 μg / mL, RNase A 100 μg / mL, EDTA 2 mM) at 37°C for 30 min in the dark. The fraction of sub-G1 cell populations was assessed using a flow cytometer.

[0125]

[0126] (16) Hoechst 33342

[0127] Cells treated with TUDCA or / and myricetin, PM 2.5 were treated. Then, the cells were stained with the DNA-specific fluorescent dye Hoechst 33342 and incubated at 37°C for 10 min. Cells with fragmented nuclei stained with Hoechst 33342 were considered apoptotic. Cell staining was visualized under a fluorescence microscope, and the percentage of apoptotic cells was quantified.

[0128]

[0129] (17) Statistical analysis

[0130] All analyses are expressed as mean ± standard error. One-way analysis of variance and Tukey's test were performed on the collected data using Sigma Stat version 3.5 (Systat Software Inc., San Jose, CA, USA). Statistical significance was defined as P < 0.05.

[0131]

[0132] 2. Results

[0133] (1) Effect of myricetin on free radical scavenging activity

[0134] To evaluate the effect of myricetin on free radical scavenging in a cell-free system, DPPH radical scavenging effect was performed.

[0135] Figure 1 shows the free radical scavenging activity of myricetin. As shown here, the DPPH radical scavenging capacity increased as the myricetin concentration increased from 1 to 40 μM, while the positive control, NAC, exhibited the highest DPPH radical scavenging capacity.

[0136] Additionally, the intracellular ROS scavenging activity was evaluated by treating cells with H2O2.

[0137] Figure 2 shows the ROS scavenging activity of myricetin. As shown here, myricetin showed a dose-dependent improvement in ROS scavenging ability, while NAC showed the highest scavenging effect.

[0138] Figure 3 shows the cytotoxic effect of myricetin on human HaCaT keratinocytes. As shown here, myricetin did not exhibit cytotoxic effects on human HaCaT keratinocytes at concentrations up to 10 μM. However, a significant cytotoxic effect was observed at a higher concentration of 20 μM. Based on these results, we decided to use a concentration of 10 μM in all subsequent experimental procedures involving myricetin. We then evaluated the ability of myricetin to directly scavenge ROS, thereby removing superoxide anions and hydroxyl radicals, using electrochemical resonance (ESR) spectroscopy.

[0139] Figure 4 demonstrates the superoxide anion scavenging effect of myricetin. As shown here, the addition of DMPO / ·OOH to the xanthine / xanthine oxidase system significantly increased the signal compared to the control, and myricetin significantly reduced this signal.

[0140] Figure 5 shows the hydroxyl radical scavenging effect of myricetin. As seen here, in the FeSO4+H2O2 system, hydroxyl radicals generated higher signal values, but myricetin reduced the hydroxyl radical signal.

[0141]

[0142] (2) Myricetin PM 2.5 Effects on macromolecular damage caused by oxidative stress induced by

[0143] PM 2.5 The ROS scavenging ability of myricetin after treatment was evaluated using H2DCFDA fluorescent dye.

[0144] Figure 6 shows the results of flow cytometry analysis using H2DCFDA fluorescent dye to measure the ROS scavenging ability of myricetin. As shown here, the results of flow cytometry analysis show that PM cells are more effective than control cells. 2.5Higher fluorescence intensity (FI) values ​​were observed in treated cells (FI:1708), and myricetin reduced the FI value to 1469.

[0145] Figure 7 is a confocal imaging image measuring the ROS scavenging ability of myricetin using H2DCFDA fluorescent dye. As shown here, confocal imaging shows that myricetin is PM 2.5 It was shown to reduce the green fluorescence intensity induced by exposure.

[0146] ROS production is associated with cellular macromolecular damage (DNA, lipids, proteins) (Juan et al., 2021). Therefore, we evaluated the effect of myricetin on cellular DNA damage using phosphorylated H2A.X as a DNA-related biomarker indicating DNA double-strand breaks (Schㆌtz et al., 2021).

[0147] Fig. 8 is PM 2.5 Here are the results of Western blot analysis showing the protective effect of myricetin against DNA damage induced by PM. As shown here, Western blot analysis showed that PM 2.5 showed an increase in phosphorylated H2A.X levels in cells exposed to PM, which was alleviated by treatment with myricetin. 2.5 It was suggested that there is a protective effect against DNA damage caused by .

[0148] Fig. 9 is PM 2.5 The results observed the effect of myricetin on protein carbonylation levels due to oxidative stress induced by PM. As shown here, 2.5 Cells treated with PM showed increased levels of protein carbonylation, a marker of protein damage due to oxidative stress, but myricetin did not. 2.5 Inhibited the production of carbonyls induced by .

[0149] Fig. 10 is PM 2.5The results observed the effects of myricetin on lipid peroxidation and cell membrane damage levels induced by PM. As shown here, 2.5 The increased blue fluorescence intensity in cells treated with , indicated higher levels of lipid peroxidation and membrane damage, which were significantly reduced by myricetin treatment.

[0150] Fig. 11 PM 2.5 This shows the effect of increasing cell viability by myricetin in cells treated with PM. As seen here, the cell viability was approximately 100% in the untreated control cells, while in the PM 2.5 Cells treated with PM showed a 58% decrease in cell viability. On the other hand, myricetin showed a 58% decrease in cell viability. 2.5 In cells treated with , cell viability was significantly increased to 76%, and NAC showed a cell viability level of 84%.

[0151] Figure 12 shows the results of trypan blue analysis to determine the effect of myricetin on increasing cell viability. As shown here, PM 2.5 Although induced cytotoxicity was confirmed, myricetin alleviated this effect.

[0152]

[0153] (3) Myricetin PM 2.5 Effects on induced mitochondrial damage and cell death

[0154] Cytoplasmic Ca 2+ As the cytosolic Ca increases, mitochondria 2+ Ca to prevent overload 2+ is rapidly absorbed. However, excessive Ca into the mitochondria 2+ Influx (mitochondrial Ca 2+ (called overload) is associated with increased ROS production and mitochondrial damage (Calvo-Rodriguez et al., 2020). Mitochondrial Ca 2+ Rhod-2 AM staining was used to evaluate the values.

[0155] Figure 13 shows mitochondrial Ca 2+ Here are the results of flow cytometry analysis to evaluate the level. As seen here, PM 2.5 Exposure to mitochondrial Ca 2+ The numbers increased, and myricetin weakened them.

[0156] Fig. 14 PM 2.5 This shows the effect of myricetin on mitochondrial ROS levels induced by PM. As seen here, myricetin 2.5 It reversed the increase in mitochondrial ROS levels induced by apoptosis. Mitochondria play a key role in the cell death signaling pathway during apoptosis (Zhang et al., 2022).

[0157] The expression levels of mitochondrial damage-related proteins were investigated using Western blot analysis.

[0158] Fig. 15 is PM 2.5 This is the result of a Western blot analysis that confirmed the expression level of proteins related to mitochondrial damage induced by PM. As shown here, myricetin 2.5 restored the levels of Bcl-2 and Mcl-1 decreased by PM. Conversely, myricetin 2.5 Inhibited the increased production of Bax and Bim induced by .

[0159] Also, PM 2.5 To further investigate the protective role of myricetin against increased mitochondrial damage, cells were stained with JC-1 dye.

[0160] Fig. 16 is PM 2.5 The JC-1 staining results investigating the protective role of myricetin against mitochondrial damage by PM are shown in Fig. 17. 2.5 The depolarization-improving effect of myricetin on mitochondrial damage caused by PM is shown here. 2.5In the exposure group, green fluorescence was increased, indicating increased mitochondrial depolarization, which was improved by pretreatment with myricetin.

[0161] Fig. 18 is PM 2.5 The recovery effect of myricetin on the cellular ATP level decreased by PM is shown here. As seen here, myricetin 2.5 Restored the reduced cellular ATP levels.

[0162] Mitochondrial damage triggers the release of pro-apoptotic factors and activates apoptotic signaling pathways, ultimately leading to cell death. Release of cytochrome c from mitochondria into the cytoplasm activates caspase-9 (Jiang et al., 2022).

[0163] Figure 19 shows the effect of myricetin on the release of mitochondrial cytochrome c into the cytoplasm. As shown here, myricetin inhibits PM 2.5 It counteracted the effect of mitochondrial cytochrome c release into the cytosol.

[0164] Figure 20 shows the inhibitory effect of myricetin on the cleavage of caspases and PARP. As shown here, PM 2.5 significantly induced cleavage of caspases and PARP. In particular, pretreatment with myricetin effectively inhibited cleavage.

[0165] Figure 21 PM of myricetin 2.5 It shows the effect of reducing cell death due to PM. As can be seen here, myricetin reduces the proportion of cells in the G1 or lower phase. 2.5 was shown to reduce cell death due to Hoechst 33342 nuclear staining compared to the control group. 2.5 It was shown that the number of apoptotic cells increased significantly in the exposure group.

[0166] Figure 22 PM of myricetin 2.5It shows the recovery effect of apoptotic bodies generated by PM. As seen here, pretreatment with myricetin 2.5 recovered apoptotic bodies generated by PM. These results show that myricetin is effective in activating HaCaT cells in PM 2.5 It is shown to effectively protect against induced mitochondrial damage and apoptosis.

[0167]

[0168] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0169]

[0170] In this way, myricetin, an effective ingredient of the composition for improving skin damage of the present invention, is PM 2.5 Oxidative stress, endoplasmic reticulum stress and mitochondrial dysfunction due to PM 2.5 It can effectively reduce cell death caused by cell exposure to PM. Therefore, the composition of the present invention comprising myricetin as an active ingredient 2.5 This suggests that it has great potential as a therapeutic candidate for treating skin damage caused by .

Claims

1. PM characterized by containing myricetin as an active ingredient 2.5 A pharmaceutical composition for improving skin damage caused by .

2. In paragraph 1, The above composition for improving skin damage is characterized in that it is a composition for inhibiting oxidative stress, endoplasmic reticulum stress and mitochondrial damage, PM 2.5 A pharmaceutical composition for improving skin damage caused by .

3. In paragraph 1, The above skin damage improvement is characterized by antioxidant, anti-aging or anti-inflammatory effects, PM 2.5 A pharmaceutical composition for improving skin damage caused by .

4. PM characterized by containing myricetin as an active ingredient 2.5 A cosmetic composition for improving skin damage caused by .

5. In paragraph 4, The above composition for improving skin damage is characterized in that it is a composition for inhibiting oxidative stress, endoplasmic reticulum stress and mitochondrial damage, PM 2.5 A cosmetic composition for improving skin damage caused by .

6. In paragraph 4, The above skin damage improvement is characterized by antioxidant, anti-aging or anti-inflammatory effects, PM 2.5 A cosmetic composition for improving skin damage caused by .

7. PM characterized by containing myricetin as an active ingredient 2.5 Functional food composition for improving skin damage caused by .

8. In paragraph 7, The above composition for improving skin damage is characterized in that it is a composition for inhibiting oxidative stress, endoplasmic reticulum stress and mitochondrial damage, PM 2.5 Functional food composition for improving skin damage caused by .

9. In paragraph 7, The above skin damage improvement is characterized by antioxidant, anti-aging or anti-inflammatory effects, PM 2.5 Functional food composition for improving skin damage caused by .

Images