KR102269087B1 - Cosmetic composition comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal for protecting skin against particulate matter - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin protection against fine dust comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient. available

Description

Cosmetic composition comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal for skin protection against fine dust containing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal protecting skin against particulate matter}

The present invention relates to a cosmetic composition for skin protection against fine dust, a quasi-drug composition, and a composition for external application for skin comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

Plum (Japanese apricot, scientific name: Prunus mume ) has long been popular as a health food among Koreans, Japanese, and Chinese. Japanese apricot extract (JAE) has several beneficial biological effects, such as reducing oxidative stress and inflammation, and enhancing innate immunity. Concentrated plum extract may prevent cardiovascular disease as an antioxidant effect on angiotensin II-induced ROS production. In addition, the triterpenoids of plum extract have antitumor and anti-inflammatory effects.

Fine dust is a very small substance that is invisible to our eyes. It is a particulate matter with a diameter of 10 μm or less that floats or scatters in the atmosphere for a long time. It includes yellow dust, fuel gas from fossil fuels, factory or automobile fumes, or cigarette smoke. . Fine dust is adsorbed to the skin, and when the sebum secreted from the skin is mixed with pollutants, fine dust, and bacteria in the fine dust, skin troubles such as acne can easily occur or become serious.

Tobacco smoke contains an abundance of toxic chemicals including reactive aldehydes that cause DNA damage and cell death. In humans, members of the aldehyde dehydrogenase (ALDH) superfamily of 19 NAD(P)+-dependent isoenzymes promote aldehyde oxidation. ALDH3A1, the most strongly induced isoenzyme in the ALDH superfamily, attenuates tobacco smoke-induced DNA damage and cytotoxicity. Numerous other ALDH species, including ALDH2 and 3A1, promote oxidation of aliphatic, aromatic and lipid peroxidation (LPO)-derived aldehydes, including 4-hydroxy-2-nonenal (4-HNE). ALDH3A1 also attenuates LPO-mediated growth inhibition, UV-ray-induced cytotoxicity, ROS-induced protein modification, and gene toxin-induced DNA damage and apoptosis.

Cigarette smoke activates DNA damage response (DDR) mediated by phosphoinositide 3-kinase-associated protein kinase (PIKK), including ataxia teleangiectasia mutated (ATM). Upon formation of DNA double-strand breaks (DSBs), ATM is activated by autophosphorylation at the serine 1981 residue and phosphorylates the serine 139 residue (γH2AX) of the H2AX variant at the chromatin proximal DSB site. γH2AX is required to relay subsequent DDR signaling and DNA repair.

Cigarette smoke harms skin health by stimulating various chemical substances contained in it. Cigarette smoke dries out the skin, lacks oxygen and nutrients, makes it brittle, and accelerates skin aging. However, the potential usefulness of plum extract in skin diseases induced by fine dust including tobacco smoke has not yet been evaluated.

Republic of Korea Patent Publication No. 10-2008-0085404

It is an object of the present invention to provide a cosmetic composition for skin protection against fine dust, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for skin protection against fine dust, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

Another object of the present invention is to provide an external composition for skin protection against fine dust, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

The present inventors isolated a compound having a cytoprotective effect against tobacco smoke-induced toxicity from a concentrated Japanese apricot extract (JAE), and as a result of structural analysis of the isolated compound, 5-hydroxymethyl-2-furaldehyde bis A novel molecule of (5-formylfurfuryl)acetal (named FA-1) was successfully identified and completed the present invention.

As one aspect for achieving the above object, the present invention provides a cosmetic composition for skin protection against fine dust comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient. to provide.

The cosmetic composition of the present invention includes 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

The cosmetic composition comprising the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal has a skin protection effect against fine dust.

In the present invention, "5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal" is a compound having the structure of Formula 1 and may be isolated from plum.

In an embodiment of the present invention, polysaccharides were precipitated in the concentrate of the plum extract with ethanol to separate, the hexane fraction was separated, and the chloroform fraction fractionated with a chloroform solvent was subjected to CPC chromatography using 5-hydroxymethyl-2-fur. The aldehyde bis(5-formylfurfuryl)acetal was isolated.

Therefore, the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal may be separated from the chloroform fraction of the plum extract, and specifically, the chloroform fraction may be separated by performing CPC chromatography. .

In the present invention, “fine dust” refers to particulate matter with a diameter of 10 μm or less that floats or scatters in the atmosphere for a long time as very small substances that are invisible to our eyes. Fossil fuels including coal and petroleum are combustion gases, and emissions from manufacturing and automobile fumes; Or, it is an air pollutant that contains cigarette smoke and is adsorbed to the skin to cause skin damage or skin aging. The fine dust may specifically be cigarette smoke.

In one embodiment of the present invention, 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal has a cytoprotective effect against tobacco smoke extract (CSE)-induced cytotoxicity in human epidermal keratinocytes. was confirmed, and it was confirmed that the phosphorylation of ATM and H2AX activated by DNA damage induced by cigarette smoke was inhibited.

That is, it was confirmed that 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal protects against tobacco smoke extract (CSE)-induced apoptosis and DNA damage in human epidermal keratinocytes.

Therefore, the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal can be used as an active ingredient in a cosmetic composition for skin protection against fine dust.

In the present invention, skin protection against fine dust may be to reduce or inhibit apoptosis induced by fine dust in skin cells.

In addition, the skin protection against the fine dust may be to inhibit DNA damage induced by fine dust, specifically, to inhibit the phosphorylation of ATM and H2AX activated by DNA damage.

In addition, in the Examples of the present invention, it was confirmed that 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal increased the expression of ALDH2D and WRN in human epidermal keratinocytes in a concentration-dependent manner, and cigarette smoke It was confirmed that the increase in the expression of WRN reduced by the extract.

Therefore, 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal of the present invention may protect the skin from fine dust by increasing the expression of ALDH2D or WRN, and more specifically, It may be to protect the skin by suppressing or alleviating the decrease in the expression of WRN induced by the

The cosmetic composition of the present invention may include, without limitation, commonly acceptable ingredients in addition to the active ingredients, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. have.

The cosmetic composition according to the present invention includes solutions, external ointments, creams, foams, nutrient lotions, softening lotions, packs, emulsions, makeup bases, essences, liquid detergents, bathing agents, sunscreen creams, sun oils, suspensions, emulsions, Paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, one or more formulations selected from the group consisting of patches and sprays, but are limited thereto no.

In addition, the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and conventional ingredients include, for example, oil, water, surfactant, humectant, lower alcohol, A thickener, a chelating agent, a colorant, a preservative, a fragrance, etc. may be appropriately mixed, but the present invention is not limited thereto. The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation.

When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures thereof may be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component. In particular, in the case of a spray, additional chlorophyll propellants such as fluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty ester, fatty acid ester of polyethylene glycol or sorbitan may be used. have.

When the formulation of the present invention is a suspension, as a carrier component a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters; Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionate, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugar, etc. may be used as carrier components. can

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, methyltaurate, sarcositate, fatty acid amide as carrier components Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters can be used.

In another aspect, the present invention provides a quasi-drug composition for skin protection against fine dust comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

The skin protection against 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal and fine dust is the same as described above.

The term "quasi-drug" as used in the present invention refers to items with a milder action than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals, for example, in the Pharmaceutical Affairs Act. According to the definition of quasi-drugs, quasi-drugs are products that are not used for medicinal purposes. Textile and rubber products used for the treatment or prevention of diseases of humans and animals, those that have a slight or no direct action on the human body, and are not instruments or machines, and similar products. , and disinfectants and insecticides to prevent infectious diseases.

The type or formulation of the quasi-drug composition of the present invention is not particularly limited, but preferably disinfectant, shower foam, gargrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

When the composition of the present invention is included in a quasi-drug for the purpose of skin protection against fine dust, the composition may be used as it is or used together with other quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use, and the quasi-drug composition according to the present invention contains the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal of 0.01 based on the total weight of the composition. It may contain ~20% by weight.

In another aspect, the present invention provides an external composition for skin protection against fine dust, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

The 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal, skin protection against fine dust, and physiologically acceptable salts are as described above.

The composition for external application for skin according to the present invention may include, but is not limited to, ointments, lotions, solubilizing burns, suspensions, emulsions, creams, gels, sprays, patches, warning agents, patches, or water pastes. It may be formulated with any base known in the art. The composition for external application for skin according to the present invention may contain the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal in an amount of 0.01 to 20% by weight based on the total weight of the composition.

The present invention relates to a cosmetic composition for skin protection against fine dust, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient. is available as

1 is a diagram showing that plum extract (JAE) increases WRN and ALDH proteins in immortalized human bronchial epithelial cells. (A) HBEC2 cells were cultured in the absence or presence of 0, 2, 4 and 6% tobacco smoke extract (CSE) for 24 h after treatment with JAE (0, 1, 2 and 4 mg/mL) for 24 h. . Cell viability was analyzed by MTT analysis. (B) Immortalized HBEC2 cells were treated with JAE (0, 1, 2 and 4 mg/mL) for 24 h and then cultured in the presence or presence of 1.5% CSE for 24 h. Immunoblot analysis was analyzed for WRN, ALDH2 and ALDH3A1 proteins. Data for two independent experiments using three triplicate samples are expressed as mean ± SEM.
2 is a view showing the purification process of FA-1 [5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal] from plum extract (JAE).
3 is a diagram showing the structure determination of FA-1 [5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal]. (A) ESI-TOFMS data of FA-1, (B) 1H NMR data (chemical shift, ppm; JH,H values, Hz), (C) 13C NMR data (chemical shift, ppm) and (D) HMBC correlation is a relationship
Figure 4 is a diagram showing that FA-1 increases ALDH and WRN proteins in a dose dependent manner and protects against tobacco smoke-induced cytotoxicity (MTT and flow cytometry) in immortalized human bronchial epithelial cells. (A) Results of expression analysis of ALDH2, ALDH3A1 and WRN treated with FA-1 [5, 25, 50, 75 and 150 nM in DMSO (dimethylsulfoxide)] or vehicle (corresponding to 1.0% volume of DMSO) Then, HBEC2 cells were cultured. (B) MTT assay showed that HBEC2 cells were incubated with NAC (2 mM), FA-1 (150 nM) and vehicle in the presence or absence of 0 and 6% CSE for 24 h and assayed for viability. Data are expressed as mean ± SEM (**p<0.01) cytotoxicity in cultured immortalized human bronchial epithelial cells. (C) As a result of flow cytometry, annexin V and propidium iodide (PI) staining were performed and apoptosis was analyzed.
5 is a diagram showing that FA-1 attenuates tobacco smoke-induced oxidative damage and DNA damage in HBEC2 cells. HBEC2 cells were incubated with FA-1 (150 nM) in the presence or absence of 2% CSE for 24 h, and immunoblot analysis was performed for phosphorylation of 4-HNE and ATM.
6 is a diagram showing that FA-1 increases ALDH2 and WRN proteins in a dose-dependent manner and attenuates smoke-induced down-regulation of WRN proteins in normal human epidermal keratinocytes. (A) ALDH2 and WRN immunoblot results. NHEK cells were cultured after treatment with various concentrations of FA-1 [5, 25, 50, 75 and 150 nM in DMSO (dimethylsulfoxide)) or vehicle (corresponding to 1.0% volume of DMSO). (B) The results of immunoblot analysis of attenuation of tobacco smoke-induced downregulation. NHEK cells were incubated with FA-1 (150 nM) and vehicle in the presence or absence of 2% CSE for 24 h.
7 is a diagram showing that FA-1 prevents tobacco smoke-induced cytotoxicity and DNA damage in normal human epidermal keratinocytes. (A) NHEK MTT (3-(4,5-dimethylthia) after treatment with FA-1 [150 nM in DMSO (dimethylsulfoxide)) for 24 h in the presence or absence of 0, 2, 4 and 6% CSE Zol-2-yl)-2,5-diphenyl tetrazolium bromide)-based cell viability. Data are expressed as mean ± SEM (**p<0.01) cytotoxicity in cultured NHEK. (B) NHEK cells were treated with 150 nM of FA-1 for 24 h in the presence or absence of 2% CSE. Immunoblot analysis was performed for phosphorylation of ATM and H2AX.
8 is a cell viability analysis result for tobacco smoke extract-induced cytotoxicity of fractions F1 to F4 obtained by refractionation of the chloroform fraction.
9 to 14 are structural analysis results of the isolated active compound FA-1. Specifically, 9 is a 1H NMR spectrum, FIG. 10 is a 13C NMR spectrum, FIG. 11 is a COSY spectrum, FIG. 12 is a TOCSY spectrum, FIG. 13 is an HMBX spectrum, and FIG. 14 is a HSQC spectrum result.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Materials and Methods>

1. Preparation of reagents and antibodies

Chemicals were purchased from Sigma Aldrich, and proteinase and phosphatase inhibitors were purchased from Thermo Scientific. Phosphorylated ATM (serine 1981), ALDH2, and ALDH3A1 antibodies were purchased from Abcam, phosphorylated H2AX antibody from Cell signaling, 4-HNE antibody from R&D Systems, and β-actin from Sigma-Aldrich.

2. Isolation and Purification of FA-1

The concentrated Prunus mume extract was purchased from Fysee Inc. in Korea and stored at -20°C or lower.

FA-1 was isolated as shown in FIG. 2 . 250 g of the concentrated plum extract was dissolved in 1 L of distilled water, and 2 L of ethanol was added to precipitate and remove polysaccharide. Then, n-hexane was added to separate the hexane fraction, and then chloroform was added to separate the chloroform fraction to obtain a chloroform fraction (9.26 g). Next, four subfractions (F1, F2, F3 and F4) were prepared from the chloroform fraction using an ODS column. The cytoprotective effects of these four subfractions (F1 to F4) on tobacco smoke extract-induced cytotoxicity were analyzed. As a result of the analysis, it was confirmed that the F2 fraction had the highest protective effect against tobacco extract (CSE)-induced cytotoxicity (FIG. 8).

To separate the active compound from the chloroform fraction, centrifugation chromatography was performed using two phases consisting of n-hexane : ethyl acetate (EtOAc) : methanol (MeOH) : water = 3: 7: 5: 6 (v/v). Graphography (CPC) was performed. To purify the active compound, the CPC eluent (F2, 815.25 mg) was subjected to preparative ODS thin layer chromatography (TLC) using 50% and 60% methanol (MeOH) solvents, monitored under UV (280 nm), FA-1 (18.53 mg) was successively isolated and purified from the F2 fraction.

3. Structural Analysis of FA-1

Since FA-1 provides effective protection against tobacco smoke extract (CSE) induced cytotoxicity, the structure of the FA-1 compound was analyzed. NMR spectra were recorded on an Agilent 600 MHz NMR spectrometer (Agilent Technologies, Santa Clara, USA) in 0.4 ml of CD3OD at 20°C. Spectra were referenced to the residual solvent signal with resonance at δH/C = 3.30/49.8 ppm (CDOD). Signals were assigned based on analysis of COSY, HSQC and HMBC spectra. MS analysis was recorded on ESI-TOFMS (micrOTOF-Q II, Bruker, Billerica, MA, USA).

4. Manufacture of tobacco smoke extract

Tobacco smoke extract (CSE) solution was prepared by purchasing a research cigarette (3R4F) from the University of Kentucky. The CSE solution was prepared by modifying the Blue and Janoff method. After putting 10 ml of sterile DMEM medium into a 50 ml plastic syringe, 40 ml of cigarette smoke was drawn into the syringe and mixed with the medium by vigorous shaking. Thereafter, it was filtered through a 0.22 μm filter to remove large particles to prepare a tobacco smoke extract. The tobacco smoke extract was designated as 100% concentration, and the tobacco smoke extract was used immediately after preparation.

5. Cell Culture and Cell Viability Analysis

Immortalized human bronchial epithelial cells (HBEC2) were provided by Dr. Shay and Minna of Southwestern Medical Center, Dallas, Texas.

Normal human epidermal keratinocytes (NHEK) were supplemented with 5 μg/L human recombinant epidermal growth factor and 50 mg/L bovine pituitary extract (Invitrogen) in plates coated with FNC coating mix (Athena ES). It was cultured in a medium free of keratinocyte serum.

All cells were grown under standard incubator conditions of _{5% CO 2} at 37°C. Experiments were performed in 12-well and 6-well tissue culture plates at a starting cell density of 10 x 10 ³ /cm ² .

Cell viability assay is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) was measured and analyzed. HBEC2 and NHEK cells were cultured in 12-well plates for 24 hours and treated with 150 nM FA-1 dissolved in DMSO for 24 hours. After treatment for 24 hours, the cells were exposed to Cigarette smoke extract (CSE) at a concentration of 2%, 4% or 6% for 24 hours. Absorbance was measured at 540 nm, and the relative cell viability of the tobacco smoke extract-treated cells compared to the vehicle control cells not treated with the tobacco smoke extract was measured.

6. Flow Cytometric analysis

Apoptosis was analyzed using flow cytometry. After treatment with FA-1 (150 nM) and exposure to tobacco smoke extract (CSE), cells were harvested by trypsinization. 10 ⁵ cells using 10 µL of propidium iodide (PI) and 5 µL of Annexin V-FITC in 1x binding buffer (0.01 M HEPES, pH 7.4; 0.14 M NaCl; 0.25 mM CaCl _{2 )} was dyed.

Cells were incubated at room temperature for 15 min in the dark and the percentage of FITC- and PI-positive cells was quantified using a FACS Canto-II flow cytometer and analyzed using Flowjo software (version 7.6.3).

7. Immunoblot Analysis

After treatment with FA-1 (150 nM) and exposure to tobacco smoke extract, cells were lysed using 1X RIPA buffer (Sigma-Aldrich) containing proteinase and phosphatase inhibitor. seafood was obtained. The protein concentration of the cell lysate was measured with a BCA protein assay reagent. Protein samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat dry milk (TBS solvent containing 0.5% Tween-20), and incubated with primary and secondary antibodies. Visualized with enhanced chemiluminescence reagent (Advansta). The blot was stripped and reprobed with an antibody against β-actin to confirm equal loading of each protein.

Relative quantification of signal intensity was determined using Image Lab software (Bio-Rad, Hercules, CA, USA) and expressed as relative concentrations.

8. Statistical Analysis.

For all comparisons involving multiple treatment groups, treatment effects were confirmed using one-way analysis of variance (ANOVA). The effect of individual treatments was evaluated using the p-value based on the calculated contrast. The results were graphically expressed as mean ± SEM, and p<0.05 was considered statistically significant.

<Result>

1. Cytoprotective effect of plum concentrate against tobacco smoke extract-induced cytotoxicity

To confirm the cytotoxic protective effect of plum extract against tobacco smoke extract, cell viability and changes in WRN, ALDH2, and ADLH3A1 protein expression were analyzed.

1 is a diagram showing the increase of WRN, ALDH2, and ADLH3A1 proteins in human bronchial epithelial cells of Japanese apricot extract (JAE).

HBEC2 cells were treated with a plum extract at concentrations of 0, 1, 2 and 4 mg/mL for 24 hours, then treated with 0, 2, 4 and 6% concentrations of tobacco smoke extract (CSE) for 24 hours and cultured. The cells were analyzed for cell viability by MTT analysis. As a result of the analysis, it was confirmed that the plum extract protected the cells from cytotoxicity caused by the tobacco smoke extract at all concentrations (FIG. 1A).

In a previous study, the present inventors reported that cigarette smoke induces cell aging and cell migration disorders through downregulation of Werner's syndrome protein (WRN). Therefore, preservation of Werner's syndrome protein (WRN) may have therapeutic effects in smoking-related diseases.

HBEC2 cells were incubated with JAE (1,2 and 4 mg/mL) or vehicle (corresponding to 1.0% volume of sterile HO), and the effect of JAE on ALDH2, ALDH3A1 and WRN proteins in vitro was also measured. Plum extract increased the expression of ALDH2, ADLH3A1 and WRN proteins in a dose-dependent manner (FIG. 1B).

That is, it was confirmed that plum extract (JAE) protects HBEC2 cells from tobacco smoke extract-induced cytotoxicity and increases ALDH2, ALDH3A1 and WRN proteins in HBEC2 cells.

2. Determination of the structure of FA-1

Since FA-1 provides effective protection against tobacco smoke extract (CSE) induced cytotoxicity, the structure of the FA-1 compound was analyzed. 3 is an NMR analysis result of FA-1. ¹ H NMR, ¹³ C NMR spectra and the main HMBC correlation of FA-1 are shown in FIG. 3 . FA-1 has the molecular formula C ₁₈ H ₁₆ O ₈ (C ₁₈ H ₂₀ NO ₈ + 378.1183 calculated for ESI-TOFMS [M + NH 4 ] ⁺ m/z 378.1188) ( FIG. 3A ).

The structure of this compound was revealed as a trimer of 5-hydroxymethyl-2-furaldehyde based on their NMR spectra (1 H NMR, 13 C NMR, COSY, HSQC and HMBC) (Supplementary Figures S2-9). In the 1H NMR spectrum, two sets of similar signals with an integral ratio of almost 2:1 were displayed. Two sets of these signals are conjugated aldehyde signals (δC 178.1, δH 9.54, s), J values of 3.2 and 3.5 Hz, two sets of olefinic methine protons binding to each other, and two pairs of olefinic quaternary carbons (δC 151.8/157.4 and 155.1). /160.2), non-equivalent (δH 4.67, 4.69) and equivalent (δH 4.47) hydroxylmethyl proton signals were analyzed as 5-hydroxy-2-furaldehyde backbones. Also, one acetal signal (δC 98.1, δH 5.81) was HMBC correlated with both furan C2 and non-identical hydroxymethyl signals. The structure of this active compound, along with other major HMBC interactions shown in Figure 3d, was identified as 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal and designated FA-1. The assignment of 1H and 13C NMR signals is shown in FIGS. 3B and C, and the NMR spectra are shown in FIGS. 8 and 9 .

3. The bronchial cytoprotective effect of 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal (FA-1) and the increase in ALDH and WRN proteins

Next, in order to analyze the cytoprotective effect of 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal (FA-1) isolated from plum extract on the tobacco smoke extract-induced toxicity, human bronchial epithelium Cell viability of cells (HBEC2) and changes in ALDH and WRN protein expression were analyzed by immunoblot.

5-Hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal [5, 25, 50, 75 and 150 nM in DMSO (dimethylsulfoxide)] or vehicle (corresponding to 1.0% volume of DMSO) After treatment with various concentrations of HBEC2 cells were cultured. FA-1 increased ALDH2, ADLH3A1 and WRN protein expression in a dose-dependent manner (Fig. 4A), i.e., the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal ALDH2 in HBEC2 cells. , ALDH3A1 and WRN proteins were improved to confirm that the effect of 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal was the same as that of plum extract (JAE).

Therefore, in subsequent experiments, FA-1 at a concentration of 150 nM was determined and used. In addition, to determine the effect of FA-1 on tobacco smoke extract (CSE) induced cytotoxicity, HBEC2 cells were treated with 2 mM N-acetyl-L-cysteine (NAC) in vehicle in the presence or absence of 6% CSE for 24 h. ) or after incubation with 150 nM of FA-1, cell viability was analyzed. As a result of the analysis, it was confirmed that 2 mM NAC had no protective effect against 6% tobacco smoke extract (CSE)-induced cytotoxicity in HBEC2. On the other hand, FA-1 showed a cytoprotective effect against 6% tobacco smoke extract (CSE)-induced cytotoxicity (FIG. 4B).

Next, propidium iodide (PI) and Annexin V staining and flow cytometry were performed to protect the cytoprotective effect of FA-1 on tobacco smoke extract (CSE)-induced apoptosis. was analyzed. As a result of the analysis, it was confirmed that 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal reduced tobacco smoke extract (CSE)-induced apoptosis ( FIG. 4C ).

That is, it was found that 150 nM of 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal increased ALDH and WRN proteins and protected against tobacco smoke extract (CSE)-induced apoptosis in HBEC2 cells. can

4. Weakening effect on oxidative damage and DNA damage of FA-1 in human bronchial epithelial cells

To determine the effect of FA-1 on inhibiting tobacco smoke extract-induced oxidative damage and DNA damage in human bronchial epithelial cells, we analyzed changes in 4-HNE and phosphorylated ATM expression.

Cigarette smoke (CS) exposure is known to increase the protein of 4-HNE, a marker of oxidative stress and DNA damage, as evidenced by increased phosphorylation of ATM. To identify FA-1 against cigarette smoke (CS)-induced oxidative stress and DNA damage, HBEC2 cells were treated with 150 nM FA-1 in the presence or absence of 2% CSE, incubated for 24 hours, and then immunoblotted. was performed to analyze the expression of 4-HNE and phosphorylated ATM.

As a result of the analysis, it was found that FA-1 attenuated the accumulation of 4-HNE protein and the phosphorylation of ATM in CSE-exposed HBEC2 cells ( FIG. 5 ). That is, it can be seen that FA-1 protects against tobacco smoke extract (CSE)-induced oxidative stress and DNA damage in cultured HBEC2 cells.

5. Regulation of ALDH2 and WRN protein expression of FA-1 in human epidermal keratinocytes

To determine the effect of FA-1 on ALDH2 and WRN proteins, various concentrations of FA-1 [5, 25, 50, 75 and 150 nM in DMSO (dimethylsulfoxide)] or vehicle (in 1.0 volume % DMSO) NHEK cells were cultured after treatment with the corresponding). FA-1 enhanced ALDH2 and WRN protein expression in NHEK cells in a dose-dependent manner ( FIG. 6A ). Due to the low production of ALDH3A1 protein in NHEK cells, ALDH3A1 was not detected. That is, it was confirmed that FA-1 protects against tobacco smoke extract (CSE)-induced cytotoxicity, improves ALDH2 and WRN proteins in HBEC2 cells, and the effect of FA-1 is the same as that of plum extract (JAE).

To determine the effect of FA-1 on tobacco smoke extract (CSE)-induced down-regulation of WRN protein, HBEC2 cells were cultured with FA-1 (150 nM) or vehicle in the presence or absence of 1.5% CSE. FA-1 attenuated CS-induced down-regulation of WRN protein in cultured HBEC2 cells ( FIG. 6B ). That is, it was confirmed that FA-1 preserves WRN protein expression and provides resistance to CS-induced downregulation.

6. Tobacco smoke extract (CSE)-induced cytotoxicity and DNA damage protective effect of FA-1 in human epidermal keratinocytes.

To determine the effect of FA-1 on tobacco smoke extract (CSE)-induced cytotoxicity, NHEK cells were treated with FA-1 [DMSO (dimethylsulfoxide) for 24 h in the presence or absence of 0, 2, 4 and 6% CSE. After treatment with 150 nM), cell viability was analyzed by MTT analysis. As a result of the analysis, it was confirmed that FA-1 had a protective effect by increasing the cell viability in a concentration-dependent manner against tobacco smoke extract (CSE)-induced cytotoxicity (FIG. 7A).

Smoking activates DNA damage responses (DDRs) mediated by phosphoinositide 3-kinase-associated protein kinase (PIKK), including ataxia teleangiectasia mutated (ATM). Upon formation of DNA double-strand breaks (DSBs), ATM is activated by autophosphorylation at the serine 1981 residue and phosphorylates the serine 139 residue (γH2AX) of the H2AX variant at the chromatin proximal DSB site.

Therefore, in order to confirm the inhibitory effect on DNA damage induced by tobacco smoke extract (CSE), changes in the phosphorylation levels of ATM and H2AX related to DNA damage were analyzed. NHEK cells were treated with 150 nM of FA-1 for 24 h in the presence or absence of 2% CSE. Immunoblot analysis was performed for phosphorylation of ATM and H2AX. FA-1 reduced tobacco smoke extract (CSE)-induced phosphorylation of ATM and H2AX ( FIG. 7B ). That is, it was confirmed that FA-1 protects against tobacco smoke extract (CSE)-induced cytotoxicity and DNA damage in cultured NHEK cells.

Claims (6)

A cosmetic composition for skin protection against fine dust, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal. The cosmetic composition according to claim 1, wherein the fine dust is cigarette smoke. The cosmetic composition according to claim 1, wherein the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal increases the expression of ALDH2 or WRN to exhibit skin protection activity. According to claim 1, wherein the cosmetic composition is a solution, external ointment, cream, foam, nourishing lotion, softening lotion, pack, soft water, emulsion, makeup base, essence, soap, liquid detergent, bath agent, sunscreen cream, sun oil , suspension, emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, one or more formulations selected from the group consisting of patches and sprays, cosmetic composition. A quasi-drug composition for skin protection against fine dust, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient. A composition for external application for skin protection against fine dust, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

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