CN111246888A - Composition for ameliorating skin disorder - Google Patents

Abstract

The invention aims to research how atmospheric pollutants influence immune response and barrier function of skin and provide a composition for effectively improving skin disorders caused by the atmospheric pollutants. Provided is a composition for ameliorating skin disorders caused by atmospheric pollutants, which contains at least 1 or more IL-8 expression inhibitors. Further, a composition for improving skin disorders caused by atmospheric pollutants, which comprises at least 1 or more of a substance promoting expression of a floodgate protein and/or a substance promoting expression of a blocking protein is provided. Also provided is a composition for ameliorating skin disorders caused by air pollutants, which contains at least 1 or more of the above-mentioned oxidative stress inhibitors. Also disclosed is a composition for ameliorating skin disorders caused by atmospheric pollutants, which comprises at least 1 or more IL-33 expression inhibitors.

Description

Composition for ameliorating skin disorder

Technical Field

The present invention relates to a composition for improving skin disorders. More particularly, the present invention relates to a composition for ameliorating skin disorders caused by atmospheric pollutants.

Background

With the development of industry and the increase of population, air pollution becomes a serious problem in the world. It has been reported that when various air pollutants such as PM2.5, exhaust gas, house dust, etc. are present, respiratory diseases such as asthma and bronchitis, and circulatory diseases such as heart disease are caused at present.

In recent years, air pollutants are concerned newly with atopic dermatitis and skin diseases caused by allergy. Patent document 1 proposes an inhibitor of skin inflammation caused by aerosol particles, which contains an extract of hippophae rhamnoides belonging to the family elaeagnus as an active ingredient. Patent document 2 proposes a skin care cosmetic containing predetermined amounts of magnesium aluminate metasilicate and octyl methoxycinnamate and/or diethylamino hydroxybenzoyl hexyl benzoate for the purpose of protecting the skin from air pollutants and the like.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2016-216366

Patent document 2: japanese patent laid-open publication No. 2017-105825.

Disclosure of Invention

However, the relationship between air pollutants and skin diseases is still unclear, and it is necessary to elucidate the molecular mechanism and to propose countermeasures.

Accordingly, it is an object of the present invention to provide a composition effective for improving skin disorders caused by atmospheric pollutants.

As a result of intensive studies to solve the above problems, the present inventors have found that a specific gene expression is accelerated or decreased, oxidative stress is increased, and tight junction is hindered due to air pollutants, and have completed the present invention.

Namely, the present invention provides the following compositions.

[1] A composition for improving skin disorder caused by atmospheric pollutants comprises at least 1 inhibitor of IL-8 expression.

[2] The composition according to [1], wherein the skin disorder is skin inflammation and/or itching.

[3] The composition according to [1] or [2], wherein the IL-8 expression inhibitor is 1 or 2 or more selected from hyaluronic acid and a salt thereof, a derivative of hyaluronic acid and a salt thereof, an artichoke extract, tranexamic acid and a salt thereof, a Sasa Veitchii leaf extract, a camellia extract, a rose extract, a perilla extract, a Scutellaria baicalensis extract, a licorice extract, a Camellia sinensis extract, an aloe leaf extract, a rose hip fruit extract, a coptis chinensis extract, a loquat leaf extract, a cherry leaf extract, a rosemary leaf extract, a Japanese sage leaf extract, a thyme extract, a carrot root extract, allantoin, Ufenamate, glycyrrhizic acid and a salt thereof, glycyrrhetinic acid and a salt thereof, stearyl glycyrrhetinate, and cholesterol.

[4] A composition for improving skin disorder caused by air pollution substances comprises at least 1 or more substances promoting expression of sluice protein (Claudin) and/or occludin.

[5] The composition according to [4], wherein the skin disorder is caused by a decrease in skin barrier function and/or immaturity.

[6] The composition according to [4] or [5], wherein the sluice protein expression-promoting substance and/or the atresia protein expression-promoting substance is 1 or 2 or more selected from the group consisting of an orange peel extract, a cowberry leaf extract, a white willow bark extract, a arnica extract, a angelica keiskei extract, a coix seed extract, a ginkgo leaf extract, a turmeric extract, a rose hip extract (rose hip extract), a scutellaria baicalensis extract, a artemisia komary extract, a chamomile extract, a perilla leaf extract, a peach seed extract, a lemon balm (Melissa) extract, a lavender extract, and a sodium salt of a condensate of N-lauroyl-L-glutamic acid and L-lysine.

[7] A composition for improving skin disorder caused by air pollutants contains at least more than 1 kind of oxidation stress inhibitor.

[8] The composition according to [7], wherein the skin disorder is at least 1 selected from wrinkles, spots, acne and sagging of the skin.

[9] The composition according to [7] or [8], wherein the oxidative stress inhibitor is 1 or 2 or more selected from the group consisting of a Scutellaria baicalensis extract, a cowberry fruit extract, hydrolyzed royal jelly, sunflower oil, peppermint, glycerol glucoside, a Actinidia polygama extract, nicotinamide, glycogen, an centella asiatica extract, a mallow extract, a houttuynia cordata extract, a Neem extract, an algae extract, a phellodendron bark extract, ascorbic acid, a Ginkgo biloba leaf extract, a Hydrangeae dulcis folium extract, a green tea extract, an aloe vera leaf extract, a hibiscus flower extract, a perilla leaf extract, a rosemary leaf extract, a sage leaf extract, a citrus extract, a chamomile extract, a licorice extract, a artichoke extract, and a eucalyptus extract.

[10] A composition for improving skin disorder caused by atmospheric pollutants comprises at least 1 inhibitor of IL-33 expression.

[11] The composition according to [10], wherein the skin disorder is at least 1 selected from the group consisting of pruritus, eczema, dermatitis, rash, urticaria and erosion.

[12] The composition according to [10] or [11], wherein the inhibitor of IL-33 expression is 1 or 2 or more selected from the group consisting of allantoin, lidocaine, isopropylmethylphenol, diphenhydramine and a salt thereof, hyaluronic acid and a salt thereof, a derivative of hyaluronic acid and a salt thereof, magnesium chloride, cholesterols, glycyrrhizic acid and a salt thereof, glycyrrhetinic acid and a salt thereof, stearyl glycyrrhetinate, and isofenvalerate.

[13] The composition according to any one of [1] to [12], wherein the air contaminant is at least 1 selected from automobile exhaust gas, urban atmospheric dust, pollen and sand dust.

According to the present invention, it is possible to effectively improve symptoms in the case of skin disorders caused by air pollutants.

Drawings

FIG. 1 is a graph showing the results of evaluation of cytotoxicity against air pollutants of NHEK (human Epidermal Keratinocyte) in test example 1. Atmospheric contaminants of 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, or 250. mu.g/mL, 500. mu.g/mL, or 1000. mu.g/mL were added to the NHEK. After 24 hours from the addition of the air contaminant, cells were visualized by Hoechst staining, and the number of cells was measured by ImageXpress. The results are expressed as mean ± standard deviation (n ═ 3).

FIG. 2 is a graph showing the results of evaluation of the effect of atmospheric pollutants on the skin (gene expression analysis) in test example 2. 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, or 250. mu.g/mL, 500. mu.g/mL, or 1000. mu.g/mL of an atmospheric pollutant was added to NHEK, 24 hours after the addition of the atmospheric pollutant, total RNA was extracted from the cells, mRNA expression of IL-1 β, IL-6, IL-8, IL-33, MMP-1, and MMP-9 was measured by qRT-PCR, and normalization was performed using GAPDH expression.the results were expressed as mean. + -. standard deviation (n. gtoreq.3). the P value was measured by Dunnett's test (Dunnett's test), and compared with the control group, the P value was expressed as average. + -. standard deviation (n.gtoreq.^*P＜0.05、^**P＜0.01、^***P < 0.001.

Fig. 3 is a graph showing the results of evaluation of the effect of atmospheric pollutants on the skin (oxidative stress) in test example 3. Atmospheric contaminants of 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, or 250. mu.g/mL, 500. mu.g/mL, or 1000. mu.g/mL were added to the NHEK. After 24 hours from the addition of the air pollutants, the oxidative stress of the cells was measured by CellROX (registered trademark) Green Reagent and ImageExpress. The results are expressed as mean ± standard deviation (n ═ 3). P value was measured by dannett test using comparison with a control group to^***P < 0.001.

FIG. 4 is a graph showing the results of evaluation of the expression level of IL-8 by an air contaminant in test example 4. Atmospheric contaminants of 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, or 250. mu.g/mL, 500. mu.g/mL, or 1000. mu.g/mL were added to the NHEK. After 24 hours from the addition of the air contaminant, the level of IL-8 secreted from NHEK into the medium was measured by ELISA and normalized by the cell number. The results are expressed as mean ± standard deviation (n ═ 3). P value was determined by dannett test using comparison with a control group to^*P＜0.05、^**P＜0.01、^***P < 0.001.

FIG. 5 is a view showing the barrier to atmospheric pollutants in test example 5Graph of the results of the functional impact evaluation (1). NHEK after confluency in culture dishes at 2mM Ca for differentiation induction²⁺Culturing under the conditions of (1). Differentiation was induced 6 days later at 2mMCa²⁺Air pollutants of 50. mu.g/mL or 1000. mu.g/mL were added under the conditions of (1). Barrier function was assessed by TER. The results are expressed as mean ± standard deviation (n ═ 3). P-value by Student's t-test, using comparison with control group, to^*P < 0.05.

Fig. 6 is a graph showing the results of evaluation (2) of the effect of atmospheric pollutants on the barrier function in test example 6. NHEK confluent in Petri dishes at 2mM Ca²⁺Under the conditions of (1) 50. mu.g/mL or 1000. mu.g/mL of an atmospheric contaminant was added to the cells. Barrier function was assessed by TER. The results are expressed as mean ± standard deviation (n ═ 3). P-value by Student's t-test, using comparison with control group, to^*P＜0.05、^**P＜0.01、^***P < 0.001.

FIG. 7 is a graph showing the results of experimental example 7, which illustrates the mechanism of the barrier formation mechanism lowering action by the air contaminant. NHEK confluent in Petri dishes at 2mM Ca²⁺Under the conditions of (1), 50. mu.g/mL or 1000. mu.g/mL of an air contaminant was added to the cells. After differentiation induction for 6 days, total RNA was extracted from the cells. mRNA expression of CLDN1 and OCLN was determined by qRT-PCR and normalized using GAPDH expression. The results are expressed as mean ± standard deviation (n ═ 3). P-value by Student's t-test, using comparison with control group, to^**P＜0.01、^***P < 0.001.

FIG. 8 is a graph showing the results of material search for inhibiting UA-induced activation of IL-8 in test example 8. Candidate compounds were added to NHEK. 24 hours after the addition of the candidate compound, UA 50. mu.g/mL and the candidate compound were added to NHEK. After 24 hours from the addition of UA, the level of IL-8 secreted from NHEK to the medium was measured by ELISA and normalized by the cell number. The results are expressed as mean ± standard deviation (n ═ 3). P values were determined by the dunnett test using comparison with the UA50 μ g/mL group to^*P＜0.05、^**P＜0.01、^***P < 0.001.

Fig. 9 is a graph showing the results of searching for a material that improves the barrier function reduction by UA in test example 9. NHEK confluent in Petri dishes at 2mM Ca²⁺Adding 50. mu.g/mL of UA and the candidate compound to the cells under the conditions of (1). Barrier function was assessed by TER. The results are expressed as mean ± standard deviation (n ═ 3). P values were determined by Student's t-test (Student's ttest) using comparison with the UA50 μ g/mL panel to^*P＜0.05、^**P < 0.01.

Fig. 10 is a graph showing the results of the experimental example 10, which explains the mechanism of improvement in barrier function of the citrus peel extract. NHEK confluent in Petri dishes at 2mM Ca²⁺Adding 50. mu.g/mL of UA and the candidate compound to the cell under the condition (1). After 4 days and 5 days of differentiation induction, total RNA was extracted from the cells. mRNA expression of CLDN1 was determined by qRT-PCR and normalized using GAPDH expression. The results are expressed as mean ± standard deviation (n ═ 3). P values were determined by Student's t-test (Student's st test) using comparison with the UA50 μ g/mL group to^**P < 0.01.

Fig. 11 is a graph showing the results of evaluation of the influence of air pollutants on the dermis via the epidermis in the two-dimensional skin model in test example 11. Air pollutants at 25. mu.g/mL and 50. mu.g/mL were added to the NHEK. After 24 hours from the addition of the atmospheric contaminants, the supernatant was recovered, and after removing the atmospheric contaminants from the supernatant by filtration through a filter, the supernatant was added to nhdf (human dermal fibroblast). After 4 days from the addition of the supernatant, MMP1 and MMP3 levels secreted from NHDF into the medium were measured by ELISA and normalized by the cell number thereof. The results are expressed as mean ± standard deviation (n ═ 3). P-value was determined by Student's t-test using comparison with control group of supernatant to^*P＜0.05、^**P＜0.01、^***P < 0.001.

Fig. 12 is a graph showing the results of evaluation of the influence of air pollutants on the dermis via the epidermis in the three-dimensional skin model in test example 12. Air pollutants 50. mu.g/mL and 500. mu.g/mL were added to the EFT400 tableOn the skin. After 3 days from the addition of the air pollutants, the levels of MMP1 and MMP3 secreted into the medium were measured by ELISA and normalized by the cell number. The results are expressed as mean ± standard deviation (n ═ 3). P value is determined by Student's t test (Student's ttest) or dannett test, using comparison with a control group, to^*P < 0.05.

Fig. 13 is a graph showing the results of evaluation of the influence of atmospheric pollutants on speckles in test example 13. Air pollutants at 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL were added to the NHEK. After 24 hours from the addition of the atmospheric contaminants, total RNA was extracted from the cells. mRNA expression of PTGS2 was determined by qRT-PCR and normalized using GAPDH expression. The results are expressed as mean ± standard deviation (n ═ 3). P value was determined by dannett test using comparison with a control group to^*P＜0.05、^**P＜0.01、^***P < 0.001.

Fig. 14 is a graph showing the results of evaluation of the influence of an air contaminant on melanocytes via the epidermis in the two-dimensional skin model in test example 14. Add 50. mu.g/mL of UA to NHEK. After 24 hours from the addition of UA, the supernatant was recovered, and after UA was removed from the supernatant by filtration through a filter, it was added to nhem (human epiermal mercacyte). After 24 hours from the addition of the supernatant, total RNA was extracted from the cells. mRNA expression of TYR was determined by qRT-PCR and normalized using GAPDH expression. The results are expressed as mean ± standard deviation (n ═ 3).

Fig. 15 is a graph showing the results of evaluation of the influence of air pollutants on melanocytes via epidermis in the three-dimensional skin model in test example 15. Air pollutants of 500. mu.g/mL and 1000. mu.g/mL were added to MEL 300A. After 24 hours from the addition of the atmospheric contaminants, total RNA was extracted from the cells. mRNA expression of PTGS2 and TYR was determined by qRT-PCR and normalized using GAPDH expression. The results are expressed as mean ± standard deviation (n ═ 3). P value was determined by dannett test using comparison with a control group to^*P＜0.05、^**P＜0.01、^***P < 0.001.

FIG. 16 is a graph showing the results of material search for suppressing oxidative stress due to air pollutants in test example 16. Candidate compounds were added to NHEK. After 24 hours from the addition of the candidate compound, 50. mu.g/mL of UA and the candidate compound were added to NHEK. 24 hours after the addition of the air pollutants, the oxidative stress of the cells was measured by CellROX (registered trademark) GreenReagent and Imageexpress. The results are expressed as mean ± standard deviation (n ═ 3).

FIG. 17 is a graph showing the results of material search for MMP1 that is inhibitory of air pollutants in test example 17. Candidate compounds were added to NHEK. After 24 hours from the addition of the candidate compound, 50. mu.g/mL of UA and the candidate compound were added to NHEK. 24 hours after UA addition, MMP1 levels secreted from NHEK into the medium were measured by ELISA and normalized by the cell number. The results are expressed as mean ± standard deviation (n ═ 3). P values were determined by the dunnett test using comparison with the UA50 μ g/mL group to^*P＜0.05、^**P＜0.01、^***P < 0.001.

FIG. 18 is a graph showing the results of a material search for suppressing an increase in IL-33 expression due to an atmospheric contaminant in test example 18. CP and candidate compound were added at 2mg/mL to NHEK. After 6 hours from the addition of CP, the mRNA expression of IL-33 was determined by qRT-PCR and normalized by the GAPDH expression measured simultaneously. The results are expressed as mean ± standard deviation (n ═ 3). P-value by means of the dunnett test, using comparison with the CP 2mg/mL (control) group, to^*P＜0.05、^**P＜0.01、^***P < 0.001.

FIG. 19 is a graph showing the results of a material search for suppressing an increase in IL-33 expression due to an atmospheric contaminant in test example 19. 100 μ g/mL GKD and candidate compound were added to NHEK. After 24 hours from the addition of GKD, the mRNA expression of IL-33 was measured by qRT-PCR and normalized by the GAPDH expression measured at the same time. The results are expressed as mean ± standard deviation (n ═ 3). P-value was determined by dannett test using comparison with GKD 100. mu.g/mL (control) group to^*P＜0.05、^**P＜0.01、^***P < 0.001.

Detailed Description

[ composition for ameliorating skin disorder ]

In one embodiment, the composition for improving skin disorders of the present invention comprises at least 1 or more IL-8 expression inhibitors. The composition for improving skin disorders of the present invention is particularly suitable for improving skin disorders caused by air pollutants.

In the present specification, the inhibitor of IL-8 expression is not particularly limited as long as it can inhibit the expression of IL-8 gene or protein in vitro, ex vivo or in vivo. The expression inhibition rate of IL-8 may be at least 1%, preferably 2%, more preferably 5%, and still more preferably 10% in the case of IL-8 gene expression or protein expression, relative to the case where the inhibitor of IL-8 expression is present in the presence of atmospheric pollutants and the case where the inhibitor of IL-8 expression is not present. The method for measuring IL-8 gene expression or protein expression can be measured by a known method, for example, as described in examples, IL-8 gene expression can be quantified by a real-time PCR method using an IL-8-specific probe, and IL-8 protein expression can be quantified by an ELISA method using an IL-8-specific antibody.

The IL-8 expression inhibitor is not particularly limited as long as it achieves the effects of the present invention, and examples thereof include hyaluronic acid and salts thereof, hyaluronic acid derivatives and salts thereof, artichoke extract, tranexamic acid and salts thereof, phyllostachys pubescens leaf extract, camellia extract, rose extract, perilla extract, scutellaria baicalensis extract, licorice extract, camellia extract, aloe leaf extract, dog rose fruit extract, coptis chinensis extract, loquat leaf extract, cherry leaf extract, rosemary leaf extract, Japanese sage leaf extract, thyme extract, carrot root extract, allantoin, fenugreek, glycyrrhizic acid and salts thereof, glycyrrhetinic acid and salts thereof, stearyl glycyrrhetinate, cholesterol, and the like. The IL-8 expression inhibitor may be used in combination with 1 or 2 or more. The inhibitor of IL-8 expression may be used in the form of a synthetic product or a commercially available product.

The inhibitor of IL-8 expression is preferably at least 1 selected from the group consisting of rose extract, hyaluronic acid and a salt thereof, a derivative of hyaluronic acid and a salt thereof, artichoke extract, tranexamic acid and a salt thereof, allantoin, ifenphenate, glycyrrhizic acid and a salt thereof, glycyrrhetinic acid and a salt thereof, stearyl glycyrrhetinate, and cholesterol, from the viewpoint of remarkably exerting the effects of the present invention.

Among the above-mentioned inhibitors of IL-8 expression, hyaluronic acid is a polymer having a basic structure (repeating unit) of glucuronic acid (GlcUA) -GlcNAc, which is formed by binding glucuronic acid (GlcNUA) and N-acetylglucosamine (GlcNAc), and is a known polymer compound that exerts a moisturizing effect.

The salt of hyaluronic acid is not particularly limited as long as it is pharmaceutically, pharmacologically or physiologically acceptable. Examples of the salt of hyaluronic acid include a salt with an organic base and a salt with an inorganic base.

The salt of glycyrrhizic acid is not particularly limited as long as it is pharmacologically (pharmaceutically) or physiologically acceptable. The salt of glycyrrhizic acid is preferably monoammonium glycyrrhizinate or dipotassium glycyrrhizinate.

Examples of the salt with an organic base include salts with organic amines such as methylamine, triethylamine, triethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, and picoline. Examples of the salt with an inorganic base include ammonium salts; alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium and magnesium; and salts with metals such as aluminum.

The source of hyaluronic acid and its salt is not particularly limited, and for example, hyaluronic acid may be obtained from cockscomb, may be derived from a microorganism, and may be a synthetic product. Among them, hyaluronic acid derived from microorganisms (biological hyaluronic acid), and salts or derivatives thereof are preferable.

The viscosity average molecular weight of hyaluronic acid and salts thereof used in the present invention is not particularly limited, and examples thereof are in the range of 0.01 to 500 ten thousand, preferably 0.1 to 400 ten thousand, more preferably 1 to 300 ten thousand, even more preferably 10 to 250 ten thousand, particularly preferably 50 to 200 ten thousand, and most preferably 100 to 170 ten thousand. Here, the viscosity average molecular weight can be determined by a known measurement method. Specifically, hyaluronic acid or its salt (dried product) was dissolved in 0.2M sodium chloride solution, and the specific temperature of 30. + -. 0 ℃ was determined by Ubbelohde viscometerIntrinsic viscosity (η) based on Laurent formula (η (intrinsic viscosity) ═ 3.6X 10^-4·M^0.78M is a viscosity average molecular weight) the intrinsic viscosity (η) was measured according to the 17 th method of general test method viscometry modified japanese pharmacopoeia, method 1: capillary viscometer method.

As hyaluronic acid and its salt, hyaluronic acid oligosaccharide can also be used. In the present specification, the hyaluronic acid oligosaccharide refers to a substance containing at least 2 saccharides having a GlcUA-GlcNAc basic structure (repeating unit) in which 1 unit of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) are bonded. The hyaluronic acid oligosaccharide is preferably a combination of repeating units having a basic structure of 1 to 10 units, and may be a salt or a derivative thereof. The hyaluronic acid oligosaccharide is not limited, and examples thereof include a 4-saccharide (HA4), a 6-saccharide (HA6), an 8-saccharide (HA8), a 10-saccharide (HA10), and a 12-saccharide (HA 12). Hyaluronic acid oligosaccharides, such as the 4 saccharide (HA4), refer to repeating units comprising the basic structure of 2 units.

Examples of the derivative of hyaluronic acid and a salt thereof include derivatives obtained by etherification, esterification, amidation, acetylation, acetalization, or the like of hydroxyl groups, carboxyl groups, and the like of hyaluronic acid and the like, hyaluronic acid decomposition products obtained by partial decomposition with an enzyme such as hyaluronidase, hyaluronic acid hydrolysis products obtained by partial decomposition with acid hydrolysis, and crosslinked hyaluronic acid obtained by crosslinking with a crosslinking agent.

As the hyaluronic acid and its salt, and hyaluronic acid derivatives and salts thereof, for example, hyaluronic acid oligosaccharides, hydrolyzed hyaluronic acid, low molecular weight hyaluronic acid, salts thereof, and derivatives thereof can be used, and among them, hyaluronic acid oligosaccharides having 4 saccharides (HA4) are more preferable.

Hyaluronic acid and salts thereof, and hyaluronic acid derivatives and salts thereof can be produced by conventionally known methods. Hyaluronic acid and its salts, and hyaluronic acid derivatives and its salts are various, and commercially available products thereof may be used in the present invention. Examples of such commercially available products include hyaluronic acid HA-LQ-RS, hyaluronic acid HA-LQ60, sodium hyaluronate HA-QA, HYALO-OLIGO, HYALOVEIL, HYALO-ZINC (manufactured by Kewpie), biological sodium hyaluronate HA12NB, biological sodium hyaluronate HA20N, sodium hyaluronate acetylated (manufactured by Kyowa), hyaluronic acid oligosaccharide 4 saccharide (HA4), 6 saccharide (HA6), 8 saccharide (HA8), 10 saccharide (HA10), 12 saccharide (HA12) (manufactured by Cosmo Bio), OLIGO-HA4, OLIGO-HA6 (manufactured by SIGMA), hyaluronic acid FCH-120, hyaluronic acid FCH-121C (manufactured by Kikkoman Chemifa), hyaluronic acid FCH-SU (manufactured by bun Chemifa), and HYsIRL CAGE SYSTEM (manufactured by Kisrl).

The aforementioned IL-8 expression inhibitor is not limited to an extract of artichoke, and is an extract obtained by subjecting artichoke (Cynara scolymus) to an extraction treatment with an extraction solvent. Artichoke (Cynara scolymus L.) is a perennial herb belonging to the genus Cynara, the family of compositae.

In the present specification, when a plant extract (also referred to as a plant extract) is used, as an extraction solvent, water (including hot water), alcohols such as methanol, ethanol, isopropanol, ethylene glycol, 1, 3-butanediol, and glycerol, esters such as ethyl acetate, ketones such as acetone and methyl ethyl ketone, nitriles such as acetonitrile, ethers such as diethyl ether and tetrahydrofuran, saturated hydrocarbons such as pentane, hexane, cyclopentane, and cyclohexane, aromatic hydrocarbons such as toluene, halogenated hydrocarbons such as dichloromethane and chloroform, other organic solvents (all of which may contain water), and the like may be used as appropriate, and any of 1 or 2 kinds of mixed solutions may be used. Among these solvents, water, ethanol, 1, 3-butanediol, or a mixed solution thereof is preferable. The extracts described in the present specification can be obtained from various raw material companies, and they are generally sold in a form including an extraction solvent, a dilution solvent, and the like. Hereinafter, the extract amount means an amount including a dry solid component, these solvents, and the like.

In the present specification, the plant extract may be a crude extract extracted from the whole plant or a necessary part of the plant (flower, capitate flower, flower bud, flower spike, leaf, branch, leaf, rhizome, root bark, fruit peel, pod, seed, etc.), or a product obtained by further refining the crude extract, or a product obtained by concentration, synthesis, or a commercially available product. The method for obtaining the plant extract is not particularly limited, and a general extraction method, a purification method, a concentration method, a synthesis method, a dry powder method, and the like can be used.

When an artichoke extract is used as the extract of a plant, it is preferably an extract of at least 1 selected from the group consisting of whole grass, flower, leaf, stem and root, and more preferably an extract of leaf. The artichoke extract can be used as a commercially available product.

When a rose extract is used as the plant extract, it is preferably an extract of at least 1 selected from the group consisting of whole herbs, fruits, flowers, leaves, stems and roots, more preferably an extract of fruits and/or leaves, and still more preferably an extract of fruits. The rose extract may be a commercially available product. The rose extract includes Rosa roxburghii extract, Rosa perfume (Rosa odorata) extract, Rosa gigantea (Rosa brecta) extract, Rosa centifolia (Rosa centifolia) extract, Rosa damascena (Rosa damascona) extract, and Rosa multiflora extract, and is preferably Rosa roxburghii extract.

In the present specification, the plant extract may be in a liquid form, or may be in a concentrated liquid form, semisolid form, solid form, or powder form by reducing or removing the liquid component by drying treatment such as reduced pressure drying, freeze drying, or spray drying, if necessary.

Tranexamic acid is also called trans-4- (aminomethyl) cyclohexane-1-carboxylic acid, and can be synthesized by a known method or can be obtained as a commercially available product.

Tranexamic acid can be used as a derivative thereof. Examples of the derivatives include cetyl tranexamate, methylaminoformic acid, ethylamide tranexamate, and the like.

Tranexamic acid can be used as a salt thereof. The salt of tranexamic acid is not particularly limited as long as it is pharmacologically (pharmaceutically) or physiologically acceptable. Examples of the salt of tranexamic acid include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salts and magnesium salts; a zinc salt; a ferric salt; an ammonium salt; salts with basic amino acids such as arginine, lysine, histidine and ornithine; and salts with amines such as monoethanolamine, diethanolamine, and triethanolamine. Among the salts of tranexamic acid, sodium salt, potassium salt, triethanolamine salt and arginine salt are preferable, and sodium salt is more preferable. A "salt" may comprise a solvate or hydrate of a salt.

Allantoin, urafenamate, glycyrrhetinic acid and salts thereof, and stearyl glycyrrhetinate are known compounds, and can be synthesized by known methods or obtained as commercially available products.

Examples of the cholesterol and a salt thereof include cholesterol, stigmasterol, lanosterol, ergosterol, and a salt thereof.

When the IL-8 expression inhibitor contains an extract, the amount of the extract may be appropriately determined depending on the type and content of other components to be added, the form of the preparation of the composition, and the like, but is not particularly limited to the total amount of the composition, and is preferably 0.00001 to 10% by mass, more preferably 0.0001 to 5% by mass, further preferably 0.001 to 2% by mass, and particularly preferably 0.01 to 1% by mass, based on the total amount of the composition. When the extract is used, the content of dry solid is preferably 0.0005 to 30% by mass, more preferably 0.001 to 20% by mass, and particularly preferably 0.01 to 10% by mass, based on the total amount of the extract.

When hyaluronic acid and salts thereof and hyaluronic acid derivatives and salts thereof are contained as the IL-8 expression inhibitor, the content of hyaluronic acid and salts thereof and hyaluronic acid derivatives and salts thereof alone is not particularly limited, and may be, for example, 0.0001 to 5% by mass, preferably 0.001 to 1% by mass, more preferably 0.005 to 0.5% by mass, and still more preferably 0.01 to 0.1% by mass.

When glycyrrhizic acid and its salt are contained as the IL-8 expression inhibitor, the content of glycyrrhizic acid and its salt alone is not particularly limited, and may be, for example, 0.0001 to 10% by mass, preferably 0.001 to 5% by mass, more preferably 0.005 to 2% by mass, and still more preferably 0.01 to 1% by mass.

When tranexamic acid or a salt thereof is contained as the IL-8 expression inhibitor, the content of tranexamic acid or a salt thereof alone is not particularly limited, and may be, for example, 0.001 to 10% by mass, preferably 0.005 to 7% by mass, more preferably 0.01 to 5% by mass, and still more preferably 1 to 3% by mass.

When allantoin is contained as the IL-8 expression inhibitor, the content of allantoin alone is not particularly limited, and may be, for example, 0.001 to 10% by mass, preferably 0.002 to 5% by mass, more preferably 0.01 to 3% by mass, and still more preferably 0.2 to 1% by mass.

When the inhibitor of IL-8 expression contains ifenprodine, the content of ifenprodine alone is not particularly limited, and may be, for example, 0.00001 to 10 mass%, preferably 0.0001 to 5 mass%, more preferably 0.01 to 7 mass%, and still more preferably 1 to 5 mass%.

When glycyrrhetinic acid and salts thereof or stearyl glycyrrhetinate is contained as the IL-8 expression inhibitor, the content of glycyrrhetinic acid and salts thereof or stearyl glycyrrhetinate alone is not particularly limited, and may be, for example, 0.00001 to 10% by mass, preferably 0.0001 to 5% by mass, more preferably 0.01 to 3% by mass, and still more preferably 0.1 to 1% by mass.

When cholesterol is contained as the IL-8 expression inhibitor, the content of cholesterol alone is not particularly limited, and may be, for example, 0.001 to 20% by mass, preferably 0.002 to 10% by mass, more preferably 0.01 to 8% by mass, and still more preferably 0.05 to 5% by mass.

In another embodiment, the composition for improving skin disorders of the present invention comprises at least 1 or more of a substance promoting expression of a floodgate protein and/or a substance promoting expression of a atresia protein.

In the present specification, the substance promoting expression of a lockin and/or a lockin is not particularly limited as long as it can promote expression of a lockin and/or a lockin gene or expression of a protein in vitro, ex vivo or in vivo. The rate of promotion of expression of the lockin and/or the occludin may be at least 1%, preferably 2%, more preferably 5%, and still more preferably 10% in the gene expression or protein expression of the lockin and/or the occludin, relative to the conditions in the presence of the atmospheric pollutant and in the absence of the lockin expression-promoting substance and/or the occludin expression-promoting substance. The method for measuring the gene expression or protein expression of the sluice protein and/or the atresia protein can be measured by a known method, for example, as described in examples, the gene expression of the sluice protein or the atresia protein can be quantified by a real-time PCR method using a probe specific to the sluice protein or the atresia protein.

The gatekeeins, atresia proteins, are membrane proteins that form a tight junction between adjacent epithelial cells.

The water gate protein may be a barrier type water gate protein (e.g., water gate protein-1, water gate protein-4, water gate protein-5, water gate protein-7, water gate protein-11, water gate protein-14, water gate protein-18, and water gate protein-19), or a channel type water gate protein (e.g., water gate protein-2, water gate protein-7, water gate protein-10, water gate protein-15, and water gate protein-16).

The sluice protein expression promoting substance and/or the atresia protein expression promoting substance is not particularly limited as long as the effect of the present invention is achieved, and examples thereof include an orange peel extract, a cowberry leaf extract, a white willow bark extract, a arnica extract, a angelica keiskei extract, a coix seed extract, a ginkgo leaf extract, a turmeric extract, a rose extract (rose hip extract), a scutellaria baicalensis extract, a artemisia inflata extract, a chamomile extract, a perilla leaf extract, a peach seed extract, a lemon balm extract, a lavender extract, and a sodium salt of a condensation product of N-lauroyl-L-glutamic acid and L-lysine. The sluice protein expression promoting substance and/or the atresia protein expression promoting substance may be used in 1 kind or 2 or more kinds in combination. The expression-promoting substance of the gatekeeper protein and/or the expression-promoting substance of the atresia protein may be a synthetic product or a commercially available product.

The sluice protein expression-promoting substance and/or the occludin expression-promoting substance is preferably an extract obtained by extracting pericarp of citrus (hereinafter, citrus pericarp extract), and more preferably a citrus peel extract, from the viewpoint of remarkably exerting the effect of the present invention.

The extract of Citrus peel is not limited, and is obtained by subjecting mature peel of Citrus unshiu Markowicz or Citrus reticulata Blanco (Rutaceae) to extraction treatment with an extraction solvent. Although not limited, the citrus peel extract is preferably a mature peel derived from citrus, from the viewpoint of remarkably exerting the effect of the present invention.

When the extract of pericarp of citrus fruit is used as the expression promoter for sluice protein and/or the expression promoter for atresia protein, the extraction solvent may be 1 or 2 kinds of arbitrary mixed solution, for example, water (including hot water), alcohols such as methanol, ethanol, isopropanol, ethylene glycol, 1, 3-butanediol, and glycerol, esters such as ethyl acetate, ketones such as acetone and methyl ethyl ketone, nitriles such as acetonitrile, ethers such as diethyl ether and tetrahydrofuran, saturated hydrocarbons such as pentane, hexane, cyclopentane, and cyclohexane, aromatic hydrocarbons such as toluene, halogenated hydrocarbons such as dichloromethane and chloroform, and other organic solvents (all of which may contain water), and the like. Among these solvents, water, ethanol, 1, 3-butanediol or a mixed solution thereof is preferable.

The pericarp extract of mandarin orange may be crude extract extracted from mature pericarp of Citrus unshiu or Citrus reticulata, or refined extract, or concentrated extract, or synthesized extract, or commercially available product. The method for obtaining the citrus peel extract is not particularly limited, and a general extraction method, a purification method, a concentration method, a synthesis method, a dry-powdering method, and the like are employed. In addition, when the tangerine peel extract is used as the tangerine peel extract, it may be a substance satisfying the items collected in the seventeenth revised japanese pharmacopoeia.

The total content of the sluice protein expression promoting substance and/or the atresia protein expression promoting substance is appropriately set according to the kind and content of other components to be blended, the formulation form of the composition, and the like, and is preferably 0.00001 mass% or more, more preferably 0.0001 mass% or more, further preferably 0.001 mass% or more, and particularly preferably 0.01 mass% or more, relative to the total amount of the composition. The total content of the sluice protein expression promoting substance and/or the atresia protein expression promoting substance is preferably 10% by mass or less, more preferably 5% by mass or less, still more preferably 2% by mass or less, and particularly preferably 1% by mass or less, based on the total amount of the composition. The total content of the sluice protein expression promoting substance and/or the atresia protein expression promoting substance is not particularly limited, and is preferably 0.00001 to 10 mass%, more preferably 0.0001 to 5 mass%, even more preferably 0.001 to 2 mass%, and particularly preferably 0.01 to 1 mass% with respect to the total amount of the composition. When the extract is used, the content of the dried solid content is preferably 0.0005 to 30% by mass, more preferably 0.001 to 20% by mass, and particularly preferably 0.01 to 10% by mass, based on the total amount of the extract.

In another embodiment, the composition for improving skin disorders of the present invention contains at least 1 or more kinds of the oxidative stress inhibitor.

The oxidative stress inhibitor is not particularly limited as long as it can inhibit the function of an oxidative stress-related factor such as MMP-1 in vitro, ex vivo or in vivo. The oxidative stress inhibitor is not particularly limited as long as it achieves the effects of the present invention, and examples thereof include a scutellaria baicalensis extract, a cowberry fruit extract, hydrolyzed royal jelly, sunflower oil, bitter mint, glycerol glucoside, a polygonum orientale extract, nicotinamide, glycogen, an centella asiatica extract, a mallow extract, a houttuynia cordata extract, a neem extract, an algae extract, a phellodendron amurense extract, ascorbic acid, a ginkgo biloba leaf extract, a camellia sinensis extract, a green tea extract, an aloe leaf extract, a hibiscus flower extract, a perilla leaf extract, a rosemary leaf extract, a sage leaf extract, a citrus extract, a chamomile extract, a licorice root extract, an artichoke extract, a eucalyptus extract, and the like. The oxidative stress inhibitor may be used in 1 kind or 2 or more kinds in combination. The oxidative stress inhibitor may be a synthetic product or a commercially available product. The method of extraction when plant extracts are used as the oxidative stress inhibitor is based on the above description of "inhibitor of IL-8 expression".

When the extract of scutellaria baicalensis is used as the extract of a plant, the extract is preferably an extract of at least 1 selected from the group consisting of whole grass, flower, leaf, stem and root, more preferably an extract of leaf and/or root, and still more preferably an extract of root. The Scutellariae radix extract can be commercially available.

When the bilberry extract is used as the extract of a plant, it is preferably an extract of at least 1 selected from the group consisting of whole grass, fruit, flower, leaf, stem and root, more preferably an extract of fruit and/or leaf, and still more preferably an extract of leaf. The cowberry fruit extract can be commercially available product.

The method for producing the hydrolyzed royal jelly is not particularly limited as long as it can be blended with foods, cosmetics, and the like. The hydrolyzed royal jelly can be produced, for example, by adding water and a proteolytic enzyme to royal jelly and reacting the mixture under heat and pressure. The hydrolyzed royal jelly can be commercially available.

The sunflower oil is not particularly limited as long as it can be blended with foods, cosmetics, and the like. The sunflower oil is preferably extracted from sunflower seeds. The sunflower oil can be obtained from commercial products.

The Mentha arvensis is an extract obtained from a plant belonging to the genus Mentha of the family Labiatae as a raw material, preferably at least 1 kind of extract selected from the group consisting of whole plant, flower, leaf, stem and root, more preferably flower and/or leaf extract, and still more preferably leaf extract. The herba Menthae can be commercially available.

The glycerol glucoside can be incorporated into pharmaceuticals, cosmetics, and the like, and can be produced by synthesis, fermentation using microorganisms, or the like, specifically, α or β forms, or a mixture thereof can be used.

When the Actinidia polygama extract is used as the extract of a plant, the extract is preferably an extract of at least 1 selected from the group consisting of whole plant, fruit, flower, leaf, stem and root, more preferably an extract of fruit and/or leaf, and still more preferably an extract of fruit. The Actinidia polygama extract can be commercially available.

The glycogen is not particularly limited as long as it can be blended with a pharmaceutical, a cosmetic, or the like. Examples of glycogen include animal glycogen derived from livers of shellfish such as scallop, abalone, oyster, mussel, pearl shell, etc., bovine and porcine livers, and plant glycogen derived from corn, barley, rice, potato, cassava, etc., preferably plant glycogen. As the glycogen, glycogen contained in a natural product produced by a conventional method may be used as it is, glycogen obtained by optionally subjecting to an enzyme treatment and then subjecting to a separation purification treatment may be used, and commercially available products may be used.

When the centella asiatica extract is used as the plant extract, it is preferably an extract of at least 1 selected from the group consisting of whole plants, fruits, flowers, leaves, stems and roots, more preferably an extract of leaves and/or stems, and still more preferably an extract of leaves. The centella asiatica extract can be obtained from commercially available products.

When the mallow extract is used as the extract of the plant, it is preferably an extract of at least 1 selected from the group consisting of whole grass, fruit, flower, leaf, stem and root, more preferably an extract of flower and/or leaf, and still more preferably an extract of flower. The mallow extract can be commercially available.

When the houttuynia cordata extract is used as the extract of the plant, the extract is preferably an extract of at least 1 selected from the group consisting of whole herbs, fruits, flowers, leaves, stems and roots, more preferably an extract of leaves and/or stems, and still more preferably an extract of leaves. The extract of houttuynia cordata can be obtained from commercial products.

When neem extract is used as the plant extract, it is preferably an extract of at least 1 selected from the group consisting of whole plant, fruit, flower, leaf, stem and root, more preferably an extract of leaf and/or stem, and still more preferably an extract of leaf. Neem extract is commercially available.

The algae extract is not particularly limited as long as it can be blended with a pharmaceutical product, a cosmetic product, or the like. The algae extract is prepared from algae, preferably at least 1 extract selected from brown algae, red algae and green algae, and more preferably their mixture. Commercially available products can be used.

When the extract is contained in the oxidation stress inhibitor, the amount of the extract is appropriately determined depending on the type and content of other components to be added, the form of the preparation of the composition, and the like, and is not particularly limited to the total amount of the composition, but is preferably 0.00001 to 10% by mass, more preferably 0.0001 to 5% by mass, further preferably 0.001 to 2% by mass, and particularly preferably 0.01 to 1% by mass, based on the total amount of the composition. When the extract is used, the content of dry solid is preferably 0.0005 to 30% by mass, more preferably 0.001 to 20% by mass, and particularly preferably 0.01 to 10% by mass, based on the total amount of the extract.

When the royal jelly is contained as the oxidative stress inhibitor, the content of the royal jelly alone is not particularly limited, and may be, for example, 0.00001 to 1 mass%, preferably 0.00005 to 0.5 mass%, more preferably 0.0001 to 0.2 mass%, and still more preferably 0.001 to 0.1 mass%.

When glycerol glucoside is contained as the oxidation stress inhibitor, the content of glycerol glucoside alone is not particularly limited, and may be, for example, 0.0001 to 10% by mass, preferably 0.001 to 5% by mass, more preferably 0.005 to 2% by mass, and still more preferably 0.01 to 1% by mass.

When nicotinamide is contained as the oxidative stress inhibitor, the content of nicotinamide alone is not particularly limited, and may be, for example, 0.005 to 20 mass%, preferably 0.01 to 10 mass%, more preferably 0.05 to 5 mass%, and still more preferably 0.1 to 1 mass%.

When glycogen is contained as the inhibitor of oxidative stress, the content of glycogen alone is not particularly limited, and may be, for example, 0.001 to 10% by mass, preferably 0.003 to 5% by mass, more preferably 0.005 to 3% by mass, and still more preferably 0.01 to 1% by mass.

When ascorbic acid is contained as the oxidation stress inhibitor, the content of ascorbic acid alone is not particularly limited, and may be, for example, 0.01 to 30% by mass, preferably 0.1 to 25% by mass, more preferably 1 to 20% by mass, and still more preferably 3 to 10% by mass.

In another embodiment, the composition for improving skin disorders of the present invention comprises at least 1 or more kinds of inhibitors of IL-33 expression.

In the present specification, the inhibitor of IL-33 expression is not particularly limited as long as it can inhibit the gene expression or protein expression of IL-33 in vitro, ex vivo or in vivo. The rate of inhibition of expression of IL-33 may be at least 1%, preferably 2%, more preferably 5%, and still more preferably 10% in the gene expression or protein expression of IL-33, relative to the conditions in the presence of atmospheric pollutants and in the absence of inhibitors of IL-33 expression. The method for measuring gene expression or protein expression of IL-33 can be measured by a known method, and for example, as described in examples, the gene expression of IL-33 can be quantified by a real-time PCR method using an IL-33-specific probe.

The IL-33 expression inhibitor is not particularly limited as long as it exerts the effects of the present invention, and examples thereof include allantoin, lidocaine, isopropylmethylphenol, diphenhydramine and salts thereof, hyaluronic acid and salts thereof, derivatives and salts of hyaluronic acid, magnesium chloride, cholesterols, glycyrrhizic acid and salts thereof, glycyrrhetinic acid and salts thereof, stearyl glycyrrhetinate, and guanyl. The inhibitor of IL-33 expression may be used in 1 type or in combination of 2 or more types. The inhibitor of IL-33 expression may be a synthetic product or a commercially available product. Of these components, the types and contents of allantoin, hyaluronic acid and its salts, hyaluronic acid derivatives and its salts, cholesterols, glycyrrhizic acid and its salts, glycyrrhetinic acid and its salts, and stearyl glycyrrhetinate are based on the above description about "inhibitors of IL-8 expression".

The salt of diphenhydramine is not particularly limited as long as it is pharmacologically (pharmaceutically) or physiologically acceptable. The diphenhydramine salt is preferably diphenhydramine hydrochloride.

When lidocaine is contained as an IL-33 expression inhibitor, the content of lidocaine alone is not particularly limited, and may be, for example, 0.001 to 10% by mass, preferably 0.002 to 7% by mass, more preferably 0.01 to 5% by mass, and still more preferably 0.2 to 2% by mass.

When isopropyl methylphenol is contained as an inhibitor of IL-33 expression, the content of isopropyl methylphenol alone is not particularly limited, and may be, for example, 0.0001 to 10% by mass, preferably 0.001 to 7% by mass, more preferably 0.01 to 5% by mass, and still more preferably 0.05 to 0.5% by mass.

When diphenhydramine or a salt thereof is contained as the IL-33 expression inhibitor, the content of diphenhydramine or a salt thereof alone is not particularly limited, and may be, for example, 0.001 to 10% by mass, preferably 0.005 to 7% by mass, more preferably 0.01 to 5% by mass, and further preferably 0.5 to 2% by mass.

When magnesium chloride is contained as an inhibitor of IL-33 expression, the content of magnesium chloride alone is not particularly limited, and may be, for example, 0.001 to 10% by mass, preferably 0.002 to 8% by mass, more preferably 0.005 to 5% by mass, and still more preferably 0.01 to 3% by mass.

In the present specification, examples of the salt of the compound include, in addition to the above-mentioned compounds, salts with alkali metal salts, alkaline earth metal salts, organic bases, and the like, and salts with sodium, potassium, calcium, magnesium, ammonium, diethanolamine, ethylenediamine, and the like. These salts can be obtained by converting a group such as a carboxyl group present in a compound into a salt by a known method. Further, there may be mentioned salts with amines such as ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris (hydroxymethyl) aminomethane, N-bis (hydroxyethyl) piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine and L-glucosamine; or salts with basic amino acids such as lysine, delta-hydroxylysine, and arginine. In addition, for example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; salts with organic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, salicylic acid, and the like; or salts with acidic amino acids such as aspartic acid and glutamic acid.

[ use ]

The composition for improving skin disorders of the present invention is particularly suitable for improving skin disorders caused by air pollutants. Examples of the air pollutants include sulfur dioxide, nitrogen dioxide, suspended particulate matter, photochemical oxidant, trichloroethylene, and the like. Further, according to the air pollution prevention law (1968), "fumes" such as sulfur oxides, nitrogen oxides, dust, cadmium, chlorine, lead, hydrogen chloride, and hydrogen fluoride, which are emission-limited from fixed sources, "general dust" and "special dust" which are scattered from deposits such as minerals, and benzene, which is identified as "special substances", are used. Carbon monoxide and hydrocarbons, which are limited in their discharge from mobile sources, belong to this category. In recent years, formaldehyde and the like, which are causative substances of ward syndrome, have been included. Further, the malodors may be regarded as a form of air pollution, and the causative substances thereof are also exemplified as air pollutants. Suspended Particulate Matter (SPM) refers to solid and liquid particles Suspended in the atmosphere, and is classified according to the size of the particles (also called aerosol particles), and also includes automobile exhaust, urban atmospheric dust, and sand dust (yellow sand, etc.). The environmental standards stipulate that PM10 has a particle size of 10 μm or less and that PM2.5 has a particle size of 2.5 μm or less. The floating particulate matter may contain carbon, nitrate, sulfate, and ammonium salts, as well as inorganic elements such as silicon, sodium, and aluminum. The floating particulate matter may contain a substance that serves as a carrier for transporting a radioactive substance such as radioactive cesium. Here, "yellow sand" generally refers to dust carried away by wind from yellow river basins, deserts, and the like, and refers to substances having a particle size of about 4 μm. In the present invention, from the viewpoint of the action on the skin, at least 1 kind selected from sulfur dioxide, nitrogen dioxide, suspended particulate matter, photochemical oxidant, trichloroethylene, smog, ordinary dust, special dust (asbestos, etc.), special matter (benzene, etc.), carbon monoxide, hydrocarbon, formaldehyde and malodor affects the skin, and particularly, automobile exhaust gas, city air dust, pollen or sand dust affects the skin.

In the present specification, the skin to be applied is not limited as long as it is a site that can come into contact with air pollutants. Although not limited thereto, the air conditioner is preferably exposed to the outside and can directly contact air pollutants, and more preferably faces, hands, feet, scalp, neck, chest, back, and the like. In addition, although not limited thereto, from another viewpoint, it is preferable that the skin is affected by contact with air pollutants and friction of clothes or the like, which may cause skin troubles to be aggravated. In addition, although not limited thereto, from another viewpoint, it is preferable that the skin is affected by contact with air pollutants and increase in humidity due to clothes, hair, and the like, which may cause skin troubles.

In the present specification, the present inventors have confirmed that the expression of IL-8 is enhanced at the gene level and the protein level in epidermal keratinocytes by contacting the skin with an atmospheric pollutant. Based on this novel finding, a composition containing at least 1 or more IL-8 expression inhibitors can be suitably used for the purpose of improving skin disorders caused by atmospheric pollutants. Examples of the skin disorder caused by air pollutants include skin inflammation, atopic dermatitis, chronic urticaria, round alopecia, skin pruritus (pruritus of skin and skin), rash, erosion, sunburn, wrinkle, speckle, acne, skin cancer, rough skin, and sensitive skin. Although not limited thereto, the present invention can be applied to skin disorders based on the above-described mechanism, preferably skin inflammation caused by air pollutants, as long as the symptoms and conditions accompany skin inflammation.

In addition, the present inventors have confirmed that the gene expression level of a fluke protein or a zonulin is reduced in epidermal keratinocytes by exposing the skin to an air pollutant (particularly, automobile exhaust gas or urban air dust). In particular, it has been newly found that the barrier function of the skin is immature or decreased, leading to accelerated barrier function failure of the skin. Based on this novel finding, a composition containing at least 1 or more of a sluice protein expression promoting substance and/or a blocking protein expression promoting substance can be suitably used for the purpose of improving skin disorders caused by atmospheric pollutants. Although not limited thereto, the present invention can be suitably used for skin diseases caused by a decrease in skin barrier function as skin disorders based on the above-described mechanism. In this specification, skin barrier function refers primarily to the ability of the stratum corneum to retain water. The state of a decreased (or low or immature) skin barrier function includes, for example, a state of low age, preferably 20 years or less, more preferably 10 years or less, still more preferably an infant (0 to 7 years), and particularly preferably an infant (0 to 2 years). Examples of the state of a decrease in skin barrier function include a case where skin roughness is caused and a case where atopic dermatitis occurs. The measurement of the skin barrier function can be performed by evaluating the state of tight junction by measurement of transepithelial resistance (TER), as described in detail in the examples of the present specification. The present invention can also be suitably used for the purpose of improving skin barrier function, the purpose of promoting tight junction formation, the purpose of normalizing/strengthening tight junction function, the purpose of promoting formation of intercellular adhesive structure, the purpose of normalizing/strengthening intercellular barrier function, the purpose of normalizing/strengthening osmotic barrier function, and the like.

In the present specification, the present inventors have confirmed that the oxidative stress is increased by bringing the skin into contact with air pollutants (particularly, automobile exhaust gas or urban air dust). Based on the knowledge that wrinkles, acne, spots, and skin sagging or worsening due to oxidative stress, a composition containing at least 1 or more of the oxidative stress suppressants can be suitably used for the purpose of improving skin disorders due to atmospheric pollutants. It is known that oxidative stress of the skin is caused by ultraviolet rays, but according to the new findings of the present inventors, it is presumed that, depending on the state of the skin, even if the skin is protected from ultraviolet rays by a sunscreen cream or the like, the oxidative stress of the skin is not sufficiently reduced in an environment where the skin is continuously exposed to air pollutants. Although not limited, the mechanism described above is suitable for at least 1 selected from wrinkles, acne, spots and sagging of the skin, more preferably at least 1 selected from wrinkles, spots and sagging, and further suitable for wrinkles and/or spots, and particularly suitable for wrinkle formation and/or spot formation, as skin disorders. In addition, it is also applicable to disturbance of skin metabolism due to oxidative stress.

In the present specification, the present inventors have confirmed that the expression of IL-33 is enhanced at the gene level in epidermal keratinocytes by contacting the skin with an air pollutant. Based on this novel finding, a composition containing at least 1 or more IL-33 expression inhibitor can be suitably used for the purpose of improving skin disorders caused by atmospheric pollutants. Examples of the skin disorder caused by air pollutants include skin inflammation, atopic dermatitis, chronic urticaria, round alopecia, skin pruritus (pruritus of skin and skin), rash, erosion, sunburn, wrinkle, speckle, acne, skin cancer, rough skin, and sensitive skin. Although not limited thereto, the present invention can be applied to skin disorders based on the above-described mechanism, preferably skin inflammation caused by air pollutants, as long as the symptoms and conditions accompany skin inflammation.

In addition, the present invention is suitably used for the following population: people who want to block air pollutants, people who want to protect skin from air pollutants, people who want to shield skin from air pollutants, people who want to moisturize skin that is dry due to air pollutants, people who are concerned about pruritus due to air pollutants, people who are sensitive to skin, people who want to adjust tight junctions of skin, people who want to repair repeated skin roughening due to air pollutants, people who want to repair daily skin roughening due to air pollutants, people who are concerned about aging (aging) of skin due to air pollutants, oxidation, wrinkles, spots, lax people, people who are stressed by air pollutants, people who feel skin pruritus due to air pollutants, people who feel very dry due to air pollutants, people who generate reddish skin due to air pollutants, people who are caused by alternate seasons to rough skin, people who have rough skin due to changes in life, people who are caused by changes in skin, A person who is concerned about air (atmosphere) in a city, a person who is concerned about pruritus due to air (atmosphere) in a city, a person who is concerned about exhaust gas of an automobile, a person who is concerned about PM10, PM2.5, a person who is concerned about pruritus due to PM10, PM2.5, a person who is concerned about yellow sand, a person who is concerned about pruritus due to yellow sand, a person who is concerned about pollen, a person who is concerned about pruritus due to pollen, a person who wants to take anti-pollution measures, a person who is concerned about smoke of cigarettes, a person who wants to shield particulate pollution, a person who wants to keep skin clean, a person who wants to take anti-oxidation of skin, a person who wants to take anti-aging of skin, a person who wants to wash away air pollutants, and the like.

[ formulation forms ]

The composition for improving skin disorders of the present invention can be used as a skin external preparation in the form of a pharmaceutical, quasi-pharmaceutical, or cosmetic. The skin external preparation may be mixed with a known base or carrier added to the skin external preparation (cosmetics, quasi-drugs, drugs) to prepare a composition as long as the effects of the present invention are not impaired.

The composition for improving skin disorders of the present invention may be in a known form such as a liquid, suspension, emulsion, cream, ointment, gel, liniment, emulsion, aerosol, powder, cataplasm, a tablet in which a sheet such as a nonwoven fabric is impregnated with a drug solution, or a stick such as lipstick, as a pharmaceutical product, quasi-pharmaceutical product, or cosmetic. Among them, it is preferably used in the form of a liquid, a suspension, an emulsion, a cream, an ointment, a gel, or an emulsion.

In particular, specific forms in the case of cosmetic compositions include basic cosmetics such as lotions, milky lotions, creams, beauty liquids, sunscreen cosmetics, face masks, hand creams, body lotions, and body creams; cleaning cosmetics such as washing fabrics, makeup removing products, bath lotions, shampoos and washing liquids; makeup cosmetics such as foundation, makeup base solution, lip gloss, lipstick, and blush; bath additives, and the like. Such a cosmetic composition is preferably intended for people who are susceptible to the effects of air pollutants, and more preferably for infants, children, people with fragile skin, sensitive skin, dry skin, wavy skin, and problem skin.

Examples of the base or carrier include hydrocarbons such as liquid paraffin, squalane, gelled hydrocarbons (such as plastic matrix), ozokerite, α -olefin oligomer, and light liquid paraffin, methyl polysiloxane, crosslinked methyl polysiloxane, highly polymerized methyl polysiloxane, cyclic silicone, alkyl-modified silicone, crosslinked alkyl-modified silicone, amino-modified silicone, polyether-modified silicone, polyglycerol-modified silicone, crosslinked polyether-modified silicone, crosslinked alkyl polyether-modified silicone, silicone-alkyl chain co-modified polyglycerol-modified silicone, polyether-modified branched silicone, polyvinyl alcohol, polyglycerol-modified branched silicone, acrylic acid-modified branched silicone, and mixtures thereofSilicone oils such as silicon, phenyl-modified silicones, and silicone resins; cellulose derivatives such as ethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose; polyvinylpyrrolidone; carrageenan; polyvinyl butyral; polyethylene glycol; II

An alkane; butanediol adipate polyester; esters such as isopropyl myristate, octyldodecyl myristate, isopropyl palmitate, cetyl palmitate, isononyl isononanoate and pentaerythritol tetrakis-2-ethylhexanoate; polysaccharides such as dextrin and maltodextrin; lower alcohols such as ethanol and isopropanol; glycol ethers such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monopropyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monopropyl ether, diethylene glycol monobutyl ether, propylene glycol monoethyl ether, propylene glycol monopropyl ether, dipropylene glycol monoethyl ether, and dipropylene glycol monopropyl ether; polyhydric alcohols such as polyethylene glycol, propylene glycol, 1, 3-butylene glycol, glycerin, and isoprene glycol; water-based base agents such as water.

The base or carrier may be used in 1 kind alone or in combination of 2 or more kinds.

The composition for improving skin disorders of the present invention may be incorporated, as needed, into various components generally used in the fields of pharmaceuticals, quasi-pharmaceuticals, and cosmetics, such as a surfactant, a moisturizing agent, a stabilizer, a stimulus-reducing agent, a blood circulation-promoting agent, a scrub agent, a thickener, a preservative, an antioxidant, a colorant, a pearl essence-imparting agent, a dispersant, a chelating agent, a pH adjuster, a perfume, an ultraviolet absorbing component, an ultraviolet scattering component, a cleansing component, an antibacterial component, an anti-inflammatory component, a firming component, a vitamin, a peptide or a derivative thereof, an amino acid or a derivative thereof, a keratolytic component, a cell activating component, and the like, in an amount and quality that do not impair the effects of the present invention. These components may be blended in 1 kind alone or in an arbitrary combination of 2 or more kinds. These components may be mixed with a known base or carrier and blended in the skin disorder-improving composition. The composition for improving skin disorders of the present invention can also be produced by treating these components according to a conventional method.

Examples of the surfactant include sorbitan fatty acid esters such as sorbitan monoisostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, diglycerin sorbitan penta-2-ethylhexanoate, and diglycerin sorbitan tetra-2-ethylhexanoate; propylene glycol fatty acid esters such as propylene glycol monostearate; hydrogenated castor oil derivatives such as polyoxyethylene hydrogenated castor oil 40(HCO-40), polyoxyethylene hydrogenated castor oil 50(HCO-50), polyoxyethylene hydrogenated castor oil 60(HCO-60), and polyoxyethylene hydrogenated castor oil 80; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene (20) sorbitan monolaurate (polysorbate 20), polyoxyethylene (20) sorbitan monostearate (polysorbate 60), polyoxyethylene (20) sorbitan monooleate (polysorbate 80), and polyoxyethylene (20) sorbitan isostearate; polyoxyethylene mono-coconut oil fatty acid glyceride; a glycerol alkyl ether; an alkyl glucoside; polyoxyalkylene alkyl ethers such as polyoxyethylene cetyl ether; amines such as stearylamine and oleylamine; silicone surfactants such as polyoxyethylene methyl polysiloxane copolymer, lauryl PEG-9 polydimethylsiloxyethyl dimethicone, and PEG-9 polydimethylsiloxyethyl dimethicone.

Examples of the moisture-retaining component include polyhydric alcohols such as glycerin, 1, 3-butylene glycol, propylene glycol, polyethylene glycol, and diglycerol trehalose; high molecular compounds such as heparin analogues, sodium chondroitin sulfate, collagen, elastin, keratin, chitin, and chitosan; amino acids such as glycine, aspartic acid and arginine; natural moisturizing factors such as sodium lactate, urea, and sodium pyrrolidone carboxylate; lipids such as ceramide, cholesterol, and phospholipid; plant extracts such as chamomile extract, witch hazel extract, green tea extract, and perilla extract. Among these moisture-retaining components, from the viewpoint of enhancing the effect of the present invention, ceramide is preferably blended, and ceramide 2 is particularly more preferably blended. It is presumed that ceramide (particularly ceramide 2) has a water-retaining function, and therefore, when it is used in combination with a functional component considered to be effective in the present invention, the amount of water in epidermal cells and dermal cells is increased, and the effect of the present invention can be improved. As shown in examples 5 and 6, in the state where the skin barrier function is normal, the influence of the atmospheric pollutants on the barrier function may be small, and the influence of the other substances may be small. Therefore, it is possible that substances such as ceramides that improve the barrier function of the skin can reduce the negative effects of atmospheric pollutants obtained according to the invention of the present application on the skin. The preferable amount is 0.000001 to 5 wt%.

Examples of the stabilizer include sodium polyacrylate, dibutylhydroxytoluene, and butylhydroxyanisole.

Examples of the irritation-reducing agent include gum arabic, polyvinylpyrrolidone, licorice extract, and sodium alginate.

Examples of the blood circulation-promoting agent include acetylcholine, itaconic alcohol, caffeine, capsaicin, cantharides tincture, γ -oryzanol, ginger tincture, zingerone, cepharanthine, swertia japonica extract, tannic acid, capsicum tincture, tolazolin, tocopherol nicotinate, benzyl nicotinate, and the like.

Examples of the scrub agent include apricot kernel powder, almond shell powder, almond kernel powder, sodium chloride granules, olive kernel powder, sea water-dried granules, candelilla wax, walnut shell powder, cherry kernel powder, coral powder, charcoal powder, hazelnut shell powder, polyethylene powder, and anhydrous silicic acid.

Examples of the thickener include guar gum, locust bean gum, carrageenan, xanthan gum, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, alkyl acrylate copolymer, polyethylene glycol, bentonite, hydroxyethyl acrylate/sodium acryloyldimethyltaurate copolymer, ammonium acryloyldimethyltaurate/vinylpyrrolidone) copolymer, and the like.

Examples of the preservative include benzoic acid, sodium benzoate, dehydroacetic acid, sodium dehydroacetate, isobutyl parahydroxybenzoate, isopropyl parahydroxybenzoate, butyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, benzyl parahydroxybenzoate, methyl parahydroxybenzoate, and phenoxyethanol.

Examples of the antioxidant include dibutylhydroxytoluene, butylhydroxyanisole, sorbic acid, sodium sulfite, ascorbic acid, isoascorbic acid, and L-cysteine hydrochloride.

Examples of the colorant include inorganic pigments and natural pigments.

Examples of the pearlescence-imparting agent include ethylene glycol distearate, ethylene glycol monostearate, and triethylene glycol distearate.

Examples of the dispersant include sodium pyrophosphate, sodium hexametaphosphate, polyvinyl alcohol, polyvinyl pyrrolidone, a crosslinked methyl vinyl ether/maleic anhydride copolymer, and an organic acid.

Examples of the chelating agent include disodium EDTA, calcium EDTA and disodium EDTA.

Examples of the pH adjuster include inorganic acids (hydrochloric acid, sulfuric acid, and the like), organic acids (lactic acid, sodium lactate, citric acid, sodium citrate, succinic acid, sodium succinate, and the like), inorganic bases (potassium hydroxide, sodium hydroxide, and the like), organic bases (triethanolamine, diisopropanolamine, triisopropanolamine, and the like), and the like.

Examples of the ultraviolet absorbing component include octyl triazone, diethylamino oxybenzoyl hexyl benzoate, dimethoxybenzylidene dioxaimidazoline octyl propionate, 2-ethylhexyl p-methoxycinnamate, tert-butyl methoxydibenzoylmethane, phenylbenzimidazole sulfonic acid, octyl methoxycinnamate, ethylhexyl methoxycinnamate, and the like.

Examples of the ultraviolet scattering component include inorganic compounds such as hydrous silicic acid, zinc silicate, cerium silicate, titanium silicate, zinc oxide, zirconium oxide, cerium oxide, titanium oxide, iron oxide, and anhydrous silicic acid, substances obtained by coating these inorganic compounds with inorganic powders such as hydrous silicic acid, aluminum hydroxide, mica, and talc, or by compounding these inorganic compounds with resin powders such as polyamide, polyethylene, polyester, polystyrene, and nylon, and substances obtained by further treating these inorganic compounds with silicone oil, fatty acid aluminum salt, and the like.

Examples of the cleansing component include alkali metal salts such as potassium laurate, potassium myristate, potassium palmitate and potassium monostearate, soaps such as alkanolamide salts and amino acid salts, amino acid surfactants such as sodium cocoyl glutamate and sodium cocoyl methyl taurate, ether sulfate salts such as sodium lauryl sulfate, ether carboxylates such as sodium lauryl ether acetate, sulfosuccinate salts such as sodium alkyl sulfosuccinate, fatty acid alkanolamides such as coconut oil fatty acid monoethanolamide and ethanolamine in coconut oil fatty acid, monoalkyl phosphate salts such as sodium lauryl phosphate and polyoxyethylene lauryl ether sodium phosphate, coconut oil fatty acid amidopropyl dimethylamino acetic acid betaine, lauryl dimethylamino acetic acid betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazole

Betaine-type amphoteric surfactants such as betaine, lauryl hydroxysulfobetaine, and lauroylamide ethyl hydroxyethyl carboxymethylbetaine hydroxypropyl sodium phosphate, amino acid-type amphoteric surfactants such as sodium laurylaminopropionate, and the like.

Examples of the antibacterial component include chlorhexidine, salicylic acid, benzalkonium chloride, rivanol, ethanol, benzethonium chloride, cresol, gluconic acid and its derivatives, iodinated polyvinylpyrrolidone, potassium iodide, iodine, isopropylmethylphenol, triclocarban, triclosan, photosensitizer No. 101, photosensitizer No. 201, paraben, phenoxyethanol, 1, 2-pentanediol, alkyldiaminoglycine hydrochloride, pyridone ethanolamine salt, miconazole, and the like.

Examples of the anti-inflammatory agent include azulene, aminocaproic acid, hydrocortisone, and the like.

Examples of the tightening component include zinc oxide, zinc sulfate, allantoin aluminum hydroxide, aluminum chloride, zinc phenolsulfonate, tannic acid, and the like.

Examples of vitamins include vitamin E such as dl- α -tocopherol, dl- α -tocopherol acetate, dl- α -tocopherol succinate, dl- α -calcium tocopherol succinate, vitamin B2 such as riboflavin, flavin mononucleotide, flavin adenine dinucleotide, riboflavin butyrate, riboflavin tetrabutyrate, riboflavin 5' -sodium phosphate, riboflavin tetranicotinate, vitamin C such as dl- α -tocopherol, benzyl nicotinate, methyl nicotinate, β -butoxyethyl nicotinate, 1- (4-methylphenyl) ethyl nicotinate, vitamin D such as ascorbyl-A, ascorbyl monostearate, ascorbyl palmitate, L-ascorbyl dipalmitate, vitamin C such as methylcetyl glycoside, calcemic acid, cholecalciferol, vitamin D such as monophosphoryl, farnesol quinone, vitamin K such as gamma-oryzanol, dibenzoylthioamine hydrochloride, thiamine hydrochloride, vitamin D-phosphate such as pyridoxine hydrochloride, vitamin D-carnitine hydrochloride, vitamin D-carnitine hydrochloride, vitamin D-phosphate, vitamin D-pyridoxine hydrochloride, vitamin D-L-ascorbyl-L-ascorbyl-L.

Examples of the peptide or its derivative include a keratin degradation peptide, a hydrolyzed keratin, a collagen, a fish-derived collagen, a telogen, a gelatin, an elastin degradation peptide, a collagen degradation peptide, a hydrolyzed collagen, a hydroxypropylammonium chloride hydrolyzed collagen, an elastin degradation peptide, a sword bean protein degradation peptide, a hydrolyzed sword bean protein, a silk protein degradation peptide, a hydrolyzed silk, sodium lauroyl-hydrolyzed silk, a soybean protein degradation peptide, a hydrolyzed soybean protein, a wheat protein degradation peptide, a hydrolyzed wheat protein, a casein degradation peptide, an acylated peptide (e.g., a palmitoyl oligopeptide, a palmitoyl pentapeptide, and a palmitoyl tetrapeptide), and the like.

Examples of the amino acid or a derivative thereof include betaine (trimethylglycine), proline, hydroxyproline, arginine, lysine, serine, glycine, alanine, phenylalanine, β -alanine, threonine, glutamic acid, glutamine, asparagine, aspartic acid, cysteine, cystine, methionine, leucine, isoleucine, valine, histidine, taurine, γ -aminobutyric acid, γ -amino- β -hydroxybutyric acid, carnitine, carnosine, creatine, and the like.

Examples of the keratolytic ingredient include lactic acid, salicylic acid-glycolic acid, gluconic acid, citric acid, malic acid, phytic acid, urea, sulfur, and the like.

Examples of the cell activating component include amino acids such as γ -aminobutyric acid and ∈ -aminocaproic acid, vitamins such as retinol, thiamine, riboflavin, pyridoxine hydrochloride and pantothenic acid, α -hydroxy acids such as glycolic acid and lactic acid, tannin, flavonoid, saponin, photosensitizer No. 301, and the like.

[ Properties ]

The pH of the composition for improving skin disorders of the present invention is not particularly limited as long as it is within a pharmaceutically, pharmacologically or physiologically acceptable range, and examples thereof include a pH of 3.0 to 9.5, preferably 3 to 8, more preferably 3 to 7, even more preferably 3 to 6, and particularly preferably 4 to 6.

The composition for improving skin disorders of the present invention can be adjusted to an osmotic pressure ratio within a range allowable for living bodies, if necessary. The appropriate osmotic pressure ratio is usually in the range of 0.5 to 5.0, more preferably 0.6 to 3.0, and still more preferably 0.7 to 2.0, depending on the site of application, dosage form, and the like. The adjustment of osmotic pressure can be performed by a method known in the art using an inorganic salt, a polyhydric alcohol, a sugar, or the like. The osmotic pressure ratio was determined based on the ratio of the osmotic pressure of the test sample to the osmotic pressure of 286mOsm (0.9 w/v% aqueous sodium chloride solution) as revised in Japanese pharmacopoeia at the seventeenth time, and the osmotic pressure was measured based on the osmotic pressure measurement method (freezing point depression method) described in the Japanese pharmacopoeia. The standard solution for measuring osmotic pressure ratio (0.9 w/v% aqueous sodium chloride solution) is prepared by drying sodium chloride (japanese pharmacopoeia standard reagent) at 500 to 650 ℃ for 40 to 50 minutes, cooling the dried product in a desiccator (silica gel), weighing 0.900g of the dried product accurately, dissolving the weighed product in purified water to prepare 100mL accurately, or by using a commercially available standard solution for measuring osmotic pressure ratio (0.9 w/v% aqueous sodium chloride solution).

The viscosity of the composition for improving skin disorders of the present invention is appropriately set depending on the kind and content of the components to be blended, the form of the preparation, the method of use, and the like, as long as the viscosity is within a pharmaceutically, pharmacologically, or physiologically acceptable range. The viscosity at 20 ℃ as measured with a rotary viscometer (RE550 type viscometer, manufactured by Toyobo industries Co., Ltd., spindle; 1 ℃ 34'. times.R 24) is preferably 1 mPas or more, more preferably 2000 mPas or more, and still more preferably 5000 mPas or more.

[ inhibitor of IL-8 expression ]

In another embodiment, the present invention may also provide an IL-8 expression inhibitor containing at least 1 or more selected from the group consisting of hyaluronic acid and a salt thereof, a derivative of hyaluronic acid and a salt thereof, an artichoke extract, tranexamic acid and a salt thereof, a phyllostachys pubescens leaf extract, a camellia extract, a rose extract, a perilla extract, a scutellaria baicalensis extract, a licorice extract, a camellia extract, an aloe leaf extract, a dog rose fruit extract, a coptis chinensis extract, a loquat leaf extract, a cherry leaf extract, a rosemary leaf extract, a violet leaf extract, a sage leaf extract, a thyme extract, a carrot root extract, allantoin, wufenfenamate, glycyrrhizic acid and a salt thereof, glycyrrhetinic acid and a salt thereof, stearyl glycyrrhetinate, and cholesterol.

The kind and content of the above components, other components, preparation form, physical properties, and the like are based on the items described above [ composition for improving skin disorders ].

[ inhibitor of IL-33 expression ]

In another embodiment, the present invention can also provide an IL-33 expression inhibitor containing at least 1 or more selected from the group consisting of allantoin, lidocaine, isopropylmethylphenol, diphenhydramine and a salt thereof, hyaluronic acid and a salt thereof, a derivative of hyaluronic acid and a salt thereof, magnesium chloride, cholesterols, glycyrrhizic acid and a salt thereof, glycyrrhetinic acid and a salt thereof, stearyl glycyrrhetinate, and isofenvalerate.

The kind and content of the above-mentioned components, other components, preparation form, physical properties, and the like are based on the items described above [ composition for improving skin disorders ].

[ other embodiments ]

In addition to the above, in other embodiments, the present invention may provide the following:

use of a composition containing at least 1 or more IL-8 expression inhibitors for producing an agent for improving skin disorders caused by air pollutants;

use of a composition comprising 1 or more selected from hyaluronic acid and a salt thereof, a derivative of hyaluronic acid and a salt thereof, an artichoke extract, tranexamic acid and a salt thereof, a phyllostachys pubescens leaf extract, a camellia extract, a rose extract, a perilla extract, a scutellaria baicalensis extract, a licorice extract, a camellia extract, an aloe leaf extract, a dog rose fruit extract, a coptis chinensis extract, a loquat leaf extract, a cherry leaf extract, a rosemary leaf extract, a Japanese sage leaf extract, a thyme extract, a carrot root extract, allantoin, urafenamate, glycyrrhizic acid and a salt thereof, glycyrrhetinic acid and a salt thereof, stearyl glycyrrhetinate, and cholesterol for the production of a skin disorder-improving agent due to an atmospheric pollutant;

use of a composition comprising at least 1 or more of a substance promoting expression of a floodgate protein and/or a substance promoting expression of a blocking protein for the manufacture of an agent for improving a skin disorder caused by an atmospheric pollutant;

use of a composition containing 1 or 2 or more selected from the group consisting of an extract of tangerine peel, an extract of cowberry leaf, an extract of white willow bark, an extract of arnica, an extract of angelica keiskei, an extract of coix seed, an extract of ginkgo biloba leaf, an extract of turmeric, an extract of rose hip, an extract of scutellaria baicalensis, an extract of artemisia princeps, an extract of chamomile, an extract of perilla leaf, an extract of peach seed, an extract of melissa officinalis, an extract of lavender, and a sodium salt of a condensate of N-lauroyl-L-glutamic acid and L-lysine for the production of an agent for improving skin disorders caused by air pollutants;

use of a composition containing at least 1 or more kinds of inhibitors of oxidative stress for producing an agent for improving skin disorders caused by air pollutants;

use of a composition containing 1 or more than 2 selected from the group consisting of a scutellaria baicalensis extract, a bilberry extract, hydrolyzed royal jelly, sunflower oil, peppermint, glycerol glucoside, polygonum multiflorum extract, nicotinamide, glycogen, a centella asiatica extract, a mallow extract, a houttuynia cordata extract, a neem extract, an algae extract, a phellodendron bark extract, ascorbic acid, a ginkgo biloba leaf extract, a camellia sinensis extract, a green tea extract, an aloe leaf extract, a hibiscus flower extract, a perilla leaf extract, a rosemary leaf extract, a sage leaf extract, a citrus extract, a chamomile extract, a licorice extract, an artichoke extract, and a eucalyptus extract in the manufacture of an agent for improving skin disorders caused by air pollutants;

use of a composition containing at least 1 or more IL-33 expression inhibitors for producing an agent for improving skin disorders caused by air pollutants;

use of a composition comprising 1 or 2 or more selected from the group consisting of allantoin, lidocaine, isopropyl methylphenol, diphenhydramine and salts thereof, hyaluronic acid derivatives and salts thereof, magnesium chloride, cholesterols, glycyrrhizic acid and salts thereof, glycyrrhetinic acid and salts thereof, stearyl glycyrrhetinate, and isofenvalerate for the production of an agent for improving skin disorders caused by air pollutants;

a method for improving skin disorders caused by atmospheric pollutants, comprising the step of applying to the skin a composition containing at least 1 or more inhibitor of IL-8 expression;

a method for improving skin disorders caused by air pollutants, comprising the step of applying to the skin a composition comprising 1 or more than 2 selected from hyaluronic acid and salts thereof, derivatives of hyaluronic acid and salts thereof, artichoke extract, tranexamic acid and salts thereof, phyllostachys pubescens leaf extract, camellia extract, rose extract, perilla extract, scutellaria extract, licorice extract, camellia extract, aloe leaf extract, dog rose fruit extract, coptis extract, loquat leaf extract, cherry leaf extract, rosemary leaf extract, Japanese sage leaf extract, thyme extract, carrot root extract, allantoin, isoxadifen, glycyrrhizic acid and salts thereof, glycyrrhetinic acid and salts thereof, stearyl glycyrrhetinate, and cholesterol;

a method for improving skin disorders caused by atmospheric pollutants, comprising the step of applying to the skin a composition containing at least 1 or more of a substance promoting the expression of a lockin and/or a substance promoting the expression of a lockin;

a method for improving skin disorders caused by atmospheric pollutants comprising the step of applying to the skin 1 or more than 2 kinds of compositions selected from the group consisting of an extract of tangerine pericarp, an extract of cowberry leaf, an extract of white willow bark, an extract of arnica montana, an extract of angelica keiskei, an extract of coix seed, an extract of ginkgo biloba, an extract of turmeric, an extract of rosa multiflora (rose hip), an extract of scutellaria baicalensis, an extract of artemisia princeps, an extract of chamomilla, an extract of perilla leaf, an extract of peach seed, an extract of melissa officinalis, an extract of lavender, and a sodium salt of a condensate of N-lauroyl-L-glutamic acid and L-lysine;

a method for improving skin disorders caused by atmospheric pollutants, comprising the step of applying to the skin a composition containing at least 1 or more kinds of inhibitors of oxidative stress;

a method for improving skin disorders due to atmospheric pollutants, comprising applying to the skin a composition containing 1 or more than 2 of Scutellaria baicalensis extract, cowberry fruit extract, hydrolyzed royal jelly, sunflower oil, Mentha piperita, glycerol glucoside, Actinidia polygama extract, nicotinamide, glycogen, centella asiatica extract, mallow extract, houttuynia cordata extract, Azadirachta indica extract, algae extract, phellodendron amurense extract, ascorbic acid, ginkgo biloba extract, Camellia sinensis extract, green tea extract, aloe vera leaf extract, hibiscus flower extract, perilla leaf extract, rosemary leaf extract, sage leaf extract, citrus extract, chamomile extract, licorice extract, artichoke extract, and eucalyptus extract;

a method for improving skin disorders caused by atmospheric pollutants, comprising the step of applying to the skin a composition containing at least 1 or more inhibitor of IL-33 expression; and the number of the first and second groups,

comprises applying a composition containing 1 or more than 2 of allantoin, lidocaine, isopropyl methylphenol, diphenhydramine and its salt, hyaluronic acid derivative and its salt, magnesium chloride, cholesterol, glycyrrhizic acid and its salt, glycyrrhetinic acid and its salt, stearyl glycyrrhetinate, and isofenvalerate to the skin, and improving skin disorder caused by air pollutants.

The kind and content of the above-mentioned components, other components, preparation form, physical properties, and the like are based on the items described above [ composition for improving skin disorders ].

Examples

The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. In addition, "%" in examples represents the dry solid content or the mass% of the essential component unless otherwise specified.

[ test example 1: evaluation of cytotoxicity based on atmospheric contaminants ]

In a 24-well plate (Cell Bind, Corning Co., Ltd.), normal Human epidermal keratinocytes (Kurabo Co., Ltd., Human epidermal Keratinocyte: NHEK) were seeded in a culture medium for proliferation of normal Human epidermal keratinocytes at a Cell number of 80000 cells/well. After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the medium was replaced with a medium containing 4 air pollutants dissolved therein at each concentration, and the culture was continued for 1 day.

As 4 air pollutants, automobile exhaust gas (VehicleExhaust Particles: VEP, NIES CRM No.8, concentration: 10. mu.g/mL, 25. mu.g/mL, or 50. mu.g/mL), city air Dust (Urban aerogels: UA, NIES CRM No.28, concentration: 10. mu.g/mL, 25. mu.g/mL, or 50. mu.g/mL), Gobi yellow sand (Gobi Kosa Dust: GKD, NIES CRM No.30, concentration: 10. mu.g/mL, 25. mu.g/mL, or 50. mu.g/mL), and Japanese cedar Pollen (Ceder Pollen: CP, product No. 10901, concentration: 250. mu.g/mL, 500. mu.g/mL, or 1000. mu.g/mL) were purchased from independent administrative agency. The concentration conditions of these 4 air pollutants are also the same in fig. 1 to 17 described later.

After the culture, Hoechst 33342(Molecular Device) was diluted in the medium and replaced with the medium in the wells. After 10 minutes of staining at room temperature, image capture (16 fields/well) was performed using ImageXpress (Molecular Device). The cell number was determined by analysis using a cell counting program. From the measurement results, the relative values were calculated for the case where air pollutants were also added, with the number of cells to which only the culture medium (control) was added being 1 (FIGS. 1A to D). The results of using VEP, UA, GKD and CP as air pollutants, respectively, are shown in FIGS. 1A-D.

As shown in fig. 1A to D, no significant decrease in the number of cells was observed at any concentration of air pollutants.

[ test example 2: evaluation of Effect of atmospheric pollutants on skin (Gene expression analysis)

NHEK was seeded in a 24-well plate (Cell Bind, Corning Inc.) at a Cell number of 80000 cells/well, after 24 hours of incubation at 37 ℃ under 5% carbon dioxide and 95% air, the medium was replaced with a medium in which 4 air pollutants were dissolved at each concentration, further 1 day of incubation was performed, after 2 washes with PBS (-) after incubation, RNA was extracted using RNeasy Mini Kit (Qiagen Co.), TOYOBO Reversar Ace RT Master Mix with gDNAremover (TOYOBO Co., Ltd.), cDNA was prepared, Premix Ex Taq (registered trademark) was used for the cDNA prepared, and analysis was performed by qRT-PCR, the gene expression of the medium alone added (control) was set to 1, and the relative values in the case where the pollutants were added (FIGS. 2A to D) were calculated by using Taq Ex Taq Gen BioHs Hs Gen BioHs Hs Gen Hs Gen No. of the samples of Taq Gen Sho Min Gen Sho-5, and Gen No. 6 was used for describing the atmospheric pollutants of Taq Gen

m1、IL-8：Hs00174103m1、IL-33：Hs00369211m1、MMP1：＿＿＿

Hs00899658m1、Hs00234579m1、CLDN1：Hs00221623m1、OCLN：

＿＿＿

Hs00170162m1。

＿

As shown in fig. 2A to D, the expression of IL-6 and IL-8, which induce inflammation by all air pollutants, is increased, and thus it is shown in the same way as other literature reports that air pollutants induce skin inflammation, on the other hand, IL-1 β, MMP1, MMP9 are increased by VEP and UA (fig. 2A, B), and IL-33 is increased by GKD and CP (fig. 2C, D), and thus different indications are given for the influence on the skin depending on the type of air pollutants, in this experimental system, VEP and UA are given to suggest that inflammation associated with oxidative stress is induced, and to participate in the development of spots, acne, etc., and it is suggested that activation of MMP by VEP and UA induces the decomposition of extracellular matrix such as collagen, and destruction of basement membrane, which are considered to participate in the formation of wrinkles and sagging, on the other hand, while GKD, CP are known to promote the expression of IL-33 by Toll-like receptor (TLR receptor), and to make skin allergy-sensitive to skin development by Th-receptor, and the like, and the skin allergy-induced by this immune cells, and the like, and the skin allergy-induced by Th-induced skin allergy.

[ test example 3: evaluation of the Effect of atmospheric contaminants on skin (oxidative stress) ]

NHEK was seeded at a Cell Bind (Corning) in a 24-well plate at a Cell number of 80000 cells/well. After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the medium was replaced with a medium containing 4 air pollutants dissolved therein at each concentration, and the culture was continued for 1 day. After the culture, the cells were washed 2 times with PBS (-), and CellROX (registered trademark) green reagent for oxidative stress detection (Thermo Fisher Scientific Co.) and Hoechst 33342 were diluted in the medium and replaced with the medium in the wells. After incubation for 30 minutes at 37 ℃, 5% carbon dioxide and 95% air, image capture was performed with ImageExpress (16 fields/well). The oxidative stress activity per cell number was measured by analysis using a fluorescence intensity measurement program and a cell count program. From the measurement results, the oxidative stress activity per cell number of the medium (control) alone was set to 1, and relative values were calculated when air pollutants were added (fig. 3A to D).

As shown in fig. 3A to D, oxidative stress was increased in NHEK by VEP and UA (fig. 3A, B). From the results thus far, it is suggested that VEP and UA induce inflammation in the skin accompanied by oxidative stress. On the other hand, it suggests that GKD and CP exert an influence on the skin through other pathways not accompanied by oxidative stress (FIG. 3C, D).

[ test example 4: evaluation of IL-8 expression level based on atmospheric contaminants

NHEK was seeded at a Cell count of 80000 cells/well in a 24-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the medium was replaced with a medium containing 4 air pollutants dissolved therein at each concentration, and the culture was continued for 1 day. After the culture, the supernatant (ELISA sample) was collected, and the expression level of IL-8 was measured using HumanIL-8/CXCL8DuoSet ELISAKit (R & D Systems). The plate from which the supernatant was removed was washed 2 times with PBS, and Hoechst 33342 was diluted in the medium and replaced with the medium in the wells. After incubation for 10 minutes at 37 ℃, 5% carbon dioxide and 95% air, image capture was performed with ImageExpress (16 fields/well). The cell number was determined by analysis using a cell counting program. Based on the measurement results, the number of cells to which only the medium was added (control) and the expression level of IL-8 per cell number were each set to 1, and relative values were calculated when air pollutants were added (FIGS. 4A to D).

As shown in FIGS. 4A to D, the protein expression level of IL-8 was increased by all air pollutants as in the case of gene expression.

[ test example 5: evaluation of Effect of atmospheric contaminants on Barrier function (1)

NHEK was seeded on a 12-well plate (12mm Transwell (registered trademark) with 0.4 μm Pore Polyester membrane insert, Sterile, Corning Co.) in a nested plate at cell number of 96000 cells/well. After culturing at 37 ℃ in an atmosphere of 5% carbon dioxide and 95% air for 3 days, the medium was changed to a differentiation induction medium (2mM Ca)²⁺Humedia-KG2) for 4 days. Thereafter, the medium was replaced with a new differentiation induction medium (2mM Ca)²⁺Humedia-KG2), cultured for 2 days, and then replaced with a differentiation-inducing medium (2mM Ca) prepared by dissolving 4 air pollutants at various concentrations²⁺Humedia-KG2), for a period of 5 days. The resistance values of the nested plates were measured from the day of addition of the air pollutants (day 9 after the start of the culture) using Millicell (registered trademark) ERS-2Voltohmmeter (Millipore Co.) (FIGS. 5A to D). In each figure, the concentrations of the air pollutants used were VEP 50. mu.g/mL, UA 50. mu.g/mL, GKD 50. mu.g/mL, and CP 1000. mu.g/mL. In each figure, the solid line shows the measurement results in the medium to which each air contaminant was added, and the broken line shows the measurement results in the medium alone (control). The concentration conditions of the air pollutants to be used are the same as those in FIGS. 6 to 7 described later.

As shown in FIGS. 5A to D, the change of TER due to air pollutants was not observed. From this result, it is suggested that the effect of the atmospheric pollutants on the barrier function of the skin is small in the case where the skin has a normal barrier function.

[ test example 6: evaluation of Effect of atmospheric contaminants on Barrier function (2)

On a nested plate of 12-well plate (12mm Transwell (registered trademark) with 0.4 μm Pore Polyester membrane insert, Sterile, Corning Co., Ltd.), NHEK was cultured in 96000 cell numbersCells/well were seeded. After culturing at 37 ℃ in an atmosphere of 5% carbon dioxide and 95% air for 3 days, the medium was replaced with a differentiation-inducing medium (2mM Ca) prepared by dissolving 4 atmospheric contaminants at various concentrations²⁺Humedia-KG2), for a period of 6 days. Meanwhile, the resistance value of the nested plates was measured by using Millicell (registered trademark) ERS-2Voltohmmeter (Millipore Co.) (FIGS. 6A to D). In each figure, the solid line shows the measurement results in the medium to which each air contaminant was added, and the broken line shows the measurement results in the medium alone (control).

As shown in fig. 6A to D, the rise of TER is inhibited by VEP and UA (fig. 6A, B). From these results, it is suggested that the skin barrier failure in the case where VEP and UA contained in the air pollutants are immature in the skin barrier and in the case where the skin barrier function is decreased is accelerated.

[ test example 7: elucidation of mechanism of action for reducing barrier formation mechanism based on atmospheric pollutants ]

NHEK was seeded at a Cell number of 84000 cells/well in a 48-well plate (Cell Bind, Corning). After culturing at 37 ℃ in an atmosphere of 5% carbon dioxide and 95% air for 3 days, the medium was replaced with a differentiation-inducing medium (2mM Ca) prepared by dissolving 4 atmospheric contaminants at various concentrations²⁺Humedia-KG2), cultured for a period of 3 days. After the culture, the cells were washed 2 times with PBS (-) and RNA was extracted using RNeasy Mini Kit (Qiagen), and then cDNA was prepared using TOYOBO ReverTra Ace qPCR RTMaster Mix with gDNASREMOVER (TOYOBO). The prepared cDNA was analyzed by qRT-PCR using Premix ExTaq (registered trademark). From the measurement results, the relative values were calculated for the case where the air pollutants were also added, with the expression of each gene being 1 in the medium (control) alone (FIGS. 7A to D). Incidentally, TaqmanProbe was purchased from Applied biosystems. Each Taqman Probe uses CLDN 1: hs00221623 _ m1, OCLN: hs00170162 _ m 1.

As shown in FIGS. 7A to D, VEP and UA reduced the expression levels of the genes constituting the tight junction, namely CLDN1 and OCLN (FIG. 7A, B). Based on the results of test example 5 (fig. 5), it is considered that VEP and UA impair the tight junction and decrease the barrier function of the skin.

[ test example 8: discovery of materials for inhibiting UA-induced activation of IL-8

NHEK was seeded at a Cell count of 80000 cells/well in a 24-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the culture medium was replaced with a medium in which the candidate compounds (dipotassium glycyrrhizinate, tranexamic acid, artichoke extract (Ichimaru Falcos), sodium hyaluronate HA12NB (Seisaku corporation), Oligo-HA4(SIGMA corporation), Rosa roxburghii extract (Ichimaru Falcos)), allantoin, ursolic acid, glycyrrhetinic acid, and cholesterol were dissolved at respective concentrations, and the culture was performed. After 24 hours of culture, the culture medium was replaced with a medium prepared by dissolving UA and the candidate compound at each concentration, and the culture was further carried out for one day. After the culture, the supernatant (ELISA sample) was collected, and the expression level of IL-8 was measured by means of a Human IL-8/CXCL8DuoSet ELISAKit (R & D Systems). The plate from which the supernatant was removed was washed 2 times with PBS, and Hoechst 33342 was diluted in the medium and replaced with the medium in the wells. After incubation for 10 minutes at 37 ℃, 5% carbon dioxide and 95% air, image capture was performed using ImageExpress (16 fields/well). The cell number was measured by analysis using a cell counting program. Based on the measurement results, the number of cells to which only the medium (control) was added and the expression level of IL-8 per cell number were set to 1, and relative values were calculated when the air contaminant and the candidate material were added (FIGS. 8A to J).

As shown in FIGS. 8A-J, glycyrrhizic acid and its salts, tranexamic acid, HA4, artichoke extract, biological hyaluronic acid, Rosa roxburghii extract, allantoin, ifenacin, glycyrrhetinic acid, and cholesterol inhibited IL-8 activation by UA. From the results, it was shown that inflammation of the skin due to urban atmospheric dust including PM2.5 and the like was suppressed.

[ test example 9: search for materials for improving barrier function reduction due to UA ]

NHEK was seeded on a 12-well plate (12mm Transwell (registered trademark) with 0.4 μm Pore Polyester membrane insert, Sterile, Corning Co.) in a nested plate at cell number of 96000 cells/well.After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 3 days, UA and a candidate compound (Citrus pericarp extract (Ichimaru Falcos Co., Ltd.)) were dissolved at various concentrations and replaced with a differentiation induction medium (2 mMCa)²⁺Humedia-KG2), cultured for a period of 7 days. The resistance values of the nested plates were measured by Millicell (registered trademark) ERS-2Voltohmmeter (Millipore Co.) on the 9 th and 10 th days from the start of culture (FIGS. 9A to B).

As shown in fig. 9A-B, TER increased based on citrus peel extract. From the results, it is suggested that the citrus peel extract improves barrier function failure due to urban atmospheric dust including PM2.5 and the like.

[ test example 10: elucidation of mechanism for improving barrier function of extract of citrus pericarp ]

NHEK was seeded at a Cell number of 84000 cells/well in a 48-well plate (Cell Bind, Corning). After culturing at 37 ℃ under 5% carbon dioxide and 95% air for 3 days, UA and a candidate compound (Citrus pericarp extract (Ichimaru Falcos Co.)) were dissolved at each concentration and then replaced with a differentiation induction medium (2mM Ca)²⁺Humedia-KG2), for 5 days or 6 days. After the incubation, the cells were washed 2 times with PBS, RNA was extracted using RNeasy Mini Kit (Qiagen), and then cDNA was prepared using TOYOBO ReverTra Ace qPCR RT Master Mix with gDNAremover (Toyo Boseki Co., Ltd.). The prepared cDNA was analyzed by qRT-PCR using Premix Ex Taq (registered trademark). From the measurement results, the relative values were calculated when the air pollutants were added, assuming that the gene expression of the medium (control) alone was 1 (FIG. 10A, B). Incidentally, Taqman Probe is purchased from Applied biosystems. Each Taqman Probe used GAPDH: hs02758991 _ g1, CLDN 1: hs00221623 _ m1 (FIG. 10A, B).

As shown in fig. 10A to B, the citrus peel extract increased expression of CLDN1 (sluice protein 1) and improved barrier function under air pollutants.

[ test example 11: evaluation of Effect of atmospheric contaminants on dermis via epidermis in two-dimensional skin model

NHEK was seeded at a Cell number of 400000 cells/well in a 6-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the medium was replaced with a medium prepared by dissolving automobile exhaust gas (VEP) or city atmospheric dust (UA) at each concentration, and the culture was further carried out for 1 day. After the culture, the supernatant was collected and air pollutants were removed by a 0.2 μm filter (Corning, 431222). At this time, in order to correct the influence not mediated through the epidermis, the same treatment was performed without adding NHEK, and the obtained supernatant was regarded as a Blank (Blank). Next, normal Human Dermal fibroblasts (Kurabo, Human der fiber: NHDF) were seeded in 48-well plates (Cell Bind, Corning) at a Cell number of 40000 cells/well using a culture Medium for Human fibroblasts (Dulbecco's modified eagle Medium (Kurabo Co.), 10% fetal bovine serum (MP Bio Co.), 1% antibacterial-antifungal agent (Gibco Co.). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the culture medium was replaced with a culture medium obtained by mixing 1:1 culture supernatant obtained by filtering human fibroblasts with a medium and a filter, and the culture was continued for a further 4 days. After the culture, the supernatant (sample for ELISA) was collected, and the expression levels of MMP1 and MMP3 were measured using Human MMP1DuoSetELISAKit (R & D Systems Co.) and Human MMP3 DuoSet ELISAKit (R & D Systems Co.). The plate from which the supernatant was removed was washed 2 times with PBS, and Hoechst 33342 was diluted in the medium and replaced with the medium in the wells. After incubation for 10 minutes at 37 ℃, 5% carbon dioxide and 95% air, image capture was performed with ImageExpress (16 fields/well). The cell number was measured by analysis using a cell counting program. Based on the measurement results, the expression levels of MMP1 and MMP3 per cell number of the blank group to which only the culture medium (blank control) was added were each set to 1, and relative values were calculated when air pollutants were added (fig. 11A to D).

As shown in fig. 11A to D, VEPs and UAs activate MMP1 and MMP3 of fibroblasts via epidermal cells, and participate in wrinkle formation.

[ test example 12: evaluation of Effect of atmospheric contaminants on dermis via epidermis in three-dimensional skin model

A reconstructed model (EFT-400, Kurabo) composed of human normal skin keratinocytes and fibroblasts was cultured in an EFT-400 medium (EFT-400 ASY, Kurabo) at 37 ℃ in an atmosphere of 5% carbon dioxide and 95% air for 24 hours. After the culture, PBS containing automobile exhaust gas or city air dust dissolved therein at each concentration was added from the upper surface of the epidermis, and further cultured for 3 days. After the culture, the medium (sample for ELISA) was collected, and the expression levels of MMP1 and MMP3 were measured using Human MMP1DuoSetELISAKit (R & D Systems Co.) and Human MMP3 DuoSet ELISAKit (R & D Systems Co.). From the measurement results, the expression levels of MMP1 and MMP3 were 1 for each well to which PBS alone (control) was added, and relative values were calculated when air pollutants were added (fig. 12A to D).

As shown in fig. 12A to D, the three-dimensional model showed an increase in the expression of MMP1 by VEP and UA and an increase in the expression of MMP1 by UA. This indicates that the air pollutants induce oxidative stress via the epidermis, thereby activating the proteolytic system of the dermis.

[ test example 13: evaluation of Effect of atmospheric contaminants on speckles ]

NHEK was seeded at a Cell count of 80000 cells/well in a 24-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the culture medium was replaced with a medium prepared by dissolving automobile exhaust or city air dust at each concentration, and the culture was further carried out for 1 day. After the culture, the cells were washed 2 times with PBS (-) and RNA was extracted using RNeasy Mini Kit (Qiagen), followed by cDNA preparation using TOYOBO ReverTra Ace qPCR RT Master Mixwith gDNAremover (Toyo Boseki Co., Ltd.). The prepared cDNA was analyzed by qRT-PCR using PremixEx Taq (registered trademark). From the measurement results, the relative values were calculated for the case where the air pollutants were also added, assuming that the gene expression of the medium (control) alone was 1 (FIG. 13A, B). Incidentally, TaqmanProbe was purchased from Applied biosystems. Each Taqman Probe used GAPDH: hs02758991 _ g1, PTGS 2: hs00153133 _ m 1.

As shown in fig. 13A, B, the expression level of PGE2 (prostaglandin E2) gene, which is produced from the epidermis to promote melanin synthesis, was increased by VEP and UA. Above, it is suggested that VEP and UA may be involved in spot formation.

[ test example 14: evaluation of Effect of atmospheric contaminants on melanocytes via epidermis in two-dimensional skin model

NHEK was seeded at a Cell number of 400000 cells/well in a 6-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the medium was replaced with a medium prepared by dissolving city atmospheric dust at each concentration, and the culture was further carried out for 1 day. After the culture, the supernatant was collected and air pollutants were removed by a 0.2 μm filter (Corning, 431222). At this time, in order to correct the influence not mediated through the epidermis, the same treatment was performed without adding NHEK, and the obtained supernatant was regarded as a blank. Next, in a 48-well plate (Cell Bind, Corning Co.), normal Human Epidermal melanocytes (Kurabo Co., German Life Ma Comp kit) were seeded using a medium specific for normal Human Epidermal melanocytes (Kurabo Co., Human Epidermal melanocytes: NHEM) at a Cell number of 40000 cells/well. After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the medium was replaced with a medium obtained by mixing a medium specific for normal human epidermal melanocytes and a culture supernatant filtered with a filter at a ratio of 1:1, and further cultured for 4 days. After the culture, the cells were washed 2 times with PBS (-) and RNA was extracted using RNeasy Mini Kit (Qiagen), followed by cDNA preparation using TOYOBO ReverTra Ace qPCR RT Master Mix with gDNAremover (Toyo Boseki Co., Ltd.). The prepared cDNA was analyzed by qRT-PCR using Premix Ex Taq (registered trademark). From the measurement results, the gene expression of the medium added only to the blank group (blank control) was set to 1, and the relative value was calculated when air pollutants were also added (FIG. 14). Incidentally, Taqman Probe is available from applied biosystems. Each Taqman Probe used GAPDH: hs02758991 _ g1, TYR: hs00165976 _ m 1.

As shown in fig. 14, the expression of melanin synthesis-related factor TYR (tyrosinase) was observed to be increased by UA.

[ test example 15: evaluation of Effect of atmospheric contaminants on melanocytes via epidermis in three-dimensional skin model

A three-dimensional model of skin (Kurabo, MEL-300A) consisting of epidermal keratinocytes and melanocytes was cultured in an epidermal model culture medium (Kurabo, EPI-100LLMM) at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours. After the culture, PBS containing automobile exhaust gas or city air dust dissolved therein at each concentration was added from the upper surface of the epidermis, and further cultured for 1 day. After the incubation, the tissue was washed with PBS and recovered, and then the tissue was crushed with a biological crusher (Nippi Co.). Thereafter, RNA was extracted using the RNeasy Tissue Mini Kit (Qiagen), and then cDNA was prepared using TOYOBOREVERTra Ace qPCR RT Master Mix with gDNAremove (Toyo Boseki Co., Ltd.). The prepared cDNA was analyzed by qRT-PCR using Premix Ex Taq (registered trademark). From the measurement results, the relative values were calculated for the case where the atmospheric pollutants were also added, with the expression of each gene being 1 in PBS alone (control) (FIGS. 15A to C). Incidentally, Taqman Probe is purchased from Applied biosystems. Each taqman probe used GAPDH: hs02758991 _ g1, PTGS 2: hs00153133 _ m1, TYR: hs00165976 _ m 1.

As shown in fig. 15A to C, the three-dimensional model also showed an increase in the expression of factors promoting melanin production, such as PGE2 and TYR, due to UA, and the data was related to the two-dimensional model, showing that UA promotes melanin production via the epidermis and participates in spot formation. In addition, for VEP, an increase in the expression of PGE2 was seen, suggesting a possible involvement in spot formation. From the above, it was shown that the formation of spots is promoted by air pollutants via the epidermis.

[ test example 16: search for Material for suppressing oxidative stress caused by air contaminants ]

NHEK was seeded at the Cell count of 15000 cells/well in a 96-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the culture medium was replaced with a medium prepared by dissolving candidate compounds (scutellaria root extract (bolus pharmacia), cowberry leaf extract (Ichimaru Falcos), hydrolyzed royal jelly (katakura chikkarinn), sunflower seed oil (RAHN), peppermint (SEDERMA), glycerol glucoside, kiwi fruit extract (bolus pharmacia), nicotinamide and glycogen) at various concentrations, and the culture was performed. After 24 hours of cultivation, the medium was replaced with a medium prepared by dissolving city air dust and candidate compounds at each concentration, and further cultivated for one day. After the culture, the cells were washed 2 times with PBS (-), and CellROX (registered trademark) green reagent for oxidative stress detection (ThermoFisher Scientific Co.) and Hoechst 33342 were diluted in the medium and replaced with the medium in the wells. After incubation for 30 minutes at 37 ℃, 5% carbon dioxide and 95% air, image capture was performed with ImageExpress (16 fields/well). The oxidative stress activity per cell number was measured by analyzing the cells by a fluorescence intensity measurement program and a cell count program. From the measurement results, the oxidative stress activity per cell number of the medium (control) alone was set to 1, and relative values were calculated when air pollutants were also added (fig. 16A to I).

As shown in fig. 16A to I, the activation of oxidative stress by scutellaria baicalensis root extract, cowberry leaf extract, hydrolyzed royal jelly, sunflower seed oil, peppermint, glycerol glucoside, kiwi fruit extract, nicotinamide, and glycogen inhibitory UA is suggested.

[ test example 17: search for materials for suppressing MMP1 caused by atmospheric pollutants ]

NHEK was seeded at the Cell count of 15000 cells/well in a 96-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the culture medium was replaced with a medium in which candidate compounds (scutellaria root extract (bokeji pharmaceutical), asparagus leaf extract (bayer), mallow flower extract (bokeji pharmaceutical), cowberry leaf extract (Ichimaru Falcos), houttuynia cordata extract (Ichimaru Falcos), neem leaf extract (Ichimaru Falcos), algae extract (Ichimaru Falcos), and bitter mint (sedermma)) were dissolved at respective concentrations, and the culture was performed. After 24 hours of cultivation, the medium was replaced with a medium prepared by dissolving city air dust and candidate compounds at each concentration, and further cultivated for one day. After the culture, the supernatant (sample for ELISA) was collected, and the expression level of MMP1 was measured using Human MMP1 DuoSeELISAKit (R & D Systems). The plate from which the supernatant was removed was washed 2 times with PBS (-), and Hoechst 33342 was diluted in the medium and replaced with the medium in the wells. After incubation for 10 minutes at 37 ℃, 5% carbon dioxide and 95% air, image capture was performed using ImageExpress (16 fields/well). The cell number was determined by analysis using a cell counting program. Based on the measurement results, the expression level of MMP1 per cell number of the medium (control) alone was set to 1, and relative values were calculated for the case where the air pollutants and the candidate materials were added (fig. 17A to H).

As shown in fig. 17A to H, it is suggested that the expression of MMP1 by UA is inhibited by scutellaria root extract, asparagus leaf extract, mallow flower extract, cowberry leaf extract, houttuynia cordata extract, neem leaf extract, algae extract, and bitter mint.

[ test example 18: search for materials that inhibit increase in IL-33 expression due to CP

NHEK was seeded at a Cell count of 80000 cells/well in a 24-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the medium was replaced with a medium prepared by dissolving the candidate compounds (allantoin, lidocaine, isopropyl methylphenol, diphenhydramine hydrochloride, diphenhydramine, sodium hyaluronate, magnesium chloride, cholesterol, dipotassium glycyrrhizinate, diphenhydramine and cholesterol simultaneously, and dipotassium glycyrrhizinate and cholesterol simultaneously) and CP at each concentration, and the culture was further cultured for 6 hours. After culturing, total RNA was extracted from the cells. IL-33 mRNA expression was determined by qRT-PCR and normalized using GAPDH expression. The results are expressed as mean ± standard deviation (n ═ 3). P value was determined by dannett test using comparison with a control group to^*P＜0.05、^**P＜0.01、^***P < 0.001. (FIGS. 18A to L).

As shown in FIGS. 18A to L, there are shown indications that allantoin, lidocaine, isopropyl methylphenol, diphenhydramine hydrochloride, diphenhydramine, sodium hyaluronate, magnesium chloride, cholesterol, dipotassium glycyrrhizinate, and the simultaneous addition of these components inhibit the increase in IL-33 expression due to CP.

[ test example 19: search for materials that inhibit increase in IL-33 expression due to GKD ]

NHEK was seeded at a Cell count of 80000 cells/well in a 24-well plate (Cell Bind, Corning). After culturing at 37 ℃ under an atmosphere of 5% carbon dioxide and 95% air for 24 hours, the medium was replaced with a medium prepared by dissolving the candidate compounds (Ufenamate, diphenhydramine, glycyrrhetinic acid, cholesterol, and lidocaine) and GKD at respective concentrations, and the culture was further carried out for 24 hours. After culturing, total RNA was extracted from the cells. IL-33 mRNA expression was determined by qRT-PCR and normalized using GAPDH expression. The results are expressed as mean ± standard deviation (n ═ 3). P value was determined by dannett test using comparison with a control group to^*P＜0.05、^**P＜0.01、^*P < 0.001. (FIGS. 19A to E).

As shown in FIGS. 19A to E, the increase in IL-33 expression due to GKD inhibition by Ufenamate, diphenhydramine, glycyrrhetinic acid, cholesterol, and lidocaine is suggested.

The following shows a prescription example. The contents in the formulation examples are all mass%.

Prescription example 1 toner

Formulation example 2 emulsion

Prescription example 3 cream

Formulation example 4O/W sunscreen gel

Formulation example 5W/O sunscreen emulsion

Formulation example 6 cleaning agent (Whole body bath foam)

Prescription example 7 cream

Prescription example 8 cream

Prescription example 9 cream

Prescription example 10 cream

Prescription example 11 cream

Prescription example 12 cream

Prescription example 13 cream

Prescription example 14 cream

Claims (13)

1. A composition for improving skin disorder caused by atmospheric pollutants comprises at least 1 inhibitor of IL-8 expression.

2. The composition of claim 1, wherein the skin disorder is skin inflammation and/or itch.

3. The composition according to claim 1 or 2, wherein the inhibitor of IL-8 expression is 1 or 2 or more selected from the group consisting of hyaluronic acid and a salt thereof, a derivative of hyaluronic acid and a salt thereof, an artichoke extract, tranexamic acid and a salt thereof, a Sasa veitchii leaf extract, a camellia extract, a rose extract, a perilla extract, a Scutellaria baicalensis extract, a licorice extract, a Camellia sinensis extract, an aloe vera leaf extract, a rose hip extract, a coptis chinensis extract, a loquat leaf extract, a cherry leaf extract, a rosemary leaf extract, a Japanese sage leaf extract, a thyme extract, a carrot root extract, allantoin, Ufenamate, glycyrrhizic acid and a salt thereof, glycyrrhetinic acid and a salt thereof, stearyl glycyrrhetinate, and cholesterol.

4. A composition for improving skin disorder caused by air pollution substances comprises at least 1 or more substances for promoting expression of water gate protein and/or substances for promoting expression of blocking protein.

5. The composition of claim 4, wherein the skin disorder is caused by a reduction and/or immaturity of skin barrier function.

6. The composition according to claim 4 or 5, wherein the sluice protein expression-promoting substance and/or the atresia protein expression-promoting substance is 1 or 2 or more selected from the group consisting of an orange peel extract, a cowberry leaf extract, a white willow bark extract, a arnica herb extract, a angelica keiskei extract, a coix seed extract, a ginkgo leaf extract, a turmeric root extract, a rosa multiflora extract (a rose hip extract), a scutellaria baicalensis extract, a artemisia inflata extract, a chamomile extract, a perilla leaf extract, a peach seed extract, a melissa officinalis extract, a lavender extract, and a sodium salt of a condensate of N-lauroyl-L-glutamic acid and L-lysine.

7. A composition for improving skin disorder caused by air pollutants contains at least more than 1 kind of oxidation stress inhibitor.

8. The composition of claim 7, wherein the skin disorder is at least 1 selected from wrinkles, spots, acne and sagging of the skin.

9. The composition according to claim 7 or 8, wherein the oxidative stress inhibitor is 1 or 2 or more selected from the group consisting of scutellaria baicalensis extract, cowberry fruit extract, hydrolyzed royal jelly, sunflower oil, bitter mint, glycerol glucoside, polygonum hydropiper extract, nicotinamide, glycogen, centella asiatica extract, mallow extract, houttuynia cordata extract, neem extract, algae extract, phellodendron amurense extract, ascorbic acid, ginkgo biloba leaf extract, camellia sinensis extract, green tea extract, aloe barbadensis leaf extract, hibiscus flower extract, perilla leaf extract, rosemary leaf extract, sage leaf extract, citrus extract, chamomile extract, licorice extract, artichoke extract, and eucalyptus extract.

10. A composition for improving skin disorder caused by atmospheric pollutants comprises at least 1 inhibitor of IL-33 expression.

11. The composition of claim 10, wherein the skin disorder is at least 1 selected from the group consisting of pruritus, eczema, dermatitis, rash, urticaria and erosion.

12. The composition according to claim 10 or 11, wherein the inhibitor of IL-33 expression is 1 or 2 or more selected from the group consisting of allantoin, lidocaine, isopropylmethylphenol, diphenhydramine and a salt thereof, hyaluronic acid and a salt thereof, a derivative of hyaluronic acid and a salt thereof, magnesium chloride, cholesterols, glycyrrhizic acid and a salt thereof, glycyrrhetinic acid and a salt thereof, stearyl glycyrrhetinate, and isofenvalerate.

13. The composition of any one of claims 1 to 12, wherein the atmospheric pollutants are at least 1 selected from automobile exhaust, urban atmospheric dust, pollen and sand dust.

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