KR101601865B1 - Method for screening harmful responses of skin by pollen house dust mite or yellow sand dust and the composition for skin external application controlling the responses - Google Patents

Abstract

The present invention provides a method for screening a substance involved in inducing skin irritation or a substance inhibiting it, a method for screening a substance that alleviates skin irritation, and a method for screening a substance that alleviates skin irritation by screening a gene whose expression amount is changed by environmental harmful factors such as pollen, house dust mite, The present invention relates to a composition for external application for skin, which contains at least one kind of extract of Ganoderma lucidum and Ganoderma lucidum as an active ingredient to alleviate harmful effects of skin caused by pollen, house dust mites or dusts, and to detoxify and release toxins.

(IL-6), IL-8 (interleukin 8), GST (interleukin 6), house dust mite, Pollen, Yellow Sand Dust, keratinocyte, Keratinocyte, Glutathione-S-Transferase), NQO1 (NADPH Quinone Oxidoreductase 1), hBD3 (human β-defensin 3)

Description

TECHNICAL FIELD [0001] The present invention relates to a method for screening harmful reactions of skin caused by pollens, dust mites or dusts, and a composition for external application for skin controlling pollens, house dust mites or yellow sand dusts, responses}

The present invention provides a method for screening a substance involved in inducing skin irritation or a substance inhibiting it, a method for screening a substance that alleviates skin irritation, and a method for screening a substance that alleviates skin irritation by screening a gene whose expression amount is changed by environmental harmful factors such as pollen, house dust mite, The present invention relates to a composition for external application for skin, which contains at least one kind of extract of Ganoderma lucidum and Ganoderma lucidum as an active ingredient to alleviate harmful effects of skin caused by pollen, house dust mites or dusts, and to detoxify and release toxins.

Skin is a part of the body that is directly exposed to the external environment. It acts as a protective layer to protect important organs of our body, as well as controlling water evaporation and protecting the body from external infections. However, even if it prevents skin penetration from the outside, exposure to ultraviolet rays or pollutants causes skin irritation such as erythema, edema, itching, and inflammatory reaction. It is known that the skin troubles caused by the environmental harmful factors are not only cosmetic problems but also the substances generated in the inflammatory reaction cause the pigmentation of the skin incidentally and promote the collapse of the elastic fibers of the skin, have.

House dust mites and pollen are one of the most common causes of allergic diseases and are distributed throughout the world and have been reported to be associated with respiratory diseases such as perennial allergic rhinitis and bronchial asthma. It has been reported that house dust mites are involved in the fibrotic process of allergic rhinitis by increasing inflammatory cytokine expression in human fibroblasts of the lungs (Yu et al., Messenger RNA Expression of the Cytokine Gene Cluster in Human Fibroblast Activated with House Dust Mite Antigen. J Clinical Otolaryngol 10: 224-230, 1999). (Jenmalm et al., 2001), and pollen also stimulates a variety of cytokines, leading to respiratory and skin disorders (Jenmalm et al., Clin Exp Allergy 31 10): 1528-35, 2001). In addition, when the yellow wind directly touches the skin, it causes allergic reaction, which is likely to cause contact dermatitis. Dry and dazzling yellowish winds take away the moisture of skin and dry skin, keratin, itching, dermatitis acne and atopy . However, the adverse effects of yellow dust on the skin have not been scientifically proven.

The keratinocytes (keratinocyte) present in the outermost layers of the skin play an important role in the initiation, regulation and regulation of the inflammatory reaction by interacting with immune cells mainly in fibroblasts, endothelial cells and dermis cells. Skin irritation is the most common adverse effect associated with subcutaneous inflammation, and clinically 70% of contact dermatitis can be explained by skin irritation. Erythema, edema, cracks, water loss, epidermal dysplasia, tickling and pain are typical symptoms of irritable or allergic contact dermatitis. This is a symptom of an abnormality in the homeostasis of the cell and an inflammatory reaction. In other words, these symptoms are the ultimate physiological findings due to the complex biochemical, neuronal, and vascular cell reactions resulting from skin irritation. Unlike allergic reactions, skin irritation differs in that it begins with a direct inflammatory response to the skin. Therefore, skin irritation occurs locally and can be defined as a reaction that occurs after irritation with a nonimmune inflammatory reaction and disappears within a few days.

Accordingly, the present inventors have found that various biological reactions that occur when keratinocytes present in the outermost layer of skin tissue are treated with the environmentally harmful factors, in order to identify skin harmful effects caused by pollen, house dust mites or dusts, , It was confirmed that the environmental harmful factors act on the keratinocytes to increase the expression of interleukin 6 and interleukin 8, which are proinflammatory factors and are involved in the development and progression of psoriasis , But also the expression of GSTT1, a toxin releasing enzyme, NQO1, an enzyme that detoxifies toxins, and the skin antimicrobial peptide hBD3, a natural immune factor, are also increased.

Accordingly, it has been found that the expression of interleukin 6 or interleukin 8 is reduced, and screening of a substance that increases expression of GSTT1, NQ01 or hBD3 enables efficient screening of a substance that alleviates skin irritation caused by environmental harmful factors.

In addition, the present inventors have found that the extract of Goryeo Plum and Kwanghwada can reduce the expression of interleukin 6 or interleukin 8 and increase the expression of GSTT1, NQ01 or hBD3, thereby alleviating the skin irritation caused by pollen, house dust mites or dusts. .

Accordingly, an object of the present invention is to provide a method for screening intracellular factors involved in inducing skin irritation caused by pollen, house dust mites or dusts and intracellular factors inhibiting them.

It is also an object of the present invention to provide a method for screening substances that control skin harmful responses caused by pollen, house dust mite or dust.

It is also an object of the present invention to provide a composition for external application for skin which contains at least one kind of sprout extract and extract of Kantai as an active ingredient to control skin harmful reaction by pollen, house dust mite or dust.

In order to accomplish the above object, the present invention provides a method for preparing a test cell, comprising the steps of: 1) treating candidate substances that regulate the expression of an intracellular factor in a test cell; 2) inducing overexpression of intracellular factors by environmental harmful factors by treating environmentally harmful factors in test cells; And 3) measuring the degree of expression of an intracellular factor in the cell of step 2) by an environmentally harmful factor. The present invention also provides a method for screening a substance that alleviates skin irritation caused by an environmentally harmful agent.

In addition, the present invention provides a composition for external application for skin which comprises at least one kind of sprout extract and extract of Kyng-bong as an active ingredient to alleviate skin harmful effects.

By using the method according to the present invention, it is possible to confirm the skin harmful action by pollen, house dust mites or dusts, and it is a proinflammatory factor which plays an important role in skin irritation caused by dust, 8 can be efficiently screened for the substance that reduces the expression of the gene. In addition, the substance that regulates the expression of GSTT1, which is an enzyme that releases toxins, NQO1, which is a toxin-decoding enzyme, and the skin antimicrobial peptide hBD3, Screening can be performed. In addition, the composition containing at least one of the extracts of Goryeo Plum and Kwangwha extract as an active ingredient is applied as a pharmaceutical or cosmetic composition for preventing skin trouble due to pollen, house dust mite or dust, reducing the pro-inflammatory factor, treating and preventing psoriasis can do.

Skin irritation occurs locally and can be defined as a nonimmune inflammatory reaction that occurs after stimulation and disappears within a few days. It is expected that skin irritation by pollen, house dust mites, or dusts will cause a similar reaction to these skin irritation responses in clinical reports.

In the present invention, in order to treat the pollutants such as pollen, house dust mites or dusts, which are major environmental harmful factors in human normal keratocyte cells, real-time reverse transcription polymerase chain reaction (PCR) we have developed a screening method which can be suitably used to elucidate the mechanism of skin irritation caused by environmentally harmful factors or the development of emollients through a reverse transcription polymerase chain reaction and a method of inducing skin irritation symptoms by environmental harmful factors.

The term " intracellular factor " used in the present invention refers to a substance whose expression amount is significantly increased by environmental harmful factors as compared with normal keratinocytes.

1) treating keratinocytes with environmental pollutants such as pollen, house dust mites or dusts; And 2) screening for a gene whose expression level is significantly changed as compared with normal keratinocyte through a reverse transcription polymerase reaction, wherein the gene is involved in inducing skin irritation caused by pollen, house dust mite or dust, or alleviating skin irritation Lt; RTI ID = 0.0 > a < / RTI >

In the present invention, it has been found that IL-6 and IL-8, which are proinflammatory factors, are increased by the skin irritation caused by pollen, house dust mites or dusts, NQO1, an enzyme that decodes, and hBD3, a skin antimicrobial peptide, which is a natural immune factor, are also increased, but the present invention is not limited thereto.

In addition, the present invention relates to a method for controlling the expression of IL-6 and IL-8 by controlling the expression of intracellular factors by environmental pollutants such as pollen, house dust mite or dustiness in mammalian keratinocytes by administering an effective amount of a skin irritation inhibitor And increases the expression of GSTT1, NQ01, hBD3, thereby providing a method for relieving skin irritation caused by environmental harmful factors.

As used herein, the term " skin irritation inhibitor " refers to an agent that inhibits the activity of IL-6 or IL-8 by environmental harmful factors, or antagonism against a mechanism that induces IL-6 or IL- The expression of GSTT1, NQ01, NQO1 and NQO1, which is an enzyme that releases toxins, and NQO1, which is a toxin-detoxifying enzyme, and the skin antimicrobial peptide hBD3, which is a natural immune factor, means a substance capable of increasing the expression amount of hBD3.

The mammal is a mammal with atopic dermatitis, acne or psoriasis, and the inhibitor is administered systemically or topically via conventional routes of administration well known in the art.

The present invention provides a method for producing a test cell, comprising the steps of: 1) treating candidate substances that regulate the expression of an intracellular factor in a test cell; 2) inducing overexpression of intracellular factors by environmental harmful factors by treating environmentally harmful factors in test cells; And 3) measuring the degree of expression of an intracellular factor in the cell of step 2) by an environmentally harmful factor. The present invention also provides a method for screening a substance that alleviates skin irritation caused by an environmentally harmful agent.

In the step 1), the test cell can be any cell capable of inducing the expression of an intracellular factor by an environmentally harmful factor. Preferably, the cell is used but is not limited thereto. In addition, it is preferable to treat the environmentally harmful agent 30 minutes before the treatment because the candidate substances that control the expression of intracellular factors should be treated first, so that they may act in the skin cells.

In addition, IL-6 and IL-8, which stimulate the cells, as well as GSTT1, NQ01 and hBD3 which alleviate the stimulation are included in the intracellular factor by the environmental harmful factor in which the expression is induced in the step 2) It is not limited.

At this time, the treatment amount of the environmentally harmful factor is preferably in the range of 25 to 50 占 퐂 / ml.

In addition, the expression level of the intracellular factor by the environmentally harmful factor in the step 3) can be confirmed by measuring by reverse transcription polymerase chain reaction (RT-PCR).

In the present invention, it has been found through the above-mentioned methods that the extracts of Goryeo Plum and Ganoderma lucidum suppress the expression of skin irritant factors and express factors inhibiting skin harmful effects, but the present invention is not limited thereto.

The present invention provides a composition for external application for skin, which contains at least one of spinach and kyngos extract as an active ingredient in order to alleviate skin irritation caused by environmental harmful factors.

It is the bark or flesh of Melia azedarach L. var. Japonica Makino. It removes roundworms, mites, and treats rubella and scabies. It is also used for skin eczema and has diuretic and antipyretic action. It has been reported that pharmacological action is effective in eliminating intestinal parasites such as tapeworms and condoms and trichomoniasis. It has a semi-tubular or tubular shape with a gray color on the outside, a longitudinally torn pattern and a transverse shrub, and a cork layer with a reddish brown color. The folded surface is yellowish white and fibrous, the quality is hard but it is easy to break. This medicine is good for young tree bark with lots of trees. Other names include Pusan, Pusan, Sukmyun, and Banzuko.

Kandonghwa (款冬花) is a flower bud of Tangshan ( Tussilago farfara L.), and it is used for dehydration, seaweed, asthma, upper respiratory tract infection. Pharmacological actions have been reported to be effective in increasing the respiratory secretion, in the action of vasodilation, respiratory excitement and light anxiety, vasoconstriction, acute bronchitis in children, bronchial asthma and sore throat. The appearance is that the bud hangs at the end of the peduncle and has an irregular rod shape. The peduncle is light colored with scaly leaves. This scaly leaf is broad ovate, pointed end, and consists of about 20 pieces of japanese shape like triangle. On the inside, there are white fluffs, and there are dyes and normal flowers. When the flower buds are cut, there are white thread-like fluffs. This medicine should be shaped like a brush, with a purple or cyan color at the base and a short peduncle. Kandonghwa is also known as Kanto, Kondo, Kanto, Tohha, Fuyu, Sodo, Kyohei, Takko, and Tiger.

The extraction method is not particularly limited, and extracts can be obtained from water or an organic solvent. The organic solvent used in the present invention may be at least one selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate and chloroform, or a mixed solvent of these organic solvents and water may be used. In the present invention, those purchased from the Korean plant extract bank were used.

The extract or suspension extract prepared by the above method is contained in an amount of 1 to 30% by weight based on the total weight of the composition.

The composition for external application for skin according to the present invention is not particularly limited to its formulation and may be selected from the group consisting of softening agents, convergent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing lotion, cleansing foam, Hair lotion, hair conditioner, hair conditioner, hair conditioner, hair lotion, hair tonic, hair nutrition lotion, hair essence, hair serum, scalp treatment, hair treatment, body lotion, body lotion, body lotion, A lotion, an ointment, a gel, a cream, a patch or a spray, and the like.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention and may be modified or modified within the spirit and scope of the present invention.

[Example 1] Expression of IL-6 and IL-8 genes in human keratinocytes by pollen, house dust mites or dusts

Human epidermal neonatal keratinocyte cells were purchased from Lonza (Inc., Walkersville, Maryland, USA) as a commercially available and recommended method, subcultured, and cultured in a CO ₂ incubator (CO ₂ incubator) at 37 ° C and 5% CO ₂ . The cell culture was cultured according to the Lonza's guidelines. 2 ml of a bullet kit (BPE (bovine pituitary extract), 0.5 ml of human epidermal growth factor (hEGF), 0.5 ml of insulin, 0.5 ml of hydrocortisone (Clonetics CC-3103) Hydrocortisone (0.5 ml), Transferrin (0.5 ml), Epinephrine (0.5 ml) and Gentamycin Suflate + Amphofericin-B (GA-1000)

In the control and experimental groups in which human keratinocytes were cultured, pollen, house dust mites or dusts were added to human keratinocyte culture media and cultured for 24 hours. Pollen and house dust mites were purchased from COSMO BIO and yellow dust samples were collected on the roof of the Pacific Technology Research Institute in Yongin, Gyeonggi - do during the DSS period. The cells were treated with 25 μg / ml of pollen, house dust mite or dust every 24 hours, washed twice with 10 ml of phosphate buffered saline (PBS), and treated with Trizol reagent (Invitrogen) , Carlsbad, Calif.) Was used to isolate total RNA (total RNA) in the cells. The isolated RNA was purified once more using the Qiagen RNeasy kit from Qiagen (Valencia, Calif., USA) and then purified using Agilent Technologies (Santa Clara, CA) ) Was used to confirm the quality of the RNA using a Bio-Analyzer 2100 model device (Agilent 2100 BioAnalyzer). CDNA was synthesized from the separated RNA using a Superscript Reverse Transcriptase (RT) II kit (Invitrogen). The cDNA was synthesized by real-time reverse transcription polymerase chain reaction (Q-RT- PCR). The expression pattern changes of IL-6 (Hs99999032_m1) and IL-8 (Hs00174103_m1) were evaluated using a TaqMan gene expression system (Applied Biosystems, Foster City, CA) , And the results are shown in Figs. 1A and 1B.

As can be seen from FIGS. 1A and 1B, pollen, house dust mites or dusts significantly increase the expression of IL-6 and IL-8 genes in human normal keratinocytes [A: untreated group, B : Treated with pollen (25 占 퐂 / ml), C: treated with house dust mite (25 占 퐂 / ml), D: treated with yellow dust (25 占 퐂 / ml).

[Example 2] Inhibition of expression of proinflammatory factors by pollen, house dust mites, and dusts in human keratinocytes upon treatment with high-grade blood or keratinization

Human epidermal neonatal keratinocyte cells were cultured in a CO ₂ incubator (CO ₂ incubator) at 37 ° C and 5% CO ₂ Cells were cultured. The cell culture was cultured according to the Lonza's guidelines. 2 ml of a bullet kit (BPE (bovine pituitary extract), 0.5 ml of human epidermal growth factor (hEGF), 0.5 ml of insulin, 0.5 ml of hydrocortisone (Clonetics CC-3103) Hydrocortisone (0.5 ml), Transferrin (0.5 ml), Epinephrine (0.5 ml) and Gentamycin Suflate + Amphofericin-B (GA-1000)

In the control and experimental groups cultured human keratinocyte, 10 ppm of high-density lipoprotein or Kwanghwanghae extract was added to human keratinocyte culture medium for pretreatment for 30 minutes in the case of Golgi herb or Ganoderma lucidum extract (purchased from Korean plant extract bank) After the addition of 25 μg / ml of pollen, house dust mites or dust samples, the cells were cultured with the cells for 24 hours. After 24 hours of treatment of the material, the cells were washed twice with 10 ml of phosphate buffer, and total RNA (total RNA) in the cells was isolated using a triazole (Invitrogen). The separated RNA was purified once more using a Qiagen RNA kit, and the quality of the RNA was confirmed using Agilent's BioAdmin 2100 model device. CDNA was synthesized from the separated RNAs using a Superscript Reverse Transcriptase (RT) II kit from Invitrogen. Real-time reverse transcription polymerase chain reaction (Q-RT- PCR). The expression patterns of IL-6 (Hs99999032_m1) and IL-8 (Hs00174103_m1) were evaluated using a TaqMan gene expression system (TaqMan® gene expression system) from Applied Biosystems. The results are shown in FIGS.

As can be seen from FIG. 2A, it is known that IL-6 gene expression, which is a proinflammatory factor that increases pollen in human normal keratinocyte cells and is important for the development and progression of psoriasis, Treated group, B: group treated with pollen (25 占 퐂 / ml), C: treated group (10ppm), D: group treated with pollen (25 占 퐂 / ml).

As shown in FIGS. 2B and 2C, the expression of IL-6 and IL-8 gene, which is a proinflammatory factor which is increased by yellow dust in human normal cornea skin cells and is important for the development and progression of psoriasis, D: Dust (25 ㎍ / ㎖) + high-purity (10ppm) treatment group. A: untreated group, B: yellow sand treated group (25 ㎍ / (10ppm) treated group, F: treated with dust (25μg / ㎖) + gout (10ppm) treated group.

Therefore, it can be seen that the method of the present embodiment can easily screen for substances that inhibit the generation of infectious agents by pollen, dust, and the like, and the onset and progression of psoriasis.

[Example 3] Confirmation of increase of gene expression of skin antimicrobial peptide, which is a toxin detoxification and emission factor and natural immune factor,

Human epidermal neonatal keratinocyte cells were cultured in a CO ₂ incubator (CO ₂ incubator) at 37 ° C and 5% CO ₂ Lt; / RTI > cells. The cell culture was cultured according to the Lonza's guidelines. 2 ml of a bullet kit (BPE (bovine pituitary extract), 0.5 ml of human epidermal growth factor (hEGF), 0.5 ml of insulin, 0.5 ml of hydrocortisone (Clonetics CC-3103) Hydrocortisone (0.5 ml), Transferrin (0.5 ml), Epinephrine (0.5 ml) and Gentamycin Suflate + Amphofericin-B (GA-1000)

In the control and experimental groups of human keratinocytes cultured human keratinocyte culture medium, 10ppm gypsum bloom extract (purchased from Korean Plant Extract Bank) was added to the goryeong bloom extract treatment group, Lt; / RTI > Twenty four hours after the treatment of the extracts, the cells were washed twice with 10 ml of phosphate buffer, and total RNA (total RNA) was isolated from the cells using a triazole (Invitrogen). The separated RNA was purified once more using a Qiagen RNA kit, and the quality of the RNA was confirmed using Agilent's BioAdmin 2100 model device. CDNA was synthesized from the separated RNAs using a Superscript Reverse Transcriptase (RT) II kit from Invitrogen. Real-time reverse transcription polymerase chain reaction (Q-RT- PCR). The expression patterns of GSTT1 (Hs00184475_m1), NQO1 (Hs01045994_m1) and hBD3 (hs00218678_m1) were evaluated using a TaqMan® gene expression system (Applied Biosystems, Inc.) and the results are shown in FIG.

As can be seen from FIG. 3, the extract of Gordonii extract increased the expression level of GSTT1, which is an enzyme that excretes toxins in human normal cornea skin cells, NQO1 which decodes toxic substances, and the skin antimicrobial peptide hBD3, which is a natural immune factor Respectively.

Therefore, it can be seen that the polyphenols have an effect of increasing toxin detoxification, excretion and expression of natural immune factors.

[Example 4] Increase of gene expression of skin antimicrobial peptide which is a natural immune factor and toxin-detoxifying factor when mixed with goryeo blood and kantouhwa

Human epidermal neonatal keratinocyte cells were cultured in a CO ₂ incubator (CO ₂ incubator) at 37 ° C and 5% CO ₂ Lt; / RTI > cells. The cell culture was cultured according to the Lonza's guidelines. 2 ml of a bullet kit (BPE (bovine pituitary extract), 0.5 ml of human epidermal growth factor (hEGF), 0.5 ml of insulin, 0.5 ml of hydrocortisone (Clonetics CC-3103) Hydrocortisone (0.5 ml), Transferrin (0.5 ml), Epinephrine (0.5 ml) and Gentamycin Suflate + Amphofericin-B (GA-1000)

In the control and experimental groups cultured human keratinocytes, human keratinocyte cultures were treated with 10 ppm of ginseng extract (purchased from Korean Plant Extract Bank) and 10 ppm (Purchased from the Korean Plant Extract Bank), and Golgi and Kwanghwha extracts were added in the same amount of 10ppm and 10ppm, respectively, and cultured for 24 hours. After 24 hours of treatment of the material, the cells were washed twice with 10 ml of phosphate buffer, and total RNA (total RNA) in the cells was isolated using a triazole (Invitrogen). The separated RNA was purified once more using a Qiagen RNA kit, and the quality of the RNA was confirmed using Agilent's BioAdmin 2100 model device. CDNA was synthesized from the separated RNAs using a Superscript Reverse Transcriptase (RT) II kit from Invitrogen. Real-time reverse transcription polymerase chain reaction (Q-RT- PCR). The expression patterns of NQO1 (Hs01045994_m1) and hBD3 (hs00218678_m1) were evaluated using a TaqMan gene expression system (TaqMan® gene expression system) from Applied Biosystems. The results are shown in FIGS. 4a and 4b.

As can be seen from FIGS. 4A and 4B, when the amount of gene expression of NQO1, which decodes a toxic substance in human normal cornea skin cells, and the skin antimicrobial peptide hBD3, a natural immune factor, is increased However, it can be confirmed that the combination of Goryeo blood and Kwanghwanghwa extract is further increased.

Therefore, it can be seen that the expression of the toxin - detoxifying agent and the natural immune factor are simultaneously increased by the synergistic effect when the extract of Goryeo Plum and Kanghwa extract is treated at the same time.

FIGS. 1A and 1B are RT-PCR analysis results showing the expression of IL-6 and IL-8 protein by house dust mite, pollen and yellow dust in human normal keratinocytes.

FIGS. 2A and 2B are graphs showing the results of treatment of progesterone and corticosteroids inhibiting the expression of harmful factors expressed by pollen and dust in human normal keratinocyte cells. These results show that IL-6 and IL-8, which are inflammation inducers by pollen, (RT-PCR) analysis confirmed the inhibition of protein expression.

FIG. 3 is a result of RT-PCR analysis in which the expression level of GSTT1, NQO1, and hBD3, which are toxin detoxification and emission factors, is increased in human normal keratinocytes.

FIGS. 4A and 4B are RT-PCR analysis results showing the increase in the expression level of NQO1 and hBD3, which are toxin detoxification and excretion factors due to the synergistic effect of spindle blood and keratinization in human normal keratinocytes.

Claims (14)

1) The test cells were injected with the skin stimulating factors interleukin 6, IL-6, interleukin 8, IL-8, glutathione S-transferase theta-1, GSTT1 ), NADPH Quinone Oxidoreductase 1 (NQ01) and human β-defensin 3 (hBD3) in the presence of a compound of the present invention. 2) treating the test cells with yellow dust as an environmental harmful factor to induce overexpression of a skin irritant factor by the environmentally harmful factor; 3) measuring the degree of expression of a skin irritant factor by environmental harmful factors in the cells of step 2); And 4) selecting the candidate substance as a skin irritant for reducing the expression of IL-6 and IL-8 in the cells and increasing the expression of GSTT1, NQ01 and hBD3 in the step 3); Wherein the skin irritation is selected from the group consisting of erythema, edema, itching, and inflammation caused by environmental harmful factors. The screening method according to claim 1, wherein the test cell in step 1) is keratinocyte. delete The screening method according to claim 1, wherein the treatment amount of the environmentally harmful factor in the step 2) is in the range of 25 to 50 μg / ml. delete The screening method according to claim 1, wherein the degree of expression of a skin irritant factor by an environmental adverse factor in the step 3) is measured by reverse transcription polymerase chain reaction (RT-PCR). delete delete A composition for external application to skin for alleviating skin irritation caused by yellow dust, which comprises an extract of Alfalfa extract as an active ingredient selected by the screening method of claim 1. [Claim 11] The composition for external application for skin according to claim 9, wherein the composition further comprises a gout extract selected by the screening method of claim 1. [ 10. The composition for external application for skin according to claim 9, wherein the extract is contained in an amount of 1 to 30% by weight based on the total weight of the composition. delete delete 10. The composition for external application for skin according to claim 9, wherein the composition alleviates at least one skin irritation selected from the group consisting of erythema, edema, itching, and inflammation caused by yellow dust.

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