KR20110070927A - Evaluation method of skin blackening tendency by ultraviolet rays - Google Patents

Abstract

The present invention relates to a method for estimating the amount of skin melanin as an indicator of the amount of Annexin II protein in the stratum corneum obtained by non-invasive means, or to a method for evaluating skin blackening tendency by ultraviolet rays.

Description

Evaluation method for skin blackening tendency by ultraviolet light {Method for evaluating tendency to skin pigmentation by ultraviolet light}

The present invention relates to a method for evaluating skin blackening tendency by ultraviolet rays.

Ultraviolet rays are concerned about many health effects such as carcinogenic effects on the skin, effects on eye diseases such as cataracts, and effects on immune function. Exposure to ultraviolet rays in the skin also causes various ultraviolet disorders. Ultraviolet rays can irritate the skin or oxidize unsaturated lipids in the skin to generate lipid radicals, producing lipid peroxide. Inflammation and oxidation caused by UV light cause tissue, cell, and DNA disorders. As a phenomenon by the ultraviolet exposure of the skin, wrinkles, blemishes, skin cancer, etc. are generally mentioned.

In recent years, there is also reported that the risk of skin cancer increases according to the difference in sensitivity to ultraviolet rays (skin color, skin type, etc.) as the dynamic data (Non-Patent Documents 1 and 2). It is also clear that the risk of malignant melanoma (melanomer), which is a type of skin cancer, increases several times due to the genetic variation of monobasic substitution of melanocortin1 receptor (Non-Patent Documents 3 and 4). Sensitivity to ultraviolet rays varies greatly depending on skin type, and is divided into types classified into six stages or based on minimum ultraviolet energy (minimum erythema: MED) that causes redness (erythema), but the former is subjective Observations and the latter carry a risk that the latter cannot be measured without exposure to ultraviolet light. The increase in ultraviolet rays is more problematic than before due to the ozone layer destruction and the like, and the sensitivity risk to ultraviolet rays for an individual needs to be appropriately evaluated.

On the other hand, in the early stages of pigmentation, commonly called blemishes, epidermal keratinocytes are stimulated by various stimuli before they are present on the skin surface, such as α-MSH, bFGF, CGRP, and endothelin. Creates and transmits the information carrier to Melanosite. After delivery to melanocytes, a large amount of melanin pigment is made in the cells. On the other hand, the turnover is abnormal due to ultraviolet rays, so that the melanin pigment produced in large quantities cannot be discharged, so it turns into a pigmented lesion. In the experiment using in vitro system, when the melanocytes were irradiated with ultraviolet rays, the expression of p73, Nup88, p27, Id1, and PCNA, which are proliferation factor markers of cells, also increased and decreased the expression of bcl-2, an apoptosis related protein. There is a report (Non Patent Literature 5). Until now, the condition of the blemishes was invaded using the biopsy skin tissue, and the expression of NT-3, ADAM9, HB-EGF, etc. in the blemishes was markedly increased. There is a report (patent document 1). In addition, as a test method for predicting the formation of blemishes on the skin, it is reported that the amount of mRNA such as MLSTD1, MOGAT1, Mcp9, Krt2-6b, etc. is measured by biopsy to determine the risk of blemish formation (Patent Documents 2, 3, 4). ). In addition, as a non-invasive method, the amount of interleukin 1 (IL-1) and interleukin 1 receptor antagonist (IL-1ra) was analyzed using a stratum corneum derived from skin collected through tape stripping or abrasion as a sample. Patent Document 5 discloses a method for evaluating quality.

Patent Document 1: Japanese Patent Application Laid-Open No. 2004-205246

Patent Document 2: Japanese Patent Application Laid-Open No. 2005-106745

Patent Document 3: Japanese Patent Application Laid-Open No. 2005-110505

Patent Document 4: Japanese Patent Application Laid-Open No. 2007-289063

Patent Document 5: Japanese Patent No. 3590708

[Non-Patent Document 1] Arch Dermatol. 2004; 140: 819-824

Non-Patent Document 2: Cancer Causes Control. 1997; 8 (2): 246-252

[Non-Patent Document 3] Invest Dermatol. 117: 294-300,2001

Non-Patent Document 4: Am.J.Hum.Genet. 66: 176-186,2000

Non-Patent Document 5: Carcinogenesis 24 (12): 1929-1934,2003

The present invention provides a method for assessing the risk of ultraviolet radiation disorders. In particular, it aims at providing the method to know the skin blackening tendency of the said subject using the skin stratum corneum collected using non-invasive means.

The main configuration of the present invention is as follows.

(1) The evaluation method of the skin blackening tendency by ultraviolet-ray which evaluates that the skin blackening tendency by the ultraviolet-ray of skin is strong according to the amount of Annexin II protein in a stratum corneum.

(2) The method of screening the component which affects skin blackening using the evaluation method of skin blackening tendency of description in (1).

(3) A method of selecting a cosmetic according to the skin blackening tendency obtained using the method for evaluating skin blackening tendency according to (2).

The present invention finds that the amount of Annexin II protein, which is a protein expressed in vivo, correlates positively with the production of melanin by UV exposure, and it is clear that the tendency to skin blackening can be evaluated.

According to the present invention, the tendency of skin blackening due to ultraviolet rays can be evaluated without actually irradiating ultraviolet rays.

Since the present invention can be evaluated using the skin stratum corneum collected using a non-invasive tape stripping means, it is safe and simple.

In addition, the evaluation method of the present invention can be used to screen components useful for suppressing skin blackening tendency by ultraviolet light or inhibiting melanin production after ultraviolet light exposure.

By this invention, the beauty of a blemish measure and a blemish prevention becomes easy. That is, the selection of cosmetics, quasi-drugs, medicines or cosmetic health foods for pigmentation sites such as blemishes and the like can provide valid information. Beauty counseling such as a blemish measure can be performed correctly.

EMBODIMENT OF THE INVENTION Hereinafter, embodiment of this invention is described in detail.

The present invention provides a method for evaluating the risk of ultraviolet ray disorders comprising measuring the amount of Annexin II protein contained in the stratum corneum.

The present invention reveals the risk of blackening of the person sourced from the stratum corneum by exposure to ultraviolet rays, based on the amount of annexin II, a protein present in the stratum corneum, collected by non-invasive means. It is.

The inventors found that there was a positive correlation between the change in melanin production after exposure and the expression amount of Annexin II, and by measuring the expression amount of Annexin II, the risk of melanin production when exposed, and the skin blackened. The extent to which it can be predicted is clearly defined.

Based on this result, by taking the expression amount of Annexin II in the stratum corneum, it is possible to take preventive measures such as the selection of a cosmetic which suppresses the effect of ultraviolet exposure. In addition, it is possible to examine the countermeasures in accordance with the anticipation of the effect of ultraviolet exposure. That is, according to this invention, the beauty of a blemish measure and a blemish prevention becomes easy. In addition, the selection of cosmetics, quasi-drugs, medicines or cosmetic health food for the pigmentation site, such as blemishes and can provide effective information therefor. Beauty counseling such as blemish measures can be performed accurately on a scientific basis.

In addition, by using an annexin II high-expression-producing cultured skin model, it is possible to screen a drug that can inhibit the production of melanin by searching for a test substance that affects the expression level of this annexin II protein.

The invention of the present invention is simple in cosmetics stores and the like, because the skin stratum corneum can be safely and easily collected by non-invasive methods such as tape stripping, and the expression level of Annexin II protein to be noted in a short time can be specified. It can be used as a cosmetic selection material on the spot. The instrument which measures the expression amount of this annexin II protein can compare and show a detection result and an indicator. Moreover, based on this result, it is also possible to display a suitable cosmetics and menu.

Annexin II is an intracellular protein with a molecular mass of 38,473 Da, which is a membrane-bound protein controlled by calcium, and two calcium ions bind to this protein. Of the two groups of annexin repeats, one binds calcium and one binds phospholipids. The protein crosslinks with actin or cytoskeleton proteins that bind to the phospholipids in the cell membrane, or activates plasminogen via a tissue plasminogen activator (t-PA). Gene sequencing information (Annexin A2, Gene 95: 243-251 (1990), BC015834). Amino acid sequence information (Annexin A2, J. Biol. Chem. 266: 5169-5176 (1991), P07355).

How to collect stratum corneum proteins from the stratum corneum

(1) Collection of stratum corneum by tape stripping method

The tape stripping method is a method of collecting a stratum corneum adhered to an adhesive face by applying an adhesive tape to the skin and peeling off the adhesive tape.

By using a commercially available adhesive tape, for example, the adhesive tape is pressed onto the human narrow part, the stratum corneum layer can be adhered to the adhesive tape and collected. Commercially available adhesive tapes include Asahi Biomed's keratin checker, Mortex's keratin layer seal, PROMOTOOL's keratin checker (disc type W, disc type G, PRO type), Integral's Corneofix, 3M's transparent tape, 3M The transparent double-stick tape manufactured by the company is mentioned.

(2) extraction buffer

An extraction buffer is used to extract the stratum corneum protein from the stratum corneum adhered to the adhesive tape. As an extraction buffer, the following compositions are mentioned, for example. Composition: PBS (-), 0.1% SDS.

The stratum corneum of the skin, which is collected by the adhesive tape, is differentiated from keratinocytes and contains many proteins of the cytoskeletal system. Therefore, in order to raise the extraction efficiency of a stratum corneum protein, it is preferable to contain a suitable surfactant in this extraction buffer. It is preferable to use SDS (sodium lauryl sulfate, CH ₃ (CH ₂ ) ₁₁ OSO ₃ Na) as the surfactant.

(3) extraction of stratum corneum protein

The adhesive tape from which the stratum corneum is collected is placed in a cylindrical container, and the stratum corneum protein can be quickly extracted by rubbing the adhesive surface using a pestle for homogenization (hereinafter referred to as "pestle") which rotates at high speed. The adhesive tape faces the adhesive face inside the cylindrical container, and puts the opposite side of the adhesive face along the inside of the cylindrical container, and puts it in the cylindrical container. By putting the adhesive tape on the opposite side of the adhesive surface along the inside of the cylindrical container, the adhesive surface can be easily rubbed with the pestle. As another method, the adhesive tape from which the stratum corneum is taken may be immersed in an extraction buffer and rubbed with a scraper to extract the stratum corneum protein, or a shaking method or an ultrasonic irradiation method may be used.

(4) Recovery of the horny layer protein extract

It is preferable that the stratum corneum protein extract is left to stand for about 1 minute, the bubbles are precipitated, and thereafter, the stratum corneum protein extract is sucked and recovered from the cylindrical container.

(5) Analysis of the amount of Annexin II protein in the stratum corneum protein

The amount of Annexin II protein in the obtained stratum corneum protein extract can be quantitatively analyzed by immunoblotting, ELISA, antibody chip or FRET.

UV skin Black flower  Assessment of trends

According to the present invention, it became clear that the amount of Annexin II protein in the stratum corneum and the skin brightness after ultraviolet irradiation of 1 MED (minimum erythema amount) had a negative correlation. In addition, it became clear that the amount of annexin II protein in the stratum corneum and the amount of melanin (N value measured by a chromometer) after 1 MED (minimum erythema amount) of ultraviolet irradiation were positively correlated.

Therefore, when the amount of annexin II protein in a stratum corneum is higher than an average value, it can be evaluated that the tendency of skin blackening by the ultraviolet-ray of skin is strong. Specifically, a person whose amount of annexin II protein in the stratum corneum is higher than the average value can be evaluated that melanin production tends to be enhanced by UV exposure, and UV disturbances such as blemishes are likely to occur.

UV skin Blackening  Screening of Inhibitors

By searching for a component that reduces the amount of Annexin II protein in the stratum corneum of the mouse or the like, an inhibitor of skin blackening caused by ultraviolet rays can be screened.

Example

1. Subject

Ten males in their twenties and thirties were enrolled. Table 1 shows the age, skin type, minimum erythema amount, L * value before ultraviolet irradiation, and N value of each subject. In addition, skin type II is a type of `` simple sunburn, slightly tanning '', and III type of `` normally sunburn, usually tanning (light tan). IV is a type that causes a slight sunburn and always tans (brown tan).

In addition, the L * value is the brightness of the L * a * b * colorimeter measured by a spectrophotometer (CM-500, manufactured by Konica Minolta Co., Ltd.), and when the L * value is large (not blackened), the L * value is If it is small, it can be evaluated as dark (blackened). In addition, N value was an index of melanin amount, and it measured using mexameterMX16 by Courage + Khazaka. The larger the N value is, the darker the skin is. The smaller the N value is, the smaller the amount of melanin can be.

2. Determination of Annexin II Protein Content in the Corneous Layer of Skin

2.1 Collection of the stratum corneum by tape stripping

Asahi Biomed Co., Ltd. keratin checker (2.5 cm x 2.5 cm) was used as the adhesive tape. A keratin checker was attached to the subject's back, lightly rubbed with a fingertip, and the skin stratum corneum was adhered to the adhesive surface of the keratin checker, and a total of five tape strips were taken from the back.

In addition, the sampling place by tape stripping was extract | collected from the ultraviolet non-irradiation site | part.

2.2 Extraction of Skin Corneal Proteins

Extraction buffer: PBS (-), 0.1% (w / v) SDS.

Extraction buffer volume: 200 μL.

Extraction vessel: A cylindrical vessel having a diameter of 11 mm.

Put the adhesive tape into the cylindrical container so that the opposite side of the adhesive surface is along the inner wall of the container, and put the extraction buffer, and put Handy pestle (TOYOBO company, code HMX-301) as a pestle to a mini cordless grinder ), The pestle was rotated at 9000 rpm, and the adhesive surface of the adhesive tape which followed the cylindrical container inner wall was rubbed. The stirring time was 90 seconds.

The skin stratum corneum protein extract was left to stand for about 1 minute, the foam was allowed to settle, and then the skin stratum corneum protein extract was aspirated and recovered from the cylindrical container.

3. Measurement of the amount of protein of the stratum corneum of the skin

The total protein contained in the stratum corneum protein extract was measured using a DC protein assay kit (BIO-RAD).

4. Determination of Annexin II Protein Content in Skin Corneal Layer Proteins

The expression level of Annexin II by immunoblotting was measured using 1 microgram amount of skin stratum corneum proteins. After separating by SDS-PAGE using a 5-15% gradient gel using 1 µg of skin stratum corneum protein of each subject, the protein was transferred to PVDF membrane and blocked with 5% skim milk. The primary antibody (annexin II polyclonal antibody: manufactured by Santa Cruz Biotechnology) was diluted 1000-fold, and the secondary antibody (HRP-rabbit anti-Goat IgG: manufactured by ZYMED Laboratories) was reacted at 10000-fold dilution, followed by ECLplus ( BD Bioscience Co., Ltd.) was used for detection. The intensity of the obtained band was quantified by image analysis software NIH-Image (NIH). This value was defined as the amount of Annexin II protein.

5. UV irradiation

Ultraviolet light of 1 MED was irradiated in a circle of about 8 mm in diameter of the subject's back. The UV irradiation apparatus used Multiple Solar Ultraviolet Simulator Model 601 manufactured by solar light company. UV intensity was set using a solar light company Erythema UV & UVA Intensity Meter Model 3D-600 v2.0.

6. Measurement of Skin Brightness

L * value of the ultraviolet irradiation site and the non-irradiation site was measured using the spectrophotometer (CM-500, the product made by Konica Minolta). Measure the L * value of the UV-irradiated and non-irradiated sites on the 2nd, 4th, 7th, 10th, and 14th day after the UV irradiation, and measure the difference between L * of the irradiated and non-irradiated sites within the same day. It was used for analysis as the amount of change in L *.

7. Measurement of Skin Melanin Level

The mexameter MX16 manufactured by Courage + Khazaka was used to measure the N value, which is an index of melanin content. N value is the reflectance of the light of wavelength 568-660 nm.

Measure the N value of the UV and non-irradiated sites on the 2nd, 4th, 7th, 10th, and 14th day after irradiation, and measure the difference between the N values of the irradiated and non-irradiated sites within the same day. It used for analysis as an amount of change of N value.

8. Measurement result of skin brightness

The amount of change in L * value after ultraviolet irradiation is shown in FIG.

The amount of change in the L * value (L * value after ultraviolet irradiation-L * value before ultraviolet irradiation) became the maximum on the second day of ultraviolet irradiation, and almost recovered on day 14 thereafter. The L * value on the 2nd day of ultraviolet irradiation is the lowest and reflects the blackening of skin.

Correlation between L * Value on Day 0 and Expression Level in Corneal Layer of Annexin II and Changes in L * Value on Days 2, 4, 7, 10, and 14 and Volume of Annexin II Protein Naturally Converted Correlation was investigated. The graph of a regression line is shown to FIGS. 2-7, and a correlation coefficient is shown in Table 2. FIG.

The amount of change in L * value on the 2nd, 10th, and 14th days of the natural logarithmic conversion of Annexin II protein indicates "significant negative correlation," and the amount of change in the L * value on the 4th and 7th days indicates a "strong negative correlation." Indicated.

From the above facts, it can be seen that the greater the amount of Annexin II protein in the stratum corneum, the stronger the tendency of L * value decrease (skin blackening) due to UV exposure. The amount of annexin II protein in the stratum corneum was found to be an indicator of the tendency of skin blackening due to UV exposure.

In general, the correlation coefficient is evaluated as follows.

            0-0.2: almost irrelevant,

          0.2-0.4: slightly correlated,

          0.4-0.7: significant correlation

            0.7 ~ 1: strong positive correlation

           0-0.2: almost irrelevant,

        -0.4 to -0.2: slightly correlated,

        -0.7 to -0.4: significant negative correlation,

          -1 to -0.7: strong negative correlation

9. Measurement result of skin melanin content

The amount of change in N value after ultraviolet irradiation is shown in FIG.

The change amount of N value (N value after ultraviolet irradiation-N value before ultraviolet irradiation) showed the high value compared with before ultraviolet irradiation (day 0) from the 4th day after ultraviolet irradiation to 14th day. The amount of melanin in the skin is large from day 4 to day 14 after the ultraviolet irradiation, reflecting blackening of the skin.

The correlation between the N value on the 0th day and the expression level in the stratum corneum of Annexin II, and the change amount and the natural value of the N value on the 2nd, 4th, 7th, 10th and 14th day based on the N value on the 0th day. The correlation of logarithmic converted Annexin II protein amount was investigated. The graph of the regression line is shown in Figs. 9 to 14 and the correlation coefficient is shown in Table 3.

After day 4, the amount of change in N-value and the amount of Annexin II protein converted by natural logarithm showed a strong positive correlation.

Therefore, an increase in the amount of annexin II protein means an increase in the amount of melanin by exposure to ultraviolet rays. In other words, the increase in Annexin II was found to increase melanin and blackening of the skin due to UV exposure. In other words, it is clear that the amount of annexin II protein reflects the tendency of the skin to darken by exposure to ultraviolet rays, and moreover, may be an index of melanin increase.

As described above, from the relationship between the change of the amount of Annexin II protein and the L * value, and the change of the amount of Annexin II protein and the N value, the amount of Annexin II protein in the stratum corneum is an indicator of the degree of blackening of the skin by UV exposure. It turned out.

Brief description of the drawings

1 is a graph showing the amount of change in L * value after ultraviolet irradiation, and the vertical axis represents the amount of change in L * value.

Fig. 2 is a graph showing the correlation between the L * value immediately before ultraviolet irradiation (day 0) and the expression level in the stratum corneum of Annexin II.

3 is a graph showing the correlation between the amount of change in L * value on the second day after ultraviolet irradiation based on the N value on day 0 and the amount of Annexin II protein converted by natural logarithm.

Fig. 4 is a graph showing the correlation between the amount of change in L * value on day 4 after ultraviolet irradiation based on the N value on day 0 and the amount of Annexin II protein converted by natural logarithm.

5 is a graph showing the correlation between the amount of change in L * value on the 7th day after ultraviolet irradiation based on the N value on day 0 and the amount of Annexin II protein converted by natural logarithm.

Fig. 6 is a graph showing the correlation between the amount of change in L * value on the 10th day after ultraviolet irradiation based on the N value on day 0 and the amount of Annexin II protein converted by natural logarithm.

7 is a graph showing the correlation between the amount of change in L * value on the 14th day after ultraviolet irradiation based on the N value on day 0 and the amount of Annexin II protein converted by natural logarithm.

8 is a graph showing the change over time of the N value after ultraviolet irradiation. The vertical axis represents the amount of change in the N value.

Fig. 9 is a graph showing the correlation between the N value immediately before ultraviolet irradiation (day 0) and the expression level in the stratum corneum of Annexin II.

FIG. 10 is a graph showing the correlation between the amount of change in N value on the second day after UV irradiation based on the value of N on day 0 and the amount of Annexin II protein converted by natural logarithm.

FIG. 11 is a graph showing the correlation between the amount of change in N value on the fourth day after ultraviolet irradiation based on the value of N on day 0 and the amount of Annexin II protein converted by natural logarithm.

12 is a graph showing the correlation between the amount of change in N value on the 7th day after ultraviolet irradiation based on the value of N on day 0 and the amount of Annexin II protein converted by natural logarithm.

FIG. 13 is a graph showing the correlation between the amount of change in N value on the 10th day after ultraviolet irradiation based on the value of N on day 0 and the amount of Annexin II protein converted by natural logarithm.

14 is a graph showing the correlation between the amount of change in N value on the 14th day after ultraviolet irradiation based on the value of N on day 0 and the amount of Annexin II protein converted by natural logarithm.

Claims (3)

The evaluation method of the skin blackening tendency by ultraviolet-ray which evaluates that the skin blackening tendency by the ultraviolet-ray of skin is strong according to the amount of Annexin II protein in a skin stratum corneum. The method of screening the component which affects skin blackening using the evaluation method of the skin blackening tendency of Claim 1. The method of selecting a cosmetics according to the skin blackening tendency obtained using the evaluation method of skin blackening tendency of Claim 2.

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