JP4473926B1 - Evaluation method of skin darkening tendency by ultraviolet rays - Google Patents

Abstract

A method for evaluating the risk of UV damage is provided. In particular, the present invention provides a method that can know the skin darkening tendency of the subject using a stratum corneum collected using noninvasive means.
A method for estimating the amount of skin melanin using the amount of annexin II protein in the stratum corneum as an index, or a method for evaluating the tendency of skin darkening due to ultraviolet rays.
[Selection] Figure 8

Description

  The present invention relates to a method for evaluating the tendency of skin darkening due to ultraviolet rays.

There are concerns about many health effects such as carcinogenic effects on the skin, effects on eye diseases such as cataracts, and immune functions. Exposure to UV rays in the skin also causes various UV damages. Ultraviolet rays cause inflammation in the skin, or oxidize unsaturated lipids in the skin to generate lipid radicals, thereby producing lipid peroxides. Such inflammation and oxidation caused by ultraviolet rays cause tissue, cell and DNA damage. Phenomena due to UV exposure of skin generally include wrinkles, spots, skin cancers and the like.
In recent years, as epidemiological data, there is a report that the risk of skin cancer increases due to differences in sensitivity to ultraviolet rays (skin color, skin type, etc.) (Non-Patent Documents 1 and 2). Furthermore, it has also been clarified that the risk level of malignant melanoma (melanoma), which is a type of skin cancer, is several times higher due to the single base substitution gene mutation of melanocortin1 receptor (Non-patent Documents 3 and 4). Sensitivity to ultraviolet rays varies greatly depending on the skin type, and it is classified according to the type classified into 6 stages, and is based on the minimum ultraviolet energy (minimum erythema amount: MED) that causes redness (erythema), but the former is subjective observation, The latter involves the risk that it cannot be measured without exposure to ultraviolet light. The increase in ultraviolet rays has become a problem more than before due to ozone layer destruction, etc., and it is necessary to appropriately evaluate the sensitivity risk to individuals for ultraviolet rays.
On the other hand, in the initial stage where pigmentation is generally said to be a stain, the epidermis keratinocytes may be signaled by α-MSH, bFGF, CGRP, endothelin, etc. by various stimuli before they appear on the skin surface. Is produced and transmitted to melanocytes. After being transmitted to melanocytes, a large amount of melanin is made in the cell. On the other hand, the turnover becomes abnormal due to ultraviolet rays, and the melanin pigment produced in large quantities cannot be discharged, so it changes to a pigmented lesion. In experiments using an in vitro system, when melanocytes were irradiated with ultraviolet rays, the expression of cell growth factor markers, p73, Nup88, p27, Id1, and PCNA, and the expression of bcl-2, an apoptosis-related protein, increased or decreased. (Non-patent document 5). So far, the state of the stain is invasively compared with the normal portion using the skin tissue by biopsy, and the expression of proteins such as NT-3, ADAM9, HB-EGF has been remarkably increased in the stain portion. (Patent Document 1). Furthermore, as a test method for predicting skin spot formation, there is a report of measuring the amount of mRNA such as MLSTD1, MOGAT1, Mcp9, Krt2-6b, etc. by biopsy to judge the risk of spot formation (Patent Documents 2, 3, 4). ). In addition, as a non-invasive method, the abundance analysis of interleukin 1 (IL-1) and interleukin 1 receptor antagonist (IL-1ra) is performed using a stratum corneum derived from the skin collected through tape stripping or abrasion as a sample. A method for evaluating skin quality is disclosed in Patent Document 5.

JP 2004-205246 A JP 2005-106745 A JP 2005-110505 A JP 2007-289063 A Japanese Patent No. 3590708 Arch Dermatol. 2004; 140: 819-824. Cancer Causes Control. 1997; 8 (2): 246-252 Invest Dermatol. 117: 294-300, 2001 Am. J. et al. Hum. Genet. 66: 176-186,2000 Carcinogenesis 24 (12): 1929-934, 2003

  The present invention provides a method for assessing the risk of UV damage. In particular, an object is to provide a method for knowing the skin darkening tendency of the subject using a stratum corneum collected using non-invasive means.

The main configuration of the present invention is as follows.
(1) Using the fact that the amount of Annexin II protein in the skin stratum corneum is positively correlated with the amount of melanin when the number of days has elapsed after UV irradiation, the amount of Annexin II protein in the skin stratum corneum is measured. A method for evaluating the tendency of skin darkening due to ultraviolet rays, wherein the tendency of skin darkening due to ultraviolet rays is evaluated.
(2) Using the fact that the amount of Annexin II protein in the stratum corneum is positively correlated with the amount of melanin when the number of days has elapsed after UV irradiation, the amount of Annexin II protein in the skin stratum corneum is affected. A method of screening for ingredients that suppress skin darkening by discriminating the ingredients to be applied.
(3) A method of selecting a cosmetic according to the skin darkening tendency obtained by using the method for evaluating skin darkening described in ( 1 ).

The present invention has found that the amount of Annexin II protein, which is a protein expressed in vivo, has a positive correlation with the production of melanin by UV exposure, and has clarified that the tendency of skin darkening can be evaluated It is.
According to the present invention, the tendency of skin darkening due to ultraviolet rays can be evaluated without actually irradiating the ultraviolet rays.
Since the present invention can be evaluated using the stratum corneum collected using a noninvasive tape stripping means, it is safe and simple.
In addition, by using the evaluation method of the present invention, it is possible to screen for a component useful for suppressing the tendency of skin darkening due to ultraviolet rays or suppressing melanin production after exposure to ultraviolet rays.
According to the present invention, it is easy to make a beauty for preventing spots and preventing spots. That is, it is possible to provide selection of cosmetics, quasi-drugs, pharmaceuticals or beauty health foods for pigmentation sites such as spots and information effective for that purpose. Beauty counseling such as anti-smudge can be performed accurately.

Hereinafter, embodiments of the present invention will be described in more detail.
The present invention provides a method for evaluating the risk of UV damage, which comprises measuring the amount of annexin II protein contained in the stratum corneum.
The invention of the present application is based on the abundance obtained by measuring annexin II, a protein present in the skin stratum corneum collected by a non-invasive technique, and the person who originated the skin stratum corneum by exposure to ultraviolet rays is blackened. It is possible to know the risk of doing.
The present inventor has found that there is a positive correlation between the change in melanin production after exposure and the expression level of annexin II, and by measuring the expression level of annexin II, the risk of melanin formation when exposed, Furthermore, it was clarified that the degree of skin darkening can be predicted.
Based on this result, knowing the expression level of annexin II in the stratum corneum, it is possible to take preventive measures such as selection of cosmetics that suppress the effects of UV exposure. In addition, measures can be considered in accordance with the expected impact of UV exposure. That is, according to the present invention, it is possible to easily make a beauty for preventing spots and preventing spots. Furthermore, it is possible to provide selection of cosmetics, quasi-drugs, pharmaceuticals or beauty health foods for pigmentation sites such as spots and information effective for that purpose. Beauty counseling, such as anti-smudge measures, can be performed accurately on a scientific basis.
Further, by using an annexin II high-expression production cultured skin model and searching for a test substance that affects the expression level of the annexin II protein, a drug capable of suppressing the production of melanin can be screened.
In the present invention, it is possible to safely and easily collect the skin stratum corneum by a invasive method such as tape stripping, and to specify the expression level of the annexin II protein of interest in the skin stratum corneum in a short time. It can be easily measured at a cosmetics department, etc., and used as a cosmetic selection material on the spot. The instrument that measures the expression level of the annexin II protein can present the detected result in comparison with the index. Furthermore, suitable cosmetics and menus can be displayed based on this result.

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

Method of collecting stratum corneum protein from skin stratum corneum (1) Extraction of stratum corneum by tape stripping method The tape stripping method is a stratum corneum that adheres to the adhesive surface by applying an adhesive tape to the skin and peeling off the adhesive tape. It is a method to collect.
By using a commercially available adhesive tape, for example, by pressing the adhesive tape against the human cheek, the stratum corneum can be adhered to the adhesive tape and collected. As commercially available adhesive tapes, Asahi Biomed's horny checker, Moritex's horny layer seal, PROMOTOOL's horny checker (disk type W, disk type G, PRO type), Integral Corneofix, 3M transparent tape, Examples thereof include a transparent double-sided tape manufactured by 3M.

(2) Extraction buffer An extraction buffer is used to extract stratum corneum protein from the stratum corneum adhered to the adhesive tape. Examples of the extraction buffer include the following compositions. Composition: PBS (-), 0.1% SDS.
The stratum corneum of skin collected with adhesive tape is a differentiated keratinocyte and contains a lot of cytoskeletal proteins. Therefore, in order to increase the extraction efficiency of the stratum corneum protein, it is preferable to contain an appropriate surfactant in this extraction buffer. As the surfactant, SDS (sodium lauryl sulfate, CH ₃ (CH ₂ ) ₁₁ OSO ₃ Na) is preferably used.

(3) Extraction of stratum corneum protein Place the adhesive tape from which the stratum corneum has been collected into a cylindrical container and quickly rub the adhesive surface with a high-speed rotating homogenization pestle (hereinafter referred to as “Pessle”). In addition, stratum corneum protein can be extracted. The adhesive tape is placed in the cylindrical container, with the adhesive surface facing the inside of the cylindrical container and the opposite surface of the adhesive surface along the inside of the cylindrical container. By placing the adhesive tape with the opposite surface 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 stratum corneum protein may be extracted by immersing an adhesive tape from which the stratum corneum has been collected in an extraction buffer and rubbing with a scraper, or using a shaking method or an ultrasonic method.

(4) Recovery of stratum corneum protein extract It is preferable that the stratum corneum protein extract is allowed to stand for about 1 minute to calm the foam, and then the stratum corneum protein extract is sucked and recovered from the cylindrical container.

(5) Analysis of Annexin II protein content in stratum corneum protein The amount of Annexin II protein in the obtained stratum corneum protein extract can be quantitatively analyzed by immunoblotting method, ELISA method, antibody chip method, FRET method. .

Evaluation of skin darkening tendency by ultraviolet rays According to the present invention, it has been clarified that the amount of annexin II protein in the stratum corneum and the skin brightness after ultraviolet irradiation of 1 MED (minimum erythema amount) have a negative correlation. It was also revealed that the amount of Annexin II protein in the stratum corneum and the amount of melanin (N value measured with a meggerometer) after irradiation with 1 MED (minimum erythema amount) had a positive correlation.
Therefore, when the amount of annexin II protein in the stratum corneum is higher than the average value, it can be evaluated that the skin darkening tendency by ultraviolet rays of the skin is strong. Specifically, it can be evaluated that a person whose annexin II protein amount in the horny layer of the skin is higher than the average value is likely to increase melanin production by UV exposure and to easily cause UV damage such as spots.

Screening for inhibitors of skin darkening caused by ultraviolet rays By searching for components that reduce the amount of annexin II protein in the stratum corneum of mice and the like, inhibitors for skin darkening caused by ultraviolet rays can be screened.

1. Subjects Ten men in their 20s and 30s were subjects. Table 1 shows the age, skin type, minimum erythema amount, L * value and N value before ultraviolet irradiation of each subject. In addition, skin type II is a type that easily causes sunburn and slightly causes suntan, and III is a type that normally causes sunburn and usually causes suntan (light brown tanning). , IV is a type of “slightly sunburn and always suntan (brown tan)”.
The L * value is the brightness of the L * a * b * color system measured with a spectrocolorimeter (CM-500, manufactured by Konica Minolta Co., Ltd.). When the L * value is large, it is bright (not blackened). When the L * value is small, it can be evaluated as dark (blackened). The N value is an index of the amount of melanin, and was measured using a Mexmeter MX16 manufactured by Course + Khazaka. The larger the N value is, the more black it is. When the N value is small, the amount of melanin is small and it can be evaluated that it is fair.

2. Measurement of annexin II protein amount in the horny layer of skin 2.1 Collection of horny layer by tape stripping A horny checker (2.5 cm × 2.5 cm) manufactured by Asahi Biomed Co., Ltd. was used as an adhesive tape. A keratin checker was affixed to the back of the subject, lightly rubbed with a fingertip to adhere the skin stratum corneum to the adhesive surface of the horny checker, and a total of five tape strips were collected from the back. In addition, the sampling location from tape stripping was sampled from the ultraviolet irradiation site.

2.2 Extraction of skin stratum corneum protein Extraction buffer: PBS (−), 0.1% (w / v) SDS.
Extraction buffer volume: 200 μL.
Extraction container: cylindrical container with a diameter of 11 mm.
Adhesive tape is put in a cylindrical container so that the surface opposite to the adhesive surface is along the inner wall of the container, an extraction buffer is placed, and a handy pestle (TOYOBO, code HMX-) is used as a pestle in a mini cordless grinder (Funakoshi code13753E). 301) was connected, the pestle was rotated at 9000 rpm, and the adhesive surface of the adhesive tape along the inner wall of the cylindrical container was rubbed. The stirring time was 90 seconds.
The skin stratum corneum protein extract was allowed to stand for about 1 minute to calm the foam, and then the skin stratum corneum protein extract was aspirated and collected from the cylindrical container.

3. Measurement of skin stratum corneum protein mass The total protein mass contained in the skin stratum corneum protein extract was measured using DC protein Assay Kit (BIO-RAD).

4). Annexin II protein mass measurement in skin stratum corneum protein The expression level of Annexin II by immunoblotting was measured using 1 μg of skin stratum corneum protein mass. After separation by SDS-PAGE using 5-15% gradient gel using 1 μg of skin horny layer protein of each subject, the protein was transferred to a PVDF membrane and blocked with 5% skim milk. The primary antibody (Annexin II polyclonal antibody: manufactured by Santa Cruz Biotechnology) was diluted 1000 times, and the secondary antibody (HRP-rabbit anti-Goat IgG: manufactured by ZYMED Laboratories) was reacted at a dilution of 10,000 times, and then ECLplus (BD Detection was performed by Bioscience). The intensity of the obtained band was quantified with image analysis software NIH-Image (manufactured by NIH). This value was defined as annexin II protein mass.

5). Ultraviolet irradiation A 1 MED ultraviolet ray was irradiated in a circle having a diameter of about 8 mm on the back of the subject. As the ultraviolet irradiation device, a Multiple Solar Ultraviolet Simulator Model 601 manufactured by solar light company was used. The ultraviolet intensity was set using Erythema UV & UVA Intensity Meter Model 3D-600 v2.0 manufactured by solar light company.

6). Measurement of skin lightness Using a spectroscopic colorimeter (CM-500, manufactured by Konica Minolta), L * values of the ultraviolet irradiation site and the non-irradiation site were measured. Measure the L * values of the irradiated and non-irradiated sites on the second, fourth, seventh, tenth, and fourteenth days before and after the irradiation, and measure the difference in L * between the irradiated and non-irradiated sites within the same day The amount of change in L * was used for analysis.

7). Measurement of skin melanin amount N value serving as an index of the amount of melanin was measured by using Mexmeter MX16 manufactured by Curage + Khazaka. The N value is the reflectance of light having a wavelength of 568 to 660 nm.
Measure the N value of the irradiated and non-irradiated sites on the second, fourth, seventh, tenth, and fourteenth days before and after the irradiation, and determine the difference in N values between the irradiated and non-irradiated sites on the same day. It was used for analysis as an N value change amount.

8). Measurement result of skin brightness FIG. 1 shows the amount of change in L * value after UV irradiation.
The amount of change in the L * value (L * value after UV irradiation-L * value before UV irradiation) became a maximum negative value on the second day of UV irradiation, and then almost recovered on the 14th day. The L * value on the second day of UV irradiation is the lowest, reflecting the darkening of the skin.
Correlation between the L * value on day 0 and the expression level of annexin II in the stratum corneum, and the amount of change in L * value on day 2, day 4, day 7, day 10, and day 14 The correlation of the natural logarithmized annexin II protein mass was investigated. The regression line graphs are shown in FIGS.
The natural logarithmized annexin II protein mass and the amount of change in L * values on days 2, 10 and 14 show a “significant negative correlation”, and the amount of change in L * values on days 4 and 7 is “strong negative” Correlation ”.
From the above, it can be seen that the greater the amount of Annexin II protein in the stratum corneum, the stronger the tendency of L * value reduction (skin darkening) due to UV exposure. It was found that the amount of annexin II protein in the stratum corneum is an index of the tendency of skin darkening due to exposure to ultraviolet rays.

In general, the correlation coefficient is evaluated as follows.
0 to 0.2: almost no correlation,
0.2-0.4: Somewhat correlated,
0.4-0.7: There is a considerable positive correlation,
0.7-1: Strong positive correlation
0--0.2: Almost no correlation,
-0.4 to -0.2: Somewhat correlated,
-0.7 to -0.4: There is a considerable negative correlation,
−1 to −0.7: Strong negative correlation

9. Measurement result of skin melanin amount The amount of change in N value after ultraviolet irradiation is shown in FIG.
The amount of change in N value (N value after ultraviolet irradiation-N value before ultraviolet irradiation) was higher than that before ultraviolet irradiation (day 0) from the 4th day to the 14th day of ultraviolet irradiation. The amount of skin melanin is large from the 4th day to the 14th day from the ultraviolet irradiation, which reflects the darkening of the skin.
Correlation between the N value on day 0 and the expression level of annexin II in the stratum corneum, and the 2nd day, 4th day, 7th day, 10th day, 14 days based on the N value on day 0 The correlation between the amount of change in N value on the day and the amount of Annexin II protein converted to natural logarithm was examined. The regression line graphs are shown in FIGS.
From the fourth day onward, the natural logarithmized annexin II protein mass and the amount of change in N value showed a strong positive correlation.
Therefore, an increase in the annexin II protein amount means an increase in the amount of melanin by exposure to ultraviolet rays. That is, when Annexin II increased, it became clear that the increase in melanin by exposure to ultraviolet rays and the darkening of skin increased. In other words, it is clear that the amount of annexin II protein reflects the tendency of skin darkening due to UV exposure and can be an index of melanin increase.

As described above, from the relationship between Annexin II protein mass and L * value change, and Annexin II protein mass and N value change, Annexin II protein mass in the skin stratum corneum is an indicator of the degree of skin darkening due to UV exposure. It has been found.

The graph which shows the variation | change_quantity of L * value after ultraviolet irradiation. The vertical axis indicates the amount of change in the L * value. Graph showing the correlation between the L * value immediately before UV irradiation (day 0) and the expression level of Annexin II in the stratum corneum Graph showing the correlation between the amount of change in L * value on the second day after ultraviolet irradiation and the natural logarithmically converted Annexin II protein amount based on the N value on Day 0 Graph showing the correlation between the amount of change in L * value on the 4th day after ultraviolet irradiation and the natural logarithmized annexin II protein amount based on the N value on the 0th day Graph showing the correlation between the amount of change in L * value on the 7th day after ultraviolet irradiation and the natural logarithmically converted Annexin II protein amount based on the N value on the 0th day Graph showing the correlation between the amount of change in L * value on the 10th day after UV irradiation and the amount of natural log-transformed Annexin II protein based on the N value on the 0th day Graph showing the correlation between the amount of change in L * value on the 14th day after ultraviolet irradiation and the natural logarithmized annexin II protein amount based on the N value on the 0th day The graph which shows the amount of time-dependent change of N value after ultraviolet irradiation. The vertical axis indicates the amount of change in N value. Graph showing the correlation between the N value immediately before UV irradiation (day 0) and the expression level of Annexin II in the stratum corneum Graph showing the correlation between the amount of change in N value on the second day after ultraviolet irradiation and the amount of natural log-transformed annexin II protein based on the N value on day 0 Graph showing the correlation between the amount of change in N value on the 4th day after UV irradiation and the natural log-transformed Annexin II protein amount based on the N value on Day 0 Graph showing the correlation between the amount of change in N value on the 7th day after UV irradiation and the natural logarithmically converted Annexin II protein amount based on the N value on Day 0 Graph showing the correlation between the amount of change in N value on the 10th day after UV irradiation and the natural logarithmically converted Annexin II protein amount based on the N value on Day 0 Graph showing the correlation between the amount of change in N value on day 14 after UV irradiation and the amount of annexin II protein converted to natural logarithm on the basis of N value on day 0

Claims (3)

Using the fact that the amount of Annexin II protein in the stratum corneum is positively correlated with the amount of melanin when the number of days has elapsed after UV irradiation, the amount of Annexin II protein in the skin stratum corneum is measured and A method for evaluating the tendency of skin darkening due to ultraviolet rays, characterized by evaluating the tendency of skin darkening due to ultraviolet rays. Using the fact that the amount of annexin II protein in the skin stratum corneum is positively correlated with the amount of melanin when the number of days has elapsed after UV irradiation , an ingredient that affects the amount of annexin II protein in the skin stratum corneum A method of screening for ingredients that are discriminated and suppress skin darkening . The method to select cosmetics according to the skin darkening tendency obtained using the evaluation method of the skin darkening tendency described in Claim 1 .