JP7190376B2 - Stratum corneum melanin observation method - Google Patents

Description

The present invention relates to a method for observing melanin in the stratum corneum of the skin.

Skin tone is one of the important cosmetic factors. Beauty counseling is performed to select recommended cosmetics based on the results of evaluating skin color and color unevenness. As a measurement method for evaluating skin color and color unevenness, in addition to measuring skin color with an instrument, the amount of melanin in the stratum corneum, which is a factor that determines skin color, and the presence and distribution of melanin are confirmed. Techniques have been taken (Patent Documents 1, 2, and 3).

The stratum corneum is located in the outermost layer among the four layers that constitute the skin, and is also called stratum corneum. In order to evaluate the amount of melanin in the stratum corneum, its state of existence, and its distribution, the stratum corneum is peeled off with an adhesive tape and collected. A method of transferring and observing melanin has been performed (Patent Documents 1, 4, 5 and 6). Observation of melanin is carried out by staining a stratum corneum sample, and is carried out by a melanin staining method using a silver salt such as silver nitrate. However, the melanin dyeing method using silver nitrate requires time and complicated operations for dyeing. In addition, since a silver salt is used, special waste liquid treatment is required.

A method for staining the stratum corneum that does not use a silver salt has also been proposed. Patent Document 7 describes dyes such as blue No. 1, purple No. 401, green No. 201, yellow No. 4, yellow No. 5, yellow No. 407, red No. 102, orange No. 205, brilliant green, hematoxylin, organic acid and alcohol. and a method for observing the stratum corneum are described.
Patent Literature 8 describes a method for identifying skin that is stained with a Congo red dye and identifies the stained stratum corneum cells with a polarizing microscope.

JP-A-11-332836 JP-A-2000-212036 JP-A-2000-212037 Japanese Patent No. 3179887 Japanese Patent No. 3212558 Japanese Patent No. 3212559 JP 2006-053117 A JP 2007-263655 A

The inventor of the present invention is studying stratum corneum melanin. In the course of this research, the present invention was accomplished as a result of examining a method for selectively microscopically observing only melanin granules in the stratum corneum.
That is, the present invention uses a method for preparing a sample of the stratum corneum, a method for evaluating the state of melanin in the stratum corneum of the skin using the prepared sample, and the observation results thereof, in which only melanin granules in the stratum corneum can be observed. An object of the present invention is to provide a method for measuring the amount of melanin in human skin.

The main configuration of the present invention is as follows.
(1) A method for preparing a stratum corneum sample for microscopic observation, which comprises treating a stratum corneum sample with a solution of a hypochlorous acid compound and/or a surfactant to make stratum corneum components other than melanin transparent.
(2) The preparation method according to (1), wherein the stratum corneum sample is obtained by a tape stripping method.
(3) The preparation method according to (1) or (2), wherein the hypochlorous acid compound is sodium hypochlorite.
(4) The preparation method according to any one of (1) to (3), wherein the surfactant is sodium dodecyl sulfate.
(5) The preparation method according to (3), wherein the concentration of sodium hypochlorite is 0.4% by mass or more.
(6) The preparation method according to (4), wherein the concentration of sodium dodecyl sulfate is 2.5 to 20% by mass.
(7) An evaluation method for evaluating the state of melanin granules in the stratum corneum of the skin by microscopic observation of the stratum corneum sample prepared by any of the methods of (1) to (6).
(8) A microscopic observation image of the stratum corneum sample prepared by any of the methods described in (1) to (6) is converted into black and white binary data, and the pixel value of the black data is obtained. A method for measuring the amount of melanin in the layer.
(9) A method of analyzing skin condition based on melanin granule amount data obtained from a microscopic observation sample of a stratum corneum sample prepared by any one of (1) to (6).

INDUSTRIAL APPLICABILITY According to the present invention, a method for preparing a stratum corneum sample, a method for evaluating the state of melanin in the stratum corneum of the skin using the prepared sample, and a method for measuring the amount of melanin using the observation results is provided. Since the method of the present invention does not use a silver salt for staining the stratum corneum sample, there is no waste disposal problem. Then, it becomes possible to prepare a microscopic observation sample of melanin in an extremely short time.
In addition, in the sample for microscopic observation obtained by the method of the present invention, only the melanin granules in the stratum corneum retain their original form, and the other tissues are homogenized and made transparent, so melanin granules can be observed. Extremely easy. Therefore, the state of melanin in the stratum corneum can be easily observed and evaluated accurately. In addition, by binarizing the observation image, only melanin can be easily measured as the number of pixels, and based on this, the amount of melanin in the stratum corneum can be measured and analyzed.

An observed image (right) of a hypochlorite-treated stratum corneum sample and an observed image (left) of an untreated stratum corneum sample. An observation image (right) of a stratum corneum sample obtained by further staining a hypochlorite-treated stratum corneum sample by the Fontana-Masson method (right) and an observation image (left) of a stratum corneum sample stained by the Fontana-Masson method are shown. It is a microscopy image obtained by directly observing the stratum corneum of four subjects. It is a microscope observation image which prepared the stratum corneum of four test subjects as an observation sample by the method of this invention. It is an image in which melanin is represented by black pixels by further converting microscopic observation images of four subjects' stratum corneum prepared as observation samples by the method of the present invention into binary data. Distilled water (H ₂ O), 40% aqueous urea solution (Urea: MP Biomedicals, Inc.), 30% by mass hydrogen peroxide solution (H ₂ O ₂ : Fujifilm Wako Pure Chemical Industries, Ltd.), 6% by mass hypochlorous acid sodium acid solution (chlorine bleach), 10% by mass sodium dodecyl sulfate (SDS) aqueous solution (MP Biomedicals, Inc.), 1N sodium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.), 12M hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) Co., Ltd.) is a microscope observation image when the stratum corneum is processed. It shows that SDS and sodium hypochlorite were effective. 1 is a microscopy image of a stratum corneum sample tested for effective concentration of SDS. 1 is a microscope observation image of a stratum corneum sample tested for effective concentration of sodium hypochlorite.

According to the present invention, a sample of the stratum corneum collected from the skin is treated with a solution of a hypochlorous acid compound or a surfactant to make the stratum corneum components other than melanin transparent, thereby leaving only melanin granules observable in the stratum corneum. Prepare the samples. This sample is characterized by observing and evaluating the state of melanin granules in the stratum corneum of the skin. Also, the data of the observation image is binarized to measure the amount of melanin granules. A more detailed description is given below.

It is preferable that the collection of the stratum corneum sample, the treatment of the stratum corneum sample, the preparation of the stratum corneum sample, the steps of preparing an observation sample for microscopic observation, and the acquisition and analysis of binary data are performed as follows.
(Step 1) A stratum corneum sample is collected from the skin using stratum corneum peeling means.
(Step 2) After the stratum corneum sample collected in step 1 is pasted on a transparent plate with the peeled surface facing upward, a hypochlorous acid solution or surfactant solution is dropped and left to stand for a certain period of time.
(Step 3) The stratum corneum sample obtained in Step 2 is washed with water.
(Step 4) The stratum corneum sample washed in step 3 is air-dried and used as a microscope observation sample.
(Step 5) Microscopic observation of the sample obtained in Step 4 is carried out for visual evaluation.
(Step 6) Acquire observation images taken with a digital camera as data.
(Step 7) The observed image obtained in step 6 is converted into black and white binarized data by image processing to obtain black and white area data.
(Step 8) Pixel values of black data are used as melanin granule data.

The stratum corneum exfoliation means in step 1 may be biopsy or a method using an adhesive tape. Considering the burden on the subject, the method using an adhesive tape is preferable. A method using an adhesive tape is called a tape stripping method.
The tape stripping method is a method in which an adhesive tape is attached to the skin and then peeled off to recover the surface layer of the skin.
As the adhesive, synthetic rubber such as natural rubber, polyisobutylene, polybutadiene, silicone rubber, polyisoprene, styrene-isoprene-styrene block copolymer, etc., synthetic resin such as polypropylene, polystyrene, acrylic acid ester copolymer, etc. are used. It is Adhesive tapes used in the tape stripping method are commercially available and may be used.

In step 2, when the exfoliated stratum corneum sample is attached to the transparent plate with the exfoliated surface facing upward, the transparent plate is preferably made of glass or acrylic suitable for microscopic observation.

The hypochlorite used in step 2 is preferably an alkali metal hypochlorite, particularly sodium hypochlorite. Hypochlorite is used as an aqueous solution. The hypochlorite concentration in the aqueous solution is 0.4-100% by weight, preferably 4-7% by weight, particularly preferably 6% by weight. The hypochlorite is preferably an aqueous solution, but may contain a surfactant as appropriate. Examples of the medium for the aqueous solution include pure water, ion-exchanged water, distilled water, and tap water, with pure water being preferred.
As the hypochlorite-containing aqueous solution used in the present invention, a commercially available chlorine bleach solution for laundry can also be used.

The pH of the hypochlorite aqueous solution used in the present invention may be determined as appropriate, but is preferably pH 8 to 12, particularly preferably pH 10 or higher, in order to promote transparency of the stratum corneum sample. Although the temperature of the hypochlorite aqueous solution is not particularly limited, it is preferably room temperature.

The amount of the hypochlorite aqueous solution used should be such that the entire surface of the stratum corneum sample is sufficiently wetted with the hypochlorite aqueous solution. Usually, 0.5 to 1 ml is dropped per test sample, and the test sample is sufficiently immersed in the hypochlorite aqueous solution.

The surfactant used in step 2 is preferably a surfactant having a protein denaturing action. Examples include Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, and sodium dodecylbenzenesulfonic acid. Among them, sodium dodecylbenzenesulfonic acid (SDS, also known as sodium lauryl sulfate), which is widely used in the field of biochemistry, is suitable.
SDS is used by dissolving it in an aqueous solution or a buffer solution such as PBS. The SDS concentration in the aqueous solution is 2.5-20% by weight, preferably 5-15% by weight, particularly preferably 10% by weight. It may be a mixed solution mixed with the hypochlorite aqueous solution. Pure water, ion-exchanged water, distilled water, tap water, physiological saline, PBS, and the like can be used as the medium for the aqueous solution.
The amount of the SDS aqueous solution used should be such that the entire surface of the stratum corneum sample is wetted with the SDS aqueous solution. Usually, 0.5 to 1 ml is dropped per test sample, and the test sample is sufficiently immersed in the SDS aqueous solution.

The washing in step 3 is for removing the hypochlorite and/or surfactant used in step 2. Repeat washing at least 10 times with a sufficient amount of water, or wash with running water. Water used for washing includes pure water, ion-exchanged water, distilled water, tap water, etc. Pure water is preferred.

In step 4, after step 3 is completed, the water content of the sample is air-dried and then used as a sample (specimen) for microscopic observation. In addition, the sample may be sealed with balsam or paraffin, which is used when preserving the sample for a long period of time.

In step 5, the sample is observed with an optical microscope, or the sample is projected onto a monitor using a videoscope or the like, and the amount, shape, size, uniformity, homogeneity, Examine the degree of aggregation, etc. In the exfoliated stratum corneum samples obtained in steps 1 to 4 above, only melanin can be observed as black spots when observed under a microscope. The fact that this black spot is melanin can be confirmed by a conventional melanin staining method as shown in Test Examples.
In this way, by directly observing melanin, which is an index of skin condition, it is possible to evaluate skin properties, skin quality, skin color, condition of spots, and the like.

In step 6, an observation image of the specimen confirmed and evaluated by the microscopic observation is selected and input into a computer as image data using a digital camera or the like.

In step 7, the captured data of the observed image acquired in step 6 is converted into black and white binarized data by image processing to acquire black and white area data. Any known image processing system can be used for image processing. As such an image processing system, "ImageJ" can be used as an appropriate image processing system in the present invention. ImageJ is an open source, public domain image processing software. A person skilled in the art can easily obtain black and white binarized data of an image according to the operating procedure of the system. By selecting a certain range of the captured image as an analysis target and confirming the number of pixels in the selected range, the melanin area ratio per total number of pixels can be calculated. In the binarization, it is preferable to measure the background gradation in advance and determine the binarization threshold based on the value in order to obtain the area by removing the background portion other than the granules.

In step 8, the pixels of the black data are counted, and the number of pixels (the number of pixels) is displayed as the area of black, and this is taken as the amount of melanin.

By going through the above steps, a method for preparing a stratum corneum sample that allows selective observation of only melanin granules in a short time and safely, and the state of melanin in the stratum corneum and the amount of melanin in the stratum corneum using the prepared sample. A method of measuring is provided. Furthermore, by binarizing the observation image, the amount of melanin in the stratum corneum can be easily measured.

Test examples are shown below to specifically describe the present invention.
<Test 1>
In this test, a test example in which melanin in the stratum corneum was directly observed by the method of the present invention is shown.
1. Test Method A 2.5 cm×2.5 cm keratin checker (Asahi Biomed Co., Ltd.) was used as an adhesive tape for exfoliating the stratum corneum of the skin.
The stratum corneum was collected from the female neck of the test subject by the tape stripping method using the aforementioned keratin checker.
The collected stratum corneum was pasted on a transparent slide glass using a mending tape, and a commercially available chlorine-based bleaching agent (Kitchen Haiter: containing 6% by mass of sodium hypochlorite) was dropped as an aqueous solution of hypochlorite. After allowing to stand at room temperature for 5 minutes, the sample was washed with purified water and air-dried. For comparison, a sample of the stratum corneum (a sample not treated with hypochlorite) was observed under a microscope under the same conditions.

2. Result In the stratum corneum sample to which the hypochlorite aqueous solution was dropped, the background of the observation screen changed to a uniform state except for melanin black spots, and the area other than melanin granules became transparent. On the other hand, in the stratum corneum sample for comparison, the cell walls and cell structures that make up the stratum corneum were observed in various shades throughout the screen, making it difficult to distinguish melanin granules.
FIG. 1 shows an observed image of the collected stratum corneum (before treatment) and an observed image of a stratum corneum sample (after treatment) treated by dropping hypochlorite aqueous solution (chlorine bleach). The granular spots appearing darker than in the surrounding uniform background in the image on the right (after processing) were melanin granules, as confirmed in Test 2 below.

<Test 2>
In order to identify whether the granules observed in Test 1 were melanin granules, melanin granules in the stratum corneum were stained by the Fontana Masson method.
1. Test method A solution of Fontana ammonium silver containing 5% silver nitrate (Muto Kagaku Co., Ltd.) was added dropwise to the stratum corneum sample collected in the same manner as in Test 1 for melanin staining. After standing at room temperature for 12 hours, melanin granules were selectively stained. After dyeing, the membrane was washed with purified water and observed under a microscope to take an observation image of melanin granules.
After microscopic observation, an aqueous solution of hypochlorite was added dropwise to the stratum corneum sample in the same manner as in Test 1 above. After standing at room temperature for 5 minutes, the sample was washed with purified water and then air-dried.

2. Results As shown in FIG. 2, melanin granules are stained black with the fontanammonium silver solution. However, proteins and cell walls in the stratum corneum sample were also stained at the same time (Fig. 2 pre-processing image).
On the other hand, when this sample was treated dropwise with an aqueous solution of hypochlorite, melanin granules could be more clearly observed (see the processed image in FIG. 2). Hypochlorite aqueous solution treatment was confirmed to have no effect on melanin granules.
Therefore, the granules identified in Test 1 were considered to be melanin granules.

<Test 3>
In order to confirm the applicability of the present invention, a test was conducted with a plurality of subjects.
1. Test method Using a 2.5 cm x 2.5 cm keratin checker (Asahi Biomed Co., Ltd.) as an adhesive tape, samples of the stratum corneum were collected from the inside of the forearm by tape stripping from four subjects with different skin tones and conditions. , the sample was pasted on a transparent slide glass for observation.
Sample names were given to the sample slide glasses for observation of the stratum corneum of four subjects, ID: 1, ID: 2, ID: 3, and ID: 4, respectively. This sample was observed with a microscope and an observed image was taken with a digital camera. Next, an aqueous solution of hypochlorite was added dropwise to this observation sample in the same manner as in Test 1 above. After standing at room temperature for 5 minutes, the sample was washed with purified water and air-dried to obtain a melanin granule observation sample.

2. Microscopic Observation Using a melanin granule observation sample, microscopic observation was performed and at the same time, photographing was performed with a digital camera.

3. Capture of Image Data and Processing of Binary Data Image data captured by a digital camera was captured by a computer, and image processing was performed using ImageJ.
The obtained image was converted to a grayscale image (0 to 256 gradations) using ImageJ. In order to determine the area by removing the background portion other than the granules, the background gradation was measured. As a result, the average of 4 images was 147. In order to exclude background noise more strictly, the threshold was set to 135, the image was binarized, and only the image of melanin granules was extracted. From this extracted image, the total number of pixels (pixels) of melanin granules contained in an image of 1500×1500 pixels was obtained.
This pixel number output was taken as the amount of melanin.

4. Results FIG. 3 shows the microscopic image of the collected stratum corneum sample, FIG. 4 shows the observed image of the stratum corneum sample treated with hypochlorite aqueous solution, and FIG. 5 shows the observed image after binary data processing. In FIG. 3, stratum corneum structure parts other than granules are also observed, but after the hypochlorite aqueous solution treatment shown in FIG. 4, the stratum corneum structure is slightly observed, but melanin granules are mainly observed. It turned out that there are many melanin granules in order of 4, 2, and 3. Furthermore, as shown in FIG. 5, by setting a threshold value and binarizing the image, the stratum corneum structure was not recognized, and an image in which only melanin granules were extracted was obtained.

The amount of melanin (pixel amount pixels) and the melanin area ratio (%) of each subject output from the binarized image were as follows.
ID: 1 11086 pixels, 0.49%
ID: 2 1489 pixels, 0.07%
ID: 3 1203 pixels, 0.05%
ID: 4 3201 pixels, 0.14%

The results of image observation after hypochlorite aqueous solution treatment and the order of melanin amount and melanin area ratio calculated from the binarized image were in good agreement, and individual differences in melanin amount could be detected.

<Test 4>
Comparison test with hypochlorite aqueous solution and other aqueous solutions 1. Test Method Using the stratum corneum samples collected in Test 1, the effects of other aqueous solutions were compared.
Aqueous solution used in the test Distilled water (H ₂ O), 40% urea aqueous solution (Urea: MP Biomedicals, Inc.), 30% by mass hydrogen peroxide water (H ₂ O ₂ : Fujifilm Wako Pure Chemical Industries, Ltd.), Sodium hypochlorite solution (chlorine bleach), 10 wt% sodium dodecyl sulfate (SDS) aqueous solution (MP Biomedicals, Inc.), 1N sodium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.), 12M hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was dropped on each stratum corneum sample and allowed to stand at room temperature for 15 minutes. Then, it was washed with purified water, air-dried, and then observed under a microscope.

2. Results FIG. 6 shows the observed images. In the image of the sample in which a sodium hypochlorite solution (chlorine bleach) and a 10% by mass SDS aqueous solution were dropped, the background of the observation screen changed to a uniform state, except for melanin black spots, and the areas other than melanin granules became transparent. On the other hand, in the other stratum corneum samples, the cell walls and cell structures that make up the stratum corneum were observed in various shades throughout the screen, making it difficult to distinguish melanin granules.

<Test 5>
Appropriate concentrations of SDS and hypochlorite, whose effects were confirmed in Test 4, were investigated.
1. Test Method Using the stratum corneum samples collected in Test 1, the optimal concentrations of hypochlorite and SDS were examined.
As hypochlorite, an aqueous solution of sodium hypochlorite (Fuji Film Wako Pure Chemical Co., Ltd.) with a concentration of 100 mass% diluted with purified water three times at a common ratio of 7 levels, and an aqueous SDS solution. Aqueous solutions with seven levels of concentration were prepared by diluting 20% with purified water at a two-fold common ratio. Each of these aqueous solutions and purified water was dropped onto the stratum corneum, allowed to stand at room temperature for 15 minutes, washed with purified water, and observed under a microscope.

2. Results FIG. 7 shows an image obtained using SDS, and FIG. 8 shows a microscopic image of sodium hypochlorite. SDS at a concentration of 5% by mass or more and sodium hypochlorite at a concentration of 1.2% by mass or more made it easy to observe melanin granules.
With 2.5% by mass of SDS and 0.4% by mass of sodium hypochlorite, melanin granules were clearly observed, although they were somewhat difficult to observe. Below this concentration there was no effect.
Therefore, the boundary values were considered to be 2.5% by mass for SDS and 0.4% by mass for sodium hypochlorite.

Claims (8)

A method for preparing a stratum corneum sample for microscopic observation, which comprises treating a stratum corneum sample with a solution of a hypochlorous acid compound and/or sodium dodecyl sulfate to make stratum corneum components other than melanin transparent. 2. The preparation method according to claim 1, wherein the stratum corneum sample is obtained by a tape stripping method. 3. The preparation method according to claim 1 or 2, wherein the hypochlorous acid compound is sodium hypochlorite. 4. The preparation method according to claim 3, wherein the concentration of sodium hypochlorite is 0.4% by mass or more. The preparation method according to any one of claims 1 to 4, wherein the concentration of sodium dodecyl sulfate is 2.5 to 20% by mass. An evaluation method for evaluating the state of melanin granules in the stratum corneum of the skin by microscopically observing the stratum corneum sample prepared by the method according to any one of claims 1 to 5 . The amount of melanin in the stratum corneum is determined by converting the microscopic observation image of the stratum corneum sample prepared by the method according to any one of claims 1 to 5 into black and white binary data, and obtaining the pixel values of the black data. how to measure. A method for analyzing skin condition based on melanin granule amount data obtained from a sample for microscopic observation of a stratum corneum sample prepared by the method according to any one of claims 1 to 5 .

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