KR101155194B1 - Quantitative analyzing method of melanin in corneocyte to visually express the distribution of melanin in the skin surface - Google Patents

Abstract

The present invention relates to a method for quantifying melanin of keratinocytes that can visually express the melanin distribution on the skin surface. More specifically, the method for quantifying melanin of keratinocytes according to the present invention, after collecting keratinocytes from the surface of the skin, performs melanin staining, then imaging them and imaging melanin spots per keratinocyte using image analysis technology. By quantifying the area of, it is possible to express visually and quantitatively the amount of melanin presented only by existing values.

Melanin Determination * Keratinocytes * Melanin Staining * Image * Melanin Spot

Description

Quantitative analyzing method of melanin in corneocyte to visually express the distribution of melanin in the skin surface}

1 is a diagram showing the melanin synthesis process.

Figure 2 is a diagram showing the melanin staining process used in the present invention.

Figure 3 is an image of the melanin distribution ratio according to the present invention.

Figure 4 is a picture showing the skin color measurement site and the stratum corneum collection site in the present invention.

5 is a diagram showing a process for quantifying melanin using the BMI software in the present invention.

6 is a diagram showing an example of calculating the area ratio of melanin spots in the BMI software in the present invention.

7 is a result of measuring the correlation between the skin color (L) and the melanin area ratio per keratinocyte measured by the spectrophotometer using the statistical program of SPSS version 10.

The present invention relates to a method for quantifying melanin of keratinocytes that can visually express the melanin distribution on the skin surface. More specifically, the method for quantifying melanin of keratinocytes according to the present invention, after collecting keratinocytes from the surface of the skin, performs melanin staining, then imaging them and imaging melanin spots per keratinocyte using image analysis technology. By quantifying the area of, it is possible to express visually and quantitatively the amount of melanin presented only by existing values.

Melanin plays an important role in protecting human skin from UV rays while determining skin color. Detailed mechanisms in which melanin is made in melanocytes (melanocytes) in the basal layer of skin are shown in FIG. 1.

Irradiation of melanocytes with UV rays increases the synthesis of various epidermal cytokines, growth factors, and hormones, which are associated with the proliferation and proliferation of melanocytes. It affects melanogenesis. When tyrosinase is activated in signaled melanin forming cells, tyrosinase forms melanin in the melanosomes. The synthesized melanin migrates to keratinocytes. Keratinocytes are constantly dividing in the basal layer of the skin to rise to the stratum corneum, at which time melanin from the surrounding melanocytes are delivered. Keratinocytes rise to the stratum corneum and become keratinocytes, and melanin is released from the skin when keratinocytes are released according to the regeneration cycle.

When the skin is irradiated with UV rays, the synthesized melanin is collected in a manner surrounding the nucleus of keratinocytes to reflect or absorb the UV rays. This plays an important role in preventing UV light from reaching the nucleus of keratinocytes and modifying the genes.

However, excessive production of melanin can make the skin look dull and cause hyperpigmenatation disorders such as freckle and freckles. Therefore, the cosmetics industry has been developing whitening products using tyrosinase inhibitors that prevent tyrosinase from functioning or substances that interfere with the mechanism by which keratinocytes receive melanin. .

In vitro tests are also conducted to confirm the whitening efficacy of the developed ingredients or cosmetics.However, the final method is to apply the skin directly to human skin and observe the changes in skin color. In these studies, there are two representative ways to use equipment that can measure skin color. The skin color is measured by melanin index and erythema index, and the skin color is evaluated by multi-wavelength skin reflectance. Applicable devices include colorimeters (Mexameter, CK, Germany) and spectrophotometers (CM-2002, Minolta, Japan).

However, the above-mentioned measuring devices are easily measurable, but have a disadvantage in that the whitening effect can only be verified with numerical results.

Therefore, the present inventors performed the melanin staining by collecting the keratin of the skin surface and visualizing it as an image to quantify the area of the melanin spot per keratinocyte by image analysis technology, which is presented only as a conventional numerical result. The present invention was completed by discovering that the amount of melanin can be expressed visually.

Accordingly, an object of the present invention is to provide a method for quantifying melanin of keratinocytes that can visually express the melanin distribution on the skin surface.

In order to achieve the above object, the method for quantifying the melanin of keratinocytes according to the present invention, (a) collecting the keratin of the skin surface; (b) performing melanin staining on the collected keratin; And (c) observing the stained keratin sample under a microscope, classifying the image by taking images, and quantifying the area of melanin spots per keratinocyte.

Hereinafter, a method for quantifying melanin of keratinocytes according to the present invention will be described in detail step by step.

(a) collecting keratin from the skin surface.

In this step, the keratin of the skin surface is collected by a tape stripping method.

(b) performing melanin staining on the collected keratin.

In this step, melanin staining is carried out by applying the Fontanna Masson staining method using the argentaffin reaction of melanin.

The process of the melanin staining is shown in step 2, and the manufacturing process of the ammonia-treated silver working solution (Ammoniacal Silver Working Solution) used in the dyeing process is shown in Table 1 below.

Manufacturing Process of Ammoniacal Silver Working Solution reagent Way 10% silver nitrate solution Add 5g of silver nitrate to 50ml of distilled water, mix well, and place in acid cleaned brown bottle Ammoniacal silver solution 4 drops of ammonia water in 25 ml of silver nitrate solution (10%) to precipitate the solution, and then leave for 4 to 24 hours to allow the precipitate to sink and the supernatant to be clear. Ammoniacal Silver Working Solution 37.5 ml of distilled water is added to 12.5 ml of ammoniacal silver solution. At this time, only the supernatant is used and filtration is performed.

(c) observing the stained keratin sample under a microscope, classifying by taking images and quantifying the area of melanin spots per keratinocyte.

After confirming that the melanin spots distributed in keratinocytes were dyed in black, the keratin sample stained in step (b) was photographed with a high resolution digital camera. The area of melanin spots per keratinocyte is then quantified using BMI image analysis software. At this time, the area ratio of melanin spot per keratinocyte is calculated by the following equation (1).

Melanin spot area ratio = Melanin spot area / keratinocyte area × 100

In addition, the area ratio of the melanin spot can be classified into 5 grades according to the melanin distribution ratio per keratinocytes as shown in Table 2, for example. In addition, the melanin distribution ratio of Table 2 is as shown in Figure 3 when the image, based on the following Table 2 and Figure 3 can be analyzed the efficacy of the cosmetics through the changed grade comparison before and after the application of the whitening product and the whitening product have.

In other words, before the application of the whitening product was a grade 5 of Table 2, but after the application of the grade 3 can be determined that the efficacy of the whitening cosmetics.

Melanin distribution ratio per keratinocyte Rating Melanin distribution ratio per keratinocyte One 0-5% melanin distribution in keratinocytes 2 5-10% melanin distribution in keratinocytes 3 10-20% melanin distribution in keratinocytes 4 20-40% melanin distribution in keratinocytes 5 More than 40% melanin distribution in keratinocytes

SPSS version 10 statistics to determine the correlation between skin color (L) and melanin area ratio per keratinocyte measured by the spectrophotometer CM-2002 (Minolta, Japan), which is generally used to measure the amount of melanin. Check using the program. Through this, it can be seen that the melanin area ratio and skin color (L) of keratinocytes have a significant correlation, and the skin color can be represented by the melanin area ratio of keratinocytes therefrom.

Hereinafter, the present invention will be described in more detail with reference to test examples, but the present invention is not limited only to these examples.

Test Example 1 Method for Measuring Skin Color (L) on Skin Surface

After washing the face with soap for 15 subjects, the skin was adapted for 15 minutes under constant temperature and humidity conditions (20 ± 2, relative humidity 40 ± 2 ° C). Next, using a spectrophotometer CM-2002 (Minolta, Japan), the cheek area indicated by the circle of Figure 4 was repeated three times. The skin color (L) was obtained by obtaining the average value of the measured values, and the results are shown in Table 3 below.

L measurement One 55.32 2 58.39 3 59.92 4 61.84 5 60.75 6 56.53 7 60.43 8 54.53 9 60.21 10 60.31 11 59.84 12 60.54 13 60.73 14 56.78 15 61.33

Test Example 2 Corneal Layer Collection and Melanin Staining Method

After the skin color measurement of Test Example 1 was performed, keratinocytes were taken from the same site as the measurement site of Test Example 1 using D-squame (Cuderm, USA) having a constant adhesiveness on 15 subjects.

Next, the collected keratin was melanin staining by the method applied to Fontanna masson staining in the process shown in FIG.

Test Example 3 Method for Quantifying Melanin Per Keratinocyte

The keratinocyte sample stained in Test Example 2 was confirmed by using a microscope (Nikon Eclipse 80i) melanin spots (spots) distributed in the keratinocytes black. This image was then taken 400 times with a high resolution digital camera and melanin spot area per keratinocyte was quantified by the steps described in FIG. 5 below on a BMI image analysis software. An example of calculating the area ratio of melanin spots on the BMI image analysis software is shown in FIG. 6, and the area ratio of melanin spots per keratinocyte is calculated according to Equation 1 shown in Table 4 below with L measurements It was.

[Equation 1]

Melanin spot area ratio = Melanin spot area / keratinocyte area × 100

From the example of FIG. 6 below, when the area ratio of melanin spot was calculated, it was calculated as "Melanin spot area / keratinocyte area x 100 = 2574 / (200 x 160) x 100 = 8.04375".

L measurement Melanin spot area ratio One 55.32 29.38 2 58.39 22.87 3 59.92 24.66 4 61.84 13.77 5 60.75 13.61 6 56.53 35.55 7 60.43 19.84 8 54.53 33.59 9 60.21 22.38 10 60.31 16.17 11 59.84 14.09 12 60.54 12.54 13 60.73 5.92 14 56.78 43.52 15 61.33 6.26

[Test Example 4] Relationship between skin color (L) value obtained by spectrophotometer and melanin area ratio per keratinocyte

Correlation analysis and regression analysis were performed using a statistical program of SPSS version 10 based on the skin color (L) and melanin area ratio per keratinocyte obtained in Test Examples 1 to 3. The results are shown in Table 5 and FIG. 7.

As shown in Table 5 and FIG. 7 below, Spearman's correlation analysis revealed that the melanin area ratio per skin color and keratinocytes had a negative correlation of −0.830. Further, a regression equation was established between the melanin area ratio and the skin color (L): "Melanin amount = 255.076-3.957 x skin color (L) [R = -0.830]". Therefore, the melanin area ratio per keratinocyte measured by the present invention has a negative correlation with the skin color (L), and thus it was confirmed that the skin color can be represented by presenting an image stained with melanin in the keratinocytes.

As described above, in the method for quantifying melanin of keratinocytes according to the present invention, after collecting keratinocytes from the surface of the skin, melanin staining is performed and then imaged and imaged. By quantifying the area of), it is possible to express visually and quantitatively the amount of melanin presented by the existing numerical values only.

Therefore, by expressing the whitening effect in the advertising of cosmetics through the melanin staining results, the general public can easily recognize the efficacy of the product. In addition, by obtaining a skin surface keratin sample of the cosmetic user after the melanin staining and giving a grade, there is an advantage that can present the user's skin melanin state change before and after use.

Claims (3)

delete (a) collecting keratin from the skin surface; (b) performing melanin staining on the collected keratin; (c) taking an image of the dyed keratin sample; (d) selecting one keratinocyte from the photographed image and converting the area of the melanin spot per keratinocyte into an area ratio per keratinocyte; (e) repeating step (d) for the other keratinocytes in the image to calculate an average value of the area ratio per keratinocyte; And (f) classifying the melanin distribution per keratinocyte into the following grades using the average value of the area ratio per keratinocyte; Rating Melanin distribution ratio per keratinocyte One 0-5% melanin distribution in keratinocytes 2 5-10% melanin distribution in keratinocytes 3 10-20% melanin distribution in keratinocytes 4 20-40% melanin distribution in keratinocytes 5 More than 40% melanin distribution in keratinocytes Method for quantifying melanin of keratinocytes comprising a. Method of quantifying melanin of keratinocytes by the method of claim 2 before and after application of whitening cosmetics, respectively, to evaluate the whitening efficacy of the whitening cosmetics by changing the melanin distribution grade.

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