KR20090053478A - Method for evaluating the scalp disruption by quantitatively analyzing and visually expressing the thickness of the stratum corneum - Google Patents

Abstract

The present invention relates to a method for evaluating the extent of damage to the scalp by measuring the thickness of the stratum corneum due to mild skin irritation under actual use conditions of scalp cleaning products such as shampoos containing a large amount of surfactant, and more specifically, To assess the extent of scalp damage by objectively and visually measuring the extent of damage to the stratum corneum using an in vivo confocal microscopy and image analysis program. will be.

Shampoos, scalp cleaning products, scalp, in vivo laser confocal microscopy, image analysis, wash-off

Description

Method for evaluating the scalp disruption by quantitatively analyzing and visually expressing the thickness of the stratum corneum}

The present invention relates to a method for evaluating the extent of damage to the scalp by measuring the thickness of the stratum corneum due to skin irritation under actual use conditions of scalp cleaning products such as shampoos containing a large amount of surfactant, and more specifically, conventional patch To improve the patch test and to evaluate the extent of scalp damage by objectively and visually measuring the extent of corneal layer damage using in vivo confocal microscopy and image analysis programs.

The skin can be roughly divided into epidermis, dermis and subcutaneous tissue. The epidermis, the outermost layer of skin, consists of four or five layers: the stratum corneum (keratin layer), the transparent layer, the granular layer, and the polar layer ( prickly layer and basal layer. The first role of the outermost stratum corneum is to suppress the evaporation of moisture from the skin to the outside, and to protect the skin against damage, substances and microorganisms caused by the external environment.

The stratum corneum is stacked vertically and consists of corneocytes of various sides and is surrounded by a matrix of lipid-enriched membranes. So it consists of a system of two zones, like a brick wall. Corneocytes do not have lipids or organelles, but are filled with structural proteins (keratin filament) and osmotically active small molecules.

The matrix of corneocytes consists of hydrophobic lipids arranged in multiple lamellar sheets. There is a lipid-bound envelope between the lamellar membrane and the corneocytes. In the cytoplasm of corneocytes, there are many keratin (mainly keratin-1 and -10) and filaggrin. Of these, keratin, the main component, is insoluble as a fibrous protein. Keratin-10 is acidic and keratin-1 is basic. About 30% of the cytoplasm of living cells consists of keratin, and nearly 90% of keratin in dead cells.

The hydrophobic and polar action of surfactants, which are found in the wash-off family of cleaning products such as shampoos, mainly serve to remove dirty substances from the surface of the skin, but have unwanted side effects on the skin. It may cause. In this way, the surfactant damages the skin by acting on the surface of the stratum corneum, which is the outermost layer of the skin. Since 70% of the dry matter content of the stratum corneum is a protein, the surfactant is likely to denature the secondary structure of the protein, which may damage the lipid ultrastructure of the stratum corneum. In addition, the delamination of the intercellular lipid lamellae leads to the separation of the corneocytes, resulting in loosening of the stratum corneum structure. Done.

A common method used to evaluate skin safety of wash-off products, such as shampoos and scalp cleaning products, is to dilute the product to a certain concentration to perform a patch test and cause erythema caused by skin ( erythema, edema and dryness. However, such a patch test method through dilution does not reflect the actual conditions of use of the cleaning product and is only affected by the total amount of the total surfactant of the product. The degree of skin irritation is determined by the amount of residual skin of the surfactant that denatures the stratum corneum by binding to the protein of the skin, rather than being washed in water after washing, rather than the total amount of the total surfactant in actual use conditions. Because. In addition, inflammatory reactions (erythema, redness and swelling, etc.) that can be visually seen in the strong irritation reactions by patch tests, etc. appear on the skin, but in the slight irritation reactions that can be caused by the actual conditions of use of the cleaning product Often there is no response. However, even these minor irritants can affect long-term cumulative or sensitive skin. In particular, the scalp is a structural feature of hair follicles and sebaceous glands are very large, so the effects of skin application or absorption of the skin irritation is very different from other parts of the skin such as forehead and face.

Weak damage to the surface of the skin under the actual conditions of use of the scalp cleaning products such as shampoos is limited by the method of visual evaluation, so to measure the transepidermal water loss (TEWL) A method of evaluating the barrier function of the skin using a measuring method is widely used. This method observes the phenomenon of passive diffusion of moisture from the inside of the skin to the outside and has been reported to be correlated with the degree of skin damage.

There is also a method using transmission electron microscopy (TEM) to measure the thickness of the stratum corneum on the skin surface. However, this method has the disadvantage of invasive biopsy of the test site.

Accordingly, the present inventors have tried to find a method that can evaluate the degree of scalp damage in order to present more scientific and objective data on the extent of damage to the stratum corneum of the skin surface that can be caused by the actual use conditions of scalp cleaning products such as shampoos, An image is taken from the skin surface to the dermis using a non-invasive method called in vivo confocal microscopy, and the thickness of the stratum corneum is measured objectively using an image analysis program. The present invention was completed by discovering that the extent of skin damage can be evaluated.

Accordingly, an object of the present invention is to provide a method that can objectively evaluate the extent of scalp damage by measuring the increase in the thickness of the stratum corneum due to the visually minor damage of the skin surface through a bioconfocal laser scanning microscope and an image analysis program. .

In order to achieve the above object, the present invention comprises the steps of: 1) obtaining a plurality of photographed images by successively photographing at regular intervals in the vertical down direction from the scalp surface to the subepidermal layer using a biological confocal laser scanning microscope; And 2) visualizing the thickness of the stratum corneum layer on the surface of the scalp by setting an analysis region of the photographed images obtained in step 1) and image processing using an image analysis program. .

The present invention also provides a method for evaluating scalp damage through a method of evaluating the scalp damage described above before applying the wash-off product for scalp cleaning; And 2) after applying the product, evaluating the degree of scalp damage by the method of evaluating the degree of scalp damage described above. .

In the present invention, it is possible to measure the image of the stratum corneum on the surface of the scalp due to minor skin damage by using the data of the confocal laser scanning microscope. The degree of scalp damage of the scalp cleaning product can be evaluated by the evaluation.

In vivo confocal microscopy used in the present invention is a non-invasive skin microimaging device that checks the state of the epidermis and dermis in a natural state without biopsy, staining, or incision of living skin. It is designed to observe dynamic changes in living tissues with high resolution and to obtain effective images of inflammatory cell infiltration and blood flow from the stratum corneum to the reticular layer of the epidermis through the transverse section in real time. .

In the present invention, the skin using a 830nm diode laser and Vivascope 1500 (Lucid Inc, NY), a commercially available confocal microscopy in vivo, having a maximum power of 25 mW at the skin surface. The horizontal image of was measured, but not limited thereto, and water immersion (30X) / oil immersion (60X) objective lense was used. The best image is obtained when the immersion index (n) is the median immersion of the epidermal layer (n = 1.34).

The thickness of the stratum corneum was determined by using an image analysis program and a data analysis program of 500 μm × 500 μm images obtained by continuously photographing the surface of the skin at 1 μm vertically from the surface of the skin using the Vivascope 1500.

The image analysis program Image J used in the present invention is image analysis software that allows easy editing, image processing, data extraction, and automatic analysis of a given image. The image obtained by using an external input device can be image-processed to change and improve to a desired form, thereby enabling data measurement and processing storage of the image.

In the present invention, in order to evaluate the extent of damage to the stratum corneum of the skin surface that may be caused by the actual use conditions of the scalp cleaning product to evaluate the extent of damage to the skin stratum corneum after treating the product for 2 weeks similar to the actual conditions of use on the scalp surface. The surface of the skin is photographed using a non-invasive method, a bio-confocal laser scanning microscope, the analysis area of the photograph is taken, and the image analysis program Image J (NIH, USA) is introduced. The extent of scalp damage according to the thickness was evaluated objectively. In addition, in order to compare the scalp surface damage caused by each product, the transdermal moisture evaporation (TEWL), which is a commonly used method, was measured and compared with the quantitative results using a bioconfocal laser scanning microscope.

Through this, it was possible to evaluate the thickness of the stratum corneum due to weak skin damage in actual use conditions of scalp cleaning products such as shampoo that could not be evaluated in the conventional patch test, and the degree of scalp damage that was dependent on the subjective evaluation by the existing subjects. Can be numerically objectified and visualized.

In addition, the stronger the irritation to the scalp, the more severe the damage, so the method of evaluating the scalp damage before and after applying the scalp wash wash off product, respectively, the difference in the resulting damage Through the scalp cleaning washoff product to evaluate the scalp irritation can be evaluated.

Through the following examples and comparative examples, the present invention provides a method for evaluating the degree of skin damage by objectively and visually measuring the thickness of the stratum corneum due to the scalp surface damage through bioconfocal laser scanning microscopy and image analysis. Although demonstrated, this invention is not limited only to these Examples.

Reference Examples 1 and 2 Application of Shampoo Products Considering Actual Use Conditions

The hair follicle and sebaceous glands were performed to reflect the actual conditions of use of Shampoo, a scalp cleansing product, to reflect the characteristics of the scalp. Nine men and women in their twenties and thirties were applied with different types of products on both sides of the head, rubbed for one minute to form bubbles, and washed well with a shower. Products were randomly applied. After the treatment for 2 weeks, 10 times a day, 5 days a week, a bioconfocal laser scanning microscope and transdermal moisture evaporation (TEWL) were taken and followed.

1. Wet the whole hair with water, apply 300µl of the product on both heads, and rub for 1 minute to make bubbles.

2. Rinse for 1 minute with a shower to remove the cleaning product.

3. The hair dryer removes much of the water remaining in the scalp and hair for 30 seconds.

4. The product was treated once daily, 5 days a week, for the last 2 weeks.

Composition state of the sample (unit: wt%) ingredient Reference Example 1 Reference Example 2 Cationic surfactant 0.5% 0.3% Anionic surfactant 30% 73% Zwitterionic surfactant 25% - Cationic polymer 0.25% - Viscosity modifier - 0.45% pH regulator 0.94% 0.08% Distilled water Remaining amount Remaining amount

[Comparative Example 1] Method for measuring the extent of damage to the stratum corneum by using a transdermal moisture evaporation (TEWL) measuring method

In order to compare the degree of skin surface damage according to scalp cleaning products such as shampoo, scalp surface was measured by using keratin moisture evaporation (TEWL).

Before and after the two-week treatment of Reference Examples 1 to 2, the same subjects were fixed on both sides of the hair on both sides of the scalp with a hairpin to expose the scalp, followed by constant temperature and humidity conditions (temperature 14 ± 4 ℃, relative humidity 30). ± 2%) for 15 minutes. VaporMeter SWL-2 (Delfin, Finland) was used to measure the transdermal moisture evaporation (TEWL) at each test site.

The difference between the percutaneous water evaporation amount (TEWL) before and after the two-week treatment of Reference Examples 1 and 2 was calculated as shown in Equation 1, and the change amount (△) of the percutaneous water evaporation amount of each test site was compared.

1 is a graph comparing the change amount? Of the transdermal moisture evaporation amount of each sample. Two weeks after the sample treatment, the skin damage of Reference Example 1 was the highest compared with Reference Example 2. The extent of skin damage by each sample was determined through the measurement of the conventional transdermal moisture evaporation (TEWL), and compared with the results of the new evaluation method according to the present invention.

Example 1 Scalp Surface Measurement Method Using a Confocal Laser Scanning Microscope and an Image Analysis Program

1) Analysis of bioconfocal laser scanning microscope

It was confirmed through Comparative Example 1 that the degree of skin damage for each sample is different. However, the method of measuring the amount of transdermal moisture evaporation can quantify the degree of skin irritation, but there is a limit that it cannot visualize the extent of skin damage.

There is also a method using transmission electron microscopy (TEM) to measure the thickness of the stratum corneum on the skin surface. However, this method has the disadvantage of invasive biopsy of the test site. Therefore, in order to overcome the limitations of the measurement of transdermal moisture evaporation and TEM method, a method that can be quantified and visualized was developed.

Skin surface was photographed using a commercially available bio-confocal laser scanning microscope, Vivascope 1500 (Lucid, USA) to quantitatively and visually observe the thickness of the stratum corneum due to minor damage to the scalp surface after using a scalp cleaning product such as shampoo. .

The imaging method using the biological confocal laser scanning microscope is as follows:

After two weeks of treatment of the sample of Comparative Example 1, the same subjects were subjected to nine subjects at the same time and fixed to both sides of the hair on both sides of the scalp with a hairpin to expose the scalp, followed by constant temperature and humidity conditions (temperature 10 ± 2 ° C, relative humidity) 30 ± 2%) for 15 minutes. First, transdermal moisture evaporation (TEWL) was measured by a vaporometer, distilled water was loaded at the center of each test site, and an adhesive ring was attached. The recording was started by loading a large-sized ultrasound transmission gel on the ring attached to the skin and fixing the lens area. Skin tissue findings were measured by scanning depth from surface to depth.

It is designed to determine the measurement start point by adjusting the Z-depth (Z-depth 0㎛) of the lens so as to shoot from the outermost layer of the skin and to scan the Z-depth of the lens vertically at regular intervals (1㎛). A VivaStack profile was run. 2 is a configuration data of scanning the scalp surface by adjusting the Z-depth of the lens. The difference between the extent of damage and the thickness of the outermost surface of the stratum corneum was not visually different for each sample. This can be seen that the results similar to the results of the method for measuring the degree of skin damage by each sample through the measurement of transdermal moisture evaporation (TEWL) of the prior art.

2) Analysis of Bioconfocal Laser Scanning Microscopy Data Using Image Analysis

An image analysis program was introduced to objectively evaluate the constituent data obtained with a bioconfocal laser scanning microscope.

The basic principle is to quantify by using the principle that the gray value of the image increases as the index value of each epidermal layer is stacked by stacking the pictures taken by the bio-confocal laser scanning microscope. Evaluation method through image analysis of the image analysis program Image J (NIH, USA) is shown in Table 2 below.

Skin irritation analysis according to the invention and the difference between Comparative Example 1 Example 1 How to measure Visual judgment, TEWL Biometric Confocal Laser Scanning Microscope Analysis method Comparison of delta values before and after the test Objectively assess the thickness of the stratum corneum by image analysis 1. Import the photographs of the stratum corneum in a vertical direction. 2. Cut each image (X-Y axis) at regular intervals and stack them vertically (Z axis) through the reconstruction process. 3. Gaussian Blur is applied to compensate the gap between Z-axis images. 4. Set the Z-axis direction using the linear tool to determine the part to measure. 5. Adjust the set scale to compensate for the actual measurement interval and the distance between each pixel. 6. Run the Plot profile to calculate the gray value by depth. 7. Copy the entire plot value. 8. Calculate the thickness of the stratum corneum using the Excel program to find the location where the gray value is the largest.

3 is a diagram illustrating an algorithm of the above-described image analysis program on a program, and shows a detailed analysis method of Embodiment 1 in order. In addition, Figure 4 shows the visualization of the thickness of the stratum corneum layer by the skin surface image and image analysis program of the biological confocal laser scanning microscope. As can be seen in Figure 4, when using the reference example 1, the change in the thickness of the stratum corneum is greater, so the keratin damage is greater and accordingly the reference example 1 can be said to be a more severe product.

The stratum corneum contains a large amount of keratin, which shows high gray values compared to other parts of the skin. The boundary between the stratum corneum and the other layer is the part where the gray value of the vertical Z axis is compared and the gray value is relatively large. The thickness of the stratum corneum was measured by calculating the change in the gray value of the vertical layer, and through this, it was possible to quantify and compare the degree of damage to the scalp surface according to the treatment sample based on a more accurate basis.

5 shows the results of quantitatively analyzing the thickness of the stratum corneum according to the shampoo type, which is a scalp cleaning product. After 2 weeks, as in the result of keratin evaporation (TEWL), it was confirmed that the thickness of keratin thick due to skin damage of Reference Example 2 was the highest. The skin irritation evaluation of scalp cleaning products considering the existing actual conditions of use has to be applied for a long time and it is difficult to judge visually as it causes a slight irritation when applied for 2 weeks. In addition, there was an existing evaluation method called TEWL measurement, but it was not possible to visualize it for easy understanding. In addition, the method of measuring the thickness of the stratum corneum on the skin surface by the TEM method has a disadvantage in that the biopsy of the test site is performed directly. However, the method of measuring thickness of the stratum corneum layer by scrutinizing the data of the noninvasive bioconfocal laser scanning microscope can visualize and objectively quantify the thickness of the stratum corneum due to skin damage. Considering the actual conditions of use

The advantage is that the test period can be shortened.

Existing skin damage evaluation method and skin damage degree evaluation method according to the present invention has the difference as shown in Table 3, through which it was confirmed that there is an efficiency of the accuracy and visualization of the evaluation method, the time reduction.

Difference between keratin exfoliation evaluation method and the existing method according to the invention Comparative Example 1 Comparative Example 2 (Prior Art) Example 1 Application method Patch test  - Wash-off processing How to measure TEWL TEM Biometric Confocal Laser Scanning Microscope Analysis method Comparison of delta values before and after the test, no visualization Tissue analysis via biopsy Visualization through objective image analysis, objective evaluation

FIG. 6 compares the results obtained by measuring the transdermal moisture evaporation (TEWL) by the method of Comparative Example 1 after treating Reference Examples 1 and 2 for 2 weeks and using the confocal laser scanning microscope by the method of Example 1. As a graph, it was confirmed that the thickness of the stratum corneum caused by the skin damage weaker than Comparative Example 1 can be visualized and quantified through the method of Example 1 according to the present invention.

1 is a graph comparing the change amount (D) of the transdermal moisture evaporation amount of each sample before and after carrying out Reference Examples 1 and 2. FIG.

2 is image data of the scalp surface scanned with a bioconfocal laser scanning microscope.

Figure 3 shows the process of measuring the thickness of the keratin after the schematic analysis method of the vertical image of the bio-confocal laser scanning microscope through the image analysis program Image J.

Figure 4 shows that the image of the in vivo confocal microscope using the image analysis program Image J to visualize the reconstructed vertically (Z-axis) to measure the thickness of the stratum corneum and measured the thickness using an Excel program.

Figure 5 is a graph showing the results of analyzing the image of the scalp surface thickness measured by a biological confocal laser scanning microscope photographed after treatment for Reference Examples 1, 2 for 2 weeks.

6 is a graph comparing the TEWL results (Comparative Example 1) and the confocal laser scanning microscope results (Example 1) after treating Reference Examples 1 and 2 for 2 weeks.

Claims (3)

1) obtaining a plurality of photographed images by successively photographing at regular intervals in a vertical downward direction from the scalp surface to the subepidermal layer using a biological confocal laser scanning microscope; And 2) visualizing the thickness of the stratum corneum layer on the scalp surface by setting an analysis region of the photographed images obtained in step 1) and performing image processing using an image analysis program; How to evaluate the degree of scalp damage comprising a. The method according to claim 1, wherein the image processing of step 2) comprises stacking the photographed images obtained in step 1) by depth, obtaining gray values of the images in the analysis region, and quantifying the change of gray values. Method for evaluating the degree of scalp damage, characterized in that it comprises the step of measuring the thickness of the stratum corneum. 1) assessing the extent of scalp damage by the method according to claim 1 before applying the scalp wash-off product; And 2) after applying the product, evaluating the degree of scalp damage by the method according to claim 1; Method for evaluating the degree of skin irritation of the scalp cleaning washoff product comprising a.

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