JP7513287B2 - Screening method for components that affect the increase or decrease of adhesion proteins between stratum corneum cells - Google Patents

Description

The present invention relates to a method for screening components that affect the increase or decrease of adhesive proteins between stratum corneum cells, and a method for preparing a stratum corneum cell sample suitable for said screening method.

Desmosomes are a representative adhesive device for keratinocytes. Desmosomes are structures involved in adhesion between epidermal cells and between stratum corneum cells, and it is known that the adhesion between epidermal cells via desmosomes works in conjunction with the cytoskeleton, which is made up of keratin fibers, to maintain a strong structure in epithelial tissue.

Increased desmosomal proteins are known to be one of the causes of deterioration of skin conditions, such as rough skin and peeling of the stratum corneum caused by sunburn or dryness.
As a method for controlling the amount of desmosomal proteins, a method has been reported in which decomposition of desmosomal proteins accumulated in the stratum corneum with proteases is used to improve acne, dandruff, and desquamation (Patent Document 1).

The constituent proteins of desmosomes are known to be desmogleins 1, 2, 3 and 4, desmocollins 1, 2 and 3, and desmoplakins 1 and 2.
Among the constituent proteins of desmosomes, the adhesive protein desmoglein 1 in particular is closely correlated with the proteolysis of desmosome components and cell detachment. Therefore, confirming the presence of desmoglein in the stratum corneum is also an evaluation of desmosome function and is extremely important in assessing the condition of the skin.

JP 7-505383 A

In view of the above, there is a need to search for components that reduce the amount of desmosomes or the adhesive proteins that make up desmosomes.
One method for screening for such components involves collecting stratum corneum cells using a tape stripping method, transferring the cells to a slide glass to prepare a stratum corneum cell sample, staining the adhesive proteins using an immunohistochemical staining method, and observing the sample under any microscope.

By the way, when transferring stratum corneum cells using the tape stripping method, components of the adhesive tape may remain on the slide glass, and may become stained and become indistinguishable from adhesive proteins, so it is necessary to clean the slide glass.
However, when the slide glass was washed, the transferred stratum corneum cells were also peeled off, making it difficult to evaluate the amount of adhesive protein present in the stratum corneum cells as an indicator, and therefore this could not be said to be a sufficient screening method.

An object of the present invention is to provide a screening method for components that affect the increase or decrease of adhesive proteins present between stratum corneum cells, including desmoglein, which is more accurate than conventional methods.
A further object of the present invention is to provide a method for preparing a keratinocyte sample for more accurately evaluating the amount of adhesion protein present in the above-mentioned screening method.

The present invention solves the above problems,
A method for screening for a component that affects an increase or decrease in adhesion protein between stratum corneum cells, comprising the steps of:
A step of collecting stratum corneum cells by tape stripping;
a transfer step of transferring the stratum corneum onto a slide glass having a positively charged surface;
a washing step of washing the slide glass;
A preparation step of adding a target component to the slide glass, staining the adhesion protein by immunohistochemical staining, and preparing a screening sample;
an image acquiring step of acquiring an image for analysis by photographing an arbitrary portion of the screening sample;
The method further includes an evaluation step of evaluating the inhibitory effect of the target component on the formation of a compact stratum corneum using the area of the stained portion in the analysis image as an index.

It is known that the cell surface has a negative charge (COO ⁻ ) due to the carboxyl groups of phospholipids.
According to the screening method of the present invention, by transferring stratum corneum cells onto a slide glass with a positively charged surface, it is possible to prevent the stratum corneum cells from being peeled off by washing, and it is possible to more accurately evaluate the amount of adhesive protein present in the stratum corneum cells.

In a preferred embodiment of the invention,
In the preparation step, a screening control sample to which the target component to be searched is not added is prepared,
The evaluation step is a step of comparing the area of the stained portion of the screening sample with the area of the stained portion of the screening control sample, and if the area of the stained portion of the screening sample is smaller, evaluating that the component to be searched for has the effect of reducing the amount of adhesive protein.
By using such a form, it is possible to more accurately screen for components that affect the increase or decrease of adhesive proteins.

In a preferred embodiment of the invention,
In the preparation step, a plurality of screening samples are prepared in which the type of the search target component is the same but the concentrations are different;
The evaluation step is a step of comparing the areas of the stained portions of a plurality of the screening samples and evaluating the optimum concentration for the effect of reducing the amount of adhesion protein.
By using such a form, it is possible not only to screen for components that have the effect of reducing the amount of adhesive protein, but also to evaluate their optimal concentration.

In a preferred embodiment of the present invention, the adhesion protein is desmoglein 1.

In a preferred embodiment of the present invention, the slide glass has NH ₃ ⁺ arranged on the surface thereof.
NH ₃ ⁺ forms an ionic bond with COO ⁻ on the cell surface, and can therefore improve the adhesiveness of stratum corneum cells to the slide glass.

In a preferred embodiment of the present invention, the washing step is a step of immersing the slide glass in xylene.
By cleaning the slide glass by immersing it in xylene, peeling of the stratum corneum cells can be further suppressed.

Further, the present invention which solves the above-mentioned problems is as follows:
A method for preparing a keratinocyte sample using a tape stripping method, comprising the steps of:
a transfer step of transferring the stratum corneum onto a slide glass having a positively charged surface;
and a cleaning step of cleaning the slide glass.
By transferring the stratum corneum onto a slide glass having a positively charged surface, it is possible to prevent detachment of the stratum corneum cells from the slide glass during the subsequent washing step.

In a preferred embodiment of the present invention, the slide glass has NH ₃ ⁺ arranged on the surface thereof.

According to the screening method of the present invention, components that affect the increase or decrease of adhesion proteins can be screened more accurately than conventional methods.
Furthermore, according to the method for preparing keratinocytes of the present invention, detachment of keratinocytes from a slide glass can be suppressed.

1 is a flowchart showing the screening method of the present embodiment. 1 is a flow chart showing the steps of preparing a keratinocyte sample in the present embodiment. 3 is a flowchart showing an evaluation process in the present embodiment. 1 shows microscopic images of a screening sample in which a wild thyme extract and a togen pear extract were added as search target components to a stratum corneum cell sample, and a screening control sample. 1 is a graph showing the area ratio of the stained portion of the screening sample when the area of the stained portion of the screening control sample is taken as 100%. 1 is a diagram (representative) showing the measurement results of the amount of desmoglein 1 in Example 2. 1 is a graph showing the measurement results of the amount of desmoglein 1 in Example 2. 1 is a graph showing the measurement results of L ^* value in Example 3. 1 is a graph showing the measurement results of a ^* value in Example 3. 1 is a graph showing the measurement results of b ^* value in Example 3. 13 is a graph showing the results of hardness measurements in Example 3. 1 is a graph showing the measurement results of the amount of desmoglein 1 in Example 3. FIG. 1 is a representative diagram showing the measurement results of the amount of desmoglein 1 in Example 3. FIG. 1 is a representative diagram showing the measurement results of the amount of desmoglein 1 in Example 4. 1 is a graph showing the measurement results of the amount of desmoglein 1 in Example 4. 13 is an optical microscope image showing the peeling of stratum corneum cells in Example 4. FIG. 1 is a representative diagram showing the measurement results of the amount of desmoglein 1 in Example 5. 1 is a graph showing the measurement results of the amount of desmoglein 1 in Example 5 (6 weeks). 1 is a graph showing the measurement results of the amount of desmoglein 1 in Example 5 (12 weeks).

Below, the screening method of the present invention will be described in detail with reference to the flowcharts in Figures 1 to 3.

The screening method of the present invention comprises a stratum corneum cell sample preparation step S10, a screening sample preparation step S20, an image acquisition step S30, and an evaluation step S40.

(1) Corneal stratum corneum cell sample preparation step FIG. 2 is a flow chart showing step S10 in detail.
Step S10 is followed by a collection step S11 in which stratum corneum cells are collected by tape stripping.
The tape stripping method is a general term for a method of exfoliating skin cells by attaching a pressure-sensitive adhesive, such as adhesive tape, to the skin and then peeling it off.
The pressure-sensitive adhesive to be used is not particularly limited, but from the viewpoint of simplicity, it is preferable to use cellophane tape.

Next, a transfer step S12 is performed in which the stratum corneum cells collected in step S11 are transferred to a slide glass whose surface is positively charged.

The surfaces of human cells such as keratinocytes are negatively charged due to the influence of carboxy groups (COO ⁻ ) of the phospholipids that form the lipid bilayer membrane.
In step S12, the stratum corneum cells are transferred to a slide glass having a positively charged surface, so that the stratum corneum cells are less likely to peel off from the slide glass due to electrostatic interaction.

Examples of the slide glass having a positively charged surface include slide glass whose surface is modified with polylysine, polyalkylamine, amino-modified silicone, and the like.
In the present invention, it is preferable to use a slide glass on whose surface NH ₃ ⁺ is arranged.
NH ₃ ⁺ forms an ionic bond with the carboxyl group of the phospholipid, making it more difficult for the stratum corneum cells to peel off from the slide glass.

As the slide glass having NH ₃ ⁺ arranged on the surface thereof, commercially available products can be used, such as "PLL coated slide glass" (manufactured by Matsunami Glass Industrial Co., Ltd.), "APS coated slide glass" (manufactured by Matsunami Glass Industrial Co., Ltd.), and "MAS coated slide glass" (manufactured by Matsunami Glass Industrial Co., Ltd.).

Next, a washing step S13 is performed in which the slide glass to which the stratum corneum cells have been transferred is washed.
The method for cleaning the slide glass is not particularly limited, and the slide glass can be cleaned using a shaking or ultrasonic cleaner.
In the present invention, it is preferable to clean the slide glass by immersing it in a cleaning agent containing an organic compound.
By adopting such a form, peeling of stratum corneum cells can be further suppressed.

Examples of cleaning agents include organic solvents such as xylene, chloroform, and ethyl acetate, but it is preferable to use xylene, which does not affect the amount of adhesive protein in stratum corneum cells.

When cleaning by immersion, the immersion time, at room temperature, is preferably 3 hours or more, more preferably 6 hours or more, even more preferably 12 hours or more, and particularly preferably 24 hours or more.

After the above steps have been carried out and the keratinocyte sample has been prepared, a screening sample preparation step S20 is then carried out.
In step S20, a component to be searched for (hereinafter referred to as a search target component) is added to the corneocyte sample.

The components to be searched for are not particularly limited, but considering that a component having the effect of reducing the amount of adhesive protein is to be applied to humans, for example, components applicable to cosmetics and components applicable to food can be the components to be searched for.
An example of such an ingredient is a plant extract.

It is preferable to dilute the component to be searched for with phosphate buffered saline (PBS) depending on the concentration to be screened.

In one embodiment of the present invention, a screening control sample may be prepared in which the component to be sought is not added.
Such an embodiment is suitable for screening a class of components that have the effect of reducing the amount of adhesion proteins.

In one embodiment of the present invention, a plurality of screening samples may be prepared in which the type of component to be searched for is the same but the concentrations are different.
Such an embodiment is suitable for screening for optimal concentrations that act to reduce the amount of adhesion protein.

In step S20, the stratum corneum cells to which the search target component has been added are stained by immunohistochemical staining.
Staining by immunohistochemical staining can be carried out by a conventional method, and a direct method, an indirect method, or a sensitization method can be arbitrarily selected depending on the type of adhesive protein.
If necessary, any blocking agent can be used.

The adhesive protein to be stained is not particularly limited as long as it is contained in keratinocytes, but desmoglein 1 is preferred.
Desmoglein 1 is closely correlated with the proteolysis of desmosomal components and cell detachment, and screening of components that affect the increase or decrease of its amount would be extremely useful.

Next, an image acquisition step S30 is performed in which an image of the stratum corneum cells stained for adhesion proteins is taken.
The image is taken using a microscope. The type of microscope is not particularly limited, and an appropriate microscope can be used depending on the immunohistochemical staining method performed in step S20.
Examples of the microscope include an optical microscope, a confocal laser microscope, an optical microscope, and an electron microscope.

In the present invention, it is preferable to use a confocal laser microscope.
By using a confocal laser microscope, it is possible to take focused images without blurring the boundaries between the stained areas (adhesion proteins) and the unstained areas (areas other than the adhesion proteins, the slide glass, etc.), thereby improving the accuracy of the screening method of the present invention, which uses the stained areas as an indicator.
When a confocal laser microscope is used, it is preferable to employ a fluorescent antibody technique as the immunohistochemical staining method.

Next, a randomly selected area within the portion of the photographed image where the keratinocytes have been transferred is enlarged and converted into a black and white image (binary image).
From the obtained image, the area value of the stained and/or non-stained parts, or the area percentage (%) of the stained and/or non-stained parts is calculated (hereinafter, when simply referring to area, it includes both the area value and the area percentage).

The above-mentioned image processing and area calculation can be carried out using image analysis software.
An example of image analysis software is "Image J (National Institutes of Health, USA)".

The area of the stained portion is preferably the average area calculated from a number of randomly selected regions.
By using the average area of multiple locations as an index, more accurate screening can be performed.

Next, an evaluation step S40 is performed in which the area obtained in step S30 is used as an index to evaluate the effect of the component being searched for on the increase or decrease of adhesive proteins.

Figure 3 shows a flowchart illustrating step S40 in detail. With reference to Figure 3, the evaluation process in one embodiment in which the area of the stained portion of the screening sample is compared with the area of the stained portion of the screening control sample will be described.

In step S41, the area At of the stained portion of the screening sample is compared with the area Ac of the stained portion of the screening control sample.
The comparison of areas may be a comparison of absolute values of the areas, or a comparison of the percentages of the areas when the area of the screening control sample is taken as 100%.
When evaluation is performed for a plurality of screening samples, it is preferable to make a comparison by regarding the area of the screening control sample as 100%.

If area At is smaller than area Ac (area At < area Ac), proceed to step S42, and the component to be searched for added to the screening sample is evaluated as a component that has the effect of reducing the amount of adhesive protein.

On the other hand, if the area At is equal to the area Ac or is greater than the area Ac (area At ≧ area Ac), the process proceeds to step S43, and the component to be searched for added to the screening sample is evaluated as a component that does not have the effect of reducing the amount of adhesive protein.

The evaluation process of one embodiment has been described with reference to Figure 3, but the evaluation process in the screening method of the present invention is not particularly limited as long as it is an evaluation of the effect on the increase or decrease of adhesion protein using the area of the stained portion of the screening sample as an index.

For example, when screening for a component that increases the amount of adhesive protein, in step S41, it is determined whether "area At > area Ac", and if the result is "yes", in step S42, it may be evaluated as "a component that has the effect of increasing the amount of adhesive protein."
Also, in step S42, it may be evaluated as "an ingredient having the effect of decreasing the amount of adhesive protein," and in step S43, it may be evaluated as "an ingredient having the effect of increasing the amount of adhesive protein."

In another embodiment, as described in step S20, a step of preparing a plurality of screening samples having different concentrations of the component to be searched for and evaluating these may be performed.
In this embodiment, the areas in multiple screening samples are compared, and if there is a specific concentration that is a threshold at which a significant effect is expressed in terms of the effect of decreasing or increasing the amount of adhesion protein, it can be evaluated that "the component to be searched for has the effect of significantly decreasing or increasing the amount of adhesion protein at a specific concentration or above."
Furthermore, for components that do not exhibit the above-mentioned effect unless the component is at a certain concentration or above, it can be evaluated that "the component being sought exhibits the effect of decreasing or increasing the amount of adhesive protein at a certain concentration or above."

The screening method of the present invention is preferably used for screening ingredients used for improving and/or preventing sensitive skin.
Here, in this specification, "sensitive skin" refers to a skin type with reduced resistance (reduced skin irritation value) to external stimuli (dryness, ultraviolet rays, application of chemical compounds).

More specifically, "sensitive skin" in this specification refers to the skin types listed in (1) and/or (2).

(1) "Skin that reacts to substances such as topical medicines, cosmetics, plants, ultraviolet rays, and metals and is prone to skin problems. Also, skin that is hypersensitive to allergens (pollen, fragrances, etc.) and irritants (alcohol, etc.)."
(2) "Skin that is temporarily more susceptible to skin problems due to lack of sleep, overwork, menstruation, seasonal changes, mental stress, etc."

In addition, in this specification, "for the improvement and/or prevention of sensitive skin" refers to the improvement of skin quality with reduced resistance to external stimuli (reduced skin irritation threshold) and/or the prevention of reduced resistance to external stimuli in skin quality (reduced skin irritation threshold).

Furthermore, the screening method of the present invention is preferably used for screening a component having an inhibitory effect on intercellular adhesion of human epidermal keratinocytes.
In this specification, the term "intercellular adhesion" refers to abnormalities in the adhesive function of the stratum corneum (progression of stratification of the stratum corneum due to increased cell adhesiveness) caused by an increase in desmosomal proteins (a group of proteins including desmoglein) (see Patent Nos. 4,197,194 and 4,002,635 for details).
In addition, in this specification, the concept of "inhibiting intercellular adhesion" includes both prevention and improvement of intercellular adhesion.

Here, by inhibiting intercellular adhesion of human epidermal keratinocytes, stratification of the stratum corneum is inhibited, and an effect of inhibiting stratum corneum desquamation can be obtained.
That is, the screening method of the present invention can be used to screen for components that have the effect of preventing or improving stratum corneum desquamation.
In this specification, the term "stratum corneum delamination" refers to the peeling off of the stratum corneum from the skin surface in a multi-layered state. Whether or not the skin is in a state where stratum corneum delamination can be confirmed by collecting the stratum corneum by tape stripping and staining and observing the collected stratum corneum.

Furthermore, as shown in the Examples described below, desmoglein 1 is present in large amounts in areas of the skin with low brightness, and it is believed that the decrease in skin brightness is due to stratification of the stratum corneum.
In view of the above findings, it can be said that a component that has the effect of reducing desmoglein is a component that has the effect of improving skin brightness.
That is, the screening method of the present invention can be used to screen for components that have the effect of improving skin lightness.
In this specification, "improving skin brightness" refers to reducing the difference in brightness between skin in a normal state and skin whose brightness has been reduced due to the excess of desmoglein.

Furthermore, as shown in the examples described below, ingredients that have a desmoglein-reducing effect have the effect of improving the skin condition to a more transparent state. In other words, the screening method of the present invention can be used to screen ingredients that have the effect of improving skin transparency.

Furthermore, as shown in the Examples described below, desmoglein 1 is present in large amounts in areas of high skin hardness, and it is believed that the cause of skin hardness is the stratification of the stratum corneum.
Based on the above findings, the effect of softening the skin can be obtained by reducing desmoglein, and thus the screening method of the present invention can be used to screen for components that have a skin softening effect.

Here, by softening the skin, other medicinal ingredients can penetrate more efficiently. Therefore, the screening method of the present invention can be used to screen ingredients that have the effect of allowing medicinal ingredients such as whitening agents to penetrate the skin more efficiently.

[Example 1]
<Step of preparing stratum corneum cell sample>
The stratum corneum was collected from the outer side of a human forearm using cellophane tape and transferred to a slide glass ("MAS Coat", Matsunami Glass Industry Co., Ltd.), and the slide glass was immersed in xylene overnight.
The slides were then washed twice with xylene for 10 minutes each, followed by washing with PBS.

<Preparation of target components>
Wild thyme extract (manufactured by Ichimaru Falcos, an extract of the aboveground parts of Wild Thyme, T. serpyllum) and Rosa roxburghii extract (manufactured by Maruzen Pharmaceuticals, an extract of the fruit of Rosa roxburghii) were diluted with PBS to concentrations of 0.1%, 0.05%, 0.025%, and 0.0125% by mass, respectively, to obtain the components to be searched for.
<Preparation of screening samples>
The stratum corneum cells were immersed in the target component overnight to obtain a sample. As a control sample, stratum corneum cells that were not immersed in the target component were prepared.

<Immunostaining test>
Each sample was washed with PBS and then incubated in 4% PFA-phosphate buffer at room temperature for 15 minutes.
Then, each sample was washed with PBS and then incubated in a 20% aqueous solution of Block Ace (DS Biopharma Medical) at room temperature for 1 hour.
A primary antibody (Anti-Desmoglein 1, Mouse-Mono) was added to each sample, and the samples were incubated in a humidified box at room temperature for 2 hours.
After each sample was washed three times with PBS for 5 minutes, a secondary antibody (Allexa Fluor@488 Goat Anti-mouse IgG) was added and incubated in a humidified box at room temperature for 1 hour.
Each sample was washed three times with PBS for 5 minutes each, washed twice with distilled water, and then sealed using Fluoromout-G (manufactured by Southern Biotech) to obtain a number of screening samples and a screening control sample.

<Image analysis>
Analytical images of the screening sample and the screening control sample were taken using a confocal laser microscope (FIG. 4).
Using image analysis software (Image J), any of four areas in the image for analysis were enlarged and converted into black and white images, the areas of the stained and non-stained parts were calculated, and the average value was calculated.
The area of the stained portion of the screening control sample was taken as 100%, and the ratio of the area of the stained portion of the screening sample (average value of four locations) was calculated. The results are shown in Table 1 and FIG.

As shown in Figures 4 and 5, the expression level of desmoglein 1 was reduced in the screening samples to which the wild thyme extract and the Togen pear extract had been added, compared to the screening control sample.
From these results, it can be evaluated that wild thyme extract and togenashi extract are ingredients that have the effect of reducing the amount of desmoglein 1.

It can also be seen that the area of the stained portion of the screening sample containing 0.1% by mass of added wild thyme extract was reduced by approximately 2.5 to 2.8 times compared to the screening samples containing 0.05, 0.025, and 0.0125% by mass of wild thyme extract.

Similarly, it can be seen that the area of the stained portion of the screening sample containing 0.1% by mass of added togenu extract was reduced by 7.6 to 8.0 times compared to the screening samples containing 0.05, 0.025, and 0.0125% by mass of the extract.

From this, it can be evaluated that wild thyme extract and togenashi extract have a significant effect of reducing the amount of desmoglein 1 when their concentrations are 0.1% by mass or more.

[Example 2]
<Step of preparing stratum corneum cell sample>
A keratinocyte sample was prepared in the same manner as in Example 1.

<Preparation of target components>
Marshmallow extract (manufactured by Jurlique, an extract of the roots of Marshmallow (Althaea Officinalis)) was diluted with PBS to a concentration of 0.25% by mass to obtain the component to be searched for.

<Preparation of screening samples>
The stratum corneum cells were immersed in the target component overnight to obtain a sample. As a control sample, stratum corneum cells that were not immersed in the target component were prepared.

<Immunostaining test>
A screening sample and a screening control sample were obtained by the same method as in Example 1.

<Image analysis>
Analytical images of the screening sample and the screening control sample were taken in the same manner as in Example 1, and the area of the stained portion of the screening sample (proportion of the area of the stained portion of the screening sample) was calculated with the area of the stained portion of the screening control sample being set as 100%. The results are shown in Table 2, Figure 6, and Figure 7.

As shown in Figures 6 and 7, the expression level of desmoglein 1 was reduced in the screening sample to which the Marshmallow extract was added, compared to the screening control sample.
From these results, it can be evaluated that Marshmallow extract is a component that has the effect of reducing the amount of desmoglein 1.

[Example 3]
Below are test results that support the relationship between differences in skin brightness and the amount of desmoglein 1 present, and the relationship between the amount of desmoglein 1 present and skin hardness.

(1) Subjects and Measurement Sites In this example, 20 women aged between 40 and 52 years old were used as subjects.
In this embodiment, the areas on the subject's face where a decrease in brightness was visually confirmed (blackish areas, see area A in FIG. 13 ) and an area in a normal state (see area B in FIG. 13 ) were selected as measurement areas.

(2) Measurement (2-1) Measurement of L ^* value, a ^* value, and b ^* value A spectrophotometer was placed on the above measurement sites, and the L ^* value, a ^* value, and b ^* value were measured by measuring each site five times.
The results are shown in Figures 8 to 10.

Regarding the L ^* value, the area where the decrease in brightness was visually confirmed (the dark area, see area A in FIG. 13) showed a significantly lower value than the area in the normal state (see area B in FIG. 13). In other words, the area where the decrease in brightness was visually confirmed (the dark area, see area A in FIG. 13) was confirmed to be darker than the area in the normal state (see area B in FIG. 13).

Regarding the a ^* value, the areas where the brightness was visually confirmed to have decreased (blackish areas, see area A in FIG. 13) showed significantly higher values than the areas in the normal state (see area B in FIG. 13). In other words, the areas where the brightness was visually confirmed to have decreased (blackish areas, see area A in FIG. 13) were confirmed to have a stronger reddish hue than the areas in the normal state (see area B in FIG. 13).

Regarding the b ^* value, the areas where the decrease in brightness was visually confirmed (blackish areas, see area A in FIG. 13) showed significantly higher values than the areas in the normal state (see area B in FIG. 13). In other words, the areas where the decrease in brightness was visually confirmed (blackish areas, see area A in FIG. 13) were confirmed to be more yellowish than the areas in the normal state (see area B in FIG. 13).

(2-2) Hardness Measurement The hardness was measured by pressing an indenter against each measurement site used in the test (2-1) above and measuring five times each.
The results are shown in Figure 11.

Regarding hardness, the areas where a decrease in brightness was visually confirmed (the dark areas, see area A in Figure 13) showed significantly lower values than the areas in a normal state (see area B in Figure 13). In other words, the areas where a decrease in brightness was visually confirmed (the dark areas, see area A in Figure 13) were confirmed to be harder than the areas in a normal state (see area B in Figure 13).

(2-3) Desmoglein 1 (Dsg1) Staining Corneal cornea cells were collected from each measurement site subjected to the above tests (2-1) and (2-2) by tape stripping and attached to a slide glass.

The slide glass to which the keratinocytes were attached was immersed in xylene overnight.
After immersion, the sample was washed and air-dried, and then allowed to stand in 4% paraformaldehyde-phosphate buffer at room temperature for 15 minutes.

After 15 minutes, the stratum corneum cells were washed with PBS and immersed in a 0.5% TritonX (Nacalai Tesque)/PBS solution at room temperature for 5 minutes.

After immersion, the sample was washed and then left to stand in an aqueous Block Ace solution (DS Biopharma Medical UK-B80) at room temperature for 1 hour.

After standing, the plate was framed with Liquid Blocker (manufactured by Daido Sangyo Co., Ltd.), a primary antibody was added, and the plate was left standing in a humid box at room temperature for 2 hours.
As the primary antibody, Anti Desmoglein 1, Mouse Mono (Dsg1 P23) Progen, 651110 stock solution (IgG concentration: 10-15 g/mL) was used.

After standing, the plate was washed with PBS, a secondary antibody was added, and the plate was left standing in a humidified box at room temperature for 1 hour.
As the secondary antibody, Allexa Fluor@488 Goat Anti-mouse IgG Invitrogen, A11001, 200-fold diluted solution in PBS was used.

After leaving it to stand, it was washed, sealed with Fluoromout-G (Southern Biotech), and dried at room temperature for 30 minutes.

After drying, the cover glass was fixed with nail polish and dried, and then (photographed) observed with a confocal laser microscope (Nikon A1) at ×20.
The results are shown in Figure 12.

Regarding the amount of desmoglein 1 (Dsg1), the areas where a decrease in brightness was visually confirmed (the dark areas, see area A in Figure 13) showed significantly higher values than the areas in a normal state (see area B in Figure 13). In other words, it was confirmed that the areas where a decrease in brightness was visually confirmed (the dark areas, see area A in Figure 13) had a higher amount of desmoglein 1 (Dsg1) than the areas in a normal state (see area B in Figure 13).

(3) Discussion As shown in Figures 8 to 10 and 12, it was found that desmoglein 1 was present in large amounts in areas of the skin with low brightness. Here, the decrease in skin brightness is thought to be due to the dense layering of the stratum corneum (stratification of the stratum corneum) caused by the low pH of the skin and low activity of serine protease that breaks down desmoglein 1.

As described above, the active ingredient of the present invention has the effect of reducing desmoglein.
In view of the above findings, it has been found that the active ingredient of the present invention has the effect of improving skin brightness by reducing desmoglein 1.

As shown in Figures 11 and 12, it was found that desmoglein 1 is present in large amounts in areas of high skin hardness. Here, the cause of skin hardness is thought to be the dense layering of the stratum corneum (stratification of the stratum corneum) due to the low pH of skin and low activity of serine protease, which breaks down desmoglein 1.

Based on the above findings, it has been found that reducing desmoglein 1 can soften the skin.
As described above, the active ingredient of the present invention has the effect of reducing desmoglein.
In other words, it was found that the active ingredient of the present invention has the effect of softening the skin by reducing desmoglein 1.

[Example 4]
The results of verifying the effect of a composition containing an ingredient having a desmoglein-reducing effect on inhibiting layer peeling are shown below.
(1) Preparation of Formulation According to the composition shown in Table 3, a cosmetic composition of Production Example 1 containing an extract of Togenashi was prepared.

(2) Subjects and Target Areas In this example, five female subjects were used as subjects. The cosmetic product of Production Example 1 was applied twice a day (morning and evening) to the inner areas of the left and right forearms of the subjects (n=10), and this was continued for 10 days.

(3) Verification of desmoglein-reducing effect Before and after (10 consecutive days) use of the cosmetic product of Production Example 1, the stratum corneum of the left and right inner forearms of each subject was collected to prepare stratum corneum cell samples according to the method described in "<Preparation process of stratum corneum cell samples>" of Example 1. Next, according to the method described in "<Immunostaining test>" of Example 1, desmoglein 1 in the stratum corneum cell samples was stained, and images for analysis before and after use of Production Example 1 were taken using a confocal laser microscope to confirm the fluorescence (staining).

The area of the fluorescent portion was calculated by taking the area of the entire image for analysis as 100%. The results are shown in Figures 14 and 15. In Figure 15, * indicates p<0.05.

As shown in Figures 14 and 15, it was confirmed that the amount of desmoglein 1 decreased by continued use of the cosmetic product of Example 1 containing Togenashi extract.

(4) Verification of the effect of inhibiting layer peeling Before and after use of the cosmetic product of Production Example 1, the stratum corneum was collected from the inside of the left and right forearms of each subject and stratum corneum cell samples were prepared according to the method described in "<Step of preparing stratum corneum cell samples>" in Example 1. Next, each stratum corneum cell sample was stained with gentian violet and brilliant green by standard methods and observed under an optical microscope.
A representative diagram of the results is shown in FIG.

16, there are multiple dark areas in the optical microscope image of the stratum corneum cells before use in Production Example 1. These dark areas are areas where the stratum corneum cells have been delaminated by the tape stripping method.
On the other hand, the stratum corneum cell sample collected after 10 days of continuous use of Production Example 1 was mostly stained lightly compared to the stratum corneum cell sample collected before use. This lightly stained area indicates that only one layer of stratum corneum cells had been peeled off, meaning that multilayer peeling had not occurred.
As described above, it was confirmed that the use of the cosmetic product of Production Example 1, which contains an ingredient that has a desmoglein-reducing effect, improves multilayer peeling.

[Example 5]
Below, the desmoglein-reducing ability of a composition containing an ingredient having a desmoglein-reducing effect was examined.

(1) Preparation of Composition A cosmetic preparation (lotion) containing wild thyme extract was prepared according to the formulation shown in Table 4 below. That is, the ingredients (a), (b), and (c) were each heated to 70° C., (c) was added to (b) to neutralize it, (a) was gradually added thereto while stirring to emulsify, the mixture was homogenized using a homogenizer, and then cooled with stirring to obtain the lotion of Production Example 2.

(2) Subjects and Measurement Sites In this example, 20 women aged between 40 and 52 years old were used as subjects.
In this embodiment, the areas (92 areas) on the face of the subject where blemishes were present were selected as the measurement areas.
The emulsion of Preparation Example 2 was applied twice a day to the areas of the skin where blemishes existed on each subject, and this was continued for 12 weeks.

(3) Measurement Before use of the emulsion of Production Example, after 6 weeks of continuous use, and after 12 weeks of continuous use, cornified cells were collected from the site of the subject where blemishes were present, and cornified cell samples were prepared according to the method described in "<Preparation of cornified cell sample>" of Example 1. Next, desmoglein 1 in each cornified cell sample was stained according to the method described in "<Immunostaining test>" of Example 1, and images for analysis before and after use of Production Example 2 were taken using a confocal laser microscope to confirm fluorescence (staining).

The area of the fluorescent portion was calculated by taking the area of the entire image for analysis as 100%. The results are shown in Tables 5 and 6, and Fig. 17 (representative), 18 and 19. Note that data after 12 weeks of using lotion was collected from 80 out of 92 spots and compared with the data before using lotion at the same spots. In Fig. 18, ** means p<0.01, and in Fig. 19, *** means p<0.001.

As shown in Tables 5-6 and Figures 17-19, it was confirmed that the amount of desmoglein 1 in the blemish area was significantly reduced by continuous use of a lotion containing wild thyme extract.

The present invention can be applied to analytical techniques related to the amount of adhesion proteins.

Claims (6)

A method for screening for a component that affects an increase or decrease in adhesion protein between stratum corneum cells, comprising the steps of:
A step of collecting stratum corneum cells by tape stripping;
a transfer step of transferring the stratum corneum cells onto a slide glass having a positively charged surface;
a washing step of washing the slide glass;
A preparation step of adding a target component to the slide glass, staining the adhesion protein by immunohistochemical staining, and preparing a screening sample;
an image acquiring step of acquiring an image for analysis by photographing an arbitrary portion of the screening sample;
and an evaluation step of evaluating the effect of the search target component on the increase or decrease of adhesive protein using the area of the stained portion in the analysis image as an index.
In the preparation step, a plurality of screening samples are prepared in which the type of the search target component is the same but the concentrations are different;
A screening method , wherein the evaluation step is a step of comparing areas of stained portions of a plurality of the screening samples and evaluating an optimal concentration for the effect of reducing the amount of adhesion protein . The transfer step is carried out using NH ₃ ⁺ The stratum corneum cells were transferred to a slide glass with NH ₃ ⁺ The screening method according to claim 1, which is a transfer step in which the step (a) forms an ionic bond with a carboxyl group of a phospholipid in the keratinocyte, thereby making the keratinocyte less likely to peel off from the slide glass. In the preparation step, a screening control sample is prepared without adding the target component to be searched for;
The screening method according to claim 1, wherein the evaluation step is a step of comparing an area of the stained portion of the screening sample with an area of the stained portion of the screening control sample, and if the area of the stained portion of the screening sample is smaller, evaluating that the component to be searched for has the effect of reducing the amount of adhesion protein. The screening method according to any one of claims 1 to 3, wherein the adhesion protein is desmoglein 1. The screening method according to any one of claims 1 to 4, wherein the slide glass has NH ₃ ⁺ arranged on the surface thereof. The screening method according to any one of claims 1 to 5, wherein the washing step is a step of immersing the slide glass in xylene.