CN113874704A

CN113874704A - Screening method of components influencing increase and decrease of stratum corneum intercellular adhesion protein

Info

Publication number: CN113874704A
Application number: CN202080037961.3A
Authority: CN
Inventors: 多田明弘; 武重史佳
Original assignee: Pola Chemical Industries Inc
Current assignee: Pola Chemical Industries Inc
Priority date: 2019-05-23
Filing date: 2020-05-22
Publication date: 2021-12-31
Also published as: SG11202112996PA; TW202101001A; WO2020235668A1; JPWO2020235668A1

Abstract

The present invention provides a method for screening a component which affects increase and decrease of an adhesion protein present between stratum corneum cells mainly comprising desmoglein more accurately than the conventional methods. A method for screening for a component that affects increase or decrease of stratum corneum intercellular adhesion protein, comprising: a collection step of collecting stratum corneum cells by a tape stripping method; a transcription step of transcribing the stratum corneum onto a glass slide having a positive charge on the surface thereof; a cleaning step of cleaning the slide glass; a preparation step of adding a component to be searched on the glass slide, staining the adhesion protein by an immunohistostaining method, and preparing a screening sample; an image acquisition step of capturing an image of an arbitrary portion of the screened sample to obtain an image for analysis; and an evaluation step of evaluating the influence of the component to be searched on the increase or decrease in the amount of the adherent protein, using the area of the stained portion in the image for analysis as an index.

Description

Screening method of components influencing increase and decrease of stratum corneum intercellular adhesion protein

Technical Field

The invention relates to a screening method of components influencing increase and decrease of stratum corneum intercellular adhesion protein and a preparation method of stratum corneum cell samples suitable for the screening method.

Background

As an adhesive bonding device for keratinocytes (keratinocyte), desmosomes are representative. Desmosomes are known to be structures involved in the adhesive connection between epidermal cells and stratum corneum cells, and the adhesive connection between epidermal cells via desmosomes maintains the firm structure of epithelial tissues by matching with cytoskeletons composed of keratin fibers.

It is known that an increase in desmoglein is one of the causes of deterioration of skin conditions, such as rough skin, sunburn, and desquamation of stratum corneum due to dryness.

As a method for controlling the amount of desmoglein, a related art has been disclosed which utilizes protease degradation of desmoglein accumulated in the horny layer to improve acne, dandruff, and desquamation (patent document 1).

The bridge constituent proteins are known as desmoglein1, 2, 3, 4, desmoglein1, 2, 3, desmoplakin 1, 2 and the like.

Among the proteins constituting the desmosome, in particular desmoglein1 of the adhesion protein has a close correlation between proteolysis of the desmosome component and cell exfoliation. Therefore, confirmation of the presence of desmoglein in the stratum corneum is also important in evaluating desmosome function and in evaluating skin condition.

Documents of the prior art

Patent document

Patent document 1 Japanese patent laid-open publication No. Hei 7-505383

Disclosure of Invention

[ problems to be solved by the invention ]

In view of the foregoing, it would be desirable to find a composition that reduces the amount of desmosomes or adhesion proteins that make up desmosomes.

As one method for screening such components, a stratum corneum cell sample can be prepared by collecting stratum corneum cells by a tape stripping method and transcribing the cells onto a slide glass, staining adherent proteins by an immunohistostaining method, and observing the cells under an arbitrary microscope.

However, when transcription of stratum corneum cells is performed using the tape stripping method, components of the adhesive tape remain on the slide, which may not be distinguished from adhering proteins after staining, and thus the slide needs to be washed.

However, the transferred stratum corneum cells were also exfoliated after washing the slide glass, and evaluation using the amount of adhesive proteins present in the stratum corneum cells as an index was difficult, and thus it was not considered to be a sufficient screening method.

The invention provides a method for screening components, wherein the components mainly comprise desmoglein and influence the increase and decrease of adhesion protein existing among corneocytes; the method is more accurate than the existing methods.

Another object of the present invention is to provide a method for preparing a stratum corneum cell sample for more accurately evaluating the amount of adhesion protein present in the above screening method.

[ means for solving the problems ]

To solve the above problems, the present invention provides a method for screening a component that affects increase or decrease of stratum corneum intercellular adhesion protein, comprising:

a collection step of collecting stratum corneum cells by a tape stripping method;

a transcription step of transcribing the stratum corneum onto a glass slide having a positive charge on the surface thereof;

a cleaning step of cleaning the slide glass;

a preparation step of adding a component to be searched on the glass slide, staining the adhesion protein by an immunohistostaining method, and preparing a screening sample;

an image acquisition step of capturing an image of an arbitrary portion of the screened sample to obtain an image for analysis; and

an evaluation step of evaluating the inhibitory effect of the component to be searched for on the formation of dense stratum corneum using the area of the stained portion in the image for analysis as an index.

It is known that the carboxyl group (COO) of cell surface is caused by phospholipid^-) But has a negative charge.

According to the screening method of the present invention, the stratum corneum cells can be prevented from being peeled off by washing by transcribing the stratum corneum cells onto a slide glass having a positive charge on the surface, and the amount of adhesive proteins present in the stratum corneum cells can be estimated more accurately.

In a preferred form of the present invention,

in the preparation step, a screening control sample to which the component to be searched is not added is prepared,

the evaluation procedure was as follows: comparing the area of the stained portion of the screening sample with the area of the stained portion of the screening control sample, and evaluating that the component to be searched has an effect of reducing the amount of the adhesion protein when the area of the stained portion of the screening sample is small.

By adopting this manner, it is possible to more accurately screen the components that affect the increase or decrease of the adhesion protein.

In a preferred form of the present invention,

in the preparation step, a plurality of screened samples of the component to be searched, which are the same in type and different in concentration, are prepared,

the evaluation procedure was as follows: comparing the areas of the stained portions of a plurality of the screened samples, the optimal concentration in the effect of reducing the amount of the adhesion protein was evaluated.

By adopting this manner, not only can a component having an effect of reducing the amount of the adhesion protein be screened, but also the optimum concentration thereof can be evaluated.

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

In a preferred embodiment of the present invention, the slide glass has NH arranged on the surface thereof₃ ⁺The slide glass of (1).

NH₃ ⁺COO with cell surface^-Ionic bonds are formed, and thus, the adhesion of stratum corneum cells to the slide can be improved.

In a preferred embodiment of the present invention, the washing step is a step of immersing the slide glass in xylene.

The peeling of stratum corneum cells can be better inhibited by soaking the slides in xylene for washing.

In order to solve the above problems, the present invention provides a method for preparing a stratum corneum cell sample by a tape stripping method, comprising:

a transcription step of transcribing the stratum corneum onto a glass slide having a positive charge on the surface thereof; and

and a cleaning step of cleaning the slide glass.

The transfer of stratum corneum onto a positively charged slide makes it possible to prevent stratum corneum cells from peeling off from the slide in the subsequent washing step.

[ Effect of the invention ]

According to the screening method of the present invention, it is possible to screen more accurately components that affect increase or decrease of adhesion proteins than in the conventional methods.

In addition, according to the method for producing stratum corneum cells of the present invention, exfoliation of stratum corneum cells on a slide glass can be suppressed.

Drawings

FIG. 1 is a flowchart showing a screening method according to the present embodiment;

FIG. 2 is a flowchart showing a procedure for preparing a stratum corneum cell sample according to the present embodiment;

fig. 3 is a flowchart showing an evaluation step in the present embodiment;

FIG. 4 is a microscope image of a screened sample and a screened control sample obtained by adding wild thyme extract and rosa roxburghii extract as components to be searched to a stratum corneum cell sample;

FIG. 5 is a graph showing the area ratio of the stained part of the screening control sample when the area of the stained part of the screening control sample is set to 100%;

FIG. 6 is a graph (representative) showing the measurement results of the amount of desmoglein1 in example 2;

FIG. 7 is a graph showing the measurement results of the amount of desmoglein1 in example 2;

fig. 8 is a graph showing the measurement results of L values in example 3;

fig. 9 is a graph showing the measurement results of a-values in example 3;

fig. 10 is a graph showing the measurement results of b values in example 3;

FIG. 11 is a graph showing the measurement results of hardness in example 3;

FIG. 12 is a graph showing the measurement results of the amount of desmoglein1 in example 3;

FIG. 13 is a graph (representative) showing the measurement results of the amount of desmoglein1 in example 3;

FIG. 14 is a graph (representative) showing the measurement results of the amount of desmoglein1 in example 4;

FIG. 15 is a graph showing the measurement results of the amount of desmoglein1 in example 4;

FIG. 16 is an optical microscope image showing the state of exfoliation of stratum corneum cells in example 4;

FIG. 17 is a graph (representative) showing the measurement results of the amount of desmoglein1 in example 5;

FIG. 18 is a graph showing the measurement results of the amount of desmoglein1 in example 5 (6 weeks);

FIG. 19 is a graph showing the measurement results of the amount of desmoglein1 in example 5 (12 weeks).

Detailed Description

The screening method of the present invention will be described in detail with reference to the flowcharts of FIGS. 1 to 3.

The screening method of the present invention includes 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) Stratum corneum cell sample preparation procedure

Fig. 2 shows a flowchart showing step S10 in detail.

In step S10, a stratum corneum cell collection step S11 is performed by a tape stripping method.

The tape peeling method is a generic name of a method of peeling skin cells by applying and peeling a pressure-sensitive adhesive represented by an adhesive tape to the skin.

The pressure-sensitive adhesive used is not particularly limited, but from the viewpoint of convenience, a transparent adhesive tape is preferably used.

Subsequently, the transfer step S12 is performed to transfer the stratum corneum cells collected in step S11 onto a slide glass having a positively charged surface.

Human cells such as stratum corneum cells, and are affected by the carboxyl group (COO) of phospholipid forming lipid bilayer membrane^-) The surface is negatively charged.

In step S12, by transcribing the stratum corneum cells onto the slide glass having a positively charged surface, the stratum corneum cells are difficult to be peeled off from the slide glass due to electrostatic interaction.

As an example of a glass slide having a positively charged surface, there can be mentioned a glass slide whose surface is modified with polylysine, polyalkylamine, amino-modified silicone, or the like.

In the present invention, it is preferable to use a surface on which NH is arranged₃ ⁺The slide glass of (1).

NH₃ ⁺Ionic bonds with the carboxyl groups of phospholipids make it more difficult for stratum corneum cells to be peeled off from the slide.

With NH arranged on the surface₃ ⁺As the slide glass, commercially available slide glass such as "PLL-coated slide glass" (manufactured by Sonlang Nitri Kabushiki Kaisha), "APS-coated slide glass" (manufactured by Sonlang Nitri Kabushiki Kaisha) and "MAS-coated slide glass" (manufactured by Sonlang Nitri Kabushiki) can be used.

Then, a washing step S13 is performed to wash the slide transcribed with the horny layer cells.

The method of cleaning the slide glass is not particularly limited, and cleaning can be performed using a vibration type or ultrasonic type cleaning machine.

In the present invention, it is preferable that the slide glass is washed by immersing it in a washing agent containing an organic compound.

By selecting this way, exfoliation of stratum corneum cells can be better inhibited.

The detergent may be an organic solvent such as xylene, chloroform, ethyl acetate, etc., preferably xylene, which does not affect the amount of adhesion proteins of stratum corneum cells.

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

After the stratum corneum cell sample is prepared through the above steps, the procedure proceeds to step S20, and a screening sample is prepared.

In step S20, a component to be searched (hereinafter, referred to as "component to be searched") is added to the stratum corneum cell sample.

The component to be explored is not particularly limited, but in consideration of application of a component having an effect of reducing the amount of adhered protein to a human, for example, a component that can be applied to cosmetics or a component that can be applied to food may be set as the component to be explored.

For example, the component may be a plant extract.

Preferably, the components to be explored are diluted with Phosphate Buffered Saline (PBS) at the concentrations used for screening.

In one embodiment of the invention, a screening control sample can also be prepared without the addition of a component to be explored.

This embodiment is suitable for screening the component species having the effect of reducing the amount of the adhesion protein.

In one embodiment of the present invention, a plurality of screening samples having the same kind of component to be searched and different concentrations may be prepared.

This embodiment is suitable for screening for an optimal concentration that acts to reduce the amount of adhering protein.

In step S20, the stratum corneum cells to which the component to be detected was added were stained by immunohistostaining.

The staining by the immunohistological staining method can be carried out by a conventional method, and the direct method, the indirect method and the sensitization method can be arbitrarily selected depending on the kind of the adherent protein.

In addition, any blocking agent may be used as necessary.

The adhesion protein to be stained is not particularly limited as long as it is included in the stratum corneum cell, but is preferably desmoglein 1.

Desmoglein1 has a close correlation between proteolysis of desmosome components and cell exfoliation, and is useful for screening components that affect the increase or decrease of the amount thereof.

Then, the image acquisition step S30 is performed to capture an image of stratum corneum cells stained with the adhesion proteins.

Image taking was performed using a microscope. The type of microscope is not particularly limited, and a suitable microscope can be used according to the immunohistological 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, a confocal laser microscope is preferably used.

By using a confocal laser microscope, an image of a focused image can be captured without blurring at the boundary between a stained portion (a adhering protein) and an unstained portion (a portion other than the adhering protein, a slide glass, or the like), and therefore the accuracy of the screening method using a stained portion as an index of the present invention can be improved.

In addition, when a confocal laser microscope is used, a fluorescent antibody method is preferably used as the above-mentioned immunohistological staining method.

Then, from the captured image, a range randomly selected in a portion where the horny layer cells are transcribed is enlarged and converted into a black-and-white image (binary image).

From the obtained image, an area value of a stained part and/or an unstained part, or an area ratio (%) of the stained part and/or the unstained part (hereinafter, simply referred to as "area", in this case including both the area value and the area ratio) is calculated.

The image processing and area calculation described above may use image analysis software.

For example, the Image analysis software may include "Image J (national institute of health, NIH)" and the like.

Preferably, the area of the dyed portion is set to an average value of the areas calculated from a plurality of randomly selected ranges.

By using the average value of the areas of a plurality of sites as an index, more accurate screening can be performed.

Next, an evaluation step S40 is performed to evaluate the influence of the component to be searched for on the increase or decrease of the adhesive protein using the area obtained in step S30 as an index.

Fig. 3 shows a flowchart showing step S40 in detail. Referring to fig. 3, an evaluation process in one embodiment for comparing the area of the stained portion of the screening sample with the area of the stained portion of the screening control sample will be described.

In step S41, the area At of the stained part of the screened sample is compared with the area Ac of the stained part of the screened sample.

The comparison of the areas may be performed by comparing absolute values of the areas, or by comparing the ratio of the areas when the area of the screening control sample is 100%.

When a plurality of screened samples are evaluated, it is preferable to perform comparison with the area of the screened control sample set as 100%.

When the area At is smaller than the area Ac (area At < area Ac), the flow proceeds to step S42, and the component to be searched for added to the selection sample is evaluated as a component having an effect of reducing the amount of the adhesion protein.

On the other hand, when the area At is the same as the area Ac or the area At is larger than the area Ac (the area At. ltoreq. the area Ac), the flow proceeds to step S43, and the component to be searched which is added to the selection sample is evaluated as a component having an effect of reducing the amount of the adherent protein.

The evaluation step of one embodiment is described with reference to fig. 3, but the evaluation step in the screening method of the present invention is not particularly limited as long as the influence on the increase or decrease of the adhered protein is evaluated using the area of the stained portion of the screened sample as an index.

For example, when a component increasing the amount of adhesive protein is to be screened, it is determined in step S41 whether or not it is "area At > area Ac", and if "YES" is the result, it may be evaluated in step S42 as "being a component having an effect of increasing the amount of adhesive protein".

In addition, it may be evaluated as "being a component having an effect of reducing the amount of the adhesive protein" in step S42 and as "being a component having an effect of increasing the amount of the adhesive protein" in step S43.

In another embodiment, as described in step S20, a process of preparing a plurality of screening samples having different concentrations of the component to be searched and evaluating them may be used.

In this embodiment, comparing the areas in a plurality of screened samples, in the effect of decreasing or increasing the amount of the adhesion protein, if there is a specific concentration for expressing a significant effect threshold, it can be evaluated as "the component to be explored has an effect of significantly decreasing or increasing the amount of the adhesion protein at a specific concentration or more".

In addition, as for the component which expresses the above-mentioned effect only at a specific concentration or more, it can be evaluated as "the component to be searched expresses the effect of reducing or increasing the amount of the adhesion protein at a specific concentration or more".

Preferably, the screening method of the present invention is used for screening a component for improving and/or preventing sensitive skin.

The term "sensitive skin" as used herein refers to a skin having reduced resistance to external irritation (dryness, ultraviolet rays, and applied compounds) (reduced skin irritation index).

More specifically, "sensitive skin" in the present specification means the skin mentioned in (1) and/or (2).

(1) Skin which is likely to cause skin troubles by reacting with substances such as external pharmaceutical preparations, cosmetics, plants, ultraviolet rays, metals, etc. And skin allergic to allergic substances (pollen, perfume, etc.) or irritant substances (alcohol, etc.) ".

(2) "skin in which skin problems are likely to temporarily occur to irritants in the case of insufficient sleep, excessive fatigue, physiological phase, season change, high mental stress, and the like".

In addition, "for improving and/or preventing sensitive skin" in the present specification means improvement of a skin having a decreased ability to resist external stimuli (a decreased skin irritation threshold) and/or prevention of a decrease in the ability of a skin to resist external stimuli (a decreased skin irritation threshold).

In addition, preferably, the screening method of the present invention is used for screening a fraction having an inhibitory effect on intercellular adhesion junctions with respect to human epidermal keratinocytes.

In the present specification, the term "intercellular adhesive junction" refers to abnormal stratum corneum adhesive junction function (increased multilayered stratum corneum due to increased cell adhesive junction) caused by an increase in desmoglein (proteome containing desmoglein) (see, if necessary, japanese patent No. 4197194 and japanese patent No. 4002635).

In addition, in the present specification, the concept of "inhibiting intercellular adhesive junctions" includes prevention and improvement of intercellular adhesive junctions.

Among them, the effect of inhibiting the multi-layer exfoliation of the horny layer can be obtained by inhibiting the intercellular adhesive connection of human epidermal horny cells to thereby inhibit the multilayering of the horny layer.

That is, the screening method of the present invention can be used for screening a component having a preventive or improving effect on delamination of stratum corneum.

In the present specification, "exfoliation of the stratum corneum" means that the stratum corneum is exfoliated from the skin surface in a state where a plurality of layers are stacked. Whether the skin is in a state of peeling the stratum corneum in multiple layers or not can be confirmed by collecting the stratum corneum by a tape peeling method, staining the collected stratum corneum and observing the stained stratum corneum.

As shown in examples described later, a large amount of desmoglein1 is present in a region with low skin brightness, and it is considered that the reduction in skin brightness is caused by the formation of multiple layers of the horny layer.

According to the above findings, the component having the effect of reducing desmoglein can be said to be a component having the effect of improving skin brightness.

That is, the screening method of the present invention can be used for screening components having an effect of improving skin brightness.

Herein, in the present specification, "improving skin brightness" means that the difference in brightness between the skin in a normal state and the skin in which the brightness is reduced due to an excess of desmoglein is reduced.

Further, as shown in examples described later, the component having the effect of reducing desmoglein has an effect of improving the skin, and the improved skin has a more transparent feeling. That is, the screening method of the present invention can be used for screening a component having an effect of improving skin transparency.

As shown in examples described later, a large amount of desmoglein1 is present in a region with high skin hardness, and it is considered that the skin hardness is mainly caused by the formation of multiple layers of the horny layer.

Based on the above findings, the effect of softening the skin can be obtained by reducing desmoglein. That is, the screening method of the present invention can be used for screening components having a skin softening effect.

Wherein the skin softening allows for more effective penetration of other pharmaceutical agent components. Therefore, the screening method of the present invention can be used for screening a component having an effect of allowing a medicinal component such as a whitening agent to more effectively permeate into the skin.

Examples

[ example 1]

< procedure for preparing stratum corneum cell sample >

Stratum corneum collected from the outside of the human forearm with a transparent tape was transcribed onto a glass slide ("MAS coating", manufactured by Sonlang Nitri Kogyo Co., Ltd.) and the glass slide was soaked in xylene overnight.

Then, the slide glass was washed with xylene for 10 minutes, twice, and then washed with PBS.

< preparation of component to be explored >

Wild Thyme extract (ground extract of Thyme (Wild Thyme, t. serpyllum), a pill of natural beauty corporation) and ROSA roxburghi extract (fruit extract of ROSA roxburghi, a pill of pharmaceutical corporation) were diluted with PBS to concentrations of 0.1 mass%, 0.05 mass%, 0.025 mass%, and 0.0125 mass%, respectively, to obtain a component to be searched.

< preparation of samples for screening >

The stratum corneum cells were soaked overnight in the composition to be explored to obtain a sample. In addition, stratum corneum cells that were not soaked in the component to be explored were prepared as control samples.

< immunostaining assay >

After washing each sample with PBS, the sample was incubated in 4% PFA-phosphate buffer at room temperature for 15 minutes.

Subsequently, each sample was washed with PBS and incubated in 20% aqueous Block ACE (DS Pharma biomedicalal) solution at room temperature for 1 hour.

Primary antibody (Anti-Desmoglein 1, Mouse-Mono) was added to each sample and incubated in a humidified chamber at room temperature for 2 hours.

The samples were washed with PBS for 5 minutes, 3 times, and then a secondary antibody (Allex Fluor @488Goat Anti-mouce IgG) was added and incubated in a humidified chamber at room temperature for 1 hour.

The samples were washed with PBS for 5 minutes, 3 times, and 2 times with distilled water, and then enclosed with Fluoromout-G (manufactured by Southern Biotech), to obtain a plurality of screening samples and screening control samples.

< image analysis >

Images of the screened sample and the screened control sample were taken separately using a confocal laser microscope for analysis (fig. 4).

Using Image analysis software (Image J), an arbitrary 4-point range in the Image for analysis was enlarged and subjected to black-and-white imaging, and the areas of the stained portion and the unstained portion were calculated, and the average value was calculated.

The ratio of the area of the stained portion of the screening control sample (average value at 4) was calculated assuming that the area of the stained portion was 100%. The results are shown in table 1 and fig. 5.

[ Table 1]

Sample(s)	Average	SD
			No additive (control sample)	100	10.98127
0.1% wild thyme	34.46502	14.89497
			0.05% wild thyme	85.26024	4.507292
0.025% wild thyme	87.75052	7.99582
			0.0125% of wild thyme	98.43656	10.21772
0.1% Roxburgh rose extractive solution BG	9.262649	8.040511
			0.05% Roxburgh rose fruit extract BG	72.48284	14.28212
0.025% Roxburgh rose extractive solution BG	74.37402	9.343535
			0.0125% Rosa roxburghii Tratt extract BG	70.43013	8.672259

As shown in fig. 4 and 5, it was found that the expression level of desmoglein1 was reduced in the screening sample to which the wild thyme extract and the rosa roxburghii tratt extract were added, as compared with the screening control sample.

From the results, it can be evaluated that wild thyme extract and rosa roxburghii extract are components having an effect of reducing the amount of desmoglein 1.

In addition, it was found that the area of the stained portion was reduced by about 2.5 times to 2.8 times in the screened sample in which the wild thyme extract was added at a concentration of 0.1 mass% as compared with the screened samples at concentrations of 0.05, 0.025 and 0.0125 mass%.

Similarly, it was found that the area of the stained portion was reduced by 7.6 to 8.0 times in the screened sample in which the rosa roxburghii tratt extract was added at a concentration of 0.1 mass% as compared with the screened samples in concentrations of 0.05, 0.025 and 0.0125 mass%.

From this, it was estimated that the wild thyme extract and the rosa roxburghii extract had the effect of significantly reducing the amount of desmoglein1 at concentrations of 0.1 mass% or more.

[ example 2]

< procedure for preparing stratum corneum cell sample >

Stratum corneum cell samples were prepared in the same manner as in example 1.

< preparation of component to be explored >

An extract of marshmallow (althia Officinalis) root, produced by jurlique corporation, was diluted with PBS to a concentration of 0.25 mass% to obtain a component to be searched.

< preparation of samples for screening >

< immunostaining assay >

Screening samples and screening control samples were obtained in the same manner as in example 1.

< image analysis >

In the same manner as in example 1, images for analysis of the screening sample and the screening control sample were taken, and the ratio 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 set to 100%. The results are shown in table 2, fig. 6 and fig. 7.

[ Table 2]

Sample receiving notebook	Expression level (%) of desmoglein1
		Without addition	100
0.25% by mass of an extract of Althaea officinalis	72.09

As shown in fig. 6 and 7, it was found that the expression level of desmoglein1 was decreased in the screening sample containing the marshmallow extract, as compared with the screening control sample.

From the results, it can be estimated that the marshmallow extract is a component having an effect of reducing the amount of desmoglein 1.

[ example 3]

The results of experiments demonstrating the correlation between the difference in skin brightness and the amount of desmoglein1 present, and the correlation between the amount of desmoglein1 present and skin firmness are shown below.

(1) Test subject and measurement site

In this example, 20 women aged 40 to 52 years were used as subjects.

In the present example, a site where the luminance of the subject's face is reduced (black portion, see site a in fig. 13) and a site in a normal state (see site B in fig. 13) are visually recognized are selected as measurement sites.

(2) Measuring

(2-1) measurement of L, a, b values

A spectrophotometer was placed at the measurement site, and the L value, a value, and b value were measured 5 times for each site.

The results are shown in fig. 8 to 10.

The value of L is clearly lower in a portion where the luminance is reduced (black portion, see portion a in fig. 13) than in a portion in a normal state (see portion B in fig. 13). That is, the portion where the luminance is reduced (black portion, see portion a in fig. 13) is visually recognized as being darker than the portion in the normal state (see portion B in fig. 13).

The value of a is clearly higher in a portion where the luminance is reduced (black portion, see portion a in fig. 13) than in a portion in a normal state (see portion B in fig. 13). That is, the portion where the luminance is reduced (black portion, see portion a in fig. 13) can be visually confirmed to have a stronger red color than the portion in the normal state (see portion B in fig. 13).

The B-value is clearly higher in the portion where the luminance is reduced (black portion, see portion a in fig. 13) than in the portion in the normal state (see portion B in fig. 13). That is, the portion where the luminance is reduced (black portion, see portion a in fig. 13) can be visually confirmed to have a stronger yellow color than the portion in the normal state (see portion B in fig. 13).

(2-2) measurement of hardness

The hardness was measured by pressing a direct-reading indentation hardness tester against each of the measurement sites used in the test of (2-1) above for 5 measurements.

The results are shown in fig. 11.

The hardness is clearly lower in the portion where the luminance is reduced (black portion, see portion a in fig. 13) than in the portion in the normal state (see portion B in fig. 13) as visually recognized. That is, the portion where the luminance is reduced (black portion, see portion a in fig. 13) is visually recognized to be harder than the portion in the normal state (see portion B in fig. 13).

(2-3) bridge core glycoprotein 1(Dsg1) staining

The stratum corneum cells at each measurement site used in the tests (2-1) and (2-2) were collected by tape stripping and adhered to a slide glass.

The slides with the adhered stratum corneum cells were soaked in xylene-evening.

After soaking, washing and air-drying, the mixture was left to stand in 4% paraformaldehyde/phosphoric acid buffer at room temperature for 15 minutes.

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

After soaking, the mixture was washed and allowed to stand in a Block ACE aqueous solution (DS Pharma biological UK-B80) at room temperature for 1 hour.

After standing, the mixture was framed with an immunohistochemical pen (manufactured by Daowu industries Co., Ltd.), primary antibody was added, and the mixture was allowed to stand in a moisture-retaining box at room temperature for 2 hours.

Anti Desmoglein1, Mouse Mono (Dsg 1P 23) Progen,651110 stock (IgG concentration: 10-15g/mL) was used as the primary antibody.

After standing, washing was performed with PBS, a secondary antibody was added, and the mixture was left standing in a humidity-retaining box at room temperature for 1 hour.

A200-fold diluted solution of Alle xa Fluor @488 coat Anti mouse IgG Invitrogen, A11001 PBS was used as the secondary antibody.

After standing, the plate was washed, sealed with Fluoromout-G (Southern Biotech) and dried at room temperature for 30 minutes.

After drying, the glass cover plate was fixed with nail polish and dried, and then used for observation (photographing) using a confocal laser microscope (Nikon a1) × 20.

The results are shown in fig. 12.

The amount of desmoglein 1(Dsg1) was clearly higher in the region where the brightness was reduced (black region, see region a in fig. 13) than in the region in the normal state (see region B in fig. 13). That is, the amount of desmoglein 1(Dsg1) was larger in the region where the decrease in brightness was visually observed (black region, see region a in fig. 13) than in the region in the normal state (see region B in fig. 13).

(3) Investigation of

As shown in fig. 8 to 10 and 12, it is found that the desmoglein1 is present in a large amount in the region of low skin brightness. Among them, the decrease in skin brightness is considered to be caused by a low pH of the skin, a low activity of serine protease which decomposes desmoglein1, and a dense overlapping of horny layers (multilayering of horny layers).

Moreover, as described above, the effective ingredient of the invention of the present application functions to reduce desmoglein.

From the above findings, the active ingredient of the present invention plays a role in improving skin brightness by reducing desmoglein 1.

As shown in FIGS. 11 to 12, it is clear that a large amount of desmoglein1 is present in the skin of a site with high hardness. Among them, it is considered that the hardness of the skin is mainly influenced by the low pH of the skin, the low activity of serine protease which decomposes desmoglein1, and the density of overlapping of the horny layer (multilayering of the horny layer).

Based on the above findings, it is found that the skin becomes soft by reducing desmoglein 1.

That is, it is known that the effective ingredient of the present invention plays a role of softening the skin by reducing desmoglein 1.

[ example 4]

The following shows the results of the validation of the effect of the composition containing the component having the effect of reducing desmoglein on inhibiting the multi-layer exfoliation.

(1) Preparation of the formulations

The cosmetic of production example 1 containing a rosa roxburghii tratt extract was prepared according to the composition described in table 3.

[ Table 3]

Components	Production example 1
		Fatty acids	40
Glycerol	19.2
		Diglycerol	3.5
Sodium carboxymethylcellulose	0.4
		Acrylic acid-dimethyl diallyl ammonium chloride-acrylamide copolymer liquid	2
Dimethyldiallylammonium chloride-acrylamide copolymer liquid	4.5
		Potassium hydroxide	8.5
Alkyl glycosides	1.5
		Rosa roxburghii extract	0.4
Water (W)	20
		Total up to	100

(2) Subject and target site

In this example, 5 women were used as subjects, and the cosmetic of production example 1 was applied to the inner sides of the left and right forearms (n ═ 10) of the subjects 2 times a day (morning and evening) for 10 days.

(3) Demonstration of desmoglein reduction

In each of the stages before and after use (after 10 consecutive days) of the cosmetic of production example 1, stratum corneum cell samples were prepared by collecting the stratum corneum on the inner left forearm and inner right forearm of each subject in accordance with the method described in "procedure for preparing stratum corneum cell sample" in example 1. Next, desmoglein1 in the stratum corneum cell sample was stained by the method described in "< immunostaining test >" of example 1, and images for analysis before and after using production example 1 were taken using a confocal laser microscope to confirm fluorescence (staining).

The area of the fluorescent portion was calculated assuming that the area of the entire image used for analysis was 100%. The results are shown in fig. 14 and 15. In fig. 15, p is < 0.05.

As shown in fig. 14 and 15, it was confirmed that the amount of desmoglein1 was reduced by continuously using the cosmetic of production example 1 containing a rosa roxburghii tratt extract.

(4) Verification of multilayer peeling inhibition

Stratum corneum cell samples were prepared by collecting stratum corneum on the inner left forearm and inner right forearm of each subject in accordance with the method described in "procedure for preparing stratum corneum cell sample" in example 1 before and after using the cosmetic of production example 1. Next, each stratum corneum cell sample was stained using gentian violet and brilliant green by a conventional method and observed with an optical microscope.

A representative graph of the results is shown in fig. 16.

As shown in fig. 16, there were a plurality of darker portions in the optical microscope image of the stratum corneum cells before production example 1. The darker part is a part where stratum corneum cells were peeled off in multiple layers by a tape peeling method.

On the other hand, in the stratum corneum cell samples collected after 10 consecutive days of use in production example 1, most of the staining was lighter than that in the stratum corneum cell samples collected before use. The shallower portion is a portion where stratum corneum cells are exfoliated by only one layer, that is, multi-layer exfoliation does not occur.

As described above, it was confirmed that by using the cosmetic of production example 1 containing a component having a desmoglein reducing effect, multi-layer exfoliation was improved.

[ example 5]

Hereinafter, the ability of a composition containing a component having a desmoglein-reducing effect to reduce desmoglein was investigated.

(1) Preparation of the composition

Cosmetics (emulsion) containing wild thyme extract were prepared according to the formulation shown in table 4 below. That is, the components (a), (b), and (c) were heated to 7O c, respectively, and (c) was added to (b) to neutralize it, and (a) was slowly added to emulsify it while stirring, and after homogenizing with a homogenizer, it was cooled while stirring, and the emulsion of production example 2 was obtained.

[ Table 4]

(2) Test subject and measurement site

In this example, 20 women aged 40 to 52 years were used as subjects.

Then, in the present embodiment, a site (at 92) where a spot is present on the face of the subject is selected as a measurement site.

The emulsion of production example 1 was applied to the spot-existing part of each subject 1 day and 2 times for 12 weeks.

(3) Measuring

Stratum corneum cell samples were prepared by collecting stratum corneum cells at the site where the spots were present in the subject, according to the method described in "stratum corneum cell sample preparation step" in example 1, at the time point of 6 weeks and at the time point of 12 weeks before the use of the emulsion of the production example. Next, desmoglein1 in each stratum corneum cell sample was stained by the method described in "< immunostaining test >" of example 1, and images for analysis before and after production example 2 were taken using a confocal laser microscope to confirm fluorescence (staining).

The area of the fluorescent portion was selected with the area of the entire image for analysis set as 100%. The results are shown in table 5, table 6, fig. 17 (representative), and fig. 18 and 19. In addition, data at 80 in spot location 93 was counted for data after 12 weeks of using the emulsion and compared with data before using the emulsion in the same location. In fig. 18, p < 0.01, and in fig. 19, p < 0.001.

[ Table 5]

Bridge core glycoprotein 1 amount (%)	Before use	Continuously used for 6 weeks
			Average	23.9	20.9
Standard deviation of	9.42	9.68
			Number of samples	n＝92	n＝92

[ Table 6]

Bridge core glycoprotein 1 amount (%)	Before use	Continuously using for 12 weeks
			Average	23.2	10.6
Standard deviation of	7.88	9.62
			Number of samples	n＝80	n＝80

As shown in tables 5 to 6 and FIGS. 17 to 19, it was confirmed that the amount of desmoglein1 in the spot portion was significantly reduced by continuously using the emulsion containing the wild thyme extract.

Industrial applicability

The present invention is applicable to an analysis method relating to the amount of adhesion protein.

Claims

1. A method of screening for a component that affects the increase or decrease of stratum corneum intercellular adhesion protein, comprising:

a cleaning step of cleaning the slide glass;

an evaluation step of evaluating the influence of the component to be searched for on the increase or decrease in the adherent protein using the area of the stained portion in the image for analysis as an index.

2. The screening method according to claim 1, wherein in the preparation step, a screening control sample to which the component to be searched is not added is prepared,

3. The screening method according to claim 1, wherein in the preparation step, a plurality of screening samples in which the component to be searched is the same in type and different in concentration are prepared,

4. Screening method according to any one of claims 1 to 3, wherein the adhesion protein is desmoglein 1(desmoglein 1).

5. The screening method according to any one of claims 1 to 4, wherein the slide glass has NH arranged on the surface thereof₃ ⁺The slide glass of (1).

6. The screening method according to any one of claims 1 to 5, wherein the washing process is a process of immersing the slide glass in xylene.

7. A method for preparing a stratum corneum cell sample by a tape stripping method, comprising:

and a cleaning step of cleaning the slide glass.

8. The preparation method according to claim 7, wherein the slide glass has NH arranged on its surface₃ ⁺The slide glass of (1).