JP2020160028A - Screening method of stress-induced skin barrier function improvement agent - Google Patents

Abstract

To provide a screening method of an improvement agent for skin barrier function deteriorated due to stress, and also to provide a more highly effective skin barrier function improvement agent.SOLUTION: A method is for screening a rough skin surface improvement agent by using, as an index, an epidermal cell differentiation-related protein reduced by addition of enkephalin or an expression level of a gene coding thereof, through utilizing a cultured epidermal cell. Furthermore, a method is for screening a rough skin surface improvement agent by using, as an index, a skin barrier function deteriorated by addition of enkephalin, through utilizing a skin three-dimensional model.SELECTED DRAWING: Figure 11

Description

The present invention relates to a method for screening an agent for improving rough skin caused by stress. Specifically, a method for screening a skin roughness improving agent caused by stress using the degree of cell differentiation of enkephalin-added epidermal cells as an index and / or the skin barrier function of a three-dimensional skin model to which enkephalin was added as an index. It relates to a rough skin improving agent caused by stress selected using the method.

It has long been said that modern society is a stressful society, but the stress that people living in modern society receive is largely due to human relationships, work, home, schoolwork, money, overcrowding of crowded trains, etc. Occupy. When this stress exceeds a certain level, the homeostasis of the living body is disrupted, causing physical and mental disorders, mental illnesses such as anxiety disorders and depression, gastric / duodenal ulcers, hypertension, lifestyle-related diseases, etc. It causes various physical illnesses. Further, it is known that in the skin, which is an organ that separates the living body from the outside world, the deterioration of the barrier function of the skin makes it more susceptible to the stimulation of the outside world and promotes rough skin.

The skin is located at the boundary between the living body and the outside world, and has a skin barrier function to prevent the ingress of substances from the outside and the evaporation of water from the inside of the skin, and plays an important role in maintaining the living body. ing. The skin is an epithelium having a thickness of 100 to 200 μm consisting of a stratum corneum, a granule cell layer, a spinous cell layer, and a basal cell layer, and the stratum corneum, which is the outermost layer of the skin, is most important for the skin barrier function. The stratum corneum is composed of keratinocytes formed by terminal differentiation of epidermal cells in the basal cell layer and intercellular lipids surrounding them. Keratinocytes are mainly composed of keratin fibers having SS bonds, contain filaggrin and natural moisturizing factor NMF, which play an important role in moisturizing function, and further consist of a cornified envelope that encloses them. On the other hand, intercellular lipids have a lamellar structure composed of ceramide, cholesterol, fatty acids and the like (Non-Patent Document 1).

The cornified envelope that encloses keratinocytes is formed by cross-linking and insolubilizing a plurality of cornified envelope precursor proteins such as involucrin and loricrin, which are produced according to the differentiation of epidermal cells, and matures. Furthermore, it is suggested that ceramide, which is an intercellular lipid, is covalently bound to a part of the cell, and that it provides a base for the lamellar structure of the intercellular lipid described above. In addition, keratinocytes are bound to each other by a corneodesmosome composed of desmoglein and desmocollin, which are intercellular adhesion molecules, and have a tough sheet structure.

For the skin barrier function, components in keratinocytes such as keratin fibers having SS bonds generated by terminal differentiation, filaggrin, natural moisturizing factor, and cornified envelope play an important role, and further, adhesion between keratinocytes. And intercellular lipids such as ceramide also play a major role. It is important that these are produced and matured normally, and that the stratum corneum is normally formed and functions.

Conventionally, as a screening method for materials for improving the deterioration of skin condition caused by psychological stress such as deterioration of skin barrier function, the skin barrier function is applied to mice by applying overcrowding stress, which is recognized as socio-psychological stress. A method of screening a material for improving the deterioration of the skin condition caused by stress by inducing rough skin such as deterioration of the skin condition has been implemented (Patent Document 1). However, since this method requires an animal experiment, the experimental operation is complicated and has an ethical problem. In addition, screening by a method of reducing stress itself by fragrance or the like has been proposed (Patent Document 2), but since these attempts require testing in humans or animals, the experimental operation is complicated and ethical. In addition, the burden on the subject is large because the process of invasively collecting the substance in the living body is required. Furthermore, in the case of humans, there is a problem that palatability has a great influence on the effect and individual differences in the effect are large.

On the other hand, since it is known that active oxygen is partially generated in the body as a result of stress loading, a method of selecting an anti-stress material by regarding the anti-oxygenizing action as an anti-stress action has been implemented (Patent Documents). 3). However, this method only improves some of the adverse effects of stress mediated by reactive oxygen species. In other words, there are many problems in conducting screening of materials for improving the deterioration of skin barrier function caused by stress, and there is no effective and simple measurement method.

It is known that psychological stress causes physiological changes in the living body, and it is known that the adrenal gland releases corticosteroid (cortisol) and the amount of adrenaline released in the brain increases. It has also been reported that endogenous opioid peptides are released and increased from the central nervous system and adrenal medulla. Opioid is a general term for substances that have an action similar to that of morphine, which is a narcotic analgesic, through binding to opioid receptors present in the nervous system, and has actions such as analgesia and induction of euphoria by narcotic-like action. Endogenous opioid peptides present in the body show similar effects, but enkephalin and its derivatives, which are a type of endogenous opioid peptides, have a lipolytic effect, as well as skin hydration and improvement of lightness, and facial wrinkles. Usefulness for skin conditions such as reducing action has been reported (Patent Document 4). All of these indicate that administration or topical administration of enkephalin or its derivatives has beneficial effects on the living body, but it has not been conventionally considered that enkephalin is involved in living body disorders, especially on the skin. The possibility of adverse effects was also not envisioned.

Kazunoibara (Hypnea flexicalis or Hypnea cervicornis) of the genus Ibaranori of the family Ibaranori is a seaweed that inhabits various parts of Japan. So far, the effect of Kazunoibara on improving rough skin caused by stress and the possibility of having some effect on the effect of enkephalin on the living body have not been investigated at all.

Japanese Patent Application Laid-Open No. 10-279505 Japanese Patent Application Laid-Open No. 2008-297554 Japanese Patent Application Laid-Open No. 8-275752 Special Table 2008-534658

Drug Delivery System, 22 (4), 424-432, 2007

In view of the above background techniques, the present inventors have investigated a method for improving the deterioration of the barrier function of the epidermis caused by stress. As a result, they found that enkephalin, which increases during psychological stress, is involved in the deterioration of skin barrier function during stress, rather than having the function of improving the deteriorated skin condition as in the known action. Specifically, we found that the amount of enkephalin in saliva, which increases during psychological stress, has a positive correlation with the evaporation of water from the skin (transepidermal water evaporation). Furthermore, it was confirmed that women who felt that their skin dryness worsened during stress had a higher amount of enkephalin in saliva than women who did not, and that there was a relationship between the deterioration of skin condition during stress and the amount of enkephalin in vivo. It was strongly suggested.

Further research revealed that enkephalin causes deterioration of skin barrier function by suppressing the differentiation of epidermal cells and inhibiting the formation of normal stratum corneum. Specifically, it was confirmed that the addition of enkephalin to cultured epidermal cells reduces the gene expression levels of differentiation-related genes in epidermal cells such as keratin 1, keratin 10, involucrin, and profilaggrin. In addition, in the three-dimensional skin model to which enkephalin was added, normal stratum corneum formation was inhibited, and immature stratum corneum was formed with thick stratum corneum and a small amount of SS bonds, which is an indicator of stratum corneum maturation. We found that the amount of transepidermal water loss in the three-dimensional skin model increased.

From the above new findings, enkephalin increased in the body during stress is carried to the epidermis of the skin by the blood, suppresses the differentiation of epidermal cells of the skin, and inhibits the normal formation of the stratum corneum, which adversely affects the skin barrier function. It was strongly suggested that Therefore, if the adverse effect of enkephalin on the skin can be suppressed, it is expected that the deterioration of the skin barrier function during stress can be suppressed and the rough skin caused by stress can be improved. Therefore, the present invention has been completed. I arrived.

An object of the present invention is to provide a method for easily screening an agent for improving rough skin caused by stress by using cultured cells without performing animal experiments or human tests, and in addition, more effective stress. An object of the present invention is to provide an agent for improving rough skin caused by stress.

Based on the above findings, the present invention is an agent for improving rough skin caused by stress by using the decreased expression level of epidermal cell differentiation-related protein or a gene encoding the same as an index when enkephalin is added to cultured epidermal cells. Is a method of screening. Furthermore, it is a method of screening for a skin roughness improving agent caused by stress by using the lowered skin barrier function as an index when enkephalin is added to a three-dimensional skin model.

That is, the present invention is as follows.
[1]
A method for screening an agent for improving rough skin caused by stress using the degree of cell differentiation of epidermal cells and / or the skin barrier function of a three-dimensional skin model as an index in the presence of enkephalin.
[2]
(1) A step of adding enkephalin and a test substance to epidermal cells and culturing them.
(2) Steps to measure the degree of epidermal cell differentiation,
(3) A method for screening a skin roughness improving agent caused by stress, in which the degree of epidermal cell differentiation obtained in step (2) is compared with the group without the addition of the test substance, and the substance that improves the degree of epidermal cell differentiation is determined to be an effective substance. ..
[3]
The method according to either [1] or [2], wherein the degree of differentiation of epidermal cells is the expression level of a protein whose expression level increases with the differentiation of epidermal cells or the expression level of a gene encoding the same.
[4]
The method according to any one of [1] to [3], wherein the protein comprises at least one selected from keratin 1, keratin 10, involucrin, loricrin, filaggrin, caspase 14, and transglutaminase.
[5]
When the expression level of the protein or its gene is increased by 10% or more as compared with these expression levels when the test substance is not added, it is judged that the test substance has an effect of improving rough skin caused by stress [1]. ] To [4].
[6]
(1) A step of adding enkephalin and a test substance to a three-dimensional skin model and culturing the skin.
(2) Steps to measure the skin barrier function of a three-dimensional skin model,
(3) A method for screening a skin roughness improving agent caused by stress, in which the skin barrier function obtained in step (2) is compared with the group without the addition of the test substance, and the substance that improves the skin barrier function is determined to be an effective substance.
[7]
An agent for improving rough skin caused by stress selected by any of the methods [1] to [5].
[8]
A stress-induced rough skin improver containing Kazunoibara extract.
[9]
An agent for improving skin barrier function deterioration caused by stress, which contains Kazunoi rose extract.
[10]
Use of Kazunoibara extract to improve rough skin caused by stress.

INDUSTRIAL APPLICABILITY The present invention provides a method for screening a rough skin improving agent capable of easily improving the deterioration of the skin barrier function caused by stress by using cultured cells without performing animal experiments or human tests. In addition, a rough skin improving agent based on a new mechanism is provided.

The figure which shows the change of the amount of enkephalin in saliva and the amount of cortisol in saliva before and after stress loading. The figure which shows the correlation between the amount of enkephalin in saliva and the amount of transepidermal water loss (TEWL). The figure which shows the relationship between the deterioration of dryness under stress and the amount of enkephalin in saliva. The figure which shows the decrease of the cell differentiation degree of the epidermal cell by the addition of enkephalin. The figure which shows the decrease of the cell differentiation degree of the skin three-dimensional model by the addition of enkephalin. The figure which shows the change of the stratum corneum morphology of the skin three-dimensional model by the addition of enkephalin. The figure which shows the change of the stratum corneum maturity (SS bond increase degree) of the skin three-dimensional model by the addition of enkephalin. The figure which shows the change of the transepidermal water loss amount of the skin three-dimensional model by the addition of enkephalin. The figure which shows the improvement of the cell differentiation suppression of the enkephalin-added epidermal cells by the addition of various plant extracts. The figure which shows the influence on the cell differentiation degree of the epidermal cells which did not add enkephalin by the addition of various plant extracts. The figure which shows the improvement of the stratum corneum morphology of the enkephalin-added skin three-dimensional model by addition of various plant extracts. The figure which shows the improvement of the transepidermal water loss amount of the enkephalin-added skin three-dimensional model in addition of the Kazunoi rose extract. The figure which shows the relationship between the type of skin external preparations used and the amount of transepidermal water loss.

Due to stress, enkephalin in the body increases. The screening method of the present invention mimics the skin affected by stress, and is a method of selecting a test substance that improves rough skin caused by stress.

One embodiment of the screening method of the present invention is a method of screening an agent for improving rough skin caused by stress by using the degree of epidermal cell differentiation of cultured epidermal cells as an index.
Specifically, it includes a step of adding enkephalin and a test substance to epidermal cells and culturing, a step of measuring to obtain the degree of epidermal cell differentiation, and a step of comparing the degree of epidermal cell differentiation and selecting an agent for improving skin roughness caused by stress. This is a screening method.

The enkephalin used in the present invention includes leucine-enkephalin (tyrosine-glycine-glycine-phenylalanine-leucine), methionine-enkephalin (tyrosine-glycine-glycine-phenylalanine-methionine), which are structures in which five amino acids are linked, and the above-mentioned enkephalin. Peptides containing the sequence and peptides derived from the enkephalin sequence can be used.

The epidermal cells used for the screening of the present invention are not particularly limited as long as they are keratinocytes, such as normal (non-cancerous) keratinocytes isolated from the epidermis of humans or animals other than humans, and immortalized keratinocyte cell lines. Can be used, and normal keratinocytes isolated from human epidermis are more preferred. As the culture form of epidermal cells, in addition to two-dimensional culture (monolayer culture), three-dimensional culture (multilayer culture) such as a three-dimensional skin model can be used, under conditions that do not inhibit the survival of epidermal cells. If so, it can be applied endlessly.

As a method for culturing epidermal cells to which enkephalin and a test substance have been added, there are no particular restrictions as long as normal culture conditions, for example, a commercially available medium for exclusive use of keratinocytes, and culture conditions that do not interfere with the execution of the screening method of the present invention Can be applied. In order to make it easier to confirm the effect of enkephalin or the test substance on the cell differentiation of epidermal cells, an operation of adding calcium ions to the medium during epidermal cell culture to induce the differentiation of epidermal cells may be performed. It is preferable to add enkephalin and a test substance to the epidermal cells to allow the cells to receive these signals, and then perform an operation for inducing differentiation of the epidermal cells by adding calcium ions.

The degree of epidermal cell differentiation is not particularly limited, but can be grasped by the expression level of a protein whose expression level increases with the differentiation of epidermal cells or the expression level of a gene encoding the same. Specifically, it can be grasped by the expression level of a protein containing at least one selected from keratin 1, keratin 10, involucrin, loricrin, filaggrin, caspase 14, and transglutaminase, or the expression level of a gene encoding the same. it can. From the viewpoint of screening accuracy, it is preferable to combine two or more kinds, for example, a combination of keratin 1 or keratin 10, and involucrin and filaggrin is preferable.

In order to use the degree of epidermal cell differentiation as an index, the result of measuring the expression level of the protein or the expression level of the gene encoding the protein by any method can be used. For example, the result of quantitatively measuring the intracellular abundance of the protein by a conventional method such as a Western blotting method, an ELISA method, a radioimmunoassy method, an immunostaining method, or a mass spectrometry method may be used. Further, the amount of the protein can be indirectly measured by measuring the metabolite of the protein. In addition, the expression level of the gene is quantitatively detected by agarose gel electrophoresis or the like by performing PCR using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer. The gene sequences encoding the various factors described above are publicly available, and those skilled in the art can appropriately design primers and use them for PCR. In addition, nucleic acid hybridization method using immobilized samples such as gene chips and arrays, RT-PCR method, real-time PCR method, subtraction method, differential display method, differential hybridization method, cross hybridization method, etc. It can be measured using a known method.

In determining the effect of improving rough skin caused by stress, it can be set according to the degree of the desired effect, but as a guide, the expression level of the protein in epidermal cells cultured for several days or the expression level thereof is encoded by adding enkephaline and a test substance. When the expression level of the gene to be used is increased as compared with these expression levels in the cells to which the test substance was not added, the expression level is preferably increased by 10% or more, more preferably 15% or more, still more preferably 20% or more. If so, it can be determined that the test substance has an effect of improving rough skin caused by stress.

Another embodiment of the screening method of the present invention is a method of screening a skin roughness improving agent caused by stress by using the skin barrier function of a three-dimensional skin model as an index.
Specifically, a step of adding enkephaline and a test substance to a three-dimensional skin model and culturing it, a step of measuring the skin barrier function of the three-dimensional skin model, comparing the skin barrier function, and selecting a skin roughness improving agent caused by stress. A screening method that includes steps.

As a three-dimensional skin model used for the screening of the present invention, normal (non-cancerous) skin cells such as keratinocytes, melanocytes, and fibroblasts collected from the skin of humans or animals other than humans are three-dimensionally cultured for several days. A three-dimensional (laminated) structure can be constructed to imitate the structure of the skin and used. Preferably, the culture period is shortened, and a state in which terminal differentiation of keratinocytes is incomplete and keratinocytes are not formed is prepared and used. For example, after sowing in a three-dimensional culture vessel such as an insert for cultured cells and culturing for 3 days, the culture medium inside the insert is removed, exposed to air, and culturing is continued for several days to construct a three-dimensional structure. The culture period after air exposure is a period during which terminal differentiation of keratinocytes is incomplete and keratinocytes are not formed, preferably about 1 to 7 days, and more preferably the shortest one day. Further, instead of the insert for cultured cells, a collagen gel containing collagen gel or fibroblast, a glass ring may be placed on the dermis extracted from the living body, and keratinocytes mixed with keratinocytes or melanocytes may be seeded in the ring. It is also possible to purchase and use a commercially available product for constructing such a form. Preferred commercial products include, for example, a three-dimensional skin model (immature stratum corneum type) MEL-301 (Kurabo Industries), a three-dimensional skin model (immature stratum corneum type) EPI-201 (Kurabo Industries Ltd.), and LabCyte EPI-Model 6D ( J-TEC), LabCite EPI-KIT (J-TEC), 3D FT Starter Kit (CELLnTEC) and the like can be used. In particular, LabCite EPI-KIT (J-TEC), which can construct a three-dimensional structure by itself, and 3D FT Starter Kit (CELLnTEC) can be more preferably used. Further, LabCite EPI-KIT (J-TEC) composed only of keratinocytes can be most preferably used.

As a method for culturing the skin three-dimensional model to which enkephaline and the test substance are added, enkephaline is added to the medium of the skin three-dimensional model prepared by the above method, or the test substance is added to the stratum corneum side of the skin three-dimensional model. However, it can be cultured for a period until the terminal differentiation of keratinocytes is completed and keratinocytes are formed. Since cell differentiation of keratinocytes is induced by air exposure, it is preferable to add the test substance to the medium and culture it. The culture period is preferably 1 to 18 days, and more preferably 7 to 14 days in which the stratum corneum is layered and the stratum corneum is sufficiently formed.

In the screening method of the present invention, the skin barrier function is selected from the stratum corneum thickness, the stratum corneum maturity (SS bond increase), the cornified envelope maturity, and the transdermal water evaporation amount formed in the skin three-dimensional model. It can be evaluated by at least one kind of measured value or observation image.
The stratum corneum is composed of keratinocytes The keratinocytes that compose the stratum corneum are mainly composed of keratin fibers having SS bonds generated by terminal differentiation, and play an important role in moisturizing function. Filaggrin and natural moisturizing factor NMF Consists of a cornified envelope that contains and encloses them. In addition, keratinocytes are bound to each other by a corneodesmosome composed of cell-cell adhesion molecules, and have a tough sheet structure. The stratum corneum exists as a boundary film between the living body and the outside of the living body having a thickness of 10 to 20 μm by overlapping many layers of this sheet structure. With such a configuration, the amount of water inside the skin is maintained and the loss of water (transdermal water evaporation) is suppressed, so that the skin barrier function is normally maintained. That is, it is important that a stratum corneum having a sufficient thickness is formed, that the constituent molecules of the stratum corneum are normally produced and matured, and that the stratum corneum is normally formed and functions.

The thickness of the stratum corneum can be calculated or evaluated by observing a section image of a three-dimensional skin model under a microscope. Specifically, the sections are stained by an arbitrary staining method capable of staining the living cell layer and the stratum corneum, such as hematoxylin-eosin staining and safranin staining, and the stratum corneum morphology is observed with an optical microscope. The thickness of the stratum corneum can be determined by immunostaining the components present in the cells using an antigen-antibody reaction and observing the morphology of the stratum corneum with a fluorescence microscope or an optical microscope, or by observing the morphology of the sections with a transmission electron microscope. A method of calculation or evaluation is used. In addition, a method of obtaining the water content distribution in the stratum corneum of a three-dimensional model by a Raman spectroscope and calculating the stratum corneum thickness from this inflection point can be used, as well as ultrasonic tomography and optical coherence tomography (optical coherence tomography). A known method can be used, such as a method of obtaining a tomographic map of a three-dimensional skin model by an Optical Coherence Tomology) or a confocal laser scanning microscope and calculating the stratum corneum thickness from the tomographic map.

The maturity of the stratum corneum (increase in SS bond) can be evaluated by a known method such as a method of staining SS bonds in a three-dimensional skin model with N- (7-dimethylamino-4-methylcuminyl) maleimide. it can. Specifically, the free SH group is protected by reacting with N-ethylmaleimide, and the SS group is cleaved with a reducing agent such as 2-mercaptoethanol or dithiothreol, and then the produced free SH group is N- ( When reacted with 7-dimethylamino-4-methylcummarinyl) maleimide, the obtained fluorescent image can be used as the abundance of SS bonds.

The cornified envelope maturity can be evaluated by a known method. Specifically, the stratum corneum is collected from a three-dimensional skin model by tape stripping or trypsin treatment, mixed with an eluate (tris-hydrochloric acid buffer containing dithiothreitol and sodium dodecyl sulfate), and centrifuged to remove the eluted components. The insoluble material is used as a cornified envelope. The involucrin contained in this is fluorescently immunostained by an antigen-antibody reaction, further stained with NileRed, and the entire area of the cornified envelope (involucrin-positive CE area + NileRed-positive) is obtained from the fluorescence observation images of involucrin and Nile Red obtained by a fluorescence microscope. By calculating the ratio of immature CE in (CE area), the degree of maturity of the cornified envelope can be evaluated.

The amount of transepidermal water loss can be measured using a known method, and a commercially available measuring device such as TEWA Meter (Courage + Khazaka), Tewiro TM24 (Courage + Khazaka), VAPO SCAN (Asahi Techno) can be used for a three-dimensional skin model. ) Can be applied and measured.

When the skin barrier function in the skin three-dimensional model cultured with the addition of enkephaline and the test substance is changed as compared with the skin barrier function in the skin three-dimensional model without the test substance, the test substance is stressed. It is judged that it has an effect of improving rough skin caused by. Specifically, in the group to which the test substance was added, the thickness of the stratum corneum, the maturity of the stratum corneum (increase in SS binding), and the maturity of the cornified envelope among the skin barrier functions increased as compared with the group to which the test substance was not added. When the amount of transepidermal water loss decreases, it can be determined that the test substance has an effect of improving rough skin caused by stress.

The rough skin improving agent or the skin barrier function lowering improving agent caused by the stress of the present invention is selected by the screening method.

The component that can be expected to have a rough skin improving effect selected by the screening method of the present invention is Kazunoi rose extract.

The Kazunoibara is a Kazunoibara (Hypnea flexicaulis or Hypnea cervicornis) of the genus Ibaranori of the family Ibaranori, and the site is not particularly limited, but for example, whole plants can be preferably used.

The preparation of the extract is not particularly limited, but the extract is extracted from low temperature to warm using, for example, various suitable organic solvents. Examples of the extraction solvent include water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol and 1,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; ethyl acetate and the like. Alkyl esters; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; one or more of alcohol halides such as dichloromethane and chloroform can be used. Among them, one or more mixed solvents of water, ethyl alcohol and 1,3-butylene glycol are particularly suitable. Furthermore, water is more preferred.

The extraction method of the extract that can be used in the present invention is not particularly limited, but in the case of normal temperature extraction, for example, if it is dried, a solvent in an amount of 1 to 1000 times, particularly 10 to 100 times the mass ratio is used. Is preferably carried out at 0 ° C. or higher, particularly 20 ° C. to 40 ° C. for 1 hour or longer, particularly 3 to 7 days. Alternatively, it may be heat-extracted at 60 to 100 ° C. for 1 hour.

Each of the extracts obtained under the above conditions may be used as it is in the extracted solution, but if necessary, it may be filtered or purified, and then concentrated or powdered as appropriate. Can be used.

The blending amount of the plant extract in the external preparation for skin of the present invention is preferably 0.0000 to 1 to 20.0% by mass in terms of evaporation and drying, and particularly preferably in the range of 0.01 to 10.0% by mass. is there.

Moreover, the test substance selected by the screening method of the present invention is not limited to the plant extract. Any compound may be used as long as it is selected by the screening method of the present invention, and extracts of fungi, animals and the like may be used in addition to plant extracts.

The rough skin improving agent of the present invention means a composition applied to the skin. For example, a composition that can be applied as a skin external preparation such as cosmetics such as lotions, milky lotions, creams, oils, makeup bases, foundations, quasi-drugs, dispersion liquids, ointments, creams, external medicines, etc. Say. It does not matter whether it is in the form of liquid, cream, powder, solid or the like.

The rough skin improving agent of the present invention is produced by a conventional method using a known base ingredient in addition to the active ingredient. Other ingredients can be blended as long as the effects of the present invention are not impaired. For example, fats and oils, waxes, hydrocarbon oils, ester oils, higher alcohols, silicone oils, UV absorbers, UV scatterers, moisturizers, surfactants, water-soluble polymers, thickeners, powders, skin protectants. , Whitening agent, wrinkle improving agent, antiaging agent, plant extract, preservative, anti-inflammatory agent, pH adjusting agent, metal ion sequestering agent, antioxidant and the like.

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

<Experiment 1> Examination of the relationship between stress load and the amount of enkephalin in the body The changes in the amount of enkephalin in the body of humans loaded with psychological stress were examined by the following procedure.
Eight men and women in their twenties and thirties who consented to the test were asked to perform a Kleperin test, and saliva was collected before and after the test. The Kleperin test is a continuous simple calculation test and is widely used as a minor psychological stress stress test. Before the Kleperin test, each subject was asked to rinse the oral cavity with water and washed, and after 5 minutes, a sponge of Sullivet (Sarstedt) was placed in the oral cavity for 90 seconds, and saliva before the Kleperin test was collected. After performing the simple calculation Kleperin test for 15 minutes, the sponge of Sullivet (Sarstedt) was placed in the oral cavity again for 90 seconds, and saliva after the Kleperin test was collected. The sponge from which saliva was collected was returned to the saliva tube, and the tube was centrifuged at 4 ° C. and 3,000 × g for 5 minutes to collect saliva. Saliva is diluted 3-fold with phosphate buffered saline (PBS (-)), the amount of enkephalin in saliva is ELISA Kit for Enkephalin (USCNLIFE), and the amount of cortisol in saliva is Salivary Crotisol ELISA Kit (Salimetrics). Measured using.
As shown in FIG. 1, the amount of enkephalin in saliva increased due to the load of slight psychological stress. At this time, there was almost no change in the amount of cortisol in saliva, suggesting that enkephalin is a substance that responds to stress much more sensitively than cortisol, which is conventionally known to increase in the body due to stress. .. It has been reported that saliva can be collected non-invasively and easily, and that the concentration of substances present in saliva (for example, cortisol and estradiol) has a strong correlation with the blood concentration, and changes in saliva substance concentration. Can be regarded as almost synonymous with the change in concentration in the living body.

<Experiment 2> Examination of the relationship between the amount of enkephalin in vivo and the skin barrier function The relationship between the amount of enkephalin in vivo and the amount of transepidermal water loss in the skin was examined by the following procedure.
Nine women in their twenties and thirties who consented to the test were treated with facial cleansers, acclimatized for 20 minutes in an environment of 22 ± 1 ° C and 50 ± 2% humidity, and then TEWA meter TM- The amount of transepidermal water loss on the face and cheeks was measured with 210 (Courage + Khazaka). In addition, the oral cavity was rinsed with water and washed, and after 5 minutes, a sponge of Sullivet (Sarstedt) was soaked in the oral cavity for 90 seconds to collect saliva. The sponge from which saliva was collected was returned to the saliva tube, and the tube was centrifuged at 4 ° C. and 3,000 × g for 5 minutes to collect saliva. Saliva is diluted 3-fold with phosphate buffered saline (PBS (-)), and the amount of enkephalin in saliva is measured by ELISA Kit for Enkephalin (USCNLIFE), and the amount of transdermal water evaporation and the amount of enkephalin in saliva The relationship was analyzed.
As shown in FIG. 2, there is a strong positive correlation between the amount of enkephalin in human saliva and the amount of transepidermal water loss in the skin, and the amount of transepidermal water evaporation tends to be higher in humans with a larger amount of in vivo enkephalin. I understand.

<Experiment 3> Examination of the relationship between the deterioration of skin condition caused by stress and the amount of enkephalin in the body The relationship between the deterioration of dryness caused by stress and the amount of enkephalin in the body was investigated by the following procedure.
A questionnaire on stress and skin condition was conducted on 27 women in their 20s and 40s who consented to the test. In addition, saliva was collected and the amount of enkephalin in saliva was measured by the same method as in <Experiment 2>, and the relationship between the skin condition during stress and the amount of enkephalin in saliva was analyzed.
As shown in FIG. 3, the group of humans who recognize that dryness worsens due to stress has a higher amount of enkephalin in saliva than the group of humans who recognize that dryness does not worsen, and is caused by stress. It is suggested that there is a relationship between the deterioration of the skin condition and the amount of enkephalin in the body.

<Experiment 4> Examination of the effect of enkephalin on the cell differentiation of epidermal cells The effect of enkephalin on the expression level of cell differentiation-related genes in epidermal cells was examined by the following procedure.
Normal human epidermal keratinocytes (BIOPREDIC) were suspended in a special medium (Medium 154CF (Gibco) medium supplemented with 0.03 mM calcium chloride and Human Kelatinocyte Growth Supplement (Gibco)) in 5 × 10 ⁴ cells / mL. The cell suspension was prepared so as to be the same, and 500 μL was seeded on a 24-well culture plate. After culturing for 2 days under humidified conditions of 37 ° C. and 5% CO ₂ /95% air, methionine-enkephalin was added to the medium to a final concentration of 10,100 nM. 37 ° C., after 24 hours of culture in a humidified conditions of _5% CO 2/95% air, the final concentration of calcium chloride solution is 37 ° C. was added to the medium so as to be 1.2 mM, of _5% CO 2/95% air humidified conditions Induced cell differentiation of keratinocytes. After culturing for 24 hours, Total RNA was extracted using Total RNA Purification Kit (Jena Bioscience). Then, reverse transcription was performed using PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the obtained cDNA as a template, the expression levels of keratin 1 (KRT1), keratin 10 (KRT10) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; used as a housekeeping gene) were measured using the following primers and enzymes. It was measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems). Primers include a sense primer for KRT1 (5'-CTTCAGGCCAAACTTGACAACTT-3'), an antisense primer (5'-TCTGAGACACTTGCTTGTAGAG-3'), a sense primer for KRT10 (5'-CAACTGGCCTTGAAACAAATTC-3'), and an antisense primer (5'). '-CTGCACACAGTAGCGACCTTCT-3'), sense primer for GAPDH (5'-CCACATCGC TCAGACACCAT-3'), and antisense primer (5'-TGACACAGGC GCCCAATA-3') were used. Power SYBR Green Master Mix (Applied Biosystems) was used for the PCR reaction, and gene expression was analyzed by the comparative Ct method. That is, the change in the gene expression level due to the addition of the test substance was determined by setting the Ct value of KRT1 and KRT10 in the control group to which no enkephalin was added to the value corrected by the Ct value of GAPDH as a relative amount.
As shown in FIG. 4, it was shown that the addition of enkephalin reduced the expression levels of the differentiation-related genes KRT1 and KRT10 in epidermal cells, and that enkephalin suppressed the cell differentiation of epidermal cells.

<Experiment 5> Examination of the effect of enkephalin on the cell differentiation of three-dimensional skin model epidermal cells The effect of enkephalin on the expression level of cell differentiation-related genes in the epidermal cells of the three-dimensional skin model was examined by the following procedure.
1. 1. Culture of Enkephalin-added Skin Three-Dimensional Model Skin three-dimensional model preparation kit Labcate EPI-KIT (J-TEC) contains normal human epidermal keratinocytes suspended in a special assay medium, and 500 μL of cell suspension is added to each cell culture insert. Sown. 1.5 mL of assay medium was dispensed into a 12-well culture plate, and cell culture inserts were immersed in each well of the 12-well culture plate and cultured for 1 day under humidified conditions of 37 ° C. and 5% CO ₂ /95% air. The medium inside the cell culture insert was carefully removed, and the cells were exposed to air to induce cell differentiation of keratinocytes. The medium of the 12-well culture plate was changed, and the cells were cultured at 37 ° C. under humidified conditions of 5% CO ₂ /95% air for 2 days. Change the medium of the 12-well culture plate, add methionine-enkephalin to the medium of each well to a final concentration of 100 nM, and change the medium containing methionine-enkephalin every few days at 37 ° C. The cells were cultured for 12 days under humidified conditions of 5% CO ₂ /95% air.
2. 2. Measurement of expression level of differentiation-related genes Using a scalpel, the three-dimensional skin model and its supporting base were separated from the cell culture insert. Total RNA was extracted from a three-dimensional skin model using the Total RNA Purification Kit (Jena Bioscience). Then, reverse transcription was performed using PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the obtained cDNA as a template, keratin 1 (KRT1), keratin 10 (KRT10), involucrin, profilaggrin (gene encoding filaggrin protein) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; used as a housekeeping gene) The expression level of was measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems) using the following primers and enzymes. Primers include a sense primer for KRT1 (5'-CTTCAGGCCAAACTTGACAACTT-3'), an antisense primer (5'-TCTGAGACACTTGCTTGTAGAG-3'), a sense primer for KRT10 (5'-CAACTGGCCTTGAAAACAAATTC-3'), and an antisense primer (5'). '-CTGCACACAGTAGCGACCTTCT-3'), Sense Primer for Involucrin (5'-GGAGAAGCAGGGAGGCACA-3'), Antisense Primer (5'-TCCAGGTGCTTGGCTTGT-3'), Sense Primer for Profilagurin (5'-GGCACTGAAAGGCAAAG-3') An antisense primer (5'-AAACCCGGATTCACCATAATTAACATCA-3'), a sense primer for GAPDH (5'-CCACATCGC TCAGACACCAT-3'), and an antisense primer (5'-TGACCAGGC GCCCAATA-3') were used. Power SYBR Green Master Mix (Applied Biosystems) was used for the PCR reaction, and gene expression was analyzed by the comparative Ct method. That is, the change in the gene expression level due to the addition of the test substance was determined by setting the Ct value of KRT1, KRT10, involucrin, and profilaggrin in the control group to which no enkephalin was added to the Ct value of GAPDH as 1, and determining it as a relative amount. It was.
As shown in FIG. 5, it was shown that the addition of enkephalin reduced the expression levels of the epidermal cell differentiation-related genes KRT1, KRT10, involucrin, and profilaggrin in the three-dimensional skin model, and enkephalin suppressed the cell differentiation of epidermal cells. It was shown again to do.

<Experiment 6> Examination of the effect of enkephalin on the morphology of the three-dimensional skin model The effect of the addition of enkephalin on the morphology of the three-dimensional skin model was confirmed by the following procedure.
1. 1. Culture of enkephalin-added skin three-dimensional model Culture was carried out in the same manner as in <Experiment 5>.
2. 2. Using an observation scalpel in the form of a stratum corneum, the three-dimensional skin model and its supporting base were separated from the cell culture insert. The three-dimensional skin model was embedded in an OCT compound (Sakura Finetech), frozen at -80 ° C, and then a three-dimensional frozen skin model section was prepared by cryostat (LEICA). After attaching the section to a slide glass and fixing it, stain it with Meyer hematoxylin staining and eosin staining so that the morphology of the skin three-dimensional model section can be easily observed, and then use an optical microscope (KEYENCE) to determine the stratum corneum morphology of the skin three-dimensional model section. Observed.
As shown in FIG. 6, it was shown that the addition of enkephalin significantly reduced the stratum corneum thickness of the three-dimensional skin model.

<Experiment 7> Examination of the effect of enkephalin on the maturity of the stratum corneum of the three-dimensional skin model The effect of the addition of enkephalin on the intracellular keratinocyte binding of the three-dimensional skin model was confirmed by the following procedure.
1. 1. Culture of enkephalin-added skin three-dimensional model Culture was carried out in the same manner as in <Experiment 5>.
2. 2. A three-dimensional skin model and its supporting base were separated from the cell culture insert using a stained keratin SS bond scalpel. The three-dimensional skin model was embedded in an OCT compound (Sakura Finetech), frozen at -80 ° C, and then a three-dimensional frozen skin model section was prepared by cryostat (LEICA). After attaching the section to a slide glass, the section is immersed in a 0.15 MN-ethylmaleimide (Wako Pure Chemical) solution prepared in phosphate buffered saline (PBS (-)) and incubated in a 37 ° C environment for 5 minutes. did. Then, the sections were immersed in 0.15 mM EDTA, 10 mM 2-mercaptoethanol solution prepared with PBS (−), and incubated in a 37 ° C. environment for 3 minutes. The SS bond was stained by immersing in a 0.01 mM N- (7-dimethylamino-4-methylcummarinyl) maleimide (Wako Pure Chemical) solution prepared with PBS (-) for 1 minute. A fluorescence image was observed with a fluorescence microscope (KEYENCE) using a filter having an excitation wavelength of 400 nm and a fluorescence wavelength of 460 nm.
As shown in FIG. 7, it was shown that the addition of enkephalin greatly reduced the keratin SS bond in the keratinocytes of the three-dimensional skin model, and that enkephalin suppressed the maturation of the stratum corneum.

<Experiment 8> Examination of the effect of enkephalin on the transepidermal water loss of the three-dimensional skin model The effect of enkephalin addition on the percutaneous water evaporation of the three-dimensional skin model was examined by the following procedure.
1. 1. Culture of enkephalin-added skin three-dimensional model Culture was carried out in the same manner as in <Experiment 5>.
2. 2. Measurement of Percutaneous Moisture Evapotranspiration The lid of the 12-well culture plate containing the cell culture insert was removed and allowed to stand at room temperature for 30 minutes to acclimatize to the air environment. Vapometer (Delfin) was brought into close contact with the cell culture insert, and the amount of transepidermal water loss of the three-dimensional skin model was measured.
As shown in FIG. 8, it was confirmed that the addition of enkephalin increased the amount of transepidermal water loss in the three-dimensional skin model.

<Example 1> Screening using the degree of cell differentiation of enkephalin-added epidermal cells as an index Improvement of rough skin caused by stress using the change in the expression level of the differentiation-related gene KRT1 of enkephalin-added epidermal cells as an index by the following procedure. The drug was screened.
1. 1. Preparation of Test Substance 40 times the mass of purified water was added to the dried plant substance, and the mixture was heated and extracted at 60 ° C. for 4 hours. Purified water was added to the dry residue of the extract so as to have a mass ratio of 1: 100 (10,000 ppm) and diluted to obtain a test substance. The plant progenitors used were whole plants of Kazunoibara, Nekoasi kelp, and Hidaka kelp.
2. 2. Screening using the expression level of the differentiation-related gene KRT1 in enkephalin-added epidermal cells as an index Medium 154CF (Gibco) containing normal human epidermal keratinocytes (BIOPREDIC) in a special medium (final concentration 0.03 mM calcium chloride and Human Keritonicite Growth Suspension (Gibco)). ) Medium), a cell suspension was prepared to a concentration of 5 × 10 ⁴ cells / mL, and 500 μL was seeded on a 24-well culture plate. After culturing for 2 days under humidified conditions of 37 ° C. and 5% CO ₂ /95% air, methionine-enkephalin and the test substance were added to the medium so that the final concentrations were 100 nM and 100 ppm, respectively. 37 ° C., after 24 hours of culture in a humidified conditions of _5% CO 2/95% air, the final concentration of calcium chloride solution is 37 ° C. was added to the medium so as to be 1.2 mM, of _5% CO 2/95% air humidified conditions Induced cell differentiation of keratinocytes. Then, Total RNA was extracted, and the gene expression level of keratin 1 was measured by the same method as in <Experiment 4>.
As shown in FIG. 9, the expression level of the differentiation-related gene KRT1 in epidermal cells decreased by the addition of enkephalin was almost unchanged in the group to which the nekoasi kelp extract and the Hidaka kelp extract were added. On the other hand, in the group to which the Kazunoibara extract was added, the expression level of KRT1 when enkephalin was added increased to the same level as the control to which enkephalin was not added, and the Kazunoibara extract improves the cell differentiation inhibitory effect of enkephalin on epidermal cells. It was shown that it can be done.

<Experimental Example 2> Examination of the effect of enkephalin-free epidermal cells on the degree of cell differentiation The effects of various test substances on the expression level of the differentiation-related gene KRT1 in epidermal cells were confirmed by the following procedure.
1. 1. Preparation of test substance The substance prepared in the same manner as in <Example 1> was used.
2. 2. Examination of the effect on the expression level of the differentiation-related gene KRT1 in the test substance Medium 154CF (Gibco) medium containing normal human epidermal keratinocytes (BIOPREDIC) in a special medium (final concentration 0.03 mM calcium chloride and Human Keratinocite Groth Suspension (Gibco)) ), A cell suspension was prepared to a concentration of 5 × 10 ⁴ cells / mL, and 500 μL was seeded on a 24-well culture plate. After culturing for 2 days under humidified conditions of 37 ° C. and 5% CO ₂ /95% air, the test substance was added to the medium to a final concentration of 100 ppm. 37 ° C., after 24 hours of culture in a humidified conditions of _5% CO 2/95% air, the final concentration of calcium chloride solution is 37 ° C. was added to the medium so as to be 1.2 mM, of _5% CO 2/95% air humidified conditions Induced cell differentiation of keratinocytes. Then, Total RNA was extracted, and the gene expression level of keratin 1 was measured by the same method as in <Experiment 4>.
As shown in FIG. 10, it was confirmed that the Kazunoibara extract reduced the expression level of the cell differentiation-related gene KRT1 in epidermal cells to which no enkephalin was added. That is, it was confirmed that the Kazunoibara extract itself did not retain the action of promoting cell differentiation of epidermal cells, and the effect confirmed in FIG. 9 was a specific action of suppressing cell differentiation by enkephalin. ..

<Example 3> Screening using the morphology of the three-dimensional skin model with enkephalin as an index Screening of a skin roughness improving agent caused by stress using the thickness of the three-dimensional skin model with enkephalin as an index according to the following procedure. Was done.
1. 1. Preparation of test substance The substance prepared in the same manner as in <Example 1> was used.
2. 2. Culture of Enkephalin-added Skin Three-Dimensional Model Skin three-dimensional model preparation kit Labcate EPI-KIT (J-TEC) contains normal human epidermal keratinocytes suspended in a special assay medium, and 500 μL of cell suspension is added to each cell culture insert. Sown. 1.5 mL of assay medium was dispensed into a 12-well culture plate, and cell culture inserts were immersed in each well of the 12-well culture plate and cultured for 1 day under humidified conditions of 37 ° C. and 5% CO ₂ /95% air. The medium inside the cell culture insert was carefully removed, and the cells were exposed to air to induce cell differentiation of keratinocytes. The medium of the 12-well culture plate was changed, and the cells were cultured at 37 ° C. under humidified conditions of 5% CO ₂ /95% air for 2 days. The medium of the 12-well culture plate was changed, and methionine-enkephalin was added to the medium of each well so that the final concentration was 100 nM, and the test substance was added to the final concentration of 100 ppm, and the temperature was 37 ° C., 5% CO ₂ /95% air. The cells were cultured under humidified conditions. The medium containing methionine-enkephalin and the test substance was exchanged every few days and cultured for 12 days.
3. 3. Observation of stratum corneum morphology The same observation as in <Experiment 6>.
As shown in FIG. 11, when compared with Nekoasi kelp extract and Hidaka kelp extract, when Kazunoibara extract and enkephalin were added, the stratum corneum thickness was significantly larger than that of the skin three-dimensional model to which only enkephalin was added. It was shown to increase.

<Example 4> Screening using the amount of transepidermal water evaporation of the enkephalin-added skin three-dimensional model as an index The rough skin caused by stress using the amount of percutaneous water evaporation of the three-dimensional skin model added with enkephalin as an index by the following procedure. Screening for improvers was performed.
1. 1. Preparation of test substance The Ibaranori extract prepared in the same manner as in <Example 1> was used.
2. 2. Culture of enkephalin-added skin three-dimensional model Culture was carried out in the same manner as in <Example 3>.
3. 3. Measurement of transdermal water evaporation amount The measurement was carried out in the same manner as in <Experiment 8>.
As shown in FIG. 12, it was confirmed that the amount of transepidermal water loss of the three-dimensional skin model increased by the addition of enkephalin was reduced to the same level as that of Control (without enkephalin) by the addition of the Kazunoibara extract.

<Example 5> Actual use test Using the Kazunoibara extract, an external preparation for skin was prepared according to the prescription shown in "Table 1", and an effect test was conducted on eight women in their 20s and 30s who consented to the test. The subjects were subjected to a stress check in advance and selected from the group of humans judged to be in the high stress group. The subjects were asked to apply the test samples of Test Example 1 and Comparative Example 1 to the half face after washing their face twice daily for one month, morning and night, and the amount of transdermal water evaporation of the facial cheeks 4 weeks after the start of use. Was measured with TEWA meter TM-210 (Courage + Khazaka). The measurement was carried out after washing the face with a face wash and acclimatizing for 20 minutes in an environment of 22 ± 1 ° C. and a humidity of 50 ± 2%.
As shown in FIG. 13, in Test Example 1 containing a component having an effect of improving rough skin due to stress selected by the screening method of the present invention, the amount of transepidermal water evaporation decreased after continuous use of the test sample as compared with Comparative Example 1. Was recognized.

Hereinafter, a formulation example using the Kazunoibara extract according to the present invention will be shown. The content is mass%. The manufacturing method is a conventional method. The Kazunoi rose extract has a dry mass.

Prescription example 1: Cream Kazunoi rose extract 0.02
Stearyl alcohol 6.00
Stearic acid 2.00
Vaseline 4.00
Squalene 9.00
Octyldodecanol 10.00
1,3-butylene glycol 6.00
Glycerin 4.00
POE (20) Cetyl Alcohol Ether 3.00
Glycerin monostearate 2.00
Antioxidant Appropriate amount Preservative Appropriate amount Purified water Total balance 100.0

Prescription example 2: Foundation Kazunoi rose extract 0.01
Talc 5.00
Sericite 8.00
Titanium oxide 5.00
Color Pigment Appropriate amount Polyglyceryl monoisostearate 3.00
Polyoxyethylene hardened castor oil 1.50
Isotridecyl isononanoate 10.00
1,3-butylene glycol 5.00
Antioxidant Appropriate amount Preservative Appropriate amount Purified water Total balance 100.00

Prescription example 3: Sunscreen Kazunoi rose extract 0.01
Titanium oxide 10.00
Zinc oxide 10.00
PEG-9 Polydimethylsiloxyethyl Dimethicone 1.50
Lauryl PEG9-polydimethylsiloxyethyl dimethicone 1.50
Cyclopentasiloxane 20.00
Dimethicone 10.00
(Dimethicone / Vinyl Dimethicone) Cross Polymer 0.50
Cetildimethicone 0.25
Glycyrrhetinic acid ester 0.05
Methylgluces-20 1.00
1,3-butylene glycol 10.00
Sodium Chloride Appropriate amount Antioxidant Appropriate amount Preservative Appropriate amount Purified water Total balance 100.00

Claims (10)

A method for screening an agent for improving rough skin caused by stress using the degree of cell differentiation of epidermal cells and / or the skin barrier function of a three-dimensional skin model as an index in the presence of enkephalin. (1) A step of adding enkephalin and a test substance to epidermal cells and culturing them.
(2) Steps to measure the degree of epidermal cell differentiation,
(3) A method for screening a skin roughness improving agent caused by stress, in which the degree of epidermal cell differentiation obtained in step (2) is compared with the group without the addition of the test substance, and the substance that improves the degree of epidermal cell differentiation is determined to be an effective substance. .. The method according to claim 1 or 2, wherein the degree of differentiation of epidermal cells is the expression level of a protein whose expression level increases with the differentiation of epidermal cells or the expression level of a gene encoding the same. The method according to any one of claims 1 to 3, wherein the protein comprises at least one selected from keratin 1, keratin 10, involucrin, loricrin, filaggrin, caspase 14, and transglutaminase. Claim that when the expression level of the protein or its gene is increased by 10% or more as compared with these expression levels when the test substance is not added, it is determined that the test substance has an effect of improving rough skin caused by stress. The method according to any one of 1 to 4. (1) A step of adding enkephalin and a test substance to a three-dimensional skin model and culturing the skin.
(2) Steps to measure the skin barrier function of a three-dimensional skin model,
(3) A method for screening a skin roughness improving agent caused by stress, in which the skin barrier function obtained in step (2) is compared with the group without the addition of the test substance, and the substance that improves the skin barrier function is determined to be an effective substance. An agent for improving rough skin caused by stress selected by the method according to any one of claims 1 to 5. A stress-induced rough skin improver containing Kazunoibara extract. An agent for improving skin barrier function deterioration caused by stress, which contains Kazunoi rose extract. Use of Kazunoibara extract to improve rough skin caused by stress.