JP2023062016A - Agents for improving skin barrier function worsened due to stress - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide agents for improving th skin roughened or the skin barrier function worsened due to stress.

SOLUTION: Provided are agents for improving th skin roughened or the skin barrier function worsened due to stress, the agents comprising extract from Hypnea flexicaulis, which is a seaweed living across Japan and belongs to Genus Hypnea, Family Hypneaceae. Provided is also the use of extract from Hypnea flexicaulis for improving the rough skin.

SELECTED DRAWING: Figure 11

COPYRIGHT: (C)2023,JPO&INPIT

Description

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

Modern society has been called a stressful society for a long time, but a large proportion of the stress that people living in modern society receive is psychological and social due to human relationships, work, family, schoolwork, money, and overcrowded trains. occupy When this stress exceeds a certain level, the homeostasis of the body breaks down, causing disorders in the mind and body or physical functions, and in addition to mental disorders such as anxiety disorders and depression, there are various diseases such as gastric and duodenal ulcers, high blood pressure, and lifestyle-related diseases. cause physical illness. In addition, it is known that the skin, which is an organ that separates the living body from the outside world, becomes more susceptible to external stimuli due to a decrease in the barrier function of the skin, which promotes rough skin.

The skin is the boundary between the living body and the outside world, and it has a skin barrier function to prevent the intrusion of substances from the outside and the evaporation of moisture from the inside of the skin, and plays an important role in the maintenance of the living body. ing. The skin is an epithelium with a thickness of 100 to 200 μm consisting of a stratum corneum, a granular cell layer, a spinous cell layer, and a basal cell layer. The stratum corneum, which is the outermost layer of the skin, is particularly important for the skin barrier function. The stratum corneum is composed of corneocytes formed by terminal differentiation of epidermal cells in the basal cell layer and intercellular lipids surrounding them. Corneocytes are mainly composed of keratin fibers with SS bonds, contain filaggrin and natural moisturizing factor NMF which play an important role in moisturizing function, and consist of a cornified envelope enveloping them. On the other hand, intercellular lipids consist of a lamellar structure composed of ceramide, cholesterol, fatty acid, etc. (Non-Patent Document 1).

Cornified envelopes enveloping corneocytes are formed and matured by insolubilizing multiple cornified envelope precursor proteins, such as involucrin and loricrin, which are produced as epidermal cells differentiate, are cross-linked by the enzyme transglutaminase. Furthermore, some of them are covalently bound with intercellular lipids such as ceramide, which has been suggested to provide a foundation for the lamellar structure of the intercellular lipids described above. Corneocytes are bound to each other by corneodesmosomes composed of intercellular adhesion molecules desmoglein and desmocholine, and form a tough sheet structure.

In addition to keratin fibers with SS bonds generated during terminal differentiation, filaggrin, natural moisturizing factors, and cornified envelopes, components in corneocytes play an important role in the skin barrier function. In addition, intercellular lipids such as ceramide also play an important role. It is important that they are produced and matured normally, and that the stratum corneum is formed normally and functions.

Conventionally, as a screening method for materials to improve the deterioration of skin conditions caused by mental stress, such as the deterioration of skin barrier function, mice are subjected to overcrowded stress, which is recognized as socio-psychological stress, to test the skin barrier function. A method of screening for a material that improves skin conditions caused by stress by inducing rough skin such as aggravation of stress has been implemented (Patent Document 1). However, since this method requires animal experiments, experimental procedures are complicated and there are ethical issues. Screening methods have also been proposed in which the stress itself is reduced by the use of scents, etc. (Patent Document 2), but these attempts require tests on humans or animals, and the experimental procedures are complicated and ethical. In addition, the need for a process of invasively collecting substances from the living body imposes a heavy burden on the subject. Furthermore, in the case of humans, there is a problem that preference has a large effect on the effect, and individual differences in the effect are large.

On the other hand, since it is also known that active oxygen is generated in part of the body as a result of stress load, a method of selecting an anti-stress material has been implemented considering the anti-oxidation effect as an anti-stress effect (Patent document 3). However, this method only ameliorate some of the ROS-mediated adverse effects of stress. In other words, there are many problems in screening for materials that improve the deterioration of skin barrier function caused by stress, and there has been no effective and simple measurement method.

It is known that psychological stress causes physiological changes in vivo, and it is known that the adrenal cortex hormone (cortisol) is released from the adrenal glands and the amount of adrenaline released in the brain is increased. It has also been reported that endogenous opioid peptides are released from the central nervous system and adrenal medulla and increased. Opioids are a general term for substances that have effects similar to morphine, a narcotic analgesic, through binding to opioid receptors present in the nervous system, and have effects such as analgesia and induction of euphoria due to narcotic-like effects. Endogenous opioid peptides present in the body also exhibit similar effects, but enkephalin and its derivatives, which are a type of endogenous opioid peptide, have lipolytic effects, skin hydration, improvement of brightness, and reduction of facial wrinkles. It has been reported to be useful for skin conditions, such as reducing action (Patent Document 4). All of these indicate that the administration or external application of enkephalins or derivatives thereof has beneficial effects on the body, but enkephalins have never been thought to be involved in the disorder of the body. The possibility of having adverse effects was also not envisioned.

Hypnea flexicaulis or Hypnea cervicornis, belonging to the genus Ibaranori of the family Ibaranaceae, is a seaweed that grows throughout Japan. So far, no investigation has been made on the effect of Kazunoi rose on improving rough skin caused by stress and on the possibility of exerting some kind of action on the effects of enkephalin on the body.

JP-A-10-279505 JP 2008-297554 A JP-A-8-275752 Special Table No. 2008-534658

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

In view of the background art described above, the present inventors have investigated methods for improving the deterioration of the epidermal barrier function caused by stress. As a result, we discovered that enkephalin, which increases during mental stress, is involved in the deterioration of the skin barrier function during stress, rather than acting to improve the deteriorated skin condition as is known. Specifically, we found that the amount of enkephalin in saliva, which increases during mental stress, is positively correlated with water loss from the skin (transepidermal water loss). Furthermore, it was confirmed that the amount of enkephalin in saliva was higher in women who felt that their skin became more dry during stress than in women who did not. strongly suggested.

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

Based on the above new findings, enkephalin, which increases in the body during stress, is transported to the epidermis of the skin in the blood, inhibits the differentiation of epidermal cells in the skin, and inhibits the normal formation of the stratum corneum, thereby adversely affecting the skin barrier function. It was strongly suggested that Therefore, if it is possible to suppress the adverse effects of enkephalin on the skin, 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. Arrived.

An object of the present invention is to provide a method for screening an agent for improving rough skin caused by stress in a simple manner using cultured cells without conducting animal experiments or tests on humans. An object of the present invention is to provide an agent for improving rough skin caused by

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

That is, the present invention is as follows.
[1]
A screening method for an agent for improving rough skin caused by stress, using as an index the degree of cell differentiation of epidermal cells and/or the skin barrier function of a three-dimensional skin model in the presence of enkephalin.
[2]
(1) adding an enkephalin and a test substance to epidermal cells and culturing them;
(2) measuring the degree of epidermal cell differentiation;
(3) A screening method for an agent for improving rough skin caused by stress by comparing the degree of epidermal cell differentiation obtained in step (2) with a test substance-free group and determining a substance that improves the degree of epidermal cell differentiation as an effective substance. .
[3]
The method according to either [1] or [2], wherein the degree of epidermal cell differentiation is the expression level of a protein whose expression level increases with 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 contains 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 increases by 10% or more compared to the expression level 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;
(2) measuring the skin barrier function of the three-dimensional skin model;
(3) A screening method for an agent for improving rough skin caused by stress, wherein the skin barrier function obtained in step (2) is compared with a test substance-free group, and a substance that improves the skin barrier function is determined as an effective substance.
[7]
An agent for improving rough skin caused by stress selected by the method according to any one of [1] to [5].
[8]
An agent for improving rough skin caused by stress, containing a Kazunoi rose extract.
[9]
An agent for improving skin barrier function deterioration caused by stress, containing a Kazunoi rose extract.
[10]
Use of Kazunoi rose extract for improving rough skin caused by stress.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for screening rough skin ameliorating agents capable of ameliorating the deterioration of skin barrier function caused by stress by using cultured cells without conducting 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 load. The figure which shows the correlation of the amount of enkephalins in saliva, and transepidermal water loss (TEWL). The figure which shows the relationship between the deterioration of the dryness at the time of stress, and the amount of enkephalins in saliva. Fig. 3 shows a decrease in the degree of cell differentiation of epidermal cells due to the addition of enkephalin. The figure which shows the reduction|decrease of the cell differentiation degree of the skin three-dimensional model by enkephalin addition. The figure which shows the change of the stratum corneum morphology of the three-dimensional skin model by enkephalin addition. The figure which shows the change of the stratum corneum maturation degree (SS bond increase degree) of the skin three-dimensional model by enkephalin addition. The figure which shows the change of the transepidermal water loss of the skin three-dimensional model by enkephalin addition. Fig. 2 shows improvement in inhibition of cell differentiation of enkephalin-added epidermal cells by addition of various plant extracts. Fig. 2 shows the effect of the addition of various plant extracts on the degree of differentiation of epidermal cells to which enkephalin was not added. FIG. 10 is a diagram showing the improvement of the stratum corneum morphology of the enkephalin-added three-dimensional skin model by the addition of various plant extracts. The figure which shows the improvement of the transepidermal water loss of the enkephalin addition skin three-dimensional model in the Kazunoi rose extract addition. The figure which shows the relationship between the kind of skin preparation for external use used, and transepidermal water loss.

Stress increases enkephalins in the body. The screening method of the present invention simulates skin affected by stress, and is a method for 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, a step of adding enkephalin and a test substance to epidermal cells and culturing them, a measuring step of obtaining the degree of epidermal cell differentiation, a step of comparing the degree of epidermal cell differentiation and selecting an agent for improving rough skin caused by stress, screening method.

The enkephalin used in the present invention is a structure in which five amino acids are linked: leucine-enkephalin (tyrosine-glycine-glycine-phenylalanine-leucine), methionine-enkephalin (tyrosine-glycine-glycine-phenylalanine-methionine), as well as the enkephalin Peptides containing sequences and peptides derived from enkephalin sequences can be used.

The epidermal cells used in the screening of the present invention are not particularly limited as long as they are keratinocytes. Normal (non-cancerous) keratinocytes isolated from the epidermis of humans or animals other than humans, immortalized keratinocyte cell lines, etc. can be used, more preferably normal keratinocytes isolated from human epidermis. The culture form of epidermal cells can be two-dimensional culture (monolayer culture) or three-dimensional culture such as a three-dimensional skin model (layered culture). If so, it can be applied indefinitely.

As a method for culturing epidermal cells to which enkephalin and a test substance are added, normal culture conditions, for example, a commercially available keratinocyte-dedicated medium are used, and culture conditions that do not interfere with the execution of the screening method of the present invention are not particularly limited. can be applied. In order to make it easier to confirm the effect of enkephalin or a test substance on cell differentiation of epidermal cells, an operation of inducing differentiation of epidermal cells by adding calcium ions to the medium during epidermal cell culture may be performed. After enkephalin and a test substance are added to epidermal cells to allow the cells to receive these signals, it is preferable to carry out an operation to induce differentiation of epidermal cells by adding calcium ions.

Although the degree of epidermal cell differentiation is not particularly limited, it can be determined by the expression level of a protein whose expression level increases with the differentiation of epidermal cells or the expression level of the 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 the gene encoding it. can. From the viewpoint of screening accuracy, a combination of two or more is preferred, for example, a combination of keratin 1 or keratin 10, involucrin, and filaggrin is preferred.

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 using any method can be used. For example, the results obtained by quantitatively measuring the amount of the protein present in cells by conventional methods such as Western blotting, ELISA, radioimmunoassay, immunostaining, and mass spectrometry may be used. Alternatively, the amount of the protein can be measured indirectly by measuring metabolites of the protein. In addition, the expression level of the gene is quantitatively detected by PCR using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer and by agarose gel electrophoresis or the like. The gene sequences encoding the various factors described above are open to the public, and those skilled in the art can appropriately design primers and subject them to PCR. In addition, nucleic acid hybridization methods using immobilized samples such as gene chips and arrays, RT-PCR methods, real-time PCR methods, subtraction methods, differential display methods, differential hybridization methods, cross-hybridization methods, 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 desired effect. When the expression level of the gene to be tested is increased compared to the expression level in cells to which the test substance was not added, the increase is preferably 10% or more, more preferably 15% or more, and still more preferably 20% or more. If so, it can be determined that the test substance has an action to improve rough skin caused by stress.

Another 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 skin barrier function of a three-dimensional skin model as an index.
Specifically, a step of adding enkephalin and a test substance to a three-dimensional skin model and culturing the three-dimensional skin model, a step of measuring the skin barrier function of the three-dimensional skin model, comparing the skin barrier functions and selecting an agent for improving rough skin caused by stress. A screening method, comprising:

As the three-dimensional skin model used for the screening of the present invention, normal (non-cancerous) skin cells such as keratinocytes, melanocytes, fibroblasts, etc. collected from the skin of humans or animals other than humans are three-dimensionally cultured for several days. It is possible to construct a three-dimensional (laminated) structure by using it to simulate the structure of the skin and use it. Preferably, the culturing period is shortened, and keratinocytes are incomplete in terminal differentiation and keratinocytes are not formed. For example, after inoculating a three-dimensional culture container such as an insert for cultured cells and culturing for three days, removing the culture medium inside the insert, exposing it to air, and continuing the culture for several days, a three-dimensional structure is constructed. The culture period after exposure to air is a period during which terminal differentiation of keratinocytes is incomplete and corneocytes are not formed. Instead of cultured cell inserts, collagen gel or fibroblast-containing collagen gel, dermis excised from the living body may be placed on a glass ring, and keratinocytes or keratinocytes mixed with melanocytes may be seeded in the ring. Moreover, it is also possible to purchase and use a commercially available product for constructing such a form. Preferable commercial products include, for example, three-dimensional skin model (immature stratum corneum type) MEL-301 (Kurabo), three-dimensional skin model (immature stratum corneum type) EPI-201 (Kurabo), LabCyte EPI-Model 6D ( J-TEC Co.), LabCyte EPI-KIT (J-TEC Co.), 3D FT Starter Kit (CELLnTEC Co.) and the like can be used. In particular, LabCyte EPI-KIT (J-TEC Co.) and 3D FT Starter Kit (CELLnTEC Co.), which can construct a three-dimensional structure by itself, can be used more preferably. Furthermore, LabCyte EPI-KIT (J-TEC Co.), which is composed only of keratinocytes, can be most preferably used.

As a method for culturing the three-dimensional skin model with the addition of enkephalin and a test substance, enkephalin is added to the medium of the three-dimensional skin model prepared by the above method, and the test substance is added in the medium or to the stratum corneum side of the three-dimensional skin model. It can then be cultured for a period of time until terminal differentiation of keratinocytes is completed and corneocytes are formed. Since keratinocytes are induced to differentiate into cells by exposure to air, it is preferable to add a test substance to the medium and culture them. The culture period is preferably 1 to 18 days, and more preferably 7 to 14 days during which corneocytes are overlaid and the stratum corneum is sufficiently formed.

In the screening method of the present invention, the skin barrier function is selected from stratum corneum thickness, stratum corneum maturity (SS bond increase), cornified envelope maturity, and transepidermal water loss amount of the stratum corneum formed in a three-dimensional skin model. It can be evaluated by at least one or more measured values or observed images.
The stratum corneum is composed of corneocytes. The corneocytes that make up the stratum corneum are mainly composed of keratin fibers with SS bonds that are generated during terminal differentiation. and consists of a cornified envelope enclosing them. Corneocytes are bound together by corneodesmosomes, which consist of intercellular adhesion molecules, and form a tough sheet structure. The stratum corneum exists as a boundary membrane between the in vivo and the ex vivo with a thickness of 10 to 20 μm by stacking many layers of this sheet structure. With such a configuration, the moisture content inside the skin is retained and the loss of moisture (transepidermal water evaporation) is suppressed, so that the skin barrier function is normally maintained. In other words, it is important that a sufficiently thick stratum corneum is formed, that its constituent molecules 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 with a microscope and then calculating or evaluating the stratum corneum thickness. Specifically, the section is stained with any staining method that can distinguish between the living cell layer and the stratum corneum, such as hematoxylin-eosin staining and safranin staining, and the morphology of the stratum corneum is observed with an optical microscope. By immunostaining the components present in the stratum corneum 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 sections with a transmission electron microscope, the thickness of the stratum corneum can be determined. A method of calculation or evaluation is used. In addition, a method of obtaining a three-dimensional model of the stratum corneum water content distribution using a Raman spectrometer and calculating the stratum corneum thickness from this inflection point can also be used, and ultrasonic tomography, optical coherence tomography ( Optical Coherence Tomography), a method of obtaining a tomographic image of a three-dimensional skin model using a confocal laser microscope, and calculating the thickness of the stratum corneum therefrom, and other known methods can be used.

Stratum corneum maturity (SS bond increase) can be evaluated by known methods such as staining SS bonds in the three-dimensional skin model with N-(7-dimethylamino-4-methylcoumarinyl)maleimide. can. Specifically, the free SH group is reacted with N-ethylmaleimide to protect it, and the SS group is cleaved with a reducing agent such as 2-mercaptoethanol or dithiothreol, and then the free SH group produced is converted to N-( When reacted with 7-dimethylamino-4-methylcoumarinyl)maleimide, the fluorescence image obtained can be taken as the abundance of SS bonds.

Cornified envelope maturity can be evaluated by a known method. Specifically, the stratum corneum is collected from the three-dimensional skin model by tape stripping or trypsin treatment, mixed with an eluate (Tris-HCl buffer containing dithiothreitol and sodium dodecyl sulfate), centrifuged, and the eluted components are removed. Let the insoluble matter which carried out was a cornified envelope. The involucrin contained in this was immunofluorescently stained by an antigen-antibody reaction, and further stained with NileRed. From the fluorescence observation image of involucrin and Nile Red obtained with a fluorescence microscope, the total area of the cornified envelope (involucrin-positive CE area + NileRed-positive Cornified envelope maturity can be evaluated by calculating the proportion of immature CE in the CE area).

The amount of transepidermal water loss can be measured using a known method. ) can be applied and measured.

When the skin barrier function in the three-dimensional skin model cultured with the addition of enkephalin and the test substance is changed compared to the skin barrier function in the three-dimensional skin model without the addition of the test substance, the test substance is stress It is judged to have an action to improve rough skin caused by Specifically, the stratum corneum thickness, stratum corneum maturity (SS bond increase), and cornified envelope maturity among the skin barrier functions increased in the group to which the test substance was added compared to the group to which the test substance was not added. If 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 stress-induced rough skin ameliorating agent or ameliorating agent for skin barrier function deterioration of the present invention is selected by the aforementioned screening method.

The component selected by the screening method of the present invention, which is expected to have an effect of improving rough skin, is an extract of Kazuro rose.

The Kazunoi rose (Hypnea flexicaulis or Hypnea cervicornis) belonging to the genus Ibaranori of the family Ibaranaceae is not particularly limited, but for example, the whole plant can be preferably used.

Although preparation of the extract is not particularly limited, for example, it is extracted using various suitable organic solvents under low temperature to high temperature. Examples of extraction solvents 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; One or more of alkyl esters; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; and halogenated alkanes such as dichloromethane and chloroform can be used. Among them, water, ethyl alcohol, 1,3-butylene glycol, or a mixed solvent of two or more of them is particularly preferable. Furthermore, water is more preferred.

The extraction method of the extract that can be used in the present invention is not particularly limited. is preferably carried out at 0° C. or higher, particularly 20° C. to 40° C. for 1 hour or longer, especially 3 to 7 days. Alternatively, heat extraction may be performed at 60 to 100° C. for 1 hour.

Each of the extracts obtained under the above conditions may be used as an extracted solution, but if necessary, it may be subjected to a treatment such as filtration or purification, and then concentrated or pulverized. can be used.

The blending amount of the plant extract in the topical skin preparation of the present invention is preferably 0.00001 to 20.0% by mass, and most preferably 0.01 to 10.0% by mass in terms of the evaporated dry matter. be.

In addition, test substances selected by the screening method of the present invention are not limited to plant extracts. Anything selected by the screening method of the present invention may be used, and in addition to plant extracts, extracts from fungi, animals, and the like, as well as compounds may be used.

The rough skin ameliorating agent of the present invention means a composition applied to the skin. For example, compositions that can be applied as topical skin preparations such as lotions, milky lotions, creams, oils, makeup bases, foundations, and other cosmetics; say. It may be in liquid, cream, powder, solid or other forms.

The rough skin ameliorating agent of the present invention is produced by a conventional method using known base ingredients in addition to active ingredients. Other ingredients can be added as long as they do not impair the effects of the present invention. For example, oils, waxes, hydrocarbon oils, ester oils, higher alcohols, silicone oils, UV absorbers, UV scattering agents, moisturizing agents, surfactants, water-soluble polymers, thickeners, powders, skin protective agents , whitening agents, anti-wrinkle agents, anti-aging agents, plant extracts, preservatives, anti-inflammatory agents, pH adjusters, sequestering agents, antioxidants and the like.

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

<Experiment 1> Investigation of relationship between stress load and amount of enkephalin in vivo Changes in the amount of enkephalin in vivo in humans subjected to mental stress were examined by the following procedure.
Eight men and women in their twenties and thirties who gave consent to the test were asked to perform the Kraepelin test, and saliva was collected before and after the test. The Kraepelin test is a series of simple arithmetic tests and is widely used as a mild psychological stress test. Each subject was asked to rinse the oral cavity with water before the Kraepelin test, and 5 minutes later, a sponge of Salivet (Sarstedt) was held in the oral cavity for 90 seconds, and saliva was collected before the Kraepelin test. After carrying out the simple calculation Kraepelin test for 15 minutes, the sponge of Salivet (Sarstedt) was again placed in the oral cavity for 90 seconds, and saliva after the Kraepelin test was collected. The saliva-collected sponge was returned to the salivet tube, and the tube was centrifuged at 4° C. and 3,000×g for 5 minutes to collect the saliva. Saliva was diluted 3-fold with phosphate-buffered saline (PBS(-)), and the amount of enkephalin in saliva was measured using ELISA Kit for Enkephalin (USCNLIFE), and the amount of cortisol in saliva was measured using Salivary Crotisol ELISA Kit (Salimetrics). was measured using
As shown in FIG. 1, the amount of enkephalin in saliva increased due to the application of slight mental 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 known to increase in the body due to stress. . Saliva can be collected noninvasively and easily, and it has been reported that the concentrations of substances present in saliva (e.g., cortisol and estradiol) are strongly correlated with blood concentrations. can be almost synonymous with concentration change in vivo.

<Experiment 2> Examination of the relationship between the amount of enkephalin in the body and the skin barrier function The relationship between the amount of enkephalin in the human body and the amount of transepidermal water loss in the skin was examined by the following procedure.
Subjects were 9 women in their 20s and 30s who gave their consent to the test. After washing their faces with facial cleanser, they were acclimatized for 20 minutes in an environment of 22 ± 1 ° C and 50 ± 2% humidity, and then TEWA meter TM- 210 (Courage+Khazaka Co.) was used to measure transepidermal water loss on the face and cheeks. In addition, the oral cavity was rinsed with water, and after 5 minutes, a sponge of Sarivet (Sarstedt) was soaked in the oral cavity for 90 seconds to collect saliva. The saliva-collected sponge was returned to the salivet tube, and the tube was centrifuged at 4° C. and 3,000×g for 5 minutes to collect the saliva. Saliva was diluted 3-fold with phosphate-buffered saline (PBS(-)), and the amount of enkephalin in saliva was measured using ELISA Kit for Enkephalin (USCNLIFE). Analyzed the relationship.
As shown in Figure 2, there is a strong positive correlation between the amount of enkephalin in saliva in humans and the amount of transepidermal water loss from the skin. I understand.

<Experiment 3> Investigation of relationship between deterioration of skin condition caused by stress and amount of enkephalin in vivo The relationship between deterioration of dryness caused by stress and amount of enkephalin in vivo was investigated by the following procedure.
A questionnaire on stress and skin conditions was conducted on 27 women in their 20s to 40s who gave their consent to the test. In addition, saliva was collected and the amount of enkephalin in saliva was measured in the same manner as in <Experiment 2>, and the relationship between the skin condition under stress and the amount of enkephalin in saliva was analyzed.
As shown in FIG. 3, in the group of humans who recognized that dryness was aggravated by stress, the amount of enkephalin in saliva was higher than that of the group of humans who recognized that dryness was not aggravated. 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 Effect of Enkephalin on Cell Differentiation of Epidermal Cells The following procedure was used to examine the influence of enkephalin on the expression levels of genes associated with cell differentiation of epidermal cells.
Normal human epidermal keratinocytes (BIOPREDIC) were suspended in a special medium (Medium 154CF (Gibco) medium containing a final concentration of 0.03 mM calcium chloride and Human Keratinocyte Growth Supplement (Gibco)) to 5×10 ⁴ cells/mL. A cell suspension was prepared so as to have a volume of 500 µL each and seeded in a 24-well culture plate. After culturing for 2 days at 37° C. in a humidified atmosphere of 5% CO ₂ /95% air, methionine-enkephalin was added to the medium to final concentrations of 10 and 100 nM. After culturing for 24 hours at 37°C under humidified conditions of 5% CO ₂ /95% air, an aqueous solution of calcium chloride was added to the medium to a final concentration of 1.2 mM, and the conditions were changed to 37°C under humidified conditions of 5% CO ₂ /95% air. to induce cell differentiation of keratinocytes. After culturing for 24 hours, total RNA was extracted using Total RNA Purification Kit (Jena Bioscience). Thereafter, reverse transcription was performed using PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the resulting 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 determined using the following primers and enzymes. It was measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems). The primers included a sense primer for KRT1 (5'-CTTCAGGCAAAACTTGACAACTT-3'), an antisense primer (5'-TCTGAGACAACTCTGCTTGGTAGAG-3'), a sense primer for KRT10 (5'-CAACTGGCCTTGAACAATCC-3'), an antisense primer (5 '-CTGCACACAGTAGCGACCTTCT-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 analysis was performed by the comparative Ct method. In other words, the change in gene expression level due to the addition of the test substance was determined as a relative amount with respect to the value obtained by correcting the Ct value of KRT1 and KRT10 in the control group to which enkephalin was not added with the Ct value of GAPDH as 1.
As shown in FIG. 4, the addition of enkephalin was shown to decrease the expression levels of epidermal cell differentiation-associated genes KRT1 and KRT10, indicating that enkephalin inhibits cell differentiation of epidermal cells.

<Experiment 5> Examination of Effect of Enkephalin on Cellular Differentiation of Three-Dimensional Skin Model Epidermal Cells The following procedure was used to examine the effect of enkephalin on expression levels of genes associated with cell differentiation of epidermal cells of a three-dimensional skin model.
1. Cultured skin three-dimensional model of enkephalin-added skin Three-dimensional model preparation kit Labcyte EPI-KIT (J-TEC) Encapsulated normal human epidermal keratinocytes were suspended in a dedicated assay medium, and 500 μL of cell suspension was added to each cell culture insert. sown. 1.5 mL of the assay medium was dispensed into each well of the 12-well culture plate, and the cell culture insert was immersed in each well of the 12-well culture plate and cultured for 1 day at 37° C. under humidified conditions of 5% CO ₂ /95% air. The medium inside the cell culture insert was carefully removed and the cells were exposed to air to induce keratinocyte cell differentiation. The medium in the 12-well culture plate was replaced and cultured for 2 days at 37°C under humidified conditions of 5% CO ₂ /95% air. The medium of the 12-well culture plate was changed, methionine-enkephalin was added to the medium in each well to a final concentration of 100 nM, and the medium was changed every 2 or 3 days at 37°C. Cultured for 12 days under humidified conditions of 5% CO ₂ /95% air.
2. Using a scalpel to measure expression levels of differentiation-related genes, the three-dimensional skin model and its supporting base were separated from the cell culture insert. Total RNA was extracted from the three-dimensional skin model using Total RNA Purification Kit (Jena Bioscience). Thereafter, reverse transcription was performed using PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the resulting cDNA as a template, keratin 1 (KRT1), keratin 10 (KRT10), involucrin, profilaggrin (genes encoding filaggrin proteins) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; used as housekeeping genes) was measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems) using the following primers and enzymes. The primers included a sense primer for KRT1 (5'-CTTCAGGCAAAACTTGACAACTT-3'), an antisense primer (5'-TCTGAGACAACTCTGCTTGGTAGAG-3'), a sense primer for KRT10 (5'-CAACTGGCCTTGAACAATCC-3'), an antisense primer (5 '-CTGCACACAGTAGCGACCTTCT-3'), sense primer for involucrin (5'-GGAGAAGCAGGAGGCACA-3'), antisense primer (5'-TCCAGGTGCTTTGGCTGT-3'), sense primer for profilaggrin (5'-GGCACTGAAAAGGCAAAGG-3'), An antisense primer (5'-AAACCCGGATTCACCATAATCA-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 analysis was performed by the comparative Ct method. In other words, the change in the gene expression level due to the addition of the test substance was calculated as a relative amount with respect to the Ct value of KRT1, KRT10, involucrin, and profilaggrin in the control group to which enkephalin was not added, which was corrected by the Ct value of GAPDH as 1. rice field.
As shown in FIG. 5, the addition of enkephalin was shown to decrease the expression levels of the differentiation-related genes KRT1, KRT10, involucrin, and profilaggrin of epidermal cells in the three-dimensional skin model, and enkephalin suppresses cell differentiation of epidermal cells. It was shown again that

<Experiment 6> Investigation of the effect of enkephalin on the morphology of the stratum corneum of the three-dimensional skin model The effect of the addition of enkephalin on the morphology of the stratum corneum of the three-dimensional skin model was confirmed by the following procedure.
1. Cultivation of three-dimensional model of enkephalin-added skin was cultured in the same manner as in <Experiment 5>.
2. A 3D skin model and its supporting base were separated from the cell culture insert using a scalpel for observing stratum corneum morphology. The three-dimensional skin model was embedded in an OCT compound (Sakura Fine Tech) and frozen at -80°C, and a frozen three-dimensional skin model section was prepared using a cryostat (LEICA). After attaching the section to a slide glass and fixing it, it was stained with Mayer's hematoxylin staining and eosin staining so that the morphology of the three-dimensional skin model section can be easily observed, and the stratum corneum layer morphology of the three-dimensional skin model section was examined with an optical microscope (KEYENCE). 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> Investigation of the effect of enkephalin on stratum corneum maturity of the three-dimensional skin model The effect of the addition of enkephalin on the keratin SS bond in the three-dimensional skin model was confirmed by the following procedure.
1. Cultivation of three-dimensional model of enkephalin-added skin was cultured in the same manner as in <Experiment 5>.
2. Using a keratin SS-bond stain scalpel, 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 Fine Tech) and frozen at -80°C, and a frozen three-dimensional skin model section was prepared using a cryostat (LEICA). After attaching the section to a slide glass, immerse the section in 0.15M N-ethylmaleimide (Wako Pure Chemical Industries) solution prepared with phosphate buffered saline (PBS(-)) and incubate at 37°C for 5 minutes. bottom. Thereafter, the sections were immersed in a 0.15 mM EDTA, 10 mM 2-mercaptoethanol solution prepared with PBS(-) and incubated at 37°C for 3 minutes. SS bonds were stained by immersing for 1 minute in a 0.01 mM N-(7-dimethylamino-4-methylcoumarinyl)maleimide (Wako Pure Chemical Industries) solution prepared in PBS(-). A fluorescence image was observed with a fluorescence microscope (KEYENCE) using a filter with an excitation wavelength of 400 nm and a fluorescence wavelength of 460 nm.
As shown in FIG. 7, addition of enkephalin significantly reduced keratin SS bonds in corneocytes in the three-dimensional skin model, indicating that enkephalin inhibits the maturation of the stratum corneum.

<Experiment 8> Investigation of the effect of enkephalin on the amount of percutaneous water loss of the three-dimensional skin model The following procedure was used to examine the effect of the addition of enkephalin on the amount of percutaneous water loss of the three-dimensional skin model.
1. Cultivation of three-dimensional model of enkephalin-added skin was cultured in the same manner as in <Experiment 5>.
2. Measurement of Percutaneous Water Loss A 12-well culture plate containing a cell culture insert was detached and allowed to stand at room temperature for 30 minutes to acclimate to the air environment. A vaporometer (Delfin) was brought into close contact with the cell culture insert to measure the percutaneous water loss of the three-dimensional skin model.
As shown in FIG. 8, it was confirmed that the addition of enkephalin increased the transepidermal water loss of the three-dimensional skin model.

<Example 1> Screening using the degree of cell differentiation of enkephalin-added epidermal cells as an index The improvement of rough skin caused by stress using changes in the expression level of the differentiation-associated gene KRT1 of the enkephalin-added epidermal cells as an index according to the following procedure. Agent screening was performed.
1. Preparation of Test Substance A 40-fold mass of purified water was added to the dried raw plant material, followed by heat extraction at 60° C. for 4 hours. A test substance was prepared by adding purified water to the dry residue of the extract at a mass ratio of 1:100 (10,000 ppm) to dilute it. The raw materials of the plants used were whole grasses of Kazunoi rose, Nekoashi kelp, and Hidaka kelp.
2. Screening using differentiation-associated gene KRT1 expression level in enkephalin-added epidermal cells Normal human epidermal keratinocytes (BIOPREDIC) were added to a special medium (final concentration of 0.03 mM calcium chloride and Human Keratinocyte Growth Supplement (Gibco) in Medium 154CF (Gibco). ) medium) to prepare a cell suspension having a concentration of 5×10 ⁴ cells/mL, and seeded in 500 μL each on a 24-well culture plate. After culturing for 2 days at 37° C. in a humidified atmosphere of 5% CO ₂ /95% air, methionine-enkephalin and the test substance were added to the medium to final concentrations of 100 nM and 100 ppm, respectively. After culturing for 24 hours at 37°C under humidified conditions of 5% CO ₂ /95% air, an aqueous solution of calcium chloride was added to the medium to a final concentration of 1.2 mM, and the conditions were changed to 37°C under humidified conditions of 5% CO ₂ /95% air. for 24 hours to induce cell differentiation of keratinocytes. Thereafter, total RNA was extracted, and the gene expression level of keratin 1 was measured in the same manner as in <Experiment 4>.
As shown in FIG. 9, the expression level of the epidermal cell differentiation-associated gene KRT1, which was decreased by the addition of enkephalin, was almost unchanged in the groups to which the Nekoashi kelp extract and the Hidaka kelp extract were added. On the other hand, in the group to which the Kazunoi rose extract was added, the expression level of KRT1 at the time of enkephalin addition increased to the same level as the control without enkephalin addition, and the Kazunoi rose extract improves the cell differentiation inhibitory effect of enkephalin on epidermal cells. was shown to be possible.

<Experimental Example 2> Investigation of the effect of enkephalin-unadded epidermal cells on the degree of cell differentiation The following procedure was used to confirm the effect of various test substances on the expression level of the epidermal cell differentiation-associated gene KRT1.
1. Preparation of test substance Prepared in the same manner as in <Example 1> was used.
2. Examination of the effect of the test substance on the expression level of the differentiation-related gene KRT1 Normal human epidermal keratinocytes (BIOPREDIC) were placed in a special medium (Medium 154CF (Gibco) medium supplemented with calcium chloride at a final concentration of 0.03 mM and Human Keratinocyte Growth Supplement (Gibco)). ) to prepare a cell suspension having a concentration of 5×10 ⁴ cells/mL, and seeded each 500 μL in 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. After culturing for 24 hours at 37°C under humidified conditions of 5% CO ₂ /95% air, an aqueous solution of calcium chloride was added to the medium to a final concentration of 1.2 mM, and the conditions were changed to 37°C under humidified conditions of 5% CO ₂ /95% air. for 24 hours to induce cell differentiation of keratinocytes. Thereafter, total RNA was extracted, and the gene expression level of keratin 1 was measured in the same manner as in <Experiment 4>.
As shown in FIG. 10, it was confirmed that the Kazuro rose extract decreased the expression level of cell differentiation-related gene KRT1 in epidermal cells to which enkephalin was not added. In other words, the Kazunoi rose extract itself does not retain the action of promoting cell differentiation of epidermal cells, and it was confirmed that the effect confirmed in FIG. 9 was a specific action on the suppression of cell differentiation by enkephalin. .

<Example 3> Screening using the stratum corneum morphology of a three-dimensional skin model with enkephalin addition as an index Screening of an agent for improving rough skin caused by stress using the thickness of the stratum corneum of a three-dimensional skin model with enkephalin addition as an index according to the following procedure. did
1. Preparation of test substance Prepared in the same manner as in <Example 1> was used.
2. Cultured skin three-dimensional model of enkephalin-added skin Three-dimensional model preparation kit Labcyte EPI-KIT (J-TEC) Encapsulated normal human epidermal keratinocytes were suspended in a dedicated assay medium, and 500 μL of cell suspension was added to each cell culture insert. sown. 1.5 mL of the assay medium was dispensed into each well of the 12-well culture plate, and the cell culture insert was immersed in each well of the 12-well culture plate and cultured for 1 day at 37° C. under humidified conditions of 5% CO ₂ /95% air. The medium inside the cell culture insert was carefully removed and the cells were exposed to air to induce keratinocyte cell differentiation. The medium in the 12-well culture plate was replaced and cultured for 2 days at 37°C under humidified conditions of 5% CO ₂ /95% air. The medium in the 12-well culture plate was replaced, and methionine-enkephalin was added to the medium in each well to a final concentration of 100 nM and the test substance to a final concentration _of 100 ppm. Cultured under humidified conditions. The cells were cultured for 12 days while changing the medium containing methionine-enkephalin and the test substance every 2 or 3 days.
3. Observation of stratum corneum morphology Observation was carried out in the same manner as in <Experiment 6>.
As shown in FIG. 11, when compared with the Nekoashi kelp extract and the Hidaka kelp extract, when the Kazunoi rose extract and enkephalin were added, the stratum corneum thickness was significantly increased compared to the three-dimensional skin model with the addition of only enkephalin. was shown to increase.

<Example 4> Screening using the amount of transepidermal water transpiration of the three-dimensional skin model with enkephalin added as an index In the following procedure, rough skin caused by stress using the amount of transepidermal water transpiration of the three-dimensional skin model with the addition of enkephalin as an index. Screening of improvers was performed.
1. Preparation of Test Substance An extract of Ibaranori prepared in the same manner as in <Example 1> was used.
2. Culture of Enkephalin-Added Three-Dimensional Skin Model Culture was performed in the same manner as <Example 3>.
3. Measurement of transepidermal water loss Measured in the same manner as in <Experiment 8>.
As shown in FIG. 12, it was confirmed that the amount of transepidermal water loss in the three-dimensional skin model, which increased due to the addition of enkephalin, decreased to the same extent as Control (no enkephalin added) due to the addition of Kazunoi rose extract.

<Example 5> Practical use test An external preparation for skin was prepared according to the formulation shown in "Table 1" using the Kazunoi rose extract, and an effect test was conducted with 8 women in their 20s and 30s who gave their consent to the test as subjects. The subjects were selected from a human group judged to be a high-stress group after conducting a stress check in advance. Subjects were asked to apply the test samples of Test Example 1 and Comparative Example 1 every day for one month, twice in the morning and at night after washing their face, to the half of their face. was measured with a TEWA meter TM-210 (Courage + Khazaka). The measurement was carried out after washing the face with a cleanser and acclimating it for 20 minutes in an environment of 22±1° C. and 50±2% humidity.
As shown in FIG. 13, in Test Example 1, in which a component having an effect of improving rough skin due to stress selected by the screening method of the present invention was blended, compared with Comparative Example 1, the amount of percutaneous water loss decreased after continuous use of the test sample. was accepted.

Hereinafter, formulation examples using the Kazunoi rose extract according to the present invention are shown. In addition, content is mass %. In addition, the manufacturing method is based on a conventional method. In addition, the Kazunoi rose extract is a dry mass.

Formulation Example 1: Cream Kazunoi rose extract 0.02
Stearyl alcohol 6.00
Stearic acid 2.00
Vaseline 4.00
Squalane 9.00
Octyldodecanol 10.00
1,3-butylene glycol 6.00
Glycerin 4.00
POE (20) cetyl alcohol ether 3.00
Glyceryl monostearate 2.00
Antioxidant Appropriate amount Preservative Appropriate amount Purified water Balance total 100.0

Formulation 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 hydrogenated castor oil 1.50
Isotridecyl isononanoate 10.00
1,3-butylene glycol 5.00
Antioxidant Appropriate amount Preservative Appropriate amount Purified water Balance total 100.00

Formulation 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) crosspolymer 0.50
Cetyl dimethicone 0.25
Glycyrrhetinic acid ester 0.05
Methylgluceth-20 1.00
1,3-butylene glycol 10.00
Sodium chloride Appropriate amount of antioxidant Appropriate amount of preservative Appropriate amount of purified water Balance total 100.00

Claims (3)

An agent for improving rough skin caused by stress, containing a Kazunoi rose extract. An agent for improving skin barrier function deterioration caused by stress, containing a Kazunoi rose extract. Use of Kazunoi rose extract for improving rough skin caused by stress.

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