KR20160127751A - Method for screening agents promoting skin barrier function and method for evaluating skin barrier function taking epidermal serine racemase and/or d-serine level as indicator - Google Patents

Abstract

A problem to be solved by the present invention is to develop a method for screening a drug having an effect of enhancing skin barrier function in an in vitro test to evaluate the barrier function in skin. By using the activity and / or expression level of serine racemase as an index, screening of a candidate drug has become possible.

Description

TECHNICAL FIELD The present invention relates to a screening method and a skin barrier function evaluation method for skin barrier function-enhancing drugs using epidermal serine racemase and / or D-serine as indicators. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a skin barrier function- RACEMASE AND / OR D-SERINE LEVEL AS INDICATOR}

The present invention relates to a screening method of a skin barrier function-enhancing drug and a method of evaluating a skin barrier function in skin.

When the barrier function of the skin is lost, the moisture of the skin is lost and foreign substances are invaded from the outside, which causes delicate skin, dry skin, rough skin, dermatitis and allergy. Therefore, development of drugs that enhance the barrier function of the skin from the viewpoint of the development of functional foods, cosmetics, or medicines is required. As a factor enhancing the skin barrier function, there is known a method of increasing the amount of intercellular lipid, or maintaining the arrangement. For example, as shown in Patent Document 1, agents for promoting intercellular lipid production have been developed have.

As an index of the skin barrier function, there can be mentioned a penetration degree to the skin using an indicator substance such as a light-to-air moisture evaporation amount (TEWL) and a pigment. The TEWL of the skin can be measured by using a device such as a vapometer. However, the measurement of the water content is greatly affected by the measurement environment such as the amount of water in the air, the flow of air, the temperature of the subject person, A method of evaluating the barrier function of the skin more stably is required. When the penetration degree of the indicator substance is used as an index of the skin barrier function, the index substance such as pigment should be applied to the skin, which has a great burden on the skin.

In order to screen a drug having an effect of increasing the barrier function of skin from a large number of candidate drugs, experiments can be carried out in vivo using TEWL as an index of skin barrier function. However, it is unstable, It has been difficult to screen drugs from a number of candidate agents that have the effect of structurally enhancing skin barrier function. On the other hand, as such a screening method, a method of evaluating the restoration and hyperactivity of the barrier function by measuring the factor of skin intrinsic property involved in restoration and hyperactivation of the barrier function by using isolated skin may be considered. However, There was no appropriate factor for easy evaluation, and the specification of such factors was required.

On the other hand, it is known that amino acids include L-isomers and D-isomers as optical isomers. The amino acids constituting living bodies are mainly L-isomers, and D-amino acids are thought to be very limited biological components such as peptide glycans of bacteria. However, as a result of recent research, it has become clear that various D-amino acids exist not only in microorganisms but also in plants and mammals as free forms or as amino acid residues constituting proteins, and exhibit various physiological functions. As free D-amino acids in mammals, D-serine and D-aspartic acid were first found. D-serine has been found to be localized in the cerebrum, hippocampus, and acts as a coreagonist of the NMDA receptor, and plays a role in the regulation of excitatory neurotransmission (Non-Patent Document 1). In addition, D-aspartic acid has been found to be localized to the pineal gland and the pineal gland and has been found to be involved in the control of hormone secretion. On the other hand, it has been clarified that D-serine, D-aspartic acid, D-alanine, D-glutamic acid and D-proline are present in the dermis and epidermis, (Non-Patent Document 2). Among them, D-serine is the D-amino acid having the highest content in the epidermis and the stratum corneum. However, as for the role that D-serine plays in the skin, it has been found that the action to alleviate the ultraviolet ray damage in fibroblasts in the dermis Patent Document 2), but it has not yet been revealed in a high-content epidermis.

Amino acid racemase is known as an enzyme involved in the production of D-amino acids, and is classified into an enzyme dependent on pyridoxal phosphate (PLP) and a racemase that does not require a donor. Although it has been reported that serine racemase in the presence of PLP-dependent serine racemase is present in mammals (Non-Patent Document 3), with regard to aspartic acid racemase, it has been reported that D-amino acids are present in eukaryotic cells including mammals In mammals, it is not known whether they are still present with racemase activity. Since D-serine is localized in the cerebrum and hippocampus, serine racemase is expressed in the brain in mammals including humans, but almost no expression has been reported in other tissues (Non-Patent Document 3) .

Patent Document 1: JP-A-2012-92032 Patent Document 2: WO2011 / 030903

Non-Patent Document 1: S.H. Snyder, et al., NeuroChem Res 25, 553 (2000) Non-Patent Document 2: Y. Tojo, Food Style 21, 2013, August, (Vo.17, No.8) Non-Patent Document 3: Molecular Brain Research, Vo 1. 125, pp. 96-104, 2004 Non-Patent Document 4: Experimental Protocol House for Development of Functional Cosmetic Material, CMC Publication, 2010, pages 214-216

In order to enhance the barrier function of the skin, it has been noted that the amount of intercellular lipid is increased or the arrangement is regulated. On the other hand, in enhancing the barrier function of the skin, it is considered that increasing the number of layers of healthy layers is more effective approach in terms of enhancing the skin barrier function physiologically or structurally. However, a drug having such an action has not been known so far, and it has been difficult to find it by screening as described above. Therefore, there has been a demand for development of a screening method of a drug that has an effect of enhancing skin barrier function physiologically or structurally.

The inventors of the present invention have conducted intensive research on D-serine whose function in the skin is not known yet, and found that D-serine contributes to the skin barrier function. The effect of D-serine on the keratinocyte cultured was investigated. It was confirmed that the cellulase activity, which is an important factor in the formation of barrier function, as well as the production of peroxidase (CRNN) and repetin, Respectively. Based on this finding, it has been found that by using the amount of D-serine in keratinocytes, and furthermore, the activity or expression level of serine racemase producing D-serine from L-serine as an index, It is possible to provide a screening method for medicines and to provide a simple and stable evaluation of the skin barrier function with less burden on the subjects, thereby leading to the present invention. By using such a screening method, it was possible to screen drugs that enhance the skin barrier function.

Specifically, the present inventors have completed the following inventions:

[1] A method for screening a skin barrier function-enhancing drug, wherein the activity or expression level of serine racemase and / or the amount of D-serine in the keratinocyte is used as an index.

[2] a step of adding a candidate agent to keratinocytes,

A step of measuring the activity or expression amount of serine racemase and / or the amount of D-serine in keratinocytes, and

From the activity or expression level of serine racemase, and / or from the amount of D-serine, a step of determining the skin barrier function hyperactivity of the candidate agent

Wherein the screening method comprises the steps of:

[3] The screening method according to item 2, further comprising a step of inducing differentiation of keratinocytes.

[4] The screening method according to item 1, wherein the screening method is performed on the keratinocytes induced by differentiation.

[5] The screening method according to any one of items 1 to 4, wherein the measurement of the amount of serine racemase expression is performed by measuring the amount of mRNA or protein of serine racemase.

[6] The screening method according to any one of items 1 to 4, wherein the serine racemase activity is measured by measuring the conversion efficiency of the substrate or measuring the amount of the D-amino acid product.

[7] A pharmaceutical composition comprising the hydrolyzed oat protein, lempuyang extract, Elephantopus mollis extract, pyridoxal phosphate and vitamin B ₆ obtained by the screening method described in items 1 to ₆ Wherein the active ingredient is one or more substances selected from the group consisting of:

[8] A method for evaluating a skin barrier function, wherein an activity or an expression amount of serine racemase from a skin sample isolated from human skin and / or a D-serine amount is used as an index.

[9] a step of measuring the activity or expression amount and / or the amount of D-serine of serine racemase in a skin sample isolated from a subject, and

A step of determining the skin barrier function of a subject from the activity or expression level of serine racemase and / or the amount of D-serine

Wherein the skin barrier function is evaluated by the method according to item 8.

[10] A method for evaluating the skin barrier function according to item 8 or 9, wherein the measurement of the expression amount of serine racemase is performed by measuring the amount of mRNA or protein of serine racemase.

[11] The skin barrier function evaluation method according to item 8 or 9, wherein the serine racemase activity is measured by measuring the conversion efficiency of the substrate by serine racemase or by measuring the amount of the D-amino acid product How to.

[12] A pharmaceutical composition, which comprises one or more substances selected from the group consisting of hydrolyzed oat protein, mulberry extract, Shirobanase kozora extract, pyridoxal phosphate and vitamin B ₆ obtained by the screening method described in items 1 to ₆ Barrier function enhancer of the skin containing.

[13] A method for administering one or a plurality of substances selected from the group consisting of hydrolyzed oat protein, mulberry extract, Shirobanase kozora extract, pyridoxal phosphate, and vitamin B ₆ to a subject having reduced skin barrier function ≪ / RTI >

[14] A method for treating a subject suffering from a decrease in serine racemase activity, the method comprising administering one or more substances selected from the group consisting of hydrolyzed oat protein, mulberry extract, Shirobanase kozora extract, pyridoxal phosphate and vitamin B ₆ ≪ / RTI >

[15] Use of hydrolyzed oat protein, mung bean extract, Shirobanaga kozora extract, pyridoxal phosphate and vitamin B ₆ for the production of serine racemase activator.

[16] Use of hydrolyzed oat protein, mung bean extract, Shirobanaga kozorina extract, pyridoxal phosphate and vitamin B ₆ for the production of skin barrier function agonists.

By using the screening method of the present invention, it becomes possible to select a drug that can improve or enhance the skin barrier function. In addition, the skin barrier function of the present invention can be determined at a cellular level or a tissue level or biochemically by the method of evaluating the skin barrier function.

Fig. 1 (A) shows a comparison of the amount of free (free) L-serine in the skin of the high TEWL and low TEWL groups. Figure 1 (B) shows a comparison of the amount of free D-serine in the skin of the high TEWL and low TEWL groups.
2 is a chromatogram showing intracellular D-serine in cultured keratinocytes before and after application of differentiation induction stimulation.
Fig. 3 (A) is a graph showing that the amount of free D-serine in the hydrolyzed oat protein solution, which is one of the candidate drugs, is increased. Fig. 3 (B) is a graph showing that the hydrolyzed oat protein solution, which is one of the candidate drugs, increased the expression of serine racemase.
4 is a graph showing that the amount of free D-serine is increased by adding pyridoxal phosphate (PLP), which is one of the candidate drugs, to cultured keratinocytes.
5 is a graph showing that D-serine increases the expression of filamentous green gene in a three-dimensional skin model.
Fig. 6 is a graph showing that D-serine increases the expression of a coenzyme gene in a three-dimensional skin model. Fig.
7 is a graph showing that D-serine increases the expression of the leptin gene in the three-dimensional skin model.

The present invention relates to a screening method of a skin barrier function-enhancing agent comprising keratinocytes, wherein one or more indicators of serine racemase expression level, activity and D-serine level are combined.

In the screening method of the present invention, it is possible to obtain a medicament having a skin barrier function-enhancing action. Here, the skin barrier function is a function to maintain the moisture of the skin or to prevent foreign matter from entering from the outside, and in particular, the stratum corneum plays a role in the skin. The skin barrier function can be measured by the TEWL, and it is thought that the higher the water evaporation amount, the lower the skin barrier function. The enhancement of the skin barrier function can be achieved, for example, by an increase in the number of layers of the stratum corneum, maturation of the keratinized outer membrane (corneoid envelope) in the stratum corneum, or increase of intercellular lipids such as cholesterol or ceramide, It is not intended to be limiting. Thus, while not intending to be bound by theory, it is believed that the agent selected by the screening method of the present invention can be applied to the skin to enhance the expression or activity of serine racemase or to increase the amount of D-serine As a result, it is considered that the skin barrier function can be exerted by exhibiting the effect of increasing the number of layers in the stratum corneum and the like.

The candidate agent used in the screening method of the present invention may be any compound, for example, a compound obtained from a recombinatorial library, or may be a mixture or an extract. As a candidate drug, for example, a compound, a mixture, or an extract library of a food material, a cosmetic material, or a pharmaceutical material may be used. According to the present invention, a medicament having a skin barrier function-enhancing action can be applied to foods such as functional foods and nutritional foods, cosmetics, medicines or quasi-drugs. The cosmetics can be applied to, for example, cosmetics, creams, emulsions, gels, essences, ointments, packs, bath salts, body soaps, shampoos, rinses, foundations and the like. More preferably, it is applied to cosmetic preparations for sensitive skin such as lotion, cream, milky lotion, serum and the like. If it is a drug or a quasi-drug, it can be applied to various ointments, creams for transdermal administration, and forms for oral administration.

In the screening method of the present invention, the activity or expression level of serine racemase and / or the amount of D-serine in keratinocytes are used as an index. As an indicator, it means that the activity or expression amount of serine racemase and / or the amount of D-serine are considered. Therefore, when a drug having a skin barrier function-enhancing effect is selected based on the activity or expression level of serine racemase and / or the amount of D-serine when the candidate drug is added, the screening method of the present invention .

The keratinocyte is a cell that constitutes about 95% of the epidermis. In vivo, keratinocyte stem cells are present in the basal layer of the epidermis, and migrate to the outer layer according to maturation to form the anolyte, granule and stratum. The keratinocytes change their properties according to their maturity. The cells are flattened in the polar or granular layer, and lipids such as ceramides are released into the cell gap, resulting in enucleation in the stratum corneum of the outermost layer. This process of maturation is called the eruption of keratinocytes. The cells having the enucleation are disappeared, the cell form is lost, and the cells are stratified into layers to finally separate from the outermost layer as dirt.

The cultured keratinocytes used in the present invention may be cells obtained from any of the basal layer, the polar layer, the granular layer, and the stratum of the epidermis, and may be stem cells such as skin epithelial stem cells, pluripotent stem cells such as ES cells, iPS Cell, EG cell, or the like. The cell obtained from the epidermis and cultured may be a primary cultured cell, a subcultured cell, or may be a cell that has been cancerized or immortalized. The cultured keratinocyte used in the present invention may be a keratinocyte, such as a human or an animal-derived coin. These keratinocyte cell lines can be obtained from public agencies or vendors.

The culture may refer to only a desired cell, or may include other cells, and may further comprise cells and a medium. Therefore, the keratinocyte culture may include only keratinocytes, keratinocytes and other cells, keratinocytes alone or keratinocytes and other cells and culture medium. Such cultures include monolayer cultures, mixed cultures, and three-dimensional cultures. The medium for culturing the keratinocytes may be any medium as long as it is capable of culturing keratinocytes. For example, various additives may be added to the medium of HuMedia-KB2 (Kurabo) or Keratinocyte-SFM (Invitrogen) But the present invention is not limited thereto. The culture conditions are usually cultured in the presence of 5% CO ₂ at 37 ° C, but it is also possible to change the range in which keratinocyte proliferation occurs. From the viewpoint of making the amount of D-serine present as a serine racemase activity, it is preferable that the medium is L-serine-rich (enriched). In the culture medium, D-amino acid is not intentionally added, and the amount of L-serine present relative to D-serine in the medium is at least 1.1 times, more preferably 10 times, still more preferably 50 times or more . For example, in a medium (KGM medium) supplemented with a HuMedia-KG growth additive set in HuMedia-KB2 (Kurabo), the amount of L-serine present in D-serine is 100 times or more.

The keratinocyte cultures can be used for experiments in which cultivation is carried out after sowing and about 50% confluence to confluent. The collection density is preferably 70% or more, and more preferably 80% or more from the viewpoint of inducing differentiation. The step of adding the candidate agent to the keratinocyte culture may be carried out by replacing the medium of the keratinocyte culture with a medium containing the candidate agent and then culturing the keratinocyte culture medium The drug may be added as it is or as a diluent.

Serine racemase is a racemic maize that is dependent on the vitamin B6 (co) enzyme type, pyridoxal 5 'phosphate (PLP). Without intending to be bound by theory, serine racemase is an enzyme capable of converting between L-serine and D-serine through the production of an anionic intermediate by withdrawing a-hydrogen of serine. In the conversion reaction between L-serine and D-serine, the enthalpy does not change before and after the reaction, and since the reaction is driven by entropy, L-serine and D-serine become equal in the equilibrium state. Since the amount of L-serine present in vivo is higher than that of D-serine, the presence of serine racemase transforms L-serine in vivo into D-serine. Since the culture medium also contains a large amount of L-form in the culture medium, L-serine is converted into D-serine by the presence of serine racemase. In the present invention, racemease may be the same as racemease expressed in brain by non-patent document 3, or may be homologous or orthologous thereto. Therefore, it refers to a substance capable of being amplified by a primer pair used in Non-Patent Document 3, and capable of being detected by an anti (anti-rachma) antibody described in Non-Patent Document 3. Regarding serine racemase, a person skilled in the art can appropriately specify the species according to the animal species from the website of a public institution such as NCBI.

In one embodiment of the present invention, the activity of serine racemase in keratinocyte is used as an index to evaluate the skin barrier function or screen the skin barrier function-enhancing drug. The activity of serine racemase may be based on the amount of D-serine produced by the conversion of L-serine contained in a normal medium in the case of not adding a substance to be an enzyme substrate, Amino acid may be based on the total amount of other D-amino acids. Furthermore, by adding a substance other than serine, which can be a substrate of serine racemase, the amount of the product based on the conversion of the additive or the conversion efficiency may be measured. It is of course possible to add the D form in the activity measurement by converting the excess optical isomer to the other optical isomer until the D form and L form are 50:50 in the serine racemase. In that case, enzyme activity can be evaluated by measuring the increase of L-serine caused by the conversion. In addition, for example, by adding a derivative of cycloserine or serine (N-methylated form, acetylated form, phosphorylated form, etc.), the enzyme activity can be evaluated by examining the conversion efficiency to the corresponding optical isomer. When the activity of serine racemase is measured based on the amount of D-serine, the presence of D-serine in skin samples isolated from the subject, or in keratinocyte cultures, i.e., in the medium, Or may be determined by an enzymatic parameter determined based on a change in the amount of D-serine present in the skin sample or in the keratinocyte culture. This method can be applied to all of the above-mentioned substances serving as substrates of enzymes. It may be indicated by determining the values of V _max and K _m as enzymatic parameters indicating the activity of the serine racemase enzyme.

In another embodiment of the present invention, the skin barrier function can be evaluated, or the skin barrier function-enhancing drug can be screened using the amount of D-serine in the keratinocyte as an index. The amount of D-serine may be measured in a skin sample isolated from a subject or in a keratinocyte culture, that is, in a medium, in a cell, or both. The method for measuring the amount of D-serine can be carried out by a known method, for example, by an HPLC method using an optical isomer separation column, a gas chromatography method, an electrophoresis method, or an enzymatic method. When the activity of serine racemase activity is measured by measuring the amount of D-serine, L-serine may be added to the keratinocyte culture to increase the amount of D-serine. When the serine racemase is subjected to the conversion reaction between L-serine and D-serine, since the enthalpy does not change before and after the reaction and the reaction is driven by entropy, the activity of serine racemase , The amount of D-serine can be increased, and the measurement of the amount of D-serine becomes easier.

In another embodiment, the expression level of serine racemase is used as an index to evaluate the skin barrier function or screen the skin barrier function antagonist. The expression level of serine racemase can be determined by measuring the protein of serine racemase by an immunological method using an anti-serine racemase antibody. As an immunological method, for example, Western blotting method, fluorescent immuno-staining method or ELISA method may be used. Antiserine racemase may be a commercially available monoclonal antibody or polyclonal antibody or may be obtained by immunizing a laboratory animal such as rabbit or mouse with a serine racemase protein or an antigen peptide to recover the antibody good.

The expression amount of serine racemase may be determined by determining the amount of mRNA of serine racemase in the cells. The measurement of mRNA may be performed by any method, for example, based on a method of quantitative PCR. The quantitative PCR method is usually carried out as follows, but is not intended to be limited thereto. MRNA is extracted from the recovered cells, and cDNA is prepared using poly T primer and reverse transcriptase. The amount of mRNA contained in the cells can be quantified by using the Syber green method or the fluorescent probe method for the prepared cDNA.

More specifically, in the screening method of the present invention,

A step of adding a candidate agent to keratinocytes,

A step of measuring the activity or expression amount of serine racemase and / or the amount of D-serine in keratinocytes, and

From the activity or expression level of serine racemase, and / or from the amount of D-serine, a step of determining the skin barrier function hyperactivity of the candidate agent

. The screening method of the present invention may further include a step of inducing differentiation of keratinocytes.

The differentiation induction step is carried out by imparting a differentiation inducing stimulus to keratinocytes. By imparting differentiation induction stimulation to the keratinocyte, it is possible to improve the flattening of the cells, the increase of the fibrous material or the granules in the cells, the obscuration of the cell gap, the stratification (especially in the case of monolayer culture) Changes in the proteins and proteins such as keratin 1, keratin 10, inbolucrin, and activation of transglutaminase occur, such as changes in morphology and cell morphology (see Journal of the Society of Obstetrics and Gynecology 90 (12), 1089-1101, http : //drmtl.org/data/090121089.pdf, Drug News Perspect 2004, 17 (2) p117). This differentiation induction stimulation may be any stimulation as long as it can cause the differentiation of keratinocytes described above. For example, calcium ion stimulation which increases the concentration of calcium ions, stimulation by phorbol-12-myristate-13-acetate Stimulation (Hawley-Nelson P, Stanley JR et al., Exp Cell Res., 137: 155-167, 1982), physical stimulation such as voltage or gaseous exposures, or stimulation by biological fluids. When stimulated by calcium ions, a calcium salt or a diluted solution thereof may be added so that the concentration of calcium ions is 0.1 mM to 10 mM as a species concentration, or may be substituted with a medium prepared so as to contain calcium ions at an intrinsic concentration . As the calcium salt to be used, for example, calcium chloride, calcium hydrogen carbonate, calcium carbonate, calcium nitrate, calcium acetate and the like are used. The upper limit of the species concentration of calcium ions is preferably 5 mM or less, more preferably 2 mM or less, from the viewpoint of not adversely affecting the cell culture. From the viewpoint of promoting differentiation induction, the lower limit of the concentration is preferably 0.5 mM or more, more preferably 1.0 mM or more.

The step of differentiation induction and the step of adding a candidate drug may be performed at the same time, or the step of adding a candidate drug may be performed after the step of differentiation induction, and the step of inducing differentiation may be performed after the step of adding a candidate drug. While not intending to be bound by theory, candidate agents can alter the timing of induction of differentiation and addition of candidate agents by the desired mechanism of action. For example, from the viewpoint of examining a drug that is antagonistic to the differentiation inducing stimulating substance, it is preferable that the adding step is performed before or simultaneously with the stimulation step. From the viewpoint of promoting and inhibiting cell changes (morphological changes, expression of various intracellular factors, etc.) after induction of differentiation, it is preferable that the addition step is performed simultaneously with or after the stimulation step. From the viewpoint of searching mechanisms such as blocking in advance, it is preferable that the stimulation process is performed after the addition process. If the addition process of the candidate drug and the stimulation process of induction of differentiation are not performed at the same time, a cell culture process of several minutes to several hours may be included between the addition process and the stimulation process.

After the stimulation of induction of differentiation and addition of the candidate agent, a cell culturing step may be further included before the step of measuring the activity or expression amount of serine racemase and / or the amount of D-serine. The period of the cell culturing process can be optionally selected depending on the kind and concentration of the differentiation inducing agent to be used and the kind of keratinocytes, but culturing can be performed for 6 hours to 1 week, for example. From the viewpoint of sufficiently expressing serine racemase, the lower limit of the culture period is preferably 12 hours or more, more preferably 1 day or more. On the other hand, the upper limit of the incubation period is not particularly defined because it differs depending on the culture method such as monolayer culture, stratified culture, and three-dimensional culture. From the viewpoint of maintaining healthy culture conditions of the cells, And more preferably within 3 days.

The screening method of the present invention may further include a damage imparting step. The damage imparting step may be to damage the cultured keratinocyte by physical or chemical stimulation. For example, it may be irradiated with ultraviolet rays, addition of a radical source such as an oxidizing agent, addition of a surfactant, And the like. It is considered that the expression of serine racemase is reduced in keratinocytes to which damage is imparted by the damage imparting process. Therefore, in the screening method for performing the damage-imparting step after the step of adding the candidate agent, when the decrease in the expression amount of racemasase or the activity of racemase and / or the decrease in the amount of D-serine is suppressed, It is possible to prevent damage to the barrier function. On the other hand, in the screening method for performing the step of adding the candidate drug after the damage-imparting step, when the amount of the decreased expression of racemase or the activity of racemase and / or the amount of D-serine increases, Improvement, or remedy of < / RTI > In the screening method that does not give damages, the candidate agent that increases the amount of racemase expression or racemease activity and / or the amount of D-serine is an agonist of skin barrier function.

As a result of the screening method of the present invention using the expression amount of serine racemase as an index on the cosmetic material library, it was found that as a drug capable of increasing the expression amount of serine racemase in the keratinocyte, the hydrolyzed oat protein, Extract, and Shirobanaga ginseng extract. When the expression amount of serine racemase is increased, the serine racemase activity is increased and the content of D-serine in each layer is increased, and skin barrier function can be renewed. Therefore, such a substance may be referred to as a serine racemase expression promoter, a serine racemase activator, or a skin barrier function agonist. The medicines screened by the screening method of the present invention are not limited to extracts derived from sperm or sirovana, but also from extracts of the related species. Similarly, the medicines screened by the screening method of the present invention have a skin barrier function-enhancing effect. Accordingly, the serine racemase expression promoter, serine racemase activator, or skin barrier function agonist of the present invention include Zingiberaceae, Zingiber plant, Compositae, and Elephantopus genus Plant extracts. Therefore, by incorporating such a substance into a cosmetic, a cosmetic material exhibiting a skin barrier function-enhancing effect can be produced.

Further, the screening method of the present invention using the serine racemase activity as an index was conducted on a cosmetic material library. As a result, pyridoxal phosphate (PLP: active form vitamin B ₆ ) could be selected for keratinocytes. When the serine racemase activity increases, the content of D-serine in each layer increases, and skin barrier function can be renewed. Thus, pyridoxal phosphate can be referred to as a serine racemase activator or a skin barrier function agonist.

The hydrolyzed oat protein used in the present invention is a hydrolyzate of a protein obtained from oven (Avena sativa). Oat proteins are extracted from, for example, seeds, leaves, roots, bran, etc., but seeds are particularly preferred. The oats are preferably dried and pulverized immediately after harvesting. The drying may be performed in sunlight or in a dryer that is generally used. Proteins are hydrolyzed by acid or enzymatic treatment during or after extraction. Oats are plants that are grown edible in Europe, North America from West Asia. The extraction solvent used in the present invention may be any solvent which is usually used for extraction. In particular, organic solvents such as alcohols such as methanol and ethanol, aqueous alcohols, acetone, and ethyl acetate may be used alone or in combination, Extraction with water may be performed.

The amount of the hydrolyzed product of the protein obtained from oats in the present invention can be arbitrarily selected within the range in which those skilled in the art can make an effect. For example, the blending amount is 0.00005 to 20.0% by mass as a dry matter in the total amount of the external-use agent. The upper limit of the blending amount is preferably 10% by mass or less, more preferably 5% by mass or less from the viewpoint of formulation and effect. The lower limit of the blending amount is preferably 0.0001 mass% or more, more preferably 0.0005 mass% or more, from the viewpoint of exhibiting sufficient skin barrier function-enhancing effect.

The plants of the genus Zingiberaceae and Zingiber used in the present invention are preferably Lempuyang (Zingiber aromaticum Mal.). Mungpu is a plant that grows especially in the dry grasslands and grasses of Indonesia. The extract used in the present invention can be obtained by immersing plant outbreaks such as leaves of plants, stems including underground stems, fruits, etc., together with an extraction solvent, heating and refluxing as occasion demands, filtering and concentrating. The extraction solvent used in the present invention may be any solvent which is usually used for extraction. In particular, organic solvents such as alcohols such as methanol and ethanol, aqueous alcohols, acetone, and ethyl acetate may be used alone or in combination, Extraction with water may be performed.

The blending amount of the extracts of Zingiberaceae and Zingiber plants in the present invention can be arbitrarily selected within the range in which those skilled in the art can exert their effects. For example, the blending amount is 0.00005 to 20.0% by mass as a dry matter in the total amount of the external-application agent. The upper limit of the blending amount is preferably 10% by mass or less, more preferably 5% by mass or less from the viewpoint of formulation and effect. The lower limit of the blending amount is preferably 0.0001 mass% or more, more preferably 0.0005 mass% or more, from the viewpoint of exhibiting sufficient skin barrier function-enhancing effect.

Compositae and Elephantopus plants used in the present invention are preferably Shirabanai kozorina (Scientific name: Elephantopus mollis). Shirobanaga Kojorina is a plant native to Venezuela in South America and widely naturalized in tropical Asia. Compositae and extracts of plants belonging to the genus Elephantopus used in the present invention can be obtained by dipping or heating the above leaves, stems, flowers, bark, seeds or fruits, , Followed by filtration and concentration. The extraction solvent used in the present invention may be any solvent which is usually used for extraction. In particular, organic solvents such as alcohols such as methanol and ethanol, aqueous alcohols, acetone, and ethyl acetate may be used singly or in combination.

In the present invention, the blending amount of the extract of Chrysanthemum (Comvitae) and the plant of the genus Elephantopus (Elephantopus) can be arbitrarily selected within the range in which those skilled in the art can exert their effects. For example, the blending amount is 0.00005 to 20.0% by mass as a dry matter in the total amount of the external-application agent. The upper limit of the blending amount is preferably 10% by mass or less, more preferably 5% by mass or less from the viewpoint of formulation and effect. The lower limit of the blending amount is preferably 0.0001 mass% or more, more preferably 0.0005 mass% or more, from the viewpoint of exhibiting sufficient skin barrier function-enhancing effect.

The pyridoxal phosphate used in the present invention is a substance used as a coenzyme of serine racemase. Pyridoxal phosphate is an active form of the pyridoxal known as vitamin B ₆ . Although not intending to be bound by theory, it is believed that pyridoxal phosphate can increase the activity of serine racemase as a co-enzyme, and D-serine produced thereby acts on keratinocytes, , Such as pilar green, koinurin, and leptin, are enhanced, thereby enhancing the skin barrier function. The vitamin B ₆ class includes pyridoxine, pyridoxine, and pyridoxamine, and is known to be a component to be incorporated in cosmetics. However, it is a water-soluble vitamin, and there is a problem in absorption rate and stability in transdermal administration. Therefore, if for the purpose of improving the percutaneous absorption of vitamin B ₆ derivatives which increase the fat-soluble is known, for example, tris-hexyl decanoic acid pyridoxine, tri-palmitic acid pyridoxine, dipal palmitic acid pyridoxine, dicarboxylic ruffle acid pyridoxine, such as the vitamin B ₆ derivative Are also included in Vitamin B ₆ . Further, for the purpose of improving the stability, pyridoxine cyclic phosphoric acid and pyridoxine hydrochloride are already known. Vitamin B ₆ as well as pyridoxal phosphate are also used as serine racemase activators or skin sensitizers. The blending amount thereof is, for example, 0.000001 to 10 mass%. The upper limit of the blending amount is preferably 10% by mass or less, more preferably 1% by mass or less from the viewpoint of formulation and effect. The lower limit of the blending amount is preferably 0.000001 mass% or more, more preferably 0.00001 mass% or more, from the viewpoint of exhibiting sufficient skin barrier function-enhancing effect.

From the activity or expression level of serine racemase, or from the amount of D-serine, in the process of determining the barrier function hyperactivity of the skin of the candidate drug, the activity or expression level of the measured serine racemase and / Amount can be determined using a table showing the relationship between a preset expression amount or activity, and / or a D-serine amount, and a barrier function hyperactivity action. These tables show that, as a control, the activity or expression level of rathemase activity and / or the amount of D-serine, the activity or expression level of serine racemase and / or the amount of D-serine measured by carrying out the same experiment without the candidate agent, Or by increasing the amount of D-serine, or by using the amount of racemase activity or expression and / or the amount of D-serine measured by carrying out the same experiment using a known reference drug. In another embodiment, the skin barrier function hyperactivity of the candidate agent can be determined by preliminarily setting a threshold value from a comparison with the control, and whether or not the threshold value is higher than a statistical significance difference. Those skilled in the art can appropriately set these threshold values in accordance with the required skin barrier function hyperactivity. As such a threshold value, for example, a threshold value of the magnification can be arbitrarily set for the activity or expression amount of serine racemase and / or the amount of D-serine in the non-drug addition control, but for example, It is also possible to set a value with a tendency difference. The statistical significance is p <0.05, and the tendency difference is p <0.1. In addition, in another embodiment, in the case of having a high expression level or activity as compared with the amount of racemase activity or expression and / or the amount of D-serine measured by carrying out the same experiment without the candidate agent as a control, It may be determined that it has a skin barrier function hyperactivity. In this case, it can be determined that the higher the expression level or activity, and / or the D-serine content, the skin barrier function hyperactivity.

In another aspect, the present invention may also be applied to a method for evaluating, determining, or discriminating a skin barrier function using the activity or expression level of serine racemase and / or the amount of D-serine as an index. Such evaluation, determination, or discrimination may be performed by measuring the D-serine content, the serine racemase activity, or the expression amount of serine racemase in a skin sample, for example, each layer sample obtained by tape stripping from the subject. The evaluation, judgment or discrimination method of the skin barrier function is usually performed by measurement of TEWL using a Vapomaeter or the like, but the TEWL depends on the condition of the skin, and the numerical value is changed by cleaning or friction, , There is no correlation between the numerical value and the health of the skin. The evaluation, determination or discrimination method of the present invention can measure the skin barrier function at the cellular level originally possessed by the skin by using the activity or expression level of serine racemase and / or the amount of D-serine as indicators . Further, since the evaluation method of the present invention determines the health of the skin barrier function at the cellular level, it is possible to predict the future skin barrier function rather than the skin barrier function at the present time. The evaluation, judgment, or discrimination of the skin barrier function can be determined using a chart or a threshold indicating the relationship between the activity or expression level of serine racemase set in advance and / or the amount of D-serine and the barrier function hyperactivity have. Such a chart or threshold value may be determined based on the amount of expression or activity obtained and / or the amount of D-serine obtained from isolated skin samples taken from a subject group having healthy skin, a subject group having rough skin, furthermore, Can be set.

It has been found that the addition of D-serine to cultured cells increases the expression of several genes. Among them, it was found that the expression of the koinyrin and leptin, which contribute to the formation of the filaggrin and the cornified cell envelope (CE), play an important role in the formation of the barrier function. Although not intending to be bound by theory, it is believed that the serine racemase activation or expression enhancer screened by the present invention can increase the D-serine content in keratinocytes, and that D-serine can bind to keratinocytes Thus, the expression of the protein involved in the barrier function is exaggerated, thereby enhancing the skin barrier function. Therefore, D-serine itself can also be used as a skin barrier function-enhancing agent. D-serine can also be referred to as an expression promoter of a filaggreen gene, a coenynthesis gene, and a leptin gene, and may also be referred to as a keratinization promoter.

Pilar green is a basic protein rich in histidine, arginine, serine, glycine, and glutamic acid, and has a function of depositing and coagulating in keratin fibers. As a result, the keratin fibers inside the keratinocytes aggregate, and the whole cells are flattened, and the keratinous fibers are changed from the granular layer to the stratum corneum and keratinized. Pilar green is expressed as a precursor in the granular cells of the epidermis, called propylaguan, and when the granular cells are keratinized, it undergoes the action of proteolytic oxygen to decompose into pilgrams. Reduction of the pilar green is considered to be a major cause of inflammation of the juvenile or atopic dermatitis, and a marked decrease in skin barrier function is seen in these symptoms.

Connine and lepetin are proteins that constitute the cornified cell envelope that supports the cell membrane of keratinocytes. Conine and lepetin are genes that are located at the chromosomal position of 1q21 in the same manner as proteins such as inbolucrin and loricrin, which are other constituents of the periphery, and are expressed in the outermost layer of the epidermis in the final stage of keratinization do. The protein that constitutes the peripheral band is involved in the function of the skin together with the keratin aggregated by the action of the pillar green.

By the screening method of the present invention, the skin barrier function-enhancing agent or the serine racemase activating agent may be incorporated in cosmetics or in pharmaceuticals. Such a cosmetic product is used in a subject having symptoms such as atopic dermatitis, lice, psoriasis, skin roughness and the like in which the skin barrier function is lowered. In addition, when formulated into pharmaceuticals, the pharmaceuticals can be used as medicinal compositions for the treatment of atopic dermatitis, asthma, psoriasis and the like.

The present invention will be described more specifically by the following examples. The present invention is not limited to this.

Example

Example 1: Verification of the relationship between D-serine content and barrier function in each layer

The subjects were 57 healthy volunteers. After washing the affected area by the prescribed method, the affected area was cleaned with the cleaning, and waited for 30 minutes under constant temperature and humidity conditions (room temperature 22 ° C, humidity 45%). Thereafter, the transdermal water volume increase (TEWL) was measured by a Vapometer (Delfin). The measurement was performed three times, and the average value was used for the subsequent analysis. Next, each of the layers at the positions where the TEWL was measured was peeled off twice using an adhesive tape, and then the amino acid was extracted in a 95% methanol aqueous solution. The optical isomers of the extracted amino acids were analyzed according to a known method (Reference: Yurika Miyoshi et al., Journal of Chromatography B, 877 (2009) 2506-2512). The outline is shown below. The extracted amino acid was subjected to fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in a borate buffer solution (pH 8.0) and acidified with trifluoroacetic acid , Then used in 2-D micro-HPLC (Shiseido). A micro-monolith ODS column (inner diameter 0.53 mm, total length 500, 750, 1000 mm, Shiseido) was used for the first dimension and an optical resolution column and a mobile phase 1.5 mm ID for each amino acid were used for the second-dimensional optical resolution. NBD-amino acid was detected by fluorescence emission (excitation wavelength 470 nm) at 530 nm. The results are shown in Table 1 and Fig.

In order to verify the relationship between D-serine content and skin barrier function in each layer, each subject was listed in order of TEWL value. The top 17 (barrier function subgroup) and the bottom 17 (barrier function group Group) were compared with each other. The significant differences between the two groups were verified by non-paired Student t-test. As a result, it was revealed that the D-serine content was significantly higher in a human having a high skin barrier. On the other hand, as a result of the comparison with L-serine, the content of L-serine in the barrier function subgroup was low and the content of L-serine was high in the barrier function superior group, but there was no significant difference. Therefore, the higher content of D-serine in the barrier function superior group is not due to the amount of L-serine, which is a precursor, but can be said to be the influence of rasemaase expression or activity.

Example 2: Variation of D-Serine Content upon Induction of Keratinocyte Differentiation

<Cell culture and drug treatment>

Human normal keratinocytes were inoculated on a 24-well multiplate at a cell density of 2.5 x 10 ⁴ cells / well and cultured in KGM medium (Kurabo) under the conditions of 37 ° C and 5% CO ₂ . After 3 days, KGM medium (test group) supplemented with 1.8 mM calcium chloride and 10 mM L-serine or KGM medium (control group) supplemented with 10 mM L-serine alone was added and cultured for 2 days.

&Lt; Analysis of free D-serine by preparation of sample for HPLC and HPLC >

The cells were washed with PBS, and 500 μL of 5% trichloroacetic acid solution was added thereto. The cells were well suspended and recovered. After ultrasonication on ice for 30 minutes, the supernatant was centrifuged to obtain supernatant. To this supernatant, 20-fold amount of methanol was added to extract the amino acid. The optical isomers of the extracted amino acids were analyzed according to a known method (Reference: Yurika Miyoshi et al., Journal of Chromatography B, 877 (2009) 2506-2512). The outline is shown below. The extracted amino acid was subjected to fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in a borate buffer solution (pH 8.0) and acidified with trifluoroacetic acid , And two-dimensional micro-HPLC (Shiseido). For the first dimension, a micro-monolith ODS column (0.53 mm in inner diameter, 750 mm in length, Shiseido) was used. In the second-step optical resolution, an optical resolution column and a mobile phase were used. NBD-amino acid was detected by fluorescence emission (excitation wavelength 470 nm) at 530 nm. The results of the test group and the control group are shown in Fig.

Example 3: Screening of candidate drugs

<Cell culture and drug treatment>

Human normal keratinocytes were inoculated on a 24-well multiplate at a cell density of 2.5 x 10 ⁴ cells / well and cultured in KGM medium (Kurabo) under the conditions of 37 ° C and 5% CO ₂ . After 3 days, the cells were exchanged with KGM medium containing 1.8 mM calcium chloride, 10 mM L-serine and a diluent of the candidate agent, and further cultured for 2 days. Seven kinds of medicines were used as candidate drugs, and 0.5% hydrolyzed oat protein solution was used as one of them.

&Lt; Analysis of free D-serine by preparation of sample for HPLC and HPLC >

The cells were washed with PBS, and 500 μL of 5% trichloroacetic acid solution was added thereto. The cells were well suspended and recovered. After ultrasonication on ice for 30 minutes, the supernatant was centrifuged to obtain supernatant. To this supernatant, 20-fold amount of methanol was added to extract the amino acid. The optical isomers of the extracted amino acids were analyzed according to a known method (Reference: Yurika Miyoshi et al., Journal of Chromatography B, 877 (2009) 2506-2512). The outline is shown below. The extracted amino acid was subjected to fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in a borate buffer solution (pH 8.0) and acidified with trifluoroacetic acid , And two-dimensional micro-HPLC (Shiseido). For the first dimension, a micro-monolith ODS column (0.53 mm in inner diameter, 750 mm in length, Shiseido) was used. In the second-step optical resolution, an optical resolution column and a mobile phase were used. NBD-amino acid was detected by fluorescence emission (excitation wavelength 470 nm) at 530 nm. The results are shown in Table 2 and Fig. 3A.

&Lt; Quantitative PCR (RT-PCR) >

Cells were washed with PBS and mRNA was extracted from the cells using MagNA Pure LC (Roche Diagnostics). Next, cDNA was synthesized using a kit of reverse transcriptase Super Script VILO (Invitrogen). This cDNA was used as a template for a real-time PCR using a fluorescent dye Cyber Green with Light Cycler (Roche Diagnostics). The PCR reaction was repeated 30 times at 95 ° C for 10 minutes, followed by amplification reaction at 95 ° C for 15 seconds, 60 ° C for 10 seconds, and 72 ° C for 10 seconds. The relative amount of gene expression was calculated from the number of cycles until a constant amount of PCR product was obtained. RT-PCR was performed on G3PDH (glyceraldehyde-3-phosphate) gene as an internal standard in addition to the serine racemase gene, and the relative expression amount of serine racemase was corrected to the relative expression amount of G3PDH To evaluate the drug. As the primers used in the RT-PCR, those described in Non-Patent Document 3 and Non-Patent Document 4 were selected and used. The results are shown in Table 2 and Fig. 3 (B).

Among the candidate drugs used for the present screening, the hydrolyzed oat protein solution showed an increase in the expression of serine racemase in the keratinocytes induced to differentiation. In addition, it was also found that the hydrolyzed oat protein solution increased D-serine content in keratinocytes induced by differentiation. Based on these results, it is possible to select the hydrolyzed oat protein solution as an effective agent for enhancing the skin barrier function.

Example 4: Screening of candidate drugs

<Cell culture and drug treatment>

Human normal keratinocytes were inoculated on a 24-well multiplate at a cell density of 2.5 × 10 ⁴ cells / well and cultured in KGM medium (Kurabo) under the conditions of 37 ° C. and 5% CO ₂ . After 3 days, the cells were exchanged with KGM medium containing 1.8 mM calcium chloride, 10 mM L-serine and a diluent of the candidate agent, and further cultured for 2 days. Twelve kinds of medicines were used as candidates, and 0.5% sperm extract and 0.5% sirovana were contained in the extracts.

&Lt; Quantitative PCR (RT-PCR) >

Cells were washed with PBS and mRNA was extracted from the cells using MagNA Pure LC (Roche Diagnostics). Next, cDNA was synthesized using a kit of reverse transcriptase Super Script VILO (Invitrogen). This cDNA was used as a template for a real-time PCR using a fluorescent dye Cyber Green with Light Cycler (Roche Diagnostics). The PCR reaction was repeated 30 times at 95 ° C for 10 minutes, followed by amplification reaction at 95 ° C for 15 seconds, 60 ° C for 10 seconds, and 72 ° C for 10 seconds. The relative amount of gene expression was calculated from the number of cycles until a constant amount of PCR product was obtained. RT-PCR was performed on the G3PDH (glyceraldehyde-3-phosphate) gene as an internal standard in addition to the serine racemase gene, and the drug was evaluated by correcting the relative expression level of serine racemase to the relative expression level of G3PDH. As the primers used in the RT-PCR, those described in Non-Patent Document 3 and Non-Patent Document 4 were selected and used. The results are shown in Table 3 below.

Among the candidate drugs used in the present screening, the extracts of Phellinus linteus and Shirobanaga kozorina showed an increase in expression of serine racemase in the keratinocytes induced by differentiation. As a result of this, the extract of Zyprei and the extract of Shirobanaga kojorina can be selected as effective agents for enhancing the skin barrier function.

Example 5: Screening of candidate drugs

<Cell culture and drug treatment>

Human normal keratinocytes were inoculated on a 24-well multiplate at a cell density of 2.5 × 10 ⁴ cells / well and cultured in KGM medium (Kurabo) under the conditions of 37 ° C. and 5% CO ₂ . After 3 days, the cells were exchanged with KGM medium containing 1.8 mM calcium chloride and cultured for 2 days. Then, the cells were exchanged with KGM medium containing 1.8 mM calcium chloride and a diluent of pyridoxal phosphate (PLP) as a candidate agent, and further cultured for 2 days.

&Lt; Analysis of free D-serine by preparation of sample for HPLC and HPLC >

The cells incubated with the pyridoxal phosphate-added culture medium were washed with PBS, and 500 μL of 5% trichloroacetic acid solution was added. The cells were well suspended and recovered. After ultrasonication on ice for 30 minutes, the supernatant was centrifuged to obtain supernatant. To this supernatant, 20-fold amount of methanol was added to extract the amino acid. The optical isomers of the extracted amino acids were analyzed according to a known method (Reference: Yurika Miyoshi et al., Journal of Chromatography B, 877 (2009) 2506-2512). The outline is shown below. The extracted amino acid was subjected to fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in a borate buffer solution (pH 8.0) and acidified with trifluoroacetic acid , Two-dimensional micro-HPLC (Shiseido). For the first dimension, a micro-monolith ODS column (0.53 mm in inner diameter, 750 mm in length, Shiseido) was used. In the second-step optical resolution, an optical resolution column and a mobile phase were used. NBD-amino acid was detected by fluorescence emission (excitation wavelength 470 nm) at 530 nm. The results of the concentration with respect to D-serine are shown in Fig. PLP, a coenzyme of serine racemase, is a drug capable of activating serine racemase and can activate skin barrier function through activation of serine racemase.

Example 6 Effect of D-Serine on Skin

<Three-dimensional culture of epidermal keratinocytes>

Human normal keratinocytes were inoculated on a polycarbonate insert (Millipore) having a diameter of 12 mm and a hole diameter of 0.4 탆, CnT-Prime and Epithelial Culture Medium (CELLnTEC Advanced Cell Systems AG) were added to the inside and outside of the insert, After culturing for 3 days in an atmosphere of 5% CO _2, the cells were replaced with Cnt-Prime 3D Barrier Medium (CELLnTEC Advanced Cell Systems AG) and cultured for 24 hours. Thereafter, the arrangement in the insert was removed, and a medium containing D-Ser at a concentration of 100 μM outside the insert, or Cnt-Prime 3D Barrier Medium without D-Ser in the control, was added and the cell surface was exposed to air . After changing the medium once every two days and culturing for 7 days, the membrane of the insert was cut out with a scalpel and immersed in Trizol Reagent (Invitrogen) to extract total RNA.

<Analysis of gene expression by Real-time PCR>

Total RNA extracted with Trizol Reagent (Invitrogen) was analyzed for RNA concentration using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific), and then cDNA was prepared using SuperScript VILO (Invitrogen). This cDNA was used as a template in a StepOne Real-time PCR System (Applied Bioscience) using platinum SYBER Green qPCR SuperMix-UDG (Invitrogen), pelagreen (FLG), connrin (CRNN), repetin RPTN) gene, and the relative expression level of the gene of mitochondrial liposome protein L19 (MRPL19) as an internal standard gene was measured. The primers used were as follows:

The relative expression level of the target gene in each group was determined by correcting the expression amount of the mitochondrial liposome protein L19 gene (MRPL19) as an internal standard (FIGS. 5 to 7). In these genes, gene expression increased significantly with the addition of D-Ser.

                         SEQUENCE LISTING <110> Shiseido Co., Ltd. <120> A Method for Screening an Agent for Enhancing Dermal Barrier        Function by Using Epidermal Serine Racemase and / or D-Serine        Content as an Index, and a Method for Evaluating Dermal Darrier        Function <130> P150102WO <150> JP 2014-43073 <151> 2014-03-05 <150> PCT / JP2014 / 055680 <151> 2014-03-05 &Lt; 150 > US 14 / 477,213 <151> 2014-09-04 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 ggctccttca ggctacattc ta 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 atctggattc ttcaggattt gc 22 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 caactgcaca gcgctcac 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 caactgcaca gcgctcac 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gctcttggct gagtttggag 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 aggttcaaga tggtttccac a 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gggatttgca ttcagagatc ag 22 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 ggaagggcat ctcgtaag 18

Claims (12)

A keratinocyte screening method for screening a skin barrier function agent, wherein the activity or expression level of serine racemase and / or the amount of D-serine is used as an index. The method according to claim 1,
A step of adding a candidate agent to keratinocytes,
A step of measuring the activity or expression amount of serine racemase and / or the amount of D-serine in keratinocytes, and
From the activity or expression level of serine racemase, and / or from the amount of D-serine, a step of determining the skin barrier function hyperactivity of the candidate agent
&Lt; / RTI &gt; The screening method according to claim 2, further comprising a step of inducing differentiation of keratinocytes. The screening method according to claim 1, wherein the screening method is performed on the keratinocytes induced by differentiation. The screening method according to any one of claims 1 to 4, wherein the measurement of the amount of serine racemase expression is performed by measuring the amount of mRNA or protein of serine racemase. 5. The method according to any one of claims 1 to 4, wherein the measurement of serine racemase activity is performed by measuring the conversion efficiency of the substrate by serine racemase or by measuring the amount of the D-amino acid product Way. A hydrolyzed oat protein, lempuyang extract, Elephantopus mollis extract, pyridoxal phosphate and vitamin B ₆ obtained by the screening method according to any one of claims 1 to 6 Wherein the active ingredient is one or more substances selected from the group consisting of: A method for evaluating a skin barrier function, wherein the activity or expression level of serine racemase from a skin sample isolated from human skin and / or the amount of D-serine is used as an index. 9. The method of claim 8,
A step of measuring the activity or expression level and / or the amount of D-serine of serine racemase in a skin sample isolated from a subject, and
A step of determining the skin barrier function of a subject from the activity or expression level of serine racemase and / or the amount of D-serine
/ RTI &gt; of a skin barrier function. 10. The method of evaluating skin barrier function according to claim 8 or 9, wherein the measurement of the amount of serine racemase expression is performed by measuring the amount of serine racemase mRNA or protein. 10. The method of claim 8 or 9, wherein the measurement of serine racemase activity is by measuring the conversion efficiency of the substrate by serine racemase or by measuring the amount of the D-amino acid product. How to evaluate. A pharmaceutical composition comprising one or a plurality of substances selected from the group consisting of hydrolyzed oat protein, mulberry extract, Shirobanaga gypsum kojorina extract, pyridoxal phosphate and vitamin B ₆ obtained by the screening method according to any one of claims 1 to ₆ Barrier function enhancer of the skin containing.

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