JP2022064914A - Screening method for preventive/ameliorating agent for keratin plug formation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening a preventive/ameliorating agent for keratin plugs, which clarifies a mechanism of keratin plug formation to inhibit a process until keratin plug formation, and prevent keratin plug formation itself to fundamentally solve the problem of keratin plug formation, and a preventive/ameliorating agent for keratin plugs obtained by the screening method.

SOLUTION: Provided is a screening method for a preventive/ameliorating agent for keratin plugs, using a generation amount of nitric oxide of cultured keratinocytes, a mass of nitrated protein, and an amount of protein complex as indicators. Also, A goishicha (registered mark) extract can be provided as a preventive/ameliorating agent for keratin plugs.

EFFECT: According to the present invention, the problem of keratin plug formation can be solved by screening a test substance having a preventive/ameliorating effect for keratin plugs by inhibiting a process until keratin plug formation. In addition, it is possible to provide a highly effective preventive/ameliorating agent for keratin plugs with a simple and inexpensive screening in a short time.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2022,JPO&INPIT

Description

The present invention relates to a method for screening a test substance having an effect of preventing / improving keratin plugs and an agent for preventing / improving keratin plugs.

A keratin plug is a white waxy mass formed mainly on the side surface of the nose, and is considered to be one of the causes of conspicuous pores. In particular, as skin problems in young people in their 20s and 30s, problems related to pores such as keratin plug formation and pore opening occupy the top ranks. Keratin plugs are composed of proteins and lipids, and the ratio is reported to be 7: 3, and proteins account for the majority (Non-Patent Document 1). Examples of proteins contained in keratin plugs include proteins derived from the stratum corneum and hair follicles, proteins derived from P. acnes and Staphylococcus aureus, proteins derived from intracellular small organs such as lysosomes, and the like. It is composed of various proteins.

As mentioned above, although some studies have been reported on the constituents of keratin plugs, there are many unclear points about the mechanism of keratin plug development. A keratin plug is sometimes described as a substance in which sebum and proteins secreted from the surrounding keratin and sebaceous glands are coagulated and developed in the pores (Patent Document 1 and Non-Patent Document 2), but these are scattered. It merely describes the state in which the constituent components are gathered and solidified, and the details of how they were formed and how they were formed are unknown.

As a method for solving problems related to keratin plugs, a method of attaching an adhesive sheet to the keratin plug and peeling it off (Patent Document 2), a method of dissolving and removing the keratin plug with a surfactant, etc. have been used so far. It has been invented (Patent Document 3).

However, all of these methods are symptomatic means of "removing", and although they can temporarily reduce keratin plugs, they do not lead to a fundamental solution to the problem of keratin plug formation. .. Therefore, in addition to removing the already formed keratotic plug, a fundamental solution that can prevent the keratin plug formation itself is desired.

In Patent Document 4, it is clarified that keratin plugs contain a large amount of a protein called keratin 17, and a test substance having an effect of reducing the amount of keratin 17 in cultured epidermal keratinocytes is selected to suppress the formation of keratin plugs. It is disclosed that it becomes possible to screen the material having. However, Patent Document 4 is a study focusing on the fact that a large amount of keratin 17 is present in comedones, not keratin 10, which is a protein derived from the stratum corneum, which is a skin debris. Since it contains a large amount of several proteins other than keratin 17, the effect of reducing the amount of keratin 17 alone is not sufficient to prevent or improve the formation of keratin plugs.

Patent Document 1 clarifies that the proteolytic enzyme cathepsin V is localized inside the keratin plug, and discloses that the promotion of the activity of the cathepsin V decomposes the keratin plug and improves the conspicuous pores. There is. However, since we are focusing on decomposing keratin plugs by promoting the activity of enzymes localized in keratin plugs, it is not based on the idea of not forming keratin plugs in the first place, but to prevent the occurrence of keratin plugs. Is difficult. In addition, Patent Document 1 shows that the amount of cathepsin V and the amount of corneodesmosin, which is a cell adhesion factor in keratin plugs, had an inverse correlation, and it is assumed that cathepsin V is a keratolytic enzyme and its activity. Cathepsination can be a means of promoting cathepsin degradation by promoting the degradation of cell-cell adhesions that originally exist in the natural state, and the formation of cathepsins is a complex of proteins that make up cathepsins. No possibility of involvement in body formation is envisioned.

From the above, the details of the mechanism by which keratin plugs are formed are completely unknown, and there is no method for solving the problem that keratin plugs are formed. In order to fundamentally solve the problem of keratin plug formation, it is necessary to inhibit the process from the absence of keratin plugs in the pores to the formation of keratin plugs.

On the other hand, protein nitration is one of the post-translational modifications of proteins caused by active nitrogen species generated in vivo, and it is formed on the benzene ring in the tyrosine residue and tryptophan residue, which are a kind of aromatic amino acids in the protein. It is added with a nitro group. The content of tryptophan residues in many proteins present in the living body is much smaller than that of tyrosine residues, and it is considered that the nitration reaction of proteins mainly occurs in tyrosine residues (non-patent literature). 3). Tyrosine nitration is the attack on the aromatic ring of tyrosine residues by peroxynitrite, nitrogen dioxide, and nitrile chloride, resulting in the covalent modification of tyrosine residues in proteins with nitro groups (non-patentable). Document 4).

It is known that nitrotyrosine in proteins accumulates in various diseases (arteriosclerosis, cerebral ischemic disease, etc.) with aging, and it has been reported to be involved in these diseases (non-patent literature). 5). In addition, it has been reported that nitrotyrosine accumulates in the skin with aging and is associated with yellowing (Patent Document 5). On the other hand, the involvement of protein nitration with keratin plug formation, which is a problem for young humans, has not been known at all.

Japanese Unexamined Patent Publication No. 2017-128560 Japanese Unexamined Patent Publication No. 11-12127 Japanese Patent Application Laid-Open No. 2015-113307 JP-A-2015-197337 Japanese Unexamined Patent Publication No. 2017-181423

Journal of Society of Cosmetic Chemists, Vol. 41 (2007) No. 4: 262-268 Cosmetics Magazine Vol. 51, No. 1, 2017 Front Chem. 2016 Jan7; 3:70 Okayama Medical Society Magazine Vol. 118 January 2007, pp. 225-234 Science. 2000 Nov 3; 290 (5493): 985-9.

In view of the above background, the present inventors have proceeded with research on keratinocyte formation, and found that the more keratinocytes there are, the more interferon gamma is present at the site where keratinocytes are predominant. It was demonstrated that it stimulates keratinocytes to promote nitric oxide production. Furthermore, they discovered that nitrated proteins are present in keratin plugs and that when the proteins are nitrated, they form a complex. It has been found that the keratotic plug contains a protein complex containing at least one of SS bond, ionic bond, and hydrophobic bond. It is this protein complex formation that causes the proteins present in the hair follicles to gather and solidify, fill the hair follicles, and make it difficult for them to naturally fall out of the pores or be removed by washing. Presented.
From these results, interferon γ promotes the production of nitric oxide, which is one of the active nitrogen species and one of the causative agents of nitric oxide, at the site where keratin plugs occur frequently, which causes protein nitration. We found the relationship that -S bonds, ionic bonds, hydrophobic bonds, etc. occur to form a complex, which leads to the formation of keratin plugs. Based on this fact, it was found that screening of a keratotic preventive / ameliorating agent can be performed by using the amount of nitric oxide produced, the amount of nitrated protein, and the amount of protein complex as indicators, and the present invention has been completed. ..

By clarifying the mechanism of keratin plug formation, inhibiting the process until keratin plug formation, and preventing keratin plug formation itself, the problem of keratin plug formation is fundamentally solved. An object of the present invention is to provide a method for screening an improving agent, and to provide a keratotic preventive / improving agent obtained by the above screening method.

By using the amount of nitric oxide produced, the amount of nitrated protein, and the amount of protein complex as indicators of cultured keratinocytes, we were able to provide a screening method for keratotic preventive / ameliorating agents. In addition, Goishicha (registered trademark) extract could be provided as a preventive / improving agent for keratin plugs. That is,
[1] The first invention is a screening method for a keratin plug preventive / ameliorating agent using the amount of nitric oxide produced in cultured keratinocytes as an index.
[2] The second invention is a screening method for a keratotic preventive / ameliorating agent using the amount of nitrated protein as an index.
[3] The third invention is a screening method for a keratotic preventive / ameliorating agent using the amount of protein complex as an index.
[4] The fourth invention is a screening method for a keratotic preventive / ameliorating agent using the amount of protein complex produced by the nitration treatment as an index.
[5] The fifth invention is the screening method according to the third or fourth invention, wherein the protein complex has at least one of SS bond, ionic bond, and hydrophobic bond.
[6] The sixth invention is a keratin plug preventive / improving agent, which comprises a Goishicha (registered trademark) extract selected by the method of the third invention or the fourth invention.

According to the present invention, the problem of keratin plug formation can be solved by screening a test substance having a keratin plug prevention / improvement effect by inhibiting the process until keratin plug formation. In addition, it is possible to provide a highly effective keratotic preventive / ameliorating agent by a simple and inexpensive screening in a short time.

The process until the formation of a keratotic plug is shown. The relationship between the number of keratin plugs and the amount of interferon gamma is shown. The amount of nitrite ion produced when interferon gamma is added in cultured keratinocytes is shown. NO2- refers to nitrite ion and is an index of the amount of nitric oxide. NO refers to nitric oxide. It is shown that the keratin plug contains a nitrated protein. It is shown that the protein forms a complex by the nitration treatment. It is shown that the proteins contained in the keratotic plug form a complex. The screening result of the test substance which has the effect of suppressing the formation of a protein complex by nitration is shown. It is shown that the increase in the number of keratin plugs decreased as a result of continuous use of a lotion containing a Goishicha (registered trademark) extract having an effect of suppressing protein complex formation by nitration for 20 days.

The present invention relates to a method for screening a test substance having an effect of preventing / improving keratin plugs and an agent for preventing / improving keratin plugs.

The prevention / improvement of keratin plugs of the present invention includes one or both of prevention / improvement of keratin plugs.
The prevention of keratin plugs of the present invention means to prevent the occurrence of keratin plugs by suppressing the process from the state without keratin plugs to the formation of keratin plugs. Further, the prevention of keratin plugs of the present invention also includes suppressing the increase of keratin plugs from the state where the keratin plugs are present.
In addition, even if there is already a keratin plug, by continuously preventing the formation of the keratin plug, the keratin plug gradually decreases as it naturally falls off from the pores and is removed by washing, so skin with many keratin plugs is present. Therefore, it leads to skin with less keratin plugs, that is, it also leads to improvement of keratin plugs. Further, the improvement of the keratin plug of the present invention is to remove the factors leading to the formation of the keratin plug from the already formed keratin plug, thereby disperse the components of the keratin plug and naturally remove or clean the keratin plug from the pores. It also includes facilitating removal by, leading from skin with many keratin plugs to skin with few keratin plugs.
For example, in the process from the absence of keratin plugs to the formation of keratin plugs, reducing nitric oxide, which is a factor in the generation of nitrated proteins, suppressing the production of nitrated proteins, and existing nitrated proteins. The reduction of protein complex formation and the suppression of protein complex formation are the prevention of keratin plugs of the present invention because they suppress the formation of keratin plugs. In addition, continuous prevention of same keratin plugs leads from skin with many keratin plugs to skin with less keratin plugs, which leads to improvement of keratin plugs in the present invention. Furthermore, after the formation of the keratotic plug, the method of reducing the nitrated protein and the method of reducing the complex correspond to the improvement of the keratotic plug of the present invention by reducing the already formed keratotic plug.

In the method for screening a keratin plug preventive / ameliorating agent using the amount of nitric oxide produced as an index of cultured keratinocytes of the present invention, cultured keratinocytes are cultured in the presence or absence of a test substance, and monooxidation in the presence of the test substance is performed. A test substance having a smaller amount of nitrogen production than the amount of nitric oxide produced in the absence of the test substance is selected as having a keratin plug prevention / improvement effect.

The screening method for a keratin plug preventive / ameliorating agent using the amount of nitric oxide produced as an index of cultured keratinocytes of the present invention is used.
Step of adding test substance to cultured keratinocytes,
The process of measuring the amount of nitric oxide produced,
It includes a step of determining the effect of preventing / improving keratin plugs of the test substance from the amount of nitric oxide produced.

In the screening method for a keratin plug preventive / ameliorating agent using the amount of nitric oxide produced in cultured keratinocytes as an index, the step of adding interferon γ to cultured keratinocytes in order to further increase the amount of nitric oxide produced in cultured keratinocytes. May include. The step of adding interferon gamma may be the same as the step of adding the test substance after sowing the keratinocyte, or may be before or after the addition of the test substance. The interferon gamma referred to here refers to a molecule that is a type of cytokine.

The cultured keratinocyte used in the present invention is a culture of keratinocyte, which is a cell type constituting about 95% of the epidermis, and produces nitric oxide or produces nitric oxide in response to applied interferon γ. As long as it is a keratinocyte that has the property of culturing, its origin and condition do not matter. As the cultured keratinocytes, in addition to normal keratinocytes isolated and cultured from the normal epidermis, strained keratinocytes can also be used. The cultured keratinocytes are preferably derived from humans, and as normal keratinocytes, commercially available products such as Epidercell Human Epidermal Keratinocyte (Kurabou) and HEK (Toyobo) can be used as strained keratinocytes such as HaCaT. The medium used for culturing may be the medium recommended by the literature and the seller for each cell type. Cultured keratinocytes are preferable when the number of passages is 1 to 3 because they have high growth activity, but even those after that can be used when growth is observed and a quantifiable level of nitric oxide production is exhibited.

The amount of nitric oxide produced in the present invention is determined by measuring the amount of nitric oxide contained in the cells of cultured keratinocytes or in the culture supernatant. The method for measuring the amount of nitric oxide is not particularly limited as long as the amount of nitric oxide contained in the cells or the culture supernatant can be grasped, and a method for directly measuring the amount of nitric oxide, in a certain manner. Either method of quantifying the product obtained by changing nitric oxide and indirectly grasping the amount of nitric oxide can be used. As a method for directly quantifying nitric oxide, for example, a Cu (II) complex of 5-methoxy-2- (1H-naphtho [2,3-d] imidazol-2-yl) phenol (MNIP) is used. There is a method of measuring the amount of nitric oxide by selectively reacting with nitrogen oxide to emit blue fluorescence, and as a method of indirectly grasping the amount of nitric oxide, nitric oxide is a medium. A commercially available kit for grasping the amount of nitric oxide by reducing nitrate ions to nitrite ions and quantifying nitrite ions among the final products nitrite ions and nitrate ions produced by undergoing hydrolysis. For example, NO2 / NO3 Assay Kit-FX (Fluorometric) to 2,3-Diaminonaphthalene Kit (DOJINDO) can be used.

In the screening method for a keratin plug preventive / ameliorating agent using the amount of nitric oxide produced in cultured keratinocytes as an index, the amount of nitric oxide produced in the presence of the test substance becomes the amount of nitric oxide produced in the absence of the test substance. A relatively small amount of test substance is selected as having a preventive / ameliorating effect on keratin plugs. For example, when the amount of nitric oxide produced in the absence of the test substance is 100%, nitric oxide production in the presence of the test substance is performed. When the amount is reduced by 10% or more, preferably 20% or more, the test substance is selected as having a keratotic preventive / ameliorating effect. More preferably, when the amount of nitric oxide produced in the absence of the test substance is 100% with the addition of interferon gamma, the amount of nitric oxide produced in the presence of the test substance is 10% or more, preferably 20. If it is reduced by% or more, the test substance is selected as having a preventive / ameliorating effect on keratin plugs.

In the screening method for a keratin plug preventive / ameliorating agent using the amount of nitrated protein of the present invention as an index, nitration of a protein by nitrogen monoxide whose production is promoted at a site where keratin plugs are predominant is one process of keratin plug formation. It is an invention based on the discovery of the present inventors, and is based on the idea that understanding the action of the test substance on the nitrated protein leads to the evaluation of the keratotic prevention / ameliorating action of the test substance. In the method for screening a keratotic preventive / ameliorating agent using the amount of nitrated protein as an index of the present invention, a test substance is applied to the protein, and the amount of nitrated protein in the presence of the test substance is the amount of nitrated protein in the absence of the test substance. Select a test substance that is smaller than the amount because it has a preventive / ameliorating effect on keratin plugs. The test substance selected as having a preventive / ameliorating effect on keratin plugs in the present invention is either a substance that suppresses the new production of nitrated proteins or a substance that reduces the abundance of existing nitrated proteins by degrading them. It can be one or both.

The screening method for a keratotic preventive / ameliorating agent using the amount of nitrated protein of the present invention as an index is
The process of adding the test substance to the protein,
The process of measuring the amount of nitrated protein,
It includes a step of determining the keratin plug prevention / improvement effect of the test substance from the amount of the nitrated protein.

The screening method for a keratin plug preventive / ameliorating agent using the amount of the nitrated protein of the present invention as an index may further include a step of nitrating the protein. The step of nitrating the protein may be simultaneous with the step of adding the test substance, or may be before or after the step of adding the test substance. Further, by utilizing proteins that have already been nitrated to a level at which a change in the degree of nitration due to the test substance can be detected, the step of nitrating them may be omitted.

The protein referred to in the present invention is used in the concept of including a single peptide or amino acid that can constitute a protein, and is described as a protein when it means a protein as a general concept. For example, commercially available protein reagents such as keratin, albumin, casein, skim milk, tyrosine, tryptophan, phenylalanine and derivatives thereof, which are aromatic amino acids that are likely to cause nitrates, or synthetic products of peptides containing them. Etc. can be used. Further, it may be a stratum corneum collected by tape stripping, an abrasive, or the like, a keratin plug collected by a sheet pack, tweezers, or the like, a cell crushed product of cultured cells such as keratinocyte, or the like.

The nitrated protein in the present invention refers to a protein having a nitro group. Examples thereof include nitrotyrosine, 3,5-dinitrotyrosine, nitrotryptophan, nitrophenylalanine, 2,4-dinitrophenylalanine and the like, and peptides or proteins containing these nitrated amino acids in their constituents.

The protein of the present invention is not particularly limited as long as the content of the nitrated protein changes due to the test substance or the nitration treatment. For example, from the viewpoint of measuring the decrease in the abundance of the existing nitrated protein by the test substance, it is preferable to use the nitrated protein, which is an already nitrated protein, and the nitro by the test substance. From the viewpoint of measuring the suppression of the suppression of new production of the converted protein, a protein having an aromatic amino acid residue that is considered to be prone to nitrate is preferable, but the content of the nitrated protein changes to a testable level. If this is the case, the nitrated protein and the protein that becomes the nitrated protein by the nitration treatment may be mixed from either of the above viewpoints.

As a method for protein nitration treatment, a known method can be used, and it may be appropriately selected according to each screening. For example, in addition to the method of allowing an active nitrogen species such as peroxynitrite to act on a protein, nitric oxide and superoxide anion are mixed, or myeloperoxidase (MPO) is mixed with NaNO2, hydrogen peroxide, etc. in a test system. A method of generating active nitrogen species and causing them to act can be used. Further, it may be nitrated using a reagent such as tetranitromethane or nitric acid that generates nitronium ions, or it may be nitrated by treatment with nitroyl chloride, nitrosoperoxycarboxylate, sodium azide, catalase or the like.

The amount of nitrated protein can be measured by a known method. For example, without particular limitation, it can be measured by an absorbance method, an immunostaining method, radioimmunoassay, ELISA, liquid chromatography, gas chromatography, mass spectrometry, NMR method, Western blotting and the like. The amount of nitrated protein may be directly measured, or for example, the amount of nitro group obtained by decomposing the nitrated protein by enzymatic treatment or the like, the nitrated peptide, or the nitrated amino acid residue. The amount may be measured and used as the amount of nitrated protein. Further, in the screening method of the present invention, when the step of nitrating the protein is added, the abundance of the nitrating reagent used for the nitrating treatment remaining in the system after the reaction, the test substance and the nitrating reagent are added. It is also possible to indirectly estimate the amount of nitrated compounds by measuring the abundance of reaction by-products produced by the reaction. For example, when the abundance of the nitration reagent in the system is larger in the presence of the test substance than in the absence of the test substance, there are more unreacted nitration reagents in the presence of the test substance, and the nitrated protein which is a reaction product. It can be estimated that the abundance of is small. In addition, in the presence of the test substance, a reaction by-product produced by the reaction between the test substance and the nitrating reagent is generated, and if this can be quantified, the abundance of the nitroated product is estimated by measuring the abundance of this reaction by-product. can do. For example, if there are more reaction by-products in the system in the presence of the test substance than in the absence of the test substance, it can be estimated that the abundance of the nitrated protein, which is a reaction product, is small.

In the method for screening a keratotic preventive / ameliorating agent using the amount of nitrated protein as an index of the present invention, a test substance in which the amount of nitrated protein in the presence of the test substance is smaller than the amount of nitrated protein in the absence of the test substance is selected. It is selected because it has a preventive / ameliorating effect on keratin plugs. If the amount is low, the test substance is selected as having a preventive / ameliorating effect on keratin plugs.

The method for screening a keratin plug preventive / ameliorating agent using the amount of protein complex of the present invention as an index is an invention in which protein complex formation by a nitrated protein found in keratin plug is one process of keratin plug formation. It is an invention based on the findings of these subjects, and is based on the idea that understanding the action of the test substance on the amount of protein complex leads to the evaluation of the preventive / ameliorating action of the test substance on keratin plugs. In the method for screening a keratotic preventive / ameliorating agent using the protein complex amount of the present invention as an index, a test substance is applied to the protein, and the protein complex amount in the presence of the test substance is the protein in the absence of the test substance. A test substance that is smaller than the amount of the complex is selected as having a preventive / ameliorating effect on keratin plugs. The test substance selected as having a preventive / ameliorating effect on keratin plugs in the present invention suppresses the formation of new protein complexes, or reduces the abundance of existing protein complexes by degrading them. It can be one or both of the things.

The screening method for a keratotic preventive / ameliorating agent using the amount of the protein complex of the present invention as an index is
The process of adding a test substance to a protein,
The process of measuring the amount of protein complex,
It includes a step of determining the keratin plug prevention / improvement prevention effect of the test substance from the amount of the protein complex.

The screening method for a keratin plug preventive / ameliorating agent using the amount of protein complex of the present invention as an index may further include a step of forming a protein complex. The step of forming the protein complex may be simultaneous with the step of adding the test substance, or may be before or after the step of adding the test substance. Further, by utilizing a protein in which a complex has already been formed to a level at which a change in the degree of complex formation due to the test substance can be detected, the step of forming the complex may be omitted. The method for forming the complex is not particularly limited as long as it is a method including a nitration treatment, but may further include a saccharification treatment, a carbonylation treatment, a heat treatment, an acid treatment, an ultraviolet irradiation treatment and the like.

The protein complex of the present invention does not refer to a substance in which proteins are simply collected, but refers to a protein complex formed by chemical bonds within or between protein molecules. Specific examples of chemical bonds include covalent bonds such as SS bonds, ionic bonds, and non-covalent bonds such as hydrophobic bonds. SS bonds are covalent bonds that occur between cysteine residues of proteins, ionic bonds are non-covalent bonds that occur between the charges of proteins due to electrostatic attraction, and hydrophobic bonds are non-polar bonds of proteins in polar solvents such as water. It is a non-covalent bond due to the attractive force generated between the regions. Proteins, peptides, and amino acids can be mixed in the protein complex of the present invention. In addition, the protein complex of the present invention includes one consisting of a single protein or a plurality of kinds of proteins.

The protein of the present invention is not particularly limited as long as it causes a change in the complex content due to the formation of the test substance or the complex. Specifically, those exemplified in [0029] [0030] can be used.

The method for measuring the amount of the protein complex of the present invention is not particularly limited, and for example, gel permeation chromatography, ultrafiltration filter, light scattering method, mass spectrometry, electrophoresis, etc. for measuring the molecular size and molecular weight of a protein are used. In the comparison between the presence / absence of the step of forming the complex and the presence / absence of the test substance, it is understood that the larger the amount containing the molecule having a large molecular size or molecular weight, the larger the amount of the complex. The method of comparison is not particularly limited as long as the difference in molecular size and molecular weight can be relatively grasped, but when the molecular size and molecular weight of the protein do not show a single value, for example, protein sample A having the same composition, When B is present, determine the molecular size or the minimum to maximum value (distribution area A) of the molecular size or molecular weight of the protein (sample A) having a relatively small amount of complex, and the amount of molecules contained in the vicinity (quantity A). From the distribution area B, determine the molecular size or the minimum to maximum value (distribution area B) of the molecular size or the molecular weight of the protein (sample B) having a relatively large amount of complex and the amount of the molecule (quantity B) contained therein. The ratio (quantity BA / quantity B) of the molecule amount (quantity BA) of the sample B in the distribution area (distribution area BA) excluding the distribution area A and the molecule amount (quantity B) in the distribution area B is used as the complex amount. Can be used. Alternatively, for example, the median or mean value of the molecular size distribution may be obtained and used in the above comparison, or within a certain standard deviation from the median value, the mean value, or a value preset based on a preliminary test or the like. The comparison may be made by determining the ratio between the samples using the amount of molecules that can be accommodated in. Furthermore, a certain amount of sample solution is applied to an ultrafiltration filter having a pore size set in advance based on a preliminary test, etc., and the protein contained in the filter-passing fraction is quantified to compare the amount of complex. May be good. In addition to the method of directly grasping the complex amount as described above, the method of calculating the complex amount by grasping the non-complex amount and indirectly grasping the complex amount should be used. Can be done.

In the method for screening a keratotic preventive / ameliorating agent using the amount of protein complex of the present invention as an index, the amount of protein complex in the presence of the test substance is smaller than the amount of protein complex in the absence of the test substance. The test substance is selected because it has a preventive / ameliorating effect on keratin plugs. Preferably, when the amount is 5% or more, the test substance is selected because it has a preventive / ameliorating effect on keratin plugs.

The test substance used in the screening method of the present invention is not particularly limited. Plant / animal-derived extracts, fungal cultures, treated products such as these enzymes, compounds or derivatives thereof can also be used as test substances, and may be in the form of powder, gel, etc. in addition to liquid. There is no problem. If it does not dissolve in the protein solution as it is, it can be used as a test substance by dissolving it by appropriately using a solubilizing agent such as a surfactant. According to the present invention, the substance selected as a keratin plug preventive / ameliorating agent can be applied to cosmetics, quasi-drugs or pharmaceuticals. As cosmetics, for example, it can be applied to, but is not limited to, lotion, milky lotion, cream, gel, beauty essence, ointment, foundation and the like.

Goishicha (registered trademark) used in the present invention is used after harvesting the leaves of Camellia sinensis, steaming them to stop the natural fermentation, and then rather while laying them in an aerobic mold, while soaking them in a tub. It is a two-stage fermented tea that is fermented by anaerobic bacteria. Since it is traditionally manufactured in Kochi prefecture, commercial products can be used.

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

The method for extracting the Goishicha (registered trademark) extract that can be used in the present invention is not particularly limited, but for example, if it is dried, a solvent in an amount of 1 to 1000 times, particularly 10 to 100 times by weight is used. In the case of room temperature extraction, it is preferably performed at 0 ° C. or higher, particularly 20 ° C. to 40 ° C. for 1 hour or longer, particularly 3 to 7 days. Further, it may be extracted by heating at 60 to 100 ° C. for 1 hour. Further, the extraction may be carried out at a temperature of 10 ° C. or lower so that the extraction solvent does not freeze, for 1 hour or more, particularly for 1 to 7 days.

The Goishicha (registered trademark) extract obtained under the above conditions may be used as it is in the extracted solution, but if necessary, it may be further subjected to a treatment such as filtration to concentrate and powder it as appropriate. It can be used properly.

The blending amount of the Goishicha (registered trademark) extract in each agent of the present invention is preferably 0.00001 to 20.0% by weight, particularly in the range of 0.001 to 10.0% by weight in terms of evaporation and drying content. Is the best.

Each agent of the present invention can be used in combination with other components as long as the effects of the present invention are not impaired.

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to this.

Experimental Example 1: Relationship between the amount of keratin plug and the amount of interferon γ in the keratin plug-prone site From 10 healthy men, a resin cylinder (diameter 7 mm, cylinder length 10) around the nose, which is the keratin plug-prone site. mm) is pressed against the skin surface inside the cylinder, and 0.1% polyoxyethylene coconut oil fatty acid sorbitan (20EO) is added to a phosphate buffer solution (PBS (-)) containing no calcium or magnesium. The mixture was washed with 0.3 mL of the above-mentioned solvent while pipetting, and the washing solution was recovered. The amount of interferon gamma contained in this washing solution was measured using a High Sensitivity ELISA kit (Invitrogen).

Subsequently, the number of keratin plugs was measured. We took advantage of the property that the substance contained in the keratin plug glows white under ultraviolet irradiation. Specifically, a photograph of the entire face was taken under ultraviolet irradiation using a skin measuring device VISIA (registered trademark) Evolution (Canfield Scientific). After that, an image of a certain area of the nose is cut out using ViX (K_OKADA), an image analysis software ImageJ (NIH) is used to extract the keratin plug portion that glows white, and the number of keratin plugs per fixed area is measured. rice field.

FIG. 2 is a diagram showing the number of keratin plugs and the amount of interferon gamma. It was shown that the group with a large amount of interferon gamma had a large amount of keratotic plug. From this experiment, it was speculated that the more keratin plugs there are, the more interferon gamma is in the area around the nose, which is the site where keratin plugs are most likely to occur.

Experimental Example 2: Human neonatal-derived epidermal keratinocytes (Kurabou) were used in the measurement test of the amount of nitric oxide when interferon gamma was added to keratinocytes. Humania-KG2 (Kurabo Industries) was used as the growth medium, and the culture environment was 37 ° C., 5% CO2 / 95% air humidification conditions. The growth medium was placed in a 25 cm2 culture flask, and the cells were cultured for 3 to 4 days and became almost saturated, and the cells were used with two passages. Inoculate 300 μL to 1.0 × 105 Cells / mL on a 48-well culture plate, incubate for 24 hours, and then use Humedia KG2 GC Attachment (Kurabo) containing 1.2 mM calcium and no phenol red added. The medium was exchanged. Then, interferon gamma was added to 1000 U / mL. Water was added to the controls. After incubating for 30 hours, the culture supernatant was collected, and the amount of nitric oxide was measured with NO2 / NO3 Assay Kit-FX (Fluorometric) to 2,3-diaminophthalene Kit- (DOJINDO).

FIG. 3 shows the amount of nitrite ion when interferon gamma is added to keratinocytes. The amount of nitrite ion is an index of the amount of nitric oxide, and it was shown that the amount of nitrite ion in the keratinocyte culture supernatant was large in the interferon gamma-added group. This experiment demonstrated that interferon gamma stimulates keratinocytes to promote nitric oxide production.

Experimental Example 3: Immunostaining of keratin plug with nitrotyrosine antibody O.D. C. It was embedded in T-compound (Sakura Finetech Japan), frozen, and an 8 μm section was prepared with a cryostat, attached to a slide glass, and immersed in 100% ethanol for fixation. Then, it was treated with 0.3% hydrogen peroxide-added methanol for 30 minutes. Subsequently, immunostaining was performed using a Vectortain ABC kit (Vector). The primary antibody was visualized with 3,3'-Diaminobenzidine (DAB; VECTOR LABORATORIES) using an anti-nitro tyrosine antibody (Stressmarq Bioscience). Then, the stained sections were observed under a microscope.

FIG. 4 shows the results of immunostaining of keratin plugs with a nitrotyrosine antibody. Immunostaining with a nitrotyrosine antibody recognizes and detects nitrotyrosine and nitrotyrosine residues in proteins, and the part where the recognition target is present is stained brown. From FIG. 4, it was found that the section of the keratotic plug contained a portion stained in brown. From this experiment, it was found that the keratotic plug contains a nitrotyrosine and / or a nitrated protein containing nitrotyrosine as an amino acid residue.

Experimental Example 4: Examination of protein complex formation by nitration treatment The relationship between protein nitration and complex formation was investigated by a method using an extrafiltration filter. 500 μL of 10 mg / mL bovine serum albumin aqueous solution and 50 μL of distilled water were added to a 1.5 mL microtube and mixed. Subsequently, 50 μL of 1% tetranitromethane-added ethanol was added, and the mixture was shaken and mixed for 10 minutes. Then, 400 μL of a 5 M aqueous urea solution was added and mixed. This was used as a nitration-treated solution. To control, 50 μL of ethanol was added instead of 1% tetranitromethane-added ethanol, and the same process was performed. These samples were placed in an ultrafiltration filter (Amicon Ultra 100 kDa, Merck) and centrifuged at 14000 xg for 30 minutes. The amount of protein in the concentrate was measured by the Bradford method to determine the protein complex content. In addition, the amount of protein in the flow-through solution was measured by the Bradford method to determine the amount of non-complex protein. In addition, the amount of protein in the concentrate was calculated from the amount of protein in the flow-through solution to determine the amount of complex. Using this, the complex amount ratio (%) and the non-complex amount ratio (%) with and without treatment were evaluated.

The complex amount ratio (%) was calculated by the following formula.

The non-complex amount ratio (%) was calculated by the following formula.

As a result of the examination of protein complex formation by the nitration treatment, the complex amount ratio (%) and the non-complex amount ratio (%) were calculated. In FIG. 5, the non-complex amount ratio (%) is shown. It was found that the nitration treatment reduced the non-complex amount ratio by 50% compared to the control. From this experiment, it was found that the protein forms a complex when it is nitrated.

Experimental Example 5: Examination of the state of the keratin plug A keratin plug around the nose was collected by a commercially available sheet pack, carefully collected with tweezers, and placed in a tube. Subsequently, 0.2 mol / L Tris-HCl buffer (pH 9.0) containing 1% Sodium dehydrate sulfate, 0.2 mol / L Dithiothreitol, and 8 mol / L Urea was added to 20 mg / mL by weight of the keratotic plug. Added. Then, the mixture was shaken at 37 ° C. for 4 days to dissolve the keratotic plug, and the solution was obtained as a keratotic plug dissolving solution. The keratin plug lysate was diluted with distilled water so that the protein content was 10 mg / mL, and the molecular weight was evaluated using SDS-PAGE and Blue native-PAGE.

By performing SDS-PAGE and Blue native-PAGE, the molecular weight of the protein can be evaluated. SDS-PAGE denatures proteins, cleaves SS bonds, ionic bonds, hydrophobic bonds, etc., and the hydrophobic part of SDS binds to the protein to run the protein as a single strand. Since native-PAGE migrates a protein in an undenatured state, it is possible to confirm the molecular weight in a state where the SS bond, the ionic bond, and the hydrophobic bond are maintained. If the molecular weight of the protein that can be confirmed by Blue native-PAGE is larger than the molecular weight of the protein that can be confirmed by SDS-PAGE, it can be said that the protein forms a complex of SS bonds, ionic bonds, and hydrophobic bonds. ..

FIG. 6 shows the state of complex formation of proteins contained in keratin plugs. In Blue native-PAGE, bands were found between 146 kDa and 242 kDa, and between 242 kDa and 480 kDa, and in SDS-PAGE, bands were formed near 50 kDa. Therefore, it can be said that the protein contained in the keratotic plug is in a state of forming a complex by SS bond, ionic bond, hydrophobic bond and the like.

Example 1: Screening method using a protein complex as an index As test substances, Goishicha (registered trademark) extract and Avocad oil (Shibahasikemifa Co., Ltd.) were used. The Goishicha (registered trademark) extract is extracted by adding 10 times the weight of a 50 V / V% ethanol aqueous solution to the dried plant bulk and heating at 60 ° C. for 4 hours. It was prepared by adding 3-butylene glycol and water in a weight ratio of 1:30:69 and diluting the mixture.

Distilled water containing 6% of monolaurin sanpolyoxyethylene sorbitan (20EO) or 50 μL of the test substance was added to 500 μL of a bovine serum albumin aqueous solution and mixed. Subsequently, 50 μL of a 1% tetranitromethane ethanol solution was added, and the mixture was shaken and mixed for 10 minutes. Then, 400 μL of a 5 M aqueous urea solution was added and mixed. This was used as a nitration-treated solution. For the control, 50 μL of ethanol was added instead of the 1% tetranitromethane ethanol solution. These samples were placed in Amicon Ultra 100 kDa and centrifuged at 14000 xg for 30 minutes. The amount of protein in the flow-through solution was quantified by the BCA method, the amount of protein in the concentrate was determined, and the complex amount ratio (%) was evaluated using this.
The complex amount ratio (%) was calculated by the following formula.

FIG. 7 shows the results of the complex formation suppression test. The amount of complex in Goishicha (registered trademark) extract was 96%, and the amount of complex when Goishicha (registered trademark) extract was added was reduced by 4% or more compared to the non-complex when not added. It was confirmed that the Goishicha (registered trademark) extract suppressed the formation of protein complex due to nitroization. On the other hand, it was found that the avocado oil had a complex amount of 100% or more and had no effect of suppressing protein complex formation by nitration.

The protein in the present invention is a concept including peptides and amino acids that can constitute a protein, but for Example 1, an ultrafiltration filter having a molecular weight size suitable for separating a peptide or an amino acid and a complex thereof is used. Needless to say, if selected, "screening for keratin plug preventive / ameliorating agents using the amount of protein complex as an index" can be performed even by using peptides and amino acids that can constitute proteins.

Example 2: Keratin plug prevention evaluation test by suppressing the formation of a nitrated protein complex For the purpose of comparing Prescription Examples 1 and 2, three subjects were asked to remove the keratin plug around the nose with a commercially available sheet-type pack. Then, after washing the face twice a day, the left and right noses were applied with the preparation shown in Formulation Example 1 or Comparative Example 1. The number of keratin plugs was measured after removing the keratin plugs at the start of the test and after applying Prescription Example 1 (test group) and Comparative Example 1 (placebo group) for 20 days.

The number of keratin plugs increased was calculated by the formula shown in "Formula".

FIG. 8 is a diagram showing the effect of preventing keratin plugs in a cosmetic solution containing an extract of Goishicha (registered trademark). In Formulation Example 1 using a lotion containing a Goishicha (registered trademark) extract having an inhibitory effect on the formation of a nitrated protein complex selected by the screening method of the present invention, a keratin plug prevention / improvement effect was observed. From the above, it can be seen that by using the protein complex obtained by the nitration treatment as an index, it is possible to screen the components having the effect of preventing / improving keratin plugs.

Example 3: Screening method using the amount of protein nitration as an index 50 μL of the test substance was added to 500 μL of a bovine serum albumin aqueous solution and mixed with shaking. Subsequently, 50 μL of a 1% tetranitromethane ethanol solution was added, and the mixture was shaken and mixed for 10 minutes. Then, 400 μL of a 5 M aqueous urea solution was added and mixed. This was used as a nitration-treated solution. For the control, 50 μL of ethanol was added instead of the 1% tetranitromethane ethanol solution. The amount of 3-nitrotyrosine in this solution was quantified using NWLSS ^TM Nitrotyrosine ELISA (NorthwestLifeScienceSpecialties). The amount of nitrotyrosine in the control to which the test substance was not added was set to 100%, and the amount of nitration was calculated from the ratio of the amount of nitrotyrosine when the test substance was added. Those in which the amount of nitrotyrosine was reduced by 10% or more as compared with the control were selected as effective.

In Example 3, the nitrotyrosine antibody ELISA was used to measure the amount of nitrated protein, but an antibody suitable for a substance contained in the nitrated protein other than nitrotyrosine, such as nitrotryptophane and nitrophenylalanine, was selected. However, it goes without saying that "screening for keratin plug preventive / ameliorating agents using the amount of nitrated protein as an index" can be performed.

Example 4: Screening method using the amount of nitric oxide as an index Human neonatal-derived epidermal keratinocytes (Kurabo Industries) contain 1.2 mM calcium and phenol red so as to be 1.0 × 10 ⁵ Cells / mL. It was suspended in the non-Humedia KG2 GC attached medium (Kurabo), and 300 μL was inoculated on a 48-well culture plate. The cells were cultured for 24 hours in an incubator at 37 ° C. under 5% CO ₂ . Then, the test substance was added. A solvent of the test substance was added to the control. Then, interferon was added to 1000 U / mL. After incubation for 30 hours, the culture supernatant was collected and the amount of nitric oxide was measured with NO ₂ / NO ₃ Assay Kit-FX (Fluorometric) to 2,3-diaminophthalene Kit- (DOJINDO). The ratio of the amount of nitric oxide in the test substance-added group was calculated, assuming that the amount of nitric oxide in the control group to which the test substance was not added was 100%. Test substances with a nitric oxide content reduced by 10% or more compared to the control were selected as effective.

According to the present invention, the problem of keratin plug formation can be solved by screening a test substance having a keratin plug prevention / improvement effect by inhibiting the process until keratin plug formation. In addition, it is possible to provide a highly effective keratotic preventive / ameliorating agent by a simple and inexpensive screening in a short time. Therefore, it is useful for the development of cosmetics for the prevention and improvement of keratin plugs and pharmaceuticals for the treatment.

Claims (4)

A screening method for keratin plug preventive / ameliorating agents using the amount of protein complex as an index. A screening method for keratin plug preventive / ameliorating agents using the amount of protein complex produced by nitration treatment as an index. The screening method according to claim 1 or 2, wherein the protein complex has at least one of SS bond, ionic bond, and hydrophobic bond. A keratin plug preventive / ameliorating agent containing a Goishicha (registered trademark) extract selected by the method of claim 1 or 2.

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