Screening method for inhibitors of abnormal elastin deposition

Abstract

To provide a screening method for substances that can suppress abnormal deposition of elastin generated in a photoaging portion, skin hardening, and a decrease in skin elasticity, and that can prevent or improve wrinkles, sagging, and a decrease in firmness of the skin accompanied with photoaging.SOLUTION: The above problem was solved by using the promotion of decomposition or suppression of production of denatured elastin as an index.EFFECT: By using the promotion of decomposition or suppression of production of denatured elastin as the index, a new substance screening method for suppressing abnormal deposition of elastin accompanied with photoaging is provided. Further, since it is possible to suppress an increase in skin hardness and a decrease in skin elasticity, a new substance screening method for preventing and improving the accompanying wrinkles, sagging formation, and a decrease in firmness is provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for screening for a substance that can prevent or improve wrinkles, sagging skin, and loss of firmness by inhibiting hardening of the skin and loss of skin elasticity caused by photoaging.

[0002] As we age, our skin loses elasticity, resulting in changes such as a loss of firmness and the formation of wrinkles and sagging. Wrinkles and sagging are particularly pronounced on areas susceptible to chronic UV exposure, such as the face. Skin aging that occurs in areas exposed to UV rays is called photoaging, and preventing and improving this is one of the biggest challenges for researchers of topical skin care products.

[0003] The dermis, the supporting tissue of the skin, is primarily composed of extracellular matrices such as collagen fibers made of collagen and elastic fibers made of elastin. Collagen fibers contribute to the strength of the skin, while elastic fibers contribute to the flexibility and resilience of the skin. It is known that photoaged dermis exhibits a decrease in collagen and abnormal elastin deposition (e.g., thickened fibers and amorphous mass accumulation). These fiber abnormalities result in a loss of skin strength and flexibility. It has been reported that abnormally deposited elastin, in particular, may contribute to a decrease in skin elasticity and an increase in dermal hardness in photoaged areas (Non-Patent Documents 1 and 2).

[0004] Elastin is known to be cleaved and degraded by extracellular matrix-degrading enzymes such as matrix metalloproteinases (MMPs), neutrophil elastase, and fibroblast elastase. However, it has been reported that abnormal deposition of elastin occurs in photoaged areas despite increased expression of extracellular matrix-degrading enzymes (Non-Patent Documents 3 and 4). The mechanism by which this abnormal deposition occurs in photoaged areas has not yet been elucidated.

[0005] Meanwhile, conventional photoaging prevention and improvement agents for preventing or improving wrinkles, sagging skin, loss of firmness, etc. have been developed to increase collagen and elastin or inhibit their degradation (Patent Documents 1-4). In particular, for elastin-targeting agents, a method of inhibiting elastin degradation by elastase has been widely used. This aims to increase the amount of elastin in the skin by inhibiting elastin degradation, thereby compensating for the loss of elasticity that occurs in photoaging sites. This method may also inhibit the abnormal deposition of elastin degradation products. However, no efficacy has been reported for elastin that has already been denatured and abnormally deposited, and conventional techniques have not known any means for inhibiting the abnormal deposition of denatured elastin. In other words, conventional techniques alone have not been able to adequately inhibit the abnormal deposition of elastin that occurs in photoaging sites, nor have they been able to fundamentally prevent or improve the progression of associated photoaging symptoms.

[0006] On the other hand, protein nitration is a type of post-translational modification of proteins caused by reactive nitrogen species generated in vivo, in which a nitro group is added to the benzene ring in the aromatic amino acid residues of tyrosine and tryptophan that make up proteins. The nitration reaction occurs when the benzene ring in the amino acid is converted to a nitronium ion (NO2 +Nitrotyrosine is produced by electrophilic substitution reactions with tryptophan, nitrite, and nitrogen dioxide radicals (Non-Patent Document 5). The tryptophan content of many proteins in the body is much lower than that of tyrosine, and nitration of proteins is thought to occur primarily at tyrosine residues (Non-Patent Document 6). Nitrotyrosine in proteins is known to affect cellular function by causing a decrease in the function of enzymes and tyrosine kinase receptors (Non-Patent Document 7). Furthermore, nitrotyrosine in proteins is known to accumulate in arteriosclerosis and cerebral ischemia, and has been reported to be involved in these diseases (Non-Patent Document 8). Nitrotyrosine is also known to exist in the skin, and nitrotyrosine present in the stratum corneum in particular is associated with skin color (Patent Document 5). However, its relationship to abnormal elastin deposition in photoaged skin was unknown. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2016-69332 [Patent Document 2] Patent Publication No. 2012-56933 [Patent Document 3] JP 2001-316221 A [Patent Document 4] Patent Publication No. 2012-207015 [Patent Document 5] Patent Publication No. 2017-181423 [Non-patent literature]

[0008] [Non-Patent Document 1] Exp. Dermatol., 2019, 28(8):914-921 [Non-patent document 2] Exp. Dermatol., 2019, 28(8):981-984 [Non-patent document 3] Biochimie, 2016, 128-129:163-73 [Non-patent document 4] J. Invest. Dermatol., 2004, 122(6):1448-55 [Non-patent document 5] Chem. Res. Toxicol., 2009, 22(5):894-898 [Non-patent document 6] Front. Chem., 2016, 3:70 [Non-Patent Document 7] Diabetes, 2008, 57(4):889-98 [Non-patent document 8] Science, 2000, 290(5493):985-9 Summary of the Invention

[0009] Under these circumstances, the present inventor has come to the realization that the prevention and improvement of photoaging skin symptoms requires the inhibition of abnormal elastin deposition. Therefore, the present inventor has considered the mechanism by which abnormal elastin deposition occurs in photoaging skin. In photoaging skin, abnormal elastin deposition occurs despite an increase in the expression level of extracellular matrix degrading enzymes. It is believed that this phenomenon occurs because, for some reason, the increased enzymes do not function normally, preventing elastin from being degraded normally. Specifically, the present inventor has come to the idea that ultraviolet exposure causes some kind of denaturation in elastin, thereby inhibiting the degradation by enzymes that would normally be increased.

[0010] Therefore, the present inventors searched for new factors involved in inhibiting the degradation of elastin in photoaged skin and found that the constituent amino acids of elastin abnormally deposited in photoaged skin are modified by nitration.Further investigations revealed that nitrated elastin has the property of promoting aggregation, acquires resistance to degradation by elastase, and furthermore, increases hardness and decreases elasticity.

[0011] In other words, we found that long-term UV exposure in photoaged areas causes denaturation of elastin, such as nitration, which leads to elastin aggregation and reduces its degradation by elastin degrading enzymes, resulting in abnormal deposition of elastin in photoaged areas. Furthermore, we found that this is one of the factors behind changes in the physical properties of elastin, such as hardening, and functional decline, such as loss of elasticity, in photoaged skin.

[0012] Furthermore, the present inventors have confirmed that elastin that has undergone post-translational modifications such as glycation and carbonylation as well as nitration acquires resistance to degradation by elastase.

[0013] Based on these new findings, we believe that by promoting the decomposition of denatured elastin or inhibiting the production of denatured elastin itself, it is possible to inhibit the abnormal deposition of elastin that occurs in photoaged areas, and to prevent or improve skin hardening and loss of skin elasticity, as well as the resulting formation of wrinkles, sagging, and loss of firmness, and have thereby arrived at the present invention. [Problem to be solved by the invention]

[0014] The present invention addresses the problem of providing a method for screening for substances that can inhibit abnormal deposition of elastin in photoaged areas, inhibit skin hardening and loss of skin elasticity, and prevent or improve skin wrinkles, sagging, loss of firmness, and the like associated with photoaging. [Means for solving the problem]

[0015] The above problem was solved by using the promotion of decomposition or inhibition of production of denatured elastin as an indicator.

[0016] The present invention provides the following screening methods. [1] Screening method for photoaging prevention and improvement agents using the amount of denatured elastin as an index [2] Screening method for agents for preventing and improving wrinkles, sagging, and loss of firmness using the amount of denatured elastin as an index [3] A method for screening an agent for inhibiting a decrease in skin elasticity and / or an agent for inhibiting skin hardening using the amount of denatured elastin as an index [4] Screening method for inhibitors of abnormal elastin deposition using the amount of denatured elastin as an index [5] The screening method according to any one of [1] to [4], wherein the indicator of the amount of denatured elastin is the promotion of degradation of denatured elastin. [6] The screening method according to [5], wherein the promotion of degradation of denatured elastin is promotion of direct degradation of denatured elastin and / or degradation by an extracellular matrix degrading enzyme. [7] The screening method according to any one of [1] to [4], wherein the indicator of the amount of denatured elastin is inhibition of production of denatured elastin. [8] The screening method according to any one of [1] to [7], wherein the modification is caused by nitration modification. [Effects of the Invention]

[0017] According to the present invention, by using the promotion of degradation or inhibition of production of denatured elastin as an indicator, a method for screening for a new substance that suppresses abnormal deposition of elastin associated with photoaging is provided. In addition, the present invention can also suppress an increase in skin hardness and a decrease in skin elasticity, thereby providing a method for screening for a new substance that prevents and improves the formation of wrinkles, sagging, and loss of firmness associated with these. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the localization of cell nuclei, nitrotyrosine, and elastin in sections of photoaged (exposed) and naturally aged (unexposed) skin. [Figure 2] FIG. 1 shows the aggregation of nitrated elastin. DETAILED DESCRIPTION OF THE INVENTION

[0019] The elastin used in the present invention is not particularly limited, and examples thereof include tropoelastin protein and elastin protein. These may be natural or artificially produced. Natural products include those extracted from vertebrates, including humans, rats, cows, rabbits, and salmon. Artificially produced elastin may be produced by transfecting tropoelastin genes derived from the above organisms into microbial cells such as Escherichia coli or yeast, or by using enzymes in extracts from Escherichia coli, wheat germ, or cultured cells in a cell-free system. Elastin may exist as a protein or may be a fibrous structure formed by crosslinking multiple elastins. For example, it may be elastic fibers such as elastic fibers and elaunin fibers recovered from the above organisms, or elastin fibers artificially crosslinked to form fibrous structures. Furthermore, these proteins or fibrous elastins may be used as they are, or may be fragmented by hydrolysis, enzymatic degradation, or other procedures. These elastins may also be chemically labeled with dyes, fluorescent substances, or the like to assess degradation.

[0020] In the present invention, denaturation refers to modifications that occur in elastin, including the addition of post-translational modifications such as nitration, glycation, and carbonylation, changes in three-dimensional structure due to misfolding, abnormal binding with other proteins and molecules, and the formation of aggregates caused by these. In the present application, elastin that has undergone one or more of the above modifications is referred to as "denatured elastin."

[0021] The method of denaturing elastin is not particularly limited. For example, methods that induce post-translational modifications such as nitration, oxidation, glycation, and carbonylation by adding a reagent to elastin, as well as methods that induce thermal denaturation by heat treatment, structural denaturation by adding an acid or alkali, photodenaturation by UV irradiation or sunlight, and age-related denaturation over time can be used. These treatments can induce changes in elastin, such as the addition of post-translational modifications such as nitration, glycation, and carbonylation, changes in three-dimensional structure due to misfolding, abnormal binding with other elastin or proteins and molecules, and the resulting formation of aggregates. It goes without saying that in addition to direct denaturation of elastin, vertebrate cells, including humans, rats, cows, rabbits, and salmon, as well as microbial cells such as Escherichia coli and yeast, which are sources of elastin, can be subjected to the above-mentioned denaturing treatments, such as reagents that induce post-translational modifications, while these organisms are still alive.

[0022] Nitration reagents used to induce post-translational modifications in elastin can be known, including the direct use of reactive nitrogen species such as peroxynitrite, or the use of a mixture of compounds capable of generating reactive nitrogen species in a test system. For example, this can be achieved by mixing nitric oxide with superoxide anion, or by mixing myeloperoxidase (MPO) with NaNO2, hydrogen peroxide, or the like. Additionally, reagents that generate nitronium ions, such as tetranitromethane and nitric acid, can also be used. Other reagents that can be used include nitroyl chloride, nitrosoperoxycarboxylate, sodium azide, and catalase. The nitration modification itself can be performed using known techniques. Using these reagents, nitro groups are introduced into tyrosine, tryptophan, phenylalanine, and cysteine ​​residues in elastin, resulting in the formation of elastin containing nitrotyrosine, dinitrotyrosine, nitrotryptophan, nitrophenylalanine, dinitrophenylalanine, nitrocysteine, and the like.

[0023] Known oxidizing reagents can be used to induce post-translational modifications in elastin, such as hydrogen peroxide, hypochlorous acid, superoxide, and hydroxyl radicals, or a mixture of compounds capable of generating reactive oxygen species in the test system. Known glycation reagents can be used, such as ribose, glucose, glyceraldehyde, and glyoxal. Carbonylation reagents can be used, such as acrolein and hydroxynonenal.

[0024] The denatured elastin used in the present invention can be elastin artificially denatured by the above-mentioned procedure, or elastin that has already been denatured without the above-mentioned denaturation treatment can also be used as the denatured elastin. Examples of denatured elastin that can be used include elastin extracted from the above-mentioned organisms that have been exposed to ultraviolet rays for a long period of time, and elastin extracted from the above-mentioned organisms that have aged over time.

[0025] Methods for measuring the amount of denatured elastin include direct measurement of denatured elastin and specific detection of denatured sites in denatured elastin and measuring the amount of denatured elastin. For example, the amount of denatured elastin can be measured directly by absorbance, fluorescence intensity, liquid chromatography (HPLC), gas chromatography, mass spectrometry, nuclear magnetic resonance (NMR), immunostaining, electrophoresis, Western blotting, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), gel permeation chromatography, ultrafiltration, protein quantification, and weight measurement. The amount of denatured elastin can also be determined by measuring the amount of nitro groups, glycated products, and carbonyl groups present in denatured elastin, as well as conformational changes due to misfolding, intermolecular crosslinks with other elastin or proteins, and the resulting formation of aggregates, using the methods described above or other well-known techniques. Aggregates refer to polymerized assemblies containing elastin fibers or elastin protein molecules or complexes, including those that have reached the point where visible precipitates are formed. The polymerization morphology of elastin polymerized assemblies includes those due to intermolecular ionic bonds, hydrophobic interactions, van der Waals forces, etc. Methods for evaluating aggregates include assessing the molecular weight of protein polymerized assemblies using electrophoresis, gel permeation chromatography, ultrafiltration, mass spectrometry, etc.; assessing the degree of aggregation using an aggregation indicator such as thioflavin T that is retained between aggregated molecules; or determining the amount of aggregates by directly assessing the change in refractive index of the solution or the occurrence of aggregated precipitates associated with aggregation using light scattering or absorbance methods; or by visually assessing the changes in molecular and fiber morphology associated with aggregation using a microscope, etc. Measurement methods are not limited to those described above.

[0026] The screening method of the present invention is a method for evaluating and selecting substances that can inhibit abnormal deposition of elastin by using the amount of denatured elastin as an index, and prevent or improve signs of photoaging such as hardening of the skin, loss of skin elasticity, and the resulting formation of wrinkles, sagging, and loss of firmness. In this application, the term "inhibitor" is used as a concept that includes "prevention and improvement."

[0027] The term "using the amount of denatured elastin as an index" in the present invention means that the amount of denatured elastin measured by any method is used as the standard for determining effectiveness.For example, if the amount of denatured elastin can be reduced compared to the non-addition group due to the presence of the test substance, it can be determined to be effective.In this case, the decrease in the amount of denatured elastin can be either due to the promotion of the degradation of existing denatured elastin or due to the inhibition of the new production of denatured elastin.

[0028] When screening for an agent that reduces the amount of denatured elastin by promoting the degradation of existing denatured elastin, the degree of degradation of denatured elastin measured using any method is used as the criterion for assessing effectiveness. For example, if the presence of a test substance can increase the degradation of denatured elastin compared to a control group, it can be determined to be effective. In a system that evaluates elastin degradation using denatured elastin, a substance that increases the degradation of denatured elastin occurring in the system depending on the presence or absence of the test substance can be selected. For example, a substance that increases the degradation of denatured elastin by preferably 3% or more, more preferably 5% or more compared to the absence of the test substance can be selected. In this case, the degradation of denatured elastin can be either direct degradation of denatured elastin or degradation by extracellular matrix degrading enzymes.

[0029] Direct degradation of denatured elastin means degradation without the intervention of enzymes or the like. Direct degradation of denatured elastin can be performed using known methods, but for example, it can be confirmed by mixing any denatured elastin with a test substance and measuring the amount of denatured elastin present or the amount of degradation products of the denatured elastin after a certain period of time. The order and timing of mixing the denatured elastin and the test substance are not important, as long as the two are present together in the test system.

[0030] Degradation of denatured elastin with an ECM-degrading enzyme can be performed using known methods. For example, enzymes such as neutrophil elastase, fibroblast elastase, and pancreatic elastase, as well as matrix metalloproteinases, serine proteases, and cysteine ​​proteases, can be used. Alternatively, cultured cells, cell lysates, or culture supernatants that produce these enzymes can be used. Degradation of denatured elastin can be confirmed by reacting the denatured elastin with the enzyme, or by adding the denatured elastin to cultured cells or mixing it with the culture supernatant and reacting, followed by measuring the amount of denatured elastin present or the amount of degradation products of the denatured elastin. The order and timing of mixing the denatured elastin, ECM-degrading enzyme, and test substance are not important, as long as they are present together in the test system.

[0031] The presence or absence and degree of degradation of denatured elastin can be measured by known methods, without particular limitation. For example, the degradation of denatured elastin can be calculated by measuring the amount of denatured elastin present or the amount of degradation products of denatured elastin. The method described above can be used to measure the amount of denatured elastin present. On the other hand, the degradation of denatured elastin can also be measured by measuring the amount of degradation products present in the system. The method for measuring the amount of degradation products of denatured elastin can be performed by known methods, without particular limitation. For example, a method can be used in which dye-bound insoluble elastin is denatured to prepare insoluble denatured elastin, followed by degradation reaction using the above-mentioned method, and the dye of the degradation product released in the supernatant can be measured by absorbance or fluorescence intensity. Alternatively, after degradation reaction using soluble denatured elastin using the above-mentioned method, the molecular weight can be measured using electrophoresis or gel permeation chromatography, etc., and substances with molecular weights smaller than the detected soluble denatured elastin can be quantified as degradation products. Furthermore, there is also a method in which denatured elastin peptide is subjected to a degradation reaction by the above-mentioned method, and then the peptide fragment degradation products present in the system are measured by HPLC. The measurement method is not limited to the above.

[0032] When screening for agents that reduce the amount of denatured elastin by inhibiting the de novo synthesis of denatured elastin, the presence or absence and degree of de novo synthesis of denatured elastin are used as the criterion for assessing effectiveness. For example, in a system in which elastin is subjected to the aforementioned denaturation treatment to generate denatured elastin, if the presence of a test substance can reduce the amount of denatured elastin present in the system compared to a control group, it can be determined to be effective. In a system for evaluating the amount of denatured elastin present, a substance that reduces the amount of denatured elastin present in the system depending on the presence or absence of the test substance can be selected. For example, a substance that reduces the amount of denatured elastin present in the system by preferably 3% or more, more preferably 5% or more, compared to the absence of the test substance can be selected. The order and timing of mixing elastin and the test substance are not important, and the denaturation treatment can be performed after the two substances coexist in the test system.

[0033] The presence or absence and degree of new formation of denatured elastin can be measured by known methods, without particular limitation. For example, the new formation of denatured elastin can be evaluated by measuring the amount of denatured elastin present. The method for measuring the amount of denatured elastin present can be the method described above.

[0034] There are no particular limitations on the test substance used. The test substance can be an extract derived from animals or plants, a fungal culture, or an enzyme-treated product thereof, or a compound or derivative thereof, and can be in liquid, powder, gel, or other form. The method for analyzing the degradation of denatured elastin and the method for analyzing the inhibition of denatured elastin production can be appropriately selected depending on the test substance. [Example]

[0035] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0036] <Experiment 1> Localization of elastin and nitrotyrosine in photoaged and naturally aged skin sections Elastin and nitrotyrosine were detected in the skin sections using the following procedure. Formalin-fixed, paraffin-embedded sections were prepared from photoaged skin (exposed area, cheek) and naturally aged skin (unexposed area, buttocks) obtained from a woman in her 90s who gave informed consent. The sections were immunostained using anti-nitrotyrosine antibody (Stressmarq) and elastin antibody (Abcam) as primary antibodies, and AlexaFluor(R) 488-labeled anti-mouse antibody (Abcam) and AlexaFluor(R) 594-labeled anti-rabbit antibody (Abcam) as secondary antibodies, and the fluorescent images were observed under a fluorescence microscope (Keyence) (10x magnification).

[0037] As shown in Fig. 1, more nitrotyrosine was detected in the dermis of the exposed skin than in the dermis of the non-exposed skin, and it was shown that nitrotyrosine was present in the extracellular matrix of the dermis in photoaged skin. In addition, nitrotyrosine in the exposed skin showed almost the same localization as elastin, suggesting the presence of nitrated elastin in photoaged skin.

[0038] <Experiment 2> Degradation of nitrated elastin protein by elastase The degradation of nitrated modified elastin by elastase was measured by the following procedure. <Preparation of peroxynitrite-treated elastin solution> Orcein-labeled insoluble elastin (SIGMA) was thoroughly suspended in Reaction Buffer (0.2 M Tris-HCl (pH 7.5), 20 mM CaCl2) to a concentration of 20 mg / mL, and peroxynitrite solution was added to concentrations of 100 μM, 300 μM, and 1 mM, followed by incubation at 37°C for 1 day. This was repeated 3 times. <F <Preparation of tetranitromethane-treated elastin solution> Orcein-labeled insoluble elastin (SIGMA) was thoroughly suspended in Reaction Buffer (0.2 M Tris-HCl (pH 7.5), 20 mM CaCl2) to a concentration of 40 mg / mL, and a 10% tetranitromethane (TNM) solution dissolved in EtOH was added to a concentration of 0.1%. After mixing for 5 minutes, an aqueous urea solution was added to a final concentration of 2 M and mixed for 5 minutes to stop the reaction. After centrifugation, the supernatant was discarded, and Reaction Buffer was added again for washing. This operation was repeated 6 times in total. After the final centrifugation, Reaction Buffer was added to the precipitate to prepare a solution. <Elastin degradation test by elastase> Neutrophil elastase (SIGMA) was added to the peroxynitrite-treated elastin solution or tetranitromethane-treated elastin solution at 0.5 units / mL, or 1 / 6 volume of fibroblast elastase solution recovered from cultured fibroblasts was added, and the mixture was incubated at 37°C for 6 days. After centrifugation, the supernatant was collected in a 96-well plate, and the amount of elastin degradation products was measured by absorbance at 595 nm. The degradation of nitrated elastin was calculated according to Equation 1, where the degradation of unmodified elastin is defined as 1.

[0039]

number

[0040] As shown in Table 1, peroxynitrite treatment and TNM treatment reduced the degradation of elastin by neutrophil elastase or fibroblast elastase compared to unmodified elastin. In other words, it was confirmed that elastase-induced degradation of nitrated elastin is suppressed.

[0041] [Table 1]

[0042] <Experiment 2> Degradation of denatured elastin protein by elastase The degradation of elastin by elastase after various denaturation treatments was measured according to the following procedure. <Preparation of various modified elastin solutions> Orcein-labeled insoluble elastin (SIGMA) was thoroughly suspended in Reaction Buffer (0.2 M Tris-HCl (pH 7.5), 20 mM CaCl2) to a concentration of 40 mg / mL, and either peroxynitrite solution was added to a concentration of 100 μM, or ribose solution was added to a concentration of 300 mM, or acrolein solution was added to a concentration of 1 mM, and the mixture was incubated at 37°C for 3 days. After centrifugation, the supernatant was discarded, and Reaction Buffer was added again to wash the mixture. This procedure was repeated three times. After the final centrifugation, Reaction Buffer was added to the sediment. <Elastin degradation test using elastase> Neutrophil elastase (SIGMA) was added to each denatured elastin solution at 0.2 units / mL and incubated at 37°C for 1 day. After centrifugation, the supernatant was collected in a 96-well plate, and the amount of elastin degradation products was measured by absorbance at 595 nm. The degradation of each denatured elastin was calculated according to Equation 2, where the degradation of undenatured elastin is set to 1.

[0043]

number

[0044] As shown in Table 2, elastin treated with nitration (peroxynitrite), glycation (ribose), or carbonylation (acrolein) all showed reduced degradation of elastin by neutrophil elastase compared to unmodified elastin. In other words, it was confirmed that degradation by elastase is inhibited in modified elastin. Of these, nitration treatment reduced elastin degradation the most.

[0045] [Table 2]

[0046] <Experiment 3> Aggregation of elastin by nitration treatment The morphological changes of peroxynitrite-treated elastin fibers were observed using the following procedure. An elastin fiber sheet (Extracellular Matrix Research Institute) was immersed in 0.2 M phosphate buffer (pH 7.5) and peroxynitrite (Dojindo) was added to a concentration of 100 μM. After incubation at 37°C for 3 days, the elastin fiber sheet was washed with distilled water, dried, and then gold was vapor-deposited. The fiber morphology was observed under a scanning electron microscope (JEOL).

[0047] As shown in Figure 2, peroxynitrite treatment caused the elastin fibers to appear bundled and united, indicating that nitration treatment causes elastin aggregation.

[0048] <Experiment 4> Changes in hardness and elasticity of elastin gel sheets due to nitration treatment The hardness and elasticity of the tetranitromethane-treated elastin gel sheet were measured according to the following procedure. An elastin gel sheet (Extracellular Matrix Research Institute, Inc.) was immersed in 0.2 M phosphate buffer (pH 7.4) and a 10% tetranitromethane (TNM) solution in EtOH was added to a concentration of 0.1%. After 3 hours of incubation at room temperature, the solution was discarded and the sheet was washed with phosphate buffer. Gel hardness (Young's modulus) was measured using a tensile tester (Extracellular Matrix Research Institute, Inc.). Total elongation and elasticity were measured using a Cutometer (Courage+Khazaka). Specifically, elasticity was calculated using the obtained Uf value (total elongation after vacuum suction) and Ur value (contraction immediately after vacuum release) according to Equation 3.

[0049]

number

[0050] As shown in Table 3, the hardness (Young's modulus) of the elastin gel sheet treated with tetranitromethane (TNM) increased, and as shown in Table 4, the elongation rate (Uf) and elasticity (Ur / Uf) decreased.

[0051] [Table 3]

[0052] [Table 4]

[0053] From the above results, it has been found that the amount of nitrated modified elastin increases in photoaged skin, and that nitration has the properties of inhibiting the degradation of elastin by elastase and promoting the aggregation of elastin.In addition, it has been found that these changes in the properties of elastin result in elastin hardening and loss of elasticity.In other words, it has been found that the denatured elastin produced by nitration modification caused by long-term exposure to UV rays reduces the degradation of elastin by elastase, contributing to the abnormal deposition of elastin in photoaged skin.In other words, it is believed that reducing the amount of denatured elastin can prevent or improve the signs of photoaging, such as skin hardening and loss of skin elasticity, and the associated wrinkles, sagging, and loss of firmness.The method of evaluating and / or selecting the promotion of degradation of denatured elastin, or the method of evaluating and / or selecting the inhibition of the production of denatured elastin, of the present invention is based on the above findings.

[0054] Example 1: Screening method for substances that promote the degradation of nitrated elastin The decomposition of nitrated elastin by elastase was measured according to the following procedure, and photoaging prevention and improvement agents were selected. <Preparation of test substance> Several dried plant materials were each extracted with 10 times the weight of 50% (v / v) ethanol in water at room temperature for one week. The dried extract residue was diluted with ethanol and water in a weight ratio of 1:50:49 to prepare the test substance. A 50% ethanol solution was used as a control. <Decomposition test of nitrated elastin> Orcein-labeled insoluble elastin (SIGMA) was thoroughly suspended in Reaction Buffer (0.2 M Tris-HCl (pH 7.5), 20 mM CaCl2) to a concentration of 20 mg / mL. Peroxynitrite solution was added to the suspension at concentrations of 0 or 100 μM and incubated at 37°C for 1 day to prepare nitrated elastin. The test substance or control substance was added to the suspension at a concentration of 100 ppm, followed by neutrophil elastase (SIGMA) at 0.5 units / mL and incubation at 37°C for 2 days. After centrifugation, the supernatant was collected in a 96-well plate, and the amount of elastin degradation products was measured by absorbance at 595 nm. The degree of decomposition of nitrated elastin by each test substance was calculated using Equation 4, with the addition of the control substance considered to be 100%. Test substances that increased decomposition by 5% or more compared to the addition of the control substance were determined to be effective ingredients.

[0055]

number

[0056] If the decomposition of nitrated elastin by elastase when the test substance is added increases by 5% or more compared to when the control substance is added, it can be determined that the test substance has sufficient effect of promoting the decomposition of nitrated elastin, and it is expected to prevent and improve photoaging symptoms such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles, sagging, and loss of firmness.

[0057] Example 2: Screening method for substances that promote the degradation of thermally denatured elastin The decomposition of thermally denatured elastin by fibroblast-derived degrading enzymes was measured according to the following procedure, and photoaging prevention and improvement agents were selected. <Preparation of test substance> Prepared in the same manner as in Example 1. <Decomposition test of thermally denatured elastin> FITC-labeled insoluble elastin (AnaSpec) was thoroughly suspended in Reaction Buffer (0.2 M Tris-HCl (pH 8.5), 20 mM CaCl2) to a concentration of 100 mg / mL, heated at 95°C for 10 minutes, and then cooled to prepare thermally denatured elastin. 500 μL of adult human dermal fibroblasts suspended in D-MEM containing 10% FBS or 500 μL of D-MEM containing 10% FBS alone was seeded into 24-well plates and cultured at 37°C under humidified conditions of 5% CO2 and 95% air for 3 days. The medium was discarded, and 300 μL of phenol red-free D-MEM containing 10% FBS was dispensed into each well. Test substances or control substances were added to each well to a concentration of 100 ppm, followed by the addition of 30 μL of the prepared thermally denatured elastin. The cells were cultured at 37°C under humidified conditions of 5% CO2 / 95% air for 3 days, and the culture supernatant was collected. The culture supernatant was centrifuged and dispensed into a 96-well plate. The amount of degradation product was measured using a fluorescent plate reader (TECAN) at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. The degradation of thermally denatured elastin for each test substance was calculated using Equation 5, with the control substance added as 100%. Test substances that increased degradation by 5% or more were determined to be effective ingredients.

[0058]

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[0059] If the decomposition of thermally denatured elastin when the test substance is added increases by 5% or more compared to when the control substance is added, it can be determined that the test substance has sufficient effect of promoting the decomposition of thermally denatured elastin, and it is expected to prevent and improve photoaging symptoms such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles, sagging, and loss of firmness.

[0060] Example 3: Screening method for decomposing substances of glycated elastin The direct degradation of glycated elastin was measured using the following procedure to select a photoaging prevention and improvement agent. <Preparation of test substance> The test substances were prepared by adding 10 times the weight of 1,3-butylene glycol solution to several dried plant materials and heating and extracting them at 50°C for 6 hours. The 1,3-butylene glycol solution was used as a control substance. <Glycated elastin degradation test> Soluble porcine elastin-A (FUJIFILM) was dissolved in 50 mM Tris-HCl buffer (pH 7.5) at a concentration of 1 mg / mL, and ribose (FUJIFILM) was added to a final concentration of 100 mM. The mixture was incubated at 60°C for 15 days to prepare glycated elastin. Test substances or control substances were added to the glycated elastin to a concentration of 1000 ppm, and the fluorescence of the glycated elastin before the reaction was measured using a fluorescence plate reader at an excitation wavelength of 370 nm and a measurement wavelength of 440 nm. After incubation at 37°C for 5 days, 200 μL of the reaction solution was dispensed into a 96-well plate, and the amount of glycated elastin after the reaction was measured using a fluorescence plate reader at an excitation wavelength of 370 nm and a measurement wavelength of 440 nm. The degradation of glycated elastin for each test substance was calculated according to Equation 6, with the addition of the control substance taken as 100%, and test substances that increased degradation by 5% or more were determined to be effective ingredients.

[0061]

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[0062] If the decomposition of glycated elastin when the test substance is added increases by 5% or more compared to when the control substance is added, it can be determined that the test substance has sufficient effect of promoting the decomposition of glycated elastin, and is expected to prevent and improve photoaging symptoms such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles, sagging, and loss of firmness.

[0063] Example 4: Screening method for substances that inhibit the formation of elastin aggregates The aggregation of elastin caused by ultraviolet irradiation was measured and photoaging prevention and improvement agents were selected according to the following procedure. <Preparation of test substance> The test substances were prepared by adding 100 times the weight of water to several dried plant substances and extracting them by heating at 80°C for 8 hours. Water was used as a control substance. <Elastin aggregation inhibition test> Tropoelastin protein (SIGMA) was dissolved in 0.2 M phosphate buffer (pH 7.5) at a concentration of 1 mg / mL, and the test substance or control substance was added to a final concentration of 100 ppm. 200 μL of the solution was then dispensed into a 96-well plate. UV-A (365 nm) was applied to the plate at 100 J / cm using a UV crosslinker (AnalytikJena). 2 After incubation at 37°C for 1 day, 10x Blue native PAGE Sample Buffer (5% (w / v) CBB-G250, 0.5 M 6-aminocaproic acid, 0.1 M Bis-Tris (pH 7.0), 50% glycerol) was added at a volume of 1 / 10 and the mixture was allowed to stand at 4°C for 10 minutes. 20 μL of each sample was applied to a 4-16% polyacrylamide gradient gel (Invitrogen). The electrophoresis chamber was filled with anode buffer (50 mM Bis-Tris (pH 7.0)) on the anode side and cathode buffer (50 mM Tricine (pH 7.0), 15 mM Bis-Tris (pH 7.0), 0.02% CBB-G250) on the cathode side. Electrophoresis was performed at 150 mA while the electrophoresis chamber was cooled. The electrophoresis was stopped midway, and the Cathode Buffer was replaced with one without CBB-G250, followed by another run. After electrophoresis, the gel was fixed and destained overnight in a fixative (50% methanol, 10% acetic acid) and then silver stained using a silver staining kit (ATTO). The gel was photographed and band intensities were quantified using ImageJ (National Institutes of Health). Tropoelastin protein has a molecular weight of approximately 70 kDa as a monomer, so the amount of aggregates was determined by summing the intensities of bands showing molecular weights at least twice this molecular weight. The amount of aggregates in the control sample was defined as 100%, and test substances that reduced the amount of aggregates by 5% or more were considered to be effective.

[0064] If the amount of elastin aggregates when the test substance is added is reduced by 5% or more compared to when the control substance is added, it can be determined that the test substance has sufficient inhibitory effect on the formation of elastin aggregates, and it is expected to prevent and improve photoaging symptoms such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles, sagging, and loss of firmness.

[0065] By using the screening method of the present invention, it is possible to select substances that prevent and improve photoaging, such as inhibitors of abnormal elastin deposition, inhibitors of skin hardening, inhibitors of reduced skin elasticity, and agents for preventing and improving wrinkles, sagging, and reduced firmness.

Claims

1. A screening method for inhibitors of abnormal elastin deposition using the amount of denatured elastin as an indicator, wherein the denaturation is at least one selected from nitration and thermal denaturation caused by exposure to ultraviolet rays and / or sunlight, and the indicator for the amount of denatured elastin is inhibition of the production of denatured elastin.

2. A screening method for inhibitors of abnormal elastin deposition using the amount of denatured elastin as an indicator, wherein the denaturation is at least one selected from nitration and thermal denaturation caused by exposure to ultraviolet light and / or sunlight, and the indicator is direct decomposition of denatured elastin and / or promotion of decomposition by extracellular matrix degrading enzymes.

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