JP7757465B2 - Screening method for inhibitors of abnormal elastin deposition - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies. (1) Publication date: September 10, 2020. (2) Publication address: http://ifscc2020.com/scientific_program.html

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

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 in 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.

The dermis, the supporting tissue of the skin, is primarily composed of extracellular matrix, 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 experiences a decrease in collagen and abnormal deposition of elastin (such as thickened fibers and accumulation of amorphous masses), and 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).

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.

On the other hand, conventional photoaging prevention and improvement agents used to prevent or improve wrinkles, sagging skin, loss of firmness, etc. have been those that aim to increase collagen and elastin or inhibit their degradation (Patent Documents 1-4). In particular, when targeting elastin, a method that inhibits the degradation of elastin by elastase is widely used. This aims to increase the amount of elastin in the skin by inhibiting its degradation, thereby compensating for the loss of elasticity that occurs in photoaged areas. This method may also be able to inhibit the abnormal deposition of elastin degradation products. However, there have been no reports of its effectiveness against elastin that has already been denatured and abnormally deposited, and conventional technology has not known any means of inhibiting the abnormal deposition of denatured elastin. In other words, conventional technology alone has not been able to sufficiently inhibit the abnormal deposition of elastin that occurs in photoaged areas, nor has it been able to fundamentally prevent or improve the progression of associated photoaging symptoms.

On the other hand, protein nitration is a type of post-translational modification caused by reactive nitrogen species generated in vivo. Nitrogen groups are attached to the benzene rings of the aromatic amino acids tyrosine and tryptophan, which make up proteins. Nitration occurs when the benzene ring in an amino acid undergoes an electrophilic substitution reaction with nitronium ions (NO ₂ ⁺ ) or nitrogen dioxide radicals formed by reactive nitrogen species (Non-Patent Document 5). The tryptophan content of many proteins present in the body is much lower than that of tyrosine, and protein nitration is thought to occur primarily at tyrosine residues (Non-Patent Document 6). Protein nitration 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 be present in the skin, and nitrotyrosine present in the stratum corneum, in particular, is associated with skin color (Patent Document 5). However, the relationship between this and abnormal elastin deposition in photoaged skin was unknown.

JP 2016-69332 A JP 2012-56933 A JP 2001-316221 A JP 2012-207015 A JP 2017-181423 A

Exp. Dermatol. , 2019, 28(8):914-921 Exp. Dermatol. , 2019, 28(8):981-984 Biochimie, 2016, 128-129:163-73 J. Invest. Dermatol. , 2004, 122(6): 1448-55 Chem. Res. Toxicol. , 2009, 22(5):894-898 Front. Chem. , 2016, 3:70 Diabetes, 2008, 57(4):889-98 Science, 2000, 290(5493):985-9

Under these circumstances, the inventors have come to the realization that inhibiting abnormal elastin deposition is important for preventing and improving photoaging skin symptoms. The inventors have therefore considered the mechanism by which abnormal elastin deposition occurs in photoaged skin. Although abnormal elastin deposition occurs in photoaged skin despite increased expression of extracellular matrix degrading enzymes, the inventors believe that this phenomenon occurs because, for some reason, the increased levels of degrading enzymes are not functioning properly, preventing elastin from being broken down normally. Specifically, the inventors have come to the idea that UV exposure causes some kind of denaturation in elastin, inhibiting its breakdown by degrading enzymes, which would normally be increased.

The inventors therefore searched for new factors involved in inhibiting the degradation of elastin in photoaged skin and discovered that the constituent amino acids of elastin abnormally deposited in photoaged skin are modified by nitration. Further investigation revealed that nitrated elastin has the property of promoting aggregation, acquires resistance to degradation by elastase, and furthermore, increases hardness and decreases elasticity.

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

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

Based on these new findings, we believe that by promoting the breakdown of denatured elastin or inhibiting the production of denatured elastin itself, we can inhibit the abnormal deposition of elastin that occurs in photoaged areas, thereby preventing or improving skin hardening and loss of skin elasticity, as well as the resulting formation of wrinkles, sagging, and loss of firmness, leading to the development of this invention.

The objective of this study is to provide a method for screening substances that can inhibit the abnormal deposition of elastin in photoaged areas, suppress skin hardening and loss of skin elasticity, and prevent or improve skin wrinkles, sagging, loss of firmness, and other problems associated with photoaging.

The above problem was solved by using the promotion of degradation or inhibition of production of denatured elastin as an indicator.

The present invention provides the following screening methods.
[1] A screening method for a photoaging prevention/improvement agent using the amount of denatured elastin as an index; [2] A screening method for a prevention/improvement agent for wrinkles, sagging, and loss of firmness using the amount of denatured elastin as an index; [3] A screening method for 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] A screening method for an agent for inhibiting abnormal elastin deposition using the amount of denatured elastin as an index; [5] A screening method according to any one of [1] to [4], wherein the index for the amount of denatured elastin is promotion of degradation of denatured elastin; [6] A 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 extracellular matrix degrading enzymes; [7] A screening method according to any one of [1] to [4], wherein the index for the amount of denatured elastin is inhibition of production of denatured elastin; [8] A screening method according to any one of [1] to [7], wherein the denaturation is caused by nitration modification.

The present invention provides a method for screening for new substances that suppress the abnormal deposition of elastin associated with photoaging by using the promotion of degraded elastin or the inhibition of its production as an indicator. In addition, the invention can also suppress an increase in skin hardness and a decrease in skin elasticity, thereby providing a method for screening for new substances that prevent and improve the associated formation of wrinkles, sagging skin, and loss of firmness.

FIG. 1 shows the localization of cell nuclei, nitrotyrosine, and elastin in sections of photoaged (exposed) and naturally aged (unexposed) skin. FIG. 1 shows the aggregation of nitrated elastin.

The elastin used in the present invention is not particularly limited, and examples 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 producing elastin in a cell-free system using enzymes in extracts of Escherichia coli, wheat germ, or cultured cells. Elastin may exist as a protein or as 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.

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

The method of denaturing elastin is not particularly limited. For example, methods that add reagents to elastin to induce post-translational modifications such as nitration, oxidation, glycation, and carbonylation, as well as methods that induce thermal denaturation by subjecting elastin to heat treatment, structural denaturation by adding acid or alkali, photodenaturation by exposure to ultraviolet light 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, proteins, or molecules, and the resulting formation of aggregates. Needless to say, in addition to directly denaturing elastin, the aforementioned denaturing treatments, such as reagents that induce post-translational modifications, can also be administered to elastin-producing vertebrates, including humans, rats, cows, rabbits, and salmon, as well as microbial cells such as Escherichia coli and yeast, while these organisms are still alive.

Nitration modification 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 NaNO ₂ , 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.

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

The denatured elastin used in the present invention can be elastin that has been artificially denatured by the procedures described above. However, elastin that has already been denatured without the denaturation treatment described above can also be used as denatured elastin. Examples of 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.

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, weight measurement, etc. Furthermore, the amount of nitro groups, glycated products, and carbonyl groups present in denatured elastin, as well as changes in three-dimensional structure due to misfolding, intermolecular crosslinks with other elastin, proteins, or molecules, and the formation of aggregates caused by these can also be measured using the methods described above or known techniques, and the amount of these can be used to determine the amount of denatured elastin. Aggregates refer to polymerized assemblies containing elastin fibers or elastin protein molecules or complexes, and include those that have reached the point where visible precipitates are formed. Polymerization forms of elastin polymerized assemblies include those due to intermolecular ionic bonds, hydrophobic interactions, van der Waals forces, etc. Methods for evaluating aggregates include methods such as evaluating the molecular weight of protein polymerized assemblies using electrophoresis, gel permeation chromatography, ultrafiltration, or a mass spectrometer; methods for evaluating the degree of aggregation using an aggregation indicator such as thioflavin T that is retained between aggregated molecules; and methods for determining the amount of aggregates by directly evaluating 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 directly evaluating the changes in molecular and fiber morphology associated with aggregation using a microscope, etc. Measurement methods are not limited to those described above.

The screening method of the present invention uses the amount of denatured elastin as an indicator to evaluate and select substances that can inhibit abnormal deposition of elastin and prevent or improve signs of photoaging such as hardening of the skin, loss of skin elasticity, and the resulting formation of wrinkles, sagging skin, and loss of firmness. In this application, the term "inhibitor" is used as a concept that encompasses "prevention and improvement."

In the present invention, the use of the amount of denatured elastin as an index means that the amount of denatured elastin present, measured using any method, is used as the standard for assessing effectiveness. For example, if the presence of the test substance can reduce the amount of denatured elastin compared to a control group, it can be determined to be effective. Note that this reduction in the amount of denatured elastin can be due to either the promotion of the breakdown of existing denatured elastin or the inhibition of the new production of denatured elastin.

When screening for agents that reduce 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, the agent can be determined to be effective. In a system that uses denatured elastin to assess elastin degradation, a substance can be selected that increases the degradation of denatured elastin that occurs in the system in the presence or absence of the test substance. For example, a substance that increases the degradation of denatured elastin by preferably 3% or more, and 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.

Direct degradation of denatured elastin means degradation without the intervention of enzymes or the like. Publicly known methods can be used for direct degradation of denatured elastin, 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 denatured elastin after a certain period of time. The order or timing in which the denatured elastin and the test substance are mixed is not important, as long as the two are able to coexist in the test system.

Known methods can be used to degrade denatured elastin with extracellular matrix-degrading enzymes. 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, extracellular matrix-degrading enzyme, and test substance are not important, as long as they are present at the same time in the test system.

The presence or absence and degree of degradation of denatured elastin can be measured by, but is not limited to, known methods. 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 for measuring the amount of denatured elastin present can be the method described above. Alternatively, the degradation of denatured elastin can 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, but is not limited to, known methods. For example, a method can be used in which dye-bound insoluble elastin is denatured to prepare insoluble denatured elastin, followed by a degradation reaction using the method described above, and the dye of the degradation product released in the supernatant can be measured by absorbance or fluorescence intensity. Alternatively, a degradation reaction can be performed using soluble denatured elastin using the method described above, followed by measuring the molecular weight using electrophoresis or gel permeation chromatography, etc., and quantifying substances with molecular weights smaller than that of the soluble denatured elastin detected as degradation products. Another method involves subjecting denatured elastin peptide to a degradation reaction using the method described above, and then measuring the peptide fragment degradation products present in the system using HPLC. Measurement methods are not limited to the above.

When screening for agents that reduce the amount of denatured elastin by inhibiting its de novo synthesis, 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 denaturing treatment to produce denatured elastin, if the presence of the test substance reduces the amount of denatured elastin compared to a control group, the agent can be determined to be effective. In a system to evaluate the amount of denatured elastin, 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, and 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; the denaturing treatment should be carried out after the two substances have coexisted in the test system.

While there are no particular limitations on the presence or absence and extent of new denatured elastin formation, it can be measured using known methods. For example, the new formation of denatured elastin can be evaluated by measuring the amount of denatured elastin present. The method described above can be used to measure the amount of denatured elastin present.

There are no particular restrictions on the test substance used. Animal and plant extracts, fungal cultures, or enzyme-treated products of these, compounds, or derivatives thereof can all be used as test substances, and they 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 selected appropriately depending on the test substance.

The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

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

As shown in Figure 1, more nitrotyrosine was detected in the dermis of sun-exposed skin than in the dermis of non-sun-exposed skin, indicating that nitrotyrosine is present in the extracellular matrix of the dermis in photoaged skin. Furthermore, nitrotyrosine in sun-exposed skin was localized in roughly the same location as elastin, suggesting the presence of nitrated elastin in photoaged skin.

<Experiment 2> Degradation of nitrated elastin protein by elastase The degradation of nitrated elastin by elastase was measured according to 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 CaCl ) to a concentration of 20 mg/mL, and peroxynitrite solution was added to concentrations of 100 μM, 300 μM, and 1 mM. The mixture was incubated at 37°C for 1 day, and this was repeated three times.
<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 CaCl ₂ ) to a concentration of 40 mg/mL, and a 10% tetranitromethane (TNM) solution 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 to wash the sample. This procedure was repeated a total of 6 times. After the final centrifugation, reaction buffer was added to the sediment to prepare a solution.
<Elastin degradation test using elastase>
Neutrophil elastase (SIGMA) was added to the peroxynitrite-treated elastin solution or tetranitromethane-treated elastin solution to a concentration of 0.5 units/mL, or 1/6 volume of a fibroblast elastase solution recovered from cultured fibroblasts was added, and the mixture was incubated for 6 days at 37°C. 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.

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 nitrated elastin inhibits degradation by elastase.

<Experiment 2> Degradation of denatured elastin protein by elastase The degradation of elastin by elastase after various denaturing 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 CaCl ₂ ) to a concentration of 40 mg/mL, and peroxynitrite solution was added to a concentration of 100 μM, or ribose aqueous solution was added to a concentration of 300 mM, or acrolein solution was added to a concentration of 1 mM, and the suspension was incubated at 37°C for 3 days. After centrifugation, the supernatant was discarded, and reaction buffer was added again to wash the suspension. 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 type of denatured elastin solution to a concentration of 0.2 units/mL, and the solution was 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 type of denatured elastin was calculated according to Equation 2, assuming that the degradation of undenatured elastin is 1.

As shown in Table 2, elastin that had been 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.

<Experiment 3> Aggregation of elastin by nitration treatment The morphological changes of elastin fibers treated with peroxynitrite were observed according to 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).

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

<Experiment 4> Changes in hardness and elasticity of elastin gel sheets due to nitration treatment The hardness and elasticity of elastin gel sheets treated with tetranitromethane were measured according to the following procedure.
Elastin gel sheets (Extracellular Matrix Research Institute, Inc.) were 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.

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

The above results revealed that nitrated, denatured elastin increases in photoaged skin, and that nitration inhibits the degradation of elastin by elastase and promotes elastin aggregation. Furthermore, the researchers found that these changes in elastin's properties result in elastin hardening and loss of elasticity. In other words, the researchers found that denatured elastin produced by nitration modification over many years of UV exposure reduces the degradation of elastin by elastase and contributes to the abnormal deposition of elastin in photoaged skin. In other words, reducing the amount of denatured elastin is believed to prevent or improve signs of photoaging, such as skin hardening and loss of skin elasticity, as well as the associated wrinkles, sagging, and loss of firmness. The methods of the present invention for evaluating and/or selecting the promotion of denatured elastin degradation, or the methods of evaluating and/or selecting the inhibition of denatured elastin production, are based on the above findings.

Example 1 Screening Method for Substances Promoting the Decomposition of Nitroelastin 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 test substances. 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 CaCl ) to a concentration of 20 mg/mL, and peroxynitrite solution was added to concentrations of 0 and 100 μM. The suspension was incubated at 37°C for 1 day to prepare nitrated elastin. To this suspension, the test substance or control substance was added to a concentration of 100 ppm, and neutrophil elastase (SIGMA) was then added to a concentration of 0.5 units/mL, followed by 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 for each test substance was calculated according to formula 4, with the addition of the control substance being taken as 100%, and test substances that increased decomposition by 5% or more compared to the addition of the control substance were determined to be effective ingredients.

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 is expected to prevent and improve symptoms of photoaging, such as inhibiting abnormal elastin deposition, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles, sagging, and loss of firmness.

Example 2 Screening method for substances that promote the degradation of thermally denatured elastin The degradation 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 CaCl ) 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 10% FBS-containing D-MEM was seeded into a 24-well plate, or 500 μL of 10% FBS-containing D-MEM alone was dispensed into each well, and the plate was cultured at 37°C under humidified conditions of 5% _CO /95% air for 3 days. The medium was discarded, and 300 μL of phenol red-free D-MEM supplemented with 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% _CO /95% air for 3 days, and the culture supernatant was collected from the plate. The culture supernatant was centrifuged and dispensed into a 96-well plate. The amount of degradation product was measured using a fluorescence plate reader (TECAN) at an excitation wavelength of 495 nm and a measurement wavelength of 515 nm. The degradation of thermally denatured elastin for each test substance was calculated using Equation 5, assuming the addition of the control substance to be 100%, and test substances that increased degradation by 5% or more were determined to be effective ingredients.

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 is expected to prevent and improve symptoms of photoaging, such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles and sagging and loss of firmness.

Example 3 Screening Method for Degrading Substances of Glycated Elastin Direct degradation of glycated elastin was measured according to the following procedure, and photoaging prevention and improvement agents were selected.
<Preparation of test substance>
Test substances were prepared by adding 10 times the weight of 1,3-butylene glycol solution to several dried plant materials and extracting them by heating at 50°C for 6 hours. 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. The test substance or control substance was 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.

If the breakdown 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 breakdown of glycated elastin, and is expected to prevent and improve symptoms of photoaging, such as inhibiting abnormal elastin deposition, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles and sagging skin and loss of firmness.

Example 4 Screening Method for Substances Inhibiting Elastin Aggregate Formation Elastin aggregation caused by ultraviolet irradiation was measured according to the following procedure, and photoaging prevention and improvement agents were selected.
<Preparation of test substance>
Test substances were prepared by adding 100 times the weight of water to each of the dried plant materials 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 dispensed into a 96-well plate. The plate was irradiated with UV-A (365 nm) at 100 J/ ^cm² using a UV crosslinker (Analytik Jena). 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 in an amount of 1/10, and the mixture was allowed to stand at 4°C for 10 minutes. 20 μL of each solution was applied to a 4-16% polyacrylamide gradient gel (Invitrogen), and the anode side of the electrophoresis tank was filled with anode buffer (50 mM Bis-Tris (pH 7.0)) and cathode side with cathode buffer (50 mM Tricine (pH 7.0), 15 mM Bis-Tris (pH 7.0), 0.02% CBB-G250). Electrophoresis was performed at 150 mA while the electrophoresis tank was cooled. The electrophoresis was stopped midway, and the cathode buffer was replaced with one that did not contain CBB-G250, and electrophoresis was performed again. After electrophoresis, the gel was immersed overnight in a fixative (50% methanol, 10% acetic acid) to fix and decolorize it, and then silver stained using a silver staining kit (ATTO). After photographing the gel, band intensities were quantified using ImageJ (National Institutes of Health). Tropoelastin protein has a molecular weight of approximately 70 kDa in its monomeric form, so the amount of aggregates was determined as the sum of the intensities of bands showing molecular weights at least twice this molecular weight. When the amount of aggregates following the addition of a control substance was defined as 100%, test substances that reduced the amount of aggregates by 5% or more were determined to be effective ingredients.

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 is expected to prevent and improve photoaging symptoms such as inhibiting abnormal elastin deposition, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles, sagging, and loss of firmness.

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 (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 rays and/or sunlight , and the indicator for the amount of denatured elastin is inhibition of the production of denatured elastin. 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.