JP2021156731A - Method for evaluating moisture retention capacity of horny layer - Google Patents

Abstract

To provide a method for evaluating moisture retention capacity of a horny layer without depending on a skin state or factors outside the horny layer in evaluation.SOLUTION: A method for evaluating moisture retention capacity of a horny layer, comprises: a step of evaluating the water retention capacity of horny layer, a barrier function of the horny layer, which is an element contributing to the water retention capacity of the horny layer, differentiation/proliferation capacity of an epidermal cell, and capacity to inhibit peroxynitrite activity, by using a mass of nitrated protein in the horny layer as an index; a step in which the mass of the nitrated protein is an amount of nitrated aromatic amino acids constituting the nitrated protein, an amount of nitrotyrosine, and a mass of nitrated protein in the hydrolyzed horny layer; and a step of evaluating the skin state of an arbitrary subject by using skin state data of a plurality of evaluated subjects as population data and comparing the population data with skin data of the subject.SELECTED DRAWING: None

Description

The present invention relates to a method for evaluating the water retention capacity of the stratum corneum.

The outermost layer of the skin, the stratum corneum, is the main protective barrier against evaporation of water, microorganisms and toxicants. In the stratum corneum, water is known to regulate physical properties such as material permeability and plasticity of the stratum corneum and the activity of enzymes that have an important effect on desquamation of stratum corneum cells on the surface of the skin (non-patented). Document 1), it is important for the skin that the stratum corneum retains water.

The ability of the stratum corneum to retain water in this way is called the stratum corneum water retention capacity. The formation of water-retaining components and the formation of a barrier accompanying the normal differentiation and proliferation of the epidermis are important for maintaining the water-retaining ability of the stratum corneum, but they deteriorate due to aging and skin troubles (Non-Patent Document 2). Research aimed at maintaining and improving the layer water retention capacity is being actively conducted. As an index of the water retention capacity of the stratum corneum, there are the amount of water in the stratum corneum and the amount of transepidermal water vapor.

As described above, the evaluation of the water retention capacity of the stratum corneum has been performed by measuring the water content of the stratum corneum. Various methods have been devised so far for measuring the water content of the stratum corneum. For example, paying attention to the fact that electrical properties such as electrical conductivity, capacitance, and dielectric constant of the stratum corneum change depending on the amount of water in the stratum corneum, the relative moisture content of the stratum corneum is measured from the measured values of the electrical properties. Devices for knowing the amount have been proposed (Patent Document 1, Non-Patent Document 3, Non-Patent Document 4). In addition, there are two methods for measuring the amount of transepidermal water evaporation, an open system and a closed system, both of which observe the water content in the space on the skin surface (Non-Patent Document 5).

These devices were useful in that the amount of water in the stratum corneum and the amount of transepidermal water evaporation could be known quickly and easily without damaging the skin. However, in the method of measuring the water content of the stratum corneum as described above, there is a large amount of blurring due to the skin condition at the time of measurement such as heat generation and sweating, and the measured value depends on the ingredients of the applied skin care cosmetics and the residue after cleaning. The measurement result is influenced by factors outside the stratum corneum, such as fluctuations in the temperature, and the directly measured amount of water content in the stratum corneum cannot be regarded as the ability to retain water in the stratum corneum. At that time, it is necessary to unify the measurement conditions of all the subjects and measure and compare them with the influence of external factors as small as possible by washing the face, acclimatization, etc. It wasn't the way to do it. In addition, the amount of percutaneous water evaporation is also easily affected by body temperature and skin deposits, and the water content in the stratum corneum is not the target of measurement in the first place. There wasn't. Therefore, the water retention capacity inherently stable in the subject's horny layer, that is, the true horny layer water content, which is not affected by temporary or transient changes in the water content of the horny layer due to sweating or application of cosmetics, can be easily obtained. There was a need for a way to know.

On the other hand, it is known that nitrated proteins are present in the skin, and the nitrated proteins are mainly produced by peroxynitrite. It is known that the production of nitrated protein in the stratum corneum causes yellowing of the skin (Patent Document 2), and peroxynitrite is involved in skin aging such as wrinkles, sagging, and pigmentation of the skin. It has been reported that (Patent Document 3). However, the relationship between peroxynitrite or nitrated protein in the skin and the ability to retain water in the stratum corneum has not been clarified at all.

Special Publication No. 63-19016 Japanese Unexamined Patent Publication No. 2017-181423 Japanese Unexamined Patent Publication No. 2002-265387

Rawlings A et al. , The effect of glycerol and Humidity on desmosome catabolism in stratum corneum. , Arch Dermatol Res, 287, 457-64, 1995. Hitoshi Masaki et al. , Changes observed in epidermal cells and stratum corneum cells with aging. , Journal of the Japanese Dermatological Association, 96 (3), 189-, 1986. Tagami Het al. , Assessment of the skin surface measurement in vivo by electrical measurement. , J Invest Dermatol, 75, 500-7, 1980. Kumiko Hashimoto et al. , Comparison of high-frequency conductivity measuring device and Conductor in measuring the water content of the stratum corneum. , Cosmetic Society Journal, 11, 252-8, 1987. Maruzen Co., Ltd. , Cosmetics Encyclopedia, edited by Japan Cosmetics Engineers Association. , 439, 2003

An object of the present invention is to provide a method for evaluating the water retention ability of the stratum corneum without being influenced by the skin condition at the time of evaluation and factors (moisture, etc.) outside the stratum corneum.

As a result of diligent studies to solve the above problems, the amount of water in the stratum corneum (water retention capacity of the stratum corneum) measured after sufficient washing, resting and acclimation has a positive correlation with the amount of nitrated protein in the stratum corneum. I found that there is.
As a result of further investigation of this phenomenon using cultured epidermal cells, it was found that the more the production of nitrated protein by peroxynitrite is promoted, the higher the cell differentiation / proliferation ability of epidermal cells and the barrier function of the stratum corneum. ..
Traditionally, protein nitration has been thought to have an adverse effect on the skin, but based on this finding, in skin with a large amount of nitrated protein in the stratum corneum, protein nitration by peroxynitrite during the process of differentiation Since it can be said that the reaction is active, the greater the amount of nitrated protein in the stratum corneum, in other words, the more the reaction of peroxynitrite to in vivo factors other than proteins is inhibited, the higher the ability of the stratum corneum to retain water. Can be evaluated as.
In addition, based on the discovery that the barrier function of the stratum corneum, the ability of epidermal cells to differentiate and proliferate, and the ability of epidermal cells to inhibit peroxynitrite activity are linked to the amount of nitrated protein, the amount of nitrated protein in the stratum corneum. We have come to the invention that the barrier function of the stratum corneum, the ability of epidermal cells to differentiate and proliferate, and the ability of epidermal cells to inhibit peroxynitrite activity can be grasped by grasping the above as an index.
In other words, by measuring the amount of nitrated protein in the stratum corneum, the ability to retain water in the stratum corneum, the barrier function of the stratum corneum, which is an element that contributes to the ability to retain water in the stratum corneum, the ability to differentiate and proliferate epidermal cells, and peroxynitrite. It was concluded that the activity-inhibiting ability could be evaluated, and the present invention was completed.
That is, the present invention has solved the above problems by using the amount of nitrated protein in the stratum corneum as an index.

By using the evaluation method of the present invention, the water retention ability of the stratum corneum can be evaluated without being affected by the skin condition at the time of evaluation.

FIG. 1 is a diagram showing the relationship between the water retention capacity of the cheek stratum corneum and the amount of nitrated protein. FIG. 2 is a diagram showing the amount of nitrated protein produced by adding peroxynitrite to cultured epidermal cells. FIG. 3 is a diagram showing altered gene expression by adding peroxynitrite to cultured epidermal cells. FIG. 4 is a diagram showing the amount of protein nitrated by peroxynitrite under different pH conditions. FIG. 5 is a diagram showing the amount of nitrated protein produced by adding peroxynitrite to cultured epidermal cells when the pH was brought close to acidity. FIG. 6 is a diagram comparing gene expression changed by adding peroxynitrite to cultured epidermal cells under different pH conditions. FIG. 7 is a diagram comparing the barrier functions of the stratum corneum changed by adding peroxynitrite to a three-dimensional skin model.

"Using the amount of nitrated protein in the stratum corneum as an index" of the present invention means that the abundance of nitrated protein in the stratum corneum is used as a criterion for determining the effect by using an arbitrary method.

The method for collecting the stratum corneum used for measuring the amount of nitrated protein in the present invention is not particularly limited, and the sample can be collected by, for example, tape stripping or rubbing the skin with cotton. Even if hair or the like other than the stratum corneum is included at the time of collection, there is no problem as long as it does not affect the measurement.

The amount of nitrated protein may be measured directly or indirectly. For example, the collected stratum corneum may be decomposed by hydrolysis treatment, enzymatic treatment, etc., and the amount of isolated nitro groups obtained or the amount of isolated nitrated amino acid residues may be measured as the amount of nitrated protein. can. For example, the nitrated amino acid includes compounds such as nitrotyrosine, 3,5-dinitrotyrosine, nitrotryptophan, nitrophenylalanine, and 2,4-dinitrophenylalanine, which can be amino acid residues constituting the nitrated protein. Further, the nitrated protein includes a protein containing a compound that can be the above-mentioned amino acid residue in the composition. Furthermore, the amount of biological material and the magnitude of biological reaction generated by the generation of nitrated protein can be quantified and grasped as the amount of nitrated protein.

The amount of nitrated protein can be measured by a known method. For example, it can be measured by absorptiometry, immunostaining, western blotting, radioimmunoassay, ELISA, liquid chromatography, gas chromatography, mass spectrometry, NMR method, autofluorescence and the like.

In the evaluation method in the present invention, it is determined that the larger the amount of nitrated protein, the higher the water retention ability of the stratum corneum, the barrier function of the stratum corneum, the differentiation / proliferation ability of epidermal cells, or the ability to inhibit peroxynitrite activity. For example, if the reference values such as the average value and median value of the subjects are set and the amount of nitrated protein is equal to or higher than the reference value, the water retention ability of the stratum corneum, the barrier function of the stratum corneum, the differentiation / proliferation ability of epidermal cells, and peroxy It can be evaluated that the ability to inhibit the activity of nitrite is high.

When quantifying the water retention capacity of the stratum corneum by measuring the amount of nitrated protein, the amount of nitrated protein of multiple persons and the water content of the stratum corneum as the water retention capacity of the stratum corneum are measured in advance as population data. By obtaining the regression curve from the scatter diagram, the water retention capacity of the stratum corneum can be obtained as a numerical value from the amount of nitrated protein of the subject. It is also possible to simply use the regression curve as a calibration curve and estimate the stratum corneum water retention capacity according to the measured amount of nitrated protein, or more accurately by performing quantile regression analysis. From the amount of nitrated protein, it is possible to determine the range of estimated water retention capacity of the stratum corneum and the probability of deviation from that range.

Generally, the water content of the stratum corneum is the amount of water contained in the stratum corneum. The water content of the stratum corneum increases when a lotion is applied or when it is wet, and decreases when the humidity is low, which is greatly affected by the environment and skin condition. In this way, the water content of the stratum corneum is the water content at the time of measuring the stratum corneum, and it has been affected by the external environment and temporary skin conditions, such as a high value when moisturizing or when it is wet. On the other hand, in the present invention, by substituting the water retention capacity of the stratum corneum with the amount of nitrated protein in the stratum corneum that correlates with this, the presence or absence of a coating material on the skin, the degree of rest of the body, and the like outside the stratum corneum can be grasped. It is possible to evaluate the original amount of water in the stratum corneum, that is, the ability of the stratum corneum to maintain the original water content, without being affected by factors.

In the present invention, the amount of nitrated protein in the stratum corneum, which is an index of the water retention capacity of the stratum corneum, increases or decreases only in a specific reaction with peroxynitrite or the like. It can be used as an index for evaluating the water retention capacity without being influenced by factors outside the stratum corneum that give a temporary fluctuation to the water content of the stratum corneum.

The peroxynitrite activity in the present invention refers to the oxidation / nitration reactivity of peroxynitrite with respect to in vivo molecules other than nucleic acids, lipids, and other proteins. In the present invention, the peroxynitrite activity of epidermal cells is the epidermis. It was clarified that it reduces the cell differentiation / proliferation ability, the barrier function of the stratum corneum, and the water retention ability of the stratum corneum.

In the evaluation of the ability to inhibit peroxynitrite activity in the present invention, the amount of nitrated protein is used as an index. The larger the amount of nitrated protein, the lower the reactivity of peroxynitrite with other than the protein, and as a result, the activity of peroxynitrite. It is judged that the protein can be inhibited.

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

<Example 1: Relationship between the amount of nitrated protein in the stratum corneum and the ability to retain water in the stratum corneum>
The subjects were 16 healthy men and women in their 20s and 40s living in Japan. Each subject kept the water content of the cheeks constant by washing the face with a face wash before measurement and resting in a room at 22 ° C. and 50% humidity for 20 minutes. The water content of the cheek stratum corneum of each subject was measured 5 times with SKICON. By making the water state of the measuring unit constant among the subjects, the water content of the stratum corneum can be regarded as the water retention capacity of the stratum corneum. The stratum corneum was collected from the test site using tape. At this time, the tape was applied to the cheeks and gently rubbed with a finger five times from the top of the tape to remove the tape. The sampling of the stratum corneum using tape was repeated 3 times at the same site. The tape with the stratum corneum was placed in a 1.5 mL tube. 1 mL of 6N hydrochloric acid was added, and the mixture was incubated at 37 ° C. for 48 hours. It was neutralized by mixing with sodium hydroxide to obtain a stratum corneum extract.

The total amount of protein (g / mL) in the stratum corneum extract was determined by the BCA method according to a conventional method. Further, the stratum corneum extract was subjected to Nitrotyrosine ELISA Kit (abcam), and the amount of nitrotyrosine (nmol / mL) was calculated. The amount of nitrotyrosine on each of the three tapes to which the stratum corneum was attached collected from the same subject was divided by the total amount of protein to determine the amount of nitrotyrosine (nmol / g) per fixed amount of protein on each tape. The amount of nitrotyrosine per protein amount of the three tapes was averaged for each subject, and the amount of nitrotyrosine per protein amount for each subject was determined.

Pearson's correlation test was performed on the relationship between the average of the measured water content of the stratum corneum and the amount of nitrotyrosine per protein amount, and it became clear that these have a positive correlation. (Fig. 1). The measurement of water content in the stratum corneum is evaluated as the ability to retain water in the stratum corneum by keeping the skin condition of all subjects constant by washing the face, acclimatizing, etc. when the subject is in a healthy state. It was shown that the higher the amount of nitrated protein, the greater the ability of the buccal stratum corneum to retain water. Further, FIG. 1 shows that the stratum corneum water retention ability can be indirectly grasped by measuring the amount of nitrated protein, and it can be said that the amount of the nitrated protein can be evaluated as the stratum corneum water retention ability.

<Example 2: Verification of action of peroxynitrite on epidermal cells>
Neonatal-derived normal human epidermal keratinocytes (KURABO) 5.0 × 104 Cells / mL dispersed in antibacterial agent-containing Humania KG2 (KURABO) were seeded at 500 μL each on a 24-well plate. Incubated at 37 ° C. under 5% CO2 for 3 days. 25 μL of peroxynitrite diluted to 2 mM (final concentration 100 μM), 1 mM (final concentration 50 μM: peroxynitrite +) and 0.5 mM (final concentration 25 μM) with 0.1 M sodium hydroxide was added. As a control (peroxynitrite-), 25 μL of 0.1 M sodium hydroxide was added. Incubated for 24 hr at 37 ° C. under 5% CO2.

50 μL of Extraction Buffer attached to the Nitro tyrosine ELISA Kit was added. After leaving it in the refrigerator for 30 minutes, the supernatant was collected and used as an extracted protein. The total protein amount (g / mL) of the extracted protein was determined by the BCA method according to a conventional method. Further, the extracted protein was subjected to a Nitrotyrosine ELISA Kit, and the amount of nitrotyrosine (nmol / mL) was calculated. The amount of nitrotyrosine (nmol / g) per constant amount of each extracted protein was determined. The amount of nitrotyrosine per fixed amount of protein was averaged for each condition, and the amount of nitrotyrosine per amount of protein for each condition was determined.

Total RNA was extracted from the cells of each well using the Total RNA Purification Kit (JenaBioscience). Using PrimeScriptTM RT Reagent Kit (TaKaRa), reverse transcription was performed to synthesize cDNA. Real-time PCR was performed, and the gene expression of p21, keratin 5 (KRT5), keratin 14 (KRT14), keratin 1 (KRT1), keratin 10 (KRT10), involucrin (INV), and loricrin (LOR) was determined by the comparative Ct method. The gene expression level of peroxynitrite + when peroxynitrite- was set to 1 was determined as "amount of change in expression by peroxynitrite 1". GAPDH was used as the housekeeping gene.

No nitrated protein production occurred in this method (Fig. 2). On the other hand, changes were observed in gene expression, and the addition of peroxynitrite increased the expression of p21 and decreased the expression of keratin 1, keratin 10, and loricrin (Fig. 3). From these results, it was found that peroxynitrite acts on substances in the body other than the protein under the condition that it does not produce nitrated protein, and suppresses cell proliferation and cell differentiation.

<Example 3: Verification of the effect of pH on nitrated protein production>
Neonatal-derived normal human epidermal keratinocytes 5.0 × 104 Cells / mL dispersed in antibacterial agent-containing Humania KG2 were seeded on a 24-well plate in an amount of 500 μL each. Incubated at 37 ° C. under 5% CO2 for 3 days. It was replaced with 100 μL of McIlvaine buffer prepared at pH 4.5, 5.5, 6.5 and 7.5. 10 μL of peroxynitrite diluted to 10 mM (final concentration 1 mM) was added. Incubated for 10 minutes at 37 ° C. under 5% CO2.

50 μL of Extraction Buffer attached to the Nitro tyrosine ELISA Kit was added. After leaving it in the refrigerator for 30 minutes, the supernatant was collected and used as an extracted protein. The total protein amount (g / mL) of the extracted protein was determined by the BCA method according to a conventional method. Further, the extracted protein was subjected to a Nitrotyrosine ELISA Kit, and the amount of nitrotyrosine (nmol / mL) was calculated. The amount of nitrotyrosine (nmol / g) per constant amount of each extracted protein was determined. The amount of nitrotyrosine per fixed amount of protein was averaged for each condition, and the amount of nitrotyrosine per amount of protein for each condition was determined.

More nitrotyrosine was produced at pH 4.5-6.5 compared to pH 7.5 (Fig. 4). From this result, peroxynitrite was more acidic than neutral. It was revealed to produce a nitrated protein.

<Example 4: Verification of action of peroxynitrite on epidermal cells 2>
Neonatal-derived normal human epidermal keratinocytes 5.0 × 104 Cells / mL dispersed in antibacterial agent-containing Humania KG2 were seeded on a 24-well plate in an amount of 500 μL each. Incubated at 37 ° C. under 5% CO2 for 3 days. 25 μL of 0.2 M hydrochloric acid (final concentration 10 mM) or distilled water was added. 25 μL of peroxynitrite diluted to 1 mM (final concentration 50 μM) was added (peroxynitrite +). As a control, 25 μL of 0.1 M sodium hydroxide was added (peroxynitrite-). Incubated for 24 hr at 37 ° C. under 5% CO2.

50 μL of Extraction Buffer attached to the Nitro tyrosine ELISA Kit was added. After leaving it in the refrigerator for 30 minutes, the supernatant was collected and used as an extracted protein. The total protein amount (g / mL) of the extracted protein was determined by the BCA method according to a conventional method. Further, the extracted protein was subjected to a Nitrotyrosine ELISA Kit, and the amount of nitrotyrosine (nmol / mL) was calculated. The amount of nitrotyrosine (nmol / g) per constant amount of each extracted protein was determined. The amount of nitrotyrosine per fixed amount of protein was averaged for each condition, and the amount of nitrotyrosine per amount of protein for each condition was determined.

Total RNA was extracted from the cells of each well using the Total RNA Purification Kit. Using the PrimeScriptTM RT Reagent Kit, reverse transcription was performed to synthesize cDNA. Real-time PCR was performed, and the gene expression of p21, keratin 5 (KRT5), keratin 14 (KRT14), keratin 1 (KRT1), keratin 10 (KRT10), involucrin (INV), and loricrin (LOR) was determined by the comparative Ct method. The gene expression level of peroxynitrite + when peroxynitrite − was set to 1 was determined as “amount of change in expression due to peroxynitrite 2”. When the "expression change amount 1 due to peroxynitrite" shown in Example 2 was set to 1, the expression increase rate of the "expression change amount 2 due to peroxynitrite" in this example was determined and shown in FIG. GAPDH was used as the housekeeping gene.

By adding peroxynitrite after adding 0.2 M hydrochloric acid to the cultured epidermal cells, nitrated protein was produced about 1.36 times as much as that of the control (Fig. 5). Furthermore, due to changes in gene expression, the expression of p21, keratin 5, and keratin 14 was decreased, and the expression of involucrin and loricrin was increased, as compared with the condition in which the nitrate protein was not produced (Fig. 6). From these results, it was found that the suppression of cell proliferation and differentiation by peroxynitrite was alleviated under the condition that nitrated protein was easily produced in epidermal cells. It is considered that this is because peroxynitrite reacts with proteins to reduce the oxidation / nitration reaction to other substances in the body. In addition, since the nitrated protein is not easily decomposed in the living body, it can be judged that the amount of the nitrated protein in the stratum corneum indicates the peroxynitrite activity inhibitory power of the entire epidermis. Furthermore, when considered together with Example 1, in skin with a large amount of stratum corneum nitrated protein, peroxynitrite generated in the process of differentiation is more efficiently inhibited by inhibiting the activity through protein nitration. It is considered that the suppression of cell differentiation and proliferation is suppressed, and as a result, the ability to retain water in the stratum corneum is increased.

<Example 5: Verification of the action of peroxynitrite on the stratum corneum barrier function>
The three-dimensional skin model LabCite EPI-MODEL 6D (J-TEC) was incubated at 37 ° C. under 5% CO2 for 7 days. During the 7-day culture period, the following treatments were performed 3 times. The medium was replaced with 25 μL of 0.2 M hydrochloric acid (HCl +) or distilled water. 25 μL of peroxynitrite / 0.1 M sodium hydroxide diluted to 1 mM (final concentration 50 μM) was added (peroxynitrite +). As a control, 25 μL of 0.1 M sodium hydroxide was added (control). Incubation was carried out at 37 ° C. under 5% CO2 for 1 hour and replaced with normal medium.

After completion of the 7-day incubation, 200 μL of a 0.1% Fluorescein sodium salt (FLUKA) aqueous solution was added, and the mixture was incubated for 2 hours at room temperature. Separate the tissue with a scalpel and use Tissue Tech O.D. C. T. It was embedded in a compound (Sakura Finetech Japan) and frozen. Sections 5 μm thick were prepared using a cryostat (Leica Microsystems) and photographed under a fluorescence microscope.

The addition of peroxynitrite promoted the permeation of the fluorescent substance, while the addition of peroxynitrite to the medium prepared by adding hydrochloric acid under weakly acidic conditions suppressed the permeation (Fig. 7). From this result, peroxynitrite lowers the barrier function of the stratum corneum and promotes the penetration of exogenous substances into the stratum corneum, but the weakly acidic conditions improve the reactivity of peroxynitrite with proteins. , It was clarified that the decrease in the barrier function of the stratum corneum can be suppressed by inhibiting the reaction with other in vivo factors.

<Example 6: Evaluation of the water retention capacity of the horny layer by determining the amount of nitrated protein in the horny layer>
The subjects were 16 healthy men and women in their 20s and 40s living in Japan. Each subject kept the water content of the cheeks constant by washing the face with a face wash before measurement and resting in a room at 22 ° C. and 50% humidity for 20 minutes. The water content of the cheek stratum corneum of each subject was measured 5 times with SKICON. The stratum corneum was collected from the test site using tape. At this time, the tape was applied to the cheeks and gently rubbed with a finger five times from the top of the tape to remove the tape. The sampling of the stratum corneum using tape was repeated 3 times at the same site. The tape with the stratum corneum was placed in a 1.5 mL tube. 1 mL of 6N hydrochloric acid was added, and the mixture was incubated at 37 ° C. for 48 hours. It was neutralized by mixing with sodium hydroxide to obtain a stratum corneum extract.

The total amount of protein (g / mL) in the stratum corneum extract was determined by the BCA method according to a conventional method. Further, the stratum corneum extract was subjected to Nitrotyrosine ELISA Kit (abcam), and the amount of nitrotyrosine (nmol / mL) was calculated. The amount of nitrotyrosine on each of the three tapes to which the stratum corneum was attached collected from the same subject was divided by the total amount of protein to determine the amount of nitrotyrosine (nmol / g) per fixed amount of protein on each tape. The amount of nitrotyrosine per protein amount of the three tapes was averaged for each subject, and the amount of nitrotyrosine per protein amount for each subject was determined.

The equation of the regression curve was obtained from the scatter plot of the amount of nitrotyrosine (nmol / g) and the water retention capacity of the stratum corneum (μS). The difference between the estimated water retention capacity of the stratum corneum obtained by applying the amount of nitrotyrosine to the equation of the regression curve and the actual water retention capacity of the stratum corneum was calculated.

In 81.25% of the subjects, the water retention capacity of the stratum corneum was an estimated value of ± 71 μS of the regression curve. From this result, for example, when the amount of nitrotyrosine in the collected stratum corneum is 1000 nmol / g, the estimated stratum corneum water retention capacity is 185 μS when applied to the regression curve, and the stratum corneum water retention capacity is 114 to 81.25%. It is 256 μS.

Claims (8)

A method for evaluating the water retention capacity of the stratum corneum using the amount of nitrated protein in the stratum corneum as an index. A method for evaluating the barrier function of the stratum corneum using the amount of nitrated protein in the stratum corneum as an index. A method for evaluating the differentiation / proliferation ability of epidermal cells using the amount of nitrated protein in the stratum corneum as an index. A method for evaluating the ability of epidermal cells to inhibit peroxynitrite activity using the amount of nitrated protein in the stratum corneum as an index. The evaluation method according to any one of claims 1 to 4, wherein the amount of nitrated protein is the amount of nitrated aromatic amino acids constituting the nitrated protein. The evaluation method according to any one of claims 1 to 5, wherein the amount of nitrated protein is the amount of nitrotyrosine constituting the nitrated protein. The evaluation method according to any one of claims 1 to 6, wherein the amount of nitrated protein is the amount of hydrolyzed stratum corneum. The skin condition data of a plurality of subjects evaluated in advance by the evaluation method according to any one of claims 1 to 7 is used as population data, and the skin of the subject is compared with the skin data of any subject. How to evaluate the condition.

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