JP2020193948A - Horny layer analysis method - Google Patents

Abstract

To provide a method for acquiring an infrared absorption spectrum of a horny layer stably with good reproducibility from an adhesive tape after peeling of a horny layer cell.SOLUTION: A method for measuring an infrared absorption spectrum of a horny layer includes: peeling a horny layer on the skin surface with a tape having a silicone adhesive layer; acquiring an infrared absorption spectrum obtained through ATR-IR measurement of an adhesive face of the tape, and an infrared absorption spectrum obtained through the ATR-IR measurement of an adhesive face of an unused tape having a silicone adhesive layer; and acquiring the spectrum of the horny layer from the difference spectrum between both spectra. The method includes calculating the estimated contribution of a water signal in all the wave number regions of the infrared absorption spectrum of the horny layer, from the estimated contribution of the water signal in a region of the horny layer having an infrared absorption spectrum of 3000 to 4000 cm-1, and subtracting the estimated contribution from the infrared absorption spectrum of the horny layer to acquire the spectrum.SELECTED DRAWING: None

Description

The present invention relates to the keratinized state of the stratum corneum, changes in the composition of stratum corneum proteins, the amount of intercellular lipids in the stratum corneum, the packing property of intercellular lipids in the stratum corneum, the amount of natural moisturizing factor (NMF) in the stratum corneum, and the composition of the stratum corneum. , The method for evaluating stratum corneum deposits.

The tissues that make up the outermost layer of the skin are called the stratum corneum. It is known that the amount and chemical state of the components constituting the stratum corneum differ depending on the difference in skin condition such as rough skin and dryness. In addition, since the stratum corneum is the main site of action of many skin care agents and cosmetics, grasping the stratum corneum constituents, the amount of stratum corneum deposits, and changes in the stratum corneum condition can be used to grasp the skin condition and skin care effect. It is useful for verification and so on.

As a method for analyzing the stratum corneum constituents on the surface layer of the stratum corneum on the skin, for example, a probe is pressed against the skin and the infrared absorption spectrum of the stratum corneum is measured by the total reflection infrared absorption method (ATR-IR method). A method for evaluating the difference in the secondary structure of a protein based on the difference in the peak shape of the amide I absorption band in the obtained spectrum is known (Patent Document 1). However, in such a method, since the absorption band of the OH angular vibration of water is superimposed on the absorption band of amide I, it is necessary to compare the peak shapes of amide I between skins having the same amount of water.

Further, there is also known a method of exfoliating the stratum corneum cells on the skin surface with an adhesive tape having a silicone-based adhesive layer and measuring the infrared absorption spectrum of the adhesive surface (Patent Document 2). In this technique, the infrared absorption spectrum of the adhesive surface of the adhesive tape before exfoliation of the stratum corneum cells is subtracted from the infrared absorption spectrum of the adhesive surface of the adhesive tape after exfoliation of the stratum corneum cells after multiplying by a coefficient. By calculating the spectral components corresponding to the exfoliated stratum corneum (hereinafter, also referred to as "exfoliated stratum corneum infrared absorption spectrum"), chemical information on the stratum corneum constituents and the stratum corneum deposits on the stratum corneum surface layer is acquired. Is. According to this technology, the infrared absorption spectrum can be obtained in the absence of water supply from the inside of the skin, so it is affected by fluctuations in water content derived from the living body compared to the case where the skin surface is directly measured by ATR-IR. There is a feature that it is possible to obtain a change in the amide I absorption band, for example, an area ratio (α / β value) of a peak in which an α-helix and a random coil, which are indicators of stratum corneum properties, are superimposed and a peak in a β sheet.

Japanese Patent No. 5756370 Japanese Unexamined Patent Publication No. 2018-105783

The present invention relates to providing a method for stably obtaining an infrared absorption spectrum of the stratum corneum from an adhesive tape after exfoliation of stratum corneum cells with good reproducibility.

In the process of examining a method for more accurately analyzing the state of the stratum corneum from the infrared absorption spectrum of the adhesive surface of the adhesive tape after exfoliation of the stratum corneum cells, the present inventors measured the measured values according to the environmental humidity. I found that it fluctuates. Specifically, the α / β value obtained from the peak of the amide I absorption band negatively correlates with the measured environmental humidity (see FIG. 4), and the evaluation of the stratum corneum state using this is affected by the measured environmental humidity. I found that there was a possibility. Then, based on the spectrum shape in the region of 3000 to 4000 cm ^-1 of the separated stratum infrared absorption spectrum, the signal component derived from water in the spectrum of the full wave number region of the infrared absorption spectrum is estimated, and the stratum corneum infrared absorption spectrum is estimated. By removing the contributing component of the water signal and correcting it to a completely dry state, the separated stratum corneum infrared absorption spectrum can be accurately obtained, and this can be used to stabilize the stratum corneum state. We succeeded in evaluating with good reproducibility.

That is, the present invention relates to the following 1) to 2).
1) An infrared absorption spectrum obtained by attaching a stratum corneum to the adhesive surface of a tape having a silicone adhesive layer and measuring the adhesive surface of the tape by ATR-IR, and an adhesive surface of an unused tape having a silicone adhesive layer. This is a method for measuring the infrared absorption spectrum of the stratum corneum, in which the infrared absorption spectrum obtained by ATR-IR measurement is obtained and the spectrum of the stratum corneum is acquired from the difference spectrum between the two. From the estimated contribution of the water signal in the region of 3000 to 4000 cm ^-1 , the estimated contribution of the water signal in the full frequency range of the infrared absorption spectrum of the stratum corneum is calculated and subtracted from the infrared absorption spectrum of the stratum corneum. How to get the spectrum by.
2) From the signal intensity and peak shape of each peak in the infrared absorption spectrum of the stratum corneum obtained by the method 1), the components constituting the stratum corneum, the properties of the stratum corneum, the amount of deposits on the surface of the stratum corneum, and A stratum corneum evaluation method for evaluating any one or more of changes in the chemical state of the stratum corneum.

According to the present invention, the influence of humidity in the measurement environment is not taken into consideration, that is, no large-scale air conditioning equipment is required, and no operation such as equilibrating the stratum corneum release tape with the environmental humidity is required. The infrared absorption spectrum of the stratum corneum can be obtained with good reproducibility. Therefore, compared to the conventional method, it is easier and more accurate to keratinize the stratum corneum, change the composition of the stratum corneum protein, the amount of intercellular lipid in the stratum corneum, the packing property of the intercellular lipid in the stratum corneum, and the natural nature of the stratum corneum. The amount of moisturizing factor (NMF), stratum corneum composition, and stratum corneum deposits can be evaluated.

Infrared absorption spectrum of human stratum corneum sheet and water. Infrared absorption spectrum of human stratum corneum sheet and water (around amide I). Infrared absorption spectrum of human stratum corneum sheet and water (high frequency range). Infrared absorption spectrum of dry stratum corneum and infrared absorption spectrum of water after tape signal correction. 73% R. H. Infrared absorption spectrum of the stratum corneum in. 73% R. H. Infrared absorption spectrum of the stratum corneum (enlarged view of the region near amide I in FIG. 3a). Relationship between α / β value and measured environmental humidity (without water signal correction). Relationship between α / β value and measured environmental humidity (with water signal correction). Effect of humidity on α / β values (without water signal correction). a1: Forearm (right), a2: Forearm (left), c1: Cheek (right), c2: Cheek (left). Effect of humidity of α / β value (with water signal correction). a1: Forearm (right), a2: Forearm (left), c1: Cheek (right), c2: Cheek (left).

As the ATR-IR spectroscope used for acquiring the infrared absorption spectrum of the present invention, a normal FT-IR and an ATR unit that can be connected to the ordinary FT-IR may be used. Further, the ATR unit may be an optical fiber type probe. Further, a system in which the ATR unit and FTIR are integrated may be used. The internal reflection element is not particularly limited, but for example, germanium, diamond, ZnSe, Si, and PIR (made of silver chloride) can be used. The number of reflections and the angle of incidence are not particularly limited as long as the total reflection conditions can be maintained. Wavenumber resolution needs to be measured in the range of 0.5 to 32 cm ^-1 , but it is preferable to measure in the range of 2 to 8 cm ^-1 where both sensitivity and S / N can be achieved.

As for the stratum corneum release tape, a silicone-based adhesive may be used for the adhesive layer. There are no particular restrictions on the material of the base film. Since the tape having a silicone adhesive layer is generally marketed for applications requiring heat resistance and chemical resistance, it is easy to obtain a tape in which Teflon (registered trademark) or aluminum is used for the base film. Since these base films are also strong, they can be easily attached to and removed from the skin, and are suitable for the present invention. However, there is no problem with base films other than Teflon (registered trademark) and aluminum.
Specifically, the tape having a silicone-based adhesive layer can be purchased from AS ONE Corporation described in Examples, 3M Company, Nitto Denko Corporation, Okado Corporation, Teraoka Seisakusho Co., Ltd., and the like. In addition, a tape having a silicone-based adhesive layer can be prepared by applying a silicone-based adhesive commercially available from Toray Dow Corning Co., Ltd., Shin-Etsu Chemical Co., Ltd., Reditex Co., Ltd., etc. to various films and non-woven fabrics. Can be done.

In the present invention, an infrared absorption spectrum (absorption spectrum A) obtained by adhering a stratum corneum to the adhesive surface of a tape having a silicone adhesive layer and measuring the adhesive surface of the tape by ATR-IR and a silicone adhesive layer are used. An infrared absorption spectrum (absorption spectrum B) obtained by ATR-IR measurement of the adhesive surface of the unused tape is obtained, and the spectrum of the stratum corneum is obtained from the difference spectrum between the two. From the estimated contribution of the water signal in the region of 3000 to 4000 cm ^-1 of the infrared absorption spectrum of the layer, the estimated contribution of the water signal in the full frequency range of the infrared absorption spectrum of the stratum corneum is calculated, and the estimated contribution of the water signal in the infrared absorption spectrum of the stratum corneum is calculated. Subtraction is performed from the infrared absorption spectrum.
As a method of adhering the stratum corneum to the silicone adhesive surface, the silicone adhesive surface may be directly pressed against the skin surface to adhere the stratum corneum, or another tape having an adhesive layer may be pressed against the skin surface for collection. The stratum corneum may be transferred to the silicone adhesive surface.
The procedure for measuring the infrared absorption spectrum of the stratum corneum of the present invention will be described below.

First, the infrared absorption spectrum (absorption spectrum B) of the tape is acquired by the following steps 1) to 3).
1) The power spectrum of air is measured in a state where air is in contact with the internal reflection element portion of the ATR-IR spectroscope.
2) The adhesive surface of an unused tape having a silicone adhesive layer is brought into contact with the internal reflection element portion of the ATR-IR spectroscope, and the power spectrum of the tape is measured.
3) The absorption spectrum (absorbance display) of the tape is obtained by dividing the power spectrum of the tape by the power spectrum of air and converting the transmittance into absorbance.

Next, the absorption spectrum (absorption spectrum A) of the adhesive surface of the tape to which the stratum corneum cells are attached is obtained by the following steps 4) to 6).
4) After the adhesive surface of the tape having the silicone adhesive layer is brought into contact with the skin, the tape is peeled off and the stratum corneum cells are attached to the adhesive surface of the tape.
5) The adhesive surface of the tape to which the stratum corneum cells are attached is attached to the internal reflection element portion of the ATR-IR spectroscope, and the power spectrum of [corneal layer + tape] is measured.
6) The absorption spectrum (absorbance display) of [horny layer + tape] is obtained by dividing the power spectrum of [horny layer + tape] by the power spectrum of air and converting the transmittance into absorbance.

Next, a difference spectrum between the absorption spectrum A and the absorption spectrum B is created. Specifically, the major absorption band derived from the silicone adhesive layer, the absorption band of the absorption band or 1000~1100cm near ^-1 around 1260 cm ^-1, either or peak intensities from both, matches As described above, the absorption spectrum A or the absorption spectrum B is multiplied by a constant value and then subtracted.
That is, the difference spectrum is created by the following steps 7) to 8) (see the reference example below).
7) appears in [corneum + Tapes absorption spectrum (absorbance Display), a major absorption band derived from the silicone adhesive layer, the absorption band near 1260 cm ^-1 or 1000～1100Cm ^-1 near the absorption band of A coefficient p that multiplies the absorption spectrum (absorbance display) of the tape by a constant so that the peak intensities of the absorption spectrum (absorbance display) of the tape corresponding to the peaks derived from either or both of the above are matched. To decide.
8) Calculate the absorption spectrum of the stratum corneum (exfoliated stratum corneum infrared absorption spectrum) by subtracting the spectrum obtained by multiplying the absorption spectrum of the tape (absorbance display) by p from the absorption spectrum (absorbance display) of [corneer layer + tape]. To do.

Next, the estimated contribution of the water signal in the infrared absorption spectrum full wave number region of the stratum corneum was calculated from the estimated contribution of the water signal in the region of 3000 to 4000 cm ^-1 of the infrared absorption spectrum of the exfoliated stratum corneum. Subtraction is performed from the infrared absorption spectrum of the stratum corneum.
The infrared absorption spectrum S _SC (ω) of the stratum corneum is a superposition of the infrared absorption spectrum S _SC-Dry (ω) of the completely dried stratum corneum and the infrared absorption spectrum S _Water (ω) of water. , Can be expressed approximately as in Equation 1 below. In the equation, q and r are coefficients and ω is the wave number.
S _SC (ω) = qS _SC-Dry (ω) + rS _Water (ω) (Equation 1)

The above equation is applied to the peak of the absorption band of 3000 to 4000 cm ^-1 to calculate the coefficients q and r, and the contribution of the water signal is estimated. The least squares method or the maximum likelihood method can be used to calculate the coefficients q and r that best reproduce the equation 1.
Then, as shown in Equation 2 below, the infrared absorption spectrum S _SC-Dry-cal (ω) of the virtual dry stratum corneum is obtained by subtracting the estimated contribution of the water signal in the full wavenumber region of the spectrum. Can be calculated.
S _SC-Dry-cal (ω) = S _SC (ω) -rS _Water (ω) (Equation 2)

Using the infrared absorption spectrum of the dried stratum corneum thus obtained, the composition change of the stratum corneum protein and the evaluation of the properties of the stratum corneum based on the peak shape analysis of the absorption band derived from amide I, the methyl group and the methylene group Amount and packing property of intercellular lipids in the stratum corneum based on analysis of peak intensity and peak shape of the absorption band based on the angular vibration of the stratum corneum, evaluation of the properties of the stratum corneum, peak intensity and peak of the absorption band based on the expansion and contraction vibration of the alkyl chain. Amount and packing property of stratum corneum intercellular lipids based on shape analysis, evaluation of stratum corneum properties, amount of natural moisturizing factor (NMF) in stratum corneum based on analysis of peak intensity of absorption band based on dissociated carboxy group , The properties of the stratum corneum can be evaluated by, for example, the following method.

The peak of the amide I may turn structure around 1668 cm ^-1, helical structure and random coil structure to α around 1649 cm ^-1, it is known that the signal of β-sheet structure in the vicinity of 1629cm ^-1 is superimposed .. By analyzing the peak shape of amide I, the proportion of these structures can be estimated, and changes in the composition and structure of the stratum corneum protein can be evaluated. For example, it is possible to evaluate the properties of the stratum corneum by dividing the peak on which the α-helix and the random coil are superimposed and the peak of the β-sheet from the peak of the amide I absorption band and obtaining the area ratio (α / β value) of both peaks. Is.

The peak of the deformation vibration of methylene, the signal of the orthorhombic structure of intercellular lipids in the vicinity of 1463cm ^-1 and near 1473cm ^-1, in the vicinity 1468Cm ^-1 signal of hexagonal structure of intercellular lipids are superimposed It has been known. By analyzing the peak shape of the angular vibration of methylene and estimating the ratio of these structures, it is possible to evaluate the change in the packing structure of intercellular lipids. It is considered that the more ortholonbic structures, the higher the barrier ability of the stratum corneum, so this analysis method can be used as an index of the stratum corneum barrier.

The peaks of expansion and contraction vibration of methylene appear around 2850 cm ^{-1 and} around 2920 cm ^-1 . It is known that these peak positions are shifted in high frequency by loosening the packing of the alkyl chain. Therefore, by reading these peak positions, changes in the packing structure of intercellular lipids can be evaluated.

There is no particular limitation on the analysis method of each peak in the infrared absorption spectrum of the stratum corneum. For example, as described in Patent Document 3, when a specific function such as a Gaussian function is applied to a known peak position and the infrared absorption spectrum of the stratum corneum is expressed by superimposing these functions, the area of each peak ( Signal strength) and width (shape) can be calculated.

To analyze changes in protein composition from the shape of the peak of amide I, calculate the first derivative spectrum and the second derivative spectrum, and use the intensity ratio at a specific wave number in the first derivative spectrum or the second derivative spectrum as an index. It can also be used.

In order to analyze the packing state of intercellular lipids from the shape of the peak of the angular vibration of the methyl group, a second derivative spectrum can be calculated and the intensity ratio in the second derivative spectrum can be used as an index.
In order to analyze the packing state of intercellular lipids from the shape of the peak of the expansion and contraction vibration of the methylene group, the peak top position of the peak in the infrared absorption spectrum of the stratum corneum may be read. At this time, the first-order differential spectrum may be calculated and the wave number position where the differential coefficient becomes zero may be read. This index can also be used as an index of the stratum corneum barrier.

The amount of natural moisturizing factor (NMF), the appearance of the spectral shape of the region of 1400～1800Cm ^-1, to then approximated by superposition of a plurality of functions such as Gaussian functions, in the vicinity of 1602 cm ^-1 and 1574 ^-1 dissociation It can be evaluated by estimating the signal strength (peak area, peak height) of the function corresponding to the type carboxy group.

The amount of stratum corneum deposits can be evaluated from the peak signal intensity (peak area, peak height) characteristic of the substance. Specifically, for example, when estimating the amount of fatty acid scum (fatty acid metal salt) corresponding to soap scum, the signal intensity (peak area, peak height) of the peak near 1575 cm- ¹ may be estimated.

From the correlation between the above various structural changes or various indicators and various skin properties (normal skin, rough skin, dry skin, various dermatitis, etc.), it is possible to evaluate the skin properties based on the various structural changes and indicators. ..

Reference Example (1) Calculation of α / β value from infrared absorption spectrum of tape peeling stratum corneum According to Japanese Patent Application Laid-Open No. 2018-105783, α correlating with skin properties from infrared absorption spectrum of tape peeling stratum corneum The / β value (the ratio of the peak area where the α-helix and the random coil are superimposed to the peak area of the β sheet) was calculated. The procedure is as follows.
1) The stratum corneum is collected from the human cheek using a tape having a silicone-based adhesive layer (AS ONE (R) tape, AS ONE Corporation).
2) The adhesive surface of the tape after peeling the stratum corneum is attached to the measurement surface of the ATR unit (iD5 ATR) attached to the FT-IR device (NICOLE iS5, Thermo Fisher Scientific), and ATR-IR measurement is performed. Let this infrared absorption spectrum be S _{tape + SC} (ω). Here, ω corresponds to the wave number.
3) In the same manner as in 2), perform ATR-IR measurement of the blank tape adhesive surface before peeling the stratum corneum. Let this infrared absorption spectrum be S _tape (ω).
4) Based on the following equation 3, the coefficient p at which the peak near 1260 cm ^-1, which is characteristic of the silicone adhesive layer, disappears most often is calculated by the least squares method, and the infrared absorption spectrum S _SC (ω) of only the stratum corneum is used. ) Is calculated.
S _SC (ω) = S _{tape + SC} (ω) -pS _tape (ω) (Equation 3)
5) Apply 23 Gaussian functions expressed by the following equations to S _SC (ω), and find the combination of coefficients k (N) that best reproduces S _SC (ω) by the least squares method. The approximate spectrum S _SC-cal (ω) of the stratum corneum is calculated. Table 1 shows the parameters and attribution of each Gaussian function at this time.

Here the peak area of the Gaussian function of 1649 cm ^-1, is defined as the alpha / beta value divided by the peak area of the Gaussian function of 1629cm ^-1.

Example 1 Examination of Moisture Correction Method In order to estimate the contribution of water to the infrared absorption spectrum of the stratum corneum, water, a human stratum corneum sheet, and a humidity control environment (0% RH, 73% RH). The infrared absorption spectrum of the tape-peeled stratum corneum was measured in. The device, measurement conditions, sample preparation method, measurement results, and water signal correction method based on the measurement results used in this measurement are described below.

(1) FT-IR device A fronter (Perkin-Elmer, TGS detector) with a universal ATR unit (diamond, 45 °) or Nicole iS5 (Thermo Fisher Scientific) with an ATR unit (iD5 ATR) attached. Using.

(2) Measurement conditions Wavenumber resolution 4 cm ^-1 , integration 4 times

(3) Sample preparation and measurement method 1) Water: Milli-Q water was used for ATR-IR measurement.
2) Human stratum corneum sheet:
It was prepared from commercially available human excised skin (Transkin (R), BIOPREDIC International). It was subjected to ATR-IR measurement in a laboratory environment.
3) Tape peeling stratum corneum:
A tape having a silicone-based adhesive layer (AS ONE (R) tape, AS ONE Corporation) was attached to the inner part of the human forearm, and the stratum corneum was collected by peeling it off. The tape peeling stratum corneum was humidity-controlled using the following two conditions and subjected to ATR-IR measurement.
i) 0% R. H. Condition: Leave in a sample bottle with nitrogen gas flowing for 1 hour.
ii) 73% R. H. Conditions: Leave overnight in a desiccator (actual measurement 25 ° C., 73% RH) adjusted with a saturated NaCl salt solution.

(4) Measurement Results The infrared absorption spectra of the human stratum corneum sheet and water are shown in FIG. 1a (entire area), FIG. 1b (around amide I region) and FIG. 1c (high wave number region).
From FIG. 1b, it can be seen that the presence of water affects the peak shape of amide I because water has absorption in the region of the stratum corneum that overlaps with amide I. Further, from FIG. 1c, it can be seen that the peak shape in the high wave frequency region (2600 to 4000 cm ^-1 ) also changes depending on the water content in the stratum corneum.
FIG. 2 shows an infrared absorption spectrum of a nitrogen flow-dried stratum corneum after tape signal correction and an infrared absorption spectrum of water calculated from Equation 3 of the above reference example. In each case, the absorbance is standardized to 0 to ^{1 in} the range of 1000 to 4000 cm ^-1 and displayed.
By comparing the regions of 3000 to 4000 cm- ¹ in FIG. 1a and FIG. 2, it can be seen that the stratum corneum sheet measured in the laboratory environment contains a certain amount of water.

(5) Water signal correction method Here, the infrared absorption spectrum _SSC (ω) of the stratum corneum obtained by removing the tape signal from the infrared absorption spectrum of the tape-peeled stratum corneum is a completely dry angle. By superimposing the infrared absorption spectrum S _SC-Dry (ω) of the layer and the infrared absorption spectrum S _Water (ω) of water, it can be approximately expressed as in Equation 1.
S _SC (ω) = qS _SC-Dry (ω) + rS _Water (ω) (Equation 1)
Here, q and r are coefficients, and ω is a wave number. In order to estimate the contribution of the water signal, Equation 1 was applied to the region of 3000 to 4000 cm ^{-1 and} the coefficients q and r were calculated by the method of least squares. Next, it was decided to subtract the water signal in Equation 1 to calculate the infrared absorption spectrum S _SC-Dry-cal (ω) of the virtual dry stratum corneum.
S _SC-Dry-cal (ω) = S _SC (ω) -rS _Water (ω) (Equation 2)
FIG. 3a shows an infrared absorption spectrum (solid line, after removing the tape signal) of the tape peeled stratum layer whose humidity was adjusted at 73%, and an infrared absorption spectrum (broken line) of the contribution of the water signal calculated from Equation 1. The infrared absorption spectrum (dotted line) after subtraction of the water signal calculated by Equation 2 is shown. FIG. 3b shows an enlarged display of the area near the amide I in FIG. 3a. From FIG. 3b, it can be seen that the peak shape of amide I changes slightly by estimating and subtracting the contribution of the water signal. The infrared absorption spectrum corrected in this way is applied to the infrared absorption spectrum analysis method represented by Equation 2 to calculate the α / β value.

Test Example 1 Analysis of the amide I absorption band of the stratum corneum measured in different seasons From January to December, a total of 1113 people were treated with adhesive tape from the face and cheeks in a room equipped with general air conditioning equipment. The stratum corneum was collected from the tape and ATR-IR measurement of the tape was performed. The measurement was performed using an infrared spectroscope iS5 (Thermo Fisher SCIENTIFIC) equipped with an ATR unit (iD5 ATR) under the conditions of resolution: 4 cm ^-1 and number of scans: 4. After removing the signal of the adhesive tape, the acquired infrared absorption spectrum shows the ratio of the signal components of the amide I absorption band of 1649 cm ^-1 and 1629 cm ^-1 (α / β) with or without correction of the infrared absorption spectrum according to Example 1. ) Was calculated, and the effect of correcting the infrared absorption spectrum according to Example 1 was verified.

Without spectral correction, the α / β value negatively correlates with the measured environmental humidity, and it was found that the value may be affected by the humidity (Fig. 4). On the other hand, in the analysis of the spectrum obtained by correcting the signal of water according to Example 1, the α / β value becomes infinitely small in dependence on the environmental humidity (FIG. 5), and the correction of the infrared absorption spectrum of the present invention is applied. It was found that this makes it possible to evaluate the amide I absorption band from which the influence of humidity is removed.

Test Example 2 Analysis of the amide I absorption band of the stratum corneum measured under different acclimation humidity conditions The stratum corneum was collected with adhesive tape from a total of 4 locations, one on each of the left and right cheeks and the left and right forearms of one male. Then, ATR-IR measurement of the tape was performed (forearm (right): a1, forearm (left): a2, cheek (right): c1, cheek (left): c2). For the measurement, an infrared spectroscope iS5 (Thermo Fisher SCIENTIFIC) equipped with an ATR unit (iD5 ATR) was used, and the resolution was 4 cm ^-1 , the number of scans was 4, the environment was room temperature 26 ° C, and the humidity was 50% RH. It was done under the conditions in the vicinity.

The measurement was carried out according to the following procedure. First, the test site was washed, acclimated at room temperature (around 50% RH), and the exfoliated stratum corneum was measured by ATR-IR to obtain an infrared absorption spectrum of the stratum corneum acclimated to room temperature. Next, those stratum corneum were transferred to a dry desiccator (actual measurement 25 ° C., 14% RH) and stored for about 1 hour, then taken out from the desiccator and immediately subjected to ATR-IR measurement, and the stratum corneum adapted to the dry environment. Infrared absorption spectrum was obtained. Then, the same stratum corneum is transferred to a desiccator (actually measured 25 ° C., 73% RH) adjusted with a saturated sodium chloride solution, stored overnight, taken out from the desiccator, and immediately subjected to ATR-IR measurement to bring it into a moist environment. An infrared absorption spectrum of the conditioned stratum corneum was obtained. Each sample was repeatedly measured 5 times in the vicinity, and the average value of the analysis values obtained by the repeated measurement was obtained.

After removing the signal of the adhesive tape, the acquired infrared absorption spectrum shows the ratio of the signal components of the amide I absorption band of 1649 cm ^-1 and 1629 cm ^-1 (α / β) with or without correction of the infrared absorption spectrum according to Example 1. ) Was calculated, and the effect of correcting the infrared absorption spectrum according to Example 1 was verified.

In the absence of spectral correction, the α / β values tended to change between acclimation conditions (Fig. 6), suggesting that the α / β values are affected by humidity. On the other hand, in the analysis of the spectrum obtained by correcting the signal of water according to Example 1, the difference between the α / β values became small between the acclimation conditions (FIG. 7), and the influence of humidity was removed by the method of the present invention. It was found that the amide I absorption band can be evaluated.

Claims (10)

An infrared absorption spectrum obtained by attaching a stratum corneum to the adhesive surface of a tape having a silicone adhesive layer and measuring the adhesive surface of the tape by ATR-IR, and ATR the adhesive surface of an unused tape having a silicone adhesive layer. This is a method for measuring the infrared absorption spectrum of the stratum corneum, in which the infrared absorption spectrum obtained by -IR measurement is acquired and the spectrum of the stratum corneum is acquired from the difference spectrum between the two. By calculating the estimated contribution of the water signal in the full frequency range of the infrared absorption spectrum of the stratum corneum from the estimated contribution of the water signal in the region of ~ 4000 cm ^-1 , and subtracting it from the infrared absorption spectrum of the stratum corneum. How to get the spectrum. Difference spectrum between infrared absorption spectrum obtained by ATR-IR measurement of the adhesive surface of the stratum corneum release tape and infrared absorption spectrum obtained by ATR-IR measurement of the adhesive surface of an unused tape having a silicone adhesive layer. creation is a major absorption band derived from the silicone adhesive layer, the absorption band of the absorption band or 1000~1100cm near ^-1 around 1260 cm ^-1, either, or as a peak intensity derived from both matches The method according to claim 1, wherein any infrared absorption spectrum is multiplied by a constant value and then subtracted. Coefficients q and r were calculated by applying the approximate expression represented by the following equation 1 to the peak of the absorption band of 3000 to 4000 cm ^-1 in the infrared absorption spectrum of the stratum corneum, and the entire infrared absorption spectrum of the stratum corneum was calculated. The method of claim 1 or 2, wherein the contribution of the water signal in the wavenumber range is estimated.
S _SC (ω) = qS _SC-Dry (ω) + rS _Water (ω) (Equation 1)
[In the equation, S _SC (ω) is the infrared absorption spectrum of the stratum corneum, S _SC-Dry (ω) is the infrared absorption spectrum of the completely dried stratum corneum, and S _Water (ω) is the infrared absorption spectrum of water. , Q and r indicate the coefficient, and ω indicates the wave number. ] From the signal intensity and peak shape of each peak in the infrared absorption spectrum of the stratum corneum obtained by the method according to any one of claims 1 to 3, the components constituting the stratum corneum, the properties of the stratum corneum, and the angle. A stratum corneum evaluation method for evaluating any one or more of the amount of deposits on the layer surface and the change in the chemical state of the stratum corneum. The method according to claim 4, wherein the compositional change of the stratum corneum protein and the properties of the stratum corneum are evaluated from the peak shape of the absorption band derived from amide I. The method according to claim 5, wherein the area ratio (α / β value) of both peaks is obtained by dividing the peak on which the α-helix and the random coil are superimposed and the peak of the β sheet from the peak of the absorption band derived from amide I. The method according to claim 4, wherein the packing property of the intercellular lipid in the stratum corneum and the property of the stratum corneum are evaluated from the peak shape of the absorption band of the angular vibration of the methylene group. The method according to claim 4, wherein the packing property of the intercellular lipid in the stratum corneum and the property of the stratum corneum are evaluated from the peak position of the absorption band of the expansion and contraction vibration of the alkyl chain. The method according to claim 4, wherein the amount of natural moisturizing factor (NMF) in the stratum corneum and the properties of the stratum corneum are evaluated from the signal intensity of the absorption band of the dissociated carboxy group. The method according to claim 4, wherein the amount of deposits on the surface of the stratum corneum is evaluated from the peak area of the absorption band characteristic of the deposits on the stratum corneum.