JP7642437B2 - Method for evaluating the moisture content of the stratum corneum of the skin - Google Patents

Description

The present invention relates to a method for evaluating the moisture content of the stratum corneum of the skin.

The tissue that constitutes the outermost layer of the skin is called the stratum corneum. It is known that the amount of water that constitutes the stratum corneum varies depending on the skin condition, such as roughness or dryness. The stratum corneum is also the main site of action of many skin care agents and cosmetics. Therefore, understanding the water content of the stratum corneum and its changes is useful for understanding the skin condition and verifying the skin care effects of skin care agents and cosmetics.
Conventionally, electrical methods such as skin contact and corneometer have been widely used to measure the moisture content of the skin. While these methods have the advantage of being able to detect changes in moisture content sensitively and easily, they also have some disadvantages, such as unclear measurement depth, inability to detect moisture content as an absolute amount, and susceptibility to factors other than water.
In order to solve these problems with moisture measurement methods, methods using confocal Raman spectroscopy (Patent Document 1) and methods using near-infrared reflectance spectroscopy (Patent Document 2) have been investigated.
However, these methods require large-scale equipment and are limited in space, making it difficult to measure moisture content easily. For this reason, a method for easily and directly evaluating the moisture content of skin has been desired.

On the other hand, attenuated total reflection infrared spectroscopy (ATR-IR), a type of infrared spectroscopy, can directly evaluate the state of keratinocytes by simply pressing the skin against an internal reflection element. However, there are still limitations to the location and environment in which the measurement can be performed, as well as the fact that the subject must assume an uncomfortable position. Therefore, it is possible to use a sample of the stratum corneum of the skin for measurement. As a simple sampling method that places a minimal burden on the subject, a method has been proposed in which the stratum corneum is collected by tape stripping, and its composition is analyzed using infrared spectroscopy based on the ATR-IR method.

One example is a method in which stratum corneum cells on the skin surface are peeled off with an adhesive tape having a silicone-based adhesive layer, and the infrared absorption spectrum of the adhesive surface is measured (Patent Document 3). This technique involves multiplying a coefficient and then subtracting the infrared absorption spectrum of the adhesive surface of the adhesive tape before the stratum corneum cells are peeled off from the infrared absorption spectrum of the adhesive surface of the adhesive tape after the stratum corneum cells are peeled off, thereby calculating the spectral component corresponding to the peeled stratum corneum (hereinafter also referred to as the "peeled stratum corneum infrared absorption spectrum"), thereby obtaining chemical information on the stratum corneum constituents and stratum corneum attachments on the stratum corneum surface. Using this method, it is possible to obtain evaluation samples regardless of the measurement location of the subject. However, it was thought that the stratum corneum collected by tape peeling in this way contains almost no water, and it was thought that this method could not be used to evaluate the moisture content in the stratum corneum.

JP 2012-50739 A JP 2018-54448 A JP 2018-105783 A

The problem to be solved by this invention is to provide a method for directly assessing the moisture content in the stratum corneum without limitations on the measurement location, using a simple method that places minimal burden on the subject.

The inventors analyzed the infrared absorption spectrum of tape-peeled stratum corneum and unexpectedly found that even tape-peeled stratum corneum, which was previously thought to contain almost no water, does contain a small amount of water. They also found that the amount of water contained in the stratum corneum immediately after peeling can be evaluated based on the peak tape of the infrared absorption spectrum in a specific wavelength range. Furthermore, they found that by properly storing the peeled tape, the water content can be evaluated in the same way as in the stratum corneum immediately after peeling, and created a method for directly evaluating the water content in the stratum corneum without restrictions on the measurement location.

The present invention provides a method for evaluating the moisture content of the stratum corneum of the skin from a peak in an infrared absorption spectrum, comprising:
(i) removing moisture from the stratum corneum obtained by tape peeling by drying treatment to prepare a reference sample of dry stratum corneum that does not contain moisture;
(ii) acquiring an absorption spectrum A of a reference sample;
(iii) peeling the stratum corneum of the skin to be measured with tape to obtain a measurement sample;
(iv) obtaining an absorption spectrum of the measurement sample;
(v') The present invention relates to a method including a step of determining an index of the amount of water contained in a measurement sample based on the peaks of the absorption spectrum A and the absorption spectrum B of water alone.

The present invention also provides a method for evaluating the moisture content of the stratum corneum of the skin from a peak in the 3800-3000 ^cm range of an infrared absorption spectrum, comprising:
(i) removing moisture from the stratum corneum obtained by tape peeling by drying treatment to prepare a reference sample of dry stratum corneum that does not contain moisture;
(ii) acquiring an absorption spectrum A of a reference sample;
(iii) peeling the stratum corneum of the skin to be measured with tape to obtain a measurement sample;
(iv) obtaining an absorption spectrum of the measurement sample;
(v) determining coefficients a and b for the spectra A and B according to (Equation 1) so that the peak at 3800 to 3000 ^cm of the measurement sample coincides with the sum of the absorption spectrum A and the absorption spectrum B of water alone;
S _SC (λ)≒a(S _dry-SC (λ))+b(S _water (λ)) (Formula 1)
S _SC (λ): Spectrum of the measured sample S _dry-SC (λ): Absorption spectrum (A) of dry keratin (reference sample)
S _water (λ): Absorption spectrum of water alone (B)
(vi) The method includes a step of determining the value of b/a as an indicator of the amount of water contained in the measured sample.

According to the present invention, by measuring the infrared absorption spectrum of the stratum corneum of skin peeled with tape, the moisture content of the stratum corneum of the skin can be evaluated in a short time with minimal burden on the subject, regardless of the place or environment of measurement. Furthermore, by appropriately storing the tape used to peel the stratum corneum, the moisture content can be evaluated in the same way as in the stratum corneum immediately after peeling.

FIG. 1 is a diagram showing a method for determining coefficients a and b relating to an absorption spectrum A of dry keratin (reference sample) and an absorption spectrum B of water alone, using Equation 1, for the spectrum of a measurement sample in the present invention. FIG. 2 is a graph showing the stratum corneum moisture value (b/a) in a low humidity environment and a high humidity environment in an example.

In the present invention, the infrared absorption spectrum can be obtained using an ATR-IR spectrometer. As the ATR-IR spectrometer, a normal FT-IR and an ATR unit that can be connected thereto may be used. The ATR unit may be an optical fiber probe. A system in which the ATR unit and the FT-IR are integrated may also be used. There is no particular limitation on the internal reflection element, and for example, germanium, diamond, ZnSe, Si, and PIR (made of silver chloride) may be used. There is also no particular limitation on the number of reflections and the angle of incidence as long as the total reflection condition can be maintained. It is necessary to measure the wave number resolution 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 sensitivity and S/N can be compatible.

The tape used for peeling off the stratum corneum has an adhesive layer on a base film, and the base film is not particularly limited as long as it is made of a material that does not absorb water, and the adhesive layer is preferably a silicone-based adhesive layer since it does not overlap with the peaks derived from water and the stratum corneum. As the tape, a tape having a silicone adhesive layer is preferable.
Tapes with silicone adhesive layers are generally commercially available for applications requiring heat resistance and chemical resistance, and therefore tapes with Teflon (registered trademark) or aluminum as the base film are readily available. These base films are strong and therefore easy to apply to and remove from the skin, making them suitable for the present invention. However, base films other than Teflon (registered trademark) and aluminum can also be used without any problems.
Specifically, tapes having a silicone-based adhesive layer can be purchased from AS ONE Corporation, as described in the Examples, as well as from 3M Corporation, Nitto Denko Corporation, Okado Co., Ltd., Teraoka Manufacturing Co., Ltd., etc. Tapes having a silicone-based adhesive layer can also be prepared by coating various films, nonwoven fabrics, etc. with commercially available silicone-based adhesives from Dow Corning Toray Co., Ltd., Shin-Etsu Chemical Co., Ltd., Resitex Co., Ltd., etc.

The stratum corneum can be attached to the silicone adhesive surface by directly pressing the silicone adhesive surface against the skin surface, or a separate tape with an adhesive layer can be pressed against the skin surface to transfer the collected stratum corneum to the silicone adhesive surface.

The procedure for the method of the present invention for evaluating the moisture content of the skin surface (stratum corneum) will be described below.
(i) The stratum corneum obtained by tape peeling is dried to remove moisture, thereby preparing a reference sample of dry stratum corneum that does not contain moisture:
Here, the stratum corneum obtained by tape peeling is a reference sample. As long as it is the same type (e.g., human, pig, etc.) as the sample (measurement sample) to be measured later, the site is not limited, but it is preferable to be the same site. For example, even if the moisture content of the cheek is to be evaluated, the stratum corneum of the arm, etc. may be used as the reference sample. In addition, the reference sample of the person to be measured is not necessarily required, and for example, if it is a human, the absorption spectrum of the reference sample is almost the same, so the reference sample of another person may be used.

The drying treatment of the stratum corneum may be carried out by any method that does not destroy the stratum corneum cells.
For example, the moisture in the stratum corneum obtained by tape peeling can be removed by methods such as leaving it for a certain period of time (about 1 hour) in a container through which dry nitrogen is flowing, blowing dry air into it, leaving it in a container with low humidity using a desiccant, allowing it to dry naturally, or drying it by reducing the air pressure inside the container.
This allows one to obtain a "reference sample" of dry, moisture-free stratum corneum.
The "reference sample" of the dry stratum corneum can also be obtained by drying the "measurement sample" used in "(iv) Obtaining the absorption spectrum of the measurement sample:".

(ii) Obtain an absorption spectrum A of a reference sample:
Infrared absorption spectra are obtained for each of the reference sample obtained above and, if necessary, water alone.
Infrared absorption spectrum is preferably measured in the range of at least 3800 to 3000 cm ⁻¹ since a peak derived from water can be obtained.
The obtained infrared absorption spectrum is designated as absorption spectrum A for the reference sample and as absorption spectrum B for water alone.
Incidentally, the absorption spectrum B, which is the infrared absorption spectrum of water alone, does not necessarily need to be measured every time, and it is sufficient to measure it as needed, for example, when calibrating the measuring device.

(iii) The stratum corneum of the skin to be measured is peeled off with tape to obtain a measurement sample:
Next, the stratum corneum of the skin to be measured is peeled off with tape to prepare a "measurement sample."
The stratum corneum can be peeled off with tape in the same manner as for the reference sample.
The absorption spectrum of the obtained measurement sample is measured immediately after acquisition, or if this is difficult, it can be measured anytime and anywhere by storing it in an environment where the state and moisture content of the stratum corneum do not change. For example, the measurement sample can be stored by pasting the tape with the stratum corneum attached to a sheet of a material that does not evaporate water, in other words, a sheet that has no water vapor permeability or has a very small water vapor permeability. The sheet of a material that does not evaporate water can be made of PET or the like, for example, a commercially available OHP sheet (OHP film product number: VF-1420N (KOKUYO), material: recycled PET film). Furthermore, the OHP sheet with the tape attached to the stratum corneum can be sandwiched between another adhesive laminate film from above and below to prevent evaporation of moisture in the stratum corneum, and can be stored for about one week. The adhesive laminate film may be made of a material that does not evaporate water, for example, a PET film or a laminate film containing an aluminum coating layer.
By storing the sample in this manner, it is possible to obtain a measurement sample at a place where there is no measurement device, and then transport it to a place where there is a measurement device and measure it. For example, it is possible to obtain a measurement sample of the stratum corneum of the skin to be measured by peeling it with tape at home, attach it to a sheet, and mail it to a testing institution to request an evaluation of the moisture content.

(iv) Obtain an absorption spectrum of the measurement sample:
The absorption spectrum of the measurement sample is measured in the same manner as described above.
The absorption spectrum may be measured immediately after obtaining the measurement sample, or may be measured after storage as described above.

(v) Determine coefficients a and b that apply to the spectra A and B according to (Equation 1) so that the peak in the range of 3800 to 3000 ^cm of the measurement sample coincides with the sum of absorption spectrum A and absorption spectrum B of water alone:
S _SC (λ)≒a(S _dry-SC (λ))+b(S _water (λ)) (Formula 1)
S _SC (λ): Spectrum of the measured sample S _dry-SC (λ): Absorption spectrum (A) of dry keratin (reference sample)
S _water (λ): Absorption spectrum of water alone (B)

That is, as shown in (Equation 1), the spectrum of the measurement sample (S _SC (λ)) can be approximately expressed as the sum of the absorption spectrum A (S _{dry- SC} (λ)) of dry stratum corneum (reference sample) and the absorption spectrum B (S _water (λ)) of water alone, as shown in the above (Equation 1) (a and b are coefficients, and λ is the wave number).
The coefficients a and b are calculated by applying the above formula to the peak of the absorption band from 3800 to 3000 ^cm . The least squares method or maximum likelihood method can be used to calculate the coefficients a and b that best reproduce (Formula 1) (see Figure 1).

(vi) The value of b/a is an index of the amount of water contained in the measurement sample:
From the obtained a and b, the value of b/a is taken as an index of the amount of water contained in the measurement sample.
b/a is a value that relatively indicates the amount of moisture contained in the measurement sample. The larger this value, the greater the moisture content, and the moisture content of the skin surface (stratum corneum) can be relatively evaluated. When comparing the b/a value with other b/a values, the absorption spectrum (A) of the same dry stratum corneum (reference sample) should be used.

The apparatus and measurement conditions used in the examples are as follows.
(1) FT-IR spectrometer:
A Universal ATR unit (Diamond, 45°) was attached to a Frontier (Perkin-Elmer, TGS detector) or a Nicole iS5 (Thermo Fisher Scientific) equipped with an ATR unit (iD5 ATR) was used.

(2) Measurement conditions:
Wave number resolution 4cm ^-1 , integration 4 times

Example 1
A tape having a silicone adhesive layer (Azflon Tape PTW13-008, AS ONE Corporation) was applied to the inner side of a human forearm, and the stratum corneum was sampled by peeling it off. This tape-peeled stratum corneum was left for 1 hour in a container in which dry nitrogen was flowing, and then removed from the container to serve as a reference sample. The reference sample removed from the container was immediately subjected to IR measurement to obtain an absorption spectrum A of the reference sample. Similarly, an absorption spectrum B of water alone was obtained.
After acclimating for 1 hour in a low humidity environment (20°C, 15%), the same tape (Azflon Tape PTW13-008, AS ONE Corporation) was applied to the inside of the forearm and peeled off to obtain the stratum corneum (measurement sample). The tape with the stratum corneum attached was immediately applied to an OHP sheet (OHP film, product number: VF-1420N (KOKUYO)) after peeling. The release tape was then peeled off from the OHP sheet and immediately subjected to IR measurement to obtain the absorption spectrum of the measurement sample.
In the above (Equation 1), the coefficients a and b applied to the spectra A and B were determined by the least squares method so that the peak of the measured sample between 3800 and 3000 cm ^-1 coincided with the sum of the absorption spectrum A of the reference sample and the absorption spectrum B of water alone.
The same measurements were also carried out under a high humidity environment (20° C., 90%).
Measurements were made at five points for each humidity, and the average value of b/a was calculated.
As a result, in a low humidity environment, b/a was 0.119.
In addition, in a high humidity environment, b/a was 0.174.
The results are shown in Figure 2. Comparing the average values of b/a, it was found that b/a was larger in a high humidity environment than in a low humidity environment, indicating a larger moisture content.

Example 2
A tape having a silicone adhesive layer (Azflon Tape PTW13-008, AS ONE Corporation) was applied to the inner side of a human forearm, and the stratum corneum was sampled by peeling it off. This tape-peeled stratum corneum was left for 1 hour in a container in which dry nitrogen was flowing, and then removed from the container to serve as a reference sample. The reference sample removed from the container was immediately subjected to IR measurement to obtain an absorption spectrum A of the reference sample. Similarly, an absorption spectrum B of water alone was obtained.
The same tape as above (Azflon Tape PTW13-008, AS ONE Corporation) was applied to the inside of the forearm and peeled off to obtain a stratum corneum sample (measurement sample). The tape with the stratum corneum attached was subjected to IR measurement immediately after peeling, and the absorption spectrum x of the measurement sample immediately after peeling was obtained. The peeled tape (measurement sample) was applied to an OHP sheet (OHP film, product number: VF-1420N (KOKUYO)) immediately after IR measurement and stored at room temperature for about 3 hours.
After storing at room temperature for about 3 hours, the release tape (measurement sample) was peeled off from the OHP sheet and immediately IR measurement was performed to obtain an absorption spectrum y of the measurement sample after storage.
For each of the absorption spectrum x immediately after peeling and the absorption spectrum y after storage, the coefficients a and b applied to the spectra A and B in the above (Equation 1) were determined by the least squares method so that the peak at ³⁸⁰⁰ to 3000 cm of the measured sample coincided with the sum of the absorption spectrum A of the reference sample and the absorption spectrum B of water alone.
These measurements were carried out for three samples ((1), (2), and (3)), and the value of b/a was calculated for each.
As a result, the values of b/a immediately after peeling were (1) 0.111, (2) 0.164, and (3) 0.157, and after storage were (1) 0.111, (2) 0.163, and (3) 0.156. These results confirmed that the moisture content of the stratum corneum of the skin can be evaluated after storage in the same way as immediately after peeling.

Claims (5)

A method for evaluating the moisture content of the skin stratum corneum from a peak in an infrared absorption spectrum, comprising:
(i) removing moisture from the stratum corneum obtained by tape peeling by drying treatment to prepare a reference sample of dry stratum corneum that does not contain moisture;
(ii) acquiring an absorption spectrum A of a reference sample;
(iii) obtaining a measurement sample of the stratum corneum of the skin to be measured by tape stripping;
(iv) obtaining an absorption spectrum of the measurement sample;
(v') A method comprising a step of determining an index of the amount of water contained in the measured sample based on the peaks of absorption spectrum A and absorption spectrum B of water alone. A method for evaluating the moisture content of the stratum corneum of the skin from a peak in the 3800-3000 cm ^-1 range of an infrared absorption spectrum, comprising:
(i) removing moisture from the stratum corneum obtained by tape peeling by drying treatment to prepare a reference sample of dry stratum corneum that does not contain moisture;
(ii) acquiring an absorption spectrum A of a reference sample;
(iii) obtaining a measurement sample of the stratum corneum of the skin to be measured by tape stripping;
(iv) obtaining an absorption spectrum of the measurement sample;
(v) determining coefficients a and b for the spectra A and B according to (Equation 1) so that the peak at 3800 to 3000 ^cm of the measurement sample coincides with the sum of the absorption spectrum A and the absorption spectrum B of water alone;
S _SC (λ)≒a(S _dry-SC (λ))+b(S _water (λ)) (Formula 1)
S _SC (λ): Spectrum of the measured sample S _dry-SC (λ): Absorption spectrum (A) of dry keratin (reference sample)
S _water (λ): Absorption spectrum of water alone (B)
(vi) A method comprising a step in which the value of b/a is an indicator of the amount of water contained in the measured sample. 3. The method for evaluating the moisture content of the stratum corneum of the skin according to claim 1 or 2, wherein the stratum corneum is peeled off using a tape using a material that does not absorb water as a base film. 4. The method for evaluating the moisture content of the stratum corneum of the skin according to any one of claims 1 to 3, wherein the stratum corneum is peeled off using a tape having an adhesive layer of a silicone-based adhesive layer. The method for evaluating the moisture content of the stratum corneum of skin according to any one of claims 1 to 4 , further comprising a step of attaching the measurement sample together with the tape to a sheet of a material that does not evaporate water and storing the same between steps (iii) and (iv).