Method for evaluating distribution of crystal structure

Abstract

To provide a distribution evaluation method of a crystal structure.SOLUTION: The method for evaluating the distribution of the crystal structure in the intercellular lipid or the pseudo-intercellular lipid, and the method for evaluating the density of the intercellular lipid or the pseudo-intercellular lipid using the method for evaluating the distribution of the crystal structure are provided. A method for evaluating the density of an intercellular lipid or a pseudo-intercellular lipid based on the ratio of the area of the liquid crystal region to the area of the entire region or a part of the region of the intercellular lipid or the pseudo-intercellular lipid, a method for evaluating the distribution of the crystal structure, a method for selecting a cosmetic and / or a supplement having an effect of improving the distribution of the crystal structure in the intercellular lipid based on the evaluation result of the skin evaluation method, and a counseling method including the method for selecting a cosmetic and / or a supplement.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the distribution of crystal structures. [Background technology]

[0002] Sensory evaluation by expert panels has been commonly used to evaluate the condition of human skin under normal conditions, when treated with cosmetics, and when skin changes due to aging. However, sensory evaluation is a qualitative evaluation method, and quantitative evaluation methods are needed as tools for discussing skin condition in more detail.

[0003] Known quantitative methods for evaluating skin condition include spectroscopic techniques such as Fourier transform infrared spectroscopy (FT-IR) (see Non-Patent Documents 1 and 2), measurement of transepidermal water loss (TEWL), and measurement of stratum corneum moisture content (see Patent Document 1).

[0004] In evaluation, the preparation of samples to be measured plays an important role, and known methods for preparing such samples include biopsy, tape stripping (see Patent Document 2), and grid stripping (see Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2019-187322 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-003413 [Patent Document 3] Patent No. 5636257 [Non-patent literature]

[0006] [Non-Patent Document 1] Mila Biochimica et al. Biophysica Acta,1778(2008),1344-1355. [Non-patent document 2] Journal of Investigative Dermatology(2010) 130,611-614. Summary of the Invention [Problem to be solved by the invention]

[0007] However, conventionally, there has been a problem in that the density of intercellular lipids or pseudo-intercellular lipids has only been grasped on average. Therefore, an object of the present invention is to provide a method for evaluating the distribution of crystal structures. [Means for solving the problem]

[0008] The gist of the present invention is as follows. The method of the present invention is a method for evaluating the distribution of crystalline structures in intercellular lipids or pseudo-intercellular lipids. In the method of evaluating the distribution of crystal structures of the present invention, the crystal structures preferably include at least one selected from the group consisting of liquid crystal, hexagonal crystal, and orthorhombic crystal. In the method of evaluating the distribution of a crystal structure of the present invention, the crystal structure preferably includes a liquid crystal and / or an orthorhombic crystal. The method of the present invention is a method for evaluating the density of intercellular lipids or pseudo-intercellular lipids using the method of the present invention for evaluating the distribution of crystal structures, and may be a method for evaluating the density of intercellular lipids or pseudo-intercellular lipids based on the ratio of the area of ​​the liquid crystal region to the area of ​​the entire region or a portion of the region of the intercellular lipid or the pseudo-intercellular lipid. The method of the present invention is a skin evaluation method that uses the method of evaluating the distribution of crystal structures of the present invention, and may be a skin evaluation method based on evaluating the distribution of crystal structures in a cornified cell region contained in a single layer of the stratum corneum. In the skin evaluation method of the present invention, it is preferable that the evaluation be based on the ratio of the area of ​​the liquid crystal region to the area of ​​the stratum corneum cell region contained in the monolayer. In the skin evaluation method of the present invention, it is preferable that the evaluation be based on the ratio of the area of ​​the rectangular crystal region to the area of ​​the stratum corneum cell region contained in the monolayer. In the skin evaluation method of the present invention, it is preferable that the evaluation be based on the ratio of the area of ​​the liquid crystal region and the area of ​​the orthorhombic crystal region to the area of ​​the stratum corneum cell region contained in the monolayer. In the skin evaluation method of the present invention, it is preferable to use a plurality of monolayers of the stratum corneum. The method of the present invention may be a method of selecting cosmetics and / or supplements that have the effect of improving the distribution of crystalline structures in intercellular lipids based on the evaluation results of the above-mentioned skin evaluation method of the present invention. In the method of selecting cosmetics and / or supplements of the present invention, it is preferable that the cosmetics and / or supplements contain a component that promotes the elongation of hydrocarbon groups of ceramides or free fatty acids, which are lipids that constitute intercellular lipids. The method of the present invention may be a counseling method that includes the above-described method for selecting the cosmetic and / or supplement of the present invention. [Effects of the Invention]

[0009] According to the present invention, the distribution of crystalline structures in intercellular lipids or pseudo-intercellular lipids can be evaluated in detail. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows a diagram and photographs illustrating the results of evaluation of Example 1 on a stratum corneum sample (sample No. 1) from the cheek region of a subject (the dashed line in the figure indicates the outer contour of the stratum corneum cell region). (A) shows the results of mapping according to wavenumber based on the results of infrared spectroscopy (wavenumber region: 2854 cm-1 to 2846 cm-1). (B) is a photograph of the stratum corneum sample observed under an optical microscope when determining the measurement location. [Figure 2]Figure 2 shows a diagram and photographs illustrating the results of evaluation of Example 1 on a stratum corneum sample (sample No. 8) from the arm of a subject (the dashed line indicates the outer contour of the stratum corneum cell region). (A) shows the results of mapping according to wavenumber based on the results of infrared spectroscopy (wavenumber region: 2854 cm-1 to 2846 cm-1). (B) is a photograph of the stratum corneum sample observed under an optical microscope when determining the measurement location. [Figure 3] Figure 3 is a schematic diagram showing the appearance of a stratum corneum sample collected from a subject by grid stripping. (A) is a plan view of the stratum corneum sample viewed from the stratum corneum cell side. (B) is an end view of the stratum corneum sample cut along line XX in (A) (the dashed lines in the figure indicate the outline of the upper and lower surfaces of the grid and the adhesive layer as seen in a cross-sectional view rather than an end view). [Figure 4] FIG. 4 is a diagram showing a schematic view of stratum corneum cells captured by infrared spectroscopic analysis using a 64×64 (128 μm×128 μm) element. [Figure 5] Figure 5 shows diagrams and photographs illustrating the results of evaluation of Reference Example 2 for stratum corneum samples (Sample No. 1 and Sample No. 8) from the cheek and arm regions of subjects. (A) is a photograph of the stratum corneum sample (Sample No. 1) observed under a microscope, which has been binarized to black and white. (B) is a photograph of the stratum corneum sample (Sample No. 8) observed under a microscope, which has been binarized to black and white. [Figure 6] FIG. 6 shows the results of evaluation of Example 1 on a stratum corneum sample (sample No. 2) from the cheek region of a subject, and in particular shows the results of mapping according to wavenumber based on the results of measurement by infrared spectroscopy (wavenumber region: 2854 cm-1 to 2846 cm-1). [Figure 7]FIG. 7 shows the results of evaluation of Example 1 on a stratum corneum sample (sample No. 3) from the cheek region of a subject, and in particular shows the results of mapping according to wavenumber based on the results of measurement by infrared spectroscopy (wavenumber region: 2854 cm-1 to 2846 cm-1). [Figure 8] FIG. 8 shows the results of evaluation of Example 1 on a stratum corneum sample (sample No. 4) from the cheek region of a subject, and in particular shows the results of mapping according to wavenumber based on the results of measurement by infrared spectroscopy (wavenumber region: 2854 cm-1 to 2846 cm-1). [Figure 9] FIG. 9 shows the results of evaluation of Example 1 on a stratum corneum sample (sample No. 5) from the cheek region of a subject, and in particular shows the results of mapping according to wavenumber based on the results of measurement by infrared spectroscopy (wavenumber region: 2854 cm-1 to 2846 cm-1). [Figure 10] FIG. 10 shows the results of evaluation of Example 1 on a stratum corneum sample (sample No. 6) from the cheek region of a subject, and in particular shows the results of mapping according to wavenumber based on the results of measurement by infrared spectroscopy (wavenumber region: 2854 cm-1 to 2846 cm-1). [Figure 11] FIG. 11 shows the results of evaluation of Example 1 on a stratum corneum sample (sample No. 7) from the cheek region of a subject, and in particular shows the results of mapping according to wavenumber based on the results of measurement by infrared spectroscopy (wavenumber region: 2854 cm-1 to 2846 cm-1). DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, a method for evaluating the distribution of a crystal structure according to an embodiment of the present invention (hereinafter also referred to as the present embodiment) will be described in detail with reference to the drawings.

[0012] (Crystal structure distribution evaluation method) The method for evaluating the distribution of crystal structures of this embodiment is a method for evaluating the distribution of crystal structures in intercellular lipids or pseudo-intercellular lipids.

[0013] The crystal structure distribution evaluation method of this embodiment may be applied to humans and non-humans (for example, pigs, cows, birds, sheep, goats, rabbits, dogs, cats, etc.).

[0014] The intercellular lipids in this embodiment may be intercellular lipids of human-derived or non-human-derived tissue, preferably intercellular lipids of the epidermis of the skin, and more preferably intercellular lipids of the stratum corneum of the epidermis. Furthermore, the pseudo-intercellular lipids in this embodiment preferably contain a mixture of free fatty acids, cholesterol, cholesterol esters, and ceramides. Examples of ceramides include ceramide [EOS], ceramide [NS], ceramide [NP], ceramide [EOH], ceramide [AS], ceramide [AP], ceramide [AH], ceramide [NH], ceramide [EOP], ceramide [NDS], ceramide [ADS], ceramide [EODS], sphingosine, phytosphingosine, sphinganine, and glycoceramide. More preferably, the pseudo-intercellular lipids in this embodiment are composed of a mixture of stearic acid, cholesterol, and ceramide [NS] (ceramide 2). The chain length and degree of unsaturation of the hydrocarbon groups contained in the free fatty acids and ceramides are not particularly limited. In this embodiment, the molar ratio in the mixture is preferably stearic acid:cholesterol:ceramide [NS] (ceramide 2) = 30-70:0-40:10-50, and particularly preferably stearic acid:cholesterol:ceramide [NS] (ceramide 2) = 30-50:20-40:20-40. Furthermore, the pseudo-intercellular lipids may be mixed with an appropriate solvent (chloroform, methanol, etc.) to obtain a pseudo-intercellular lipid sample, which may be subjected to measurement. In this case, the concentration of the pseudo-intercellular lipids in the mixture may be 0.1 mmol / L to 100 mmol / L, and preferably 1 mmol / L to 10 mmol / L.

[0015] The measurement method that can be used in this embodiment is not particularly limited, but various spectroscopic methods may be used, such as infrared spectroscopy (IR), X-ray diffraction (XRD), and Raman spectroscopy (NRS), with infrared spectroscopy (IR) being preferred.

[0016] In conventional evaluation methods, due to issues such as the accuracy of measurement techniques, the density of intercellular lipids or pseudo-intercellular lipids could only be captured on average, and low-density regions could not be measured, making it difficult to evaluate the distribution of crystalline structures in detail. For example, measuring transepidermal water loss (TEWL) and stratum corneum moisture content are known as methods for evaluating skin condition, but these are indirect methods for evaluating the distribution of crystalline structures. Furthermore, it has become clear that there is no clear correlation between transepidermal water loss (TEWL) and stratum corneum moisture content, and therefore these methods are not effective for evaluating the distribution of crystalline structures.

[0017] On the other hand, in the method for evaluating the distribution of crystal structure of this embodiment, a highly accurate measurement technique is used to evaluate the crystal structure of a single layer or a thin layer, thereby making it possible to evaluate in detail the distribution of crystal structure in intercellular lipids or pseudo-intercellular lipids. Specifically, the method for evaluating the distribution of crystal structures according to this embodiment can calculate the ratio of the crystal structures, and can evaluate the localization or uneven distribution of liquid crystal regions.

[0018] Here, examples of the crystal structure include liquid crystal, hexagonal, and orthorhombic crystals. These may be used alone, in combination of two or more, or in combination of all three. In this embodiment, it is preferable that the crystal structure for which distribution evaluation is performed includes a liquid crystal. Liquid crystal is a state in which the density of intercellular lipids or pseudo-intercellular lipids is low, and has been attracting attention in the prior art, but only in terms of averaged information, and information regarding its distribution has not been grasped. By using liquid crystal as the target of distribution evaluation, it is possible to evaluate the distribution of the crystal structure in more detail. Although it cannot be said that liquid crystals are included in the crystal structure academically, in this specification they are included in the crystal structure. In this embodiment, it is preferable that the crystal structure to be subjected to distribution evaluation includes an orthorhombic crystal. The orthorhombic crystal is in a state where the density of intercellular lipids or pseudo-intercellular lipids is high. By using an orthorhombic crystal as the target of distribution evaluation, it becomes possible to evaluate the distribution of the crystal structure in more detail. In this embodiment, it is also preferable that the crystal structures to be subjected to distribution evaluation include liquid crystal and orthorhombic crystal. By subjecting liquid crystal and orthorhombic crystal to distribution evaluation, knowledge can be obtained about the relationship between liquid crystal, which is a state in which the density of intercellular lipids or pseudo-intercellular lipids is low, and orthorhombic crystal, which is a state in which the density of intercellular lipids or pseudo-intercellular lipids is high (for example, the respective proportions of liquid crystal and orthorhombic crystal, and the ratio of the proportions of both), and it becomes possible to evaluate the distribution of the crystal structure in more detail.

[0019] In this embodiment, the liquid crystal, hexagonal crystal, or orthorhombic crystal may be analyzed and / or identified according to various spectroscopic methods. When infrared spectroscopy (IR) is used, the 2850 cm band originating from the CH symmetric stretching vibration is -1 The wavenumber region around 1460 cm originates from the CH2 symmetric in-plane bending vibration. -1 ~1480cm -1 The wavenumber region around 2920 cm originates from the CH2 antisymmetric stretching vibration. -1 In the wavenumber region around this range, significant differences are found in the peak positions of the liquid crystal, hexagonal crystal, and orthorhombic crystal.

[0020] 2850cm -1 When using a wavenumber region around 2848 cm, for example, in a chart showing wavenumber on the horizontal axis and infrared absorption intensity on the vertical axis, -1 More than 2854cm -1 The following wavenumber range may be used: 2851 cm -1 More than 2854cm -1 The region where the liquid crystal is -1 Over 2851cm -1 The region where the crystal structure is hexagonal is defined as the region where the crystal structure is hexagonal. -1 More than 2850cm -1The region where the crystal structure is less than 100% may be considered to be an orthorhombic region.

[0021] 1460cm originating from CH2 symmetric in-plane bending vibration -1 ~1480cm -1 When using the wavenumber region around 1460 cm in the second derivative spectrum, -1 over 1480cm -1 The full width at half maximum of two peaks in the following wavenumber range may be used, and the full width at half maximum is 4 cm -1 Over 9cm -1 The area where the full width at half maximum is less than 9 cm is the liquid crystal area. -1 More than 11cm -1 The region where the full width at half maximum is less than 11 cm is considered to be the hexagonal crystal region. -1 Over 13cm -1 The region where: The second derivative spectrum is a spectrum in which the horizontal axis indicates wavenumber and the vertical axis indicates infrared absorption intensity, and is obtained by taking the derivative of the spectrum and processing it to emphasize the peak positions of the constituent spectral bands.

[0022] In addition, instead of the full width at half maximum of the region, the full width at 15% of the maximum peak height (FW-15) derived from the CH2 symmetric in-plane bending vibration may be used. When the full width at 15% of the maximum peak height is used, the full width is 6 cm. -1 Over 12cm -1 The area where the total width is less than 12cm is the liquid crystal area. -1 Over 13cm -1 The area where the total width is less than 13 cm is the hexagonal crystal area. -1 More than 14cm -1 The region where:

[0023] In this embodiment, from the viewpoint of achieving high-precision measurement, the size of one element is preferably 4 μm×4 μm or less, more preferably 2 μm×2 μm or less, and particularly preferably 1 μm×1 μm or less. In the example shown in FIGS. 1 and 2, the size of one element is 2 μm×2 μm.

[0024] In this embodiment, when infrared spectroscopy is used as the measurement method, it is preferable to use an FPA detector equipped with multiple detectors in order to achieve high-precision measurement. The number of elements in the FPA detector is not particularly limited, but elements with a size of 64 μm × 64 μm or more are preferred, and elements with a size of 128 μm × 128 μm or more are more preferred.

[0025] In this embodiment, the wave number of the maximum peak in the chart observed in each element may be the wave number in that element. Here, the chart observed in each element may be a chart obtained by adding the charts of liquid crystal, hexagonal crystal, and orthorhombic crystal according to their respective proportions.

[0026] In this embodiment, various crystal structures may be mapped to evaluate the distribution of the crystal structures. For example, the crystal structures may be color-coded according to their type or according to their wavenumber range (see FIGS. 1 and 2).

[0027] In this embodiment, the density of the intercellular lipids or pseudo-intercellular lipids can be evaluated based on the ratio of the area of ​​the liquid crystal region to the area of ​​the entire region or part of the region of the intercellular lipids or pseudo-intercellular lipids. When the above ratio is relatively low, preferably 30% or less, more preferably 20% or less, and particularly preferably 10% or less, the density of intercellular lipids or pseudo-intercellular lipids can be evaluated as high, and when the above ratio is relatively high, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more, the density of intercellular lipids or pseudo-intercellular lipids can be evaluated as low. In this embodiment, the density of the intercellular lipids or pseudo-intercellular lipids can be evaluated based on the ratio of the area of ​​the rectangular crystal regions to the area of ​​the entire region or part of the region of the intercellular lipids or pseudo-intercellular lipids. When the above ratio is relatively high, preferably 15% or more, more preferably 20% or more, and particularly preferably 30% or more, the density of intercellular lipids or pseudo-intercellular lipids can be evaluated as high, and when the above ratio is relatively low, preferably 10% or less, more preferably 5% or less, and particularly preferably 3% or less, the density of intercellular lipids or pseudo-intercellular lipids can be evaluated as low. The above ratio may be the average of values ​​measured by arbitrarily selecting three measurement fields for one sample. Furthermore, the entire region of the intercellular lipids refers to the region defined by the outer contours of the cells present in the measurement field of view, and a portion of the region of the intercellular lipids refers to a portion of the entire region of the intercellular lipids that is of particular interest. Here, if there are multiple cells present in the measurement field of view, the region defined by the outer contours of the region where multiple cells gather may be considered to be the entire region of the intercellular lipids. Furthermore, the entire region of the pseudo-intercellular lipids refers to the region of the pseudo-intercellular lipids present in the measurement field of view, and a partial region of the pseudo-intercellular lipids refers to a partial region of particular interest among the entire region of the pseudo-intercellular lipids.

[0028] In this embodiment, the distribution of crystalline structures can be evaluated based on the proportion of the area of ​​the liquid crystal regions to the area of ​​the entire region or a portion of the region of the above-mentioned intercellular lipid or pseudo-intercellular lipid, and the proportion of the area of ​​the orthorhombic crystal regions to the area of ​​the entire region or a portion of the region of the above-mentioned intercellular lipid or pseudo-intercellular lipid. Specifically, the distribution of crystalline structures can be evaluated based on the proportion of the area of ​​the orthorhombic crystal regions to the area of ​​the entire region or a portion of the region of the intercellular lipid or pseudo-intercellular lipid to the proportion of the area of ​​the liquid crystal regions to the area of ​​the entire region or a portion of the region of the intercellular lipid or pseudo-intercellular lipid (proportion of the area of ​​the orthorhombic crystal regions / proportion of the area of ​​the liquid crystal regions). When the above ratio is relatively high, preferably 0.5 or more, more preferably 1.5 or more, and particularly preferably 3 or more, the density of intercellular lipids or pseudo-intercellular lipids can be evaluated as high, and when the above ratio is relatively low, preferably 0.3 or less, more preferably 0.15 or less, and particularly preferably 0.1 or less, the density of intercellular lipids or pseudo-intercellular lipids can be evaluated as low.

[0029] In this embodiment, it is preferable to evaluate the distribution of the crystal structure based on whether the liquid crystal domains are localized or unevenly distributed in the entire region or part of the intercellular lipid or pseudo-intercellular lipid. If the liquid crystal domains are unevenly distributed, the distribution of the crystal structure can be evaluated as being favorable.

[0030] (Skin evaluation method) Hereinafter, in this embodiment, the method for evaluating the distribution of crystal structures of this embodiment can be used on human or non-human skin to form a skin evaluation method. In such a method, the skin may be evaluated based on the evaluation of the distribution of crystal structures.

[0031] In this specification, a single layer of the stratum corneum refers to a stratum corneum sample collected by a suitable method such as grid stripping in order to obtain one layer of the stratum corneum, and refers to a sample having an average of 1 to 1.4 layers throughout the sample. Strictly speaking, the stratum corneum sample does not necessarily have to have a single layer of stratum corneum cells throughout the sample, and some areas may have two or more layers of stratum corneum cells.

[0032] In this embodiment, the skin can be evaluated by evaluating the ratio of crystalline structure in the stratum corneum cell region contained in a single layer of the stratum corneum based on the ratio of the area of ​​the liquid crystal region to the area of ​​the intercellular lipid region. When the above ratio is relatively low, preferably 30% or less, more preferably 20% or less, and particularly preferably 10% or less, the density of intercellular lipids in the skin can be evaluated as high, and when the above ratio is relatively high, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more, the density of intercellular lipids in the skin can be evaluated as low. In this embodiment, the skin can be evaluated by evaluating the ratio of crystalline structure in the stratum corneum cell region contained in a single layer of the stratum corneum based on the ratio of the area of ​​the rectangular crystal region to the area of ​​the intercellular lipid region. When the above ratio is relatively high, preferably 15% or more, more preferably 20% or more, and particularly preferably 30% or more, the density of intercellular lipids in the skin can be evaluated as high, and when the above ratio is relatively low, preferably 10% or less, more preferably 5% or less, and particularly preferably 3% or less, the density of intercellular lipids in the skin can be evaluated as low. The above ratio may be the average of values ​​measured multiple times (e.g., about 10 times) in a randomly selected measurement field for one sample. If there are multiple stratum corneum cells in one measurement field, the ratio may be calculated by measuring one or more (e.g., about 10) randomly selected stratum corneum cells. The intercellular lipid region refers to the region defined by the outer contours of the cells present in the measurement field of view (see Figures 1 and 2). Here, if there are multiple cells present in the measurement field of view, the region defined by the outer contours of the area where multiple cells gather may be considered to be the intercellular lipid region.

[0033] In this embodiment, the skin can be evaluated by evaluating the ratio of the crystalline structure based on the ratio of the area of ​​the liquid crystal region to the area of ​​the intercellular lipid region described above and the ratio of the area of ​​the orthorhombic crystal region to the area of ​​the intercellular lipid region described above. Specifically, the distribution of the crystalline structure can be evaluated based on the ratio of the area of ​​the orthorhombic crystal region to the area of ​​the intercellular lipid region to the area of ​​the liquid crystal region to the area of ​​the intercellular lipid region (area ratio of the orthorhombic crystal region / area ratio of the liquid crystal region). When the above ratio is relatively high, preferably 0.5 or more, more preferably 1.5 or more, and particularly preferably 3 or more, the density of intercellular lipids in the skin can be evaluated as high, and when the above ratio is relatively low, preferably 0.3 or less, more preferably 0.15 or less, and particularly preferably 0.1 or less, the density of intercellular lipids in the skin can be evaluated as low.

[0034] In this embodiment, it is preferable to evaluate the skin by evaluating the distribution of crystalline structures in the stratum corneum cell region contained in a single layer of the stratum corneum based on whether the liquid crystal regions are localized or unevenly distributed in the intercellular lipid region. When the liquid crystal domains are unevenly distributed, it can be evaluated that the distribution of the crystal structure is favorable.

[0035] In the skin evaluation method of this embodiment, it is preferable to use multiple monolayers of the stratum corneum. Specifically, in this embodiment, it is preferable to evaluate each of the multiple monolayers separately. Also, in this embodiment, it is preferable to use multiple monolayers stacked in the thickness direction of the stratum corneum, and it is preferable to prepare the multiple monolayers by sequentially peeling off the multiple monolayers stacked in the thickness direction of the stratum corneum.

[0036] Methods for preparing multiple monolayers include grid stripping, tape stripping, etc., and grid stripping is preferred.

[0037] The number of layers that can be used is not particularly limited, but may be 2 to 20. The lower limit may be 3 or more, 5 or more, or 10 or more.

[0038] The detailed procedure for grid stripping is not particularly limited and may be performed according to procedures described in known literature (e.g., Japanese Patent No. 5636257, etc.), but it is preferable to perform grid stripping so that 10 or more stratum corneum cells are collected in one grid stripping session in order to enhance the effects of the present invention.

[0039] In the skin evaluation method of this embodiment, if the ratio of the number of stratum corneum monolayers in which the ratio of the area of ​​the liquid crystal domain to the area of ​​the intercellular lipid domain in the stratum corneum cell region is equal to or greater than a predetermined level to the number of stratum corneum monolayers measured does not fall within a predetermined range, the skin condition may be evaluated as poor. For example, when the entire stratum corneum is measured, if the ratio of the number of stratum corneum monolayers in which the ratio of the area of ​​the liquid crystal domain to the area of ​​the intercellular lipid domain in the stratum corneum cell region is equal to or greater than a predetermined level to the total number of stratum corneum layers does not fall within a predetermined range, the skin condition may be evaluated as poor. In the skin evaluation method of this embodiment, if the ratio of the number of stratum corneum monolayers in which the ratio of the area of ​​the cubic crystalline region to the area of ​​the intercellular lipid region in the stratum corneum cell region is equal to or less than a predetermined level to the number of stratum corneum monolayers measured does not fall within a predetermined range, the skin condition may be evaluated as poor. For example, when the entire stratum corneum is measured, if the ratio of the number of stratum corneum monolayers in which the ratio of the area of ​​the cubic crystalline region to the area of ​​the intercellular lipid region in the stratum corneum cell region is equal to or less than a predetermined level to the total number of stratum corneum layers does not fall within a predetermined range, the skin condition may be evaluated as poor. In the skin evaluation method of this embodiment, if the ratio of the number of stratum corneum monolayers in which the ratio of the area of ​​the orthorhombic crystal domains to the area of ​​the intercellular lipid domains in the stratum corneum cell region to the area of ​​the liquid crystal domains to the area of ​​the intercellular lipid domains in the stratum corneum cell region is equal to or less than a predetermined level to the number of stratum corneum monolayers measured is not within a predetermined range. For example, when the entire stratum corneum is measured, if the ratio of the number of stratum corneum monolayers in which the ratio of the area of ​​the orthorhombic crystal domains to the area of ​​the intercellular lipid domains in the stratum corneum cell region to the area of ​​the liquid crystal domains to the area of ​​the intercellular lipid domains in the stratum corneum cell region is equal to or less than a predetermined level to the total number of stratum corneum layers measured is not within a predetermined range, the skin condition may be evaluated as not being good.

[0040] This skin evaluation method makes it possible to grasp the distribution of the crystalline structure of the stratum corneum for each layer, and to obtain information on the density of intercellular lipids in the depth direction of the stratum corneum, which has not been clarified by conventional techniques. Based on this density information in the depth direction, it becomes possible to observe the gaps in moisture, so to speak, and provide a skin evaluation method that focuses on a new aspect.

[0041] This skin evaluation method can also be used as a means of confirming the repair effect of a barrier function improving agent on intercellular lipids by understanding the distribution state of the crystalline structure of intercellular lipids before and after use in a formulation use test. In this case, it is more desirable for the formulation to contain lipids that constitute intercellular lipids, but other lipids such as phospholipids can also be used. Furthermore, as an example of applying this skin evaluation method, the use of deuterium-labeled lipids in formulations allows for the determination of the crystal structure distribution when the deuterated lipids are incorporated into the crystal structure lattice. Generally, peaks derived from the CD2 (D represents deuterium) of deuterated lipids are observed at different positions than peaks derived from the CH2 of undeuterated lipids. Therefore, peak positions of symmetric stretching vibrations of intercellular lipids can be observed at different positions (wavenumber regions) in deuterated lipids than in undeuterated lipids. Therefore, the barrier repair effect of the formulation can be more clearly determined as distribution information. Specifically, it is possible to confirm that the orientation of deuterated lipids in formulations applied to the liquid crystal region within the crystal structure lattice results in a denser crystal structure. Note that, like the peak positions derived from the CH2, the peak positions derived from the CD2 also shift depending on the density of intercellular lipids.

[0042] (How to Select Cosmetics and / or Supplements) In this embodiment, a method can be configured to select cosmetics and / or supplements that have the effect of improving the distribution of crystalline structures in intercellular lipids based on the evaluation results of the skin evaluation method of this embodiment. In such a method, for example, the skin evaluation method of this embodiment is performed on a subject, and if the subject's skin condition is evaluated as poor, cosmetics and / or supplements to be used by the subject may be selected that are highly effective in improving the distribution of crystalline structures in intercellular lipids. Examples of cosmetics include cosmetics, external skin preparations classified as pharmaceuticals or quasi-drugs, etc. Examples of supplements include oral supplements, such as foods for specified health uses, foods with nutrient functions, and foods with functional claims. Cosmetics and / or supplements that improve the distribution of crystalline structures in intercellular lipids preferably contain ingredients that promote the elongation of hydrocarbon groups in ceramides or free fatty acids, which are lipids that make up intercellular lipids. Examples of such ingredients include rice-derived extracts that enhance gene expression of the fatty acid elongase ELOVL.

[0043] (Counseling method) In this embodiment, a counseling method can also be configured that includes the method for selecting the cosmetic and / or supplement of this embodiment. In such a method, for example, when counseling a subject about their skin condition, the method for selecting cosmetics and / or supplements of this embodiment is carried out, i.e., the skin evaluation method of this embodiment is carried out on the subject, and when the subject's skin condition is evaluated as poor, cosmetics and / or supplements to be used by the subject that are highly effective in improving the distribution of crystalline structures in intercellular lipids are selected, and the selected cosmetics and / or supplements are recommended to the subject.

[0044] The embodiments of the present invention have been described above with reference to the drawings, but the above embodiments can be modified as appropriate, and the present invention is not limited to the above-described exemplary embodiments. [Example]

[0045] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples in any way.

[0046] (Sample preparation) Grid stripping was performed on the cheek and arm of one subject (an adult male in his 30s) as described below, and stratum corneum samples were prepared from each area.

[0047] The adhesive used was an adhesive manufactured by Sanyo Chemical Industries, Ltd. As a support, a grid for electron microscope observation having circular edges and a lattice-shaped mesh portion as shown in FIG. 3(A) was used. It was confirmed using an optical microscope that the holes in the grid were not blocked by the adhesive. In order to volatilize the solvent in the adhesive layer, the support having the adhesive layer was left overnight under reduced pressure.

[0048] The surface dirt on the subject's skin (cheek and arm areas) was wiped off with dry gauze. The adhesive layer formed on the support was attached to the subject's skin, the support was pressed down, and then the support was peeled off to collect stratum corneum cells (thickness: approximately 500 nm) on the support as shown in Figure 3. When observed under an optical microscope, the collected keratinocytes were found to be a single layer (specifically, a 1.0 layer) and firmly fixed on the support without sagging, reproducing the state of skin in vivo. The stratum corneum sample from the cheek area was designated Sample No. 1, and the stratum corneum sample from the arm area was designated Sample No. 8.

[0049] Figure 3 is a schematic diagram showing the appearance of a stratum corneum sample collected from a subject by grid stripping. (A) is a plan view of the stratum corneum sample viewed from the stratum corneum cell side. (B) is a cross-sectional view of the stratum corneum sample cut along line XX in (A) (the dashed lines in the figure indicate the outline of the upper and lower surfaces of the grid and the adhesive layer as seen in a cross-sectional view rather than an end view).

[0050] In addition, grid stripping was performed on the cheeks of six subjects (adult women aged 20 to 60) using the same method as used to prepare the stratum corneum sample from the cheeks of Sample No. 1, and stratum corneum samples from the cheeks (Sample No. 2 to Sample No. 7) were prepared. The thickness of the stratum corneum cells collected on the support for each sample was approximately 500 nm. When observed under an optical microscope, the collected stratum corneum cells were a single layer (specifically, a 1.0 layer) and were firmly fixed on the support without sagging, reproducing the condition of skin in vivo.

[0051] A pseudo-intercellular lipid was prepared as Sample No. 9 as follows. Ceramide [NS], cholesterol, and stearic acid were prepared in a 1:1:1 molar ratio and mixed with a 2:1 chloroform / methanol solution to prepare a mixture with a lipid concentration of 10 mmol / L. Thin films were prepared using the Bangham method. The mixture was hydrated above the phase transition temperature for 30 minutes, followed by 5 minutes of sonication to form vesicles. The resulting lipid aggregates were used as the pseudo-intercellular lipid sample.

[0052] Example 1 [Measurement by infrared spectroscopy] The prepared stratum corneum samples (Sample No. 1 to Sample No. 8) were measured using an infrared spectroscopic imaging system: Vertex70 + Hyperion300 (Bruker Optex) as the measuring device, and a mercury cadmium telluride (MCT) (FPA)-Focal Plane Array (MCT) with 64 x 64 (128 μm x 128 μm) elements as the detector, with a wavenumber resolution of 4 μm. -1 Infrared spectroscopic analysis was carried out under the following measurement conditions.

[0053] The location of the collected keratinocytes on the support was confirmed using an optical microscope, and a hole that was covered near the center by the collected keratinocytes and was covered by 50% or more by the keratinocytes was selected.The position of the selected hole relative to the central mark was confirmed to determine the measurement location. The support on which the stratum corneum cells were arranged was placed on the sample stage of an infrared spectroscopic measuring device with the stratum corneum cells facing upward, and the support was fixed to the sample stage with a double-sided adhesive.

[0054] The support on which the keratinocytes were placed was subjected to infrared spectroscopy analysis. The results of the infrared spectroscopy were subjected to Fourier transformation to measure the intensity at each wave number.

[0055] In Example 1, in each element, the wave number corresponding to the symmetric stretching vibration of CH2 was 2854 cm -1 ~2846cm -1 The infrared absorption intensity was measured in the wave number region of The wave number in the element was determined as the wave number of the maximum peak of infrared absorption in the element.

[0056] FIG. 4 is a diagram showing a schematic view of stratum corneum cells captured by infrared spectroscopic analysis using a 64×64 (128 μm×128 μm) element.

[0057] [mapping] Each element was color-coded according to the wavenumber at each element, and mapping was performed on the stratum corneum cells in the measurement field (see Figures 1 and 2).

[0058] FIG. 1 shows a diagram and photographs showing the results of the evaluation of Example 1 on a stratum corneum sample (Sample No. 1) from the cheek region of a subject (in the figure, the dashed line indicates the outer contour of the stratum corneum cell region). (A) shows the results of measurements by infrared spectroscopy (wavenumber region: 2854 cm -1 ~2846cm -1 (B) is a photograph of the stratum corneum sample observed under an optical microscope when determining the measurement location of the stratum corneum sample. FIG. 2 shows a diagram and photographs showing the results of the evaluation of Example 1 on a stratum corneum sample (sample No. 2) taken from the arm of a subject (the dashed line in the figure indicates the outer contour of the stratum corneum cell region). (A) shows the results of measurements by infrared spectroscopy (wavenumber region: 2854 cm -1~2846cm -1 (B) is a photograph of the stratum corneum sample observed under an optical microscope when determining the measurement location of the stratum corneum sample.

[0059] 6 to 11 are diagrams showing the results of evaluation of Example 1 on stratum corneum samples (samples No. 2 to No. 7 in FIGS. 6 to 11, respectively) from the cheeks of test subjects, and in particular, the results of measurements by infrared spectroscopy (wavenumber region: 2854 cm -1 ~2846cm -1 10 is a diagram showing the results of mapping according to wavenumber based on the results of (10).

[0060] [Calculation of average wave number] For the stratum corneum cells in the measurement field, the average wave number (cm) of the entire stratum corneum cell area -1 ) was calculated.

[0061] [Calculation of liquid crystal area ratio] The contours of three stratum corneum cells present in the measurement field were confirmed, and the areas defined by the outer contours of Samples No. 1 and No. 8 were determined as the stratum corneum cell areas, as shown in Figures 1 and 2. Furthermore, observation under an optical microscope confirmed that intercellular lipids were present throughout the entire stratum corneum cell region, and therefore the stratum corneum cell region was defined as the region of intercellular lipids. The liquid crystal region was determined based on the results of the above mapping. -1 ~2854cm -1 The region is the liquid crystal region. The ratio of the liquid crystal area to the intercellular lipid area was calculated for each stratum corneum cell region. Ten measurement fields were randomly selected, and this was performed for one or more stratum corneum cells present in each measurement field, and the average of these ratios was calculated. The results are shown in Table 1.

[0062] The prepared pseudo-intercellular lipid sample (Sample No. 9) was subjected to infrared spectroscopic measurements in the same manner as the stratum corneum samples (Samples No. 1 to No. 8), followed by mapping, calculation of the average wavenumber, and calculation of the liquid crystal domain ratio. The results are shown in Table 1.

[0063] In addition, the prepared stratum corneum samples (Sample No. 1 to Sample No. 8) and the prepared pseudo-intercellular lipid sample (Sample No. 9) The wavenumber corresponding to the CH2 symmetric in-plane bending vibration is 1460 cm -1 ~1480cm -1 Infrared spectroscopy was performed in the same manner as in the above [Measurement by infrared spectroscopy], and the second derivative spectrum was measured at 1460 cm -1 over 1480cm -1 For the full width at half maximum of the two peaks in the following wavenumber regions, mapping, calculation of the average full width at half maximum, and calculation of the liquid crystal region ratio were performed in the same manner as in the above-mentioned [Mapping], [Calculation of the average wavenumber], and [Calculation of the liquid crystal region ratio]. The results are shown in Table 1.

[0064] Furthermore, for the prepared stratum corneum samples (Sample No. 1 to Sample No. 8) and the prepared pseudo-intercellular lipid sample (Sample No. 9), The wavenumber corresponding to the CH2 symmetric in-plane bending vibration is 1460 cm -1 ~1480cm -1 Infrared spectroscopy was performed in the same manner as in the above [Measurement by infrared spectroscopy], and the second derivative spectrum was measured at 1460 cm -1 over 1480cm -1 For the full width at 15% of the maximum peak height in the following wavenumber range, mapping, the average of the full width at 15% of the maximum peak height, and the liquid crystal region ratio were calculated in the same manner as in the above-mentioned [Mapping], [Calculation of the average wavenumber], and [Calculation of the liquid crystal region ratio]. The results are shown in Table 1.

[0065] (Comparative Example 1) [Transepidermal water loss (TEWL) measurement] The transepidermal water loss (g / m) of each of the prepared stratum corneum samples (Sample No. 1 to Sample No. 8) was measured using a Delfin Technologies Vapometer. 2 ·h) was measured. The evaluation was made according to the following criteria. · Judgment criteria (score: judgment criteria) ◎ (Very good): Less than 8 〇 (Good): 8 or more and less than 11 △ (bad): 11 or more but less than 15 × (very bad): 15 or more

[0066] [Measurement of stratum corneum moisture content] The moisture content (μS) of the stratum corneum samples prepared (Sample No. 1 to Sample No. 8) was measured using Yayoi's SKICON-200EX. The evaluation was made according to the following criteria. · Judgment criteria (score: judgment criteria) ◎ (Very good): 100 μS or more Good: 75 μS or more and less than 100 μS △ (bad): 50 μS or more and less than 75 μS × (very bad): Less than 50 μS

[0067] (Reference example 1) [Visual evaluation by expert panel] For the prepared stratum corneum samples (Sample No. 1 to Sample No. 8), a panel of 10 experts visually observed the skin of the subjects from a distance of 50 cm in front of the subjects and evaluated their skin condition. The evaluation was made by comprehensively judging transparency, moisture, the presence or absence of dryness symptoms such as flaking, and clear observation of texture according to the following criteria: ◎ (very good), ○ (good), △ (bad), × (very bad).

[0068] (Reference example 2) [Microscopic evaluation (texture evaluation)] For the prepared stratum corneum samples (Sample No. 1 to Sample No. 8), the surfaces of the samples were observed using a microscope (RH-8800 manufactured by Hirox) for the subjects from whom the samples to be evaluated were collected, and the skin condition was evaluated. The evaluation was made according to the following criteria. · Judgment criteria (score: judgment criteria) ◎ (Very good): The texture is even, and the skin grooves and ridges are clearly defined. Good (Good): The texture is even, but the skin grooves are slightly shallow and the skin mounds are becoming flat. △ (bad): The texture is uneven and the skin grooves are shallow × (very bad): Texture cannot be distinguished

[0069] Figure 5 shows diagrams and photographs illustrating the results of evaluation of Reference Example 2 for stratum corneum samples (Sample No. 1 and Sample No. 8) from the cheek and arm regions of subjects. (A) is a photograph of the stratum corneum sample (Sample No. 1) observed under a microscope, which has been binarized to black and white. (B) is a photograph of the stratum corneum sample (Sample No. 8) observed under a microscope, which has been binarized to black and white. In Figure 5, the streaky areas (white areas) are skin grooves, and the other areas (black areas) are skin ridges. The stratum corneum sample from the arm shown in Figure 5(B) has a more uniform texture than the stratum corneum sample from the cheek shown in Figure 5(A).

[0070] [Table 1]

[0071] By comparing Example 1 with Comparative Example 1 and Reference Examples 1 and 2, it was shown that the state of the intercellular lipid packing structure of the stratum corneum (which cannot be captured by conventional skin evaluation methods) can be captured in more detail, and the actual state of the skin's barrier function can be objectively and correctly evaluated. A detailed analysis of the evaluation of Samples No. 1 to No. 7, which were made using stratum corneum derived from the cheek, is as follows. It can be said that the smaller the proportion of the liquid crystal region in Example 1, the higher the evaluation of the skin condition in Reference Examples 1 and 2. It can be said that the larger the proportion of the orthorhombic region in Example 1, the higher the evaluation of the skin condition in Reference Examples 1 and 2. It can be said that the larger the ratio of the area proportion of the orthorhombic region to the area proportion of the liquid crystal region in Example 1, the higher the evaluation of the skin condition in Reference Examples 1 and 2. On the other hand, it cannot necessarily be said that the smaller the TEWL of Comparative Example 1, the higher the evaluation of the skin condition in Reference Examples 1 and 2. In particular, among Samples No. 5 to No. 7, the evaluation of the skin condition in Reference Examples 1 and 2 was higher for Samples No. 5 and No. 7 and lower for Sample No. 6, whereas the evaluation of the TEWL of Comparative Example 1 was almost the same for Samples No. 5 to No. 7. From the above analysis, it can be said that for samples in which the TEWL value is within a range below a predetermined threshold, evaluation by TEWL may not be able to accurately evaluate skin conditions such as texture, whereas evaluation by the distribution of crystal structures in Example 1 can accurately evaluate skin conditions even for subjects whose skin conditions are above a certain level. [Industrial Applicability]

[0072] According to the present invention, the distribution of crystalline structures in intercellular lipids or pseudo-intercellular lipids can be evaluated in detail.

Claims

1. A method for evaluating the distribution of crystalline structures in intercellular lipids or pseudo-intercellular lipids.

2. 2. The method for evaluating a distribution of crystal structures according to claim 1, wherein the crystal structure includes at least one selected from the group consisting of liquid crystal, hexagonal crystal, and orthorhombic crystal.

3. The method for evaluating a distribution of a crystal structure according to claim 2 , wherein the crystal structure includes a liquid crystal and / or an orthorhombic crystal.

4. A method for evaluating the density of intercellular lipids or pseudo-intercellular lipids using the method for evaluating the distribution of crystal structures according to any one of claims 1 to 3, Based on the ratio of the area of ​​the liquid crystal region to the area of ​​the entire region or a part of the region of the intercellular lipid or the pseudo-intercellular lipid, A method for assessing the density of intercellular lipids or pseudo-intercellular lipids.

5. A skin evaluation method using the method for evaluating the distribution of crystal structures according to any one of claims 1 to 3, Based on the evaluation of the distribution of crystalline structures in the corneocyte region contained in the stratum corneum monolayer, Skin evaluation method.

6. The skin evaluation method according to claim 5 , based on the ratio of the area of ​​the liquid crystal region to the area of ​​the stratum corneum cell region contained in the monolayer.

7. The skin evaluation method according to claim 5 , based on the ratio of the area of ​​the rectangular crystal region to the area of ​​the stratum corneum cell region contained in the monolayer.

8. The skin evaluation method according to claim 5 , based on the ratio of the area of ​​the liquid crystal region and the area of ​​the orthorhombic crystal region to the area of ​​the stratum corneum cell region contained in the monolayer.

9. The skin evaluation method according to any one of claims 6 to 8, wherein a plurality of monolayers of the stratum corneum are used.

10. A method for selecting cosmetics and / or supplements that have the effect of improving the distribution of crystalline structures in intercellular lipids, based on the evaluation results of the skin evaluation method according to any one of claims 6 to 9.

11. The method for selecting cosmetics and / or supplements according to claim 10, wherein the cosmetics and / or supplements contain a component that promotes the elongation of hydrocarbon groups of ceramides or free fatty acids, which are lipids that constitute intercellular lipids.

12. A counseling method, comprising a method for selecting the cosmetic and / or supplement according to claim 10 or 11.

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