JP2013044525A - Evaluation method of property of horny layer of skin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of noninvasively, quickly and easily evaluating a property of a horny layer of the skin.SOLUTION: The evaluation method of the property of the horny layer of the skin in the present invention includes: measuring an infrared absorption spectrum by infrared spectroscopy with the horny layer of the skin of a human being as an object; dividing a superimposed peak of an α helix and a random coil and a peak of a β sheet from the peak of an amide I absorption band derived from a protein secondary structure in the infrared absorption spectrum; calculating an area of the superimposed peak of the α helix and the random coil and a peak area of the β sheet and calculating a peak area ratio β/α defined by [the peak area of the β sheet]/[the area of the superimposed peak of the α helix and the random coil]; and evaluating the property of the horny layer of the skin with the value of the calculated ratio β/α as an index.

Description

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

  In order to recommend products that are most suitable for customers in the sale of cosmetics and skin cleansing products, services that measure the moisture content, oil content, color tone, form, etc. of the customer's skin are widely used in stores. Also, in the process of research and development of cosmetics and skin cleansers, quantifying the state of skin by instrumental measurement can make more effective cosmetics and skin cleansers without taking a long development period. It is indispensable for development. For this reason, various technical developments for objectively evaluating the moisture content, oil content, color tone, form, etc. of the skin have been carried out.

  For example, Patent Document 1 proposes a method for evaluating skin flexibility and elasticity using an oxidized protein in the stratum corneum of skin as an index. In this method, the horny layer oxidized protein is detected by specifically fluorescently labeling the carbonyl group of the oxidized protein in the stratum corneum sample collected from the skin and detecting the fluorescence.

  It has also been proposed to change the secondary structure of protein in the stratum corneum of human skin by acrolein treatment and measure the intensity change in the infrared absorption spectrum of the amide I absorption band, which is the wavenumber region derived from skin protein. (Refer nonpatent literature 1).

JP 2006-349372 A

Iwai I, Ikuta K, Murayama K, Hirao T, Int. J. Cosmet. Sci., 2008 Feb; 30 (1): 41-6

  However, each of the above-described techniques is not a non-invasive method, and there is room for improvement in that the measurement requires time.

  Accordingly, an object of the present invention is to provide a method for evaluating the properties of the stratum corneum of the skin, which can eliminate the drawbacks of the prior art described above.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the properties of the horny layer of the skin such as texture are correlated with the structure of the protein constituting the horny layer, thereby completing the present invention. It came to.

The present invention has been made on the basis of the above findings, and an infrared absorption spectrum is measured by infrared spectroscopy for the stratum corneum of human skin,
From the peak of the amide I absorption band derived from the protein secondary structure in the infrared absorption spectrum, the peak where the α helix and the random coil are superimposed and the peak of the β sheet are divided,
Calculate the peak area of α helix and random coil superimposed and the peak area of β sheet, and the peak defined by [β sheet peak area] / [α helix and random coil overlap peak area] Calculate the area ratio β / α,
The present invention provides a method for evaluating the properties of the horny layer of the skin, which evaluates the properties of the horny layer of the skin for purposes other than medical practice, using the calculated ratio β / α as an index.

  According to the present invention, the properties of the stratum corneum of skin can be easily evaluated non-invasively and quickly.

FIG. 1 is a schematic diagram showing the structure of a total reflection absorption spectrum measurement probe suitably used in the method of the present invention. 2A to 2D are schematic views showing various aspects of the measurement head unit in the total reflection absorption spectrum measurement probe shown in FIG. FIG. 3 is a schematic view of a total reflection infrared absorption spectrum measuring apparatus including the total reflection absorption spectrum measuring probe shown in FIG. FIG. 4 is a diagram showing an infrared absorption spectrum of the stratum corneum of skin. FIG. 5 is a diagram showing a state in which the peak of the amide I absorption band derived from the protein secondary structure in the infrared absorption spectrum shown in FIG. 4 is divided. FIG. 6 is a diagram showing the result of measuring the infrared absorption spectrum of the stratum corneum of human forearm skin as a model experiment and the calculation result of curve fitting of the infrared absorption spectrum. FIGS. 7A to 7E are graphs showing the relationship between the ratio β / α and the amount of water transpiration at each part of the body.

  The present invention will be described below based on preferred embodiments with reference to the drawings. In the method of the present invention, an infrared absorption spectrum is measured by infrared spectroscopy using a stratum corneum of human skin as a measurement target. For the measurement of the infrared absorption spectrum, a known apparatus capable of measuring the surface of human skin can be used without particular limitation. As such an apparatus, for example, an apparatus provided with a total reflection absorption spectrum measuring probe shown in FIG.

  In FIG. 1, reference numeral 1 denotes an infrared optical fiber for guiding infrared light a and total reflection light b attenuated by absorption by skin. As the infrared optical fiber 1, one formed of chalcogenite, one formed of a mixed crystal of AgCl and AgBr, one made of AgCl alone, one made of AgBr alone, or the like can be used.

  A part of the infrared optical fiber 1 is curved, whereby the function of an ATR (attenuated total reflection) prism is given to the infrared optical fiber 1 itself. The curved infrared optical fiber 1 forms a convex measuring head portion 2 as shown in FIG.

  The core layer of the infrared optical fiber in the measurement head unit 2 is exposed. By bringing the exposed core layer into close contact with the skin, the infrared light guided to this portion is totally reflected at the interface with the skin. Then, the totally reflected light attenuated by the absorption of the skin is again guided to the infrared optical fiber, and the infrared absorption spectrum of the skin is measured.

  The shape of the measurement head unit 2 may be any shape as long as the function of the ATR prism is exhibited and the skin surface can be sufficiently contacted. For example, in addition to a smooth loop shape as shown in FIG. 2 (a), a square shape having two corners as shown in FIG. 2 (b), a sharp shape as shown in FIG. 2 (c), Or what was wound by the coil shape etc. as shown in FIG.2 (d) can be used.

  Returning to FIG. 1 again, the infrared optical fiber 1 only needs to be exposed at the measurement head unit 2, and the other part is usually covered with a clad layer and is stored and held by the holding member 3. Preferably it is. Examples of the holding member 3 include a tube or a cylindrical sheath formed of insulating polyvinyl chloride, fluororesin, synthetic rubber, or the like.

  Reference numeral 4 in FIG. 1 denotes a support that supports the measurement head. The support 4 is installed on the side opposite to the contact surface with the skin in the measurement head unit 2 where the convex curvature of the tip of the measurement head unit is negative, that is, the measurement head unit 2 in which the infrared optical fiber 1 is convex. The material of the support 4 is not particularly limited as long as it does not damage the infrared optical fiber 1 and the skin. For example, an elastic material such as a foaming agent, soft rubber, or urethane can be used.

  FIG. 3 shows a schematic diagram of a total reflection infrared absorption spectrum measuring apparatus provided with the probe shown in FIG. In the figure, reference numeral 5 denotes an infrared light irradiation unit for generating and irradiating infrared light, and reference numeral 6 denotes an interferometer unit. Reference numeral 7 denotes a detection unit for detecting infrared light reflected from the skin. Reference numeral 8 denotes a calculation unit, which is provided with calculation means for correcting fluctuations in absorbance. In this measuring apparatus, infrared light is guided from the infrared light irradiation unit 5 to the measurement head unit 2 through the infrared optical fiber 1. The infrared light totally reflected at the interface between the infrared optical fiber probe and the skin is again guided to the infrared optical fiber 1 and reaches the detection unit 7. The infrared light that has reached the detection unit 7 is converted into an electric signal, and the electric signal is processed in the calculation unit 8 to calculate an infrared absorption spectrum. As the infrared light irradiation part 5, the thing of the structure similar to the infrared light irradiation part of a general infrared spectrometer is mentioned. That is, a combination of an infrared light source and an interferometer, and a combination of an infrared light source and a spectroscope are included.

Details of the probe and the total reflection infrared absorption spectrum measuring apparatus having the above structure are described in, for example, Japanese Patent Application Laid-Open No. 2008-241593 related to the earlier application of the present applicant. Moreover, it can replace with the apparatus described in the said gazette, and can also use the apparatus as described in Unexamined-Japanese-Patent No. 2009-109282 based on the previous application of this applicant. Furthermore, a commercial item can also be used as said probe. Examples of such commercially available products include PIR optical fiber type IR-ATR probes (manufactured by Systems Engineering Co., Ltd.).
The same measurement can be performed using a probe of the type in which an ATR prism is attached to the tip of an infrared optical fiber. Examples of the material of the prism at this time include Ge, ZnSe, diamond, Si, and quartz.

  The measurement is generally performed in an environment of 20 to 30 ° C. and 30 to 60% RH, but is not limited thereto. As will be described later, no habituation of the subject is required for the measurement. Further, it is not necessary to perform any pretreatment on the measurement site.

The IR-ATR (infrared attenuated total reflection) spectrum of human skin measured using the above probe and the total reflection infrared absorption spectrum measurement apparatus is as shown in FIG. 4, for example. As shown in the figure, in the infrared absorption spectrum, a peak is observed in the vicinity of 1744 cm ⁻¹ on the highest wavenumber side. This peak is derived from the CO stretching vibration in the OCO of the ester type fatty acid among the sebum in the skin. The peak near 1711 cm ⁻¹ observed next to it is derived from CO stretching vibration in COOH of free fatty acids among sebum in the skin. A large peak near 1651 cm ⁻¹ observed on the lower wavenumber side than this peak is attributed to the amide I absorption band derived from the protein secondary structure in the skin. A peak around 1603 cm ⁻¹ derived from natural moisturizing factor (NMF) is observed on the lower wave number side than this peak.

In the infrared absorption spectrum shown in FIG. 4, a large peak near 1651 cm ⁻¹ belonging to the amide I absorption band derived from the protein secondary structure is actually a plurality of peaks derived from a plurality of protein secondary structures. Is superimposed. Specifically, as shown in FIG. 5, the peak attributed to the amide I absorption band is obtained by superimposing a plurality of peaks including α-helix and β-sheet of protein secondary structure. In the present invention, the peak attributed to the amide I absorption band and the peak of α helix and random coil superimposed and the peak of β sheet are divided, and the area ratio β / α of these peaks, that is, the peak of β sheet [Area] / [Area of peak where α helix and random coil are superimposed] is calculated. The present inventors have found that the value of this ratio β / α is an index reflecting the properties of the stratum corneum of the skin. The ratio β / α reflects the secondary structure of the proteins that make up the stratum corneum.

  There is no particular limitation on the means for dividing the peak attributed to the amide I absorption band derived from the protein secondary structure to the peak where α helix and random coil are superimposed and the peak of β sheet, and a conventionally known means should be used appropriately. Can do. Examples of such means include a curve fitting method, a difference spectrum method, and a spectrum synthesis method. Particularly preferred means in the present invention is the curve fitting method.

  The curve fitting method is a method for quantitatively estimating the contribution of each signal component by expressing the infrared absorption spectrum of the horny layer of the skin by superimposing an appropriate function. By using the curve fitting method, the absorption band of each component can be separated, and the exact position and area of each absorption band can be calculated. Examples of the function form used in the curve fitting method include a Gaussian function, a Lorentz function, a Forked function, and the like. The skin is composed of proteins, lipids, NMF, etc., and its infrared absorption spectrum is extremely complex, but it is possible to express its spectral shape well by superimposing appropriate functions (for example, Gaussian functions). .

  In order to actually perform curve fitting, commercially available numerical analysis software may be used, or dedicated software may be created. As commercially available software, for example, IGOR Pro 6.0 (Hulinks Co., Ltd.) and Origin 8.0 (Light Stone Co., Ltd.) can be used. For example, in IGOR Pro 6.0, which is now widely used in the field of spectroscopy, linear, polynomial, sine, exponential, double exponential, Gaussian, Lorentz, as built-in regression functions for regression analysis (same as curve fitting) Hill's differential equation, sigmoid, lognormal, Gaussian 2D (two-dimensional Gaussian peak), and polynomial 2D (two-dimensional polynomial) are prepared. Read the infrared absorption spectrum you want to analyze into IGOR Pro 6.0, place the required number of arbitrary functions (for example, Gaussian functions) at wave number positions where the spectral shape can be best expressed, and fix parameters (for example, peak position and peak) Width, etc.) and parameters to be made variable (for example, peak height). After that, if the regression analysis function is executed, this software will output a variable parameter value group that minimizes the sum of squared errors at each data point of the spectrum synthesized by superimposing the function and the measured infrared absorption spectrum. .

When creating dedicated software, for example, it can be created on Visual Basic 6.0 or Visual C ++, which are typical programming languages. As in the case of the commercially available software described above, software that approximates the shape of the infrared absorption spectrum to be analyzed by least square approximation by superimposing a nonlinear function such as a Gaussian function is described. As the least square approximation method, for example, by using the least square Taylor differential correction method, a variable parameter that minimizes the sum of square errors at each data point of the spectrum synthesized by superimposing the function and the actually measured infrared absorption spectrum. A value group can be calculated. For the programming method of the least square Taylor differential correction method, the following books are helpful.
"Waveform data processing for scientific measurement", Minami, CQ publisher "Experimental data analysis by least squares", Nakagawa, Koyanagi, University of Tokyo Press "Numerical Recipes in C" by H. w. Press, S. A. Teukolsky, W. T. Vetterling, and B. P. Flannery, Cambrige University Press (1988)
Japanese translation: “Recipes for numerical computation in C language”, Technical Review

When the above-mentioned means are employed to divide the peak attributed to the amide I absorption band derived from the protein secondary structure into the peak of α helix and random coil and the peak of β sheet, the α helix and random coil The superimposed peak is attributed around 1649 cm ⁻¹ , and the β sheet peak is attributed near 1629 cm ⁻¹ . Then, the areas of these peaks are calculated, and the peak area ratio β / α is calculated. A specific method for calculating the peak area ratio β / α is as described below. In the following explanation, human forearm skin is measured as a model experiment.

The infrared absorption spectrum is measured by, for example, the following apparatus configuration.
(1) FTIR: Nicolet 380 (trade name), manufactured by Thermo Scientific (2) Optical fiber coupling unit: FiberMate (trade name), manufactured by Systems Engineering (3) PIR optical fiber type IR-ATR probe: manufactured by Systems Engineering (4) Computer for device control and data processing

  IR-ATR measurement of human forearm skin was performed using the apparatus configured as described above. The measurement results are as shown in FIG. Usually, the analysis depth of IR-ATR measurement is about 1 μm, which is sufficiently shallower than the stratum corneum of general human skin (about 15 μm). Therefore, the IR-ATR spectrum of the skin obtained with this device configuration can be regarded as the IR-ATR spectrum of the stratum corneum.

The region of 1000-1800 cm ^{−1 in} the obtained IR-ATR spectrum was fitted by a function expressed by superposition of 30 Gauss functions. This function is expressed by the following equation (1).

  Table 1 below shows parameters corresponding to the peak positions and peak widths of the 30 Gaussian functions used for fitting in the above equation (1). Fitting was performed by the least square Taylor differential correction method described in the above-mentioned “waveform data processing for scientific measurement”. The fitting program was written in Visual Basic 6.0.

FIG. 6 shows the result of fitting together with the measured IR-ATR spectrum. The strong absorption band near 1650 cm ⁻¹ in the figure is an absorption band called amide I of the protein. In this fitting, the absorption band of amide I has three Gaussian functions (center position: 1668c).
m ⁻¹ , 1649 cm ⁻¹ , 1629 cm ⁻¹ ). Generally absorption and absorption of the amide bond of a protein took α- helical structure in the vicinity of 1649 cm ^-1, it is said to be absorption and absorption of the amide bond of a protein took a random coil structure is superimposed, 1629cm ^- Absorption near ¹ is said to be absorption of an amide bond of a protein having a β-sheet structure. Therefore the area of the Gaussian function of 1629cm ^-1, a ratio alpha / beta in the area of the Gaussian function of 1649 cm ^-1, was calculated using the following equation (2).
α / β = (C (1629 cm ⁻¹ ) × W (1629 cm ⁻¹ )) / (C (1649 cm ⁻¹ ) × W (1649 cm ⁻¹ )) (2)
(Where C (1629 cm ⁻¹ ) and C (1649 cm ⁻¹ ) represent the coefficients of the Gaussian function at each wave number, and W (1629 cm ⁻¹ ) and W (1649 cm ⁻¹ ) are Gauss at each wave number. Represents a parameter that reflects the width of the function.

In general, the IR spectrum of skin shows a shape in which lipids, NMF (amino acids) and the like are superimposed on a signal mainly composed of a protein as a main component. Detailed investigations have already been made on the position of infrared absorption of proteins, lipids, amino acids, etc. and their vibration modes, and many findings have been accumulated. Based on these findings, the present inventors examined the superposition of a minimum number of Gaussian peaks that can express the IR spectrum of the skin in the region of 1000 to 1800 cm ⁻¹ , and as shown in Table 1 below. A combination of 30 Gaussian functions reflecting 30 vibration modes could be selected.
Next, the combination of the peak width and peak position of these 30 Gaussian functions was set as a constant, and fitting to the skin IR spectrum was performed by the least square method using each peak intensity as a variable. The combination of peak width and peak position was repeatedly examined so that the residual sum of squares of the measured spectrum and simulation spectrum at this time was minimized. Table 1 below shows combinations of peak widths and peak positions of 30 Gaussian functions that can best express the IR spectrum of the skin in the region of 1000 to 1800 cm ⁻¹ by conducting this examination on various skin IR spectra. The indicated parameter group was obtained.

  Based on the above method, FIG. 7 shows the result of examining the relationship between the ratio β / α obtained from the infrared absorption spectrum measured for a specific part of the subject's body and the properties of the stratum corneum at the part. (A) to (e). In the figure, attention is paid to the amount of moisture transpiration, which is one of the properties of the stratum corneum. Moreover, the specific measurement site | parts in the figure are a cheek part, a palm part, a finger part, a shin part, and a buttocks part. The number of subjects is 40. As is apparent from the results shown in FIGS. 7A to 7E, it can be seen that there is a positive correlation between the ratio β / α and the amount of water transpiration, although there is some variation depending on the measurement site. From these results, it is understood that the amount of water transpiration can be evaluated based on the value of the ratio β / α obtained from the measurement of the infrared absorption spectrum. Specifically, it can be evaluated that the greater the ratio β / α, the greater the amount of moisture transpiration, and the smaller the ratio β / α, the less the amount of moisture transpiration.

  The amount of water transpiration is a measure of the degree of water transpiration from the skin to the outside of the body. Therefore, a small amount of moisture transpiration means that the skin moisture barrier ability is high. Therefore, according to the present invention, the moisture barrier ability as the property of the horny layer of the skin can be evaluated based on the value of the ratio β / α.

  The amount of moisture transpiration is measured by the following method. As an apparatus, an apparatus is used in which two pieces of an upper humidity sensor and a lower humidity sensor are installed inside a cylindrical body that is open at the top and bottom. The lower open end of the cylindrical body is brought into airtight contact with the skin of the subject, and the moisture concentration in the cylindrical body is measured with the upper humidity sensor and the lower humidity sensor in this state. Then, the amount of moisture transpiration per unit time and unit area is determined from the difference between the measured values of the two humidity sensors. This measurement is based on the fact that the moisture concentration decreases from the lower part of the cylinder to the upper part. As a specific apparatus, a commercially available device, for example, a sealed moisture evaporation measuring device (Evaporator) or a moisture transpiration measuring device (Tevameter) can be used. In addition, when measuring with such a conventional apparatus, there is a problem that the measurement location is limited and stable data is difficult to obtain in the first place because it is easily affected by the external environment such as wind. Therefore, the measurement may be carried out using a moisture transpiration measuring device that solves this problem, for example, a device described in JP-A-2001-190502 or JP-A-10-286238.

  As is clear from the measurement method described above, the evaluation method of the present invention does not require habituation of the subject. Therefore, if a calibration curve of the relationship between the ratio β / α and the amount of water transpiration is prepared in advance, the acclimation of the subject is achieved. The amount of moisture transpiration from the skin and the barrier ability of the skin can be predicted by a simple operation of simply measuring the ratio β / α without performing it.

  Further, according to the present invention, not only the skin barrier ability using the amount of moisture transpiration from the skin as an index, but also the skin texture as the property of the stratum corneum can be evaluated.

  As is clear from the above description, the method of the present invention is suitably used for purposes other than medical practice such as the development of cosmetics and pharmaceuticals based on the evaluation of the properties of the stratum corneum and the selection of appropriate cosmetics in face-to-face sales of cosmetics. It is done.

DESCRIPTION OF SYMBOLS 1 Infrared optical fiber 2 Measuring head part 3 Holding member 4 Support body 5 Infrared light irradiation part 6 Interferometer part 7 Detection part 8 Calculation part

Claims (3)

Measure the infrared absorption spectrum by infrared spectroscopy for the stratum corneum of human skin,
From the peak of the amide I absorption band derived from the protein secondary structure in the infrared absorption spectrum, the peak where the α helix and the random coil are superimposed and the peak of the β sheet are divided,
Calculate the peak area of α helix and random coil superimposed and the peak area of β sheet, and the peak defined by [β sheet peak area] / [α helix and random coil overlap peak area] Calculate the area ratio β / α,
A method for evaluating the properties of the horny layer of the skin, wherein the property of the horny layer of the skin is evaluated for purposes other than medical practice, using the calculated ratio β / α as an index.   The evaluation method according to claim 1, wherein the amount of water transpiration as a property of the horny layer of the skin is evaluated based on the value of the ratio β / α.   The evaluation method according to claim 2, wherein the larger the value of the ratio β / α, the larger the amount of water transpiration, and the smaller the value of the ratio β / α, the smaller the amount of water transpiration.