Method for evaluating condition of skin, and screening method

Abstract

Provided is a method for evaluating the condition of skin, the method comprising: a step for irradiating the skin of interest with excitation light of an electron carrier, and measuring the fluorescence lifetime of the electron carrier in a granular layer by using a multi-photon scanning laser biological microscope; a step for analyzing the degree of oxidative phosphorylation of the electron carrier in the granular layer on the basis of the fluorescence lifetime of the electron carrier; and a step for evaluating the condition of the skin in accordance with the degree of the oxidative phosphorylation in the granular layer. In the method, the electron carrier is at least one component selected from among nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and reduced flavin adenine dinucleotide (FADH2).

Description

Skin condition evaluation method and screening method

[0001] The present invention relates to a method for evaluating a skin condition and a screening method.

[0002] It is known that epidermal cell differentiation and cell proliferation differ in pigmented areas compared to normal, non-pigmented areas. Although attempts have been made to visualize the epidermal and dermal structures inside the skin non-invasively, there have been no examples of quantitatively visualizing the metabolic state of the epidermis in spatiotemporal terms in an actual pigmented area under non-invasive conditions.

[0003] A method for visualizing the distribution of melanin in biological tissue has been reported so far as a method for providing accurate and reliable discrimination of melanin in biological tissue without damaging the biological tissue (see, for example, Patent Document 1).

[0004] Furthermore, as a method for non-invasively and directly quantifying the collagen state inside the skin, such as the dermis layer, a method has been reported in which the density of collagen inside the skin is quantified by detecting second high-frequency generated light (see, for example, Patent Documents 2 and 3).

[0005] JP 2009-142597 A JP 2009-236610 A JP 2012-235804 A

[0006] An object of the present invention is to provide a method for evaluating skin condition that can non-invasively evaluate skin condition and provide a new index for evaluating dark spots.

[0007] A method for evaluating skin condition according to one aspect of the present invention as a means for solving the above-mentioned problems comprises the steps of: irradiating the skin of a subject to be measured with excitation light for an electron carrier; measuring the fluorescence lifetime of the electron carrier in the granular layer using a multiphoton scanning laser biomicroscope; analyzing the degree of oxidative phosphorylation of the electron carrier in the granular layer based on the fluorescence lifetime of the electron carrier; and evaluating the skin condition according to the degree of oxidative phosphorylation in the granular layer, wherein the electron carrier is selected from the group consisting of nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), reduced flavin adenine dinucleotide (FADH), and phospholipids. ２ ) is at least one of the following.

[0008] According to one aspect of the present invention, it is possible to provide a method for evaluating skin condition that can non-invasively evaluate skin condition and provide a new index for evaluating spots.

[0009] FIG. 1 is a schematic diagram illustrating a method for measuring the fluorescence lifetime of an electron carrier in the granular layer. FIG. 2 is a schematic diagram illustrating an example of a multi-photon scanning laser biomicroscope that can be used in the skin condition evaluation method of this embodiment. FIG. 3 is an external photograph showing the measurement site of one subject in an example. FIG. 4 is a tomographic image of the granular layer obtained by observing the non-blemish site in FIG. 3 with a multi-photon scanning laser biomicroscope. FIG. 5 is a tomographic image of the granular layer obtained by observing the blemish site in FIG. 3 with a multi-photon scanning laser biomicroscope. FIG. 6 is a diagram illustrating a target region set in a tomographic image of the granular layer in an example. FIG. 7 is a graph showing the relationship between the fluorescence lifetime and the number of fluorescence detection events in a non-pigmented site. FIG. 8 is a graph showing the relationship between the fluorescence lifetime and the number of fluorescence detection events in a pigmented site. 9 is a graph showing the distribution of A2 / A1 values ​​of all subjects, which is the ratio (A2 / A1) of the fluorescence lifetime amplitude A2 derived from NAD(P)H molecules with a short fluorescence lifetime to the fluorescence lifetime amplitude A1 derived from NAD(P)H molecules with a long fluorescence lifetime. ａｖ T of all subjects in (Int)ａｖ 10 is a graph showing the distribution of (Int) values.

[0010] (Method for Evaluating Skin Condition) The method for evaluating skin condition of the present embodiment includes a step of irradiating the skin to be measured with excitation light for an electron carrier and measuring the fluorescence lifetime of the electron carrier in the granular layer using a multiphoton scanning laser biomicroscope (measurement step), a step of analyzing the level of oxidative phosphorylation of the electron carrier in the granular layer based on the fluorescence lifetime of the electron carrier (analysis step), and a step of evaluating the skin condition in accordance with the level of oxidative phosphorylation in the granular layer (evaluation step), and further includes other steps.

[0011] The electron carriers include nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and reduced flavin adenine dinucleotide (FADH ２ ) is at least one of the following.

[0012] Non-invasive visualization and investigation of the metabolic state, such as cell proliferation and differentiation state, within the skin is important for evaluating skin condition and is thought to lead to the development of products for treating pigmentation and sunscreen products, as well as support for skin evaluation. As mentioned above, attempts have been made to visualize epidermal and dermal structures by non-invasively detecting melanin, collagen, and the like within the skin. However, no examples have been reported in which the metabolic state of the epidermis has been visualized spatiotemporally and quantitatively in an actual pigmented area under non-invasive conditions. Furthermore, although changes in metabolites within the epidermis due to UV and aging have been experimentally investigated, no examples have been reported in which non-invasive live imaging of changes in metabolites in the epidermis at pigmented areas has been used to provide in vivo insights into pigmented areas.

[0013] The present inventors measured and analyzed the fluorescence lifetime of the electron carrier in the granular layer of skin in pigmented and non-pigmented areas, and found that the level of metabolism of oxidative phosphorylation based on the fluorescence lifetime of the electron carrier differs between pigmented and non-pigmented areas and is associated with pigmented and non-pigmented areas. Therefore, the present inventors discovered that skin condition can be evaluated based on the level of oxidative phosphorylation in the granular layer, enabling non-invasive evaluation of skin condition and providing a new index for evaluating pigmented areas, leading to the completion of the present embodiment.

[0014] Until now, the degree and distribution of melanin deposition and epidermal cell division and differentiation have been used as the main indicators for diagnosing pigmentation sites and targeting drugs. However, these are merely indicators that represent the pigmentation phenotype. The skin condition evaluation method of the present embodiment can provide an indicator based on the fluorescence lifetime of electron carriers as a new pigmentation evaluation indicator that enables non-invasive evaluation of the metabolic state of cells in the granular layer, i.e., the level of oxidative phosphorylation, which is upstream of cell division and differentiation that cause pigmentation, and can contribute to non-invasive evaluation and prediction of skin condition.

[0015] <Measuring Step> The measuring step is a step of irradiating the skin of the measurement subject with excitation light for an electron carrier and measuring the fluorescence lifetime of the electron carrier in the granular layer using a multiphoton scanning laser biomicroscope, and can be suitably performed using a multiphoton scanning laser biomicroscope as a measuring means.

[0016] The skin to be measured can be selected appropriately depending on the purpose, but is preferably human skin, and more preferably at least one of the temple, cheek, back of the hand, and arm. The skin to be measured may also be at least one of cultured skin tissue extracted from a human, reconstituted cultured skin tissue, and artificial cultured skin tissue, and can be suitably used in the screening method described below.

[0017] -Electron Carrier- Examples of the electron carrier include nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and reduced flavin adenine dinucleotide (FADH). ２ ) and NADH and NADPH are preferred in that desired excitation light can be used in available multiphoton scanning laser biological microscopes. Here, either NADH or NADPH may be collectively referred to as NAD(P)H or NAD(P)H molecule. Also, FAD and FADH ２ These are sometimes collectively referred to as FAD.

[0018] Intracellular NAD(P)H has the property of absorbing light having an excitation wavelength (the wavelength of the excitation spectrum having a peak at approximately 340 nm) and emitting fluorescence (the fluorescence spectrum having a peak at approximately 450 nm). The time it takes for the fluorescence emitted from NAD(P)H to decay (i.e., the fluorescence lifetime) varies depending on the form of NAD(P)H, with protein-bound NAD(P)H having a long fluorescence lifetime of 2.5 ns to 4 ns and free NAD(P)H having a short fluorescence lifetime of 0.1 ns to 0.5 ns.

[0019] Furthermore, FAD has the property of absorbing light having an excitation wavelength (a wavelength of an excitation spectrum having a peak at about 450 nm) and emitting fluorescence (a fluorescence spectrum having a peak at about 525 nm).

[0020] Based on these properties of the electron carrier, the fluorescence lifetime of the electron carrier is measured in the measurement step, and the state of the electron carrier such as NADH and the degree of oxidative phosphorylation can be analyzed in the analysis step described below.

[0021] Fig. 1 is a schematic diagram illustrating a method for measuring the fluorescence lifetime of an electron carrier in the granular layer. As shown in Fig. 1, skin is irradiated with electron carrier excitation light 1 using ultrashort pulsed light, and the fluorescence 2 generated from the electron carrier is detected over time, thereby making it possible to measure the fluorescence lifetime of the electron carrier, particularly in the granular layer of the epidermis 4. In Fig. 1, reference numeral 3 denotes the stratum corneum, and reference numeral 5 denotes the dermis.

[0022] As the multi-photon scanning laser biomicroscope, for example, MPTcompact (manufactured by JenLab), DermaInspect (manufactured by JenLab), MPTflex (manufactured by JenLab), etc. can be suitably used.

[0023] A multiphoton scanning laser biomicroscope can noninvasively obtain tomographic images of skin tissue at a desired depth from the epidermis to the upper dermis by scanning the laser irradiation in the plane direction and depth, thereby enabling specific measurement and analysis of the granular layer, which is the target of the skin condition evaluation method of this embodiment. Furthermore, by using a femtosecond laser (e.g., wavelength: 780 nm) to irradiate pulsed light having a wavelength twice the excitation wavelength of the electron carrier, two-photon excitation is induced, allowing measurement of strong signals across the entire fluorescence spectrum. Furthermore, a near-infrared laser, which has excellent permeability through biological tissue, can noninvasively obtain microscopic images of skin tissue several hundred micrometers deep.

[0024] The excitation light for the electron carrier can be appropriately selected depending on the purpose, as long as it has a wavelength capable of exciting the electron carrier. However, the wavelength is preferably a wavelength that generates multiphoton excitation, such as a wavelength that generates two-photon excitation (a wavelength approximately twice the excitation wavelength). Furthermore, 700 nm to 1000 nm is preferred in terms of low light absorption and scattering by biological tissue. Among these wavelengths, wavelengths that generate two-photon excitation (a wavelength approximately twice the excitation wavelength, approximately 780 nm for NAD(P)H and approximately 900 nm for FAD) are more preferred in terms of enabling more non-invasive measurement of skin tissue and efficient measurement of fluorescent signals.

[0025] The fluorescence lifetime of the electron carrier can be measured by fluorescence lifetime imaging microscopy (FLIM). Specifically, the decay time (fluorescence lifetime) of the fluorescence emitted from the electron carrier can be measured using the multiphoton scanning laser biomicroscope and a femtosecond laser. As the multiphoton scanning laser biomicroscope, for example, an MPTcompact or an MPTflex can be used.

[0026] Fig. 2 is a schematic diagram showing an example of a multi-photon scanning laser biomicroscope that can be used in the skin condition evaluation method of this embodiment. The multi-photon scanning laser biomicroscope 100 shown in Fig. 2 includes a femtosecond laser light source 101, a high-speed galvanometer scanner 102, beam splitters 103 and 104, a piezoelectric element 105 for Z-axis fine movement, an objective lens 106, filters 107, 108, 109, and 120, an autofocus (AF) 121, a photon counter 122, and an SHG detector 123.

[0027] The femtosecond laser light source is capable of outputting laser light (IR light) with an output power of 1 mW to 50 mW and a pulse width of 90 fs to 200 fs or less. A wavelength-tunable type (710 nm to 920 nm) with a mode-locking frequency of 50 MHz to 80 MHz is more preferred. The wavelength of the femtosecond laser light source is preferably 700 nm to 1000 nm, which is less absorbed and scattered by biological tissue. A wavelength capable of two-photon excitation (approximately 760 nm to 780 nm for NAD(P)H and approximately 900 nm for FAD) is more preferred, as it can sufficiently excite electron carriers present in biological tissue (shorter wavelengths are preferable) and minimizes damage to biological skin (longer wavelengths are preferable).

[0028] The laser light emitted from the femtosecond laser light source 101 passes through a high-speed galvanometer scanner 102 for scanning on the XY plane in the granular layer of the skin 124 to be measured, and is focused by an objective lens 106 to a single point at a predetermined depth in the skin tissue so that the granular layer of the skin 124 to be measured can be detected.

[0029] The fluorescence emitted from the electron carrier present in the granular layer passes through the objective lens 106 and the beam splitter 103 and is detected by the photon counter 122 .

[0030] In order to detect as many photons as possible, it is preferable not to limit the wavelength during detection by a filter or the like. However, if there is a risk that light that is easily mistaken for fluorescence of the electron carrier may be generated from substances other than the electron carrier present in the granular layer (e.g., collagen or melanin), it is preferable to install filters 107 and 108 such as bandpass filters between the beam splitter 103 and the photon counter 122 to remove this easily mistaken light.

[0031] For example, when collagen may be present in biological tissue, second harmonic generation (SHG) light is generated from collagen molecules in the biological tissue, and since this SHG light may be mistaken for the fluorescence of the electron carrier, it is preferable to install a bandpass filter (450±40 nm) as a filter to remove this SHG light. Furthermore, from the viewpoint of preventing noise derived from melanin, it is preferable to analyze the fluorescence lifetime of the electron carrier using the fluorescence lifetime of melanin (<100 ns) as a cutoff value.

[0032] A fluorescence decay curve of the electron carrier is created based on the detected fluorescence data of the electron carrier, and the fluorescence decay curve (τ) is approximated to a quadratic decay function shown in the following equation to determine four parameters related to the fluorescence lifetime (fluorescence lifetime (τ1, τ2), amplitude relative to the fluorescence lifetime (A1, A2)).

[0033] τ(t): Fluorescence decay function t: Time τ1: Fluorescence lifetime derived from an electron carrier with a long fluorescence lifetime τ2: Fluorescence lifetime derived from an electron carrier with a short fluorescence lifetime A1: Fluorescence lifetime amplitude derived from an electron carrier with a long fluorescence lifetime A2: Fluorescence lifetime amplitude derived from an electron carrier with a short fluorescence lifetime

[0034] <Analysis step> The analysis step is a step of analyzing the degree of oxidative phosphorylation of the electron carrier in the granular layer based on the fluorescence lifetime of the electron carrier, and can be suitably performed using analysis software (e.g., SymphoTime, PicoQuant) of the multiphoton scanning laser biomicroscope as an analysis means.

[0035] Examples of methods for analyzing the level of oxidative phosphorylation of the electron carrier in the granular layer based on the fluorescence lifetime of the electron carrier include the following: In a tomographic image of the granular layer obtained by the multiphoton scanning laser biomicroscope, a region of cells that does not contain melanin and that is not a nucleus is selected as one region of interest (ROI), and multiple ROIs (e.g., 5 ROIs) are set. For each ROI, a model fit is performed for two components: a fluorescence lifetime amplitude A1 derived from a protein-bound electron carrier with a long fluorescence lifetime, and a fluorescence lifetime amplitude A2 derived from a free electron carrier with a short fluorescence lifetime. The obtained fluorescence lifetime amplitude A1 and fluorescence lifetime amplitude A2 can be used as indicators to analyze the level of oxidative phosphorylation. Analysis conditions include, for example, cells with a high model explanation rate (explanation rate X ２ = 0.9 to 1.4), the peak top time can be limited to the range of the NAD(P)H molecule (short fluorescence lifetime: 0.14 ns to 0.52 ns, long fluorescence lifetime: 2.65 ns to 3.69 ns) for analysis.

[0036] Here, from the viewpoint of preventing noise derived from melanin, it is preferable to select cells that do not contain melanin when selecting an ROI and to perform the analysis using the fluorescence lifetime of melanin as a cutoff value (e.g., <100 ns). Autofluorescence derived from melanin can be observed and recognized as clumps of melanin accumulation in autofluorescence images of epidermal cells near the basal layer. Furthermore, histologically, the intracellular distribution of melanin is rarely observed in the granular layer just below the stratum corneum, even under a conventional optical microscope. From the above, it is possible to select cells that do not contain melanin in the granular layer.

[0037] - Degree of oxidative phosphorylation - The degree of oxidative phosphorylation can be determined using as an index the fluorescence lifetime amplitude A1 derived from an electron carrier having a long fluorescence lifetime and the fluorescence lifetime amplitude A2 derived from an electron carrier having a short fluorescence lifetime, and is preferably determined using at least one of the following (1) and (2) as an index, and more preferably using the following (1) and (2): (1) the ratio (A2 / A1) of the fluorescence lifetime amplitude A2 derived from the electron carrier having a short fluorescence lifetime to the fluorescence lifetime amplitude A1 derived from the electron carrier having a long fluorescence lifetime, and (2) the intensity-weighted average fluorescence lifetime T based on the fluorescence intensity of the electron carrier ａｖ (Int)

[0038] As described above, electron carriers with long fluorescence lifetimes correspond to protein-bound electron carriers, and a high fluorescence lifetime amplitude A1 derived from electron carriers with long fluorescence lifetimes indicates that oxidative phosphorylation (OXPHOS) metabolism is dominant. On the other hand, electron carriers with short fluorescence lifetimes correspond to free electron carriers, and a high fluorescence lifetime amplitude A2 derived from electron carriers with short fluorescence lifetimes indicates that glycolytic metabolism is dominant. Therefore, when compared with the control, a relatively high ratio (A2 / A1) indicates a relatively low oxidative phosphorylation metabolism, and a relatively low ratio (A2 / A1) indicates a relatively high oxidative phosphorylation metabolism.

[0039] Furthermore, the intensity-weighted mean fluorescence lifetime T based on the fluorescence intensity of the electron carrier ａｖ (Int) indicates the average value of the fluorescence lifetime weighted by the fluorescence intensity of the electron carrier, and serves as an index showing whether the fluorescence lifetime amplitude is leaning toward the fluorescence lifetime amplitude A1 or the fluorescence lifetime amplitude A2.

[0040] In a normal epidermal layer structure, it is known that glycolysis is dominant in the basal layer where cell differentiation occurs, while oxidative phosphorylation metabolism is dominant in the granular layer, which rises to the upper layer due to skin metabolism (turnover).

[0041] As is clear from the following examples, when comparing a spotted area and a non-spotted area in the same measurement subject, the ratio (A2 / A1) of the spotted area is relatively high compared to the nearby non-spotted area, and the T of the spotted area isａｖ The relatively low (Int) values ​​indicated a decrease in the metabolism of oxidative phosphorylation in the granular layer of the pigmented area, and it was found that the decrease in the metabolism of oxidative phosphorylation is associated with the risk of metabolic disorders such as metabolic disorders of the skin due to the decrease in metabolism, or with pigmentation.

[0042] <Evaluation Step> The evaluation step is a step of evaluating the skin condition according to the level of oxidative phosphorylation in the granular layer. The skin condition of the subject can be evaluated based on the index indicating the level of oxidative phosphorylation obtained in the analysis step. The skin condition is preferably a metabolic disorder such as a skin metabolic disorder caused by oxidative phosphorylation metabolism (decrease or increase) in oxidative phosphorylation metabolism, and more preferably pigmentation, in relation to oxidative phosphorylation metabolism.

[0043] Here, pigmentation is a general term for inflammatory pigmentation caused by inflammation of the skin due to sunburn, acne, injury, etc., as well as pigmented lesions such as freckles, dull skin, and melasma, and refers to pigmentation that has resulted in spots. For example, when inflammation occurs in the skin, the stimulation causes melanocytes to produce melanin, and if this melanin is not properly excreted, the remaining melanin on the skin becomes spots. Melanin pigment plays a role in protecting against ultraviolet rays, but if melanin pigment is produced in excess due to various causes and the turnover cycle is disrupted, the melanin that would normally peel off remains there, resulting in pigmentation (spots). In this specification, pigmentation may also be referred to as spots, and these terms are synonymous.

[0044] As mentioned above, if the ratio (A2 / A1) is relatively low compared to the control, ａｖ When the (Int) value is relatively high, the metabolism of oxidative phosphorylation is considered to be normal, and when the level of oxidative phosphorylation in the granular layer is high, the skin condition can be evaluated as good. On the other hand, when the ratio (A2 / A1) is relatively high compared to the control, ａｖ When the (Int) value is relatively low, a decrease in the metabolism of oxidative phosphorylation is observed, and when the level of oxidative phosphorylation in the granular layer is low, the skin condition can be evaluated as poor.

[0045] Here, the comparison with the control can be selected appropriately depending on the purpose, and examples include comparison of adjacent areas such as a spot area and a non-spot area in the same measurement subject, or comparison of the same area in the same measurement subject before and after treatment.

[0046] The "relatively low" ratio (A2 / A1) can be selected appropriately depending on the purpose, but is preferably significantly lower than the control value when a paired t-test is performed on the values ​​of the non-spotted area and the spotted area of ​​all subjects. Also, the "relatively high" ratio (A2 / A1) can be selected appropriately depending on the purpose, but is preferably significantly higher than the control value when a paired t-test is performed on the values ​​of the non-spotted area and the spotted area of ​​all subjects.

[0047] T ａｖ The "relatively high" (Int) value can be selected appropriately depending on the purpose, but it is preferable that the value is significantly higher than the control value when a paired t-test is performed on the values ​​of the non-spotted area and the spotted area of ​​all subjects. ａｖ The (Int) value being "relatively low" can be selected appropriately depending on the purpose, but it is preferably significantly lower than the control value when a paired t-test is performed on the values ​​of the non-spotted areas and spotted areas of all subjects.

[0048] (Screening Method) The screening method of this embodiment is a method for screening cosmetic raw materials using the above-described skin condition evaluation method of this embodiment.

[0049] The cosmetic raw materials include cosmetic raw materials that improve skin conditions.

[0050] - Measurement target - The skin to be measured can be appropriately selected depending on the purpose, but preferred are human skin such as that of the temple, cheek, back of the hand, or arm; and skin culture tissues such as cultured skin tissue extracted from a human, reconstructed cultured skin tissue, and artificial cultured skin tissue.

[0051] In the evaluation step, the skin condition of the subject is evaluated based on an index indicating the level of oxidative phosphorylation by comparison with a control. The comparison with the control can be appropriately selected depending on the purpose, and examples include comparison of adjacent sites, such as a treated site and an untreated site, on the same subject, or comparison of the same site before and after treatment on the same subject.

[0052] Here, "treatment" means applying a cosmetic raw material that is a candidate for screening to the skin of a subject. The application method can be selected appropriately depending on the purpose, and examples include a method of applying a composition containing the cosmetic raw material to the skin of the subject, and a method of immersing a cultured skin tissue in a composition containing the cosmetic raw material. The treatment may be performed once or multiple times, and can be selected appropriately depending on the purpose.

[0053] By comparing with a control, if the level of oxidative phosphorylation in the granular layer is high, the skin condition can be evaluated as being good, and cosmetic ingredients evaluated as being good in skin condition can be screened as cosmetic ingredients that improve skin condition.

[0054] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0055] [Measurement subjects] Eighteen Asian subjects (aged 30s to 60s; 4 men and 14 women, a total of 18 subjects; Phototypes II to V) with age spots (senile lentigo) were photographed at their age spots and non-age spots using a multiphoton scanning laser biomicroscope (MPTCompact, manufactured by JenLab), and the fluorescence lifetime was measured using fluorescence lifetime microscopy (FLIM).

[0056] Measurement sites were limited to the temples and cheeks of the face. A comparison was made between the pigmented areas and nearby non-pigmented areas of the same subject. Light with a wavelength of 780 nm, capable of two-photon excitation of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), was irradiated onto the measurement sites. Of the skin layer structures at the measurement sites, measurements in the granular layer were used for analysis.

[0057] [Analysis Method] SymphoTime (PicoQuant) was used as the analysis software.

[0058] To analyze the metabolic state of the granular layer, a region of cells that did not contain noise and melanin and that was not part of the nucleus was selected as one region of interest (ROI). For each of the five ROIs, a model fit was performed for two components: the fluorescence lifetime amplitude A1 derived from NAD(P)H molecules with a long fluorescence lifetime, and the fluorescence lifetime amplitude A2 derived from NAD(P)H molecules with a short fluorescence lifetime.

[0059] In this case, cells with a high explanatory rate of the model (explanation rate X ２ = 0.9 to 1.4) and the peak top time was limited to the range of the NAD(P)H molecule (short fluorescence lifetime: 0.14 ns to 0.52 ns, long fluorescence lifetime: 2.65 ns to 3.69 ns).

[0060] The fluorescence lifetime of melanin (<100 ns) was used as a cutoff value, and the following indices (1) and (2) were calculated: (1) the ratio (A2 / A1) of the fluorescence lifetime amplitude A2 derived from NAD(P)H molecules with a short fluorescence lifetime to the fluorescence lifetime amplitude A1 derived from NAD(P)H molecules with a long fluorescence lifetime, and (2) the intensity-weighted average fluorescence lifetime T based on the fluorescence intensity of NAD(P)H molecules. ａｖ (Int) The average values ​​of these indices were compared in five ROIs of the pigmented area and the adjacent non-pinched area of ​​the same subject.

[0061] Representative examples of measurements shown in Figures 3 to 7 will be used for explanation. Figure 3 is a photograph showing the appearance of the measurement site of one subject (subject in his 50s) in the example. Figures 4 and 5 are tomographic images of the granular layer of the non-blemish site 10 (Figure 4) and the blemish site 11 (Figure 5) in Figure 3, observed with a multiphoton scanning laser biomicroscope.

[0062] As shown in Figure 3, a non-spotted area 10 and a spotted area 11 on the temples and cheeks of the face were set as measurement areas for each subject, and tomographic images of the granular layer of the non-spotted area 10 and the spotted area 11 were obtained by measuring using a multiphoton scanning laser biomicroscope, as shown in Figures 4 and 5.

[0063] Figure 6 is a diagram for explaining the regions of interest (ROIs) set in the tomographic images of the granular layer in the examples. In Figure 6, the highlighted areas indicate ROIs, and each ROI is numbered. For the analysis of the metabolic state of the granular layer, as shown in Figure 6, a region of cells that do not contain melanin and that is not a nucleus was selected as one ROI, and five ROIs were set for each measurement site. A two-component model fit was performed, and the ratio (A2 / A1) and T were calculated as the average values ​​of the five ROIs at each measurement site. ａｖ (Int) was calculated.

[0064] 7 and 8 are graphs showing the relationship between the fluorescence lifetime and the number of fluorescence detection events in the five ROIs of the non-pigmented and pigmented regions, respectively. In FIGS. 7 and 8, the x-axis shows the fluorescence lifetime, and the y-axis shows the number of times that a fluorescence detection event occurred in the five ROIs (number of events). On the x-axis, a fluorescence lifetime amplitude A2 derived from NAD(P)H molecules with a short fluorescence lifetime is detected in the short fluorescence lifetime (peak top time: 0.14 ns to 0.52 ns), and a fluorescence lifetime amplitude A1 derived from NAD(P)H molecules with a long fluorescence lifetime is detected in the long fluorescence lifetime (peak top time: 2.65 ns to 3.69 ns). Furthermore, the intensity-weighted average fluorescence lifetime T based on the fluorescence intensity of the NAD(P)H molecules is ａｖ (Int) indicates the average value of the fluorescence lifetime weighted by the fluorescence intensity of the NAD(P)H molecule, and serves as an index showing whether the value leans toward A1 or A2.

[0065] As described above, NAD(P)H molecules with long fluorescence lifetimes correspond to protein-bound NAD(P)H molecules, and a high fluorescence lifetime amplitude A1 derived from NAD(P)H molecules with long fluorescence lifetimes indicates that oxidative phosphorylation (OXPHOS) metabolism is dominant. On the other hand, NAD(P)H molecules with short fluorescence lifetimes correspond to free NAD(P)H molecules, and a high fluorescence lifetime amplitude A2 derived from NAD(P)H molecules with short fluorescence lifetimes indicates that glycolytic metabolism is dominant.

[0066] Furthermore, it is known that in a normal epidermal layer structure, glycolysis is dominant in the basal layer where cell differentiation occurs, while oxidative phosphorylation metabolism is dominant in the granular layer, which rises to the upper layer due to skin metabolism (turnover). It is possible that metabolism is not normal in the granular layer at the site of pigmented spots.

[0067] 9 is a graph showing the measurement results of the ratio (A2 / A1) of the fluorescence lifetime amplitude A2 derived from NAD(P)H molecules with a short fluorescence lifetime to the fluorescence lifetime amplitude A1 derived from NAD(P)H molecules with a long fluorescence lifetime. ａｖ 10 is a graph showing the measurement results of (Int).

[0068] 9 and 10, the open graphs show non-spotted areas (non-pigmented areas), the black graphs show spotted areas (pigmented areas), and * indicates that the p-value in the t-test for 18 subjects was <0.005 when comparing a spotted area and a non-spotted area in the same subject as a pair. The vertical axis in FIG. 9 shows the ratio (A2 / A1), and the vertical axis in FIG. 10 shows the ratio (A2 / A1). ａｖ (Int).

[0069] From the results in Figure 9, when comparing the pigmented and non-pigmented areas of the same subject, the ratio (A2 / A1) of the pigmented area was relatively higher than the ratio (A2 / A1) of the nearby non-pigmented area, and this was significant in 18 subjects (paired t-test, p<0.0005).

[0070] From the results of FIG. 10, by comparing the pigmented and non-pigmented areas of the same subject, it is clear that the T ａｖ (Int) value is the T of the nearby non-stained area ａｖ (Int) values ​​were relatively low and were significant in 18 subjects (paired t-test, p<0.0005).

[0071] These results indicate that when the ratio (A2 / A1) of the pigmented area is relatively high compared to the ratio (A2 / A1) of the surrounding non-pigmented area, it can be evaluated that a decrease in the metabolism of oxidative phosphorylation is observed. ａｖ (Int) value is the T of the nearby non-stained area ａｖIt was found that when the value is relatively low relative to the (Int) value, it can be evaluated that a decrease in oxidative phosphorylation metabolism is observed.

[0072] From the above, it was found that the metabolic level of oxidative phosphorylation based on the fluorescence lifetime of the electron carrier differs between pigmented and non-pinched areas and is associated with pigmented and non-pinched areas. Therefore, it is possible to evaluate skin condition according to the level of oxidative phosphorylation in the granular layer, enabling non-invasive evaluation of skin condition and providing a new index for evaluating pigmented areas.

[0073] Although the present invention has been described above based on specific embodiments and examples, these embodiments and examples are presented only as examples, and the present invention is not limited to the above embodiments and examples. Various changes, modifications, substitutions, deletions, additions, combinations, etc. are possible within the scope of the disclosure of the present invention.

[0074] This application claims priority based on Japanese Patent Application No. 2024-003897, filed on January 15, 2024, the entire contents of which are incorporated herein by reference.

[0075] REFERENCE SIGNS LIST 1 Excitation light 2 Fluorescence 3 Stratum corneum 4 Epidermis 5 Dermis 10 Non-spotted area 11 Spotted area 100 Multi-photon scanning laser biomicroscope 101 Femtosecond laser light source 103 Beam splitter 106 Objective lens 107 Filter 122 Photon counter 124 Skin to be measured

Claims

1. A step of irradiating the skin to be measured with excitation light of an electron carrier, and measuring the fluorescence lifetime of the electron carrier in the granular layer by a multiphoton scanning laser microscope; a step of analyzing the degree of oxidative phosphorylation of the electron carrier in the granular layer based on the fluorescence lifetime of the electron carrier; and a step of evaluating the skin condition according to the degree of oxidative phosphorylation in the granular layer, wherein the electron carrier is nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), and flavin adenine dinucleotide (FAD), reduced flavin adenine dinucleotide (FADH ２ ), or at least any one of them, and a method for evaluating the skin condition.

2. The method for evaluating skin condition according to claim 1, wherein the skin condition is metabolic disorder.

3. The method for evaluating skin condition according to claim 1, wherein the skin condition is pigmentation.

4. The method for evaluating skin condition according to claim 1, wherein the electron carrier is nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH).

5. The degree of the oxidative phosphorylation is based on (1) the ratio (A2 / A1) of the fluorescence lifetime amplitude A2 derived from the electron carrier with a short fluorescence lifetime to the fluorescence lifetime amplitude A1 derived from the electron carrier with a long fluorescence lifetime, and (2) the intensity-weighted average fluorescence lifetime T ａｖ (Int), and the method for evaluating the skin condition according to claim 1, wherein at least one of them is used as an index.

6. The method for evaluating skin condition according to claim 1, wherein the evaluating step is to evaluate that the skin condition is good when the degree of oxidative phosphorylation in the granular layer is high.

7. The method for evaluating skin condition according to claim 1, wherein the skin to be measured is at least one of the forehead, cheek, nail, and arm.

8. A screening method characterized by screening cosmetic raw materials using the method for evaluating skin condition according to any one of claims 1 to 7.

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