A method for evaluating the overall strength of skin using phenylalanine in the stratum corneum as an index

Abstract

To provide a simple, objective method for evaluating a subject's skin condition using less invasive means.SOLUTION: A method for evaluating a subject's total skin quality includes the steps of: (a) measuring content of phenylalanine in a horny layer of the subject's skin; (b) evaluating one or more physiological state indices selected from a group consisting of antioxydation power in the subject's body, skin complexion, and autonomic nervous function of the subject according to the phenylalanine content obtained in step (a); and (c) evaluating total skin quality of the subject based on an evaluation result obtained in step (b).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the overall strength of skin using phenylalanine in the stratum corneum as an index. [Background technology]

[0002] In the field of cosmetics, it is extremely useful to objectively evaluate skin conditions by measuring physiological indices using physical instruments, rather than relying solely on sensory evaluation.

[0003] Representative widely known physiological indicators include, for example, evaluating the skin condition based on the antioxidant capacity in the subject's body (Non-Patent Document 1), evaluating the skin condition based on the complexion of the subject's skin (Non-Patent Document 2), and evaluating the skin condition based on the subject's autonomic nervous function (Non-Patent Document 3).

[0004] However, measuring antioxidant capacity in the body requires the collection of biological samples, such as urine or blood samples, which are burdensome for the subject. In addition, these samples are easily affected by the food and behavior immediately before the test, so dietary and behavioral restrictions are also necessary to accurately understand the subject's condition.

[0005] Skin color is generally measured by capturing and analyzing images of the skin. However, this method requires specialized equipment, which means that subjects must be present at the test site. Furthermore, skin color is easily affected by environmental factors such as temperature and humidity, as well as the previous meal and behavior. In order to accurately measure the subject's condition, dietary and behavioral restrictions must be imposed, and the subject must be allowed to acclimate for a certain period of time in a constant temperature and humidity room.

[0006] Autonomic nervous function is generally measured by analyzing fingertip pulse waves. However, this method also requires specialized equipment, so the subject must be present at the test site for the measurement. Furthermore, because the autonomic nervous system is significantly affected by the subject's previous behavior and mental state, the test requires the subject to rest for a certain period of time before and during the measurement, and an environment suitable for measurement that takes into account sounds, smells, etc.

[0007] Therefore, there is a need for a method for easily and objectively evaluating the condition of a subject's skin using less invasive means. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Journal of the Japan Mibyo System Association, 2007,13(1):10-13 [Non-patent document 2] J.Soc.Cosmet.Chem.Japan.,2004,38(3):202-210 [Non-patent document 3] J Otolaryngol Jpn 100.,1997:457-466 Summary of the Invention

[0009] The present invention provides a method for easily and objectively assessing the condition of a subject's skin by less invasive means.

[0010] The present inventors have surprisingly found that by measuring the phenylalanine content in the stratum corneum of a subject's skin, it is possible to evaluate one or more physiological indicators selected from the group consisting of antioxidant capacity, skin color, and autonomic nervous function in the subject, thereby enabling evaluation of the overall strength of the subject's skin. The present invention is based on these findings.

[0011] According to the present invention, the following inventions are provided. (1) A method for evaluating the overall strength of the skin of a subject, comprising: (a) measuring the content of phenylalanine in the stratum corneum of the skin of the subject; (b) evaluating one or more physiological state indicators selected from the group consisting of antioxidant capacity in the body, skin color, and autonomic nervous function in the subject based on the phenylalanine content obtained in step (a); and (c) evaluating the overall strength of the subject's skin based on the evaluation results obtained in step (b); The method comprising: (2) Step (a) (a1) preparing a stratum corneum derived from the subject by applying and removing tape to the skin of the subject; and (a2) measuring the content of phenylalanine in the stratum corneum obtained in step (a1); The method according to (1), comprising: (3) The method according to (1) or (2), wherein the indicator of antioxidant capacity in the body is antioxidant potential (PAO) in the blood. (4) The method according to any one of (1) to (3), wherein the index of skin color is the blood oxygen saturation index (Hb SO2 Index) of the arm or cheek. (5) The method according to any one of (1) to (4), wherein the index of autonomic nervous function is autonomic nervous activity (CVRR). (6) An evaluation kit for use in the method according to any one of (1) to (5), comprising an instrument or reagent for measuring the phenylalanine content in the stratum corneum of the skin. (7) A method for evaluating one or more physiological state indicators selected from the group consisting of antioxidant capacity in the body, skin color, and autonomic nervous function in a subject, comprising: (a) measuring the content of phenylalanine in the stratum corneum of the skin of the subject; and (b) evaluating one or more physiological state indicators selected from the group consisting of antioxidant capacity in the body, skin color, and autonomic nervous function in the subject based on the phenylalanine content obtained in step (a). The method comprising: (8) Step (a) (a1) preparing a stratum corneum derived from the subject by applying and removing tape to the skin of the subject; and (a2) measuring the content of phenylalanine in the stratum corneum obtained in step (a1); The method according to (7), comprising: (9) The method according to (7) or (8), wherein the indicator of antioxidant capacity in the body is antioxidant potential (PAO) in the blood. (10) The method according to any one of (7) to (9), wherein the index of skin color is the blood oxygen saturation index (Hb SO2 Index) of the arm or cheek. (11) The method according to any one of (7) to (10), wherein the index of autonomic nervous function is autonomic nervous activity (CVRR). (12) An evaluation kit for use in the method according to any one of (7) to (11), comprising an instrument or reagent for measuring the phenylalanine content in the stratum corneum of the skin.

[0012] According to the present invention, there is provided a method for easily and objectively evaluating the skin condition of a subject using a minimally invasive means. Furthermore, according to the present invention, there is also provided a method for easily and objectively evaluating a physiological index for evaluating the skin condition of a subject using a minimally invasive means. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the correlation between the phenylalanine content in the stratum corneum of the subject's skin and the antioxidant potential (PAO) in the blood. [Figure 2] FIG. 2 shows the correlation between the phenylalanine content in the stratum corneum of the subject's skin and the blood oxygen saturation index (Hb SO2 Index) of the arm. [Figure 3] FIG. 3 shows the correlation between the phenylalanine content in the stratum corneum of the subject's skin and the cheek blood oxygen saturation index (Hb SO2 Index). [Figure 4] FIG. 4 shows the correlation between the phenylalanine content in the stratum corneum of the subject's skin and autonomic nervous activity (CVRR). Specific Description of the Invention

[0014] According to the present invention, there is provided a method for evaluating the overall strength of the skin of a subject, comprising: (a) measuring the phenylalanine content in the stratum corneum of the skin of the subject; (b) evaluating one or more physiological state indicators selected from the group consisting of antioxidant capacity in the body, skin color, and autonomic nervous function of the subject based on the phenylalanine content obtained in step (a); and (c) evaluating the overall strength of the skin of the subject based on the evaluation results obtained in step (b).

[0015] The subject of the present invention refers to mammals such as, but not limited to, humans, primates including chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, horses, sheep and goats, and rodents such as mice and rats, and is preferably a human. The subject of the present invention is preferably a healthy individual. Here, a healthy individual refers to an individual who is not suffering from a disease, such as a skin disease such as atopic dermatitis.

[0016] The term "skin overall strength" used in the present invention refers to the subject's physical and mental condition at the time of measurement, which is expressed as a score of 0 to 100. A higher score indicates a better mental and physical condition of the subject that constitutes healthy skin, while a lower score indicates a worse mental and physical condition of the subject that constitutes healthy skin. The skin overall strength score is calculated by evaluating and predicting physiological indicators such as antioxidant capacity, skin complexion, and autonomic nervous function based on the phenylalanine content in the stratum corneum of the skin. Depending on this skin overall strength score, appropriate skin care and lifestyle recommendations may be established and advice provided to the subject.

[0017] Generally, skin is divided from the outside into the epidermis, dermis, and subcutaneous tissue, and the epidermis is further divided into the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale depending on the degree of cellular differentiation. The stratum corneum in this invention refers to the stratum corneum. Normally, the stratum corneum is mainly composed of keratinocytes, and has a structure in which keratinocytes are layered in multiple layers. The degree of layering of keratinocytes and the thickness of the stratum corneum vary depending on the part of the body.

[0018] Step (a) of the present invention is not particularly limited as long as it is a step of measuring the phenylalanine content in the stratum corneum of the skin of a subject, but preferably includes (a1) a step of preparing a stratum corneum derived from the subject by applying and removing tape to the skin of the subject, and (a2) a step of measuring the phenylalanine content in the stratum corneum obtained in step (a1). In step (a1) of the present invention, it is preferable to prepare a stratum corneum derived from the subject by applying and removing tape to the skin multiple times at the same location on the subject's body. It is more preferable to prepare a stratum corneum derived from the subject by applying and removing tape to the skin once to ten times at the same location on the subject's body, and more preferably to prepare a stratum corneum derived from the subject by applying and removing tape five times. The tape obtained by applying and removing tape to the skin is not particularly limited, but it is preferable to prepare a stratum corneum derived from the subject, excluding the tape obtained by the first and second application and removal. For example, if a tape is applied to and removed from the skin five times, it is preferable to prepare the tape that has been applied and removed from the skin the third to fifth times, excluding the tape that has been applied and removed the first and second times, as the stratum corneum derived from the subject.

[0019] The measurement of the phenylalanine content in the present invention may be carried out by known methods, including, but not limited to, methods using instruments such as an amino acid analyzer, HPLC or LC-MS, fluorometry, etc. According to one embodiment of the present invention, the method of the present invention is carried out in vitro.

[0020] The method of the present invention can evaluate one or more physiological state indicators selected from the group consisting of antioxidant capacity in the body, skin complexion, and autonomic nervous function in a subject based on the phenylalanine content.Furthermore, the overall strength of the subject's skin can be evaluated based on the evaluation results of one or more physiological state indicators selected from the group consisting of antioxidant capacity in the body, skin complexion, and autonomic nervous function.

[0021] In the present invention, "antioxidant power in the body" refers to the amount of hydrogen peroxide (H2O2), hydroxyl radicals, and OH radicals present in the body. - , superoxide, O 3- The term "antioxidant" refers to the ability to eliminate free radicals and reactive oxygen species, such as erythrocytes, erythrocytes, and erythrocytes. Substances with antioxidant potential are generally called antioxidants and function to remove excess free radicals and reactive oxygen species. While these free radicals and reactive oxygen species have the ability to eliminate bacteria and other pathogens that invade the body, their excessive production is known to oxidize the human body and potentially contribute to arteriosclerosis, cancer, lifestyle-related diseases, aging, and other conditions. According to the method of the present invention, the antioxidant potential of the body can be evaluated based on the phenylalanine content in the stratum corneum of a subject's skin. The indicator of the antioxidant potential of the body evaluated in this manner may be, for example, an antioxidant potential (PAO) measurement, a measurement using the CUPRAC method, the DPPH method, the FRAP method, the ORAC method, the TRAP method, or other methods, as a measure of the reducing ability of antioxidants contained in a biological sample. The antioxidant potential (PAO) measurement is preferably used. The antioxidant potential (PAO) measurement is measured, for example, using the "PAO" antioxidant potential measurement kit (manufactured by Nikken Seil Co., Ltd., Japan Institute for Aging Control).

[0022] In the present invention, "skin color" refers to the degree to which skin is bright and reddish in a healthy, non-pathological state, and does not include redness due to skin abnormalities such as inflammation. Skin color can be measured by known methods, such as measuring color difference or measuring the blood oxygen saturation index (HbSO2 index). In the present invention, the blood oxygen saturation index (HbSO2 index) refers to the proportion of hemoglobin (Hb) in blood bound to oxygen. A high value of this index indicates a bright red color for blood, whereas a low value indicates a dark red color, which is reflected in skin color. According to the method of the present invention, skin color can be evaluated based on the phenylalanine content in the stratum corneum of a subject's skin. The index of skin color evaluated in this manner is preferably the blood oxygen saturation index (HbSO2 index). The site on the subject where skin color is evaluated is not particularly limited, but is preferably the arm or cheek. The blood oxygen saturation index (Hb SO2 Index) is measured, for example, using a spectrophotometer.

[0023] According to the method of the present invention, autonomic nervous function can be evaluated based on the content of phenylalanine in the stratum corneum of the skin of a subject. The index of autonomic nervous function evaluated in this manner is preferably autonomic nervous activity. In the present invention, autonomic nervous activity refers to an index indicating the intensity of sympathetic and parasympathetic nervous activity, with a higher value of autonomic nervous activity indicating a higher intensity of autonomic nervous activity. Autonomic nervous activity may be calculated based on the pulse wave of the subject, and is the coefficient of variation of the RR interval (CVRR), which represents the magnitude of variation over a given period of time in the interval between the R wave and the next R wave (RR interval) in an electrocardiogram. Autonomic nervous activity (CVRR) is measured, for example, using a pulse wave measuring device.

[0024] The present invention also provides an evaluation kit for carrying out the method of the present invention, which comprises an instrument or reagent for measuring the phenylalanine content in the stratum corneum of the skin of the subject described above. [Example]

[0025] The present invention will be described in detail based on the following examples, but the present invention is not limited to these examples. Contents are expressed in mass % unless otherwise specified.

[0026] Measurement of phenylalanine content in the stratum corneum The phenylalanine content in the stratum corneum was measured by a method including the following steps.

[0027] (Recovery of stratum corneum from human skin) The stratum corneum of the skin on the inner forearms of 48 subjects was peeled using stratum corneum tape (22 mm diameter, D-Squame™ Standard Sample Disc, D100, CuDerm Co., USA). Five strips of stratum corneum tape were taken from each site, and the third to fifth layers counting from the surface were used for analysis.

[0028] (protein quantification) Using a SquameScan 850A (Heiland electronic GmbH), the protein content per unit area (μg / cm) of the stratum corneum tape was measured after peeling off the third to fifth layers of the stratum corneum. 2 ) was measured by near-infrared spectroscopy.

[0029] (Pretreatment of test specimens) The tape from which the third to fifth stratum corneum layers had been peeled was placed in a screw tube (20 mL), 0.2 mL of 5% ethanol solution was added, and the mixture was sonicated at room temperature for 10 minutes. The supernatant was used as a stratum corneum extract.

[0030] (Preparation of standard solutions) A standard solution was prepared by adjusting the amino acid mixture standard solution type H (Fujifilm Wako Pure Chemical Industries, Ltd.) so that the concentration of each amino acid was 2.5 μmol / mL.

[0031] (Preparation of internal standard solution) An internal standard solution was prepared using the accessories of the APDS Tag (trademark) Wako Amino Acid Internal Standard Mixture Kit (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0032] (Preparation of standard samples) 155 μL of APDS Tag™ Wako borate buffer solution, 20 μL of standard solution, 20 μL of internal standard solution, and 5 μL of APDS Tag test solution included in the APDS Tag™ Wako Amino Acid Internal Standard Mixture Kit (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed, stirred for a few seconds using a vortex, reacted at 60°C for 15 minutes, and cooled to room temperature. This was used as a standard sample and subjected to LC-MS / MS measurement under the analytical conditions shown in Table 1 below.

[0033] (Preparation of test specimens) 155 μL of APDS Tag™ Wako borate buffer, 20 μL of stratum corneum extract, 20 μL of internal standard solution, and 5 μL of APDS Tag test solution included in the APDS Tag™ Wako Amino Acid Internal Standard Mixture Kit (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed, stirred for a few seconds using a vortex, reacted at 60°C for 15 minutes, and cooled to room temperature. This was used as the test sample and subjected to LC-MS / MS measurement under the analytical conditions shown in Table 1 below.

[0034] [Table 1]

[0035] (Data Analysis) Using the peak area ratio (amino acid / internal standard) of the standard sample, a calibration curve was created through the origin, and the peak area ratio (analyte / internal standard) of the test sample was applied to the calibration curve to calculate the phenylalanine content of the test sample. The obtained phenylalanine content was corrected by the protein content of the stratum corneum tape, and the obtained value was used as the final quantitative value (phenylalanine content in the stratum corneum). The final quantitative value was calculated using the following formula: "phenylalanine content in the test sample (μmol / mL)" x 0.2 (volume of extraction solvent mL) / 3.7994 (tape area cm 2 ) / "Protein content of stratum corneum tape (μg / cm 2 ) was calculated.

[0036] Measurement of antioxidant capacity in blood The antioxidant potential in the blood of 48 subjects was evaluated. Blood was drawn from the subjects, left at room temperature for 30 minutes, and then centrifuged (2000g, 10 minutes) to obtain serum. The total serum antioxidant potential (Potential Antioxidant) of the obtained serum was measured using the antioxidant potential measurement kit "PAO" (manufactured by Nikken Seil Co., Ltd., Japan Institute for Aging Control), and the obtained value was taken as the antioxidant potential in the blood. The measurement procedure was carried out by referring to the attached manual of each kit.

[0037] Measurement of blood color (Hb SO2 Index) on the arms and cheeks The blood oxygen saturation index (HbSO2 Index (%)) was measured on the cheeks and inner forearms of 48 subjects to evaluate their skin color. The HbSO2 Index was measured by directly contacting a spectrophotometer (CM-700d, manufactured by Konica Minolta) with the subject's cheek and inner forearm, and the measurement results were analyzed using analysis software (CM-SA, manufactured by Konica Minolta). Measurements were performed seven times on the same location on each subject. The measurements with the highest or lowest HbSO2 Index were excluded, and the average of the five values ​​was calculated. This value was used as the skin color of each subject's arm and cheek.

[0038] Measurement of autonomic nervous function (CVRR) The autonomic nervous function (CVRR) of 48 subjects was evaluated based on pulse wave information. Measurement and analysis of pulse wave information was performed using BITAS-PULSEADVANCE (Shiseido version, manufactured by Human and Scientific Research Institute). The pulse wave sensor of BITAS-PULSEADVANCE was attached to the subject's finger, and the subject's pulse wave was measured for 70 seconds while they were resting. After the measurement was completed, the measurement results were analyzed using BITAS-PULSEADVANCE, and the value obtained was used as the subject's autonomic nervous function (CVRR).

[0039] statistical processing We investigated the correlation between the phenylalanine content in the stratum corneum and blood antioxidant activity (PAO), complexion in the arms and cheeks (Hb SO2 Index), and autonomic nervous function (CVRR). The correlations between the phenylalanine content in the stratum corneum and PAO, the phenylalanine content in the stratum corneum and Hb SO2 Index in the arms, the phenylalanine content in the stratum corneum and Hb SO2 Index in the cheeks, and the phenylalanine content in the stratum corneum and CVRR were plotted on scatter plots, and correlation coefficients were calculated. The results are shown in Table 2 and Figures 1-4.

[0040] [Table 2]

[0041] These results indicate that all four physiological indices show a weak positive correlation with phenylalanine content. Therefore, by measuring the phenylalanine content in the stratum corneum of a subject's skin, it is possible to evaluate the antioxidant potential in the blood (PAO), the complexion of the arms and cheeks (Hb SO2 Index), and autonomic nervous function (CVRR), and it is also possible to evaluate the overall strength of the skin based on these evaluation results.

Claims

1. 1. A method for providing one or more evaluation indexes selected from the group consisting of antioxidant capacity (PAO) measurement value, blood oxygen saturation index (Hb SO2 Index), and autonomic nervous activity rate (CVRR) in a subject, comprising: (a) measuring the content of phenylalanine in the stratum corneum of the skin of the subject; and (b) providing one or more evaluation indexes selected from the group consisting of a measured antioxidant potential (PAO), a blood oxygen saturation index (Hb SO2 Index), and a CVRR in the subject based on the phenylalanine content obtained in step (a). The method comprising:

2. Step (a) (a1) preparing a stratum corneum derived from the subject by applying and removing tape to the skin of the subject; and (a2) measuring the content of phenylalanine in the stratum corneum obtained in step (a1); The method of claim 1 , comprising:

3. A method according to claim 1 or 2, wherein the antioxidant capacity (PAO) measurement value is a blood antioxidant capacity (PAO) measurement value.

4. A method according to any one of claims 1 to 3, wherein the blood oxygen saturation index (Hb SO2 Index) is a blood oxygen saturation index (Hb SO2 Index) of the arm or cheek.

5. An evaluation kit for use in the method according to any one of claims 1 to 4, comprising an instrument or reagent for measuring the phenylalanine content in the stratum corneum of the skin.

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