JP2023092196A - Method for evaluating total skin quality using phenylalanine in skin horny layer as indicator - Google Patents

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 The present invention relates to a method for evaluating total skin strength using phenylalanine in the stratum corneum of the skin as an index.

In the cosmetics field, it is very useful to objectively evaluate skin conditions by measuring physiological indices using physical instruments, without relying solely on sensory evaluation.

As the physiological index, for example, evaluation of the skin condition based on the antioxidant power in the body of the subject (Non-Patent Document 1), and evaluation of the skin condition based on the complexion of the subject's skin (Non-Patent Document 2). , evaluation of the skin condition based on the autonomic nerve function of the subject (Non-Patent Document 3), etc. are widely known as typical methods.

On the other hand, the measurement of the antioxidant capacity in the body requires the collection of biological samples by methods such as urine collection and blood collection, which impose a heavy burden on the subject. In addition, since these samples are easily affected by the previous meal and behavior, dietary and behavioral restrictions are also required to accurately determine the subject's condition.

The complexion of the skin is generally measured by a method of acquiring and analyzing an image of the skin, or the like. However, since this method requires special equipment, it is necessary for the subject to visit the test site for the measurement. In addition, the skin color is easily affected by the environment such as temperature and humidity, the meal immediately before, and the behavior. It also needs to be acclimated.

Autonomic nerve function is generally measured by a method of analyzing fingertip pulse waves, or the like. However, this method also requires dedicated equipment, so the subject must visit the test site for the measurement. In addition, since the autonomic nervous system is greatly affected by the behavior and mental state immediately before the measurement, it is necessary to have an environment suitable for the measurement considering the subject's rest for a certain period of time before the measurement, the subject's rest during the measurement, and sounds and smells. becomes.

Therefore, there is a demand for a method of easily and objectively evaluating the skin condition of a subject by less invasive means.

Japan Mibyou System Society Journal, 2007, 13(1): 10-13 J. Soc. Cosmet. Chem. Japan. , 2004, 38(3):202-210 J Otolaryngol Jpn 100. , 1997:457-466

The present invention provides a method for easily and objectively evaluating the skin condition of a subject by less invasive means.

Surprisingly, the present inventors found that by measuring the content of phenylalanine in the stratum corneum of the skin of a subject, it was found that the group consisting of antioxidant power in the body, complexion of the skin, and autonomic nerve function in the subject. It has been found that one or more indicators of physiological condition selected from can be assessed, thereby assessing the overall strength of the skin of said subject. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
(1) A method for evaluating the overall strength of the skin of a subject,
(a) measuring the content of phenylalanine in the stratum corneum of the skin of the subject;
(b) based on the content of phenylalanine obtained in step (a), one or more physiological states selected from the group consisting of antioxidant capacity in the body, complexion of the skin, and autonomic nerve function in the subject; and (c) evaluating the overall strength of the skin of the subject based on the evaluation results obtained in step (b).
(2) step (a) is
(a1) a step of preparing a stratum corneum derived from the subject obtained by applying and peeling a tape to the skin of the subject; and (a2) content of phenylalanine in the stratum corneum obtained in step (a1) The method according to (1), comprising the step of measuring
(3) The method according to (1) or (2), wherein the index of antioxidant capacity in the body is blood antioxidant capacity (PAO).
(4) The method according to any one of (1) to (3), wherein the index of skin complexion 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 nerve function is autonomic nerve 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 content of phenylalanine in the stratum corneum of the skin. kit.
(7) A method for evaluating one or more physiological condition indicators selected from the group consisting of antioxidant capacity in the body, skin complexion, and autonomic nerve function in a subject,
(a) measuring the content of phenylalanine in the stratum corneum of the skin of the subject; and (b) based on the content of phenylalanine obtained in step (a), the antioxidant capacity in the body of the subject. , evaluating one or more indicators of physiological condition selected from the group consisting of skin complexion and autonomic nerve function.
(8) step (a) is
(a1) a step of preparing a stratum corneum derived from the subject obtained by applying and peeling a tape to the skin of the subject; and (a2) content of phenylalanine in the stratum corneum obtained in step (a1) The method according to (7), comprising the step of measuring
(9) The method according to (7) or (8), wherein the index of antioxidant capacity in the body is blood antioxidant capacity (PAO).
(10) The method according to any one of (7) to (9), wherein the index of skin complexion 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 nerve function is autonomic nerve 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. kit.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for easily and objectively evaluating the skin condition of a subject by a less invasive means. In addition, according to the present invention, it is possible to provide a method for easily and objectively evaluating a physiological index for evaluating the skin condition of a subject by a less invasive means.

FIG. 1 shows the correlation between the content of phenylalanine in the stratum corneum of the skin of a subject and the antioxidant capacity (PAO) in blood. FIG. 2 represents the correlation between the content of phenylalanine in the stratum corneum of the skin of a subject and the blood oxygen saturation index (Hb SO2 Index) of the arm. FIG. 3 represents the correlation between the content of phenylalanine in the stratum corneum of the skin of a subject and the blood oxygen saturation index (Hb SO2 Index) of the cheek. FIG. 4 represents the correlation between the content of phenylalanine in the stratum corneum of the skin of a subject and autonomic nerve activity (CVRR).

Specific description of the invention

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

Subjects of the present invention include, but are not limited to, humans, primates including chimpanzees, pet animals such as dogs and cats, livestock animals such as cattle, horses, sheep and goats, and rodents such as mice and rats. It means a mammal such as a genus, preferably a human. Also, the subject of the present invention is preferably a healthy subject. Here, healthy subjects mean those who are not suffering from diseases such as skin diseases such as atopic dermatitis.

The overall skin power of the present invention is the mental and physical condition of healthy skin of a subject at the time of measurement, and is represented by a score of 0 to 100. A higher score means that the subject's mental and physical conditions that constitute healthy skin are better, and a lower score means that the subject's mental and physical conditions that constitute healthy skin are poorer. The overall skin strength score is calculated by evaluating and predicting physiological indicators such as antioxidant capacity in the body, skin complexion, and autonomic nerve function based on the phenylalanine content in the stratum corneum of the skin. . Depending on the overall skin power score, setting, advice, or the like may be performed on appropriate skin care and lifestyle of the subject.

In general, the skin is divided into epidermis, dermis, and subcutaneous tissue from the outside, and the epidermis is further divided into stratum corneum, stratum granulosum, stratum spinosum, and stratum basale depending on the degree of cell differentiation. The stratum corneum in the present invention means stratum corneum. Normally, the stratum corneum is mainly composed of stratum corneum cells, and has a structure in which the stratum corneum cells are stacked in many layers. The degree of stratum corneum cell overlap and the thickness of the stratum corneum differ depending on each part of the body.

The step (a) of the present invention is not particularly limited as long as it is a step of measuring the content of phenylalanine in the stratum corneum of the skin of a subject. and (a2) measuring the content of phenylalanine in the stratum corneum obtained in step (a1). In the step (a1) according to the present invention, it is preferable to prepare the subject-derived stratum corneum by applying and peeling the tape to the skin a plurality of times at the same place on the body of the subject. It is more preferable to prepare the subject-derived stratum corneum by applying and peeling the tape at the same place on the subject's body 1 to 10 times, and performing 5 times to prepare the subject-derived stratum corneum. It is more preferable to prepare. The tape obtained by sticking to and peeling from the skin is not particularly limited, but excluding the tapes obtained by the first and second sticking and peeling, the stratum corneum derived from the subject should be prepared. is preferred. For example, when the tape is applied and removed from the skin 5 times, the tape that can be applied and removed 3 to 5 times is removed from the tape that can be applied and removed 1st and 2nd times. It is preferable to prepare as the stratum corneum from which it originated.

The measurement of the phenylalanine content of the present invention may be carried out by a known method, and is not limited, but for example, an amino acid analyzer, a method using an instrument such as HPLC or LC-MS, a fluorescence analysis method, or the like. can be done by According to one embodiment of the invention, the method of the invention is performed in vitro.

According to the method of the present invention, one or more indicators of physiological condition selected from the group consisting of antioxidant capacity in the body, complexion of the skin, and autonomic nerve function are evaluated in a subject based on the phenylalanine content. be able to. In addition, it is also possible to evaluate the overall strength of the subject's skin from the evaluation results of one or more physiological condition indicators selected from the group consisting of antioxidant capacity in the body, complexion of the skin, and autonomic nerve function.

In the present invention, the term "antioxidant power in the body" refers to the ability to eliminate free radicals such as hydrogen peroxide H ₂ O ₂ and hydroxyl radicals, OH ⁻ , superoxide, O ³⁻ , and active oxygen present in the body. means. Substances having antioxidative power are generally called antioxidants, and have the function of removing excess free radicals and active oxygen. These free radicals and active oxygen have the effect of eliminating bacteria that have invaded the body, but on the other hand, when they are generated excessively, they oxidize the human body and cause arteriosclerosis, cancer, lifestyle-related diseases, aging, etc. It is known that it can be According to the method of the present invention, the antioxidant capacity in the body can be evaluated based on the phenylalanine content in the stratum corneum of the skin of the subject. The index of the antioxidant capacity in the body evaluated in this way is, for example, the antioxidant capacity (PAO) measurement value, the CUPRAC method, the DPPH method, the FRAP method as the reducing ability of the antioxidant contained in the biological sample. , the ORAC method, the TRAP method, etc., and preferably the antioxidant capacity (PAO) measurement value. This antioxidant capacity (PAO) measurement value is measured using, for example, an antioxidant capacity measurement kit "PAO" (manufactured by Nikken Zile Co., Ltd., Japan Institute for Aging Control).

In the present invention, the term "complexion of the skin" means the degree to which the skin is bright and reddish in a healthy state, not in a pathological state. For example, the skin is reddish due to skin abnormalities such as inflammation. not included in this case. The complexion of the skin is measured according to known methods such as color difference measurement and blood oxygen saturation index (Hb SO2 Index) measurement. The blood oxygen saturation index (Hb SO2 Index) in the present invention means the ratio of hemoglobin (Hb) in the blood bound to oxygen. and is reflected in skin color. According to the method of the present invention, the complexion of the skin can be evaluated based on the phenylalanine content in the stratum corneum of the skin of the subject. The index of skin complexion evaluated in this way is preferably the blood oxygen saturation index (Hb SO2 Index). The site of the subject to be evaluated for skin complexion is not particularly limited, but is preferably the arm or cheek. The blood oxygen saturation index (Hb SO2 Index) is measured using, for example, a spectrophotometer.

According to the method of the present invention, autonomic nerve function can be evaluated based on the phenylalanine content in the stratum corneum of the skin of a subject. The index of autonomic nerve function evaluated in this way is preferably the autonomic nerve activity level. The autonomic nerve activity in the present invention means an index indicating the intensity of activity of the sympathetic nerve and the parasympathetic nerve, and the higher the value of the autonomic nerve activity, the higher the intensity of the autonomic nerve activity. The autonomic nerve activity may be calculated based on the pulse wave of the subject, and the RR interval variation coefficient representing the magnitude of variation in the interval (RR interval) between the R wave and the next R wave in the electrocardiogram over a predetermined time (Coefficient of Variation of RR-interval; CVRR). Autonomic nerve activity (CVRR) is measured using, for example, a plethysmograph.

The present invention also provides an evaluation kit for carrying out the method of the present invention, comprising the instrument or reagent for measuring the phenylalanine content in the stratum corneum of the skin of a subject as described above.

The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples. Unless otherwise specified, the contents are shown in % by mass.

Measurement of phenylalanine content in stratum corneum Phenylalanine content in stratum corneum was measured by a method including the following steps.

(Recovery of stratum corneum in human skin)
The stratum corneum of the skin on the inside of the forearm of 48 subjects was peeled off using a stratum corneum tape (diameter 22 mm, D-Squame (trademark) Standard Sample Disc, D100, CuDerm Co., USA). Five stratum corneum tapes were collected from each site, and the 3rd to 5th layers counted from the surface layer were analyzed.

(protein quantification)
Using SquameScan 850A (Heiland electronic GmbH), the protein content (μg/cm ² ) per unit area of the stratum corneum tape from which the 3rd to 5th stratum corneum was peeled off was measured by near-infrared spectrometry. .

(Pretreatment of test specimen)
The tape from which the 3rd to 5th stratum corneum has been peeled off is placed in a screw tube (20 mL), 0.2 mL of a 5% ethanol aqueous solution is added, and ultrasonication is performed at room temperature for 10 minutes, and the supernatant is treated as a stratum corneum extract. bottom.

(Preparation of standard solution)
Amino acid mixture standard solution H type (Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared so that each amino acid concentration was 2.5 μmol/mL to prepare a standard solution.

(Preparation of internal standard solution)
An internal standard solution was prepared using the accessories of the APDS tag (trademark) Amino Acid Internal Standard Mixture Kit for Wako (Fuji Film Wako Pure Chemical Industries, Ltd.).

(Preparation of standard sample)
APDS tag (trademark) Borate buffer for Wako 155 μL, standard solution 20 μL, internal standard solution 20 μL, APDS tag (trademark) accessory for Wako amino acid internal standard mixture kit (Fujifilm Wako Pure Chemical Industries, Ltd.) 5 μL of the test solution was mixed, stirred with Vortex for several seconds, reacted at 60° C. for 15 minutes, cooled to room temperature, and used as a standard specimen, and subjected to LC-MS/MS measurement under the analysis conditions shown in Table 1 below.

(Preparation of test specimen)
APDS Tag (trademark) borate buffer for Wako 155 μL, stratum corneum extract 20 μL, internal standard solution 20 μL, APDS Tag (trademark) Amino Acid Internal Standard Mixture Kit for Wako (Fujifilm Wako Pure Chemical Industries, Ltd.) accessories 5 μL of the APDS tag test solution was mixed, stirred with Vortex for several seconds, reacted at 60° C. for 15 minutes, cooled to room temperature, and used as a test specimen, and subjected to LC-MS/MS measurement under the analysis conditions shown in Table 1 below. .

(data analysis)
Using the peak area ratio of the standard sample (amino acid / internal standard), create a calibration curve through the origin, apply the peak area ratio of the test sample (Analyte / internal standard) to the calibration curve, in the test sample The content of phenylalanine was calculated. 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 (the content of phenylalanine in the stratum corneum). The final quantitative value is calculated by the following formula, "content of phenylalanine in test specimen (μmol / mL)" × 0.2 (amount of extraction solvent mL) / 3.7994 (tape area cm ² ) / "protein of stratum corneum tape content (μg/cm ² )”.

Measurement of antioxidant activity in blood The antioxidant activity in blood of 48 subjects was evaluated. Blood was collected from a subject, left at room temperature for 30 minutes, and then centrifuged (2000 g, 10 minutes) to obtain serum. The serum total antioxidant capacity (Potential Anti Oxidant) in the obtained serum was measured using the antioxidant capacity measurement kit "PAO" (manufactured by Nikken Zile Co., Ltd., Japan Institute for Aging Control), and the obtained value was measured in the blood. of antioxidant power. The measurement procedure was carried out with reference to the package insert of each kit.

Measurement of complexion (Hb SO2 Index) in arms and cheeks Blood oxygen saturation index (Hb SO2 Index (%)) was measured in cheeks and inner forearms of 48 subjects to evaluate skin complexion. The Hb SO2 Index is measured by directly contacting a spectrophotometer (CM-700d, manufactured by Konica Minolta Co., Ltd.) with the subject's cheek and inner forearm, and the measurement results are analyzed using analysis software (CM-SA, manufactured by Konica Minolta Co., Ltd.). obtained by analyzing with The measurement was performed 7 times on the same part of each subject. The average value of each of the 5 measurements, excluding the measurement at which the Hb SO2 Index was maximum or minimum, was calculated, and the obtained value was used as the complexion of the skin on the arms and cheeks of each subject. bottom.

Autonomic nerve function (CVRR) measurement Autonomic nerve function (CVRR) was evaluated based on the pulse wave information of 48 subjects. Pulse wave information was measured and analyzed using BITAS-PULSEADVANCE (Shiseido version, manufactured by Human and Science Research Institute). A pulse wave sensor portion of BITAS-PULSEADVANCE was attached to the subject's finger, and the subject's pulse wave in a resting state was measured for 70 seconds. After the measurement was completed, the measurement results were analyzed by BITAS-PULSEADVANCE, and the obtained value was defined as the subject's autonomic nerve function (CVRR).

Statistical Processing Correlations between the antioxidant capacity in blood (PAO), complexion in arms and cheeks (Hb SO2 Index), and autonomic nerve function (CVRR) and the content of phenylalanine in the stratum corneum were investigated. Phenylalanine content in stratum corneum and PAO, Phenylalanine content in stratum corneum and Hb SO2 index in arm, Phenylalanine content in stratum corneum and Hb SO2 index in cheek, and Phenylalanine content in stratum corneum and CVRR, respectively, and the correlation coefficient was calculated. The results are shown in Table 2 and Figures 1-4.

From the above results, it was found that all of the above four physiological indices show a weak positive correlation with the phenylalanine content. Therefore, by measuring the phenylalanine content in the stratum corneum of the skin of the subject, it is possible to evaluate the antioxidant capacity in the blood (PAO), the complexion in the arms and cheeks (Hb SO2 Index), and the autonomic nerve function (CVRR). Also, it was shown that the total power of the skin can be evaluated based on these evaluation results.

Claims (12)

A method for evaluating the overall strength of skin in a subject, comprising:
(a) measuring the content of phenylalanine in the stratum corneum of the skin of the subject;
(b) based on the content of phenylalanine obtained in step (a), one or more physiological states selected from the group consisting of antioxidant capacity in the body, complexion of the skin, and autonomic nerve function in the subject; and (c) evaluating the overall strength of the skin of the subject based on the evaluation results obtained in step (b). step (a) is
(a1) a step of preparing a stratum corneum derived from the subject obtained by applying and peeling a tape to the skin of the subject; and (a2) content of phenylalanine in the stratum corneum obtained in step (a1) 2. The method of claim 1, comprising measuring the . 3. The method according to claim 1 or 2, wherein the index of antioxidant capacity in the body is blood antioxidant capacity (PAO). 4. The method according to any one of claims 1 to 3, wherein the index of skin complexion is the blood oxygen saturation index (Hb SO2 Index) of the arm or cheek. The method of any one of claims 1-4, wherein the indicator of autonomic function is autonomic activity (CVRR). An evaluation kit for use in the method according to any one of claims 1 to 5, comprising an instrument or reagent for measuring the content of phenylalanine in the stratum corneum of the skin. A method for evaluating one or more physiological condition indicators selected from the group consisting of antioxidant capacity in the body, skin complexion, and autonomic nerve function in a subject,
(a) the step of measuring the content of phenylalanine in the stratum corneum of the skin of the subject; and (b) based on the content of phenylalanine obtained in step (a), the antioxidant power in the body of the subject. , evaluating one or more indicators of physiological condition selected from the group consisting of skin complexion and autonomic nerve function. step (a) is
(a1) a step of preparing a stratum corneum derived from the subject obtained by applying and peeling a tape to the skin of the subject; and (a2) content of phenylalanine in the stratum corneum obtained in step (a1) 8. The method of claim 7, comprising measuring the . 9. The method according to claim 7 or 8, wherein the index of antioxidant capacity in the body is blood antioxidant capacity (PAO). The method according to any one of claims 7 to 9, wherein the index of skin complexion is the blood oxygen saturation index (Hb SO2 Index) of the arm or cheek. A method according to any one of claims 7 to 10, wherein the indicator of autonomic function is autonomic activity (CVRR). An evaluation kit for use in the method according to any one of claims 7 to 11, comprising an instrument or reagent for measuring the content of phenylalanine in the stratum corneum of the skin.

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