Method for non-invasive evaluation of aging degree of skin

Abstract

To provide a method for non-invasive evaluation of an aging degree of skin.SOLUTION: A method for non-invasive evaluation of an aging degree of skin includes the steps of: 1) applying a solvent-containing three-dimensional structure having a liquid retaining property and a liquid releasing property to skin; 2) collecting the solvent in the three-dimensional structure to obtain an extraction liquid; 3) concentrating the extraction liquid; and 4) measuring an amount of 8-hydroxy deoxyguanosine in the concentrated liquid; wherein the measured amount of 8-hydroxy deoxyguanosine is used as an indicator.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for non-invasively assessing the degree of skin aging. [Background technology]

[0002] The skin is the outermost layer of the human body and is easily susceptible to external stimuli. Therefore, reactive oxygen species are generated in the skin due to external factors such as ultraviolet rays and chemicals. The generated reactive oxygen species oxidize and damage DNA, cell membrane lipids, intracellular and extracellular proteins, and sugars, thereby reducing the cells' inherent functions. This results in various diseases and aging phenomena (Non-Patent Document 1).

[0003] 8-Hydroxydeoxyguanosine (8-hydroxy-2'-deoxyguanosine) is a substance in which the 8th position of deoxyguanosine, a component of DNA, is hydroxylated due to oxidative damage caused by reactive oxygen species. It is known as a marker of DNA oxidative damage (Non-Patent Document 2). The resulting 8-hydroxydeoxyguanosine is excised from DNA by the action of gene repair enzymes and excreted outside the cell, with most of it being excreted outside the body in urine. 8-Hydroxydeoxyguanosine has previously been measured in urine, serum, and peripheral leukocytes and used to assess oxidative stress. These measurements are known to fluctuate depending on lifestyle factors such as exercise and diet, as well as stress and disease. On the other hand, skin is expected to be strongly affected by external stimuli such as ultraviolet light and chemicals, and therefore may differ from measurements in urine, serum, and peripheral leukocytes used to assess oxidative stress within the body. Although a non-invasive method for measuring the amount of 8-hydroxydeoxyguanosine in skin is desirable, free 8-hydroxydeoxyguanosine is present in extremely small amounts in skin, making measurement difficult. There have been no reports of non-invasive measurement of the amount of 8-hydroxydeoxyguanosine in skin. A method for staining surgically excised skin with an anti-8-hydroxydeoxyguanosine antibody has been reported (Non-Patent Document 3).

[0004] Conventional methods for assessing the degree of skin aging have been reported, including methods for evaluating wrinkles, blemishes, changes in color, moisturizing ability, and elasticity, and methods for assessing the amount of accumulated advanced glycation end products (Non-Patent Document 4), a method in which a stimulant is added to a collagen gel embedded with skin fibroblasts collected by punch biopsy or the like and changes in the force generated by the cells are measured (Patent Document 1), a method using the measured value of blood vitamin D concentration as an index (Patent Document 2), and a method in which keratinous skin is collected by tape stripping and the lactic acid content of the keratinous skin is measured (Patent Document 3). However, no method is known for non-invasively quantifying the amount of 8-hydroxydeoxyguanosine in the skin and using the resulting value as an index for assessing the degree of skin aging. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2004-354159 [Patent Document 2] Patent Publication No. 2016-216435 [Patent Document 3] Patent Publication No. 2024-033289 [Non-patent literature]

[0006] [Non-Patent Document 1] Oleoscience Vol. 18 No. 3 (2018) 121-129 [Non-patent document 2] Surgery, Metabolism and Nutrition, Vol. 52, No. 2 (2018) 141-144 [Non-patent document 3] J.Invest.Dermat Vol.107 No.5(1996)733-737 [Non-patent document 4] J.Soc.Cosmet.Chem.Vol.53 No.2(2019)83-90 Summary of the Invention Problem to be solved by the invention

[0007] An object of the present invention is to provide a method for non-invasively evaluating the degree of skin aging. [Means for solving the problem]

[0008] The inventors conducted extensive research to solve this problem and discovered that the amount of 8-hydroxydeoxyguanosine in skin can be measured by applying a liquid-retaining and liquid-repelling three-dimensional structure to the skin, soaking it in a solvent, and then recovering the solvent from the three-dimensional structure to obtain an extract of skin components. The extract is then concentrated and the amount of 8-hydroxydeoxyguanosine in the concentrate is measured. Furthermore, the measured amount of 8-hydroxydeoxyguanosine is higher in people in their 40s and 50s than in their 20s and 30s, and is also higher in exposed areas than in non-exposed areas. Therefore, the inventors discovered that the measured amount of 8-hydroxydeoxyguanosine can be used as an index to noninvasively and objectively evaluate the degree of skin aging, leading to the completion of the present invention.

[0009] That is, the present invention includes the following inventions. (1) A method for non-invasively assessing the degree of skin aging, comprising: 1) a step of soaking a three-dimensional structure having liquid-retaining and liquid-repelling properties in a solvent and applying the structure to the skin; 2) a step of obtaining an extract by recovering the solvent in the three-dimensional structure; 3) a step of concentrating the extract; and 4) a step of measuring the amount of 8-hydroxydeoxyguanosine in the concentrate, and the measured amount of 8-hydroxydeoxyguanosine is used as an indicator to assess the degree of skin aging. (2) A method for evaluating the degree of skin aging, characterized in that the three-dimensional structure having liquid-retaining and liquid-repelling properties described in (1) is one or more materials selected from the group consisting of absorbent cotton, nonwoven fabric, paper, and cloth. (3) A method for evaluating the degree of skin aging, characterized in that the solvent according to (1) is water. (4) A method for evaluating the degree of skin aging, characterized in that in the step of concentrating the extract described in (1), the extract is concentrated to 1 / 10,000 to 1 / 100 of the liquid volume of the solvent contained in the three-dimensional structure. [Effects of the Invention]

[0010] The present invention provides a method for assessing the degree of skin aging noninvasively by impregnating a three-dimensional structure with liquid-retaining and liquid-repelling properties with a solvent, applying the structure to the skin, recovering the solvent from the three-dimensional structure to obtain an extract of skin components, concentrating the extract, measuring the amount of 8-hydroxydeoxyguanosine in the concentrate, and using the measured amount of 8-hydroxydeoxyguanosine as an index. Furthermore, the present invention is also useful as a means for verifying the effectiveness of pharmaceuticals, quasi-drugs, cosmetics, and foods intended to inhibit skin aging. DETAILED DESCRIPTION OF THE INVENTION

[0011] The method of the present invention for non-invasively assessing the degree of skin aging comprises the steps of: 1) soaking a three-dimensional structure having liquid-retaining and liquid-repelling properties in a solvent and applying the structure to the skin; 2) obtaining an extract by recovering the solvent in the three-dimensional structure; 3) concentrating the extract; and 4) measuring the amount of 8-hydroxydeoxyguanosine in the concentrate, and assessing the degree of skin aging using the measured amount of 8-hydroxydeoxyguanosine as an index.

[0012] In the present invention, 8-hydroxydeoxyguanosine (8-hydroxy-2'-deoxyguanosine) is a substance in which the 8th position of deoxyguanosine, a component of DNA, is hydroxylated. It is also referred to as 8-oxo-2'-deoxyguanosine, 2'-deoxy-8-hydroxyguanosine, 7,8-dihydro-8-oxo-2'-deoxyguanosine, etc. Abbreviations such as 8-OHdG and 8-oxo-dG are also used.

[0013] In the present invention, "non-invasive" means not damaging a living body or causing any burden such as pain or suffering. Removing a part of the skin by surgical operation and using the removed specimen for evaluation is an invasive technique and is therefore not included in the present invention.

[0014] In the present invention, "aging degree" refers to physiological changes in the skin caused by the generation of reactive oxygen species in the skin due to aging or exposure to ultraviolet light, which damages DNA. The method for assessing the aging degree in the present invention evaluates the aging degree of the skin based on the finding that the amount of 8-hydroxydeoxyguanosine in the skin increases with aging or exposure to ultraviolet light. Therefore, in the evaluation, the amount of 8-hydroxydeoxyguanosine in the skin of a subject to be assessed for the aging degree is measured, and the aging degree is evaluated based on the measured amount of 8-hydroxydeoxyguanosine.

[0015] In the present invention, the degree of skin aging can be evaluated, for example, by comparing the amount of 8-hydroxydeoxyguanosine in a subject measured by the above-mentioned method with a predetermined reference amount. The reference amount can be determined using age as an index, for example, by averaging values ​​for a young group, an elderly group, or a specific age group. Alternatively, the amount of 8-hydroxydeoxyguanosine can be determined using UV exposure as an index, by averaging values ​​for exposed and non-exposed areas, or for average daily UV exposure times. For example, if the amount of 8-hydroxydeoxyguanosine in a subject is higher than the reference amount, the degree of aging can be evaluated as high; if it is lower, the degree of aging can be evaluated as low. Alternatively, a reference amount can be defined for each age group, and if the amount of 8-hydroxydeoxyguanosine is higher than the reference amount for the age group corresponding to the subject's actual age, the degree of aging can be evaluated as high or advanced, whereas if it is lower than the reference amount, the subject can be evaluated as low or not advanced. Furthermore, when verifying the effectiveness of pharmaceuticals, quasi-drugs, cosmetics, and foods intended to inhibit skin aging, the amount of 8-hydroxydeoxyguanosine before and after use or ingestion of the product is used as the base line, and if there is a decrease in the amount of 8-hydroxydeoxyguanosine, it can be evaluated that the degree of aging has decreased and aging has been inhibited.

[0016] The subject in the present invention may be a human, or may be a non-human mammal such as a monkey, chimpanzee, dog, cat, cow, pig, rabbit, mouse, rat, or guinea pig.

[0017] The liquid-retaining property in the present invention refers to the property of retaining 1 / 100 to 1 / 1 volume of solvent when the solvent is added to the object.

[0018] In the present invention, the term "lyotropy" refers to the property of an object that retains a solvent in an amount of 1 / 100 to 1 / 1 of its volume, and that allows the object to expel the solvent in an amount of 1 / 100 to 1 / 1 of the retained solvent by a process such as squeezing, suction, or centrifugation.

[0019] The three-dimensional structure of the present invention is an object that, when attached to the skin, has a volume of 0.1 to 100 cm in width and 0.1 to 100 cm in depth on a surface parallel to the skin and a volume of 0.001 to 10 cm in thickness perpendicular to the skin, preferably a volume of 1 to 15 cm in width, 1 to 15 cm in depth, and 0.005 to 3 cm in thickness, and more preferably a volume of 4 to 7 cm in width, 4 to 7 cm in depth, and 0.05 to 1.5 cm in thickness.

[0020] The three-dimensional structure having liquid-retaining and liquid-releasing properties in the present invention refers to absorbent cotton, nonwoven fabric, paper, cloth, and porous sponges such as natural sponge and polyurethane foam, made from fibers such as cotton, linen, wool, silk, polyester, nylon, acrylic, polyurethane, cupra, rayon, glass wool, and pulp, which have gaps in their structure that retain liquid and from which the retained liquid can be removed by squeezing, suction, centrifugation, or the like. Although not particularly limited, preferred are cosmetic cotton puffs, face masks, absorbent cotton, towels, filter paper, kitchen paper, and the like, which adhere to the skin when they absorb liquid and can be deformed to fit the shape of the skin.

[0021] A plurality of three-dimensional structures having liquid-retentive and liquid-repellent properties according to the present invention may be attached as necessary, and these may be combined and extracted for measurement.

[0022] The solvent in the present invention is not particularly limited as long as it dissolves 8-hydroxydeoxyguanosine, but water is preferred because it is gentle on the skin. The solvent may be used alone or in combination of two or more.

[0023] The amount of solvent in the present invention can be freely selected within the range of the amount of liquid that can be retained by the three-dimensional structure having liquid-retaining and liquid-releasing properties. However, if the amount of liquid is too small, it will be difficult to fill the entire three-dimensional structure with the solvent and to remove the liquid after extraction, and even if the amount of liquid is increased, there is a possibility that the solvent will drip. Therefore, the amount of solvent is preferably 1 / 100 to 1 / 1 volume, and more preferably 1 / 50 to 1 / 2 volume, relative to the volume of the structure.

[0024] The extraction time for skin components in the present invention is not particularly limited, but if the extraction time for skin components is too long, the solvent will dry out and the extract will not be able to be squeezed out, and if the extraction time is too short, the skin components will not be sufficiently extracted, so 3 to 30 minutes is preferable, and 5 to 15 minutes is even more preferable.

[0025] The method for extracting the extract from the three-dimensional structure having liquid-retaining and liquid-syringing properties of the present invention is not particularly limited, and examples include squeezing, suction, centrifugation, etc. After recovering the extract, in order to prevent 8-hydroxydeoxyguanosine from remaining in the three-dimensional structure, a solvent may be added to the three-dimensional structure from which the extract has been extracted, and the solvent may be removed again and combined with the extract previously extracted.

[0026] The concentration ratio in the present invention is not particularly limited, but if the concentration is insufficient, the 8-hydroxydeoxyguanosine concentration in the concentrate will be low and below the measurement limit, so it is preferable to concentrate it to about 1 / 10,000 to 1 / 100 of the liquid volume of the solvent retained in the three-dimensional structure.The concentration method is not particularly limited, but a method in which the sample is once dried by freeze-drying or the like and then redissolved in a small amount of water is preferred, as this allows the concentration ratio of the sample to be controlled.

[0027] In the present invention, the method for measuring 8-hydroxydeoxyguanosine in the extract is not particularly limited, and any method such as ELISA, HPLC, or LC / MS / MS can be used for measurement.

[0028] EXAMPLES Next, examples will be given to explain the present invention in detail, but the present invention is not limited to these examples. [Example]

[0029] Measurement of 8-hydroxydeoxyguanosine levels in non-sun-exposed and sun-exposed skin using cotton puffs The inner upper arms (non-exposed area) and face (exposed area) of 25 subjects were washed with facial cleanser and lightly wiped dry. Four cotton puffs (6.5cm x 5cm x 0.7cm, Menard Japan Cosmetics) were soaked evenly with 3mL of ultrapure water and placed on each of the inner upper arms and face, respectively, and pressed tightly against the skin. After 10 minutes, the four cotton puffs were transferred to a 20mL syringe, and the extract was squeezed into a spitz tube. Another 15mL of ultrapure water was added to the cotton puffs remaining in the syringe, and the ultrapure water was squeezed out from the cotton puffs and combined with the extract squeezed into the spitz tube. The extract in the spitz tube was then lyophilized to remove water and redissolved in 100µL of ultrapure water to obtain a concentrate. The concentrate was measured using a high-sensitivity 8-OHdG Check ELISA (Nikken Zaile) to calculate the amount of 8-hydroxydeoxyguanosine per area of ​​skin used for extraction.

[0030] Table 1 shows the comparison of 8-hydroxydeoxyguanosine levels between non-exposed and exposed areas. These results demonstrate that the amount of 8-hydroxydeoxyguanosine in the skin can be measured. Furthermore, the amount of 8-hydroxydeoxyguanosine was higher in exposed areas than in non-exposed areas. 8-hydroxydeoxyguanosine is released extracellularly when DNA is damaged by UV rays and the damaged genes are repaired. Therefore, the higher amount of 8-hydroxydeoxyguanosine measured in exposed areas reflects the amount of DNA damage in the skin. Since DNA damage reduces skin function and leads to aging, it was demonstrated that the measured amount of 8-hydroxydeoxyguanosine can be used as an indicator to evaluate the degree of aging. Furthermore, although this test used an ELISA kit with a detection limit of 0.125 ng / mL, without concentration, the value was below the detection limit and measurement was not possible. In addition, keratin was collected from the same area using the tape stripping method by repeatedly peeling the keratin 10 times with 10 pieces of adhesive tape (2 cm x 2 cm, Nichiban). The 10 pieces were combined and the amount of 8-hydroxydeoxyguanosine in the keratin was measured, but it was below the detection limit and could not be measured.

[0031] [Table 1] [Example]

[0032] Measurement of 8-hydroxydeoxyguanosine levels in the skin of people in their 20s-30s and 40s-50s using cotton puffs Twenty-five subjects in their 20s to 50s were divided into two groups: 13 in their 20s to 30s and 12 in their 40s to 50s. After washing the inside of their upper arms with facial cleanser and gently wiping them dry, four cotton puffs (6.5cm x 5cm x 0.7cm, Menard Japan Cosmetics) soaked evenly with 3mL of ultrapure water were placed on the inside of their upper arms and pressed tightly against the skin. After 10 minutes, the four cotton puffs were transferred to a 20mL syringe, and the extract was squeezed into a Spitz tube. 15mL of ultrapure water was added to the cotton puffs remaining in the syringe, and the ultrapure water was squeezed out from the cotton puffs and combined with the extract squeezed into the Spitz tube. The extract in the Spitz tube was then lyophilized to remove water and redissolved in 100µL of ultrapure water to obtain a concentrated solution. The concentrate was measured using a highly sensitive 8-OHdG Check ELISA (Nikken Zaile), and the amount of 8-hydroxydeoxyguanosine per area of ​​skin used for extraction was calculated.

[0033] Table 2 shows the results of a comparison of 8-hydroxydeoxyguanosine levels between people in their 20s and 30s and those in their 40s and 50s. It was found that 8-hydroxydeoxyguanosine levels were higher in people in their 40s and 50s compared to people in their 20s and 30s. It is thought that with aging, the activity of antioxidant enzymes against reactive oxygen species produced in the skin declines, making DNA damage more likely to occur due to reactive oxygen species. Therefore, the higher levels of 8-hydroxydeoxyguanosine measured in people in their 40s and 50s reflect the amount of DNA damage in the skin. Furthermore, because DNA damage reduces skin function and leads to aging, it has been shown that the measured levels of 8-hydroxydeoxyguanosine can be used as an indicator to evaluate the degree of aging.

[0034] [Table 2] [Industrial Applicability]

[0035] The present invention can provide a method for non-invasively evaluating the degree of skin aging.

Claims

1. A method for non-invasively evaluating the degree of skin aging, comprising: 1) a step of soaking a three-dimensional structure having liquid-retaining and liquid-repelling properties in a solvent and applying the structure to the skin; 2) a step of recovering the solvent in the three-dimensional structure to obtain an extract; 3) a step of concentrating the extract; and 4) a step of measuring the amount of 8-hydroxydeoxyguanosine in the concentrate, and the method evaluates the degree of skin aging using the measured amount of 8-hydroxydeoxyguanosine as an index.

2. A method for evaluating the degree of skin aging, characterized in that the three-dimensional structure having liquid-retaining and liquid-repelling properties described in claim 1 is one or more materials selected from the group consisting of absorbent cotton, nonwoven fabric, paper, and cloth.

3. 2. The method for evaluating the degree of skin aging according to claim 1, wherein the solvent is water.

4. A method for evaluating the degree of skin aging, characterized in that in the step of concentrating the extract according to claim 1, the extract is concentrated to 1 / 10,000 to 1 / 100 of the liquid volume of the solvent contained in the three-dimensional structure.

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