Skin characteristics evaluation method

Abstract

To provide a method for evaluating skin characteristics around the eyes that enables prediction of the formation of fine lines, and furthermore to develop cosmetics that prevent the formation of fine lines and improve skin texture based on the mechanism involved in such an evaluation method.SOLUTION: ADAMTSL6 has been discovered as an elastic fiber formation cofactor, and by using ADAMTSL6 as an indicator, it is possible to evaluate skin characteristics around the eyes. We also provide a screening method for agents that improve skin characteristics around the eyes using ADAMTSL6 as an indicator, as well as a novel skin characteristic improving agent.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for evaluating skin properties using ADAMTSL6, which promotes elastic fiber formation, as an index, and a method for screening for an agent for improving skin properties using ADAMTSL6 as an index, and further relates to the use of a component selected by such a screening method. [Background technology]

[0002] Fine lines around the eyes are also called crepe lines, and are wrinkles formed in the shallow layers of the skin. The cause of fine lines is a decrease in moisture retention, and the formation of fine lines is promoted by dryness, ultraviolet rays, and aging. Fine lines are closely related to skin texture (Non-Patent Document 1), and fine lines are formed when the skin texture becomes rough and the skin grooves become deep. The mesh-like pattern formed by the skin grooves and skin ridges on the skin surface is called skin texture, and when the skin texture is fine and uniform, it is considered to have a regular texture. A stratum corneum with a regular texture has a high moisture retention function and is soft, and the occurrence of fine lines is suppressed. On the other hand, fine lines are formed when the skin texture becomes rough and large, and the skin grooves become deep. It has been shown that elastic fibers in the dermis layer are involved in the formation of skin texture (Patent Document 1: JP 2012-117827 A). In particular, elastic fibers that run perpendicular to the epidermal surface are thought to line the epidermis and are directly involved in the formation of skin texture. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2012-117827 A [Non-patent literature]

[0004] [Non-Patent Document 1] Skin Research and Technology 2015; 21: 184-191 [Non-Patent Document 2] JBC Vol.285, No. 7 pp.4870-4882 Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors aim to provide a method for evaluating skin properties that enables prediction of the formation of fine wrinkles, and further to develop cosmetics that prevent the formation of fine wrinkles and smooth skin texture based on the mechanisms involved in such an evaluation method. [Means for solving the problem]

[0006] In attempting to elucidate factors in the dermis that are involved in skin properties, the present inventors focused on factors whose abundance changes significantly with age, and searched for factors involved in the formation of elastic fibers, discovering a factor called ADAMTSL6 that strongly co-localizes with oxytalan fibers and elaunin fibers. Further research by the present inventors demonstrated that ADAMTSL6 is involved in the formation of elastic fibers in the skin, and discovered ADAMTSL6 as a cofactor for elastic fiber formation. Thus, the present invention relates to the following: [1] A method for evaluating skin condition using ADAMTSL6 gene expression level or protein level as an indicator. [2] The evaluation method according to item 1, wherein the evaluation includes predicting future formation of fine lines. [3] A method for screening skin quality improving agents using the gene expression level or protein level of ADAMTSL6 as an indicator. [4] Culturing fibroblasts in a medium containing the candidate component; Measuring the gene expression level or protein level of ADAMTSL6 in a culture cultured in a medium containing the candidate component; and comparing the measured gene expression level with a control, and determining that the added candidate ingredient is a skin property improving agent when the gene expression level or protein level of ADAMTSL6 is enhanced; The method according to item 3, comprising: [5] The method according to item 4, wherein the control is a threshold determined from the gene expression level or protein level of ADAMTSL6 in a fibroblast culture cultured in a medium not supplemented with the candidate component. [6] The method according to item 4 or 5, wherein the skin property improving agent can be at least one agent selected from the group consisting of an ADAMTSL6 expression promoter, a fibrillin 1 fiber formation promoter, an oxytalan fiber formation promoter, a fine wrinkle improving agent, a fine wrinkle prevention agent, and a skin texture improving agent. [7] An agent for promoting the expression of the ADAMTSL6 gene, comprising at least one extract selected from the group consisting of camellia seed extract, soybean extract, longan seed extract, Cysticerass tamaricifolia extract, and peach seed extract. [8] A microfibril formation promoter comprising the ADAMTSL6 gene expression promoter described in item 7. [9] An agent for promoting the formation of oxytalan fibers, comprising an agent for promoting the expression of the ADAMTSL6 gene described in item 7.

[10] A skin condition improving agent comprising the ADAMTSL6 gene expression promoter described in item 7.

[11] A microfibril formation promoter comprising at least one extract selected from the group consisting of camellia seed extract, soybean extract, longan seed extract, Cysticera tamaricifolia extract, and peach seed extract. Effect of the Invention

[0007] Using ADAMTSL6 as an indicator makes it possible to evaluate skin properties and screen for skin property improving agents. [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows fluorescence microscopy images of age-related changes in ADAMTSL6 protein levels. Skin samples from the buttocks (non-exposed area) and the eye area (exposed area) were used. [Diagram 2]FIG. 2 is a set of fluorescence micrographs showing the localization of ADAMTSL6 and fibrillin-1, and the localization of ADAMTSL6 and elastin. [Diagram 3] FIG. 3 is a set of fluorescence micrographs showing the localization of ADAMTSL6 and fibrillin-1 in ADAMTSL6 knockout dermal fibroblasts. [Figure 4] Figure 4 is a graph showing the change in gene expression level of ATAMTSL6 (THSD4) when camellia seed extract (A), soybean extract (B), (C) longan seed extract, (D) Cysticeira tamaricifolia extract, and (E) peach seed extract were added to dermal fibroblasts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention relates to a method for evaluating skin properties using ADAMTSL6 as an index. The present invention also relates to a method for screening a skin property improving agent using ADAMTSL6 as an index. Furthermore, the present invention also relates to use of a component screened by the screening method.

[0010] The skin properties evaluated by the present invention are those in areas where fine wrinkles occur, particularly in exposed areas such as the face. Areas where fine wrinkles occur include the eye area, such as the lower eyelid and upper eyelid, and the skin properties in these areas are evaluated. Skin properties include firmness and texture, or may be a combination of firmness and texture. Firmness refers to a state in which the elasticity and moisture retention function of the skin are maintained, and in a firm state, the formation of fine wrinkles is suppressed. Texture is a structure composed of skin grooves and skin ridges that appear on the epidermis, which scatters light and contributes to transparency, etc. The skin around the eyes is thin, and when the firmness around the eyes is lost and the skin dries, fine wrinkles occur. Fine wrinkles are wrinkles that mainly occur in the epidermis around the eyes, and their formation mechanism is different from deep dermal wrinkles such as those around the eyes. Fine wrinkles can be formed when the skin pattern composed of skin grooves and skin ridges becomes rough and the skin grooves become deep. The skin grooves and ridges are formed by the lining of elastic fibers running perpendicular to the basement membrane in the dermis layer, and a decrease in the elastic fibers running perpendicular to the basement membrane leads to loss of firmness around the eyes, which leads to the development of fine wrinkles. Firmness around the eyes can be measured using a cutometer or a derma torque meter, and texture can be measured using a microscope. The present invention makes it possible to evaluate skin properties from the perspective of the inside of skin tissue by using ADAMTSL6, a factor that promotes the formation of elastic fibers, as an index. This makes it possible to predict the formation of fine wrinkles in the future.

[0011] ADAMTSL6 is a gene belonging to the ADAMTS superfamily, and is also known as THSD4. ADAMTSL is an abbreviation for "A disintegrin and metalloproteinase with thrombospondin motifs-like" protein, and has been reported to bind to fibrillin 1 and promote the formation of microfibrils (Non-Patent Document 2: JBC Vol. 285, No. 7 pp. 4870-4882). ADAMTSL6 is widely distributed in the epidermis and dermis, but is particularly accumulated in elastic fibers running perpendicular to the basement membrane (Figure 2). ADAMTSL6 changes little with age in the skin of non-exposed areas (buttocks), while it changes with age in the skin of exposed areas (lower eyelids), and its expression decreases especially from the 30s to the 40s (Figure 1). The present inventors' research has shown that ADAMTSL6 colocalizes with fibrillin-1 and elastin that compose elastic fibers, but does not coincide with the localization of all fibrillin-1 and elastin. It is colocalized with fibrillin-1 and elastin in elastic fibers that run perpendicular to the basement membrane just below the basement membrane in the dermis, but is not colocalized with fibrillin-1 and elastin present in the deeper layers of the dermis (Figure 2). Thus, ADAMTSL6 is not distributed throughout the elastic fibers, and is distributed in particular in oxytalan fibers just below the basement membrane and elaunin fibers connected to oxytalan fibers, but is not distributed in deeper fibers (Figure 2). siRNA knockdown experiments in dermal fibroblast cultures showed that the formation of fibrillin-1 fibers was suppressed (Figure 3). These results and knowledge from well-known techniques indicate that ADAMTSL6 is released from dermal fibroblasts into the extracellular matrix, is involved in the formation of microfibrils in the extracellular matrix, and contributes to the formation of elastic fibers, particularly oxytalan fibers and elaunin fibers.

[0012] The elastic fibers that line the basement membrane have different properties depending on whether they are located just below the basement membrane or deeper. The elastic fibers just below the basement membrane are called oxytalan fibers, while those deeper are called elaunin fibers. These elastic fibers are made up of elastin and microfibrils. Oxytalan fibers contain less elastin and are thin fibers, while elaunin fibers contain more elastin and form thicker fibers.

[0013] Microfibrils are elastic fibers, also called fine fibers. They are mainly composed of fibrillin and may contain multiple component proteins such as LTBP (latent transforming growth factor beta binding protein), MAGP (microfibril-associated glycoprotein), MFAP (microfibril-associated protein), emylin, and fibulin. Microfibrils are usually thin fibers, but when elastin binds to microfibrils, they become thicker fibers.

[0014] Elastin is synthesized as a precursor, tropoelastin, which aggregates around and between microfibrils and forms elastic fibers by cross-linking. It is found in connective tissues and can be found in arteries, lungs, and the dermis. Oxytalan fibers present just below the basement membrane contain less elastin and form thin fibers, while elaunin fibers present deeper in the body contain more elastin and are thicker.

[0015] Oxytalan fibers are a type of elastic fiber and are a fibrous component that creates mechanical strength in connective tissues in living organisms. Elastic fibers are found in abundance in organs and tissues that are subject to mechanical pressure, such as the skin, blood vessels, lungs, and ligaments. Oxytalan fibers mainly contain microfibrils and elastin, but the elastin content is low and they are mainly composed of thin microfibrils. Oxytalan fibers exist vertically from just below the basement membrane and line the basement membrane. When observing the internal structure of the skin at the skin ridge of the texture, the basement membrane in the area corresponding to the skin ridge is lined with many oxytalan fibers, while the basement membrane in the area corresponding to the skin groove is not lined with oxytalan fibers, and it is believed that the skin ridge and skin groove structures that make up the texture are formed by oxytalan fibers that line the basement membrane. In addition, by backing the basement membrane, it is possible to bring firmness to the skin surface. The epidermis around the eyes is particularly thin, and the influence of the backing structure of the basement membrane is strongly evident. Oxytalan fibers are connected to elaunin fibers at deeper sites. Elaunin fibers are usually thicker than oxytalan fibers. ADAMTSL6 is localized to oxytalan and elaunin fibers, but is particularly abundant in oxytalan fibers. Without intending to be limited by theory, ADAMTSL6 is thought to assist in the maturation of fibrillin-1 into microfibrils and also contribute to the maintenance of oxytalan fibers. Therefore, ADAMTSL6 can also be called an elastic fiber cofactor.

[0016] In one embodiment of the present invention, the present invention relates to a method for evaluating firmness of the eye area using ADAMTSL6 as an index. In particular, the gene expression or protein amount of ADAMTSL6 can be used as an index. Since ADAMTSL6 contributes to the formation of oxytalan fibers, it can be determined that the higher the gene expression or protein amount of ADAMTSL6, the better the firmness of the eye area. By comparing with one or more predetermined reference values, the firmness can be evaluated into multiple stages.

[0017] Eye firmness is particularly involved in the formation of fine lines and / or fine wrinkles around the eyes. Therefore, the method for evaluating eye firmness according to the present invention can also evaluate the possibility of fine lines forming now or in the future. Therefore, the present invention may also relate to a method for evaluating the possibility of fine lines forming using ADAMTSL6 as an indicator. Subjects with low gene expression or protein amount of ADAMTSL6 are more likely to form fine lines in the future.

[0018] The gene expression or protein amount of ADAMTSL6 can be measured in the obtained skin sample. In one example, the protein of ADAMTSL6 can be quantified in a skin tissue section using an anti-ADAMTSL6 antibody. The antibody-based method can be any method known in the art, such as Western blotting or immunostaining. In another example, the gene expression amount is determined by quantifying the amount of ADAMTSL6 mRNA in the skin sample. The gene expression amount can usually be determined based on a known method, such as real-time PCR.

[0019] In a further aspect of the present invention, the present invention also relates to a screening method for an agent for improving eye-area skin properties using ADAMTSL6 as an index. The gene expression level or protein level of ADAMTSL6 can be used as an index. More specifically, the screening method of the present invention is as follows: Culturing fibroblasts in a medium supplemented with the candidate component; Measuring the gene expression or protein amount of ADAMTSL6 in a culture cultured in a medium containing the candidate component; and A step of comparing the measured gene expression level or protein level with a control, and determining the added candidate ingredient as an eye skin property improving agent when the gene expression or protein level of ADAMTSL6 is enhanced. The eye skin property improving agent thus screened can promote the formation of oxytalan fiber and / or elaunin fiber just below the basement membrane through the promotion of ADAMTSL6 expression, thereby smoothing the skin texture, inhibiting the formation of fine wrinkles, and improving the firmness of the eye area. Therefore, the eye skin property improving agent can also be called an ADAMTSL6 expression promoter, a microfibril formation promoter, an oxytalan fiber formation promoter, a fine wrinkle improving agent, a fine wrinkle prevention agent, a skin texture improving agent, and an eye firmness improving agent. By carrying out such a screening method, camellia seed extract, soybean extract, longan seed extract, cystoseira tamarisifolia extract, and peach seed extract were screened as eye skin property improving agents (Figure 4).

[0020] The fibroblasts are particularly dermal fibroblasts. The dermal fibroblasts may be primary cultures or established cells. The fibroblasts may be cultured alone, or may be a three-dimensional cultured skin model cultured together with other cells that constitute the skin, such as keratinocytes, or may be organ cultures. The control may be a gene expression level or protein level in a culture that differs only in that the candidate component is not added. The gene expression level or protein level of the control may be measured by conducting an experiment in parallel with an experiment in which the candidate component is added, or may be determined in advance.

[0021] Candidate components used in the screening method of the present invention can be any library of cosmetic materials, food materials, pharmaceutical materials, etc. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or may be synthesized compounds and prepared extracts.

[0022] The plant extracts described in this specification can be obtained by a conventional method, for example, by immersing or refluxing the plant with an extraction solvent at room temperature or at high temperature, followed by filtration and concentration. As the extraction solvent, any solvent that is usually used for extraction can be used, for example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer, or organic solvents such as alcohols such as ethanol, propylene glycol, 1,3-butylene glycol, glycerin, hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc., can be used alone or in combination. Preferably, a mixed solvent of water and alcohol, such as 1,3-butylene glycol, is used as the solvent. The extract obtained by extraction with the above solvent can be used as it is, or the extract can be concentrated by, for example, freeze-drying, and if necessary, it can be used after removing impurities using an adsorption method, for example, an ion exchange resin, or after adsorption on a column of porous polymer (e.g., Amberlite XAD-2), eluted with a desired solvent, and further concentrated.

[0023] Camellia seed extract is an extract extracted from the seeds of Camellia japonica native to Japan, and is expressed as the display name / INCI name: Camellia seed extract / CAMELLIA JAPONICA SEED EXTRACT in the display name used for displaying all ingredients of cosmetics specified by the Japan Cosmetic Industry Association and the international display name according to the INCI name (International Nomenclature of Cosmetic Ingredients). It can be prepared by crushing camellia seeds and extracting them with water, alcohol or a mixed solution thereof. The camellia seeds may be fresh or dried, or may be fermented. As the alcohol, ethanol, propylene glycol, glycerin or butylene glycol is used. More preferably, it can be extracted with a mixture of water and alcohol, particularly water and ethanol, water and glycerin, or water and 1,3-butylene glycol in any ratio, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and more preferably 50:50. Camellia seed extract is blended at a concentration of 0.0001% to 1%, more preferably 0.01% to 0.3%.

[0024] Soybean extract is an extract extracted from soybean (Glycine soja) seeds, and is represented by the international display name / INCI name: soybean seed extract / GLYCINE SOJA (SOYBEAN) SEED EXTRACT. It can be prepared by crushing soybean seeds and extracting them with water, alcohol, or a mixed solution thereof. The soybean seeds may be raw or dried, or may be fermented. As the alcohol, ethanol, propylene glycol, glycerin, or butylene glycol is used. More preferably, it can be extracted with a mixture of water and alcohol, particularly water and ethanol, water and glycerin, or water and 1,3-butylene glycol, in any ratio, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and more preferably 50:50. The soybean seed extract is blended at a concentration of 0.001% to 1%, more preferably 0.01% to 1%.

[0025] Longan seed extract is an extract extracted from the seeds of longan (Dimocarpus longan L.) native to South Asia to Southeast Asia, and is represented by the International Name / INCI Name: Longan Seed Extract / NEPHELIUM LONGANA SEED EXTRACT. It can be prepared by crushing longan seeds and extracting them with water, alcohol or a mixed solution thereof. The seeds may be raw or dried, or may be fermented. As the alcohol, ethanol, propylene glycol, glycerin or butylene glycol is used. More preferably, it can be extracted with a mixture of water and alcohol, particularly water and ethanol, water and glycerin, or water and 1,3-butylene glycol in any ratio, for example, a mixture of 10:90 to 90:10, preferably a mixture of 30:70 to 70:30, and more preferably a mixture of 50:50. Longan seed extract is formulated at a concentration of 0.001% to 1%, more preferably 0.01% to 1%.

[0026] Cystoseira tamariscifolia extract is an extract extracted from rainbow algae (Cystoseira tamariscifolia), a type of brown algae that grows in the coastal areas of Europe, and is represented by the international display name / INCI name: Cystoseira Tamariscifolia Extract. It can be prepared by crushing the main body of rainbow algae and extracting it with water, alcohol, or a mixed solution thereof. The main body of rainbow algae may be raw or dried, or may be fermented. As the alcohol, ethanol, propylene glycol, glycerin, or butylene glycol is used. More preferably, it can be extracted with a mixed solution of water and alcohol, particularly water and ethanol, water and glycerin, or water and 1,3-butylene glycol, in any ratio, for example, a mixed solution of 10:90 to 90:10, preferably a mixed solution of 30:70 to 70:30, and more preferably a mixed solution of 50:50. Cystoseira tamaricifolia extract is blended at a concentration of 0.001% to 1%, and more preferably 0.01% to 1%.

[0027] Peach seed extract is an extract extracted from the seeds of peach (Prunus persica) native to China, and is represented by the international display name / INCI name: Peach seed extract / Prunus Persica (Peach) Kernel Extract. It can be prepared by crushing peach seeds and extracting them with water, alcohol or a mixed solution thereof. The seeds may be raw or dried, or may be fermented. As the alcohol, ethanol, propylene glycol, glycerin, or butylene glycol is used. More preferably, it can be extracted with a mixture of water and alcohol, particularly water and ethanol, water and 1,3-butylene glycol, or water and glycerin in any ratio, for example, a mixture of 10:90 to 90:10, preferably a mixture of 30:70 to 70:30, and more preferably a mixture of 50:50. The peach seed extract is blended at a concentration of 0.001% to 1%, more preferably 0.01% to 1%.

[0028] In the present invention, the term "eye skin quality improving agent", "ADAMTSL6 expression promoter", "eye firmness improving agent", "microfibril fiber formation promoter", "oxytalan fiber formation promoter", "fine wrinkle improving agent", "fine wrinkle prevention agent", or "texture improving agent" can be used interchangeably. Without intending to be limited by theory, the eye skin quality improving agent selected in the screening method of the present invention promotes the expression of ADAMTSL6 in dermal fibroblasts. ADAMTSL6 associates with fibrillin 1 in the dermis and promotes the formation of microfibrils. The microfibrils further incorporate elastin to form oxytalan fibers, which line the basement membrane from the dermis, thereby improving texture and improving or preventing fine wrinkles.

[0029] In the present invention, the skin quality improving agent for the eye area, the ADAMTSL6 expression promoter, the eye firmness improving agent, the microfibril fiber formation promoter, the oxytalan fiber formation promoter, the fine wrinkle improving agent, the fine wrinkle prevention agent, or the texture improving agent may each be incorporated into a cosmetic, a pharmaceutical, or a quasi-drug, and may also be incorporated into a food, for example, a nutritional supplement such as a supplement. These drugs may be administered orally or parenterally, for example, transdermally. When administered transdermally, they can be formulated into a skin topical preparation. The skin topical preparation is not particularly limited as long as it can be applied to the skin, and any formulation such as a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. can be applied. When formulated into a skin topical preparation, a base and excipients commonly used in skin topical preparations, such as a preservative, emulsifier, pH adjuster, etc., may be used. When incorporated into cosmetics, they can be incorporated into facial or body cosmetics such as lotions, milky lotions, beauty essences, creams, lotions, packs, essences, gels, etc., makeup cosmetics such as foundations, makeup bases, concealers, etc., and even bath additives, etc. By using cosmetics, medicines, and quasi-drugs containing the components of the present invention, it is possible to maintain or improve skin texture and maintain or improve firmness around the eyes through the promotion of oxytalan fiber formation, thereby improving or preventing fine wrinkles.

[0030] The concentration of the eye skin quality improving agent, ADAMTSL6 expression promoter, eye firmness improving agent, microfibril fiber formation promoter, oxytalan fiber formation promoter, fine wrinkle improving agent, fine wrinkle prevention agent, or texture improving agent of the present invention can be selected arbitrarily from the viewpoint of exerting the desired effect, for example, the effect of maintaining or improving the firmness of the eye, or suppressing or improving fine wrinkles. From the viewpoint of blending as a skin external preparation, the extract of the present invention can be further blended with a commercially available extract at 0.0005% to 0.5%. From the viewpoint of fully exerting the effect, it can be blended preferably at 0.001% or more, and more preferably at 0.005% or more. From the viewpoint of avoiding a strong odor, it can be blended preferably at 0.1% or less, and more preferably at 0.05% or less. The commercially available extract is usually blended at a concentration of about 0.1 to 10%, preferably about 0.5 to 5%.

[0031] All documents mentioned in this specification are incorporated herein by reference in their entirety. The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention can be modified, for example, by adding, deleting, and replacing the constituent elements of the present invention, without departing from the spirit of the present invention. EXAMPLES

[0032] Example 1: Changes in ADAMTSL6 protein levels with age Skin samples were obtained by punch biopsy from the buttocks and corners of the eyes of subjects in their 20s, 30s, 40s, and 60s. The obtained skin samples were fixed using the AMeX fixation method (reference: Am J Pathol, 1986, 125-3, p431-435), embedded in paraffin, and 4 μm thick skin sections were obtained. After the usual deparaffinization process, the skin sections were incubated overnight at 4°C with the primary antibody (anti-ADAMTSL6 antibody (Proteintech Group Inc., 20619-1-AP) diluted 200-fold in blocking solution (PBC containing 12% bovine serum albumin). After washing with PBS, the sections were incubated for 1 hour in the dark at room temperature with the secondary antibody, fluorescently labeled donkey anti-rabbit IgG antibody (Lifetechnologies, R37119) diluted 200-fold. The secondary antibody was washed off with PBS. Finally, the mounting medium Vectashield (Vector Laboratories) containing DAPI was added, and the slide sample was mounted with a cover glass and observed under a fluorescent microscope (Figure 1).

[0033] Example 2: Localization of ADAMTSL6 Skin samples were obtained from the buttocks of subjects by punch biopsy, fixed using the AMeX fixation method, embedded in paraffin, and skin sections with a thickness of 4 μm were obtained. After deparaffinization, the skin sections were incubated overnight at 4°C with primary antibodies (anti-ADAMTSL6 antibody (Proteintech, 20619-1-AP) and anti-elastin antibody (Abcam, ab77804), (anti-ADAMTSL6 antibody (Proteintech, 20619-1-AP) and anti-fibrillin 1 antibody (Thermo Fisher Scientific)) diluted 200-fold in blocking solution (PBC containing 12% bovine serum albumin). After washing with PBS, the sections were incubated for 1 hour in the dark at room temperature with secondary antibodies, fluorescently labeled donkey anti-rabbit / mouse IgG (Lifetechnologies, R37119) and fluorescently labeled donkey anti-mouse IgG (Lifetechnologies, R37114), diluted 200-fold. The secondary antibodies were washed off with PBS. Finally, the sections were mounted in Vectashield mounting medium (Vector) containing DAPI. After adding 100% ethanol (Fujitsu Laboratories) and sealing the slide sample with a cover glass, the sample was observed under a fluorescence microscope (Figure 2).

[0034] Example 3: Knockdown of ADAMTSL6 ADAMTSL-6 gene expression was suppressed in cultures of dermal fibroblasts (Biopredic International) using siRNA (Thermo Fisher Scientific, Silencer select® siRNA). 1 × 10 dermal fibroblasts were cultured in a 1-well culture plate. 5Cells were seeded at 1000x1000 cells / well and cultured overnight at 37°C and 5% CO2. siRNA was introduced by adding a mixture of siRNA and transfection reagent (Lifetechnologies, 13778) to the cells at 1mL / well, and the cells were cultured for 3 days. Quantitative PCR was performed on the siRNA-introduced cells to confirm that the expression of the target gene was suppressed (data not shown). Dermal fibroblast cultures were stained with anti-ADAMTSL-6 antibody (Proteintech) and anti-fibrillin-1 antibody (Thermo Fisher Scientific) as primary antibodies, and fluorescently labeled donkey anti-rabbit / mouse IgG (Lifetechnologies, R37119) and fluorescently labeled donkey anti-mouse IgG antibody (Lifetechnologies, R37114) as secondary antibodies (Figure 3). When ADAMTSL-6 gene expression was suppressed, fibrillin-1 fiber formation was suppressed.

[0035] Example 4: Screening of candidate drugs that increase ADAMTSL-6 expression 7x10 fibroblasts 4The cells were seeded at 1mL per well in a 12-well plate and cultured at 37°C under 5% CO2 for 48 hours. Candidate drugs were added: 0.07% Camellia seed extract (butylene glycol extraction), 0.3% soybean extract (butylene glycol extraction), 0.1% longan seed extract (butylene glycol extraction), 0.1% Schistoseira tamarisifolia extract (glycerin extraction), and 0.1% peach seed extract (ethanol extraction). After 24 hours of culture at 37°C under 5% CO2, the cells were harvested. DMSO was used to dilute the candidate drugs. For the control group, the cells were harvested after culturing under the same conditions except that no extract was added. After washing with PBS, RNA was extracted from the harvested cells using RNeasy Mini Kit (Qiagen) and reverse transcription was performed using Super Script VILO (Thermo Fisher Scientific). The nucleic acid after reverse transcription was subjected to quantitative PCR using the following primer set, Platinum® SYBR® green qRCR SuperMix-UDG with ROX (Thermo Fisher Scientific), and StepOne® plus (Applied Biosystems). The results are shown in Figure 4. When camellia extract, soybean extract, longan seed extract, Schistoseira tamarisifolia extract, and peach seed extract were added, ADAMTSL-6 gene expression was enhanced, and these components were screened as ADAMTSL-6 expression promoters. The gene expression of THSD4 when camellia extract and soybean extract were added was measured using glyceraldehyde 3-phosphate dehydrogenase (G3PDH) as an internal standard gene. The gene expression of THSD4 when longan seed extract, Cystoseira tamaricifolia extract, and peach seed extract were added was measured using β-actin (ACTB) as an internal standard gene. The ADAMTSL-6 expression promoter can be used as an eye firmness improver, fibrillin 1 fiber formation promoter, oxytalan fiber formation promoter, elaunin fiber formation promoter, fine wrinkle formation inhibitor, fine wrinkle improvement agent, fine wrinkle prevention agent, and skin texture improver. [Table 1]

Claims

1. A method for evaluating skin condition using the gene expression level or protein level of ADAMTSL6 as an indicator.

2. The method of claim 1 , wherein the evaluation includes predicting future fine line formation.

3. A method for screening a skin quality improving agent using the gene expression level or protein level of ADAMTSL6 as an index.

4. Culturing fibroblasts in a medium supplemented with the candidate component; Measuring the gene expression level or protein level of ADAMTSL6 in a culture cultured in a medium containing the candidate component; and comparing the measured gene expression level with a control, and determining that the added candidate component is an eye area skin property improving agent when the gene expression level or protein level of ADAMTSL6 is enhanced; The method of claim 3 , comprising:

5. The method according to claim 4 , wherein the control is a threshold value determined from the gene expression level or protein level of ADAMTSL6 in a fibroblast culture cultured in a medium to which the candidate component is not added.

6. The method according to claim 4, wherein the eye area skin property improving agent can be at least one agent selected from the group consisting of ADAMTSL6 expression promoters, fibrillin 1 fiber formation promoters, oxytalan fiber formation promoters, fine wrinkle improving agents, fine wrinkle prevention agents, and texture improving agents.

7. An expression promoter for the ADAMTSL6 gene, comprising at least one extract selected from the group consisting of camellia seed extract, soybean extract, longan seed extract, Cystoseira tamaricifolia extract, and peach seed extract.

8. A microfibril formation promoter comprising the expression promoter of the ADAMTSL6 gene according to claim 7.

9. An agent for promoting the formation of oxytalan and elaunin fibers, comprising the agent for promoting the expression of the ADAMTSL6 gene according to claim 7.

10. An agent for improving skin properties around the eyes, comprising the ADAMTSL6 gene expression promoter according to claim 7.

11. A microfibril formation promoter comprising at least one extract selected from the group consisting of camellia seed extract, soybean extract, longan seed extract, Cystoseira tamaricifolia extract, and peach seed extract.

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