Agent for inhibiting or improving skin aging caused by ultraviolet exposure and method for inhibiting or improving skin aging

Abstract

To provide novel inhibiting or improving agent for skin aging caused by UV exposure and a method for inhibiting or improving skin aging, which can effectively inhibit / improve the accumulation of calcium (calcification) in the dermis tissue caused by exposing the skin to sunlight, particularly ultraviolet rays.SOLUTION: The inhibiting or improving agent for skin aging caused by UV exposure is an inhibiting or improving agent that inhibits or improves calcification in the dermis caused by ultraviolet exposure, the agent comprising as active ingredients at least one component selected from the group consisting of vitamins K, bisphosphonate, okadaic acid, acetazolamide, furosemide, phenylalanine, leucine, homoarginine, levamisole, mead acid, and oligogalacturonic acid, as well as deep ocean water.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel agent for inhibiting or ameliorating skin aging caused by exposure to ultraviolet light and a method for inhibiting or ameliorating skin aging, and in particular to a novel agent for inhibiting or ameliorating skin aging and a method for inhibiting or ameliorating skin aging that reduce calcification that accumulates in skin fibroblasts (NHDF) when the skin is exposed to ultraviolet light. [Background technology]

[0002] In addition to calcification occurring in the human skeleton, calcification can also occur in other biological tissues. This is called ectopic calcification, and for example, calcification in the tunica media of blood vessels is known. It is also known that alkaline phosphatase (ALP), phosphorus transporter (Pit-1), inflammatory enzyme phospholipase A2 (PLA2), osteocalcin (OCN), osteopontin (OPN), and other factors are involved in vascular calcification.

[0003] On the other hand, it is also known that ultraviolet rays have a significant effect on skin aging, and that long-wavelength ultraviolet rays in particular cause calcification in NHDF (Non-Patent Document 1). Such skin calcification changes the physical properties of the skin and leads to aging.

[0004] JP 2015-017081 A (Patent Document 1) discloses an anti-aging agent containing coenzyme Q10 and pomegranate processed products, which is said to have the effect of promoting the proliferation of NHDF and be useful in preventing or improving skin aging phenomena as an anti-aging agent that prevents skin aging.

[0005] Furthermore, JP 2016-523982 A (Patent Document 2) discloses an oil-in-water emulsion comprising: a) an aqueous phase containing, as a skin anti-aging compound: i) more than about 0.5%, preferably more than about 1.2%, and even more preferably more than about 1.5% of a neutralized acid salt form, the acid being preferably selected from the group consisting of salicylic acid, glycolic acid, and combinations thereof, and even more preferably a neutralized salt form of salicylic acid; and ii) about 2% by weight to about 0.001% by weight, preferably about 1.5% by weight to about 0.1% by weight, and even more preferably about 1.0% by weight to about 0.5% by weight of a gum; and b) an oil phase; wherein the chemical state and physical uniformity of the oil-in-water emulsion are substantially unchanged by exposure to a temperature of about 50°C for 14 days.

[0006] Furthermore, Japanese Patent Application Laid-Open No. 9-136824 (Patent Document 3) discloses an anti-aging skin agent that prevents skin aging, primarily an external skin agent that inhibits collagen cross-linking in the dermis, which contains an extract extracted from the genus Lavender of the Lamiaceae family as an active ingredient and further contains an ultraviolet protection agent.

[0007] However, conventional topical skin preparations do not inhibit or reduce calcification caused by ultraviolet rays that accumulates in NHDF in the dermis of the skin, and do not inhibit or improve skin aging by inhibiting or reducing calcium accumulation in NHDF in the dermis, making it difficult to sufficiently inhibit or improve skin aging.

[0008] Therefore, there is a need for agents that can inhibit or improve skin aging by inhibiting or improving calcium accumulation (calcification) in the dermis when the skin is exposed to ultraviolet rays, thereby preventing or improving skin aging. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Publication No. 2015-017081 [Patent Document 2] Special Publication No. 2016-523982 [Patent Document 3] Japanese Patent Application Publication No. 9-136824 [Non-patent literature]

[0010] [Non-Patent Document 1] "Inhibitory effect of deep seawater on calcification of normal human fibroblasts induced by UVA irradiation," Deep Ocean Water Research, 18(1), 1-7, 2017 (Deep Ocean Water Utilization Society, October 30, 2017) Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to provide a novel agent for inhibiting or ameliorating skin aging caused by exposure to ultraviolet light, and a novel method for inhibiting or ameliorating skin aging, which can effectively inhibit or ameliorate calcium accumulation (calcification) in dermal tissue and NHDF present therein due to exposure of skin to sunlight, particularly ultraviolet light. [Means for solving the problem]

[0012] The present inventors have discovered that calcium (calcification) accumulated in the dermal tissue and the NHDF present therein due to exposure of the skin to ultraviolet rays can be inhibited or improved by applying a combination of deep sea water and specific ingredients.

[0013] PurpleThe agent for inhibiting or improving skin aging caused by exposure to ultraviolet rays is an agent for inhibiting or improving calcification in the dermis, and is characterized by containing, as active ingredients, at least one component selected from the group consisting of vitamin K, bisphosphonate, okadaic acid, acetazolamide, furosemide, phenylalanine, leucine, homoarginine, levamisole, mead acid, and oligogalacturonic acid, and deep sea water. In particular, the present invention The agent for inhibiting or improving skin aging caused by exposure to ultraviolet rays is the above An agent for inhibiting or improving skin aging caused by exposure to ultraviolet rays, the active ingredient of which is at least one component selected from the group consisting of vitamin K, bisphosphonate, and homoarginine, and deep seawater. Yes ED mineral water The present invention is an agent for inhibiting or improving skin aging caused by exposure to ultraviolet rays, characterized by:

[0014] In addition, the present invention The method for inhibiting or improving skin aging caused by exposure to ultraviolet rays comprises inhibiting or improving calcification in the dermis caused by exposure to ultraviolet rays, the above A method for inhibiting or ameliorating skin aging caused by exposure to ultraviolet rays, characterized by bringing an agent for inhibiting or ameliorating skin aging into contact with calcified dermal tissue to reduce calcification in the dermal tissue. [Effects of the Invention]

[0015] The agent for inhibiting or improving skin aging caused by UV exposure of the present invention can effectively inhibit and improve calcium accumulation (calcification) in dermal tissue, particularly in the NHDF present therein, caused by exposure of the skin to sunlight, particularly UV rays, and therefore can prevent skin hardening and loss of skin elasticity, thereby inhibiting and improving skin aging.

[0016] Furthermore, the method of the present invention for inhibiting or ameliorating skin aging caused by exposure to ultraviolet light can effectively inhibit or reduce calcification in NHDF by bringing the agent of the present invention for inhibiting or ameliorating skin aging into contact with calcified dermal tissue.

[0017] Therefore, by using deep sea water and specific ingredients as active ingredients, it is possible to apply it to cosmetics such as lotions, emulsions, creams, essences, packs, foundations, and body lotions that are applied directly to the skin, and furthermore, by using ingredients that are not toxic to humans, it is expected that it will be applied to health foods, etc. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a graph showing the relationship between the amount of ultraviolet light irradiation and the calcification of cultured NHDFs. [Figure 2] FIG. 1 is a photograph showing the state of calcification and accumulation of cultured NHDFs after ultraviolet irradiation. [Figure 3] FIG. 1 shows the relationship between the amount [concentration] of deep ocean water and surface seawater added to cultured NHDF and the degree of calcification inhibition. [Figure 4] FIG. 1 shows the relationship between ultraviolet irradiation and the expression level of the PiT-1 gene. [Figure 5] FIG. 1 shows the relationship between ultraviolet irradiation and the expression level of the PLA2 gene. [Figure 6] FIG. 1 shows the relationship between ultraviolet irradiation and the expression level of the OCN gene. [Figure 7] FIG. 1 shows the relationship between ultraviolet irradiation and the expression level of the OPN gene. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in detail based on the following embodiments. The agent for inhibiting or ameliorating skin aging caused by exposure to ultraviolet rays of the present invention is an agent for inhibiting or ameliorating calcification in the dermis, and contains at least one component selected from the group consisting of vitamin K, bisphosphonate, okadaic acid, acetazolamide, furosemide, phenylalanine, leucine, homoarginine, levamisole, mead acid, and oligogalacturonic acid, and deep sea water as active ingredients. Preferably, the active ingredient is at least one component selected from the group consisting of vitamin K, bisphosphonate, and homoarginine, combined with deep sea water.

[0020] The agent of the present invention for inhibiting or improving skin aging caused by exposure to ultraviolet light contains deep sea water as an active ingredient. Deep ocean water is seawater at depths of 200m or more, located deeper than the euphotic zone (euphotic zone), where organic matter production by photosynthesis is almost nonexistent and decomposition is predominant. Its resource properties, currently known to date, include low temperature and cleanliness, and minerals and salts are also attracting attention. There are currently 15 intake facilities in nine prefectures, including Ito City (Izu Akazawa) in Shizuoka Prefecture, as intake points for collecting deep sea water (according to the Deep Sea Water Utilization Society website, as of February 2017). Any of the deep sea water collected from these intake points can be used in the present invention, and it is also possible to use deep sea water collected from points other than the above. Particularly preferred is Izu Akazawa deep seawater, which is pumped up from a depth of approximately 800 m off the coast of Izu Akazawa and has a microorganism abundance ratio of approximately one thousandth that of surface water. Furthermore, the Izu Akazawa deep seawater is located near the Izu Peninsula, where the Kuroshio Current flows northeastward in the ocean surface layer, and the Izu-Ogasawara Arc, which is made up of a group of high submarine volcanoes on the seafloor southeast of the Izu Peninsula, making it possible to extract clean, uncontaminated deep seawater, despite being the closest location to the Tokyo metropolitan area and other large urban areas, and can therefore be effectively applied in the present invention.

[0021] The "deep sea water" in the present invention includes seawater from a depth of 200 m or more, as well as water obtained by electrodialysis of deep sea water (ED salt water, ED mineral water), water treated with reverse osmosis membrane filtration (RO membrane), water treated with ion exchange membranes, and any treated water that is a liquid containing minerals equivalent to those contained in deep sea water, all of which are included in the concept of "deep sea water" in the present invention and can be suitably used. The deep-sea water preferably used in the present invention is particularly preferably deep-sea water that has been subjected to ion-selective treatment to reduce the ions contained in the collected deep-sea water to a specific ratio, since it is desirable to consider, for example, irritation to the skin due to salt content, etc. The method for reducing the ions contained in the raw deep-sea water to a specific ratio is not particularly limited, and ion-selective treatment can be performed using, for example, known reverse osmosis membrane filtration, electrodialysis, ion exchange membrane methods, etc. Furthermore, any other known method for ion-selective treatment of raw deep-sea water can also be used. For example, an ion-selective treatment method may involve using an electrodialysis apparatus (ion exchange membrane method) "Astome Corporation's Acilyzer 25 type, 100 pairs x 3 stages continuous type (seawater desalination specification)" to desalinate and concentrate ionic substances in raw deep-sea water by using an ion exchange membrane and direct current. Furthermore, as disclosed in Japanese Patent Laid-Open Publication No. 2000-159655, a method may be used in which deep-sea water is desalinated using a reverse osmosis membrane apparatus (Toyobo Co., Ltd., HR5155) and then desalted using an electrodialysis apparatus (Asahi Glass Co., Ltd., Selemion CMV, Selemion ASV), or a reverse osmosis membrane desalination method as described in Japanese Patent Laid-Open Publication No. 2010-274214. As for the ion-selective treatment, it is desirable to subject deep seawater to ion-selective treatment so that the ratio of contained potassium ions, sodium ions, calcium ions, magnesium ions, and chloride ions becomes, for example, K:Na:Ca:Mg:Cl=about 1:30 to 70:10 to 40:40 to 150:200 to 500. Specifically, it is particularly desirable to treat deep seawater so that, for example, per 100 g of deep seawater, the content is about 0 to 0.1 g of K, about 0.01 to 0.50 g of Na, about 0.01 to 0.04 g of Ca, about 0.05 to 0.5 g of Mg, and about 0.1 to 1.0 g of Cl.

[0022] Furthermore, the agent for inhibiting or improving skin aging caused by exposure to ultraviolet rays of the present invention contains, in addition to the deep sea water, at least one active ingredient selected from the group consisting of vitamin K, bisphosphonate, okadaic acid, acetazolamide, furosemide, phenylalanine, leucine, homoarginine, levamisole, mead acid, and oligogalacturonic acid.

[0023] Vitamin K is a fat-soluble vitamin and is an essential component for activating vitamin K-dependent proteins. It is involved in blood coagulation and tissue calcification in animals, and vitamin K deficiency is known to result in bleeding tendencies. Vitamin K, chemically speaking, is a derivative of 2-methyl-1,4-naphthoquinone, and naturally occurs as K1 and K2 (menaquinone). Menaquinone (MK) includes many compounds with different isoprenoid side chain lengths and modifications, and all of these vitamin K species can be used.

[0024] Examples of bisphosphonates include etidronate, clodronate, tiludronate, bamidronate, neridronate, olbadronate, alendronate, tiludronate, incadronate, risedronate, minodronate, and zoledronate. Examples of salts include sodium salts, potassium salts, calcium salts, and magnesium salts. Disodium etidronate is preferably used, and one or more of these may be used in combination.

[0025] Okadaic acid is a toxin produced by toxic dinoflagellates and can be effectively used in the present invention. Mead acid is one of the omega-9 fatty acids and can be effectively used in the present invention. Oligogalacturonic acid is an oligomer of uronic acid in which galactose is oxidized, and can be effectively used in the present invention.

[0026] In addition to the above vitamin Ks, bisphosphonates, okadaic acid, mead acid, and oligogalacturonic acid, examples of active ingredients that can be used in combination with deep sea water include so-called diuretic ingredients such as acetazolamide and furosemide, unique amino acids such as phenylalanine, leucine, and homoarginine, and levamisole and oligogalacturonic acid. Levamisole is a type of nematode anthelmintic and is known as an inhibitor of alkaline phosphatase activity.

[0027] The ratio of the combination of deep sea water and at least one component selected from the group consisting of vitamin K, bisphosphonate, okadaic acid, acetazolamide, furosemide, phenylalanine, leucine, homoarginine, levamisole, mead acid, and oligogalacturonic acid is deep sea water:component=1:1×10 by mass. -9 ~20, preferably 1:1 × 10 -8 ~1×10, more preferably 1:1×10 -7 It is desirable that the ratio be 1 to 5 in order to effectively exhibit the effects of the present invention.

[0028] As described above, by combining deep seawater with at least one active ingredient selected from the group consisting of vitamin K, bisphosphonate, okadaic acid, acetazolamide, furosemide, phenylalanine, leucine, homoarginine, levamisole, mead acid, and oligogalacturonic acid, calcium accumulation (calcification) in the dermal tissue, particularly in the NHDF present, due to skin exposure to sunlight, particularly ultraviolet rays, can be effectively inhibited and improved. This prevents skin hardening and loss of skin elasticity, and prevents the formation of wrinkles and sagging, as well as rough, dry skin.

[0029] Other ingredients may also be contained as long as they do not impair the effects of the present invention. Examples of such other ingredients include known ultraviolet absorbers and ultraviolet blockers, whitening agents, anti-inflammatory agents, animal and vegetable oils, waxes, polyhydric alcohols, fluorinated oils, higher alcohols, higher fatty acids, other surfactants such as nonionic surfactants, anionic surfactants, and amphoteric surfactants, antioxidants, fragrances, preservatives, and pH adjusters.

[0030] The method of the present invention for inhibiting or ameliorating skin aging caused by exposure to ultraviolet light involves contacting calcified dermal tissue, particularly present NHDF, with an agent for inhibiting or ameliorating skin aging of the present invention, which comprises as active ingredients at least one composition selected from the group consisting of vitamin Ks, bisphosphonates, okadaic acid, acetazolamide, furosemide, phenylalanine, leucine, homoarginine, levamisole, mead acid, and oligogalacturonic acid, and deep-sea water, to inhibit or ameliorate calcification in the dermis caused by exposure to ultraviolet light, thereby reducing calcification in the NHDF.

[0031] The method for bringing the agent for inhibiting or ameliorating skin aging of the present invention into contact with calcified dermal tissue and the NHDF present therein is not particularly limited, and can be carried out, for example, by applying the agent for inhibiting or ameliorating skin aging of the present invention directly to the skin or by diluting and applying it.

[0032] For example, the agent for inhibiting or improving skin aging of the present invention can be applied in the form of cosmetics such as lotion, emulsion, cream, essence, pack, foundation, and body lotion that are applied directly to the skin. Furthermore, by using ingredients that are not toxic to humans, it is expected that the compound can be used in health foods and other products. [Example]

[0033] Preferred examples will be described below, but the present invention is not limited to these. (reagents, equipment, etc.) ·NaCl (special grade, Wako Pure Chemical) KCl (special grade, Kanto Chemical) ·CaCl2 (special grade, Wako Pure Chemical) ·MgCl2·6H2O (special grade, domestic chemical) Methanol (special grade, Wako Pure Chemical Industries) PBS(-) (Ca, Mg-free isotonic phosphate buffer solution for cell culture, Nissui Pharmaceutical) Eagle MEM (for cell culture, Nissui Pharmaceutical) L(+) glutamine (special grade, Wako Pure Chemical Industries) Sodium bicarbonate (special grade, Wako Pure Chemical Industries) ·3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (for biochemistry, Dojindo Chemical) Trypsin-EDTA (Trypsin-EDTA (0.25%), phenol red, Gibco) ·Fetal Bovine Serum (Biological Industries Ltd., hereinafter FBS) Deep sea water (DSW: Izu Akazawa, 34°50'19"N, 139°08'11"E, 800m depth, stored at 4°C after intake) Surface seawater (SSW: Izu Akazawa, depth 0m, stored at 4℃ after intake) Calcified nodule staining kit (Cosmo Bio Co., Ltd., Calcified nodule staining kit, Code No. AK21) Hexadecylpyridinium chloride hydrate (special grade, Wako Pure Chemical Industries, Ltd.) ·Triton X-100 (MP Biomedicals, Inc.) UVA lamp (FL15BLB, Toshiba) Microplate reader (MODEL 550, BIO-RAD) Vitamin K2 (Vit-K2, product name: K2 Oil M-1500, manufacturer: J-Oil Mills Co., Ltd.) Etidronic acid disodium (ETA.2Na, product name: etidronate disodium hydrate, manufacturer: Tokyo Chemical Industry Co., Ltd.) Homoarginine (product name: L-homoarginine hydrochloride, manufacturer: Tokyo Chemical Industry Co., Ltd.)

[0034] (1) Cultured NHDF and culture Of the ultraviolet A rays (long wavelength) and ultraviolet B rays (short wavelength) that reach the ground, the cultured NHDFs were irradiated with ultraviolet A rays (UVA), which penetrate to the dermis. The cultured NHDFs were prepared as follows. Human skin-derived cultured fibroblast NHDF (RGB RCB0222, RIKEN BioResource Center) was seeded onto one culture dish (φ90, Nippon Genetic) and cultured until confluent. After that, the cultured NHDF was detached using trypsin-EDTA and subcultured onto two new culture dishes. These were then seeded onto a 96-well microplate (for cultured cells, Iwaki) for the calcification test. All cultured NHDFs were cultured at 37°C in 5% CO 2 , and growth and pre-culture of the cultured NHDFs were carried out using Eagle's MEM medium containing 10% (v / v) FBS.

[0035] (2) Calcification of cultured NHDFs by UVA irradiation The cultured NHDFs were suspended in Eagle's MEM containing 10% (v / v) FBS and plated in a 96-well microplate (for cultured cells, Iwaki) at 2 × 10 4 The cells were seeded at 200 μL / well and pre-cultured for 2 days. After pre-culture, the medium was removed and washed twice with 50 μL / well of PBS(-), and then completely replaced with 50 μL / well of fresh PBS(-). The irradiation energy from a stand with a UVA lamp installed in parallel to this was 0.5 mW / cm 2 (UV METER Model UV-340, custom-made) and adjust the distance from the light source to the cultured NHDFs so that the concentration was 0.4 to 1.2 J / cm 2 In the range of 0.2J / cm 2 The UVA dose was varied step by step.

[0036] After irradiation, 100 μL / well of Eagle's MEM containing 10% (v / v) FBS, 20 μL / well of 20 mM CaCl2 aqueous solution, 10 μL / well of 20 mM MgCl2·6H2O aqueous solution, and 25 μL / well of PBS(-) were added, and purified water was added to bring the total volume to 200 μL / well, and the cells were cultured for 24 hours.

[0037] After the culture, the degree of calcification of the cultured NHDF was measured using the calcification staining kit. Specifically, the medium was removed from the cultured NHDFs that had been cultured for 24 hours after UVA irradiation, and the cultured NHDFs were washed twice with 50 μL of fresh PBS(-) added per well. After that, 50 μL of cold methanol was added per well, and the cultured NHDFs were left to stand on ice for 30 minutes to fix them.

[0038] After fixation, the methanol was removed, and the cultured NHDFs were washed twice with 50 μL / well of purified water and then air-dried. After air drying, 50 μL of the prepared staining solution was added to each well using a calcification staining kit, and the plate was left to stand at room temperature for 30 minutes for staining. After staining, the staining solution was removed, and the washing solution provided with the kit was prepared and added at 50 μL per well to wash away nonspecific staining dye.

[0039] After these procedures, the 96-well plate in which the calcium deposits were specifically stained was thoroughly dried at room temperature, and 100 μL of 10% (v / v) cetylpyridinium chloride solution was added to each well. The dye was extracted at 37°C for 1 hour, and the absorbance at 570 nm (hereinafter referred to as OD) was measured. 570 ) was measured and used as the degree of calcification. The degree of calcification of cultured NHDFs not irradiated with UVA, measured by the above procedure, was set at 1.0, and the degree of calcification induced by UVA irradiation was expressed as a relative value (n=6). The results are shown in Figure 1.

[0040] (3) Calcification by UVA irradiation In the absence of UVA irradiation, the cultured NHDFs hardly calcified. However, as shown in Figure 1, when UVA irradiation was not performed, the calcification rate was increased to 0.4-0.8 J / cm 2 A dose-dependent increase in the degree of calcification was observed within the range of irradiation doses.

[0041] In addition, unirradiated and 0.8 J / cm 2 Micrographs of calcium accumulation in cultured NHDF after irradiation are shown in Figure 2 as Figure 2(1) and Figure 2(2), respectively. The microscopic photograph in Figure 2 was taken using the following equipment. Upright microscope (BX-51, Olympus) Epifluorescence illuminator (BX-RFA, Olympus) Microscope digital camera (DP71, Olympus)

[0042] (4) Application of deep sea water (DSW) to calcified cultured NHDF In the above (calcification of cultured NHDF by UVA irradiation), 0.8 J / cm 2 DSW was added to cultured NHDFs that had been irradiated to induce calcification to a final concentration of 0-2.5% (v / v) (in a CO2 incubator (5% CO2, 37°C)), and the effect on the calcification level of the cultured NHDFs that developed after 24 hours of culture was examined. As a control for comparison with DSW, cultured NHDFs were used that had been cultured and treated in the same manner except that SSW was added instead of DSW (n=5). The results are shown in Figure 3.

[0043] As shown in Figure 3, when DSW was added to cultured NHDFs in which calcification had been induced after UVA irradiation, both SSW and DSW inhibited the calcification of the cultured NHDFs in a concentration-dependent manner, but DSW showed a greater inhibitory effect on calcification than SSW. However, it can be seen that DSW is not sufficient to inhibit or improve skin calcification.

[0044] (5) Relationship between UVA irradiation and ALP activity, OCN gene, OPN gene, PiT-1 gene, and PLA2 gene The following tests were conducted to evaluate the relationship between trends in ALP activity, OCN gene, OPN gene, PiT-1 gene, PLA2 gene, etc. and calcification accumulated in the skin due to UV irradiation. The relationship between the ALP activity index, an enzyme involved in calcium phosphate formation in the induction of mineralization by ultraviolet light, and the incubation time after UVA irradiation was investigated. As a result, an increase in ALP activity, which is associated with tissue mineralization, was observed in cultured NHDFs 2 hours after UVA irradiation (a relative value of approximately 3.2 compared to ALP activity in the unirradiated state), and an even higher activity index (also approximately 5.5) was maintained from 4 to 6 hours after irradiation. 4 to 7 show changes in the expression level of each osteogenesis-related gene during calcification induction by ultraviolet light.

[0045] The specific test method is outlined below. The cultured NHDFs prepared in the above section (Cultivation of NHDFs and Culture) were placed in a cell culture dish (φ30, Nippon Genetic) at 6.0 × 10 5 The cells were seeded at 100 cells / dish and pre-cultured for 2 days. After pre-culture, the medium was removed and washed once with 1 mL / dish of PBS(-), and then completely replaced with 1 mL / dish of fresh PBS(-). According to the above (calcification of cultured NHDF by UVA irradiation), the irradiation dose was 0.5 J / cm 2 UVA was irradiated so that After irradiation, 1 mL / dish of Eagle's MEM containing 10% (v / v) FBS, 200 μL / dish of 20 mM CaCl2 aqueous solution, 100 μL / dish of 20 mM MgCl2·6H2O aqueous solution, and 250 μL / dish of PBS(-) were added, and purified water was added to bring the total volume to 2 mL / dish for evaluation culture.The cultured NHDFs after 0.5 hours of culture were subjected to PLA2 gene expression analysis, and the cultured NHDFs after 24 hours of culture were subjected to OPN, OCN, and PiT-1 expression analysis.

[0046] The medium components were removed from the dish containing the cultured NHDF, and 1.0 mL of lysis buffer was added using a micropipette, followed by pipetting several times to dissolve the cultured NHDF. The entire cultured NHDF lysate was transferred to a 1.5 mL tube, 0.2 mL of chloroform was added, and the mixture was vortexed for 10 seconds. After centrifugation at 12,000 × g for 10 minutes at 4°C, 500 μL of the upper layer (aqueous phase) was collected and transferred to a new 1.5 mL tube. An equal volume (500 μL) of 2-propanol was added, mixed by inversion, and then allowed to stand for 10 minutes at 4°C. After centrifugation at 12,000 × g for 10 minutes at 4°C, the supernatant was discarded, and the gel-like RNA precipitate was dissolved in 200 μL of nuclease-free water. 20 μL of 3 M sodium acetate (pH 5.2) and 200 μL of 2-propanol were added and mixed, and then the mixture was allowed to stand at 4° C. for 10 minutes. After centrifugation at 12,000×g for 10 minutes at 4° C., 250 μL of 75% ethanol was added to the RNA precipitate to wash away any guanidine salts that had been carried over. After the supernatant was completely removed, the mixture was air-dried, and 50 μL of nuclease-free water was added to completely dissolve the RNA precipitate, to prepare an RNA solution.

[0047] To measure the concentration and purity of the RNA solution, the RNA solution was diluted with TE buffer and measured at a wavelength of 260 nm (A 260 ) and 280 nm (A 280 The absorbance at 1000 kJ / min was measured. The RNA concentration (μg / mL) was calculated by A260 x 40, and the purity was calculated by A 260 / A 280 A 260 / A 280 A pH of ≥ 1.8 confirmed that the RNA solution was highly pure with little protein contamination.

[0048] A reverse transcription reaction was carried out using the ReverTra Ace qPCR RT Kit (Toyobo) according to the kit's instructions to prepare a cDNA solution. Specifically, the required amounts of 2.0 μL of 5x RT Buffer, 0.5 μL of RT Enzyme Mix, and 0.5 μL of Primer Mix were prepared per reaction, and 3.0 μL was dispensed into 0.2 mL tubes. 1 μg of RNA solution was then dispensed into each 0.2 mL tube, with nuclease-free water added to make a total of 7.0 μL per reaction. The 0.2 mL tube was placed in a thermal cycler (MyCycler, BIO-RAD) and heated at 37°C for 15 minutes and at 98°C for 5 minutes, and then cooled to 4°C to prepare a cDNA solution.

[0049] Quantitative polymerase chain reaction (qPCR) was performed using THUNDERBIRD qPCR Mix (Toyobo) according to the kit's instructions, and cDNA quantification of each gene was performed. Specifically, the required amounts of THUNDERBIRD SYBR qPCR Mix (5.0 μL), forward primer (10 μM) (0.3 μL), reverse primer (10 μM) (0.3 μL), 50×ROX reference dye (0.2 μL), and sterile water (2.2 μL) were prepared for each reaction, and then 8.0 μL of each was dispensed into 0.1 mL tubes, to which 2.0 μL of 10-fold diluted cDNA solution was added. PCR was performed using a real-time PCR device (StepOnePlus, Applied Biosystems) under the following conditions: 1 minute at 95°C, followed by 40 cycles of 95°C for 30 seconds and 60°C for 30 seconds. Furthermore, melting curve analysis was performed after PCR to confirm the absence of nonspecific amplification.

[0050] The base sequences of the analyzed genes and the primers used in the chain reaction are as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 5'-CATTGATGGCAACAATATCCACTT-3' 3'-GGTTATGCTGGTTTAGGCAAC-5' OCN; 5'-GGCAGCGAGGTAGTGAAGAG-3' 3'-GGTCAAGACGAGGAGAGGTC-5' OPN; 5'-ACAGCCGTGGGAAGGACAGTTA-3' 3'-GTAAGGCTACACTAACTATCAGTCC-5' PiT-1; 5'-GCCAAAGTGAGCGAAACCATCC-3' 3'-CGATACAAACCAAGACGACACACC-5' PLA2; 5'-ATGAAGACCCTCCTACTGTTGG-3' 3'-TCAACTGCTGTCCTTTCCTTCG-5' Quantitative analysis was performed using Ct (Threshold Cycle) values, and relative evaluation was performed using the Ct value of GAPDH as an internal standard.

[0051] FIG. 4 shows the results for the expression levels of the PiT-1 gene, FIG. 5 shows the results for the PLA2 gene, FIG. 6 shows the results for the OCN gene, and FIG. 7 shows the results for the OPN gene. In Figures 4, 6, and 7, the expression level of each gene 24 hours after irradiation is shown relative to the expression level of unirradiated genes, which is set to 1. In Figure 5, the expression level of the PLA2 gene 0.5 hours after irradiation is shown relative to the expression level of unirradiated genes, which is set to 1.

[0052] The results in Figures 4 to 7 show that the expression levels of the phosphate transporter gene PiT-1 gene, which is a calcification-inducing gene in the skin after UV exposure and is active in the early stages of calcification, and the PLA2 gene both increased by more than three-fold in cultured NHDFs due to UVA irradiation. Furthermore, the expression level of the OCN gene, which increases in the final stage of bone formation, increased by approximately 1.6 times in the cultured NHDFs compared to unirradiated cases, indicating that the OPN gene was also increased.

[0053] This is presumably because calcium accumulation in cultured NHDF due to UVA irradiation promoted the intracellular production of monophosphate due to increased ALP activity, and calcium phosphate accumulated in the cultured NHDF due to a chain reaction of calcium mobilization into the cytoplasm caused by the initiation of an inflammatory response by PLA2.Therefore, it is possible to evaluate calcification in cultured NHDF by measuring the expression levels of PiT-1, PLA2, OCN, OPN, etc. in these cultured NHDF.

[0054] (6) Application of the agent for inhibiting or improving skin aging of the present invention The agent for inhibiting or ameliorating skin aging of the present invention is administered by the above-mentioned ultraviolet irradiation (1.2 J / cm 2The cultured NHDF calcified by the above-mentioned method was contacted with a liquid mixture prepared by blending ED mineral water with at least one component selected from the group consisting of vitamin Ks, bisphosphonates, okadaic acid, acetazolamide, furosemide, phenylalanine, leucine, homoarginine, levamisole, mead acid, and oligogalacturonic acid in various ratios. Specifically, as a representative example, a liquid containing at least one component selected from the group consisting of vitamin K (vitamin K2), bisphosphonate (disodium etidronate), and homoarginine, and ED mineral water was used. As a result, it was found that the agent for inhibiting or improving skin aging and the method for inhibiting or improving skin aging of the present invention can significantly reduce the degree of calcification in cultured NHDF that has been calcified by ultraviolet irradiation.

[0055] The deep sea water used was deep sea water (from Izu Akazawa) pumped up from a depth of approximately 800 m off the coast of Izu Akazawa, and subjected to ion-selective treatment using the following method to produce electrodialyzed deep sea water (deep sea water (ED mineral water)) (electrical conductivity: 12.5 mS / cm). Specifically, using an electrodialysis device "Astro Corporation, Acilyzer 25 type, 100 pairs x 3 stages continuous type (seawater desalination specifications)", deep seawater from Akazawa, Izu, was subjected to electrodialysis (ion exchange membrane method) using an ion exchange membrane to apply a direct current, thereby desalination and concentration of ionic substances in the raw deep seawater, and ion-treated deep seawater (ED mineral water) was obtained through the desalination process. Such deep sea water (ED mineral water) has the component composition shown in Table 2 below. The ion contents of K, Na, Ca, and Mg in the deep seawater were measured using a multi-type ICP emission spectrometer (Shimadzu Corporation), and the Cl content was measured using a high-performance liquid chromatograph (Shimadzu Corporation).

[0056] [Table 1]

[0057] Specifically, the experiment was carried out as follows. In the procedure of "(2) Calcification of cultured NHDF by UVA irradiation" above, UVA was irradiated at 1.2 J / cm 2 Cultured NHDFs calcified by irradiation were used. 10% (v / v) FBS-containing Eagle's MEM was added at 100 μL / well, 20 mM CaCl2 aqueous solution at 20 μL / well, 20 mM MgCl2·6H2O aqueous solution at 10 μL / well, PBS(-) at 25-40 μL / well, and the following evaluation sample containing vitamin K2, etidronate disodium, or homoarginine. Purified water was added to bring the total volume to 200 μL / well, and the mixture was cultured at 37°C in 5% CO2 for 24 hours to prepare the evaluation medium. 1) Vitamin K2 content evaluation sample: Vitamin K2 0.01 μM + ED mineral water was added to the evaluation medium so that the final concentration in the medium was 0.5% by mass (ED mineral water: vitamin K2 = 1:1.3 × 10 -6 (mass ratio)) Vitamin K2 0.01 μM + ED mineral water was added to the evaluation medium so that the final concentration in the medium was 2.5% by mass (ED mineral water: vitamin K2 = 1:2.6 × 10 -7 (mass ratio)) Vitamin K2 10.00 μM + ED mineral water was added to the evaluation medium so that the final concentration in the medium was 0.5% by mass (ED mineral water: vitamin K2 = 1:1.3 x 10 -3 (mass ratio)) Vitamin K2 10.00 μM + ED mineral water was added to the evaluation medium so that the final concentration in the medium was 2.5% by mass (ED mineral water: vitamin K2 = 1:2.6 × 10 -4 (mass ratio)) 2) Etidronic acid disodium-containing evaluation sample: Etidronate disodium 0.01 μM + ED mineral water was added to the evaluation medium so that the final concentration in the medium was 0.5% by mass (ED mineral water: etidronate disodium = 1:5.0 × 10 -5 (mass ratio)) Etidronate disodium 0.01 μM + ED mineral water was added to the evaluation medium so that the final concentration in the medium was 2.5% by mass (ED mineral water: etidronate disodium = 1:1.0 × 10 -5 (mass ratio)) Etidronate disodium 0.1 μM + ED mineral water was added to the evaluation medium so that the final concentration in the medium was 2.5% by mass (ED mineral water: etidronate disodium = 1:1.0 × 10 -4 (mass ratio)) 3) Homoarginine-containing evaluation sample: Homoarginine 0.1 mM + ED mineral water was added to the evaluation medium so that the final concentration in the medium was 0.5% by mass (ED mineral water: homoarginine = 1:4.4 x 10 -1 (mass ratio)) Homoarginine 0.1 mM + ED mineral water was added to the evaluation medium to give a final concentration of 2.5% by mass (ED mineral water: homoarginine = 1:8.8 x 10 -2 (mass ratio)) Homoarginine 1.00mM + ED mineral water was added to the evaluation medium so that the final concentration was 0.5% by mass (ED mineral water: homoarginine = 1:4.4 (mass ratio)) Homoarginine 1.00 mM + ED mineral water was added to the evaluation medium so that the final concentration was 2.5% by mass (ED mineral water: homoarginine = 1:8.8 x 10 -1 (mass ratio))

[0058] For comparison, as shown in Table 1 below, evaluation media were used that did not contain the vitamin K2-containing evaluation sample, the etidronate disodium-containing evaluation sample, or the homoarginine-containing evaluation sample, as well as those that contained ED mineral water, and those that contained vitamin K2, disodium etidronate, or homoarginine.

[0059] After the culture, the degree of calcification of the cultured NHDF was measured by Alizarin Red-S staining using the calcification staining kit described above. Specifically, the medium was removed from the cultured NHDFs that had been cultured for 24 hours after UVA irradiation, and the cultured NHDFs were washed twice with 50 μL of fresh PBS(-) added per well. After that, 50 μL of cold methanol was added per well, and the cultured NHDFs were left to stand on ice for 30 minutes to fix them.

[0060] After fixation, the methanol was removed, and the cultured NHDFs were washed twice with 50 μL / well of purified water and then air-dried. After air drying, 50 μL of the prepared Alizarin Red-S staining solution included in the calcified nodule staining kit (Cosmo Bio Co., Ltd., Calcified nodule staining kit, Code No. AK21) was added to each well in accordance with the operating instructions of the staining kit, and the cells were left to stand at room temperature for 30 minutes for staining. After staining, 50 μl / well of AK21 buffer solution provided with the staining kit was added to wash away nonspecific staining dye, and the stained NHDF cells were washed twice.

[0061] The 96-well plate in which the calcium deposits were specifically stained by these procedures was thoroughly air-dried at room temperature, and then 100 μl of 10% cetylpyridinium chloride solution was added to each well. The dye was extracted at 37°C for 10 minutes, and the absorbance at 570 nm (hereinafter referred to as OD 570 ) was measured and used as the calcification degree (n=6). The results are shown in Table 2 below.

[0062] [Table 2]

[0063] (Calcification due to UVA irradiation) As shown in Figure 1, the cultured NHDFs hardly calcified under UVA non-irradiation conditions, but under UVA irradiance of 1.2 J / cm 2 An increase in the degree of calcification was observed with irradiation at this workload. 1) Suppression of calcification by combined use of vitamin K2 and ED mineral water UVA 1.2J / cm 2The degree of calcification in the evaluation medium without vitamin K2 or ED mineral water was set as 1 (control), and the inhibitory effect of each evaluation sample was expressed as a relative value. As can be seen from Table 1 above, 0.5% ED mineral water did not inhibit calcification. Furthermore, when 0.01 μM or 10.00 μM vitamin K2 was added, the calcification of cultured NHDF cells was slightly inhibited, but not sufficiently. On the other hand, it was found that the combined use of 0.01 μM vitamin K2 and 0.5% or 2.5% ED mineral water, or the combined use of 0.00 μM vitamin K2 and 0.5% or 2.5% ED mineral water, significantly suppressed the calcification of NHDF cells.

[0064] 2) Suppression of calcification by combined use of etidronate disodium and ED mineral water From Table 1 above, it was found that when 0.01 μM or 0.10 μM of etidronate disodium was added, calcification of cultured NHDF cells was slightly inhibited, but not sufficiently. On the other hand, it was found that the combined use of 0.01 μM etidronate disodium and 0.5% or 2.5% ED mineral water, or the combined use of 0.1 μM etidronate disodium and 2.5% ED mineral water, significantly inhibited the calcification of NHDF cells.

[0065] 3) Calcification suppression by the combined use of homoarginine and ED mineral water From Table 1 above, it was found that when 0.1 mM or 1.00 mM homoarginine was added, it was difficult to inhibit the calcification of cultured NHDF cells. On the other hand, it was found that the combined use of 0.1 mM homoarginine and 0.5% or 2.5% ED mineral water, or the combined use of 1.00 mM homoarginine and 0.5% or 2.5% ED mineral water, significantly inhibited the calcification of NHDF cells.

[0066] From the above, it can be seen that by combining deep seawater with, as representative examples, vitamin K, bisphosphonates, and homoarginine, the calcification in the dermal tissue, in which calcium accumulates (calcification) due to skin exposure to ultraviolet rays, and the calcification in the NHDF present therein, can be significantly improved, and skin aging can be inhibited and improved extremely effectively. Although vitamin K, bisphosphonates, and homoarginine have been given as representative examples, the use of okadaic acid, acetazolamide, furosemide, phenylalanine, leucine levamisole, mead acid, and oligogalacturonic acid in place of or in addition to these ingredients also significantly improves calcification in dermal tissue. [Industrial Applicability]

[0067] The present invention can inhibit and improve skin aging caused by dermal tissue, in which calcium accumulates (calcification) due to exposure of the skin to sunlight, particularly ultraviolet rays, and in particular by the NHDF present therein, and can be applied by incorporating it into cosmetics and health foods.

Claims

1. An agent for inhibiting or improving skin aging caused by ultraviolet exposure by inhibiting or improving calcification in the dermis caused by ultraviolet exposure, characterized in that the agent for inhibiting or improving skin aging caused by ultraviolet exposure contains, as active ingredients, at least one component selected from the group consisting of vitamin Ks, bisphosphonates, and homoarginine, and ED mineral water, which is deep sea water.

2. A method for inhibiting or ameliorating skin aging caused by ultraviolet exposure by inhibiting or ameliorating calcification in the dermis due to ultraviolet exposure, comprising contacting the agent for inhibiting or ameliorating skin aging described in claim 1 with calcified dermal tissue and skin fibroblasts present therein, thereby reducing calcification in the skin fibroblasts.

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