Mycosporine-containing composition

Abstract

The present invention addresses the problem of providing a composition that can be substituted for a synthetic ultraviolet absorber and that has an exceptional skin-problem-suppressing effect due to a component that absorbs ultraviolet rays of a wide range of wavelengths and is stable.　A mycosporine (MYC) is used as an active ingredient of a composition for suppressing skin problems and / or a composition for suppressing active oxygen. The MYC preferably includes one or more substances selected from mycosporine-glutaminol-glucoside (MGGnol), mycosporine-glutamicol-glucoside (MGGcol), or mycosporine-glutamine (MGn). "Skin problems" may refer to problems triggered by ultraviolet irradiation or problems that occur irrespective of ultraviolet irradiation.

Description

Mycosporine-containing composition

[0001] The present disclosure relates to compositions containing mycosporines (MYCs) and provides new uses for MYCs.

[0002] Skin exposed to ultraviolet rays can develop various undesirable conditions (skin troubles), such as inflammation and pigmentation. For this reason, sunscreen cosmetics containing ultraviolet absorbers and ultraviolet scattering agents are widely used. Currently, most of the ultraviolet absorbers used in sunscreen cosmetics are synthetic, and generally cover both UV-B and UV-A. There are concerns about synthetic ultraviolet absorbers, such as the generation of active oxygen through photolysis, allergies and itching, and environmental pollution (Non-Patent Documents 1 and 2).

[0003] Therefore, there is a demand for natural UV absorbers, and their development is underway. Mycosporine-like amino acids (MAAs), one of these, are naturally occurring substances known to efficiently absorb UV rays such as UV-A and UV-B, and are expected to be used as UV absorbers added to sunscreen cosmetics and other products. For example, MAAs (including Porphyra 334 and shinorine) extracted from red algae have already been commercialized (Non-Patent Document 3). However, the production of such MAAs is often unstable and susceptible to weather and other factors, making it difficult to obtain them in large quantities on a stable basis. Furthermore, a method for producing shinorine using genetic engineering has been proposed (Patent Document 1), but consumer resistance to genetic engineering can mean that commercialization has yet to be achieved. Furthermore, MAAs that are expected to have UV absorption capabilities, such as shinorine, have absorption wavelengths in the UV-A2 range (320-340 nm), which does not cover the UV-B or UV-A1 wavelength ranges (340-400 nm), making them insufficient as UV absorbers (Non-Patent Document 1). In particular, Non-Patent Document 2 points out that conventional MAAs are not effective as UV absorbers. Furthermore, Porphyran 334 is sensitive to heat (Non-Patent Document 4), and shinorine discolors during storage (Patent Document 2). These MAAs have stability issues, posing quality challenges when commercializing them.

[0004] MAAs have been reported to have various effects, including antioxidant properties, inhibition of DNA damage, inhibition of lipid peroxidation, and inhibition of advanced glycation end products. However, they are not known to have any inhibitory effect on tyrosinase, which is involved in pigmentation (Non-Patent Document 5). These effects have been evaluated in cell tests and tests using mice, but there have been almost no examples of evaluation in human tests (Non-Patent Document 2).

[0005] Meanwhile, various functionalities of mycosporines (MYCs), which are similar to MAAs (and are sometimes treated as part of the same group), have also been investigated. MYCs is a general term for biologically produced compounds that have a cyclohexenone or cyclohexenimine skeleton and UV absorption ability. "Bioproduced" may also include compounds that are produced by or obtainable from aquatic organisms such as algae and shellfish, or microorganisms such as yeast and mold. Specific examples include mycosporine glutaminol glucoside (MGGnol; Mycosporine-glutaminol-glucoside), mycosporine glutamicol glucoside (MGGcol; Mycosporine-glutamicol-glucoside), mycosporine glutamine (MGn; Mycosporine-glutamine), mycosporine glutamic acid (MGa; Mycosporine-glutamic acid), mycosporine glycine (MG; Mycosporine-glycine), and coremin A. It has been reported that MG and coremin have various effects on the skin, such as erythema suppression, fibroblast proliferation, and damaged skin healing, but the credibility of these effects is low, and the beneficial effects resulting from their UV absorption ability have not been substantiated (Non-Patent Documents 6-7, Patent Document 3). Furthermore, like MAAs, there has been no method for mass-producing MYCs, and they have stability issues (Non-Patent Document 8). Regarding MGGnol, it has been reported that the survival rate of epidermal cells to which it is added is improved by UV-B irradiation, that it promotes fibroblast proliferation, collagen production, and prostaglandin E in epidermal cells. ２ (PGE ２ It has been reported that it has effects such as inhibiting the production of hydroxybenzoates and quenching singlet oxygen (Patent Document 4, Non-Patent Document 9). However, because epidermal cells do not contain keratin, it cannot be said that the effects of its application to human skin have been properly evaluated.

[0006] Japanese Patent No. 5927593 Japanese Patent No. 6719267 Japanese Patent Publication No. 2007-16004 Japanese Patent No. 5913988

[0007] Nao Mori, Cosmetology Research Reports 27,64-67 (2019) KP Lawrence, et al., Current Medical Chemistry 25,5512-5527 (2018) S. Daniel, et al., Cosmetic and Toiletries Manufacture worldwide, 139-143 (2004) M. Yoshiki et al., Food Chemistry, 113, 1127-1132 (2009) K. Hakuto et al., Marine Drugs ,14,222,120-137 (2019) A. Torres, et al., Eur. J. Biochem 271,780-784 (2004) HJ Suh, et al., Photochem photobiol 78(2),109-113 (2003) S. Ito, et al., Tetrahedron Lett, 28,2429-2430(1977)M. Moline, et al., RADIATION RESEARCH 175, 44-50 (2011)

[0008] In view of the above circumstances, an object of the present disclosure is to provide a composition that has an excellent effect of suppressing skin troubles and that contains stable components that can replace synthetic ultraviolet absorbers and absorb ultraviolet rays over a wide range of wavelengths.

[0009] Previously, the inventors of the present disclosure invented a method for mass-producing MYCs by culturing black yeast and then efficiently extracting and manufacturing it (Patent Application Nos. 2023-2010, 2023-2008, and 2023-2009). These inventions established the prospect of industrial production of MYCs, making commercialization a realistic goal. While conducting extensive research to solve the above-mentioned problems, the inventors of the present disclosure focused on the possibility of industrial production of the aforementioned MYCs. They evaluated the functionality of MYCs using human skin slices and three-dimensional human skin models, and found that MYCs suppresses skin problems caused by various mechanisms and can be an active ingredient in compositions for suppressing skin problems. The effects of MYCs discovered by the inventors of the present disclosure are described below. The inventors of the present disclosure have discovered that MYCs, by absorbing UV-B, suppress erythema and inflammation, suppress pigmentation such as darkening and age spots, and suppress the generation of reactive oxygen species (ROS), thereby suppressing oxidative stress. Carbonyl proteins (CPs) are the footprint of oxidative stress. In other words, the occurrence of CP generation indicates a state of exposure to oxidative stress. Oxidative stress is involved in wrinkles, age spots, skin aging, and other conditions. Suppressing oxidative stress prevents these conditions. CP generation causes yellowing and dryness of the skin, and UV-A irradiation of CPs generates reactive oxygen species, so suppressing CPs is highly significant. Furthermore, the inventors of the present disclosure had previously believed that the skin trouble suppression effect of MYCs was due to the absorption of UV-B by MYCs, particularly MGGnol, MGGcol, and MGn, but surprisingly discovered that these MYCs also suppress oxidative stress and achieve the above-mentioned effect through a pathway involving the absorption of singlet oxygen, a type of reactive oxygen generated by UV-A. Furthermore, the inventors of the present disclosure also discovered that MYCs suppress skin inflammation and oxidative stress even in the absence of ultraviolet irradiation because they have anti-inflammatory and reactive oxygen scavenging effects.

[0010] That is, the present disclosure includes the following inventions. The term "MYCs" may include a single type or multiple types of mycosporines. [1] A composition for suppressing skin troubles, containing mycosporines (MYCs). [2] A composition for suppressing active oxygen, containing mycosporines (MYCs). [3] The composition according to [1] or [2], wherein the MYCs includes one or more types selected from mycosporine glutaminol glucoside (MGGnol; Mycosporine-glutaminol-glucoside), mycosporine glutamicol glucoside (MGGcol; Mycosporine-glutamicol-glucoside), and mycosporine glutamine (MGn; Mycosporine-glutamine). [4] The composition according to any one of [1] to [3], wherein the MYCs contain at least MGGnol and MGGcol. [5] The composition according to any one of [1] to [3], wherein the total amount of MGGnol, MGGcol, and MGn is 0.001 wt % or more of the total composition. [6] The composition according to any one of [1] to [3], wherein the total amount of MGGnol, MGGcol, and MGn is 0.1 μg / cm ２ The composition according to any one of [1] to [5], which is applied to the skin for days or more. [7] The composition according to [1], wherein the skin trouble is one or more selected from erythema, inflammation, darkening / blemishes, and phenomena caused by oxidative stress on the skin. [8] The composition according to [1], wherein the skin trouble is skin trouble caused by ultraviolet irradiation or skin trouble not caused by ultraviolet irradiation. [9] The composition according to any one of [1] to [8], which is a composition for external use on the skin.

[10] The composition according to any one of [1] to [8], wherein the total amount of MGGnol, MGGcol, and MGn is 0.1 μg / cm ２

[11] The composition according to [9], which is applied to the skin for at least 1 day.

[12] The composition according to [9], which is for suppressing erythema and / or inflammation in the skin, and which contains 0.1 μg / cm as the total amount of MGGnol, MGGcol, and MGn. ２

[12] The composition according to [9], which is applied to the skin for at least 1 day.

[13] The composition according to [9], which is for inhibiting darkening and / or blemishes on the skin, and which contains 0.5 μg / cm as the total amount of MGGnol, MGGcol, and MGn. ２

[13] The composition according to [9], which is applied to the skin for at least 1 day.

[14] The composition according to [9], which is for suppressing phenomena caused by oxidative stress in the skin, and which contains 0.1 μg / cm as the total amount of MGGnol, MGGcol, and MGn. ２

[14] The composition according to [9], which is a sunscreen cosmetic.

[0011] The present disclosure provides a composition that exhibits excellent inhibitory effects against various skin troubles caused by UV irradiation, as well as various skin troubles caused independently of UV irradiation. This disclosure is particularly significant in that it suppresses inflammation and ROS levels in the skin even in steady-state conditions without UV irradiation, thereby enabling not only UV resistance but also the suppression of various everyday skin troubles. The present disclosure also provides a composition that suppresses reactive oxygen species (ROS). Since reactive oxygen species cause oxidative stress, their suppression is significant. Furthermore, MYCs, such as MGGnol, MGGcol, and MGn, are typically water-soluble and do not decompose or disappear even at high temperatures, allowing for autoclave sterilization. Furthermore, unlike common synthetic UV absorbers, MYCs do not photodecompose and do not generate reactive oxygen species, making them a suitable alternative to synthetic UV absorbers. This makes them ideal for incorporation into compositions for topical skin applications, such as cosmetics. In particular, the incorporation of preservatives and synthetic UV absorbers into the composition can be reduced, potentially meeting the needs of users with chemical sensitivities.

[0012] Graph showing ROS levels in cells to which MYCs was applied, with and without UV-A irradiation, in Example 5.

[0013] The composition of the present disclosure essentially contains MYCs as an active ingredient. In a preferred embodiment, the MYCs comprises one or more selected from MGGnol, MGGcol, and MGn, and in a more preferred embodiment, the MYCs comprises at least MGGnol and MGGcol.

[0014] MYCs may be commercially available, or may be produced by any method from a microorganism that produces MYCs. Specific examples of the production method include culturing a microorganism that produces MYCs and extracting MYCs from the microorganism. The microorganism from which MYCs is extracted may be a single strain or multiple strains.

[0015] Microorganisms that produce MYCs are not particularly limited, but examples of microorganisms that produce MGGnol and / or MGGcol include black yeasts of the order Dothideales and family Dothioraceae that produce MGGnol and / or MGGcol. Generally, yeasts with black body color are called black yeasts. In addition to those classified into the Dothideales order, Dothioraceae family, black yeasts also include those belonging to the Moniliella genus of the Basidiomycota phylum or the Exophiala genus of the Chaetothyriales order, Ferpotrichiellaceae family (de Hoog et al., Atlas of clinical fungi, 2nd edition: Utrecht: centraalubureauvoor Schimmelcultures; (2000)), and the black yeast referred to here may be one classified into the family Dothioraceae in the order Dothideales. Specifically, it may be a black yeast belonging to the genus Aureobasidium, the genus Hormonema, or the genus Hortaea. Here, black yeast belonging to the genus Hormonema may also include those whose teleomorphs belong to the genus Sydowia. Anamorphs of the genus Sydowia correspond to the genus Hormonema, and the difference is whether they reproduce sexually or asexually in the same strain (N.A. Yurlava et al., Studies in Mycology, 43, 63-69 (1999); de Hoog et al., Atlas of clinical fungi, 2 ndEdition: Utrecht: centraalubureauvoor Schimmelcultures; (2000)), and when cultured in liquid, fungi described as belonging to the genus Sydowia can grow in a yeast-like cell shape, and can be distinguished from anamorphs of the genus Hormonema (G.S. de Hoog et al., Antonie van Leeuwenhoek., 65, 41-54 (1994)). Therefore, in this specification, both species will be referred to collectively. Furthermore, although anamorphs belonging to the genus Aureobasidium and anamorphs belonging to the genus Hormonema are closely related, the two can be clearly distinguished molecularly phylogenetically (Non-Patent Document 7).

[0016] Specific examples of black yeasts of the family Dothioraceae in the order Dothideales that produce MGGnol and / or MGGcol are not particularly limited, and any strain can be used. Examples of black yeasts of the genus Aureobasidium include Aureobasidium pullulans, Aureobasidium subglaciale, Aureobasidium aerium, Aureobasidium microstictum, and Aureobasidium proteae. Specific examples of the bacterial strain include Aureobasidium pullulans strains NRBC 6353, NRBC 100716, NRBC 106987, NRBC 4464, NRBC 7757, NRBC 108784, and NRBC 114573 (all of which are available from the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan)). In addition, the IDF-23 strain (NPMD deposited strain, accession number FERM P-22141) described in Patent Document 1 may also be used. Examples of black yeasts of the genus Hormonema include Hormonema macrosporum and Hormonema dematioides. Specific strains include NRBC 107026, NRBC 107027, NRBC 107028, NRBC 5982, and NRBC 32065 (all available from NPMD), which are assigned as Sydowia polyspora (teleomorphs) whose anamorphs correspond to Hormonema macrosprum. Examples of black yeasts of the genus Hortaea include Hortaea wirneckii. Specific examples of the strain include Hortaea wirneckii strains NRBC 4875 and NRBC 6407 (both available from NPMD).

[0017] Furthermore, the microorganism that produces MGn is not particularly limited, and examples thereof include fungi belonging to the genus Hormonema that produce MGn. Examples of fungi belonging to the genus Hormonema include Hormonema macrosporum and Hormonema dematioides. Specific examples of the fungi include the Hormonema macrosporum strains NSK102 (NITE ABP-03776), NSK33 (NITE ABP-03775), and NSK6 (NITE ABP-03774).

[0018] The Hormonema macrosporum strains NSK102 (NITE ABP-03776), NSK33 (NITE ABP-03775), and NSK6 (NITE ABP-03774) are novel fungi isolated from nature (Patent Application No. 2023-2008). When these fungi were cultured in liquid and their cell shapes were observed under an optical microscope, yeast-type cells were observed. In addition, to investigate the genetic properties of these fungi, the D1 / D2 region base sequence of 28SrRNA was identified by conventional methods, and the 28SrRNA gene base sequence of each Hormonema macrosporum was further analyzed using DB-FU15.0 (Techno Suruga Laboratory, Japan) and the International Base Sequence Database (DDBJ / ENA / GenBank) database (search date July 29, 2022). The analysis software ENKIv3.2 (Techno Suruga Laboratory, Japan) was used to perform a homology search of the base sequence by BLAST analysis. During the analysis, a phylogenetic tree was estimated using the neighbor-joining method, and the Kimura-2-parameter was used as a base substitution model. The reliability of the tree was evaluated by the bootstrap method (1000 repetitions). As a result, all three strains were identified as Hormonema macrosporum (anamorph) or Sydowia polysopra (telemorph).

[0019] The Hormonema macrosporum NSK102 strain has been applied for international deposit under the Budapest Treaty at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) and has been assigned the accession number NITE ABP-03776 (Date of receipt: October 20, 2022). The Hormonema macrosporum NSK33 strain has been applied for international deposit under the Budapest Treaty at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) and has been assigned the accession number NITE ABP-03775 (Date of receipt: October 20, 2022). The Hormonema macrosporum NSK6 strain has been applied for international deposit under the Budapest Treaty at the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture), and has been assigned the receipt number NITE ABP-03774 (received date: October 20, 2022).

[0020] An example of a method for producing MYCs from a microorganism is described below, but the method for obtaining MYCs is not limited to this. In the culture step for culturing a microorganism that produces MYCs, any medium can be used as long as it allows the microorganism to grow well. This medium can contain components such as a carbon source, a nitrogen source, inorganic salts, and, if necessary, amino acids suitable for microbial growth. Examples of carbon sources that can be used include sugars such as glucose, starch, and blackstrap molasses, organic acids, and sodium acetate. Sugars such as glucose are particularly useful. Examples of nitrogen sources include ammonium salts, yeast extract, corn steep liquor, and peptone. Examples of inorganic salts that can be used include magnesium salts, calcium salts, phosphate salts, iron salts, and copper salts. Typically, a liquid medium in which these components are mixed and dissolved in water can be used. The pH can generally be within the ranges of 2 to 9, 3 to 8, or 3.5 to 7.5. Microbial growth can be inhibited if the pH is too high or too low.

[0021] In the culturing step, the culture temperature may be any temperature that allows the microorganism to grow well, and may be, for example, 15 to 40°C, 18 to 35°C, or 20 to 30°C. If the culture temperature is too low, the growth rate will be insufficient, while if the culture temperature is too high, abnormalities in the biosynthetic pathway are likely to occur. In the culturing step, the culture time is not particularly limited, but may be 72 to 240 hours, or 96 to 168 hours. Furthermore, light irradiation may or may not be performed in the culturing step.

[0022] After the culturing step, an extraction step is carried out in which a MYCs fraction is extracted from a microorganism that produces MYCs into a solvent. Here, extraction is likely to be efficient by satisfying the following (a) and / or (b). However, if the following (c) is satisfied in the culturing step, efficient extraction is possible without necessarily carrying out the step that satisfies (a) or (b). That is, one or more selected from the following (a), (b), and (c) may be satisfied. (a) In the extraction step, the MYCs fraction is extracted into a solvent at 40°C or higher. (b) Prior to the extraction step, a heating step is carried out in which microorganisms are recovered from the culture solution after the culturing step and the recovered microorganisms are heated. (c) In the culturing step, the microorganisms are cultured in a medium containing glucose and yeast extract and / or cultured without light irradiation.

[0023] After the culturing step, the microbial cells may be collected from the culture medium prior to the extraction step. The collection of the microbial cells can be carried out by centrifugation, filtration, etc. The collected microbial cells may be dried using a dry heat dryer, freeze dryer, vacuum dryer, flash dryer, etc.

[0024] In this production method, (a) when extracting the MYCs fraction with a solvent at 40°C or higher, the microorganism is contacted with the extraction solvent. A polar solvent may be used as the extraction solvent, or a polar solvent containing primarily water may be used. Examples of such polar solvents include polar organic solvents such as water, lower alcohols (methanol, ethanol, butyl alcohol, isopropanol, butylene glycol, etc.), and acetone. Examples of polar solvents containing primarily water include water, or aqueous solutions of polar organic solvents such as lower alcohols (methanol, ethanol, butyl alcohol, isopropanol, butylene glycol, etc.) or acetone. Water alone may also be used. A solvent "containing primarily water" may contain 80% by weight or more, 85% by weight or more, or 90% by weight or more of water relative to the total extraction solvent. The higher the water content in the extraction solvent, the more efficient the extraction can be expected. That is, when using an aqueous solution of a polar organic solvent such as a lower alcohol as the extraction solvent, the concentration may be 20% by weight or less, and organic solvents may be substantially eliminated. Here, "substantially no organic solvent is used" means that the concentration of the polar organic solvent in the aqueous solution used as the extraction solvent is 1% by weight or less, 0.1% by weight or less, or 0.01% by weight or less. By not using a polar organic solvent substantially in the extraction, a step of volatilizing the polar organic solvent from the obtained extract may be unnecessary.

[0025] The temperature of the extraction solvent when extracting the MYCs fraction into the solvent can be 40°C or higher and 95°C or lower, 50°C or higher and 90°C or lower, or 60°C or higher and 85°C or lower. If the temperature of the extraction solvent is too low, the extraction efficiency is likely to decrease. If the temperature of the extraction solvent is too high, problems with the stability of the desired components are likely to occur. Alternatively, the extraction solvent may be heated to 90°C or higher, 100°C or higher and 135°C or lower, 110°C or higher and 130°C or lower, or 120°C or higher and 125°C or lower using an autoclave or other device. Heating can inhibit the activity of undesired enzymes contained in the cells. If the heating temperature is too low, the inhibition of the activity of undesired enzymes may be insufficient, while if the heating temperature is too high, problems with the stability of the desired components are likely to occur. Alternatively, the pressure may be increased to 0.10 MPa or higher, 0.12 MPa or higher, 0.15 MPa or higher, or 0.20 MPa or higher. Pressurization makes it easier to inhibit the activity of undesired enzymes contained in the cells. If the pressure is insufficient, the activity of unwanted enzymes contained in the cells may not be sufficiently inhibited. From the viewpoint of safety, the pressure may be set to 1.00 MPa or less.

[0026] The extraction time can be 15 minutes or more and 120 minutes or less, 60 minutes or more and 110 minutes or less, or 90 minutes or more and 100 minutes or less. The longer the extraction time, the greater the extraction amount. The upper limit of the extraction time can be, for example, about 120 minutes. If the extraction time is too long, undesirable impurities are likely to be contained. During the extraction process, the microorganism in contact with the extraction solvent may be stirred or shaken as appropriate, or may be left to stand.

[0027] After the culturing step, the microorganisms may not be recovered from the culture medium, and the culture medium after the culturing step may be subjected to an extraction step either directly or after appropriate dilution, thereby (a) extracting the MYCs fraction into a solvent at 40°C or higher. In this case, the culture medium may be heated to 40°C or higher, or 50°C or higher. Heating the culture medium can be expected to ensure efficient extraction. The culture medium usually contains a polar solvent, particularly water. The culture medium after the culturing step may be diluted, for example, twice or more in volume, with water, a buffer solution, a medium, or the like and then subjected to the extraction step. Diluting the culture medium with water, a buffer solution, a medium, or the like prior to use in the extraction step can improve operability. For example, if the culture medium has a high viscosity, diluting it with a buffer solution, a medium, or the like prior to use in the extraction step may reduce retention in the device. Alternatively, the culture medium may be used directly in the extraction step. Using the culture medium directly can contribute to reducing extraction costs.

[0028] In the production method disclosed herein, instead of or prior to (a) above, a heating step may be carried out (b) before the extraction step, in which the microorganisms recovered from the culture medium after the culture step are heated. In this step, the following conditions can be used to damage or disrupt the microorganisms, thereby improving the efficiency of MYCs extraction, or to inhibit the activity of undesired enzymes in the microbial cells, thereby improving the recovery amount of MYCs. Heating of the microorganisms in the heating step may be carried out under conditions selected from the following: (i) temperature, (ii) time, and (iii) pressure. (i) The temperature to which the microorganisms are exposed may be 40° C. or higher and 200° C. or lower, 50° C. or higher and 190° C. or lower, 60° C. or higher and 180° C. or lower, 70° C. or higher and 170° C. or lower, 80° C. or higher and 160° C. or lower, 90° C. or higher and 150° C. or lower, 100° C. or higher and 140° C. or lower, 105° C. or higher and 130° C. or lower, 110° C. or higher and 130° C. or lower, 115° C. or higher and 130° C. or lower, 120° C. or higher and 130° C. or lower, or 125° C. or higher and 130° C. In this case, if the temperature to which the microorganisms are exposed is too low, the inhibition of the activity of undesired enzymes may be insufficient, and if the temperature to which the microorganisms are exposed is too high, problems with the stability of desired components may occur.

[0029] (ii) The time for heating the microorganism may be 2 hours to 6 hours, 3 hours to 5 hours, or 4 hours to 5 hours. The heating time may be the time required to reach and maintain a desired heating temperature. If the heating time for the microorganism is too short, the inhibition of undesired enzyme activity may be insufficient, and if the heating time for the microorganism is too long, problems with the stability of the desired components may occur. (iii) The pressure applied to the microorganism, calculated as the average pressure during the heating period, may be 0.01 MPa to 0.10 MPa, 0.02 MPa to 0.08 MPa, or 0.03 MPa to 0.05 MPa. If the pressure applied to the microorganism is too low, the inhibition of undesired enzyme activity may be insufficient, and if the pressure applied to the microorganism is too high, problems with the stability of the desired components may occur.

[0030] Conditions (i) to (iii) may be arbitrarily selected and combined, and particularly, at least (i) and (ii) may be combined. In this case, heating at a high temperature for a certain period of time can simultaneously perform sterilization, thereby inactivating enzymes in the microorganisms and potentially improving the recovery amount of MYCs. Furthermore, (iii) disrupts the microorganisms, which may further improve the extraction efficiency of MYCs. The heating step may also be performed under conditions that result in the drying of the microorganisms. For example, the heating step may be performed under one or more conditions selected from (i) a temperature of 105°C or higher, (ii) a heating time of 2 hours or longer, and (iii) a pressure of 0.10 MPa or lower, or may be performed under conditions of (i) a temperature of 105°C or higher, (ii) a heating time of 2 hours or longer, and (iii) a pressure of 0.10 MPa or lower.

[0031] In the production method disclosed herein, when an extraction step is performed using a microorganism after the heating step (b), the extraction may be performed under the conditions described in (a), or at a lower extraction temperature. Specifically, the same extraction solvent as described in (a) may be used. However, the temperature of the extraction solvent when extracting the MYCs fraction into the solvent may be 5°C or higher, 10°C or higher, or 25°C (room temperature) or higher. The conditions described in (a) may be used, i.e., 40°C to 70°C, 50°C to 90°C, or 60°C to 100°C. Alternatively, the extraction may be performed using an autoclave or other device, at 90°C to 150°C, 100°C to 140°C, 110°C to 130°C, or 120°C to 125°C. The extraction time may be 15 minutes or longer, 60 minutes or longer, or 90 minutes or longer. The longer the extraction time, the greater the extraction amount. The upper limit of the extraction time may be, for example, about 120 minutes. During the extraction step, the microorganisms in contact with the extraction solvent may be appropriately stirred, shaken, or left to stand.

[0032] In this way, by exposing the microorganisms and / or the culture solution containing them to high temperature conditions during or before the extraction step, it is possible to kill the microorganisms and / or inactivate enzymes in the microorganisms that have a degrading effect on MYCs, thereby improving the recovery yield of MYCs. The high temperature conditions to which the culture solution is exposed can be, for example, 40°C or higher or 50°C or higher. Therefore, the survival rate of the microorganisms after the extraction step may be 0% to 20%, 0% to 10%, or 0% to 5% after the culture step. Note that while it is sufficient for the survival rate after the extraction step to be within the above range, if the survival rate of the microorganisms is low even before or during the extraction step, it is expected that the degradation of MYCs will be suppressed and the recovery yield will be improved.

[0033] After the extraction step, the resulting extract may be subjected to removal of contaminants, fractionation, purification, etc., using well-known methods.

[0034] The composition of the present disclosure preferably contains MGGnol, MGGcol, and MGn in total at 0.001 wt% or more of the total composition, more preferably 0.01 wt% or more, and even more preferably 0.05 wt% or more. Furthermore, the composition of the present disclosure more preferably contains MGGnol at 0.0001 wt% or more of the total composition, more preferably 0.001 wt% or more, and even more preferably 0.005 wt% or more. Of the total MYCs contained in the composition of the present disclosure, MGGnol may preferably account for 10 wt% or more, more preferably 20 wt% or more, and even more preferably 30 wt% or more. By setting the content of MYCs within the above range, the desired effect can be easily achieved and flexibility in formulation design can be ensured. The content can be appropriately adjusted according to the administration route and the type of composition to be contained, as described below.

[0035] The content of MYCs can be measured by a conventional method. Generally, MYCs have ultraviolet absorption ability due to a structure having cyclohexanone as a central ring. Therefore, the presence of MYCs in a composition and the amount and concentration of MYCs in the composition can be confirmed by measuring the wavelength of light in the ultraviolet region, for example, by measuring the ultraviolet absorption of MYCs in the composition with a spectrophotometer (Patent No. 5913988, Buscot et al., Mycol. Res, 95(6):752-754(1991)). The amount of MGGnol can be determined by measuring the absorbance (ABS) at a maximum absorption wavelength of 310 nm and determining the molar extinction coefficient of 25,000 M －１ cm －１ (Patent No. 5913988). MGGcol can be calculated by measuring the absorbance at a maximum absorption wavelength of 310 nm and determining the molar absorption coefficient of 25,000 M －１ cm －１(N. Wada et al., Antioxidants, 4, 603-646 (2015)). The maximum absorption wavelength of MGn has been reported to be 310 nm (N. Wada et al., Antioxidants, 4, 603-646 (2015)). However, the molar absorption coefficient has not been reported. Therefore, the molar absorption coefficient of mycosporin glutamic acid (MGa), which has almost the same separation amount and a similar structure, 20900 M －１ cm －１ (N. Wada et al., Antioxidants, 4, 603-646 (2015)) can be used instead for calculation.

[0036] The compositions of the present disclosure may also contain MYCs other than the aforementioned MYCs or other MAAs. Other MAAs containing other MYCs may include compounds having a central ring of cyclohexanone or cyclohexenimine, or compounds having various functional groups, such as amino acids, bound to the central ring.More specifically, mycosporine-hydroxyglutamicol, gadusol, deoxygadusol, mycosporine-glycine, mycosporine-taurine, mycosporine-alanine, mycosporine-serine, mycosporine-serinol, mycosporine-2-glycine, palythinol, palythenic acid acid), mycosporine-methylamine-threonine, mycosporine-glycine-valine, palythine, Asterina-330, shinorine, porphyra-334, euhalothece-362, mycosporine-ornithine, mycosporine-lysine, mycosporine-glutamic acid-glycine Mycosporine-methylamine-serine, mycosporine-taurine, palythene, palythine-serine, palythine-serine-sulfate, usujirene, and the like.

[0037] The composition of the present disclosure can suppress skin troubles. In the present disclosure, skin troubles refer to phenomena caused by erythema, inflammation, darkening / blemishes, and oxidative stress on the skin.

[0038] One of the factors that contribute to these effects is the ability of MYCs to absorb light over a wide range of wavelengths, including mid-wavelength ultraviolet (UV-B: 280-320 nm) and long-wavelength ultraviolet (UV-A: 320-400 nm). MyCs's absorption of UV-B can suppress erythema and inflammation, pigmentation such as darkening and age spots, and the generation of reactive oxygen species (ROS), thereby suppressing oxidative stress. Carbonyl proteins (CPs) are the footprint of oxidative stress. In other words, the generation of CPs indicates exposure to oxidative stress. Oxidative stress is involved in wrinkles, age spots, and skin aging, and therefore suppressing oxidative stress can prevent these conditions. CP generation causes yellowing and dryness of the skin, and UV-A irradiation of CPs generates reactive oxygen species, so inhibiting CPs is highly significant. Furthermore, the inventors of the present disclosure had previously believed that the skin trouble suppression effect of MYCs was due to the absorption of UV-B by MYCs, particularly MGGnol, MGGcol, and MGn. However, they surprisingly discovered that these MYCs can also suppress oxidative stress and achieve the aforementioned effect through a pathway involving the absorption of singlet oxygen, a type of reactive oxygen generated by UV-A.

[0039] Furthermore, one of the reasons for the skin trouble suppression effect of the composition of the present disclosure is that MYCs MGG and MGn absorb singlet oxygen generated by UV-A, thereby suppressing oxidative stress. Skin troubles caused by UV-A are caused over a longer period of time than those caused by UV-B, and the damage persists, causing skin aging to progress gradually. For this reason, it has been difficult to take effective measures against these problems until now, but the present disclosure may make it possible to resolve these problems.

[0040] Furthermore, one of the reasons why the composition of the present disclosure has the effect of suppressing skin troubles is that MYCs has anti-inflammatory and active oxygen scavenging properties, even in the absence of UV irradiation (i.e., regardless of the UV absorption ability of MYCs), thereby suppressing skin inflammation and oxidative stress.

[0041] From the above, the composition of the present disclosure can suppress both skin troubles caused by ultraviolet irradiation and skin troubles that are not caused by ultraviolet irradiation.

[0042] Therefore, MYCs, particularly one or more selected from MGGnol, MGGcol, and MGn, can be an active ingredient of a composition for suppressing skin troubles. Here, skin troubles refer to erythema, inflammation, darkening / blemishes, and phenomena caused by oxidative stress on the skin. In the examples described below, it is shown that MYCs can suppress the production of cytokine IL-1α and PGE2, which are indicators of inflammation, regardless of whether or not UV-A and UV-B irradiation is performed. ２ It has been shown that the amount of PGE produced was suppressed. ２ MYCs reflects inflammation induced by oxidative stress, such as UV irradiation, and, together with nitric oxide radicals, is involved in the generation of erythema, specifically reddening the skin through vasodilatory effects. Therefore, these examples support the erythema suppression and anti-inflammatory effects. Furthermore, the examples described below demonstrate that MYCs suppresses CP levels in a three-dimensional skin model irradiated with UV-B. Therefore, these examples support the oxidative stress suppression effect. Furthermore, the examples described below demonstrate that MYCs suppresses darkening and age spots in a skin model irradiated with UV-B, supporting this effect. Furthermore, the examples described below demonstrate that ROS levels were suppressed in three-dimensional skin models or cells to which MYCs was applied, regardless of whether they were irradiated with UV-A or UV-B. This suggests that ROS production was suppressed and that the generated ROS was eliminated (removed) by MYCs.

[0043] The present inventors are the first to discover that MYCs can suppress skin troubles regardless of the presence or absence of UV irradiation. Conventionally, it has been assumed that MYCs and MAAs have UV absorption ability and can protect the skin by UV protection based on their chemical structures, but the fact that they can suppress various skin troubles even in the absence of UV exposure was completely unimaginable from the prior art.

[0044] In this specification, "suppressing" skin troubles may include preventing skin troubles from occurring in the future and preventing the worsening of skin troubles that have already occurred. "Preventing the worsening of skin troubles that have already occurred" may include preventing the severity of skin troubles from increasing and delaying the severity of skin troubles from increasing.

[0045] Furthermore, as described above, one or more selected from MGGnol, MGGcol, and MGn can particularly suppress the amount of ROS and can therefore serve as an active ingredient in a composition for suppressing reactive oxygen. In this specification, "suppression" of reactive oxygen may include both a reduction in the amount of ROS produced and the elimination (removal) of the produced ROS.

[0046] The composition of the present disclosure may be used for therapeutic or non-therapeutic purposes. "Non-therapeutic use" refers to a practice that does not include medical treatment, i.e., a concept that does not include therapeutic treatment of the human body. Examples include health promotion and cosmetic procedures. When the composition of the present disclosure is used for non-therapeutic purposes, it is administered to a healthy subject. When the composition of the present disclosure is used for non-therapeutic purposes, it may be used to suppress skin problems and / or suppress reactive oxygen species in a healthy subject. When the composition of the present disclosure is used for non-therapeutic purposes, it may be used to prevent diseases or symptoms caused by inflammation, erythema, skin darkening, pigmentation, oxidative stress, etc., due to ultraviolet exposure. When the composition of the present disclosure is used for therapeutic purposes, it may be administered to an unhealthy subject. Such unhealthy subjects include those with diseases or symptoms caused by inflammation, erythema, skin darkening, pigmentation, oxidative stress, etc., due to ultraviolet exposure, and the composition may be used to suppress or alleviate these diseases or symptoms.

[0047] Diseases and symptoms caused by inflammation, erythema, skin darkening, pigmentation, oxidative stress, and the like due to UV exposure include, for example, skin cancer and autoimmune diseases such as systemic lupus erythematosus and Sjögren's syndrome (see, for example, Non-Patent Document 2, T. Ogura et al., Jpn. J. Clin. Immunol., 37(1), 25-32 (2014), Yoichiro Hamasaki, Journal of the Japanese Society of Clinical Dermatology, 33(4), 483-491 (2016)). Skin cancer is thought to be caused by oxidative stress due to long-term UV exposure, resulting in a weakened immune system and so-called photoaging. Specific examples of skin cancer include actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and melanoma. Autoimmune diseases are multifactorial diseases, and their onset is a complex combination of genetic predisposition and environmental factors. Excessive oxidative stress caused by environmental factors such as UV exposure can lead to inflammation, tissue damage due to apoptosis induction, and the induction of autoimmune responses, resulting in autoimmune pathology. Systemic lupus erythematosus and Sjögren's syndrome are also known as major autoimmune diseases, and eliminating the undesirable phenomena caused by UV exposure is important for preventing these diseases.

[0048] Another aspect of the present invention is the use of MYCs in the manufacture of a composition for suppressing skin troubles and / or a composition for suppressing active oxygen. Another aspect of the present invention is the use of MYCs in suppressing skin troubles and / or suppressing active oxygen. Another aspect of the present invention is MYCs used for suppressing skin troubles and / or suppressing active oxygen. Another aspect of the present invention is a method for suppressing skin troubles and / or suppressing active oxygen, comprising administering (ingesting) MYCs to an animal.

[0049] The timing of administration (ingestion) of the composition of the present disclosure is not particularly limited and can be selected appropriately depending on the condition of the subject to be administered.

[0050] The administration (ingestion) amount of the composition of the present disclosure may be appropriately selected depending on the age, sex, condition, other conditions, etc. of the subject to be administered (ingested). The administration (ingestion) amount of the composition of the present disclosure is not particularly limited as the administration (ingestion) amount of MYCs, but in consideration of the desired effect and safety, when administered transdermally in the form of a topical skin composition described below, for example, the total amount of MGGnol, MGGcol, and MGn per skin surface area in an adult is 0.1 μg / cm. ２ days or more, more preferably 0.2 μg / cm ２ Days or more, 0.4μg / cm ２ Days or more, 0.8μg / cm ２ Days or more, 1.6μg / cm ２ Days or more, 3.2μg / cm ２ Days or more, 6.4μg / cm ２ The administration period can be 1 to 3 days or more. There is no particular upper limit to the dose.

[0051] The dosage may be adjusted appropriately depending on the skin trouble to be targeted. For example, when the purpose is to suppress erythema and / or inflammation in the skin, the total amount of MGGnol, MGGcol, and MGn is 0.1 μg / cm ２ For the purpose of suppressing darkening and / or blemishes on the skin, the total amount of MGGnol, MGGcol and MGn may be 0.5 μg / cm ２ For the purpose of suppressing phenomena caused by oxidative stress in the skin, the total amount of MGGnol, MGGcol and MGn can be 0.1 μg / cm ２ It can be more than one day.

[0052] Regardless of the dosage or duration of administration (ingestion), the composition of the present disclosure can be administered once or multiple times a day. In addition to a single administration (ingestion), it can also be administered (ingested) continuously or intermittently for a period of several weeks or more, or several months or more.

[0053] The subject to which the composition of the present disclosure is administered (ingested) is not particularly limited, and examples thereof include mammals. Mammals include humans, dogs, cats, etc. Mammals in particular include humans. Humans may be of any age, such as infants, young children, children, adults, middle-aged and elderly people, and the elderly. Furthermore, there are no particular limitations on gender. Furthermore, the UV-B absorption ability of the MYCs described above is exerted as long as MYCs is present on the cell surface upon administration, and therefore the skin condition or skin type of the subject to be administered is not particularly important.

[0054] The route of administration (ingestion) of the composition of the present disclosure is not particularly limited and may include transdermal, oral, nasal, inhalation, intravenous injection, etc., but is usually transdermal. Here, "administration" may be replaced with "ingestion." When administering (ingesting) via transdermal administration, it is preferably a topical skin composition. The topical skin composition may be in any form as long as it is applied topically to the skin, but preferred examples include cosmetics, quasi-drugs, pharmaceuticals, etc., with cosmetics being more preferred and sunscreen cosmetics being particularly preferred. When the composition of the present disclosure is in the form of cosmetics, it is used for the aforementioned non-therapeutic purposes, and when the composition of the present disclosure is in the form of a quasi-drug or pharmaceutical, it is used for the aforementioned therapeutic purposes. The area to which the topical skin composition is applied is not particularly limited, but is usually the face, limbs, neck, or décolleté.

[0055] The formulation of the topical skin composition is not particularly limited, and examples thereof include lotion formulations, emulsion formulations such as lotions and creams, oil formulations, gel formulations, packs, cleansers, etc. The topical skin composition may be either a leave-on type or a leave-off type.

[0056] When the composition of the present disclosure is in the form of a topical skin composition, in addition to MYCs, ingredients that are typically used in the formulation of cosmetics, quasi-drugs, pharmaceuticals, etc. can be optionally blended in the composition during production. Examples of such optional components include hydrocarbons such as squalane, petrolatum, and microcrystalline wax; esters such as jojoba oil, carnauba wax, and octyldodecyl oleate; triglycerides such as olive oil, beef tallow, and coconut oil; fatty acids such as stearic acid, oleic acid, and retinoic acid; higher alcohols such as oleyl alcohol, stearyl alcohol, and octyldodecanol; anionic surfactants such as sulfosuccinate esters and polyoxyethylene alkyl sulfate sodium; amphoteric surfactants such as alkyl betaine salts; cationic surfactants such as dialkylammonium salts; nonionic surfactants such as sorbitan fatty acid esters, fatty acid monoglycerides, polyoxyethylene adducts thereof, polyoxyethylene alkyl ethers, and polyoxyethylene fatty acid esters; polyhydric alcohols such as polyethylene glycol, glycerin, and 1,3-butanediol; thickening / gelling agents; antioxidants; ultraviolet scattering agents; colorants; preservatives; and powders.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, and representative exemplary methods and materials are described herein.

[0058] Where a range of values ​​is provided, it is understood that each value between the upper and lower limits of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated or intervening value in that stated range is encompassed within the scope of the invention set forth in this disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed within the scope of the invention set forth in this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention set forth in this disclosure.

[0059] The present disclosure is not limited to the particular embodiments described. Because the scope of the invention(s) set forth in this disclosure will be limited only by the appended claims, it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0060] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described herein has distinct components and features that may be readily separated from or combined with the features of any of the other several embodiments without departing from the inventive scope and spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible and consistent.

[0061] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the inventions presented in this disclosure are not entitled to antedate the cited publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0062] The following examples are provided for the purpose of illustrating various aspects of the present disclosure and are not intended to limit the disclosure in any manner. Those skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples, together with the methods described herein, are presently representative of embodiments and are illustrative and are not intended as limitations on the scope of the present disclosure. Modifications therein and other uses encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

[0063] The invention disclosed in this disclosure will be described below based on examples, but the invention disclosed in this disclosure is not limited to these examples. Note that unless otherwise specified, units are by weight.

[0064] <Reference Example> Preparation of MYCs Sample Microorganisms (Aureobasidium pullulans and / or Hormonema macrosporum) that produce MGGnol, MGGcol, and / or MGn were cultured by the method described above, and MYCs was extracted from the cultured cells.

[0065] The concentration of each MYCs in the obtained extract was measured by HPLC. Specifically, using a Shimadzu Corporation HPLC system, a Lichrospher (registered trademark) 100RP-18 column (particle size 5 μm, inner diameter 4 mm x length 250 mm, manufactured by Merck) equipped with a guard column LiChrospher (registered trademark) 100 RP-18 (5 μm) LiChroCART (registered trademark) 4-4 was used. The mobile phase (aqueous solution of 0.5% methanol and 0.2% acetic acid), flow rate 0.7 mL / min, column oven temperature 35 ° C., injection volume 10 μL, and a PDA detector were used to measure 250 nm to 400 nm. A peak showing a maximum absorption wavelength was observed near 310 nm, and this was considered to be the peak of MAAs. Quantitation was performed at a detection wavelength of 310 nm. Three types of MYCs samples were prepared and obtained, and the compositions of MGGnol, MGGcol, and MGn contained therein are shown in Table 1.

[0066]

[0067] Example 1: Study of anti-inflammatory and erythema suppression effects (1) Preliminary study of the culture method and ultraviolet irradiation dose of the three-dimensional skin model To evaluate the response of the three-dimensional skin model to UV-B, the irradiation dose at which the cells do not die was investigated. A comparison was also made with the results using skin cells from the prior art (Patent Document 4). Sample MGG-E was adjusted with PBS(-) to MYCs concentrations of 0.07, 0.14, and 0.35% by weight to prepare a MYCs solution. The three-dimensional skin model used was LabCyte EPI-MODEL24 from Japan Tissue Engineering Co., Ltd. Using the attached assay medium, the model was cultured in a 24-well plate at a culture temperature of 37°C and CO ２ The cells were then conditioned at 5% UV for 24 hours. UV irradiation was performed using a UV lamp (TL 20W / 12 RS, manufactured by PHILIPS Lighting). The UV lamp irradiation intensity was measured using a UVX Radiometer manufactured by Analytik Jena. The irradiation dose was 400 mJ / cm. ２ , 600 mJ / cm ２After acclimation, 30 μL of PBS(−) or MYCs solution was applied to the stratum corneum side of the LabCyte EPI-MODEL24 three-dimensional skin model in one well, and the three-dimensional skin model was incubated at 37°C and CO ２ , and 5% for 24 hours. Then, 400 or 600 mJ / cm ２ After 24 hours of irradiation, the PBS(-) or MYCs solution on the stratum corneum was washed away with 500 μL of PBS(-), and cell viability was measured using the AlamarBlue Assay. Note that the amount of MYCs in 30 μL of a 0.07% MYCs solution was 2.1 μg, and the area of ​​the well was 0.32 cm. ２ Therefore, the application amount of MYCs is 6.6 μg / cm ２ 400, 600 mJ / cm ２ Regardless of the irradiation dose, cells survived in the presence of MYCs.

[0068] (2) Study of anti-inflammatory and erythema suppression effects IL-1α is always retained intracellularly and is easily released outside the cells by the stimulus of ultraviolet irradiation. Because it can be detected quickly and in large amounts, it can be widely evaluated as an inflammatory mediator (T. Hirano, et al., Dermatol 106, 1102-1107 (1996)). ２ As mentioned above, is involved in the generation of erythema and is an indicator of inflammation caused by oxidative stress such as ultraviolet radiation (Non-Patent Document 2). As mentioned above, a three-dimensional skin model was prepared. As discussed in (1), 600 mJ / cm ２ The cell viability did not decrease even at an irradiation dose of 600 mJ / cm. ２ The PGE released into the medium 24 hours after irradiation was selected and tested. ２ The amount of IL-1α was measured using a Prostaglandin E2 EIA Monoclonal Kit (manufactured by Cayman), and the amount of IL-1α was measured using a Human IL-1 alpha / IL-1F1 Quantikine ELISA Kit (manufactured by R&D).

[0069] Table 2 shows the IL-1α and PGE per well of the three-dimensional skin model. ２ The production amounts of 600 mJ / cm ２ IL-1α increased from 6 pg / 3D to 11 pg / 3D. ２ The ion beam energy also increased from 25 to 47 pg / 3D. ２ Under the conditions of ２ When 30 μL of the 0.07% MYC solution and 0.14% MYCs solution prepared from sample MGG-E in (1) was applied to one well from the stratum corneum side, IL-1α and PGE ２ IL-1α decreased in a MYCs concentration-dependent manner. When MYCs was added at 0.14%, IL-1α was lower than in the control, demonstrating that MYCs has an anti-inflammatory effect independent of UV-B irradiation.

[0070]

[0071] Example 2: Examination of the inhibitory effect on skin darkening. Human skin was excised from a Caucasian woman, 39 years old, with a BMI of 28, and cut into 14 mm diameter pieces (surface area 1.5 cm). ２ The skin was placed on the intercell and placed in a well containing the medium. The tissue was incubated at 37°C and CO ２ The mice were cultured for 24 hours at 5% MYCs. The MGG-K solution was adjusted with PBS(-) to a MYCs content of 0.7% to prepare a MYCs solution. The amount of MYCs solution applied was 22.7 μg / cm2 of skin surface area. ２ The test plots were as follows: control (no irradiation throughout the entire period), test plot 4: UV-B irradiation (irradiation throughout the entire period), test plot 5: MYCs addition before and after UV-B irradiation, test plot 6: addition during UV-B irradiation, and test plot 7: addition before, during, and after UV-B irradiation (throughout the entire period) (see Table 3). There were three samples in each test plot.

[0072] A silicone washer was placed on the skin, and 50 μL of PBS(-) or MYCs solution was added. After 24 hours, the PBS(-) or MYCs solution was removed, and the skin was washed three times with 1 mL of PBS(-). Intercellular and PBS(-) remaining on the skin surface were removed. Fresh PBS(-) or MYCs solution was dropped into the silicone washer, and the skin was transferred to a plate containing PBS(-). After that, the skin was exposed to 300 mJ / cm using an ultraviolet lamp (TL 20W / 12 RS, PHILIPS Lighting). ２ The cells were irradiated with 10 ... The average L* value within a certain area of ​​three samples in each test plot was calculated.

[0073]

[0074] The results are shown in Table 4. A decrease in L*, an index of skin brightness, indicates skin darkening. In experimental groups 4 and 5, UV-B irradiation significantly reduced skin brightness (L* value) by approximately 10%. In other words, skin darkening occurred. In experimental groups 6 and 7, L* values ​​decreased by approximately 2% compared to the control, but this was not significant. It was found that applying 50 μL of 0.7% MYCs solution to the skin during irradiation inhibited skin darkening. Adding MYCs before the first UV-B irradiation, followed by washing and irradiation, did not have any inhibitory effect on darkening, suggesting that the presence of MYCs in the skin during UV irradiation is necessary for darkening inhibition.

[0075]

[0076] Example 3: Study on the inhibitory effect on skin damage caused by reactive oxygen species Evaluation was performed with reference to the method of Katsuyama et al. (Katsuyama, Yushi, et al., Journal of the Japanese Society of Cosmetic Science, 39, 89-94 (2015)). The three-dimensional skin model used was LabCyte EPI-MODEL24 from Japan Tissue Engineering Co., Ltd. Using the attached assay medium, the model was incubated in a 24-well plate at 37°C and CO ２ After the acclimation, 30 μL of PBS(-) or sample MGG-K (adjusted to 0.15% MYCs), sample MGG-EX (adjusted to 0.15% MYCs), or sample MGG-E (adjusted to 0.14% MYCs) was applied to the stratum corneum side of the LabCyte EPI-MODEL 24 cells per well. ２ After 24 hours, the PBS(-) or each MYCs solution on the stratum corneum was removed and washed with PBS(-). After applying 30 μL of freshly prepared PBS(-) or each MYCs solution to the stratum corneum side, the cells were exposed to 600 mJ / cm UV-B lamp (TL 20W / 12 RS) (PHILIPS Lighting). ２ The samples were irradiated with UV-B for 10 min. Each test group consisted of four samples. After UV-B irradiation, the PBS(-), MGG-K, or MGG-EX solution on the stratum corneum was removed, and the stratum corneum surface was washed with PBS(-). ROS levels in the epidermal tissue were evaluated by chemiluminescence detection using 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one hydrochloride (MCLA) (Tokyo Chemical Industry Co., Ltd.). The detector used was Night OWL (registered trademark) (Berthold Technologies). Image processing was performed using Image J.

[0077] The results are shown in Table 5. A higher gray value indicates a higher ROS level. Similar to many literature reports, UV-B irradiation increased the ROS level by 4%. Addition of MYCs reduced the ROS level by approximately 10% compared to UV-B irradiation. Surprisingly, the control ROS level, i.e., the amount of ROS that is constantly generated in biological reactions even without UV-B irradiation, was also reduced by addition of MYCs.

[0078]

[0079] Example 4: Study of the inhibitory effect of carbonyl proteins produced by UV exposure The three-dimensional skin model used was LabCyte EPI-MODEL24 from Japan Tissue Engineering Co., Ltd. The attached assay medium was used in a 24-well plate, and the culture temperature was 37°C, CO ２ After the acclimation, 30 μL of PBS(-) or sample MGG-K (adjusted to 0.15% MYCs), sample MGG-EX (adjusted to 0.15% MYCs), or sample MGG-E (adjusted to 0.14% MYCs) was applied to each well from the stratum corneum side of the LabCyte EPI-MODEL 24. The cells were incubated at 37°C and CO ２ After 24 hours, the PBS(-) or each MYCs solution on the stratum corneum was removed and washed with PBS(-). After applying 30 μL of freshly prepared PBS(-) or each MYCs solution to the stratum corneum side, the cells were exposed to 600 mJ / cm UV-B light using a UV-B lamp (TL 20W / 12 RS, PHILIPS Lighting). ２The stratum corneum was irradiated with UV-B. Four samples were used in each test group. After UV-B irradiation, the PBS(-) or each MYCs solution was removed from the stratum corneum, and the stratum corneum surface was washed with PBS(-). 30 μL of freshly prepared PBS(-) or each MYCs solution was applied to the stratum corneum side and then cultured for 24 hours. After 24 hours, the PBS(-) or MGG-K or MGG-EX solution from the stratum corneum was removed, and the stratum corneum surface was washed with PBS(-). The epidermal tissue from the three-dimensional cultured skin model was removed from the culture cup, frozen, and then thinly sectioned using OCT compound (Sakura Finetech Japan Co., Ltd.). Carbonyl proteins (CPs) in thin sections were detected using Fluorescein-5-thiosemicarbazide (Invitrogen) (see T. Mizutani, et al., Journal of the Japanese Society of Cosmetic Science, 41, 101-105 (2017)). CP levels were quantified using fluorescent image analysis. Corneocytometry 2 (CIEL) image analysis software was used.

[0080] Table 6 shows the relative amount of CP to the control. UV-B irradiation increased CP by 1.27 times. Addition of MYCs suppressed the increase in CP.

[0081]

[0082] Example 5: Study of intracellular ROS levels with and without UV-A irradiation Normal human epidermal keratinocytes (Kurabo) were used. They were cultured using standard culture medium (HuMedia-KG2). For ultraviolet irradiation, an ultraviolet UV-A lamp (FL20SBLB 20W, Toshiba) was used. A MYCs solution prepared by diluting sample MGG-EX with PBS (-) was added to the cells and allowed to stand for 24 hours. After 24 hours, the MYCs solution was added again together with riboflavin. In the presence of MYCs and riboflavin, 6 J / cm ２The cells were irradiated with UV-A at 1000 kJ / cm², and intracellular ROS levels immediately after irradiation were detected using a cell-permeable redox-sensitive fluorescent probe, DCFDA (2,7-Dichlorofluoroscin Diacetate). Cellular protein levels were quantified using a BCA Protein Assay Kit (Thermo Scientific®). Three samples were used for each test group. The results are shown in Figure 1 and Tables 7 (without UV-A irradiation) and 8 (without UV-A irradiation). Protein levels were used as an indicator of cell number. As shown in the table, no changes in protein levels were observed with MYCs application or UV-A irradiation. The figures show the amount of intracellular ROS generated without UV-A irradiation. ROS decreased with increasing MYCs concentration. Even at a low concentration of 0.006%, MYCs significantly reduced ROS, and at 0.05%, MYCs reduced ROS to half of the control level. Furthermore, with UV-A irradiation, ROS increased 1.5-fold even in the control. With UV-A irradiation, MYCs at 0.003%, a lower concentration than without irradiation, significantly reduced ROS. ROS decreased with increasing MYCs concentration. At 0.05%, MYCs reduced ROS to approximately one-third of the UV-A control level. Application of MYCs reduced intracellular ROS in a concentration-dependent manner, regardless of UV-A irradiation.

[0083]

[0084]

Claims

1. A composition for suppressing skin problems, containing mycosporines (MYCs).

2. A composition for suppressing active oxygen, containing mycosporines (MYCs).

3. The composition described in claim 1 or 2, wherein the MYCs comprises one or more selected from mycosporine glutaminol glucoside (MGGnol; Mycosporine-glutaminol-glucoside), mycosporine glutamicol glucoside (MGGcol; Mycosporine-glutamicol-glucoside), and mycosporine glutamine (MGn; Mycosporine-glutamine).

4. The composition according to claim 3, wherein the MYCs contains at least MGGnol and MGGcol.

5. The composition according to claim 3, containing MGGnol, MGGcol and MGn in a total amount of 0.001% by weight or more of the total composition.

6. The total amount of MGGnol, MGGcol and MGn is 0.1 μg / cm ２ The composition according to claim 3, which is applied to the skin for a period of at least one day.

7. The composition according to claim 1, wherein the skin trouble is one or more selected from the group consisting of erythema, inflammation, darkening / blemishes, and phenomena caused by oxidative stress in the skin.

8. The composition according to claim 1, wherein the skin trouble is skin trouble caused by ultraviolet light exposure or skin trouble that is not caused by ultraviolet light exposure.

9. The composition according to claim 1 or 2, which is a composition for external use on the skin.

10. The total amount of MGGnol, MGGcol and MGn is 0.1 μg / cm ２ The composition according to claim 9, which is applied to the skin for a period of days or more.

11. For suppressing erythema and / or inflammation in the skin, the total amount of MGGnol, MGGcol and MGn is 0.1 μg / cm ２ The composition according to claim 9, which is applied to the skin for a period of days or more.

12. For inhibiting darkening and / or spots on the skin, the total amount of MGGnol, MGGcol and MGn is 0.5 μg / cm ２ The composition according to claim 9, which is applied to the skin for a period of days or more.

13. For suppressing phenomena caused by oxidative stress in the skin, the total amount of MGGnol, MGGcol and MGn is 0.1 μg / cm ２ The composition according to claim 9, which is applied to the skin for a period of days or more.

14. The composition according to claim 9, which is a sunscreen cosmetic.

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