Composition containing mycosporines

Abstract

The present application provides a composition which has excellent skin problem inhibiting effect by a component which can replace a synthetic ultraviolet absorber, absorbs ultraviolet rays of a wide range of wavelengths, and is stable. A mycosporine (MYC) is used as an effective component of a skin problem inhibiting composition and / or a reactive oxygen species inhibiting composition. The MYC preferably contains one or two or more selected from the group consisting of mycosporine-glutaminol-glucoside (MGGnol), mycosporine-glutamicol-glucoside (MGGcol), and mycosporine-glutamine (MGn). The skin problem can be a skin problem caused by ultraviolet irradiation, or a skin problem not caused by ultraviolet irradiation.

Description

Technical Field

[0001] This disclosure relates to compositions containing mycosporins (MYCs), providing new uses for MYCs. Background Technology

[0002] Skin exposed to ultraviolet (UV) radiation can develop various adverse conditions (skin problems) such as inflammation and pigmentation. Therefore, sunscreen cosmetics containing UV absorbers and UV scatterers are widely used.

[0003] Currently, most UV absorbers used in sunscreen cosmetics are synthetic, typically covering both UV-B and UV-A. Concerns exist regarding synthetic UV absorbers, including the potential for photodegradation leading to reactive oxygen species, allergies, itching, or environmental pollution (Non-Patent Literature 1, Non-Patent Literature 2).

[0004] Therefore, natural ultraviolet absorbers are needed, and their development is underway.

[0005] Mycosporine-like amino acids (MAAs) are naturally occurring substances known to efficiently absorb ultraviolet rays such as UV-A and UV-B, and their application as ultraviolet absorbers added to sunscreen cosmetics is anticipated.

[0006] For example, MAAs extracted from red algae (including Porphyra 334 and Shinorine) have been commercialized (Non-Patent Literature 3). However, the production of these MAAs is largely unstable due to factors such as climate, making it difficult to obtain them in large quantities in a stable manner.

[0007] In addition, a method for producing Shinorine through gene recombination technology has been proposed (Patent Document 1), but consumers are sometimes resistant to gene recombination technology, and it has not yet been commercialized.

[0008] Furthermore, MAAs with expected UV absorption capabilities, such as Shinorine, have an absorption wavelength in the UV-A2 range (320–340 nm), but do not cover the UV-B and UV-A1 wavelength regions (340–400 nm), making them insufficient as UV absorbers (Non-Patent Literature 1). In particular, Non-Patent Literature 2 points out that the effectiveness of conventional MAAs as UV absorbers cannot be confirmed. In addition, Porphyran 334 is not heat-resistant (Non-Patent Literature 4), and Shinorine stains during storage (Patent Literature 2), which presents stability problems, thus leading to quality issues during product commercialization.

[0009] Various effects have been reported regarding the functions of MAAs, including, as speculated, antioxidant activity, DNA damage inhibition, lipid peroxidation inhibition, and inhibition of terminal glycation products. However, it is believed that they do not inhibit tyrosinases associated with pigment deposition (Non-Patent Literature 5).

[0010] These effects have been evaluated through cell experiments or experiments using mice, but there are very few examples of evaluation through human trials (Non-Patent Literature 2).

[0011] In addition, various functionalities of mycosporins (MYCs), which are similar to MAAs (and sometimes treated as the same group), have also been studied.

[0012] MYCs are a general term for compounds with a cyclohexenone or cyclohexeneimine skeleton, ultraviolet absorption capabilities, and bio-production capabilities. Bio-production can include production by aquatic organisms such as algae and shellfish, as well as microorganisms such as yeast and molds, or can be obtained from them. Specific examples include: mycosporine-glutaminol-glucoside (MGGnol), mycosporine-5-hydroxy-4-aminovaleric acid-glucoside (MGGcol), mycosporine-glutamine (MGn), mycosporine-glutamic acid (MGa), mycosporine-glycine (MG), and Collemin A.

[0013] Regarding MG and Collemin, various skin-related effects have been reported, such as erythema inhibition, fibroblast proliferation, and skin damage healing. However, the reliability of these effects is low. Furthermore, the beneficial effects caused by ultraviolet absorption have not been confirmed (Non-Patent Literature 6-7, Patent Literature 3). In addition, previously, MYCs and MAAs also lacked methods for mass production or had stability issues (Non-Patent Literature 8).

[0014] Regarding MGGnol, reports indicate that it enhances the survival rate of epidermal cells exposed to UV-B irradiation, promotes fibroblast proliferation, promotes collagen production, inhibits prostaglandin E2 (PGE2) production in epidermal cells, and quenches singlet oxygen (Patent Document 4, Non-Patent Document 9). However, since epidermal cells lack keratin, it cannot be said that its effects when actually applied to human skin have been accurately evaluated.

[0015] Existing technical documents Patent documents Patent Document 1: Japanese Patent No. 5927593 Patent Document 2: Japanese Patent No. 6719267 Patent Document 3: Japanese Patent Application Publication No. 2007-16004 Patent Document 4: Japanese Patent No. 5913988 Non-patent literature Non-patent literature 1: Mori Nao, cosmetology research report 27, 64-67 (2019) Non-patent literature 2: KP Lawrence, et al., Current Medical Chemistry 25, 5512-5527 (2018) Non-patent literature 3: S. Daniel, et al., Cosmetic and Toiletries Manufactureworldwide, 139-143 (2004) Non-patent literature 4: M. Yoshiki et al., Food Chemistry, 113, 1127-1132 (2009) Non-patent literature 5: K. Hakuto et al., Marine Drugs, 14, 222, 120-137 (2019) Non-patent literature 6: A. Torres, et al., Eur. J. Biochem 271, 780-784 (2004) Non-patent literature 7: HJ Suh, et al., Photochem photobiol 78(2),109-113(2003) Non-patent literature 8: S. Ito, et al., Tetrahedron Lett, 28, 2429-2430 (1977) Non-patent literature 9: M. Moline, et al., RADIATION RESEARCH 175, 44-50 (2011) Summary of the Invention

[0016] The technical problem that the invention aims to solve In view of this situation, the object of this disclosure is to provide a composition that, through a stable component that can replace synthetic ultraviolet absorbers and absorbs ultraviolet light over a wide range of wavelengths, has excellent skin problem suppression effects.

[0017] Technical solutions for solving technical problems First, the inventors of this disclosure invented a method for the mass production and efficient extraction of MYCs through the cultivation of black yeast (Japanese Patent Application 2023-2010, Japanese Patent Application 2023-2008, Japanese Patent Application 2023-2009). These inventions paved the way for the industrial production of MYCs, making their commercialization a reality.

[0018] In the course of repeated and in-depth research to solve the aforementioned technical problems, the inventors of this disclosure focused on the industrial production feasibility of MYCs and evaluated the functionality of MYCs using human skin slices and three-dimensional human skin models. The results showed that MYCs can inhibit skin problems caused by various mechanisms, thus becoming an effective ingredient in compositions for inhibiting skin problems. The effects of MYCs discovered by the inventors of this disclosure are described below.

[0019] The inventors of this disclosure have discovered that by absorbing UV-B through MYCs, erythema, inflammation, pigmentation such as melanin and spots, and the generation of reactive oxygen species (ROS) can be inhibited, thereby suppressing oxidative stress. Carbonyl proteins (CPs) are the footprint of oxidative stress. That is, the occurrence of CP generation indicates exposure to oxidative stress. Oxidative stress is associated with wrinkles, spots, and skin aging. These can be prevented by inhibiting oxidative stress. The generation of CP causes yellowing and dryness of the skin, and UV-A generates reactive oxygen species by irradiating CP; therefore, inhibiting CP is of great significance.

[0020] Furthermore, the inventors of this disclosure believe that the skin problem suppression effect brought about by MYCs is due to the effect of MYCs, especially MGGnol and MGGcol, MGn absorbing UV-B. However, they also found that, surprisingly, these MYCs can also suppress oxidative stress and achieve the above-mentioned effect by absorbing one of the reactive oxygen species generated by UV-A, namely singlet oxygen.

[0021] Furthermore, the inventors of this disclosure have also discovered that MYCs have anti-inflammatory and reactive oxygen species removal effects even in the absence of ultraviolet radiation, thus inhibiting skin inflammation and oxidative stress.

[0022] That is, this disclosure includes the following invention. It should be noted that the term "MYCs" may include a single type or multiple types of mycospores.

[0023] [1] A composition for suppressing skin problems, which contains mycosporins (MYCs).

[0024] [2] A composition for inhibiting reactive oxygen species, comprising mycotoxins (MYCs).

[0025] [3] The composition according to [1] or [2], wherein the MYCs comprises one or more of mycosporine-glutaminol-glucoside (MGGnol), mycosporine-5-hydroxy-4-aminovaleric acid-glucoside (MGGcol), and mycosporine-glutamine (MGn).

[0026] [4] The composition according to any one of [1] to [3], wherein the MYCs contain at least MGGnol and MGGcol.

[0027] [5] The composition according to any one of [1] to [3], wherein the composition contains MGGnol, MGGcol and MGn in an aggregate of 0.001% by weight or more of the whole composition.

[0028] [6] The composition according to any one of [1] to [5], wherein the composition is 0.1% based on the total amount of MGGnol, MGGcol, and MGn. m g / cm 2 Apply an amount sufficient for one day to the skin before use.

[0029] [7] According to the composition of [1], wherein the skin problem is selected from one or more of erythema, inflammation, melanosis, spots and phenomena caused by oxidative stress in the skin.

[0030] [8] The composition according to [1], wherein the skin problem is a skin problem caused by ultraviolet radiation or a skin problem not caused by ultraviolet radiation.

[0031] [9] The composition according to any one of [1] to [8], wherein the composition is a topical skin composition.

[0032]

[10] The composition according to [9], wherein the total amount of the composition, MGGnol, MGGcol, and MGn, is 0.1. m g / cm 2 Apply an amount sufficient for one day to the skin before use.

[0033]

[11] The composition according to [9], wherein the composition is used to inhibit erythema and / or inflammation in the skin, wherein the composition is 0.1% of the total amount of MGGnol, MGGcol, and MGn. m g / cm 2 Apply an amount sufficient for one day to the skin before use.

[0034]

[12] The composition according to [9], wherein the composition is used to inhibit melaninization and / or spots in the skin, The composition is 0.5% based on the total amount of MGGnol, MGGcol, and MGn. m g / cm 2 Apply an amount sufficient for one day to the skin before use.

[0035]

[13] The composition according to [9], wherein the composition is used to inhibit phenomena in the skin caused by oxidative stress. The composition is 0.1% based on the total amount of MGGnol, MGGcol, and MGn. m g / cm 2 Apply an amount sufficient for one day to the skin before use.

[0036]

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

[0037] Invention Effects According to this disclosure, a composition is provided that exhibits excellent inhibitory effects on various skin problems caused by ultraviolet (UV) radiation and various skin problems not caused by UV radiation. In particular, it inhibits inflammation and ROS levels in the skin even in a stable state without UV radiation, thus promising not only anti-UV effects but also inhibitory effects on various everyday skin problems. In this respect, this disclosure is significant.

[0038] Furthermore, according to this disclosure, a composition for inhibiting reactive oxygen species (ROS) is provided. Reactive oxygen species are a major cause of oxidative stress, therefore their inhibition is meaningful.

[0039] Furthermore, MYCs such as MGGnol, MGGcol, and MGn are typically water-soluble and will not disappear due to decomposition even under high-temperature conditions, thus allowing for sterilization using an autoclave. Unlike typical synthetic UV absorbers, MYCs do not undergo photodecomposition and therefore do not generate reactive oxygen species, making them a preferred alternative to synthetic UV absorbers. Therefore, they are suitable for incorporation into cosmetic and other topical skin compositions. In particular, because the inclusion of preservatives and synthetic UV absorbers can be controlled within the composition, the needs of users with chemical allergies can be met. Attached Figure Description

[0040] Figure 1 This is a graph showing the intracellular ROS levels of MYCs applied during and without UV-A irradiation in Example 5. Detailed Implementation

[0041] The compositions disclosed herein must contain MYCs as active ingredients. In a preferred embodiment, the MYCs comprise one or more of MGGnol, MGGcol, and MGn; in a more preferred embodiment, the MYCs comprise at least MGGnol and MGGcol.

[0042] MYCs can be purchased commercially or manufactured by microorganisms that produce MYCs through any method.

[0043] As a specific manufacturing method, MYCs can be obtained by culturing microorganisms that produce MYCs and extracting MYCs from these microorganisms.

[0044] The microorganism used as the extraction source can be a single strain or multiple strains.

[0045] There is no particular limitation on the microorganisms that produce MYCs. For example, black yeast, belonging to the family Dothioraceae in the order Dothideales, is an example of a microorganism that produces MGGnol and / or MGGcol. It should be noted that yeasts with black cell color are generally called black yeast. Besides yeasts classified as belonging to the family Dothioraceae in the order Dothideales, black yeast also includes yeasts belonging to the genus *Moniliella* in the phylum Basidiomycota or the genus *Exophiala* in the family Ferpotrichiellaceae in the order Chaetothyriales (de Hoog et al., Atlas of clinical fungi, 2). ndedition: Utrecht: centraalubureauvoor Schimmelcultures; (2000)), the black yeast referred to herein can be a yeast classified as belonging to the family Aureobasidiumceae in the order Aureobasidium. Specifically, it can be a black yeast belonging to the genera Aureobasidium, Hormonema, or Hortaea.

[0046] Here, black yeasts belonging to the genus *Hormonema* can also include black yeasts with a sexual form belonging to the genus *Sydowia*. The asexual form of *Sydowia* corresponds to that of *Hormonema*, and within the same strain, there are differences in whether reproduction is sexual or asexual (NAYurlava et al., *Studies in Mycology*, 43, 63-69 (1999), de Hoog et al., *Atlas of Clinical Fungi*, 2000). nd edtion:Utrecht:centraalubureauvoor Schimmelcultures; (2000)), if strains described as Sydowia are also cultured in liquid, the cell shape sometimes grows to resemble yeast and can be identified as asexual forms of Hormonema (GS de Hoog et al., Antonie van Leeuwenhoek., 65, 41-54 (1994)), therefore both are described in general in this specification.

[0047] Furthermore, although asexual fungi belonging to the genus *Briestros* and asexual fungi belonging to the genus *Hormonema* are closely related, they can be clearly distinguished through molecular systematic analysis (Non-Patent Literature 7).

[0048] As a specific example of a black yeast belonging to the family Microtylosceae in the order Entomomycetes that produces MGGnol and / or MGGcol, there are no particular limitations, and any strain can be used.

[0049] Examples of black yeasts in the genus *Aureobasidium* include *Aureobasidium pullulans*, *Aureobasidium subglaciale*, *Aureobasidium aerium*, *Aureobasidium microstictum*, and *Aureobasidium proteae*. Specific strains include *Aureobasidium pullulans* strains NRBC 6353, NRBC 100716, NRBC 106987, NRBC 4464, NRBC 7757, NRBC 108784, and NRBC 114573 (all of which can be obtained from the National Patent Microbial Collection Center (NPMD) of the National Technical Evaluation Society (NPMD) (Address: Room 122, 2-5-8 Kamisamare, Kisarazu City, Chiba Prefecture, Japan, 292-0818). Alternatively, the IDF-23 strain (NPMD deposited strain, accession number FERMP-22141) described in Patent Document 1 can also be used.

[0050] Examples of black yeasts in the genus *Hormonema* include *Hormonema macrosporum* and *Hormonemadematioides*. Specific strains include, for example, *NRBC* strains 107026, 107027, 107028, 5982, and 32065 (all obtainable from NPMD), which were transferred as the sexual form of *Sydowia polyspora*, corresponding to the asexual form of *Hormonema macrosporum*.

[0051] Regarding black yeasts of the genus Hortaea, examples include Hortaea wirneckii. Specific strains include NRBC 4875 and NRBC 6407 of Hortaea wirneckii (both obtainable from NPMD).

[0052] Furthermore, there are no particular limitations on the microorganisms that produce MGn; fungi belonging to the genus *Hormonema* that produce MGn can be cited. For example, fungi belonging to the genus *Hormonema* include *Hormonema macrosporum* and *Hormonema dematioides*. Specific strains include, for example, *Hormonema macrosporum* strains NSK102 (NITE ABP-03776), NSK33 (NITE ABP-03775), and NSK6 (NITE ABP-03774).

[0053] The NSK102 strain (NITE ABP-03776), NSK33 strain (NITE ABP-03775), and NSK6 strain (NITE ABP-03774) of *Hormonema macrosporum* were novel fungi isolated from nature (Japan Patent Application 2023-2008). These fungi were cultured in liquid, and cell morphology was observed using an optical microscope, revealing yeast-like cells. Furthermore, to investigate the genetic nature of these fungi, the D1 / D2 region base sequence of the 28S rRNA was identified using standard methods. Then, for the 28S rRNA gene base sequences of each *Hormonema macrosporum*, homology was searched using BLAST analysis software ENKIv3.2 (Techno Suruga Laboratory, Japan) in DB-FU15.0 (Techno Suruga Laboratory, Japan) and the international base sequence database (DDBJ / ENA / GenBank) (search date July 29, 2022). In the analysis, the phylogenetic tree was estimated using the neighbor-joining method, with the Kimura-2-parameter as the base substitution model. The reliability of the tree structure was evaluated using a bootstrapping method (1000 iterations). The results showed that all three strains were identified as *Hormonema macrosporum* (asexual) or *Sydowia polysopra* (sexual).

[0054] The NSK102 strain of Hormonema macrosporum was filed for international deposit under the Budapest Treaty at the National Institute for Technical Evaluation of Patent Microorganisms (NPMD) (Postcode: 292-0818, Address: Room 122, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan), with the receiving number NITE ABP-03776 (receiving date: October 20, 2022).

[0055] The NSK33 strain of Hormonema macrosporum was submitted for international deposit under the Budapest Treaty at the National Institute for Technical Evaluation of Patent Microorganisms (NPMD) (Postcode: 292-0818, Address: Room 122, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan), with the receiving number NITE ABP-03775 (receiving date: October 20, 2022).

[0056] The NSK6 strain of Hormonema macrosporum was submitted for international deposit under the Budapest Treaty at the National Institute for Technical Evaluation of Patent Microorganisms (NPMD) (Postcode: 292-0818, Address: Room 122, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan), with the receiving number NITE ABP-03774 (receiving date: October 20, 2022).

[0057] The following is an example of a method for producing MYCs by microorganisms, but the methods for obtaining MYCs are not limited to this.

[0058] In the cultivation process of microorganisms that produce MYCs, the culture medium used need only be able to support the good growth of these microorganisms. A culture medium containing carbon sources, nitrogen sources, inorganic salts, and amino acids suitable for microbial growth as needed can be used. As carbon sources, sugars such as glucose, starch, and molasses, organic acids, and sodium acetate can be used, especially sugars such as glucose. As nitrogen sources, ammonium salts, yeast extract, corn steep liquor, and peptone can be used. As inorganic salts, magnesium salts, calcium salts, phosphates, iron salts, and copper salts can be used. A liquid culture medium prepared by mixing and dissolving these components in water is usually used.

[0059] In addition, the pH can usually be set in the range of 2–9, 3–8, or 3.5–7.5. A pH that is too high or too low may inhibit the growth of microorganisms.

[0060] During the cultivation process, the cultivation temperature only needs to be suitable for the healthy growth of the aforementioned microorganisms, such as 15–40°C, 18–35°C, or 20–30°C. If the cultivation temperature is too low, the growth rate will be insufficient; if the cultivation temperature is too high, abnormalities may occur in the biosynthetic pathway.

[0061] There is no particular time limit for the cultivation process; it can be 72 to 240 hours or 96 to 168 hours.

[0062] In addition, during the cultivation process, light exposure can be provided or not.

[0063] Following the culturing process, an extraction process is performed to extract MYC fractions from the microorganisms that produce MYCs into a solvent.

[0064] Here, during extraction, efficiency is easily achieved by satisfying (a) and / or (b) below. However, if (c) below is satisfied during the cultivation process, efficient extraction can be achieved even if the process satisfying (a) or (b) is not required. That is, one or more of (a), (b), and (c) below can be satisfied.

[0065] (a) In the extraction process, MYCs fractions are extracted in a solvent at a temperature above 40°C.

[0066] (b) Prior to the extraction process, a heating process is performed to recover microorganisms from the culture medium after the culturing process and to heat the recovered microorganisms.

[0067] (c) In the cultivation process, the culture is carried out using a culture medium containing glucose and yeast extract, and / or without light irradiation.

[0068] Microbial cells can be recovered from the culture medium after the culturing process and before the extraction process. Microbial cell recovery can be carried out through methods such as centrifugation and filtration. The recovered microbial cells can be dried using dry heat dryers, freeze dryers, vacuum dryers, or airflow dryers.

[0069] In this manufacturing method, (a) when extracting MYCs fractions in a solvent at a temperature above 40°C, the microorganisms are brought into contact with the extraction solvent.

[0070] As the extraction solvent, polar solvents or polar solvents mainly containing water can be used. Examples of such polar solvents include water, lower alcohols (methanol, ethanol, butanol, isopropanol, butanediol, etc.), and acetone. Examples of polar solvents mainly containing water include water, aqueous solutions of lower alcohols (methanol, ethanol, butanol, isopropanol, butanediol, etc.), or acetone, or water alone. As a solvent "mainly containing water," a solvent containing 80% or more, 85% or more, or 90% or more of water as the total extraction solvent can be used. 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, its concentration can be 20% or less, or no organic solvent can be used at all. Here, "no organic solvent used at all" means that the concentration of the polar organic solvent in the aqueous solution used as the extraction solvent is 1% or less, 0.1% or less, or 0.01% or less. By substantially avoiding the use of polar organic solvents in the extraction process, it is possible to eliminate the need for a step that causes them to evaporate from the obtained extract.

[0071] When extracting MYC fractions into a solvent, the temperature of the extraction solvent should be above 40°C and below 95°C, or above 50°C and below 90°C, or above 60°C and below 85°C. If the extraction solvent temperature is too low, the extraction efficiency will easily decrease. If the extraction solvent temperature is too high, the stability of the desired component may be compromised. Alternatively, an autoclave or similar device can be used to heat the solvent to above 90°C, above 100°C and below 135°C, above 110°C and below 130°C, or above 120°C and below 125°C. Heating can inhibit the activity of unwanted enzymes contained in the cells. If the heating temperature is too low, it may be insufficient to inhibit the activity of unwanted enzymes; if the heating temperature is too high, the stability of the desired component may be compromised. Alternatively, pressure can be applied to above 0.10 MPa, above 0.12 MPa, above 0.15 MPa, or above 0.20 MPa. Applying pressure can easily inhibit the activity of unwanted enzymes contained in the cells. At this point, if the pressure is insufficient, it may be inadequate to inhibit the action of unwanted enzymes contained in the cell. From a safety perspective, the pressure can be set below 1.00 MPa.

[0072] Additionally, extraction times can be set to 15 minutes or more but less than 120 minutes, 60 minutes or more but less than 110 minutes, or 90 minutes or more but less than 100 minutes. Longer extraction times generally result in higher extraction yields. For example, an upper limit of approximately 120 minutes can be set for extraction time. Excessive extraction time can lead to the inclusion of unwanted impurities. During the extraction process, the substance that brings the microorganisms into contact with the extraction solvent can be appropriately stirred or shaken, or it can be allowed to stand.

[0073] After the culturing process, the microorganisms can be retained in the culture medium without recovery, and the culture medium after the culturing process can be directly or appropriately diluted and used for the extraction process, thereby performing (a) extraction of MYC fractions in a solvent above 40°C. In this case, the culture medium can be heated to above 40°C or above 50°C. By preheating the culture medium, efficient extraction can be expected. The culture medium typically contains polar solvents, especially water.

[0074] The culture medium after the culturing process can be diluted to more than twice its volume with water, buffer solution, or culture medium before being used in the extraction process. Diluting the culture medium with water, buffer solution, or culture medium before use in the extraction process improves operability. For example, when the culture medium has high viscosity, diluting it with buffer solution or culture medium before use in the extraction process can sometimes reduce its retention in the apparatus. Alternatively, the culture medium can be used directly in the extraction process. Direct use of the culture medium can help reduce extraction costs.

[0075] In the manufacturing method disclosed herein, a heating step can be performed instead of (a) above, or before the extraction step in (b), to heat the microorganisms recovered from the culture medium after the culturing step. In this step, the microorganisms can be damaged or broken down under certain conditions, thereby improving the extraction efficiency of MYCs, or the action of undesirable enzymes within the microorganisms can be inhibited, thereby increasing the recovery amount of MYCs.

[0076] The heating of microorganisms in the heating process can be carried out under conditions selected from (i) temperature, (ii) time and (iii) pressure.

[0077] (i) The temperature at which the microorganisms are exposed can be set to 40°C or higher and below 200°C, 50°C or higher and below 190°C, 60°C or higher and below 180°C, 70°C or higher and below 170°C, 80°C or higher and below 160°C, 90°C or higher and below 150°C, 100°C or higher and below 140°C, 105°C or higher and below 130°C, 110°C or higher and below 130°C, 115°C or higher and below 130°C, 120°C or higher and below 130°C, or 125°C or higher and below 130°C. In this case, if the temperature of the exposed microorganisms is too low, it may be insufficient to inhibit the action of undesirable enzymes; if the temperature of the exposed microorganisms is too high, the stability of the desired components may be compromised.

[0078] (ii) The heating time for the microorganisms can be set to more than 2 hours but less than 6 hours, more than 3 hours but less than 5 hours, or more than 4 hours but less than 5 hours. The heating time can be the time to reach and maintain any heating temperature. In this case, if the heating time for the microorganisms is too short, it may not be sufficient to inhibit the action of undesirable enzymes; if the heating time for the microorganisms is too long, the stability of the desired components may be compromised.

[0079] (iii) The pressure applied to the microorganism, calculated as the average pressure during the exposure to heating time, can be 0.01 MPa or more and 0.10 MPa or less, 0.02 MPa or more and 0.08 MPa or less, or 0.03 MPa or more and 0.05 MPa or less. In this case, if the pressure applied to the microorganism is too low, it may be insufficient to inhibit the action of undesirable enzymes, and if the pressure applied to the microorganism is too high, the stability of the desired component may be problematic.

[0080] Conditions (i) to (iii) can be arbitrarily selected and combined, and in particular, at least (i) and (ii) can be combined. In this case, sterilization can be performed simultaneously by heating at high temperature for a certain time, thereby inactivating the enzymes in the microorganisms and expecting a further increase in the recovery of MYCs. In addition, through (iii), the microorganisms are broken down, thus further improving the extraction efficiency of MYCs. It should be noted that the heating process can also be performed under conditions where the microorganisms are dry. For example, the heating process can 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 more, and (iii) a pressure of 0.10 MPa or less, or under conditions where (i) a temperature of 105°C or higher, (ii) a heating time of 2 hours or more, and (iii) a pressure of 0.10 MPa or less.

[0081] In the manufacturing method disclosed herein, when using microorganisms after the heating step (b) for the extraction step, the process can be carried out under the conditions described in (a) or at a low extraction temperature.

[0082] Specifically, the extraction solvent can be the same as the solvent described in (a). The temperature of the extraction solvent used to extract the MYCs fraction can be above 5°C, above 10°C, or above 25°C (room temperature). Alternatively, it can be the conditions described in (a), i.e., above 40°C and below 70°C, above 50°C and below 90°C, or above 60°C and below 100°C. It can also be heated to above 90°C and below 150°C, above 100°C and below 140°C, above 110°C and below 130°C, or above 120°C and below 125°C using an autoclave or similar device.

[0083] In addition, the extraction time can be 15 minutes or more, 60 minutes or more, or 90 minutes or more. The longer the extraction time, the easier it is to increase the extraction yield. For example, the upper limit of the extraction time can be set to about 120 minutes. During the extraction process, the substance obtained by contacting the microorganisms with the extraction solvent can be appropriately stirred, either by shaking or by letting it stand.

[0084] Thus, by exposing the microorganisms and / or the culture medium containing them to high temperatures during or before the extraction process, the microorganisms can be killed and / or the enzymes that decompose MYCs within the microorganisms can be inactivated, thereby increasing the recovery rate of MYCs. The high temperature conditions for exposing the culture medium can be, for example, 40°C or 50°C or higher.

[0085] Therefore, the survival rate of the aforementioned microorganisms after the extraction process can be above 0% and below 20%, above 0% and below 10%, or above 0% and below 5% after the culture process. It should be noted that as long as the survival rate after the extraction process falls within the above range, it is acceptable. However, if the survival rate of microorganisms is also low before or during the extraction process, it is possible to inhibit the decomposition of MYCs and expect to increase the recovery rate.

[0086] After the extraction process, the obtained extract can be subjected to impurity removal, grading, and purification using known methods.

[0087] The composition disclosed herein preferably contains MGGnol, MGGcol, and MGn in an aggregate of 0.001% by weight or more of the total composition, more preferably 0.01% by weight or more, and even more preferably 0.05% by weight or more.

[0088] Furthermore, the composition disclosed herein more preferably contains 0.0001% by weight or more of MGGnol as a whole composition, more preferably 0.001% by weight or more, and even more preferably 0.005% by weight or more.

[0089] In addition, in the total amount of MYCs contained in the composition disclosed herein, MGGnol is preferably 10% by weight or more, more preferably 20% by weight or more, and even more preferably 30% by weight or more.

[0090] By setting the content of MYCs within the above range, the desired effect can be easily obtained, and the freedom of formulation design can be ensured.

[0091] It should be noted that the above content can be adjusted appropriately according to the route of administration and the composition contained herein.

[0092] It should be noted that the content of MYCs can be determined by conventional methods.

[0093] Typically, MYCs possess ultraviolet absorption capabilities due to their structure with cyclohexanone as the central ring. Therefore, the confirmation of the presence of MYCs in a composition and the determination of the amount and concentration of MYCs in the composition can be performed using the wavelength of light in the ultraviolet region. For example, this can be done by measuring the ultraviolet absorption of MYCs in the composition using a spectrophotometer (Japanese Patent No. 5913988, Buscot et al., Mycol. Res, 95(6):752-754(1991)). Regarding the amount of MGGnol, the absorbance (ABS) at the maximum absorption wavelength of 310 nm can be measured, based on a molar absorptivity of 25000 M. -1 cm -1(Calculated using Japanese Patent No. 5913988). Regarding MGGcol, the absorbance at the maximum absorption wavelength of 310 nm can be measured, based on a molar absorptivity of 25000 M. -1 cm -1 (N. Wada et al., Antioxidants, 4, 603-646 (2015)) The molar absorptivity was calculated using the molar absorptivity of MGn, which is approximately the same molecular weight and has a similar structure as spore-forming glutamic acid (MGa), at 20900 nm. -1 cm -1 (N. Wada et al., Antioxidants, 4, 603-646 (2015)) was calculated as a substitute.

[0094] The compositions disclosed herein may also contain MYCs other than those described above, 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 bonded to the aforementioned central ring.

[0095] More specifically, examples include: Mycosporine-3,5-dihydroxy-4-aminovaleric acid, Gadusol, deoxygadusol, Mycosporine-glycine, Mycosporine-taurine, Mycosporine-alanine, Mycosporine-serine, Mycosporine-serinol, Mycosporine-2-glycine, Palythinol, and Palythenic. 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, etc.

[0096] The compositions disclosed herein can inhibit skin problems. In this disclosure, skin problems refer to erythema, inflammation, hyperpigmentation, spots, and phenomena caused by oxidative stress in the skin.

[0097] One of the main reasons for this effect is that MYCs have the ability to absorb a wide range of wavelengths of light, including mid-wavelength ultraviolet (UV-B: 280–320 nm) and long-wavelength ultraviolet (UV-A: 320–400 nm).

[0098] By absorbing UV-B through MYCs, erythema and inflammation can be suppressed, as well as pigmentation such as melanin and spots. It can also inhibit the generation of reactive oxygen species (ROS), thereby suppressing oxidative stress. Carbonyl proteins (CPs) are the footprint of oxidative stress; that is, CP generation indicates exposure to oxidative stress. Oxidative stress is associated with wrinkles, spots, and skin aging; therefore, these can be prevented by inhibiting oxidative stress. CP generation causes yellowing and dryness of the skin, and UV-A generates ROS through CP irradiation; therefore, inhibiting CP is of great significance.

[0099] Furthermore, the inventors of this disclosure believe that the skin problem suppression effect brought about by MYCs is due to the effect of MYCs, especially MGGnol and MGGcol, MGn absorbing UV-B. However, it was also found that, surprisingly, these MYCs can also suppress oxidative stress and achieve the above-mentioned effect by absorbing one of the reactive oxygen species generated by UV-A, namely singlet oxygen.

[0100] In addition, one of the main reasons for the skin-promoting effect of the compositions disclosed herein is that MGG and MGn, as MYCs, absorb singlet oxygen generated by UV-A, thereby inhibiting oxidative stress.

[0101] Skin problems caused by UV-A are more persistent and cause longer-lasting damage than those caused by UV-B, leading to slower skin aging. Therefore, effective countermeasures have been difficult to implement until now, but this disclosure enables their elimination.

[0102] Furthermore, regarding the skin problem-inhibiting effect of the composition disclosed herein, even in the absence of ultraviolet radiation (i.e., regardless of the ultraviolet absorption capacity of MYCs), the main reason for the inhibition of skin inflammation and oxidative stress is that MYCs have anti-inflammatory and reactive oxygen species removal effects.

[0103] As can be seen from the above, the composition disclosed herein can inhibit skin problems caused by ultraviolet radiation as well as skin problems that do not occur through ultraviolet radiation.

[0104] Therefore, MYCs, especially one or more selected from MGGnol, MGGcol, and MGn, can be effective ingredients in compositions for inhibiting skin problems. Here, skin problems refer to erythema, inflammation, hyperpigmentation, spots, and phenomena caused by oxidative stress in the skin.

[0105] It should be noted that, in the embodiments described later, it was shown that MYCs inhibited the cytokine IL-1, an inflammatory marker, in both the presence and absence of UV-A and UV-B irradiation. αProduction levels, specifically PGE2 production levels. It should be noted that PGE2 reflects inflammation caused by oxidative stress such as ultraviolet radiation, and also participates in erythema formation along with nitric oxide free radicals; specifically, it reddens the skin through vasodilation. Therefore, this example demonstrates the inhibitory and anti-inflammatory effects on erythema.

[0106] Furthermore, in the embodiments described later, it is shown that CP levels were suppressed by MYCs in a three-dimensional skin model irradiated with UV-B. Therefore, this embodiment confirms the inhibitory effect of oxidative stress.

[0107] Furthermore, in the embodiments described later, it was shown that MYCs suppressed melanin spots in a skin model irradiated with UV-B, confirming the effect.

[0108] Furthermore, in the embodiments described later, it was shown that ROS levels were suppressed in both the three-dimensional skin models and cells in which MYCs were applied, regardless of the presence or absence of UV-A and UV-B irradiation. This indicates that ROS production was suppressed and the generated ROS was eliminated (removed) by MYCs.

[0109] The inventors of this invention have discovered for the first time that MYCs can suppress skin problems regardless of whether there is UV exposure. Previously, it was speculated that MYCs and MAAs possessed UV absorption capabilities based on their chemical structure and could protect the skin by defending against UV rays. However, the ability to suppress various skin problems even without UV exposure was something that could not have been conceived from existing technology.

[0110] In this specification, "inhibition" of skin problems can include preventing subsequent skin problems and preventing the worsening of existing skin problems. "Preventing the worsening of existing skin problems" can include preventing the severity of skin problems from increasing and delaying the increase in the severity of skin problems.

[0111] In addition, as mentioned above, one or more of MGGnol, MGGcol and MGn can suppress ROS levels, and therefore can also be effective components of compositions for suppressing reactive oxygen species.

[0112] In this specification, “inhibition” of reactive oxygen species can include a reduction in the amount generated and the elimination (removal) of the generated ROS.

[0113] The compositions disclosed herein may be used for therapeutic purposes or for non-therapeutic purposes.

[0114] "Non-therapeutic purposes" refers to actions that do not involve medical procedures, that is, actions that do not involve the treatment of the human body. Examples include health enhancement and cosmetic procedures.

[0115] When the compositions of this disclosure are used for non-therapeutic purposes, they are administered to healthy subjects. When the compositions of this disclosure are used for non-therapeutic purposes, they can be used to inhibit skin problems and / or suppress reactive oxygen species in healthy individuals. Additionally, when the compositions of this disclosure are used for non-therapeutic purposes, they can be used to prevent diseases and symptoms caused by exposure to ultraviolet radiation, such as inflammation, erythema, skin melaninization, pigmentation, and oxidative stress.

[0116] When the compositions of this disclosure are used for therapeutic purposes, they can be administered to unhealthy individuals. These unhealthy individuals are those with diseases or symptoms caused by exposure to ultraviolet radiation, such as inflammation, erythema, skin melaninization, spot formation, and oxidative stress, and the compositions can be used to suppress or alleviate these diseases or symptoms.

[0117] As diseases and symptoms caused by exposure to ultraviolet (UV) radiation, such as inflammation, erythema, skin melaninization, spot formation, and oxidative stress, examples include skin cancer, systemic lupus erythematosus, and autoimmune diseases such as Sjögren syndrome (see Non-Patent Literature 2, T. Ogura et al., Jpn. J. Clin. Immunol., 37(1), 25-32 (2014), and Hamasaki Yoichiro, Nichirin Skin Journal, 33(4), 483-491 (2016)). It is believed that skin cancer is caused by oxidative stress from long-term UV exposure, resulting in decreased immunity and so-called photoaging. Specifically, examples include actinic keratosis, acanthosis nigra, basal cell carcinoma, and melanoma. Autoimmune diseases are multifactorial, with a complex relationship between genetic and environmental factors. As an environmental factor, excessive oxidative stress caused by exposure to ultraviolet radiation can lead to inflammation, tissue damage induced by apoptosis, and the initiation of autoimmune responses, resulting in an autoimmune disorder. Systemic lupus erythematosus and Sjögren's syndrome are known to be major autoimmune diseases. Therefore, avoiding adverse effects caused by ultraviolet radiation exposure is crucial for preventing these diseases.

[0118] Another aspect of the present invention is the use of MYCs in the manufacture of compositions for inhibiting skin problems and / or compositions for inhibiting reactive oxygen species.

[0119] Another aspect of the invention is the use of MYCs in inhibiting skin problems and / or inhibiting reactive oxygen species.

[0120] Another aspect of the present invention is MYCs for inhibiting skin problems and / or inhibiting reactive oxygen species.

[0121] Another aspect of the present invention is a method for inhibiting skin problems and / or inhibiting reactive oxygen species, which includes administering (ingesting) MYCs to animals.

[0122] The timing of administration (uptake) of the compositions disclosed herein is not particularly limited and can be appropriately selected according to the condition of the recipient.

[0123] The dosage of the composition disclosed herein can be appropriately selected based on the age, sex, condition, and other conditions of the recipient.

[0124] Regarding the dosage (uptake) of the compositions disclosed herein, there is no particular limitation on the dosage (uptake) of MYCs. Considering the desired efficacy and safety, for example, in the case of transdermal administration as a topical skin composition described later, in adults, the dosage per unit skin surface area, for example, based on the total amount of MGGnol, MGGcol, and MGN, is preferably 0.1. m g / cm 2 For days or more, 0.2 is preferred. m g / cm 2 More than 1 day, 0.4 m g / cm 2 More than 1 day, 0.8 m g / cm 2 More than 1 day, 1.6 m g / cm 2 More than 3.2 days m g / cm 2 More than 1 day, 6.4 m g / cm 2 More than 10 days. In addition, there is no specific upper limit to the dosage.

[0125] Additionally, the dosage can be adjusted appropriately based on the skin condition of the target individual. For example, in cases where the aim is to suppress erythema and / or inflammation in the skin, the total amount of MGGnol, MGGcol, and MGn can be set to 0.1. m g / cm 2 More than 10 days. When the purpose is to inhibit melaninization and / or spots in the skin, the total amount of MGGnol, MGGcol, and MGn can be set to 0.5. m g / cm 2 More than 1 day. Furthermore, when the aim is to suppress oxidative stress-induced phenomena in the skin, the total amount of MGGnol, MGGcol, and MGn can be set to 0.1. m g / cm 2 More than one day.

[0126] It should be noted that, regardless of the amount or duration of administration, the compositions disclosed herein can be administered once daily or in multiple divided doses. Furthermore, in addition to single administration, administration can be performed continuously or intermittently for periods of several weeks or months.

[0127] There are no particular limitations on the recipients (ingestors) of the compositions disclosed herein, and mammals may be cited as examples. Examples of mammals include humans, dogs, and cats. Humans are particularly noteworthy. Humans may be infants, toddlers, children, adults, middle-aged and elderly individuals, or the elderly of any age. Furthermore, there are no particular limitations on gender. Additionally, the UV-B absorption capacity of the aforementioned MYCs is effective as long as the MYCs are present on the cell surface after administration; therefore, there are no particular limitations on the skin condition or skin type of the recipient.

[0128] The routes of administration (uptake) for the compositions disclosed herein are transdermal, oral, nasal, inhalation, intravenous injection, etc., without particular limitation, but transdermal is generally preferred. It should be noted that "administration" here can be replaced with "uptake".

[0129] When administration is carried out via transdermal delivery (ingestion), it is preferable to formulate a topical skin composition.

[0130] As a topical skin composition, there is no particular limitation as long as it is applied to the skin topically. Examples of preferred formulations include cosmetics, quasi-pharmaceuticals, and pharmaceuticals, with cosmetics being more preferred, and sunscreen cosmetics being particularly preferred. It should be noted that when the composition disclosed herein is a cosmetic, it is used for the aforementioned non-therapeutic purposes; when the composition disclosed herein is a quasi-pharmaceutical or pharmaceutical, it is used for the aforementioned therapeutic purposes.

[0131] There are no particular limitations on the application site of the topical composition to the skin, but it is usually applied to the face, limbs, neck, and the area from the neck to the chest (decollete).

[0132] Examples of dosage forms for topical skin components include lotions, emulsions such as lotions and creams, oils, gels, masks, and cleansers, without any particular limitation.

[0133] In addition, as a topical skin composition, it can be either a no-rinse type or a wash-off type.

[0134] When the compositions disclosed herein are formulated into topical skin compositions, during their manufacture, in addition to MYCs, any ingredients commonly used in the formulation of cosmetics, quasi-medicines, pharmaceuticals, etc., may be added.

[0135] As an arbitrary component, it can be combined with hydrocarbons such as squalane, petrolatum, and microcrystalline wax; esters such as jojoba oil, carnauba wax, and octyl dodecyl oleate; triglycerides such as olive oil, tallow, and coconut oil; fatty acids such as stearic acid, oleic acid, and retinoic acid; higher alcohols such as oleyl alcohol, stearyl alcohol, and octyl dodecyl alcohol; anionic surfactants such as sulfosuccinate and sodium polyoxyethylene alkyl sulfate; amphoteric surfactants such as alkyl betaine salts; cationic surfactants such as dialkylammonium salts; nonionic surfactants such as sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, polyoxyethylene alkyl ethers, and polyoxyethylene fatty acid esters; polyols such as polyethylene glycol, glycerol, and 1,3-butanediol; thickeners and gelling agents; antioxidants; ultraviolet scattering agents; dyes; preservatives; and powders.

[0136] Unless otherwise defined, all technical and scientific terms used in this specification shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All methods and materials similar to or equivalent to those described in this specification may also be used in the implementation or testing of this disclosure, and representative illustrative methods and materials are described herein.

[0137] When a range of values ​​is provided, it should be understood that each value between the upper and lower limits of that range (unless the context explicitly indicates otherwise, up to one-tenth of the unit of the lower limit) and any other recorded value within or within that recorded range are included in the scope of the invention disclosed herein. The upper and lower limits of these smaller ranges can be independently included within smaller ranges, which are also included in the scope of the invention disclosed herein, becoming the object of any specifically excluded boundary within the recorded range. Where a recorded range includes one or both of the boundaries, the range excluding any or both of the boundaries it includes is also included in the invention disclosed herein.

[0138] This disclosure is not limited to the specific manner described. The scope of the invention shown in this disclosure is considered to be defined only by the appended patent claims, and therefore it should be understood that the terminology used in this specification is merely for describing specific manners and not for limitation.

[0139] As will be apparent to those skilled in the art from reading this disclosure, each of the various methods described herein, without departing from the scope and spirit of the invention, has individual constituent elements and features that are readily separable from or readily combined with any feature of the other various methods. Any method cited can be implemented in the order of the cited events, or in any other logically possible and non-contradictory order.

[0140] All publications and patents referenced in this specification are referenced hereby by reference in a specific and separate manner, referring to each individual publication or patent by reference. Methods and / or materials referenced in a publication for the purpose of disclosure and description thereof are also referenced hereby by reference. Any reference to a publication that was published prior to the filing date should not be construed as an admission of no right to any publication prior to the referenced date. Furthermore, the publication date provided may differ from the current publication date and may require separate verification.

[0141] The following embodiments are provided for illustrative purposes and various methods of this disclosure are given, and are not intended to limit the disclosure in any way. Those skilled in the art will readily understand that they are well suited for carrying out the purposes mentioned in this disclosure, obtaining the mentioned end goals and advantages, and carrying out the purposes, end goals and advantages originally present in this specification. These embodiments, together with the methods described in this specification, represent current methods and are illustrative, not intended to limit the scope of this disclosure. Those skilled in the art will conceive of variations and other uses included within the spirit of this disclosure as defined in the patent claims.

[0142] Example The invention described below is based on embodiments, but the invention is not limited to these embodiments. It should be noted that, unless otherwise specified, units are based on weight.

[0143] <Reference Example> Preparation of MYCs Samples Using the methods described above, microorganisms (Budding Short-stalked Pleurotus and / or Hormonema macrosporum) that produce MGGnol, MGGcol, and / or MGn are cultured, and MYCs are extracted from the cultured cells.

[0144] The concentrations of each MYC in the extract were determined by HPLC. Specifically, an HPLC system from Shimadzu Corporation was used, employing a LiChrospher (registered trademark) 100 RP-18 (5) column with a protective column. m m)LiChroCART (registered trademark) 4-4 Lichrospher (registered trademark) 100 RP-18 column (particle size 5) m (m, inner diameter 4mm × length 250mm, manufactured by Merck), mobile phase (aqueous solution of 0.5% methanol and 0.2% acetic acid), flow rate 0.7mL / min, column oven temperature 35℃, injection volume 10 mAn L-type PDA detector was used to measure wavelengths from 250 nm to 400 nm. A peak representing the maximum absorption wavelength was identified near 310 nm and was taken as the peak of the MAAs. Quantification was performed at the detection wavelength of 310 nm.

[0145] Preparation: Three MYCs samples were obtained, and the composition of MGGnol, MGGcol and MGn contained in them is shown in Table 1.

[0146] [Table 1]

[0147] <Example 1> Study on anti-inflammatory and erythema-inhibiting effects (1) Preliminary study on the cultivation method and ultraviolet irradiation of the three-dimensional skin model To evaluate the response of a three-dimensional skin model to UV-B, an irradiation dose that would not cause cell death was investigated. Furthermore, results were compared with those obtained using skin cells obtained using existing techniques (Patent Document 4).

[0148] MYCs solutions were prepared by adjusting the MGG-E sample with PBS(-) to achieve MYCs concentrations of 0.07, 0.14, and 0.35% by weight.

[0149] The three-dimensional skin model was created using a LabCyte EPI-MODEL24 from Japan Tissue Engineering Co., Ltd. Using the included assay medium, the skin was acclimated for 24 hours in 24-well plates at 37°C, CO2, and 5%. Ultraviolet irradiation was performed using a UV lamp (TL 20W / 12 RS, manufactured by PHILIPS Lighting). The irradiation intensity was measured using a UVX Radiometer from Analytik Jena. The irradiation dose was expressed as 400 mJ / cm². 2 600mJ / cm 2 Adjust the irradiation time in this way.

[0150] After acclimatization, apply PBS(-) or MYCs solution 30g from the stratum corneum side of the LabCyte EPI-MODEL24. m L was applied to a three-dimensional skin model in one well and cultured for 24 hours at 37°C, CO2, and 5%. Then, it was used to achieve a concentration of 400 mJ / cm². 2 Or 600mJ / cm 2 Irradiate with UV-B in this manner. After 24 hours of irradiation, use 500... mCells were washed with PBS (-) to remove PBS (-) or MYCs solution from the stratum corneum, and cell viability was determined using the AlamarBlue Assay. It should be noted that MYCs 0.07% solution, 30 m The MYCs value in L is 2.1. m g, the area of ​​the pore is 0.32 cm². 2 Therefore, the application volume of MYCs is 6.6. m g / cm 2 .

[0151] At 400 mJ / cm 2 600mJ / cm 2 At any irradiation level, the cells survived in the presence of MYCs.

[0152] (2) Studies on anti-inflammatory and erythema-inhibiting effects IL-1 α Normally contained within cells, it easily leaks out through stimulation such as ultraviolet radiation. Because it can be detected rapidly and in large quantities, it can be widely evaluated as an inflammatory mediator (T. Hirano, et al., Dermatol 106, 1102-1107 (1996)). Furthermore, as mentioned above, PGE2 is associated with erythema formation and is an indicator of inflammation induced by oxidative stress such as ultraviolet radiation (Non-Patent Literature 2).

[0153] As described above, a three-dimensional skin model was prepared. As studied in (1), even at 600 mJ / cm²... 2 Even with higher irradiation levels, cell survival did not decrease; therefore, 600 mJ / cm² light irradiation, which has a greater impact on cell viability, was chosen. 2 Experiments were conducted. The amount of PGE2 and IL-1 effluxed into the culture medium 24 hours after irradiation was measured using the Prostaglandin E2, EIA Monoclonal Kit (manufactured by Cayman). α The levels were measured using the Human IL-1 alpha / IL-1F1 Quantikine ELISA Kit (manufactured by R&D Company).

[0154] Table 2 shows the IL-1 levels for each three-dimensional skin model in one well. α And the amount of PGE2 produced.

[0155] Through 600mJ / cm 2 Irradiation, IL-1 α The concentration of PGE2 increased from 6 pg / 3D to 11 pg / 3D. Similarly, PGE2 increased from 25 pg / 3D to 47 pg / 3D. This indicates that at 600 mJ / cm²...2 IL-1 under the condition α Both PGE2 and PGE2 increased to approximately 2 times. The 0.07% MYC solution and 0.14% MYCs solution prepared from sample MGG-E in (1) were applied from the stratum corneum side into well 1 for 30 minutes. m L, resulting in IL-1 α PGE2 decreases depending on MYC concentration.

[0156] When 0.14% MYCs are added, IL-1 α The results were lower than the control, confirming that it has an anti-inflammatory effect regardless of the amount of UV-B irradiation.

[0157] [Table 2]

[0158] <Example 2> Study on the inhibitory effect on skin melaninization A 39-year-old Caucasian woman with a BMI of 28 had her skin thinly sliced ​​into pieces 14 mm in diameter (1.5 cm² surface area). 2 Thickness 400±100 m m, for experimental use. Skin was placed in an intercell, positioned in a well containing culture medium, and cultured for 24 hours at 37°C and 5% CO2.

[0159] MGG-K was adjusted to a MYCs concentration of 0.7% using PBS(-) to prepare the MYCs solution. The application amount of the MYCs solution, calculated as 22.7 g / unit skin surface area, was determined by the amount of MYCs. m g / cm 2 .

[0160] The experimental zones were set as follows: control (no irradiation throughout the entire period), experimental zone 4: UV-B irradiation (irradiation throughout the entire period), experimental zone 5: MYCs added before and after UV-B irradiation, experimental zone 6: added during UV-B irradiation, and experimental zone 7: added before, during, and after UV-B irradiation (throughout the entire period) (refer to Table 3). The number of samples in each experimental zone was set to 3.

[0161] Place a silicone pad on the skin and add 50 μL of PBS(-) or MYCs solution. m L. After 24 hours, remove the PBS(-) or MYCs solution and wash three times with 1 mL of PBS(-). Remove any residual PBS(-) between units and on the skin surface. Add fresh PBS(-) or MYCs solution to the silicone pad and transfer the skin to the PBS(-) plate. Then, irradiate with a UV lamp (TL 20W / 12 RS, manufactured by PHILIPS Lighting) at 300 mJ / cm². 2UV-B irradiation was performed. After irradiation, the PBS(-) or MYCs solution was removed. Then, the cells were placed back into plates containing culture medium for incubation. The same procedure was repeated for two days, for a total of three UV-B irradiations. After the final irradiation, the cells were incubated while visually confirming the melanization of the skin. 72 hours after the third irradiation, the skin was retrieved from the cell and photographed using a colorimetric chart (Casmatch; manufactured by Bear Medic).

[0162] Referring to the brightness index by Gerg. G. Hillebrand et al. The method for evaluating skin darkening (Gerg. G. Hillebrand, et al., J. Dermatol. Sci. 27, S42-S52 (2001)) involves image correction of the captured images according to Casmatch's instructions, and calculation using Photoshop. Value. Calculate the area within a certain range of the three samples in each test area. The average value.

[0163] [Table 3]

[0164] The results are shown in Table 4.

[0165] As an indicator of brightness A decrease in the number of 'a' indicates skin darkening.

[0166] Test areas 4 and 5 were irradiated with UV-B, and the brightness ( The value decreased by approximately 10%, showing a significant difference. That is, the skin darkened.

[0167] On the other hand, compared with the control, test areas 6 and 7 The value decreased by about 2%, but the difference was not statistically significant. This indicates that adding 50% [unclear - possibly a specific substance or ingredient] to the skin during irradiation... m A 0.7% MYCs solution inhibited skin melaninization. No melaninization inhibition effect was observed after MYCs addition prior to the first UV-B irradiation, during cleansing, after irradiation, and during MYCs addition again. This suggests that the presence of MYCs in the skin during UV irradiation is necessary for melaninization inhibition.

[0168] [Table 4]

[0169] <Example 3> Study on the inhibitory effect of reactive oxygen species on skin damage The evaluation was conducted with reference to the methods of Katsuyama et al. (Katsuyama, et al., Journal of the Japan Society of Fragrance and Cosmetics, 39, 89-94 (2015)).

[0170] The three-dimensional skin model was created using the LabCyte EPI-MODEL24 from Japan Tissue Engineering Co., Ltd. Using the included assay medium, the samples were acclimated for 24 hours in 24-well plates at 37°C, CO2, and 5%. After 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) were applied to each well from the stratum corneum side of the LabCyte EPI-MODEL24. m L was cultured at 37℃, CO2, and 5% for 24 hours. After 24 hours, the PBS(-) or MYCs solution on the stratum corneum was removed, and the cells were washed with PBS(-). Freshly prepared PBS(-) or MYCs solution was applied from the stratum corneum side for 30 minutes. m After L, irradiation with a UV-B lamp (TL 20W / 12RS) (PHILIPS Lighting) at 600mJ / cm² was performed. 2 UV-B. The number of samples in each test area was set to 4.

[0171] Following UV-B irradiation, PBS(-), MGG-K, or MGG-EX solutions were removed from the stratum corneum, and the stratum corneum surface was washed with PBS(-). ROS levels in the epidermal tissue were evaluated using chemiluminescence detection with 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazole[1,2-a]pyrazin-3-one hydrochloride (MCLA) (Tokyo Kasei Corporation). The detector used was NightOWL (registered trademark) (manufactured by Berthold Technologies). Image processing was performed using ImageJ.

[0172] The results are shown in Table 5. Higher gray values ​​indicate higher ROS levels. As reported in many studies, UV-B irradiation increased ROS levels by 4%. Adding MYCs reduced ROS levels by approximately 10% compared to UV-B irradiation. Surprisingly, the addition of MYCs also reduced ROS levels in the control group—the amount of ROS frequently generated in organismal responses even without UV-B irradiation.

[0173] [Table 5]

[0174] <Example 4> Study on the inhibitory effect of carbonyl proteins generated by exposure to ultraviolet light The three-dimensional skin model was constructed using the LabCyte EPI-MODEL24 from Japan Tissue Engineering Co., Ltd. Using the included assay medium, the samples were acclimated for 24 hours in 24-well plates at 37°C, CO2, and 5%. After acclimation, 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) were applied to one well from the stratum corneum side of the LabCyte EPI-MODEL24. m L was cultured at 37℃, CO2, and 5% for 24 hours. After 24 hours, the PBS(-) or MYCs solution on the stratum corneum was removed, and the cells were washed with PBS(-). Freshly prepared PBS(-) or MYCs solution was applied from the stratum corneum side for 30 minutes. m After L, irradiation with a UV-B lamp (TL 20W / 12 RS) (PHILIPS Lighting) at 600mJ / cm² was performed. 2 UV-B. The number of samples in each test area was set to 4.

[0175] After UV-B irradiation, remove the PBS(-) or MYCs solution from the stratum corneum, and wash the stratum corneum surface with PBS(-). Apply freshly prepared PBS(-) or MYCs solution from the stratum corneum side for 30 minutes. m After L, incubate for 24 hours. After 24 hours, remove PBS(-) or MGG-K or MGG-EX solution from the stratum corneum and wash the stratum corneum surface with PBS(-).

[0176] The epidermal tissue of the three-dimensional cultured skin model was removed from the culture cup, and frozen blocks were made using OCT Compound (manufactured by Sakura Finetek Japan Co., Ltd.), and thin sections were prepared.

[0177] Carbonyl proteins (CP) in thin sections were detected using fluorescein-5-aminothiourea (manufactured by Invitrogen) (see T. Mizutani, et al., Journal of the Japanese Society of Perfume and Cosmetology, 41, 101-105 (2017)). CP levels were then numerically analyzed using fluorescence image analysis. Corneocytemetry-2 (manufactured by CIEL Co., Ltd.) was used for image analysis.

[0178] The relative amounts of CP compared to the control are shown in Table 6. CP increased 1.27-fold with UV-B irradiation. The increase in CP was inhibited by the addition of MYCs.

[0179] [Table 6]

[0180] <Example 5> Study on intracellular ROS levels under UV-A irradiation or non-irradiation conditions Cells were normal human epidermal keratinocytes (manufactured by Kurabo). They were cultured using standard culture medium (HuMedia-KG2). UV irradiation was performed using a UV-A lamp (FL20SBLB 20W, manufactured by TOSHIBA). A MYCs solution prepared by diluting the 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 along with riboflavin. Irradiation was performed at 6 J / cm² under the coexistence of MYCs and riboflavin. 2 UV-A radiation was used, and intracellular ROS levels immediately after irradiation were detected using a cell-permeable redox-sensitive fluorescent probe, DCFDA (2,7-dichlorofluorescein diacetate). Additionally, cellular protein levels were quantified using the BCA Protein Assay Kit (Thermo Scientific). Three samples were used in each experimental area.

[0181] The results are shown in Figure 1 Tables 7 (without UV-A irradiation) and 8 (without UV-A irradiation) show the results. Protein mass was used as an indicator of cell number. As shown in the tables, no changes in protein mass were identified due to MYC application and UV-A irradiation. Intracellular ROS production is shown in the absence of UV-A irradiation. ROS decreased with increasing MYC concentration. Even at a low concentration of 0.006% MYC, ROS was significantly reduced, and at 0.05% MYC, ROS was reduced to half that of the control.

[0182] Furthermore, even in the control group, ROS increased 1.5-fold under UV-A irradiation. UV-A irradiation significantly reduced ROS at a low concentration of 0.003% MYCs compared to the unirradiated group. ROS decreased with increasing MYC concentration. At 0.05% MYCs, ROS decreased to approximately one-third of that in the UV-A irradiated control. With the application of MYCs, intracellular ROS decreased in a concentration-dependent manner, independent of UV-A irradiation.

[0183] [Table 7]

[0184] [Table 8] PCT / RO / 134 form

Claims

1. A composition for inhibiting skin problems, characterized in that, It contains mycotoxins, namely MYCs.

2. A composition for inhibiting reactive oxygen species, characterized in that, It contains mycotoxins, namely MYCs.

3. The composition according to claim 1 or 2, wherein, The MYCs contain one or more of the following: mycosporine-glutaminol-glucoside (MGGnol), mycosporine-5-hydroxy-4-aminovaleric acid-glucoside (MGGcol), and mycosporine-glutamine (MGn).

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

5. The composition according to claim 3, wherein, The composition contains MGGnol, MGGcol and MGn in a total weight of more than 0.001% of the whole composition.

6. The composition according to claim 3, wherein, The composition is 0.1% based on the total amount of MGGnol, MGGcol, and MGn. μ g / cm 2 Apply an amount sufficient for one day to the skin before use.

7. The composition according to claim 1, wherein, The skin problem is selected from one or more of the following: erythema, inflammation, darkening / spots, and phenomena caused by oxidative stress.

8. The composition according to claim 1, wherein, The skin problems mentioned are those caused by ultraviolet radiation or those not caused by ultraviolet radiation.

9. The composition according to claim 1 or 2, wherein, The composition is a topical skin composition.

10. The composition according to claim 9, wherein, The composition is 0.1% based on the total amount of MGGnol, MGGcol, and MGn. μ g / cm 2 Apply an amount sufficient for one day to the skin before use.

11. The composition according to claim 9, wherein, The composition is used to inhibit erythema and / or inflammation in the skin. The composition is 0.1% based on the total amount of MGGnol, MGGcol, and MGn. μ g / cm 2 Apply an amount sufficient for one day to the skin before use.

12. The composition according to claim 9, wherein, The composition is used to inhibit melaninization and / or spots in the skin. The composition is 0.5% based on the total amount of MGGnol, MGGcol, and MGn. μ g / cm 2 Apply an amount sufficient for one day to the skin before use.

13. The composition according to claim 9, wherein, The composition is used to inhibit phenomena in the skin caused by oxidative stress. The composition is 0.1% based on the total amount of MGGnol, MGGcol, and MGn. μ g / cm 2 Apply an amount sufficient for one day to the skin before use.

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

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