Ultraviolet absorber, antioxidant, anti-saccharification agent, external preparation for skin, cosmetic, method for producing compound, and compound

Abstract

Provided is a novel ultraviolet absorber, antioxidant, or anti-saccharification agent. An ultraviolet absorber, an antioxidant or an anti-saccharification agent which contains, as an active ingredient, at least one compound selected from the group consisting of a compound represented by formula (1) and a compound represented by formula (2).

Description

Technical Field

[0001] This disclosure relates to a method for manufacturing an ultraviolet absorber, an antioxidant, an anti-glycation agent, a topical skin agent, a cosmetic, and a compound, as well as the compound itself. Background Technology

[0002] Patent document 1 describes a compound called Junsainoside A as a useful novel compound contained in water shield.

[0003] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2014-31361 Summary of the Invention The problem the invention aims to solve The inventors of this application have previously studied ultraviolet-absorbing substances derived from cyanobacteria. Among the ultraviolet-absorbing substances biosynthesized by cyanobacteria are known spore-like amino acids and pseudocladin.

[0004] The technical problem to be solved by this disclosure is to provide at least one of the following: novel ultraviolet absorbers, antioxidants, anti-glycation agents, topical skin agents, cosmetics, novel manufacturing methods related to compounds, and novel compounds.

[0005] means for solving problems The inventors of this application, while exploring substances different from spore-forming amino acids and pseudocladin, focused on the edible cyanobacterium *Suizenji*, a species endemic to Japan. Furthermore, during analysis of the extract of *Suizenji*, a substance with strong absorption in the ultraviolet range was newly discovered, ultimately leading to the development of the technology disclosed herein. This disclosure can be implemented in the following manner.

[0006] [1] An ultraviolet absorber, antioxidant or anti-glycation agent, wherein at least one of the compounds represented by the following formula (1) and the compounds represented by the following formula (2) is an active ingredient.

[0007] [Chemical Formula 1]

[0008] [Chemical Formula 2]

[0009] [2] A topical skin agent or cosmetic containing at least one of the compounds represented by formula (1) and the compounds represented by formula (2).

[0010] [3] A method for manufacturing a compound, comprising extracting at least one of the compounds represented by formula (1) and the compounds represented by formula (2) from cyanobacteria.

[0011] [4] The cyanobacteria are dried before extracting the compound according to the method for manufacturing the compound described in [3].

[0012] [5] A method for manufacturing a compound, wherein a solution of the compound represented by formula (2) above is irradiated with light to obtain the compound represented by formula (1) above.

[0013] [6] A compound selected from at least one of the groups consisting of derivatives of the compound represented by formula (1) and derivatives of the compound represented by formula (2).

[0014] [7] An ultraviolet absorber, antioxidant or anti-glycation agent, wherein the compound described in [6] is the active ingredient.

[0015] [8] A topical skin preparation or cosmetic containing the compound described in [6]. Attached Figure Description

[0016] Figure 1 This is a chromatogram of the extract of Suizenji cyanobacteria by HPLC analysis.

[0017] Figure 2 This is a diagram used to illustrate the hypothesis regarding the stability of Saclipin B.

[0018] Figure 3 These are the absorption spectra of Saclipin A and Saclipin B.

[0019] Figure 4 This is a graph showing the main HMBC and NOE correlations of Saclipin A.

[0020] Figure 5 This is a graph showing the main HMBC and NOE correlations of Saclipin B.

[0021] Figure 6 This is a chromatogram of saclipin A solution analyzed by HPLC before and after light irradiation.

[0022] Figure 7 This is a graph showing the changes in the antioxidant activity of Saclipin A, Saclipin B, Ascorbic Acid, and Trolox over time.

[0023] Figure 8 This is a graph showing the inhibitory effect on the glycation of elastin.

[0024] Figure 9 This is a graph showing the inhibitory effect on the glycation reaction of collagen.

[0025] Figure 10 This is a chromatogram of saclipin A and saclipin B analyzed by HPLC before and after methyl esterification.

[0026] Figure 11 These are the absorption spectra of saclipin A and saclipin B before and after methyl esterification.

[0027] Figure 12 This is a diagram showing the results of a biocompatibility test on human skin fibroblasts in newborns.

[0028] Figure 13 This is a graph showing the results of a biocompatibility test on human keratinocytes. Detailed Implementation

[0029] The following is a detailed description of this embodiment. Furthermore, in this specification, unless otherwise specified, references to numerical ranges using "~" include both the lower and upper limits. For example, in the reference "10 to 20", both the lower limit "10" and the upper limit "20" are included. That is, "10 to 20" means the same as "10 or more and 20 or less". Additionally, in this specification, the upper and lower limits of each numerical range can be arbitrarily combined.

[0030] 1. Compounds represented by formula (1) and compounds represented by formula (2) This disclosure relates to a compound represented by the following formula (1).

[0031] [Chemical Formula 3]

[0032] The compound represented by formula (1) is (10E,12Z,14E)-9,16-Dioxooctadeca-10,12,14-trienoicacid in IUPAC nomenclature. The molecular weight of this compound is 306. The inventors of this application have named this compound saclipin B.

[0033] Suizenji cyanobacteria (Aphanothece sacrum) is widely known as a species of cyanobacterium endemic to Japan, confirmed to inhabit only a portion of Kyushu. Currently, it is only grown naturally at a farm along the Konkomawa River in Asakura City, Fukuoka Prefecture, and distributed as food.

[0034] The inventors of this application analyzed the extract of Suizenji cyanobacteria and confirmed the presence of a substance with strong absorption in the ultraviolet range. The substance with strong absorption in the ultraviolet range was separated and purified by preparative HPLC, and the structure of the purified product was analyzed. The results identified the known compound of formula (2) and the compound of formula (1) above.

[0035] [Chemical Formula 4]

[0036] The compound represented by formula (2) is (10E,12E,14E)-9,16-Dioxooctadeca-10,12,14-trienoic acid in IUPAC nomenclature. The molecular weight of this compound is 306. The inventors of this application have named this compound saclipin A. Saclipin A and saclipin B have a geometric isomer (cis-trans isomer) relationship. Furthermore, although the compound of formula (2) is a known compound, there are no reports of its presence in Suizenji cyanobacteria or its physiological activity.

[0037] Figure 1 This is a chromatogram of the extract of *Suizenji* cyanobacteria, obtained by HPLC analysis. The HPLC analysis conditions are described below. Furthermore, Figure 1 In the study, the peak with a retention time of 10 min to 12 min was the peak of saclipin A, and the peak with a retention time of 12 min to 14 min was the peak of saclipin B.

[0038] [Measurement conditions] Detection wavelength: 320nm Column: Inerteil ODS-P 3μm, 4.6×33+4.6×150mm Elution buffer: 50% Acetonitrile (isocratic elution) Thermodynamically, it is generally believed that in organic compounds with carbon-carbon double bonds, the trans (E-type) form is more stable than the cis (Z-type) form. Surprisingly, not only trans (E-type) saclipin A but also cis (Z-type) saclipin B is found in *Suizenji* cyanobacteria. The reason for the stable existence of saclipin B is unclear, but... Figure 2 As shown, saclipin B is likely stabilized in its enol form due to hydrogen bonding. From a stability point of view, saclipin B is a compound suitable for a wide range of applications.

[0039] In *Suizenji* cyanobacteria, the ratio of saclipin A to saclipin B is typically around 75:35 to 90:10. The inventors of this application have obtained a novel insight into the isomerization reaction from saclipin A to saclipin B via light irradiation. The light irradiation treatment will be described later. The ratio of saclipin A to saclipin B can be appropriately designed according to the intended use, etc. For example, the ratio of saclipin A to saclipin B can be 70:30 to 5:95, or 50:50 to 8:92, or 30:70 to 10:90.

[0040] As mentioned above, saclipin B can be extracted from Suizenji cyanobacteria. Additionally, saclipin A can also be extracted from Suizenji cyanobacteria. Methods for manufacturing saclipin B and saclipin A will be described later. However, the methods for manufacturing saclipin B and saclipin A are not limited to these. Saclipin B and saclipin A can be synthesized using known chemical synthesis methods, or they can be manufactured by reacting or processing substances obtained from natural sources.

[0041] 2. Utilization of compounds (Part 1) The first aspect of this disclosure is an ultraviolet absorber, antioxidant, or anti-glycation agent, wherein at least one of the compounds represented by formula (1) and formula (2) above is used as an active ingredient. Hereinafter, the compounds represented by formula (1) and at least one of the compounds represented by formula (2) above will also be referred to as saclipin B and / or saclipin A. In addition, saclipin B and saclipin A will not be distinguished and will be collectively referred to as saclipin.

[0042] Saclipin B exhibits a maximum absorption at 319 nm in the ultraviolet wavelength region, with a molar absorptivity of 30,555 MΩ. -1 cm -1 Saclipin A exhibits maximum absorption in the ultraviolet wavelength region of 315 nm to 316 nm, with a molar absorptivity of 26,454 MΩ. -1 cm -1 Saclipin B and / or saclipin A possess sufficient ultraviolet absorption properties and are useful as active ingredients in ultraviolet absorbers. The molar absorptivity of Saclipin B at its maximum absorption value is higher than that of saclipin A. Therefore, ultraviolet absorbers preferably contain at least saclipin B.

[0043] There are no particular restrictions on the form of ultraviolet (UV) absorbers. UV absorbers are suitable for use as topical skin agents, cosmetics, food, pharmaceuticals, and quasi-pharmaceuticals. UV absorbers, especially when applied to the skin surface, are highly effective in exerting their effects. Therefore, they are particularly suitable as topical skin agents or cosmetics in the aforementioned forms.

[0044] In addition, saclipin B and / or saclipin A have antioxidant properties and are useful as active ingredients in antioxidants. Saclipin B has higher free radical scavenging activity than saclipin A. Therefore, antioxidants preferably contain at least saclipin B.

[0045] Saclipin B and saclipin A are slow-acting antioxidants that maintain their antioxidant activity for a longer period compared to ascorbic acid and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid). The following is a detailed description. In the determination of ABTS free radical scavenging activity in the following examples, the IC50 value was measured 20 minutes after the antioxidant was added to the reaction solution. 50 The value is set to α, and the IC value after 40 minutes is... 50 With the value set as β, the β / α values ​​for saclipin B, saclipin A, ascorbic acid, and Trolox are as follows. Ascorbic acid and Trolox have β / α values ​​greater than 0.95, while saclipin B and / or saclipin A have β / α values ​​less than 0.8. Furthermore, β / α values ​​are generally less than 1, and the lower the value (less than 1), the more likely it is to be considered an indicator of free radical scavenging activity during the period from 20 minutes to 40 minutes.

[0046] The β / α value of Saclipin B is 0.74. The β / α value of Saclipin A is 0.78. The β / α ratio of ascorbic acid is 0.95. Trolox's β / α value is 1.00. There are no particular restrictions on the form of antioxidants. Antioxidants can be formulated in suitable forms for use in food and pharmaceuticals. Based on these forms, they can scavenge reactive oxygen species in the body after oral administration, thus promoting health. Furthermore, by incorporating antioxidants into topical skin agents, cosmetics, or quasi-pharmaceuticals, not only can the oxidation of the contained ingredients be prevented, but anti-aging effects on the skin and other components can also be expected.

[0047] Furthermore, saclipin B and / or saclipin A possess anti-glycation properties and are useful as active ingredients in anti-glycation agents. For example, the usefulness of saclipin B and / or saclipin A as anti-glycation agents inhibiting the glycation reaction of elastin has been confirmed. Saclipin B exhibits higher activity in inhibiting the glycation reaction of elastin than saclipin A. As an anti-glycation agent inhibiting the glycation reaction of elastin, saclipin B is preferred. Additionally, the usefulness of saclipin B and / or saclipin A as anti-glycation agents inhibiting the glycation reaction of collagen has been particularly confirmed. Saclipin A exhibits higher activity in inhibiting the glycation reaction of collagen than saclipin B. As an anti-glycation agent inhibiting the glycation reaction of collagen, saclipin A is preferred. Moreover, it is known that in the glycation reactions of various proteins, at least a portion involves a common reaction. It is also speculated that Saclipin B and / or saclipin A may be useful as anti-glycation agents that inhibit the glycation of proteins other than elastin and collagen.

[0048] There are no particular restrictions on the form of anti-glycation agents. These agents are suitable for use in food and pharmaceuticals. Based on this form, they can inhibit the glycation of proteins in blood vessel walls or skin tissue in vivo, preventing arteriosclerosis and skin aging. Furthermore, by including anti-glycation agents in topical skin agents, cosmetics, or quasi-pharmaceuticals, effects such as preventing skin aging can be expected.

[0049] To the extent that the quantity and quality of the effects of this disclosure are not impaired, the ultraviolet absorber, antioxidant, or anti-glycation agent may appropriately contain, in addition to saclipin B and saclipin A, components commonly used in ultraviolet absorbers, antioxidants, or anti-glycation agents. There is no particular limitation on the total amount of saclipin B and saclipin A in the ultraviolet absorber, antioxidant, or anti-glycation agent. From the viewpoint of ensuring manageability, ease of application, ease of consumption, and effectiveness, when the total mass of the agent is set as 100% by mass, the total amount of the aforementioned saclipin B and saclipin A can be from 0.0001% by mass to 100% by mass.

[0050] The dosage of oral medications in food, medicine, etc., can be appropriately adjusted according to factors such as the age, weight, gender, and condition of the user. For example, the intake or dosage of oral medications, converted to the dry weight of saclipin B and saclipin A, is, for example, 0.0001 mg to 100 mg per day for an adult weighing 60 kg, but is not limited to this range. As needed, the above intake can be divided into one or more doses per day, for example, divided into 2 to 3 separate doses.

[0051] There are no particular restrictions on the dosage of topical skin agents, cosmetics, or quasi-medicinal products. Generally, an appropriate amount can be applied to the skin or other external dermal surfaces several times a day, such as by rubbing or sprinkling. Examples of application for topical skin agents or cosmetics will be explained later.

[0052] 3. Utilization of compounds (part 2) The second aspect of this disclosure is a topical skin agent or cosmetic containing at least one of the compounds represented by formula (1) and the compounds represented by formula (2) above.

[0053] When Saclipin B and / or saclipin A are used as topical skin preparations, the preparations may also contain excipients, base agents, emulsifiers, solvents, stabilizers, etc. There are no particular restrictions on excipients, carriers, and additives, as long as they are commonly used and physiologically or pharmaceutically permissible; their types and compositions can be appropriately modified. Examples of dosage forms include ointments, liquid preparations, sprays, tablets, powders, and granules.

[0054] There is no particular limit to the total amount of saclipin B and saclipin A in topical skin preparations. From the viewpoint of ensuring shelf life, ease of application, and effectiveness, when the total mass of the topical skin preparation is set as 100% by mass, the total amount of saclipin B and saclipin A in the topical skin preparation can be 0.00001% by mass to 10% by mass.

[0055] When saclipin B and / or saclipin A are used in cosmetics, the cosmetics may also contain ingredients commonly used as cosmetic ingredients, such as surfactants, oily components, moisturizers, film-forming agents, pigments, fragrances, etc. Examples of cosmetic forms include toners, creams, lotions, gels, aerosols, serums, masks, cleansers, foundations, powders, and various color cosmetics (lipsticks, blushes, etc.).

[0056] There are no particular restrictions on the total amount of saclipin B and saclipin A in cosmetics. From the perspective of ensuring storeability, handleability, and effectiveness, if the overall mass of the cosmetic is set at 100% by mass, the total amount of saclipin B and saclipin A in the cosmetic can be 0.00001% by mass to 10% by mass.

[0057] 4. Methods for manufacturing compounds (Part 1) The third aspect of this disclosure is a method for manufacturing a compound, wherein at least one of the compounds represented by formula (1) and formula (2) above is extracted from cyanobacteria.

[0058] As an example of a method for manufacturing a compound, a method for drying cyanobacteria and extracting the compound from the dried cyanobacterial material will be described. Furthermore, the drying of cyanobacteria is an arbitrary process and can be performed as needed. Below, a method for manufacturing a compound using *Suizenji cyanobacteria* as an example of a cyanobacterium will be described in detail.

[0059] There are no particular restrictions on the drying conditions for Suizenji cyanobacteria. The drying method is also not particularly limited; both natural drying and artificial drying methods are acceptable. Examples of natural drying methods include air drying, shade drying, and sun drying. Examples of artificial drying methods include heat drying, hot air drying, vacuum drying, and freeze drying. From the viewpoint of improving drying efficiency and inhibiting the inactivation of enzymes related to the synthesis of saclipin B and saclipin A, the drying temperature is preferably 10°C to 60°C, more preferably 15°C to 50°C, and even more preferably 20°C to 40°C. The drying time can be appropriately determined according to the selected drying method and the moisture state of the Suizenji cyanobacteria. In the case of natural drying, for example, it can be half a day to several days. In the case of artificial drying, for example, it can be 1 minute to 48 hours. Commercially available dried Suizenji cyanobacteria products can be used as the dried product.

[0060] Compared to keeping Suizenji cyanobacteria moist, drying Suizenji cyanobacteria increases the production of saclipin B and saclipin A. The mechanism is not yet clear, but one possible reason is that drying the surviving Suizenji cyanobacteria induces the production of saclipin B and saclipin A through drying stress. However, this disclosure is not limited to this theoretical explanation.

[0061] There are no particular limitations on the extraction method for the compounds. As an example of the extraction method, a method for obtaining saclipin B and saclipin A by pulverizing Suizenji blue algae, adding an extraction solvent, and stirring is described. Furthermore, pulverization and stirring are arbitrary processes, as long as sufficient yield can be obtained. For example, saclipin B and / or saclipin A can also be extracted by immersing Suizenji blue algae in the extraction solvent and allowing it to stand.

[0062] The pulverization of *Suizenji* cyanobacteria can be performed using methods such as a stirrer, an ultrasonic pulverizer, or a bead-type pulverizer. Various methods can be used in combination. For example, when pulverizing dried *Suizenji* cyanobacteria, the dried material can be pulverized using a stirrer, followed by ultrasonic treatment in an extraction solvent. Alternatively, when directly pulverizing *Suizenji* cyanobacteria that is encapsulated in an agar matrix and forms clusters, the moist *Suizenji* cyanobacteria* can be homogenized using a stirrer, followed by ultrasonic treatment in an extraction solvent. The frequency of ultrasonic treatment can be, for example, from 15 kHz to 50 kHz. The temperature of ultrasonic treatment can be, for example, between 4°C and 40°C. The duration of ultrasonic treatment can be, for example, from 5 minutes to 60 minutes.

[0063] As extraction solvents, alcohols such as methanol, ethanol, propanol, and butanol can be used; diols such as 1,3-butanediol, glycerol, and propylene glycol; esters such as ethyl acetate and butyl acetate; ethers such as diethyl ether, propyl ether, isopropyl ether, tetrahydrofuran, and dioxane; halogenated hydrocarbons such as dichloromethane and chloroform; ketones such as acetone, methyl ethyl ketone, diethyl ketone, and cyclohexanone; and solvents such as n-hexane, cyclohexane, petroleum ether, and water. These solvents can be used alone or in mixtures of two or more.

[0064] There are no particular restrictions on extraction conditions. The extraction temperature can be, for example, between 4°C and 40°C. The extraction time can be, for example, between 5 minutes and 6 hours. The amount of extraction solvent added relative to 10g of dried *Suizenji* algae can be between 10mL and 100mL.

[0065] The extract can be obtained by solid-liquid separation from the residue, such as through centrifugation, filtration, suction, or pressing. The saclipin B and saclipin A obtained as the extract can be used directly, or the solvent can be removed by appropriate concentration or distillation, and used as extracts or powders. Furthermore, depending on the needs, one or more appropriate separation and purification methods, such as chromatography, can be combined for purification.

[0066] 5. Methods for manufacturing compounds (part 2) The fourth aspect of this disclosure is a method for manufacturing a compound by irradiating a solution of the compound represented by formula (2) above with light to obtain the compound represented by formula (1) above.

[0067] By irradiating a solution of Saclipin A with light, saclipin A can be isomerized to saclipin B. The solvent for the Saclipin A solution is not particularly limited. Examples of solvents include methanol, ethanol, or acetonitrile. The extraction solvents described above can also be used. Furthermore, the extract described above can also be used as a solution of saclipin A. That is, the method for producing this compound can also involve irradiating saclipin A from Suizenji cyanobacteria with light.

[0068] There are no particular restrictions on the light irradiation conditions. The illuminance can be, for example, between 4000 lux and 10000 lux. The temperature during light irradiation can be, for example, between 4°C and 40°C. The irradiation time can be, for example, between 12 hours and 48 hours. There are no particular restrictions on the isomerization rate based on light irradiation. The isomerization rate is typically below 92%, and can be appropriately adjusted within a range of, for example, between 10% and 92% to achieve the desired saclipin A to saclipin B ratio.

[0069] 6. Derivative compounds The fifth aspect of this disclosure is a compound selected from the group consisting of derivatives of the compound represented by formula (1) and derivatives of the compound represented by formula (2). In the following description, the derivatives of the compound represented by formula (1) are also referred to as saclipin B derivatives, and the derivatives of the compound represented by formula (2) are also referred to as saclipin A derivatives.

[0070] In this disclosure, a saclipin B derivative refers to a derivative modified with one or more functional groups present in saclipin B, and having the same or higher functionality as saclipin B. A saclipin A derivative refers to a derivative modified with one or more functional groups present in saclipin A, and having the same or higher functionality as saclipin A. The aforementioned "functionality" is not particularly limited, and may include, for example, ultraviolet absorption, antioxidant properties, and anti-glycation properties.

[0071] Saclipin B derivatives are preferably selected from one or more of the group consisting of an ester derivative obtained by reacting a carboxyl group at the end of saclipin B with an alcohol, an amide derivative obtained by reacting a carboxyl group at the end of saclipin B with an amine, and an ammonium salt obtained by reacting a carboxyl group at the end of saclipin B with ammonium. Saclipin A derivatives are preferably selected from one or more of the group consisting of an ester derivative obtained by reacting a carboxyl group at the end of saclipin A with an alcohol, an amide derivative obtained by reacting a carboxyl group at the end of saclipin A with an amine, and an ammonium salt obtained by reacting a carboxyl group at the end of saclipin A with ammonium. The type of carboxyl derivative group in the saclipin B derivative and / or the saclipin A derivative can be appropriately selected according to the application, dosage form, etc.

[0072] Carboxyl derivative groups can be represented as, for example, -COOR 1 -CONH2, -CONHR 2 -CONR 3 R 4 -COONH4, -COONH3R 5 Here, R 1 R 2 R 3 R 4 R 5 Each saclipin B and saclipin A is an independent hydrocarbon group with 1 to 10 carbon atoms and can have a functional group. Saclipin B with a -COOCH3 group in its carboxyl derivative group and saclipin A with a -COOCH3 group are also referred to as methylated saclipin B and methylated saclipin A, respectively. Methylated saclipin B and methylated saclipin A ensure equivalent UV absorption to saclipin B and saclipin A, while also improving hydrophobicity. Such saclipin B derivatives and / or saclipin A derivatives are more suitable for use in mixtures with hydrophobic components such as oily components compared to saclipin B and / or saclipin A.

[0073] The fifth aspect of this disclosure includes ultraviolet absorbers, antioxidants, or anti-glycation agents with Saclipin B derivatives and / or saclipin A derivatives as active ingredients. The description of the ultraviolet absorbers, antioxidants, or anti-glycation agents in the fifth aspect can be directly applied by replacing "saclipin A" and "saclipin B" in "2. Use of the compound (1)" above with "saclipin A derivative" and "saclipin B derivative," respectively.

[0074] The fifth method of this disclosure includes a topical skin preparation or cosmetic containing a Saclipin B derivative and / or a saclipin A derivative. The description of the topical skin preparation or cosmetic of the fifth method can be directly applied by replacing "saclipin A" and "saclipin B" in "3. Use of the compound (2)" above with "saclipin A derivative" and "saclipin B derivative".

[0075] Example The present disclosure will now be described in more detail through examples. However, the scope of the present disclosure is not limited to these examples.

[0076] 1. Extraction and separation of compounds The extraction and separation of compounds from Suizenji cyanobacteria were carried out according to the following steps.

[0077] Specifically, 10g of dried *Suizenji* cyanobacteria (product name "Jusenmo," sold by "Endo Kinkawado," postal code 838-0031, Asakura City, Fukuoka Prefecture 2949) was pulverized using an IFM-C20G (Iwatani) grinder. The resulting powder was transferred to a 50ml tube, and 30ml of methanol was added. The mixture was then ultrasonically treated using a Model UR-200P (TOMY Seiko) ultrasonic treatment device. The resulting suspension was centrifuged at 4°C and 2330×g for 15 minutes, and the supernatant was transferred to a new tube. 20ml of methanol was added to the remaining particles, and the mixture was ultrasonically treated and centrifuged in the same manner. The supernatants were combined. The supernatant was then dried using a VC-36R (TAITEC) centrifugal concentrator under light-shielded conditions. The dried material was dissolved in 5ml of 55% (v / v) acetonitrile containing 0.1% (v / v) formic acid, and then passed through a 0.22μm filter. The sample was fed to a preparative HPLC system using Inertsil ODS-3 columns (10 μm particle size; 50 mm × 20 mm inner diameter, GL Sciences) and Inertsil ODS-3 columns (10 μm particle size; 250 mm × 20 mm inner diameter, GL Sciences). 55% (v / v) acetonitrile containing 0.1% (v / v) formic acid was used as the mobile phase, and the system was run at a flow rate of 6.0 mL / min. Elution of the compound was monitored at a detection wavelength of 320 nm. In this preparative HPLC, saclipin A and saclipin B eluted as the same fraction. After lyophilizing the fraction containing the target compound, the dried sample was dissolved in 50% (v / v) acetonitrile and fed to a preparative HPLC system using a YMC carotenoid column (5 μm particle size; 250 mm × 10 mm inner diameter). 50% (v / v) acetonitrile was used as the mobile phase, and the system was run at a flow rate of 1.0 mL / min. The elution of the compounds was monitored at a detection wavelength of 320 nm. In this preparative HPLC, saclipin A was eluted first, followed by saclipin B. The fractions were freeze-dried to obtain pale green dried samples of saclipin A and saclipin B. From 10 g of dried *Suizenji* cyanobacteria, 7.0 mg–10 mg of saclipin A and 1.5 mg–3.0 mg of saclipin B were obtained. The content of saclipin A in dried *Suizenji* cyanobacteria was higher than that of saclipin B.

[0078] 2. Determination of the structures of saclipin A and saclipin B The obtained dried sample was dissolved in methanol, and absorption spectroscopy analysis and precise mass analysis were performed. 1 H and 13C-NMR spectroscopy determination. The absorption spectra of Saclipin A and Saclipin B were measured... Figure 3 As shown. Figure 3 In the various charts, the horizontal axis represents wavelength (nm) and the vertical axis represents relative absorbance.

[0079] (1) saclipinA [Physicochemical data] Maximum absorption (methanol solution): 316 nm Molar absorptivity: 26,454 M -1 cm -1 Precise ESI MS data: calcd for C 18 H 25 O4 ([MH]) - ): m / z 305.1758; Found: m / z305.1758 Fragment ions from precision ESI MS: m / z 287.1654, 249.1497, 135.0815, 125.0971 1 H-NMR and 13 C-NMR (CD3OD): [Table 1]

[0080] [Two-dimensional NMR] The results of HMBC (Heteronuclear multiple-bond correlation spectroscopy) analysis are as follows: Figure 4 The arrow indicates a correlation of HMBC between protons and carbon. Additionally, NOESY (Nuclear Overhauser Effect Spectroscopy) analysis revealed... Figure 4 The dashed line indicates the NOE correlation.

[0081] Based on the above physicochemical data, the chemical structure of saclipin A obtained from dried Suizenji cyanobacteria was determined.

[0082] (2) saclipinB [Physicochemical data] Maximum absorption (methanol solution): 319 nm Molar absorptivity: 30,555 M -1 cm -1 Precise ESIMS data: calcd for C 18 H 25 O4 ([MH]) - ): m / z 305.1758; Found: m / z305.1757 Fragment ions from precise ESIMS: m / z 287.1655, 249.1496, 135.0815, 125.0971 1 H-NMR and 13 C-NMR (CD3OD): [Table 2]

[0083] The result of HMBC (Heteronuclear multiple-bond correlation spectroscopy) analysis is that... Figure 5 The arrow indicates a correlation of HMBC between protons and carbon. Additionally, the NOESY (Nuclear overhausereffect spectroscopy) analysis revealed... Figure 5 The dashed line indicates the NOE correlation.

[0084] Based on the above physicochemical data, the chemical structure of saclipin B obtained from dried Suizenji cyanobacteria was determined.

[0085] 3. Induction of saclipin A and saclipin B through drying treatment Saclipin A and saclipin B can be induced by drying fresh Suizenji cyanobacteria.

[0086] 50g of fresh *Suizenji* algae collected from the Kōgan River was mixed with 10ml of river water and homogenized using an IFM-C20G (Iwatani) grinder. The homogenized sample was aliquoted into 5.0g portions and placed in plastic trays, then dried in a DY400 (Yamato Scientific) drying oven set to 30°C under dark conditions. A control experiment was also conducted, maintaining the sample in a moist state while sealed in a plastic bag. Water was added to the dried sample to bring the weight back to the initial value, followed by the addition of methanol to achieve a final concentration of 80% (v / v). Saclipin was then extracted by thorough ultrasonic treatment using a Model UR-200P (TOMY Seiko) ultrasonic treatment device. The ultrasonically treated sample was centrifuged at 4°C, 2330×g for 15 minutes, and the resulting supernatant was used for HPLC analysis to quantify the saclipin content. The control sample without drying treatment contained 0.005 mg / g and 0.001 mg / g of saclipin A and saclipin B per wet weight, respectively. On the other hand, the samples treated with drying treatment showed significantly increased contents of 0.056 mg / g and 0.008 mg / g, respectively. These results indicate that saclipin A and saclipin B are substances induced by the drying process of *Suizenji* cyanobacteria.

[0087] 4. Generation of saclipin B via photoisomerization Saclipin A, purified from dried cyanobacteria from Suizenji Temple, was dissolved in methanol, ethanol, or acetonitrile. The solution was then irradiated with 6000 lux of white fluorescent light for at least 24 hours. Subsequently, HPLC analysis was performed under the following conditions to calculate the amounts of saclipin A and saclipin B, and to calculate the isomerization rate of saclipin A. The chromatogram of the saclipin A solution before light irradiation is shown below. Figure 6 The chromatogram of saclipin A solution after light irradiation is shown on the left. Figure 6 Right side.

[0088] [Measurement conditions] Detection wavelength: 320nm Column: Inerteil ODS-P 3μm, 4.6×33+4.6×150mm Eluent: 50% Acetonitrile (isocratic elution) Approximately 90% of dissolved saclipin A was converted to saclipin B. The ratio of saclipin A to saclipin B remained unchanged for at least 96 hours after treatment in the dark following light irradiation. This result indicates that saclipin B is generated from saclipin A via photoisomerization.

[0089] 5. Assay of the physiological activities of saclipin A and saclipin B (1) Experimental Example 1: Evaluation of antioxidant activity (1.1) Determination of ABTS free radical scavenging activity To evaluate the antioxidant activity of Saclipin A and saclipin B, the free radical scavenging activity of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was determined.

[0090] An ABTS radical solution was prepared by mixing a 7 mM aqueous solution of ABTS and a 2.45 mM aqueous solution of potassium persulfate in the dark at 25°C for 16 hours. The ABTS radical solution was diluted with ethanol to achieve an absorbance of approximately 1.0 at 734 nm. 50 μL of this diluted ABTS radical solution was mixed with 50 μL of an ethanol solution of the test sample. The mixture was kept in the dark at room temperature for 20 or 40 minutes, and the absorbance at 734 nm was then measured. The final concentrations of the sample were adjusted to 0 mM, 0.06 mM, 0.12 mM, 0.18 mM, 0.24 mM, 0.30 mM, 0.60 mM, 1.20 mM, and 1.80 mM. Ascorbic acid and Trolox were used as standard samples. The inhibition rate was calculated using the following equation.

[0091] Inhibitory activity (%) = ((Ab-As) / Ab) × 100 Here, Ab and As represent the absorbance of the blank sample and the test sample, respectively. The 50% inhibition concentration (IC50) is calculated based on the measurement results. 50 The values ​​are shown in Table 3.

[0092] The results of the antioxidant activity assay are as follows.

[0093] [Table 3]

[0094] According to the IC shown in Table 3 50 The results confirmed that both saclipin A and saclipin B exhibited free radical scavenging activity against ABTS free radicals. It was determined that saclipin B had higher activity than saclipin A.

[0095] (1.2) Evaluation of the changes in the antioxidant activity of saclipin over time According to the experimental results in Table 3, the scavenging rate of ABTS free radicals by saclipin increased after 40 minutes compared to after 20 minutes of reaction, indicating that saclipin is a gradually active antioxidant. Therefore, the change in free radical scavenging over time was determined using a time-series experiment. In the time-series experiment, the decrease in absorbance after adding the test sample was continuously recorded for 2900 seconds at 25℃ and 734nm. In the time-series experiment, the final reaction concentrations of saclipin A, saclipin B, ascorbic acid, and Trolox were adjusted to 0.60 mM, 0.18 mM, 6 μM, and 6 μM, respectively.

[0096] The results of the time series experiment are as follows.

[0097] It was determined that the free radical scavenging reaction was still proceeding 2900 seconds after both Saclipin A and saclipin B were added to the reaction system. Figure 7 (A) and (B) in the sample. Ascorbic acid and Trolox, the antioxidants used as standard samples, undergo free radical scavenging reactions immediately after addition and then have almost no effect. Figure 7 (C) and (D) in the results. Based on these results, it is determined that both saclipin A and saclipin B have free radical scavenging effects and are slow-acting antioxidants.

[0098] (2) Experimental Example 2: Evaluation of anti-glycation activity To evaluate the anti-glycation activity of Saclipin A and Saclipin B, their effects on the glycation of elastin and collagen were investigated.

[0099] The glycation effects of saclipin on elastin and collagen were evaluated using an elastin anti-glycation assay kit (model: AAS-AGE-K05, COSMO BIO Co., Ltd.) and a collagen anti-glycation assay kit (model: AK71, COSMO BIO Co., Ltd.). The analyses were performed using saclipin A and saclipin B purified from *Suizenji* algae according to the manufacturer's instructions. However, since saclipin is not soluble in the accompanying aqueous buffer, it was dissolved in dimethyl sulfoxide (DMSO) for the experiments. The final reaction concentrations of the samples were adjusted to 0 mM, 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM. The results are shown below. Figure 8 and Figure 9 As shown.

[0100] The results of the anti-glycation activity assay are as follows.

[0101] As an inhibitory effect on the glycation of elastin, saclipin A has an IC50 value of [missing information]. 50 The value is 2.5 mM. This is comparable to the IC50 of the known glycation inhibitor, aminoguanidine. 50 The value (1.5 mM) indicates that saclipin A inhibits the glycation of elastin. Figure 8 Although Saclipin B is slightly less active than saclipin A, it inhibited the saccharification reaction by approximately 45% at a concentration of 4 mM. On the other hand, such as Figure 9 As shown, saclipin exhibits a stronger inhibitory effect on collagen glycation. The IC50 of aminoguanidine... 50 The value is 2.4 mM, compared to the IC values ​​of saclipin A and saclipin B. 50 The values ​​were 1.9 mM and 0.9 mM, respectively. These results clearly demonstrate that both saclipin A and saclipin B function as glycation inhibitors of elastin and collagen, which are components of the dermis.

[0102] 6. Formation of saclipin A and saclipin B derivatives Ssaclipin A and saclipin B derivatives were generated using a fatty acid methyl esterification kit (model 06482-04, Nacalai Tesque Co., Ltd.). Analysis was performed using purified saclipin A and saclipin B according to the manufacturer's instructions. However, reagent C was used instead of reagent B. Reagent C is used to methylate free fatty acids by reacting them with methanol in the presence of an acidic catalyst. After adding the extraction reagent and mixing, the upper layer, separated into two layers, was collected.

[0103] The components obtained from the supernatant were analyzed by reversed-phase HPLC. Saclipin A and saclipin B were also analyzed by reversed-phase HPLC under the same conditions. The results are shown in... Figure 10 . Figure 10The upper half of the chromatogram shows the chromatograms of saclipin A and saclipin B, while the lower half shows the chromatogram of the upper layer components. In the upper half of the chromatogram, the peak "SacA" with a retention time of 12-15 min represents saclipin A, and the peak "SacB" with a retention time of 15-18 min represents saclipin B. If saclipin A and saclipin B are methylated, their hydrophobicity increases, resulting in a longer retention time in reversed-phase HPLC analysis. In the lower half of the chromatogram of the upper layer components, peaks were identified at retention times of 40-50 min and 60-70 min. This indicates that saclipin A and saclipin B are methylated. The respective peaks are labeled "SacA_methyl ester" and "SacB_methyl ester".

[0104] Absorption spectra of saclipin A (SacA), saclipin B (SacB), methylated saclipin A (SacA methyl ester), and methylated saclipin B (SacB methyl ester) were obtained using the methods described in "2. Structural Determination of Saclipin A and Saclipin B" above. The results are shown in... Figure 11 . Figure 11 In the various charts, the horizontal axis represents wavelength (nm), and the vertical axis represents relative absorbance. The maximum value for saclipin A is 317 nm. The maximum value for methylated saclipin A is 316 nm. The maximum value for saclipin B is 319 nm. The maximum value for methylated saclipin B is 319 nm.

[0105] These results show that methylated saclipin A has the same UV absorption capacity as saclipin A. Furthermore, methylated saclipin B has the same UV absorption capacity as saclipin B.

[0106] It is speculated that the functionality of Saclipin A and Saclipin B primarily stems from the presence of carbon-carbon double bonds. The carbon-carbon double bond sites remain unchanged in methylated Saclipin A and Saclipin B. Therefore, it is reasonable that methylated Saclipin A and Saclipin B can ensure equivalent UV absorption capacity to their respective derivatives. In other words, by analogy, as long as Saclipin A and Saclipin B derivatives possess carbon-carbon double bond sites, their functionality, excluding UV absorption capacity, can also be ensured to be equivalent to that of Saclipin A and Saclipin B.

[0107] 7. Biocompatibility test To investigate the cytotoxicity of Saclipin A and saclipin B, MTT assays were performed on (1) neonatal human skin fibroblasts and (2) human keratinocytes.

[0108] (1) MTT assay for neonatal human skin fibroblasts Neonatal human skin fibroblasts (NHDF) were purchased from ATCC. The neonatal human skin fibroblasts were processed at a concentration of 1×10⁻⁶. 4 Cells were seeded per well into 96-well plates. Neonatal human skin fibroblasts were cultured overnight in DMEM medium at 37°C and 5% CO2. DMEM medium was prepared by adding 10% fetal bovine serum, 100 μg / mL penicillin, 100 units / mL streptomycin, and 10 mM HEPES to DMEM (Dulbecco's Modified Eagle Medium). Saclipin A or saclipin B at concentrations of 0.1 μM, 0.36 μM, 1.0 μM, 3.6 μM, and 10.0 μM were added to each cell line. As a control, 1% ethanol was added to the cells. Then, 0.5 mg / mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added, and the cells were cultured at 37°C and 5% CO2 for 20 hours. After dissolving all methyl sulfoxide crystals in DMSO (dimethyl sulfoxide), the absorbance of each sample solution at 540 nm was measured to determine the cell viability. The results are as follows: Figure 12 As shown.

[0109] Figure 12The upper part is a graph showing the results of cell viability (%). The labels on the horizontal axis indicate the samples corresponding to the bars in the graph. For example, the label "saclipin A" "10.00" indicates that cells were treated with 10.0 μM saclipin A. The vertical axis represents the cell viability when the cell viability of cells treated with 1% ethanol is set to 100%.

[0110] according to Figure 12 The upper part of the chart shows that saclipin A at any concentration is not significantly more cytotoxic than 1% ethanol. Similarly, saclipin B at any concentration is not significantly more cytotoxic than 1% ethanol.

[0111] Figure 12 The lower half, from left to right, shows microscope images of "cells treated with 1% ethanol", "cells treated with 10.0 μM saclipin A", and "cells treated with 10.0 μM saclipin B".

[0112] Cells treated with 10.0 μM Msaclipin A had the same appearance as cells treated with 1% ethanol, with no abnormalities observed. This indicates that saclipin A has high biocompatibility. Cells treated with 10.0 μM saclipin B had the same appearance as cells treated with 1% ethanol, with no abnormalities observed. This indicates that saclipin B has high biocompatibility.

[0113] (2) MTT assay for human keratinocytes As human keratinocytes, PSVK1 cells (JCRB1093, JCRB Cell Bank, National Research and Development Corporation Pharmaceutical Foundation) were used. healthy Nutrition Research Institute, Japan). In this experiment, cells were cultured using keratinocyte culture medium (Thermo Fisher Scientific) supplemented with 0.05 mg / mL bovine pituitary extract, 0.005 μg / mL epidermal growth factor, 100 units / mL penicillin, and 100 μg / mL streptomycin (Gibco), and the same method as described above for "(1) MTT assay for neonatal human skin fibroblasts" was used. The results are shown in Figure 13 .

[0114] Figure 13The upper part is a graph showing the results of cell viability (%). The labels on the horizontal axis indicate the sample corresponding to each bar. For example, the label "saclipin A" "10.00" indicates that cells were treated with 10.0 μM saclipin A. The vertical axis represents the cell viability when the cell viability of cells treated with 1% ethanol is set to 100%.

[0115] according to Figure 13 The upper part of the chart shows that saclipin A at any concentration is not significantly more cytotoxic than 1% ethanol. Similarly, saclipin B at any concentration is not significantly more cytotoxic than 1% ethanol.

[0116] Figure 13 The lower half, from left to right, shows microscope images of "cells treated with 1% ethanol", "cells treated with 10.0 μM saclipin A", and "cells treated with 10.0 μM saclipin B".

[0117] Cells treated with 10.0 μM saclipin A had the same appearance as cells treated with 1% ethanol, with no abnormalities observed. This indicates that saclipin A has high biocompatibility. Cells treated with 10.0 μM saclipin B had the same appearance as cells treated with 1% ethanol, with no abnormalities observed. This indicates that saclipin B has high biocompatibility.

[0118] 8. Summary According to this embodiment, a novel ultraviolet absorber, antioxidant, anti-glycation agent, topical skin agent, and cosmetic can be provided. According to this embodiment, a novel manufacturing method related to the compound can be provided. According to this embodiment, a novel compound can be provided.

Claims

1. A UV absorber, antioxidant, or anti-glycation agent, wherein, At least one of the compounds represented by formula (1) and formula (2) below is used as an active ingredient. 。 2. A topical skin agent or cosmetic, wherein, Containing at least one of the compounds represented by formula (1) and formula (2) below, 。 3. A method for manufacturing a compound, wherein, Extract at least one of the compounds represented by formula (1) and formula (2) from cyanobacteria. 。 4. The method for manufacturing the compound according to claim 3, wherein, The cyanobacteria are dried before the compound is extracted.

5. A method for manufacturing a compound, wherein, Irradiating a solution of the compound represented by formula (2) with light yields the compound represented by formula (1). 。 6. A compound selected from at least one of the group consisting of derivatives of a compound represented by formula (1) and derivatives of a compound represented by formula (2). 。 7. An ultraviolet absorber, antioxidant, or anti-glycation agent, wherein the compound of claim 6 is the active ingredient.

8. A topical skin agent or cosmetic comprising the compound of claim 6.

Images