Ultraviolet absorber, antioxidant, Anti-glycation agent, external preparation for skin, cosmetic, method for producing compound, and compound

Abstract

Provided are a novel ultraviolet absorber, a novel antioxidant, or a novel anti-glycation agent.　The ultraviolet absorber, the antioxidant, or the anti-glycation agent contains, as an active ingredient, at least one compound selected from among compounds represented by formula (1) and compounds represented by formula (2).

Description

Ultraviolet absorber, antioxidant, anti-glycation agent, topical skin preparation, cosmetic, method for producing compound, and compound

[0001] The present disclosure relates to ultraviolet absorbers, antioxidants, anti-glycation agents, topical skin preparations, cosmetics, methods for producing compounds, and compounds.

[0002] Patent Document 1 describes a compound called Junsaiinoside A as a useful novel compound contained in Junsai.

[0003] JP 2014-31361 A

[0004] The present inventors have been conducting research into ultraviolet absorbing substances derived from cyanobacteria. Well-known ultraviolet absorbing substances biosynthesized by cyanobacteria include mycosporine-like amino acids and scytonemin.

[0005] The present disclosure aims to achieve at least one of the following: to provide a novel ultraviolet absorber, antioxidant, anti-glycation agent, topical skin preparation, or cosmetic; to provide a novel method for producing a compound; and to provide a novel compound.

[0006] In searching for substances different from mycosporine-like amino acids and scytonemin, the present inventors focused on Aphanothece sacrum, an edible cyanobacterium endemic to Japan. Analysis of an extract from Aphanothece sacrum revealed the presence of a substance with strong absorption in the ultraviolet region, leading to the development of the technology of the present disclosure. The present disclosure can be realized in the following forms.

[0007] [1] An ultraviolet absorber, antioxidant, or anti-glycation agent containing, as an active ingredient, at least one compound represented by the following formula (1) and at least one compound represented by the following formula (2): [2] A skin topical preparation or cosmetic containing at least one compound represented by the above formula (1) and the above formula (2). [3] A method for producing a compound, comprising extracting at least one compound represented by the above formula (1) and the above formula (2) from cyanobacteria. [4] A method for producing a compound according to [3], comprising drying the cyanobacteria before extracting the compound. [5] A method for producing a compound, comprising irradiating a solution of a compound represented by the above formula (2) with light to obtain the compound represented by the above formula (1). [6] At least one compound selected from the group consisting of a derivative of the compound represented by the above formula (1) and a derivative of the compound represented by the above formula (2). [7] An ultraviolet absorber, antioxidant, or anti-glycation agent containing the compound according to [6] as an active ingredient. [8] A skin topical preparation or cosmetic containing the compound according to [6].

[0008] 1 is a chromatogram obtained by HPLC analysis of an extract of Aphanothece saclipa. This is a diagram for explaining a hypothesis regarding the stability of Saclipin B. This is an absorption spectrum of Saclipin A and Saclipin B. This is a diagram showing the main HMBC correlation and NOE correlation of Saclipin A. This is a diagram showing the main HMBC correlation and NOE correlation of Saclipin B. This is a chromatogram obtained by HPLC analysis of a saclipin A solution before and after light irradiation. This is a graph showing the time course of the antioxidant activity of Saclipin A, Saclipin B, ascorbic acid, and Trolox. This is a graph showing the inhibitory effect on the glycation reaction of elastin. This is a graph showing the inhibitory effect on the glycation reaction of collagen. This is a chromatogram obtained by HPLC analysis of saclipin A and saclipin B before and after methyl esterification. 1 shows the absorption spectra of saclipin A and saclipin B before and after methyl esterification. 2 shows the results of a biocompatibility test on neonatal human skin fibroblasts. 3 shows the results of a biocompatibility test on human keratinocytes.

[0009] This embodiment will be described in detail below. In this specification, when a numerical range is described using "to" it is assumed that the range includes both the lower limit and the upper limit unless otherwise specified. For example, the expression "10 to 20" includes both the lower limit "10" and the upper limit "20". In other words, "10 to 20" has the same meaning as "10 or more and 20 or less". In addition, in this specification, the upper limit and lower limit of each numerical range can be combined in any way.

[0010] 1. Compounds represented by formula (1) and formula (2) The present disclosure relates to compounds represented by the following formula (1):

[0011] The compound represented by formula (1) is (10E,12Z,14E)-9,16-Dioxooctadeca-10,12,14-trienoic acid in the IUPAC nomenclature system. The molecular weight of this compound is 306. The inventors of the present application have named this compound saclipin B.

[0012] Aphanothece sacrum is a cyanobacterium endemic to Japan, and has only been confirmed to inhabit parts of Kyushu. Currently, it grows wild only at a farm using the Kogane River in Asakura City, Fukuoka Prefecture, and is distributed as a food product.

[0013] The present inventors analyzed the extract of Aphanothece sacrum and found that it contains a substance that has strong absorption in the ultraviolet region. The substance was isolated and purified by preparative HPLC, and structural analysis of the purified product revealed that it was a known compound of formula (2) below and the compound of formula (1) above.

[0014] The compound represented by formula (2) is (10E,12E,14E)-9,16-Dioxooctadeca-10,12,14-trienoic acid in the IUPAC nomenclature system. The molecular weight of this compound is 306. The inventors of the present application have named this compound saclipin A. Saclipin A and saclipin B are geometric isomers (cis-trans isomers). Although the compound of formula (2) is a known compound, its presence in Aphanothece sacrum and its physiological activity have not been reported.

[0015] Figure 1 shows a chromatogram of an extract of Aphanothece saccharin analyzed by HPLC. The measurement conditions for the HPLC analysis are as follows. In Figure 1, the peak at a retention time of 10 to 12 minutes is the peak of saclipin A, and the peak at a retention time of 12 to 14 minutes is the peak of saclipin B. [Measurement conditions] Detection wavelength: 320 nm Column: Interteil ODS-P 3 μm, 4.6 × 33 + 4.6 × 150 mm Eluent: 50% acetonitrile (isocratic)

[0016] Thermodynamically, it is generally said that organic compounds with a carbon-carbon double bond are more stable in the trans form (E form) than in the cis form (Z form). It is surprising that Aphanothece saclipin B exists in addition to the trans form (E form) of saclipin A. The reason why saclipin B can exist stably is unclear, but as shown in Figure 2, it is possible that saclipin B is stabilized by hydrogen bonding in its enol form. From the viewpoint of stability, saclipin B is a useful compound for various applications.

[0017] In A. saclipinum, the ratio of saclipin A to saclipin B is usually about 75:35 to 90:10. The present inventors have discovered that light irradiation promotes the isomerization reaction of saclipin A to saclipin B. The light irradiation treatment will be described later. The ratio of saclipin A to saclipin B can be appropriately designed depending on the intended use. For example, the ratio of saclipin A to saclipin B can be 70:30 to 5:95, 50:50 to 8:92, or 30:70 to 10:90.

[0018] As described above, saclipin B can be obtained by extraction from Amorphophallus saclipinus. Similarly, saclipin A can be obtained by extraction from Amorphophallus saclipinus. Methods for producing saclipin B and saclipin A will be described later. However, the methods for producing saclipin B and saclipin A are not limited thereto. Saclipin B and saclipin A may be synthesized by known chemical synthesis methods, or they can be produced by subjecting substances obtained from natural products as raw materials to treatment such as reaction.

[0019] 2. Use of Compound (Part 1) A first aspect of the present disclosure is an ultraviolet absorber, antioxidant, or anti-glycation agent containing, as an active ingredient, at least one compound selected from the group consisting of a compound represented by the above formula (1) and a compound represented by the above formula (2). Hereinafter, the compound represented by the above formula (1) and at least one compound selected from the group consisting of a compound represented by the above formula (2) will also be referred to as saclipin B and / or saclipin A. Furthermore, saclipin B and saclipin A will also be collectively referred to simply as saclipin, without distinction.

[0020] Saclipin B has an absorption maximum at 319 nm in the ultraviolet wavelength range, and the molar extinction coefficient at the absorption maximum is 30,555 M －１ cm －１ Saclipin A has an absorption maximum in the ultraviolet wavelength range of 315 nm to 316 nm, and the molar extinction coefficient at the absorption maximum is 26,454 M －１ cm －１Saclipin B and / or saclipin A have sufficient ultraviolet absorbing activity and are useful as active ingredients of ultraviolet absorbers. Saclipin B has a higher molar extinction coefficient at the absorption maximum than saclipin A. Therefore, it is preferable that the ultraviolet absorber contains at least saclipin B.

[0021] The form of the ultraviolet absorber is not particularly limited. The ultraviolet absorber is suitable for use as an external preparation for skin, a cosmetic, a food or drink, a pharmaceutical, a quasi-drug, or the like. The ultraviolet absorber can effectively exert its effects, particularly when applied to the skin surface. Therefore, among the above forms, it is particularly suitable for use as an external preparation for skin or a cosmetic.

[0022] Furthermore, saclipin B and / or saclipin A have antioxidant properties and are useful as active ingredients of antioxidants. Saclipin B has higher radical scavenging activity than saclipin A. Therefore, it is preferable that the antioxidant contains at least saclipin B.

[0023] Saclipin B and saclipin A are slow-acting antioxidants that can maintain antioxidant activity for a longer period of time than ascorbic acid and Trolox (6-hydroxy-2,5,7,8-tetramethylchromato-2-carboxylic acid). Specific explanations are given below. In the measurement of ABTS radical scavenging activity in the Examples below, the IC 20 minutes after adding the antioxidants to the reaction solution was ５０ The value is taken as α, and the IC ５０ When the value is β, the β / α values ​​of saclipin B, saclipin A, ascorbic acid, and Trolox are as follows: Ascorbic acid and Trolox have β / α values ​​of 0.95 or more, while saclipin B and / or saclipin A have β / α values ​​of 0.8 or less. The β / α value is usually 1 or less, and a value less than 1 is an indicator of the progress of the radical scavenging reaction between 20 and 40 minutes. β / α value of Saclipin B = 0.74 β / α value of Saclipin A = 0.78 β / α value of ascorbic acid = 0.95 β / α value of Trolox = 1.00

[0024] The form of the antioxidant is not particularly limited. The form of the antioxidant is suitable for use in foods, beverages, and pharmaceuticals. In such a form, the antioxidant can eliminate active oxygen in the body when orally ingested, thereby exhibiting a health-promoting effect. Furthermore, by incorporating the antioxidant into topical skin preparations, cosmetics, or quasi-drugs, it is expected to have effects such as preventing the oxidation of the contained ingredients and preventing aging of the skin, etc.

[0025] Furthermore, saclipin B and / or saclipin A have an anti-glycation effect and are also useful as active ingredients of anti-glycation agents. For example, saclipin B and / or saclipin A have been confirmed to be useful as anti-glycation agents that inhibit the glycation of elastin. Saclipin B has a stronger activity of inhibiting the glycation of elastin than saclipin A. As an anti-glycation agent that inhibits the glycation of elastin, it is preferable to include saclipin B. Furthermore, saclipin B and / or saclipin A have been confirmed to be particularly useful as anti-glycation agents that inhibit the glycation of collagen. Saclipin A has a stronger activity of inhibiting the glycation of collagen than saclipin B. As an anti-glycation agent that inhibits the glycation of collagen, it is preferable to include saclipin A. It is known that at least some common reactions are involved in the glycation reactions of various proteins. Saclipin B and / or saclipin A are presumed to be useful as anti-glycation agents that inhibit the glycation reactions of proteins other than elastin and collagen.

[0026] The form of the anti-glycation agent is not particularly limited. The form of the anti-glycation agent is suitable for use as a food or drink or a pharmaceutical. In such a form, oral ingestion of the anti-glycation agent inhibits glycation of proteins in blood vessel walls and skin tissues in the living body, thereby preventing hardening of blood vessels and deterioration of skin. Furthermore, by incorporating the anti-glycation agent into topical skin preparations, cosmetics, or quasi-drugs, it is expected to have effects such as preventing aging of the skin, etc.

[0027] The ultraviolet absorber, antioxidant, or anti-glycation agent may contain, in addition to saclipin B and saclipin A, appropriate components used in conventional ultraviolet absorbers, antioxidants, anti-glycation agents, etc., within quantitative and qualitative ranges that do not impair the effects of the present disclosure. The total amount of saclipin B and saclipin A in the ultraviolet absorber, antioxidant, or anti-glycation agent is not particularly limited. From the viewpoints of ensuring ease of storage, ease of application, ease of drinking, and other handleability, as well as efficacy, the total amount of saclipin B and saclipin A may be 0.0001% by mass to 100% by mass, where the mass of the entire agent is taken as 100% by mass.

[0028] The dosage of oral preparations in the form of foods, beverages, pharmaceuticals, etc. can be varied as appropriate depending on factors such as the age, weight, sex, and condition of the recipient. For example, the intake or administration amount of an oral preparation is, for example, 0.0001 mg to 100 mg per day for a human adult weighing 60 kg, calculated as the dry mass of saclipin B and saclipin A, but is not limited to this range. If necessary, the above intake amount can be taken once or several times per day, for example, in divided doses, divided into two or three times. The dosage of topical skin preparations, cosmetics, or quasi-drugs is not particularly limited. Typically, an appropriate amount can be applied to the outer skin, such as the skin, several times a day, for example, by painting. Examples of application of topical skin preparations or cosmetics will be described later.

[0029] 3. Use of Compound (Part 2) A second aspect of the present disclosure is a skin external preparation or cosmetic containing at least one compound selected from the compound represented by formula (1) and the compound represented by formula (2).

[0030] When saclipin B and / or saclipin A are used as a topical skin preparation, the topical skin preparation may further contain an excipient, a base, an emulsifier, a solvent, a stabilizer, etc. The excipients, carriers, and additives are not particularly limited as long as they are commonly used and physiologically or pharmaceutically acceptable, and their types and compositions can be appropriately changed. Examples of dosage forms include ointments, liquids, sprays, sheets, powders, and dusts.

[0031] The total amount of saclipin B and saclipin A in the topical skin preparation is not particularly limited. From the viewpoints of ensuring storage stability, ease of application, and efficacy, the total amount of saclipin B and saclipin A in the topical skin preparation can be 0.00001% by mass to 10% by mass, where the mass of the entire topical skin preparation is 100% by mass.

[0032] When saclipin B and / or saclipin A are used as a cosmetic, the cosmetic may further contain ingredients commonly used as cosmetic ingredients, such as surfactants, oils, moisturizers, film-forming agents, colorants, fragrances, etc. Examples of the form of the cosmetic include lotions, creams, emulsions, gels, aerosols, essences, packs, cleansers, foundations, dusting powders, and various makeup products (lipstick, blush, etc.).

[0033] The total amount of saclipin B and saclipin A in the cosmetic is not particularly limited. From the viewpoint of ensuring storage stability, ease of handling, and efficacy, the total amount of saclipin B and saclipin A in the cosmetic can be 0.00001% by mass to 10% by mass, where the mass of the entire cosmetic is 100% by mass.

[0034] 4. Compound Production Method (Part 1) A third aspect of the present disclosure is a compound production method that involves extracting at least one compound selected from the group consisting of the compound represented by formula (1) and the compound represented by formula (2) from cyanobacteria.

[0035] As an example of a method for producing a compound, a method of drying cyanobacteria and extracting a compound from the dried cyanobacteria will be described. Note that drying of cyanobacteria is an optional process and may be performed as needed. Below, a specific description will be given of a method for producing a compound using Aphanothece sacrum as an example of cyanobacteria.

[0036] The conditions for drying A. saccharinum are not particularly limited. The drying method is not particularly limited and may be either natural drying or artificial drying. Natural drying methods include air drying, shade drying, and sun drying. Artificial drying methods include heat drying, hot air drying, vacuum drying, and freeze drying. From the viewpoint of improving drying efficiency and suppressing the inactivation of enzymes involved in the synthesis of saclipin B and saclipin A, the drying temperature is preferably 10°C or higher and 60°C or lower, more preferably 15°C or higher and 50°C or lower, and even more preferably 20°C or higher and 40°C or lower. The drying time can be appropriately determined depending on the selected drying method, the moisture state of the A. saccharinum, and the like. In the case of natural drying, the drying time can be, for example, half a day to several days. In the case of artificial drying, the drying time can be, for example, 1 minute to 48 hours or lower. Commercially available dried processed A. saccharinum products may be used as the dried product.

[0037] By drying A. saclipin B, the yield of A. saclipin B and A can be increased compared to when A. saclipin B is kept wet. Although the mechanism is unclear, it is speculated that one reason is that when A. saclipin B is dried while still alive, the production of A. saclipin B and A is induced by drought stress. However, the present disclosure is not limited by this speculation.

[0038] The method for extracting the compounds is not particularly limited. As an example of a compound extraction method, a method will be described in which A. saclipinum is crushed, an extraction solvent is added, and the mixture is stirred to obtain saclipin B and saclipin A as an extract. Crushing and stirring are optional treatments, and as long as a sufficient yield is obtained, for example, A. saclipinum may be immersed in an extraction solvent and allowed to stand to extract saclipin B and / or saclipin A.

[0039] Amorphophallus saccharinus can be crushed using, for example, a mixer, an ultrasonic crusher, a bead crusher, or the like. Amorphophallus saccharinus may be crushed using a combination of multiple means. For example, when crushing dried Amorphophallus saccharinus, the dried product may be pulverized using a mixer or the like, and then subjected to ultrasonic treatment in an extraction solvent to crush the product. Furthermore, when crushing Amorphophallus saccharinus that forms colonies wrapped in an agar matrix, the wet Amorphophallus saccharinus may be homogenized using a mixer or the like, and then subjected to ultrasonic treatment in an extraction solvent to crush the product. The ultrasonic treatment frequency may be, for example, 15 kHz to 50 kHz. The ultrasonic treatment temperature may be, for example, 4°C or higher and 40°C or lower. The ultrasonic treatment time may be, for example, 5 minutes to 60 minutes.

[0040] Examples of extraction solvents that can be used include alcohols such as methanol, ethanol, propanol, and butanol; glycols such as 1,3-butylene glycol, glycerin, and propylene glycol; esters such as ethyl acetate and butyl acetate; ethers such as ethyl 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; hexane, cyclohexane, petroleum ether, and water. These solvents can be used alone or in combination.

[0041] The extraction conditions are not particularly limited. The extraction temperature can be, for example, 4°C or higher and 40°C or lower. The extraction time can be, for example, 5 minutes to 6 hours. The amount of extraction solvent added can be 10 mL to 100 mL per 10 g of dry weight of Aphanothece sacrum.

[0042] The extract can be obtained by, for example, centrifugal separation, filtration, suction, squeezing, etc., followed by solid-liquid separation from the residue. Saclipin B and saclipin A obtained as the extract can be used as is, or can be used in the form of an extract or powder after appropriate concentration or solvent removal. Furthermore, if necessary, they can be purified by one or a combination of two or more suitable separation and purification means, such as chromatography.

[0043] 5. Method for Producing Compound (Part 2) A fourth aspect of the present disclosure is a method for producing a compound, comprising irradiating a solution of a compound represented by the above formula (2) with light to obtain a compound represented by the above formula (1).

[0044] 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 the solvent include methanol, ethanol, and acetonitrile. The above-mentioned extraction solvent may also be used as the solvent. Furthermore, the above-mentioned extract may also be used as the Saclipin A solution. That is, the method for producing the present compound may involve irradiating Saclipin A derived from Aphanothece saclipin with light.

[0045] The conditions for light irradiation are not particularly limited. The illuminance of light irradiation can be, for example, 4,000 lux or more and 10,000 lux or less. The temperature during light irradiation can be, for example, 4°C or more and 40°C or less. The light irradiation time can be, for example, 12 hours or more and 48 hours or less. The isomerization rate due to light irradiation is not particularly limited. The isomerization rate is usually 92% or less and can be appropriately adjusted, for example, within the range of 10% to 92% to achieve the desired ratio of saclipin A to saclipin B.

[0046] 6. Derivative Compound A fifth aspect of the present disclosure is at least one compound selected from the group consisting of derivatives of the compound represented by the above formula (1) and derivatives of the compound represented by the above formula (2). In the following description, the derivatives of the compound represented by the above formula (1) are also referred to as saclipin B derivatives, and the derivatives of the compound represented by the above formula (2) are also referred to as saclipin A derivatives.

[0047] In the present disclosure, a saclipin B derivative refers to a derivative in which one or more functional groups present in saclipin B have been modified, and has functionality equivalent to or greater than that of saclipin B. A saclipin A derivative refers to a derivative in which one or more functional groups present in saclipin A have been modified, and has functionality equivalent to or greater than that of saclipin A. The above-mentioned "functionality" is not particularly limited, and examples include ultraviolet absorption, antioxidant properties, anti-glycation properties, etc.

[0048] The saclipin B derivative is preferably one or more selected from the group consisting of an ester derivative obtained by reacting the carboxyl group present at the terminal of saclipin B with an alcohol, an amide derivative obtained by reacting the carboxyl group present at the terminal of saclipin B with an amine, and an ammonium salt obtained by reacting the carboxyl group present at the terminal of saclipin B with ammonium. The saclipin A derivative is preferably one or more selected from the group consisting of an ester derivative obtained by reacting the carboxyl group present at the terminal of saclipin A with an alcohol, an amide derivative obtained by reacting the carboxyl group present at the terminal of saclipin A with an amine, and an ammonium salt obtained by reacting the carboxyl group present at the terminal of saclipin A with ammonium. The type of carboxyl group derivative in the saclipin B derivative and / or saclipin A derivative can be appropriately selected depending on the intended use, dosage form, etc.

[0049] The carboxyl group derivative group is, for example, —COOR １ , -CONH ２ , -CONHR ２ , -CONR ３ R ４ , -COONH ４ , -COONH ３ R ５ where R １ , R ２ , R ３ , R ４ , R ５are each independently a hydrocarbon group having 1 to 10 carbon atoms which may have a functional group. ３ saclipinB having -COOCH ３ Saclipin A having the formula (I) is also referred to as methyl-esterified saclipin B or methyl-esterified saclipin A. Methyl-esterified saclipin B and methyl-esterified saclipin A can enhance hydrophobicity while maintaining UV absorbency equivalent to that of saclipin B and saclipin A. Such saclipin B derivatives and / or saclipin A derivatives are more suitable for use as a mixture with a hydrophobic component such as an oil than saclipin B and / or saclipin A.

[0050] A fifth aspect of the present disclosure includes an ultraviolet absorber, antioxidant, or anti-glycation agent containing a saclipin B derivative and / or a saclipin A derivative as an active ingredient. The description of the ultraviolet absorber, antioxidant, or anti-glycation agent of the fifth aspect applies as is to the above "2. Use of Compound (Part 1)" by replacing "saclipin A" and "saclipin B" with "saclipin A derivative" and "saclipin B derivative."

[0051] A fifth aspect of the present disclosure includes a skin topical preparation or cosmetic containing a saclipin B derivative and / or a saclipin A derivative. The explanation of the skin topical preparation or cosmetic of the fifth aspect applies as is, with the above description in "3. Use of Compound (Part 2)" being replaced with "saclipin A derivative" and "saclipin B derivative."

[0052] The present disclosure will be described in more detail below with reference to examples, although the scope of the present disclosure is not limited to these examples.

[0053] 1. Extraction and Isolation of Compounds Extraction and isolation of compounds from A. sacchariflorus was performed as follows. Specifically, 10 g of dried A. sacchariflorus (product name: "Jusen-Koke," sold by Endo Kinsendo, 2949 Yanaga, Asakura City, Fukuoka Prefecture, 838-0031) was crushed using a crusher mill IFM-C20G (Iwatani). The resulting powder was transferred to a 50 ml tube, and then 30 ml of methanol was added. The mixture was sonicated using an ultrasonicator Model UR-200P (Tomy Seiko). The resulting suspension was centrifuged at 4°C and 2330 × g for 15 minutes, and the resulting supernatant was transferred to another new tube. 20 ml of methanol was added to the remaining pellet, and the mixture was sonicated and centrifuged as above. The resulting supernatant was also combined. The supernatant was then dried in a centrifugal concentrator VC-36R (Taitec) under light-protected conditions. The dried material was dissolved in 5 ml of 55% (v / v) acetonitrile containing 0.1% (v / v) formic acid and then passed through a 0.22 μm pore size filter. This sample was subjected to preparative HPLC using an Inertsil ODS-3 column (10 μm particle size; 50 mm i.d. × 20 mm, GL Sciences) connected to an Inertsil ODS-3 column (10 μm particle size; 250 mm i.d. × 20 mm, GL Sciences). The mobile phase was 55% (v / v) acetonitrile containing 0.1% (v / v) formic acid, and the flow rate was 6.0 ml / min. Compound elution was monitored at a detection wavelength of 320 nm. In this preparative HPLC, saclipin A and saclipin B eluted in the same fraction. Fractions containing the target compound were lyophilized, and the dried sample was dissolved in 50% (v / v) acetonitrile and subjected to preparative HPLC using a YMC carotenoid column (particle size 5 μm; inner diameter 250 mm x 10 mm). The mobile phase was 50% (v / v) acetonitrile, and the column was operated at a flow rate of 1.0 ml / min. Compound elution was monitored at a detection wavelength of 320 nm. In this preparative HPLC, saclipin A eluted first, followed by saclipin B. Each fraction was lyophilized to obtain pale green dried samples of saclipin A and saclipin B.From a 10 g sample of dried A. saclipin A, 7.0 mg to 10 mg and 1.5 mg to 3.0 mg of saclipin B were obtained, respectively. The content of saclipin A in dried A. saclipin B was higher than that of saclipin B.

[0054] 2. Structural determination of saclipin A and saclipin B The obtained dried sample was dissolved in methanol, and absorption spectrum analysis, accurate mass analysis, １ H and １３ C-NMR spectra were measured. The absorption spectra of Saclipin A and Saclipin B are shown in Figure 3. In each graph in Figure 3, the horizontal axis represents wavelength (nm) and the vertical axis represents relative absorbance.

[0055] (1) saclipin A [Physicochemical data] Absorption maximum (methanol solution): 316 nm Molar extinction coefficient: 26,454 M －１ cm －１ Precision ESI MS data: calcd for C １８ H ２５ O ４ ([M-H] － ): m / z 305.1758; Found: m / z 305.1758 Precision ESI MS fragment ions: m / z 287.1654, 249.1497, 135.0815, 125.0971 １ H-NMR and １３ C-NMR (CD ３ OD):

[0056] [Two-dimensional NMR] As a result of HMBC (Heteronuclear multiple-bond correlation spectroscopy) analysis, a proton-carbon HMBC correlation was observed as shown by the arrow in Figure 4. Furthermore, as a result of NOESY (Nuclear overhauser effect spectroscopy) analysis, an NOE correlation was observed as shown by the dashed line in Figure 4.

[0057] From the above physicochemical data, the chemical structure of saclipin A obtained from dried Aphanothece saclipin was determined.

[0058] (2) Saclipin B [Physicochemical data] Absorption maximum (methanol solution): 319 nm Molar extinction coefficient: 30,555 M －１ cm －１ Precision ESI MS data: calcd for C １８ H ２５ O ４ ([M-H] － ): m / z 305.1758; Found: m / z 305.1757 Precision ESI MS fragment ions: m / z 287.1655, 249.1496, 135.0815, 125.0971 １ H-NMR and １３ C-NMR (CD ３ OD):

[0059] As a result of HMBC (Heteronuclear multiple-bond correlation spectroscopy) analysis, a proton-carbon HMBC correlation was observed, as shown by the arrow in Figure 5. Furthermore, as a result of NOESY (Nuclear overhauser effect spectroscopy) analysis, an NOE correlation was observed, as shown by the dashed line in Figure 5. From the above physicochemical data, the chemical structure of saclipin B obtained from dried Aphanothece saclipin B was determined.

[0060] 3. Induction of saclipin A and saclipin B by drying. Saclipin A and saclipin B can be induced by drying raw A. 50 g of fresh A. saclipin collected from the Kogane River was mixed with 10 ml of river water and homogenized using a crusher IFM-C20G (Iwatani). The homogenized sample was divided into 5.0 g portions, placed on plastic trays, and dried in a DY400 drying oven (Yamato Scientific) set at 30°C under dark conditions. A control experiment was also conducted in which the samples were kept moist while sealed in plastic bags. Water was added to the dried sample to match the weight before drying, and then methanol was added to a final concentration of 80% (v / v). Saclipin was then extracted by sufficient sonication using a Model UR-200P ultrasonicator (Tomy Seiko). The sonicated samples were centrifuged at 2330 × g for 15 minutes at 4 °C, and the resulting supernatant was subjected to HPLC analysis to quantify saclipin. While the saclipin A and saclipin B contents in the control sample without drying were 0.005 mg / g and 0.001 mg / g, respectively, the drying treatment significantly increased the contents to 0.056 mg / g and 0.008 mg / g, respectively. These results demonstrate that saclipin A and saclipin B are substances induced during the drying process of Aphanothece saccharin.

[0061] 4. Production of saclipin B by photoisomerization Purified saclipin A from dried A. saclipin was dissolved in methanol, ethanol, or acetonitrile, and the solution was irradiated with 6000 lux of white fluorescent light for 24 hours or more. The amount of saclipin A and saclipin B was then calculated by HPLC analysis under the following measurement conditions, and the isomerization rate of saclipin A was calculated. The chromatogram of the saclipin A solution before light irradiation is shown on the left in Figure 6, and the chromatogram of the saclipin A solution after light irradiation is shown on the right in Figure 6. [Measurement conditions] Detection wavelength: 320 nm Column: Inerteil ODS-P 3 μm, 4.6 x 33 + 4.6 x 150 mm Eluent: 50% acetonitrile (isocratic)

[0062] Approximately 90% of the dissolved saclipin A was converted to saclipin B. The ratio of saclipin A to saclipin B did not change for at least 96 hours after light irradiation and subsequent dark treatment. This result demonstrated that saclipin B was produced from saclipin A by a photoisomerization reaction.

[0063] 5. Measurement of Physiological Activity of Saclipin A and Saclipin B (1) Test Example 1: Evaluation of Antioxidant Activity (1.1) Measurement of ABTS Radical Scavenging Activity To evaluate the antioxidant activity of Saclipin A and Saclipin B, the radical scavenging activity of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was measured. An ABTS radical solution was prepared by mixing a 7 mM aqueous solution of ABTS with a 2.45 mM aqueous solution of potassium persulfate at 25°C for 16 hours in the dark. The ABTS radical solution was diluted with ethanol to an absorbance of approximately 1.0 at 734 nm. 50 μL of this diluted ABTS radical solution was mixed with 50 μL of a test sample in ethanol. The mixture was incubated at room temperature in the dark for 20 or 40 minutes, and then the absorbance at 734 nm was measured. The final reaction concentrations of the samples 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: Inhibitory activity (%) = ((Ab - As) / Ab) × 100, where Ab and As represent the absorbance of the blank sample and test sample, respectively. The 50% inhibitory concentration (IC) calculated from the measurement results was used as the 50% inhibitory concentration (IC ５０ ) values ​​are shown in Table 3.

[0064] The results of the measurement of antioxidant activity are as follows:

[0065] ICs shown in Table 3 ５０ From these values, it was confirmed that both saclipin A and saclipin B exhibited radical scavenging activity against the ABTS radical. It was also found that saclipin B had higher activity than saclipin A.

[0066] (1.2) Evaluation of the Time Course of the Antioxidant Activity of Saclipin The experimental results in Table 3 show that the ABTS radical scavenging rate by saclipin increased after 40 minutes of reaction compared to 20 minutes, suggesting that saclipin is a gradually functioning antioxidant. Therefore, the time course of radical scavenging was measured using a time course experiment. In the time course experiment, the decrease in absorbance after the addition of the test sample was continuously recorded for 2900 seconds at 25°C and 734 nm. In the time course 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.

[0067] The results of the time-course experiment are as follows. It was found that the radical scavenging reaction of both saclipin A and saclipin B was still progressing even 2,900 seconds after their addition to the reaction system (Fig. 7(A) and (B)). The antioxidants ascorbic acid and Trolox used as standard samples showed almost no effect after the radical scavenging reaction occurred immediately after their addition (Fig. 7(C) and (D)). These results demonstrate that both saclipin A and saclipin B have radical scavenging activity and are slow-acting antioxidants.

[0068] (2) Test 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. The effects of saclipin on the glycation of elastin and collagen were evaluated using an elastin anti-glycation assay kit (product number: AAS-AGE-K05, Cosmo Bio Co., Ltd.) and a collagen anti-glycation assay kit (product number: AK71, Cosmo Bio Co., Ltd.), respectively. The assay was performed using saclipin A and saclipin B purified from Aphanothece saclipin according to the manufacturer's protocol. However, since saclipin was not soluble in the aqueous buffer provided, it was dissolved in dimethyl sulfoxide (DMSO) before use. 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 measurement results are shown in Figures 8 and 9.

[0069] The results of measuring the anti-glycation activity are as follows: The IC of saclipin A was measured as the inhibitory effect on the glycation reaction of elastin. ５０ The IC value of aminoguanidine, a known glycation inhibitor, was 2.5 mM. ５０ The IC value of aminoguanidine was comparable to that of saclipin A (1.5 mM), demonstrating that saclipin A inhibits the glycation reaction of elastin (Fig. 8). Although the activity of saclipin B was slightly weaker than that of saclipin A, it inhibited approximately 45% of the glycation reaction at a concentration of 4 mM. On the other hand, as shown in Fig. 9, saclipin showed a stronger inhibitory effect on collagen glycation. ５０ The IC value for saclipin A and saclipin B was 2.4 mM. ５０ The values ​​were 1.9 mM and 0.9 mM, respectively. These results demonstrate that both saclipin A and saclipin B function as inhibitors of the glycation of elastin and collagen, which are components of the dermis.

[0070] 6. Production of saclipin A derivatives and saclipin B derivatives Saclipin A derivatives and saclipin B derivatives were produced using a fatty acid methylation kit (product number 06482-04, Nacalai Tesque, Inc.). The assay was carried out using purified saclipin A and saclipin B according to the manufacturer's protocol. However, reagent B was not used, and only reagent C was used. Reagent C is a reagent that methylates free fatty acids by reacting free fatty acids with methanol in the presence of an acid catalyst. After adding and mixing the extraction reagent, the mixture separated into two layers, and the upper layer was collected.

[0071] The components obtained by separating the upper layer were subjected to reverse-phase HPLC analysis. Saclipin A and saclipin B were also subjected to reverse-phase HPLC analysis under the same conditions. The results are shown in Figure 10. The upper panel of Figure 10 is a chromatogram of saclipin A and saclipin B, and the lower panel is a chromatogram of the upper layer components. In the upper chromatogram, the peak "SacA" at a retention time of 12 min to 15 min is the peak of saclipin A, and the peak "SacB" at a retention time of 15 min to 18 min is the peak of saclipin B. When saclipin A and saclipin B are methyl esterified, their hydrophobicity increases, and their retention times in reverse-phase HPLC analysis become longer. In the chromatogram of the upper layer component in the lower panel, peaks were observed at retention times of 40 min to 50 min and 60 min to 70 min. This indicated that saclipin A and saclipin B were methyl esterified. The respective peaks are labeled "SacA_methyl ester" and "SacB_methyl ester."

[0072] Absorption spectra were obtained for "saclipin A (SacA)," "saclipin B (SacB)," "methyl-esterified saclipin A (SacA methyl ester)," and "methyl-esterified saclipin B (SacB methyl ester)" by the method described above in "2. Determination of the structures of saclipin A and saclipin B." The results are shown in FIG. 11. In each graph in FIG. 11, the horizontal axis represents wavelength (nm) and the vertical axis represents relative absorbance. The maximum point of saclipin A was 317 nm. The maximum point of methyl-esterified saclipin A was 316 nm. The maximum point of saclipin B was 319 nm. The maximum point of methyl-esterified saclipin B was 319 nm.

[0073] These results demonstrate that methyl-esterified saclipin A has an ultraviolet absorbing ability equivalent to that of saclipin A. Furthermore, it has been demonstrated that methyl-esterified saclipin B has an ultraviolet absorbing ability equivalent to that of saclipin B.

[0074] It is presumed that the functionality of saclipin A and saclipin B is mainly due to the moiety having a carbon-carbon double bond. In methyl-esterified saclipin A and methyl-esterified saclipin B, the moiety having a carbon-carbon double bond remains unchanged. Therefore, it is reasonable that methyl-esterified saclipin A and methyl-esterified saclipin B were able to ensure UV absorption ability equivalent to that of saclipin A and saclipin B. In other words, it is presumed that saclipin A derivatives and saclipin B derivatives can ensure functionality equivalent to that of saclipin A and saclipin B with respect to functionality other than UV absorption ability, as long as they have a moiety having a carbon-carbon double bond.

[0075] 7. Biocompatibility Test To examine the cytotoxicity of Saclipin A and Saclipin B, an MTT test was performed on (1) neonatal human dermal fibroblasts and (2) human keratinocytes. (1) MTT test on neonatal human dermal fibroblasts Neonatal human dermal fibroblasts (NHDF) were purchased from ATCC. Neonatal human dermal fibroblasts were cultured at 1 x 10 ４ Neonatal human dermal fibroblasts were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg / mL penicillin, 100 units / mL streptomycin, and 10 mM HEPES at 37°C and 5% CO ２ The cells were cultured overnight under the conditions described above. 0.1 μM, 0.36 μM, 1.0 μM, 3.6 μM, or 10.0 μM of saclipin A or saclipin B was added to each cell. As a control, 1% ethanol was added to the cells. Next, 0.5 mg / mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added, and the cells were incubated at 37°C and 5% CO ２ The cells were cultured under these conditions for 20 hours. After dissolving all the formazan crystals in DMSO (dimethyl sulfoxide), the absorbance of each sample solution was measured at 540 nm to determine the cell viability. The results are shown in Figure 12.

[0076] The upper graph in Figure 12 is a graph showing the results of cell viability (%). The labels on the horizontal axis represent the samples corresponding to the bar graphs. For example, the labels "saclipin A" and "10.00" indicate that cells were treated with 10.0 μM saclipin A. The vertical axis represents cell viability when the cell viability of cells treated with 1% ethanol is set at 100%. From the upper graph in Figure 12, it can be seen that saclipin A was not more cytotoxic than 1% ethanol at any concentration. Similarly, saclipin B was not more cytotoxic than 1% ethanol at any concentration.

[0077] The bottom row of Figure 12 shows, from left to right, micrographs of "cells treated with 1% ethanol," "cells treated with 10.0 μM saclipin A," and "cells treated with 10.0 μM saclipin B." The "cells treated with 10.0 μM saclipin A" had the same appearance as the "cells treated with 1% ethanol," and no abnormalities were observed. This suggests that saclipin A has high biocompatibility. The "cells treated with 10.0 μM saclipin B" had the same appearance as the "cells treated with 1% ethanol," and no abnormalities were observed. This suggests that saclipin B has high biocompatibility.

[0078] (2) MTT Test on Human Keratinocytes PSVK1 cells (JCRB1093, JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, Japan) were used as human keratinocytes. In this test, the cells were cultured in keratinocyte 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). The test was performed in the same manner as in "(1) MTT Test on Neonatal Human Dermal Fibroblasts" above. The results are shown in Figure 13.

[0079] The upper graph in Figure 13 is a graph showing the results of cell viability (%). The labels on the horizontal axis represent the samples corresponding to each bar graph. For example, the labels "saclipin A" and "10.00" indicate that cells were treated with 10.0 μM saclipin A. The vertical axis represents cell viability when the cell viability of cells treated with 1% ethanol is set at 100%. From the upper graph in Figure 13, it can be seen that saclipin A was not more cytotoxic than 1% ethanol at any concentration. Similarly, saclipin B was not more cytotoxic than 1% ethanol at any concentration.

[0080] The bottom row of Figure 13 shows, from left to right, micrographs of "cells treated with 1% ethanol," "cells treated with 10.0 μM saclipin A," and "cells treated with 10.0 μM saclipin B." The "cells treated with 10.0 μM saclipin A" had the same appearance as the "cells treated with 1% ethanol," and no abnormalities were observed. This suggests that saclipin A has high biocompatibility. The "cells treated with 10.0 μM saclipin B" had the same appearance as the "cells treated with 1% ethanol," and no abnormalities were observed. This suggests that saclipin B has high biocompatibility.

[0081] 8. Summary According to this example, it was possible to provide novel ultraviolet absorbers, antioxidants, anti-glycation agents, topical skin preparations, and cosmetics. According to this example, it was possible to provide novel manufacturing methods for compounds. According to this example, it was possible to provide novel compounds.

Claims

1. An ultraviolet absorber, antioxidant, or anti-glycation agent comprising at least one compound represented by the following formula (1) and the following compound represented by the following formula (2) as an active ingredient. 【Chemistry 1】 【Chemistry 2】

2. A topical skin preparation or cosmetic containing at least one compound represented by the following formula (1) and the following compound represented by the following formula (2). 【Transformation 3】 【Chemistry 4】

3. A method for producing compounds, comprising extracting at least one compound represented by the following formula (1) and the following compound represented by the following formula (2) from cyanobacteria. 【Transformation 5】 【Transformation 6】

4. A method for producing a compound according to claim 3, wherein the cyanobacteria are dried before extraction of the compound.

5. A method for producing a compound, comprising irradiating a solution of the compound represented by the following formula (2) with light to obtain the compound represented by the following formula (1). 【Transformation 7】 【Transformation 8】

6. At least one compound selected from the group consisting of derivatives of the compound represented by the following formula (1) and derivatives of the compound represented by the following formula (2). 【Chemistry 9】 【Chemistry 10】

7. A derivative of the compound represented by formula (1) is a derivative in which the carboxyl group of the compound represented by formula (1) is converted to -COOR 1, -CONH 2, -CONHR 2, -CONR 3R 4, -COONH 4, or -COONH 3R 5 (wherein R 1, R 2, R 3, R 4, and R 5 are each independently hydrocarbon groups having 1 to 10 carbon atoms), The compound according to claim 6, wherein a derivative of the compound represented by formula (2) is a derivative in which the carboxyl group of the compound represented by formula (2) is converted to -COOR 1, -CONH 2, -CONHR 2, -CONR 3R 4, -COONH 4, or -COONH 3R 5 (wherein R 1, R 2, R 3, R 4, and R 5 are each independently hydrocarbon groups having 1 to 10 carbon atoms).

8. A derivative of the compound represented by formula (1) is a derivative in which the carboxyl group of the compound represented by formula (1) is converted to -COOCH3, The compound according to claim 6, wherein the derivative of the compound represented by formula (2) is a derivative in which the carboxyl group of the compound represented by formula (2) is converted to -COOCH3.

9. An ultraviolet absorber, antioxidant, or anti-glycation agent comprising the compound described in claim 6 as an active ingredient.

10. A topical skin preparation or cosmetic containing the compound described in claim 6.

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