JP6614448B2 - Anti-photoaging agent - Google Patents

Description

  The present invention relates to an anti-photoaging agent.

Sun rays are known to have various effects on human skin. Sunlight contains light of various wavelengths. Of the sun rays, it is said that ultraviolet rays A (UVA), ultraviolet rays B (UVB), and near infrared rays have an adverse effect on the skin.
Although UVA (wavelength 320 nm to 400 nm) has a lower energy amount than UVB (wavelength 280 nm to 320 nm), it reaches the dermis deep in the skin. Long-term skin exposure to UVA causes photoaging such as wrinkles and reduced elasticity.

  More specifically, when the skin is irradiated with UVA, the expression of matrix metalloproteinase (MMP), which is a collagenase, is increased in the dermis inside the epidermis. For this reason, degradation of collagen forming the dermis proceeds. Moreover, when UVA is irradiated to the skin, elastin and hyaluronic acid, which are matrix components that form the dermis together with collagen, are also reduced (see, for example, Non-Patent Document 1). A decrease in matrix components causes a decrease in wrinkles and skin elasticity.

  UVA also penetrates glass that UVB cannot penetrate. For this reason, UVA also reaches the human skin in the room. For these reasons, in recent years, the importance of suppressing adverse effects on the skin caused by UVA has increased.

Conventionally, an external preparation for skin containing an ultraviolet shielding agent has been used in order to avoid the adverse effects of ultraviolet rays on the skin. As the ultraviolet shielding agent, various organic or inorganic ultraviolet absorbers and / or ultraviolet scattering agents are known.
For example, Patent Document 1 describes a cosmetic comprising a combination of titanium oxide particles and zinc oxide particles coated with silica, alumina, or alumina / silica.
Patent Document 2 describes an ultraviolet absorbent composition containing an ultraviolet absorbing component extracted from a bacterium belonging to the genus Methylobacterium which is a plant-inhabiting microorganism.

JP 2002-154915 A JP2013-127027A

Journal of Japanese Dermatological Association 122 (14), 3717-3723,2012

Conventionally, there has been no anti-photoaging agent that has a function of shielding the skin from UVA and physiologically suppresses photoaging of the skin caused by exposing the skin to UVA.
This invention is made | formed in view of the said situation, and makes it a subject to provide the anti-photoaging agent which suppresses the photoaging of the skin which arises by exposing skin to UVA.

In order to solve the above-mentioned problems, the present inventors paid attention to a compound represented by the following formula (I) having UVA absorption ability, and conducted intensive studies. As a result, the present inventors have found that the compound represented by the formula (I) has an action of suppressing the photoaging of the skin caused by exposing the skin to UVA, and completed the present invention. .
That is, the present invention adopts the following configuration.

[1] An anti-photoaging agent comprising a compound represented by the formula (I) or a salt thereof.

(In Formula (I), R ₁ and R ₂ are each independently a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or a linear or branched structure having 1 to 5 carbon atoms. Represents an alkoxy group having 1 to 5 carbon atoms, or a linear or branched alkenyl group having 1 to 5 carbon atoms, R ₃ represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or 1 to 5 carbon atoms. X represents a hydrogen atom or a linear or branched sugar chain having 1 to 10 sugar molecules.)

[2] A compound represented by the formula (I) or a salt thereof, a compound represented by the formula (II) or a salt thereof, wherein R ₁ and R ₂ in the formula (I) are methyl groups and R ₃ is a hydrogen atom The anti-photoaging agent according to [1].

[3] In Formula (I), R ₁ and R ₂ are methyl groups, R ₃ is a hydrogen atom, and X is a linear sugar chain having 5 sugar molecules. Photoaging agent.

  The anti-photoaging agent of the present invention contains a compound represented by the formula (I) or a salt thereof. Thus, when the skin coated with the anti-photoaging agent of the present invention is exposed to UVA, it promotes gene expression of collagen, elastin, and hyaluronic acid as matrix components and matrix metalloproteinase as a collagen degrading enzyme. Suppresses the expression of a gene that produces (MMP). For this reason, in the skin to which the anti-photoaging agent of the present invention is applied, when exposed to UVA, the matrix component in the dermis is less likely to decrease, and wrinkles and a decrease in elasticity associated with the decrease in the matrix component are suppressed. Photoaging is prevented.

  The anti-photoaging agent of the present invention has UVA absorption ability. Therefore, the anti-photoaging agent of this invention shields skin from UVA when the skin which apply | coated this is exposed to UVA, and suppresses the photoaging of the skin by exposure to UVA effectively.

It is the graph which showed the relationship between the ultraviolet absorption intensity and the elution time in the HPLC analysis of the crudely purified product obtained in the examples.

Hereinafter, the present invention will be described in detail.
"Anti-photoaging agent"
The anti-photoaging agent of this embodiment contains a compound represented by the formula (I) or a salt thereof as an active ingredient.

(In Formula (I), R ₁ and R ₂ are each independently a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or a linear or branched structure having 1 to 5 carbon atoms. Represents an alkoxy group having 1 to 5 carbon atoms, or a linear or branched alkenyl group having 1 to 5 carbon atoms, R ₃ represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or 1 to 5 carbon atoms. X represents a hydrogen atom or a linear or branched sugar chain having 1 to 10 sugar molecules.)

In the formula (I), R ₁ is a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a C _{1 to} 5 carbon atom. A linear or branched alkenyl group, preferably a linear alkyl group having 1 to 5 carbon atoms, a methoxy group, or a vinyl group, and more preferably an alkyl group having 1 to 2 carbon atoms. Preferably, it is a methyl group.

R ₂ represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, a linear or branched group having 1 to 5 carbon atoms. It is preferably a linear alkenyl group having 1 to 5 carbon atoms and a methoxy group, more preferably an alkyl group having 1 to 2 carbon atoms, and most preferably a methyl group. preferable.
In addition, when R ₂ is a linear or branched alkenyl group having 1 to 5 carbon atoms, if it is a —CH ₂ —C ₄ H ₇ group, it has an influence on the conjugation of electrons related to the ultraviolet absorption ability. It is preferable because there are few.

R ₃ is a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkenyl group having 1 to 5 carbon atoms, a hydrogen atom or an alkyl having 1 to 2 carbon atoms. It is preferably a group, more preferably a hydrogen atom or a methyl group, and most preferably a hydrogen atom.

In formula (I), when R ₃ is a hydrogen atom, the carboxyl group possessed by the compound represented by formula (I) may form a salt. When the carboxyl group of the compound forms a salt, it is preferably a carboxylic acid alkali metal salt or a carboxylic acid amine salt, more preferably a carboxylic acid alkali metal salt, and a carboxylic acid sodium salt. Most preferably it is.

  X is a hydrogen atom or a linear or branched sugar chain having 1 to 10 sugar molecules, preferably a hydrogen atom or a linear sugar chain having 1 to 10 sugar molecules, A linear sugar chain having 3 to 8 sugar molecules is more preferable, and a linear sugar chain having 5 or 5 sugar molecules is more preferable.

The compound represented by the formula (I) or a salt thereof is particularly a compound represented by the formula (II) or a salt thereof, wherein R ₁ and R ₂ are methyl groups, and R ₃ and X are hydrogen atoms, or R _It is preferable that ₁ and R ₂ are methyl groups, R ₃ is a hydrogen atom, and X is a straight-chain sugar chain having 5 sugar molecules or a salt thereof. The sugar molecule may be pyranose or furanose, but is preferably pyranose.

R ₁ and R ₂ are methyl groups, R ₃ is a hydrogen atom, X is a linear sugar chain having 5 sugar molecules, or a salt thereof, X is 1,4-glycosidically bonded to each other A compound represented by the formula (III), which is a sugar chain consisting of five hexoses (pyranose) or a salt thereof, and a sugar chain consisting of five hexoses (pyranose) in which X is 1,6-glycosidically bonded to each other A compound represented by a formula (IV) or a salt thereof, wherein X in the formula (I) is a linear sugar chain composed of five hexoses (pyranose), and the hexoses are 1,4-glycosides A compound or a salt thereof which is a sugar chain in which a bonded part and a 1,6-glycoside bonded part are mixed is preferable. A compound represented by formula (III) or a salt thereof, a compound represented by formula (IV) or a salt thereof, and X in formula (I) is a linear sugar chain composed of five hexoses (pyranose) A compound or a salt thereof, which is a sugar chain in which a portion where hexoses are bonded with 1,4-glycosides and a portion where 1,6-glycosides are bonded, is selected from these. Two or more kinds may exist as a mixture in which an arbitrary ratio is mixed.

The compound represented by the formula (II) or a salt thereof may exist as an inner salt in an equilibrium state represented by the formula (V). More specifically, in the formula (V), a structure (N = C—C) between a nitrogen atom double-bonded to a carbon atom and another nitrogen atom in the compound represented by the formula (II) or a salt thereof. = C−N) is in an equilibrium state (N ⁺ = C−C = C−N⇔N−C = C−C = N ⁺ ).

Also in the compound represented by the formula (I), the formula (III), the formula (IV) or a salt thereof, the (N = C—C═C—N) moiety in the formula is in an equilibrium state (N ⁺ = C -C = C-N 分子 N-C = C-C = N ⁺ (see formula (V))) may be present as an inner salt.
In addition, the compound represented by the formula (I) or a salt thereof has a plurality of stereoisomers. The compound represented by the formula (I) or a salt thereof includes all these stereoisomers.

Since the compound or salt thereof contained in the anti-photoaging agent of the present embodiment has the structure represented by the formula (I), it has solubility in an aqueous solvent, and light of the skin caused by exposing the skin to UVA. It is excellent in the function which suppresses aging and the function which absorbs the ultraviolet-ray in a UVA area | region.
The ultraviolet absorptivity of the compound represented by the formula (I) or a salt thereof is determined by the conjugated system in the formula (I) (the structure between the nitrogen atom double-bonded to the carbon atom and the nitrogen atom bonded to R ₁ ( N = C−C = C−N)) is presumed to contribute. Although the detailed mechanism is unknown, by adjusting pH or the like, the part of (N = C—C = C—N) in the formula (I) is in an equilibrium state (N ⁺ = C—C = C−N⇔N− C = C-C = N ⁺ (see formula (V))) and participates in the conjugation of electrons, and is considered to exhibit ultraviolet absorbing ability.

In the compound represented by formula (I) or a salt thereof, R ₁ , R ₂ , R ₃ , and X are all arranged at positions that do not affect the above-described electron conjugation. For this reason, the excellent ultraviolet absorptivity is similarly obtained regardless of R ₁ , R ₂ , R ₃ , and X in the compound represented by the formula (I) or a salt thereof.

"Method for producing compound or salt thereof"
The compound represented by the formula (I) or a salt thereof contained in the anti-photoaging agent of the present embodiment can be produced, for example, by the production method shown below.
That is, a step of culturing microorganisms growing on a plant to obtain microbial cells (first step), a step of extracting microbial cells with a solvent to obtain an extract (second step), and formula (I) from the extract And a step (third step) of recovering the compound represented by the formula:

(First step)
The microorganisms cultured in this embodiment are grown on plants.
The type of plant from which the microorganism is collected is not particularly limited. Examples of plant types include wheat ears, strawberry leaves, evening primrose petals, and rice leaf sheaths. These plants are preferable because many microorganisms of the genus Methylobacterium that produce the compound represented by the formula (I) or a salt thereof are grown.
Examples of the method for collecting microorganisms that grow on the plant from the plant include a method of immersing the plant in a phosphate buffer solution and grinding it in a mortar to obtain a grinding solution containing the microorganisms.

A conventionally known method can be used as a method for culturing microorganisms. Specifically, as a method for culturing microorganisms, a liquid culture method, a solid culture method, a liquid culture method or a solid culture method may be used, and the types of cells to be cultured. It can be appropriately determined according to the like.
Examples of the medium used for culturing the microorganism include standard agar medium, L (Lennox) medium, LB (Luria Bertani) medium, NB (Nutrient Broth) medium, PD (potato dextrose) medium, PPD (potato peptone dextrose). ) Medium, TB (Terrific broth) medium, etc. can be used.

In the present embodiment, as an example of a microorganism culturing method, a case where the following method is used will be described as an example.
First, a microorganism collected from a plant is cultured by a solid culture method, and each microbial cell contained in the microorganism collected from the plant is separated and recovered. Specifically, a grinding liquid containing microorganisms collected from a plant by the above method is applied (smeared) on the surface of a solid medium and cultured to form colonies. Conventionally known conditions can be adopted as culture conditions for microorganisms in the case of using a solid medium. Specifically, for example, aerobic conditions can be set at 25 ° C. for 3 to 7 days.

Next, the cells forming single colonies are collected by a method of scraping the single colonies appearing on the surface of the solid medium, and the individual cells contained in the microorganisms collected from the plant are separated.
Each bacterial cell recovered from a single colony that appears on the surface of the solid medium may be applied to the surface of a new solid medium and cultured (pure culture) for each bacterial cell, if necessary, and recovered. .

Next, the presence or absence of ultraviolet absorbing ability is examined by spectrocolorimetric method, absorptiometric method, etc. for each bacterial cell separated and recovered from the microorganisms collected from the plant in this way.
Next, among the separated and recovered cells, cells having ultraviolet absorbing ability are identified. As a method for identifying a bacterial cell, a conventionally known method such as a method of identifying based on the base sequence of an rRNA gene can be used.

  In the present embodiment, among the identified bacterial cells, the bacterial cells of the genus Methylobacterium are cultured by the liquid culture method. A microorganism belonging to the genus Methylobacterium produces a compound represented by the formula (I) or a salt thereof. Among microorganisms belonging to the genus Methylobacterium, the WI-182 strain (strain name), the W-213 strain (strain name), the f11 strain (strain name), and the 24N-25 strain (strain name) described later are used. Is preferred. These strains are microorganisms that grow on plants, can be increased by culture, and can efficiently produce the compound represented by the formula (I) or a salt thereof. In particular, WI-182 strain (strain name) is preferable because it can be easily increased by culture.

Next, the cells of the genus Methylobacterium separated and recovered from the microorganisms collected from the plant by the above method are cultured by a liquid culture method. When the cells isolated from the microorganisms collected from the plant contain multiple types of Methylobacterium, the highest UV-absorbing ability from the multiple types of Methylobacterium It is preferable to select cells and culture them by a liquid culture method.
In the present embodiment, microorganisms collected from plants are cultured and separated and collected by an individual culture method, and among the obtained bacterial cells, only the cells of the genus Methylobacterium having ultraviolet absorption ability are cultured by a liquid culture method. To do. For this reason, the microbial cell which has the ultraviolet absorption ability contained in the microorganisms extract | collected from the plant can be increased efficiently.

Conventionally known conditions can be employed as the culture conditions for the bacterial cells when using a liquid medium. Specifically, for example, aerobic conditions can be set at 25 ° C. for 3 to 7 days.
In the liquid culture method, in order to increase the number of cells efficiently, the liquid medium containing the cells may be cultured while being stirred or shaken, or air is supplied to the liquid medium containing the cells.
Bacteria obtained by culturing using a liquid medium can be recovered using, for example, a centrifugation method, a filtration method, or the like. The cells recovered from the liquid medium may be frozen and vacuum dried, and then extracted with a solvent in the next step, or may be extracted with a solvent in the next step while being recovered.

  In addition, the cells cultured in a liquid medium may be further cultured using a new liquid medium as necessary in order to secure a sufficient amount of the compound represented by formula (I) or a salt thereof. May be recovered from.

(Second step)
Next, the cells of the genus Methylobacterium collected by culturing in the first step are extracted with a solvent to obtain an extract. Examples of the method for obtaining the extract include the following methods. First, a solvent is added to the cells and stirred, and the compound represented by the formula (I) or a salt thereof is extracted from the cells in the solvent. Next, the solvent containing the cells after extraction is filtered to obtain an extract as a filtrate. Thereafter, the extract is concentrated, frozen and vacuum dried to obtain an extract.
In the present embodiment, examples of the solvent used for bacterial cell extraction include alcohol or a mixed solution of alcohol and water. Among these, as a solvent, it is preferable to use methanol, ethanol, isopropyl alcohol, or a mixed solution of these and water, and it is more preferable to use a mixed solution of methanol and water.

(Third step)
Next, a step of recovering the compound represented by the formula (I) or a salt thereof, which is the target product, from the extract obtained in the second step.
In the present embodiment, alkali treatment and purification treatment are performed as a step of recovering the target product from the extract. The alkali treatment may be performed before the purification treatment, after the purification treatment, or may be performed together with the purification treatment.

  The alkali treatment is carried out by bringing the extract into contact with an alkaline solution and further extracting the target product from the extract. The pH of the alkaline solution is preferably 9.0 to 14.0, more preferably 10.0 to 13.0, and still more preferably 11.0 to 12.0. Examples of the alkaline solution include a mixed solution of aqueous ammonia and methanol.

  As a purification method, a conventionally known method can be used, and examples thereof include anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography. It is done. The purification treatment may be performed by a mixed mode chromatography method in which the above chromatography methods are mixed, or may be performed by a method in which different types of chromatography methods are performed a plurality of times. Among the methods described above, the purification method is particularly preferably a cation exchange chromatography method using Porata Pak Rxn CX manufactured by WATERS as a column and a hydrophobic chromatography method using Sunrise C28 manufactured by Chromanic Technologies as a column.

  Examples of the method of performing the alkali treatment together with the purification treatment include a method of performing the above-described chromatography method using the above-mentioned alkaline solution. Specifically, it is preferable to use a cation exchange chromatography method using Porata Px Rxn CX manufactured by WATERS as a column and recovering components eluted with a mixed solution of aqueous ammonia and methanol as an alkaline solution.

  By performing the above steps, the compound represented by the formula (I) or a salt thereof, which is the target product, is obtained.

  The target product recovered by the production method of the present embodiment is represented by the formula (I) using LC / MS (liquid chromatography mass spectrometry) analysis, NMR (nuclear magnetic resonance) analysis, X-ray crystal structure analysis, and the like. Can be confirmed.

The LC / MS analysis can be performed, for example, under the following conditions.
As LC (liquid chromatography), LCSolution manufactured by Shimadzu Corporation, PDA detector (SPD-M10A), column temperature 40 ° C., flow rate 1.0 ml / minm, as mobile phase, ammonium formate, formic acid aqueous solution, Any one or more selected from formic acid methanol solutions can be used. MS (mass spectrometry) can be performed according to an ESI (electrospray ionization) ion trap method.
The NMR analysis can be performed in heavy water using, for example, AVANCE500 manufactured by BRUKER BIOSPIN.

The compound represented by the formula (I) or a salt thereof has ultraviolet absorbing ability and solubility in an aqueous solvent. In particular, the compound represented by the formula (I) or a salt thereof has a high absorption peak in the wavelength region of 350 to 360 nm, and is excellent in ultraviolet absorbing ability in the UVA region.
In addition, the compound of the present embodiment or a salt thereof is a mixed solution of any one or more of methanol, ethanol, propanol, acetonitrile, ethylene glycol, propylene glycol, and butanediol and water, an alkali metal, an alkaline earth metal salt, Since it is excellent in solubility in an aqueous solvent such as water, a mixture of at least one of an organic acid and an amino acid and water, when used as a material for an anti-photoaging agent, it can be easily blended using an aqueous solvent.

  Moreover, according to the manufacturing method of the compound or its salt of this embodiment, the compound or its salt represented by highly purified formula (I) can be manufactured. Therefore, for example, when the compound represented by the formula (I) obtained by the production method of the present embodiment or a salt thereof is used as a material for an anti-photoaging agent, the compound represented by the formula (I) or a salt thereof is used. It is preferable because defects caused by impurities contained are unlikely to occur.

In addition, the manufacturing method of the compound represented by Formula (I) or its salt is not limited to said method.
For example, by chemically synthesizing the target product manufactured by the manufacturing method of this embodiment as a raw material, any one of R ₁ , R ₂ , R ₃ , and X in the formula (I) You may manufacture the compound or its salt represented by the formula (I) from which 1 or more differs.
In addition, the compound represented by the formula (I) or a salt thereof is chemically used only as a raw material by using only components that are not derived from nature without using those extracted from cells obtained by culturing microorganisms that grow on plants. May be synthesized.

  The compound represented by the formula (I) or the salt thereof contained in the anti-photoaging agent of the present embodiment may be only one type or two or more types. The anti-photoaging agent of this embodiment may contain only the compound represented by the formula (I) and may not contain a salt of the compound represented by the formula (I), or may be represented by the formula (I). Contains only a salt of the compound and may not contain the compound represented by formula (I), or contains both the compound represented by formula (I) and the salt of the compound represented by formula (I) But you can. When the compound represented by the formula (I) or the salt thereof contained in the anti-photoaging agent is two or more, the combination and ratio can be appropriately selected according to the purpose.

In addition to the compound represented by the formula (I) or a salt thereof, the anti-photoaging agent of the present embodiment may be a pharmaceutically acceptable carrier or the like as long as it does not impair the effects of the present invention. These components may be contained at a general concentration.
The pharmaceutically acceptable carrier is not particularly limited, and examples thereof include excipients, binders, disintegrants, lubricants, emulsifiers, stabilizers, diluents, thickeners, wetting agents, pH adjusting agents, Oils, solvents for injections, etc. can be used.

  Other components are not particularly limited, for example, moisturizers, feel improvers, surfactants, polymer compounds, thickening / gelling agents, solvents, propellants, antioxidants, reducing agents, oxidizing agents, Preservatives, antibacterial agents, chelating agents, pH adjusters, acids, alkalis, powders, inorganic salts, UV absorbers, whitening agents, vitamins and their derivatives, anti-inflammatory agents, anti-inflammatory agents, hair growth agents, blood circulation promoters , Stimulants, hormones, anti-wrinkle agents, anti-aging agents, squeeze agents, cooling sensation agents, warming sensation agents, wound healing promoters, stimulation relieving agents, analgesics, cell activators, plant / animal / microbe extracts, antipruritic agents Agent, exfoliating / dissolving agent, antiperspirant, refreshing agent, astringent, enzyme, nucleic acid, fragrance, pigment, colorant, dye, pigment, water, metal-containing compound, unsaturated monomer, polyhydric alcohol, high Molecular additive, anti-inflammatory analgesic, antifungal, antihistamine, hypnotic sedative, mental health Agent, antihypertensive agent, antihypertensive diuretic, antibiotic, anesthetic, antibacterial agent, antiepileptic agent, coronary vasodilator, herbal medicine, adjuvant, wetting agent, astringent, thickener, tackifier, antipruritic Agent, keratin softening release agent, ultraviolet ray blocking agent, antiseptic disinfectant, metal soap and the like.

  Examples of pharmaceutically acceptable carriers and other ingredients include, for example, the 16th revised Japanese Pharmacopoeia, Cosmetic Raw Material Standards Second Edition Commentary (Edited by the Japanese Official Church, Yakuji Nipposha, 1984), non-cosmetic raw material standards Ingredient standards (supervised by the Ministry of Health and Welfare Pharmacy Examination Division, Yakuji Nippo, 1993), supplements for ingredients outside the standard of cosmetic ingredients (supervised by the Ministry of Health and Welfare Pharmacy Examination Division, Pharmacy Daily, 1993) Supervised by the Bureau of Public Affairs, Yakuji Nippo, 1993), Cosmetic Raw Material Dictionary (Nikko Chemicals, 1991), International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol. General raw materials described in 1-4, by CTFA and the like can be used. More specifically, for example, each raw material described in JP 2014-114289 A can be mentioned.

  The anti-photoaging agent of this embodiment is a mixture of the compound represented by the formula (I) or a salt thereof and other components contained as necessary according to a conventional method (for example, a method described in the Japanese Pharmacopoeia). And can be manufactured by formulation.

  Specific examples of anti-aging agents include creams, lotions, lotions, emulsions, foundations, packs, foams, skin cleansers, extracts, plasters, ointments, spirits, suspensions, tinctures. And dosage forms such as agents, poultices, liniments, and aerosols.

  The anti-photoaging agent of this embodiment contains a compound represented by the formula (I) or a salt thereof. Thus, the anti-photoaging agent of the present embodiment promotes gene expression of the matrix components collagen, elastin, and hyaluronic acid when the skin to which the anti-photoaging agent is applied is exposed to UVA. It suppresses the expression of a gene that produces a certain matrix metalloprotease (MMP). For this reason, when the skin to which the anti-photoaging agent of this embodiment is applied is exposed to UVA, the matrix component in the dermis is less likely to decrease, and wrinkles and elasticity decreases due to the decrease in the matrix component are suppressed. , Photoaging is prevented.

  Moreover, the anti-photoaging agent of this embodiment has UVA absorption ability. Therefore, the anti-photoaging agent of the present embodiment shields the skin from UVA when the skin to which it is applied is exposed to UVA, and effectively suppresses photoaging of the skin due to exposure to UVA. .

In addition, the conventional ultraviolet shielding agent prevents photoaging due to ultraviolet rays by a function of absorbing and scattering ultraviolet rays physicochemically, and the skin caused by exposing the skin to UVA. It did not have a function of physiologically suppressing photoaging itself.
In contrast, when the anti-photoaging agent of the present embodiment is applied to the skin, not only the skin is shielded from UVA, but also the effect of physiologically suppressing skin aging caused by exposing the skin to UVA is obtained. It is done. Furthermore, due to the function of promoting gene expression of collagen, elastin and hyaluronic acid as anti-photoaging agents, the matrix component increases in the dermis after the skin is exposed to UVA, and the effect of improving aging can be expected. The above effect is also exhibited when the anti-photoaging agent of this embodiment is applied to the skin after being exposed to UVA.

  In addition, since the anti-photoaging agent of the present embodiment contains a compound represented by the formula (I) having a solubility in an aqueous solvent or a salt thereof, it can be produced without using a hydrophobic solvent, and variously depending on applications. Can be made into various dosage forms.

  As a specific composition of the anti-photoaging agent of this embodiment, the composition of the composition example 1-composition example 8 shown in Table 1-Table 5 is mentioned, for example.

Hereinafter, the present invention will be described in detail by way of examples. The present invention is not limited by the following examples.
<Example>
“1. Collecting microorganisms”
Strawberry leaves were immersed in a 10 mM phosphate buffer and ground in a mortar to obtain a ground solution containing microorganisms.

“2. Culture of microorganisms”
The ground solution containing microorganisms obtained in “1. Collecting microorganisms” was applied to the surface of a standard agar medium (manufactured by Difco) and cultured at 25 ° C. for 5 days under aerobic conditions. Then, the single colony which appeared on the surface of the standard agar medium was scraped off, and the bacterial cells forming the single colony were separated and recovered.
Next, each microbial cell recovered from a single colony was applied to the surface of a plurality of NB (Nutrient Broth) agar plates (manufactured by Difco) and cultured at 25 ° C. for 6 days under aerobic conditions. Thereafter, the colonies that appeared on the surface of the agar plate medium were scraped off, and the cells were collected.

Next, each bacterial cell separated and recovered from the microorganisms collected from the plant in this manner was examined for the presence or absence of ultraviolet absorbing ability by absorptiometry.
Next, among the separated and recovered bacterial cells, the bacterial cell having the highest ultraviolet absorption ability was identified. The bacterial cells were identified based on the base sequence of the rRNA gene. As a result, the cells were of the genus Methylobacterium. This microbial cell was named WI-182 strain.

Next, two agar plate media from which the cells of the genus Methylobacterium (WI-182 strain) were obtained were prepared and suspended by adding 10 ml of sterile purified water to each agar plate medium. Obtained. Suspensions obtained from two agar plate media were mixed to prepare a mixed solution, each 2 ml was added to seven 100 ml PD media (manufactured by Difco), and cultured at 25 ° C. for 90 hours under aerobic conditions.
600 ml was collected from a culture cultured in 7 PD media and added to 30 L of PD media (Difco). Then, 15 L / min of air was supplied to the liquid medium while stirring the liquid medium containing the bacterial cells using a stirrer at a rotational speed of 400 rpm, and cultured at 25 ° C. for 7 days.

“3. Recovery of bacterial cells”
28 kg of the culture cultured as described above (liquid medium containing the cultured cells) was centrifuged at a rotational speed of 8000 rpm for 20 minutes to collect wet cells. Thereafter, the collected cells were frozen and vacuum-dried to obtain 51 g of cells.

“4. Extraction from microorganisms”
To 45 g of the microbial cells (WI-182 strain) obtained as described above, 2250 ml of a mixed solution of water and methanol (water: methanol (volume ratio) = 2: 8) was added, and the three-one at 25 ° C. for 1 hour. Extraction was performed by stirring with a motor (registered trademark) stirring blade at a rotation speed of 140 rpm. Next, the solvent containing the cells after the extraction was suction filtered to collect the extract as a filtrate. Thereafter, the recovered extract was concentrated with a rotary evaporator, frozen and vacuum dried to obtain 12.2 g of extract.

"5. Recovery of target product from extract (1)"
As shown below, the extract was purified by cation exchange chromatography using an alkaline solution.
First, an extract was added to methanol and dissolved to obtain a methanol solution. Next, the methanol solution was passed through a column (PoraPak Rxn CX manufactured by WATERS) to adsorb the extract on the ion exchange resin. Next, a mixed solution of ammonia water (28% by mass) and methanol (ammonia water: methanol (volume ratio) = 5: 95 (pH 11.2)) as an alkaline solution is passed through the column to obtain an ion exchange resin. From the adsorbed extract, components dissolved in the alkaline solution were eluted to obtain an eluate.
Thereafter, the eluate was concentrated on a rotary evaporator, frozen and vacuum dried to recover 1.36 g of a tan compound as a crude product.

"6. Recovery of target product from extract (2)"
The crude product obtained as described above was purified using high performance liquid chromatography (HPLC) (LCSolution manufactured by Shimadzu Corporation, PDA (photodiode array) detector (SPD-M10A)). The conditions were as follows.

Column: Sunrise C28 (particle diameter of the substrate packed in the column: 5 μm, column inner diameter: 10 mm, column length: 250 mm, manufactured by Chromanic Technologies)
Column temperature: 40 ° C
Flow rate: 5.0 ml / min
Mobile phase A: 10 mM ammonium formate, 0.2% formic acid
Solvent Water mobile phase B: 10 mM ammonium formate, 0.2% formic acid
Solvent Mixed solution of water and methanol (methanol: water (volume ratio) = 95: 5)
Gradient condition: 0 to 7 min Fixed at 100% mobile phase A
7 to 15 min Mobile phase A: Mobile phase B = 100: 0 to Mobile phase A: Mobile phase B = 24: 76 linear gradient
15-25min Fixed at 95% mobile phase B
25 to 35 min Fixed at 100% mobile phase A

  The results of high performance liquid chromatography analysis are shown in FIG. FIG. 1 is a graph showing the relationship between the ultraviolet absorption intensity and elution time of the crude product obtained in the examples. As shown in FIG. 1, the crude product obtained in the examples had elution times of 3.3 min, 4.3 min, 5.2 min, 16.8 min, and 17.6 min when measured at a UV measurement wavelength of 360 nm. A major peak was detected at the position. It was confirmed that the peaks at elution times of 4.3 min, 16.8 min, and 17.6 min show high absorption peaks in the wavelength region of 350 to 360 nm.

"Photoaging-related gene expression promotion effect"
The crude product obtained in the examples was purified with an ion exchange column, and the fraction eluted with 5% aqueous ammonia was collected, and then the solvent was removed with an evaporator to obtain a purified solid. This purified product is represented by the compound represented by the formula (II) and the formula (V) when measured at the UV measurement wavelength of 360 nm described in the previous section from the area ratio of the high performance liquid chromatography (HPLC) analysis result. It is estimated that it contains 64% of the main peak with an elution time of 4.3 min corresponding to the compound and 36% of other compounds (mass ratio).
Next, the effect of promoting the expression of photoaging-related genes (collagen gene, elastin gene, hyaluronic acid synthase (HAS3) gene) was examined for the obtained purified product by the method described below.

(Sample 1)
Normal human fibroblasts (NB1RGB cells) manufactured by RIKEN BioResource Center are seeded in a plastic petri dish at a seeding density of 10,000 cells / cm ² , and Dulbecco's Modified Eagle (DMEM) medium manufactured by Sigma containing 10% fetal bovine serum. Incubated for 24 hours.
Subsequently, the NB1RGB cells were irradiated with 15 J / cm ² of UVA using a UVA irradiation apparatus manufactured by UVP. Thereafter, the purified product dissolved in 60% ethanol is added to a plastic petri dish so that the concentration of the purified product contained in the culture solution in the DMEM medium is 0.01% by mass, and further cultured for 24 hours. Sample 1 was obtained.

(Sample 2)
After irradiation with UVA, Sample 2 was obtained in the same manner as Sample 1, except that 60% ethanol was added to the plastic petri dish instead of the purified product dissolved in 60% ethanol.
(Sample 3)
Sample 3 was obtained in the same manner as Sample 2, except that UVA was not irradiated.

  Total RNA (ribonucleic acid) was extracted from each of the samples 1 to 3, and cDNA (complementary deoxyribonucleic acid) was synthesized. Subsequently, quantitative real-time PCR (polymerase chain reaction) was performed using the above cDNA as a template, and the expression levels of collagen gene, elastin gene, and HAS3 gene in NB1RGB cells of Sample 1 to Sample 3 were quantified.

Moreover, the expression level of the internal standard gene in the NB1RGB cells of Sample 1 to Sample 3 was quantified in the same manner as the expression levels of the collagen gene, elastin gene, and HAS3 gene in NB1RGB cells of Sample 1 to Sample 3. As an internal standard gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used.
Then, based on the expression level of the GAPDH gene in the NB1RGB cells of Sample 1 to Sample 3, the expression levels of the collagen gene, elastin gene, and HAS3 gene in the NB1RGB cells are standardized, and the collagen gene, elastin gene of Sample 3, The expression level (relative value) when the expression level of each gene of the HAS3 gene was 1.00 was calculated. The results are shown in Table 6.

  As a GAPDH gene amplification primer, GAPDH (ID: HA067812) manufactured by Takara Bio Inc. was used. Moreover, as a collagen gene amplification primer, COLA1 (ID: HA181818) manufactured by Takara Bio Inc. is used, and as an elastin gene amplification primer, ELA (ID: CH000581) manufactured by Takara Bio Inc. is used as an HAS3 gene. As an amplification primer, (ID: HA095624) manufactured by Takara Bio Inc. was used.

As shown in Table 6, in sample 1 in which the purified product was added to a plastic petri dish containing NB1RGB cells, the expression levels of the collagen gene, elastin gene, and HAS3 gene were different from those in sample 3 that had not been irradiated with UVA. Compared to more.
On the other hand, in the sample 2 to which the purified product is not added, the expression levels of the collagen gene, elastin gene, and HAS3 gene are reduced compared to the sample 3 not irradiated with UVA.

"MMP-1 gene expression suppression effect"
By the method shown below, the matrix metalloproteinase-1 (MMP-1) gene expression inhibitory effect of the purified product was examined.

  Total RNA (ribonucleic acid) was extracted from each of the samples 1 to 3, and cDNA (complementary deoxyribonucleic acid) was synthesized. Subsequently, quantitative real-time PCR (polymerase chain reaction) was performed using the above cDNA as a template, and the expression level of the MMP-1 gene in the NB1RGB cells of Samples 1 to 3 was quantified.

Based on the expression level of the GAPDH gene in the NB1RGB cells of the samples 1 to 3, the expression level of the MMP-1 gene in the NB1RGB cells was standardized, and the expression level of the MMP-1 gene in the sample 3 was set to 1.00. The expression level (relative value) was calculated. The results are shown in Table 7.
As the MMP-1 gene amplification primer, MMP-1 (ID: HA205024) manufactured by Takara Bio Inc. was used.

  As shown in Table 7, in the sample 1 in which the purified product was added to the plastic petri dish containing NB1RGB cells, the MMP-1 gene caused by UVA irradiation was compared with the sample 2 in which the purified product was not added. Is expressed less.

Claims (3)

An anti-photoaging agent comprising a compound represented by the formula (I) or a salt thereof.

(In Formula (I), R ₁ and R ₂ are each independently a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or a linear or branched structure having 1 to 5 carbon atoms. Represents an alkoxy group having 1 to 5 carbon atoms, or a linear or branched alkenyl group having 1 to 5 carbon atoms, R ₃ represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or 1 to 5 carbon atoms. X represents a hydrogen atom or a linear or branched sugar chain having 1 to 10 sugar molecules.) The compound represented by the formula (I) or a salt thereof is a compound represented by the formula (II) or a salt thereof in which R ₁ and R ₂ in the formula (I) are methyl groups and R ₃ is a hydrogen atom. Item 2. The antiphotoaging agent according to Item 1.

The anti-photoaging agent according to claim 1, wherein in formula (I), R ₁ and R ₂ are methyl groups, R ₃ is a hydrogen atom, and X is a linear sugar chain having 5 sugar molecules. .

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