JPWO2017051875A1 - UV-absorbing compound or salt thereof, method for producing the same, external preparation for skin, cosmetic - Google Patents

Abstract

A compound represented by the formula (I) or a salt thereof. (In Formula (I), R ₁ and R ₂ are 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, carbon, A linear or branched alkenyl group having 1 to 5. R ₃ is a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or a linear or branched group having 1 to 5 carbon atoms. X is a hydrogen atom, a linear or branched sugar chain having 1 to 10 sugar molecules.)
[Chemical 1]

Description

The present invention relates to a compound having an ability to absorb ultraviolet rays or a salt thereof, a production method thereof, an external preparation for skin, and a cosmetic.
This application claims priority based on Japanese Patent Application No. 2015-187181 filed in Japan on September 24, 2015, the contents of which are incorporated herein by reference.

Ultraviolet rays contained in sunlight have various effects on human skin and the like. Specifically, various harmful effects due to ultraviolet rays are known, such as causing blemishes and wrinkles on the skin to promote aging, causing allergic reactions, and causing carcinogenesis.
Ultraviolet rays contained in sunlight are roughly classified into ultraviolet A waves (UVA) having a wavelength of 320 to 400 nm, ultraviolet B waves (UVB) having a wavelength of 280 to 320 nm, and ultraviolet C waves (UVC) having a wavelength of 200 to 280 nm.

  Conventionally, various ultraviolet absorbers are used to protect human skin from ultraviolet rays. For example, as an ultraviolet absorber having UVB absorption ability, there are chemically synthesized agents such as salicylic acid derivatives, p-aminobenzoic acid, benzophenone derivatives and the like. Further, as an ultraviolet absorber having UVA absorption ability, there is a chemically synthesized material such as avobenzene.

  Moreover, as an ultraviolet absorber having UVA absorption ability, yangonin obtained by extraction from a plant belonging to the family Pepperaceae (for example, see Patent Document 1), an extract obtained from a Gramineae family plant (for example, see Patent Document 2). 2-amino-3- (β-D-glucopyranosyloxy) -γ-oxobenzenebutanoic acid extracted from human and primate ocular lenses has been reported (for example, see Patent Document 3). .

  As an ultraviolet absorber having a UVB absorption ability produced by marine bacteria, a compound collected from a culture of microorganisms belonging to the genus Perradibacter (Patent Document 4) is known. In addition, a compound collected from a culture of a microorganism belonging to the genus Thermoactinomyces (Patent Document 5) is known as an ultraviolet absorber having a UVC absorption ability produced by marine bacteria.

  Patent Document 6 discloses an ultraviolet absorbent composition derived from a microorganism that inhabits the plant surface and having an absorption peak of an ultraviolet absorption spectrum in the UVA region.

JP-A-9-67238 Special table 2012-516311 gazette JP-A-8-3183 JP 2000-016976 A JP 2006-160662 A JP2013-127027A

It is well known that UVA penetrates deeper into the skin and promotes skin aging than UVB and UVC. For this reason, there is a high social interest in protecting human skin and the like from UVA, and there is a great demand for UV absorbers having UVA absorption ability.
Moreover, it is desirable that the ultraviolet absorber used as a material such as a skin external preparation is a compound having solubility in an aqueous solvent.

This invention is made | formed in view of the said situation, and makes it a subject to provide the novel compound which has UVA absorptivity, and the solubility with respect to an aqueous solvent, and its manufacturing method.
Moreover, this invention makes it a subject to provide the skin external preparation and cosmetics containing the novel compound which has the solubility with respect to UVA absorption ability and an aqueous solvent.

In order to solve the above-mentioned problems, the present inventors have intensively studied.
As a result, the inventors succeeded in identifying and producing a novel compound having UVA absorption ability and solubility in an aqueous solvent, and completed the present invention.
That is, the present invention adopts the following configuration.

[1] A compound having a UV-absorbing ability 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] The compound or salt thereof according to [1], wherein the compound represented by formula (I) or a salt thereof is a compound represented by formula (II) or a salt thereof.

[3] The compound according to [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. Or its salt.

[4] A method for producing the compound or salt thereof according to any one of [1] to [3],
Culturing microorganisms growing on plants to obtain microbial cells;
Extracting the cells with a solvent to obtain an extract;
Recovering the compound represented by the formula (I) or a salt thereof from the extract.

[5] The method for producing a compound or a salt thereof according to [4], wherein the microbial cell belongs to the genus Methylobacterium.

[6] A skin external preparation containing the compound or salt thereof according to any one of [1] to [3].
[7] A cosmetic comprising the compound or salt thereof according to any one of [1] to [3].

The compound of the present invention or a salt thereof has ultraviolet absorbing ability and solubility in an aqueous solvent. In particular, the compound of the present invention or a salt thereof is excellent in ultraviolet absorbing ability in the UVA region.
Further, since the compound of the present invention or a salt thereof is excellent in solubility in an aqueous solvent, it can be easily formulated using an aqueous solvent when used as a material for cosmetics and skin external preparations.
Since the cosmetic and external preparation for skin of the present invention contains the compound of the present invention or a salt thereof, it has excellent UVA absorption ability and can be produced without using a hydrophobic solvent.

2 is a graph showing the relationship between ultraviolet absorption intensity and elution time in the HPLC analysis of the crude product obtained in Example 1. FIG. 2 is a graph showing the relationship between the mass-to-charge ratio of the purified compound obtained in Example 1 and detection sensitivity (relative intensity). 2 is a diagram showing the results of X-ray crystal structure analysis of the purified compound obtained in Example 1. FIG. 2 is a graph showing the relationship between ultraviolet absorption intensity and elution time in HPLC analysis of the purified compound obtained in Example 1. FIG. 4 is a graph showing the relationship between the mass-to-charge ratio of the purified compound obtained in Example 2 and detection sensitivity (relative intensity). 4 is a graph showing the relationship between ultraviolet absorption intensity and elution time in HPLC analysis of the purified compound obtained in Example 2. 4 is a graph showing the relationship between the mass-to-charge ratio of the crude product obtained in Example 3 and detection sensitivity (relative intensity).

Hereinafter, the present invention will be described in detail.
"Compound having ultraviolet absorbing ability or salt thereof"
The compound of the present embodiment or a salt thereof is a novel compound represented by the following 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.)

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. Since there are few, it is preferable.

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 of the present embodiment or a salt thereof has a structure represented by the formula (I), it has solubility in an aqueous solvent and is excellent in ultraviolet absorbing ability in the UVA region.
The ultraviolet absorptivity in 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 having ultraviolet absorbing ability”
The compound of this embodiment or its salt can be manufactured by the manufacturing method shown below, for example.
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 in which the plant is immersed in a phosphate buffer and ground in a mortar to obtain a ground solution containing the microorganism.

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 (Nutient 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 the 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 collected using, for example, a centrifugal separation 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 can 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, 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 above-described purification methods, the cation exchange chromatography method using Porata Pak Rxn CX manufactured by WATERS as the column and the hydrophobic chromatography method using Sunrise C28 manufactured by Chromanic Technologies as the column are particularly preferable.

  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 PorataPak Rxn CX manufactured by WATERS as a column and recovering components eluted with a mixed solution of aqueous ammonia and methanol, which is 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 / min, mobile phase as 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 of the present embodiment represented by the formula (I) or a salt thereof has ultraviolet absorption 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 one or more of organic acids and amino acids and water, it can be easily formulated using an aqueous solvent when used as a material for cosmetics and skin external preparations. .

  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 cosmetics and skin external preparations, the compound represented by the formula (I) or a salt thereof It is preferable because defects caused by impurities contained in the salt hardly 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.

"External preparation for skin, cosmetics"
The skin external preparation and cosmetics of this embodiment contain the compound represented by the formula (I) or a salt thereof. The compound represented by the formula (I) or a salt thereof contained in the external preparation for skin and cosmetics of this embodiment may be only one type or two or more types. The skin external preparation and cosmetics of this embodiment may contain only the compound represented by the formula (I) and may not contain the salt of the compound represented by the formula (I). It may contain only the salt of the compound represented, and may not contain the compound represented by formula (I), or it may contain both the compound represented by formula (I) and the salt of the compound represented by formula (I). You may do it. When there are two or more compounds represented by the formula (I) or salts thereof contained in the external preparation for skin and cosmetics, the combination and ratio can be appropriately selected depending on the purpose.

In addition to the compound represented by the formula (I) or a salt thereof, the external preparation for skin and cosmetics are components usually used in the external preparation for skin or cosmetics as long as they do not impair the effects of the present invention. Other components may be contained at a general concentration.
Examples of the components that are usually used in the external preparation for skin or cosmetics include ingredients such as raw materials described in existing raw material specifications or official documents, pharmaceutically acceptable carriers, additives, etc. as external preparations for skin. . These other components may be used individually by 1 type, and may use 2 or more types together.

  Examples of skin external preparations and cosmetics include, for example, treatment, hair pack, hair foam, hair mousse, hair spray, hair mist, hair wax, hair gel, water grease, set lotion, color lotion, hair tonic, hair liquid, pomade, Cosmetics for hair such as tics, hair creams, hair blows, split coats, hair oils, hair color after treatments, perm after treatments, hair manicures, hair restorers; lotions, soft lotions, astringent lotions, cleansing lotions, Multi-layer lotion, emulsion, emollient lotion, moisture lotion, milky lotion, nourishing lotion, nourishing milk, skin moisture, moisture emulsion, makeup lotion, elbow , Hand lotion, body lotion, cream, emollient cream, nourishing cream, nourishing cream, burnishing cream, moisture cream, night cream, makeup cream, base cream, pre-makeup cream, gel, moisture gel, whitening essence, liposome serum , Basic cosmetics such as liposome lotion; makeup powders such as white powder, dusting powder, foundations, makeup base, lipstick, lip gloss, blusher, eyeliner, mascara, eye shadow, eyebrow; perfume, perfume, parfum Fragrance cosmetics such as eau de parfum, eau de toilette, eau de cologne, perfume, fragrance powder, body lotion, bath oil; body powder, deodorant lotion, deodorant pa Loaders, deodorant spray, deodorant sticks, deodorant cosmetics, bug spray, insect repeller body cosmetics such as Ra; ointments, plasters, lotions, liniments, liquid liniments and the like.

  Examples of skin external preparations and cosmetics include emulsified types such as oil-in-water (O / W) type, water-in-oil (W / O) type, W / O / W type, and O / W / O type; Polymer type; Solid; Liquid; Kneaded; Stick; Volatile oil; Powder; Water; Jelly; Gel; Paste; Cream; Sheet; Film; Mist; Spray; Examples include lamellar form; foam form; flake form and the like.

  The external preparation for skin and cosmetics can be produced by blending the compound represented by the formula (I) or a salt thereof, and other components as required, and preparing it. The skin external preparation and cosmetic can be produced in the same manner as known skin external preparations and cosmetics, except that the compound represented by the formula (I) or a salt thereof is blended.

  The skin external preparation and cosmetics of this embodiment contain the compound represented by the formula (I) having excellent UVA absorption ability or a salt thereof. Therefore, the external preparation for skin and cosmetics of this embodiment are highly effective in protecting human skin and the like from UVA. Moreover, since the skin external preparation and cosmetics of this embodiment contain the compound or its salt represented by Formula (I) which has the solubility with respect to an aqueous solvent, it can be manufactured without using a hydrophobic solvent, and according to a use. Can be made into various dosage forms.

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>
“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, 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 at a rotational speed of 140 rpm using a motor stirring blade. 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 of the crude product obtained in Example 1 and the elution time. As shown in FIG. 1, the crude product obtained in Example 1 was eluted at 3.3 min, 4.3 min, 5.2 min, 16.8 min, 17.6 min when measured at a UV measurement wavelength of 360 nm. A major peak was detected at the position of. It was confirmed that the peak of elution time 4.3min, 16.8min, 17.6min shows a high absorption peak in the wavelength region of 350 to 360nm.

  In addition, the peak elution fraction with an elution time of 4.3 min, which gave the strongest UV absorption result in HPLC analysis, was collected. The collected eluate was concentrated under reduced pressure using a rotary evaporator, frozen, and vacuum dried to recover a yellowish white compound (target product) as a purified compound.

"Liquid chromatography mass spectrometry (LC / MS) analysis of purified compounds"
The purified compound of Example 1 obtained as described above was subjected to LC / MS analysis to determine the molecular weight.
LC (Liquid Chromatography) is “6. Recovery of target substance from extract” except that the column inner diameter is 4.6 mm, the flow rate is 1.0 mL / min, and the gradient conditions are changed as follows. (2) "was performed under the same conditions as in HPLC.

Gradient condition: 0 to 10 min Fixed at 100% mobile phase A
10-20 min Mobile phase A: Mobile phase B = 100: 0 Mobile phase A: Mobile phase B = 5: 95 linear gradient
20 ~ 23min Fixed at 95% mobile phase B
23-30min Fixed at 100% mobile phase A

MS (mass spectrometry) is an electrospray ionization (ESI) ion trap, positive and negative detection mode, source voltage (positive mode 3.5 kV, negative mode -3.0 kV), capillary temperature 275 ° C., source heater temperature The measurement was performed using a Thermo Fisher Scientific Orbitrap Elite at 450 ° C.
The result is shown in FIG. FIG. 2 is a graph showing the relationship between the mass-to-charge ratio of the purified compound obtained in Example 1 and the detection sensitivity (relative intensity). From FIG. 2, it was confirmed that the molecular weight of the purified compound obtained in Example 1 was 302.

"Nuclear magnetic resonance (NMR) analysis of purified compounds"
NMR analysis of the purified compound of Example 1 obtained as described above was performed. As an NMR apparatus, an AVANCE 500 manufactured by BRUKER BIOSPIN was used and analyzed in heavy water. The results are shown below.

¹ H-NMR (500 MHz)
δ ppm 2.36 (s, 3H), 3.41 (s, 3H), 3.78 (dd, 1H), 3.84 (dd, 1H), 3.90 (dd, 1H) 4.00 (s, 1H) 4.30 (dd, 1H) 5.42 (d, 1H),
¹³ C-NMR (126 MHz):
δppm 18.0 (q), 45.4 (q), 64.0 (t), 64.1 (d), 71.7 (d), 71.7 (d), 76.2 (d), 94 0.0 (d), 121.7 (s), 156.9 (s), 159.9 (s), 171.4 (s)

"X-ray crystal structure analysis of purified compounds"
8 mg of the purified compound of Example 1 obtained as described above was weighed into a 10 mL glass-made pitch glass, and 0.2 mL of ultrapure water was added and completely dissolved. Next, 1 mL of ethanol was gently added to the upper layer along the wall of the vessel and allowed to stand in the laboratory for 2 days to precipitate crystals.
The obtained crystal was subjected to X-ray crystal structure analysis using Rigaku R-AXIS RAPID II. As a result, the molecular formula was found to be C ₁₂ H ₁₈ N ₂ O ₇ . The X-ray crystal structure analysis result of the purified compound obtained in Example 1 is shown in FIG.

  Obtained in Example 1 from the results of “liquid chromatography mass spectrometry (LC / MS) analysis of purified compound”, “nuclear magnetic resonance (NMR) analysis of purified compound”, and “X-ray crystal structure analysis of purified compound” above. It was confirmed that the purified compound was a compound represented by the formula (V).

"HPLC analysis of purified compounds"
The purified compound obtained as described above was analyzed using HPLC (LC Solution manufactured by Shimadzu Corporation, PDA detector (SPD-M10A)).
The result is shown in FIG. FIG. 4 is a graph showing the relationship between the ultraviolet absorption intensity of the purified compound obtained in Example 1 and the elution time.
As shown in FIG. 4, in the purified compound obtained in Example 1, a peak was detected at an elution time of 4.1 min when measured at a UV measurement wavelength of 360 nm. This peak was confirmed to show a high absorption peak in the wavelength region of 350 to 360 nm.

"Solubility test of purified compounds"
About the refinement | purification compound obtained as mentioned above, the solubility test with respect to water, methanol, and acetonitrile was done by the method shown below.
5 mg of the purified compound obtained in Example 1 was accurately weighed, 0.5 ml of each of the solvents 1 to 7 shown in Table 1 was added and stirred for 30 seconds, and then filtered to obtain 1 to 7 dissolved solutions. . Next, 100 μl of each of the obtained 1-7 lysates was put in a 96-well microplate, and an ultraviolet absorption spectrum was measured with a microplate reader (INFINITE200 PRO manufactured by TECAN). The ultraviolet absorption spectrum was measured by a spectrum scanning method in which wavelengths in the 200 to 600 nm region were continuously measured at 10 nm intervals.

And about the light absorbency of wavelength 360nm of each solution 1-7, the light absorbency value (absorbance ratio) of each solution when the light absorbency of the solution using 100% water (solvent 1) is 1.000. ) The results are shown in Table 1.
As shown in Table 1, the higher the proportion of water in the solvent, the greater the absorbance value. From this, it was found that the solubility of the purified compound in water was high.

<Example 2>
The crude purified product obtained in Example 1 was subjected to HPLC analysis in the same manner as in “6. Recovery of target product from extract (2)” in Example 1, and the second UV absorption intensity in HPLC analysis. The elution fraction of the peak (see FIG. 1) with an elution time of 16.8 min, which gave a strong result, was collected. Then, the fraction eluted was concentrated under reduced pressure using a rotary evaporator, frozen and vacuum dried to recover a yellowish white compound.

Thereafter, the yellowish white compound obtained in Example 2 was subjected to HPLC in the same manner as in “6. Recovery of target product from extract (2)” in Example 1 except that the gradient conditions were as follows. It refine | purified in.
Gradient condition: 0 to 13 min Fixed at 100% mobile phase A
13-18min Mobile phase B fixed at 5%
18-20min fixed at 10% mobile phase B
20-22min Fixed with mobile phase B15%
22 to 23 min Mobile phase A: Mobile phase B = 85: 15: Mobile phase A: Mobile phase B = 5: 95 linear gradient
23-26min Mobile phase B fixed at 95%
The liquid obtained by fixing and recovering with a mobile phase A of 100% for 26 to 33 min was concentrated under reduced pressure using a rotary evaporator, frozen and vacuum dried to recover a yellow compound which was a purified compound.

The purified compound obtained in Example 2 was subjected to “liquid chromatography mass spectrometry (LC / MS) analysis of the purified compound” in the same manner as in Example 1 except that the gradient conditions were changed as follows.
Gradient condition: 0 to 10 min Fixed at 100% mobile phase A
10 to 15 min Mobile phase B fixed at 5%
15 to 17 min Mobile phase B fixed at 10%
17-19min Mobile phase B fixed at 15%
19 to 20 min Mobile phase A: Mobile phase B = 85: 15 to Mobile phase A: Mobile phase B = 5: 95 linear gradient
20 ~ 23min Fixed at 95% mobile phase B
Fixed for 23 to 30 minutes with mobile phase A of 100%. The results are shown in FIG. FIG. 5 is a graph showing the relationship between the mass-to-charge ratio of the purified compound obtained in Example 2 and the detection sensitivity (relative intensity). From FIG. 5, it was confirmed that the molecular weight of the purified compound obtained in Example 2 was 1112.

  The purified compound obtained in Example 2 was subjected to “nuclear magnetic resonance (NMR) analysis of the purified compound” in the same manner as in Example 1. The results are shown below.

¹ H-NMR (500 MHz)
δppm 2.36 (s, 3H), 3.35 (t, 1H), 3.41-3.58 (m, 16H), 3.65 (t, 1H), 3.73-3.80 (m, 10H), 3.90-4.09 (m, 10H), 4.47 (dd, 1H), 4.65 (d, 1H), 4.80-4.90 (m, 4H), 5.42 (D, 1H),
¹³ C-NMR (126 MHz):
δ ppm 18.0 (q), 45.4 (q), 63.4 (t), 63.5 (t), 63.6 (t), 63.7 (t), 64.1 (d), 71 .5 (d), 71.7 (d), 71.9 (t), 72.1 (d), 72.3 (d), 74.7 (d), 76.7 (d), 78. 3 (d), 78.5 (d), 78.6 (d), 78.7 (d), 78.9 (d), 79.1 (d), 79.3 (d), 84.1 (D), 84.9 (d), 85.0 (d), 86.1 (d), 94.0 (d), 104.1 (d), 104.5 (d), 104.8 ( d), 105.3 (d), 106.0 (d), 121.7 (s), 156.8 (s), 160.0 (s)

From the results of the above-mentioned “liquid chromatography mass spectrometry (LC / MS) analysis of the purified compound” and “nuclear magnetic resonance (NMR) analysis of the purified compound”, the purified compound obtained in Example 2 is represented by the formula (I). It was confirmed that R 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.
Further, from the results of the above-mentioned “liquid chromatography mass spectrometry (LC / MS) analysis of the purified compound” and “nuclear magnetic resonance (NMR) analysis of the purified compound”, the purified compound obtained in Example 2 has the formula (III ) And / or a compound represented by formula (IV).

Next, the purified compound obtained in Example 2 was subjected to “HPLC analysis of the purified compound” in the same manner as in Example 1.
The result is shown in FIG. 6 is a graph showing the relationship between the ultraviolet absorption intensity and the elution time of the purified compound obtained in Example 2. FIG.
As shown in FIG. 6, in the purified compound obtained in Example 2, a peak was detected at an elution time of 21.25 min when measured at a UV measurement wavelength of 360 nm. This peak was confirmed to show a high absorption peak in the wavelength region of 350 to 360 nm.

Next, the purified compound obtained in Example 2 was subjected to a “purification compound solubility test” in the same manner as in Example 1.
As a result, also in the purified compound obtained in Example 2, the higher the proportion of water in the solvent, the greater the absorbance value. From this, it was found that the solubility of the purified compound in water was high.

<Example 3>
“1. Collecting microorganisms”
Wheat leaves were immersed in a 10 mM phosphate buffer and ground in a mortar to obtain a ground liquid containing microorganisms.
Thereafter, the steps from “2. Culture of microorganism” to “5. Recovery of target product from extract (1)” were performed in the same manner as in Example 1.

In Example 3, each bacterial cell obtained by separation and recovery in “2. Microbial culture” was examined for the presence or absence of ultraviolet absorbing ability in the same manner as in Example 1, and each bacterial cell isolated and recovered in the same manner as in Example 1. A cell having the highest UV-absorbing ability was identified. As a result, the cells were of the genus Methylobacterium. This microbial cell was named W-213 strain.
In addition, by performing “5. Recovery of target product from extract (1)”, a tan compound, which was a crude product similar in appearance to the crude product obtained in Example 1, was obtained.

The crude purified product obtained in Example 3 was subjected to LC / MS analysis in the same manner as in “Liquid chromatography mass spectrometry (LC / MS) analysis of purified compound” in Example 1.
The result is shown in FIG. FIG. 7 is a graph showing the relationship between the mass-to-charge ratio of the crude product obtained in Example 3 and detection sensitivity (relative intensity). From FIG. 7, it was confirmed that the crudely purified product obtained in Example 3 contains a compound having a molecular weight of 302.

The crude product obtained in Example 3 was obtained in Example 1 based on the fact that the identified cells were of the genus Methylobacterium and the results of LC / MS analysis of the obtained crude product. It is considered to be the same as the crude product.
The crude compound obtained in Example 3 was subjected to “6. Recovery of target product from extract (2)” in Example 1 to obtain a purified compound. The “HPLC analysis of the purified compound” in Example 1 was performed on the purified compound of Example 3 thus obtained. As a result, similar to the purified compound of Example 1, a peak was detected at an elution time of 4.1 min when measured at a UV measurement wavelength of 360 nm. This peak was confirmed to show a high absorption peak in the wavelength region of 350 to 360 nm. Further, the purified compound obtained in Example 3 was subjected to the same test as the “purified compound solubility test” in Example 1. As a result, it was confirmed that the higher the proportion of water in the solvent, the higher the absorbance value and the higher the solubility in water.

<Example 4>
In “1. Collecting microorganisms”, except that primrose petals were used instead of strawberry leaves, the same procedure as in Example 1 was performed, except that “1. Collecting microorganisms” to “5. The steps up to “Recovery (1)” were performed.
Each bacterial cell obtained by separation and recovery in “2. Microbial culture” in Example 4 was examined for the presence or absence of ultraviolet absorbing ability in the same manner as in Example 1, and each bacterial cell isolated and recovered in the same manner as in Example 1. A cell having the highest UV-absorbing ability was identified. As a result, the cells were of the genus Methylobacterium. This microbial cell was named f11 strain.
In addition, by performing “5. Recovery of target product from extract (1)”, a tan compound, which was a crude product similar in appearance to the crude product obtained in Example 1, was obtained.

<Example 5>
In “1. Collecting microorganisms”, except for using a rice leaf sheath instead of strawberry leaves, the same procedure as in Example 1 was performed, except that “1. Collecting microorganisms” to “5. The steps up to (1) ”were performed.
In Example 5, each bacterial cell obtained by separation and recovery in “2. Microbial culture” was examined for the presence or absence of ultraviolet absorption ability in the same manner as in Example 1, and each bacterial cell isolated and recovered in the same manner as in Example 1. A cell having the highest UV-absorbing ability was identified. As a result, the cells were of the genus Methylobacterium. This microbial cell was named 24N-25 strain.
In addition, by performing “5. Recovery of target product from extract (1)”, a tan compound, which was a crude product similar in appearance to the crude product obtained in Example 1, was obtained.

  Next, the crude purified product obtained in Example 4 and Example 5 was subjected to HPLC analysis in the same manner as in “6. Recovery of target product from extract (2)” in Example 1, respectively. As a result, in both Example 4 and Example 5, the peak with the strongest ultraviolet absorption intensity was obtained at the elution time of 4.3 min in the HPLC analysis.

The crude product obtained in Example 4 and Example 5 was obtained in Example 1 based on the fact that the identified bacterial cells belong to the genus Methylobacterium and the results of HPLC analysis of the obtained crude product. It is considered to be the same as the obtained crude product.
Further, the crude compound obtained in Example 4 and Example 5 was subjected to “6. Recovery of target product from extract (2)” in Example 1 to obtain a purified compound. . The “HPLC analysis of the purified compound” in Example 1 was performed on the purified compounds of Example 4 and Example 5 thus obtained. As a result, similar to the purified compound of Example 1, a peak was detected at an elution time of 4.1 min when measured at a UV measurement wavelength of 360 nm. This peak was confirmed to show a high absorption peak in the wavelength region of 350 to 360 nm. In addition, the purified compound obtained in Example 4 and Example 5 was subjected to the same test as the “purified compound solubility test” in Example 1. As a result, it was confirmed that the higher the proportion of water in the solvent, the higher the absorbance value and the higher the solubility in water.

  In Examples 1 to 5, Table 2 shows the plants from which microorganisms were collected, the molecular weight of the purified compound, the structure of the purified compound, the ultraviolet absorption ability, and the solubility. In Table 2, the ultraviolet absorption ability “◯” means having an absorption ability in an ultraviolet A wave (UVA) region having a wavelength of 320 to 400 nm. Further, the solubility “◯” means having solubility in water.

  As described above, the compound of the present invention or a salt thereof has an ultraviolet absorbing ability in the UVA region and solubility in an aqueous solvent, and is suitable as a material for cosmetics and skin external preparations.

  The compound of the present invention or a salt thereof can be expected to be used as an ultraviolet absorber in cosmetics, skin external preparations and the like.

Claims (7)

The compound or its salt which has the ultraviolet absorptivity represented by Formula (I).

(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 or salt thereof according to claim 1, wherein the compound represented by formula (I) or a salt thereof is a compound represented by formula (II) or a salt thereof.

The compound or salt thereof 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. . It is a manufacturing method of the compound or its salt as described in any one of Claims 1-3,
Culturing microorganisms growing on plants to obtain microbial cells;
Extracting the cells with a solvent to obtain an extract;
Recovering the compound represented by the formula (I) or a salt thereof from the extract.   The method for producing a compound or a salt thereof according to claim 4, wherein the microbial cell belongs to the genus Methylobacterium.   The skin external preparation containing the compound or its salt as described in any one of Claims 1-3.   Cosmetics containing the compound or its salt as described in any one of Claims 1-3.

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