JP5305842B2 - Involucrin expression promoter - Google Patents

Description

  The present invention relates to an involucrin expression promoter and a stratum corneum formation promoter.

For improving skin moisture retention, preventing and improving rough skin, and preventing and improving skin aging such as wrinkle formation and reduction of texture, it works on skin epidermal cells, promotes keratinization of epidermal cells, and is healthy It is recommended that the formation of a stratum corneum is promoted to make the stratum corneum barrier function for protecting the external stimulus and the living body healthy.
For the soundness of this stratum corneum barrier function, it is said that it is important to maintain the stratum corneum water content by maintaining the water content of the stratum corneum. As a cause of the decrease in the stratum corneum barrier function, it is considered that the turnover is disturbed due to the effects of aging, drying, ultraviolet rays and the like, and the formation of stratum corneum cells and the structure of intercellular lipids are abnormal. It is known to cause skin problems such as various skin diseases and rough skin.

Here, the turnover refers to the repeated proliferation of keratinocytes (epidermal keratinocytes) in the basal layer, the process of keratinization, and the detachment of the stratum corneum. The keratinocytes form basal cells, spinous cells, granule cells, and stratum corneum cells while sequentially keratinizing outward. And the protein which comprises a cornified envelope (CE) is synthesize | combined from the spiny layer upper layer to the granule layer. Further, in the process of reaching the stratum corneum, substrate proteins such as involucrin and loricrin are bound to the cell membrane of keratinocytes by the enzyme transglutaminase to form insolubilized CE. Furthermore, it is known that ceramide and the like are covalently bonded to insolubilized CE and form the basis of the stratum corneum barrier function.
Involucrin of the above substrate proteins is a constituent protein located in the outermost layer of the coneified envelope as described above (Non-patent Document 1), and is not only cross-linked with proteins of other coneified envelopes, loricrin and the like, It is known to be covalently bonded to ceramide and involved in the formation of an intercellular lipid lamellar structure (Non-patent Document 2). Moreover, involucrin is produced in the vicinity of the cell membrane specifically in the differentiation process of epidermal cells, and is produced in the previous stage of formation of a confined envelope (Non-patent Document 3), and is attracting attention as an epidermal cell differentiation marker.

Conventionally, a skin trouble such as rough skin due to a decrease in the stratum corneum barrier function has been solved by supplementing the stratum corneum barrier function with a cream containing ceramide or the like. However, the improvement of CE of stratum corneum cells is not sufficient, and the development of a component that promotes the formation of healthy CE is desired. In particular, the development of a component that promotes the expression of involucrin is desired.
Examples of the effects of promoting the expression of involucrin that forms CE include, for example, Mesua Ferrea L. (patent document 1) of Guttiferrae and Craterellus cornucopioides (patent document 1). Reference 2) is mentioned.

  On the other hand, alkyl (C10-C14) phosphate esters or salts thereof are known as low-irritant cleaning active ingredients (Patent Document 3), and linear alkyl (C12-C22) phosphate esters. And β-branched alkyl (carbon number 12 to 22) phosphate ester mixtures or salts thereof are known as surface active ingredients and aqueous emulsion ingredients (Patent Document 4). These alkyl phosphate esters or salts thereof Is not known to have an involucrin expression promoting action or stratum corneum formation promoting action.

JP 2005-213187 A JP 2005-89389 A JP-A-53-26806 JP-A-11-152292 Steinert, PM. Et al., J. Biol. Chem., 270 (30), p17702-17711 (1995) Nemes, Z., Proc. Natl. Acad. Sci. USA, 96 (15), p8402-8407 (1999) Steinert, PM. Et al., J. Biol. Chem., 272 (3), p2021-2030 (1997)

  The present invention relates to an involucrin expression promoter and a stratum corneum formation promoter that can promote involucrin expression and promote keratinization of horny layer cells.

  The present inventors have examined the promotion of involucrin expression. As a result, the alkyl or alkoxyalkyl phosphate represented by the following formula (1) or a salt thereof has excellent involucrin expression promoting action and stratum corneum formation promoting action. And it discovered that it was useful as an involucrin expression promoter and a stratum corneum formation promoter, and completed this invention.

  That is, the present invention relates to the following 1) to 4).

1) The following formula (1):
R-OPO ₃ H ₂ (1)
[R is an alkyl or alkoxyalkyl group having 12 to 24 carbon atoms]
The involucrin expression promoter which uses the compound or its salt represented by these as an active ingredient.

2) The following formula (1):
R-OPO ₃ H ₂ (1)
[R is an alkyl or alkoxyalkyl group having 12 to 24 carbon atoms]
A stratum corneum formation accelerator comprising a compound represented by the formula or a salt thereof as an active ingredient.

  3) The compound represented by the formula (1) is 16-methyloctadecylphosphoric acid, 17-methyloctadecylphosphoric acid, 11- (2-methylbutoxy) undecylphosphoric acid, octadecylphosphoric acid, hexadecylphosphoric acid, tetradecylphosphoric acid The involucrin expression promoter according to 1) or the stratum corneum formation promoter according to 2) above, which is an acid or dodecyl phosphate.

  4) 16-methyloctadecyl phosphate, 17-methyloctadecyl phosphate, 11- (2-methylbutoxy) undecyl phosphate or a salt thereof.

  According to the involucrin expression promoter and the stratum corneum formation promoter of the present invention, it has excellent involucrin production promoting ability, and promotes the formation of a healthy stratum corneum by promoting cornification of the stratum corneum cells, The skin barrier function can be made healthy.

In the formula (1), the alkyl group having 12 to 24 carbon atoms represented by R is preferably one having 12 to 20 carbon atoms.
Further, the carbon chain of the alkyl group may be either linear or branched.

  Preferable specific examples of the alkyl group represented by R include, for example, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, heicosyl group, docosyl group, Tricosyl group, tetracosyl group, 2-hexyldecyl group, 2-heptylundecyl group, 2-octyldodecyl group, 3,7,11-trimethyldodecyl group, 3,7,11,15-tetramethylhexadecyl group, iso Examples include stearyl, 16-methylstearyl group, 17-methylstearyl group, and among them, 16-methylstearyl group, 17-methylstearyl group, dodecyl group, tetradecyl group, hexadecyl group and octadecyl group are preferable.

On the other hand, in the formula (1), as the alkoxyalkyl group having 12 to 24 carbon atoms represented by R, those having 14 to 20 carbon atoms are preferable, and those having 16 to 18 carbon atoms are more preferable.
Moreover, what contains one containing ether oxygen atom is preferable.
A more preferable alkoxyalkyl group is R ¹ —O—R ² — (wherein R ¹ represents an alkyl group having 2 to 10 carbon atoms, and R ² represents an alkylene group having 10 to 21 carbon atoms). What is shown.

Here, the alkyl group having 2 to 10 carbon atoms of R ¹ may be either linear or branched, but is preferably a branched chain.
Preferable specific examples of the alkyl group represented by R ¹ include, for example, ethyl group, n-propyl group, isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, 2-methylbutyl group, 4-methylbutyl. Group, sec-amyl group, tert-amyl group, isoamyl group, anteisoamyl group, n-hexyl group, 4-methylhexyl group, 5-methylhexyl group, 5-methylheptyl group, 6-methylheptyl group, 6- Examples thereof include a methyloctyl group, a 7-methyloctyl group, a 7-methylnonyl group, and an 8-methylnonyl group.

In addition, the alkylene group having 10 to 21 carbon atoms of R ² may be linear or branched, but is preferably linear. Moreover, as an alkylene group of R < ² >, a C10-C13 thing is preferable.
Preferable specific examples of the alkylene group represented by R ² include, for example, a decylene group, an undecylene group, a dodecylene group, a tridecylene group, a tetradecylene group, a pentadecylene group, a hexadecylene group, a heptadecylene group, an octadecylene group, a nonadecylene group, and an eicosylene group. And henecosylene group.

  A more suitable alkoxyalkyl group includes, for example, an 11- (2-methylbutoxy) undecyl group.

  The salt of the compound represented by the formula (1) may be any salt that is pharmaceutically acceptable. For example, an alkali metal salt such as a lithium salt, a sodium salt, or a potassium salt, an alkaline earth such as a calcium salt, or a magnesium salt. Metal salt, alkylamine salts such as trimethylamine and triethylamine and quaternary ammonium salts, alkanolamine salts such as triethanolamine, diethanolamine and monoethanolamine, or amino acid salts such as lysine, histidine and arginine.

  In the compound represented by the formula (1) in the present invention, R is a 16-methyloctadecyl group, 17-methyloctadecyl group, 11- (2-methylbutoxy) undecyl group is a novel compound. These compounds can be produced in accordance with a known phosphoric acid esterification method. For example, an alcohol (R—OH) corresponding to the target compound is phosphorylated to form the above-mentioned salt as necessary. It can be obtained by neutralizing with an alkali. Examples of the phosphoric acid esterifying reagent include phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, polyphosphoric acid, water and phosphoric anhydride, phosphoric acid and phosphoric anhydride (Experimental Chemistry Course 1, Organic Compounds) Synthesis I, p206-210, Chemical Dojinsha). The obtained compound may be appropriately separated and purified by a known method.

  As shown in Examples below, the compound represented by the formula (1) of the present invention or a salt thereof exhibits an action of promoting the expression of the involucrin gene, thereby promoting the expression of involucrin and further promoting the formation of the stratum corneum. Thus, the skin barrier function can be made healthy. For this reason, the compound represented by the formula (1) or a salt thereof is expected to improve the moisture retention ability of the skin and prevent skin aging such as rough skin prevention, wrinkle formation, texture reduction, and pore conspicuousness. it can.

  Therefore, the compound represented by the formula (1) of the present invention or a salt thereof is used as an involucrin expression promoter or a stratum corneum formation promoter because an involucrin expression promoting action and a stratum corneum formation promoting action were observed. And can be used to produce an involucrin expression promoter or stratum corneum formation promoter. The involucrin expression promoter or stratum corneum formation promoter can be used as cosmetics, quasi drugs, pharmaceuticals, and the like.

When the involucrin expression promoter or stratum corneum formation promoter of the present invention is used as cosmetics, quasi-drugs, pharmaceuticals, etc., in the form of a skin external preparation, specifically, ointment, emulsified cosmetic, cream, milky lotion It is particularly preferable to use it in various forms such as a lotion and a gel. The external preparation for skin may be used on the surface of the skin after depilation and after removal of the stratum corneum, in addition to being used on the normal skin surface.
Such a preparation can be obtained by mixing and dispersing directly or together with a pharmaceutically acceptable carrier and then processing into a desired form by a general production method. Such various types of cosmetics, quasi-drugs, pharmaceuticals, etc., are plant extracts that contain the compound represented by the formula (1) of the present invention or a salt thereof alone or in these cosmetics. , Oily bases such as vegetable oils and animal oils, analgesics / anti-inflammatory agents, analgesics, bactericides / disinfectants, astringents, emollients, hormones, vitamins, moisturizers, UV absorbers, alcohols, chelating agents, pH adjusters , Preservatives, thickeners, pigments, fragrances and the like can be prepared by appropriately blending them within a range that does not interfere with the effects of the present invention.

  The content of the compound represented by the formula (1) of the present invention or a salt thereof in the cosmetic, quasi-drug or pharmaceutical is generally preferably 0.001 to 20% by mass, It is more preferable to set it to -5 mass%.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Production Example 1: Production of 16-methyloctadecyl phosphate disodium salt (Compound 1)
14.3 g (59.5 mmol) of 15-pentadecanolide and 24.8 g (98.0 mmol) of a 32% hydrogen bromide / acetic acid solution were placed in a 100 mL autoclave protected with Teflon (registered trademark), purged with nitrogen, sealed, and sealed at 120 ° C. In an oil bath and stirred for 16 hours. After cooling, 14 mL of ion exchange water was added, and the mixture was transferred to a separatory funnel using 200 mL of hot hexane. After washing with ion-exchanged water, the extract was dried over magnesium sulfate, filtered, and crystallized from n-hexane to obtain 17.1 g of 15-bromopentadecanoic acid (yield 90%).

  Next, 8.0 g (24.90 mmol) of 15-bromopentadecanoic acid synthesized above and 39.2 mg (0.15 mmol) of triphenylphosphine (Kanto Chemical) were added and dried under reduced pressure. Under an argon atmosphere, 107.2 mg (0.75 mmol) of copper (I) bromide (Aldrich) and 10 mL of anhydrous tetrahydrofuran were added to dissolve the raw materials. At room temperature, 54.9 mL (74.70 mmol, 1.36N tetrahydrofuran solution) of sec-butylmagnesium bromide was added dropwise over 1 hour. After stirring for 1 hour, 100 mL of 1N aqueous hydrochloric acid solution was added, and the mixture was extracted twice with 200 mL of hexane. After washing twice with 100 mL of ion exchange water, it was dried over magnesium sulfate. Filtration and concentration under reduced pressure gave 6.91 g (yield 93%) of 16-methyloctadecanoic acid.

Next, 3.21 g (10.75 mmol) of 16-methyloctadecanoic acid was dissolved in 100 mL of anhydrous diethyl ether and cooled to 10 ° C. or lower under a nitrogen atmosphere, and then 0.6650 g (17.52 mmol) of LiAlH ₄ was gradually added. After stirring at 10 ° C. or lower for 1 hour and 15 minutes, 100 mL of 1N aqueous hydrochloric acid and 100 mL of hexane were added. After removing the aqueous layer, the membrane was washed twice with 100 mL of ion exchange water and then dried over magnesium sulfate. Filtration, concentration under reduced pressure, and purification by silica gel column chromatography (hexane-ethyl acetate) gave 2.18 g (yield 71%) of 16-methyloctadecanol.

  Next, 1.90 g (12.39 mmol) of phosphorus oxychloride is dissolved in 2 mL of anhydrous tetrahydrofuran, cooled to 10 ° C. or less under a nitrogen atmosphere, and then mixed with 2.18 g (8.77 mmol) of 16-methyloctadecanol and 5 mL of anhydrous tetrahydrofuran. The solution was added dropwise, and 1.10 g (10.53 mmol) of toluethylamine was further added dropwise. After stirring at 10 ° C. or lower for 2 hours, the white precipitate was filtered off, and the filtrate was gradually charged with 0.73 g (8.77 mmol) of 48% aqueous sodium hydroxide and 50 g of ion-exchanged water, and stirred at 50 ° C. for 1 hour. . After 100 mL of diethyl ether was added, it was washed sequentially with 50 mL of saturated saline and 50 mL of ion-exchanged water, and then poured into 150 mL of cold acetone. The precipitated crystals were filtered and washed, then dried under reduced pressure, and 16-methyloctadecyl phosphate (C19- aP) 2.63 g of sodium salt (yield 78%) was obtained. Of these, 508.8 mg (1.31 mmol) was dissolved in 50 g of ion-exchanged water, 109.2 mg (1.31 mmol) of 48% sodium hydroxide was added dropwise, stirred at 50 ° C. for 30 minutes, and then freeze-dried for 24 hours. -530.1 mg (99% yield) of methyl octadecyl phosphate disodium salt was obtained.

IR (cm ^-1 , ATR method): 2922,2853,1470, 1126,1105,996
¹ H-NMR (D ₂ O, ppm): 0.87-0.90 (m, 6H), 1.14-1.21 (m, 2H), 1.33 (m, 26H), 1.64 (m, 2H), 3.80 (m, 2H)

Production Example 2: Production of 11- (2-methylbutoxy) undecyl phosphate sodium salt (Compound 2)
16.70 g (189.45 mmol) of 2-methyl-1-butanol and 8.50 g (151.49 mmol) of potassium hydroxide were stirred at 80 ° C. for 1 hour to dissolve. Further, 10.05 g (37.90 mmol) of dissolved 11-bromoundecanoic acid was gradually added dropwise. After stirring at 100 ° C. for 6 hours, the mixture was cooled at room temperature. After removing the aqueous layer, the membrane was washed twice with 100 mL of ion exchange water and then dried over magnesium sulfate. Filtration, concentration under reduced pressure, and purification by silica gel column chromatography (hexane-ethyl acetate) gave 7.61 g (74% yield) of 11- (2-methylbutoxy) undecanoic acid.

  Next, production was carried out in the same manner as in Production Example 1 using 2.46 g (9.03 mmol) of 11- (2-methylbutoxy) undecanoic acid instead of 16-methyloctadecanoic acid, and 11- (2-methylbutoxy) undecanol 2.16 g (93% yield) was obtained. Next, production was carried out in the same manner as in Production Example 1 using 1.0 g (3.87 mmol) of 11- (2-methylbutoxy) undecanol instead of 16-methyloctadecanol, and 11- (2-methylbutoxy) undecyl phosphate 1.0 g of sodium salt (yield 71%) was obtained.

IR (cm ^-1 , ATR method): 2920,2850,1462,1112,1051,948
¹ H-NMR (D ₂ O, ppm): 0.72-0.75 (m, 6H), 0.98 (m, 1H), 1.11-1.28 (m, 16H), 1.28 (m, 1H), 1.41-1.49 (m, 4H), 3.08 (t, J = 8Hz, 2H), 3.15 (dd, J = 8Hz, 2Hz, 1H), 3.24 (dd, J = 8Hz, 2Hz, 1H), 3.28 (t, J = 8Hz, 2H) , 3.68-3.70 (m, 2H)

Production Example 3: Production of 17-methyloctadecylphosphate arginine salt (compound 3)
Prepared in the same manner as in Production Example 1 from 3.01 g (9.37 mmol) of 15-bromopentadecanoic acid and 2.88 g (23.43 mmol) of iso-propyl bromide to obtain 2.28 g of 17-methyloctadecanoic acid (yield 86%). It was. Next, instead of 16-methyloctadecanoic acid, production was carried out in the same manner as in Production Example 1 using 3.00 g (10.05 mmol) of 17-methyloctadecanoic acid, and 2.62 g of 17-methyloctadecanol (yield 92%) From 17-methyloctadecanol 1.0 g (3.51 mmol), 17-methyloctadecylphosphate arginine salt 1.56 g (yield 83%) was obtained.

IR (cm ^-1 , ATR method): 2918,2849,1641,1058,1038
¹ H-NMR (D ₂ O, ppm): 0.71 (d, J = 6Hz, 6H), 0.99-1.20 (m, 26H), 1.35-1.75 (m, 4H), 3.07 (t, J = 6Hz, 2H ), 3.50 (t, J = 6Hz, 1H), 3.66 (m, 2H)

Comparative Example 1: Production of 3-methylbutyl phosphate arginine salt (Comparative Compound 1)
Manufacture in the same manner as in Production Example 1 using 1.0 g (3.51 mmol) of 3-methylbutanol instead of 16-methyloctadecanol to obtain 1.56 g of 3-methylbutyl phosphate arginine salt (yield 83%). It was.

IR (cm ^-1 , ATR method): 2918,2849,1641,1058,1038
¹ H-NMR (D ₂ O, ppm): 0.71 (d, J = 6Hz, 6H), 0.99-1.20 (m, 26H), 1.35-1.75 (m, 4H), 3.07 (t, J = 6Hz, 2H ), 3.50 (t, J = 6Hz, 1H), 3.66 (m, 2H)

Comparative Example 2: Production of 6-methyloctyl phosphate arginine salt (Comparative Compound 2)
Manufacture in the same manner as in Production Example 1 using 1.0 g (3.51 mmol) of 6-methyloctanol instead of 16-methyloctadecanol to obtain 1.56 g of 3-methyloctyl phosphate arginine salt (yield 83%). It was.

IR (cm ^-1 , ATR method): 2918,2849,1641,1058,1038
¹ H-NMR (D ₂ O, ppm): 0.71 (d, J = 6Hz, 6H), 0.99-1.20 (m, 26H), 1.35-1.75 (m, 4H), 3.07 (t, J = 6Hz, 2H ), 3.50 (t, J = 6Hz, 1H), 3.66 (m, 2H)

Example 1: Keratinocyte keratinization enhancing action Monolayer cultured normal human epidermal cells (frozen NHEK (F) Cascade Biologics) were cultured in EpiLife-KG2 medium (KURABO). Inoculate epidermal cells in a 12-well plate at 1 x 10 ⁴ cells / cm ² and incubate at 37 ° C under 5% CO ₂ for 24 hours. Then, replace with epithelial growth factor and bovine pituitary extract. Each test substance was added to a final concentration of 1 μM and 10 μM. Cells were dissolved in ISOGEN ^(R) (Nippon Gene Co., Ltd.) were collected 24 hours after the addition, RNA was prepared according to the attached protocol. From the obtained RNA using ^{TaqMan (R) High Capacity cDNA Transcription} Kit (Applied Biosystems Cat.No.4374966) was synthesized cDNA. Using the obtained cDNA as a template, the expression level of the involucrin gene was quantified by quantitative real-time PCR. As an internal standard gene, the expression level of Ribosomal Protein LargePO whose expression was not changed by addition of the compound was quantified, and the expression level of the involucrin gene was normalized. For PCR reactions, TaqMan ^(R) Universal PCR Master Mix (Applied Biosystems Cat.No. 4304437) and TaqMan ^(R) Gene Expression Assays (Applied Biosystems Cat.No.) are used as primers and probes for gene amplification and detection. It was. Note Assay ID of TaqMan ^(R) Gene Expression Assays corresponding to each gene involucrin: Hs00846307_s1, Ribosomal Protein LargePO: a Hs99999902_m1.

  In addition, as the test substance, dodecyl phosphate disodium salt (compound 4) obtained by neutralizing the above compounds 1 to 3, dodecyl phosphate (MAP-20H: Kao Corporation) with 2 equivalents of sodium hydroxide, Tetradecyl phosphate (MAP-40H: Kao Corp.) neutralized with 2 equivalents of sodium hydroxide tetradecyl phosphate disodium salt (compound 5), hexadecyl phosphate (MAP-60H: Hexadecyl phosphate disodium salt (compound 6) and octadecyl phosphate (MAP-80H: Kao Corporation) obtained by neutralizing Kao Corporation with 2 equivalents of sodium hydroxide Octadecyl phosphate disodium salt (compound 7) obtained by neutralization with sodium oxide, and the above comparative compounds 1 and 2 were used as comparative controls.

  The expression level was determined when the untreated control was taken as 100%. The results are shown in Table 1.

  As is apparent from Table 1, Compounds 1 to 7 have an excellent inboliclin expression promoting action and are thought to have an excellent stratum corneum formation promoting action.

  Formulation Example 1 prepared by a conventional method using Compound 1 among the compounds represented by Formula (1) is shown below.

Formulation Example 1
Compound 1 2.0%
Glycerin 5.0%
Dipropylene glycol 4.0%
Polyoxyethylene isocetyl ether
(20EO) 1.0%
Clove extract 1.0%
Ethanol 8.0%
Preservative Appropriate amount Perfume Appropriate amount Buffer agent Appropriate amount

Claims (4)

The following formula (1):
R-OPO ₃ H ₂ (1)
[R is an alkyl or alkoxyalkyl group having 12 to 24 carbon atoms]
The involucrin expression promoter which uses the compound or its salt represented by these as an active ingredient. The following formula (1):
R-OPO ₃ H ₂ (1)
[R is an alkyl or alkoxyalkyl group having 12 to 24 carbon atoms]
A stratum corneum formation accelerator comprising a compound represented by the formula or a salt thereof as an active ingredient.   The compound represented by the formula (1) is 16-methyloctadecyl phosphate, 17-methyloctadecyl phosphate, 11- (2-methylbutoxy) undecyl phosphate, octadecyl phosphate, hexadecyl phosphate, tetradecyl phosphate or The involucrin expression promoter according to claim 1 or the stratum corneum formation promoter according to claim 2, which is dodecyl phosphate.   16-methyl octadecyl phosphoric acid, 17-methyl octadecyl phosphoric acid, 11- (2-methylbutoxy) undecyl phosphoric acid or a salt thereof.