JP2015221755A - Cornified envelope protein production promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cornified envelope protein production promoter that acts on an epidermal cell to promote the production of cornified envelope protein.SOLUTION: A cornified envelope protein production promoter comprises ceramide that is an ester body of sphingoid and fatty acid having 9 or more carbon atoms as an active ingredient.

Description

  The present invention relates to a cornified envelope protein production promoter.

The skin is a highly complex tissue that serves as a barrier function against physical, chemical and biological attacks. This plays an important role in protecting against dehydration and controlling body temperature. This barrier function is constituted by the stratum corneum located in the outermost layer of the epidermis.
The stratum corneum structure is known to be a structure in which intercellular lipids are buried between corneocytes. Intercellular lipids contain ceramide, cholesterol, and fatty acids, and form a high-density lamellar structure. . And, when the lipid composition changes due to factors such as age, health, and environmental conditions, there is a concern that the barrier function is lowered. For this reason, it is attempted to improve the skin performance by locally applying components contained in intercellular lipids for the purpose of suppressing a decrease in barrier function.
Ceramide is known to have a barrier function when applied to the skin stratum corneum. For example, a skin treatment composition containing ceramide, cholesterol and free fatty acid has been proposed (Patent Document 1).
On the other hand, cosmetics containing soy extract and chlorappa bamboo extract have been proposed for the purpose of improving and preventing dry skin, wrinkles, rough skin, and atopic dermatitis by promoting the differentiation process of epidermal cells. Although an example containing sugar ceramide is described as the cosmetic, a plant-derived component soybean extract or chlorappatake extract has a function of promoting the differentiation process of epidermal cells (Patent Document 2, 3).

As a function of ceramide, it is reported that transcription factor PPAR (peroxisome proliferator-responsive receptor) is activated by ceramide (phytotype) in hepatoma cells (HepG2) and mouse fibroblasts (NIH / 3T3). However, all of the ceramides studied are synthetic ceramides having an aliphatic group having 2 carbon atoms (Non-patent Document 1).
Moreover, it is described that application of a PPAR activator to epidermal cells promotes cell differentiation (Non-patent Document 2).

If the epidermal cells are not sufficiently formed of the confined envelope protein, the physical strength of the stratum corneum becomes abnormal. For example, in rough skin with hyperproliferation, atopic dermatitis, and the like, dysplasia of the cornified envelope protein has been observed (Non-patent Document 3).
Therefore, Cornified envelope proteins, such as involucrin and transglutaminase, are closely related to maintaining the physical strength of the stratum corneum and maintaining the protective function in epidermal cells, and are very important proteins.

Special table 2010-505886 Special table 2010-529960 JP 2005-089389 A

Lipids Health Dis. 2011 Aug 24; 10: 150. Dermatoendocrinol. 2011 Apr; 3 (2): 113-8. Exp Dermatol 2001 Feb; 10 (1): 35-44.

  It is known that ceramide forms a physical barrier as an intercellular lipid component of the stratum corneum and has a barrier function. On the other hand, the action of promoting the production of specific proteins in normal epidermal cells located inside the stratum corneum has not been fully elucidated. As for ceramide, although synthetic ceramides having an aliphatic group having 2, 6, and 8 carbon atoms have been studied, at present, sufficient knowledge has not been obtained.

  An object of the present invention is to provide a novel cornified envelope protein production promoter that acts on epidermal cells to promote the production of cornified envelope protein.

  As a result of detailed studies, the present inventors have found that ceramide, which is an ester of a fatty acid having 9 or more carbon atoms and a sphingoid, promotes the production of a lining protein called a cornified envelope protein in keratinocytes. As a result, the present invention has been completed.

The present invention is as follows.
[1] A cornified envelope protein production promoter containing ceramide, which is an ester of a sphingoid and a fatty acid having 9 or more carbon atoms, as an active ingredient.
[2] The cornified envelope protein production promoter according to [1], wherein the ceramide that is an ester of a sphingoid and a fatty acid having 9 or more carbon atoms is a human ceramide.
[3] The cornified envelope protein production promoter according to [1] or [2], wherein the sphingoid is phytosphingosine.
[4] The cornified envelope according to any one of [1] to [3], which is a dispersion composition containing ceramide, which is an ester of a sphingoid and a fatty acid having 9 or more carbon atoms, in dispersed particles. Protein production promoter.
[5] The cornified envelope protein production promoter according to any one of [1] to [4], further comprising oleic acid.
[6] The cornified envelope protein production promoter according to any one of [1] to [5], wherein the cornified envelope protein is at least one protein selected from involucrin and transglutaminase.

  ADVANTAGE OF THE INVENTION According to this invention, it can act on an epidermis cell and can provide the production promoter of a novel coneified envelope protein which accelerates | stimulates the production of a coneified envelope protein.

6 is a graph showing the expression level of transglutaminase protein in monolayer cultured cells according to Example 4. 6 is a graph showing the expression level of involucrin protein in the epidermis model according to Example 5. 10 is a graph showing the expression level of transglutaminase protein in the epidermis model according to Example 6.

The cornified envelope protein production promoter of the present invention (hereinafter sometimes referred to as “protein production promoter”) is ceramide (hereinafter referred to as “specific ceramide”) which is an ester of sphingoid and a fatty acid having 9 or more carbon atoms. As an active ingredient.
The enhanced envelope protein production promoter of the present invention acts on epidermal cells and promotes the production of coneified envelope protein, thereby activating the epidermal cells and expected to improve dry skin, wrinkles and rough skin. The

In the present specification, the term “process” is not limited to an independent process, and even if it cannot be clearly distinguished from other processes, the term “process” is used as long as the intended action of the process is achieved. included.
In addition, in this specification, “to” indicates a range including numerical values described before and after that as a minimum value and a maximum value, respectively.
Furthermore, in this specification, the amount of each component in the composition is the total amount of the plurality of substances present in the composition unless there is a specific indication when there are a plurality of substances corresponding to each component in the composition. means.
The present invention will be described below.

<Ceramide which is an ester of sphingoid and fatty acid having 9 or more carbon atoms>
The ceramide contained as an active ingredient in the production promoter for the cornified envelope protein of the present invention is an ester of sphingoid and a fatty acid having 9 or more carbon atoms.
Ceramide is an ester of sphingoid and a fatty acid, and examples of the sphingoid constituting the specific ceramide include dihydrosphingosine, sphingosine, phytosphingosine, 6-dihydroxysphingosine and the like.
The fatty acid having 9 or more carbon atoms constituting the specific ceramide is preferably a fatty acid having an aliphatic group having 9 to 30 carbon atoms, more preferably a fatty acid having 16 to 24 carbon atoms.
The fatty acid may be a fatty acid having a linear aliphatic group, an unsaturated fatty acid containing a carbon-carbon double bond or a carbon-carbon triple bond in the carbon chain, and an α-hydroxy fatty acid. Alternatively, it may be a ω-hydroxy fatty acid having a structure in which a linear fatty acid such as linoleic acid or stearic acid is ester-bonded to the ω end of the linear ω-hydroxy saturated fatty acid.

The specific ceramide according to the present invention is not particularly limited as long as it is an ester of a sphingoid and a fatty acid having 9 or more carbon atoms.
The specific ceramide is preferably a human ceramide having the same structure as that contained in human epidermal cells, or a similar structure, and the human ceramide is more preferably phytoceramide which is an ester of phytosphingosine and a fatty acid. .
Although the operation of the present invention is not clear, it is estimated as follows. When ceramide, which is an ester of sphingoid and a fatty acid having 9 or more carbon atoms, is applied to the stratum corneum, human ceramide contained in intercellular lipids existing between the stratum corneum cells of the stratum corneum or other coexisting cells Compared to ceramides having other structures, it is easy to pass through the stratum corneum because of its good affinity for interstitial substances. For this reason, it is considered that it is easy to reach a site that contributes to protein production in epidermal cells. Furthermore, even when the specific ceramide reaches the epidermal cells, it is considered that the protein production promoting effect can be efficiently obtained due to the affinity to the living body.
On the other hand, in fatty acids that form ester bodies with sphingoids, ceramides are obtained by forming ester bodies with sphingoids for fatty acids having carbon chains of less than 9, for example, carbon numbers 2, 6, and 8. Even so, the ceramide obtained cannot unexpectedly exhibit the effects of the present invention.

The specific ceramide may be a natural ceramide or a synthetic ceramide, and the origin of a synthetic product, an extract or the like is not limited. The specific ceramide in the present invention is a natural ceramide to be described later and a compound having this as a basic skeleton, including a ceramide that is an ester of a sphingoid and a fatty acid having 9 or more carbon atoms. It is a general term for ceramide.
In addition, the specific ceramide may have a carbon-carbon multiple bond in the molecule in order to impart solubility in a range not impairing the effects of the present invention, and a hydrophobic group is introduced to improve permeability. Or may have a substituent. Examples of the substituent that can be introduced include an alkyl group, an aryl group, an aralkyl group, an amino group, a nitro group, a sulfonyl group, a phosphoric acid group, a hydroxyl group, an acyloxy group, and an alkoxy group.
Hereinafter, the specific ceramide in this invention is explained in full detail.

(Natural ceramide)
In the present invention, the natural ceramide means a human ceramide having the same structure or a similar structure as ceramide present in the stratum corneum of human skin.
Hereinafter, the natural ceramide usable in the present invention will be described in detail.

  Examples of natural ceramides that can be suitably used as ceramides in the present invention include Ceramide 1, Ceramide 4, Ceramide 9, Ceramide 2, Ceramide 3, Ceramide 3B, Ceramide 5, Ceramide 6, Ceramide 7, Ceramide 8, Ceramide 10, etc. Can be mentioned.

  In addition, the specific ceramide is modified according to the purpose, such as introducing a double bond in the molecule to impart solubility or introducing a hydrophobic group to impart permeability. Can also be used.

These ceramides having a general structure called a natural type are natural products (extracts) and ceramides obtained by a microbial fermentation method, but may further contain synthetic products and animal-derived ceramides.
These ceramides are preferably natural (D (-)) optically active substances. The relative configuration of the specific ceramide used in the present invention is a natural configuration, and may be a mixture of a ceramide having a natural configuration and a ceramide having another non-natural configuration.
Especially, it is preferable to use more natural type optically active substances from a viewpoint of an effect.

  Such natural type ceramide is also available as a commercial product. For example, Ceramide I, Ceramide III, Ceramide IIIA, Ceramide IIIB, Ceramide IIIC, Ceramide VI (above, manufactured by Cosmo Farm), Ceramide TIC-001 ( Takasago Inc.), CERAMIDE II (Quest International), DS-Ceramide VI, DS-CLA-Phytoceramide, C6-Phytoceramide, DS-ceramide Y3S (DOOSAN), CERAMIDE2 (Cedera), "Ceramide 3 [Trade name, manufactured by Evonik Co., Ltd.], “ceramide 6” (trade name, manufactured by Evonik Co., Ltd.), and the like.

(Synthetic ceramide)
A ceramide analog is synthesized by imitating the structure of ceramide. Examples of such known compounds of synthetic ceramide include synthetic ceramides (3-1) and (3-2) as shown in the following structural formula.

  When synthetic ceramide is applied, it can be used in the present invention as long as it is synthesized by imitating the structure of natural ceramide or sugar-modified ceramide, and the fatty acid constituting ceramide has 9 or more carbon atoms. A synthetic ceramide synthesized by mimicking the structure of natural ceramide is preferable. Synthetic ceramide is also available as a commercial product. For example, the exemplary compound (3-2) (cetyl hydroxyprolymph rumitamide) is available as “CeramideBio” (trade name) manufactured by symrise Inc.

  In the following, preferred examples of fatty acids constituting the specific ceramide useful as an active ingredient of a production promoter for a confined envelope protein in the present invention will be listed, but the present invention is not limited thereto.

  Examples of sphingoids that form ester forms with the above fatty acids and can become specific ceramides are given.

Specific ceramides include esters of dihydrosphingosine [DS] and fatty acid [N] (hereinafter referred to as ceramide [NDS]), esters of dihydrosphingosine [DS] and α-hydroxy fatty acid [A] (hereinafter referred to as ceramide [ ADS], ester of phytosphingosine [P] and fatty acid [N] (hereinafter referred to as ceramide [NP]), ester of phytosphingosine [P] and α-hydroxy fatty acid [A] (hereinafter referred to as ceramide) (Referred to as [AP]) and the like. Among them, ceramide [NP] and ceramide [AP] included in phytoceramide are more preferable.
In the following, as a typical specific ceramide that can be used in the present invention, a compound that is an ester of a sphingosine having 18 carbon atoms and a linear fatty acid having 18 carbon atoms is listed. In the following compounds, the ester bond site is indicated by a broken line.

  Examples of the human ceramide preferably used in the present invention include ceramide 3, ceramide 3B, ceramide 6, and ceramide 10. As typical ceramides, the structures of ceramide 3 and ceramide 3B are shown below.

  The protein production promoter of the present invention only needs to contain at least one selected from ceramide, which is an ester of sphingoid and a fatty acid having 9 or more carbon atoms, as an active ingredient, and contains two or more. May be. Since the specific ceramide generally has a high melting point and high crystallinity, it is preferable to use two or more kinds in combination from the viewpoint of improving dispersion stability and handleability.

  The epidermal cells on which the protein production promoter of the present invention acts are normal epidermal cells unless otherwise specified, and the normal epidermal cells are non-cancerous epidermal cells. Epidermal cells include keratinocytes, melanocytes, Langerhans cells, α-dendritic cells, and Merkel cells.

  The protein production-promoting agent of the present invention can act on normal epidermal cells to promote the production of a confined envelope protein. As a result, the production of a lining protein called a cornified envelope protein is promoted in epidermal cells. Cornified envelope protein is also referred to as a lining protein. By increasing the number of coneified envelope protein, the cell membrane is maintained in a good state and contributes to the formation of an intercellular lipid lamellar structure.

<Cornified envelope protein>
In the stratum corneum structure, the important elements for the barrier function are keratin filaggrin lysate in keratinocytes, cell membranes thickened by the binding of a lining protein called a cornified envelope protein, and stratum corneum cells. Examples include interstitial lipid layers.
In the present invention, it has been found that among these various important elements, the Cornified envelope protein is excellent in the barrier function. The Cornified envelope protein is preferably at least one protein selected from involucrin and transglutaminase.

(Involucrin)
Involucrin is a constituent protein located in the outermost layer in the cornified envelope (Steinert, PM. Et al J Biol Chem 1995 Jul 28; 270 (30): 17702-11).
Involucrin is not only cross-linked by other proteins such as loricrin and enzymes such as transglutaminase, but also covalently binds to ceramide, an intercellular lipid, and is very important for the formation of intercellular lipid lamellar structures. (Nemes, Z., Proc Natl Acad Sci USA 1999 Jul 20; 96 (15): 8402-7). Involucrin is produced specifically in the vicinity of the cell membrane in the differentiation process of epidermal cells and in the pre-stage of the formation of the cornified envelope protein, and is considered to be an epidermal cell differentiation marker.

(Transglutaminase)
Transglutaminase (TGase) catalyzes a reaction that forms an isopeptide bond between a glutamic acid residue and a lysine residue of a protein in a calcium-dependent manner. This physiological significance is broad with blood coagulation, cell death, morphogenesis, and enhancement of extracellular matrix. Although TGase has been reported to have 9 types of isozymes in humans, TGase1 and TGase3 are mainly present in epidermal cells. The formation of the skin epidermis is achieved by the differentiation of deep undifferentiated progenitor cells that are pushed into the surface as keratinized epidermal cells. Along with this differentiation, intracellular proteins are covalently crosslinked by TGase, resulting in hardness (keratinization) and function as a strong barrier against the outside. TGase in epidermal cells exists in an inactive precursor form at an undifferentiated stage, is limitedly decomposed with differentiation, and changes to an active form to be active (Shitaka Hitomi, “Protein-crosslinking enzyme in transdermal skin (trans Analysis of activation mechanism of glutaminase) ”).

  By applying the protein production promoter of the present invention to epidermal cells, it is possible to increase the production amount of the cornified envelope protein in the epidermal cells as compared with the case where it is not applied. Therefore, the protein production promoter of the present invention can be expected to have effects such as improvement of skin turnover by activating epidermal cells, prevention of skin dryness, prevention of wrinkles due to drying, improvement of rough skin, and the like.

The measuring method of the production amount of the confined envelope protein in the epidermal cells is not particularly limited, and a known method can be adopted. Specifically, a protein is extracted from a cultured cell or a commercially available three-dimensional epidermis model to which the protein production promoter of the present invention has been added, a method for analysis such as spectrophotometry and immunostaining, a nucleic acid is extracted, and related Examples include a method of subjecting the gene to analysis such as PCR.
An example of the extraction and analysis method will be described with reference to a method for measuring protein production in a monolayer culture system. Details of the measurement conditions will be described in Examples.

Normal human foreskin-derived epidermal keratinocytes are cultured using a medium, the cells are detached, redispersed in an assay medium or the like, and seeded. Then, after culturing for 1 day, the medium is replaced with a medium supplemented with 1% of a ceramide solution or the like, which is a test substance dissolved in dimethyl sulfoxide (DMSO), and further cultured for 2 days. After washing with PBS, 0.02% Tween-PBS containing protease inhibitor cocktail is added, and the cells are detached and collected. The collected cells are allowed to stand at 4 ° C. overnight, and then sonicated in a water bath for 30 minutes to extract proteins. Centrifugation is performed, the supernatant is collected, and the amount of protein produced can be measured by a method of quantifying the amount of protein using Protein Quantification Kit-Rapid (manufactured by DOJINDO).
Examples of cultured cells include normal human foreskin-derived epidermal keratinocytes (Life Technologies), normal human epidermis keratinocytes (Kurabo), and the like.
Examples of commercially available three-dimensional skin models include LaboCyte Epimodel (J-TEC), EPI-200 (Kurabo), and TESTSKIN (TOYOBO).

  When the protein production promoter of the present invention is applied to epidermal cells, the amount of the specific ceramide contained in the protein production promoter is preferably 0.01 mg to 100,000 mg per gram of protein in normal epidermal cells, preferably 0.1 mg to 100 mg. 10000 mg is more preferable, and 1 mg to 1000 mg is more preferable.

When the protein production promoter of the present invention is used in the form of a composition containing a specific ceramide such as a dispersion composition, the content of the specific ceramide in the composition varies depending on the dosage form.
In general, the content of the specific ceramide is preferably 0.0005% by mass to 10% by mass, more preferably 0.001% by mass to 5% by mass, and still more preferably based on the total mass of the composition. It is 0.005 mass%-1 mass%.

When the protein production promoter of the present invention is used, the form of the protein production promoter is not particularly limited. In addition to the ceramide described above, it may be in the form of a composition containing other components (carriers acceptable as pharmaceuticals or cosmetics, or other optional components as required) described later.
Examples of the form of the composition include an oil composition, a dispersion composition, and a powder composition. The oil composition, the dispersion composition, and the powder composition can be prepared according to a known method.
In the conventional method of using ceramide, an effect can be obtained by providing a barrier function in the stratum corneum. Therefore, various dosage forms can be selected as long as they can be applied to the stratum corneum as an application site. On the other hand, in the cornified envelope protein production promoter of the present invention, ceramide preferably acts on normal epidermal cells through the stratum corneum, and passes through the stratum corneum to reach the epidermal cells. It is preferable to set it as the aspect for expressing efficiently the production promotion function of an envelope protein.
As one embodiment of the preparation that easily reaches the epidermal cells, for example, an embodiment containing ceramide as dispersed particles in the dispersion composition can be mentioned. At this time, it is preferable to reduce the particle size of the dispersed particles from the viewpoint of permeability when applied to the skin.
Moreover, the aspect which contains transdermal absorbents, such as urea, in a dispersion composition is also applicable in the range which does not impair the effect of the protein production promoter of this invention.

  The other components contained in the composition containing the protein production promoter of the present invention can be appropriately selected according to the form, purpose, etc. of the composition. Preferable examples of other components include a functional oily component, an emulsifier, and other additive components.

<Dispersion composition>
The dispersion composition which is one of the preferred embodiments of the protein production promoter of the present invention will be described.
In the present invention, as one embodiment for improving the permeability of the specific ceramide and sufficiently exhibiting the function, there can be mentioned a dispersion composition as described below.

Examples of the dispersion composition include an embodiment containing specific ceramide and containing dispersed particles which are oil phase components dispersed in an aqueous phase as an oil phase, and an aqueous phase component as a dispersion medium. (Hereinafter, the dispersed particles that are the oil phase containing the specific ceramide may be referred to as “specific ceramide-containing particles”.)
The specific ceramide-containing particles may contain other oil components.
Hereinafter, components other than the specific ceramide that can be contained in the dispersion composition will be described.

(Other oily ingredients)
When the protein production promoter of the present invention takes the form of a dispersion composition, the specific ceramide-containing particles (oil phase) are dispersed in the aqueous phase. The oil phase contains at least one selected from an oily component different from the specific ceramide (hereinafter sometimes referred to as “other oily component”) and a solvent, and at least one of the oily component and the solvent. It can take a form in which oil droplet-like dispersed particles containing a specific ceramide are present. In addition, when taking the form which contains components other than specific ceramide in a dispersed particle, the average particle diameter of the specific ceramide containing particle | grains in this invention means the average particle diameter of the dispersed particle which consists of an oil phase containing specific ceramide.

  The “other oily component” in the present invention refers to an oily component that does not separate from the specific ceramide at room temperature, and the “solvent” refers to a solvent that can dissolve the specific ceramide, such as alcohol.

Examples of other oily components that can be used in combination include the following functional oily components.
(Functional oil component)
The functional oil component is not particularly limited as long as it is an oil-soluble component that does not dissolve in an aqueous medium but dissolves in an oil medium, and those having physical properties or functionality according to the purpose are appropriately selected and used. Can do.
Other oily ingredients are usually UV absorbers, anti-inflammatory agents, moisturizers, hair protectors, whitening agents, anti-staining agents, cell activators, emollients, keratolytic agents, antistatic agents, fat-soluble vitamins, etc. Are used.
More specifically, the functional oil component as the usable oil component includes a radical scavenger containing oil-soluble vitamins such as carotenoid and tocopherol, and fats and oils such as coconut oil.
Here, the functional oily component means an oily component that can be expected to exert a desired physiological action in a living body when it exists in a living body.

(water)
When the protein production promoter of the present invention takes the form of a dispersion composition, the aqueous phase as the continuous phase can contain water. The specific ceramide-containing dispersed particles exist as a dispersed phase in an aqueous phase as a continuous phase.
The water used in the present invention is preferably water with few impurities such as pure water and ion exchange water.

  The water phase only needs to contain water, but may contain components other than water, such as water-soluble components and components miscible with water. Components other than water that can be contained in the aqueous phase are not particularly limited, and examples thereof include water-soluble known components that can be usually added to the continuous phase in the production of the dispersion composition.

(Dispersant or emulsifier)
In order to improve the dispersion stability of the ceramide-containing particles, the dispersion composition may contain a known dispersant or emulsifier such as lecithin, fatty acid, surfactant, polymer dispersant, salt and the like.

(Other additive components)
In addition to the other oily components, water, and dispersants described above, the dispersion composition may appropriately contain additive components that are usually used in the fields of pharmaceuticals, functional foods, cosmetics, and the like depending on the purpose. it can. The other additive component can be contained in the protein production promoter of the present invention as an oil-soluble or water-soluble additive component depending on its characteristics.

Other additive components include, for example, polyhydric alcohols such as glycerin and 1,3-butylene glycol; glucose, fructose, lactose, maltose, sucrose, pectin, copper carrageenan, locust bean gum, guar gum, hydroxypropyl guar gum, xanthan gum , Karaya gum, tamarind seed polysaccharide, gum arabic, gum tragacanth, hyaluronic acid, sodium hyaluronate, sodium chondroitin sulfate, dextrin, etc .; sugar alcohols such as sorbitol, mannitol, maltitol, lactose, maltotriitol, xylitol Inorganic salts such as sodium chloride and sodium sulfate; casein, albumin, methylated collagen, hydrolyzed collagen, water-soluble collagen, gelatin, etc. Protein: glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, glutamic acid, cystine, methionine, lysine, hydroxylysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, acetylhydroxyproline, etc. Amino acids and derivatives thereof; synthetic polymers such as carboxyvinyl polymer, sodium polyacrylate, polyvinyl alcohol, polyethylene glycol, ethylene oxide / propylene oxide block copolymer; water-soluble cellulose derivatives such as hydroxyethyl cellulose and methyl cellulose; Can be mentioned.
The other additive components described above may be included, for example, as a functional component, an excipient, a viscosity modifier, a radical scavenger, or the like based on the function.
When the protein production promoter of the present invention takes the form of a dispersion composition, for example, various medicinal ingredients, pH adjusters, pH buffers, UV absorbers, preservatives, fragrances, colorants, etc. Other additives used in the application can be used in combination.

<Method for producing dispersion composition>
The formation mode of the specific ceramide-containing particles is not particularly limited. 1) A mode in which the specific ceramide-containing particles (oil phase) are previously formed as solid particles and then dispersed in a dispersion medium (water phase). ) Heat the specific ceramide to a molten state, or dissolve it in an appropriate solvent to make it liquid, then add and disperse it in the aqueous phase, and then cool it down to room temperature or remove the solvent By this, the aspect etc. which form specific ceramide containing particle | grains in a system are mentioned.
The specific ceramide may form dispersed particles alone as an oil phase. However, from the viewpoint of dispersion stability, the specific ceramide is compatible with other oil components or dissolved in an organic solvent to form an oil phase. The method of preparation is preferred.

  The dispersion composition containing specific ceramide-containing particles is a dispersion composition of specific ceramide-containing particles containing specific ceramide-containing particles as an oil phase dispersed in an aqueous phase, and includes an oil phase component containing at least a specific ceramide, water It can be obtained by a production method including stirring and mixing the phase components by a conventional method.

  In preparing the oil phase, a water-soluble organic solvent can be preferably used. The water-soluble organic solvent refers to an organic solvent having a solubility in water at 25 ° C. of 10% by mass or more. From the viewpoint of the stability of the resulting dispersion composition, the solubility of the water-soluble organic solvent used for preparing the oil phase in water is preferably 30% by mass or more, and more preferably 50% by mass or more.

  The water-soluble organic solvent may be used alone, or a plurality of water-soluble organic solvents may be used in combination. Further, water and a water-soluble organic solvent may be used in combination. When used in combination with water, the water-soluble organic solvent is preferably contained at least 50% by volume or more, more preferably 70% by volume or more.

  The water-soluble organic solvent is preferably used for preparing an oil phase by mixing an oil phase component when preparing a dispersion composition containing a specific ceramide in the production of the dispersion composition. Is preferably removed.

Examples of water-soluble organic solvents include methanol, ethanol, 1-propanol, 2-propanol, 2-butanol, acetone, tetrahydrofuran, acetonitrile, methyl ethyl ketone, dipropylene glycol monomethyl ether, methyl acetate, methyl acetoacetate, N-methylpyrrolidone Dimethyl sulfoxide, ethylene glycol, 1,3-butanediol, 1,4-butanediol, propylene glycol, diethylene glycol, triethylene glycol and the like, and mixtures thereof.
Among these, ethanol, a mixed solvent of ethanol and water, propylene glycol, acetone and the like are preferable, and a mixed solvent of ethanol and ethanol and water is more preferable.

The method for preparing the dispersion composition by mixing the water phase component and the oil phase component containing the specific ceramide is not particularly limited, and a known dispersion method or emulsification method can be appropriately used. From the viewpoint of reducing the particle size of the dispersed particles, a high pressure emulsification method in which a shearing force of 100 MPa or more is applied, a jet injection method in which an oil phase component is directly injected into a water phase component, or the like may be used.
From the viewpoint of further reducing the particle size of the specific ceramide-containing particles and further improving the dispersion stability and storage stability, the oil phase component and the water phase component are each independently made to have a cross-sectional area of 1 μm ² to 1 mm ^2. It is also preferable to use a method using a micromixer in which each is independently passed through the microchannel and then combined and mixed.
At this time, the viscosity of the aqueous phase is preferably 30 mPa · s or less from the viewpoint of making the specific ceramide-containing particles fine.

The ratio (mass) of the oil phase and the aqueous phase in preparing the dispersion composition is not particularly limited, but is 0.1 / 99.9 to 50 as the oil phase / water phase ratio (mass%). / 50 is preferable, 0.5 / 99.5 to 30/70 is more preferable, and 1/99 to 20/80 is more preferable.
By setting the oil phase / water phase ratio in the above range, it is preferable because the active ingredient is sufficiently contained and practically sufficient dispersion stability can be obtained.

(Particle size of specific ceramide-containing particles)
The volume average particle size of the specific ceramide-containing particles is preferably 500 nm or less, more preferably 2 nm or more and 150 nm or less, and 3 nm from the viewpoint of improving the stability of the specific ceramide-containing particles and the permeability to the stratum corneum. It is more preferably 150 nm or less, still more preferably 5 nm or more and 120 nm or less, and most preferably 5 nm or more and 100 nm or less.

The particle diameter of the dispersed particles in the present invention can be measured with various commercially available particle size distribution analyzers, but the dynamic light scattering method is applied from the particle diameter range and ease of measurement.
As a commercially available measuring device using dynamic light scattering, Nanotrac UPA (Nikkiso Co., Ltd.), dynamic light scattering type particle size distribution measuring device LB-550 (Horiba, Ltd.), a concentrated particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.) etc. are mentioned.

The particle diameter of the dispersed particles in the present invention is a value measured using a dynamic light scattering particle size distribution measuring device LB-550 (Horiba, Ltd.), and specifically, a value measured as follows. Is adopted.
That is, the particle diameter is measured by using a quartz cell after diluting with pure water so that the concentration of the oil component contained in the sample fractionated from the dispersion composition according to the present invention is 1% by mass. Do. The particle diameter can be obtained as a volume average diameter when the sample refractive index is 1.600, the dispersion medium refractive index is 1.333 (pure water), and the viscosity of the pure water is set as the viscosity of the dispersion medium.

Since the specific ceramide has high crystallinity, even if it is prepared as a dispersion composition, it becomes milky or precipitates when diluted or mixed with general skin preparations such as functional oils or cosmetic ingredients. May occur.
In the present invention, the dispersion composition of the embodiment described below can be applied as an example for improving the permeability of the specific ceramide and sufficiently exhibiting the function.

The embodiment of the dispersion composition detailed below is referred to as the “first aspect” of the dispersion composition.
As a first aspect of the dispersion composition, (A) specific ceramide-containing dispersed particles containing specific ceramide and dispersed in an aqueous phase as an oil phase, and (B) at least one fatty acid selected from fatty acid and fatty acid salt A component, (C) a first polyhydric alcohol having an IOB of 2.2 or more and a total content in the composition of 3 to 20% by mass, and an IOB of 2.0 or less And a polyhydric alcohol component containing a second polyhydric alcohol whose total content in the composition is 0 or 3% by mass or less, and the total content of the surfactant with respect to the total amount of the dispersion composition Examples thereof include a dispersion composition in which the amount is 0 or 1% by mass or less, and the total content of the specific ceramide is 3.0 times or more the total content of the fatty acid component.

  (B) The fatty acid component is preferably at least one selected from the group consisting of lauric acid, isostearic acid, oleic acid, γ-linolenic acid, α-linolenic acid and salts thereof, from the viewpoint of dispersion stability, Oleic acid is particularly preferred.

  When a fatty acid salt is applied as the fatty acid component, the salt structure constituting the fatty acid salt includes metal salts such as sodium and potassium, basic amino acid salts such as L-arginine, L-histidine and L-lysine, and triethanolamine. And alkanolamine salts. Although the kind of salt is suitably selected according to the kind of fatty acid used, etc., metal salt such as sodium salt is preferable from the viewpoint of solubility and dispersibility.

  In the dispersion composition according to the present invention, the fatty acid component only needs to be contained in an amount that allows the specific ceramide to be satisfactorily dispersed. From the viewpoint of storage stability and permeability, the total mass of the specific ceramide in the composition Is preferably 3.0 times or more of the total mass of the fatty acid component, more preferably 3.0 times or more and 100 times or less, and still more preferably 4.0 times or more and 20 times or less. By setting it as the above range, separation and precipitation of excess fatty acid can be suppressed, and fixing of the fatty acid component to the specific ceramide dispersed particles becomes sufficient, which is preferable.

  Further, from the viewpoint of reducing the size of the dispersed particles in the dispersion composition and improving the transparency of the composition, the content of the fatty acid component is 0.00001% by mass or more in terms of mass relative to the total composition. The content is preferably 0% by mass or less, and more preferably 0.00005% by mass to 2.0% by mass.

(C) As a polyhydric alcohol component, IOB is 2.2 or more, the 1st polyhydric alcohol whose total content in a composition is 3 mass% or more and 20 mass% or less, and IOB is 2.0 or less. And the 2nd polyhydric alcohol whose total content in a composition is 0 or 3 mass% or less is included. In the present invention, among the two types of polyhydric alcohol components having different IOBs, the first polyhydric alcohol having a high IOB is contained in a larger amount than the second polyhydric alcohol having a low IOB. Aggregate generation of dispersed particles is effectively suppressed, and dispersion stability is improved.
In the present invention, “polyhydric alcohol” means a substance having two or more hydroxyl groups in a compound and liquid at 25 ° C.

Here, IOB (Inorganic Organic Balance) is well known as a value representing the ratio between the inorganic value and the organic value required based on the organic conceptual diagram, and represents the degree of polarity of the oily base. I). ["Prediction of Organic Compounds and Organic Conceptual Diagram", Fujita (Chemical Domain 11-10), 1957, p. 719-725, “Emulsion prescription design based on organic conceptual diagram”, Nippon Emulsion Co., Ltd., Yaguchi, 1985, p. 98] is specifically obtained by the following formula (I).
IOB = inorganic value (IV) / organic value (OV) (I)

In the present invention, the first polyhydric alcohol has an IOB of 2.2 or more. From the viewpoint of dispersed particle stability, the IOB is preferably 2.4 or more, more preferably 2.8 or more, and most preferably 3.2 or more.
Examples of the first polyhydric alcohol having an IOB of 2.2 or more include propylene glycol (IOB = 3.33), glycerin (IOB = 5.00), 1,3-butylene glycol (IOB = 2.5), di Examples thereof include glycerin (IOB = 3.5) and the like, or a combination of two or more thereof. Of these, glycerin is preferable.

The second polyhydric alcohol is a compound having an IOB of 2.0 or less. The second polyhydric alcohol preferably has an IOB of 1.0 to 2.0, and most preferably 1.5 to 2.0, from the viewpoint of particle stability.
Such second polyhydric alcohols include isoprene glycol (IOB = 2.00), dipropylene glycol (IOB = 1.83), ethoxydiglycol (diethylene glycol monoethyl ether) (IOB = 1.63) pliers. Mention may be made of lenglycol (IOB = 2.00), hexylene glycol (IOB = 1.82) and the like, or a combination of two or more thereof.

  Content in the dispersion composition of a 1st polyhydric alcohol is 3 mass% or more and 20 mass% or less. When the content of the first polyhydric alcohol is 3% by mass or less, the moisturizing effect by the first polyhydric alcohol cannot be expected. Moreover, since content of a 1st polyhydric alcohol is 20 mass% or more, particle stability deteriorates and a transparent feeling falls, it is unpreferable. The content of the first polyhydric alcohol is more preferably 5.0% by mass to 15.0% by mass.

Content in the dispersion composition of a 2nd polyhydric alcohol is 3 mass% or less. By setting it as 3 mass% or less, the solution stability of a dispersion composition is not impaired. From the viewpoint of particle stability, the content of the second polyhydric alcohol is preferably 0.0% by mass to 2.0% by mass, and preferably 0.0% by mass to 1.0% by mass. More preferably, it is particularly preferably 0, that is not contained in the dispersion composition.
Further, from the viewpoint of solution stability, the content of the second polyhydric alcohol is preferably 80% by mass or less, more preferably 50% by mass or less of the total content of the first polyhydric alcohol. 0 is particularly preferable.

The content of the first polyhydric alcohol in the dispersion composition is preferably 3 to 1000 times the total mass of the specific ceramide, preferably 5 to 1000 times, from the viewpoint of providing stability and moisture retention. More preferably, it is more preferably 10 times to 1000 times.
The first polyhydric alcohol is at least one selected from glycerin and 1,3-butylene glycol, and the volume average particle diameter of the dispersed particles containing the specific ceramide contained in the dispersion composition is 500 nm or less. Is a preferred embodiment.

  Such a dispersion composition is obtained by mixing an oil phase component containing at least a specific ceramide and an aqueous phase component at a temperature of 40 ° C. or lower to obtain a specific ceramide dispersion having a polyhydric alcohol component as an aqueous phase component. In addition, it can be obtained by a production method including mixing the obtained specific ceramide dispersion and an aqueous composition.

The dispersion composition in the first aspect may contain a surfactant not included in the fatty acid component.
The content of the surfactant is 0, or 1% by mass or less of the total mass of the dispersion composition. As the surfactant, a nonionic surfactant is preferable, and the content of the nonionic surfactant in the dispersion composition may be 10 to 100 times the total mass of the specific ceramide.
Examples of nonionic surfactants include glycerin fatty acid ester, organic acid monoglyceride, polyglycerin fatty acid ester, propylene glycol fatty acid ester, polyglycerin condensed ricinoleic acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, polyoxyethylene sorbitan Examples include fatty acid esters.
A nonionic surfactant can be contained as an oil phase component in the dispersion composition.
When the dispersion composition contains a nonionic surfactant, the content of the nonionic surfactant is 1% by mass or less with respect to the total mass of the dispersion composition from the viewpoint of miniaturization of the dispersed particles, Preferably it is 0.5 mass% or less, and it is preferable that it is 0.3 mass% or more.

Among such nonionic surfactants, polyglycerin fatty acid esters are preferable from the viewpoint of emulsion stability, and polyglycerin fatty acid esters having an HLB of 10 to 16 are more preferable.
Surfactants such as polyglycerin fatty acid esters having an HLB of 10 or more and 16 or less can greatly reduce the oil / water phase interfacial tension, and as a result, the specific ceramide-containing particles contained in the dispersion composition as an oil phase The particle size of can be reduced.

Here, HLB is a balance between hydrophilicity and hydrophobicity that is usually used in the field of surfactants, and a commonly used calculation formula such as the Kawakami formula can be used. In the present invention, the following Kawakami equation is adopted.
HLB = 7 + 11.7 log (M _w / M _O )
Here, M _w molecular weight of the hydrophilic groups, M _O is the molecular weight of the hydrophobic group.

  Moreover, you may use the numerical value of HLB described in the catalog etc. Further, as can be seen from the above formula, a surfactant having an arbitrary HLB value can be obtained by utilizing the additivity of HLB.

  Also in the first aspect of the dispersion composition, as described above, it is preferable that the specific ceramide-containing particles are dispersed in an aqueous phase to constitute an oil phase. Further, in addition to the specific ceramide in the dispersed oil phase, at least one selected from other oil components and solvents is contained, and oil droplets containing the specific ceramide in at least one of the oil components and the solvent It can take a form in which various dispersed particles exist.

  As the “other oil component”, “solvent”, and “other additive component” that can be used in the dispersion composition, the “other oil component”, “solvent”, and “other additive component” described above are the same. Can be exemplified.

<Method for Producing Dispersion Composition (First Aspect)>
As the formation mode of the specific ceramide-containing particles in the first mode, 1) In addition to the mode in which the specific ceramide-containing particles (oil phase) are formed in advance as solid particles and then dispersed in the dispersion medium (water phase), 2) specific The ceramide is heated to a molten state, or dissolved in an appropriate solvent to form a liquid, then added to the aqueous phase and dispersed, and then cooled to room temperature or the solvent is removed. An embodiment in which specific ceramide-containing particles are formed in the system can be mentioned. The specific ceramide may be prepared alone as an oil phase, but it is preferable to prepare the oil phase by dissolving it with other oily components or dissolving it in an organic solvent.

The dispersion composition containing the specific ceramide-containing particles in the first aspect is a dispersion of a specific ceramide containing specific ceramide-containing particles dispersed in an aqueous phase as an oil phase and a fatty acid component that is an oil phase component or an aqueous phase component. It is a composition and can be obtained by a production method comprising mixing an oil phase component containing at least a specific ceramide and an aqueous phase component at a temperature of 40 ° C. or lower.
According to this method, since the oil phase component and the aqueous phase component are mixed at a temperature of 40 ° C. or less, the oil phase component dissolves well, and a specific ceramide dispersion excellent in stability over time and storage stability is obtained. Can be obtained.
In the dispersion composition in the first aspect, the volume average particle diameter of the specific ceramide-containing dispersion particles is sufficiently small, so that a transparent dispersion composition can be obtained.

  In preparing the oil phase, a water-soluble organic solvent can be preferably used. Examples of the water-soluble organic solvent that can be used in the present invention include the water-soluble organic solvents described above, and preferred embodiments are also the same.

  The water-soluble organic solvent may be used alone, or a plurality of water-soluble organic solvents may be used in combination. Further, water and a water-soluble organic solvent may be used in combination. When used in combination with water, the water-soluble organic solvent is preferably contained at least 50% by volume or more, more preferably 70% by volume or more.

  The water-soluble organic solvent is preferably used for preparing the oil phase by mixing the oil phase component when preparing the dispersion composition of the specific ceramide in the production of the dispersion composition, and after mixing with the water phase It is preferably removed.

  When preparing a dispersion composition containing the specific ceramide as dispersed particles, the temperature at the time of mixing the oil phase component and the aqueous phase component is 40 ° C. or less. Although the temperature of 40 degrees C or less at the time of mixing should just be able to be achieved when mixing an oil phase component and an aqueous phase component, the area | region set by the applied mixing (emulsification) method can be changed suitably. In the method using a micromixer, the temperature in at least the region immediately before mixing and immediately after dispersion may be 40 ° C. or lower.

For mixing the water phase component and the oil phase component, a known method such as a high pressure emulsification method in which a shearing force of 100 MPa or more is applied, or a jet injection method in which the oil phase component is directly injected into the water phase component may be used.
From the viewpoint of the particle size, dispersion stability, and storage stability of the specific ceramide-containing particles, the oil phase component and the water phase component are each independently provided in a microchannel having a cross-sectional area of 1 μm ² to 1 mm ² at the narrowest portion. It is preferable to use a method using a micromixer that is mixed and mixed after being independently passed.
At this time, the viscosity of the aqueous phase is preferably 30 mPa · s or less from the viewpoint of making the specific ceramide-containing particles fine.

  As a method for producing a dispersion composition containing a specific ceramide, for example, a) an aqueous phase is prepared using an aqueous medium (such as water) containing a fatty acid salt (if present), and b) an oil containing at least the specific ceramide An oil phase is prepared using phase components, and c) the oil phase and the aqueous phase are mixed and dispersed by a method described in detail later using a micromixer, and the volume average particle diameter is 1 nm. A step of obtaining a specific ceramide dispersion (emulsion) containing specific ceramide-containing particles (dispersed particles) of 100 nm or less is mentioned.

The ratio (mass) of the oil phase and the aqueous phase in preparing the dispersion is not particularly limited, but is 0.1 / as in the oil phase / water phase ratio (mass%) described above. 99.9-50 / 50 is preferable, 0.5 / 99.5-30 / 70 is more preferable, and 1 / 99-20 / 80 is further more preferable.
By setting the oil phase / water phase ratio in the above range, it is preferable because the specific ceramide as an active ingredient is sufficiently contained and practically sufficient dispersion stability can be obtained.

(Micro mixer)
In the production of a dispersion composition containing specific ceramide as dispersed particles, the volume average particle size of the dispersed particles is smaller, for example, in order to form stably as dispersed particles of 1 nm or more and 100 nm, an oil phase component, an aqueous phase component, It is preferable to adopt a manufacturing method in which each is independently passed after passing through a microchannel having a cross-sectional area of the narrowest portion of 1 μm ² or more and 1 mm ² or less.

  The mixing of the oil phase component and the water phase component is preferably mixing by counterflow collision from the viewpoint of obtaining finer dispersed particles.

The most suitable device for mixing by countercurrent collision is a counterimpact micromixer. A micromixer mainly mixes two different liquids in a minute space, one liquid is an organic solvent phase containing a functional oil component such as a specific ceramide, and the other liquid is an aqueous solution. It is an aqueous phase.
When a micromixer is applied to emulsion preparation with a small particle size, which is one of the microchemical processes, the heat generation is relatively low and less heat is generated, and the particle size is uniform compared to the usual stirring emulsification dispersion method and high-pressure homogenizer emulsification dispersion. In addition, it is easy to obtain a good emulsion or dispersion having excellent storage stability. This is the most suitable method for emulsification including natural components that are susceptible to thermal degradation.

  In the first aspect described above, the specific ceramide is dispersed using a fatty acid component instead of an emulsifier such as a surfactant. For this reason, it is possible to improve the permeability of the specific ceramide to the keratin than when an emulsifier such as a surfactant is used, and thus improve the reach of the specific ceramide to the epidermal cells in the lower layer of the keratin. be able to.

  Another embodiment of the method for producing a dispersion composition containing the specific ceramide includes a dispersion composition obtained by the production method shown below. Hereinafter, this embodiment is referred to as a second aspect.

<Method for producing dispersion composition (second embodiment)>
The method for producing a dispersion composition in the present embodiment includes a mixture (hereinafter, referred to as a specific ceramide, a nonionic surfactant, and a polyhydric alcohol in an amount of 10 times to 12000 times the mass of the specific ceramide. (Also referred to as “pre-dispersion liquid”) in a state heated to 100 ° C. or higher to obtain a coarse dispersion (hereinafter also referred to as “coarse dispersion preparation step”), the coarse dispersion and water Alternatively, a composition containing water (hereinafter also referred to as “water”) is mixed and then subjected to high-pressure emulsification treatment (hereinafter, appropriately referred to as “high-pressure emulsification treatment step”). The dispersion composition obtained by the production method of the second aspect of the dispersion composition can also be applied to the present invention.

In the method for producing a dispersion composition according to the second aspect, a mixture obtained by combining a specific ceramide, a nonionic surfactant, and a specific amount of a polyhydric alcohol in advance is heated to 100 ° C. or higher, and the specific ceramide is added. After the dispersion treatment in the melted state, the resulting coarse dispersion is mixed with water, etc., and subjected to high-pressure emulsification treatment, so that the specific ceramide in a state that the surface state of the particles is easy to penetrate into the skin. Dispersed particles can be obtained.
The dispersion composition containing the fine specific ceramide dispersed particles exhibits significantly superior skin permeability as compared with the dispersion composition containing the specific ceramide-containing dispersed particles having the same particle diameter produced by the conventional method. .

The reason is not clear, but the present inventor presumes as follows.
In general, from the viewpoint of the permeability of the specific ceramide-containing dispersed particles to the skin, it is considered that the smaller the particle size of the specific ceramide-containing dispersed particles, the better. In the production method according to the second aspect, a liquid containing a combination of a specific ceramide, a nonionic surfactant, and a specific amount of a polyhydric alcohol is heated to 100 ° C. or higher to melt the specific ceramide. After the dispersion treatment in the state, the resulting coarse dispersion is mixed with water, etc., and high-pressure emulsification treatment is performed, thereby forming specific ceramide dispersed particles that are fine and in which the surface state of the particles can easily penetrate into the keratin. It is thought to be done.

The polyhydric alcohol used for the preparation of the pre-dispersion solution is preferably at least one selected from the group consisting of glycerin and diol compounds.
The content of the polyhydric alcohol is 10 times to 12000 times the amount of the specific ceramide, preferably 30 times to 1000 times, and more preferably 50 times to 500 times. preferable.

The specific ceramide, the nonionic surfactant, and the polyhydric alcohol need only be mixed. The specific ceramide, the nonionic surfactant, and the polyhydric alcohol may be mixed at one time, or these components may be divided into several parts and mixed. The mixing method is not particularly limited, and examples thereof include mixing by stirring.
The pre-dispersion solution may contain other components other than the specific ceramide, the nonionic surfactant, and the polyhydric alcohol as necessary. About another component, it is as having described in the above-mentioned 1st aspect etc., For example, components, such as cholesterol, are mentioned.

  In the coarse dispersion preparation step, the pre-dispersion solution is heated to 100 ° C. or higher from the viewpoint of melting the specific ceramide. The temperature for heating the pre-dispersion solution is preferably 100 ° C. or higher and 200 ° C. or lower, more preferably 105 ° C. or higher and 140 ° C. or lower. When the pre-dispersion solution is heated at a temperature exceeding 140 ° C., it may be oxidatively decomposed depending on the type of specific ceramide, heating conditions, and the like.

  Means for dispersing the pre-dispersion solution to obtain a coarse dispersion is not particularly limited, and a general stirring device can be used. Examples of the stirrer include a magnetic stirrer, a household mixer, a paddle mixer, an impeller mixer, a homomixer, a disper mixer, and an ultra mixer.

  The time for the dispersion treatment is not particularly limited, and can be appropriately set according to the type of the stirring device, the composition of the mixture (liquid before dispersion treatment), and the like.

<High pressure emulsification process>
The high-pressure emulsification treatment step is a step of performing high-pressure emulsification treatment after mixing the coarse dispersion obtained in the coarse dispersion preparation step with water or the like.
In the high-pressure emulsification treatment step, a molten coarse dispersion of the specific ceramide and water are mixed and subjected to high-pressure emulsification treatment to obtain a dispersion composition containing the specific ceramide-containing dispersed particles.

From the viewpoint of preventing bumping, the temperature of the coarse dispersion when mixing with water or the like is preferably set to 100 ° C. or less, and more preferably set to 90 ° C. to 100 ° C.
The temperature of water or the like is not particularly limited, but is preferably set to 40 ° C. to 90 ° C. and more preferably set to 50 ° C. to 80 ° C. from the viewpoint of miniaturization of the specific ceramide-containing dispersed particles. preferable.

The coarse dispersion and water may be mixed at one time, or may be mixed while adding the other little by little, and the coarse dispersion is stirred from the viewpoint of refinement of the specific ceramide dispersion particles. A method of adding water or the like is preferable. Examples of the mixing method include mixing by stirring.
The mixing ratio of the coarse dispersion and water or the like is not particularly limited, but from the viewpoint of miniaturization of the specific ceramide dispersed particles, the coarse dispersion / water equivalent ratio (mass basis) is 1/20 to 10 Is preferably 1/10, more preferably 1/10 to 5/1, and still more preferably 1/2 to 2/1.
In addition, the ratio of the dispersed phase to the continuous phase in the specific ceramide dispersion composition obtained by the production method of the present invention is such that the coarse dispersion and water are fine from the specific ceramide dispersed particles and are stable over time. , The dispersed phase / continuous phase ratio (mass basis) is preferably 1/1000 to 1/5, more preferably 1/100 to 1/10. It is more preferable to mix so that it may become 50-1 / 10.

  In the present invention, “high-pressure emulsification treatment” means a dispersion treatment in which a shearing force (treatment pressure) of 50 MPa or more is applied to the material to be dispersed. From the viewpoint of miniaturization of the specific ceramide dispersed particles, the shearing force applied to the dispersion is preferably 100 MPa or more, more preferably 180 MPa or more, and further preferably 200 MPa or more. In the case of a commercially available apparatus, the upper limit value is preferably 300 MPa or less from the viewpoint of temperature rise and pressure resistance.

  The means for the high-pressure emulsification treatment is not particularly limited, and a general high-pressure emulsification apparatus can be used. As high pressure emulsifiers, there are ULTIMIZER HJP-25005 (manufactured by Sugino Machine Co., Ltd.), microfluidizer (manufactured by Microfluidic Corporation), nanomizer (manufactured by Yoshida Kikai Kogyo Co., Ltd., gorin type homogenizer (manufactured by APV), Lanier type. Homogenizer (manufactured by Lanier), high-pressure homogenizer (manufactured by Niro Soabi), homogenizer (manufactured by Sanwa Kikai Co., Ltd.), high-pressure homogenizer (manufactured by Izumi Food Machinery Co., Ltd.), ultra-high pressure homogenizer (manufactured by Ika) A high pressure homogenizer may be mentioned.

  The temperature during the high-pressure emulsification treatment is preferably set to 20 ° C. to 80 ° C., more preferably 40 ° C. to 70 ° C., from the viewpoint of miniaturization of the specific ceramide dispersed particles.

  Although the frequency | count of performing a high pressure emulsification process may be 1 time, in order to improve the uniformity of the whole liquid, it is preferable to perform a high pressure emulsification process 2 times or more, and it is more preferable to perform 2-5 times.

  In the high-pressure emulsification treatment step, from the viewpoint of miniaturization of the specific ceramide dispersion particles, it is preferable to perform dispersion treatment by applying ultrasonic waves after mixing the coarse dispersion and water and before high-pressure emulsification treatment. A general ultrasonic dispersion apparatus can be used for the dispersion treatment by applying ultrasonic waves.

  As an ultrasonic dispersing apparatus, ultrasonic homogenizer US-600, US-1200T, RUS-1200T, MUS-1200T (above, manufactured by Nippon Seiki Seisakusho), ultrasonic processor UIP2000, UIP-4000, UIP-8000, UIP-16000 (above, manufactured by Heelscher) and the like. These ultrasonic dispersion apparatuses can be used at a frequency of 25 kHz or less, preferably 15 kHz to 20 kHz.

<Other processes>
In the method for producing a dispersion composition applicable to the present invention exemplified above, other steps other than the above-described dispersion treatment may be included as necessary within a range not impairing the effects of the present invention.
Examples of other processes include a sterilization process.

The protein production promoter of the present invention can act on the epidermis cells as an active ingredient to promote the production of a confined envelope protein.
Moreover, it can use suitably for pharmaceuticals, cosmetics, etc., such as a skin external preparation, by setting it as the dispersion composition which contains the specific ceramide which is a preferable aspect of a protein production promoter in a dispersion particle.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them. Unless otherwise specified, “%” and “parts” are based on mass.

[Example 1]
(Preparation of human-type ceramide-containing dispersion composition A)
Each component of the following composition 1 was mixed and stirred for about 30 minutes to prepare a human-type ceramide-containing dispersion composition A.
When the particle diameter of the ceramide-containing dispersed particles in the dispersion composition A was measured by the method described above, the volume average particle diameter was several hundred nm to several tens of μm.

(Composition 1) (Content (%))
Ceramide 3B [human ceramide] 0.9%
Ceramide 6 [human ceramide] 1.1%
Oleic acid 0.4%
Glycerin 1.43%
1,3-butanediol 1.43%
Sodium hydroxide Appropriate amount Pure water Remaining amount

[Example 2]
(Dispersion composition containing human nanoceramide as dispersed particles (dispersion composition B))
Each component described in the following oil phase liquid 1 composition was stirred at room temperature for about 30 minutes to prepare oil phase liquid 1.
The aqueous phase liquid 1 was prepared by heating each component described in the following “Aqueous phase liquid 1 composition” to pure water at about 30 ° C., and sufficiently stirring and dissolving.

<1 composition of oil phase liquid>
Ceramide 3B [human ceramide] 0.9 part Ceramide 6 [human ceramide] 1.1 part Oleic acid (melting point: 14 ° C.) 0.2 part Ethanol [water-soluble organic solvent] 97.8 parts

<Aqueous phase 1 composition>
Pure water 97.14 parts Glycerin 1.43 parts 1,3-butanediol 1.43 parts Sodium hydroxide Suitable amount

  The resulting oil phase liquid 1 (oil phase) and aqueous phase liquid 1 (aqueous phase) were mixed at a ratio (mass ratio) of 1: 7 using a collision type KM micromixer 100/100. Thus, a ceramide dispersion 1 at 30 ° C. was obtained. The conditions for using the micromixer are as follows.

-Microchannel-
Oil phase side microchannel Cross-sectional shape / width / depth / length = rectangle / 70 μm / 100 μm / 10 mm
Water phase side microchannel Cross-sectional shape / width / depth / length = rectangle / 490 μm / 100 μm / 10 mm
-Flow rate-
The aqueous phase is 21.0 ml / min. At a flow rate of 3.0 ml / min. Were introduced at a flow rate of 5 μm and micromixed.

(Preparation of dispersion composition B)
The obtained ceramide dispersion 1 was repeatedly desolvated until the ethanol concentration became 0.1% by mass or less using “Evapol (CEP-lab)” manufactured by Okawara Seisakusho, so that the ceramide concentration was 2.0 mass. The dispersion composition B containing dispersed particles containing specific ceramide having a pH of 7.5 was obtained. Here, the ceramide concentration is the content of the specific ceramide based on the total mass of the dispersion composition B.
When the particle size of the ceramide-containing dispersed particles in the dispersion composition B was measured by the method described, the volume average particle size was about 20 nm.

In addition, the detail of each component used in Example 1 and Example 2 is as follows.
Ceramide 3B [Ceramide IIIB: trade name, EVONIK Corporation]
Ceramide 6 [Ceramide VI: Trade name, EVONIK Corporation]
Oleic acid (melting point: 14 ° C.) [LUNAC-OV: trade name, Kao Corporation]
Ethanol [Traceable 99: Trade name, Nippon Alcohol Sales Co., Ltd.]
Glycerin [Glycerin: trade name, Kao Corporation]
1,3-butanediol (butanediol: trade name, Daicel Corporation)
0.1 molar sodium hydroxide [1N NaOH: trade name, Wako Pure Chemical Industries, Ltd.]

[Example 3-1]
(Monolayer culture system: measurement of involucrin protein production)
Normal human foreskin-derived epidermal keratinocytes (HKDn) were converted to Epilife (0.06 mmol Ca2 +, 0.2 ug / L EGF, 20 mg / L insulin, 1 mg / L hydrocortisone, 4 ml / L BPE, 100 mg / L gentamicin, 100 ug / L Using an amphotericin-containing medium (manufactured by Kurabo Industries, Inc.), the medium was cultured at 37 ° C. under 5% CO ₂ until it was 80% confluent.
After exfoliation by trypsin treatment, it was redispersed in assay medium (DMEM • HAM-F12 (3: 1), 5% fetal calf serum; manufactured by Japan Tissue Engineering Co., Ltd.), and 2.5 × 10 ⁴ cells / cm ² sowing. After culturing at 37 ° C. under 5% CO ₂ for 1 day, the test substance (1: 1 (molar ratio) mixture of human ceramide: ceramide 3B and ceramide 6) dissolved in DMSO has a ceramide concentration of 5 μmol. The medium was replaced with the medium added in an amount and cultured at 37 ° C. under 5% CO ₂ for 2 days. After washing with phosphate buffered saline (PBS), 0.02% Tween-PBS containing protease inhibitor cocktail (complete; manufactured by Roche Applied Science) was added at 0.5 ml / well, and the cells were added. Stripped and collected.

The collected cells were allowed to stand overnight at 4 ° C. and then sonicated in a water bath for 30 minutes to extract proteins. Centrifugation was performed at 4 ° C. and 3000 × g for 15 minutes, and the supernatant was collected. The amount of protein was quantified using Protein Quantification Kit-Rapid (manufactured by DOJINDO).
Western blotting was performed using an anti-human involucrin antibody (manufactured by Sigma-aldrich) using the recovered protein extract. The results of Western blotting were quantified using image analysis software (Fuji Film Co., Ltd .: Multi Gauge). In addition, the amount of involucrin protein produced in a standard sample to which no specific ceramide was added was quantified in the same manner. In the column of “Protein production promoting effect” in Table 1, the increase rate of the protein production amount in the sample containing the specific ceramide when the protein production amount in the standard sample not containing the specific ceramide is defined as 100 is described.

Moreover, the promotion effect was evaluated from the increase rate of protein production according to the following criteria. The results are shown in the “determination” column of Table 2.
(Standard)
A: Increase rate of production amount is 50% or more B: Increase rate of production amount is 20% or more and less than 50% C: Increase rate of production amount is 10% or more and less than 20% D: Increase rate of production amount is less than 10%

[Example 3-2 to Example 3-3]
Example 3-1 except that a 1: 1 molar ratio mixture of ceramide 3B and ceramide 6 was added to the medium so that ceramide 3 and ceramide 3B, which are human ceramides, each had a ceramide concentration of 5 μmol. Thus, the increase rate of the amount of involucrin protein produced was calculated. Moreover, the protein production promotion effect was evaluated from the increase rate. The results are shown in Table 1.

[Example 3-4]
Involucrin was prepared in the same manner as in Example 3-1, except that a 1: 1 molar ratio mixture of ceramide 3B and ceramide 6 was added to the medium so as to give a ceramide concentration of 10 μmol instead of ceramide 10 as human ceramide. The rate of increase in protein production was calculated. Moreover, the protein production promotion effect was evaluated from the increase rate. The results are shown in Table 1.

[Example 3-5]
A 1: 1 molar ratio mixture of ceramide 3B and ceramide 6 was used as a dispersion composition obtained in Example 2 (volume containing human ceramide ceramide 3B and ceramide 6 in a mass ratio of 0.9: 1.1). The increase rate of the amount of involucrin protein produced was calculated in the same manner as in Example 3-1, except that it was added to the medium so that the ceramide concentration was 0.6 μmol. Moreover, the protein production promotion effect was evaluated from the increase rate. The results are shown in Table 1.

  From the results in Table 1, it was revealed that the amount of involucrin protein produced increased by applying human ceramide in monolayer cultured cells. Among human ceramides, it can be seen that ceramides 3, 3B and 6 which are phytoceramides can obtain a good protein production promoting effect even at a low concentration as compared with ceramide 10 which is dihydroceramide. Furthermore, from the comparison between Example 3-1 and Example 3-5, even when the same kind of specific ceramide is used, the protein production promoting effect is further improved by reducing the particle size of the ceramide-containing particles. I understand.

Example 4
(Monolayer culture system: measurement of transglutaminase protein production)
In the test substance (human ceramide) dissolved in DMSO used in Example 3, a 1 μmol solution (wherein μmol is abbreviated as “μM” in FIG. 1) and a medium supplemented with 1% of a 5 μmol solution, respectively. The protein extract was recovered in the same manner as in Example 3 except that it was used.
Using the collected protein extract, the amount of transglutaminase protein was measured using Transglutaminase 1, Keratinocyte ELISA Kit (manufactured by Uscn Life Science Inc.). The results are shown in FIG.
From the results of FIG. 1, it was revealed that the expression level of transglutaminase protein is increased by applying human-type ceramide in monolayer cultured cells.

Example 5
(Epidermal model: measurement of involucrin protein production)
A three-dimensional epidermis model (manufactured by J-TEC) was pre-cultured in the attached assay medium for 1 day at 37 ° C. and 5% CO ₂ .
50 μl of 1% human ceramide coarse dispersion (dispersion composition A obtained in Example 1) or 1% human ceramide nanodispersion (dispersion composition B obtained in Example 2) was applied from the stratum corneum side. The cells were cultured for 3 days under 5% CO ₂ .
The cultured skin tissue was washed twice with 100 μl of PBS, peeled off from the culture cup, and added with 0.5 ml / well of 0.02% Tween-PBS containing a protease inhibitor cocktail (complete; manufactured by Roche Applied Science). Was peeled and collected.
The collected cells were allowed to stand overnight at 4 ° C. and then treated for 2 minutes with a bead crusher to extract proteins. Centrifugation was performed at 4 ° C. and 3000 × g for 15 minutes, and the supernatant was collected. The amount of protein was quantified using Protein Quantification Kit-Rapid (manufactured by DOJINDO).
Using the recovered protein, the amount of involucrin protein was measured in the same manner as in Example 3. The relative values when the control (water) is 100% are shown in FIG.
From the results shown in FIG. 2, it was revealed that the expression level of involucrin protein is increased by applying the human ceramide dispersion in the epidermis model. Furthermore, it has been clarified that the effect is improved by making the volume average particle diameter of the specific ceramide-containing particles nano.

Example 6
(Epidermal model: measurement of transglutaminase protein production)
Using the protein extract collected in Example 3, the amount of transglutaminase protein was measured by the method described in Example 4. The relative values when the control (water) is 100% are shown in FIG.
From the results shown in FIG. 3, it was revealed that the expression level of the transglutaminase protein is increased by applying the human ceramide dispersion in the epidermis model. Furthermore, it has been clarified that the effect is improved by making the volume average particle diameter of the specific ceramide-containing particles nano.

Example 7
Preparation of lotion Using the protein production accelerator of the present invention (dispersion composition B obtained in Example 2), a lotion having the following composition 1 was prepared by a conventional method (total amount: 100% by mass).
[Composition 1]
(Ingredients) [Content:%]
Dispersion Composition B (Dispersion Composition B obtained in Example 2: the same applies hereinafter) 5.0
Glycerin 5.0
1,3-butanediol 6.5
Polyoxyethylene sorbitan monolaurate 1.2
Ethanol 3.0
Carboxyvinyl polymer 0.2
Sodium citrate 1.0
Collagen 1.0
Methyl paraoxybenzoate 0.05
N-acetylhydroxyproline 1.0
Purified water remaining

Example 8
Preparation of lotion A lotion having the following composition 2 was prepared by a conventional method (total amount: 100% by mass).
[Composition 2] [Content (%)]
Dispersion composition B 1.0
Dipotassium glycyrrhetinate 1.0
Dipropylene glycol 4.0
Polyoxyethylene methyl glucoside 1.0
1,3-butylene glycol 4.0
Polyethylene glycol 1.0
Ethanol 2.0
Polyoxyethylene hydrogenated castor oil (60 EO) 0.2
Phenoxyethanol 0.3
Glycerin mono-2-ethylhexyl ether 0.2
Oryzanol 0.01
Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals) 0.03
Alpha lipoic acid 0.5
Cystine 0.5
N-acetyl-L-hydroxyproline 1.0
Water-soluble collagen 1.0
Hydrolyzed collagen 1.0
Seaweed extract (1) 1.0
Astaxanthin-containing emulsion composition (krill extract) 0.2
[Composition prepared by the method described below]
Lycopene-containing emulsion composition [composition prepared by the method described below] 0.1
Phosphoric acid-L-ascorbyl magnesium 1.0
Sodium hyaluronate 0.2
Citric acid 1.0
Sodium citrate appropriate amount water remaining

<Preparation of an astaxanthin-containing emulsion composition>
The following components were dissolved for 1 hour while heating at 70 ° C. to obtain an aqueous phase composition A.
・ Sucrose stearate (HLB = 16) 33.0 g
・ Decaglyceryl monooleate (HLB = 12) 67.0 g
・ Glycerin 450.0g
・ Pure water 300.0g

The following components were dissolved for 1 hour while heating at 70 ° C. to obtain an oil phase composition A.
・ Krill extract 15.0 g
・ Mix tocopherol (Riken Vitamin, Riken E Oil 800) 32.0g
・ Medium chain fatty acid glyceride (Kao Coconard MT) 93.0g
・ Lecithin (manufactured by Riken Vitamin, Recion P, derived from soybean) 10.0 g

The aqueous phase composition A obtained above was stirred with a homogenizer (model name: HP93, manufactured by SMT Co., Ltd.) while maintaining the temperature at 70 ° C. (10000 rpm), and the oil phase composition A was added to the aqueous phase composition A. Addition to obtain a pre-emulsion.
Subsequently, the obtained preliminary emulsified product was cooled to about 40 ° C., and high pressure emulsification was performed at 200 MPa using an optimizer HJP-25005 (manufactured by Sugino Machine Co., Ltd.). Then, it filtered with the micro filter with an average hole diameter of 1 micrometer, and prepared the astaxanthin containing emulsion composition (astaxanthin content rate: 0.3 mass%).

  The obtained astaxanthin emulsion composition was diluted to 1% by mass with MilliQ water, and the particle size of the dispersed particles was measured using a particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.). It was.

  Even if the krill extract is replaced with product name: Astax-ST (Marin Daio Co., Ltd.) or product name: ASTOTS-S Takeda Paper Co., Ltd. (Hematococcus alga extract), an equivalent emulsion composition is obtained. Can be obtained.

<Preparation of lycopene-containing emulsion composition>
[Oil phase composition B]
・ Tomato oleoresin 1.14g
(Sunbright, Lyc-O-Mato 15% (containing 15% by mass of lycopene))
・ Lecithin (manufactured by Riken Vitamin, Recion P, derived from soybean) 1.0g
・ Medium chain fatty acid glyceride (Kao Coconard MT) 12.8g

[Aqueous composition B]
・ Decaglyceryl oleate-10 8.0 g
(HLB = 12.0, manufactured by Nikko Chemicals, Decaglyn 1-OV)
・ Sucrose stearate 2.0g
(Mitsubishi Chemical Foods, Ryoto Sugar Ester S-1670)
・ Glycerin 45.0g
・ Remaining amount up to 100g of purified water

Each component used in the aqueous phase composition B is weighed in a container according to the above composition, heated and mixed with stirring in a constant temperature bath at 70 ° C., confirmed to be well mixed, held at 70 ° C., An aqueous phase composition B was obtained.
Also, each component used in the oil phase composition B was weighed in a container according to the above composition, heated and mixed for 5 minutes while stirring on a hot plate at 150 ° C., and confirmed to be well mixed. Phase composition B was obtained.
The obtained aqueous phase composition B was added to the oil phase composition B, mixed with stirring, and dispersed for a predetermined time using an ultrasonic homogenizer to obtain a coarse dispersion.
Thereafter, the obtained coarse dispersion was further emulsified at a high pressure of 200 MPa using an ultrahigh pressure emulsifier (Altimizer, manufactured by Sugino Machine Co., Ltd.), and a lycopene-containing composition (lycopene content: 0.17 mass). %) Was prepared.

The obtained lycopene-containing emulsion composition was diluted by 1% by mass with milliQ water, and the particle size of the dispersed particles was measured using a particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.). It was.
The lotion of Example 8 was prepared by appropriately using the astaxanthin-containing emulsion composition and the lycopene emulsion composition prepared by the above method.

Example 9
Preparation of cream A cream having the following composition 3 was prepared by a conventional method (total amount: 100% by mass).
[Composition 3] [Content (%)]
Dispersion composition B 1.0
Arbutin 2.0
Dipotassium glycyrrhetinate 1.0
Phosphoric acid-L-ascorbyl magnesium 0.1
1,2-pentanediol 3.0
Dipropylene glycol 7.0
Concentrated glycerin 5.0
Polyethylene glycol 6000 [Molecular weight: 6000] 1.0
Sodium hyaluronate 0.5
Trimethylglycine 0.5
1,3-butylene glycol 3.0
Xanthan gum 0.5
Acrylic acid / alkyl methacrylate copolymer 0.7
Squalane 0.5
Shea fat 1.0
Sara honey bee 1.0
Behenyl alcohol 1.0
Glyceryl monostearate 2.0
Polyoxyethylene glyceryl isostearate 1.0
Tocopherol 0.5
Astaxanthin 0.2
Water-soluble collagen 1.0
Hydrolyzed collagen solution (from fish) 1.0
N-acetyl-L-hydroxyproline 1.0
Oryzanol 0.01
Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals) 0.03
Polyoxyethylene hydrogenated castor oil (60 EO) 0.2
Camellia extract 0.01
Lecithin 0.1
Sucrose fatty acid ester 0.1
Polyglyceryl monooleate 0.1
Citric acid 0.7
Sodium citrate appropriate amount Phenoxyethanol 0.3
Purified water remaining

Example 10
Preparation of jelly-like serum A jelly-like serum having the following composition 4 was prepared by a conventional method (total amount: 100 mass%).
[Composition 4] [Content (%)]
Haematococcus alga extract 0.1
Astaxanthin-containing emulsion composition (krill extract)
[Composition prepared by the method described above] 0.2
Ceramide 3, ceramide 6 mixture (molar ratio 1: 1) 1.0
Dispersion composition B 1.0
Hydrolyzed collagen 1.0
Acetylhydroxyproline 1.0
Ethylhexyl glycerin 0.1
Oleic acid 0.5
1,3-butylene glycol 1.0
Glycerin 2.0
Sucrose 0.1
Polyglyceryl oleate-10 0.1
Polyglyceryl stearate-2 0.1
Phenoxyethanol 0.2
Collagen 1.0
Oryzanol 0.01
Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals) 0.03
Polyoxyethylene hydrogenated castor oil (60 EO) 0.2
Camellia extract 0.01
Lecithin 0.1
Citric acid 1.0
Sodium citrate appropriate amount (PEG-240 / decyltetradeceth-20 / HDI) copolymer 0.3
Damask rose flower oil Trace amount Fragrance amount Purified water Remaining amount

Claims (6)

  Cornified envelope protein production promoter containing ceramide, which is an ester of sphingoid and a fatty acid having 9 or more carbon atoms, as an active ingredient.   The ceramide, which is an ester of a sphingoid and a fatty acid having 9 or more carbon atoms, is a human-type ceramide.   The spawning agent according to claim 1 or 2, wherein the sphingoid is phytosphingosine.   The enhanced production of a cornified envelope protein according to any one of claims 1 to 3, which is a dispersion composition containing ceramide, which is an ester of a sphingoid and a fatty acid having 9 or more carbon atoms, in dispersed particles. Agent.   The cornified envelope protein production promoter according to any one of claims 1 to 4, further comprising oleic acid.   The cornified envelope protein production promoter according to any one of claims 1 to 5, wherein the cornified envelope protein is at least one protein selected from involucrin and transglutaminase.