Skin barrier function improving agent, ceramide production promoter, cosmetic composition, skin external preparation, and oral administration preparation

Abstract

This skin barrier function improving agent contains cytidylic acid or a precursor thereof as an active ingredient.

Description

Skin barrier function improver, ceramide production promoter, cosmetic composition, skin external preparation, and oral administration agent

[0001] The present invention relates to a skin barrier function improver, a ceramide production promoter, a cosmetic composition, a skin external preparation, and an oral administration agent. The present invention also relates to a method for improving the skin barrier function and a method for promoting the production of ceramides.

[0002] In modern society where the aging population is advancing, not only healthy longevity but also the improvement of QOL (Quality of Life) has become an issue. Skin beauty in the anti-aging field has attracted increasing attention because it is extremely important for improving the QOL of the elderly.

[0003] The skin gently wraps the internal living tissues, and its surface is covered by the stratum corneum, a thin barrier membrane derived from the epidermis, so that it can be kept in a relatively constant state in any environment. Epidermal cells differentiate into stratum corneum cells, which adhere to each other and stack up to form a soft stratum corneum similar to a polyethylene film, with a thickness of 10 to 20 microns. Every day, old stratum corneum cells are peeled off from the skin surface as dirt, while new stratum corneum cells are replenished from below to maintain a certain thickness.

[0004] In the stratum corneum of the epidermis covering the outermost layer, cholesterol, free fatty acids, and ceramides are the main lipids, forming a multi-layer (lamellar) membrane structure between the cells of the stratum corneum. The molar ratio of these lipids is 1:1:1. Since the molecular weight of ceramides is larger than that of cholesterol and free fatty acids, the weight ratio of ceramides accounts for 50% of the total. That is, ceramides are the main constituent components of the stratum corneum.

[0005] An important function of ceramides is to act as a component of the epidermal substance permeation barrier. Ceramides have a substance permeation defense wall function that prevents excessive water evaporation, leakage of in-vivo components, and invasion of foreign substances. A decrease in body temperature due to excessive water evaporation reduces the metabolic function of the living body. The epidermal permeation barrier also contributes indirectly as a heat retention barrier.

[0006] Human stratum corneum ceramides exhibit a high diversity of molecular species, and their fatty acid chain lengths range from C16 to C26. However, ceramides with longer chain lengths are more important for barrier function. It has been reported that an increase in the proportion of ceramides with relatively short fatty acids such as C16 and C18 reduces barrier function, while an increase in the proportion of ceramides with long fatty acids such as C20 and C24 enhances barrier function (Non-Patent Literature 1).

[0007] Another important role of ceramides is that of lipid mediators (signaling lipids). Sphingomyelin is synthesized using ceramides and phosphatidylcholine as raw materials. Microbial infection, oxidative stress, cytokines, and other stimuli activate sphingomyelinase, which hydrolyzes the phosphorylcholine group from sphingomyelin, producing ceramides. The ceramides produced by hydrolysis are rapidly hydrolyzed by ceramidase to sphingosine, which is then phosphorylated by sphingosine kinase to sphingosine-1-phosphate (Non-Patent Literature 2). Sphingosine-1-phosphate is known to induce the production of inflammatory cytokines in epidermal keratinocytes (Non-Patent Literature 3, 4).

[0008] Concepts that are often confused with skin barrier function include "moisture" and "emollient," which are concepts related to hydration. "Moisture" refers to the smooth spread of highly hygroscopic, water-soluble components on the skin's surface, maintaining skin moisture. Water-soluble components are sometimes called "humectants." "Emollient" refers to the smooth spread of oil-soluble components on the skin's surface, preventing water evaporation from the skin, and maintaining skin moisture. While the concepts of hydration, represented by "moisture" and "emollient," are achieved on the outside of the skin by topical agents, improving skin barrier function involves penetrating the epidermis, activating keratinocytes, and increasing the amount of intercellular lipids such as ceramides, thereby enhancing the ability of keratinocytes to retain moisture within the skin. (Non-patent Literature 5) Therefore, the two are fundamentally different concepts.

[0009] Transepidermal water loss (TEWL) is used as an indicator of stratum corneum barrier function (Non-patent Literature 6). Transepidermal water loss measures the amount of water that evaporates from the skin, and a lower value indicates a higher stratum corneum barrier function.

[0010] In recent years, there has been growing interest in methods to improve the skin's barrier function.

[0011] Non-patent document 5 describes Rice Power (registered trademark) No. 11, a representative skin barrier function improving agent. Rice Power No. 11 has a general moisturizing effect, but it also has a skin barrier function improving effect that enhances the skin's moisture retention capacity by penetrating the epidermis, activating keratinocytes, and increasing the amount of intercellular lipids such as ceramides.

[0012] Cytidylic acid is a type of nucleotide and is a substance widely found in living organisms and food, making it a highly safe ingredient.

[0013] Whether or not cytidylic acid has an effect on improving skin barrier function has not been evaluated to date.

[0014] Journal of Dermatological Science 60, 47-50 (2010) Biochemistry Vol. 84 No. 2 (2012) pp. 92-101 PLOS ONE Vol. 9, Issue 2, e89402 (2014) Journal of Investigative Dematology 139, 1743-1752 (2019) Chemistry and Biology Vol. 57 No. 2 (2019) pp. 129-133 Journal of the Japan Society of Colour Material, 88

[12] , 412-415 (2015)

[0015] The object of the present invention is to provide a novel and highly safe skin barrier function improving agent, cosmetic composition, topical skin preparation, and oral administration preparation that has a skin barrier function improving effect. Another object of the present invention is to provide a novel and highly safe ceramide production promoting agent, cosmetic composition, topical skin preparation, and oral administration preparation that has a ceramide production promoting effect.

[0016] As a result of diligent research to achieve the above objective, the inventors of the present invention have, to their surprise, discovered for the first time that cytidylic acid or its precursors have the properties of improving skin barrier function and promoting the production of ceramides, leading to the present invention.

[0017] In other words, the present invention is a skin barrier function improving agent containing cytidylic acid or its precursor as an active ingredient. Furthermore, the present invention is a cosmetic composition, topical skin preparation, or oral administration preparation for improving skin barrier function, containing cytidylic acid or its precursor as an active ingredient. Furthermore, the present invention is a ceramide production promoting agent containing cytidylic acid or its precursor as an active ingredient. Moreover, the present invention is a cosmetic composition, topical skin preparation, or oral administration preparation for promoting ceramide production, containing cytidylic acid or its precursor as an active ingredient.

[0018] The skin barrier function improving agent, cosmetic composition, topical skin preparation, and oral administration preparation of the present invention have a skin barrier function improving effect, and are novel and highly safe. Furthermore, the ceramide production promoting agent, cosmetic composition, topical skin preparation, and oral administration preparation of the present invention have a ceramide production promoting effect, and are novel and highly safe.

[0019] Figure 1 shows the quantitative analysis results of TEWL in the three-dimensional cultured epidermis of Example 1. In the figure, the vertical axis represents the TEWL (g / m³) for each sample. 2The horizontal axis represents the number of culture days as defined in Example 1. The graphs shown with dotted lines and white circles show the TEWL measurement results for a control sample with a CMP,2Na concentration of 0 (w / v%), the graphs shown in light gray show the TEWL measurement results for a sample with a CMP,2Na concentration of 0.25 (w / v%), and the graphs shown in dark gray show the TEWL measurement results for a sample with a CMP,2Na concentration of 0.50 (w / v%). Error bars represent the standard error, *: p < 0.05, and the cross symbol (cross-shaped symbol) represents *: p < 0.10. Figure 2 shows the cell viability in the three-dimensional cultured epidermis of Example 1. In the figure, the vertical axis represents the cell viability in each sample, and the horizontal axis represents the CMP,2Na concentration in each sample. The graph labeled "control" represents the measurement results of a control sample with a CMP,2Na concentration of 0 (w / v%), while the graphs labeled "0.25" and "0.50" represent the measurement results of samples with CMP,2Na concentrations of 0.25 (w / v%) and 0.50 (w / v%), respectively. Error bars represent the standard error. Figure 3 shows the results of the HPTLC analysis of three-dimensional cultured epidermis in Example 2. In the figure, "Cholesterol" refers to cholesterol, "Free fatty acid" refers to free fatty acids, and "Ceramide" refers to ceramides. The horizontal axis, labeled "control," represents the four consecutive samples with a control CMP,2Na concentration of 0 (w / v%). The graphs labeled "0.25% CMP,2Na" and "0.50% CMP,2Na" represent the measurement results of four consecutive samples with CMP,2Na concentrations of 0.25 (w / v%) and 0.50 (w / v%), respectively. Figure 4 shows the quantitative analysis results of ceramides in three-dimensional cultured epidermis in Example 2. In the figure, the horizontal axis represents the CMP,2Na concentration in each sample. The horizontal axis, labeled "control," represents the quantitative results of the control sample with a CMP,2Na concentration of 0 (w / v%). The graphs labeled "0.25" and "0.50" represent the quantitative results of the samples with CMP,2Na concentrations of 0.25 (w / v%) and 0.50 (w / v%), respectively.The vertical axis, "Ceramide content (% of control)," represents the relative amount (%) of ceramides when the amount of ceramides in the control group is set to 100. Error bars indicate the standard error, and * indicates p < 0.05. Figure 5 shows the results of quantitative analysis of ceramide content by fatty acid type in the three-dimensional cultured epidermis of Example 3. In the figure, the vertical axis represents the amount of substance (pmol) of each fatty acid, and the horizontal axis represents the various fatty acids measured. The various fatty acids are notated by c (number of carbon atoms):(number of unsaturated bonds). For example, stearic acid, which has 18 carbon atoms and 0 unsaturated bonds, is notated as C18:0, and linoleic acid, which has 18 carbon atoms and 1 unsaturated bond, is notated as C18:1. In the figure, the graph on the far left, shown in dark gray, represents the results of a control sample with a CMP,2Na concentration of 0 (w / v%), the graph in the center, shown in gray, represents the results of a sample with a CMP,2Na concentration of 0.25 (w / v%), and the graph on the far right, shown in light gray, represents the results of a sample with a CMP,2Na concentration of 0.50 (w / v%). Error bars represent the standard error, * represents p < 0.05, and the cross symbol represents p < 0.10. Figure 6 shows the quantitative analysis results of phosphatidylcholine in Example 4. In the figure, the vertical axis represents the amount of phosphatidylcholine (pmol), and the horizontal axis represents the CMP,2Na concentration in each sample. The horizontal axis labeled "control" represents the quantitative analysis results of the control sample with a CMP,2Na concentration of 0 (w / v%), while the graphs labeled "0.25" and "0.50" represent the quantitative analysis results of the samples with a CMP,2Na concentration of 0.25 (w / v%) and 0.50 (w / v%), respectively. Error bars represent the standard error. Figure 7 shows the quantitative analysis results of sphingomyelin in Example 5. In the figure, the vertical axis represents the amount of sphingomyelin (pmol), and the horizontal axis represents the CMP,2Na concentration in each sample. The horizontal axis labeled "control" represents the quantitative analysis results of the control sample with a CMP,2Na concentration of 0 (w / v%), while the graphs labeled "0.25" and "0.50" represent the quantitative analysis results of the samples with a CMP,2Na concentration of 0.25 (w / v%) and 0.50 (w / v%), respectively. Error bars represent the standard error.Figure 8 shows the quantitative analysis results of sphingosine in Example 5. In the figure, the vertical axis represents the amount of sphingosine (pmol), and the horizontal axis represents the CMP,2Na concentration in each sample. The "control" on the horizontal axis represents the quantitative analysis results of the control sample with a CMP,2Na concentration of 0 (w / v%), and the graphs labeled "0.25" and "0.50" represent the quantitative analysis results of samples with a CMP,2Na concentration of 0.25 (w / v%) and 0.50 (w / v%), respectively. Error bars represent the standard error, and * represents p < 0.05. Figure 9 shows the quantitative analysis results of sphingosine monophosphate in Example 5. In the figure, the vertical axis represents the amount of sphingosine monophosphate (pmol), and the horizontal axis represents the CMP,2Na concentration in each sample. The horizontal axis, "control," represents the quantitative analysis results of a control sample with a CMP,2Na concentration of 0 (w / v%). The graphs labeled "0.25" and "0.50" represent the quantitative analysis results of samples with CMP,2Na concentrations of 0.25 (w / v%) and 0.50 (w / v%), respectively. Error bars represent the standard error.

[0020] The present invention relates to a skin barrier function improving agent, a cosmetic composition or topical preparation for improving skin barrier function, and an oral preparation, all containing cytidylic acid or its precursor as an active ingredient.

[0021] In this specification, "skin barrier function improving agent" is a concept that encompasses compositions containing cytidylic acid or its precursor as an active ingredient, and further containing other ingredients. Similarly, in this specification, "topical skin preparation" and "oral administration preparation" are concepts that encompass compositions containing cytidylic acid or its precursor as an active ingredient, and further containing other ingredients. Furthermore, in this specification, "cosmetic composition" contains cytidylic acid or its precursor as an active ingredient, and further contains other ingredients.

[0022] In this specification, cytidylic acid (cytidine monophosphate, cytidine 5'-phosphate, CMP) is the compound indicated by CAS registration number 63-37-6. In this specification, when cytidylic acid is mentioned, salts of cytidylic acid are also included.

[0023] In this specification, when the mass of cytidylic acid is stated, it shall be expressed as the mass converted to disodium cytidylic acid salt (CMP, 2Na). When the concentration (%) of a cytidylic acid solution is stated, unless otherwise specified, it shall be expressed as mass-volume percentage concentration (w / v%), and the mass of cytidylic acid shall be expressed as the mass converted to CMP, 2Na. When a salt other than the disodium salt is selected, or in the case of a free acid that does not form a salt, the amount of cytidylic acid shall be used as the basis, and the mass shall be expressed as the mass converted to CMP, 2Na.

[0024] In this specification, a cytidylic acid precursor means a compound that can be metabolized to cytidylic acid. Whether or not a compound is included as a cytidylic acid precursor is determined by whether or not it is known to be converted to cytidylic acid. Specifically, cytidine diphosphate and cytidine triphosphate, which are known to be broken down to cytidylic acid by the action of ectonucleotidases, etc. (Isao Matsuoka, "Ectonucleotidases in the Nervous System", Clinical Chemistry 33:11-18, 2004), and cytidine and cytosine, which are known to be phosphorylated to cytidylic acid by the action of kinases (A. Orengo, "Regulation of enzymic activity by metabolites. I. Uridine-cytidine kinase of Novikoff ascites rat tumor", J Biol Chem. 1969 Apr 25;244(8):2204-9.) are exemplified as cytidylic acid precursors in this specification.

[0025] In this specification, the skin barrier function improving effect of the present invention is evaluated by quantifying the amount of TEWL (Non-Patent Literature 6). The specific quantification method is described in the examples below.

[0026] Examples of cytidylic acid or its precursors in the present invention include cytidine, cytosine, cytidylic acid, cytidine diphosphate, and cytidine triphosphate, as described above. Among these, cytidylic acid is preferred.

[0027] As mentioned above, the concept of cytidylic acid in this invention includes salts. Examples of cytidylic acid salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; basic amino acid salts such as arginine and lysine; ammonium salts such as ammonium salt and tricyclohexylammonium salt; and various alkanolamine salts such as monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, diisopropanolamine salt and triisopropanolamine. Among these, alkali metal salts such as sodium salt are preferred. Specifically, examples of alkali metal salts include monosodium cytidylate and disodium cytidylate, with disodium cytidylate being preferred from the standpoint of ease of handling.

[0028] The concentration of cytidylic acid or its precursor in the skin barrier function improving agent, cosmetic composition, topical skin preparation, and oral administration preparation of the present invention is preferably 0.25 (w / v%) or higher, as an increase in the content of long-chain ceramides of C22 or higher is observed, and an improvement in skin barrier function is seen, as described in the examples below. In particular, the concentration of cytidylic acid or its precursor is preferably 0.50 (w / v%) or higher, as this significantly increases the ceramide content and substantially improves skin barrier function. Furthermore, since the skin barrier function improving effect is concentration-dependent, the concentration of cytidylic acid or its precursor is preferably 0.75 (w / v%) or higher, and more preferably 1.00 (w / v%) or higher. In addition, for ease of handling when used as various preparations or compositions, the concentration of cytidylic acid or its precursor is preferably 10.00 (w / v%) or lower.

[0029] The skin barrier function improving agent, cosmetic composition, topical skin preparation, and oral administration preparation of the present invention may use cytidylic acid or its precursor as the active ingredient alone, or two or more types of cytidylic acid or its precursors may be used simultaneously, or cytidylic acid or its precursors may be used in combination with other active ingredients.

[0030] There are no particular restrictions on the origin of cytidylic acid or its precursor in the present invention, but those derived from natural products such as yeast, bacteria, fish and shellfish, animals, and plants are preferred.

[0031] The skin barrier function improving agent, cosmetic composition, topical skin preparation, and oral administration preparation of the present invention may be prepared in various forms by combining the above-mentioned components with pharmaceutically or cosmetically acceptable bases or carriers. Conventionally known pharmaceutically or cosmetically acceptable bases or carriers can be used. In particular, in the case of cosmetic compositions or topical skin preparations, various known components used in topical compositions applied to the skin or mucous membranes, such as cosmetics and topical pharmaceuticals / quasi-drugs, may be incorporated as needed. Examples of such components include surfactants, dyes (dyes, pigments), fragrances, preservatives, bactericides (antibacterial agents), thickeners, antioxidants, chelating agents, cooling agents, deodorants, moisturizers, UV absorbers, UV scattering agents, vitamins, plant extracts, skin astringents, anti-inflammatory agents, whitening agents, cell activators, vasodilators, blood circulation promoters, and skin function enhancers.

[0032] Specific examples of surfactants among the above components include anionic surfactants such as higher fatty acid soaps, alkyl sulfate esters, polyoxyethylene alkyl ether sulfates, alkyl ether phosphate esters, N-acyl amino acid salts, and acyl N-methyl taurate salts; cationic surfactants such as alkyltrimethylammonium chloride and dialkyldimethylammonium chloride; amphoteric surfactants such as alkyldimethylaminoacetic acid betaine, alkylamidedimethylaminoacetic acid betaine, and 2-alkyl-N-carboxy-N-hydroxyimidazolinium betaine; and nonionic surfactants such as polyoxyethylene type, polyhydric alcohol ester type, and ethylene oxide / propylene oxide block copolymer. Furthermore, surfactants belonging to polymer surfactants or natural surfactants can also be used without particular limitation.

[0033] Examples of preservatives include ethyl parahydroxybenzoate, salicylic acid, and sorbic acid. Examples of thickeners include xanthan gum, sodium carboxymethylcellulose, and carboxyvinyl polymer. Examples of chelating agents include sodium ethylenediaminetetraacetic acid salt, phosphoric acid, and citric acid.

[0034] Specific examples of moisturizers include polyethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, xylitol, sorbitol, maltitol, chondroitin sulfate, hyaluronic acid, mucoitin sulfate, carotenoid acid, atelocollagen, sodium lactate, bile salts, dl-pyrrolidone carboxylate, short-chain soluble collagen, diglycerin (EO) PO adduct, Rosa rugosa extract, Achillea millefolium extract, and Melilotus extract.

[0035] Specific examples of vitamins include vitamin A oil, retinol, retinyl acetate, retinyl palmitate, and other vitamin A compounds; vitamin B2 compounds such as riboflavin, riboflavin butyrate, and flavin adenine nucleotides; vitamin B6 compounds such as pyridoxine hydrochloride and pyridoxine dioctanoate; vitamin C compounds such as L-ascorbic acid, L-ascorbic acid dipalmitate, L-ascorbic acid-2-sulfate sodium, and dl-α-tocopherol-L-ascorbic acid phosphate dipotassium; Examples include pantothenic acids such as calcium iodide, D-pantothenyl alcohol, pantothenyl ethyl ether, and acetylpantothenyl ethyl ether; vitamin D compounds such as ergocalciferol and cholecalciferol; nicotinic acids such as nicotinic acid, benzyl nicotinate, and nicotinamide; vitamin E compounds such as dl-α-tocopherol, dl-α-tocopherol acetate, dl-α-tocopherol nicotinate, and dl-α-tocopherol succinate; and vitamin P and biotin.

[0036] The skin barrier function improving agent of the present invention can be used as an additive in cosmetics, topical pharmaceuticals, or quasi-drugs for topical use. Furthermore, the cosmetic composition or topical skin preparation of the present invention is used in a form applied to the skin (e.g., by application or spraying). Specifically, the cosmetic composition or topical skin preparation of the present invention is used as cosmetics, topical pharmaceuticals, or quasi-drugs for topical use. Of these, cosmetics are preferred from the viewpoint of being able to improve the skin barrier function on a daily basis. Such cosmetics or topical preparations include hair tonics or hair growth stimulants, or various hair cosmetics such as shampoos, conditioners, and hair lotions (including tonics and liquids) that are effective in nourishing or growing hair.

[0037] The form of the cosmetic composition or topical skin preparation of the present invention is not particularly limited as long as it is applicable to the skin or mucous membranes. Examples include paste, mousse, gel, liquid, emulsion, suspension, cream, ointment, sheet, aerosol, spray, and liniment. In particular, when used as a cosmetic, examples include lotions; emulsions such as emollient emulsions, milky lotions, nourishing emulsions, and cleansing emulsions; and creams such as emollient creams, massage creams, cleansing creams, and makeup creams. Furthermore, when used as a hair product such as a hair tonic or hair growth agent, examples include tonics, hair creams, hair lotions, aerosols (sprays), mousses, shampoos, conditioners, and liquids.

[0038] The cosmetic composition or topical skin preparation of the present invention can be used as a cosmetic, topical drug, or quasi-drug by direct application, spraying, or patching to the skin or mucous membranes. The proportion of use can be appropriately selected according to the user (age, sex, purpose, severity of symptoms in the affected area, etc.) and is not particularly limited. For example, an effective amount to improve the skin's barrier function can be administered transdermally to the skin once a day to five times a day.

[0039] The amount of the cosmetic composition or topical skin preparation of the present invention to be applied is preferably 1 mg or more per application, more preferably 1 to 1000 mg, even more preferably 5 to 800 mg, and particularly preferably 10 to 500 mg, in terms of the amount of cytidylic acid or its precursor.

[0040] The oral formulation of the present invention can be used in practical applications as a pharmaceutical, supplement, enteral nutritional supplement, etc. In this case, cytidylic acid or its precursor can be formulated alone or in combination with formulation aids, etc.

[0041] The oral preparation of the present invention can take the form of tablets, granules, capsules, powders, solutions, syrups, emulsions, and the like.

[0042] In the oral administration agent of the present invention, in addition to the active ingredient (cytidylic acid or its precursor) of the present invention, optional formulation adjuvants such as excipients, binders, disintegrants, lubricants, flavoring and odor-correcting agents, solubilizing agents, suspending agents, coating agents, etc. can be appropriately combined and used according to their respective dosage forms.

[0043] Although the appropriate dosage (dosage or intake amount) of the oral administration agent of the present invention varies depending on the age, body weight, degree of symptoms, etc. of the subject, and the administration or intake method, etc., the appropriate dosage per administration is preferably 1 to 1000 mg.

[0044] As is clear from the content described so far, the present invention is an invention based on finding that cytidylic acid is suitable for use in a new application as a skin barrier function improving agent.

[0045] As one aspect of the present invention, it is a method for improving skin barrier function by administering a skin barrier function improving agent containing cytidylic acid as an active ingredient.

[0046] The concentration of cytidylic acid or its precursor in the method for improving skin barrier function of the present invention is preferably 0.25 (w / v%) or more because an increase in the content of long-chain ceramides with C22 or more is observed and an improvement in skin barrier function is seen. In particular, in terms of a significant increase in the ceramide content and a significant improvement in skin barrier function, the concentration of cytidylic acid or its precursor is preferably 0.50 (w / v%) or more. Further, since the skin barrier function improving effect is exerted in a concentration-dependent manner, the concentration of cytidylic acid or its precursor is preferably 0.75 (w / v%) or more, and more preferably 1.00 (w / v%) or more. Also, from the ease of handling when used as various agents or compositions, etc., the concentration of cytidylic acid or its precursor is preferably 10.00 (w / v%) or less.

[0047] As one aspect of the present invention, it is a promoter for the production of ceramides, a cosmetic composition for promoting the production of ceramides, a skin external preparation, and an oral administration agent containing cytidylic acid or its precursor as an active ingredient.

[0048] In this specification, "ceramide production promoter" refers to a composition containing cytidylic acid or its precursor as an active ingredient, and also includes other components. Furthermore, in this specification, "ceramides" refers to a concept that includes ceramides and ceramide glycosides such as glucosylceramide, galactosylceramide, and lactosylceramide. Of these, ceramides with 22 or more carbon atoms are defined in this specification as "long-chain ceramides." It has been reported that barrier function improves as the proportion of ceramides with long-chain fatty acids increases (Non-Patent Literature 1), and therefore, long-chain ceramides are thought to play an important role in skin barrier function among ceramides.

[0049] In this specification, the ceramide production-promoting effect of the present invention is evaluated by processing the results of high-performance thin-layer chromatography (HPTLC) using image processing software to quantify the amount of ceramides. In this specification, the long-chain ceramide production-promoting effect of the present invention is evaluated by quantifying the ceramide content by fatty acid type using multiphospholipid analysis.

[0050] The ceramide production promoter of the present invention promotes the production of ceramides, as described in the examples below. As will be clear from the examples below, the ceramide production promoter of the present invention promotes the production of long-chain ceramides among ceramides. At least a part of the skin barrier function improving effect of the present invention can be explained by the ceramide production promoting effect of the ceramide production promoter of the present invention.

[0051] In one embodiment of the present invention, the concentration of cytidylic acid or its precursor in the long-chain ceramide production promoter is preferably 0.25 (w / v%) or higher, as described in the examples below. Furthermore, since the long-chain ceramide production promoting effect is expected to be concentration-dependent, the concentration of cytidylic acid or its precursor is preferably 0.50 (w / v%) or higher, more preferably 0.75 (w / v%) or higher, and even more preferably 1.00 (w / v%) or higher. In addition, for ease of handling when used as various agents or compositions, the concentration of cytidylic acid or its precursor is preferably 10.00 (w / v%) or lower.

[0052] The concentration of cytidylic acid or its precursor in the ceramide production promoter of the present invention is preferably 0.50 (w / v%) or higher, as described in the examples below. Furthermore, since the ceramide production promoting effect is expected to be concentration-dependent, the concentration of cytidylic acid or its precursor is preferably 0.75 (w / v%) or higher, and more preferably 1.00 (w / v%) or higher.

[0053] One aspect of the present invention is a ceramide production promoter containing cytidylic acid or its precursor as an active ingredient, which promotes the production of ceramides and suppresses the production of sphingosine and sphingosine monophosphate.

[0054] The ceramide production promoter of the present invention promotes the production of ceramides, but surprisingly, it does not promote the production of lipid mediators (signaling lipids) such as sphingosine and sphingosine-1-phosphate, which are produced by the degradation of ceramides; rather, it inhibits their production. As shown in the examples described later, the present invention promotes the production of ceramides and phosphatidylcholine, and also promotes the production of sphingomyelin, which is synthesized from ceramides and phosphatidylcholine. Naturally, it would be expected that the amount of sphingosine and sphingosine-1-phosphate produced by the degradation of sphingomyelin would also increase, but the ceramide production promoter of the present invention inhibits the production of lipid mediators such as sphingosine and sphingosine-1-phosphate. In other words, the ceramide production promoter of the present invention is thought to have the effect of promoting the production of ceramides, phosphatidylcholine, and sphingomyelin, while also having the opposite effect of inhibiting the degradation of sphingomyelin.

[0055] As described above, the ceramide production promoter of the present invention has the effect of suppressing the degradation of sphingomyelin, and therefore can suppress the production of lipid mediators and the production of inflammatory cytokines.

[0056] The ceramide production promoter, cosmetic composition, topical skin preparation, and oral administration preparation of the present invention may use cytidylic acid or its precursor as the active ingredient alone, or two or more types of cytidylic acid or its precursors may be used simultaneously, or cytidylic acid or its precursors may be used in combination with other active ingredients. Note that other components usable in the ceramide production promoter, cosmetic composition, topical skin preparation, and oral administration preparation of the present invention are the same as those in the skin barrier function improving agent, cosmetic composition, topical skin preparation, and oral administration preparation of the present invention, and therefore their description is omitted.

[0057] The present invention will be described below with reference to examples, but the present invention is not limited in any way by these examples.

[0058] (Example 1) Evaluation of epidermal barrier formation ability using TEWL measurement as an indicator To evaluate the epidermal barrier function in actual epidermis, the TEWL of 3D cultured epidermis was measured.

[0059] The experiment was conducted according to the following procedure.

[0060] Three-dimensional cultured epidermis (LabCyte EPI MODEL 24 6D, manufactured by J TEC) was placed in a 24-well plate with 500 μL of the included assay medium added, and acclimatized at 37°C for 48 hours. TEWL was measured using VAPOSCAN (AS-VT100, manufactured by Nippon Ash) (Day 0). 50 μL of sample-containing PBS was added from the stratum corneum side and cultured for 7 hours. After removing the sample with a sterile cotton swab, the cells were cultured for a further 17 hours, and TEWL was measured using VAPOSCAN (Day 1). Thereafter, culture was continued daily while measuring TEWL (Days 2-4). The CMP and 2Na concentrations in each sample were 0, 0.25, and 0.50 (w / v%). After the completion of culture, cell viability was measured using Alamar Blue. Furthermore, all of the above procedures were performed in a series of three for each concentration group, and the average value was taken.

[0061] Figure 1 shows the quantitative analysis results of TEWL, and Figure 2 shows the results of cell viability measurement.

[0062] Quantitative analysis of TEWL revealed a tendency for TEWL levels to decrease on Day 4 with 0.25% CMP, 2Na treatment. Furthermore, 0.50% CMP, 2Na treatment significantly reduced TEWL levels on Day 2, and a tendency for further reduction in TEWL levels was observed on Day 3 and Day 4. On the other hand, no change was observed in cell viability after the end of culture with any of the treatments. From this, it became clear that the decrease in TEWL levels due to CMP, 2Na treatment is due to an improvement in the barrier function of the three-dimensional cultured epidermis.

[0063] The results of this embodiment clearly demonstrate that CMP,2Na has an effect of improving skin barrier function.

[0064] (Example 2) Evaluation of the effect of promoting ceramide production by HPTLC analysis of 3D cultured epidermis In order to investigate the mechanism by which CMP improves skin barrier function, HPTLC analysis was performed on 3D cultured epidermis to evaluate its effect of promoting ceramide production.

[0065] Three-dimensional cultured epidermis (LabCyte EPI MODEL 24 6D JTEC) was placed in a 24-well plate with 500 μL of the included assay medium added, and acclimatized at 37°C for 48 hours. After adding 50 μL of sample-containing PBS from the stratum corneum side and culturing for 7 hours, the sample was removed with a sterile cotton swab and cultured for another 3 days. After culturing, lipids were extracted from the three-dimensional cultured epidermis using a chloroform:methanol (2:1) mixture and HPLC analysis was performed. Ceramides were quantified using ImageJ (National Institutes of Health, USA). The CMP,2Na concentrations in each sample were 0, 0.25, and 0.50 (w / v%). All procedures were performed in four consecutive samples for each concentration group, and the average value was taken.

[0066] Figure 3 shows the results of the HPLC analysis, and Figure 4 shows the results of quantifying ceramides based on those results.

[0067] Quantitative analysis of ceramides by HPLC revealed a significant increase in ceramide content after treatment with 0.50% CMP, 2Na. This indicates that CMP, 2Na has an effect of promoting ceramide production in the epidermis of the skin.

[0068] (Example 3) Analysis of ceramide fatty acid profiles by lipidomics analysis In Example 2, it was shown that CMP promotes the production of ceramides. Therefore, in order to analyze the ceramides promoted by CMP in more detail, lipidomics analysis was performed to analyze the fatty acid profiles of the ceramides.

[0069] Three-dimensional cultured epidermis (LabCyte EPI MODEL 24 6D, manufactured by JTEC) was placed in a 24-well plate with 500 μL of the included assay medium added, and acclimatized overnight at 37°C. 50 μL of sample-containing PBS was added from the stratum corneum side and cultured for 7 hours. After removing the sample with a sterile cotton swab, the culture was continued for another 17 hours. The above sample processing was repeated, and culture was continued for 5 days. The three-dimensional cultured epidermis was harvested and subjected to lipidomics analysis (Multiphospholipid Analysis Lipidome Labs). The CMP, 2Na concentrations in each sample were 0, 0.25, and 0.50 (w / v%). The procedure was performed in three consecutive trials for each concentration group, and the average value was taken.

[0070] Figure 5 shows the results of quantitative analysis of ceramide content by fatty acid type.

[0071] Lipidomic analysis revealed no significant changes in ceramides of short-chain fatty acids with C20 or less. However, in ceramides of long-chain fatty acids with C22 or more, a significant increase in the average content was observed in both 0.25% CMP, 2Na treatment and 0.50% CMP, 2Na treatment. In particular, a statistically significant increase was observed in ceramides of C24.

[0072] The results of this embodiment revealed that CMP,2Na has the effect of promoting the production of long-chain ceramides with C22 or more. Since long-chain ceramides are particularly important for skin barrier function, it is thought that this effect resulted in an improvement in skin barrier function.

[0073] In Example 2, the 0.25% CMP, 2Na treated sample did not show a ceramide production-promoting effect, but an increase in long-chain ceramide content was observed. This suggests that the skin barrier function improvement effect observed in Example 1 by the 0.25% CMP, 2Na treatment may be due to an increase in the proportion of long-chain ceramides among the ceramides produced.

[0074] (Example 4) Analysis of phosphatidylcholine by lipidomics analysis In this example, quantitative analysis of phosphatidylcholine was performed by lipidomics analysis.

[0075] In the lipidomics analysis of Example 3, phosphatidylcholine, a major phospholipid, was analyzed.

[0076] The results of quantifying the phosphatidylcholine content are shown in Figure 6.

[0077] Analysis of phosphatidylcholine revealed an increase in the average content after treatment with 0.50% CMP, 2Na. This indicates that CMP, 2Na promotes the production of phosphatidylcholine, a major phospholipid, in the epidermis of the skin.

[0078] (Example 5) Analysis of ceramide metabolites by lipidomics analysis In this example, quantitative analysis of ceramide metabolites was performed by lipidomics analysis.

[0079] In the lipidomics analysis of Example 3, ceramide metabolites, namely sphingomyelin synthesized from ceramides and phosphatidylcholine, sphingosine produced by the degradation of ceramides, and sphingosine monophosphate synthesized from sphingosine, were analyzed.

[0080] As a result of quantifying ceramide metabolites, the quantitative analysis results for sphingomyelin are shown in Figure 7, the quantitative analysis results for sphingosine are shown in Figure 8, and the quantitative analysis results for sphingosine monophosphate are shown in Figure 9.

[0081] Analysis of ceramide metabolites revealed that the average content of sphingomyelin, synthesized from ceramides and phosphatidylcholine, increased with 0.25% CMP, 2Na treatment, and increased more significantly with 0.50% CMP, 2Na treatment. On the other hand, the content of sphingosine, produced by the degradation of ceramides, decreased significantly with 0.25% CMP, 2Na treatment, and decreased more significantly and dramatically with 0.50% CMP, 2Na treatment. Furthermore, the average content of sphingosine monophosphate, synthesized from sphingosine, decreased with 0.25% CMP, 2Na treatment, and decreased more significantly with 0.50% CMP, 2Na treatment.

[0082] The results of this embodiment revealed that CMP,2Na promotes the production of not only ceramides and the major phospholipid phosphatidylcholine in the epidermis of the skin, but also sphingomyelin synthesized from them. On the other hand, it was revealed that it exerts the opposite effect, inhibiting the production of sphingosine and sphingosine-1-phosphate, which are produced by the degradation of sphingomyelin and ceramides. Since sphingosine and sphingosine-1-phosphate are lipid mediators known to induce inflammation in epidermal keratinocytes, it is thought that the present invention also exerts an anti-inflammatory effect by suppressing their production.

[0083] Accordingly, the present invention can be in the following embodiments: [1] A skin barrier function improving agent containing cytidylic acid or its precursor as an active ingredient. [2] The skin barrier function improving agent according to [1], wherein the concentration of cytidylic acid or its precursor is 0.25 to 10.00 (w / v%). [3] A cosmetic composition, topical skin preparation, or oral preparation for improving skin barrier function, containing cytidylic acid or its precursor as an active ingredient. [4] The cosmetic composition, topical skin preparation, or oral preparation according to [3], wherein the concentration of cytidylic acid or its precursor is 0.25 to 10.00 (w / v%). [5] A method for improving skin barrier function using a cosmetic composition, topical skin preparation, or oral preparation containing cytidylic acid or its precursor as an active ingredient. [6] The method for improving skin barrier function according to [5], wherein the amount of the cosmetic composition, topical skin preparation, or oral administration preparation applied is 1 to 1000 mg per application, converted to the amount of cytidylic acid or its precursor. [7] A ceramide production promoter containing cytidylic acid or its precursor as an active ingredient. [8] The ceramide production promoter according to [7], wherein the ceramides are long-chain ceramides. [9] The ceramide production promoter according to [7] or [8], wherein the concentration of cytidylic acid or its precursor is 0.25 to 10.00 (w / v%).

[10] The ceramide production promoter according to [8], which inhibits the production of sphingosine and sphingosine monophosphate.

[11] A cosmetic composition, topical skin preparation, or oral administration preparation for promoting ceramide production, containing cytidylic acid or its precursor as an active ingredient.

[12] A method for promoting the production of ceramides by using a cosmetic composition, a topical skin preparation, or an oral preparation containing cytidylic acid or a precursor thereof as an active ingredient.

Claims

1. A skin barrier function improving agent containing cytidylic acid or its precursor as an active ingredient.

2. The skin barrier function improving agent according to claim 1, wherein the concentration of cytidylic acid or its precursor is 0.25 to 10.00 (w / v%).

3. A cosmetic composition, topical skin preparation, or oral preparation containing cytidylic acid or its precursor as an active ingredient for improving skin barrier function.

4. The cosmetic composition, topical skin preparation, or oral preparation according to claim 3, wherein the concentration of cytidylic acid or its precursor is 0.25 to 10.00 (w / v%).

5. A method for improving skin barrier function using a cosmetic composition, topical skin preparation, or oral preparation containing cytidylic acid or its precursor as an active ingredient.

6. The method for improving skin barrier function according to claim 5, wherein the amount of the cosmetic composition, topical skin preparation, or oral preparation applied is 1 to 1000 mg per application, converted to the amount of cytidylic acid or its precursor.

7. A ceramide production promoter containing cytidylic acid or its precursor as an active ingredient.

8. The ceramide production promoter according to claim 7, wherein the ceramides are long-chain ceramides.

9. The ceramide production promoter according to claim 7 or 8, wherein the concentration of the cytidylic acid or its precursor is 0.25 to 10.00 (w / v%).

10. A ceramide production promoter according to claim 8, which suppresses the production of sphingosine and sphingosine monophosphate.

11. A cosmetic composition, topical skin preparation, or oral preparation containing cytidylic acid or its precursor as an active ingredient for promoting the production of ceramides.

12. A method for promoting the production of ceramides by using a cosmetic composition, topical skin preparation, or oral preparation containing cytidylic acid or its precursor as an active ingredient.

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