Composition for promoting ceramide production or aquaporin 3 production

Abstract

This composition for promoting ceramide production or aquaporin-3 production contains a residue obtained by extracting a pigment from safflower.

Description

Composition for promoting ceramide production or aquaporin 3 production

[0001] The present invention relates to a composition for promoting ceramide production or aquaporin 3 production.

[0002] Ceramide exists as a main component of intercellular lipids in the skin and is known as a substance that functions as a barrier to prevent water evaporation from inside the skin. The search for useful components that promote ceramide production is underway (Patent Documents 1 and 2).

[0003] Aquaporin (AQP) proteins are known as proteins that selectively permeate water on the cell membrane. In humans, the existence of 13 types of AQP is known, and mainly aquaporin 3 (AQP3) exists in epidermal cells. AQP3 is important for improving the physiological barrier function of the skin by promoting the transport of water and glycerol. By promoting AQP3 production, an effective improvement in the barrier function can be expected (Patent Documents 3 and 4).

[0004] Japanese Patent Application Laid-Open No. 2004-210743, Japanese Patent Application Laid-Open No. 2010-150237, Japanese Patent Application Laid-Open No. 2004-168732, Japanese Patent Application Laid-Open No. 2009-256271

[0005] Substances that promote ceramide production are known, but it is desired to expand the options in this technical field and broaden the possibilities for new research and applications by providing novel substances to replace the known substances. Also, substances that promote AQP3 production are known, but it is desired to expand the options in this technical field and broaden the possibilities for new research and applications by providing novel substances to replace the known substances.

[0006] As a result of intensive studies to solve the above problems, the present inventors have found that a substance that promotes ceramide production or AQP3 production can be produced using the residue after extracting pigments from safflower, and thus have reached the present invention.

[0007] In other words, the present invention relates to at least the following: [Invention A1] A composition for promoting ceramide production or aquaporin 3 production, comprising a residue obtained by extracting a pigment from safflower. [Invention A2] The composition according to Invention A1, wherein the residue is an extract obtained by extracting from the residue obtained by extracting a pigment from safflower using an organic solvent. [Invention A3] The composition according to Invention A2, wherein the organic solvent is an aliphatic compound containing one or more hydroxyl groups. [Invention A4] A cosmetic, cosmetic additive, or humectant containing the composition according to any one of Inventions A1 to A3. [Invention A5] A method for producing a composition for promoting ceramide production or aquaporin 3 production, comprising the step of extracting an extract from the residue after extracting a pigment from safflower using an organic solvent. [Invention A6] The method according to Invention A5, wherein the organic solvent is an aliphatic compound containing one or more hydroxyl groups. [Invention A7] A method for producing a cosmetic, cosmetic additive, or humectant, comprising carrying out the method according to Invention A5 or A6.

[0008] The present invention also includes the following embodiments. [Invention B1] A composition for promoting ceramide production or aquaporin 3 production, comprising an extract obtained using an organic solvent from the residue after extracting pigment from safflower, wherein the residue is not fermented by yeast and not decomposed by enzymes after the extraction, and the extract is not fermented by yeast and not decomposed by enzymes. [Invention B2] The composition according to Invention B1, wherein the organic solvent is an aliphatic compound containing one or more hydroxyl groups. [Invention B3] A cosmetic, cosmetic additive, or humectant comprising the composition according to Invention B1 or B2. [Invention B4] A method for producing a composition for promoting ceramide production or aquaporin 3 production, comprising the step of extracting an extract using an organic solvent from the residue after extracting pigment from safflower, the method not comprising the steps of fermenting the residue with yeast, decomposing the residue with enzymes, fermenting the extract with yeast, or decomposing the extract with enzymes. [Invention B5] The method according to Invention B4, wherein the organic solvent is an aliphatic compound containing one or more hydroxyl groups. [Invention B6] A method for producing a cosmetic, a cosmetic additive, or a humectant, comprising carrying out the method according to Invention B4 or B5.

[0009] Furthermore, the present invention includes the following embodiments. [Invention C1] A ceramide production promoter or aquaporin 3 production promoter containing an extract obtained using an organic solvent from the residue obtained by extracting pigment from safflower, wherein the residue is not fermented by yeast and not decomposed by enzymes after the extraction, and the extract is not fermented by yeast and not decomposed by enzymes. [Invention C2] The ceramide production promoter or aquaporin 3 production promoter according to Invention C1, wherein the organic solvent is an aliphatic compound containing one or more hydroxyl groups. [Invention C3] A cosmetic, cosmetic additive, or humectant containing the ceramide production promoter or aquaporin 3 production promoter according to Invention C1 or C2. [Invention C4] A method for producing a ceramide production promoter or an aquaporin 3 production promoter, comprising the step of extracting an extract from the residue after extracting a pigment from safflower using an organic solvent, wherein the method does not include the steps of fermenting the residue with yeast, decomposing the residue with enzymes, fermenting the extract with yeast, or decomposing the extract with enzymes. [Invention C5] The method according to Invention C4, wherein the organic solvent is an aliphatic compound containing one or more hydroxyl groups. [Invention C6] A method for producing a cosmetic, a cosmetic additive, or a humectant, comprising carrying out the method according to Invention C4 or C5.

[0010] This application provides the present invention, which is a novel composition for promoting ceramide production or AQP3 production. In one embodiment, the present invention is a cosmetic, cosmetic additive, or humectant containing the above composition.

[0011] This graph shows that the expression of AQP3 mRNA is significantly enhanced by the safflower extract of the present invention. This graph shows that the expression of NQO1 mRNA is significantly enhanced by the safflower extract of the present invention. This graph shows that the expression of GCLC mRNA is significantly enhanced by the safflower extract of the present invention. This graph shows that the expression of SPTLC1 mRNA is significantly enhanced by the safflower extract of the present invention. This graph shows that the safflower extract of the present invention has a high ceramide production promoting effect. This graph shows that the safflower extract of the present invention significantly increases the moisture content of the stratum corneum.

[0012] The composition of the present invention contains a residue obtained by extracting pigment from safflower.

[0013] In preparing the residue for safflower pigment extraction, the method for extracting the pigment from safflower is not particularly limited and can be carried out by known methods.

[0014] For extracting the pigment, safflower flowers may be used, dried safflower (kouka) may be used as a crude drug, or red safflower paste may be used.

[0015] Safflower can be used in its raw state for extraction, or it may be subjected to processing such as chopping, drying, or grinding before extraction. Extraction is carried out by immersion in the extraction solvent. Stirring or homogenization in the extraction solvent may be performed to improve extraction efficiency. The extraction temperature and time can be selected as appropriate.

[0016] The solvent used to extract the pigment is not particularly limited. For example, water, physiological saline, phosphate buffer, or phosphate-buffered physiological saline can be used individually or in any combination of two or more in any mixing ratio. Water alone may be used, or a solvent such as ethanol that is easily removed after extraction may be used, either alone or in any mixture with water in any mixing ratio. Preferably, a solvent containing 50% by weight or more of water (i.e., a solvent that does not contain organic solvents or a mixed solvent containing less than 50% by weight of organic solvents) is used. The solvent may contain acidic or basic components.

[0017] Residue after pigment extraction The residue contained in the composition of the present invention may be the residue obtained by extracting pigment from safflower itself, or it may be obtained by further purification steps such as extraction or filtration from the said residue.

[0018] For further extraction, it is preferable to use an organic solvent (i.e., a solvent containing an organic solvent), and it is preferable to use a solvent containing 50% by weight or more of an organic solvent (i.e., an organic solvent alone or a mixed solvent containing 50% by weight or more of an organic solvent). The solvent may also contain an acid or a basic component. As the organic solvent, an alcohol, i.e., an aliphatic compound containing one or more hydroxyl groups (for example, monohydric alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, or polyhydric alcohols such as propylene glycol, dipropylene glycol, 1,3-butylene glycol, 1,2-pentylene glycol, glycerin) is preferred.

[0019] The extract obtained from the residue after pigment extraction using an organic solvent may be incorporated into the final composition as is, or it may be incorporated after further purification steps such as extraction or filtration from the said extract.

[0020] It is preferable that the residue after pigment extraction, or the extract obtained from said residue using an organic solvent, is not subjected to a yeast fermentation process or an enzymatic decomposition process between the time it is obtained and when it is incorporated into the final product. However, this does not exclude unavoidable and very slight fermentation or decomposition caused by yeast or enzymes in the air.

[0021] In one embodiment, the composition of the present invention can be used as an active ingredient in cosmetics, cosmetic additives, or moisturizers. Depending on the application, the formulation (the proportion of the composition and the types and proportions of other ingredients) can be appropriately selected from conventional formulation examples in accordance with the common technical knowledge of those skilled in the art. For example, the composition of the present invention can be used in the formulation of conventional topical skin preparations. The type or form of the cosmetic of the present invention can be applied as, for example, lotion, emulsion, cream, ointment, patch, pack, cleanser, lipstick, lip balm, foundation, blush, eyeshadow, eyebrow, etc. Furthermore, the composition of the present invention can be used in topical pharmaceuticals, quasi-drugs, etc. for the skin, etc.

[0022] Other Components: The compositions, cosmetics, cosmetic additives, and humectants of the present invention may, as necessary and within the limits that do not impair the effects of the present invention, appropriately incorporate various other components, such as mineral oil, alcohols, UV absorbers, moisturizing ingredients, aqueous components, antioxidants, cosmetic ingredients, preservatives, water-soluble polymers, film-forming agents, color-fastening agents, defoaming agents, fragrances, etc., to impart various effects.

[0023] Other components may be added at any stage in the production of the composition of the present invention.

[0024] Examples of mineral oils include petrolatum, synthetic waxes, and microcrystalline waxes.

[0025] Examples of alcohols include natural monoalcohols such as ethanol and phenoxyethanol, synthetic monoalcohols such as 2-hexyldecanol, and polyhydric alcohols such as ethylene glycol and pentylene glycol.

[0026] Examples of UV absorbers include benzophenone-based, PABA-based, cinnamic acid-based, and salicylic acid-based UV absorbers, as well as 4-tert-butyl-4'-methoxydibenzoylmethane and oxybenzone.

[0027] Examples of moisturizing ingredients include proteins, mucopolysaccharides, collagen, elastin, and keratin.

[0028] The aqueous component can be any water or any water-soluble component. In addition to water, examples include glycerols such as glycerin, diglycerin, and polyglycerin, and plant extracts such as aloe vera, witch hazel, cucumber, lemon, lavender, and rose.

[0029] Examples of antioxidants include α-tocopherol and ascorbic acid.

[0030] Examples of beauty ingredients include vitamins, anti-inflammatory agents, and herbal medicines.

[0031] Examples of preservatives include parahydroxybenzoic acid esters and phenoxyethanol.

[0032] The composition of the present invention promotes ceramide production. This can be confirmed by known methods, for example, by the methods described in the examples of this specification.

[0033] Promoting AQP3-producing gene expression: The composition of the present invention promotes the expression of a gene that produces AQP3, thereby promoting AQP3 production. The expression of the AQP3-producing gene can be confirmed by known methods, for example, by the method described in the examples of this specification.

[0034] NQO1 Production Promotion: In one embodiment, the compositions of the present invention may also promote the expression of a gene that produces NAD(P)H quinone dehydrogenase 1 (NQO1), thereby promoting the production of NQO1. The expression of the NQO1-producing gene can be confirmed by known methods, for example, by the methods described in the examples herein.

[0035] In one embodiment, the compositions of the present invention can also promote the expression of genes that produce glutamate cysteine ​​ligase catalytic subunit (GCLC), thereby promoting GCLC production. The expression of GCLC-producing genes can be confirmed by known methods, for example, by the methods described in the examples herein.

[0036] In one embodiment, the composition of the present invention can also promote the expression of a gene that produces serine palmitoyltransferase, long chain base subunit 1 (SPTLC1). This promotes the production of SPTLC1. The expression of the SPTLC1-producing gene can be confirmed by known methods, for example, by the method described in the examples herein.

[0037] Method for Manufacturing the Composition of the Present Invention The composition, cosmetics, etc. of the present invention can be manufactured by commonly used methods. For example, a solid cosmetic can be manufactured by heating and mixing all the components, degassing, filling into a mold or container, and then cooling and solidifying it.

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0039] Pigment Extraction from Safflower: Safflower cakes obtained from the Yamagata Prefecture Safflower Producers' Association were soaked in water and kneaded to extract the yellow pigment. The process was repeated, changing the water as needed, until no more yellow liquid came out. The safflower cakes, from which no more yellow liquid came out, were then soaked in a weak alkaline solution (wood ash water, straw ash water, 5% potassium carbonate solution) and kneaded to extract the red pigment. The process was repeated, changing the weak alkaline solution as needed, until the safflower cakes turned a color similar to wet straw scraps, to obtain the safflower cake residue.

[0040] Processing of the residue after pigment extraction: 50 g of the residue obtained after extracting pigment from safflower-derived red rice cakes was immersed in 1 kg of pentylene glycol for 3 hours. This was filtered and purified to obtain a safflower flower extract sample to be used in the tests described later. As can be seen from the above description, in this specification, "safflower flower extract" refers to the extract obtained from the residue after obtaining the pigment, not the extract obtained when obtaining the pigment from safflower flowers.

[0041] Aquaporin 3 gene-expression-promoting NHEK cells (Normal Human Epidermal Keratinocytes) were seeded in a 96-well plate. KG2 (Kurabo) was used as the culture medium for seeding. The above safflower extract sample was filtered and sterilized, then mixed with Kurabo KB2 (KB2) at a concentration of 0.3%, and evaluation substance samples of different concentrations were prepared using pentylene glycol in a 3-fold dilution series (i.e., 0.3%, 0.1%, and 0.03%). In addition, a negative control sample was prepared by adding pentylene glycol to KG2 medium to a final concentration of 0.3%. After 24 hours of incubation following seeding of NHEK cells in KG2 medium, the evaluation substance samples of each concentration and the negative control sample were added to the NHEK cells. qPCR was performed 24 hours after sample addition to check for genetic changes. For the preparation of the reverse transcript used in qPCR, SuperPrep® II Cell Lysis & RT Kit for qPCR (Toyobo) was used, and the reverse transcription reaction was performed according to the protocol provided by the manufacturer. The resulting reverse transcript was used as a template, and THUNDERBIRD® SYBR® qPCR Mix (Toyobo) was used as the qPCR reagent to perform gene expression analysis of AQP3 and Peptidylprolyl Isomerase A (PPIA) as an internal standard. The results of the AQP3 mRNA measurement are shown in Figure 1. As can be seen from these results, it was found that the expression of AQP3 mRNA was significantly enhanced with the safflower extract of the present invention.

[0042] NQO1 mRNA expression was expressed and its expression level was measured using the same method as described above for the promotion and confirmation of NQO1 gene expression and aquaporin 3 gene expression. The measurement results are shown in Figure 2. As can be seen from these results, it was found that NQO1 mRNA expression was significantly enhanced by the safflower extract of the present invention.

[0043] By the same experimental procedures as those for promoting the expression of the GCLC gene, promoting the expression of the aquaporin 3 gene, and confirmation, the expression of GCLC mRNA was induced, and its expression level was measured. The measurement results are shown in Fig. 3. As can be seen from these results, it was confirmed that the expression of GCLC mRNA was significantly enhanced by the safflower flower extract of the present invention.

[0044] By the same experimental procedures as those for promoting the expression of the SPTLC1 gene, promoting the expression of the aquaporin 3 gene, and confirmation, the expression of SPTLC1 mRNA was induced, and its expression level was measured. The measurement results are shown in Fig. 4. As can be seen from these results, it was confirmed that the expression of SPTLC1 mRNA was significantly enhanced by the safflower flower extract of the present invention.

[0045] Human epidermal tissue culture cups containing human epidermal tissue in LabCyte EPI-MODEL 24 (J-TEC) were placed in each well of an assay plate dispensed with 500 μL of assay medium (J-TEC), and cultured in a CO2 incubator. The above safflower extract sample was diluted to 0.1% with HEPES buffer (Kurabo) to prepare the evaluation sample. Pentylene glycol (Symrise) was diluted to 0.1% with HEPES buffer to prepare the negative control sample. After 24 hours of culture, the medium was replaced with fresh medium, and 50 μL of the prepared sample (evaluation substance sample or negative control sample) was exposed to each tissue. After 1 hour, the samples were removed, the surface of each tissue was washed three times with 50 μL of HEPES buffer, and then incubated for 24 hours. This series of operations was performed for 4 days. After washing on the fourth day, the bottom of the culture cup was immersed in Trypsin / EDTA solution (Kurabo) for 1 hour to collect the stratum corneum from the cup. A ceramide sample was obtained by extraction, lipid fractionation, and concentration. Specifically, 3 mL of methanol and 100 μL of 1 ppm CERAMIDE III (Evonik) were added to the stratum corneum collected above. After sonication for 3 minutes, the methanol was completely evaporated under reduced pressure, and 1 mL of chloroform was added and sonication was performed for 1 minute. This was the total lipid sample. Sep-Pak Silica 3 cc Vac Cartridge (Waters) was conditioned with 10 mL of chloroform / methanol (99.5 / 0.5 volume ratio) followed by 10 mL of chloroform, and then 1 mL of the total lipid sample was injected into the cartridge and retained in the substrate. After cleaning by passing 10 mL of chloroform through it, 10 mL of methanol was passed through it to obtain the ceramide fraction. The ceramide fraction was completely dried under reduced pressure, and 1 mL of methanol was added and sonication was performed for 1 minute. This was collected in a vial, 100 μL of 1 ppm Ceramide Bio(Symrise) was added, and then it was dried to nitrogen. After drying, it was redissolved in 100 μL of heptane / chloroform / methanol (95 / 2.5 / 2.5 volume ratio) to obtain a ceramide sample. The total amount of ceramide in the obtained ceramide sample was quantified using LC-MS. The quantification results are shown in Figure 5.As can be seen from these results, it has been confirmed that the safflower extract of the present invention has a high ceramide production promoting effect.

[0046] Lip balms containing the safflower extract sample of the present invention, formulated according to the formulations described in Table 1, and a negative control lip balm were prepared. Next, the moisture content of the stratum corneum of the lips was measured using a Corneometer CM825 (Courage+Khazaka) at a total of 12 points: 3 points on the upper left, 3 points on the lower left, 3 points on the upper right, and 3 points on the lower right of the lips, for six subjects with no bias in gender or age, before applying the lip balm. After measurement, the negative control lip balm was applied to the upper and lower left sides of the lips, and the lip balm of the present invention was applied to the upper and lower right sides. Subsequently, the lip balm was applied at times when the subjects felt that no lip balm was present, such as upon waking, after lunch, and before going to bed, and the application state was maintained for one week. During this time, the use of other lip cosmetics was avoided. After one week, the lip balm was wiped off, and the moisture content was measured again, and the rate of change in moisture content from the start to the end of the test was compared. The results are shown in Table 1 and Figure 6. As these results show, the lip balm containing safflower extract of the present invention significantly increased the moisture content of the stratum corneum.

[0047] According to the present invention, it is possible to provide a cosmetic composition having excellent ceramide production ability, and therefore it can be used in the cosmetics industry and the like.

Claims

1. A composition for promoting ceramide production or aquaporin 3 production, comprising an extract obtained using an organic solvent from the residue obtained by extracting pigment from safflower, wherein the residue has not been fermented by yeast or decomposed by enzymes after the extraction, and the extract has not been fermented by yeast or decomposed by enzymes.

2. The composition according to claim 1, wherein the organic solvent is an aliphatic compound containing one or more hydroxyl groups.

3. A cosmetic, cosmetic additive, or humectant containing the composition described in claim 1 or 2.

4. A method for producing a composition for promoting ceramide production or aquaporin 3 production, comprising the step of extracting an extract from the residue after extracting pigment from safflower using an organic solvent, wherein the method does not include the steps of fermenting the residue with yeast, decomposing the residue with enzymes, fermenting the extract with yeast, or decomposing the extract with enzymes.

5. The method according to claim 4, wherein the organic solvent is an aliphatic compound containing one or more hydroxyl groups.

6. A method for producing a cosmetic, a cosmetic additive, or a humectant, comprising carrying out the method described in claim 4 or 5.

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