JP2016023155A - Ceramide production promoter and skin external preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase an amount of ceramide in skin.SOLUTION: A ceramide production promoter comprises glycogen.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a ceramide production promoter and a skin external preparation.

Skin ceramides are known to improve strong moisturizing action and skin barrier function. To increase skin ceramide, 1) Apply a topical skin preparation such as cosmetics containing the ceramide body to the skin, 2) Apply a topical skin preparation containing a substance that can be expected to promote ceramide production in the skin. 3) It has been proposed to ingest the ceramide body orally (Non-Patent Documents 1 to 5)
It is difficult for the ceramide body to be absorbed from the skin, and it is considered that the oral ceramide intake method does not lead to an increase in the amount of ceramide in the skin because it is decomposed in the digestive tract.

Newsl. Chem. Soc. Japam Dov. Colloid Surf Chem. Vol. 32 No. 4 p. 2-6, 2007 J. Soc. Cosmet. Chem. Jpn. Vol. 43, No. 4 2009 JATAFF J. Vol. 2 No. 3 p.24-27, 2014 Cosmetic Stage Vol. 7 No. 5 p. 46-50, 2013 Skin Vol. 31 No. Suppl 6 p. 107-119, 1989

  An object of the present invention is to promote ceramide production in the skin and increase the amount of ceramide.

The present invention provides the following ceramide production promoter and skin external preparation.
Item 1. Ceramide production promoter consisting of glycogen.
Item 2. Item 2. The ceramide production promoter according to Item 1, which promotes ceramide production in the skin by being applied to the skin.
Item 3. Item 3. The ceramide production promoter according to Item 1 or 2, wherein the amount of ceramide synthase is increased and / or the amount of ceramide is increased by decreasing the amount of ceramide-degrading enzyme expressed.
Item 4. Item 4. The ceramide production promoter according to items 1 to 3, wherein the glycogen is enzymatically synthesized glycogen (ESG).
Item 5. A topical skin preparation for promoting ceramide production in the skin, including glycogen.
Item 6. Item 6. The topical skin preparation according to Item 5, comprising 0.2% to 2% by weight of glycogen.
Item 7. Item 7. The external preparation for skin according to Item 5 or 6, wherein the glycogen is ESG.

  Ceramide, the main component of skin epidermal lipid, is an intercellular lipid that plays an important role in both moisture retention and barrier function of the skin. It has been reported so far that ceramide is decreased in the skin epidermis in dry skin diseases such as atopic dermatitis and in which the barrier function has been decreased, and decreased with aging. It has been reported that ceramide is effective in improving the barrier function in the skin epidermis in such dry skin diseases. According to the present invention, ceramide synthesis of keratinocytes can be promoted and the amount of ceramide can be increased. If glycogen is applied to the skin (three-dimensional skin model), it penetrates into the stratum corneum. By applying an external preparation containing glycogen, the glycogen that reaches the keratinocytes stimulates the keratinocytes, Promotes ceramide production. As a result, the amount of ceramide produced in the skin is increased, the moisture of the skin and the skin barrier function are improved, and it is effective for dry skin and rough skin.

Effects of ESG on ceramide synthesis in cultured keratinocytes Effect of promoting ceramide synthesis by ESG in cultured 3D skin model Detection amount of genes related to ceramide metabolism by glycogen. (A) Ceramide degradation system (sphingomyelin synthase (SMS1, SMS2)); (B) Ceramide synthesis system (sphingomyelinase (aSMase)) Ceramide metabolism diagram

  The glycogen used in the present invention is at least one selected from the group consisting of natural glycogen and glycogen synthesized from sugar or starch using an enzyme. Natural glycogen is abundant in the liver and muscle in animals, and there are also a wide variety of other species such as shellfish such as oysters, scallops and squirts, plants such as sweet corn, and microorganisms such as cyanobacteria. The physicochemical characteristics and content of glycogen vary somewhat depending on the species of production, the extraction method, and the season and origin. On the other hand, ESG is higher in purity than natural glycogen and has a large molecular weight. Therefore, ESG is more preferable as the glycogen used in the present invention.

ESG, for example, is a method in which sucrose phosphorylase (EC 2.4.1.7) and α-glucan phosphorylase (EC 2.4.1.1) are allowed to act on sugar (Ryoyama, K .; Kidachi, Y .; Yamaguchi, H .; Kajiura, H .; Takata, H. (2004) Biosci Biotechnol Biochem 68, 2332-2340), and a method of allowing a branching enzyme (EC 2.4.1.18) to act on short-chain amylose (Kajiura, H .; Kakutani, R .; Akiyama, T Takata, H .; Kuriki, T. (2008) Biocatal. Biotransform., 26, 133-140). ESG synthesized by these methods is known to have chemical and physical structures similar to those of natural glycogen (Takata, H .; Kajiura, H .; Furuyashiki, T .; Kakutani, R. ; Kuriki, T. (2009) Carbohydr. Res., 344, 654-659., Kajiura, H .; Takata, H .; Kuriki, T .; Kitamura, S. (2010) Carbohydr. Res., 345, 817 -824), these enzymatically synthesized glycogens can also be used as the glycogen of the present invention. The molecular weight of glycogen is preferably about 1 × 10 ⁶ to 2 × 10 ⁷ , but higher molecular weight or lower molecular weight can be widely used.

  Glycogen has a structure in which glucose is mainly linked by α-1,4 bonds and partially branched by α-1,6 bonds. This ESG mainly contains a large amount of components having a molecular weight of 1 to 10 million. In addition, since it is a naturally derived component and is present in various organs of the human body, it is highly safe and can be used continuously for a long period of time. It can use suitably for the cosmetics composition used for.

  The active ingredient of the ceramide production promoter is glycogen. Since glycogen is degraded in the digestive tract when orally administered, parenteral administration is desirable. In order to promote ceramide production in the skin, it is desirable to apply glycogen as an external preparation for skin. However, ceramide production in the skin can also be promoted by administration by subcutaneous or intradermal injection.

  The external preparation for skin may be in the form of cosmetics such as emulsions, lotions, creams, foundations, aerosols, packs, lipsticks, body powders, scalp treatments, patches, ointments, plasters, and poultices. , Creams, tapes, patches, lotions, liquids, tics, gels, and other pharmaceutical or quasi-drug forms.

  The skin external preparation of the present invention contains glycogen. The amount of glycogen is 0.01 to 20% by weight, more preferably 0.1 to 10% by weight, still more preferably 0.2 to 2% by weight, based on the total amount of the external preparation for skin. It is preferable.

  Ceramide has effects such as moisturizing, softening, whitening, anti-inflammatory, antioxidant and blood circulation promotion on human skin, and the skin external preparation and ceramide production promoter of the present invention are expected to have such effects based on ceramide. it can.

  Examples of the ceramide produced by the ceramide production promoter and the skin external preparation of the present invention include ceramide 1, ceramide 2, ceramide 3, ceramide 6 and the like, and particularly ceramide 2.

  In addition to glycogen, the skin external preparation of the present invention includes other components usually used in cosmetics and pharmaceuticals / quasi drugs, such as lower alcohols, silicones, fats, ester oils, sterols and their derivatives.・ Oil components such as hydrocarbons, oils, antioxidants, surfactants, moisturizers, wetting agents, fragrances, water, colorants, powders, drugs, chelating agents, pH adjusters, emulsifiers / solubilizers, increases Viscous / gelling agent, antiseptic, bactericidal agent, acid / alkali, UV absorber, anti-inflammatory agent, whitening agent, solvent, exfoliating / dissolving agent, anti-inflammatory agent, cooling agent, astringent, polymer powder, Hydroxy acids / vitamins and derivatives thereof, saccharides and derivatives thereof, organic acids, enzymes, inorganic powders, and the like can be appropriately blended as necessary. Furthermore, edetate disodium, edetate trisodium, sodium citrate, sodium polyphosphate, sodium metaphosphate, sequestering agents such as gluconic acid, preservatives such as methylparaben, ethylparaben, butylparaben, caffeine, tannin, Verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, Whitening agents such as ascorbic acid glucoside, arbutin, and kojic acid, and sugars such as glucose, fructose, mannose, sucrose, and trehalose can be appropriately blended.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
(1) Keratinocyte culture method Keratinocytes were cultured according to the following protocol.

The keratinocytes used were human-derived normal epidermal keratinocytes.
-Seed keratinocytes at 1.5x 10 ⁵ cells / well in Day0 6well plate-Add Day3 ESG sample to culture solution (0.2 wt% or 2 wt%)
・ Day10 lipid extraction ・ Culture medium: gibco EpiLife growth factor (+)
・ (Change medium every other day)

(2) Lipid Extraction Method Lipids containing ceramide were extracted from Day 10 keratinocytes cultured in (1) above according to the following protocol.
・ Wash with PBS-1mL (twice)
・ After adding 300μL of distilled water, peel off cells with a scraper and collect.
・ Add 300 μL of distilled water and collect the rest. 600μL in total
・ MeOH 1500μL, CHCl ₃ 750μL → Vortex for 2 minutes → CHCl ₃ 750μL → Vortex → H ₂ O 750μL → Vortex → 2000rpm, 5min → Chloroform layer (lower layer) sampling ・ Solid to solidify lipid

(3) Thin layer chromatography method (ceramide separation method)
About the lipid extracted by said (2), the ceramide was isolate | separated with the following thin layer chromatography methods.
・ Thin layer Merck Silica Gel60 HPT LC Plates (10x10)
・ Developing solution Chloroform: Methanol: Acetic acid (190: 9: 1) Developed twice (30-35min x2)
・ Coloring method: Spray a mixed aqueous solution of 10% copper sulfate and 8% phosphoric acid, dry, and then bake in 180 ° C for 15 min. ・ Std3 adjustment: Mix of Ceramide2,3,6 (equal amount of 10 mg / mL) Mixing) Std2: Std3 diluted 3 times Std1: Std2 diluted 3 times ・ Sample preparation: Sample in Control, ESG addition group: 25 μL of lipid extracted in (2) above was added to chloroform: methanol (2: 1) solution Vortexed and spot volume: 3μL

  The results are shown in Figure 1. control means no ESG added. From the results shown in FIG. 1, it was revealed that ceramide in keratinocytes increases in a concentration-dependent manner by adding ESG to the medium.

Example 2
(1) Three-dimensional model skin culture method Epi200 manufactured by MatTek Corporation was used as the three-dimensional model skin. Epi200 is similar to living human skin tissue, and has a basal layer, a granular layer, a spiny layer, and a horny layer of the basic epidermis structure, and has an intercellular lipid composition similar to that of human skin. Higher reproducibility than human skin has been reported.

Three-dimensional model skin culture was performed according to the following protocol.
・ Day0 culture start ・ Day1 1h After pre-incubate at 37 ℃, add ESG sample in the cup (0.2 wt% or 2 wt%)
-Day2 sample collection, lipid collection Lipid collection was performed in the same manner as in Example 1 (2) and (3) to separate ceramide. The results are shown in FIG.

  In the three-dimensional model skin culture, ceramide was found to increase in a concentration-dependent manner by adding ESG to the medium as compared with the control without ESG addition, as in the case of the cultured keratinocytes of Example 1. 24 hours after the addition of 2% ESG, ceramide synthesis in the three-dimensional skin increased about 1.5 times.

Example 3
(1) Culture method Keratinocytes were cultured according to the following protocol.

The keratinocytes used were human-derived normal epidermal keratinocytes.
・ Keratinocyte culture Medium: gibco EpiLife Growth factor (+)
・ Day3 ESG (2% by weight) sample added ・ Day10 RNA extraction ・ Medium: gibco EpiLife Growth Factor (+)
・ (Change medium every other day)

(2) RNA extraction method About the keratinocyte obtained by said (1), RNA was extracted according to the following procedures.
Kit use: QIAGEN RNeasy Mini Kit (250)
Wash x1 with PBS
Add 350 μL Buffer RLT, transfer to an eppen to detach the cells, and suspend with Voltex or Pipette
Move to QIAshredder, 13000 rpm x 2min
Filtrate recovery. Add 350 μL of 70% EtOH, pipette, and drop onto RNeasy spin column.
Add 700 μL of RW1 to the centrifuged column and centrifuge. Add 500 μL of RPE to the column and centrifuge. After 500 μL of RPE was again dropped onto the column and centrifuged, distilled water was finally added and collected after centrifugation.
The obtained RNA was converted to DNA using High-Capacity cDNA Reverse Transcription Kits (ABI). For real-time PCR, use the converted DNA, suspend Power SYBR Green PCR Master Mix (Applied Biosystems), various primers (Forward, Reverse), and distilled water, and use Applied Biosystems 7500 Fast Real-Time PCR System. And analyzed.

  The expression levels of mRNA were compared for the ceramide degradation system (sphingomyelin synthase (SMS1, SMS2)) and ceramide synthesis system (sphingomyelin degrading enzyme (aSMase)). Each expression level was corrected using GAPDH as an internal standard. The results are shown in FIG. From the results in FIG. 3, it was revealed that ESG increases the amount of ceramide in the cell by suppressing the expression level of the ceramide degradation system and increasing the expression level of the ceramide synthesis system.

Claims (7)

Ceramide production promoter consisting of glycogen. 2. The ceramide production promoter according to claim 1, which promotes the production of ceramide in the skin by being applied to the skin. The ceramide production promoter according to claim 1 or 2, wherein the ceramide production promoter is increased by increasing the expression level of ceramide synthase and / or decreasing the expression level of ceramide degrading enzyme. The ceramide production promoter according to claim 1, wherein the glycogen is enzymatically synthesized glycogen (ESG). A topical skin preparation for promoting ceramide production in the skin, including glycogen. The skin external preparation according to claim 5, comprising 0.2% by weight to 2% by weight of glycogen. The skin external preparation according to claim 5 or 6, wherein the glycogen is ESG.