JP4713765B2 - Skin basement membrane application composition - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
The present invention relates to a skin basement membrane utilization composition containing, as an active ingredient, a specific compound having an activity of promoting the production of laminin 5, which is a kind of extracellular matrix protein present in the skin basement membrane.
[0002]
[Prior art]
Laminin 5 (also known as kalinin, epiligrin, and nisin) is identified as a kind of laminin molecular species present in the skin basement membrane, and has a molecular weight of 380 to 490 kDa composed of three subunits of α3 chain, β3 chain, and γ2 chain. It is a glycoprotein (Carter, WG, et al., Cell., 65, 599-610, 1991., Rousselle, P., et al., J. Cell Biol., 114, 567-576, 1991). , Verrando, P., et al., Biochem. Biophys. Acta, 942, 45-56, 1988.). Laminin 5 is produced by epidermal cells, promotes adhesion of epidermal cells, binds the epidermal cells to the basement membrane, binds to type VII collagen, and is also involved in the binding of the dermis to the basement membrane (Rousselle, P , Et al., J. Cell Biol., 114, 567-576, 1991., Rousselle, P., et al., J. Cell Biol., 138, 719-728, 1997., Chen, M.,. et al., J. Invest. Dermatol., 112, 177-183, 1999). Mutations in the laminin 5 gene cause hereditary epidermolysis bullosa (Herlitz's junctional epidermolysis bullosa), which is a severe genetic disease characterized by epidermis-dermis separation and blister formation. (Aberdam, D., et al., Nat. Genet., 6, 299-304, 1994., Pulkkinen, L., et al., Genomics). , 24, 357-360, 1994., Kiviriko, S., et al., Hum. Mol. Genet., 4, 959-962, 1995.). In addition, laminin 5 has an activity of promoting epidermal cell migration, and expression of laminin 5 gene and its receptor gene is increased at the wound healing site of skin, suggesting that it is involved in wound healing. (Verrndo, P., et al., Lab. Invest., 71, 567-574, 1994., Ryan, MC, J. Biol. Chem., 269, 22779-22787, 1994.). As described above, laminin 5 is responsible for the connection between the epidermis and the dermis and is important for maintaining a normal skin structure. When the skin is damaged, laminin 5 promotes the migration of epidermal cells, It seems to work for healing.
[0003]
On the other hand, in the aged skin, duplication and flattening of the basement membrane have occurred, suggesting that structural changes in the basement membrane may cause skin aging (Lavker, R., et al., J. Invest. Dermatol., 73, 59-389, 1979., Lavker, R., et al., Dermatol. Clin., 4, 379-389, 1986.). In addition, in the aging human dermal fibroblasts, the expression of type IV collagen, which is a major component of the skin basement membrane, is decreased, suggesting that the activity of the skin basement membrane decreases with ageing. (Olsen, D., et al., J. Invest. Dermatol., 93, 127-131, 1989.). Therefore, it has been expected that reduction of skin function can be improved by promoting the production of laminin 5 in epidermal cells and preventing or repairing structural changes in the skin basement membrane due to aging.
[0004]
From the above, laminin 5 itself, preparations derived from soybeans having laminin 5 production-promoting activity and lysophospholipids as active ingredients, skin external preparations and skin utilization compositions having skin activation effects Products have been developed (JP-A-10-147515, JP-A-11-343226, JP-A-11-52704, JP-A-2000-226308).
[0005]
By the way, the compounds of phenylpropanoids have been used in cosmetics and skin external preparations for various reasons. For example, since phenylpropanoid compounds have ultraviolet absorption activity, tyrosinase inhibitory activity, and melanin production inhibitory activity, isoferulic acid and / or a derivative thereof (Patent No. 1928802), ferulic acid amide derivative (Patent No. 1945459) Gazette), isoferulic acid and / or its salt and ascorbic acid and / or its derivative and polyhydric alcohol (Japanese Patent No. 2533377), isoferulic acid and / or its salt and antioxidant (Japanese Patent No. 2533774), isoferula Acids and / or salts thereof and polyhydric alcohols (Patent No. 2533375), isoferulic acid and / or salts thereof and organic acids and / or salts thereof (Patent No. 2533776), ferulic acids and / or salts thereof and organics Acid and / or Its salt (Japanese Patent No. 2564139), caffeic acid glycoside (Japanese Patent No. 2997358), ferulic acid-2,3-dihydroxypropyl and / or its salt (Japanese Patent Laid-Open No. 6-157272) are contained as active ingredients. Cosmetics and topical skin preparations have been developed. It has been found that ferulic acid and / or ferulic acid ester promotes differentiation of normal human fibroblasts, and cell differentiation promoting agents, hair growth / hair growth agents, and anti-aging agents for skin containing these as active ingredients have been developed. (Unexamined-Japanese-Patent No. 5-310526). Caffeic acid, ferulic acid, and coniferyl alcohol have been found to have an active oxygen scavenging action, and cosmetics, pharmaceutical compositions and edible compositions containing these active oxygen scavengers as active ingredients have been developed (Japanese Patent Laid-Open No. Hei 9 (1994)). 7-300412). An antioxidant containing ferulic acid ester as an active ingredient has been developed (Japanese Patent Laid-Open No. 9-40613). A phenylpropanoid compound has been found to have an effect of suppressing phototoxicity caused by sunlight ultraviolet rays and air pollutants, and cosmetics and foods and drinks containing these phototoxicity inhibitors as active ingredients have been developed (Japanese Patent Application Laid-Open No. 2000-2000). -319154).
[0006]
[Problems to be solved by the invention]
However, an external preparation for skin containing laminin 5 as an active ingredient has problems in stability, skin permeability, etc. because laminin 5 is a very high molecular weight glycoprotein, and does not necessarily have a sufficient skin utilization effect. I can't expect it. In addition, preparations derived from soybeans and lysophospholipids do not have sufficient activity to promote the production of laminin 5, and skin utilization compositions containing them as active ingredients cannot necessarily be expected to have a sufficient skin activation effect.
[0007]
On the other hand, phenylpropanoid compounds are known to have the above-mentioned activities, but by focusing on the skin basement membrane component and promoting the production of that component, it is intended to activate the skin basement membrane and thus the entire skin. No attempt has been made.
[0008]
[Means for Solving the Problems]
The inventors examined the activity of various compounds to promote the production of laminin 5 in epidermal cells. As a result, it was found that cinnamic acid, cinnaldehyde, eugenol, coniferyl alcohol, caffeic acid and chlorogenic acid significantly promote laminin 5 production, and the present invention was completed.
[0009]
That is, the present invention
Composition for promoting production of laminin 5 containing one or more compounds selected from cinnamic acid, cinnaldehyde, eugenol, coniferyl alcohol, caffeic acid and chlorogenic acid and / or a salt thereof and / or a glycoside thereof object,
About.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Cinnamic acid, cinnamic aldehyde, eugenol, coniferyl alcohol, caffeic acid, and chlorogenic acid are one of a group of compounds that exist in nature, and linear 3-carbon (C3, propane) is present in the benzene nucleus (C6, phenyl). It is a combination. These compounds can be obtained directly or indirectly from a wide range of biological sources, but may be chemically synthesized.
[0011]
In nature, transcinnamic acid (p-coumaric acid in Gramineae) is produced by deamination of the amino acid phenylalanine (tyrosine in Gramineae) in vascular plants. Transcinnamic acid is oxidized to p-coumaric acid and further oxidized to caffeic acid. Caffeic acid is methylated to ferulic acid. Angiosperms can further oxidize ferulic acid to 5-hydroxyferulic acid and then methylate to synaptic acid, but not fern or gymnosperms. In nature, the phenolic hydroxyl groups of these acids are partially methylated, and the carboxyl groups are sequentially reduced to the corresponding aldehyde, alcohol, or olefin (Natural Product Chemistry, 4th revised edition, edited by Hiroshi Mitsuhashi, Nanedo).
[0012]
Caffeic acid is represented by the following formula (1) and has an inhibitory effect on histamine release from mast cells and an inhibitory activity on 5-lipoxygenase and leukotriene production (Natural Drug Dictionary, Takuma Okuda, 1986, Yodogawa Shoten). It exists as a constituent of chlorogenic acid, mugwort and 3,5-di-O-caffeylquinic acid, which is a main component of homologous plants, and other so-called caffeine tannins contained in coffee beans, yarrow and hypericum. It is also free and present in a wide range of plants such as tobacco leaves, sweet potatoes and pear leaves. It is obtained by heating a mixture of 3,4-dihydroxybenzaldehyde, acetic anhydride and potassium acetate (Parkin reaction). It is also produced by acid hydrolysis of chlorogenic acid.
[0013]
[Chemical 1]
[0016]
Cinnamic acid is represented by the following formula (3), and esters thereof are used as perfumes and pharmaceuticals (Natural Drug Dictionary, Takuma Okuda, 1986, Yodogawa Shoten). Usually, cinnamic acid refers to the trans form, and the cis form is called arrocinic acid. The cis form is unstable and tends to be a transformer form. It exists as a free acid or ester in resins such as cassia oil, perubalsum, and trubalsum. It can be obtained by oxidizing cinnamaldehyde or heating a mixture of benzaldehyde, acetic anhydride and potassium acetate (Perkin reaction).
[0017]
[Chemical 3]
[0018]
Keihialdehyde is represented by the following formula (4) and has a strong peculiar aroma and sweetness, so it is used in food flavors such as confectionery (Natural Drug Dictionary, Takuma Okuda, 1986, Yodogawa Shoten). It is also used for pharmaceuticals and cosmetics. It is contained in the bark and leaves of Kay, and it is the main component of scented oil and cassia oil, and it is also contained in myrrh oil and patchouli oil. Obtained by condensing benzaldehyde and acetaldehyde with alkali.
[0019]
[Formula 4]
[0020]
Eugenol is represented by the following formula (5) and is used as a raw material for producing vanillin, a fragrance, a bactericidal agent, an antiseptic and the like (Natural Drug Dictionary, Takuma Okuda, Yodogawa Shoten). In addition to being abundant in clove oil, it is a phenolic essential oil component widely present in essential oils of plants. It can be obtained by alkaline extraction of glove oil and cinnamon oil.
[0021]
[Chemical formula 5]
[0022]
Coniferyl alcohol is represented by the following formula (6) and is present as a glycoside coniferin in the conifer formation layer and its surrounding parenchyma, comfrey root, sugar beet, asparagus, etc. (Natural drug dictionary, Takuo Okuda) Hen, 1986, Yodogawa Shoten). It becomes a lignin-like substance by dehydrogenative polymerization in the plant body. Lignin has been recognized to have antitumor effects, liver catalase activity promoting effects and the like. It is obtained by allowing the enzyme hydrolase emulsin to act on coniferin.
[0023]
[Chemical 6]
[0024]
Chlorogenic acid is represented by the following formula (7) and has the effect of promoting gastric juice and bile secretion in addition to the central nervous excitability (natural drug dictionary, Takuma Okuda, 1986, Yodogawa Shoten). Antioxidant action (Japan Women's University, Professor Nguyen et al., 1995) and cancer cell metastasis inhibitory action (Tokyo University of Agriculture and Technology, Prof. Yagasaki et al., 2000) have been reported. Although isolated from coffee beans for the first time, it is widely distributed in the fruits and leaves of dicotyledonous plants, and many of them are particularly found in plants such as eggplants, asteraceae, and ceraceae. It is obtained by esterifying caffeic acid and quinic acid.
[0025]
[Chemical 7]
Any compound is commercially available and can be used.
[0026]
According to the present invention, cinnamic acid, cinnamic aldehyde, eugenol, coniferyl alcohol, caffeic acid, chlorogenic acid promotes the production of laminin 5 in epidermal keratinocytes.
[0027]
The compounds in the present invention can be used even in a state where they are dissolved using various solvents. For example, it can be used in a dissolved state using water or an alcohol such as ethanol or methanol, a polyhydric alcohol such as propylene glycol or 1,3-butylene glycol, an organic solvent such as ether, acetone or ethyl acetate. These may be used alone or in combination of two or more.
[0030]
【Example】
EXAMPLES Next, although an Example is given and this invention is further demonstrated, this invention is not limited to these Examples.
[0031]
Preparation Preparation of Compound-Containing Liquid: Cinnamic acid, cinnaldehyde, eugenol, coniferyl alcohol, caffeic acid, ferulic acid, and chlorogenic acid (all Sigma-Aldrich Co.) were prepared at 10 mg / ml with 99.5% ethanol.
[0032]
Test results 1. Measurement test of laminin 5 producing ability in epidermal keratinocytes;
As the epidermal keratinocytes and medium (KGM), fetal-derived normal human epidermal keratinocytes and Keratinocyte Growth Medium Bullet Kit (Asahi Techno Glass) were used. The cells were cultured with KGM in a 37 ° C.-5% CO ₂ incubator. In this experiment, cells having passage numbers 3 to 5 were used.
[0033]
Epidermal keratinocytes subcultured with KGM were peeled off from the culture dish using a trypsin / EDTA solution, and then trypsin was neutralized using a trypsin neutralization solution. Cells were collected by centrifugation, resuspended in KGM, seeded in a 96-well plate at 2 × 10 ⁴ cells / well, and cultured for 24 hours. Cinnamic acid, cinnamic aldehyde, eugenol, coniferyl alcohol, caffeic acid, ferulic acid, and chlorogenic acid were treated with 100 μg / ml and further cultured for 24 hours, and the cell culture supernatant was collected. After removing floating cells by centrifuging at 800 rpm for 5 minutes, the cell debris was removed by centrifuging at 15000 rpm for 30 minutes and used as a sample for ELISA.
[0034]
The cell culture supernatant was appropriately diluted with a phosphate buffer solution {PBS (−)} and adsorbed on a 96-well ELISA plate at 37 ° C. for 2 hours. After removing the cell culture supernatant, the cells were immersed in a blocking solution {PBS (−) containing 1% bovine serum albumin (BSA)} and blocked at 37 ° C. for 1 hour. After washing with a washing solution {PBS (-)} containing 0.05% polyoxyethylene (20) sorbitan monolaurate (Wako Pure Chemical Industries, Ltd.)}, the primary antibody solution {for laminin γ2 chain prepared to 5 mg / ml with the washing solution Monoclonal antibody (clone D4B5) (Mizushima, H., et al., Horm. Res., 50, 7-14, 1998.)} was added and reacted at 37 ° C. for 2 hours. After washing, a secondary antibody {biotinylated anti-mouse immunoglobulin G (Vector laboratories) prepared to 1 mg / ml with a washing solution} was added and reacted at room temperature for 1 hour. After washing, an enzyme solution {alkaline phosphatase avidin D (Vector laboratories) prepared to 1 mg / ml with a washing solution} was added and reacted at room temperature for 1 hour. After washing, a substrate solution {0.75 M Tris-HCl (pH 10.3) containing 1 mg / ml p-nitrophenyl phosphate (ICN Biomedicals, Inc.)} was added, reacted at 37 ° C. for 30 minutes, and then at 405 nm. Absorbance was measured.
The results are shown in Table 1.
[0035]
[Table 1]
The production of laminin 5 when each compound was treated was evaluated with the untreated control as 100%. As a result, all compounds significantly promoted laminin 5 production. In addition, epidermal growth factor (EGF) (Sigma-Aldrich Co.) (Mizushima, H., et al., J. Biochem., 120, 1196-1202, known to promote the production of laminin 5 1996.) was also significantly promoted in the production of laminin 5 when treated with 50 ng / ml.
[0036]
Reference test results Measurement test of light injury suppression effect;
(1) Measurement test of the effect of suppressing photodamage of human epidermal keratinocytes; after culturing human epidermal keratinocytes with KGM, using a trypsin / EDTA solution, the adherent cells are peeled off from the culture dish, and then a trypsin neutralizing solution. Was used to neutralize trypsin. After collecting the cells by centrifugation, the cells were resuspended in KGM to 2 × 10 ⁵ cells / ml. This cell suspension was treated with 100 μg / ml ferulic acid and 5 μg / ml laminin 5 neutralizing antibody (Chemicon, cloneP3H9-2), either alone or in combination. In this experiment, a laminin 5 neutralizing antibody was used in order to examine whether the effect of ferulic acid on photodamage suppression depends on the promotion of laminin 5 production. This cell suspension was seeded at 1 ml / well in a 24-well plate and cultured for 24 hours. UVB was irradiated at 0, 75 and 125 mJ / cm ² and further cultured for 24 hours. After washing with PBS (−) to remove floating cells, the cells were fixed with 5% glutaraldehyde aqueous solution at room temperature for 15 minutes. After washing with tap water, fluorescent staining solution {5 μg / ml Hoechst 33342 (Sigma-Aldrich Co.) and 0.001% polyoxyethylene (10) octylphenyl ether (Wako Pure Chemical Industries, Ltd., aqueous solution)} at room temperature, Treated in the dark for 1.5 hours. After washing with tap water, the cell viability was evaluated by measuring the fluorescence intensity using a fluorescence plate reader.
[0037]
The results are shown in FIG. The effect of inhibiting light damage was evaluated with no treatment of ferulic acid and laminin 5 neutralizing antibody, and 100% UVB non-irradiation. In the case of non-UVB irradiation, the cell viability was comparable to that in the case of no treatment in either case where ferulic acid and laminin 5 neutralizing antibody were treated alone or in combination. On the other hand, when UVB was irradiated at 75 and 125 mJ / cm ² , the cell viability was significantly increased when ferulic acid was treated as compared to no treatment. The cell viability when treated with a combination of ferulic acid and laminin 5 neutralizing antibody was similar to that of no treatment. Therefore, it is thought that the photodamage suppression effect of ferulic acid is caused by the promotion of laminin 5 production.
[0038]
(2) Measurement test of the photoinjury inhibitory effect of a three-dimensional human skin model;
The human skin three-dimensional model was cultured using TESTSKIN (LSE-high) (Toyobo) according to the protocol. Medium containing ferulic acid (100 μg / ml) was added onto the tissue in the assay ring and cultured for 24 hours. UVB was irradiated at 0, 120, 240 and 360 mJ / cm ² and further cultured for 24 hours. The tissue culture solution was collected and centrifuged at 15000 rpm for 30 minutes to remove tissue pieces.
[0039]
Cytotoxicity was measured by measuring lactate dehydrogenase (LDH) activity released from cells damaged by the cell membrane using this tissue culture solution. The LDH activity was measured using an LDH-cytotoxicity test kit (Wako Pure Chemical Industries). A tissue culture solution appropriately diluted with PBS (-) was dispensed into a 96-well reaction plate, and then the color developing solution was treated and reacted at room temperature for 20 minutes. After processing the reaction stop solution, the absorbance at 560 nm was measured with a microplate reader to evaluate cytotoxicity.
[0040]
The results are shown in FIG. The cytotoxicity rate in the case of no treatment with ferulic acid and no irradiation with UVB was evaluated as 100%. When UVB was not irradiated, the cytotoxicity rate was almost the same regardless of the presence or absence of ferulic acid treatment. On the other hand, when UVB was irradiated, the cytotoxic rate increased as the dose of UVB increased, but when ferulic acid was treated, the cytotoxicity was significantly reduced compared to no treatment.
[0041]
Reference Formulation Example 1. Cream production;
(Composition) (Content)
(A) stearyl alcohol 6.0% stearic acid 2.0% hydrogenated lanolin 4.0% squalane 9.0% octyldodecanol 10.0% POE (25) cetyl alcohol ether 3.0% glyceryl monostearate 2 0.0% Ferulic acid 0.1% Preservative Appropriate amount Perfume Appropriate amount (B) 1,3 Butylene glycol 6.0% PEG 15004.0% Purified water Residues (Preparation method) A and B are each heated to 70 ° C. Add A to B, make the emulsified particles uniform with a homomixer, deaerate, filter and cool.
[0042]
Reference formulation example 2 Manufacture of tablets;
(Composition) (Content)
Ferulic acid 20.0% Lactose 65.0% Corn starch 14.0% Guar gum 1.0%
[0043]
【The invention's effect】
Cinnamic acid, cinnamic aldehyde, eugenol, coniferyl alcohol, caffeic acid, and chlorogenic acid promote the production of laminin 5 and thus promote the structure maintenance and functional improvement of the skin basement membrane.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a reference diagram showing the effect of inhibiting photodamage when human epidermal keratinocytes are treated with ferulic acid and laminin 5 neutralizing antibody.
FIG. 2 is a reference diagram showing the effect of suppressing photodamage when ferulic acid is treated on a three-dimensional human skin model.

Claims (1)

Composition for promoting production of laminin 5 containing one or more compounds selected from cinnamic acid, cinnaldehyde, eugenol, coniferyl alcohol, caffeic acid, chlorogenic acid and / or a salt thereof and / or a glycoside thereof object.