JP7061766B2 - Composition for promoting DJ-1 protein production - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Date of publication June 21, 2017 Publication address http: // www. sfrrj2017. org / pdf / sfrrj2017_Abstracts. pdf

Application of Article 30, Paragraph 2 of the Patent Act Date: June 28, 2017 Meeting name 70th Annual Meeting of the Japanese Society of Oxidative Stress

The present invention relates to a composition that promotes the production of DJ-1 protein and a composition that enhances resistance to external stimuli to skin cells.

The DJ-1 protein is a known bioactive protein that is present in a wide range of human cells, including nerve cells and skin cells, and consists of 189 amino acids. The DJ-1 protein is an oncogene product and is known to be associated with familial Parkinson's disease (Non-Patent Document 1).
Furthermore, the DJ-1 protein is known to have an effect of suppressing nerve cell death due to oxidative stress. That is, when DJ-1 protein is injected into Parkinson's disease model rats injected with 6-hydroxydopamine that gives oxidative stress at the same time as or after injection of 6-hydroxydopamine, dopamine neuronal cell death is suppressed and behavioral abnormalities occur. It is known to be improved (Patent Document 1).
Patent Document 1 describes that DJ-1 has an effect of suppressing nerve cell death due to oxidative stress. Then, we propose a compound that binds to and activates DJ-1.

The DJ-1 protein is also known to be involved in skin resistance. By measuring the amount of DJ-1 protein in the stratum corneum of the skin, the present inventors can use it as an index of resistance to the occurrence of redness when exposed to ultraviolet rays (Patent Document 2), and the history of exposure to ultraviolet rays. It is clarified that it can be used as an evaluation index (Patent Document 3) or can be used as an evaluation index of hair growth state (Patent Document 4).

International Publication No. 2007/060886 Japanese Patent No. 5775538 Japanese Patent No. 5775540 Japanese Unexamined Patent Publication No. 2017-9426

Biochemical and Biophysical Research Communications 473 (2016) 87-91 Experimental Neurology. 2002, 175; 303-317

The present invention was discovered in the process of searching for a substance that promotes the production of DJ-1 protein involved in the resistance of the skin to external stimuli. In addition, the present inventors found that in the process of investigating the effect of ultraviolet rays on skin cells, cells in which DJ-1 protein was increased had enhanced ultraviolet resistance.
That is, it is an object of the present invention to provide a new composition that promotes DJ-1 protein production in skin cells, and to provide a composition for imparting ultraviolet resistance to skin cells.

The present invention has the following configuration.
(1) A composition for promoting DJ-1 protein production containing curcumin and / or resveratrol as an active ingredient.
(2) An external skin preparation for promoting DJ-1 protein production, which comprises the composition according to (1).
(3) A composition for enhancing the resistance to external stimuli of skin cells containing the composition according to (1).
(4) The composition according to (3), wherein the external stimulus is due to ultraviolet rays.
(5) An agent for improving resistance to the occurrence of redness (redness) induced by exposure to ultraviolet rays.

The present invention provides a composition that promotes the production of DJ-1 protein. The composition of the present invention is useful as a skin external preparation. Further, by increasing the DJ-1 protein in the skin cells, particularly the stratum corneum cells, the resistance of the cells to external stimuli is enhanced, and the inflammatory reaction induced by ultraviolet rays can be suppressed. Therefore, resistance to inflammation and redness induced by exposure to ultraviolet rays can be improved.

It is a graph which shows the DJ-1 protein production promotion effect of curcumin in Test Example 1. It is a graph which shows the DJ-1 protein production promotion effect of resveratrol in Test Example 1. It is a graph which shows the DJ-1 protein production promotion effect of curcumin in Test Example 2. It is a graph which shows the DJ-1 protein production promotion effect of resveratrol in Test Example 2. It is a graph which shows the effect of curcumin on the epidermal cell viability in Test Example 3. It is a graph which shows the effect which resveratrol has on the epidermal cell viability in Test Example 3.

Hereinafter, the present invention will be described in detail.
The present application is an invention relating to a composition for promoting DJ-1 protein production containing curcumin and / or resveratrol as an active ingredient.

As described above, the human DJ-1 protein is a protein that is highly expressed in human skin cells and nerve cells.
By promoting the production of DJ-1 protein in the skin, it can be expected to increase the amount of DJ-1 protein in the skin and increase the resistance of the skin to external stimuli.

The composition for promoting DJ-1 protein production of the present invention contains curcumin and / or resveratrol as an active ingredient for promoting DJ-1 protein production.

Curcumin, which is the active ingredient of the composition of the present invention, is a general term for known yellow polyphenol compounds contained in turmeric (turmeric, scientific name Curcuma longa) and the like. It is used as a colorant for spices and foods, and its safety is well known.
The curcumins used in the present invention refer to compounds classified as curcumin or curcumin derivatives, including, but not limited to, curcumin, demethoxycurcumin, bisdemethoxycurcumin, cis-trans-curcumin, and cyclocurcumin. Of these, curcumin is particularly preferable. Further, it may be a turmeric extract extracted from the rhizome of a plant turmeric. Turmeric extract is commercially available as a raw material for health foods and supplements, and such commercially available products can also be used.

Resveratrol is a kind of stilbenoid (stilbenoid derivative) polyphenol, and the strain name is also called 3,5,4'-trihydroxy-trans-stilbene. Resveratrol functions as a phytoalexin in some plants and is known as an antioxidant contained in grape skins and the like. Resveratrol forms a dimer and is also called ε-viniferin. Glycosides also exist, and in the present invention, these are collectively referred to as resveratrols. Among the resveratrols, the monomeric resveratrol is preferable. Resveratrols can be obtained from grape skins and red wine, peanut red skins, melinjo (gunetum), and Japanese knotweed roots. It is sold as a raw material for health foods, and it can also be used.

Examples of the composition for promoting DJ-1 protein production of the present invention include pharmaceuticals, quasi-drugs, cosmetics, foods, and beverages. The administration route can be either oral administration or transdermal administration. Of these, transdermal administration is particularly preferable, and it is preferable to adopt the form of a transdermal composition such as cosmetics and external medicines for skin. The active ingredient can also reach the skin by oral administration or the like.
In order to prepare a composition for promoting DJ-1 protein production, for example, in the case of an external preparation, curcumin and / or resveratrol are added to 0.001 to 0.1% by mass per mass of the external preparation in terms of curcumin. Alternatively, by containing 0.01 to 1% by mass per mass of the external preparation in terms of resveratrol, the production of DJ-1 protein in skin cells can be promoted when externally applied by a method such as application to the skin. In order to confirm the effect of promoting the production of DJ-1 protein, a skin stratum corneum sample is collected by a tape stripping method or the like, extracted according to the operation method described in Patent Document 2, and measured by an ELISA method or the like. do.

It is used in the formulation of ordinary foods, pharmaceuticals, cosmetics, etc. in order to prepare the composition for promoting DJ-1 protein production containing the curcumins and / or resveratrols of the present invention into a human-administered product. It can contain any component. Such optional components include excipients such as lactose and sucrose, binders such as starch, cellulose, arabic gum and hydroxypropyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose and the like as long as they are oral administration compositions. Disintegrants, surfactants such as soybean lecithin and sucrose fatty acid esters, sweeteners such as martitol and sorbitol, acidulants such as citric acid, buffers such as phosphates, film-forming agents such as shelac and zein, Lubricants such as talc and wax, flow promoters such as light anhydrous citric acid and dry aluminum hydroxide gel, diluting agents such as physiological saline and glucose aqueous solution, flavoring agents, coloring agents, bactericides, preservatives, etc. A fragrance or the like can be preferably exemplified. For transdermally administered compositions, hydrocarbons such as squalane, vaseline and microcrystalin wax, esters such as jojoba oil, carnauba wax and octyldodecyl oleate, triglycerides such as olive oil, beef fat and coconut oil, and stearers. Fatty acids such as acid, oleic acid and retinoic acid, lower alcohols such as ethanol and isopropanol, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinic acid ester and sodium polyoxyethylene alkyl sulfate. , Amphoteric surfactants such as alkylbetaine salts, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, Nonionic surfactants such as polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil, polyhydric alcohols such as dipropylene glycol, polyethylene glycol, glycerin, 1,3-butanediol and 1,2-pentanediol, It can contain a thickening / gelling agent, an antioxidant, an ultraviolet absorber, a coloring agent, an antiseptic, a powder and the like. The dosage form is preferably in the form of an external preparation such as a cosmetic, a coating agent, or a patch.

Hereinafter, a test example confirming the effect of curcumin and resveratrol on promoting the production of DJ-1 protein will be shown, and the effect of the composition of the present invention will be specifically described.

<DJ-1 protein production promotion test using cultured human skin stratum corneum cells>
1. 1. Test Example 1
(1) Test method HaCaT cells (human epidermal keratinized cell line) were seeded and cultured on a 6-well plate, and after 2 days, curcumin was added to 0, 5, and 10 μM, and the cells were cultured for 3 hours. Similarly, resveratrol was added to 0, 20, 40 μM and cultured for 3 hours.
After the culture was completed, the cells were collected, the cells were destroyed by a conventional method, and the amount of DJ-1 protein in the cells was detected by Western blotting. In addition, the intracellular actin protein was similarly detected by Western blotting, and the amount of DJ-1 protein with respect to the amount of actin protein was calculated, which was used as the amount of DJ-1 protein produced.

(2) Results Figure 1 shows the results of the production promotion test using curcumin, and FIG. 2 shows the results of the production promotion test using resveratrol.
It was confirmed that curcumin and resveratrol act on HaCaT cells and promote DJ-1 protein production.

2. 2. Test Example 2 (Extended treatment time to cells to 6 hours)
(1) Test method Normal human epidermal keratinized cells were seeded and cultured on a 6-well plate, and after 2 days, curcumin was added to 0, 1, 3, 10, and 30 μM, and the cells were cultured for 6 hours. Similarly, resveratrol was added to 0, 0.001 and 0.01%, and the cells were further cultured for 6 hours.
After the culture was completed, the cells were collected, the cells were destroyed by a conventional method, and the amount of DJ-1 protein in the cells was detected by Western blotting. The expression level of DJ-1 protein in cells to which no sample was added was set to 1 and expressed as a ratio to the expression level.

(2) Results Figure 3 shows the results of the production promotion test using curcumin, and FIG. 4 shows the results of the production promotion test using resveratrol.
Unlike the HaCaT cells used in Test Example 1, the results are normal human epidermal keratinocytes, but both are epidermal keratinocytes, and even when the treatment time of curcumin and resveratrol is extended, the results are against human skin cells. It was confirmed that it acts and promotes DJ-1 protein production.
In addition, resveratrol and curcumin can be used as resistance improving agents for external stimuli such as ultraviolet rays because they improve resistance to external stimuli such as ultraviolet rays.

3. 3. Test Example 3 (Effect on cell viability)
(1) Test method Normal human epidermal keratinized cells were seeded on a 35 mm dish, and curcumin or resveratrol was added 1 day later. Curcumin was added at 0, 1, 3 μM, and resveratrol was added at 0, 0.0001, 0.001%. After 24 hours, the cells were washed with PBS (−), replaced with HANKS solution, and irradiated with UVB 15 mJ / cm ² . The cell viability was expressed as a ratio to which the fluorescence intensity of the UVB-free and sample-free cells was set to 1 by performing an AlamarBlue assay 24 hours after irradiation.

(2) Results Figure 5 shows the cell viability measurement results by curcumin, and FIG. 6 shows the cell viability measurement results by resveratrol.
Both curcumin and resveratrol improved cell viability after UV irradiation. In particular, the cell viability when 0.001% by mass of resveratrol was added exceeded that without UVB irradiation.
Therefore, it was revealed that curcumin and resveratrol improve resistance to external stimuli such as ultraviolet rays to skin epidermal cells.

Claims (1)

A composition for promoting DJ-1 protein production containing curcumin and / or resveratrol as an active ingredient (however, for promoting the biological defense ability of melanin, for preventing photoaging, for sunscreen, and for suppressing the expression of NF-κB). Uses, cell death suppression use, skin aging prevention use, α-synuclein fibril formation and elongation suppression use, Parkinson's disease agent, antioxidant use) .

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