JP2023094400A - Screening method of anti-aging agent - Google Patents

Abstract

To provide a method for screening an anti-aging agent.SOLUTION: A screening method of an anti-aging agent which has a change of endoplasmic reticulum stress in a cell as an index, where the cell is an epidermal cell or a dermal fibroblast. Preferably, the screening method has a reduction of endoplasmic reticulum stress in the cell as an index, more preferably, the screening method includes the steps of: adding a test substance to the cell; and selecting a test substance in which the endoplasmic reticulum stress in the cell to which the test substance is added is lower than the endoplasmic reticulum stress in the cell to which the test substance is not added.SELECTED DRAWING: None

Description

The present invention relates to a method of screening anti-aging agents.

The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrix such as collagen and elastin, which are outside these cells and support the skin structure. In young skin, the proliferation of fibroblasts is active, and the interaction of skin tissue such as fibroblasts and extracellular matrix components maintains homeostasis, ensuring moisture retention, flexibility, elasticity, etc. The appearance is maintained in a fresh state with tension and luster.

However, due to factors such as aging and ultraviolet rays, collagen, elastin, etc., which are the main constituents of the extracellular matrix, are degraded or their production is reduced. As a result, the moisturizing function and elasticity of the skin are reduced, and abnormal exfoliation of keratin occurs, so that the skin loses its firmness and luster, and exhibits aging symptoms such as rough skin and wrinkles. Thus, changes associated with aging of the skin, ie, wrinkles, dullness, changes in texture, loss of elasticity, etc., are associated with reductions in matrix components such as collagen and elastin.
It is known that such a decrease in matrix components is caused by the secretion of proteolytic enzymes such as matrix metalloprotease and neutrophil elastase from skin cells, neutrophils and the like in the skin.

In recent years, a component that suppresses neutrophil elastase activity has been discovered, and various cosmetics containing elastase inhibitors as active ingredients to prevent or improve the formation of wrinkles and sagging have been disclosed (Patent Documents 1 to 4). ).

Japanese Patent Application Laid-Open No. 2002-205950 JP 2009-191043 A JP 2013-006815 A JP 2019-081726 A

In today's world where consumers are increasingly yearning for anti-aging, the demand for cosmetics to prevent or improve wrinkle formation, dullness, deterioration of texture, deterioration of elasticity, etc. is increasing. ing. In addition to cosmetics with a mechanism that inhibits proteolytic enzymes such as elastase and matrix metalloprotease as described above, new approaches to prevent and improve aging such as wrinkle formation are required.
Accordingly, an object of the present invention is to provide a technique effective in preventing and improving aging symptoms such as wrinkle formation by means of a new mechanism of action.

The present inventors have found that protein density in all skin layers (epidermis/dermis) decreases when endoplasmic reticulum stress increases. Furthermore, we found that endoplasmic reticulum stress increased in epidermal cells and dermal fibroblasts when facial pressure was applied to the skin.
Expression pressure is the pressure applied to the skin when the skin contracts and collides with each other or when the skin stretches when making a facial expression. When the protein density in all layers of the skin decreases, the skin loses its elasticity and tension, and exhibits aging symptoms such as wrinkle formation, dullness, decreased texture, and decreased elasticity. Therefore, the present inventors considered that facial pressure causes an increase in endoplasmic reticulum stress, which in turn causes aging symptoms such as wrinkle formation.
Based on these findings, the inventors completed a method of screening anti-aging agents using reduction of endoplasmic reticulum stress as an indicator.

That is, the present invention is as follows.
[1] A screening method for an anti-aging agent, using changes in endoplasmic reticulum stress in cells as an index,
A screening method, wherein the cells are epidermal cells or dermal fibroblasts.
[2] The screening method of [1], wherein reduction in endoplasmic reticulum stress is used as an index.
[3] adding a test substance to cells, and selecting a test substance that reduces the endoplasmic reticulum stress in cells to which the test substance has been added as compared with the endoplasmic reticulum stress in cells to which the test substance has not been added; The screening method of [1] or [2], comprising the step of [4] The screening according to [3], further comprising the step of inducing endoplasmic reticulum stress in the step of adding the test substance to the cells, or before or after the step of adding the test substance to the cells. Method.
[5] The screening method of [4], wherein the step of inducing endoplasmic reticulum stress is a step of adding an endoplasmic reticulum stress-inducing agent to the cells.
[6] In the step of selecting the test substance, the expression level of the endoplasmic reticulum stress marker in cells to which the test substance was added is smaller or greater than the expression level of the stress marker in cells to which the test substance was not added. The screening method according to any one of [3] to [5], wherein a test substance is selected.
[7] In the step of selecting the test substance, the expression level of the endoplasmic reticulum stress marker in cells to which the test substance was added is smaller than the expression level of the stress marker in cells to which the test substance was not added. and select
The endoplasmic reticulum stress marker is CHOP,
The screening method according to any one of [3] to [6], wherein the expression level of the endoplasmic reticulum stress marker is the expression level of CHOP protein or the expression level of the gene encoding CHOP.
[8] The screening method according to any one of [1] to [7], wherein the anti-aging agent is an anti-wrinkle agent.
[9] A method of designing a composition, comprising the step of performing the screening method according to any one of [1] to [8], and the step of containing the substance selected by the above step.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to search for a component that is effective as an anti-aging agent suitable for inclusion in cosmetics.

Graph showing relative values of CHOP gene expression levels in epidermal cells and dermal fibroblasts when contraction pressure is applied. Graph showing relative values of protein amounts per cell in epidermal cells and dermal fibroblasts when tunicamycin was added. Graph showing the relative expression level of each gene in epidermal cells and dermal fibroblasts when tunicamycin was added. Graph showing relative values of CHOP gene expression levels in epidermal cells and dermal fibroblasts when tunicamycin and a test substance were added. A graph showing relative values of CHOP gene expression levels in epidermal cells when a test substance was added. A graph showing relative values of CHOP gene expression levels in dermal fibroblasts when a test substance was added.

The screening method of the present invention uses changes, preferably reductions, in endoplasmic reticulum stress in epidermal cells or dermal fibroblasts as an indicator.
As mentioned above, when endoplasmic reticulum stress increases in epidermal cells and dermal fibroblasts, protein density such as extracellular matrix in all skin layers decreases, resulting in wrinkle formation, dullness, decreased texture, decreased elasticity, etc. causes aging symptoms. Therefore, a substance that has the effect of changing the endoplasmic reticulum stress level in epidermal cells and dermal fibroblasts can be an anti-aging agent that can prevent, treat, or improve aging symptoms such as wrinkles.

Endoplasmic reticulum stress refers to a state in which proteins are no longer folded normally in the lumen of the endoplasmic reticulum and accumulate as defective proteins when cells are exposed to various internal or external environmental changes. Known factors that cause endoplasmic reticulum stress include nutritional starvation, disturbance of intracellular calcium concentration, hypoxia, expression of mutant proteins, and viral infection (Journal of Japanese Biochemical Society 90 (1): 51-59 ( 2018)).

Specifically, the screening method of the present invention includes a step of adding a test substance to cells, and comparing the endoplasmic reticulum stress in cells to which the test substance has been added with the endoplasmic reticulum stress in cells to which the test substance has not been added. and selecting test substances that are low in

The screening method of the present invention preferably further comprises the step of inducing endoplasmic reticulum stress in the step of adding the test substance to the cells, or before or after the step of adding the test substance to the cells.
As described above, when facial pressure is applied to the skin, endoplasmic reticulum stress increases in epidermal cells and dermal fibroblasts. Therefore, a substance that has the effect of reducing endoplasmic reticulum stress when endoplasmic reticulum stress is induced can serve as an anti-aging agent that prevents, treats, or improves aging symptoms such as wrinkles caused by facial pressure.
Methods for inducing endoplasmic reticulum stress include known methods such as a method of applying pressure load and/or stretching load to cells, and a method of adding an endoplasmic reticulum stress inducer to cells.

That is, the screening method of the present invention comprises the steps of adding a test substance to cells, applying pressure load and/or stretching load to the cells, and reducing endoplasmic reticulum stress in the cells to which the test substance was added. A step of selecting a test substance that has a low endoplasmic reticulum stress compared to the endoplasmic reticulum stress in the infected cells may be included.
The pressure load is applied until the long diameter of the cells is preferably reduced by 10 to 30%, more preferably by 15 to 25%. The stretching load is applied until the major axis of the cell increases by preferably 10-30%, more preferably 15-25%. The time for applying pressure load and extension load is preferably 30 minutes to 10 hours, more preferably 1 hour to 5 hours. When both the pressure load and the extension load are applied, the frequency of applying the pressure load or the extension load is preferably 0.1 to 1.5 Hz. When applying only the pressure load, the frequency of applying the pressure load is preferably 0.1 to 1.5 Hz. When applying only the extension load, the frequency of applying the extension load is preferably 0.5 to 1.0 Hz.
Means for applying pressure load and extension load are not particularly limited, and any means can be adopted. For example, after cells are seeded in an elastic culture vessel, pressure load can be applied to the lateral direction of the cells by repeating stretching and unstretching of the culture vessel.

The step of inducing endoplasmic reticulum stress may be a step of adding an endoplasmic reticulum stress-inducing agent to the cells.
Further, the screening method of the present invention comprises the step of adding a test substance and an endoplasmic reticulum stress-inducing agent to cells, and the endoplasmic reticulum stress in the cells to which the test substance has been added is the endoplasmic reticulum stress in the cells to which the test substance has not been added. selecting a test substance that is low compared to the .

Endoplasmic reticulum stress can be induced by an endoplasmic reticulum stress inducer. An endoplasmic reticulum stress inducer is a drug that specifically inhibits an endoplasmic reticulum protein. A known endoplasmic reticulum stress-inducing agent can be used, and examples thereof include tunicamycin, sapsigargin and the like.
The final concentration of the endoplasmic reticulum stress inducer added to the cells is preferably 0.1 ng/ml to 2000 ng/ml, more preferably 0.5 ng/ml to 1500 ng/ml, and 1 ng/ml to 1000 ng. /ml is more preferred. By setting the final concentration within the above range, induction of endoplasmic reticulum stress is facilitated.

Reduction of endoplasmic reticulum stress can be evaluated by known methods, such as measuring the expression level of an endoplasmic reticulum stress marker or the expression level of a gene encoding an endoplasmic reticulum stress marker protein, or by introducing an endoplasmic reticulum stress-responsive luminescent plasmid. It can be evaluated by luminescence measurement.
An endoplasmic reticulum stress marker refers to a biomarker for detecting endoplasmic reticulum stress. The endoplasmic reticulum stress marker may be any endoplasmic reticulum stress marker whose expression level increases or decreases due to endoplasmic reticulum stress.
As the endoplasmic reticulum stress marker, a known endoplasmic reticulum stress marker whose expression level changes due to endoplasmic reticulum stress can be used, and an endoplasmic reticulum stress marker protein can be mentioned. Examples include proteins such as CHOP, GRP78, ATF4.

CHOP (C/EBP homologous protein, also known as GADD153 or DDIT3) is a transcription factor present downstream of the endoplasmic reticulum stress pathway. CHOP is known to be significantly expressed by endoplasmic reticulum stress and induce apoptosis in various cells (Frontiers in Immunology, 2019 January 04, Volume 9, Article 3083).

GRP78 (also known as HSPA5 (heat shock protein 5)) is a member of the HSP70 (Heat Shock Protein 70) family, also called "immunoglobulin heavy chain-binding protein (BiP)". and is a regulator of the endoplasmic reticulum stress pathway. GRP78 is known to be involved in protein assembly and folding in the endoplasmic reticulum.

ATF4 (activating transcription factor 4) is a transcription factor that is expressed by endoplasmic reticulum stress, and is known to induce transcription of genes involved in amino acid metabolism and antioxidant stress.

When the endoplasmic reticulum stress marker is an endoplasmic reticulum stress marker whose expression level increases due to endoplasmic reticulum stress, the screening method of the present invention preferably comprises the steps of adding a test substance and an endoplasmic reticulum stress-inducing agent to cells, and selecting a test substance in which the expression level of the endoplasmic reticulum stress marker in cells to which the is added is smaller than the expression level of the stress marker in cells to which the test substance is not added. In other words, in the step of selecting a test substance, a test substance is selected in which the expression level of the endoplasmic reticulum stress marker in the cells to which the test substance was added is lower than the expression level of the stress marker in the cells to which the test substance was not added. preferably.
When the endoplasmic reticulum stress marker is an endoplasmic reticulum stress marker whose expression level increases due to endoplasmic reticulum stress, for example, when the endoplasmic reticulum stress marker is selected from CHOP gene, GRP78 gene, and ATF4 gene, expression of these stress markers Suppression correlates with a reduction in endoplasmic reticulum stress, which in turn correlates with a reduction in endoplasmic reticulum stress and promotion of anti-aging. Therefore, a test substance that suppresses the expression of these endoplasmic reticulum stress markers by adding the test substance to cells can be selected as a component having an endoplasmic reticulum stress-reducing effect and an anti-aging effect.

The degree of decrease in the expression level of the endoplasmic reticulum stress marker is preferably as long as the expression level of the endoplasmic reticulum stress marker in cells to which the test substance has been added is smaller than the expression level in cells to which the test substance has not been added. can be considered small when it is 90% or less 12 to 24 hours after the addition of the test substance, more preferably 80% or less, still more preferably 70% or less, and most preferably 50% or less. In some cases.

When the endoplasmic reticulum stress marker is an endoplasmic reticulum stress marker whose expression level decreases due to endoplasmic reticulum stress, the screening method of the present invention preferably comprises the steps of adding a test substance and an endoplasmic reticulum stress-inducing agent to cells, and The expression level of the endoplasmic reticulum stress marker in the cells to which is added is greater than the expression level of the stress marker in cells to which the test substance is not added, selecting a test substance. In other words, in the step of selecting a test substance, a test substance is selected in which the expression level of the endoplasmic reticulum stress marker in cells added with the test substance is greater than the expression level of the stress marker in cells not added with the test substance. preferably.
When the endoplasmic reticulum stress marker is an endoplasmic reticulum stress marker whose expression level decreases due to endoplasmic reticulum stress, promotion of expression of the endoplasmic reticulum stress marker correlates with a decrease in endoplasmic reticulum stress, resulting in a decrease in endoplasmic reticulum stress, Furthermore, it correlates with promotion of anti-aging. Therefore, a test substance that promotes the expression of these endoplasmic reticulum stress markers by adding the test substance to cells can be selected as a component having an endoplasmic reticulum stress-reducing effect and an anti-aging effect.

The degree of increase in the expression level of the endoplasmic reticulum stress marker is preferably as long as the expression level of the endoplasmic reticulum stress marker in cells to which the test substance has been added is greater than the expression level in cells to which the test substance has not been added. 12 to 24 hours after addition of the test substance, it can be considered large when it is 110% or more, more preferably 120% or more, and still more preferably 130% or more.

The amount of the test substance to be added is not particularly limited as long as it is within a concentration range in which significant cytotoxicity does not occur. For example, the test substance can be added so that the final concentration of the test substance is preferably 0.01 to 10.00% by mass, more preferably 0.05 to 5.00% by mass.

The expression level of the endoplasmic reticulum stress marker can be measured using any method. For example, a DNA fragment having a sequence that specifically binds to the sequence of the gene encoding the endoplasmic reticulum stress marker protein is used as primers to perform PCR and perform quantitative detection. The nucleotide sequences of genes encoding known endoplasmic reticulum stress marker proteins such as CHOP, GRP78, and ATF4 have been published, and those skilled in the art can design primers as appropriate and subject them to PCR. .
Alternatively, for example, the intracellular abundance of the endoplasmic reticulum stress marker protein may be quantitatively measured by a conventional method and used as the expression level of the endoplasmic reticulum stress marker.

The screening method of the present invention is usually an in vitro screening method.
Cultured cells can also be used as the cells to which the test substance is added.

In the present invention, "wrinkles" include so-called wrinkles, which are groove-shaped forms formed on the surface of the skin, as well as deformation of the skin due to loss of firmness and elasticity, such as sagging, which is deformation of the skin in the direction of gravity. is a broad term for
In the present invention, "aging" is a phrase that means changes associated with aging of the skin such as wrinkles, dullness, changes in texture and loss of elasticity.
Also, the anti-aging agent is preferably an anti-wrinkle agent. Furthermore, the anti-aging agent selected by the screening method of the present invention can also be used as an endoplasmic reticulum stress-reducing agent.

A test substance targeted by the screening method of the present invention may be a pure substance, an extract derived from animals or plants, or a mixture thereof.
Animal and plant-derived extracts shall mean not only animal- or plant-derived extracts themselves, but also fractions of extracts, purified fractions, extracts or fractions, and solvent-removed purified products. Plant-derived extracts include wild or grown plants, extracts using those sold as raw materials for herbal medicines, commercially available extracts, and the like.
In the extraction operation, in addition to using the whole plant part, parts such as the plant body, the ground part, the rhizome part, the tree trunk, the leaf part, the stem part, the flower, the flower bud, the fruit, etc. can be used. Alternatively, it is preferable to cut into small pieces to improve the extraction efficiency. Extraction solvents include water, alcohols such as ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. One or more selected from polar solvents such as polar solvents can be exemplified as suitable. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of the part used for extraction of a plant or the like or its dried product, and at room temperature for several days, at a temperature near the boiling point. If possible, a method of immersing for several hours, cooling to room temperature, optionally removing insoluble matter and/or solvent, and fractionating and purifying by column chromatography or the like can be mentioned.

The present specification also provides a method of designing a composition, comprising the step of performing the screening method of the present invention, and the step of containing a substance (anti-aging agent) selected by said step. Such compositions include transdermally administered compositions (e.g., cosmetics, quasi-drugs, external skin preparations such as pharmaceuticals) and orally administered/ingested compositions (e.g., food and drink, pharmaceutical Oral compositions such as quasi-drugs and pharmaceuticals).
Moreover, the dosage form of the composition is not particularly limited. For example, formulations designed as cosmetics include commonly known lotion formulations, emulsion formulations, essence formulations, cream formulations, powder-containing formulations, and the like.

When designing a composition containing an anti-aging agent selected by the screening method of the present invention, the content (blended amount) of the anti-aging agent is usually 0.00001% by mass or more, preferably 0.0001% by mass. Above, more preferably 0.001% by mass or more, usually 80% by mass or less, preferably 30% by mass or less, more preferably 10% by mass or less. By setting it as the said range, it can be set as the composition which shows an anti-aging effect suitably.
Moreover, the number of anti-aging agents contained in the composition may be one or two or more.

When designing a composition containing an anti-aging agent selected by the screening method of the present invention, it may be designed so as to appropriately incorporate necessary components according to the above-described aspects and uses.
For example, when it is designed as a cosmetic composition, it is possible to incorporate a wide range of ingredients that are commonly used in cosmetics, and there are no restrictions on the dosage form or application.
Hereinafter, components that can be contained in cosmetics when mainly applied to cosmetics will be described.

Active ingredients include other anti-aging agents, whitening ingredients, anti-inflammatory ingredients, extracts derived from animals and plants, and the like. In addition, it may be mixed with the anti-aging agent selected by the screening method of the present invention.

Other anti-aging agents are not particularly limited as long as they are generally used in cosmetics. For example, sodium trifluoride isopropyloxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate, nicotinamide, vitamin A or derivatives thereof (retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopheryl retinoate, retinol palmitate , retinol acetate, etc.), benzyl ursolic acid, ursolic acid phosphate, betulinic acid benzyl ester, and benzylic acid phosphate.
The content of other anti-aging agents in cosmetics is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 1 to 5% by mass.

The whitening component is not particularly limited as long as it is commonly used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethylascorbic acid, tranexamic acid, arbutin, ellagic acid, kojic acid, linoleic acid, nicotinamide, 5,5′-dipropylbiphenyl-2, 2′-diol, disodium 5′-adenylate, cetyl tranexamate, potassium 4-methoxysalicylate, hydroquinone, pantothenic acid, panthenol and the like.
The content of the whitening component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 0.3 to 5% by mass.

The extract derived from animals and plants is not particularly limited as long as it is generally used in pharmaceuticals, cosmetics, foods, and the like. For example, akebi extract, asunaro extract, asparagus extract, avocado extract, amacha extract, almond extract, arnica extract, aloe extract, aronia extract, apricot extract, ginkgo biloba extract, Indian mushroom extract, fennel extract, udo extract, ageitsu extract, eleuthero extract , Trillium extract, Scutellaria root extract, Phellodendron extract, Coptis extract, Panax ginseng extract, Hypericum extract, Dead nettle extract, Orange extract, Kakyoku extract, Kakon extract, Chamomile extract, Carrot extract, Kawara mugwort extract, Glycyrrhiza extract, Kiwi extract, Cucumber extract, Guava extract, Kujin extract, Gardenia extract, Kumazasa extract, Clara extract, Walnut extract, Grapefruit extract, Black rice extract, Chlorella extract, Mulberry extract, Gerbera extract, Alpinia extract, Gentian extract, Gennoshoko extract, Black tea extract, Burdock extract, Rice extract, Fermented rice extract, fermented rice bran extract, rice germ oil, cowberry extract, salvia extract, soapwort extract, bamboo grass extract, hawthorn extract, sansha extract, Japanese pepper extract, shiitake extract, rhubarb extract, rhizome extract, perilla extract, linden extract, meadowsweet extract, peony Extract, ginger extract, calamus root extract, white birch extract, horsetail extract, stevia extract, fermented stevia, ivy extract, hawthorn extract, elderberry extract, yarrow extract, mint extract, sage extract, mallow extract, cnidium extract, assembly extract, sage extract, rhubarb extract, soybean extract, taisou extract, thyme extract, dandelion extract, tea extract, clove extract, chimp extract, sweet tea extract, capsicum extract, angelica extract, calendula officinalis extract, tonin extract, spruce extract, dokudami extract , Tomato extract, Natto extract, Carrot extract, Garlic extract, Novara extract, Hibiscus extract, Bakumondou extract, Lotus extract, Parsley extract, Birch extract, Hamamelis extract, Hinoki extract, Loquat extract, Coltsfoot extract, Butterbur extract, Bukuryo extract, Butcher bloom extract, grape extract, grape seed extract, luffa extract, safflower extract, peppermint extract, bodaiju extract, botan extract, hop extract, pine extract, mayonara extract, horse chestnut extract, skunk cabbage extract, soapberry extract, melissa extract, mozuku extract, peach extract, cornflower extract , eucalyptus extract, yuzu extract, lily extract, coix seed extract, mugwort extract, lavender extract, green tea extract, apple extract, rooibos tea extract, litchi extract, lettuce extract, lemon extract, forsythia extract, astragalus extract, rose extract, rosemary extract, roma Extracts such as chamomile extract, royal jelly extract and burnet extract are preferred.
The content of the animal or plant-derived extract in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 1 to 5% by mass.
The content of the animal or plant-derived extract in the food is usually 0.01 to 80% by mass, preferably 0.1 to 50% by mass, more preferably 1 to 30% by mass.

Anti-inflammatory components include clarinone, glabridin, glycyrrhizic acid, glycyrrhetinic acid, pantothenyl alcohol and the like, preferably glycyrrhizic acid and its salts, alkyl glycyrrhetinate and its salts, and glycyrrhetinic acid and its salts.
The content of the anti-inflammatory ingredient in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 1 to 5% by mass.

Polar oil, volatile hydrocarbon oil, etc. are mentioned as an oily component.
Polar oils include synthetic ester oils such as isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyldecyl dimethyloctanoate, and lactic acid. Cetyl, myristyl lactate, lanolin acetate, 2-ethylhexyl isononanoate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycol di-2-ethylhexanoate, dipentaerythritol fatty acid ester, monoisostearate N- Alkyl glycol, neopentyl glycol dicaprate, diisostearyl malate, glyceryl di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentane tetra-2-ethylhexanoate Mention may be made of erythritol, glyceryl tri-2-ethylhexanoate, trimethylolpropane triisostearate.

In addition, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, tri-2-heptyl undecanoic acid glyceride, castor oil fatty acid methyl ester, oleic oil, cetostearyl alcohol, acetoglyceride, palmitic acid 2 -heptylundecyl, diisobutyl adipate, N-lauroyl-L-glutamic acid-2-octyldodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyl myristate Decyl, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebacate, 2-ethylhexyl succinate, ethyl acetate, butyl acetate, amyl acetate, triethyl citrate, octyl methoxycinnamate and the like.

In addition, as natural oils, avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg yolk oil, sesame oil, persic oil, wheat germ oil, sasanqua oil, castor oil, linseed oil, Safflower oil, cottonseed oil, perilla oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, sinagiri oil, Japanese pungent oil, jojoba oil, germ oil, triglycerin, glyceryl trioctanoate, glyceryl triisopalmitate etc.

Volatile hydrocarbon oils include isododecane, isohexadecane and the like.

Surfactants include fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, anionic surfactants such as alkyl sulfate triethanolamine ether, stearyltrimethylammonium chloride, benzalkonium chloride, laurylamine oxide. cationic surfactants such as betaine surfactants (alkylbetaine, amidobetaine, sulfobetaine, etc.), imidazoline amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy 2 sodium salts, etc.), amphoteric surfactants such as acylmethyl taurine,
Sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acid esters (glyceryl monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl Ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbit fatty acid esters (POE-sorbit monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoiso stearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), Pluronic types, POE/POP alkyl ethers (POE/POP 2-decyltetradecyl ether, etc.), tetronics, POE castor oil/hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, alkyl glucosides, etc. nonionic surfactants, and the like.

Polyhydric alcohols include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4 -hexylene glycol, 1,2-hexanediol, 1,2-octanediol and the like.

Thickeners include guar gum, quince seed, carrageenan, galactan, gum arabic, pectin, mannan, starch, xanthan gum, curdlan, methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, methylhydroxypropylcellulose, chondroitin sulfate, dermatan sulfate, glycogen, Heparan sulfate, hyaluronic acid, sodium hyaluronate, tragacanth gum, keratan sulfate, chondroitin, mucoitin sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, dextran, kerato sulfate, locust bean gum, succinoglucan, caroninic acid, chitin, chitosan, carboxymethyl Chitin, agar, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, alkyl-modified carboxyvinyl polymer, sodium polyacrylate, polyethylene glycol, bentonite and the like.

As the powders, powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, and barium sulfate, which may be surface-treated, may be used. Inorganic pigments such as red iron oxide, yellow iron oxide, black iron oxide, cobalt oxide, ultramarine blue, Prussian blue, titanium oxide, and zinc oxide that may be treated, mica titanium that may be treated, fish phosphorus foil, Pearl agents such as bismuth oxychloride, optionally laked Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5, Red No. 505, Red No. 230, Red No. 223 No., Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, Red No. 204 and other organic pigments, polyethylene powder, polymethyl methacrylate, nylon powder, and organic powders such as organopolysiloxane elastomers.

Examples of UV absorbers include para-aminobenzoic acid UV absorbers, anthranilic acid UV absorbers, salicylic acid UV absorbers, cinnamic acid UV absorbers, benzophenone UV absorbers, sugar UV absorbers, 2-(2 UV absorbers such as '-hydroxy-5'-t-octylphenyl)benzotriazole, 4-methoxy-4'-t-butyldibenzoylmethane, and the like.

Cosmetics can be produced by treating and blending the aforementioned ingredients according to conventional methods.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

<Reference Example 1> Effect of facial pressure on endoplasmic reticulum stress in epidermal cells and dermal fibroblasts 500 μL of normal human epidermal cells suspended in HuMedia KG2 medium and Dulbecco's Modified Eagle medium containing 10% Fatal Bovine Serum 500 μL of normal human dermal fibroblasts suspended in , were seeded (200,000 cells/mL) in silicon stretch chambers (STB-CH-4W, Strex Inc.). After culturing at 37° C., the cells were cultured while repeatedly applying a contractile pressure load (approximately 20% contraction) at a frequency of 1 Hz for 3 hours. After culturing, cells were collected using RLT Buffer (Qiagen), and RNA was extracted using RNeasy MiniKit (Qiagen). RT-PCR was performed by a standard method using SuperScript VILO cDNA Synthesize Kit (Thermo Fisher Scientific Co., Ltd.) and QuantiTect SYBR Green PCR Kit (Qiagen Co., Ltd.) to measure the amount of mRNA of the CHOP gene. For comparison, the amount of CHOP gene mRNA (control) in epidermal cells and dermal fibroblasts cultured in a chamber without application of contractile pressure was also measured.

FIG. 1 shows the relative values of the mRNA expression level of the CHOP gene when the control mRNA expression level is set to "1". Both epidermal cells and dermal fibroblasts showed an increase in the expression level of CHOP due to the application of continuous contraction pressure, suggesting that the application of facial pressure induces endoplasmic reticulum stress.

<Reference Example 2> Effect of endoplasmic reticulum stress on protein production 500 μL of normal human epidermal cells and normal human dermal fibroblasts were seeded in a 24-well plate (50000 cells/well of epidermal cells/well, 30000 cells/well of dermal fibroblasts). well), epidermal cells in HuMedia KG2 medium, dermal fibroblasts in Dulbecco's Modified medium containing 10% Fatal Bovine Serum
Each was cultured at 37° C. for 12 hours in Eagle medium. After culturing, tunicamycin (TUM) was added to a final concentration of 400 ng/ml for epidermal cells and tunicamycin (TUM) to a final concentration of 100 ng/ml for dermal fibroblasts.
After that, it was cultured at 37° C. for 24 hours. After culturing, the cells were collected using a trypsin solution, and the number of cells was counted by a conventional method.
In addition, the collected cells were dissolved in a buffer of DC Protein Assay (manufactured by Bio-Rad), a protein amount measuring reagent, and the protein concentration was measured by a conventional method using a plate reader to determine the amount of protein per cell.
For comparison, the amount of protein per cell in epidermal cells and dermal fibroblasts cultured without adding tunicamycin (control) was also measured.

FIG. 2 shows the protein amount per cell when tunicamycin was added as a relative value when the control was set to "100". In both epidermal cells and dermal fibroblasts, the amount of protein per cell when tunicamycin was added decreased compared to when it was not added. These results indicated that endoplasmic reticulum stress decreased the amount of intracellular protein.

<Reference Example 3> Identification of proteins whose expression level is reduced by endoplasmic reticulum stress 500 μL of normal human epidermal cells and normal human dermal fibroblasts were each seeded in a 24-well plate (50000 cells/well of epidermal cells, dermal fibroblasts 30000 cells/well), epidermal cells in HuMedia KG2 medium, dermal fibroblasts in Dulbecco's Modified medium containing 10% Fatal Bovine Serum
Each was cultured at 37° C. for 12 hours in Eagle medium. After incubation, tunicamycin (TUM) at a final concentration of 1000 ng/ml was added to each.
After that, it was cultured at 37° C. for 24 hours. After culturing, cells were collected using RLT Buffer (Qiagen), and RNA was extracted using RNeasy MiniKit (Qiagen). SurePrint G3 Human GE microarray 8x60K Ver. 3.0 (Agilent Co.) was used to carry out a DNA microarray according to a conventional method, and comprehensively analyzed the mRNA expression levels of the genes.
For comparison, the mRNA expression levels of genes in epidermal cells and dermal fibroblasts cultured with the same amount of dimethyl sulfoxide (DMSO) added instead of tunicamycin were also analyzed (control).

Fig. 3 shows the relative expression level of each gene when tunicamycin was added to the expression level of each gene when tunicamycin was not added, with respect to the genes for which a significant difference in expression level was observed. indicate a value.

In epidermal cells, the mRNA expression levels of HJURP gene, UHRF1 gene, MKI67 gene, and CDCA3 gene decreased when tunicamycin was added compared to when tunicamycin was not added. All of these genes are related to cell proliferation, and it is known that an increase in the amount of change in these genes promotes cell proliferation.

In dermal fibroblasts, the mRNA expression levels of COL3A1 gene, COL4A1 gene, COL11A2 gene, COL5A3 gene, COL10A1 gene, COL27A1 gene, COL13A1 gene, and COL6A6 gene were higher when tunicamycin was added than when tunicamycin was not added. decreased. All of these genes are genes related to collagen production, and it is known that the expression of these genes promotes collagen production.

The above results showed that endoplasmic reticulum stress decreased the expression level of cell growth-related factors in epidermal cells and decreased the expression level of collagen in dermal fibroblasts.

<Example 1> Screening of test substances that reduce endoplasmic reticulum stress when endoplasmic reticulum stress is induced By the following procedure, anti-aging agents were screened using reduction of endoplasmic reticulum stress as an indicator.
500 μL of normal human epidermal cells and normal human dermal fibroblasts were each seeded in a 24-well plate (50000 cells/well of epidermal cells, 30000 cells/well of dermal fibroblasts), the epidermal cells were HuMedia KG2 medium, and the dermal fibroblasts were is Dulbecco's Modified with 10% Fatal Bovine Serum
Each was cultured at 37° C. for 12 hours in Eagle medium. After culturing, tunicamycin (TUM) at a final concentration of 100 ng/ml and a test substance at a final concentration of 0.10 to 1.00% by mass were added to form a test group, and the same amount of the solvent was added in place of each test substance. was used as a control group and cultured at 37°C for 24 hours. After culturing, mRNA was extracted from the collected cells in the same manner as in Reference Example 1, RT-PCR was performed using the synthesized cDNA, and the mRNA expression level of the CHOP gene was measured.

In FIG. 4, the mRNA expression level of the CHOP gene in epidermal cells and dermal fibroblasts is a relative value when the same expression level in epidermal cells and dermal fibroblasts without the test substance added (control) is set to "1". Indicated.
In epidermal cells, when the saxifrage extract at a final concentration of 0.50% by mass was added, it was found that the expression of the CHOP gene was suppressed in 74.5% of the control, and this extract imparted facial pressure. It was suggested that it has the effect of reducing the endoplasmic reticulum stress at the time of aging, resulting in an anti-aging effect.
In the dermal fibroblasts, it was found that the expression of the CHOP gene was suppressed in 41.6% of the control when the larvae extract was added at a final concentration of 0.50% by mass. It was suggested that it has the effect of reducing endoplasmic reticulum stress when applied, and as a result exhibits an anti-aging effect.

<Example 2> Screening for test substances that reduce endoplasmic reticulum stress when endoplasmic reticulum stress is induced or not In the following procedure, anti-aging agents were screened using reduction of endoplasmic reticulum stress as an index.
500 μL of normal human epidermal cells and normal human dermal fibroblasts were each seeded in a 24-well plate (50000 cells/well of epidermal cells, 30000 cells/well of dermal fibroblasts), the epidermal cells were HuMedia KG2 medium, and the dermal fibroblasts were is Dulbecco's Modified with 10% Fatal Bovine Serum
Each was cultured at 37° C. for 12 hours in Eagle medium. After culturing, each test substance at the final concentration shown in Table 1 was added to the epidermal cells and dermal fibroblasts to form each test group, and 50% by mass of 1,3-butylene glycol was added in place of the test substance. A group to which the same amount was added was used as a control group, which was cultured at 37°C for 24 hours. However, in the test group in which the corn extract was added as the test substance to the dermal fibroblasts, tunicamycin (TUM) at a final concentration of 100 ng/ml was also added at the same time as tunicamycin was added.
After culturing, mRNA was extracted from the collected cells in the same manner as in Reference Example 1, RT-PCR was performed using the synthesized cDNA, and the mRNA expression level of the CHOP gene was measured.

FIG. 5 shows the mRNA expression level of CHOP gene in epidermal cells as a relative value when the same expression level in epidermal cells to which no test substance was added (control) was defined as "1".
In addition, FIG. 6 shows the mRNA expression level of the CHOP gene in dermal fibroblasts as a relative value when the same expression level in dermal fibroblasts to which no test substance was added (control) was set to "1".

In epidermal cells, the saxifrage extract suppressed the expression of the CHOP gene in 82.5% of the control, indicating that this extract induces endoplasmic reticulum stress in epidermal cells even when endoplasmic reticulum stress is not induced. It was suggested that it has a reducing action and as a result exhibits an anti-aging effect.

In dermal fibroblasts, when endoplasmic reticulum stress was not induced, gardenia extract was 40.3%, linden extract was 49.7%, eucalyptus extract was 35.5%, assembly extract was 91.0%, saxifrage Extract: 90.1%, Reishi extract: 61.2%, Lemongrass extract: 71.3%, Lonicera caerulea juice: 59.1%, Burdock extract: 68.7%, and Birch extract: 68.0% It was confirmed that the expression of the CHOP gene was suppressed up to %. It was suggested that these test substances have the effect of reducing endoplasmic reticulum stress in dermal fibroblasts even when endoplasmic reticulum stress is not induced, resulting in anti-aging effects.
In addition, it was found that the expression of the CHOP gene was suppressed in 63.8% of the controls when the extract was added at the time of endoplasmic reticulum stress induction. Similar to Example 1, it was suggested that this extract has the effect of reducing endoplasmic reticulum stress in dermal fibroblasts when facial pressure is applied, resulting in an anti-aging effect.

INDUSTRIAL APPLICABILITY The present invention provides a novel screening method for anti-aging agents, using reduction of endoplasmic reticulum stress as an index. This makes it possible to search for materials that can be useful in the design and manufacture of anti-aging cosmetics and the like, and is industrially useful.

Claims (9)

A screening method for an anti-aging agent, wherein changes in endoplasmic reticulum stress in cells are used as an index,
A screening method, wherein the cells are epidermal cells or dermal fibroblasts. 2. The screening method of claim 1, wherein said change is reduction. adding a test substance to cells; and selecting a test substance in which the endoplasmic reticulum stress in cells to which the test substance has been added is lower than the endoplasmic reticulum stress in cells to which the test substance has not been added. , the screening method according to claim 1 or 2. 4. The screening method according to claim 3, further comprising the step of inducing endoplasmic reticulum stress in the step of adding the test substance to the cells, or before or after the step of adding the test substance to the cells. 5. The screening method according to claim 4, wherein the step of inducing endoplasmic reticulum stress is the step of adding an endoplasmic reticulum stress-inducing agent to the cells. In the step of selecting the test substance, the expression level of the endoplasmic reticulum stress marker in the cells to which the test substance was added is smaller or larger than the expression level of the stress marker in the cells to which the test substance was not added. The screening method according to any one of claims 3 to 5, which selects. In the step of selecting the test substance, a test substance is selected in which the expression level of the endoplasmic reticulum stress marker in cells to which the test substance has been added is lower than the expression level of the stress marker in cells to which the test substance has not been added. ,
The endoplasmic reticulum stress marker is CHOP,
The screening method according to any one of claims 3 to 6, wherein the expression level of the endoplasmic reticulum stress marker is the expression level of CHOP protein or the expression level of the gene encoding CHOP. The screening method according to any one of claims 1 to 7, wherein the anti-aging agent is an anti-wrinkle agent. A method of designing a composition, comprising the step of performing the screening method according to any one of claims 1 to 8, and the step of containing a substance selected by said step.

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