Cytoskeleton repair agent

Abstract

To provide a composition for repairing a cytoskeletal disorder.SOLUTION: A cytoskeleton repair agent contains at least one component selected from sweet almond oil, a Rosa rugosa flower extract, hazelnut oil, and a Ruscus aculeatus extract as an active ingredient. The Rosa rugosa flower extract and the Ruscus aculeatus extract are water-soluble. The cytoskeleton repair agent is applied to cells having a cytoskeletal disorder caused by the presence of cortisol, and rearranges filamentous actin present in the cells, thereby repairing the cytoskeleton, wherein the cells are human dermal fibroblasts.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cytoskeleton repair agent for repairing a disordered cytoskeleton and a screening method thereof. [Background technology]

[0002] For maintaining cell functions, it is important to maintain normal cytoskeletal morphology.

[0003] Actin is a type of protein that constitutes the eukaryotic cytoskeleton, and its polymerization state affects the stability and mobility of the cytoskeleton. Using this, methods have been developed to induce cell death by promoting actin polymerization for the treatment of tumors. For example, Patent Document 1 describes peptides and polypeptides that induce actin polymerization in cells to cause cell growth inhibition and / or cell death. It also describes a method for treating tumors by introducing this peptide or polypeptide into cells to induce actin polymerization in cells and cause growth inhibition and / or cell death. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2004-516006 Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a technique for repairing cytoskeletal disorders.

[0006] However, as mentioned above, only the induction of actin polymerization has been investigated so far, and no research has been conducted on techniques for rearranging actin that has been polymerized within cells. [Means for solving the problem]

[0007] The present invention, which solves the above-mentioned problems, provides a cytoskeleton repair agent comprising at least one component selected from sweet almond oil, rosehip flower extract, hazelnut oil, and butcher's broom extract. According to the present invention, a disturbed cytoskeleton can be repaired.

[0008] In a preferred embodiment of the present invention, the cytoskeleton repair agent contains at least one component selected from sweet almond oil, rosehip flower extract, hazelnut oil, and butcher's broom extract in a total amount of 0.05% by mass or more based on the entire cytoskeleton repair agent. According to the present invention, a disturbed cytoskeleton can be repaired.

[0009] In addition, the present invention, which solves the above-mentioned problems, is a screening method for cytoskeletal repair components, which includes an application step of applying a test substance to a model cell with a disrupted cytoskeleton, and a selection step of selecting a test substance that has been shown to have the effect of repairing the cytoskeleton as a cytoskeletal repair component. According to the present invention, it is possible to screen for components capable of repairing a disordered cytoskeleton.

[0010] In a preferred embodiment of the present invention, a test substance that improves the uniformity of intracellular actin distribution is selected as the cytoskeleton repair component in the selection step. Effect of the Invention

[0011] According to the present invention, it is possible to provide a cytoskeleton repair agent that repairs cytoskeleton disorders and a screening method for the same. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows that the presence of cortisol disrupts the cytoskeleton. [Diagram 2] FIG. 1 shows actin distribution when model cells with disrupted cytoskeleton were cultured in the presence of a water-soluble test substance. [Diagram 3] FIG. 1 shows actin distribution when model cells with disrupted cytoskeleton were cultured in the presence of an oil-soluble test substance. [Figure 4] FIG. 1 shows the concentration dependence of the cytoskeleton repair ability of cytoskeleton repair components. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the present invention, "cytoskeleton disturbance" means that the distribution of actin within the cell becomes uneven due to internal and / or external factors, causing changes in the cytoskeleton, resulting in a loss of cell morphology compared to that of normal cells. Here, the term "internal factor" refers to a factor that promotes actin polymerization due to a phenomenon occurring within the cell, such as the excessive production of a particular substance. Furthermore, an "external factor" is a factor that promotes actin polymerization due to a stimulus from outside the cell, such as excessive secretion of a particular substance.

[0014] In the present invention, "repairing the cytoskeleton" means bringing the disordered cytoskeleton closer to the state of the cytoskeleton of a normal cell.

[0015] For example, when actin polymerizes within a cell and becomes distributed in the form of thick filaments, the cytoskeleton becomes disorganized. In addition, the cytoskeleton can be repaired by rearranging the thick filamentous actin present in the cell.

[0016] The cytoskeleton repair agent of the present invention contains at least one component selected from sweet almond oil, rosehip flower extract, hazelnut oil, and butcher's broom extract. The above components may be used alone or in combination of two or more.

[0017] The above-mentioned components may be extracted by known methods from plants that grow wild or are cultivated in Japan, or from plants that are sold in Japan as raw materials for herbal medicines, or commercially available extracts may be purchased and used.

[0018] Of the above components, when sweet almond oil and hazelnut oil are extracted, they can be extracted by known methods such as low-temperature pressing (cold pressing), high-temperature pressing, and solvent extraction.

[0019] Among the above-mentioned components, when extracting the rosehip flower extract and butcher's broom extract, they can be extracted by the following method. The raw materials for extraction, rugosa rose flowers and butcher's blooms (which may be any of above-ground parts, rhizomes, seeds, fruits, and petals), are preferably crushed or chopped in advance to improve extraction efficiency. The extract is prepared by adding 1 to 30 parts by mass of a solvent to 1 mass of the raw material for extraction or its dried product, and immersing for several days at room temperature or for several hours at a temperature near the boiling point. After immersion, the mixture is cooled to room temperature and insoluble matter is removed as desired to obtain a rugosa rose flower extract and butcher's bloom extract.

[0020] As the extraction solvent for extracting the rosehip flower extract and the butcher's broom extract, a polar solvent is preferable, and suitable examples thereof include water, alcohols such as ethanol, isopropyl alcohol, butanol, polyhydric alcohols such as 1,3-butanediol, polypropylene glycol, ketones such as acetone, methyl ethyl ketone, and ethers such as diethyl ether, tetrahydrofuran, etc. Only one type of extraction solvent may be used, or two or more types may be used in combination.

[0021] When the cytoskeleton repair agent of the present invention is an oil-based agent, it is preferable to use sweet almond oil and / or hazelnut oil.On the other hand, when the agent is a water-based agent, it is preferable to use Rugosa rose flower extract and / or butcher's broom extract.

[0022] At least one component selected from the above sweet almond oil, rosehip flower extract, hazelnut oil, and butcher's broom extract is preferably present in a total amount of 0.05% by mass or more, more preferably 0.1% by mass or more, based on the entire cytoskeleton repair agent. By adjusting the content of the above component to be equal to or higher than the lower limit, a cytoskeleton repair effect can be expected. In addition, the upper limit of the content of at least one component selected from the above sweet almond oil, rosehip flower extract, hazelnut oil, and butcher's broom extract in the entire cytoskeleton repair agent can be set appropriately depending on the dosage form, etc., but is preferably 3 mass% or less, and more preferably 1 mass% or less.

[0023] The cytoskeleton repair agent of the present invention can be used to improve the function of cells.

[0024] The cytoskeleton repair agent of the present invention is preferably in the form of an external preparation. Suitable examples of external preparations include cosmetics, quasi-drugs, and pharmaceuticals. Furthermore, physical properties such as fluidity and viscoelasticity can be appropriately adjusted depending on the form.

[0025] The cytoskeleton repair agent of the present invention is preferably used as a cosmetic. Examples of the cosmetic include skin care cosmetics such as lotion and milky lotion, body care cosmetics such as body lotion and body cream, and UV care cosmetics such as sunscreen. For the purpose of repairing the cytoskeleton, it is preferable to use the cosmetic as a skin care cosmetic that is used continuously.

[0026] When the cytoskeleton repair agent of the present invention is used as a cosmetic, ingredients that are usually used in cosmetics can be blended therein as long as they do not impair the effects of the present invention. For example, whitening ingredients, wrinkle improving ingredients, anti-inflammatory ingredients, etc. can be included.

[0027] The whitening ingredient is not particularly limited as long as it is one that is generally used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethyl ascorbic acid, tranexamic acid, arbutin, 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2-(triphenylmethyloxy)ethanol, 2-(triphenylmethylamino)ethanol, 2-(triphenylmethyloxy)ethylamine, triphenylmethylamine, triphenylmethanol, triphenylmethane and aminodiphenylmethane, N-(o-toluoyl)cysteic acid, N-(m-toluoyl)cysteic acid, N-(p-toluyl)cysteic acid, N-(p-methoxybenzoyl)cysteic acid, N-benzoyl-serine, N-(p-methyl)-cysteic acid ... Benzoyl)serine, N-(p-ethylbenzoyl)serine, N-(p-methoxybenzoyl)serine, N-(p-fluorobenzoyl)serine, N-(p-trifluoromethylbenzoyl)serine, N-(2-naphthoyl)serine, N-(4-phenylbenzoyl)serine, N-(p-methylbenzoyl)serine methyl ester, N-(p-methylbenzoyl)serine ethyl ester, N-(2-naphthoyl)serine methyl ester, N-benzoyl-O-methylserine, N-(p-methylbenzoyl)-O-methylserine, N-(p-methylbenzoyl)-O-acetylserine, N-(2-naphthoyl)-O-methylserine, panthenol, niacinamide, etc. These whitening ingredients can be either commercially available or synthetic. The content of the whitening ingredient is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, and more preferably 0.01 to 5% by mass (in the case of an extract, the dry mass) based on the total mass of the cosmetic.

[0028] The wrinkle improving ingredient is not particularly limited as long as it is one that is generally used in cosmetics. For example, vitamin A or its derivatives include retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoate, retinol palmitate, retinol acetate, ursolic acid benzyl ester, ursolic acid phosphate, betulinic acid benzyl ester, benzilic acid phosphate, trifluoroisopropyloxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate Na, and niacinamide. The content of the wrinkle improving ingredient is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, and more preferably 0.01 to 5% by mass (in the case of an extract, dry mass) based on the total amount of the cosmetic.

[0029] Examples of anti-inflammatory ingredients include clarinone, glabridin, glycyrrhizinic acid, glycyrrhetinic acid, pantothenyl alcohol, etc., and preferable examples include glycyrrhizinic acid and its salts, alkyl glycyrrhetinates and their salts, as well as glycyrrhetinic acid, niacinamide, tranexamic acid and its salts, etc. The content of the anti-inflammatory ingredient is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass (dry mass in the case of an extract) based on the total mass of the cosmetic.

[0030] In addition to the above-mentioned whitening, wrinkle improving and anti-inflammatory ingredients, extracts derived from animals and plants (excluding sweet almond oil, rosehip flower extract, hazelnut oil and butcher broom extract), moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid and sodium lactate, 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, inorganic pigments such as cobalt oxide, ultramarine, Prussian blue and zinc oxide which may be surface-treated, composite pigments such as iron oxide titanium dioxide sintered body which may be surface-treated, pearling agents such as titanium mica, fish phosphorus foil and bismuth oxychloride which may be surface-treated, Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5 and Red No. 50 which may be laked. organic pigments such as No. 5, Red 230, Red 223, Orange 201, Red 213, Yellow 204, Yellow 203, Blue 1, Green 201, Purple 201, and Red 204; organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomer; lower alcohols such as ethanol and isopropanol; vitamin A or a derivative thereof, vitamin B group such as vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or a derivative thereof, vitamin B12, and vitamin B15 or a derivative thereof; vitamin E group such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate; vitamin D group, vitamin H, pantothenic acid, pantethine, and pyrroloquinoline quinone.

[0031] The present invention also relates to a method for screening for cytoskeletal repair components. The screening method of the present invention includes an application step of applying a test substance to a model cell with a disrupted cytoskeleton, and a selection step of selecting a test substance that has been shown to have the effect of repairing the cytoskeleton as a cytoskeleton repair component.

[0032] The term "model cells with a disrupted cytoskeleton" refers to cells in a state where the "cytoskeleton is disrupted" as defined above, i.e., cells in which actin has polymerized into thick filaments due to internal and / or external factors and the distribution of actin within the cells has become uneven.

[0033] A "model cell with a disrupted cytoskeleton" can be obtained, for example, by adding a component that causes non-uniform distribution of actin in a commercially available cell. Furthermore, the type of cells used in the screening of the present invention is not particularly limited, and for example, normal human fibroblasts, normal human keratinocytes, etc. can be used.

[0034] For example, it is known that the presence of cortisol causes uneven distribution of actin and disrupts the cytoskeleton (for example, "Effects of Hydrocortisone on the Function and Morphology of Cultured Human Skin Fibroblasts - In Particular, Effects on Proliferative Ability, Cytoskeleton, and Fibronectin -", Egawa Masaaki, Journal of the Japanese Dermatological Association, Vol. 96, No. 12, 1986, pp. 1259-1273). Therefore, model cells with disrupted cytoskeleton can be obtained by culturing cells in a medium containing cortisol. When cortisol is used, the above-mentioned model with a disrupted cytoskeleton can be obtained by culturing cells in a medium containing cortisol at a concentration of, for example, 50 μg / mL.

[0035] In the above-mentioned application step, the test substance is applied to a previously prepared model cell having a disrupted cytoskeleton. There are no particular limitations on the test substance, and extracts extracted from animals and plants, and synthesized compounds can be used as test substances for screening.

[0036] The application step may be carried out in any manner that allows the test substance to act on the model cells having a disrupted cytoskeleton. For example, the application step may be carried out by culturing the model cells having a disrupted cytoskeleton in a medium containing the test substance. The application step can also be carried out by culturing model cells with a disrupted cytoskeleton in a medium containing no test substance and administering the test substance at an appropriate timing.

[0037] In the selection step, a test substance that is found to have an effect of repairing the cytoskeleton when applied to a model cell with a disturbed cytoskeleton is selected as a cytoskeleton repair component. For example, when the actin distribution within cells is observed after application of a test substance, if the uniformity of the actin distribution is improved compared to a model cell with a disrupted cytoskeleton, the test substance can be selected as a cytoskeleton repair component.

[0038] Observation of intracellular actin distribution can be carried out by known methods, for example, by fixing cells by known methods, staining intracellular actin, and then observing the cells under a microscope.

[0039] When observed under a microscope, the actin distribution in a model cell with a disrupted cytoskeleton is compared with the actin distribution in the cells after administration of the test substance, and if the cells after administration of the test substance have less thick fibrous actin, it can be determined that the uniformity of the actin distribution has been improved. EXAMPLES

[0040] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0041] <Test Example 1> In this test example, it was verified that cortisol causes uneven distribution of actin and disrupts the cytoskeleton.

[0042] The test method is as follows. (1) Normal human dermal fibroblasts (NHDFs) were cultured at 5.0 × 10 3The cells were seeded in the chamber at 20 cells / well and cultured for 24 hours in D-MEM medium (1 mL / well) containing 10% FBS and antibiotics. (2) The medium was removed, and 500 μL of fresh cortisol-containing medium (cortisol concentrations: 0, 1, 10, and 50 μg / mL) was added per well and cultured for 24 hours. Cortisol was added by diluting stock cortisol solution with 100% ethanol to the designated concentration. (3) The medium was removed, the cells were washed with PBS(-), and then fixed with 4% paraformaldehyde (room temperature, 30 minutes). (4) After washing with PBS(-), 0.5% TritonX-100 / PBS was added and the mixture was allowed to stand at room temperature for 10 minutes. (5) 10% Block Ace was added and allowed to stand for 30 minutes, after which antibody was added and allowed to stand at room temperature for 30 minutes. (6) After washing with PBS(-) (5-minute wash x 3 times), the plate was washed twice with ultrapure water. (7) Actin was stained with phalloidin, rhodamine conjugate, and Actin-stain 535 (Cosmo Bio Co., Ltd.), and cell nuclei were stained with DAPI Fluoromount-G (registered trademark), respectively. After mounting and drying, the uniformity of actin distribution within the cells was observed under a microscope. The photographs taken under a microscope are shown in FIG.

[0043] As shown in FIG. 1, when cells were cultured in a medium without added cortisol (cortisol concentration: 0 μg / mL), actin was observed to be distributed within the cells without any specific directionality. On the other hand, when cells were cultured in a medium with a cortisol concentration of 50 μg / mL, thick striped actin with a specific orientation was observed to be distributed within the cells. It can also be seen that the boundary between inside and outside the cell is becoming blurred. This test example demonstrated that actin is distributed in the form of thick fibers within cells, causing disruption of the cytoskeleton.

[0044] <Test Example 2> In this test example, a test substance was administered to model cells with a disturbed cytoskeleton, and the uniformity of actin distribution was observed to screen for cytoskeleton repair components.

[0045] The test method is as follows. (1) Normal human dermal fibroblasts, 5.0 × 10 3 The cells were seeded in the chamber at 37.2 × 10 cells / well and cultured for 24 hours in D-MEM medium (1 mL / well) containing 10% FBS and antibiotics. (2) The medium was removed, and 500 μL of cortisol-containing medium (cortisol concentration: 50 μg / mL) was added per well and cultured for 24 hours. (3) The medium was removed, and 500 μL of medium containing the test substance was added per well and cultured for 24 hours. The concentration of the test substance was determined based on the cytotoxicity of the test substance. (4) The medium was removed, the cells were washed with PBS(-), and then fixed with 4% paraformaldehyde (room temperature, 10 minutes). (5) After washing with PBS(-), 0.5% TritonX-100 / PBS was added and the plate was left to stand at room temperature for 10 minutes. (6) 10% Block Ace was added and allowed to stand for 30 minutes, after which antibody was added and allowed to stand at room temperature for 30 minutes. (7) After washing with PBS(-) (5 minutes x 3 times), the plate was washed twice with ultrapure water. (8) Actin is conjugated with phalloidin and rhodamine conjugate. The cell nuclei were stained with Actin-stain 535 (Cosmo Bio Co., Ltd.), DAPI, Fluoromount-G (registered trademark), and then mounted and dried, and the uniformity of intracellular actin distribution was observed under a microscope. Photographs taken under a microscope are shown in FIG. 2 and FIG.

[0046] As shown in Figure 2, in cells cultured in medium containing rosehip flower extract and butcher's broom extract, the thick fibrous actin seen in cells cultured in a cortisol-containing medium (model cells with a disrupted cytoskeleton) was reduced, and the uniformity of actin distribution within the cells was improved. On the other hand, in cells cultured in media containing other test substances, thick filamentous actin was not reduced, and there was no change in the uniformity of actin distribution within the cells.

[0047] As shown in Figure 3, in cells cultured in medium supplemented with sweet almond oil and hazelnut oil, the thick filamentous actin seen in cells cultured in a cortisol-containing medium (model cells with a disrupted cytoskeleton) was reduced, and the uniformity of actin distribution within the cells was improved. On the other hand, in cells cultured in media containing other test substances, thick filamentous actin was not reduced, and there was no change in the uniformity of actin distribution within the cells.

[0048] As described above, rosehip flower extract, butcher's broom extract, sweet almond oil, and hazelnut oil improve the uniformity of actin distribution within cells, and therefore the above four components were selected as cytoskeleton repair components.

[0049] <Test Example 3> In this test example, the concentration dependency of the cytoskeleton repair ability of Rugosa rose flower extract, butcher's broom extract, sweet almond oil, and hazelnut oil, which were selected as cytoskeleton repair components in Test Example 2 above, was tested.

[0050] In (3) of the above Test Example 2, the test substances were rosehip flower extract, butcher's broom extract, sweet almond oil and hazelnut oil, and the concentrations of each component in the culture medium were 0%, 0.05%, 0.1% and 0.3%, respectively. Except for this, the uniformity of actin distribution within the cells was evaluated in the same manner as in Test Example 2. The results are shown in Figure 4.

[0051] As shown in Figure 4, it was found that the rosehip flower extract and sweet almond oil at 0.05% or more, and the butcher's broom extract and hazelnut oil at 0.1% or more, showed the effect of improving the uniformity of actin distribution within the cells. [Industrial Applicability]

[0052] According to the present invention, it is possible to provide a composition that repairs cytoskeletal disorders and a screening method for the same.

Claims

[Claim 1] Contains at least one active ingredient selected from sweet almond oil, rosehip flower extract, hazelnut oil, and butcher's broom extract, The above-mentioned Rugosa rose flower extract and the above-mentioned butcher's broom extract are water-soluble, It is applied to cells whose cytoskeleton has been disrupted by the presence of cortisol, and repairs the cytoskeleton by rearranging the filamentous actin present in the cells. A cytoskeleton repair agent, wherein the cells are human dermal fibroblasts.

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