Proteasome activation enhancer

Abstract

To provide a novel composition for enhancing proteasome activity in tendon cells.SOLUTION: Provided is a proteasome activity enhancer in tendon cells, containing rice bran extract hydrolysate as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a proteasome activity enhancer containing a hydrolysate of a rice bran extract as an active ingredient. [Background technology]

[0002] Recent research has confirmed that intracellular proteasomes are involved in the aging of human cells. Proteasomes are distributed in both the cytoplasm and nucleus of eukaryotic cells, and are huge enzyme complexes that degrade proteins within cells. For example, proteasomes are known to degrade proteins (crosslinked, glycated, or carbonylated proteins) that have been degraded by ultraviolet rays or active oxygen, preventing the accumulation of degraded proteins within cells, and contributing to the maintenance and improvement of skin cell function. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent Publication No. 2016-124827 Summary of the Invention [Problem to be solved by the invention]

[0004] In view of the prior art, the present inventors have conducted extensive research to find a novel composition having a proteasome activity enhancing effect. As a result, they have found that a hydrolysate of rice bran extract has a proteasome activity enhancing effect in tendon cells.

[0005] That is, an objective of the present invention is to provide a novel composition that enhances proteasome activity in tendon cells. [Means for solving the problem]

[0006] The present invention, which solves the above-mentioned problems, provides a proteasome activity enhancer in tendon cells, which contains a hydrolysate of rice bran extract as an active ingredient. According to the present invention, proteasome activity can be enhanced in tendon cells.

[0007] In a preferred embodiment, the present invention is an agent for increasing the expression level of mimecan in tendon cells. According to the present invention, the expression level of mimecan in tendon cells can be increased.

[0008] In a preferred embodiment of the invention, the tendon cells are tendon cells present in the retinaculum cutis. According to the present invention, proteasome activity can be enhanced in tendon cells present in the retinaculum cutis.

[0009] In a preferred embodiment, the present invention is an agent for improving retinaculum cutis. According to the present invention, the retina cutis can be improved.

[0010] In a preferred embodiment, the present invention is an agent for improving skin conditions associated with aging. According to the present invention, it is possible to improve skin conditions associated with aging.

[0011] In a preferred embodiment of the present invention, the skin condition is one or more selected from the group consisting of increased wrinkles, sagging skin, loss of firmness, and loss of skin elasticity. According to the present invention, it is possible to improve one or more symptoms selected from the group consisting of increased wrinkles, sagging skin, loss of firmness, and loss of skin elasticity.

[0012] In a preferred embodiment, the present invention is an external skin preparation. Effect of the Invention

[0013] According to the present invention, a novel composition that enhances proteasome activity in tendon cells can be provided. [Brief description of the drawings]

[0014] [Figure 1] FIG. 13 shows the effect of a rice bran extract hydrolysate on enhancing proteasome activity in tendon cells. [Diagram 2] FIG. 1 shows the inhibition of skin support-related gene expression by inhibition of proteasome activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] <Proteasome activity enhancer in tendon cells> The proteasome activity enhancer in tendon cells of the present invention will be described in detail below.

[0016] <Active ingredients> The agent for enhancing proteasome activity in tendon cells of the present invention contains a hydrolysate of rice bran extract as an active ingredient.

[0017] There are no particular limitations on the rice bran used in the present invention, and brown rice, germinated brown rice, polished rice, processed rice, pigmented rice (black rice, purple rice, red rice, etc.), or white bran and / or red bran contained in brown rice can be used. As for the type of rice, any of non-glutinous rice, glutinous rice, etc. may be used. In addition, as the processed rice, there may be mentioned anti-allergy rice, low protein rice (e.g. low glutelin rice), enriched rice (e.g. γ-aminobutyric acid rice), etc.

[0018] In the present invention, an extract is first prepared using the above-mentioned rice bran. The preparation of the extract can be carried out by washing the rice bran with water, drying, shredding or crushing it as necessary, and then contacting it with an extraction solvent by a conventional method such as immersion, countercurrent extraction, steam distillation, subcritical extraction or supercritical extraction. The extract is then subjected to a hydrolysis treatment as described below.

[0019] Examples of extraction solvents include water; lower alcohols such as methanol, ethanol, and propanol; polyhydric alcohols such as ethylene glycol, propylene glycol, 1,3-butylene glycol, and glycerin; esters such as ethyl acetate, butyl acetate, and methyl propionate; ketones such as acetone and methyl ethyl ketone; ethers such as ethyl ether and isopropyl ether; and hydrocarbon solvents such as n-hexane, toluene, and chloroform, and these may be used alone or in combination of two or more.

[0020] Among these extraction solvents, the use of water or a mixed solvent of water and lower alcohols or water and polyhydric alcohols is preferred in the present invention, since it can be widely applied to topical skin preparations (cosmetics or quasi-drugs).

[0021] In the present invention, when preparing the rice bran extract solution, it is preferable to adjust the pH appropriately using an alkali adjuster or acidity adjuster. Examples of alkali adjusters include sodium salts such as sodium hydroxide and sodium carbonate, and potassium salts such as potassium hydroxide. Among these, sodium hydroxide and sodium carbonate are preferred. Examples of acidity adjusters include inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as acetic acid, lactic acid, malic acid, and citric acid.

[0022] The extract obtained by the above-mentioned treatment is subjected to a hydrolysis treatment before or after the extraction, or in parallel with the extraction. The hydrolysis treatment may be performed using an acid, an alkali, or an enzyme, and is preferably performed using an enzyme.

[0023] Enzymes used in the hydrolysis treatment include protease, amylolytic enzyme, cellulose decomposing enzyme, and lipolytic enzyme. These enzymes may be used alone or in combination depending on the plant species.

[0024] Examples of proteolytic enzymes include actinases such as actinase, pepsins such as pepsin, trypsins such as trypsin and chymotrypsin, papains such as papain and chymopapain, peptidases such as glycylglycine peptidase, carboxypeptidase, and aminopeptidase, bromelain, and conjugated protease derived from microorganisms [e.g., Neurase (manufactured by Amano Enzyme Co., Ltd.)]. When using proteolytic enzymes, any of the above enzymes may be used alone or in combination. Examples of starch-degrading enzymes that can be used include α-amylase, β-amylase, glucoamylase, and β-galactosidase. When using starch-degrading enzymes, any of the above enzymes may be used alone or in combination. Examples of cellulose-degrading enzymes include cellulase, hemicellulase, and pectinase. When using cellulose-degrading enzymes, any of the above enzymes may be used alone or in combination. Examples of lipolytic enzymes include lipase. When carrying out hydrolysis treatment with an enzyme, one or more enzymes selected from the above enzyme groups may be used in combination.

[0025] The hydrolysis treatment using enzymes is carried out by adding one or more of the enzymes described above to the rice bran extract solution described above and carrying out the enzymatic reaction under conditions near the optimal pH and optimal temperature of the enzyme used. When using a combination of two or more enzymes, the two or more enzymes may be allowed to act simultaneously or sequentially with or without changing the reaction conditions depending on the characteristics of the enzymes used. The amount of enzyme used is preferably in the range of 0.001 to 50 parts by weight per enzyme per 100 parts by weight of the solid content of the rice bran extract solution. The time of the enzymatic treatment varies depending on the type of enzyme used, but is generally in the range of 0.5 to 24 hours.

[0026] After completion of hydrolysis using the above enzymes, the enzymes are inactivated by an appropriate method, for example, by heating the enzyme-treated solution to 80° C. or higher, to obtain an enzyme-treated hydrolyzate solution.

[0027] More specifically, for example, as in the technique described in JP-A-2022-138725, a rice bran extract hydrolyzed solution obtained by the following production example can also be used. Example of rice bran extract hydrolyzate solution production 1500g of 0.1M lactic acid solution was added to 500g of rice bran, stirred to thoroughly mix the rice bran and lactic acid solution, and then left to stand at room temperature for one day. Insoluble matter was then removed by filtration, and the filtrate was treated with papain. The enzyme treatment was carried out by using 1.2mg of enzyme, at the optimum pH for the enzyme, and keeping the mixture at 80°C for one hour. The insoluble matter resulting from the treatment was filtered off, and the filtrate was adjusted to pH 6.5 with phytic acid, yielding 731g of a pale yellow, transparent rice bran extract hydrolyzed solution (solids concentration 3.7%).

[0028] In addition, the hydrolysate of the rice bran extract according to the present invention may be a commercially available product.

[0029] ≪Applications≫ The hydrolysate of rice bran extract can be used to enhance proteasome activity in tendon cells. That is, the present invention can be in the form of a proteasome activity enhancer in tendon cells, which contains a hydrolysate of rice bran extract as an active ingredient. The present invention can also be in the form of a composition for enhancing proteasome activity in tendon cells, comprising a hydrolysate of rice bran extract as an active ingredient.

[0030] Here, the method for detecting whether or not proteasome activity is enhanced in tendon cells is not particularly limited, and any method that can be appropriately selected by a person skilled in the art can be used.

[0031] As shown in the Examples below, it was found that the expression level of mimecan in tendon cells was reduced by inhibiting proteasome activity. Therefore, it is considered that the expression level of mimecan in tendon cells can be increased by enhancing proteasome activity. Therefore, the hydrolysate of rice bran extract, which has the effect of enhancing proteasome activity in tendon cells, can be used to increase the expression level of mimecan in tendon cells. Furthermore, the present invention can be in the form of a proteasome activity enhancer in tendon cells, which contains a hydrolysate of rice bran extract as an active ingredient and is intended to increase the expression level of mimecan in tendon cells.

[0032] Tendon cells are also present in the retinaculum cutis and play an important role in the structural formation of the retinaculum cutis. Therefore, in the present invention, tendon cells are preferably tendon cells present in the retinaculum cutis.

[0033] The dermal support (Retinacula cutis) plays an important role in maintaining the shape of the skin.

[0034] It is also known that the density of the retinaculum cutis correlates with age and the degree of sagging skin. Furthermore, it is known that mimecan, a protein that makes up the retinaculum, is a component that decreases with age, and that a decrease in mimecan causes wrinkles and sagging skin. Therefore, the present invention can be in the form of a proteasome activity enhancer in tendon cells for improving Retinacula cutis, which contains a hydrolysate of rice bran extract as an active ingredient.

[0035] The present invention can also be used to improve skin conditions associated with aging. That is, the present invention can be in the form of a proteasome activity enhancer in tendon cells, which contains a hydrolysate of rice bran extract as an active ingredient, for improving skin conditions associated with aging. The skin condition is preferably one or more selected from the group consisting of increased wrinkles, sagging skin, loss of firmness, and loss of skin elasticity.

[0036] ≪Dosage / Dosage≫ The agent for enhancing proteasome activity in tendon cells of the present invention can be administered to mammals, including humans.

[0037] The content of the hydrolysate of the rice bran extract of the present invention relative to the proteasome activity enhancer in the tendon cells of the present invention is typically 0.00001 mass% or more, preferably 0.0001 mass% or more, more preferably 0.0003 mass% or more, and even more preferably 0.01 mass% or more, based on the dry mass.

[0038] In addition, the content of the hydrolysate of the rice bran extract of the present invention relative to the proteasome activity enhancer in the tendon cells of the present invention is typically 80 mass% or less, preferably 50 mass% or less, more preferably 30 mass% or less, even more preferably 10 mass% or less, and even more preferably 5 mass% or less, based on the dry mass.

[0039] The agent according to the present invention is preferably in the form of an external preparation for skin, which can be produced by using known methods and devices.

[0040] Suitable examples of external skin preparations include cosmetics, quasi-drugs, and medicines, with cosmetics and quasi-drugs being more preferred since they can be used on a daily basis. Cosmetics can be processed into the form of lotions, emulsions, gels, creams, ointments, etc. Specific examples include lotions, milky lotions, creams, gels, packs, hair creams, sprays, sun care products, etc., and particularly preferred examples include lotions, milky lotions, and creams.

[0041] The content of the rice bran extract hydrolysate of the present invention in the topical skin preparation is usually 0.00001 mass% or more, preferably 0.0001 mass% or more, and more preferably 0.0003 mass% or more, based on the dry mass.

[0042] Furthermore, the content of the hydrolysate of the rice bran extract of the present invention in the topical skin preparation is usually 80% by mass or less, preferably 50% by mass or less, more preferably 30% by mass or less, more preferably 10% by mass or less, more preferably 1% by mass or less, more preferably 0.1% by mass, and even more preferably 0.05% by mass or less, based on the dry mass.

[0043] When the present invention is in the form of an external skin preparation, whitening ingredients, wrinkle-reducing ingredients, anti-inflammatory ingredients, extracts derived from animals and plants other than rice bran, and ingredients normally used in cosmetics other than the active ingredient may be blended as optional ingredients within a range that does not impair the proteasome activity enhancing effect. These optional ingredients may be commercially available and used, or may be synthesized by a known method and used. Each optional ingredient may have two or more effects (for example, a whitening effect and an anti-wrinkle effect).

[0044] When the present invention is used as an external preparation for skin, it is also preferable that the present invention be in a form labeled with the use "for enhancing proteasome activity."

[0045] The above-mentioned "representation" includes all representations that have the function of informing consumers of the above-mentioned uses. In other words, any representation that can recall or infer the above-mentioned uses falls under the above-mentioned "representation", regardless of the purpose of the representation, the content of the representation, the object or medium on which it is displayed, etc. In addition, the phrase "labeled" means that there is a labeling action intended to cause the viewer to associate the label with a skin topical agent, etc. (product).

[0046] The act of displaying the intended use is preferably one that enables consumers to directly recognize the intended use.Specific examples include the act of describing the intended use on the product or its packaging according to the present invention, and the act of describing the intended use in advertisements for the product, price lists, or transaction documents (including those provided by electromagnetic means). EXAMPLES

[0047] The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these examples.

[0048] <Test Example 1> Effect of enhancing proteasome activity Human tendon-derived tenocytes were cultured in a black 96-well plate at 8.0 × 10 3 The cells were seeded at 10 cells / well and cultured at 37°C in a 5% CO2 environment for 24 hours. 24 hours after seeding, hydrolysate of rice bran extract was added to a final concentration of 0.5%, and the culture was continued for another 24 hours.

[0049] Then, the cell culture supernatant was removed and each well was washed with Tris-HCL buffer ((pH 8.0): 12 mM trishydroxymethylaminomethane, 5 mM EDTA, 150 mM NaCl, hydrochloric acid). Then, 100 μL of cell lysis solution (Tris-HCL buffer (pH 8.0) containing 1% TritonX-100: 12 mM trishydroxymethylaminomethane, 5 mM EDTA, 150 mM NaCl, hydrochloric acid) was added to the wells for measuring total substrate degradation ability and 90 μL to the wells for measuring non-proteasome activity, and the cells were disrupted to prepare a crude enzyme solution. 10 μL of proteasome activity inhibitor solution (cell lysis solution containing 2 mM epigallocatechin gallate) was added to the wells for measuring non-proteasome activity, and the wells were left to stand at room temperature for 10 minutes.

[0050] 10 μL of a buffer containing 65 μM Suc-Leu-Leu-Val-Tyr-AMC as a substrate was added to each plate, and the mixture was allowed to react for 1 hour at 37° C. After the reaction was completed, the fluorescence intensity (excitation wavelength: 355 nm, fluorescence wavelength: 460 nm) was measured using a fluorescent plate reader (ARVO X4, PerkinElmer).

[0051] Similarly, a black plate was prepared without cell disruption treatment, to which the extract was added, and the cell supernatant was removed and each well was washed with PBS(-). 100 μL of PBS containing Hoechst33342 was added and reacted at 37°C for 30 minutes. After the reaction was completed, the fluorescence intensity (excitation wavelength: 355 nm, fluorescence wavelength: 460 nm) was measured using a fluorescent plate reader (ARVO X4, PerkinElmer) to measure the fluorescence intensity due to DNA staining.

[0052] The non-proteasome activity measurement results were subtracted from the total substrate degradation measurement results, and the value obtained was standardized by the fluorescence intensity of DNA staining to obtain the proteasome activity. The same procedure as above was also carried out for the case where no sample was added but PBS(-) was added (Control), and the relative value of the proteasome activity when each sample was added to the proteasome activity obtained here was calculated, and this was defined as the proteasome activity rate (%). The results are shown in Table 1.

[0053] [Table 1]

[0054] As shown in Table 1, it was confirmed that the hydrolysate of the rice bran extract of the present invention has the effect of enhancing proteasome activity in tendon cells.

[0055] In addition, in the present invention, the proteasome activity enhancing effect was confirmed in tendon cells cultured for 24 hours after the addition of the hydrolysate of rice bran extract. Therefore, it was found that the hydrolysate of rice bran extract according to the present invention can maintain the proteasome activity enhancing effect in tendon cells for a long period of time.

[0056] Furthermore, in the present invention, the rice bran extract hydrolysate was added to a final concentration of 0.5% and the proteasome activity enhancing effect was confirmed in tendon cells cultured. Therefore, it was found that the rice bran extract hydrolysate according to the present invention has the proteasome activity enhancing effect in tendon cells even at a low concentration.

[0057] <Test Example 2> Inhibition of skin support-related gene expression by inhibiting proteasome activity Human tendon-derived tenocytes were cultured in a collagen I-coated 24-well plate at 3.0 × 10 4 The cells were seeded at 10 cells / well and cultured at 37°C in a 5% CO2 environment for 24 hours. 24 hours after seeding, MG-132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, a proteasome activity inhibitor) diluted in DMSO was added to the cells at 100 nM, and the cells were further cultured for 72 hours, after which RNA was collected from the cells. The control (NC) was subjected to the same conditions as above except that MG-132 was not added.

[0058] The collected RNA was analyzed using real-time PCR to evaluate the expression of the OGN gene (mimecan-related gene). Specifically, the gene expression level in cells to which MG-132 was not added was used as a standard, and the gene expression level after the addition of MG-132 was calculated. Table 2 shows the ratio of the gene expression level after the addition of MG-132, assuming that the gene expression level in cells to which MG-132 was not added is "1.00."

[0059] [Table 2]

[0060] As shown in Table 2, it was confirmed that the expression of the OGN gene in tendon cells was inhibited by inhibiting proteasome activity. This shows that the expression level of mimecan in tendon cells can be increased by enhancing proteasome activity. [Industrial Applicability]

[0061] According to the present invention, a composition capable of enhancing proteasome activity can be provided.

Claims

1. A proteasome activity enhancer in tendon cells, containing the active ingredient hydrolysate of rice bran extract.

2. The proteasome activity enhancer according to claim 1 for increasing the expression level of mimecan in tendon cells.

3. The proteasome activity enhancer according to claim 1 or 2, wherein the tendon cells are tendon cells present in a retinacula cutis.

4. The proteasome activity enhancer according to claim 1 or 2, for improving retinacula cutis.

5. The proteasome activity enhancer according to claim 1 or 2, for improving skin conditions associated with aging.

6. The proteasome activity enhancer according to claim 1 or 2, wherein the skin condition is one or more selected from the group consisting of increased wrinkles, sagging skin, loss of firmness, and loss of skin elasticity.

7. The proteasome activity enhancer according to claim 1 or 2, which is an external preparation for skin.

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