Proteasome activation enhancer

Abstract

To provide a novel functional material with proteasome activity enhancement effect, consisting of natural product-derived ingredients.SOLUTION: The present invention is a proteasome activity enhancer comprising, as active ingredients, one or two kinds of an enzymatic hydrolysate of a rice bran extract and an enzymatic hydrolysate of a rice leaf extract.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a proteasome activity enhancer containing, as an active ingredient, an enzymatic hydrolysate of a rice bran extract or an enzymatic hydrolysate of rice leaves. [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. 2000-264834 [Patent Document 2] Patent Publication No. 2013-103906 DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0004] In view of the prior art, the present inventors have conducted extensive research to find a naturally occurring component that enhances proteasome activity, and as a result have found that an enzymatic hydrolysate of a rice bran extract and an enzymatic hydrolysate of a rice leaf extract have the effect of enhancing proteasome activity.

[0005] Previously, it has been reported in Patent Document 1 that an enzymatic hydrolysate of rice bran extract has an inhibitory effect on intracellular tyrosinase activity, and in Patent Document 2 that an enzymatic hydrolysate of rice leaves has an inhibitory effect on tyrosinase activity and an effect of promoting collagen synthesis, but it has not been reported that a hydrolysate of rice bran extract or an enzymatic hydrolysate of rice leaves has an effect of enhancing proteasome activity. [Means for solving the problem]

[0006] The present invention relates to a proteasome activity enhancer comprising, as an active ingredient, an enzymatic hydrolysate of a rice bran extract or an enzymatic hydrolysate of a rice leaf extract. Effect of the Invention

[0007] The present invention relates to a proteasome activity enhancer containing an enzymatic hydrolysate of rice bran extract or an enzymatic hydrolysate of rice leaves as an active ingredient, and according to the present invention, it is possible to provide an external skin preparation that enhances proteasome activity in skin cells and exerts the effect of maintaining and improving the function of skin cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Preferred embodiments of the present invention will now be described in detail.

[0009] The "rice bran" used in the present invention is not particularly limited, 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.

[0010] The "rice" used in the present invention refers to rice of the genus Oryza in the family Poaceae, and any rice that is taxonomically classified as Oryza sativa can be used. In the present invention, the part of rice used is preferably the leaves or the whole plant including the leaves. In addition, the "leaves" used are preferably leaves before ear emergence, and the leaves may be subjected to heat treatment before extraction treatment.

[0011] In the present invention, in order to obtain the enzymatic hydrolysate of rice bran and rice leaves, the rice bran and rice leaves are firstly cut into small pieces or crushed as necessary after being dried or as such, and then brought into contact with an extraction solvent for extraction. The extraction can be carried out by contacting with the extraction solvent according to a conventional method such as a soaking method.

[0012] 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, and these may be used alone or in combination of two or more kinds.

[0013] Among the extraction solvents, hydrophilic solvents such as water, lower alcohols, and polyhydric alcohols are preferred in the present invention from the standpoint of skin irritation and efficacy, and also because they can be widely applied to topical skin preparations.

[0014] In preparing the extract, the pH is not particularly limited, but is generally preferably in the range of 3 to 9. In this sense, if necessary, an alkalinity regulator such as sodium hydroxide, sodium carbonate, or potassium hydroxide, or an acidity regulator such as citric acid, hydrochloric acid, phosphoric acid, or sulfuric acid may be added to the extraction solvent to adjust the pH to a desired level.

[0015] The extraction conditions such as extraction temperature and extraction time vary depending on the type and pH of the solvent used, but for example, when water or 1,3-butylene glycol, or a mixture of water and 1,3-butylene glycol is used as the solvent, the extraction temperature is preferably in the range of 0° C. to 80° C. The extraction time is preferably in the range of 1 to 168 hours (1 hour to 1 week).

[0016] Next, the rice bran extract and rice leaf extract obtained by the above method are subjected to hydrolysis treatment using enzymes. As the enzyme, protease is preferable for the rice bran extract, and protease, pectinase and cellulosease are preferable for the rice leaf extract.

[0017] 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, aminopeptidase, bromelain, neurase, etc.

[0018] Examples of pectic enzymes include pectinase, pectin depolymerase, pectin demethoxylase, pectin lyase, pectin esterase, and polygalacturonase.

[0019] Examples of cellulose decomposition enzymes include cellulase, hemicellulase, agarase, mannase, chitinase, chitosanase, carrageenase, alginase, fucoidanase, inulase, xylanase, and ligninase.

[0020] The enzymatic hydrolysates of the rice bran extract and rice leaf extract prepared as described above may be used as ingredients of topical skin preparations as they are after generally adjusting the pH to 3 to 8, or may be used at a desired concentration by concentrating under reduced pressure, etc. Also, they may be dried by a conventional method such as spray drying.

[0021] Examples of topical skin preparations (cosmetics, quasi-drugs, topical medicines) containing the enzymatic hydrolysate of rice bran extract or the enzymatic hydrolysate of rice leaf extract, which are the active ingredients of the present invention, include milky lotions, creams, lotions, essences, packs, lipstick, foundations, sheet masks, liquid foundations, makeup press powders, blushers, face powders, facial cleansers, body shampoos, hair shampoos, soaps and other cleansing cosmetics, hair growth agents, and bath additives, but are not limited to these.

[0022] The amount of the enzymatic hydrolysate of rice bran extract or the enzymatic hydrolysate of rice leaf extract of the present invention to be incorporated in a skin external preparation for skin care is generally 0.00001 to 5.0% by weight, preferably 0.0001 to 1.0% by weight, in terms of the solid content of each extract. When incorporated in a skin external preparation for hair, the amount is generally 0.00001 to 5.0% by weight (solid content weight %, the same applies below), preferably 0.0001 to 3.0% by weight, in terms of the solid content of each extract.

[0023] When the enzymatic hydrolysate of rice bran extract or the hydrolysate of rice leaf extract according to the present invention is incorporated into topical skin preparations (cosmetics, quasi-drugs, topical medicines, etc.), the following optional ingredients used in topical skin preparations may be incorporated. Suitable examples of optional components include hydrocarbons such as squalane, petrolatum, and microcrystalline wax; esters such as jojoba oil, carnauba wax, and octyldodecyl oleate; triglycerides such as olive oil, beef tallow, and coconut oil; fatty acids such as stearic acid, oleic acid, and retinoic acid; higher alcohols such as oleyl alcohol, stearyl alcohol, and octyldodecanol; anionic surfactants such as sulfosuccinate esters and sodium polyoxyethylene alkyl sulfates; amphoteric surfactants such as alkyl betaine salts; cationic surfactants such as dialkyl ammonium salts; nonionic surfactants such as sorbitan fatty acid esters, fatty acid monoglycerides, polyoxyethylene adducts thereof, polyoxyethylene alkyl ethers, and polyoxyethylene fatty acid esters; polyhydric alcohols such as polyethylene glycol, glycerin, and 1,3-butanediol; thickening and gelling agents; antioxidants; ultraviolet protection agents such as ultraviolet absorbing agents and ultraviolet scattering agents; coloring agents; preservatives; pH adjusters; powders; and various active ingredients.

[0024] The present invention will now be described in more detail with reference to Production Examples, Formulation Examples and Test Examples, but the present invention is not limited thereto. In the following, all parts mean parts by weight and all % mean % by weight.

[0025] Manufacturing Example 1. Preparation of enzymatic hydrolysate of rice bran extract 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℃ for one hour. The insoluble matter resulting from the treatment was filtered off, and the filtrate was adjusted to pH 6.5 with an alkaline pH adjuster, yielding 730g of a pale yellow, transparent rice bran extract hydrolyzed solution (solids concentration 3.5%).

[0026] Preparation Example 2. Preparation of enzymatic hydrolysate of rice leaf extract 1000g of purified water was added to 200g of dried and ground rice leaves immediately before heading (booting stage), and extraction was performed at 80°C for 1 hour, followed by filtration to obtain 550g of a pale yellow, transparent rice leaf extract solution (solid concentration 2.5%). 0.025g of pectinase was added to 500g of the obtained extract solution, and hydrolysis was performed at 40°C for 4 hours. The solution was then heated at 90°C for 1 hour to inactivate the enzyme, followed by filtration to obtain 460g of a pale yellow, transparent enzymatic hydrolysate solution of rice leaf extract (solid concentration 2.8%).

[0027] Test Example 1. Effect of enhancing proteasome activity Normal human epidermal cells (NHEK) were cultured at 8 × 10 in a 96-well microplate containing HumediaKG2 (Kurabo). 3The cells were seeded in one well and pre-cultured at 37°C and 5.0% CO2 for one day, after which the enzymatic hydrolysate of the rice bran extract from Production Example 1 was added as a sample solution. The sample solution was added so that the enzymatic hydrolysate of the rice bran extract from Production Example 1 was 0.5% of the total volume of the medium. After the sample solution was added, the cells were further cultured for two days under the same conditions as the pre-culture. Then, the cell culture supernatant of each test group was removed, and the cells were disrupted using 100 μL of cell lysis solution (Tris-HCL buffer (pH 8.0) containing 1% Tween 20: 12 mM trishydroxymethylaminomethane, 5 mM EDTA, 150 mM NaCl, hydrochloric acid) to prepare a crude enzyme solution. 50 μL of the crude enzyme solution was placed on a separate plate, and 10 μL of a proteasome activity inhibitor solution (cell lysis solution containing 2 mM epigallocatechin gallate) was added thereto, and this was used to measure non-proteasome activity. In addition, 10 μL of cell lysate was added to the remaining crude enzyme solution to adjust the volume, and this was used for measuring the total substrate degradation ability. 10 μL of buffer containing 65 μM Suc-Leu-Leu-Val-Tyr-AMC as a substrate was added to each plate, and the mixture was reacted at 37 ° C for 1 hour. After the reaction was completed, the fluorescence intensity (excitation wavelength: 355 nm, fluorescence wavelength: 460 nm) was measured using a fluorescent plate reader (Fluoroscan Ascent, Thermo Labsystems). The value obtained by subtracting the non-proteasome activity measurement result from the total substrate degradation ability measurement result was taken as the proteasome activity. The same operation as above was also performed for the case where PBS (-) was added instead of the sample solution of Production Example 1 and no sample was added (Control: 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 taken as the proteasome activity rate (%).

[0028] Human dermis-derived fibroblasts, NB1RGB, were cultured in a 96-well microplate containing 0.5% NCS-containing Eagle's minimum essential medium at 1 × 10 4The cells were seeded at 100 μL / well and pre-cultured for 1 day under conditions of 37°C and 5.0% CO2, and then the enzymatic hydrolysate of rice leaves from Production Example 1 was added as a sample solution. The sample solution was added so that the enzymatic hydrolysate of rice leaf extract from Production Example 2 was 0.5% as a final concentration relative to the total volume of the medium. After the sample solution was added, the cells were further cultured for 3 days under the same conditions as the pre-culture. Thereafter, the cell culture supernatant of each test group was removed, and the cells were disrupted using 100 μL of cell lysis solution (Tris-HCL buffer (pH 8.0) containing 1% Tween 20: 12 mM trishydroxymethylaminomethane, 5 mM EDTA, 150 mM NaCl, hydrochloric acid) to prepare a crude enzyme solution. 50 μL of the crude enzyme solution was separated into a separate plate, and 10 μL of a proteasome activity inhibitor solution (cell lysis solution containing 2 mM epigallocatechin gallate) was added thereto, and this was used for measuring non-proteasome activity. In addition, 10 μL of cell lysate was added to the remaining crude enzyme solution to adjust the volume, and this was used for measuring the total substrate degradation ability. 10 μL of buffer containing 65 μM Suc-Leu-Leu-Val-Tyr-AMC as a substrate was added to each plate, and the mixture was reacted at 37 ° C for 1 hour. After the reaction was completed, the fluorescence intensity (excitation wavelength: 355 nm, fluorescence wavelength: 460 nm) was measured using a fluorescent plate reader (Fluoroscan Ascent, Thermo Labsystems). The value obtained by subtracting the non-proteasome activity measurement result from the total substrate degradation ability measurement result was taken as the proteasome activity. The same operation as above was also performed for the case where PBS (-) was added instead of the sample solution of Production Example 1 and no sample was added (Control: 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 taken as the proteasome activity rate (%).

[0029] The results of Test Example 1 are shown in Table 1. [Table 1] JPEG2025020603000001.jpg39140

[0030] [Table 2] JPEG2025020603000002.jpg38141

[0031] As shown in Tables 1 and 2, it was confirmed that the enzymatic hydrolysate of the rice bran extract according to Production Example 1 of the present invention has an effect of enhancing proteasome activity in epidermal cells, and the enzymatic hydrolysate of the rice leaf extract according to Production Example 2 of the present invention has an effect of enhancing proteasome activity in fibroblasts. Thus, by blending the enzymatic hydrolysate of the rice bran extract and the rice leaf hydrolysate according to the present invention as active ingredients, it is possible to provide an external skin preparation that has an effect of maintaining and improving the functions of epidermal cells and fibroblasts.

[0032] Formulation example 1. Lotion [Ingredients] Part Roman chamomile oil 0.1 Enzymatic hydrolysate of Production Example 1 1.0 Enzymatic hydrolysate of Production Example 2 1.0 Glycerin 5.0 1,3-Butylene glycol 1.0 Water-soluble collagen 0.01 Phenoxyethanol 1.0 Citric acid (appropriate amount) Purified water (enough to make the total amount 100 parts)

[0033] Formulation example 2: Cream [Ingredients] Part Olive oil 3.0 Squalane 3.0 Enzymatic hydrolysate of Production Example 1 1.0 Enzymatic hydrolysate of Production Example 2 1.0 Lactic acid fermented rice 2.0 Hydrogenated Lecithin 0.5 Carboxyvinyl polymer 0.3 Sodium alginate 0.2 Water-soluble collagen 0.1 1,3-butylene glycol 0.1 Phenoxyethanol 0.5 Diglycerin 0.3 Citric acid (appropriate amount) Purified water (enough to make the total amount 100 parts)

Claims

[Claim 1] A proteasome activity enhancer comprising, as active ingredients, one or both of an enzymatic hydrolysate of rice bran extract and an enzymatic hydrolysate of rice leaf extract.

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