JP7634217B2 - Skin preparations - Google Patents

Description

The present invention relates to a functional material derived from rice bran that removes proteins damaged by oxidation reactions in the body, and to a skin care product that contains the same.

It has been known that when proteins in cells are oxidized or glycated due to factors such as sunlight (ultraviolet rays), active oxygen, and chemicals in the air, the function of the cells is reduced. It has been suggested that when this phenomenon occurs in skin cells, it may cause dryness or dullness of the skin. For this reason, ingredients for use in cosmetics that prevent protein glycation in skin cells have been proposed.

However, these conventional ingredients have problems in terms of safety to the skin and other parts of the body, and effectiveness when actually applied to the body. For example, Patent Documents 1 and 2 disclose anti-glycation ingredients derived from rice or rice bran, but there is a demand for more effective ingredients.

Patent Publication No. 2010-209003 Patent Publication No. 2018-095618

In view of the problems with the conventional technology, the present inventors have newly discovered that proteolytic enzyme-treated rice bran inhibits the occurrence of protein glycation and enhances the enzyme activity that breaks down oxidized proteins, and have thus completed the present invention.

The present invention is a skin care product containing as an active ingredient rice bran with a polishing rate of 90% or more treated with a proteolytic enzyme.

The present invention provides an external skin preparation that contains as an active ingredient a protease-treated product of rice bran with a polishing rate of 90% or more, and that can provide an external skin preparation that maintains a healthy cellular environment based on the active ingredient's ability to inhibit the occurrence of protein glycation and activate oxidative protease.

FIG. 1 is a graph showing the inhibitory effect of the protease-treated rice bran according to the present invention on the generation of advanced glycation end products (AGE). FIG. 2 is a graph showing the effect of enhancing oxidative protease activity of the protease-treated rice bran according to the present invention.

From the viewpoint of effectiveness, the rice bran used in the present invention has a polishing ratio of 90% or more, and more preferably 95.0% or more. Here, the polishing ratio refers to the weight ratio of white rice (rice from which the bran, germ, and other outer layers have been removed, including white rice used to make rice koji) to brown rice (see National Tax Agency Notification No. 8, November 22, 1989, "Standards for Labeling the Manufacturing Method and Quality of Sake").

In the present invention, an extract is prepared using the rice bran described above. The extract can be prepared by washing the part of each plant to be extracted in advance, if necessary, drying, shredding or crushing, and then contacting it with an extraction solvent by a conventional method such as immersion, countercurrent extraction, steam distillation or supercritical extraction. The extract is then treated with a proteolytic enzyme as described below.

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, which may be used alone or in combination of two or more.

Among these extraction solvents, the use of water or a mixed solvent of water and lower alcohols or polyhydric alcohols is preferred in the present invention, since it can be widely applied to skin external preparations (cosmetics or quasi-drugs) and food and beverages. When using a mixed solvent, the mixing ratio of lower alcohol or polyhydric alcohol to water is generally 70% by weight or less, preferably 50% by weight or less.

There are no particular limitations on the pH, temperature, or time required to prepare the extract, but it is preferable to select them appropriately depending on the part of the plant to be extracted.

For example, in the present invention, when preparing a rice bran extract solution, it is preferable to adjust the pH using an alkali adjuster or acidity adjuster as appropriate. 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.

The extract obtained by the above-mentioned treatment is treated with a protease before or after the extraction, or in parallel with the extraction. Examples of protease 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 a complex protease derived from a microorganism [e.g., Neurase (manufactured by Amano Enzyme Co., Ltd.)]. When using a protease, any of the above enzymes may be used alone or in combination. Furthermore, a combination of enzymes such as amylolytic enzymes (e.g., α-amylase, β-amylase, glucoamylase, or β-galactosidase), cellulose-decomposing enzymes (e.g., cellulase, hemicellulase, pectinase), and lipolytic enzymes (lipase, etc.) may be used before or after the treatment with the protease, or in parallel with the treatment.

The enzyme treatment is carried out by adding one or more of the enzymes to the plant extract solution and carrying out the enzyme reaction under conditions near the optimum pH and temperature for 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 100 parts by weight of the solid content of the plant extract solution, more preferably in the range of 0.1 to 10 parts by weight. The time of the enzyme treatment varies depending on the type of enzyme used, but is generally in the range of 0.5 to 24 hours, and preferably in the range of 1 to 6 hours.

After the enzyme treatment using the above enzymes is completed, the enzymes are inactivated using an appropriate method, such as heating the enzyme treatment solution to 80°C or higher, to obtain an enzyme-treated hydrolysate solution.

The protease-treated product prepared as described above may be used as a cosmetic ingredient as is, generally after adjusting the pH to 3 to 8, or may be used at the desired concentration by concentrating under reduced pressure. The extract may also be dried by standard methods such as spray drying.

When the enzyme-treated product of the present invention is incorporated into a skin topical preparation, in addition to the enzyme-treated product as an essential component, ingredients normally used in cosmetics, such as oily ingredients, surfactants (synthetic and natural), moisturizing agents, anti-inflammatory agents, thickeners, preservatives and germicides, anti-acne agents, cell activators, powder ingredients, ultraviolet absorbers, antioxidants, whitening agents, anti-wrinkle agents, pigments, fragrances, etc., can be appropriately incorporated as necessary.

Examples of oily components include olive oil, jojoba oil, castor oil, soybean oil, rice oil, rice germ oil, coconut oil, palm oil, cocoa oil, meadowfoam oil, shea butter, tea tree oil, avocado oil, macadamia nut oil, bergamot oil, lavender oil, rose oil, bergamot oil, chamomile oil, and other plant-derived oils and fats such as squalane; vitamin A oil; animal-derived oils and fats such as mink oil and turtle oil; waxes such as beeswax, carnauba wax, rice wax, and lanolin; liquid paraffin, petrolatum, and paraffin wax. , squalane, and other hydrocarbons; myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, cis-11-eicosenoic acid, and other fatty acids; lauryl alcohol, cetanol, pantothenyl alcohol, stearyl alcohol, and other higher alcohols; isopropyl myristate, isopropyl palmitate, butyl oleate, 2-ethylhexyl glyceride, and higher fatty acid octyldodecyl (octyldodecyl stearate, etc.), as well as synthetic esters and synthetic triglycerides.

Examples of surfactants include nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyoxyethylene sorbitol fatty acid esters; fatty acid salts, alkyl sulfates, alkylbenzene sulfonates, polyoxyethylene alkyl ether sulfates, polyoxyethylene fatty amine sulfates, polyoxyethylene alkyl phenyl ether sulfates, polyoxyethylene alkyl ether phosphates, α-sulfonated fatty acid alkyl ester salts, polyoxyethylene Anionic surfactants such as phenyl alkyl phenyl ether phosphate; cationic surfactants such as quaternary ammonium salts, primary to tertiary fatty amine salts, trialkyl benzyl ammonium salts, alkyl pyridinium salts, 2-alkyl-1-alkyl-1-hydroxyethyl imidazolinium salts, N,N-dialkyl morpholinium salts, and polyethylene polyamine fatty acid amide salts; amphoteric surfactants such as N,N-dimethyl-N-alkyl-N-carboxymethyl ammonio betaine, N,N,N-trialkyl-N-alkylene ammonio carboxybetaine, N-acylamidopropyl-N', N'-dimethyl-N'-β-hydroxypropyl ammonio sulfobetaine, etc. can be used.

As emulsifiers and/or emulsifier aids, stevia derivatives such as enzyme-treated stevia, saponin or its derivatives, casein or its salts (sodium, etc.), sugar and protein complexes, sucrose or its esters, lactose, soy-derived water-soluble polysaccharides, soy-derived protein and polysaccharide complexes, lanolin or its derivatives, cholesterol, stevia derivatives (enzyme-treated stevia, etc.), silicates (aluminum, magnesium, etc.), carbonates (calcium, sodium, etc.), saponin and its derivatives, lecithin and its derivatives (hydrogenated lecithin, etc.), lactic acid bacteria fermented rice, lactic acid bacteria fermented germinated rice, lactic acid bacteria fermented grains (wheat, beans, millet, etc.), etc. can also be blended.

Examples of moisturizing agents include glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, polyethylene glycol, sorbitol, xylitol, sodium pyrrolidone carboxylate, and the like, as well as sugars such as trehalose and raffinose, mucopolysaccharides (e.g., hyaluronic acid and its derivatives, hyaluronic acid fermentation liquid, chondroitin and its derivatives, heparin and its derivatives, etc.), elastin and its derivatives, collagen and its derivatives, collagen peptides, NMF-related substances, lactic acid, urea, higher fatty acid octyldodecyl, seaweed extract, estradiol, various amino acids and their derivatives.

Examples of thickeners include components derived from brown algae, green algae, or red algae, such as alginic acid, agar, carrageenan, and fucoidan; polysaccharides such as pectin and aloe polysaccharides; gums such as tragacanth gum, locust bean gum, xanthan gum, and guar gum; cellulose derivatives such as carboxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; synthetic polymers such as carboxyvinyl polymers, alkyl-modified carboxyvinyl polymers, polyvinyl alcohol, polyvinylpyrrolidone, and acrylic acid/methacrylic acid copolymers; hyaluronic acid and its derivatives; polyglutamic acid and its derivatives, and polyacrylic acid.

Anti-inflammatory agents include allantoin, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, β-glycyrrhetinic acid, stearyl glycyrrhetinate, ε-aminocaproic acid, d-camphor, dl-camphor, zinc oxide, panthenol, pyridoxine hydrochloride, and riboflavin or its derivatives.

Examples of preservatives and disinfectants include urea; benzoic acid or its salts, paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate; phenoxyethanol, dichlorophene, hexachlorophene, chlorhexidine hydrochloride, benzalkonium chloride, salicylic acid, sodium salicylate, zinc pyrithione, benzalkonium chloride, ethanol, undecylenic acid, phenols, and alkane bromide. These include kilisoquinolinium, resorcinol, jamal (imidazolidinyl urea), isopropyl methylphenol, triclosan, trichlorocarbanide, trichlorohydroxydiphenol ether, hinokitiol, 1,2-pentanediol, propanediol, concentrated benzalkonium chloride solution 50, essential oils such as peppermint oil and eucalyptus oil, bark distillate, fermented radish liquid, ethanol derived from plants such as sugar cane and corn, or 1,3-butylene glycol.

Cell activators include pantothenyl alcohol, menthol, dl-menthol, and gamma-oryzanol.

Anti-acne agents include sulfur, salicylic acid or its salts, photosensitizer No. 201, pyridoxine dicaprylate, etc.

Examples of powder components include sericite, titanium oxide, talc, kaolin, bentonite, zinc oxide, magnesium carbonate, magnesium oxide, zirconium oxide, barium sulfate, silicic anhydride, mica, nylon powder, polyethylene powder, silk powder, cellulose-based powder, grain powder (rice, wheat, corn, millet, etc.), and bean powder (soybean, adzuki bean, etc.).

Examples of ultraviolet absorbers include ethyl paraaminobenzoate, ethylhexyl paradimethylaminobenzoate, amyl salicylate and its derivatives, 2-ethylhexyl paramethoxycinnamate, octyl cinnamate, oxybenzone, 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonate, 4-tert-butyl-4-methoxybenzoylmethane, 2-(2-hydroxy-5-methylphenyl)benzotriazole, urocanic acid, ethyl urocanate, aloe extract, etc.

Examples of antioxidants include butyl hydroxyanisole, butyl hydroxytoluene, propyl gallate, carotenoids such as astaxanthin, vitamin E and its derivatives (e.g., tocopherol acetate, tocopherol nicotinate), vitamin A and its derivatives (retinol palmitate, etc.), etc.

Examples of skin whitening agents include t-cycloamino acid derivatives, kojic acid and its derivatives, ascorbic acid and its derivatives, hydroquinone or its derivatives, ellagic acid and its derivatives, nicotinic acid and its derivatives, resorcinol derivatives, tranexamic acid and its derivatives, 4-methoxysalicylic acid potassium salt, magnolignan (5,5'-dipropyl-biphenyl-2,2'-diol), hydroxybenzoic acid and its derivatives, vitamin E and its derivatives, α-hydroxy acid, AMP (adenosine monophosphate, adenosine monophosphate), etc., which may be used alone or in combination.

Examples of the kojic acid derivatives include kojic acid esters such as kojic acid monobutyrate, kojic acid monocaprate, kojic acid monopalmitate, and kojic acid dibutyrate, kojic acid ethers, and kojic acid sugar derivatives such as kojic acid glucoside. Examples of the ascorbic acid derivatives include ascorbic acid ester salts such as sodium L-ascorbic acid 2-phosphate, magnesium L-ascorbic acid 2-phosphate, sodium L-ascorbic acid 2-sulfate, and magnesium L-ascorbic acid 2-sulfate, L-ascorbic acid 2-glucoside, and L-ascorbic acid 5-glucoside. Ascorbic acid sugar derivatives such as coside, ascorbyl tocopheryl maleate, ascorbyl tocopheryl phosphate K, myristyl 3-glyceryl ascorbate, and caprylyl 2-glyceryl ascorbate; 6-acylated products of these ascorbic acid sugar derivatives (the acyl group is a hexanoyl group, an octanoyl group, a decanoyl group, or the like); L-ascorbic acid tetraisopalmitate, L-ascorbic acid tetralaurate, and other L-ascorbic acid tetrafatty acid esters; 3-O-ethyl ascorbic acid, L-ascorbic acid-2-phosphate-6-O-sodium palmitate, and glyceryl ascorbate. or its acylated derivatives, ascorbic acid glycerin derivatives such as bisglyceryl ascorbic acid, L-ascorbic acid aminopropyl phosphate, hyaluronic acid derivatives of L-ascorbic acid, 3-O-D lactose-L-ascorbic acid, isostearyl ascorbyl phosphate, etc.; hydroquinone derivatives include arbutin (hydroquinone-β-D-glucopyranoside), α-arbutin (hydroquinone-α-D-glucopyranoside), etc.; tranexamic acid derivatives include tranexamic acid esters (for example, tranexamic acid lauryl ester, tranexamic acid hexadecyl ester, tranexamic acid cetyl ester, etc.); Examples of the resorcinol derivatives include 4-n-butylresorcinol, 4-isoamylresorcinol, etc. Examples of the 2,5-dihydroxybenzoic acid derivatives include 2,5-diacetoxybenzoic acid, 2-acetoxy-5-hydroxybenzoic acid, 2-hydroxy-5-propionyloxybenzoic acid, etc. Examples of the nicotinic acid derivatives include nicotinamide, benzyl nicotinate, etc. Examples of the α-hydroxy acids include lactic acid, malic acid, succinic acid, citric acid, α-hydroxyoctanoic acid, etc.

In addition, examples of physiologically active ingredients include placenta extract, mulberry extract, saxifrage extract, perilla extract, rice bran extract or hydrolysate thereof, white mustard extract or hydrolysate thereof, fermented white mustard, peony extract or hydrolysate thereof, lactic acid bacteria extract, bifidobacteria extract, barley extract, lotus seed extract or hydrolysate thereof, fermented lotus seed, Job's tears hydrolysate, fermented Job's tears seed, fermented royal jelly, soybean sprout extract, sake lees extract or ceramide contained therein, fermented sake lees, Pandanus amaryllis extract, Arcangelisia flava extract, Calendula officinalis extract, chamomile extract, coral grass extract, rice leaf extract or or hydrolysates thereof, eggplant (lotus, long eggplant, Kamo eggplant, rice eggplant, etc.) extract or hydrolysate thereof, apricot fruit extract, seaweed extract such as Eucalyptus tschonoskii, extract of marine flowering plants such as Zostera marina, fermented soy milk, jellyfish water, rice extract or hydrolysate thereof, fermented rice extract, germinated rice extract or hydrolysate thereof, fermented germinated rice, black soybean extract or hydrolysate thereof, brown sugar extract or fermented product thereof, extract of Damask rose flower, ylang-ylang flower extract, extract of bamboo shoot skin, linoleic acid and its derivatives or processed products (e.g. liposomal linoleic acid, etc.), flavonoids or glycosides derived from citrus plants, collagen and its derivatives derived from animals or fish, elastin and its derivatives Conductors, animal or fish-derived proteoglycans, glycyrrhizic acid and its derivatives (dipotassium salt, etc.), t-cycloamino acid derivatives, vitamin A and its derivatives, allantoin, diisopropylamine dichloroacetate, gamma-amino-beta-hydroxybutyric acid, gentian extract, licorice extract, ginseng extract, Panax ginseng (white ginseng) extract, fermented Panax ginseng, camellia extract, Mitsuishikonbu extract, loofah extract, Ulva pertusa extract, peach extract, plum extract, peach kernel extract, kiwi extract, sunflower extract, juazeiro extract, pau d'arco bark extract, daylily extract or fermented product, hibiscus flower extract or fermented product, hago Examples include lomogonia extract, cherimoya extract, mango extract, mangosteen extract, funori extract, oolong tea extract, red fuki extract, striated lanthanum extract, sweet potato extract, Japanese pepper peel or seed coat extract or its hydrolysate, safflower flower extract, Casablanca extract, sweet potato extract or its fermented product, guava leaf extract, artemisia capillaris extract, clove extract, olive leaf extract, horse chestnut extract, ginger extract, vanilla extract, dokudami extract, banpeiyu extract, aloe extract, aloe vera extract, tulip extract, centella asiatica extract, fig flower extract, apple extract, white asparagus extract, pueraria root extract, and party ginseng extract or its hydrolysate.

The application site of the skin topical preparation (including cosmetics and quasi-drugs) containing the enzyme-treated product of the present invention can be any skin including the scalp, and there are no particular limitations. Therefore, it can be used in milky lotions, creams, lotions, essences, ointments, packs, poultices, skin cleansers (such as soaps), facial cleansers, shampoos, rinses, treatments, body creams, lip cosmetics, various makeup cosmetics, bath additives, etc.

The present invention will now be described in more detail with reference to manufacturing examples, test examples, and formulation examples, but the present invention is not limited thereto. In the following, all parts are by weight, and all percentages are by weight.

Manufacturing Example 1. Preparation of the enzyme-treated rice bran product (1) according to the present invention 1500 g of 0.1 M lactic acid solution was added to 500 g of rice bran (polished rice ratio 90%), and the rice bran and the lactic acid solution were thoroughly mixed by stirring, and then the mixture was left at room temperature for one day. Next, insoluble matter was removed by filtration, and the filtrate was treated with papain. The enzyme treatment was carried out by using 1.2 mg of enzyme, and maintaining the mixture at 80°C for 1 hour at the optimum pH of the enzyme. The insoluble matter generated by the treatment was filtered off, and the filtrate was adjusted to pH 6.5 with phytic acid to obtain 730 g of a pale yellow transparent enzyme-treated rice bran product solution (solid concentration 3.60%).

Production Example 2. Preparation of enzyme-treated rice bran product (2) according to the present invention 735 g of enzyme-treated rice bran solution (solid concentration 3.64%) was obtained by the same process as in Production Example 1, except that rice bran with a polishing ratio of 95% was used instead of the rice bran (polished ratio 90%) in Production Example 1.

Comparative Production Examples 1 to 4. Preparation of Enzyme-treated Rice Bran Products for Comparison Instead of the rice bran (polishing ratio 90%) in Production Example 1, rice bran with the following polishing ratios was used to obtain enzyme-treated rice bran solutions obtained by the same process as in Production Example 1. That is, a rice bran enzyme-treated solution (solid concentration: 3.40%) obtained using rice bran with a polishing ratio of 10% was used as Comparative Production Example 1, a rice bran enzyme-treated solution (solid concentration: 3.46%) obtained using rice bran with a polishing ratio of 30% was used as Comparative Production Example 2, a rice bran enzyme-treated solution (solid concentration: 3.50%) obtained using rice bran with a polishing ratio of 50% was used as Comparative Production Example 3, and a rice bran enzyme-treated solution (solid concentration: 3.53%) obtained using rice bran with a polishing ratio of 70% was used as Comparative Production Example 4. Enzyme-treated solutions were prepared.

Test Example 1. Evaluation of the effect of inhibiting the generation of advanced glycation end products (AGE) In this test, the effect of inhibiting the generation of AGE, i.e., the effect of inhibiting protein glycation, was evaluated by the generation of fluorescence from BSA (bovine serum albumin) via glucose. First, 40 μL of each of the rice bran enzyme-treated products of Production Examples 1 and 2 according to the present invention and the rice bran enzyme-treated products of Comparative Production Examples 1 to 4, 40 μL of 50 mg/mL BSA aqueous solution, 40 μL of 2.5 M glucose aqueous solution, and 80 μL of PBS (-) solution were mixed and stirred to prepare a sample solution. Here, each of the rice bran enzyme-treated products was prepared so that the final concentration of the solution in the sample solution was 2.0%. Next, the sample solution was left to stand at 50°C for 7 days, and after 7 days, the fluorescence generation (excitation: 355 nm, emission: 460 nm: fluorescence microplate reader (Fluoroscan Ascent, Thermo Fisher Scientific)) was measured for each sample solution. The same procedure was also carried out for a control in which purified water was used instead of the sample solution and no sample was added. The relative values (%) of the fluorescence measurement values of each sample solution to the fluorescence measurement values obtained here were calculated and used as the protein glycation rate (%).

The results of Test Example 1 are shown in Figure 1. As shown in Figure 1, it was confirmed that the rice bran enzyme-treated products of Production Examples 1 and 2 according to the present invention have a significantly superior inhibitory effect on the generation of advanced glycation end products (AGEs) compared to the rice bran enzyme-treated products of Comparative Production Examples 1 to 4. This suggests that the rice bran enzyme-treated products of Production Examples 1 and 2 according to the present invention can exert a cellular environment healthifying effect.

Test Example 2. Evaluation of promotion of oxidized protein hydrolase activity In this test example, the activity promotion effect of oxidized protein hydrolase (OPH), an "acyl amino acid releasing enzyme" that breaks down oxidized proteins, was evaluated.
First, human dermis-derived fibroblasts NB1RGB were seeded at 1× ¹⁰⁴ cells/well in a 96-well microplate containing 0.5% NCS-containing Eagle's minimum essential medium, and pre-cultured for 1 day under conditions of 37°C and 5.0% _CO2. Then, the enzyme-treated product solution of Production Example 1 and the enzyme-treated product solutions of Comparative Production Examples 1 to 5 were added to the medium as sample solutions, and cultured for another 3 days under the same conditions. Here, each sample solution was added so that the final concentration of the solution was 2.0% relative to the amount of medium. Next, the medium was removed, and 100 μL/well of 0.5% Triton X-100 solution was added, and the cells were disrupted by leaving it to stand for 5 minutes. Then, 50 μL/well of Ac-Ala-pNA (BACHEM L-1640) diluted with PBS(-) to 3 mM was added as a substrate, and incubated at 37°C for 3 hours. Then, the absorbance at 405 nm (ABS405nm) was measured using a microplate reader (Model 680, Bio-Rad). The same procedure was also performed on wells in which cells were not seeded, and the value obtained by subtracting the ABS405nm value of the wells in which cells were not seeded from the ABS405nm value of each test group (ΔABS405nm) was used as the OPH activity of each test group.
In addition, after the cells in the other plate were cultured, they were washed once with PBS(-) and then 100 μL/well of hoechst33342 reagent diluted 100-fold with PBS(-) was added and incubated at 37°C for 1 hour to fluorescently stain DNA. Then, the fluorescence intensity (excitation: 355 nm, emission: 460 nm: fluorescence microplate reader (Fluoroscan Ascent, Thermo Fisher Scientific)) was measured to determine the amount of DNA. The OPH activity per cell was calculated by dividing the OPH activity value (ΔABS405 nm) of each sample-added area obtained here by the DNA amount (excitation: 355 nm, emission: 460 nm). The OPH activity per cell in each sample-added area was expressed as a relative value, with the OPH activity per cell value in the control area, to which PBS(-) was added instead of the sample solution, taken as 100.

As shown in Figure 2, it was confirmed that the rice bran enzyme-treated products of Production Examples 1 and 2 according to the present invention have a significantly superior effect of enhancing OPH activity compared to the rice bran enzyme-treated products of Comparative Production Examples 1 to 4. This confirmed that they have an effect of improving the cellular environment.

Formulation Example 1. Lotion [Ingredients] Parts Lavender oil 1.0
Glyceryl Caprylate 3.0
Polyglyceryl-10 Lauryl Acid 3.0
Methylparaben 0.1
Rice bran enzyme-treated product of Production Example 1 2.0
White ginseng extract 2.0
Fermented ginseng extract 2.0
Fermented sweet potato extract 1.0
Guava Leaf Extract 1.0
Water-soluble collagen 1.0
Hydrolyzed Collagen 1.0
Eggplant extract 1.0
Strawberry Flower Extract 1.0
Glycerin 2.0
1,3-butylene glycol 5.0
Sodium citrate 0.2
Purified water (enough to make the total amount 100 parts)

Formulation Example 2. Lotion A lotion was obtained with the same composition as Formulation Example 1, except that the rice bran enzyme-treated product of Production Example 1 in Production Example 2 was used in place of the rice bran enzyme-treated product of Production Example 1 in Formulation Example 1.

Formulation Example 3. Emulsion [Ingredients] Parts Squalane 5.0
Hexalan 3.0
Wild rose oil 1.0
Camellia oil 1.5
Polyglyceryl laurate 1.0
Lipophilic glyceryl stearate 1.0
Bisabolol 1.0
Behenyl alcohol 3.0
Hydrogenated soy lecithin 1.5
Rice bran enzyme-treated product of Production Example 1 2.0
White ginseng extract 2.0
Fermented ginseng extract 2.0
L-Ascorbic acid-2-glucoside 2.0
Fermented sweet potato extract 1.0
Shell ginger leaf extract 1.0
Guava Leaf Extract 1.0
Licorice extract 1.0
Parthenium ginseng hydrolyzed extract 1.0
Water-soluble collagen 1.0
Hydrolyzed Collagen 1.0
Potassium hydroxide 0.5
Glycerin 3.0
1,3-butylene glycol 2.0
Carboxymethyl cellulose 0.3
Xanthan gum 0.2
Sodium hyaluronate 0.01
Purified water (enough to make the total amount 100 parts)

Formulation Example 4. Milky lotion A milky lotion was obtained with the same composition as in Formulation Example 1, except that the rice bran enzyme-treated product of Production Example 1 in Production Example 2 was used in place of the rice bran enzyme-treated product of Production Example 1 in Formulation Example 3.

Formulation Example 5. Milky lotion A milky lotion was obtained with the same composition as Formulation Example 4, except that tranexamic acid (1.0 part) was used instead of L-ascorbic acid-2-glucoside and potassium hydroxide used in Formulation Example 3 above.

Formulation Example 6. Milky lotion A milky lotion was obtained with the same composition as Formulation Example 4, except that arbutin (3.0 parts) was used instead of L-ascorbic acid-2-glucoside and potassium hydroxide used in Formulation Example 3 above.

Formulation Example 7. Milky lotion A milky lotion was obtained with the same composition as Formulation Example 4, except that nicotinamide (3.0 parts) was used instead of L-ascorbic acid-2-glucoside and potassium hydroxide used in Formulation Example 3.

Formulation Example 8. Cream [Ingredients] Parts Olive oil 5.0
Jojoba oil 5.0
Meadowfoam oil 5.0
Squalane 5.0
Behenyl alcohol 2.0
Palmitic acid 2.5
Lactic acid fermented rice 2.0
Hydrogenated lecithin 0.5
Glyceryl stearate 3.0
Rice bran enzyme-treated product of Production Example 1 2.0
White ginseng extract 2.0
Fermented ginseng extract 2.0
Fermented sweet potato extract 1.0
Shell ginger leaf extract 1.0
Guava Leaf Extract 1.0
Hydrolyzed rice extract 2.0
Water-soluble collagen 1.0
Hydrolyzed Collagen 1.0
Xanthan gum 1.0
Purified water (enough to make the total amount 100 parts)

Formulation Example 9. Cream A cream was obtained with the same composition as Formulation Example 8, except that the rice bran enzyme-treated product of Production Example 1 in Production Example 2 was used in place of the rice bran enzyme-treated product of Production Example 1 in Production Example 8.

Formulation Example 10. Facial cleanser [Ingredients] Parts Coconut oil 2.0
Stearyl stearate 2.0
Glyceryl Caprylate 2.0
Enzyme-treated product of Production Example 1 2.0
White ginseng extract 2.0
Fermented ginseng extract 2.0
Water-soluble collagen 1.0
Glycerin 2.0
Fig Bark Extract 1.0
Job's Tears Seed Extract 1.0
Clove extract 1.0

Claims (2)

An inhibitor of the generation of advanced glycation end products , the active ingredient of which is rice bran with a polishing rate of 90% or more treated with proteolytic enzymes. An oxidative protease activity enhancer containing as an active ingredient a protease-treated product of rice bran with a polishing rate of 90% or more.