Skin preparations

Abstract

To provide a novel active ingredient that can be blended in an external preparation for skin and is effective in exhibiting skin-whitening effect or the like.SOLUTION: An external preparation for skin contains Lilium japonicum-derived yeast culture extract, or concentrate or dry powder thereof, lactic acid bacteria-fermented products of Nelumbo nucifera, belonging to Nelumbonaceae, Nelumbo, and yeast-fermented products of Hemerocallis fulva var. fulva and / or Hemerocallis fulva var. kwanso, belonging to Liliaceae, Hemerocallis, as active ingredients.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition that combines a yeast culture extract and a plant fermentation product, which can be incorporated into an external preparation for the skin (including the scalp). [Background technology]

[0002] In the past, research and development has been conducted on ingredients derived from natural products as active ingredients to be incorporated into topical skin preparations. However, when used as active ingredients in topical skin preparations, these ingredients derived from natural products have issues in terms of efficacy, stability, etc. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0003] As a result of intensive research aimed at solving the above problems, the inventors have newly discovered that a combination of a yeast culture extract, a fermented product of a lotus plant, and a daylily plant has synergistic effects such as whitening and antioxidant properties, and is useful as an active ingredient in an external skin preparation.

[0004] Conventionally, the use of yeast (e.g., Saccharomyces cerevisiae) as a whitening ingredient in external skin preparations has been known, for example, from Patent Documents 1 and 2. In addition, the incorporation of a fermented product of a plant of the genus Lotus or a fermented product of a plant of the genus Daylily in a cosmetic preparation has been known, for example, from Patent Documents 3 and 4. However, it was not known that a combination of a culture extract of yeast derived from a Japanese lily, a lactic acid bacteria fermented product of a plant of the genus Lotus, and a yeast fermented product of a plant of the genus Daylily has synergistic effects such as whitening and antioxidant properties, and is useful as an active ingredient in external skin preparations. [Patent Document 1] Patent Publication No. 2001-354570 [Patent Document 2] Patent Publication No. 2002-234828 [Patent Document 3] Patent Publication No. 2005-298489 [Patent Document 4] Patent Publication No. 2008-050326 [Means for solving the problem]

[0005] The present invention is a skin topical preparation containing as active ingredients a culture extract or a concentrate or dried powder thereof of yeast derived from a lily of the valley, a lactic acid bacteria fermentation product of lotus belonging to the genus Nelumbo in the family Nelumbo, and a yeast fermentation product of day lily and / or day lily in the genus Helianthus in the family Liliaceae. Effect of the Invention

[0006] The present invention provides an external skin preparation having exceptionally excellent whitening and antioxidant effects, etc., by combining and blending as active ingredients a culture extract or a concentrate or dried powder thereof of yeast derived from Japanese lily, a lactic acid bacteria fermentation product of lotus belonging to the genus Nelumbo in the family Nelumbo, and a yeast fermentation product of day lily and / or hepatica in the genus Hepatica in the family Liliaceae. [Brief description of the drawings]

[0007] [Figure 1] 1 shows the results of a test evaluating the composition according to the present invention for its inhibition of steam cell factor (SCF) synthesis. [Diagram 2] 1 shows the results of a test evaluating the prostaglandin E2 (PGE2) synthesis inhibition of the composition according to the present invention. [Diagram 3] 1 shows the results of a test evaluating the melanin synthesis inhibition of the composition according to the present invention. [Figure 4] 1 shows the results of a test evaluating the enhancement of expression of heparan sulfate proteoglycan (HSPG) genes by the composition according to the present invention. [Diagram 5] 1 shows the results of a test evaluating the suppression of expression of Hepatocyte Growth Factor (HGS) by the composition according to the present invention. [Figure 6] 1 shows the results of a test evaluating the SOD-like activity of the composition according to the present invention. [Figure 7] 1 shows the results of a test evaluating the glycation inhibition effect of a composition according to the present invention. [Figure 8] 1 shows the results of a test evaluating the composition according to the present invention for its ability to inhibit ETBR synthesis in melanocytes. [Figure 9] 1 shows the results of a test evaluating the composition according to the present invention for its ability to inhibit MC1R synthesis in melanocytes. BEST MODE FOR CARRYING OUT THEINVENTION

[0008] The present invention will be described in detail below. The yeast used in the present invention is a yeast of the genus Saccharomyces, and is derived from the flowers of Lilium japonicum, which belongs to the genus Lilium in the family Liliaceae. An example of the yeast derived from the flowers of Lilium japonicum is the yeast "Saccharomyces cerevisiae" (Patent Microorganisms Deposit Center Accession Number NITE P-01947) collected from the flowers of Lilium japonicum, which is described in Patent 6175697.

[0009] The yeast culture extract or its concentrate or dry product can be prepared as follows: The yeast culture extract can be produced by a method of culturing yeast in a culture medium, a hydrolysis method in which the cultured yeast is solubilized with an acid or alkali to solubilize the cell components, an autolysis method using protease or the like present in the yeast cell, an enzymatic method using an enzyme such as a protease, or a combination of these methods.

[0010] The carbon source for culturing yeast is not particularly limited, and examples of the carbon source include glucose, fructose, galactose, and oligosaccharides and polysaccharides having these as constituent sugars. In addition to the carbon source, a nitrogen source may be added, and examples of the nitrogen source include amino acids and peptones.

[0011] The yeast culture temperature is in the range of 25° C. to 40° C., and preferably in the range of 28° C. to 35° C. The pH is in the range of 3.0 to 8.0, and preferably in the range of 3.5 to 7.0.

[0012] A concentrate of a yeast culture extract can be obtained by concentrating a yeast culture liquid or a liquid obtained by a hydrolysis method, a yeast autolysis method or an enzymatic method using a vacuum concentrator or the like.

[0013] Furthermore, a dried yeast culture extract can be obtained by drying a yeast culture solution or a concentrated solution thereof by a vacuum drying method, a spray drying method or the like.

[0014] The dried yeast culture extract may be dried by adding an excipient for the purpose of chemical stability and low hygroscopicity. The excipient may be a sugar such as starch, glucose, crystalline cellulose, lactose, dextrin, or a sugar alcohol such as sorbitol, xylitol, erythritol, mannitol, maltitol, lactitol, or the like, but any substance may be used as long as it can be mixed with the yeast culture extract to form a dry powder.

[0015] The plant used as the raw material for the lotus fermented product of the present invention is the lotus (Nelumbo nucifera) belonging to the genus Nelumbo in the family Nelumbonaceae, and the part used for fermentation is its seeds.

[0016] In addition, the plant of the genus Daylily of the family Liliaceae used as the fermented material in the present invention is Hemerocallis fulva var. fulva or Hemerocallis fulva var. kwanso, or a mixture of the two, and the parts used for fermentation are the buds or the petals and buds.

[0017] The fermentation treatment can be carried out as follows. First, as the assimilation source for fermentation, a plant part (seed, bud or petal) itself (hereinafter sometimes referred to as the plant body) may be used, or an extract obtained by extracting the plant body with an appropriate medium may be used. When an extract is used, it is also possible to carry out fermentation while still containing the plant body, without removing the plant body to be extracted by solid-liquid separation. Here, the plant may be fresh, or may have been dried or semi-dried in advance. In terms of form, it is also possible to use the plant as it is after it has been harvested.

[0018] In the present invention, the microorganisms used for fermenting lotus seeds are lactic acid bacteria, for example, lactic acid bacteria of the genus Lactobacillus such as Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, and Lactobacillus delbrueckii; lactic acid bacteria of the genus Carnobacterium such as Carnobacterium divergens and Carnobacterium piscicola; lactic acid bacteria of the genus Carnobacterium such as Leuconostoc mesenteroides, Leuconostoc lactis, and Leuconostoc citreum. lactic acid bacteria of the genus Leuconostoc such as citreum; lactic acid bacteria of the genus Streptococcus such as Streptococcus faecalis and Streptococcus pyogenes; lactic acid bacteria of the genus Enterococcus such as Enterococcus caseliflavus and Enterococcus sulfreus; lactic acid bacteria of the genus Lactococcus such as Lactococcus plantarum and Lactococcus rafinolactis; lactic acid bacteria of the genus Veissella such as Weissella confusa and Weissella kandleri; Atopobium Lactic acid bacteria of the genus Atopobium, such as Atopobium minutum and Atopobium parvulus;Examples of lactic acid bacteria include Vagococcus genus lactic acid bacteria such as Vagococcus fluvialis and Vagococcus salmoninarum; Pediococcus genus lactic acid bacteria such as Pediococcus damnosus and Pediococcus pentosaceus.

[0019] In the present invention, the yeast used for fermenting the buds or the buds and petals of Helianthus oryzae can be, for example, yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, Saccharomyces awamori, Saccharomyces chevalieri, Saccharomyces carlsbergensis, Saccharomyces bayonus, yeasts of the genus Galactomyces, yeasts of the genus Lachancea (Lachancea kluyveri, etc.), Torulaspora delbruekii, Torulaspora fermentati, Torulaspora spp., and the like. yeasts of the genus Torulaspora such as Torulaspora rosei, yeasts of the genus Zygosaccharomyces such as Zygosaccharomyces rouxii, Zygosaccharomyces soya, Zygosaccharomyces sake, Zygosaccharomyces miso, and Zygosaccharomyces lactis, yeasts of the genus Zygosaccharomyces such as Candida versatilis, Candida etchellsii, Candida kefyr, Candida sake, Candida scotti, yeasts of the genus Candida, such as Aureobasidium pullulans, Aureobasidium mansonii, Aureobasidium microstictum, etc.Furthermore, the yeast according to the present invention may be any of sake yeast, wine yeast, brewer's yeast, yeast derived from plant flowers (rose, lily, cherry blossom, etc.), and yeast derived from the sea.

[0020] When the above-mentioned suspension or extract is fermented by a microorganism, it is necessary to remove unwanted bacteria that hinder fermentation by sterilization before the fermentation step. As a method for sterilizing and removing the unwanted bacteria, a method of washing the fermentation material with sterilizing ethanol or the like in advance and then suspending it in a sterile solvent such as sterile water may be used, or the fermentation material may be suspended in a solvent and then sterilized by heat sterilization or the like. As the heat sterilization treatment, a heat sterilization method such as an autoclave sterilization method in which the suspension is heated at 120 to 130°C for 10 to 20 minutes or an intermittent sterilization method in which the suspension is kept at 80 to 90°C for 60 to 120 minutes and is repeated once a day for 2 to 3 days is generally used.

[0021] The sterilized suspension is placed in a fermentation tank and inoculated with microorganisms for fermentation. The inoculum size is 10 7 ~10 8 If the inoculum amount is more than the above range, the fermentation time will not change much, while if it is less than the above range, it will take a long time to complete the fermentation, which is not preferable.

[0022] The fermentation temperature is generally in the range of 5 to 50°C, preferably in the range of 20 to 40°C, which is the optimum temperature for growth of each microorganism (for example, 30 to 40°C for lactic acid bacteria and 25 to 30°C for yeast). The number of days for fermentation is generally in the range of 1 to 10 days, preferably 2 to 5 days, at the optimum temperature. If the number of days for fermentation is shorter than the above general range, the fermentation does not proceed sufficiently and the effectiveness of the fermented product tends to decrease, while if the number of days is longer than 10 days, not only will no further increase in effectiveness be observed, but coloring and an increase in fermentation odor will occur, both of which are undesirable.

[0023] When preparing a fermented product, in order to more effectively utilize the components of the target use site as a source of assimilation for the lactic acid bacteria or yeast, the suspension or extract solution may be subjected to a hydrolysis treatment with an acid, alkali or enzyme (such as a sugar-degrading enzyme, a fiber-degrading enzyme, a protein-degrading enzyme or a lipolytic enzyme) before or simultaneously with inoculation thereof.

[0024] The extract, hydrolysate or fermentation product prepared as described above may be used as it is after generally adjusting the pH to 3 to 9, or may be used at a desired concentration by concentrating under reduced pressure, etc. Also, it may be dried by a conventional method such as spray drying.

[0025] In addition, the extract, hydrolysate or fermentation product prepared as described above may be refrigerated for a certain period of time in order to improve storage stability, and the supernatant may then be used.

[0026] The composition of the yeast culture extract or its concentrate or dried product according to the present invention combined with the above-mentioned plant fermentation product can be incorporated into external skin preparations (cosmetics, quasi-drugs, external medicines). Examples of external skin preparations include milky lotions, creams, lotions, essences, packs, lipsticks, foundations, liquid foundations, makeup press powders, blushers, face powders, face washes, body shampoos, shampoos for the scalp and hair, hair conditioners, shampoos or tonics for hair growth and nourishment, cleansing cosmetics such as soaps, and bath agents, but the present invention is not limited thereto.

[0027] In addition to the composition of the present invention, the skin topical preparation may contain, as necessary, ingredients used in skin topical preparations, such as oily ingredients, surfactants (synthetic and natural), moisturizing agents, thickeners, preservatives and disinfectants, powder ingredients, ultraviolet absorbers, antioxidants, colorants, fragrances, etc. Furthermore, as long as the efficacy and features of the composition of the present invention are not impaired, there is no problem in combining and formulating other physiologically active ingredients.

[0028] Examples of oily components include plant-derived oils such as lotus oil, olive oil, jojoba oil, castor oil, soybean oil, rice oil, rice bran oil, rice germ oil, coconut oil, chamomile oil, palm oil, cocoa oil, meadowfoam oil, bergamot oil, rosehip oil, coffee seed oil, lambender oil, shea butter, tea tree oil, avocado oil, macadamia nut oil, vanilla oil, and plant-derived squalane; animal-derived oils such as mink oil and turtle oil; waxes such as beeswax, carnauba wax, rice wax, and lanolin; liquid paraffin, Examples of the fatty acids include petrolatum, paraffin wax, squalane, etc.; myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, eicosenoic acid, etc.; higher alcohols such as lauryl alcohol, cetanol, stearyl alcohol, etc.; synthetic esters and synthetic triglycerides such as isopropyl myristate, isopropyl palmitate, butyl oleate, cetyl isooctanoate, 2-ethylhexyl glyceride, and higher fatty acid octyldodecyl (e.g., octyldodecyl stearate), etc.

[0029] Examples of the surfactant include nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyethylene glycol 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, and α-sulfonated fatty acid alkyl esters. Examples of usable surfactants include anionic surfactants such as quaternary ammonium salts, primary to tertiary fatty amine salts, trialkylbenzylammonium salts, alkylpyridinium salts, 2-alkyl-1-alkyl-1-hydroxyethylimidazolinium salts, N,N-dialkylmorpholinium salts, and polyethylenepolyamine fatty acid amide salts; and amphoteric surfactants such as N,N-dimethyl-N-alkyl-N-carboxymethylammoniobetaine, N,N,N-trialkyl-N-alkyleneammoniocarboxybetaine, and N-acylamidopropyl-N',N'-dimethyl-N'-β-hydroxypropylammoniosulfobetaine.

[0030] Further, as the emulsifier or emulsification aid, 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, water-soluble polysaccharides derived from soybeans, soybean-derived protein and polysaccharide complexes, lanolin or its derivatives, cholesterol, stevia derivatives (stevia enzyme fermentation products, 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.

[0031] Examples of moisturizing agents include glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, polyethylene glycol, sorbitol, xylitol, sodium pyrrolidone carboxylate, 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate copolymer liquid, and further include sugars such as trehalose, mucopolysaccharides (e.g., hyaluronic acid and its derivatives, chondroitin and its derivatives, heparin and its derivatives, etc.), tuberose polysaccharides, elastin and its derivatives, collagen and its derivatives, NMF-related substances, hydrolyzed conchiolin protein, hydrolyzed silk, Sphingomonas culture, sphingoglycolipids, ceramide, lactic acid, urea, higher fatty acid octyldodecyl, seaweed extract, citrus-derived flavonoids or their glycosides, Bletilla serrata root (white extract), and various amino acids and their derivatives.

[0032] Further, examples of thickening agents include components derived from brown algae, green algae, or red algae, such as alginic acid, agar, carrageenan, and fucoidan; Bletilla serrata root (white) extract; polysaccharides such as pectin, locust bean gum, aloe polysaccharide, and Alcaligenes polysaccharide; gums such as xanthan gum, tragacanth gum, roast bean gum, and guar gum; cellulose derivatives such as carboxymethyl cellulose and hydroxyethyl cellulose; synthetic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, mastic resin, and acrylic acid / methacrylic acid copolymer; hyaluronic acid and its derivatives; and polyglutamic acid and its derivatives.

[0033] Examples of preservatives and disinfectants include urea; paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate; phenoxyethanol, dichlorophene, hexachlorophene, chlorhexidine hydrochloride, benzalkonium chloride, salicylic acid, ethanol, undecylenic acid, phenols, jamal (imidazolidinyl urea), polyphosphoric acid, propanediol, 1,2-pentanediol, various essential oils, bark distillate, radish fermentation liquid, and ethanol or 1,3-butylene glycol derived from plants such as sugar cane.

[0034] 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, powder of grains (rice, wheat, corn, millet, etc.), powder of beans (soybeans, adzuki beans, etc.), and the like.

[0035] 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, and aloe extract.

[0036] Examples of the antifoaming agent include ethanol, isopropanol, disiloxane, dimethylpolycyclohexane, dimethicone silicate, trisiloxane, silylated silica, dimethicone, trimethylsiloxysilicate, and DPG isobornyl ether.

[0037] Examples of antioxidants include butylhydroxyanisole, butylhydroxytoluene, propyl gallate, extract of Brussels sieboldii, extract of Bletilla serrata root, extract of peony root, vitamin E and its derivatives (e.g., vitamin E nicotinate, vitamin E linoleate, etc.).

[0038] Examples of the chelating agent include trisodium ethylenediaminehydroxyethyltriacetate, edetic acid or its salts, gluconic acid, phytic acid, sodium polyphosphate, sodium metaphosphate, and tetrasodium hydroxyethanediphosphonate.

[0039] Examples of pH adjusters include citric acid or salts thereof, lactic acid or salts thereof, glycolic acid, succinic acid, hydrochloric acid, monoethanolamine, diethanolamine, triethanolamine, sodium hydroxide, and potassium hydroxide.

[0040] The present invention will now be described in more detail with reference to Production Examples, Test Examples and Examples, but the present invention is not limited thereto. In the following, all parts mean parts by weight, and all % means % by weight.

[0041] Production Example 1. Preparation of yeast culture extract (1) 300g of preculture solution of yeast (Saccharomyces cerevisiae) derived from Japanese lily, which had been cultivated in the same medium, was added to 2700g of sterilized GP liquid medium, and cultivated for 24 hours at 30℃ with aeration. After heat sterilization, the pH was adjusted to 8.5 with an aqueous solution of sodium hydroxide, and the mixture was heated at 90℃ for 3 hours with stirring. After adjusting the pH of this liquid, it was filtered to obtain 1610g of yeast culture extract (solid concentration 1.13%).

[0042] Preparation Example 2. Preparation of yeast culture extract (2) 300g of preculture solution of yeast (Saccharomyces cerevisiae) derived from Japanese lily, which had been cultivated in the same medium, was added to 2700g of sterilized GP liquid medium and cultivated for 24 hours at 30℃ with aeration. After heat sterilization, the pH was adjusted to 2.5 with an aqueous hydrochloric acid solution and heated at 90℃ for 3 hours with stirring. After adjusting the pH of this liquid, it was filtered to obtain 1215g of yeast culture extract (solid concentration 1.07%).

[0043] Preparation Example 3. Preparation of yeast culture extract (3) 300g of preculture solution of yeast (Saccharomyces cerevisiae) derived from Japanese lily, which had been cultivated in the same medium, was added to 2700g of sterilized GP liquid medium and cultivated for 24 hours with aeration at 30℃. After heat sterilization, it was heated at 90℃ for 3 hours. After adjusting the pH of this liquid, it was filtered to obtain 1540g of yeast culture extract (solid concentration 0.62%).

[0044] Preparation Example 4. Preparation of yeast culture extract (4) The yeast culture extract prepared by the method shown in Production Example 1 was concentrated to obtain 17 g of yeast culture extract powder. A small amount of water was added to this to dissolve it, and then a small amount of ethanol and 833 g of D-mannitol were added, followed by vacuum drying with stirring to obtain 850 g of D-mannitol-excipient yeast culture extract powder.

[0045] Production Example 5. Fermented lotus seeds 100 g of lotus seeds (with the astringent skin removed) were crushed and 1900 g of purified water was added to prepare a suspension, which was then sterilized by heating. 8 The cells were inoculated at 1000 / mL and cultured stationarily for 3 days at 37° C. under a nitrogen stream. After the culture was completed, the culture was sterilized by heating and filtered to obtain 1,415 g of a lotus seed lactic acid bacteria fermentation solution (solid concentration 2.53%).

[0046] Production Example 6: Fermented Day Lily Flower 150 g of finely chopped flowers (including buds and petals) of Day Lily were mixed with 1500 g of purified water and extracted at 40°C for 3 hours. The extracted suspension was then sterilized by heating, and 100 ml of yeast (Saccharomyces cerevisiae) was added.8 The yeast was inoculated at 1000 / mL and cultured at 30°C for 3 days under a nitrogen stream. After the culture was completed, the yeast was sterilized by heating, filtered, and deodorized and decolorized to obtain 1,110 g of a brown, transparent yeast fermentation solution (solid concentration 4.0%). This was diluted 3 times with purified water to obtain the yeast fermentation solution.

[0047] Example 1. Preparation of the composition Equal amounts of the extract solution of Production Example 1, the fermentation product solution of Production Example 5, and the fermentation product solution of Production Example 6 were mixed, and the insoluble matter was filtered off to obtain 2920 g of a brown, transparent extract solution mixture (solid concentration 2.53%).

[0048] The composition according to Example 1 prepared as described above was used as present invention sample 1 and its effectiveness was evaluated as follows. As comparative samples for present invention sample 1, the yeast culture extract of Production Example 1 was used as Comparative Sample 1, the lotus seed fermented product of Production Example 5 was used as Comparative Sample 2, and the fermented product of day lily flower parts of Production Example 6 was used as Comparative Sample 3.

[0049] Test Example 1. Inhibitory effect on SCF synthesis in epidermal cells Normal human skin-derived epidermal cells (NHEK) were cultured at 8 × 10 in a 96-well microplate containing HuMedia KG2 medium (Kurabo). 3 After pre-cultivation for 1 day under conditions of 37°C and 5.0% CO2, HuMedia KG2 medium containing the present invention sample 1 and comparative samples 1 to 3 was added as a sample solution. The concentration of each sample was adjusted so that the final concentration of the solution in the medium was 1.2%. Then, a UV-B lamp (TL20W / 12RS, manufactured by Philips) was used to illuminate the bottom of the incubator at approximately 10 mJ / cm2. 2 The cells were then exposed to UV light for 10 min and cultured for an additional 2 days under the same conditions. The SCF expressed on the cell membrane was determined as follows. That is, after removing and washing the culture supernatant, the cells were fixed and SCF on the cell membrane surface was detected using an anti-SCF antibody labeled with HRP (horseradish peroxidase). The detection result was determined by measuring the absorbance at 450 nm of the reaction solution obtained by the reaction of the substrate with HRP, and the amount of SCF was determined. For comparison, a control group was also set up in which the cells were cultured in a medium not containing the above sample solution, and the amount of SCF was also measured for the UV-irradiated control and the non-UV-irradiated control. The results were shown as relative values, with the SCF value in the UV-irradiated control taken as 100%.

[0050] The results of Test Example 1 are shown in FIG. As shown in Figure 1, it was confirmed that the composition according to the present invention (Sample 1 of the present invention) has a significantly superior effect of inhibiting SCF synthesis compared to Comparative Samples 1 to 3. SCF is a protein expressed on the surface of epidermal cells, and when it binds to a receptor in melanocytes, it promotes melanin synthesis, suggesting that the composition according to the present invention exhibits a significantly superior whitening effect by inhibiting SCF synthesis.

[0051] Test Example 2: Prostaglandin E2 (PGE2) Inhibitory Effect Test Normal human epidermal keratinocytes (NHEK) were suspended in a dedicated medium Humedia-KG2 (Kurabo) and plated at 4 × 10 3 After seeding the cells in one well and culturing at 37°C for one day, the composition according to the present invention (sample 1 of the present invention) was added as a sample solution to the medium so that the final concentration of the solution was 0.3%, 0.45%, 0.6%, 0.9%, or 1.2% relative to the total volume of the medium, and the medium was further cultured for 24 hours. Next, 100 mJ / cm2 was applied from the bottom of the culture vessel. 2After irradiating with ultraviolet B rays of 10 ...

[0052] The results of Test Example 2 are shown in FIG. As shown in Figure 2, it was confirmed that the composition according to the present invention (Sample 1 of the present invention) has a significantly superior prostaglandin E2 inhibitory effect in a concentration-dependent manner. Since prostaglandins are inflammatory factors that stimulate the synthesis of melanin in pigment cells, it is suggested that the composition according to the present invention exerts a significantly superior whitening effect by inhibiting prostaglandin E2.

[0053] Test Example 3. Tyrosinase activity inhibition test Normal keratinocytes (NHEK) were cultured in a 24-well plate containing Humedia KG2 medium (Kurabo Industries, Ltd.) at 6 × 10 4 The next day, the supernatant was replaced with PBS(-) and exposed to UV light (50 mJ / cm 2 After UV irradiation, the supernatant was replaced with Humedia KB2 medium (Kurabo Industries, Ltd.) and the culture was continued. Next, the culture supernatant was collected after one day of culture (UV-irradiated supernatant). For comparison, a group was set up that was not UV-irradiated, and the other operations were carried out in the same way, and the culture supernatant was also collected (non-UV-irradiated supernatant). On the other hand, normal melanocytes NHEM were cultured in a 96-well microplate containing DermaLife medium (Kurabo Industries, Ltd.) at 5 × 10 3After seeding in each well, pre-cultured for 1 day under conditions of 37°C and 5.0% CO2, the composition according to the present invention (Sample 1 of the present invention) was added as a sample solution so that the final concentration of the solution was 0.3%, 0.45%, 0.6%, 0.9%, and 1.2% relative to the total volume of the medium, and the UV-irradiated supernatant of the keratinocytes was also added. After culturing for 3 days, the supernatant was discarded, washed once with PBS(-), and a surfactant (Triton X-100) and 5 mM L-dopa solution were added to react at 37°C, and the dopa value was measured at a wavelength of 490 nm using a microplate reader (Model 450, Bio-Rad). For comparison, a control (UV-irradiated control) was also set in which PBS(-) was added instead of the sample solution and the supernatant was irradiated with UV light, and a control (UV-unirradiated control) was also set in which PBS(-) was added instead of the sample solution and the supernatant was not irradiated with UV light. The test results were calculated as the relative values ​​of the DOPA values ​​in the non-UV control group and the groups to which each sample was added, with the DOPA value in the UV-irradiated control group being set at 100, and each value was expressed as the tyrosinase activity rate (%).

[0054] The results of Test Example 3 are shown in FIG. As shown in FIG. 3, it was confirmed that the composition of the present invention (sample 1 of the present invention) has a significantly superior tyrosinase activity inhibitory effect in a concentration-dependent manner, suggesting that the composition of the present invention exhibits a significantly superior whitening effect.

[0055] Test Example 4. Genetic Analysis Fibroblast NB1RGB cells were cultured in a 24-well plate at 6 x 10 4The cells were seeded per well. After 24 hours of culture, the composition according to the present invention (sample 1 of the present invention) was added as a sample solution, and ultraviolet irradiation was performed from the bottom. Culture was further performed for 24 hours. Here, sample 1 of the present invention was added so that the final concentration as a solution was 0.3%, 0.45%, 0.6%, 0.9%, and 1.2%. Then, the cells in each test group were recovered with 500 μL of Trizol reagent (Invitrogen). 100 μL of chloroform was added to the recovered cells, stirred and mixed, and centrifuged at 12,500 rpm and 4° C. for 15 minutes, and then 200 μL of the aqueous layer was separated. 250 μL of isopropanol was added to the recovered aqueous layer, stirred and mixed, and centrifuged at 12,500 rpm and 4° C. for 10 minutes to obtain a precipitate of total RNA. 1 mL of 75% cold ethanol was added to the total RNA, stirred and washed, and centrifuged at 12,500 rpm and 4° C. for 10 minutes to recover the precipitate. The collected total RNA was subjected to reverse transcription reaction using a specified kit (PrimeScript RT reagent Kit with gDNA Erase (Perfect Real Time) (Takara Bio)) to synthesize cDNA. Using the synthesized cDNA as a sample, expression of the target gene and expression of the internal standard β-actin gene were detected using Thermal Cycler Dice Real Time System Single (Takara Bio) and SYBR Premix Ex TaqTM II (Perfect Real Time) (Takara Bio). The test results were calculated by calculating the relative expression level of the target gene to the expression level of the β-actin gene. In this Test Example 4, expression of the heparan sulfate proteoglycan gene (HSPG) and expression of the hepatocyte growth factor (HGF) gene were evaluated.

[0056] The results of Test Example 4 are shown in FIGS. As shown in Figure 4, it was confirmed that the composition according to the present invention (sample 1 of the present invention) has a significantly excellent effect of enhancing the expression of HSPG genes, the expression of which is reduced by ultraviolet light, in a concentration-dependent manner. Since heparan sulfate proteoglycan (HSPG) is known to play a role in blocking melanin synthesis stimulants from the dermis, it is suggested that the composition according to the present invention maintains the health of the basement membrane by enhancing the expression of HSPG genes, thereby exhibiting a significantly excellent whitening effect.

[0057] Furthermore, as shown in Figure 5, it was confirmed that the composition of the present invention (invention sample 1) had an exceptionally excellent effect of suppressing HGF gene expression in a concentration-dependent manner. Since HGF is a melanin synthesis stimulator, it is suggested that the composition of the present invention exerts an exceptionally excellent skin whitening effect by suppressing HGF gene expression.

[0058] Test Example 5. Evaluation test of SOD-like activity A test solution was prepared by mixing 0.15 mL of 1M Tris-HCl buffer, 0.30 mL of 1 mM ethylenediaminetetraacetic acid disodium salt solution, 0.30 mL of 1 mM xanthine solution, 0.20 mL of 0.75 mM nitroblue tetrazolium solution, 0.10 mL of the composition according to the present invention (invention sample 1), and 1.90 mL of purified water. The concentrations of the inventive sample 1 were adjusted so that the final concentrations in the test solution were 0.3%, 0.45%, 0.6%, 0.9%, and 1.2%. In addition, a mixed solution (control) having the same composition as the above test solution was prepared, except that 0.10 mL of 30% 1,3-butylene glycol was used instead of the inventive sample 1 in the test solution. In addition, a mixed solution (positive control) was prepared having the same composition as the above test solution, except that 0.10 mL of a 0.875 Unit / mL superoxide dismutase solution was used in place of the inventive sample 1 (0.10 mL) in the test solution. The above test solutions or the mixtures without sample were incubated at 37℃, and then 0.05mL of 1Unit / mL xanthine oxidase solution was added. After a certain time (5 minutes), the absorbance of each test solution at 570nm (an index of the amount of superoxide anion in the test solution) was measured. The results were shown as a relative value (%) of the absorbance of each test solution when the absorbance of the control mixture was taken as 100.

[0059] The results of Test Example 5 are shown in FIG. As shown in FIG. 6, it was confirmed that the composition according to the present invention (Invention Sample 1) had significantly superior SOD-like activity in a concentration-dependent manner.

[0060] Test Example 6: Evaluation test of glycation inhibition effect In this test, the effect of inhibiting the generation of AGE, that is, the effect of inhibiting protein glycation, was evaluated by the generation of fluorescence from BSA (albumin) via glucose. First, 40 μL of the composition according to the present invention (sample 1 of the present invention), 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. The sample solutions were prepared so that the final concentrations of sample 1 of the present invention were 0.3%, 0.45%, 0.6%, 0.9%, and 1.2%. Next, the sample solutions were 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. In addition, the same procedure was performed for a control where a 30% 1,3-butylene glycol solution was used instead of the inventive sample 1, and the relative values ​​(%) of the fluorescence measurement values ​​of each sample solution to the fluorescence measurement values ​​obtained here were calculated to obtain the protein glycation rate (%).

[0061] The results of Test Example 6 are shown in FIG. As shown in FIG. 7, it was confirmed that the composition according to the present invention (Sample 1 of the present invention) has an exceptionally excellent glycation inhibitory effect in a concentration-dependent manner.

[0062] Test Example 7. Evaluation test of the effect of inhibiting ETBR synthesis in melanocytes In Test Example 7, the effect of inhibiting the synthesis of ETBR, which is a receptor for endothelin-1 (ET-1), known as a melanocyte stimulating factor, is evaluated. Normal human epidermal melanocytes were cultured in a 96-well microplate at 1 × 10 cells using DermaLife (registered trademark) [manufactured by Kurabo] containing a proliferation additive. 4 After seeding at 1000 cells / well and pre-cultivating for 1 day under conditions of 37°C and 5.0% CO2, the composition according to the present invention (Sample 1 of the present invention) was adjusted to a prescribed concentration (final concentration as a solution of 0.3%, 0.45%, 0.6%, 0.9%, 1.2%) (registered trademark) [Kurabo] was added. Then, ultraviolet light of about 15 mJ / cm2 was irradiated from the bottom of the culture vessel using a UV-B lamp (TL20W / 12RS manufactured by Philips). Then, the cells were cultured for another 2 days under the same conditions. Then, immunodetection was performed using an ETBR antibody. That is, after washing with PBS(-), the cells were fixed by treatment with a 15% neutral buffered formalin solution for 30 minutes, permeabilized with a 0.2% Triton X-100 solution for 1 hour, blocked by treatment with a 5-fold diluted Blocking One P (Nacalai Tesque) solution for 2 hours, and then the ETBR antibody was added and the cells were left to stand at 4°C for a whole day and night. After that, the plate was washed with PBS(-), a fluorescently labeled secondary antibody was added, and the plate was left to stand in the dark for a certain period of time. After washing with PBS(-), the plate was measured for fluorescence intensity. First, the fluorescent label of the secondary antibody (Alexa Fluor546) was measured at Ex=544nm and Em=590nm (fluorescence microplate reader (Fluoroscan Ascent, Thermo Fisher Scientific)), and then DNA staining with Hoechst33342 was performed, and measurements were made at Ex=355nm and Em=460nm. The degree of ETBR generation was calculated by dividing the fluorescence intensity of Alexa Fluor546 in each test group by the fluorescence intensity of Hoechst33342. The same operation as above was performed for the case where PBS(-) was added instead of the sample solution and no sample was added (control), and the relative value of the degree of ETBR generation when each sample was added to the degree of ETBR generation in the ultraviolet irradiated group was calculated, and this was taken as the amount of ETBR generation (%).

[0063] The results of Test Example 7 are shown in Figure 8. As shown in Figure 8, it was confirmed that the composition according to the present invention (Sample 1 of the present invention) has a remarkably excellent effect of inhibiting ETBR synthesis in melanocytes in a concentration-dependent manner. This makes it difficult for stimulation by endothelin to be transmitted, thereby inhibiting melanin synthesis by endothelin and leading to the effect of preventing pigmentation.

[0064] Test Example 8. Evaluation test of inhibition of MC1R synthesis in melanocytes In Test Example 8, the effect of inhibiting the synthesis of MC1R, the receptor for melanocyte-stimulating hormone αMSH, was evaluated. Normal human epidermal melanocytes were cultured in a 96-well microplate at 1 × 10 cells using DermaLife (registered trademark) [manufactured by Kurabo] containing a proliferation additive. 4After seeding at 100 cells / well and pre-cultivating for 1 day under conditions of 37°C and 5.0% CO2, DermaLife (registered trademark) [Kurabo] was added, which had been adjusted to a specified concentration (as a solution). After that, ultraviolet light of about 15 mJ / cm2 was irradiated from the bottom of the incubator using a UV-B lamp (TL20W / 12RS, Philips). After that, the cells were cultured for another 2 days under the same conditions. After that, immunodetection was performed using MC1R antibody. That is, after washing with PBS(-), the cells were fixed by treating with 15% neutral buffered formalin solution for 30 minutes, permeabilized with 0.2% Triton X-100 solution for 1 hour, blocked by treating with 5-fold diluted Blocking One P (Nacalai Tesque) solution for 2 hours, added with MC1R antibody, and left to stand at 4°C for a day and night. After that, the cells were washed with PBS(-), and a fluorescently labeled secondary antibody was added and left to stand for a certain period of time in the dark. After washing with PBS(-), the fluorescence intensity was measured. First, the fluorescent label of the secondary antibody (Alexa Fluor 546) was measured at Ex = 544 nm, Em = 590 nm (fluorescence microplate reader (Fluoroscan Ascent, Thermo Fisher Scientific)), and then DNA staining with Hoechst 33342 was performed and measured at Ex = 355 nm, Em = 460 nm. The degree of MC1R production was calculated by dividing the fluorescence intensity of Alexa Fluor 546 in each test group by the fluorescence intensity of Hoechst 33342. The same operation as above was performed for the case where PBS (-) was added instead of the sample solution and no sample was added (control), and the relative value of the degree of MC1R production when each sample was added to the degree of MC1R production in the ultraviolet irradiated group was calculated and defined as the amount of MC1R produced (%).

[0065] The results of Test Example 8 are shown in Figure 9. As shown in Figure 9, it was confirmed that the composition according to the present invention (Sample 1 of the present invention) has a remarkably excellent effect of inhibiting MC1R synthesis in melanocytes in a concentration-dependent manner. This makes it difficult for stimulation by αMSH to be transmitted, thereby inhibiting melanin synthesis by αMSH and leading to the effect of preventing pigmentation.

[0066] Formulation example 1. Lotion [Ingredients] Part Jojoba oil 1.0 Polyoxyethylene (5.5) Cetyl Alcohol 5.0 Methylparaben 0.1 Yeast culture extract of Preparation Example 1 1.0 Fermented product of Production Example 5 1·0 Fermentation product of Production Example 6 1·0 Glycerin 5.0 1,3-Butylene glycol 5.0 Sodium citrate 0.2 Purified water (enough to make the total amount 100 parts)

[0067] Formulation example 2: Lotion A lotion was obtained in the same manner as in Formulation Example 1, except that 1.0 part of the yeast culture extract of Production Example 2 was used instead of the yeast culture extract of Production Example 1 contained in Formulation Example 1.

[0068] Formulation example 3: Lotion A lotion was obtained in the same manner as in Formulation Example 1, except that 2.0 parts of the yeast culture extract of Production Example 3 was used instead of the yeast culture extract of Production Example 1 contained in Formulation Example 1.

[0069] Formulation example 4. Emulsion [Ingredients] Part Squalane 5.0 Hexalan 3.0 Jojoba oil 1.0 Camellia oil 1.5 Polyoxyethylene (20) Sorbitan Monostearate 1.0 Lipophilic glyceryl stearate 1.0 Hydrogenated soy lecithin 1.5 Yeast culture extract of Preparation Example 1 0.5 Fermentation product of Production Example 5 0.5 Fermentation product of Production Example 6 0.5 Glycerin 3.0 1,3-Butylene glycol 2.0 Carboxymethylcellulose 0.3 Xanthan gum 0.2 Sodium hyaluronate 0.01 Purified water (enough to make the total amount 100 parts)

[0070] Formulation example 5. Cream [Ingredients] Part Olive oil 5.0 Jojoba oil 5.0 Squalane 5.0 Behenyl Alcohol 2.0 Palmitic acid 2.5 Yeast culture extract powder of Preparation Example 2 0.5 Fermentation product of Production Example 5 0.5 Fermentation product of Production Example 6 0.5 Lactic acid fermented rice 2.0 Hydrogenated Lecithin 0.5 Carboxyvinyl polymer 0.3 Sodium alginate 0.2 Seaweed extract 2.0 Methylparaben 0.15 Ethylparaben 0.03 Purified water (enough to make the total amount 100 parts)

[0071] Formulation example 7. Liquid foundation [Ingredients] Part Stearic acid 2.4 Propylene glycol monostearate 2.0 Cetostearyl alcohol 0.2 Liquid Lanolin 2.0 Liquid Paraffin 3.0 Isopropyl myristate 8.5 Propylparaben 0.05 Yeast culture extract powder of Preparation Example 3 0.5 Fermentation product of Production Example 5 0.5 Fermentation product of Production Example 6 0.5 Sodium carboxymethylcellulose 0.2 Bentonite 0.5 Propylene glycol 4.0 Triethanolamine 1.1 Methylparaben 0.1 Titanium dioxide 8.0 Talc 4.0 Color pigment (appropriate amount) Purified water (enough to make the total amount 100 parts)

[0072] Formulation example 8. Body shampoo [Ingredients] Part Sodium N-lauroylmethylalanine 25.0 Potassium coconut oil fatty acid solution (40%) 26.0 Coconut oil fatty acid diethanolamide 3.0 Methylparaben 0.1 Yeast culture extract powder of Preparation Example 1 0.5 Fermentation product of Production Example 5 0.5 Fermentation product of Production Example 6 0.5 1,3-Butylene glycol 2.0 Purified water (enough to make the total amount 100 parts)

[0073] Formulation example 9. Hair shampoo [Ingredients] Part Sodium N-cocoyl methyl taurate 10.0 Sodium polyoxyethylene (3) alkyl ether sulfate 20.0 Lauryl dimethylaminoacetate betaine 10.0 Coconut oil fatty acid diethanolamide 4.0 Methylparaben 0.1 Citric acid 0.1 Yeast culture extract powder of Preparation Example 1 0.5 Fermentation product of Production Example 5 0.5 Fermentation product of Production Example 6 0.5 1,3-Butylene glycol 2.0 Purified water (enough to make the total amount 100 parts)

[0074] Formulation example 10. Hair conditioner [Ingredients] Part Polyoxyethylene (10) hydrogenated castor oil 1.0 Distearyldimethylammonium chloride 1.5 Stearyltrimethylammonium chloride 2.0 Glyceryl 2-ethylhexanoate 1.0 Cetanol 3.0 Stearyl alcohol 1.0 Methylparaben 0.1 Yeast culture extract powder of Preparation Example 4 0.5 Fermentation product of Production Example 5 0.5 Fermentation product of Production Example 6 0.5 1,3-Butylene glycol 5.0 Purified water (enough to make the total amount 100 parts)

[0075] Formulation example 11. Sheet mask A sheet mask is obtained by impregnating a nonwoven fabric with the following components. [Ingredients] Part Glycerin 3.0 1,3-Butylene glycol 2.0 Methylparaben 0.2 Citric acid 0.1 Sodium citrate 0.3 Xanthan gum 1.0 Water-soluble collagen 1.0 Sodium hyaluronate 1.0 Yeast culture extract of Preparation Example 1 0.5 Fermentation product of Production Example 5 0.5 Fermentation product of Production Example 6 0.5 Purified water (enough to make the total amount 100 parts)

Claims

[Claim 1] The skin topical preparation contains as active ingredients a culture extract or a concentrate or dried powder thereof of yeast derived from Japanese lily, a lactic acid bacteria fermentation product of lotus seeds belonging to the Nelumbo genus of the Nelumbo family, and a yeast fermentation product of the flower parts of day lilies and / or day lilies belonging to the Hemerocallis genus of the Liliaceae family.

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