JP7610262B2 - Skin preparations - Google Patents

Description

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

Traditionally, research and development has been conducted on ingredients derived from natural products as active ingredients to be incorporated into topical skin preparations. However, these naturally derived ingredients have had issues with efficacy, stability, and other aspects when used as active ingredients in topical skin preparations.

As a result of intensive research aimed at solving the above problems, the present inventors have newly discovered that a combination of yeast culture extract, a fermented product of a plant of the genus Lotus, and a plant of the genus Daylily has synergistic effects such as whitening and antioxidant effects, and is useful as an active ingredient in a skin care product.

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 a yeast derived from a lily of the valley, 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 Publication No. 2001-354570 Patent Publication No. 2002-234828 Patent Publication No. 2005-298489 Patent Publication No. 2008-050326

The present invention is a skin topical agent containing as active ingredients a culture extract or concentrate or dried powder 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 day lily in the genus Helianthus in the family Liliaceae.

The present invention provides a skin care product with outstanding whitening and antioxidant effects by combining and blending as active ingredients a culture extract or concentrate or dried powder 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 day lily in the genus Helianthus in the family Liliaceae.

1 shows the results of a test evaluating the composition according to the present invention for its inhibition of synthesis of SCF (Steam Cell Factor). 1 shows the results of a test evaluating the prostaglandin E2 (PGE2) synthesis inhibition of the composition according to the present invention. 1 shows the results of a test evaluating the melanin synthesis inhibition of the composition according to the present invention. 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. 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. 1 shows the results of a test evaluating the SOD-like activity of a composition according to the present invention. 1 shows the results of a test evaluating the glycation inhibition effect of a composition according to the present invention. 1 shows the results of a test evaluating the composition according to the present invention for its ability to inhibit ETBR synthesis in melanocytes. 1 shows the results of a test evaluating the composition according to the present invention for its ability to inhibit MC1R synthesis in melanocytes.

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 yeast derived from Lilium japonicum flowers 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.

The yeast culture extract or its concentrate or dried product can be prepared as follows. The yeast culture extract can be produced by culturing yeast in a culture medium, a hydrolysis method in which the cultured yeast is solubilized with acid or alkali to dissolve the cell components, an autolysis method that uses proteolytic enzymes present in the yeast cell, an enzymatic method that uses enzymes such as proteolytic enzymes, or a combination of these methods.

The carbon source used in culturing yeast is not particularly limited, and examples of the carbon source include glucose, fructose, or galactose, as well as oligosaccharides and polysaccharides that contain these sugars. In addition to the carbon source, a nitrogen source may also be added, and examples of the nitrogen source include amino acids and peptones.

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

A concentrate of yeast culture extract can be obtained by concentrating yeast culture liquid or a liquid obtained by hydrolysis method, yeast autolysis method or enzymatic method using a vacuum concentrator, etc.

A dried yeast culture extract can be obtained by drying a yeast culture solution or a concentrated solution thereof using a vacuum drying method, a spray drying method, or the like.

The dried yeast culture extract may also be dried with an excipient added for the purposes of chemical stability and low hygroscopicity. The excipient may be a sugar such as starch, glucose, crystalline cellulose, lactose, or dextrin, or a sugar alcohol such as sorbitol, xylitol, erythritol, mannitol, maltitol, or lactitol, but any substance that can be mixed with the yeast culture extract to form a dry powder may be used.

The plant used as the raw material for the lotus fermentation 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.

The plant of the genus Hemerocallis and 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.

The fermentation process can be carried out as follows. First, as the source of assimilation for fermentation, the plant parts (seeds, buds, or petals) themselves (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.

In the present invention, the microorganisms used for fermenting lotus seeds are lactic acid bacteria, such as 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. citreum, etc.; Streptococcus faecalis, Streptococcus pyogenes, etc.; Enterococcus caseliflavus, Enterococcus sulfreus, etc.; Lactococcus plantarum, Lactococcus rafinolactis, etc.; Weissella confusa, Weissella kandleri, etc.; Atopobium Examples of lactic acid bacteria include lactic acid bacteria of the genus Atopobium, such as Atopobium minutum and Atopobium parvulus; lactic acid bacteria of the genus Vagococcus, such as Vagococcus fluvialis and Vagococcus salmoninarum; and lactic acid bacteria of the genus Pediococcus, such as Pediococcus damnosus and Pediococcus pentosaceus.

In the present invention, the yeast used for fermenting the buds or the buds and petals of Helianthus oryzae includes, 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, Examples of yeasts include yeasts of the genus Candida, such as Aureobasidium scottii, and yeasts of the genus Aureobasidium, such as Aureobasidium Pullulans, Aureobasidium mansonii, and Aureobasideium microstictum. 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.

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

The sterilized suspension is placed in a fermentation tank, and the microorganisms are inoculated into it to cause fermentation. The appropriate amount of microorganism to be inoculated is ¹⁰⁷ to ¹⁰⁸ cells/mL. If the inoculation amount is greater than the above range, the fermentation progress time is almost unchanged, whereas if it is less than the above range, it takes a long time to complete the fermentation, which is undesirable.

The fermentation temperature is generally in the range of 5 to 50°C, preferably in the range of 20°C to 40°C, which is the optimum temperature for growth of each microorganism (e.g., 30°C to 40°C for lactic acid bacteria, and 25°C to 30°C for yeast). The number of fermentation days is generally in the range of 1 to 10 days, preferably 2 to 5 days, at the optimum temperature. If the fermentation period 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 period is longer than 10 days, not only will no further increase in effectiveness be observed, but coloring and an increased fermentation odor will occur, both of which are undesirable.

When preparing a fermented product, in order to make the components of the target used part more effectively available as a source of assimilation for the lactic acid bacteria or yeast, the suspension or extract solution may be subjected to a hydrolysis treatment using 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 at the same time as inoculating them.

The extract, hydrolysate or fermentation product prepared as described above may be used as is after generally adjusting the pH to 3 to 9, or may be concentrated under reduced pressure or the like to a desired concentration. It may also be dried by standard methods such as spray drying.

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

The composition of the present invention, which is a combination of the yeast culture extract or its concentrate or dried product 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, make-up 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 even bath additives, but the present invention is not limited to these.

In addition to the composition of the present invention, the topical skin preparation may contain, as necessary, ingredients used in topical skin preparations, such as oily ingredients, surfactants (synthetic and natural), moisturizers, thickeners, preservatives and disinfectants, powder ingredients, UV absorbers, antioxidants, colorants, fragrances, etc. Furthermore, as long as the effectiveness and characteristics of the composition of the present invention are not impaired, there is no problem in combining it with other physiologically active ingredients.

Here, 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, These include hydrocarbons such as petrolatum, paraffin wax, and squalane; fatty acids such as myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, and eicosenoic acid; higher alcohols such as lauryl alcohol, cetanol, and stearyl alcohol; synthetic esters and synthetic triglycerides such as isopropyl myristate, isopropyl palmitate, butyl oleate, cetyl isooctanoate, 2-ethylhexyl glyceride, and higher fatty acid octyldodecyl (octyldodecyl stearate, etc.).

Examples of surfactants 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. Anionic surfactants such as tert-butyl ether salts and polyoxyethylene alkyl phenyl ether phosphates; cationic 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 polyethylene polyamine 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 can be used.

In addition, the emulsifier or emulsifier aid may be a stevia derivative such as enzyme-treated stevia, saponin or its derivative, casein or its salt (sodium, etc.), sugar and protein complex, sucrose or its ester, lactose, soy-derived water-soluble polysaccharide, soy-derived protein and polysaccharide complex, lanolin or its derivative, cholesterol, stevia derivative (stevia enzyme fermentation product, etc.), silicate (aluminum, magnesium, etc.), carbonate (calcium, sodium, etc.), saponin and its derivative, lecithin and its derivative (hydrogenated lecithin, etc.), lactic acid bacteria fermented rice, lactic acid bacteria fermented germinated rice, lactic acid bacteria fermented grains (wheat, beans, millet, etc.), etc.

Moisturizing agents include, for example, 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 (for example, 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.

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 (white algae) 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 carboxymethylcellulose and hydroxyethylcellulose; 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.

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, fermented radish broth, and ethanol or 1,3-butylene glycol derived from plants such as sugar cane.

Powder components include, for example, 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.), 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 antifoaming agents include ethanol, isopropanol, disiloxane, dimethylpolycyclohexane, dimethicone silicate, trisiloxane, silica silylate, dimethicone, trimethylsiloxysilicate, and DPG isobornyl ether.

Antioxidants include, for example, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, extract of Brussels sieboldii, extract of Bletilla serrata root, extract of peony root, vitamin E and its derivatives (for example, vitamin E nicotinate, vitamin E linoleate, etc.).

Examples of chelating agents include trisodium ethylenediamine hydroxyethyl triacetate, edetic acid or its salts, gluconic acid, phytic acid, sodium polyphosphate, sodium metaphosphate, and tetrasodium hydroxyethane diphosphonate.

Examples of pH adjusters include citric acid or its salts, lactic acid or its salts, glycolic acid, succinic acid, hydrochloric acid, monoethanolamine, diethanolamine, triethanolamine, sodium hydroxide, potassium hydroxide, etc.

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

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

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

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

Production 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 dissolve the concentrate, 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.

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 heat sterilized. Lactic acid bacteria (Lactobacillus plantarum) were inoculated into this suspension at ¹⁰⁸ cells/mL and cultured at 37°C for 3 days under a nitrogen stream. After the culture was completed, the mixture was heat sterilized and the culture solution was filtered to obtain 1415 g of a solution of lactic acid bacteria fermented lotus seeds (solid concentration 2.53%).

Production Example 6. Fermented Day Lily Flowers 150 g of shredded Day Lily flowers (including buds and petals) were mixed with 1500 g of purified water and extracted at 40° C. for 3 hours. The extracted suspension was then heat sterilized, and yeast (Saccharomyces cerevisiae) was inoculated at 10 ⁸ cells/mL and cultured at 30° C. for 3 days under a nitrogen stream. After the culture was completed, the mixture was heat sterilized, filtered, deodorized, and decolorized to obtain 1110 g of a brown, transparent yeast fermentation solution (solid concentration 4.0%). This was diluted 3 times with purified water to obtain a yeast fermentation solution.

Example 1. Preparation of composition The extract solution of Preparation Example 1, the fermentation product solution of Preparation Example 5, and the fermentation product solution of Preparation Example 6 were mixed in equal amounts, and the insoluble matter was filtered off to obtain 2920 g of a brown, transparent extract solution mixture (solid content concentration: 2.53%).

The composition according to Example 1 prepared as described above was used as Sample 1 of the present invention and its effectiveness was evaluated as follows. In addition, as comparative samples for Sample 1 of the present invention, the yeast culture extract of Production Example 1 was used as Comparative Sample 1, the fermented lotus seed product of Production Example 5 was used as Comparative Sample 2, and the fermented flower part of Day Lily of the Valley ...

Test Example 1. Inhibitory effect on SCF synthesis in epidermal cells Normal human skin-derived epidermal cells (NHEK) were seeded at 8 x ¹⁰³ cells/well in a 96-well microplate containing HuMedia KG2 medium (Kurabo Industries, Ltd.), and pre-cultured for 1 day under conditions of 37°C and 5.0% _CO2 , and then 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 in the medium was 1.2%. Then, the cells were irradiated with ultraviolet light at about 10 mJ/ ^cm2 from the bottom of the incubator using a UV-B lamp (TL20W/12RS, Philips Corporation), and further cultured for 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 the 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 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 control irradiated with ultraviolet light (UV-irradiated control) and the control not irradiated with UV (UV-unirradiated control). The results were expressed as relative values, with the SCF value in the UV-irradiated control taken as 100%.

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. Since SCF is a protein expressed on the surface of epidermal cells, and when this binds to a receptor in melanocytes, it promotes melanin synthesis, it is suggested that the composition according to the present invention exhibits a significantly superior whitening effect by inhibiting SCF synthesis.

Test Example 2. Prostaglandin E2 (PGE2) Inhibitory Effect Test Normal human epidermal cells (NHEK) were suspended in a special medium Humedia-KG2 (Kurabo) and seeded in a 96-well plate at 4 x ¹⁰³ cells/well, and then cultured at 37°C for 1 day. Then, the composition according to the present invention (Sample 1 of the present invention) was added to the medium 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 medium was further cultured for 24 hours. Next, the bottom of the culture vessel was irradiated with 100mJ/ ^cm2 ultraviolet B waves, and after further culture for 2 days, the amount of PGE2 secreted into the culture supernatant was measured using a PGE2 measurement kit (Caiman Chemical). For comparison, a control (UV-irradiated control) was prepared by adding PBS(-) instead of the sample solution and irradiating with ultraviolet light, and a non-irradiated control (non-UV-irradiated control) was prepared by adding PBS(-) instead of the sample solution and not irradiating with ultraviolet light. The test results were calculated by calculating the relative amount of PEG2 in the non-UV-irradiated control and each sample-added group, with the amount of PEG2 in the UV-irradiated control being taken as 100, and each value was expressed as the PGE2 synthesis rate (%).

The results of Test Example 2 are shown in FIG.
As shown in Fig. 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 prostaglandin is an inflammatory factor that stimulates 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.

Test Example 3. Tyrosinase activity inhibition test Normal keratinocytes NHEK were seeded at 6 x ¹⁰⁴ cells/well in a 24-well plate containing Humedia KG2 medium (Kurabo Industries, Ltd.), and the next day the supernatant was replaced with PBS (-) and UV irradiation (50 mJ/ ^cm2 ) was performed. 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 1 day of culture (UV-irradiated supernatant). In addition, a section was set up for comparison where UV irradiation was not performed, and the other operations were performed in the same manner, and the culture supernatant was also collected (UV-unirradiated supernatant).
On the other hand, normal melanocytes NHEM were seeded in a 96-well microplate containing DermaLife medium (Kurabo) at 5 x ¹⁰³ cells/well, and pre-cultured for one day under conditions of 37°C and 5.0% _CO2. Then, the composition according to the present invention (Sample 1 of the present invention) was added as a sample solution to the total volume of the medium at final concentrations of 0.3%, 0.45%, 0.6%, 0.9%, and 1.2% as a solution, and the UV-irradiated supernatant of the keratinocytes was also added. After culturing for three days, the supernatant was discarded, washed once with PBS(-), and a surfactant (Triton X-100) and 5 mM L-dopa solution were added and reacted at 37°C. The dopa value was measured at a wavelength of 490 nm using a microplate reader (Model 450, Bio-Rad). For comparison, a control was also prepared in which PBS(-) was added instead of the sample solution and the supernatant was irradiated with ultraviolet light (UV-irradiated control), and a control was also prepared in which PBS(-) was added instead of the sample solution and the supernatant was not irradiated with ultraviolet light (non-UV-irradiated control).The test results were calculated by calculating the relative values of the DOPA values in the non-UV-irradiated control and each sample-added group, with the DOPA value in the UV-irradiated control being taken as 100, and each value was expressed as the tyrosinase activity rate (%).

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.

Test Example 4. Genetic Analysis Fibroblast cells NB1RGB were seeded in a 24-well plate at 6 x ¹⁰⁴ cells/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 light was irradiated from the bottom. Culture was continued for another 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 collected with 500 μL of Trizol reagent (Invitrogen). 100 μL of chloroform was added to the collected cells, stirred and mixed, and centrifuged at 12,500 rpm and 4 ° C for 15 minutes, and then 200 μL of only the aqueous layer was collected. 250 μL of isopropanol was added to the collected 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 recovered total RNA was reverse transcribed 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, the expression of the target gene and the 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, the expression of the heparan sulfate proteoglycan gene (HSPG) and the expression of the Hepatocyte Growth Factor (HGF) gene were evaluated.

The results of Test Example 4 are shown in FIG.
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 rays, 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 integrity of the basement membrane by enhancing the expression of HSPG genes, thereby exhibiting a significantly excellent whitening effect.

As shown in FIG. 5, the composition according to the present invention (sample 1 of the present invention) was confirmed to have a significantly superior effect of suppressing HGF gene expression in a concentration-dependent manner. Since HGF is a melanin synthesis stimulant, it is suggested that the composition according to the present invention exerts a significantly superior whitening effect by suppressing the expression of the HGF gene.

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. 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 (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.

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.

Test Example 6. Evaluation of the effect of inhibiting glycation 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 (albumin) via glucose. First, 40 μL of the composition according to the present invention (sample 1 of the present invention), 40 μL of a 50 mg/mL BSA solution, 40 μL of a 2.5 M glucose solution, and 80 μL of a 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 present invention sample 1, and the relative value (%) of the fluorescence measurement value of each sample solution to the fluorescence measurement value obtained here was calculated and defined as the protein glycation rate (%).

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 (invention sample 1) has a significantly excellent glycation inhibitory effect in a concentration-dependent manner.

Test Example 7. Test to evaluate 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 stimulatory factor for melanocytes, is evaluated.
Normal human epidermal melanocytes were seeded in a 96-well microplate at 1 x ¹⁰⁴ cells/well using a proliferation additive-containing DermaLife (registered trademark) [Kurabo], and pre-cultured for one day under conditions of 37°C and 5.0% _CO2 , and then the composition according to the present invention (invention sample 1) was added at a specified concentration (final concentration as a solution: 0.3%, 0.45%, 0.6%, 0.9%, 1.2%) (registered trademark) [Kurabo]. Then, ultraviolet light of about 15 mJ/cm2 was irradiated from the bottom of the incubator using a UV-B lamp (TL20W/12RS, Philips). Then, the cells were cultured for another two days under the same conditions. Then, immunological detection was performed using an ETBR antibody. That is, after washing with PBS(-), the cells were fixed by treating with 15% neutral buffered formalin 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 ETBR antibody, and left at 4°C for a day and night. Then, washed with PBS(-), fluorescently labeled secondary antibody was added and further left in the dark for a certain period of time. After washing with PBS(-), the fluorescence intensity was measured. First, the fluorescent label of the secondary antibody (Alexa Fluor546) was measured at Ex=544nm, Em=590nm (fluorescence microplate reader (Fluoroscan Ascent, Thermo Fisher Scientific)), then DNA staining with Hoechst33342 was performed, and measurements were made at Ex=355nm, Em=460nm. The degree of ETBR formation was calculated by dividing the fluorescence intensity of Alexa Fluor 546 in each test group by the fluorescence intensity of Hoechst 33342. The same procedure as above was performed for a control group in which PBS(-) was added instead of the sample solution, and the relative value of the degree of ETBR formation when each sample was added to the degree of ETBR formation in the UV-irradiated group was calculated, and this was taken as the amount of ETBR formed (%).

The results of Test Example 7 are shown in Figure 8. As shown in Figure 8, it was confirmed that the composition of the present invention (Sample 1 of the present invention) has a significantly superior 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.

Test Example 8. Test to evaluate inhibition of MC1R synthesis in melanocytes In Test Example 8, the effect of inhibiting the synthesis of MC1R, the receptor for αMSH, a melanocyte-stimulating hormone, was evaluated.
Normal human epidermal melanocytes were seeded in a 96-well microplate at 1 x ¹⁰⁴ cells/well using DermaLife (registered trademark) [Kurabo] containing a proliferation additive, and pre-cultured for one day under conditions of 37°C and 5.0% _CO2 , after which DermaLife (registered trademark) [Kurabo] prepared to a prescribed concentration (as a solution) of the sample solution was added. Then, ultraviolet light of about 15 mJ/cm2 was irradiated from the bottom of the incubator using a UV-B lamp (TL20W/12RS, Philips). Then, the cells were cultured for another two days under the same conditions. Then, immunodetection was performed using MC1R antibody. That is, after washing with PBS(-), the cells were fixed by treatment with 15% neutral buffered formalin solution for 30 minutes, permeation treatment with 0.2% Triton X-100 solution for one hour, blocking treatment with 5-fold diluted Blocking One P (Nacalai Tesque) solution for two hours, MC1R antibody was added, and the cells were left to stand at 4°C for one 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 fluorescence intensity of Alexa Fluor546 in each test group was divided by the fluorescence intensity of Hoechst33342 to determine the degree of MC1R production. The same procedure was performed for the case where PBS(-) was added instead of the sample solution (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 determined, and this was taken as the amount of MC1R produced (%).

The results of Test Example 8 are shown in Figure 9. As shown in Figure 9, it was confirmed that the composition of 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.

Formulation Example 1. Lotion [Ingredients] Parts 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
Fermented 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)

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.

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.

Formulation Example 4. Emulsion [Ingredients] Parts 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
Carboxymethyl cellulose 0.3
Xanthan gum 0.2
Sodium hyaluronate 0.01
Purified water (enough to make the total amount 100 parts)

Formulation Example 5. Cream [Ingredients] Parts 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)

Formulation Example 7. Liquid Foundation [Ingredients] Parts 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 (total amount is 100 parts)

Formulation Example 8. Body Shampoo [Ingredients] Parts N-Lauroylmethylalanine Sodium 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)

Formulation Example 9. Hair Shampoo [Ingredients] Parts Sodium N-coconut oil fatty acid 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)

Formulation Example 10. Hair conditioner [Ingredients] Parts 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)

Formulation Example 11. Sheet Mask A sheet mask is obtained by impregnating a nonwoven fabric with the following ingredients.
[Ingredients] Parts 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 (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.