JP2021024805A - External preparation for skin - Google Patents

Abstract

To provide an external preparation for skin that is derived from a natural product and comprises a plurality of active ingredients that exert a synergistic effect on whitening.SOLUTION: An external preparation for skin comprises a lactic acid bacterium fermented product of Arnica montana, which is a plant in Asteraceae (Compositae) family, Arnica genus, or an extract thereof, and a melanin production inhibitor or a tyrosinase activity inhibitor as active ingredients.SELECTED DRAWING: None

Description

The present invention relates to an external preparation for skin containing an Arnica fermented product and a melanin production inhibitor or a tyrosinase activity inhibitor as active ingredients.

Conventionally, skin aging, dryness, rough skin, stains, and buckwheat have been caused by internal factors such as inactivation of cell proliferation and differentiation with aging, decreased hormone secretion, and decreased amount of extracellular matrix components, and the sun. It is known that this is a phenomenon caused by complex entanglement of external factors such as cell / tissue damage or inflammation caused by active oxygen induced by light (ultraviolet rays).

Here, active oxygen, which is an external factor of skin aging, not only directly damages skin cells, but also denatures or cross-links extracellular matrix components and the like, resulting in the formation of wrinkles and a decrease in skin elasticity, and further. Is also known to cause various damages to the skin, such as inducing abnormal deposition of melanin pigments and causing spots and freckles.

Conventionally, in order to prevent skin aging and keep the skin in a healthy and youthful state, the use of various active ingredients has been proposed, and the skin containing these anti-aging ingredients, moisturizing ingredients and ingredients that improve age spots. External preparations are on the market. For example, antioxidants such as vitamin C, vitamin E, superoxide dismutase, catalase; tyrosinase activity inhibitors such as glycyrrhizic acid; various UV absorbers; α-hydroxycarboxylic acid, placenta extract, γ-amino Cell-activating components such as -β-hydroxybutyric acid; extracellular matrix components such as collagen, elastin, hyaluronic acid; moisturizers such as urea. Arbutin, linoleic acid, kojic acid, and the like are known as substances that suppress the occurrence of pigmentation such as skin spots and freckles, and are widely used as active ingredients of melanin production inhibitors.

However, these conventional ingredients have problems in terms of stability, safety, or effectiveness when actually applied to the skin when viewed as a compounding raw material for an external preparation for skin.

As a result of diligent studies on the above-mentioned problems of the prior art, the present inventors have found that Arnica montana, which is a plant of the Asteraceae (Compositae) Arnica genus, or an extract thereof, is a fermented lactic acid bacterium and an inhibitor of melanin production. Alternatively, they have found that the combination of tyrosinase activity inhibitors has an excellent synergistic effect of whitening effect, and have completed the present invention.

Conventionally, Patent Document 1 also discloses a skin external preparation containing a fermented product of Arnica as an active ingredient. However, it has not been known that a synergistic effect of whitening is exhibited by combining an Arnica fermented product with a melanin production inhibitor or a tyrosinase activity inhibitor.
JP-A-2019-038760

The present invention is an external preparation for skin containing a lactic acid bacterium fermented product of Arnica montana, which is a plant of the genus Arnica of the family Asteraceae (Compositae), and an agent for suppressing melanin production or an agent for suppressing tyrosinase activity.

According to the present invention, it is possible to provide a skin external preparation that exerts a synergistic effect of whitening by combining a fermented lactic acid bacterium of Arnica derived from a natural product with a melanin production inhibitor or a tyrosinase activity inhibitor.

Hereinafter, preferred embodiments of the present invention will be described in detail.
In the present invention, "Arnica" refers to Arnica (Arnica montana), which is a plant of the Asteraceae (Compositae) Arnica genus (Arnica). Examples of the site of use include whole plants, leaves, flowers, stems, roots, grains and the like. When the flower part is used, the flowering time and size are not particularly limited, and any one of petals, calyxes, etc. or all of them may be used.

The method for obtaining the fermented product according to the present invention is shown below.
In the present invention, examples of lactic acid bacteria used for fermentation of Arnica include Lactobacillus plantarum, L. brevis, L. casei, Lactobacillus delbrueckii and the like (Lactobacillus plantarum). Lactobacillus genus lactic acid bacteria; Carnobacterium divergens, Carnobacterium piscicola and other Carnobacterium genus lactic acid bacteria; Leuconostoc mesenteroides, Leuconostoc citreum Lactobacillus of the genus Leuconostoc such as (Leuconostoc citreum); Lactobacillus of the genus Streptococcus such as Streptococcus faecalis, Streptococcus pyogenes; Entercoccus case Lactobacillus genus Enterococcus; lactic acid bacteria of the genus Lactococcus such as Lactococcus plantarum, Lactococcus rafinolactis; Weissella confusa, Weissella confusa, Weissella candoureri Lactobacillus genus; Lactobacillus genus Atopobium such as Atopobium minutum, Atopobium parvulus; Vagococcus fluvialis, Lactobacillus vulvialis, Lactobacillus vulcacus Diococcus Damnosus (Pedioco) Examples include lactic acid bacteria of the genus Pediococcus such as ccus damnosus) and Pediococcus pentosaceus, and lactic acid bacteria of marine origin such as Marinelactobacillus phychrotolerans.

A preferable specific example of the method for fermenting the above plant using the above lactic acid bacterium is as follows. First, the fermentation materials of these plants are immersed or suspended in a fermentation medium to prepare a suspension for fermentation. In this case, the plant may be used as it is, or may be used after being dried or semi-dried in advance. Further, as the shape, the collected material can be used as it is, but the fermentation efficiency can be improved by shredding or pulverizing the material.

As the fermentation medium for suspending the fermentation material, water or water and lower alcohols (methanol, ethanol, propanol, etc.) or glycols (ethylene glycol, propylene glycol (propanediol), 1,3-butylene glycol, glycerin) Etc.), and sugars such as glucose, fructose, and shoe cloth may be added to these media, but the fact that microorganisms are most likely to exert their effects and that the fermentation material is a plant. The single use of water is most preferable in the sense that it avoids the formation of fermentation by-products due to the presence of other assimilating components.

This suspension of fermented material is sterilized to remove germs that interfere with fermentation before it is subjected to the fermentation process. In this case, as a sterilization removal method, a method may be used in which the fermented material is previously washed and sterilized with sterilizing ethanol or the like and then suspended in a sterile medium such as sterile water, or the fermented material is suspended in the medium and then suspended. A method of heat sterilizing the turbid liquid may be used. Examples of the heat sterilization method include an autoclave sterilization method in which the suspension is heated at 120 to 130 ° C. for 10 to 20 minutes, and holding the suspension at 80 to 90 ° C. for 60 to 120 minutes once a day for 2 to 3 times. Intermittent sterilization, which repeats for days, is commonly used.

Next, this sterilized suspension is placed in a fermentation tank, and microorganisms are inoculated into the suspension to carry out fermentation treatment. The appropriate amount of microorganisms to be inoculated is 10 ⁷ to 10 ⁸ cells / mL. Even if the inoculation amount is larger than the above range, the fermentation progress time is almost unchanged, while if it is less than the above range, it takes a long time to complete the fermentation, which is not preferable.

The fermentation temperature is generally in the range of 5 to 50 ° C, preferably in the range of 35 ° C to 40 ° C, which is the optimum temperature for growth of lactic acid bacteria. 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 number of fermentation days is shorter than the above general range, fermentation tends to be insufficient and the effectiveness of the fermented product tends to decrease, while even if it is longer than 10 days, no further increase in effectiveness is observed. Not only that, coloring and an increase in fermented odor occur, which is not preferable.

In performing the above fermentation treatment, in order to make the components of the plant more effectively utilized by the lactic acid bacteria, the above suspension is performed before or during the inoculation, or in some cases, during the fermentation after the inoculation. An enzyme may be added to the liquid to hydrolyze the plant, which is a fermentation material, with the enzyme. In this case, as the enzyme, as described above, at least one enzyme selected from proteolytic enzymes, glycolytic enzymes, pectic degrading enzymes and lipid degrading enzymes can be used.

As for the treatment conditions such as pH, temperature, and time, if the enzyme treatment is performed before fermentation, it is preferable to perform the treatment for 1 to 24 hours near the optimum pH and temperature of the enzyme to be used. If it is carried out in parallel with the fermentation, the conditions may be the same as those of the fermentation.

When the above fermentation treatment is completed, heat sterilize the fermented liquid at 80 to 100 ° C. for about 10 to 120 minutes for sterilization of microorganisms and, if combined with enzyme treatment, also for inactivation of the enzyme. Apply treatment. The fermented liquor that has been sterilized is separated into a liquid phase as it is, or generally and preferably by a solid-liquid separation means such as filtration or centrifugation, and if necessary, the pH is set to the pH range of ordinary cosmetics. The pH is adjusted to a certain pH 4 to 9, and if necessary, the pH is adjusted to an appropriate concentration by dilution or concentration, and then used as a compounding raw material for cosmetics. Further, in some cases, the liquid phase after solid-liquid separation may be solidified according to a conventional method such as a spray-drying method or a freeze-drying method, and further pulverized into a powder form if necessary.

In the present invention, examples of the melanin production inhibitor or tyrosinase activity inhibitor to be combined with the fermented product include the following.
Kodiic acid and its derivatives, ascorbic acid and its derivatives, hydroquinone or its derivatives, ellagic acid and its derivatives, nicotinic acid and its derivatives, resorcinol derivatives, tranexamic acid and its derivatives, 4-methoxysalicylic acid potassium salt, magnolignan (5, 5'-dipropyl-biphenyl-2, 2'-diol), hydroxybenzoic acid and its derivatives, vitamin E and its derivatives, α-hydroxy acids, AMP (adenosine monophosphate, adenosine monophosphate), t-cycloamino acids Examples include one or more selected from derivatives, salicylic acid extracts, chamomile extracts, rice bran extract hydrolysates, yukinoshita extracts and white potato extracts or hydrolysates thereof.

Examples of the ascorbic acid derivative include ascorbic acid esters such as ascorbic acid monobutyrate, ascorbic acid monocaplate, ascorbic acid monopalmitate, and ascorbic acid dibutyrate, ascorbic acid ethers, and ascorbic acid sugar derivatives such as ascorbic acid glucoside. However, examples of the ascorbic acid derivative include L-ascorbic acid-2-phosphate ester sodium, L-ascorbic acid-2-phosphate ester magnesium, L-ascorbic acid-2-sulfate sodium ester, and L-ascorbic acid-2. -Ascorbic acid ester salts such as magnesium sulfate, L-ascorbic acid-2-glucoside, L-ascorbic acid-5-glucoside, ascorbic acidcopheryl maleic acid, ascorbic acidcopheryl phosphate K, myristyl 3-glyceryl ascorbic acid, caprylyl Ascorbic acid sugar derivatives such as 2-glyceryl ascorbic acid, 6-position acylated products of those ascorbic acid sugar derivatives (acyl groups are hexanoyl group, octanoyl group, decanoyle group, etc.), L-ascorbic acid tetraisopalmitic acid ester, L- L-ascorbic acid tetrafatty acid esters such as ascorbic acid tetralauric acid ester, 3-O-ethylascorbic acid, L-ascorbic acid-2-phosphate-6-O-palmitate sodium, glyceryl ascorbic acid or acylation thereof Derivatives, glycerin ascorbic acid derivatives such as bisglyceryl ascorbic acid, aminopropyl L-ascorbic acid phosphate, hyaluronic acid derivatives of L-ascorbic acid, 3-OD lactose-L-ascorbic acid, isostearyl ascorbic acid, etc. However, as the hydroquinone derivative, albutin (hydroquinone-β-D-glucopyranoside), α-arbutin (hydroquinone-α-D-glucopyranoside) and the like are used, and as the tranexamic acid derivative, a tranexamic acid ester (for example, tranexamic acid lauryl ester, etc.) Examples thereof include tranexamic acid hexadecyl ester, tranexamic acid cetyl ester or a salt thereof), an amide form of tranexamic acid (for example, methylamide tranexamic acid), and examples of the resorcinol derivative include 4-n-butylresorcinol and 4-isoamylresorsinol. As the 2,5-dihydroxybenzoic acid derivative, for example, 2,5-diacetoxybenzoic acid, 2-acetoxy-5-hydroxybenzoic acid, 2-hydroxy-5-propionylo, etc. Xybenzoic acid and the like, nicotinic acid derivatives such as nicotinamide and benzyl nicotinate, and α-hydroxy acids include, for example, lactic acid, malic acid, succinic acid, citric acid, α-hydroxyoctanoic acid and the like. ..

Examples of skin external preparations (including cosmetics and non-medicinal products) to which the present invention is applied include emulsions, creams, lotions, essences, packs, lipsticks, foundations, liquid foundations, makeup press powders, and blushers. Examples thereof include white powder, facial cleansers, body shampoos, slimming agents, hair shampoos, soaps, etc., and hair growth agents, bathing agents, etc., but of course, are not limited thereto.

The blending amount of the fermented product according to the present invention in the external preparation for skin (cosmetics and quasi-drugs) is generally 0.002 to 1.0% by weight (solid content weight) in the case of basic cosmetics as its solid content. %, The same shall apply hereinafter), in the case of make-up cosmetics, it is generally in the range of 0.002 to 1.0% by weight, and in the case of cleaning cosmetics, it is generally in the range of 0.002 to 10.0% by weight.

In addition to the fermented product and melanin production inhibitor or tyrosinase activity inhibitor according to the present invention, skin external preparations (cosmetics and non-medicinal products) include components usually used in skin external compositions, such as oily components. Surface active agents (synthetic, natural products), moisturizers, thickeners, preservatives / bactericides, powder components, UV absorbers, antioxidants, pigments, fragrances, etc. may be added as needed. it can. Further, as long as the effectiveness and characteristics of the extract, hydrolyzate or fermented product according to the present invention are not impaired, other physiologically active ingredients may be combined and blended at all.

Here, examples of the oily component include olive oil, jojoba oil, paraffin oil, soybean oil, rice oil, rice germ oil, palm oil, palm oil, cacao oil, meadowfoam oil, sheer butter, tea tree oil, and avocado oil. , Macadamia nut oil, bergamot oil, lavender oil, rose oil, bergamot oil, chamomile oil and other plant-derived oils and fats such as squalane; animal-derived oils and fats such as mink oil and turtle oil; honey wax, carnauba wax, rice wax , Lanorin and other waxes; liquid paraffin, vaseline, paraffin wax, squalane and other hydrocarbons; myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, cis-11-eicosenoic acid and other fatty acids; lauryl alcohol , Cetanol, higher alcohols such as stearyl alcohol; synthetic esters such as isopropyl myristate, isopropyl palmitate, butyl oleate, 2-ethylhexyl glyceride, higher fatty acid octyldodecyl (octyldodecyl stearate, etc.) and synthetic triglycerides. Can be mentioned.

Examples of the surfactant include polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene hydrogenated castor oil, and poly. Nonionic surfactants such as oxyethylene sorbitol fatty acid ester; fatty acid salt, alkyl sulfate, alkylbenzene sulfonate, polyoxyethylene alkyl ether sulfate, polyoxyethylene fatty amine sulfate, polyoxyethylene alkyl phenyl ether sulfate, Anionic surfactants such as polyoxyethylene alkyl ether phosphate, α-sulfonated fatty acid alkyl ester salt, polyoxyethylene alkyl phenyl ether phosphate; quaternary ammonium salt, primary to tertiary fatty amine salt, tri Cationic surfactants such as alkylbenzylammonium salt, alkylpyridinium salt, 2-alkyl-1-alkyl-1-hydroxyethylimidazolinium salt, N, N-dialkylmorphonium salt, polyethylene polyamine fatty acid amide salt; N-dimethyl-N-alkyl-N-carboxymethylammoniobetaine, N, N, N-trialkyl-N-alkylene ammoniocarboxybetaine, N-acylamide propyl-N', N'-dimethyl-N'- Amphoteric surfactants such as β-hydroxypropylammoniosulfobetaine can be used.

Emulsifiers and / or emulsifying aids are derived from stevia derivatives such as enzyme-treated stevia, saponins or derivatives thereof, casein or salts thereof (sodium, etc.), sugar-protein complexes, sucrose or esters thereof, lactose, soybeans. Water-soluble polysaccharides, soybean-derived protein-polysaccharide complexes, lanolin or derivatives thereof, cholesterol, stevia derivatives (stevia enzyme-treated products, etc.), silicates (aluminum, magnesium, etc.), carbonates (calcium, sodium, etc.) saponins and The derivative, lecithin and its derivative (hydrogenated lecithin, etc.), lactic acid bacterium fermented rice, lactic acid bacterium fermented sprouted rice, lactic acid bacterium fermented grain (wheat, beans, miscellaneous grains, etc.) and the like can also be blended.

As a moisturizer, examples of the moisturizer include glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, polyethylene glycol, sorbitol, xylitol, sodium pyrrolidone carboxylate and the like, and saccharides such as trehalose and raffinose. , Mucopolysaccharides (eg, hyaluronic acid and its derivatives, hyaluronic acid fermented liquid, chondroitin and its derivatives, heparin and its derivatives, etc.), elastin and its derivatives, collagen and its derivatives, collagen peptides, NMF-related substances, lactic acid, urea , Higher fatty acid octyldodecyl, seaweed extract, silane root (white and) extract, various amino acids and derivatives thereof.

Examples of the thickener include ingredients derived from brown algae, green algae or red algae such as alginic acid, agar, carrageenan and fucoidan; silane root (white and) extracts; polysaccharides such as pectin and aloe polysaccharides; tragant gum, locust bean gum, etc. Gum such as xanthan gum and guar gum; Cellulose derivatives such as carboxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose; Synthesis of carboshikivinyl polymer, alkyl-modified carboxyvinyl polymer, polyvinyl alcohol, polyvinylpyrrolidone, alginic acid / methacrylic acid copolymer and the like Polymers; hyaluronic acid and its derivatives; polyglutamic acid and its derivatives and the like.

Examples of preservatives and bactericides include urea; paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate; phenoxyethanol, dichlorophenol, hexachlorophene, chlorhexidine hydrochloride, and benza chloride. Derived from plants such as luconium, salicylic acid, ethanol, undesyleneic acid, phenols, jamar (imidazodeini ruurea), 1,2-pentanediol, propanediol, various essential oils, bark dry distillate, radish fermented liquid, sugar cane, corn, etc. Ethanol or 1,3-butylene glycol and the like.

Powder components include, for example, cericite, titanium oxide, talc, kaolin, bentonite, zinc oxide, magnesium carbonate, magnesium oxide, zirconium oxide, barium sulfate, silicic anhydride, mica, nylon powder, polyethylene powder, silk powder, etc. There are cellulose-based powders, grains (rice, wheat, corn, millet, etc.) powders, beans (soybeans, azuki, etc.) powders, and the like.

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

Examples of the antioxidant include butylhydroxyanisole, butylhydroxytoluene, propyl gallate, vitamin E and its derivatives (for example, vitamin E nicotinate, vitamin E linoleate, etc.).

Physiologically active ingredients include, for example, placenta extract, perilla extract, shakuyaku extract or its hydrolyzate, lactic acid bacterium fermented rice, purple kib extract, hass seed fermented product, ginseng extract or its hydrolyzate, and honeybee hydrolyzate. Examples thereof include fermented honeybee seeds, fermented hibiscus, royal jelly fermented product, liquor lees extract or ceramide contained therein, liquor lees fermented product, pandanus amaririfolius extract, arcangelicia flava extract and the like. In addition, coral grass extract, rice leaf extract or its hydrolyzate, eggplant (has, long eggplant, Kamo eggplant, rice eggplant, etc.) extract or its hydrolyzate, apricot fruit extract, catamen giraffe, etc. Seaweed extract, marine flowering plant extract such as amamo, soymilk fermented product, jellyfish water, rice extract or its hydrolyzate, rice fermented extract, sprouted rice extract or its hydrolyzate, sprouted rice fermentation Products, black bean extract or its hydrolyzate, damask rose flower extract, bamboo shoot skin extract, linoleic acid and its derivatives or processed products (eg, liposomal linoleic acid, etc.), animal or fish-derived collagen and Its derivatives, elastin and its derivatives, glycyrrhizic acid and its derivatives (dipotassium salt, etc.), t-cycloamino acid derivatives, vitamin A and its derivatives, allantin, diisopropylamine dichloroacetate, γ-amino-β-hydroxybutyric acid, gentiana extract , Lantern extract, Carrot extract, Otane carrot extract or fermented product thereof, Red ginseng extract, Mitsuishikonbu extract, Hechima extract, Anaaosa extract, Peach extract, Peach seed extract, Kiwi extract, Juazeiro extract, Paudalco bark extract, daily grass extract or fermented product, hagoromogusa extract, cherimoya extract, mango extract, mangostin extract, funori extract, dragon tea extract, Benifuki extract, silane extract, sansho peel Or seed coat extract or hydrolyzate, Benibana flower extract, Casablanca extract, sweet potato extract or fermented product thereof, sunflower extract, Guava leaf extract, dokudami extract, evening white yuzu extract, aloe extract, There are fig flower extract, apple extract, white asparagus extract and the like.

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

Production example 1. Preparation of Arnica fermented product An extract suspension was prepared by adding 300 g of purified water and 1 g of glucose to 10 g of dried Arnica flower part, and sterilized by heating at 80 to 90 ° C. for 1 hour. Sterile extracted suspension lactic acid bacteria (Lactobacillus plantarum) were inoculated 10 ⁸ / mL, under nitrogen flow, allowed to stand for 3 days of culture at 37 ° C.. After completion of the culture, the culture solution was sterilized by heating and filtered to obtain 213 g of a lactic acid bacterium fermented product solution (solid content concentration 0.70%).

Production example 2. Preparation of hydrolyzate of rice bran extract 1500 g of 0.1 M lactic acid aqueous solution was added to 500 g of rice bran, and the mixture was sufficiently mixed with rice bran and lactic acid aqueous solution, and then allowed to stand at room temperature for 1 day. The insoluble material was then filtered off and the filtrate was treated with papain. The enzyme treatment was carried out by using 1.2 mg of the enzyme and holding it at 80 ° C. for 1 hour at the optimum pH of the enzyme. The insoluble matter produced by the treatment was filtered off, and the filtrate was adjusted to pH 6.5 with phytic acid to obtain 730 g of a pale yellow transparent rice bran extract hydrolyzed solution (solid content concentration 3.65%).

Test example 1. Suppressive effect on melanin synthesis Cultured B16 mouse melanoma cells B16-F10 were ^{seeded in a 24-well microplate at 2.4 × 10 4} cells / well, in RPMI1640 medium containing 10% FBS, under the conditions of _{37 ° C and 5.0% CO 2 for 1 day.} Pre-cultured. Next, the sample solution shown in Table 1 and theophylline-containing medium adjusted to a final concentration of 1 mM were added to RPMI1640 medium containing 10% FBS, and the cells were cultured under the same conditions for 2 days. The culture was then removed, 200 μL of 1N NaOH / 10% dimethylsulfoxide solution was added per hole, sealed and incubated at 50 ° C. for 2 hours to lyse the cells. 100 μL of this solution was transferred to another 96-well microplate, and the melanin level was measured at a wavelength of 490 nm using a microplate reader (Model680, manufactured by Bio-Rad). On the other hand, 5 μL of the solution in which the same cells were lysed was transferred to another 96-well microplate, and 200 μL of Dye Reagent Concentrate (Biorad) solution diluted 5-fold with purified water was added to the microplate reader (Model 680, Biorad). The absorbance at a wavelength of 570 nm was measured using (manufactured by the same company). Another known amount of bovine serum albumin (manufactured by Sigma) was serially diluted, and the amount of protein equivalent to albumin was measured from the calibration curve obtained by the same operation. The obtained absorbance was gradually adjusted by the amount of protein to determine the amount of melanin per protein.
In the case of no sample addition (Control) in which 30% BG was added instead of the sample solution, the same operation as above was performed, and the amount of melanin per protein at the time of adding each sample to the amount of melanin per protein obtained here was performed. The relative value of was calculated and used as the melanin synthesis rate (%).

Table 1 shows the sample solutions used in Test Example 1. Regarding the fermented product of Production Example 1 and the hydrolyzate of Production Example 2, the final concentration as a solution with respect to the medium is shown.
[Table 1]

The test results of Test Example 1 are shown in Table 2. As shown in Table 2, it was found that the present invention has a remarkably excellent synergistic effect of suppressing melanin synthesis.
[Table 2]

Test example 2. ^{Evaluation of suppression of tyrosinase activity Human epidermal cells NHEK (NB) were seeded in 2 × 10 3} cells / hole in a 24-well microplate containing HuMedia KG2 medium (manufactured by Kurabo Industries Ltd.) under the conditions of 37 ° C and 5.0% CO _2. Pre-cultured for 1 day. Next, a sample prepared with HuMedia KB2 medium (HuMedia KG2 from which cell growth factors were removed, manufactured by Kurabo Industries Ltd.) so as to have tranexamic acid of 10 mM was added, and the cells were cultured under the same conditions for another day. Next, the medium is removed, washed once with PBS (-), then PBS (-) is added, and about 50 mJ / cm ^{2 is added from the bottom of the incubator using a UV-B lamp (TL20W / 12RS manufactured by Philips).} UV irradiation was performed. The supernatant was then replaced with HuMedia KB2 medium and culture was continued.
On the other hand, on the same day, B16 mouse melanoma cells B16-F10 were ^{seeded in 4 × 10 3} cells / hole in a 96-well microplate and pre-seeded in RPMI1640 medium containing 10% FBS under the conditions of _{37 ° C. and 5.0% CO 2 for 1 day.} It was cultured. The next day, using the culture supernatant of epidermal cells (24 hours after irradiation with ultraviolet rays) as a medium, the solution of the combination of Production Example 1 and Production Examples 1 and 2 shown in Table 3 was used as a culture system for B16 cells. 100 μL / hole was added, and the cells were cultured under the same conditions for 3 days. In parallel, a group to which the same concentration of 30% BG solution was added instead of the sample solution was set as a negative control, and the cells were cultured under the same conditions. Next, the culture solution was removed, a surfactant (Triton X-100) and a 5 mM L-dopa solution were added, and the reaction was carried out at 37 ° C., and then a microplate reader (Model 680, manufactured by Biorad) was used. , The dopa value was measured at a wavelength of 490 nm. Furthermore, the relative value of the dopa value of each sample addition group to the dopa value of the negative control group was determined and used as the tyrosinase activity rate (%).

Table 3 shows the concentration of the sample solution used in Test Example 2. Regarding the fermented product of Production Example 1 and the hydrolyzate of Production Example 2, the final concentration as a solution with respect to the medium is shown.
[Table 3]

The test results of Test Example 4 are shown in Table 4. As shown in Table 4, it was found that the present invention has a remarkably excellent synergistic effect of suppressing tyrosinase activity.
[Table 4]

Prescription example 1. Toner [Ingredients] Part Olive oil 1.0
Polyoxyethylene (5.5) cetyl alcohol 5.0
Butylparaben 0.1
Fermented product of Production Example 1 5.0
Tranexamic acid 2.0
Ethanol 5.0
Glycerin 5.0
1,3-butylene glycol 5.0
Potassium hydroxide Appropriate amount Purified water Total amount is 100 parts Fragrance Appropriate amount

Prescription example 2. Emulsion [Ingredients] Part Liquid paraffin 6.0
Hexalan 4.0
Jojoba oil 1.0
Polyoxyethylene (20) sorbitan monostearate 1.0
Oil-based glyceryl stearate 1.0
Soy lecithin 1.5
Fermented product of Production Example 1 3.0
L-ascorbic acid-2-glucoside 2.0
Potassium hydroxide 0.5
Glycerin 3.0
1,3-butylene glycol 2.0
Carboxymethyl cellulose 0.3
Sodium hyaluronate 0.01
Amount that makes the total amount of purified water 100 copies Appropriate amount of fragrance

Prescription example 3. Emulsion Emulsion was obtained in the same manner as in Formulation 3 except that 3.0 parts of arbutin was used instead of 2.0 parts of L-ascorbic acid-2-glucoside and 0.5 part of potassium hydroxide among the components of Formulation Example 2. It was.

Prescription example 4. Emulsion Emulsion was prepared in the same manner as in Formulation 3 except that 2.0 parts of tranexamic acid was used instead of 2.0 parts of L-ascorbic acid-2-glucoside and 0.5 part of potassium hydroxide among the components of Formulation Example 2. Obtained.

Prescription example 5. Emulsion Emulsion in the same manner as in Formulation 3 except that 3.0 parts of nicotinamide is used instead of 2.0 parts of L-ascorbic acid-2-glucoside and 0.5 part of potassium hydroxide among the components of Formulation Example 2. Got

Prescription example 6. Emulsion Same as in Formulation Example 3 except that 2.0 parts of ascorbic tetrahexyl decanoate is used instead of 2.0 parts of L-ascorbic acid-2-glucoside and 0.5 parts of potassium hydroxide in the components of Formulation Example 2. Emulsion was obtained.

Prescription example 7. Emulsion In the components of Formulation Example 2, 3.0 parts of 3-O-ethylascorbic acid is used instead of 2.0 parts of L-ascorbic acid-2-glucoside and 0.5 part of potassium hydroxide. A milky lotion was obtained in the same manner.

Prescription example 8. Emulsion [Ingredients] Part Liquid paraffin 6.0
Hexalan 4.0
Jojoba oil 1.0
Polyoxyethylene (20) sorbitan monostearate 1.0
Oil-based glyceryl stearate 1.0
Soy lecithin 1.5
Fermented product of Production Example 1 3.0
Arbutin 2.0
L-ascorbic acid-2-glucoside 2.0
Potassium hydroxide 0.5
Glycerin 3.0
1,3-butylene glycol 2.0
Carboxymethyl cellulose 0.3
Sodium hyaluronate 0.01
Amount that makes the total amount of purified water 100 copies Appropriate amount of fragrance

Prescription example 9. Emulsion An emulsion was obtained in the same manner as in Formulation 7, except that 2.0 parts of tranexamic acid was used instead of 2.0 parts of arbutin among the components of Formulation Example 8.

Prescription example 10. Emulsion An emulsion was obtained in the same manner as in Formulation 10, except that 2.0 parts of a hydrolyzate of rice bran extract was used instead of 2.0 parts of arbutin in the components of Formulation Example 8.

Claims (2)

An external preparation for skin containing a fermented lactic acid bacterium of Arnica montana, which is a plant of the genus Arnica of the Asteraceae family, and a melanin production inhibitor or a tyrosinase activity inhibitor as active ingredients. The external preparation for skin according to claim 1, further comprising a hydrolyzate of a rice bran extract.