JP7664595B2 - Skin preparations - Google Patents

Description

The present invention relates to a skin care product that contains a plant extract as an active ingredient.

It has been known that ultraviolet rays, chemical substances, allergens, etc. stimulate living cells, causing the deterioration of their constituent components and promoting the synthesis of cytokines and active oxygen. It is also known that the altered cellular components, cytokines, or active oxygen induce inflammation and abnormal deposition of melanin pigment in cells, resulting in age spots, freckles, melasma, etc.

To date, various ultraviolet absorbers, antioxidants, and anti-inflammatory agents have been proposed for the purpose of improving skin health and preventing and improving aging, and cosmetics, foods, beverages, and medicines containing these have been marketed. For example, ultraviolet absorbers such as cinnamic acid derivatives and benzophenone derivatives; antioxidants such as vitamin C, vitamin E, and catalase; and anti-inflammatory agents such as glycyrrhizinic acid or its salts, allantoin, and tranexamic acid have been proposed, but there is a demand for safer antioxidants and anti-inflammatory agents derived from natural products.

Although plant extracts have been proposed as active ingredients that can be added to cosmetics, there have been issues with efficacy and safety.

JP 61-024522 A JP 04-305519 A

In view of the above problems, the present inventors have conducted intensive research to discover new active ingredients derived from natural products. As a result, they have newly discovered that an extract of saffron (Crocus sativus) of the genus Crocus in the family Iridaceae has the effect of suppressing gene expression of factors that contribute to the production of inflammatory substances (cytokines) induced by external stimuli such as ultraviolet rays, and is useful as an active ingredient in external skin preparations such as cosmetics.

The present invention is an external skin preparation containing, as an active ingredient, an extract of saffron (Crocus sativus) of the genus Crocus in the family Iridaceae.
The present invention is a skin external preparation containing an extract of saffron (Crocus sativus) of the genus Crocus in the family Iridaceae as an active ingredient, and is characterized in that the extract contains Kaempferol 3-O-beta-Sophoroside.

The present invention provides an external skin preparation containing an extract of saffron (Crocus sativus) of the genus Crocus in the family Iridaceae as an active ingredient, and provides an external skin preparation that protects the skin from external stimuli such as ultraviolet rays based on the effects of the active ingredient, saffron extract.

In the present invention, saffron (Crocus sativus) of the genus Crocus in the family Iridaceae is used as the extraction material for the plant extract. The whole plant, flowers, leaves, stems, stamens, pistils, and roots can be used for extraction, but the whole plant or flowers are preferred. When flowers are used, the stamens and/or pistils may be included.

The preparation of a plant extract containing the active ingredient of the present invention can be carried out by first washing the plant to be extracted with water, if necessary, to remove foreign matter, leaving it as is or drying it, and then shredding or crushing it if necessary, and contacting it with an extraction solvent according to a conventional method such as immersion, countercurrent extraction, or steam distillation. In addition, a supercritical extraction method may be used in the present invention.

Extraction solvents used in the extraction process include water; lower alcohols such as methanol, ethanol, and propanol; polyhydric alcohols such as ethylene glycol, propylene glycol, 1,3-butylene glycol, and glycerin; esters such as ethyl acetate, butyl acetate, and methyl propionate; ketones such as acetone and methyl ethyl ketone; ethers such as ethyl ether and isopropyl ether; and hydrocarbon solvents such as n-hexane, toluene, and chloroform, which may be used alone or in combination of two or more.

When preparing the extract, there is no particular limit to the pH of the extract, but it is generally preferable to set the pH in the range of 3 to 9. The pH can be adjusted by adding an alkali adjuster such as sodium hydroxide, sodium carbonate, or potassium hydroxide, or an acid adjuster such as citric acid, hydrochloric acid, phosphoric acid, or sulfuric acid to the extraction solvent.

The extraction conditions such as extraction temperature and time vary depending on the type and pH of the solvent used, the size of the plant material, etc., but for example, in the case of the immersion method using methanol or ethanol, or a mixed solvent of water and a lower alcohol or polyhydric alcohol as the extraction solvent, the extraction temperature is in the range of 0 to 80°C. The extraction time is preferably in the range of 1 hour to 7 days for cold extraction at 4°C, 1 hour to 3 days for medium temperature extraction around 40°C, and 1 hour to 24 hours for high temperature extraction at 70 to 80°C. In the case of the immersion method, the bath ratio is generally 1 to 200 times, preferably 1 to 100 times, the amount of solvent relative to the plant material by weight.

The extract according to the present invention is also characterized by containing "Kaempferol 3-O-beta-Sophoroside" as an active ingredient.

On the other hand, the extract according to the present invention is preferably prepared to have a high concentration of Kaempferol 3-O-beta-Sophoroside by combining one or more of the following: activated carbon treatment, ion exchange resin treatment, synthetic adsorbent, silica gel, and recrystallization treatment. Activated carbon may be obtained by treating raw materials such as pine trees, bamboo, coconut shells, walnut shells, and other plant materials, as well as coal and petroleum materials, with these raw materials as raw materials, using a physical method such as a high-temperature carbonization method using gases such as steam, carbon dioxide, and air, or by treating the raw materials with chemicals such as zinc chloride, and then heating them to make them porous. Examples of ion exchange resins include weakly basic anion exchange resins, strongly acidic cation exchange resins, strongly basic ion exchange resins, and weakly acidic cation exchange resins. Examples of synthetic adsorbents include non-ionic resins such as chelating resins, strongly acidic cation adsorption resins, styrene-divinylbenzene copolymers, and methacrylic acid ester polymers. By using any of these treatments, compounds that cause coloring problems (pigments such as anthocyanins) and compounds that cause skin irritation are removed from the extract. Compounds that cause safety issues such as coloring and skin irritation (pigments such as anthocyanins) are removed. By using the above treatments, it is preferable that the compounds are contained in the extract at a concentration of 0.01% to 0.1%.

In the present invention, topical skin preparations include cosmetics, quasi-drugs, and topical medicines, such as milky lotions, creams, lotions, essences, packs, lipstick, foundations, liquid foundations, make-up press powders, blushers, face powders, facial cleansers, 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 extract 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), moisturizers, thickeners, preservatives and disinfectants, powder ingredients, UV absorbers, antioxidants, colorants, fragrances, etc. Furthermore, as long as the effectiveness and characteristics of the extract, hydrolysate, or fermented product of the present invention are not impaired, there is no problem in combining and formulating 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, Bletilla serrata root (white) extract, 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.

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

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

Examples of physiologically active ingredients include placenta extract, mulberry extract, saxifrage extract, perilla extract, rice bran extract or hydrolysate thereof, white mustard extract or hydrolysate thereof, fermented white mustard, peony extract or hydrolysate thereof, lactic acid bacteria fermented rice, barley extract, lotus seed extract or hydrolysate thereof, fermented lotus seed product, Job's tears hydrolysate, fermented Job's tears seed product, fermented royal jelly product, sake lees extract or ceramides contained therein, fermented sake lees product, Pandanus amaryllifolius Roxb. extract, Arcangelicia flava Merrilli extract, and chamomile extract. In addition, coral grass extract, rice leaf extract or hydrolysate thereof, eggplant (lotus, long eggplant, Kamo eggplant, rice eggplant, etc.) extract or hydrolysate thereof, apricot fruit extract, seaweed extract such as Eucalyptus tschonoskii, extract of marine flowering plants such as Zostera marina, fermented soy milk, jellyfish water, rice extract or hydrolysate thereof, fermented rice extract, germinated rice extract or hydrolysate thereof, fermented germinated rice, black bean extract or hydrolysate thereof, brown sugar extract or fermented product thereof, extract of Damask rose flower, ylang-ylang flower extract, extract of bamboo shoot skin, linoleic acid and its derivatives or processed products (e.g. liposomal linoleic acid, etc.) ), collagen and its derivatives derived from animals or fish, elastin and its derivatives, glycyrrhizic acid and its derivatives (dipotassium salt, etc.), t-cycloamino acid derivatives, vitamin A and its derivatives, allantoin, diisopropylamine dichloroacetate, gamma-amino-β-hydroxybutyric acid, gentian extract, licorice extract, carrot extract, ginseng extract or its fermented product, camellia extract, red ginseng extract, Mitsuishi Laminaria extract, loofah extract, Ulva pertusa extract, peach extract, plum extract, peach kernel extract, kiwi extract, sunflower extract, Zizyphus joazeiro) extract, pau d'arco bark extract, daylily extract or fermented product, hibiscus flower extract or fermented product, wormwood extract, cherimoya extract, mango extract, mangosteen extract, funori extract, oolong tea extract, hongfuki extract, sliver extract, sweet potato extract, Japanese pepper peel or seed coat extract or hydrolyzate, safflower flower extract, Casablanca extract, sweet potato extract or its fermented product, guava leaf extract, olive leaf extract, horse chestnut extract, ginger extract, vanilla extract, dokudami extract, banpeiyu extract, aloe extract, aloe vera extract, fig flower extract, apple extract, white asparagus extract, red clover extract, etc.

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

Production Example 1. Preparation of extract (1)
Saffron flowers were dried, and 2000g of purified water was added to 100g of the dried product, followed by soaking at 40°C for 2 hours. This was filtered to obtain 1750g of a brown, transparent saffron flower crude extract. The saffron flower crude extract was subjected to a styrene-divinylbenzene copolymer, washed with purified water and a 20w/w% aqueous ethanol solution, and then eluted with 1,3-butylene glycol. This was filtered, and purified water was added to obtain 4700g of a yellow, transparent saffron flower extract (solid content: 0.10%).

Test Example 1. Quantitative analysis of Kaempferol 3-O-beta-Sophoroside <Quantitative method>
1 g of the extract of Preparation Example 1 was precisely weighed out, and the mobile phase was added to make exactly 10 mL to prepare a sample solution. 10 μL of this sample solution was taken and tested by liquid chromatography under the following test conditions. The peak area of the obtained chromatogram was calculated, and the concentration (%) of Kaempferol 3-O-beta-Sophoroside was calculated from the calibration curve prepared in advance.
Kaempferol 3-O-beta-Sophoroside concentration in this product (%) = C/sample amount (g) x 10
C: Kaempferol 3-O-beta-Sophoroside concentration (w/v%) calculated from the calibration curve
<Creating a calibration curve>
Accurately weigh out about 5 mg of Kaempferol 3-O-beta-Sophoroside, add the mobile phase to make exactly 50 mL, and use this as the standard stock solution. Accurately take 1.5 mL of the standard stock solution, add the mobile phase to make exactly 10 mL, and use this as the standard solution. Take 10 μL of the standard stock solution and the standard solution, and perform the test by liquid chromatography under the above test conditions. A calibration curve was created by plotting the peak area of the obtained chromatogram on the vertical axis and the Kaempferol 3-O-beta-Sophoroside concentration (w/v%) on the vertical axis.
<Test conditions>
Detector: ultraviolet spectrophotometer (measurement wavelength: 254 nm)
Column: A stainless steel tube having an inner diameter of 4.6 mm and a length of 25 cm is packed with 5 μm octadecylsilylated silica gel for liquid chromatography.
Column temperature: constant temperature around 35° C. Mobile phase: 0.05% phosphoric acid aqueous solution/acetonitrile mixture (17:3)
Flow rate: 0.8 mL per minute (retention time of Kaempferol 3-O-beta-Sophoroside is approximately 17 minutes)

As a result of Test Example 1, it was confirmed that the extract according to the Production Example contained 0.043% Kaempferol 3-O-beta-Sophoroside. In the present invention, for example, by adjusting the extraction process of Production Example 1, it is preferable that the extract contains 0.01% to 0.1% Kaempferol 3-O-beta-Sophoroside.
[Table 1]

Test Example 2. Evaluation test of the effect of suppressing the expression of inflammation-related genes by ultraviolet light Normal human epidermal cells were prepared at 6 x ¹⁰⁴ cells/mL using growth additive-containing HuMediaKG2 [manufactured by Kurabo Industries, Ltd.], 1 mL of the cells were seeded on a 24-well plate, and cultured at 37°C under 5% _CO2 and saturated water vapor. After one day of culture, a culture medium containing the extract of Production Example 1 (the extract of Production Example 1 was added so that the final concentration as a solution was 2% relative to the total volume of the culture medium) was added and cultured. As a comparative control, a test group (control group) was set in which a culture medium containing only a 30% 1,3-butylene glycol aqueous solution (the final concentration of 30% 1,3-butylene glycol (30-BG) relative to the total volume of the culture medium was adjusted to 2%) was added instead of the extract of Production Example 1. After one day of culture, the cells in the test group were irradiated with about 50 mJ/ ^cm2 ultraviolet light from the bottom of the incubator using a UV-B lamp (TL20W/12RS, Philips) for 24 hours and then collected with 0.5 mL of Trizol reagent (Invitrogen). 100 μL of chloroform (Wako Pure Chemical Industries, Ltd.) was added to the collected cells, stirred and mixed, and centrifuged at 12,500 rpm and 4 ° C for 15 minutes using a centrifuge (TOMY/MX-160), and 200 μL of the aqueous layer was collected. 250 μL of isopropanol (Wako Pure Chemical Industries, Ltd.) 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% ethanol was added to the total RNA, stirred and washed, and then centrifuged at 12,500 rpm and 4 ° C for 10 minutes to collect the precipitate. The collected total RNA was subjected to reverse transcription reaction using a specified kit [PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio)] to synthesize cDNA. The synthesized cDNA was used as a sample, and the expression of various genes and the expression of the internal standard ACTB gene were detected using Thermal Cycler Dice (registered trademark) Real Time System Single (Takara Bio) and SYBR (registered trademark) Premix Ex TaqTM II (Tli RNaseH Plus) [Takara Bio]. Here, ACTB (β-actine) is one of the housekeeping genes (genes that are expressed in a constant amount in many tissues and cells, are constantly expressed, and are essential for the maintenance and proliferation of cells), and since its expression level is always constant, it is used as an internal standard in PCR experiments. The test results were obtained by comparing the expression levels of each gene in each test group when the expression level of the ACTB gene was constant. In this test system, the expression level of each gene in the control group was set to 100, and the relative value of the expression level of the gene in the other test groups was calculated.

The results of Test Example 2 are shown in Table 2.
[Table 2]

As shown in Table 2, it was confirmed that the present invention significantly suppresses the expression of factors involved in skin inflammation [chemokine CXCL16, interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-1 (IL-1α), chemokine CCL2, and HMGB1 (high mobility group box 1)]. This suggests that the present invention has the effect of suppressing skin inflammation.

Test Example 3. Evaluation test of the effect of suppressing HMGB1 (High Mobility Group Box 1) HMGB1 is a nuclear factor that promotes the transcription of NF-kB, which is secreted at the early stage of inflammation, and in this Test Example 3, the effect of suppressing the production of this factor was evaluated.
On the first day, epidermal cells (NHEK) and fibroblasts (NB1RGB) were seeded in a 96-well plate, and on the second day, a medium containing the extract of Production Example 1 was added to each culture system. On the third day, each cell was irradiated from the bottom with UVB (50 mJ/cm ² ). 24 hours after irradiation, the cultured cell supernatant was collected and reacted with an ELISA kit to measure the absorbance (450 nm), and the amount of HMGB1 (ng/mL) was calculated from the calibration curve. A group containing 30% 1,3-butylene glycol instead of the extract of Production Example 1 was used as a control, and the absorbance was measured when the group was irradiated with UV light and when it was not irradiated with UV light, and a relative value was calculated when the amount of HMGB1 in the control group without UV light irradiation was set at 100.

The test results for epidermal cells in Test Example 3 are shown in Table 3.
[Table 3]

The test results for fibroblasts in Test Example 3 are shown in Table 4.
[Table 4]
As shown in Tables 3 and 4, it was confirmed that the extract of Production Example 1 inhibits UV-induced HMGB1 production in epidermal cells and fibroblasts, suggesting an effect of inhibiting early inflammation by inhibiting the transcription of NF-kB.

Test Example 4. Test to evaluate the inhibitory effect on ICAM-1 (intercellular adhesion molecule-1) ICAM-1 is an adhesion factor present in vascular endothelial cells and involved in the migration of leukocytes, and in this Test Example 3, the inhibitory effect on the production of this factor was evaluated.
On the first day, fibroblasts (NB1RGB) were seeded in a 96-well plate, and on the second day, a medium containing the extract of Production Example 1 was added. On the third day, UVA (20 mJ) was irradiated from the bottom. 24 hours after irradiation, the cultured cell supernatant was collected. Vascular endothelial cells (HUVEC) were seeded in a 96-well plate, and the following day, the above-recovered fibroblast supernatant was added and cultured. After 6 days of culture, the cells were fixed and blocked, and reacted with anti-ICAM-1 antibody as the primary antibody and fluorescently labeled secondary antibody in that order. After washing and removing the secondary antibody, the fluorescence intensity (Ex = 544 nm, Em = 590 nm) was measured. Furthermore, DNA was fluorescently stained with hoechst33342, and then the fluorescence intensity (Ex = 355 nm, Em = 460 nm) was measured to calculate the amount of ICAM-1 per DNA. A group containing 30% 1,3-butylene glycol instead of the extract of Production Example 1 was used as a control, and the fluorescence intensity was measured when exposed to ultraviolet light and when not exposed to ultraviolet light, and a relative value was calculated with the amount of ICAM-1 in the control without ultraviolet light exposure taken as 100.

The evaluation results of Test Example 4 are shown in Table 5.
[Table 5]

As shown in Table 5, it was confirmed that the extract of Production Example 1 inhibits the production of ICAM-1 induced by ultraviolet light. This suggests an effect of inhibiting the worsening of inflammation caused by leukocyte infiltration.

Formulation Example 1. Lotion [Ingredients] Parts Olive oil 1.0
Polyoxyethylene (5.5) Cetyl Alcohol 5.0
Butylparaben 0.1
Extract of Preparation Example 1 5.0
Ethanol 5.0
Glycerin 5.0
1,3-butylene glycol 5.0
Potassium hydroxide (as needed) Purified water (enough to make the total 100 parts) Fragrance (as needed)

Formulation Example 2. Emulsion [Ingredients] Parts Liquid paraffin 6.0
Hexalan 4.0
Jojoba oil 1.0
Polyoxyethylene (20) sorbitan monostearate 1.0
Lipophilic glyceryl stearate 1.0
Soy lecithin 1.5
Extract of Preparation 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
Purified water (enough to make the total amount 100 parts) Flavoring (as needed)

Formulation Example 3. Milky Lotion A milky lotion was obtained in the same manner as in Formulation Example 3, except that 3.0 parts of arbutin were used in place of 2.0 parts of L-ascorbic acid-2-glucoside and 0.5 parts of potassium hydroxide in the ingredients of Formulation Example 2.

Formulation Example 4. Milky Lotion A milky lotion was obtained in the same manner as in Formulation Example 3, except that, among the ingredients of Formulation Example 2, 2.0 parts of L-ascorbic acid-2-glucoside and 0.5 parts of potassium hydroxide were replaced with 2.0 parts of tranexamic acid.

Formulation Example 5. Milky Lotion A milky lotion was obtained in the same manner as in Formulation Example 3, except that, among the ingredients of Formulation Example 2, 2.0 parts of L-ascorbic acid-2-glucoside and 0.5 parts of potassium hydroxide were replaced with 3.0 parts of nicotinamide.

Formulation Example 6. Lotion [Ingredients] Parts Extract of Production Example 1 10.0
Ethanol 10.0
Glycerin 3.0
1,3-butylene glycol 2.0
Methylparaben 0.2
Citric acid 0.1
Sodium citrate 0.3
Carboxyvinyl polymer 0.1
Xanthan gum 0.1
Fragrance Appropriate amount Potassium hydroxide Appropriate amount Purified water (total amount is 100 parts)

Formulation Example 7. Essence [Ingredients] Parts Ethanol 2.0
Glycerin 5.0
1,3-butylene glycol 5.0
Methylparaben 0.1
Hyaluronic acid 0.1
Extract of Preparation Example 1 5.0
Citric acid 0.3
Sodium citrate 0.6
Purified water (enough to make the total amount 100 parts)

Example 8. 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
Extract of Preparation Example 1 5.0
Sodium carboxymethylcellulose 0.2
Bentonite 0.5
Propylene glycol 4.0
Triethanolamine 1.1
Methylparaben 0.1
Purified water (to make the total amount 100 parts) Titanium oxide 8.0
Talc 4.0
Color pigment (appropriate amount)

Formulation Example 9. 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
Extract of Preparation Example 1 5.0
1,3-butylene glycol 2.0
Purified water (enough to make the total amount 100 parts)

Formulation Example 10. 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
Extract of Preparation Example 1 2.0
1,3-butylene glycol 2.0
Purified water (enough to make the total amount 100 parts)

Example 11. 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 3.2
Stearyl alcohol 1.0
Methylparaben 0.1
Extract of Preparation Example 1 2.0
1,3-butylene glycol 5.0
Purified water (enough to make the total amount 100 parts)

Claims (2)

A skin topical agent containing as its active ingredient an extract of saffron (Crocus sativus) petals (excluding the mixed solvent extract consisting of betaine, lactic acid and water) from the Crocus genus of the Iridaceae family. A skin topical preparation characterized in that the petal extract according to claim 1 contains Kaempferol 3-O-beta-Sophoroside (excluding Kaempferol 3-O-lactyl-Sophoroside).