JP7836035B2 - Topical skin preparations - Google Patents

Description

Applicable under Article 30, Paragraph 2 of the Patent Law. Date of dispatch: May 24, 2019. Publication name: Program and abstracts of the 19th Annual Meeting of the Japanese Society for Anti-Aging Medicine. Publisher: Secretariat of the 19th Annual Meeting of the Japanese Society for Anti-Aging Medicine.

Applicable under Article 30, Paragraph 2 of the Patent Law, November 1, 2018. https://saishunkan.co.jp/labo/report/shuji02/index.html

This invention relates to an extract of Panax ginseng (C.A. Meyer), a plant belonging to the genus Panax in the family Araliaceae, which can be incorporated into topical skin preparations (including those for the scalp).

Conventionally, techniques for using ginseng or its extracts as active ingredients in topical skin preparations are known, as described in, for example, Patent Documents 1 to 6. However, conventional ginseng extracts have problems in terms of efficacy and safety.
Japanese Patent Publication No. 2000-212059 Japanese Patent Publication No. 2003-160497 Japanese Patent Publication No. 2005-194246 Japanese Patent Publication No. 2009-537466 International Public Gazette WO2014/132430 Japanese Patent Publication No. 2014-237633

As a result of diligent research to solve the above problems, the present inventors have newly discovered that an extract obtained from Panax ginseng (Panax CA Meyer), belonging to the genus Panax of the family Araliaceae, and containing at least ginsenosides Rg ₃ and Rg ₅ , has a whitening effect, an anti-aging effect, and/or an effect of repairing cell damage caused by ultraviolet rays, and is useful as an active ingredient in topical skin preparations. Furthermore, they have newly discovered that ginsenoside Rg ₅ is also useful as a whitening ingredient.

The present invention relates to a topical skin whitening agent containing a ginseng extract obtained from Panax ginseng CA Meyer, a plant belonging to the genus Panax in the family Araliaceae, and containing ginsenoside Rg ₃ and ginsenoside Rg ₅ , as an active ingredient.
The present invention relates to an anti-aging topical skin preparation containing an extract of Panax ginseng (Panax ginseng CA Meyer), which belongs to the genus Panax of the family Araliaceae, and which contains ginsenoside Rg ₃ and ginsenoside Rg ₅ as an active ingredient.
The present invention relates to a topical skin preparation for repairing cell damage caused by ultraviolet light, which contains a ginseng extract obtained from Panax ginseng CA Meyer, a plant belonging to the genus Panax of the family Araliaceae, and which contains ginsenoside Rg ₃ and ginsenoside Rg ₅ as an active ingredient.
The present invention relates to a skin whitening agent comprising ginsenoside Rg ₅ .

According to the present invention, a ginseng extract containing ginsenoside Rg ₃ and ginsenoside Rg ₅ provides a skin-whitening, anti-aging, and UV damage repair effect, making it possible to provide a topical skin preparation for whitening, anti-aging, or repairing cell damage caused by ultraviolet rays. Furthermore, ginsenoside Rg ₅ can be provided as a skin-whitening ingredient for topical application.

This figure shows the results of a skin whitening effect evaluation test. This figure shows the results of a skin whitening effect evaluation test. This figure shows the results of a skin whitening effect evaluation test. This figure shows the results of a skin whitening effect evaluation test. This figure shows the results of a cell repair test after UV irradiation. This figure shows the evaluation results of intracellular glutathione levels after UV irradiation. This figure shows the evaluation results of intracellular glutathione levels after UV irradiation. This figure shows the results of the fibroblast activation test.

The plant used in this invention is Panax ginseng (Panax ginseng C.A. Meyer), belonging to the genus Panax in the family Araliaceae. The root and fibrous roots are particularly preferred as the extractant part of the Panax ginseng.

In the present invention, a ginseng extract containing ginsenosides Rg ₃ and Rg ₅ is obtained by subjecting ginseng roots (particularly fibrous roots are preferred) to a treatment such as heating. In the present invention, Rg ₃ is preferably present in an amount of 20 ppm or more, more preferably 40 ppm or more, and even more preferably 60 ppm or more. Ginsenoside Rg ₅ is preferably present in an amount of 10 ppm or more, more preferably 15 ppm or more, and even more preferably 25 ppm or more in the extract.

Furthermore, in the extract according to the present invention, the content ratio of ginsenoside Rg ₃ to ginsenoside Rg ₅ is preferably 2:1 to 4:1 by weight.

The ginseng extract containing the above-mentioned ginsenosides Rg ₃ and Rg ₅ can be obtained, for example, by heat treatment. The heating temperature conditions are 100°C to 121°C for 1 hour or more, and the heated ginseng is extracted with water, lower alcohol, polyhydric alcohol, or a mixture thereof to obtain a ginseng extract containing amounts of ginsenosides Rg ₃ and Rg ₅ that exhibit whitening effects, anti-aging effects, and effects of repairing cell damage caused by ultraviolet rays. Furthermore, as will be described later, from the viewpoint of whitening effect, it is preferable that the amount of ginsenosides Rg ₁ and Rb ₁ in the extract be smaller. It is preferable that it be at least less than the amount of ginsenoside Rg ₃ .

From the viewpoint of extracting the active ingredient and ensuring safety, a mixed solvent of water and a polyhydric alcohol (ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.) is preferred as the extraction solvent. The mixing ratio of water to polyhydric alcohol is preferably 70:30 to 10:90.

The extract obtained by the method described above may be incorporated into a topical skin preparation as a liquid extract, or as a dry powder.

The extract or dried product thereof according to the present invention can be incorporated into topical skin preparations (cosmetics, quasi-drugs, topical pharmaceuticals). Examples of topical skin preparations include, but are not limited to, emulsions, creams, lotions, essences, masks, lipsticks, foundations, liquid foundations, makeup press powders, blushes, face powders, facial cleansers, body shampoos, scalp and hair shampoos, hair conditioners, hair growth and hair nourishment shampoos or tonics, cleansing cosmetics such as soaps, and bath additives.

The amount of the extract, concentrate, or dried product according to the present invention is, in terms of solid content, in the range of 0.0001 to 10.0% by weight (solid content by weight, the same applies hereinafter) for basic cosmetics, in the range of 0.0001 to 5.0% by weight for makeup cosmetics, and in the range of 0.0001 to 20.0% by weight for cleansing cosmetics.

When using the extract according to the present invention in a topical skin preparation, components suitable for topical skin preparations, such as oily components, surfactants (synthetic and natural), emulsifiers, moisturizers, thickeners, preservatives/bactericides, defoaming agents, powder components, antioxidants, chelating agents, pH adjusters, pigments, fragrances, etc., can be appropriately added as needed. Furthermore, there is no problem in combining it with other physiologically active components, as long as the effectiveness and characteristics of the ginseng extract or its concentrate or dried product according to the present invention are not impaired.

Here, the oily components include, for example, lotus oil, rose oil, rosehip oil, olive oil, jojoba oil, castor oil, soybean oil, rice oil, rice bran oil, rice germ oil, coconut oil, chamomile oil, Roman chamomile oil, palm oil, cocoa oil, meadowfoam oil, bergamot oil, rosehip oil, coffee arabic seed oil, lavender oil, shea butter, geranium oil, pine seed oil, tea tree oil, avocado oil, macadamia nut oil, vanilla oil, plant-derived oils such as squalane; animal-derived oils such as mink oil and turtle oil; beeswax, carnauba wax, rice wax. Examples include waxes such as custard and lanolin; hydrocarbons such as liquid paraffin, 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; and synthetic esters and triglycerides such as isopropyl myristate, isopropyl palmitate, butyl oleate, cetyl isooctanoate, 2-ethylhexylglyceride, and higher fatty acid octyldodecyl (e.g., octyldodecyl stearate).

Furthermore, 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 alkylphenyl ether sulfates, polyoxyethylene alkyl ether phosphates, and α-sulfonated fatty acid alkyl ethers. Anionic surfactants such as telates and polyoxyethylene alkylphenyl 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-dialkylmorphonium salts, and polyethylene polyamine fatty acid amide salts; and amphoteric surfactants such as N,N-dimethyl-N-alkyl-N-carboxymethylammonium betaine, N,N,N-trialkyl-N-alkyleneammonium carboxybetaine, and N-acylamidopropyl-N',N'-dimethyl-N'-β-hydroxypropylammonium sulfobetaine can be used.

Furthermore, emulsifiers or emulsifying aids may include stevia derivatives such as enzyme-treated stevia, saponins or their derivatives, casein or its salts (such as sodium), sugar-protein complexes, sucrose or its esters, lactose, water-soluble polysaccharides derived from soybeans, complexes of soybean-derived proteins and polysaccharides, lanolin or its derivatives, cholesterol, stevia derivatives (such as enzyme-treated stevia), silicates (such as aluminum and magnesium), carbonates (such as calcium and sodium), saponins and their derivatives, lecithin and its derivatives (such as hydrogenated lecithin), lysolecithin or its derivatives, lactic acid bacteria-fermented rice, lactic acid bacteria-fermented germinated rice, lactic acid bacteria-fermented grains (such as wheat, beans, and other grains), etc.

Furthermore, examples of humectants include glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, polyethylene glycol, sorbitol, xylitol, sodium pyrrolidone carboxylate, 2-methacryloyloxyethyl phosphorylcholine/butyl methacrylate copolymer solution, and other sugars such as trehalose, Tremella fuciformis polysaccharides, mucopolysaccharides (e.g., hyaluronic acid and its derivatives, chondroitin and its derivatives, heparin and its derivatives, etc.), tuberose polysaccharides, elastin and its derivatives, collagen and its derivatives, NMF-related substances, hydrolyzed conchiolin, hydrolyzed silk, Sphingomonas culture, sphingoglycolipids, ceramides (human-type ceramides, yuzu ceramides, etc.), lactic acid, urea, higher fatty acid octyldodecyl, seaweed extracts, Bletilla striata root extract, various amino acids and their derivatives.

Furthermore, examples of thickening agents include components derived from brown, green, or red algae such as alginic acid, agar, carrageenan, and fucoidan; Bletilla striata root extract; polysaccharides such as pectin, locust bean gum, aloe polysaccharide, and Alcaligenes-producing polysaccharide; gums such as xanthan gum, tragacanth gum, roasted 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.

Furthermore, preservatives and disinfectants include, for example, urea; parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate; phenoxyethanol, dichlorophene, hexachlorophene, chlorhexidine hydrochloride, benzalkonium chloride, salicylic acid, ethanol, undecylenic acid, phenols, jamal (imidazodinyl urea), polyphosphate, propanediol, 1,2-pentanediol, various essential oils, bark distillates, radish ferment filtrate, and plant-derived ethanol or 1,3-butylene glycol from sugarcane, etc.

Furthermore, powder components include, for example, sericite, titanium dioxide, talc, kaolin, bentonite, zinc oxide, magnesium carbonate, magnesium oxide, zirconium oxide, barium sulfate, anhydrous silicic acid, mica, nylon powder, polyethylene powder, silk powder, cellulose-based powders, grain powders (rice, wheat, corn, millet, etc.), and legume powders (soybeans, adzuki beans, etc.).

Furthermore, examples of UV absorbers include ethyl para-aminobenzoate, ethylhexyl para-dimethylaminobenzoate, amyl salicylate and its derivatives, 2-ethylhexyl para-methoxycinnamate, octyl cinnamate, oxybenzone, 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonate, 4-tert-butyl-4-methoxybenzoylmethane, 2-(2-hydroxy-5-methylphenyl)benzotriazole, urocanic acid, ethyl urocanate, and aloe extract.

Furthermore, examples of antifoaming agents include ethanol, isopropanol, disiloxane, dimethylpolycyclosane, dimethicone silica, trisiloxane, silylated silica, dimethicone, trimethylsiloxysilicate, and DPG isobornyl ether.

Furthermore, antioxidants include, for example, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, astaxanthin, extract of Callicarpa japonica, extract of Bletilla striata root, extract of Paeonia lactiflora, vitamin E and its derivatives (e.g., vitamin E nicotinate, vitamin E linoleate, etc.).

Furthermore, examples of chelating agents include trisodium ethylenediamine hydroxyethyl triacetate, EDTA or its salts, gluconic acid, phytic acid, sodium polyphosphate, sodium metaphosphate, and tetrasodium hydroxyethanediphosphonate.

Examples of pH adjusting agents 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.

Furthermore, examples of 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, potassium 4-methoxysalicylate, magnolignan (5,5'-dipropyl-biphenyl-2,2'-diol), hydroxybenzoic acid and its derivatives, vitamin E and its derivatives, α-hydroxy acids, and AMP (adenosine monophosphate, adenosine monophosphate). These may be used individually or in combination.

Furthermore, examples of kojic acid derivatives include kojic acid esters such as kojic acid monobutyrate, kojic acid monocaprate, kojic acid monopalmitate, and kojic acid dibutyrate, kojic acid ethers, and kojic acid sugar derivatives such as kojic acid glucoside. Examples of ascorbic acid derivatives include ascorbic acid ester salts such as L-ascorbic acid-2-phosphate sodium, L-ascorbic acid-2-phosphate magnesium, L-ascorbic acid-2-sulfate sodium, and L-ascorbic acid-2-sulfate magnesium, as well as L-ascorbic acid-2-glucoside and L-ascorbic acid-5- Ascorbic acid sugar derivatives such as glucoside, ascorbyl tocopheryl maleate, ascorbyl tocopheryl potassium phosphate, myristyl 3-glyceryl ascorbate, caprylyl 2-glyceryl ascorbate, 6-acyl derivatives of these ascorbic acid sugar derivatives (acyl groups include hexanoyl, octanoyl, and decanoyl groups), L-ascorbic acid tetra fatty acid esters such as L-ascorbic acid tetraisopalmitate and L-ascorbic acid tetralaurate, 3-O-ethyl ascorbic acid, L-ascorbic acid-2-phosphate-6-O-palmitate sodium, and glyceryl ascorbate. Examples of hydroquinone derivatives include acids or their acylated derivatives, ascorbic acid glycerin derivatives such as bisglyceryl ascorbic acid, L-ascorbic acid aminopropyl phosphate, hyaluronic acid derivatives of L-ascorbic acid, 3-O-D lactose-L-ascorbic acid, isostearyl ascorbyl phosphate, etc. Examples of hydroquinone derivatives include arbutin (hydroquinone-β-D-glucopyranoside), α-arbutin (hydroquinone-α-D-glucopyranoside), etc. Examples of tranexamic acid derivatives include tranexamic acid esters (e.g., tranexamic acid lauryl ester, tranexamic acid hexadecyl ester, tranexamic acid cetyl ester). Examples include tranexamic acid esters or their salts, tranexamic acid amides (e.g., tranexamic acid methylamide), etc. Resorcinol derivatives include, for example, 4-n-butylresorcinol and 4-isoamylresorcinol. 2,5-dihydroxybenzoic acid derivatives include, for example, 2,5-diacetoxybenzoic acid, 2-acetoxy-5-hydroxybenzoic acid, and 2-hydroxy-5-propionyloxybenzoic acid. Nicotinic acid derivatives include, for example, nicotinamide and benzyl nicotinate. α-hydroxy acids include, for example, lactic acid, malic acid, succinic acid, citric acid, and α-hydroxyoctanoic acid.

Furthermore, as physiologically active ingredients, for example, placenta extract, mulberry bark extract, saxifrage extract, perilla extract, rice bran extract or its hydrolysate, white mustard extract or its hydrolysate, white mustard ferment, peony extract or its hydrolysate, lactic acid bacteria extract, bifidobacteria extract, beautyberry extract, lotus seed extract or its hydrolysate, lotus seed ferment, Job's tears hydrolysate, Job's tears seed ferment, royal jelly ferment, soybean sprout extract, sake lees extract or ceramide contained therein, sake lees ferment, Pandanus amaryllis extract, Arcangelicia flava extract, calendula extract, chamomile extract, coral grass extract, rice Leaf extract or hydrolysate thereof, eggplant (lotus, long eggplant, Kamo eggplant, American eggplant, etc.) extract or hydrolysate thereof, apricot fruit extract, seaweed extract such as Eucommia ulmoides, marine flowering plant extract such as Eucommia gracilis, soy milk ferment, jellyfish water, rice extract or hydrolysate thereof, rice fermentation extract, germinated rice extract or hydrolysate thereof, germinated rice ferment, black bean extract or hydrolysate thereof, brown sugar extract or ferment, Damask rose flower extract, ylang-ylang flower extract, bamboo shoot peel extract, linoleic acid and its derivatives or processed products (e.g., liposomal linoleic acid), collagen and its derivatives derived from animals or fish, elastin and its derivatives, animal or fish derived Proteoglycans, glycyrrhizic acid and its derivatives (such as dipotassium salts), t-cycloamino acid derivatives, vitamin A and its derivatives, allantoin, diisopropylamine dichloroacetate, γ-amino-β-hydroxybutyric acid, gentian extract, licorice extract, ginseng extract, Panax ginseng (white ginseng) extract, Panax ginseng ferment, camellia extract, kelp extract, loofah extract, Ulva extract, peach extract, plum extract, peach kernel extract, kiwi extract, sunflower extract, juazeiro extract, Pau d'arco bark extract, daylily extract or ferment, hibiscus flower extract or ferment, rhododendron extract These include cherimoya extract, mango extract, mangosteen extract, seaweed extract, oolong tea extract, red bell pepper extract, orchid extract, sweet potato extract, sansho pepper peel or seed coat extract or its hydrolysate, safflower extract, Casablanca lily extract, sweet potato extract or its fermented product, guava leaf extract, Artemisia capillaris extract, clove extract, olive leaf extract, horse chestnut extract, ginger extract, vanilla extract, Houttuynia cordata extract, pomelo extract, aloe extract, aloe vera extract, tulip extract, centella asiatica extract, fig flower extract, apple extract, white asparagus extract, kudzu extract, Codonopsis pilosula extract or its hydrolysate, etc.

Next, the present invention will be described in more detail with reference to manufacturing examples, test examples, and embodiments, but the present invention is not limited to these. In the following, all parts refer to parts by weight, and all percentages refer to weight percent.

Preparation Example 1. Preparation of Panax Ginseng Extract (1) 10 g of Panax ginseng root hairs were immersed in 40 g of purified water and heated at 100°C to 120°C for 1 hour. The heated root hairs were extracted with 50% butylene glycol to obtain 86 g of Panax ginseng extract (solid content concentration 1.7%).

Manufacturing Example 2. Preparation of Panax Ginseng Extract (2) 10 g of Panax ginseng root hairs were immersed in 40 g of purified water and heated at 100°C to 120°C for 2 hours. The heated root hairs were extracted with 50% butylene glycol to obtain 80 g of Panax ginseng extract (solid content concentration 2.0%).

Manufacturing Example 3. Preparation of Panax Ginseng Extract (3) 10 g of Panax ginseng root hairs were immersed in 40 g of purified water and heated at 100°C to 120°C for 3 hours. The heated root hairs were extracted with 50% butylene glycol to obtain 90 g of Panax ginseng extract (solid content concentration 2.3%).

Manufacturing Example 4. Preparation of Panax Ginseng Extract (4) 100 g of Panax ginseng root hairs were immersed in 500 g of purified water and heated at 100°C to 115°C for 2 hours. After heating, the root hairs were immersed in purified water to obtain 1000 g of Panax ginseng extract (solid content concentration 2.0%).

Manufacturing Example 5. Preparation of Panax Ginseng Extract (5) 20 g of Panax ginseng root hairs were immersed in 80 g of purified water and heated at 100°C to 115°C for 4 hours. The heated root hairs were extracted with 75% butylene glycol to obtain 219 g of Panax ginseng extract (solid content concentration 1.8%).

Comparative Production Example 1. Preparation of Panax Ginseng Extract 10 g of Panax ginseng root hairs were immersed in 40 g of purified water and extracted at 40°C to obtain 105 g of Panax ginseng extract (solid content concentration 1.45%).

Test Example 1. Quantitative Test Method for Ginsenosides _Rg3 and _Rg5 10 μL of Production Examples 1-5 and Comparative Production Example 1 were taken and tested by liquid chromatography under the following test conditions. The peak areas of the obtained chromatograms were determined, and the concentrations of ginsenosides _Rg3 and _Rg5 were determined from a pre-prepared calibration curve.
(1) Test conditions Detector: UV absorbance spectrophotometer (measurement wavelength: 203 nm)
Column: A stainless steel tube with an inner diameter of 4.6 mm and a length of 15 cm was used, packed with 5 μm octadecylsilylated silica gel for liquid chromatography.
Column temperature: 30°C
Mobile phase A: Acetonitrile Mobile phase B: Water Mobile phase delivery: The concentration gradient was controlled by changing the mixing ratio of mobile phase A and mobile phase B as shown in Table 1.
[Table 1]
(2) Preparation of Calibration Curve First, 5 mg of ginsenoside Rg ₃ was weighed out and 90% acetonitrile aqueous solution was added to make 25 mL of standard stock solution. Next, 2.5 mL and 5.0 mL of ginsenoside Rg ₃ standard stock solution were accurately taken, and mobile phase was added to each to make exactly 10 mL of standard solution. Then, 10 μL of the standard stock solution and 10 μL of the standard solution were taken and tested by liquid chromatography under the test conditions described above. A calibration curve was created by plotting the peak area of the obtained chromatogram on the x-axis and the concentration of ginsenoside Rg ₃ (w/v ppm) on the y-axis. For Rg ₅ , 5 mg was weighed out and 90% acetonitrile aqueous solution was added to make 25 mL of standard stock solution. Next, 1.0 mL, 2.5 mL, and 5.0 mL of ginsenoside Rg ₅ standard stock solution were accurately taken, and mobile phase was added to each to make exactly 10 mL of standard solution. Then, 10 μL of the standard solution was taken and tested by liquid chromatography under the aforementioned test conditions. A calibration curve was created by plotting the peak area of the obtained chromatogram on the x-axis and the concentration of ginsenoside Rg ₅ (w/v ppm) on the y-axis.

Table 2 shows the measurement results of ginsenosides Rg ₃ and Rg ₅ contained in the extracts of Production Examples 1 to 5 and the extract of Comparative Production Example 1.
[Table 2]

Test Example 2. Melanin Production Inhibition Evaluation Test (1) Culture of B16 Cells B16 cells (mouse melanoma cells) were cultured in DMEM (Dulbecco's modified Eagle's medium (Sigma-Aldrich, St Louis, MO, USA)) containing 10% FBS (fetal bovine serum), 100 units/ml penicillin, and 100 μg/ml streptomycin (Wako, Osaka, Japan) at 5% _CO2 and a temperature of 37°C.
(2) Test Solution Treatment Using the same culture method as in (1) above, B16 cells were seeded at a rate of 5 × ^10⁴ cells/well in a 6-well multiplate. After 24 hours, a mixed solution of 3-isobutyl-1-methylxanthine (IBMX) as a melanin inducer, the test solution, and 2 mL of DMEM containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin was prepared in each well, and the culture medium was replaced. The cells were then cultured at 5% _CO₂ and a temperature of 37°C. As test solutions, extracts from Production Examples 1 to 3 and their comparative control (extract from Comparative Production Example 1) were prepared to a concentration of 1.0% (as a solution), and extracts from Production Examples 4 and 5 and their comparative control (extract from Comparative Production Example 1) were prepared to a concentration of 0.5% (as a solution). Furthermore, a control (-IBMX) was set up when the culture medium was cultured without IBMX, and instead of the test solution, 0.75% or 0.5% 1,3-butylene glycol (BG) was added. Additionally, a control (+IBMX) was set up when IBMX was added, and 0.75% or 0.5% BG was added instead of the test solution.
(3) Measurement of melanin content 72 hours after sample processing, the culture medium was removed, and each well was washed with 2 mL of PBS. Then, 50 μL of 1% TritonX-100 (SIGMA) and 250 μL of 1N NaOH were added in that order, mixed thoroughly, and collected in 1.5 mL tubes. The collected tubes were incubated at 98°C for 30 minutes to prepare cell lysates. The lysates were mixed thoroughly and 80 μL/well was added to a 96-well multiplate, and the absorbance at 405 nm was measured using a microplate reader (ARVO ^™ X3).
(4) Measurement of protein content The cell lysate prepared in (3) above was analyzed using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manual. The absorbance at 560 nm was measured using a microplate reader (ARVO ^™ X3).
(5) Evaluation of Melanin Production Melanin and protein amounts were calculated using the method described above, and melanin production was evaluated from the melanin/protein ratio. The evaluation value was calculated as a relative value with the control (-IBMX) set to 100.

The results of Test Example 2 are shown in Figures 1 and 2. As shown in Figures 1 and 2, the extracts from Production Examples 1 to 5 were confirmed to exhibit significantly superior melanin production inhibitory effects compared to the extract from Comparative Production Example 1.

Test Example 3. Whitening Evaluation Test of Ginsenosides _Rg3 , _Rg5 , _Rb1 , and _Rg1 Instead of using the manufacturing examples 1-5 and comparative manufacturing example 1 of Test Example 2 as test solutions, solutions of ginsenosides _Rg3 , _Rg5 , _Rb1 , and _Rg1 were used. Dimethyl sulfoxide (DMSO) was used as the solvent to dissolve IBMX. A control (-IBMX) was set up when the culture medium was cultured with DMSO added instead of ginsenosides, without the addition of IBMX. Furthermore, a control (+IBMX) was set up when the culture medium was cultured with IBMX and DMSO was added instead of the test solution. The final concentration of DMSO was adjusted to 0.1%.

The results of Test Example 3 are shown in Figures 3 and 4. As shown in Figure 3, Rg ₃ and Rg ₅ exhibited a melanin production inhibitory effect at low concentrations, while as shown in Figure 4, Rb ₁ and Rg ₁ were confirmed to promote melanin production.

Test Example 4: Evaluation Test of Cell Damage Repair Caused by Ultraviolet Radiation (1) Cell (HaCaT) Culture HaCaT cells (human keratinocyte cells) were cultured in DMEM (Dulbecco's modified Eagle's medium (Sigma-Aldrich, St Louis, MO, USA)) containing 5% FBS (fetal bovine serum), 100 units/mL penicillin, and 100 μg/mL streptomycin (Wako, Osaka, Japan) at 5% _CO2 and a temperature of 37°C.
(2) Test Solution Treatment HaCaT cells were seeded at a rate of 1 × ^10⁵ cells/well in a 6-well multiplate using the same culture method as in (1) above. After 24 hours, a mixed solution of the test solution and 2 mL of DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin was prepared in each well, replaced with the culture medium, and cultured at 5% _CO₂ and 37°C. Here, as test solutions for evaluating cell viability repair, production examples 1 to 3 and their comparison target (extract from comparative production example 1) were adjusted to a concentration of 0.5% (as a solution). In addition, as test solutions for evaluating the promotion of intracellular glutathione production, extracts from production examples 1, 2, and 5 and their comparison target (extract from comparative production example 1) were adjusted to a concentration of 1.0% (as a solution). Furthermore, a control was set when cells were cultured in a culture medium to which 0.25% or 1.0% BG was added instead of the test solution.
(3) UV-B treatment (HaCaT cells)
Five hours after treatment with the test solution, the samples were exposed to UV-B (60 mJ) using a UV-B irradiator (TL20W/12RS lamp, Dermaray 200; Muranaka). UV intensity was measured using an ultraviolet radiation sensor (UVX-31; TGK). After UV-B exposure, the samples were cultured at 5% _CO2 and 37°C. A control group (UV-) that was not irradiated with UV-B was set up for comparison.
(4) Cell viability evaluation (HaCaT cells)
The Cell Counting Kit-8 (CCK8; Dojindo) was used, following the manual. The following modifications were made from the manual. 24 hours after UV-B exposure, the culture medium was removed, and the CCK8 solution was added to each well. The cells were incubated at 5% _CO2 and 37°C for 1 hour. The CCK8 solution was collected in a 96-well plate, and the absorbance at 450 nm was measured using a microplate reader (ARVO ^™ X3). The evaluation values were calculated as relative values with the control group (UV-) without UV-B irradiation set to 100.
(5-1) Glutathione (GSH) measurement (HaCaT cells)
After 24 hours of UV-B exposure, the culture medium was removed, and each well was washed with 2 mL of PBS. 300 μL of 0.05% Trypsin-EDTA (Gibco) was added, and the mixture was incubated at 5% _CO2 , 37°C, for 5 minutes. 300 μL of DMEM was added and the mixture was collected in a 1.5 mL tube. 500 μL of PBS was then added to each well, collected in the same tube, centrifuged (2000 rpm, 3 minutes), and the supernatant was removed. 1 mL of PBS was added, centrifuged (2000 rpm, 3 minutes), and the supernatant was removed. 250 μL of 0.5% Triton-X100 was added, thoroughly vortexed and sonicated, centrifuged (13200 rpm, 10 minutes), and the supernatant was collected. The collected supernatant was subjected to GSSG/GSH Quantification using the GSSG/GSH Quantification Kit (Dojindo) according to the manual. The absorbance at 405 nm was measured using a microplate reader (ARVO ^™ X3).
(5-2) Protein level measurement (HaCaT cells)
The supernatant collected in the "Glutathione Measurement" described above was analyzed using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manual. The absorbance at 560 nm was measured using a microplate reader (ARVO ^™ X3).
(5-3) Glutathione level evaluation (HaCaT cells)
Glutathione (GSH) and protein levels were calculated using the method described above, and glutathione levels were evaluated from the glutathione/protein ratio. The evaluation values were calculated as relative values with the control group (UV-) without UV-B irradiation set to 100.

The results of Test Example 4 are shown in Figures 5 to 7. As shown in Figure 5, the extract according to the present invention was confirmed to exhibit a remarkable effect in repairing cells damaged by ultraviolet light compared to Comparative Production Example 1. Furthermore, as shown in Figures 6 and 7, the extract according to the present invention was also confirmed to have a remarkable effect in restoring intracellular glutathione levels reduced by ultraviolet irradiation, compared to Comparative Production Example 1.

Test Example 5. Evaluation Test of Fibroblast Activation Effect Human dermal fibroblasts NB1RGB were seeded at a rate of 1 × ^10⁴ cells/well in a 96-well microplate containing 0.5% NCS-containing Eagle Minimum Essential Medium. After pre-culturing for 1 day at 37°C and 5.0% _CO₂ , extracts from Production Examples 4, 5, and Comparative Production Example 1 were added to the medium as sample solutions to a concentration of 0.5% (as solution), and cultured for a further 3 days under the same conditions. Next, the medium was removed, 0.03% MTT was added, and the culture was held at 37°C for 1 hour. The resulting formazan was extracted with isopropanol, and the MTT value was measured at a wavelength of 570–630 nm using a microplate reader (Model 680, Bio-Rad). The same procedure was performed for the control case (no sample added), where PBS(-) was added instead of the sample solution. The relative values of the MTT values obtained with each sample added were then calculated and expressed as the fibroblast MTT activity rate (%). In addition, to confirm that the test system was functioning correctly, the same test was performed when 100 mM glucose was added as a positive control instead of the sample solution.

The results of Test Example 5 are shown in Figure 8. As shown in Figure 8, the extract according to the present invention was confirmed to exhibit a significant fibroblast-activating effect compared to Comparative Production Example 1.

Formula Example 1. Lotion [Ingredients] Lavender Oil 1.0
Glyceryl caprylate 3.0
Polyglyceryl-10 Laurate 3.0
Methylparaben 0.1
Extract from Production Example 1 2.0
White Ginseng Extract 2.0
Ginseng Fermented Extract 2.0
Sweet potato fermented extract 1.0
Guava leaf extract 1.0
Water-soluble collagen 1.0
Hydrolyzed collagen 1.0
Water eggplant extract 1.0
Strawberry flower extract 1.0
Glycerin 2.0
1,3-Butylene glycol 5.0
Sodium citrate 0.2
Purified water: an amount that makes the total volume 100 parts

Formulation Example 2. Lotion A lotion was obtained in the same manner as in Formulation Example 1, except that 2.0 parts of the extract from Production Example 2 were used instead of the extract from 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 extract from Production Example 3 were used instead of the extract from Production Example 1 contained in Formulation Example 1.

Formulation Example 4. Lotion A lotion was obtained in the same manner as in Formulation Example 1, except that 2.0 parts of the extract from Production Example 4 were used instead of the extract from Production Example 1 contained in Formulation Example 1.

Formulation Example 5. Lotion A lotion was obtained in the same manner as in Formulation Example 1, except that 2.0 parts of the extract from Production Example 5 were used instead of the extract from Production Example 1 contained in Formulation Example 1.

Formula Example 6. Emulsion [Ingredients] Squalane 5.0
Hexaran 3.0
Rosehip oil 1.0
Camellia oil 1.5
Polyglyceryl laurate 1.0
Lipophilic glyceryl stearate 1.0
Bisabolol 1.0
Behenyl alcohol 3.0
Hydrogenated soy lecithin 1.5
Extract from manufacturing example 5: 2.0
White Ginseng Extract 2.0
Ginseng Fermented Extract 2.0
L-ascorbic acid-2-glucoside 2.0
Sweet potato fermented extract 1.0
Alpinia zerumbet leaf extract 1.0
Guava leaf extract 1.0
Licorice extract 1.0
Codonopsis pilosula hydrolyzed extract 1.0
Water-soluble collagen 1.0
Hydrolyzed collagen 1.0
Potassium hydroxide 0.5
Glycerin 3.0
1,3-Butylene glycol 2.0
Carboxymethylcellulose 0.3
Xanthan gum 0.2
Sodium hyaluronate 0.01
Purified water: an amount that makes the total volume 100 parts

Formula Example 7. Cream [Ingredients] Olive oil 5.0
Jojoba oil 5.0
Meadowfoam oil 5.0
Squalane 5.0
Behenyl alcohol 2.0
Palmitic acid 2.5
Lactic acid fermented rice 2.0
Hydrogenated lecithin 0.5
Glyceryl stearylate 3.0
Extract from Manufacturing Example 1 2.0
White Ginseng Extract 2.0
Ginseng Fermented Extract 2.0
Sweet potato fermented extract 1.0
Alpinia zerumbet leaf extract 1.0
Guava leaf extract 1.0
Hydrolyzed rice extract 2.0
Water-soluble collagen 1.0
Hydrolyzed collagen 1.0
Xanthan gum 1.0
Purified water: an amount that makes the total volume 100 parts

Formulation Example 8. Cream A cream was obtained in the same manner as in Formulation Example 1, except that 2.0 parts of the extract from Production Example 2 were used instead of the extract from Production Example 1 contained in Formulation Example 7.

Formulation Example 9. Cream A cream was obtained in the same manner as in Formulation Example 1, except that 2.0 parts of the extract from Production Example 3 were used instead of the extract from Production Example 1 contained in Formulation Example 7.

Formulation Example 10. Cream A cream was obtained in the same manner as in Formulation Example 11, except that 2.0 parts of the extract from Production Example 4 were used instead of the extract from Production Example 1 contained in Formulation Example 7.

Formula Example 11. Facial Cleanser [Ingredients] Coconut Oil 2.0
Stearyl stearylate 2.0
Glyceryl caprylate 2.0
Extract from Manufacturing Example 1 2.0
White Ginseng Extract 2.0
Ginseng Fermented Extract 2.0
Water-soluble collagen 1.0
Glycerin 2.0
Fig bark extract 1.0
Job's tears seed extract 1.0
Clove extract 1.0

Claims (3)

A melanin production inhibitor in pigment cells , containing a ginseng extract obtained from Panax ginseng (Panax CA Meyer), a plant belonging to the genus Panax in the family Araliaceae, and containing ginsenoside Rg3 and ginsenoside Rg5 as the active ingredient. An inhibitor of the decrease in glutathione in epidermal cells caused by ultraviolet light , containing an extract of Panax ginseng (Panax CA Meyer), which belongs to the genus Panax in the family Araliaceae, and which contains ginsenoside Rg3 and ginsenoside Rg5, as the active ingredient. A fibroblast activator containing a ginseng extract as an active ingredient, obtained from Panax ginseng (Panax CA Meyer), a plant belonging to the genus Panax in the family Araliaceae, and containing ginsenoside Rg3 and ginsenoside Rg5.