JP2018090576A - Skin-whitening agent, method for producing skin-whitening agent and cosmetics - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a skin-whitening agent having excellent skin-whitening action and safety.SOLUTION: A skin-whitening agent contains licorice extract extracted from Glycyrrhiza uralensis Fisher, and licorice flavonoid that is extracted from Glycyrrhiza glabra Linne and works with the licorice extract to exhibit a skin-whitening action. The licorice extract is 0.0012-0.0045% based on the total amount, and the licorice flavonoid is 0.000125-0.0005% based on the total amount.SELECTED DRAWING: Figure 6

Description

  The present invention relates to a whitening agent, a method for producing a whitening agent, and a cosmetic. Specifically, the present invention relates to a whitening agent excellent in whitening action and safety, a method for producing a whitening agent, and a cosmetic.

  Conventionally, various whitening agents have been added as raw materials for cosmetics. As the action of the whitening agent, suppression of production of melanin that causes pigmentation on the skin, or pale bleaching of the produced melanin has been reported.

  The production of melanin is caused by the activation of pigment cells (melanocytes) existing in the skin, and by the action of the enzyme tyrosinase biosynthesized in the pigment cells, it passes through an intermediate from tyrosine, which is a kind of amino acid. It is said that melanin is formed.

  Various plant extracts are used as raw material components having a whitening action, and for example, ascorbic acid, kojic acid, Erica extract, mulberry leaf extract and the like are known.

  In addition, the tyrosinase expression inhibitory action which acts on the production of melanin was also reported to the extract from the root of the licorice plant (Glycyrrhiza), for example, the tyrosinase expression inhibitor described in Patent Document 1 Has been proposed. Patent Document 1 discloses an extract from bamboo shoots and licorice roots.

JP 2004-352697 A

  However, in the conventional whitening agents including the tyrosinase expression inhibitor disclosed in Patent Document 1, there is almost no mention of cytotoxicity and skin irritation at a concentration at which whitening action is observed. It can be said that the collateral is insufficient.

  Moreover, in the tyrosinase expression inhibitor of Patent Document 1, there is a whitening agent using a combination of extracts derived from completely different types of plant materials, such as a combination of bamboo shoot extract and licorice root extract, but it belongs to the same genus. There have been almost no reports on whitening agents using two or more kinds of extracts extracted from plants belonging to the genus, for example, licorice plants by various methods. This point can be considered for improving the whitening effect and improving the irritation to the skin.

  The present inventors have made synergistic whitening by combining extracts extracted from two species of licorice plants of different species, more particularly, larva (Glycyrrhiza uralensis Fisher) and Spanish licorice (Glycyrrhiza glabra Linne). It has been found that a whitening agent having an action and suppressing cytotoxicity and irritation to the skin can be produced.

  The present invention has been made in view of the above points, and an object thereof is to provide a whitening agent excellent in whitening action and safety, a method for producing a whitening agent, and a cosmetic.

  In order to achieve the above object, the whitening agent of the present invention contains a licorice extract extracted from licorice and a licorice flavonoid extracted from licorice having a different species from the extraction origin of the licorice extract.

  Here, with the licorice extract extracted from licorice, a whitening effect derived from the components contained in the licorice extract can be obtained.

  Moreover, the whitening action of the licorice flavonoid derived from the licorice of the seed | species different from a licorice extract can be acquired with the licorice flavonoid extracted from the licorice whose seeds differ from the extraction origin of the licorice extract.

  In addition, when licorice extract is extracted from the roots of Ural licorice with a mixture of water and ethanol, and licorice flavonoids are extracted from Spanish licorice roots and rhizomes with absolute ethanol, water from the Ural licorice root is water-soluble. The oil-soluble component and the oil-soluble component are extracted, and the oil-soluble component is extracted from the roots and rhizomes of Spanish licorice.

  In addition, when the licorice extract contains 0.5 mg / mL or more of liquiritin, a moisturizing effect can be imparted to the whitening agent.

  The licorice extract has a content of 0.0012 to 0.0045% on the basis of the total amount, and the licorice flavonoid has a content of 0.000125 to 0.0005% on the basis of the total amount. An excellent and synergistic whitening effect can be obtained. Moreover, it can be set as the whitening agent with low irritation to skin.

  In order to achieve the above object, the whitening agent of the present invention is extracted from licorice extract extracted from Glycyrrhiza uralensis Fisher and Spanish licorice (Glycyrrhiza glabra Linne). Contains licorice flavonoids that work and have a whitening effect.

  Here, licorice extract was extracted from Glycyrrhiza uralensis Fisher, and licorice flavonoids were extracted from Spanish licorice (Glycyrrhiza glabra Linne), rather than the whitening effect that the two types of extracts produce alone. An excellent synergistic whitening effect can be obtained. Moreover, it can be set as the whitening agent with low irritation | stimulation to skin.

  Further, when the content of licorice extract is 0.0045% based on the total amount, and the content of licorice flavonoid is 0.0005% based on the total amount, the whitening effect of the two kinds of extracts is obtained. A sufficient synergistic effect can be obtained. That is, compared with the whitening effect of a single extract, the whitening effect of a mixture of two types of extracts can be greatly improved. Moreover, the mixture which mixed two types of extracts can be made into a thing with little reduction effect of protein amount (low irritation to skin).

  Moreover, in order to achieve said objective, the manufacturing method of the whitening agent of this invention WHEREIN: The licorice extract from the licorice which is a 1st plant body, the 2nd plant body from which a seed differs from a 1st plant body A step of extracting licorice flavonoids from licorice.

  Here, by extracting the licorice extract from the licorice that is the first plant body, a component having a whitening effect contained in the licorice extract can be obtained.

  Also, by extracting licorice flavonoids from licorice, which is a second plant body having a different species from the first plant body, licorice flavonoids derived from licorice of a different species from the licorice extract can be obtained.

  Moreover, when the 1st plant body is Ural licorice (Glycyrrhiza uralensis Fisher), the component which shows the whitening effect contained in Ural licorice can be obtained.

  Further, when the second plant body is Spanish licorice (Glycyrrhiza glabra Linne), licorice flavonoids derived from Spanish licorice can be obtained.

  In order to achieve the above object, the method for producing the whitening agent of the present invention cooperates with a licorice extract from Glycyrrhiza uralensis Fisher and a licorice extract from Spanish licorice (Glycyrrhiza glabra Linne). And a step of extracting licorice flavonoids having a whitening effect.

  Here, we extract licorice extract from Glycyrrhiza uralensis Fisher and extract two kinds of licorice flavonoids which have whitening effect in cooperation with licorice extract from Spanish licorice (Glycyrrhiza glabra Linne) It is possible to obtain a mixture of extracts having a synergistic whitening effect which is superior to the whitening effect produced by the product alone. Moreover, a mixture with a low protein amount reducing effect (low irritation to skin) can be obtained.

  In order to achieve the above object, the cosmetic of the present invention comprises a licorice extract extracted from licorice having a content of 0.05% based on the total amount, and a content of 0.00001% based on the total amount. Licorice flavonoids extracted from licorice whose seeds are different from the licorice extract, arbutin, tranexamic acid, wetting agent, chelating agent, thickener, oil agent, silicone, and surfactant And a stabilizer, a pH adjuster, and a preservative.

  Here, with the licorice extract extracted from licorice, a whitening effect derived from the components contained in the licorice extract can be obtained.

  Moreover, the whitening action of the licorice flavonoid derived from the licorice of the seed | species different from a licorice extract can be acquired with the licorice flavonoid extracted from the licorice whose seeds differ from the extraction origin of the licorice extract.

  In addition, a synergistic whitening effect that is superior to the whitening effect that two kinds of extracts are produced by licorice flavonoids extracted from licorice extract extracted from licorice and licorice flavonoids extracted from licorice extract derived from different species. Can be obtained. Moreover, it can be set as the whitening agent with low irritation | stimulation to skin.

  Further, arbutin can impart a whitening effect to the cosmetic.

  In addition, tranexamic acid can impart an anti-inflammatory effect to cosmetics.

The whitening agent and cosmetic according to the present invention are excellent in whitening action and safety.
Moreover, the manufacturing method of the whitening agent which concerns on this invention is a method which can provide the whitening agent excellent in the whitening effect | action and safety | security.

It is a graph which shows the result of the protein amount of B16 cell per well of Comparative Examples 1-4 and 10. It is a graph which shows the result of the protein amount of B16 cell per well of Comparative Example 1 and Comparative Examples 4-7 and 11. It is a graph which shows the result of the melanin amount per protein amount of Comparative Examples 1-4. It is a graph which shows the result of the melanin amount per protein amount of Comparative Example 1 and Comparative Examples 4-7. It is a graph which shows the result of the protein amount of B16 cell per well of Examples 1-3, Comparative Example 1, and Comparative Example 8. It is a graph which shows the result of the melanin amount per protein amount of Examples 1-3, Comparative Example 1, and Comparative Example 8.

  Hereinafter, the extraction process of the whitening agent to which the present invention is applied will be shown. In addition, the extraction process shown below is an example to the last, and it cannot be overemphasized that it can change a setting suitably in the range which can extract a licorice extract and a licorice flavonoid.

(1) Extraction of licorice extract The licorice extract was extracted by the following steps. As an extraction object, a root of Glycyrrhiza uralensis Fisher was prepared, and this was finely chopped. As an extract, primary ethanol (purity 99.9%) and water were mixed to prepare 70% ethanol. Licorice was added to this extraction solvent (70% ethanol), and immersion extraction was performed over 5 to 10 days.

  The mixture after immersion extraction was filtered through a filtration filter (pore size: about 2 μm), and a filter aid having a mass concentration of 1% based on the amount of filtrate was added. This was stirred at room temperature at a rotation speed of 150 rpm for 30 minutes, and further filtered through a filtration filter (pore diameter: about 1 μm) to obtain a filtrate. The filtrate was stored refrigerated for 3 days or longer to precipitate insolubles in the filtrate.

  The filtrate after refrigerated storage was filtered through a membrane filter (pore size 0.2 μm), and ultrafiltration was further performed. The filtrate at this time was analyzed for liquiritin content, and it was confirmed that about 0.5 mg / mL liquiritin was contained. The filtrate was further filtered through a membrane filter (pore size 0.2 μm) to obtain a licorice extract.

(2) Extraction of licorice flavonoids Licorice flavonoids were extracted by the following steps. As the extraction object, the roots and rhizomes of Spanish licorice (Glycyrrhiza glabra Linne) were prepared and used by finely chopping them. As an extraction liquid, primary ethanol (purity 99.9%) was weighed and used as an extraction solvent. Licorice was added to this extraction solvent, and extraction was carried out at room temperature with stirring all day and night. The extract after stirring was roughly filtered with a filter cloth to remove the extraction residue, and the filtrate was filtered with a filtration filter (pore size: about 1 μm). The filtrate after filtration was concentrated under reduced pressure to remove the precipitated crystalline insoluble matter, and the supernatant was transferred to another eggplant-shaped flask when a certain amount of concentration had progressed. Because the residue of the eggplant-shaped flask after concentration contains the crystalline residue of the target product, wash the eggplant-shaped flask with ethanol for recovery, and combine the supernatant recovered with ethanol with the supernatant after the first concentration Then, concentration under reduced pressure was performed again. The concentrated residue was washed with ethanol for recovery and concentrated under reduced pressure. In order to dry the concentrate, the vacuum concentrate was put in a drier while being placed in an eggplant-shaped flask, and dried overnight.

  Ethyl acetate was added to the dried concentrate and extracted with ethyl acetate while stirring all day and night. The extract was filtered with a filtration filter (pore size: about 0.5 μm), and the filtrate was transferred to another eggplant-shaped flask and concentrated under reduced pressure. The vacuum concentrate was placed in a dryer and dried overnight. The dried concentrate was transferred to a mortar and ground with a pestle to obtain a powder. The powder body was sieved to obtain a uniform powder body. This powder was used as licorice flavonoid.

  An example of the whitening agent of the present invention includes a licorice extract obtained in the above-described process and a licorice flavonoid. By adding these two components as a whitening agent to cosmetics, a synergistic whitening effect can be obtained. Moreover, the whitening agent containing these two components has low irritation to the skin.

  Hereinafter, an example of the composition of the cosmetics containing the whitening agent to which the present invention is applied as a characteristic component will be shown. The whitening agent to which the present invention is applied can be used as a raw material for cosmetics.

  The cosmetics shown here have weight ratios based on the total amount of concentrated glycerin: 5.0%, 1,3-butylene glycol: 3.0%, 1,2-pentanediol: 2.0%, diethylenetriaminepentaacetic acid: 0.2%, diethylenetriaminepentaacetic acid pentasodium solution: 0.05%, carboxyvinyl polymer: appropriate amount, glyceryl tri-2-ethylhexanoate: 2.5%, methylpolysiloxane: 2.5%, decaglyceryl monomyristate : 0.9%, lipophilic glyceryl monostearate: 0.8%, beeswax wax: 0.8%, behenyl alcohol: 0.8%, natural vitamin E: 0.05%, vegetable squalane: 1.8 %, Squalane: 0.45%, sodium hydroxide: appropriate amount, sodium pyrosulfite: 0.008%, phenoxyethanol: 0.3%, glycerin 2-ethylhexyl ether: 0.05%, licorice extract (derived from Ural licorice): 0.05%, licorice flavonoid (derived from Spanish licorice): 0.00001%, arbutin: appropriate amount, tranexamic acid: appropriate amount, purified water: The composition includes a remaining amount. With the above composition, a gel-like cosmetic is obtained.

  Concentrated glycerin, 1,3-butylene glycol, 1,2-pentanediol and glycerin mono-2-ethylhexyl ether are wetting agents and humectants.

  Diethylenetriaminepentaacetic acid and diethylenetriaminepentaacetic acid pentasodium solution are chelating agents.

  Carboxyvinyl polymer is a thickener and imparts an appropriate viscosity to cosmetics.

  Glyceryl tri-2-ethylhexanoate, white beeswax, behenyl alcohol, vegetable squalane and squalane have a moisturizing effect and are components used as an emollient.

  Methylpolysiloxane is a kind of silicone having an antifoaming action, and becomes a feel improving agent for cosmetics.

  Decaglyceryl monomyristate and lipophilic glyceryl monostearate are surfactants.

  Natural vitamin E and sodium pyrosulfite are stabilizers and function as antioxidants.

  Sodium hydroxide is a pH adjuster. Phenoxyethanol is a preservative.

  Arbutin and tranexamic acid are one of the characteristic components that impart whitening action. Tranexamic acid also imparts an anti-inflammatory effect.

  Purified water is a base for cosmetics and adjusts the total amount of cosmetics to 100% including the various components described above. In addition to the raw materials described above, additives such as plant extracts and fragrances can be added to adjust the amount of purified water.

  Here, in this cosmetic, each compounding raw material is not limited to what was mentioned above, Each component can be suitably changed with the function to give to cosmetics and the kind of cosmetics. One example is described in detail below.

  In this cosmetic, in addition to arbutin, as whitening characteristic components, for example, ellagic acid, chamomile extract, kojic acid, tranexamic acid, vitamin C derivative, placenta extract, lucinol, linoleic acid, t-cycloamino acid, 4-methoxysalicylic acid A potassium salt or the like may be employed or added additionally.

  In the present cosmetic, other than tranexamic acid, for example, allantoin, dipotassium glycyrrhizinate or the like can be used or additionally blended as a whitening characteristic component or a component imparting an anti-inflammatory effect.

  In this cosmetic, in addition to concentrated glycerin, 1,3-butylene glycol, 1,2-pentanediol and glycerin mono-2-ethylhexyl ether, as a wetting agent / humectant, for example, diglycerin, sorbitol, propylene glycol, polyethylene glycol 400, polyethylene glycol 600, dipropylene glycol, isoprene glycol, etc. may be adopted or additionally blended.

  In this cosmetic, in addition to diethylenetriaminepentaacetic acid and diethylenetriaminepentaacetic acid pentasodium solution, for example, etidronic acid, ethylenediaminetetraacetic acid or a salt thereof, L-aspartic acid diacetic acid, nitrilotriacetic acid or a salt thereof, L- It is also possible to employ glutamic acid diacetic acid or the like, or to add it additionally.

  In this cosmetic, in addition to the carboxyvinyl polymer, as a thickener, for example, (acrylates / alkyl acrylate (C10-30)) crosspolymer, (hydroxyethyl acrylate / acryloyldimethyltaurine Na) copolymer, (acryloyldimethyltaurine) Ammonium / VP) copolymer, Acrylates copolymer Na, Acrylates / Beheneth methacrylate-25) copolymer, natural polymer (thickener) carrageenan, agar, locust bean gum, xanthan gum, tragacanth gum, guar gum, gellan gum, tamarind gum, etc. It is also possible to adopt or additionally blend.

  In this cosmetic, in addition to glyceryl tri-2-ethylhexanoate, plant squalane and squalane, as an emollient, for example, isopropyl palmitate, isopropyl myristate, isopropyl isostearate, cetyl octanoate, isononyl isononanoate, ethylhexyl palmitate Hydrogenated polyisobutene, tri (caprylic acid / capric acid) glyceryl, cetyl palmitate and the like can be adopted or additionally blended.

  In this cosmetic, in addition to white beeswax, as an emollient, for example, carnauba wax, candelilla wax, microcrystalline wax, paraffin wax, microcrystalline wax, montan wax, ozokerite, etc. are used or additionally blended It is also possible.

  In this cosmetic, in addition to behenyl alcohol, for example, stearyl alcohol, cetanol, cetostearyl alcohol, cetearyl alcohol, myristyl alcohol, etc., which are higher alcohols, can be employed or additionally blended as an emollient.

  In the present cosmetic, in addition to methylpolysiloxane, for example, cyclopentasiloxane, trisiloxane, dimethiconol, phenyltrimethicone, dimethicone, etc. can be adopted or additionally blended as silicone.

  In this cosmetic, in addition to decaglyceryl monomyristate and glyceryl monostearate, surfactants such as lecithin, lecithin derivatives, lecithin, hydrogenated lecithin, propylene glycol fatty acid ester, stearic acid PG (SE), stearic acid PG as glyceryl fatty acid ester, glyceryl myristate, glyceryl stearate, sorbitan stearate, glyceryl oleate, glyceryl distearate, glyceryl isostearate as polyglyceryl fatty acid ester, polyglyceryl stearate-2 , Polyglyceryl-2 oleate, polyglyceryl-2 isostearate, polyglyceryl-4 stearate, polyglyceryl-4 oleate, polyglyceryl-6 laurate Polyglyceryl-6 myristate, polyglyceryl-6 myristate, polyglyceryl-6 stearate, polyglyceryl oleate-6, polyglyceryl laurate-10, polyglyceryl-10 myristate, polyglyceryl-10 isostearate, polyglyceryl-10 oleate, linoleic acid Polyglyceryl-10, polyglyceryl-10 distearate, polyglyceryl-10 diisostearate, polyglyceryl tristearate, polyglyceryl trioleate as polyoxyethylene glyceryl fatty acid ester, PEG-5 glyceryl stearate, PEG stearate 15 glyceryl, PEG-5 glyceryl oleate, PEG-15 glyceryl oleate, PEG-20 g chicken isostearate Ceryl as sorbitan fatty acid ester, coconut fatty acid sorbitan, sorbitan palmitate, sorbitan stearate, sorbitan sesquistearate, sorbitan tristearate, sorbitan isostearate, sorbitan sesquiisostearate, sorbitan oleate, sorbitan sesquioleate, triolein Acid sorbitan as polyoxyethylene sorbitan acid fatty acid ester, PEG-20 sorbitan cocoate, polysorbate 40, PEG-6 sorbitan stearate, polysorbate 65, PEG-20 sorbitan isostearate, polysorbate 80, PEG-6 sorbitan oleate, polysorbate 85 As polyoxyethylene sorbite fatty acid ester, sorbate laurate-6, tetras Solvate Tearate-60, Solves tetraoctoleate-6, Solves tetraoleate-30, Solves tetraoleate-40, Solves tetraoleate-60, Solves tetraisostearate-30 Polyoxyethylene hydrogenated castor oil: PEG-5 Hydrogenated castor oil, PEG-10 hydrogenated castor oil, PEG-20 hydrogenated castor oil, PEG-30 hydrogenated castor oil, polyoxyethylene sterol / hydrogenated sterol, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene Alkyl ether, polyoxyethylene alkyl ether phosphoric acid / phosphate, and polyethylene glycol fatty acid ester may be employed or additionally blended.

  In this cosmetic, in addition to natural vitamin E and sodium pyrosulfite, as stabilizers, for example, dl-α-tocopherol, vitamin C (ascorbic acid), dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate , Sodium sulfite, sulfur dioxide, cysteamine HCl, cysteine, etc. may be employed or added additionally.

  In this cosmetic, in addition to sodium hydroxide, examples of pH adjusters include inorganic acids (hydrochloric acid, sulfuric acid, etc.), organic acids (lactic acid, sodium lactate, citric acid, sodium citrate, succinic acid, and sodium succinate). Inorganic bases (potassium hydroxide, sodium hydroxide, etc.), organic bases (triethanolamine, diisopropanolamine, triisopropanolamine, etc.) can be employed or added additionally.

  In this cosmetic, other than phenoxyethanol, for example, benzoic acid, sodium benzoate, paraoxybenzoic acid ester, benzalkonium chloride, phenoxyethanol, isopropylmethylphenol, dehydroacetic acid, sodium dehydroacetate, etc. Additional blending is also possible.

  In addition, in this cosmetic, 0.05% licorice extract and 0.00001% licorice flavonoid are blended, but the concentration of licorice extract and licorice flavonoid is not limited to this. It can be set as appropriate according to the type and the effect of whitening.

  In addition, cosmetics containing a whitening agent to which the present invention is applied are not limited to gel cosmetics, but are different types of cosmetics such as face wash, lotion, cosmetic liquid, cream, milky lotion, cleansing, pack, etc. It is also possible to mix with the material.

As described above, the whitening agent and the cosmetics to which the present invention is applied have excellent whitening action and safety.
Moreover, the manufacturing method of the whitening agent to which this invention is applied is a method which can provide the whitening agent excellent in the whitening effect | action and safety | security.

  Examples of the present invention will be described below.

  First, two species to be combined were selected from a plurality of licorice plants. In the following experiment, licorice extract and licorice flavonoid were extracted from 7 species of genus licorice, and the mixture was tested for tyrosinase activity inhibition to evaluate the melanin synthesis inhibitory effect. .

(1) Kinds of licorice plants used in the experiment Sample 1: G. uralensis
Sample 2: Spanish licorice (G. glabra)
Sample 3: Dog Licorice (G. pallidiflora)
Sample 4: Xinjiang (G. inflata)
Sample 5: American daylily (G. lepidota)
Sample 6: No Japanese name (G. bucharica)
Sample 7: No Japanese name (G. macedonica)

(2) Sample preparation of licorice extract used in the experiment Samples 1 to 7 and 70 w / w% ethanol were mixed and immersed, and filtered through a 0.45 μm filter. Further, each sample after filtration was diluted with 70 w / w% ethanol at the dilution rate shown in Table 1 below, in accordance with G. bucharica, which had the least amount of evaporation component during immersion. The dilution of each sample was adjusted by adding 70 w / w% ethanol so that the final concentration was 0.1% as shown in Table 4 when testing tyrosinase activity inhibition. It was.

(3) Preparation of licorice flavonoid sample used in the experiment Samples 1 to 7 and primary ethanol (purity 99.9%) were mixed and immersed, and filtered through a 0.45 μm filter. The filtered sample was dried at 60 ° C. for 2 days. Furthermore, 20 ml of ethyl acetate was dissolved in the dried concentrate, filtered through a 0.45 μm filter, and dried at 50 ° C. for 2 days. To this dried product (licorice flavonoid), primary ethanol (purity 99.9%) was added at the ratio shown in Table 2, and the solution was adjusted so as to be a 1% licorice flavonoid solution based on the total amount. The licorice flavonoid 1% solution of each sample was subjected to primary ethanol (purity 99.9%) so that the final concentration would be the concentration shown in Table 4 (0.0025%) when testing tyrosinase activity inhibition. Was added to adjust the concentration.

(4) Test of Tyrosinase Inhibitory Activity In a reaction in which tyrosinase, which is a melanin synthase, produces dopachrome, which is an intermediate of melanin biosynthesis, from L-DOPA, which is a substrate for melanin synthesis, Each sample was added in combination with the contents shown in 4, and the inhibition rate inhibiting the production of dopachrome was measured.
The contents of the test are as follows. Table 4 shows the combination of each sample and also shows the inhibition rate as a test result.

1. With the contents of (2) and (3) described above, licorice extracts and licorice flavonoids of each species of licorice were prepared. In addition, tyrosinase (diluted to 40 U / mL at the time of the test) and L-DOPA stored frozen were adjusted immediately before the start of the test. Since tyrosinase was stored at a concentration 50 times the concentration used for the test, it was diluted 50 times with a buffer and stored at room temperature. The required amount of L-DOPA (12.5 mM) was preheated at 37 ° C.
2. According to the procedure described in Table 3 below, a sample (sample), water (solvent), Phosphate (pH 6.8) (buffer), tyrosinase (enzyme) were added to a 96-well plate, and A ( Control), B (control blank), C (sample) and D (sample blank) were adjusted.
3. After adjusting so that it might become AD, the 96-well plate was fully stirred with the vortex for plates. In addition, C (sample) here is a single component or a mixture of licorice extract and licorice flavonoid extracted from samples 1 to 7 prepared with the contents described in (2) and (3) above.
4. Incubate at 37 ° C for 10 minutes.
5. 50 μL of L-DOPA was applied to all of AD.
6). Using an absorptiometer, apply L-DOPA at Abs 475 nm and check the absorbance after 5 minutes. Abs 475 nm is the absorption maximum wavelength of dopachrome which is an intermediate of melanin biosynthesis.
7). The inhibition rate of each sample was calculated from A to D using the following formula.
Inhibition rate = (1− (C−D) / (A−B)) × 100
A to D are calculated by subtracting the absorbance value immediately after the start of measurement (0 minute) from the absorbance value after 5 minutes from the start of measurement. For example, for the value of A, the value of A (Abs 5 minutes−Abs 0 minutes) is adopted.

(5) Combination of licorice plants About the contents shown below, the licorice extract has a final concentration of 0.1% and the licorice flavonoid has a final concentration of 0.0025%.
Comparative Example 100: Ural licorice extract only Comparative Example 101: Spanish licorice extract only Comparative Example 102: Dog licorice extract only Comparative Example 103: Xinjiang licorice extract only Comparative Example 104: American licorice extract only Comparative Example 105: G. bucharica licorice extract only comparative example 106: G. macedonica licorice extract only comparative example 110: Ural licorice flavonoid only comparative example 111: Spanish licorice flavonoid only comparative example 112: canine licorice flavonoid only comparative example 113: Xinjiang licorice flavonoid only comparative example 114: American licorice flavonoid only Comparative Example 115: G. bucharica licorice flavonoid only Comparative Example 116: G. macedonica licorice flavonoid only Comparative Example 120: Ural licorice extract + Ural licorice flavonoid Example 4 Uralcan Eucalyptus extract + Spanish licorice flavonoid comparative example 121: Ural licorice extract + dog licorice flavonoid comparative example 122: Ural licorice extract + Xinjiang licorice flavonoid comparative example 123: Ural licorice extract + American licorice flavonoid comparative example 124: Ural licorice extract + G. bucharica licorice flavonoid comparative example 125: Ural licorice extract + G. macedonica licorice flavonoid comparative example 130: Spanish licorice extract + Ural licorice flavonoid comparative example 131: Spanish licorice extract + Spanish licorice flavonoid comparative example 132: Spanish licorice extract Product + dog licorice flavonoid comparative example 133: Spanish licorice extract + Xinjiang licorice flavonoid comparative example 134: Spanish licorice extract + American licorice flavo Id comparative example 135: Spanish licorice extract + G. bucharica licorice flavonoid comparative example 136: Spanish licorice extract + G. macedonica licorice flavonoid comparative example 140: Dog licorice extract + Ural licorice flavonoid comparative example 141: Dog licorice extract + Spanish licorice flavonoid comparison Example 142: Dog licorice extract + dog licorice flavonoid comparative example 143: Dog licorice extract + Xinjiang licorice flavonoid comparative example 144: Dog licorice extract + American licorice flavonoid comparative example 145: Dog licorice extract + G. bucharica licorice flavonoid comparative example 146: Dog licorice extract + G. macedonica licorice flavonoid comparative example 150: Xinjiang licorice extract + Ural licorice flavonoid comparative example 151: Xinjiang licorice extract + spanish Licorice flavonoid comparative example 152: Xinjiang licorice extract + dog licorice flavonoid comparative example 153: Xinjiang licorice extract + Xinjiang licorice flavonoid comparative example 154: Xinjiang licorice extract + American licorice flavonoid comparative example 155: Xinjiang licorice flavonoid extract + G. bucharica Licorice flavonoid comparative example 156: Xinjiang licorice extract + G. macedonica licorice flavonoid comparative example 160: American licorice extract + Ural licorice flavonoid comparative example 161: American licorice extract + Spanish licorice flavonoid comparative example 162: American licorice extract + dog licorice Flavonoid comparative example 163: American licorice extract + Xinjiang licorice flavonoid comparative example 164: American licorice extract + American licorice flavonoid comparative example 165: American licorice Elephant extract + G. bucharica licorice flavonoid comparative example 166: American licorice extract + G. macedonica licorice flavonoid comparative example 170: G. bucharica licorice extract + Ural licorice flavonoid comparative example 171: G. bucharica licorice flavonoid comparative example 171: G. bucharica licorice flavonoid comparative example Example 172: G. bucharica licorice extract + dog licorice flavonoid comparison example 173: G. bucharica licorice extract + Xinjiang licorice flavonoid comparison example 174: G. bucharica licorice extract + American licorice flavonoid comparison example 175: G. bucharica licorice extraction + G. bucharica licorice flavonoid comparative example 176: G. bucharica licorice extract + G. macedonica licorice flavonoid comparative example 180: G. macedonica licorice flavonoid comparative example 181: G. macedonica licorice flavonoid comparative example 181 Comparative licorice flavonoid 182: G. macedonica licorice extract + dog licorice flavonoid comparative example 183: G. macedonica licorice extract + Xinjiang licorice flavonoid comparative example 184: G. macedonica licorice extract + American licorice flavonoid comparative example 185: G macedonica licorice extract + G. bucharica licorice flavonoid Comparative Example 186: G. macedonica licorice extract + G. macedonica licorice flavonoid

From the results shown in (5) and Table 4 above, for the licorice extract and licorice flavonoids extracted from seven kinds of licorice plants, the combination of the Ural licorice extract shown in Example 4 and the extract of Spanish licorice flavonoid The inhibition rate was the highest, indicating that it has excellent tyrosinase inhibitory activity. More specifically, the inhibition rate of Example 4 showed a value superior to the inhibition rate exhibited by a single Urachanthus extract and a single Spanish licorice flavonoid. Further, from the results of Example 4, Comparative Example 120 and Comparative Example 131, the inhibition rate of Example 4 is superior to the mixture of licorice extract and licorice flavonoid obtained from the same kind of licorice plant, and became. Furthermore, comparison was made with plant species belonging to the genus genus other than Ural licorice and Spanish licorice. From the results of Example 4 and other comparative examples, a combination of licorice extract derived from Ural licorice and licorice flavonoid derived from Spanish licorice Was the best combination.
In the following, the whitening action and irritation were evaluated with a focus on the liquorice extract derived from Ural licorice and the licorice flavonoid derived from Spanish licorice.

(6) Sample components Samples containing the components shown in Tables 5 to 7 were prepared. The numerical values described in Tables 5 to 7 are mass percent concentrations (wt%) in the solution. Examples 1-3 and Comparative Examples 1-7, 10 and 11 are all prepared using 1% DMSO as a solvent. As the licorice extract and licorice flavonoid contained in the examples and comparative examples, those obtained in the extraction process described in the above-mentioned “Mode for Carrying Out the Invention” are used. Examples 1 to 3 are samples containing licorice extract and licorice flavonoids in solution. Comparative Example 1 is a sample containing only 1% DMSO. Comparative Example 4 and Comparative Example 8 are positive controls that have a low protein amount reducing effect (low irritation) and a cell melanin synthesis inhibitory effect (tyrosinase activity inhibition). Comparative Example 4 is kojic acid. Comparative Example 8 is arbutin.

(7) Evaluation of single components of licorice extract and licorice flavonoids For the prepared Comparative Examples 1 to 7, 10 and 11, the following evaluation was performed for irritation. Moreover, the following evaluation was performed about irritation | stimulation and whitening effect regarding Comparative Examples 1-7.
After seeding B16 cells (mouse B16 melanoma cells. Melanin producing cells) in a 24-well plate, a medium containing each sample of Comparative Example was added, and the amount of protein and the amount of melanin were measured 2 days after the culture. The cultured cells were dissolved in 1N NaOH containing 10% DMSO, and the absorbance at 490 nm was measured to determine the amount of melanin. Further, the protein content of the cell lysate was measured by the Bradford method. From the measured melanin amount and protein amount, the melanin amount per protein amount was calculated. The amount of protein and the amount of melanin per amount of protein were expressed as a ratio when the value of Comparative Example 1 was set to 100% with Comparative Example 1 (1% DMSO) as a control. Those having a low ratio of protein amount were judged to be highly irritating because of protein degradation and the like. Moreover, the thing with a low ratio of the amount of melanins per protein amount shall be judged that the production suppression ability of melanin is high.

  The results of the protein amount are shown in FIG. 1 and FIG. The vertical axis in each figure represents the amount of protein (%) of B16 cells per well (ratio when the value of Comparative Example 1 is 100%). FIG. 1 shows the results of Comparative Examples 1 to 4 and 10, and FIG. 2 shows the results of Comparative Examples 1, 4 to 7 and 11.

  Comparative Examples 2 and 3 which are samples of licorice extract alone and Comparative Examples 5 to 7 which are samples of licorice flavonoids only have a protein amount of 80% or more and low irritation. In Comparative Example 10 and Comparative Example 11, the amount of protein was a low value of 60% or less. From this result, it was confirmed that licorice extract and licorice flavonoids became stronger in irritation when the concentration was increased with a single component. For this reason, it was confirmed that the licorice extract is preferably set to 0.0045% or less and the licorice flavonoid is preferably set to 0.0005% or less from the viewpoint of suppressing irritation.

  The result of the amount of melanin per protein amount is shown in FIG.3 and FIG.4. The vertical axis in each figure is the amount of melanin per protein amount (%) (ratio when the value of Comparative Example 1 is 100%). FIG. 3 shows the results of Comparative Examples 1 to 4, and FIG. 4 shows the results of Comparative Example 1 and Comparative Examples 4 to 7.

  Comparative Example 2 which is a sample of licorice extract alone has a value of 77.3% and Comparative Example 3 has a value of 65.0%, and Comparative Example 4 (kojic acid which is a positive control) has a remarkable ability to inhibit melanin production (44 .8%), the melanin production inhibitory ability was low. In addition, Comparative Examples 5 to 7, which are samples of only licorice flavonoids, all showed a numerical value of 80% or more, and the melanin production inhibitory ability was even lower than Comparative Examples 2 and 3.

(8) Evaluation of mixed components of licorice extract and licorice flavonoid With respect to the prepared Examples 1 to 3 and Comparative Examples 1 and 8, the following evaluations were performed for stimulating and whitening effects. In addition, although not shown in Tables 7 to 9 and the figures, the synergistic effect of the whitening action of the mixture on samples higher in concentration than licorice extract 0.0045% and higher in concentration than licorice flavonoid 0.0005%, Evaluation was performed.
After seeding B16 cells in a culture petri dish (or 24-well plate), the medium containing each sample of Examples 1 to 3 and Comparative Examples 1 and 8 was added the next day, and the cells were recovered by trypsin treatment after 3 days of culture. Then, the amount of melanin and the amount of protein were measured. The cultured cells were dissolved in 1N NaOH containing 10% DMSO, and the absorbance at 490 nm was measured to determine the amount of melanin. Further, the protein content of the cell lysate was measured by the Bradford method. From the measured melanin amount and protein amount, the melanin amount per protein amount was calculated. The amount of protein and the amount of melanin per amount of protein were expressed as a ratio when the value of Comparative Example 1 was set to 100% with Comparative Example 1 (1% DMSO) as a control.

  The result of protein amount is shown in FIG. The vertical axis in FIG. 5 is the protein amount (%) of B16 cells per petri dish (ratio when the value of Comparative Example 1 is 100%).

  The numerical value was 97.8% in Example 1 and 89.5% in Example 2, and the irritation was low. Moreover, although Example 3 is a numerical value of 79.0% and a numerical value is lower than Examples 1 and 2, it is a numerical value of about 80% and the irritation | stimulation result was low.

  The result of the melanin amount per protein amount is shown in FIG. The vertical axis in FIG. 6 is the amount of melanin per protein amount (%) (ratio when the value of Comparative Example 1 is 100%).

  The numerical value was 51.8% in Example 1 and 46.5% in Example 2, showing a higher ability to suppress melanin production than licorice extract or licorice flavonoid at the same concentration. Furthermore, Example 3 was a numerical value of 37.2% and showed a melanin production inhibitory ability superior to that of Comparative Example 8 (Arbutin) which is a positive control. In Example 3, when compared with the value of the ability to suppress melanin production with a single component, the value “65.0%” of licorice extract (0.0045%: Comparative Example 3), licorice flavonoid (0.0005%) : The difference from the average value “74.1%” calculated from the value “83.2%” of Comparative Example 7) was a large value “36.9 (74.1-37.2)”. From this point, it became clear that the mixture in which each component has the ratio of Example 3 exhibits a superior synergistic effect than the whitening effect exhibited by a single component.

  Moreover, about the synergistic effect in melanin production suppression ability, it evaluated about the sample set to the value higher than the ratio of Example 3 in each component. In the sample in which each component was at a higher concentration than the values in Example 3 (licorice extract: 0.0045%, licorice flavonoid: 0.0005%), it was regarded as a synergistic effect from the whitening effect exhibited by a single component. The growth of the ability to suppress melanin production was insufficient. In particular, in the sample of 0.018% licorice extract and 0.001% licorice flavonoid, and in the sample of 0.018% licorice flavonoid and 0.002% licorice flavonoid, a mixture of the whitening effect exhibited by a single component Significant improvement in whitening effect was not confirmed.

Claims (5)

Licorice extract extracted from Glycyrrhiza uralensis Fisher,
A whitening agent containing a licorice flavonoid extracted from Spanish licorice (Glycyrrhiza glabra Linne) and having a whitening effect in cooperation with the licorice extract. The licorice extract has a content of 0.0045% based on the total amount,
The whitening agent according to claim 1, wherein the content of the licorice flavonoid is 0.0005% based on the total amount. The whitening agent according to claim 1 or 2, wherein the licorice extract contains 0.5 mg / mL or more of liquiritin. A method for producing a whitening agent comprising a step of extracting a licorice extract from Glycyrrhiza uralensis Fisher and a licorice flavonoid having a whitening effect in cooperation with the licorice extract from Spanish licorice (Glycyrrhiza glabra Linne).   Cosmetics containing the whitening agent according to claim 1.