JP2005281228A - Whitening cosmetic - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare a whitening cosmetic containing a sea mud extract having high tyrosinase inhibiting activity and produced by purifying an extract of white sea mud in Milky Way of Urukthapel island of the Republic of Palau. <P>SOLUTION: This whitening cosmetic contains the sea mud extract obtained by purifying the hot water extract of sea mud in the Urukthapel island of the Republic of Palau and exhibiting ≥80% tyrosinase inhibition activity at an aqueous solution concentration of 100μg/mL. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cosmetic that has an effect of whitening human skin and smoothing the skin, and has high safety.

  In the skin, the production of melanin proceeds by sunlight, various chemical substances, or physical stimulation such as scratching, and melanin is deposited on the skin and becomes black. Severe pigmentation and black skin disease often seen in melanin spots, freckles, atopic dermatitis patients, etc. cause not only cosmetic problems but also mental distress and affect daily life. There is also. Melanin pigments are produced in melanocytes (melanocytes) between the epidermis and dermis, and the produced melanin diffuses to neighboring cells by osmotic action. In the biochemical melanogenesis reaction in this melanocyte, tyrosine, which is one of the essential amino acids, becomes dopaquinone by the action of the enzyme tyrosinase. After continuous oxidation reaction from dopaquinone, dopachrome is generated, and then melanin is produced. Monomers 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) are produced and polymerized with each other, and finally, the black pigment melanin of the copolymer Is generated. Therefore, if the action of tyrosinase, which is the first stage of the reaction, can be suppressed, melanin production can be effectively suppressed.

  Currently, kojic acid, arbutin, hydroquinone, vitamin C, bovine placenta extract, mulberry bark extract, licorice extract and the like are known as whitening compounds having tyrosinase inhibitory activity (for example, Patent Document 1). reference). Many cosmetics and pharmaceuticals containing these whitening compounds are on the market. However, beef placenta extract, which has been used for whitening cosmetics for many years, was banned due to the problem of mad cow disease, and was further banned because kojic acid was pointed out to be carcinogenic. In addition, the inhibitory effect of vitamin C and its derivatives on tyrosinase activity is relatively low. Hydroquinone has an irreversible whitening effect and strong skin irritation, and its use has been restricted due to safety problems. Recently, it has been found to be carcinogenic, and its use as a cosmetic ingredient is prohibited. Most plant extracts show a relatively high tyrosinase activity inhibitory effect only at high farming levels, and hardly show an inhibitory effect at low concentrations. Under such circumstances, development of a safe and useful whitening agent is awaited.

In recent years, it has been reported that white water mud hot water, hot alcohol, or a mixture of hot water and hot alcohol in the Milky area of the Republic of Palau has a tyrosinase inhibitory activity. Application to this is attempted (see Patent Document 2). However, none of the whitening effects of this extract is yet satisfactory.
Japanese Patent Laid-Open No. 06-016532 Japanese Patent Laid-Open No. 2002-338452

  An object of the present invention is to provide a whitening cosmetic containing a sea mud extract having a highly active tyrosinase inhibitory activity obtained by refining an extract of white sea mud in the direction of Milky Island in the island of Urkutaburu, Palau. That is.

The present inventors attempted purification for the purpose of separating and refining the whitening active ingredient of the sea mud hot water extract. As a result, it was found that this fraction was remarkably superior to the conventional product in the whitening effect. The inventors have further studied and completed the present invention.
That is, the present invention includes (1) a sea mud extract obtained by purifying a sea mud hot water extract of Urkturbul Island in the Republic of Palau, and exhibiting a tyrosinase inhibitory activity of 80% or more at an aqueous solution concentration of 100 μg / mL. Whitening cosmetics, characterized by
(2) Whitening cosmetics according to (1) above, characterized by containing 0.001 to 5.0% by weight of a sea mud extract, and (3) Sea mud hot water extraction of Urkutaburu Island in the Republic of Palau A tyrosinase inhibitor characterized by containing a sea mud extract obtained by purifying a product and exhibiting tyrosinase inhibitory activity of 80% or more at an aqueous solution concentration of 100 μg / mL,
About.

Since the sea mud extract blended in the whitening cosmetic composition of the present invention has a markedly improved tyrosinase inhibitory activity compared to the hot water extract of sea mud, melanin production can also be significantly suppressed.
Since the whitening cosmetic composition of the present invention contains a highly active tyrosinase inhibitory active ingredient, pigmentation due to melanin production, for example, pigmentation after sunburn, pigmentation caused by changes in hormone balance, flat nevus, freckles, or cosmetics Prevents rashes, acne, burns, atopic dermatitis or pigmentation that occurs after inflammation due to external stimuli such as nylon towels and brushes used to wash the body, senile pigmentation, or pigmentation that occurs after laser treatment, etc. Can be suppressed. In addition, the whitening cosmetic of the present invention is not only excellent in the melanin production inhibitory effect compared to the conventional products, but also retains the whitening effect of skin such as whitening, prevention of rough skin, prevention of skin, etc. possessed by sea mud. Yes.
The sea mud extract blended in the whitening cosmetic composition of the present invention is different from the sea mud hot water extract of Urkutaburu Island of the Republic of Palau. Since the preparation is not separated by a salt, it is easy to make a preparation and a whitening cosmetic that is stable over time can be produced.

The sea mud on the island of Urkutaburu in Palau (sometimes abbreviated simply as “sea mud” in this specification) is the white sea mud in the direction of Milky on the island of Urkutaburu in Palau, Washed with water if necessary, removed unnecessary materials, and heat-sterilized if necessary. The sea mud is considered to have been deposited by fossilizing corals and the like.
The sea mud extract used in the whitening cosmetic composition of the present invention is an extract collected from the sea mud by means such as extraction, purification or separation, and exhibits an tyrosinase inhibitory activity of 80% or more at an aqueous solution concentration of 100 μg / mL. If so, regardless of the purification or separation or extraction method, any of them can be used as the sea mud extract of the present invention. The aqueous solution concentration refers to the concentration of the sea mud extract in the reaction solution for measuring tyrosinase inhibitory activity, and dimethyl sulfoxide can be added to the reaction solution as long as it does not affect the measurement. Or a solubilizing agent such as ethanol may be contained. In the present invention, the term “purification” means that the tyrosinase inhibitory activity is increased by further adding means to the sea mud hot water extract. The degree of purification is such that the concentration of an aqueous solution of a test substance exhibiting a tyrosinase inhibition rate equivalent to that of a sea mud hot water extract before purification is usually reduced to about 1/10 or less, preferably about 1/10 to 1 / 10,000. It is preferable. In the present specification, the “fraction” means a dried product obtained by eluting the components of the hot water extract with various separation means and removing the solvent (for example, a vacuum dried product or a freeze-dried product). Say.

  The purification of the sea mud extract is carried out by using a hot water extract of the sea mud as an indicator of tyrosinase inhibitory activity, for example, various types of chromatography (molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, affinity chromatography). Chromatography, high performance liquid chromatography, partition chromatography, etc.), ultrafiltration or electrophoresis, or a combination thereof. For example, as shown in FIG. 1, silica gel (adsorption) column chromatography and molecular sieve column chromatography can be combined and purified. The seam used in the present invention is obtained by removing a part or all of the elution solvent from the eluate in chromatography by vacuum concentration or / and freeze-drying or / and spray-drying, etc., and having the tyrosinase inhibitory activity. Can be mud extract. The hot water extract used as a raw material is added to sea mud with water, such as purified water, distilled water or tap water, and heated to about 70 to 100 ° C., preferably about 100 ° C. for about 1 hour or more, preferably Is obtained by heat extraction for about 1 hour. The ratio of sea mud to water is about 2 to 10 parts by weight, preferably about 3 to 5 parts by weight, with respect to 1 part by weight of sea mud. The hot water extract thus obtained is filtered, and the solvent of the filtrate is removed by vacuum concentration or / and freeze-drying, and / or spray drying, etc. to obtain a sea mud hot water extract. The tyrosinase inhibitory activity of the sea mud hot water extract is 2 mg / mL, and the inhibition rate is about 70 to 80%.

The above-mentioned sea mud hot water extract dissolved in water is first eluted using a mixed solvent of chloroform / methanol / water [6: 4: 1 (V / V)], for example, by silica gel column chromatography, Elute with methanol. The fraction d eluted with methanol is collected. As the silica gel, for example, silica gel 60N (63-210 μm; manufactured by Kanto Chemical Co., Inc.) can be used. The tyrosinase inhibitory activity of the fraction d is about 90% of the inhibition rate at half the concentration (1 mg / mL) of the sea mud hot water extract.
Next, the fraction d is eluted by molecular sieve chromatography using a methanol / water [1: 1 (V / V)] mixed solvent. As resin used for molecular sieve chromatography, Sephadex LH-20 (made by Tosoh Corporation) etc. can be used, for example. A part of the eluate is silica gel thin layer chromatography (hereinafter abbreviated as TLC), using a mixed solvent of chloroform / methanol / water / acetic acid [3: 3: 1: 0.1 (V / V)]. The eluate is fractionated while coloring with ninhydrin reagent and luminescence at UV254 mm as indicators.
Fraction d3 having a high tyrosinase inhibitory activity is further eluted by silica gel chromatography using a mixed solvent of chloroform / methanol / water [5: 4: 1 (V / V)]. The mixture is developed using a mixed solvent of chloroform / methanol / water [2: 4: 1 (V / V)], and the eluate is fractionated using coloration by the ninhydrin reagent as an index. Fraction d3-1 not colored with ninhydrin reagent, fraction d3-2 showing a colored spot at Rf value 0.3, fraction d3-3 showing a colored spot at Rf values 0.2 and 0.4 After elution, the eluate is changed to a chloroform / methanol / water [4: 4: 1 (V / V)] mixed solvent and further eluted. Fraction d3-4 eluted with chloroform / methanol / water [4: 4: 1 (V / V)] mixed solvent is collected. As silica gel, for example, silica gel 60N (63-210 μm; manufactured by Kanto Chemical Co., Inc.) can be used. The tyrosinase inhibitory activities of fractions d3-3 and d3-4 are both 100 μg / mL in aqueous solution and the inhibition rate is 80% or more, and can be used as a sea mud extract used in the present invention.

  Tyrosinase catalyzes the hydroxylation reaction from tyrosine to DOPA (L-dopa), which is the first stage of melanin production, and the oxidation reaction from DOPA to DOPAquinone, which is the next stage. The tyrosinase inhibitory activity can be measured by a method known per se. That is, when the tyrosinase solution is added to the DOPA solution and the reaction proceeds, the colorless and transparent solution turns orange. In tyrosinase inhibitory activity measurement, tyrosinase inhibitory activity can be quantified by measuring the phenomenon of this color change by adding a test substance by absorbance. The concentration of DOPA is usually preferably about 0.03% by mass. The reaction is preferably performed in the presence of a buffer solution (for example, a phosphate buffer solution, pH of about 6) and heated at about 25 ° C. The concentration of tyrosinase is preferably about 0.05 mg / mL (about 100 U / mL). The reaction time is about 5 minutes. Absorbance is preferably measured at 475 nm.

Since the sea mud extract has high tyrosinase inhibitory activity, it can be applied as a tyrosinase inhibitor to an external preparation for skin, particularly whitening cosmetics.
The sea mud extract can be provided in dosage forms such as lotions, emulsions, gels, creams, ointments and the like. Also, skin cosmetics such as lotion, milky lotion, cream and pack, makeup base lotion, makeup base cream, makeup cosmetics such as emulsion, cream or ointment type foundation, eye color and teak color, hand cream It can also be provided as body cosmetics such as leg creams and body lotions. Moreover, it can also provide as a bath agent etc.
The whitening cosmetic composition of the present invention can be produced by, for example, dissolving or stirring a sea mud extract in water or / and a cosmetic base according to a cosmetic production method known per se. For the stirring, any method known per se, for example, stirring with a kiln or mixer, paddle stirring, stirring with ultrasonic waves, stirring using magnetic force, or the like can be used as appropriate. As a compounding quantity of the sea mud extract in whitening cosmetics, about 0.001-5 mass% is preferable with respect to the total mass of cosmetics, Most preferably, it is about 0.005-2 mass%.

  Examples of the cosmetic base of the whitening cosmetic of the present invention include, for example, anionic surfactants, amphoteric surfactants, nonionic surfactants, thickeners, oils, powders (pigments, resins, pigments), and fragrances. , Moisturizers, physiologically active ingredients, ultraviolet absorbers, solvents, antioxidants, chelating agents, preservatives, neutralizers / pH adjusters, insect repellents, enzymes, and the like.

Examples of the anionic surfactant include alkyl sulfates, cocoyl methyl taurine salts, fatty acid salts, coconut oil fatty acid salts thereof, alkyl phosphates, and phosphates and sulfates of polyoxyethylene alkyl ethers.
Examples of amphoteric surfactants include alkylbetaines, phosphobetaines, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and their lysates, as well as phosphophatidic acid and its salts.
Nonionic surfactants include, for example, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene cetyl ether, sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil and polyglycerin fatty acid ester. Can be mentioned.

  Examples of the thickener include acrylic amide and derivatives thereof, carboxyvinyl polymer, alkyl-modified carboxyvinyl polymer, cellulose, keratin and collagen or derivatives thereof, calcium alginate, pullulan, agar, gelatin, tamarind seed polysaccharide, xanthan gum, carrageenan , High methoxyl pectin, polyvinyl alcohol, low methoxyl pectin, guar gum, gum arabic, crystalline cellulose, natural rubber latex, arabinogalactan, karaya gum, tragacanth gum, alginic acid, albumin, casein, curdlan, gellan gum and dextran.

  Examples of the oil include higher alcohols such as cetanol, isostearyl alcohol, lauryl alcohol, hexadecyl alcohol, behenyl alcohol and octyldodecanol, fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, undecylenic acid and oleic acid, myristic Myristyl acid, hexyl laurate, octyldodecyl myristate, cetyl ethylhexanoate, glyceryl tri-2-ethylhexanoate, decyl oleate, isopropyl myristate, hexyldecyl dimethyloctanoate, glyceryl monostearate, glyceryl trioctanoate, palmitic acid Esters such as cetyl, diethyl phthalate, ethylene glycol monostearate and octyl oxystearate, cholesteryl stearate Cholesterol esters such as cholesteryl oleate and branched fatty acid cholesteryl, hydrocarbons such as liquid paraffin, petrolatum, squalane, phospholipids such as lanolin, reduced lanolin, hydrogenated soybean phospholipid, wax such as beeswax and carnauba wax, jojoba oil, mink oil And oils such as cacao butter, palm oil, palm kernel oil, camellia oil, sesame oil, castor oil and olive oil, and silicone oils such as dimethylpolysiloxane, cyclic dimethylpolysiloxane and methylphenylpolysiloxane.

  Examples of the powder include red 201, yellow 4, blue 1, black 401 and other dyes, yellow 4 Al lake, yellow 203 Ba lake and other lake dyes, nylon powder, silk powder, silicone powder, Cellulose powder, silicone elastomer spherical powder, resin such as polyethylene powder, yellow iron oxide, red iron oxide, chromium oxide, carbon black, colored pigments such as ultramarine and bitumen, white pigments such as zinc oxide and titanium oxide, talc, mica Body pigments such as sericite and kaolin, pearl pigment pigments such as titanium mica, metal salts such as barium sulfate, calcium carbonate, magnesium carbonate and magnesium silicate, inorganic powders such as silica and alumina, bentonite, smectite and boron nitride Etc. There are no particular restrictions on the shape of these powders (spherical, rod-like, needle-like, plate-like, indeterminate, flake-like, spindle-like, etc.).

Examples of the humectant include glycerin, xylitol, sorbitol, dipropylene glycol, butylene glycol, propylene glycol, polyethylene glycol 200 to 600, polyoxyethylene methyl glucoside, maltitol and mannitol.
Examples of the physiologically active component include glycyrrhizic acid and its salt, diphenhydramine hydrochloride, placental extract, arbutin, collagen, allantoin, and dl-α-tocopherol. Examples of the solvent include polyoxyethylene cetyl ether, purified water and lower alcohol.
Examples of the ultraviolet absorber include oxybenzone, phenyl salicylate, sinoxate and 2- (2-hydroxy-5-methylphenyl) benzotriazole.

Examples of the antioxidant include catechol, dl-α-tocopherol acetate, dibutylhydroxytoluene and butylhydroxyanisole.
Examples of the chelating agent include edetic acid and its salts, citric acid and its salts, and the like.
Examples of preservatives include benzoic acid and its salts, isopropylmethylphenol, chlorhexidine hydrochloride, orthophenylphenol, chlorhexidine gluconate, chlorcresol, chlorphenesin, chlorobutanol, sorbic acid and its salts, dehydroacetic acid and its salts, paraoxy And ethylene benzoate and halocarban.
Examples of the neutralizing agent / pH adjusting agent include sodium hydroxide, potassium hydroxide, hydrochloric acid, diethanolamine and diisopropanolamine.
Examples of insect repellents include diethyl toluamide.
Examples of the enzyme include lysozyme chloride.

In addition, other whitening components can be added to the whitening cosmetic of the present invention. Examples of the whitening component include ascorbic acid and its salts, cysteine, glutathione and its salts or esters thereof, N-acylated glutathione, hydroquinone and its salts, ferulic acid and its salts, isoferulic acid and its salts, caffeic acid and its Salts, resorcinols such as 4-n-butylresorcinol, ergoic acid and its salts, placenta extract, caffeine, tannin, verapamil, tranexamic acid, licorice extract, glabrizine, tocopherol acetate, glycyrrhizic acid, arbutin, kojic acid, Examples include ellagic acid, chamomile extract, linoleic acid, oleic acid, linolenic acid and the like.
Hereinafter, the present invention will be described more specifically with reference to examples and test examples. However, it goes without saying that the present invention is not limited to these examples and the like.

Whitening pack
A:
Sea mud extract 3.0 parts by weight Titanium oxide 2.0 parts by weight Polyoxyethylene (20E.0) cetyl ether 1.0 part by weight 1,3 butylene glycol 10.0 parts by weight Glycerin 10.0 parts by weight Methyl paraoxybenzoate 0.1 parts by weight B:
Polyvinyl alcohol 1.0 part by weight Purified water 72.9 parts by weight Preparation method: A is heated at 50 ° C. and mixed uniformly. B dissolves uniformly at room temperature. While stirring A, gradually add B and mix uniformly. Cool with stirring, stop stirring at 35 ° C. and leave to stand as a pack agent.

Whitening cream A:
Squalane 25.0 parts by weight Jojoba oil 5.0 parts by weight Beeswax 5.0 parts by weight Cetanol 3.0 parts by weight Monoglyceryl stearate 2.0 parts by weight B:
Polyoxyethylene (20 mol) sorbitan monostearate 3.0 parts by weight Sea mud extract 1.0 part by weight Glycerol 7.0 parts by weight 1,3 Butylene glycol 7.0 parts by weight Methyl paraoxybenzoate 0.3 parts by weight 41.7 parts by weight of purified water Production method: A and B are heated, and while stirring, B is gradually added to A and cooled.

Whitening emulsion A:
Decaglyceryl monomyristate 2.1 parts by weight Hydrogenated soybean phospholipid 0.6 part by weight Stearic acid 0.5 part by weight Behenyl alcohol 1.6 part by weight Cetyl palmitate 0.6 part by weight α-olefin oligomer 6.0 part by weight Cetyl 2-ethylhexanoate 6.0 parts by weight Glyceryl tri-2-ethylhexanoate 6.0 parts by weight Sea mud extract 0.2 parts by weight Glycerin 5.0 parts by weight Paraoxybenzoic acid 0.3 parts by weight B:
Purified water 54.2 parts by weight C:
Carboxyvinyl polymer 0.1 parts by weight Purified water 10.0 parts by weight D:
Sodium hydroxide 0.04 parts by weight Purified water 6.76 parts by weight Preparation method: Both A and B are heated and dissolved at 80 ° C., B is gradually added to A while stirring with a homomixer, and emulsified at 5,000 rpm for 5 minutes. To do. Add C and D with paddle stirring, cool, stop stirring at 40-35 ° C., and let stand.

Whitening lotion A:
Dipotassium glycyrrhizinate 0.2 parts by weight Citric acid 0.1 parts by weight Sodium citrate 0.3 parts by weight Purified water 82.99 parts by weight B:
Tetraoleic acid P0E (60) sorbitol 0.9 parts by weight Sorbitan monooleate 0.1 parts by weight Olive oil 0.1 parts by weight Dipropylene glycol 5.0 parts by weight Sea mud extract 0.01 parts by weight Ethanol 10.0 parts by weight Part 0.3 parts by weight of paraoxybenzoic acid Preparation method: Both A and B are heated and dissolved at 50 ° C., and B is gradually added to A while stirring and solubilized. Cool with stirring, stop stirring at 30 ° C. and let stand.

Whitening facial cleanser A:
Lauric acid 2.0 parts by weight Myristic acid 17.0 parts by weight Palmitic acid 4.0 parts by weight Stearic acid 4.0 parts by weight Palm oil fatty acid potassium 8.0 parts by weight Palm oil fatty acid diethanolamide 3.0 parts by weight N-cocoyl Methyl taurine sodium 10.0 parts by weight Concentrated glycerin 5.0 parts by weight 1,3-butylene glycol 5.0 parts by weight Sea mud extract 0.5 parts by weight Paraoxybenzoic acid 0.3 parts by weight B:
Potassium hydroxide 5.5 parts by weight Tesodium edetate 0.2 parts by weight Purified water 35.5 parts by weight Preparation method: A is heated and dissolved at 80 ° C. B dissolves at room temperature. Slowly add B to A while stirring and dissolve uniformly. Cool with stirring, stop stirring at 35 ° C. and let stand.

Test example 1
Tyrosinase inhibitory activity Tyrosinase separated and purified from mushrooms was purchased from Sigma and used. Tyrosinase was dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) to give a 0.1 mg / mL (200 U / mL) solution, which was used as the tyrosinase solution. To the test tube, 0.5 mL of the test solution and 0.5 mL of the substrate L-dopa solution (0.03% by mass, dissolved in 1/15 M phosphate buffer, pH 6) were added and heated at 25 ° C. for 10 minutes. Next, 0.5 mL of tyrosinase solution was added, and after heating at 25 ° C. for 5 minutes, the absorbance was measured at 475 nm (D1). The absorbance obtained by performing the same operation without adding a substrate was D2, and the absorbance obtained by performing the same operation without adding a test solution was D3, and the inhibition rate was determined by the following equation.
Inhibition rate (%) = ((D3-D2)-(D1-D2)) / (D3-D2) × 100

Test example 2
Separation of sea mud extract (1) Sea mud hot water extract 3 kg of purified water was added to 1 kg of sea mud, heated at 100 ° C. for 1 hour, and then filtered. The filtrate was concentrated under reduced pressure, freeze-dried, water was removed, and a sea mud hot water extract was extracted. The yield based on sea mud at this time was 4%. The tyrosinase inhibitory activity of the sea mud hot water extract was 74% at a concentration of 2 mg / mL.

  (2) The sea mud hot water extract obtained in (1) is dissolved in water, and silica gel [silica gel 60N (63-210 μm; manufactured by Kanto Chemical Co., Ltd.)] column chromatography (diameter 6 cm, length 50 cm). First, elution was performed using a mixed solvent of chloroform / methanol / water [6: 4: 1 (V / V)], and a part of the eluate of each fraction was subjected to TLC with chloroform / methanol / water [6 : 4: 1 (V / V)] using a mixed solvent. A fraction having an Rf value of 0.6 to 1 when colored with a ninhydrin reagent is fraction a (yield based on sea mud hot water extract (hereinafter simply referred to as yield): 0.8%), Fraction b (yield: 6.56%) having an Rf value of 0.5 to 0.6 and Fraction c (yield: 43.9%) having an Rf value of 0.1 to 0.2 ). The tyrosinase inhibition rates at an aqueous solution concentration of 1 mg / mL for each fraction were 40.0%, 58.6%, and 63.3%, respectively. The silica gel chromatography is then eluted with methanol. The eluate eluted with the methanol was collected and used as fraction d (yield: 20.8%). The tyrosinase inhibition rate of the fraction d at a concentration of 1 mg / mL was 89. %Met. The fraction a and the fraction b were further separated and purified by ODS column chromatography and silica gel column chromatography [eluent: hexane / ethylene acetate = 1: 2 (V / V)] to obtain 3-hydroxybutane. The acid was collected. The tyrosinase inhibition rate of 3-hydroxybutanoic acid 100 μg / mL was about 40%.

(3) Subsequently, the fraction d10 g was separated from methanol / water [1: 1 (V / V) by molecular sieve chromatography (diameter 3.5 cm, length 42 cm) using Sephadex LH-20 (manufactured by Tosoh Corporation). Elution was performed using a mixed solvent. After elution of the eluate of volume volume (about 150 mL), about 15 mL each was dispensed into a test tube, sequentially numbered, and elution no. It was. A part of the fractionated eluate was developed by TLC using a mixed solvent of chloroform / methanol / water / acetic acid [3: 3: 1: 0.1 (V / V)], colored with ninhydrin reagent and UV254 mm. Were fractionated into six fractions shown in Table 1 by luminescence at, and were designated as fractions d1 to d6. The yield of each fraction and the tyrosinase inhibition rate of 100 μg / mL are shown in Table 1.

(4) Fraction d3 having high tyrosinase inhibitory activity was further purified by silica gel chromatography (diameter 2 cm, length 15 cm) using a chloroform / methanol / water [5: 4: 1 (V / V)] mixed solvent. Eluted. The eluate was developed by TLC using a mixed solvent of chloroform / methanol / water [2: 4: 1 (V / V)], and three fractions were obtained by coloring with a ninhydrin reagent. Fraction d3-1 (fraction not colored with ninhydrin reagent, yield: 0.04%), d3-2 (Rf value 0.3, yield: 0.11%), d3-3 (Rf value) 0.2 and 0.4, yield: 0.046%). The tyrosinase inhibition rate of 100 μg / mL of each fraction was 33%, 45.2%, and 90.6%, respectively.
Next, the eluate is replaced with a mixed solvent of chloroform / methanol / water [4: 4: 1 (V / V)] and further eluted. The fraction eluted with a mixed solvent of chloroform / methanol / water [4: 4: 1 (V / V)] was collected and used as fraction d3-4. The yield of fraction d3-4 was 0.77%, and the tyrosinase inhibition rate at 100 μg / mL was 84.8%.
The sea mud hot water extract was purified to obtain fraction d3-3 and fraction d3-4, which are sea mud extracts having a tyrosinase inhibition rate of 100% or more at 80% or more. It was found that these sea mud extracts were purified to have a tyrosinase inhibitory activity equivalent to that of the sea mud hot water extract in an amount of about 1/20 that of the sea mud hot water extract.

  Since the whitening cosmetic composition of the present invention contains a sea mud extract having a high tyrosinase inhibitory activity, it is useful as a cosmetic composition capable of preventing and suppressing pigmentation due to melanin production.

FIG. 1 is a diagram showing a method for purifying a sea mud extract from a sea mud hot water extract.

Claims (3)

  A whitening cosmetic comprising a sea mud extract obtained by refining a sea mud hot water extract of Urkutaburu Island in the Republic of Palau, and exhibiting tyrosinase inhibitory activity of 80% or more at an aqueous solution concentration of 100 μg / mL.   The whitening cosmetic composition according to claim 1, comprising 0.001 to 5.0% by mass of a sea mud extract. A tyrosinase inhibitor characterized by containing a sea mud extract obtained by purifying a sea mud hot water extract from Urkturbul Island in the Republic of Palau, and exhibiting a tyrosinase inhibitory activity of 80% or more at an aqueous solution concentration of 100 μg / mL.

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