TWI666029B - Use of albumin-based compound for producing skin-whitening external preparation - Google Patents

Abstract

A whitening cosmetic containing albumin compounds. Albumin compounds are compounds in which a sulfur atom is added to albumin or an albumin derivative. The albumin-based compound has a plurality of sulfur atoms at each site. The method for producing an albumin-based compound includes a step of performing an addition reaction between sulfide and albumin or an albumin derivative. Albumin compounds are contained in whitening cosmetics.

Description

Use of albumin compounds for manufacturing whitening skin external preparation

The invention relates to a whitening cosmetic and a method for producing albumin compounds contained therein.

As a whitening cosmetic, for example, a skin external preparation for whitening is known (Patent Document 1). This skin external preparation for whitening contains, as an active ingredient, a melanin production inhibitor such as glutathione in protein nano particles that have been subjected to an enzyme cross-linking treatment as necessary.

In addition, sodium sulfide is known to have a whitening effect by suppressing the production of melanin, and sodium sulfide is also considered to be applied to cosmetics.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open Publication No. 2008-247814

In the preparation of the active ingredient contained in the whitening skin external preparation described in Patent Document 1, protein nano particles are used as described above. The protein nanoparticle is prepared in multiple steps. In order to obtain an effective ingredient, a melanin production inhibitor needs to be contained in the protein nano particles. Therefore, the preparation of the active ingredient contained in the skin external preparation for whitening is complicated.

On the other hand, when sodium sulfide (Na ₂ S) is dissolved in a solvent to be in a solution state, it becomes hydrogen sulfide (H ₂ S), and about half of the physiological pH quickly becomes a gas and disappears. Therefore, a useful substance as a carrier of a sulfur atom is sought, and development of a cosmetic containing a melanin production inhibiting component (active ingredient) in a stable state is desired.

The present invention has been made in view of the foregoing problems, and an object thereof is to provide a whitening cosmetic having excellent stability of effective ingredients. Furthermore, the object is to provide a method for producing an active ingredient contained in a whitening cosmetic by a simple procedure.

The whitening cosmetic of the present invention contains an albumin-based compound. The albumin-based compound is a compound in which a sulfur atom is added to albumin or an albumin derivative. The albumin-based compound has a plurality of sulfur atoms at each site.

The manufacturing method of this invention is a manufacturing method of an albumin type compound. The albumin-based compound is included in the whitening cosmetic. The production method of the present invention includes a step of performing an addition reaction between sulfide and albumin or an albumin derivative. [Effect of the invention]

The whitening cosmetic of the present invention not only has excellent whitening effect, but also has excellent stability and safety of active ingredients, and has an odor suitable for the cosmetic. Moreover, according to the manufacturing method of this invention, the active ingredient contained in a whitening cosmetics can be manufactured with a simple process.

Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited in any way by the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention. In addition, the repeated description may be appropriately omitted, but this does not limit the gist of the invention. In addition, "classes" are sometimes added to the names of organic compounds to collectively refer to organic compounds and their derivatives.

<First embodiment: albumin-based compound and method for producing the same> The first embodiment relates to albumin-based compound and method for producing the same. The albumin-based compound is contained in a whitening cosmetic as an active ingredient that imparts an effect of inhibiting the production of melanin.

As described above, sulfides such as sodium sulfide have a whitening effect by suppressing the production of melanin. Therefore, they have attracted attention as effective ingredients for whitening cosmetics. A solvent is usually used in the preparation of cosmetics, but in a solution where the sulfide is dissolved in the solvent, the sulfide becomes hydrogen sulfide. Although the hydrogen sulfide in the solution maintains the ability to suppress melanin production, it easily disappears by itself. Approximately half of the hydrogen sulfide in a physiological pH (optimal pH for cosmetics) solution quickly becomes a gas and disappears.

Therefore, it is discussed to prepare a stable state albumin compound instead of hydrogen sulfide which is easy to disappear, and to mix it as an active ingredient into a whitening cosmetic.

Albumin-based compounds are compounds in which sulfur atoms are added to albumin or an albumin derivative, and each site has a plurality of sulfur atoms. The method for producing an albumin-based compound includes a step of subjecting a sulfide to an addition reaction of albumin or an albumin derivative.

1. Albumin or albumin derivative Albumin or albumin derivative is a component that functions as a carrier of a sulfur atom.

The type of albumin or albumin derivative is not particularly limited. Albumin is any of serum albumin and genetically modified albumin. From the viewpoint of easy availability, it is desirable to use serum albumin. The serum albumin includes, for example, human serum albumin, bovine serum albumin, or chicken serum albumin. From the viewpoint of higher safety, human serum albumin is particularly desirable. In the case of genetic recombination, dimer or multimerized albumin is also contained. In addition, not only albumin itself, but also albumin derivatives that have been chemically modified with albumin can be used. Albumin or an albumin derivative may be used alone or in combination of two or more.

The molecular weight of albumin or an albumin derivative is not particularly limited. For example, in the case of serum albumin, it is desirable to use serum albumin of 66,000 to 66500. In the case of multimerization, the particle size is preferably about 100 nm.

2. The type of the sulfide is not particularly limited. Examples of the sulfide include sodium sulfide and propylene sulfide (specifically, diallyl sulfide or disulfide (2-propylene) represented by the general formula: Na ₂ Sn (wherein n represents an integer of 1 to 6). )), Or sulfur (for example, sulfur represented by S ₈ ). From the viewpoint of easy availability, it is desirable to use sodium sulfide in the sulfide. In terms of good addition reaction to albumin or albumin derivatives, and easy addition of sulfur atoms to albumin or albumin derivatives, sodium sulfide is preferably sodium polysulfide (in the above general formula, n represents a compound having an integer of 2 to 6). From the viewpoint of obtaining an albumin-based compound having higher stability and more excellent melanin production suppressing ability, Na ₂ S ₂ , Na ₂ S _3, or Na ₂ S ₄ is particularly desirable among sodium polysulfides. The sulfides may be used singly or in combination of two or more kinds.

3． Addition reaction step The reaction of albumin or albumin derivative with sulfide is an addition reaction. The reaction ratio between albumin or albumin derivative and sulfide (albumin or albumin derivative: sulfide) is not particularly limited, but in terms of mole ratio, it is preferably 0.1: 1 to 0.6: 1 , More preferably 0.2: 1 to 0.5: 1. If the amount of albumin or albumin derivative is 0.1 mol or more relative to 1 mol concentration of sulfide, unreacted sulfide can be easily removed. When the amount of albumin or albumin derivative is 0.6 mol or less relative to the 1 mol concentration of the sulfide, an albumin compound having higher stability and more excellent melanin production suppressing ability can be obtained.

The addition reaction of sulfide and albumin or albumin derivative can be performed by, for example, heating the albumin or albumin derivative and sulfide, or shaking the albumin or albumin derivative and sulfide. method. A particularly desirable method is a method of heating albumin or an albumin derivative and a sulfide, because this method does not require special equipment and can make it easier to react.

In the method for heating albumin or an albumin derivative and a sulfide, sulfide is added to the albumin or an albumin derivative, and the reaction temperature and reaction time are adjusted in a pH-adjusted buffer solution to make the albumin or Albumin derivatives react with sulfides.

There is no particular limitation on the type of the buffer solution in the heating method, and examples thereof include a phosphate buffer solution, a Torris buffer solution, a phthalic acid buffer solution, a citric acid buffer solution, a succinic acid buffer solution, or an acetic acid buffer solution. From the viewpoint of easily promoting the reaction between albumin or an albumin derivative and a sulfide, the pH of the buffer solution is preferably 7.0 or more and 7.8 or less, and more preferably 7.2 or more and 7.6 or less.

In the heating method, the reaction temperature of albumin or an albumin derivative and a sulfide is preferably 30 ° C or higher and 50 ° C or lower, and more preferably 32 ° C or higher and 45 ° C or lower. When the reaction temperature is 30 ° C or higher, the reaction between albumin or an albumin derivative and a sulfide is easily promoted. If the reaction temperature is 50 ° C or lower, there is no possibility that the albumin or the albumin derivative is degraded such as degradation or aggregation.

In the heating method, the reaction time between the albumin or the albumin derivative and the sulfide is preferably from 4 to 10 hours, and more preferably from the viewpoint of facilitating the reaction between the albumin or the albumin derivative and the sulfide. It is 5 hours or more and 8 hours or less.

In the method of oscillating albumin or an albumin derivative and a sulfide, sulfide is added to the albumin or an albumin derivative, and the pH is adjusted in a buffer solution whose pH is adjusted to adjust the oscillation speed and reaction time. The protein or albumin derivative reacts with the sulfide.

The type of the buffer is not particularly limited in the shaking method, and the buffers exemplified in the heating method described above can be used. Similarly to the heating method described above, the pH of the buffer solution is preferably 7.0 or more and 7.8 or less, and more preferably 7.2 or more and 7.6 or less.

In the shaking method, the shaking speed of albumin or the albumin derivative and the sulfide is not limited as long as it can promote the reaction between the albumin or the albumin derivative and the sulfide, and is preferably adjusted to about 30 times per minute or more 180 Times per minute or less.

In the shaking method, the reaction time of the albumin or albumin derivative with the sulfide is preferably 4 hours or more and 10 hours or less, and more preferably 5 in terms of facilitating the reaction between the albumin or the albumin derivative and the sulfide. More than 8 hours and less than 8 hours.

In addition, the albumin, albumin derivative, and sulfide can be oscillated using, for example, a general oscillator, and as long as it is appropriately adjusted, for example, the above-mentioned oscillation speed may be used.

By the above-mentioned heating method or shaking method, an sulfide can be subjected to an addition reaction with albumin or an albumin derivative, for example, in the presence of an oxidant (specifically, nitric oxide or hydrogen peroxide) or a metal ion When the sulfide is subjected to an addition reaction with albumin or an albumin derivative, the addition reaction can be promoted and the reaction efficiency can be improved.

It is particularly desirable to perform the addition reaction in the presence of metal ions, because it can further promote the addition reaction, further improve the reaction efficiency, and easily obtain albumin compounds with sufficient sulfur atoms to be added. The specific situation that the reaction efficiency is improved due to the presence of metal ions is not clear. It can be considered that the metal ions attracted sulfur atoms in the sulfide, thereby promoting the addition of sulfur atoms to albumin or albumin derivatives. The metal ions may be used singly or in combination of two or more kinds. Among the metal ions, divalent metal ions are desirable in terms of more significant effects and convenient handling. Examples of the divalent metal ion include Fe ^{2 +} , Mg ^{2 +} , Ca ^{2 +,} or Cu ^{2 +} .

When the addition reaction is performed in the presence of metal ions, the molar ratio is preferably 0.2: 1 to 2: 1 in terms of the ratio of metal ions to sulfide (metal ion: sulfide), and more preferably It is 0.5: 1 to 1.5: 1. The effect of improving the reaction efficiency can be fully exhibited if the substance mass of the metal ion is 0.2 Molar concentration or more with respect to the 1 mol concentration of the sulfide. When the amount of metal ions is 2 mol or less relative to the 1 mol concentration of sulfide, the addition reaction will not be hindered by the oxidation reaction caused by the excessive metal ions, such as the typical Fenton reaction.

When the addition reaction is performed in the presence of metal ions, for example, hot spring water can be used. Hot spring water generally contains a plurality of divalent metal ions as described above. Therefore, the hot spring water is appropriately selected according to the type of metal ions contained. For example, it is desirable to adjust the use ratio of the hot spring water to the sulfide so that the ratio of the metal ion to the sulfide reaches the above-mentioned molar ratio range. That is, the hot spring water is appropriately selected according to the type of metal ions contained, and the ratio of sulfide to metal ions is taken into consideration to determine the ratio of use of the hot spring water to the sulfide, and then the buffer solution with adjusted pH is mixed with the hot spring water . By adding a sulfide to albumin or an albumin derivative in this mixed solution, the sulfide can be subjected to an addition reaction with albumin or an albumin derivative in the presence of a metal ion.

4． After confirming the completion of the reaction between albumin or albumin derivative and sulfide in other steps, the gel can be filtered (for example, by using gel filtration column chromatography) to remove unreacted sulfide, thereby enabling Purification to obtain the target albumin compounds.

5． Albumin-based compounds Albumin-based compounds are compounds in which sulfur atoms are added to albumin or albumin derivatives, and each site has multiple sulfur atoms.

In the case of albumin or an albumin derivative, it originally had an SH group derived from cysteine and the SH group of cysteine was oxidized. It becomes a cysteine adduct. However, albumin or cysteine-addition albumin and its derivatives having only SH groups do not exert melanin production inhibitory effects. Therefore, even if such albumin or an albumin derivative is blended, a whitening cosmetic having excellent whitening effect cannot be obtained.

An albumin-based compound is a compound in which a sulfur atom is further added to a sulfur atom in an SH group and an S-S bond in albumin or an albumin derivative, and a compound such as an SSH group, an SSSH group, or an S-S-S bond is formed. The specific situation is not clear, but in albumin or albumin derivatives, when the sulfur atom is added to the sulfur atom in this way, multiple sulfur atoms are supported by the carrier, so that there is no effect in albumin or albumin derivatives. Melanin produces an inhibitory effect. In addition, unlike sulfur compounds, albumin compounds having sulfur atoms (hereinafter, also referred to as polysulfide additions) further added to sulfur atoms have physiological pHs that remain stable.

The albumin-based compound preferably has 2 or more and 6 or less sulfur atoms per site, and more preferably has 2 or more and 4 or less sulfur atoms per site. If a sulfur atom is not added to albumin or an albumin derivative, and there is only one sulfur atom at each site in an albumin compound, the melanin production inhibitory effect will not be as described above. When an albumin compound has less than 6 sulfur atoms per site, a sulfur atom transfer reaction hardly occurs, and sufficient melanin is provided to suppress the effect. Therefore, the albumin compound is easily applicable to whitening cosmetics. In addition, it is not clear whether the sulfur atom transfer reaction in the albumin compounds is due to the environment (temperature, humidity, etc.) where the albumin compounds are located. Therefore, the number of sulfur atoms in one part of the albumin-based compound is preferably six or less as described above, but it is not limited thereto.

In albumin or albumin derivatives, the position and number of SH groups or S-S bonds are determined. However, in albumin compounds, the location and number of polysulfide addition sites have not been determined. The specific situation is not clear, and it can be considered that the position and the number of polysulfide addition sites are appropriately changed according to the reaction conditions of the addition reaction. In addition, it is considered that the position of the polysulfide addition site in the albumin-based compound is changed relative to the position of the SH group or the S-S bond in the albumin or the albumin derivative.

In the albumin compounds, the number of sulfur atoms present in each polysulfide addition site may be the same or different at each site. That is, for example, the same number of sulfur atoms can be added to each SH group in albumin or an albumin derivative, and different numbers of sulfur atoms can be added according to each SH group.

The sulfur atom (sulfane sulfur) in the albumin-based compound can be detected by, for example, a fluorescent probe.

Sulfane Sulfur compounds are a general term for sulfur molecules that are trapped in sulfur atoms such as SS ^* -S, SS ^* -S ^* -S, etc. The detection of sulfane sulfur uses a fluorescent probe for sulfane sulfur detection. . The fluorescent probe for sulfane sulfur detection is ideally a non-destructive fluorescent probe capable of detecting sulfane sulfur without damaging cells or tissues. Examples of such a non-destructive sulfane sulfur detection fluorescent probe include SSP2 or SSP4. SSP2 and SSP4 have one molecule (SSP2) or two molecules (SSP4) of thiosalic acid having a nucleophilic functional group, and is composed of fluorescent yellow, which is a fluorescent dye, in which thiosalic acid has a nucleophilic functional group. In the state where the phenolic hydroxyl group of the fluorescent dye is protected by thiosalic acid, SSP2 and SSP4 hardly emit fluorescence, but when thiosalic acid reacts with sulfane sulfur, the reactants undergo a rapid intramolecular cyclization reaction, 3H-1,2-benzodithiol-3-one is released to form a phosphor. Therefore, by measuring the fluorescence intensity based on this phosphor, and comparing with the fluorescence intensity of SSP2 or SSP4, sulfane sulfur can be detected.

The albumin compounds according to the first embodiment and the method for producing the albumin compounds have been described above. The melanin of the albumin-based compound obtained by the production method according to the first embodiment is excellent in suppressing melanin, has stability and safety, and has an odor suitable as a cosmetic ingredient. The production method according to the first embodiment can produce such an albumin compound in a simple step.

<Second embodiment: Whitening cosmetics> The second embodiment relates to whitening cosmetics. Whitening cosmetics contain albumin compounds. The albumin-based compound is an albumin-based compound according to the first embodiment, which has an excellent melanin production suppressing effect, stability, and safety, and has an odor suitable as a cosmetic ingredient.

The albumin compounds contained in the whitening cosmetics may be used singly or in combination of two or more different ones.

The blending amount of the albumin-based compound in the whitening cosmetic can be appropriately adjusted according to the type (morphology) of the target whitening cosmetic, and is not particularly limited. For example, with respect to 100 parts by mass of the whitening cosmetic material, the blending amount of the albumin-based compound is preferably 0.1 to 20 parts by mass, and more preferably 0.12 to 0.3 parts by mass, in terms of solid content. When the compounding amount of the albumin-based compound is 0.133 parts by mass or more, the whitening effect of the albumin-based compound can be fully exerted.

In addition to blending albumin-based compounds in the whitening cosmetic, for example, additives commonly used in cosmetics can be appropriately blended. Examples of the additives include fats and oils, surfactants, humectants, whitening agents, pH adjusters, tackifiers, preservatives, antioxidants, ultraviolet light absorbers, pigments, detergents, emulsifiers, excipients, or fragrances.

Examples of fats and oils include vegetable fats (specifically, mobile paraffin, paraffin, cetyl alcohol, avocado oil, olive oil, jojoba oil, or coconut oil), animal fats (specifically, tallow, lard, Horse fat, turtle oil, mink oil, duck oil (Perselin oil or squalane), or synthetic fats (specifically, methyl polysiloxane, eicosanediol, caprylic acid glyceride, Caprylic acid glyceride, glycerol triisopalmitate or silicone oil).

Examples of the surfactant include anionic surfactants (specifically, sodium lauryl sulfate, triethanolamine lauryl sulfate or diethanolammonium laurate), and cationic surfactants (specifically, trimethyl stearate chloride). Ammonium, cetyltrimethylammonium chloride or alkyldimethylbenzylammonium chloride), or non-ionic surfactants (specifically, glycerol monostearate, sorbitan monohard Fatty acid esters, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene hardened ramie oil, sucrose esters, or fatty amines).

Examples of the humectants include synthetic humectants (specifically, glycerin, propylene glycol, 1,3-butanediol, pentafluorenone carboxylic acid soda or panthenol-S-sulfonate), or natural humectants (specifically In terms of hyaluronic acid, collagen, elastin, placenta extract, royal jelly, microbial fermentation broth, chitin, polyglucosamine, or pectin).

Examples of the whitening agent include compounds other than albumin-based compounds, such as osmic acid, ascorbic acid, or placenta extract.

Examples of the pH adjusting agent include citric acid or sodium citrate.

Tackifiers include, for example, carboxymethylcellulose, oxymetavin, hydroxyethylcellulose, polycarboxyvinyl, polyvinyl alcohol, tragacanth, sodium alginate, or red algal gum (ie, carrageenan).

Examples of the preservative include parabens (specifically, methyl parabens, ethyl parabens, propyl parabens, or butyl parabens), 2-phenoxy Ethanol, ethanol or dehydroacetic acid.

Examples of the antioxidant include vitamin E, dibutylhydroxytoluene (BHT), or butylhydroxyanisole (BHA).

Examples of the ultraviolet light absorber include 4-imidazole acrylic acid, 4-imidazole ethyl acrylate, hydroxybenzophenone, octyl salicylate, dihydroxyphenylphenyl ketone, p-aminobenzoic acid, and octyl p-dimethylaminobenzoate ( Octyl dimethyl p-amino benzoic acid) or octyl methoxycinnamate.

Examples of the pigment include red iron powder, iron oxide yellow, black iron oxide, titanium oxide, nylon powder, sericite, mica, or talc.

The cleaning agent is, for example, sodium lauryl sulfate.

An emulsifier is, for example, soybean lecithin oil.

An excipient is, for example, sodium sulfate.

The blending amount of the additives can be appropriately adjusted according to the type (morphology) of the target whitening cosmetic, and is not particularly limited as long as it does not hinder the effect of the albumin-based compound.

The method for preparing the whitening cosmetic is not particularly limited, and a method for preparing an ordinary cosmetic may be adopted, and for example, it may be appropriately determined in accordance with the form described later.

The form of the whitening cosmetic is not particularly limited. Considering the excellent whitening effect of whitening cosmetics, the forms of whitening cosmetics are, for example, basic cosmetics (specifically, creams, lotions, toners, cleansers or masks), or beauty cosmetics (specifically, lipstick, powder) Foundation or liquid foundation).

The whitening cosmetics according to the second embodiment have been described above. The whitening cosmetic according to the second embodiment includes an albumin-based compound as an active ingredient. The albumin-based compound is excellent in melanin production suppressing effect, stability, and safety, and has an odor suitable for the cosmetic. With the whitening cosmetic material according to the second aspect, it is possible to suppress pigmentation, freckles, and the like caused by accumulation of melanin, improve dull skin tone, and maintain whitening skin. [Example]

Hereinafter, the present invention will be described in more detail through examples. In addition, the present invention is not limited at all by the scope of the examples.

Production Example 1 (Production of albumin compounds) In a phosphate buffer solution (pH 7.4), Na ₂ S (78 μg, 1 mM) was added to defatted human serum albumin (molecular weight: 66,500, 20 mg, 300 μM), and heated to 37 It was allowed to react at 7 ° C for 7 hours to add a sulfur atom to human serum albumin. Regarding the product after the addition reaction, the gel was filtered under the following conditions to remove unreacted sulfides, thereby obtaining the target albumin compound-1 (9.5 mg). [Conditions of gel filtration] Mobile phase: Phosphate buffer flow rate: 10 mL / min Temperature: 25 ° C

Production Examples 2 to 4 (Production of albumin-based compounds) Except Na ₂ S ₂ (110 μg, 1 mM, Production Example 2), Na ₂ S ₃ (142 μg, 1 mM, Production Example 3), or Na ₂ S ₄ ( 174 μg, 1 mM, Production Example 4) Instead of Na ₂ S (78 μg, 1 mM), in the same manner as in the production of Albumin Compound-1, Albumin Compound-2 (9.4 mg, Production Example 2) was obtained. , Albumin-based compound-3 (9.5 mg, Production Example 3) or albumin-based compound-4 (10.5 mg, Production Example 4).

Test Example 1 (Detane Sulfide Detection) 检测 Sulfane Sulfur was detected by a fluorescent probe. SSP2 (fluorescent dye: fluorescent yellow) was used as the fluorescent probe for sulfane sulfur detection.

Cetyltrimethylammonium bromide (1 mM) was dissolved in a phosphate buffer solution (pH 7.4), and any one of albumin compounds 1-4 was added to the solution so that the molar concentration was 20 μM. Prepare the sample. This sample was reacted with a fluorescent indicator obtained by dissolving SSP2 (5 μM) in dimethylarsine at room temperature (25 ° C.) for 10 minutes, and then a spectrofluorimeter (manufactured by JASCO Corporation, FP-8200) to measure the fluorescence intensity of the reactants (excitation wavelength: 457nm, fluorescence wavelength: 490-580nm). The fluorescence intensity of each reactant including the fluorescence intensity of SSP2 is shown below.

Fluorescence intensity (unit-free) SSP2 512 Reactant 1 (albuminoid-1) 1135 Reactant 2 (albuminoid-2) 1219 Reactant 3 (albuminoid-3) 1534 Reactant 4 (white Protein compounds-4) 3380

As described above, the fluorescence intensity of the samples of albumin compounds 1-4 by reaction with SSP2 is about 2-7 times that of SSP2. In this way, it was confirmed that all of the albumin compounds 1-4 had sulfane sulfur in the molecule. Therefore, it can be seen that the albumin compounds 1-4 are all compounds in which sulfur atoms are added to human serum albumin, and each has a plurality of sulfur atoms at each position. In addition, it can be seen that all of the albumin compounds-1 to 4 are stable at physiological pH.

Second test example (confirmation of melanin production inhibitory effect) 2-1. B16 mouse melanoma cells were seeded in 24-well plates and measured in absorbance and photographed with a light microscope in Dulbecco'S Modified Eagle'S Medium (hereinafter referred to as DMEM (+)). The cells were prepared at 37 ° C and 5% CO ₂ . The cells were cultured for 24 hours to make them 2.5 × 10 ⁴ cells / well. Next, the preculture medium was exchanged with DMEM (+) (albuminoid: 20 μM, tyrosine: 0.4 mM, NH4Cl: 10 mM) to which albumin-type compound-2 was added, and the mixture was incubated at 37 ° C for 5 minutes. % CO ₂ was cultured for 72 hours.

In addition, instead of DMEM (+) to which albumin-based compound-2 was added, those cultured in DMEM (+) with phosphate buffer (pH7.4) were prepared, and commercially available whitening lotions (bear Fruit glycosides: those cultured in DMEM (+) with a final concentration of 1.10 mM and ascorbic acid: 591 μM) were used as a control group.

Next, the cultured B16 mouse melanoma cells adhered to the cannula were washed with a phosphate buffer solution (pH 7.4), and then 200 μL of an aqueous sodium hydroxide solution (1N) was added. The cultured B16 mouse melanoma cells were cultured at 60 ° C. for 1 hour, and the cell lysate was recovered. The absorbance of the cell lysate at a wavelength of 405 nm was measured using a spectrophotometer (manufactured by Bio-Rad, model 680 μ measuring cup). The results are shown in Figure 1. In addition, 405 nm is the absorption wavelength of melanin. Furthermore, the B16 mouse melanoma cells (400 times) after culture were photographed using a single-unit fluorescence microscope (manufactured by Keyence Corporation, BZ-X700, hereinafter referred to as a fluorescence microscope). The results are shown in Figure 2.

Figure 1 shows the absorbance of cell lysate cultured in DMEM (+) supplemented with phosphate buffer solution and cell lysate cultured in DMEM (+) supplemented with albumin compound-2 at a wavelength of 405 nm. Bar chart. In Figure 1, the vertical axis represents the absorbance (unitless). The smaller the absorbance, the less melanin is produced.

As shown in FIG. 1, the absorbance of albumin-based compound-2 at a wavelength of 405 nm is approximately 1/2 of the absorbance of a phosphate buffer solution as a control group at a wavelength of 405 nm.

Figure 2 (a) is a SEM photograph of B16 mouse melanoma cells cultured in phosphate-buffered DMEM (+) culture; Figure 2 (b) is a DMEM (+) commercially available whitening lotion ) SEM photograph of B16 mouse melanoma cells; Figure 2 (c) is a SEM photograph of B16 mouse melanoma cells cultured in DMEM (+) supplemented with albumin-2. In the SEM photograph of FIG. 2, the black spots are locations where melanin is generated.

As shown in FIG. 2, when a commercially available whitening lotion is used, less melanin is generated than when a phosphate buffer solution is used. However, the use of albumin-type compound-2 produced almost no melanin. Compared with phosphate buffered liquid phase, it significantly inhibited the production of melanin, and even compared with commercially available whitening lotions, it also significantly inhibited the production of melanin.

Therefore, it can be seen from FIG. 1 and FIG. 2 that an albumin compound having sulfur atoms added to albumin and having a plurality of sulfur atoms at each site has a very excellent melanin production suppressing effect.

2-2. Measurement of melanin production amount B16 mouse melanoma cells were seeded in a 24-well plate and pre-cultured with DMEM (+) at 37 ° C and 5% CO ₂ for 24 hours to make them 2.5 × 10 ⁴ cells / hole. Next, the pre-cultured medium and DMEM (+) (albumin-based compound: 20 μM, tyrosine: 0.4 mM, NH ₄ Cl: 10 mM) to which any of the albumin-based compounds _1-4 were added were performed. Exchange and incubate for 72 hours at 37 ° C and 5% CO ₂ .

In addition, instead of DMEM (+) to which albumin compounds were added, DMEM (+) cultures to which delipidated human serum albumin used in Production Examples 1 to 4 were prepared were used as a control group.

Next, the cultured B16 mouse melanoma cells adhered to the cannula were washed with a phosphate buffer solution (pH 7.4), and then 200 μL of an aqueous sodium hydroxide solution (1N) was added. The cells were cultured at 60 ° C. for 1 hour, the cultured B16 mouse melanoma cells were lysed, and the cell lysate was recovered. Using the same spectrophotometer used in the above "2-1. Measurement of Absorbance and Photographing of Optical Microscopy", the absorbance of the cell lysate at a wavelength of 405 nm was measured to calculate the melanin in the B16 mouse melanoma cells after culture. Generated. The results are shown in Figure 3.

FIG. 3 is a bar graph showing the amount of melanin production in B16 mouse melanoma cells cultured in each cell lysate. In Fig. 3, the vertical axis represents the amount of melanin produced (mg) in the cell lysate (1 mL).

As shown in Fig. 3, the amount of melanin produced by the albumin compounds 1-4 was lower than that of the defatted human serum albumin as a control group. From this, it can be seen that an albumin compound having a sulfur atom added to albumin and having a plurality of sulfur atoms per site has an excellent melanin production suppressing effect compared to albumin.

In particular, albumin-based compounds-2, albumin-based compounds-3, and albumin-based compounds- _{4 are} subjected to addition reactions of defatted human serum albumin with Na ₂ S ₂ , Na ₂ S ₃ and Na ₂ S ₄ respectively. The amount of melanin produced is very small. From this, it can be known that the albumin compounds obtained by the addition reaction of sulfide, especially sodium polysulfide such as Na ₂ S ₂ , Na ₂ S ₃ or Na ₂ S ₄ , with albumin have More excellent melanin produces an inhibitory effect.

Third Test Example (Measurement of Protein Concentration) 黑色 B16 mouse melanoma cells cultured in the same manner as in "2-2. Measurement of Melanin Production Amount" described above in the second test example were washed twice with physiological saline. As above, absorb as much water as possible from the sleeve. After 200 μL of sodium hydroxide was added thereto, the cell extract was transferred to a microtube and cultured at 60 ° C. for 2 hours. The protein solution of the cultured supernatant was recovered into a new microtube and used as a sample.

In addition, a BSA (bovine serum albumin) standard protein solution (protein concentration: 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, or 0.125 mg / mL) was prepared as the standard solution.

In the test tube, take 10 μL of each sample, 10 μL of each standard solution, or 10 μL of background solution, and add 100 μL of the protein quantification reagent (diquinolinecarboxylic acid reagent) and stir uniformly, and heat at 37 ° C for 30 minutes. After confirming that the temperature of the solution in the test tube was reduced to room temperature (25 ° C), the absorbance was measured using a spectrophotometer used in "2-1. Measurement of Absorbance and Photographing of Optical Microscope" in the second test example described above.

The absorbance of the background solution at a wavelength of 540 nm was set to 0.00. Next, the absorbance of each standard solution at a wavelength of 540 nm was measured to prepare a standard straight line. After measuring the absorbance of each sample at a wavelength of 540 nm, the protein concentration of each sample was determined using the standard straight line of the standard solution. The protein concentration of each sample is shown below.

Protein concentration (mg / mL) Control group (human serum albumin) 0.81 Sample 1 (albuminoid-1) 0.84 Sample 2 (albuminoid-2) 0.88 Sample 3 (albuminoid-3) ) 0.97 Sample 4 (Albumin-4) 0.95

Fourth test example (confirmation of effect of metal ion generation) 4-1. Detection of sulfane sulfur First, measurement samples 1a and 2a were prepared. Defatted human serum albumin (20 mg, 300 μM) was dissolved in a mixed solution of a phosphate buffer solution (pH 7.4) and ferric chloride (Fe ^{3 +} concentration: 1 mM) to prepare a measurement sample 1 a. Degreased human serum albumin (20mg, 300μM) was dissolved in phosphate buffer and copper (Cu ^{2 +} concentration: 1 mM) of the chloride mixture to prepare the measurement specimen 2a.

Next, measurement samples 1b and 2b were prepared. To a mixture of phosphate buffer solution (pH 7.4) and ferric chloride (Fe ^{3 +} concentration: 1 mM), add Na ₂ S (78 μg, 1 mM) to defatted human serum albumin (20 mg, 300 μM), and The mixture was heated to 37 ° C for 7 hours to add a sulfur atom to human serum albumin. About the product after the addition reaction, the gel was filtered under the same conditions as in Production Example 1 to remove unreacted sulfides to prepare a measurement sample 1b. Except phosphate buffer and copper chloride: a mixture of (Cu ^{2 +} concentration of 1 mM) is used instead of phosphate buffer (pH7.4) and ferric chloride (Fe ^{3 +} concentration: 1 mM) than a mixture, by The measurement sample 2b is prepared by the same method as that of the measurement sample 1b.

The measurement samples 1a and 2a and the measurement samples 1b and 2b were subjected to detection of sulfane sulfur by the same fluorescent probe as in Test Example 1 described above.

That is, the reaction between the measurement sample and the fluorescent indicator in which SSP2 (5 μM) was dissolved in dimethylarsine was carried out at room temperature (25 ° C.) for 10 minutes, and then the fluorescence intensity of the reactant was measured with a spectrofluorometer. The results are shown in Figure 4.

Fig. 4 is a bar graph showing the fluorescence intensities of the measurement samples 1a and 2a and the measurement samples 1b and 2b. In Fig. 4, the vertical axis represents the fluorescence intensity (unitless).

As shown in FIG. 4, the fluorescence intensity of the measurement sample 1b is higher than that of the measurement sample 1a, and the fluorescence intensity of the measurement sample 2b is higher than that of the measurement sample 2a. From this, it can be known that reacting human serum albumin with sodium sulfide in the presence of metal ions promotes the addition reaction of sulfur atoms to human serum albumin.

Further, the difference between the fluorescence intensity of the measurement sample 2b and the fluorescence intensity of the measurement sample 2a is much larger than the difference between the fluorescence intensity of the measurement sample 1b and the fluorescence intensity of the measurement sample 1a. It can be that, in particular based divalent abundance ratio Cu ^{2 +} Fe ^{3 +} trivalent much easier to promote the presence of an addition reaction of sulfur atom to a human serum albumin.

4-2. The measurement of the amount of melanin produced was performed by mixing hot spring water with a pH and components shown in Tables 1 and 2 described below in a proportion of 66.7% by mass to a mixed solution obtained with a phosphate buffer solution (pH 7.4). Serum albumin (20 mg, 300 μM) was added with Na ₂ S ₂ (110 μg, 1 mM) and heated to 37 ° C. for 7 hours to add sulfur atoms to human serum albumin. Regarding the product after the addition reaction, the gel was filtered under the same conditions as in Production Example 1 to remove unreacted sulfides, and albumin compounds-2A to 2J (10 to 18 mg) were obtained. Further, Table 1 and Table 2, the amount of each component other than containing radon hot spring water per 1kg (mg), containing the amount of radon hot spring water per 1kg (× ^{10 -} 10 Ci).

【Table 1】

【Table 2】

B16 mouse melanoma cells were seeded in a 24-well plate and pre-cultured with DMEM (+) at 37 ° C. and 5% CO ₂ for 24 hours to 2.5 × 10 ⁴ cells / well. Next, the pre-cultured medium was exchanged with DMEM (+) (albumin-based compound: 20 μM, tyrosine: 0.4 mM, NH ₄ Cl: 10 mM) to which any of the albumin-based compounds 2A to 2J was added. , And cultured at 37 ° C and 5% CO ₂ for 72 hours.

In addition, instead of DMEM (+) to which any of the albumin compounds-2A to 2J was added, those cultured in DMEM (+) to which the defatted human serum albumin used in Production Example 1 was added were prepared as a control group. . In addition, instead of DMEM (+) to which any of the albumin-based compounds-2A to 2J was added, they were also prepared together with those cultured in DMEM (+) to which the albumin-based compound-2 (made without hot spring water) was added.

Next, the cell lysate was recovered in the same manner as "2-2. Measurement of melanin production amount" in the second test example, and "2-1. Measurement of absorbance and photographing of an optical microscope photograph" were used in the second test example. The spectrophotometer was used to measure the absorbance of the cell lysate at a wavelength of 405 nm, and the amount of melanin produced in the melanoma cells of the B16 mouse after the culture was calculated. The results are shown in Figure 5.

FIG. 5 is a bar graph showing the amount of melanin production in B16 mouse melanoma cells cultured in each cell lysate. In Fig. 5, the vertical axis represents the absorbance (450 nm) in the cell lysate (1 mg / mL).

As shown in Fig. 5, even in the albumin-based compounds -2A to 2J, it is clear that not only the albumin-based compounds -2C to 2E and 2F to 2J produce melanin in comparison with the defatted human serum albumin as a control group. It is very small, and the amount of melanin produced is very small compared with the albumin compound-2 produced without using hot spring water. From this, it can be seen that human serum albumin and sodium sulfide are reacted in the presence of hot spring water containing various metal ions, sulfur atoms are more fully added, and albumin compounds having an excellent melanin production inhibitory effect are obtained.

Fifth Test Example (Confirmation of Safety) First, a whitening cosmetic material containing an albumin compound was prepared. Each component other than the following albumin-based compounds was prepared at the following blending amount (in terms of solid content), and then heated to 50 ° C. with stirring and mixing to make it uniform. After the mixture was cooled to room temperature (25 ° C) and the albumin-based compound was further mixed, cream A was obtained.

Ingredients blending amount (parts by mass) albumin compounds-2 0.133 emulsified wax 5 jojoba oil 15 净化 purified water residual amount (total) 100

Furthermore, cream B was obtained by the same method as preparation of cream A, except that albumin-based compound-2 D was blended in place of the above-mentioned albumin-based compound-2. In addition, the cream of the control group was obtained by the same method as that of the cream A except that the above-mentioned albumin-based compound-2 was not blended.

The cream A was evaluated based on the survival rate of three-dimensionally cultured skin cells in accordance with the method described in the Economic Development Cooperation Agency (OECD guidelines for testing of chemical substances) TG439 "in vitro skin irritation: recombinant human epidermal test". , Cream B and the safety of the control group. The results are shown in Figure 6.

Fig. 6 is a bar graph showing the cell survival rate in each cream. In Fig. 6, the vertical axis represents the cell survival rate (%). When the cell survival rate exceeds 50%, it is determined that there is no skin irritation.

As shown in FIG. 6, cream A and cream B had cell survival rates exceeding 50% in the same manner as the cream of the control group. From this, it can be known that neither the albumin-based compound-2 produced without hot spring water nor the albumin-based compound 2D produced in the presence of hot spring water has no skin irritation and high safety.

In addition, neither the albumin-based compound-2 nor the albumin-based compound-2D has a hydrogen sulfide odor, so there is no unpleasant odor. Therefore, neither Cream A doped with Albumin Compound-2 nor Cream B doped with Albumin Compound-2D did not have an unpleasant odor.

Sixth Test Example (Measurement of intracellular ROS and NO) The fluorescence reagents CM-H ₂ DCF-DA and CM-H ₂ DCF-DA were used to measure intracellular reactive oxygen species (ROS) and nitric oxide (NO). Fluorescence intensity.

B16 mouse melanoma cells were seeded in a 96-well plate, and pre-cultured with DMEM (+) at 37 ° C and 5% CO ₂ for 24 hours to 1 × 10 ⁴ cells / well.

Fluorescent reagent CM-H ₂ DCF-DA was added to the culture medium until the concentration reached 5 μM, and it was reacted at 37 ° C. for 30 minutes to take it into the cells. After the reaction, the supernatant (supernatant) was removed, and then albumin-based compound-4 was added. The same culture medium was prepared as a sample to which a phosphate buffer (pH 7.4) and defatted human serum albumin were added, and this was used as a control group. Each sample was irradiated with ultraviolet light having a wavelength of 254 nm for 10 minutes. Then, the excitation light having a wavelength of 485 nm was irradiated, and the fluorescence intensity at a wavelength of 535 nm was measured.

Then, the fluorescent reagent DAF-FM-DA was added to the culture medium until the concentration reached 10 μM, and the reaction was carried out at 37 ° C. for 30 minutes, and the cells were taken as a suspension sample for nitric oxide (NO) measurement. After the reaction, the medium was replaced with a base albumin-based compound-4 solution. In the same medium, a sample substituted with a phosphate buffer solution (pH 7.4) and a defatted human serum albumin solution was prepared as a control group. These samples were irradiated with ultraviolet light having a wavelength of 254 nm for 10 minutes. The obtained sample was irradiated with excitation light having a wavelength of 485 nm to measure the fluorescence intensity at a wavelength of 535 nm.

Figures 7 (a) and 7 (b) are bar graphs showing the average fluorescence intensity due to intracellular reactive oxygen species and nitric oxide, respectively. The lower the fluorescence intensity, the less active oxygen (ROS) and nitric oxide (NO) contained in the cell.

As shown in FIG. 7 (a), when using defatted human serum albumin, the fluorescence intensity based on active oxygen is also reduced compared to the case using a phosphate buffer. However, it can be seen that if the albumin-based compound-4 is used, the fluorescence intensity based on active oxygen is greatly reduced. Compared with the case of adding a phosphate buffer, the active oxygen is significantly reduced, Compared to defatted human serum albumin, there is also a decrease in active oxygen.

In addition, as shown in FIG. 7 (b), when using defatted human serum albumin, the fluorescence intensity based on nitric oxide was increased compared to the case using a phosphate buffer. However, it can be seen that if the albumin-based compound-4 is used, the fluorescence intensity based on nitric oxide is greatly reduced, and compared with the case where phosphate buffer solution and defatted human serum albumin are added, nitric oxide dramatically decrease.

FIG. 8 is a schematic diagram illustrating a mechanism for generating a melanin production mechanism. It is thought that the skin produces melanin as described below.

First, tyrosine in the skin is converted to duba pigment by the oxidative enzyme tyrosinase. Due to the action of tyrosinase, duba pigment is converted into duba tincture. Dopamine is highly reactive. Through various reactions in the body, melanin with a stable structure is eventually produced. Melanin is the cause of skin browning.

It is known that tyrosinase is activated by intracellular reactive oxygen species (ROS) and nitric oxide (NO). It is considered that although active oxygen and nitric oxide increase due to ultraviolet rays, as long as the active oxygen and nitric oxide can be reduced, the activation of tyrosinase can be suppressed, and the production of melanin can be suppressed.

As shown in Figures 7 (a) and 7 (b), it can be considered that the albumin compounds with added sulfur atoms inhibit the production of active oxygen and nitric oxide, thereby reducing the production of melanin and having whitening effect.

The whitening cosmetic material according to the present invention is suitable for use as a basic cosmetic or a cosmetic cosmetic.

FIG. 1 is a bar graph showing the absorbance of the cell lysate at a wavelength of 405 nm in the second test example. Figures 2 (a), 2 (b), and 2 (c) are optical microscope photographs of B16 mouse melanoma cells after culture in the second test example, respectively. Figure 3 is a bar graph showing the amount of melanin production in B16 mouse melanoma cells cultured in cell lysate in the second test example. Figure 4 is a bar graph showing the fluorescence intensity of the test sample in the fourth test example. Figure 5 is a bar graph showing the amount of melanin produced in the melanoma cells of the B16 mouse after culturing in the cell lysate in the fourth test example. Figure 6 is a bar graph showing the cell survival rate in the cream in the fifth test example. Figures 7 (a) and 7 (b) are bar graphs showing the average fluorescence intensity in the sixth test example, respectively. Figure 8 is a schematic diagram illustrating the inhibition of melanin by albumin compounds.

Claims (8)

An albumin-based compound for use in the manufacture of a skin-whitening external preparation, comprising: reacting a sulfide with albumin or an albumin derivative, and producing a sulfur atom added to the albumin or the albumin derivative A step of an albumin-based compound of the present invention, wherein the albumin-based compound has a plurality of sulfur atoms per position. The use of the albumin-based compound according to item 1 of the scope of the patent application for the manufacture of a skin-whitening external preparation, wherein the albumin is serum albumin. The use of the albumin-based compound described in item 1 of the scope of the patent application for the manufacture of a whitening skin external preparation, wherein the sulfide is sodium polysulfide. The use of the albumin-based compound according to item 3 of the scope of the patent application for the manufacture of a whitening skin external preparation, wherein the sodium polysulfide is Na ₂ S ₂ , Na ₂ S ₃ or Na ₂ S ₄ . The use of the albumin-based compound described in item 1 of the scope of the patent application for the manufacture of a whitening skin external preparation, wherein the manufacturing method is to derivatize the sulfide with the albumin or the albumin by heating. The product undergoes an addition reaction. The use of the albumin-based compound according to any one of claims 1 to 5 for the manufacture of a whitening skin external preparation, wherein the sulfide and the white are in the presence of metal ions. The protein or the albumin derivative undergoes an addition reaction. The use of the albumin-based compound according to item 6 of the application for manufacturing whitening skin external preparations, wherein the metal ion is a divalent metal ion. The use of the albumin-based compound according to item 6 of the scope of the patent application for the manufacture of a whitening skin external preparation, wherein the metal ion is derived from a hot spring component.