JPH0930953A - Dermal preparation for external use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a safe and stable dermal preparation for external use, capable of reducing the dark skin, dermal stains or ephelides of the skin or pigmentation due to sunburn, taking preventing and prophylactic measures for the aging of the skin based on the oxidizing reaction and having low irritation to the skin. SOLUTION: This dermal preparation for external use is obtained by blending 0.001-10wt.% ester of cystine selected from esters represented by the formula (R1 and R2 are each H, a 1-20C alkyl, an alkenyl or an alkylphenyl, with the proviso that R1 and R2 are not simultaneously H), phospholipid esters, esters of sphingosin and its derivative, glycolipid esters, sugar esters and sterol esters as an active ingredient. The cystine ester of the formula has whitening actions on melanin pigment and inhibiting actions on tryrosinase activities and tyrosinase biosynthesis and cystine is capable of sufficiently attaining melanocytes in the epidermis, dermis and a basal layer of the epidermis. The cystine is esterified to thereby improve both the solubility in solvents and the stability of a pharmaceutical preparation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is a blend of an ester of cystine to improve skin tone, dark spots, freckles,
Alternatively, the present invention relates to a skin external preparation that improves pigmentation due to sunburn, prevents or prevents skin aging due to oxidation reaction, has low skin irritation, and is safe and stable.

[0002]

2. Description of the Related Art Cystine is known as an amino acid which has a whitening effect by inhibiting tyrosinase activity and tyrosinase biosynthesis. It is also known that it has an antioxidant effect of capturing active oxygen species in the living body and prevents or prevents skin aging. Therefore, a skin external preparation containing cystine is expected as an effective preparation having both a whitening effect and an antiaging effect.

However, since cystine has a property of being poorly soluble in water and oil, it is difficult to mix it with a skin external preparation, and even if it is mixed in a solid state, it is difficult to be percutaneously absorbed. Was there. Therefore, in order to improve the solubility of cystine, it is known to use N, N'-diacetylcystine dimethyl as an external preparation for skin (JP-A-6-12).
8143). However, since this N, N'-diacetylcystine dimethyl has a water-soluble property, it has low transdermal permeability and cell membrane permeability, and in order to exert an effective whitening effect and antioxidant effect, It was necessary to blend a fairly high concentration into the external preparation base. Therefore, p of external preparation
H may change and affect the stability of the formulation,
It is difficult to obtain a preferable whitening agent and antiaging skin external preparation because of irritation to the skin.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems and is a safe and stable external preparation for skin having no skin irritation or side effects and exhibiting a very excellent whitening effect and antiaging effect. The purpose is to provide. That is,
It is possible to enhance the percutaneous and cell membrane permeability of cystine, which has a whitening effect and an anti-aging effect, so that it can effectively reach the basal layer of the epidermis.

[0005]

Therefore, in the present invention, in order to enhance the transdermal and cell membrane permeability of cystine, an attempt was made to use it by esterification. As a result, it was found that, by esterifying the carboxylic acid of cystine and mixing it, the percutaneous permeability of these is remarkably increased, and the tyrosinase activity or the biosynthesis inhibitory action and the antioxidant action in the same amount are surprisingly improved. It was Therefore, a small amount of the cystine ester may be added to the base of the external preparation for skin, and the influence on the formulation stability of the external preparation is small, and there is no safety problem such as skin irritation.

[0006] The ester of cystine is not particularly limited as long as it has a high cell membrane affinity, but it is represented by the general formula (1)

Preferable examples include the ester represented by [Chemical formula 1], phospholipid ester, sphingosine ester, glycolipid ester, sugar ester, and sterol ester.

Examples of the cystine ester represented by the above general formula (1) include straight chain aliphatic esters such as cystine octyl ester, branched aliphatic esters such as cystine isostearyl ester, and phenyl such as cystine phenylethyl ester. Examples thereof include unsaturated aliphatic esters such as alkyl ester and cystine oleyl ester.

Examples of the phospholipid used for the phospholipid ester include glycerophospholipids such as phosphatidic acid, lysophosphatidic acid, lecithin, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and phosphatidylcholine. A cystinyl group is introduced into the 1-position or 2-position of these glycerophospholipids by a transesterification reaction. Cystine can be efficiently ester-bonded to the 1-position or 2-position of the phospholipid by the transesterification reaction with phospholipase A1 or phospholipase A2.

Further, as the phospholipid used for the phospholipid ester, sphingolipids such as sphingomyelin, ceramide phosphoethanolamine and ceramide phosphoinositol can be used.

Esters of sphingosine and its derivatives are represented by the following general formula (2).

Embedded image

The glycolipid used for the glycolipid ester may be either a glyceroglycolipid or a glycosphingolipid. Examples of glyceroglycolipids include monoacylglycosylglycerol, diacylgalactosylglycerol, and glucosamylphosphatidylglycerol. Examples of glycosphingolipids include cerebrosides, sulfatides, ceramide oligohyxosides, globosides, and the like.
Gangliosides and the like.

The sugar used for the sugar ester is preferably a monosaccharide or an oligosaccharide having about 2 to 3 sugars from the viewpoint of cell membrane affinity. For the purposes of the present invention, erythrose,
Tetroses such as threose, pentoses such as arabinose, xylose and ribose; hexoses such as galactose, glucose, mannose, psicose and fructose; amino sugars such as N-acetylgalactosamine, N-acetylglucosamine and mannosamine; maltose;
Examples include disaccharides such as lactose, sucrose and cellobiose, and trisaccharides such as cellotriose.

The sugar ester of cystine can be synthesized by esterifying cystine, reacting this with sugar in the presence of an alkali catalyst such as sodium hydroxide, and transesterification. When the alcohol by-product is removed from the system, the reaction can proceed efficiently.

The sterol used for the sterol ester may be of animal origin, plant origin, or synthetic origin. Examples of such sterols include cholesterol, lanosterol, dihydrosterol, and mixtures thereof such as isocholesterol, phytosterol, sitosterol, stigmasterol, campesterol, desmosterol, 7-dehydrocholesterol, and reduced products of these sterols. it can.

The sterol ester of cystine can be carried out by a conventionally known esterification reaction. For example, a mixture of cystine and sterol of about 1 is obtained in the absence of a catalyst or in the presence of a catalyst such as paratoluenesulfonic acid or tin chloride.
It may be heated to about 00 to 250 ° C. The end point of this reaction can be known by measuring the acid value.

The above-mentioned cystine ester is blended in a usual skin external preparation base to prepare a skin external preparation. The blending amount is 0.001 to 1 considering the effective concentration in the preparation and the stability of the preparation.
About 10% by weight is suitable. As the form of the external preparation,
It can take various forms such as lotions, emulsions, creams and ointments. In addition, it can be provided as a cosmetic for aging prevention and whitening such as lotion, beauty essence, and emulsion.

[0017]

The melanin pigment whitening action, tyrosinase activity inhibiting action and tyrosinase biosynthesis inhibiting action of the cystine ester used in the present invention are shown below. Cystine esters include cystine-2-isopropyl ester as an alkyl ester, cystinyl phosphatidic acid having a cystinyl group introduced at the 2-position as a phospholipid ester, cystinyl glucoside as a sugar ester, and cystine cholesterol ester as a sterol ester. Was used. For comparison, cystine was measured for a similar effect.

For melanin pigment whitening action, cystine ester was dissolved in 95% by weight of ethanol to prepare a sample, which was added to a suspension of mouse B16 melanoma cells (cell number: about 50,000) and cultured for 3 days. Then separate the cells,
The color was measured and evaluated. The difference in L value was determined using a system treated in the same manner without the addition of cystine ester as a control and is shown in FIG. Compared to the system with cystine added, the difference in L value was 3 times or more in the system with cystine ester added.
The cell membrane permeability was improved and the melanin pigment whitening effect was remarkably increased.

The tyrosinase activity inhibitory action was obtained by dissolving cystine ester in 95% by weight ethanol to prepare a sample, and using tyrosinase (50,000 units, manufactured by Sigma) diluted 600 times with purified water as an enzyme solution, and using 50 mg of tyrosine. Was dissolved in 100 ml of purified water and evaluated as a substrate solution. The final concentration of cystine ester is 1.0μ
It was added in a stepwise manner in the range of M to 100 mM.

The enzyme reaction was carried out using a sample solution of 2 ml, 1 / 15M.
Phosphate buffer (pH 6.8) 2 ml, substrate solution 0.5 m
1 and 0.5 ml of the enzyme solution were mixed and incubated at 37 ° C. for 1 hour. After the reaction, the absorbance (As) at 475 nm was measured. Further, the same reaction was performed in a system in which purified water was added instead of the sample solution and in a system in which purified water was added instead of the substrate solution, and the absorbance (Ab and A0) in each case was measured. The tyrosinase activity inhibition rate was calculated by the mathematical formula 1.

[Equation 1]

The tyrosinase biosynthesis inhibitory effect was evaluated as follows. 95% by weight of the above cystine ester
The ethanol solution was added to a suspension of mouse B16 melanoma cells (the number of cells was about 50,000), the cells were cultured for 3 days, and the tyrosinase activity in the cells was measured by the following method. That is, 1 / 15M phosphate buffer (pH
6.8) 0.5 ml of a 1.0 wt% aqueous dopa solution in 2 ml
And 0.5 ml of the cultured cell solution were mixed and incubated at 37 ° C. for 1 hour, and then the absorbance at 405 nm (A
s) was measured. As a control, only a 95% by weight aqueous solution of ethanol was similarly added and cultured, and similarly the cultured cell solution and the dopa aqueous solution were incubated to obtain the absorbance (A
b) was measured, and the inhibition rate of tyrosinase biosynthesis was calculated by Formula 2. The final concentration of cystine ester is 1.0 μM
It was added in a stepwise manner in the range of ˜100 mM.

[Equation 2]

A dose response curve was prepared for each sample, and the 50% inhibitory concentration (ID50) was determined and summarized in Table 1. In Table 1, the ID50 for the tyrosinase activity inhibitory action and tyrosinase biosynthesis inhibitory action of cystine ester is 1/58 of cystine alone.
It becomes 1/27 and 1/63 to 1/24, and by esterifying cystine, when added at the same concentration, the tyrosinase activity inhibitory action and the tyrosinase biosynthesis inhibitory action are significantly improved.

[Table 1]

The cytotoxic protective action of cystine ester by active oxygen species can be evaluated by exposing the human fibroblasts to reactive oxygen species and using the cell survival rate at that time as an index. The cultured human fibroblasts were treated with cystine ester, washed, and exposed to reactive oxygen species (superoxide and hydroxyl radical) with a hypoxanthine-xanthine oxidase system. The cell survival rate after exposure was measured by the MTT reduction method.

As a result, as shown in FIG. 2, when treated with cystine ester having excellent cell membrane permeability,
It showed a good cell viability of 80%. On the other hand, when treated with non-esterified cystine, cell viability was low and cell viability was as low as less than 40%.

[0025]

The present invention will be described in more detail with reference to Examples.

The formulations of Examples 1 to 4 and Comparative Example 1 as the essence oil according to the present invention are shown in Table 2. (1)-(8)
Is heated and mixed at 70 ° C., and then cooled to room temperature to prepare.

[Table 2]

Table 3 shows the formulations of Examples 5 to 8 and Comparative 2 as the beauty essence according to the present invention. Add the ingredients from (4) to (9) to (3)
After homogenizing by dissolving in (1), (1), (2) and (10) are sequentially added to prepare.

[Table 3]

Examples 9 to 12 as emulsions according to the present invention
The formulations of Comparative Example 3 are shown in Table 4. The oil phase components (1) to (5) are mixed and heated to 75 ° C. to dissolve and homogenize. Meanwhile (6),
The aqueous phase components (13) and (17) are mixed and dissolved, and the mixture is heated to 75 ° C., and the above oil phase component is added to carry out preliminary emulsification. After adding (14), emulsify uniformly with a homomixer, and add (15) to adjust pH. After cooling, the components (8) to (11) are dissolved in (7), added, mixed and homogenized at 40 ° C.

[Table 4]

Table 5 shows the formulations of Examples 13 to 16 and Comparative Example 4 as the W / O emulsion type cream according to the present invention.
The oil phase components (1) to (8) are mixed and heated to 75 ° C. to dissolve and homogenize. On the other hand, the aqueous phase components (9) to (11) are mixed and dissolved, heated to 75 ° C., added to the above oil phase components, and uniformly emulsified by a homomixer. After cooling at 40 ℃, (13) ~ (17)
Dissolve the ingredients in (12) and add, mix, and homogenize.

[Table 5]

Table 6 shows the formulations of Examples 17 to 20 and Comparative Example 5 as the O / W type emulsion ointment according to the present invention.
The oil phase components (1) to (4) are mixed and heated to 75 ° C to dissolve and homogenize. The oil phase component is added to (5) heated to 75 ° C. and emulsified. After cooling, the components (7) to (11) are dissolved in (6), added, mixed and homogenized at 40 ° C.

[Table 6]

For the examples of the present invention, the stability of the formulation was 2
It was observed in a 5 ° C constant temperature bath. The state after 1 month, 3 months, and 6 months was evaluated by the presence or absence of separation and crystal precipitation. The results are shown in Table 7 when the separation and the precipitation of the crystals were not observed, and when the separation and the precipitation of the crystals were observed. Precipitation of crystals was already observed after 1 month in Comparative Examples 2 and 3 which were cosmetic liquids and emulsions, and separation and precipitation of crystals were also observed after 3 months in Comparative Examples 1, 4 and 5. However, in the examples, there was no sample in which separation and crystals were precipitated even after 6 months, and it was clear that the stability of the preparation was improved.

[Table 7]

With respect to the examples of the present invention, the anti-aging effect on the skin was examined by evaluating the anti-wrinkle effect on the skin. The samples subjected to the test are described in Examples 1, 2, 3,
4, 5, 10, 15, 20 and Comparative Examples 1-5. Five hairless mice were made into one group, and the examples and comparative examples of the present invention were applied to the back once a day for each group.
UVA was irradiated for 50 weeks / square cm / week for 50 weeks, and the occurrence of wrinkles was evaluated by visual observation over time. The wrinkle occurrence status is “no occurrence; 0 point”, “slight wrinkle slightly occurs; 1 point”, “minor wrinkle clearly occurs; 2 points”, “medium wrinkle occurs; 3 points” , "Deep wrinkles occur; 4 points"
Was scored, and the average score of each group was calculated and shown in Table 8. At that time, a group to which purified water was applied was used as a control.

[Table 8]

Table 8 shows that the wrinkles were remarkably suppressed in each of the application groups to which the cystine ester was added, and even after irradiation with UVA for 50 weeks, the formation of minute wrinkles was observed in each group. Was only admitted.
On the other hand, in Comparative Examples 1 to 5 containing cystine, a slight wrinkle suppressing effect was recognized as compared with the control, but clear wrinkle formation was observed in all, and the formed wrinkles were almost moderate. Had reached the depth of.

Subsequently, a usage test was conducted on the examples and comparative examples of the present invention. The tested samples are Examples 1, 2, 3, 4, 6, 11, 16, 17 and Comparative Examples 1 to 1.
5 A group consisting of 20 panelists, whose main symptoms were decreased skin elasticity and pigmentation such as spots, freckles, and sunburn, were allowed to use the examples and comparative examples on the face and hands blindly for each group. And the change of pigmentation was observed and evaluated. The period of use was 6 months from April to October. For skin elasticity, "rise",
Each grade was evaluated with 4 grades of "slightly increased", "no change" and "decrease", and with respect to the whitening effect, 3 grades of "improved", "slightly improved" and "no change". The number of panelists is shown in Table 9.

[Table 9]

As is clear from Table 9, all the panelists using the examples containing cystine ester were found to have an increase in skin elasticity and an improvement in pigmentation. On the other hand, among the panelists using Comparative Examples 1 to 5, there are some panelists whose elasticity was slightly increased, but no panelists were found to have a clear increase in elasticity, and no panelists were found to have a clear improvement in whitening effect. No change was observed in 35 to 50% of panelists. From the above results, it can be seen that by esterifying cystine and blending it, the antiaging effect and the whitening effect are dramatically improved as compared with the case where cystine is blended.

No panelists complained of skin irritation such as pain and itching, or skin symptoms such as allergic reaction in any of the groups used in the above-mentioned use period. In addition, no deterioration of the emulsified state, no sedimentation and deterioration of the components were observed.

[0037]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, by adding a small amount of cystine ester, it is possible to provide a skin external preparation which exhibits a very excellent whitening effect and antiaging effect. In the external preparation for skin according to the present invention, cystine, which is an active ingredient, can successfully reach melanocytes in the epidermis, dermis and basal layer of epidermis, and exhibits excellent anti-aging effect and whitening effect at a low concentration. Therefore, there is no possibility that skin irritation or sensitization is exhibited. Also, by esterifying cystine, the solubility in the solvent is improved,
The stability of the formulation was improved.

[Brief description of drawings]

FIG. 1 is a view showing a cystine ester used in the present invention and a melanin pigment whitening action of cystine.

FIG. 2 is a diagram showing cystine ester used in the present invention and a cytotoxic protective action of cystine by reactive oxygen species.

[Explanation of symbols]

 1. Cystine-2-isopropyl ester 2. Cysteinyl phosphatidic acid 3. Cystinyl glucoside 4. Cystine cholesterol ester 5. Cystine

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication location A61K 7/00 A61K 7/00 W 31/22 ADA 31/22 ADA 31/66 31/66 31/70 31 / 70

Claims (3)

[Claims] 1. An external preparation for skin, which comprises an ester of cystine. 2. An ester of cystine has the general formula (1): 2. One or more selected from the group consisting of ester, phospholipid ester, ester of sphingosine and its derivative, glycolipid ester, sugar ester, and sterol ester represented by: External skin preparation. 3. The external preparation for skin according to claim 1, wherein the external preparation for skin is a cosmetic.