Tyrosinase activity inhibitor and external preparation for skin

Abstract

A tyrosinase activity inhibitor is prepared by combining (A) a metal (zinc, cobalt or iron) chelate compound of -lipoyl amino acid or its pharmaceutically acceptable salt with (B-1) a human manganese SOD or (B-2) an ascorbic acid compound such as ascorbic acid and ascorbyl glucoside. A ratio (R1) of the (B-1) component content to the (A) component content is preferably 0.001 to 1,500, when R1 is defined as R1=[Unit concentration of (B-1) component (unit/mL)/Concentration of (A) component (g/mL)], and a ratio (R2) of the (A) component content to the (B-2) component content is preferably 0.0001 to 1,000, when R2 is defined as R2=[Concentration of (A) component (m/mL)/Concentration of (B-1) component (mg/mL)]. An external preparation for skin contains the (A) component and the (B-1) component or the (B-2) component as effective ingredients for inhibiting tyrosinase activity.

Claims

1. A tyrosinase activity inhibitor comprising a combination of (A) histidine dithiooctanamide (Na/zinc) ##STR00003## and (B-2) at least one of ascorbyl glucoside and salts thereof, wherein, when the tyrosinase activity inhibitor is a combination of said (A) component and said (B-2) component, the tyrosinase activity inhibitor has a ratio (R2) of the (A) component content to the (B-2) component content calculated based on the following formula being from 0.05 to 2.15
R2=[Concentration of (A) component (g/mL)/Concentration of (B-2) component (mg/mL)].

2. An external preparation for skin comprising the tyrosinase activity inhibitor according to claim 1.

3. The external preparation for skin according to claim 2, wherein the external preparation for skin is a cosmetic.

4. The external preparation for skin according to claim 2, wherein a content of the (A) component is 110.sup.5 to 1% by mass, and a content of the (B-2) component is 0.01 to 3.0% by mass.

5. The external preparation for skin according to claim 2, wherein a dosage form is selected from solid, paste, mousse, gel, powder, solution, micro-emulsion, emulsion, powder dispersion and multilayer.

6. The external preparation for skin according to claim 5, wherein the dosage form is an emulsion.

7. The external preparation for skin according to claim 6, wherein the emulsion is O/W (oil-in-water type), W/O (water-in-oil type), W/O/W (water-in-oil-in-water type) or O/W/O (oil-in-water-in-oil type).

Description

EXAMPLE

(1) Hereinafter, the present invention will be further described with reference to specific examples. However, the present invention is not limited by these examples. The content of each ingredient is expressed in % by mass unless otherwise specified.

(2) <Test for Evaluating Inhibition of Tyrosinase Activity>

(3) A test solution having a volume of 2.0 mL was prepared by adding 0.1 mL of tyrosinase (derived from mushrooms, manufactured by Sigma, 100 units/mL) to a sample for evaluation, and then 0.1 M phosphate buffer solution having a pH of 6.5 was added. The test solution was incubated in a thermostat at 37 C. for 10 minutes, and then 1 mL of DOPA (3,4-dihydroxy-L-phenylalanine, 0.03% solution) was added. After 2 minutes of the addition, the test solution was subjected to measurement of absorbance at 475 nm.

(4) Thus-obtained absorbance value was defined as D1. As a control, the same measurement was conducted on a test solution in which each sample was replaced with purified water, and the obtained absorbance value was defined as D2. An inhibition rate of tyrosinase activity was calculated by the following formula.
Inhibition rate of tyrosinase activity (%)=(D2D1)/D2100

Reference Example 1

(5) Test for Evaluating Inhibition Ability of Tyrosinase Activity on Histidine Dithiooctanamide (Na/Zinc)

(6) As an -lipoylamino acid derivative for evaluation, histidine dithiooctanamide (Na/zinc) was used which is commercially available from Oga Research Co., Ltd. as DM-His. Zn and has a molecular weight of 430.84. Test solutions containing histidine dithiooctanamide (Na/zinc) in a content of 1.08 g/mL (2.5 M), 2.15 g/mL (5 M), 3.23 g/mL (7.5 M) or 4.31 g/mL (10 M) were prepared, and the inhibition rate of tyrosinase activity on each test solution was measured according to the above test method. The results are as follows.

(7) TABLE-US-00001 Concentration of histidine Inhibition rate of dithiooctanamide (Na/zinc) tyrosinase activity (%) 1.08 g/mL 13.2 2.15 g/mL 25.3 3.23 g/mL 53.6 4.31 g/mL 68.9

(8) These results indicate that the inhibition rate of tyrosinase activity increases with increase of the content of histidine dithiooctanamide (Na/zinc). Based on the data, histidine dithiooctanamide (Na/zinc) was confirmed to be effective to inhibit tyrosinase activity. However, the ability to inhibit tyrosinase activity was not yet sufficient.

Reference Example 2

(9) Test for Evaluating Inhibition Ability of Tyrosinase Activity on Human Manganese SOD

(10) As a human manganese SOD for evaluation, yeast extract (SOD) was used which is commercially available from Biox Technologies, and has a concentration of 3,000 units/mL. Test solutions containing human manganese SOD in a content of 3 units/mL, 15 units/mL or 150 units/mL were prepared, and the inhibition rate of tyrosinase activity on each test solution was measured according to the above test method. The results are as follows.

(11) TABLE-US-00002 Concentration of human Inhibition rate of tyrosinase manganese SOD activity (%) 3 units/mL 4.53 15 units/mL 3.74 150 units/mL 4.66

(12) These results indicate that the inhibitory effect of tyrosinase activity does not increase even when the concentration of human manganese SOD is increased, and sufficient properties to inhibit tyrosinase activity cannot be expected when human manganese SOD is used solely.

Reference Example 3

(13) Test for Evaluating Inhibition Ability of Tyrosinase Activity on Ascorbyl Glucoside

(14) As an ascorbic acid derivative for evaluation, ascorbyl glucoside AS-G commercially available from Hayashibara Co., Ltd. was used. Test solutions containing ascorbyl glucoside in a content of 5 mg/mL, 10 mg/mL or 20 mg/mL were prepared, and the inhibition rate of tyrosinase activity on each test solution was measured according to the above test method. The results are as follows.

(15) TABLE-US-00003 Concentration of Inhibition rate of tyrosinase ascorbyl glucoside activity (%) 5 mg/mL 8.4 10 mg/mL 15.4 20 mg/mL 26.8

(16) These results indicate that, when ascorbyl glucoside is used solely, the inhibition rate of tyrosinase activity increases with increase of the concentration of ascorbyl glucoside, but the inhibitory effect of tyrosinase activity is insufficient as compared with histidine dithiooctanamide (Na/zinc).

Example 1

(17) Test solutions were prepared according to Reference Example 1 and Reference Example 2 except that histidine dithioctanamide (Na/zinc) used in Reference Example 1 and human manganese SOD used in Reference Example 2 were used in combination so as to have proportions shown in Table 1, and the inhibition rate of tyrosinase activity was measured according to the above test method. Histidine dithioctanamide (Na/zinc) and human manganese SOD are respectively referred to as (A) component and (B-1) component in Table 1. Table 1 shows an inhibition rate of tyrosinase activity of each test solution as well as a degree of improvement as compared with a test solution containing histidine dithioctanamide (Na/zinc) solely. Table 1 also shows the results obtained in Reference Examples 1 and 2.

(18) TABLE-US-00004 TABLE 1 (B-1) component (unit/mL) Composition 0 3 15 150 (A) component (g/mL I II III I II III I II III I II III 0.0 4.5 3.7 4.7 1.08 0 13.2 1.00 2.78 22.0 1.67 13.89 20.6 1.56 138.90 16.0 1.21 2.15 0 25.3 1.00 1.40 43.3 1.71 6.98 65.7 2.60 69.80 61.1 2.42 3.23 0 53.6 1.00 0.93 72.7 1.36 4.64 90.7 1.69 46.40 78.1 1.46 4.31 0 68.9 1.00 0.70 93.3 1.35 3.48 95.9 1.39 34.80 97.3 1.41 I R1: Ratio of content of (B) component to content of (A) component II Inhibition rate of tyrosinase activity (%) III Inhibition rate of tyrosinase activity in the case of conbining human manganese SOD/Inhibition rate of tyrosinase activity in the case without addition of human manganese SOD

(19) As seen from these results, the ability to inhibit the tyrosinase activity of histidine dithioctanamide (Na/zinc) is remarkably improved by using histidine dithiooctanamide (Na/zinc) and human manganese SOD in combination.

Comparative Example 1

(20) An ability to inhibit tyrosinase activity was measured in the same manner as Example 1 except that human manganese SOD was replaced by 0.5% by volume aqueous solution of copper/zinc SOD (1), DISMUTIN BTJ, which is available from DSM Nutrition and has a concentration of 50,000 PIU/mL. The aqueous solution copper/zinc SOD (1) had a concentration of 250 PIU/mL. The concentration of histidine dithiooctanamide (Na/zinc) was 4.31 g/mL. PIU indicates an enzyme unit measured by the pyrogallol autoxidation method.

(21) As a result, the inhibition rate of tyrosinase activity was 38% that is between 16.1% in the case of using copper/zinc SOD (1) solely and 68.9% in the case of using histidine dithioctanamide (Na/zinc) solely. As seen from the data, copper/zinc SOD (1) has no function to improve the ability to inhibit tyrosinase activity of histidine dithiooctanamide (Na/zinc).

Comparative Example 2

(22) An ability to inhibit tyrosinase activity was measured in the same manner as Comparative Example 1 except that copper/zinc SOD (1) was replaced by 0.5% volume aqueous solution of another copper/zinc SOD (2), SOD yeast extract that is available from Aria Co., Ltd. The inhibition rate of tyrosinase activity was 75.9% that is higher than 68.9% in the case of using histidine dithioctanamide (Na/zinc) solely, but does not reach 95.9% obtained in the present invention at all.

Comparative Example 3

(23) Histidine dithiooctanamide (Na/zinc) was replaced by kojic acid, ascorbic acid derivative (ascorbyl glucoside) or arbutin which are known as a compound having the ability to inhibit tyrosinase activity, and an ability to inhibit tyrosinase activity in combination of each compound and histidine dithiooctanamide (Na/zinc) was evaluated according to Example 1. Human manganese SOD was added to become 150 units/mL. As to the other compounds, kojic acid, ascorbic acid derivative (ascorbyl glucoside) and arbutin were added to be 1.2% by mass, 2.0% by mass and 3.0% by mass, respectively.

(24) The inhibition rate of tyrosinase activity in the case of using kojic acid solely was 45.6%, and one in the case of using kojic acid in combination with human manganese SOD was 47.4%. As seen from the comparison, there was hardly any effect due to the combination of both components. The inhibition rate of tyrosinase activity in the case of using ascorbic acid derivative solely was 21.9%, and one in the case of using ascorbic acid derivative in combination with human manganese SOD was 25.6%. As seen from the comparison, there was very limited improvement due to the combination of both components. The inhibition rate of tyrosinase activity in the case of using arbutin solely was 71.3%, and one in the case of using arbutin in combination with human manganese SOD was 69.7%. As seen from the comparison, there was no effect due to the combination of both components.

(25) From the results of Example 1 and Comparative Examples 1 to 3 as described above, it can be understood that (1) the inhibition rate of tyrosinase activity is greatly improved only when using -lipoyl amino acid derivative and human manganese SOD in combination thereof, and (2) the inhibition rate of tyrosinase activity cannot be improved when using, in place of -lipoyl amino acid derivative, other compounds having the ability to inhibit tyrosinase activity in combination with human manganese SOD.

Example 2

(26) <Preparation of Lotion>

(27) A lotion was prepared according to the formulation and procedure indicated below.

(28) Procedure:

(29) To purified water, a moisturizer, an anti-fading agent, a buffer, histidine dithiooctanamide (Na/zinc) and human manganese SOD were added, and they were dissolved at room temperature to obtain a water phase. On the other hand, an alcoholic phase was obtained by adding an emollient agent, a surfactant, a preservative and a fragrance to ethanol at room temperature. The resultant alcoholic phase is added to the water phase.

(30) Formulation (% by Mass):

(31) TABLE-US-00005 Humectant: 1,3-Butylene glycol 6.0 Glycerol 5.0 PEG 4000 3.0 Emollient agent: olive oil 0.5 Surfactant: POE (20) sorbitan monostearate 1.5 POE (5) oleyl alcohol ether 0.3 Ethanol: 10.0 Perfume: Appropriate amount Coloring agent: Appropriate amount Preservative: Appropriate amount Buffer: Appropriate amount Anti-fading agent: Appropriate amount Histidine dithiooctanamide (Na/zinc) *1 0.5 Human manganese SOD *2 1.0 Purified water: Balance *1: DM-His. Zn (available from Oga Research Co., Ltd.) *2: Yeast extract (SOD) (available from Biox Technology Co., Ltd.)

Example 3

(32) <Preparation of Oil-in-Water Emulsion Cream>

(33) A cream was prepared according to the formulation and procedure indicated below.

(34) Procedure:

(35) A water phase was prepared by adding a moisturizer and an alkali to purified water and heating the mixture to 70 C. After heating and melting an oil component, a surfactant, a preservative and an antioxidant were added to the oil component and the mixture was adjusted to 70 C. to obtain an oil phase. The oil phase was added to the water phase, and resultant mixture was homogenized using a homomixer, and then cooled. Finally, histidine dithiooctanamide (Na/zinc), human manganese SOD and a fragrance dissolved in a small amount of purified water were added to the mixture, mixed and degassed.

(36) Formulation (% by Mass):

(37) TABLE-US-00006 Oil component: Stearic acid 8.0 Stearyl alcohol 4.0 Butyl stearate 6.0 Humectant: Propylene glycol 5.0 Surfactant: Glyceryl monostearate 2.0 Alkali: Potassium hydroxide 0.4 Preservative: Appropriate amount Antioxidant: Appropriate amount Fragrance: Appropriate amount Histidine dithiooctanamide (Na/zinc) *1 0.1 Human manganese SOD *2 0.1 Purified water: Balance

Example 4

(38) <Preparation of W/O/W (Water-in-Oil-in Water) Type Cream>

(39) A cream was prepared according to the formulation and procedure indicated below.

(40) Procedure:

(41) (1) Step for Preparing an Intermediate Water-in-Oil Type Emulsion

(42) A water phase was prepared by adding a moisturizer, sodium chloride and histidine dithiooctanamide (Na/zinc) to purified water and mixing at 50 C. After heating and melting an oil component, a surfactant was added to the oil component and then the mixture was adjusted to 50 C. After addition of the water phase to the mixture, the resultant mixture was homogenized using Disper mixer, and then cooled to obtain an intermediate water-in-oil type emulsion.

(43) (2) Step for Preparing a Water-in-Oil-in Water Type Cream Using the Intermediate Water-in-Oil Type Emulsion

(44) A water phase was prepared by adding a moisturizer to purified water and heating the mixture to 75 C. After heating and melting an oil component, the intermediate water-in-oil type emulsion prepared in Step (1), a surfactant, a preservative and an antioxidant were added to the oil component and the mixture is adjusted to 75 C. The mixture was added to the water phase, and the resultant mixture was homogenized using Disper mixer. Finally, human manganese SOD and a fragrance dissolved in a small amount of purified water were added to the mixture, mixed and degassed.

(45) Formulation (% by Mass):

(46) TABLE-US-00007 (1) Intermediate water-in-oil emulsion Oil Squalane 0.8 component: Ethylhexyl palmitate 0.5 Surfactant: Hexaglyceryl polyricinoleate 0.15 Diglyceryl monoisostearate 0.05 Humectant: Glycerin 5.0 Sorbitol 0.05 Purified water: 6.0 Sodium chloride 0.2 Histidine dithiooctanamide (Na/zinc) *1 0.1 (2) Water-in-oil-in water type cream Oil component: Squalane 5.0 Ethylhexyl palmitate 2.0 Microcrystalline wax 2.0 Petrolatum 2.0 Behenyl alcohol 3.0 Intermediate water-in-oil emulsion 12.85 Surfactant: Glyceryl monostearate 2.0 Sodium stearoyl glutamate 0.5 Humectant: 1,3-Butylene glycol 5.0 Preservative: Appropriate amount Antioxidant: Appropriate amount Fragrance: Appropriate amount Human manganese SOD *2 0.1 Purified water: Balance

Example 5

(47) <Preparation of Cleansing Foam>

(48) A cleansing foam was prepared according to the formulation and procedure indicated below.

(49) Procedure:

(50) An oil phase was prepared by heating and melting a mixture of a fatty acid, an emollient agent and a humectant at 70 C. After dissolving an alkali in purified water, the oil phase was added to the alkaline aqueous solution with stirring. The resultant mixture was sufficiently neutralized, and then a surfactant was added to the mixture. After conducting a mixing, the mixture was cooled. To the mixture, a chelating agent, a fragrance, histidine dithiooctanamide (Na/zinc) dissolved in a small amount of purified water and human manganese SOD were added, and then the resultant mixture was homogenized, cooled and defoamed.

(51) Formulation (% by Mass):

(52) TABLE-US-00008 Oil component: Stearic acid 12.0 Mirystic acid 14.0 Lauric acid 5.0 Emollient agent: Jojoba oil 3.0 Alkali: Potassium hydroxide 5.0 Humectant: 70% Sorbitol aqueous solution 15.0 Glycerin 10.0 1,3-Butylene glycol 10.0 Surfactant: PEG (20) glyceryl monostearate 2.0 Acyl methyl taurine 4.0 Chelating agent: Appropriate amount Fragrance: Appropriate amount Histidine dithiooctanamide (Na/zinc) *1: 0.5 Human manganese SOD *2: 0.5 Purified water: Balance

Example 6

(53) <Preparation of Liquid Foundation>

(54) A liquid foundation was prepared according to the formulation and procedure indicated below.

(55) Procedure:

(56) The powder ingredients shown in the following formulation were thoroughly mixed and pulverized, and then added to the oil phase shown in the formulation. Next, the oil phase was homogenized using a homomixer, and then the water phase shown in the formulation was added to the oil phase, and the resultant mixture was treated with a homomixer. Subsequently, histidine dithiooctanamide (Na/zinc) dissolved in a small amount of purified water and human manganese SOD were added to the mixture, and the resultant mixture was homogenized and defoamed.

(57) Formulation (% by Mass):

(58) TABLE-US-00009 Powder: Hydrophobized talc 7.0 Hydrophobized titanium dioxide 12.0 Hydrophobized anhydrous silicic acid 2.0 Nylon powder 4.0 Hydrophobize color pigment 2.0 Oil phase: Decamethylcyclopentasiloxane 30.0 Rosin pentaerythritol ester 1.5 Polyoxyethylene-modified 1.5 dimethylpolysiloxane Water phase: Purified water: just Balance 1,3-Butylene glycol 4.0 Ethanol 7.0 Histidine dithiooctanamide (Na/zinc) *1: 0.1 Human manganese SOD*2: 1.0

Example 7

(59) <Preparation of Sunscreen Milky Lotion>

(60) A sunscreen milky lotion was prepared according to the formulation and procedure indicated below.

(61) Procedure:

(62) Each of the oil phase and the water phase shown in the following formulation was heated to 70 C. After titanium dioxide was sufficiently dispersed in the oil phase, the water phase was added to the oil phase while performing a treatment using a homogenizer. After completion of homogenization, the mixture was cooled. Next, histidine dithiooctanamide (Na/zinc) dissolved in a small amount of purified water and human manganese SOD were added to the mixture, and the resultant mixture was homogenized and defoamed.

(63) Formulation (% by Mass):

(64) TABLE-US-00010 Water phase: Purified water: Balance 1,3-Butylene glycol 5.0 Oil phase: Octyl p-methoxycinnamate 5.0 Oxybenzone 3.0 4-tert-butyl-4-methoxydibenzoylmethane 1.0 Hydrophobized titanium dioxide 3.0 Squalane 20.0 Decamethylcyclopentasiloxane 20.0 Polyoxyethylene-modified 3.0 dimethylpolysiloxane Organo-modified montmorillonite 1.5 Preservative: Appropriate amount Fragrance: Appropriate amount Histidine dithiooctanamide (Na/zinc) *1: 1.0 Human manganese SOD *2: 1.0

Example 8

(65) <Evaluation Test for Inhibition of Tyrosinase Activity in Combination of Histidine Dithiooctanamide (Na/Zinc) and Ascorbyl Glucoside>

(66) Test solutions were prepared according to Reference Example 1 and Reference Example 3 except that histidine dithioctanamide (Na/zinc) used in Reference Example 1 and ascorbyl glucoside used in Reference Example 3 were used in combination so as to have proportions shown in Table 2, and the inhibition rate of tyrosinase activity was measured according to the above test method. Histidine dithioctanamide (Na/zinc) and ascorbyl glucoside are respectively referred to as (A) component and (B-2) component in Table 2. Table 2 shows an inhibition rate of tyrosinase activity of each test solution as well as a degree of improvement as compared with a test solution containing histidine dithioctanamide (Na/zinc) solely. Table 2 also shows the results obtained in Reference Examples 1 and 3.

(67) TABLE-US-00011 TABLE 2 (B-2) component (mg/mL) Coponent 0 5.0 10.0 20.0 (A) component (g/mL) I II III I II III I(R) II III I(R) II III 0 0 8.4 0 15.4 0 26.8 1.08 13.2 1.00 0.22 31.9 1.48 0.11 45.5 1.59 0.05 85.7 2.14 2.15 25.3 1.00 0.43 89.2 2.65 0.22 92.5 2.27 0.11 100.0 1.92 I R2: Ratio of content of (A) component to content of (B) component II Inhibition rate of tyrosinase activity (%) III Tyrosinase activity inhibition rate in the case of using both components/Sum of tyrosinase activity inhibition rate in the case of using each component alone

(68) As seen from these results, the combination of histidine dithiooctanamide (Na/zinc) and ascorbyl glucoside results in a remarkable improvement of the ability to inhibit tyrosinase activity as compared with the case of using each compound solely. For example, when using 2.15 g/mL of histidine dithiooctanamide (Na/zinc) or 20.0 mg/mL of ascorbyl glucoside, the inhibition rate of tyrosinase activity was 25.3% or 26.8%, respectively, while, when using both compounds in combination, the inhibition rate of tyrosinase activity was 100%. This value corresponds to 1.92 times the sum of inhibition rates of tyrosinase activity (i.e. 52.1%) when each of both compounds is used solely.

Examples 9 to 11 and Comparative Examples 4 to 5

(69) <Evaluation Test for Inhibition of Tyrosinase Activity in Combination of Histidine Dithiooctanamide (Na/Zinc) and Ascorbic Acid Derivatives>

(70) An ability to inhibit tyrosinase activity was measured in the same manner as Example 8 except that ascorbyl glucoside used as (B-2) component was replaced by magnesium ascorbyl phosphate (5 mg/mL), sodium ascorbyl phosphate (5 mg/mL) or ascorbic acid (0.01 mg/mL). The concentration of histidine dithiooctanamide (Na/zinc) was 2.15 g/mL. For comparison, compositions containing 3-O-ethylascorbic acid (2.5 mg/mL) or di-sodium ascorbyl sulfate (2.5 mg/mL) were also tested in the same manner. Table 3 shows an inhibition rate of tyrosinase activity of each test solution as well as a degree of improvement as compared with a case using each compound solely. Table 3 also shows the results obtained in Reference Examples 1.

(71) TABLE-US-00012 TABLE 3 (A) component: 2.15 g/mL II (%) I (B) component Combination of (B-2) component (R2) only (A) and (B) III Reference 25.3 1.00 Example 1 Example 9 Magnesium ascorbyl phosphate 0.43 11.1 89.1 2.45 Example 10 Sodium ascorbyl phosphate 0.43 15.6 93.4 2.28 Example 11 Ascorbic acid 215 28.7 100.0 1.85 Comparative 3-O-ethylascorbic acid 0.86 29.9 63.0 1.14 Exmple 4 Comparative Disodium ascorbyl sulfate 0.86 40.5 40.8 0.62 Exmple 5 R2: Ratio of amount of (A) component to amount of (B-2) component Inhibition rate of tyrosinase activity (%) Tyrosinase activity inhibition rate in the case of using both components/Sum of tyrosinase activity inhibition rate in the case of using each component alone

(72) As seen from these results, the ability to inhibit the tyrosinase activity of histidine dithioctanamide (Na/zinc) is remarkably improved by using histidine dithiooctanamide (Na/zinc) and ascorbic acid, magnesium ascorbyl phosphate or sodium ascorbyl phosphate in combination as compared with a case using each compound solely. On the other hand, when 3-O-ethylascorbic acid or disodium ascorbyl sulfate is used, sufficient ability to inhibit tyrosinase activity could not be obtained even when histidine dithiooctanamide (Na/zinc) is used in combination.

Comparative Example 6

(73) An ability to inhibit tyrosinase activity was measured in the same manner as Example 8 except that ascorbyl glucoside was replaced by an aqueous solution of pyridoxine hydrochloride (vitamin B6) having a concentration of 5 mg/mL. The concentration of histidine dithiooctanamide (Na/zinc) was 2.15 g/mL. Results are shown in Table 4.

(74) TABLE-US-00013 TABLE 4 Pyridoxine hydrochloride (mg/mL) Composition 0 5.0 (g/mL) II III II III (A) 0.0 42.9 component 2.15 25.3 1.00 51.1 0.75 II Inhibition rate of tyrosinase activity (%) III Tyrosinase activity inhibition rate in the case of using both components/Sum of tyrosinase activity inhibition rate in the case of using each component alone

(75) As seen from the results, even when histidine dithiooctanamide (Na/zinc) and pyridoxine hydrochloride are used in combination, there is hardly improvement on the ability to inhibit tyrosinase activity as compared with the case using each compound solely.

Comparative Example 7

(76) An ability to inhibit tyrosinase activity was measured in the same manner as Example 8 except that histidine dithiooctanamide (Na/zinc) was replaced by an aqueous solution of -lipoic acid (available from Tateyama Kasei) having a concentration of 4.31 g/mL. The concentration of ascorbyl glucoside was 5 mg/mL. Results are shown in Table 5.

(77) TABLE-US-00014 TABLE 5 Ascorbyl glycoside (mg/mL) Composition 0 5.0 -lipoic acid (g/mL) I II (%) III I II (%) III 0.0 0.0 8.4 4.31 0.7 1.00 0.86 9.8 1.07 I R2: Ratio of content of -lipoic acid to content of (B-2) component II Inhibition rate of tyrosinase activity (%) III Tyrosinase activity inhibition rate in the case of using both components/Sum of tyrosinase activity inhibition rate in the case of using each component alone

(78) As seen from the results, in the case of -lipoic acid, there is no sufficient improvement on the ability to inhibit tyrosinase activity even when ascorbyl glucoside is used in combination.

Example 12

(79) <Preparation of Lotion>

(80) A lotion was prepared according to the formulation and procedure indicated below.

(81) Procedure:

(82) To purified water, a moisturizer, an anti-fading agent, a buffer, histidine dithiooctanamide (Na/zinc), ascorbyl glucoside and a pH adjuster were added, and they were dissolved at room temperature to obtain a water phase. On the other hand, an alcoholic phase was obtained by adding an emollient agent, a surfactant, a preservative and a fragrance to ethanol at room temperature. The resultant alcoholic phase was added to the foregoing water phase.

(83) Formulation (% by Mass):

(84) TABLE-US-00015 Humectant: 1,3-Butylene glycol 6.0 Glycerin 5.0 PEG 4000 3.0 Emollient agent: olive oil 0.5 Surfactant: POE (20) sorbitan monostearate 1.5 POE (5) oleyl alcohol ether 0.3 Ethanol: 10.0 Fragrance: Appropriate amount Coloring agent: Appropriate amount Preservative: Appropriate amount Buffer: Appropriate amount Anti-fading agent: Appropriate amount Histidine dithiooctanamide (Na/zinc) *1 0.5 Ascorbyl glucoside 2.0 pH adjuster: Appropriate amount Purified water: Balance *1: DM-His. Zn (available from Oga Research Co., Ltd.)

Example 13

(85) <Preparation of Oil-in-Water Cream>

(86) An oil-in-water cream was prepared according to the formulation and procedure indicated below.

(87) Procedure:

(88) A water phase was prepared by adding moisturizer and alkali to purified water and heating to 70 C. while mixing the mixture. After heating and melting oil components, surfactant, preservative and antioxidant were added to the oil component and the mixture was adjusted to 70 C. to obtain an oil phase. The oil phase was added to the water phase, and resultant mixture was homogenized using a homomixer, and then cooled. Finally, histidine dithiooctanamide (Na/zinc), fragrance and ascorbyl glucoside dissolved in a small amount of purified water were added to the mixture, mixed and degassed.

(89) Formulation (% by Mass):

(90) TABLE-US-00016 Oil component: Stearic acid 8.0 Stearyl alcohol 4.0 Butyl stearate 6.0 Humectant: Propylene glycol 5.0 Surfactant: Glyceryl monostearate 2.0 Alkali: Potassium hydroxide 0.4 Preservative: Appropriate amount Antioxidant: Appropriate amount Fragrance: Appropriate amount Histidine dithiooctanamide (Na/zinc) *1 0.1 ascorbyl glucoside 0.1 pH adjuster: Appropriate amount Purified water: Balance

Example 14

(91) <Preparation of W/O/W (Water-in-Oil-in-Water) Type Cream>

(92) A cream was prepared according to the formulation and procedure indicated below.

(93) Procedure:

(94) (1) Step for Preparing an Intermediate Water-in-Oil Type Emulsion

(95) A water phase was prepared by adding a moisturizer, sodium chloride and histidine dithiooctanamide (Na/zinc) to purified water and mixing at 50 C. After heating and melting oil components, a surfactant was added to the oil component and the mixture was adjusted to 50 C. The water phase was added to the mixture, and the resultant mixture was homogenized using Disper mixer, and then cooled to obtain an intermediate water-in-oil type emulsion.

(96) (2) Step for Preparing a Water-in-Oil-in Water Type Cream Using the Intermediate Water-in-Oil Type Emulsion

(97) A water phase was prepared by adding a moisturizer to purified water and heating to 75 C. while mixing the mixture. After heating and melting oil components, the intermediate water-in-oil type emulsion prepared in Step (1), a surfactant, a preservative and an antioxidant were added to the oil components and the mixture is adjusted to 75 C. The mixture was added to the water phase, and the resultant mixture was homogenized using Disper mixer and then cooled. Finally, ascorbyl glucoside and a pH adjuster dissolved in a small amount of purified water and a fragrance were added to the mixture, mixed and degassed.

(98) Formulation (% by Mass):

(99) TABLE-US-00017 (1) Intermediate water-in-oil emulsion Oil component: Squalane 0.8 Ethylhexyl palmitate 0.5 Surfactant: Hexaglyceryl polyricinoleate 0.15 Diglyceryl monoisostearate 0.05 Humectant: Glycerin 5.0 Sorbitol 0.05 Purified water: 6.0 Sodium chloride 0.2 Histidine dithiooctanamide (Na/zinc) *1 0.1 (2) Water-in-oil-in water type cream Oil component: Squalane 5.0 Ethylhexyl palmitate 2.0 Microcrystalline wax 2.0 Petrolatum 2.0 Behenyl alcohol 3.0 Intermediate water-in-oil emulsion indicated as 12.85 (1) above Surfactant: Glyceryl monostearate 2.0 Sodium stearoyl glutamate 0.5 Humectant: 1,3-Butylene glycol 10.0 Preservative: Appropriate amount Antioxidant: Appropriate amount Fragrance: Appropriate amount Ascorbyl glucoside 0.5 Purified water: Balance

Example 15

(100) <Preparation of Cleansing Foam>

(101) A cleansing foam was prepared according to the formulation and procedure indicated below.

(102) Procedure:

(103) An oil phase was prepared by heating and melting a mixture of a fatty acid, an emollient agent and a humectant at 70 C. After dissolving an alkali in purified water, the oil phase was added to the alkaline aqueous solution with stirring. The resultant mixture was sufficiently neutralized, and then a surfactant was added to the mixture. After conducting a mixing, the mixture was cooled and followed by addition of a chelating agent, a fragrance, Histidine dithiooctanamide (Na/zinc), a pH adjuster and ascorbyl glucoside dissolved in a small amount of purified water, and the resultant mixture was homogenized, cooled and defoamed.

(104) Formulation (% by Mass):

(105) TABLE-US-00018 Oil component: Stearic acid 12.0 Mirystic acid 14.0 Lauric acid 5.0 Emollient agent: Jojoba oil 3.0 Alkali: Potassium hydroxide 5.0 Humectant: 70% Sorbitol aqueous solution 15.0 Glycerin 10.0 1,3-Butylene glycol 10.0 Surfactant: PEG (20) glyceryl monostearate 2.0 Acyl methyl taurine 4.0 Chelating agent: Appropriate amount Fragrance: Appropriate amount Histidine dithiooctanamide (Na/zinc) *1: 0.5 Ascorbyl glucoside 0.5 pH adjuster Appropriate amount Purified water: Balance

Example 16

(106) <Preparation of Liquid Foundation>

(107) A liquid foundation was prepared according to the formulation and procedure indicated below.

(108) Procedure:

(109) The powder ingredients shown in the following formulation were thoroughly mixed and pulverized, and then added to the oil phase shown in the formulation. Next, the oil phase was homogenized using a homomixer, and then the water phase shown in the formulation was added to the oil phase, and the resultant mixture was treated with a homomixer. Subsequently, histidine dithiooctanamide (Na/zinc), a pH adjuster and sodium ascorbate dissolved in a small amount of purified water were added to the mixture, and then the resultant mixture was homogenized and defoamed.

(110) Formulation (% by Mass):

(111) TABLE-US-00019 Powder: Hydrophobized talc 7.0 Hydrophobized titanium dioxide 12.0 Hydrophobized anhydrous silicic acid 2.0 Nylon powder 4.0 Hydrophobized color pigment 2.0 Oil phase: Decamethylcyclopentasiloxane 30.0 Rosin pentaerythritol ester 1.5 Polyoxyethylene-modified 1.5 dimethylpolysiloxane Water phase: Purified water: Balance 1,3-Butylene glycol 4.0 Ethanol 7.0 Histidine dithiooctanamide (Na/zinc) *1: 0.1 Sodium ascorbate 1.0 pH adjuster Appropriate amount

Example 17

(112) <Preparation of Sunscreen Milky Lotion>

(113) A sunscreen milky lotion was prepared according to the formulation and procedure indicated below.

(114) Procedure:

(115) Each of the oil phase and the water phase shown in the following formulation was heated to 70 C. After titanium dioxide was sufficiently dispersed in the oil phase, the water phase was added to the oil phase while performing a treatment using a homogenizer. After completion of homogenization, the mixture was cooled. Next, histidine dithiooctanamide (Na/zinc), a pH adjuster and magnesium ascorbyl phosphate dissolved in a small amount of purified water were added to the mixture, and then the resultant mixture was homogenized and defoamed.

(116) Formulation (% by Mass):

(117) TABLE-US-00020 Water phase: Purified water: Balance 1,3-Butylene glycol 5.0 Oil phase: Octyl p-methoxycinnamate 5.0 Oxybenzone 3.0 4-tert-butyl-4-methoxydibenzoylmethane 1.0 Hydrophobized titanium dioxide 3.0 Squalane 20.0 Decamethylcyclopentasiloxane 20.0 Polyoxyethylene-modified 3.0 dimethylpolysiloxane Organo-modified montmorillonite 1.5 Preservative: Appropriate amount Fragrance: Appropriate amount Histidine dithiooctanamide (Na/zinc) *1: 1.0 Magnesium ascorbyl phosphate: 3.0 pH adjuster Appropriate amount

INDUSTRIAL APPLICABILITY

(118) The tyrosinase activity inhibitor of the present invention has a remarkably improved ability to inhibit tyrosinase activity as compared with the case where the -lipoic acid derivative is used solely. Hence, it is useful as a material for preparing cosmetics, quasi-drugs and the like. The external preparation for skin containing the tyrosinase activity inhibitor can effectively inhibit tyrosinase activity, and is useful as a cosmetic product and quasi-drugs which are excellent in skin-whitening effect.