KR100432449B1 - Composition Including Ketoconazole of External Application for Skin-whitening - Google Patents

Abstract

A topical composition for ketoconazole-containing skin whitening excellent in safety and excellent in whitening effect is disclosed. It is a novel whitening application that inhibits the activity of tyrosinase, the main enzyme of melanin pigment production, and suppresses the production of melanin pigment, by adding 0.01 to 5.00% by weight of ketoconazole as an active ingredient based on the total amount of the composition. It has excellent safety, no side effects, and has melanin pigment inhibitory effect to prevent pigmentation of skin due to blemishes, freckles and sunburn, and it has excellent skin whitening effect.

Description

Composition Including Ketoconazole of External Application for Skin-whitening}

The present invention relates to an external preparation composition for ketoconazole-containing skin whitening, and more particularly, to the production of melanin by containing ketoconazole, which acts to inhibit the activity and production of tyrosinase, the main enzyme of melanin pigment production, as an active ingredient. It relates to an external composition for skin whitening that can provide excellent skin whitening effect by inhibiting the.

Human skin color is genetically determined by the concentration and distribution of melanin in skin cells, but may also be influenced by environmental or physiological conditions such as exposure to sun UV rays, the use of female hormones, pregnancy, cosmetics, fatigue and stress. The melanin pigment is deposited on the skin and blackening of the skin may progress. Due to these causes, pigmented diseases such as blemishes, freckles, etc. that occur on the face have emerged as a major problem of female beauty.

The mechanism of skin blackening is as follows. An internal and external factor activates an enzyme called tyrosinase, which acts on tyrosine, a type of amino acid present in skin tissue, producing dopa and then dopaquinone. Through a non-enzymatic autooxidation process, a polymer called melanin is synthesized in melanocytes in melanocytes, which are skin pigment cells, and are transferred to keratinocytes, which are skin keratinocytes.

As the causes and mechanisms of skin blackening have been revealed, melanin is inhibited by incorporating substances having an inhibitory effect of tyrosinase, an enzyme involved in skin blackening, into cosmetics or medicines or inhibiting some reactions during the production of melanin. To reduce the production of the skin to improve the whitening, blemishes, freckles and the like are commonly used. Representative materials used for this purpose are tocopherol, vitamins, ascorbic acid (ascorbic acid), kojic acid (kojic acid), hydroquinone (hydroquinone) and the like. However, ascorbic acid is not suitable as a melanogenesis inhibitor due to its low tyrosinase activity inhibitory effect and low stability of the molecule itself. Kojic acid has a strong inhibitory effect on tyrosinase activity, but has a problem in stability such as discoloration and decrease in titration due to changes over time when combined with pharmaceuticals, and because of high skin irritation, it denatures melanin pigment or melanocytes. Have In addition, although hydroquinone is recognized for its effectiveness, its use is generally limited due to safety issues because of its sensitivity to skin.

Ketoconazole (aka 1-acetyl-4- [4-[[2- (2,4-dichlorophenyl) -2- (1H-imidazole-1-ylmethyl) -1,3-dioxolan-4-yl] -methoxy] phenyl] piperazine) has been reported in Korean Patent Publication No. 98-0020952 as an antifungal agent, in addition to Candida, the use of imidazole derivatives econazol and ketoconazole as a therapeutic agent for skin diseases caused by Aspergillus, Cryptococcus, etc. There is a bar. In addition, Korean Patent Publication No. 98-0706130 discloses the use as a shampoo composition effective to treat and relieve itching, dandruff. However, the application of ketoconazole to the melanin inhibitory action, in particular as a whitening agent, is not known at all.

Accordingly, the present inventors tested ketoconazole's melanin production inhibitory effect and tyrosinase inhibitory effect, and found that ketoconazole is a substance that can be used as an active ingredient in high-efficiency external preparations for skin whitening. That is, the inventors of the present invention, while researching a substance that has a skin whitening effect that can see a visible effect in a short time without side effects in removing the blemishes on the skin completely, ketoconazole, which is a kind of imidazole derivative, has an excellent melanin inhibitory effect and The inventors have found that they have a tyrosinase inhibitory effect and have completed the present invention as a novel use of ketoconazole.

Accordingly, an object of the present invention is to provide a ketoconazole-containing external preparation composition for skin whitening by containing ketoconazole having a melanin production inhibitory effect and a tyrosinase activity inhibitory effect as an active ingredient.

Another object of the present invention is to provide an external preparation for skin whitening containing the above ketoconazole as an active ingredient.

Figure 1 is a graph showing the results of measuring the cell non-toxic tyrosinase inhibitory effect according to the concentration of ketoconazole and hydroquinone.

In order to achieve the above object, the present invention provides a topical composition for skin whitening containing 0.01 to 5.00% by weight of ketoconazole as an active ingredient based on the total amount of the composition.

Another object of the present invention described above is achieved by an external preparation for skin whitening containing the above external preparation for skin whitening as an active ingredient.

The present inventors investigated the effects of ketoconazole's tyrosinase activity inhibitory activity, B16 melanoma cell line's tyrosinase activity inhibitory activity, melanin production inhibitory activity, and confirmed its whitening effect and melanin production inhibitory effect. It was completed.

Hereinafter, the present invention will be described in more detail.

The blending amount of the ketoconazole is 0.01-5.00% by weight based on the total weight of the external preparation composition. If the amount of the ketoconazole is less than 0.01% by weight, the whitening effect is not sufficiently exhibited. If the amount of the ketoconazole is less than 5.00% by weight, there is a problem in skin safety, and thus the amount of the ketoconazole is in the above-mentioned range. Preferably it is 0.1 to 2.0% by weight.

The external preparation for skin whitening of the present invention, in addition to the above-mentioned essential components, components commonly used in external preparations for skin, such as cosmetics and medicines, for example, other whitening agents, moisturizing agents, antioxidants, oily ingredients, ultraviolet absorbers, surfactants, thickeners, alcohols The oils, powder ingredients, colorants, flavors, aqueous ingredients, water, various skin nutrients and the like may be mixed and used in appropriate amounts as necessary.

As a method for use as an external preparation for skin whitening, all formulations used in conventional external preparations for skin such as creams, ointments, solutions, lotions, and packs are applicable.

The stability test of ketoconazole presented in the present invention was performed in the following order. First, ketoconazole powder was dissolved in dimethyl sulfoxide (DMSO) and diluted while adjusting the concentration to prepare a ketoconazole sample. Then, as the experimental cell line, the mouse-derived B16 melanoma cell line was selected and cultured. Ketoconazole samples were treated in the cultured B16 melanoma cell line, followed by ketoconazole and hydroquinone cells by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium, Sigma Co., USA] Toxicity was compared.

The effect of ketoconazole, which inhibits tyrosinase enzyme activity at the cellular level, and the effect of ketoconazole, which inhibits melanin production, were confirmed, and the inhibitory ability of the cell nontoxic tyrosinase activity of hydroquinone and ketoconazole, known as the whitening agent, was confirmed. Analyzes were compared for efficacy.

Hereinafter, specific examples for the stability test will be described in detail with reference to Examples and Experimental Examples. Samples used in the examples and experimental examples below were prepared and used according to the following experimental examples, unless otherwise specified.

<Example 1>

Preparation of Ketoconazole Samples

To prepare a ketoconazole sample to be applied in the present invention, ketoconazole (manufactured by Sigma Co., Ltd.) was dissolved in dimethylsulfoxide, and this solution was diluted in a medium to adjust the concentration.

Experimental Example 1

Cytotoxicity Measurement of Ketoconazole and Hydroquinone

To compare the cytotoxicity between ketoconazole and hydroquinone, a known whitening substance, the viability of B16 melanoma cells was measured by MTT assay. MTT (Sigma, USA) quantification is a test method to determine the effect of ketoconazole and hydroquinone on the cell viability, adjusted to 1 × 10 ⁴ cells in each well and 200μL divided into 96 microwell plate and 24 After hours, ketoconazole and hydroquinone at appropriate concentrations were treated and incubated for 48 hours.

Five wells of the control group and the experimental group were made into one group, and MTT was added to a final concentration of 100 µg / ml and incubated for 4 hours, and then the culture medium was discarded and washed with phosphate buffer (pH 6.8). After dissolving the purple insoluble formazan produced by living cells in 200 μl of dimethyl sulfoxide, the absorbance was measured at 570 nm with a spectrophotometer. The relative absorbance of the control group was calculated as cell viability (%), and the degree of cytotoxicity was calculated by the following formula ( It was calculated according to 1), and the results are shown in Table 1.

Cytotoxicity (%) = 100-(absorbance of each sample / absorbance of control) × 100 --- (1)

Comparative Analysis of Cytotoxicity between Ketoconazole and Hydroquinone sample Cytotoxicity (%) Control - Ketoconazole (1 μg / ml) - Ketoconazole (10 μg / ml) 1.19% Hydroquinone (1 μg / ml) 21.66% Hydroquinone (10 μg / ml) 94.67%

As shown in Table 1, the cytotoxicity of the cultured melanoma cells of ketoconazole and hydroquinone showed cytotoxicity of 94.67% at 10 μg / ml due to severe cytotoxicity, but 10 μg of ketoconazole. It is shown that the cytotoxicity is 1.19% even at a / ml concentration, indicating that ketoconazole is a very safe substance compared to hydroquinone, a known whitening substance.

Experimental Example 2

Inhibitory Effects of Ketoconazole on Tyrosinase Activity at the Cell Level

Inhibition of tyrosinase activity at the cellular level was investigated by adding ketoconazole to the culture solution of B-16 mouse melanoma cells. B16 mouse melanoma cells derived from the mouse were used by the Korea Cell Line Bank. Cultivation was performed using a DMEM medium containing 10% fetal bovine serum (product made by Haas) in a carbon dioxide incubator (37 ° C., 5%) and incubating 10 ⁵ cells per culture vessel and incubating for one day.

After 24 hours of incubation, the ketoconazole sample solution was added, and the culture was continued for 2 days, and the effect of inhibiting tyrosinase activity was measured by the following method. First, harvested melanocytes to make cell infiltrates, add 100 μl of cell lysate (1% Triton X-100, 10 mM sodium phosphate, pH 7.0, 0.1 mM PMSF) and destroy the cells by shaking occasionally for 30 minutes at 4 ° C. After centrifugation, the supernatant was taken and used as a Tyrosinase activity measurement solution. 100 μl of 100 mM sodium phosphate (pH 7.0) was added to 50 μl supernatant, and kept in a water bath at 30 ° C. for 5 minutes. The change was observed for 1 hour. At this time, using the buffer solution instead of ketoconazole as a control to measure the absorbance by the same method to calculate the inhibition rate of tyrosinase activity. Tyrosinase activity inhibition rate was calculated according to the following formula (2), the results are shown in Table 2.

Tyrosinase activity inhibition rate (%) = 100-(absorbance of each sample / reaction absorbance of the control) × 100 --- (2)

Inhibitory effect of ketoconazole on tyrosinase activity at the cellular level sample Inhibition Rate (%) Control - Ketoconazole (1 μg / ml) 19.39% Ketoconazole (10 μg / ml) 40.75% Hydroquinone (1 μg / ml) 46.33% Hydroquinone (10 μg / ml) 92.29%

As can be seen in Table 2, it can be seen that ketoconazole has a tyrosinase activity inhibitory activity against melanoma cells in cultured mice. Hydroquinone has strong tyrosinase activity inhibitory ability, whereas cytotoxicity is severe, ketoconazole is a safe substance having inhibitory effect on tyrosinase activity without showing cytotoxicity.

Experimental Example 3

Inhibitory Effects of Ketoconazole on Melanogenesis

Ketoconazole was added to the culture of melanoma cells to test the whitening effect at the cellular level (Lotan R., Lotan D., Cancer Res., 40: 3345-3350, 1980). Ketoconazole and hydroquinone were added to the culture medium of B-16 melanoma cells at each concentration and incubated for 3 days. The cells were then trypsinized to remove the melanin after centrifugation. 300 μl of 1N sodium hydroxide added with 10% DMSO was added thereto to treat melanin at 80 ° C. for 1 hour, and the absorbance was measured at 475 nm. The relative melanin production relative to the control group was calculated as inhibition rate (%). It is shown in Table 3 below. Melanin production inhibition was calculated according to the following formula (3).

Melanin production inhibition rate (%) = 100-(absorbance of each sample / absorbance of control) × 100 --- (3)

Inhibitory Effects of Ketoconazole at the Cell Level on Melanin Production sample % Inhibition Control - Ketoconazole (1 μg / ml) 16.28% Ketoconazole (10 μg / ml) 38.24% Hydroquinone (1 μg / ml) 63.64% Hydroquinone (10 μg / ml) 97.73%

As can be seen in Table 3, it can be seen that ketoconazole has melanin production inhibitory ability against melanoma cells in cultured mice. Hydroquinone has a strong melanin production inhibitory ability, whereas cytotoxicity is severe, ketoconazole has a high melanin production inhibitory effect without showing cytotoxicity.

Experimental Example 4

Inhibition of Tyrosinase Activity by Cytotoxicity of Ketoconazole and Hydroquinone

Hydroquinone, known as a conventional whitening agent, has excellent whitening efficacy but severe cytotoxicity, whereas ketoconazole has a whitening effect without cytotoxicity. Therefore, we compared and analyzed the cellular nontoxic tyrosinase activity inhibition rate except for the enzyme inhibition rate which is secondary to the decrease in survival rate due to the cytotoxicity of ketoconazole and hydroquinone. Inhibition rate of cell nontoxic tyrosinase activity was calculated according to the following formula (4) and the results are shown in FIG.

Inhibition rate of cell nontoxic tyrosinase activity (%) = Inhibition rate of tyrosinase activity (%) × [Cell survival rate (%) / 100] --- (4)

As shown in FIG. 1, ketoconazole has a higher inhibitory effect on tyrosinase activity at higher concentrations, but hydroquinone has a higher cytotoxicity at higher concentrations, which results in lower cell viability and a marked decrease in efficacy at 10 ㎍ / ml. And it was found.

In summary, the ketoconazole has an excellent pigmentation inhibitory effect by inhibiting tyrosinase activity and melanin production without cytotoxicity.

Hereinafter, the whitening medicines containing ketoconazole (Example 2 and Comparative Example 1) and the whitening medicines not containing (compared with the above) were confirmed that the tyrosinase inhibitory effect and the melanin production inhibitory effect were excellent. After preparing Example 2), the stability test and the inhibition of hyperpigmentation of skin were tested.

<Example 2 and Comparative Examples 1 and 2>

It was prepared as shown in Table 4 as a prescription of the cream in medicine. In Example 2, a cream containing ketoconazole was prepared, and in Comparative Example 1, a cream was prepared according to a formulation containing ketoconazole and containing sodium hydrogen sulfite as in Example 1, and in Comparative Example 2 from Example 1 A cream containing ascorbic acid was prepared, excluding ketoconazole and sodium hydrogen sulfite.

Cream composition (unit: weight) Compounding ingredient Example 2 Comparative Example 1 Comparative Example 2 Ketoconazole 1.0 1.0 - Ascorbic acid - - 1.0 Sodium bisulfite - 0.1 - ethanol 10.0 10.0 10.0 Stearic acid 2.0 2.0 2.0 Methylparaben 0.25 0.25 0.25 Mineral oil 6.0 6.0 6.0 Propylene glycol 5.0 5.0 5.0 antiseptic a very small amount a very small amount a very small amount incense a very small amount a very small amount a very small amount Purified water to 100 to 100 to 100

Experimental Example 5

Stability Test of Pharmaceutical Compositions

In order to determine the stability of the pharmaceutical composition of the present invention was performed for the cream according to Example 2 and the cream according to Comparative Examples 1 and 2 as follows.

Each of the two test materials was capped in a clear glass container and then discolored while standing at room temperature and observed at 1, 2, 4 and 6 weeks. The results are shown in Table 5 below.

Stability Test of Pharmaceutical Compositions Test substance 1 day 2 weeks 4 Weeks 6 Weeks Example 2 Light white Light white Light white Light white Comparative Example 1 Light white Light white Light white Light white Comparative Example 2 Light white Light white Brown mum

As shown in the results of Table 5, it can be seen that the test substance containing ketoconazole is not significantly discolored even after 6 weeks of storage at room temperature to prevent oxidation.

Experimental Example 6

Skin pigmentation inhibitory effect

Twenty healthy men and women were selected to attach aluminum foil in two rows of six holes with a diameter of 7 mm in the lower part of both arms, and 60m / cm ² light was irradiated using ORIEL solar simulator 1000W at a distance of 10 cm from the arm. . At this time, the irradiation site was washed with 70% ethanol aqueous solution before irradiation. Prepared according to Example 1 twice a day from 3 days before irradiation to 3 weeks after irradiation with a sample containing ketoconazole and prepared according to Comparative Example 2 in a pair containing a base containing ascorbic acid and no ketoconazole. The same line was applied. In each case, the degree of pigmentation at the time of use of the cream according to Example 2 and Comparative Example 2 was visually determined, and Example 2 had a noticeable effect, a slight effect, and no effect compared to Comparative Example 2. Evaluated in stages, the results are shown in Table 6.

Skin pigmentation inhibition effect (unit: person) Experimental substance A noticeable effect Slight effect no effect Example 2 7 9 4 Comparative Example 2 0 4 16

As shown in the results of Table 6, the pharmaceutical composition containing ketoconazole prepared according to Example 2 showed a whitening effect for 16 out of 20 subjects, a pharmaceutical composition containing ascorbic acid instead of ketoconazole Compared with the whitening effect, ketoconazole is safe and effective in improving skin and freckles or skin whitening agent.

Hereinafter, on the basis of the above examples, it will be exemplified a pharmaceutical of various formulations that can provide a skin lightening effect by containing ketoconazole, but the composition of the present invention is not limited to the following preparation examples.

<Example 3>

A skin external ointment was prepared using the ingredients and blending ratios shown in Table 7.

Compounding ingredient Content (% by weight) Ketoconazole 1.0 Prepayment 5.0 Diethylsebagate 8.0 Squalane 5.0 Propylene glycol 5.0 Vaseline 75.5 Sodium benzoate Quantity Total 100

Experimental Example 7

Whitening effect of the skin ointment of the present invention on blemishes

Thirty people with blemishes regardless of age and gender were applied to the affected area twice daily for 60 days, and then applied to the affected area at the composition ratio shown in Example 3, and then every 10 days. The state was observed. The results are shown in Table 8.

Decontamination effect of the external preparation for skin whitening of the present invention on blemishes (unit: persons) case Very useful utility Slightly useful Availability (%) 30 7 14 6 90

As shown in Table 8 above, the usefulness is the result of a combination of very useful, useful, and slightly useful. Therefore, the external preparation for skin whitening of the present invention is very excellent in the removal of blemishes, that is, the whitening effect.

<Example 4>

The liquid formulation was prepared with the component and compounding ratio shown in Table 9.

Ingredient Content (% by weight) Ketoconazole 1.0 ethanol 75 Purified water to 100

Example 5

The milk lotion was prepared with the ingredients and blending ratios shown in Table 10.

Ingredient Content (% by weight) Ketoconazole 0.5 Propylene glycol 5.4 glycerin 4.0 Triethanolamine 1.2 Tocopheryl Acetate 3.0 Liquid paraffin 5.0 Squalane 3.0 Macadamia Nut Oil 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.0 Carboxy Vinyl Polymer 1.0 Incense, preservative a very small amount Purified water to 100

<Example 6>

A pack agent was prepared at a component and blending ratio as shown in Table 11.

Compounding ingredient Content (% by weight) Ketoconazole 0.5 Polyvinyl alcohol 15.0 glycerin 5.0 Propylene glycol 4.0 ethanol 8.0 Polyoxyethylene oleylethyl 1.0 Methyl paraoxybenzoate 0.2 Pigment Quantity incense Quantity Purified water to 100

As described in the above examples and examples, the present invention provides a novel use of ketoconazole as a whitening agent, the use of which is known as an antifungal agent. As a result of measuring the inhibitory activity of tyrosinase activity, melanin production inhibitory activity, and cell non-toxic tyrosinase activity of ketoconazole, ketoconazole, which has been proven to have whitening effect and melanin production inhibitory effect, has excellent safety, no side effect, and within a short time. You can see that the whitening effect can be seen. Therefore, such an external preparation composition for skin whitening and an external preparation including the same may be very usefully applied in the cosmetic and pharmaceutical manufacturing industries.

Although the above has been described with reference to the preferred embodiments of the present invention, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention as set forth in the claims below. It will be appreciated.

Claims (6)

An external preparation composition for skin whitening containing 0.01 to 5.00 wt% of ketoconazole as an active ingredient based on the total weight of the composition. The external preparation composition for skin whitening according to claim 1, wherein the ketoconazole content is 0.01 to 2.00 wt% based on the total weight of the composition. An external preparation for skin whitening containing 0.01 to 5.00% by weight of ketoconazole as an active ingredient based on the total weight of the composition. The external preparation for skin whitening according to claim 3, wherein the content of ketoconazole is 0.01 to 2.00 wt% based on the total weight of the composition. The method of claim 3, wherein the external preparation includes a whitening agent, a moisturizing agent, an antioxidant, an oily ingredient, an ultraviolet absorber, a surfactant, a thickener, an alcohol, a powder ingredient, a colorant, a perfume, an aqueous ingredient, water, and various skin nutrients. External preparation for skin whitening, characterized in that. The external preparation for skin whitening according to claim 3, wherein the external preparation for skin whitening has any one of a skin external cream, an ointment, a liquid, a lotion, and a pack.