KR100680585B1 - Composition for skin whitening containing deoxymannojirimycin - Google Patents

Abstract

A skin whitening composition with excellent whitening effect is provided to be able to effectively inhibit melanogenesis without side effects by comprising deoxymannojirimycin, which is mannosidase inhibitor, only or the deoxymannojirimycin and deoxynojirimycin in combination. The composition for skin whitening comprises deoxymannojirimycin as an effective ingredient. The composition further comprises deoxynojirimycin as an effective ingredient. Regarding the total weight of the composition, 0.001-20 wt.% of the effective ingredient is contained.

Description

Composition for skin whitening containing deoxymannojirimycin}

1 is a graph showing the effect of deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ) on melanin production of melanocytes.

2 is a photograph taken by collecting the melanosite treated with DMJ and DNJ.

3 is a graph showing that the melanin production inhibitory effect is increased when the DMJ and DNJ at the same time.

4 is a photograph taken by collecting the melanosite treated with the DMJ and DNJ at the same time.

5 is a protein electrophoresis picture showing the effect of DMJ and DNJ on glycosylation of tyrosinase.

Figure 6 is a protein electrophoresis picture showing the effect on the glycosylation of tyrosinase when simultaneous treatment with DMJ and DNJ.

The present invention relates to a skin whitening composition containing only deoxymannojirimycin (DMJ) as an active ingredient, or simultaneously containing deoxymannojirimycin and deoxynojirimycin (DNJ).

Melanin, which determines human skin color, is made from skin cells called melanocytes and migrates to epidermal cells called keratinocytes. Here, melanin plays an important role such as forming a hat-like structure around the nucleus to protect genes from ultraviolet rays and to remove free radicals to protect intracellular proteins. There are no enzymes that break down melanin in vivo, but they are removed by falling off the skin as keratinocytes fall off the epidermis. However, if more melanin is produced than necessary, it causes hyperpigmentation, such as blemishes, freckles, and spots, which leads to cosmetically bad results. In addition, as the leisure population increases, the number of people who enjoy working outside increases the need to prevent melanin pigmentation caused by ultraviolet rays. Therefore, it was necessary to develop a whitening agent that prevents excessive melanin production and many efforts have been made.

Until now, the development of whitening agents has been mainly focused on finding a substance that reduces melanin by inhibiting the activity of tyrosinase, an essential and most important enzyme in melanin biosynthesis. The whitening agent thus developed includes kojic acid, arbutin, glutathione, vitamin A, vitamin C, etc., but it does not have a satisfactory whitening effect for consumers and has a limited problem due to its side effects. Therefore, it is urgent to develop a whitening agent that is more effective and safer than ever before.

On the other hand, tyrosinase, an enzyme that synthesizes melanin, is a glycoprotein, and is produced through glycosylation, a glycoprotein synthesis process in vivo. Many enzymes are involved in the glycosylation process, the most important of which are mannosidase and glucosidase. Deoxynojirimycin (DNJ) is known to inhibit the enzymatic activity of glucosidase and inhibit melanin production of tyrosinase (Japanese Patent No. 1987-241966). However, there is no report that deoxymannojirimycin (DMJ), a mannosidase inhibitor, inhibits melanogenesis of tyrosinase. In particular, the synergistic melanin production inhibitory effect that occurs when the DMJ and DNJ at the same time has not been studied yet.

Accordingly, the present inventors have studied to provide a new whitening agent having excellent whitening effect but no side effects, deoxymannojirimycin (DMJ) alone, or deoxymannojirimycin and deoxynojirimycin It was found that (deoxynojirimycin, DNJ) can effectively inhibit melanin production without side effects, and completed the present invention.

Therefore, an object of the present invention is a composition for skin whitening containing deoxymannojirimycin (DMJ) alone or deoxymannojirimycin and deoxynojirimycin (DNJ) simultaneously as an active ingredient. To provide.

In order to achieve the above object, the composition for skin whitening according to the present invention is characterized in that it contains deoxymannojirimycin alone, or deoxymannojirimycin and deoxynojirimycin simultaneously as an active ingredient.

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

Deoxymannojirimycin and deoxynojirimycin contained in the composition for skin whitening of the present invention as an active ingredient inhibits the glycosylation of tyrosinase to prevent melanin production and thereby exhibit a whitening effect.

The active ingredient contained in the composition for skin whitening according to the present invention is contained in an amount of 0.001 to 20% by weight based on the total weight of the composition. This is because the effect can not be expected at less than 0.001% by weight, there is a difficulty in the safety or formulation formulation above 20% by weight.

The composition for skin whitening according to the present invention is not particularly limited in its formulation, but specifically Cosmetic formulations include lotion, nutrition cream, nutrition lotion, massage cream, nutrition essence and the like.

Hereinafter, although an Example and a test example are given and this invention is demonstrated in detail, this invention is not limited only to these examples.

Test Example 1 Determination of the Effects of Deoxymannojirimycin (DMJ) and Deoxynojirimycin (DNJ) on Melanin Production in Human Melanoma Cells

Minimum HM3KO cells (Y. Funasaka, Department of dermatology, Kobe university school of medicine, 5-1 Kusunoki-cho 7-chrome, Chuo-ku, Kobe 650, Japan), human melanoma cells Incubated in Essential Medium (MEM) medium at 37 ℃, 5% CO ₂ conditions. The cultured cells were spread over a 75 T flask so that the number of cells was 3 × 10 ⁵ per flask, and the cells were transferred to a new medium containing the sample from the next day after waiting for the cells to adhere to the wall. DMJ and DNJ were 1.0, 0.5, and 0.25 mM, respectively. In addition, the control group used a medium containing a vehicle (ethanol: propylene glycol: water = 5: 3: 2) used to dissolve the sample, a medium containing 3.5 mM kojic acid as a positive control group Was used. The new medium containing the sample was changed every 1 to 2 days and cultured until the cells were filled in the flask. When the cells were grown, they were collected and photographed for color comparison with the control group. After completely dissolving the cells with 1N NaOH, the absorbance at 490 nm was measured to determine the amount of melanin. When the amount of melanin in the control group is 100%, the relative melanin amount is calculated and expressed as a graph compared to the control group. The results are shown in FIGS.

Meanwhile, using the same method as above, 0.15, and 0.39 mM of DMJ and DNJ, and 0.13, 0.27, and 0.54 mM of DMJ + DNJ, respectively, were used to calculate the amount of melanin in% relative to the control. It was. The results are shown in Figures 3 to 4 below.

From the results of FIG. 1 below, kojic acid 3.5 mM reduced melanin production by 20.55%, while deoxymannojirimycin (DMJ) 1, 0.5, 0.25 mM inhibited melanogenesis of melanocytes by 63.80, 56.61, and 48.15%, respectively. Showed. This is a higher inhibitory effect than the same concentration of deoxynojirimycin (DNJ). In addition, it can be seen from the results of FIG. 2 that the apparent color of melanocytes treated with deoxymannojirimycin (DMJ) became white compared to deoxynojirimycin (DNJ) at the same concentration as the control.

In addition, from the results of Figures 3 and 4, it was confirmed that melanin production inhibitory synergistically increased significantly when simultaneously treated with deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ). In other words, when melanocytes treated with 0.15 mM of deoxymannojirimycin (DMJ) and 0.39 mM of deoxynojirimycin (DNJ) are hardly inhibited in melanin production, deoxymannojirimycin (DMJ) 0.15 Simultaneous treatment of mM and deoxynojirimycin (DNJ) 0.39 mM confirmed that melanin production was inhibited by about 71.70%.

On the other hand, as a result of relative quantification of the total protein amount between the test groups, deoxymannojirimycin (DMJ), deoxynojirimycin (DNJ) and kojic acid showed no cytotoxicity at all experimental concentrations.

Test Example 2 Determination of the Effect of Deoxymannojirimycin (DMJ) and Deoxynojirimycin (DNJ) on Glycosylation of Tyrosinase in Human Melanoma Cells

After HM3KO cells were grown in the same manner as in Test Example 1, the cells were collected and placed in a cell lysis solution (lysis buffer: 2% CHAPS in 50 mM Hepes and 200 mM NaCl, pH 7.5, protease inhibitors) and sonicated by ultrasound. In this case, 1.0, 0.5 and 0.25 mM of DMJ and DNJ were used, 0.15 and 0.39 mM of DMJ and DNJ, and 0.13, 0.27 and 0.54 mM of DMJ + DNJ, respectively. The cell wash was centrifuged at 4 ° C. and 12000 rpm for 10 minutes to separate and remove unbroken cells and melanin (melanin), and only the supernatant was taken. Endoglycosidase H (125 units) was added to the supernatant (protein amount: 20 mg), and the enzyme was reacted at 37 ° C. for 1 hour, and the proteins in the supernatant were separated by size under electrophoresis. Tyrosinase among the proteins isolated by electrophoresis can be confirmed by Western blotting using an antibody.

Tyrosinase, in which sugar residues are normally formed, appears as a glycoprotein of about 80 kD due to the enzymatic degradation of sugar residues by endoglycosidase H, and is an enzyme of glucosidase or mannosidase that is involved in the glycosylation process. Tyrosinase, in which activity is inhibited and a normal sugar residue is not formed, is hydrolyzed by endoglycosidase H, resulting in a protein of about 60 kD. The results are shown in FIGS. 5 to 6.

From the results of FIG. 5, the endo H + shows that the tyrosinase of the control group is large in size (about 80 kD) because the sugar is not degraded by endoglycosidase, but deoxymannojirimycin (DMJ). ), The tyrosinase of melanocytes treated with 0.25, 0.5, and 1 mM was found to be small (about 60 kD) in size as the sugar concentration was increased by endoglycosidase. In addition, it was confirmed that such deoxymannojirimycin (DMJ) tyrosinase glycosylation inhibitory effect is greater than the same concentration of deoxynojirimycin (DNJ). This is consistent with the result that deoxymannojirimycin (DMJ) inhibits melanin production more than deoxynojirimycin (DNJ). On the other hand, it was confirmed that the total tyrosinase expression level was not changed at all test concentrations (Endo H-). From the above results, it was found that deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ) are involved in glycoprotein formation of tyrosinase and inhibit melanin production.

In addition, when the deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ) are simultaneously treated at a concentration that does not inhibit the glycosylation of tyrosinase from the results of FIG. 6, glyco of tyrosinase It was confirmed that the sillation was suppressed.

[Examples 1 and 2 and Comparative Example 1]

To the composition of Tables 1 and 2 and Comparative Example 1 skin whitening in the composition of Table 1 was prepared as a nutrient essence formulation.

In Examples 1 to 2, raw materials 1 to 6 in Table 1 were mixed, dissolved at 70 ° C. to form an aqueous phase, and raw materials 7 to 14 were dissolved at 70 ° C. to obtain an oil phase. Next, the oil phase was added to the water phase, stirred with a homomixer (Tokushu Kika, Japan) to emulsify first, and the raw material 15 was added to increase. After removing bubbles, the mixture was cooled to room temperature.

On the other hand, in the manufacturing method of the comparative example 1, the raw materials 1-6 of Table 1 were mixed, it melt | dissolved at 70 degreeC, it was made into the water phase, and the raw materials 8-14 were melted at 70 degreeC, and it was set as the oil phase. Next, the oil phase was added to the water phase, stirred with a homomixer (Tokushu Kika, Japan) to emulsify first, and the raw material 15 was added to increase. After removing bubbles, the mixture was cooled to room temperature.

ingredient Example 1 Example 2 Comparative Example 1 One Purified water To 100 To 100 To 100 2 glycerin 8.0 8.0 8.0 3 Butylene glycol 4.0 4.0 4.0 4 Hyaluronic acid extract 5.0 5.0 5.0 5 Beta Glucan 7.0 7.0 7.0 6 Camomer 0.1 0.1 0.1 7 DMJ 0.5 0.25 - DNJ - 0.25 - 8 Caprylic Capric Triglycerides 8.0 8.0 8.0 9 Squalane 5.0 5.0 5.0 10 Cetearyl Glucoside 1.5 1.5 1.5 11 Sorbitan stearate 0.4 0.4 0.4 12 Cetearyl Alcohol 1.0 1.0 1.0 13 antiseptic Quantity Quantity Quantity 14 incense Quantity Quantity Quantity 15 Triethanolamine 0.1 0.1 0.1

[Test Example 3]

In order to confirm the whitening effect of the compositions prepared in Examples 1 and 2 and Comparative Example 1, after attaching an opaque tape with six holes 1.5 cm in diameter to the upper forearm of the subject to 12 healthy men, Induced blackening of the skin by irradiating 1.5 ~ 2 times of ultraviolet light (UVB) of the minimum erythema dose of each subject, and applying the test materials two months after applying the test substances Measured. Each test substance was corrected twice daily (morning, evening) the composition of Comparative Example 1 and Examples 1-2. The determination of the effect was determined by obtaining a "L" value representing the darkness of the skin (unburned Korean skin color generally indicates a value of 50-70). If the test substance is effective, the L value is gradually increased, and the comparison between the test substances is the ΔL value, that is, the value of the final L minus the initial L value (ΔL) (final L value-application of the test substance). Previous value). The larger the ΔL value, the greater the whitening effect. The results are shown in Table 2 below.

Example 1 Example 2 Comparative Example 1 DL 2.05 2.95 1.43

As shown in Table 2, the ΔL values of the compositions of Examples 1 to 2 containing deoxymannojirimycin (DMJ) or deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ) are It was 2.05 and 2.95, respectively, which was higher than that of Comparative Example 1, 1.43. In particular, in the case of Example 2 treated with deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ) together, whitening higher than that of Example 1 treated with deoxymannojirimycin (DMJ) alone Effect.

As can be seen from the above, the composition for skin whitening according to the present invention contains deoxymannojirimycin alone or deoxymannojirimycin and deoxynojirimycin simultaneously as an active ingredient to produce melanin without side effects. It can be effectively suppressed.

Claims (3)

A skin whitening composition containing deoxymannojirimycin as an active ingredient. The composition for skin whitening according to claim 1, wherein the composition further contains deoxynojirimycin as an active ingredient. The composition for skin whitening according to claim 1 or 2, wherein the active ingredient is contained in an amount of 0.001 to 20% by weight based on the total weight of the composition.

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