KR20030013738A - Skin whitening composition containing n,n'-dilinoleylcystamine as melanogenesis modulator - Google Patents

Abstract

PURPOSE: Provided is a skin whitening composition containing as active ingredient N,N'-dilinoleylcystamine of the formula(I) as a melanogenesis modulator to inhibit the production of melanin and consequently to have skin whitening effect. CONSTITUTION: The skin whitening composition characteristically contains as active ingredient N,N'-dilinoleylcystamine of the formula(I) as a melanogenesis modulator in an amount of 0.00001-10 wt.% based on total weight of the composition. The N,N'-dilinoleylcystamine is excellent in the inhibition of the production of melanin, therefore the composition is useful as a skin whitening agent.

Description

Skin whitening composition containing N, N'-dilinoleylcystamine as melanogenesis modulator}

The present invention is a derivative of cystamine and linoleic acid represented by the following formula (I), which has been conventionally used as an adjuvant, that is, dilinolyl cystamine (N, N'-Dilinoleylcystamine; C ₂₁ H ₃₉ NOS: Molecular weight: 353.61) The present invention relates to a skin whitening composition having a whitening effect by inhibiting melanin production.

[Formula 1]

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 degrade melanin in vivo, but they are removed by falling off the skin as keratinocytes fall off the epidermis. However, when more melanin is produced than necessary, it causes hyperpigmentation such as blemishes, freckles, and spots, resulting in cosmetic consequences. Also, the increase in leisure population increases the number of people who enjoy working outside. There is an increasing demand to prevent melanin pigmentation. Therefore, it was necessary to develop a whitening agent that prevents excessive melanin production, and many efforts have been made for this purpose.

Until now, the development of whitening agents has been mainly to find a substance that reduces the amount of melanin by inhibiting the activity of tyrosinase, an enzyme that is essential and most important in melanin biosynthesis. The developed whitening agents include kojic acid, arbutin, glutathione, vitamin A, vitamin C, etc., but they do not have a satisfactory whitening effect and have a limited use due to side effects such as causing skin irritation. Therefore, there is an urgent need for development of a whitening agent that is more effective and safer than ever before.

According to the above needs, the present inventors have studied to develop a whitening agent having no side effects and excellent efficacy. As a result, dilinolyl cystamine (N, N'-Dilinoleylcystamine), which has been used as an immune enhancer, has an excellent whitening effect and no side effects. The present invention has been accomplished by discovering that it can be used as a whitening agent.

Therefore, it is an object of the present invention to provide a new skin whitening composition which is excellent in whitening effect and has no side effects.

1A and 1B are photographs measuring the effect of dirinolysistamine on melanin production in human melanoma cells, where 1a is a control and 1b is a diolysylcystamine treated group.

Figures 2a and 2b is a photograph of the tyrosinase expression of HM3KO cells using an antibody against tyrosinase, 2a is a picture showing the separation of proteins on a 10% SDS gel, 2b is tyrosinase Photo shows the expression of.

<Description of Drawing Symbols>

a: control, b: cystamine treated group, c: dilinolyl cystamine treated group

Figure 3 is a graph showing the results of visual determination of the whitening effect measured after treating the test material for 8 weeks using brown guinea pigs.

Figure 4 is a graph showing the average value of the visual determination results of the whitening effect measured after treatment with the test material for 8 weeks using a brown guinea pig.

Figure 5 is a graph showing the whitening effect measured by the chromatometer after treatment with the test material for 8 weeks using a brown guinea pig for each week.

Figure 6 is a graph showing the average value of the whitening effect measured using a chromameter after treating the test material for 8 weeks using a brown guinea pig.

7A and 7B are photographs of the test material treatment site after treating the test material for 8 weeks using the brown guinea pig, 7a is a picture 1 week after the test material treatment, and 7b is 4 weeks after the test material treatment. It is a photograph.

<Description of Drawing Symbols>

a: untreated test material, b: control

c: hydroquinone (HQ) treatment unit (182 mM)

d: dilinolyl cystamine treatment (14 mM)

e: treatment of cystamine (14 mM) and linoleic acid (14 mM) mixture

In order to achieve the above object, the composition for skin whitening according to the present invention is characterized in that it contains dilinolyl cystamine (N, N'-Dilinoleylcystamine).

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

Dilinolyl cystamine, an active ingredient of the composition of the present invention, is known to be effective as an adjuvant.

[Formula 1]

In the present invention, dilinolylcystamine, which has been used for the above-mentioned applications, reduces the expression of tyrosinase to one-third of normal in the whitening experiment using melanoma cells, and in the experiment using guinea pigs, the skin color is changed by ultraviolet rays. The blackening is clearly inhibited without causing any side effects, unlike dilinolylcytamine treated with linoleic acid and cystamine, and the effect is similar at much lower concentrations than hydroquinone (HQ), which is used as a whitening agent. It is to find a degree, to provide a composition for skin whitening containing it as an active ingredient.

Dilinolyl cystamine in the skin whitening composition of the present invention is preferably contained in an amount of 0.00001 to 10% by weight based on the total weight of the composition. This is a content determined in consideration of the minimum content that can produce the effect, the degree of dissolution of the substance in the composition, side effects and the like.

The composition for skin whitening according to the present invention is not particularly limited, but may be formulated in the form of cosmetics such as external skin preparations, supple cosmetics, nourishing cosmetics, nutrition lotions, massage creams, essences, and packs.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited to these examples.

Test Example 1 Effect on Melanin Production in Human Melanoma Cells

Minimum Essential containing 10% fetal bovine serum from HM3KO cells (Y. Funasaka, Department of dermatology, Kobe university school of medicine, 5-1 Kusunoki-cho 7-chrome, Chuo-ku, Kobe650, Japan) Incubated in Medium (MEM) at 37 ℃, 5% CO ₂ conditions. The cells thus cultured are spread over 75 flasks with a cell count of 3 × 10 ^{5 for} each flask, and the cells are allowed to stick to the base wall overnight. After confirming that the cells adhered well, the medium was changed to a new medium containing 10 ppm of dilinolyl cystamine. As a control, a medium containing DMSO used to dissolve dilinolylcystamine was used. In this way, every two to three days, the cells were transferred to a new medium containing samples, and the cells were incubated until the flask was filled. When the cells were grown, the cells were collected and compared with the cell color of the control group and the dilinolyl cystamine treatment. The results are shown in FIGS. 1A and 1B.

It can be seen from FIG. 1A and FIG. 1B that the color of the dilinolylcystamine treated group (FIG. 1B) is lighter than that of the control (FIG. 1A).

Experimental Example 2 Observation of Tyrosinase Expression of HM3KO Cells Using an Antibody to Tyrosinase

A buffer containing 1% of Nonidet P-40 was added to the cells, disrupted by ultrasonication, and centrifuged at 12,000 rpm for 10 minutes. The supernatant was taken and boiled for 5 minutes, and electrophoresis was performed by adding 20 µg of protein to 10 g of SDS gel. The proteins thus separated were transferred to a nitrocellulose membrane. Tyrosinase antibodies purchased from Novocastra (Novocastra Laboratories Ltd, UK) were diluted to 500: 1 and treated on nitrocellulose membranes. ECL was used to identify the protein bound to the antibody. The results are shown in Figures 2a and 2b.

As a result of quantifying the amount of tyrosinase at 570 nm using a densitometer from FIG. 2b, the cells treated with dilinolylcystamine showed 357.4 and the control was 1070.6. As a result, it can be seen that the amount of tyrosinase is reduced to about one third by dilinolylcytamine.

[Test Example 3] Whitening animal experiment using brown guinea pig

Experimental Method

The hair on the back of the brown guinea pig having a weight of about 500 g was removed, anesthetized with pentobarbital (30 mg / kg), and then irradiated with 500 mJ ultraviolet rays. The irradiation site should be a circle about 1cm in diameter. In the same way, the same site was irradiated with ultraviolet rays three times a week. After the last UV irradiation, the sample was applied 5 L per circle from the next day to morning and evening. Samples are made by dissolving in a vehicle (ethanol: propylene glycol = 8: 2). The color change of the burned area is measured by using a chromameter once a week and the method of observing the difference in color with the eyes and giving a relative value, and taking a picture of the irradiation site It was.

<Experimental Result 1>

During the 8 weeks from the start of the application (week 0), the values of 0.5, 1, 2, and 3 are relatively different depending on the degree of whitening effect based on the untreated (rounded, burned area). Given. FIG. 3 shows the average values obtained by visually judging each of the four experimental animals, and FIG. 4 shows the average values of the determination values for eight weeks. Dilinolyl cystamine-coated circles (dilinolyl cystamin- gu) showed a clear whitening effect for 2-3 weeks compared to the control (vehicles with a circle). Unlike quinone (HQ), side effects were hardly seen during sample application (see FIG. 3).

As a result of gross evaluation for 8 weeks, dilinolyl cystaminocytes showed a whitening effect of about 2.3 times higher than that of the control and 0.8 times higher than the HQ at a very low concentration (see FIG. 4).

Experimental Result 2

The degree of shading of each application site was quantified using a chromameter for 8 weeks from week (0 week) starting application. The measurements were averaged three times in the range not to deviate from the inside of the circle to which the sample was applied. FIG. 5 shows chromatographs of four experimental animals, and the average value for each sample is shown from 0 to 8 weeks, and FIG. 6 shows the average of the measured values over 8 weeks for each sample. Dilinolyl cystamine shows a whitening effect that continues to increase from two weeks after the application as shown in the visual determination results presented above (see FIG. 5). Looking at the results of the eight weeks in Figure 6 it can be seen that also showed a higher value than the non-treated, control, HQ.

Experimental Result 3

Results of photographing for 8 weeks are shown in FIGS. 7A and 7B. The photograph of the application site | part after 1 week of sample application | coating (FIG. 7A) and 4 weeks after application | coating (FIG. 7B) is shown. If you look at the site where you applied dilinolylcytamine, you noticed that it became noticeably white at 4 weeks. On the other hand, when cystamine and linoleic acid are applied together, the dead skin comes off from 1 week and the skin becomes red, and after 4 weeks, it becomes much darker than the control.

Test Example 4 Whitening Effect Test on Human Skin

The overall experimental method is the same as in the animal level efficacy evaluation of Example 3 above. After attaching an opaque tape with 6 holes of 1.5cm diameter to the upper forearm of 12 subjects, 12 1.5 times of UVB of each subject's minimum erythema (Minimal Erythema Dose) Was irradiated to induce blackening of the skin, the test materials were applied, and after 2 months, the contrast of the skin was measured using a chromameter. Each test substance was prepared by applying a skin external preparation (lotion) according to the following Comparative Examples and Examples twice a day (morning, evening) every day.

<Example 1 and Comparative Example 1> nutrition lotion

ingredient Example 1 Comparative Example 1 1. Purified water to 100 to 100 2. Glycerin 8.0 8.0 3. Butylene Glycol 4.0 4.0 4. Hyaluronic Acid Extract 5.0 5.0 5. Beta Glucan 7.0 7.0 6. Carbomer 0.1 0.1 7. Dilinolylcytamine 1.0 - 8. Caprylic Capric Triglycerides 8.0 8.0 9. Squalane 5.0 5.0 10. Cetearyl Glucoside 1.5 1.5 11.Sorbitan Stearate 0.4 0.4 12. Cetearyl Alcohol 1.0 1.0 13. Preservative Quantity Quantity 14. Incense Quantity Quantity 15. Triethanolamine 0.1 0.1

<Production method>

1) The raw materials 1-6 were mixed, melt | dissolved at 70 degreeC, and it was set as the water phase.

2) The raw materials 7-14 were melt | dissolved at 70 degreeC, and it was set as the oil phase.

3) The oil phase was added to the aqueous phase, stirred with a homomixer (Tokushu Kika, Japan) to emulsify first, and the raw material 15 was added to increase. After removing the bubbles, the nutrient lotion was prepared by cooling to room temperature.

The determination of the effect was determined by obtaining an "L" value representing the skin's contrast as in animal testing (unburned Korean skin color generally ranges between 50 and 70). When the test substance is applied, the L value is gradually increased, and the comparison between the test substances is expressed as ΔL value (final L value-value before application of the test substance). The larger the ΔL value, the greater the whitening effect.

As a result of the experiment, as can be seen from Table 1, Example 1 containing dilinolyl cystamine was superior to Comparative Example 1 containing no dilinolyl cystamine.

Test substance Whitening effect (ΔL) Example 1 2.89 Comparative Example 1 1.25

[Test Example 5] Skin irritation measurement

The patch test was performed to observe the degree of primary skin stimulation and cumulative stimulation of the nutrient lotion of Example 1. That is, the nutrition lotion of Example 1 was correctly applied to the forearm of each of five healthy male and female subjects for 14 days and the degree of stimulation was observed. As a result, the nutrition lotion of Example 1 had no primary stimulus and almost no cumulative stimulus.

As can be seen from the above, dilinolyl cystamine is excellent in inhibiting the production of melamine, and thus can be usefully used as a whitening agent.

Claims (2)

Dilinolyl cystamine (N, N'-Dilinoleylcystamine; C ₂₁ H ₃₉ NOS; Molecular weight: 353.61) of the general formula (I). [Formula 1] The composition for skin whitening according to claim 1, wherein the dilinolyl cystamine is contained in an amount of 0.00001 to 10% by weight based on the total weight of the composition.