KR100458701B1 - Whitening makeup composition containing sphingolipid or sphingolipid metabolites - Google Patents

Abstract

The present invention relates to a whitening cosmetic composition containing sphingolipid, sphingolipid and its metabolism that inhibits DNA synthesis to inhibit the growth and development of melanocytes, as well as inhibits the activity of tyrosinase to reduce melanin formation. The composition prepared by containing the product C ₂ -ceramide or C ₆ -ceramide has an excellent whitening function.

Description

Whitening cosmetic composition containing sphingolipid or its metabolite {WHITENING MAKEUP COMPOSITION CONTAINING SPHINGOLIPID OR SPHINGOLIPID METABOLITES}

[Industrial use]

The present invention relates to a whitening cosmetic composition containing sphingolipid or a metabolite thereof. More specifically, whitening cosmetics comprising sphingolipid or its metabolite C ₂ -ceramide or C ₆ -ceramide to inhibit the proliferation and division of melanocytes and inhibit the activity of tyrosinase to reduce melanin formation To a composition.

[Prior art]

The pigments that determine human skin color include melanin, hemoglobin and carotenoids, and the various colors of human skin, hair and eyes are determined by these pigments. The most important pigment that determines the color of the skin is melanin, the skin color is determined by the nature and degree of distribution of melanin.

In humans, skin and hair pigments protect against the harmful effects of sunlight (especially ultraviolet rays). For example, people with poor pigmentation are very sensitive to sunlight and are prone to burns (ALBINOS), and are at high risk of developing skin cancer even at a young age. Short wavelength ultraviolet light (290-320 nm) and carcinogens form oxygen radicals in the skin, which are known to attack skin cells and age the skin.

The main function of melanin is to remove oxygen radicals and protect the skin from the damage thereby. Therefore, high levels of melanin mean that it has an effective response system to protect the skin from physical and chemical toxic substances. However, from the human aesthetic point of view, freckles and blemishes formed by melanin are negative factors.

Factors that promote the production of melanin include sunlight (ultraviolet rays) as well as hormones such as estrogen, prostaglandin and threomyo sin. The melanin production and extinction cycle produces melanosomes when the melanocytes are stimulated, in which the amino acid tyrosine contained in the cell binds to tyrosinase, which is activated by stimulation. . In this case, tyrosine is combined with oxygen and oxidized, and tyrosinase should be combined with copper. If the skin lacks copper, it will interfere with normal melanogenesis. Stimulation such as ultraviolet light not only directly affects melanocytes, but also affects the stimulating hormone system (MSH; Melanocyte Stimulating Hormone). The amino acid tyrosine contained in the melanocytes is changed into a substance called dopa by the action of an oxidase called tyrosinase, and then into a substance called dopa-quinone by the action of an enzyme called dopaoxidase. Melanin is produced. This melanin is circulated in skin cells and with the epidermal peeling, melanin is lost and disappeared. The melanin production mechanism is characterized by a reaction involving only one enzyme, tyrosinase, which inhibits the tyrosinase activity and thus prevents melanin production, thus expecting a whitening effect.

As a method of suppressing melanin production in whitening cosmetics can be divided into the following largely. One method is to block the ultraviolet rays to remove the main cause of melanin production, this method can contain a light scattering agent or a light blocking agent as a cosmetic composition can be expected good results. Another method is to inhibit melanin production by inhibiting the synthesis of core carbohydrates required for tyrosinase to be active, such as gluco samine. Another method is to interfere with the function of tyrosinase, the only enzyme involved in the production of melanin, such as kojic acid or arbutin. Another method is to disrupt cell division because it has specific toxicity against melanocytes, which produce melanin, such as hydroquinone. Another method is to reduce the melanin produced to decolorize.

Ceramide derivatives, such as ceramide or sphingosine-1-phosphate (SPP), also known as spingolipids, are major lipids that form the membrane structure of higher cells such as animals, plants, and yeast. Many studies are being conducted as bioactive functions such as differentiation and role as intermediate transporter of signaling system have been revealed. In particular, as the sphingolipids, which are present on the skin layer of human skin tissue (stratum comium) and inhibit moisture evaporation, have been discovered, various types of natural or synthetic sphingolipids have begun to be used as functional compositions of cosmetics. .

Ceramide (ceramide) is a very important component in the outer skin of the skin epidermal layer, and plays an important role in forming and preserving the lipid impermeable membrane having water retention capacity of the skin. In addition, the efficacy of cosmetics by controlling the growth and division of melanocytes (melanocyte) to further improve the healthy skin structure and tissues and to strengthen damaged skin recovery and protection.

Sphingolipids and their metabolites are involved in cell growth and division. For example, ceramide, a metabolite of sphingolipid, stimulates cell division in fusion inactive Swiss 3T3 fibroblasts (Olivera et al., 1994) and HL-60 by 1α-25-dihydroxyvitamin D3 Reducing monocyte proliferation of cells (Okazaki et al., 1989) and reducing the division of cell line DJM-1 associated with keratinocytes (Kawita et al., 1994). As mentioned above, there have been many studies on the effectiveness of ceramide on a wide range of cells, but few studies on the action of ceramide on melanocytes.

Therefore, studies on the action on melanocytes using the effectiveness of sphingolipids and metabolites, ceramides, or studies on whitening cosmetic compositions containing the sphingolipids or their metabolites are required.

Accordingly, an object of the present invention is a whitening cosmetic composition containing the following C ₂ -ceramide or C ₆ -ceramide, which is a sphingolipid or a metabolite thereof which inhibits the proliferation and division of melanocytes and inhibits the melanin response to reduce melanin production. C ₂ -ceramide (Formula: N-Acetyl-D-erythro-sphingosine) C ₆ -ceramide (Formula N-Hexanoyl-D-erythro-sphingosine)

1 is a diagram showing the inhibitory effect of [³ H] thymidine uptake in Mel-Ab melanin cells according to the degree of administration of C₂-ceramide, the metabolite of sphingolipid of the present invention,

Figure 2 is a diagram showing the effect of [³ H] thymidine uptake (2a) and cell proliferation inhibitory effect (2b) in melanoma cells G361 according to the degree of administration of the metabolite C₂-ceramide of the present invention sphingolipid,

Figure 3 is a diagram showing the effect of inhibiting Mel-Ab melanin cell growth according to the degree of administration of C₂-ceramide, the metabolite of the present invention sphingolipid,

Figure 4 is a diagram showing the inhibitory effect of tyrosinase activity according to the degree of administration of the C₂- ceramide metabolite of the present invention sphingolipid,

5 is a diagram showing whether C 2 -ceramide, a metabolite of sphingolipid of the present invention, is directly involved in tyrosinase inhibition.

In order to achieve the above object, the present invention is a whitening cosmetic containing C ₂ -ceramide or C ₆ -ceramide which is a sphingolipid or its metabolite which inhibits the proliferation and division of melanocytes and inhibits the melanin response to reduce melanin production. To provide a composition.

The sphingolipid metabolite in the above is preferably cell-permeable active-ceramide (active-ceramide) having a short chain (C ₂ or C ₆ ) short chain (short chain).

Hereinafter, the present invention will be described in detail.

The material [methyl- [H] thymidine used in the present invention was purchased from Amersham Pharmaia Bioech (Piscataway, NJ, USA), and active-ceramide and SPP were supplied from Alexis (SanDiego, CA, USA), active in the present invention. C2-ceramide (N-acetyl-D-erythro-sphingosine) in ceramide was used. ffa-BSA (fatty acid-free bovine serum albumin), 12- O- tertradecanoylphorbol-13-acetate (TPA), cholera toxin (CT), ribonuclease, propidium iodide, yeast acid, synthetic melanin, L-DOPA, mushroom tyrosinase, MTT [3- (4,5-demethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] were purchased from Sigma (St. Louis, MO, USA). Ceramide and SPP were administered intracellularly with 0.4% ffa-BSA.

The present invention sphingolipids or its metabolite, C ₂ - ceramide or C ₆ - ceramide or C ₆ - - sphingolipids or its metabolite, C ₂ in the whitening cosmetic composition containing the ceramide amount of a skin cosmetic composition of the ceramide is Although not limited, 0.01 to 30.0% by weight is most preferred.

The whitening cosmetic composition containing the present invention sphingolipid or its metabolite C ₂ -ceramide or C ₆ -ceramide can be prepared as a skin contact material in contact with the skin such as cosmetics, detergents and fibers.

Considering that the main factor of melanin formation is ultraviolet rays, the composition of the present invention may include a sunscreen or a light scattering agent such as a benzophenone-based sunscreen in addition to the reduction and decolorization function of melanin to obtain a white and clean whitening effect.

The whitening cosmetic composition containing the sphingolipid and its metabolite C ₂ -ceramide or C ₆ -ceramide of the present invention is a common ingredient formulated in general skin cosmetics, such as oil, water, surfactant, humectant, and lower alcohol. It is possible to apply and mix as much as necessary a thickener, a chelating agent, a pigment, a preservative, a fragrance | flavor, etc. In the present invention, the skin cosmetic is used for the skin, for example, a cosmetic, such as a lotion, milky lotion, cream, pack, essence, and the application in particular to the whitening cosmetics.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are only one preferred embodiment provided to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1 Cell Proliferation Inhibition Experiment

In order to measure the cell proliferation inhibitory effect of the whitening cosmetic composition containing sphingolipid of the present invention, C 2 -ceramide was administered to Mel-Ab cells and human melanoma cell line G361 cells to confirm the cell proliferation inhibitory effect.

1. Cell Culture

Human epidermal melanocytes were isolated from the penile epidermis of adolescents according to Eisinger and Marko (Eisinger, M., and O.Marko: Proc. Natl. Acad. Sci. USA, 79: 2018-22). Orodlund) was cultured with 5% FBS, 13 ng / mL bovine pituitary extract (BPE), 10 ng / mL TPA, 1 ng / mL human recombinant fibroblast growth gene (Gibco BRL), penicillin-styreptomycin each of 1000 U / mL, 1000 ㎍ / mL was added to the prepared; Sigma Co.), and then was stored at 5% CO _2, incubator of 37 ℃ in the laboratory stored at. Mel-Ab cells were treated with 5% CO ₂ , 10% fetal bovine serum (FBS), 100 nM TPA, 1 nM CT, 50 μg / mL streptomycin and 50 μg / mL penicillin at 37 ° C. Melanoma cells G361 were cultured in RPMI supplemented with 10% FBS, 50 μg / mL streptomycin, 50 μg / mL penicillin at 5% CO ₂ , 37 ° C.

2. Inhibition of Proliferation of Mel-Ab Cells

Mel-Ab melanocytes cultured in the same manner as in Example 1-2 and the experimental group incubated with C₂-ceramide for 72 hours and Mel-Ab melanocytes not treated with C₂-ceramide as control group Mel-Ab cells The degree of inhibition of proliferation was measured. After incubation as described above, the culture medium was removed and each well with 10% ethanol was colored for 5 minutes at room temperature in place of 0.5 mL of 0.1% crystal violet and washed four times. The crystal violet remaining in the cells was extracted in 1.0 mL of 95% ethanol and the absorption was measured by a crystal violet assay measured spectrophotometrically at 590 nm using an ELISA reader (Dooley et al., 1994).

As a result, the effect of inhibiting cell proliferation according to the degree of administration of C₂-ceramide was shown in FIG. 1. In the experimental group treated with C₂-ceramide at the concentration of 10 uM, [³ H] thymidine binding was reduced by 52% compared to the control group, but it did not affect the concentration below 1 uM.

3. Inhibition of Proliferation of G361 Cells

The melanoma cell line G361 of humans prepared as in Example 1-1. Was measured in the same manner as in Example 1-2. Experimental results As shown in FIG. 2, DNA synthesis exhibited by [³ H] pyridine binding showed a marked decrease in the experimental group administered at a C 2 -ceramide concentration of 1-10 uM compared to the control group.

In this example, it was confirmed that the administration of C 2 -ceramide, a metabolite of sphingolipid, reduced DNA synthesis and inhibited the proliferation and division of melanocytes.

Example 2 Experiment for Inhibiting ERK and Akt / PKB Activity of Melanocytes

To determine whether C₂-ceramide has anti-proliferative effect on melanocytes by regulating the activity of ERK and Akt / PKB, each of the melanocytes cultured as in Example 1-1 above with C₂-ceramide three times each Western blot analysis was performed over. As shown in FIG. 3, ERK1 and ERK2 were inactivated 60 minutes after adding ceramide to melanocytes, whereas Akt / PKB was inactivated after 3 hours or more. Through this, it was confirmed that C ₂ -ceramide inhibited the proliferation of melanocytes by inactivating ERK and Akt / PKB, and through this, melanin formation was suppressed, thereby showing a whitening effect.

Example 3 Tyrosinase Activity Inhibition Experiment

In this example, in order to test the inhibition of tyrosinase activity, the experiments of Koji Tomita, etc., which are conventional methods, were slightly modified using the C₂-ceramide treatment group and the yeast acid treatment group. This experimental method is a method of calculating the tyrosinase activity inhibition rate by measuring the amount of melanin production by adding melanin-producing material tyrosine to the extract to be tested. Melanocytes were plated at a density of 2000 cells / well on a 96-well plate, the cells were washed with PBS, lysed in 1% Triton-X / PBS (90 μl / well), and frozen at −80 ° C. for 30 minutes. . After thawing, 10 μl of 10 nM L-DOPA was added to each well, mixed, and incubated at 37 ° C. for 1 hour. Absorption was measured at 475 nm and experimental values were normalized to mushroom tyrosinase. In addition, the cell-free assay system was used to determine whether C₂-ceramide directly acts on tyrosinase.

As shown in FIG. 4, tyrosinase activity was decreased according to the degree of administration of C₂-ceramide, which was accompanied by a decrease in melanin levels. The effect of C₂-ceramide (1-100 uM) on inhibition of tyrosinase activity was much stronger than that of 1-100 uM yeast acid, because C₂-ceramide inhibits melanin synthesis in melanocytes after modulating tyrosinase activity. .

Experimental results for confirming that C₂-ceramide directly inhibits tyrosinase As shown in FIG. 5, yeast acid significantly inhibited tyrosinase activity in the cell-free system, but C₂-ceramide did not inhibit tyrosinase activity. Through this, it was confirmed that C₂-ceramide inhibits activity by indirectly acting on tyrosinase.

Examples 4 to 6 Preparation of Creamy Emulsion

For whitening clinical experiments of the whitening cosmetic composition containing the sphingolipid and the metabolite of the present invention, the aqueous phase and oil phase components were heated to 75 ° C. according to the respective compositions as shown in Table 1 below. After mixing and emulsifying the components, the neutralizing agent was added and cooled to 40 ℃, and then added to the flavor and additives to prepare four creamy emulsions.

TABLE 1

As mentioned above, the whitening cosmetic composition containing the sphingolipid or its metabolite C ₂ -ceramide or C ₆ -ceramide of the present invention inhibits DNA synthesis in melanocytes and inhibits the growth and development of cells. It inhibits tyrosinase activity and reduces melanin formation, so it has an excellent effect on whitening activity.

Claims (7)

A whitening cosmetic composition containing C ₂ -ceramide or C ₆ -ceramide as an active ingredient that inhibits the growth and development of melanocytes and inhibits melanin formation. The whitening cosmetic composition according to claim 1, which contains 0.01 to 30.0 wt% of the C ₂ -ceramide or C ₆ -ceramide. delete The whitening cosmetic composition according to claim 1, wherein the whitening cosmetic composition contains C ₂ -ceramide or C ₆ -ceramide formulated in any one of lotion, milky lotion, cream, pack, cosmetic liquid. delete delete delete