WO2004045573A1

WO2004045573A1 - Composition for skin whitening containing n-acetylophytosphingosine

Info

Publication number: WO2004045573A1
Application number: PCT/KR2003/002474
Authority: WO
Inventors: Chang Seo Park; Jin Wook Kim; You-A Hwang; Mee Kyung Park; Young Sook Yoo; She Yoon Yi
Original assignee: Doosan Corporation
Priority date: 2002-11-19
Filing date: 2003-11-18
Publication date: 2004-06-03
Also published as: KR100956698B1; AU2003280880A1; KR20040043645A

Abstract

The present invention relates to a composition containing N-acetylphytosphingosine for skin whitening through the inhibition of the activity of tyrosinase or peeling of hyperpigmented skin cells. The composition is effective in inhibiting the synthesis of melanine by repressing the activity of tyrosinase and thereby preventing skin pigment-related symptoms such as discoloration, freckle, senile chromelasma, hyperpigmentation. Also, the composition is effective in treating skin pigment-related symptoms such as discoloration, freckle, senile chromelasma, hyperpigmentation by peeling hyperpigmented skin cells. Furthermore, the composition can be used in cosmetic and pharmaceutical field.

Description

COMPOSITION FOR SKIN WHITENING CONTAINING N-ACETYLPHYTOSPHΓNGOSINE

TECHNICAL FIELD The present invention relates to a sphingolipid inhibiting the synthesis of melanin in the melanocytes which play a key role in determining the color of human skin. More particularly, the present invention relates to the effective use of N- acetylphytosphingosine, which is a acetylated derivative of phytosphingosine, in whitening cosmetics and pharmaceuticals for the prevention and treatment of discoloration, freckles, senile chromelasma and hyperpigmentation due to the potential inhibitory functions of N-acetylphytosphingosine on melanin synthesis and tyrosinase activity.

BACKGROUND ART Hyperpigmentation of the skin is a severe psychological burden to the affected individuals from a dermatological viewpoint, and further negatively affects their social activities. Indeed, there is a recent social trend that white and fair skin is regarded as a criterion of beauty. In this connection, studies on whitening agents are being actively undertaken. Recent research on whitening agents has been conducted toward the control of various factors involved in the melanin synthetic pathways, e.g., blocking of UV light, control of cytokines or hormones, regulation of internal signals in the melanocytes, inhibition of tyrosinase activity, inhibition of melanosome migration, rapid desquamation of melanin-pigmented skin cells, etc. It is well known that sphingolipids are involved in intracellular signal transmission and play a critical role in cellular growth, differentiation and death. It is also known that ceramides, which are the most widely found lipids in the skin, inhibit moisture vaporization from the skin and prevent the progress of aging and skin diseases. Further, phytosphingosine as a long-chain base of the sphingolipids, or an acetylated derivative thereof is known to have excellent antibacterial and anti- inflammatory effects and is involved in ceramide biosynthesis within the skin. Although a great deal of research on ceramide and sphingolipid derivatives in the keratinocytes has been conducted, no research has been conducted on the synthesis of melanin.

Kim et al. first conducted their research associated with melanin synthesis using sphingolipid derivatives (D. S. Kim, Cellular signaling 14 (2002) 779-785). As a result, they found that a ceramide derivative (C2 ceramide) exhibits better inhibitory effects on melanin synthesis at a concentration of 1-1 OμM than kojic acid, and reported the finding that sphingosine-1 -phosphate inhibits melanin synthesis by regulating the signal transmission processes of melanin synthesis.

DISCLOSURE OF THE INVENTION

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for inhibiting the activity of tyrosinase using a sphingolipid derivative, thereby preventing melanin hyperpigmentation of the skin and thus preventing skin pigment-related symptoms such as discoloration, freckles, senile chromelasma and hyperpigmentation.

It is another object of the present invention to provide a method for treating skin pigment-related symptoms such as discoloration, freckles, senile chromelasma and hyperpigmentation by peeling hyperpigmented skin cells. It is yet another object of the present invention to provide a skin composition for external use having the above-mentioned inhibitory and treating effects.

According to the present invention, the above objects can be accomplished by a composition for inhibiting the activity of tyrosinase which comprises N- acetylphytosphingosine as an active ingredient, represented by Formula 1 below:

Formula 1

In accordance with another aspect of the present invention, there is provided a composition for peeling hypeφigmented skin cells which comprises N- acetylphytosphingosine of Formula 1 as an active ingredient.

In accordance with another aspect of the present invention, there is provided a composition for skin whitening which comprises N-acetylphytosphingosine of Formula 1 as an active ingredient. The present invention is characterized in that the compositions are those for external use, in particular, cosmetic compositions, and are preferably prepared into cosmetic formulations such as softening lotions, nutritive lotions, massage creams, gels, nutritive creams, packs and skin patches for external use.

The compositions of the present invention preferably comprise N- acetylphytosphingosine in an amount of 0.005-10% by weight.

The compositions of the present invention may be prepared into formulations for transdermal administration such as lotions, ointments, gels, creams, patches and nebulas.

In accordance with another aspect of the present invention, there is provided a method for inhibiting the activity of tyrosinase using N-acetylphytosphingosine of Formula 1. In accordance with yet another aspect of the present invention, there is provided a method for promoting the peeling of hypeφigmented skin cells using N- acetylphytosphingosine of Formula 1.

BRIEF DESCRD?TION OF THE DRAWINGS The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Fig. 1 is the result of RT-PCR (reverse transcription polymerase chain reaction) analysis showing the influence of N-acetylphytosphingosine used in the present invention on the expression of the TRP-2 (tyrosinase related protein 2) gene;

Fig. 2 is the result of RT-PCR (reverse transcription polymerase chain reaction) analysis showing the influence of N-acetylphytosphingosine used in the present invention on the expression of the TRP-1 (tyrosinase related protein 1) gene; and

Fig. 3 is the result of RT-PCR (reverse transcription polymerase chain reaction) analysis showing the influence of N-acetylphytosphingosine used in the present invention on the expression of the tyrosinase gene.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be described in more detail. The color of human skin is determined by various skin components. Of these skin components, melanin produced in the melanocytes is most responsible for skin color. The skin melanocytes are regulated by various genetic characteristics according to race so as to exhibit the characteristic skin colors of different races. When the skin is stimulated by many factors including UV light, stress, cytokines and melanin synthesis promoting factors such as melanocyte-stimulating hormone (MSH), etc., melanin is increasingly synthesized, migrates to adjacent keratinocytes through the melanosome and is finally peeled off from the skin (desquamation).

In general, temporary change in skin color is naturally returned to the original skin color through the mechanism as described above with the lapse of time. However, when exposed to sunlight for a long period of time, the skin may irreversibly become tanned.

Melanin is synthesized in the melanosome of the melanocyte, which originates from the endoplasmic reticulum. Due to the internal structure of the melanosome, the synthesized melanin is regularly accumulated in the melanosome. Tyrosinase is an enzyme converting tyrosine to melanin. Tyrosinase is synthesized in the Golgi apparatus and then migrates to the melanosome. In premature melanosomes, while tyrosine goes through oxidation by the action of the tyrosinase, the biosynthesis of melanin begins. The premature melanosomes where melanin synthesis begins undergo 4 growth stages until they develop into mature melanosomes. The mature melanosomes migrate to adjacent keratinocytes. At this time, the color of human skin is determined by the number, size and distribution of melanin in the keratinocytes. The melanosomes migrate from the stratum basale of the epidermis to the keratinocytes and then migrate to the stratum corneum of the skin while protecting the keratinocytes. During migration, the melanosomes are degraded, but the melanin is not degraded and is finally desquamated from the skin. The tyrosinase playing the most important role in the biosynthesis of melanin is synthesized in the Golgi apparatus and undergoes glycosylation. After the glycosylated tyrosinase migrates to the melanosomes, it is converted to its active state by physphorylation. Melanin synthesis begins with the oxidation of tyrosine as the substrate by the activated tyrosinase. The tyrosine is converted to dihydroxyphenylalanine (DOPA) by tyrosinase, and then converted to DOPA quinone by the same enzyme. The DOPA quinone binds with glutathione or cysteines to produce cysteinyl DOPA, which is further converted to pheomelanin. Alternatively, the DOPA quinone is converted to DOPA chrome to produce 5,6-dihydroxyindole, which is converted to indole-5,6-quinone by the action of tyrosinase. The indole-5,6-quinone is finally converted to eumelanin. As is well known, tyrosinase is involved not only in the initial synthetic step but also in the final synthetic step of melanin and thus plays the most important role in the synthesis of melanin. Accordingly, many substances having inhibitory effects on melanin synthesis have been developed focusing on the inhibition of tyrosinase activity. The compositions of the present invention comprise N-acetylphytosphingosine as an active ingredient to inhibit tyrosinase activity.

The eumelanin and pheomelanin synthesized in the melanocytes are present in various ratios depending on race or body sites. These ratios between the eumelanin and pheomelanin represent different colors of human skin. The eumelanin is black or brown, and the pheomelanin is orange, both of which are synthesized from two amino acids, i.e. tyrosine and cysteine, respectively.

The melanin functions to protect the skin from UV light and to protect skin organs lying beneath the hypodermis. The melanin also captures reactive oxygen and free radicals and thus protects proteins and nucleic acids. In spite of these beneficial functions, abnormal formation of melanin and pigmentation of the skin cause skin conditions such as discoloration, freckles and skin pigment anomalies.

When the compositions of the present invention comprising 0.005-10% by weight of N-acetylphytosphingosine as an active ingredient are pharmaceutical compositions for transdermal administration, they are preferably administered in an appropriate amount twice daily.

The present inventors measured melanin synthesis and tyrosinase activity using phytosphingosine derivatives, which can be easily prepared by yeast fermentation, and as a result discovered that an acetylated derivative, N-acetylphytosphingosine among the phytosphingosine derivatives exhibits better inhibitory effects on melanin synthesis and tyrosinase activity than kojic acid, even at much lower concentration (1/1,000). Further, the present inventors found that N-acetylphytosphingosine exhibits better inhibitory effects on melanin synthesis and tyrosinase activity than C2 ceramide (having a sphingosine backbone structure) and sphingosine-1 -phosphate reported by Kim et al. Furthermore, the present inventors found that when human skin cells were artificially hypeφigmented and then a cream prepared from the composition according to the present invention was applied on the hypeφigmented skin cells, the hypeφigmented skin cells were peeled off in a short time.

Hereinafter, the present invention will be described in more detail with reference to the following preferred examples. However, these examples are given for the piupose of illustration and are not to be construed as limiting the scope of the invention.

Examples Some preferred creams (Examples 1 to 4) containing N-acetylphytosphingosine at various concentrations were prepared to have the compositions shown in Table 1 below. For comparison with the creams of Examples 1 to 4, a cream of Comparative Example 1 was prepared as a negative control to have the composition shown in Table 1 below.

Table 1

In order to demonstrate the whitening effects of N-acetylphytosphingosine and the compositions of the present invention, the present inventors conducted the following experiments.

Experimental Example 1 : Inhibitory effect of N-acetylphytosphingosine on melanin synthesis>

Phytosphingosine, N-acetylphytosphingosine and tetraacetylphytosphingosine were chosen as sphingolipids, and their inhibitory effect on melanin synthesis were evaluated. The phytosphingosine derivatives were synthesized by acetylation of phytosphingosine prepared by yeast fermentation. The sphingolipids were dissolved in DMSO (dimethylsulfoxide) before use. As a cell line used in the present invention, B16F10 mouse melanoma cells were used. The cell line was incubated in DMEM (Dulbecco's Modified Eagle Medium) in a 5% CO₂ incubator at 37°C supplemented with 10% FBS (fetal bovine serum) and antibiotics.

The melanin content was measured in accordance with the following procedure. First, the B16F10 mouse melanoma cells were inoculated on a 24- ell multi-plate in an amount of 2 x 10³ cells/well, and then incubated in a 5% CO₂ incubator for 24 hours. Following pre-incubation, the sphingolipids were diluted in serum-free DMEM (Dulbecco's Modified Eagle Medium). After the cell line was treated with the compounds at various concentrations, it was cultured for 96 hours. The cultures were washed with PBS, and 0.85N KOH was added thereto. After the resulting cultures were dissolved by sonication, the light absorbance was measured at 475nm to determine the total melanin content. Eumelanin and pheomelanin were fractionally quantified using high speed chromatography. The obtained values were expressed as percentage relative to the control. The results are shown in Table 2 below. The values shown in Table 2 represent the formation rates of melanin, i.e. formation percentages (%) relative to that of the untreated group.

Table 2

As can be seen from the data shown in Table 2, the group treated with 5μM N- acetylphytosphingosine exhibited remarkable inhibitory effects on melanin synthesis by a decrease of about 60% when compared to the untreated group. However, it was observed that phytosphingosine and tetraacetylphytosphingosine increased melanin synthesis. Since the inhibitory effect of kojic acid on melanin synthesis was observed at a high concentration ranging from 500μM to lmM, it is concluded that it is meaningless to directly compare the inhibitory effect of kojic acid on melanin synthesis with those of the test compounds used in the present invention. The above experimental results reveal that N-acetylphytosphingosine exhibits better inhibitory effect on melanin synthesis than C2 ceramide and sphingosine- 1 -phosphate reported by Kim et al.

Experimental Example 2: Inhibitory effect of N-acetylphytosphingosine on tyrosinase activity>

In order to determine whether N-acetylphytosphingosine is effective as a tyrosinase inhibitor, the present inventors conducted the following experiment.

In this Experimental Example, phytosphingosine, N-acetylphytosphingosine and tetraacetylphytosphingosine chosen as sphingolipids were dissolved in DMSO

(dimethylsulfoxide) before use, as Experimental Example 1. As a cell line used in the present invention, B16F10 mouse melanoma cells were used. The cell line was incubated in DMEM (Dulbecco's Modified Eagle Medium) in a 5% CO₂ incubator at

37°C supplemented with 10% FBS (fetal bovine serum) and antibiotics. The inhibitory effect of the compounds on tyrosinase activity was measured in accordance with the following procedure. First, the B16F10 mouse melanoma cells were inoculated on a 96- well multi-plate in an amount of 1 x 10⁴ cells/well, and then incubated in a 5% CO₂ incubator for 24 hours. Following pre-incubation, the test compounds were diluted in serum-free DMEM (Dulbecco's Modified Eagle Medium). After the cell line was treated with the compounds at various concentrations, it was cultured for 96 hours. The respective cultures were washed with PBS twice, and 1% Triton X-100/PBS was added thereto. After the resulting cultures were dissolved by sonication, 50μl of lOmM L-Dopa which was previously prepared at 37°C was added thereto. The mixtures were reacted at 37°C for 30 minutes. The light absorbance of the mixtures was measured at 475nm. The results are shown in Table 3 below. The values shown in Table 2 represent the activity of tyrosinase, i.e. activity percentages (%) relative to that of the untreated group.

Table 3

As can be seen from the data shown in Table 3, the group treated with 5μM N- acetylphytosphingosine exhibited remarkable inhibitory effects on tyrosinase activity by a decrease of about 50% when compared to the untreated group. However, it was observed that phytosphingosine and tetraacetylphytosphingosine increased tyrosinase activity. Since the inhibitory effect of kojic acid on tyrosinase activity was observed at a high concentration ranging from 500μM to ImM, like the inhibitory effect on melanin synthesis, it is concluded that it is meaningless to directly compare the inhibitory effect of kojic acid on tyrosinase activity with those of the test compounds used in the present invention.

Experimental Example 3: Confirmation of inhibitory mechanism of N- acetylphytosphingosine on tyrosinase activity> In order to confirm the inhibitory mechanism of N-acetylphytosphingosine on tyrosinase activity, the present inventors conducted the following experiment.

The expression of TRP-1 (tyrosinase related protein 1) and TRP-2 (tyrosinase related protein 2) was evaluated by RT-PCR (reverse transcription polymerase chain reaction) to confirm the inhibitory mechanism of N-acetylphytosphingosine on tyrosinase activity. B16F10 mouse melanoma cells were treated with N- acetylphytosphingosine at concentrations of 1 and 5μM, and then cultured for 0, 48 and 72 hours. Total RNA was extracted using TRIZOL and RT-PCR was performed.

The results obtained associated with TRP-2 are shown in Fig. 1. Lane 1 represents the results obtained before culture, lane 2 represents the results obtained after treating with lμM N-acetylphytosphingosine for 48 hours, lane 3 represents the results obtained after treating with 5μM N-acetylphytosphingosine for 48 hours, lane 4 represents the results obtained after treating with lμM N-acetylphytosphingosine for 72 hours, and lane 5 represents the results obtained after treating with 5μM N- acetylphytosphingosine for 72 hours, respectively. The results obtained associated with TRP-1 are shown in Fig. 2. As shown in

Fig. 1, lane 1 represents the results obtained before culture, lane 2 represents the results obtained after treating with lμM N-acetylphytosphingosine for 48 hours, lane 3 represents the results obtained after treating with 5μM N-acetylphytosphingosine for 48 hours, lane 4 represents the results obtained after treating with lμM N- acetylphytosphingosine for 72 hours, and lane 5 represents the results obtained after treating with 5μM N-acetylphytosphingosine for 72 hours, respectively.

The results obtained associated with tyrosinase are shown in Fig. 3. As shown in Figs. 1 and 2, lane 1 represents the results obtained before culture, lane 2 represents the results obtained after treating with lμM N-acetylphytosphingosine for 48 hours, lane 3 represents the results obtained after treating with 5μM N-acetylphytosphingosine for 48 hours, lane 4 represents the results obtained after treating with lμM N- acetylphytosphingosine for 72 hours, and lane 5 represents the results obtained after treating with 5μM N-acetylphytosphingosine for 72 hours, respectively.

As shown in Figs. 1 to 3, N-acetylphytosphingosine did not inhibit the expression of the tyrosinase gene. Based on this finding, it is expected that N- acetylphytosphingosine would have direct inhibitory effects on tyrosinase activity.

Experimental Example 4: Whitening effect of creams according to the present invention on human skin> In order to confirm whether the compositions of the present invention exhibit whitening effects on the human skin, the present inventors conducted the following experiment. The whitening effects of the creams of Examples 1 to 4 were measured. For comparison, the whitening effect of the cream of Comparative Example 1 was measured. Opaque tapes having a hole of 1.5cm in diameter were adhered to each arm of

20 healthy men and women, and UV light (UVB) at an intensity 1.5-2 times higher than the minimal erythema dose was irradiated to induce skin darkness. Thereafter, the creams of Examples 1 to 4 and Comparative Example 1 were applied, and then changes in the brightness were measured using a colorimeter after 2 months. The test compounds were applied twice daily (in the morning and evening for two months). The whitening effects of the creams were evaluated by L value representing skin brightness, which is calculated by the equation below:

ΔL = L value measured on final day after application of a test compound - L value before application of the test compound

The results are shown in Table 4 below.

Table 4

Comparative Example 1 Example 1 Example 2 Example 3 Example 4

ΔL 1.3 2.11 2.52 2.87 3.54

As is apparent from Table 4, it was confirmed that as the concentration of N- acetylphytosphingosine increased, the whitening effect was shown to be excellent. This result suggests that N-acetylphytosphingosine can effectively peel off skin cells hypeφigmented by UV light irradiation.

As apparent from the above description, since N-acetylphytosphingosine used in the present invention inhibits melanin synthesis and tyrosinase activity, skin pigment- related symptoms such as discoloration, freckles, senile chromelasma and hypeφigmentation can be prevented. In addition, since N-acetylphytosphingosine peels hypeφigmented skin cells, skin pigment-related symptoms such as discoloration, freckles, senile chromelasma and hypeφigmentation can be treated. Furthermore, since N-acetylphytosphingosine has peeling effects on hypeφigmented skin cells as well as whitening effects, it can be effectively used in the cosmetic and pharmaceutical industries.

Although the preferred embodiments of the present invention have been disclosed for illustrative puφoses, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

WHAT IS CLAIMED IS:

1. A composition for inhibiting the activity of tyrosinase which comprises N- acetylphytosphingosine as an active ingredient.

2. A composition for peeling hypeφigmented skin cells which comprises N- acetylphytosphingosine as an active ingredient.

3. A composition for skin whitening which comprises N- acetylphytosphingosine as an active ingredient.

4. The composition according to any one of claims 1 to 3, wherein the composition is one for external use.

5. The composition according to claim 4, wherein the composition is a cosmetic composition.

6. The composition according to any one of claims 1 to 3, wherein the composition comprises N-acetylphytosphingosine in an amount of 0.005-10% by weight.

7. The composition according to claim 5, wherein the cosmetic composition is prepared into a cosmetic formulation selected from softening lotions, nutritive lotions, massage creams, nutritive creams, gels, packs and skin patches.

8. The composition according to any one of claims 1 to 3, wherein the composition is prepared into a formulation for transdermal administration selected from lotions, ointments, gels, creams, patches and nebulas.

9. A method for inhibiting the activity of tyrosinase using N- acetylphytosphingosine.

10. A method for promoting the peeling of hypeφigmented skin cells using N- acetylphytosphingosine.