Cosmetic composition for preventing or ameliorating skin damage caused by ultraviolet light

Abstract

Provided are a cosmetic composition for preventing or ameliorating a ultraviolet-induced skin damage including mitochondria-targeted vitamin E as an active ingredient, as well as a method for protecting skin from ultraviolet-induced skin damage or for ameliorating an ultraviolet-induced skin damage using the same compound.

Claims

1. A cosmetic composition for preventing or ameliorating an ultraviolet-induced skin damage, comprising the compound of Formula 1 as an active ingredient in an amount of 0.5 M to 10 M based on the total weight of the composition: ##STR00003##

2. The cosmetic composition according to claim 1, wherein the ultraviolet-induced skin damage is burns, erythema, or pigmentation, which is generated in the epidermis or dermis by ultraviolet.

3. A method for protecting skin from ultraviolet-induced skin damage or for ameliorating an ultraviolet-induced skin damage comprising applying a cosmetic composition to a subject in need thereof, the cosmetic composition comprising an effective amount of the compound of Formula 1 in an amount of 0.5 M to 10 M based on the total weight of the composition: ##STR00004##

4. The method according to claim 3, wherein the ultraviolet-induced skin damage is burns, erythema, or pigmentation, which is generated in the epidermis or dermis by ultraviolet.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows that the compound of Formula 1 (MVE) protected dermal fibroblasts from UVB. FIG. 1A shows that vitamin E (VE) slightly increased dermal fibroblast survival with UVB irradiation (200 mJ), while MVE decreased dermal fibroblast survival at 10100 M concentration. FIG. 1B shows that MVE increased fibroblast proliferation at 1001000 nM concentration. FIG. 1C shows that MVE protected dermal fibroblasts from UVB at 101000 nM concentration. **p<0.01

(2) FIG. 2 shows that MVE altered expression of extracellular matrix proteins. FIG. 2A shows that MVE significantly increased mRNA expression of collagen and down-regulated that of matrix metalloproteinase 1 (MMP1) in dermal fibroblasts. FIG. 2B shows that mRNA levels of collagen were reduced by UVB but attenuated by MVE treatment and that mRNA levels of MMP1 were induced by UVB but reduced by MVE treatment. FIG. 2C shows that MVE altered protein levels of collagen and MMP1 in fibroblasts.

(3) FIG. 3 shows that MVE reduced production of reactive oxygen species (ROS). FIG. 3a shows the cytosolic ROS levels measured by using DCF-CA (green). UVB increased the fluorescent signal intensity of DCF-DA in fibroblasts, while MVE (1000 nM) attenuated the signal intensity of DCF-DA. FIG. 3b shows the mitochondria ROS production measured by using mito-Sox (red). Likewise, UVB increased the fluorescent signal intensity of mito-Sox in fibroblasts, while MVE (1000 nM) reduced the signal intensity of mito-Sox. Scale bar=100 m, **p<0.01

(4) FIG. 4 shows that MVE protected HaCaT cells from UVB. FIG. 4A shows that MVE significantly increased HaCaT cell proliferation in a dose-dependent manner. FIG. 4B shows that MVE increased UVB-reduced cell viability. FIG. 4C shows that UVB increased p53 protein levels in HaCaT cells, while MVE attenuated UVB-induced p53 levels in HaCaT cells. *p<0.05, **p<0.01

(5) FIG. 5 shows that MVE reduced production of ROS. FIG. 5a shows the cytosolic ROS level measured by using DCF-CA (green). UVB increased the fluorescent signal intensity of DCF-DA in HaCaT cells, while MVE (1000 nM) attenuated the signal intensity of DCF-DA. FIG. 5b shows the mitochondria ROS production measured by using mito-Sox (red). UVB increased the fluorescent signal intensity of mito-Sox in HaCaT cells, while MVE (1000 nM) reduced the signal intensity of mito-Sox. Scale bar=100 m, **p<0.01

BEST MODE

(6) As used herein, the expression ultraviolet-induced skin damage refers to the damages caused by interactions between skin cells and ultraviolet coming into contact with the skin. Said interactions include DNA damages caused by ultraviolet, increase of reactive oxygen species, and variations or apoptosis of cells induced therefrom. The symptoms of ultraviolet-induced skin damage include burns, erythema, pigmentation, etc., which are generated in the epidermis or dermis by ultraviolet.

(7) And also, the term preventing used herein refers to blocking, delaying, or to delaying skin damages by protecting the skin from ultraviolet-induced skin damages. Accordingly, the expressions preventing a ultraviolet-induced skin damage and protecting a ultraviolet-induced skin damage have the same meaning.

(8) And also, the term ameliorating used herein refers to healing ultraviolet-induced skin damages; inhibiting the progress and/or deterioration of symptoms to stop progressing damages, although complete healing is not provided; or inducing some or all of the symptoms to the direction of healing.

(9) The present invention provides a cosmetic composition for preventing or ameliorating a ultraviolet-induced skin damage, comprising a compound of Formula 1 as an active ingredient:

(10) ##STR00002##

(11) The compound of Formula 1 is a known material and may be prepared according to known methods. For example, the compound of Formula 1 may be prepared according to the methods described in prior arts, such as Mao G et al., (2011) The British journal of nutrition 106: 87-95, Mao G et al., The Journal of nutrition 140: 1425-1431, Makoto Matsui, Journal of Japan Oil Chemists' Society, Vol. 45, no. 9, p 821-890 (1996), Robin A. J. Smith et al., Eur. J. Biochem. 263, 709-716 (1999), and the like.

(12) The cosmetic composition of the present invention may be in various forms and the forms are not limited. That is, the cosmetic composition of the present invention may be in conventional cosmetic composition forms such as cream, pack, lotion, essence, cleansing water, foundation, makeup base, and the like. The cosmetic composition may be formulated, along with a carrier conventionally used in the field of cosmetic composition, according to conventional methods.

(13) It has been found by the present invention that the treatments of human dermal fibroblasts (HDF) and immortalized human keratinocyte cell line (HaCaT) with a low concentration of the compound of Formula 1 (e.g., 1 M or less) exhibits a protective effect against ultraviolet (especially UVB), while the treatments with a high concentration thereof decrease the cell survival rate; and thus that the compound of Formula 1 shows dual actions. Therefore, the amounts of the compound of Formula 1 in the unit is cosmetic composition may range from 0.1 to 100 M, preferably 0.5 to 10 M, based on the total weight of the composition. Of course, the amount may vary depending on the severities of ultraviolet-induced skin damage.

(14) The present invention will be described in further detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

(15) In the following examples, MVE (the compound of Formula 1) was synthesized as described previously (Mao G et al., (2011) The British journal of nutrition 106: 87-95; Mao G et al., The Journal of nutrition 140: 1425-1431). Briefly, trimethylhydroquinone was reacted with myrcene in the presence of (+)-10-camphorsulfonic acid to provide chroman-6-ol, which was protected as the acetate. Oxidative cleavage of the olefin (cat. OsO.sub.4, NMO followed by NaIO.sub.4), reduction (NaBH.sub.4), and deprotection (K.sub.2CO.sub.3, MeOH) led to the alcohol, which was converted to an iodide. Treatment of the iodide with triphenylphosphine furnished MVE (the compound of Formula 1).

(16) 1. Methods

(17) (1) Cell Culture

(18) Human dermal fibroblasts (HDFs) and immortalized keratinocyte cell line (HaCaT) were cultured using DMEM (low, high glucose, Hyclone, Thermo Scientific, Logan, Utah, USA) supplemented with 10% FBS (Gibco, Invitrogen, Carlsbad, Calif., USA), 1% penicillin, and streptomycin (Gibco) at 37 C. with 5% CO.sub.2 in a humidified atmosphere (Kim W S et al., (2009) Journal of dermatological science 53: 96-102). Said cultures were performed by exchanging with fresh media every two days.

(19) (2) Proliferation Assay and Viability Analysis

(20) HDFs (310.sup.4 cells/well) and HaCaT (410.sup.4 cells/well) were seeded in 6-well plates. Cells were treated with various concentrations of MVE. Cells were then incubated for 48 hours and the MTT assay was performed. MTT solution (5 mg/ml in phosphate buffered saline) was added to each well at 1/20 of the media volume, incubated for 2 hours, and then supernatant was removed. Dimethyl sulfoxide (DMSO) was then added to dissolve formazan crystals, and the absorbance was measured at 595 nm using an ELISA reader (TECAN, Grodig, Austria).

(21) (3) Cellular and Mitochondrial ROS Generation Assay

(22) Cellular ROS generation was measured using 2,7-dichlorodihydrofluorescein diacetate (DCF-DA, Molecular Probes, Eugene, Oreg., USA), as described previously (Hye Kim J et al., Stem cells 33: 542-556). Similarly, mitochondrial ROS (mtROS) generation was measured using Mito-Sox (Molecular Probes). Cells were seeded in 6-well plates in 0.2% FBS and cultured overnight. MVE (100 nM) was added with or without DCF-DA (20 M) or Mito-Sox (5 M). Each well was imaged every 10 minutes for 40 minutes under standard incubation conditions using an IncuCyte ZOOM microscope placed inside an incubator. Image-based analysis of fluorescence intensity was carried out using IncuCyte software (Essen Bioscience, MI, USA).

(23) (4) Western Blot Analysis

(24) Cells (210.sup.5 cells/ml) were seeded in a 60 mm dish and cultured to 80% confluence. Cells were treated with MVE. Cells were then lysed with 1RIPA buffer (50 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1% Triton-X100, pH 7.4, 1% SDS, 50 mM NaF, 1 mM Na.sub.3VO.sub.4, 5 mM Dithiothreitol, 1 mg/ml Leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Sample protein (40 g) was separated in 10-12% SDS-polyacrylamide gels by electrophoresis. Proteins were transferred to PVDF membranes and incubated with antibodies to collagen (1:1000 rabbit source, Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and matrix metalloproteinase 1 (MMP1, 1:1000 rabbit source, Santa Cruz Biotechnology). Membranes were then washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology). Blots were reacted with western reagent (ECL; Millipore Billerica, Ann Arbor, Mass., USA) and exposed to X-ray film.

(25) (5) Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

(26) Total RNA of HDFs and HaCaT was extracted with Trizol reagent followed by reverse transcription to cDNA. The following oligonucleotides were used as primers: collagen type I [5-TAGGGTCTAGACATGTTCAGCTTTGT-3 (SEQ ID NO: 1) and 5-GTGATTGGTGGGATGTCTTCGT-3 (SEQ ID NO: 2)], MMP-1 [5-AGATGTGGAGTGCCTGATGT-3 (SEQ ID NO: 3) and 5-AGCTAGGGTACATCAAAGCC-3 (SEQ ID NO: 4)], and control GAPDH [5-CGAGATCCCTCCAAAATCAA-3 (SEQ ID NO: 5) and 5-TGTGGTCATGAGTCCTCCCA-3 (SEQ ID NO: 6)]. PCR was carried out in a total volume of 30 l for PCR amplification of cDNA that was reverse-transcribed from the total RNA. After initial denaturation at 95 C. for 5 minutes, amplification was performed in 35 cycles for 30 seconds at 95 C., 20 seconds at 54 C., and 30 seconds at 72 C. This was followed by a final extension at 72 C. for another 10 minutes. GAPDH mRNA level was used for sample standardization.

(27) (6) Statistical Analysis

(28) Statistical significance was determined using a Wilcoxon signed-rank test or a Student's t-test. P<0.05 was considered statistically significant. Statistical analysis was performed using SPSS 18.0 (SPSS, IBM Corp, Armonk, N.Y., USA).

(29) 2. Results

(30) (1) MVE Protected Dermal Fibroblasts from UVB

(31) When dermal fibroblasts were treated with Vitamin E and MVE having benzopyran in the range from 0.1 to 100 M, vitamin E slightly increased survival of dermal fibroblasts from UVB irradiation (200 mJ) at 0.1 to 100 M. However, MVE decreased fibroblast survival in a concentration-dependent manner at the 10 to 100 M concentrations, while MVE increased fibroblast survival in a concentration-dependent manner at the 0.1 and 1 M concentrations (FIG. 1A). These results shows that MVE at high concentrations functions as a cytotoxic agent inhibiting cell survival in response to UVB irradiation, while MVE at low concentrations functions as a cytoprotective agent protecting cells from UVB irradiation. Based on these results, the protective effect of MVE was also studied in nM concentration ranges. As the results thereof, MVE increased fibroblast proliferation in the 1001000 nM range (FIG. 1B, p<0.01). In addition, MVE protected fibroblasts from UVB in the 101000 nM range (FIG. 10, p<0.01). These results indicate that MVE protects fibroblasts from UVB and is more effective that vitamin E.

(32) (2) MVE Altered Expression of Extracellular Matrix Proteins

(33) The present inventors examined whether MVE altered the mRNA or protein levels of extracellular matrix (ECM) proteins, such as collagen type I and matrix metalloproteinase 1 (MMP1), in fibroblasts. MVE increased collagen mRNA expression and down-regulated that of MMP1 in dermal fibroblasts (FIG. 2A). The mRNA level of collagen was reduced by UVB irradiation, but attenuated by MVE treatment (FIG. 2B). The mRNA level of MMP1 was increased by UVB, but reduced by MVE treatment (FIG. 2B). In addition, MVE altered the protein levels of collagen and MMP1 in fibroblasts (FIG. 2C).

(34) (3) MVE Reduced Production of Reactive Oxygen Species

(35) Because UVB reportedly generates reactive oxygen species (ROS) and induced apoptosis in skin, ROS levels were measured after MVE treatment. First, the cytosolic ROS level was measured using DCF-CA (green, FIG. 3a). UVB increased the fluorescent signal intensity of DCF-DA in fibroblasts, while MVE (1000 nM) attenuated the signal intensity of DCF-DA (p<0.01). Mitochondria ROS production was measured using mito-Sox (red, FIG. 3b). UVB increased the fluorescent signal intensity of mito-Sox in fibroblasts, while MVE (1000 nM) reduced the signal intensity of mito-Sox (p<0.01). These results indicate that MVE protects dermal fibroblasts from UVB via reducing ROS generation.

(36) (4) MVE Protected HaCaT Cells from UVB

(37) The protective effects of MVE in epidermal skin cells were also investigated using the HaCaT cell line. MVE significantly increased proliferation of HaCaT cells in a dose-dependent manner (FIG. 4A, p<0.05). In addition, MVE increased the UVB-reduced cell viability (FIG. 4B, p<0.01). UVB induced p53 protein levels in HaCaT cells, while MVE reduced p53 levels in HaCaT cells (FIG. 4C).

(38) (5) MVE Reduced Production of Reactive Oxygen Species

(39) ROS levels were also measured after MVE treatment in HaCaT calls. The cytosolic ROS level was measured using DCF-CA. UVB increased the fluorescent signal intensity of DCF-DA in HaCaT cells, while MVE (1000 nM) attenuated the signal intensity of DCF-DA (FIG. 5a, p<0.01). Mitochondrial ROS production was also measured using mito-Sox. UVB increased the fluorescent signal intensity of mito-Sox in HaCaT cells, while MVE (1000 nM) reduced the signal intensity of mito-Sox (FIG. 5b, p<0.01). These results indicate that MVE protects dermal fibroblasts from UVB via reducing ROS generation.

(40) 3. Discussion

(41) The present study investigated the protective effects of MVE against UVB in dermal fibroblasts and epidermal HaCaT cells. MVE increased the proliferation and survival of fibroblasts at low concentration (i.e., nM ranges). In addition, MVE increased collagen production and downregulated MMP1 expression. MVE also increased the proliferation and survival of HaCaT cells. UVB increased ROS production in fibroblasts and HaCaT cells, while MVE decreased ROS production in these cells. These results collectively suggest that low dose MVE protects skin from UVB irradiation. Therefore, MVE can be used as a cosmetic raw material for blocking ultraviolet.

(42) Vitamin E is a group of compounds that include both tocopherols and tocotrienols. As a fat-soluble antioxidant, vitamin E inhibits the production of ROS when fat undergoes oxidation. Vitamin E acts as a peroxyl radical scavenger, preventing the propagation of free radicals in tissues. It is known that vitamin E protects dermal fibroblasts and epidermal keratinocytes from UVB. For example, -tocotrienol protected HaCaT keratinocytes from UVB induced inflammation (Shibata A et al., (2010) Journal of agricultural and food chemistry 58: 7013-7020). And also, D-alpha-tocopherol prevented UVB-induced DNA damage in human epidermis (Placzek M et al., (2005) The Journal of investigative dermatology 124: 304-307). Vitamin E is included as antioxidants in many sunscreens and lotions.

(43) In the present study, MVE showed a dual mode of actions. That is, at low concentrations (<1 M), MVE protected dermal fibroblasts and epidermal HaCaT cells from UVB via scavenging ROS production (FIG. 1A). However, MVE inhibited the survival of dermal fibroblasts at high concentrations (>1 M). Although the mechanism of action is not yet clarified, MVE acts as an antioxidant at low concentrations and as a prooxidant at high concentrations in normal cells.

(44) In summary, it has been found by the present invention that MVE exhibits remarkably excellent ultraviolet blocking activity, as compared to vitamin E commonly having a benzopyran moiety in its molecule. Low concentration MVE protected dermal fibroblasts and epidermal HaCaT cells from UVB irradiation by scavenging ROS in these cells. MVE also increased collagen production and decreased MMP1 expression. Therefore, MVE can be developed and used for cosmetic raw materials to replace vitamin E.