Method for skin-whitening using composition containing resveratryl triglycolate

Abstract

The present invention relates to a method for skin-whitening using a composition containing resveratryl triglycolate as an active ingredient, and more specifically, relates to a composition for skin-whitening containing resveratryl triglycolate or a cosmetically or pharmaceutically acceptable salt thereof as an active ingredient, a method for skin-whitening including applying or administering a composition containing resveratryl triglycolate or an acceptable salt thereof as an active ingredient to a subject, and a resveratryl triglycolate compound which has a skin-whitening effect, since the resveratryl triglycolate can inhibit melanin synthesis and brighten skin tone.

Claims

1. A compound represented by Formula 1 below: ##STR00003##

2. A cosmetic composition comprising the compound of claim 1.

3. A pharmaceutical composition comprising the compound of claim 1.

4. A method for skin-whitening comprising: applying or administering a composition comprising resveratryl triglycolate, represented by Formula I below, or an acceptable salt thereof as an active ingredient to a subject: ##STR00004##

5. The method of claim 1, wherein the resveratryl triglycolate or an acceptable salt thereof is comprised at from 0.01 wt % to 5 wt %.

6. The method of claim 1, wherein the composition is a cosmetic composition.

7. The method of claim 1, wherein the composition is a pharmaceutical composition.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the chemical structures of resveratryl triglycolate (RTG), glycolic acid (GA), and resveratryl triacetate (RTA).

(2) FIG. 2 shows effects of arbutin, resveratrol, resveratryl triglycolate (RTG), GA, and RTA on cell viability. Cells were treated with the five test compounds at the indicated concentrations for 48 hours. The data are shown as percentages of controls (meanSE, n=3).

(3) FIG. 3 shows effects of arbutin, resveratrol, resveratryl triglycolate (RTG), GA, and RTA on melanin synthesis in HEMs. The values for intra- and extracellular melanin levels were normalized with respect to total protein content. The data are shown as percentage of vehicle control (meanSE, n=3). # p<0.05 vs. control. *p<0.05 vs. L-tyrosine treatment alone.

(4) FIG. 4 shows effects of arbutin, resveratrol, resveratryl triglycolate (RTG), GA, and RTA on the mRNA expression corresponding to melanogenic enzymes in HEMs. HEMs were pre-treated with the test compounds at the indicated concentrations for 60 minutes, and then stimulated by adding 4.0 mM of L-tyrosine for 48 hours. The total cellular RNA was applied to qPCR analysis. The mRNA expression levels of TYR, TYRP1, and DCT were normalized with respect to values of GAPDH mRNA levels. The data are shown as percentage of vehicle control (meanSE, n=3). # p<0.05 vs. control. *p<0.05 vs. L-tyrosine treatment alone.

(5) FIG. 5 shows effects of arbutin, resveratrol, resveratryl triglycolate (RTG), GA, and RTA on melanogenic enzyme expression in HEMs. HEMs were pre-treated with the test compounds at the indicated concentrations for 60 minutes, and then stimulated by adding 4.0 mM of L-tyrosine for 48 hours. Whole cell lysates were applied to western blot analysis. The protein expression levels for TYR, TYRP1, and DCT were normalized with respect to values of GAPDH protein levels. The data are shown as percentage of vehicle control (meanSE, n=3). # p<0.05 vs. control. *p<0.05 vs. L-tyrosine treatment alone.

(6) FIG. 6 shows effects of arbutin, resveratrol, resveratryl triglycolate (RTG), GA, and RTA on the expression of MITF mRNA and protein in HEMs. HEMs were pre-treated with the test compounds at the indicated concentrations for 60 minutes, and then stimulated by adding 4.0 mM of L-tyrosine for 48 hours. The total cellular RNA was applied to qPCR analysis. The MITF expression levels were normalized with respect to values of GAPDH. The data are shown as percentage of vehicle control (meanSE, n=3). # p<0.05 vs. control. *p<0.05 vs. L-tyrosine treatment alone.

(7) FIG. 7 shows changes of visual assessment following 8 consecutive weeks' application of the products (meanSD, *p<0.05 vs. before treatment, .sup.p<0.05 vs. control group).

(8) FIG. 8 shows changes of melanin index following 8 consecutive weeks' application of the products (meanSD, *p<0.05 vs. before treatment, .sup.p<0.05 vs. control group).

(9) FIG. 9 shows changes of skin lightness (L*value) following 8 consecutive weeks' application of the products (meanSD, *p<0.05 vs. before treatment, .sup.p<0.05 vs. control group).

(10) FIG. 10 shows changes of skin color (ITA value) following 8 consecutive weeks' application of the products (meanSD, *p<0.05 vs. before treatment, .sup.p<0.05 vs. control group).

BEST MODE

(11) Hereinafter, the present invention will be described in more detail with reference to the following examples, comparative examples, and experimental examples. However, the following examples, comparative examples, and experimental examples are provided for illustrative purposes only, and the scope of the present invention should not be limited thereto in any manner.

Example 1

(12) Synthesis of Resveratryl Triglycolate (RTG)

(13) Tetrahydropyran (THP)-protected glycolic acid (GA) was synthesized in the same manner as described in the reference (Costin G E, Faseb J 2007, 21:976-94). Esterification of resveratrol and THP-protected GA yielded (E)-5-(4-(2-(tetrahydro-2H-pyran-2-yloxy)acetoxy)styryl)-1,3-phenylene bis(2-(tetrahydro-2H-pyran-2-yloxy)acetate), using (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC) as a dehydration agent, and 4-dimethylaminopyridine (DMAP) as a catalyst. The thus-obtained product was dissolved in dioxane and mixed with 2 N HO/ether to prepare resveratryl triglycolate as a white solid. Every step was confirmed by .sup.1H and .sup.13C NMR spectroscopy and mass spectrometry, and resveratryl triglycolate was obtained as an amorphous powder having the following properties. The properties are shown in Table 1.

(14) UV (EtOH) .sub.max (log ), 294 (4.24) nm; ESIMS (positive mode) m/z425 [M+Na].sup.+, 403 [M+H].sup.+, 345 [MC2H2O2+H].sup.+, 287 [MC4H4O4+H].sup.+, (negative mode) m/z 401 [MH].sup., 344 [MC2H2O2H].sup., 285 [MC4H4O4H].sup.; .sup.1H-NMR (700 MHz, DMSO-d6); .sup.13C-NMR (175 MHz, DMSO-d6)

(15) A chemical identification of resveratryl triglycolate (RTG), was verified based on MS and NMR data. As a result resveratryl triglycolate (RTG), showed an [M+H].sup.+ ion peak at m/z 403 of the positive mode ESI-MS, corresponding to the molecular formula C.sub.20H.sub.18O.sub.9. The .sup.1H- and .sup.13C-NMR spectra data showed the presence of one set of ortho-coupled aromatic protons assignable to a p-substituted benzene unit [.sub.H 7.66 (2H, d, J=9.1 Hz, H-2, 6) and .sub.C 127.5 (C-2, 6); .sub.H 7.18 (2H, d, J=9.1 Hz, H-3, 5) and .sub.C 121.7 (C-3, 5)], three meta-coupled aromatic protons on a 1,3,5-trisubstituted benzyl moiety [.sub.H 7.33 (2H, d, J=1.4 Hz, H-2, 6) and .sub.C 116.8 (C-2, 6); .sub.H 6.95 (1H, t, J=1.4 Hz, H-4) and .sub.C 114.4 (C-4)], one pair of trans olefinic double bonds [.sub.H 7.36 (1H, d, J=16.1 Hz, H-8) and .sub.C 129.3 (C-8); .sub.H 7.25 (1H, d, J=16.1 Hz, H-7) and .sub.C 126.4 (C-7)], and three sets of GA moieties [.sub.H 4.33 (4H, d, J=6.3 Hz, H-2, 2) and 5.64 (2H, d, J=6.3 Hz, OH-2, 2), .sub.C 171.0 (C-1, 1) and 59.35 (C-2, 2); .sub.H 4.31 (2H, d, J=6.3 Hz, H-2) and 5.60 (1H, d, J=6.3 Hz, OH-2), .sub.C 171.2 (C-1) and 59.37 (C-2)].

(16) All protonated carbons were confirmed by analysis of the heteronuclear single quantum coherence (HSQC) spectrum. The results indicated that the compound may have a resveratrol skeleton with three GA groups. Additionally, the locations of these GA groups were confirmed by observation of heteronuclear multiple bond correlation (HMCB). From H-3/5 (.sub.H 7.18) to C-1 (.sub.C 171.2), from H-4 (.sub.H 6.95) to C-1 and 1 (.sub.C 171.0), and from H-2/6 (.sub.H 7.33) to C-1 and 1 (.sub.C 171.0). These correlations indicate three GA groups of C-3, 5, and 4 respectively. Thus, the structure of the compound is shown to be 3, 5, 4-triglycolate-trans-stilbene or resveratryl triglycolate.

(17) TABLE-US-00001 TABLE 1 RTG position .sub.H mult., (J Hz) .sub.C mult. HMBC 1 139.2 s 2 7.33 d (1.4) 116.8 d 1, 3, 5, 6, 7, 1 3 150.6 s 4 6.95 t (1.4) 114.4 d 2, 3, 5, 6, 1, 1 5 150.6 s 6 7.33 d (1.4) 116.8 d 1, 2, 3, 5, 7, 1 7 7.25 d (16.1) 126.4 d 1, 1, 8, 2, 6, 8 7.36 d (16.1) 129.3 d 1, 1, 7, 2, 6 1 133.9 s 2 7.66 d (9.1) 127.5 d 8, 3, 4, 5, 6 3 7.18 d (9.1) 121.7 d 1, 4, 5, 1 4 149.6 s 5 7.18 d (9.1) 121.7 d 1, 3, 4, 1 6 7.66 d (9.1) 127.5 d 8, 2, 3, 4, 5 1 171.0 s 2 4.33 d (6.3) 59.35 t 1 1 171.0 s 2 4.33 d (6.3) 59.35 t 1 1 171.2 s 2 4.31 d (6.3) 59.37 t 1 2-OH 5.64 t (6.3) 1, 2 2-OH 5.64 t (6.3) 1, 2 2-OH 5.60 t (6.3) 1, 2 .sup.aMeasured at 700 and 175 MHz; obtained in DMSO-d.sub.6 with TMS as an internal standard. The assignments were based on .sup.1H-.sup.1H COSY, HSQC, and HMBC experiments.

Example 2

(18) Evaluation of Cytotoxicity of Resveratryl Triglycolate (RTG)

Example 2-1

(19) Cell Culture

(20) Human epidermal melanocytes (HEM) derived from neonatal human foreskins were purchased from Cascade Biologics (Portland, Oreg., USA). The HEMs were cultivated in Medium 254 supplemented with human melanocyte growth supplements (Cascade Biologics) and antibiotics.

Example 2-2

(21) Method of Analysis

(22) In order to analyze the cytotoxicity of resveratryl triglycolate (RTG), the resveratryl triglycolate (RTG) prepared in Example 2-1 was used, and the cytotoxicity was analyzed by trypan blue exclusion assay using arbutin, resveratrol, glycolic acid (GA), and resveratryl triacetate (RTA) as test compounds or comparative test compounds.

(23) First, arbutin, resveratrol, resveratryl triglycolate (RTG), GA, and RTA were treated in HEMs at concentrations in the range of 3 M to 1000 M, detached from the culture plates by trypsinization, and then harvested by centrifugation at 1200g for 3 minutes. The cells were then suspended in the culture medium and mixed with 0.1% trypan blue solution (Sigma-Aldrich) at a 1:1 ratio. The numbers of stained dead cells and unstained live cells were counted three times using a hemocytometer.

Example 2-3

(24) Evaluation of Cytotoxicity

(25) As a result of evaluation of the cytotoxicity, as shown in FIG. 2, resveratrol, resveratryl triglycolate (RTG), and RTA showed the same level of cytotoxicity as HEMs, barely showed cytotoxicity at concentrations below 30 M, and showed cytotoxicity at concentrations above 30 M. Arbutin and GA showed no cytotoxicity at concentrations of up to 1000 M.

Example 3

(26) Effect of Resveratryl Triglycolate on Inhibiting Melanin Synthesis

Example 3-1

(27) Methods

(28) In order to examine the effects of resveratryl triglycolate on melanin synthesis using HEMs, arbutin, resveratrol, resveratryl triglycolate (RTG), GA, and RTA were treated at concentrations of 3 M and 10 M for 60 minutes, and then stimulated with 4.0 mM L-tyrosine for 48 hours. The extracellular melanin levels were evaluated by measuring the absorbance of the conditioned media at a wavelength of 490 nm, while the intracellular melanin levels were evaluated after extracting melanin from the cells using 0.1 M NaOH at 60 C. for 60 minutes. The values for melanin content were normalized with respect to the protein content in each sample. The protein content was evaluated using the Bio-Rad DC assay (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Example 3-2

(29) Effects of Inhibiting Melanin Synthesis

(30) Effects of resveratryl triglycolate (RTG) on inhibiting melanin synthesis were examined after adding 4 mM L-tyrosine, which stimulates melanin synthesis in HEMs, and as a result, as shown in FIG. 3, the intra- and extracellular melanin levels were increased by L-tyrosine treatment, and these increases were reduced by resveratryl triglycolate (RTG). These increases were also reduced by resveratrol and RTA, although the decreases were not as large as those by resveratryl triglycolate. Arbutin and GA showed no significant effect on melanin synthesis at concentrations of up to 10 M.

Example 4

(31) Effects of Melanogenic Enzymes of Resveratryl Triglycolate (RTG) on Inhibiting mRNA Expression

Example 4-1

(32) qRT-PCR Analysis

(33) In order to examine an effect of resveratryl triglycolate (RTG) on melanogenic enzyme expression in L-tyrosine-stimulated HEMs, first, the total mRNA was extracted, and the mRNA expression levels of TYR, TYRP1, and DCT were analyzed through qRT-PCR.

(34) RNA was extracted using an RNeasy kit (Qiagen, Valencia, Calif., USA), and 1 g of mRNA was prepared by reverse transcription using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, Calif. USA). PCR was conducted in a reaction mixture solution (20 L) containing SYBR Green PCR Master Mix (Applied Biosystems), 60 g of cDNA, and 2 pmol of gene-specific primer sets (Macrogen, Seoul), using a StepOnePlus Real-Time PCR System (Applied Biosystems). The primers used are shown in Table 2, primers of SEQ ID NO: 1 and SEQ ID NO: 2 were used for tyrosinase (TYR, GenBank accession number NM000372.3) extraction, the primers of SEQ ID NO: 3 and SEQ ID NO: 4 were used for tyrosinase-related protein 1 (TRP1, NM000550.1) extraction, and the primers of SEQ ID NO: 5 and SEQ ID NO: 6 were used for DOPA chrome tautomerase (DCT, NM001922.3) extraction. The reactions were conducted using the following protocol: the initial incubation at 50 C. for 2 minutes, DNA polymerase activation at 95 C. for 10 minutes, 40 cycles of denaturation and annealing at 95 C. for 15 seconds, and extension at 60 C. for 1 minute.

(35) TABLE-US-00002 TABLE21 SEQ ID Gene Primer Sequence NO Tyrosinase forward 5-GATGAGTACATGGGAGGTCAGC-3 1 (TYR) primer reverse 5-GTACTCCTCCAATCGGCTACAG-3 2 primer tyrosinase- forward 5-GCTCCAGACAACCTGGGATA-3 3 related primer protein1 reverse 5-TCAGTGAGGAGAGGCTGGTT-3 4 (TYRP1) primer DOPAchrome forward 5-AGATTGCCTGTCTCTCCAGAAG-3 5 tautomerase primer (DCT) reverse 5-CTTGAGAATCCAGAGTCCCATC-3 6 primer

Example 4-2

(36) Effects of Melanogenic Enzymes on Inhibiting mRNA Expression

(37) Effects of resveratryl triglycolate (RTG) on mRNA expression of melanogenic enzymes were analyzed, as a result, as shown in FIG. 4, the mRNA expression levels of the melanogenic enzymes, TYR, TYRP1, and DCT, were increased by L-tyrosine treatment, and these increases were reduced by resveratryl triglycolate (RTG). These increases were also reduced by resveratrol and RTA, yet the decreases were not as large as those by resveratryl triglycolate (RTG). Additionally, arbutin and GA showed no effect on melanin synthesis at concentrations of up to 10 M.

Example 5

(38) Effects of Melanogenic Enzyme of Resveratryl Triglycolate (RTG) on Protein Expression

Example 5-1

(39) Western Blot Analysis

(40) In order to examine an effect of resveratryl triglycolate (RTG) on melanogenic enzyme expression, western blotting of HEM cell lysates was conducted. First, cells were lysed in a lysis buffer (lysis buffer, 10 mM Tris-Cl pH 7.2, 150 mM NaCl, 5 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% deoxycholate) supplemented with a 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche, Mannheim, Germany). After the lysates were mixed with Laemmli sample buffer, the proteins were denatured by heating at 95 C. for 5 minutes. Then the proteins were separated from gel containing 10% polyacrylamide by polyacrylamide gel electrophoresis (SDS-PAGE). Once separated by electrophoresis, the proteins were transferred to polyvinylidene difluoride membrane (Amersham Pharmacia, Little Chalfont, UK). The membranes were incubated overnight at 4 C. in a solution containing primary antibodies, and subsequently in a solution containing secondary antibodies conjugated with horseradish peroxidase (Cell Signaling, Danvers, Mass., USA) at room temperature for 1 hour. The primary antibodies of tyrosinase (TYR), TRP1, DCT, MITF, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). The immunoreactive bands were detected by using a picoEPD Western Reagent kit (ELPIS-Biotech, Daejeon, Korea), and densitometric analysis was performed using the NIH Image program.

Example 5-2

(41) Effects of Melanogenic Enzymes on Protein Expression

(42) Effects of resveratryl triglycolate (RTG) on melanogenic enzyme expression were analyzed, as a result, as shown in FIG. 5, the protein expression levels of TYR, TYRP1, and DCT, were increased by L-tyrosine treatment, and these increases were reduced by treatment of resveratryl triglycolate, RTA, or resveratrol. Resveratryl triglycolate showed the largest decreases, and resveratrol and RTA reduced the protein expression levels of TYR, TYRP1, and DCT as well, although the decreases were not as large as those by resveratryl triglycolate. Additionally, arbutin and GA showed no effect on protein expression of TYR, TYRP1, and DCT.

(43) Additionally, in order to examine the mechanism of melanogenic enzyme regulations of resveratryl triglycolate, the expression levels of mRNA and protein of microphthalmia-associated transcription factor (MITF) were examined if affected by resveratryl triglycolate. As a result, as shown in FIG. 6, the expression levels of mRNA and protein were both increased, and these increases were largely reduced by resveratryl triglycolate. These increases were also reduced by resveratrol and RTA, although the decreases were not as large as those by resveratryl triglycolate. Additionally, arbutin and GA showed no effect on MITF expression at concentrations of up to 10 M.

Example 6

(44) Evaluation of Human Body Skin-Whitening Effect

Example 6-1

(45) Selection of Test Subjects

(46) The clinical test was conducted with 22 female subjects aged 18 to 60 who met the eligibility criteria, but not the exclusion criteria. Those who had a Fitzpatrick skin type of III or IV and were interested in participating in the test were provided with information about the purpose and the methods of the test as well as expected efficacies and adverse reactions, signed a consent form, and then participated in the test.

(47) Volunteers who met the following eligibility criteria were selected as a test subject group.

(48) (1) Women aged 18 to 60 with a skin type of III or IV, (2) Volunteers who fully understood and agreed to the purpose and content of the experiment and voluntarily signed the written consent form before the experiment, (3) Those observable and traceable for the follow-up period

(49) Additionally, as the exception criteria, volunteers with a specific skin disease were excluded, and the subjects were not allowed to use any functional cosmetics or pharmaceutical products other than the test products.

Example 6-2

(50) Skin Characteristics of the Subjects

(51) The skin characteristics of the subjects were investigated by conducting a survey, and the results are as follows (Table 3 and Table 4).

(52) TABLE-US-00003 TABLE 3 Skin characteristics of volunteers (n = 22) Item Classification Frequency (N) Percentage (%) Age 20s 2 9.09 30s 5 22.73 40s 15 68.18 Skin type Dry 6 27.27 Normal 11 50.00 Oily 0 0.00 Dry and oily 5 22.73 Problematic 0 0.00

(53) TABLE-US-00004 TABLE 4 Skin condition of volunteers by skin physiological factors (n = 22) Frequency Percentage Item Classification (N) (%) Hydration Sufficient 1 4.55 Normal 12 54.55 Deficient 9 40.91 Sebum Glossy 3 13.64 Normal 14 63.64 Deficient 5 22.73 Surface Smooth 3 13.64 Normal 17 77.27 Rough 2 9.09 Thickness Thin 6 27.27 Normal 16 72.73 Thick 0 0.00 Duration of UV Less than 1 hr 5 22.73 exposure 1 to 3 hrs 15 68.18 More than 3 hrs 2 9.09 Occurrence frequency Little 0 0.00 of hyper-pigmentation Normal 15 68.18 Well 7 31.82 Smoking No 22 100.00 Less than 10 pieces 0 0.00 More than 10 pieces 0 0.00 Irritability Yes 3 13.64 No 19 86.36 Stinging Yes 0 0.00 No 22 100.00 Adverse reaction Yes 0 0.00 No 22 100.00

Example 6-3

(54) Induction of Artificial Pigmentation

(55) Artificial pigmentation was induced by having forearms exposed to UVA+B, determining a minimal erythema dose (MED), and then having the forearms exposed to UV(UVA+B) 2 to 4.5 times stronger than MED.

Example 6-4

(56) Test Products and Method of Use

(57) As test products, product C containing 0.4% resveratryl triglycolate as an active ingredient, product B containing grape stem extracts, and product A containing no active ingredient were used. The composition of each product is as follows (Table 5).

(58) TABLE-US-00005 TABLE 5 Rx Name of Component Product A Product B Product C A Water 56.94 3.82 53.42 Phenoxyethanol 0.3 0.3 0.3 1,2-Hexanediol 2 2 2 EDTA-4Na 0.08 0.08 0.08 A.sub.1 Grape stem extract 50 A.sub.2 Na-Hyaluronate (1%) 5 5 5 Distilled water 5 5 5 A.sub.3 Cabopol #940(2%) 0.34 0.4 0.4 Distilled water 17 20 20 B 1.3-B.G 4 4 4 Polysorbate 60 0.9 RTG 0.4 C Glycerine 2 2 2 Polysorbate 60 1.8 1.8 0.9 Perfume 0.1 0.1 0.1 D Hydrolyzed collagen 0.1 0.1 0.1 E Triethanolamine 0.34 0.4 0.4 Distilled water 5 5 5 TOTAL 100 100 100

(59) As a method of product use, the test subject group was made to apply each of products A, B, and C on the test area (forearm), twice daily for 8 weeks after the artificial pigmentation. The subjects were divided into three groups through block randomization. Group A applied the products in the order of A, B, and C from the top, and group B applied the products in the order of B, C, and A from the top, whereas group C applied the products in the order of C, A, and B from the top. The test was conducted in three groups, but the results of product A (test group) and C (control group) were described as below. The result of the product B was omitted due to its low relevance to the present patent.

Example 6-5

(60) Analysis of Visual Evaluation of Artificial Pigmented Area of Skin

(61) Visual assessment was independently conducted by two testers, rating the degree of pigmentation on a scale from 0 to 10 (0, bright and transparent; 9, dark and dull, in increments of 0.5), and then averaging the values.

(62) Observations were compared and analyzed before and after each evaluation point, the degree of pigmentation for both the test group and the control group showed significant improvement after two weeks of use of the products (p<0.05, Table 6, FIG. 7).

(63) TABLE-US-00006 TABLE 6 Statistical analysis of visual assessment Group Week N Mean.sup.1 SD p-value.sup.2 Control Before 22 7.05 0.49 (A) 2 W 22 6.82 0.42 0.000* 4 W 22 6.53 0.40 0.000* 6 W 22 5.83 0.47 0.000* 8 W 22 4.98 0.65 0.000* Test Before 22 7.11 0.41 (C) 2W 22 6.93 0.44 0.002* 4 W 22 6.48 0.39 0.000* 6 W 22 5.73 0.45 0.000* 8 W 22 4.84 0.67 0.000* .sup.1Decrement of the mean-value represents improvement of hyperpigmentation. .sup.2Significantly different at *p < 0.05 compared with before treatment.

(64) Additionally, observations of the pigmentations were compared and analyzed before and after each evaluation point. The pigmentation for the test group showed a significant improvement after 6 weeks and 8 weeks of product use, compared to the control group (p<0.05, Table 7, FIG. 7).

(65) TABLE-US-00007 TABLE 7 Statistical analysis of visual assessment between test and control groups Group 2 W 4 W 6 W 8 W Test vs. Control 0.463 0.102 0.028* 0.012* *Significantly different at p < 0.05 compared with control group.

Example 6-6

(66) Melanin Index Analysis using Absorbance and Reflection

(67) Mexameter MX18 (C+K, Germany) was used for melanin index measurement. This is a tool to measure the levels of melanin and hemoglobin, key factors of skin color determination, based on absorbance and reflection. The skin absorption rates of each wavelength were digitalized and measured as melanin index (MI) and erythema index (EI). These measurements were repeated three times on the pigmented skin area and averaged.

(68) Observations of the MI were compared and analyzed before and after each evaluation point, and the MI for the both test group and control group showed a significant improvement after two weeks of product use (p<0.05, Table 8, FIG. 8).

(69) TABLE-US-00008 TABLE 8 Statistical analysis of melanin index by absorption and reflection Group Week N Mean.sup.1 SD p-value.sup.2 Control Before 22 204.89 34.43 (A) 2 W 22 191.08 33.64 0.000* 4 W 22 188.41 32.49 0.000* 6 W 22 179.73 31.45 0.000* 8 W 22 172.38 32.62 0.000* Test Before 22 204.67 34.33 (C) 2 W 22 194.55 34.12 0.000* 4 W 22 187.94 35.49 0.000* 6 W 22 168.96 32.18 0.000* 8 W 22 163.17 32.77 0.000* .sup.1Decrement of the mean-value represents decrease of melanin index. .sup.2Significantly different at *p < 0.05 compared with before treatment.

(70) Additionally, observations of the MI were compared and analyzed before and after each evaluation point. The MI for the test group showed a significant improvement after 6 weeks and 8 weeks of product use compared to the control group (p<0.05, Table 9, FIG. 8).

(71) TABLE-US-00009 TABLE 9 Statistical analysis of melanin index between test and control groups Group 2 W 4 W 6 W 8 W Test vs. Control 0.027 0.885 0.000* 0.000* *Significantly different at p < 0.05 compared with control group.

Example 6-7

(72) Analysis of Skin Luminance (L*value) and Individual Typology Angle (ITA value)

(73) Spectrophotometer CM-2500d (Minolta, Japan) was used to measure skin lightness and an individual typology angle. This is a tool to measure spectral reflection of object color in L*, a*, and b* parameters, the color coordinates of CIE, by measuring tristimulus values. L* represents the lightness, and a* and b* represent the color and the chroma. a* indicates the red while a* indicates the green, and b* indicates yellow while b* indicates blue. The values of L*, a*, and b* toward the middle indicate achromatic colors while those to the opposite directions indicate high chromaticity. These measurements were repeated three times on the pigmented skin area, and the average value was used to analyze the L* value. The ITA value was analyzed using the following equation. L*: Luminance parameters (brightness) a*: Chrominance parameters (green-to-red) b*: Chrominance parameters (blue-to-yellow) ITA=[Arc Tangent ((L*50)/b*)]180/3.14159

(74) Additionally, for the L* value using spectral reflection, observations were compared and analyzed before and after each evaluation point. The L* value for the test group showed a significant improvement after 6 weeks and 8 weeks of product use compared to the control group (p<0.05, Table 11, FIG. 9).

(75) TABLE-US-00010 TABLE 10 Statistical analysis of skin lightness (L* value) by spectral reflectance Group Week N Mean.sup.1 SD p-value.sup.2 Control Before 22 61.20 2.30 (A) 2 W 22 62.53 2.19 0.000* 4 W 22 62.86 2.23 0.000* 6 W 22 63.54 2.36 0.000* 8 W 22 63.88 2.10 0.000* Test Before 22 61.05 2.56 (C) 2 W 22 62.23 2.20 0.002* 4 W 22 62.88 2.26 0.000* 6 W 22 64.08 2.51 0.000* 8 W 22 64.59 2.38 0.000* .sup.1Increment of the mean-value represents improvement of skin lightness (L* value). .sup.2Significantly different at *p < 0.05 compared with before treatment.

(76) Additionally, for the L* value using spectral reflection, observations were compared and analyzed before and after each evaluation point. The L* value for the test group showed a significant improvement after 6 weeks and 8 weeks of product use compared to the control group (p<0.05, Table 11, FIG. 9).

(77) TABLE-US-00011 TABLE 11 Statistical analysis of skin lightness (L* value) between test and control groups Group 2 W 4 W 6 W 8 W Test vs. Control 0.234 0.344 0.000* 0.000* *Significantly different at p < 0.05 compared with control group.

(78) Meanwhile, for the ITA value using spectral reflection, observations were compared and analyzed before and after each evaluation point. The ITA value for the both test group and the control group showed a significant improvement after two weeks of product use (p<0.05, Table 12, FIG. 10).

(79) TABLE-US-00012 TABLE 12 Statistical analysis of skin color (ITA value) by spectral reflectance Group Week N Mean.sup.1 SD p-value.sup.2 Control Before 22 29.47 5.16 (A) 2 W 22 31.74 5.00 0.000* 4 W 22 32.83 5.25 0.000* 6 W 22 34.72 5.40 0.000* 8 W 22 35.97 5.38 0.000* Test Before 22 29.11 6.02 (C) 2 W 22 31.54 5.11 0.001* 4 W 22 33.11 5.53 0.000* 6 W 22 36.22 6.07 0.000* 8 W 22 37.54 5.95 0.000* .sup.1Increment of the mean-value represents improvement of skin color (ITA value). .sup.2Significantly different at *p < 0.05 compared with before treatment.

(80) Additionally, for the ITA value using spectral reflection, observations were compared and analyzed before and after each evaluation point. The ITA value for the test group showed a significant improvement after 6 weeks and 8 weeks of product use compared to the control group (p<0.05, Table 13, FIG. 10).

(81) TABLE-US-00013 TABLE 13 Statistical analysis of skin lightness (L* value) between test and control groups Group 2 W 4 W 6 W 8 W Test vs. Control 0.659 0.174 0.000* 0.000* *Significantly different at p < 0.05 compared with control group.

Example 6-8

(82) Evaluation of Skin Safety

(83) Medical examinations, observations of the test conductor, evaluations of subjects' subjective degree of skin stimulus, and objective skin stimulus were conducted at each evaluation point, and no adverse reactions were observed in all subjects during the entire experiment period (Table 14).

(84) TABLE-US-00014 TABLE 14 Skin adverse reactions 2 W 4 W 6 W 8 W Symptom A/C A/C A/C A/C Subjective Itching 0 0 0 0 initation Prickling 0 0 0 0 Tickling 0 0 0 0 Burning 0 0 0 0 Stinging 0 0 0 0 Stiffness 0 0 0 0 Tightening 0 0 0 0 etc. 0 0 0 0 Objective Erythema 0 0 0 0 Irritation Edema 0 0 0 0 Scale 0 0 0 0 Papule 0 0 0 0 etc. 0 0 0 0 Total number of subjects 0 0 0 0 A; Control group, C; Test group

(85) Based on the above description, it should be understood by one of ordinary skill in the art that other specific embodiments may be employed in practicing the invention without departing from the technical idea or essential features of the present invention. In this regard, the above-described examples are for illustrative purposes only, and the invention is not intended to be limited by these examples. The scope of the present invention should be understood to include all of the modifications or modified forms derived from the meaning and scope of the following claims or its equivalent concepts, rather than the above detailed description.