PEPTIDE WITH AMIDATED CARBOXY TERMINUS HAVING MELANOGENESIS INHIBITORY ACTIVITY AND COMPOSITION COMPRISING SAME

Abstract

The present invention relates to a peptide with amidated carboxy terminus having melanogenesis inhibitory activity and a composition comprising same, and more specifically, to a peptide with amidated (—NH.sub.2) carboxy terminus, composed of one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9 to 18, and a pharmaceutical composition, cosmetic composition, and food composition for the treatment of pigmentation diseases comprising same as an active ingredient.

Claims

1. A peptide with amidated (—NH.sub.2) carboxy terminus comprising one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 9 to 18.

2. The peptide of claim 1, wherein the peptide includes one amino acid sequence selected from the group consisting of SEQ ID NO: 14 to 18.

3. The peptide of claim 1, wherein the peptide has melanogenesis inhibitory activity.

4. A pharmaceutical composition for preventing or treating pigmentation diseases comprising at least one peptide with amidated (—NH.sub.2) carboxy terminus selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 9 to 18 as an active ingredient.

5. A cosmetic composition comprising at least one peptide with amidated (—NH.sub.2) carboxy terminus selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 9 to 18.

6. A food composition comprising at least one peptide with amidated (—NH.sub.2) carboxy terminus selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 9 to 18.

7. The pharmaceutical composition of claim 4, wherein the pigmentation diseases are at least one selected from the group consisting of melasma, freckles, dark circles, lentigos, age spots, melanocytic nevus, cafe au lait spots, Ota nevus, blue nevus, hyperpigmented nevus, pigmentation of nipples, pigmentation of labia minora and blemishes.

8. The composition of claim 5, wherein the composition is used for whitening.

9. The composition of claim 6, wherein the composition includes one form selected from the group consisting of a functional food, a nutritional supplement, a health functional food and food additives.

10. A use of at least one peptide with amidated (—NH.sub.2) carboxy terminus for preparing a formulation for preventing or treating pigmentation diseases selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 9 to 18.

11. A method for treating pigmentation diseases, the method comprising: administering an effective amount of a composition containing at least one peptide with amidated (—NH.sub.2) carboxy terminus selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 9 to 18 into a subject in need thereof.

12. The composition of claim 6, wherein the composition is used for whitening.

Description

DESCRIPTION OF THE DRAWINGS

[0076] FIG. 1a is a view showing results of positional scanning of synthetic peptide combinatorial library (PS-SPCL) with regard to intracellular melanogenesis. B16-F10 cells, which are a murine melanoma cell line, were stimulated with a vehicle control group or a corresponding peptide pool, after which a content of melanin was measured. Each panel shows results obtained from a tetrapeptide pool, of which amino acid was identified, and a sequence of X and O in the upper part of the panel indicates positional properties of amino acid of the tetrapeptide pool. Each of positions O is determined as one of 20 L-amino acids and the rest of five Xs include a mixture of 20 L-amino acids. FIG. 1b is a view showing an effect on B16-F10 cell viability as test results of MTT assay for each peptide combination. [FIG. 1a: representing a p value compared to cells stimulated with α-MSH only, and FIG. 1b: representing a p value compared to control group]

[0077] FIG. 2a is a view showing experimental results representing an effect of each tetrapeptide on cellular melanin biosynthesis. A cell-based assay of FIG. 2a was performed by treating B16-F10 cells with a vehicle or a peptide at a certain concentration, then stimulating the resultant cells with 100 nM of α-MSH, then measuring absorbance at 475 nm in 72 hours later, and then measuring a content of melanin. The amino acid sequences described in FIG. 2a are all described in a direction from amino terminus to carboxy terminus. An amino functional group (—NH.sub.2) represented at a peptide terminus represents an amino functional group added by amidation of a carboxy terminus, which is used in peptide synthesis. FIG. 2b is a view showing an effect of each tetrapeptide on B16-F10 cell viability as test results of MTT assay for each tetrapeptide. [FIG. 2a: representing a p value compared to cells stimulated with α-MSH only, and FIG. 2b: representing a p value compared to control group]

[0078] FIG. 3a is a view showing experimental results representing an effect of D3, D5 and D9 tetrapeptides on a melanin content of B16-F10 cells. FIG. 3a shows a melanin content of cells which are treated with a peptide at a concentration of 10-30 μM and stimulated with 100 nM of α-MSH for 72 hours. The melanin content was normalized with regard to a total protein content. FIG. 3b is a view showing an effect of each tetrapeptide on B16-F10 cell viability as test results of MTT assay for D3, D5 and D9 tetrapeptides. In FIGS. 3a and 3b, coumaric acid and arbutin were used as a positive control group. [FIG. 3a: representing a p value compared to cells stimulated with α-MSH only, and FIG. 3b: representing a p value compared to control group]FIG. 4a shows an effect of peptides, in which a sequence of two or three amino acids is defined. A cell-based assay of FIG. 4a was performed by treating B16-F10 cells with a vehicle or a peptide at a certain concentration, then stimulating the resultant cells with 100 nM of α-MSH, then measuring absorbance at 475 nm in 72 hours later, and then measuring a content of melanin. FIG. 4b is a view showing an effect of each peptide on B16-F10 cell viability as test results of MTT assay for peptides, in which a sequence of two or three amino acids is defined. [FIG. 4a: representing a p value compared to cells stimulated with α-MSH only, and FIG. 4b: representing a p value compared to control group]

[0079] FIG. 5a is a view showing experimental results representing an effect of glycine derivatives on a melanin content of B16-F10 cells. FIG. 5a shows a melanin content of cells which are treated with glycine derivatives at various concentrations and stimulated with 100 nM of α-MSH for 72 hours. The melanin content was normalized with regard to a total protein content. FIG. 5b is a view showing an effect of each peptide on B16-F10 cell viability as test results of MTT assay for peptides, in which a sequence of glycine derivatives is defined. In FIGS. 5a and 5b, coumaric acid and arbutin were used as a positive control group. [FIG. 5a: representing a p value compared to cells stimulated with α-MSH only, and FIG. 5b: representing a p value compared to control group]

[0080] FIG. 6a is a view showing experimental results representing an effect of tetrapeptide D3, tripeptide E5, dipeptide F1 and monopeptide G1 on a content of extracellular and intracellular melanins in HEMs cell lines stimulated with α-MSH. FIG. 6b is a view showing an effect of each peptide on HEMs cell viability as test results of MTT assay for HEMs cell lines. [FIG. 6a: representing a p value compared to cells stimulated with α-MSH only, and FIG. 6b: representing a p value compared to control group]

[0081] FIG. 7a is a view showing an effect on B16-F10 cell viability as test results of MTT assay for glycinamide-HCl. FIG. 7b is a view showing experimental results representing an effect of glycinamide-HCl on a melanin content of B16-F10 cells. [FIG. 7a: representing a p value compared to control group, and FIG. 7b: representing a p value compared to cells stimulated with α-MSH only]

[0082] FIG. 8a is a view showing an effect of glycinamide-HCl on activity of tyrosinase (TYR) with regard to B16-F10 cell lines stimulated with α-MSH. FIG. 8b shows a protein expression level of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrometautomerase (DCT) and β-actin. [representing a p value compared to cells stimulated with α-MSH only]

[0083] FIGS. 9a and 9b show an effect of glycinamide-HCl on a phosphorylation level of cAMP-responsive element binding protein (CREB) and extracellular signal-regulated kinase (ERK), and a protein level of microphthalmia-associated transcription factor (MITF) with regard to B16-F10 cell lines stimulated with α-MSH. [FIG. 9a: representing a p value compared to groups, and FIG. 9b: representing a p value compared cells stimulated with α-MSH only]

[0084] FIG. 10 shows peptide sequences having the melanogenesis inhibitory activity identified in the present invention.

MODE FOR INVENTION

[0085] Hereinafter, the present invention will be described in more detail.

[0086] However, the following Examples are provided only for the purpose of illustrating the present invention, and thus the content of the present invention is not limited thereto.

Experimental Method

[0087] 1. Peptide Synthesis

[0088] A synthetic tetrapeptide combinatorial library package was purchased from Peptron (Daejeon, the Republic of Korea). A library includes four positional sub-libraries, that is, OXXX—NH.sub.2, XOXX—NH.sub.2, XXOX—NH.sub.2, and XXXO—NH.sub.2. In this case, each of positions O includes one of 20 L-amino acids, and a position X includes an equimolar mixture of 20 L-amino acids. Peptides of the library were synthesized through a C-terminus amidation reaction.

[0089] Additionally, a peptide pool and each peptide were prepared by using a peptide custom-made service of Peptron Co. (Daejeon, Korea). In an example, a peptide, which was chemically synthesized by using a carboxy terminus amidation, was used. A type or purity of peptide was confirmed by using mass spectrometry (MS) and high performance liquid chromatography (HPLC).

[0090] 2. Cell Culture

[0091] Cells were cultured in a humidified incubator at 37° C. and 5% CO.sub.2. Murine melanoma B16-F10 cell lines were cultured in Dulbecco' Modified Eagle Medium containing 10% fetal bovine serum and antibiotics (100 U/mL of penicillin, 0.1 mg/mL of streptomycin and 0.25 μg/mL of amphotericin B), which were purchased from the American Type Culture Collection (Manassas, Va., USA). A human epidermal melanocyte (HEM), derived from a pigmented human neonatal foreskin, was purchased from Cascade Biologics (Portland, Oreg., USA) and cultured in medium 254 containing human melanocyte growth supplements (Cascade Biologics) and antibiotics.

[0092] 3. Cell Viability

[0093] Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. B16-F10 cell lines were treated with test peptides at various concentrations for 72 hours. The cells were washed with PBS and cultured in 100 μl of culture fluid, to which 1 mg/mL of MTT (Amresco, Solon, Ohio, USA) was added, for three hours. After that, the culture fluid was removed therefrom and formazan accumulated inside the cells was extracted with 100 μl of dimethyl sulfoxide (DMSO). Absorbance of the extracted solution was measured at 595 nm by using SPECTROstar Nano microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).

[0094] 4. Melanogenesis Inhibitory Activity

[0095] A melanin formation inhibitory effect of the peptide to be analyzed was confirmed by using B16-F10 cell lines and HEM. The B16-F10 cells were treated with test peptides at various concentrations and stimulated for 72 hours by using 100 nM of α-MSH. Hexapeptide B6 (Phe-Ser-His-His-Leu-Gly-NH.sub.2) (SEQ ID NO: 20), p-coumaric acid and arbutin were used as a control group. A level of extracellular melanin was measured by using a conditioned medium in which cells were previously cultured. Intracellular melanin was extracted at 60° C. for 60 minutes by using 1.0 M NaOH. A content of melanin was spectroscopically measured by measuring absorbance at 475 nm and resultant values were normalized with regard to a total protein content of cells by using Bio-Rad DC assay.

[0096] 5. TYR Activity Assay

[0097] B16-F10 cells were treated with glycinamide-HCl for 60 minutes and stimulated for 24 hours by using 100 nM of α-MSH. The cells were dissolved in 10 mM of cold Tris-HCl buffer solution (pH 7.4) containing 120 mM of sodium chloride, 25 mM of potassium chloride, 2.0 mM of ethylene glycol tetraacetic acid, 1.0 mM of ethylenediaminetetraacetic acid, 0.5% triton X-100, protease inhibitor cocktail (Roche, Mannheim, Germany). Cytolysate was centrifuged at 13,000×g at 4° C. for 15 minutes to obtain a cell-free extract therefrom. TYR activity was measured by using L-tyrosine and L-3,4-dihydroxyphenylalanine. A reaction mixture (200 μL) includes 100 mM of sodium phosphate (pH 6.8), 1.0 mM of L-tyrosine, 42 μM of L-3,4-dihydroxy phenylalanine and a cell-free extract (40 μg of protein), which was cultured at 37° C. A change in absorbance was measured at 475 nm by using Spectrostar Nano microplate reader.

[0098] 6. Western Blotting

[0099] Western blotting was performed as described in a document [Study on phenolic compounds and novel peptides inhibiting UVB- and PM10-induced MMP1 expression and melanogenesis in skin cells. PhD Thesis 2017. Kyungpook National University, Korea]. B16-F10 cells were dissolved in 10 mM of cold Tris-HCl buffer solution (pH 7.2) containing 150 mM of NaCl, 5 mM of EDTA, 0.1% sodium dodecyl sulfate (SDS), 1% triton X-100, 1% deoxycholate, 1 mM of phenylmethylsulphonyl fluoride and protease inhibitor cocktail (Roche). A part of cytolysate (30 μg of protein) was mixed with Laemmli sample buffer, then heated at 95° C. for five minutes to be denaturalized, and then was subject to electrophoresis in 10% SDS-polyacrylamide gel. After the electrophoresis, protein was transferred from gel to polyvinylidene difluoride membrane (Amersham Pharmacia, Little Chalfont, UK). Primary antibodies for TYR, TYRP1, DCT, MITF and @-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Primary antibodies for CREB, phospho-CREB (Ser133), extracellular signal-regulated kinase (ERK) and phospho-ERK (Thr202/Tyr204) were purchased from Cell Signaling (located in Danvers, Mass., USA). The membrane was cultured overnight at 4° C. along with the primary antibodies and cultured at the room temperature for one hour along with secondary antibodies (Cell Signaling) bound with horseradish peroxidase. A protein band was visualized by using picoEPD Western Reagent kit (Elpis-Biotech, Daejeon, South Korea), and a depth of image was analyzed by using National Institutes of Health ImageJ program.

[0100] 7. Statistical Analysis

[0101] Data are indicated as mean±SD of three independent experiments. Experimental results were statistically analyzed through one-way ANOVA by using SigmaStat v.3.11 software (Systat Software Inc., San Jose, Calif., USA), and then all the experimental groups were compared with a single control group through Dunnett test. If a p value was less than 0.05, the value itself was indicated in a graph.

Example 1: Identification of Peptide Having Melanogenesis Inhibitory Activity Using PS-SPCL

[0102] Screening using PS-SPCL was performed by treating B16-F10 melanoma cells, which had been stimulated with α-MSH, with all the peptide pools of synthetic tetrapeptide combinatorial library, and then observing an effect on melanogenesis (FIG. 1a).

[0103] As expected, α-MSH increased a melanin content of the B16-F10 cells, which was then weakened in various degrees by various tetrapeptide pools at a concentration of 1.0 mM. Out of those peptide pools, a tetrapeptide pool particularly having an excellent effect of inhibiting melanogenesis was confirmed. According to the above experimental results and an amino acid sequence of the identified peptide pool, a peptide pool having an arginine residue at a first position of an amino acid residue significantly inhibited melanogenesis in the cells stimulated with α-MSH. Similarly, peptide pools having phenylalanine or leucine at a second position, cysteine or tryptophan at a third position, and glycine, arginine or cysteine at a fourth position showed a significant effect of inhibiting melanin synthesis. Thus, a sequence of tetrapeptide having an anti-melanogenesis effect was predicted as follows according to positional scanning results: (Arg)-(Phe/Leu)-(Cys/Trp)-(Gly/Arg/Cys)-NH.sub.2 (SEQ ID NO: 21).

Example 2: Melanogenesis Inhibitory Effect of Individual Tetrapeptide

[0104] According to PS-SPCL results of the preceding example, eight individual tetrapeptides having a certain amino acid at each position of amino acid sequence were synthesized (FIGS. 2a and 3a).

[0105] These tetrapeptides include Arg at a first position; Phe or Leu at a second position; Cys or Trp at a third position; and Gly or Arg at a fourth position from an amino terminus, and were named as D1 to D8 (D1 peptide, RFCG-NH.sub.2, SEQ ID No: 1; D2 peptide, RFCR—NH.sub.2, SEQ ID NO: 2; D3 peptide, RFWG-NH.sub.2, SEQ ID NO: 3; D4 peptide, RFWR—NH.sub.2, SEQ ID NO: 4; D5 peptide, RLWG-NH.sub.2, SEQ ID NO: 5; D6 peptide, RLWR-NH.sub.2, SEQ ID NO: 6; D7 peptide, RLCG-NH.sub.2, SEQ ID NO: 7; and D8 peptide, RLCR-NH.sub.2, SEQ ID NO: 8). These individual peptides were evaluated for melanin inhibitory effect at concentrations of 30 μM and 100 μM. Hexapeptide B6 (Phe-Ser-His-His-Leu-Gly-NH.sub.2) (SEQ ID NO: 20) was used as a control group. (Seok J K, Lee S W, Choi J, Kim Y M, Boo Y C. Identification of novel antimelanogenic hexapeptides via positional scanning of a synthetic peptide combinatorial library. Experimental Dermatology. 2017 August; 26(8):742-744). As shown in FIG. 2a, tetrapeptide D3 (Arg-Phe-Trp-Gly-NH.sub.2) (SEQ ID NO: 2) and D5 (Arg-Leu-Trp-Gly-NH.sub.2) (SEQ ID NO: 4) excellently inhibited melanogenesis in the cells, which were stimulated with α-MSH. Hexa-peptide B6 showed a melanogenesis inhibitory effect at 100 μM only.

[0106] The present inventors have found that a sequence of tetrapeptides D3 (Arg-Phe-Trp-Gly-NH.sub.2) (SEQ ID NO: 2) and D5 (Arg-Leu-Trp-Gly-NH.sub.2) (SEQ ID NO: 4) is similar to a sequence of α-MSH, that is, a part of Ac-SYSMEHFRWGKPV-NH.sub.2 (SEQ ID NO: 19), which is FRWG, and thus included tetrapeptide D9 (FRWG-NH.sub.2, SEQ ID NO: 9) in the following experiment. The present inventors have further confirmed anti-melanogenesis activity with regard to D3, D5 and D9 peptides according to the same method by using coumaric acid and arbutin as a positive control. When making a comparison based on a molar concentration, it was confirmed that not only tetrapeptides D3 and D5 but also tetrapeptide D9 have melanogenesis inhibitory activity at a concentration lower than that of p-coumaric acid and arbutin (See FIG. 3a).

Example 3: Melanogenesis Inhibitory Effect of Short Peptide

[0107] In the preceding example, it was confirmed that a peptide having an amino acid sequence of D3, D5 and D9 has the most powerful melanogenesis inhibitory effect. A melanogenesis inhibitory effect and applicability thereof were further examined with regard to a peptide having a sequence shorter than that of the above tetrapeptide, which was expected to be more advantageous in terms of convenience, economic efficiency and the like of peptide synthesis (FIG. 4a).

[0108] To that end, E1 to E8 peptides were further synthesized: E1 (RFW-NH.sub.2, SEQ ID NO: 10); E2 (RFG-NH.sub.2, SEQ ID NO: 11); E3 (RLG-NH.sub.2, SEQ ID NO: 12); E4 (RLW-NH.sub.2, SEQ ID NO: 13); E5 (FWG-NH.sub.2, SEQ ID NO: 14); E6 (LWG-NH.sub.2, SEQ ID NO: 15); E7 (RWG-NH.sub.2, SEQ ID NO: 16); and F1 (WG-NH.sub.2, SEQ ID NO: 17). In case of the above E1 to F1 peptides, a carboxy terminus was amidated according to a chemical synthesis method, and an effect on melanogenesis was evaluated at a concentration of 30 μM to 100 μM.

[0109] As shown in FIG. 4a, the above peptides for additional analysis all showed an excellent melanin inhibitory effect. Out of the tested tripeptides, E5 (Phe-Trp-Gly-NH.sub.2) (SEQ ID NO: 14), E6 (Leu-Trp-Gly-NH.sub.2) (SEQ ID NO: 15) and E7 (Arg-Trp-Gly-NH.sub.2) (SEQ ID NO: 16) containing Trp-Gly-NH.sub.2 showed melanogenesis inhibitory activity higher than that of other tripeptides. Further, it was confirmed that dipeptide F1 (Trp-Gly-NH.sub.2 (SEQ ID NO: 17), tryptophanyl glycinamide) also has an effect of melanogenesis inhibitory activity.

Example 4: Melanogenesis Inhibitory Effect of Glycine Derivatives

[0110] The present inventors have further confirmed that glycinamide (Gly-NH.sub.2, SEQ ID NO: 18, G1 peptide) maintains anti-melanogenesis activity, while free glycine and acetyl glycinamide have no activity.

[0111] As shown in FIG. 5a, when making a comparison based on a molar concentration, it was assumed that a melanogenesis inhibitory effect of glycinamide is similar to that of p-coumaric acid and remarkably more excellent than that of arbutin.

Example 5: Melanogenesis Inhibitory Effect on HEM Cell Line

[0112] To more clearly confirm experimental results of B16-F10 cells, an additional analysis was performed by using HEM cell lines (human epidermal melanocyte cells).

[0113] As shown in FIG. 6a, a content of intracellular and extracellular melanins in HEMs was increased by α-MSH treatment, and an increase in the melanin content was weakened by tetrapeptide D3, tripeptide E5, dipeptide F1 and monopeptide G1.

[0114] HEMs cell viability was not affected by peptide treatment (FIG. 6b).

[0115] Thus, it was clearly confirmed that the peptide of the present invention is a melanogenesis inhibitor which is safely available without cytotoxicity.

Example 6: Efficacy Test of Glycinamide-HCl

Example 6-1: Cytotoxicity and Melanogenesis Inhibitory Activity of Glycinamide-HCl

[0116] Cytotoxicity and melanogenesis inhibitory activity of glycinamide-HCl were more specifically confirmed by using glycinamide-HCl, which was an edible type of salt, while showing an effect of glycinamide.

[0117] As shown in FIG. 7, glycinamide-HCl did not cause any cytotoxicity in the B16-F10 cells up to 300 μM. It was confirmed that glycinamide-HCl reduced a level of intracellular and extracellular melenins in the cells stimulated with α-MSH at 25-100 μM.

Example 6-2: Measurement of TRY Activity and Related Protein Levels of Glycinamide-HCl

[0118] As shown in FIG. 8a, it was confirmed that α-MSH increased TYR activity and this change was significantly weakened by glycinamide-HCl. Further, α-MSH increased a protein level of tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1), but did not increase a protein expression level of dopachrometautomerase (DCT) or β-actin. However, in case of being treated with glycinamide-HCl, a protein level of TYR, TYRP1 and DCT was significantly reduced both in the presence and in the absence of α-MSH.

[0119] It was confirmed that glycinamide-HCl controls an expression level of enzymes related to melanin biosynthesis such as tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT, also called tyrosinase-related protein 2 (TYRP2)), etc., thus effectively inhibiting melanogenesis.

Example 6-3: Effect of Glycinamide-HCl on CREB, ERK Phosphorylation and MITF Protein Expression Levels

[0120] It is known that an α-MSH-MC1R path induces a TYR expression by activating CREB and MITF. Thus, it was examined whether glycinamide-HCl affects a signaling path or not.

[0121] As shown in FIG. 9a, phosphorylation of CREB (Ser) was significantly increased in just 20 minutes after stimulation with α-MSH, and reached a maximum level in 60 minutes later as measured by western blotting. In the meantime, a total CREB value was not changed. α-MSH also increased phosphorylation of ERK (Thr and Tyr), but any significant change was not observed at a level of total ERK. Glycinamide-HCl inhibited phosphorylation of CREB, which had been stimulated with α-MSH, but did not affect phosphorylation of ERK.

[0122] Further, as shown in FIG. 9b, a-MSH increased a level of MITF expression, but was weakened by glycinamide-HCl.

[0123] Thus, it might be confirmed that glycinamide-HCl inhibits phosphorylation of CREB and ERK to reduce a protein expression of MITF, thereby effectively inhibiting melanogenesis.

Example 7: Measurement of Cell Viability

[0124] After the B16-F10 cells and HEM were treated with the peptides of Examples 1 to 4 according to the same method, cell viability was measured and the results thereof were summarized in FIGS. 1b, 2b, 3b, 4b, 5b, 6b and 7a respectively.

[0125] In case of being treating with the peptide pool of Example 1 at a concentration of 1.0 mM, cell viability thereof was somewhat decreased depending on the peptide pool in some cases. However, it was determined that a melanogenesis inhibitory effect of a selected peptide pool is not attributable to a decrease in cell viability (FIG. 1b).

[0126] Out of individual peptides of Examples 2 to 4, some individual peptides somewhat decreased cell viability in some cases. However, it was also determined that a melanogenesis inhibitory effect of a selected peptide pool is not attributable to a decrease in cell viability. (FIGS. 2b to 5b) In addition, it was confirmed that D3, D5, F1 and G1 peptides have almost no cytotoxicity to HEM cell lines, which were additionally experimented (FIG. 6b).

[0127] Thus, it was confirmed that the peptides according to the present invention may be safely and effectively used due to low cytotoxicity.

INDUSTRIAL APPLICABILITY

[0128] As described above, the peptide having melanogenesis inhibitory activity, which has been identified in the present invention, may be effectively used in developing medicine and medical supplies, cosmetic materials or functional foods for more safely and effectively preventing or treating pigmentation diseases.