Identification of small peptides and glycinamide that inhibit melanin synthesis using a positional scanning synthetic peptide combinatorial library.

BACKGROUND: Antimelanogenic peptides are potentially useful to treat hyperpigmentation, but many peptides have limited application because of high cost and/or low activity. OBJECTIVES: To identify small and potent peptide inhibitors of cellular melanin synthesis that are useful for cosmetic and medical applications. METHODS: A positional scanning synthetic tetrapeptide combinatorial library was used for screening of potentially active peptides. Antimelanogenic activities of the peptide pools and individual peptides were evaluated in B16-F10 melanoma cells and human epidermal melanocytes treated with alpha-melanocyte-stimulating hormone (α-MSH). RESULTS: Predicted active tetrapeptide sequences were R-(F/L)-(C/W)-(G/R)-NH2 . Of the individual tetrapeptides tested, D3 (RFWG-NH2 ) and D5 (RLWG-NH2 ) exhibited high antimelanogenic activities. Tetrapeptide D9 (FRWG-NH2 ) with a sequence identical to that of a portion of α-MSH also showed antimelanogenic activity. Of the tripeptides tested, E5 (FWG-NH2 ), E6 (LWG-NH2 ) and E7 (RWG-NH2 ) were relatively more active. Dipeptide F1 (WG-NH2 ) and monopeptide G1 (G-NH2 , glycinamide) retained activity, but G2 (Ac-G-NH2 ) and G3 (glycine) did not. The antimelanogenic activities of peptides D3, E5, F1 and G1 were verified in α-MSH-stimulated human epidermal melanocytes. Commercially available G-NH2 ·HCl suppressed the phosphorylation levels of cAMP-responsive element binding protein, protein levels of microphthalmia-associated transcription factor and tyrosinase, l-tyrosine hydroxylase activity of tyrosinase, and the melanin levels in stimulated cells. CONCLUSIONS: Small peptides, including glycinamide and tryptophanyl glycinamide, are potent antimelanogenic agents with potential value for the treatment of skin hyperpigmentation.

Human skin-depigmenting effects of resveratryl triglycolate, a hybrid compound of resveratrol and glycolic acid.

OBJECTIVES: Resveratryl triglycolate (RTG) is a hybrid compound derived by the esterification of resveratrol with glycolic acid. This compound has been previously shown to inhibit cellular melanin synthesis in vitro. This study aimed to examine the in vivo skin-depigmenting efficacy of RTG in human participants. METHODS: In total, 22 women aged between 25 and 49 years with Fitzpatrick skin type III or IV were enrolled. Their forearms were exposed to UV to induce artificial pigmentation. The test product containing 0.4% RTG or the control product was applied twice daily for up to 8 weeks after the artificial pigmentation. The participants visited the research centre every 2 weeks and were subjected to skin assessments. RESULTS: Visual assessment of pigmentation degree and instrumental analysis of melanin index, skin lightness (L* value) and skin colour (individual typology angle, ITAo ) indicated enhanced depigmentation of the skin in the test group, compared with the control group, in Weeks 6 and 8 (P < 0.05). No adverse skin reactions were observed in any of the participants during the entire test. CONCLUSION: This study demonstrated the skin-depigmenting effects of RTG in human participants.

Antimelanogenic effects of luteolin 7-sulfate isolated from Phyllospadix iwatensis Makino.

BACKGROUND: Abnormal deposition of melanin may cause an aesthetic skin problem; therefore, the control of unwanted excessive melanin synthesis is the major goal of cosmetic research. OBJECTIVES: To identify novel tyrosinase (TYR) inhibitors from marine plants and examine their cellular antimelanogenic effects. METHODS: The extracts of 50 marine plants endemic to Korea were screened against human TYR. Active constituents were then isolated from the selected plant extracts that showed potential and their chemical structures elucidated. Furthermore, their antimelanogenic effects were examined using murine melanoma B16/F10 cells and human epidermal melanocytes (HEM). RESULTS: Among the tested extracts, that of Phyllospadix iwatensis Makino exhibited the strongest human TYR inhibitory activity. The active constituents were purified from the butanol fraction of the P. iwatensis extract and identified as hispidulin 7-sulfate and luteolin 7-sulfate. Luteolin 7-sulfate inhibited human TYR more strongly than hispidulin 7-sulfate, luteolin, hispidulin and arbutin. Furthermore, luteolin 7-sulfate showed lower cytotoxicity than luteolin in both B16/F10 cells and HEM. Luteolin 7-sulfate attenuated cellular melanin synthesis more effectively in B16/F10 cells and HEM stimulated by α-melanocyte-stimulating hormone and l-tyrosine than arbutin. CONCLUSIONS: This study demonstrates that luteolin 7-sulfate isolated from P. iwatensis is a human TYR inhibitor with advantageous antimelanogenic properties, and would be useful for development as a therapeutic agent for the control of unwanted skin pigmentation.

A proposal of a standardized protocol to evaluate waterproof effect of eyeliner and mascara.

OBJECTIVE: Eye make-up products must have waterproofing properties to make sure that their colours do not smudge or wash away easily and remain intact despite water or perspiration. Until now, most research has focused on composition and components of make-up products and not on the level of waterproof. This study aimed to find methods to assess the waterproof degree of eyeliners and mascaras and determine the suitability of these methods. METHODS: Twenty female subjects were selected to test the waterproof of eyeliners, whereas 20 sets of false eyelashes were used to evaluate the waterproof of mascaras. For evaluating water-resistant properties, after test sites where eyeliners and mascaras were applied were immersed in water and natural drying for over 20 min (not artificial drying by drier etc.), L* value of the eyeliners applied on the forearm before and after the immersions, and intensity analysis values of mascaras applied on the false eyelashes were used to calculate the mean percentage waterproof removal ratio (%WPR). A product was hypothesized to be water resistant if the value for the mean %WPR was ≤50%. RESULTS: The non-waterproof eyeliners were not waterproof if their mean %WPR was >50%, whereas the waterproof eyeliners were waterproof if their mean %WPR was <50%. For mascaras, the mean %WPR was <50% after 1- to 2-h marks after immersion in water for both non-waterproof and waterproof products. After 3-4 h, the mean %WPR for the non-waterproof mascaras was >50%, rendering them not waterproof, whereas the mean %WPR for the waterproof mascaras was <50%, making them waterproof. CONCLUSION: We have evaluated the waterproof properties by analysing photographed images of the test sites where eyeliners and mascaras were applied. Results of the comparison between non-waterproof and waterproof eyeliners and mascaras, and the methods used, in particular, will be found useful in evaluating waterproof of other make-up products.

Ucma, a direct transcriptional target of Runx2 and Osterix, promotes osteoblast differentiation and nodule formation.

OBJECTIVE: Runt-related transcription factor 2 (Runx2) and Osterix (Osx) are the master transcription factors in bone formation. Nonetheless, genes acting downstream of both Runx2 and Osx have yet to be fully characterized. Here, we investigate the downstream targets of both Runx2 and Osx in osteoblasts. MATERIALS AND METHODS: DNA microarray analysis was conducted on calvarial RNA from wild-type, Runx2 heterozygous, Osx heterozygous, and Runx2/Osx double heterozygous embryos. Expression and transcriptional responses of the selected target gene were analyzed in MC3T3-E1 osteoblastic cells. RESULTS: The expression of unique cartilage matrix-associated protein (Ucma) was decreased in Runx2/Osx double heterozygous embryos. In contrast, Ucma expression was increased in osteoblasts overexpressing both Runx2 and Osx. Ucma expression was initiated mid-way through osteoblast differentiation and continued throughout the differentiation process. Transcriptional activity of the Ucma promoter was increased upon transfection of the cells with both Runx2 and Osx. Runx2-and Osx-mediated activation of the Ucma promoter was directly regulated by Runx2-and/or Sp1-binding sites within its promoter. During osteoblast differentiation, the formation of mineralized nodules in Ucma-overexpressing stable clones occurred earlier and was more enhanced than that in the mock-transfected control. Mineralized nodule formation was strongly augmented in the cells cultured in a medium containing secretory Ucma proteins. CONCLUSION: Ucma is a novel downstream gene regulated by both Runx2 and Osx and it stimulates osteoblast differentiation and nodule formation.

A quantitative evaluation method of skin texture affected by skin ageing using replica images of the cheek.

OBJECTIVE: Skin texture is a fine structure of skin surface where the hill and furrow were crossed to form a star shape. This study was performed to establish a quantitative evaluation method of skin texture affected by skin ageing using replica images of the cheek. METHODS: After producing replicas of the left cheek areas of 80 female subjects, representative replica images were chosen to establish six-level facial skin texture index. Using this new index, skin texture of different-aged subjects was visually assessed by multiple examiners. The number of star configurations was also analysed using the same replica images. Other factors contributing to skin texture, such as skin elasticity, roughness, dermal density, moisture and gloss, were also analysed. RESULTS: The concordance between skin texture scores evaluated by three researchers was high (0.896), and there was a high correlation between skin texture score and age (r = 0.642). The number of star configurations showed high correlations with skin texture scores (r = 0.753) and with age (r = 0.776). Skin texture scores were highly correlated with skin roughness and dermal density, but not with moisture, gloss and elasticity. CONCLUSION: This study suggests that visual grading of skin texture score based on new facial skin texture index and quantification of star configurations will be useful in evaluating skin ageing.

Screening of plant extracts for human tyrosinase inhibiting effects.

Screening for tyrosinase (TYR) inhibitors potentially useful for control of skin pigmentation has been hampered by the limited availability of human TYR. To overcome this hurdle, we have established human embryonic kidney (HEK293)-TYR cells that constitutively express human TYR. In the current study, we assayed human TYR inhibition activities of 50 plant extracts using the lysates of transformed HEK293-TYR cells. The strongest inhibition of human TYR was shown by the extract of Vaccinium bracteatum Thunberg, followed by the extract of Morus bombycis Koidzumi. The former extract did not inhibit mushroom TYR activity whereas significant inhibition was observed with the latter extract, demonstrating the importance of using human TYR in the screening for human TYR inhibitors. Upon liquid-liquid partitioning of the extract from V. bracteatum, the active constituents were enriched in the ethyl acetate fraction, and the subsequent preparatory thin-layer chromatography identified p-coumaric acid (PCA) as the main active constituent. The hypo-pigmentation of PCA was verified in the MelanoDerm™ Skin Model. This study demonstrates that transformed HEK293-TYR cells could expedite the discovery of human TYR-specific inhibitors from natural sources which might be useful in the control of skin pigmentation.

Effects of a new mild shampoo for preventing hair loss in Asian by a simple hand-held phototrichogram technique.

This study was conducted to evaluate the effects of a commercially available shampoo in Korean subjects with alopecia using a simple hand-held phototrichogram technique. Forty-four subjects with alopecia were enrolled and forty subjects continued for 16 weeks. In the test group, total hair counts increased significantly at weeks 8 and 16, and the number of shedding hair significantly decreased at week 16. Terminal hair counts significantly increased at week 8. In the control group, hair thickness and the number of vellus hairs significantly decreased at week 16. The number of total hairs significantly increased in the test group than in the control group at weeks 8 and 16. The number of shedding hairs significantly decreased in the test group than in the control group at week 16. Visual assessment using clinical digital images showed that the number of total hairs appeared to increase although there was no statistical significance. In this study, it was found that the test shampoo could prevent hair loss.

Effects of p-coumaric acid on erythema and pigmentation of human skin exposed to ultraviolet radiation.

BACKGROUND: It has been recently recognized that p-coumaric acid (PCA) is a strong inhibitor of cellular melanogenesis. AIM: To evaluate the erythema-suppressive and skin-lightening effects of PCA after topical application to human skin. METHODS: The control and PCA cream products were applied twice daily to the skin of the forearm of 21 subjects before and after ultraviolet (UV) irradiation to determine whether they could prevent erythema formation and pigmentation. The cream products were also applied to different areas only after the induction of erythema or pigmentation to determine whether they could have a depigmenting effect. RESULTS: A 7-day application of control and PCA cream products before UV irradiation decreased UV-induced erythema formation by 31% and 77%, respectively, compared with untreated skin. When the PCA cream was applied after UV irradiation, its effects on skin colour or pigmentation were less remarkable. However, the melanin index was significantly decreased at the sites treated with PCA cream for 70 days compared with control sites, and the Individual Typology Angle (ITA°) value was increased significantly. Of the 21 subjects, 2 had mild adverse skin reactions to both the PCA and control creams. CONCLUSION: These results suggest that PCA cream can reduce UV-induced erythema formation and subsequent pigmentation in human skin.

p-Coumaric acid, a constituent of Sasa quelpaertensis Nakai, inhibits cellular melanogenesis stimulated by alpha-melanocyte stimulating hormone.

BACKGROUND: Recent study has demonstrated that Sasa quelpaertensis (Korean name, Jeju-Joritdae) extracts inhibit cellular melanogenesis implicating potential use in the control of skin pigmentation. OBJECTIVES: The purpose of the present study was to elucidate the active constituents of this plant inhibiting melanogenesis and the associated mechanism. METHODS: The effect of the plant-derived materials on melanin production and/or tyrosinase expression was examined in murine melanoma B16/F10 cells and neonatal human melanocytes. RESULTS: When tested in melanoma B16/F10 cells treated with the alpha-melanocyte stimulating hormone (alpha-MSH), the aqueous ethanol extract of S. quelpaertensis culm inhibited the cellular melanogenesis more effectively than its leaf extract. A major active compound was isolated from the culm extract by solvent fractionation and column chromatography, and identified to be p-coumaric acid by spectroscopic and chromatographic analyses. The compound (p-coumaric acid) inhibited alpha-MSH-stimulated cellular melanogenesis more effectively than arbutin or other structurally similar compounds including 3-(4-hydroxyphenyl) propionic acid, cinnamic acid and caffeic acid. It also attenuated alpha-MSH-dependent increase of tyrosinase protein. The antimelanogenic effect of p-coumaric acid was also verified in neonatal human melanocytes. CONCLUSIONS: The present study identified p-coumaric acid as a main constituent of S. quelpaertensis inhibiting cellular melanogenesis. Because of its structural similarity, p-coumaric acid may interfere with l-tyrosine action in the control of tyrosinase expression in response to alpha-MSH.

Mechanisms of melanogenesis inhibition by 2,5-dimethyl-4-hydroxy-3(2H)-furanone.

BACKGROUND: Increased production and accumulation of melanin is characteristic of a large number of skin diseases, including acquired hyperpigmentation such as melasma, postinflammatory melanoderma and solar lentigo. Thus, there is a increasing need for the development of depigmenting agents. OBJECTIVES: To evaluate the depigmenting capacity of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and to elucidate the mechanisms by which it inhibits alpha-melanocyte-stimulating hormone (alpha-MSH)-induced melanogenesis in B16 melanoma cells in vitro. METHODS: Several experiments were performed in B16 melanoma cells. We studied melanin content, tyrosinase activity and cAMP production, and performed cAMP response element (CRE) luciferase reporter assay and Western blots for proteins involved in melanogenesis. RESULTS: The melanin content and tyrosinase activity induced by alpha-MSH were inhibited significantly by DMHF. To clarify the mechanism of the depigmenting property of DMHF, we examined the involvement of DMHF in cAMP signalling induced by alpha-MSH. In CRE luciferase reporter assay, CRE reporter activation induced by alpha-MSH was inhibited by DMHF. Additionally, although DMHF did not inhibit cAMP production by alpha-MSH, both CRE binding protein (CREB) phosphorylation and the reduction of glycogen synthase kinase-3beta phosphorylation by alpha-MSH were blocked by DMHF. These data suggest that DMHF inhibits the downstream step of cAMP production induced by alpha-MSH, consequently inhibiting melanogenesis. This suggestion was further confirmed by the fact that the increased production levels of microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein-1 induced by alpha-MSH were all reduced by DMHF in B16 melanoma cells. CONCLUSIONS: Our study shows that DMHF inhibits alpha-MSH-induced melanogenesis by suppressing CREB phosphorylation, which is induced by protein kinase A, and suggests that DMHF may be an effective inhibitor of hyperpigmentation.

Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress.

Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.