Inhibitory effects of a novel ascorbic derivative, disodium isostearyl 2-O-L-ascorbyl phosphate on melanogenesis

We investigated the inhibitory effects of a novel amphiphilic ascorbic derivative, disodium isostearyl 2-O-L-ascorbyl phosphate (VCP-IS-2Na), synthesized from a hydrophilic ascorbic derivative, sodium-2-O-L-ascorbyl phosphate (VCP-Na), on melanogenesis in cultured human melanoma cells, normal human melanocytes, and three-dimensional cultured human skin models. Melanin synthesis in melanoma cells treated with VCP-IS-2Na at 300 muM and melanocytes treated with VCP-IS-2Na at 100 muM decreased to 23% and 52% of that in non-treated cells, respectively, and the cell viability was not affected. VCP-IS-2Na also significantly suppressed the cellular tyrosinase activity of melanoma cells and melanocytes. Melanin synthesis in human skin models was evaluated by macro- and microscopic observations of its pigmentation and quantitative measurements of melanin. Treatment of the human skin models with 1.0% VCP-IS-2Na did not inhibit cell viability, while melanin synthesis was decreased to 21% of that in the control. In contrast, L-ascorbic acid (VC) and VCP-Na did not seem to inhibit melanin synthesis and cellular tyrosinase activity. These results indicate that VCP-IS-2Na may be an effective whitening agent for the skin, and we expect the application of VCP-IS-2Na in the cosmetic industry.     

Inhibitory Effect of Elaeagnus umbellata Fractions on Melanogenesis in α-MSH-Stimulated B16-F10 Melanoma Cells

When skin is exposed to UV radiation, melanocytes produce melanin. Excessive melanin production leads to skin pigmentation, which causes various cosmetic and health problems. Therefore, the development of safe, natural therapeutics that inhibit the production of melanin is necessary. Elaeagnus umbellata (EU) has long been widely used as a folk medicinal plant because of pharmacological properties that include anti-ulcer, antioxidant, and anti-inflammatory properties. In this study, we investigated the antioxidant activity and melanogenesis inhibitory effects of EU fractions in B16-F10 melanoma cells. EU fractions showed a dose-dependent increase in antioxidant activity in radical scavenging activity. In addition, we evaluated the effect of EU fractions on tyrosinase activity and melanogenesis in α-melanocyte-stimulating hormone-induced B16-F10 melanoma cells. EU was noncytotoxic at 12.5–50 μg/mL. EU fractions effectively inhibited tyrosinase activity and melanogenesis, suppressed the phosphorylation of CREB and ERK involved in the melanogenesis pathway, and down-regulated expression of melanogenesis-related proteins. Interestingly, the anti-melanogenesis effect was most effective at a concentration of 50 μg/mL EU, and the effects of the fractions were superior to those of the extract. Therefore, our study suggests that EU has potential as a safe treatment for excessive pigmentation or as a natural ingredient in cosmetics.     

The Combination of Niacinamide,Vitamin C,and PDRN Mitigates Melanogenesis by Modulating Nicotinamide Nucleotide Transhydrogenase

Nicotinamide nucleotide transhydrogenase (NNT) is involved in decreasing melanogenesis through tyrosinase degradation induced by cellular redox changes. Nicotinamide is a component of coenzymes, such as NAD⁺, NADH, NADP⁺, and NADPH, and its levels are modulated by NNT. Vitamin C and polydeoxyribonucleotide (PDRN) are also known to decrease skin pigmentation. We evaluated whether a mixture of nicotinamide, vitamin C, and PDRN (NVP-mix) decreased melanogenesis by modulating mitochondrial oxidative stress and NNT expression in UV-B-irradiated animals and in an in vitro model of melanocytes treated with conditioned media (CM) from UV-B-irradiated keratinocytes. The expression of NNT, GSH/GSSG, and NADPH/NADP⁺ in UV-B-irradiated animal skin was significantly decreased by UV-B radiation but increased by NVP-mix treatment. The expression of NNT, GSH/GSSG, and NADPH/NADP⁺ ratios decreased in melanocytes after CM treatment, although they increased after NVP-mix administration. In NNT-silenced melanocytes, the GSH/GSSG and NADPH/NADP⁺ ratios were further decreased by CM compared with normal melanocytes. NVP-mix decreased melanogenesis signals, such as MC1R, MITF, TYRP1, and TYRP2, and decreased melanosome transfer-related signals, such as RAB32 and RAB27A, in UV-B-irradiated animal skin. NVP-mix also decreased MC1R, MITF, TYRP1, TYRP2, RAB32, and RAB27A in melanocytes treated with CM from UV-irradiated keratinocytes. The expression of MC1R and MITF in melanocytes after CM treatment was unchanged by NNT silencing. However, the expression of TYRP1, TYRP2, RAB32, and RAB27A increased in NNT-silenced melanocytes after CM treatment. NVP-mix also decreased tyrosinase activity and melanin content in UV-B-irradiated animal skin and CM-treated melanocytes. In conclusion, NVP-mix decreased mitochondrial oxidative stress by increasing NNT expression and decreased melanogenesis by decreasing MC1R/MITF, tyrosinase, TYRP1, and TYRP2.     

DDX3 Upregulates Hydrogen Peroxide-Induced Melanogenesis in Sk-Mel-2 Human Melanoma Cells

DDX3 is a DEAD-box RNA helicase with diverse biological functions through multicellular pathways. The objective of this study was to investigate the role of DDX3 in regulating melanogenesis by the exploring signaling pathways involved. Various concentrations of hydrogen peroxide were used to induce melanogenesis in SK-Mel-2 human melanoma cells. Melanin content assays, tyrosinase activity analysis, and Western blot analysis were performed to determine how DDX3 was involved in melanogenesis. Transient transfection was performed to overexpress or silence DDX3 genes. Immunoprecipitation was performed using an antityrosinase antibody. Based on the results of the cell viability test, melanin content, and activity of tyrosinase, a key melanogenesis enzyme, in SK-Mel-2 human melanoma cells, hydrogen peroxide at 0.1 mM was chosen to induce melanogenesis. Treatment with H₂O₂ notably increased the promoter activity of DDX3. After treatment with hydroperoxide for 4 h, melanin content and tyrosinase activity peaked in DDX3-transfected cells. Overexpression of DDX3 increased melanin content and tyrosinase expression under oxidative stress induced by H₂O₂. DDX3 co-immunoprecipitated with tyrosinase, a melanogenesis enzyme. The interaction between DDX3 and tyrosinase was strongly increased under oxidative stress. DDX3 could increase melanogenesis under the H₂O₂-treated condition. Thus, targeting DDX3 could be a novel strategy to develop molecular therapy for skin diseases.     

Development of 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent

A stable derivative of kojic acid, 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one (Kojyl-APPA), was synthesized in good yield. The effects of Kojyl-APPA on tyrosinase activity and melanin synthesis were investigated. Kojyl-APPA showed tyrosinase inhibition effect (30%) in situ, but not in vitro. Kojyl-APPA inhibited tyrosinase activity significantly at 24 h after treatment in normal human melanocytes. It means that Kojyl-APPA is not a direct inhibitor of tyrosinase itself, but it is converted to a potential inhibitor kojic acid enzymatically in cells. In addition, Kojyl-APPA decreased melanin content to 75% of control in melanoma cells and decreased neomelanin synthesis to 43% of control in normal human melanocytes. Its permeation through skin increased by about 8 times as compared with kojic acid.     

The Human Melanocyte as a Particular Target for UVA Radiation and an Endpoint for Photoprotection Assessment

The induction of DNA breaks by UVA (320-400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin-related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

Effects of ultraviolet irradiation on melanogenesis from tyrosine, Dopa and dopamine: a matrix-assisted laser desorption/ionization mass spectrometric study

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry experiments were applied to study the influence of ultraviolet (UV) irradiation in melanogenesis. Samples were prepared starting from three different precursors, tyrosine, Dopa and dopamine, in the presence or absence of tyrosinase, the enzyme responsible for the synthesis of melanin. Enzymatic reactions were carried out for 10, 30, 60 and 120 min under UV irradiation at 365 nm, and aliquots were then immediately ultrafiltered and lyophilized. Samples obtained by irradiation of tyrosine solution revealed the formation of 5,6-dihydroxyindole (DHI) oligomers up to pentamers at 120 min; the reaction kinetics were markedly enhanced in the presence of tyrosinase. In the case of Dopa, UV irradiation favored melanogenesis only in the presence of the enzyme; in this case, many reaction pathways were activated, originating various oligomeric species of Dopa, DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Conversely, when dopamine was used as tyrosinase substrate under UV light, mechanisms of melanogenesis different from those generated by simple enzymatic reaction without irradiation were not activated, as the same oligomeric species were present.     

Phospholipases Induce Melanogenesis in Organ-Cultured Skin

Guinea pig skin becomes more pigmented following exposure to UV rays. This melanization was accompanied by enhanced intensity of tyrosinase-staining and increased number of tyrosinase-positive melanocytes (MEL^ty+), with resultant enhancement of melanin synthesis. To clarify the regulatory mechanism for melanization following UV irradiation, organ-cultured guinea pig skins have been used to examine their melanogenic responses to exogenous stimulation. This organ culture system responded well to UV irradiation, by increasing melanogenic activity. Also, in this system, phospholipases (PL), arachidonic acid, interleukin-1α and melanocytestimulating hormone, but not endothelin-1 or phosphatidylinositol-specific PLC (PI-PLC), stimulated melanogenesis to various extents as indicated by the number of MEL^ty+ and morphological changes. Among them, the PLA₂ and PLD were found to have a potent stimulatory property for melanocytes. They might affect melanocytes directly or indirectly through an effect on keratinocytes. These results suggest that PLA₂ and PLD play a key role in epidermal hyperpigmentation after UV irradiation or inflammation.     

Dawson结构的多金属氧酸盐对酪氨酸酶的抑制作用

按文献方法合成了两种Dawson结构的多金属氧酸盐,并对其结构进行紫外光谱和红外光谱分析。以H_6[P_2Mo_(18)O_(62)]和H_8[P_2Mo_(17)Cr(OH_2)O_(61)](简写为P_2Mo_(18)和P_2Mo_(17)Cr)为效应物,采用紫外分光光度法和酶动力学方法研究两种Dawson结构的多金属氧酸盐效应物对蘑菇酪氨酸酶二酚酶活性的抑制作用。结果表明,P_2Mo_(18)和P_2Mo_(17)Cr对酪氨酸酶二酚酶均具有显著的抑制效果,测定抑制酪氨酸酶活力下降50%的抑制浓度(IC_(50))分别为(0.482±0.009)mmol/L和(0.503±0.011)mmol/L。动力学分析表明,P_2Mo_(18)和P_2Mo_(17)Cr对酪氨酸酶的抑制作用均表现为可逆的竞争型,抑制常数K_I分别为0.212和0.249 mmol/L。其中,综合考虑IC_(50)值和抑制常数,P_2Mo_(18)对酪氨酸酶二酚酶活性的抑制效果略优于P_2Mo_(17)Cr。     

THE CHEMICAL TRANSFORMATION OF THE CATECHOLAMINES INDUCED BY UV IRRADIATION*

Abstract— The conversion of catecholamines exposed to ultraviolet irradiation has been investigated spectrophotometricaly. The UV activation of the molecule is a direct effect and the activation is not dependent upon the presence of metals. The transformation, which includes mdolization and polymerization requkes the presence of oxygen. The rate of the oxidative transformation is of the same order of magnitude in 100 per cent oxygen as in air, provided a metal chelating agent is present.
At neutral pH photoactivated adrenaline is oxidized to adrenochrome and further polymerized to melanin. At alkaline pH U.V. irradiation results in the formation of conversion products of adrenaline, different from those observed at neutral pH, and the melanin production is low.
When noradrenaline is subjected to U.V. irradiation at neutral pH the processes of oxidation and polymerization takes place at a much slower rate than that observed for adrenaline. The radiation effect on noradrenaline at alkaline pH is characterized by increased melanin formation.
Radicals derived from U.V. irradiation of H₂O₂ convert the catecholamines to different products, and no melanin formation can be recorded.
The sequence of reactions leading from the activated catecholamines to indolic structures, and the participation of the indolic structures in melanogenesis from the catecholamines is discussed.     

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, R_conj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of R_conj by O^- has been found to be more efficient than the less selective OH radical. Peroxidation of R_conj is shown to be somewhat slower, ( k _R+ O₂˜ 3 × 10⁸ M ^-1 s^-1), than O₂ reactions with radicals in oleate ( k _R+ O₂= 1 × 10⁹ M ^-1 s^-1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2 k RO₁˜ 10⁷ M ^-1 s^-1). The superoxide radical, O^-₂, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.     

RATE CONSTANTS FOR INTERACTION OF 1O21Δg) WITH AZIDE ION IN WATER

Abstract— The rate constant for quenching of ¹O₂ by azide ion in water was determined to be (5.0 ± 0.4) × 10⁸ M ⁻¹ s⁻¹ using a variety of sensitizers (including humic acids) and ¹O₂ acceptors. The apparent second-order rate constant decreases with pH below pH 5.5 in accordance with the protonation of azide ion to form hydrazoic acid (p K _a= 4.6). Quenching by hydrazoic acid is at least 2 orders of magnitude slower than by azide ion. Greater than 99% of all interactions between ¹O₂ and azide ion involve physical quenching rather than chemical reaction. Humic acid triplets are not significantly quenched by azide ion at concentrations less than 2 m M , allowing azide ion quenching to be used as a diagnostic test for the intermediacy of ¹O₂ in photosensitized oxidations in natural surface waters.