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Antioxidant and Anti-Melanogenic Activities of Hyssopus officinalis Extracts
Abstract
Hyssopus officinalis is a herbaceous plant of the genus Hyssopus. Due to its properties as an antiseptic, cough reliever and expectorant, it is commonly used as an aromatic herb and medicinal plant. This study was performed to investigate the anti-oxidative and anti-melanogenic properties of Hyssopus officinalis extracts (HE) using in vitro assays and cell culture systems. As a result, HE showed higher DPPH and ABTS radicals scavenging activity in a dose-dependent manner. Also, HE inhibited the prodution of intracellular ROS and melanin contents in B16F10 melanoma cell as well as tyrosinase activity. We also found that HE inhibit mRNA expression of MITF, tyrosinase and TRP-2 gene. These findings suggest that HE may be beneficial for preventing oxidative damage and melanogenesis of skin.
... The results showed that the mentioned extract inhibits the production of intracellular reactive oxygen species and melanin in B16F10 melanoma cells, as well as the activity of tyrosinase. These findings suggest that the hyssop extract may be beneficial in preventing oxidative damage and melanogenesis in the skin, calling for further research in this direction [141]. ...
The aim of this review is to evaluate the potential of Hyssopus officinalis L., commonly known as hyssop, in addressing specific
dermatological concerns based on existing research evidence. The review presents an analysis of the composition of essential
oils and extracts derived from Hyssopus officinalis L. It encompasses a comprehensive examination of the pharmacological
activities associated with hyssop herb essential oils/extracts, particularly in the context of dermatological concerns. Existing
studies suggest promising potential for hyssop essential oil/extracts in addressing various skin conditions, including microbial
skin infections, inflammatory diseases and oxidative stress-related skin damage. There are also data on potential cytotoxic,
antigenotoxic and antimelanogenic effects. Furthermore, this review highlights the predominant compounds present in hyssop
essential oil and extracts, elucidating their role as potential carriers of pharmacological activity. In the conclusion, the key
findings of the review are summarized, emphasizing the promising attributes of Hyssopus officinalis L. for skin health.
However, it also underscores the importance of addressing existing gaps in research and conducting clinical trials and additional
studies to ensure the efficacy and safety of hyssop herb in dermatological applications.
... Human whole blood cells (WBC) were incubated with different concentrations of essential oils (12.5, 5 and 2.5 µg/mL) or methanol extracts of hyssop herb (100, 200 and 400 µg/mL) for 30 min at 37 • C. Tested concentrations were selected on the basis of the literature data [53,54]. The samples were treated in parallel with a negative control of PBS for 30 min at 37 • C, as well as with a positive control of hydrogen peroxide (H 2 O 2 , 50 µM) for 20 min at 4 • C and PBS for 30 min at 37 • C. A concentration of 50 µM H 2 O 2 was the lowest one that induced a statistically significant increase of DNA damage in the tested cells. ...
Hyssopus officinalis L. is a well-known aromatic plant used in traditional medicine and the food and cosmetics industry. The aim of this study is to assess the antioxidant, genotoxic, antigenotoxic and cytotoxic properties of characterized hyssop essential oils and methanol extracts. Chemical composition was analyzed by gas chromatography - mass spectrometry (GC-MS) and liquid chromatography with diode array detection and mass spectrometry (LC-DAD-MS), respectively. Antioxidant activity was examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) tests; genotoxic and antigenotoxic activity were examined by the comet assay, while cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide dye (MTT) test against tumor cell lines (SW480, MDA-MB 231, HeLa) and non-transformed human lung fibroblast cell lines (MRC-5). The essential oils were rich in monoterpene hydrocarbons (e.g., limonene; 7.99–23.81%), oxygenated monoterpenes (1,8-cineole; 38.19–67.1%) and phenylpropanoids (methyl eugenol; 0.00–28.33%). In methanol extracts, the most abundant phenolics were chlorogenic and rosmarinic acid (23.35–33.46 and 3.53–17.98 mg/g, respectively). Methanol extracts expressed moderate to weak antioxidant activity (DPPH IC50 = 56.04–199.89 µg/mL, FRAP = 0.667–0.959 mmol Fe2+/g). Hyssop preparations significantly reduced DNA damage in human whole blood cells, induced by pretreatment with hydrogen peroxide. Methanol extracts exhibited selective and potent dose- and time-dependent activity against the HeLa cell line. Results of the current study demonstrated notable H. officinalis medicinal potential, which calls for further investigation.
... Various medicinal herbs such as Codonopsis lanceolata [18], Kaempferia galanga [19], Hominis Placenta [20], and Hyssopus officinalis [21] Have been examined for their effects on skin whitening and wrinkles. In this study, to determine whether Galgeungyulpitang has inhibitory activity on elastase (which is involved in the maintenance of skin elasticity) and inhibition of melanin biosynthesis was studied. ...
Background This study was designed to investigate the potential effects of Galgeungyulpitang for whitening and elasticity treatment by examining its effect on melanoma cells. Methods The effects of Galgeungyulpitang on B16/F10 melanoma cell viability, production of melanin, tyrosinase and elastase, were investigated. Cell viability was measured by colorimetric assay that assesses cell metabolic activity (MTT assay). Melanin was measured by Hosei’s method, tyrosinase was measured by Yogi’s method and elastase was measured by James’s method. Results At concentrations higher than 500 μg/mL Galgeungyulpitang, cell viability was significantly reduced (p ≤ 0.05). At concentrations of 500 μg/mL and lower, morphological changes were not observed. The rate of melanin synthesis was significantly reduced to 73.49% ± 2.92% at a concentration of 500 μg/mL Galgeungyulpitang compared with untreated cells (p < 0.05). Extracellular tyrosinase production was not significantly decreased in vitro, however, intracellular tyrosinase production was significantly reduced to 76.06% ± 2.17% when treated with Galgeungyulpitang at a concentration of 500 μg/mL compared with the control (p < 0.05). Elastase Type 1 production was significantly reduced to 74.98% ± 3.24% and 69.62% ± 4.66% at concentrations of 250 and 500 μg/mL Galgeungyulpitang, respectively (p < 0.05). Elastase Type 4 production was significantly reduced to 72.77% ± 3.52% at concentrations of 250 and 500 μg/mL (p < 0.05). Conclusions The results in this study showed that Galgeungyulpitang may inhibit melanin and tyrosinase synthesis, and inhibit elastase production, suggesting that Galgeungyulpitang may be potentially beneficial for skin whitening and loss of skin elasticity treatments.
... However, research is ongoing to discover raw materials that exhibit better properties for preventing hyperpigmentation effects. Recently, there have been studies published on the effects of Pueraria [9], Siberian Sibiricum [10], and Hyssopus officinalis [11] on melanogenesis. ...
Background The purpose of this study was to investigate the effects of wild ginseng pharmacopuncture on melanin production in B16/F10 murine melanoma cells. Methods To determine the effect of wild ginseng pharmacopuncture solution on B16/F10 cells, cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) method. To observe B16/F10 cell growth, death, and morphological changes, Trypan blue solution was used. The Hosoi method was used to investigate the effect of wild ginseng pharmacopuncture solution on melanin production. The Martinez-Esparza method was used to investigate the effect of wild ginseng pharmacopuncture solution on tyrosinase activity. To determine the pathway involved in the melanogenesis in cells exposed to wild ginseng pharmacopuncture solution, a cell-free tyrosinase was used. Results Following treatment with 200 μL of wild ginseng solution, the cell survival rate was 76.32 ± 2.45% which significantly decreased with higher concentrations (μL) of wild ginseng (up to 200 μL). When 100 μL of wild ginseng was used, the cell survival rate was 89.95 ± 2.07%. No morphological changes or abnormalities were observed in the B16/F10 murine melanoma cells as observed in the Trypan blue test. Melanin production was significantly reduced to 72.17 ± 3.74% at 100 μL. Using 100 μL of wild ginseng solution, tyrosinase activity was significantly decreased to 80.15 ± 1.05%. Wild ginseng pharmacopuncture solution reduced melanin production both directly and indirectly. Conclusions This study suggests that wild ginseng pharmacopuncture solution may be effective in inhibiting melanin production. Further studies are needed to determine safe and effective clinical applications.
Background
Although various natural ingredients and formulated reagents are used as ingredients in cosmetics, it is important to have alternatives to animal tests, such as in vitro tests of toxicity, as animal testing of cosmetics is prohibited in many countries.Objectives
Here, cytotoxicity, genotoxicity, and embryotoxicity on zebrafish embryos were evaluated to confirm the safety of ingredients formulated with 30 extracts from natural resources used as cosmetic ingredients. The blended ingredients were tested by dividing them into mixtures of 25 natural products (NMA), 30 natural products (NMB), and concentrated NMB (NMC), depending on the number of blended natural components and their application.ResultsFirst, in vitro, HaCaT cells were treated with concentrations of 0 to 20% for 24 h to measure cell viability. DNA damage within the IC20 value was measured by comet assay. In the comet assay, 10 mM H2O2 was used as a positive control. Additionally, zebrafish embryos were used as a substitute for conventional animal testing to observe natural mixture toxicity. The olive tail moment (OTM) were not affected by NMA, NMB, or NMC in comparison with the positive control versus three representatives within the IC20 value concentration of each mixture. Zebrafish embryo results were analysed in a manner similar to the cytotoxicity test results. NMA mixtures showed no cytotoxicity, genotoxicity, or toxicity on zebrafish embryos. Other mixtures showed non-toxicity within the representatively concentrated range.Conclusion
Therefore, the 3 mixtures containing Hyssopus officinalis, Morus alba, Engraulis japonicus, and 27 other extracts were considered to be not significantly toxic in HaCaT cell and zebrafish, so it is suggested that they could be used as cosmetic ingredients.
The aim of this study was to determine the effects of adlay extract on melanin production and the antioxidant characteristics of the extract. The seeds were extracted by the supercritical fluid CO2 extraction (SFE) method. The effect of adlay extract on melanin production was evaluated using mushroom tyrosinase activity assay, intracellular tyrosinase activity, antioxidant properties and melanin content. Those assays were performed spectrophotometrically. In addition, the expression of melanogenesis-related proteins was determined by western blotting. The results revealed that the adlay extract suppressed intracellular tyrosinase activity and decreased the amount of melanin in B16F10 cells. The adlay extract decreased the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2). The extract also exhibited antioxidant characteristics such as free radical scavenging capacity and reducing power. It effectively decreased intracellular reactive oxygen species (ROS) levels in B16F10 cells. We concluded that the adlay extract inhibits melanin production by down-regulation of MITF, tyrosinase, TRP-1 and TRP-2. The antioxidant properties of the extract may also contribute to the inhibition of melanogenesis. The adlay extract can therefore be applied as an inhibitor of melanogenesis and could also act as a natural antioxidant in skin care products.
The kinetic behavior of the melanin biosynthesis pathway from L-tyrosine up to dopachrome has been studied from experimental and simulation assays. The reaction mechanism proposed is based on a single active site of tyrosinase. The diphenolase and monophenolase activities of tyrosinase involve one single (oxidase) and two overlapped (hydroxylase and oxidase) catalytic cycles, respectively. The stoichiometry of the pathway implies that one molecule of tyrosinase must accomplish two turnovers in the hydroxylase cycle for each one in the oxidase cycle. Furthermore, the steady-state rates of dopachrome production and O2 consumption from tyrosine and L-dopa, also fulfill the stoichiometry of the pathway: VO2T/VDCT = 1.5 and VO2T/VDCD = 1.0, where T represents L-tyrosine, DC represents dopachrome, and D represents L-dopa. It has been ascertained by high performance liquid chromatography that in the steady-state, a quantity of dopa is accumulated ([D]ss) which fulfills the constant ratio [D]ss = R[T]0. Taking this ratio into account, an analytical expression has been deduced for the monophenolase activity of tyrosinase. In this expression kcatT congruent to (2/3)k3(K1/K2)R, revealing that kcatT is not a true catalytic constant, since it also depends on equilibrium constants and on the experimental R = 0.057. This low value explains the lower catalytic efficiency of tyrosinase on tyrosine than on dopa, (VmaxT/KmT)/(VmaxD/KmD) congruent to (2/3)R, since a significant portion of tyrosinase is scavenged from the catalytic turnover as dead-end complex EmetT in the steady-state of the monophenolase activity of tyrosinase.
Melanocytes are cells of neural crest origin. In the human epidermis, they form a close association with keratinocytes via their dendrites. Melanocytes are well known for their role in skin pigmentation, and their ability to produce and distribute melanin has been studied extensively. One of the factors that regulates melanocytes and skin pigmentation is the locally produced melanocortin peptide alpha-MSH. The effects of alpha-MSH on melanogenesis are mediated via the MC-1R and tyrosinase, the rate-limiting enzyme in the melanogenesis pathway. Binding of alpha-MSH to its receptor increases tyrosinase activity and eumelanin production, which accounts for the skin-darkening effect of alpha-MSH. Other alpha-MSH-related melanocortin peptides, such as ACTH1-17 and desacetylated alpha-MSH, are also agonists at the MC-1R and could regulate melanocyte function. Recent evidence shows that melanocytes have other functions in the skin in addition to their ability to produce melanin. They are able to secrete a wide range of signal molecules, including cytokines, POMC peptides, catecholamines, and NO in response to UV irradiation and other stimuli. Potential targets of these secretory products are keratinocytes, lymphocytes, fibroblasts, mast cells, and endothelial cells, all of which express receptors for these signal molecules. Melanocytes may therefore act as important local regulators of a range of skin cells. It has been shown that alpha-MSH regulates NO production from melanocytes, and it is possible that the melanocortins regulate the release of other signalling molecules from melanocytes. Therefore, the melanocortin signaling system is one of the important regulators of skin homeostasis.
The microphthalamia-associated transcription factor (MITF) is an integral transcriptional regulator in melanocyte, the lineage from which melanoma cells originate. This basic-helix-loop-helix-leucine-zipper (bHLHzip) protein is critical for melanocyte cell-fate choice during commitment from pluripotent precursor cells in the neural crest. Its role in differentiation pathways has been highlighted by its potent transcriptional and lineage-specific regulation of the three major pigment enzymes: tyrosinase, Tyrp1, and Dct as well as other pigmentation factors. However, the cellular functions of MITF seem to be wider than differentiation and cell-fate pathways alone, since melanocytes and melanoma cells appear to require an expression of this factor. Here, we discuss the transcriptional networks in which MITF is thought to reside and describe signaling pathways in the cell which impinge on MITF. Accumulating evidence supports the notion that MITF is involved in survival pathways during normal development as well as during neoplastic growth of melanoma.
The microphthalmia-associated transcription factor Mitf plays a critical role in regulating many aspects of melanocyte biology. It is required for melanoblast and postnatal melanocyte survival, regulates proliferation, and activates genes associated with differentiation such as tyrosinase and related genes involved in melanogenesis. Identifying the signals that regulate Mitf expression is crucial if we are to understand how cells of the melanocyte lineage respond to environmental cues. Here we show that the Mitf promoter is induced by lipid signalling via the p38 stress-activated kinase pathway that is also activated by a wide range of receptors as well as UV irradiation. Signalling via p38 leads to increased phosphorylation and activation of cyclic adenosine monophosphate response element-binding (CREB) that binds and activates the Mitf promoter via the cyclic adenosine monophosphate (cAMP) response element. Moreover, we also show that activation of p38 mediated by lipids is potentiated by inhibition of the PI3kinase pathway but not by inhibition of protein kinase A (PKA). The results identify a mechanism in which stress signalling via p38 leads to activation of CREB, enhanced Mitf expression and consequently increased tyrosinase expression. The results are relevant for the regulation of melanocytes by Mitf, but also raise the possibility that lipid mediated activation of p38 signalling may represent a potential therapy for vitiligo.
The signalling pathways involved in melanogenesis have been partially elucidated. The role of melanotropin peptides, α-MSH and ACTH, in melanocyte differentiation and in the regulation of melanogenesis is now widely recognized. Their melanogenic effects appear to be mediated through the up-regulation of the cAMP pathway by the melanocortin-1 receptor and the activation of adenylate cyclase. The molecular mechanisms involve the subsequent phosphorylation and activation of PKA, followed by CREB which binds to the promoter of the transcription factor Microphthalmia, and upregulates its transcription. The elevated expression of Microphthalmia increases its binding to the specific E-box and M-box present in the tyrosinase promoter, resulting in an increased expression of this enzyme and the up-regulation of melanin synthesis. LEF-1 and β-catenin also regulate the expression of Microphthalmia and play a key-role in pigment cell differentiation. Other transcription factors such as Brn2, TBX2, PAX3, Sox10 are also involved in these processes. cAMP modulates several other signalling pathways involved in the regulation of melanogenesis. The cAMP-induced melanogenesis and dendricity are partially mediated by the inhibition of the phosphatidylinositol-3-kinase/p70S6 kinase pathway. In addition, cAMP activates a melanocyte-specific pathway leading to MAP kinase activation. These new advances lead to a better understanding of the natural photoprotective mechanisms of the skin. In the future, they should help to the development of treatments for pigmentary disorders, and more efficient prevention strategies against sun-induced carcinogenesis of human skin.
The purpose of this study was to investigate the antioxidant and skin whitening effects of Rhamnus yoshinoi extracts. Rhamnus yoshinoi was extracted with 100% ethanol and water. The antioxidative and skin whitening effects of extracts were determined by in vitro assays using the 1,1-diphenyl-2- picrylhydrazyl (DPPH) method and inhibitory effects against tyrosinase activity and melanogenesis in B16F1 melanoma cells. The radical scavenging activities of the Rhamnus yoshinoi extracts were tested by DPPH assay and showed high DPPH radical scavenging activities (SC50; 21.6 ppm in EtOH, 40.5 ppm in water). As for tyrosinase inhibitory activity, the Rhamnus yoshinoi ethanol extract had the highest inhibition activity (IC50; 256.3 ppm). In B16F1 mouse melanoma cells, the Rhamnus yoshinoi ethanol extract significantly inhibited melanin synthesis by 53.36% at the concentration of 50 ppm. These results suggest that Rhamnus yoshinoi ethanol extract has significant antioxidant activity and whitening activity.
The purpose of this study was to investigate how herb extracts may improve blood circulation. Twenty-six extracts from nine different herbs (marjoram, lavender, dill, rosemary, hyssop, rose, lemon balm, pineapple sage, and echinacea) were evaluated for their anti-hypertensive effects via angiotensin I converting enzyme (ACE) inhibition. Their cholesterol-lowering effects via hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibition and their fibrinolytic activity via fibrin-plate method were also evaluated. Both water extraction of rose flowers and 70% EtOH extraction of pineapple sage leaves effectively reduced the ACE activity with inhibition rates of 133.8% and 91.2%, respectively. Similarly, both water and 70% EtOH extracts of rose flowers strongly inhibited the enzymatic activity of HMG-CoA reductase by 48.9% and 80.5%, respectively. Water and 70% EtOH extracts of rose flowers also showed relatively high fibrinolytic activity. Based on these observations, rose flower extracts can be developed as a functional tool for use in the improvement of blood circulation.
In this study, the potent skin-whitening effects of octaphlorethol A (OPA) isolated from Ishige foliacea was investigated through inhibitory effect of melanin synthesis and tyrosinase activity in alpha-melanocyte stimulating hormone (α-MSH) induced B16F10 melanoma cells. OPA markedly inhibited melanin synthesis and tyrosinase activity in a concentrationdependent manner. We also found that OPA decreased microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein -1 and -2 (TRP-1 and TRP- 2) protein expressions. Moreover, OPA reduces p38 MAPK protein levels and activates extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinases (JNK) protein expressions in B16F10 cells. A specific ERK inhibitor PD98059 significantly blocks OPAinhibited melanin synthesis and tyrosinase activity, whereas a p38MAP and JNK inhibitor had no effect. These findings provide evidence demonstrating that the anti-melanogenic effect of OPA is mediated through the activation of ERK signal pathway in B16F10 cells. These results indicate that OPA has the potential to be used as a melanogenesis inhibitor in the food and cosmetics industry.
Tyrosinase is one of the key enzymes essential for melanogenesis. The control of its activity rests in part at the level of transcriptional regulation. The 5’ promoter regions of the human, mouse, chicken, quail, snapping turtle, and frog tyrosinase sequences have been isolated and the mechanisms regulating the activity of these sequences are beginning to be elucidated. This review provides an update on the following aspects of tyrosinase gene regulation: basal promoter elements that determine the site of transcription initiation for RNA polymerase II; the cis-acting elements and DNA-binding factors that mediate melanocyte-specific expression of the tyrosinase gene; promoter elements involved in the temporal control of tyrosinase gene expression; additional elements that may be required to achieve wild-type levels of gene expression; and specific elements that may be required for modulation of tyrosinase gene expression in response to humoral factors or external stimuli that are known to influence the amounts of melanin synthesized by fully differentiated melanocytes. The wild type expression of tyrosinase is the result of the interaction of many different factors and it is becoming evident that certain elements and factors play more than one role in this process.
Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.
The color of mammalian hair, skin, and eyes results mainly from the secretory products of melanocytes. These secretory products consist of a wide range of melanin pigments with different structures and compositions. These include black or brown nitrogenous eumelanins; yellow or reddish brown, sulfur-containing pheomelanins, e.g., the trichochromes of low molecular weight; and other pigments whose chemical and physical properties are intermediate between those of typical eumelanins and pheomelanins. Despite the evident differences in molecular size and general properties, all these pigments are biogenetically related, and they arise from a common mmetabolic pathway in which dopaquinone is the key intermediate. The current state of knowledge on the molecular mechanisms governing the etabolic fate of dopaquinone in melanocytes is discussed with special reference to the role of such sulfhydryl compounds as cysteine and glutathione in melanogenesis.
It was found that kojic acid, which is used in cosmetics for its excellent whitening effect, inhibits catecholase activity of tyrosinase in a non-classical manner. A decrease in the initial velocity to a steady-state inhibited velocity can be observed over a few minutes. This time-dependence, which is unaltered by prior incubation of the enzyme with the inhibitor, is consistent with a first-order transition. The kinetic data obtained correspond to those for a postulated mechanism that involves the rapid formation of an enzyme inhibitor complex that subsequently undergoes a relatively slow reversible reaction. Kinetic parameters characterizing this type of inhibition were evaluated by means of nonlinear regression of product accumulation curves.
Kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone), a fungal metabolic product, has increasingly been used as a skin-depigmenting agent in skin care products marketed in Japan since 1988. In order to determine its frequency of sensitization, during 1 year from October 1992 to September 1993, we performed patch testing with it in 220 female patients with suspected cosmetic-related contact dermatitis. Of the 220 patients, 8 used at least 1 skin care product containing kojic acid, 5 of whom reacted to kojic acid as well as to 1 or more their own products containing 1% kojic acid, but not to their other products not containing it, and 3 of whom were negative to kojic acid and all their own products. Patch testing with kojic acid in the remaining group of 212 patients, who had not previously used skin care products containing it, was negative without exception. The 5 kojic-acid-sensitive patients, aged 34 to 58 years, developed facial dermatitis 1-12 months after starting application of kojic-acid-containing products. Kojic acid is considered to have high sensitizing potential, as a comparatively high frequency of contact sensitivity was observed in patients using products containing it (5 out of 8).
Several genes critical to the enzymatic regulation of melanin production in mammals have recently been cloned and mapped to the albino, brown and slaty loci in mice. All three genes encode proteins with similar structures and features, but with distinct catalytic capacities; the functions of two of those gene products have previously been identified. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, while the slaty locus encodes tyrosinase-related protein 2 (TRP2), an enzyme with a single specific, but distinct, function as DOPAchrome tautomerase. Although the brown locus, encoding TRP1, was actually the first member of the tyrosinase gene family to be cloned, its catalytic function (which results in the production of black rather than brown melanin) has been in general dispute. In this study we have used two different techniques (expression of TRP1 in transfected fibroblasts and immunoaffinity purification of TRP1 from melanocytes) to examine the enzymatic function(s) of TRP1. The data demonstrate that the specific melanogenic function of TRP1 is the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to a carboxylated indole-quinone at a down-stream point in the melanin biosynthetic pathway. This enzyme activity appears to be essential to the further metabolism of DHICA to a high molecular weight pigmented biopolymer.
The present study was carried out to investigate the abundance of tyrosinase and related proteins (TRP-1 and TRP-2) in human epidermis and their relationship to melanin type. Positive immunocytochemical staining was seen for all three proteins in epidermal melanocytes. For each protein the numbers of positively stained melanocytes were similar in all subjects studied irrespective of skin type. Following 5 daily suberythemal doses of UVB the melanocytes were larger, more dendritic, and increased in number. With TRP-1 and TRP-2 the increase in number in response to UVB was unrelated to skin type and, hence, with melanin type but with tyrosinase there was a much greater increase in skin types III and IV than in skin type I and II. The enhanced numbers of tyrosinase-positive melanocytes were accompanied by increased staining intensity, suggesting a greater expression of tyrosinase in the melanocytes from skin types III and IV compared with skin types I and II. This increase in tyrosinase could be related to the greater levels of eumelanin found in skin types III and IV, and this is in keeping with the view that higher levels of tyrosinase are associated with the production of eumelanin than phaeomelanin.
Mice with mutations at the microphthalmia (mi) locus have some or all of the following defects: loss of pigmentation, reduced eye size, failure of secondary bone resorption, reduced numbers of mast cells, and early onset of deafness. Using a transgenic insertional mutation at this locus, we have identified a gene whose expression is disrupted in transgenic animals. This gene encodes a novel member of the basic-helix-loop-helix-leucine zipper (bHLH-ZIP) protein family of transcription factors, is altered in mice carrying two independent mi alleles (mi and miws), and is expressed in the developing eye, ear, and skin, all anatomical sites affected by mi. The multiple spontaneous and induced mutations available at mi provide a unique biological resource for studying the role of a bHLH-ZIP protein in mammalian development.
Tyrosinase is the key enzyme in pigment synthesis, initiating a cascade of reactions which convert the amino acid tyrosine to the melanin biopolymer. Two other tyrosinase-related proteins (TRP) are known, TRP-1 (probably DHICAoxidase) and TRP-2 (DOPAchrome tautomerase). These proteins show about 40% homology, and recent results have indicated that the genes might be derived from a common ancestor. We will discuss recent findings on genomic organization, and on the proteins and their presumed function, which is important for eumelanin synthesis in mouse and man.
The development of melanocytes, which are pigment-producing cells responsible for skin, hair, and eye color, is absolutely dependent on the action of the microphthalmia basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor (Mi); mice lacking a functional Mi protein are entirely devoid of pigment cells. Mi has been shown to activate transcription of the tyrosinase, TRP-1, TRP-2, and QNR-71 genes through specific E-box elements, most notably the highly conserved M box. We investigated the mechanism which enables Mi to be recruited specifically to a restricted subset of E boxes in target promoters while being prevented from binding E-box elements in other promoters. We show both in vitro and in vivo that the presence of a T residue flanking a CATGTG E box is an essential determinant of the ability of Mi to bind DNA, and we successfully predict that the CATGTG E box from the P gene would not bind Mi. In contrast, no specific requirement for the sequences flanking a CACGTG E box was observed, and no binding to an atypical E box in the c-Kit promoter was detected. The relevance of these observations to the control of melanocyte-specific gene expression was highlighted by the fact that the E-box elements located in the tyrosinase, TRP-1, TRP-2, and QNR-71 promoters without exception possess a 5' flanking T residue which is entirely conserved between species as diverse as man and turtle. The ability of Mi to discriminate between different E-box motifs provides a mechanism to restrict the repertoire of genes which are likely to be regulated by Mi and provides insight into the ability of bHLH-LZ transcription factors to achieve the specificity required for the precise coordination of transcription during development.
To discover safe and effective topical skin-lightening agents, we have evaluated alkyl esters of the natural product gentisic acid (GA), which is related to our lead compound methyl gentisate (MG), and four putative tyrosinase inhibitors, utilizing mammalian melanocyte cell cultures and cell-free extracts. Desirable characteristics include the ability to inhibit melanogenesis in cells (IC50 < 100 microg/mL) without cytotoxicity, preferably due to tyrosinase inhibition. Of the six esters synthesized, the smaller esters (e.g. methyl and ethyl) were more effective enzyme inhibitors (IC50 approximately 11 and 20 microg/mL, respectively). For comparison, hydroquinone (HQ), a commercial skin "bleaching" agent, was a less effective enzyme inhibitor (IC50 approximately 72 microg/mL), and was highly cytotoxic to melanocytes in vitro at concentrations substantially lower than the IC50 for enzymatic inhibition. Kojic acid was a potent inhibitor of the mammalian enzyme (IC50 approximately 6 microg/mL), but did not reduce pigmentation in cells. Both arbutin and magnesium ascorbyl phosphate were ineffective in the cell-free and cell-based assays. MG at 100 microg/mL exhibited a minimal inhibitory effect on DHICA oxidase (TRP 1) and no effect on DOPAchrome tautomerase (TRP-2), suggesting that MG inhibits melanogenesis primarily via tyrosinase inhibition. MG and GA were non-mutagenic at the hprt locus in V79 Chinese hamster cells, whereas HQ was highly mutagenic and cytotoxic. The properties of MG in vitro, including (1) pigmentation inhibition in melanocytes, (2) tyrosinase inhibition and selectivity, (3) reduced cytotoxicity relative to HQ, and (4) lack of mutagenic potential in mammalian cells, establish MG as a superior candidate skin-lightening agent.
The pigment melanin in human skin is a major defense mechanism against ultraviolet light of the sun, but darkened skin color, which is the result of increased and redistributed epidermal melanin, could be a serious aesthetic problem. Epidemiologically, it is well known that the consumption of green tea may help prevent cancers in humans and also reduce several free radicals including peroxynitrite. In the present study, to assess the efficacy of the inhibition of mushroom tyrosinase (monophenol monooxygenase EC 1.14.18.1), ten kinds of Korean traditional teas were screened for their tyrosinase inhibitory activity. Green tea was the strongest inhibitor, and the major active constituents in the tea are (-)-epicatechin 3-O-gallate (ECG), (-)-gallocatechin 3-O-gallate (GCG), and (-)-epigallocatechin 3-O-gallate (EGCG). All are catechins with gallic acid group as an active site. The kinetic analysis for inhibition of tyrosinase revealed a competitive nature of GCG with this enzyme for the L-tyrosine binding at the active site of tyrosinase.
Compelling evidence has been gathered indicating that pro-opiomelanocortin peptides, alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH), through the cyclic AMP pathway, play a pivotal role in melanocyte differentiation and in the regulation of melanogenesis. Recently, the molecular events linking cAMP to melanogenesis up-regulation have been elucidated. This cascade involves the activation of protein kinase A and CREB transcription factor, leading to the up-regulation of the expression of Microphthalmia associated transcription factor (MITF). MITF has been found mutated in patients with Waardenburg syndrome 2A, and plays a crucial role in melanocyte development. MITF binds and activates melanogenic gene promoters, thereby increasing their expression which results in an increased melanin synthesis. Beyond this simplified scheme, It appears that melanogenic gene expression is controlled by a complex network of regulation involving other transcription factors such as Brn2, TBX2, PAX3 and SOX10. Further studies are required to better understand the respective roles of these factors in the regulation of melanin synthesis. In addition, other intracellular signaling pathways, like the phosphatidyl inositol 3-kinase pathway, as well as the molecular cascade of events governed by the small GTP-binding protein Rho, seem to be involved in the regulation of melanogenesis and melanocyte dendricity. Finally, it should be mentioned that cAMP activates a melanocyte-specific pathway leading to MAP kinase activation. MAP kinase, ERK2, phosphorylates MITF, thereby targeting the transcription factor to proteasomes for degradation. Thus, in addition to the complex transcriptional regulation, melanogenesis is also subjected to a post-translational regulation that controls MITF or tyrosinase function. Taken together, these complex molecular processes would finally allow a fine tuning of melanocyte differentiation leading to melanin synthesis.
A combination of techniques, including high-performance liquid chromatography (HPLC), spectrophotometric measurements, and a novel method for quantifying melanosome morphology, were applied to the analysis of melanin content and composition in highly pigmented (Fitzpatrick type V and VI) human skin. We found that total epidermal melanin content is significantly elevated in photoexposed type V and VI skin (approximately 1.6 x), while analysis of individual melanin components suggests that pheomelanin content increases only slightly, whereas 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-eumelanin and to a greater extent 5,6-dihydroxyindole (DHI)-eumelanin content are both markedly elevated. Analysis of the relative composition of epidermal melanin in these subjects revealed that DHI-eumelanin is the largest single component (approximately 60-70%), followed by DHICA-eumelanin (25-35%), with pheomelanin being a relatively minor component (2-8%). Moreover, there was a comparative enrichment of DHI-eumelanin at photoexposed sites, with a corresponding decline in the relative contributions from DHICA-eumelanin and pheomelanin. There was also a good correlation and close agreement between the concentration of spheroidal melanosomes determined by morphological image analysis and the concentration of pheomelanin determined by a combination of HPLC and spectrophotometric analysis (r = 0.89, P < 0.02). This study demonstrates the usefulness of melanosome morphology analysis as a sensitive new method for the quantification of melanin composition in human skin. The data also suggest that DHI-eumelanin formation is the dominant pathway for melanin synthesis in heavily pigmented (Fitzpatrick V and VI) skin types in vivo, and is the favoured pathway when melanin production is increased in chronically photoexposed skin.
Lipids, particularly sphingolipids, are emerging as novel regulators of cellular activity. A placental total lipid fraction (PTLF), the total lipid prepared from an hydroalcoholic extract of fresh term human placenta, was previously shown to have a pigment-inducing activity in an animal model. The PTLF contains sphingolipids which stimulate DNA synthesis and melanin formation with marked morphological changes in B16F10 melanoma cells. In order to identify the mechanism underlying the increased melanin synthesis, B16F10 cells were treated with PTLF to assess the catalytic activities of tyrosinase (i.e. tyrosine hydroxylase and DOPA oxidase), the key regulatory enzyme of melanin synthesis. Tyrosine hydroxylase (estimated by the release of (3)H(2)O) as well as DOPA oxidase (measured spectrophotometrically and also in non-denaturing gels), was stimulated significantly by PTLF. Western blot analysis demonstrated an increase in the expression of tyrosinase, tyrosinase related proteins 1 and 2 (TRP1 and TRP2) at the protein level and RT-PCR analysis revealed stimulated transcription of tyrosinase, TRP1 and TRP2 mRNAs in PTLF-treated B16F10 cells. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PTLF-induction of tyrosinase activity with a corresponding decrease in melanogenesis. In all cases, the response to PTLF was similar to that induced by alpha-melanocyte stimulating hormone, a well-known stimulator of melanogenesis. Thus, these results provide the basis of action of PTLF stimulated melanogenesis in B16F10 cells showing that this placental extract is a strong inducer of pigmentation at the transcriptional and translational levels.
Most of the melanin pigmentary disorders are cosmetically important and have a strong impact on the quality of life of affected individuals. This article examines recent advances in the treatment of melanin pigmentary disorder including hypermelanosis and hypomelanosis. The development of laser technologies has completed the use of the increasing number of bleaching agents in treating hyperpigmented lesions. The treatment of hypomelanotic disorders is still often disappointing, but new therapeutic options provide encouraging results.
Inhibition of tyrosinase by green tea components
- J K No
- D Y Soung
- Y J Kim
- K H Shim
- Y S Jun
- S H Rhee
- T Yokizawa
- H Y Chung
Isolation of inhibitory components on tyrosinase activity from the bark of Paeonia moutan
- S H Lee
- J S Park
- S Y Kim
- J J Kim
- S R Chung
S. H. Lee, J. S. Park, S. Y. Kim, J. J. Kim, and S.
R. Chung, Isolation of inhibitory components on
tyrosinase activity from the bark of Paeonia moutan,
J. Pharm. Soc. Korea, 42(4), 353 (1998).