Modulation of melanogenesis by aloesin: a competitive inhibitor of tyrosinase

Aloesin, [2-acetonyl-8-beta-d-glucopyranosyl-7-hydroxy-5-methylchromone], a compound isolated from the Aloe plant, is shown in these studies to modulate melanogenesis via competitive inhibition of tyrosinase. Aloesin inhibits purified tyrosinase enzyme and specifically inhibits melanin production in vitro. Enzyme kinetics studies using normal human melanocyte cell lysates and cell-based melanin production demonstrated that aloesin is a competitive inhibitor of tyrosinase from mushroom, human and murine sources. Tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (DOPA) oxidase activities of tyrosinase from normal human melanocyte cell lysates were inhibited by aloesin in a dose dependent manner. In a percutaneous absorption study a finite dose of aloesin penetrated the skin slowly and was recovered primarily in the surface wash. Aloesin shows promise as a pigmentation-altering agent for cosmetic or therapeutic applications.     

Identifying 6,7,4'-trihydroxyisoflavone as a potent tyrosinase inhibitor

A known biotransformed compound, 6,7,4'-trihydroxyisoflavone, was identified as a potent tyrosinase inhibitor. It inhibited mushroom tyrosinase with an IC50 value of 9.2 microM, which is six times the anti-tyrosinase activity of kojic acid (IC50 = 54.4 microM). The inhibition kinetics, analyzed by Lineweaver-Burk plots, indicated 6,7,4'-trihydroxyisoflavone to be a competitive inhibitor of tyrosinase when L-tyrosine was used as a substrate. Its biosynthesis precursors and analogs, including glycitein, daidzein, and genistein, showed little anti-tyrosinase activity. The results suggest that hydroxyl groups at the C-6 and C-7 positions of the isoflavone skeleton might play an important role in the expression of tyrosinase inhibitory activity.     

Kojic acid-tripeptide amide as a new tyrosinase inhibitor

Twenty two kojic acid-tripeptide amides were prepared using a solid-phase Fmoc/tBu strategy with Rink Amide SURE(R) resin. To effectively obtain kojic acid-tripeptide amide conjugates, the coupling conditions of kojic acid to the tripeptide on the resin were optimized. The tyrosinase inhibitory activity of kojic acid-tripeptide amides and the effect of the amino acid sequence on the activity were compared with those of kojic acid-tripeptide acids. The stability of kojic acid-tripeptide amides were then compared with those of kojic acid and kojic acid-tripeptides acids. As a consequence, kojic acid-FWY-NH(2) proved to be the best compound, with the highest inhibitory activity, which was maintained over different storage times under various temperatures and pHs.     

Gnetol as a potent tyrosinase inhibitor from genus Gnetum

Gnetol (2,3',5',6-tetrahydroxy-trans-stilbene), a naturally occurring compound particularly found in the genus Gnetum, had a strong inhibitory effect on murine tyrosinase activity. Gnetol (IC50, 4.5 microM) was stronger than kojic acid (IC50, 139 microM) as a standard inhibitor for murine tyrosinase activity. Moreover, gnetol significantly suppressed, melanin biosynthesis in murine B16 melanoma cells.     

Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO)

Background

Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase.

Methods

The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo.

Results

MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling.

Conclusions

Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders.

General significance

MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders.     

Statistically designed enzymatic hydrolysis for optimized production of icariside II as a novel melanogenesis inhibitor

Three kinds of prenylated flavonols, icariside I, icariside II, and icaritin, were isolated from an icariin hydrolysate and their effects on melanogenesis evaluated based on mushroom tyrosinase inhibition and quantifying the melanin contents in melanocytes. Although none of the compounds had an effect on tyrosinase activity, icariside II and icaritin both effectively inhibited the melanin contents with an IC50 of 10.53 and 11.13 MM, respectively. Whereas icariside II was obtained from a reaction with beta-glucosidase and cellulase, the icariin was not completely converted into icariside II. Thus, for the high-purity production of icariside II, the reaction was optimized using the response surface methodology, where an enzyme concentration of 5.0 mg/ml, pH 7, 37.5 degrees C;, and 8 h reaction time were selected as the central conditions for the central composite design (CCD) for the enzymatic hydrolysis of icariin into icariside II using cellulase. Empirical models were developed to describe the relationships between the operating factors and the response (icariside II yield). A statistical analysis indicated that all four factors had a significant effect (p<0.01) on the icariside II production. The coefficient of determination (R2) was good for the model (0.9853), and the optimum production conditions for icariside II was an enzyme concentration of 7.5 mg/ml, pH 5, 50 degrees C, and 12 h reaction time. A good agreement between the predicted and experimental data under the designed optimal conditions confirmed the usefulness of the model. A laboratory pilot scale was also successful.     

Mammalian melanogenesis: tyrosinase versus peroxidase involvement, and activation mechanisms

A study of the enzymes functioning in murine melanogenesis was carried out on tissue homogenates of the black mouse. Several major points were resolved: (a) while the enzyme peroxidase is capable of converting tyrosine to melanin in vitro, it is not responsible for observed melanogenesis in the mouse, (b) a proteolytic activation system for tyrosinase, such as that described for amphibian skin, does not seem to function in mammalian tyrosinase activation, and (c) tyrosinase activity in normal murine tissues can be stimulated with a variety of treatments.     

L-mimosine a slow-binding inhibitor of mushroom tyrosinase

It was found that L-mimosine is a slow-binding inhibitor of L-DOPA oxidation by mushroom tyrosinase. This inhibition is characterized by a prolonged transient phase. A mechanism is postulated according to the kinetic data.     

Hen egg white lysozyme as an inhibitor of mushroom tyrosinase

We report a kinetics study on hen egg white lysozyme's (HEWL) inhibitory effect on mushroom tyrosinase catalysis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) or L-tyrosine. For the first time, we demonstrate HEWL as a robust inhibitor against mushroom tyrosinase in catalysis of both substrates. The kinetics pattern matches a mixed (mostly non-competitive) partial inhibition. Ki and ID50 value of HEWL are more than 20-fold lower than that of kojic acid, a well-known chemical inhibitor of mushroom tyrosinase. Ki, alpha value and beta value, are almost identical in both experiments (L-DOPA and L-tyrosine as substrates, respectively), which suggests this common inhibition mechanism affects both steps. The inhibitory effect increases as both proteins were mixed and pre-incubated for less than 1 h. HEWL-depletion only removed about half of the inhibitory effect. Here we propose a novel function of HEWL, which combines the reversible inhibition and the irreversible inactivation toward mushroom tyrosinase. Discovery of HEWL as an inhibitor to mushroom tyrosinase catalysis may be commercially valuable in the food, medical and cosmetic industries.     

Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase

Effect of a series of 1-phenylthioureas 1a-k and 1,3-disubstituted thioureas 2a-k were evaluated against melanin formation in melanoma B16 cell line and mushroom tyrosinase. Inhibitory activity of tyrosinase of 1-phenylthioureas 1a-k is parallel to their melanogenic inhibition. Thus, the melanogenic inhibition in melanoma B16 cells of 1-phenylthioureas could be the result of inhibition of tyrosinase. However, 1,3-diaryl or 1-phenyl-3-alkylthioureas, 2a-k, appears as melanogenic inhibitor without inhibition of tyrosinase. The molecular docking study of 1e and 2b to binding pocket of tyrosinase provided convincing explanation regarding the necessity of direct connection of planar phenyl to thiourea unit without N'-substitution of phenylthioureas 1 as tyrosinase inhibitor and 2 as non-tyrosinase inhibitor.     

Isolation of 6-hydroxy-L-tryptophan from the fruiting body of Lyophyllum decastes for use as a tyrosinase inhibitor

ABSTRACT

Tyrosinase is the key enzyme that controls melanin formation. We found that a hot water extract of the lyophilized fruiting body of the fungus Lyophyllum decastes inhibited tyrosinase from Agaricus bisporus. The extract was fractionated by ODS column chromatography, and an active compound was obtained by purification through successive preparative HPLC using an ODS and a HILIC column. Using spectroscopic data, the compound was identified to be an uncommon amino acid, 6-hydroxytryptophan. 6-Hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan were prepared through a Fenton reaction from L-tryptophan and D-tryptophan, respectively. The active compound was determined to be 6-hydroxy-L-tryptophan by comparison of their circular dichroism spectra and retention time on HPLC analysis of the N^α-(5-fluoro-2,4-dinitrophenyl)-L-leuciamide derivative with those of 6-hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan. A Lineweaver–Burk plot of the enzyme reaction in the presence of 6-hydroxy-L-tryptophan indicated that this compound was a competitive inhibitor. The IC₅₀ values of 6-hydroxy-L-tryptophan was 0.23 mM.     

Resveratrol,a remarkable inhibitor of ribonucleotide reductase

Resveratrol, a natural phytoalexin found in grapes, is well known for its presumed role in the prevention of heart disease, associated with red wine consumption. We show here that it is a remarkable inhibitor of ribonucleotide reductase and DNA synthesis in mammalian cells, which might have further applications as an antiproliferative or a cancer chemopreventive agent in humans.     

Resveratrol as a Therapeutic Agent for Neurodegenerative Diseases

Excess production of reactive oxygen species in the brain has been implicated as a common underlying risk factor for the pathogenesis of a number of neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and stroke. In recent years, there is considerable interest concerning investigation of antioxidative and anti-inflammatory effects of phenolic compounds from different botanical sources. In this review, we first describe oxidative mechanisms associated with stroke, AD, and PD, and subsequently, we place emphasis on recent studies implicating neuroprotective effects of resveratrol, a polyphenolic compound derived from grapes and red wine. These studies show that the beneficial effects of resveratrol are not only limited to its antioxidant and anti-inflammatory action but also include activation of sirtuin 1 (SIRT1) and vitagenes, which can prevent the deleterious effects triggered by oxidative stress. In fact, SIRT1 activation by resveratrol is gaining importance in the development of innovative treatment strategies for stroke and other neurodegenerative disorders. The goal here is to provide a better understanding of the mode of action of resveratrol and its possible use as a potential therapeutic agent to ameliorate stroke damage as well as other age-related neurodegenerative disorders.     

Microorganisms as a source of tyrosinase inhibitors: a review

Tyrosinase is the main enzyme responsible for enzymatic browning of fruits post-harvest and melanogenesis in mammals, an undesirable phenomenon. This encouraged researchers to seek potent tyrosinase inhibitors for application in the food and cosmetics industries. Despite an increased knowledge of tyrosinase inhibitors from plants and synthetic sources in the past few years, inhibitors of microbial origin are under-explored. Thus, this article surveys tyrosinase inhibitors produced by microorganisms and hence, serves as an updated database of tyrosinase inhibitors from microbial sources.     

Activation of melanogenesis by vacuolar type H(+)-ATPase inhibitors in amelanotic, tyrosinase positive human and mouse melanoma cells

In this study, we describe the activation of melanogenesis by selective vacuolar type H(+)-ATPase inhibitors (bafilomycin A1 and concanamycin A) in amelanotic human and mouse melanoma cells which express tyrosinase but show no melanogenesis. Addition of the inhibitors activated tyrosinase within 4 h, and by 24 h the cells contained measurable amounts of melanin. These effects were not inhibited by cycloheximide (2 microgram/ml) which is consistent with a post-translational mechanism of activation. Our findings suggest that melanosomal pH could be an important and dynamic factor in the control of melanogenesis in mammalian cells.     

2-hydroxy-4-isopropylbenzaldehyde, a potent partial tyrosinase inhibitor

Chamaecin (2-hydroxy-4-isopropylbenzaldehyde) was synthesized and tested for its tyrosinase inhibitory activity. It partially inhibits the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an IC(50) of 2.3 microM. The inhibition kinetics analyzed by Dixon plots found that chamaecin is a mixed type inhibitor. This inhibition may come in part from its ability to form a Schiff base with a primary amino group in the enzyme.     

3-hydroxykynurenine as a substrate/activator for mushroom tyrosinase

3-Hydroxykynurenine is a tryptophan metabolite with an o-aminophenol structure. It is both a tyrosinase activator and a substrate, reducing the lag phase, stimulating the monophenolase activity, and being oxidized to xanthommatin. In the early stage of monophenol hydroxylation, catechol accumulation takes place, whereas 3-hydroxykynurenine is substantially unchanged and no significant amounts of the o-quinone are produced. These results suggest an activating action of 3-hydroxykynurenine toward o-hydroxylation of monophenols. 3-Hydroxykynurenine could therefore well act as a physiological device to control phenolics metabolism to catechols and quinonoids.     

Evidence for tyrosinase as a beta1,6 branch containing glycoprotein: substrate of GnT-V

Tyrosinase is a rate-limiting enzyme in mammalian melanogenesis, and is known as a glycoprotein. Post-translational processing of mammalian tyrosinase is required for its folding, sorting, and for enzymatic activity. Here we show for the first time that the mammalian tyrosinase has beta1,6-branched N-glycan structure that can be recognized by binding with specific lectin Leukoagglutinating phytohematoagglutinin (L-PHA). Further, this specific glycoconjugate structure has been shown to have a function relationship in melanin synthesis.