Synthesis of novel azo compounds containing 5(4H)-oxazolone ring as potent tyrosinase inhibitors

Six new azo dyes containing of 5(4H)-oxazolone ring were prepared by diazotization of 4-aminohippuric acid and coupling with N,N-dimethylaniline, 1-naphthol and 2-naphthol and condensation with 4-fluoro benzaldehyde or 4-trifluoromethoxy benzaldehyde. The new compounds were fully characterized by spectroscopic techniques. All synthesized compounds exhibited high tyrosinase inhibitory behavior. The results of mushroom tyrosinase inhibition assays indicate that the 4-trifluoromethoxy derivatives have high degrees of inhibition and N,N-dimethylaniline derivatives are better for tyrosinase inhibition than 1-naphthol and 2-naphthol derivatives. All synthesized azo compounds (4a-4f) showed the most potent mushroom tyrosinase inhibition, comparable to that of Kojic acid and l-mimosine, as reference standard inhibitors. © 2013 Elsevier Ltd. All rights reserved

Formulation of Liquorice Root Extract (Glycyrrhiza Glabra L.) as Skin Whitening Cream

Liquorice root extract (Glycyrrhiza glabra L.) contains glabridin, an isoflavane as inhibitors of tyrosinase. This enzyme is responsible in melanin synthesis. The aim of this research was to determine the tyrosinase inhibition activity of liquorice root extract and to formulate into a cream with a variety of emulsifier agent glyceryl monostearate. Liquorice root was macerated using ethanol 96%, invitro tyrosinase inhibition assay was conducted using kojic acid as positive control in 96-well plate. The physical quality parameters of the cream were also evaluated. The results showed that liquorice root extract inhibits tyrosinase with the IC50 126.75 µg/mL. Creams containing 1.01% liquorice root extract were yellowish white, aromatics odour, soft texture, homogen and no segregation in O/W emulsion type. It also showed plastic thixotropic rheological property, viscosity of (2800±0.00) – (4000±0.00) Ps, spreadability of (3029.72±0.81) – (3531.79±6.15)mm2, droplet size of (60.00±0.00) – (65.12±0.01)μm, pH of (4.55±0.03)–(4.63±0.04) and inhibited tyrosinase 10.14 - 19.30%. It can be concluded that the formula with 0.1% of glyceryl monostearate was the best formula that conforms physical quality test and potentially to be developed as a skin whitening cream

Mechanistic studies of anti-hyperpigmentary compounds: elucidating their inhibitory and regulatory actions.

Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol

Tyrosinase and phenolic pressor amines

Basic to the consideration of the action of tyrosinase on the oxidation of phenolic pressor amines are the observations of Keilin and Mann (16) and of Nelson and his coworkers (17-19) that show that different preparations may vary considerably in their relative actions on monophenols and o-diphenols. Both of these types of activity appear to belong to the same enzyme complex, as they bear a proportionality to the same copper content. However, since the activities vary with the purity and method of purification, each enzyme preparation must be defined in terms of both monophenolase and o-diphenolase activities. This was done in the present studies, and modifications of previously described preparative methods were required to retain a reasonable proportioning of such activities in purified preparations

Interaction of human heat shock protein 70 with tumor-associated peptides

Molecular chaperones of the heat shock protein 70 (Hsp70) family play a crucial role in the presentation of exogenous antigenic peptides by antigen-presenting cells (APCs). In a combined biochemical and immunological approach, we characterize the biochemical interaction of tumor-associated peptides with human Hsp70 and show that the strength of this interaction determines the efficacy of immunological cross-presentation of the antigenic sequences by APCs. A fluorescein-labeled cytosolic mammalian Hsc70 binding peptide is shown to interact with human Hsp70 molecules with high affinity (K(d)=0.58 mu M at 25 degrees C). Competition experiments demonstrate weaker binding by Hsp70 of antigenic peptides derived from the tumor-associated proteins tyrosinase (K(d)=32 mu M) and melanoma antigen recognized by T cells (MART-1) (K(d)=2.4 mu M). Adding a peptide sequence (pep70) with high Hsp70 binding affinity (K(d)=0.04 mu M) to the tumor-associated peptides enables them to strongly interact with Hsp70. Presentation of tumor-associated peptides by B cells resulting in T cell activation in vitro is enhanced by Hsp70 when the tumor-associated peptides contain the Hsp70 binding sequence. This observation has relevance for vaccine design, as augmented transfer of tumor-associated antigens to APCs is closely linked to the vaccine's efficacy of T cell stimulation

Glutathione (GSH) conjugates with dopamine (DA)-derived quinones to form reactive or non-reactive GSH-conjugates

In this study we demonstrate for the first time that GSH could rapidly conjugate with dopamine (DA)-derived DA-o-quinones without enzymatic catalysis to form short-lived intermediate GSH-conjugates (2-S-GSH-DA-o-quinone and 5-S-GSH-DA-o-quinone). These intermediate GSH-conjugates are unstable and would finally form reactive or non-reactive GSH-conjugates dependent on ambient reductive forces. Under insufficient reductive forces, the intermediate GSH-conjugates could cyclize spontaneously to form reactive 7-S-GSH-aminochrome (7-S-GSH-AM). The 7-S-GSH-AM is so reactive that it could further react with another GSH to form 4,7-bi-GSH-5,6-dihydroindole. Its reactivity could also abrogate tyrosinase activity in solutions. In addition, the 7-S-GSH-AM could further undergo internal rearrangement to form non-reactive 7-S-GSH-5,6-dihydroindole. From these novel findings, we propose two detrimental positive feedback loops involving accelerated DA oxidation, increased GSH consumption and impaired GSH detoxification efficiency, as the underlying chemical explanation for dopaminergic neuron degeneration in Parkinson's disease

Catalytic molecularly imprinted polymer membranes: Development of the biomimetic sensor for phenols detection

Portable biomimetic sensor devices for the express control of phenols content in water were developed. The synthetic binding sites mimicking active site of the enzyme tyrosinase were formed in the structure of free-standing molecularly imprinted polymer membranes. Molecularly imprinted polymer membranes with the catalytic activity were obtained by co-polymerization of the complex Cu (II)–catechol–urocanic acid ethyl ester with (tri)ethyleneglycoldimethacrylate, and oligourethaneacrylate. Addition of the elastic component oligourethaneacrylate provided formation of the highly cross-linked polymer with the catalytic activity in a form of thin, flexible, and mechanically stable membrane. High accessibility of the artificial catalytic sites for the interaction with the analyzed phenol molecules was achieved due to addition of linear polymer (polyethyleneglycol Mw 20,000) to the initial monomer mixture before the polymerization. As a result, typical semi-interpenetrating polymer networks (semi-IPNs) were formed. The cross-linked component of the semi-IPN was represented by the highly cross-linked catalytic molecularly imprinted polymer, while the linear one was represented by polyethyleneglycol Mw 20,000. Extraction of the linear polymer from the fully formed semi-IPN resulted in formation of large pores in the membranes’ structure. Concentration of phenols in the analyzed samples was detected using universal portable device oxymeter with the oxygen electrode in a close contact with the catalytic molecularly imprinted polymer membrane as a transducer. The detection limit of phenols detection using the developed sensor system based on polymers–biomimics with the optimized composition comprised 0.063 mM, while the linear range of the sensor comprised 0.063–1 mM. The working characteristics of the portable sensor devices were investigated. Storage stability of sensor systems at room temperature comprised 12 months (87%). As compared to traditional methods of phenols detection the developed sensor system is characterized by simplicity of operation, compactness, an

Mechanism underlying synergic activation of Tyrosinase promoter by MITF and IRF4

Background: The transcription factor interferon regulatory factor 4 (IRF4) was identified to be involved in human pigmentation by genome-wide association studies (GWASs). The rs12203592-[T/C], which is located in intron 4 of IRF4, shows the strongest link to these pigmentation phenotypes including freckling, sun sensitivity, eye and hair color. Previous studies indicated a functional cooperation of IRF4 with Microphthalmia-associated transcription factor (MITF), a causing gene of Waardenburg syndrome (WS), to synergistically trans-activate Tyrosinase (TYR). However, the underlying mechanism is still unknown. Methods: To investigate the importance of DNA binding in the synergic effect of IRF4. Reporter plasmids with mutant TYR promoters was generated to locate the IRF4 DNA binding sites in the Tyrosinase minimal promoter. By building MITF and IRF4 truncated mutations plasmids, the necessary regions of the synergy functions of these two proteins were also located. Results: The cooperative effect between MITF and IRF4 was specific for TYR promoter. The DNA-binding of IRF4 was critical for the synergic function. IRF4 DNA binding sites in TYR promoter were identified. The Trans-activation domains in IRF4 (aa134-207, aa300-420) were both important for the synergic function, whereas the auto-mask domain (aa207-300) appeared to mask the synergic effect. Mutational analysis in MITF indicated that both DNA-binding and transcriptional activation domains were both required for this synergic effect. Conclusions: Here we showed that IRF4 potently synergized with MITF to activate the TYR promoter, which was dependent on DNA binding of IRF4. The synergic domains in both IRF4 and MITF were identified by mutational analysis. This identification of IRF4 as a partner for MITF in regulation of TYR may provide an important molecular function for IRF4 in the genesis of melanocytes and the pathogenic mechanism in WS

Tyrosinase inhibitory and antioxidant activity by bromophenols from the alga Odonthalia corymbifera

In the course of our search for tyrosinase inhibitors and antioxidants, six known bromophenol dimers were purified from methanol extract of the red alga Odonthalia corymbifera. The compounds were identified by comparison with published spectroscopic data. These bromophenols were categorized into symmetric and asymmetric dimers. Among them, the tetrabrominated dimers displayed more potent tyrosinase inhibition than the tribrominated ones. Especially, the asymmetric tetrabrominated compound showed strong inhibition. These results suggest that number of bromine substitution and orientation of bromine and phenolic hydroxy groups are important factors of tyrosinase inhibitory potency. The bromophenols were also investigated for antioxidant activities by using DPPH and ABTS radical scavenging, CUPRAC and FRAP metal reducing and copper chelation assays. All dimers showed comparable antioxidant activities to the positive controls examined. Symmetric dimers displayed relatively higher antioxidant activities than asymmetric ones