Tyrosinase Research Articles

Investigation of the Efficacy of Benzylidene-3-methyl-2-thioxothiazolidin-4-one Analogs with Antioxidant Activities on the Inhibition of Mushroom and Mammal Tyrosinases.

Based on the fact that substances with a β-phenyl-α,β-unsaturated carbonyl (PUSC) motif confer strong tyrosinase inhibitory activity, benzylidene-3-methyl-2-thioxothiazolidin-4-one (BMTTZD) analogs 1-8 were prepared as potential tyrosinase inhibitors. Four analogs (1-3 and 5) inhibited mushroom tyrosinase strongly. Especially, analog 3 showed an inhibitory effect that was 220 and 22 times more powerful than kojic acid in the presence of l-tyrosine and l-dopa, respectively. A kinetic study utilizing mushroom tyrosinase showed that analogs 1 and 3 competitively inhibited tyrosinase, whereas analogs 2 and 5 inhibited tyrosinase in a mixed manner. A docking simulation study indicated that analogs 2 and 5 could bind to both the tyrosinase active and allosteric sites with high binding affinities. In cell-based experiments using B16F10 cells, analogs 1, 3, and 5 effectively inhibited melanin production; their anti-melanogenic effects were attributed to their ability to inhibit intracellular tyrosinase activity. Moreover, analogs 1, 3, and 5 inhibited in situ B16F10 cellular tyrosinase activity. In three antioxidant experiments, analogs 2 and 3 exhibited strong antioxidant efficacy, similar to that of the positive controls. These results suggest that the BMTTZD analogs are promising tyrosinase inhibitors for the treatment of hyperpigmentation-related disorders.

Discovery of (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide among N-substituted cinnamamide derivatives as a novel cosmetic ingredient for hyperpigmentation

Hyperpigmentation disorders may result from inappropriate melanin deposition and/or excessive melanin synthesis. They are classified mainly as aesthetic problems, but they can significantly affect human health by decreasing self-esteem. There are available only limited treatment options for hyperpigmentation disorder, among others, cosmetic products applied topically. Depigmenting ingredients were found to be ineffective and characterized by various side effects. As a result, many efforts are made to discover novel, potent, and safe melanogenesis inhibitors for possible use in topical cosmetic depigmenting formulations. Cinnamic acid derivatives constitute a widely tested group for that purpose. This article reports research in the group of N-alkyl cinnamamide derivatives (un)substituted in phenyl ring. Among tested series, (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide (compound 21) showed the most promising inhibitory properties in mushroom tyrosinase assay (IC50 = 36.98 ± 1.07 µM for monophenolase activity, IC50 = 146.71 ± 16.82 µM for diphenolase activity) and melanin production inhibition in B16F10 mouse melanoma cell line at concentration 6.25 µM resulting probably from decreasing of Tyr, Mitf, Tyrp-1, and Tyrp-2 genes expression. This compound also showed melanin production inhibitory properties in pigmented reconstructed human epidermis when used in 1 % and 2 % solutions in 50 % PEG400. In vitro evaluation of its safety profile showed no cytotoxicity to human keratinocytes HaCaT, human skin fibroblasts BJ, and human primary epidermal melanocytes HEMa, no mutagenicity in the Ames test, no genotoxicity in micronucleus test, no phototoxicity, as well as no skin irritation potential tested in PEG400 solution. This compound was also shown to penetrate across the epidermis to reach the possible site of action. The performed research led to classify (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide as a novel potential depigmenting cosmetic ingredient.

Two Coffee Diterpenes, Kahweol and Cafestol, Inhibit Extracellular Melanogenesis: An In Vitro Pilot Study

Hyperpigmentation skin disorders are marked by an abnormal accumulation or export of melanin pigment synthesized within melanocytes and pose a significant aesthetic concern. The search for novel natural compounds that exhibit pharmacological potential for treating pigmentation disorders is growing. In this study, kahweol (KW) and cafestol (CFS), two structural analogs of coffee diterpenes, were evaluated and compared for their effects on melanogenesis using B16F10 mouse melanoma cells and primary human melanocytes derived from Asian and African American skin. To the best of our knowledge, there are no reports of the effects of KW and CFS on melanogenesis yet. We first screened nontoxic concentrations of both compounds using an MTS assay after 72 h incubations and subsequently tested their effects on melanin synthesis and export. Cellular tyrosinase activity and cell-free mushroom tyrosinase activity were assayed to study the mechanisms of melanogenesis suppression. Human melanocytes from a moderately pigmented donor (HEMn-MP cells) and from a darkly pigmented donor (HEMn-DP cells) were next examined, and effects on cellular viability, melanin content, cellular tyrosinase activity, and melanin export (quantitated via dendricity) were similarly examined for both compounds. Our results show that KW and CFS did not significantly affect intracellular melanin content but suppressed extracellular melanin in B16F10 cells and dendritic parameters in human melanocytes, indicating their unique capacity to target extracellular melanogenesis and melanin export. Although KW showed a greater extracellular melanogenesis inhibitory capacity in B16F10 cells, in both primary melanocyte cells, CFS emerged as a potent inhibitor of melanin export compared to KW. Together, these results reveal novel modes of action of both compounds and indicate a promise to use CFS as a novel candidate for treating hyperpigmentation disorders of the human skin for clinical and cosmetic use. Additional research is necessary to shed light on the molecular pathways and the efficacy of melanogenesis inhibition by CFS in 3D human skin equivalents and in vivo studies.

Exploring phytochemical tyrosinase inhibitors against melanoma skin cancer

Tyrosinase enzyme is the vital enzyme for synthesis of melanin pigment in melanocytes, though its dysregulated activity may cause over production of the pigment causing a skin cancer melanoma. Every year the cases of Melanoma is increasing by 1%. To control the hyperpigmentation, tyrosinase activity is to be inhibited in these cells. An attempt was made to find phytochemicals acting as an inhibitor for tyrosunase activity, thus reducing the synthesis of melanin pigment. Four plants were randomly selected namely Tacoma stans, Murraya paniculata, Galphimia gracilis and Mansoa alliacea for the work and the ethanol leaf extract were used to determine their inhibitory influence on freshly extracted and purified mushroom tyrosinase enzyme. Enzyme activity and product L-DOPA formation was estimated through UV-Visible Spectrophotometer and found to be 3.85U/min/ml and 0.018 mg/ml respectively. Murraya paniculata leaf extract shows the highest tyrosinase inhibitory action of 80.61% followed by Tacoma stans, Galphimia gracilis and Mansoa alliacea with74.71%, 72.95% and 71.99% respectively. Thus, the work conclude that the phytochemicals have the efficiency to control melanoma risk in human.. KEYWORDS :Mushroom Tyrosinase, Tyrosinase Inhibition, Melanoma, phytochemicals, Enzyme Activity

Synthesis and evaluation of 3-hydroxyquinolin-2(1H)-one derivatives as inhibitors of tyrosinase

The tyrosinase (TYR) enzyme catalyses sequential reactions in the melanogenesis pathway: l-tyrosine is oxidised to yield L-3,4-dihydroxyphenylalanine (l-dopa), which in turn is converted to dopaquinone. These two reactions are the first two steps of melanin biosynthesis and are rate limiting. The accumulation or overproduction of melanin may cause skin hyperpigmentation and inhibitors of TYR are thus of interest to the cosmeceutical industry. Several TYR inhibitors are used to treat skin hyperpigmentation, however, some are ineffective and possess questionable safety profiles. This emphasises the need to develop novel TYR inhibitors with better safety and efficacy profiles. The small molecule, 3-hydroxycoumarin, has been reported to be a good potency TYR inhibitor (IC50 = 2.49 µM), and based on this, a series of eight structurally related 3-hydroxyquinolin-2(1H)-one derivatives were synthesised with the aim to discover novel TYR inhibitors. The results showed that four of the derivatives inhibited TYR from the champignon mushroom Agaricus bisporus (abTYR) with IC50 < 6.11 µM. The most potent inhibitor displayed an IC50 value of 2.52 μM. Under the same conditions, the reference inhibitors, thiamidol and kojic acid, inhibited abTYR with IC50 values of 0.130 and 26.4 μM, respectively. Based on the small molecular structures of the active 3-hydroxyquinolin-2(1H)-one inhibitors which are amenable to structure optimisation, it may be concluded that this class of compounds are good leads for the design of TYR inhibitors for cosmeceutical applications.

Germacrone, isolated from Curcuma wenyujin, inhibits melanin synthesis through the regulation of the MAPK signaling pathway.

Abnormal melanin synthesis causes hyperpigmentation disorders, such as chloasma, freckles, and melanoma, which are highly multiple and prevalent. There were few reports on the anti-melanogenic effect of Curcuma wenyujin Y.H. Chen et C. Ling, and the bioactive compound has not been elucidated as well. The study aims to investigate the anti-melanogenic effect of C. wenyujin, and identify the bioactive compound, and further explore its underlying mechanism. Our results showed that the Petroleum ether fraction extracted from C. wenyujin rhizome had a significant anti-melanogenic effect, and germacrone isolated from it was confirmed as the major bioactive compound. To our data, germacrone significantly inhibited tyrosinase (TYR) activity, reduced melanosome synthesis, reduced dendrites formation of B16F10 cells, and melanosome transport to keratinocytes. Moreover, germacrone effectively decreased the hyperpigmentation in zebrafish and the skin of guinea pigs in vivo. Western-blot analysis showed that germacrone down-regulated the expression of TYR, TRP-1, TRP-2, Rab27a, Cdc42, and MITF proteins via the activation of the MAPK signaling pathway. Taken together, germacrone is an effective bioactive compound for melanogenesis inhibition. Our studies suggest that germacrone may be considered a potential candidate for skin whitening.

P30 Exposure to particulate matter induces structural and pigmentary changes to human skin

Abstract Introduction and aims The majority of the world’s population reside in places with high levels of air pollution. The impact of air pollution on human health has been extensively studied and demonstrates a significant correlation between particulate matter (PM) and risk of cardiovascular and respiratory diseases. However, little is known about the impact of air pollution on human skin health. Here, we develop an ex vivo skin model to investigate cutaneous changes induced by the topical application of PM. Methods PM (0, 0.5, 3, 6, 10, 20 and 40 μg mL−1) was topically applied to Genoskin® NativeSkin Access® (Genoskin, Salem, MA, USA) human ex vivo skin samples of White European ancestry (n = 3) for 8 days, with reapplication on day 4. Resultant samples were assessed using haematoxylin and eosin staining (for morphometric analyses), immunofluorescence for Ki67 and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) (to determine cellular turnover), while pigmentary changes were analysed by colourimetry, Warthin–Starry (WS) staining and immunohistochemistry for the melanin markers tyrosinase (TYR), gp100 and c-kit. Results Morphological assessment revealed that epidermal thickness was significantly reduced following exposure to higher doses of PM (r = −0.9163, P &amp;lt; 0.01). In addition, there was reduced epidermal cell proliferation (Ki67) and increased apoptosis (TUNEL) at higher doses of PM. Exposure to PM resulted in a dose-dependent decrease in individual typology angle, which is indicative of skin colour becoming darker (r = −0.7895, P &amp;lt; 0.05). Furthermore, WS staining demonstrated significantly increased epidermal melanin (r = 0.9583, P &amp;lt; 0.01) and significantly increased expression of melanin markers TYR (P &amp;lt; 0.01), gp100 (P &amp;lt; 0.001) and c-kit (P &amp;lt; 0.01) in response to PM exposure. Conclusions This study confirms that PM induces pigmentary changes to ex vivo human skin by increasing epidermal melanin. Increased expression of c-kit in response to PM exposure suggests that increased pigmentation occurs owing to the upregulation of the microphthalmia-associated transcription factor signalling pathway. Further investigation of the mechanistic drivers of increased pigmentation is warranted.

Whitening and antioxidant activities of Inonotus sanghuang Extract and development of primary whitening gel products.

Polysaccharides and flavonoids have excellent antioxidant properties and tyrosinase inhibitory effects. In this paper, the antioxidant capacity of Inonotus sanghuang extract and its inhibition kinetics on mushroom tyrosinase were investigated to determine the preparation process of Inonotus sanghuang primary whitening gel. By conducting experimental studies on the effects of water extract and alcohol extract of Inonotus sanghuang on antioxidant capacity and tyrosinase activity, their inhibitory ability and types of inhibitory effects were determined. The single factor experiment was used to determine the preparation process of Sanghuang primary whitening gel. This study has proven that the extract of Inonotus sanghuang possesses antioxidant and tyrosinase inhibitory capabilities. It also identified the preparation process for the primary whitening gel of Inonotus sanghuang, thereby providing a theoretical and experimental basis for the development of whitening products utilising Inonotus sanghuang.

Chemical composition and biological activities of essential oil of the Malaysian endemic Syzygium variolosum (King) Chantar. & J.Parn

This study was designed to investigate the chemical composition of the essential oil of Syzygium variolosum (King) Chantar. & J.Parn. and their cytotoxicity, acetylcholinesterase, antityrosinase, and anti-inflammatory activities. In total, 32 chemical components were identified in the essential oil, which made up 98.9%. The essential oil is mainly composed of β-elemene (20.2%), bicyclogermacrene (13.5%), viridiflorol (11.1%), globulol (8.6%), and selin-11-en-4α-ol (5.3%). Acetylcholinesterase, antityrosinase, and anti-inflammatory activities were evaluated with the Ellman method, mushroom tyrosinase, and lipoxygenase enzymes, respectively, while cytotoxicity was assessed using an MTT assay. The results showed that essential oil gave significant percentage inhibition (I%: acetylcholinesterase 35.2%, antityrosinase 42.5%, lipoxygenase 48.6%). Furthermore, the essential oil exhibited cytotoxicity against three cancer cell lines, HepG2, MCF7, and A549, with IC50 values ranging from 90.2 to 95.2 μg/mL. The current study highlights the potential of the use of essential oils as an alternative to the development of pharmaceutical antichemopreventives or cosmetics.

Diversity in Pyracantha fortuneana fruits maturity stages enables discrepancy in the phenolic compounds, antioxidant activity, and tyrosinase inhibitory activity.

Pyracantha fortuneana (P. fortuneana) fruit is a wild fruit that is popular because of its delicious taste and numerous nutrients, and phenolic compounds are considered to be the main bioactive components in P. fortuneana fruits. However, the relationship between phenolic compounds and their antioxidant and tyrosinase (TYR) inhibitory activities during the ripening process is still unclear. The study compared the influence of the five developmental stages on the accumulation of phenolic compounds, antioxidant activity, and TYR inhibitory activity in the fruits of P. fortuneana. The compounds were identified by offline two-dimensional liquid chromatography-electrochemical detection (2D-LC-ECD) combined with liquid chromatography-tandem mass spectrometry, and the main active ingredients were quantified. The results showed that stage II had higher total phenolic and flavonoid content, as well as higher antioxidant and TYR inhibitory activity, but the total anthocyanin content was lowest at this stage. A total of 30 compounds were identified by 2D-LC-ECD. Orthogonal partial least squares discriminant analysis screened out six major potential markers, including phenolic acids, procyanidins, and flavonoids. In addition, it was found that caffeoylquinic acids, procyanidins, and flavonoids were higher in stage II than in stages I, III, IV, and V, whereas anthocyanins accumulated gradually from stages III to V. Therefore, this study suggests that the changes in antioxidant and TYR inhibitory activities of P. fortuneana during the five developmental stages may be due to the transformation of procyanidins, caffeoylquinic acids, and phenolic glycosides into other forms during the fruit maturation process. Practical Application: Differences in chemical constituents, antioxidant, and tyrosinase inhibitory activities in fruit maturity stages of P. fortuneana were elucidated to provide reference for rational harvesting and utilization of the fruits and their bioactive components. These findings are expected to provide a comprehensive assessment of the bioactive profile and guide the food industrial production.

In vitro Differentiation of Melanocyte Stem Cells Derived from Vitiligo Patients into Functional Melanocytes

BACKGROUND: Melanocyte stem cells (MelSCs) residing in the hair follicle bulge act as melanocyte reservoir for skin and hair, and may serve as an autologous source for treating vitiligo. Therefore, the study aimed to evaluate the in vitro differentiation ability of MelSCs derived from vitiligo patients into melanin-producing melanocytes for potential cellular therapy.METHODS: MelSCs from the vitiliginous (V-MelSCs), non-vitiliginous (NV-MelSCs) regions of vitiligo patients, as well as from control subjects (C-MelSCs) were established for evaluating their differentiation potential into melanin producing cells. The differentiation abilities were compared at the cellular and molecular levels. MelSCs were differentiated in vitro into induced-melanocytes (iMCs) by supplementing the culture medium with melanogenic factors. iMCs were analyzed by quantitative polymerase chain reaction (qPCR) for the expression of key melanogenic markers, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT), microphthalmia-associated transcription factor (MITF), c-KIT and S100. iMCs were also stained with L-DOPA to assess TYR activity in cells. Intracellular melanin content in iMCs was evaluated and compared among the vitiligo and control groups.RESULTS: MelSCs induced into iMCs displayed morphological changes with longer dendrites and prominent nuclei. iMCs stained positive for L-DOPA with an average intracellular melanin content of 30 pg/cell. iMCs expressed key melanogenic genes and the relative expression did not differ significantly among the groups.CONCLUSION: NV-MelSCs were unaltered by disease pathogenesis and capable of differentiating into melanocytes compared to V-MelSCs. Hence, these cells might offer a reliable source of melanocytes for vitiligo repigmentation therapy by autologous cellular transplantation.KEYWORDS: melanocyte stem cells, differentiation, melanocytes, vitiligo, in vitro

Anti-Melanogenic Effects of Takifugu flavidus Muscle Hydrolysate in B16F10 Melanoma Cells and Zebrafish.

Abnormal melanogenesis can lead to hyperpigmentation. Tyrosinase (TYR), a key rate-limiting enzyme in melanin production, is an important therapeutic target for these disorders. We investigated the TYR inhibitory activity of hydrolysates extracted from the muscle tissue of Takifugu flavidus (TFMH). We used computer-aided virtual screening to identify a novel peptide that potently inhibited melanin synthesis, simulated its binding mode to TYR, and evaluated functional efficacy in vitro and in vivo. TFMH inhibited the diphenolase activities of mTYR, reducing TYR substrate binding activity and effectively inhibiting melanin synthesis. TFMH indirectly reduced cAMP response element-binding protein phosphorylation in vitro by downregulating melanocortin 1 receptor expression, thereby inhibiting expression of the microphthalmia-associated transcription factor, further decreasing TYR, tyrosinase related protein 1, and dopachrome tautomerase expression and ultimately impeding melanin synthesis. In zebrafish, TFMH significantly reduced black spot formation. TFMH (200 μg/mL) decreased zebrafish TYR activity by 43% and melanin content by 52%. Molecular dynamics simulations over 100 ns revealed that the FGFRSP (T-6) peptide stably binds mushroom TYR via hydrogen bonds and ionic interactions. T-6 (400 μmol/L) reduced melanin content in B16F10 melanoma cells by 71% and TYR activity by 79%. In zebrafish, T-6 (200 μmol/L) inhibited melanin production by 64%. TFMH and T-6 exhibit good potential for the development of natural skin-whitening cosmetic products.

SERS-Based Microneedle Biosensor for In Situ and Sensitive Detection of Tyrosinase.

Tyrosinase (TYR) emerges as a key enzyme that exerts a regulatory influence on the synthesis of melanin, thereby assuming the role of a critical biomarker for the detection of melanoma. Detecting the authentic concentration of TYR in the skin remains a primary challenge. Distinguished from ex vivo detection methods, this study introduces a novel sensor platform that integrates a microneedle (MN) biosensor with surface-enhanced Raman spectroscopy (SERS) technology for the in situ detection of TYR in human skin. The platform utilized dopamine (DA)-functionalized gold nanoparticles (Au NPs) as the capturing substrate and 4-mercaptophenylboronic acid (4-MPBA)-modified silver nanoparticles (Ag NPs) acting as the SERS probe. Here, the Au NPs were functionalized with mercaptosuccinic acid (MSA) for DA capture. In the presence of TYR, DA immobilized on the MN is preferentially oxidized to dopamine quinone (DQ), a process that results in a decreased density of SERS probes on the platform. TYR concentration was detected through variations in the signal intensity emitted by the phenylboronic acid. The detection system was able to evaluate TYR concentrations within a linear range of 0.05 U/mL to 200 U/mL and showed robust anti-interference capabilities. The proposed platform, integrating MN-based in situ sensing, SERS technology, and TYR responsiveness, holds significant importance for diagnosing cutaneous melanoma.

Determination of the New SNP g.939A&gt;G of the TYR Gene in Abnormal Coat Color in Bali Cattle

This study aimed to assess the presence of single nucleotide polymorphisms (SNPs) in the exon 1 region of the tyrosinase (TYR) gene in Bali cattle with abnormal coat color. This study analyzed 43 Bali cattle, 26 individuals with typical or standard coat color, and 17 individuals with albinism. The genetic diversity of the TYR gene was determined via direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. The sequence data of the TYR gene were scrutinized using BioEdit and MEGA10 software to detect single nucleotide polymorphisms (SNPs) related to different types of coat color features in Bali cattle. The genetic diversity information was derived from computations performed using PopGen 1.32 software. The results showed that exon 1 of the TYR gene was affected by the new SNP g.939A&gt;G, which is polymorphic in Bali cattle. In conclusion, the SNP c.939A&gt;G can be further analyzed for use as a candidate-assisted selection (MAS) for abnormal coat color in Bali cattle.

Deciphering the molecular pathway of an asiaticosiderich fraction of Centella asiatica as an anti-melanogenesis agent

Introduction: Melanin is a defense against UV radiation; however, it leads to significant cosmetic issues mainly melasma and hyperpigmentation. This study evaluated the potential effect of ethyl acetate (EtOAc) fraction of Centella asiatica extract in vitro inhibition activity against tyrosinase (TYR). Bioinformatics and in silico experiments were also employed to predict molecular pathways of asiaticoside, as the main active compound. Methods: Centella asiatica was extracted with ethanol and then fractionated with EtOAc. The fraction was tested in vitro for TYR inhibitory activity, and its active compounds were investigated using thin-layer chromatography (TLC). After obtaining the online database of the genes related to pigmentation and melanogenesis in the skin, the genes affected by asiaticoside were determined by the Venn diagram. The top 10 target proteins, underlying molecular pathways, got from CytoHubba, were further studied to figure out their molecular pathway. The molecular docking was conducted on two selected protein targets. Results: EtOAc fraction of C. asiatica extract demonstrated strong TYR inhibitory activity with an IC50 of 18.85 μg/mL. TLC profiling of the EtOAc fraction revealed the Rf value of 0.28 for the standard, Rf value of 0.26, 0.21, and 0.15 for the extract, and Rf value of 0.26 and 0.15 for the fraction. Asiaticoside inhibited melanogenesis by elaborating many molecular pathways involving keratinocytes, melanocytes, fibroblast, and endothelial cells by elaborating cytokine, growth factor, extracellular matrix, and melanin degradation enzyme Conclusion: Asiaticoside-rich C. asiatica fraction has the potential as an anti-melanogenesis agent through its TYR inhibitory activity and many molecular pathways.

Synthesis and Characterization of Metronidazole and Heterocycle Ester Dyads for Tyrosinase Inhibitory Activity

Tyrosinase is an important copper-based enzyme mainly involved in skin pigmentation. The inhibition of the tyrosinase enzyme attracts importance in cosmetic and medicinal chemistry industry for its applications in skin whitening and anti-browning agents for humans as well as in food, agriculture industries. Imidazole based Metronidazole and its derivatives are widely accepted drug for wide range of diseases. Therefore, the present report involves the synthesis of heterocyclic derivatives of metronidazole and investigation of its efficacy towards the tyrosinase inhibitory activity. A series of metronidazole esters were synthesized and their chemical structures were confirmed using spectral techniques like, infrared spectroscopy (IR), proton nuclear magnetic resonance (1H-NMR), and liquid chromatography-mass spectrometry (LC-MS). All the compounds were evaluated for its tyrosinase inhibitory activity by oxidation of 3,4-dihydroxyphenylalanine in the presence of the synthesized esters(I-VIII) with kojic acid as standard. Among the synthesized compounds, VII (isonicotinic ester) and VIII (quinoline ester) demonstrated significant activity IC50 values 92.5 and 91.8µM respectively. Further molecular docking experiments were carried out for the synthesized compounds with 2y9w protein exhibited greater number of physical interactions for compounds VII and VIII than the other compounds in the series confirming the mushroom tyrosinase activity.

Design and synthesis of novel dihydropyridine- and benzylideneimine-based tyrosinase inhibitors.

Tyrosinase (TYR) inhibitors are very significant as they inhibit enzyme tyrosinase activity, and its inhibition is vital for skin care, anticancer medication, and antibrowning of fruits and vegetables. This work presents a novel and economical route for the preparation of new synthetic tyrosinase inhibitors using amlodipine (4). The novel conjugates 6 (a-o) were designed, synthesized, and characterized by spectroscopic analyses, including Fourier transform infrared and low- and high-resolution mass spectroscopy. The purified compound 4 was refluxed with various aldehydes and ketones 5 (a-o) for 5-8h in methanol at 60°C-90°C. This research modified the drug in a step-by-step manner to develop therapeutic properties as a tyrosinase inhibitor. The structures of synthesized ligands 6 (a-o) were established based on spectral and analytical data. The synthesized compounds 6 (a-o) were screened against tyrosinase enzyme. Kojic acid was taken as standard. All the prepared compounds 6 (a-o) have good inhibition potential against the enzyme tyrosinase. Compounds 6o, 6b, 6f, and 6k depicted excellent antityrosinase activity. Compound 6k, with an IC50 value of 5.34 ± 0.58µM, is as potent as the standard kojic acid (IC50 6.04 ± 0.11µM), standing out among all synthesized compounds 6 (a-o). The in silico studies of the conjugates 6 (a-o) were evaluated via PatchDock. Compound 6k showed a binding affinity score of 8,999 and an atomic contact energy (ACE) value of -219.66kcal/mol. The structure-activity relationship illustrated that the presence of dihydropyridine nuclei and some activating groups at the ortho and para positions of the benzylideneimine moiety is the main factor for good tyrosinase activity. The compound 6k could be used as a lead compound for drug modification as a tyrosinase inhibitor for skin care, anticancer medication, and antibrowning for fruits and vegetables.

Enhancement of preference, catalytic activity and thermostability of polyphenol oxidase from Rosa Chinensis by semi-rational engineering

Improvement of activity and property in polyphenol oxidases (PPOs) by protein engineering may contribute to synthesis of theaflavin (TF1) in industrial processes. Here, the potential noncatalytic residues of PPO from Rosa chinensis (RcPPO) were selected by sequence alignment, structural analysis and surface residues prediction and the mutants were gained by semi-rational site-directed mutagenesis. Mutants L173F, A174K and N334I possessed 10.48, 9.74 and 7.13 times increases respectively in preference for diphenols over monophenols. Enzymatic activity, half-life at 40 and 50 °C of the most active mutant RcPPO-FKI increased to 2.67, 2.62 and 3.37 times respectively in comparison with wild type, implying that activity and thermostability of RcPPO through mutation were improved. The improvement of activity and thermostability in the mutant should be ascribed to more hydrogen bonds formed by the conserved catalytic histidine residues with substrate, reduced fluctuation of the loop regions and increased hydrogen network of the mutational amino acids, revealed by molecular dynamics simutation and molecular docking. Moreover, the yield of TF1 was improved by catalysis of mutant RcPPO-FKI with higher synthetic efficiency of TF1 in comparison with some reported catalysts and as a promising alternative to commercial mushroom tyrosinase. PPOs with enhanced performances provide the robust candidate for industrial application.

A turn-on fluorescence probe for imaging tyrosinase at the wound site in broken tail of zebrafish

Tyrosinase (TYR) is a copper-containing oxidase that affects the synthesis of melanin in the human body, which is regulate to the pigmentation of the skin. Nevertheless, abnormal expression of TYR can lead to albinism, vitiligo and other skin diseases. Excessive accumulation of TYR is a marker of melanoma cancer and an important factor leading to pigmentation during wound healing, freckles and browning of fruits and vegetables. Efficient tracking of TYR is of significance for studying its pathophysiological mechanism. Herein, we synthesized a benzindole-based fluorescent probe Pro-OH to detect TYR in living cells and zebrafish. The probe displayed a high selectivity and sensitivity in distinguishing TYR from other analytes with the low detection limit of 1.024 U/mL. Importantly, Pro-OH was successfully used to imagine TYR at the wound site of broken tail of zebrafish.

Ultrasensitive detection of tyrosinase with click reaction-combined dark-field imaging platform

Tyrosinase (TYR) is an essential oxidase that is responsible for the regulation of multiple physiological processes and diseases. Achieving the trace and reliable detection of TYR in complex biological samples is of great significance for the diagnosis of TYR-related diseases, but which faces a great challenge. In this study, we developed an ingenious and powerful method for the ultrasensitive detection of TYR by click reaction-combined dark-field microscopy. This method begins with the formation of cuprous ions (Cu+) based on the reduction of copper ions (Cu2+) by ascorbic acid (AA). Subsequently, the formed Cu+ can catalyze the crosslinking between azide- and alkyne-functionalized gold nanoparticles, causing a significant red-shift in the scattering spectrum. However, AA can chelate with TYR, which inhibits the generation of Cu+ and subsequent click reaction, thus achieving TYR-controlled scattering spectral shift. The proposed sensing platform shows a good linear detection range of 0.01–0.8 U/L with a low detection limit of 0.003 U/L, which is three orders of magnitude lower than the best performance of TYR sensing probes reported to date. Most importantly, the strategy has the ability to reliably and accurately detect TYR in serum sample, suggesting its potential clinical application in diagnosing TYR-related diseases. This visual sensing platform offers promising prospects for future research in enzymatic analysis and biomedical diagnostics.