Diphenolase Activity Of Mushroom Tyrosinase Research Articles

In silico and in vitro studies of novel derivatives of tyrosol and raspberry ketone as the mushroom tyrosinase inhibitors.

Tyrosinase is the key enzyme for melanin biosynthesis. Overproduction and deposition of this pigment cause different problems in various industries including agriculture and food. Finding safe tyrosinase inhibitors thus attracts great research interest. The goal of this study is evaluation of inhibitory potencies of some novel synthetic derivatives of tyrosol and raspberry ketone on diphenolase activity of mushroom tyrosinase. The ligands inhibited enzyme activity and compound 4-(2-(4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl)ethyl)phenol (1d) exhibited the most inhibitory potency (77% inhibition, IC50=0.32µmol L-1) via the mixed inhibition mode. This compound was also safe according to the results of in vitro analyses. The enzyme-ligands interactions were theoretically and experimentally investigated using molecular docking and fluorescence quenching approaches, respectively. Modes of quenching and related parameters were also determined and molecular docking data showed that the ligands bind to important sites of the enzyme. These compounds, especially 1d, can be suggested as efficient candidates for further investigations.

Design, synthesis, in silico ADME, DFT, molecular dynamics simulation, anti-tyrosinase, and antioxidant activity of some of the 3-hydroxypyridin-4-one hybrids in combination with acylhydrazone derivatives

Tyrosinase is the rate-limiting enzyme in synthesizing melanin. Melanin is responsible for changing the color of fruits and vegetables and protecting against skin photo-carcinogenesis. Herein, some of the hybrids of 3-hydroxypyridine-4-one and acylhydrazones were designed and synthesized to study the anti-tyrosinase and antioxidant activities. The diphenolase activity of mushroom tyrosinase using L-DOPA assayed the inhibitory effects, and the antioxidant activity was assessed using DPPH free radical. The synthesized derivatives were confirmed using 1H-NMR, 13C-NMR, IR, and Mass spectroscopy. Among analogs, compound 5h bearing furan ring with IC50=8.94 μM was more potent than kojic acid (IC50=16.68 μM). The pharmacokinetic profile of the compounds showed that the tested compounds had suitable oral bioavailability and drug-likeness properties. The molecular docking studies showed that compound 5h was located in the tyrosinase-binding site. Also, the molecular dynamics simulation was performed on compound 5h, proving the obtained molecular docking results. At the B3LYP/6-31 + G** level of theory, the reactivity descriptors for 5 g and 5h were investigated using DFT calculations. Also, IR frequency was calculated to verify DFT results with experimental data. The electrostatic potential energy of the surface and the HOMO and LUMO molecular orbitals were also studied. It agrees with experimental results that the 5h is a soft molecule and ready for chemical reaction with other interacting molecules. Communicated by Ramaswamy H. Sarma

Cinnamic acid derivatives linked to arylpiperazines as novel potent inhibitors of tyrosinase activity and melanin synthesis

A novel series of twenty-seven cinnamides constituted by cinnamic acid derivatives liked to 1-aryl piperazines were synthesized and evaluated for their potential inhibitory diphenolase activity of mushroom tyrosinase. Among them, the presence of a 3-chloro-4-fluorophenyl moiety at the N-1 position of piperazine ring was essential for a potent tyrosinase inhibitory effect, with the 3-nitrocinnamoyl (19p) and 2-chloro-3-methoxycinnamoyl (19t) derivatives as the most potent compounds of the series, with IC50 of 0.16 and 0.12 μM, respectively, resulting much active than kojic acid, whose IC50 value was 17.76 μM. In general, all compounds characterized by the presence of a 1-(3-chloro-4-fluorophenyl)piperazine moiety showed an excellent potency, and the nature, position and number of the substituents on the aryl of the cinnamic acid did not affect significantly the anti-tyrosinase activity. The molecular docking to the active site of the enzyme has been also performed to investigate the nature of enzyme-inhibitor interactions. Furthermore, for selected highly active compounds, their ability to inhibit melanogenesis in the A375 human melanoma cells and in vivo zebrafish model was also evaluated. One of the most potent compounds of series (19t) significantly reduced the pigmentation of zebrafish at 50 μM, unfortunately showing 100% mortality in the Fish Embryo Acute Toxicity (FET) test at the same concentration, Moreover, the zebrafish assay reveals that also compound 19r (IC50:0.51 μM against mushroom tyrosinase) effectively reduces melanogenesis with no acute toxicity effects and it could be proposed as potential candidate to treat tyrosinase-mediated hyperpigmentation.

Evaluation of quercetin omega-6 and -9 esters on activity and structure of mushroom tyrosinase: Spectroscopic and molecular docking studies.

Quercetin is one of the most ubiquitous dietary flavonoids widely distributed in plants and foods of plant origin, and is a potent tyrosinase inhibitor. Quercetin fatty esters could lead to an improve in quercetin lipophilicity which could positively affect its pharmacological activity. In this study, the inhibitory effect of two novel esters of quercetin-linoleic acid (ligand A) and quercetin-oleic acid (ligand B) has been investigated on structure and diphenolase activity of mushroom tyrosinase (MT) by experimental and molecular docking techniques. The inhibitory kinetics study using UV-visible spectrophotometry in the presence of its substrate 3,4-dihydroxyphenylalanine (L-dopa), revealed that both esters successfully inhibit the activity of tyrosinase and reduce the formation of dopaquinone. Results showed that the binding of ligands to MT induced rearrangement and conformational changes of the enzyme. Thermodynamic parameters of these interactions (Ka , ∆G°, ∆H° and ∆S°) were obtained at pH=6.8 and temperatures of 298 and 310K. Molecular docking studies further was applied to calculation of binding energies (ΔGbA =-21.84kJ/mol, ΔGbB =-20.92kJ/mol), inhibition constant values (KIA =160µM, KIB =220µM) and the special binding site. It can be deduced that ligands act as a potential tyrosinase inhibitor and it was found that the best possible interaction condition with binding modes visualize was achieved by ligand A and exhibited the potent tyrosinase inhibitory activity. These findings may be helpful to understand the inhibition mechanism of quercetin fatty acids esters on tyrosinase and provide a convenient screening method to differentiate phenolic tyrosinase inhibitors. PRACTICAL APPLICATIONS: Bioavailability and antioxidant activity of conjugated fatty acids with their bioequivalence in several biological effects and metabolic processes such as beta-oxidation from various forms has been reported to be highly variable and useful. Quercetin shows beneficial role in human health, but its biological effects in vivo is limited by poor bioavailability, low skin permeability and solubility. This study design new tyrosinase inhibitors which helpful to functional research of unsaturated fatty acid esters in the treatment of inflammatory diseases and hyperpigmentation disorders. In addition, undesirable enzymatic browning of plant derived-foods by tyrosinase causes a decrease in market value and economic loss of food products. The results suggest that the conjugation of quercetin with linoleic and oleic acids resulted in novel stronger tyrosinase inhibitors which may have therapeutic applications and replacement of toxic tyrosinase inhibitors and contribute as anti- browning agents in food, cosmetic and pharmaceutical industry.

The Hull of Ripe Pistachio Nuts (Pistacia vera L.) as a Source of New Promising Melanogenesis Inhibitors.

In the present study an acidified methanol pistachio hull extract was investigated for antioxidant and inhibitory effects on melanin biosynthesis by in vitro and in vivo assays. The chromatographic analysis revealed that cyanidin-3-O-galactoside represents the main compound (98.37%). The pistachio hull extract efficiently inhibits the mono and diphenolase activity of mushroom tyrosinase (IC50= 141.07 and 116.08μg/mL, respectively) and it was able, thanks to its strong antioxidant and free-radical scavenging activities, to hinder the L-DOPA auto-oxidation in a concentration-dependent manner (125-500μg/mL). Results of in vivo assay showed that the treatment with pistachio hull extract (10μg/mL) reduced pigmentation in zebrafish embryos at early stages of development (60.01% of inhibition vs control). In conclusion, these findings suggest that the ripe pistachio hull may be considered as a promising source of antioxidant and skin whitening agents for the development of new products useful in preventing the pigmentation disorders in humans and/or to improve the food quality.

Kinetic, spectroscopic and computational docking study of the inhibitory effect of the pesticides 2,4,5-T, 2,4-D and glyphosate on the diphenolase activity of mushroom tyrosinase

The inhibitory effect of 2,4,5-T, 2,4-D, glyphosate and paraquat on the diphenolase activity of mushroom tyrosinase for oxidation of L-DOPA has been investigated by kinetic measurements, fluorescence spectroscopy and computational docking analysis. 2,4,5-T and 2,4-D inhibit the diphenolase activity of the enzyme following a competitive mechanism, while glyphosate is a mixed inhibitor according to Lineweaver-Burk kinetic analysis. The inhibitory activity follows the order glyphosate >2,4,5-T > 2,4-D with IC50 values of 65, 90 and 106 μM, respectively. Intrinsic tyrosinase fluorescence quenching and computational docking analysis suggest that 2,4,5-T and 2,4-D interact with the active site of the enzyme through hydrophobic interactions, while glyphosate also interacts with external residues of the active site of the enzyme by hydrogen bonding and hydrophilic interactions inducing conformational changes in the protein structure.

Structure⁻Activity Relationship of the Tyrosinase Inhibitors Kuwanon G, Mulberrofuran G, and Albanol B from Morus Species: A Kinetics and Molecular Docking Study.

Kuwanon G (KG) and benzofuran flavonoids such as mulberrofuran G (MG) and albanol B (AB) isolated from Morus sp. are reported to exhibit anti-Alzheimer’s disease, anti-inflammatory, fungicidal, anti-cancer, anti-bacterial, and anti-tyrosinase properties. We investigated the inhibition of mono- and diphenolase activity of mushroom tyrosinase by KG, MG, and AB. KG and MG displayed acceptable inhibition activity compared to kojic acid. AB did not show any activity up to 350 µM. MG displayed six-fold higher inhibition of l-tyrosine oxidation (IC50 = 6.35 ± 0.45 µM) compared to kojic acid (IC50 = 36.0 µM). Kinetic studies revealed that KG and MG inhibited monophenolase activity of tyrosinase in a competitive manner. Docking simulations of KG and MG demonstrated favorable binding energies with amino acid residues of the active sites of tyrosinase. Our investigation of the structure-activity relationship of the fused benzofuran flavonoids (MG vs. AB) implicated the methyl cyclohexene ring moiety in tyrosinase inhibition. The enzyme substrate and relative structural analyses demonstrated that KG and MG from Morus sp. could be useful natural tyrosinase inhibitors in foods or cosmetics.

Design and synthesis of novel bis-hydroxychalcones with consideration of their biological activities

In the present study, eight new substituted bis-hydroxychalcones for the first time were designed, synthesized, purified and fully characterized. Then their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The concentration of compounds to scavenge 50% of ABTS (IC50) values were calculated for all the synthesized compounds. IC50 values for 2,2-azinobis (3-ethylbenzothiazoline-sulfonate) assay (ABTS) were in the range of 0.130–0.39 mM. Synthesized compounds possessed an inhibitory effect on tyrosinase activity, too. All of the products were much more active than E and C vitamins. The high antioxidant activity of products could be due to the longer conjugated system which can stabilize the free radical by resonance through a longer system. The concentration of compounds to scavenge 50% of LDOPA (IC50) values were in the range of 0.2–0.6 mM for the tyrosinase inhibition screening Products 4a, 4c, 4d, 4e and 4g which have inhibitory properties comparable to kojic acid (KA). Molecular modeling studies have been performed to achieve insight into the binding mode of the synthesized compounds to the tyrosinase enzyme active site.

Novel hydroxypyridinone derivatives containing an oxime ether moiety: Synthesis, inhibition on mushroom tyrosinase and application in anti-browning of fresh-cut apples

A range of hydroxypyridinone derivatives were synthesized starting from kojic acid. Among them, 10 and 11 were found to possess the strongest inhibitory effect on monophenolase activity of mushroom tyrosinase, having IC50 values of 2.04 and 1.60μM, respectively. The IC50 values of 10 and 11 for the inhibition of diphenolase activity of mushroom tyrosinase were determined as 13.89 and 7.99μM, respectively. Investigation of the inhibitory mechanism of these two compounds indicated that the inhibition was reversible and of a competitive-uncompetitive mixed type. The KI and KIS values of 10 were determined to be 24.84 and 32.54μM, respectively, and the corresponding values for 11 being 18.07 and 21.34μM, respectively. The effect of 11 on the browning process of fresh-cut apples was evaluated by measuring the color change and browning index. The results indicated that 11 had a significant effect on controlling the browning of fresh-cut apple slices.

Comprehensive kinetic and structural studies of different flavonoids inhibiting diphenolase activity of mushroom tyrosinase

The effect of 4 flavonoids on the diphenolase activity of mushroom tyrosinase was studied using spectroscopic approach. Analysis of kinetic data demonstrated that flavonoids cause a reversible inhibition of the enzyme activity. Further study showed that gallic acid acted as noncompetitive inhibitor, whereas chrysin, naringin and quercetin inhibited the diphenolase activity of mushroom tyrosinase in a competitive fashion. Comparison of the inhibition constants revealed that the strength with which the inhibitors acted on the enzyme activity was ranking as follows: chrysin (Ki 7.90 mM) < quercetin (Ki 7.44 mM) < naringin (Ki 3.04 mM) < gallic acid (Ki 1.5 mM). These data, therefore, suggest that gallic acid is the most potent inhibitor of the enzyme compared to the other flavonoids used.

Design and synthesis of thiourea derivatives with sulfur-containing heterocyclic scaffolds as potential tyrosinase inhibitors

Tyrosinase is a key enzyme during the production of melanins in plants and animals. A class of novel N-aryl-N′-substituted phenylthiourea derivatives (3a–i, 6a–k) were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed some 4,5,6,7-tetrahydro-2-[[(phenylamino)thioxomethyl]amino]-benzo[b]thiophene-3-carboxylic acid derivatives (3a–i) exhibited moderate inhibitory potency on diphenolase activity of tyrosinase. When the scaffold of 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid was replaced with 2-(1,3,4-thiadiazol-2-yl)thio acetic acid, the inhibitory activity of compounds (6a–k) against tyrosinase was improved obviously; especially, the inhibitory activity of compound 6h (IC50=6.13μM) is significantly higher than kojic acid (IC50=33.3μM). Moreover, the analysis on inhibition mechanism revealed that compound 6h might plays the role as a noncompetitive inhibitor.

Isolation of Sulfuretin and Butin from Rhus verniciflua Stokes Using Medium-pressure Liquid Chromatography and their Tyrosinase Inhibitory Effects

The aim of this study was to separate antityrosinase compounds of the ethyl acetate fraction from Rhus verniciflua Stokes using medium pressure liquid chromatography. Among the different fractions, the Fr.6 fraction showed the highest antityrosinase capacity (96.5%), followed by the Fr.5 fraction (85.6%). The Fr.1 fraction showed the lowest antityrosinase capacity (12.4%). Bioactivity-guided fractionation of Fr.5.5 and Fr.6.4 led to the isolation and identification of butin and sulfuretin. Then the inhibitory effects of butin and sulfuretin on the monophenolase and diphenolase activity of mushroom tyrosinase were investigated. The results showed that butin and sulfuretin can act as potent inhibitors of monophenolase and diphenolase activities of the enzyme, and the IC50 of the butin and sulfuretin were 16.0 μmol/L and 13.64 μmol/L, respectively. The lag period of the enzyme was obviously lengthened; it was estimated to be 1 min in the absence of inhibitor, extended to 26 min in the presence of 185 μmol/L of butin, and 6 min in the presence of 111.1 μmol/L of sulfuretin. A kinetic analysis showed that butin and sulfuretin are competitive inhibitors. The results revealed that the butin and sulfuretin took up the loci of the substrate combined with enzyme, or blocked the anionic initiation by eliminating free radicals, thus weakening the catalytic reaction of oxidation of L-dopa.

Inhibitory Effects of Physalis alkekengi L., Alcea rosea L., Bunium persicum B. Fedtsch. and Marrubium vulgare L. on Mushroom Tyrosinase.

The key enzyme in the process of melanin biosynthesis is tyrosinase. Skin hyperpigmentation and browning of foods are undesirable phenomena which tyrosinase represents. Therefore, tyrosinase inhibitors have been used increasingly for medicinal and cosmetic products. In this study, inhibitory effects of four plants including: physalis alkekengi L., Alcea rosea L., Bunium persicum B. Fedtsch. and Marrubium vulgare L. on diphenolase activity of mushroom tyrosinase were evaluated. The inhibitory activities of hydroalcoholic extracts of plants against oxidation of L-Dopa (as a substrate) by mushroom tyrosinase were investigated. The hydroalcoholic extract of P. alkekengi showed the most tyrosinase inhibitory effect with IC50 of 0.09 mg/mL vs. 0.38, 0.38 and 2.82 mg/mL of B. persicum, A. rosea and M. vulgare, respectively. M. vulgare exhibited uncompetitive inhibition and other plants showed mixed type inhibition on mushroom tyrosinase. All plants could inhibit mushroom tyrosinase, but more investigations on human tyrosinase and clinical studies are needed.

Inhibitory Effect of the Ethyl Acetate Fraction of Ethanol Extract from Rhus verniciflua Stokes Wood on the Activity of Mushroom Tyrosinase

Solvent extracts of Rhus verniciflua Stokes wood were made using decompressing inner ebullition, and a Box-Behnken design was used to optimize extraction conditions to produce an extract that inhibited tyrosinase activity. The chemical compositions and inhibition rates were determined in extracts made with petroleum ether, ethyl acetate, n-butanol, and an aqueous fractionation. The ethyl acetate fraction had the highest total phenolic content and inhibition rates. The main flavonoids in this fraction were 0.531% fisetin, 7.582% fustin, 0.848% sulfuretin, and 0.272% butein. The effects of the extract on the monophenolase and diphenolase activity of mushroom tyrosinase were studied using the Lineweaver-Burk equation to determine the effect of the extract on inhibition of tyrosinase activity. The results showed that the extract inhibited both the monophenolase and diphenolase activity of the enzyme. The IC50 of the ethyl acetate extract was 308 μg/mL, with the lag period of the enzyme being obviously lengthened; it was estimated to be 2.45 min in the absence of the inhibitor and extended to 9.63 min in the presence of 500 μg/mL of extract. The ethyl acetate extract acted as a mixed type inhibitor. The KI was less than the KIS, which demonstrates that the [ESI] is less stable than [EI], suggesting that the extract could easily combine with free enzyme in the enzyme catalysis system, thus affecting enzyme catalysis on the substrate.

Design, synthesis and biological evaluation of hydroxy- or methoxy-substituted 5-benzylidene(thio) barbiturates as novel tyrosinase inhibitors

Here a new class of hydroxy- or methoxy-substituted 5-benzylidene(thio)barbiturates were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that several compounds had more potent tyrosinase inhibitory activities than the widely used tyrosinase inhibitor kojic acid (IC50=18.25μM). In particular, 3′,4′-dihydroxylated 1e was found to be the most potent inhibitor with IC50 value of 1.52μM. The inhibition mechanism analysis revealed that the potential compounds 1e and 2e exhibited such inhibitory effects on tyrosinase by acting as the irreversible inhibitors. Structure–activity relationships’ (SARs) analysis also suggested that further development of such compounds might be of interest.

Microwave‐Assisted Synthesis and Tyrosinase Inhibitory Activity of Chalcone Derivatives

A series of chalcones and their derivatives were synthesized, and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that some of the synthesized compounds exhibited significant inhibitory activity, and four compounds exhibited more potent tyrosinase inhibitory activity than the reference standard inhibitor kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one). Specifically, 1-(-1-(4-methoxyphen- yl)-3-phenylallylidene)thiosemicarbazide (18) exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value of 0.274 μM. The inhibition mechanism analysis of 1-(-1-(2,4-dihydroxyphenyl)-3-phenylallylidene) thiosemicarbazide (16) and 1-(-1-(4-methoxyphenyl)-3-phenylallylidene) thiosemicarbazide (18) demonstrated that the inhibitory effects of the two compounds on the tyrosinase were irreversible. Preliminary structure activity relationships' analysis suggested that further development of such compounds might be of interest.

Biological evaluation of coumarin derivatives as mushroom tyrosinase inhibitors

A series of coumarin derivatives were synthesised and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that some of the synthesised compounds exhibited significant inhibitory activities. Especially, 2-(1-(coumarin-3-yl)ethylidene)hydrazinecarbothioamide bearing thiose-micarbazide group exhibited the most potent tyrosinase inhibitory activity with IC50 value of 3.44μM. The inhibition mechanism analysis of 2-(1-(coumarin-3-yl)-ethylidene)hydrazinecarbothioamide and 2-(1-(6-chlorocoumarin-3-yl)ethylidene)-hydrazinecarbothioamide demonstrated that the inhibitory effects of the compounds on the tyrosinase were irreversible. Preliminary structure activity relationships’ (SARs) analysis suggested that further development of such compounds might be of interest.

Wheat bran globulins: Competitive inhibitors of mushroom tyrosinase

The inhibitory capacity of the globulin fraction of wheat bran against the diphenolase activity of mushroom tyrosinase, using l-tyrosine as substrate, was evaluated. Enzyme kinetics was monitored in the presence of globulin solutions by measuring the absorbance at 475nm. Lineweaver-Burk plots were drawn in order to determine Vmax, Km, and type of inhibition. Results showed that globulins from wheat bran competitively inhibited, the activity of mushroom tyrosinase with a KI of 0.79%(w/v). The degree of inhibition was 24% at 2 mM of the substrate L-tyrosine.

Inhibitory effects of naphthols on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1), a copper-containing multifunctional oxidase, was known to be a key enzyme for biosynthesis in fungi, plants and animals. In this work, the inhibition properties α-naphthol and β-naphthol toward the activity of tyrosinase have been evaluated, and the effects of α-naphthol and β-naphthol on monophenolase and diphenolase activity of tyrosinase have been investigated. The results showed that both α-naphthol and β-naphthol could potently inhibit both monophenolase activity and diphenolase activity of mushroom tyrosinase, and that β-naphthol exhibited stronger inhibitory effect against tyrosinase than α-naphthol. For monophenolase activity, β-naphthol could not only lengthen the lag time but also decrease the steady-state activity, while α-naphthol just only decreased the steady-state activity. For diphenolase activity, both α-naphthol and β-naphthol displayed revisible inhibition. Kinetic analyses showed that both α-naphthol and β-naphthol were competetive inhibitors.

The Inhibitory Effect of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) on the Monophenolase and Diphenolase Activities of Mushroom Tyrosinase

In the present work, we investigated the effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the monophenolase and diphenolase activity of mushroom tyrosinase. The results showed that diflunisal and indomethacin inhibited both monophenolase and diphenolase activity. For monophenolase activity, the lag time was extended in the presence of diflunisal. In the presence of indomethacin, the lag time did not change. IC50 values of monophenolase activity were estimated to be 0.112 mM (diflunisal) and 1.78 mM (indomethacin). Kinetic studies of monophenolase activity revealed that both diflunisal and indomethacin were non-competitive inhibitors. For diphenolase activity, IC50 values were estimated to be 0.197 mM (diflunisal) and 0.509 mM (indomethacin). Diflunisal and indomethacin were also found to be non-competitive diphenolase inhibitors.