Inhibitory effects of 4-vinylbenzaldehyde and 4-vinylbenzoic acid on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones which form brown or black pigments. Here, the inhibitory effects of 4-vinylbenzaldehyde and 4-vinylbenzoic acid on the activity of mushroom tyrosinase have been investigated. The results showed that both 4-vinylbenzaldehyde and 4-vinylbenzoic acid could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, 4-vinylbenzoic acid could lengthen the lag time, but 4-vinylbenzaldehyde could not. Both 4-vinylbenzaldehyde and 4-vinylbenzoic acid decreased the steady-state activity, and the IC50 values were estimated as 93 microM and 3.0 mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of 4-vinylbenzaldehyde was stronger than that of 4-vinylbenzoic acid, and the IC50 values were estimated as 23 microM and 0.33 mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were mixed-II type; their inhibition constants were also determined and compared.     

Inhibitory effects of Cefazolin and Cefodizime on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, the effects of Cefazolin and Cefodizime on the activity of mushroom tyrosniase have been studied. The results showed that the Cephalosporin antibacterial drugs (Cefazolin and Cefodizime) could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, Both Cefazolin and Cefodizime could lengthen the lag time and decrease the steady-state activities, and the IC₅₀ values were estimated as 7.0 mM and 0.13 mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of Cefodizime was obviously stronger than that of Cefazolin, and the IC₅₀ values were estimated as 0.02 mM and 0.21 mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were competitive and mixed-type, respectively. Their inhibition constants were also determined and compared. The research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors and also under the application field of Cephalosporins.     

Inhibitory effects of hinokitiol on tyrosinase activity and melanin biosynthesis and its antimicrobial activities

The inhibitory effects of hinokitiol, a constituent of the woody oils isolated from Cupressaceae heartwood, on mushroom tyrosinase and melanin formation in B16 melanoma cells as well as its antimicrobial activity were investigated. Our results showed that hinokitiol could strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition was reversible. The IC₅₀ values were estimated as 9.67?μM for monophenolase activity and 0.21?μM for diphenolase activity. The lag time of the monophenolase activity was not obviously lengthened by the compound. Kinetic analyses showed that the inhibition mechanism of hinokitiol was a mixed-type inhibition of the diphenolase activity. Hinokitiol effectively inhibited both cellular tyrosinase activity and melanin biosynthesis in B16 melanoma cells with significant cytotoxicity. Furthermore, it was found that hinokitiol could inhibit the proliferation of Salmonella enteritidis, Escherichia coli, Bacillus subtilis, Staphyloccocus aureus, Klebsiella pneumoniae, and Ralstonia solanacearum to different extents. This research may widen the use of hinokitiol in the fields of food preservation, depigmentation, and insecticide use.     

Inhibitory effects of fluorobenzaldehydes on the activity of mushroom tyrosinase

The effects of fluorobenzaldehydes (2-,3- and 4-fluorobenzaldehyde) on the activity of mushroom tyrosinase have been studied. The results show that fluorobenzaldehydes can strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition is reversible. The IC₅₀ values were estimated as 1.62 mM, 1.06 mM and 0.16 mM for diphenolase activity and as 1.35 mM, 1.18 mM and 1.05 mM for monophenolase activity, respectively. The lag time of the monophenolase was obviously lengthened by these three fluorobenzaldehydes. When the concentration of inhibitors reached 2.0 mM, the lag time was lengthened from 33 s to 142 s, 168 s and 190 s, respectively. Kinetic analyses show that the inhibition mechanism of 2-fluorobenzaldehyde on the diphenolase was competitive inhibition of the diphenolase activity, and that of 3-fluorobenzaldehyde and 4-fluorobenzaldehyde were of a mixed-type. The inhibition constants for these three fluorobenzaldehydes on the diphenolase were determined and compared.     

Inhibitory Effects of 4-Halobenzoic Acids on the Diphenolase and Monophenolase Activity of Mushroom Tyrosinase

The effects of 4-halobenzoic acids (4-fluorobenzoic acid, 4-chlorobenzoic acid, and 4-bromobenzoic acid) on the activity of mushroom tyrosinase have been studied. The results show that 4-halobenzoic acids can strongly inhibit both monophenolase activity and diphenolase activity of the enzyme, and the inhibition displays a reversible course. The IC50 values were estimated as 0.26, 0.20, and 0.18 mM for diphenolase activity and as 1.03, 0.75, and 0.60 mM for monophenolase activity, respectively. Kinetic analyses show that the inhibition mechanism of all three 4-halobenzoic acids is noncompetitive inhibition to the diphenolase activity, and the inhibition constants (K1) were determined to be 0.25, 0.20, and 0.17 mM, respectively. The lag time of the monophenolase was obviously lengthened by these three 4-halobenzoic acids. When the concentration of inhibitors reached 1.4 mM, the lag time was lengthened from 30 s to 120, 125, and 150 s, respectively.     

Kinetic study of monophenol and o-diphenol binding to oxytyrosinase

The complex reaction mechanism of tyrosinase involves three enzymatic forms, two overlapping catalytic cycles and a dead-end complex. The deoxytyrosinase form binds oxygen with a high degree of affinity, μM. The mettyrosinase and oxytyrosinase forms bind monophenols and o-diphenols, although the former is inactive on monophenols. Analytical expressions for the catalytic and Michaelis constants of tyrosinase towards phenols and o-diphenols have been derived. Thus, the Michaelis constant of tyrosinase towards monophenols and o-diphenols are related with the catalytic constants for monophenols and o-diphenols , respectively, and with the binding rate constants of the oxytyrosinase form with these substrates (k₊₄ and k₊₆, respectively), by means of the expressions and . From these expressions, we calculate the values of the binding rate constant of oxytyrosinase to the substrates (monophenols and o-diphenols) for tyrosinases from different biological sources, and reveal that the o-diphenols bind more rapidly to oxytyrosinase than the monophenols. In addition, a new kinetic constant (the Michaelis constant for o-diphenol in the monophenolase activity), is derived and determined. Thus, it has been shown that tyrosinase has apparently higher affinity towards o-diphenols in its monophenolase than in its diphenolase activity.     

Purification and comparative characterization of monophenolase and diphenolase activities from a wild edible mushroom (Macrolepiota gracilenta)

Polyphenol oxidases (PPO) are very important enzymes group in many industrial applications, especially in food, medicine and cosmetics. PPO from Macrolepiota gracilenta, a wild edible mushroom, was purified using a Sepharose 4B-l-tyrosine-p-amino benzoic acid affinity column and characterized in terms of mono- and diphenolase activity. The highest activities for pure enzyme were observed in the presence of PHPPA and DHPPA for monophenolase and diphenolase, respectively. The enzyme showed pH optimum values at 7.0 and 5.0, respectively, for monophenolase and diphenolase activities. K_m values calculated as 0.8 mM for monophenolase and 1 mM for diphenolase activity at the presence of PHPPA and DHPPA as substrate, respectively. V_max values were calculated as 2000 U/mg protein for both activity. Monophenolase and diphenolase activities were conserved approximately 40% and 60%, respectively, in their optimum pH at 4 °C after 5 day incubation. The activities were inhibited most effectively by thiourea. The data obtained from this study showed that this enzyme could be useful for some industrial purposes.     

Inhibitory effect of 4-cyanobenzaldehyde and 4-cyanobenzoic acid on mushroom (Agaricus bisporus) tyrosinase

Mushroom tyrosinase (EC 1.14.18.1), a copper containing oxidase, catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the current study, the effects of 4-cyanobenzaldehyde and 4-cyanobenzoic acid on the monophenolase and diphenolase activities of mushroom tyrosinase have been studied. The results show that 4-cyanobenzaldehyde and 4-cyanobenzoic acid can inhibit both the monophenolase activity and the diphenolase activity of mushroom tyrosinase. The lag phase of tyrosine oxidation catalyzed by the enzyme was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. 1.0 mM 4-cyanobenzaldehyde and 4-cyanobenzoic acid can lengthen the lag phase from 78 s to 134 and 115 s, respectively. Both 4-cyanobenzaldehyde and 4-cyanobenzoic acid can lead to reversible inhibition of the enzyme. The IC50 values of 4-cyanobenzaldehyde and 4-cyanobenzoic acid were estimated as 0.62 and 2.45 mM for monophenolase and as 0.72 and 1.40 mM for diphenolase, respectively. A kinetic analysis shows that 4-cyanobenzaldehyde and 4-cyanobenzoic acid are mixed-type inhibitors for the diphenolase. The apparent inhibition constants for 4-cyanobenzaldehyde and 4-cyanobenzoic acid binding with both the free enzyme and the enzyme-substrate complex have been determined and compared.     

In Vitro Effect of Ozagrel on Mushroom Tyrosinase

This investigation, in vitro, shows that ozagrel, an antithrombotic drug, inhibited both monophenolase and diphenolase activities of mushroom tyrosinase when l-tyrosine and l-DOPA were assayed spectrophotometrically, respectively. The IC₅₀ values, for monophenolase and diphenolase activities, were 1.35 and 3.45 mM, respectively. Ozagrel was estimated to be a reversible mixed-type inhibitor of diphenolase activity with the constants (K _S1, K _S2, K _i1, and K _i2) determined to be 2.21, 3.89, 0.454, and 0.799 mM, repectively. Increasing ozagrel concentrations provoked longer lag periods as well as a concomitant decrease in the monophenolase activity. Inhibition experiment demonstrated that ozagrel bound the enzyme at a site distincted from the substrate active site, but it bound to either E (Enzyme) or ES (Enzyme-Substrate) complex.     

Discovery of a new type of scaffold for the creation of novel tyrosinase inhibitors

Tyrosinase is known as the key enzyme for melanin biosynthesis, which is effective in preventing skin injury by ultra violet (UV). In past decades, tyrosinase has been well studied in the field of cosmetics, medicine, agriculture and environmental sciences, and a lot of tyrosinase inhibitors have been developed for their needs. Here, we searched for new types of tyrosinase inhibitors and found phenylbenzoic acid (PBA) as a unique scaffold. Among three isomers of PBA, 3-phenylbenzoic acid (3-PBA) was revealed to be the most potent inhibitor against mushroom tyrosinase (IC₅₀ = 6.97 μM, monophenolase activity; IC₅₀ = 36.3 μM, diphenolase activity). The kinetic studies suggested that the apparent inhibition modes for the monophenolase and diphenolase activities were noncompetitive and mixed type inhibition, respectively. Analyses by in silico docking studies using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid group of the 3-PBA could adequately bind to two cupric ions in the tyrosinase. To prove this hypothesis, we examined the effect of modification of the carboxylic acid group of the 3-PBA on its inhibitory activity. As expected, the esterification abrogated the inhibitory activity. These observations suggest that 3-PBA is a useful lead compound for the generation of novel tyrosinase inhibitors and provides a new insight into the molecular basis of tyrosinase catalytic mechanisms.     

Inhibition of tyrosinase activity by polyphenol compounds from Flemingia philippinensis roots

Flemingia philippinensis is used as a foodstuff or medicinal plant in the tropical regions of China. The methanol (95%) extract of the roots of this plant showed potent tyrosinase inhibition (80% inhibition at 30 μg/ml). Activity-guided isolation yielded six polyphenols that inhibited both the monophenolase (IC₅₀ = 1.01–18.4 μM) and diphenolase (IC₅₀ = 5.22–84.1 μM) actions of tyrosinase. Compounds 1–6 emerged to be three new polyphenols and three known flavanones, flemichin D, lupinifolin and khonklonginol H. The new compounds (1–3) were identified as dihydrochalcones which we named fleminchalcones (A–C), respectively. The most potent inhibitor, dihydrochalcone (3) showed significant inhibitions against both the monophenolase (IC₅₀ = 1.28 μM) and diphenolase (IC₅₀ = 5.22 μM) activities of tyrosinase. Flavanone (4) possessing a resorcinol group also inhibited monophenolase (IC₅₀ = 1.79 μM) and diphenolase (IC₅₀ = 7.48 μM) significantly. In kinetic studies, all isolated compounds behaved as competitive inhibitors. Fleminchalcone A was found to have simple reversible slow-binding inhibition against monophenolase.     

Tyrosinase inhibitory effect and inhibitory mechanism of tiliroside from raspberry

Tiliroside was found to inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag time of tyrosine oxidation catalyzed by mushroom tyrosinase was obviously lengthened; 0.337?mM of tiliroside resulted in the lag time extension from 46.7?s to 435.1?s. A kinetic analysis shown that tiliroside was a competitive inhibitor for monophenolase and diphenolase with K_i values of 0.052?mM and 0.26?mM, respectively. Furthermore, tiliroside showed 34.5% (p?<?0.05) inhibition of intracellular tyrosinase activity and 54.1% (p?<?0.05) inhibition of melanin production with low cytotoxicity on B16 mouse melanoma cells at 0.168?mM. In contrast, arbutin displayed 9.1% inhibition of cellular tyrosinase activity and 29.5% inhibition of melanin production at the same concentration. These results suggested that tiliroside was a potent tyrosinase inhibitor and might be used as a skin-whitening agent and pigmentation medicine.     

Inhibitory effects of fluorobenzaldehydes on the activity of mushroom tyrosinase

The effects of fluorobenzaldehydes (2-,3- and 4-fluorobenzaldehyde) on the activity of mushroom tyrosinase have been studied. The results show that fluorobenzaldehydes can strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition is reversible. The IC50 values were estimated as 1.62 mM, 1.06 mM and 0.16 mM for diphenolase activity and as 1.35 mM, 1.18 mM and 1.05 mM for monophenolase activity, respectively. The lag time of the monophenolase was obviously lengthened by these three fluorobenzaldehydes. When the concentration of inhibitors reached 2.0 mM, the lag time was lengthened from 33 s to 142 s, 168 s and 190 s, respectively. Kinetic analyses show that the inhibition mechanism of 2-fluorobenzaldehyde on the diphenolase was competitive inhibition of the diphenolase activity, and that of 3-fluorobenzaldehyde and 4-fluorobenzaldehyde were of a mixed-type. The inhibition constants for these three fluorobenzaldehydes on the diphenolase were determined and compared.     

Synthesis and antimycobacterial activity of 7-O-substituted-4-methyl-2H-2-chromenone derivatives vs Mycobacterium tuberculosis

A series of 7-O-alkoxy-4-methylumbelliferone derivatives were prepared using a convenient one step synthesis. Additionally the bromo- and azido derivatives 7-O-(4-bromobutoxy)-, 7-O-(6-bromohexyloxy)- and 7-O-(6-azidohexyloxy)-4-methylumbelliferone derivatives were prepared. In vitro evaluation of antimycobacterial activity determined % inhibition and MIC vs M. tuberculosis H₃₇Rv with toxicity (IC₅₀) assessed in VERO cells. The coumarins with longer alkyl chains (nonyl and decyl) showed the optimum inhibitory activity in this series (MIC 3.13?μg/mL) and IC₅₀>10?μg/mL.     

Dual Effects of Alpha-Arbutin on Monophenolase and Diphenolase Activities of Mushroom Tyrosinase

The effects of α-arbutin on the monophenolase and diphenolase activities of mushroom tyrosinase were investigated. The results showed that α-arbutin inhibited monophenolase activity but it activated diphenolase activity. For monophenolase activity, IC₅₀ value was 4.5 mmol·L⁻¹ and 4.18 mmol·L⁻¹ of α-arbutin could extend the lag time from 40.5 s to 167.3 s. Alpha- arbutin is proposed to be regarded as a triphenolic substrate by the enzyme during catalyzation, leading to the suicide inactivation of the active site of tyrosinase. For diphenolase activity, α-arbutin acted as an activator and its activation mechanism was mixed type activation. To reveal such activation, it should be mainly refered to the conformational changes in tyrosinase caused by the interaction of α-arbutin with residues located at the entrance to the active site, and the decrease of the effect of suicide inactivation.     

A novel synthetic compound, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (MHY773) inhibits mushroom tyrosinase

As part of continued efforts for the development of new tyrosinase inhibitors, (Z)-5-(substituted benzylidene)-2-iminothiazolidin-4-one derivatives (1a – 1l) were rationally synthesized and evaluated for their inhibitory potential in vitro. These compounds were designed and synthesized based on the structural attributes of a β-phenyl-α,β-unsaturated carbonyl scaffold template. Among these compounds, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (1e, MHY773) exhibited the greatest tyrosinase inhibition (IC₅₀ = 2.87 μM and 8.06 μM for monophenolase and diphenolase), and outperformed the positive control, kojic acid (IC₅₀ = 15.59 and 31.61 μM). The kinetic and docking studies demonstrated that MHY773 interacted with active site of tyrosinase. Moreover, a melanin quantification assay demonstrated that MHY773 attenuates α-melanocyte-stimulating hormone (α-MSH) and 3-isobutyl-1-methylxanthine (IBMX)-induced melanin contents in B16F10 melanoma cells. Taken together, these data suggest that MHY773 suppressed the melanin production via the inhibition of tyrosinase activity. MHY773 is a promising for the development of effective pharmacological and cosmetic agents for skin-whitening.     

Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents

A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC₅₀ of 0.10?mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC₅₀ of 0.18?mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4?μg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents.     

Synthesis of novel 3,5-diaryl pyrazole derivatives using combinatorial chemistry as inhibitors of tyrosinase as well as potent anticancer,anti-inflammatory agents

In the present article, we have synthesized a combinatorial library of 3,5-diaryl pyrazole derivatives using 8-(2-(hydroxymethyl)-1-methylpyrrolidin-3-yl)-5,7-dimethoxy-2-phenyl-4H-chromen-4-one (1) and hydrazine hydrate in absolute ethyl alcohol under the refluxed conditions. The structures of the compounds were established by IR, ¹H NMR and mass spectral analysis. All the synthesized compounds were evaluated for their anticancer activity against five cell lines (breast cancer cell line, prostate cancer cell line, promyelocytic leukemia cell line, lung cancer cell line, colon cancer cell line) and anti-inflammatory activity against TNF-α and IL-6. Out of 15 compounds screened, 2a and 2d exhibited promising anticancer activity (61–73% at 10 μM concentration) against all selected cell lines and IL-6 inhibition (47% and 42% at 10 μM concentration) as in comparison to standard flavopiridol (72–87% inhibition at 0.5 μM) and dexamethasone (85% inhibition at 1 μM concentration), respectively. Cytotoxicity of the compounds checked using CCK-8 cell lines and found to be nontoxic to slightly toxic. Out of 15, four 3,5-diaryl pyrazole derivatives exhibiting potent inhibitory activities against both the monophenolase and diphenolase actions of tyrosinase. The IC₅₀ values of compounds (2a, 2d, 2h and 2l) for monophenolase inhibition were determined to range between 1.5 and 30 μM. Compounds 2a, 2d, 2h and 2l also inhibited diphenolase significantly with IC₅₀ values of 29.4, 21.5, 2.84 and 19.6 μM, respectively. All four 3,5-diaryl pyrazole derivatives were active as tyrosinase inhibitors (2a, 2d, 2h and 2l), and belonging to competitive inhibitors. Interestingly, they all manifested simple reversible slow-binding inhibition against diphenolase.     

New antihyperglycemic, α-glucosidase inhibitory,and cytotoxic derivatives of benzimidazoles

Glycosidases play an important role in a wide range of physiological and pathological conditions, and have become potential targets for the discovery and development of agents useful for the treatment of diseases such as diabetes, cancer, influenza, and even AIDS. In this study, several benzimidazole derivatives were prepared from o-phenylenediamine and aromatic and heteroaromatic carboxaldehydes in very good yields, using PdCl₂(CH₃CN)₂ as the most efficient catalyst. Synthesized compounds were assayed for their activity on yeast and rat intestinal α-glucosidase inhibition and cytotoxic activity against colon carcinoma cell line HT-29. Compound 3e exhibited 95.6% and 75.3% inhibition of yeast and rat intestinal α-glucosidase enzyme, while showing 74.8% cytotoxic activity against the HT-29 cell line at primary screening concentrations of 2.1?mM for yeast and rat intestinal α-glucosidase enzyme and 0.2?mM for cytotoxic activity against the HT-29 cell line, respectively. Compound 3c displayed 76% and 34.4% inhibition of yeast and rat intestinal α-glucosidase enzyme, and 80.4% cytotoxic activity against the HT-29 cell line at similar primary screening concentrations. The IC₅₀ value for the most potent intestinal α-glucosidase inhibitor compound 3e was found to be 99.4?μM. The IC₅₀ values for the most active cytotoxic compounds 3c and 3e were 82?μM and 98.8?μM, respectively. Both compounds displayed significant antihyperglycemic activity in starch-induced postprandial hyperglycemia in rats. This is the first report assigning yeast and rat intestinal α-glucosidase enzyme inhibition, cytotoxic activity against the HT-29 cell line, and antihyperglycemic activity to benzimidazole compounds 3c and 3e.     

Isolation and biological activities of 3-hydroxy-4(1H)-pyridone

3-Hydroxy-4(4H)-pyridone (3,4-DHP), a degraded product of mimosine [β-[N-(3-hydroxy-4-oxypyridyl)]-α-aminopropionic acid], is known to cause goiters, loss of hair, and infertility in animals, but limits of 3,4-DHP on separation and purification have prevented efforts on investigating other toxicity and biological properties of 3,4-DHP. By this study, a novel and simple isolation of 3,4-DHP was developed either from Leucaena leaves using an ion-exchanged resin or mimosine degraded in high temperature (110°C, 6?h). The inhibition of mimosine on the growth of barnyardgrass was approximately fourfold higher (IC₅₀?=?0.04?mg?g^?1) than that of 3,4-DHP (IC₅₀?=?0.15?mg?g^?1). In general, the antifungal activity of mimosine is much stronger than that of 3,4-DHP, but it differs depending on the kind of fungi. The 1,1-diphyenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of 3,4-DHP, in contrast with the growth inhibitory activity, is about fourfold stronger [EC₅₀?=?2.4?mg?g^?1 gallic acid equivalent (GAE)] than that of mimosine [EC₅₀?=?10.3?mg?g^?1 GAE]. This study is the first to report on the herbicidal, antifungal, and antioxidant activities of 3,4-DHP.