Effect of Maltol on the Oxidation of DL-DOPA,Dopamine, N-Acetyldopamine (NADA), and Norepinephrine by Mushroom Tyrosinase

Maltol (3-hydroxy-2-methyl-4H-pyran-4-one) appears to inhibit the rate of oxidation of DL-DOPA, dopamine, NADA and epinephrine by tyrosinase when assayed spectropho-tometrically but not when assayed polarographically. Maltol has an effect on the spectrum of product(s) formed when each catecholamine was oxidized by tyrosinase showing that maltol hastens the disappearance of the quinones, possibly by conjugating with them. Indeed, at relatively high concentrations, maltol prevented the conversion of DL-DOPA, dopamine, and norepinephrine to their corresponding melanins via tyrosinase.     

Aspergillus oryzae laeA Regulates Kojic Acid Synthesis Genes

Kojic acid synthesis genes regulation was investigated in Aspergillus oryzae. Our results indicate that kojic acid production was lost in the laeA disruption strain, but was recovered in the LaeA complement strain. Real-time PCR also confirmed that expression of kojic acid biosynthesis genes decreased in the laeA disruption strain, indicating that these genes are under the control of LaeA.     

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

1,2,3-Triazole-based kojic acid analogs as potent tyrosinase inhibitors: Design,synthesis and biological evaluation

A series of kojic acid-derived compounds 6a-p bearing aryloxymethyl-1H-1,2,3-triazol-1-yl moiety were designed by modifying primary alcoholic group of kojic acid as tyrosinase inhibitors. The target compounds 6a-p were synthesized via click reaction. All compounds showed very potent anti-tyrosinase activity (IC₅₀s = 0.06–6.80 µM), being superior to reference drug, kojic acid. In particular, the naphthyloxy analogs 6o and 6p were found to be 31–155 times more potent than kojic acid. The metal-binding study of selected compound 6o revealed that the prototype compound possesses metal-chelating ability, particularly with Cu²⁺ ions. The promising compounds 6o and 6p had acceptable safety profile as demonstrated by cytotoxicity assay against melanoma (B16) cell line and Human Foreskin Fibroblast (HFF) cells.     

Solid-phase synthesis of kojic acid-tripeptides and their tyrosinase inhibitory activity, storage stability, and toxicity

A small library of kojic acid-tripeptides (Ko-X1X2X3) was prepared by solid-phase parallel synthesis and assayed to evaluate their tyrosinase inhibitory activity. Most of the kojic acid-tripeptides showed better activities than kojic acid. Kojic acid-FWY was the best compound, and it exhibited 100-fold tyrosinase inhibitory activity compared with kojic acid. In addition, their storage stabilities were approximately 15 times higher and their toxicity was lower than that of kojic acid.     

N-Acetyl-L-Tyrosine (NAT) as a Substrate for Mushroom Tyrosinase

N-acetyl tyrosine (NAT) is hydroxylated by mushroom tyrosinase and the N-acetyl dopa formed is oxidized by the enzyme to N-acetyl dopaquinone (λ_max= 390 ± 10 nm). H₂O₂ and NH₂OH each shortened the lag period of NAT hydroxylation by the enzyme. H₂O₂ had an effect on the changes with time in the spectrum of product(s) formed and on the spectrum of the final product(s) obtained when NAT was hydroxylated by mushroom tyrosinase, in a manner suggesting that H₂O₂ converts N-acetyl dopaquinone to a pink-violet product(s) (λ_max= 490 nm), whereas such a product(s) was not formed in the absence of H₂O₂. A pink-violet product(s) (λ_max 490 ± 20 nm) was also formed when NAT was hydroxylated by mushroom tyrosinase in the presence of NH₂OH or para amino benzoic acid (PABA), probably as a result of an interaction between N-acetyl dopaquinone and NH₂OH or PABA forming mono- or di-oximes. Kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) inhibited effectively the rate of NAT hydroxylation by mushroom tyrosinase in the absence or presence of H₂O₂. When NAT was oxidized by the enzyme in the absence of kojic acid, N-acetyl dopaquinone was formed at once and a shoulder at 490–530 nm appeared later. Under identical conditions but in the presence of kojic acid, a yellow product(s), characterized by a peak at 320±10 nm, was detected, suggesting that N-acetyl dopaquinone oxidizes kojic acid to the yellow product(s). Maltol(3-hydroxy-2-methyl-4H-pyran-4-one), a γ-pyrone derivative structurally related to kojic acid, also inhibited the rate of NAT hydroxylation by mushroom tyrosinase. The addition of maltol at the plateau phase of the reaction resulted in an immediate decline in absorbance at 400 nm, suggesting that maltol conjugates with N-acetyl dopaquinone, yielding a product(s) characterized by a lower extinction coefficient at 400 nm than that of N-acetyl dopaquinone alone. The final brown-red product(s) formed when NAT was hydroxylated by mushroom tyrosinase was bleached in the presence of ascorbic acid or H₂O₂.     

Dopachrome conversion activity in Aedes aegypti: Significance during melanotic encapsulation of parasites and cuticular tanning

Phenol oxidase (PO) and dopachrome conversion enzyme (DCE) were partially purified from Aedes aegypti larvae by ammonium sulfate fractionation. PO from A. aegypti functions in the hydroxylation of monophenols (e.g., tyrosine and tyramine) to their related o-diphenols, and the oxidation of o-diphenols (e.g., l-dopa, dopamine, N-acetyldopamine) to their respective o-quinones. Partially purified DCE showed high specificity toward dopachrome generated from dopa with the l-configuration. The combined effects of PO and DCE significantly accelerated melanization pathways when l-dopa was used as substrate. Significant DCE activity also was detected in hemolymph samples from adult, female A. aegypti, and undoubtedly plays a role in melanotic encapsulation reactions.     

Studies on Kojic Acid and its Related γ-Pyrone Compounds

The reactions of kojic acid and its related γ-pyrones with anhydrous hydrazine have been investigated. Kojic acid and hydrazine gave 3,6-dihydroxy methyl-4-охо-1,4-dihydro- pyridazine and 3-hydroxymethyl-pyrazolyl-(5)-glycoloyl-hydrazone, respectively, in 65% and 21% yields. The same reaction occured in the case of allomaltol and pyromeconic acid and gave the analogous results. On the other hand, 5-methoxykojic acid was allowed to react with hydrazine and afforded 1-amino-2-hydroxymethyl-5-methoxy-γ-pyridone and α[3-hydroxymethyl-pyrazolyl-(5)]-α-methoxy-acetaldehyde-hydrazone, respectively. The structural elucidation of these products could be fully substantiated by chemical evidences and spectroscopic data. The mechanisms for the reactions are also discussed.     

Laccase-mediated functionalization of chitosan by caffeic and gallic acids for modulating antioxidant and antimicrobial properties

A new strategy for the functionalization of chitosan with caffeic acid (CA) or gallic acid (GA) using laccase from Trametes versicolor is presented for the first time, yielding a product with modulated antioxidant and antimicrobial properties. UV-vis spectroscopy coupled to HPLC-SEC analysis and cyclic voltammetry kinetic studies showed that laccase catalyzes the oxidation of phenolic acids to electrophilic o-quinones, which undergo new oligomer/polymer-forming structures originated by C-C coupling between the benzene rings and C-O-C coupling involving phenolic side-chains. Furthermore, pH tunable reactions/interactions of the laccases oxidized o-quinones with nucleophilic amino groups of chitosan were determined with FTIR and ¹H NMR spectroscopy's. The highest antioxidant activity was found to be for chitosan modified with phenolic acids at pH 4.5, exhibit also an increased activity against Escherichia coli and Listeria monocytogenes compared to untreated chitosan.     

Oxidative addition of quinones to planar cobalt(II) dithiolato,dithioacetylacetonato and Schiff-base complexes

The reactivity of ortho- and para-quinones with various four-coordinated planar Co(II) complexes was investigated. The o-quinones add oxidatively, producing Co(III) complexes containing chelated o-semiquinone radical-anions. No coordination of the fifth ligand in the axial position is involved in these reactions. The reaction between o-quinones and Co(II) dithiolates represents the first known example of oxidative addition to Co(II) dithiolato complexes. All observed oxidative additions are reversible; the position of the equilibrium depends strongly on the nature of the equatorial ligand. The extent of radical adduct formation decreases in the order: dithioacetylacetonate > Schiff bases > dithiolates. When redox potentials of the reacting species are changed in a way that makes simple electron transfer between Co(II) complexes and quinones thermodynamically possible, the reduction of quinones to free semiquinone radical-anions becomes competitive with the oxidative addition. In the case of p-quinones, only electron transfer is observed if the thermodynamic conditions are met. The structural factors determining quinone reactivity are briefly discussed.     

花色苷的酶降解

综述了降解花色苷的酶类及其降解机理的研究进展.降解花色苷的酶有花色苷酶、多酚氧化酶、过氧化物酶和果胶酶.花色苷酶和果胶酶均能水解花色苷糖苷键产生花色素和糖,花色素很不稳定,因吡喃烊环极易开环可自发转换成无色衍生物.花色苷不能直接作为PPO或POD的底物;PPO和POD氧化、降解花色苷须依赖具邻二酚结构的其他酚类的存在,...     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

LEVELS OF NOREPINEPHRINE AND DOPAMINE IN MOUSE BRAIN REGIONS FOLLOWING MICROWAVE INACTIVATION-RAPID POST-MORTEM DEGRADATION OF STRIATAL DOPAMINE IN DECAPITATED ANIMALS

—The effects of 2 methods of killing on norepinephrine and dopamine in mouse brain regions were examined. One method utilized decapitation, while the other method utilized heating with microwave irradiation concentrated on the head. The norepinephrine and dopamine contents of the cerebellum, medulla-pons, midbrain, diencephalon, hippocampus, corpus striatum, and cerebral cortex were determined by methods using liquid chromatography with electrochemical detection. Dopamine content in striatum was also quantitated by the method of gas chromatography with mass fragmentography. A significantly lower value for decapitated animals, as compared to the microwave heated group, was found only for dopamine exclusively in the striatum. Activities of the enzymes tyrosine hydroxylase, DO PA decarboxylase, monoamine oxidase, and catechol-o-methyltransferase in the striatum were also examined. These enzymes were totally inactivated by the microwave heating, except catechol-o-methyltransferase which was decreased approx 80%. These results support either (1) the existence of a substantial pool of dopamine in the striatum with a very rapid turnover rate or (2) a decapitation-related release and destruction of striatal dopamine. Measurements of 3-methoxytyramine in the striatum exhibit post-mortem increases corresponding to the decreases of dopamine. Use of the rapid tissue enzyme inactivation technique suggests that in vivo levels of this O-methylated dopamine metabolite are an order of magnitude lower than the results normally obtained after killing by decapitation.     

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?μM 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?μM 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.     

Synthesis of aryl pyrazole via Suzuki coupling reaction,in vitro mushroom tyrosinase enzyme inhibition assay and in silico comparative molecular docking analysis with Kojic acid

Aryl pyrazoles are well recognized class of heterocyclic compounds found in several commercially available drugs. Owing to their significance in medicinal chemistry, in this current account we have synthesized a series of suitably substituted aryl pyrazole by employing Suzuki cross-coupling reaction. All compounds were evaluated for inhibition of mushroom tyrosinase enzyme both in vitro and in silico. Compound 3f (IC₅₀ = 1.568 ± 0.01 µM) showed relatively better potential compared to reference kojic acid (IC₅₀ = 16.051 ± 1.27 µM). A comparative docking studies showed that compound 3f have maximum binding affinity against mushroom tyrosinase (PDBID: 2Y9X) with binding energy value (−6.90 kcal/mol) as compared to Kojic acid. The 4-methoxy group in compound 3f shows 100% interaction with Cu. Compound 3f displayed hydrogen binding interaction with His61 and His94 at distance of 1.71 and 1.74 Å which might be responsible for higher activity compared to Kojic acid.     

Aspergillus oryzae laeA regulates kojic acid synthesis genes

Kojic acid synthesis genes regulation was investigated in Aspergillus oryzae. Our results indicate that kojic acid production was lost in the laeA disruption strain, but was recovered in the LaeA complement strain. Real-time PCR also confirmed that expression of kojic acid biosynthesis genes decreased in the laeA disruption strain, indicating that these genes are under the control of LaeA.     

Synthesis of tyrosinase inhibitory (4-oxo-4H-pyran-2-yl)acrylic acid ester derivatives

Melanogenesis is a physiological process that results in the production of melanin pigment. However, excessive accumulations of epidermal pigmentation can cause various hyperpigmentary disorders such as, melasma and age spots. Kojic acid and hydroxylated cinnamic acid derivatives are known to inhibit tyrosinase, a key component of melanin biosynthesis. Pyronyl-acrylic acid esters 3a–i, which share structural features of kojic acid and hydroxylated cinnamic acid, were prepared and their abilities to inhibit tyrosinase and melanin production were evaluated. Of the esters synthesized, 3e and 3h, which derived from diethylene glycol moieties were found to inhibit melanin production by ca. 20% at 20 μg/ml, whereas kojic acid at 200 μg/ml inhibited melanin production by 15.8%.     

Multiple effect of hydroxylamine on mushroom tyrosinase

Mushroom tyrosinase is affected by hydroxylamine (NH₂OH) in several ways. At relatively low concentrations (up to 33 mM) NH₂OH shortens the lag period of tyrosine hydroxylation. The o-dihydroxyphenolase activity of mushroom tyrosinase is slightly stimulated by short exposure to relatively low concentrations ofNH₂OH (1.5 mM). Relatively high concentrations ofNH₂OH (above 20 mM) inhibit the o-dihydroxyphenolase activity of the enzyme and lowers the extent of final pigment production. Preincubation of mushroom tyrosinase with different concentrations ofNH₂OH for different times results in the inactivation of the enzyme. The rate of inactivation occurred much faster under anaerobic than under aerobic conditions. It was also found that NH₂OH changes the spectra of o-quinones prepared chemically or of products formed during the oxidation of o-dihydroxyphenols by mushroom tyrosinase. These spectral changes were attributed to the formation of oximes (mono- or dioximes) as a result of an interaction between o-quinones and NH₂OH. The apparent inhibition exerted by NH₂OH on the o-dihydroxyphenolase activity of mushroom tyrosinase is, in part, due to spectral changes in pigmented product formation and, in part, due to the inactivation of the enzyme by NH₂OH.