Selective growth inhibitor toward human intestinal bacteria derived from Pulsatilla cernua root

Among 21 medicinal plants, the growth-inhibiting activity of Pulsatilla cernua root-derived materials toward human intestinal bacteria was examined by using an impregnated paper disk method. The biologically active components of P. cernua roots were characterized as 4-hydroxy-3-methoxycinnamic acid and 3,4-dihydroxycinnamic acid by spectroscopic analysis. The activity was compared with that of six commercially available cinnamic acid derivatives trans-cinnamaldehyde, trans-cinnamic acid, cinnamyl alcohol, 2-methoxycinnamic acid, 3-methoxycinnamic acid, and 4-methoxycinnamic acid. The growth responses varied with each bacterial strain tested. Two isolated compounds revealed a potent inhibition against Clostridium perfringens, and moderate to weak activity against Escherichia coli was exhibited by 4-hydroxy-3-methoxycinnamic acid. Weak or no inhibitory activity was obtained against the bifidobacteria or Lactobacillus acidophilus. The inhibitory effect was much more pronounced in C. perfringens and E. coli as compared to B. adolescentis, B. bifidum, B. fragilis, B. longum, or L. acidophilus. Cinnamaldehyde exhibited a strong growth-inhibiting activity, but no inhibition was observed from treatments with trans-cinnamic acid, cinnamyl alcohol, 2-methoxycinnamic acid, 3-methoxycinnamic acid, and 4-methoxycinnamic acid. These results may be an indication of at least one of the pharmacological actions of P. cernua root.     

Competitive inhibition of mushroom tyrosinase by 4-substituted benzaldehydes

A kinetic study of the inhibition of mushroom tyrosinase by 4-substituted benzaldehydes showed that these compounds behave as classical competitive inhibitors, inhibiting the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) by mushroom tyrosinase (o-diphenolase activity). The kinetic parameter (K(I)) characterizing this inhibition was evaluated for all of the seven compounds assayed. Cuminaldehyde showed the most potent inhibitory activity (K(I) = 9 microM). It also inhibited the oxidation of L-tyrosine by mushroom tyrosinase (o-monophenolase activity) in a competitive manner. The corresponding kinetic parameter for this inhibition was evaluated (K(I) = 0.12 mM).     

Solvent-free lipase-catalyzed preparation of long-chain alkyl phenylpropanoates and phenylpropyl alkanoates

An enzymatic method was developed for the preparation of medium- or long-chain alkyl 3-phenylpropenoates (alkyl cinnamates), particularly alkyl hydroxy- and methoxy-substituted cinnamates such as oleyl p-coumarate and oleyl ferulate. The various alkyl cinnamates were formed in high to moderate yield by lipase-catalyzed esterification of cinnamic acid and its analogues with fatty alcohols in vacuo at moderate temperatures in the absence of drying agents and solvents. Immobilized Candida antarctica lipase B was the most effective biocatalyst for the various esterification reactions. The relative esterification activities were of the following order: dihydrocinnamic > cinnamic > 3-methoxycinnamic > dihydrocaffeic approximately 3-hydroxycinnamic > 4-methoxycinnamic > 2-methoxycinnamic > 4-hydroxycinnamic > ferulic approximately 3,4-dimethoxycinnamic > 2-hydroxycinnamic acid. With respect to the position of the substituents at the phenyl moiety, the esterification activity increased in the order meta > para > ortho. Rhizomucor miehei lipase demonstrated moderate esterification activity. Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-hydroxyphenyl)propyl oleate, were also obtained in high yield by esterification of fatty acids with the corresponding 3-phenylpropan-1-ols.     

Flavonols from saffron flower: tyrosinase inhibitory activity and inhibition mechanism.

A common flavonol, kaempferol, isolated from the fresh flower petals of Crocus sativus L. (Iridaceae) was found to inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an ID(50) of 67 microgram/mL (0.23 mM). Interestingly, its 3-O-glycoside derivatives did not inhibit this oxidation. The inhibition kinetics analyzed by a Lineweaver-Burk plot found kaempferol to be a competitive inhibitor, and this inhibitory activity presumably comes from its ability to chelate copper in the enzyme. This copper chelation mechanism can be applicable for all of the flavonols as long as their 3-hydroxyl group is free. However, quercetin, kaempferol, and galangin each affect the oxidation of L-tyrosine in somewhat different ways.     

Tyrosinase inhibitory activity of citrus essential oils

Thirteen kinds of citrus essential oils and their volatile flavor constituents were investigated for tyrosinase inhibitory activity. Eureka lemon, Lisbon lemon, Keraji, and Kiyookadaidai significantly inhibited the oxidation of L-dihydroxy phenylalanine (L-DOPA) by mushroom tyrosinase. Citral and myrcene among volatile flavor constituents of citrus essential oils exhibited tyrosinase inhibitory activities with Ki values of 0.318 and 2.38 mM, respectively. The inhibition kinetics analyzed by a Lineweaver-Burk plot indicated that citral is a noncompetitive inhibitor and myrcene is a competitive inhibitor. These results indicated that citral and myrcene are responsible for the tyrosinase inhibitory activity of citrus essential oils.     

Kinetics of mushroom tyrosinase inhibition by quercetin

The effects of quercetin on the activity of mushroom tyrosinase were studied. The equilibrium constants for this inhibitor binding with the enzyme molecule were established. The inhibition mechanism obtained from Lineweaver-Burk plots show that quercetin is a competitive inhibitor. In the time course of the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by the enzyme in the presence of different concentrations of quercetin, the rate decreased with increasing time until a straight line was approached. The inhibition of tyrosinase by quercetin is a slow and reversible reaction with residual enzyme activity. The microscopic rate constants were determined for the reaction of quercetin with the enzyme.     

Molecular design of antibrowning agents

Tyrosinase inhibitory and antioxidant activity of gallic acid and its series of alkyl chain esters were investigated. All inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase. However, gallic acid and its short alkyl chain esters were oxidized as substrates yielding the colored oxidation products. In contrast, the long alkyl chain esters inhibited the enzyme activity without being oxidized. This indicates that the carbon chain length is associated with their tyrosinase inhibitory activity, presumably by interacting with the hydrophobic protein pocket in the enzyme. On the other hand, the esters, regardless their carbon chain length, showed potent scavenging activity on the autoxidation of linoleic acid and 1,1-diphenyl-2-p-picryhydrazyl (DPPH) radical, suggesting that the alkyl chain length is not related to the activity. The effects of side-chain length of gallates in relation to their antibrowning activity are studied.     

Isolation and tyrosinase inhibitory effects of polyphenols from the leaves of persimmon, Diospyros kaki

The main polyphenols were isolated from the leaves of six selected persimmon cultivars. Seven compounds were obtained by reverse-phase HPLC, and their structures were elucidated by multiple NMR measurements. These compounds are hyperoside, isoquercitrin, trifolin, astragalin, chrysontemin, quercetin-3-O-(2'-O-galloyl-β-D-glucopyranoside) (QOG), and kaempferol-3-O-(2'-O-galloyl-β-D-glucopyranoside) (KOG). Their inhibitory activity was tested against tyrosinase for the oxidation of L-DOPA, and only chrysontemin showed inhibitory activity. To investigate the differences of their inhibitory effects, the tyrosinase inhibitory activities of their aglycons, cyanidin, quercetin, and kaempferol, were also tested. As a result, it was confirmed that the most influential moiety for tyrosinase inhibition was the 3',4'-dihydroxy groups of the catechol moiety. Moreover, the tyrosinase inhibitory activity of chrysontemin, which was identified in persimmon leaves for the first time, is supported by a simulated model of chrysontemin docking into mushroom tyrosinase.     

Inhibitory effect of red koji extracts on mushroom tyrosinase

Red koji has been recognized as a cholesterol-lowering diet supplement because of it contains fungi metabolites, monacolins, which reduce cholesterol synthesis by inhibiting HMG-CoA reductase. In this study, water extracts of red koji were loaded onto a C(18) cartridge, and the acetonitrile eluate was collected as test fraction. Red koji water extracts and its C(18) cartridge acetonitrile eluent had total phenols concentrations of 5.57 and 1.89 mg/g of red koji and condensed tannins concentrations of 2.71 and 1.20 mg/g of red koji, respectively. Both exhibited an antioxidant activity and an inhibitory activity to mushroom tyrosinase. The higher antioxidant activity of the red koji acetonitrile eluent was due to the existence of a high percentage of condensed tannins. The results from the kinetic study for inhibition of mushroom tyrosinase by red koji extracts showed that the compounds in the extracts competitively inhibited the oxidation of tyrosine catalyzed by mushroom tyrosinase with an ID(50) of 5.57 mg/mL.     

Inhibitory effects of (S)- and (R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acids on tyrosinase activity

The inhibition of (R)-, (S)-, and (+/-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acids (HTCCA) on mushroom tyrosinase was evaluated. All HTCCAs inhibited the tyrosinase activity. The ID(50) values were 1.88, 1.84, and 1.88 for the (R)-, (S)-, and (+/-)-HTCCAs, respectively. The inhibition kinetics analyzed by Hanes-Woolf plots indicated that both (R)- and (S)-HTCCAs are competitive inhibitors of the tyrosinase, with K(i) values of 0.83 and 0.61 mM, respectively. Dimethyl sulfoxide (DMSO) was also tested for its direct inhibitory activity against the tyrosinase and its potential influence on the tyrosinase inhibitory effects of (R)- and (S)-HTCCAs. DMSO, a widely used solvent for tyrosinase inhibitors, was found to dose-dependently inhibit the tyrosinase activity. Addition of DMSO in a tyrosinase digest containing either (R)- or (S)-HTCCA further dose-dependently reduced the tyrosinase activity. These data indicated a potential to use a HTCCA as a tyrosinase inhibitor in food, cosmetic, and medicinal products and a need to improve the solvent system for the studies of tyrosinase inhibitions.     

Effects of mushroom tyrosinase on anisaldehyde

Anisaldehyde (p-methoxybenzaldehyde) was previously reported to inhibit the tyrosinase-catalyzed oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) noncompetitively as long as the enzyme activity was monitored by measuring dopachrome formation. However, anisaldehyde did not inhibit this oxidation if a longer reaction time was observed, although it suppressed the initial rate of oxidation to a certain extent. Anisaldehyde significantly suppressed the rate of enzymatic oxidation of L-tyrosine.     

Two potent suicide substrates of mushroom tyrosinase: 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone

The inhibitory characteristics of two isoflavone metabolites, 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone, on mushroom tyrosinase were investigated. The two isoflavones were isolated from soygerm koji and inhibited both monophenolase and diphenolase activities of tyrosinase. Their inhibition type was demonstrated to be irreversible inhibition by preincubation and recovery experiments. By using HPLC analysis, it was found that mushroom tyrosinase could catalyze the two isoflavones. These results revealed that the two isoflavones belonged to suicide substrates of mushroom tyrosinase. The partition ratios between molecules of suicide substrate in the formation of product and in the inactivation of enzyme were determined to be 81.7 +/- 5.9 and 35.5 +/- 3.8 for 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone, respectively. From kinetic studies, maximal inactivation rate constants and Michaelis constants were 0.79 +/- 0.08 and 1.01 +/- 0.04 min(-1) and 18.7 +/- 2.31 and 7.81 +/- 0.05 microM for 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone, respectively, when L-DOPA was used as the enzyme substrate. Structure analysis comparing the inactivating activity between the two isoflavones and their structure analogues showed that not only the 7,8-dihydroxyl groups but also the isoflavone skeleton of the two isoflavones played an important role in inactivating tyrosinase activity. The present study demonstrated that 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone are potent suicide substrates of mushroom tyrosinase.     

Constituents of the leaves of Peucedanum japonicum Thunb. and their biological activity

In the course of our study on the isolation and structure determination of constituents in tropical plants, we focused on Peucedanum japonicum Thunb., belonging to the family Umbelliferae. In this study, a new C(13) norisoprenoid glucoside, (3S)-O-beta-d-glucopyranosyl-6-[3-oxo-(2S)-butenylidenyl]-1,1,5-trimethylcyclohexan-(5R)-ol (1), and two new phenylpropanoid glucosides, 3-(2-O-beta-d-glucopyranosyl-4-hydroxyphenyl)propanoic acid (3) and methyl 3-(2-O-beta-d-glucopyranosyl-4-hydroxyphenyl)propanoate (4), were isolated from the n-butanol soluble fraction of this plant's leaves, together with five known compounds. The structures of these compounds were determined on the basis of spectroscopic evidence. In addition, all isolated compounds were examined for scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radical and inhibitory activity against mushroom tyrosinase. These results suggested that 2-(4-hydroxy-3-methoxyphenyl)propane-1,3-diol (7) and 3-O-beta-d-glucopyranosyl-2-(4-hydroxy-3-methoxyphenyl)propanol (8) showed an appreciable activity in both assay systems.     

Inhibition of enzymatic browning of chlorogenic acid by sulfur-containing compounds

The antibrowning activity of sodium hydrogen sulfite (NaHSO(3)) was compared to that of other sulfur-containing compounds. Inhibition of enzymatic browning was investigated using a model browning system consisting of mushroom tyrosinase and chlorogenic acid (5-CQA). Development of brown color (spectral analysis), oxygen consumption, and reaction product formation (RP-UHPLC-PDA-MS) were monitored in time. It was found that the compounds showing antibrowning activity either prevented browning by forming colorless addition products with o-quinones of 5-CQA (NaHSO(3), cysteine, and glutathione) or inhibiting the enzymatic activity of tyrosinase (NaHSO(3) and dithiothreitol). NaHSO(3) was different from the other sulfur-containing compounds investigated, because it showed a dual inhibitory effect on browning. Initial browning was prevented by trapping the o-quinones formed in colorless addition products (sulfochlorogenic acid), while at the same time, tyrosinase activity was inhibited in a time-dependent way, as shown by pre-incubation experiments of tyrosinase with NaHSO(3). Furthermore, it was demonstrated that sulfochlorogenic and cysteinylchlorogenic acids were not inhibitors of mushroom tyrosinase.     

2-羟基-4-甲氧基苯甲醛和2-羟基-4-甲氧基苯甲酸对菜粉蝶多酚氧化酶的抑制作用

测定了2羟-基-4-甲氧基苯甲醛和2羟-基-4-甲氧基苯甲酸两种效应物对菜粉蝶多酚氧化酶(po lypheno lox idase,简称PPO,EC.1.14.18.1)催化L-多巴(L-DOPA)氧化活力的抑制作用。结果表明,这两种效应物对该酶的活性均有明显的抑制作用,抑制中浓度(IC50)分别为2.71和5.66 mm o l/L,抑制常数分别为2.82和3.35 mm o l/L;2羟-基-4-甲氧基苯甲醛对该酶的抑制作用显著高于2羟-基-4-甲氧基苯甲酸的;这两种抑制剂对酶的抑制作用机理完全不同,2羟-基-4-甲氧基苯甲醛对酶的作用表现为混合型抑制,而2羟-基-4-甲氧基苯甲酸对酶的作用表现为竞争性抑制。研究结果为设计以该酶为靶标的杀虫剂提供理论依据。     

Effects of thymol on mushroom tyrosinase-catalyzed melanin formation

The novel inhibitory mechanism of thymol (2-isopropyl-5-methylphenol) on dopachrome formation by mushroom tyrosinase (EC 1.14.18.1) was identified. The UV-vis spectrum and oxygen consumption assays showed dopachrome formation using L-tyrosine as a substrate was suppressed by thymol. This inhibitory activity was reversed by the addition of a well-known radical scavenger, butylated hydroxyanisole (BHA). Further investigations using N-acetyl-L-tyrosine as a substrate with HPLC analysis suggested that thymol inhibits chemical redox reactions between dopaquinone and leukodopachrome instead of enzymatic reaction. This redox inhibitory activity of thymol was examined by using a model redox reaction with L-dihydroxyphenylalanine (L-DOPA) and p-benzoquinone. Thymol successfully inhibited oxidation of L-DOPA to dopaquinone, coupled with reduction of p-benzoquinone. Hence, the suppression of dopachrome formation by thymol is due to the inhibition of conversion of leukodopachrome to dopachrome. The antioxidant property of thymol is a key characteristic for the inhibitory mechanism of melanin synthesis.     

Tyrosinase inhibitory activity of the olive oil flavor compounds.

A series of alpha,beta-unsaturated aldehydes, otherwise known as (2E)-alkenals, characterized from the olive Olea europaea L. (Oleaceae) oil flavor was found to inhibit the oxidation of L-3, 4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase, and the inhibition kinetics analyzed by a Lineweaver-Burk plot found that they are noncompetitive inhibitors. The inhibition mechanism presumably comes from their ability to form a Schiff base with a primary amino group in the enzyme. In addition, the hydrophobic alkyl chain length from the hydrophilic enal group seems to relate to their affinity to the enzyme, and this results in their inhibitory potency.     

Structure-antifungal activity relationship of cinnamic acid derivatives

A structure-antifungal activity relationship (SAR) study of 22 related cinnamic acid derivatives was carried out. Attention was focused on the antifungal activities exhibited against Aspergillus flavus, Aspergillus terreus, and Aspergillus niger. (E)-3-(4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid (16) exhibited antifungal activity against A. niger, comparable to that of miconazole and a significant antifungal effect against A. flavus and A. terreus as well. A structure-activity relationship (SAR) study of related cinnamic acid derivatives has allowed a model to be proposed for the recognition of the minimal structural requirements for the antifungal effect in this series.     

Tyrosinase inhibitor from black rice bran

The inhibitor of tyrosinase activity in black rice bran was investigated. The methanol extract from black rice bran was re-extracted with hexane, chloroform, ethyl acetate, or water. The ethyl acetate extract had the most potent inhibition against tyrosinase activity by 80.5% at a concentration of 0.4 mg/mL. Inhibitory compound in the ethyl acetate fraction was isolated by silica gel column chromatography, and identified as protocatechuic acid methyl ester (compound 1) by GC, GC-MS, IR, and 1H and 13C NMR spectroscopy. Compound 1 inhibited 75.4% of tyrosinase activity at a concentration of 0.50 micromol/mL. ID(50) (50% inhibition dose) value of compound 1 was 0.28 micromol/mL. To study the structure-activity relationship, protocatechuic acid (2), vanillic acid (3), vanillic acid methyl ester (4), isovanillic acid (5), isovanillic acid methyl ester (6), veratric acid (7), and veratric acid methyl ester (8) were also assayed.     

Kinetics of activation of latent mushroom (Agaricus bisporus) tyrosinase by benzyl alcohol.

A latent isoform of Agaricus bisporus tyrosinase has been isolated and activated by benzyl alcohol, one of the major volatile compounds in mushrooms of this genus. The progress curve that describes the activation process reached the steady-state rate (V(ss)) after a lag period (tau). The rate of active tyrosinase formation was calculated by coupling the oxidation of o-diphenols to the activation process. V(ss) depended on benzyl alcohol, o-diphenol, and latent tyrosinase concentrations. The lag period depended on benzyl alcohol concentrations but not on o-diphenol and enzyme concentrations. The size of the latent mushroom tyrosinase was 67 kDa, determined by SDS-PAGE and Western blotting assays. This size was not modified after activation by benzyl alcohol. The presence of a lag period and the lack of change of the molecular mass of the protein after activation could indicate a slow conformational change of the protein to render the final active form. The values of the kinetic constants V(max) and K(m) on the o-diphenols 4-tert-butylcatechol, L-DOPA, and dopamine were different between the latent tyrosinase activated by benzyl alcohol and the commercial tyrosinase. They might indicate that a different final active tyrosinase, depending on the activator used, could arise.