The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones

The effect of the pH on antioxidant properties of a series of hydroxyflavones was investigated. The pKa of the individual hydroxyl moieties in the hydroxyflavones was compared to computer-calculated deprotonation energies. This resulted in a quantitative structure activity relationship (QSAR), which enables the estimation of pKa values of individual hydroxyl moieties, also in hydroxyflavones for which these pKa values are not available. Comparison of the pKa values to the pH-dependent antioxidant profiles, determined by the TEAC assay, reveals that for various hydroxyflavones the pH-dependent behavior is related to hydroxyl moiety deprotonation, resulting in an increase of the antioxidant potential upon formation of the deprotonated forms. Comparison of these experimental results to computer calculated O-H bond dissociation energies (BDE) and ionization potentials (IP) of the nondeprotonated and the deprotonated forms of the various hydroxyflavones indicates that especially the parameter reflecting the ease of electron donation, i.e., the IP, and not the BDE, is greatly influenced by the deprotonation. Based on these results it is concluded that upon deprotonation the TEAC value increases (radical scavenging capacity increases) because electron-, not H*-, donation becomes easier. Taking into account that the mechanism of radical scavenging antioxidant activity of the neutral form of the hydroxyflavones is generally considered to be hydrogen atom donation, this implies than not only the ease of radical scavenging, but also the mechanism of antioxidant action changes upon hydroxyflavone deprotonation.     

Antioxidant properties of 4-quinolones and structurally related flavones

Neurodegenerative disorders are frequently associated with increased oxidative damage to the brain as a result of free radicals produced by cellular respiration. The onset and progression of neurodegeneration may therefore be curbed by exogenous hydrogen-donating antioxidant moieties such as the naturally occurring flavonoids. A series of 2-phenylquinolin-4(1H)-ones was synthesised and displayed moderate to high antioxidant activity when compared to structurally related flavones and quinolines. Activity of the hydroxy-2-phenylquinolin-4(1H)-ones (8-10) was established in reducing ferrous ions and diminishing hydrogen peroxide and hydroxyl radical production, in the FRAP (1.41-97.71% Trolox equivalents), ORAC (9.18-15.27 μM Trolox equivalents at 0.00 1mM) and TBARS (0.05-0.72 nmol MDA/mg tissue) assays, respectively. The results indicated that the additional hydrogen donating groups on the synthesised 2-phenylquinolin-4(1H)-one series increased antioxidant activity.     

Antioxidant activities of carotenoids: quantitative relationships between theoretical calculations and experimental literature data

Quantitative structure activity relationships (QSARs) are described for the antioxidant activity of series of all-trans carotenoids. The antioxidant activity of the carotenoids is characterised by literature data for (i) their relative ability to scavenge the ABTS^·+ radical cation, reflected by the so-called trolox equivalent antioxidant capacity (TEAC) value, (ii) their relative rate of oxidation by a range of free radicals, or (iii) their capacity to inhibit lipid peroxidation in multilamellar liposomes, leading to a decrease in formation of thiobarbituric acid reactive substances (TBARS). All these antioxidant values for radical scavenging action correlate quantitatively with computer-calculated ionisation potentials of the carotenoids. These correlations are observed both when the ionisation potential is calculated as the negative of the energy of the highest occupied molecular orbital (-E(HOMO)) of the molecule, or as the relative change in heat of formation (ΔΔHF) upon the one-electron oxidation of the carotenoids.

The calculations provide a theoretical assay able to characterise the intrinsic electron donating capacity of an antioxidant, in hydrophilic, hydrophobic or artificial membrane environment.     

The effect of catechol O-methylation on radical scavenging characteristics of quercetin and luteolin--a mechanistic insight

The biological effect of flavonoids can be modulated in  vivo due to metabolism. The O-methylation of the catechol group in the molecule by catechol O-methyl transferase is one of the important metabolic pathways of flavonoids. In the present study, the consequences of catechol O-methylation for the pH-dependent radical scavenging properties of quercetin and luteolin were characterized both experimentally and theoretically. Comparison of the pKa values to the pH-dependent TEAC profiles reveals that O-methylation not only affects the TEAC as such but also modulates the effect of changing pH on this radical scavenging activity due to an effect on the pKa for deprotonation. The pH-dependent TEAC curves and computer calculated electronic parameters: bond dissociation energy (BDE) and ionisation potential (IP) even indicate that O-methylation of the luteolin catechol group affects the radical scavenging potential only because it shifts the pKa for deprotonation. O-Methylation of the quercetin catechol moiety affects radical scavenging capacity by both an effect on the pKa, and also by an effect on the electron and hydrogen atom donating properties of the neutral (N) and the anionic (A) form of the molecule. Moreover, O-methylation of a catechol OH-group in quercetin and luteolin has a similar effect on their TEAC profiles and on calculated parameters as replacement of the OH-group by a hydrogen atom. Altogether, the results presented provide new mechanistic insight in the effect of catechol O-methylation on the radical scavenging characteristics of quercetin and luteolin.     

Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604.

A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-hydroxybenzoate.     

Novel diphenylalkyl piperazine derivatives with high affinities for the dopamine transporter

The novel diphenyl piperazine derivatives containing the phenyl substituted aminopropanol moiety, including 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1, which were modified at the connective between the diphenyl and piperazine moieties, have been found to be potent dopamine uptake inhibitors. To study the further structure-activity relationship (SAR) of these compounds, a new series was synthesized, with modifications at the 2-hydroxy-3-phenylaminopropyl moiety of 1. The series was evaluated for dopamine transporter (DAT) binding affinity with [3H]GBR12935 in rat striatal membranes. Most of the compounds showed moderate to high DAT binding affinities and some were approximately equivalent in activity to compound 1 or GBR12909 as a dopamine uptake inhibitor, with IC(50) values of nanomolar range. The SAR suggested that on exhibiting a potent interaction with the DAT, there is probably a steric limitation around the benzene ring of the phenylamino moiety of 1, allowing only small-sized substituents with the exception of basic moieties at the 4-position. In addition, the SAR at the 3-amino-2-propanol moiety of 1 suggested that either the nitrogen atom with an electron donating substituent or the unsubstituted nitrogen atom and also the hydroxy group are desirable for elicitation of a potent DAT binding affinity.     

Estrogen A-ring structure and antioxidative effect on lipoproteins

The oxidative modification of lipoprotein particles is an important step in atherogenesis. Estrogens are known to be powerful antioxidants independently of their binding to the estrogen receptors and the hormonal functions. We explored the structural determinants for the antioxidant activity of a large number of estrogen derivatives (n=43) in an aqueous lipoprotein solution in vitro by monitoring formation of conjugated dienes. Our results indicate that estrogen derivatives with an unsubstituted A-ring phenolic hydroxyl group with one or two adjacent methoxy groups provide strongest antioxidant protection of low density lipoprotein (LDL) and high density lipoprotein (HDL). The electron donating methoxy groups may enhance the antioxidant effect by weakening the phenolic OH bond and providing stability to the formed phenoxyl radical. With some exceptions, compounds completely lacking unsubstituted hydroxyl groups in the A-ring exhibited no antioxidant effect, e.g. the most hydrophilic "tetrol" compound with three unsubstituted A-ring hydroxyl groups had no antioxidant effect. Moreover, additional hydroxyl groups in the B-, C- or D-ring seemed to weaken the antioxidant effect. Accordingly, both the presence of unsubstituted hydroxyl groups and adjacent substituents, as well as the lipophilicity of the derivatives determine the antioxidant activity of estrogen derivatives in aqueous lipoprotein solutions.     

Enantioselectivity of 3-amino-2-oxazolidinone derivatives with potential psychotropic activity on cellulose tris(4-methylbenzoate) chiral selector

The behavior of a series of 3-amino-2-oxazolidinone derivatives with a potential hypnotic activity on achiral (octadecylsilane) and chiral (cellulose tris(4-methylbenzoate)) stationary phases was examined. The compounds differed in the composition of a substituted aromatic ring containing different substituents in different positions. It was possible to resolve all the compounds with selectivity 1.11 < or = alpha < or = 2.74. The enantiodifferentiating power of substituents was correlated to their electron donating ability and position in the aromatic ring.     

Synthesis and Biological Evaluation of Novel Coumarins with tert‐Butyl and Terpene Substituents

Coumarins with terpene and tert‐butyl substituents were synthesized via Pechmann condensation reaction. New derivatives were investigated in different model system for the exhibition of antioxidant, radical scavenging and membrane‐protective activities. It has been found that 4‐methylcoumarin derivatives with monoterpene moieties exhibit high antioxidant activities. The most active and promising for further investigations is 5‐hydroxy‐6,8‐diisobornyl‐4‐methylcoumarin, containing two isobornyl substituents on the benzopyran ring.     

Antioxidant activity and protective effect on DNA strand scission of Rooibos tea (Aspalathus linearis)

Rooibos tea (Aspalathus linearis) was extracted by refluxing with water and 75% ethanol as a solvent. Antioxidant activity and protective effect on DNA strand scission were investigated by using different antioxidant assay systems and DNA strand nicking assay, respectively. 75% Ethanol extract has higher content of total soluble phenolics and flavonoid than water extract. Antioxidant activities such as hydrogen donating capacity and scavenging activity of hydrogen peroxide were higher in 75% ethanol extract than in water extract except the rate constant with hydroxyl radical. Peroxyl radical induced DNA strand scission was prevented by both 75% ethanol and water extract and hydroxyl radical induced DNA strand scission was not. This result indicates that total soluble phenolics, specially flavonoid, of Rooibos tea are responsible for several kinds of antioxidant activities and preventive activity on peroxyl radical induced DNA strand scission.     

高分子量褐藻多酚抗氧化性质研究

采用DPPH体系、羟基自由基体系、烷基自由基引发的亚油酸氧化体系、超氧阴离子自由基体系对海黍子高分子量褐藻多酚抗氧化性质进行了研究,发现其清除DPPH活性、清除羟基自由基活性、抑制烷基自由基引发的亚油酸氧化的活性分别为77％、27％和21％,但它不具有清除超氧阴离子自由基的能力。褐藻多酚具有供氢能力,其连三羟基的供氢能力大于间三羟基,清除羟基自由基与其苯环提供的羟基自由基加成位点的数量有关。     

Metabolism of 2-, 3- and 4-hydroxybenzoates by soil isolates Alcaligenes sp. strain PPH and Pseudomonas sp. strain PPD

Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3- and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O(2) uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3- and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O(2) uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3- or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.     

Synthesis and radical-scavenging activity of C-methylated fisetin analogues

The radical-scavenging reaction of fisetin, a natural antioxidant found in strawberries, is known to proceed via hydrogen transfer to produce a fisetin radical intermediate. Thus, introduction of an electron-donating group into the fisetin molecule is expected to stabilize the radical, leading to enhanced radical-scavenging activity. In this study, fisetin derivatives in which methyl substituents were introduced at the ortho positions relative to the catechol hydroxyl groups were synthesized and their radical scavenging activities were evaluated and compared with that of the parent fisetin molecule. Among the methyl derivatives, 5′-methyl fisetin, in which the inherent planar structure of fisetin was retained, exhibited the strongest radical scavenging activity. Introduction of methyl substituents may be effective for the enhancement of various biological activities of antioxidants, particularly radical-scavenging activity.     

Antioxidant properties of bucillamine: possible mode of action

The antioxidant properties of Bucillamine (BUC), a di-thiol compound used for treatment of rheumatoid arthritis (RA) and its possible mode of action, were investigated. BUC exhibits potent antioxidant activity similar to those of trolox and ascorbic acid. It reduces the stable free radical diphenyl-2-picrylhydrazyl (DPPH) with IC(50) of 18.5+/-0.1 micromol, its relative antioxidant activity by the ferric reducing ability (FRAP) is 2.07+/-0.01 mM and by the trolox equivalent antioxidant capacity (TEAC), 1.46+/-0.05 mM. However, its superoxide and apparent hydroxyl radical scavenging activities are low (IC(50) at millimolar concentrations). We found that BUC is a strong iron (II) and copper (II) chelator. This finding is very important since these metal ions are significantly higher in RA patients and may be involved in oxidative stress-induced damage. Our study suggests that BUC is a potent antioxidant which exerts its beneficial therapeutic activities in RA patients by metal chelation rather than by scavenging free radical species.     

Concerning the rate limiting step in the hydrolysis of substituted phenyl acetates by acetylcholinesterase

Kinetic parameters of acetylcholinesterase catalyzed hydrolysis of substituted phenyl acetates under the conditions of S ? E and S ? E, were analyzed in terms of the Hammett plot. In both cases, the slope of the line changes from negative for the electron withdrawing substituents to positive for the electron donating substituents. It is suggested that formation of a hydrogen bonded tetrahedral intermediate may be rate limiting in the hydrolysis of some substrates by acetylcholinesterase.     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

Heterocyclic β-keto sulfide derivatives of carvacrol: Synthesis and copper (II) ion reducing capacity

Sixteen β-keto sulfide derivatives of carvacrol (4–19) incorporating phenyl or N, O and S heterocyclic moieties were synthesized in three steps. The relationships between heterocyclic structure and cupric, Cu(II), ion reducing antioxidant capacity (CUPRAC) were examined. Nine of the compounds (8–9 and 13–19) showed better CUPRAC activity than trolox at neutral pH, with trolox equivalent antioxidant capacity (TEAC) coefficients ranging between 1.20 and 1.75. Two derivatives (11–12) showed comparable reducing capacity to trolox, with TEAC values of 0.95 for 11 and 1.02 for 12. Compounds 8–9 and 11–19 were more effective at reducing the Cu(II) ion than ascorbic acid and the parent compound, carvacrol. The most effective antioxidants were those containing an oxadiazole, thiadiazole or triazole moiety. In particular, the methyl thiadiazole derivative (15) had the highest Cu(II) ion reducing capacity, with a TEAC coefficient of 1.73.     

Synthesis and in vitro antioxidant activity study of some new piperazinyl flavone compounds

Flavones exhibit a variety of beneficial effects and are well known for their medicinal importance in several diseases, including cardiovascular, neurodegenerative and cancer. The inclusion of the piperazine ring to the flavone backbone is an important strategy in drug discovery but only a few studies have synthesized piperazinyl flavone compounds to test their biological activity. While there is a major focus on the antioxidant properties of drugs in therapy of several diseases of inflammatory origin, we synthesized a series of the novel piperazinyl flavone analogues bearing the phenyl ring with different substituents. The analogues were evaluated for in vitro antioxidant activity against superoxide anion radical, hydroxyl radical, 2,2‐diphenyl‐1‐picrylhydrazyl radical, and hydrogen peroxide scavenging properties. The total antioxidant status based on the absorbance of the 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) radical cation (ABTS^+?) and total antioxidant capacity using the Fe(III)‐ferrozine complex were also monitored. The results of the above studies showed that the compounds synthesized were found possessed moderate radical scavenging potential, and that their interaction with reactive oxygen species is complex and depends on their structural conformation and the type of substituent R in the piperazine ring being attached. Best antiradical activity were found for the compounds with methoxy groups on the phenyl ring of substituent R, whereas the presence of methoxy or trifluoromethyl groups in substituent R resulted in higher ABTS^+? and ion Fe(III) reduction. These compounds are promising molecules to be used for their antioxidant properties and may be regarded, after improvement of the antioxidant potential, to control diseases of free radical etiology.     

Structure-activity relationship on (+/-)-nantenine derivatives in antiserotonergic activities in rat aorta

A series of nine (+/-)-nantenine derivatives were synthesized and assayed for their pharmacological activities by using tension in aorta and binding experiments in rat brain membrane. Replacing a methyl group with a hydrogen ((+/-)-nornantenine) and an ethyl group at a nitrogen atom ((+/-)-ethylnornantenine) or introducing a hydroxyl group at the alpha/beta position of C-4 or displacement of a methoxy moiety at the C-1 position with a hydroxyl ((+/-)-domesticine) of (+/-)-nantenine decreased the affinity. Moreover, changing a methyl group of (+/-)-domesticine to hydrogen at a nitrogen atom ((+/-)-nordomesticine) caused loss of the activities. These results suggest that a methyl group at a nitrogen atom and a methoxy moiety at C-1 play important roles in the development of the antiserotonergic activity. Molecular modeling analysis of the interaction between the 5-HT2A receptor and (+/-)-nantenine suggested that electron lone pairs of N-6 and of the oxygen atom of the methoxy group at C-1 are important in forming a hydrogen bond to Asp155 and Asn343 of the 5-HT2A receptor, respectively.