Radical scavenging properties of genistein

The reactivity of genistein toward reactive radical species has been investigated by means of pulse radiolysis. The values of rate constants, respectively 2.3 x 10(10) M(-1)s(-1) and 1.3 x 10(10) M(-1)s(-1) for the reaction with hydroxyl radical at pH 8.3 and 3.0, are close to diffusion limit indicating that genistein is a potent hydroxyl radical scavenger. The reactivity of genistein towards one-electron oxidants has also been investigated. The rate constants k = 4.6 x 10(9) M(-1)s(-1) (pH 8.3) and 6.7 x 10(8) M(-1)s(-1) (pH 7.6) have been determined for the reaction of genistein with *N3 and Br2*- radicals, respectively. For both oxidants the rate constants at pH 3 does not exceed 10(8) M(-1)s(-1). The differences in reactivity of genistein towards the oxidants at different acidity of the solution have been assumed to arise from the acid-base equilibria of genistein. The dissociation constants for genistein (pKa: 7.2, 10.0, and 13.1) have been evaluated spectroscopically. The influence of acid-base equilibria on bond dissociation energy and ionization potential for genistein has also been investigated by means of DFT calculations. It has been concluded on the basis of these calculations that monoanionic form of genistein existing at physiological pH is more powerful radical scavenger than the neutral molecule.     

Radical scavenging properties of polyamines

Di- and polyamines are effective scavengers of free radicals generated in a number of chemical and in vitro enzyme systems. Free radical production was quantified spectrophotometrically using nitroblue tetrazolium and cytochrome c or by electron spin resonance. Levels of superoxide radical formed either enzymatically with xanthine oxidase or chemically from riboflavin or pyrogallol were significantly inhibited by spermine, spermidine, putrescine and cadaverine at 10 and 50 mM. The more reactive hydroxyl radical generated by the Fenton reaction was also effectively scavenged by di- and polyamines. In addition, the production of superoxide radical by senescing microsomal membranes was inhibited by di- and polyamines, as was the superoxide-dependent conversion of 1- aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The efficacy of polyamine-scavenging appears to be correlated with the extent of amination suggesting the involvement of amino groups. It is also apparent that some of the physiological effects of polyamines, in particular their propensity to inhibit lipid peroxidation and retard senescence, may be attributable to their radical-scavenging capability.     

Lipid radicals: properties and detection by spin trapping

Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe2+/LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were similar to those of alkoxyl radical adducts, which were independently synthesized in model experiments using Fe3+-catalyzed nucleophilic addition of methanol or t-butanol to the respective spin trap.     

Radical scavenging and NO-releasing properties of selected beta-adrenoreceptor antagonists

It is claimed that novel beta-adrenolytic drugs possess superior antioxidant properties as compared to classical selective or non-selective beta-adrenoceptor antagonists. Here we tested this notion by analyzing radical scavenging properties of selected beta-adrenolytic drugs and their ability to release nitric oxide in biological preparations. Selective beta1-adrenolytics such as nebivolol, atenolol, metoprolol and non-selective beta-adrenolytics with alpha1-receptor blocking properties such as carvedilol and labetalol were chosen for analysis. NO-releasing properties of nebivolol and carvedilol distinguished third generation beta-adrenolytics from their older counterparts while the reactivity towards hydroxyl and peroxyl radicals discerns only carvedilol but not nebivolol. Thus, superior clinical efficacy of third generation beta-adrenolytics may be related to their ability to release NO rather then to their direct antioxidant properties.     

Novel multi-functional nitrones for treatment of ischemic stroke

Ischemic stroke resulting from obstruction of blood vessels is an enormous public health problem with urgent need for effective therapy. The co-administration of thrombolytic/antiplatelet agent and neuroprotective agent improves therapeutic efficacy and agent possessing both thrombolytic/antiplatelet and antiradical activities provides a promising strategy for the treatment of ischemic stroke. We have previously reported a novel compound, namely TBN, possessing both antiplatelet and antiradical activities, showed significant neuroprotective effect in a rat stroke model. We herein report synthesis of a series of new pyrazine derivatives, and evaluation of their biological activities. Their mechanisms of action were also investigated. Among these new derivatives, compound 21, armed with two nitrone moieties, showed the greatest neuroprotective effects in vitro and in vivo. Compound 21 significantly inhibited ADP-induced platelet aggregation. In a cell free antiradical assay, compound 21 was the most effective agent in scavenging the three most damaging radicals, namely (·)OH, O(2)(·-) and ONOO(-).     

New heteroaryl nitrones with spin trap properties: Identification of a 4-furoxanyl derivative with excellent properties to be used in biological systems

A new series of heteroaryl nitrones, 1–7, bearing furoxanyl and thiadiazolyl moieties, were evaluated for their free radical-trapping properties. The physicochemical characterization by electron paramagnetic resonance (EPR) demonstrated its capability to trap and stabilize oxygen-, carbon-, sulfur-, and nitrogen-centered free radicals. The 4-furoxanyl nitrone 3 (FxBN), α(Z)-(3-methylfuroxan-4-yl)-N-tert-butylnitrone, showed appropriate solubility in aqueous solution and taking into account that this physicochemical property is very important for biological applications, we studied it deeply in terms of its trapping and kinetic behaviors. For this, kinetic studies of the hydroxyl adduct decay gave rate constants k_ST of 1.22 × 10¹⁰ dm³ mol^?1 s^?1 and half-live up to 7200 s at physiological pH, without any artifactual signals. The ability of FxBN to directly traps and stabilizes superoxide free radical, with a half-life of 1620 s at physiological pH, was also demonstrated. Besides, FxBN-hydroxyl and -superoxide adducts exhibited distinct and characteristic EPR spectral patterns. Finally, we confirmed the ability of FxBN to act as spin trap in a specific biological system, that is, in the free radical production of experimental anti-trypanosomatid drugs using Trypanosoma cruzi microsomes as biological system. Moreover, previous observations of low FxBN toxicity transform it in a good candidate for in vivo spin trapping.     

ESR characterization of thallium(III)-mediated nitrones oxidation

We characterized the reactions between Tl (III) and the usual nitrone spin traps (DMPO, POBN, and PBN). The ESR signal obtained with DMPO corresponded to DMPOX which was avoided neither by the enzymes superoxide dismutase or catalase, nor by the hydroxyl radical scavengers’ mannitol, ethanol, or methanol. Only when methanol concentration rose to 90% (v/v), a significant decrease in DMPOX formation was observed while no detectable DMPOX signal was present at 100% methanol solution. POBN rendered an unexpected adduct, while no oxidation products were obtained with PBN. Together, the experimental evidence demonstrates that Tl(III)-supported DMPO oxidation is not mediated by oxygen reactive species. Instead, we propose that Tl(III) interacts with the probe, favoring the addition of water. The intermediate is susceptible to be further oxidized by Tl(III) yielding a keto group. Kinetic studies showed that Tl(III) disappearance rate was higher (1.6 times) than DMPOX formation rate, and equal to Tl(I) formation rate, suggesting a stoichiometry 1.5:1 for Tl(III):DMPOX and 1:1 for Tl(III):Tl(I). The process, an example of the Forrester-Hepburn’s mechanism, was demonstrated for DMPO while the reactions of Tl(III) with POBN and PBN are still under elucidation.     

2-substituted-3H-indol-3-one-1-oxides: preparation and radical trapping properties

A series of 2-alkyl and 2-aryl substituted-3H-indol-3-one-1-oxides was prepared and evaluated for its radical trapping properties. Spin trapping and electron paramagnetic resonance experiments demonstrate the ability of these indolone-1-oxides to trap hetero- and carbon-centered radicals. The most stable spin adducts (lifetime of several hours) are obtained with 2-alkyl substituted nitrones, the 2-ethyl-5,6-dioxolo-3H-indolone-1-oxide, 5e and the 2-secbutyl-3H-indolone-1-oxide, 5f. These two nitrones are also sensitive to redox reactions in solution. Therefore this indolone-1-oxide series lacking a β-hydrogen atom gives rise to highly stable adducts with free radicals.     

The role of the imidazolyl nitrogen atoms of histidine-12 in ribonuclease S.

S-peptide (residues 1--14) analogues in which the active histidine-12 residue is replaced by Npi-methyl-L-histidine, Ntau-methyl-L-histidine and beta-(pyrid-3-yl)-L-alanine were synthesized and tested for their capacity to bind to S-protein and to activate it. The results show that both imidazolyl nitrogen atoms are required for optimal catalytic functioning, Ntau being essential to the catalytic reaction itself, Npi playing a role in keeping the imidazole ring in the correct position.     

The control of the enzymes degrading histidine and related imidazolyl derivatives in Pseudomonas testosteroni

1. The induction of the enzymes for the degradation of l-histidine, imidazolylpropionate and imidazolyl-l-lactate in Pseudomonas testosteroni was investigated. 2. The activities of histidine ammonia-lyase, histidine-2-oxoglutarate aminotransferase and urocanase are consistent with these enzymes being subject to co-ordinate control under most growth conditions. However, a further regulatory mechanism may be superimposed for histidase alone under conditions where degradation of histidine must take place for growth to occur. 3. Experiments with a urocanase(-) mutant show that urocanate is an inducer for the enzymes given above and also for N-formiminoglutamate hydrolyase and N-formylglutamate hydrolase. 4. N-Formiminoglutamate hydrolase and N-formylglutamate hydrolase are also induced by their substrates, and it is suggested that these two enzymes may be different gene products from those expressed in the presence of urocanate. 5. Induction of the enzyme system for the oxidation of imidazolylpropionate is dependent on exposure of cells to this compound.     

PASS-predicted design, synthesis and biological evaluation of cyclic nitrones as nootropics

Out of 400 virtually designed imidazoline N-oxides, five cyclic nitrones were selected on the basis of PASS prediction as potent nootropics and were evaluated for their biological activities in albino mice. The selected N-alkyl and aryl-substituted nitrones were found to be excellent nootropics. A series of lead compounds acting as cognition enhancers have been provided, which can be further exploited in search of such New Chemical Entities (NCEs).     

Immuno-spin trapping of DNA radicals

The detection of DNA radicals by immuno-spin trapping (IST) is based on the trapping of radicals with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), forming stable nitrone adducts that are then detected using an anti-DMPO serum. DNA radicals are very reactive species, and because they are paramagnetic they have previously been detected only by electron spin resonance (ESR) with or without spin trapping, which is not available in most bioresearch laboratories. IST combines the simplicity, reliability, specificity and sensitivity of spin trapping with heterogeneous immunoassays for the detection of DNA radicals, and complements existing methods for the measurement of oxidatively generated DNA damage. Here we have used IST to demonstrate that DMPO traps Cu(II)-H(2)O(2)-induced DNA radicals in situ and in real time, forming DMPO-DNA nitrone adducts, but preventing both 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) formation and DNA fragmentation. We also applied IST to detect DNA radicals in rat hepatocytes exposed to Cu(II) and H(2)O(2) under nonlethal conditions.     

Investigation of 1,3-dipolar cycloaddition reactions of unsaturated nitrones in ionic liquids

The effect of the media (achiral and chiral ionic liquids) on the stereochemistry of intramolecular 1,3-dipolar cycloaddition reactions of D-galactose-derived omega-unsaturated nitrones, leading to bicyclic isoxazolidines, has been investigated.     

2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: evaluation of the spin trapping properties

The 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide (EMPO), an easily prepared pyrroline-N-oxide has been tested as a free radical scavenger. Spin adducts of superoxide, hydroxyl radical, and other free radicals were characterized in phosphate buffer at pH 7.0 and 5.6. At pH 7 in phosphate buffer, the EMPO/O(2)(-*) spin adduct was estimated to be about five times more persistent than its DMPO analogue. Furthermore, its decay does not produce the EMPO/HO&z.rad; adduct.     

Scavenging and antioxidant properties of compounds synthesized by carotenogenic yeasts stressed by heavy metals--EPR spin trapping study

Free radical scavenging and antioxidant activities of metabolites produced by carotenogenic yeasts of Rhodotorula sp. and Sporobolomyces sp. grown under heavy metal presence were studied using various EPR experiments. The thermally initiated decomposition of K(2)S(2)O(8) coupled with EPR spin trapping was shown to be the best choice to characterize antioxidant properties of yeast's samples. EPR spectroscopy revealed that yeast walls showed higher ability to scavenge free radicals than those from inside the cells. Since carotenogenic yeast differ to each other in resistance against the heavy metals due to their individual protective system, quenching properties and antioxidant activities of carotenogenic yeasts were modulated by Ni(2+) or Zn(2+) ions variously.     

Anti-cancer activity of nitrones in the ApcMin/+ model of colorectal cancer

Abstract

The nitrones of α-phenyl-tert-butyl nitrone (PBN) and 4-hydroxyl-PBN (4-OH-PBN) that have anti-cancer activity in models of liver cancer and glioblastomas were tested in the ApcMin/+ mouse model. Mice were administered PBN and 4-OH-PBN in drinking water and intestinal tumour size and number assessed after 3–4 months. Throughout the experiment, contrast-enhanced magnetic resonance imaging (MRI) was used to monitor colon tumours. MRI data showed a time-dependent significant increase in total colonic signal intensity in sham-treated mice, but a significant decrease for PBN-treated mice and slight decrease for 4-OHPBN treated mice, probably due to the limited water solubility of 4-OH-PBN. Final pathological and percentage survival data agreed with the MRI data. PBN had little effect on oxaliplatin-mediated killing of HCT116 colon cancer cells and caused only a slight decrease in the amount of active fraction caspase 3 in oxaliplatin-treated cells. PBN has significant anti-cancer activity in this model of intestinal neoplasia.     

Catalytic action of comicelles containing imidazolyl and hydroxyl groups in the hydrolysis of enantiomeric esters

The rate constants for hydrolysis of the enantiomers of amino acid p-nitrophenyl esters catalyzed by bifunctional comicellar catalysts containing the imidazolyl and hydroxyl groups have been determined at pH 7.30, 0.02 m phosphate buffer, and 25°C. The kinetic analysis suggests a reaction scheme which involves acylation followed by deacylation at the imidazolyl group. Although no appreciable cooperative catalytic efficiencies are observed between the bifunctional groups in the acylation step, it is found that the deacylation rates are thus accelerated by surfactant hydroxyl groups, and some of the stereoselective acyl transfer reaction occurs from the imidazolyl to the hydroxyl group in optically active comicellar systems.