Erythrinaline alkaloids from the flowers and pods of Erythrina lysistemon and their DPPH radical scavenging properties

Fourteen different erythrinaline alkaloids have been isolated from the flowers and pods of Erythrina lysistemon with four being reported for the first time in nature and five for the first time in this species and the rest having been re-isolated. The new compounds are (+)-11beta-hydroxyerysotramidine (1), (+)-11beta-methoxyerysotramidine (2), (+)-11beta-hydroxyerysotrine N-oxide (4) and (+)-11beta-hydroxyerysotrine (8). (+)-11alpha-Hydroxyerysotrine N-oxide (3), earlier misidentified as erythrartine N-oxide (beta-hydroxyerysotrine N-oxide 4), was also re-isolated along with four other alkaloids. Correct identification of compounds 4 and 8 was aided by the fact that the two sets of C-11 epimers 3, 4 and 8, 9 were both isolated in this study thus making it easier to identify and assign the individual epimers. (+)-Erythristemine (14) was found distributed in most of the plant parts investigated. Preliminary work on the crude chloroform/methanol (1:1) showed moderate toxicity to brine shrimp (LC50 23 ppm) and moderate (IC50 86 microg/ml) radical scavenging properties against stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The DPPH radical scavenging properties of the isolated compounds were assessed using TLC autographic and spectrophotometric assays whereupon only compounds 11 (1 microg; 90 microg/ml) and 12 (0.1 microg; 160 microg/ml) showed any notable activity. It appears the two compounds are slow reacting and do not reach steady state conditions within the standard half an hour time frame but only seemed to have reached steady state conditions after 4 h.     

The effect of pH on the oxidation of bovine serum albumin by hypervalent myoglobin species

Bovine serum albumin (BSA) was used as a probe for the oxidation of proteins by hypervalent myoglobin species in solutions with pH from 5.3 to 7.7. The reaction between perferrylmyoglobin, *MbFe(IV)=O, and BSA was studied by activating metmyoglobin with equimolar amounts of hydrogen peroxide in the presence of BSA. A minor pH dependence was observed as judged from the formation of BSA-centered radicals, which were monitored at room temperature by electron spin resonance spectroscopy, and the formation of dityrosine. The reaction between ferrylmyoglobin, MbFe(IV)=O, and BSA was pH-dependent. BSA-centered radicals and dityrosine were formed in low levels at neutral pH and increased at low pH to the same levels as observed in the reaction of *MbFe(IV)=O with BSA. The present results demonstrate that protein-centered radicals can be formed from the non-radical MbFe(IV)=O under mildly acidic conditions, and this should be taken into account when considering oxidation in cellular compartments of low pH and in meat-related products.     

Ethanolamine plasmalogen and cholesterol reduce the total membrane oxidizability measured by the oxygen uptake method

To investigate the effects of ethanolamine plasmalogen, phosphatidylethanolamine, cholesterol, and alpha-tocopherol on the oxidizability of membranes, various large unilamellar vesicles (LUVs) including these lipids and antioxidant were examined for their total membrane oxidizabilities, evaluated as R(p)/R(i)(1/2) value (where R(p) is rate of oxygen consumption and R(i)(1/2) is the square root of rate of chain initiation) by the oxygen uptake method with water-soluble radical initiator and inhibitor. Incorporation of bovine brain ethanolamine plasmalogen (BBEP) into vesicles as well as cholesterol led to lower the total membrane oxidizability dose-dependently. The effect of BBEP was more efficient in the presence of cholesterol in vesicles. On the other hand, diacyl counterpart, egg yolk phosphatidylethanolamine, and a typical radical scavenger, alpha-tocopherol, had no effect on the membrane oxidizability. Alpha-tocopherol only prolonged an induction period dose-dependently in the present oxidizing system, suggesting a novel antioxidant mechanism of ethanolamine plasmalogens besides the action of scavenging radicals.     

Aminoacetone induces loss of ferritin ferroxidase and iron uptake activities

Aminoacetone (AA) is a threonine and glycine metabolite overproduced and recently implicated as a contributing source of methylglyoxal (MG) in conditions of ketosis. Oxidation of AA to MG, NH4+, and H

2

O

2

has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by copper- and iron ion-catalyzed reactions with oxygen. We previously demonstrated that AA-generated O2•al (AA

•

) induce dose-dependent Fe(II) release from horse spleen ferritin (HoSF); no reaction occurs under nitrogen. In the present study we further explored the mechanism of iron release and the effect of AA on the ferritin apoprotein. Iron chelators such as EDTA, ATP and citrate, and phosphate accelerated AA-promoted iron release from HoSF, which was faster in horse spleen isoferritins containing larger amounts of phosphate in the core. Incubation of apoferritin with AA (2.5-50 mM, after 6 h) changes the apoprotein electrophoretic behavior, suggesting a structural modification of the apoprotein by AA-generated ROS. Superoxide dismutase (SOD) was able to partially protect apoferritin from structural modification whereas catalase, ethanol, and mannitol were ineffective in protection. Incubation of apoferritin with AA (1-10 mM) produced a dose-dependent decrease in tryptophan fluorescence (13-30%, after 5 h), and a partial depletion of protein thiols (29% after 24 h). The AA promoted damage to apoferritin produced a 40% decrease in apoprotein ferroxidase activity and an 80% decrease in its iron uptake ability. The current findings of changes in ferritin and apoferritin may contribute to intracellular iron-induced oxidative stress during AA formation in ketosis and diabetes mellitus.     

Oxidative stress,mitochondrial dysfunction,and epilepsy

Epilepsy is a common and heterogeneous neurological disorder arising from biochemical and molecular events that are incompletely understood. To effectively manage epilepsies, it is important to understand the mechanisms underlying both seizure-induced brain damage as well as seizure initiation. Oxidative stress is emerging as a mechanism that may play an important role in the etiology of seizure-induced neuronal death. Conversely, epileptic seizures are a common occurrence in mitochondrial diseases arising from defects in oxidative phosphorylation. This review focuses on the emerging role of oxidative stress and mitochondrial dysfunction both as a consequence and cause of epileptic seizures.     

Oxidative deamination by hydrogen peroxide in the presence of metals

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H 2 O 2 /Cu 2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O 2 on the oxidative deamination of benzylamine by H 2 O 2 . The formation of benzaldehyde was the greatest with Cu 2+ , and catalysis occurred with Co 2+ , VO 2+ , and Fe 3+ . The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O 2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O 2 /Cu 2+ and hemoglobin/H 2 O 2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H 2 O 2 /Cu 2+ system is discussed.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P_M, F and F) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F but when F is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P_M is produced at high pH with CO/O₂, no EPR-detectable radicals are formed. Conversion of the CO/O₂-generated P_M into F when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F determined optically. It is concluded that low pH causes a change of protonation pattern in P_M which induces partial electron redistribution and tryptophan cation radical formation in F. These protonation changes may mimic a key step of the proton translocation process.     

The mediation of veratryl alcohol in oxidations promoted by lignin peroxidase: the lifetime of veratryl alcohol radical cation

The kinetics of decay of veratryl alcohol radical cation, generated by cerium(IV) ammonium nitrate induced oxidation of veratryl alcohol, have been followed spectrophotometrically in a stopped-flow apparatus. In acidic aqueous acetonitrile the radical cation was found to decay by a first-order process, due to deprotonation from the alpha-carbon leading to an alpha-hydroxybenzyl radical with the rate constant of 17.1+/-0.5 s(-1). This value is in full agreement with those obtained by pulse radiolysis studies but much lower than the value (1.2x10(3) s(-1)) indirectly determined by EPR experiments. The implications of these results with respect to the possible role of veratryl alcohol as a mediator in the oxidative biodegradation of lignin catalysed by lignin peroxidase are discussed.     

Stereoselective synthesis of a C-glycosylic compound (a "methyl C-glycoside") through a regioselective free-radical ring-opening reaction. A single-crystal X-ray structure determination

Readily available 3,4,6-tri-O-acetyl-D-glucal was converted to 2,6-anhydro-5,7-O-benzylidene-1,3,4-trideoxy-D-arabino-hept-3-enitol, a methyl C-glycosylic compound. Cyclopropanation of 4,6-O-benzylidene-D-glucal, followed by tributylstannyl radical-mediated regioselective ring opening of the 1,2-cyclopropano sugar led to a 2,6-anhydro-1-deoxyheptose, (a "methyl C-beta-D-glycoside"). The stereochemistry of the 1,2-cyclopropano sugar and the "methyl C-glycoside" were confirmed by single-crystal X-ray diffraction studies.     

Interaction between sodium ascorbate and dopamine

The interaction between sodium ascorbate and dopamine was investigated by three different parameters: radical intensity, prooxidant action, and cytotoxicity induction. Sodium ascorbate and dopamine produced the doublet and quartet ESR signals under alkaline conditions (pH 8.0–9.5), respectively. Addition of increasing concentrations of sodium ascorbate completely scavenged the dopamine radical and replaced the latter with its own radical. Similarly, dopamine slightly, but significantly reduced the radical intensity of sodium ascorbate. These two compounds stimulated the methionine oxidation and hydrogen peroxide generation in culture medium, but in combination, their stimulation activities were weakened. Both of these two compounds dose-dependently reduced the viable cell number of human oral squamous carcinoma HSC-4 cells, and their cytotoxic activity was significantly reduced by catalase. When these two compounds were mixed together before adding to HSC-4 cells, both of their cytotoxic activities were diminished. The present study demonstrates the interaction between sodium ascorbate and dopamine, which might modify their biological activities and generation of nerve disorders such as Parkinson’s disease.