Raquel Bridi

Antiinflammatory and antinociceptive activities of extracts and isolated compounds from Stachytarpheta cayennensis

The alcoholic and n-butanolic extracts of dried leaves of Stachytarpheta cayennensis (L.C. Rich) Vahl (Verbenaceae) was assessed in antiinflammatory and antinociceptive models. Intraperitoneal pretreatment with the dried extracts at doses ranging from 100 to 200 mg/kg, significantly inhibited carrageenin inducing edema formation. The active extracts were then fractionated and monitored with the same bioassay. The iridoid ipolamiide and the phenylethanoid glycoside acteoside were isolated from the active fraction and showed inhibitory effect on histamine and bradykinin induced contractions of guinea-pig ileum. The compounds also showed in vivo antiinflammatory activity when administered orally to rats mainly in the fourth hour after the administration of the phlogistic agent (70.22% and 93.99%, respectively). These results indicate that S. cayennensis shows antiinflammatory properties which seems to be due, at least partly, to the inhibition of bradyknin and histamine. The extracts also exhibited antinociceptive activity measured by the hot-plate test both i.p. and p.o. in doses ranging from 100 to 300 mg/kg.

Induction of oxidative stress in rat brain by the metabolites accumulating in maple syrup urine disease

Maple syrup urine disease (MSUD) is an inherited disorder caused by deficiency of branched‐chain l‐2‐keto acid dehydrogenase complex activity. Affected patients present severe brain dysfunction manifested as convulsions, coma, psychomotor delay and mental retardation. However, the underlying mechanisms of these neurological findings are virtually unknown. In this study, we tested the in vitro effect of l‐leucine, l‐isoleucine and l‐valine, the amino acids accumulating in MSUD, on the lipid peroxidation parameters chemiluminescence and thiobarbituric acid‐reactive substances (TBA‐RS), as well as on total radical‐trapping antioxidant potential (TRAP) and total antioxidant reactivity (TAR) in cerebral cortex from 30‐day‐old rats. l‐Leucine significantly increased chemiluminescence and TBA‐RS measurements and markedly decreased TRAP and TAR values. l‐Isoleucine increased chemiluminescence and decreased TRAP measurements, but TAR and TBA‐RS levels were not altered by the amino acid. Finally, TRAP measurement was diminished by l‐valine. The results indicate a stimulation of lipid peroxidation and a reduction of brain capacity to efficiently modulate the damage associated with an increased production of free radicals by the branched‐chain amino acids (BCAAs) accumulated in MSUD. It is therefore tempting to speculate that oxidative stress may be implicated in the brain damage found in MSUD patients.

Experimental hyperphenylalaninemia provokes oxidative stress in rat brain

Tissue accumulation of L-phenylalanine (Phe) is the biochemical hallmark of human phenylketonuria (PKU), an inherited metabolic disorder clinically characterized by mental retardation and other neurological features. The mechanisms of brain damage observed in this disorder are poorly understood. In the present study we investigated some oxidative stress parameters in the brain of rats with experimental hyperphenylalaninemia. Chemiluminescence, total radical-trapping antioxidant potential (TRAP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were measured in the brain of the animals. We observed that chemiluminescence is increased and TRAP is reduced in the brain of hyperphenylalaninemic rats. Similar data were obtained in the in vitro experiments using Phe at various concentrations. CAT activity was significantly inhibited by Phe in vitro and in vivo, whereas GSH-Px activity was reduced in vivo but not in vitro and SOD activity was not altered by any treatment. The results indicate that oxidative stress may be involved in the neuropathology of PKU. However, further studies are necessary to confirm and extend our findings to the human condition and also to determine whether an antioxidant therapy may be of benefit to these patients.

α-keto acids accumulating in maple syrup urine disease stimulate lipid peroxidation and reduce antioxidant defences in cerebral cortex from young rats

Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder caused by deficiency of branched-chain α-keto acid dehydrogenase complex activity which leads to tissue accumulation of the branched-chain α-keto acids (BCKAs) α-ketoisocaproic acid (KIC), α-ketoisovaleric acid (KIV) and α-keto-β-methylvaleric acid (KMV) and their respective amino acids. Neuropathologic findings characteristic of the disease are cerebral edema and atrophy, whose pathophysiology is poorly known. In the present study, we investigated the in vitro effect of BCKAs on various parameters of oxidative stress, namely chemiluminescence (CL), thiobarbituric acid–reactive substances (TBA-RS), total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), and the activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in cerebral cortex of 30-day-old rats. The major effects observed were with KIC, which significantly increased CL and TBA-RS measurements, decreased TRAP and TAR values, and markedly inhibited GPx activity. KMV and KIV increased CL and decreased TRAP and TAR values. In contrast, these compounds did not affect CAT and SOD activities. Taken together, it was shown that: the BCKAs studied stimulated lipid peroxidation and reduced the brain antioxidant defences, suggesting an increased production of free radicals. In case the in vitro effects here detected also occur in vivo in MSUD, it can be presumed that oxidative stress might contribute, at least in part, to the brain damage found in the affected patients.

Evaluation of the mechanisms involved in leucine-induced oxidative damage in cerebral cortex of young rats.

Maple syrup urine disease (MSUD) is a metabolic disorder caused by the deficiency of the activity of the mitochondrial enzyme complex branched-chain l-2-keto acid dehydrogenase. The metabolic block results in tissue and body fluid accumulation of the branched-chain amino acids leucine (Leu), isoleucine and valine, as well as of their respective α-keto acids. Neurological sequelae are usually present in MSUD, but the pathophysiologic mechanisms of neurotoxicity are still poorly known. It was previously demonstrated that Leu elicits oxidative stress in rat brain. In the present study we investigated the possible mechanisms involved in Leu-induced oxidative damage. We observed a significant attenuation of Leu-elicited increase of thiobarbituric acid-reactive substances (TBA-RS) measurement when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus trolox, dithiothreitol, glutathione, and superoxide dismutase, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of Nω-nitro-l-arginine methyl ester or catalase (CAT) did not affect TBA-RS values. We also demonstrated an inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and gluthathione peroxidase, as well as a significant reduction in the membrane-protein thiol content from mitochondrial enriched preparations. Furthermore, dichlorofluorescein levels were increased although not significantly by Leu. Taken together, our present data indicate that an unbalance between free radical formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage.

A chemically-induced acute model of maple syrup urine disease in rats for neurochemical studies.

We report a chemically-induced model of maple syrup urine disease (MSUD) in 10- and 30-day-old rats produced by subcutaneous administration of a branched-chain amino acids (BCAA) pool along with the analyses of plasma and brain amino acid levels by HPLC at 0-120 min after administration. We observed an increase of plasma leucine (Leu), isoleucine (Ile) and valine (Val) concentrations in both 10- and 30-day-old rats. These increases were accompanied by a concomitant reduction of plasma concentrations of methionine (Met), phenylalanine (Phe), tyrosine (Tyr), histidine (His), alanine (Ala), lysine (Lys), and ornithine (Orn) in 10-day-old rats and of Met, Phe, Tyr, tryptophan (Trp), and Orn in 30-day-old rats. These results are similar to those observed in MSUD patients during crises, when plasma levels of large neutral amino acids (LNAA) are also reduced when BCAA concentrations are increased. In the brain, increased concentrations of Leu, Ile and Val were achieved in 10-day-old rats at all times after injection. In contrast, no differences in cerebral concentrations of BCAA were observed in 30-day-old rats. In conclusion, the present MSUD model, using 10- rather than 30-day-old rats, has a similar amino acid profile to that of MSUD untreated patients and is suitable to investigate the mechanisms of brain damage characteristic of this disorder.

Essential oils from five southern Brazilian species of Myrcia (Myrtaceae)

Abstract Essential oils from fresh leaves of five Myrcia species collected in the southern region of Brazil were obtained by hydrodistillation and analyzed by GC and GC/MS. Seventy compounds representing 87.5% to 97.0% of the oils were identified. Two samples comprised mainly of monoterpenes with linalool being the most abundant compound in M. acuminatissima oil (22.3%), whereas α- and β-pinenes represented 36.3% of M. bombycina oil. The three other oil samples were found to be rich in sesquiterpenes. The oil of M. fallax contained a high percentage of α-bisabolol (83.8%), while M. glabra oil was characterized by the presence of selinenes (15.2%) and valerianol (13.2%). Finally, the oil of M. multiflora was found to be the most complex with germacrene D (8.7%) and β-caryophyllene (7.5%) being the two major constituents.

The ORAC (oxygen radical absorbance capacity) index does not reflect the capacity of antioxidants to trap peroxyl radicals

In the present work we demonstrate from kinetic studies that under the experimental conditions proposed for the ORAC protocol, ORAC values do not correlate with the capacity of antioxidants to trap peroxyl radicals (ROO˙), suggesting a dominant role of alkoxyl radicals (RO˙) in the assay.

ANTIOXIDANT ACTIVITY IN SOUTHERN BRAZIL HYPERICUM SPECIES

The present study was conducted to assess the antioxidant activity of Hypericum species endemic to South Brazil, H. caprifoliatum, H. carinatum, H. myrianthum and H. polyanthemum. The free radical scavenging properties of plant extracts were evaluated employing different methodologies, including the bleaching of a stable free radical (2,2-diphenyl-1-picrylhydrazyl, DPPH) and the peroxyl reactivity indexes TRAP (Total Reactive Antioxidant Potential) and ORAC-pyrogallol red (Oxygen Radicals Absorbance Capacity). A fair correlation was found between total phenol content determined by Folin-Ciocalteau and DPPH consumption, both in crude methanol and n-hexane extracts. In particular, H. myrianthum and H. caprifoliatum showed the highest TRAP and ORACpyrogallol red values, respectively. This would imply that H. myrianthum contains a larger amount of antioxidants of lower reactivity.

Redox-implications associated with the formation of complexes between copper ions and reduced or oxidized glutathione.

Binding of copper by reduced glutathione (GSH) is generally seen as a mechanism to lower, if not abolish, the otherwise high electrophilicity and redox activity of its free ions. In recent years, however, this concept has been contradicted by new evidence revealing that, rather than stabilizing free copper ions, its binding to GSH leads to the formation of a Cu(I)-[GSH]2 complex capable of reducing molecular oxygen into superoxide. It is now understood that, under conditions leading to the removal of such radicals, the Cu(I)-[GSH]2 complex is readily oxidized into Cu(II)-GSSG. Interestingly, in the presence of a GSH excess, the latter complex is able to regenerate the superoxide-generating capacity of the complex it originated from, opening the possibility that a GSH-dependent interplay exists between the reduced and the oxidized glutathione forms of these copper-complexes. Furthermore, recent evidence obtained from experiments conducted in non-cellular systems and intact mitochondria indicates that the Cu(II)-GSSG complex is also able to function in a catalytic manner as an efficient superoxide dismutating- and catalase-like molecule. Here we review and discuss the most relevant chemical and biological evidence on the formation of the Cu(I)-[GSH]2 and Cu(II)-GSSG complexes and on the potential redox implications associated with their intracellular occurrence.