Oleanolic acid promotes orofacial antinociception in adult zebrafish (Danio rerio) through TRPV1 receptors.

This study aimed to evaluate the antinociceptive effect of oleanolic acid using adult zebrafish models of orofacial pain. Acute nociception was induced by formalin, capsaicin, cinnamaldehyde, menthol, acidified saline or glutamate (cutaneous modes) and hypertonic saline (corneal model). In another set of experiments, animals were pre-treated with naloxone, L-NAME, methylene blue, ketamine, camphor, HC-030031, mefenamic acid, ruthenium red or amiloride to investigate the mechanism of antinociception. The involvement of central afferent C-fibers was also investigated. A molecular docking was performed using the TRPV1 channel. Motor activity was evaluated with the open field test. Pre-treatment with oleanolic acid significantly reduced nociceptive behavior associated with acute pain. Antinociception was effectively inhibited by ruthenium red and capsaicin-induced desensitization. Presence of trpv1 was confirmed by RT-PCR in cerebral tissue of zebrafish. In line with in vivo experiments, docking studies indicated that oleanolic acid may interact with TRPV1. Results confirm the potential pharmacological relevance of oleanolic acid as an inhibitor of orofacial nociception mediated by TRPV1.

Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method.

Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123-128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 µg of polygalacturonic acid, and 45 µg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities.

Ruthenium red-catalyzed degradation of peroxides can prevent mitochondrial oxidative damage induced by either tert-butyl hydroperoxide or inorganic phosphate.

We have recently shown that ruthenium red, a non-competitive inhibitor of the mitochondrial Ca2+ uniporter, can reduce tert-butyl hydroperoxide via a Fenton-type reaction. In respiring mitochondrial preparations containing tert-butyl hydroperoxide, redox cycling of ruthenium red occurs and causes the amplification of methyl radical generation (Meinicke, A. R., Zavan, S. S., Ferreira, A. M. C., Vercesi, A. E., and Bechara, E. J. H. (1996) Arch. Biochem. Biophys. 328, 239-244). In this study we show that ruthenium red can act as an antioxidant preventing mitochondrial damage when the respiratory chain is reduced or when ascorbate is present. Ruthenium red can catalyze the degradation of hydrogen peroxide into H2O and O2. We show here that ruthenium red prevents both accumulation of mitochondrial generated H2O2 and swelling in the presence of the Ca2+ ionophore A23187. Under these conditions the damage induced by Ca2+ ions and either tert-butyl hydroperoxide or inorganic phosphate is promoted by mitochondrial-generated reactive oxygen species. Swelling induced by phenylarsine oxide, a thiol cross-linker, by a mechanism independent of free radicals is not inhibited by ruthenium red. These data provide evidence that the antioxidant behavior of ruthenium red under our conditions is due to its ability to destroy peroxides, which is related to its redox cycling and is prevalent over the Fenton-type reaction.