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Neem fruit (Azadirachta indica A. Juss.) are popularly used to treat infections, diarrhea, fever, bronchitis, skin diseases, infected burns and hypertension. Although the antinociceptive and anti-inflammatory potential of A. indica has already been investigated in experimental models of pain and inflammation in mice, the current research is the first to report the evaluation of the capacity of A. indica fruit ethanolic extract (EtFrNeem) in acute pain attenuation using the adult zebrafish (Danio rerio) as an alternative model to the use in rodents. EtFrNeem was submitted to antioxidant action, preliminary chemical prospecting, FT-IR and determination of phenol and flavonoid content tests. Subsequently, EtFrNeem was tested for acute nociception and abdominal inflammation, locomotor activity, and acute toxicity in adult zebrafish. Possible neuromodulation mechanisms were also evaluated. EtFrNeem showed low antioxidant activity, but was shown to be rich in flavonoids. EtFrNeem showed no anti-inflammatory action, did not alter the locomotor system, and it was not toxic. However, EtFrNeem significantly reduced the nociceptive behavior induced by formalin, glutamate and acidic saline, when compared to the control group. These effects of EtFrNeem were significantly similar to those of morphine, used as a positive control. The antinociceptive effect of EtFrNeem was inhibited by naloxone, ketamine and amiloride. EtFrNeem has the pharmacological potential for acute pain treatment and this effect is modulated by the opioid system, NMDA receptors and ASICs channels. These results lead us to studies of isolation and characterization of EtFrNeem bioactive principles, using adult zebrafish as an experimental model., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.) more...

A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu 2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and R free values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.) more...