The objectives of this work were to carry out a comparative chemical study and to evaluate the antinociceptive and anti-inflammatory activities of ethanol extracts (EtOHE) and vanilic acid (VA) from cultivated and wild Amburana cearensis A.C. Smith (Fabaceae), an endangered species used in Northeast Brazil for the treatment of asthma. The HPLC analysis of EtOHE, showed that coumarin (CM) and VA were the major constituents from the cultivated plant, while in the extract from the wild plant the major constituents were amburoside A (AMB) and CM. Pharmacological tests were performed with male Swiss mice or male Wistar rats acutely administered with 100-400mg/kg, p.o. of EtOHEs or 12.5-50mg/kg, p.o. of VA. EtOHEs from A. cearensis with 4, 7 or 9 months of cultivation significantly inhibited, from 32 to 64%, both phases of the formalin test in mice. Similar results were observed with the EtOHE from the wild species. VA significantly reduced both phases of the formalin test. This effect was partially reversed by naloxone. EtOHE from cultivated or wild A. cearensis inhibited the carrageenan (Cg)-induced mice paw edema. Furthermore, VA inhibited the paw edema and the leukocyte migration in rat peritoneal cavity induced by Cg. On the other hand, it did not inhibit the edema and the increase of vascular permeability induced by dextran in the rat paw. All together, these results indicate that the EtOHE from cultivated A. cearensis exhibit similar chemical and pharmacological profiles, as related to the wild plant. VA is, at least partially, responsible for these pharmacological effects. Its antinociceptive effect occurs by a mechanism partly dependent upon the opioid system, while the anti-inflammatory action was manifested in inflammatory processes dependent on polymorphonuclear cells and are probably related to the VA inhibition of cytokines as observed by others.

The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMPα-II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMPα-II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMPα-II cleaves Aα-chain of fibrinogen followed by Bβ-chain, and does not show any effect on the γ-chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30–50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMPα-II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMPα-II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMPα-II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders.

Abstract:A myotoxin phospholipase A 2 homologue, BmooMtx, was isolated from the venom of Bothrops moojeni by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. SDS-PAGE showed the enzyme to be a monomer with a molecular weight of 16,500. BmooMtx induced release of creatine kinase and morphological analyses indicated that it provoked an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 hours after injection. Anti-BmooMTx antibodies partially neutralized the myotoxic activity of BmooMtx and crude B. moojeni venom, as judged by determination of plasma creatine kinase levels and histological evaluation of skeletal muscle in mice. Anti-BmooMTx antibodies were effective in reducing the plasma creatine kinase levels of crude B. alternatus and B. leucurus venoms, evidencing immunological cross-reactivity between BmooMTx and other bothropic venoms. Intraplantar (i.pl.) injection of BmooMtx (1 to 15 μg/animal) caused a doseand time-dependent hyperalgesia and edematogenic responses. Dexamethasone (0.4 mg/kg), meloxicam (2 mg/kg) and promethazine (5 mg/kg) markedly reduced the hyperalgesia. Our data suggest that these drugs may likely serve as complementary therapies in cases of accidents with Bothrops moojeni, provided that such pharmacological treatments are administered immediately after the incident.