RESUMEN
In Brazil, there are species of snakes that become involved in accidents and cause serious health problems to the inhabitants, highlighting the genus Bothrops for being responsible for approximately 90% of accidents reported annually. In the northern region of the country, this genus is responsible for the largest number of accidents, especially among rural dwellers. These populations invest in alternative treatments for with the purpose of improving the symptoms caused by snakebites. The species Mauritia flexuosa L. f., known as buriti, is traditionally used for the treatment of envenomation by snakes. Aim of the study This study aimed to evaluate the antiophidic potential of the oil of Mauritia flexuosa L. f. for Bothrops moojeni H. venom, confronting cultural and scientific knowledge. Materials and methods The physicochemical properties were determined, and the components present in the oil, extracted from fruit pulp, were analyzed by Gas Chromatography Coupled with Mass Spectrometry. The in vitro inhibitory capacity of the oil for phospholipase, metalloprotease and serine protease activities was investigated. In the in vivo studies, male Swiss mice were used to evaluate the effect of oil on lethality and toxicity, and hemorrhagic, myotoxic and edematogenic activities were assessed. Results GC‒MS analysis identification of 90.95% of the constituents of the oil, with the main components being 9-eicosenoic acid, (Z)- (34.54%), n-hexadecanoic acid (25.55%) and (E)-9-octadecenoic acid ethyl ester (12.43%). For the substrates, the outcomes indicate that the oil inhibited the activity of the main classes of toxins present in Bothrops moojeni H. venom (VBm) at the highest dose tested (0.5 μL), with inhibition of 84% for the hydrolysis of the selective substrate for serine protease and inhibition of 60% for the hydrolysis of substrates for PLA2 and metalloproteases. The antiophidic activity in vivo was evaluated with two concentrations of the oil: 1.5 mg, the dosage the population, diluted in mineral oil to a volume of 1 tablespoon and 15 mg, administered by gavage 30 min before poisoning and at time zero (concomitant to poisoning), and both concentrations administered by gavage in combination with topical use at time zero. The bleeding time in the group treated with oil at a concentration of 15 mg administered at time zero was significantly lower than that in the control group (p < 0.05). However, a greater inhibition of bleeding time was observed when local application was combined with the gavage treatment at both concentrations tested at time zero (p < 0.05). In the myotoxicity test, oil was efficient in reducing the myotoxic effects induced by the venom at the two concentrations tested, with gavage administration at time zero and gavage plus topical administration at time zero (p < 0.05). Conclusions The data obtained show that the oil is safe to use at the concentrations studied and contains fatty acids that may collaborate for cellular-level repair of the injuries caused by Bm poisoning. The in vitro and in vivo experiments showed that oil inhibits the main proteolytic enzymes present in the venom and that it has important activities to control the local effects caused by bothropic venom.
RESUMEN
Snakebite accidents are a serious public health problem neglected in tropical countries. In Brazil, the Northern Region has the largest number of cases and snakes from Lachesis genus are responsible for the most serious accidents and the highest number of deaths. Victims of Lachesis envenoming have bite site damage, bleeding and, in severe cases, hypotension. Proteomic and transcriptomic studies report the presence of bradykinin potentiating peptide (BPPs) in L. muta venom, however, there are no data in the literature describing their purifications and identifications. The objective of this work is to identify and characterize angiotensin-converting enzyme inhibitors in the low molecular weight portion of Lachesis muta venom. For this lyophilized L. muta venom was dissolved in ammonium acetate buffer and filtered by centrifugation for size fractionating, using a 10 kDa centrifugal filter unit. The low molecular weight fraction was then chromatographed by RP-HPLC using a C18 column. Twelve fractions were collected and four were tested in fluorimetric assays using ACE as an enzyme and Abz-FRK(Dnp)P-OH as substrate. These fractions present peaks with retention time similar to the peaks that contained BPPs of other snake venoms. In addition, four fractions presented 100% inhibition of the catalytic activity of ACE over the FRET substrate. Based on these results, and due to other data in the literature describing the presence of BPPs in L. muta venom, is possible that it may contain BPPs. For confirmation of this hypothesis, the next step will be to perform analysis by mass spectrometry aiming to determine the primary sequences of the peptides present in each fraction.
Os acidentes ofídicos são um grave problema de saúde pública negligenciado em países tropicais. No Brasil, a Região Norte apresenta o maior número de casos e as serpentes do gênero Lachesis são responsáveis pelos acidentes mais graves e com maior número de óbitos. Vítimas do envenenamento por Lachesis apresentam danos no local da picada, hemorragias e em casos graves apresentam hipotensão arterial. Estudos proteômicos e trancriptômicos descrevem a presença de peptídeos potenciadores de bradicinina (BPPs) no veneno de Lachesis muta, porém não há dados na literatura descrevendo o isolamento e/ou suas identificações. O objetivo deste trabalho é identificar e caracterizar inibidores da enzima conversora de angiotensina (ECA) na porção de baixa massa molecular do veneno de Lachesis muta. Para isso o veneno liofilizado de Lachesis muta foi dissolvido em tampão acetato de amônio e filtrado por centrifugação para o fracionamento de tamanho, usando uma membrana de corte de 10 kDa. A porção de baixa massa molecular foi fracionada por RP-HPLC usando uma coluna C-18. Doze frações foram coletadas e quatro foram testadas em ensaios fluorimétricos utilizando a ECA como enzima e o Abz-FRK(Dnp)P-OH como substrato. Das quatro frações selecionadas, duas se destacaram devido à alta intensidade dos picos, um em 13,65 minutos de retenção e outro em 14 minutos de retenção, com 44% e 45% concentração de solução B, respectivamente. As frações de interesse apresentam picos eluídos nos mesmos tempos de retenção que os BPPs de outros venenos de serpentes, sugerindo, juntamente com dados da literatura, que eles podem conter BPPs semelhantes. As quatro frações inibiram em 100% a atividade catalítica da ECA sobre o substrato Abz-FRK(Dnp)P-OH. Por essas razões e devido outros dados na literatura descreverem a presença BPPs no veneno de L. muta, acreditamos que as quatro frações selecionadas contenham BPPs. Para a confirmação o próximo passo será realizar a análise por espectrometria de massas.