Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.

sPLA2 from Crotalus durissus terrificus venom, free of crotapotin (Cdt sPLA2), purified and isolated sPLA2, was able to significantly increase lipid peroxidation, which occurred simultaneously with increased arachidonic acid (AA) metabolism. In addition, MDA and AA levels were elevated at 15 min after Cdt sPLA2 injection and after peak edema (negative control). Thus, oxidative stress and ROS play important roles in the inflammation induced by Cdt sPLA2. On the other hand, edema induced by sPLA2 involves the direct and indirect mobilization of arachidonic acid by the involvement of phosphokinase C (PKC) and phospholipase C (PLC), which indirectly stimulates cytosolic PLA2 (cPLA2). We also observed that the specific antivenin against Cdt venom had no significant effect on the neutralization of induced edema compared to the natural products 5-caffeine-linoleic acid (5CQA) and dexamethasone (AACOCF3). Our results also indicate that there was improvement in the inhibition of edema of natural polyphenolic compounds compared to antivenin or inhibition of the enzymatic activity of sPLA2 due to the fact that 5CQA is a potent antioxidant compound. Thus, our results show a clear correlation between increased arachidonic acid metabolism and oxidative stress.

Compound 8-C-rhamnosyl apigenin (8CR) induced a moderate reduction in the enzymatic activity of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus and cytosolic phospholipase A2 (cPLA2), but the compound also significantly inhibited the enzymatic activity of the enzyme cyclooxygenase. In vitro assays showed that the compound induced a slight change in the secondary structure of sPLA2 from Crotalus durissus terrificus snake venom. In vivo assays were divided into two steps. In the first step, the 8CR compound was administered by intraperitoneal injections 30 min prior to administration of sPLA2. In this condition, 8CR inhibited edema and myonecrosis induced by the sPLA2 activity of Crotalus durissus terrificus in a dose-dependent manner by decreasing interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), prostaglandin E2 (PGE2), and lipid peroxidation. This has been demonstrated by monitoring the levels of malondialdehyde (MDA) in rat paws after the course of edema induced by sPLA2. These results, for the first time, show that sPLA2 of Crotalus durissus terrificus venom induces massive muscle damage, as well as significant edema by mobilization of cyclooxygenase enzymes. Additionally, its pharmacological activity involves increased lipid peroxidation as well as TNF-α and IL-1β production. Previous administration by the peritoneal route has shown that dose-dependent 8CR significantly decreases the enzymatic activity of cyclooxygenase enzymes. This resulted in a decrease of the amount of bioactive lipids involved in inflammation; it also promoted a significant cellular protection against lipid peroxidation. In vivo experiments performed with 8CR at a concentration adjusted to 200 μg (8 mg/kg) of intraperitoneal injection 15 min after sPLA2 injection significantly reduced sPLA2 edema and the myotoxic effect induced by sPLA2 through the decrease in the enzymatic activity of cPLA2, cyclooxygenase, and a massive reduction of lipid peroxidation. These results clearly show that 8CR is a potent anti-inflammatory that inhibits cyclooxygenase-2 (COX-2), and it may modulate the enzymatic activity of sPLA2 and cPLA2. In addition, it was shown that Crotalus durissus terrificus sPLA2 increases cell oxidative stress during edema and myonecrosis, and the antioxidant properties of the polyphenolic compound may be significant in mitigating the pharmacological effect induced by sPLA2 and other snake venom toxins.

Ellagitannins constitute the largest group of hydrolyzable tannins of plants, and, from this group, casuarictin (Casu) was identified in some plant species. However, to our knowledge, no investigation of secretory phospholipase A2 (sPLA2) inhibition by Casu has been performed yet. Casuarictin was isolated by chromatography n-butanol (n-BuOH) partition of Laguncularia racemosa leaves. The pharmacological and biological effects of Casu were evaluated on isolated sPLA2 from the rattlesnake (Crotalus durissus terrificus) and using a plant bacterial strain. The compound was able to form a protein complex consisting of a stable sPLA2 + Casu complex. Analyses carried out with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) revealed that the molecular mass of sPLA2 increased from 14,425.62 to 15,362.74 Da. The enzymatic activity of the sPLA2 + Casu complex was significantly lower than that of native sPLA2. Besides, molecular interactions of Casu with sPLA2 were able to virtually abolish the native edematogenic effect as well as myonecrosis induced by the protein when injected 10 min after sPLA2. Therefore, Casu may be considered a potential anti-inflammatory that can be used to treat edema and myonecrosis induced by serine-secreting phospholipase A2. In addition, the compound also showed great antimicrobial potential.

Bioaffinity capturing of molecules allows the discovery of bioactive compounds and decreases the need for various stages in the natural compound isolation process. Despite the high selectivity of this technique, the screening and identification methodology depends on the presence of a protein to capture potential ligands. However, some proteins, such as snake secretory phospholipase A2 (sPLA2), have never been investigated using this approach. The purpose of this study was to evaluate the use of a new method for screening natural compounds using a bioaffinity-guided ultrafiltration method on Crotalus durissus terrificus sPLA2 followed by HPLC-MS to identify the compounds, and this method could be used to discover new anti-inflammatory compounds from the various organisms originating from biodiversity. Different extracts were selected to evaluate their ability to inhibit sPLA2 activity. The extracts were incubated with sPLA2 and the resulting mixture was ultrafiltrated to elute unbound components. The resulting compounds were identified by HPLC-MS. We identified hispidulin as one of the components present in the Moquiniastrum floribundum leaf and evaluated the ability of this isolated compound to neutralize the inflammatory activity of sPLA2 from Crotalus durissus terrificus.

Resumo: O pau-rosa é uma das espécies com maior potencial econômico atualmente na Amazônia brasileira. Desta árvore é extraído óleo valorizado pela indústria de perfumaria, tanto que sua exploração levou a espécie à ameaça de extinção. Com a diminuição do recurso, a produção do óleo oscilou de 46 a 2 toneladas ano-1 nos últimos 20 anos, aumentando o preço de venda de USD 40.00 kg-1 a USD 320.00 kg-1 neste mesmo período. Esta valorização tem estimulado o plantio da espécie, que se depara com a limitada disponibilidade de mudas para comercialização. Diante disto, o objetivo desta pesquisa foi analisar o mercado de sementes e mudas de pau-rosa no Amazonas, identificando o número de produtores, produção e demanda média anual, preços praticados, perfil dos clientes e dificuldades na produção. Os produtores foram identificados através de pesquisas na internet e contatados para elucidar questões mais específicas da cadeia produtiva. Foi verificado que atualmente apenas três empreendedores comercializavam mudas da espécie com certa regularidade, totalizando a média de produção de 20.000 mudas/ano com preço de venda entre R$ 10,00 e R$ 15,00. É evidente que este mercado representa uma oportunidade promissora de negócio, que poderá obter maior êxito caso sejam estabelecidos novos arranjos produtivos.

ResumoO estado de São Paulo é o terceiro maior produtor de uvas do Brasil e o maior produtor nacional de uvas de mesa, sendo a variedade Niágara Rosada (Vitis labrusca) a mais plantada no estado. O objetivo do trabalho foi verificar a viabilidade econômica do cultivo de Niágara Rosada. O projeto foi dimensionado para uma área de três hectares implantada no município de Piracicaba, SP. As plantas foram conduzidas no sistema em espaldeira em uma densidade de 5000 plantas ha -1 . O fluxo de caixa foi dimensionado para um período de 13 anos. AbstractSão Paulo State is the third largest grapevine producer in Brazil and the largest national producer of table grapes. Niagara Rosada (Vitis labrusca) is the most planted cultivar in the State. This study aimed to verify the economic feasibility of Niagara Rosada production. The project was designed for an area of tree hectares to be implemented in Piracicaba, SP. Plants were conducted in vertical trellis system with 5000 plants ha -1 . The cash flow was dimensioned for a period of 13 years. Costs with implantation and maintenance of the vineyard, bank financing, payment of the Rural Worker Support Fund, and the opportunity cost of land were estimated. The minimum acceptable rate of return [MARR] used was 11.45%. The economic indicators calculated were Net Present Value [NPV], Internal Rate of Return [IRR], simple payback, discounted payback, and benefit-cost ratio. Positive values of NPV (R$ 398,951.36) and benefit-cost ratio (1.81) were obtained. The IRR estimated was 27.34%, higher than the MARR. The simple payback and the discounted payback were five years and six years, respectively, indicating that the period of returning the invested capital was lower than the project period. The estimated economic indicators demonstrated the financial feasibility of the project.

Ao longo da história da humanidade as doenças de plantas desencadearam mudanças de hábitos e, mais recentemente, ao aumento nos custos de produção, devido à necessidade de controle e perdas de produção. Tornou-se imprescindível o emprego de técnicas para a correta identificação das doenças, implicando diretamente, no controle adequado das mesmas. Diante do exposto, o objetivo do presente trabalho foi estudar a viabilidade econômica da instalação de uma clínica de diagnose de doenças de plantas na região de Santa Maria, Rio Grande do Sul. Para tanto, foram apurados dados de demanda e tipo de análises necessárias para a identificação dos principais agentes patogênicos causadores de doenças em plantas. Junto a empresas especializadas, foram levantados os custos de equipamentos e reagentes para a execução das mesmas. O VPL obtido foi de R$ 1.894.882,59, ou seja, este valor está sendo agregado em relação a minha melhor opção de investimento. O Payback foi de 3,06 anos e a TIR foi de 61%. A análise de sensibilidade mostrou que exceto no caso da vida útil ser inferior a 6 anos, o VPL foi positivo em todos os casos, demonstrando que os dados apurados estão dentro da realidade e que o projeto continua viável mesmo que os parâmetros sofram uma variação considerável. Com base nos coeficientes de rentabilidade Valor Presente Líquido, Payback e Taxa Interna de Retorno pode-se concluir que o empreendimento em análise é viável, apresentando alta rentabilidade sobre o capital investido.