Toxicity assessment of carvacrol and its acetylated derivative in early staged zebrafish (Danio rerio): Safer alternatives to fipronil-based pesticides?

Fipronil is a broad-spectrum pesticide presenting high acute toxicity to non-target organisms, particularly to aquatic species. Natural compounds stand out as promising alternatives to the use of synthetic pesticides such as fipronil. Thus, our study aimed to compare the toxicity of carvacrol (natural), acetylcarvacrol (semisynthetic), and fipronil (synthetic) to early staged zebrafish. We conducted a series of toxicity assays at concentrations ranging from 0.01 µM to 25 µM for fipronil and 0.01 µM to 200 µM for carvacrol and acetylcarvacrol, depending on the assay, after 7-days post-fertilization (dpf). The potency (EC50) of fipronil was ∼1 µM for both deformities and mortality at 7 dpf, whereas EC50 was >50 µM for carvacrol and >70 µM for acetylcarvacrol. Fipronil at 0.1 and 1 µM caused a decrease in body length and swim bladder area of larvae at 7dpf, but no difference was observed for either carvacrol or acetylcarvacrol. Based upon the visual motor response test, fipronil induced hypoactivity in larval zebrafish at 1 µM and acetylcarvacrol induced hyperactivity at 0.1 µM. Anxiolytic-type behaviors were not affected by any of these chemicals. All chemicals increased the production of reactive oxygen species at 7 dpf, but not at 2 dpf. Genes related to swim bladder inflation, oxidative stress, lipid metabolism, and mitochondrial activity were measured; only fipronil induced upregulation of atp5f1c. There were no changes were observed in oxygen consumption rates of fish and apoptosis. Taken together, our data suggest that carvacrol and its derivative may be safer replacements for fipronil due to their lower acute toxicity.

Comparative toxicological evaluation of carvacrol, acetylcarvacrol anda fipronil-based pesticide in human blood cells.

Plant-derived chemicals are promising substances to control arthropod pests, although synthetic ones are still the most frequently used. Thus, comparative toxicological studies are needed to determine if natural substances are safe alternatives to replace the use of synthetic chemicals. This study aimed to compare the toxicity of carvacrol (natural origin), acetylcarvacrol (semi-synthetic) and a fipronil-based pesticide (synthetic). We assessed the effects of these chemicals on hemolytic activity, erythrocytes morphology and leucocyte viability using whole blood from human subjects. Additionally, DNA damage was evaluated through comet and DNA fragmentation assays. Fipronil and carvacrol caused hemolysis at concentrations ranging from 0.5 to 2.0%, whereas acetylcarvacrol did not cause hemolysis at 0.5 and 0.75%. Fipronil and carvacrol caused severe alterations in erythrocytes' morphology at 2%, such as ghost erythrocytes, elliptocyte-like shape and rouleau-like shape, presenting only 3.3 and 8.3% normal cells, respectively, at this concentration. However, 73.3% erythrocytes incubated with 2% acetylcarvacrol exhibited normal morphology. Fipronil considerably reduced leucocytes viability, decreasing it to 78% at 2%. Carvacrol and acetylcarvacrol showed no differences in leucocyte viability for 0.5 to 1.0%, but a decrease was observed for 2% carvacrol. The comet assay showed similar DNA damage for fipronil and carvacrol, but it was significantly lower for 1 and 2% acetylcarvacrol. Incubation with genomic DNA showed that only fipronil caused fragmentation of this molecule. Thus, we conclude that carvacrol and fipronil can present similar toxicity at higher concentrations. However, acetylation of carvacrol significantly reduced its toxicity to human blood cells compared with the other chemicals.

Achyrocline satureioides (Lam) DC extracts acting as enzyme modulators: digestion, inflammation, and hemostasis / Extratos de Achyrocline satureioides (Lam) DC atuando como moduladores enzimáticos: digestão, inflamação e hemostasia

Achyrocline satureioides is popularly known for its richness in phenolic compounds and medicinal properties (anti-inflammatory, analgesic, and hepatoprotective). The present study aimed at broadening the knowledge about the pharmacological potential exerted by the aqueous and ethanolic extracts of A. satureioides. These extracts were characterized by HPLC and tested for their modulatory action on phospholipases A2 and proteases of snake venoms. In addition, they were tested on the activities of digestive enzymes. Snake venoms were used as tools since they have enzymes with high functional and structural homology to human enzymes. The results demonstrate that the extracts of A. satureioides act as enzymatic inhibitors or potentiators, interfering in processes related to the hemostasis, such as coagulation and thrombus dissolution. In addition, the anti-genotoxic activity and inhibitions exerted on digestive enzymes suggests their potential use in the prevention and/or treatment of several pathologies. New studies could provide information on how the compounds present in the extracts and the different enzymes interact.

A Achyrocline satureioides é popularmente conhecida por sua riqueza em compostos fenólicos e por suas propriedades medicinais (anti-inflamatória, analgésica e hepatoprotetora). No presente estudo, com o objetivo de ampliar o conhecimento sobre o potencial farmacológico exercido por esses extratos, os extratos aquoso e etanólico de A. satureioides foram caracterizados por HPLC e testados quanto à sua ação modulatória sobre as fosfolipases A2 e proteases de peçonhas de serpentes. Além disso, também foram testados em atividades de enzimas digestivas. As peçonhas de serpentes foram usadas como ferramentas por apresentarem enzimas com alta homologia funcional e estrutural às humanas. Os resultados demonstram que os extratos de A. satureioides atuam como inibidores ou potencializadores enzimáticos, interferindo em processos relacionados à hemostasia, como coagulação e dissolução do trombo. Além do mais, destacam seu potencial antigenotóxico e as inibições exercidas sobre as enzimas digestivas direcionando seu potencial de uso na prevenção e/ou tratamento de diversas patologias. Novos estudos poderão fornecer informações sobre os mecanismos de interação entre os compostos presentes nos extratos e as diferentes enzimas.

Thrombolysis, clotting, and genotoxic activities modulated by essential oils extracted from Lippia alba

Abstract The composition and pharmacological properties of Lippia alba (Mill.) (L. alba) (Verbenaceae) flower and leaf essential oils (EO) were determined in this study. The major constituents in the flower EO were geranial (49.83%) and neral (32.75%), and in the leaf EO were geranial (38.06%), neral (31.02%), and limonene (18.03%). Flower EO inhibited thrombolysis induced by Bothrops moojeni (B. moojeni) and Lachesis muta muta (L. muta muta) venoms (0.05-1.2 µL mL-1). When tested against L. muta muta venom, the protective effect was smaller in both EO. The EOs prolonged the clotting time induced by L. muta muta venom and a procoagulant effect was observed on B. moojeni. In the comet assay, the flower EO presented anti-genotoxic action (damage frequency of only 11.6 - 34.9%) against the L. muta muta venom. The positive control (Doxorubicin) and the venom alone presented a damage frequency of 80.3% and 70.7%, respectively. The flower EO protected DNA from damage induced by L. muta muta venom. L. alba leaf and flower EOs presented anti-genotoxic action

Metabolites from Induratia spp. modulating key enzymes in human hemostasis.

Induratia spp. fungi have been poorly evaluated for their non-volatile secondary metabolites. In the present work, we evaluated the effects of non-volatile secondary metabolites released into the culture medium by Induratia spp. upon toxic alterations induced by Bothrops spp. venoms. B. atrox venom phospholipase was inhibited by Induratia spp. around 12 and 16%. The extracts of the two strains inhibited 12-25% of the hemolysis induced by B.moojeni venom. Thrombolysis was inhibited by 30-60% by the compounds present in both extracts. The coagulation induced by B. moojeni venom was prolonged by 26-41 s by the action of the extracts of I. coffeana. The fungal extracts did not exert any cytotoxic effect, nor did they induce any alteration in the other evaluated substrates show the potential use of non-volatile metabolites produced by the fungi evaluated as enzyme modulators, especially for proteases with a fundamental role in human hemostasis.

Gallic acid anti-myotoxic activity and mechanism of action, a snake venom phospholipase A2 toxin inhibitor, isolated from the medicinal plant Anacardium humile.

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a KD of 9.146 × 10-7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA2, thus proposing a new mechanism of PLA2 inhibition, and presenting more evidence of GA's potential as an antivenom compound.

Vanillic acid as phospholipase A2 and proteases inhibitor: In vitro and computational analyses.

Enzymatic inhibition by natural compounds may represent a valuable adjuvant in snakebite serum therapy. The objective in this work was to evaluate possible in vitro interactions between vanillic acid and enzymes from Bothrops spp. and Crotalus durissus terrificus venoms, and also suggest a theory as how they interact based on molecular docking. Vanillic acid inhibited the phospholipase activity induced by Bothrops alternatus (∼25% inhibition); the caseinolytic activity induced by Bothrops atrox (∼30%), Bothrops jararacussu (∼44%), and C. d. terrificus (∼33%); the fibrinogenolysis induced by B. jararacussu, B. atrox, and C. d. terrificus (100%); the serine protease activity induced by Bothrops moojeni (∼45%) and Bothrops jararaca (∼66%); the hemolytic activity induced by B. moojeni (∼26%); the thrombolysis activity induced by B. atrox (∼30%) and B. jararacussu (∼20%); and the thrombotic activity induced by C. d. terrificus (∼8%). The compound was also capable of delaying the coagulation time in citrated plasma by 60, 35, and 75 Sec, when incubated with B. moojeni, B. atrox, and B. jararaca, respectively. The results obtained expand the possibilities for future pharmaceutical use of vanillic acid, considering the high homology degree among human and snake venom phospholipases A2 and proteases (involved in chronic inflammatory diseases). Also, this compound can be used as adjuvant to improve currently available treatments for ophidism victims.

Polybia occidentalis and Polybia fastidiosa venom: a cytogenotoxic approach of effects on human and vegetal cells.

The venoms of wasps are a complex mixture of biologically active compounds, such as low molecular mass compounds, peptides, and proteins. The aim of the study was to evaluate the action of wasp venoms, Polybia occidentalis and Polybia fastidiosa, on the DNA of human leukocytes and on the cell cycle and genetic material of the plant model Lactuca sativa L. (lettuce). The cultured leukocytes were treated with the venoms and then evaluated by the comet assay. On another assay, seeds were exposed to a venom solution; the emitted roots were collected and the occurrence of cell cycle alterations (CCAs) and DNA fragmentation were evaluated by agarose gel electrophoresis and TUNEL assay. The results demonstrated that the venom of both wasps induces several CCAs and reduces the mitotic index (MI) on treated cells. They induced damage on human leukocytes DNA. High frequencies of fragments were observed in cells exposed to P. occidentalis venom, while those exposed to P. fastidiosa showed a high frequency of non-oriented chromosome. Both venoms induced the occurrence of various condensed nuclei (CN). This alteration is an excellent cytological mark to cell death (CD). Additionally, CD was evidenced by positive signals in TUNEL assay, by DNA fragmentation in agarose gel electrophoresis with vegetal cells, and by DNA fragmentation of the human leukocytes evaluated. Furthermore, human leukocytes exposed to the venom of P. fastidiosa had high rate of damage. The data demonstrate that both vegetal and human cells are adequate to evaluate the genotoxicity induced by venoms.

In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victim’s local effects and time to heal.

Eucalyptus grandis and Eucalyptus microcorys Essential Oils: in vitro Modulation of Hemostasis and Genotoxic Activity

Abstract The genus Eucalyptus present high content of essential oil (EO). This study evaluated the pharmacological properties of Eucalyptus grandis (EG) and Eucalyptus microcorys (EM) EOs. The major component in both EOs was 1,8-Cineole. Both essential oils prevented thrombus dissolution and reduced clotting, hemolysis, and genotoxicity induced by snake venoms. 50% (EM) and 73% (EG) were the greatest inhibitions obtained in the thrombolytic assay - thrombolysis induced by B. moojeni venom. Increases in clotting time were also observed, with values considered significant between 10-27 seconds. Lysis values 50% lower than the negative control were observed in both EOs. The EOs also protected fibrinogenolysis induced by snake venom. EM EO was more effective in reducing venom-induced DNA fragmentation in the comet assay, with arbitrary unit values 66.15% lower than the positive control. These oils present wide application potential considering the pharmacological properties observed in this study.

Enzymatic Modulators from Induratia spp.

In the present work, ethyl acetate extracts, consisting of non-volatile compounds, from the culture of endophytic fungi isolated from coffee plants, Induratia coffeana and Induratia yucatanensis, were prospected in enzyme modulation tests that act in human hemostasis. Dry extracts of the fungi were diluted in dimethyl sulfoxide p.a. 99.9% (DMSO), and then tested. Bothrops atrox venom was used as an enzyme source and tool to induce the activities. Prior to the evaluation of the activities, incubations of the extracts with the venom were performed in the proportions 1: 0.01, 1: 0.25, 1: 0.5, and 1: 1 (venom: extract; mass: mass). The extracts of all fungi promoted a significant increase in the clotting time induced by the venom, which was even longer when the extracts were previously incubated with the citrated plasma. The activity of phospholipases A2 did not significantly change when evaluated in the presence of fungal extracts. However, the evaluated extracts inhibited proteases by 73% and 30% in the thrombolytic and caseinolytic tests, respectively. In addition, the extracts did not induce cytotoxicity on human erythrocytes when evaluated in the absence of the venom. Thus, it is possible to suggest the presence of specific interactions between molecules present in extracts of Induratia spp. and venom proteases, highlighting non-volatile metabolites as promising sources of compounds of medical and scientific interest.

Catechin and epicatechin as an adjuvant in the therapy of hemostasis disorders induced by snake venoms.

Snake toxins, such as phospholipases A2 and proteases, are used as research tools to evaluate biological activities and to understand physiopathological processes of natural compounds better. In the present study, the phenolic compounds catechin and epicatechin were incubated with snake venoms to evaluate their inhibition against different substrates. Catechin and epicatechin exerted inhibitions between 20% and 95% on the activity of phospholipases A2 present in the venom of Bothrops alternatus. In the hemolytic activity, catechin exerted inhibitions between 20% and 25% in all proportions evaluated on the B. jararacussu venom, whereas epicatechin inhibited 20% of the venom activity. Coagulation induced by B. atrox and B. jararacussu venoms was significantly inhibited by catechin and epicatechin, where the time for coagulation was two to three times higher after previous incubation of the venoms with the compounds. The most significant inhibitions for the proteolytic activity on casein were 17% and 27%, respectively, by both compounds. Catechin inhibited serine protease activity induced by B. atrox venom by 64% and epicatechin by 65%. Regarding B. atrox-induced thrombolysis, catechin exerted 40% inhibition and epicatechin around 30%. The fibrinogen proteolysis was completely inhibited by catechin acting on the B. atrox venom in the proportion of 1:1 and by epicatechin on B. jararacussu venom. Catechin and epicatechin showed promising inhibitory action on proteases and phospholipases A2 . Therefore, these compounds can be explored as an adjuvant for serum therapy or pharmaceutical purposes, once they act on homologous enzymes that are present in humans.

Mitochondriotropic action and DNA protection: Interactions between phenolic acids and enzymes.

The protective action of caffeic (CA) and syringic (SA) acids on the genotoxicity exercised by snake venoms was investigated in this study. Molecular interactions between phenolic acids and the enzyme succinate dehydrogenase were also explored. In the electrophoresis assay, SA did not inhibit the genotoxicity induced by the venom. However, CA partially inhibited DNA degradation. In the comet assay, SA and CA exerted an inhibitory effect on the venom-induced fragmentation. Succinate dehydrogenase presented, in computational analyzes, favorable energies to the molecular bond to both the malonic acid and the phenolic compounds evaluated. In the enzymatic activity assays, SA inhibited succinate dehydrogenase and interfered in the interaction of malonic acid. Meanwhile, CA potentiated the inhibition exerted by the malonic acid. The results suggest transient interactions between toxins present in venoms and phenolic acids, mainly by hydrogen interactions, which corroborate with the data from previous works.

Current Anti-Inflammatory Therapies and the Potential of Secretory Phospholipase A2 Inhibitors in the Design of New Anti-Inflammatory Drugs: A Review of 2012 - 2018.

The inflammatory process is a natural self-defense response of the organism to damage agents and its action mechanism involves a series of complex reactions. However, in some cases, this process can become chronic, causing much harm to the body. Therefore, over the years, many anti-inflammatory drugs have been developed aiming to decrease the concentrations of inflammatory mediators in the organism, which is a way of controlling these abnormal chain reactions. The main target of conventional anti-inflammatory drugs is the cyclooxygenase (COX) enzyme, but its use implies several side effects. Thus, based on these limitations, many studies have been performed, aiming to create new drugs, with new action mechanisms. In this sense, the phospholipase A2 (PLA2) enzymes stand out. Among all the existing isoforms, secretory PLA2 is the major target for inhibitor development, since many studies have proven that this enzyme participates in various inflammatory conditions, such as cancer, Alzheimer and arthritis. Finally, for the purpose of developing anti-inflammatory drugs that are sPLA2 inhibitors, many molecules have been designed. Accordingly, this work presents an overview of inflammatory processes and mediators, the current available anti-inflammatory drugs, and it briefly covers the PLA2 enzymes, as well as the diverse structural array of the newest sPLA2 inhibitors as a possible target for the production of new anti-inflammatory drugs.

Enzymes modulation by dried grape pomace from the manufacture of wines and juices

The processing of grapes for the manufacture of juices and wines, generates large quantities of by-products rich in metabolites with antioxidant, antimicrobial, anti-inflammatory and cicatrizing activities. The high homology between human enzymes and snake venoms makes the latter valuable laboratory tools for the study of pathophysiological processes. Proteases and phospholipases A2 act in processes related to hemostasis and inflammatory response. Thus, in this work, dried pomace obtained from grape (Isabel, Niagara, Bordô, BRS Violeta and Blend cultivars) processing were evaluated on phospholipase, proteolytic, hemolytic and thrombolytic activities induced by snakes venoms and the content of phenolic compounds and minerals was evaluated. The dried pomace exerted inhibitory and potentiating actions in all analyzed activities. The enzymatic modulators present in the evaluated dried pomace have potential for therapeutic use, although their broad characterization is still necessary, in order to define adequate amounts and formulations to obtain efficacy and safety in their use.

Inhibition by essential oils of Mentha viridis and Mentha pulegium (Lamiaceae) in proteolysis, fibrinogenolysis and coagulation caused by venomous snakes

Snake venoms are widely used as laboratory tools for studies of physiological, pharmacological and toxicological mechanisms. Venoms used here are rich sources of several classes of proteases that act on factors of the coagulation cascade, fibrinogenolysis and fibrinolysis, altering the hemostatic processes, and phospholipases A2 which are involved mainly in inflammatory and clotting processes. Natural products such as essential oils are made up of active ingredients with wide application in the food, pharmaceutical and cosmetic industries. Thus, this study evaluates the essential oils from Mentha viridis and Mentha pulegium on coagulation, fibrinogenolysis and degradation of azocasein, induced by Bothrops sp and Lachesis muta muta venoms. These oils were achieved by hydrodistillation and presented, respectively, as the main constituents linalool (40.70 %), carvone (13.52 %) and α-terpinene (8.56 %); pulegone (50.01 %), menthol (31.90 %) and menthone (16.56 %). The essential oils were previously incubated with Bothrops alternatus venom, for two different times, then plasma was added and time was recorded. The M. viridis oil presented greater anticlotting potential. Meanwhile, the M. pulegium oil presented anticlotting or proclotting activity dependent on the dose tested. The incubation time also influenced the effect of the oils on the coagulation time. At azocaseinolytic assay, the oil from M. pulegium reduced the activity for all evaluated venoms. The highest inhibition was of 39.99 %; on activity induced by B. jararacussu, M. viridis reduced the activity in 57.72 %. On B. moojeni, the major inhibition observed was of 74.67 %. The fibrinogenolysis induced by B. moojeni venom was totally inhibited by both oils in the evaluated proportions. The results show the presence in oils of protease inhibitors, considering serine and metalloproteases (acting on clotting factors or with hemorrhagic activity), as well as phospholipase A2 (enzymes involved in inflammation and clotting processes) inhibitors of wide application in medical and biotechnology areas.

Los venenos de serpientes son ampliamente utilizados como herramientas de laboratorio para estudios de mecanismos fisiológicos, farmacéuticos y toxicológicos. Los venenos son recursos ricos en diferentes clases de proteasas que actúan sobre los factores de la cascada de coagulación, fibrinogenólisis y fibrinólisis, alterando los procesos homeostáticos y las fosfolipasas A2, los cuales están involucrados en procesos inflamatorios y de coagulación. Los productos naturales como los aceites esenciales se componen de ingredientes activos de amplia aplicación en las industrias alimentaria, farmacéutica y cosmética. El presente estudio evalúa los aceites esenciales de Mentha viridis y Mentha pulegium sobre la coagulación, fibrinogenólisis y degradación de azocaseína, inducida por los venenos de Bothrops sp. y Lachesis muta muta. Los aceites esenciales fueron obtenidos por hidrodestilación y sus componentes principales fueron identificados respectivamente como el linalool (40.70 %), carvona (13.52 %) y α-terpineno (8.56 %); pulegone (50.01 %), mentol (31.9 %) y mentona (15.56 %). Los aceites esenciales fueron previamente incubados con veneno de Bothrops alternatus, durante dos tiempos diferentes, luego se agregó el plasma y fue registrado el tiempo. El aceite de M. viridis presentó el más grande potencial anticoagulante. Mientras tanto, el aceite de M. pulegium presentó actividad anticoagulante o procoagulante dependiendo de la dosis evaluada. El tiempo de incubación también influyó en el efecto de los aceites sobre el tiempo de coagulación. En el ensayo azocaseínolitico, el aceite de M. pulegium redujo la actividad para todos los venenos evaluados. La inhibición más alta inducida para el veneno de B. jararacussu fue de 39.99 %, para M. viridis la actividad fue reducida en 57.72 %, mientras que para B. moojeni, la mayor inhibición observada fue de 74.67 %. La fibrinogenólisis inducida por el veneno de B. moojeni fue totalmente inhibida por ambos aceites en las proporciones evaluadas. Los resultados muestran la presencia de inhibidores de proteasas en los aceites, como las serinas y metaloproteasas (actuando sobre los factores de coagulación o sobre la actividad hemorrágica), como también fosfolipasas A2 (enzimas involucradas en los procesos de inflamación y coagulación), los cuales son inhibidores de amplia aplicación en áreas médicas y biotecnológicas.

Exploring the structural and functional aspects of the phospholipase A2 from Naja spp.

Naja spp. venom is a natural source of active compounds with therapeutic application potential. Phospholipase A2 (PLA2) is abundant in the venom of Naja spp. and can perform neurotoxicity, cytotoxicity, cardiotoxicity, and hematological disorders. The PLA2s from Naja spp. venoms are Asp 49 isoenzymes with the exception of PLA2 Cys 49 from Naja sagittifera. When looking at the functional aspects, the neurotoxicity occurs by PLA2 called ß-toxins that have affinity for phosphatidylcholine in nerve endings and synaptosomes membranes, and by α-toxins that block the nicotinic acetylcholine receptors in the neuromuscular junctions. In addition, these neurotoxins may inhibit K+ and Ca++ channels or even interfere with the Na+/K+/ATPase enzyme. The disturbance in the membrane fluidity also results in inhibition of the release of acetylcholine. The PLA2 can act as anticoagulants or procoagulant. The cytotoxicity exerted by PLA2s result from changes in the cardiomyocyte membranes, triggering cardiac failure and hemolysis. The antibacterial activity, however, is the result of alterations that decrease the stability of the lipid bilayer. Thus, the understanding of the structural and functional aspects of PLA2s can contribute to studies on the toxic and therapeutic mechanisms involved in the envenomation by Naja spp. and in the treatment of pathologies.

Jabuticaba (Plinia jaboticaba) skin extracts as inhibitors of phospholipases A2 and proteases.

The phenolic extracts of jabuticaba skin flour (JSF) were characterized by HPLC, and evaluated for their modulating action upon phospholipases A2 and proteases of snake venom, aiming at their possible use in the treatment of the various diseases associated with the action of venom toxins. Two types of extracts were prepared from JSF: aqueous and methanolic. These extracts, evaluated at different ratios, (venom: extract, m/m), significantly inhibited the phospholipase activity induced by the venom of Bothrops moojeni and Crotalus durissus terrificus, except for Bothrops atrox venom. The greatest hemolysis inhibitory action was observed for the methanolic extract, when incubated with venoms of B. moojeni and C. durissus terrificus, with inhibitions between 21 and 100%. Thrombolysis induced by venoms of B. moojeni and C. durissus terrificus was inhibited by both extracts, ranging from 32 to 83% and 51 to 83% for the aqueous and methanolic extracts, respectively. Both extracts extended coagulation time, induced by the venoms of B. moojeni and Lachesis muta muta. Inhibitory actions are related to phenolic compounds, such as gallic, syringic and p-coumaric acids, besides catechin, epigallocatechin gallate, epicatechin; resveratrol and quercetin, present in the extracts of jabuticaba skin flour, confirming their potential for nutraceutical use.

Effects of cooling rates on the quality of Prochilodus lineatus (Valenciennes, 1836) sperm.

This study aims to investigate the effect of different cooling rates on the semen cryopreservation of curimba (Prochilodus lineatus). Nineteen ejaculates were obtained from adults males and cryopreserved at 15°C/min (CR15), 30°C/min (CR30) (controlled temperature inside and outside straw, speed was stable during freezing) and direct freezing in liquid nitrogen vapour (~35.6°C/min) (CRNV). The straws were thawed and seminal parameters evaluated. DNA fragmentation through the comet assay was assessed. A fresh sperm sample was not frozen and used for analyses. Data were submitted to an analysis of variance (ANOVA), and means were compared by Scott-Knott test (p < 0.05) using the R Software. Mean motility percentage was 100%, and motility duration was 39.5 ± 5.7 s for the fresh sperm (subjective analysis); 58.9 ± 8.0% and 24.5 ± 5.7 s for CR15; 64.8 ± 4.8% and 26.5 ± 7.1 s for CR30; and 50.1 ± 16% and 25.7 ± 4.7 s for CRNV, respectively. Motility percentages were higher and equal between CR15 and CR30 compared to CRNV (p < 0.05). Some sperm motion kinetics, namely average path velocity (VAP) and straight line velocity (VAS), were higher for CR30 (p < 0.05), while curvilinear velocity (VCL) and velocity progression (PRO) were lower for CRNV (p < 0.05). Straightness (STR) and wobble (WOB) were the same among treatments (p > 0.05). Sperm morphology results indicated higher means for total morphological sperm alterations in CRNV. All cooling rates caused sperm DNA fragmentation, although CR30 provided a less harmful effect. This is the first report for cryopreserved P. lineatus sperm preserved under different controlled cooling rates. The cooling rate of 30°C/min is indicated for the cryopreservation of this fish sperm as it led to the lowest detrimental spermatozoa effects.

Molecular interactions between p-coumaric acid and snake venom toxins.

A large number of natural compounds, such as phenolic compounds, have been scientifically evaluated in the search for enzyme inhibitors. The interactions between the phenolic compound p-coumaric acid and the enzymes present in snake venoms (used as research tools) were evaluated in vitro and in silico. The p-coumaric acid was able to inhibit 31% of the phospholipase activity induced by Bothrops alternatus venom, 27% of the hemolytic activity induced by B. moojeni, 62.5% of the thrombolytic activity induced by B. jararacussu, and approximately 27% of the activity thrombosis induced by Crotalus durissus terrificus. Previous incubation of p-coumaric acid with the venoms of B. atrox and B. jararacussu increased the coagulation time by 2.18 and 2.16-fold, respectively. The activity of serine proteases in B. atrox and B. jararacussu venoms was reduced by 60% and 66.34%, respectively. Computational chemistry analyses suggests the specific binding of p-coumaric acid to the active site of proteases through hydrogen and hydrophobic interactions. The phenolic compound evaluated in this work has great potential in therapeutic use to both prevent and treat hemostatic alterations, because the venom proteins inhibited by the p-coumaric acid have high homology with human proteins that have a fundamental role in several pathologies.