Biological Activities of Species of the Genus Clusia L (Clusiaceae): A General Approach.

The genus Clusia L. is highly diverse in Central and South America, comprising about 300 species, including trees and shrubs, hemiepiphytes, epiphytes, and lianas. This genus deserves attention due to its wide range of biological activities. Clusia belongs to the Clusiaceae family, chemically characterized by the presence of xanthones, benzophenones, flavonoids, coumarins, terpenoids, and other substances with bioactive activity already described. This review aims to highlight the biological activity associated to extracts and isolated substances from species of the Clusia genus, including anti-HIV, antimicrobial, antioxidant, antinociceptive, antitumor, leishmanicidal, modulator of inflammatory processes, neutralization of toxic effects caused by snake bites, and others. This review gathered information on biological activities associated with different types of extracts and isolated substances of the genus Clusia, traditional use, chemical profile, and biological properties of plants of the genus, published in the last 23 years (1998 to 2021) and that can provide support for future research. The paper aims to provide an overview of existing knowledge about the biological properties of the genus Clusia plant species.

Alkaloids from Siparuna (Siparunaceae) are predicted as the inhibitors of proteolysis and plasma coagulation caused by snake venom and potentially counteract phospholipase A2 activity of Bothrops jararaca.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation (SBE) is the world's most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE. AIM OF THE STUDY: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics. MATERIALS AND METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform. RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species. CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Effect of Seaweed-Derived Fucoidans from Undaria pinnatifida and Fucus vesiculosus on Coagulant, Proteolytic, and Phospholipase A2 Activities of Snake Bothrops jararaca, B. jararacussu, and B. neuwiedi Venom.

BACKGROUND: Snakebite envenomation (SBE) causes diverse toxic effects in humans, including disability and death. Current antivenom therapies effectively prevent death but fail to block local tissue damage, leading to an increase in the severity of envenomation; thus, seeking alternative treatments is crucial. METHODS: This study analyzed the potential of two fucoidan sulfated polysaccharides extracted from brown seaweeds Fucus vesiculosus (FVF) and Undaria pinnatifida (UPF) against the fibrinogen or plasma coagulation, proteolytic, and phospholipase A2 (PLA2) activities of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom. The toxicity of FVF and UPF was assessed by the hemocompatibility test. RESULTS: FVF and UPF did not lyse human red blood cells. FVF and UPF inhibited the proteolytic activity of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom by approximately 25%, 50%, and 75%, respectively, while all venoms led to a 20% inhibition of PLA2 activity. UPF and FVF delayed plasma coagulation caused by the venoms of B. jararaca and B. neuwiedi but did not affect the activity of B. jararacussu venom. FVF and UPF blocked the coagulation of fibrinogen induced by all these Bothropic venoms. CONCLUSION: FVF and UPF may be of importance as adjuvants for SBE caused by species of Bothrops, which are the most medically relevant snakebite incidents in South America, especially Brazil.

Triazoles with inhibitory action on P2X7R impaired the acute inflammatory response in vivo and modulated the hemostatic balance in vitro and ex vivo.

OBJECTIVE: The present study aimed to investigate five triazole compounds as P2X7R inhibitors and evaluate their ability to reduce acute inflammation in vivo. MATERIAL: The synthetic compounds were labeled 5e, 8h, 9i, 11, and 12. TREATMENT: We administered 500 ng/kg triazole analogs in vivo, (1-10 µM) in vitro, and 1000 mg/kg for toxicological assays. METHODS: For this, we used in vitro experiments, such as platelet aggregation, in vivo experiments of paw edema and peritonitis in mice, and in silico experiments. RESULTS: The tested substances 5e, 8h, 9i, 11, and 12 produced a significant reduction in paw edema. Molecules 5e, 8h, 9i, 11, and 12 inhibited carrageenan-induced peritonitis. Substances 5e, 8h, 9i, 11, and 12 showed an anticoagulant effect, and 5e at a concentration of 10 µM acted as a procoagulant. All derivatives, except for 11, had pharmacokinetic, physicochemical, and toxicological properties suitable for substances that are candidates for new drugs. In addition, the ADMET risk assessment shows that derivatives 8h, 11, 5e, and 9i have high pharmacological potential. Finally, docking tests indicated that the derivatives have binding energies comparable to the reference antagonist with a competitive inhibition profile. CONCLUSIONS: Together, the results indicate that the molecules tested as antagonist drugs of P2X7R had anti-inflammatory action against the acute inflammatory response.

Anticoagulant and antiplatelet activity of aqueous extracts of Citrus sinensis and Lippia alba : interactions and potential for the development of antithrombotics.

Citrus sinensis and Lippia alba are herbal medicines widely used in the form of tea (infusion, decoction), which ethanolic extracts have already shown great anticoagulant activity in vitro . For this reason, they seem to be excellent candidates for the development of new antithrombotics and also have the potential to interact with them. The aim of this study was to evaluate the activity of aqueous extracts in blood coagulation and platelet aggregation, in addition to analysing the micromolecular composition of these species. Thrombin generation test (TGT) by the Calibrated Automated Thrombogram method and Platelet Aggregation Test by turbidimetry were performed to evaluate the biological activities, while the chemical composition was qualitatively evaluated using high-performance liquid chromatography. Aqueous extracts were elaborated according to the folk use. All extracts were effective in reducing thrombin formation in TGT. Infusion of L. alba and infusion and decoction of C. sinensis at a concentration of 0.6 mg/ml significantly reduced platelet aggregation induced by ADP, and only the decoction of L. alba at the same concentration was able to significantly reduce collagen-induced platelet aggregation. The presence of phenylpropanoids and flavonoids in C. sinensis and L. alba extracts was verified. Furthermore, hesperidin was identified in C. sinensis through coinjection. C. sinensis and L. alba are rich in phenolics and demonstrated an in-vitro effect on important processes of haemostasis (blood coagulation, platelet agreggation), corroborating the potential of C. sinensis and L. alba for the development of antithrombotics and interact with them.

Utilization of gallic acid to inhibit some toxic activities caused by Bothrops jararaca or B. jararacussu snake venoms.

Snake bite envenoming is a serious public health issue, affecting thousands of people worldwide every year, especially in rural communities of tropical and subtropical countries. Injection of venom into victims may cause hemorrhaging, blood coagulation imbalance, inflammation, pain, edema, muscle necrosis, and eventually, death. The official validated treatment recommended by governments is the administration of antivenom that efficiently prevents morbidity and mortality. However, this therapy does not effectively neutralize the local effects of Viperidae venoms which constitute one of the leading causes of disability or amputation of the affected limb. Thus, bioprospecting studies seeking for alternative therapies to complement antivenom should be encouraged, especially those investigating the blockage of local venomic toxicity. Plants produce a great diversity of metabolites with a wide range of pharmacological and biological properties. Therefore, the objective of this study was to assess the utilization of gallic acid, which is widely found in plants, against some toxic in vitro (coagulation, proteolytic, and hemolytic) or in vivo (edematogenic, hemorrhagic, and lethal) activities of Bothrops jararaca or B. jararacussu venom. Gallic acid was incubated with B. jararaca or B. jararacussu venom (incubation protocol), after which, in vitro or in vivo assays were performed. Additionally, a gel containing gallic acid was developed and topically applied over the skin of mice after injection of B. jararaca or B. jararacussu venom (treatment protocol), and then, a hemorrhagic assay was carried out. As a result, gallic acid inhibited the toxic activities, with variable efficacy, and the gallic acid gel neutralized B. jararaca or B. jararacussu venom-induced hemorrhagic activity. Gallic acid was devoid of in vitro toxicity as shown through a hemocompatibility test. Thus, these findings demonstrate the potential of gallic acid in the development of an alternative agent to treat victims of snake bites inflicted by Bothrops species.

Study on the synthesis and structure-activity relationship of 1,2,3-triazoles against toxic activities of Bothrops jararaca venom.

Snakebite envenoming is a health concern and has been a neglected tropical disease since 2017, according to the World Health Organization. In this study, we evaluated the ability of ten 1,2,3-triazole derivatives AM001 to AM010 to inhibit pertinent in vitro (coagulant, hemolytic, and proteolytic) and in vivo (hemorrhagic, edematogenic, and lethal) activities of Bothrops jararaca venom. The derivatives were synthesized, and had their molecular structures fully characterized by CHN element analysis, Fourier-transform infrared spectroscopy and Nuclear magnetic resonance. The derivatives were incubated with the B. jararaca venom (incubation protocol) or administered before (prevention protocol) or after (treatment protocol) the injection of B. jararaca venom into the animals. Briefly, the derivatives were able to inhibit the main toxic effects triggered by B. jararaca venom, though with varying efficacies, and they were devoid of toxicity through in vivo, in silico or in vitro analyses. However, it seemed that the derivatives AM006 or AM010 inhibited more efficiently hemorrhage or lethality, respectively. The derivatives were nontoxic. Therefore, the 1,2,3-triazole derivatives may be useful as an adjuvant to more efficiently treat the local toxic effects caused by B. jararaca envenoming.

Synthesis, characterization and utilization of a new series of 1,2,3-triazole derivatives to neutralize some toxic activities of Bothrops jararaca snake venom

Abstract Snake envenomation is a public health problem, and while serum therapy prevents death, the local effects of venoms can lead to amputations or morbidities. Thus, alternative treatments deserve attention. In this study, we tested eight derivatives of 1,2,3-triazole against some toxic activities of Bothrops jararaca venom. The derivatives were synthesized, and their structures analyzed by infrared and nuclear magnetic resonance. After that, the ability of compounds to inhibit hemolysis, coagulation, proteolysis, hemorrhaging, edema, and lethal activities of B. jararaca venom was investigated. The derivatives were incubated with B. jararaca venom (incubation protocol), administered before (prevention protocol) or after (treatment protocol) injecting venom into the mice. Then, hemorrhaging assay occurred. As a result, most of the derivatives inhibited the activities, even if they were incubated, injected before or after B. jararaca venom. However, the derivatives TRI 07 and TRI 18 seemed to be the most efficient in impairing hemorrhaging. The derivatives showed a low drug score of toxicity based on an in silico technique. Therefore, the derivatives fulfilled physicochemical and biological requirements to become drugs, and they may be a brand new initiative for designing antivenom molecules to complement antivenom therapy to efficiently block tissue necrosis or any other local effects.

Synergism of in vitro plasmodicidal activity of phospholipase A2 isoforms isolated from panamanian Bothrops asper venom.

Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A2 (PLA2s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA2s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA2s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA2-like. Acidic PLA2s catalyzed the degradation of all substrates evaluated; however, for the basic PLA2s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA2s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum, while the basic PLA2s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA2 and Lys49-PLA2 were associated with one another, denoting synergistic action between these PLA2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA2s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents.

The plant Stryphnodendron adstringens (Mart.) Coville as a neutralizing source against some toxic activities of Bothrops jararacussu snake venom.

Snakebites produce several toxic effects in victims, such as hemorrhage, tissue necrosis, hemostatic, renal, or cardiotoxic alterations, inflammation, and death. To counteract these symptoms, antivenom is the official treatment. Although such therapy prevents death, it does not efficiently neutralize necrosis or other local effects, leading to amputation or morbidities of the affected limb. Therefore, the search for better and more efficient therapies deserves attention; further, plants have been used to ameliorate a number of diseases and medical conditions, including snakebites, for many years. Thus, the aim of this work was to evaluate the antivenom effect of the crude extract, fractions (aqueous and diethyl acetate), and subfractions derived from the aqueous fraction (P1, P2, P3, and P4) of the plant Stryphnodendron adstringens against in vitro (coagulation and proteolytic) and in vivo (edema, hemorrhage, and myotoxic) activities caused by Bothrops jararacussu venom. Overall, all extracts inhibited the toxic effect of B. jararacussu venom, but with different potencies, regardless of whether plant samples were incubated together with venom or injected before or after venom injection into animals; the crude extract and aqueous fraction were found to be the most effective. Indeed, phytochemical and mass spectrometry analysis of S. adstringens samples revealed the presence of flavonols, tannins, and saponins. In conclusion, the plant S. adstringens may represent a promising natural source of molecules to treat the toxic effects associated with envenomation by B. jararacussu snakebites.

Potential use of extract of the plant Schwartiza brasiliensis (choisy) bedell ex gir.-Cañas against the toxic effects of the venom of Bothrops jararaca or B. jararacussu.

Envenomation by snakes is a worldwide health public issue, and antivenoms are less efficient in neutralizing local toxic effects. Thus, more efficient therapies to treat patients deserve attention, and plants have been extensively tested. So, the aim of this work was to evaluate the effect of the aqueous fraction of the plant Schwartzia brasiliensis to inhibit some toxic activities of Bothrops jararaca or B. jararacussu venom. S. brasiliensis inhibited coagulant, hemolytic, proteolytic, hemorrhagic, edematogenic, and lethal activities of both venoms, regardless if plant was mixed together with venoms or injected after them as well as the route of administration (intravenous, oral or subcutaneous) of the plant. The S. brasiliensis extract showed no toxicity to mice or red blood cells. Thus, S. brasiliensis may be useful as an alternative treatment for snakebite envenomation and aid antivenom therapy to neutralize relevant toxic activities in patients bitten by Bothrops species.

Utilization of the Plant Clusia Fluminensis Planch & Triana Against Some Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snake Venom Toxic Activities.

BACKGROUND: In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. OBJECTIVE: The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. METHODS: The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. RESULTS: All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. CONCLUSION: Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.

Chemical structure and snake antivenom properties of sulfated agarans obtained from Laurencia dendroidea (Ceramiales, Rhodophyta).

Aqueous and KCl-soluble polysaccharides were extracted from Laurencia dendroidea (Rhodomelaceae, Ceramiales) and their chemical profile was accessed by anion-exchange chromatography, chemical and spectroscopic analyses. The homogeneous agaran DHS-4 (181.3 × 103 g. mol-1, 21.3% of NaSO3) presents A units mostly 2-sulfated (18.9 mol%), nonsubstituted (15.3 mol%) and 6-O-methylated (10.1 mol%), while B units are l-sugars composed predominantly by galactose 6-sulfate precursor units (19.2 mol%) and 3,6-anhydrogalactose (13.8 mol%), besides non-precursor galactose 6-sulfate units bearing d-xylose substituents on C-3 (8.1 mol%). The crude KCl-soluble DHS agaran (20.5% of NaSO3) inhibited proteolysis and hemolysis induced by Lachesis muta and Bothrops jararaca venoms. DHS was able to inhibit up to 75% the L. muta venom hemorrhagic effect and to reduce the lethality displayed by B. jararaca venom, increasing the mice survival time up to 3 times. Therefore, this agaran has high potential to be used as an additional tool to treat snakebite envenomation.

Protective effect of Myrsine parvifolia plant extract against the inflammatory process induced by Bothrops jararaca snake venom.

Accidents involving snakes from the genus Bothrops sp. constitute the most important cause of snake envenomation in Brazil. The Myrsine genus has been reported to be used in folk medicine against snakebites. In this work, the phytochemical profiles and ability of extracts from Myrsine parvifolia leaves to reduce the inflammatory process (edema, vascular permeability increase and leukocyte migration) induced by Bothrops jararaca venom were investigated in vivo. Chemical compounds were identified by chromatographic and spectroscopy techniques. Total polyphenol, tannin, and flavonoid contents were determined by spectrophotometric methods. Swiss male mice received an oral administration of extracts (100 mg/kg) in different protocols. Paw edema, intraperitoneal vascular permeability and pleurisy models in mice were used to evaluate the antiophidic potential of the extracts. Paw edema was induced by subplantar injection of B. jararaca venom and quantified as the increase in paw volume. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. Leukocyte migration was assessed by total and differential counts in the pleural cavity washes. Myricetin, myricetin-3-O-ß-arabinopyranoside, quercetin and kaempferol were isolated from the ethyl acetate extract and identified as the primary compounds of the dichloromethane extract. Terpenes and fatty acids were identified in the hexane and dichloromethane extracts. The pretreated group with hydroethanolic and dichloromethane extract reduced total edema (40 and 52%, respectively), vascular permeability increase (32.4 and 32.2%, respectively) and leukocyte influx into the pleural cavity (42 and 39%, respectively), while the group treated with hexane extract showed only reduced edema (37%) induced by B. jararaca venom. The hydroethanolic extract showed better results in all of the tests performed and was also administered by the protocol of post-poisoning, showing maintenance of paw edema reduction and cell migration. These data indicate a potential anti-inflammatory activity of M. parvifolia in poisoning by B. jararaca, especially to reduce local poison effects.

Potential Utilization of a Polysaccharide from the Marine Algae Gayralia oxysperma, as an Antivenom for Viperidae Snakebites.

Worldwide, snakebites have serious implications for human health. The administration of antivenom is the official treatment used to reverse the toxic activities of envenomation. However, this therapy is not efficient to treat the local effects, leading to the amputation or deformity of affected limbs. As such, alternative treatments are needed. Here, we analyze the ability of a polysaccharide from the green marine alga Gayralia oxysperma (Go3) to inhibit the effects of venom from Bothrops jararaca and Lachesis muta. B. jararaca or L. muta venoms were incubated together with sulfated heterorhamnans from Go3, and the in vitro (coagulation, proteolytic, and hemolytic) and in vivo (hemorrhagic, myotoxic, edematogenic, and lethal) activities of venoms were assessed. Additionally, Go3 was injected before and after the injection of venoms, and the toxic activities were further tested. When incubated with the venoms, Go3 inhibited all activities, though results varied with different potencies. Moreover, Go3 neutralized hemorrhagic, myotoxic, and edematogenic activities when injected before or after injection with B. jararaca and L. muta venom. Go3 also blocked the coagulation of plasma in mice caused by the venoms in an ex vivo test. Therefore, Go3 has the potential to be used as antivenom for B. jararaca and L. muta bites, notably exhibiting higher efficacy on L. muta venom.

Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties.

BACKGROUND: Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall). METHODS: The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities. RESULTS: All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C. gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C. gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells. CONCLUSION: The cnidarian extracts analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms; these results may contribute to elucidate the possible mechanisms of interaction between cnidarian extracts and snake venoms.

Oscillatory shear stress induces hemostatic imbalance in healthy men.

INTRODUCTION: In vitro and animal model studies have demonstrated that oscillatory shear can trigger vascular hemostasis and remodeling. However, the roles of hemodynamic forces in vascular human biology are not well understood. This study aimed to determine the effects of increasing oscillatory shear stress (OSS) on coagulation/fibrinolysis factors and matrix metalloproteinase-9 activity in healthy subjects. MATERIALS AND METHODS: Ten healthy males (35 ±â€¯7 years) underwent a 30-minute dominant forearm cuff occlusion (75 mm Hg) to exacerbate OSS in the brachial artery. Blood flow was quantified (Doppler ultrasound), and plasma samples were obtained from both arms at rest and during the last 30 s of cuff occlusion on the dominant arm. A proximal cuff (40 mm Hg, close to axilla) was also occluded to facilitate venous blood biomarker trapping. RESULTS: The retrograde shear rate and oscillatory shear index were increased and the mean shear rate, mean blood velocity, and mean blood flow were decreased in the cuffed arm (p < 0.05 vs. baseline and non-cuffed arm). Cuff occlusion induced increases in platelet microparticle release (p = 0.05 vs. baseline), prothrombin time (p < 0.05 vs. baseline and non-cuffed arm), tissue plasminogen activator (p < 0.01 vs. baseline and non-cuffed arm), plasminogen activator inhibitor-1 (p < 0.02 vs. baseline and non-cuffed arm), and matrix metalloproteinase-9 activity (p = 0.01 vs. baseline). No significant changes were found in the non-cuffed arm throughout the protocol. CONCLUSIONS: Exacerbation of OSS induced in vivo disturbances in platelet microparticle release, coagulation-fibrinolysis, and matrix metalloproteinase-9 activity in healthy individuals. These are potential mechanisms involved in OSS-mediated endothelial dysfunction.

Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming.

Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.

Anti-platelet aggregation activity of two novel acidic Asp49-phospholipases A2 from Bothrops brazili snake venom.

Phospholipases A2 (PLA2s) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLA2s from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLA2s was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ​​of 13,894 and 13,869Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLA2s using acid PLA2BthA-I-PLA2 from B. jararacussu template showed high quality. Both acidic PLA2s showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100µg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10µg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLA2s biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads.