Nanoemulsion containing Jatropha gossypiifolia leaf extract reduces dermonecrosis induced by Bothrops erythromelas venom and accelerates wound closure.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Jatropha gossypiifolia, popularly known as "pinhão-roxo", is distributed throughout Brazil, is commonly employed for topical or oral administration in treating wounds, inflammations, and snake bites. Given the significant impact of snakebites on public health and the limitations of antivenom, coupled with the diverse molecular composition of this plant species, investigating its healing and antidermonecrotic capacities is relevant. AIM OF THE STUDY: This study aimed to develop a topical nanoemulsion incorporating the hydroethanolic extract of J. gossypiifolia leaves, to evaluate its therapeutic potential, particularly in terms of its efficacy in wound healing and inhibition of dermonecrosis induced by B. erythromelas venom (BeV). MATERIAL AND METHODS: The extract of J. gossypiifolia (JgE) leaves was obtained by maceration and remaceration. The phytochemical analysis was conducted and J. gossypiifolia nanoemulsion (JgNe) was obtained, characterized and assessed for stability. The cytotoxicity was determined in normal cells (erythrocytes and 3T3) using hemolytic assay and cell viability assay using crystal violet staining. The antioxidant activity was evaluated by the reduction of ABTS and DPPH radicals. The evaluation of wound healing was conducted in vivo following treatment with JgNe, wherein the percentage of wound closure and inflammatory mediators. The skin irritation test was assessed in vivo by applying JgNe directly to the animal's skin. In vitro, the antivenom capacity was evaluated through enzymatic inhibition assays (phospholipase A2 and hyaluronidase) of BeV. Additionally, the in vivo antidermonecrotic activity of JgNe was evaluated by measuring the reduction of the dermonecrotic halo. RESULTS: The HPLC-DAD analysis identified flavonoids, specifically vitexin, luteolin derivatives and apigenin derivatives. In addition, 95.08 ± 5.46 mg of gallic acid/g of extract and 137.92 ± 0.99 mg quercetin/g extract, was quantified. JgNe maintained stability over a 4-week period. Moreover, JgE and JgNe demonstrated no cytotoxicity in human erythrocytes and murine fibroblasts at tested concentrations (32.25-250 µg/mL). Additionally, exhibited significant antioxidant activity by reducing ABTS and DPPH radicals. The treatment with JgNe did not induce skin irritation and accelerated wound healing, with significant wound closure observed from 5th day and reduction in nitrite levels, myeloperoxidase activity, and cytokine. Both JgE and JgNe demonstrated in vitro inhibition of the phospholipase and hyaluronidase enzymes of BeV. Moreover, JgNe exhibited antidermonecrotic activity by reducing the dermonecrotic halo caused by BeV after 24 h. CONCLUSIONS: JgNe and JgE exhibited no cytotoxicity at the tested concentrations. Additionally, our findings demonstrate that JgNe has the ability to accelerate wound closure and reduce dermonecrosis caused by BeV, indicating to be promising formulation for complementary therapy to antivenom treatment.

Potential low-impact immunogen for the production of anti-bothropic serum: Bothrops alternatus venom treated with Na2EDTA.

This study proposes an alternative method using Na2EDTA to neutralize B. alternatus venom and using it as an immunogen from the start of inoculation to minimize side effects and enhance antivenom production. To achieve this, 1.8 mg/mL of B. alternatus venom (B.aV) was treated with Na2EDTA, and any extra chelate was eliminated by filtering the resulting solution through a Sephadex G-25 column. Two groups of BALB/c mice were immunized subcutaneously on days 1, 15 and 30 with B.aV/Na2EDTA (45, 90, 135 µg/mouse) or B.aV (15, 30, 45 µg/mouse), respectively. Both formulations were emulsified with Freund's adjuvant (complete first and incomplete-booster). Blood samples were collected from each mouse on days 14, 29, 41, and 50 post-first immunization, and serum was separated for antibody detection. Animals were then sacrificed and lungs removed for histological analysis (hematoxylin-eosin). Immunoblotting analysis revealed that the sera from mice inoculated with B.aV/Na2EDTA (anti-B.aV/Na2EDTA) recognized the major venom proteins (20-66 kDa) similarly to the sera from mice inoculated with B.aV (anti-B.aV). The enzyme-linked immunosorbent assay results indicated that the anti-B.aV/Na2EDTA had a higher titer (5.76 × 104) than those the anti-B.aV (1.92 × 104). Additionally, sera from animals immunized with B.aV/Na2EDTA significantly neutralized proteolytic, indirect hemolytic and coagulant activity (p < 0.05). Finally, histological examination of the lungs of mice inoculated with B.aV/Na2EDTA showed normal appearance, while animals inoculated with B.aV showed interstitial lung injury (p < 0.05). In conclusion, the B.aV/Na2EDTA formulation, free of excess Na2EDTA, proved to be a promising candidate as an immunogen for antivenom production.

Analysis of Hemostatic Effect and Safety of Local Spray Treatment With Hemocoagulase Bothrops Atrox for Injection After Resection of Colon Polyps.

Context: Postoperative bleeding after resection of colon polyps (CPs) is an extremely common adverse event with endoscopic treatment. Hemocoagulase Bothrops Atrox (HBA) is a newly discovered hemostatic substance that contains thrombin-like and coagulation kinase-like enzymes. However, research is lacking about its use for the treatment of intestinal polyps. Objective: The study intended to examine the hemostatic efficacy and safety of a local spray treatment with HBA, derived from HBA for injection, after CP resection, to provide a new hemostatic method, support HBA's use, and provide evidence for clinical decision making. Design: The research team performed a randomized controlled study. Setting: The study took place at the Affiliated Hospital of Hebei University in Baoding, Hebei, China. Participants: Participants were 200 patients with CP who received treatment at the hospital between December 2020 and December 2022. Intervention: The research team divided participants into two groups with 100 participants each, an intervention group and a control group, using the random number expression method. For hemostasis, the intervention group received a local spray treatment that used HBA for injection, and the control group received metal-clip closure or electrocoagulation. Outcome Measures: The research team measured: (1) the hemostatic efficacy; (2) clinical outcomes-time to hemostasis, hemostasis rate, rebleeding rate, and incidence of late postoperative bleeding; (3) at baseline and at 24h postintervention, the coagulation function-prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB); (4) at baseline and at 24h postintervention, PLT parameters-platelet count (PLT), procalcitonin (PCT), and mean platelet volume (MPV); (5) economic effects-total number of participants with hemostasis, hospital days, and total hospital costs; and (6) adverse reactions. Results: The total hemostatic efficacy for the intervention group was significantly higher than that of the control group (P = .027), and the time to hemostasis was significantly shorter (P < .001) and the hemostasis rate, rebleeding rate, and incidence of late postoperative bleeding were all significantly lower than those of the control group, at P = .009, P = .009, and P = .048, respectively. In addition, the intervention group's postoperative PT, TT, APTT, FIB, and MPV were all significantly lower than those of the control group (all P < .05), while its PLT and PCT were significantly higher than those of the control group (both P < .05). The intervention group's total number of participants with hemostasis, participants with hemostasis, hospital days, and total cost were significantly lower than those of the control group (all P < .05), while no significant difference existed between the groups in the incidence of adverse effects (P > .05). Conclusions: HBA has an excellent hemostatic effect on intestinal polypectomy, with convenient use and high safety. In the future, popularizing the use of HBA in the treatment of intestinal polypectomy can not only effectively guarantee the postoperative safety of patients but also could reduce their economic burden and improve the quality of clinical medical services.

Effects of Mauritia flexuosa L. f. buriti oil on symptoms induced by Bothrops moojeni snake envenomation.

ETHNOPHARMACOLOGICAL RELEVANCE: 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.

Morbidity survey of the history of snakebites in different communities in the alto Juruá, western Brazilian Amazon.

The Alto Juruá region has a rich diversity of venomous snakes, and snakebites represent an important cause of morbidity. The present study was carried out in five communities in the Alto Juruá region, and aimed to evaluate the history of snakebites in terms of which snakes were involved and what was the victims' conduct in relation to first aid and prevention. Between 2017 and 2019, semi-structured interviews were applied to 260 residents, 56 (21.53%) of whom had a history of snakebites. The profile of the victims is 73.2% male, a resident of rural areas who develops activities mainly in agriculture and extractivism and who has a low level of education. Just over half of the snakebites occurred during the rainy season (52.8%) and the majority during the day (77%). Bothrops bites were the most frequently reported (90.3%) and B. atrox was the most mentioned snake (jararaca and surucucu) for being responsible for snakebites (80.8%). Approximately 87.5% of the bites occurred to the lower limbs, and 48.2% of the victims were barefoot at the time of the bite. Inadequate first-aid procedures were reported, such as using tourniquets and consuming the drink known as "Específico Pessoa", not drinking water, and treatment with traditional therapy (phytotherapics, zootherapeutics and faith healers), as well as not seeking hospital care. Thus, the need to facilitate access to hospital units in these communities and health education (prevention and first aid) are essential in order to minimize this situation.

Structural and functional studies of a snake venom phospholipase A2-like protein complexed to an inhibitor from Tabernaemontana catharinensis.

Snake envenomation is an ongoing global health problem and tropical neglected disease that afflicts millions of people each year. The only specific treatment, antivenom, has several limitations that affects its proper distribution to the victims and its efficacy against local effects, such as myonecrosis. The main responsible for this consequence are the phospholipases A2 (PLA2) and PLA2-like proteins, such as BthTX-I from Bothrops jararacussu. Folk medicine resorts to plants such as Tabernaemontana catharinensis to palliate these and other snakebite effects. Here, we evaluated the effect of its root bark extract and one of its isolated compounds, 12-methoxy-4-methyl-voachalotine (MMV), against the in vitro paralysis and muscle damage induced by BthTX-I. Secondary and quaternary structures of BthTX-I were not modified by the interaction with MMV. Instead, this compound interacted in an unprecedented way with the region inside the toxin hydrophobic channel and promoted a structural change in Val31, loop 58-71 and Membrane Disruption Site. Thus, we hypothesize that MMV inhibits PLA2-like proteins by preventing entrance of fatty acid into the hydrophobic channel. These data may explain the traditional use of T. catharinensis extract and confirm MMV as a promising candidate to complement antivenom or a structural guide to develop more effective inhibitors.

The anti-snake activity of Nectandra angustifolia flavonoids on phospholipase A2: In vitro and in silico evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Lauraceae family includes Nectandra angustifolia a species widely used in the folk medicine of South America against various maladies. It is commonly used to treat different types of processes like inflammation, pain, and snakebites. Snakes of the Bothrops genus are responsible for about 97% of the ophidic accidents in northeastern Argentina. AIM OF THE STUDY: To evaluate the anti-snake activity of the phytochemicals present in N. angustifolia extracts, identify the compounds, and evaluate their inhibitory effect on phospholipase A2 (PLA2) with in vitro and in silico assays. METHODS: Seasonal variations in the alexiteric potential of aqueous, ethanolic and hexanic extracts were evaluated by inhibition of coagulant, haemolytic, and cytotoxic effects of B. diporus venom. The chemical identity of an enriched fraction obtained by bio-guided fractioning was established by UPLC-MS/MS analysis. Molecular docking studies were carried out to investigate the binding mechanisms of the identified compounds to PLA2 enzyme from snake venom. RESULTS: All the extracts inhibited venom coagulant activity. However, spring ethanolic extract achieved 100% inhibition of haemolytic activity. Bio-guide fractioning led to an enriched fraction (F4) with the highest haemolytic inhibition. Five flavonoids were identified in this fraction; molecular docking and Molecular Dynamics (MD) simulations indicated the binding mechanisms of the identified compounds. The carbohydrates present in some of the compounds had a critical effect on the interaction with PLA2. CONCLUSION: This study shows, for the first time, which compounds are responsible for the anti-snake activity in Nectandra angustifolia based on in vitro and in silico assays. The results obtained in this work support the traditional use of this species as anti-snake in folk medicine.

Photobiomodulation therapy on local effects induced by juvenile and adult venoms of Bothrops alternatus.

Bothrops snake envenomation is characterized by severe local manifestations such as pain, edema, inflammation, hemorrhage, and myonecrosis. Furthermore, it is described that venom from juvenile and adult snakes may have differences in their composition that can lead to differences in the evolution of the clinical manifestation of the victim. Photobiomodulation (PBM) has been shown to be an effective adjuvant therapy to serum therapy to reduce the local effects induced by bothropic snake venom. This study evaluated the effect of PBM on the local reaction, after Bothrops alternatus snake venom (BaV) injection, in its juvenile (BaJV) and adult (BaAV) stages. Balb/C mice were injected with the juvenile or adult venoms of BaV or saline solution (control group). PBM at a wavelength of 660 nm, 100 mW, 0.33 W/cm2, 40 s, and a 0.028 cm2 beam was applied transcutaneous to a single point with a radiant exposure of 4 J/cm2, 30 min after venom injection. Edema, inflammatory infiltrate, hyperalgesia, and myonecrosis were analyzed. Both venoms induced significant edema and myonecrosis in the gastrocnemius muscle. Hyperalgesia in the mice paw and a prominent leukocyte infiltrate into the peritoneum were also observed. PBM significantly reduced all evaluated parameters. In conclusion, PBM treatment was effective in reducing the local effects induced by B. alternatus venom at different stages of snake development and could be a useful tool as an adjuvant treatment for bothropic envenomation.

Topical gel containing phenolic-rich extract from Ipomoea pes-capre leaf (Convolvulaceae) has anti-inflammatory, wound healing, and antiophidic properties.

The growing use of phytotherapy in clinical practice arouses interest in studies using medicinal plants as active ingredients for new medicines. Ipomoea pes-caprae has a wide medicinal use in the treatment of inflammatory disorders, skin wounds, stings, and painful rheumatic processes. Assayed in this study are the physicochemical characterization of a gel developed with this extract and the evaluation of its anti-inflammatory and healing efficacy, in addition to its antiedematogenic action on Bothrops snake envenoming in mice. The qualitative and quantitative analyses of the hydroethanolic extract by mass spectrometry showed 18 phenolic compounds, highlighting a high content of chlorogenic acid (0.92 µg/g), neochlorogenic acid (6.07 µg/g), and isochlorogenic acid (0.80 µg/g) compounds. The formulation was stable in relation to the physical-chemical characteristics during the time of analysis and was considered safe for topical treatment in animals, causing no skin irritation. Although the results have shown an absence of activity in the model of ear edema induced by croton oil (acute inflammation), the herbal gel efficiently inhibited carrageenan paw edema and chronic ear edema induced by multiple applications of croton oil, which may indicate the possible performance under the kinin pathway such as bradykinin, histamine, and serotonin. Wound healing in the group treated with the I. pes-caprae gel was accelerated compared with the placebo group, also confirmed through histological data. Edema induced by Bothrops erythromelas snake venom was efficiently reduced in the treatment with I. pes-caprae gel associated with the antibothropic-crotalic serum, whereas the antivenom alone was not effective. This approach presents a promising formulation based on I. pes-caprae with potential therapeutic use for inflammatory disorders.

The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni.

(1) Background: The amino acid sequence elucidation of peptides from the gas phase fragmentation mass spectra, de novo sequencing, is a valuable method for the identification of unknown proteins complementary to Edman sequencing. It is increasingly used in shot-gun mass spectrometry (MS)-based proteomics experiments. We review the current state-of-the-art and use the identification of an unknown snake venom protein targeting the human tissue factor (TF) as an example to describe the analysis process based on manual spectrum interrogation. (2) Methods: The immobilized TF was incubated with a crude B. moojeni venom solution. The potential binding partners were eluted and further purified by gel electrophoresis. Edman degradation was performed to elucidate the N-terminus of the 31 kDa protein of interest. High-resolution MS with collision-induced dissociation was employed to generate peptide fragmentation spectra. Sequence tags were deduced and used for searches in the NCBI and Uniprot databases. Protein matches from the snake species were further validated by target MS/MS. (3) Results: Sequence tag D [K/Q] D [I/L] VDD [K/Q] led to a snake venom serine protease (SVSP) from lancehead B. jararaca (P81824). With target MS/MS, 24% of the SVSP sequence were confirmed; an additional 41% were tentatively assigned by data-independent MS. Edman sequencing provided information for 10 N-terminal amino acid residues, also confirming the match to SVSP. (4) Conclusions: The identification of unknown proteins continues to be a challenge despite major advances in MS instrumentation and bioinformatic tools. The main requirement is the generation of meaningful, high-quality MS peptide fragmentation spectra. These are used to elucidate sufficiently long sequence tags, which can subsequently be submitted to searches in protein databases. This basic method does not require extensive bioinformatics because peptide MS/MS spectra, especially of doubly-charged ions, can be analysed manually. We demonstrated the procedure with the elucidation of SVSP. While de novo sequencing quickly indicates the correct protein group, the validation of the entire protein sequence of amino acid-by-amino acid will take time. Reasons are the need to properly assign isobaric amino acid residues and modifications. With the ongoing efforts in genomics and transcriptomics and the availability of ever more data in public databases, the need for de novo MS sequencing will decrease. Still, not every animal and plant species will be sequenced, so the combination of MS and Edman sequencing will continue to be of importance for the identification of unknown proteins.

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.

A First Look at the Inhibitory Potential of Urospatha sagittifolia (Araceae) Ethanolic Extract for Bothrops atrox Snakebite Envenomation.

Bothrops atrox snakebites are a relevant problem in the Amazon basin. In this biodiverse region, the ethnomedicinal approach plays an important role as an alternative to antivenom therapy. Urospatha sagittifolia (Araceae) is a plant used for this purpose; however, its neutralizing properties have not been scientifically accessed. To fill this gap, we investigated the ability of U. sagittifolia to modulate the catalytic activity of Bothrops atrox venom, and their toxic consequences, such as local damage and lethality. The venom profile of B. atrox was assessed by chromatography and electrophoresis. Inhibition of the three main enzymatic and medically important toxins from the venom was evaluated using synthetic substrates and quantified by chromogenic activity assays. Additionally, the neutralization of lethality, hemorrhage and edema were investigated by in vivo assays. The possible interactions between venom proteins and plant molecules were visualized by polyacrylamide gel electrophoresis. Finally, the phytochemical constituents present in the ethanolic extract were determined by qualitative and quantitative analyses. The ethanolic extract reduced the activity of the three main enzymes of venom target, achieving ranges from 19% to 81% of inhibition. Our in vivo venom neuralizations assays showed a significant inhibition of edema (38.72%) and hemorrhage (42.90%). Additionally, lethality was remarkably counteracted. The highest extract ratio evaluated had a 75% survival rate. Our data support the biomedical value of U. sagittifolia as a source of natural enzyme inhibitors able to neutralize catalytically active B. atrox venom toxins and their toxic effects.

The repellent activity evaluation of the ethanolic extract of the tipi herb against the Bothrops moojeni serpents.

The serpent study involves physiological, behavioral and interaction factors with an extremely dynamic and complex environment. The relation of these animals with biodiversity is still quite preliminary, little studied and sustained by popular practices related to traditional medicine, esoteric and mystical currents. Therefore, this study proposed to know about the effect of 80% crude alcoholic extract of Petiveria alliacea L., popularly known as Tipi Herb, and its effects associated with the repellency of the Bothrops moojeni serpent. A complex of boxes was built for the observation of the snake's displacement, carried out for a total period of six days, the first three for defining the exploratory pattern and the other two for testing the alcoholic extract of Tipi, and finally on the sixth day, one cycle with the presence of a live attractant as bait. It was observed that the animal had no inhibition behavior and moved through the complex in a way like what occurs in nature and showed behavior to avoid remaining in the presence of the extract both with the presence of a live attractant or without it, preferring to move in the other compartments. The results show that this proposed tool is valid for the study of displacement of Bothrops moojeni snakes, in the same way that it added values on the importance of cerrado biome biodiversity and on the popular medicine practices of Tipi Herb, since the repellent effect on the snake was significant in the two tests performed.

Inhibition of enzymatic activities of Bothrops asper snake venom and docking analysis of compounds from plants used in Central America to treat snakebite envenoming.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenoming is a public health problem of high impact in Central America. Bothrops asper, known as barba amarilla, terciopelo, and equis, is the snake species responsible for most snakebites in Central America. In this region, there is a long-standing tradition on the use of plants in the management of snakebites, especially in indigenous communities. Ethnomedical use of Eryngium foetidum L., Neurolaena lobata (L.) Cass. and Pimenta dioica (L.) Merr. to treat snakebite envenoming has been reported in Belice, Guatemala, Nicaragua, and Costa Rica. Extracts of the leaves of these plants have shown anti-venom activities in in vitro assays in previous studies. AIM OF THE STUDY: To assess the ability of organic fractions from these three plants to inhibit enzymatic activities associated with toxicity of the venom of B. asper, and to study, by docking analysis, the interaction of metalloproteinase and phospholipases A2 (PLA2) from B. asper venom with secondary metabolites previously described in these plants. MATERIALS AND METHODS: Organic fractions were obtained from these three plant species and their ability to neutralize proteolytic, PLA2 and in vitro coagulant activities of B. asper venom was assessed. A phytochemical analysis was carried out in these fractions. The interaction of secondary metabolites previously described in these plants with three toxins from B. asper venom (a metalloproteinase, a PLA2 and a PLA2 homologue) was investigated by docking analysis. RESULTS: The inhibitory activity of plants was mainly concentrated in their polar fractions. Acetonic fraction from P. dioica was the most active against PLA2 activity, while the acetonic fraction of E. foetidum completely inhibited the proteolytic activity of the venom. Coagulant activity was partially inhibited only by the acetone and ethyl acetate fractions of P. dioica. Phytochemical analysis of the most bioactive fractions identified flavonoids, saponins, essential oils, coumarins, alkaloids, tannins and sesquiterpene lactones. Docking analysis revealed high affinity interactions of several secondary metabolites of these plants with residues in the vicinity of the catalytic site of these enzymes and, in the case of PLA2 homologue myotoxin II, in the hydrophobic channel. CONCLUSIONS: Various fractions from these plants have inhibitory activity against enzymatic actions of B. asper venom which are directly associated with toxicological effects. Docking analysis showed structural evidence of the interaction of secondary metabolites with three toxins. These observations provide support to the potential of these plants to inhibit relevant toxic components of this snake venom.

Neutralization of toxic activities of Bothrops asper snake venom by ethnomedicinal plants used in Central America, with emphasis in Guatemala: a review / Neutralización de actividades tóxicas del veneno de la serpiente Bothrops asper por plantas etnomedicinales utilizadas en Centroamérica, con énfasis en Guatemala: Revisión

There are few scientific studies that explore the use of medicinal plants for snakebite envenoming in Central America, although plant-based therapies have been traditionally used in the region. This work reviews the studies conducted in Central America to assess the ability of extracts obtained from plants of local ethnomedical use to inhibit toxic activities of the venom of Bothrops asper, the snake responsible for approximately half of the snakebite envenomings in these countries. The search prioritized the description of the plants used in Guatemala, since most of the studies described in this work were conducted in that country, although references to other countries are included. Information concerning secondary metabolites and other pharmacological activities of these plant species, relevant to the treatment of snakebites, was also described. The literature search was conducted in the Google Scholar, PubMed and Scopus databases and completed with locally available literature. It was found that extracts of 12 plant species inhibited the hemorrhagic effect of the venom and three neutralized the edema-forming activity, while inhibition of proteolytic and phospholipase A2 (PLA2) activities was achieved by three and one plant species, respectively. Only Brownea rosa-de-monte was able to effectively counteract the in vitro coagulant effect of the venom. Some plant extracts screened in Guatemala demonstrated procoagulant or anti-thrombin intrinsic effects that might aggravate the coagulopathy induced by the venom. These findings underscore the need of carrying out scientific studies aimed to validate the inhibitory potential of Central American plant extracts and their metabolites against B. asper venom.

Pocos estudios científicos han explorado el uso de plantas medicinales para el tratamiento del envenenamiento ofídico en Centroamérica, a pesar de que las terapias basadas en plantas son de uso tradicional en la región. Este trabajo recopiló información sobre los estudios realizados en Centroamérica para evaluar la capacidad de extractos de plantas de uso etno-médico para inhibir las actividades tóxicas del veneno de Bothrops asper, la serpiente responsable de aproximadamente la mitad de los envenenamientos ofídicos en Centroamérica. La búsqueda priorizó la descripción de plantas utilizadas en Gua-temala, ya que la mayoría de los estudios aquí descritos fueron realizados en ese país. También se incluyó la descripción de los metabolitos secundarios y otras actividades farmacológicas de las especies evaluadas, que podrían explicar su uso como antiofídicos. La búsqueda de literatura se realizó en las bases de datos de Google Scholar, PubMed, Scopus, y se completó con literatura disponible localmente. Se determinó que 12 extractos de plantas inhibieron el efecto hemorrágico del veneno y tres el efecto edematígeno; la actividad proteolítica fue inhibida por extractos de tres especies y la fosfolipasa A2 (PLA2) por una especie. Solamente Brownea rosa-de-monte demostró inhibir efectivamente el efecto coagulante del veneno in vitro. Algunos extractos de las plantas tamizadas en Guatemala demostraron efectos procoagulantes o anti-trombina intrínsecos, que podrían agravar las alteraciones inducidas por el veneno en la coagulación. Estos hallazgos enfatizan la necesidad de validar el potencial de extractos de plantas centroamericanas y sus metabolitos secundarios para neutralizar el veneno de B. asper.

Oral Tolerance Induction by Bothrops jararaca Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms.

Oral tolerance is defined as a specific suppression of cellular and humoral immune responses to a particular antigen through prior oral administration of an antigen. It has unique immunological importance since it is a natural and continuous event driven by external antigens. It is characterized by low levels of IgG in the serum of animals after immunization with the antigen. There is no report of induction of oral tolerance to Bothrops jararaca venom. Here, we induced oral tolerance to B. jararaca venom in BALB/c mice and evaluated the specific tolerance and cross-reactivity with the toxins of other Bothrops species after immunization with the snake venoms adsorbed to/encapsulated in nanostructured SBA-15 silica. Animals that received a high dose of B. jararaca venom (1.8 mg) orally responded by showing antibody titers similar to those of immunized animals. On the other hand, mice tolerized orally with three doses of 1 µg of B. jararaca venom showed low antibody titers. In animals that received a low dose of B. jararaca venom and were immunized with B. atrox or B. jararacussu venom, tolerance was null or only partial. Immunoblot analysis against the venom of different Bothrops species provided details about the main tolerogenic epitopes and clearly showed a difference compared to antiserum of immunized animals.

Photobiomodulation induces murine macrophages polarization toward M2 phenotype.

Photobiomodulation using light-emitting diode (LED) treatment has analgesic and anti-inflammatory effects which can be an effective therapeutic associated with serum therapy for local treatment of snakebites. Here we explored the effects of LED treatment on isolated macrophage under Bothrops jararacussu venom. Results showed that LED induced IL-6 and TNF-α genes down-regulation and, TGF and ARG1 genes up-regulation which indicates a polarization of macrophages to an M2 phenotype contributing to both tissue repair and resolution of inflammation.

Phytochemical composition, antisnake venom and antibacterial activities of ethanolic extract of Aegiphila integrifolia (Jacq) Moldenke leaves.

Snakebites are considered a major neglected tropical disease, resulting in around 100,000 deaths per year. The recommended treatment by the WHO is serotherapy, which has limited effectiveness against the toxins involved in local tissue damage. In some countries, patients use plants from folk medicines as antivenoms. Aegiphila species are common plants from the Brazilian Amazon and are used to treat snakebites. In this study, leaves from Aegiphila integrifolia (Jacq) Moldenke were collected from Roraima state, Brazil and its ethanolic extract was evaluated through in vitro and in vivo experiments to verify their antiophidic activity against Bothrops atrox crude venom. The isolated compounds from A. integrifolia were analyzed and the chemical structures were elucidated on the basis of infrared, ultraviolet, mass, 1H and 1³C NMR spectrometry data. Among the described compounds, lupeol (7), betulinic acid (1), ß-sitosterol (6), stigmasterol (5), mannitol (4), and the flavonoids, pectolinarigenin (2) and hispidulin (3), were identified. The ethanolic extract and flavonoids (2 and 3) partially inhibited the proteolytic, phospholipase A2 and hyaluronidase activities of B. atrox venom, and the skin hemorrhage induced by this venom in mice. Antimicrobial activity against different bacteria was evaluated and the extract partially inhibited bacterial growth. Thus, taken together, A. integrifolia ethanolic extract has promising use as an antiophidic and antimicrobial.

Exploring the ability of low-level laser irradiation to reduce myonecrosis and increase Myogenin transcription after Bothrops jararacussu envenomation.

Envenoming caused by snakebites is a very important neglected tropical disease worldwide. The myotoxic phospholipases present in the bothropic venom disrupt the sarcolemma and compromise the mechanisms of energy production, leading to myonecrosis. Photobiomodulation therapy (PBMT) has been used as an effective tool to treat diverse cases of injuries, such as snake venom-induced myonecrosis. Based on that, the aim of this study was to analyze the effects of PBMT through low-level laser irradiation (904 nm) on the muscle regeneration after the myonecrosis induced by Bothrops jararacussu snake venom (Bjssu) injection, focusing on myogenic regulatory factors expression, such as Pax7, MyoD, and Myogenin (MyoG). Male Swiss mice (Mus musculus), 6-8-week-old, weighing 22 ± 3 g were used. Single sub-lethal Bjssu dose or saline was injected into the right mice gastrocnemius muscle. At 3, 24, 48, and 72 h after injections, mice were submitted to PBMT treatment. When finished the periods of 48 and 72 h, mice were euthanized and the right gastrocnemius were collected for analyses. We observed extensive inflammatory infiltrate in all the groups submitted to Bjssu injections. PBMT was able to reduce the myonecrotic area at 48 and 72 h after envenomation. There was a significant increase of MyoG mRNA expression at 72 h after venom injection. The data suggest that beyond the protective effect promoted by PBMT against Bjssu-induced myonecrosis, the low-level laser irradiation was able to stimulate the satellite cells, thus enhancing the muscle repair by improving myogenic differentiation.

Actions of Cannabis sativa L. fixed oil and nano-emulsion on venom-induced inflammation of Bothrops moojeni snake in rats.

BACKGROUND: Bothrops moojeni snake venom (VBm) has toxins that cause pronounced tissue damage and exacerbated inflammatory reaction. Cannabis sativa L. is a plant species that produces an oil (CSO) rich in unsaturated fatty acids. Nano-emulsions have several advantages, such as better stability and higher penetrating power in membranes. Therefore, this study evaluated the effect of a nano-emulsion based on this herbal derivative (NCS) against VBm-induced inflammation in Wistar rats. METHODS: The CSO and NCS were submitted to physicochemical characterization. The inflammatory process was induced by the VBm (0.10 mg/kg) as follows: rat paw edema, peritonitis, analysis of leukocyte infiltrate in gastrocnemius muscle of rats and formation of granulomatous tissue. RESULTS: No significant changes were observed when the NCS was submitted to the centrifugation and thermal stress tests. There was no phase separation, changes in density (0.978 ± 0.01 g/cm3) and viscosity (0.889 ± 0.15). The droplet diameter ranged from 119.7 ± 065 to 129.3 ± 0.15 nm and the polydispersity index ranged from 0.22 ± 0.008 to 0.23 ± 0.011. The results showed that treatments with CSO (200 and 400 mg/kg) and NCS (100 mg/kg) were able to decrease significantly (p < 0.001) the formation of edema and granulomatous tissue. The CSO and NCS groups significantly attenuated (p < 0.001) the recruitment of inflammatory cells in the tests for peritonitis and leukocyte infiltrate. The histopathological analysis of the gastrocnemius muscle showed a reduction in tissue damage caused by VBm. CONCLUSION: The results obtained in this study showed anti-inflammatory activity of the CSO which may be due to a high UFA content. The nanosizing, as evidenced by the incorporation of the CSO in the NCS improved the effect and opens the perspective for the obtainment of a nanomedicine in which a kinetic stable phytotherapic can be used at low doses.