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.

Epidemiological study in Brazil: Scorpion sting cases in Natal, Rio Grande do Norte.

Scorpion sting accidents are a public health problem in the state of Rio Grande do Norte, Brazil. The increasing and high incidence of cases in urban areas reveals the importance of studies to determine the epidemiological profile and the spatial distribution of these accidents. This is a retrospective study that describes and analyzes the cases of scorpion stings in the city of Natal, Rio Grande do Norte, Northeast Brazil, from 2007 to 2018. Data from the Information System database of Notifiable Diseases (SINAN) were obtained from the Secretary of Health of Rio Grande do Norte. 31,368 accidents due to scorpion stings were reported, more frequently in urban areas of Natal, whose Human Development Index is low. The cases occurred predominantly in hot and humid regions, mainly affecting women aged between 30 and 60 years. Most individuals sought medical attention within 3 h of the incident. The severity and mortality of the injured individuals varied according to the area of occurrence, age of the patient, and the local and systemic symptoms presented. Pain, numbness, and edema were the most frequent local symptoms, and systemic symptoms were frequently described as headache, hyperthermia and sweating. Therefore, scorpionism in the city of Natal is an environmental and public health problem, with a significant growth trend (p < 0.05). Through the data collected on the spatial distribution and risks, this approach allows the creation of effective control strategies to prevent accidents.

From Benznidazole to New Drugs: Nanotechnology Contribution in Chagas Disease.

Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi. Benznidazole and nifurtimox are the two approved drugs for their treatment, but both drugs present side effects and efficacy problems, especially in the chronic phase of this disease. Therefore, new molecules have been tested with promising results aiming for strategic targeting action against T. cruzi. Several studies involve in vitro screening, but a considerable number of in vivo studies describe drug bioavailability increment, drug stability, toxicity assessment, and mainly the efficacy of new drugs and formulations. In this context, new drug delivery systems, such as nanotechnology systems, have been developed for these purposes. Some nanocarriers are able to interact with the immune system of the vertebrate host, modulating the immune response to the elimination of pathogenic microorganisms. In this overview of nanotechnology-based delivery strategies for established and new antichagasic agents, different strategies, and limitations of a wide class of nanocarriers are explored, as new perspectives in the treatment and monitoring of Chagas disease.

Temperature effect in the inhibition of PLA2 activity of Bothrops brazili venom by Rosmarinic and Chlorogenic acids, experimental and computational approaches.

Myotoxicity caused by snakebite envenoming emerges as one of the main problems of ophidic accidents as it is not well neutralized by the current serum therapy. A promising alternative is to search for efficient small molecule inhibitors that can act against multiple venom components. Phospholipase A2 (PLA2) is frequently found in snake venom and is usually associated with myotoxicity. Thus it represents an excellent target for the search of new treatments. This work reports the effect of temperature in the inhibition of catalytic properties of PLA2 from Bothrops brazili venom by Rosmarinic (RSM) and Chlorogenic (CHL) acids through experimental and computational approaches. Three temperatures were evaluated (25, 37 and 50 °C). In the experimental section, enzymatic assays showed that RSM is a better inhibitor in all three temperatures. At 50 °C, the inhibition efficiency decayed significantly for both acids. Docking studies revealed that both ligands bind to the hydrophobic channel of the protein dimer where the phospholipid binds in the catalytic process, interacting with several functional residues. In this context, RSM presents better interaction energies due to stronger interactions with chain B of the dimer. Molecular dynamics simulations showed that RSM can establish selective interactions with ARG112B of PLA2, which is located next to residues of the putative Membrane Disruption Site in PLA2-like structures. The affinity of RSM and CHL acids towards PLA2 is mainly driven by electrostatic interactions, especially salt bridge interactions established with residues ARG33B (for CHL) and ARG112B (RSM) and hydrogen bonds with residue ASP89A. The inability of CHL to establish a stable interaction with ARG112B was identified as the reason for its lower inhibition efficiency compared to RSM at the three temperatures. Furthermore, extensive structural analysis was performed to explain the lower inhibition efficiency at 50 °C for both ligands. The analysis performed in this work provides important information for the future design of new inhibitors.Communicated by Ramaswamy H. Sarma.

Cationic PLGA Nanoparticle Formulations as Biocompatible Immunoadjuvant for Serum Production and Immune Response against Bothrops jararaca Venom.

Snakebite envenoming represents a worldwide public health issue. Suitable technologies have been investigated for encapsulated recombinant or native proteins capable of inducing an effective and long-lasting adaptive immune response. Nanoparticles are colloidal dispersions that have been used as drug delivery systems for bioactive biological compounds. Venom-loaded nanoparticles modulate the protein release and activate the immune response to produce specific antibodies. In this study, biocompatible cationic nanoparticles with Bothrops jararaca venom were prepared to be used as a novel immunoadjuvant that shows a similar or improved immune response in antibody production when compared to a conventional immunoadjuvant (aluminum hydroxide). We prepared stable, small-sized and spherical particles with high Bothrops jararaca venom protein association efficiency. The high protein loading efficiency, electrophoresis, and zeta potential results demonstrated that Bothrops jararaca venom is adsorbed on the particle surface, which remained as a stable colloidal dispersion over 6 weeks. The slow protein release occurred and followed parabolic diffusion release kinetics. The in vivo studies demonstrated that venom-loaded nanoparticles were able to produce an immune response similar to that of aluminum hydroxide. The cationic nanoparticles (CNp) as carriers of bioactive molecules, were successfully developed and demonstrated to be a promising immunoadjuvant.

Phytochemical analysis and preclinical toxicological, antioxidant, and anti-inflammatory evaluation of hydroethanol extract from the roots of Harpalyce brasiliana Benth (Leguminosae).

ETHNOPHARMACOLOGICAL RELEVANCE: Harpalyce brasiliana Benth (Leguminosae) is a shrub endemic to Brazil, popularly known as "snake's root." This species is used in folk medicine for the treatment of inflammation and snakebites. However, up to now there is no scientific research to justify its popular use. The study aimed to characterize the phytochemical profile of the hydroethanol extract from the roots of H. brasiliana (Hb), to evaluate its antioxidant and anti-inflammatory potential, as well as to investigate its cytotoxicity and acute toxicity. MATERIALS AND METHODS: The extract was obtained by maceration method using a solution of ethanol:water (70: 30, v/v). The phytochemical profile was obtained by liquid chromatography coupled to mass spectrometry. The cytotoxicity of extract (31-2000 µg/mL) was evaluated in vitro, by the 3-methyl-[4-5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method using murine macrophage and fibroblast cell lines (RAW 247.6 and 3T3, respectively) and by the hemolytic assay. For the in vivo acute toxicity, the extract (2000 mg/kg) was administered and after 14 days the weight (body and organs) and hematological and biochemical parameters were analyzed. Chemical free radical scavenging effect of the extract (125-2000 µg/mL) was investigated through diphenylpicryl hydrazine reduction, total antioxidant capacity, reducing power, hydroxyl radical scavenging, and iron and copper chelating assays. In vitro anti-inflammatory effect of the extract (125, 500, and 2000 µg/mL) was demonstrated through of nitric oxide (NO) analyzed in lipopolysaccharides stimulated RAW 264.7 cells. In vivo anti-inflammatory activities were evaluated in carrageenan-induced paw edema and zymosan-air-pouch models, with gavage administration (post-treatment) of extract at 100, 200, and 400 mg/kg. For the first animal model, the anti-edematogenic activity and myeloperoxidase (MPO) levels were investigated, while in the zymosan-air-pouch model the leukocyte number, MPO, total protein and pro-inflammatory cytokine (IL-1ß, IL-6, and TNF-α) levels were quantified. In addition, the oxidative parameters such as malondialdehyde (MDA) and reduced glutathione (GSH) were determined. RESULTS: The phytochemical profile revealed the presence of 20 compounds, mainly prenylated and geranylated pterocarpans. The extract demonstrated no cytotoxicity in erythrocytes, macrophages and fibroblasts cells at the tested concentrations, as well as no sign of toxicity and mortality or significant alterations on the hematological and biochemical parameters in the acute toxicity model. The extract was also able to neutralize chemical free radicals, with copper and iron chelating effect. For the NO dosage, the extract evidenced the reduction of expression of NO after the administration of the extract (500 and 2000 µg/mL). The edematogenic model revealed a decrease in paw edema and MPO level, while the zymosan-air-pouch model evidenced a reduction of leukocyte number (especially of polymorphornuclears), MPO production, and total protein and cytokine levels, and demonstrated the antioxidant effect through a decrease in MDA and increase in GSH parameters. CONCLUSION: This approach demonstrates for the first time that Hb is not cytotoxic, has low acute toxicity, and possesses antioxidant and anti-inflammatory properties in preclinical analyses, corroborating its popular use.

In silico and in vitro structure-stability-function relationship of analog peptides of Stigmurin and its antibacterial and antibiofilm activities.

Multidrug-resistant bacterial infections are a threat to public health worldwide, which boosts the urgent need for pharmacological research for new drugs. Although the peptides without disulfide bridges from scorpions have shown antimicrobial action, usually their toxicity hamper their pharmacological application. Stigmurin is a non-hemolytic cationic peptide from Tityus stigmurus venom with antibacterial effect and toxicity on normal cells. In this approach, the conformational changes and stability of two Stigmurin analog peptides, named StigA8 and StigA18, were evaluated by circular dichroism, as well as the mechanism of interaction with bacterial membranes in silico. In addition, the in vitro and in vivo antibacterial activity and the action against the biofilm formed by multidrug-resistant Staphylococcus aureus were investigated. StigA8 (+4) and StigA18 (+5) revealed the ability to change their structural conformation depending on the medium composition, and high stability at different temperatures and pH conditions. Both analog peptides showed greater ability to interact with bacterial membranes in silico when compared to the native one. StigA8 and StigA18 demonstrated low hemolytic action, with non-toxic effect on G. mellonella larvae up to 120 mg/kg. StigA8 and StigA18 presented a broad spectrum of antibacterial action in vitro, especially against multidrug-resistant clinical isolates. The analog peptides (7.5 µM) also reduced the biofilm biomass of multidrug-resistant S. aureus, as well as increased the larval survival of the Galleria mellonella infected larvae. Therefore, StigA8 and StigA18 showed a beneficial potential in the treatment of bacterial infections, constituting promising bioactive components for the development of new antimicrobial agents.

Antiophidic potential of chlorogenic acid and rosmarinic acid against Bothrops leucurus snake venom.

Bothrops leucurus is responsible for most cases of snakebite in Northeast Brazil; however, this species is not included in the pool of venoms used in antivenom production in Brazil. The serotherapy has logistical and effectiveness limitations, which stimulates the search for therapeutic alternatives. Chlorogenic acid and rosmarinic acid present several biological activities, but their antiophidic potential has been poorly explored. Thus, the aim of this approach was to evaluate the potential inhibitory effects of these compounds on B. leucurus venom. Initially, the enzymatic inhibition of toxins was evaluated in vitro. Then, anti-hemorrhagic, anti-myotoxic, and anti-edematogenic assays were performed in vivo, as well analysis of several biochemical markers and hemostatic parameters. In addition, the interaction of inhibitors with SVMP and PLA2 was investigated by docking analysis. Results revealed that compounds inhibited in vitro the enzymatic activities and venom-induced edema, with a decrease in both myeloperoxidase and interleukin quantification. The inhibitors also attenuated the hemorrhagic and myotoxic actions and mitigated changes in serum biochemical and hemostatic markers, as well as decreased lipid peroxidation in liver and kidney tissues. Docking analysis revealed attractive interactions of both inhibitors with the zinc-binding site of SVMP and, in the case of PLA2, chlorogenic acid showed a similar inhibition mechanism to that described for rosmarinic acid. The results evidenced the antiophidic potential of both compounds, which showed higher efficiency than antivenom serum. Thus, both inhibitors are promising candidates for future adjuvants to be used to complement antivenom serotherapy.

The potential of phenolic acids in therapy against snakebites: A review.

Ophidism is a serious health problem worldwide and is included in the World Health Organization's (WHO's) list of Neglected Tropical Diseases. Although snakebite envenoming requires emergency treatment, currently the only treatment recommended by WHO is serotherapy, which has some disadvantages such as low access to the rural population, low effectiveness in neutralizing local effects, and high cost. In this context, new alternatives for the treatment of snakebites are required. The use of plant-derived compounds to inhibit the effects caused by snake venoms has been the object of a number of studies in recent years. This review aims to provide an up-to-date overview of the use of phenolic acids with therapeutic application against envenomation by snakes of different species. In this sense, structural analysis in silico and biological activities in vivo and in vitro were reported. The acids were subdivided into derivatives of benzoic and cinnamic acids, with derivatives of cinnamic acids being the most studied. Studies have revealed that these compounds are capable of inhibiting local and systemic effects induced by envenomation, and structural analyses indicate that the acids interact with important sites responsible for the action of toxins. Thus, it was reported that phenolic acids showed antiophidic potential, providing insights for future research to develop complementary drugs for the treatment of snakebites.

Purification of chitosanases produced by Bacillus toyonensis CCT 7899 and functional oligosaccharides production.

Chitooligosaccharides (COS) have a great potential to be used by pharmaceutical industry due to their many biological activities. The use of enzymes to produce them is very advantageous, however it still faces many challenges, such as discovering new strains capable to produce enzymes that are able to generate bioactive oligosaccharides. In the present study a purification protein protocol was performed to purify chitosanases produced by Bacillus toyonensis CCT 7899 for further chitosan hydrolysis. The produced chitooligosaccharides were characterized by mass spectroscopy (MS) and their antiedematogenic effect was investigated through carrageenan-induced paw edema model. The animals were treated previously to inflammation by intragastric route with COS at 30, 300 and 600 mg/kg. The purification protocol showed a good performance for the chitosanases purification using 0.20 M NaCl solution to elute it, with a 9.54-fold purification factor. The treatment with COS promoted a decrease of paw edema at all evaluated times and the AUC0-4h, proving that COS produced showed activity in acute inflammation like commercial anti-inflammatory Dexamethasone (corticosteroid). Therefore, the strategy used to purification was successfully applied and it was possible to generate bioactive oligosaccharides with potential pharmacological use.

Gel formulated with Bryophyllum pinnatum leaf extract promotes skin wound healing in vivo by increasing VEGF expression: A novel potential active ingredient for pharmaceuticals.

Bryophyllum pinnatum (Crassulaceae) is used in traditional medicine for treating skin wounds. In our previous study, a topical gel containing B. pinnatum aqueous leaf extract showed a preclinical anti-inflammatory effect in in vivo acute edema models. In continuation, the present study aims to evaluate the phytochemical content and the stability of a formulation in gel containing B. pinnatum aqueous leaf extract and its healing properties and mechanism of action through an experimental model of induction of skin wounds in rats and in vitro assays. The animals were treated topically for 7 or 14 days with a formulation in gel containing extract at 5% or a placebo or Fibrinase® in cream. In addition, to establish some quality control parameters, the total phenolic content (TPC), total flavonoid content (TFC), and a study focusing on the phytochemical and biological stability of a gel for 30 days at two different conditions (room temperature and 40°C/75% RH) were performed. Gel formulation containing extract showed a TPC and TFC of 2.77 ± 0.06 mg of gallic acid/g and 1.58 ± 0.03 mg of quercetin/g, respectively. Regarding the stability study, the formulation in gel showed no significant change in the following parameters: pH, water activity, chromatographic profile, and the content of the major compound identified in the extract. The gel formulation containing extract stimulated skin wound healing while reducing the wound area, as well as decreasing the inflammatory infiltrate, reducing the levels of IL-1ß and TNF-α, and stimulating angiogenesis with increased expression of VEGF, an effect similar to Fibrinase. In conclusion, the gel formulation containing extract exhibited relevant skin wound healing properties and, therefore, has the potential to be applied as a novel active ingredient for developing wound healing pharmaceuticals.

PA-Int5: An isatin-thiosemicarbazone derivative that exhibits anti-nociceptive and anti-inflammatory effects in Swiss mice.

Pain and inflammation are symptoms of various diseases, and they can be modulated by different pathways, thus highlighting the importance of investigating the therapeutic effects of novel compounds. Previous studies have shown that isatin-thiosemicarbazone exhibits antitumor, antifungal antibacterial and other biological properties. Based on the wide range of biological effects of these compounds, the aim of the present study was to investigate the central nervous system (CNS) performance, and the anti-nociceptive and anti-inflammatory activity of (Z)-2-(5-nitro-2-oxoindolin-3-ilidene)-N-hydroazinecarbothioamide (PA-Int5) in treated mice. Three doses of PA-Int5 were tested orally (1.0, 2.5 and 5.0 mg/kg) in the nociceptive and inflammatory animal models. Additionally, the potential sedative effects of PA-Int5 (5 mg/kg, oral gavage) were investigated using an open field and rotarod tests, to exclude any possible unspecific effects of the nociceptive assays. Anti-nociceptive activity was assessed using the acetic acid-induced abdominal contortion and formalin tests, whereas anti-inflammatory activity was assessed using a carrageenan-induced paw edema and zymosan-induced air-pouch models. PA-Int5 (5 mg/kg) induced anti-nociceptive activity in the abdominal contortion model. In the formalin test, PA-Int5 (at 2.5 and 5 mg/kg) reduced nociception in the second phase. At the higher dose tested, PA-Int5 did not affect spontaneous locomotion or motor coordination. The data revealed that at all doses tested, the compound significantly reduced paw edema following carrageenan administration. In the zymosan-induced air-pouch model, PA-Int5 potently inhibited leukocyte migration and protein levels at the site of inflammation. When combined, the results revealed, for the first time, that PA-Int5 exhibited anti-nociceptive and anti-inflammatory activities, and highlights its potential, as well that of other derivatives, as novel candidates for pain relief.

Antimicrobial Activity of Chitosan Oligosaccharides with Special Attention to Antiparasitic Potential.

The global rise of infectious disease outbreaks and the progression of microbial resistance reinforce the importance of researching new biomolecules. Obtained from the hydrolysis of chitosan, chitooligosaccharides (COSs) have demonstrated several biological properties, including antimicrobial, and greater advantage over chitosan due to their higher solubility and lower viscosity. Despite the evidence of the biotechnological potential of COSs, their effects on trypanosomatids are still scarce. The objectives of this study were the enzymatic production, characterization, and in vitro evaluation of the cytotoxic, antibacterial, antifungal, and antiparasitic effects of COSs. NMR and mass spectrometry analyses indicated the presence of a mixture with 81% deacetylated COS and acetylated hexamers. COSs demonstrated no evidence of cytotoxicity upon 2 mg/mL. In addition, COSs showed interesting activity against bacteria and yeasts and a time-dependent parasitic inhibition. Scanning electron microscopy images indicated a parasite aggregation ability of COSs. Thus, the broad biological effect of COSs makes them a promising molecule for the biomedical industry.

Antifungal and Antibiofilm Activities of B-Type Oligomeric Procyanidins From Commiphora leptophloeos Used Alone or in Combination With Fluconazole Against Candida spp.

Commiphora leptophloeos (Burseraceae) is a medicinal plant native to Brazil which is popularly used for treating oral and vaginal infections. There has been no scientific evidence pointing to its efficacy in the treatment of these infections. Thus, this study sought to investigate the cytotoxic, antifungal, and antibiofilm activity of C. leptophloeos against Candida spp. and to isolate, identify, and quantify the content of B-type oligomeric procyanidins (BDP) in the extract of C. leptophloeos stem bark. The extract and the n-butanol fraction were obtained by maceration and liquid-liquid partition, respectively. Phytochemical analysis performed by HPLC-PDA/ELSD and FIA-ESI-IT-MS/MS allowed the identification and quantification of BDP in the samples. The application of centrifugal partition chromatography helped isolate BDP, which was identified by 1H NMR and MS analyses. Candida spp. reference strains and clinical isolates (including fluconazole-resistant strains) derived from the blood cultures of candidemic patients and the vaginal secretion of patients with vulvovaginal candidiasis were used for evaluating the antifungal and antibiofilm effects. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were determined by the microdilution technique, and biofilm inhibition was evaluated through crystal violet and XTT assays. The combined action of BDP with fluconazole was determined by the checkerboard method. The extract, the n-butanol fraction, and the BDP exhibited antifungal activity with MIC values ranging from 312.5 to 2500 µg/mL and were found to significantly reduce the biofilm formed in all the Candida strains investigated. BDP showed a fungicidal potential against strains of Candida spp. (especially against fluconazole-resistant strains), with MIC and MFC values ranging from 156.2 to 2500 µg/mL. In addition, the combined application of BDP and fluconazole produced synergistic antifungal effects against resistant Candida spp. (FICI = 0.31-1.5). The cytotoxic properties of the samples evaluated in human erythrocytes through hemolytic test did not show hemolytic activity under active concentrations. The findings of the study show that C. leptophloeos has antifungal and antibiofilm potential but does not cause toxicity in human erythrocytes. Finally, BDP, which was isolated for the first time in C. leptophloeos, was found to exhibit antifungal effect against Candida spp. either when applied alone or in combination with fluconazole.

TanP: A Multifunctional Anionic Peptide From Tityus stigmurus Scorpion Venom.

Anionic peptides of scorpions are molecules rich in aspartic and/or glutamic acid residues and correspond to a class of peptides without disulfide bonds that are still little explored. TanP is a linear anionic peptide (50 amino acid residues and net charge -20) present in the venom gland of the scorpion, Tityus stigmurus, with chelating properties for Cu2+ ion and immunomodulatory properties. The therapeutic application of chelating molecules is related to cases of acute or chronic intoxication by metals, neurodegenerative diseases, hematological diseases, healing of skin wounds, cardiovascular diseases, and cancer. In this approach, the chelating activity of TanP was evaluated in relation to new metal ions (Fe2+ and Zn2+) of biological importance, as well as its antioxidant, hemostatic, immunomodulatory, and healing potential, aiming to expand the biological and biotechnological potential of this peptide. TanP (25 µM) was able to form stable complexes with Fe2+ in a ratio of 1:5 (TanP: Fe2+). Theoretical results suggest that TanP can work as a sensor to identify and quantify Fe2+ ions. The fluorescence intensity of TanP (1.12 µM) decreased significantly after the addition of Fe2+, obtaining the highest ratio 1: 7.4 (TanP: Fe2+) that led to the lowest fluorescence intensity. For Zn2+, no relevant spectral change was noted. TanP (50 µM) showed a maximum of 3% of hemolytic activity, demonstrating biocompatibility, as well as exhibiting a 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity of above 70% at all the concentrations tested (1-25 µM), and 89.7% iron-chelating activity at 25 µM and 96% hydroxyl radical-scavenging activity at 73.6 µM. In addition, TanP (12.5 and 25 µM) revealed an anticoagulant effect, prolonging the clotting time in prothrombin time and activated partial thromboplastin time assays, with no fibrinogenolytic activity. TanP (12.5 and 25 µM) induced the release of TNF-α by murine macrophages, in the absence of lipopolysaccharides, with a concentration-dependent increase and also stimulated the migration of 3T3 cells in the in vitro healing assay. Thus, TanP revealed a multifunctional potential, being useful as a prototype for the development of new therapeutic and biotechnological agents.

NMR three-dimensional structure of the cationic peptide Stigmurin from Tityus stigmurus scorpion venom: In vitro antioxidant and in vivo antibacterial and healing activity.

Infectious diseases and the rapid development of pathogens resistant to conventional drugs are a serious global public health problem, which motivates the search for new pharmacological agents. In this context, cationic peptides without disulfide bridges from different species of scorpion venom have been the target of scientific studies due to their multifunctional activities. Stigmurin is a linear peptide composed of 17 amino acid residues (Phe-Phe-Ser-Leu-Ile-Pro-Ser-Leu-Val-Gly-Gly-Leu-Ile-Ser-Ala-Phe-Lys-NH2), which is present in the venom gland of the scorpion Tityus stigmurus. Here we present investigations of the in vitro antioxidant action of Stigmurin together with the in vivo antibacterial and healing activity of this peptide in a wound infection model induced by Staphylococcus aureus. In addition, we have reports for the first time of the three-dimensional structure determined by NMR spectroscopy of a peptide without disulfide bridges present in scorpion venom from the Tityus genus. Stigmurin showed hydroxyl radical scavenging above 70 % at 10 µM and antibiotic action in the skin wound, reducing the number of viable microorganisms by 67.2 % on the 7 day after infection. Stigmurin (1 µg / µL) increased the retraction rate of the lesion, with wound area reduction of 43 % on the second day after skin injury, which indicates its ability to induce tissue repair. Stigmurin in trifluoroethanol:water exhibited a random conformation at the N-terminus region (Phe1 to Pro6), with a helical structure from Ser7 to Phe16. This structural information, allied with the multifunctional activity of Stigmurin, makes it an attractive candidate for the design of novel therapeutic agents.

Self-Assembled Cationic-Covered Nanoemulsion as A Novel Biocompatible Immunoadjuvant for Antiserum Production Against Tityus serrulatus Scorpion Venom.

This study assesses the efficacy of different nanoemulsion formulations as new and innovative adjuvants for improving the in vivo immunization against the Tityus serrulatus scorpion venom. Nanoemulsions were designed testing key-variables such as surfactants, co-solvents, and the influence of the temperature, which would be able to induce the phase transition from a liquid crystal to a stable nanoemulsion, assessed for four months. Additionally, cationic-covered nanoemulsion with hyper-branched poly(ethyleneimine) was prepared and its performance was compared to the non-cationic ones. The physicochemical properties of the selected nanoemulsions and the interactions among their involved formulation compounds were carefully monitored. The cytotoxicity studies in murine macrophages (RAW 264.7) and red blood cells were used to compare different formulations. Moreover, the performance of the nanoemulsion systems as biocompatible adjuvants was evaluated using mice immunization protocol. The FTIR shifts and the zeta potential changes (from -18.3 ± 1.0 to + 8.4 ± 1.4) corroborated with the expected supramolecular anchoring of venom proteins on the surface of the nanoemulsion droplets. Cell culture assays demonstrated the non-toxicity of the formulations at concentrations less than 1.0 mg/mL, which were able to inhibit the hemolytic effect of the scorpion venom. The cationic-covered nanoemulsion has shown superior adjuvant activity, revealing the highest IgG titer in the immunized animals compared to both the non-cationic counterpart and the traditional aluminum adjuvant. In this approach, we demonstrate the incredible potential application of nanoemulsions as adjuvants, using a nanotechnology platform for antigen delivery system on immune cells. Additionally, the functionalization with hyper-branched poly(ethyleneimine) enhances this recognition and improves its action in immunization.

Biology, venom composition, and scorpionism induced by brazilian scorpion Tityus stigmurus (Thorell, 1876) (Scorpiones: Buthidae): A mini-review.

Scorpionism is a serious public health problem in various regions of the world. In Brazil, a high number of accidents by scorpions have been reported. From 2014 to 2018, about 547,000 cases were recorded, resulting in 466 deaths. The scorpion Tityus stigmurus is the predominant species in the northeast of Brazil, being responsible for most scorpionism cases in this region. With the aid of the transcriptomic approach of the venom gland of this species, components as neurotoxins, antimicrobials, metal chelating peptides and hypotensins, have been identified and characterized in silico, showing different biologic activity in vitro. In addition, the neuronal, pancreatic, renal, and enzymatic effects have been demonstrated for the crude T. stigmurus venom. Therefore, the T. stigmurus scorpion venom constitutes a rich arsenal of bioactive molecules with high potential for therapeutic and biotechnological application.

Chitosan Film Containing Mansoa hirsuta Fraction for Wound Healing.

Chitosan films entrapped with the Mansoa hirsuta fraction (CMHF) was developed as a new dressing for wound care. The chromatographic profile of the M. hirsuta fraction (MHF) was evaluated by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and the results showed that MHF is rich in acid triterpenes. Physicochemical characterization of the films prepared using the solvent casting method was performed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetry (TGA), differential scanning calorimetry (DCS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and mechanical properties. CMHF exhibited characteristic bands of both chitosan and MHF, revealing a physical mixture of both. CMHF presented an amorphous nature, thermostability, and dispersion of MHF in the chitosan matrix, resulting in a rough structure. Incorporation of M. hirsuta fraction into chitosan matrix favorably enhanced the mechanical performance and films thickness. The in vivo wound treatment with CMHF for seven days showed a characteristic area of advanced healing, re-epithelization, cell proliferation, and collagen formation. Furthermore, wound closure reached 100% contraction after 10 days of treatment with modulation of interleukins. The incorporation of M. hirsuta fraction into chitosan films was advantageous and showed great potential for stimulating wound repair and regeneration.

Mangaba (Hancornia speciosa Gomes) fruit juice decreases acute pulmonary edema induced by Tityus serrulatus venom: Potential application for auxiliary treatment of scorpion stings.

Scorpionism represents a serious public health problem due to its increasing incidence. In Brazil, Tityus serrulatus is a species of major medical importance, especially in children and the elderly, as envenomation may induce serious acute pulmonary edema. "Mangaba" (Hancornia speciosa Gomes) fruit juice is popularly used in the treatment of several inflammatory disorders. The objective of this study was to analyze the chemical composition of fruit juice of H. speciosa by HPLC-DAD-MS/MS, as well as to evaluate its anti-inflammatory potential and antioxidant activity, and analyze the biochemical and hematological parameters in acute pulmonary edema induced by T. serrulatus venom (TsV) in mice. Mice were challenged with TsV (30 µg/kg, subcutaneously) and were treated with dexamethasone (2 mg/kg, intraperitoneally) or fruit juice (pre- or post-treatment protocols, by intra-gastric route at 100 and 200 mg/kg), and 2 h later were anesthetized for blood, lung, and kidney collection, for several biochemical analyses. Results showed that the juice decreased edema, myeloperoxidase levels, vascular permeability, and production of cytokines (IL-1ß, IL-6, and TNF-α) in lung tissue. Also, the juice reduced the concentration of nitrite and malondialdehyde oxidative stress markers in renal tissue. Amylase, lactate dehydrogenase, aspartate aminotransferase, and creatine kinase seric levels were reduced when the animals were treated with the juice. HPLC-DAD-MS/MS analysis identified 13 phenolic derivatives. The results suggest that the juice was able to decrease the inflammatory effects induced by T. serrulatus, demonstrating that the use of juice can be relevant for the treatment of scorpion stings.