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.

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.

Production and Characterization of Chitooligosaccharides: Evaluation of Acute Toxicity, Healing, and Anti-Inflammatory Actions.

The search for promising biomolecules such as chitooligosaccharides (COS) has increased due to the need for healing products that act efficiently, avoiding complications resulting from exacerbated inflammation. Therefore, this study aimed to produce COS in two stages of hydrolysis using chitosanases derived from Bacillus toyonensis. Additionally, this study aimed to structurally characterize the COS via mass spectrometry, to analyze their biocompatibility in acute toxicity models in vivo, to evaluate their healing action in a cell migration model in vitro, to analyze the anti-inflammatory activity in in vivo models of xylol-induced ear edema and zymosan-induced air pouch, and to assess the wound repair action in vivo. The structural characterization process pointed out the presence of hexamers. The in vitro and in vivo biocompatibility of COS was reaffirmed. The COS stimulated the fibroblast migration. In the in vivo inflammatory assays, COS showed an antiedematogenic response and significant reductions in leukocyte migration, cytokine release, and protein exudate. The COS healing effect in vivo was confirmed by the significant wound reduction after seven days of the experiment. These results indicated that the presence of hexamers influences the COS biological properties, which have potential uses in the pharmaceutical field due to their healing and anti-inflammatory action.

Potent and Broad-Spectrum Antimicrobial Activity of Analogs from the Scorpion Peptide Stigmurin.

Scorpion venom constitutes a rich source of biologically active compounds with high potential for therapeutic and biotechnological applications that can be used as prototypes for the design of new drugs. The aim of this study was to characterize the structural conformation, evaluate the antimicrobial activity, and gain insight into the possible action mechanism underlying it, for two new analog peptides of the scorpion peptide Stigmurin, named StigA25 and StigA31. The amino acid substitutions in the native sequence for lysine residues resulted in peptides with higher positive net charge and hydrophobicity, with an increase in the theoretical helical content. StigA25 and StigA31 showed the capacity to modify their structural conformation according to the environment, and were stable to pH and temperature variation-results similar to the native peptide. Both analog peptides demonstrated broad-spectrum antimicrobial activity in vitro, showing an effect superior to that of the native peptide, being non-hemolytic at the biologically active concentrations. Therefore, this study demonstrates the therapeutic potential of the analog peptides from Stigmurin and the promising approach of rational drug design based on scorpion venom peptide to obtain new anti-infective agents.

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.

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.

Safety evaluation of aqueous extract from Eugenia uniflora leaves: Acute and subacute toxicity and genotoxicity in vivo assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Eugenia uniflora Linn (Myrtaceae) is the native species of Brazil. The leaves of this species are used in folk medicine to treat different inflammatory and gastrointestinal disorders. However, research on the safety of using E. uniflora leaves has been poorly explored. AIM OF THE STUDY: This approach aims to investigate the phytochemical composition as well as the acute, subacute toxicity, and in vivo genotoxic profile of the aqueous extract of E. uniflora leaves. MATERIALS AND METHODS: The chemical composition of E. uniflora leaf extract was determined by Fingerprint by High-Performance Thin Layer and High-Performance Liquid Chromatography. The acute toxicity in vivo was evaluated for 14 days after the administration of E. uniflora leaves extract (2000 mg/kg). For the evaluation of subacute toxicity, mice were daily treated for 28 days with E. uniflora extract (250, 500, or 1000 mg/kg). Signs of behavioral toxicity and biochemical and hematological alterations, including the multiple organ toxicities were investigated. In addition, the micronucleus assay was used to evaluate the in vivo genotoxicity of the leaves extract in murine erythrocytes. RESULTS: The phytochemical analysis showed the majority presence of phenolic compounds (gallic acid, ellagic acid, and myricitrin). Single or repeated doses of the aqueous extract of E. uniflora leaves did not reveal any signs of in vivo toxicity. Daily doses of the extract for 28 days induced a slight reduction in cholesterol and triglyceride levels. Furthermore, E. uniflora leaves extract (1000-2000 mg/kg) showed no genetic damage in the micronucleus assay, indicating the absence of genotoxicity of the herbal species. CONCLUSION: The aqueous extract of E. uniflora leaves showed a predominance of phenolic compounds, with non-toxic and non-genotoxic action in vivo. This approach sheds light on the chemical composition of the leaves of E. uniflora and suggests a high margin of safety in the popular use of the leaves of this plant species.

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.

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.

Antivenom Production against Bothrops jararaca and Bothrops erythromelas Snake Venoms Using Cross-Linked Chitosan Nanoparticles as an Immunoadjuvant.

In Brazil, envenomation by snakes of the genus Bothrops is clinically relevant, particularly for the species Bothrops jararaca and B. erythromelas. The most effective treatment for envenomation by snakes is the administration of antivenoms associated with adjuvants. Novel adjuvants are required to reduce side effects and maximize the efficiency of conventional serum and vaccine formulations. The polymer chitosan has been shown to have immunoadjuvant properties, and it has been used as a platform for delivery systems. In this context, we evaluated the potential immunoadjuvant properties of chitosan nanoparticles (CNPs) loaded with B. jararaca and B. erythromelas venoms in the production of sera against these venoms. Stable CNPs were obtained by ionic gelation, and mice were immunized subcutaneously for 6 weeks with 100 µL of each snake venom at concentrations of 5.0 or 10.0% (w/w), encapsulated in CNPs or associated with aluminium hydroxide (AH). The evaluation of protein interactions with the CNPs revealed their ability to induce antibody levels equivalent to those of AH, even with smaller doses of antigen. In addition, the CNPs were less inflammatory due to their modified release of proteins. CNPs provide a promising approach for peptide/protein delivery from snake venom and will be useful for new vaccines.

Analogs of the Scorpion Venom Peptide Stigmurin: Structural Assessment, Toxicity, and Increased Antimicrobial Activity.

Scorpion venom is a rich source of biologically active components and various peptides with high-potential therapeutic use that have been characterized for their antimicrobial and antiproliferative activities. Stigmurin is a peptide identified from the Tityus stigmurus venom gland with high antibacterial and antiproliferative activities and low toxicity. Amino acid substitutions in peptides without a disulfide bridge sequence have been made with the aim of reducing their toxicity and increasing their biological activities. The purpose of this study was to evaluate the structural conformation and structural stability, as well as antimicrobial, antiproliferative, and hemolytic activities of two peptide analogs to Stigmurin, denominated StigA6 and StigA16. In silico analysis revealed the α-helix structure for both analog peptides, which was confirmed by circular dichroism. Data showed that the net charge and hydrophobic moment of the analog peptides were higher than those for Stigmurin, which can explain the increase in antimicrobial activity presented by them. Both analog peptides exhibited activity on cancerous cells similar to the native peptide; however, they were less toxic when tested on the normal cell line. These results reveal a potential biotechnological application of the analog peptides StigA6 and StigA16 as prototypes to new therapeutic agents.

Structure and in vitro activities of a Copper II-chelating anionic peptide from the venom of the scorpion Tityus stigmurus.

Anionic Peptides are molecules rich in aspartic acid (Asp) and/or glutamic acid (Glu) residues in the primary structure. This work presents, for the first time, structural characterization and biological activity assays of an anionic peptide from the venom of the scorpion Tityus stigmurus, named TanP. The three-dimensional structure of TanP was obtained by computational modeling and refined by molecular dynamic (MD) simulations. Furthermore, we have performed circular dichroism (CD) analysis to predict TanP secondary structure, and UV-vis spectroscopy to evaluate its chelating activity. CD indicated predominance of random coil conformation in aqueous medium, as well as changes in structure depending on pH and temperature. TanP has chelating activity on copper ions, which modified the peptide's secondary structure. These results were corroborated by MD data. The molar ratio of binding (TanP:copper) depends on the concentration of peptide: at lower TanP concentration, the molar ratio was 1:5 (TanP:Cu2+), whereas in concentrated TanP solution, the molar ratio was 1:3 (TanP:Cu2+). TanP was not cytotoxic to non-neoplastic or cancer cell lines, and showed an ability to inhibit the in vitro release of nitric oxide by LPS-stimulated macrophages. Altogether, the results suggest TanP is a promising peptide for therapeutic application as a chelating agent.

Stigmurin and TsAP-2 from Tityus stigmurus scorpion venom: Assessment of structure and therapeutic potential in experimental sepsis.

Microbial resistance to conventional antibiotics is a public health problem worldwide, motivating the search for new therapeutic alternatives in varied natural sources. Cationic peptides without disulfide bridges from scorpions have been targeted in this context, mainly due to their multifunctional action and the limited ability of microorganisms to develop resistance against them. The present study was focused on Stigmurin and TsAP-2, cationic peptides found in the transcriptome of the venom gland from the scorpion Tityus stigmurus. The aims were: to assess the secondary structure of TsAP-2 and the structural stability of both peptides by circular dichroism; to evaluate their antiproliferative effect, and antimicrobial activities in vitro, ex vivo and in vivo; and to investigate their therapeutic potential in a murine model of polymicrobial sepsis. Stigmurin and TsAP-2 secondary structures responded similarly to environment polarity changes, and were stable to temperature and pH variation. Both peptides showed antiproliferative effect on tumor cells. TsAP-2 showed lower cytotoxicity to normal cells, and had a mitogenic activity on murine macrophages. Stigmurin demonstrated bactericidal and bacteriostatic activity, depending on the microorganism, whereas TsAP-2 had bactericidal action upon different bacterial strains analyzed. Both peptides were able to reduce leukocyte migration, TNF-α levels and microorganism load in the peritoneal cavity after induction of experimental sepsis, decreasing inflammation in the lung and cecum of septic animals. TsAP-2 also reduced the release of nitric oxide in the peritoneal cavity. Taken together, these data suggest that Stigmurin and TsAP-2 are structurally stable molecules and are efficient in the control of the infectious focus in polymicrobial sepsis, with potential use as a prototype for the rational design of novel therapeutic agents.