Modulation of hNav by Tst1, a ß-toxin purified from the scorpion Tityus stigmurus.

Scorpion venoms are known as a rich mixture of components, including peptides that can interact with different ion channels, particularly voltage-gated potassium channels (Kv), calcium channels (Cav) and sodium channels (Nav), essential membrane proteins for various physiological functions in organisms. The present work aimed to characterize the modulation of hNa+-channels by Tst1, a peptide purified from the venom of Tityus stigmurus, using whole-cell patch clamp. Tst1 at 100 nM provoked current inhibition in Nav 1.3 (85.23%), Nav 1.2 (67.26%) and Nav 1.4 (63.43%), while Nav 1.1, 1.5, 1.6, and 1.7 were not significantly affected. Tst1 also shifted the voltage of activation and steady-state inactivation to more hyperpolarized states and altered the recovery from inactivation of the channels, reducing repetitive firing of cells, which was more effective in Nav 1.3. Tst1 also demonstrated that the effect on Nav 1.3 is dose-dependent, with an IC50 of 8.79 nM. Taken together, these results confirmed that Tst1, the first Tityus stigmurus NaScTx assayed in relation to Nav channels, is a ß-toxin, as was previously suggested due to its amino acid sequence. KEY CONTRIBUTION: First ß-toxin purified from the venom of Tityus stigmurus scorpion broadly characterized in hNa+-channels.

Anti-Inflammatory Activity of Bullfrog Oil Polymeric Nanocapsules: From the Design to Preclinical Trials.

BACKGROUND: Although bullfrog oil (BFO) exerts anti-inflammatory effects, it has undesirable properties limiting its use. METHODOLOGY: BFO nanocapsules (BFONc) were produced through nanoprecipitation, and their physicochemical and morphological properties were characterized. To evaluate the biocompatibility of the formulation, a mitochondrial activity evaluation assay was conducted, and cell uptake was assessed. The in vitro anti-inflammatory activity was evaluated by measuring reactive oxygen species (ROS), nitric oxide (NO), type-6 interleukin (IL-6), and tumor necrosis factor (TNF) levels. The in vivo anti-inflammatory effect was assessed by quantifying myeloperoxidase (MPO) levels using the carrageenan-induced paw edema model. RESULTS: BFONc showed a particle size of 233 ± 22 nm, a polydispersity index of 0.17 ± 0.03, and a zeta potential of -34 ± 2.6mV. BFONc revealed remarkable biocompatibility and did not induce changes in cell morphology. Furthermore, BFONc decreased ROS levels by 81 ± 4%; however, NO level increased by 72 ± 18%. TNF and IL-6 levels were reduced by approximately 10% and 90%, respectively. Significant in vivo anti-inflammatory activity was observed compared to dexamethasone. MPO levels were reduced up to 2 MPOs/mg. CONCLUSION: Taken together, the results pointed out the remarkable biocompatibility and anti-inflammatory effects of BFONc.

Bullfrog Oil Reduces the Carrageenan-induced Edema in Wistar Rats by in vitro Reduction of Inflammatory Mediators.

Bullfrog oil (BFO) is a natural product from the adipose tissue of the amphibian Rana catesbeiana Shaw, a bio-product rich in polyunsaturated fatty acids, which claims anti-inflammatory activity. The objective of this work was to evaluate the cytotoxicity and the anti-inflammatory activity of BFO using in vivo and in vitro assays. Thus, the in vitro cytotoxicity was assessed by the MTT assay. Additionally, the in vivo anti-inflammatory activity was performed by the carrageenan-induced paw edema model in Wistar rats, followed by histological analysis. Moreover, the BFO effect on inflammatory pathways was investigated by in vitro evaluation of the nitric oxide (NO) synthesis, and type-6 interleukin (IL-6) and tumor-necrosis-factor (TNF) levels. In vivo experiments showed that BFO administered by intragastric route produced a significant anti-inflammatory effect, which was as substantial as indomethacin, the positive control. Histopathological analysis confirmed these results, showing the absence of the edema and minimal signs of inflammation in the paws of rats treated with BFO. The MTT results showed that BFO at all tested concentrations had no toxic effect against a macrophage cell line, not affecting the cell viability. In addition, after 48 hours of treatment, the BFO itself and its blend with Cetiol®-V (1:1v/v) at 200 µg.mL-1 were able to reduce the NO synthesis, and the IL-6 and TNF levels up to 35 ± 2%, 40 ± 6%, and 12 ± 3%, respectively. Therefore, these results provide unprecedented scientific evidence of the anti-inflammatory effect of BFO, suggesting its potential as a new candidate for the development of pharmaceutical products with anti-inflammatory activity.

Kalanchoe laciniata and Bryophyllum pinnatum: an updated review about ethnopharmacology, phytochemistry, pharmacology and toxicology

Abstract The species Kalanchoe laciniata (L.) DC. and Bryophyllum pinnatum (Lam) Pers. are native from Brazil and Madagascar, respectively. Both belonging to the Crassulaceae family and being widely used by population as a natural anti-inflammatory agent. These species have similar leaf morphology and for this reason, they are known by the same popular name as " saião " or " coirama ". Several studies have been published involving different parts and preparations of these species. Therefore, this review aims to provide an update overview about the traditional uses, chemical constitution, pharmacology and toxicology of K. laciniata and B. pinnatum species. An extensive literature review was conducted in different scientific databases. Various chemical constituents have been identified in extracts from different parts of K. laciniata and B. pinnatum , being flavonoids the major compounds. They have been traditionally used to treat inflammation, microbial infection, pain, respiratory diseases, gastritis, ulcers, diabetes and cancer tumors. Non-clinical in vitro assays evaluated mainly the antimicrobial and antioxidant activities, while in vivo assays evaluated the leishmanicide, anti-inflammatory and immunomodulatory activities. Regarding toxicity, few studies have been conducted for the two species. The information reported in this work might contribute to the recognition of the importance of K. laciniata and B. pinnatum species, as well as to direct further studies. (AU)

Self-Assembled Benznidazole-Loaded Cationic Nanoparticles Containing Cholesterol/Sialic Acid: Physicochemical Properties, In Vitro Drug Release and In Vitro Anticancer Efficacy.

Cationic polymeric nanoparticles (NPs) have the ability to overcome biological membranes, leading to improved efficacy of anticancer drugs. The modulation of the particle-cell interaction is desired to control this effect and avoid toxicity to normal cells. In this study, we explored the surface functionalization of cationic polymethylmethacrylate (PMMA) NPs with two natural compounds, sialic acid (SA) and cholesterol (Chol). The performance of benznidazole (BNZ) was assessed in vitro in the normal renal cell line (HEK-293) and three human cancer cell lines, as follows: human colorectal cancer (HT-29), human cervical carcinoma (HeLa), and human hepatocyte carcinoma (HepG2). The structural properties and feasibility of NPs were evaluated and the changes induced by SA and Chol were determined by using multiple analytical approaches. Small (<200 nm) spherical NPs, with a narrow size distribution and high drug-loading efficiency were prepared by using a simple and reproducible emulsification solvent evaporation method. The drug interactions in the different self-assembled NPs were assessed by using Fourier transform-infrared spectroscopy. All formulations exhibited a slow drug-release profile and physical stability for more than 6 weeks. Both SA and Chol changed the kinetic properties of NPs and the anticancer efficacy. The feasibility and potential of SA/Chol-functionalized NPs has been demonstrated in vitro in the HEK-293, HepG2, HeLa, and HT-29 cell lines as a promising system for the delivery of BNZ.

Tailoring microstructural, drug release properties, and antichagasic efficacy of biocompatible oil-in-water benznidazol-loaded nanoemulsions.

This study explored the transition of lamellar-type liquid crystal (LLC) to biocompatible oil-in-water nanoemulsions able to modify benznidazole (BNZ) release and target the drug to cells infected with the T. cruzi parasite. Three cosolvents (2methylpyrrolidone [NMP], polyethylene glycol [POL], and propylene glycol [PRO] were tested to induce the transition of anisotropic LLC systems to isotropic nanoemulsions. Mixtures of soy phosphatidylcholine with sodium oleate stabilized the dispersions of medium chain triglyceride in water. Rheological measurements, polarized microscopy, and small angle X-ray scattering demonstrated that there is a phase transition from LLC to desired nanoemulsions. These small and narrow droplet-sized nanocarriers exhibited some advantages and promising features, such as the enhanced BNZ aqueous solubility and slow drug release rate. In vitro cell biocompatibility of formulations was assessed in the Vero E6 and SiHa cell lines. Drug-loaded nanoemulsions inhibited the epimastigote growth of the T. cruzi parasite (IC50 0.208 ±â€¯0.052 µg mL-1) and reduced its infective life form trypomastigote (IC50 0.392 ±â€¯0.107 µg mL-1). The oil-in-water nanoemulsions were demonstrated as promising biocompatible liquid drug delivery systems capable of improving the BNZ trypanocidal activity for the treatment of Chagas disease.

Getting the Jump on the Development of Bullfrog Oil Microemulsions: a Nanocarrier for Amphotericin B Intended for Antifungal Treatment.

Amphotericin B (AmB), a potent antifungal drug, presents physicochemical characteristics that impair the development of suitable dosage forms. In order to overcome the AmB insolubility, several lipid carriers such as microemulsions have been developed. In this context, the bullfrog oil stands out as an eligible oily phase component, since its cholesterol composition may favor the AmB incorporation. Thus, the aim of this study was to develop a microemulsion based on bullfrog oil containing AmB. Moreover, its thermal stability, antifungal activity, and cytotoxicity in vitro were evaluated. The microemulsion formulation was produced using the pseudo-ternary phase diagram (PTPD) approach and the AmB was incorporated based on the pH variation technique. The antifungal activity was evaluated by determination of minimal inhibitory concentration (MIC) against different species of Candida spp. and Trichosporon asahii. The bullfrog oil microemulsion, stabilized with 16.8% of a surfactant blend, presented an average droplet size of 26.50 ± 0.14 nm and a polydispersity index of 0.167 ± 0.006. This system was able to entrap AmB up to 2 mg mL-1. The use of bullfrog oil as oily phase allowed an improvement of the thermal stability of the system. The MIC assay results revealed a growth inhibition for different strains of Candida spp. and were able to enhance the activity of AmB against T. asahii. The microemulsion was also able to reduce the AmB toxicity. Finally, the developed microemulsion showed to be a suitable system to incorporate AmB, improving the system's thermal stability, increasing the antifungal activity, and reducing the toxicity of this drug.

Monitoring structural features, biocompatibility and biological efficacy of gamma-irradiated methotrexate-loaded spray-dried microparticles.

In this study, biodegradable and biocompatible gamma irradiated poly-(dl-lactide-co-glycolide) (PLGA) spray-dried microparticles were prepared aiming to improve the efficacy of methotrexate (MTX). The experimental design included three formulations of microparticles containing distinct drug amount (9%, 18%, and 27% w/w) and three distinct gamma irradiation dose (15kGy, 25kGy, and 30kGy). The physicochemical and drug release properties of the microparticles supported their biocompatibility and biological efficacy studies in different cell lines. The irradiation induced slight changes in the spherical shape of the microparticles and the formation of free radicals was dependent on the drug loading. However, the amorphous character, particle size, drug loading, and drug release rate of the microparticles were preserved. The drug release data from all microparticles formulation were evaluated by using four drug kinetic models and by comparison of their similarity factor (f2). The gamma irradiation did not induce changes in the biocompatibility of PLGA microparticles and in the biological activity of the MTX-loaded microparticles. Finally, the spray-dried MTX-loaded PLGA microparticles enhanced the efficacy of the drug in the human cervical cancer cells (SiHa cell line). This study demonstrated the feasibility of the gamma irradiated spray dried PLGA microparticles for prolonged release of MTX, supporting a promising antitumor-drug delivery system for parenteral (subcutaneous) or pulmonary use.

Designing structural features of novel benznidazole-loaded cationic nanoparticles for inducing slow drug release and improvement of biological efficacy.

Several polymers have been investigated for producing cationic nanocarriers due to their ability to cross biological barriers. Polycations such as copolymers of polymethylmethacrylate are highlighted due to their biocompatibility and low toxicity. The purpose of this study was to produce small and narrow-sized cationic nanoparticles able to overcome cell membranes and improve the biological activity of benznidazole (BNZ) in normal and cancer cells. The effect of composition and procedure parameters of the used emulsification-solvent evaporation method were controlled for this purpose. The experimental approach included particle size, polydispersity index, zeta potential, atomic force microscopy (AFM), attenuated total reflectance Fourier transforms infrared spectroscopy (ATR- FTIR), drug loading efficiency, and physical stability assays. Spherical and stable (over six weeks) sub 150nm cationic nanoparticles were optimized, with the encapsulation efficiency >80%. The used drug/copolymer ratio modulated the slow drug release, which was adjusted by the parabolic diffusion mathematical model. In addition, the ability of the cationic nanoparticles improve the BNZ uptake in the normal kidney cells (HEK 293) and the human colorectal cancer cells (HT 29) demonstrate that this novel BNZ-loaded cationic has great potential as a chemotherapeutic application of benznidazole.

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.

Characterization of TistH, a multifunctional peptide from the scorpion Tityus stigmurus: Structure, cytotoxicity and antimicrobial activity.

The presence of bioactive peptides in animal venoms has been targeted in scientific research for assessing biological activities, as well as mechanisms of action. A recent study by our group observed hypotensive action of TistH (Tityus stigmurus Hypotensin), a peptide deduced from the transcriptome of T. stigmurus venom gland. The present study aims to analyze TistH structure properties and to evaluate its toxicity on normal and tumor cells, its in vitro antimicrobial activity, as well as its inflammatory effect. Circular dichroism analyses of TistH showed a general predominance of α-helix conformation in TFE (20-70%) and structural stability to pH variations. TistH was not cytotoxic to normal cell lines (3T3, RAW and HEK), and also not to cancer cell lines (HeLa, B16, 786-0, SiHa and HepG2). The peptide did not present inflammatory activity up to 6 h after administered subcutaneously to Swiss mice. It was observed that concentrations of 4-1024 µg/mL of TistH produced no inhibition against the bacteria Staphylococcus aureus, Staphylococcus epidermidis e Pseudomonas aeruginosa. The results of antifungal assays showed a moderate activity of TistH against Candida albicans strain LM-108 and the filamentous fungus Trichophyton rubrum LM-640, with growth inhibition at a concentration of 1024 µg/mL. In contrast, the peptide presented a greater activity (MIC 128 µg/mL) against C. albicans LM-106, Candida tropicalis ATCC 13308 and Aspergillus flavus strains LM-247 and LM-26, fungi that cause oral and vaginal infections, candidiasis and respiratory allergies, respectively. The present data contribute to a better understanding of TistH and its possible use as a bioactive compound. This multifunctional peptide is capable of acting as anti-hypertensive, as well as to inhibit the growth of fungal strains, having low toxicity, which suggests its safety for using as a pharmacological agent.

Phytochemical study and anti-inflammatory and antioxidant potential of Spondias mombin leaves

Abstract Spondias mombin L., Anacardiaceae, is a plant native of Brazil, where it is known as "cajá". In order to find a potential application for this native species, the anti-inflammatory and antioxidant effects were investigated. The anti-inflammatory activity was evaluated using the in vivo model carrageenan-induced peritonitis in mice. The in vitro antioxidant potential as well the cytotoxicity against 3T3 fibroblast cells also were evaluated. Through High Performance Liquid Chromatography-diode array detector analysis, an analytic method was developed and validated. It allowed the identification and quantification of ellagic acid and chlorogenic acid in hydroethanolic extract of S. mombin leaves. This extract showed anti-inflammatory effect at 100, 200, 300 and 500 mg/kg, however, the ethyl acetate fraction, at 200 mg/kg, showed the highlighted results. Ellagic acid and chlorogenic acid (2.5, 5 and 10 mg/kg) also inhibited the leukocyte migration to the site of inflammation. The extract, fractions and compounds showed significant antioxidant potential when evaluated in different assays. The results shown in this work suggest the anti-inflammatory potential of the leaf extract of S. mombim on peritonitis model induced by carrageenan, it was also observed antioxidant properties associated with an absence of cytotoxicity in cell culture. Further in vivo studies are required to confirm the anti-inflammatories action of S. mombin and its possible anti-inflammatory mechanisms of action.

Homology modeling, vasorelaxant and bradykinin-potentiating activities of a novel hypotensin found in the scorpion venom from Tityus stigmurus.

In a recent work by our group involving a transcriptomics approach applied to the venom glands from Tityus stigmurus we identified a new family of peptides called Hypotensins (TSTI0006C) (Almeida et al., 2012). The cluster TSTI0006C was analyzed in the main 25 amino acid residues and named T. stigmurus Hypotensin (TistH), showing a molecular mass of 2.7 kDa, an absence of cysteines and the presence of two C-terminal proline residues, which are a bradykinin-potentiating peptide (BPP) signature. Here, we describe the homology modeling of the three-dimensional structure of TistH. In addition, we evaluated the cardiovascular effects elicited by TistH in normotensive rats. Firstly, TistH showed no cytotoxic effect on horse erythrocyte. Furthermore, in normotensive rats TistH was able to potentiate the hypotensive action of bradykinin (BK) and induced a vasorelaxant effect in mesenteric artery rings by endothelium-dependent release of nitric oxide (NO) and demonstrated independent inhibition of angiotensin converting enzyme (ACE). Our data can contribute to a better understanding of the structural and functional characteristics of TistH and suggest its potential use in cardiovascular diseases.