Matheus F. Fernandes-Pedrosa

Structural basis for metal ion coordination and the catalytic mechanism of sphingomyelinases d

Sphingomyelinases D (SMases D) from Loxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-Å resolution using the “quick cryo-soaking” technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (α/β)8 barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg2+ ion, which is coordinated by Glu32, Asp34, Asp91, and solvent molecules. In the proposed acid base catalytic mechanism, His12 and His47 play key roles and are supported by a network of hydrogen bonds between Asp34, Asp52, Trp230, Asp233, and Asn252.

Profiling the resting venom gland of the scorpion Tityus stigmurus through a transcriptomic survey

BackgroundThe scorpion Tityus stigmurus is widely distributed in Northeastern Brazil and known to cause severe human envenoming, inducing pain, hyposthesia, edema, erythema, paresthesia, headaches and vomiting. The present study uses a transcriptomic approach to characterize the gene expression profile from the non-stimulated venom gland of Tityus stigmurus scorpion.ResultsA cDNA library was constructed and 540 clones were sequenced and grouped into 153 clusters, with one or more ESTs (expressed sequence tags). Forty-one percent of ESTs belong to recognized toxin-coding sequences, with transcripts encoding antimicrobial toxins (AMP-like) being the most abundant, followed by alfa KTx- like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Our analysis indicated that 34% of the transcripts encode “other possible venom molecules”, which correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins. Fifteen percent of ESTs are similar to cellular transcripts. Sequences without good matches corresponded to 11%.ConclusionsThis investigation provides the first global view of gene expression of the venom gland from Tityus stigmurus under resting conditions. This approach enables characterization of a large number of venom gland component molecules, which belong either to known or non yet described types of venom peptides and proteins from the Buthidae family.

Jatropha gossypiifolia L. (Euphorbiaceae): A Review of Traditional Uses, Phytochemistry, Pharmacology, and Toxicology of This Medicinal Plant

Jatropha gossypiifolia L. (Euphorbiaceae), widely known as “bellyache bush,” is a medicinal plant largely used throughout Africa and America. Several human and veterinary uses in traditional medicine are described for different parts and preparations based on this plant. However, critical reviews discussing emphatically its medicinal value are missing. This review aims to provide an up-to-date overview of the traditional uses, as well as the phytochemistry, pharmacology, and toxicity data of J. gossypiifolia species, in view of discussing its medicinal value and potential application in complementary and alternative medicine. Pharmacological studies have demonstrated significant action of different extracts and/or isolated compounds as antimicrobial, anti-inflammatory, antidiarrheal, antihypertensive, and anticancer agents, among others, supporting some of its popular uses. No clinical trial has been detected to date. Further studies are necessary to assay important folk uses, as well as to find new bioactive molecules with pharmacological relevance based on the popular claims. Toxicological studies associated with phytochemical analysis are important to understand the eventual toxic effects that could reduce its medicinal value. The present review provides insights for future research aiming for both ethnopharmacological validation of its popular use and its exploration as a new source of herbal drugs and/or bioactive natural products.

Triethanolamine stabilization of methotrexate-β-cyclodextrin interactions in ternary complexes.

The interaction of methotrexate (MTX) with beta-cyclodextrin (β-CD) in the presence of triethanolamine (TEA) was investigated with the aim to elucidate the mechanism whereby self-assembly cyclodextrin systems work in association with this third component. Solubility diagram studies showed synergic increment of the MTX solubility to be about thirty-fold. Experiments using 2D ROESY and molecular modeling studies revealed the inclusion of aromatic ring III of the drug into β-CD cavity, in which TEA contributes by intensifying MTX interaction with β-CD and stabilizes MTX:β-CD:TEA ternary complex by electrostatic interaction. The maintenance of these interactions in solid phase was also studied in ternary MTX:β-CD:TEA and comparisons were made with freeze dried binary MTX:β-CD and physical mixtures. FTIR studies evidenced that MTX–β-CD interaction remained in solid ternary complexes, which was also supported by thermal (differential scanning calorimetry (DSC), thermogravimetric analysis (TG)/first derivative of TG analysis (DTG) and C,N,H elementary analysis) and structural (X-ray diffraction analysis, (XRD)) studies, mainly regarding the increment of drug stability. The efficient in vitro drug dissolution studies successfully demonstrated the contribution of ternary complexes, which highlights the importance of this possible new raw material for further applications in drug delivery systems.

Aqueous leaf extract of Jatropha gossypiifolia L. (Euphorbiaceae) inhibits enzymatic and biological actions of Bothrops jararaca snake venom.

Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A2 (Lys49 PLA2). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that it may be used as a new source of bioactive molecules against bothropic venom.

Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin.

A new antimicrobial peptide, herein named Stigmurin, was selected based on a transcriptomic analysis of the Brazilian yellow scorpion Tityus stigmurus venom gland, an underexplored source for toxic peptides with possible biotechnological applications. Stigmurin was investigated in silico, by circular dichroism (CD) spectroscopy, and in vitro. The CD spectra suggested that this peptide interacts with membranes, changing its conformation in the presence of an amphipathic environment, with predominance of random coil and beta-sheet structures. Stigmurin exhibited antibacterial and antifungal activity, with minimal inhibitory concentrations ranging from 8.7 to 69.5μM. It was also showed that Stigmurin is toxic against SiHa and Vero E6 cell lines. The results suggest that Stigmurin can be considered a potential anti-infective drug.

Serum production against Tityus serrulatus scorpion venom using cross-linked chitosan nanoparticles as immunoadjuvant.

Several species of scorpions are known to cause accidents which can lead to death, most of them belonging to the genus Tityus. Tityus serrulatus is considered the most dangerous scorpion in South America. In Brazil, T. serrulatus is responsible for serious accidents, including deaths, which occur mainly with children and elderly people. Anti-scorpion sera are routinely produced by various institutions, and suitable technologies have been investigated for encapsulation and release recombinant or native proteins capable of inducing antibody production. In this context, biocompatible and biodegradable polymers, such as chitosan, have been employed for this purpose. This study aimed to obtain a protein release system for the peptides or proteins from T. serrulatus, based on cross-linked chitosan nanoparticles (CN) in order to generate a new model of immunization in animals, and consequently a potentially novel polyclonal serum, namely an anti-T. serrulatus venom. CN were successfully obtained by ionic gelation using the polyanion tripolyphosphate (TPP), which demonstrated a suitable particle size of about 200 nm, with maximum encapsulation efficiency (100%) and enhanced antigen-specific antibody titers of 72%. The serum production data revealed that CN were equipotent to aluminum hydroxide, the traditional adjuvant for immunization. This study demonstrates that chitosan nanoparticles are a promising and safe system for peptide/protein delivery for T. serrulatus scorpion.

Ipomoea asarifolia neutralizes inflammation induced by Tityus serrulatus scorpion venom

ETHNOPHARMACOLOGICAL RELEVANCE Envenoming caused by scorpion sting is a serious public health problem. In Brazil, 13,038 accidents caused by venomous animals have been reported. Of this total, 53% of the cases and 14 deaths were caused by scorpions. Furthermore, Tityus serrulatus (Buthidae) is the most dangerous scorpion due to the high toxicity of its venom. The treatment is the common supportive therapy and the serum therapy, but some people do not have access to both therapies and seek healing through the use of medical plants. AIM OF THE STUDY This study evaluated the ability of the crude extract and fractions from the leaves of Ipomoea asarifolia in neutralizing the main biological effects caused by Tityus serrulatus envenoming in mice. MATERIALS AND METHODS BALB/c mice were pretreated (i.v.) with 100 μλ of aqueous extracts and fractions dichloromethane, ethyl acetate, and n-butanol (CH₂Cl₂, EtOAc, and n-BuOH, respectively) of Ipomoea asarifolia, rutin or saline. Then, the animals received 100 μλ (i.p.) of venom of Tityus serrulatus (0.8 mg/kg). After six hours, the peritoneal lavage was performed with PBS and the number cells were determined using a Neubauer chamber. The supernatants were collected for determination of cytokines, such as IL-6, IL-12, and IL-1β. RESULTS The aqueous extract, fractions and rutin, at all doses, significantly reduced cell migration, which was endorsed by the reduction of the levels of certain cytokines. CONCLUSION This is the first study that demonstrated the potential effect of Ipomoea asarifolia against inflammation caused by Tityus serrulatus venom, suggesting that these extracts and/or their bioactive molecules, especially the flavonoid rutin, have potential use in the therapy of this envenomation.

Anti-inflammatory activity of aqueous extract and bioactive compounds identified from the fruits of Hancornia speciosa Gomes (Apocynaceae)

BackgroundHancornia speciosa Gomes (Apocynaceae), popularly known as “mangabeira,” has been used in folk medicine to treat inflammatory disorders, hypertension, dermatitis, diabetes, liver diseases and gastric disorders. Although the ethnobotany indicates that its fruits can be used for the treatment of ulcers and inflammatory disorders, only few studies have been conducted to prove such biological activities. This study investigated the anti-inflammatory properties of the aqueous extract of the fruits of H. speciosa Gomes as well as its bioactive compounds using in vivo experimental models.MethodsThe bioactive compounds were identified by High Performance Liquid Chromatography coupled with diode array detector (HPLC-DAD) and Liquid Chromatography coupled with Mass Spectrometry (LC-MS). The anti-inflammatory properties were investigated through in vivo tests, which comprised xylene-induced ear edema, carrageenan-induced peritonitis and zymosan-induced air pouch. The levels of IL-1β, IL-6, IL-12 and TNF-α were determined using ELISA.ResultsRutin and chlorogenic acid were identified in the extract as the main secondary metabolites. In addition, the extract as well as rutin and chlorogenic acid significantly inhibited the xilol-induced ear edema and also reduced the cell migration in both carrageenan-induced peritonitis and zymosan-induced air pouch models. Reduced levels of cytokines were also observed.ConclusionThis is the first study that demonstrated the anti-inflammatory activity of the extract of H. speciosa fruits against different inflammatory agents in animal models, suggesting that its bioactive molecules, especially rutin and chlorogenic acid are, at least in part, responsible for such activity. These findings support the widespread use of Hancornia speciosa in popular medicine and demonstrate that its aqueous extract has therapeutical potential for the development of herbal drugs with anti-inflammatory properties.

Aqueous extract from Ipomoea asarifolia (Convolvulaceae) leaves and its phenolic compounds have anti-inflammatory activity in murine models of edema, peritonitis and air-pouch inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE Ipomoea asarifolia (Desr.) Roem. and Schult.(Convolvulaceae), popularly known as salsa or salsa-brava, is a plant of which the decoction of leaves is used in folk medicine to treat various inflammatory disorders such of dermatitis, scabies, symptoms of syphilis, skin ulcers and external wounds. However, little is known about possible compounds and mechanisms of action of the plant to support the activities reported by popular use. AIM OF THE STUDY The study aimed to identify bioactive molecules present in the crude extract of I. asarifolia leaves and investigate the anti-inflammatory potential of this extract in different experimental in vivo models to improve the understanding on that activity. MATERIAL AND METHODS Aqueous extract of I. asarifolia leaves was prepared by decoction (1:10 m/v) and its chromatographic profile was obtained by high performance liquid chromatography coupled with diode array detector (HPLC-DAD) and liquid chromatography diode array detector coupled with mass spectrometry (LC-DAD-MS). The potential anti-inflammatory activity of the extract was assessed using the following in vivo models: xylene-induced ear edema (20, 30 and 40mg/kg), evaluating the degree of edema formation; carrageenan-induced peritonitis (10, 20 and 30mg/kg), evaluating leukocyte migration and cytokine levels (IL-1β, IL-6, IL-12 and TNF-α) at 4h; zymosan-induced air pouch inflammation (20, 30 and 40mg/kg), evaluating the kinetics of leukocyte migration by total and differential counts at 6, 24 and 48h. The same tests were conducted using pure compounds identified in the aqueous extract from I. asarifolia leaves in different doses for each experimental model. RESULTS The compounds identified in the aqueous extract of I. asarifolia leaves by HPLC-DAD and LC-DAD-MS were rutin, chlorogenic acid and caffeic acid. The extract significantly reduced ear edema induced by xylene (81%, 85% and 86% for doses of 20, 30 and 40mg/kg, respectively, p<0.001), as well as cell migration in experimental models of peritonitis (70%, 78% and 83% for doses of 10, 20 and 30mg/kg, respectively, p<0.001) and air pouch inflammation (58%, 67% and 53% for doses of 20, 30 and 40mg/kg, respectively, p<0.001). In addition, the extract demonstrated the ability to significantly inhibit the production of cytokines IL-1β, IL-6, IL-12 and TNF-α (p<0.001). The secondary metabolites tested (rutin, chlorogenic acid and caffeic acid) also showed the ability to significantly (p<0.001) decrease the parameters analyzed above. CONCLUSION This is the first study to identify and confirm these phenolic compounds in I. asarifolia leaves extract and to suggest that these compounds contribute to the anti-inflammatory activity in vivo, as reported by ethnomedicinal use of this plant. Through the different experimental models performed, we can conclude that the results obtained with the aqueous extract from I. asarifolia leaves support its popular use for the treatment of inflammatory disorders.