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.

Toxicological and pharmacological effects of pentacyclic triterpenes rich fraction obtained from the leaves of Mansoa hirsuta.

Mansoa hirsuta is a medicinal plant native to the Brazilian semi-arid region. This approach aimed to investigate the in vitro and in vivo toxicity and anti-inflammatory and analgesic actions of the M. hirsuta fraction (MHF). In vitro cell viability was assessed in 3T3 cells. In vivo, the acute toxicity test, a single dose of the MHF was administered. For the subchronic toxicity test, three doses of were administered for 30 days. Locomotion and motor coordination were assessed using open field and rota-rod. The anti-inflammatory activity was evaluated in carrageenan-induced paw edema and zymosan-induced air-pouch models. Myeloperoxidase (MPO) and total proteins were also measured. The antinociceptive activity MHF was determined using acid acetic-induced abdominal writhing and formalin models. In the cytotoxicity assay, MHF showed no significative impairment of cell viability and in the acute toxicity study, did not cause mortality or signs of toxicity. Repeated exposure to MHF did not cause relevant toxicological changes. The evaluation in the open field test showed that the MHF did not alter the locomotor activity and there was no change in motor coordination and balance of animals. MHF significantly reduced edema, MPO production, the migration of leukocytes and protein leakage. In addition, MHF reduced abdominal writhing and significantly inhibited the first and second stage of the formalin test. The results of this study indicated that MHF has an anti-inflammatory and analgesic potential without causing acute or subchronic toxic effects and it can be a promising natural source to be explored.

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.

Mass spectrometry characterization of Commiphora leptophloeos leaf extract and preclinical evaluation of toxicity and anti-inflammatory potential effect.

ETHNOPHARMACOLOGICAL RELEVANCE: Commiphora leptophloeos (Mart.) J.B. Gillett (Burseraceae) is a medicinal plant native from the brazilian northeast caatinga biome, known popularly as "imburana" or "imburana-de-cambão". The leaves of C. leptophloeos are widely used in folk medicine in the treatment of various inflammatory disorders. However, there is no scientific evidence to justify their popular use. AIM OF THE STUDY: This approach aimed to characterize the phytochemical profile of hydroethanolic leaf extract, as well as evaluate the anti-inflammatory and antioxidant potential activity and to investigate the acute toxicity with pre-clinical in vitro and in vivo methodologies. MATERIALS AND METHODS: The phytochemical profile was characterized by UPLC-MS and FIA-ESI-IT-MS/MS. The in vitro anti-inflammatory potential the hydroethanolic extract of C. leptophloeos (1, 10, 100 and 200 µg/mL) was investigated by lipopolysaccharide (LPS) induced nitric oxide assay, in order to analyze the potential decrease of nitric oxide (NO) production. For carrageenan-induced paw edema and zymosan-induced air pouch models, the extract (100, 200 and 400 mg/kg) was administrated by intragastric gavage (i.g.) route and used for evaluating the anti-inflammatory effect in vivo. Related to the first animal model, the antiedematogenic activity and myeloperoxidase (MPO) levels could be investigated. In addition, the zymosan-induced air pouch model allowed the analyses of leukocytes migration, total MPO, malondialdehyde (MDA) and cytokines (TNF-α and IL-10) levels. The toxicity in vitro of the extract (1, 10, 100 and 200 µg/mL) was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acute toxicity in vivo was tested using the extract at 2000 mg/kg by i. g. route. RESULTS: The phytochemical analyses of C. leptophloeos leaf extract pointed the presence of six glycosylated flavonoids, identified as orientin, isoorientin, vitexin and isovitexin, quercetrin and isoquercitrin. A decrease of NO in vitro was noticed by the use of the extract in the LPS-induced nitric oxide assay and an expressive reduction of the paw-edema followed by a decrease of myeloperoxidase activity at doses of 200 and 400 mg/kg. The zymosan-induced air pouch model indicated that the extract, in all doses, significantly reduced the leukocytes migration, total protein concentration, MPO and MDA levels. The levels of cytokines were verified by the administration of extract in this model, revealing a lower of TNF-α level and an increase of the IL-10 production. In the toxicity study, the MTT assay evidenced no cytotoxicity of the tested concentrations and acute toxicity in vivo test did not result in any sign of toxicity and mortality or significant changes on the biochemical parameters. CONCLUSION: Based on these results, is possible suggest that the anti-inflammatory activity revealed in this approach can be related to the modulating the level of cytokine, decrease of TNF-α, increase of IL-10 in vivo and also the inhibition of the production of nitric oxide RAW 264.7 activated by LPS. These results demonstrate the potential anti-inflammatory effect C. leptophloeos leaf extrat in inflammatory in vivo models, supporting its use in folk medicine for treatment of inflammatory diseases. Finally, glycosylated flavonoids can be responsible, at least in part, for this effect.

Tailoring structural properties of spray-dried methotrexate-loaded poly (lactic acid)/poloxamer microparticle blends.

Drug delivery systems can overcome cancer drug resistance, improving the efficacy of chemotherapy agents. Poly (lactic acid) (PLA) microparticles are an interesting alternative because their hydrophobic surface and small particle size could facilitate interactions with cells. In this study, two poloxamers (PLX 407 and 188) were applied to modulate the structural features, the drug release behavior and the cell viability from spray-dried microparticles. Five formulations with different PLA: PLX blend ratio (100:0, 75:25, 50:50, 25:50, and 0:100) were well-characterized by SEM, particle size analysis, FTIR spectroscopy, differential scanning calorimetry (DSC), and X-ray diffraction analysis (XRD). The spray-dried microparticles showed higher drug loading, spherical-shape, and smaller particle size. The type of poloxamer and blend ratio affected their structural and functional properties such as morphology, crystallinity, blend miscibility, drug release rate, and cell viability. The methotrexate (MTX), a model drug, was loaded in amorphous spray-dried microparticles. Moreover, the drug release studies demonstrated that PLX induced a leaching-effect of MTX from PLA: PLX blends, suggesting the formation of MTX/PLX micelles in aqueous medium. This finding was better established by cell viability assays. Therefore, biocompatible PLA: PLX blends showed promising in vitro results, and further in vivo studies will be performed to evaluate the performance of this chemotherapeutic agent.

Molecular basis of Tityus stigmurus alpha toxin and potassium channel kV1.2 interactions.

The Tityus stigmurus scorpion is widely distributed in the Northeast of Brazil and is the main causal agent of human envenoming. The venom produced by this scorpion includes neurotoxins, which are peptides belonging to Family 2 toxins and are able to interact with ion channels. The KTx subfamily displays selectivity and affinity for Kv channel subtypes and the result of this interaction is the blockade of potassium channels, impairing vital functions. We report the optimized structural model of a transcript encoding a potassium channel blocker toxin from T. stigmurus. LC-MS analysis confirmed the presence of the toxin in the venom and the three-dimensional structure was obtained by computational homology modeling and refined by molecular dynamic simulations. Furthermore, docking simulations were performed using a Shaker kV-1.2 potassium channel from rats as receptor model and the contacts were identified revealing which amino acid residues and interactions could be involved in its blockade. These residues were mapped and their contact and electrostatic interactions were evaluated revealing the influence of positive lysine residues and the additional contribution of an asparagine to the stabilization of the complex, bringing new insights into the mechanism of action of this toxin.

Intestinal Anti-Inflammatory Activity of the Aqueous Extract from Ipomoea asarifolia in DNBS-Induced Colitis in Rats.

Inflammatory bowel disease is triggered by an uncontrolled immune response associated with genetic, environmental, and intestinal microbiota imbalance. Ipomoea asarifolia (IA), popularly known as "salsa" or "brave salsa", belongs to the Convolvulaceae family. The aim of this approach was to study the preventive effect of IA aqueous extract in 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rats. Rats pretreated with IA extract or sulfasalazine (SSZ) received intracolonic instillation of DNBS in 50% ethanol (v/v). IA extract presented a protective effect against intestinal inflammation, with improvement in the disease activity index and macroscopic damage. IA or SSZ significantly reduced myeloperoxidase activity, and also down-regulation of the gene expression of JNK1, NF-κß-p65, STAT3, and decreased levels of TNFα, IL-1ß, and increased IL-10, associated with a significant improvement of oxidative stress, in addition to a reduction in MDA and an increase of glutathione in colonic tissue. The protective effect of the extract was also confirmed in histological evaluation, showing preservation of the colonic cytoarchitecture. Immunohistochemical analysis revealed down-regulation of NF-κß-p65, iNOS, IL-17, and up-regulation of SOCs-1 and MUC-2. IA extract presents antioxidant and anti-inflammatory intestinal properties, and proved to be a potential application for preventing damage induced by DNBS.

Spondias tuberosa (Anacardiaceae) leaves: profiling phenolic compounds by HPLC-DAD and LC-MS/MS and in vivo anti-inflammatory activity.

Spondias tuberosa is a medicinal plant used by several local communities in northeast Brazil to treat infections, digestive disorders and inflammatory conditions. The study aimed to identify and quantify the major phenolic in hydroethanolic extract of leaves from S. tuberosa and to evaluate its anti-inflammatory potential. The chemical profile of extract was analyzed by HPLC-DAD and HPLC-MS. The in vivo anti-inflammatory activity was investigated in carrageenan-induced hind paw edema and peritonitis models in mice. Identified and quantified through HPLC-DAD or HPLC-MS analyses of S. tuberosa extract were the following compounds: chlorogenic acid, caffeic acid, rutin and isoquercitrin. The inflammatory response to carrageenan was significantly reduced in both models by S. tuberosa extract. In hind paw edema, the edematogenic response was reduced by up to 63.6% and the myeloperoxidase activity was completely inhibited. In the peritonitis model, the total cell migration into the peritoneal cavity was reduced by up to 65%. The results obtained give evidence of the anti-inflammatory action of S. tuberosa and suggest the potential therapeutic benefit of this plant on inflammatory conditions. The chlorogenic acid, caffeic acid, rutin and isoquercitrin identified and quantified in S. tuberosa leaves enable us to suggest that these compounds could be used as chemical markers for quality control of derivative products from this species. Copyright © 2016 John Wiley & Sons, Ltd.

Neutralizing effects of Mimosa tenuiflora extracts against inflammation caused by Tityus serrulatus scorpion venom.

Scorpion bite represents a significant and serious public health problem in certain regions of Brazil, as well as in other parts of the world. Inflammatory mediators are thought to be involved in the systemic and local immune response induced by Tityus serrulatus scorpion envenomation. The aim of this study was to evaluate the effect of extracts of Mimosa tenuiflora on model envenomation. In mice, the envenomation model is induced by Tityus serrulatus venom. Previous treatment of mice with fractions from M. tenuiflora was able to suppress the cell migration to the peritoneal cavity. The treatment of mice with M. tenuiflora extracts also decreased the levels of IL-6, IL-12, and IL-1ß. We concluded that the administration of the extract and fractions resulted in a reduction in cell migration and showed a reduction in the level of proinflammatory cytokines. This study demonstrates, for the first time, the anti-inflammatory effect of aqueous extract from the Mimosa tenuiflora plant on T. serrulatus venom.

Production of enzymes by Paenibacillus chitinolyticus and Paenibacillus ehimensis to obtain chitooligosaccharides.

Obtaining oligosaccharides from chitosan has been the focus of several studies in the pharmaceutical, chemical, food, and medical areas, due to their functional properties. Here, we evaluated the production potential of biologically functional chitooligosaccharides using enzymes extracts produced by Paenibacillus chitinolyticus and Paenibacillus ehimensis. After 48 h of fermentation, these microorganisms were able to produce chitosanases, which generated oligomers with a degree of polymerization between dimers and hexamers. The maximum conversion of chitosan to oligomers was 99.2 %, achieved after 12 h incubation of chitosan with enzymes produced by P. ehimensis. The chitooligosaccharides generated were capable of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical, reaching a maximum scavenging rate of 61 and 39 % when produced with P. ehimensis and P. chitinolyticus enzymes, respectively. The use of these enzymes in the crude form could facilitate their use in industrial applications.

Molecular approaches for structural characterization of a new potassium channel blocker from Tityus stigmurus venom: cDNA cloning, homology modeling, dynamic simulations and docking.

Potassium channels are involved in the maintenance of resting membrane potential, control of cardiac and neuronal excitability, neurotransmitters release, muscle contractility and hormone secretion. The Tityus stigmurus scorpion is widely distributed in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hypoesthesia, edema, erythema, paresthesia, headaches and vomiting. Most potassium channel blocking peptides that have been purified from scorpion venoms contain 30-40 amino acids with three or four disulfide bridges. These peptides belong to α-KTx subfamily. On the other hand, the ß-KTx subfamily is poorly characterized, though it is very representative in some scorpion venoms. A transcriptomic approach of T.stigmurus scorpions developed by our group revealed the repertoire of possible molecules present in the venom, including many toxins of the ß-KTx subfamily. One of the ESTs found, named TSTI0003C has a cDNA sequence of 538 bp codifying a mature protein with 47 amino acid residues, corresponding to 5299 Da. This ß-KTx peptide is a new member of the BmTXKß-related toxins, and was here named TstKMK. The three-dimensional structure of this potassium channel toxin of the T. stigmurus scorpion was obtained by computational modeling and refined by molecular dynamic simulations. Furthermore, we have made docking simulations using a Shaker kV-1.2 potassium channel from rats as receptor model and proposed which amino acid residues and interactions could be involved in its blockade.

Role of matrix metalloproteinases in HaCaT keratinocytes apoptosis induced by loxosceles venom sphingomyelinase D.

Envenomation by the spider Loxosceles (brown spider) can result in dermonecrosis and severe ulceration. We have previously shown that Loxosceles sphingomyelinase D (SMaseD), the enzyme responsible for these pathological effects, induced expression of matrix metalloproteinase-9 (MMP-9), which is possibly one of the main factors involved in the pathogenesis of the cutaneous loxoscelism. The aim of this study was to further investigate the molecular mechanisms triggered by Loxosceles SMaseD involved in the initiation of the dermonecrotic lesion, using HaCaT cultures, a human keratinocyte cell line, as an in vitro model for cutaneous loxoscelism. We show here that SMaseD from Loxosceles spider venom induces apoptosis in human keratinocytes, which is associated with an increased expression of metalloproteinase-2 and -9, and that the use of metalloproteinase inhibitors, such as tetracycline, may prevent cell death and potentially may prevent tissue destruction after envenomation.