Monocrotaline induces acutely cerebrovascular lesions, astrogliosis and neuronal degeneration associated with behavior changes in rats: A model of vascular damage in perspective.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites playing an important role as phytotoxins in the plant defense mechanisms and can be present as contaminant in the food of humans and animals. The PA monocrotaline (MCT), one of the major plant derived toxin that affect humans and animals, is present in a high concentration in Crotalaria spp. (Leguminosae) seeds and can induce toxicity after consumption, characterized mainly by hepatotoxicity and pneumotoxicity. However, the effects of the ingestion of MCT in the central nervous system (CNS) are still poorly elucidated. Here we investigated the effects of MCT oral acute administration on the behavior and CNS toxicity in rats. Male adult Wistar were treated with MCT (109 mg/Kg, oral gavage) and three days later the Elevated Pluz Maze test demonstrated that MCT induced an anxiolytic-like effect, without changes in novelty habituation and in operational and spatial memory profiles. Histopathology revealed that the brain of MCT-intoxicated animals presented hyperemic vascular structures in the hippocampus, parahippocampal cortex and neocortex, mild perivascular edema in the neocortex, hemorrhagic focal area in the brain stem, hemorrhage and edema in the thalamus. MCT also induced neurotoxicity in the cortex and hippocampus, as revealed by Fluoro Jade-B and Cresyl Violet staining, as well astrocyte reactivity, revealed by immunocytochemistry for glial fibrillary acidic protein. Additionally, it was demonstrated by RT-qPCR that MCT induced up-regulation on mRNA expression of neuroinflammatory mediator, especially IL1ß and CCL2 in the hippocampus and cortex, and down-regulation on mRNA expression of neurotrophins HGDF and BDNF in the cortex. Together, these results demonstrate that the ingestion of MCT induces cerebrovascular lesions and toxicity to neurons that are associated to astroglial cell response and neuroinflammation in the cortex and hippocampus of rats, highlighting CNS damages after acute intoxication, also putting in perspective it uses as a model for cerebrovascular damage.

Antitumor and antiangiogenic effects of Tonantzitlolone B, an uncommon diterpene from Stillingia loranthacea.

Natural products have played a pivotal role for the discovery of anticancer drugs. Tonantzitlolones are flexibilan-type diterpenes rare in nature; therefore, few reports have shown antiviral and cytotoxic activities. This study aimed to investigate the in vivo antitumor action of Tonantzitlolone B (TNZ-B) and its toxicity. Toxicity was evaluated in mice (acute and micronucleus assays). Antitumor activity of TNZ-B (1.5 or 3 mg/kg intraperitoneally - i.p.) was assessed in Ehrlich ascites carcinoma model. Angiogenesis and reactive oxygen species (ROS) and nitric oxide (NO) production were also investigated, in addition to toxicological effects after 7-day treatment. The LD50 (lethal dose 50%) was estimated at around 25 mg/kg (i.p.), and no genotoxicity was recorded. TNZ-B reduced the Ehrlich tumor's volume and total viable cancer cell count (p < 0.001 for both). Additionally, TNZ-B reduced peritumoral microvessel density (p < 0.01), suggesting antiangiogenic action. Moreover, a decrease was observed on ROS (p < 0.05) and nitric oxide (p < 0.001) levels. No significant clinical findings were observed in the analysis of biochemical, hematological, and histological (liver and kidney) parameters. In conclusion, TNZ-B exerts antitumor and antiangiogenic effects by reducing ROS and NO levels and has weak in vivo dose-repeated toxicity. These data contribute to elucidate the antitumor action of TNZ-B and point the way for further studies with this natural compound as an anticancer drug.

Bioactive compounds of Copaifera sp. impregnated into three-dimensional gelatin dressings.

This study investigates the immersion impregnation process of the copaiba oleoresin and leaf extract into SpongostanTM gelatin dressings to be used in wound healing treatment. Copaiba oleoresin and leaf extract were characterized by spectroscopic analyses in order to confirm the identity of bioactive compounds and their compatibility with dressing material. Their antibacterial properties were evaluated and oleoresin activity against Escherichia coli and Staphylococcus aureus bacteria was confirmed while the leaf extract showed activity against S. aureus. Solubility assays in organic solvents revealed that copaiba oleoresin is miscible into dichloromethane, while leaf extract showed a 20 g/ml solubility coefficient at 35 °C in the same solvent. These miscibility and solubility conditions were selected for the impregnation process. Using the organic solvent immersion method, 11 mg of copaiba oleoresin and 19 mg of leaf extract were impregnated into 1 cm3 of 3D matrix. The main bioactives from copaiba products, such as ß-caryophyllene and lupeol, were tracked in the gelatin dressing. DSC and TGA assays showed no thermal changes in the samples after impregnation. Furthermore, the spatial organization of foam structure of the dressings was preserved after superficial distribution of oleoresin, as well as amorphous-like particulate deposition of leaf extract. The main compound of copaiba oleoresin, ß-caryophyllene, which exhibits well-known anti-inflammatory activities, and the main compound of copaiba leaf extract, lupeol, also an anti-inflammatory agent, were successfully impregnated using organic solvent in wound dressings and are promising for further application on tissue wound healing. Graphical Abstract.

Sambucus australis Modulates Inflammatory Response via Inhibition of Nuclear Factor Kappa B (NF-kB) in vitro.

Medicinal plants have long been used as an alternative to traditional drugs for the treatment of inflammatory conditions due to the classical side effects and restricted access of various commercially available drugs, such as steroids (GCs) and nonsteroidal anti-inflammatory drugs (NSAIDs). Sambucus australis is a Brazilian herb that is commonly used to treat inflammatory diseases; however, few studies have examined the use of this species in the treatment of inflammatory conditions. The present study aims to evaluate the potential anti-inflammatory activity of S. australis in vitro. We established spleen cell cultures stimulated with pokeweed mitogen (PWM) to evaluate the production of proinflammatory cytokines, such as IL-4, IL-5, IFN-y, and IL-10 (by ELISA), and the expression of the transcription factor NF-kB (by RT-PCR). In addition, we evaluated the levels of nitric oxide in macrophage cultures and the membrane-stabilizing activity of S. australis methanolic extract (EMSA). Treatment with EMSA at concentrations of 100, 50, 25 and 12.5 µg/ml significantly decreased IL-4 (p<0.001) and IL-5 (p<0.001) levels. Treatment with 100 µg/ml EMSA reduced IFN-у (p<0.001) levels. Moreover, at 100 mg/ml, EMSA also increased IL-10 production and reduced NF-kB expression (p<0.01). In macrophage cultures stimulated with LPS, EMSA decreased nitric oxide levels (p<0.001) at all concentrations tested (100, 50, 25 and 12.5 µg/ml). Additionally, EMSA had a protective effect in the erythrocyte membrane stabilization assay. Taken together, these results suggest that S. australis has anti-inflammatory potential in vitro, characterized by the reduction of both inflammatory cytokines and the expression of NF-kB along with the up-regulation of IL-10.

In vitro antileishmanial and antitrypanosomal activity of compounds isolated from the roots of Zanthoxylum tingoassuiba

Abstract Five coumarins (5,7,8-trimethoxycoumarin (1), sabandin (2), cubreuva lactone (3), 5,7-dimethoxycoumarin (5) and braylin (6)), seven furoquinoline alkaloids (isopimpinelin (4), pteleine (7), maculine (8), skimianine (10), robustine (11), y-fagarine (12) and dictamine (13) and the furofuran type lignin syringaresinol (9)) have been identified for the first time in the roots of Zanthoxylum tingoassuiba A. St.-Hil., Rutaceae. Pure compounds 1, 6, 9, 12 were tested against Leishmania amazonensis parasites and epimastigotes forms of Trypanosoma cruzi. All the tested products displayed an antiparasitic activity similar to that of the positive controls (benznidazole and amphotericin B). Compound 9 was the most active against both parasites with IC50 values of 11.98 µM and 7.55 µM against L. amazonensis and T. cruzi, respectively.

Autophagy protects against neural cell death induced by piperidine alkaloids present in Prosopis juliflora (Mesquite).

Prosopis juliflora is a shrub that has been used to feed animals and humans. However, a synergistic action of piperidine alkaloids has been suggested to be responsible for neurotoxic damage observed in animals. We investigated the involvement of programmed cell death (PCD) and autophagy on the mechanism of cell death induced by a total extract (TAE) of alkaloids and fraction (F32) from P. juliflora leaves composed majoritary of juliprosopine in a model of neuron/glial cell co-culture. We saw that TAE (30 µg/mL) and F32 (7.5 µg/mL) induced reduction in ATP levels and changes in mitochondrial membrane potential at 12 h exposure. Moreover, TAE and F32 induced caspase-9 activation, nuclear condensation and neuronal death at 16 h exposure. After 4 h, they induced autophagy characterized by decreases of P62 protein level, increase of LC3II expression and increase in number of GFP-LC3 cells. Interestingly, we demonstrated that inhibition of autophagy by bafilomycin and vinblastine increased the cell death induced by TAE and autophagy induced by serum deprivation and rapamycin reduced cell death induced by F32 at 24 h. These results indicate that the mechanism neural cell death induced by these alkaloids involves PCD via caspase-9 activation and autophagy, which seems to be an important protective mechanism.

In vitro antibacterial effects of Zanthoxylum tingoassuiba root bark extracts and two of its alkaloids against multiresistant Staphylococcus aureus

ABSTRACT The emergence of multiresistant strains of bacteria reinforces the need to search for new compounds able to combat resistant organisms. Medicinal plants are a great resource of bioactive substances, providing the possibility of obtaining molecules with potential antimicrobial activity. The aim of the present study is the evaluation of the antibacterial activity of extracts and alkaloids isolated from the root bark of Zanthoxylum tingoassuiba A. St.-Hil., Rutaceae, against four resistant clinical isolates and Staphylococcus aureus ATCC 25923. The dichloromethane and methanol extracts were fractionated by chromatography on silica gel, leading to the isolation of dihydrocheleryhtrine and N-methylcanadine, identified by Nuclear Magnetic Resonance spectroscopy. The antibacterial activity of the extracts and isolated compounds was evaluated by the disc diffusion method and the minimum inhibitory concentration was determined. The dichloromethane extract was the most active against all the tested strains and the two pure alkaloids were more active than the extracts. The anti-MRSA activity of the two benzophenanthridine alkaloids is demonstrated for the first time in this study. These compounds appear as potential leads for the development of new anti-MRSA compounds and could be responsible for the antibacterial activity, justifying the ethnobotanical use of Z. tingoassuiba and other species for the treatment of various infectious diseases.

Autophagy protects against neural cell death induced by piperidine alkaloids present in Prosopis juliflora (Mesquite)

ABSTRACT Prosopis juliflora is a shrub that has been used to feed animals and humans. However, a synergistic action of piperidine alkaloids has been suggested to be responsible for neurotoxic damage observed in animals. We investigated the involvement of programmed cell death (PCD) and autophagy on the mechanism of cell death induced by a total extract (TAE) of alkaloids and fraction (F32) from P. juliflora leaves composed majoritary of juliprosopine in a model of neuron/glial cell co-culture. We saw that TAE (30 µg/mL) and F32 (7.5 µg/mL) induced reduction in ATP levels and changes in mitochondrial membrane potential at 12 h exposure. Moreover, TAE and F32 induced caspase-9 activation, nuclear condensation and neuronal death at 16 h exposure. After 4 h, they induced autophagy characterized by decreases of P62 protein level, increase of LC3II expression and increase in number of GFP-LC3 cells. Interestingly, we demonstrated that inhibition of autophagy by bafilomycin and vinblastine increased the cell death induced by TAE and autophagy induced by serum deprivation and rapamycin reduced cell death induced by F32 at 24 h. These results indicate that the mechanism neural cell death induced by these alkaloids involves PCD via caspase-9 activation and autophagy, which seems to be an important protective mechanism.

Pyranochromones from Dictyoloma vandellianum A. Juss and Their Cytotoxic Evaluation.

One new chromone 3,3-dimethylallylspatheliachromene methyl ether (1), as well as five known chromones, 6-(3-methylbut-2-enyl) allopteroxylin methyl ether (2), 6-(3-methylbut-2-enyl) allopteroxylin (3), 3,3-dimethylallylspatheliachromene (4), 5-O-methylcneorumchromone K (5) and spatheliabischromene (6), two alkaloids, 8-methoxy-N-methylflindersine (7) and 8-methoxyflindersine (8), and two limonoids, limonin diosphenol (9) and rutaevin (10), were isolated from Dictyoloma vandellianum A. Juss (Rutaceae). Cytotoxic activities towards tumor cell lines B16-F10, HepG2, K562 and HL60 and non-tumor cells PBMC were evaluated for compounds 1 - 6. Compound 1 was the most active showing IC50 values ranging from 6.26 to 14.82 µg/ml in B16-F10 and K562 cell lines, respectively, and presented IC50 value of 11.65 µg/ml in PBMC cell line.

Juliprosopine and juliprosine from prosopis juliflora leaves induce mitochondrial damage and cytoplasmic vacuolation on cocultured glial cells and neurons.

Prosopis juliflora is a shrub largely used for animal and human consumption. However, ingestion has been shown to induce intoxication in animals, which is characterized by neuromuscular alterations induced by mechanisms that are not yet well understood. In this study, we investigated the cytotoxicity of a total alkaloid extract (TAE) and one alkaloid fraction (F32) obtained from P. juliflora leaves to rat cortical neurons and glial cells. Nuclear magnetic resonance characterization of F32 showed that this fraction is composed of a mixture of two piperidine alkaloids, juliprosopine (majority constituent) and juliprosine. TAE and F32 at concentrations between 0.3 and 45 µg/mL were tested for 24 h on neuron/glial cell primary cocultures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed that TAE and F32 were cytotoxic to cocultures, and their IC50 values were 31.07 and 7.362 µg/mL, respectively. Exposure to a subtoxic concentration of TAE or F32 (0.3-3 µg/mL) induced vacuolation and disruption of the astrocyte monolayer and neurite network, ultrastructural changes, characterized by formation of double-membrane vacuoles, and mitochondrial damage, associated with changes in ß-tubulin III and glial fibrillary acidic protein expression. Microglial proliferation was also observed in cultures exposed to TAE or F32, with increasing levels of OX-42-positive cells. Considering that F32 was more cytotoxic than TAE and that F32 reproduced in vitro the main morphologic and ultrastructural changes of "cara torta" disease, we can also suggest that piperidine alkaloids juliprosopine and juliprosine are primarily responsible for the neurotoxic damage observed in animals after they have consumed the plant.

Extraction with supercritical fluid and comparison of chemical composition from adults and young leaves of Zanthoxylum tingoassuiba

Plants differ in metabolism during their life cycle. In species used as phytotherapics, these changes determine the quality and effectiveness of the product. The aim of this study to evaluate the differences of chemical extracts obtained with supercritical CO2 from young and adult leaves of Zanthoxylum tingoassuiba St.-Hil., Rutaceae, a species used in the folk medicine in Brazil. The chemical composition of the extracts was elucidated by spectroscopic analysis and comparison with literature data. The results showed difference in the composition of the leaves from Z. tingoassuiba and allowed the determination of parameters for the extraction of α-bisabolol and furanocoumarins in this vegetal matrix.

Antinociceptive effects of Abarema cochliacarpos (B.A. Gomes) Barneby & J.W.Grimes (Mimosaceae) / Efeito antinociceptivo de Abarema cochliacarpos (B.A. Gomes) Barneby & J.W.Grimes (Mimosaceae)

In this study, we investigated the analgesic activity of crude aqueous and methanol extracts obtained from Abarema cochliacarpos bark in mice, and analyzed its phytochemical profile. All the extracts exhibited analgesic properties against the writhing test in mice, but the aqueous and methanol extracts were more active, and more potent than two known analgesic and anti-inflammatory drugs used as reference. They were also active against the capsaicin-model, but inactive when evaluated in the hot-plate test. Phytochemical studies revealed the presence of saponins, catechins, tannins, phenols and anthraquinones.

No presente trabalho foram avaliados a atividade antinociceptiva e o perfil fitoquímico dos extratos aquosos e metanólico produzidos com a casca do caule de Abarema cochliacarpos, uma espécie de Mata Atlântica com diversas indicações populares. Todos os extratos apresentaram atividade analgésica quando avaliados pelo teste das contorções abdominais induzidas pelo ácido acético via intraperitonial, apresentando respostas superiores às drogas usadas como referência, bem como no modelo da dor induzida por capsaicina. A avaliação fitoquímica demonstrou a presença de saponinas, catequinas, taninos, fenóis e antraquinonas.