Ouratea spectabilis and its Biflavanone Ouratein D Exert Potent Anti-inflammatory Activity in MSU Crystal-induced Gout in Mice.

Gouty arthritis (GA) is an inflammatory arthritis triggered by the deposition of monosodium urate monohydrate (MSU) crystals, causing pain, inflammation, and joint damage. Several drugs are currently employed to manage acute flares of GA, but they either have limited effectiveness or induce severe adverse reactions. Ouratea spectabilis is traditionally used in Brazil to treat gastric ulcers and rheumatism. The ethanolic extract of O. spectabilis stems (OSpC) and four biflavanones (ouratein A - D) isolated thereof were evaluated in a murine model of GA induced by the injection of MSU crystals. The underlying mechanism of action of ouratein D was investigated in vitro in cell cultures by measurement of IL-1ß levels by ELISA and Western blot analysis. The administration of OSpC (10, 30 or 100 mg/Kg, p. o.) reduced the migration of total inflammatory cells, monocytes, and neutrophils and diminished the levels of IL-1ß and CXCL1 in the synovial tissue. Among the tested compounds, only ouratein D (1 mg/Kg) reduced the migration of the inflammatory cells and it was shown to be active up to 0.01 mg/Kg (equivalent to 0.34 nM/Kg, p. o.). Treatment of pre-stimulated THP-1 cells (differentiated into macrophages) or BMDMs with ouratein D reduced the release of IL-1ß in both macrophage lines. This biflavanone reduced the activation of caspase-1 (showed by the increase in the cleaved form) in supernatants of cultured BMDMs, evidencing its action in modulating the inflammasome pathway. The obtained results demonstrate the anti-gout properties of O. spectabilis and point out ouratein D as the bioactive component of the assayed extract.

Digitoxigenin presents an effective and selective antileishmanial action against Leishmania infantum and is a potential therapeutic agent for visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hampered mainly by drug toxicity, their high cost, and parasite resistance. Drug development is a long and pricey process, and therefore, drug repositioning may be an alternative worth pursuing. Cardenolides are used to treat cardiac diseases, especially those obtained from Digitalis species. In the present study, cardenolide digitoxigenin (DIGI) obtained from a methanolic extract of Digitalis lanata leaves was tested for its antileishmanial activity against Leishmania infantum species. Results showed that 50% Leishmania and murine macrophage inhibitory concentrations (IC50 and CC50, respectively) were of 6.9 ± 1.5 and 295.3 ± 14.5 µg/mL, respectively. With amphotericin B (AmpB) deoxycholate, used as a control drug, values of 0.13 ± 0.02 and 0.79 ± 0.12 µg/mL, respectively, were observed. Selectivity index (SI) values were of 42.8 and 6.1 for DIGI and AmpB, respectively. Preliminary studies suggested that the mechanism of action for DIGI is to cause alterations in the mitochondrial membrane potential, to increase the levels of reactive oxygen species and induce accumulation of lipid bodies in the parasites. DIGI was incorporated into Pluronic® F127-based polymeric micelles, and the formula (DIGI/Mic) was used to treat L. infantum-infected mice. Miltefosine was used as a control drug. Results showed that animals treated with either miltefosine, DIGI, or DIGI/Mic presented significant reductions in the parasite load in their spleens, livers, bone marrows, and draining lymph nodes, as well as the development of a specific Th1-type response, when compared with the controls. Results obtained 1 day after treatment were corroborated with data corresponding to 15 days after therapy. Importantly, treatment with DIGI/Mic induced better parasitological and immunological responses when compared with miltefosine- and DIGI-treated mice. In conclusion, DIGI/Mic has the potential to be used as a therapeutic agent to protect against L. infantum infection, and it is therefore worth of consideration in future studies addressing VL treatment.

Inhibition of the sphingosine-1-phosphate pathway promotes the resolution of neutrophilic inflammation.

Sphingosine-1-phosphate (S1P) is an important sphingolipid derived from plasma membrane and has a known role in productive phase of inflammation, but its role in neutrophil survival and resolution phase of inflammation is unknown. Here, we investigated the effects of inhibition of S1P receptors and the blockade of S1P synthesis in BALB/c mice and human neutrophils. S1P and S1PR1-3 receptors expression were increased in cells from the pleural cavity stimulated with LPS. Using different antagonists of S1PRs and inhibitors of different steps of the metabolic pathway of S1P production, we show that S1P and its receptors are involved in regulating neutrophil survival and resolution of inflammation in the pleural cavity. Given the role of the S1P-S1PR axis in resolution of inflammation, we sought to identify whether blockade at different levels of the sphingosine-1-phosphate synthesis pathway could affect neutrophil survival in vitro. Inhibitors of the S1P pathway were also able to induce human neutrophil apoptosis. In addition, blockade of S1P synthesis or its receptor facilitated the efferocytosis of apoptotic neutrophil. Taken together, our data demonstrate a fundamental role for S1P in regulating the outcome of inflammatory responses, and position S1P-S1PR axis as a potential target for treatment of neutrophilic inflammation.

(3,3″)-Linked Biflavanones from Ouratea spectabilis and Their Effects on the Release of Proinflammatory Cytokines in THP-1 Cells.

Ouratea spectabilis is an arborous species traditionally used in Brazil as an anti-inflammatory agent. Four new (3,3″)-linked biflavanone O-methyl ethers, named ouratein A (1), B (2), C (3), and D (4), were isolated from the bark extract of the species. Ouratein A (1) is an enantiomer of neochamagesmine A, which has never been described before. The structures were elucidated by extensive spectroscopic data analyses, whereas their absolute configurations were defined by electronic circular dichroism data. Ouratein D (4) inhibited in vitro the release of the pro-inflammatory cytokine CCL2 by lipopolysaccharide-stimulated THP-1 cells (IC50 of 3.1 ± 1.1 µM), whereas TNF and IL-1ß release were not reduced by any of the biflavanones. These findings show ouratein D (4) as a selective CCL2 inhibitor, which may have potential for the development of new anti-inflammatory agents to prevent or treat cardiovascular diseases.

The Cyclitol L-(+)-Bornesitol as an Active Marker for the Cardiovascular Activity of the Brazilian Medicinal Plant Hancornia speciosa.

The cyclitol bornesitol is the main constituent of the leaves from the antihypertensive medicinal plant Hancornia speciosa. This study aimed to investigate the ability of bornesitol to reduce blood pressure and its mechanism of action. Normotensive Wistar rats were divided into control group and bornesitol groups treated intravenously with bornesitol (0.1, 1.0 and 3.0 mg/kg). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded in non-anesthetized awake animals. Nitric oxide (NO) and angiotensin-converting enzyme (ACE) were measured in plasma by using colorimetric methods. Vascular reactivity study was performed in rat aorta rings and the involvement of nitric oxide synthase (NOS), calcium-calmodulin complex and phosphatidylinositol-3-kinase (PI3K)/Akt pathway in the vasodilator effect was investigated. Administration of bornesitol significantly reduced the SBP, increased the plasmatic level of nitrite, and decreased ACE activity in normotensive rats. In the rat aorta, bornesitol induced endothelium-dependent vasodilatation, which was abolished by NOS blockade. While calcium-calmodulin complex inhibition decreased the vasodilator effect of bornesitol, the inhibition of PI3K/Akt pathway did not alter it. Bornesitol reduced the blood pressure by a mechanism involving an increased production or bioavailability of NO, inhibition of ACE, and by an endothelium- and NO-dependent vasodilator effect. The present results support the use of bornesitol as an active marker for the cardiovascular activity of Hancornia speciosa.

Cytotoxic effects of the cardenolide convallatoxin and its Na,K-ATPase regulation.

Cardenolides are cardiac glycosides, mostly obtained from natural sources. They are well known for their inhibitory action on the Na,K-ATPase, an effect that regulates cardiovascular alterations such as congestive heart failure and atrial arrhythmias. In recent years, they have also sparked new interest in their anticancer potential. In the present study, the cytotoxic effects of the natural cardenolide convallatoxin (CON) were evaluated on non-small cell lung cancer (A549 cells). It was found that CON induced cytostatic and cytotoxic effects in A549 cells, showing essentially apoptotic cell death, as detected by annexin V-propidium iodide double-staining, as well as changes in cell form. In addition, it prompted cell cycle arrest in G2/M and reduced cyclin B1 expression. This compound also increased the number of cells in subG1 in a concentration- and time-dependent manner. At a long term, the reduction of cumulative population doubling was shown along with an increase of ß-galactosidase positive cells and larger nucleus, indicative of senescence. Subsequently, CON inhibited the Na,K-ATPase in A549 cells at nM concentrations. Interestingly, at the same concentrations, CON was unable to directly inhibit the Na,K-ATPase, either in pig kidney or in red blood cells. Additionally, results of docking calculations showed that CON binds with high efficiency to the Na,K-ATPase. Taken together, our data highlight the potent anticancer effects of CON in A549 cells, and their possible link with non-classical inhibition of Na,K-ATPase.

Production of the Cytotoxic Cardenolide Glucoevatromonoside by Semisynthesis and Biotransformation of Evatromonoside by a Digitalis lanata Cell Culture.

Recent studies demonstrate that cardiac glycosides, known to inhibit Na+/K+-ATPase in humans, have increased susceptibility to cancer cells that can be used in tumor therapy. One of the most promising candidates identified so far is glucoevatromonoside, which can be isolated from the endangered species Digitalis mariana ssp. heywoodii. Due to its complex structure, glucoevatromonoside cannot be obtained economically by total chemical synthesis. Here we describe two methods for glucoevatromonoside production, both using evatromonoside obtained by chemical degradation of digitoxin as the precursor. 1) Catalyst-controlled, regioselective glycosylation of evatromonoside to glucoevatromonoside using 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide as the sugar donor and 2-aminoethyldiphenylborinate as the catalyst resulted in an overall 30 % yield. 2) Biotransformation of evatromonoside using Digitalis lanata plant cell suspension cultures was less efficient and resulted only in overall 18 % pure product. Structural proof of products has been provided by extensive NMR data. Glucoevatromonoside and its non-natural 1-3 linked isomer neo-glucoevatromonoside obtained by semisynthesis were evaluated against renal cell carcinoma and prostate cancer cell lines.

Mansoins C-F, Oligomeric Flavonoid Glucosides Isolated from Mansoa hirsuta Fruits with Potential Anti-inflammatory Activity.

Continued investigation of the polyphenolic pool of the fruits of Mansoa hirsuta afforded four additional members of the new class of glucosylated oligomeric flavonoids comprising a flavanone core linked to 1,3-diarylpropane C6-C3-C6 units. The structures and absolute configurations of mansoins C-F (3-6) were established by analysis of NMR and electronic circular dichroism data. Mansoin C (3) was identified as a diglucosylated heterodimer, whereas mansoins D (4), E (5), and F (6) were identified as triglucosylated heterotrimers, isomeric with mansoin A (1). Mansoin F (6) inhibited TNF-α release by lipopolysaccharide-stimulated THP-1 cells (IC50 of 19.3 ± 1.3 µM) and, as with mansoin A (1), reduced the phosphorylation levels of p-65-NF-κB, when assayed at 50 µM. These results indicate that the potential anti-inflammatory properties of mansoin F (6) are probably due to inhibition of the NF-κB pathway and inhibition of TNF-α release.

Anti-TNF-α Activity of Brazilian Medicinal Plants and Compounds from Ouratea semiserrata.

Several plant species are used in Brazil to treat inflammatory diseases and associated conditions. TNF-α plays a pivotal role on inflammation, and several plant extracts have been assayed against this target, both in vitro and in vivo. The effect of 11 Brazilian medicinal plants on TNF-α release by LPS-activated THP-1 cells was evaluated. The plant materials were percolated with different solvents to afford 15 crude extracts, whose effect on TNF-α release was determined by ELISA. Among the evaluated extracts, only Jacaranda caroba (Bignoniaceae) presented strong toxicity to THP-1 cells. Considering the 14 non-toxic extracts, TNF-α release was significantly reduced by seven of them (inhibition > 80%), originating from six plants, namely Cuphea carthagenensis (Lythraceae), Echinodorus grandiflorus (Alismataceae), Mansoa hirsuta (Bignoniaceae), Ouratea semiserrata (Ochnaceae), Ouratea spectabilis and Remijia ferruginea (Rubiaceae). The ethanol extract from O. semiserrata leaves was fractionated over Sephadex LH-20 and RP-HPLC to give three compounds previously reported for the species, along with agathisflavone and epicatechin, here described for the first time in the plant. Epicatechin and lanceoloside A elicited significant inhibition of TNF-α release, indicating that they may account for the effect produced by O. semiserrata crude extract.

Ursolic acid incorporation does not prevent the formation of a non-lamellar phase in pH-sensitive and long-circulating liposomes.

Ursolic acid (UA) is a triterpene found in different plant species that has been shown to possess significant antitumor activity. However, UA presents a low water solubility, which limits its biological applications. In this context, our research group has proposed the incorporation of UA in long-circulating and pH-sensitive liposomes (SpHL-UA).These liposomes, composed of dioleylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethylene glycol2000 (DSPE-PEG2000), were shown to be very promising carriers for UA. Considering that the release of UA from SpHL-UA and its antitumor activity depend upon the occurrence of the lamellar to non-lamellar phase transition of DOPE, in the present work, the interactions of UA with the components of the liposomes were evaluated, aiming to clarify their role in the structural organization of DOPE. The study was carried out by differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS) under low hydration conditions. DSC studies revealed that DOPE phase transition temperatures did not shift significantly upon UA addition. On the other hand, in SAXS studies, a different pattern of DOPE phase organization was observed in the presence of UA, with the occurrence of the cubic phase Im3m at 20 °C and the cubic phase Pn3m at 60 °C. These findings suggest that UA interacts with the lipids and changes their self-assembly. However, these interactions between the lipids and UA were unable to eliminate the lamellar to non-lamellar phase transition, which is essential for the cytoplasmic delivery of UA molecules from SpHL-UA.

TNF-α inhibition elicited by mansoins A and B, heterotrimeric flavonoids isolated from Mansoa hirsuta.

Mansoins A (1) and B (2) isolated from the fruits of Mansoa hirsuta represent new glucosylated heterotrimeric flavonoids with a flavanone core linked to two 1,3-diarylpropane C6-C3-C6 units. Their structures and absolute configurations were established by analysis of their NMR and electronic circular dichroism spectroscopic data. Compounds 1 and 2 inhibited TNF-α release by LPS-stimulated THP-1 cells with different potencies, with mansoin B (2) being active at lower concentrations than mansoin A (1) (IC50 values 20.0±1.4 and 48.1±1.8 µM, respectively). These results indicate potential anti-inflammatory properties for this structural type of oligoflavonoids, especially for mansoin B (2).

Lithothamnion muelleri controls inflammatory responses, target organ injury and lethality associated with graft-versus-host disease in mice.

Lithothamnion muelleri (Hapalidiaceae) is a marine red alga, which is a member of a group of algae with anti-inflammatory, antitumor, and immunomodulatory properties. The present study evaluated the effects of treatment with Lithothamnion muelleri extract (LM) in a model of acute graft-versus-host disease (GVHD), using a model of adoptive splenocyte transfer from C57BL/6 donors into B6D2F1 recipient mice. Mice treated with LM showed reduced clinical signs of disease and mortality when compared with untreated mice. LM-treated mice had reduced tissue injury, less bacterial translocation, and decreased levels of proinflammatory cytokines and chemokines (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5)). The polysaccharide-rich fraction derived from LM could inhibit leukocyte rolling and adhesion in intestinal venules, as assessed by intravital microscopy. LM treatment did not impair the beneficial effects of graft-versus-leukaemia (GVL). Altogether, our studies suggest that treatment with Lithothamnion muelleri has a potential therapeutic application in GVHD treatment.

Isolation and HPLC quantitation of kaurane-type diterpenes and cinnamic acid derivatives of long-term stored leaves of Mikania laevigata and Mikania glomerata.

The leaves of Mikania laevigata and Mikania glomerata are used in Brazil to treat respiratory affections, being kaurane-type diterpenes and coumarin considered as the bioactive compounds. The present study reports an investigation on the HPLC-DAD profiles and contents of coumarin (1), trans-o-coumaric (2), kaurenoic (3), benzoylgrandifloric (4) and cinnamoylgrandifloric (5) acids in dried leaves of Mikania species stored in dark room under controlled conditions. Excepting 2, the constituents were isolated and purified to be employed as reference compounds. The samples were analyzed at three monthly intervals up to 18 months for M. laevigata and 12 months for M. glomerata. trans-o-Coumaric was not detected in both, whereas 1 occurred only in M. laevigata. The concentrations of the assayed constituents did not vary significantly within the evaluated period (p < 0.05), for both species. In contrast, changes in the chromatographic profiles and spectral purity of peaks from 3, 4 and 5 were detected in samples of both Mikania stored for three months, while the coumarin profile in M. laevigata modified after six months of storage. The evaluation of chromatographic profiles based on spectral purity analyses of selected peaks was shown to be a more robust tool to access chemical stability of Mikania samples than the quantitation of chemical markers' contents.

Seasonal variation on the contents of coumarin and kaurane-type diterpenes in Mikania laevigata and M. glomerata leaves under different shade levels.

Coumarin (1) and kaurane-type diterpenes are considered the bioactive constituents of Mikania glomerata and M. laevigata, used in Brazil to treat respiratory affective disorders. The seasonal variation of 1, ortho-coumaric acid (2), benzoylgrandifloric acid (3), cinnamoylgrandifloric acid (4), and kaurenoic acid (5) in leaves of both species, cultivated in full sunlight and under shade levels of 40 and 80%, was quantified by HPLC. Compound 2 was detected solely in M. laevigata in concentrations below the limit of quantification. Coumarin was not found in M. glomerata, whereas its concentration reached 0.94±0.24% (w/w) in M. laevigata farmed in summer under 80% shading. Both Mikania species produced higher amounts of kaurane diterpenes when cultivated in plenty of sunlight. Hence, maximum contents of 1 are reached in M. laevigata cultivated under high shading, but with reduced concentrations of 3-5. Conversely, M. glomerata should be cultivated under full sunlight and harvested in winter for highest concentrations of kaurane-type diterpenes.

Oral sub-chronic treatment with Terminalia phaeocarpa Eichler (Combretaceae) reduces liver PTP1B activity in a murine model of diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE: The endemic Brazilian medicinal plants of the genus Terminalia (Combretaceae), popularly known as capitão, comprising the similar species Terminalia phaeocarpa Eichler and Terminalia argentea, are traditionally and indistinguishably used in the country to treat diabetes. AIM OF THE STUDY: The present work investigated the effect of 28 days of treatment with the crude ethanolic extract (CEE) and its derived ethyl acetate fraction (EAF) from T. phaeocarpa leaves in a mice model of diabetes. MATERIALS AND METHODS: Streptozotocin-nicotinamide-fructose diabetic model was used to evaluate the antidiabetic activity of 28 days of treatment with the CEE and EAF from the leaves of T. phaeocarpa and metformin as a positive control. Serum levels of total cholesterol, triglycerides, uric acid, ALP, AST, and ALT were measured with specific commercial kits and glucose with a strip glucometer. The thiobarbituric acid method measured the liver MDA level, while a colorimetric assay measured the GSH level and PTP1B activity. A UPLC-DAD profile was obtained to identify the main polyphenolic compound in the EAF. RESULTS: Treatment with CEE and EAF reduced plasma glucose in diabetic mice. At the end of the treatment, the plasma glucose level was significantly lower in EAF-treated (100 mg/kg) diabetic mice (106.1 ± 13.7 mg/dL) than those treated with 100 mg/kg CEE (175.2 ± 20.9 mg/dL), both significantly lower than untreated diabetic mice (350.4 ± 28.1 mg/dL). The serum levels of total cholesterol, triglycerides, uric acid, ALP, AST, and ALT were significantly reduced in diabetic mice treated with CEE and EAF. In the livers of diabetic mice, the treatment with CEE and EAF reduced MDA levels and the activity of the enzyme PTP1B (96.9 ± 3.7%, 113.8 ± 2.8%, and 134.8 ± 4.6% for CEE-, EAF-treated, and untreated diabetic mice, respectively). Galloylpunicalagin was the main polyphenol observed in the EAF of T. phaeocarpa. CONCLUSION: The present results demonstrate the significant antidiabetic effect of CEE and EAF of T. phaeocarpa and their reduction on the markers of liver dysfunction in diabetic mice. Moreover, the antidiabetic activity of T. phaeocarpa might be associated with lowering the augmented activity of the PTP1B enzyme in the liver of diabetic mice.

Antiadhesive activity of polysaccharide-rich fractions from Lithothamnion muelleri.

Red seaweeds are known sources of polysaccharides, some of which possess antiadhesive properties by inhibition of P-selectin-mediated leukocyte rolling. We here report the chemical composition and the antiadhesive activity of polysaccharide-rich fractions from the red alga Lithothamnion muelleri (Hapalidiaceae). The crude fractions enriched in polysaccharides B1 and B2 were obtained, respectively, by sequential extraction with 1% and 2% (w/v) Na2CO3 solution, at 60 degrees C. Fractionation of B1 and B2 by gel permeation chromatography afforded three polysaccharide-rich fractions each, whose compositions were characterized by chemical analysis (total contents of carbohydrates, proteins, sulfate, and uronic acid); their molecular weights were estimated by high-performance gel permeation chromatography (HPGPC). The antiadhesive activity of B1-derived fractions was assayed by visualizing lipopolysaccharides-induced leukocyte rolling under intravital miscroscopy. The intravenous injection of fractions B1a and B1b in mice, at the dose of 10 mg/kg body weight, reduced leukocyte rolling by approximately 90%; fucoidan (10 mg/kg body weight) employed as positive control induced a similar response. Therefore, the sulfated polysaccharides of L. muelleri deserve further evaluation as potential templates for the development of new anti-inflammatory agents.

Bioguided chemical characterization of pequi (Caryocar brasiliense) fruit peels towards an anti-diabetic activity.

Pequi fruit peels are an underexploited source of polyphenols. The anti-diabetic potential of an extract and fractions from the peels were evaluated in a panel of assays. The extract and fractions thereof inhibited the release of cytokines involved in insulin resistance - TNF, IL-1ß, and CCL2 - by lipopolysaccharide-stimulated THP-1 cells. The ethyl acetate fraction inhibited in vitro α-glucosidase (pIC50 = 4.8 ± 0.1), an enzyme involved in the metabolization of starch and disaccharides to glucose, whereas a fraction enriched in tannins (16C) induced a more potent α-glucosidase inhibition (pIC50 = 5.3 ± 0.1). In the starch tolerance test in mice, fraction 16C reduced blood glucose level (181 ± 10 mg/dL) in comparison to the vehicle-treated group (238 ± 11 mg/dL). UPLC-DAD-ESI-MS/MS analyses disclosed phenolic acids and tannins as constituents, including corilagin and geraniin. These results highlight the potential of pequi fruit peels for developing functional foods to manage type-2 diabetes.

In vitro and in vivo antileishmanial activity of ß-acetyl-digitoxin, a cardenolide of Digitalis lanata potentially useful to treat visceral leishmaniasis.

Current treatments of visceral leishmaniasis face limitations due to drug side effects and/or high cost, along with the emergence of parasite resistance. Novel and low-cost antileishmanial agents are therefore required. We report herein the antileishmanial activity of ß-acetyl-digitoxin (b-AD), a cardenolide isolated from Digitalis lanata leaves, assayed in vitro and in vivo against Leishmania infantum. Results showed direct action of b-AD against parasites, as well as efficacy for the treatment of Leishmania-infected macrophages. In vivo experiments using b-AD-containing Pluronic® F127 polymeric micelles (b-AD/Mic) to treat L. infantum-infected mice showed that this composition reduced the parasite load in distinct organs in more significant levels. It also induced the development of anti-parasite Th1-type immunity, attested by high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and specific IgG2a antibodies, in addition to low IL-4 and IL-10 contents, along with higher IFN-γ-producing CD4+ and CD8+ T-cell frequency. Furthermore, low toxicity was found in the organs of the treated animals. Comparing the therapeutic effect between the treatments, b-AD/Mic was the most effective in protecting animals against infection, when compared to the other groups including miltefosine used as a drug control. Data found 15 days after treatment were similar to those obtained one day post-therapy. In conclusion, the results obtained suggest that b-AD/Mic is a promising antileishmanial agent and deserves further studies to investigate its potential to treat visceral leishmaniasis.

TITLE: Activité antileishmaniale in vitro et in vivo de la ß-acétyl-digitoxine, un cardénolide de Digitalis lanata potentiellement utile pour traiter la leishmaniose viscérale. ABSTRACT: Les traitements actuels de la leishmaniose viscérale font face à des limitations dues aux effets secondaires des médicaments et/ou à leur coût élevé, ainsi qu'à l'émergence d'une résistance parasitaire. Des agents antileishmaniaux nouveaux et peu coûteux sont donc nécessaires. Nous rapportons ici l'activité antileishmaniale de la ß-acétyl-digitoxine (b-AD), un cardénolide isolé à partir de feuilles de Digitalis lanata, mesurée in vitro et in vivo contre Leishmania infantum. Les résultats ont montré une action directe de la b-AD contre les parasites, ainsi qu'une efficacité pour le traitement des macrophages infectés par Leishmania. Des expériences in vivo utilisant des micelles polymériques Pluronic® F127 contenant de la b-AD (b-AD/Mic) pour traiter des souris infectées par L. infantum ont montré que cette composition réduisait à des niveaux plus significatifs la charge parasitaire dans différents organes, ainsi que le développement d'une immunité antiparasitaire de type Th1, attestée par les taux élevés d'IFN-γ, d'IL-12, de TNF-α, de GM-CSF, de nitrite et d'anticorps IgG2a spécifiques, en plus des faibles taux d'IL-4 et IL-10, ainsi qu'une fréquence plus élevée des cellules T CD4+ and CD8+ productrices d' IFN-γ. De plus, une faible toxicité a été trouvée dans les organes des animaux traités. En comparant l'effet thérapeutique des traitements, b-AD/Mic était le plus efficace pour protéger les animaux contre l'infection, par rapport aux autres groupes comprenant la miltefosine utilisée comme contrôle médicamenteux. Les données trouvées 15 jours après le traitement étaient similaires à celles obtenues un jour après le traitement. En conclusion, les résultats obtenus suggèrent que b-AD/Mic est un agent antileishmanial prometteur et mérite des études supplémentaires pour étudier son potentiel à traiter la leishmaniose viscérale.

Evaluation of Brazilian plants on cancer chemoprevention targets in vitro.

Cancer is a leading cause of death worldwide. Cancer chemoprevention is one of the promising strategies to decrease its incidence and both plant extracts and natural products may constitute sources of new chemoprevention agents. Some Brazilian species popularly used to treat inflammatory conditions were selected for evaluation for cancer chemoprevention. A total of 32 extracts/fractions from Hancornia speciosa, Davilla elliptica, Jacaranda caroba, Mansoa hirsuta, Remija ferrugina, Solanum paniculatum and Xyris pterygoblephara, along with a mixture of ursolic and oleanolic acids obtained from J. caroba and a dihydroisocoumarin isolated from aerial parts of X. pterygoblephara were tested for their cancer chemoprevention activity [inhibition of 12-O-tetradecanoyl-13-acetate (TPA)-mediated NF-kappaB activation, ornithine decarboxylase (ODC) and cyclooxygenase-1 (COX-1); induction of antioxidant response element (ARE)]. Several extracts/fractions were active in more than one assay and those from H. speciosa, M. hirsuta and J. caroba mediated strong responses with NF-kappaB, COX-1 and ARE, respectively.

Determination of l-(+)-bornesitol, the hypotensive constituent of Hancornia speciosa, in rat plasma by LC-MS/MS and its application on a pharmacokinetic study.

Hancornia speciosa is a medicinal plant with proven antihypertensive activity. The cyclitol l-(+)-bornesitol is the main constituent of its leaves and is a potent inhibitor of the angiotensin-converting enzyme. We herein investigated the pharmacokinetic properties of bornesitol administered orally to Wistar rats, as well as bornesitol permeation in Caco-2 cells. Bornesitol was isolated and purified from an ethanol extract of H. speciosa leaves. An ultra-high performance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated to quantify bornesitol in rat plasma based on Multiple Reaction Monitoring, using pentaerythritol as an internal standard. Pharmacokinetics was evaluated by the administration of single doses via intravenous in bolus (3 mg/kg) and gavage (3, 15 and 25 mg/kg). Bornesitol permeation was assayed in a transwell Caco-2 cells model, tested alone, or combined with rutin, or as a constituent of H. speciosa extract, using a developed and validated UPLC-ESI-MS/MS method. All assayed validation parameters (selectivity, residual effect, matrix effect, linearity, precision, accuracy and stability of analyte in plasma and solution) for the bioanalytical method met the acceptance criteria established by regulatory guidelines. Bornestiol reached peak plasma concentration within approximately 60 min after oral administration with a half-life ranging from 72.15 min to 123.69 min. The peak concentration and area under the concentration-time curve of bornesitol did not rise proportionally with the increasing doses, suggesting a non-linear pharmacokinetics in rats and the oral bioavailability ranged from 28.5%-59.3%. Bornesitol showed low permeability in Caco-2 cells, but the permeability apparently increased when it was administered either combined with rutin or as a constituent of H. speciosa extract. In conclusion, bornesitol was rapidly absorbed after a single oral administration to rats and followed a non-linear pharmacokinetics. The obtained data will be useful to guide further pre-clinical development of bornesitol-containing herbal preparations of H. speciosa as an antihypertensive agent.