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.

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.

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.

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.

(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.

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.

Antiviral activity of Myracrodruon urundeuva against rotavirus

ABSTRACT Myracrodruon urundeuva Allemão, Anacardiaceae, is a medicinal plant widely found in Brazil, especially in the northern region. In our previous study, the ethanolic extract from leaves of M. urundeuva showed antiviral activity against simian rotavirus SA-11. Here, the crude extract was subjected to fractionations in order to subsequently work with more concentrated and pure bioactive compounds, which were analyzed by TLC and HPLC methods to support a better understanding of their virucidal effect. The antiviral activity was evaluated using a rotavirus infection model in MA-104 cells treated with the maximum non-cytotoxic concentration of the crude extract and its fractions. Data were expressed as the percentage inhibition of viral replication calculated by the inhibition of cytopathic effect in the treated cells compared to untreated controls after 48 h of incubation. First, we conducted a fractionation, generating five fractions (F1–F5) which were submitted to antiviral assay. Then, the fraction that showed the highest virucidal effect (F3, PI = 75%) was subjected to a larger partition, yielding eighteen subfractions, which were submitted to new antiviral assays. Terpenes, flavonoids and tannins were the major secondary metabolites detected by TLC analysis in F3. SF1, a flavonoid-enriched fraction, showed the strongest in vitro activity against rotavirus (PI = 92%), preventing cytopathic effect. Chromatographic profiles were obtained by HPLC for the crude extract and SF1, the most potent subfraction. Overall, our data point to the potential anti-rotavirus activity of flavonoid-enriched fraction (SF1) of M. urundeuva leaves, corroborating the traditional use of this species to treat diarrhea and broadening our perspectives on in vivo assays in mice with SF1 isolated or associated with other fractions.

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.

Evaluation of the Effects of Some Brazilian Medicinal Plants on the Production of TNF- α and CCL2 by THP-1 Cells.

Several plant species are traditionally used in Brazil to treat various inflammatory diseases. Tumor necrosis factor- (TNF-) α and chemokine (C-C motif) ligand 2 (CCL2) are key inflammatory mediators in diseases like rheumatoid arthritis and atherosclerosis, respectively; nevertheless, only a few extracts have been assayed against these targets. We herein report the effect of 19 plant extracts on TNF-α and CCL2 release by lipopolysaccharide- (LPS-) stimulated THP-1 cells, a human monocytic leukemia cell line, along with their radical scavenging activity on DPPH. The extracts of Caryocar brasiliense, Casearia sylvestris, Coccoloba cereifera, and Terminalia glabrescens inhibited TNF-α production in a concentration-dependent manner. Fractionation of these extracts potentiated the anti-TNF-α effect, which was shown to concentrate in polar fractions, mainly composed by polyphenols. Significant CCL2 inhibition was elicited by Lippia sidoides and Terminalia glabrescens extracts, whose fractionation resulted in highly active low polar fractions. All assayed extracts showed strong radical scavenging activity, but antioxidant activity did not correlate with inhibition of TNF-α or CCL2 production. Our results allowed identifying extracts with selective capacity to block cytokine production; therefore, further purification of these extracts may yield molecules that could be useful in the treatment of chronic inflammatory diseases.

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).

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.

Endothelium-dependent vasorelaxation in rat thoracic aorta by Mansoa hirsuta D.C.

The vasodilator effect of the ethanolic extract of Mansoa hirsuta leaves (EEF) was assayed in rat aortic rings. EEF produced a concentration-dependent vasodilatation (pIC(50)=5.1+/-0.2), which was absent in endothelium-denuded vessels. The vasodilator effect of EEF was similar to a standardized ethanolic extract of Hancornia speciosa Gomes (pIC(50)=5.1+/-0.1). The endothelium-dependent vasodilatation induced by EEF was abolished by L-NAME (100 microM), a nitric oxide (NO) synthase inhibitor, but not by indomethacin (10 microM; pIC(50)=4.9+/-0.2), a cyclooxygenase inhibitor. The concentration-response curve of EEF was not modified by the addition of superoxide dismutase (SOD; 300 U/ml). In addition, EEF (50 microg/ml) displaced the 3-morpholino-sidnonimine (SIN-1; p<0.05) concentration-effect curve to the left, as well as SOD (300 U/ml). These findings lead us to conclude that EEF induces a NO- and endothelium-dependent vasodilatation in rat aortic preparations, and that this effect is, at least in some extent, due to an increase in the NO bioavailability as consequence of its antioxidant activity. The HPLC-DAD profile recorded for EEF indicates the presence of four major peaks with close retention times, exhibiting similar UV spectra with wavelength maxima compatible with heterogeneous proanthocyanidins.