Hydroxytakakiamide and Other Constituents from a Marine Sponge-Associated Fungus Aspergillus fischeri MMERU23, and Antinociceptive Activity of Ergosterol Acetate, Acetylaszonalenin and Helvolic Acid.

An unreported prenylated indole derivative hydroxytakakiamide (4) was isolated, together with the previously described ergosterol (1), ergosterol acetate (2), and (3R)-3-(1H-indol-3-ylmethyl)-3, 4-dihydro-1H-1,4-benzodiazepine-2,5-dione (3), from the column fractions of the crude ethyl acetate extract of the culture of a marine sponge-associated fungus, Aspergillus fischeri MMERU 23. The structure of 4 was elucidated by the interpretation of 1D and 2D NMR spectral data and high-resolution mass spectrum. The absolute configuration of the stereogenic carbon in 3 was proposed to be the same as those of the co-occurring congeners on the basis of their biogenetic consideration and was supported by the comparison of its sign of optical rotation with those of its steroisomers. The crude ethyl acetate extract and 2 were evaluated, together with acetylaszonalenin (5) and helvolic acid (6), which were previously isolated from the same extract, for the in vivo antinociceptive activity in the mice model. The crude ethyl acetate extract exhibited antinociceptive activity in the acetic acid-induced writhing and formalin tests, while 2, 5, and 6 displayed the effects in the late phase of the formalin test. On the other hand, neither the crude ethyl acetate extract nor 2, 5, and 6 affected the motor performance of mice in both open-field and rotarod tests. Additionally, docking studies of 2, 5, and 6 were performed with 5-lipoxygenase (5-LOX) and phosphodiesterase (PDE) enzymes, PDE4 and PDE7, which are directly related to pain and inflammatory processes. Molecular docking showed that 6 has low affinity energy to PDE4 and PDE7 targets while retaining high affinity to 5-LOX. On the other hand, while 2 did not display any hydrogen bond interactions in any of its complexes, it achieved overall better energy values than 6 on the three antinociceptive targets. On the other hand, 5 has the best energy profile of all the docked compounds and was able to reproduce the crystallographic interactions of the 5-LOX complex.

Antiplasmodial activity of coumarins isolated from Polygala boliviensis: in vitro and in silico studies.

Polygala boliviensis is found in the Brazilian semiarid region. This specie is little chemically and biologically studied. Polygala spp. have different metabolites, especially coumarins. Studies indicate that coumarins have antimalarial potential, denoting the importance of researching new active compounds from plants, since the resistance of Plasmodium strains to conventional therapy has increased. The present study aimed to evaluate the antiplasmodial activity of auraptene and poligalen against a chloroquine-resistant strain of Plasmodium falciparum. Coumarins were isolated from P. boliviensis by open column chromatography and identified by Nuclear Magnetic Resonance Spectroscopy. A cytotoxicity assay was carried out using MTT test, and the in vitro antiplasmodial activity was evaluated using the W2 strain. The antiplasmodial activity results found were IC50=0.171 ± 0.016 for auraptene and 0.164 ± 0.012 for poligalen; the selectivity indexes were 78.71 and 609.76, respectively. Inverse virtual screening in the BRAMMT database by OCTOPUS 1.2 was applied to coumarins to find potential P. falciparum targets and showed higher affinity energy of auraptene for purine nucleoside phosphorylase (PfPNP) and of poligalen for dihydroorotate dehydrogenase (PfDHODH). Molecular Dynamics studies (MD and MM-GBSA) approach were applied to calculate binding energies against selected P. falciparum targets and showed that all coumarins were stable at the binding site during simulations. Furthermore, energies were favorable for complexation. This is the first report of auraptene in P. boliviensis species and of in vitro antiplasmodial activity of auraptene and poligalen. In silico studies indicated that the mechanism of action of coumarins is the inhibition of PfPNP and PfDHODH.Communicated by Ramaswamy H. Sarma.

Assessment of in vitro anthelmintic activity and bio-guided chemical analysis of BRS Boyrá pineapple leaf extracts.

Species of the Bromeliaceae are known for their pharmacological actions, including anthelmintic effects. The aim of this study was to investigate the in vitro anthelmintic activity of extracts and fractions of BRS Boyrá pineapple leaf against the eggs and infective larvae of gastrointestinal nematodes (Trichostrongylidae) of goats and to identify the compounds involved in this activity. Crude methanol, hexane, dichloromethane, ethyl acetate and residual hydromethanol extracts were investigated by quantitative analysis of phenolic and flavonoid contents, antioxidant activity, anthelmintic activity against gastrointestinal nematodes of goats. The extracts were submitted to chromatographic methods for substance isolation and spectrometric techniques to identify their structures. The anthelmintic activity was performed by in vitro assays with eggs and larvae of nematodes obtained from naturally infected donor goats. All extracts contained phenolic (2.22-14.12 g of gallic acid equivalent per 100 g of dry extract) and flavonoid compounds (0.13-1.45 g of quercetin equivalent per 100 g of dry extract). Bio-guided fractionation of the BRS Boyrá pineapple leaves showed high antioxidant activity (EC50 for DPPH of 2.16-21.38 mg mL-1 and inhibition of co-oxidation of ß-carotene of 36.40-74.86%) and anthelmintic activity (15.69-100% inhibition of egg hatching). The ethyl acetate extract exhibited greatest activity in all assays. Through chromatographic column analysis it was possible to isolate three substances: ß-sitosterol and stigmasterol mixture in dichloromethane and hexane extracts, identified by NMR and p-coumaric acid in the ethyl acetate extract, identified by HPLC-DAD. The isolated p-coumaric acid exhibited high ovicidal effect against goat gastrointestinal nematodes (IC50: 0.12 mg mL-1) and can be considered the active substance of the ethyl acetate extract. This study revealed for the first time that the pineapple BRS Boyrá possesses inhibitory activity against gastrointestinal nematodes (Haemonchus spp., Oesophagostomum spp. and Trichostrongylus spp.), and that p-coumaric acid is an important bioactive.

Evaluation of anti-inflammatory, antinociceptive and biological activities of Cenostigma macrophyllum standardized extracts and determination and quantification of the main metabolites.

The stem barks and leaves of Cenostigma macrophyllum are used in Brazilian folk medicines in the treatment of stomach and intestinal diseases. However, there are no reports of chromatographic methods used to evaluate the bioactives of its standardized extracts and for biological evaluation. An analytical method was developed and validated for simultaneous determination and quantification of the bioactive phenolics gallic acid, methyl gallate, ellagic acid and, the biflavonoids agathisflavone and amentoflavone in the leaves and stem bark of C. macrophyllum. HPLC operating conditions were optimized and the parameters such as selectivity, linearity, precision, accuracy, LOD, LOQ and, robustness of the method were also evaluated. Robustness was evaluated using a multivariate optimization technique. Linear relationships within the range of investigated concentrations were observed with their correlation coefficients greater than 0.9991. The method was validated for repeatability (RSD ≤ 2.88%), intermediate precision (RSD ≤ 3.38%) with recovery between 84.12 and 106.64% and the RSD less than 3.40% and proved to be robust. Besides, antioxidant, acetylcholinesterase inhibition, anti-inflammatory and antinociceptive activities of the standardized hydromethanolic extracts of leaves and stem bark of this species were evaluated. The method was successfully applied in the quantification of the gallic acid, methyl gallate, ellagic acid, agathisflavone and amentoflavone of standardized extracts. The results showed the present method developed was simple, sensitive, reproducible, accurate and precise. The standardized hydromethanolic extracts of leaves and stem bark of C. macrophyllum showed antioxidant activity (EC50 69.09 and 83.06 µg mL-1), acetylcholinesterase inhibition (52.23 and 83.36%) and they were able to inhibit the formalin-induced nociception and also reduced the edema formations at 100 mg kg-1 doses. The anti-inflammatory potentials were evaluated by the decrease of the Cg-induced neutrophils migrations at the same doses.

Asemeia ovata (Polygalaceae): Quantitative determination and evaluation in silico of identified substances by HPLC-DAD.

BACKGROUND: In Brazil, the Asemeia genus has 19 species (12 endemic) and 2 varieties (both endemic) and some of them are found in semi-arid Bahia. OBJECTIVE: The objective of this study was to quantitatively determine identified substances by HPLC-DAD in Asemeia ovata extracts and to predict their biological activities in silico. METHOD: The quantification method by HPLC-DAD has been validated according to the guidelines of the International Conference of Harmonization. The prediction in silico activities was made by Target Fishing methods (TF), followed by docking by the program DOCK 6.7 and assessment of interaction profiles for Protein-Ligand Interaction Profiler server. RESULTS: It was possible to identify and quantify using HPLC-DAD substances: rutin, luteolin-7-O-glucoside, caffeic acid, p-coumaric acid and trans-ferulic acid. The ChemProt 2.0 server was selected for TF method, which has shown potential activity of compounds on molecular targets such as Carbonic anhydrase 12, epidermal growth factor receptor and sodium-glucose cotransporter 2. CONCLUSION: This work provides new results for the species both from a biological and chemical point of view, and has interesting potential to be discovered with the prospect of further studies.