benzofurans and eupomatenoid-5

We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages.. The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed.. EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form.. Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

Topics: Bacterial Proteins; Benzofurans; Citrate (si)-Synthase; Electrophoresis, Gel, Two-Dimensional; Genes, Bacterial; Humans; Ketol-Acid Reductoisomerase; Microbial Sensitivity Tests; Microscopy, Electron, Scanning; Mycobacterium tuberculosis; Phenols; Phosphoglycerate Kinase; Protein Interaction Domains and Motifs; Proteome; Proteomics; Tandem Mass Spectrometry; Time Factors; Tuberculosis

Natural products remain an important source of new drugs, including anticancer drugs. Recently, our group reported the anticancer activity of eupomatenoid-5 (eup-5), a neolignan isolated from Piper regnellii (Miq.) C. DC. var. regnellii leaves. In vitro studies demonstrated that MCF-7 (breast) and 786-0 (kidney) were among the cancer cell lines most sensitive to eup-5 treatment. The current results demonstrate that mitochondrial membrane depolarization and generation of reactive oxygen species are implicated in eup-5-mediated cytotoxic effects on these cancer cells lines. In MCF-7 cells, eup-5 led to phosphatidylserine externalization and caspase activation, whereas the same did not occur in 786-0 cells. Scanning electron microscopy revealed a reduction of microvilli density, as well as cell morphology alterations. Moreover, treated MCF-7 cells exhibited well-characterized apoptosis alterations, while treated 786-0 cells exhibited characteristics of programmed necroptosis process. These findings support the possibility that different mechanisms may be targeted by eup-5 in cell death response.

Topics: Antineoplastic Agents, Phytogenic; Benzofurans; Caspases; Cell Death; Cell Line, Tumor; Humans; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Phenols; Phosphatidylserines; Piper; Plant Leaves; Superoxides

Department of Clinical Analysis and Biomedicine, State University of Maringa, Maringa, Parana, Brazil.. To evaluate the in vitro interaction between eupomatenoid-5 (EUP-5), extracted from Piper solmsianum C. DC. var. solmsianum, and first-line anti-tuberculosis drugs against Mycobacterium tuberculosis H₃₇Rv and 20 clinical isolates.. Resazurin drugs combination microtiter assay (REDCA) was performed to determine the interaction between EUP-5 and isoniazid, rifampicin (RMP) and ethambutol (EMB).. Synergism was observed in M. tuberculosis H₃₇Rv and eight clinical isolates with EUP-5+RMP, and in M. tuberculosis H₃₇Rv and 17 clinical isolates with EUP-5+EMB combinations.. EUP-5 is a promising compound for further studies on the development of anti-tuberculosis drugs.

Topics: Antitubercular Agents; Benzofurans; Drug Resistance, Multiple, Bacterial; Drug Synergism; Ethambutol; Genotype; Isoniazid; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Phenols; Phytotherapy; Piper; Plant Extracts; Plant Leaves; Plants, Medicinal; Rifampin

The present study determined the anti-Mycobacterium tuberculosis activities of supercritical CO2 extracts, neolignans eupomatenoid-5 (1), conocarpan (4) and eupomatenoid-3 (7) and their derivatives (2, 3, 5, 6, and 8) from Piper regnellii, as well as their cytotoxicities. The supercritical CO2 extract from leaves was purified by chromatographic methods, yielding compounds (1), (4) and (7), which were identified by (1)H NMR and comparison with literature data. Anti-M. tuberculosis activity (H37Rv and clinical isolates) was evaluated using a resazurin microtiter assay plate (REMA) to determine the MIC. The cytotoxicity assay was carried out in macrophages J774G.8 by sulforhodamine B colorimetric assay. The supercritical CO2 extracts from leaves and stems, and compound (4) showed activity against M. tuberculosis (MIC 15.6 μg/ml). Compound (1) showed the best activity (MIC 1.9 μg/ml), with good SI. Compounds (7) and (8) showed low activity against M. tuberculosis H37Rv. The derivative compounds did not show increased anti-M. tuberculosis activity. This is the first report, to our knowledge, to describe neolignans from P. regnellii with activity against M. tuberculosis, and compound (1) is a potential candidate for future antituberculosis drugs.

Topics: Animals; Antitubercular Agents; Benzofurans; Cell Line; Lignans; Macrophages; Magnetic Resonance Spectroscopy; Mice; Microbial Sensitivity Tests; Molecular Structure; Mycobacterium tuberculosis; Phenols; Piper; Plant Extracts; Toxicity Tests

The antifungal activity of extracts obtained from Piper regnellii with supercritical carbon dioxide was tested against yeast and filamentous fungi. The most active extract was obtained from leaves extracts of P. regnellii at 40°C and 25 MPa, featuring a minimal inhibitory concentration of 3.9 μg/mL against Trichophyton mentagrophytes. Neolignans eupomatenoid-3, eupomatenoid-5, eupomatenoid-6 and conocarpan were present in all extracts. The results indicate the possibility of further studies on the use of extracts of P. regnellii obtained by supercritical extraction, as potential sources of bioactive compounds for use in medicine.

Topics: Antifungal Agents; Benzofurans; Chromatography, Supercritical Fluid; Microbial Sensitivity Tests; Phenols; Piper; Plant Extracts; Plant Leaves; Trichophyton

Our group assays natural products that are less toxic and more effective than available nitroheterocycles as promising therapeutic options for patients with Chagas disease. Our previous study reported the trypanocidal activity of eupomatenoid-5, a neolignan isolated from the leaves of Piper regnellii var. pallescens, against the three main parasitic forms of Trypanosoma cruzi. The present study further characterizes the biochemical and morphological alterations induced by this compound to elucidate the mechanisms of action involved in the cell death of T. cruzi. We show that eupomatenoid-5 induced oxidative imbalance in the three parasitic forms, especially trypomastigotes, reflected by a decrease in the activity of trypanothione reductase and increase in the formation of reactive oxygen species (ROS). A reduction of mitochondrial membrane potential was then triggered, further impairing the cell redox system through the production of more ROS and reactive nitrogen species. Altogether, these effects led to oxidative stress, reflected by lipid peroxidation and DNA fragmentation. These alterations are key events in the induction of parasite death through various pathways, including apoptosis, necrosis, and autophagy.

Topics: Benzofurans; Cell Death; Chagas Disease; Free Radicals; Humans; Membrane Potential, Mitochondrial; Mitochondria; NADH, NADPH Oxidoreductases; Oxidative Stress; Phenols; Piper; Plant Extracts; Plant Leaves; Reactive Oxygen Species; Trypanosoma cruzi

Despite numerous studies with the Piper genus, there are no previous results reporting in vitro or in vivo Piper regnellii (Miq.) C. DC. var. regnellii anticancer activity. The aim of this study was to investigate P. regnellii in vitro and in vivo anticancer activity and further identify its active compounds. In vitro antiproliferative activity was evaluated in 8 human cancer cell lines: melanoma (UACC-62), breast (MCF7), kidney (786-0), lung (NCI-H460), prostate (PC-3), ovary (OVCAR-3), colon (HT29), and leukemia (K-562). Total growth inhibition (TGI) values were chosen to measure antiproliferative activity. Among the cell lines evaluated, eupomatenoid-5 demonstrated better in vitro antiproliferative activity towards prostate, ovary, kidney, and breast cancer cell lines. In vivo studies were carried out with Ehrlich solid tumor on Balb/C mice treated with 100, 300, and 1000 mg/kg of P. regnellii leaves dichloromethane crude extract (DCE), with 30 and 100 mg/kg of the active fraction (FRB), and with 30 mg/kg of eupomatenoid-5. The i. p. administration of DCE, FRB, and eupomatenoid-5 significantly inhibited tumor progression in comparison to control mice (saline). Therefore, this study showed that neolignans of Piper regnellii have promising anticancer activity. Further studies will be undertaken to determine the mechanism of action and toxicity of these compounds.

Topics: Animals; Antineoplastic Agents; Benzofurans; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Female; Humans; Lignans; Male; Mice; Mice, Inbred BALB C; Phenols; Piper; Plant Extracts; Plant Leaves

Because of its severe side effects and variable efficacy, the current treatment for Chagas disease is unsatisfactory. Natural compounds are good alternative chemotherapeutic agents for the treatment of this infection. Recently, our group reported the antiproliferative activity and morphological alterations in epimastigotes and intracellular amastigotes of Trypanosoma cruzi treated with eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. Here, we demonstrate that eupomatenoid-5 exhibited activity against trypomastigotes, the infective form of T. cruzi (EC₅₀ 40.5 μM), leading to ultrastructural alteration and lipoperoxidation in the cell membrane. Additionally, eupomatenoid-5 induced depolarization of the mitochondrial membrane, lipoperoxidation and increased G6PD activity in epimastigotes of T. cruzi. These findings support the possibility that different mechanisms may be targeted, according to the form of the parasite, and that the plasma membrane and mitochondria are the structures that are most affected in trypomastigotes and epimastigotes, respectively. Thus, the trypanocidal action of eupomatenoid-5 may be associated with mitochondrial dysfunction and oxidative damage, which can trigger destructive effects on biological molecules of T. cruzi, leading to parasite death.

Topics: Benzofurans; Chagas Disease; Glucose-6-Phosphate; Humans; Hydrogen Peroxide; Lignans; Lipid Peroxidation; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Oxidative Stress; Phenols; Phosphogluconate Dehydrogenase; Piper; Plant Extracts; Plant Leaves; Trypanocidal Agents; Trypanosoma cruzi

Infection with Leishmania spp. causes a disease with multifaceted clinical manifestations in humans. The treatment for leishmaniasis is dependent on a limited range of drugs. Here we investigated the antileishmanial activity of eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. We showed that eupomatenoid-5 had a dose-dependent activity during 72h of treatment, exhibiting IC(50) of 9.0microg/mL and 13.0microg/mL for promastigote and axenic amastigote forms, respectively, and IC(50) of 5.0microg/mL for intracellular amastigote forms of Leishmania amazonensis. When L. amazonensis was treated with eupomatenoid-5, it underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Among the alterations was the appearance of intense exocytic activity in the region of the flagellar pocket, myelin-like figures, and vacuoles in the cytoplasm of parasites treated with 9.0microg/mL. Cells treated with 25.0microg/mL showed a very large structure, apparently an extension of the endoplasmic reticulum. Also, mitochondrial swelling was detected at this concentration, indicating damage and significant change in this organelle. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa and it is not toxic to macrophages. Our studies indicated that eupomatenoid-5 might be a potential new drug for the treatment of leishmaniasis, because this compound displays interesting antileishmanial activity in vitro against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis.

Topics: Animals; Antiprotozoal Agents; Benzofurans; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Leishmania; Leishmaniasis; Macrophages; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Microscopy, Electron, Transmission; Parasitic Sensitivity Tests; Phenols; Piper; Plant Leaves

Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck (Piperaceae) is a medicinal plant traditionally used in Brazil to treat infectious diseases. The extracts obtained of the leaves from P. regnellii were investigated for their antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). The ethyl acetate extract presented a good activity against MRSA, with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 16 microg/mL. Based on this finding, the ethyl acetate extract was fractionated by silica gel column chromatography into nine fractions. The hexane fraction was active against MRSA (MIC at 4 microg/mL). Further column chromatography separation of the hexane fraction afforded the pure compound eupomatenoid-5. The structure of the compound was established by spectral data (1H and 13C NMR HSQC, HMBC, gNOE, IR and MS). Eupomatenoid-5 was the only compound active on the bacterium. The antibacterial property of P. regnellii extract provides preliminary scientific validation for the traditional medicinal use of this plant. The active compound eupomatenoid-5 should be further studied in animal models to verify in vivo efficacy and toxicity.

Topics: Acetates; Anti-Bacterial Agents; Benzofurans; Chromatography, Gel; Lignans; Methicillin-Resistant Staphylococcus aureus; Microscopy, Electron, Scanning; Phenols; Piper; Plant Extracts; Plant Leaves; Silica Gel; Silicon Dioxide

Eupomatenoid-5, a compound isolated from leaves of Piper regnellii var. pallescens, showed antiprotozoal activity against the epimastigote proliferative stages and intracellular amastigote forms of Trypanosoma cruzi Y strain. Eupomatenoid-5, at 7.0 microg/ml (50% growth inhibition concentration) produced morphological changes in epimastigote forms of the parasite, such as intense cytoplasmic vacuolization, mitochondrial swelling, kinetoplast alteration, presence of myelin-like figures, and mitochondrial damage, as observed by transmission electron microscopy. In amastigote forms in LLCMK(2) cells, at 7.0-microg/ml concentration, the compound induced a decrease in the number of cells with internalized parasites and in the number of internalized parasites per LLCMK(2) cell, compared with untreated cells. Furthermore, intense cytoplasmic vacuolization and autophagic vacuoles were observed in T. cruzi intracellular amastigotes after 72 h of incubation. Scanning electron microscopy confirmed the alterations in the shape of the parasites.

Topics: Animals; Benzofurans; Microscopy, Electron, Scanning; Phenols; Piper; Plant Leaves; Trypanocidal Agents; Trypanosoma cruzi

Activity-guided fractionation of the dichloromethane extract from leaves of Piper fulvescens, using an agar overlay bioautographic method, led to the isolation of three antifungal neolignans identified as conocarpan, eupomatenoid 5 and eupomatenoid 6. The minimal inhibitory concentration of these three neolignans against five fungi strains were determined. Conocarpan showed the widest activity, whereas eupomatenoid 6 was the most active against dermatophytes.

Topics: Antifungal Agents; Benzofurans; Candida albicans; Cryptococcus neoformans; Furans; Medicine, Traditional; Microbial Sensitivity Tests; Microsporum; Paraguay; Phenols; Piperaceae; Plant Extracts; Plant Leaves; Saccharomyces cerevisiae; Trichophyton