Primary targets of the sesquiterpene lactone deoxymikanolide on Trypanosoma cruzi.

BACKGROUND: Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model. PURPOSE: Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets. METHODS: The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine. RESULTS: Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy. CONCLUSION: Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.

Assessment of sesquiterpene lactones isolated from Mikania plants species for their potential efficacy against Trypanosoma cruzi and Leishmania sp.

Four sesquiterpene lactones, mikanolide, deoxymikanolide, dihydromikanolide and scandenolide, were isolated by a bioassay-guided fractionation of Mikania variifolia and Mikania micrantha dichloromethane extracts. Mikanolide and deoxymikanolide were the major compounds in both extracts (2.2% and 0.4% for Mikania variifolia and 21.0% and 6.4% for Mikania micrantha respectively, calculated on extract dry weight). Mikanolide, deoxymikanolide and dihydromikanolide were active against Trypanosoma cruzi epimastigotes (50% inhibitory concentrations of 0.7, 0.08 and 2.5 µg/mL, for each compound respectively). These sesquiterpene lactones were also active against the bloodstream trypomastigotes (50% inhibitory concentrations for each compound were 2.1, 1.5 and 0.3 µg/mL, respectively) and against amastigotes (50% inhibitory concentrations for each compound were 4.5, 6.3 and 8.5 µg/mL, respectively). By contrast, scandenolide was not active on Trypanosoma cruzi. Besides, mikanolide and deoxymikanolide were also active on Leishmania braziliensis promastigotes (50% inhibitory concentrations of 5.1 and 11.5 µg/mL, respectively). The four sesquiterpene lactones were tested for their cytotoxicity on THP 1 cells. Deoxymikanolide presented the highest selectivity index for trypomastigotes (SI = 54) and amastigotes (SI = 12.5). In an in vivo model of Trypanosoma cruzi infection, deoxymikanolide was able to decrease the parasitemia and the weight loss associated to the acute phase of the parasite infection. More importantly, while 100% of control mice died by day 22 after receiving a lethal T. cruzi infection, 70% of deoxymikanolide-treated mice survived. We also observed that this compound increased TNF-α and IL-12 production by macrophages, which could contribute to control T. cruzi infection.

Mode of Action of the Sesquiterpene Lactones Psilostachyin and Psilostachyin C on Trypanosoma cruzi.

Trypanosoma cruzi is the causative agent of Chagas' disease, which is a major endemic disease in Latin America and is recognized by the WHO as one of the 17 neglected tropical diseases in the world. Psilostachyin and psilostachyin C, two sesquiterpene lactones isolated from Ambrosia spp., have been demonstrated to have trypanocidal activity. Considering both the potential therapeutic targets present in the parasite, and the several mechanisms of action proposed for sesquiterpene lactones, the aim of this work was to characterize the mode of action of psilostachyin and psilostachyin C on Trypanosoma cruzi and to identify the possible targets for these molecules. Psilostachyin and psilostachyin C were isolated from Ambrosia tenuifolia and Ambrosia scabra, respectively. Interaction of sesquiterpene lactones with hemin, the induction of oxidative stress, the inhibition of cruzipain and trypanothione reductase and their ability to inhibit sterol biosynthesis were evaluated. The induction of cell death by apoptosis was also evaluated by analyzing phosphatidylserine exposure detected using annexin-V/propidium iodide, decreased mitochondrial membrane potential, assessed with Rhodamine 123 and nuclear DNA fragmentation evaluated by the TUNEL assay. Both STLs were capable of interacting with hemin. Psilostachyin increased about 5 times the generation of reactive oxygen species in Trypanosoma cruzi after a 4h treatment, unlike psilostachyin C which induced an increase in reactive oxygen species levels of only 1.5 times. Only psilostachyin C was able to inhibit the biosynthesis of ergosterol, causing an accumulation of squalene. Both sesquiterpene lactones induced parasite death by apoptosis. Upon evaluating the combination of both compounds, and additive trypanocidal effect was observed. Despite their structural similarity, both sesquiterpene lactones exerted their anti-T. cruzi activity through interaction with different targets. Psilostachyin accomplished its antiparasitic effect by interacting with hemin, while psilostachyin C interfered with sterol synthesis.

The sesquiterpene lactone polymatin B from Smallanthus sonchifolius induces different cell death mechanisms in three cancer cell lines.

A 8ß-angeloyloxy-9α-hydroxy-14-oxo-acanthospermolide and five known melampolide sesquiterpene lactones (uvedalin, enhydrin, polymatin B, sonchifolin, and fluctuanin) were isolated from the leaves of Smallanthus sonchifolius. The compounds were identified by 1D-, 2D-NMR, HRMS, IR and UV analyses. In vitro cytotoxicity assays (MTT) showed that these sesquiterpene lactones display poor cytotoxic effects on peripheral blood mononuclear cells (PBMC) of healthy human subjects, whereas a strong cytotoxicity was observed in leukemia and pancreas cancer cells. For the mechanism of action of polymatin B, oxidative stress seems to be involved. Interestingly, reactive oxygen species (ROS) formation mainly induced different effects: apoptosis in CCRF-CEM cells, necroptosis in CEM-ADR5000 cells through induction of RIP1K, neither apoptosis nor necroptosis in MIA-PaCa-2 cells. Additionally, cells also died partly by necrosis.

Natural terpenoids from Ambrosia species are active in vitro and in vivo against human pathogenic trypanosomatids.

Among the natural compounds, terpenoids play an important role in the drug discovery process for tropical diseases. The aim of the present work was to isolate antiprotozoal compounds from Ambrosia elatior and A. scabra. The sesquiterpene lactone (STL) cumanin was isolated from A. elatior whereas two other STLs, psilostachyin and cordilin, and one sterol glycoside, daucosterol, were isolated from A. scabra. Cumanin and cordilin were active against Trypanosoma cruzi epimastigotes showing 50% inhibition concentrations (IC50) values of 12 µM and 26 µM, respectively. Moreover, these compounds are active against bloodstream trypomastigotes, regardless of the T. cruzi strain tested. Psilostachyin and cumanin were also active against amastigote forms with IC50 values of 21 µM and 8 µM, respectively. By contrast, daucosterol showed moderate activity on epimastigotes and trypomastigotes and was inactive against amastigote forms. We also found that cumanin and psilostachyin exhibited an additive effect in their trypanocidal activity when these two drugs were tested together. Cumanin has leishmanicidal activity with growth inhibition values greater than 80% at a concentration of 5 µg/ml (19 µM), against both L. braziliensis and L. amazonensis promastigotes. In an in vivo model of T. cruzi infection, cumanin was more active than benznidazole, producing an 8-fold reduction in parasitemia levels during the acute phase of the infection compared with the control group, and more importantly, a reduction in mortality with 66% of the animals surviving, in comparison with 100% mortality in the control group. Cumanin also showed nontoxic effects at the doses assayed in vivo, as determined using markers of hepatic damage.

Antipoliovirus Activity of the Organic Extract of Eupatorium buniifolium: Isolation of Euparin as an Active Compound.

The antiviral activity of the organic extract (OE) of Eupatorium buniifolium against poliovirus type 1 was determined by in vitro assays with an effective concentration 50 (EC50) of 23.3 ± 3.3 µg/mL. Bioassay-guided fractionation of the OE allowed the isolation of an active principle that was identified by spectroscopic methods ((1)H- and (13)C-NMR, EI-MS, UV, and IR spectroscopy) as the benzofuran euparin. The plaque reduction assay in Vero cells was used to assess the antiviral activity of euparin against poliovirus types 1, 2, and 3 with EC50 values of 0.47, 0.12, and 0.15 µg/mL, respectively. Moreover, this compound showed high selectivity indexes of 284.9, 1068, and 854.7, respectively. In order to identify the mechanism by which euparin exerts its antiviral activity, the virucidal effect, the pretreatment of Vero cells, and the time of action on one viral replication cycle were evaluated. Results obtained demonstrated that euparin exerts its effect during the early events of the replication cycle, from the virus adsorption to cells up to the first twenty minutes after infection. This is the first report on the presence of euparin in E. buniifolium and its antiviral activity.

In vitro antiviral activity of plant extracts from Asteraceae medicinal plants.

BACKGROUND: Due to the high prevalence of viral infections having no specific treatment and the constant appearance of resistant viral strains, the development of novel antiviral agents is essential. The aim of this study was to evaluate the antiviral activity against bovine viral diarrhea virus, herpes simplex virus type 1 (HSV-1), poliovirus type 2 (PV-2) and vesicular stomatitis virus of organic (OE) and aqueous extracts (AE) from: Baccharis gaudichaudiana, B. spicata, Bidens subalternans, Pluchea sagittalis, Tagetes minuta and Tessaria absinthioides. A characterization of the antiviral activity of B. gaudichaudiana OE and AE and the bioassay-guided fractionation of the former and isolation of one active compound is also reported. METHODS: The antiviral activity of the OE and AE of the selected plants was evaluated by reduction of the viral cytopathic effect. Active extracts were then assessed by plaque reduction assays. The antiviral activity of the most active extracts was characterized by evaluating their effect on the pretreatment, the virucidal activity and the effect on the adsorption or post-adsorption period of the viral cycle. The bioassay-guided fractionation of B. gaudichaudiana OE was carried out by column chromatography followed by semipreparative high performance liquid chromatography fractionation of the most active fraction and isolation of an active compound. The antiviral activity of this compound was also evaluated by plaque assay. RESULTS: B. gaudichaudiana and B. spicata OE were active against PV-2 and VSV. T. absinthioides OE was only active against PV-2. The corresponding three AE were active against HSV-1. B. gaudichaudiana extracts (OE and AE) were the most selective ones with selectivity index (SI) values of 10.9 (PV-2) and > 117 (HSV-1). For this reason, both extracts of B. gaudichaudiana were selected to characterize their antiviral effects. Further bioassay-guided fractionation of B. gaudichaudiana OE led to an active fraction, FC (EC50 = 3.1 µg/ml; SI = 37.9), which showed antiviral activity during the first 4 h of the viral replication cycle of PV-2 and from which the flavonoid apigenin (EC50 = 12.2 ± 3.3 µM) was isolated as a major compound. CONCLUSIONS: The results showed that, among the species studied, B. gaudichaudiana seemed to be the most promising species as a source of antiviral agents.

Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana.

Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on the host's cells. In this work, we have investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promastigotes. We first observed that the antiproliferative effect of mexicanin I (Mxc), dehydroleucodine (DhL), psilostachyin (Psi), and, at lesser extent, psilostachyin C (Psi C) is blocked by 1.5 mM reduced glutathione. The reducing agent was also able to reverse the early effect of the compounds, suggesting that lactones may react with intracellular sulfhydryl groups. Moreover, we have shown that all the sesquiterpene lactones, except Psi C, significantly decreased the endogenous concentration of glutathione within the parasite. Consistent with these findings, the active sesquiterpene lactones increased between 2.7 and 5.4 times the generation of ROS by parasites. These results indicate that the induction of oxidative stress is at least one of the mechanisms of action of DhL, Mxc, and Psi on parasites while Psi C would act by another mechanism.

In vitro evaluation of antiprotozoal and antiviral activities of extracts from Argentinean Mikania species.

The aim of this study was to investigate the antiprotozoal and antiviral activities of four Argentinean Mikania species. The organic and aqueous extracts of Mikania micrantha, M. parodii, M. periplocifolia, and M. cordifolia were tested on Trypanosoma cruzi epimastigotes, Leishmania braziliensis promastigotes, and dengue virus type 2. The organic extract of M. micrantha was the most active against T. cruzi and L. braziliensis exhibiting a growth inhibition of 77.6 ± 4.5% and 84.9 ± 6.1%, respectively, at a concentration of 10 µg/ml. The bioguided fractionation of M. micrantha organic extract led to the identification of two active fractions. The chromatographic profile and infrared analysis of these fractions revealed the presence of sesquiterpene lactones. None of the tested extracts were active against dengue virus type 2.

Problemática sanitaria y social de la enfermedad de Chagas. Aporte de la medicina tradicional argentina / Health and social problems of Chagas disease. Contribution of traditional Argentine medicine

A más de cien años de su descubrimiento la enfermedad de Chagas sigue siendo una realidad cuyo panorama inicial no solo no se ha modificado sino que se ha visto complicado por el agregado de cambios ecológicos y de transformación social. La enfermedad de Chagas no solo involucra los conocidos trastornos biológicos y sanitarios sino que está íntimamente relacionada con las condiciones de desarrollo económico y social de vastas zonas endémicas de toda Latinoamérica. Por ende, su erradicación constituye un problema en el que entran en juego factores sanitarios, sociales, económicos y políticos. En este trabajo se discuten algunos de estos aspectos y se concluye que es solo por medio de una acción multidisciplinaria e integrada sobre diferentes focos: vector, pacientes, comunidades, profesionales de la salud y comunidades educativa y científica, que es posible encontrar una solución, siempre en el marco de una política clara y continua por parte de un Estado responsable. La ciencia y la tecnología pueden hacer una gran contribución para la erradicación de esta enfermedad abandonada. No solo desde disciplinas como la Química, Farmacia, Biología Molecular y las áreas bioquímicas,sino desde otras disciplinas, como la Etnomedicina. La enfermedad de Chagas afecta también a muchas de las comunidades aborígenes que habitan las regiones endémicas y son consideradas comunidades de alto riesgo. Esas comunidades tienen una cultura propia y un sistema de salud shamánica, fuertemente ligado a creencias religiosas, con lengua, cultura y cosmovisión diferentes. Poseen un conocimiento ancestral sobre el uso medicinal de las plantas que crecen en su entorno. Este conocimiento, así como la gran biodiversidad de las regiones tropicales y subtropicales donde esta enfermedad ocurre, constituyen un recurso muy valioso como fuente de moléculas activas contra el Trypanosoma cruzi, el protozoo productor de la enfermedad.

Psilostachyin C: a natural compound with trypanocidal activity.

In this study, the antiprotozoal activity of the sesquiterpene lactone psilostachyin C was investigated. This natural compound was isolated from Ambrosia scabra by bioassay-guided fractionation and was identified by spectroscopic techniques. Psilostachyin C exerted in vitro trypanocidal activity against Trypanosoma cruzi epimastigotes, trypomastigotes and amastigotes, with 50% inhibitory concentration (IC(50)) values of 0.6, 3.5 and 0.9 µg/mL, respectively, and displayed less cytotoxicity against mammalian cells, with a 50% cytotoxic concentration (CC(50)) of 87.5 µg/mL. Interestingly, this compound induced ultrastructural alterations, as seen by transmission electron microscopy, in which vacuolisation and a structural appearance resembling multivesicular bodies were observed even at a concentration as low as 0.2 µg/mL. In an in vivo assay, a significant reduction in the number of circulating parasites was found in T. cruzi-infected mice treated with psilostachyin C for 5 days compared with untreated mice (7.4 ± 1.2 × 10(5)parasites/mL vs. 12.8 ± 2.0 × 10(5)parasites/mL) at the peak of parasitaemia. According to these results, psilostachyin C may be considered a promising template for the design of novel trypanocidal agents. In addition, psilostachyin C inhibited the growth of Leishmania mexicana and Leishmania amazonensis promastigotes (IC(50)=1.2 µg/mL and 1.5 µg/mL, respectively).

Trypanocidal and leishmanicidal activities of sesquiterpene lactones from Ambrosia tenuifolia Sprengel (Asteraceae).

Bioassay-guided fractionation of the organic extract of Ambrosia tenuifolia Sprengel (Asteraceae) led to the isolation of two bioactive sesquiterpene lactones with significant trypanocidal and leishmanicidal activities. By spectroscopic methods ((1)H- and (13)C-nuclear magnetic resonance, distortionless enhancement by polarization transfer, correlated spectroscopy, heteronuclear multiple-quantum coherence, electron impact-mass spectrometry, and infrared spectroscopy), these compounds were identified as psilostachyin and peruvin. Both compounds showed a marked in vitro trypanocidal activity against Trypanosoma cruzi epimastigotes with 50% inhibitory concentration (IC(50)) values of less than 2 microg/ml. Psilostachyin exerted a significant in vitro activity against the trypomastigote forms of T. cruzi (IC(50), 0.76 microg/ml) and was selected for in vivo testing. Psilostachyin-treated mice had a survival of 100% and lower parasitemia values than control mice. Both compounds were also tested on Leishmania sp. promastigotes: psilostachyin (IC(50), 0.12 microg/ml) and peruvin (IC(50), 0.39 microg/ml) exerted significant leishmanicidal activities. This is the first time that the trypanocidal and leishmanicidal activities of these compounds have been reported. The selectivity index (SI) was employed to evaluate the cytotoxic effect of lactones on T lymphocytes. Although the SIs of both compounds were high for T. cruzi epimastigotes, psilostachyin was more selective against trypomastigotes (SI, 33.8) while peruvin showed no specificity for this parasite. Both compounds presented high selectivity for Leishmania spp. The results shown herein suggest that psilostachyin and peruvin could be considered potential candidates for the development of new antiprotozoal agents against Chagas' disease and leishmaniasis.

Trypanocidal and leishmanicidal activities of flavonoids from Argentine medicinal plants.

In vitro trypanocidal and leishmanicidal activities of the flavonoids hispidulin, from Ambrosia tenuifolia, and santin, from Eupatorium buniifolium, are reported. A sensitive technique that takes advantage of ((3)H)thymidine uptake by dividing trypanosomatids has been adjusted for quantification of the parasiticidal effect of the natural products. The IC(50) values for hispidulin and santin on Trypanosoma cruzi epimastigotes were 46.7 and 47.4 muM, respectively. On trypomastigotes, the IC(50) values were 62.3 microM for hispidulin and 42.1 microM for santin. Hispidulin was more active than santin on promastigotes of Leishmania mexicana (IC(50) = 6.0 microM versus 32.5 microM). No cytotoxic activity was observed on lymphoid cells, making hispidulin and santin potential lead compounds for the development of new natural drugs. This is the first report on the trypanocidal and leishmanicidal activities of these flavonoids and on the presence of santin in E. buniifolium.

A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template-primer.

Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template-primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.