A paraguayan toad Rhinella schneideri preparation based on Mbya tradition increases mitochondrial bioenergetics with migrastatic effects dependent on AMPK in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In Paraguay, healers from the Mbya culture treat cancer with a recipe prepared with the native toad Rhinella schneideri. However, the chemical composition and biological effects of the recipe remain unknown. AIM OF THE STUDY: The aim is to determine the composition of the traditional preparation made using the toad R. schneideri and to evaluate its effect on human breast cancer (BC) cells. MATERIALS AND METHODS: The metabolites contained in the preparation were concentrated using XAD-7 resin, and the concentrate was analyzed by HPLC-MS/MS. The effect of the preparation was assessed in normal (MCF10F) and BC cells (MDA-MB-231 and MCF7). The mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) levels, and cell cycle progression were determined by flow cytometry. The oxygen consumption rate (OCR) was measured by Clark electrode, and fibronectin-dependent migration in normoxia and hypoxia-like conditions were evaluated by transwell assay. RESULTS: From the Amberlite-retained extract from the preparation, 24 compounds were identified, including alkaloids, amino acids, bufadienolides, and flavonoids, among others. The crude extract (CE) did not affect cell cycle progression and viability of BC cell lines. Moreover, it did not make cancer cells more sensitive to the cytotoxic effect of the chemotherapeutics doxorubicin and teniposide. On the other hand, the CE reduced the menadione-induced ROS production and increased NADH, Δψm, and the OCR. Respiratory complexes I and III as well as ATP synthase levels were increased in an AMPK-dependent manner. Moreover, the CE inhibited the migration of BC cells in normoxia and a hypoxia-like condition using CoCl2 as a HIF1α-stabilizing agent. This latter effect involved an AMPK-dependent reduction of HIF1α levels. CONCLUSIONS: The Paraguayan toad recipe contains metabolites from the toad ingredient, including alkaloids and bufadienolide derivatives. The CE lacks cytotoxic effects alone or in combination with chemotherapeutics. However, it increases mitochondrial bioenergetics and inhibits the cancer cell migration in an AMPK-dependent manner in BC cells. This is the first report of the in vitro anticancer effect of a traditional Rhinella sp. toad preparation based on Mbya tradition.

Helietta apiculata: a tropical weapon against Chagas disease.

The present study pretends to evaluate the in vivo efficacy of the crude chloroform bark extract of Helietta apiculata, then the activity will be compared with the reference drug, benznidazole, in acute Trypanosoma cruzi infected mice when administered by oral route. The chloroformic extract of Helieta apiculata was administered by oral route at 5, 10 and 50 mg/kg daily for two weeks. This study has shown a moderate efficacy of the H. apiculata bark extract in reducing T. cruzi parasitaemia in 42 to 54% after a monitoring of 60 days post-infection and when compared with control groups. Concerning mice mortality, only two only two mice died, one from the control group and the other one from the group threated with 10 mg of the chlorofom extract of H. apiculata, suggesting the potential of H. apiculta extracts as a safe and inexpensive treatment of Chagas disease.

Multi-Anti-Parasitic Activity of Arylidene Ketones and Thiazolidene Hydrazines against Trypanosoma cruzi and Leishmania spp.

A series of fifty arylideneketones and thiazolidenehydrazines was evaluated against Leishmania infantum and Leishmania braziliensis. Furthermore, new simplified thiazolidenehydrazine derivatives were evaluated against Trypanosoma cruzi. The cytotoxicity of the active compounds on non-infected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their anti-parasitic effects. Seven thiazolidenehydrazine derivatives and ten arylideneketones had good activity against the three parasites. The IC50 values for T. cruzi and Leishmania spp. ranged from 90 nM-25 µM. Eight compounds had multi-trypanocidal activity against T. cruzi and Leishmania spp. (the etiological agents of cutaneous and visceral forms). The selectivity of these active compounds was better than the three reference drugs: benznidazole, glucantime and miltefosine. They also had low toxicity when tested in vivo on zebrafish. Trying to understand the mechanism of action of these compounds, two possible molecular targets were investigated: triosephosphate isomerase and cruzipain. We also used a molecular stripping approach to elucidate the minimal structural requirements for their anti-T. cruzi activity.

The Paraguayan Rhinella toad venom: Implications in the traditional medicine and proliferation of breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Toads belonging to genus Rhinella are used in Paraguayan traditional medicine to treat cancer and skin infections. AIM OF THE STUDY: The objective of the study was to determine the composition of venoms obtained from three different Paraguayan Rhinella species, to establish the constituents of a preparation sold in the capital city of Paraguay to treat cancer as containing the toad as ingredient, to establish the effect of the most active Rhinella schneideri venom on the cell cycle using human breast cancer cells and to assess the antiprotozoal activity of the venoms. METHODS: The venom obtained from the toads parotid glands was analyzed by HPLC-MS-MS. The preparation sold in the capital city of Paraguay to treat cancer that is advertised as made using the toad was analyzed by HPLC-MS-MS. The effect of the R. schneideri venom and the preparation was investigated on human breast cancer cells. The antiprotozoal activity was evaluated on Leishmania braziliensis, L. infantum and murine macrophages. RESULTS: From the venoms of R. ornata, R. schneideri and R. scitula, some 40 compounds were identified by spectroscopic and spectrometric means. Several minor constituents are reported for the first time. The preparation sold as made from the toad did not contained bufadienolides or compounds that can be associated with the toad but plant compounds, mainly phenolics and flavonoids. The venom showed activity on human breast cancer cells and modified the cell cycle proliferation. The antiprotozoal effect was higher for the R. schneideri venom and can be related to the composition and relative ratio of constituents compared with R. ornata and R. scitula. CONCLUSIONS: The preparation sold in the capital city of Paraguay as containing the toad venom, used popularly to treat cancer did not contain the toad venom constituents. Consistent with this, this preparation was inactive on proliferation of human breast cancer cells. In contrast, the toad venoms of Rhinella species altered the cell cycle progression, affecting the proliferation of malignant cells. The findings suggest that care should be taken with the providers of the preparation and that the crude drug present a strong activity towards human breast cancer cell lines. The antiprotozoal effect of the R. schneideri venom was moderate while the venom of R. ornata was devoid of activity and that of R. scitula was active at very high concentration.

Bond-based bilinear indices for computational discovery of novel trypanosomicidal drug-like compounds through virtual screening.

Two-dimensional bond-based bilinear indices and linear discriminant analysis are used in this report to perform a quantitative structure-activity relationship study to identify new trypanosomicidal compounds. A data set of 440 organic chemicals, 143 with antitrypanosomal activity and 297 having other clinical uses, is used to develop the theoretical models. Two discriminant models, computed using bond-based bilinear indices, are developed and both show accuracies higher than 86% for training and test sets. The stochastic model correctly indentifies nine out of eleven compounds of a set of organic chemicals obtained from our synthetic collaborators. The in vitro antitrypanosomal activity of this set against epimastigote forms of Trypanosoma cruzi is assayed. Both models show a good agreement between theoretical predictions and experimental results. Three compounds showed IC50 values for epimastigote elimination (AE) lower than 50 µM, while for the benznidazole the IC50 = 54.7 µM which was used as reference compound. The value of IC50 for cytotoxicity of these compounds is at least 5 times greater than their value of IC50 for AE. Finally, we can say that, the present algorithm constitutes a step forward in the search for efficient ways of discovering new antitrypanosomal compounds.

Zanthoxylum chiloperone leaves extract: first sustainable Chagas disease treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum chiloperone var. angustifolium Engl. (Rutaceae) stem bark is used traditionally in Paraguay for its antiparasitic properties. Canthin-6-one is main compound isolated from Zanthoxylum chiloperone var angustifolium with broad spectrum antifungal, leishmanicidal and trypanocidal activities. AIM OF THE STUDY: The qualitative and quantitative characterization and the isolation of main alkaloidal components of different organs of Zanthoxylum chiloperone are investigated by HPLC-UV-MS. The in vitro biological activity of each extract against trypomastigote and amastigote forms of Trypanosoma cruzi parasites were evaluated, then comparison the in vivo efficacy of the ethanolic leaves extract of Zanthoxylum chiloperone with reference drug, benznidazole, in acute Trypanosoma cruzi infected mice when administered by oral route. We have also evaluated the mutagenic and cytotoxic activity of the main component of Zanthoxylum chiloperone, i.e. canthin-6-one, by mouse bone marrow micronucleus test. MATERIALS AND METHODS: The compositions of the ethanol extracts obtained after the maceration process were studied by HPLC-UV-MS methods. The quantitation analysis was performed by external standard method, using a calibration curve constructed utilizing solutions containing different concentrations of the reference samples. The anti-trypomastigote activity was evaluated by the lysis effect on mouse blood trypomastigotes (Y strain Trypanosoma cruzi). The anti-amastigote Trypanosoma cruzi activity was evaluated by a modified colorimetric method with chlorophenol red-ß-d-galactopyranoside (CPRG). The cytotoxicity of extracts and compounds was performed on NCTC 929 cells. The in vivo efficacy of the ethanolic leaves extract of Zanthoxylum chiloperone and benznidazole, in acute Trypanosoma cruzi (two different strains) was evaluated in Trypanosoma cruzi infected mice; the drugs were administered by oral route. The mortality rates were recorded and parasitaemias in control and treated mice were determined once weekly for 70 days. The mutagenic and cytotoxic activity of the main component of Zanthoxylum chiloperone, canthin-6-one, by mouse bone marrow micronucleus test. RESULTS: Canthin-6-one was the main compound of stem and root bark and 5-methoxy-canthin-6-one in leaves and fruits. The ethanolic leaves extract, canthin-6-one and benznidazole presented, approximately, the same level of in vitro activity against trypomastigote and amastigote forms of Trypanosoma cruzi. We have also evaluated the mutagenic and cytotoxic effects of canthin-6-one by micronucleus test in mice. This test showed any mutagenic and cytotoxic damages. The effects of oral or subcutaneous treatments at 10 mg/kg daily for 2 weeks with the ethanolic extract of leaves of Zanthoxylum chiloperone were examined in Balb/c mice infected acutely with Trypanosoma cruzi (CL or Y strain) and compared with benznidazole at 50 mg/kg for 2 weeks. In these experiments, 70 days after infection, parasitaemia and serological response were significantly reduced with the oral ethanolic extract treatment compared with reference drug. CONCLUSIONS: This study have shown the efficacy of the leaves extract of Zanthoxylum chiloperone in reducing Trypanosoma cruzi parasitaemia in vivo assays and could be welcomed by scientific and rural communities of Paraguay because it could help them towards the use of local resources to treat an endemic infection, Chagas disease, affecting 20% of the population of this country.

In vitro and in vivo antitrypanosomatid activity of 5-nitroindazoles.

Previously, we have identified a series of 5-nitroindazoles with good antiprotozoal activities, against Trypanosoma cruzi epimastigotes and Trichomonas vaginalis. Most of them have shown very low unspecific toxicity on macrophage cell lines. In the present work, we assayed these compounds on T. cruzi bloodstream trypomastigotes and Leishmania promastigotes (Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum). Derivatives 1, 2, 7 and 8 displayed remarkable trypanocidal activity (>80% lysis) equivalent to gentian violet. Derivatives 2 and 10, as Pentamidine, caused the complete lysis of promastigotes of Leishmania. An oxidative stress-mediated mechanism of action was confirmed for derivatives 1, 10 and 12 on T. cruzi epimastigotes. Supported by the in vitro activities, derivatives 1 and 2 were submitted to in vivo assays using an acute model of Chagas' disease and a short-term treatment. None of the animals treated with derivatives 1 and 2 died, unlike the untreated control and Benznidazole groups.

Phytochemical and antiprotozoal activity of Ocotea lancifolia.

Thirteen known isoquinoline alkaloids were isolated from Ocotea lancifolia, popularly known as << canela pilosa >> in Brasil and << laurel né >> by the Guarani people which means smell laurel. Their activities against the promastigote forms of three Leishmania strains and the bloodstream form of Trypanosoma cruzi were evaluated, as well as their hepatocytotoxicity. Among them, the noraporphine alkaloid (-) caaverine has shown the most interesting antiprotozoal activity against Leishmania and T. cruzi parasites.

Antifungal compounds from Zanthoxylum chiloperone var. angustifolium.

An alkaloidal extract of the stem barks of Zanthoxylum chiloperone var. angustifolium exhibited antifungal activity against Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes var. interdigitale using a TLC bioautographic method. Bioassay-guided fractionation of this extract resulted in the isolation of two active compounds identi fi ed as canthin-6-one and 5-methoxycanthin-6-one. Canthin-6-one exhibited a broad spectrum of activities against Aspergillus fumigatus, A. niger, A. terreus, Candida albicans, C. tropicalis, C. glabrata, Cryptococcus neoformans, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon beigelii, Trichosporon cutaneum and Trichophyton mentagrophytes var. interdigitale with MICs values between 5.3 and 46 micro mol/L. 5-methoxy-canthin-6-one was active against only Trichophyton mentagrophytes var. interdigitale with a MIC value of 12.3 micro mol/L.

Situaciòn de la transfusiòn de sangre inocua en el Paraguay durante el periodo 1990-1994 / Annual reports 1996 / Situation of the tranfusion of inocue blood in Paraguay during the period of 1990-1994

La transmisiòn de enfermedades infecciosas por vìa transfusional ha adquirido cada vez màs importancia debido a la gran migraciòn de contingentes poblacionales de paìses endèmicos a otros no endèmicos, agravada por la falta de control serològico en los bancos de sangre. Con el propòsito de aportar mayor informaciòn sobre la mayorìa de bancos de sangre en el Paraguay el presente trabajo revisa la informaciòn pre-existente sobre seroprevalencia de las enfermedades serocontroladas en bancos pùblicos y privados. Ademàs, realiza una descripciòn de datos actualizados sobre la administraciòn de sangre inocua en el Paraguay.

Situaciòn de la transfusiòn de sangre inocua en el Paraguay durante el periodo 1990-1994 / Annual reports 1996 / Situation of the tranfusion of inocue blood in Paraguay during the period of 1990-1994

La transmisiòn de enfermedades infecciosas por vìa transfusional ha adquirido cada vez màs importancia debido a la gran migraciòn de contingentes poblacionales de paìses endèmicos a otros no endèmicos, agravada por la falta de control serològico en los bancos de sangre. Con el propòsito de aportar mayor informaciòn sobre la mayorìa de bancos de sangre en el Paraguay el presente trabajo revisa la informaciòn pre-existente sobre seroprevalencia de las enfermedades serocontroladas en bancos pùblicos y privados. Ademàs, realiza una descripciòn de datos actualizados sobre la administraciòn de sangre inocua en el Paraguay

Inhibiciòn de la agregaciòn plaquetaria por extractos de plantas de uso medicinal en Paraguay / Annual reports 1995 / Inhibition of platelet aggregation by medicinal plants usein Paraguay

Con el propòsito de identificar extractos y luego aislar productos on actividad inhibidoria sobre la agregaciòn plaquetaria in vitro fueron probados 21 extractos alcohòlicos de plantas antitrombòlicos y en transtornos circulatorios e inmunològicos. La inhibiciòn de la agregaciòn se determinò mediante el mètodo turbo-densitomètrico (coagulòmetro AccuStasisTM 1000) midiendo el tiempo de agregaciòn del plasma rico en plaquetas inducido por adenosina 5 difosfato (ADP), colàgeno y epinefrina a las concentraciones iniciales de 2x10-4mo1/L, 2 mg/ml y 1x10-4mo1/L respectivamente y como control positivo de la inhibiciòn de la agregaciòn plaquetaria se usò aspirina a la concentraciòn de 0,1g/ml. La evaluaciòn cualitativa se realizò por apreciaciòn microscòpica (A 100x) de frotis teñidos con Giemsa del agregado plaquetario o nò por los extractos y los controles. Del screening resultò activo el extracto de una planta medicinal perteneciente a la familia Myrtaceae a la concentraciòn de 272ug/ml con los porcentajes de inhibiciòn de 96 por ciento a la concentraciòn de 0,18x10-4mo1/L de ADP y 94,4 por ciento a la concentraciòn de 0,045x10-4mo1/L de ADP. Tambièn resultò activo el extracto de una planta medicinal perteneciente a la familia Flacourtiaceae con un porcentaje de inhibiciòn de 92 por ciento al ser inducido por ADP a la concentraciòn de 0,045x10-4mo1/L. Este estudio indica el potencial de algunos extractos estudiados y justifica el aislamiento de compuestos puros de los extractos activos con mejores propiedades.

Inhibiciòn de la agregaciòn plaquetaria por extractos de plantas de uso medicinal en Paraguay / Annual reports 1995 / Inhibition of platelet aggregation by medicinal plants usein Paraguay

Con el propòsito de identificar extractos y luego aislar productos on actividad inhibidoria sobre la agregaciòn plaquetaria in vitro fueron probados 21 extractos alcohòlicos de plantas antitrombòlicos y en transtornos circulatorios e inmunològicos. La inhibiciòn de la agregaciòn se determinò mediante el mètodo turbo-densitomètrico (coagulòmetro AccuStasisTM 1000) midiendo el tiempo de agregaciòn del plasma rico en plaquetas inducido por adenosina 5 difosfato (ADP), colàgeno y epinefrina a las concentraciones iniciales de 2x10-4mo1/L, 2 mg/ml y 1x10-4mo1/L respectivamente y como control positivo de la inhibiciòn de la agregaciòn plaquetaria se usò aspirina a la concentraciòn de 0,1g/ml. La evaluaciòn cualitativa se realizò por apreciaciòn microscòpica (A 100x) de frotis teñidos con Giemsa del agregado plaquetario o nò por los extractos y los controles. Del screening resultò activo el extracto de una planta medicinal perteneciente a la familia Myrtaceae a la concentraciòn de 272ug/ml con los porcentajes de inhibiciòn de 96 por ciento a la concentraciòn de 0,18x10-4mo1/L de ADP y 94,4 por ciento a la concentraciòn de 0,045x10-4mo1/L de ADP. Tambièn resultò activo el extracto de una planta medicinal perteneciente a la familia Flacourtiaceae con un porcentaje de inhibiciòn de 92 por ciento al ser inducido por ADP a la concentraciòn de 0,045x10-4mo1/L. Este estudio indica el potencial de algunos extractos estudiados y justifica el aislamiento de compuestos puros de los extractos activos con mejores propiedades

Disuasíon de alimenticia y efectos insecticidas de extratos de plantas en Lutzomyia longipalpis / Annual Reports 1992 / Feeding deterrency and insecticidal efects of plant extracts on Lutzomyia longipalpis

Papers impregnated with seventeen extracts and three essential oils derived from nineteen plants species were assayed for insecticidal effect on Lutzomyia longipalpis sandflies. Five extracts singnificantly increased after aplication to hamsters for repellency/antifeedant effects on the flies. Bixa orellana seed extact and Bulnesia sarmientoi essential oil elicited a strong feeding inhibition at 2.50-1.25 Mg/cm2 skin

Un metodo de tamizaje de productos naturales en insecto triatominos / Annual Reports 1992 / A Screening method for natural products on triatomine bugs

A simple method for screening natural products on triatomine bugs was developed using fourth instar Rhodnius neglectus nymphs as an insect model. The statistical method of randomized blocks and randomized blocks with a dichotomous variable were compared and the minimum sample size for screening purposes was established. Tests were made on 94 plant extracts from 49 species, 28 of which are traditionally regarded as having some effect on arthropods or parasitic helminths. 50 mg of crude hexane or ethanol extract was topically applied to each insect. The effect on mortality and moulting was assesed. Ten plant species cuasede caused an increased mortality (16.0 percent to 52.3 precent) compared with untreated controls. Moulting inhibition ranged from 22.2 percent to 33.3 percent and was related to insecticidal effect in most cases. Extracts from two Meliaceae plants belonging to the genera Melia and Cabralea were among the most actived tested, causing 32.5 percent and 45 percent mortality, respectively. Extracts from a Salvia species killed 52.3 percent of the bugs. Ten of the 13 plants showing activity are used by the rural population in various parts of Latin America for arthropod control or as vermifuges

Actividad tripanocida de productos naturales contra formas sanguineas tripomasrigotes de Trypanosoma Cruzi / Trypanocidal activity of natural products against blood Trypomastigote forms of Trypanosoma cruzi / Annual Reports 1991

Blood trnasfusion is the second most importnatmechanism of transmission of Chagas'disease in both endemic and non-endemic areas. In Latin America, more than 20,000 cases of transmission of the disease, trough blood transfusion, may be occurring. Gentian Violet is currently the only available trypanicidal agent used for treating blood before transfusion. The main disadvantages of this treatment are the time required to allow drug action before transfusion and the coloring of the blood. In vitro trypanocidal effect of plant extracts and that of pure compounds are assessed against trypomastigote blood forms of T. cruzi. The induction of sister chromatid exchange (SCE) in human lypocytes was performed to evaluate mutagenic effects of these compounds. Eight compounds had a high activity against blood forms of T. cruzi at a concentration of 250 mg/ml; namely, daphnandrine, limacine, pheantine, daphnoline and cocsuline. Plumbagin (a naphthoquinone) also showed high trypanicidal activity and has previously been recognized as a leismaniacidal, bactericidal and fungicidal, compound. Daphnoline showed low values for both LC50 (8mg/ml) and LC90 (50mg/ml) and was the only tested compound which did not induce sister chromatid exchange below 10 mg/ml. Therefore, deeper evaluations of these compounds should be considered