Synergism of in vitro plasmodicidal activity of phospholipase A2 isoforms isolated from panamanian Bothrops asper venom.

Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A2 (PLA2s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA2s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA2s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA2-like. Acidic PLA2s catalyzed the degradation of all substrates evaluated; however, for the basic PLA2s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA2s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum, while the basic PLA2s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA2 and Lys49-PLA2 were associated with one another, denoting synergistic action between these PLA2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA2s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents.

First Molecular Report of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) guyanensis in Paraguayan Inhabitants Using High-Resolution Melt-PCR.

American tegumentary leishmaniasis is an endemic anthropozoonosis undergoing expansion on the American continent. The disease is caused by several Leishmania species and it is manifested as cutaneous and mucocutaneous leishmaniasis. In this study, we evaluate the viability of high-resolution melt polymerase chain reaction (HRM-PCR) analysis to differentiate four closely related Leishmania species as a routine tool for the diagnosis of leishmaniasis. For this purpose, biopsy specimens from cutaneous and mucocutaneous lesions were taken from 132 individuals from endemic and non-endemic areas for leishmaniasis. Each sample was processed for parasitological, histopathological, and molecular analysis. Positive biopsy samples were analyzed by HRM-PCR of a 144-bp heat-shock protein (hsp70) gene fragment, and new cases were confirmed by sequencing. Of the 132 samples analyzed, 36 (27%) were positive for Leishmania spp., of which 86% were from cutaneous lesions and 14% from mucocutaneous lesions. We identified Leishmania (Viannia) braziliensis (84%), Leishmania (Leishmania) infantum (13%), and Leishmania (Leishmania) amazonensis (3%) in cutaneous lesions, and L. (V.) braziliensis (40%), L. (L.) infantum (20%), L. (L.) amazonensis (20%), and Leishmania (Viannia) guyanensis (20%) in mucocutaneous lesions. The main purpose of this research was to report for the first time in Paraguay the presence of L. (L.) amazonensis and L. (V.) guyanensis in patients with cutaneous and mucocutaneous lesions, using the HRM-PCR technique. In addition, we report the presence of additional new cases of L. (L.) infantum in cutaneous lesions.

BmajPLA2-II, a basic Lys49-phospholipase A2 homologue from Bothrops marajoensis snake venom with parasiticidal potential.

Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100µg/mL. The venom and BmajPLA2-II presented IC50 of 0.14±0.08 and 6.41±0.64µg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64±7.94 and >150µg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.

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.

BmajPLA(2)-II, a basic Lys49-phospholipase A(2) homologue from Bothrops marajoensis snake venom with parasiticidal potential

Snake venoms contain various proteins, especially phospholipases A(2) (PLA(2)s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA(2) from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA(2)-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA(2)-II as a basic Lys49 PLA(2) homologue, compatible with other basic snake venom PLA(2)s (svPLA(2)), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA(2)-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100 mu g/mL. The venom and BmajPLA(2)-II presented IC50 of 0.14 +/- 0.08 and 6.41 +/- 0.64 mu g/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64 +/- 1 7.94 and >150 mu g/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.

Phenolic composition and antiparasitic activity of plants from the Brazilian Northeast "Cerrado".

This work describes the antiparasitic and cytotoxic activities of three plant species from the Cerrado biome, Northeastern Brazil. Significant antiparasitic inhibition was observed against Trypanosoma cruzi (63.86%), Leishmania brasiliensis (92.20%) and Leishmania infantum (95.23%) when using ethanol extract from leaves of Guazuma ulmifolia Lam. (Malvaceae), at a concentration of 500 µg/mL. However, low levels of inhibition were observed when assessing leishmanicidal and trypanocidal (Clone CL-B5) activities of crude ethanol extracts from leaves and bast tissue of Luehea paniculata (Malvaceae) and leaves and bark of Prockia crucis (Salicaceae) at a concentration of 500 µg/mL. The extracts revealed the presence of phenolic acids such as gallic acid, chlorogenic acid, caffeic acid and rosmarinic acid, as well as flavonoids such as rutin, luteolin, apigenin and quercetin - the latter detected only in G. ulmifolia. G. ulmifolia extract displayed higher leishmanicidal activity probably due to the presence of quercetin, a potent known leishmanicidal compound. A cytotoxicity test indicated values over 50% at the highest concentration (1000 µg/mL) for all natural products, which were considered cytotoxic. This points out the need for further tests to enable future in vivo trials, including antineoplastic activity on human tumor cells.

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.