Abstract Chagas disease, caused by Trypanosoma cruzi, poses a significant global health burden. Current treatment relies solely on benznidazole and nifurtimox, both with limited efficacy and significant adverse effects. In vitro phenotype-based screening enables identification of alternative treatments. While epimastigote-based screening is not suitable for primary drug discovery, it provides valuable complementary data for compound evaluation. However, the lack of standardized experimental conditions across studies compromises reproducibility and inter-laboratory comparability. Here we optimized experimental conditions for T. cruzi epimastigote drug susceptibility assays. T. cruzi epimastigotes (strain Y) were cultured under systematically varied conditions to optimize medium composition, serum supplementation, medium replacement frequency, inoculum density, and treatment exposure time. Epimastigote growth peaked on day 8, declining thereafter. Low initial parasite inoculum showed reduced growth and high variability, while high parasite densities formed aggregates. Growth rates were similar across media, but Roswell Park Memorial Institute (RPMI) medium could promote trypomastigote-like forms. Fetal bovine serum (FBS) enhanced growth rates with positive correlation between serum concentration and proliferation. Three-day medium replacement maintained >98% epimastigotes. Benznidazole exhibited an IC₅₀ of 16.61 ± 0.38 µM under optimized conditions. Optimal conditions include: Liver Infusion Tryptose or Brain Heart Infusion medium with 10% FBS, medium replacement every three days, 1 × 106 parasites/mL inoculum, and 72-hour treatment at 28 °C. The assay demonstrated robustness across microplate designs, producing consistent results with both U-bottom and flat-bottom 96-well plates. Under these optimized conditions, benznidazole serves as an effective positive control.

Introducción: La enfermedad de Chagas, causada por Trypanosoma cruzi, afecta entre 6 y 8 millones de personas en todo el mundo. Los tratamientos actuales plantean problemas de seguridad. La variabilidad del tamizaje fenotípico in vitro en cuanto a cepas del parásito, estadios del ciclo vital y condiciones experimentales dificultan la comparación de resultados e impiden el desarrollo de nuevos tratamientos. Objetivo: Identificar condiciones experimentales críticas en el modelo de epimastigotes de T. cruzi y proponer criterios para armonizar los procedimientos experimentales. Metodología: Análisis de 63 artículos de investigación (106 ensayos) de revistas indexadas (2008-2023), sobre actividad antiparasitaria frente a epimastigotes. Resultados: Los estudios utilizan diversas formas evolutivas, principalmente amastigotes y epimastigotes. A pesar de avances en las técnicas de cultivo, establecer condiciones uniformes para todos los estadios sigue siendo inviable. Proponemos focalizar formas parasitarias específicas. Conclusiones: Para armonizar condiciones experimentales, proponemos optimizar: cepas, medio de cultivo (y enriquecimiento), parámetros de incubación (temperatura y frecuencia de sustitución del medio). Del mismo modo, las condiciones de prueba farmacológica, como concentración del inóculo infectante, especificaciones de placa de cultivo, tiempo de exposición al tratamiento y método de cuantificación del parásito, facilitan comparación de resultados y aceleran el desarrollo de nuevos tratamientos.

Background: The study of medicinal plants has made it possible to develop products and drugs for the treatment of different diseases. Several plants in Colombia have a history of popular use for the treatment of malaria. The objective of this work was to provide information on the antiplasmodic and phytochemical activity of five neotropical native plants with a folk use for the treatment of malarial.Methods: The ethanolic extract of each species was obtained by percolation method and characterized by TLC, HPLC, and 1H-NMR. The in vitro antiplasmodial activity was evaluated against Plasmodium falciparum (strain FCR-3, chloroquine-resistant).Results: Ethanolic extracts of Ambelania duckey, Cecropia metensis, Cecropia membranacea, and Verbena littoralis showed no activity. However, Curarea toxicofera extract exhibited an IC50 of 7.6 ± 3.9 μg/mL and was classified as moderately active. Furthermore, hemolytic activity was assayed, none of the extracts were tested positive. A preliminary phytochemical study was carried out using tube analysis, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and hydrogen nuclear magnetic resonance (1H-NMR). Steroids or triterpenes, and phenolic compounds were detected by TLC in all extracts. These findings were confirmed by characteristic aliphatic and aromatic signals in 1H-NMR spectra, corresponding to triterpenes and phenolics, respectively. Additionally, alkaloids were extracted from C. toxicofera and detected by aromatic signals in 1H-NMR spectra.Conclusion: ethanolic extract of C. toxicofera showed antiplasmodial activity (IC50 of 7.6 ± 3.9 μg/mL); this activity may be due to the bisbenzylisoquinoline alkaloids. The ethanol extracts of A. duckey, C. membranacea, C. metensis, and V. littoralis did not present antiplasmodial activity.

Background The chagra is the agroforestry system adapted to the characteristics of the Amazon region. Recently, there has been a reported loss of biodiversity and traditional knowledge associated with the chagras. This paper characterizes the cultivators, exploring knowledge and expressed value perception in the context of the Amazonian chagra of an indigenous community; also, this prioritizes species, under the optics of commercial opportunity. Methods A semi-structured instrument was applied to 14 volunteers, asking about marketing preferences and use values of the species; later, a floristic inventory and prioritization workshop was developed. Results Sixty-two percent of the participants were 50 years or older at the time of the interview. Open conversations showed that traditional knowledge is a matter of practice; and is maintained mainly by the older "grandfathers". Thirty-eight species, belonging to 28 different families, were reported, showing considerable diversity. Seventy-nine percent of the participants consider the Leticia market and sales to tourists as the main marketing scenarios. Conclusions The Ziora-Amena community centralizes the handling of chagras in the community's older adults, who transmit their traditional knowledge to new generations through oral tradition. Indicators of preference, use, and abundance highlight the food species. The perception of the trade stakeholder encourages research and development of endemic species, with health properties or ingredients for industry, which represent an opportunity of high added value for the region.

Background: Neglected Infectious Diseases control is an urgent need in endemic areas, but it's not a priority in the commercial interests of the pharmaceutical industry. Chagas disease (caused by Trypanosoma cruzi) is the parasitic disease of greatest impact in Latin America. Medicinal plants continue to be an affordable front-line option in endemic areas. Thus, we aimed to advance in the pharmacological evaluation of four medicinal plants traditionally used in the Amazon against parasitic infections. Materials and Methods: The plants were collected in the Amazon region of Colombia. The dry plant material was submitted to water percolation extraction. Extracts were tested in vitro against Trypanosoma cruzi (epimastigotes), and cytotoxicity was assessed against HepG2 and MRC5 cells. Finally, the general profile of metabolites present in the extracts was studied by thin-layer chromatography. Results: In vitro, against T. cruzi, extracts of Ambelania duckei and Curarea toxicofera shows concentration-dependent inhibition (IC50 of 221+/-29 and 50+/-5 mu g/mL respectively), comparable with benznidazole (IC50: 0.7 mu g/mL); while Abuta grandifolia and Aspidosperma excelsum exhibited IC50's > 500 mu g/mL. All extracts showed no cytotoxicity against HepG2 and MRC5 cells. Yields of extraction were between 3.2 and 9.5% and preliminary phytochemical profile showed flavonoids and steroids in all extracts. Conclusion: Promising plants, traditionally used to treat other protozoan infections, could be assessed against T. cruzi. C. toxicofera exhibits good activity against epimastigotes of T. cruzi, being a species that can reasonably be considered for bioassay-guided antitrypanosomal fractionation.

Background & objectives: In Colombian Amazonia, Uitoto indigenous people use a preparation of Curarea toxicofera (Menispermaceae) to prevent and treat malaria. To open the way for the production of a standardized herbal remedy, we compared the activity of the traditional preparation with laboratory preparations.Methods: People were interviewed on their mode of use and preparation of what is considered the best remedy against fevers in this area. The herbal remedy was prepared according to the healer’s recommendations. The plant was also submitted to continuous distillation and percolation extraction. The preparations were then tested against Plasmodium falciparum, in vitro. Traditional preparation and extract obtained by percolation were tested on Plasmodium berghei infected mice. Chemical profiles were also explored by thin-layer chromatography.Results: Yields of extraction were around 7% in the preparations (percolation was the most efficient). The phytochemical profile showed a mix of steroids, flavonoids and alkaloids qualitatively similar in all preparations. In vitro, the extracts showed inhibitory concentration 50 <10μg/mL: the traditional preparation was almost three times less active than laboratory preparations. In vivo, percolation was also more active than traditional preparation, inhibiting 78% of the parasite growth at 400mg/kg/day by oral route.Interpretation & conclusion: Pharmacological activities suggest that both the original remedy (prepared according to traditional pharmacopeia) and the extracts obtained by percolation extraction exhibit relevant antiparasitic activity. C. toxicofera should therefore be considered for the elaboration of an improved traditional medicine by implementing toxicological studies and carefully following quality control guidelines for its preparation.

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.

Ethnopharmacological relevance: In the Leticia-Amazonas area, Uitoto indigenous people use a preparation of Curarea toxicofera (Wedd) Barneby & Krukoff (Menispermaceae) alone or combined with prescribed medications to prevent and treat malaria. Aim of study: To determine the in vitro and in vivo antiplasmodial activity of traditional preparations of Curarea toxicofera alone and in combination with classical antimalarials. Material and Methods: The traditional preparation was evaluated in vitro against P. falciparum FCR3 CQ resistant strain, alone and combined. The preparation was further administered orally alone or combined with chloroquine and artesunate in mice infected with Plasmodium berghei ANKA strain on the four-day antimalarial test model. Results: The herbal remedy used alone was able to significantly decrease the parasitemia both in vitro (IC50 7.3 mu g/ml) and in vivo (ED50 328 mg/Kg) but it was less active than chloroquine (IC50 0.29 mu g/ml in vitro and ED50 2.3 mg/Kg/day in vivo), and than artesunate (IC50 0.002 mu g/ml and ED50 3.7 mg/Kg/day). Interestingly it presented synergism with chloroquine in vitro (Combination Index: 0.39) and in vivo; and was additive with artesunate in vitro (Combination Index: 0.94) and in vivo. Conclusion: The traditional preparation showed potential as an antimalarial and, when used in combination, does not negatively affect the efficacy of the drugs evaluated. Pre-clinical studies should be conducted with a standardized preparation to confirm its efficacy and safety alone and in combination with chloroquine and artesunate.

Design, synthesis, structure-activity relationship, cytotoxicity studies, in silico drug-likeness, genotoxicity screening, and in vivo studies of new 1-aryl-3-substituted propanol derivatives led to the identification of nine compounds with promising in vitro (55, 56, 61, 64, 66, and 70-73) and in vivo (66 and 72) antimalarial profiles against Plasmodium falciparum and Plasmodium berghei. Compounds 55, 56, 61, 64, 66 and 70-73 exhibited potent antiplasmodial activity against chloroquine-resistant strain FCR-3 (IC50s < 0.28 μM), and compounds 55, 56, 64, 70, 71, and 72 showed potent biological activity in chloroquine-sensitive and multidrug-resistant strains (IC50s < 0.7 μM for 3D7, D6, FCR-3 and C235). All of these compounds share appropriate drug-likeness profiles and adequate selectivity indexes (77 < SI < 184) as well as lack genotoxicity. In vivo efficacy tests in a mouse model showed compounds 66 and 72 to be promising candidates as they exhibited significant parasitemia reductions of 96.4% and 80.4%, respectively. Additional studies such as liver stage and sporogony inhibition, target exploration of heat shock protein 90 of P. falciparum, targeted delivery by immunoliposomes, and enantiomer characterization were performed and strongly reinforce the hypothesis of 1-aryl-3-substituted propanol derivatives as promising antimalarial compounds.

The in vivo efficacy of potential antimalarials is usually evaluated by direct microscopic determination of the parasitaemia of Plasmodium-infected mice on Giemsa-stained blood smears. This process is time-consuming, requires experienced technicians and is not automatable. Therefore, we optimized a SYBR Green I (SYBRG I) fluorescence-based assay to fluorometers commonly available in many research laboratories. This technique was originally developed to assess parasitaemia in humans by cytometry. We defined optimal conditions with Plasmodium berghei-infected mice, standard lysis buffer (Tris, EDTA, saponin and Triton), whole blood cells and 2 h staining incubation with SYBRG I 2X. The fluorescence background generated by uninfected whole blood cells was low (around 4.6%), and the linearity high (r 2 = 0.96), with parasitaemia ranging from 1.4 to 60%. The Bland–Altman plot showed a strong correlation between SYBRG I and Giemsa gold standard method; Z′-factor was >0.5. These findings suggest that our fluorescence-based assay is suitable for in vivo antimalarial drug assessment in a malaria murine model. It can help to overcome the human bias found with microscopic techniques.

Synthesis of new 1-aryl-3-substituted propanol derivatives followed by structure-activity relationship, in silico drug-likeness, cytotoxicity, genotoxicity, in silico metabolism, in silico pharmacophore modeling, and in vivo studies led to the identification of compounds 22 and 23 with significant in vitro antiplasmodial activity against drug sensitive (D6 IC50 ≤ 0.19 μM) and multidrug resistant (FCR-3 IC50 ≤ 0.40 μM and C235 IC50 ≤ 0.28 μM) strains of Plasmodium falciparum. Adequate selectivity index and absence of genotoxicity was also observed. Notably, compound 22 displays excellent parasitemia reduction (98 ± 1%), and complete cure with all treated mice surviving through the entire period with no signs of toxicity. One important factor is the agreement between in vitro potency and in vivo studies. Target exploration was performed; this chemotype series exhibits an alternative antimalarial mechanism.

El modelo farmacológico de cultivo in vitro de P. falciparum es crucial en el tamizaje inicial de sustancias o extractos de plantas con posible actividad antiplasmodial. La densidad parasitaria puede determinarse mediante variadas metodologías, sin embargo, se han descrito numerosas ventajas y desventajas asociadas a cada una de ellas. Se evaluaron el tiempo de incubación necesario para la tinción y el uso de cultivos asincrónicos o sincrónicos en busca de valores óptimos; evidenciando un tiempo óptimo de 2 h, y límites de detección y cuantificación, menores en cultivos asincrónicos. Empleando las cepas FCR3 y FCB2 se evidenció un ruido de fondo de 12% y 38% respectivamente; la linealidad mostró una buena correlación, r2 de 0,9644 (FCR3) y 0,9841 (FCB2) y una pendiente de 1761,8 y 852,4 respectivamente. Además, se comprobó había concordancia entre los métodos, fluorométrico con SYBR Green I (SYBRG I) y microscópico con Giemsa, con diferencia media de 0,00002% y 0,09109% para FCR3 y FCB2 respectivamente. Los límites de detección y cuantificación fueron 0,5% y 1,5% de parasitemia. El factor Z con FCB2 fue 0,376, en tanto que con FCR3 alcanzó 0,702. La concentración inhibitoria 50 (CI50) frente a P. falciparum FCR3, generada por cloroquina (CQ) fue 0,37 mcg/mL por microscopía y 0,35 mcg/mL por fluorometría. Nuestros hallazgos sugieren que el ensayo de fluorescencia con SYBRG I, empleando fluorómetros comúnmente disponibles en muchos laboratorios, es preciso, robusto, rápido y exacto; para la evaluación in vitro de sustancias o extractos con posible actividad antiplasmodial.

Antiplasmodial and haemolytic activity of ethanolic extracts and fractions obtained from Cecropia membranacea Trecul. and Cecropia metensis Cuatrec. (syn. Cecropia peltata var. candida Velasquez)

The in vitro susceptibilities of 30 isolates of Plasmodium vivax to a number of antimalarials (chloroquine [CQ], mefloquine, amodiaquine, quinine, and artesunate [AS]) were evaluated. The isolates came from the region of Urabá in Colombia, in which malaria is endemic, and were evaluated by the schizont maturation test. The 50% inhibitory concentration (IC50) was 0.6 nM (95% confidence interval [CI], 0.3 to 1.0 nM) for artesunate, 8.5 nM (95% CI, 5.6 to 13.0 nM) for amodiaquine, 23.3 nM (95% CI, 12.4 to 44.1 nM) for chloroquine, 55.6 nM (95% CI, 36.8 to 84.1 nM) for mefloquine, and 115.3 nM (95% CI, 57.7 to 230.5 nM) for quinine. The isolates were classified according to whether the initial parasites were mature or immature trophozoites (Tfz). It was found that the IC50s for chloroquine and artesunate were significantly different in the two aforementioned groups (P < 0.001). The IC50s of CQ and AS were higher in the isolates from mature Tfz (CQ, 39.3 nM versus 17 nM; AS, 1.4 nM versus 0.3 nM), and 10% of the isolates showed lower susceptibilities to one of the antimalarial drugs, 13.3% to two antimalarial drugs, and 3.3% to more than three antimalarial drugs. It should be highlighted that despite the extensive use of chloroquine in Colombia, P. vivax continues to be susceptible to antimalarials. This is the first report, to our knowledge, showing in vitro susceptibilities of P. vivax isolates to antimalarials in Colombia.

We report here our investigations on plants belonging to the botanical genus Aspidosperma (Apocynaceae) that are used to treat malaria and/or fevers in Brazil, aiming to prepare standardized alkaloid extracts for phytomedicines development. Ethanol and alkaloid extracts of A. parvifolium and A. subincanum barks were prepared and their in vitro antiplasmodial activity was evaluated against Plasmodium falciparum (W2 strain) by the pLDH method. Bioguided fractionation of the active alkaloid extracts was undertaken. A study on antiplasmodial activity versus alkaloids content variation from three A. subincanum specimens that were collected in different regions was carried on by HPLC-UV. Ethanol and alkaloid extracts from A. parvifolium and A. subincanum were evaluated in toxicological studies in male and female Swiss mice and male Wistar rats (n = 10); a control group was included in the experiments. At the end of treatment, animals were sacrificed, hematology and serum biochemical analyses were performed. Fractionation of alkaloid extracts from A. parvifolium and A. subincanum guided by in vitro assays against P. falciparum (W2 strain) led to the isolation of the indole alkaloids uleine (Oliveira et al., 2010) and N-demethyluleine. HPLC-UV and LC-UV-MS/ESI analyses of the extracts disclosed uleine as the major constituent, its content reaching 70.7% in the alkaloid extract from A. subincanum (ASALB) which was more potent than uleine and N-demethyluleine themselves in the in vitro assays, as can be inferred from the selectivity indexes in relation to HepG2 cells (SI = CC50/IC50) of ASALB (100), uleine (44) and N-demethyluleine (21). Moreover, a significative variation on the contents of these two alkaloids was observed for the same plant species when collected in different regions (Paula, 2014). Both ethanol and alkaloid extracts from A. parvifolium bark were relatively safe, producing minor changes in the parameters evaluated, especially in smaller doses, thus making feasible their future use in phase I clinical trials. On the other hand, high toxicity of both extracts from A. subincanum bark was observed in doses >200 mg/kg. The present results point to the necessity of rigorous investigation of traditional antimalarial plants including in vitro studies for identification of active natural products, standardization of extracts to be submitted to pre-clinical toxicological studies and controlled clinical trials. Our results disclose that efficient and safe phytomedicines might be developed from standardized crude or semi-purified extracts of Aspidosperma species containing the active chemical entities.