Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites.

Artemisinin-based combination therapies (ACT) are the frontline treatments against malaria worldwide. Recently the use of traditional infusions from Artemisia annua (from which artemisinin is obtained) or Artemisia afra (lacking artemisinin) has been controversially advocated. Such unregulated plant-based remedies are strongly discouraged as they might constitute sub-optimal therapies and promote drug resistance. Here, we conducted the first comparative study of the anti-malarial effects of both plant infusions in vitro against the asexual erythrocytic stages of Plasmodium falciparum and the pre-erythrocytic (i.e., liver) stages of various Plasmodium species. Low concentrations of either infusion accounted for significant inhibitory activities across every parasite species and stage studied. We show that these antiplasmodial effects were essentially artemisinin-independent and were additionally monitored by observations of the parasite apicoplast and mitochondrion. In particular, the infusions significantly incapacitated sporozoites, and for Plasmodium vivax and P. cynomolgi, disrupted the hypnozoites. This provides the first indication that compounds other than 8-aminoquinolines could be effective antimalarials against relapsing parasites. These observations advocate for further screening to uncover urgently needed novel antimalarial lead compounds.

New method for screening anti-Leishmania compounds in plants extracts by HPTLC-bioautography.

Leishmania genus is responsible for leishmaniasis, a group of diseases affecting 12 million people in the tropical and subtropical zone. Currently, the few drugs that are available to treat this disease are expensive and cause many side effects. Searching for new therapeutics from plant species seems to be a promising path. This work proposes an original HPTLC test against parasites, in particular on Leishmania infantum, to screen new molecules from plant extracts. The technique uses protozoa transformed to express the luciferase gene to observe the bioautogram in bioluminescence. We have developed two different test protocols based on the two dimorphic stages of the parasite. The free promastigote stage, and an intracellular stage parasitizing macrophage cells called the amastigote stage. These two stages only survive under extremely different conditions which required the development of two very different test protocols. For the promastigote free stage of the protozoa, the direct bioautography technique was chosen while for the intracellular amastigote stage, bioautography by immersion (agar overlay) was required. Amphotericine B was chosen as the reference compound for this assay. The development of each of these two tests made it possible to clearly detect areas of activity on the bioautogram, allowing a rapid and inexpensive screening of the antiparasitic properties of molecules in natural extracts.

Screening of FDA-Approved Drugs Using a MERS-CoV Clinical Isolate from South Korea Identifies Potential Therapeutic Options for COVID-19.

Therapeutic options for coronaviruses remain limited. To address this unmet medical need, we screened 5406 compounds, including United States Food and Drug Administration (FDA)-approved drugs and bioactives, for activity against a South Korean Middle East respiratory syndrome coronavirus (MERS-CoV) clinical isolate. Among 221 identified hits, 54 had therapeutic indexes (TI) greater than 6, representing effective drugs. The time-of-addition studies with selected drugs demonstrated eight and four FDA-approved drugs which acted on the early and late stages of the viral life cycle, respectively. Confirmed hits included several cardiotonic agents (TI > 100), atovaquone, an anti-malarial (TI > 34), and ciclesonide, an inhalable corticosteroid (TI > 6). Furthermore, utilizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we tested combinations of remdesivir with selected drugs in Vero-E6 and Calu-3 cells, in lung organoids, and identified ciclesonide, nelfinavir, and camostat to be at least additive in vitro. Our results identify potential therapeutic options for MERS-CoV infections, and provide a basis to treat coronavirus disease 2019 (COVID-19) and other coronavirus-related illnesses.

The Efficacy of Hydroalcoholic Extracts of Prosopis farcta Against Leishmania major

Objective: Cutaneous leishmaniasis caused by Leishmania major (L. major) is an endemic disease in Iran. The current reference drugs, including Glucantime, possess high toxicity in addition to some side-effects. Therefore, there is a growing interest in exploring biomedical plants. The goal of the present study was to evaluate the anti-leishmanial activity and cytotoxicity of hydroalcoholic extracts from Prosopis farcta (P. farcta) over promastigote and amastigote forms. Methods: This study was performed at the Iran Birjand University of Medical Sciences, during the year 2019. In this study, the hydroalcoholic extracts of the stems, leaves (LE) and fruits (FE) of P. farcta were obtained. The anti-leishmanial activity was assessed against leptomonad promastigotes and intracellular amastigotes of L. major. The cytotoxicity of these extracts was determined in murine macrophages. Results: The FE and LE of P. farcta demonstrated a significant leishmanicidal effect against L. major promastigotes with an IC50 of 0.9 mg/mL and 1.1 mg/mL, respectively. The FE showed the most anti-leishmanial activity and presented with the highest index of selectivity (SI=14.6) as an anti-leishmanial product. Infected macrophages treated using the FE showed a reduction in parasite burden by 97.3%. Conclusion: The results of the present study demonstrated the leishmanicidal activity of P. farcta on both promastigotes and intracellular amastigotes. There is a need for performing comprehensive studies on relevant animal models and to access the effects of active components of P. farcta extract on the growth of L. major.

In vitro analyses of Artemisia extracts on Plasmodium falciparum suggest a complex antimalarial effect.

Dried-leaf Artemisia annua L. (DLA) antimalarial therapy was shown effective in prior animal and human studies, but little is known about its mechanism of action. Here IC50s and ring-stage assays (RSAs) were used to compare extracts of A. annua (DLAe) to artemisinin (ART) and its derivatives in their ability to inhibit and kill Plasmodium falciparum strains 3D7, MRA1252, MRA1240, Cam3.11 and Cam3.11rev in vitro. Strains were sorbitol and Percoll synchronized to enrich for ring-stage parasites that were treated with hot water, methanol and dichloromethane extracts of DLA, artemisinin, CoArtem™, and dihydroartemisinin. Extracts of A. afra SEN were also tested. There was a correlation between ART concentration and inhibition of parasite growth. Although at 6 hr drug incubation, the RSAs for Cam3.11rev showed DLA and ART were less effective than high dose CoArtem™, 8 and 24 hr incubations yielded equivalent antiparasitic results. For Cam3.11, drug incubation time had no effect. DLAe was more effective on resistant MRA-1240 than on the sensitive MRA-1252 strain. Because results were not as robust as observed in animal and human studies, a host interaction was suspected, so sera collected from adult and pediatric Kenyan malaria patients was used in RSA inhibition experiments and compared to sera from adults naïve to the disease. The sera from both age groups of malaria patients inhibited parasite growth ≥ 70% after treatment with DLAe and compared to malaria naïve subjects suggesting some host interaction with DLA. The discrepancy between these data and in-vivo reports suggested that DLA's effects require an interaction with the host to unlock their potential as an antimalarial therapy. Although we showed there are serum-based host effects that can kill up to 95% of parasites in vitro, it remains unclear how or if they play a role in vivo. These results further our understanding of how DLAe works against the malaria parasite in vitro.

Anti-Leishmania activity of extracts from Piper cabralanum C.DC. (Piperaceae).

Species of Piperaceae are known by biological properties, including antiparasitic such as leishmanicidal, antimalarial and in the treatment of schistosomiasis. The aim of this work was to evaluate the antileishmania activity, cytotoxic effect, and macrophage activation patterns of the methanol (MeOH), hexane (HEX), dichloromethane (DCM) and ethyl acetate (EtOAc) extract fractions from the leaves of Piper cabralanum C.DC. The MeOH, HEX and DCM fractions inhibited Leishmanina amazonensis promastigote-like forms growth with a half maximal inhibitory concentration (IC50) of 144.54, 59.92, and 64.87 µg/mL, respectively. The EtOAc fraction did not show any relevant activity. The half maximal cytotoxic concentration (CC50) for macrophages were determined as 370.70, 83.99, 113.68 and 607 µg/mL for the MeOH, HEX and DCM fractions, respectively. The macrophage infectivity was concentration-dependent, especially for HEX and DCM. MeOH, HEX and DCM fractions showed activity against L. amazonensis with low cytotoxicity to murine macrophages and lowering infectivity by the parasite. Our results provide support for in vivo studies related to a potential application of P. cabralanum extract and fractions as a promising natural resource in the treatment of leishmaniasis.

Chemicals sorbed to environmental microplastics are toxic to early life stages of aquatic organisms.

Microplastics are ubiquitous in aquatic ecosystems, but little information is currently available on the dangers and risks to living organisms. In order to assess the ecotoxicity of environmental microplastics (MPs), samples were collected from the beaches of two islands in the Guadeloupe archipelago, Petit-Bourg (PB) located on the main island of Guadeloupe and Marie-Galante (MG) on the second island of the archipelago. These samples have a similar polymer composition with mainly polyethylene (PE) and polypropylene (PP). However, these two samples are very dissimilar with regard to their contamination profile and their toxicity. MPs from MG contain more lead, cadmium and organochlorine compounds while those from PB have higher levels of copper, zinc and hydrocarbons. The leachates of these two samples of MPs induced sublethal effects on the growth of sea urchins and on the pulsation frequency of jellyfish ephyrae but not on the development of zebrafish embryos. The toxic effects are much more marked for samples from the PB site than those from the MG site. This work demonstrates that MPs can contain high levels of potentially bioavailable toxic substances that may represent a significant ecotoxicological risk, particularly for the early life stages of aquatic animals.

IP3 receptor-mediated Ca2+ release from acidocalcisomes regulates mitochondrial bioenergetics and prevents autophagy in Trypanosoma cruzi.

In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIP3R) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIP3R is a Ca2+ release channel gated by IP3 when expressed in DT40 cells knockout for all vertebrate IP3 receptors, and is required for Ca2+ uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O2 consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIP3R by CRISPR/Cas9 genome editing caused: a) a reduction in O2 consumption rate and citrate synthase activity; b) decreased mitochondrial Ca2+ transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIP3R overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIP3R-mediated acidocalcisome Ca2+ release on cell bioenergetics in T. cruzi.

Mode of action of a formulation containing hydrazones and saponins against leishmania spp. Role in mitochondria, proteases and reinfection process.

Toxicity and poor adherence to treatment that favors the generation of resistance in the Leishmania parasites highlight the need to develop better alternatives. Here, we evaluated the in vitro effectiveness of hydrazone derived from chromanes 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (TC1) and 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide (TC2) and the mixture of triterpene saponin hederagenin-3-O-(3,4-O-diacetyl-ß-D-xylopyranosyl-(1à3)-a-L- rhamnopyranosyl-(1à2)-a-L-arabinofuranoside, hederagenin-3-O-(3,4-O-diacetyl-a-L- arabinopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside and, hederagenin-3-O-(4-O-acetyl-ß-D-xylopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside from Sapindus saponaria (SS) on L. braziliensis and L. pifanoi. Mixtures of TC1 or TC2 with saponin were formulated for topical application and the therapeutic effectiveness was evaluated in the model for cutaneous leishmaniasis (CL) in golden hamster. The mode of action of these compounds was tested on various parasite processes and ultrastructural parasite modifications. TC1, TC2 and SS showed moderate cytotoxicity when tested independently but toxicity was improved when tested in combination. The compounds were more active against intracellular Leishmania amastigotes. In vivo studies showed that combinations of TC1 or TC2 with SS in 1:1 ratio (w/w) cured 100% of hamsters with no signs associated with toxicity. The compounds did cause changes in the mitochondrial activity of the parasite with a decrease in ATP levels and depolarization of membrane potential and overproduction of reactive oxygen species; nevertheless, these effects were not related to alterations in membrane permeability. The phagolysosome ultrastructure was also affected impacting the survival of Leishmania but the function of the lysosome nor the pH inside the phagolysosome did not change. Lastly, there was a protease inhibition which was directly related to the decrease in the ability of Leishmania to infect and multiply inside the macrophage. The results suggest that the combination of TC1 and TC2 with SS in a 1:1 ratio is capable of curing CL in hamsters. This effect may be due to the ability of these compounds to affect parasite survival and the ability to infect new cells.

Naphthylisoquinoline alkaloids, validated as hit multistage antiplasmodial natural products.

The discovery and development of multistage antimalarial drugs targeting intra-erythrocytic asexual and sexual Plasmodium falciparum parasites is of utmost importance to achieve the ambitious goal of malaria elimination. Here, we report the validation of naphthylisoquinoline (NIQ) alkaloids and their synthetic analogues as multistage active antimalarial drug candidates. A total of 30 compounds were tested, of which 17 exhibited IC50 values <1 µM against drug-sensitive P. falciparum parasites (NF54 strain); 15 of these retained activity against a panel of drug-resistant strains. These compounds showed low in vitro cytotoxicity against HepG2 cells, with selectivity indices of >10. The tested compounds showed activity in vitro against both early- and late-stage P. falciparum gametocytes while blocking male gamete formation (>70% inhibition of exflagellation at 2 µM). Additionally, five selected compounds were found to have good solubility (≥170 µM in PBS at pH 6.5), while metabolic stability towards human, mouse, and rat microsomes ranged from >90% to >7% after 30 min. Dioncophylline C (2a) emerged as a front runner from the study, displaying activity against both asexual parasites and gametocytes, a lack of cross-resistance to chloroquine, good solubility, and microsomal stability. Overall, this is the first report on the multistage activity of NIQs and their synthetic analogues including gametocytocidal and gametocidal effects induced by this class of compounds.

In Vitro Drug Efficacy Testing Against Trypanosoma brucei.

The recent endorsement of fexinidazole by the European Medicines Agency for the treatment of human African trypanosomiasis has demonstrated the high predictive value of cell-based assays for parasite chemotherapy. Here we describe three in vitro drug susceptibility tests with Trypanosoma brucei that have served as the basis for the identification of fexinidazole as a promising lead: (1) a standard assay with end-point measurement to determine drug efficacy; (2) a wash-out assay to test for reversibility and speed of drug action; (3) isothermal microcalorimetry for real-time measurement of onset of drug action and time to kill. Together, these assays allow to estimate pharmacodynamic parameters in vitro and to devise appropriate treatment regimens for subsequent in vivo experiments.

Probing the Open Global Health Chemical Diversity Library for Multistage-Active Starting Points for Next-Generation Antimalarials.

Most phenotypic screens aiming to discover new antimalarial chemotypes begin with low cost, high-throughput tests against the asexual blood stage (ABS) of the malaria parasite life cycle. Compounds active against the ABS are then sequentially tested in more difficult assays that predict whether a compound has other beneficial attributes. Although applying this strategy to new chemical libraries may yield new leads, repeated iterations may lead to diminishing returns and the rediscovery of chemotypes hitting well-known targets. Here, we adopted a different strategy to find starting points, testing ∼70,000 open source small molecules from the Global Health Chemical Diversity Library for activity against the liver stage, mature sexual stage, and asexual blood stage malaria parasites in parallel. In addition, instead of using an asexual assay that measures accumulated parasite DNA in the presence of compound (SYBR green), a real time luciferase-dependent parasite viability assay was used that distinguishes slow-acting (delayed death) from fast-acting compounds. Among 382 scaffolds with the activity confirmed by dose response (<10 µM), we discovered 68 novel delayed-death, 84 liver stage, and 68 stage V gametocyte inhibitors as well. Although 89% of the evaluated compounds had activity in only a single life cycle stage, we discovered six potent (half-maximal inhibitory concentration of <1 µM) multistage scaffolds, including a novel cytochrome bc1 chemotype. Our data further show the luciferase-based assays have higher sensitivity. Chemoinformatic analysis of positive and negative compounds identified scaffold families with a strong enrichment for activity against specific or multiple stages.

Study of the in vitro and in vivo antileishmanial activities of nimodipine in susceptible BALB/c mice.

BACKGROUND & OBJECTIVES: Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL), however, treatment failures are frequent. Nimodipine, a calcium channel blocker is known to show promising antiprotozoal effects. Here, we investigated the antileishmanial effect of Nimodipine in both in vitro and in vivo BALB/c mice model of CL. We also compared the in vivo effect with amphotericin B and meglumine antimoniate in the experimental CL mice model. METHODS: Colorimetric alamar blue assay and J774 A.1 mouse macrophage cells were used to determine the effect of nimodipine on promastigotes and amastigotes viability, respectively. Then, the in vivo activity of nimodipine was compared to that of conventional therapies in both the early and established courses of Leishmania major infection in susceptible non-healing BALB/c mice. RESULTS: Nimodipine was highly active against promastigotes and amastigotes of L. major with IC50 values of 49.40 and 15.03 µM, respectively. In the early model, the combination therapy with meglumine antimoniate and nimodipine showed no parasites in the spleen or footpad of animals. The footpad thickness was significantly lower in mice treated with either nimodipine (1 mg/kg or 2.5 mg/kg) or amphotericin B compared to the control group in the established lesions model. However, no complete remission was observed in the footpad lesion of any of the treatment groups (nimodipine, amphotericin B, meglumine antimoniate, and combination therapy). INTERPRETATION & CONCLUSION: The effect of nimodipine was comparable to that of amphotericin B and meglumine antimoniate in early and established CL lesion models. Since nimodipine is more cost-effective than conventional therapies, our results merit further investigation in other animal models and voluntary human subjects.

Characterization of acaricide resistance in Rhipicephalus microplus populations infesting cattle in northeastern India and assessment of local plant extracts for tick management.

In the present study, the tick isolates were collected from Assam state, of northeastern region (NER) and characterized using in vitro bioassay, biochemical and molecular assays. Comparing LC50 value of susceptible IVRI-I and larvae of field isolates, revealed that RF against deltamethrin was highest for Morigaon (MGN = 21.8) and lowest for Sonitpur (SNP = 3.3) isolate. The RF against cypermethrin was highest for Nagaon (NGO = 5.0) and lowest for Barpeta (BPT = 1.2) isolate. Against coumaphos, the highest RF of 4.5 was calculated for BPT (4.5) and lowest for NGO (1.3) isolate. While using adults based assay, highest RF of 24.68 against deltamethrin and lowest RF of 4.96 was determined for MGN and SNP isolate, respectively. In contrast to the results obtained using larvae, against cypermethrin, highest RF was recorded for Kamrup Metropolitan (KMP) while it was NGO isolate using larvae. In case of coumaphos, both larvae and adults of BPT isolate were also highly resistant and lowest RF was detected in SNP (2.30) isolate. All the isolates were susceptible to ivermectin. A significant correlation (p < 0.01) between deltamethrin resistance and higher expression of glutathioneS-transferase was observed while no correlation with esterase and monooxygenase enzymes activity was noted. For the development of possible ecofriendly control measure, different accessions of Argemone mexicana and Datura metel plant species were collected, extracted and screened against adult ticks. Two accessions, NEA-03 and NED-06 collected from Amlighat and Diphu (East Karbi Anglong) were more than 90 % effective. Further dose response study of these accessions determined the LC50 values of 4.86 and 3.96 %, respectively.The resistance status of the collected tick isolates was compared with the data generated from other regions having higher livestock population and possibility of exploitation of identified plant species for the development of natural antitick product is discussed.

In ovo exposure of fathead minnow (Pimephales promelas) to selenomethionine via maternal transfer and embryo microinjection: A comparative study.

Selenium (Se) is an essential trace element of concern that is known to contaminate aquatic ecosystems as a consequence of releases from anthropogenic activities. Selenium is of particular toxicological concern for egg-laying vertebrates as they bioaccumulate Se through the diet and deposit excess Se to embryo-offspring via maternal transfer, a process which has been shown to result in significant teratogenic effects. The purpose of the present study was to determine and compare the in ovo effects of Se exposure on early development of a laboratory model fish species native to North American freshwater systems, the fathead minnow (Pimephales promelas), through two different exposure routes, maternal transfer and microinjection. For maternal transfer studies, fathead minnow breeding groups (3 females: 2 males) were exposed to diets containing Se-background levels (1.21 µg Se/g food, dry mass [dm]) or environmentally relevant concentrations of selenomethionine (SeMet; 3.88, 8.75 and 26.5 µg Se/g food dm) and bred for 28 days. Embryos were collected at different time points throughout the study to measure Se concentrations and to assess teratogenicity in embryos. While exposure to dietary Se did not negatively affect fecundity among treatment groups, the lowest treatment group (3.88 µg Se/g food dm) produced on average the most embryos per day, per female. The maternal transfer of excess Se occurred rapidly upon onset of exposure, reaching steady-state after approximately 14 days, and embryo Se concentrations increased in a dose-dependent manner. The greatest concentrations of maternally transferred Se significantly increased the total proportion of deformed embryo-larval fathead minnows but did not impact hatchability or survival. In a second study, fathead minnow embryos were injected with SeMet at concentrations of 0.00 (vehicle control), 9.73, 13.5 and 18.9 µg Se/g embryo dm. Microinjection of SeMet did not affect hatchability but significantly increased the proportion of deformed embryo-larval fish in a dose-dependent manner. There was a greater proportion of deformed fathead minnows at embryo Se concentrations of 18.9 µg Se/g embryo dm when exposed via microinjection versus maternal transfer at concentrations of 28.4 µg Se/g embryo dm. However, the findings suggest that both exposure routes induced analogous developmental toxicities in early life stage fish at Se concentrations between 9.73 and 13.5 µg Se/g embryo dm. Overall, this study demonstrated that microinjection has utility for studying the effects of Se in embryo-larval fish and is a promising method for the study of early life stage Se exposure in egg-laying vertebrates.

The effects of Deepwater Horizon crude oil on ammonia and urea handling in mahi-mahi (Coryphaena hippurus) early life stages.

Many ecologically important fishes, including mahi-mahi (Coryphaena hippurus), and their offspring were directly exposed to crude oil following the Deepwater Horizon (DWH) oil spill. Early life stage fish are especially vulnerable to the toxicity of crude oil-derived polycyclic aromatic hydrocarbons (PAHs). In teleosts, yolk sac proteins are the main energy source during development and are usually catabolized into ammonia or urea among other byproducts. Although excretion of these waste products is sensitive to oil exposure, we know little about the underlying mechanisms of this process. In this study, we examined the effects of crude oil on ammonia and urea handling in the early life stages of mahi. Mahi embryos exposed to 30-32 µg L-1 ∑PAH exhibited increased urea excretion rates and greater accumulation of urea in the tissues before hatch suggesting that ammonia, which is highly toxic, was converted into less-toxic urea. Oil-exposed embryos (6.3-32 µg L-1 ∑PAH) displayed significantly increased tissue ammonia levels at 42 hpf and upregulated mRNA levels of ammonia transporters (Rhag, Rhbg and Rhcg1) from 30 to 54 hpf. However, despite increased accumulation and higher expression of ammonia transporters, the larvae exposed to higher ∑PAH (30 µg L-1 ∑PAH) showed reduced ammonia excretion rates after hatch. Together, the increased production of nitrogenous waste reinforces previous work that increased energy demand in oil-exposed embryos is fueled, at least in part, by protein metabolism and that urea synthesis plays a role in ammonia detoxification in oil-exposed mahi embryos. To our knowledge, this study is the first to combine physiological and molecular approaches to assess the impact of crude-oil on both nitrogenous waste excretion and accumulation in the early life stages of any teleosts.

Effect of Indigofera oblongifolia on the Hepatic Oxidative Status and Expression of Inflammatory and Apoptotic Genes during Blood-Stage Murine Malaria.

Malaria is a dangerous disease spread across several countries. Recent studies have focused on medicinal plants to discover alternative agents to the currently used drugs for malaria treatment. Here, we investigated the potential role of Indigofera oblongifolia leaf extract (IE) on hepatic inflammation in mice with Plasmodium chabaudi-infected erythrocytes. Female C57BL/6 mice were divided into three groups. The first group served as a control noninfected group, while the second and third groups were intraperitoneally injected with 106 erythrocytes parasitized by P. chabaudi. Mice from the third group were treated daily with a dose of 100 mg/kg of IE for 7 days. IE significantly reduced the number of leukocytes and apoptotic cells. The numbers of CD68-positive cells decreased in the livers of mice from the treatment group. Moreover, IE raised the hepatic antioxidant levels (glutathione and catalase) and reduced the levels of hepatic oxidative stress markers (malondialdehyde, nitric oxide, and reactive oxygen species). IE regulated some functions of the genes related to immune responses, including apoptotic genes (B-cell lymphoma-2, Bax, and caspase-3) and cytokine genes (interleukin-1ß (IL-1ß), IL-6, interferon-γ, and tumor necrosis factor-α). Therefore, IE exerts significant effects against malaria and protects the liver from injury caused by P. chabaudi via antioxidant and anti-inflammatory ways.

Flavopereirine-An Alkaloid Derived from Geissospermum vellosii-Presents Leishmanicidal Activity In Vitro.

Chemotherapy is limited in the treatment of leishmaniasis due to the toxic effects of drugs, low efficacy of alternative treatments, and resistance of the parasite. This work assesses the in vitro activity of flavopereirine on promastigote cultures of Leishmania amazonensis. In addition, an in silico evaluation of the physicochemical characteristics of this alkaloid is performed. The extract and fractions were characterized by thin-layer chromatography and HPLC-DAD, yielding an alkaloid identified by NMR. The antileishmanial activity and cytotoxicity were assayed by cell viability test (MTT). The theoretical molecular properties were calculated on the Molinspiration website. The fractionation made it possible to isolate a beta-carboline alkaloid (flavopereirine) in the alkaloid fraction. Moreover, it led to obtaining a fraction with greater antileishmanial activity, since flavopereirine is very active. Regarding the exposure time, a greater inhibitory effect of flavopereirine was observed at 24 h and 72 h (IC50 of 0.23 and 0.15 µg/mL, respectively). The extract, fractions, and flavopereirine presented low toxicity, with high selectivity for the alkaloid. Furthermore, flavopereirine showed no violation of Lipinski's rule of five, showing even better results than the known inhibitor of oligopeptidase B, antipain, with three violations. Flavopereirine also interacted with residue Tyr-499 of oligopeptidase B during the molecular dynamics simulations, giving a few insights of a possible favorable mechanism of interaction and a possible inhibitory pathway. Flavopereirine proved to be a promising molecule for its antileishmanial activity.

Toxicity of phenolic compounds to entomopathogenic nematodes: A case study with Heterorhabditis bacteriophora exposed to lentisk (Pistacia lentiscus) extracts and their chemical components.

Insects show adaptive plasticity by ingesting plant secondary compounds, such as phenolic compounds, that are noxious to parasites. This work examined whether exposure to phenolic compounds affects the development of insect parasitic nematodes. As a model system for parasitic life cycle, we used Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade) grown with Photorhabdita luminescens supplemented with different concentrations of plant phenolic extracts (0, 600, 1200, 2400 ppm): a crude ethanol extract of lentisk (Pistacia lentiscus) or lentisk extract fractionated along a scale of hydrophobicity with hexane, chloroform and ethyl acetate; and flavonoids (myricetin, catechin), flavanol-glycoside (rutin) or phenolic acids (chlorogenic and gallic acids). Resilience of the nematode to phenolic compounds was stage-dependent, with younger growth stages exhibiting less resilience than older growth stages (i.e., eggs < young juveniles < young hermaphrodites < infective juveniles < mature hermaphrodites). At high concentrations, all of the phenolic compounds studied were lethal to eggs and young juveniles. The nematodes were able to survive in the presence of medium and low concentrations of all studied compounds, but very few of those treatments allowed for reproduction beyond the infective juvenile stage and, at low concentrations, the crude 70% ethanol extract, chloroform and hexane extracts, and myricetin were associated with some impaired reproduction. The ethyl-acetate fraction and gallic acid were extremely lethal to the young stages and allowed almost no development beyond the infective juvenile stage. We conclude that exposure of infective juveniles to phenolics before they infect insects and post-infection exposure of other nematode developmental stages may affect the initiation of the infection, suggesting that the chemistry of dietary phenolics may limit H. bacteriophora's infection of insects.

Repurposing of an old drug: In vitro and in vivo efficacies of buparvaquone against Echinococcus multilocularis.

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the lethal disease alveolar echinococcosis. Current chemotherapeutic treatment options are based on benzimidazoles (albendazole and mebendazole), which are insufficient and hence alternative drugs are needed. In this study, we screened the 400 compounds of the Medicines for Malaria Venture (MMV) Pathogen Box against E. multilocularis metacestodes. For the screen, we employed the phosphoglucose isomerase (PGI) assay which assesses drug-induced damage on metacestodes, and identified ten new compounds with activity against the parasite. The anti-theilerial drug MMV689480 (buparvaquone) and MMV671636 (ELQ-400) were the most promising compounds, with an IC50 of 2.87 µM and 0.02 µM respectively against in vitro cultured E. multilocularis metacestodes. Both drugs suggested a therapeutic window based on their cytotoxicity against mammalian cells. Transmission electron microscopy revealed that treatment with buparvaquone impaired parasite mitochondria early on and additional tests showed that buparvaquone had a reduced activity under anaerobic conditions. Furthermore, we established a system to assess mitochondrial respiration in isolated E. multilocularis cells in real time using the Seahorse XFp Analyzer and demonstrated inhibition of the cytochrome bc1 complex by buparvaquone. Mice with secondary alveolar echinococcosis were treated with buparvaquone (100 mg/kg per dose, three doses per week, four weeks of treatment), but the drug failed to reduce the parasite burden in vivo. Future studies will reveal whether improved formulations of buparvaquone could increase its effectivity.