Schistosoma mansoni: In vitro schistosomicidal activity of piplartine

Schistosomiasis is one of the world’s greatly neglected tropical diseases, and its control is largely dependent on a single drug, praziquantel. Here, we report the in vitro effect of piplartine, an amide isolated from Piper tuberculatum (Piperaceae), on Schistosoma mansoni adult worms. A piplartine concentration of 15.8 μM reduced the motor activity of worms and caused their death within 24 h in a RPMI 1640 medium. Similarly, the highest sub-lethal concentration of piplartine (6.3 μM) caused a 75% reduction in egg production in spite of coupling. Additionally, piplartine induced morphological changes on the tegument, and a quantitative analysis carried out by confocal microscopy revealed an extensive tegumental destruction and damage in the tubercles. This damage was dose-dependent in the range of 15.8–630.2 μM. At doses higher than 157.6 μM, piplartine induced morphological changes in the oral and ventral sucker regions of the worms. It is the first time that the schistosomicidal activity has been reported for piplartine.     

In Vitro Anthelminthic Activity and Ultrastructural Analysis of Barbatic Acid against Schistosomulae and Juvenile Worms of Schistosoma mansoni**

Schistosomiasis affects about 260 million people worldwide and the search for new schistosomicidal compounds is urgent. In this study we evaluated the in vitro effect of barbatic acid against schistosomulae and young worms of Schistosoma mansoni. The barbatic acid was evaluated through the bioassay of motility and mortality, cellular viability and ultrastructural analysis of juvenile stages through Scanning Electron Microscopy. Barbatic acid showed a schistosomicidal effect against schistosomulae and young worms of S. mansoni after 3 h of exposure. At the end of 24 h, barbatic acid showed 100 %, 89.5 %, 52 % and 28.5 % of lethality for schistosomulae at the concentrations of 200, 100, 50 and 25 μM, respectively. For young worms, barbatic acid showed 100 % and 31.7 % of lethality at the concentrations of 200 and 100 μM, respectively. Motility changes were observed at all sublethal concentrations. There was a significant reduction in the viability of young worms after exposure to barbatic acid at 50, 100 and 200 μM. Extensive damage to the schistosomulae and young worm's tegument, was observed from 50 μM. This report provides data showing the schistosomicidal effect of barbatic acid on schistosomulae and young worms of S. mansoni, causing death, motility changes and ultrastructural damage to worms.     

Antiprotozoal,Schistosomicidal, and Antimicrobial Activities of the Essential Oil from the Leaves of Baccharis dracunculifolia

Baccharis dracunculifolia DC. (Asteraceae), popularly known as ‘alecrim do campo’, is a native plant from Brazil used in folk medicine as febrifuge, anti‐inflammatory, antiseptic, and to treat skin sores. Also, B. dracunculifolia is the most important plant source of the Brazilian green propolis, which is recognized for its antiseptic and antiprotozoal activities. This study aimed at investigating the in vitro antiprotozoal, schistosomicidal, and antimicrobial activities of the essential oil from the leaves of B. dracunculifolia. The essential oil was obtained by hydrodistillation and analyzed by GC and GC/MS, which allowed the identification of 14 compounds, mainly oxygenated sesquiterpenes, such as (E)‐nerolidol (33.51%) and spathulenol (16.24%). The essential oil showed activity against promastigote forms of Leishmania donovani, with IC₅₀ values of 42 μg/ml. The essential oil displayed high activity in the schistosomicidal assay, since all pairs of Schistosoma mansoni adult worms were dead after incubation with the essential oil (10, 50, and 100 μg/ml). B. dracunculifolia essential oil was neither cytotoxic against Vero cells, nor active in the antimicrobial and antiplasmodial assays.     

Evaluation of the in vitro activity of dermaseptin 01, a cationic antimicrobial peptide, against Schistosoma mansoni

Schistosomiasis is a neglected tropical disease that remains a considerable public health problem worldwide. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we examined the in vitro effects of dermaseptin 01 (DS 01), an antimicrobial peptide found in the skin secretion of frogs of the genus Phyllomedusa, on Schistosoma mansoni adult worms. DS 01 at a concentration of 100 μg/ml reduced the worm motor activity and caused the death of all worms within 48 h in RPMI 1640 medium. At the highest sublethal concentration of antimicrobial peptide (75 μg/ml), a 100% reduction in egg output of paired female worms was observed. Additionally, DS 01 induced morphological alterations on the tegument of S. mansoni, and a quantitative analysis carried out by confocal microscopy revealed extensive destruction of the tubercles in a dose-dependent manner over the concentration range of 50-200 μg/ml. It was the first time that an anthelmintic activity towards schistosomes has been reported for a dermaseptin.     

Chemical composition and in vitro schistosomicidal activity of the essential oil of Plectranthus neochilus grown in Southeast Brazil

The chemical composition and the in vitro schistosomicidal effects of the essential oil of Plectranthus neochilus (PN-EO) grown in Southeast Brazil was studied. β-Caryophyllene (1; 28.23%), α-thujene (2; 12.22%), α-pinene (3; 12.63%), β-pinene (4; 6.19%), germacrene D (5; 5.36%), and caryophyllene oxide (6; 5.37%) were the major essential oil constituents. This chemical composition differed from that previously reported for specimens harvested in Africa. Concerning the in vitro schistosomicidal activity against adult Schistosoma mansoni worms, PN-EO was considered to be active, but less effective than the positive control praziquantel (PZQ) in terms of separation of coupled pairs, mortality, decrease in the motor activity, and tegumental alterations. However, PN-EO caused an interesting dose-dependent reduction in the number and the percentage of developed S. mansoni eggs. These results suggest that PN-EO might be very promising for the development of new schistosomicidal agents.     

Anthelmintic Activity In Vivo of Epiisopiloturine against Juvenile and Adult Worms of Schistosoma mansoni

Schistosomiasis is a serious disease currently estimated to affect more that 207 million people worldwide. Due to the intensive use of praziquantel, there is increasing concern about the development of drug-resistant strains. Therefore, it is necessary to search for and investigate new potential schistosomicidal compounds. This work reports the in vivo effect of the alkaloid epiisopiloturine (EPI) against adults and juvenile worms of Schistosoma mansoni. EPI was first purified its thermal behavior and theoretical solubility parameters charaterised. In the experiment, mice were treated with EPI over the 21 days post-infection with the doses of 40 and 200 mg/kg, and 45 days post-infection with single doses of 40, 100 and 300 mg/kg. The treatment with EPI at 40 mg/kg was more effective in adult worms when compared with doses of 100 and 300 mg/kg. The treatment with 40 mg/kg in adult worms reduced parasite burden significantly, lead to reduction in hepatosplenomegaly, reduced the egg burden in faeces, and decreased granuloma diameter. Scanning electron microscopy revealed morphological changes to the parasite tegument after treatment, including the loss of important features. Additionally, the in vivo treatment against juvenile with 40 mg/kg showed a reduction of the total worm burden of 50.2%. Histopathological studies were performed on liver, spleen, lung, kidney and brain and EPI was shown to have a DL50 of 8000 mg/kg. Therefore EPI shows potential to be used in schistosomiasis treatment. This is the first time that schistosomicidal in vivo activity of EPI has been reported.     

Studies on the in vitro effects of bithionol and menichlopholan on flukes of Clonorchis sinensis, Metagonimus takahashii and Paragonimus miyazakii.

The present study was carried out in order to obtain information on the action of bithionol and menichlopholan on Clonorchis sinensis, Metagonimus takahashii and Paragonimus miyazakii. The drugs inhibited the motility of the newly excysted juveniles and adult worms in vitro. Moreover, the in vitro treatment slightly decreased lactate dehydrogenase activity in homogenates of adult worms. The drug treatments of adult worms in vitro also inhibited the activity of fumarate reductase, malic enzyme, phosphogluconate dehydrogenase and succinate dehydrogenase in the homogenate. In addition to this, these drugs caused morphological alterations in the ultrastructure of the tegument of the flukes.     

Exploration of novel in vitro assays to study drugs against Trichuris spp

Though trichuriasis is a significant public health problem, few effective drugs are available underscoring the need for new drug therapies. For the evaluation of trichuricidal activity of test compounds in vitro an accurate, reliable, sensitive, fast and cheap drug sensitivity assay is essential. The aim of the present investigation was to evaluate the performance of different in vitro drug sensitivity assays in comparison to the standard motility assay. Trichuris muris L4 larvae or adult worms were isolated from the intestinal tract from infected female C57BL/10 mice and incubated in the presence of ivermectin, levamisole and nitazoxanide (200, 100 and 50 μg/ml) for 72 h. The health status of the worms was either evaluated microscopically using a motility scale from 0 to 3 (motility assay), by examination of absorbance or emission in response to metabolic activity (MTT (Thiazolyl Blue Tetrazolium Bromide) and Alamar Blue assay), through analysis of absorbance of an enzyme-substrate reaction (acid phosphatase activity assay), by measuring the noise amplitudes (isothermal microcalorimetry and xCELLigence System) or the heat flow (isothermal microcalorimetry) of T. muris. The Alamar Blue assay, xCELLigence and microcalorimetry compared favorably to the standard motility assay. These three assays precisely determined the trichuricidal activity of the three test drugs. The acid phosphatase and the MTT assays showed a poorer performance than the motility assay. In conclusion, the colorimetric Alamar Blue in vitro assay is a good alternative to the motility assay to study drug effects against T. muris L4 and adults, since it is easy to perform, precise and of low cost.     

Synthetic Aurones: New Features for Schistosoma mansoni Therapy

In this work, two synthetic aurones revealed moderate schistosomicidal potential in in vitro and in vivo assays. Aurones ( 1 ) and ( 2 ) promoted changes in tegument integrity and motor activity, leading to death of adult Schistosoma mansoni worms in in vitro assays. When administered orally (two doses of 50 mg/kg) in experimentally infected animals, synthetic aurones ( 1 ) and ( 2 ) promoted reductions of 56.20 % and 57.61 % of the parasite load and stimulated the displacement towards the liver of the remaining adult worms. The oogram analysis revealed that the treatment with both aurones interferes with the egg development kinetics in the intestinal tissue. Seeking an action target for compounds ( 1 ) and ( 2 ), the connection with NTPDases enzymes, recognized as important therapeutic targets for S. mansoni, was evaluated. Molecular docking studies have shown promising results. The dataset reveals the anthelmintic character of these compounds, which can be used in the development of new therapies for schistosomiasis.     

Schistosoma mansoni: lack of correlation between praziquantel-induced intra-worm calcium influx and parasite death

The schistosomicidal activity of praziquantel (PZQ) is accompanied by a large influx of calcium into the worms, suggesting that this phenomenon could be the source of the observed muscular contraction, surface disruption and eventual death of the parasite. We have incubated live adult schistosomes in a medium containing radioactive calcium and we were able to confirm that PZQ does indeed stimulate calcium entry into the parasite. An even higher calcium uptake, however, occurred in schistosomes exposed to PZQ after pre-incubation with cytochalasin D, a condition that suppresses PZQ schistosomicidal effects and allows the complete survival of the parasites. The calcium blockers nicardipine and nifedipine also failed to prevent the calcium influx induced by PZQ. Similarly, a large calcium influx occurred in 28-day-old worms exposed to PZQ, in spite of the fact that these immature worms are largely insensitive to the schistosomicidal effects of the drug. Schistosomes incubated overnight with radioactive calcium and PZQ and then returned to normal medium, retained a calcium content higher than worms pre-incubated with cytochalasin D, but the difference could be a consequence—rather than a cause—of schistosomicidal effects. These results suggest that calcium accumulation by itself, at least as measured in whole parasites maintained in vitro, may not represent an exhaustive explanation for the schistosomicidal effects of PZQ.     

Schistosoma mansoni: In vitro schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula

Schistosomiasis is one of the most important parasitic infections in humans that occur in many tropical and subtropical countries. Currently, the control of schistosomiasis rests with a single drug, praziquantel, which is effective against adult worms but not the larval stages. Recent studies have shown that piplartine, an amide isolated from plants of the genus Piper (Piperaceae), reveals interesting antischistosomal properties against Schistosoma mansoni adult worms. Here, we report the in vitro antischistosomal activity of piplartine on S. mansoni schistosomula of different ages (3h old and 1, 3, 5, and 7days old), and examine alterations on the tegumental surface of worms by means of confocal laser scanning microscopy. Piplartine at a concentration of 7.5μM caused the death of all schistosomula within 120h. The lethal effect occurred in a dose-dependent manner and was also dependent on the age of the parasite. Microscopy observation revealed extensive tegumental destruction, including blebbing, granularity, and a shorter body length. This report provides the first evidence that piplartine is able to kill schistosomula of different ages and reinforce that piplartine is a promising compound that could be used for the development of new schistosomicidal agent.     

Fungal transformation and schistosomicidal effects of pimaradienoic Acid

The schistosomicidal effects of pimaradienoic acid (PA) and two derivatives, obtained by fungal transformation in the presence of Aspergillus ochraceus, were investigated. PA was the only compound with antischistosomal activity among the three diterpenes studied, with the ability to significantly reduce the viability of the parasites at concentrations ranging from 25 to 100?μM. PA also promoted morphological alterations of the tegument of Schistosoma mansoni, separated all the worm couples, and affected the production and development of eggs. Moreover, this compound was devoid of toxicity toward human fibroblasts. In a preliminary in vivo experiment, PA at a dose of 100?mg/kg significantly diminished the number of parasites in infected Balb/c mice. Taken together, these results show that PA may be potentially employed in the discovery of novel schistosomicidal agents, and that diterpenes are an important class of natural compounds for the investigation of agents capable of fighting the parasite responsible for human schistosomiasis.     

Sativa seeds against Schistosoma mansoni different stages

The schistosomicidal properties of Nigella sativa seeds were tested in vitro against Schistosoma mansoni miracidia, cercariae, and adult worms. Results indicate its strong biocidal effects against all stages of the parasite and also showed an inhibitory effect on egg-laying of adult female worms. In the present work we also studied the effects of crushed seeds on some antioxidant enzymes; which have a role in protection of adult worms against host oxidant killing; as well as some enzymes of glucose metabolism; which have a crucial role in the survival of adult worms inside their hosts. The data revealed that the used drug induce an oxidative stress against adult worms which indicated by a decrease in the activities of both antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and glutathione reductase and enzymes of glucose metabolism, hexokinase and glucose-6-phosphate dehydrogenase. Disturbing of such enzymes of adult worms using N. sativa seeds could in turn render the parasite vulnerable to damage by the host and may play a role in the antischistosomal potency of the used drug.     

Schistosomicidal Activity of Dihydrobenzofuran Neolignans

We have evaluated the antischistosomal activity of synthetic dihydrobenzofuran neolignans (DBNs) derived from (±)‐trans‐dehydrodicoumaric acid dimethyl ester ( 1 ) and (±)‐trans‐dehydrodiferulic acid dimethyl ester ( 2 ) against adult Schistosoma mansoni worms in vitro. Compound 4 ((±)‐trans‐4‐O‐acetyldehydrodiferulic acid dimethyl ester) displayed the most promising activity; at 200 μm , it kills 100 ± 0% of worms after 24 h, which resembles the result achieved with praziquantel (positive control) at 1.56 μm . The hydrogenation of the double bond between C7′ and C8′, the introduction of an additional methyl group at C3′, and a double bond between C7 and C8 decreased the schistosomicidal activity of DBNs. On the other hand, the presence of the acetoxy group at C4 played an interesting role in this activity. These results demonstrated the interesting schistosomicidal potential of DBNs, which could be further exploited.     

Comparison of novel and existing tools for studying drug sensitivity against the hookworm Ancylostoma ceylanicum in vitro

The motility assay is the current gold standard for evaluating drug effects on hookworm larvae and adults, however, among other drawbacks the assay is time consuming, and prone to individual subjectivity. We evaluated six alternative in vitro assays, namely the feeding inhibition assay, the colourimetric AlamarBlue?, MTT formazan and acid phosphatase activity assays, as well as isothermal calorimetry and the xCELLigence System using Ancylostoma ceylanicum third-stage larvae, stimulated third-stage larvae and adults. The performances of the assays were compared to the motility assay using three standard drugs: albendazole, levamisole and ivermectin (100-1 μg/ml). None of the assays investigated offered an advantage over the motility assay, because they were all inapplicable to third-stage larvae, which were presumably metabolically and physically too inactive. Among all assays tested the xCELLigence System performed best on adult worms as the test was accurate, simple, required a minimal number of worms and offered the possibility for conducting a medium-throughput screening.     

Cardamonin,a schistosomicidal chalcone from Piper aduncum L. (Piperaceae) that inhibits Schistosoma mansoni ATP diphosphohydrolase

Background: Schistosomiasis is one of the world's major public health problems, and praziquantel (PZQ) is the only available drug to treat this neglected disease with an urgent demand for new drugs. Recent studies indicated that extracts from Piper aduncum L. (Piperaceae) are active against adult worms of Schistosoma mansoni, the major etiological agent of human schistosomiasis.Purpose: We investigated the in vitro schistosomicidal activity of cardamonin, a chalcone isolated from the crude extract of P. aduncum. Also, this present work describes, for the first time, the S. mansoni ATP diphosphohydrolase inhibitory activity of cardamonin, as well as, its molecular docking with S. mansoni ATPDase1, in order to investigate its mode of inhibition.Methods: In vitro schistosomicidal assays and confocal laser scanning microscopy were used to evaluate the effects of cardamonin on adult schistosomes. Cell viability was measured by MTT assay, and the S. mansoni ATPase activity was determined spectrophotometrically. Identification of the cardamonin binding site and its interactions on S. mansoni ATPDase1 were made by molecular docking experiments.Results: A bioguided fractionation of the crude extract of P. aduncum was carried out, leading to identification of cardamonin as the active compound, along with pinocembrin and uvangoletin. Cardamonin (25, 50, and 100 µM) caused 100% mortality, tegumental alterations, and reduction of oviposition and motor activity of all adult worms of S. mansoni, without affecting mammalian cells. Confocal laser scanning microscopy showed tegumental morphological alterations and changes on the numbers of tubercles of S. mansoni worms in a dose-dependent manner. Cardamonin also inhibited S. mansoni ATP diphosphohydrolase (IC₅₀ of 23.54 µM). Molecular docking studies revealed that cardamonin interacts with the Nucleotide-Binding of SmATPDase 1. The nature of SmATPDase 1–cardamonin interactions is mainly hydrophobic and hydrogen bonding.Conclusion: This report provides evidence for the in vitro schistosomicidal activity of cardamonin and demonstrated, for the first time, that this chalcone is highly effective in inhibiting S. mansoni ATP diphosphohydrolase, opening the route to further studies of chalcones as prototypes for new S. mansoni ATP diphosphohydrolase inhibitors.     

Anthelmintic Effects of the Essential Oil of Fennel (Foeniculum vulgare Mill., Apiaceae) against Schistosoma mansoni

Foeniculum vulgare Mill . (Apiaceae), known as fennel, is a widespread aromatic herbaceous plant, and its essential oil is used as additive in the food, pharmaceutical, cosmetic, and perfume industries. The in vitro antischistosomal activity and cytotoxic effects against V79 cells of the essential oil of F. vulgare cultivated in southeastern Brazil (FV‐EO) was investigated. The FV‐EO was obtained by hydrodistillation and characterized by GC‐FID and GC/MS analyses. (E)‐Anethole (69.8%) and limonene (22.5%) were identified as the major constituents. Its anthelmintic activity against Schistosoma mansoni was evaluated at concentrations of 10, 50, and 100 μg/ml, and it was found to be active against adult S. mansoni worms, although it was less effective than the positive control praziquantel (PZQ) in terms of separation of the coupled pairs, mortality, and decreased motor activity. However, FV‐EO elicited an interesting dose‐dependent reduction in the number of S. mansoni eggs. On their own, (E)‐anethole and the limonene enantiomers were much less effective than FV‐EO and PZQ. An XTT‐cytotoxicity‐based assay evidenced no FV‐EO cytotoxicity against V79 cells. In summary, FV‐EO displayed moderate in vitro schistosomicidal activity against adult S. mansoni worms, exerted remarkable inhibitory effects on the egg development, and was of low toxicity.     

Brugia malayi: In vitro effects of ivermectin and moxidectin on adults and microfilariae

The effect of ivermectin and moxidectin on the motility of Brugia malayi adults and microfilariae and on the fertility of B. malayi females was examined. Motility was reduced in adults after exposure to both drugs and worms were non-motile and dead within eight days. The motility of microfilariae was significantly reduced at all drug concentrations and ceased at concentrations of 2500 and 5000 μg/mL. The motility of microfilariae released by females was reduced after exposure to both drugs, however ivermectin had a greater effect at concentrations between 170 and 5000 μg/mL. Both drugs reduced the number of microfilariae released by females and within four days their release was inhibited. The presence of the bacterial endosymbiont Wolbachia was examined in adults and microfilariae after exposure to increasing concentrations of ivermectin and moxidectin. A decrease in wsp expression was correlated with increasing drug concentration.     

Lack of correlation between schistosomicidal and anticholinergic properties of hycanthone and related drugs

Visual observation of the motor activity of Schistosoma mansoni kept in vitro showed an increase of activity in the presence of hycanthone (HC). In addition, HC caused a delay in the paralytic effects of carbachol. Similar results were observed in the presence of oxamniquine (OXA). The same pattern of motor activity, however, was shown by HC-resistant worms, by Schistosoma japonicum, and by worms exposed to drug precursors (lucanthone and UK-3883), which are not schistosomicidal in vitro. Other analogs with in vitro killing activity (IA-4 and IA-4 N-oxide) showed minimal anticholinergic effects. The anticholinergic effects of HC and OXA were quickly reversible in vitro and in vivo, whereas their antischistosomal effects are irreversible and delayed. Incubation of schistosomes with high concentrations of carbachol or with anticholinergic drugs failed to compete with the schistosomicidal effects of HC. These results are viewed as contradictory to the hypothesis that HC kills schistosomes by blocking their acetylcholine receptors.