Senna plant generates reactive oxygen species (ROS) and induces apoptosis in Hymenolepis diminuta.

Like mammalian cells, helminth parasites are equipped with an array of enzymatic anti-oxidant system which has an adaptive strategy to cope up with several conditions of stress that arise from host immune response or drug treatment. Earlier, we had reported that three species of Senna, viz. S. alata, S. alexandrina and S. occidentalis leaf extracts caused severe morphological and biochemical alterations in the zoonotic parasite Hymenolepis diminuta. To understand whether the leaf extracts of the three species of Senna have any effect on the enzymatic anti-oxidant system in H.diminuta or not, the present study was investigated on the mechanism of action of these leaf extracts on the anti-oxidant system of the parasite. The viability of the parasite was assessed by MTT reduction assay, chromatin condensation through Hoechst staining of tissue and DNA fragmentation assay, and the oxidative enzymes of the parasite were estimated biochemically. Activity of superoxide dismutase, catalase, glutathione S- transferase and glutathione peroxidase were found to be increased in all the treated parasites from that of the control, with S. alata showed the highest increased amongst the three plant species in all the enzymes, at 331.0 %, 215.4 %, 85.4 % and 65.5 % respectively. Upliftment of apoptotic protein CED-3, CED-4 and EGL-1 and down regulation of anti-apototic protein CED-9 was visualised in all treated paraites. The redox imbalance triggered by these leaf extracts resulted in the activation of apoptotic pathway that led to death of the parasite. Our results demonstrated that the leaf extracts of the three Senna plant species could open new insight for an affordable natural anthelmintic with high efficacy and less toxicity.

Timing of sub-lethal insecticide exposure determines parasite establishment success in an insect-helminth model.

Environmental toxicants are pervasive in nature, but sub-lethal effects on non-target organisms and their parasites are often overlooked. Particularly, studies on terrestrial hosts and their parasites exposed to agricultural toxicants are lacking. Here, we studied the effect of sequence and timing of sub-lethal exposures of the pyrethroid insecticide alpha-cypermethrin on parasite establishment using the tapeworm Hymenolepis diminuta and its intermediate insect host Tenebrio molitor as a model system. We exposed T. molitor to alpha-cypermethrin (LD20) before and after experimental H. diminuta infection and measured the establishment success of larval tapeworms. Also, we conducted in vitro studies quantifying the direct effect of the insecticide on parasite viability. Our results showed that there was no direct lethal effect of alpha-cypermethrin on H. diminuta cysticercoids at relevant concentrations (LD10 to LD90 of the intermediate host). However, we observed a significantly increased establishment of H. diminuta in beetles exposed to alpha-cypermethrin (LD20) after parasite infection. In contrast, parasite establishment was significantly lower in beetles exposed to the insecticide before parasite infection. Thus, our results indicate that environmental toxicants potentially impact host-parasite interactions in terrestrial systems, but that the outcome is context-dependent by enhancing or reducing parasite establishment depending on timing and sequence of exposure.

Functional Imaging of Neurotransmitters in Hymenolepis diminuta Treated with Senna Plant Through Light and Confocal Microscopy.

Previous studies have shown the anthelmintic efficacy of Senna alata, Senna alexandrina and Senna occidentalis on the zoonotic parasite Hymenolepis diminuta through microscopic studies on morphological structure. The present study is based on the light and confocal microscopic studies to understand if Senna extracts affect neurotransmitter activity of the parasites. A standard concentration (40 mg/mL) of the three leaf extracts and one set of 0.005 mg/mL concentration of the reference drug praziquantel were tested against the parasites, keeping another set of parasites in phosphate buffer saline as a control. Histochemical studies were carried out using acetylthiocholine iodide as the substrate and acetylcholinesterase as the marker enzyme for studying the expression of the neurotransmitter of the parasite and the staining intensity was observed under a light microscope. Immunohistochemical studies were carried out using anti serotonin primary antibody and fluorescence tagged secondary antibody and observed using confocal microscopy. Intensity of the stain decreases in treated parasites compared with the control which implies loss of activity of the neurotransmitters. These observations indicated that Senna have a strong anthelmintic effect on the parasite model and thus pose as a potential anthelmintic therapy.

Coinfection with Hymenolepis nana, Hymenolepis diminuta, Giardia intestinalis, and Human Immunodeficiency Virus: A Case Report with Complex Immunologic Interactions.

AbstractWe describe the case of a 43-year-old human immunodeficiency virus-infected man receiving combined antiretroviral therapy and coinfected with Hymenolepis nana, Hymenolepis diminuta, and Giardia intestinalis, presenting as chronic diarrhea and critical weight loss. Immunological aspects of these interactions are reviewed.

Anthelmintic effects of Oroxylum indicum stem bark extract on juvenile and adult stages of Hymenolepis diminuta (Cestoda), an in vitro and in vivo study.

Worldwide, traditional usage of herbal medicines is a common practice to treat various parasitic infections. In India, bark decoction of Oroxylum indicum (L.) Kurz. (Bignoniaceae) is used as a traditional medicine to cure intestinal-helminthic infections. This study investigated the anthelmintic efficacy of methanolic bark extract of O. indicum on Hymenolepis diminuta (Cestoda), using both in vitro and in vivo methods. Utilizing a mini-questionnaire, first, we collected information about the pattern of anthelmintic use of this plant. Later, in vitro efficacy of extract was tested at 10, 20 and 30 mg/ml on both the artificially excysted juveniles and adult H. diminuta worms. Herein, scanning electron microscopy (SEM) was also utilized to determine the possible effects of extract on tegumental surfaces of juvenile and adult cestode. In vivo, extract was tested at 250, 500 and 1000 mg/kg in H. diminuta albino rat model, against juvenile and adult cestode. Praziquantel (PZQ) served as reference drug in anthelmintic assays. The acute toxicity of extract was determined as per the OECD guidelines. The field questionnaire data revealed that 78 % of people in the area use O. indicum stem bark against intestinal helminths, and of these, 75 % of people also believed it highly efficacious anthelmintic remedy. In vitro testing of extract revealed significant effects on juvenile worms, and 30 mg/ml of extract caused mortality of juveniles at the initial period (0.25 ± 0.00 h). Conversely, PZQ (1 mg/ml) showed paralysis and mortality of juvenile cestodes in 0.44 ± 0.04 and 1.11 ± 0.06 h, respectively. As determined by SEM, in vitro exposure to extract showed substantial effects on both juveniles and adult worms in the form of wrinkled scolex, distorted tegument and eroded microtriches. In vivo study revealed better efficacy of extract against juveniles than adult stages of parasite. Treatment of rats with 1000 mg/kg of extract caused 79.3 % reduction in EPG counts and 70.8 % of reduction in worm counts, which compared well to PZQ efficacy. In acute toxicity study, administration of a single oral dose of 2000 mg/kg of extract to mice did not cause any signs of toxicity or mortality within 2-week observation. Hence, the LD50 of extract was estimated to be greater than 2000 mg/kg. In conclusion, the bark extract of O. indicum possesses concentration-dependent effects against the larval and adult H. diminuta worms, and thus the extract holds promise in the control of intestinal helminthiases. This also supports the common belief of people about the reputation of this plant as anthelmintic in traditional medicine. There is a need to standardize the formulations of this plant for their possible use as standardized herbal anthelmintic agents in the future.

The anthelmintic efficacy of natural plant cysteine proteinases against the rat tapeworm Hymenolepis diminuta in vivo.

Hymenolepis diminuta is a natural parasite of the common brown rat Rattus norvegicus, and provides a convenient model system for the assessment of the anthelmintic activity of novel drugs against cestodes. The experiments described in this paper indicate that treatment of rats infected with H. diminuta with a supernatant extract of papaya latex, containing a mixture of four cysteine proteinases, was moderately efficacious, resulting in a significant, but relatively small, reduction in worm burden and biomass. However, faecal egg output was not affected by treatment. In our experiments these effects were only partially dose-dependent, although specific inhibition by E-64 confirmed the role of cysteine proteinases as the active principles in papaya latex affecting worm growth but not statistically reducing worm burden. Data collected for a further 7 days after treatment indicated that the effects of papaya latex supernatant on worm loss and on worm growth were not enhanced. Our findings provide a starting point for further refinement in formulation and delivery, or assessment of alternative natural plant-derived cysteine proteinases in efforts to develop these naturally occurring enzymes into broad-spectrum anthelmintics, with efficacy against cestodes as well as nematodes.

In vivo anthelmintic activity of Carex baccans and its active principle resveratrol against Hymenolepis diminuta.

Anthelmintic resistance against most of the commercial drugs is a great threat to humans as well as the veterinary live stocks. Hence, new treatment strategies to control helminth infections are essential at this hour. Carex baccans Nees has been traditionally used by Jaintia tribes in Northeast India to get rid of intestinal worm infections. Therefore, the present study was conducted to evaluate in vivo cestocidal activity of root tuber extract of C. baccans and its active component resveratrol against the zoonotic cestode Hymenolepis diminuta in the experimental model rat. The cestocidal activity was determined by monitoring the eggs per gram (EPG) counts in faeces of different treated groups. The result showed that the highest dose of the plant extract (50 mg/kg) and resveratrol (4.564 mg/kg body weight) has significant anthelmintic efficacy against H. diminuta. Crude extract of the plant as well as resveratrol reduced EPG count (56.012 and 46.049 %) and also resulted in decreased worm burden by 44.287 and 31.034 %, respectively. The efficacy of the crude extract and resveratrol can be compared to the reference drug praziquantel. The results exhibits considerable cestocidal potential of root tuber crude extract of C. baccans and resveratrol and justify its folklore use.

Efficacy of condensed tannins against larval Hymenolepis diminuta (Cestoda) in vitro and in the intermediate host Tenebrio molitor (Coleoptera) in vivo.

Natural anti-parasitic compounds in plants such as condensed tannins (CT) have anthelmintic properties against a range of gastrointestinal nematodes, but for other helminths such effects are unexplored. The aim of this study was to assess the effects of CT from three different plant extracts in a model system employing the rat tapeworm, Hymenolepis diminuta, in its intermediate host, Tenebrio molitor. An in vitro study examined infectivity of H. diminuta cysticercoids (excystation success) isolated from infected beetles exposed to different concentrations of CT extracts from pine bark (PB) (Pinus sps), hazelnut pericarp (HN) (Corylus avellana) or white clover flowers (WC) (Trifolium repens), in comparison with the anthelmintic drug praziquantel (positive control). In the in vitro study, praziquantel and CT from all three plant extracts had dose-dependent inhibitory effects on cysticercoid excystation. The HN extract was most effective at inhibiting excystation, followed by PB and WC. An in vivo study was carried out on infected beetles (measured as cysticercoid establishment) fed different doses of PB, HN and praziquantel. There was a highly significant inhibitory effect of HN on cysticercoid development (p=0.0002). Overall, CT showed a promising anti-cestodal effect against the metacestode stage of H. diminuta.

The anthelmintic efficacy of natural plant cysteine proteinases against two rodent cestodes Hymenolepis diminuta and Hymenolepis microstoma in vitro.

Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections. We examined the effects of CPs on two rodent cestodes, Hymenolepis diminuta and H. microstoma in vitro. Our data showed that naturally occurring mixtures of CPs, such as those found in papaya latex, and relatively pure preparations of fruit bromelain, papain and stem bromelain, were active in vitro against both juvenile, artificially excysted scoleces, as well as against adult worms of both rodent cestodes. Significant dose-dependent reduction in motility, ultimately leading to death of the worms, was observed with both species, and against both freshly excysted scoleces and 14-day old pre-adult worms. The most effective was fruit bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=63 and 74 µM, respectively, and for pre-adult worms=199 and 260 µM, respectively). The least effective was stem bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=2855 and 2772 µM, respectively, and for pre-adult worms=1374 and 1332 µM, respectively) and the efficacies of papaya latex supernatant and papain were between these extremes. In all cases these values are higher than those reported previously for efficacy of CPs against intestinal nematodes, and in contrast to nematodes, all CPs were effective against cestodes in the absence of exogenous cysteine in incubation media. The CPs appeared to attack the tegument resulting in generalised erosion mainly on the strobila. The scolex was more resistant to CP attack but nevertheless some damage to the tegument on the scolex was detected.

Cassia alata L: potential role as anthelmintic agent against Hymenolepis diminuta.

The efficacy of Cassia alata against cestode Hymenolepis diminuta was evaluated in our study. Worms were maintained between rat model and beetle. Air-dried leaves of C. alata were subjected to ethanol extraction. Different concentrations (10-80 mg/ml) of the extract were prepared in phosphate buffer saline and tested on the parasite in vitro. Parasites treated with C. alata showed a decrease in motility with an increase in concentrations and complete immobilization took lesser time compared to control. The paralyzed parasites were further processed for electron microscopic studies. Ultrastructural micrographs revealed swelling of the tegument and blebbing on the tegumental surface throughout the body accompanied with destruction of microtriches and changes such as shrinkage in the scolex region. Depletion of parenchyma cells and destruction in the connective tissues along with sparsely cytoplasmic cytons were also observed, and these observations are similar with worms treated with a known drug praziquantel. These results may suggest that the plant leaves could be considered for controlling helminth infection and can represent a step forward in the search for alternative anthelmintic drug.

In vivo anthelmintic activity of Clerodendrum colebrookianum Walp., a traditionally used taenicidal plant in Northeast India.

Although there are several effective drugs available for the treatment of intestinal helminths, the fact remains that they continue to remain out of reach to a vast majority of people in the world, especially in developing countries. On the other hand, there are a great many herbal remedies that are effective against common intestinal worms and are easily available to common people in developing countries. Clerodendrum colebrookianum Walp. (Lamiaceae) is a perennial shrub which is native to South and Southeast Asia. Traditionally, the leaves of this plant are used by the indigenous people of Northeast India as a remedy for the treatment of intestinal tapeworm infections. The aim of this study was to evaluate and authenticate the anthelmintic efficacy of C. colebrookianum leaf extract in experimentally induced Hymenolepis diminuta (a zoonotic tapeworm) infections in Wistar rats. The efficacy of the plant extract was assessed by monitoring the eggs per gram of faeces (EPG) counts and worm recovery rates of experimental animals, following treatment with the leaf extract of this plant at three different doses, i.e. 200, 400 and 800 mg/kg body weight, each given singly for 5 days. The results obtained revealed that the leaf extract of C. colebrookianum possesses a dose-dependent efficacy against the larval, immature and adult stages of H. diminuta. However, the efficacy of the extract was found to be considerably high only against the adult stages of the parasite. For this stage, a single 800-mg/kg dose of extract, given for 5 days, resulted into 68.42% reduction in the EPG counts and 62.50% reduction in the worm counts in the extract-treated group of animals, as compared to the control. The reference drug, praziquantel (5 mg/kg, single dose), however, showed slightly better efficacy and caused 95.16 and 87.00% reductions in the EPG and worm counts of treated animals, respectively. Unlike the adult stages, the efficacy of the plant extract was recorded to be comparatively low against the larval and immature stages of the parasite, as the treatment of animals with the highest dose of extract (800 mg/kg) could cause only 37.50 and 54.00% reductions in worm counts, respectively. The experimental evidence obtained in this study suggests that leaves of C. colebrookianum possess significant anthelminthic properties and supports their use against intestinal tapeworm infections in traditional medicine.

The inability of tapeworm Hymenolepis diminuta and fluke Dicrocoelium dendriticum to metabolize praziquantel.

Biotransformation enzymes can, to a certain extent, protect parasitic worms against the toxic effects of anthelmintics and can contribute to drug-resistance development. The objective of our work was (1) to find and identify phase I and II metabolites of the anthelmintic praziquantel (PZQ) formed by the lancet fluke (Dicrocoelium dendriticum) and the rat tapeworm (Hymenolepis diminuta) and (2) to compare PZQ metabolites in helminths with PZQ biotransformation in rat as host species. Ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, mitochondria-like and microsomes-like fractions (prepared from homogenates of adult worms or from rat liver homogenate) were incubated with 10 and 100 µM PZQ. Liquid/liquid extraction was used for samples during in vitro experiments. In the ex vivo study, living D. dendriticum and H. diminuta adults were incubated in RPMI-1640 medium in the presence of 50 nM or 100 nM PZQ for 24h. After incubation, the worms were removed from the medium and homogenized. Homogenates of worms, medium from the incubation of worms or rat hepatocytes and rat urine (collected during 24h after oral PZQ administration) were separately extracted using solid-phase extraction. The results showed that both D. dendriticum and H. diminuta enzymatic systems are not able to metabolize PZQ. On the other hand, thirty one different phase I and four phase II PZQ metabolites were detected in rat samples using UHPLC/MS/MS analyses. These results show that our experimental helminths, as the members of tapeworm and fluke groups of parasites, are not able to deactivate PZQ, and that the biotransformation enzymes of the studied helminths do not contribute to PZQ-resistance.

Anticestodal activity of Adhatoda vasica extract against Hymenolepis diminuta infections in rats.

AIM OF THIS STUDY: Adhatoda vasica Nees has been commonly used in the indigenous system of medicine of Naga tribes in India for curing intestinal worm infections. In this study the anticestodal efficacy of Adhatoda vasica leaf extract was evaluated using Hymenolepis diminuta-rat experimental model. MATERIALS AND METHODS: The anticestodal efficacy of leaf extract was determined by monitoring the eggs per gram (EPG) of faeces counts and percentage worm recovery rates following treatment with methanol leaf extract of this plant to different groups of rats harbouring immature and mature Hymenolepis diminuta infections. RESULTS: The result indicated 800 mg/kg double dose of extract has profound efficacy against mature worms, where the EPG count was reduced by 79.57% and percentage worm recovery rate by 16.60%. These effects were better than treatment with 5 mg/kg single dose of praziquantel, the standard drug. In case of efficacy against immature worms, the extract showed a significant reduction in worm recovery rate (from 100% in control to 20.00% at 800 mg/kg dose of extract). CONCLUSION: The study shows that the leaf extract of Adhatoda vasica possesses significant anticestodal efficacy and supports its use in the folk medicine.