Bishnupada Roy

In vitro anthelmintic activity of root-tuber extract of Flemingia vestita, an indigenous plant in Shillong, India

Abstract The in vitro activity of root-tuber-peel extract of Flemingia vestita, an indigenous plant consumed by the natives in Northeast India, was tested against helminth parasites. Live parasites (nematode: Ascaris suum from pigs, A. lumbricoides from humans, Ascaridia galli and Heterakis gallinarum from domestic fowl; cestode: Raillietina echinobothrida from domestic fowl; trematode: Paramphistomum sp. from cattle) were collected in 0.9 % physiological buffered saline (PBS) and maintained at 37 ± 1 °C. In vitro treatment of the parasites with the crude extract (50 mg/ml) in PBS revealed complete immobilization of the trematode and cestode in about 43 and 20 min, respectively. However, the cuticle-covered nematodes did not show any change in physical activity and remained viable even after a long period of exposure to the extract. Exposure of R. echinobothrida to genistein (0.5 mg/ml), an active principle isolated from the root-tuber peel, caused spontaneous loss of movement (paralysis) in 4.5 h, which was slower than the time required for praziquantel, the reference flukicide and cestodicide. The treated parasites showed structural alteration in their tegumental architecture. This study suggests the vermifugal activity of this plant extract against trematodes and cestodes.

Effect of root-tuber extract of Flemingia vestita, a leguminous plant, on Artyfechinostomum sufrartyfex and Fasciolopsis buski: a scanning electron microscopy study.

Abstract The tegumental surface of Artyfechinostomum sufrartyfex as viewed under the scanning electron microscope revealed the presence of double rows of spines in the collar. The dorsal surface (6–8 rows) and the ventral surface are provided with posteriorly directed spines. The normal body surface of Fasciolopsis buski shows posteriorly directed scales throughout the ventral surface; the dorsal surface is free of any scales but has domed, coarsely distributed papillae. When treated in vitro with ethanol root-tuber extract of Flemingia vestita, an indigenous medicinal plant in Meghalaya, India, at a concentration of 5, 10, and 20 mg/ml phosphate-buffered saline (PBS), A. sufrartyfex became paralyzed within 1.1–1.4, 0.8–1.0, and 0.3–0.5 h, respectively. Following similar treatment, F. buski took 3.0–3.6, 1.5–2.0, and 0.6–0.8 h, respectively, to reach a paralytic state. Oxyclozanide B.P. was used as the reference drug and paralyzed the worm, taking slightly less time than the crude extract for both species of flukes. Stereoscanning observations on the tegumental surface of treated (20 mg extract/ml PBS) A. sufrartyfex revealed sloughing off of most of the spines or their deformation as well as wrinkles and rupture of the general tegument. Severe tegumental alterations and deformities were also displayed by F. buski exposed to 20 mg extract/ml PBS.

Effect of Lysimachia ramosa (Primulaceae) on helminth parasites: motility, mortality and scanning electron microscopic observations on surface topography.

The alcoholic extract of Lysimachia ramosa Wall (Primulaceae) was tested in vitro against helminth parasites, Fasciolopsis buski and Ascaris suum, from porcine hosts and Raillietina echinobothrida from domestic fowl. The live adult parasites, collected from a freshly autopsied host, were exposed to different concentrations (5-50mg) of the test plant extract in physiological phosphate-buffered saline (PBS) having 0.1% dimethyl sulphoxide (DMSO) at 37+/-1 degrees C. The treated parasites revealed complete inactivation and flaccid paralysis that was followed by death at varying periods of time. A dose-dependent loss of motility and mortality was observed in all the treated parasites. Scanning electron microscopic observations revealed conspicuous deformity of the surface architecture in all the parasites exposed to the test plant extract. The general tegument in F. buski showed shrinkage and loss of scale-like spines; proglottides all along the strobilar length in R. echinobothrida appeared shrunken and deformed and the cuticular surface of A. suum appeared disorganised, having lost transverse striations. The botanicals of the test plant seem to be effective against all the three types of helminth parasites.

Anthelmintic efficacy of ethanolic shoot extract of Alpinia nigra on tegumental enzymes of Fasciolopsis buski, a giant intestinal parasite

Ethanolic shoot extract of Alpinia nigra, a traditionally used anthelmintic medicinal plant among the Tripuri tribes of north-east India, was tested in vitro to determine its anthelmintic efficacy in gastrointestinal trematode Fasciolopsis buski, using alterations in the activity of vital tegumental enzymes viz. acid phosphatase (AcPase), alkaline phosphatase (AlkPase) and adenosine triphosphatase (ATPase). Live adult F. buski treated in vitro with different concentrations of the plant extract showed significant decline in the visible stain histochemically and enzyme activities. Quantitatively, the total enzyme activity of AcPase, AlkPase and ATPase was found to be reduced by 45, 41 and 43%, respectively compared to the control. The reference drug, praziquantel also showed more or less similar effect like that of the plant extract. The results suggest that the tegumental enzymes of the parasite may be an important target of action for active component(s) of A. nigra, which appears to act transtegumentally.

Ultrastructural observations on tegumental surface of Raillietina echinobothrida and its alterations caused by root-peel extract of Millettia pachycarpa.

Millettia pachycarpa Benth (Leguminosae) has a usage in traditional medicine system practiced among the Lushai tribes of Mizoram, a state in North East India, who customarily consume the aqueous extract of the root peel of the plant to get rid of intestinal worm infections. The crude ethanol, methanol, and acetone fractions of the plant were assayed against Raillietina echinobothrida, the intestinal cestode parasite of domestic fowl, to authenticate the putative anthelmintic efficacy and cestocidal potential in particular of the plants. In vitro exposure of the worm to the extract at a concentration of 25 mg/mL phosphate buffered saline (at 37°C ± 1°C) revealed distortion and disruption of mitochondria, nucleus, nucleolus, nuclear membrane, basal lamina, and tegumental vacuolization in the distal cytoplasm leading to scar formation in the surface. The possible use of the plant as a potential anthelmintic against cestode parasite is discussed. Microsc. Res. Tech., 2008.

Ultrastructural observations on Fasciolopsis buski and its alterations caused by shoot extract of Alpinia nigra

The ultrastructural alterations in the tegument of Fasciolopsis buski in response to incubation in the alcoholic extract of Alpinia nigra were evaluated using transmission electron microscopy. The body tegument of the trematode is composed of an external syncytial layer, musculature, and an inner layer containing tegumental cells. The syncytium comprises various organelles like mitochondria, lysosomes, and tegumentary bodies of the type 2 kind with rare sighting of the type 1. Severe distortion and disorganization of the tegument was revealed in the parasite exposed to the A. nigra extract in the current study. The extent of vacuolization was such that vacuoles proceeded down to the basal lamina causing the syncytium to separate from the tegument at different places. There was depletion of parenchyma material and loss of connecting tubules running down from the syncytium to the tegumental cells causing the cells to be deprived of any proper boundaries. Microsc. Res. Tech., 2009.

Seasonal prevalence of some zoonotic trematode infections in cattle and pigs in the north-east montane zone in India

Seasonal variations in the prevalence of Fasciola gigantica and Eurytrema pancreaticum in cattle, and Opisthorchis noverca, Artyfechinostomum malayanum, Fasciolopsis buski and Gastrodiscoides hominis in pigs, were studied post-necropsy over a 1 year period in a humid, subtropical north-east hilly region in India. The overall prevalence rate was 53.02% in cattle (n = 960) and 12.92% in pigs (n = 960). Fasciola gigantica and E. pancreaticum occurred throughout the year with peaks during cold winter months. Both species showed a high intensity of infection in winter and a low intensity during summer and autumn. The rate of infection due to A. malayanum, Fasciolopsis buski and G. hominis rose to a peak during June-September and thereafter declined to a low level (November-March). Except for the month of February, O. noverca occurred throughout the year, with the highest rate of infection in late autumn and winter. The abundance of infection due to A. malayanum, Fasciolopsis buski and G. hominis was high during late summer and early autumn. Opisthorchis noverca showed a higher density during late autumn and winter.

Molecular characterization of pouched amphistome parasites (Trematoda: Gastrothylacidae) using ribosomal ITS2 sequence and secondary structures.

Members of the family Gastrothylacidae (Trematoda: Digenea: Paramphistomata) are parasitic in ruminants throughout Africa and Asia. In north-east India, five species of pouched amphistomes, namely Fischoederius cobboldi, F. elongatus, Gastrothylax crumenifer, Carmyerius spatiosus and Velasquezotrema tripurensis, belonging to this family have been reported so far. In the present study, the molecular phylogeny of these five gastrothylacid species is derived using the second internal transcribed spacer (ITS2) sequence and secondary structure analyses. ITS2 sequence analysis was carried out to see the occurrence of interspecific variations among the species. Phylogenetic analyses were performed for primary sequence data alone as well as the combined sequence-structure information using neighbour-joining and Bayesian approaches. The sequence analysis revealed that there exist considerable interspecific variations among the various gastrothylacid fluke species. In contrast, the inferred secondary structures for the five species using minimum free energy modelling showed structural identities, in conformity with the core four-helix domain structure that has been recently identified as common to almost all eukaryotic taxa. The phylogenetic tree reconstructed using combined sequence-structure data showed a better resolution, as compared to the one using sequence data alone, with the gastrothylacid species forming a monophyletic group that is well separated from members of the other family, Paramphistomidae, of the amphistomid flukes group. The study provides the molecular characterization based on primary sequence data of the rDNA ITS2 region of the gastrothylacid amphistome flukes. Results also demonstrate the phylogenetic utility of the ITS2 sequence-secondary structure data for inferences at higher taxonomic levels.

Resveratrol induced structural and biochemical alterations in the tegument of Raillietina echinobothrida

The root tuber of Carex species has been used as an anthelmintic medicine for intestinal helminthic infections in the Northeast region of India. The main compound present in the root tuber of the genus Carex is resveratrol. Therefore, the present study was conducted to evaluate the anthelmintic effects of resveratrol in Raillietina echinobothrida by using motility observation, electron microscopy, histochemical and biochemical analysis. Resveratrol causes complete inactivation and flaccid paralysis of the cestode, followed by death. The treated parasites also exhibited extensive distortion of the surface fine topography and decrease in the activities of major tegumental enzymes compared to that of control parasite. Ultrastructural alterations include changes in cell organelles present in the tegument and sub-tegumental cyton. Extensive alterations in the surface topography of the treated parasites resulted in a decrease in the activities of tegumental enzyme which suggest that, resveratrol may be useful as a therapeutic agent to treat cestode parasites.

In vitro anthelmintic efficacy of Carex baccans (Cyperaceae): ultrastructural, histochemical and biochemical alterations in the cestode, Raillietina echinobothrida

The aqueous juice of the root extract of Carex baccans (Family: Cyperaceae) is used as an anthelmintic in Meghalaya, India. The present investigation was carried out to evaluate the extent of ultrastructural, histochemical and biochemical alterations caused by the plant derived component(s) on Raillietina echinobothrida, a cestode parasite of domestic fowl. Live tapeworms, collected from the freshly slaughtered host, were exposed to different concentrations of the crude ethanolic root extract of C. baccans for varying time durations. The treated parasites revealed complete inactivation and flaccid paralysis leading to death; they were processed for ultrastructural, histochemical and biochemical observations, as soon as paralysis set in. Compared to controls, the treated parasites showed extensive distortion and destruction of the surface fine topography of the tegument, erosion of microtriches, disruption of muscle layers, intense vacuolization of tegumental and subtegumental layers, swelling and vacuolization of mitochondria and a significantly reduced activity of tegumental enzymes like AcPase and AlkPase. Phytochemicals from the root of C. baccans seem to be effective against soft-bodied cestode parasites and need to be characterized and identified.