Tegumental Enzymes Research Articles

Evidences of in vivo cytotoxic and apoptotic potential of anthelmintic phytochemical kaempferol derivative isolated from Lysimachia ramosa (Wall. ex Duby) in Wistar rats

BackgroundLysimachia ramosa (Wall. ex Duby) is a traditionally used medicinal plant in Meghalaya, a northeastern state of India. The people use the leaves of the plant to cure gastrointestinal worm infection. Kaempferol derivative, isolated from the leaves of the plant L. ramosa, was found to be the active anthelmintic compound in previous studies. The phytochemical present in the leaves causes alterations in the ultrastructure of the tegument of cestode, its glycogen content and tegumental enzymes. However, it has been found in the review of the literature that phytoproduct should be consumed in a limited dose during longtime treatment period to avoid any potential ill effects on the health of the consumers as many plant-based products are found to be toxic beyond a safe dose limit. Thereby, it becomes necessary to check the potential toxic effects of the active compound taking an animal model. Hence, the present study had been carried out to evaluate the toxicity of the active component on Wistar rats to find out the safe dose of the test compound for oral consumption through observations, if any, on chromosomal abnormalities, ultrastructural changes in bone marrow cells and apoptosis in liver and kidney.ResultsThe result shows kaempferol derivative causes a significant rise in chromosomal abnormalities (2.83 ± 0.19%, 3.83 ± 0.11%) and a significant decline in mitotic indices (3.17 ± 0.02%, 3.10 ± 0.01%) upon treatment with 500 mg and 1000 mg/kg b.w. dosages. At this level, the number of micronucleated erythrocytes increases significantly in the animals (2.33 ± 0.33%, 3.15 ± 0.24%). Intercellular integrity and eosinophil in the bone marrow are observed to be deformed and damaged on treatment with 1000 mg compound/kg b.w. of animals. Apoptotic indices are seen to be increased significantly (3.12 ± 0.18%, 6.17 ± 0.17%) and (2.43 ± 0.13%, 4.17 ± 0.17%) for the liver and kidney, respectively, in the animals of higher-dose treatment groups.ConclusionsThe compound, kaempferol derivative in the present study, has been found to have cytotoxic and pro-apoptotic effects on the test animals beyond a certain dose, if consumed for longer period of time. Therefore, it can be concluded that kaempferol derivative extracted from Lysimachia ramosa should be tested for future trials on animals/humans at dose below 250 mg/kg body weight.

Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis.

Globally, traditional knowledge systems are a powerhouse of information which can revolutionise the world, if decoded accurately and logically. Plant-based ethno-traditional and folklore curatives/medicines has a firm basis in the psyche of the common masses of West Bengal and Holarrhena pubescens is a representative example of it. This article communication on depicting the anthelmintic efficacy of ethanolic extract and Ethyl acetate fraction of the stem bark of Holarrhena pubescens against the cestode Raillietina spp. through efficacy studies, ultra-structural observations, histochemical and biochemical analysis on some tegumental enzymes i.e., Acid Phosphatase (AcPase), Alkaline Phosphatase (AlkPase), Adenosine Triphosphatase (ATPase) and 5'-Nucleotidase (5'-Nu) along with Gray Level Co-occurrence Matrix (GLCM) analysis of histochemical study. Praziquantel was used as the reference drug. Investigations revealed 10mg/ml dosage of crude extract was the most efficacious dose and amongst the fractions the ethyl acetate fraction showed the most anthelmintic property. Ultrastructural studies through Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) clearly depicted the damage in head, sucker, proglottids, proximal and distal cytoplasm (DC), microtriches (MT), basal lamina (BL), nuclear membrane (NM), and, nucleolus (NL) in the treated worms. Histochemical studies revealed decrease in staining intensity for all the tegumental enzymes in the treated worms compared to control. The GLCM analysis strongly supported the result of histochemical studies. Biochemical studies revealed marked reduction in enzyme activity in the treated worms with maximum reduction in the activity of 5'- Nu (77.8%) followed by ATPase (63.17%).

Worms With Armor: Tegumental Adaptations of Stomach Fluke, Dinurussp. (Trematoda: Hemiuridae)—Ultrastructural Characteristics

The stomach is a hostile environment rarely utilized as a habitat by parasites, but stomach flukes are unique helminths that have adapted to survive in this acidic environment. Unlike the flatworms, stomach flukes have a cylindrical body like blood flukes. We hypothesized that, in addition to suckers, stomach flukes are equipped with structures to prevent them from being ripped loose by peristaltic movements and that they have secretory glands to resist the pH of the niche. In the present study, we collected Dinurus sp. (Trematoda: Hemiuridae) from dolphinfish Coryphaena hippurus to understand the tegumental structure and host–parasite interactions. We used ultrastructural and histochemical techniques to investigate the functions of the tegument. The prosoma (proper body) was equipped with densely packed frill‐like tegumental plications with rough and smooth lobes, while the ecsoma (tail) was without plications. Smooth‐lobed prosomal plications had pores (5 μm) that were secretory in function. Secretory granules were delivered from the underlying type‐2 tegumental cells through cytoplasmic connections. Electron‐dense granules coalesced within these cells and were secreted by holocrine method. They were also periodic acid‐Schiff (PAS) and Alcian blue positive. Similarly, PAS positive mucus cells were observed in the gastric pits of the host stomach. Rough‐lobed plications of the flukes enable them to cling onto the mucin. In Dinurus, a glycocalyx forms the outer limit of the tegument. Below the glycocalyx is a stream of cytoplasm devoid of mitochondria, suggesting the tegument is for protection and not for absorption. Under similar conditions, tegumental pores of the ecsoma secreted serine proteinase, which hydrolases gelatin but is incapable of hydrolysis in the presence of serine protease inhibitors. Although additional studies are needed to confirm these initial observations, our study demonstrated for the first time that tegumental plications of stomach fluke Dinurus sp. have mechanical and secretory, not absorptive, functions. Related helminths, blood flukes (schistosomes) are medically important digeneans that cause human diseases. Further, tegumental enzymes in the native form are too scarce for analysis in blood flukes. Unlike blood flukes, Dinurus are relatively large (few mm). Based on the year‐round availability, larger size, and nature of the tegument, we propose Dinurus sp. as an excellent study model for tegumental investigations, such as serine proteinase enzyme analysis.

Alteration in the Tegumental Enzymes of Hymenolepis diminuta by Senna spp

Alteration in the Tegumental Enzymes of Hymenolepis diminuta by Senna spp

An in vitro confirmation of the ethonopharmacological use of Senna plants as anthelmintic against rumen fluke Paramphistomum gracile

BackgroundParamphistomosis is a pathogenic disease of domesticated ruminants, causing great economic loss in dairy industry and meat production. It is considered as a neglected tropical disease with highest prevalence throughout tropical and subtropical regions, particularly in Africa, Asia, Europe, and Australia. There are few trematocidal drugs available in the market. Most are resistant and have elevated side effects. Therefore, alternative trematocidal drugs need to discover. This study was conducted to evaluate three plants leaf extracts (from Senna alata, S. alexandrina, and S. occidentalis) as effective remedies against gastrointestinal trematode parasite (Paramphistomum gracile) of ruminants. Live adult parasites were collected in 0.1 M phosphate-buffered saline (PBS) from fresh autopsied goat’s rumen. Parasites were incubated in leaf extracts of S. alata, S. alexandrina and, S. occidentalis individually and in combination (1:1) ratio at 37 ± 1°C. Treatment media contain extracts at different concentrations (10, 20 and 40 mg/mL) in 10 mL of 0.1 M PBS with 1% dimethylsulphoxide (DMSO). Parasites in control group were incubated in PBS without extract. The efficacy of three Senna extracts was evaluated on the basis of dose-dependent motility and mortality of the trematode. Immediately after paralysis, all treated parasites were collected for histology, SEM and biochemical study.ResultsDose-dependent efficacy was observed in terms of motility and time of mortality in all treated parasites after exposure in various concentrations of the Senna plant extracts. S. occidentalis and S. alexandrina showed better efficacy in combination than comparing with individual treatment groups. Histological study and scanning electron microscopic observations revealed conspicuous deformity of surface architecture in all treated parasites. Scanning electron micrographs also revealed shrinkage, vacuolization, infoldings and blebbings on the body surface of treated worms. Activities of tegumental enzymes were inhibited in all treatment groups compared to control.ConclusionThe overall findings from this study revealed that all three Senna leaf extracts individually and in combination showed potential antitrematocidal activity against Paramphistomum gracile by damaging body tegument and neural propagation. Thus, this study confirmed that all three Senna extracts can be considered as a potential drug-like candidate in indigenous system of traditional medicine against trematode infections in livestock.Graphical abstract

Biochemical and ultrastructural changes in Raillietina echinobothrida in vitro exposed to extract of Lysimachia ramosa.

Lysimachia ramosa (Primulaceae) is a traditionally used medicinal plant, leaves extract of which is being widely used by the Jaintia tribes of Meghalaya, India for controlling helminthiasis. Preliminary investigation carried out on helminth parasites revealed that the crude extract of the plant causes deformity in the surface topography leading to death of the parasites. Therefore, the present study was conducted to identify the specific fraction of the crude leaf extract of the plant responsible for cestocidal efficacy, through biochemical and ultrastructural studies in Raillietina echinobothrida exposed to crude extract and its different fractions namely hexane, chloroform, ethyl acetate and n-butanol. A dose dependent efficacy, with highest rate of mortality among n-butanol exposed parasites was recorded. The treated parasites exhibited complete erosion of microtriches from the tegument, disintegration of muscle bundles, cellular organelles, plasma membrane, nuclear membrane, nucleolus and vacuolization of mitochondria was also observed. Observations on histochemical distribution of some important tegumental enzymes like adenosine triphosphatase (ATPase), alkaline phosphatase (AlkPase), acid phosphatase (AcPase) and 5'Nucleotidase (5'-Nu) revealed a marked diminished stain intensity in the tegument of R. echinobothrida exposed to the crude extract and n-butanol fraction of the crude extract compared to the control. Highest reduction (77.93%) in the activity of ATPase was observed when the parasites exposed to 6mg n-butanol fraction/ml of PBS. The results suggest that these enzymes act as target for anthelmintic stress caused by the phytochemicals present in the plant.

Genistein: is the multifarious botanical a natural anthelmintic too?

Genistein (4',5,7-trihydroxyisoflavone) is naturally present in plants of the soy family and is known to have various pharmacological activities, such as anti-cancer, anti-diabetic, anti-oxidant, etc. The phytoestrogen is one of the major isoflavones found in some medicinal plants having anthelmintic properties. This review describes the putative role of genistein as an anthelmintic, which has been tested on some helminth parasites in vitro. Genistein has been shown to cause paralysis and alterations in the tegument and tegumental enzymes (acid phosphatase, alkaline phosphatase, adenosine triphosphatase, and 5'-nucleotidase) of helminth parasites. Alterations in the activities of several enzymes associated with the coordination system (specifically non-specific esterases, acetylcholine esterase, and nitric oxide synthase), and changes in the concentration of nitric oxide, cGMP, free amino acid pool, and tissue ammonia are observed in helminth parasites treated with genistein. The phytoestrogen also affects the carbohydrate metabolism by altering the activities of key enzymes involved in glycogen- and glucose-metabolism of a cestode parasite. Considering the significance of phosphoenolpyruvate carboxykinase (PEPCK) in glycolysis of the cestode parasite, Ki of the phytoestrogen for PEPCK in the parasite has been determined, and molecular docking of genistein into the active site of the enzyme has also been described. The potential beneficial role of genistein as a natural alternative in management of helminth parasites needs to be further explored, particularly considering its in vivo efficacy and pharmacokinetics.

α-Viniferin-Induced Structural and Functional Alterations in Raillietina echinobothrida, a Poultry Tapeworm.

α-Viniferin, an active component of the plant Carex baccans L., is known for its anticancer, antidiabetic, and anti-inflammatory properties. In Northeast India, different tribes traditionally consume C. baccans to control intestinal helminth infections. Therefore, the present study was carried out to assess the extent of tegumental alteration caused by α-viniferin in Raillietina echinobothrida, a widely prevalent poultry helminth in northeast India. Helminths were exposed in vitro to various doses of α-viniferin (50, 100, and 200 µM/mL of physiological buffered saline) and their motility and mortality were recorded. Stereoscan observations on the parasite exposed to the active compound showed extensive distortion and destruction of the surface fine topography of the tegument compared with controls. The compound also caused extensive damage to the tegument by disintegration of microtriches, disorganization of muscle bundles, and loss of cellular organelles combined with distortion and disruption of the plasma membrane, nuclear membrane, nucleolus, mitochondrial membrane, and cristae. Histochemical and biochemical studies carried out parasites exposed to α-viniferin revealed a decline in the activity of vital tegumental enzymes like acid phosphatase, alkaline phosphatase, and adenosine triphosphatase. Extensive structural and functional alterations observed in the treated parasites are indicative of efficient cestocidal activity of the compound.

Evaluation of anthelmintic activity of ethanolic and aqueous leaf extracts of Azadirachta indica on phosphatases in Gastrothylax indicus

Because of increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against helminth parasite in sheep. The present study was performed to evaluate in vitro anthelmintic activity of plant Azadirachta indica on rumen amphistome of sheep.Anthelmintic activity of ethanolic and aqueous extracts of A. indica leaves in comparison with albendazole ( positive control ) and PBS (negative control) was evaluated through in vitro studies using worm motility inhibition and tegumental enzyme assays. Gastrothylax indicus was exposed in vitro to ethanolic and aqueous extracts(6.25 to 50 mg) of neem leaf to evaluate its anthelmintic efficacy. Mortality was observed at 2.14± 0.28h and 1.71± 0.21h for ethanolic and aqueous extracts respectively for the parasite at the highest test concentration of the plant extract. Anthelmintic albendazole was observed to cause mortality instantly (0.08±0.01h) at 80 μg/ml. Tegumental enzymes of the parasite ALP, ACP, ATPase and G-6-pase were found to be suppressed both by ethanolic and aqueous extracts . Enzyme kinetic studies showed inhibition to be non- competitive in case of ALP with both the extracts whereas for ACP it was found to be non- competitive with ethanolic and competitive with aqueous extract. Albendazole showed competitive inhibition in case of ALP and non-competitive with ACP.

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.

Echinococcus granulosus tegumental enzymes as in vitro markers of pharmacological damage: A biochemical and molecular approach

Cystic echinococcosis is a chronic, complex, and neglected disease. Novel therapeutical tools are needed to optimize human treatment. A number of compounds have been investigated, either using in vitro cultured parasites and/or applying in vivo rodent models. Although some of these compounds showed promising activities in vitro, and to some extent also in the rodent models, they have not been translated into clinical applications. Membrane enzyme activities in culture supernatants of treated protoscoleces with calcium modulator drugs and anthelmintic drugs were measured and provided an indication of compound efficacy. This work describes for the first time the detection of alkaline phosphatase, gamma-glutamyl-transpeptidase and acetylcholinesterase activities in supernatants of in vitro treated Echinococcus granulosus protoscoleces. Marked differences on the enzymatic activities in supernatants from drug treated cultures were detected. We demonstrated that those genes that show the highest degree of conservation when compared to orthologs, are constitutively and highly expressed in protoscoleces and metacestodes. Due to high sensibility and the lack of activity in supernatants of intact protoscoleces, gamma-glutamyl-transpeptidase is proposed as the ideal viability marker during in vitro pharmacological studies against E. granulosus protoscoleces.

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.

Potentilla fulgens (Family Rosaceae), a medicinal plant of north-east India: a natural anthelmintic?

The cestode parasite, Raillietina echinobothrida and the trematode, Gastrothylax crumenifer were exposed to the ethanolic root peel extract of Potentilla fulgens, an antiparasitic local medicinal plant of Meghalaya, India, to evaluate the anthelmintic efficacy of the plant. The parasites were incubated in 1, 5, 10, 20, 50 and 100mg crude alcoholic extract per ml of phosphate buffered saline (PBS) at a temperature of 37±1°C. Paralysis and death were observed at 2.00±0.05 and 2.80±0.06h for the cestode and 1.21±0.06 and 2.18±0.04h for the trematode parasites at the highest test concentration of the plant extract. The commercial anthelmintic, Praziquantel (PZQ) showed higher activity at the tested concentration (0.02mg/ml). To further investigate the efficacy of the plant extract, vital tegumental enzymes of the parasite viz. Acid phosphatase (AcPase), Alkaline phosphatase (AlkPase) and Adenosine triphosphatase (ATPase) were studied. Quantitatively, the total enzyme activity of AcPase, AlkPase and ATPase was found to be reduced significantly by 69.20, 66.43 and 29.63% for R. echinobothrida and 47.96, 51.79 and 42.63% for G. crumenifer, respectively compared to the respective controls; histochemical study also showed reduction in the visible staining of the enzymes. The reference drug, PZQ also showed more or less similar effect like that of the plant extract. The result suggests that phytochemicals of P. fulgens have anthelmintic potential.

Antiparasitic activity of methanolic extract of Acacia oxyphylla (Leguminosae) against Raillietina echinobothrida

Acacia oxyphylla has been used traditionally by the natives of Mizoram against intestinal worm infections. In the present study, the crude methanolic extract of the plant was tested in vitro on the cestode parasite Raillietina echinobothrida to evaluate its potential anthelmintic efficacy. The test parasites were exposed to varying concentrations of the plant extract in phosphate buffered saline and they all slipped into a flaccid paralytic state at varying periods of time post incubation. Ultrastructural observations on the paralysed worms revealed wide scale destruction of the parasite tegument with intense vacuolization of the syncytium and swellings of the basal lamina accompanied by deformities in the cell organelles. To determine the exact mode of action of the botanicals on the parasite body surface, the tegumental enzymes viz. acid phosphatase (AcPase), alkaline phosphatase (AlkPase) and adenosine triphosphatase (ATPase) were investigated. A prominent decrease in the phosphatase activity, in comparison to the untreated control parasites was noticeable. In view of the alterations in the structural and functional integrity of the tegument, occurring in the treated parasites, it may be assumed that the changes transpire because of a compromise in the permeability of the tegument under the influence of the test plant-derived active chemical principles.

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.

Effects of phytochemicals of Flemingia vestita (Fabaceae) on glucose 6-phosphate dehydrogenase and enzymes of gluconeogenesis in a cestode ( Raillietina echinobothrida)

The crude root-peel extract of Flemingia vestita, containing genistein as the major isoflavone, has a vermifugal/vermicidal effect. It acts by causing flaccid paralysis accompanied by alterations in the activities of several tegumental enzymes and other metabolic activities in the fowl tapeworm, Raillietina echinobothrida. To elucidate the mode of action of the putative phytochemicals on energy metabolism, crude root-peel extract, pure genistein and praziquantel were tested on glucose 6-phosphate dehydrogenase (G6PDH) and enzymes of gluconeogenesis—pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and fructose 1,6-bisphosphatase (FBPase)—in R. echinobothrida. The activities of G6PDH, PEPCK and FBPase were largely restricted to the cytosolic fraction, while PC was confined to the mitochondrial fraction. Following treatments, the G6PDH activity was decreased by 23–31%, whereas the activities of PC and PEPCK were increased by 32–44% and 44–49%, respectively. There was no significant effect by any of the treatments on FBPase activity. We hypothesize that the phytochemicals from F. vestita, genistein in particular, influence the key enzymes of these pathways, which is perhaps a function of high energy demand of the parasite under anthelmintic stress.

Anthelmintic efficacy of Flemingia vestita (Leguminoceae): Genistein-induced alterations in the activity of tegumental enzymes in the cestode, Raillietina echinobothrida

The crude root-tuber peel extract of Flemingia vestita, an indigenous leguminous plant of Meghalaya, and its active chemical component, i.e. genistein, have shown paralyzing effects on soft-bodied helminth parasites of trematode and cestode groups. With a view to investigating the mode of action of the plant-derived component, its effect on the activity of tegumental enzymes of the parasite was studied. Acid phosphatase (AcPase), alkaline phosphatase (AlkPase), adenosine triphosphatase (ATPase) and 5′-nucleotidase (5′-Nu) are predominantly distributed in the tegument, subtegument, and somatic musculature. After exposure to the crude extract (50 mg/ml of the incubation medium) or genistein (0.5 mg/ml), a pronounced decline in the visible stain intensity was noticeable indicating very little or no activity in these sites. Quantitatively the activity of AcPase, AlkPase, ATPase and 5′-Nu was found to be suppressed by 97, 95, 88, and 57%, respectively, following genistein treatment. The reference drug, praziquantel (0.01 mg/ml) also caused a reduction in the enzymatic activities, somewhat at par with the genistein treatment. The results suggest that the tegumental enzymes of the parasite may be an important target of action for genistein, which appears to act transtegumentally.