In vitro antimicrobial efficacy of Centratherum anthelminticum seeds extracts

The in vitro antimicrobial efficacy of different extracts of seeds of Centratherum anthelminticum was studied by the filter paper disk method against several human pathogenic bacteria and fungi. Some of the extracts showed significant effect over tested bacteria and fungi.     

Potential antifilarial activity of fruit extracts of Ficus racemosa Linn. against Setaria cervi in vitro

Effect of alcoholic and aqueous extracts of the fruits of F. racemosa Linn., on the spontaneous movements of both the whole worm and nerve muscle preparation of Setaria cervi and on the survival of microfilariae in vitro was studied. Alcoholic as well as aqueous extracts caused inhibition of spontaneous motility of whole worm and nerve muscle preparation of Setaria cervi characterized by increase in amplitude and tone of contractions. Initial stimulatory effect was not observed with aqueous extract on whole worm preparation, while effect of alcoholic extract on whole worm and nerve muscle preparation was characterized by an increase in amplitude and tone of contractions followed by paralysis. The concentrations required to inhibit the movement of the whole worm and nerve muscle preparation for alcoholic extract of fruits of F. racemosa were 250 and 50 microg/ml, respectively, whereas aqueous extract caused inhibition of the whole worm and nerve muscle preparation at 350 and 150 microg/ml, respectively, suggesting a cuticular barrier. Both alcoholic and aqueous extracts caused death of microfilariae in vitro. LC50 and LC90 were 21 and 35 ng/ml, respectively for alcoholic, which were 27 and 42 ng/ml for aqueous extracts.     

Structure elucidation by NMR spectroscopy of a new acetylated saponin from Centratherum anthelminticum

A new glycosylated triterpene has been isolated from the seeds of Centratherum anthelminticum, a medicinally important plant. The structural analysis of its acetylated derivative was performed by 1H, 13C NMR, 1H-1H COSY, HMQC, HMBC and DEPT spectroscopy. The saponin was shown to contain hederagenin and six sugar residues forming two glycosyl chains. The complete structure of the saponin was established as 3-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl]-28-O-[beta-D-glucuronopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->3)-beta-D-glucopyranosyl]-hederagenin.     

Histochemical enzyme pattern of microfilaria of Setaria cervi

The present study deals with the histochemical localization of glucose-6-phosphatase, malic dehydrogenase and aldolase in the microfilaria of Setaria cervi. Marked activity of glucose-6-phosphatase was observed in the cephalic cells, excretory and anal pores, G-cells and Innenk?rper. Malic dehydrogenase activity was noted throughout the body (including cuticle) of the microfilaria except for Innenk?rper. Intense aldolase activity was observed in the excretory pore and G-cells only. Muscle cells and anal pore were negative for this enzyme.     

Scanning electron microscopy of Setaria cervi adult male worms

Scanning electron microscopy studies of adult Setaria cervi male worms showed the presence of horn-like lateral appendages and characteristic striated bands on the ventral side of the tail. All other features were indistinguishable from other members of the genus Setaria.     

Albendazole induces apoptosis in adults and microfilariae of Setaria cervi

Setaria cervi, a filarial nematode of cattle, inhabits in the peritoneal cavity and has been used as a suitable model for screening antifilarial agents. Albendazole (ABZ), a tubulin-disrupting benzimidazole (BZ) and a potent microfilaricide binds to β-tubulin, is causing structural impairment of cytoskeleton and worm death. Our present study has revealed that exposure of microfilaria (Mf) and adult to gradually increasing concentration of ABZ leads to a dose-dependent gradual impairment of their motility followed by early death in vitro. We found extreme cellular disturbances in ABZ-treated worms characterized by nucleosomal DNA laddering and chromatin condensation. However, in the treated Mf no nucleosomal DNA laddering was found although presence of TUNEL reactive DNA was evident, thus indicating an apoptotic pathway independent of DNA fragmentation. We present data from molecular studies to provide evidence for ABZ-induced apoptosis in Mf and adult worms of S. cervi.     

Identification and characterization of a selenium-dependent glutathione peroxidase in Setaria cervi

Setaria cervi a bovine filarial parasite secretes selenium glutathione peroxidase during in vitro cultivation. A significant amount of enzyme activity was detected in the somatic extract of different developmental stages of the parasite. Among different stages, microfilariae showed a higher level of selenium glutathione peroxidase activity followed by males then females. However, when the activity was compared in excretory secretory products of these stages males showed higher activity than microfilariae and female worms. The enzyme was purified from female somatic extract using a combination of glutathione agarose and gel filtration chromatography, which migrated as a single band of molecular mass approximately = 20 kDa. Selenium content of purified enzyme was estimated by atomic absorption spectroscopy and found to be 3.5 ng selenium/microg of protein. Further, inhibition of enzyme activity by potassium cyanide suggested the presence of selenium at the active site of enzyme. This is the first report of identification of selenium glutathione peroxidase from any filarial parasite.     

Enzymes of PEP-succinate pathway in Setaria cervi and effect of anthelmintic drugs

Localization of different enzymes of PEP-succinate pathway has been done in Setaria cervi, a bovine filarial worm. Succinate dehydrogenase and fumarate reductase were localized in mitochondria rich particulate fraction while all other enzymes were cytosolic. The in vitro effect of certain antifilarial/anthelmintic agents on these enzymes was also investigated. Sumarmin, at low concentration, could cause a marked inhibition of most of the enzymes of this pathway. Centperazine, an antifilarial drug being developed by CDRI showed significant inhibitory action on pyruvate kinase, lactate dehydrogenase, fumarase and succinate dehydrogenase while CDRI compound 72/70 showed significant inhibition of PEP-carboxykinase activity. Diethylcarbamazine and levamisole, however, were found to be more or less ineffective at lower concentrations against all the enzymes of this pathway.     

Voltage activated calcium channels in somatic muscle of filarial nematode Setaria cervi

Acetylcholine (Ach), levamisole and pyrantel pamoate all cause stimulation of spontaneous rhythmic movements of whole worm and nerve muscle preparation of filarial nematode Setaria cervi. These stimulant effects are manifested only in the presence of available Ca2+ or extracellular Ca2+. Electrical stimulation of nerve muscle preparation of Setaria cervi elicited depolarization and increase in amplitude and tone of contractions. Electrical current stimulates Ca2+ entry leading to depolarization and during the phase of depolarization addition of any of the three stimulants viz. Ach, levamisole or pyrantel pamoate fails to elicit any response on nerve muscle preparation. The findings indicate that electrical stimulation, excitatory neurotransmitter Ach and stimulant anthelmintics levamisole and pyrantel pamoate all produce their stimulant effect by triggering entry of Ca2+ into the muscle cell. Further, blocking the calcium channels by nifedepine and thereby the entry of Ca2+ into the cells blocks the stimulant effect of Ach levamisole and pyrantel pamoate.     

Effect of anthelmintics and phenothiazines on adenosine 5'-triphosphatases of filarial parasite Setaria cervi

S. cervi showed particulate bound Ca2+ ATPase and Na+,K(+)-ATPase activities while Mg2+ ATPase was detected in traces. ATPase of S. cervi was also differentiated from the nonspecific p-nitrophenyl phosphatase activity. Female parasite and microfilariae exhibited higher Ca2+ ATPase and Na+,K(+)-ATPase activities than the male adults and the enzyme Na+,K(+)-ATPase was mainly concentrated in the gastrointestinal tract of the filarial parasite. Na+,K(+)-ATPase of the filariid was ouabain-sensitive while Ca2(+)-ATPase activity was regulated by concentration of Ca2+ ions and inhibited by EGTA. Phenothiazines, viz. trifluoperazine, promethazine and chlorpromazine caused significant inhibition of Ca2+ ATPase and Na+,K(+)-ATPase. Diethylcarbamazine was a potent inhibitor of these ATPases. Mebendazole, levamisole and centperazine also caused significant inhibition of the ATPases indicating this enzyme system as a common target for the action of anthelmintic drugs.     

Setaria cervi: immunoprophylactic potential of glutathione-S-transferase against filarial parasite Brugia malayi

Glutathione-S-transferase (GST) has been detected in the adult female Setaria cervi, a bovine filarial parasite. The role of S. cervi GST antigen in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell mediated reaction as well as in situ inoculation of filarial parasites within a microchamber in Mastomys. The immune sera from glutathione-S-transferase immunized Mastomys promoted the adherence of peritoneal exudate cells to B. malayi microfilariae and infective larvae in vitro inducing 80.7 and 77.6% cytotoxicity, respectively in 72 h. In the microchambers implanted in the immunized Mastomys host cells could migrate and adhere to the microfilariae and infective larvae and induced 77.8 and 75% cytotoxicity to B. malayi microfilariae and infective larvae in 72 h, respectively. These results suggest that native GST from S. cervi is effective in inducing protection against heterologous B. malayi filarial parasite and thus has potential in immunoprophylaxis.     

Purification and biochemical characterization of cytosolic glutathione-S-transferase from filarial worms Setaria cervi

The present study reports the purification and characterization of GST from cytosolic fraction of Setaria cervi. GST activity was determined in various subcellular fractions of bovine filarial worms S. cervi (Bubalus bubalis Linn.) and was found to be localized mainly in the cytosolic and microsomal fractions. The soluble enzyme from S. cervi was purified to homogeneity using a combination of salt precipitation, centrifugation, cation exchange and GSH-Sepharose affinity chromatography followed by ultrafiltration. SDS-PAGE analysis revealed a single band and activity staining was also detected on PAGE gels. Gel filtration and MALDI-TOF studies revealed that the native enzyme is a homodimer with a subunit molecular mass of 24.6 kDa. Comparison of kinetic properties of the parasitic and mammalian enzymes revealed significant differences between them. The substrate specificity and inhibitor profile of cytosolic GST from S. cervi appeared to be different from GST from mammalian sources.     

Partial purification and characterization of acetylcholinesterase isozymes from adult bovine filarial parasite Setaria cervi

Filariasis is a major health problem, affecting millions of people in tropical and sub-tropical regions of the world. The isolation and characterization of parasite-specific enzyme targets is essential for developing effective control measures against filariasis. Acetylcholinesterase (AchE, E.C. 3.1.1.7), an important enzyme of neuromuscular transmission is found in a number of helminths including filarial parasites and may be playing a role in host-parasite interactions. Earlier, we demonstrated the presence of two isozymes of AchE, different from the host enzyme in the human (Brugia malayi) and bovine (Setaria cervi) filarial parasites. In the present study, two isozymes of AchE (pAchE1 and pAchE2) were isolated from S. cervi adults and characterized biochemically and immunochemically. The AchE was partially purified on Con-A Sepharose column and then subjected to preparative polyacrylamide gel electrophoresis (PAGE) for separation of the isozymes. The AchE activity was localized by the staining of gel and the isozymes were isolated from the PAGE strips by electroelution. Both isozymes preferentially utilized acetylcholine iodide as substrate and were strongly inhibited by the true AchE inhibitor (BW284c51), suggesting that they were true AchE. The polyclonal antibodies produced against the isozymes showed significant cross-reactivity with B. malayi AchE, but not against the host enzyme. These findings suggested that both the isozymes were biochemically (in terms of their substrate specificity and inhibitor sensitivity) and immunochemically similar, but different from the host enzyme.     

Setaria cervi: kinetic studies of filarial glutathione synthetase by high performance liquid chromatography

The bovine filarial worm Setaria cervi was found to have abundance of glutathione synthetase (GS; EC 6.3.2.3) activity, the enzyme being involved in catalysing the final step of glutathione (GSH) biosynthesis. A RP-HPLC method involving precolumn derivatization with o-phthalaldehyde has been followed for the estimation of GS activity in crude filarial preparations. Subcellular fractionation of the enzyme was undertaken and it was confirmed to be a soluble protein residing mainly in cytosolic fraction. Attempts to determine the Km value for L-gamma-glutamyl-L-cysteine gave a distinctly nonlinear double-reciprocal plot in which data obtained at relatively high dipeptide concentrations (>1 mM) extrapolate to a Km value of about 400 microM whereas data obtained at lower concentrations (<0.1 mM) extrapolate to a value of about 33 microM. Km was determined to be around 950 and 410 microM for ATP and glycine, respectively. The effect of various amino acids was studied on enzyme activity at 1mM concentration. L-cystine caused a significant enzyme inhibition of 11%. Preincubation with N-ethylmaleimide also resulted in significant inhibition of GS activity.     

Differential neuroprotective activity of two different grape seed extracts

Glutamate excitotoxicity is one of the major events that takes place during various neurotoxic injuries such as brain ischemia. We prepared grape seed extracts, from two different varieties, containing high amounts of polyphenols but little resveratrol. Their neuroprotective effects were investigated using primary culture of neonatal mouse hippocampal neurons treated with an excitotoxic concentration of glutamate. Koshu, a white, local variety of V. vinifera, alleviated the acute inactivation of Erk1/2 and dendrite retraction in cultured hippocampal neurons exposed to a toxic concentration of glutamate (1.0 ng/ml). By contrast, Muscat Bailey A, a red, hybrid variety (Muscat Humburg × Bailey), failed to show any neuroprotective effect. Unlike brain-derived neurotrophic factor and other neuroprotective cytokines, Koshu extract did not induce Akt phosphorylation. Koshu extract also augmented neuron survival rate 24 hours after glutamate toxicity. The comparison of polyphenols between the two samples by liquid chromatography/time-of-flight mass spectrometry demonstrated that Koshu had higher amounts of low molecular weight polyphenols along with several Koshu-specific procyanidin oligomers. These data suggest the presence of high affinity molecular targets for polyphenols in hippocampal neurons, which induce neuroprotective effects in a manner different from BDNF, and the importance of low molecular weight polyphenols and/or procyanidin oligomers for neuroprotection.     

Polyphenol contents and antioxidant activity of soybean seed extracts

The antioxidant activity and contents of various polyphenol classes in the seeds of 20 soybean hybrids were evaluated. Total polyphenols, tannins and proanthocyanidins were determined after extraction of plant material with 70% aqueous acetone. In addition, flavonoid content was determined. Antioxidant activity of seed extracts was evaluated by DPPH free radical scavenging activity assay. A positive linear correlation between antioxidant activity and contents of total phenols, tannins and proanthocyanidins was established. The highest antioxidant activity was observed in the extracts of hybrids which have higher levels of all polyphenol classes examined. The most of the single-cross hybrids were poor in tannins which recommend them as good source for ensiled livestock feed. Results suggested that polyphenol content should be considered as an important feature of the soybean seed.