Prasanta Saini

Effect of ferulic acid from Hibiscus mutabilis on filarial parasite Setaria cervi: molecular and biochemical approaches.

In the reported work the in vitro activity of a methanolic extract of leaves of Hibiscus mutabilis (Malvaceae) against bovine Setaria cervi worms has been investigated. Bioassay-guided fractionation led to isolation of ferulic acid from ethyl acetate fraction. The crude extract and ferulic acid, the active molecule, showed significant microfilaricidal as well as macrofilaricidal activities against the microfilaria (L(1)) and adult of S. cervi by both a worm motility and MTT reduction assay. The findings thus provide a new lead for development of a filaricidal drug from natural products. To examine the possible mechanism of action of ferulic acid, the involvement of apoptosis in adult worms of S. cervi was investigated. We found extreme cellular disturbances in ferulic acid-treated adult worms characterized by chromatin condensation, in situ DNA fragmentation and nucleosomal DNA laddering. In this work we are reporting for the first time that ferulic acid exerts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants (GSH, GST and SOD) of the filarial nematode S. cervi. Our results have provided experimental evidence supporting that ferulic acid causes an increased proapoptotic gene expression and decreased expression of anti-apoptotic genes simultaneously with an elevated level of ROS and gradual dose dependent decline of parasitic GSH level. We also observed a gradual dose dependent elevation of GST and SOD activity in the ferulic acid treated worms.

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.

Evidence of reactive oxygen species (ROS) mediated apoptosis in Setaria cervi induced by green silver nanoparticles from Acacia auriculiformis at a very low dose.

Green synthesis of silver nanomaterial plays a pivotal role in the growing field of nanotechnology. Development of anti-parasitic drugs from plant metabolites has been in regular practice from the ancient period but most of them were discarded due to their inefficiency to control diseases effectively. At present, nanoparticles are used for developing anti-parasitic therapy for their unique properties such as smallest in size, bio-ability, bio-compatibility and penetration capacity into a cell. The present study aims at synthesis of silver nanoparticles (AgNPs) by using funicles extract of Acacia auriculiformis and tests its efficacy as antifilarial. Experimental evidence show that AgNPs are effective at a very low concentration compared to crude plant extracts. Synthesis of these nanoparticles is a single-step, biogenic, cost effective and eco-friendly process. Synthesized nanoparticles were characterized by UV-Vis spectroscopy, TEM, SAED, FTIR, EDX, FESEM and Z-potential. The antifilarial efficacy of AgNPs was tested against different life cycle stages of bovine filarial parasite Setaria cervi by morphological study, motility assessment and viability assay. These nanoparticles are found to have antifilarial activity with LC50 of 5.61 μg/mL and LC90 of 15.54 μg/mL against microfilaria of S. cervi. The microscopic findings and the detailed molecular studies confirmed that green synthesized AgNPs were effective enough to induce apoptosis through up regulation of ROS (reactive oxygen species).

Antifilarial effect of ursolic acid from Nyctanthes arbortristis: molecular and biochemical evidences.

A bio-assay guided fractionation and purification approach was used to examine in vitro antifilarial activities of the crude methanolic extract of Nyctanthes arbortristis as well as fractions and isolated compound. From ethyl-acetate fraction we isolated and identified a triterpenoid compound which has been characterized as ursolic acid (UA) by HPLC and NMR data. We are reporting for the first time isolation and identification of UA from the leaves of N. arbortristis. The crude extract and UA showed significant micro- as well as macrofilaricidal activities against the oocyte, microfilaria and adult of Setaria cervi (S. cervi) by dye exclusion test and MTT reduction assay. Significant microfilaricidal activity of UA was further proved against mf of W. bancrofti by viability assay. The findings thus provide a new lead for development of a suitable filaricide from natural products. The molecular mechanism of UA was investigated by performing TUNEL, Hoechst staining, Annexin V-Cy3, flow cytometric analysis and DNA fragmentation assay. Differential expressions of pro- and anti-apoptotic genes were observed at the transcription and translational levels in a dose-dependent manner. Depletion in the worm GSH level and elevation in the parasite GST, SOD and super oxide anion indicated the generation of ROS. In this investigation we are reporting for the first time that UA acts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants like GSH, GST and SOD of S. cervi.

A double-blind controlled field trial of doxycycline and albendazole in combination for the treatment of bancroftian filariasis in India

In a placebo controlled field trial, the effects of doxycycline (200mg/day) for 23 days followed by doxycycline (200mg/day) in combination with albendazole (ABZ) (400mg/day) for 7 days on depletion of Wolbachia endobacteria from Wuchereria bancrofti and microfilaricidal activity were studied in 68 patients (34 males and 34 females) from West Bengal, India. The drugs in combination (i.e., doxycycline+ABZ) provided the best efficacy by totally eliminating the circulating microfilaria (mf) (in 42% cases) on day 365 with (99.8%, P<0.05) suppression even on day 365 post-treatment compared to both exclusive doxycycline (69%, P<0.05) and ABZ (89%, P<0.05) groups. Thus, our results have established that a 30-day course of doxycycline in combination with a 7-day course of ABZ is sufficient to ensure long-term reduction in mf level by depleting Wolbachia from worm tissues. Doxycycline combined with ABZ led to a greater reduction in mf density in blood at 4 months (post-treatment) in comparison to doxycycline or ABZ alone. There were significant differences between the three treatments after 12 months (post-treatment). Further, the impact of a 7-day regimen of ABZ was surprisingly good in reducing mf compared to doxycycline-alone group. Adverse reactions were mild. A 30-day course of doxycycline and ABZ in combination is a safe and well-tolerated treatment for lymphatic filariasis with significant activity against microfilaremia.

Molecular evidence on the occurrence of co-infection with Pichia guilliermondii and Wuchereria bancrofti in two filarial endemic districts of India

BackgroundLymphatic filariasis (LF), a vector-borne parasitic disease, is endemic in several parts of India and mostly affects the poor or those with a low-income. The disease results in huge numbers of morbidities, disabilities, and deaths every year. Association of co-infection with other pathogens makes the condition more severe. Although co-infection is becoming a growing area of research, it is yet to emerge as a frontier research topic in filarial research specifically. This study reports the occurrence of a fungal infection in a large number of patients suffering from bancroftian filariasis in two districts of West Bengal, India.MethodsNocturnal blood samples from filarial patients containing parasites and fungus were initially co-cultured, and further the fungus was isolated and characterized. Molecular identification of the isolate was carried out by PCR-based selective amplification and sequencing of highly-conserved D1/D2 region of 26S rDNA, whereas pathogenicity was determined by amplification of the RPS0 gene. A phylogenetic tree was constructed to study the relationship between the isolate and common pathogenic yeasts. The isolate was studied for antibiotic sensitivity, whereas morphological characterization was performed by microscopic techniques.ResultsThe isolate was identified as Pichia guilliermondii and this fungus was found to exist in co-infection with Wuchereria bancrofti in filarial patients. The fungus showed resistance to azole antifungals, griseofulvin, and, amphotericin B, whereas significant susceptibility was evident in cases of nystatin and cycloheximide. A total of 197 out of 222 patients showed this co-infection.ConclusionThis study revealed, for the first time, that P. guilliermondii exists as a co-infection in microfilaraemic individuals living in a filarial endemic zone. The findings are important and have relevance to human health, especially for filarial patients.

Carbohydrate polymer inspired silver nanoparticles for filaricidal and mosquitocidal activities: A comprehensive view.

The carbohydrate polymer inspired silver nanoparticles (AgNPs) are designed and synthesized through ultrasound assisted green process using unique combination of a biomolecule (tyrosine) and a natural polymer (starch). A comprehensive mechanistic study on the reactive oxygen species (ROS) mediated filaricidal (against Setaria cervi) and mosquitocidal (against second and fourth instar larvae of Culex quinquefasciatus) activities of AgNPs has been made for the first time for controlling filariasis by taking care of both filariid and its vector. The mechanism may help in formulating antifilarial drug based on carbohydrate polymer inspired AgNPs. The role of carbohydrate polymer in inspiring bioactivity of AgNPs has been looked into and its activities have been compared with the commercially available AgNPs. Cytotoxicity of AgNPs on macrophages of Wistar rat has been evaluated to ensure its selectivity towards filariid and larvae.

In vitro antifilarial activity of Azadirachta indica aqueous extract through reactive oxygen species enhancement.

OBJECTIVE To evaluate an aqueous preparation from the Azadirachta indica leaves (AEA) against Setaria cervi (S. cervi), a model filarial parasite. METHOD In vitro efficacy of AEA was evaluated against S. cervi through estimation of relative motility value, dye exclusion test and MTT assay. Visible morphological alterations were monitored using conventional microscopic techniques in microfilariae and haematoxylin-eosin stained sections of AEA-treated adults. RESULTS Enhancement of reactive oxygen species in S. cervi treated with AEA was established through alteration in the activity of glutathione S-transferase, superoxide dismutase, catalase, peroxidase and level of superoxide anion and reduced glutathione. CONCLUSION In vitro filaricidal activity of AEA is possibly through disturbing redox homeostasis by down-regulating and altering the level of some key antioxidants and regulatory enzymes like reduced glutathione, glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxidase of S. cervi.

Phenolics and Terpenoids; the Promising New Search for Anthelmintics: A Critical Review.

Ailments caused by helminth parasites are global causing different types of clinical complications with permanent and long term morbidity in humans. Although huge advances have been made in medical sciences the effectiveness of available anthelmintics are still quite limited. Starting from the 50s, most importance was given to synthetic compounds for developing remedies from them, however, the traditional knowledge of medicine of different countries continued to provide us clues against this widespread health problem. Natural products or structural analogs with diverse structures are always been the major sources for discovering new therapeutics and in recent past different active compounds have also been identified form these plant sources having anthelmintic properties. Although compounds of diverse chemical nature and classes were identified most active ones belong to either phenol or terpene in broad chemical nature. The mechanism of action of these phytotherapeutics is usually multi-targeted and can act against the helminth parasites through diverse spectrum of activities. In this reviewwe summarized the effective anthelmintics belong to either phenolics or terpenoids and highlighted the major way of their effectiveness. This also highlights the recent development of new therapeutic strategies against helminth parasites in the light of recent advances of knowledge. In addition, developing efficient strategies to promote apoptosis and disturbing redox status in them by natural products can provide us a clue in antifilarial drug developmental research and crucial unmet medical need.

An approach toward optimization of the influential growth determinants of opportunistic yeast isolate Pichia guilliermondii

ABSTRACT The present study reports statistical optimization of growth conditions of an opportunistic fungal strain Pichia guilliermondii, isolated from the blood of patients suffering from bancroftian filariasis. Seven key determinants, namely, primary inoculums size (%), volume (mL) and pH of media, serum proportion, temperature (°C), incubation time (hr), and agitation speed (rpm) that influence in vitro growth of the pathogen were optimized statistically using response surface methodology (RSM). RSM with seven factors and two-level Box–Behnken design was employed for designing experimental run, prediction of case statistics, suitable exploration of quadratic response surfaces, and constructing a second-order polynomial equation. Analysis of variance (ANOVA) showed that primary inoculums size, volume of culture media, temperature, incubation time, and agitation speed exert most significant influence over fungal growth. The RSM study predicted that optimum fungal growth can be obtained using 10% primary inoculums size in 100 mL culture media with pH 6.0, 6.28% serum, 32.5°C temperature, and 24 hr of incubation, alongside agitation speed at 400 rpm. The desirability of the optimized growth model for P. guilliermondii is 99.123%, which indicated its accuracy and acceptability. Finally, the optimized growth module illustrated in the study could be useful in improving in vitro growth of clinically important P. guilliermondii.