Exploration of antifilarial activity of gold nanoparticle against human and bovine filarial parasites: A nanomedicinal mechanistic approach.

The present work seeks to explore the antifilarial activity of biopolymer functionalized gold nanoparticles (AuNPs) against human filarial parasite (Wuchereria bancrofti) through Nrf2 signaling for the first time. A natural polymer, chitosan is used along with Terminalia chebula extract to synthesize AuNPs following the principles of green chemistry. The probable mode of action of AuNPs as filaricidal agent has been investigated in detail using model filarial parasite, Setaria cervi (bovine parasite). Biopolymers inspired AuNPs exhibit superior antifilarial activity against both human and bovine filarial parasites, and are able to induce oxidative stress and apoptotic cell death in filarial parasites mediated through mitochondria. AuNPs also alter the Nrf2 signaling. In addition, the synthesized nanomaterials appear to be nontoxic to mammalian system. Thus the present mechanistic study, targeting human filarial parasites, has the potential to increase the therapeutic prospects of AuNPs to control lymphatic filariasis in the upcoming days.

Development of chitosan based gold nanomaterial as an efficient antifilarial agent: A mechanistic approach.

The gold nanoparticles (AuNPs) have been synthesized biogenically by using black pepper (Piper nigrum) extract according to the principles of green chemistry in presence and absence of a biopolymer, chitosan. A comprehensive study (up to cellular level) on the antifilarial (against Setaria cervi) activity of AuNPs has been made for the first time with a view to use it clinically. The bioactivity of biopolymer capped biogenic AuNP increases significantly compared to simple biogenic AuNP. The biopolymer plays an important role in inspiring AuNP through its inherent positive charges and hydrophobicity. The developed nanomaterial boosts the production of ROS (reactive oxygen species) and misbalances the antioxidant parameters of parasites such as GSH, GST, GPx, SOD and catalase. The produced ROS ultimately induces oxidative stress, which leads to apoptotic cell death in filarial worms. The synthesized nanomaterials exhibit negligible toxicity towards human PBMCs. The present study may serve as a fruitful platform to explore biopolymer capped gold nanoparticles as efficient antifilarial therapeutics.

Metabolic Inhibitors as Antiparasitic Drugs: Pharmacological, Biochemical and Molecular Perspectives.

BACKGROUND: Human diseases caused by the infectious parasites have been one of the major problems throughout the evolutionary journey. Protozoan and metazoan parasitic infections result in a large number of deaths, disabilities and socio-economic loss worldwide to date. Despite the best efforts for developing suitable antiparasitics, these infections take a massive toll on human health. The prevalence of emerging resistance to the existing drugs, lack of efficacy and toxic side effects are as added complications. Being enlisted under 'neglected' category, serious diseases like leishmaniasis, filariasis, trypanosomiasis etc. have failed to draw attention of the governments as well as the pharmaceutical companies. Thus, target specific as well as cost-convenient therapy needs to be employed for the treatment of these diseases and selective targeting of metabolic pathways appears to be the most promising mean. METHODS: In this context, quality works have been explored for screening either anti-metabolic drugs or selective targets in different groups of parasites. Moreover, complete genome sequencing and metabolomic profiling have provided the initiatives to search for new lethal targets in parasites. RESULTS: New metabolic targets are being reported from different organelles and other sub-cellular compartments of parasites such as mitochondrion, kinetoplast, apicoplast, glycosome, hydrogenosome, acidocalcisome, plasma membrane, cytoskeleton, etc. Herein, unique findings achieved in identifying new antimetabolic drugs or targets and studying their molecular mode of actions have been reviewed by incorporating existing and upcoming approaches. CONCLUSION: Considering the alarming scenario of diseases caused by parasites globally, this paper provides a comprehensive review to the scientific community on the development of novel interventions based on metabolic targets to combat the challenges posed by parasites.

Diospyros perigrena bark extract induced apoptosis in filarial parasite Setaria cervi through generation of reactive oxygen species.

CONTEXT: Lymphatic filariasis is a major neglected tropical disease. Diospyros perigrena Gurke (Ebenaceae) was selected for antifilarial chemotherapy because of unavailability of proper medicine. In India, different parts of this plant were used for the treatment of diabetes, diarrhea, dysentery, cholera, mouth ulcers, and wounds. OBJECTIVE: The present study was undertaken to access antifilarial potential and mechanism of action of n-butanol extract (NBE) of D. perigrena stem bark on Setaria cervi Rudolphi (Onchocercidae). MATERIALS AND METHODS: In vitro efficacy and apoptotic mechanism were evaluated by Hoechst, TUNEL, DNA fragmentation assay, pro- and anti-apoptotic gene expression in NBE (250, 125, 62.5, 31.25, and 15.6 µg/ml)-treated S. cervi after 24 h of incubation. Reactive oxygen species (ROS) up-regulation was also determined by GSH, GST, SOD assays, and super oxide anion level. RESULTS: Significant in vitro antifilarial activity of NBE was found 50% inhibitory concentration (IC50): adult = 57.6 µg/ml, microfilariae (mf) = 56.1 µg/ml, and lethal dose (LD100) in mf is 187.17 µg/ml) after 24 h of treatment. NBF-induced apoptosis was proved by Hoechst, TUNEL, RT-PCR, and Western blot method. NBF (250 µg/ml) decreased the level of GSH (17.8%) and GST (65.4%), increased SOD activity (1.42-fold) and super oxide anion production (1.32-fold) in the treated parasite which culminated into ROS up-regulation. DISCUSSION AND CONCLUSION: NBE induced apoptosis in different life cycle stages of S. cervi. In future, a detailed study of NBF will give us a novel antifilarial compound which will be used for antifilarial chemotherapy.

Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria.

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.

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.

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

BACKGROUND: Lymphatic 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. METHODS: Nocturnal 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. RESULTS: The 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. CONCLUSION: This 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.

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.

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.

Molecular evidence of curcumin-induced apoptosis in the filarial worm Setaria cervi.

Curcumin (diferuloyl methane) is a major curcuminoid from Curcuma longa that exhibits various pharmacological effects and has shown multiple beneficial activities. Our understanding of its anticarcinogenic and other activities occurring through curcumin-induced apoptosis in several cancer cells has greatly expanded in recent years. Lymphatic filariasis is a worldwide health problem causing global disability in humans and is caused by filarial nematodes. Development of efficient strategies to promote programmed cell death in filarial worms remains a key challenge for anti-filarial drug developing research and a crucial unmet medical need. In this study, we have taken molecular and biochemical approaches toward understanding the molecular basis for curcumin-mediated anti-filarial activity in the filarial nematode Setaria cervi. Results of MTT assay showed that curcumin causes a significant reduction in viability of Mf and adults and thus acts as a potent macro- and micro-filaricidal agent. Hoechst staining, TUNEL staining, showed several apoptotic nuclei in different parts of curcumin-treated adults. At 25 µM concentration it showed chromosomal DNA fragmentation in adult worms. Our results indicate that curcumin decreases protein and mRNA expression levels of anti-apoptotic gene ced-9 and enhances both the levels of pro-apoptotic genes ced-3 and ced-4 in a dose-dependent manner. All these observations ascertained the apoptogenicity of curcumin at a minimum concentration of 50 µM in this filarial worm. Furthermore, we showed that curcumin causes depletion of parasitic glutathione level, enhances the activities of glutathione S-transferase and superoxide dismutase and stimulates rapid generation of reactive oxygen species (ROS). Here, we present molecular evidence on curcumin-induced apoptosis in the filarial nematode S. cervi with probable involvement of ROS in a caspase-dependent manner.

Evidence for Wolbachia symbiosis in microfilariae of Wuchereria bancrofti from West Bengal, India.

Wolbachia are symbiotic endobacteria that infect the majority of filarial nematodes, including Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus. Recent studies have suggested that Wolbachia are necessary for the reproduction and survival of filarial nematodes and have highlighted the use of antibiotic therapy such as tetracycline/doxycycline as a novel method of treatment for infections caused by these organisms. Before such therapy is conceived and implemented on a large scale, it is necessary to assess the prevalence of the endosymbiont in W. bancrofti from different geographical locations. We present data from molecular and electron microscopic studies to provide evidence for Wolbachia symbiosis in W. bancrofti microfilariae collected from two districts (Bankura and Birbhum) of West Bengal, India.