Santi P. Sinha Babu

Salicylic acid-induced suppression of Meloidogyne incognita infestation of okra and cowpea

Salicylic acid (SA) applied as 10 mM foliar spray to okra (Abelmoschus esculentus) cv. Purbani Kranti and cowpea (Vigna unguiculata) cv. Pusa Ruby plants 24 h before inoculation of roots with Meloidogyne incognita juveniles, reduced infestation. Salicylic acid had no direct influence on plant growth and did not kill nematodes in an in vitro test. It induced increased accumulation of a 14 kDa pathogenesis-related protein (PR-1) in roots of both non-inoculated and inoculated salicylic acid-sprayed okra but not in the treated leaves. Salicylic acid sprays enhanced phenylalanine ammonia lyase (PAL) activity in roots of both non-inoculated and inoculated cowpea, estimated at 15 days after inoculation. Infected roots had higher PAL activity than roots of the corresponding non-inoculated treatments. Both PAL activity and SA-induced resistance gradually declined with plant age after treatment.

TLR2 and TLR4 mediated host immune responses in major infectious diseases: a review

During the course of evolution, multicellular organisms have been orchestrated with an efficient and versatile immune system to counteract diverse group of pathogenic organisms. Pathogen recognition is considered as the most critical step behind eliciting adequate immune response during an infection. Hitherto Toll-like receptors (TLRs), especially the surface ones viz. TLR2 and TLR4 have gained immense importance due to their extreme ability of identifying distinct molecular patterns from invading pathogens. These pattern recognition receptors (PRRs) not only act as innate sensor but also shape and bridge innate and adaptive immune responses. In addition, they also play a pivotal role in regulating the balance between Th1 and Th2 type of response essential for the survivability of the host. In this work, major achievements rather findings made on the typical signalling and immunopathological attributes of TLR2 and TLR4 mediated host response against the major infectious diseases have been reviewed. Infectious diseases like tuberculosis, trypanosomiasis, malaria, and filariasis are still posing myriad threat to mankind. Furthermore, increasing resistance of the causative organisms against available therapeutics is also an emerging problem. Thus, stimulation of host immune response with TLR2 and TLR4 agonist can be the option of choice to treat such diseases in future.

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.

Oxidative stress plays major role in mediating apoptosis in filarial nematode Setaria cervi in the presence of trans-stilbene derivatives

Lymphatic filariasis, affecting around 120 million people in 80 countries worldwide, is an extremely painful disease and caused permanent and long term disability. Owing to its alarming prevalence there is immediate need for development of new therapeutics. A series of trans-stilbene derivatives were synthesized using aqueous reaction condition showing potential as antifilarial agents demonstrated in vitro. MTT reduction assay and dye exclusion test were performed to evaluate the micro and macrofilaricidal potential of these compounds. Amid 20 trans-stilbene derivatives together with Resveratrol (RSV), a multifunctional natural product was screened; nine compounds (28, 29, 33, 35, 36, 38, 39, 41 and 42) have showed promising micro and macrofilaricidal activities and four of them (28, 39, 41 and 42) showed better effectiveness than RSV. In the treated parasites apoptosis was established by DNA laddering, in situ DNA fragmentation and FACS analysis. The generation of ROS in the treated parasites was indicated by the depletion in the level of GSH, GR and GST activity and elevation of SOD, catalase, GPx activity and superoxide anion and H2O2 level. Along with the ROS generation and oxidative stress, the decreased expression of anti-apoptotic ced-9 gene and increased expression of nematode specific pro-apoptotic genes, egl-1, ced-4 and ced-3 at the level of transcription and translation level; the up-regulation of caspase-3 activity and involvement of caspase-8,9,3, cytochrome-c and PARP were also observed and which denotes the probable existence of both extrinsic and intrinsic pathways apoptosis in parasitic nematodes. This observation is reported first time and thus it confirmed the mode of action and effectiveness of the compounds. Further, the comparative bioavailability-pharmacokinetics studies showed that compound 28 possesses comparable properties with Ivermectin. This study will certainly intensify our understanding of the pharmacological importance of trans-stilbenes as an anti-filarial agent.

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.

Design and green synthesis of polymer inspired nanoparticles for the evaluation of their antimicrobial and antifilarial efficiency

Polymer inspired silver nano particles (AgNPs), endowed with high biochemical potency and very low cytotoxicity, were designed and synthesized through a tyrosine controlled green process. In contrary to other conventional techniques, the process was so optimized so that the least amount of reducing and stabilizing agents could generate the maximum possible amount of stable nanoparticles. The role of tyrosine as reducing as well as stabilizing agent was found to be dependent on the level of its concentration. Biocompatible polymers inspired the stability and biological activity of the synthesized nanoparticles to a great extent. The formation of AgNPs was monitored spectroscopically (UV-Vis) both in the presence and absence of biocompatible polymers. Biochemical effects of the polymer stabilized metal nano particles were studied in terms of antiparasitic (antifilarial), antibacterial and antifungal activities. The particles were found to be strongly interactive with the principle cellular bio-molecule (viz. DNA and protein) which might be the reason behind their bioactivity. Particularly against filarial parasites, the synthesized AgNPs were able to induce apoptosis through increasing the level of key regulator of cell death and fragmentation of genomic DNA. Among the biocompatible polymers used, chitosan improved the stability and bio-activity of the composites significantly as evident from the bioactivity and molecular studies like Circular Dichorism (CD) spectroscopic analysis as well.

Evidence of apoptosis in Raillietina echinobothrida induced by methanolic extracts of three traditional medicinal plants of Northeast India

The therapeutic benefits of medicinal plants in terms of anthelmintic properties are known since time immemorial in India, particularly among natives of the Northeast India. However, only sporadic and scarce reports on scientific validation of these plants are available. The present study was conducted on the cestode Raillietina echinobothrida, to establish whether the anthelmintic activity of Potentilla fulgens, Alpinia nigra and Millettia pachycarpa was mediated by apoptosis or not. Light microscopic observation following MTT assay revealed the highest percentage of inhibition of viability among the worms by methanol extract of M. pachycarpa (89.33%), followed by A. nigra (65%) and P. fulgens (37%). Ultrastructural observations revealed swelling of mitochondria, disruption of mitochondrial membrane, vacuolization of mitochondria, appearance of apoptotic bodies in the cytoplasm, disintegration of nuclear membrane and nucleolus were very common throughout the tegument. DAPI stained specimens showed typical morphology of apoptosis, like nuclear condensation and fragmentation in the extracts treated parasites. A decrease in mitochondrial membrane potential was also recorded in the treated groups. Confirmatory TUNEL assay and DNA fragmentation assay of the extracts treated parasites also confirmed the apoptotic nature of cell death and is concluded to be responsible for paralysis and death of the parasite.

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.