Browsing by Author "Sambamurthy Chandrasekaran"

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Alcoholic fractions F5 and F6 from Withania somnifera leaves show a potent antileishmanial and immunomodulatory activities to control experimental visceral leishmaniasis
(Frontiers Media S.A., 2017) Sambamurthy Chandrasekaran; Jalaja Veronica; Shyam Sundar; Radheshyam Maurya
Visceral leishmaniasis (VL) causes fatal life-threatening disease, if left untreated. The current drugs have various limitations; hence, natural products from medicinal plants are being focused in search of new drugs to treat leishmaniasis. The aim of the present study was to evaluate the antileishmanial and immunomodulatory activities of F5 and F6 alcoholic fractions from Withania somnifera leaves and purified withaferin-A in Leishmania donovani-infected peritoneal macrophages and BALB/c mice. We observed that F5 (15 μg/mL), F6 (10 μg/mL), and withaferin-A (1.5 μM) reduce amastigote count in peritoneal macrophages and induce reactive oxygen species and significant decrease in IL-10 mRNA expression compared to control upon treatment. Subsequently, in vivo study mice were treated with F5 (25 and 50 mg/kg b.wt.), F6 (25 and 50 mg/kg b.wt.) orally, and withaferin-A (2 mg/kg b.wt.) intraperitoneally for 10 consecutive days and a drastic reduction in parasite burden in both spleen and liver were observed. The treatment resulted in the reduction in IL-10, IL-4, and TGF-β mRNA expression and a significant increase in IFN-γ/IL-10 expression ratio in the treated group compared to control. The humoral response of these alcoholic fractions and withaferin-A shows increased IgG2a levels when compared with IgG1 in treated mice. Taken together, our result concludes that withanolides in alcoholic fractions demonstrate a potent antileishmanial and immunomodulatory activities in experimental VL. © 2017 Chandrasekaran, Veronica, Sundar and Maurya.
An in vitro study of apoptotic like death in Leishmania donovani promastigotes by withanolides
(2013) Sambamurthy Chandrasekaran; Alti Dayakar; Jalaja Veronica; Shyam Sundar; Radheshyam Maurya
The aim of this study was to isolate and evaluate the withanolides in inducing apoptotic like death in Leishmania donovani in vitro. Withanolides were fractionated and isolated from the leaves of Withania somnifera and LC-MS/MS analysis of two fractions namely, F5 and F6 of ethanolic extracts, obtained through column chromatography with silica gel, was performed. The antileishmanial effect of withanolides on L. donovani promastigotes was assessed in vitro using PI dye exclusion test. The effect of withanolides on promastigote morphology was determined by scanning electron microscopy. To understand their mode of action against L. donovani, DNA fragmentation, quantification of parasites at sub G0/G1 phase, determination of phosphatidylserine externalization, measurement of reactive oxygen species (ROS) and mitochondrial membrane potential (Ψm) were done. Results showed that LC-MS/MS analysis confirmed the presence of withanolides in isolated fractions. Treatment with withanolides resulted in morphological alterations from spindle to round shape and loss of flagella/cell integrity in promastigotes. Moreover, it induced DNA nicks, cell cycle arrest at sub G0/G1 phase and externalization of phosphatidylserine in dose and time dependent manner via increase in ROS and decrease in Ψm. Results of this study indicate that withanolides induce apoptotic like death through the production of ROS from mitochondria and disruption of Ψm in promastigotes of L donovani. © 2013 Elsevier Ireland Ltd.
Exploring the inhibitory activity of Withaferin-A against Pteridine reductase-1 of L. donovani
(Taylor and Francis Ltd, 2016) Sambamurthy Chandrasekaran; Jalaja Veronica; Ravi Kumar Gundampati; Shyam Sundar; Radheshyam Maurya
Withaferin A is an abundant withanolide present in Withania somnifera leaves and to some extent in roots. It has been known for its profound anti-cancer properties, but its role in counteracting the Leishmania donovani infection has to be explored. Pteridine reductase 1 (PTR1) is involved in pteridine salvage and an important enzyme for the parasite growth, which could be targeted for the development of an efficient antileishmanial drug. We employed molecular docking studies to identify the binding mode of withaferin A with PTR1 in silico. We further cloned, expressed, and purified PTR1 of L. donovani and performed the enzyme kinetics using the Michaelis–Menten equation and enzyme inhibition studies with withaferin A by plotting the Lineweaver–Burk graph, which followed an uncompetitive mode of inhibition. We also showed the inhibition of the enzyme in the crude lysate of treated parasites. Thus, our study contributes towards understanding the mode of action of withaferin A against L. donovani parasite. © 2015 Informa UK Limited, trading as Taylor & Francis Group.
In silico and in vitro studies: Tryparedoxin Peroxidase inhibitor activity of methotrexate for antileishmanial activity
(2013) Ravi Kumar Gundampati; Shraddha Sahu; Avinash Kumar Srivastava; Sambamurthy Chandrasekaran; Parameswara Rao Vuddanda; Rajesh Kumar Pandey; Radheshyam Maurya; Sanjay Singh; Medicherla V. Jagannadham
In order to understand the mechanism of molecular interactions at the active site of Tryparedoxin Peroxidase (Try P), homology modeling and docking studies were performed. We generated a Three-Dimensional (3D) model of target protein based on the Crystal structure of Leishmania Major Try PI (PDB ID: 3TUE) using modeler software. Docking analysis was carried out to study the effects of methotrexate on Tryparedoxin Peroxidase (Try P). Inhibition of the Tryparedoxin peroxidase interaction has become a new therapeutic strategy in treating leishmaniasis. Docking analysis was carried out to study the effects of methotrexate on Tryparedoxin Peroxidase (TryP). Tryparedoxin peroxidase of Trypanosomatidae family functions as antioxidant through their peroxidase and peroxynitrite reductase activities. The theoretical docking study, conducted on a sample previously reported for anti-cancer properties of Methotrexate at the binding site of 3D models of Tryparedoxin Peroxidase of Leishmania braziliensis (L. braziliensis Try P) examine interaction energy. Our studies indicate that Methotrexate displays potent activity against Try P with lowest binding energy and RMSD values to be -14.5879 Kcal/Mol and 2.0 A. The results of the present study clearly demonstrated the Tryparedoxin Peroxidase inhibitory activity by methotrexate in in silico docking analysis and in vitro assay which contributes towards understanding the mechanism of antileishmanial activity. © 2013 Science Publication.
In vitro and in vivo evaluation of anti-leishmanial and immunomodulatory activity of Neem leaf extract in Leishmania donovani infection
(Academic Press Inc., 2015) Alti Dayakar; Sambamurthy Chandrasekaran; Jalaja Veronica; Shyam Sundar; Radheshyam Maurya
The toxicity and emergence of resistance to available chemical drugs against visceral leishmaniasis is evoking to explore herbal treatment. One such attempt with the Neem is being reported here. The current study is primarily focused to evaluate the anti-leishmanial effects of Neem leaf extracts. Among which, ethyl acetate fraction (EAF) alone was found to exhibit leishmanicidal effect validated through cytotoxicity assay and estimated its IC50 to be 52.4μg/ml on the promastigote stage. Propidium iodide (PI) staining of dead cells substantiated the aforementioned activity. Carboxy fluorescein-diaceate succinimidyl ester (CFSE) staining of promastigotes has affirmed its anti-proliferation activity. The characteristic features such as DNA fragmentation, reduced mitochondrial membrane potential, increased sub G0/G1 phase parasites and increased reactive oxygen species (ROS) production in EAF treated promastigotes indicate the apoptosis like death. In addition, the reduced parasite burden both in vitro (viz.~45% in human monocytic leukemia cell line (THP-1) and ~50% in peripheral blood mononuclear cells) and in vivo (spleen and liver) provides the evidence for its anti-leishmanial activity on amastigote stage. The increase of ROS levels in THP-1 and nitric oxide (NO) production from J774.1 cell line (mouse macrophages) upon EAF treatment was evidenced for oxidative killing of intracellular amastigotes. Active immunomodulatory activity at m-RNA level (viz. upregulation of Th1 cytokines, and downregulation of Th2 cytokines) both in vitro and in vivo was also shown to be exhibited by EAF. Liquid chromatography-tandemmass spectrometry (LC-MS/MS) analysis of EAF revealed the presence of 14 compounds. © 2015 Elsevier Inc.
Iron superoxide dismutase contributes to miltefosine resistance in Leishmania donovani
(Blackwell Publishing Ltd, 2019) Jalaja Veronica; Sambamurthy Chandrasekaran; Alti Dayakar; Moodu Devender; Vijay Kumar Prajapati; Shyam Sundar; Radheshyam Maurya
The emergence of drug-resistant Leishmania is the major challenge to management of visceral leishmaniasis (VL) in areas in which this parasite is endemic. Miltefosine has been widely used against VL, but the emergence of resistant strains could impose a significant threat in the near future. The present study used high-throughput proteomics to determine whether proteins are differentially expressed in miltefosine-resistant (BHU875) and -sensitive (DD8) Leishmania donovani strains. Comparative proteomic analysis revealed up-regulation of iron superoxide dismutase (FeSODA) in the resistant BHU875 strain compared to the drug-sensitive DD8 strain. In accordance with the proteomic data, BHU875 showed higher FeSODA enzymatic activity relative to the sensitive strain. Molecular characterization of BHU875 parasites in which the gene encoding FeSODA was silenced demonstrated that drug sensitivity was restored and the intracellular survival of the parasite was lowered. This suggests that FeSODA activity plays a part in miltefosine resistance. Our study provides a drug target that could be used to overcome miltefosine resistance or help in rational redesigning of miltefosine-based therapy to combat Leishmania infection. © 2019 Federation of European Biochemical Societies
Molecular docking study on the interaction between trypanothione reductase and mangiferin for antileishmanial activity
(2013) Ravi Kumar Gundampati; Sambamurthy Chandrasekaran; Medicherla V. Jagannadham
Mangiferin was found to bind at active site of Leishmania infantum Try R with lowest binding energy and RMSD values to be -9.16 Kcal/Mol and 1.98 respectively. Docking analysis of Try R with ligand enabled us to identify specific residues viz. Phe-203, Glu-202, Asp-218, Pro-336, Try-221 and Phe-270, within the Try R binding pocket to play an important role in ligand binding affinity. The availability of Try R built model, together with insights gained from docking analysis will promote the rational design of potent and selective Try R inhibitor as antileishmanial therapeutic. The study contributes towards understanding mechanism of antileshmanial effect of the mangiferin. We have surveyed the available literature to summarize the inhibition of Try R activity of this natural compound. Thus on the basis of our in silico studies we hypothesize that this compound into mangiferin can be inhibitory effect on against leishmaniasis.