Abstract
Visceral leishmaniasis (VL) is a life-threatening protozoal infection chiefly impinging the rural and poor population in the tropical and sub-tropical countries. The deadly affliction is rapidly expanding after its association with AIDS, swiftly defying its status of a neglected disease. Despite successful formulation of vaccine against canine leishmaniasis, no licensed vaccine is yet available for human VL, chemotherapy is in appalling state, and the development of new candidate drugs has been painfully slow. In face of lack of proper incentives, immunostimulatory plant preparations owing antileishmanial efficacy bear potential to rejuvenate awful antileishmanial chemotherapy. We have earlier reported profound leishmanicidal activity of Piper nigrum hexane (PNH) seeds and P. nigrum ethanolic (PNE) fractions derived from P. nigrum seeds against Leishmania donovani promastigotes and amastigotes. In the present study, we illustrate that the remarkable anti-promastigote activity exhibited by PNH and PNE is mediated via apoptosis as evidenced by phosphatidylserine externalization, DNA fragmentation, arrest in sub G0/G1 phase, loss of mitochondrial membrane potential and generation of reactive oxygen species. Further, P. nigrum bioactive fractions rendered significant protection to L. donovani infected BALB/c mice in comparison to piperine, a known compound present in Piper species. The substantial therapeutic potential of PNH and PNE was accompanied by elicitation of cell-mediated immune response. The bioactive fractions elevated the secretion of Th1 (INF-γ, TNF-α, and IL-2) cytokines and declined IL-4 and IL-10. PNH and PNE enhanced the production of IgG2a, upregulated the expression of co-stimulatory molecules CD80 and CD86, augmented splenic CD4+ and CD8+ T cell population, induced strong lymphoproliferative and DTH responses and partially stimulated NO production. PNH and PNE were devoid of any hepatic or renal toxicity. These encouraging findings merit further exploration of P. nigrum bioactive fractions as a source of potent and non-toxic antileishmanials.
Highlights
Leishmaniasis comprises poverty associated neglected vectorborne diseases that are responsible for considerable morbidity and mortality all over the world
Major findings emanating from our work are that the remarkable antileishmanial activity exhibited by Piper nigrum hexane (PNH) and (PNE) fractions in vitro was interceded via apoptosis and the in vivo therapeutic potential was accompanied by Th1 stimulation without any potential toxicity
Induction of apoptosis by PNH and P. nigrum ethanolic (PNE) in L. donovani promastigotes was evidenced by PS externalization detected by annexin-propidium iodide (PI) co-staining, DNA fragmentation demonstrated by terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL) assay and cell cycle analysis, depolarization of MMP/ m by JC-1 staining and generation of reactive oxygen species (ROS) by H2DCFDA staining
Summary
Leishmaniasis comprises poverty associated neglected vectorborne diseases that are responsible for considerable morbidity and mortality all over the world. AmB, constitutes second line of drugs and serves as the first choice of treatment in cases where patients become unresponsive to antimonials. More toxic but less common side-effects include hypokalaemia, nephrotoxicity, myocarditis, and even death. All these adverse toxicities coupled with high cost, limit the choice of AmB as a favorable treatment for leishmaniasis despite its tremendous efficacy. Lipid formulations of AmB non-toxic, are expensive which is a crucial drawback as leishmaniasis-inflicted major population is rural and extremely poor. Newer drugs such as miltefosine (oral agent) and paromomycin have been registered for use in India against VL.
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