Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo

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.

Cinnamomum cassia exhibits antileishmanial activity against Leishmania donovani infection in vitro and in vivo

“…NISCAIR/RHMD/Consult/-2010-11/1440/38). The extraction was performed as described previously by us [25] with few modifications. In brief, 100 g of powdered bark was immersed in 500 ml of dichloromethane (DCM) for 24 h, followed by four consecutive washes with half the volume of DCM at 24 h interval.…”

“…Pentamidine (a known antileishmanial drug) served as positive control (500 μg ml -1 ) and dimethyl sulphoxide (DMSO, 0.25%) was taken as solvent control (maximum concentration used to solubilize the extracts) to ascertain any unspecific parasite death. Following treatment, culture aliquots from all the groups were taken at every 24 h for 7 days, erythrosin B (0.2%, 1:1) stained, and counted in a haemocytometer under phase contrast microscope (40X) to ascertain the cell density [25]. To obtain photomicrographs, treated and untreated cells were erythrosin B stained and observed under oil immersion at 100 X using a phase contrast microscope.…”

“…The drug was withdrawn by washing the promastigotes twice (3000×g, 10 min, 4°C) with incomplete M199 (without FBS) and all the samples were re-incubated in fresh M199 (complete media, with 10% FBS) at 22°C. Post 96 h of incubation, culture aliquots were taken and cell density determined [25].…”

“…PS externalization as a marker of apoptosis was studied in treated and untreated L . donovani promastigotes as described previously [25]. Briefly, stationary phase promastigotes were adjusted to a cell density of 2×10 6 cells ml -1 and subjected to treatment with IC 50 concentration of CBD or pentamidine (taken at equivalent concentration).…”

“…After confirmation of infection, infected mice (n = 5) were randomly assorted into different groups namely INF; infection control (infected mice that only received PBS), VC; vehicle control (that received 0.25% DMSO in PBS, the highest concentration used to dissolve the test fractions), CBD50 (CBD at 50 mg/kg bw) and CBD100 (CBD at 100 mg/kg bw) in parallel with uninfected control animals (normal group). AmB was used as a positive control in all the in vivo experiments and was reconstituted in sterile milli Q water containing 5% dextrose as described previously [25]. Treatment was carried out for ten days daily, the test fractions were administered orally and AmB (5 mg/kg bw) was administered intravenously.…”