L. donovani strain AG83 (MHOM/IN/83/AG83) was maintained in vivo in BALB/c mice. The promastigote form of the parasites was cultured in Medium 199 (M199), pH 7.4 supplemented with penicillin G sodium (100 U・ml⁻¹), streptomycin sulfate (100 µg・ml⁻¹), HEPES (25 mM) and 10% heat-inactivated FBS (complete M199). Log phase promastigotes were subcultured every 72 - 96 h, in the same medium at 22˚C, inoculum being 2 × 10⁶ cells・ml⁻¹ [7] .

P. nigrum dried fruits were purchased locally, and authenticated by Dr. H.B. Singh (voucher no. NISCAIR/ RHMD/Consult/-2010-11/1440/38), Taxonomist, NISCAIR, CSIR, New Delhi. The dried fruits were thoroughly washed with water, shade dried and ground to powder form. The powdered plant material (100 g) was sequentially extracted with solvents of increasing polarity, including hexane, ethanol and water by percolation. The powder was initially soaked in hexane (500 ml) and four washes (250 ml each, at 24 h interval) were performed, followed by extraction in ethanol and water in a similar way. Filtrate at each step was passed through Whatman filter paper (No. 1), pooled and then concentrated to dryness under vacuum by rotary evaporation at 35˚C for hexane (PNH) and ethanolic (PNE) extracts and lyophilized to obtain the aqueous (PNA) extract. The dried extracts were stored at −20˚C until used for bioassay.

Growth kinetics assay was performed to appraise the anti-leishmanial efficacy of P. nigrum extracts. Freshly transformed L. donovani promastigotes (2 × 10⁶ cells・ml⁻¹) were incubated at 22˚C in the presence or absence of test extracts (PNH, PNE and PNA) at 500 µg・ml⁻¹. Piperine, one of the known constituents of Piper species and pentamidine, a standard antileishmanial drug, (positive control used for in vitro studies) were also tested at 500 µg・ml⁻¹ for all the experiments. DMSO (0.5%), which was used to solubilize the extracts, was taken as solvent control to determine any unspecific parasite death. The bioactivity of the extracts was assessed by enumerating the viable parasites under phase contrast microscope (40×) after every 24 h for 7 days. Erythrosin B stain (0.2%, 1:1) was used to distinguish live and dead cells. In a parallel set of experiment, untreated and treated parasites were harvested, washed twice with M199 and finally resuspended in fresh complete M199 at 22˚C. After 96 h, the cell viability was determined by counting the motile, viable, flagellated cells under the microscope [8] .

Exponential phase promastigotes (2 × 10⁶ ml⁻¹) were incubated in triplicates with the bioactive extracts, piperine, pentamidine or medium alone at serial three fold dilutions (500 to 2.05 µg・ml⁻¹). After 96 h, parasite survival was assessed at different concentrations of the drugs by enumerating the live promastigotes and percent viability calculated according to the formula:

IC₅₀, the concentration that decreased parasite growth by 50% was determined by graphical extrapolation. In parallel, at 96 h, the parasites were harvested, washed twice (3000 ×g, 10 min, 4˚C) with phosphate buffered saline (PBS, 0.02 M, pH 7.4), fixed in 80% chilled ethanol, stained with erythrosin B and observed under Nikon Eclipse 80i microscope (100× objective) for morphological alterations. Since the cells were fixed prior to staining, erythrosin B was not used to distinguish between live and dead cells but for staining the cells for optimal imaging.

Leishmania infected macrophage cells (RAW 264.7) were used to test intracellular efficacy of the bioactive extracts. Macrophages (5 × 10⁶ ml⁻¹, 100 µl per well) in RPMI-1640 medium were plated on round cover slips in 24-well plates. The cells were allowed to adhere for 24 h in carbon dioxide incubator with 5% CO₂ at 37˚C. Non adherent cells were removed followed by infection with stationary phase promastigotes, maintaining a Leishmania: macrophage ratio of 10:1. After 24 h, the non-phagocytosed promastigotes were washed with serum-free RPMI 1640 and the infected macrophages were incubated with RPMI 1640 (infected control) or with serial four-fold dilutions of PNH, PNE, piperine (200 to 3.12 µg・ml⁻¹) or pentamidine (100 to 0.39 µg・ml⁻¹) in triplicates at 37˚C. After 48 h, the cover slips were washed with PBS, dried, fixed with chilled methanol (analytical grade) and giemsa-stained. At least 200 macrophages per cover slip from triplicate cultures were counted to calculate the percentage of infected macrophages. Percent amastigote infectivity was determined using the formula:

IC₅₀, the concentration of drug that is cytotoxic to 50% of the amastigotes, was obtained by plotting the graph of percent infectivity versus drug concentrations tested [9] . NO was estimated from supernatants of treated and untreated macrophages by Griess reaction [10] .

To assess the cytotoxicity, the macrophages (2 × 10⁶ ml⁻¹) in RPMI 1640 were seeded (200 µl・well⁻¹) and left for adherence. After 24 h, the non-adherent macrophages were removed by washing with serum-free RPMI and the adherent macrophages were incubated with the bioactive extracts at serial two fold dilutions (500 to 7.81 µg・ml⁻¹) in triplicates for 48 h. MTT reagent (5 mg・ml⁻¹, 50 µl per well) was added for a further 4 - 6 h, following which the formazan crystals were dissolved in isopropanol:dimethylsulfoxide (1:1) and the absorbance taken in ELISA plate reader at 570 nm. The absorbance by macrophages in the absence of any treatment was considered to be 100%. CC₅₀, the cytotoxic concentration at which 50% of macrophages are viable was calculated according to the formula:

Selectivity index (SI) as a measure of specificity of the leishmanicidal effect of the extracts against internalized parasites over the host macrophages was determined as the ratio of CC₅₀ to IC₅₀ against intramacrophagic amastigotes.

The secondary metabolites from PNH and PNE were characterized by GC-MS using a Shimadzu QP2010 with a DB-5 column (30 m, film 0.25 mm, ID 0.25 mm). The temperature of the column was increased gradually from 45˚C to 270˚C at 5˚C・min⁻¹, and the injector and detector temperature for the analysis was about 250˚C. Helium was used as the carrier gas at a flow rate of 1.5 ml・min⁻¹. Identification of the chemical constituents was based on correlation of the recorded mass spectra with those obtained from WILEY8.LIB and NIST08.LIB library spectra provided with the software of the GC-MS system [8] .

All experiments were performed at least thrice in triplicate. The results shown are from one of three independent experiments and expressed as mean ± standard error of mean (SEM) of the samples in triplicate. Statistical analysis was performed using Graph-Pad software and statistical significance was calculated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Differences were considered statistically significant at P < 0.05.

PNH was the most potent in inducing parasite death. PNE also exhibited killing of L. donovani promastigotes but was less effectual than PNH. Pentamidine, completely arrested the parasite growth after 72 h of incubation (Figure 1(a)). No cell death was apparent in PNA, parasite control, and 0.5% DMSO (solvent control) treated parasites. Piperine initially induced marginal reduction in the promastigote proliferation rate after which the parasites expanded normally.

The cytocidal (lethal) or cytostatic (growth inhibitory) effect of P. nigrum extracts was confirmed in a growth reversibility assay. Analysis of parasite count after 96 h revealed negligible reversion of growth in PNH treated parasites, indicating that the mode of PNH induced killing was essentially leishmanicidal (Figure 1(b)). However, low parasite count observed in case of PNE treatment reflected its cytostatic effect. High parasite counts were observed in parasites receiving no treatment (parasite control) or upon treatment with 0.5% DMSO (solvent

control) or PNA whereas in pentamidine treated parasites, there was absolutely no reversion in growth of L. donovani promastigotes.

Morphological changes in L. donovani promastigotes in the presence or absence of Piper extracts were examined microscopically (100×) after 96 h. The photomicrographs depicted that the control untreated parasites retained their long flagella, slender and elongated shape, and were highly motile (Figure 2(a)). PNH treated promastigotes however, had an atypical appearance owing to shortening of flagella, cell shrinkage and consequent circularization. Similar observations were found after pentamidine treatment whereas such changes were less pronounced in PNE treated samples. No such morphological alterations occurred in piperine treated promastigotes.

To assess IC₅₀ concentration of the bioactive extracts of P. nigrum, parasites were incubated for 96 h with different concentrations of the extracts (2.05 to 500 µg・ml⁻¹) and control was set without any treatment. PNH and PNE induced dose dependent inhibition of parasite growth with IC₅₀ value corresponding to 31.6 and 37.83 µg・ml⁻¹, respectively (Figure 2(b), Table 1).

The anti-leishmanial activity of PNH and PNE was tested against intracellular amastigotes in L. donovani

SI of bioactive extracts was determined as a measure of their toxicity against RAW 264.7 macrophages. SI = CC₅₀ against macrophages/IC₅₀ against amastigotes, SE = Standard error.

infected RAW 264.7 cells in comparison to pentamidine (100 to 0.39 µg・ml⁻¹), owing to its cytotoxicity. Percent inhibition for each group (PNH, PNE, piperine, and pentamidine) was plotted in a concentration-response curve to calculate the IC₅₀ as elaborated in methods. PNH and PNE exhibited a dose dependent reduction in parasite load with IC₅₀ of 14.63 and 18.83 µg・ml⁻¹, respectively. The IC₅₀ of pentamidine was found to be 0.84 µg・ml⁻¹ and that of piperine was achieved at 41.33 µg・ml⁻¹ (Figure 3, Table 1), which is indicative of its moderate antileishmanial activity.

Since NO is one of the major effector microbicidal molecules responsible for clearance of Leishmania amastigotes, we probed the role of NO in PNH and PNE induced death. We found that our bioactive extracts along with piperine and pentamidine failed to generate NO in L. donovani infected RAW macrophages (P > 0.05, data not shown).

To assess the safety of bioactive extracts of P. nigrum against macrophages, the RAW 264.7 cells were incubated with increasing concentrations of PNH and PNE (0 to 500 µg・ml⁻¹). In order to examine the specificity of these extracts against Leishmania amastigotes over host macrophages, the SI was calculated as a ratio of CC₅₀ against macrophages to IC₅₀ against amastigotes. PNH was found to be most selective with SI of 25.95 (Table 1). PNE exhibited SI = 10.91, whereas piperine was non-toxic against RAW macrophages.

GC-MS profiling of PNH and PNE was performed to identify the leishmanicidal plant secondary metabolites in these bioactive extracts. PNH constituted 52 compounds, the most abundant being trans-β-caryophyllene (22.28%). The other constituents included β-bisabolene (6.08%), tetrahydropiperine (5.66%), 9Z-9-tetra-decen- al (5.32%), α-copaene (4.76%), 3-amino-4-piperonyl-5-pyrazolone (4.14%), 1,1-dimethyldecahydronap-thalene (3.42%), 14-methyl-8-hexadcyn-1-ol (3.04%), δ-cadinol (2.21%), 1-methyldecahydronapthalene (2.75%), α-caryophyllene (2.06%), 1-(12-octadecanoyl) pyrrolidine (2.03%), δ-cadinene (1.81%), α-selinene (1.80%), β-elemene (1.62%) and elemol (1.37%). Piperine content of PNH was observed to be 8.65% (Supplementary

File 1). However, PNE was dominated by piperine (70.36%) though other components such as δ-(sup 9)-cis oleic acid (3.71%), piperoylpiperidine (3.70%), piperyline (3.02%), 3-amino-4-iperonyl-5-pyrazolone (2.13%), tetrahydropiperine (1.64%), trans-β-caryophyllene (1.33%) were also detected (Supplementary File 2).