The plant materials were collected from Manipur, North-East India, 920m from sea level, longitude 93°58″ and latitude 24°44″ in March, 2012. The plant was identified by the taxonomist of the institute and had given the accession number as IBSD/Z-42-23. Fresh rhizomes were collected and washed thoroughly with tap water. These were cut into 5–6mm slices and put into the Clevenger type oil extractor. Oil samples were analyzed by GC-FID on a Agilent 5975 C inert XL MSD. The oil was dried over anhydrous sodium sulphate and stored at 4±2°C. The oil was analyzed by GC–MS on a Varian CP-3800 GC coupled to a Varian Saturn 2000 MS/MS. The GC was equipped with a DB-5 fused silica capillary column (30m×0.25mm, with film thickness of 0.25μm) operated using the following conditions: injector temperature, 240°C, column temperature, 60–240°C at 3°C/min, then held at 240°C for 5min; carrier gas, He; injection volume, 1μL (splitless). The MS mass ranged from 40 to 650 m/z, filament delay of 3min, target TIC of 20,000, a prescan ionization time of 100μs, an ion trap temperature of 150°C, manifold temperature of 60°C, and a transfer line temperature of 170°C. The constituents of the oil were identified using retention times, Kovats indices and mass spectra. Confirmed integrated peaks were then used for the percentage of each chemical constituent present in the essential oil. Kovats indices were calculated using the equation: KI(x)=100[(logRT(x)−logPz)/(logRT(Pz+1)−logRT(Pz)], where RT(Pz)≤RT(x)≤RT(Pz+1), and P4, …, P25 are n paraffins.3,4

The anti-proliferative effect of zerumbone was estimated on L. donovani AG83 (MHOM/IN/1983/AG83) as per the guidelines of biosafety committee of West Bengal State University. Promastigotes were transformed from splenic intracellular amastigotes of infected BALB/c mice in complete M199 medium (Invitrogen) supplemented with 1% penicillin–streptomycin (Invitrogen) and 10% FCS (GIBCO) at requisite temperature (22°C). To estimate the percentage of inhibition, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) micro method was used. Briefly, promastigotes cultures were incubated with or without (control) increasing concentrations of zerumbone (0.1–50μM) for 48h in a 96-well flat-bottom plate (200μL per well; BD Falcon) in complete M199 medium. After 48h of incubation at 22°C, MTT (10mg/mL, 10μL per well) was added to each well and the plates were incubated for another 4h at 37°C. The reaction was then stopped with acidic isopropanol (0.4mL 10N HCl in 100mL isopropanol, 100μL per well), and the absorbance was measured at 595nm.5 The 50% inhibitory concentration of zerumbone had been determined from the plot of percent inhibition against increasing concentrations. Cytotoxic effect was also evaluated on PHA (5μg/mL) stimulated murine splenocytes (1×106cells per well) cells without (control) or with increasing concentrations of zerumbone (0.1–50μM).

2.5×106cells/mL exponential phase L. donovani AG83 promastigotes were incubated for 24h and 48h respectively in complete M199 medium in the presence or absence of 50% inhibitory concentration of zerumbone on promastigotes at 22°C. After washing with 1× PBS, the cells were fixed in 45% ethanol (diluted in 1× PBS), treated with 500μg/mL RNAse A and then suspended in 0.5M sodium citrate containing 69μM PI.6 Acquisition was performed using a flow cytometer (BD FACSVerse™, BD Biosciences, USA) and the data were analyzed using Flowing software 2.5.

In order to study the apoptosis inducing capacity of zerumbone in promastigotes, the treated cells were stained with Annexin V-PE and 7-AAD as per manufacturer's instruction (BD Pharmingen). Briefly, 2×106log phase promastigotes were incubated with IC50 concentration of zerumbone in triplicate for 24h and 48h respectively. They were washed twice with cold PBS and resuspended in 1× binding buffer at a concentration of 1×106mL−1. 100μL of the samples was transferred to a fresh tube and Annexin V-PE (5μL), 7-AAD (5μL) were added, incubated for 15min at RT in the dark. 400μL binding buffer was added and cells were acquired in a flow cytometer (BD FACSVerse™, BD Biosciences, USA) and analyzed using Flowing 2.5 version software.5,7

In Leishmania, oxidative stress has been suggested to be responsible for the apoptotic process.5,6 To estimate the level of ROS, the cell permeant probe H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) was used and analyzed by flow cytometer as described previously.5 H2DCFDA is a non-fluorescent dye which is converted into a fluorescent DCF (2′,7′-dichlorofluorescein) in the presence of proper oxidants inside the cells. Promastigotes were treated with IC50 concentration of zerumbone and the induction of ROS had been estimated at 1h, 3h, 5h and 12h by incubating with H2DCFDA (20μM) at room temperature for 20min in dark. H2O2 has been used for positive control.6

The chromatin condensation and lipid accumulation in zerumbone-treated promastigotes were detected under confocal microscope after staining with DAPI (2μg/mL) and Nile Red (10μg/mL) as described earlier.5–8 Images were obtained using an Olympus confocal laser scanning microscope (Model: IX81) and analyzed by Olympus fluoview version 3.0 viewer software.

As elevation of ROS is related with the increase in lipid peroxides, we were keen to check the state of lipid peroxidation in treated promastigotes. L. donovani promastigotes (107) were treated with IC50 concentration of zerumbone for 1h, 3h and 5h respectively. The cell-pellet was dissolved in 2mL of 15% SDS-PBS solution. The fluorescence intensities of the total fluorescent lipid peroxidation products were measured with excitation at 360nm and emission at 430nm and expressed as relative fluorescence units with respect to quinine sulfate (1mg/mL in 0.5M H2SO4).6

Control and treated promastigotes (2×106cells) were fixed with 2.5% gluteraldehyde (Sigma Aldrich), dehydrated in ethanol, critical point-dried in CO2, mounted on stubs, sputtered with a thin gold layer5,6 and observed under a scanning electron microscope (Model: ZEISS EVO-MA 10).

Peritoneal macrophages were isolated from thioglycolate (i.p., 4% (w/v), 1.0mL/mouse) elicited peritoneal lavage of 6–8 weeks old male BALB/c mice as per the guidelines of institutional animal ethics committee of West Bengal State University. Cells were allowed to adhere in 8-chambered slides (105cells per well) in complete RPMI-1640 at 37°C in 5% CO2 environment for 4h. The subsequent steps of washing (3× PBS) were performed to move out the nonadherent cells and granulocytes and then the cultures were continued for another 48h without any manipulation.9,10 Adhered resting macrophages were infected with stationary phase of promastigotes (1:10), incubated for 6h, washed (2× PBS) to remove the uningested promastigotes and cultured overnight in complete RPMI-1640 at 37°C in 5% CO2 environment. Cells were washed (3×) and incubated for additional 48h in the presence or absence of graded concentrations of zerumbone. Prechilled methanol-fixed cells were stained with Giemsa, and examined under phase contrast microscope. At least 400 macrophages were examined for each set. Anti-leishmanial activity was determined by calculating the number of amastigotes per 100 macrophages.9,11

Statistical analyses for all experiments were performed by one-way ANOVA followed by post hoc Holm–Sidak test with the program Sigma Plot.

The yield of essential oil was 0.12%. GC–MS analyses of the essential oil led to the identification of ten major compounds accounting for the 98.4% of the oil. Zerumbone (75.2%), α-caryophyllene (7.1%), camphene (5.1%), eucalyptol (2.4%), and camphor (3.0%) were the major components of the oil were identified in oil samples by Kovat analysis and comparison of mass spectra with those reported in the NIST mass spectra database (Supplementary Fig. 1). Compounds were quantified by performing area percentage calculations based on the total combined FID area. The percentage of a peak is a percentage relative to all other constituents integrated in the FID chromatogram. The differences in chemical composition of essential oil of the present study and previous research may be because of the geographic and climatic factors, chemo types, drying conditions and mode of distillation. Zerumbone was isolated in pure form and its structure was confirmed by 1H NMR, 13C NMR, DEPT, HR-ESIMS and comparison with literature data.

Zerumbone was found to inhibit the growth of Leishmania promastigotes dose dependently, in vitro. At a concentration of 10μM, zerumbone inhibited the growth of L. donovani AG83 promastigotes approximately by 53.43%. Interestingly, the 50% inhibitory concentration of zerumbone (9.36μM) could only inhibit the proliferation of PHA induced murine splenocytes by 5.75% even at 96h (Fig. 1).

Fig. 1.

Anti-proliferative effect of zerumbone against L. donovani-promastigotes and PHA induced murine splenocytes with different concentrations of zerumbone (0.1–50μM) as determined by MTT at 48h and 96h respectively. Each point corresponds to the mean±SD of at least three experiments. Statistical significance was determined by one-way ANOVA followed by Holm–Sidak post hoc test (*p<0.004, **p<0.001, ***p<0.01 vs control).

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The treated promastigotes appeared rounded with loss of flagella with porous cell membrane (Fig. 2B) in comparison to the flagellated and slender promastigotes of the control culture (Fig. 2A).

Fig. 2.

Scanning electron microscopy was performed to determine severe alterations in the morphology of zerumbone treated L. donovani-promastigotes in comparison to control culture. Images are representative profile of at least three independent experiments.

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Another prominent effect resulting from the treatment of the promastigotes with zerumbone was the accumulation of lipid droplets in the cytoplasm (Fig. 3B and C), probably resulting from the accumulation of lipid precursors due to the drastic alteration of the sterol content in the parasite membrane. The alteration in lipid contents as evidenced from the deposition of lipid in the cytosol might also be correlated with plasma membrane integrity, leading to apoptosis.

Fig. 3.

Accumulation of cytosolic lipid in zerumbone-treated L. donovani AG83 promastigotes as evident by nile red staining (magnification: 100×, zoom: 3.4×). (A) Phase contrast; (B) fluorescence; (C) phase contrast–fluorescence merge. Zerumbone treatment significantly increased lipid droplets accumulation (white arrows) in the cytosol in comparison to the untreated cells. Images are representative profile of at least three independent experiments.

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At first, we have shown that the cell cycle progression of promastigotes was arrested at the sub-G0/G1 time dependently. At 24h, the proportion of cells in the sub-G0/G1 was found only 2.9% in comparison to 1.6% of the control culture. Zerumbone further increased sub-G0/G1 cells from 1.6% (untreated) to 9.9% (treated) at 48h accompanied by a decrease in the number of cells in G0/G1 from 59.7% to 50.5% (Fig. 4A).

Fig. 4.

(A) Cell cycle progression in zerumbone treated promastigotes was assessed flow cytometrically by Propidium Iodide staining. Zerumbone induced cell death in promastigotes at 48h by increasing the proportion of sub G0–G1 cells in comparison to control culture. Data are representative of at least three independent experiments. (B) Zerumbone caused the externalization of phosphatidyl serine as estimated by Annexin V and 7-AAD incorporation assay. The proportions of cells at early apoptotic (Annexin V+ 7AAD−) and late apoptotic phase (Annexin V+ 7AAD+) were increased time dependently following zerumbone treatment. Values represent the percentage of positive cells at the respective quadrants. Data are representative of three independent experiments.

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A significant step of apoptosis is the translocation of phosphatidyl serine from the inner to the outer leaflet of the plasma membrane.12 The externalization of phosphatidyl serine residues was observed in 1.39% (early apoptotic; Annexin V only) and in 0.19% (late apoptotic; Annexin V+ 7AAD+) of untreated promastigotes at 48h. After the treatment with zerumbone (IC50 concentration) for 48h, the percentage of early as well as late apoptotic cells was increased significantly with respect to untreated cells. The percentage of Annexin V positive cells (early apoptotic) increased to 15.7% at 48h. The percentage of Annexin V positive 7AAD stained cells (late apoptotic) increased to 7.58% at 48h with respect to the untreated cells (Fig. 4B).

DAPI was used to measure DNA condensation in promastigotes. DAPI staining showed discrete nuclei (blue spots) in untreated promastigotes whereas the treated promastigotes were observed to have blebbed nuclei and condensed chromatin material (Fig. 5B and C).

Fig. 5.

Zerumbone induced DNA condensation as visualized after DAPI staining at 48h. Images were taken using an Olympus fluoview confocal microscope (Model: IX81) and analyzed by Olympus fluoview ver.3.0 viewer software (magnification: 100×, zoom: 3.4×). (A) Phase contrast; (B) fluorescence; (C) phase contrast–fluorescence merge. In untreated promastigotes, the nuclei (K: kinetoplast; N: nucleus) appeared as distinct blue structure, whereas zerumbone treated promastigotes showed blebbed nuclei and condensed chromatin material (white arrow). Images are representative profile of at least three experiments.

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We found that 50% inhibitory concentration of zerumbone against promastigotes could increase the level of ROS time dependently resulting in oxidative damage of the promastigotes (Fig 6A). The mean fluorescence intensity (MFI) of ROS generation in treated promastigotes increased time dependently in comparison to control culture (Fig. 6A).

Fig. 6.

(A) Zerumbone induced ROS production in L. donovani AG83 promastigotes. ROS generation in treated promastigotes has been measured using H2DCFDA at 1, 3, 5 and 12h. Treatment of promastigotes with IC50 concentration of zerumbone revealed an elevation of intracellular ROS time dependently. However, pretreatment of promastigotes with the antioxidant NAC before treatment with zerumbone abrogated ROS generation in each time point. Each point corresponds to the mean±SD of at least three experiments. Statistical significance was determined by one-way ANOVA followed by Holm–Sidak post hoc test (*p<0.004, ***p<0.002, ****p<0.005 vs control; **p<0.001 vs treatment). (B) Zerumbone increased the level of lipid peroxidation time dependently. The total fluorescent lipid peroxidation products was quantified with excitation at 360nm and emission at 430nm and expressed as relative fluorescence units with respect to quinine sulfate (1mg/mL in 0.5M H2SO4) by a spectrofluorometer at 1, 3 and 5h. Each point corresponds to the mean±SD of at least three experiments in duplicate. Statistical significance was determined by one-way ANOVA followed by Holm–Sidak post hoc test (*p<0.04, **p<0.002, ***p<0.005 vs control).

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Zerumbone elevated the level of lipid peroxides in a time dependent manner after 1h of treatment and reached to maximum level at 12h [control vs treatment – 492.14 vs 696.55] (Fig. 6B).

Zerumbone was found effective against intracellular amastigotes and the 50% inhibitory concentration was estimated with the treatment of 5μM of zerumbone at 48h (Fig. 7).

Fig. 7.

Zerumbone inhibited the proliferation of intracellular amastigotes with an IC50 of only 5μM. Thioglycolate-elicited peritoneal macrophages of BALB/c mice were infected in vitro with L. donovani AG83 promastigotes. After established infection, cells were incubated with or without (control) graded concentrations (0.5–10μM) of zerumbone at 37°C in 5% CO2 environment for the determination of anti-leishmanial activity on intracellular amastigotes. Each point corresponds to the mean±SD of at least three experiments in duplicate. Statistical significance was determined by one-way ANOVA followed by Holm–Sidak post hoc test (*p<0.001, **p<0.002 vs infected control).

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