Induction of apoptosis by zerumbone isolated from Zingiber zerumbet (L.) Smith in protozoan parasite Leishmania donovani due to oxidative stress

In the present context of emergence of resistance aligned with the conventional anti-leishmanial drugs and occasional treatment failure compelled us to continue the search for replaceable therapeutic leads against Leishmaniainfection. Various ginger spices of the Zingiberaceae family are widely used as spices, flavouring agents, and medicines in Southeast Asia because of their unique flavour as well as due to their medicinal properties. Zerumbone, a natural component of Zingiber zerumbet (L.) Smith, has been studied for its pharmacological potential as antiulcer, antioxidant, anticancer, and antimicrobial. In this study, we have shown that zerumbone could induce ROS mediated apoptosis in Leishmania donovani promastigotes and also found effective in reducing intracellular amastigotes in infected-macrophages. We emphasized the potential of zerumbone to be employed in the development of new therapeutic drugs against L. donovaniinfection and provided the basis for future research on the application of transitional medicinal plants.

Keywords
Leishmania donovani; Zingiber zerumbet; Zerumbone; Anti-leishmanial; ROS

Authorship

Debarati Mukherjee

Cellular Immunology and Experimental Therapeutics Laboratory, Department of Zoology, West Bengal State University, Barasat, West Bengal, IndiaWest Bengal State UniversityIndiaBarasat, West Bengal, IndiaCellular Immunology and Experimental Therapeutics Laboratory, Department of Zoology, West Bengal State University, Barasat, West Bengal, India

1 These author contributed equally to this work.

Chingakham Brajakishor Singh

Institute of Bioresource and Sustainable Development, Imphal, Manipur, IndiaInstitute of Bioresource and Sustainable DevelopmentIndiaImphal, Manipur, IndiaInstitute of Bioresource and Sustainable Development, Imphal, Manipur, India

1 These author contributed equally to this work.

Somaditya Dey

2 Present address: Post Graduate Department of Zoology, Barasat Government College, Barasat, West Bengal, India.

Supratim Mandal

3 Present address: PG Department of Microbiology, APC College, New Barrackpore, West Bengal, India.

Joydip Ghosh

Suvadip Mallick

Aabid Hussain

Ningombam Swapana

Department of Chemistry, S. Kula Women's College, Nambol 795134, Manipur, IndiaS. Kula Women's CollegeIndiaNambol 795134, Manipur, IndiaDepartment of Chemistry, S. Kula Women's College, Nambol 795134, Manipur, India

Samir Anis Ross

National Center for Natural Product Chemistry and Department of Bimolecular Sciences, School of Pharmacy, University of Mississippi, Mississippi, USAUniversity of MississippiUSAMississippi, USANational Center for Natural Product Chemistry and Department of Bimolecular Sciences, School of Pharmacy, University of Mississippi, Mississippi, USA

Chiranjib Pal ^*Corresponding author at: Cellular Immunology and Experimental Therapeutics Laboratory, Department of Zoology, West Bengal State University, Barasat, DT: 24 PGS (N), Kolkata 700126, West Bengal, India.cpcu.immunology@gmail.com

Corresponding author at: Cellular Immunology and Experimental Therapeutics Laboratory, Department of Zoology, West Bengal State University, Barasat, DT: 24 PGS (N), Kolkata 700126, West Bengal, India.cpcu.immunology@gmail.com

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (7)

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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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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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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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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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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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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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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).

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).

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.

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.

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.

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.

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).

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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