Induction of apoptosis by zerumbone isolated from Zingiber zerumbet (L.) Smith in protozoan parasite Leishmania donovani due to oxidative stress | The Brazilian Journal of Infectious Diseases

array:24 ["pii" => "S1413867015002032" "issn" => "14138670" "doi" => "10.1016/j.bjid.2015.10.002" "estado" => "S300" "fechaPublicacion" => "2016-01-01" "aid" => "527" "copyright" => "Elsevier Editora Ltda.. All rights reserved" "copyrightAnyo" => "2015" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Braz J Infect Dis. 2016;20:48-55" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 2373 "formatos" => array:3 [ "EPUB" => 207 "HTML" => 1482 "PDF" => 684 ] ] "itemSiguiente" => array:19 [ "pii" => "S141386701500210X" "issn" => "14138670" "doi" => "10.1016/j.bjid.2015.10.005" "estado" => "S300" "fechaPublicacion" => "2016-01-01" "aid" => "531" "copyright" => "Elsevier Editora Ltda." "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Braz J Infect Dis. 2016;20:56-60" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 1902 "formatos" => array:3 [ "EPUB" => 271 "HTML" => 995 "PDF" => 636 ] ] "en" => array:11 [ "idiomaDefecto" => true "cabecera" => "Original Article" "titulo" => "Clinical and bacteriological characteristics of invasive pneumococcal disease after pneumococcal 10-valent conjugate vaccine implementation in Salvador, Brazil" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "56" "paginaFinal" => "60" ] ] "contieneResumen" => array:1 [ "en" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Carolina Regis Leite, Jailton Azevedo, Vivian Santos Galvão, Otávio Moreno-Carvalho, Joice Neves Reis, Cristiana Nascimento-Carvalho" "autores" => array:6 [ 0 => array:2 [ "nombre" => "Carolina Regis" "apellidos" => "Leite" ] 1 => array:2 [ "nombre" => "Jailton" "apellidos" => "Azevedo" ] 2 => array:2 [ "nombre" => "Vivian Santos" "apellidos" => "Galvão" ] 3 => array:2 [ "nombre" => "Otávio" "apellidos" => "Moreno-Carvalho" ] 4 => array:2 [ "nombre" => "Joice Neves" "apellidos" => "Reis" ] 5 => array:2 [ "nombre" => "Cristiana" "apellidos" => "Nascimento-Carvalho" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S141386701500210X?idApp=UINPBA00003Y" "url" => "/14138670/0000002000000001/v1_201601240048/S141386701500210X/v1_201601240048/en/main.assets" ] "itemAnterior" => array:19 [ "pii" => "S1413867015002019" "issn" => "14138670" "doi" => "10.1016/j.bjid.2015.09.011" "estado" => "S300" "fechaPublicacion" => "2016-01-01" "aid" => "525" "copyright" => "Elsevier Editora Ltda." "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Braz J Infect Dis. 2016;20:41-7" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 1876 "formatos" => array:3 [ "EPUB" => 214 "HTML" => 980 "PDF" => 682 ] ] "en" => array:12 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "Resistance patterns, prevalence, and predictors of fluoroquinolones resistance in multidrug resistant tuberculosis patients" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "41" "paginaFinal" => "47" ] ] "contieneResumen" => array:1 [ "en" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 666 "Ancho" => 1665 "Tamanyo" => 88296 ] ] "descripcion" => array:1 [ "en" => "Inclusion and exclusion of study patients. MDR-TB, multidrug resistant tuberculosis; XDR-TB, extensive drug resistant tuberculosis." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Nafees Ahmad, Arshad Javaid, Syed Azhar Syed Sulaiman, Long Chiau Ming, Izaz Ahmad, Amer Hayat Khan" "autores" => array:6 [ 0 => array:2 [ "nombre" => "Nafees" "apellidos" => "Ahmad" ] 1 => array:2 [ "nombre" => "Arshad" "apellidos" => "Javaid" ] 2 => array:2 [ "nombre" => "Syed Azhar Syed" "apellidos" => "Sulaiman" ] 3 => array:2 [ "nombre" => "Long Chiau" "apellidos" => "Ming" ] 4 => array:2 [ "nombre" => "Izaz" "apellidos" => "Ahmad" ] 5 => array:2 [ "nombre" => "Amer Hayat" "apellidos" => "Khan" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1413867015002019?idApp=UINPBA00003Y" "url" => "/14138670/0000002000000001/v1_201601240048/S1413867015002019/v1_201601240048/en/main.assets" ] "en" => array:21 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "Induction of apoptosis by zerumbone isolated from Zingiber zerumbet (L.) Smith in protozoan parasite Leishmania donovani due to oxidative stress" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "48" "paginaFinal" => "55" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Debarati Mukherjee, Chingakham Brajakishor Singh, Somaditya Dey, Supratim Mandal, Joydip Ghosh, Suvadip Mallick, Aabid Hussain, Ningombam Swapana, Samir Anis Ross, Chiranjib Pal" "autores" => array:10 [ 0 => array:3 [ "nombre" => "Debarati" "apellidos" => "Mukherjee" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "1" "identificador" => "fn1" ] ] ] 1 => array:3 [ "nombre" => "Chingakham Brajakishor" "apellidos" => "Singh" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] 1 => array:2 [ "etiqueta" => "1" "identificador" => "fn1" ] ] ] 2 => array:3 [ "nombre" => "Somaditya" "apellidos" => "Dey" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "2" "identificador" => "fn2" ] ] ] 3 => array:3 [ "nombre" => "Supratim" "apellidos" => "Mandal" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "3" "identificador" => "fn3" ] ] ] 4 => array:3 [ "nombre" => "Joydip" "apellidos" => "Ghosh" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 5 => array:3 [ "nombre" => "Suvadip" "apellidos" => "Mallick" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 6 => array:3 [ "nombre" => "Aabid" "apellidos" => "Hussain" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 7 => array:3 [ "nombre" => "Ningombam" "apellidos" => "Swapana" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 8 => array:3 [ "nombre" => "Samir Anis" "apellidos" => "Ross" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "d" "identificador" => "aff0020" ] ] ] 9 => array:4 [ "nombre" => "Chiranjib" "apellidos" => "Pal" "email" => array:1 [ 0 => "cpcu.immunology@gmail.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:4 [ 0 => array:3 [ "entidad" => "Cellular Immunology and Experimental Therapeutics Laboratory, Department of Zoology, West Bengal State University, Barasat, West Bengal, India" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Institute of Bioresource and Sustainable Development, Imphal, Manipur, India" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Department of Chemistry, S. Kula Women's College, Nambol 795134, Manipur, India" "etiqueta" => "c" "identificador" => "aff0015" ] 3 => array:3 [ "entidad" => "National Center for Natural Product Chemistry and Department of Bimolecular Sciences, School of Pharmacy, University of Mississippi, Mississippi, USA" "etiqueta" => "d" "identificador" => "aff0020" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "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." ] ] ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0020" "etiqueta" => "Fig. 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 2005 "Ancho" => 2948 "Tamanyo" => 276916 ] ] "descripcion" => array:1 [ "en" => "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." ] ] ] "textoCompleto" => "IntroductionLeishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Treatment of leishmaniasis is based on pentavalent antimonials, drugs developed more than 50 years ago that are toxic and prone to drug resistance. Several drug screening of natural compounds have been successful in discovering novel compounds for treating some parasitic diseases. Extracts obtained from plants, as well as pure compounds including terpenoids, flavonoids (quercetin, rotenone) have been reported to possess significant antiprotozoal activities. Plants and natural products remain as the ideal resource in search for drug discovery because of their unique structural diversity and promising long term safety records.<a class="elsevierStyleCrossRef" href="#bib0075">1</a>Zingiber zerumbet (L.) Smith (awapuhi), also known as shampoo ginger (Malay=lempoyang) or pinecone ginger is a vigorous species of the ginger family with leafy stems growing to about 1.2m (3.9ft) tall. It is found in many tropical countries. The rhizomes of Z. zerumbet have been used as food flavouring and appetizers in various cuisines while the rhizome extracts have been used in herbal medicine. In Hawaii, the fresh rhizomes were used as medicine for indigestion and other ailments. For a toothache or a cavity, the cooked and softened ‘awapuhi’ rhizome was pressed into the hollow and left for as long as was needed. To ease a stomach ache, the ground and strained rhizome material is mixed with water and drunk. Zerumbone was identified as a monocyclic sesquiterpene moiety [2,6,10-cy-cloundecatrien-1-one, 2,6,9,9-tetramethyl-,(E,E,E)-] of the essential component in rhizomes of Z. zerumbet (L.) Smith, shows a variety of physiological effects e.g. anti-cancer, HIV inhibitory, anti-inflammatory, anti-viral effects.<a class="elsevierStyleCrossRef" href="#bib0080">2</a> Recently, our neighbouring group indicated the anti-leishmanial effect of essential oil and zerumbone from Z. zerumbet (L.) Smith against Leishmania donovani promastigotes.<a class="elsevierStyleCrossRef" href="#bib0085">3</a> In this study, we have shown that zerumbone could induce apoptosis by disrupting oxidative axis and also effectively inhibited the intracellular amastigotes, pathogenic stage of the parasite in mammalian host.Materials and methodsExtraction of essential oil and Purification of zerumboneThe 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.<a class="elsevierStyleCrossRefs" href="#bib0085">3,4</a>Parasites maintenance and viability assayThe 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.<a class="elsevierStyleCrossRef" href="#bib0095">5</a> 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).Analysis of cell cycle progression in L. donovani promastigotes2.5×106cells/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.<a class="elsevierStyleCrossRef" href="#bib0100">6</a> Acquisition was performed using a flow cytometer (BD FACSVerse™, BD Biosciences, USA) and the data were analyzed using Flowing software 2.5.Externalization of phosphatidyl serineIn 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.<a class="elsevierStyleCrossRefs" href="#bib0095">5,7</a>Estimation of reactive oxygen speciesIn Leishmania, oxidative stress has been suggested to be responsible for the apoptotic process.<a class="elsevierStyleCrossRefs" href="#bib0095">5,6</a> 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.<a class="elsevierStyleCrossRef" href="#bib0095">5</a> 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.<a class="elsevierStyleCrossRef" href="#bib0100">6</a>Detection of chromatin condensation and cytoplasmic lipid droplet accumulationThe 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.<a class="elsevierStyleCrossRefs" href="#bib0095">5–8</a> Images were obtained using an Olympus confocal laser scanning microscope (Model: IX81) and analyzed by Olympus fluoview version 3.0 viewer software.Measurement of total lipid peroxidationAs 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).<a class="elsevierStyleCrossRef" href="#bib0100">6</a>Detection of the change in morphology by scanning electron microscopyControl 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 layer<a class="elsevierStyleCrossRefs" href="#bib0095">5,6</a> and observed under a scanning electron microscope (Model: ZEISS EVO-MA 10).Anti-proliferative activity on intracellular amastigotesPeritoneal 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.<a class="elsevierStyleCrossRefs" href="#bib0115">9,10</a> 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.<a class="elsevierStyleCrossRefs" href="#bib0115">9,11</a>Statistical analysesStatistical analyses for all experiments were performed by one-way ANOVA followed by post hoc Holm–Sidak test with the program Sigma Plot.ResultsAnalysis of phytochemicalsThe 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 inhibited the proliferation of L. donovani promastigotesZerumbone 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 (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 1</a>).<elsevierMultimedia ident="fig0010"></elsevierMultimedia>Zerumbone induced morphological alterations in L. donovani promastigotesThe treated promastigotes appeared rounded with loss of flagella with porous cell membrane (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 2</a>B) in comparison to the flagellated and slender promastigotes of the control culture (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 2</a>A).<elsevierMultimedia ident="fig0015"></elsevierMultimedia>Zerumbone caused lipid accumulation in L. donovani promastigotesAnother prominent effect resulting from the treatment of the promastigotes with zerumbone was the accumulation of lipid droplets in the cytoplasm (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 3</a>B 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.<elsevierMultimedia ident="fig0020"></elsevierMultimedia>Zerumbone altered the cell cycle progression and induced externalization of phosphatidyl serine in L. donovani promastigotesAt 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% (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 4</a>A).<elsevierMultimedia ident="fig0025"></elsevierMultimedia>A significant step of apoptosis is the translocation of phosphatidyl serine from the inner to the outer leaflet of the plasma membrane.<a class="elsevierStyleCrossRef" href="#bib0130">12</a> 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 (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 4</a>B).Zerumbone caused DNA condensation in L. donovani promastigotesDAPI 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 (<a class="elsevierStyleCrossRef" href="#fig0030">Fig. 5</a>B and C).<elsevierMultimedia ident="fig0030"></elsevierMultimedia>Zerumbone induced the oxidative stress in L. donovani promastigotesWe 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 (<a class="elsevierStyleCrossRef" href="#fig0035">Fig. 6</a>A).<elsevierMultimedia ident="fig0035"></elsevierMultimedia>Zerumbone increased the level of lipid peroxidation in L. donovani promastigotesZerumbone 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] (<a class="elsevierStyleCrossRef" href="#fig0035">Fig. 6</a>B).Zerumbone inhibited the intracellular amastigotes in infected macrophagesZerumbone was found effective against intracellular amastigotes and the 50% inhibitory concentration was estimated with the treatment of 5μM of zerumbone at 48h (<a class="elsevierStyleCrossRef" href="#fig0040">Fig. 7</a>).<elsevierMultimedia ident="fig0040"></elsevierMultimedia>DiscussionZerumbone is a naturally occurring dietary compound, present in many natural foods consumed today. The compound derived from several plant species of the Zingiberaceae family that has been found to possess multiple biomedical properties, such as antiproliferative, antioxidant, anti-inflammatory, and anticancer activities.<a class="elsevierStyleCrossRef" href="#bib0080">2</a> However, evidence of efficacy is sparse against protozoan infection to support therapeutic claims to identify future uses against L. donovani infection. In the present study we successfully analyzed the nature of zerumbone-mediated cell death in L. donovani promastigotes and the possible key cellular mediators involved in the death cascade. Our initial observation that the zerumbone was effective against promastigotes but substantially non-toxic towards murine splenocytes (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 1</a>) made us curious for further in depth study. Morphological structure as observed through SEM has also authenticated the cytotoxic nature of zerumbone against Leishmania promastigotes (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 2</a>B and C) which can be correlated with the unnatural lipid accumulation on treatment (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 3</a>B and C). Interestingly, we found that zerumbone could increase the sub-G0/G1 (dead cells) up to 9.9% from 1.6% as in control promastigotes (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 4</a>A), concomitantly caused the externalization of phosphatidyl serine (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 4</a>B) in promastigote plasma membrane which is a crucial step in the process of apoptosis.<a class="elsevierStyleCrossRef" href="#bib0120">10</a> However, recently an interesting study raised the question on relevance of Annexin V binding assay in detecting apoptosis in Leishmania as they showed that the promastigotes lack phosphatidyl serine and Annexin V can also bind other lipids, including phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol.<a class="elsevierStyleCrossRef" href="#bib0135">13</a> Accordingly, we enquired the effect of zerumbone on Leishmania chromatin condensation and nuclear blebbing, the hallmark of apoptosis. The debate regarding the induction of apoptosis by zerumbone has been resolved and further confirmed by the DNA condensation in promastigotes (<a class="elsevierStyleCrossRef" href="#fig0030">Fig. 5</a>B and C). Looking into the mechanism, we found that the oxidative stress (<a class="elsevierStyleCrossRef" href="#fig0035">Fig. 6</a>A) followed by an increase in the level of lipid peroxidation (<a class="elsevierStyleCrossRef" href="#fig0035">Fig. 6</a>B) in zerumbone-treated promastigotes might involve the alteration of mitochondrial membrane potential leading to apoptosis.<a class="elsevierStyleCrossRef" href="#bib0140">14</a> The ultimate conviction came true when we found that zerumbone inhibited the clinically important morphs of L. donovani in mammalian host, the intracellular amastigotes in macrophages (<a class="elsevierStyleCrossRef" href="#fig0040">Fig. 7</a>). In conclusion, our findings indicate that zerumbone induced ROS-mediated apoptosis in L. donovani promastigotes and further pharmacological studies on this particular anti-leishmanial efficacy, in vivo, against L. donovani infection appear promising.FundingThis work was supported by Department of Biotechnology, Government of India through a collaborative project between WBSU, Barasat, West Bengal and IBSD, Imphal, Manipur (Project ref: BT/217/NE/TBP/2011, dated 15.12.2011).Ethical approvalApproved.Conflicts of interestThe authors declare no conflicts of interest." "textoCompletoSecciones" => array:1 [ "secciones" => array:11 [ 0 => array:3 [ "identificador" => "xres599235" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec613640" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 3 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:10 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Extraction of essential oil and Purification of zerumbone" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Parasites maintenance and viability assay" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Analysis of cell cycle progression in L. donovani promastigotes" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Externalization of phosphatidyl serine" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Estimation of reactive oxygen species" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Detection of chromatin condensation and cytoplasmic lipid droplet accumulation" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Measurement of total lipid peroxidation" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "Detection of the change in morphology by scanning electron microscopy" ] 8 => array:2 [ "identificador" => "sec0055" "titulo" => "Anti-proliferative activity on intracellular amastigotes" ] 9 => array:2 [ "identificador" => "sec0060" "titulo" => "Statistical analyses" ] ] ] 4 => array:3 [ "identificador" => "sec0065" "titulo" => "Results" "secciones" => array:9 [ 0 => array:2 [ "identificador" => "sec0070" "titulo" => "Analysis of phytochemicals" ] 1 => array:2 [ "identificador" => "sec0075" "titulo" => "Zerumbone inhibited the proliferation of L. donovani promastigotes" ] 2 => array:2 [ "identificador" => "sec0080" "titulo" => "Zerumbone induced morphological alterations in L. donovani promastigotes" ] 3 => array:2 [ "identificador" => "sec0085" "titulo" => "Zerumbone caused lipid accumulation in L. donovani promastigotes" ] 4 => array:2 [ "identificador" => "sec0090" "titulo" => "Zerumbone altered the cell cycle progression and induced externalization of phosphatidyl serine in L. donovani promastigotes" ] 5 => array:2 [ "identificador" => "sec0095" "titulo" => "Zerumbone caused DNA condensation in L. donovani promastigotes" ] 6 => array:2 [ "identificador" => "sec0100" "titulo" => "Zerumbone induced the oxidative stress in L. donovani promastigotes" ] 7 => array:2 [ "identificador" => "sec0105" "titulo" => "Zerumbone increased the level of lipid peroxidation in L. donovani promastigotes" ] 8 => array:2 [ "identificador" => "sec0110" "titulo" => "Zerumbone inhibited the intracellular amastigotes in infected macrophages" ] ] ] 5 => array:2 [ "identificador" => "sec0115" "titulo" => "Discussion" ] 6 => array:2 [ "identificador" => "sec0120" "titulo" => "Funding" ] 7 => array:2 [ "identificador" => "sec0130" "titulo" => "Ethical approval" ] 8 => array:2 [ "identificador" => "sec0135" "titulo" => "Conflicts of interest" ] 9 => array:2 [ "identificador" => "xack201801" "titulo" => "Acknowledgements" ] 10 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2015-06-20" "fechaAceptado" => "2015-10-01" "PalabrasClave" => array:1 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec613640" "palabras" => array:5 [ 0 => "Leishmania donovani" 1 => "Zingiber zerumbet" 2 => "Zerumbone" 3 => "Anti-leishmanial" 4 => "ROS" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "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 Leishmania infection. 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. donovani infection and provided the basis for future research on the application of transitional medicinal plants." ] ] "NotaPie" => array:3 [ 0 => array:3 [ "etiqueta" => "1" "nota" => "These author contributed equally to this work." "identificador" => "fn1" ] 1 => array:3 [ "etiqueta" => "2" "nota" => "Present address: Post Graduate Department of Zoology, Barasat Government College, Barasat, West Bengal, India." "identificador" => "fn2" ] 2 => array:3 [ "etiqueta" => "3" "nota" => "Present address: PG Department of Microbiology, APC College, New Barrackpore, West Bengal, India." "identificador" => "fn3" ] ] "apendice" => array:1 [ 0 => array:1 [ "seccion" => array:1 [ 0 => array:4 [ "apendice" => "The following are the supplementary data to this article:<elsevierMultimedia ident="fig0005"></elsevierMultimedia>" "etiqueta" => "Appendix A" "titulo" => "Supplementary data" "identificador" => "sec0145" ] ] ] ] "multimedia" => array:8 [ 0 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1168 "Ancho" => 1510 "Tamanyo" => 88082 ] ] "descripcion" => array:1 [ "en" => "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)." ] ] 1 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1018 "Ancho" => 750 "Tamanyo" => 51356 ] ] "descripcion" => array:1 [ "en" => "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." ] ] 2 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Fig. 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 2005 "Ancho" => 2948 "Tamanyo" => 276916 ] ] "descripcion" => array:1 [ "en" => "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." ] ] 3 => array:7 [ "identificador" => "fig0025" "etiqueta" => "Fig. 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 2123 "Ancho" => 2961 "Tamanyo" => 366916 ] ] "descripcion" => array:1 [ "en" => "(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." ] ] 4 => array:7 [ "identificador" => "fig0030" "etiqueta" => "Fig. 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 1961 "Ancho" => 2957 "Tamanyo" => 185455 ] ] "descripcion" => array:1 [ "en" => "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." ] ] 5 => array:7 [ "identificador" => "fig0035" "etiqueta" => "Fig. 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 1131 "Ancho" => 2910 "Tamanyo" => 143143 ] ] "descripcion" => array:1 [ "en" => "(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)." ] ] 6 => array:7 [ "identificador" => "fig0040" "etiqueta" => "Fig. 7" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr7.jpeg" "Alto" => 1185 "Ancho" => 1523 "Tamanyo" => 63914 ] ] "descripcion" => array:1 [ "en" => "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)." ] ] 7 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Supplementary Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => false "mostrarDisplay" => true "figura" => array:1 [ 0 => array:4 [ "imagen" => "mmc1.jpeg" "Alto" => 2115 "Ancho" => 2500 "Tamanyo" => 244562 ] ] "descripcion" => array:1 [ "en" => "GC Spectra of essential oil of Z. zerumbet (the upper spectra is the standard hydrocarbon while the lower portion of the spectra is the oils)." ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:14 [ 0 => array:3 [ "identificador" => "bib0075" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Calpain and reactive oxygen species targets Bax for mitochondrial permeabilization and caspase activation in zerumbone induced apoptosis" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:3 [ 0 => "P. Sobhan" 1 => "M. Seervi" 2 => "L. Deb" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1371/journal.pone.0059350" "Revista" => array:5 [ "tituloSerie" => "PLoS ONE" "fecha" => "2013" "volumen" => "8" "paginaInicial" => "e59350" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/23593137" "web" => "Medline" ] ] ] ] ] ] ] ] 1 => array:3 [ "identificador" => "bib0080" "etiqueta" => "2" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Attractive reactivity of a natural product, zerumbone" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:1 [ 0 => "T. Kitayama" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1271/bbb.100532" "Revista" => array:6 [ "tituloSerie" => "Biosci Biotechnol Biochem" "fecha" => "2011" "volumen" => "75" "paginaInicial" => "199" "paginaFinal" => "207" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/21307568" "web" => "Medline" ] ] ] ] ] ] ] ] 2 => array:3 [ "identificador" => "bib0085" "etiqueta" => "3" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Chemical composition and biological activity of the essential oil of rhizome of Zingiber zerumbet (L.) Smith" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "C.B. Singh" 1 => "B.C. Saikhom" 2 => "K.H. Lenin" 3 => "S. Ningombam" 4 => "C. Charles" 5 => "A.R. Samir" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:5 [ "tituloSerie" => "J Pharmacog Phytochem" "fecha" => "2014" "volumen" => "3" "paginaInicial" => "130" "paginaFinal" => "133" ] ] ] ] ] ] 3 => array:3 [ "identificador" => "bib0090" "etiqueta" => "4" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Dual extraction of essential oil and podophyllotoxin from creeping juniper (Juniperus horizontalis)" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "C.L. Cantrell" 1 => "V.D. Zheljazkov" 2 => "C.R. Carvalho" 3 => "T. Astatkie" 4 => "E.A. Jeliazkova" 5 => "L. Rosa" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1371/journal.pone.0106057" "Revista" => array:5 [ "tituloSerie" => "PLoS ONE" "fecha" => "2014" "volumen" => "9" "paginaInicial" => "e106057" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/25203255" "web" => "Medline" ] ] ] ] ] ] ] ] 4 => array:3 [ "identificador" => "bib0095" "etiqueta" => "5" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Protective effect of Croton caudatus Geisel leaf extract against experimental visceral leishmaniasis induces proinflammatory cytokines in vitro and in vivo" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:3 [ 0 => "S. Dey" 1 => "D. Mukherjee" 2 => "S. Chakraborty" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1016/j.exppara.2015.09.011" "Revista" => array:6 [ "tituloSerie" => "Exp Parasitol" "fecha" => "2015" "volumen" => "151–152" "paginaInicial" => "84" "paginaFinal" => "95" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26420465" "web" => "Medline" ] ] ] ] ] ] ] ] 5 => array:3 [ "identificador" => "bib0100" "etiqueta" => "6" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "A novel triterpene from Astraeus hygrometricus induces reactive oxygen species leading to death in Leishmania donovani" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:3 [ 0 => "S. Mallick" 1 => "S. Dey" 2 => "S. Mandal" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:5 [ "tituloSerie" => "Fut Microbiol" "fecha" => "2015" "volumen" => "10" "paginaInicial" => "763" "paginaFinal" => "789" ] ] ] ] ] ] 6 => array:3 [ "identificador" => "bib0105" "etiqueta" => "7" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Selective inhibition of Leishmania donovani by active extracts of wild mushrooms used by the tribal population of India: an in vitro exploration for new leads against parasitic protozoans" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:3 [ 0 => "S. Mallick" 1 => "A. Dutta" 2 => "S. Dey" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1016/j.exppara.2014.01.002" "Revista" => array:6 [ "tituloSerie" => "Exp Parasitol" "fecha" => "2014" "volumen" => "138" "paginaInicial" => "9" "paginaFinal" => "17" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/24440295" "web" => "Medline" ] ] ] ] ] ] ] ] 7 => array:3 [ "identificador" => "bib0110" "etiqueta" => "8" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "In vitro activities of ER-119884 and E5700, two potent squalene synthase inhibitors, against Leishmania amazonensis: antiproliferative, biochemical, and ultrastructural effects" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "J.C.F. Rodrigues" 1 => "J.L. Concepcion" 2 => "C. Rodrigues" 3 => "A. Caldera" 4 => "J.A. Urbina" 5 => "W. Souza" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1128/AAC.01616-07" "Revista" => array:5 [ "tituloSerie" => "Antimicrob Agents Chemother" "fecha" => "2008" "volumen" => "52" "paginaInicial" => "4098" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/18765694" "web" => "Medline" ] ] ] ] ] ] ] ] 8 => array:3 [ "identificador" => "bib0115" "etiqueta" => "9" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Mycobacterium indicus pranii (Mw)-mediated protection against visceral leishmaniasis: involvement of TLR4 signalling" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:3 [ 0 => "A. Adhikari" 1 => "S. Majumder" 2 => "S. Banerjee" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1093/jac/dks315" "Revista" => array:6 [ "tituloSerie" => "J Antimicrob Chemother" "fecha" => "2012" "volumen" => "67" "paginaInicial" => "2892" "paginaFinal" => "2902" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22879460" "web" => "Medline" ] ] ] ] ] ] ] ] 9 => array:3 [ "identificador" => "bib0120" "etiqueta" => "10" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:3 [ 0 => "F. Nomura" 1 => "S. Akashi" 2 => "Y. Sakao" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:6 [ "tituloSerie" => "J Immunol" "fecha" => "2000" "volumen" => "164" "paginaInicial" => "3476" "paginaFinal" => "3479" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/10725699" "web" => "Medline" ] ] ] ] ] ] ] ] 10 => array:3 [ "identificador" => "bib0125" "etiqueta" => "11" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Protective therapy with novel chromone derivative against Leishmania donovani infection induces Th1 response in vivo" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:3 [ 0 => "S. Mallick" 1 => "A. Dutta" 2 => "J. Ghosh" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1159/000330856" "Revista" => array:6 [ "tituloSerie" => "Chemotherapy" "fecha" => "2011" "volumen" => "57" "paginaInicial" => "388" "paginaFinal" => "393" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22024637" "web" => "Medline" ] ] ] ] ] ] ] ] 11 => array:3 [ "identificador" => "bib0130" "etiqueta" => "12" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:4 [ 0 => "I. Vermes" 1 => "C. Haanen" 2 => "H. Steffens-Nakken" 3 => "C. Reutelingsperger" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:6 [ "tituloSerie" => "J Immunol Methods" "fecha" => "1995" "volumen" => "184" "paginaInicial" => "39" "paginaFinal" => "51" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/7622868" "web" => "Medline" ] ] ] ] ] ] ] ] 12 => array:3 [ "identificador" => "bib0135" "etiqueta" => "13" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Leishmania promastigotes lack phosphatidylserine but bind Annexin V upon permeabilization or miltefosine treatment" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:3 [ 0 => "A. Weingärtner" 1 => "G. Kemmer" 2 => "F.D. Müller" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1371/journal.pone.0042070" "Revista" => array:5 [ "tituloSerie" => "PLoS ONE" "fecha" => "2012" "volumen" => "7" "paginaInicial" => "e42070" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22870283" "web" => "Medline" ] ] ] ] ] ] ] ] 13 => array:3 [ "identificador" => "bib0140" "etiqueta" => "14" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Induction of apoptosis by the medium-chain length fatty acid lauric acid in colon cancer cells due to induction of oxidative stress" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:4 [ 0 => "J.K. Fauser" 1 => "G.M. Matthews" 2 => "A.G. Cummins" 3 => "G.S. Howarth" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1159/000356067" "Revista" => array:6 [ "tituloSerie" => "Chemotherapy" "fecha" => "2013" "volumen" => "59" "paginaInicial" => "214" "paginaFinal" => "224" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/24356281" "web" => "Medline" ] ] ] ] ] ] ] ] ] ] ] ] "agradecimientos" => array:1 [ 0 => array:4 [ "identificador" => "xack201801" "titulo" => "Acknowledgements" "texto" => "The authors are indebted to Vice Chancellor, West Bengal State University and Director, Institute of Bioresource and Sustainable Development (IBSD) for providing them the research infrastructures for this work. We are also thankful to the Director, CU BD Centre of Excellence for Nanobiotechnology, CRNN and DBT-IPLS, University of Calcutta for Flow Cytometry, SEM and Confocal Microscopy facility." "vista" => "all" ] ] ] "idiomaDefecto" => "en" "url" => "/14138670/0000002000000001/v1_201601240048/S1413867015002032/v1_201601240048/en/main.assets" "Apartado" => array:4 [ "identificador" => "36721" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Original article" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/14138670/0000002000000001/v1_201601240048/S1413867015002032/v1_201601240048/en/main.pdf?idApp=UINPBA00003Y&text.app=https://bjid.org.br/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1413867015002032?idApp=UINPBA00003Y"]

Statistics

Original article

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

Debarati Mukherjeea,1, Chingakham Brajakishor Singhb,1, Somaditya Deya,2, Supratim Mandala,3, Joydip Ghosha, Suvadip Mallicka, Aabid Hussaina, Ningombam Swapanac, Samir Anis Rossd, Chiranjib Pala,

Corresponding author

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.

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

b Institute of Bioresource and Sustainable Development, Imphal, Manipur, India

c Department of Chemistry, S. Kula Women's College, Nambol 795134, Manipur, India

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

Read

5538

Times
was read the article

2194

Total PDF

3344

Total HTML

Share statistics

 array:24 [
  "pii" => "S1413867015002032"
  "issn" => "14138670"
  "doi" => "10.1016/j.bjid.2015.10.002"
  "estado" => "S300"
  "fechaPublicacion" => "2016-01-01"
  "aid" => "527"
  "copyright" => "Elsevier Editora Ltda&#46;. All rights reserved"
  "copyrightAnyo" => "2015"
  "documento" => "article"
  "crossmark" => 1
  "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/"
  "subdocumento" => "fla"
  "cita" => "Braz J Infect Dis. 2016;20:48-55"
  "abierto" => array:3 [
    "ES" => true
    "ES2" => true
    "LATM" => true
  ]
  "gratuito" => true
  "lecturas" => array:2 [
    "total" => 2373
    "formatos" => array:3 [
      "EPUB" => 207
      "HTML" => 1482
      "PDF" => 684
    ]
  ]
  "itemSiguiente" => array:19 [
    "pii" => "S141386701500210X"
    "issn" => "14138670"
    "doi" => "10.1016/j.bjid.2015.10.005"
    "estado" => "S300"
    "fechaPublicacion" => "2016-01-01"
    "aid" => "531"
    "copyright" => "Elsevier Editora Ltda&#46;"
    "documento" => "article"
    "crossmark" => 1
    "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/"
    "subdocumento" => "fla"
    "cita" => "Braz J Infect Dis. 2016;20:56-60"
    "abierto" => array:3 [
      "ES" => true
      "ES2" => true
      "LATM" => true
    ]
    "gratuito" => true
    "lecturas" => array:2 [
      "total" => 1902
      "formatos" => array:3 [
        "EPUB" => 271
        "HTML" => 995
        "PDF" => 636
      ]
    ]
    "en" => array:11 [
      "idiomaDefecto" => true
      "cabecera" => "<span class="elsevierStyleTextfn">Original Article</span>"
      "titulo" => "Clinical and bacteriological characteristics of invasive pneumococcal disease after pneumococcal 10-valent conjugate vaccine implementation in Salvador&#44; Brazil"
      "tienePdf" => "en"
      "tieneTextoCompleto" => "en"
      "tieneResumen" => "en"
      "paginas" => array:1 [
        0 => array:2 [
          "paginaInicial" => "56"
          "paginaFinal" => "60"
        ]
      ]
      "contieneResumen" => array:1 [
        "en" => true
      ]
      "contieneTextoCompleto" => array:1 [
        "en" => true
      ]
      "contienePdf" => array:1 [
        "en" => true
      ]
      "autores" => array:1 [
        0 => array:2 [
          "autoresLista" => "Carolina Regis Leite, Jailton Azevedo, Vivian Santos Galv&#227;o, Ot&#225;vio Moreno-Carvalho, Joice Neves Reis, Cristiana Nascimento-Carvalho"
          "autores" => array:6 [
            0 => array:2 [
              "nombre" => "Carolina Regis"
              "apellidos" => "Leite"
            ]
            1 => array:2 [
              "nombre" => "Jailton"
              "apellidos" => "Azevedo"
            ]
            2 => array:2 [
              "nombre" => "Vivian Santos"
              "apellidos" => "Galv&#227;o"
            ]
            3 => array:2 [
              "nombre" => "Ot&#225;vio"
              "apellidos" => "Moreno-Carvalho"
            ]
            4 => array:2 [
              "nombre" => "Joice Neves"
              "apellidos" => "Reis"
            ]
            5 => array:2 [
              "nombre" => "Cristiana"
              "apellidos" => "Nascimento-Carvalho"
            ]
          ]
        ]
      ]
    ]
    "idiomaDefecto" => "en"
    "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S141386701500210X?idApp=UINPBA00003Y"
    "url" => "/14138670/0000002000000001/v1_201601240048/S141386701500210X/v1_201601240048/en/main.assets"
  ]
  "itemAnterior" => array:19 [
    "pii" => "S1413867015002019"
    "issn" => "14138670"
    "doi" => "10.1016/j.bjid.2015.09.011"
    "estado" => "S300"
    "fechaPublicacion" => "2016-01-01"
    "aid" => "525"
    "copyright" => "Elsevier Editora Ltda&#46;"
    "documento" => "article"
    "crossmark" => 1
    "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/"
    "subdocumento" => "fla"
    "cita" => "Braz J Infect Dis. 2016;20:41-7"
    "abierto" => array:3 [
      "ES" => true
      "ES2" => true
      "LATM" => true
    ]
    "gratuito" => true
    "lecturas" => array:2 [
      "total" => 1876
      "formatos" => array:3 [
        "EPUB" => 214
        "HTML" => 980
        "PDF" => 682
      ]
    ]
    "en" => array:12 [
      "idiomaDefecto" => true
      "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>"
      "titulo" => "Resistance patterns&#44; prevalence&#44; and predictors of fluoroquinolones resistance in multidrug resistant tuberculosis patients"
      "tienePdf" => "en"
      "tieneTextoCompleto" => "en"
      "tieneResumen" => "en"
      "paginas" => array:1 [
        0 => array:2 [
          "paginaInicial" => "41"
          "paginaFinal" => "47"
        ]
      ]
      "contieneResumen" => array:1 [
        "en" => true
      ]
      "contieneTextoCompleto" => array:1 [
        "en" => true
      ]
      "contienePdf" => array:1 [
        "en" => true
      ]
      "resumenGrafico" => array:2 [
        "original" => 0
        "multimedia" => array:7 [
          "identificador" => "fig0005"
          "etiqueta" => "Fig&#46; 1"
          "tipo" => "MULTIMEDIAFIGURA"
          "mostrarFloat" => true
          "mostrarDisplay" => false
          "figura" => array:1 [
            0 => array:4 [
              "imagen" => "gr1.jpeg"
              "Alto" => 666
              "Ancho" => 1665
              "Tamanyo" => 88296
            ]
          ]
          "descripcion" => array:1 [
            "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Inclusion and exclusion of study patients&#46; MDR-TB&#44; multidrug resistant tuberculosis&#59; XDR-TB&#44; extensive drug resistant tuberculosis&#46;</p>"
          ]
        ]
      ]
      "autores" => array:1 [
        0 => array:2 [
          "autoresLista" => "Nafees Ahmad, Arshad Javaid, Syed Azhar Syed Sulaiman, Long Chiau Ming, Izaz Ahmad, Amer Hayat Khan"
          "autores" => array:6 [
            0 => array:2 [
              "nombre" => "Nafees"
              "apellidos" => "Ahmad"
            ]
            1 => array:2 [
              "nombre" => "Arshad"
              "apellidos" => "Javaid"
            ]
            2 => array:2 [
              "nombre" => "Syed Azhar Syed"
              "apellidos" => "Sulaiman"
            ]
            3 => array:2 [
              "nombre" => "Long Chiau"
              "apellidos" => "Ming"
            ]
            4 => array:2 [
              "nombre" => "Izaz"
              "apellidos" => "Ahmad"
            ]
            5 => array:2 [
              "nombre" => "Amer Hayat"
              "apellidos" => "Khan"
            ]
          ]
        ]
      ]
    ]
    "idiomaDefecto" => "en"
    "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1413867015002019?idApp=UINPBA00003Y"
    "url" => "/14138670/0000002000000001/v1_201601240048/S1413867015002019/v1_201601240048/en/main.assets"
  ]
  "en" => array:21 [
    "idiomaDefecto" => true
    "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>"
    "titulo" => "Induction of apoptosis by zerumbone isolated from <span class="elsevierStyleItalic">Zingiber zerumbet</span> &#40;L&#46;&#41; Smith in protozoan parasite <span class="elsevierStyleItalic">Leishmania donovani</span> due to oxidative stress"
    "tieneTextoCompleto" => true
    "paginas" => array:1 [
      0 => array:2 [
        "paginaInicial" => "48"
        "paginaFinal" => "55"
      ]
    ]
    "autores" => array:1 [
      0 => array:4 [
        "autoresLista" => "Debarati Mukherjee, Chingakham Brajakishor Singh, Somaditya Dey, Supratim Mandal, Joydip Ghosh, Suvadip Mallick, Aabid Hussain, Ningombam Swapana, Samir Anis Ross, Chiranjib Pal"
        "autores" => array:10 [
          0 => array:3 [
            "nombre" => "Debarati"
            "apellidos" => "Mukherjee"
            "referencia" => array:2 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0005"
              ]
              1 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">1</span>"
                "identificador" => "fn1"
              ]
            ]
          ]
          1 => array:3 [
            "nombre" => "Chingakham Brajakishor"
            "apellidos" => "Singh"
            "referencia" => array:2 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">b</span>"
                "identificador" => "aff0010"
              ]
              1 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">1</span>"
                "identificador" => "fn1"
              ]
            ]
          ]
          2 => array:3 [
            "nombre" => "Somaditya"
            "apellidos" => "Dey"
            "referencia" => array:2 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0005"
              ]
              1 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">2</span>"
                "identificador" => "fn2"
              ]
            ]
          ]
          3 => array:3 [
            "nombre" => "Supratim"
            "apellidos" => "Mandal"
            "referencia" => array:2 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0005"
              ]
              1 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">3</span>"
                "identificador" => "fn3"
              ]
            ]
          ]
          4 => array:3 [
            "nombre" => "Joydip"
            "apellidos" => "Ghosh"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0005"
              ]
            ]
          ]
          5 => array:3 [
            "nombre" => "Suvadip"
            "apellidos" => "Mallick"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0005"
              ]
            ]
          ]
          6 => array:3 [
            "nombre" => "Aabid"
            "apellidos" => "Hussain"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0005"
              ]
            ]
          ]
          7 => array:3 [
            "nombre" => "Ningombam"
            "apellidos" => "Swapana"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">c</span>"
                "identificador" => "aff0015"
              ]
            ]
          ]
          8 => array:3 [
            "nombre" => "Samir Anis"
            "apellidos" => "Ross"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">d</span>"
                "identificador" => "aff0020"
              ]
            ]
          ]
          9 => array:4 [
            "nombre" => "Chiranjib"
            "apellidos" => "Pal"
            "email" => array:1 [
              0 => "cpcu&#46;immunology&#64;gmail&#46;com"
            ]
            "referencia" => array:2 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0005"
              ]
              1 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">&#42;</span>"
                "identificador" => "cor0005"
              ]
            ]
          ]
        ]
        "afiliaciones" => array:4 [
          0 => array:3 [
            "entidad" => "Cellular Immunology and Experimental Therapeutics Laboratory&#44; Department of Zoology&#44; West Bengal State University&#44; Barasat&#44; West Bengal&#44; India"
            "etiqueta" => "a"
            "identificador" => "aff0005"
          ]
          1 => array:3 [
            "entidad" => "Institute of Bioresource and Sustainable Development&#44; Imphal&#44; Manipur&#44; India"
            "etiqueta" => "b"
            "identificador" => "aff0010"
          ]
          2 => array:3 [
            "entidad" => "Department of Chemistry&#44; S&#46; Kula Women&#39;s College&#44; Nambol 795134&#44; Manipur&#44; India"
            "etiqueta" => "c"
            "identificador" => "aff0015"
          ]
          3 => array:3 [
            "entidad" => "National Center for Natural Product Chemistry and Department of Bimolecular Sciences&#44; School of Pharmacy&#44; University of Mississippi&#44; Mississippi&#44; USA"
            "etiqueta" => "d"
            "identificador" => "aff0020"
          ]
        ]
        "correspondencia" => array:1 [
          0 => array:3 [
            "identificador" => "cor0005"
            "etiqueta" => "&#8270;"
            "correspondencia" => "<span class="elsevierStyleItalic">Corresponding author at</span>&#58; Cellular Immunology and Experimental Therapeutics Laboratory&#44; Department of Zoology&#44; West Bengal State University&#44; Barasat&#44; DT&#58; 24 PGS &#40;N&#41;&#44; Kolkata 700126&#44; West Bengal&#44; India&#46;"
          ]
        ]
      ]
    ]
    "resumenGrafico" => array:2 [
      "original" => 0
      "multimedia" => array:7 [
        "identificador" => "fig0020"
        "etiqueta" => "Fig&#46; 3"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr3.jpeg"
            "Alto" => 2005
            "Ancho" => 2948
            "Tamanyo" => 276916
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Accumulation of cytosolic lipid in zerumbone-treated <span class="elsevierStyleItalic">L&#46; donovani</span> AG83 promastigotes as evident by nile red staining &#40;magnification&#58; 100&#215;&#44; zoom&#58; 3&#46;4&#215;&#41;&#46; &#40;A&#41; Phase contrast&#59; &#40;B&#41; fluorescence&#59; &#40;C&#41; phase contrast&#8211;fluorescence merge&#46; Zerumbone treatment significantly increased lipid droplets accumulation &#40;white arrows&#41; in the cytosol in comparison to the untreated cells&#46; Images are representative profile of at least three independent experiments&#46;</p>"
        ]
      ]
    ]
    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Leishmaniasis&#44; a parasitic disease caused by protozoa of the genus <span class="elsevierStyleItalic">Leishmania</span>&#44; affects more than 12 million people worldwide&#46; Treatment of leishmaniasis is based on pentavalent antimonials&#44; drugs developed more than 50 years ago that are toxic and prone to drug resistance&#46; Several drug screening of natural compounds have been successful in discovering novel compounds for treating some parasitic diseases&#46; Extracts obtained from plants&#44; as well as pure compounds including terpenoids&#44; flavonoids &#40;quercetin&#44; rotenone&#41; have been reported to possess significant antiprotozoal activities&#46; Plants and natural products remain as the ideal resource in search for drug discovery because of their unique structural diversity and promising long term safety records&#46;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">1</span></a><span class="elsevierStyleItalic">Zingiber zerumbet</span> &#40;L&#46;&#41; Smith &#40;<span class="elsevierStyleItalic">awapuhi</span>&#41;&#44; also known as shampoo ginger &#40;Malay<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">lempoyang</span>&#41; or pinecone ginger is a vigorous species of the ginger family with leafy stems growing to about 1&#46;2<span class="elsevierStyleHsp" style=""></span>m &#40;3&#46;9<span class="elsevierStyleHsp" style=""></span>ft&#41; tall&#46; It is found in many tropical countries&#46; The rhizomes of <span class="elsevierStyleItalic">Z&#46; zerumbet</span> have been used as food flavouring and appetizers in various cuisines while the rhizome extracts have been used in herbal medicine&#46; In Hawaii&#44; the fresh rhizomes were used as medicine for indigestion and other ailments&#46; For a toothache or a cavity&#44; the cooked and softened &#8216;<span class="elsevierStyleItalic">awapuhi</span>&#8217; rhizome was pressed into the hollow and left for as long as was needed&#46; To ease a stomach ache&#44; the ground and strained rhizome material is mixed with water and drunk&#46; Zerumbone was identified as a monocyclic sesquiterpene moiety &#91;2&#44;6&#44;10-cy-cloundecatrien-1-one&#44; 2&#44;6&#44;9&#44;9-tetramethyl-&#44;&#40;E&#44;E&#44;E&#41;-&#93; of the essential component in rhizomes of <span class="elsevierStyleItalic">Z&#46; zerumbet</span> &#40;L&#46;&#41; Smith&#44; shows a variety of physiological effects e&#46;g&#46; anti-cancer&#44; HIV inhibitory&#44; anti-inflammatory&#44; anti-viral effects&#46;<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">2</span></a> Recently&#44; our neighbouring group indicated the anti-leishmanial effect of essential oil and zerumbone from <span class="elsevierStyleItalic">Z&#46; zerumbet</span> &#40;L&#46;&#41; Smith against <span class="elsevierStyleItalic">Leishmania donovani</span> promastigotes&#46;<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">3</span></a> In this study&#44; we have shown that zerumbone could induce apoptosis by disrupting oxidative axis and also effectively inhibited the intracellular amastigotes&#44; pathogenic stage of the parasite in mammalian host&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Extraction of essential oil and Purification of zerumbone</span><p id="par0010" class="elsevierStylePara elsevierViewall">The plant materials were collected from Manipur&#44; North-East India&#44; 920<span class="elsevierStyleHsp" style=""></span>m from sea level&#44; longitude 93&#176;58&#8243; and latitude 24&#176;44&#8243; in March&#44; 2012&#46; The plant was identified by the taxonomist of the institute and had given the accession number as IBSD&#47;Z-42-23&#46; Fresh rhizomes were collected and washed thoroughly with tap water&#46; These were cut into 5&#8211;6<span class="elsevierStyleHsp" style=""></span>mm slices and put into the Clevenger type oil extractor&#46; Oil samples were analyzed by GC-FID on a Agilent 5975 C inert XL MSD&#46; The oil was dried over anhydrous sodium sulphate and stored at 4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The oil was analyzed by GC&#8211;MS on a Varian CP-3800 GC coupled to a Varian Saturn 2000 MS&#47;MS&#46; The GC was equipped with a DB-5 fused silica capillary column &#40;30<span class="elsevierStyleHsp" style=""></span>m<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>0&#46;25<span class="elsevierStyleHsp" style=""></span>mm&#44; with film thickness of 0&#46;25<span class="elsevierStyleHsp" style=""></span>&#956;m&#41; operated using the following conditions&#58; injector temperature&#44; 240<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; column temperature&#44; 60&#8211;240<span class="elsevierStyleHsp" style=""></span>&#176;C at 3<span class="elsevierStyleHsp" style=""></span>&#176;C&#47;min&#44; then held at 240<span class="elsevierStyleHsp" style=""></span>&#176;C for 5<span class="elsevierStyleHsp" style=""></span>min&#59; carrier gas&#44; He&#59; injection volume&#44; 1<span class="elsevierStyleHsp" style=""></span>&#956;L &#40;splitless&#41;&#46; The MS mass ranged from 40 to 650 <span class="elsevierStyleItalic">m&#47;z</span>&#44; filament delay of 3<span class="elsevierStyleHsp" style=""></span>min&#44; target TIC of 20&#44;000&#44; a prescan ionization time of 100<span class="elsevierStyleHsp" style=""></span>&#956;s&#44; an ion trap temperature of 150<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; manifold temperature of 60<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; and a transfer line temperature of 170<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The constituents of the oil were identified using retention times&#44; Kovats indices and mass spectra&#46; Confirmed integrated peaks were then used for the percentage of each chemical constituent present in the essential oil&#46; Kovats indices were calculated using the equation&#58; KI&#40;<span class="elsevierStyleItalic">x</span>&#41;<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>100&#91;&#40;log<span class="elsevierStyleHsp" style=""></span>RT&#40;<span class="elsevierStyleItalic">x</span>&#41;<span class="elsevierStyleHsp" style=""></span>&#8722;<span class="elsevierStyleHsp" style=""></span>log<span class="elsevierStyleHsp" style=""></span>Pz&#41;&#47;&#40;log<span class="elsevierStyleHsp" style=""></span>RT&#40;Pz<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>1&#41;<span class="elsevierStyleHsp" style=""></span>&#8722;<span class="elsevierStyleHsp" style=""></span>log<span class="elsevierStyleHsp" style=""></span>RT&#40;Pz&#41;&#93;&#44; where RT&#40;Pz&#41;<span class="elsevierStyleHsp" style=""></span>&#8804;<span class="elsevierStyleHsp" style=""></span>RT&#40;<span class="elsevierStyleItalic">x</span>&#41;<span class="elsevierStyleHsp" style=""></span>&#8804;<span class="elsevierStyleHsp" style=""></span>RT&#40;Pz<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>1&#41;&#44; and P4&#44; &#8230;&#44; P25 are <span class="elsevierStyleItalic">n</span> paraffins&#46;<a class="elsevierStyleCrossRefs" href="#bib0085"><span class="elsevierStyleSup">3&#44;4</span></a></p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Parasites maintenance and viability assay</span><p id="par0015" class="elsevierStylePara elsevierViewall">The anti-proliferative effect of zerumbone was estimated on <span class="elsevierStyleItalic">L&#46; donovani</span> AG83 &#40;MHOM&#47;IN&#47;1983&#47;AG83&#41; as per the guidelines of biosafety committee of West Bengal State University&#46; Promastigotes were transformed from splenic intracellular amastigotes of infected BALB&#47;c mice in complete M199 medium &#40;Invitrogen&#41; supplemented with 1&#37; penicillin&#8211;streptomycin &#40;Invitrogen&#41; and 10&#37; FCS &#40;GIBCO&#41; at requisite temperature &#40;22<span class="elsevierStyleHsp" style=""></span>&#176;C&#41;&#46; To estimate the percentage of inhibition&#44; the 3-&#40;4&#44;5-dimethylthiazol-2-yl&#41;-2&#44;5-diphenyltetrazolium bromide &#40;MTT&#41; micro method was used&#46; Briefly&#44; promastigotes cultures were incubated with or without &#40;control&#41; increasing concentrations of zerumbone &#40;0&#46;1&#8211;50<span class="elsevierStyleHsp" style=""></span>&#956;M&#41; for 48<span class="elsevierStyleHsp" style=""></span>h in a 96-well flat-bottom plate &#40;200<span class="elsevierStyleHsp" style=""></span>&#956;L per well&#59; BD Falcon&#41; in complete M199 medium&#46; After 48<span class="elsevierStyleHsp" style=""></span>h of incubation at 22<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; MTT &#40;10<span class="elsevierStyleHsp" style=""></span>mg&#47;mL&#44; 10<span class="elsevierStyleHsp" style=""></span>&#956;L per well&#41; was added to each well and the plates were incubated for another 4<span class="elsevierStyleHsp" style=""></span>h at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The reaction was then stopped with acidic isopropanol &#40;0&#46;4<span class="elsevierStyleHsp" style=""></span>mL 10<span class="elsevierStyleHsp" style=""></span>N HCl in 100<span class="elsevierStyleHsp" style=""></span>mL isopropanol&#44; 100<span class="elsevierStyleHsp" style=""></span>&#956;L per well&#41;&#44; and the absorbance was measured at 595<span class="elsevierStyleHsp" style=""></span>nm&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">5</span></a> The 50&#37; inhibitory concentration of zerumbone had been determined from the plot of percent inhibition against increasing concentrations&#46; Cytotoxic effect was also evaluated on PHA &#40;5<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;mL&#41; stimulated murine splenocytes &#40;1<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>cells per well&#41; cells without &#40;control&#41; or with increasing concentrations of zerumbone &#40;0&#46;1&#8211;50<span class="elsevierStyleHsp" style=""></span>&#956;M&#41;&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Analysis of cell cycle progression in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes</span><p id="par0020" class="elsevierStylePara elsevierViewall">2&#46;5<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>cells&#47;mL exponential phase <span class="elsevierStyleItalic">L&#46; donovani</span> AG83 promastigotes were incubated for 24<span class="elsevierStyleHsp" style=""></span>h and 48<span class="elsevierStyleHsp" style=""></span>h respectively in complete M199 medium in the presence or absence of 50&#37; inhibitory concentration of zerumbone on promastigotes at 22<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; After washing with 1&#215; PBS&#44; the cells were fixed in 45&#37; ethanol &#40;diluted in 1&#215; PBS&#41;&#44; treated with 500<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;mL RNAse A and then suspended in 0&#46;5<span class="elsevierStyleHsp" style=""></span>M sodium citrate containing 69<span class="elsevierStyleHsp" style=""></span>&#956;M PI&#46;<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">6</span></a> Acquisition was performed using a flow cytometer &#40;BD FACSVerse&#8482;&#44; BD Biosciences&#44; USA&#41; and the data were analyzed using Flowing software 2&#46;5&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Externalization of phosphatidyl serine</span><p id="par0025" class="elsevierStylePara elsevierViewall">In order to study the apoptosis inducing capacity of zerumbone in promastigotes&#44; the treated cells were stained with Annexin V-PE and 7-AAD as per manufacturer&#39;s instruction &#40;BD Pharmingen&#41;&#46; Briefly&#44; 2<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>log phase promastigotes were incubated with IC<span class="elsevierStyleInf">50</span> concentration of zerumbone in triplicate for 24<span class="elsevierStyleHsp" style=""></span>h and 48<span class="elsevierStyleHsp" style=""></span>h respectively&#46; They were washed twice with cold PBS and resuspended in 1&#215; binding buffer at a concentration of 1<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>mL<span class="elsevierStyleSup">&#8722;1</span>&#46; 100<span class="elsevierStyleHsp" style=""></span>&#956;L of the samples was transferred to a fresh tube and Annexin V-PE &#40;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41;&#44; 7-AAD &#40;5<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; were added&#44; incubated for 15<span class="elsevierStyleHsp" style=""></span>min at RT in the dark&#46; 400<span class="elsevierStyleHsp" style=""></span>&#956;L binding buffer was added and cells were acquired in a flow cytometer &#40;BD FACSVerse&#8482;&#44; BD Biosciences&#44; USA&#41; and analyzed using Flowing 2&#46;5 version software&#46;<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">5&#44;7</span></a></p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Estimation of reactive oxygen species</span><p id="par0030" class="elsevierStylePara elsevierViewall">In <span class="elsevierStyleItalic">Leishmania</span>&#44; oxidative stress has been suggested to be responsible for the apoptotic process&#46;<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">5&#44;6</span></a> To estimate the level of ROS&#44; the cell permeant probe H<span class="elsevierStyleInf">2</span>DCFDA &#40;2&#8242;&#44;7&#8242;-dichlorodihydrofluorescein diacetate&#41; was used and analyzed by flow cytometer as described previously&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">5</span></a> H<span class="elsevierStyleInf">2</span>DCFDA is a non-fluorescent dye which is converted into a fluorescent DCF &#40;2&#8242;&#44;7&#8242;-dichlorofluorescein&#41; in the presence of proper oxidants inside the cells&#46; Promastigotes were treated with IC<span class="elsevierStyleInf">50</span> concentration of zerumbone and the induction of ROS had been estimated at 1<span class="elsevierStyleHsp" style=""></span>h&#44; 3<span class="elsevierStyleHsp" style=""></span>h&#44; 5<span class="elsevierStyleHsp" style=""></span>h and 12<span class="elsevierStyleHsp" style=""></span>h by incubating with H<span class="elsevierStyleInf">2</span>DCFDA &#40;20<span class="elsevierStyleHsp" style=""></span>&#956;M&#41; at room temperature for 20<span class="elsevierStyleHsp" style=""></span>min in dark&#46; H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> has been used for positive control&#46;<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">6</span></a></p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Detection of chromatin condensation and cytoplasmic lipid droplet accumulation</span><p id="par0035" class="elsevierStylePara elsevierViewall">The chromatin condensation and lipid accumulation in zerumbone-treated promastigotes were detected under confocal microscope after staining with DAPI &#40;2<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;mL&#41; and Nile Red &#40;10<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;mL&#41; as described earlier&#46;<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">5&#8211;8</span></a> Images were obtained using an Olympus confocal laser scanning microscope &#40;Model&#58; IX81&#41; and analyzed by Olympus fluoview version 3&#46;0 viewer software&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Measurement of total lipid peroxidation</span><p id="par0040" class="elsevierStylePara elsevierViewall">As elevation of ROS is related with the increase in lipid peroxides&#44; we were keen to check the state of lipid peroxidation in treated promastigotes&#46; <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes &#40;10<span class="elsevierStyleSup">7</span>&#41; were treated with IC<span class="elsevierStyleInf">50</span> concentration of zerumbone for 1<span class="elsevierStyleHsp" style=""></span>h&#44; 3<span class="elsevierStyleHsp" style=""></span>h and 5<span class="elsevierStyleHsp" style=""></span>h respectively&#46; The cell-pellet was dissolved in 2<span class="elsevierStyleHsp" style=""></span>mL of 15&#37; SDS-PBS solution&#46; The fluorescence intensities of the total fluorescent lipid peroxidation products were measured with excitation at 360<span class="elsevierStyleHsp" style=""></span>nm and emission at 430<span class="elsevierStyleHsp" style=""></span>nm and expressed as relative fluorescence units with respect to quinine sulfate &#40;1<span class="elsevierStyleHsp" style=""></span>mg&#47;mL in 0&#46;5<span class="elsevierStyleHsp" style=""></span>M H<span class="elsevierStyleInf">2</span>SO<span class="elsevierStyleInf">4</span>&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">6</span></a></p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Detection of the change in morphology by scanning electron microscopy</span><p id="par0045" class="elsevierStylePara elsevierViewall">Control and treated promastigotes &#40;2<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>cells&#41; were fixed with 2&#46;5&#37; gluteraldehyde &#40;Sigma Aldrich&#41;&#44; dehydrated in ethanol&#44; critical point-dried in CO<span class="elsevierStyleInf">2</span>&#44; mounted on stubs&#44; sputtered with a thin gold layer<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">5&#44;6</span></a> and observed under a scanning electron microscope &#40;Model&#58; ZEISS EVO-MA 10&#41;&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Anti-proliferative activity on intracellular amastigotes</span><p id="par0050" class="elsevierStylePara elsevierViewall">Peritoneal macrophages were isolated from thioglycolate &#40;i&#46;p&#46;&#44; 4&#37; &#40;w&#47;v&#41;&#44; 1&#46;0<span class="elsevierStyleHsp" style=""></span>mL&#47;mouse&#41; elicited peritoneal lavage of 6&#8211;8 weeks old male BALB&#47;c mice as per the guidelines of institutional animal ethics committee of West Bengal State University&#46; Cells were allowed to adhere in 8-chambered slides &#40;10<span class="elsevierStyleSup">5</span><span class="elsevierStyleHsp" style=""></span>cells per well&#41; in complete RPMI-1640 at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in 5&#37; CO<span class="elsevierStyleInf">2</span> environment for 4<span class="elsevierStyleHsp" style=""></span>h&#46; The subsequent steps of washing &#40;3&#215; PBS&#41; were performed to move out the nonadherent cells and granulocytes and then the cultures were continued for another 48<span class="elsevierStyleHsp" style=""></span>h without any manipulation&#46;<a class="elsevierStyleCrossRefs" href="#bib0115"><span class="elsevierStyleSup">9&#44;10</span></a> Adhered resting macrophages were infected with stationary phase of promastigotes &#40;1&#58;10&#41;&#44; incubated for 6<span class="elsevierStyleHsp" style=""></span>h&#44; washed &#40;2&#215; PBS&#41; to remove the uningested promastigotes and cultured overnight in complete RPMI-1640 at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in 5&#37; CO<span class="elsevierStyleInf">2</span> environment&#46; Cells were washed &#40;3&#215;&#41; and incubated for additional 48<span class="elsevierStyleHsp" style=""></span>h in the presence or absence of graded concentrations of zerumbone&#46; Prechilled methanol-fixed cells were stained with Giemsa&#44; and examined under phase contrast microscope&#46; At least 400 macrophages were examined for each set&#46; Anti-leishmanial activity was determined by calculating the number of amastigotes per 100 macrophages&#46;<a class="elsevierStyleCrossRefs" href="#bib0115"><span class="elsevierStyleSup">9&#44;11</span></a></p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Statistical analyses</span><p id="par0055" class="elsevierStylePara elsevierViewall">Statistical analyses for all experiments were performed by one-way ANOVA followed by post hoc Holm&#8211;Sidak test with the program Sigma Plot&#46;</p></span></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Results</span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Analysis of phytochemicals</span><p id="par0060" class="elsevierStylePara elsevierViewall">The yield of essential oil was 0&#46;12&#37;&#46; GC&#8211;MS analyses of the essential oil led to the identification of ten major compounds accounting for the 98&#46;4&#37; of the oil&#46; Zerumbone &#40;75&#46;2&#37;&#41;&#44; &#945;-caryophyllene &#40;7&#46;1&#37;&#41;&#44; camphene &#40;5&#46;1&#37;&#41;&#44; eucalyptol &#40;2&#46;4&#37;&#41;&#44; and camphor &#40;3&#46;0&#37;&#41; 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 &#40;Supplementary Fig&#46; 1&#41;&#46; Compounds were quantified by performing area percentage calculations based on the total combined FID area&#46; The percentage of a peak is a percentage relative to all other constituents integrated in the FID chromatogram&#46; The differences in chemical composition of essential oil of the present study and previous research may be because of the geographic and climatic factors&#44; chemo types&#44; drying conditions and mode of distillation&#46; Zerumbone was isolated in pure form and its structure was confirmed by <span class="elsevierStyleSup">1</span>H NMR&#44; <span class="elsevierStyleSup">13</span>C NMR&#44; DEPT&#44; HR-ESIMS and comparison with literature data&#46;</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Zerumbone inhibited the proliferation of <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes</span><p id="par0065" class="elsevierStylePara elsevierViewall">Zerumbone was found to inhibit the growth of <span class="elsevierStyleItalic">Leishmania</span> promastigotes dose dependently&#44; <span class="elsevierStyleItalic">in vitro</span>&#46; At a concentration of 10<span class="elsevierStyleHsp" style=""></span>&#956;M&#44; zerumbone inhibited the growth of <span class="elsevierStyleItalic">L&#46; donovani</span> AG83 promastigotes approximately by 53&#46;43&#37;&#46; Interestingly&#44; the 50&#37; inhibitory concentration of zerumbone &#40;9&#46;36<span class="elsevierStyleHsp" style=""></span>&#956;M&#41; could only inhibit the proliferation of PHA induced murine splenocytes by 5&#46;75&#37; even at 96<span class="elsevierStyleHsp" style=""></span>h &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 1</a>&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Zerumbone induced morphological alterations in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes</span><p id="par0070" class="elsevierStylePara elsevierViewall">The treated promastigotes appeared rounded with loss of flagella with porous cell membrane &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 2</a>B&#41; in comparison to the flagellated and slender promastigotes of the control culture &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 2</a>A&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Zerumbone caused lipid accumulation in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes</span><p id="par0075" class="elsevierStylePara elsevierViewall">Another prominent effect resulting from the treatment of the promastigotes with zerumbone was the accumulation of lipid droplets in the cytoplasm &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 3</a>B and C&#41;&#44; probably resulting from the accumulation of lipid precursors due to the drastic alteration of the sterol content in the parasite membrane&#46; The alteration in lipid contents as evidenced from the deposition of lipid in the cytosol might also be correlated with plasma membrane integrity&#44; leading to apoptosis&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Zerumbone altered the cell cycle progression and induced externalization of phosphatidyl serine in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes</span><p id="par0080" class="elsevierStylePara elsevierViewall">At first&#44; we have shown that the cell cycle progression of promastigotes was arrested at the sub-G<span class="elsevierStyleInf">0</span>&#47;G<span class="elsevierStyleInf">1</span> time dependently&#46; At 24<span class="elsevierStyleHsp" style=""></span>h&#44; the proportion of cells in the sub-G<span class="elsevierStyleInf">0</span>&#47;G<span class="elsevierStyleInf">1</span> was found only 2&#46;9&#37; in comparison to 1&#46;6&#37; of the control culture&#46; Zerumbone further increased sub-G<span class="elsevierStyleInf">0</span>&#47;G<span class="elsevierStyleInf">1</span> cells from 1&#46;6&#37; &#40;untreated&#41; to 9&#46;9&#37; &#40;treated&#41; at 48<span class="elsevierStyleHsp" style=""></span>h accompanied by a decrease in the number of cells in G<span class="elsevierStyleInf">0</span>&#47;G<span class="elsevierStyleInf">1</span> from 59&#46;7&#37; to 50&#46;5&#37; &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 4</a>A&#41;&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia><p id="par0085" class="elsevierStylePara elsevierViewall">A significant step of apoptosis is the translocation of phosphatidyl serine from the inner to the outer leaflet of the plasma membrane&#46;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">12</span></a> The externalization of phosphatidyl serine residues was observed in 1&#46;39&#37; &#40;early apoptotic&#59; Annexin V only&#41; and in 0&#46;19&#37; &#40;late apoptotic&#59; Annexin V&#43; 7AAD&#43;&#41; of untreated promastigotes at 48<span class="elsevierStyleHsp" style=""></span>h&#46; After the treatment with zerumbone &#40;IC<span class="elsevierStyleInf">50</span> concentration&#41; for 48<span class="elsevierStyleHsp" style=""></span>h&#44; the percentage of early as well as late apoptotic cells was increased significantly with respect to untreated cells&#46; The percentage of Annexin V positive cells &#40;early apoptotic&#41; increased to 15&#46;7&#37; at 48<span class="elsevierStyleHsp" style=""></span>h&#46; The percentage of Annexin V positive 7AAD stained cells &#40;late apoptotic&#41; increased to 7&#46;58&#37; at 48<span class="elsevierStyleHsp" style=""></span>h with respect to the untreated cells &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 4</a>B&#41;&#46;</p></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Zerumbone caused DNA condensation in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes</span><p id="par0090" class="elsevierStylePara elsevierViewall">DAPI was used to measure DNA condensation in promastigotes&#46; DAPI staining showed discrete nuclei &#40;blue spots&#41; in untreated promastigotes whereas the treated promastigotes were observed to have blebbed nuclei and condensed chromatin material &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 5</a>B and C&#41;&#46;</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Zerumbone induced the oxidative stress in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes</span><p id="par0095" class="elsevierStylePara elsevierViewall">We found that 50&#37; inhibitory concentration of zerumbone against promastigotes could increase the level of ROS time dependently resulting in oxidative damage of the promastigotes &#40;Fig 6A&#41;&#46; The mean fluorescence intensity &#40;MFI&#41; of ROS generation in treated promastigotes increased time dependently in comparison to control culture &#40;<a class="elsevierStyleCrossRef" href="#fig0035">Fig&#46; 6</a>A&#41;&#46;</p><elsevierMultimedia ident="fig0035"></elsevierMultimedia></span><span id="sec0105" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Zerumbone increased the level of lipid peroxidation in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes</span><p id="par0100" class="elsevierStylePara elsevierViewall">Zerumbone elevated the level of lipid peroxides in a time dependent manner after 1<span class="elsevierStyleHsp" style=""></span>h of treatment and reached to maximum level at 12<span class="elsevierStyleHsp" style=""></span>h &#91;control vs treatment &#8211; 492&#46;14 vs 696&#46;55&#93; &#40;<a class="elsevierStyleCrossRef" href="#fig0035">Fig&#46; 6</a>B&#41;&#46;</p></span><span id="sec0110" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Zerumbone inhibited the intracellular amastigotes in infected macrophages</span><p id="par0105" class="elsevierStylePara elsevierViewall">Zerumbone was found effective against intracellular amastigotes and the 50&#37; inhibitory concentration was estimated with the treatment of 5<span class="elsevierStyleHsp" style=""></span>&#956;M of zerumbone at 48<span class="elsevierStyleHsp" style=""></span>h &#40;<a class="elsevierStyleCrossRef" href="#fig0040">Fig&#46; 7</a>&#41;&#46;</p><elsevierMultimedia ident="fig0040"></elsevierMultimedia></span></span><span id="sec0115" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Discussion</span><p id="par0110" class="elsevierStylePara elsevierViewall">Zerumbone is a naturally occurring dietary compound&#44; present in many natural foods consumed today&#46; The compound derived from several plant species of the Zingiberaceae family that has been found to possess multiple biomedical properties&#44; such as antiproliferative&#44; antioxidant&#44; anti-inflammatory&#44; and anticancer activities&#46;<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">2</span></a> However&#44; evidence of efficacy is sparse against protozoan infection to support therapeutic claims to identify future uses against <span class="elsevierStyleItalic">L&#46; donovani</span> infection&#46; In the present study we successfully analyzed the nature of zerumbone-mediated cell death in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes and the possible key cellular mediators involved in the death cascade&#46; Our initial observation that the zerumbone was effective against promastigotes but substantially non-toxic towards murine splenocytes &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 1</a>&#41; made us curious for further in depth study&#46; Morphological structure as observed through SEM has also authenticated the cytotoxic nature of zerumbone against <span class="elsevierStyleItalic">Leishmania</span> promastigotes &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 2</a>B and C&#41; which can be correlated with the unnatural lipid accumulation on treatment &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 3</a>B and C&#41;&#46; Interestingly&#44; we found that zerumbone could increase the sub-G<span class="elsevierStyleInf">0</span>&#47;G<span class="elsevierStyleInf">1</span> &#40;dead cells&#41; up to 9&#46;9&#37; from 1&#46;6&#37; as in control promastigotes &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 4</a>A&#41;&#44; concomitantly caused the externalization of phosphatidyl serine &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 4</a>B&#41; in promastigote plasma membrane which is a crucial step in the process of apoptosis&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">10</span></a> However&#44; recently an interesting study raised the question on relevance of Annexin V binding assay in detecting apoptosis in <span class="elsevierStyleItalic">Leishmania</span> as they showed that the promastigotes lack phosphatidyl serine and Annexin V can also bind other lipids&#44; including phosphatidylcholine&#44; phosphatidylethanolamine and phosphatidylinositol&#46;<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">13</span></a> Accordingly&#44; we enquired the effect of zerumbone on <span class="elsevierStyleItalic">Leishmania</span> chromatin condensation and nuclear blebbing&#44; the hallmark of apoptosis&#46; The debate regarding the induction of apoptosis by zerumbone has been resolved and further confirmed by the DNA condensation in promastigotes &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 5</a>B and C&#41;&#46; Looking into the mechanism&#44; we found that the oxidative stress &#40;<a class="elsevierStyleCrossRef" href="#fig0035">Fig&#46; 6</a>A&#41; followed by an increase in the level of lipid peroxidation &#40;<a class="elsevierStyleCrossRef" href="#fig0035">Fig&#46; 6</a>B&#41; in zerumbone-treated promastigotes might involve the alteration of mitochondrial membrane potential leading to apoptosis&#46;<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">14</span></a> The ultimate conviction came true when we found that zerumbone inhibited the clinically important morphs of <span class="elsevierStyleItalic">L&#46; donovani</span> in mammalian host&#44; the intracellular amastigotes in macrophages &#40;<a class="elsevierStyleCrossRef" href="#fig0040">Fig&#46; 7</a>&#41;&#46; In conclusion&#44; our findings indicate that zerumbone induced ROS-mediated apoptosis in <span class="elsevierStyleItalic">L&#46; donovani</span> promastigotes and further pharmacological studies on this particular anti-leishmanial efficacy&#44; <span class="elsevierStyleItalic">in vivo</span>&#44; against <span class="elsevierStyleItalic">L&#46; donovani</span> infection appear promising&#46;</p></span><span id="sec0120" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Funding</span><p id="par0115" class="elsevierStylePara elsevierViewall">This work was supported by Department of Biotechnology&#44; <span class="elsevierStyleGrantSponsor" id="gs1">Government of India</span> through a collaborative project between WBSU&#44; Barasat&#44; West Bengal and IBSD&#44; Imphal&#44; Manipur &#40;Project ref&#58; BT&#47;217&#47;NE&#47;TBP&#47;2011&#44; dated 15&#46;12&#46;2011&#41;&#46;</p></span><span id="sec0130" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Ethical approval</span><p id="par0125" class="elsevierStylePara elsevierViewall">Approved&#46;</p></span><span id="sec0135" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Conflicts of interest</span><p id="par0120" class="elsevierStylePara elsevierViewall">The authors declare no conflicts of interest&#46;</p></span></span>"
    "textoCompletoSecciones" => array:1 [
      "secciones" => array:11 [
        0 => array:3 [
          "identificador" => "xres599235"
          "titulo" => "Abstract"
          "secciones" => array:1 [
            0 => array:1 [
              "identificador" => "abst0005"
            ]
          ]
        ]
        1 => array:2 [
          "identificador" => "xpalclavsec613640"
          "titulo" => "Keywords"
        ]
        2 => array:2 [
          "identificador" => "sec0005"
          "titulo" => "Introduction"
        ]
        3 => array:3 [
          "identificador" => "sec0010"
          "titulo" => "Materials and methods"
          "secciones" => array:10 [
            0 => array:2 [
              "identificador" => "sec0015"
              "titulo" => "Extraction of essential oil and Purification of zerumbone"
            ]
            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Parasites maintenance and viability assay"
            ]
            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "Analysis of cell cycle progression in L&#46; donovani promastigotes"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Externalization of phosphatidyl serine"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "Estimation of reactive oxygen species"
            ]
            5 => array:2 [
              "identificador" => "sec0040"
              "titulo" => "Detection of chromatin condensation and cytoplasmic lipid droplet accumulation"
            ]
            6 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Measurement of total lipid peroxidation"
            ]
            7 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "Detection of the change in morphology by scanning electron microscopy"
            ]
            8 => array:2 [
              "identificador" => "sec0055"
              "titulo" => "Anti-proliferative activity on intracellular amastigotes"
            ]
            9 => array:2 [
              "identificador" => "sec0060"
              "titulo" => "Statistical analyses"
            ]
          ]
        ]
        4 => array:3 [
          "identificador" => "sec0065"
          "titulo" => "Results"
          "secciones" => array:9 [
            0 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "Analysis of phytochemicals"
            ]
            1 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "Zerumbone inhibited the proliferation of L&#46; donovani promastigotes"
            ]
            2 => array:2 [
              "identificador" => "sec0080"
              "titulo" => "Zerumbone induced morphological alterations in L&#46; donovani promastigotes"
            ]
            3 => array:2 [
              "identificador" => "sec0085"
              "titulo" => "Zerumbone caused lipid accumulation in L&#46; donovani promastigotes"
            ]
            4 => array:2 [
              "identificador" => "sec0090"
              "titulo" => "Zerumbone altered the cell cycle progression and induced externalization of phosphatidyl serine in L&#46; donovani promastigotes"
            ]
            5 => array:2 [
              "identificador" => "sec0095"
              "titulo" => "Zerumbone caused DNA condensation in L&#46; donovani promastigotes"
            ]
            6 => array:2 [
              "identificador" => "sec0100"
              "titulo" => "Zerumbone induced the oxidative stress in L&#46; donovani promastigotes"
            ]
            7 => array:2 [
              "identificador" => "sec0105"
              "titulo" => "Zerumbone increased the level of lipid peroxidation in L&#46; donovani promastigotes"
            ]
            8 => array:2 [
              "identificador" => "sec0110"
              "titulo" => "Zerumbone inhibited the intracellular amastigotes in infected macrophages"
            ]
          ]
        ]
        5 => array:2 [
          "identificador" => "sec0115"
          "titulo" => "Discussion"
        ]
        6 => array:2 [
          "identificador" => "sec0120"
          "titulo" => "Funding"
        ]
        7 => array:2 [
          "identificador" => "sec0130"
          "titulo" => "Ethical approval"
        ]
        8 => array:2 [
          "identificador" => "sec0135"
          "titulo" => "Conflicts of interest"
        ]
        9 => array:2 [
          "identificador" => "xack201801"
          "titulo" => "Acknowledgements"
        ]
        10 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2015-06-20"
    "fechaAceptado" => "2015-10-01"
    "PalabrasClave" => array:1 [
      "en" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec613640"
          "palabras" => array:5 [
            0 => "<span class="elsevierStyleItalic">Leishmania donovani</span>"
            1 => "<span class="elsevierStyleItalic">Zingiber zerumbet</span>"
            2 => "Zerumbone"
            3 => "Anti-leishmanial"
            4 => "ROS"
          ]
        ]
      ]
    ]
    "tieneResumen" => true
    "resumen" => array:1 [
      "en" => array:2 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">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 <span class="elsevierStyleItalic">Leishmania</span> infection&#46; Various ginger spices of the Zingiberaceae family are widely used as spices&#44; flavouring agents&#44; and medicines in Southeast Asia because of their unique flavour as well as due to their medicinal properties&#46; Zerumbone&#44; a natural component of <span class="elsevierStyleItalic">Zingiber zerumbet</span> &#40;L&#46;&#41; Smith&#44; has been studied for its pharmacological potential as antiulcer&#44; antioxidant&#44; anticancer&#44; and antimicrobial&#46; In this study&#44; we have shown that zerumbone could induce ROS mediated apoptosis in <span class="elsevierStyleItalic">Leishmania donovani</span> promastigotes and also found effective in reducing intracellular amastigotes in infected-macrophages&#46; We emphasized the potential of zerumbone to be employed in the development of new therapeutic drugs against <span class="elsevierStyleItalic">L&#46; donovani</span> infection and provided the basis for future research on the application of transitional medicinal plants&#46;</p></span>"
      ]
    ]
    "NotaPie" => array:3 [
      0 => array:3 [
        "etiqueta" => "1"
        "nota" => "<p class="elsevierStyleNotepara" id="npar0005">These author contributed equally to this work&#46;</p>"
        "identificador" => "fn1"
      ]
      1 => array:3 [
        "etiqueta" => "2"
        "nota" => "<p class="elsevierStyleNotepara" id="npar0010">Present address&#58; Post Graduate Department of Zoology&#44; Barasat Government College&#44; Barasat&#44; West Bengal&#44; India&#46;</p>"
        "identificador" => "fn2"
      ]
      2 => array:3 [
        "etiqueta" => "3"
        "nota" => "<p class="elsevierStyleNotepara" id="npar0015">Present address&#58; PG Department of Microbiology&#44; APC College&#44; New Barrackpore&#44; West Bengal&#44; India&#46;</p>"
        "identificador" => "fn3"
      ]
    ]
    "apendice" => array:1 [
      0 => array:1 [
        "seccion" => array:1 [
          0 => array:4 [
            "apendice" => "<p id="par0140" class="elsevierStylePara elsevierViewall">The following are the supplementary data to this article&#58;<elsevierMultimedia ident="fig0005"></elsevierMultimedia></p>"
            "etiqueta" => "Appendix A"
            "titulo" => "Supplementary data"
            "identificador" => "sec0145"
          ]
        ]
      ]
    ]
    "multimedia" => array:8 [
      0 => array:7 [
        "identificador" => "fig0010"
        "etiqueta" => "Fig&#46; 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr1.jpeg"
            "Alto" => 1168
            "Ancho" => 1510
            "Tamanyo" => 88082
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Anti-proliferative effect of zerumbone against <span class="elsevierStyleItalic">L&#46; donovani</span>-promastigotes and PHA induced murine splenocytes with different concentrations of zerumbone &#40;0&#46;1&#8211;50<span class="elsevierStyleHsp" style=""></span>&#956;M&#41; as determined by MTT at 48<span class="elsevierStyleHsp" style=""></span>h and 96<span class="elsevierStyleHsp" style=""></span>h respectively&#46; Each point corresponds to the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD of at least three experiments&#46; Statistical significance was determined by one-way ANOVA followed by Holm&#8211;Sidak post hoc test &#40;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#44; &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#44; &#42;&#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;01 vs control&#41;&#46;</p>"
        ]
      ]
      1 => array:7 [
        "identificador" => "fig0015"
        "etiqueta" => "Fig&#46; 2"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr2.jpeg"
            "Alto" => 1018
            "Ancho" => 750
            "Tamanyo" => 51356
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Scanning electron microscopy was performed to determine severe alterations in the morphology of zerumbone treated <span class="elsevierStyleItalic">L&#46; donovani</span>-promastigotes in comparison to control culture&#46; Images are representative profile of at least three independent experiments&#46;</p>"
        ]
      ]
      2 => array:7 [
        "identificador" => "fig0020"
        "etiqueta" => "Fig&#46; 3"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr3.jpeg"
            "Alto" => 2005
            "Ancho" => 2948
            "Tamanyo" => 276916
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Accumulation of cytosolic lipid in zerumbone-treated <span class="elsevierStyleItalic">L&#46; donovani</span> AG83 promastigotes as evident by nile red staining &#40;magnification&#58; 100&#215;&#44; zoom&#58; 3&#46;4&#215;&#41;&#46; &#40;A&#41; Phase contrast&#59; &#40;B&#41; fluorescence&#59; &#40;C&#41; phase contrast&#8211;fluorescence merge&#46; Zerumbone treatment significantly increased lipid droplets accumulation &#40;white arrows&#41; in the cytosol in comparison to the untreated cells&#46; Images are representative profile of at least three independent experiments&#46;</p>"
        ]
      ]
      3 => array:7 [
        "identificador" => "fig0025"
        "etiqueta" => "Fig&#46; 4"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr4.jpeg"
            "Alto" => 2123
            "Ancho" => 2961
            "Tamanyo" => 366916
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">&#40;A&#41; Cell cycle progression in zerumbone treated promastigotes was assessed flow cytometrically by Propidium Iodide staining&#46; Zerumbone induced cell death in promastigotes at 48<span class="elsevierStyleHsp" style=""></span>h by increasing the proportion of sub G<span class="elsevierStyleInf">0</span>&#8211;G<span class="elsevierStyleInf">1</span> cells in comparison to control culture&#46; Data are representative of at least three independent experiments&#46; &#40;B&#41; Zerumbone caused the externalization of phosphatidyl serine as estimated by Annexin V and 7-AAD incorporation assay&#46; The proportions of cells at early apoptotic &#40;Annexin V&#43; 7AAD&#8722;&#41; and late apoptotic phase &#40;Annexin V&#43; 7AAD&#43;&#41; were increased time dependently following zerumbone treatment&#46; Values represent the percentage of positive cells at the respective quadrants&#46; Data are representative of three independent experiments&#46;</p>"
        ]
      ]
      4 => array:7 [
        "identificador" => "fig0030"
        "etiqueta" => "Fig&#46; 5"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr5.jpeg"
            "Alto" => 1961
            "Ancho" => 2957
            "Tamanyo" => 185455
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Zerumbone induced DNA condensation as visualized after DAPI staining at 48<span class="elsevierStyleHsp" style=""></span>h&#46; Images were taken using an Olympus fluoview confocal microscope &#40;Model&#58; IX81&#41; and analyzed by Olympus fluoview ver&#46;3&#46;0 viewer software &#40;magnification&#58; 100&#215;&#44; zoom&#58; 3&#46;4&#215;&#41;&#46; &#40;A&#41; Phase contrast&#59; &#40;B&#41; fluorescence&#59; &#40;C&#41; phase contrast&#8211;fluorescence merge&#46; In untreated promastigotes&#44; the nuclei &#40;K&#58; kinetoplast&#59; N&#58; nucleus&#41; appeared as distinct blue structure&#44; whereas zerumbone treated promastigotes showed blebbed nuclei and condensed chromatin material &#40;white arrow&#41;&#46; Images are representative profile of at least three experiments&#46;</p>"
        ]
      ]
      5 => array:7 [
        "identificador" => "fig0035"
        "etiqueta" => "Fig&#46; 6"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr6.jpeg"
            "Alto" => 1131
            "Ancho" => 2910
            "Tamanyo" => 143143
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">&#40;A&#41; Zerumbone induced ROS production in <span class="elsevierStyleItalic">L&#46; donovani</span> AG83 promastigotes&#46; ROS generation in treated promastigotes has been measured using H<span class="elsevierStyleInf">2</span>DCFDA at 1&#44; 3&#44; 5 and 12<span class="elsevierStyleHsp" style=""></span>h&#46; Treatment of promastigotes with IC<span class="elsevierStyleInf">50</span> concentration of zerumbone revealed an elevation of intracellular ROS time dependently&#46; However&#44; pretreatment of promastigotes with the antioxidant NAC before treatment with zerumbone abrogated ROS generation in each time point&#46; Each point corresponds to the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD of at least three experiments&#46; Statistical significance was determined by one-way ANOVA followed by Holm&#8211;Sidak post hoc test &#40;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#44; &#42;&#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;002&#44; &#42;&#42;&#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;005 vs control&#59; &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001 vs treatment&#41;&#46; &#40;B&#41; Zerumbone increased the level of lipid peroxidation time dependently&#46; The total fluorescent lipid peroxidation products was quantified with excitation at 360<span class="elsevierStyleHsp" style=""></span>nm and emission at 430<span class="elsevierStyleHsp" style=""></span>nm and expressed as relative fluorescence units with respect to quinine sulfate &#40;1<span class="elsevierStyleHsp" style=""></span>mg&#47;mL in 0&#46;5<span class="elsevierStyleHsp" style=""></span>M H<span class="elsevierStyleInf">2</span>SO4&#41; by a spectrofluorometer at 1&#44; 3 and 5<span class="elsevierStyleHsp" style=""></span>h&#46; Each point corresponds to the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD of at least three experiments in duplicate&#46; Statistical significance was determined by one-way ANOVA followed by Holm&#8211;Sidak post hoc test &#40;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;04&#44; &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;002&#44; &#42;&#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;005 vs control&#41;&#46;</p>"
        ]
      ]
      6 => array:7 [
        "identificador" => "fig0040"
        "etiqueta" => "Fig&#46; 7"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr7.jpeg"
            "Alto" => 1185
            "Ancho" => 1523
            "Tamanyo" => 63914
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Zerumbone inhibited the proliferation of intracellular amastigotes with an IC<span class="elsevierStyleInf">50</span> of only 5<span class="elsevierStyleHsp" style=""></span>&#956;M&#46; Thioglycolate-elicited peritoneal macrophages of BALB&#47;c mice were infected <span class="elsevierStyleItalic">in vitro</span> with <span class="elsevierStyleItalic">L&#46; donovani</span> AG83 promastigotes&#46; After established infection&#44; cells were incubated with or without &#40;control&#41; graded concentrations &#40;0&#46;5&#8211;10<span class="elsevierStyleHsp" style=""></span>&#956;M&#41; of zerumbone at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in 5&#37; CO<span class="elsevierStyleInf">2</span> environment for the determination of anti-leishmanial activity on intracellular amastigotes&#46; Each point corresponds to the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD of at least three experiments in duplicate&#46; Statistical significance was determined by one-way ANOVA followed by Holm&#8211;Sidak post hoc test &#40;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#44; &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;002 vs infected control&#41;&#46;</p>"
        ]
      ]
      7 => array:7 [
        "identificador" => "fig0005"
        "etiqueta" => "Supplementary Fig&#46; 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => false
        "mostrarDisplay" => true
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "mmc1.jpeg"
            "Alto" => 2115
            "Ancho" => 2500
            "Tamanyo" => 244562
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">GC Spectra of essential oil of <span class="elsevierStyleItalic">Z&#46; zerumbet</span> &#40;the upper spectra is the standard hydrocarbon while the lower portion of the spectra is the oils&#41;&#46;</p>"
        ]
      ]
    ]
    "bibliografia" => array:2 [
      "titulo" => "References"
      "seccion" => array:1 [
        0 => array:2 [
          "identificador" => "bibs0005"
          "bibliografiaReferencia" => array:14 [
            0 => array:3 [
              "identificador" => "bib0075"
              "etiqueta" => "1"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Calpain and reactive oxygen species targets Bax for mitochondrial permeabilization and caspase activation in zerumbone induced apoptosis"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:3 [
                            0 => "P&#46; Sobhan"
                            1 => "M&#46; Seervi"
                            2 => "L&#46; Deb"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1371/journal.pone.0059350"
                      "Revista" => array:5 [
                        "tituloSerie" => "PLoS ONE"
                        "fecha" => "2013"
                        "volumen" => "8"
                        "paginaInicial" => "e59350"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/23593137"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            1 => array:3 [
              "identificador" => "bib0080"
              "etiqueta" => "2"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Attractive reactivity of a natural product&#44; zerumbone"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:1 [
                            0 => "T&#46; Kitayama"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1271/bbb.100532"
                      "Revista" => array:6 [
                        "tituloSerie" => "Biosci Biotechnol Biochem"
                        "fecha" => "2011"
                        "volumen" => "75"
                        "paginaInicial" => "199"
                        "paginaFinal" => "207"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/21307568"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            2 => array:3 [
              "identificador" => "bib0085"
              "etiqueta" => "3"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Chemical composition and biological activity of the essential oil of rhizome of <span class="elsevierStyleItalic">Zingiber zerumbet</span> &#40;L&#46;&#41; Smith"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "C&#46;B&#46; Singh"
                            1 => "B&#46;C&#46; Saikhom"
                            2 => "K&#46;H&#46; Lenin"
                            3 => "S&#46; Ningombam"
                            4 => "C&#46; Charles"
                            5 => "A&#46;R&#46; Samir"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:5 [
                        "tituloSerie" => "J Pharmacog Phytochem"
                        "fecha" => "2014"
                        "volumen" => "3"
                        "paginaInicial" => "130"
                        "paginaFinal" => "133"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            3 => array:3 [
              "identificador" => "bib0090"
              "etiqueta" => "4"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Dual extraction of essential oil and podophyllotoxin from creeping juniper &#40;<span class="elsevierStyleItalic">Juniperus horizontalis</span>&#41;"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "C&#46;L&#46; Cantrell"
                            1 => "V&#46;D&#46; Zheljazkov"
                            2 => "C&#46;R&#46; Carvalho"
                            3 => "T&#46; Astatkie"
                            4 => "E&#46;A&#46; Jeliazkova"
                            5 => "L&#46; Rosa"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1371/journal.pone.0106057"
                      "Revista" => array:5 [
                        "tituloSerie" => "PLoS ONE"
                        "fecha" => "2014"
                        "volumen" => "9"
                        "paginaInicial" => "e106057"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/25203255"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            4 => array:3 [
              "identificador" => "bib0095"
              "etiqueta" => "5"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Protective effect of <span class="elsevierStyleItalic">Croton caudatus</span> Geisel leaf extract against experimental visceral leishmaniasis induces proinflammatory cytokines in vitro and in vivo"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:3 [
                            0 => "S&#46; Dey"
                            1 => "D&#46; Mukherjee"
                            2 => "S&#46; Chakraborty"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1016/j.exppara.2015.09.011"
                      "Revista" => array:6 [
                        "tituloSerie" => "Exp Parasitol"
                        "fecha" => "2015"
                        "volumen" => "151&#8211;152"
                        "paginaInicial" => "84"
                        "paginaFinal" => "95"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26420465"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            5 => array:3 [
              "identificador" => "bib0100"
              "etiqueta" => "6"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "A novel triterpene from <span class="elsevierStyleItalic">Astraeus hygrometricus</span> induces reactive oxygen species leading to death in <span class="elsevierStyleItalic">Leishmania donovani</span>"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:3 [
                            0 => "S&#46; Mallick"
                            1 => "S&#46; Dey"
                            2 => "S&#46; Mandal"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:5 [
                        "tituloSerie" => "Fut Microbiol"
                        "fecha" => "2015"
                        "volumen" => "10"
                        "paginaInicial" => "763"
                        "paginaFinal" => "789"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            6 => array:3 [
              "identificador" => "bib0105"
              "etiqueta" => "7"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Selective inhibition of <span class="elsevierStyleItalic">Leishmania donovani</span> by active extracts of wild mushrooms used by the tribal population of India&#58; an in vitro exploration for new leads against parasitic protozoans"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:3 [
                            0 => "S&#46; Mallick"
                            1 => "A&#46; Dutta"
                            2 => "S&#46; Dey"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1016/j.exppara.2014.01.002"
                      "Revista" => array:6 [
                        "tituloSerie" => "Exp Parasitol"
                        "fecha" => "2014"
                        "volumen" => "138"
                        "paginaInicial" => "9"
                        "paginaFinal" => "17"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/24440295"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            7 => array:3 [
              "identificador" => "bib0110"
              "etiqueta" => "8"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "In vitro activities of ER-119884 and E5700&#44; two potent squalene synthase inhibitors&#44; against <span class="elsevierStyleItalic">Leishmania amazonensis</span>&#58; antiproliferative&#44; biochemical&#44; and ultrastructural effects"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "J&#46;C&#46;F&#46; Rodrigues"
                            1 => "J&#46;L&#46; Concepcion"
                            2 => "C&#46; Rodrigues"
                            3 => "A&#46; Caldera"
                            4 => "J&#46;A&#46; Urbina"
                            5 => "W&#46; Souza"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1128/AAC.01616-07"
                      "Revista" => array:5 [
                        "tituloSerie" => "Antimicrob Agents Chemother"
                        "fecha" => "2008"
                        "volumen" => "52"
                        "paginaInicial" => "4098"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/18765694"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            8 => array:3 [
              "identificador" => "bib0115"
              "etiqueta" => "9"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "<span class="elsevierStyleItalic">Mycobacterium indicus pranii</span> &#40;Mw&#41;-mediated protection against visceral leishmaniasis&#58; involvement of TLR4 signalling"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:3 [
                            0 => "A&#46; Adhikari"
                            1 => "S&#46; Majumder"
                            2 => "S&#46; Banerjee"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1093/jac/dks315"
                      "Revista" => array:6 [
                        "tituloSerie" => "J Antimicrob Chemother"
                        "fecha" => "2012"
                        "volumen" => "67"
                        "paginaInicial" => "2892"
                        "paginaFinal" => "2902"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22879460"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            9 => array:3 [
              "identificador" => "bib0120"
              "etiqueta" => "10"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Cutting edge&#58; endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:3 [
                            0 => "F&#46; Nomura"
                            1 => "S&#46; Akashi"
                            2 => "Y&#46; Sakao"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:6 [
                        "tituloSerie" => "J Immunol"
                        "fecha" => "2000"
                        "volumen" => "164"
                        "paginaInicial" => "3476"
                        "paginaFinal" => "3479"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/10725699"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            10 => array:3 [
              "identificador" => "bib0125"
              "etiqueta" => "11"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Protective therapy with novel chromone derivative against <span class="elsevierStyleItalic">Leishmania donovani</span> infection induces Th1 response in vivo"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:3 [
                            0 => "S&#46; Mallick"
                            1 => "A&#46; Dutta"
                            2 => "J&#46; Ghosh"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1159/000330856"
                      "Revista" => array:6 [
                        "tituloSerie" => "Chemotherapy"
                        "fecha" => "2011"
                        "volumen" => "57"
                        "paginaInicial" => "388"
                        "paginaFinal" => "393"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22024637"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            11 => array:3 [
              "identificador" => "bib0130"
              "etiqueta" => "12"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "A novel assay for apoptosis&#46; Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:4 [
                            0 => "I&#46; Vermes"
                            1 => "C&#46; Haanen"
                            2 => "H&#46; Steffens-Nakken"
                            3 => "C&#46; Reutelingsperger"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:6 [
                        "tituloSerie" => "J Immunol Methods"
                        "fecha" => "1995"
                        "volumen" => "184"
                        "paginaInicial" => "39"
                        "paginaFinal" => "51"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/7622868"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            12 => array:3 [
              "identificador" => "bib0135"
              "etiqueta" => "13"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "<span class="elsevierStyleItalic">Leishmania</span> promastigotes lack phosphatidylserine but bind Annexin V upon permeabilization or miltefosine treatment"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:3 [
                            0 => "A&#46; Weing&#228;rtner"
                            1 => "G&#46; Kemmer"
                            2 => "F&#46;D&#46; M&#252;ller"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1371/journal.pone.0042070"
                      "Revista" => array:5 [
                        "tituloSerie" => "PLoS ONE"
                        "fecha" => "2012"
                        "volumen" => "7"
                        "paginaInicial" => "e42070"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22870283"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            13 => array:3 [
              "identificador" => "bib0140"
              "etiqueta" => "14"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Induction of apoptosis by the medium-chain length fatty acid lauric acid in colon cancer cells due to induction of oxidative stress"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:4 [
                            0 => "J&#46;K&#46; Fauser"
                            1 => "G&#46;M&#46; Matthews"
                            2 => "A&#46;G&#46; Cummins"
                            3 => "G&#46;S&#46; Howarth"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1159/000356067"
                      "Revista" => array:6 [
                        "tituloSerie" => "Chemotherapy"
                        "fecha" => "2013"
                        "volumen" => "59"
                        "paginaInicial" => "214"
                        "paginaFinal" => "224"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/24356281"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
          ]
        ]
      ]
    ]
    "agradecimientos" => array:1 [
      0 => array:4 [
        "identificador" => "xack201801"
        "titulo" => "Acknowledgements"
        "texto" => "<p id="par0130" class="elsevierStylePara elsevierViewall">The authors are indebted to Vice Chancellor&#44; West Bengal State University and Director&#44; Institute of Bioresource and Sustainable Development &#40;IBSD&#41; for providing them the research infrastructures for this work&#46; We are also thankful to the Director&#44; CU BD Centre of Excellence for Nanobiotechnology&#44; CRNN and DBT-IPLS&#44; University of Calcutta for Flow Cytometry&#44; SEM and Confocal Microscopy facility&#46;</p>"
        "vista" => "all"
      ]
    ]
  ]
  "idiomaDefecto" => "en"
  "url" => "/14138670/0000002000000001/v1_201601240048/S1413867015002032/v1_201601240048/en/main.assets"
  "Apartado" => array:4 [
    "identificador" => "36721"
    "tipo" => "SECCION"
    "en" => array:2 [
      "titulo" => "Original article"
      "idiomaDefecto" => true
    ]
    "idiomaDefecto" => "en"
  ]
  "PDF" => "https://static.elsevier.es/multimedia/14138670/0000002000000001/v1_201601240048/S1413867015002032/v1_201601240048/en/main.pdf?idApp=UINPBA00003Y&text.app=https://bjid.org.br/"
  "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1413867015002032?idApp=UINPBA00003Y"
]

Article information

ISSN: 14138670
Original language: English

DOI:10.1016/j.bjid.2015.10.002

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 October	27	19	46
2024 September	30	19	49
2024 August	33	26	59
2024 July	66	41	107
2024 June	38	27	65
2024 May	27	34	61
2024 April	38	32	70
2024 March	38	25	63
2024 February	35	14	49
2024 January	30	18	48
2023 December	29	33	62
2023 November	45	29	74
2023 October	25	46	71
2023 September	26	43	69
2023 August	20	13	33
2023 July	30	17	47
2023 June	32	15	47
2023 May	53	19	72
2023 April	23	12	35
2023 March	58	36	94
2023 February	35	20	55
2023 January	17	6	23
2022 December	53	23	76
2022 November	51	30	81
2022 October	50	29	79
2022 September	35	36	71
2022 August	20	24	44
2022 July	26	48	74
2022 June	16	25	41
2022 May	17	26	43
2022 April	37	43	80
2022 March	26	43	69
2022 February	43	33	76
2022 January	40	30	70
2021 December	32	37	69
2021 November	44	45	89
2021 October	33	24	57
2021 September	17	32	49
2021 August	27	26	53
2021 July	27	23	50
2021 June	35	27	62
2021 May	35	49	84
2021 April	49	55	104
2021 March	31	32	63
2021 February	23	13	36
2021 January	40	21	61
2020 December	41	29	70
2020 November	19	20	39
2020 October	27	22	49
2020 September	32	22	54
2020 August	24	10	34
2020 July	20	16	36
2020 June	24	10	34
2020 May	50	15	65
2020 April	22	10	32
2020 March	26	23	49
2020 February	108	52	160
2020 January	23	12	35
2019 December	20	11	31
2019 November	15	23	38
2019 October	11	12	23
2019 September	23	25	48
2019 August	37	38	75
2019 July	25	25	50
2019 June	20	19	39
2019 May	55	43	98
2019 April	24	21	45
2019 March	26	26	52
2019 February	21	20	41
2019 January	13	13	26
2018 December	25	21	46
2018 November	65	34	99
2018 October	173	47	220
2018 September	38	21	59
2018 July	1	1	2
2018 June	2	0	2
2018 May	12	7	19
2018 April	38	14	52
2018 March	20	9	29
2018 February	25	17	42
2018 January	13	9	22
2017 December	30	17	47
2017 November	35	10	45
2017 October	24	8	32
2017 September	34	13	47
2017 August	25	8	33
2017 July	28	12	40
2017 June	26	13	39
2017 May	47	13	60
2017 April	30	9	39
2017 March	30	9	39
2017 February	18	4	22
2017 January	13	6	19
2016 December	89	19	108
2016 November	39	9	48
2016 October	56	11	67
2016 September	90	4	94
2016 August	27	5	32
2016 July	17	9	26
2016 May	1	0	1
2016 April	1	0	1
2016 March	1	0	1
2016 February	1	0	1
2016 January	2	0	2

Show all

Indexed in:

Follow us:

Statistics

Indexed in:

Follow us:

Statistics

Subscribe to our newsletter