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Sep 6, 2012 - Department of Biotechnology, Jamia Hamdard. 8. (Hamdard University), New Delhi 110 062, India. 9. 2. Institute of Nuclear Medicine and Allied ...
Journal of Medical Microbiology Papers in Press. Published September 6, 2012 as doi:10.1099/jmm.0.049387-0

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Artemisia annua leaves and seeds mediate programmed cell death in Leishmania donovani

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Mohammad Islamuddin, 1 Abdullah Farooque, Dinkar Sahal 3 and Farhat Afrin 1

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B. S. Dwarakanath,

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Parasite Immunology Lab. Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India 2 Institute of Nuclear Medicine and Allied Sciences, Timarpur, Delhi 110 052, India 3 International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India

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Corresponding Author: Farhat Afrin Parasite Immunology Lab. Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India. Email: [email protected], [email protected]

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Abstract

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Leishmaniasis is one of the major tropical parasitic diseases that ranges in severity from the self-

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healing cutaneous lesions to fatal visceral manifestations. On one hand, there is no vaccine

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available against visceral leishmaniasis (VL) (also known as Kala-azar in India) and on the other

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hand drug resistance, variable efficacy, toxicity and parenteral administration are the main

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drawbacks of current antileishmanial drugs. We report that n-hexane fraction of Artemisia annua

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leaves (AAL) and seeds (AAS) possess significant antileishmanial activity against Leishmania

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donovani promastigotes with GI50 of 14.4 and 14.6 μg ml─1, respectively and the IC50 against

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intracellular amastigotes was found to be 6.6 and 5.05 µgml─1, respectively. Changes in the

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morphology of promastigotes and growth reversibility analysis following treatment confirmed

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the leishmanicidal effect of the active fractions, which presented no cytotoxic effect on

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mammalian cells. Furthermore, the anti-leishmanial activity was mediated via apoptosis as

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evidenced by externalization of phosphatidylserine, in situ labelling of DNA fragments by

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terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle

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arrest at the sub-G0/G1 phase. High performance thin layer chromatography (HPTLC) fingerprint

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showed that the content of artemisinin in the crude bioactive extracts (~1.4 μg 100μg─1 n-hexane

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fractions) was too low to account for the observed antileishmanial activity. Characterization of

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the active constituents by Gas chromatography-Mass spectroscopy GC-MS showed that α−

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amyrinyl acetate, β- amyrine and derivatives of artemisinin were the major constituents in AAL

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and cetin, einecs and artemisinin derivatives in AAS. Our findings indicate the presence of

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antileishmanial compounds besides artemisinin in the n-hexane fraction of A. annua leaves and

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seeds.

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INTRODUCTION: Leishmaniasis, a complex spectrum of diseases, is currently a global health

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problem occurring as cutaneous, mucocutaneous or visceral forms. Visceral leishmaniasis (VL),

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or kala-azar, caused by Leishmania donovani, is often associated with marked suppression of the

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host’s cell-mediated immune response, leading to severe morbidity and mortality, if left

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untreated (Halder et al., 1983). VL is also considered as an opportunistic infection among

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immunocompromised patients around the world (Albrecht et al., 1996). Treatment of VL mainly

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relies on pentavalent antimonials and second line drugs such as amphotericin B and pentamidine.

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However, resistance to antimonial chemotherapy, coupled with high cost, toxicity and parenteral 1

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route of administration, is a matter of great concern in the endemic regions of Bihar, India

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(Sundar et al., 2000). In view of the ongoing challenge to introduce new antileishmanial drugs,

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traditional medicinal plants are expected to provide valuable leads (Ahua et al., 2007; Sarkar et

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al., 2008; Misra et al., 2009; Mokoka et al., 2011). According to World Health Organization

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(WHO), approximately 80% of the world inhabitants rely on traditional medicines for their

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healthcare (WHO, 2000). Artemisia annua L (Asteraceae), a traditional medicinal plant has been

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extensively used as an anti-malarial (Willcox et al., 2007 and Atemnkeng et al., 2009) and anti-

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cancer agent. Recently, the efficacy of artemisinin (one of the constituent of A. annua, A. indica,

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and A. dracunculus) against promastigotes and amastigotes of L. donovani have been reported

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(Sen et al., 2007). The antileishmanial activity of ethanolic extracts of the leaves of A. indica was

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investigated in exponential phase promastigotes from six strains responsible for cutaneous,

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mucocutaneous or visceral leishmaniasis and the IC50 value ranged from 210 µg ml─1 to 580 µg

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ml─1 (Ganguly et al., 2006). Flavonoids from A. annua as antioxidants and their potential

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synergy with artemisinin against malaria and cancer have been observed (Ferreira et al., 2010).

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In this study, we delineated the putative mechanisms contributing to leishmanicidal activity of

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the bioactive fractions from A. annua leaves and seeds with the aim of developing effective

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herbal remedy for the treatment of VL that may complement or synergise with the conventional

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drugs against VL.

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Abbreviations: AAL, n-hexane fraction of Artemisia annua leaves; AAS, n-hexane fraction of

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Artemisia annua seeds; MFI, mean fluorescence intensity; PCD, programmed cell death; PI,

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propidium

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deoxynucleotidyltransferase mediated dUTP nick end labelling.

iodide;

FITC,

fluorescein

isothiocyanate;

TUNEL,

terminal

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METHODS:

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Materials. M199 medium, propidium iodide (PI) and RNase were obtained from Sigma-Aldrich,

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fetal bovine serum (FBS) from Gibco-BRL, DMSO from SRL, methanol from Merck and

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Annexin V-FITC and the ApoDirect kits were from Roche Inc., Basel, Switzerland. All other

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chemicals were from Sigma-Aldrich unless stated.

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Parasite culture: L. donovani strain AG83 was a kind gift from Dr. Nahid Ali, IICB, Kolkata

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and was maintained in vivo in BALB/c mice. Promastigotes were maintained in medium M199 2

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supplemented with 10% heat inactivated fetal bovine serum (FBS), supplemented with 100U

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ml─1 penicillin G sodium, 100 µg ml─1 streptomycin sulphate at 220C and sub-cultured every 72h

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in the same medium at an average density of 2x106 cells ml─1 (Afrin et. al. 2002).

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Plant material and extraction: Fresh A. annua leaves and dried seeds with floral parts were

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collected from the Herbal Garden of Jamia Hamdard, washed and air dried in shade at room

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temperature. The dried plant materials were separately ground in an electric grinder. The

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powdered leaves and seeds were extracted with solvents of increasing polarity (n-hexane, ethanol

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and water). The residue from the n-hexane extraction was dried and extracted successively with

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ethanol and water. All the fractions were concentrated to dryness under reduced pressure at 350C

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using a rotary evaporator. The semisolid paste from the organic solvents was further

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concentrated to dryness in vacuum dessicator and rotary vacuum concentrator. The aqueous

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extract was lyophilized, and all the extracts stored at -200 C until use. Stock solutions of 25 mg

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ml─1 were prepared aseptically in DMSO (cell culture grade) except aqueous (in 20 mM

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phosphate buffered saline, pH 7.2 PBS) and diluted further in culture medium (1 mg ml─1) to

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achieve a final DMSO concentration not exceeding 0.2%.

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Growth kinetics assay: Promastigotes of L. donovani AG83 (2x106 cells ml─1) were incubated

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in the presence of test extracts (n-hexane, ethanol and aqueous of A. annua leaves and seeds),

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artemisinin and artesunate in M199 containing 10% FBS (complete medium) at a concentration

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of 100 µg ml─1. Pentamidine (100 µg ml─1) served as the reference drug while 0.2% DMSO,

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which represented the highest concentration in the test extracts, was used as solvent control.

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Parasites in media alone were taken as control. The viable parasites were enumerated for 7 days

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using a phase contrast microscope under 40X objective (Afrin et al., 2001).

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Reversibility assay: To confirm the leishmanicidal effect of the active fractions of A. annua

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leaves and seeds, treated and untreated parasites after 7 days of incubation were washed twice

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with incomplete medium and finally resuspended in complete media and cultured at 220C for a

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further 96 h. The viability of the parasites was ascertained microscopically (Afrin et al., 2001).

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Analysis of cellular morphology: Morphology of the promastigotes was evaluated after 96 h of

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treatment with the test extracts, pentamidine or 0.2% DMSO and photomicrographs were taken

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at 400X magnification under phase contrast microscope (Dutta et al., 2007b). 3

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Determination of 50% growth inhibition (GI50): To determine the GI50 (the concentration of

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extract that inhibits 50% growth of parasites), promastigotes at a density of 2x106 cells ml─1 were

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incubated in triplicate with or without the most active fractions of AAL and AAS at serial three

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fold dilutions starting from 100 µg ml─1 for 96 h. Pentamidine served as the reference drug and

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artemisinin as the known bioactive compound of A. annua.

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Antileishmanial activity in an ex-vivo macrophage-amastigote model: To evaluate the effects

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of the AAL and AAS on the intracellular L. donovani amastigote forms, the J774 macrophage

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cell line (2.5x105 cells ml─1) was allowed to adhere to the glass coverslips in RPMI 1640

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medium supplemented with 10% FBS and cultured for 12 h at 370C in 5% CO2. Adherent

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macrophages were infected with stationary growth phase promastigotes using a cell to parasite

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ratio 1:10 and incubated at 370C for 12 h. Subsequently non-phagocytosed parasites were

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removed by gentle washing, and infected macrophages were incubated with AAL and AAS (0-

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100 µg ml─1) for an additional 48 h. The coverslips were fixed in methanol and geimsa-stained

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for microscopic evaluation of amastigote viability. At least 100 macrophages per coverslip were

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analyzed and the inhibitory concentration that decreases growth of amastigotes by 50% (IC50)

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was determined (Dutta et al., 2008).

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Visualization of phosphatidylserine (PS) exposure by Annexin-V fluorescein isothiocyanate

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(FITC)/ Propidium iodide (PI) binding of cells:

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Externalization of PS in outer membrane of extract-treated promastigotes was measured by

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double staining for Annexin-V and PI with minor modifications (Paris et. al. 2004). Briefly,

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exponential phase promastigotes (2x106 cells ml─1) were incubated with AAL and AAS (the

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most active fractions) at a concentration of 100 µg ml─1 for 72 h. Pentamidine served as the

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reference drug while parasites without any treatment were taken as control. Untreated and treated

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parasites were washed twice in cold PBS and centrifuged at 14,000xg for 10min. The pellet was

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resuspended in Annexin V-FLUOS labeling solution (100 µl, containing both Annexin-V and

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PI), as per the manufacturer’s instruction. After 15min of incubation in the dark at 260C, 400 µl

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of incubation buffer was added, mixed and acquired using a BD LSR-II flow cytometer and

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analysed using FLOWJO software.

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In a parallel experiment, promastigotes stained with Annexin V-FITC and PI, were analyzed

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using a fluorescence microscope (Data not shown).

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Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL)

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assay: DNA fragmentation was detected by catalytically incorporating FITC-dUTP at the 3’-

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hydroxyl end of the fragmented DNA using in situ cell death detection kit (Roche Inc., Basel,

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Switzerland) as per the manufacturer’s instructions. Briefly, exponential phase promastigotes

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(2x106 cells ml─1) were incubated for 72 h with 100 µg ml─1 of AAL and AAS or pentamidine

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(positive control) or with media alone (parasite control) or 0.2%DMSO (solvent control). After

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72 h, the cells were washed, fixed with 4% paraformaldehyde on ice for 1 h, washed with PBS

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and incubated with 3% H2O2 in methanol for 10min at 250C. This was followed by washing with

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PBS and the cells were then permeabilized with freshly prepared chilled 0.1% Triton X-100 for

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5min on ice. Cells were washed twice with PBS, after which 50µl of reaction mixture containing

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TdT and FLUOS labeled dUTP was added for 1 h at 370C, washed and finally resuspended in

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PBS for acquisition in a BD LSR-II flow cytometer, and subsequent analysis using FLOWJO

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software. Histogram analysis of FL-1H (x- axis; FITC fluorescence) was recorded to show the

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shift in fluorescence intensity compared with the unstained cell population used as negative

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control (Dutta et al., 2007a).

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In a parallel experiment, the stained samples were visualized under a high-resolution

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fluorescence microscope, and the images captured and processed (Misra et al., 2008)

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Cell cycle analysis: The potency of the extracts for damaging DNA was detected through flow

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cytometry by propidium iodide (PI) staining that binds stoichiometrically to the DNA (Dutta et

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al., 2007b). Log phase promastigotes (2x106 cells ml─1) were treated with AAL and AAS (100

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µg ml─1) for 72 h at 220C, washed twice with PBS, fixed with 80% chilled ethanol and kept at

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40C for 24 h. The cells were then washed with PBS and the pellet resuspended in 500µl DNase

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free RNase (200 µg ml─1) for 1h at 370C. The cells were stained with PI (50 µg ml─1) and

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incubated in the dark for 20 min at 40C. Data acquisition was carried out using a BD LSR-II flow

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cytometer and analyzed using FLOWJO software.

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Cytotoxicity to mammalian cells: Macrophages were collected by peritoneal lavage from

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starch-stimulated mice. The peritoneal cells were collected in RPMI-1640 medium (incomplete), 5

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centrifuged at 2500 rpm for 10 min at 40C, washed twice and finally resuspended in complete

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medium. Macrophages at a cell density of 8x105 cells ml─1 were incubated with AAL and AAS

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at varying concentrations for 48 h in a CO2 incubator (5% CO2, 370C). Pentamidine as a

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reference drug and 0.2% DMSO (which represented the highest concentration of DMSO required

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to dissolve the test extracts) was used as solvent control. Macrophages without any treatment

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were taken as control. The cells were observed under a phase contrast microscope and viability

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ascertained after trypan blue staining.

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Phytochemical estimation of the extracts by HPTLC: The artemisinin content in the different

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fractions (n-hexane, ethanol and aqueous) of A. annua leaves and seeds were analyzed by high

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performance thin layer chromatography (HPTLC) (Widmer et. al. 2007). Different fractions

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were applied on a silica coated TLC plate (10x20 cm). The plate was air dried and developed

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with benzene: ethyl acetate: glacial acetic acid (9: 0.8: 0.2) as the mobile phase in a Camag twin

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trough chamber. Standard artemisinin was used to generate a calibration curve for quantification.

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The densitometric determination of artemisinin was carried out after derivatization with

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anisaldehyde reagent. Scanning and quantification of spots were performed at 450nm in an

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absorbance/reflectance mode with Camag TLC scanner 3 using a Win CATS 1.3.2 software.

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GC-MS analysis of AAL and AAS: The chemical composition and characterization of active

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compounds from AAL and AAS was analyzed by a gas chromatography-mass spectrometry

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(GC-MS) using a SHIMADZU QP2010 with a DB-5 column (30 m, film 0.25 μm, ID 0.25 mm).

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The temperature of the column was programmed from 450C to 2700C at 50C min─1, and the

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injector or detector temperature for the analysis was about 2500C. Helium was used as the carrier

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gas at a flow rate of 1.5 ml min─1. The mass spectrometer was operated in an electron impact

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ionization mode with 70eV energy. The identification of the chemical constituents was based on

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matching their recorded mass spectra with those obtained from the WILEY8.LIB and

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NIST08.LIB library spectra provided by the software of GC-MS system (Bagavan et al 2011).

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Statistical analysis. Antileishmanial activity against promastigotes and amastigotes were

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expressed as GI50 and IC50, respectively by linear regression analysis. All values reported are

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mean ± SE of samples in triplicate and are from one of three independent experiments. P ≤ 0.05

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were considered significant. 6

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RESULTS:

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In vitro leishmanicidal activity of test extracts by growth kinetics assay: All fractions of A.

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annua leaves and seeds were assayed for their cytotoxicity against L. donovani promastigotes.

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The test extracts exhibited time dependent killing of promastigotes at a concentration of 100

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μgml─1. The n-hexane extracts of seeds (AAS) and leaves (AAL) exhibited highest cytotoxicity.

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No viable parasites were observed after 2, 3 and 5 days of incubation with pentamidine, AAS

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and AAL, respectively. In case of artemisinin (100 µg ml─1), the growth of parasites initially

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slowed down but eventually expanded after 96 h as compared to control. No cell death was

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observed in artesunate and solvent control (Fig. 1). The untreated parasites also proliferated at a

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normal rate.

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Alteration of cellular morphology of extract-treated promastigotes: Visual inspection by

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phase contrast microscopy revealed the onset of cell shrinkage and cytoplasmic condensation

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upon treatment of promastigotes with AAL and AAS (100 µg ml─1) (Fig. 2) marked by complete

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circularization of almost all the cells 96 h post-treatment. Similar morphological changes were

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observed in the pentamidine treated parasites. Microscopic study indicated that the effect is

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leishmanicidal rather than leishmanistatic.

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Growth reversibility assay of extract-treated promastigotes: To confirm the lethal effect of

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AAL and AAS for promastigotes, treated and untreated parasites (from growth kinetics study)

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were washed and re-suspended in fresh media and their viability ascertained microscopically

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after 96 h of incubation. No viable parasites were detected in case of prior incubation with AAL

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and AAS as also observed with pentamidine, confirming their leishmanicidal effect. Artemisinin-

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treated parasites reverted to late log phase and resembled untreated control parasites (Fig. 3).

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Evaluation of GI50 of n-hexane fractions against promastigotes: The viability of

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promastigotes after treatment with AAL and AAS was evaluated using a modified GI50 assay.

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Treatment of promastigotes with n-hexane fractions demonstrated a dose dependent inhibition of

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parasite growth with GI50 achieved at 14.4 µg ml─1 and 14.6 µg ml─1 in AAL and AAS,

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respectively. The established antileishmanial drug pentamidine, used as positive control showed

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a similar trend in dose dependent parasite killing with GI50 of 1.1 µg ml─1 (Fig. 4). The effect of 7

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DMSO (0.2%), representative of highest concentration of diluent present in the test extracts (100

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µg ml─1), showed no loss of parasite viability.

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Anti-amastigote activity of AAL and AAS: In Leishmania infection, the promastigotes

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transform into amastigotes within the phagolysosomal vacuoles of macrophages. Accordingly,

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the antileishmanial activity of AAL and AAS was tested against intracellular amastigotes in L.

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donovani infected J774 macrophage cell line. Giemsa-stained cover slips were analysed

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microscopically for the presence of phagocytosed amastigotes within the macrophages. The IC50

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value of AAL and AAS for L. donovani amastigotes was found to be 6.6 µg ml─1 and 5.05 µg

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ml─1 respectively, which is close to IC50 value of pentamidine (1 µg ml─1) (Fig. 5)

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Externalization of phosphatidylserine in promastigotes: In metazoan and unicellular cells,

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translocation of the phosphatidylserine (PS) from the inner to the outer leaflet of the plasma

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membrane occurs during programmed cell death (PCD) (Mehta and Shaha, 2004; Sudhandiran

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and Shaha, 2003; Koonin and Aravind, 2002). Annexin-V, a Ca2+ dependent phospholipid

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binding protein with affinity for PS, is routinely used to label its externalization. Since annexin-

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V can also label necrotic cells following the loss of membrane integrity, simultaneous addition of

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PI, which does not permeate cells with an intact plasma membrane, allows discrimination

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between apoptotic cells (Annexin-V +ve, PI –ve), secondary necrotic cells (Annexin-V +ve, PI

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+ve), necrotic cells (Annexin-V -ve, PI +ve) and live cells (both Annexin-V -ve, PI -ve).

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Accordingly, in order to study whether the mechanism of cell death triggered by the n-hexane

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fractions of leaves and seeds is via apoptosis or necrosis, treated and untreated promastigotes

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were double stained with FLUOS-conjugated annexin-V and PI. A significant percentage (3.1

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and 6.6) of promastigotes treated with AAL and AAS, respectively stained positive for annexin-

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V, compared to only 0.4 % in artemisinin-treated and untreated cells (Fig. 6). This was

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comparable with apoptosis triggered by pentamidine (3.2%) that served as positive control

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indicating that the leishmanicidal activity of AAL and AAS is via apoptosis.

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Nuclear DNA fragmentation in AAS and AAL -treated promastigotes by in situ labeling of

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DNA fragments via a TUNEL assay: One of the hallmarks of apoptotic cell death is the

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degradation of nuclear DNA into oligonucleosomal units. DNA nicking resulting from the

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exposure to n-hexane fractions of leaves and seeds was detected using a TUNEL assay in which

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the proportion of DNA nicks was quantified by measuring the binding of dUTP-FLUOS to the 8

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nicked end via TdT. Promastigotes treated with 100 µg ml─1 of AAL and AAS for 72 h appeared

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yellowish green under fluorescence microscope representing incorporation of dUTP-labelled

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with FLUOS, thus indicating nicking of DNA (Fig. 7b).

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The occurrence of DNA nicking was also detected by quantifying the binding of dUTP-FLUOS

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to the nicked end via TdT, as the proportion of DNA nicks is directly proportional to the

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fluorescence obtained from dUTP-FLUOS. Treated promastigotes after 72 h showed an increase

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in nuclear DNA fragmentation as evidenced from dUTP-FLUOS binding. The mean

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fluorescence intensity (MFI) of untreated (control) and treated fractions (AAL, AAS, artemisinin

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and pentamidine) were found to be 611, 13083, 15296, 433 and 24289, respectively (Fig. 7a),

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thus providing strong evidence for apoptosis in L. donovani promastigotes.

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Artemisia annua (n-hexane fractions) induced apoptosis causing an increase in the sub G0-

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G1 population: Flow cytometric analysis after cell permeabilization and labeling with PI was

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used to quantify the percentage of pseudohypodiploid cells. In a given cell, the amount of PI

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correlates with the DNA content, and accordingly, DNA fragmentation in apoptotic cells

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translates into a sub G0-G1 peak (Sarkar et. al., 2008). Promastigotes treated with AAL and AAS

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(100 µg ml─1) for 24 and 72 h caused cell cycle arrest at sub G0-G1 phase, as analysed by flow

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cytometry (Fig. 8). The proportion of cells in control was 5.52% in the sub G0-G1 phase, which

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increased to 19.90%, 19.76% and 16.35% in promastigotes, treated with AAL, AAS and

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pentamidine, respectively. Only a slight increase (5.91%) in the sub G0-G1 cell population was

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observed upon treatment with artemisinin. This increase in cell proportion in the sub G0-G1

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phase corroborated our findings that AAL and AAS induced apoptosis in promastigotes causing

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DNA fragmentation.

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Cytotoxicity of bioactive fractions on mammalian macrophages: Peritoneal macrophages

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were isolated from mice to check the adverse side effects of the bioactive fractions, using

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pentamidine as the reference drug. The cytotoxicity assay revealed that there was no adverse

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toxicity of AAL and AAS even at 200 µg ml─1 on mammalian macrophages (Fig. 9).

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Determination of artemisinin content in AAL and AAS by HPTLC fingerprinting: The

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artemisinin content in the n-hexane fractions of A. annua leaves and seeds was quantified by

9

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HPTLC fingerprinting. The content of artemisinin in 100 µg of AAL and AAS was found to be

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1.447 µg and 1.336µg, respectively (Table. 1).

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Composition of AAL and AAS: The major chemical constituents of AAL and AAS that may

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have contributed to leishmanicidal activity were identified by GC-MS analysis. α−amyrinyl

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acetate (28.17%), β-amyrine (20.87%), dihydroartemisinin 3-desoxy (4.91%), deoxyartemisinin

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(4.29%) and arteannuin (1.34%) were predominantly present in AAL while cetin (15.23%)

303

einecs (9.97%), dihydroartemisinin 3-desoxy (7.77%), deoxyartemisinin (5.15%) and arteannuin

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(1.45%) were revealed in AAS by comparison of mass spectral data and retention times as

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presented (Fig. 10a and 10b and Supplementary Tables 2(a) and 2(b)).

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Discussion:

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The leishmanicidal activity of miltefosine (Verma and Dey, 2004), amphotericin B (Lee et al.,

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2002) and camptothecin (Sen et al., 2004) have been reported to be mediated by apoptosis. The

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efficacy of artemisinin against promastigotes of L. donovani has recently been reported (Sen et

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al., 2007). However, in our study, we have shown that, artemisinin and artesunate exhibit partial

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growth inhibitory activity against L. donovani promastigotes at a concentration of 100µg ml─1, in

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contrast to the significant leishmanicidal effect mediated by the n-hexane fractions of A. annua

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leaves (AAL) and seeds (AAS). To elucidate the mode of action of AAL and AAS against

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promastigotes of L. donovani, we have used biochemical and morphological approaches, and

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have demonstrated that AAL and AAS induced programmed cell death that share several

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phenotypic features with metazoan apoptosis as also reported by Debrabant et al. (2003). These

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apoptotic correlates include PS exposure, DNA nicking as evidenced by TUNEL assay and cell

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cycle arrest at sub G0-G1 phase. Taken together, we have conclusively demonstrated that AAL

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and AAS induce PCD in promastigotes that notably share some, but not all of the classical

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features of apoptosis, observed in the higher eukaryotes. The bioactive fractions (AAL and AAS)

322

had no observable cytotoxicity on mammalian macrophages even at 200 µg ml─1.

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HPTLC fingerprint profile showed that the content of artemisinin in the bioactive fractions was

324

too low (~1.4 μg 100 μg─1 n-hexane fractions) to account for the observed antileishmanial

325

activity. GC-MS analysis revealed α−amyrinyl acetate, β-amyrine, dihydroartemisinin 3-desoxy, 10

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deoxyartemisinin and arteannuin as the major active constituents in AAL while cetin, einecs,

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dihydroartemisinin 3-desoxy, deoxyartemisinin and arteannuin were predominantly present in

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AAS. Synthetic dihydroartemisinin acetal derivatives (dimer and trimer) have also been reported

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by others to exhibit antileishmanial, antimalarial, anticancer and antifungal activities (Slade et al.

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2009). Our studies further indicate that the leishmanicidal activity of AAL may be attributed to

331

the presence of α- amyrinyl acetate, β- amyrine and derivatives of artemisinin

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(dihydroartemisinin 3-desoxy, arteannuin and deoxyartemisinin) while cetin, einecs and the

333

artemisinin derivatives may have contributed to the observed effect in AAS. Our findings

334

demonstrate promising antileishmanial activity of AAL and AAS that is mediated by

335

programmed cell death and, accordingly, the active constituents may have potential as a source

336

of novel agents for the treatment of leishmaniasis.

337 338

ACKNOWLEDGEMENTS: This work received financial assistance from the Department of

339

Biotechnology (DBT) India. Mohammad Islamuddin, is the recipient of Senior Research

340

Fellowship from the DBT, Government of India.

341 342 343

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13

A. annua

Total artemisinin content (%)

Fractions

Artemisinin (%)

Leaves

0.082

Seeds

0.088

n-hexane ethanol aqueous n-hexane ethanol aqueous

1.447 0.834 0.078 1.336 0.803 0

Artemisinin content (μg) per 100μg of each fraction 1.447 0.834 0.078 1.336 0.803 0

Antileishmanial activity

Bioactive Bioactive -

451 452

Table 1. Artemisinin content in different fractions of A. annua leaves and seeds

14

A. annua leaves

100

n-hexane fraction ethanolic fraction aqueous fraction Pentamidine

)

1

─l

m lsl ec 10 6 01 x( yt is n e d 1 et is ar aP 0.1

Artemisinin Artesunate Parasite control 0

1

2

3

4 5 Time (days)

6

7

8

A. annua seeds

100

)

1

─l

m lsl ec 10 6 01 x( yt is n e d 1 et is ar aP 0.1

0

1

2

3

4 5 Time (days)

6

7

Fig. 1. Analysis of anti-promastigote activity of A. annua leaf and seed fractions. Exponential phase promastigotes (2x106 cells ml─1) of L. donovani were incubated with 100 µg ml─1 of each fraction for different time points as described in Methods. Each point corresponds to the mean ± SE of triplicate samples and is representative of one of three independent experiments.

8

Control

Artesunate

Pentamidine

Artemisinin

AAS

AAL

Fig. 2. Analysis of cellular morphology of AAL and AAS treated promastigotes. Exponential phase promastigotes (2x106 cells ml─1) were incubated with 100 µg ml─1 n-hexane fractions of A. annua leaves (AAL) and seeds (AAS) for 96 h and analysed by light microscopy (400X) as described in Methods.

50 1) 40 ─l m sll ec30 6 01 x(20 yt is n e10 d et is ar aP 0

A. annua leaves

50

)

1

─l 40

A. annua seeds

m sll ec 6 30 01 x( yt 20 is n e d 10 et is ar aP 0

Fig.3. Reversibility analysis of test extract-treated promastigotes revealed no reversion of growth in n-hexane fraction of leaves (AAL), seeds (AAS) and pentamidine treated samples. Each bar represents mean ± SE of triplicate samples and the data is representative of one of three independent experiments.

120

AAL

100

AAS

)l or tn 80 oc f o 60 % ( yt ili ba 40 i V

Pentamidine Artemisinin

20 0

0

20

40

60 Concentration (µg ml─1)

80

100

120

Fig. 4. Estimation of GI50 of AAL and AAS against promastigotes. Parasites (2x106 cells ml─1) were incubated with serial three-fold dilutions of AAL and AAS (starting at 100 µg ml─1) for 96 h and viability was determined microscopically as described in Methods. Each point corresponds to the mean ± SE of triplicate samples and data is from one of three independent experiments.

120

AAL AAS Pentamidine

100

)l or tn 80 oc f o 60 % ( yt ili ba 40 i V 20 0

0

20

40

60 Concentration (µg ml─1)

80

100

120

Fig. 5. Effects of AAL and AAS on Leishmania donovani intracellular amastigote forms. J774 macrophage cell line after infection with promastigotes was incubated with serial three-fold dilutions of AAL and AAS (starting at 100 µg ml─1) for 48 h and viability was determined microscopically as described in Materials and Methods. Each point corresponds to the mean ± SE of triplicate samples.

Fig. 6. Externalization of phosphatidylserine in AAL and AAS treated promastigotes. Parasites were incubated with AAL, AAS, artemisinin or pentamidine (100 µg ml─1) for 72 h, co-stained with PI and annexin V-FITC and analysed by flow cytometry as described in Methods. This is a representative profile of three independent experiments.

st n u o C

b

d

e

c

e

c

b

a

d a

a= Control b= Artemisinin c= AAL d= AAS e= Pentamidine

dUTP-FITC

(a)

(b)

Fig. 7. Analysis of TUNEL positivity in AAL and AAS treated promastigotes. Exponential phase promastigotes were incubated with AAL or AAS (100 µg ml─1) for 72 h. Cells were fixed, stained with dUTP-FITC in the presence of TdT and analysed by flow cytometry (a) and, observed under a fluorescence microscope (b) as described in Methods.

24 h

72 h Sub G0/G1: 5.38% G1: 65.88% S:12.19% G2/M: 15.55%

Sub G0/G1: 15.50% G1: 71.03% S:3.95% G2/M: 8.36%

Sub G0/G1: 5.52% G1: 71.94% S:6.26% G2/M: 14.23%

Sub G0/G1: 19.90% G1: 63.74% S:9.72% G2/M: 7.74

Sub G0/G1: 6.43% G1: 81.25% S:3.83% G2/M: 7.37%

Sub G0/G1: 19.76% G1: 66.45% S: 6.04% G2/M: 6.67%

Sub G0/G1: 3.47% G1: 66.50% S:11.42% G2/M: 16.82%

Sub G0/G1: 5.91% G1: 66.80% S: 9.21% G2/M: 16.44

Sub G0/G1: 7.00% G1: 74.91% S: 4.68 G2/M: 11.36%

Sub G0/G1: 16.35% G1: 72.7% S: 5.75% G2/M: 4.74%

Control

AAL

AAS

Artemisinin

Pentamidine

Fig. 8. Analysis of the cell-cycle status of AAL and AAS-treated promastigotes. Exponential-phase promastigotes were incubated with AAL and AAS (100µg ml─1) for 24 and 72 h, fixed in chilled methanol, probed with PI and acquired using a LSR-II flow cytometer for subsequent analysis using a FLOWJO software as described in Methods.

Fig.9. Adverse toxicity of test extracts on mammalian macrophages. Macrophages from peritoneal cavity of mice were incubated for 72 h at 370C in CO2 incubator with increasing concentrations of test extracts or pentamidine and viability was ascertained. Each bar represents mean ± SE of triplicate samples and data is representative of one of three independent experiments.

AAS

Peak Intensity

AAL

Retention Time

Fig. 10. Chromatogram of GC-MS analysis of AAL and AAS. GC-MS analysis was performed using SHIMADZU QP2010 with a DB-5 column (30 m, film 0.25 μm, ID 0.25 mm), helium was used as the carrier gas at a flow rate of 1.5 ml min─1. Chemical constituents were identified using WILEY8.LIB and NIST08.LIB library