Journal of Medical Microbiology Papers in Press. Published September 6, 2012 as doi:10.1099/jmm.0.049387-0
1 2
Artemisia annua leaves and seeds mediate programmed cell death in Leishmania donovani
3 4 5 6
Mohammad Islamuddin, 1 Abdullah Farooque, Dinkar Sahal 3 and Farhat Afrin 1
2
B. S. Dwarakanath,
2
7 8 9 10 11 12
1
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
13 14 15 16
Corresponding Author: Farhat Afrin Parasite Immunology Lab. Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India. Email:
[email protected],
[email protected]
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 0
32 33
Abstract
34
Leishmaniasis is one of the major tropical parasitic diseases that ranges in severity from the self-
35
healing cutaneous lesions to fatal visceral manifestations. On one hand, there is no vaccine
36
available against visceral leishmaniasis (VL) (also known as Kala-azar in India) and on the other
37
hand drug resistance, variable efficacy, toxicity and parenteral administration are the main
38
drawbacks of current antileishmanial drugs. We report that n-hexane fraction of Artemisia annua
39
leaves (AAL) and seeds (AAS) possess significant antileishmanial activity against Leishmania
40
donovani promastigotes with GI50 of 14.4 and 14.6 μg ml─1, respectively and the IC50 against
41
intracellular amastigotes was found to be 6.6 and 5.05 µgml─1, respectively. Changes in the
42
morphology of promastigotes and growth reversibility analysis following treatment confirmed
43
the leishmanicidal effect of the active fractions, which presented no cytotoxic effect on
44
mammalian cells. Furthermore, the anti-leishmanial activity was mediated via apoptosis as
45
evidenced by externalization of phosphatidylserine, in situ labelling of DNA fragments by
46
terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle
47
arrest at the sub-G0/G1 phase. High performance thin layer chromatography (HPTLC) fingerprint
48
showed that the content of artemisinin in the crude bioactive extracts (~1.4 μg 100μg─1 n-hexane
49
fractions) was too low to account for the observed antileishmanial activity. Characterization of
50
the active constituents by Gas chromatography-Mass spectroscopy GC-MS showed that α−
51
amyrinyl acetate, β- amyrine and derivatives of artemisinin were the major constituents in AAL
52
and cetin, einecs and artemisinin derivatives in AAS. Our findings indicate the presence of
53
antileishmanial compounds besides artemisinin in the n-hexane fraction of A. annua leaves and
54
seeds.
55
INTRODUCTION: Leishmaniasis, a complex spectrum of diseases, is currently a global health
56
problem occurring as cutaneous, mucocutaneous or visceral forms. Visceral leishmaniasis (VL),
57
or kala-azar, caused by Leishmania donovani, is often associated with marked suppression of the
58
host’s cell-mediated immune response, leading to severe morbidity and mortality, if left
59
untreated (Halder et al., 1983). VL is also considered as an opportunistic infection among
60
immunocompromised patients around the world (Albrecht et al., 1996). Treatment of VL mainly
61
relies on pentavalent antimonials and second line drugs such as amphotericin B and pentamidine.
62
However, resistance to antimonial chemotherapy, coupled with high cost, toxicity and parenteral 1
63
route of administration, is a matter of great concern in the endemic regions of Bihar, India
64
(Sundar et al., 2000). In view of the ongoing challenge to introduce new antileishmanial drugs,
65
traditional medicinal plants are expected to provide valuable leads (Ahua et al., 2007; Sarkar et
66
al., 2008; Misra et al., 2009; Mokoka et al., 2011). According to World Health Organization
67
(WHO), approximately 80% of the world inhabitants rely on traditional medicines for their
68
healthcare (WHO, 2000). Artemisia annua L (Asteraceae), a traditional medicinal plant has been
69
extensively used as an anti-malarial (Willcox et al., 2007 and Atemnkeng et al., 2009) and anti-
70
cancer agent. Recently, the efficacy of artemisinin (one of the constituent of A. annua, A. indica,
71
and A. dracunculus) against promastigotes and amastigotes of L. donovani have been reported
72
(Sen et al., 2007). The antileishmanial activity of ethanolic extracts of the leaves of A. indica was
73
investigated in exponential phase promastigotes from six strains responsible for cutaneous,
74
mucocutaneous or visceral leishmaniasis and the IC50 value ranged from 210 µg ml─1 to 580 µg
75
ml─1 (Ganguly et al., 2006). Flavonoids from A. annua as antioxidants and their potential
76
synergy with artemisinin against malaria and cancer have been observed (Ferreira et al., 2010).
77
In this study, we delineated the putative mechanisms contributing to leishmanicidal activity of
78
the bioactive fractions from A. annua leaves and seeds with the aim of developing effective
79
herbal remedy for the treatment of VL that may complement or synergise with the conventional
80
drugs against VL.
81
Abbreviations: AAL, n-hexane fraction of Artemisia annua leaves; AAS, n-hexane fraction of
82
Artemisia annua seeds; MFI, mean fluorescence intensity; PCD, programmed cell death; PI,
83
propidium
84
deoxynucleotidyltransferase mediated dUTP nick end labelling.
iodide;
FITC,
fluorescein
isothiocyanate;
TUNEL,
terminal
85 86
METHODS:
87
Materials. M199 medium, propidium iodide (PI) and RNase were obtained from Sigma-Aldrich,
88
fetal bovine serum (FBS) from Gibco-BRL, DMSO from SRL, methanol from Merck and
89
Annexin V-FITC and the ApoDirect kits were from Roche Inc., Basel, Switzerland. All other
90
chemicals were from Sigma-Aldrich unless stated.
91
Parasite culture: L. donovani strain AG83 was a kind gift from Dr. Nahid Ali, IICB, Kolkata
92
and was maintained in vivo in BALB/c mice. Promastigotes were maintained in medium M199 2
93
supplemented with 10% heat inactivated fetal bovine serum (FBS), supplemented with 100U
94
ml─1 penicillin G sodium, 100 µg ml─1 streptomycin sulphate at 220C and sub-cultured every 72h
95
in the same medium at an average density of 2x106 cells ml─1 (Afrin et. al. 2002).
96
Plant material and extraction: Fresh A. annua leaves and dried seeds with floral parts were
97
collected from the Herbal Garden of Jamia Hamdard, washed and air dried in shade at room
98
temperature. The dried plant materials were separately ground in an electric grinder. The
99
powdered leaves and seeds were extracted with solvents of increasing polarity (n-hexane, ethanol
100
and water). The residue from the n-hexane extraction was dried and extracted successively with
101
ethanol and water. All the fractions were concentrated to dryness under reduced pressure at 350C
102
using a rotary evaporator. The semisolid paste from the organic solvents was further
103
concentrated to dryness in vacuum dessicator and rotary vacuum concentrator. The aqueous
104
extract was lyophilized, and all the extracts stored at -200 C until use. Stock solutions of 25 mg
105
ml─1 were prepared aseptically in DMSO (cell culture grade) except aqueous (in 20 mM
106
phosphate buffered saline, pH 7.2 PBS) and diluted further in culture medium (1 mg ml─1) to
107
achieve a final DMSO concentration not exceeding 0.2%.
108
Growth kinetics assay: Promastigotes of L. donovani AG83 (2x106 cells ml─1) were incubated
109
in the presence of test extracts (n-hexane, ethanol and aqueous of A. annua leaves and seeds),
110
artemisinin and artesunate in M199 containing 10% FBS (complete medium) at a concentration
111
of 100 µg ml─1. Pentamidine (100 µg ml─1) served as the reference drug while 0.2% DMSO,
112
which represented the highest concentration in the test extracts, was used as solvent control.
113
Parasites in media alone were taken as control. The viable parasites were enumerated for 7 days
114
using a phase contrast microscope under 40X objective (Afrin et al., 2001).
115
Reversibility assay: To confirm the leishmanicidal effect of the active fractions of A. annua
116
leaves and seeds, treated and untreated parasites after 7 days of incubation were washed twice
117
with incomplete medium and finally resuspended in complete media and cultured at 220C for a
118
further 96 h. The viability of the parasites was ascertained microscopically (Afrin et al., 2001).
119
Analysis of cellular morphology: Morphology of the promastigotes was evaluated after 96 h of
120
treatment with the test extracts, pentamidine or 0.2% DMSO and photomicrographs were taken
121
at 400X magnification under phase contrast microscope (Dutta et al., 2007b). 3
122
Determination of 50% growth inhibition (GI50): To determine the GI50 (the concentration of
123
extract that inhibits 50% growth of parasites), promastigotes at a density of 2x106 cells ml─1 were
124
incubated in triplicate with or without the most active fractions of AAL and AAS at serial three
125
fold dilutions starting from 100 µg ml─1 for 96 h. Pentamidine served as the reference drug and
126
artemisinin as the known bioactive compound of A. annua.
127
Antileishmanial activity in an ex-vivo macrophage-amastigote model: To evaluate the effects
128
of the AAL and AAS on the intracellular L. donovani amastigote forms, the J774 macrophage
129
cell line (2.5x105 cells ml─1) was allowed to adhere to the glass coverslips in RPMI 1640
130
medium supplemented with 10% FBS and cultured for 12 h at 370C in 5% CO2. Adherent
131
macrophages were infected with stationary growth phase promastigotes using a cell to parasite
132
ratio 1:10 and incubated at 370C for 12 h. Subsequently non-phagocytosed parasites were
133
removed by gentle washing, and infected macrophages were incubated with AAL and AAS (0-
134
100 µg ml─1) for an additional 48 h. The coverslips were fixed in methanol and geimsa-stained
135
for microscopic evaluation of amastigote viability. At least 100 macrophages per coverslip were
136
analyzed and the inhibitory concentration that decreases growth of amastigotes by 50% (IC50)
137
was determined (Dutta et al., 2008).
138 139
Visualization of phosphatidylserine (PS) exposure by Annexin-V fluorescein isothiocyanate
140
(FITC)/ Propidium iodide (PI) binding of cells:
141
Externalization of PS in outer membrane of extract-treated promastigotes was measured by
142
double staining for Annexin-V and PI with minor modifications (Paris et. al. 2004). Briefly,
143
exponential phase promastigotes (2x106 cells ml─1) were incubated with AAL and AAS (the
144
most active fractions) at a concentration of 100 µg ml─1 for 72 h. Pentamidine served as the
145
reference drug while parasites without any treatment were taken as control. Untreated and treated
146
parasites were washed twice in cold PBS and centrifuged at 14,000xg for 10min. The pellet was
147
resuspended in Annexin V-FLUOS labeling solution (100 µl, containing both Annexin-V and
148
PI), as per the manufacturer’s instruction. After 15min of incubation in the dark at 260C, 400 µl
149
of incubation buffer was added, mixed and acquired using a BD LSR-II flow cytometer and
150
analysed using FLOWJO software.
4
151
In a parallel experiment, promastigotes stained with Annexin V-FITC and PI, were analyzed
152
using a fluorescence microscope (Data not shown).
153
Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL)
154
assay: DNA fragmentation was detected by catalytically incorporating FITC-dUTP at the 3’-
155
hydroxyl end of the fragmented DNA using in situ cell death detection kit (Roche Inc., Basel,
156
Switzerland) as per the manufacturer’s instructions. Briefly, exponential phase promastigotes
157
(2x106 cells ml─1) were incubated for 72 h with 100 µg ml─1 of AAL and AAS or pentamidine
158
(positive control) or with media alone (parasite control) or 0.2%DMSO (solvent control). After
159
72 h, the cells were washed, fixed with 4% paraformaldehyde on ice for 1 h, washed with PBS
160
and incubated with 3% H2O2 in methanol for 10min at 250C. This was followed by washing with
161
PBS and the cells were then permeabilized with freshly prepared chilled 0.1% Triton X-100 for
162
5min on ice. Cells were washed twice with PBS, after which 50µl of reaction mixture containing
163
TdT and FLUOS labeled dUTP was added for 1 h at 370C, washed and finally resuspended in
164
PBS for acquisition in a BD LSR-II flow cytometer, and subsequent analysis using FLOWJO
165
software. Histogram analysis of FL-1H (x- axis; FITC fluorescence) was recorded to show the
166
shift in fluorescence intensity compared with the unstained cell population used as negative
167
control (Dutta et al., 2007a).
168
In a parallel experiment, the stained samples were visualized under a high-resolution
169
fluorescence microscope, and the images captured and processed (Misra et al., 2008)
170
Cell cycle analysis: The potency of the extracts for damaging DNA was detected through flow
171
cytometry by propidium iodide (PI) staining that binds stoichiometrically to the DNA (Dutta et
172
al., 2007b). Log phase promastigotes (2x106 cells ml─1) were treated with AAL and AAS (100
173
µg ml─1) for 72 h at 220C, washed twice with PBS, fixed with 80% chilled ethanol and kept at
174
40C for 24 h. The cells were then washed with PBS and the pellet resuspended in 500µl DNase
175
free RNase (200 µg ml─1) for 1h at 370C. The cells were stained with PI (50 µg ml─1) and
176
incubated in the dark for 20 min at 40C. Data acquisition was carried out using a BD LSR-II flow
177
cytometer and analyzed using FLOWJO software.
178
Cytotoxicity to mammalian cells: Macrophages were collected by peritoneal lavage from
179
starch-stimulated mice. The peritoneal cells were collected in RPMI-1640 medium (incomplete), 5
180
centrifuged at 2500 rpm for 10 min at 40C, washed twice and finally resuspended in complete
181
medium. Macrophages at a cell density of 8x105 cells ml─1 were incubated with AAL and AAS
182
at varying concentrations for 48 h in a CO2 incubator (5% CO2, 370C). Pentamidine as a
183
reference drug and 0.2% DMSO (which represented the highest concentration of DMSO required
184
to dissolve the test extracts) was used as solvent control. Macrophages without any treatment
185
were taken as control. The cells were observed under a phase contrast microscope and viability
186
ascertained after trypan blue staining.
187
Phytochemical estimation of the extracts by HPTLC: The artemisinin content in the different
188
fractions (n-hexane, ethanol and aqueous) of A. annua leaves and seeds were analyzed by high
189
performance thin layer chromatography (HPTLC) (Widmer et. al. 2007). Different fractions
190
were applied on a silica coated TLC plate (10x20 cm). The plate was air dried and developed
191
with benzene: ethyl acetate: glacial acetic acid (9: 0.8: 0.2) as the mobile phase in a Camag twin
192
trough chamber. Standard artemisinin was used to generate a calibration curve for quantification.
193
The densitometric determination of artemisinin was carried out after derivatization with
194
anisaldehyde reagent. Scanning and quantification of spots were performed at 450nm in an
195
absorbance/reflectance mode with Camag TLC scanner 3 using a Win CATS 1.3.2 software.
196
GC-MS analysis of AAL and AAS: The chemical composition and characterization of active
197
compounds from AAL and AAS was analyzed by a gas chromatography-mass spectrometry
198
(GC-MS) using a SHIMADZU QP2010 with a DB-5 column (30 m, film 0.25 μm, ID 0.25 mm).
199
The temperature of the column was programmed from 450C to 2700C at 50C min─1, and the
200
injector or detector temperature for the analysis was about 2500C. Helium was used as the carrier
201
gas at a flow rate of 1.5 ml min─1. The mass spectrometer was operated in an electron impact
202
ionization mode with 70eV energy. The identification of the chemical constituents was based on
203
matching their recorded mass spectra with those obtained from the WILEY8.LIB and
204
NIST08.LIB library spectra provided by the software of GC-MS system (Bagavan et al 2011).
205 206
Statistical analysis. Antileishmanial activity against promastigotes and amastigotes were
207
expressed as GI50 and IC50, respectively by linear regression analysis. All values reported are
208
mean ± SE of samples in triplicate and are from one of three independent experiments. P ≤ 0.05
209
were considered significant. 6
210 211
RESULTS:
212
In vitro leishmanicidal activity of test extracts by growth kinetics assay: All fractions of A.
213
annua leaves and seeds were assayed for their cytotoxicity against L. donovani promastigotes.
214
The test extracts exhibited time dependent killing of promastigotes at a concentration of 100
215
μgml─1. The n-hexane extracts of seeds (AAS) and leaves (AAL) exhibited highest cytotoxicity.
216
No viable parasites were observed after 2, 3 and 5 days of incubation with pentamidine, AAS
217
and AAL, respectively. In case of artemisinin (100 µg ml─1), the growth of parasites initially
218
slowed down but eventually expanded after 96 h as compared to control. No cell death was
219
observed in artesunate and solvent control (Fig. 1). The untreated parasites also proliferated at a
220
normal rate.
221
Alteration of cellular morphology of extract-treated promastigotes: Visual inspection by
222
phase contrast microscopy revealed the onset of cell shrinkage and cytoplasmic condensation
223
upon treatment of promastigotes with AAL and AAS (100 µg ml─1) (Fig. 2) marked by complete
224
circularization of almost all the cells 96 h post-treatment. Similar morphological changes were
225
observed in the pentamidine treated parasites. Microscopic study indicated that the effect is
226
leishmanicidal rather than leishmanistatic.
227
Growth reversibility assay of extract-treated promastigotes: To confirm the lethal effect of
228
AAL and AAS for promastigotes, treated and untreated parasites (from growth kinetics study)
229
were washed and re-suspended in fresh media and their viability ascertained microscopically
230
after 96 h of incubation. No viable parasites were detected in case of prior incubation with AAL
231
and AAS as also observed with pentamidine, confirming their leishmanicidal effect. Artemisinin-
232
treated parasites reverted to late log phase and resembled untreated control parasites (Fig. 3).
233
Evaluation of GI50 of n-hexane fractions against promastigotes: The viability of
234
promastigotes after treatment with AAL and AAS was evaluated using a modified GI50 assay.
235
Treatment of promastigotes with n-hexane fractions demonstrated a dose dependent inhibition of
236
parasite growth with GI50 achieved at 14.4 µg ml─1 and 14.6 µg ml─1 in AAL and AAS,
237
respectively. The established antileishmanial drug pentamidine, used as positive control showed
238
a similar trend in dose dependent parasite killing with GI50 of 1.1 µg ml─1 (Fig. 4). The effect of 7
239
DMSO (0.2%), representative of highest concentration of diluent present in the test extracts (100
240
µg ml─1), showed no loss of parasite viability.
241
Anti-amastigote activity of AAL and AAS: In Leishmania infection, the promastigotes
242
transform into amastigotes within the phagolysosomal vacuoles of macrophages. Accordingly,
243
the antileishmanial activity of AAL and AAS was tested against intracellular amastigotes in L.
244
donovani infected J774 macrophage cell line. Giemsa-stained cover slips were analysed
245
microscopically for the presence of phagocytosed amastigotes within the macrophages. The IC50
246
value of AAL and AAS for L. donovani amastigotes was found to be 6.6 µg ml─1 and 5.05 µg
247
ml─1 respectively, which is close to IC50 value of pentamidine (1 µg ml─1) (Fig. 5)
248
Externalization of phosphatidylserine in promastigotes: In metazoan and unicellular cells,
249
translocation of the phosphatidylserine (PS) from the inner to the outer leaflet of the plasma
250
membrane occurs during programmed cell death (PCD) (Mehta and Shaha, 2004; Sudhandiran
251
and Shaha, 2003; Koonin and Aravind, 2002). Annexin-V, a Ca2+ dependent phospholipid
252
binding protein with affinity for PS, is routinely used to label its externalization. Since annexin-
253
V can also label necrotic cells following the loss of membrane integrity, simultaneous addition of
254
PI, which does not permeate cells with an intact plasma membrane, allows discrimination
255
between apoptotic cells (Annexin-V +ve, PI –ve), secondary necrotic cells (Annexin-V +ve, PI
256
+ve), necrotic cells (Annexin-V -ve, PI +ve) and live cells (both Annexin-V -ve, PI -ve).
257
Accordingly, in order to study whether the mechanism of cell death triggered by the n-hexane
258
fractions of leaves and seeds is via apoptosis or necrosis, treated and untreated promastigotes
259
were double stained with FLUOS-conjugated annexin-V and PI. A significant percentage (3.1
260
and 6.6) of promastigotes treated with AAL and AAS, respectively stained positive for annexin-
261
V, compared to only 0.4 % in artemisinin-treated and untreated cells (Fig. 6). This was
262
comparable with apoptosis triggered by pentamidine (3.2%) that served as positive control
263
indicating that the leishmanicidal activity of AAL and AAS is via apoptosis.
264
Nuclear DNA fragmentation in AAS and AAL -treated promastigotes by in situ labeling of
265
DNA fragments via a TUNEL assay: One of the hallmarks of apoptotic cell death is the
266
degradation of nuclear DNA into oligonucleosomal units. DNA nicking resulting from the
267
exposure to n-hexane fractions of leaves and seeds was detected using a TUNEL assay in which
268
the proportion of DNA nicks was quantified by measuring the binding of dUTP-FLUOS to the 8
269
nicked end via TdT. Promastigotes treated with 100 µg ml─1 of AAL and AAS for 72 h appeared
270
yellowish green under fluorescence microscope representing incorporation of dUTP-labelled
271
with FLUOS, thus indicating nicking of DNA (Fig. 7b).
272
The occurrence of DNA nicking was also detected by quantifying the binding of dUTP-FLUOS
273
to the nicked end via TdT, as the proportion of DNA nicks is directly proportional to the
274
fluorescence obtained from dUTP-FLUOS. Treated promastigotes after 72 h showed an increase
275
in nuclear DNA fragmentation as evidenced from dUTP-FLUOS binding. The mean
276
fluorescence intensity (MFI) of untreated (control) and treated fractions (AAL, AAS, artemisinin
277
and pentamidine) were found to be 611, 13083, 15296, 433 and 24289, respectively (Fig. 7a),
278
thus providing strong evidence for apoptosis in L. donovani promastigotes.
279
Artemisia annua (n-hexane fractions) induced apoptosis causing an increase in the sub G0-
280
G1 population: Flow cytometric analysis after cell permeabilization and labeling with PI was
281
used to quantify the percentage of pseudohypodiploid cells. In a given cell, the amount of PI
282
correlates with the DNA content, and accordingly, DNA fragmentation in apoptotic cells
283
translates into a sub G0-G1 peak (Sarkar et. al., 2008). Promastigotes treated with AAL and AAS
284
(100 µg ml─1) for 24 and 72 h caused cell cycle arrest at sub G0-G1 phase, as analysed by flow
285
cytometry (Fig. 8). The proportion of cells in control was 5.52% in the sub G0-G1 phase, which
286
increased to 19.90%, 19.76% and 16.35% in promastigotes, treated with AAL, AAS and
287
pentamidine, respectively. Only a slight increase (5.91%) in the sub G0-G1 cell population was
288
observed upon treatment with artemisinin. This increase in cell proportion in the sub G0-G1
289
phase corroborated our findings that AAL and AAS induced apoptosis in promastigotes causing
290
DNA fragmentation.
291
Cytotoxicity of bioactive fractions on mammalian macrophages: Peritoneal macrophages
292
were isolated from mice to check the adverse side effects of the bioactive fractions, using
293
pentamidine as the reference drug. The cytotoxicity assay revealed that there was no adverse
294
toxicity of AAL and AAS even at 200 µg ml─1 on mammalian macrophages (Fig. 9).
295
Determination of artemisinin content in AAL and AAS by HPTLC fingerprinting: The
296
artemisinin content in the n-hexane fractions of A. annua leaves and seeds was quantified by
9
297
HPTLC fingerprinting. The content of artemisinin in 100 µg of AAL and AAS was found to be
298
1.447 µg and 1.336µg, respectively (Table. 1).
299
Composition of AAL and AAS: The major chemical constituents of AAL and AAS that may
300
have contributed to leishmanicidal activity were identified by GC-MS analysis. α−amyrinyl
301
acetate (28.17%), β-amyrine (20.87%), dihydroartemisinin 3-desoxy (4.91%), deoxyartemisinin
302
(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
304
(1.45%) were revealed in AAS by comparison of mass spectral data and retention times as
305
presented (Fig. 10a and 10b and Supplementary Tables 2(a) and 2(b)).
306 307
Discussion:
308
The leishmanicidal activity of miltefosine (Verma and Dey, 2004), amphotericin B (Lee et al.,
309
2002) and camptothecin (Sen et al., 2004) have been reported to be mediated by apoptosis. The
310
efficacy of artemisinin against promastigotes of L. donovani has recently been reported (Sen et
311
al., 2007). However, in our study, we have shown that, artemisinin and artesunate exhibit partial
312
growth inhibitory activity against L. donovani promastigotes at a concentration of 100µg ml─1, in
313
contrast to the significant leishmanicidal effect mediated by the n-hexane fractions of A. annua
314
leaves (AAL) and seeds (AAS). To elucidate the mode of action of AAL and AAS against
315
promastigotes of L. donovani, we have used biochemical and morphological approaches, and
316
have demonstrated that AAL and AAS induced programmed cell death that share several
317
phenotypic features with metazoan apoptosis as also reported by Debrabant et al. (2003). These
318
apoptotic correlates include PS exposure, DNA nicking as evidenced by TUNEL assay and cell
319
cycle arrest at sub G0-G1 phase. Taken together, we have conclusively demonstrated that AAL
320
and AAS induce PCD in promastigotes that notably share some, but not all of the classical
321
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.
323
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
326
deoxyartemisinin and arteannuin as the major active constituents in AAL while cetin, einecs,
327
dihydroartemisinin 3-desoxy, deoxyartemisinin and arteannuin were predominantly present in
328
AAS. Synthetic dihydroartemisinin acetal derivatives (dimer and trimer) have also been reported
329
by others to exhibit antileishmanial, antimalarial, anticancer and antifungal activities (Slade et al.
330
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
332
(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
References:
344 345 346
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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
