Journal of Medical Microbiology (2009), 58, 1058–1066
DOI 10.1099/jmm.0.009290-0
Pro-apoptotic effect of the landrace Bangla
Mahoba of Piper betle on Leishmania donovani
may be due to the high content of eugenol
Pragya Misra,1 Awanish Kumar,1 Prashant Khare,1 Swati Gupta,1
Nikhil Kumar2 and Anuradha Dube1
Correspondence
1
Anuradha Dube
2
anuradha_dube@hotmail.com
Received 7 January 2009
Accepted 9 April 2009
Division of Parasitology, Central Drug Research Institute (CDRI), Lucknow, India
Betel Vine Biotechnology Laboratory, National Botanical Research Institute, Lucknow, India
In the absence of effective and safe treatment for visceral leishmaniasis or Kala-azar – a
devastating parasitic disease caused by Leishmania donovani – the search for anti-leishmanial
agents from natural resources in common use is imperative. Recently, the comparative in vitro
anti-leishmanial activity of methanolic extracts from two landraces of Piper betle – P. betle
landrace Bangla Mahoba (PB-BM) and P. betle landrace Kapoori Vellaikodi (PB-KV) – has been
reported. Here, the putative pathway responsible for death induced by the effective extract of
PB-BM methanolic extract in promastigotes, as well as the intracellular amastigote form of L.
donovani, was assessed using various biochemical approaches. It was found that PB-BM was
capable of selectively inhibiting both stages of Leishmania parasites by accelerating apoptotic
events by generation of reactive oxygen species targeting the mitochondria without any
cytotoxicity towards macrophages. The study was extended to determine the presence or
absence of activity of the methanolic extract of PB-BM and PB-KV on the basis of differences in
essential oil composition present in the extract assessed by GC and MS. The essential oil from
PB-BM was found to be rich in eugenol compared with that from PB-KV. The anti-leishmanial
efficacy of PB-BM methanolic extract mediated through apoptosis is probably due to the higher
content of eugenol in the active landrace. This observation emphasizes the need to extend studies
related to traditional medicines from bioactive plants below the species level to the gender/
landrace level for better efficacy and reproducibility.
INTRODUCTION
Leishmaniasis comprises a group of infectious diseases with
worldwide distribution causing severe morbidity or/and
fatality, of which visceral leishmaniasis (VL) (or Kala-azar)
caused by the protozoan parasite Leishmania donovani is
the most devastating. For various reasons, current therapies
are not proving very effective. The first line of drugs for VL,
such as pentavalent antimonials, sodium stibogluconate
and meglumine antimoniate, have variable efficacy and
have severe side effects (Thakur et al., 1984). Amphotericin
B and pentamidine, the second-line drugs used clinically,
Abbreviations: FITC, fluorescein isothiocyanate; FIU, fluorescence
intensity unit; GFP, green fluorescent protein; H2DCFDA, 29,79dichlorodihydrofluorescein diacetate; IC50, concentration inhibiting cell
growth by 50 %; KI, Kovats index; MFI, mean fluorescence intensity; PBBM, Piper betle landrace Bangla Mahoba; PB-BME, PB-BM essential oil;
PB-BMM, PB-BM methanolic extract; PB-KV, Piper betle landrace
Kapoori Vellaikodi; PB-KVE, PB-KV essential oil; PB-KVM, PB-KV
methanolic extract; PI, propidium iodide; ROS, reactive oxygen species;
TUNEL, terminal deoxyribonucleotidyl transferase-mediated dUTP nick
end labelling; VL, visceral leishmaniasis.
1058
have limited efficacy and are very toxic (Iwu et al., 1994).
Moreover, cases of drug resistance are also on the rise
(Croft, 2001). Due to these problems, interest in the study
of ethano-medicines as a source of new chemotherapeutic
compounds with comparable or better activities and
minimal side effects has increased in recent years. India is
rich in traditional medicinal plant species, providing
opportunities to explore and exploit this resource for
various diseases and metabolic disorders. Piper betle Linn.
(Piperaceae) is a well-known medicinal plant grown widely
in the humid climate of South-East Asia. Globally, more
than 600 million people consume P. betle daily in one form
or the other. It is also known to be dioecious with more
than a hundred landraces reported. Differences between
landraces in terms of leaf shape, size and chlorophyll
content along with characterization based on random
amplification of polymorphic DNA have been reported
(Kumar et al., 2006; Verma et al., 2004) . The leaves are
known for their antimicrobial, anti-inflammatory and
antifungal activities (Ramji et al., 2002). The leaves inhibit
carcinogen-induced tumours of the oral cavity and
009290 G 2009 SGM Printed in Great Britain
P. betle induces apoptosis in Leishmania
mammary tissue (Rao, 1984). Protective single/combined
treatment with betel leaf and turmeric against methyl(acetoxymethyl)nitrosamine-induced hamster oral carcinogenesis has been observed. In addition, the leaves exert
chemopreventive effects against lung and forestomach
tumours induced by N9-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (Padma et al.,
1989).
The medicinal importance of P. betle along with its high
level of consumption in areas endemic for leishmaniasis
prompted us to evaluate the anti-leishmanial efficacy of
this natural product. The in vitro anti-leishmanial activity
of the methanolic extracts from leaves of P. betle landrace
Bangla Mahoba (PB-BM) and P. betle landrace Kapoori
Vellaikodi (PB-KV) has been evaluated previously against
L. donovani promastigotes and intracellular amastigotes by
our group, and a potent anti-leishmanial activity was
observed only for PB-BM methanolic extract (PB-BMM)
(Tripathi et al., 2006). The current study investigated the
anti-leishmanial mode of action of PB-BMM by using
various biochemical assays. In addition, possible explanations for the different activities obtained for both PB-BMM
and PB-KV methanolic extract (PB-KVM) are discussed
based on the composition of their essential oils.
METHODS
source and interface was kept at 250 uC. Identification of the
constituents in the oil was carried out by comparing Kovats index
(KI) values and peak enrichment with authentic samples, and by
comparing the mass spectra of eluted compounds with those in the
(USA) National Bureau of Standards (NBS) and National Institute of
Standards and Technology (NIST)/Wiley libraries.
Parasites. The promastigotes of L. donovani (MHOM/IN/80/Dd8)
were grown in RPMI 1640 supplemented with 10 % heat-inactivated
fetal bovine serum, 100 U penicillin ml21 and 100 mg streptomycin
ml21 at 26 uC (all from Sigma). Transgenic parasites expressing green
fluorescent protein (GFP) (Singh & Dube, 2004) were cultured in the
presence of 100 mg geneticin ml21 (G 418 sulphate; Sigma).
Isolation of amastigotes from a macrophage cell line. J774A.1
macrophages (108 cells) in 50 ml culture flasks (Nunc) were infected
with promastigotes at an m.o.i. of 10 and incubated at 37 uC in 5 %
CO2 for 8–12 h, after which the cells were washed three times with
PBS (pH 7.2) and complete Dulbecco’s minimal essential medium
was added. Infected macrophages were harvested by centrifugation at
2000 g for 10 min. The pellet was resuspended in 1 ml PBS and
passed repeatedly through a 26-gauge sterile needle to facilitate the
release of amastigotes by forced bursting of the macrophages. The
released amastigotes were purified using Percoll (Sigma) densitygradient centrifugation (Chang, 1980). Briefly, amastigotes released
from the macrophages were centrifuged at 800 g for 10 min to
remove tissue debris. The supernatant was centrifuged at 1600 g for
15 min at 4 uC. The pellet was resuspended in 5 ml 45 % Percoll in
2 mM EDTA in PBS and layered over a cushion of 2 ml 90 % Percoll.
The gradient was centrifuged at 3500 g at 4 uC for 1 h in a swing-out
rotor. The amastigotes were collected from the interphase of the 45–
90 % step gradient and washed with PBS.
Plant material and preparation of extract. P. betle landraces were
grown at the National Botanical Research Institute, Lucknow, India,
by obligate vegetative propagation to eliminate the variability that can
arise from crops grown from seed. Mature leaves of the landraces BM
and KV were harvested, washed and patted dry in the shade until
there was no change in their dry weight. The dried leaves were
pulverized to a fine powder and extracted in methanol until there was
no further green colour in the extract. Pooled extract was
concentrated in a Rota Vapour and further dried under vacuum.
Both plants yielded 50 g methanol extract (kg dry leaf powder)21.
Extraction of essential oils. Distillation was carried out for about
12 h for 2 days. The oil was recovered in ether, dried and weighed.
The PB-BM essential oil (PB-BME) and the PB-KV essential oil (PBKVE) represented 0.20 and 0.10 % of the fresh leaf weight,
respectively.
GC and GC-MS analyses of the essential oils. GC analysis was
carried out by using a PerkinElmer AutoSystem XL chromatograph
equipped with a flame-ionization detector and a PE-5 column
(60 m60.32 mm, 0.25 mm film thickness). The column oven
temperature was maintained initially at 100 uC for 1 min and then
programmed at an increase rate of 3 uC min21 up to 280 uC. The
column head pressure was maintained at 10 p.s.i. with a split ratio of
1 : 30 and the carrier gas used was hydrogen. The injector and detector
temperatures were 250 and 280 uC, respectively.
GC-MS analysis was carried out on a PerkinElmer XL system attached
to a TerboMass. GC-MS analysis was performed on an Equity-5
column (60 m60.32 mm, 0.25 mm film thickness). The column oven
temperature was initially held at 70 uC for 2 min and then
programmed at an increase rate of 3 uC min21 up to 250 uC, with
a 2 min hold. The column head pressure was maintained at 10 p.s.i.
with a split ratio of 1 : 30. The carrier gas used was helium. Ionization
was by electron impact at 70 eV, and the temperature of the injector,
http://jmm.sgmjournals.org
Activity against promastigotes and intracellular amastigotes.
Exponential-phase transgenic GFP-expressing promastigotes (106
cells ml21) were added to a 48-well culture plate (CellStar) and
treated with different concentrations of miltefosine (a standard
antiprotozoal drug used for the treatment of VL, used here as a
reference drug), as well as with PB-BMM and PB-KVM. Untreated
cells served as a control. J774A.1 macrophages (105 cells per well)
cultured in 24-well plates were infected with late-exponential-phase
GFP-expressing promastigotes at an m.o.i. of 10 and incubated at
37 uC in 5 % CO2 for 8–12 h. Wells were washed to remove nonphagocytosed parasites. Cells supplemented with complete medium
were treated with different concentrations of extract, as well as
with miltefosine. Both treated promastigotes and intracellular
amastigotes were removed at different time intervals (48–72 h),
washed in PBS and analysed by acquiring 10 000 cells in a
FACSCalibur flow cytometer equipped with a 15 mV 488 nm aircooled argon laser with excitation at 488 nm and emission at
515 nm, using CellQuest software (Becton Dickinson). Inhibition
of parasite growth was determined by comparing the mean
fluorescence intensity (MFI) of drug-treated parasites with that
of untreated parasites.
Cytotoxicity assay. The in vitro cytotoxicity of PB-BMM against
J774A.1 macrophages was assessed by a colorimetric 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
reduction assay. Cells (105 ml21) were incubated with various
concentrations of PB-BMM at 37 uC in 5 % CO2 for 48 h. MTT was
added at a concentration of 400 mg ml21 and the cells were further
incubated for 3 h at 24 uC. The cells were centrifuged at 1000 g, the
pellets were dissolved in DMSO and the absorbance was read at
540 nm. The mean percentage of viable cells after treatment was
calculated relative to the control, and results were expressed as the
concentration inhibiting cell growth by 50 % (IC50).
1059
P. Misra and others
DNA condensation study by propidium iodide (PI) staining.
DNA condensation following treatment with 11.2 mg PB-BMM ml21
(IC50) was observed by staining untreated and treated promastigotes
with PI as described by Rybczynska et al. (2001). Briefly, untreated
and treated promastigotes were fixed in 4 % paraformaldehyde on to
poly-L-lysine-coated slides. Slides were washed twice with PBS to
remove non-adherent cells. Adherent cells were permeabilized with
0.2 % Triton X-100 for 1 min and washed twice with PBS. They were
then incubated with 10 mg PI ml21 for 2 min. Cells were then
observed using a high-resolution fluorescence camera (Leica DFC320)
mounted on a Leica DM5000B microscope and images were
processed as described above. At least 20 microscopic fields were
observed for each sample.
Double staining with annexin V and PI. Externalization of
phosphatidylserine on the outer membrane of treated and untreated
promastigotes as well as on intracellular amastigotes was measured by
the binding of annexin V and PI as described by Mehta & Shaha
(2004). Briefly, parasites were incubated with 10 mg PB-BMM ml21
for different time periods. Treated and untreated promastigotes and
purified amastigotes (Chang, 1980), suspended in annexin V binding
buffer (BD Biosciences), were incubated with annexin V and PI
following the manufacturer’s instructions for 15 min in the dark at
20–25 uC. Acquisition and analysis were carried out on a
FACSCalibur flow cytometer using CellQuest software.
Estimation of reactive oxygen species (ROS) levels. To evaluate
the generation of ROS in promastigotes and infected macrophages
following treatment with PB-BMM, the cell-permeant probe 29,79dichlorodihydrofluorescein diacetate (H2DCFDA) was used
(Duranteau et al., 1998). H2DCFDA is a non-polar compound that
readily diffuses into cells, where it is hydrolysed to the nonfluorescent derivative dichlorodihydrofluorescein and is thereby
trapped within the cells. In the presence of a proper oxidant,
dichlorodihydrofluorescein is oxidized to the highly fluorescent 29,79dichlorofluorescein. Cells treated with PB-BMM (at the IC50) for
different time periods were resuspended in 500 ml RPMI 1640 and
labelled with 10 mM H2DCFDA for 15 min in the dark. Cells were
analysed for intracellular ROS by using a FACSCalibur flow cytometer
with CellQuest software.
Measurement of nitric oxide (NO) production in infected
macrophages. NO detection was carried out as follows. Briefly,
treated and untreated cells were incubated with the fluorescent probe
4,5-diaminofluorescein-2 diacetate (1 mM; Molecular Probes) following the manufacturer’s protocol for 30 min in the dark and acquired
on a FACSCalibur flow cytometer (excitation 488 nm, emission
535 nm). Data were analysed by CellQuest software and results were
expressed as MFI.
Mitochondrial membrane potential determination. The mitochondrial membrane potential (DYm) was monitored by using JC-1
dye as a probe (Dey & Moraes, 2000). JC-1 is a cationic mitochondrial
vital dye that is lipophilic and becomes concentrated in the
mitochondria in proportion to DYm: more dye accumulates in
mitochondria with a greater DYm and ATP-generating capacity
(Sudhandiran & Shaha, 2003). The dye exists as a monomer at low
concentrations (emission 530 nm) but forms J-aggregates (emission
590 nm) at higher concentrations. Briefly, both treated promastigotes
and isolated amastigotes were collected after treatment with PB-BMM
for various time periods and incubated for 7 min with 10 mM JC-1 at
37 uC, washed and resuspended in medium. The ratio of fluorescence
at 590 to 530 nm was considered to be the relative DYm value.
DNA fragmentation assay. Fragmentation of chromatin to units of
single or multiple nucleosomes that form the nucleosomal DNA
ladder in agarose gel is an established hallmark of programmed cell
1060
death or apoptosis (Bortner et al., 1995). Total cellular DNA from
treated and untreated isolated amastigotes was isolated by a published
procedure (Das et al., 2001). Briefly, pellets of untreated and treated
cells (107) were treated with sarcosyl detergent lysis buffer [50 mM Tris/
HCl (pH 7.5), 10 mM EDTA, 0.5 % (w/v) sodium N-lauryl sarcosine]
and proteinase K (15.6 mg ml21) and incubated overnight at 50 uC.
The lysates were then extracted with phenol/chloroform/isoamyl
alcohol (25 : 24 : 1) and centrifuged at 16 000 g for 5 min. To the
upper phase, 0.3 M sodium acetate and 100 % ethanol (twice the
volume) were added, and the mixture was kept overnight at 220 uC.
The sample was centrifuged at 16 000 g for 10 min. The DNA pellet was
washed with 0.5 ml 70 % ethanol and solubilized in TE [10 mM Tris/
HCl (pH 8.0), 1 mM EDTA]. RNase A (0.3 mg ml21) treatment was
carried out for 1 h at 37 uC. Extracted DNA was quantified spectrophotometrically at 260/280 nm. A total of 10 mg DNA was mixed with
tracking dye and run on a 1 % agarose gel containing ethidium
bromide in TAE buffer [40 mM Tris/acetate (pH 8.0), 1 mM EDTA].
Gels were run for 2 h at 50 V and visualized under UV light.
In situ labelling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling
(TUNEL) assay. In situ detection of DNA fragments by a TUNEL
assay was performed by using a DeadEnd fluorometric TUNEL
system (Promega). Briefly, promastigotes and intracellular amastigotes (16106) isolated from macrophages treated or not with PBBMM were fixed in 4 % formaldehyde and permeabilized with 0.2 %
Triton X-100 followed by incubation with buffer containing
nucleotide mix following the manufacturer’s protocol. Cells stained
by TUNEL assay were analysed in a FACSCalibur flow cytometer.
Green fluorescence, gated on forward and side light scatter, was
determined using a band-pass filter (525±10 nm).
Statistical analysis. The data are presented as means±SD. The
statistical significance of differences in percentage expression between
treated and untreated groups was analysed by one-way analysis of
variance using SigmaStat (version 2.03) software.
RESULTS
Essential oil composition in PB-BM and PB-KV
The amount of essential oil produced by PB-BM was higher
than that obtained from PB-KV (0.2 and 0.1 % of fresh leaf
weight, respectively). The composition of the essential oils
PB-BME and PB-KVE was studied by GC and GC-MS
analyses, and the major constituents are shown in Table 1.
PB-BME was found to be richer in eugenol and other
phenols than PB-KVE (73.01and 27.15 % eugenol, respectively). cis-Methylisoeugenol and trans-isoeugenol co-elute in
one peak but can be identified based on KI values, peak
enrichment and comparison with the mass spectra of
authentic samples. However, they were not detected by GC
or GC-MS analysis in either of the essential oils.
Determination of IC50 of PB-BMM against
L. donovani promastigotes and intracellular
amastigotes
PB-BMM treatment showed a dose-dependent inhibitory
effect on promastigotes and intracellular transgenic
amastigotes. The IC50 values of PB-BMM for promastigotes
and intracellular amastigotes were 11.2±1.23 and
Journal of Medical Microbiology 58
P. betle induces apoptosis in Leishmania
Table 1. Major constituents of PB-BME and PB-KVE
Compound
n-Hexanol
a-Thujene
Sabinene
b-Myrcene
a-Phellandrene
a-Terpinene
p-Cymene
Limonene
b-Phellandrene
b-Ocimene
cis-Ocimene
Linalool
a-Thujone
trans-Sabinenehydrate
Decanal
Geraniol
Safrole
Eugenol
cis-Isoeugenol
cis-Methylisoeugenol+
trans-isoeugenol
a-Copaene
b-Elemene
Methyl eugenol
b-Caryophyllene
a-Humlene
c-Muurolene
Germacrene D
Eugenol acetate
Methyl chavicol
Chavicol
d-Cadinene
Total
KI value
PB-KVE PB-BME
(%)
(%)
870
931
978
993
1007
1019
1029
1032
1034
1036
1045
1102
1104
1107
1207
1255
1295
1357
1363
1382
–
0.05
0.29
0.14
–
0.04
0.04
–
–
0.06
0.05
0.04
–
–
0.73
0.24
46.28
27.15
3.44
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
73.01
3.41
–
1386
1396
1407
1431
1461
1482
1484
1534
1198
1253
1526
0.32
0.07
2.84
2.56
1.43
–
2.89
–
–
–
–
88.66
–
–
–
–
–
–
–
7.89
0.19
1.93
1.71
88.14
9.31±0.53 mg ml21, respectively. This was comparable to
the IC50 of the standard drug miltefosine (10 and 5 mg
ml21, respectively). No anti-leishmanial activity was
observed for PB-KVM, even at a 50-fold higher concentration, against either form of the parasite. Thus, further
studies were conducted using landrace PB-BMM.
Cytotoxicity of PB-BMM
PB-BMM was found to be devoid of any cytotoxic effect
towards macrophages, even at a concentration of 100 mg
ml21, which is many fold higher than the IC50 of PB-BMM
against L. donovani promastigotes and intracellular amastigotes (data not shown).
Condensation of nuclear material
Condensation of nuclear material, a primary event in
apoptosis-like cell death, was studied using PI. Cells with
http://jmm.sgmjournals.org
condensed nuclei exhibit brighter red fluorescence than
non-condensed nuclei. PB-BMM-treated promastigotes
showed bright red fluorescent spots compared with a dull
red generalized fluorescence in untreated cells (Fig. 1a).
Externalization of phosphatidylserine in
promastigotes and intracellular amastigotes after
exposure to PB-BMM
During apoptosis in metazoan and unicellular cells,
phosphatidylserine is translocated from the inner side to
the outer layer of the plasma membrane, which can be
measured by double staining with annexin V and PI. A
significant number of promastigotes (60.29 %) treated with
PB-BMM for 48 h stained positive for annexin V compared
with 6.4 % in untreated cells (Fig. 1b, ii and i, respectively).
The effect of PB-BMM was comparable to miltefosine
(74.43 %; Fig. 1b, iii) (Paris et al., 2004).
Harvested amastigotes from treated and untreated infected
macrophages were stained with annexin V conjugated to
fluorescein isothiocyanate (FITC). At 48 h, a significant
number of amastigotes recovered from cells treated with
PB-BMM or miltefosine stained positive for annexin V
(64.89 and 70.32 %; Fig. 1b, v and vi, respectively)
compared with controls at 24 h (8.02 %; Fig. 1b, iv).
PB-BMM-induced generation of ROS in
promastigotes and infected macrophages
H2DCFDA, a non-polar compound, is converted on
oxidation to the highly fluorescent 29,79-dichlorofluorescein and this property has been utilized to monitor ROS
generation. PB-BMM treatment of promastigotes led to a
significant increase in ROS up to 24 h (fluorescence
intensity units (FIU): 0 h, 6.18±0.56; 6 h, 15.33±0.59;
12 h, 27.39±1.52; 24 h, 35.64±1.06; Fig. 2a). An increase
in ROS was also observed in infected macrophages
following treatment with PB-BMM (FIU: 0 h,
20.29±1.15; 6 h, 26.8±1.8; 12 h, 46.29±2.4; 24 h,
109.7±5.9; Fig. 2b).
PB-BMM-stimulated NO production in infected
macrophages
PB-BMM treatment caused a significant increase in
diaminofluorescein-mediated fluorescence in infected
macrophages (MFI: 0 h, 93.36±2.12; 12 h, 198.6±3.35;
24 h, 267.7±14.35; Fig. 2c), indicating the generation of
NO.
PB-BMM-induced time-dependent loss of DYm in
promastigotes and isolated amastigotes
PB-BMM-induced time-dependent changes in DYm were
monitored at 6, 12 and 24 h post-treatment. A significant
drop in DYm of 44.9 % was observed 6 h post-treatment
(590 : 530 nm ratio: control 6.28±0.49; drug-treated
1061
P. Misra and others
Fig. 1. Induction of apoptosis after PB-BMM treatment. (a) Analysis of DNA condensation in promastigotes treated with PBBMM (10 mg ml”1) for 24 h by PI staining. ( i) Untreated promastigotes; (ii) PB-BMM-treated promastigotes. Condensed nuclei
are indicated by arrows. The results are representative of three independent experiments. (b) Externalization of
phosphatidylserine following PB-BMM (IC50) treatment. The lower-left quadrant indicates the percentage of unstained cells,
whilst the upper left shows PI-positive cells, the lower right shows annexin V-stained cells and the upper right shows PI- and
annexin-V-positive cells. (i) Untreated promastigotes; (ii) PB-BMM-treated promastigotes; (iii) miltefosine-treated promastigotes; (iv) untreated amastigotes; (v) PB-BMM-treated amastigotes; (vi) miltefosine-treated amastigotes. The results shown are
representative of at least three experiments.
3.46±0.57; Fig. 3a), which increased to 62 % after 12 h of
treatment (control 5.19±0.32; drug-treated 1.95±0.2;
Fig. 3a). A further drop in DYm of 71 % at 24 h (control
(b)
50
(c)
150
300
***
***
30
20
***
***
***
***
200
100
MFI
40
FIU (530 nm)
FIU (530 nm)
(a)
5.12±0.24; drug-treated 1.46±0.14; Fig. 3a) was observed,
indicating that PB-BMM caused sustained hypopolarization of the mitochondrial membrane in promastigotes.
***
50
100
10
0
6
12
24
0
6
12
Time (h)
24
0
12
24
Fig. 2. PB-BMM induces the generation of ROS (a, b) and NO (c). Increase in ROS generation in promastigotes (a) and
infected macrophages (b) after treatment with PB-BMM for 6, 12 and 24 h. (c) NO generation in infected macrophages after
treatment with PB-BMM for 0, 12 and 24 h. Each bar represents MFI±SD. Grey bars, untreated; hatched bars, PB-BMM
treated. Asterisks indicate significant differences between treated and untreated groups: *, P,0.05; **, P,0.01; ***, P,0.001.
1062
Journal of Medical Microbiology 58
MFI (590: 530 nm fluorescence)
P. betle induces apoptosis in Leishmania
(a)
15
(b)
15
10
10
5
***
6
***
5
***
0
**
12
***
***
0
24
6
12
24
Time (h)
Fig. 3. Decrease in DYm in promastigotes (a) and isolated amastigotes (b) following treatment with PB-BMM for the indicated
times and staining with the potentiometric probe JC-1 (10 mM). DYm values are expressed as the ratio of 590 : 530 nm
fluorescence. Each bar represents the MFI±SD. Grey bars, untreated; hatched bars, PB-BMM treated. Asterisks indicate
significant differences between treated and untreated groups: *, P,0.05; **, P,0.01; ***, P,0.001.
Whilst monitoring DYm in intracellular amastigotes, the
macrophage mitochondria might interfere and lead to
erroneous data. Isolation of the amastigotes from macrophages after drug treatment and measurement of DYm
could have been performed afterwards, but in this case the
preparation time would have contributed to alterations in
the DYm (Sudhandiran & Shaha, 2003). Therefore,
isolated and purified amastigotes were treated with PBBMM for various time periods, and changes in DYm were
monitored. The decrease in DYm due to PB-BMM
treatment after 6 h was 26 % (590 : 530 nm ratio: control
11.09±0.7; drug-treated 8.16±0.61; Fig. 3b) followed by a
further decrease to 43.2 % at 12 h post-treatment (control
9.86±0.65; drug-treated 5.6±0.56; Fig. 3b). PB-BMM
treatment extended the decrease in DYm to 59 % after 24 h
(control 10.20±1.02; drug-treated 4.18±0.36; Fig. 3b).
isolated amastigotes showed clear fragmentation of the
genomic DNA into oligonucleosomal fragments in the
characteristic ladder form seen during apoptosis, compared
with untreated cells (Fig. 4a).
DISCUSSION
Oligonucleosomal DNA fragmentation in isolated
amastigotes following treatment with PB-BMM
Several plants have been shown to possess interesting antileishmanial activities, validating their use in folk medicine
(Rocha et al., 2005). In our earlier studies, we reported
gender/landrace-based differences in the anti-leishmanial
activity of the methanolic extract of P. betle, a dioecious
plant (Tripathi et al., 2006). It was observed that PB-BMM
showed potent anti-leishmanial activity, whereas no activity
was observed with PB-KVM. Moreover, selective elimination of the parasite without affecting host macrophage cells
led us to evaluate the mode of death induced in both forms
of the parasite. As apoptosis can cause the selective killing of
parasites without affecting the entire population (Debrabant
et al., 2003), this mode of cell death was studied in PBBMM-treated L. donovani parasites. Interesting information
regarding the phenomenon of apoptosis in Leishmania has
been obtained in promastigotes (Paris et al., 2004), but has
been less studied in intracellular amastigotes, the form
responsible for disease pathogenesis, due to its intracellular
localization in splenic macrophages and the tedious
methods required for their isolation, along with low yield.
Studies carried out by Sarkar et al. (2008) were based on the
anti-leishmanial activity of P. betle leaves (without assigning
any particular landrace) and its mode of action against the
non-pathogenic (promastigote) form. Therefore, observations of mechanisms by which effective drugs induce their
anti-leishmanial effect against intracellular amastigotes
would help to develop rational strategies for evaluation of
the efficacy of preparations from plants.
DNA laddering is a classical sign of apoptosis.
Oligonucleosomal DNA fragmentation analysis of treated
To clarify the PB-BMM mode of action against L.
donovani, we demonstrated that PB-BMM-induced cell
Nuclear DNA fragmentation in promastigotes and
intracellular amastigotes following PB-BMM
treatment by in situ labelling of DNA fragments by
a TUNEL assay
DNA fragmentation, a hallmark of apoptosis in metazoan
and unicellular cells, was assessed by in situ labelling of DNA
fragments by a TUNEL assay. Flow cytometric analysis of
promastigotes and intracellular amastigotes isolated from
macrophages treated or not with PB-BMM and probed with
a TUNEL assay showed an increase in the number of cells
staining positive for TUNEL. TUNEL-positive cells are
represented by increased forward scatter. This indicated a
greater degree of staining for fragmented DNA (nearly 60 %
in promastigotes, Fig. 4b, c; 48 % in amastigotes, Fig. 4d, e).
http://jmm.sgmjournals.org
1063
P. Misra and others
Fig. 4. Detection of PB-BMM-induced DNA fragmentation. (a) DNA fragmentation in isolated amastigotes after treatment.
(b–e) Flow cytometric analysis after TUNEL staining of: (b) untreated promastigotes, (c) PB-BMM-treated promastigotes at
24 h post-treatment, (d) amastigotes isolated from untreated macrophages and (e) amastigotes isolated from PB-BMM-treated
macrophages at 24 h post-treatment.
death in L. donovani shares several phenotypic features
with other cases of metazoan apoptosis (Debrabant et al.,
2003), including phosphatidylserine exposure, PI staining,
in situ TUNEL staining of nicked DNA and oligonucleosomal DNA fragmentation (Das et al., 2001). During
programmed cell death in metazoan and unicellular cells,
phosphatidylserine is transferred from the inner side to the
outer layer of the plasma membrane. The apoptotic nature
of death induced by PB-BMM in both promastigotes and
intracellular amastigotes was therefore confirmed by
double staining with annexin V–FITC and PI, as annexin
V, a Ca2+-dependent phospholipid-binding protein with
affinity for phosphatidylserine, is routinely used to
demonstrate externalization of phosphatidylserine.
Annexin V–FITC labelling experiments were also performed for cells treated with PB-KVM to further confirm
our finding that this landrace/gender of plant showed no
activity against the parasite studied. The results of this
experiment, similar to those of the untreated control,
further confirmed that there are gender/landrace-based
differences in the activity of methanolic extracts from P.
betle landraces (data not shown).
P. betle has been shown to possess antioxidant activity, but
observation that metabolic activation of some plant
extracts can lead to the production of toxic pro-oxidants
and cause cell injury under different conditions (Cao et al.,
1997) led us to evaluate whether PB-BMM could induce
oxidative stress in promastigotes as well as in infected
macrophages. It is well established that ROS generation in
cells following drug treatment can direct the cells towards
apoptosis (Chipuk & Green, 2005). The increase in ROS
after exposure to PB-BMM followed by death of the
parasite suggested that PB-BMM-mediated generation of
1064
ROS by promastigotes and amastigote-infected macrophages was responsible for their apoptotic death.
Generation of NO after drug treatment in infected
macrophages further indicated the involvement of ROS
in amastigote death. NO generated in vitro by NOdonating compounds has been shown to induce DNA
fragmentation (Holzmuller et al., 2002).
Further studies were conducted to determine the changes
occurring after oxidative stress that were responsible for
apoptotic cell death. Earlier studies have established that
the mitochondrion is a possible target of ROS-induced
apoptosis in promastigotes and intracellular amastigotes,
occurring via the loss of DYm (Sudhandiran & Shaha,
2003). In this study, a sharp decrease in DYm indicated
that mitochondrial dysfunction occurred, which initiated
the changes required for the cell to enter the apoptosis-like
pathway following treatment with PB-BMM. Apart from
the mitochondrial dysfunction studies, all other studies
related to PB-BMM inducement of apoptosis-like death
were performed in intracellular amastigotes. Therefore, it
was necessary to confirm whether death in isolated
amastigotes occurred by the same method or not. The
observation of a DNA ladder after treatment showed that
isolated amastigotes also showed apoptosis-like cell death.
The observation of an apoptotic mode of cell death
induced in Leishmania parasites by P. betle, and the fact
that this effect was ‘landrace specific’ as PB-KVM was noneffective against Leishmania, led us to evaluate the
differences in essential oil composition of both landraces
to identify possible active components responsible for the
anti-leishmanial activity. GC and GC-MS of the essential
oil confirmed a significantly higher content of eugenol
Journal of Medical Microbiology 58
P. betle induces apoptosis in Leishmania
along with eugenyl acetate in PB-BME compared with PBKVE. Other active phenols such as anethol, chavicol,
chavicol acetate and hexadecanoic acid/methyl benzoate
were present in PB-BME but absent in the ineffective PBKVE. The effectiveness of eugenol in stimulating apoptosis
is known in human melanomas (Kim et al., 2006).
Moreover, it is known that eugenol at lower concentrations
acts as an anti-oxidant and anti-inflammatory agent,
whereas at higher concentrations it can work as a prooxidant, resulting in enhanced production of tissuedamaging free radicals (Suzuki et al., 1985; Wright et al.,
1995). PB-BME was found to have a significantly higher
eugenol content (approx. threefold higher than PB-KVE),
and apoptosis induced by PB-BMM was found to be
mediated via ROS generation. This finding, along with
the fact that eugenol at a higher concentration enhances the
generation of ROS and induces apoptosis, supports the
theory that the anti-leishmanial efficacy of PB-BMM is
likely to be due to the higher eugenol content. UedaNakamura et al. (2006) demonstrated the leishmanicidal
activity of the eugenol-rich essential oil derived from
Ocimum gratissimum, which further supports our observation. The presence of other active phenols in PB-BME
may also contribute to the higher efficacy of PB-BMM.
The present findings indicate that P. betle leaves possess
bioactive components, and further identification, characterization, purification and biological evaluation of these
are in progress. As the raw leaves are consumed by many
people who inhabit areas with a high incidence of
leishmaniasis, the use this folk medicine may have
therapeutic merit and deserves to be explored further.
This study has provided a novel rationale for extending the
search for bioactive plants below the species level to ensure
better efficacy and reproducibility.
ACKNOWLEDGEMENTS
Das, M., Mukherjee, S. B. & Shaha, C. (2001). Hydrogen peroxide
induces apoptosis-like death in Leishmania donovani promastigotes.
J Cell Sci 114, 2461–2469.
Debrabant, A., Lee, N., Bertholet, S., Duncan, R. & Nakhasi, H. L.
(2003). Programmed cell death in trypanosomatids and other
unicellular organisms. Int J Parasitol 33, 257–267.
Dey, R. & Moraes, C. T. (2000). Lack of oxidative phosphorylation and
low mitochondrial membrane potential decrease susceptibility to
apoptosis and do not modulate the protective effect of Bcl-xL in
osteosarcoma cells. J Biol Chem 275, 7087–7094.
Duranteau, J., Chandel, N. S., Kulisz, A., Shao, Z. & Schumacker,
P. T. (1998). Intracellular signaling by reactive oxygen species during
hypoxia in cardiomyocytes. J Biol Chem 273, 11619–11624.
Holzmuller, P., Sereno, D., Cavaleyra, M., Mangot, I., Daulouede, S.,
Vincendeau, P. & Lemesre, J. L. (2002). Nitric oxide-mediated
proteasome-dependent oligonucleosomal DNA fragmentation in
Leishmania amazonensis amastigotes. Infect Immun 70, 3727–
3735.
Iwu, M. M., Jackson, J. E. & Schuster, B. G. (1994). Medicinal plants
in the fight against leishmaniasis. Parasitol Today 10, 65–68.
Kim, G. C., Choi, D. S., Lim, J. S., Jeong, H. C., Kim, I. R., Lee, M. H. &
Park, B. S. (2006). Caspases-dependent apoptosis in human
melanoma cell by eugenol. Korean J Anat 39, 245–253.
Kumar, N., Gupta, S. & Tripathi, A. N. (2006). Gender-specific
responses of Piper betle L. to low temperature stress: changes in
chlorophyllase activity. Biol Plant 50, 705–708.
Mehta, A. & Shaha, C. (2004). Apoptotic death in Leishmania
donovani promastigotes in response to respiratory chain inhibition:
complex II inhibition results in increased pentamidine cytotoxicity.
J Biol Chem 279, 11798–11813.
Padma, P. R., Lalitha, V. S., Amonkar, A. J. & Bhide, S. V. (1989).
Anticarcinogenic effect of betel leaf extract against tobacco carcinogens. Cancer Lett 45, 195–202.
Paris, C., Loiseau, P. M., Bories, C. & Breard, J. (2004). Miltefosine
induces apoptosis-like death in Leishmania donovani promastigotes.
Antimicrob Agents Chemother 48, 852–859.
Ramji, N., Ramji, N., Iyer, R. & Chandrasekaran, S. (2002). Phenolic
antibacterials from Piper betle in the prevention of halitosis.
J Ethnopharmacol 83, 149–152.
Rao, A. R. (1984). Modifying influences of betel quid ingredients on
We express our sincere gratitude to the Director of the CDRI for his
keen interest and for providing facilities for the experiments. We are
also thankful to Dr Girish Chandra Uniyal, Scientist, Central Institute
of Medicinal and Aromatic Plants, Lucknow, India, for GC-MS
analysis. This study has been given CDRI communication no. 7443.
REFERENCES
Bortner, C. D., Oldenburg, N. B. & Cidlowski, J. A. (1995). The role of
DNA fragmentation in apoptosis. Trends Cell Biol 5, 21–26.
Cao, G., Sofic, E. & Prior, R. L. (1997). Antioxidant and prooxidant
behavior of flavonoids: structure–activity relationships. Free Radic
Biol Med 22, 749–760.
Chang, K. P. (1980). Human cutaneous Leishmania in a mouse
macrophage line: propagation and isolation of intracellular parasites.
Science 209, 1240–1242.
Chipuk, J. E. & Green, D. R. (2005). Do inducers of apoptosis trigger
caspase-independent cell death? Nat Rev Mol Cell Biol 6, 268–275.
Croft, S. L. (2001). Monitoring drug resistance in leishmaniasis. Trop
Med Int Health 6, 899–905.
http://jmm.sgmjournals.org
B(a)P-induced carcinogenesis in the buccal pouch of hamster. Int J
Cancer 33, 581–586.
Rocha, L. G., Almeida, J. R., Macedo, R. O. & Barbosa-Filho, J. M.
(2005). A review of natural products with antileishmanial activity.
Phytomedicine 12, 514–535.
Rybczynska, M., Spitaler, M., Knebel, N. G., Boeck, G., Grunicke, H.
& Hofmann, J. (2001). Effects of miltefosine on various biochemical
parameters in a panel of tumor cell lines with different sensitivities.
Biochem Pharmacol 62, 765–772.
Sarkar, A., Sen, R., Saha, P., Ganguly, S., Mandal, G. & Chatterjee, M.
(2008). An ethanolic extract of leaves of Piper betle (Paan) Linn
mediates its antileishmanial activity via apoptosis. Parasitol Res 102,
1249–1255.
Singh, N. & Dube, A. (2004). Short report: fluorescent Leishmania:
application to anti-leishmanial drug testing. Am J Trop Med Hyg 71,
400–402.
Sudhandiran, G. & Shaha, C. (2003). Antimonial-induced increase
in intracellular Ca2+ through non-selective cation channels in the
host and the parasite is responsible for apoptosis of intracellular
Leishmania donovani amastigotes. J Biol Chem 278, 25120–
25132.
1065
P. Misra and others
Suzuki, Y., Sugiyama, K. & Furuta, H. (1985). Eugenol-mediated
superoxide generation and cytotoxicity in guinea pig neutrophils. Jpn
J Pharmacol 39, 381–386.
Ueda-Nakamura, T., Mendonça-Filho, R. R., Morgado-Dı́az, J. A.,
Korehisa Maza, P., Prado Dias Filho, B., Aparı́cio Garcia Cortez, D.,
Alviano, D. S., Rosa Mdo, S., Lopes, A. H. & other authors (2006).
Thakur, C. P., Kumar, M., Singh, S. K., Sharma, D., Prasad, U. S.,
Singh, R. S., Dhawan, P. S. & Achari, V. (1984). Comparison of
Antileishmanial activity of eugenol-rich essential oil from Ocimum
gratissimum. Parasitol Int 55, 99–105.
regimens of treatment with sodium stibogluconate in kala-azar. Br
Med J (Clin Res Ed) 288, 895–897.
Verma, A., Kumar, N. & Ranade, S. A. (2004). Genetic diversity
amongst landraces of a dioecious vegetatively propagated plant,
betelvine (Piper betle L.). J Biosci 29, 319–328.
Tripathi, S., Singh, N., Shakya, S., Dangi, A., Bhattacharya, S. M.,
Dube, A. & Kumar, N. (2006). Landrace/gender-based differences in
Wright, S. E., Baron, D. A. & Heffner, J. E. (1995). Intravenous eugenol
phenol and thiocyanate contents and biological activity in Piper betle
L. Curr Sci 91, 746–749.
causes hemorrhagic lung edema in rats: proposed oxidant mechanisms. J Lab Clin Med 125, 257–264.
1066
View publication stats
Journal of Medical Microbiology 58