Research Paper

Tamarindus indica Bark Extract and a Bioactive Fraction

Induce Apoptosis in HeLa and PA-1 Cells
R. SHIRISHA AND K. N. VARALAKSHMI*
Department of Biotechnology, Center for Post Graduate Studies, Jain University, Bangalore 560 011 India

Shirisha and Varalakshmi: Anticancer Activity of Tamarindus indica Bark

The aim of the current study was to evaluate the anticancer potential of tamarind bark and its bioactive
fraction on HeLa and PA-1 cell lines. Tamarind bark extract was prepared with dichloromethane as the
solvent using Soxhlet apparatus and treated on HeLa and PA-1 cell lines to determine the anticancer
potential by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Further, bioassay guided
fractionation of the extract was carried out using thin layer chromatography. In vitro assays like fluorescence
microscopy, DNA fragmentation analysis, caspase-9 activity assay and flow cytometry analyses were carried
out to determine the mechanism of anticancer activity of the selected bioactive fraction. The fraction was
further characterized by gas chromatography-mass spectrometry. Tamarind bark dichloromethane extract
was found to be cytotoxic to HeLa and PA-1 cells. Bioassay guided fractionation indicated that the fourth
fraction of the extract possessed the bioactive component. Further, in vitro assays demonstrated that the
bioactive fraction induced caspase-9-mediated apoptosis in the cells and was able to reduce the total cell
count as evidenced by flow cytometry analyses. The gas chromatography-mass spectrometry analysis of the
bioactive fraction indicated the presence of cantharidin, an anticancer compound earlier reported from the
blister beetles. It can be concluded that the presence of cantharidin might be responsible for inhibition of
proliferation and induction of apoptosis in the cancer cells.

Key words: Apoptosis, anticancer, cantharidin, caspase, tamarind bark extract

Cancer is one of the most dreaded diseases as mortality from the Chinese blister beetles, Mylabris phalerata or
associated is very high despite the availability of M. cichorii, displays antitumor activity and induces
modern treatment modalities[1,2]. Hence, research apoptosis in many types of tumor cells. It can be
towards novel, more effective and tumor-specific poisonous to humans if taken internally. Externally it
treatment has gained momentum during the past is a potent vesicant. Properly dosed and applied, the
decades. In this pursuit, several medicinal and dietary same properties have been used for effective topical
plants have provided many anticancer compounds till medications for some conditions, such as treating
date[3,4]. Tamarindus indica L. commonly known as patients with infection of the skin. It has been used as
tamarind is one of the most important dietary plants an anticancer agent by the Chinese for the treatment
in which all parts have some nutritional or medicinal of hepatoma and esophageal carcinoma since a long
value. It has a history that dates back to ancient tribal time[12]. Numerous other studies have also shown that
people who used to prepare decoctions of the parts and cantharidin has induced cytotoxic effects on cancer
use it for medication. It was reported that tamarind cells[13]. According to reports from several epidemiologic
bark and leaves were used to treat wounds in West and studies, chronic inflammatory diseases are usually
East Africa[5]. Fruit and leaf have laxative property, associated with increased cancer risk[14,15]. Due to the
paste of dried seeds were used to set fractured bones, existence of causal relationship between inflammation
and decoction of the bark and leaves were used to treat and cancer and the reported antiinflammatory property
jaundice[6-8]. The bark of tamarind tree was reported as
to have antiinflammatory, analgesic and wound healing
properties[9,10]. In West Africa, the macerated fresh bark This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
of the young twigs was used both as a purgative and to allows others to remix, tweak, and build upon the work non-commercially,
relieve abdominal pain[11]. as long as the author is credited and the new creations are licensed under
the identical terms
Cantharidin is a colorless, odourless terpene secreted
Accepted 01 November 2016
by many species of blister beetles. Cantharidin, isolated Revised 26 September 2016
Received 06 June 2016
*Address for correspondence
E-mail: [email protected] Indian J Pharm Sci 2016;78(6):725-731

November-December 2016 Indian Journal of Pharmaceutical Sciences 725

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of tamarind bark, in the current study we aimed to chromatography (TLC):
analyse the anticancer potential of tamarind bark, also
To identify the bioactive fraction, TLC was used. TLC
to identify the biochemical mechanism and the active
plates (TLC silica gel 60 F254) were procured from
component responsible for this activity.
Merck Specialties Private Limited, Mumbai. Different
MATERIALS AND METHODS combinations of solvents with different concentrations
were tried and the best solvent combination was
Cell lines: used for separating the fractions from tamarind bark
extract[18]. The fractions were collected by preparative
HeLa and PA-1 cell lines were procured from National
TLC, dissolved in methanol and were dried. The dried
Centre for Cell Sciences (NCCS), Pune. HeLa cells
fractions were again tested for cytotoxicity by MTT
were grown in Dulbecco’s modified eagles medium
assay and the fraction with highest cytotoxic property
(HiMedia Laboratories Pvt Ltd) and PA-1 cell lines
was selected as the bioactive fraction.
were grown in minimum essential medium containing
10% fetal bovine serum (HiMedia Laboratories Pvt Fluorescence microscopy:
Ltd). Cells were maintained below passage 20 and used
HeLa and PA-1 cells were cultured in 25 cm2 culture
in experiments during the linear phase of growth. Both
flasks and treated with different concentrations of
the cell lines were used for the cytotoxicity tests.
tamarind bark bioactive fraction for 24 h. A control
Preparation of the crude extract and screening for was maintained simultaneously. After incubation for
cytotoxicity: the indicated time period, the control and treated cells
were harvested separately, washed with PBS. The cells
Tamarind bark was collected from Indian Institute of
were treated with acridine orange (100 µg/ml) and
Science (IISc), Bengaluru, India and dried under the
ethidium bromide (100 µg/ml) stain prepared in the
shade. The dried bark was ground to a fine powder and
ratio 1:1, mounted on slides and were observed under a
50 g of the powder was packed in a Whatman filter paper
fluorescence microscope[19].
and extracted in a Soxhlet apparatus, using the solvent
dichloromethane[16]. The extract was concentrated DNA fragmentation analysis:
using a rotary evaporator, dried and stored at 4° till
HeLa and PA-1 cells (2×104 cells/ml) were treated with
further use. Stock solution of 1 mg/ml of the extract
different concentrations of tamarind bark bioactive
was prepared with dimethyl sulphoxide (DMSO) and
fraction and incubated for 24 h. simultaneously, a
later diluted to desired concentrations using phosphate
control was maintained. Cells were washed with
buffered saline (PBS).
PBS, trypsinised, centrifuged and the cell pellet was
MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl lysed in buffer containing 10 mM Tris HCl, 10 mM
tetrazolium bromide] assay: EDTA, 0.5% Triton X-100. To avoid RNA and protein
contamination, 200 µg/ml of RNase and 200 µg/ml
MTT assay was performed to determine the cytotoxic
proteinase K were added. DNA was precipitated with
effect of tamarind bark extract on cancer cell lines[17]. ice cold ethanol and suspended in Tris-EDTA solution.
Approximately 2×104 cells/well were seeded in 96 well Samples were resolved using 0.8% agarose gel and
microplates and incubated at 37° for 24 h. Different visualized under a UV transilluminator[20].
concentrations of the sample were added and the
microplates were further incubated for 24, 48 and 72 Caspase-9 activity assay:
h. After specified period of incubation, 20 µl of MTT The assay was performed using caspase-9 apoptosis
was added and incubated in the dark for 3 h. After 3 detection kit (G-Biosciences) according to the
h, 200 µl of DMSO was added to each well and the manufacturer’s instructions. Cells were treated with 5
absorbance was recorded at 540 nm in an enzyme µg/ml of tamarind bark bioactive fraction for 24 h and
linked immune sorbent assay (ELISA) plate reader simultaneously a control was maintained. After 24 h,
(LISA Plus). Percent inhibition was calculated using cells were collected by trypsinisation and treated with
the formula, Inhibition (%)=(1–ODs/OD)×100, where, 50 µl of lysis buffer. The lysate was frozen and thawed
ODs was the optical density of the sample and OD was 3 times for the even suspension of the cells in the buffer.
the optical density of the control. The suspension was centrifuged and the supernatant
was collected. Fifty microliters of caspase assay buffer
Bioassay guided fractionation by thin layer
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containing 1 M DTT (dithiothreitol) was added to the plant. Several medicinal properties were reported for T.
lysate. A total of 5 µl of AFC conjugated substrate (50 indica. It has broad spectrum of antibacterial activity.
µM final concentration) was added and incubated at The methanol extract of the leaf of this plant was
37° for 2 h. Absorbance was recorded every 15 min found to possess strong antibacterial activity[23]. The
at 405 nm in an ELISA plate reader. Percent caspase seed and pericarp of tamarind have been reported to
activity was calculated using the following formula, contain phenolic antioxidant compounds. Extracts
OD control/sample-OD blank/OD blank×100 from all parts of T. indica exhibit good antioxidant
activities[24]. Tamarind is often used for the treatment
Annexin V apoptosis assay: of cuts, wounds and abscesses. The bark or leaves are
Annexin V coupled with propidium iodide (PI) staining most commonly used, is applied externally on the spot,
is the most common approach used for the detection of either as a decoction or as a powder or poultice, alone
apoptosis in a flow cytometer[21]. The cell population or in combination with other species[25]. T. indica bark
undergoing apoptosis, necrosis and also the population has been traditionally used for the treatment of pain.
of viable cells can be analysed from this assay. HeLa Petroleum ether extract of the bark had significant
cells at a concentration of 1×106 cells/ml were seeded analgesic activity in mice models. The presence of sterols
in a culture flask and after 24 h treated with tamarind and triterpenes were reported in the bark extract[26]. But
bark violet fraction and incubated further for 24 h. The to the best of our knowledge, anticancer activity of T.
cells later were harvested by trypsinisation, stained indica bark has not been reported in literature. Because
with Annexin V and PI and analysed with the help of a of the causal relationship between inflammation and
flow cytometer. cancer and the reported antiinflammatory activity of
tamarind bark, in the current study we tried to analyse
Gas chromatography-mass spectrometry (GC-MS) the anticancer activity of tamarind bark extract.
analysis:
When HeLa cells were treated with 5 µg/ml of the
The bioactive fraction of tamarind bark was subjected dichloromethane extract of tamarind bark, percent cell
to GC-MS analysis. For GC-MS analysis, Thermo viability decreased to 65.4% and as the concentration
GC-Trace Ultra version 5.0, equipped with Thermo of the extract increased to 75 µg/ml and the % viability
MS DSQ II mass spectrometer were used. DB 5-MS decreased further to 45.2% (fig. 1a). As the incubation
Capillary Standard non polar column was used with of treatment was increased to 72 h, a drastic decrease
dimensions 30×0.25 mm and film thickness 0.25 μm. in the percent viability of the cells was observed with a
Helium gas was used as a carrier gas with a flow rate mere 26.3% at 75 µg/ml treatment. A similar effect was
of 1 ml/min. The oven temperature initially was 70° observed even in the case of PA-1 cells.
which was gradually increased to 260° at the rate of 6°/
As the extract demonstrated promising results, we
min. 1 μl of the sample was injected to the column and
attempted to fractionate the components in the tamarind
the mass spectra were obtained within the mass range
bark by TLC. The solvent combination of toluene:ethyl
of 50-650 atomic mass unit (AMU). The mass spectra acetate (9:1) as the mobile phase, we could get 7
thus obtained were compared with the known mass distinct fractions (results not shown). Among these
spectra from NIST library and the molecular weight the 4th fraction was cytotoxic to HeLa and PA-1 cells.
and structure of the compounds were determined[22]. When HeLa and PA-1 cells were treated with this
Statistical analysis: bioactive fraction for 24, 48 and 72 h, there was a
decrease in the percentage viability of the cells in a
All experiments were carried out in triplicates. The dose and time dependent manner (fig. 1b). From the
results were calculated as mean±standard error (SE) dose response curve, the IC50 value of this bioactive
values. Statistical significance was calculated using fraction was calculated as 7.5 µg/ml on HeLa and 0.1
one way analysis of variance (ANOVA) and Dunnett’s µg/ml on PA-1 cells. As this fraction had exhibited
multiple comparison tests. The values P<0.05 were antiproliferative properties on both the cancer cell
taken as significant. lines, we wanted to analyze its mechanism of action
by observing the morphology of the cells through
RESULTS AND DISCUSSION
fluorescence microscopy. Here, we could notice nuclear
The current study demonstrated the anticancer effects disintegration of the treated cells as compared to the
of tamarind bark, an important nutritive and medicinal control cells. Viable cells with intact membrane were
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Fig. 1: Effect of tamaind bark extract and bioactive fraction on HeLa and PA-1 cells.
a. Effect of tamarind bark dichloromethane extract on HeLa and PA-1 cells. b. effect of tamarind bark bioactive fraction on HeLa
and PA-1 cell lines at incubation times of ■ 24; ■ 48; ■ 72 h.

Fig. 2: Fluorescence microscopic pictures of the cells.

a. Control HeLa cells, b. HeLa cells treated with the bioactive fraction, c. PA-1 cells treated with the bioactive fraction.

fluorescing bright green and treated cells were bright

orange in colour (fig. 2). The results clearly suggested
that the bioactive fraction had brought about apoptotic
changes in the cells.
DNA fragmentation is one of the characteristics
of apoptotic cells. The apoptogenic activity of the
bioactive fraction was further supported from the
DNA patterns after agarose gel electrophoresis. When
observed under the UV transilluminator, DNA from
the treated cells appeared as a smear, whereas the DNA
from the control cells was an intact band (fig. 3).
Caspase-9 is an initiator caspase that activates all the
procaspases thereby bringing about apoptosis in the
cells. An increase in caspase-9 activity in cancer cells
can be regarded as an indication of cells undergoing Fig. 3: DNA fragmentation analysis of HeLa and PA-1 cells
treated with the bioactive fraction.
apoptosis. When we analysed the activity of caspase-9
Lanes: 1. DNA from control HeLa cells, 2. DNA from treated
in the cells treated with the bioactive fraction, it was HeLa cells, 3. ladder DNA, 4. DNA from treated PA-1 cells, 5.
observed that the caspase activity in HeLa cells the DNA from control PA-1 cells.
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activity was 51.2% and in PA-1 cells it was 75% (fig. 60% in the bioactive fraction treated flasks from 3% in
4). The caspase-9 activity increased to 77.3% in HeLa the control flasks (fig. 5). Our cell cycle analysis results
cells and 104% in PA-1 cells after 45 min. When provided a strong evidence for the apoptotic induction
the time of incubation was increased to 90 min, the of cell death by the bioactive fraction.
caspase-9 activity increased to 89.8% in HeLa cells and
As the results of all these assays provided proof for
137.5% in PA-1 cells. As compared to the control cells,
the anticancer potential of the bioactive fraction from
the increase in caspase-9 activity was 83.7% in HeLa tamarind bark, we further proceeded to characterize
and 125.7% in PA- 1 cells. This gave further evidence
for the induction of apoptosis in the treated HeLa and
PA-1 cells by this fraction.
Irresponsive to cell cycle signals is one of the
mechanisms employed by cancer cells while evading
apoptosis and resulting in rapid cell proliferation.
Therefore, quantitative analysis of cell cycle has become
a major criterion for the determination of cell death.
To determine the effect of the bioactive fraction on the
different stages of cell cycle, we subjected the treated
cancer cells to cell cycle analysis by flow cytometry
after Annexin V/PI staining. We found that live cells
Fig. 4: Caspase-9 activity in HeLa and PA-1 cells treated with
were 17% in treated flasks as compared to the control the bioactive fraction.
flask where it was 95.9%. Apoptotic cells increased to ■ 0 min; ■ 45; ■ 90 min

Fig. 5: Annexin V/PI based flow cytometry analysis of HeLa cells.

a and b: Control cells, c and d: HeLa cells treated with the bioactive fraction.
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Fig. 6: GC-MS analysis of tamarind bark violet fraction.

a. Chromatogram of tamarind bark violet fraction, b. mass spectrum of tamarind bark bioactive fraction. Arrow indicates a
fragment ion of molecular weight of 196.1 corresponding to cantharidin.

the compound responsible for this bioactivity through might be responsible for the observed anticancer
GC-MS analysis. The GC-MS analysis of the bioactive activity of tamarind bark bioactive fraction. To the
fraction indicated the presence of a major peak at best of our knowledge, this is the first report of
retention time 10.75 min (fig. 6a). The mass spectral cantharidin from tamarind bark, which possibly could
analysis coupled with library search for this peak be the contributing compound towards the anticancer
has resulted in a major fragment with 100% relative activity observed from this plant source. We conclude
abundance having a mass to charge ratio (m/z) of 83 by saying that cantharidin might be responsible for the
as the base peak (fig. 6b) and another fragment ion antiproliferative and apoptogenic activity of tamarind
with a mass to charge ratio of 196.1. This value was bark and this common plant holds promise towards
corresponding to the molecular weight of cantharidin future cancer treatment strategies.
as per the library search results. Cantharidin was earlier Acknowledgements:
reported for its anticancer activity from the blister
beetle M. phalerat[27]. The authors are grateful to the management, Jain Group
of Institutions for the infrastructural facilities provided.
Hence, we assume that the presence of this compound

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