Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 568148, 16 pages
Thujone-RichFraction of Thujaoccidentalis
Demonstrates Major Anti-Cancer Potentials:
EvidencesfromInVitro StudiesonA375 Cells
Raktim Biswas,1Sushil Kumar Mandal,1Suman Dutta,1Soumya Sundar Bhattacharyya,1
Naoual Boujedaini,2andAnisurRahman Khuda-Bukhsh1
1Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India
2Boiron Laboratory, 20 rue de la Lib´ eration., Sainte-Foy-Les-Lyon (69110), France
Correspondence should be addressed to Anisur Rahman Khuda-Bukhsh, prof email@example.com
Received 17 November 2009; Accepted 9 April 2010
Copyright © 2011 Raktim Biswas et al.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Crude ethanolic extract of Thuja occidentalis (Fam: Cupressaceae) is used as homeopathic mother tincture (TOΦ) to treat various
ailments, particularly moles and tumors, and also used in various other systems of traditional medicine. Anti-proliferative and
apoptosis-inducing properties of TOΦ and the thujone-rich fraction (TRF) separated from it have been evaluated for their
possible anti-cancer potentials in the malignant melanoma cell line A375. On initial trial by S-diphenyltetrazolium bromide assay,
both TOΦ and TRF showed maximum cytotoxic effect on A375 cell line while the other three principal fractions separated by
chromatography had negligible or no such effect, because of which only TRF was further characterized and subjected to certain
C10H16O with a molecular weight of 152. Exposure of TRF of Thuja occidentalis to A375 cells in vitro showed more cytotoxic, anti-
proliferative and apoptotic effects as compared with TOΦ, but had minimal growth inhibitory responses when exposed to normal
cells (peripheral blood mononuclear cell). Furthermore, both TOΦ and TRF also caused a significant decrease in cell viability,
induced inter-nucleosomal DNA fragmentation, mitochondrial transmembrane potential collapse, increase in ROS generation,
and release of cytochrome c and caspase-3 activation, all of which are closely related to the induction of apoptosis in A375 cells.
Thus, TRF showed and matched all the anti-cancer responses of TOΦ and could be the main bio-active fraction. The use of TOΦ
in traditional medicines against tumors has, therefore, a scientific basis.
Thuja occidentalis (Fam: Cupressaceae), commonly known
as Arbor vitae or white cedar, is an ornamental tree
grown in Europe . It is used as a medicinal plant in
various forms of traditional medicines like folk medicine,
homeopathy, and so forth. for treatment of bronchial
catarrh, enuresis, cystitis, psoriasis, uterine carcinomas,
amenorrhea and rheumatism [2–5]. In homeopathy, the
crude ethanolic extract of T. occidentalis is used as mother
tincture (TOΦ). Thuja is also occasionally used for treating
diseases of skin, blood, gastrointestinal tract, kidney, brain,
warty excrescences, spongy tumors, and so forth. and
claimed to have pronounced remedial effects. The extract
has been reported to enhance the antibody response to
sheep blood cells . Protective effect of T. occidentalis
has also been reported against radiation-induced toxicity
in mice . Ameliorative effect of T. occidentalis has also
been suggested in preventing congestive heart disease .
Therefore, it gained attention of pharmacologists to study
the major constituents of the dried herbal substances of T.
occidentalis [9–11]. Some 31 compounds were identified in
T. occidentalis “globosa” and 27 in T. occidentalis “gracilis”
. Although some minor differences exist in the actual
ingredients of several varieties of Thuja, generally the major
comprise essential oil (1.4–4%) as the principal constituent.
Other constituents include coumarins (p-coumaric acid,
Umbelliferone) flavonoids (Catechine, Gallocatechine, etc.),
tannic acid, polysaccharides and proteins . The essential
oil of the fresh leaves (related to the monoterpene fraction)
contains 65% thujone, 8% isothujone and fenchone each,
2 Evidence-Based Complementary and Alternative Medicine
5% sabines and 2% a-pinen as the main monoterpenes.
Among the constituents of the dried herbal substances,
it is not known precisely as to which fraction(s) of the
extract is(are) the most bioactive agent(s), particularly in
respect of its (their) anti-tumor or anti-cancer activities. In
homeopathic literature , TOΦ has been reported to be
effective against various forms of skin diseases, particularly
in the treatment of moles and papillomas. Homeopathic
TOΦ has been reported earlier to have cytotoxic effect in
Dalton’s lymphoma ascites (DLA), Ehrlich ascites carcinoma
(EAC), and lung carcinoma L929 . Dubey and Batra also
reported hepato-protective activities  and antioxidant
activity  of T. occidentalis in CCL4-treated liver damage
in rats. However, to the best of our knowledge, anti-
cancer potentials of TOΦ or any of its major fractions had
not been tested earlier in skin cancer cell line A375 by
utilizing some widely acceptable parameters of study, like
S-diphenyltetrazolium bromide (MTT) assay, Trypan blue
exclusion assay and lactate dehydrogenase (LDH) activity-
based cytotoxicity assays, fluorescence microscopy, comet
assay, DNA fragmentation assay, analysis of changes in
morphological features of cells and in mitochondrial mem-
brane potential, ROS production, flow cytometric analysis,
immunofluorescence study and western blot analysis.
Thus, the hypotheses to be tested were:
(i) whether TOΦ and all chromatographically separated
fractions had potential anti-cancer effects in A375
(ii) if any fraction could be demarcated as the most
biologically active one;
(iii) if, it is possible, to chemically characterize this
(iv) whether the cytotoxic, anti-proliferative and apop-
totic effects of the fraction, vis-` a-vis TOΦ, could be
compared with focus on its possible mechanism of
(v) if it is possible to assess the overall anti-cancer
potentials of TOΦ in relation to its preferential
of different relevant assays including the possible
2.1. Sources of the Homeopathic Drugs. Thuja occidentalis
Φ was procured from Dr Willmer Schwabe India Pvt. Ltd
(a subsidiary of Schwabe International GmbH, Germany),
Noida, India. The drug was prepared by following the
procedure as laid down in the Pharmacopeia of India .
2.2. Chromatographic Separation of the TOΦ. The solvent
was removed and the residue was dissolved in a minimum
volume of ethanol and mixed with silica gel (60–120 mesh)
column pre-equilibrated in petroleum ether. The column
was then eluted sequentially with 100mL volume of each of
(i) petroleum ether–ethyl acetate (9:1, v/v), (ii) petroleum
ether–ethyl acetate (7:3, v/v); (iii) petroleum ether–ethyl
acetate (3:7, v/v), and (iv) ethanol (90%v/v) . A375 cells
were treated with fractions for preliminary screening.
2.3. Chemicals and Reagents. Dulbecco’s modified Eagle
medium (DMEM), fetal bovine serum (FBS), penicillin,
streptomycin, neomycin (PSN) antibiotic, trypsin and
ethylenediaminetetraaceticacid (EDTA) were purchased
from Gibco BRL (Grand Island, NY, USA). Tissue culture
plastic wares were obtained from BD Bioscience (USA).
All organic solvents used were of HPLC grade. Propidium
iodide (PI), acrydine orange (AO), 4?,6?-diamidino-2 phenyl
indole (DAPI), 3-(4,5-dimethyl-thiazol-2-yl)-2, MTT and all
other chemicals used were purchased from Sigma Chemical
Co. (St. Louis, MO, USA).
was collected from National Centre for Cell Science, Pune,
India. Cells were cultured in DMEM supplemented with
10% heated inactivated FBS and 1% antibiotic (PSN) and
maintained at 37◦C with 5% CO2in a humidified incubator.
Cells were harvested with 0.025% trypsin and 0.52mM
numbers and allowed to adhere for 24h before treatment.
2.5. MTT Assay. The human melanoma A375 cells were
of 1 × 106cells per well. After 24h of incubation, they were
treated with various concentrations of T. occidentalis Φ to
of cell death was nearly 50%.
A375 cells were further treated with various concen-
trations of thujone-rich fraction (TRF) followed by 24h
of incubation. The control received no drug. After the
incubation 10μL of MTT solution (5mgmL–1) was added to
each well. The intracellular formazan crystals formed were
solubilized with acidic isopropanol and the absorbance of
the solution was measured at 595nm  using an ELISA
reader (Multiscan EX, Thermo Electron Corporation, USA).
The relative percentage of viability was calculated as follows:
Relative percentage viability
?OD of drug-treated sample
OD of control sample
2.6. Trypan Blue Exclusion Assay (Cytotoxicity Assay). For
cytotoxicity assessment, trypan blue exclusion assay was
performed . After treatment with TRF (0–200μgmL–1)
for 24h, A375 cells were stained with 0.4% trypan blue and
100 cells were counted at various fields in haemocytometer
for each experiment. The level of cytotoxicity was calculated
as per the following formula:
Relative percentage of cytotoxicity
?Totalcells −Viable cells
Evidence-Based Complementary and Alternative Medicine3
2.7. LDH Activity-Based Cytotoxicity Assay. LDH activity
was assessed using a standardized kinetic determination kit
(Enzopak, Recon,India).LDHactivitywasmeasuredin both
floating dead cells and viable adherent cells. The floating
cells were collected from culture medium by centrifugation
(1000g) at 4◦C for 5min, and the LDH content from
the pellets was used as an index of apoptotic cell death
(LDHp) . The LDH released in the culture supernatant
[designated as extracellular LDH (LDHe)] was used as an
index of necrotic death, and the LDH present in the adherent
viable cells was designated as intracellular LDH (LDHi).
The percentages of apoptotic and necrotic cell deaths were
calculated as follows:
2.8. Observation of Morphological Changes. Cells plated in
six-well culture plates (2 × 105cells per well) in DMEM
supplemented with 10% FBS for 24h were treated with or
without TRF at a specified concentration. After 24h, the cells
were observed under inverted phase contrast microscope
(Axiscope plus 2, Zeiss, Germany) and photographs were
2.9. Fluorescence Microscopy. To determine the live apoptotic
cells, cells treated for 24h were stained separately with
10μgmL–1of DAPI and with acridine orange:ethidium
bromide (AO/EB) . After 24h, the control and treated
cells were stained with acridine orange (50μgmL–1) and
ethidium bromide (50μgmL–1) mixture. Then the cells were
analyzed under fluorescence microscope (Axiscope plus 2,
Zeiss, Germany) and representative photographs were taken
for further quantitative analysis.
2.10. Analysis of Changes in Mitochondrial Membrane Poten-
tial. The changes in mitochondrial membrane potential
of the treated cells were determined using a fluorescent
probe, Rhodamine 123 . Briefly, 5μL of Rhodamine 123
(1mmol L–1) were added to the cells (2 × 105). Then the
cells were incubated for 15min. Next, the cells were washed
with PBS and observed under a fluorescence microscope
(Axioscope plus 2, Zeiss) and photographs were taken.
The mitochondrial membrane potential changes were
also determined by flow cytometer (FACS caliber, BD
Bioscience) . Briefly, cells after treatment were washed
with ice-cold PBS before incubation with Rhodamine 123
(5mmolL–1) in darkness for 15min at room temperature.
The fluorescence emissions were analysed with a flow
2.11. Analysis of ROS Production. Cells after treatment were
suspended in growth medium and incubated with DCFDA
were washed with PBS and analyzed under fluorescence
microscope and photographs were taken.
To evaluate the intra-cellular ROS level, the cells were
incubated with 10 μM DCFDA for 30 min at room temper-
ature. After washing the cells twice with PBS, the intensity
of DCFDA fluorescence was determined by flow cytometer
with an excitation wavelength of 480nm and an emission
wavelength of 530nm.
2.12. Flow Cytometric Analysis. A375 cells were harvested
and washed once in cold phosphate-buffer saline (PBS). Cell
pellets were fixed in 3% paraformaldehyde and suspended in
1mL of PI solution containing 50mgmL–1PI, 0.1% (w/v)
sodium citrate and 0.1% (v/v) Triton X-100. Cells were
incubated at 4◦C in the dark for 15min and analyzed by
a flow cytometer .
from inner cell membrane to outer cell membrane (a char-
acteristic feature of cells undergoing apoptosis), cells were
subjected to flow cytometric analysis after staining with
Annexin V-FITC and PI .
and washed in ice-cold PBS. The cell suspension was mixed
with an equal amount of 1% low melting agarose kept at
37◦C. Immediately after mixing, 100μL of the suspension
was pipetted on to microscope slide pre-coated with normal
melting agarose, then covered with cover slip and placed
on a glass tray on ice. Then the slides were immersed
in cold lysis solution (2.5M Nacl, 100mM EDTA, 10mM
Tris, PH-10, with freshly added 1% Triton X-100 and 2%
DMSO) followed by incubation at 4◦C for at least 1h. The
electrophoresis in weak alkali (0.03M NaOH, 1mM EDTA,
PH-12) at 1Vcm–1and 30mA for 15min was preceded by
a 20min immersion of the slides in electrophoresis buffer
to promote chromatin unwinding. After electrophoresis the
slides were neutralized in 0.05M Tris buffer. Then DNA
was stained with ethidium bromide (50μgmL–1) for 10min,
washed in distilled water and examined in a fluorescence
microscope (Lyca, USA) .
2.14. DNA Fragmentation Assay. The cells were grown to
70% confluence and treated with various concentrations
of TRF for 24h. Following this treatment, the cells were
washed twice with phosphate-buffered saline [10mM Tris
(pH 7.5), 150mM NaCl, 5mM MgCl2 and 0.5% Triton
X-100], left on ice for 15min, and pelleted by centrifu-
gation (1000 rpm) at 4◦C. The pellet was incubated with
DNA lysis buffer [10mM Tris (pH 7.5), 400mM NaCl,
1mM EDTA and 1% Triton X-100] for 30min on ice
and then centrifuged at 15000g at 4◦C. The supernatant
that was obtained was incubated overnight with RNase
K (0.1mgmL–1) for 2h at 37◦C. DNA was extracted using a
ethanol. The DNA precipitate was centrifuged at 15000g
and 4◦C for 15min, and the pellet was air-dried and
dissolved in 20μL of Tris–EDTA buffer [10mM Tris–HCl
(pH 8.0) and 1mM EDTA]. The total amount of DNA was
resolved over a 1.5% agarose gel, containing 0.3mgmL–1
4 Evidence-Based Complementary and Alternative Medicine
Table 1: One-way ANOVA for effect of different fractions of TOΦ
on viability of A375 cells as measured by MTT assay.
Source of variation
Effect of different fractions of TOΦ on
viability of A375 cells as measured by
3 55 679.278
∗P < .05.
Cell viability (%)
Figure 1: Effect of different fractions of TOΦ on viability of A375
cells as measured by MTT assay. Fraction 4 appeared to show
maximum inhibitory effect against A375 cells while Fractions 1
and 2 had no effect and Fraction 3 had little effect on the viability
of A375 cells. Results are expressed as mean ± SEM of three
independent experiments and analyzed using one-way ANOVA. P-
value < .05 was considered statistically significant.
ethidium bromide in 1× TAE buffer. The bands were
visualized under an UV transilluminator followed by digital
2.15. Immunofluorescence Study. Cells were plated in six-
well culture plates and allowed to adhere for 24h before
treatment. At the end of the treatments, cells were washed
with PBS and fixed in 3% paraformaldehyde for 1h. The
cells were permeabilized with 0.2% CHAPS in PBS for 2min
and were blocked in 2% BSA with 0.2% Tween-20 for 30min
a 1:400 dilution of specific primary antibodies (anti-Bcl-2,
anti-Bax, anti-cytochrome c and anti-caspase-3) and further
incubated for 1h with a 1:2000 dilution of goat anti-mouse
IgG-FITCasa secondaryantibody. Immunofluorescencewas
2.16. Preparation of Cell Lysates. Lysates were prepared to
examine the expression of Bax, Bcl2, cytochrome c, caspase-
3. A375 cells were washed twice with PBS and then lysed in
ice-cold lysis buffer containing 50mM HEPES (pH 7.4), 1%
Triton-X 100, 1mM EDTA, 2mM sodium orthovanadate,
100mM Sodium fluoride, 10μgmL–1leupeptin, and 1mM
PMSF, 10μgmL–1aprotinin. After 60 min of incubation on
ice, the cells were swelled and then centrifuged at 12000g
10 25 50100 125150 200 250
Cell viability (%)
Cell viability (%)
Figure 2: (a) Effect of different concentrations TRF on viability of
A375 cells as measured by MTT assay. Histogram shows 50% of cell
on PBMC was measured in the mentioned dose of TRF that showed
minimal cytotoxic effects (∼14%) even at the highest dose. Results
are expressed as mean ± SEM of three independent experiments.
Data are means ± SD from three different measurements and were
analyzed using one-way ANOVA. P < .05 compared to the control
group (zero concentration). Con = control.
for 20min. The protein content of the supernatant was
determined by Bradford assay with bovine serum albumin
as standard and then was stored at −20◦C until analysis
2.17. Western Blot Analysis. Both adherent and floating cells
were collected, and then western blot analysis was carried
out. Equal amounts of lysate protein were run on 12.5%
SDS–PAGE and electrophoretically transferred to PVDF
membrane . After blocking, the blots were incubated
with specific primary antibodies (anti-Bcl-2, anti-Bax, anti-
cytochrome c and anti-caspase-3) overnight at 4◦C and
further incubated for 2h with a 1:2000 dilution of goat anti-
mouse IgG-ALP as a secondary antibody. Bound antibodies
were developed by BCIP-NBT and quantification of proteins
was done by densitometry using image analyzer (Gel Doc
System; Ultra Lum, USA). The same membranes were also
immunoblotted against β-actin (house keeping gene) for
Evidence-Based Complementary and Alternative Medicine5
Cell viability (%)
Cell death (%)
Cell death (%)
Figure 3: (a) Effect of T. occidentalis Φ and different concentrations of TRF on viability of A375 cells as measured by MTT assay. Treatment
of A375 cells with 100 and 200μgmL–1for 24h resulted in ∼42 and 59% cell deaths, respectively, where TOΦ had ∼32% cell death. (b)
Cytotoxic effect of TOΦ and different concentrations of TRF on A375 cells as determined by trypan blue exclusion method. An amount of
100 and 200μgmL–1concentrations of TRF showed more cytotoxic effect compared with that of TOΦ for 24h. (c) Cells treated with TOΦ
and various doses of TRF (50, 100, 200μgmL–1) for 24h were measured by LDH activity-based assay. The ratio of apoptotic cells increased
from 23.59% at 50μgmL–1to 68.83% at 200μgmL–1for TRF, but that of necrotic cells was still negligible in the presence of 200μgmL−1
TRF. Data are each the mean ± SD (bars) from three independent experiments. Data are means ± SD from three different measurements
and were analyzed using one-way ANOVA. P < .05 compared to the control group (zero concentration). Con = control.∗= P < .05,∗∗= P <
2.18. Statistical Analysis. Statistical analysis was performed
by the Student’s t-test for the significance of difference
between the data of control and drug-treated cells. Data were
obtained fromrepresentative experiments with triplicate and
were expressed as mean ± standard error (SE). Additionally,
< .05 was considered to be significant (Tables 1–4).
3.1. Chromatographically Separated Fractions. Four major
fractions were obtained of which only Fraction 4 appeared
to show strong inhibition of A375 cell viability, while the
other fractions had no (Fractions 1 and 2) or little (Fraction
3) effect. For this, only Fraction 4 which was eluted with
90% (v/v) ethanol was selected for further detailed study.
Differences in cell viability between the Fractions 4 and 1,
Fractions 2 or 3 were found to be statistically significant (P <
.05) (Figure 1).
3.2. Viability of A375 Cells. MTT assay was conducted on the
A375 cell treated with different concentrations of TOΦ. The
results revealed 50% of cell death at 226.18μgmL–1of TOΦ
(Figure 2(a)). The cell cytotoxicity on normal peripheral
blood mononuclear cell (PBMC) was also measured in the
mentioned dose of TRF that showed minimal cytotoxic
effects(∼14%)evenatthehighestdose(Figure 2(b)).Table 2
shows the results of the statistical analysis done by one way
MTT assay showed that TRF had significantly stronger
inhibitory effects (P < .05) on proliferation of A375 cells.
Treatment of A375 cells with 100 and 200μgmL–1for
24h resulted in ∼42 and 59% cell deaths, respectively
6 Evidence-Based Complementary and Alternative Medicine
(a) (b) (c)
Figure 4: Morphology of control cells (a) and TOΦ-treated cells (b) Panels (c), (d), and (e) denotes morphology of cells treated with
different concentrations of TRF (50, 100, and 200μgmL–1), respectively, for 24h observed under a phase contrast microscope showing (10×
magnification). Cell rounding, cytoplasmic blebbing and detachment along with cell shrinkage were observed in treated cells.
Figure 5: Cells were stained with DAPI ((a)= control, (b)= TOΦ, (c)= 50μgmL–1(d)= 100μgmL–1, (e)= 200μgmL–1) and viewed using
fluorescence microscope (10× magnification). Photographs demonstrate brightly stained apoptotic nuclei in TRF-treated cells. Note that
the number of apoptotic nuclei was increased with the increasing concentrations of TRF where no or negligible number of apoptotic nuclei
was observed in the control cells.
Evidence-Based Complementary and Alternative Medicine7
Number of fluorescent cells
Figure 6: Quantitative data on DAPI staining of A375 cells treated
with TOΦ and different concentrations of TRF represented by
histogram. A positive dose-response was noted as number of
fluorescent cells increases with the increase in concentration of TRF
(50, 100, and 200μgmL–1). Con = control.
Table 2: One-way ANOVA for effect of different concentrations
TRF on viability of A375 cells as measured by MTT assay and
Cytotoxic effect of
tions TPΦ on viability
of A375 cells as
measured by MTT
Cytotoxicity of TRF on
PBMC as determined
by trypan blue staining
Sig. dfF Sig.
∗P < .05.
8 35 856.993 0.000∗
Table 3: One-way ANOVA for effect of T. occidentalis Φ and
different concentrations of TRF on viability of A375 cells as
measured by MTT assay, cytotoxic effect of TOΦ and different
concentrations of TRF on A375 cells as determined by trypan blue
Cytotoxic effect of
TOΦ and different
concentrations of TRF
on viability of A375
cells as measured by
Cytotoxic effect of
TOΦ and different
concentrations of TRF
on A375 cells as
determined by trypan
blue exclusion method
∗P < .05.
3 81638.983 0.000∗
3 68772.131 0.000∗
(Figure 3(a)). The cell cytotoxicity in response to treatment
results demonstrate that one of the causes of A375 cell death
induced by TRF could be attributed to apoptosis. The results
were statistically significant at various levels (P < .05 through
Table 4: One-way ANOVA of LDH activity of TOΦ and various
doses of TRF (50, 100, 200μgmL–1) on A375 cells for 24h.
Source of variation
LDH activity-based assay of cells
treated with TOΦ and various doses
∗P < .05.
P < .01). The statistical data of ANOVA have been shown in
To further characterize TRF-induced A375 cell death,
the ratios of LDH release from viable cells, floating dead
cells, and the culture medium were compared (Figure 3(c)).
There was a significant (P < .05 to P < .01) increase in the
ratio of apoptotic cells from 23.59% at 50μgmL–1to 68.83%
at 200μgmL–1for TRF, but that of the necrotic cells was
still negligible in the presence of 200μg mL–1TRF. Table 4
summarizes the statistical findings of ANOVA performed to
identify if the differences were significant among the means
of different groups.
3.3. Morphological Changes. The morphological changes of
A375 cells treated with TRF were also observed. Results
revealed that morphological changes (specifically for chro-
matin condensation and cell shrinkage) were typical of
apoptosis. These changes were apparent at 24h, and became
common in ∼50% or more of the treated A375 cells after
A375 cells to shrink, round up and detach from the culture
dish. The effect was more pronounced in the cells treated
with the highest dose of 200μgmL–1(Figure 4).
3.4. Fluorescence Microscopy. The untreated A375 cells did
not take positive staining with DAPI (Figure 5) and showed
no cells with visible chromatin condensation. However, with
different concentrations of treatment, cells with chromatin
condensation appeared to increase in number along with the
increase in dose (Figure 6).
The cells stained with AO/EB in untreated culture
(Figure 7) showed intact chromatin without fragmentation;
while there were nearly 39% cells with fragmented and
condensed chromatin visible at 100μgmL–1TRF-treated
culture. In the 200μgmL–1TRF-treated culture; nearly 50%
cells were fragmented and condensed (Figure 8).
3.5. Mitochondrial Membrane Potential. As compared to the
control (Figure 9(a)), A375 cells showed greenish stain in
greater intensity. However, in the TOΦ and TRF-treated
cells (Figures 9(b)–9(e)) reddish stain was more prevalent,
indicating mitochondrial potential depolarization, and that
8 Evidence-Based Complementary and Alternative Medicine
(a) (b) (c)
Figure 7: Cells were stained with acridine orange and ethidium bromide ((a) = control, (b) = TOΦ, (c)–(e) = TRF; (c) = 50μg mL–1, (d) =
100μg mL–1, (e) = 200μgmL–1) and viewed using fluorescence microscope (10× magnification). Photographs demonstrate brightly stained
50 100 200
Number of fluorescent cells
Figure 8: Quantitative data on acridine orange and ethidium
bromide staining of A375 cells treated with TOΦ and different
concentrations of TRF represented by histogram. Number of
fluorescent cells increases with the increase in concentration of TRF
(50, 100, and 200μgmL–1). Con = control.
was more pronounced in the higher dose (Figure 9(e)).
Quantitative data obtained by flow cytometry (Figures 9(f)–
9(j), resp.) would also support the mitochondrial membrane
potential depolarization convincingly.
3.6. Reactive Oxygen Species Generation. In the TRF-
untreated control (Figure 10(a)), the A375 cells did not take
up much stain, showing less generation of reactive oxygen
species (ROS). TOΦ showed intense staining as compared to
the control (Figure 10(b)). In contrast, there were number of
cells that showed more intense staining with the increase in
dose (Figures 10(c)–10(e)) signifying more amount of ROS
The quantitative data generated by flow cytometry
(Figures 10(f)–10(j)) would corroborate the same findings.
3.7. Flowcytometric Analysis. To further confirm whether the
cause of A375 cell death induced by TRF was apoptosis,
flow cytometric analysis was performed. Incubation of
fixed and permeabilized cells with fluorochrome PI results
in quantitative PI binding with total cellular DNA, and
the fluorescence intensity of PI-labeled cells was propor-
tional to DNA contents (Figure 11). Apoptotic nuclei with
hypodiploid DNA correspond to the sub G0/G1 peak.
The maximal increase in the frequency of apoptotic cells
was observed upon 24h treatment with 200μgmL–1TRF
(Figure 11(d)). These results demonstrate that the treatment
with TRF at 200μgmL–1induced A375 cell apoptosis
The percentages of apoptosis in TRF (200μgmL−1)
treated cells were found to be 34.69% as compared with
a very low percentage of apoptotic cells (8.23%) in the
untreated cell (Figure 12; Table 5).
3.8. Comet Assay. In comet assay, the nuclei were intact
and round, without any fragmented DNA (Figure 13(a)) in
the control. In the treated cultures (Figures 13(b)–13(e)),
there was a linear increase in comet lengths (Figure 14) with
the increase in concentrations from 50, 100 to 200μgmL–1,
Evidence-Based Complementary and Alternative Medicine9
Figure 9: A375 cells ((a) = control) showed greenish stain with higher mitochondrial membrane potential. In the cells treated with TOΦ
(b) and different concentrations of TRF cells ((c)–(e)) reddish stain depicts lower mitochondrial membrane potential. ((f)–(j)) shows the
histogram of mitochondrial membrane potential depolarization measured by flow cytometry.
10Evidence-Based Complementary and Alternative Medicine
(a) (b) (c)
Figure 10: Induction of ROS produced in A375 cells (a)= control, (b)= TOΦ, (c)= 50μgmL–1, (d)= 100μgmL–1, (e)= 200μgmL–1) treated
with TOΦ and different concentrations of TRF for 24h. The generation of ROS was monitored by fluorescence microscopy. Lower panel
((f)–(j)) shows the histogram of intracellular ROS measured by flow cytometry.
Evidence-Based Complementary and Alternative Medicine 11
Figure 11: Flow cytometric analysis of the cell cycle distribution of A375 cells with or without TRF for 24h. Histogram shows TRF-induced
A375 cell death by apoptosis. Number of apoptotic cells increased with the increase of dose. ((a)= Control, (b)= TOΦ, (c)= 100μg/mL TRF,
(d)= 200μg/mL TRF).
showing signs of more DNA damage with extended comet
also tested by agarose gel electrophoresis. Figure 15 indicates
a significant increase in inter-nucleosomal DNA fragmenta-
tion of A375 cells. When the DNA isolated from TRF-treated
cells was subjected to agarose gel electrophoresis, a DNA
ladder characteristic of apoptotic DNA was observed in the
cells treated with different concentrations.
3.10. Immunofluorescence Study. With the increase in dose
of TRF treatment, there was an increase in fluorescence
intensity of Bax, cytochrome c and caspase-3 and decrease
in fluorescence of Bcl-2 (Figure 16) consistent with the
immunoblot data (Figure 17).
12Evidence-Based Complementary and Alternative Medicine
Figure 12: Flow cytometric analysis of TOΦ and TRF on A375 cells after staining with Annexin V-FITC and PI. Figure (d) represents more
apoptotic cells at the lower right quadrant (early) at 200μg/mL dose. ((a)= Control, (b)= TOΦ, (c)= 100μg/mL TRF, (d) = 200μg/mL TRF).
Table 5: Quantitative evaluation of apoptosis through Annexin V-
FITC staining method.
LL = lower left panel, LR = lower right panel.
3.11. Immunoblot Analysis. Along with the increase in dose
of TRF, the expression levels of Bax, cytochrome c in
cytosolic fraction and caspase-3 in the total cell lysate were
increased while the expression of Bcl-2 was downregulated
Results of the present study would indicate that the homeo-
pathic drug T. occidentalis Φ had four chromatographically
separated fractions of which the thujone-rich fraction was
found to be the most bioactive (anti-cancer, pro-apoptotic)
component. Unfortunately, despite our best effort, the oily
nature of this fraction did not allow us to purify it to 100%,
for which we were able to perform only mass spectrometry
of TRF. The mass spectrophotometric data obtained by us
Evidence-Based Complementary and Alternative Medicine 13
Figure 13: Photomicrographs show the control A375 cells (a) with round shaped nuclei and panels (b), (c), (d), and (e) show nuclei with
comet tails when treated with TOΦ and different concentrations of TRF, respectively.
Figure 14: Histogram of the comet tail of A375 cells. Con=control,
2nd column = TOΦ, 3rd = 50μg/mL, 4th = 100μg/mL, 5th =
supported the published data on the chemical structure of
the C10H16O with a molecular weight 152 as reported by
Anti-cancer activity of the alcoholic extract of T. occi-
dentalis had earlier been reported by Sunila and Kuttan
 from their in vivo studies in rats. In our present in
vitro study involving several protocols, we confirm the pro-
apoptotic and anti-cancer potential of T. occidentalis mother
tincture. Additionally, it was revealed that the thujone-
rich component was possibly the key bioactive compound
showing its promising anti-cancer potentials in the skin
cancer cell line A375, a fact which had not earlier been
50 100 200
Figure 15: DNA fragmentation assay of A375 cells treated with
different concentrations of TRF for 24h. Lane (1) media, lane (2)
control, lane (3) TOΦ, lane (4) 50μgmL–1, lane (5) 100μgmL–1;
lane (6) 200μgmL–1-treated TRF, respectively.
reported (Figure 18). Furthermore, the anti-cancer activity
was mediated through activation of pro-apoptotic signal-
ing via the activation of Bax, caspase-3 and cytochrome
14Evidence-Based Complementary and Alternative Medicine
Figure 16: Immunofluorescence staining pictures of A375 cells following ((a)–(c)) Bax activation, ((d)–(f)) Bcl-2 deactivation, ((g)–(i))
cytochrome c activation and ((j)–(l)) caspase-3 activation.
Evidence-Based Complementary and Alternative Medicine 15
Figure 17: Western blot analysis of Bax, Bcl-2, caspase-3,
cytochrome c and β-actin lane (1) Control; lane (2) TOΦ, lane (3)
50μgmL–1, lane (4) 100μgmL–1; lane (5) 200μgmL–1-treated TRF,
respectively. The expressions of Bax, caspase-3 and cytochrome c
were up-regulated with the increasing concentrations of TRF where
Bcl-2 was down regulated.
Cell cycle arrest
at sub G1
Figure 18: Simple schematic representation of effect of TRF and
TOΦ on A375 cell line.
We determined the optimum dose through a range
finding trial. We also studied the probable cytotoxic effects
of both TOΦ and the TRF on a relative basis. The result
revealed that the 220.18μgmL–1of TOΦ could produce
50% cell death in A375 while that dose was relatively non-
toxic to PBMC. Correspondingly, of the three doses of 50,
100, 200μgmL–1of TRF used, 200μgmL–1TRF showed the
maximum effect surpassing the effect of treatment with the
TOΦ as revealed from the result of the several protocols.
Thus, the TRF appeared to be more potent anti-cancer agent
when treated alone, a fact which may be of therapeutic
importance in drug design and development of anti-cancer
drug from natural sources.
In recent years, the use of complementary and alternative
medicine (CAM) and other traditional medicines (TM)
is increasingly becoming popular [31–37]. However, many
people are hesitant to use such drugs either because they
are not scientifically validated for their action or because the
mechanism of their action is not properly known. Therefore,
search should be on for finding out safe, affordable and
efficient natural plant products that are experimentally
proven to be effective and are relatively non-toxic in nature,
because most of the orthodox anti-cancer drugs used in
cancer therapy are toxic and have adverse side-effects. Thus,
studies pinpointing confirmed efficacy of a particular frac-
tion or compound among several found in crude extract are
important for therapeutic purposes. Although the efficacy
of TRF and TOΦ has presently been tested against an in
vitro cancer cell line, it is very likely that the result can be
extrapolated in animal or in human. However, to determine
this, more experiments should be carried out on in vivo
animal models, which will hopefully be taken up in the next
phase of our program using mice as a model.
Boiron Laboratory, 20 rue de la Lib´ eration, Sainte-Foy-Les-
Lyon (69110), France.
Sincere thanks are due to Dr Philippe Belon, Ex-Director,
Boiron Laboratory for his kind cooperation and encour-
agements. The authors express their sincere thanks to Dr
P.K. Das, Former Director, Central Vector Control Research
Station, for kindly going through the manuscript and for his
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