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Synergistic growth inhibitory effects of Phyllanthus emblica and Terminalia bellerica extracts with conventional cytotoxic agents: Doxorubicin and cisplatin against human hepatocellular carcinoma and lung cancer cells

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To examine the growth inhibitory effects of Phyllanthus emblica (P. emblica) and Terminalia bellerica (T. bellerica) extracts on human hepatocellular carcinoma (HepG2), and lung carcinoma (A549) cells and their synergistic effect with doxorubicin or cisplatin. HepG2 and A549 cells were treated with P. emblica and T. bellerica extracts either alone or in combination with doxorubicin or cisplatin and effects on cell growth were determined using the sulforhodamine B (SRB) assay. The isobologram and combination index (CI) method of Chou-Talalay were used to evaluate interactions between plant extracts and drugs. P. emblica and T. bellerica extracts demonstrated growth inhibitory activity, with a certain degree of selectivity against the two cancer cell lines tested. Synergistic effects (CI < 1) for P. emblica/doxorubicin or cisplatin at different dose levels were demonstrated in A549 and HepG2 cells. The T. bellerica/cisplatin or doxorubicin also showed synergistic effects in A549 and HepG2 cells. In some instances, the combinations resulted in antagonistic effects. The dose reduction level was different and specific to each combination and cell line. The growth inhibitory activity of doxorubicin or cisplatin, as a single agent, may be modified by combinations of P. emblica or T. bellerica extracts and be synergistically enhanced in some cases. Depending on the combination ratio, the doses for each drug for a given degree of effect in the combination may be reduced. The mechanisms involved in this interaction between chemotherapeutic drugs and plant extracts remain unclear and should be further evaluated.
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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2008 March 14; 14(10): 1491-1497
www.wjgnet.com
World Journal of Gastroenterology
ISSN 1007-9327
wjg@wjgnet.com © 2008 WJG. All rights reserved.
Synergistic growth inhibitory effects of
Phyllanthus
emblica
and
Terminalia bellerica
extracts with conventional
cytotoxic agents: Doxorubicin and cisplatin against human
hepatocellular carcinoma and lung cancer cells
Khosit Pinmai, Sriharut Chunlaratthanabhorn, Chatri Ngamkitidechakul, Noppamas Soonthornchareon,
Chariya Hahnvajanawong
www.wjgnet.com
LIVER CANCER
Khosit Pinmai, Sriharut Chunlaratthanabhorn,
Department of
Preclinical Science, Faculty of Medicine, Thammasat University,
Pathumthani 12120, Thailand
Chatri Ngamkitidechakul,
Department of Biochemistry, Faculty
of Medicine, Chiang Mai University, Muang, Chiang Mai 50200,
Thailand
Noppamas Soonthornchareon,
Department of Pharmacognosy,
Faculty of Pharmacy, Mahidol University, Bangkok 10400,
Thailand
Chariya Hahnvajanawong,
Department of Microbiology,
Faculty of Medicine, Khon Kaen University, Khon Kaen 40002,
Thailand
Author contributions:
Khosit P contributed to this work; Khosit
P, Chatri N, and Chariya H designed research; Chariya H provided
SRB technique; Khosit P performed research; Sriharut C, Chatri N
and Noppamas S contributed new reagents/analytic tools; Khosit P,
and Chariya H analyzed data; and Khosit P, and Chariya H wrote
the paper.
Supported by
research grants from Thammasat University,
Thailand
Correspondence to: Khosit Pinmai,
Department of Preclinical
Science, Faculty of Medicine, Thammasat University, Paholyotin
Rd., Amphur Klongluang, Pathumthani 12120,
Thailand. all_sants@hotmail.com
Telephone:
+66-292-69736
Fax:
+66-292-69711
Received:
August 29, 2007
Revised:
January 25, 2008
Abstract
AIM:
To examine the growth inhibitory effects of
Phyllanthus emblica
(
P. emblica
) and
Terminalia bellerica
(
T. bellerica
) extracts on human hepatocellular carcinoma
(HepG2), and lung carcinoma (A549) cells and their
synergistic effect with doxorubicin or cisplatin.
METHODS:
HepG2 and A549 cells were treated with
P. emblica
and
T. bellerica
extracts either alone or in
combination with doxorubicin or cisplatin and effects on
cell growth were determined using the sulforhodamine
B (SRB) assay. The isobologram and combination index
(CI) method of Chou-Talalay were used to evaluate
interactions between plant extracts and drugs.
RESU LT S :
P. em bl i c a
a nd
T. be l l e ri ca
e x t ra c ts
demonstrated growth inhibitory activity, with a certain
degree of selectivity against the two cancer cell lines
tested. Synergistic effects (CI < 1) for
P. emblica
/
doxorubicin or cisplatin at different dose levels were
demonstrated in A549 and HepG2 cells. The
T. bellerica
/
cisplatin or doxorubicin also showed synergistic effects
in A549 and HepG2 cells. In some instances, the
combinations resulted in antagonistic effects. The
dose reduction level was different and specic to each
combination and cell line.
CONCLUSION:
The growth inhibitory activity of
doxorubicin or cisplatin, as a single agent, may be
modified by combinations of
P. emblica
or
T. bellerica
extracts and be synergistically enhanced in some cases.
Depending on the combination ratio, the doses for each
drug for a given degree of effect in the combination may
be reduced. The mechanisms involved in this interaction
between chemotherapeutic drugs and plant extracts
remain unclear and should be further evaluated.
© 2008
WJG
. All rights reserved.
Key words:
Cisplatin; Doxorubicin; Liver cancer;
Phyllanthus emblica
; Synergistic effect;
Terminalia
bellerica
Peer reviewers:
Toru Ishikawa, MD, Department of Gastro-
enterology, Saiseikai Niigata Second Hospital, Teraji 280-7,
Niigata, Niigata 950-1104, Japan; Akihito Tsubota, Assistant
Professor, Institute of Clinical Medicine and Research, Jikei
University School of Medicine, 163-1 Kashiwa-shita, Kashiwa,
Chiba 277-8567, Japan; Luis Rodrigo, Professor, Gastroenterology
Service, Hospital Central de Asturias, c/ Celestino Villamil, s.n.,
Oviedo 33.006, Spain
Pinmai K, Chunlaratthanabhorn S, Ngamkitidechakul C,
Soonthornchareon N, Hahnvajanawong C. Synergistic growth
inhibitory effects of Phyllanthus emblica and Terminalia bellerica
extracts with conventional cytotoxic agents: Doxorubicin and
cisplatin against human hepatocellular carcinoma and lung cancer
cells. World J Gastroenterol 2008; 14(10): 1491-1497 Available
from: URL: http://www.wjgnet.com/1007-9327/14/1491.asp DOI:
http://dx.doi.org/10.3748/wjg.14.1491
INTRODUCTION
Cancer is the third leading cause of death worldwide,
preceded by cardiovascular and infectious diseases.
Doxorubicin and cisplatin represent the current standard
chemotherapeutic drugs for treatment of cancers.
Although there are many therapeutic strategies including
chemotherapy to treat cancer, high systemic toxicity
and drug resistance limit the successful outcomes in
most cases. Accordingly, several new strategies are being
developed to control and treat cancer. One such approach
could be a combination of an effective phytochemicals
with chemotherapeutic agents, which when combined,
would enhance efcacy while reducing toxicity to normal
tissues.
The notion has evolved that natural products frequent-
ly exert a valuable role in broadening the scope of disease
intervention strategies used by drug designers. Several
herbs and plants with diversied pharmacological proper-
ties are known to be rich sources of chemical constituents
that may have potential for the prevention and/or treat-
ment of several human cancers
[1-4]
.
P. emblica
L. (Euphorbiaceae) known as emblic my-
robalan, a shrub or tree, is widely used in folk medicine in
Southeast Asia. This plant exhibits a variety of pharma-
cological effects including anti-inammatory, anti-pyretic,
anti-oxidant, anti-carcinogenic, and anti-mutagenic ef-
fects
[5-7]
. The active principles or extracts of
P. emblica
have
demonstrated anti-proliferative effects in several cancer
cell lines both
in vitro
and
in vivo
[7,8]
. The anti-tumor activity
of
P. emblica
extract was attributed to its ability to interfere
with cell cycle regulation
via
the inhibition of cdc 25 phos-
phatase and partial inhibition of cdc 2 kinase activity
[8]
.
T. bellerica
(Combretaceae), known as belleric myroba-
lan, is a large deciduous tree, common to the plains and
lower hills of Southeast Asia. This plant exhibits several
pharmacological effects including anti-bacterial, anti-malar-
ial, anti-fungal, anti-HIV, anti-oxidant, and anti-mutagenic
effects
[9-12]
.
T. bellerica
extract exhibited anti-proliferative
effects in several cancer cell lines including Shiongi 115,
breast cancer MCF-7, prostate cancer PC-3 and DU-145
cells
[13]
. Phytochemical studies have shown that
T. bellerica
contains a variety of chemical components, including gallic
acid
[14,15]
. Furthermore, recent studies have demonstrated
the cytotoxic activity of gallic acid in human leukemia
HK-63, HOS-1 cell, HSC-2, and HL-60 cell lines
[16-19]
. Gal-
lic acid has also been shown to induce apoptotic cell death
in HSC-2 and HL-60 cells
[16,18]
.
These ndings prompted us to test the anti-prolifera-
tive activity of
P. emblica
and
T. bellerica
extracts on human
lung carcinoma A549, and human hepatocellular carcino-
ma HepG2 cell lines, and to investigate whether it can syn-
ergize the inhibitory action of doxorubicin and cisplatin,
the standard chemotherapeutic drugs.
MATERIALS AND METHODS
Plant extracts and chemotherapeutic drugs
The dried fruits of
T. bellerica
and
P. emblica
were collected
from Srakeaw and Nan Provinces in northeastern and
northern Thailand, respectively. The voucher specimens
were identified and kept by Associate Professor Dr.
Noppamas Soonthornchareon, Department of Pharma-
cognosy, Faculty of Pharmacy, Mahidol University.
The dried fruits of medicinal plant were obtained and
extracted with boiling water. Then, the aqueous solution
was separated from the plant residues by ltration. After
that, the aqueous solution was spray-dried. For testing, the
extracts were dissolved in media and diluted to the desired
concentrations.
Doxorubicin and Cisplatin (Platinol) were purchased
from Ebewe (Austria) and Bristol-Myers Squibb (USA),
respectively. The drugs were dissolved in water for
injection at a concentration of 2 mg/mL. Both drugs were
kept in aliquots at 4
. Serial dilutions of these drugs
were performed in culture media, immediately before
each experiment, in order to obtain the required final
concentrations.
Cell culture
The human lung carcinoma (A549) cell was kindly provided
by the Chulabhorn Research Institute. The human
hepatocellular carcinoma (HepG2) cell (ATCC HB-8065)
was obtained from the National Cancer Institute, Bangkok,
Thailand. The HepG2, and A549 cells were cultured in
RPMI 1640 medium (Hyclone, Logan, UT, USA). Both
cell lines were supplemented with 10% heat inactivated
fetal bovine serum (Hyclone), 100 U/mL penicillin and
100
µ
g/mL streptomycin (Gibco BRL, Grand Island, NY).
All cell lines were maintained at 37
in a 5% CO
2
incubator
and the media were changed twice weekly.
In vitro cytotoxicity assay
The sulforhodamine B (SRB) assay was performed to
assess growth inhibition using a colorimetric assay, which
estimates cell number indirectly by staining total cellular
protein with the dye SRB
[20]
.
Briefly, 100
µ
L/well of cell suspensions (0.5-2.0 ×
10
5
cells/mL)
were seeded in 96-well microtiter plates
and incubated at 37
to allow for cell attachment. After
24 h, the cells were treated with the extract by adding
100
µ
L/well of each concentration in triplicate to obtain a
nal concentration of 0.8, 4, 12.5, 25, 50, 100, 200, 400, and
1000
µ
g/well for the extracts; 0.029, 0.058, 0.116, 0.174, and
0.348
µ
g/mL for the doxorubicin; and, 0.1, 0.2, 0.5, 1.0, 2.5,
and 5.0
µ
g/mL for the cisplatin.
The plates were incubated for 1 h (d 0) and 72 h (d 3)
at 37
. At the end of each exposure time, the medium
was removed. The cells were fixed with 20% (w/v)
trichloroacetic acid (TCA, Fluka, Buchs, Switzerland) at 4
for 1 h, stained for 30 min with 0.4% (w/v) SRB (Sigma,
St. Louis, MO, USA) dissolved in 1% acetic acid (Sigma)
for 30 min, and washed four times with 1% acetic acid.
The protein-bound dye was solubilized with 10 mmol/L
Tris base, pH 10 (Sigma).
The absorbance (OD) of each well was read on an
ELISA plate reader (Amersham, Buckinghamshire, UK) at
492 nm. Percentage of cell survival was calculated using the
formula: Percentage cell survival = [(OD test sample at d 3 -
OD d 0)/(OD control at d 3 - OD d 0)] × 100.
Dose-response curves were plotted, and 50% growth
inhibitory concentrations of extracts or drugs (IC
50
)
were calculated through computation with the CalcuSyn
software program (Biosoft, Cambridge, UK). By
comparing the growth inhibitory effect with normal cells,
1492 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol March 14, 2008 Volume 14 Number 10
www.wjgnet.com
proliferation of two cancer cell lines and Vero cells were
determined using the SRB assay. All cell lines were growth-
inhibited in a dose-dependent manner after exposure to
the plant extracts (Figure 1). The IC
50
values for
P. emblica
extract ranged from 12.18
±
5.83 to 157.86
±
14.90
µ
g/mL
and for
T. bellerica
extract ranged from 27.07
±
2.19 to
238.70
±
8.45
µ
g/mL (Table 1).
When the activities of
P. emblica
and
T. bellerica
extracts
against the cancer cell lines were compared with that against
Vero cells and expressed as the ratio of IC
50
values, the
P. emblica
extract had significantly different ratios of 13.0
and 5.2 against the A549 and HepG2 cells (
P
< 0.01),
respectively. The
T. bellerica
extract also showed signicantly
different ratios of 8.8 and 2.9 against the A549 and HepG2
cells (
P
< 0.01), respectively (Table 1). It appears these two
plant extracts were selectively toxic against the two cancer
cell lines tested, motivating further work to determine
the selectivity of the extract was expressed as the extract
shown to be toxic to specic types of cancer cell lines.
Evaluation of drug interaction
Combination assays were performed using appropriate
concentrations of
P. emblica
and
T. bellerica
extracts (4,
8, 16, 24, and 48
µ
g/mL for A549 cells, and 25, 50, 75,
100, and 200
µ
g/mL for HepG2 cells) with appropriate
concentrations of doxorubicin (0.029, 0.058, 0.116, 0.174,
and 0.348
µ
g/mL) or cisplatin (0.1, 0.2, 0.3, 0.4, and 0.8
µ
g/mL). Cells treated with the same nal concentrations
of the extracts or chemotherapeutic drugs alone were also
examined. Cell growth inhibition was determined using
the SRB assay, as previously described. In the assessment
of synergism, the combination index (CI) method (i.e.,
additivity or antagonism) according to Chou and Talalay
was used
[21]
.
The CIs were calculated by the Chou-Talalay equation
[21]
,
which takes into account both the potency (
D
m
or IC
50
) and
shape of the dose-effect curve. The general equation for the
classic isobologram (CI = 1) is given by:
CI =
(
D
)
1
/(
D
x
)
1
+
(
D
)
2
/(
D
x
)
2
(A) where (
D
x
)
1
and (
D
x
)
2
in the denominators are
the doses (or concentrations) of
D
1
(drug #1, for example,
the extract) and
D
2
(drug #2, for example, the doxorubicin)
alone that gives
x
% inhibition, whereas (
D
)
1
and (
D
)
2
in
the numerators are the doses of D
1
and D
2
in combination
that also inhibits
x
% (i.e.
,
isoeffective). The (
D
x
)
1
and (
D
x
)
2
can be readily calculated from the Median-effect equation
of Chou
et al
[22]
:
D
x
=
D
m
[
f
a
/(1 -
f
a
)]
1/m
(B) where
D
x
is the
median-effect dose obtained from the anti-log of the X-in-
tercept of the median-effect plot, X-log (
D
)
versus
, Y
=
log
[
f
a
/(1 -
f
a
)], or
D
m
= 10-
(Y-intercept)/m
,
f
a
is the fraction affected
by dose
D
(e.g.
,
0.5 if cell growth is inhibited by 50%) and
m
is the slope of the median-effect plot. From (
D
m
)
1
, and (
D
x
)
2
and
D
1
+
D
2
, an isobologram can be constructed based on
Eq. A: as CI < 1 indicates synergism; CI = 1 indicates an
additive effect; and CI > 1 indicates antagonism.
For conservative, mutually nonexclusive isobolograms
of two agents, a third term, (
D
)
1
(
D
)
2
/(
D
x)
1
(
D
x)
2
, is added
to Eq. A. For simplicity, the third term is usually omitted,
and thus the mutually exclusive assumption or classic
isobologram is indicated. In this study, the CI values
obtained from classic (mutually exclusive) calculations are
given.
The dose-reduction index (DRI) denes the extent (folds)
of dose reduction possible in a combination, for a given
degree of effect, compared with the dose of each drug
alone: (DRI)
1
= (D
x
)
1
/(D)
1
and (DRI)
2
= (D
x
)
2
/(D)
2
. The
relationship between DRI and CI is, therefore, expressed as:
CI = (D
x
)
1
/(D)
1
+ (D
x
)
2
/(D)
2
= 1/(DRI)
1
+ 1/(DRI)
2
Statistical analysis
The data were analyzed using the SPSS software version
11.0. The IC
50
of plants against cancer cells were compared
with IC
50
of normal cells and calculated by the Student’s
t
-test. Differences were considered signicant at
P
< 0.01.
RESULTS
Effect of herbal extracts on cell growth
The effects of
P. emblica
and
T. bellerica
extracts on the
Concentration (
µ
g/mL)
0 20 40 60 80 100 120
Cell growth (%)
120
100
80
60
40
20
0
-20
-40
Cell growth (%)
Concentration (
µ
g/mL)
0 200 400 600 800 1000 1200
120
100
80
60
40
20
0
-20
-40
A
B
Figure 1 Cytotoxic effects of different concentrations of P. emblica (A) and
T. bellerica (B) extracts on cancer cell lines. The percentage of cell growth was
measured using the SRB staining assay. Each value represents the mean ± SE of
three independent experiments.
Table 1 IC
50
values of individual herbal extracts in human
cancer cells and normal cells
Extract IC
50
value (
µ
g/mL)
A549
b
HepG2
b
Vero (Normal)
P. emblica 12.18 ± 5.83 30.47 ± 6.67 157.86 ± 14.90
T. bellerica 27.07 ± 2.19 82.39 ± 27.09 238.70 ± 8.45
Data are shown as mean ± SD from three separate experiments.
b
P < 0.01
represents the statistical significance between normal cell (Vero) and each
cancer cell (A549 and HepG2).
Pinmai K
et al
. Synergistic effects of myrobalan with cytotoxic drugs 1493
www.wjgnet.com
HepG2
A549
HepG2
A549
the combination effect of these plant extracts with
chemotherapeutic drugs.
Effect of chemotherapeutic drugs on cell growth
Using the SRB assay, the effects of doxorubicin and cis-
platin on the proliferation of two cancer cell lines were
determined. All cell lines were growth-inhibited in a dose-
dependent manner (Figure 2). The IC
50
values for doxo-
rubicin
vs
cisplatin ranged from 0.170 ± 0.006 to 0.511 ±
0.025
µ
g/mL
vs
1.04 ± 0.21 to 1.05 ± 0.18
µ
g/mL, respec-
tively (Table 2).
Combination effects of herbal extracts with
chemotherapeutic drugs on cell growth
The combination effects of
P. emblica
and
T. bellerica
ex-
tracts with doxorubicin and cisplatin in the A549 and
HepG2 cell lines, as represented by the DRI, the CI and
the dose-effect levels of cell growth inhibition (IC
50
-IC
90
),
are summarized in Table 3.
The data were also examined using median effect analy-
sis to determine the type of interactions which occurred, i.e.
antagonism (CI > 1), additivity (CI = 1) or synergism (CI < 1)
(Figures 3 and 4). With A549 cells, the combinations were
synergistic at medium and high dose levels (IC
75
and IC
90
)
for the
P. emblica
/doxorubicin and
P. emblica
/cisplatin com-
binations (Table 3, Figures 3 and 4). By contrast, the syner-
gistic and additive effects were recorded at low and medium
dose levels (IC
50
and IC
75
) for the
T. bellerica
/cisplatin com-
bination
(Table 3, Figure 4). In HepG2 cells, combinations
were synergistic at low and medium dose levels (IC
50
and
IC
75
) for the
P. emblica
/doxorubicin combination or at a low
level (IC
50
) for the
T. bellerica
/doxorubicin combination (Table
3, Figure 3). All other combinations had antagonistic effects.
The DRI showed a considerable dose reduction for
herbal extracts and drugs used as a result of their synergism
(Table 3). When using synergistic drug combinations at
corresponding dose levels, the DRI indicated that the con-
centration of doxorubicin necessary to inhibit the growth
of 50% of cancer cells (IC
50
) could be decreased 1.64-fold
(A549,
P. emblica
/doxorubicin) to 4.69-fold (HepG2,
P. em-
blica
/doxorubicin), and the IC
90
could be reduced 2.59-fold
(A549,
P. emblica
/cisplatin) to 2.60-fold (A549,
P. emblica
/
doxorubicin; Table 3). The dose reduction level was differ-
ent and specic to each combination and cell line.
DISCUSSION
In this study, the growth inhibitory activity of the
P. emblica
and
T. bellerica
extracts and the chemotherapeutic drugs
doxorubicin and cisplatin were investigated in A549, and
HepG2 cells. Our results indicate that both plant extracts
and chemotherapeutic drugs mediated signicant growth
inhibitory effects on both cell lines tested in a dose-depen-
dent manner (Tables 1 and 2, Figure 1 and 2).
P. emblica
extract combined with doxorubicin or cispla-
tin resulted in synergistically enhanced growth inhibitory
activity at different dose levels in A549 and HepG2 cell
lines. The synergistic effects were also demonstrated when
T. bellerica
extract was combined with doxorubicin or cis-
paltin in HepG2 and A549 cells. The signicance of this
nding lies in the fact that doxorubicin and cisplatin are
well-known cancer therapeutic agents, but cause high tox-
icity to normal tissues during cancer therapy
[23,24]
.
Several studies have shown that doxorubicin has harmful
effects on health (i.e., immunosuppression and secondary
cardiomyopathy) and can lead to the development of pri-
mary and secondary drug resistance in tumor cells thereby
limiting the clinical success of cancer chemotherapy
[25,26]
.
Recent reports show that combination (rather than single-
agent) chemotherapy is a superior modality and that natu-
rally occurring dietary supplements with known anti-cancer
activity could be used in combination chemotherapy to re-
duce the systemic toxicity of chemotherapeutic agents
[27,28]
.
Our study provides corroborative evidence as it showed
that
P. emblica
and
T. bellerica
extracts were selectively toxic
against two cancer cell lines and that, in combination with
doxorubicin and cisplatin, produced an increased growth
inhibitory effect in both A549 and HepG2 cells. Calcula-
120
100
80
60
40
20
0
-20
-40
Cell growth (%)
Concentration (
µ
g/mL)
0 1 2 3 4 5 6
Cell growth (%)
Concentration (
µ
g/mL)
0.0 0.5 1.0 1.5 2.0
100
80
60
40
20
0
A
B
Figure 2 Cytotoxic effects of different concentrations of doxorubicin (A) and
cisplatin (B) on cancer cell lines. The percentage of cell growth was measured
using SRB staining assay. Each value represents the mean ± SE of three
independent experiments.
Table 2 IC
50
values of chemotherapeutic drugs in human cancer
cells
Drug IC
50
value (
µ
g/mL ± SD)
A549 HepG2
Doxorubicin 0.170 ± 0.006 0.511 ± 0.025
Cisplatin 1.04 ± 0.21 1.05 ± 0.18
Data are shown as mean ± SD from three separate experiments.
1494 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol March 14, 2008 Volume 14 Number 10
www.wjgnet.com
HepG2
A549
HepG2
A549
tion of the DRI at the IC
50
demonstrated possible reduc-
tions in doxorubicin concentrations for the drug combina-
tions ranging from 1.64-fold (
P. emblica
+ doxorubicin in
A549) to 4.69-fold (
P. emblica
+ doxorubicin in HepG2).
Table 3 Dose-effect relationships of extracts and drug combinations in human cancer cell lines
Cell line
Single extracts, drugs
and combinations
Parameters CI value at DRI value at
D
m
(
µ
g/mL)
m r
IC
50
IC
75
IC
90
IC
50
IC
75
IC
90
A549 P. emblica 36.75 0.71 0.99 2.67 3.90 5.67
Doxorubicin 0.164 0.78 0.99 1.64 2.07 2.60
(D)
1
+ (D)
2
(4:0.029) 13.75 + 0.100 0.93 0.99 0.98 0.74 0.56
T. bellerica 17.78 1.57 0.86 1.26 0.85 0.57
Doxorubicin 0.169 0.87 0.99 1.66 1.95 2.30
(D)
1
+ (D)
2
(4:0.029) 14.11 + 0.102 1.00 1.00 1.40 1.69 2.18
P. emblica 18.75 0.75 0.95 1.65 2.63 4.19
Cisplatin 0.596 0.99 0.96 2.09 2.33 2.59
(D)
1
+ (D)
2
(40:1) 11.38 + 0.285 1.09 0.98 1.08 0.81 0.62
T. bellerica 30.36 0.95 0.97 1.76 1.87 1.99
Cisplatin 1.228 1.36 1.00 2.85 2.15 1.62
(D)
1
+ (D)
2
(40:1) 17.26 + 0.431 1.00 1.00 0.92 1.00 1.12
HepG2 P. emblica 157.82 2.65 0.98 1.47 1.26 1.07
Doxorubicin 0.584 1.04 0.96 4.69 7.58 12.23
(D)
1
+ (D)
2
(25:0.029) 107.23 + 0.124 1.92 0.94 0.89 0.93 1.02
T. bellerica 150.34 3.11 0.95 1.37 1.07 0.84
Doxorubicin 0.569 1.04 0.95 4.48 7.09 11.24
(D)
1
+ (D)
2
(25:0.029) 109.66 + 0.127 1.83 0.97 0.95 1.07 1.28
P. emblica 111.59 2.18 0.99 1.24 1.08 0.94
Cisplatin 0.839 0.91 0.95 2.32 4.08 7.19
(D)
1
+ (D)
2
(250:1) 90.395 + 0.362 1.72 0.96 1.24 1.17 1.20
T. bellerica 111.02 2.26 1.00 1.19 1.01 0.85
Cisplatin 0.881 1.13 0.98 2.36 3.26 4.49
(D)
1
+ (D)
2
(250:1) 93.30 + 0.373 1.68 0.99 1.26 1.30 1.40
Dose-effect relationships were calculated by the median-effect equation. D
m
median-effect dose (concentration in
µ
g/mL that inhibits cell growth by 50%), m
shape of the dose-effect curve (where m = 1, m > 1, and m < 1 indicate hyperbolic, sigmoidal, and negative sigmoidal curves, respectively), r linear correlation
coefcient of the median effect plot (indicates conformity of data). CI was calculated by Chou and Talalay’s CI equation. CI < 1, CI = 1, and CI > 1 indicate
synergism, additive effect, and antagonism, respectively. D
m
and m values for single drugs, extracts and their combinations were used in the equations Dx
= Dm [f
a
/(1) f
a
)]
1/m
and CI = (D)
1
/(Dx)
1
+ (D)
2
/(Dx)
2
. f
a
fraction affected by D (e.g. 0.9 if cell growth is inhibited by 90%), (D)
1
and (D)
2
combined doses of
extract 1 and drug 2 for x% inhibition, (Dx)
1
and (Dx)
2
doses of the single extract 1 and drug 2 for x% inhibition. DRI dose reduction index was measured by
comparing the doses required to reach a given degree of inhibition when using the drug as a single agent and in combination.
2.5
2.0
1.5
1.0
0.5
0.0
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
3.0
2.0
1.0
0.0
A549
Phyllanthus emblica Terminalia bellerica
Fractional effectFractional effect
Combination index
Combination index
0.0 0.2 0.4 0.6 0.8 1.00.0 0.2 0.4 0.6 0.8 1.0
1.5
1.0
0.5
0.0
Fractional effect
HepG2
Combination index
Combination index
1.5
1.0
0.5
0.0
Fractional effect
DC
BA
Figure 3 Combination index (CI) vs
fraction affected (f
a
) plots obtained
from the median-effect analysis
pro gr a m (C alc uSy n, Bi os o ft ,
Cambridge, UK). (A) and (B) A549;
(C) and (D) HepG2. Curves with
solid lines are computer simulated
f
a
-CI plots, based on the parameters
(m and D
m
values) for doxorubicin
and herbal extract combinations.
Circles are actual combination data
points. CI < 1, = 1 and > 1 indicates
synergism, additive effect and
antagonism, respectively.
Pinmai K
et al
. Synergistic effects of myrobalan with cytotoxic drugs 1495
www.wjgnet.com
This finding supports our hypothesis that combinations
of plant extracts and chemotherapeutic agents allow a re-
duction in the dosage of the latter (i.e.,
doxorubicin and
cisplatin), yet retaining the benets but minimizing the cy-
totoxic effects, thus enhancing therapeutic efcacy.
The mechanism of action is unclear and, possibly,
multiple compounds in the herbal extracts are involved
[29]
.
Plant derived polyphenols, including tannins and gallic acid,
were reported to be the main constituents in
P. emblica
and
T. bellerica
[13,30]
. Marienfeld
et al
[31]
reported that tannic acid
(TA) could inhibit the malignant cholangiocyte growth both
in vitro
and
in vivo
and also enhance sensitivity of Mz-ChA-1
cholangiocarcinoma cells to camptothecin cytotoxicity
which might involve an effect on xenobiotic metabolism.
In addition, TA also acts as an inhibitor of the glutathione
conjugate export pump. As a result, the sensitivity of tumor
cells to anticancer drugs was increased
[32,33]
. This study
agreed with the study of Sandhya and Mishra
[34]
which
showed the cytotoxic response of human breast cancer cell
lines to Triphala, which contained
T. bellerica
,
P. emblica
and
T. chebula
extracts. It might be possible that
T. bellerica
and
P. emblica
extracts could induce ROS in the induction of
apoptosis. Apart from the effect of tannic acid, the cytotoxic
effect and apoptosis induction of gallic acid in several cancer
cell lines have been reported
[13,34,35]
. It was demonstrated
that the effect of gallic acid on cancer cell line particularly
lung cancer cells involved caspase activation and oxidative
processes
[35]
. In addition, gallic acid, a major component of
T. bellerica
[14,15]
has the capacity to induce apoptosis
[35]
and
increase the efficacy of cisplatin in combined treatment
of mice transplanted with LL-2 lung cancer cells
[36]
. These
ndings suggest that in the synergistic activity of
P. emblica
or
T. bellerica
extracts and doxorubicin it seem to be possible
that the extracts could induce cytotoxic to tumor cells by a
ROS mediated mechanism in cancer cells but less in normal
cell
[37]
as well as inhibit glutathione conjugate export pump
as a result, the extracts would increase the sensitivity of
A549 and HepG2 cells to doxorubicin and cisplatin.
0.0 0.2 0.4 0.6 0.8 1.0
1.5
1.0
0.5
0.0
Terminalia bellerica
Fractional effect
Combination index
0.0 0.2 0.4 0.6 0.8 1.0
Combination index
2.0
1.5
1.0
0.5
0.0
Fractional effect
D
0.0 0.2 0.4 0.6 0.8 1.0
3.0
2.0
1.0
0.0
Fractional effect
HepG2
Combination index
C
B
3.0
2.0
1.0
0.0
0.0 0.2 0.4 0.6 0.8 1.0
A549
Phyllanthus emblica
Fractional effect
Combination index
A
Figure 4 Combination index (CI) vs
fraction affected (f
a
) plots obtained
from the median-effect analysis
program (CalcuSyn, Biosoft, Cambri-
dge, UK). (A) and (B) A549; (C)
and (D) HepG2. Curves with solid
lines are computer simulated f
a
-CI
plots, based on parameters (m and
D
m
values) for cisplatin and herbal
extract combinations. Circles are
actual combination data points. CI < 1,
= 1 and > 1 indicates synergism,
additive effect and antagonism,
respectively.
In summary, our results demonstrate that combinations
of
P. emblica
or
T. bellerica
extracts with doxorubicin or
cisplatin in A549 and HepG2 cell lines have a better effect
than either agent alone. Further studies are needed to
assess the underlying mechanism(s), signal transduction
pathways, leading to growth inhibition induced by single
agents and combinations both
in
vitro
and
in
vivo
. A positive
outcome of such studies would be increased efficacy
of existing chemotherapies with reduced toxicity to the
normal tissues in treatment of human lung carcinoma and
hepatocellular carcinoma.
ACKNOWLEDGMENTS
We thank Dr. Noppamas Soonthornchareon, Department
of Phar macognosy, Faculty of Phar macy, Mahidol
University, for providing the herbal extracts, Dr. Chariya
Hahnvajanawong guidance during the preparation of the
manuscript, and Mr. Bryan Roderick Hamman for assistance
with the English-language presentation.
COMMENTS
Background
Belleric myrobalan (Terminalia bellerica) and emblic myrobalan (Phyllanthus
emblica), are constituents of herbal formulation (Triphala) and is widely used in
folk medicine in Southeast Asia. Both extracts have been shown to inhibit cancer
cell growth.
Research frontiers
Currently, a variety of effective phytochemicals have been tested in cancer
treatment, which are used alone, or in combination with chemotherapeutic
agents of treatment. The combinations of P. emblica or T. bellerica extracts with
doxorubicin or cisplatin have a synergistic growth effect against some cancer
cell lines. Depending on the combination ratio, the doses for each drug for a
given degree of effect in the combination may be reduced. The mechanism of
this synergistic effect is unclear and, possibly, multiple compounds in the herbal
extracts are involved.
Innovations and breakthroughs
In this report, we demonstrate that combinations of P. emblica or T. bellerica
1496 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol March 14, 2008 Volume 14 Number 10
www.wjgnet.com
COMMENTS
extracts with doxorubicin or cisplatin in A549 and HepG2 cell lines have a better
effect than either agent alone.
Applications
P. emblica and T. bellerica extracts appear to be an effective against hepatocellular
carcinoma and lung cancer cells and less toxic against normal cells. A combination
of an effective P. emblica and T. bellerica extracts with chemotherapeutic agents
which combined may be enhancing efficacy while reducing toxicity to normal
tissues.
Peer review
This is a very nice experimental study about the interaction between drugs
and plant extracts performed in vitro. In this study, the authors investigated the
combination effects of P. emblica and T. bellerica extracts with conventional
cytotoxic agents against human cancer cells. The paper shown a synergistic effect
of P. emblica and T. bellerica extracts with doxorubicin and cisplatin against human
hepatocellular carcinoma and lung cancer cells.
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S- Editor
Li DL
L- Editor
Rippe RA
E- Editor
Liu Y
Pinmai K
et al
. Synergistic effects of myrobalan with cytotoxic drugs 1497
www.wjgnet.com
... It contains several phytochemical constituents such as glucoside (bellericanin) [22], gallo-tannic acid, coloring matter, resins and a greenish-yellow oil [23], ellagic acid, gallic acid, lignans (termilignan and thannilignan), 7-hydroxy 3'4' (methylenedioxy) flavone and anolignan B [24], tannins, ethyl gallate, galloyl glucose and chebulagic acid, phyllemblin, β-sitosterol, mannitol, glucose, fructose and rhamnose [18,25]. Different parts of Terminalia bellerica have been recognized as capable of inducing diverse pharmacological response for exampleimmunological activity [24], β-lactamase inhibitor activity [26], immune response In vitro [24], wound healing activity [27], analgesic activity, antihypertensive effect [28], Anti-diarrheal activity [29], anti-salmonella activity [30], anti-microbial activity [31], antimicrobial and toxicity studies [32], antimutagenic activity [33], antipyretic activity [34], hepatoprotective activity [35], antithrombotic and thrombolytic activity [36], anti-spasmodic and bronchodilatory properties [37], antibiofilm activity [38], acute and sub-acute toxicities [39], antiulcer activity [40], antioxidant activity [23], anticancer activity [41] etc. ...
... P. emblica fruit has antibacterial and antioxidant [6], blood pressure lowering [7], and gastrointestinal wound healing activities [8], as well as protective effects on certain chemotherapy drugs [9]. The effects of T. bellirica include reducing fever and pain [10], antidepressant effects [11], lowering blood sugar [12], and inhibiting cancer cell growth [13]. ...
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