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Pharm Biol, 2013; 51(6): 753–759
! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.764538

ORIGINAL PAPER

Antitumor activity and antioxidant property of Curcuma caesia against

Ehrlich’s ascites carcinoma bearing mice
Indrajit Karmakar1, Narayan Dolai1, R. B. Suresh Kumar1, Biswakanth Kar1, Sudipendra Nath Roy2, and
Pallab Kanti Haldar1
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1
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India and 2National Institute of Pharmaceutical Education
and Research, Kolkata, West Bengal, India

Abstract Keywords
Context: Curcuma caesia Roxb. (Zingiberaceae), commonly known as ‘‘Kala Haldi’’ in Bengali, has Biochemical estimation, black turmeric,
been traditionally used for the treatment of cancer, bruises, inflammation and as an cytotoxicity, hematological parameters
aphrodisiac.
Objective: To evaluate the antitumor activity and antioxidant status of the methanol extract of History
Curcuma caesia (MECC) rhizomes on Ehrlich’s ascites carcinoma (EAC)-treated mice.
Materials and methods: In vitro cytotoxicity assay of MECC was evaluated by using Trypan blue Received 15 June 2012
method. Determination of in vivo antitumor activity was performed after 24 h of EAC cells Revised 20 November 2012
(2  106 cells/mouse) inoculation; MECC (50 and 100 mg/kg i.p.) was administered daily for nine Accepted 6 January 2013
consecutive days. On day 10, half of the mice were sacrificed and the rest were kept alive for Published online 25 March 2013
assessment of increase in lifespan. Antitumor effect of MECC was assessed by the study of
For personal use only.

tumor volume, tumor weight, viable and non-viable cell count, hematological parameters and
biochemical estimations. Furthermore, antioxidant parameters were assayed by estimating liver
and kidney tissue enzymes.
Results: MECC showed direct cytotoxicity (IC50 90.70  8.37 mg/mL) on EAC cell line. MECC
exhibited significant (p50.01) decrease in tumor volume, tumor weight, viable cell count and
percentage increased the lifespan (57.14 and 88.09%) of EAC-treated mice. Hematological
profile, biochemical estimation, tissue antioxidant assay significantly (p50.01) reverted to
normal level in MECC-treated mice.
Conclusion: MECC possesses potent antitumor activity that may be due to its direct cytotoxic
effect or antioxidant properties. Further research is in progress to find out the active principle(s)
of MECC for its antitumor activity.

Introduction (Chandrashekhar et al., 2011). Indeed, molecules derived

from natural sources including plants, marine organisms and
Cancer represents one of the largest causes of death through-
micro-organisms, have played, and continue to play, a
out the world. It already affects over six million lives annually
dominant role in the discovery of leads for the development
and nearly 10 million new cases are diagnosed globally every
year (Abdullaev, 2002). Moreover, incidences of cancer keep
of conventional drugs for the treatment of most human 13
20
diseases (Cragg & Newman, 2005).
increasing on a global scale. There is only one plausible
The Ehrlich tumor was initially described as a spontaneous
explanation for this: conventional medicine does not know the
murine mammary adenocarcinoma (Dolai et al., 2012a). It is a
causes for cancer nor how this disease spreads. Because of this,
rapidly growing carcinoma with very aggressive behavior and
there is no effective cancer therapy available and the disease
is able to grow in almost all strains of mice. In ascites form, it
keeps expanding on a global scale (Rath, 2001).
has been used as a transplantable tumor model to investigate
Although many synthetic anticancer agents are used in the
the antitumor effects of several substances (Haldar et al.,
treatment of cancer, side effects and emergence of synthetic
2010).
drug resistant cancer cells among patients limits their use
Curcuma caesia Roxb. (Zingiberaceae), commonly known
(Karthikeyan et al., 2007). The limitation of synthetic
as ‘‘Kala Haldi’’ in Bengali and ‘‘Black turmeric’’ in English,
drugs highlights the pivotal need of anticancer agents
is a perennial herb found throughout the Himalayan region,
from natural products, particularly medicinal plants
Northeast and Central India. It is also sparsely found in Papi
Hills of East Godavari, root hills of the Himalayas and the
North Hill forest of Sikkim (Karmakar et al., 2011c). The
Correspondence: Dr. Pallab Kanti Haldar, Department of Pharmaceutical
Technology, Jadavpur University, Kolkata 700032, West Bengal, India. rhizomes are traditionally used in the treatment of hemor-
Tel: þ91 9433230566. E-mail: pallab_haldar@rediffmail.com rhoids, leprosy, asthma, cancer, fever, wounds, vomiting,
 754 I. Karmakar et al.
menstrual disorder, anthelmentic, aphrodisiac, gonorrheal
discharges and inflammation (Amalraj et al., 1989; Singh &
Jain, 2003). The rhizome paste is applied on bruises, contu-
sions, and rheumatic pains. The rhizome is also used in
dysentery, diarrhea and cough as an aromatic and as a source of
D-camphor (Craker & Simon, 1996; Sarangthem & Haokip,
2010). Previous bioactivity studies have been revealed that
plant rhizome possesses antifungal activity (Banerjee &
Nigam, 1976), anxiolytic and CNS depressant activities
(Karmakar et al., 2011a), neuropharmacological assessment
(Karmakar et al., 2011c), anti-asthmatic, smooth muscle
relaxant activity (Arulmozhi et al., 2006) and free radical
scavenging activity against reactive oxygen and nitrogen
species (Karmakar et al., 2011b). Presence of curcuminoids,
1,8-cineole, camphor, ar-turmeone, phenolics, flavonoids,
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protein and alkaloids were reported in the rhizomes of
C. caesia (Paliwal et al., 2011; Sarangthem & Haokip,
2010). However, antitumor activity of its rhizomes has not
yet been scientifically explored; therefore the main objective of
present study is to evaluate antitumor and antioxidant status of
the methanol extract of C. caesia (MECC) on Ehrlich ascites
carcinoma (EAC) cell treated in Swiss albino mice.
Materials and methods
Plant material and preparation of extract
The rhizome of C. caesia was collected from the upper hill
For personal use only.
region of Sikkim in the month of September 2010 and
identified by Dr. A. K. Panda, Taxonomist; Regional
Research Institute (Ay), Gangtok, India and the voucher
specimen (No. HPI/IK-01) was deposited in the Department
of Pharmacology, Himalayan Pharmacy Institute, Sikkim,
India for future reference. First, the collected rhizome was
shade-dried at room temperature for 7 d and then powdered in
a mechanical grinder. Next, the powdered plant material
(225 g) was extracted by methanol using Soxhlet extraction
apparatus. The solvent was completely removed under
reduced pressure in a rotary evaporator. The concentrated
extract was obtained by lyophilization and stored in vacuum
desiccators (20  C) for further use. The yield of the methanol
extract was about 21.51% w/w.
Phytochemical analysis
The preliminary phytochemical analysis and planar chroma-
tographic studies of MECC was done for qualitative analysis
by using the standard method (Harborne, 1998).
Drugs and chemicals
Chemicals used were Trypan blue, 5-fluorouracil (Merck
Limited, Mumbai, India). trichloroacetic acid, thiobarbituric
acid, phenazine methosulphate, reduced nicotinamide adenine
dinucleotide, nitroblue tetrazolin and dithionitrobenzene.
They were obtained from Sigma-Aldrich, Bangalore, India.
All the chemicals and reagents were used in highest analytical
grade.
Animals
Male Swiss albino mice (20–25 g) of 8 weeks of age were
used for the experiment. The mice were grouped and housed
Pharm Biol, 2013; 51(6): 753–759
in polyacrylic cages (38 cm  23 cm  10 cm) with not more
than six animals per cage. The animals were maintained under
standard laboratory conditions (temperature 25–30  C and
55–60% relative humidity with dark/light cycle 14/10 h) and
were allowed free access to standard dry pellet diet and water
ad libitum. The mice were acclimatized to laboratory
conditions for 7 d before commencement of the experiment.
All the procedures described were reviewed and approved by
the University Animal Ethical Committee (367001/C/
CPCSEA).
Acute toxicity
MECC was administered orally to male Swiss albino mice
to evaluate the acute toxicity as per the reported method
(Lorke, 1983).
Transplantation of tumor cell
EAC cells were obtained from Chittaranjan National Cancer
Institute (CNCI, Kolkata, India). The EAC cells were
maintained in vivo in Swiss albino mice by intraperitoneal
transplantation of 2  106 cells per mouse after every
10 days and it is used for both in vivo and in vitro study
(Haldar et al., 2010).
Assay for in vitro cytotoxicity
In vitro short term cytotoxicity of MECC was assayed by
using Trypan blue exclusion method (Dolai et al., 2012a). At
first, we have prepared different concentration of MECC
solution as 250, 500, 1000, 1500, 2000 and 3000 mg/mL.
Then, 1  106 EAC cells were suspended in 0.1 mL of
phosphate buffered saline (PBS, 0.2 M, pH 7.4) and mixed
with 100 mL of various aforementioned concentrations of
MECC. Final concentration (25, 50, 100, 150, 200 and 300
mg/mL) of MECC was adjusted by PBS and incubated at
37  C for 3 h. After the completion of incubation, the viability
of the cells was determined using Trypan blue dye (0.4% in
normal saline) and the percentage of cytotoxicity was
determined by calculating % inhibition and IC50 values.
Treatment schedule for assessment of in vivo
antitumor potential
Swiss albino mice (20–25 g) were divided into five groups
(n ¼ 12). All the animals in each groups except Group-I
received EAC cells [2  106 cells/mouse intraperitoneally
(i.p.)]. This was taken as day ‘‘0’’. Group-I served as normal
saline control (5 mL/kg i.p.) and group-II served as EAC
control. After 24 h of EAC transplantation, Groups-III and -IV
received MECC at a dose of 50 and 100 mg/kg i.p. for nine
consecutive days, respectively. Group-V received reference
drug 5-FU (20 mg/kg i.p.) for nine consecutive days (Bala
et al., 2010). After administration of the last dose, six mice
from each group were kept fasting for 18 h and blood was
subsequently collected by direct cardiac puncture for the
estimation of hematological and determination of serum
biochemical parameters. The animals were then sacrificed for
collection of liver and kidney tissues to check the different
antioxidant parameters. The rest of the animals, in each group
were kept alive and given food and water ad libitum to
 DOI: 10.3109/13880209.2013.764538
evaluate percentage increase in their life span to determine the
mean survival time (MST).
Antitumor activity of MECC was assessed by observation
of changes with respect to the following (tumor volume,
tumor weight, percentage increase in life span (%ILS), viable/
non-viable tumor cell count, hematological parameters,
biochemical parameters and tissue antioxidant assay) param-
eters (Haldar et al., 2010).
Determination of tumor volume and tumor weight
The ascites fluid was collected from the peritoneal cavity. The
volume was measured in a graduated centrifuge tube and
expressed in milliliter (mL).
The tumor weight was measured by weighing the mice
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before and after the collection of the ascetic fluid from
peritoneal cavity and expressed in grams (g).
Percentage increase in life span
The effect of MECC on percentage increased in life span was
calculated on the basis of the mortality rates of the
experimental mice, expressed as:
MST in days ¼ ðFirst death þ last deathÞ=2
ILS ð%Þ ¼ ½ðMST of the treated group
=MST of the control groupÞ  1  100:
For personal use only.
Estimation of viable/non-viable tumor cell count
The ascites fluid was taken in a white blood cell (WBC)
pipette and diluted to 20 times with PBS. A drop of the
diluted cell suspension was then placed on the Neubauer’s
counting chamber and the number of cells counted.
The viability and non-viability of the cells were
checked by Trypan blue dye exclusion assay method. The
cells were stained with Trypan blue (0.4% in normal saline)
dye. The cells that did not take up the dye were viable and
those that took the dye were nonviable. The viable and
nonviable cells were determined by using the following
formula:
Cell count ¼ ðNumber of cells  dilution factorÞ
=ðArea  thickness of liquid filmÞ:
Determination of hematological parameters
Collected blood was used for the estimation of hemoglobin
(Hb), red blood cell (RBC) and WBC count by standard
procedures (D’Armour et al., 1965; Wintrobe et al., 1961).
Estimation of biochemical parameters
The blood samples were allowed to clot and the serum was
separated by centrifugation at 5000 rpm for 10 min. Serum
was utilized for the estimation of various biochemical
parameters such as total protein, serum glutamic oxaloacetate
transaminase (SGOT), serum glutamic pyruvic transaminase
(SGPT) and alkaline phosphatase (ALP). All analyzes were
performed by using commercially available kits (Span
Diagnostics Ltd, Surat, India).
Antitumor and antioxidant properties of Curcuma caesia 755
Determination of tissue antioxidant assay parameters
The tissue antioxidant assay was performed with liver and
kidney tissues and evaluate by measuring the level of total
protein (Span Diagnostics kit), protein carbonylation (PC)
(Abraham et al., 1999), lipid peroxidation (Ohkawa et al.,
1979), the amount of enzymatic catalase (CAT) (Aebi, 1974)
and superoxide dismutase (SOD) (Kakkar et al., 1984) and
nonenzymatic antioxidant system such as reduced glutathione
(GSH) (Ellman, 1959).
Statistical analysis
All the experimental data are expressed as the mean  SEM.
Data was statistically analyzed by using one way analysis of
variance followed by Dunnett’s post hoc test by Instat
software (version 4). p Values of 50.01 were considered
statistically significant.
Results
Phytochemical analysis
MECC extract indicated the presence of phenols and tannins
(ferric chloride test), terpenoids (Liberman Burchard test),
saponins (froth test), alkaloids (Dragendroff’s test), flavon-
oids (Shinoda test) and volatile oil (spot test).
Acute toxicity
The MECC was found to be safe in male Swiss albino mice up
to the dose of 3000 mg/kg body weight per oral.
In vitro cytotoxicity
From the in vitro cytotoxicity study, it is clear that MECC
showed direct cytotoxic effect on the EAC cell line, with an
IC50 value of 90.70  8.37 mg/mL (Figure 1).
Tumor growth and survival parameters
Antitumor activity of MECC against EAC tumor bearing
mice was assessed by the parameters such as tumor volume,
tumor weight, cell count (viable and non-viable), MST and
%ILS. The tumor volume, tumor weight and viable cell count
were found to be significantly (p50.01) increased and non-
viable cell count declined significantly (p50.01) in EAC
control animals, when compared with normal control animals
(Table 1). Administration of MECC at doses of 50 and
80
60
% Inhibition
40
IC50 = 90.7 ± 8.37 µg/mL
20
0
0 100 200 300 400
Concentration (µg/mL)
Figure 1. Cytotoxic effect of MECC on in vitro EAC cell line. Values
are mean  S.E.M.; where n ¼ 3.
 756 I. Karmakar et al. Pharm Biol, 2013; 51(6): 753–759

Table 1. Effect of MECC on tumor volume, tumor weight, total cell count, viable and nonviable cell count, MST and %ILS in EAC bearing mice.

Parameters EAC control EAC þ MECC (50 mg/kg) EAC þ MECC (100 mg/kg) EAC þ 5-FU (20 mg/kg)
Tumor volume (mL) 2.75  0.13 1.40  0.13* 0.91  0.05* 0.53  0.05*
Tumor weight (g) 3.21  0.10 1.08  0.08* 0.83  0.06* 0.57  0.04*
Total cell (107 cell/mL) 9.30  0.43 4.00  0.13* 3.89  0.05* 3.81  0.09*
Viable cell (107 cell/mL) 8.92  0.42 2.78  0.18* 0.94  0.08* 0.60  0.08*
Nonviable cell (107 cell/mL) 0.38  0.04 1.22  0.07* 2.96  0.08* 3.21  0.09*
Viable % 95.91 69.50 24.16 15.75
Nonviable % 4.09 30.50 76.09 84.25
MST (days) 21 33 39.5 43
% ILS 00 57.14 88.09 104.76

Values are represented as mean  SEM, where n ¼ 6. *p50.01 for treated groups versus EAC control group.

Table 2. Effect of MECC on hematological parameters in EAC bearing mice.

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Parameters Normal control EAC control EAC þ MECC (50 mg/kg) EAC þ MECC (100 mg/kg) EAC þ 5-FU (20 mg/kg)
6 a, b, b,
RBC (cell  10 /mL) 5.89  0.20 2.93  0.11 * 4.81  0.23 * 5.09  0.70 * 5.19  0.20b,*
WBC (cell  103/mL) 5.16  0.32 10.97  0.68a,* 7.53  0.36b,* 6.16  0.27b,* 5.09  0.33b,*
Hb (g/dL) 11.48  0.98 4.80  0.29a,* 7.98  0.41b,* 9.86  0.41b,* 10.35  1.16b,*

Values are represented as mean  SEM, where n ¼ 6.

a
EAC control group versus normal group.
b
Treated groups versus EAC control group, *p50.01.

100 mg/kg significantly (p50.01) decreased the tumor compared to normal control mice. Administration of MECC
volume and viable cell count. Non-viable cell count was at the doses of 50 and 100 mg/kg significantly (p50.01)
significantly (p50.01) higher in MECC treated animals as attenuated the PC of both liver and kidney tissues (Figure 3a
For personal use only.

compared to EAC control animals. Furthermore, the MST was
increased to 33  0.80 (% ILS ¼ 57.14) and 39.5  0.51 (%
ILS ¼ 88.09) on administration of MECC 50 and 100 mg/kg,
respectively.
Hematological parameters
There was significantly (p50.01) elevated levels of WBC and
a significant (p50.01) reduction of RBC and Hb levels in
EAC control group as compared to the normal control group
(Table 2). Administration of MECC at doses of 50 and
100 mg/kg significantly (p50.01) reduced WBC count in
respect to EAC control group. RBC count and Hb content
were found to be significantly (p50.01) restored to the
normal levels. These results implied the protective effect of
MECC on the hematological profile of EAC bearing mice.
Biochemical parameters
Biochemical parameters like SGPT, SGOT and ALP indicates
the significant (p50.01) elevated levels of liver functional
enzymes in the serum of the EAC-treated group with respect to
normal animals. The total protein content was found to be
significantly (p50.01) decreased in the EAC control group
was compared to the normal control group. Administration of
MECC significantly (p50.01) increased the total protein
content as compared with the EAC control mice (Figure 2a).
Treatment with MECC (50 and 100 mg/kg) in EAC bearing
mice significantly (p50.01) decreased the SGOT, SGPT and
ALP levels as compared to EAC control groups (Figure 2b–d).
Tissue antioxidant assay parameters
Total protein content and PC were significantly (p50.01)
raised in both liver and kidney in EAC control mice when
and b) as compared to EAC control mice.
Lipid peroxidation results in the formation of reactive
oxygen species and subsequently elevates the level of
malondialdehyde (MDA) which induces cancer in liver and
kidney tissues. In the present study, the MDA level was
significantly (p50.01) increased in EAC control animals
when compared with normal control animals. Interestingly,
treatment with MECC significantly (p50.01) reduced
the MDA levels as compared with EAC control group
(Figure 3c).
The levels of CAT, reduced GSH and SOD were signifi-
cantly (p50.01) decreased in EAC control group when
compared with the normal control group. Administration of
MECC (50 and 100 mg/kg) significantly (p50.01) raised the
CAT, reduced GSH and SOD levels as compared with EAC
control animals (Figure 3d–f).
Discussion
The Ehrlich ascites tumor implantation induces a local
inflammatory reaction, with increasing vascular permeability,
which results in an intense edema formation, cellular
migration, and a progressive ascites fluid accumulation
(Bala et al., 2010). The ascites fluid is essential for tumor
growth, since it constitutes a direct nutritional source for
tumor cells (Dolai et al., 2012a). The present study showed
that MECC significantly decreased the tumor volume, tumor
weight, viable tumor cell and increased the non-viable tumor
cell and lifespan of the EAC bearing control mice. The
reliable criteria for judging the value of any anticancer drug
are prolongation of lifespan (Dolai et al., 2012b). The
observed increase in the lifespan of tumor bearing mice by
reduction of nutritional fluid volume and seization of the
 DOI: 10.3109/13880209.2013.764538 Antitumor and antioxidant properties of Curcuma caesia 757
(a) Normal control MECC 50 mg/kg (b) Normal control MECC 50 mg/kg
EAC control MECC 100 mg/kg EAC control MECC 100 mg/kg
15 150
5-FU 20 mg/kg 5-FU 20 mg/kg
a, #
b, *
b, *
10 100 b, *
b, *

IU/L
g/dl

b, *
a, # b, *
5 50

0 0

(c) Normal control MECC 50 mg/kg (d) Normal control MECC 50 mg/kg
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MECC 100 mg/kg EAC control MECC 100 mg/kg

EAC control
80 200
a, # 5-FU 20 mg/kg
a, # 5-FU 20 mg/kg

60 150
b, * b, *

KA Unit
b, * b, *
IU/L

40 b, * 100 b, *

20 50

0 0
For personal use only.

Figure 2. Effect of MECC on serum biochemical parameters such as total protein count (a), SGOT (b), SGPT (c) and ALP (d) in EAC bearing mice.
Values are represented as mean  SEM, where n ¼ 6. aEAC control group versus normal control group, #p50.01. bAll treated groups versus EAC
control group, *p50.01.

tumor growth is a positive result and further corroborates the
antitumor potential of MECC.
Usually in cancer chemotherapy, the major problems are
myelosuppression and anemia. The anemia encountered in
tumor bearing mice is mainly due to reduction in RBC or Hb
percentage, and this may occur either due to iron deficiency
or due to hemolytic or myelopathic conditions (Haldar et al.,
2011). Pharmacotherapy with MECC replenishes the RBC
and Hb content to the normal levels. The WBC count was
reduced as compared with that of EAC control mice.
These indicating parameters revealed that MECC exerted
less toxic effect to the hemopoietic system and plausibly had
selective affinity to the tumor cell and thereby it could
maintain the normal hematological profile. It is evident from
the result that MECC possesses a protective action on
hemopoietic system.
Enzymes in serum have been studied as both early possible
indicators of neoplasia and as an aid in monitoring the
progression and regression of disease. In certain circum-
stances serum enzymes can be carcinogenic and may
engender hepatoxicity (Dolai et al., 2012b). Elevated levels
of serum parameters, i.e., SGPT, SGOT and ALP which are
indicative of impaired liver functions can be clearly seen due
to hepatotoxicity associated after 9 d of inoculation with EAC
cells (Haldar et al., 2011). The analysis of changes in serum
total protein in malignancy is in itself a means of
studying abnormality in the protein metabolism in this
condition. Treatment with the MECC restored the elevated
biochemical parameters more or less to normal range,
indicating the protection of the tumor cell induced hepato-
toxicity by MECC.
Carbonylation of protein often leads to a loss of protein
function, which is considered a widespread marker of severe
oxidative stress, damage and disease-derived protein dys-
function. Significantly elevated plasma level of PC is found in
various types of cancer including hematological malignancies
and neurodegenerative diseases as well as in human cell lines
(Dalle-Donne et al., 2006). The proposed mechanism for high
levels of PC is that myeloid cells are the major source of free
radicals which lead to irreversible protein oxidation in the
cells (Dolai et al., 2012a). Administration of MECC restored
the increased PC levels more or less to normal range
signifying the antioxidant and free radical scavenging prop-
erty of MECC.
Oxidative stress may lead to damage of the macromol-
ecules such as lipids and can induce lipid peroxidation in vivo
(Dolai et al., 2012a). In EAC bearing mice, the level of lipid
peroxide in liver was significantly raised, which was however
reduced near to normal level in MECC treated animal groups.
This reflects the decline in free radical production and
subsequent reduction in oxidative stress, which are the main
risk factors of the ailment. GSH, a potent inhibitor of
neoplastic process plays an important role as an endogenous
antioxidant system that is found particularly in high concen-
tration in liver and is known to have key function in the
protective process (Haldar et al., 2010). The level of reduced
GSH was depleted in cancer bearing mice, which may be due
to its utilization, by the excessive amount of free radicals.
 758 I. Karmakar et al. Pharm Biol, 2013; 51(6): 753–759

Normal control MECC 50 mg/kg Normal control MECC 50 mg/kg

(a) (b)
EAC control MECC 100 mg/kg EAC control MECC 100 mg/kg
8 5-FU 20 mg/kg 10 a,# 5-FU 20 mg/kg
a,#
8 a,#
6 b,* b,*
a,# b,*

mM/cm
6 b,* b,*
g/dl

4 b,* b,*
b,* 4 b,*
b,*
b,*
b,*
2 2

0 0
Liver Kidney Liver Kidney

(c) Normal control MECC 50 mg/kg (d) Normal control MECC 50 mg/kg
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EAC control MECC 100 mg/kg 20 EAC control MECC 100 mg/kg
200 5-FU 20 mg/kg 5-FU 20 mg/kg
a,#

KU/min/mg protein
15 b,*
150 b,*
nM/mg-protein

b,*
b,* a,#
10 b,*
100 b,* b,*
b,* a,# b,*
b,* b,*
b,* 5 a,#
50

0 0
Liver Kidney Liver Kidney
For personal use only.

(e) Normal control MECC 50 mg/kg (f) Normal control MECC 50 mg/kg
EAC control MECC 100 mg/kg EAC control MECC 100 mg/kg
20 5-FU 20 mg/kg 10 5-FU 20 mg/kg
b,*
b,*
mg GSH/mg protein

15 b,* 8 b,*
mUnit/mg protein

b,*
b,* b,* b,*
b,* 6
b,
10 b,* b,* *
a,# 4
a,#
5 a,# a,#
2

0 0
Liver Kidney Liver Kidney

Figure 3. Effect of MECC on tissue antioxidant defense parameters like total protein count (a), protein oxidation (b), lipid peroxidation (c), CAT (d),
reduce GSH (e) and SOD (f) in EAC bearing mice. Values are represented as mean  SEM, where n ¼ 6. aEAC control group versus normal control
group, #p50.01. bAll treated groups versus EAC control group, *p50.01.

Treatment with MECC was found to increase the GSH
content in the liver and kidney as compared to EAC control
animals.
Cells are also equipped with enzymatic antioxidant
mechanisms that play an important role in the elimination
of free radicals. SOD is involved in the clearance of
superoxide. The inhibition of SOD activities as a result of
tumor growth was reported (Gupta et al., 2000) and similar
findings were observed in our present results in EAC bearing
mice. The administration of MECC at both doses significantly
recovered the SOD level towards normal level. On the other
hand, the free radical scavenging enzyme CAT is present in
all oxygen-metabolizing cells and its function is to provide a
direct defense against the potentially damaging reactivities of
superoxide and hydrogen peroxide. The inhibition of CAT
activities as a result of tumor growth has also been reported
(Dolai et al., 2012a). Similar findings were observed in the
present investigation with EAC bearing mice. The adminis-
tration of MECC at both doses increased the CAT levels,
which along with the restoration of lipid peroxide and GSH
contents to near normal indicate the antioxidant and free
radical scavenging property of MECC.
Conclusion
The present investigation is quite encouraging as it explored
the potential antitumor activity of MECC presumably
potentiated by its direct cytotoxic effect and antioxidant
property. It was assumed that attenuation of oxidative stress in
different tissues in EAC bearing mice decreased the viability
 DOI: 10.3109/13880209.2013.764538
of EAC cells. Phytochemical study showed the presence of
steroids, tannins, saponins, volatile oil, proteins alkaloids and
flavonoids in MECC; these compounds have been known to
possess potent antitumor properties. Finally, our findings
suggest that the MECC exhibits potential antitumor and
antioxidant activities which enlighten a novel source of
phytomedicines in free radical and tumor biology. Further
investigations are in progress in our laboratory to identify the
active principle(s) responsible for these antitumor and anti-
oxidant activities.
Acknowledgements
The authors greatly acknowledge Department of
Pharmaceutical Technology, Jadavpur University, Kolkata.
Pharmaceutical Biology Downloaded from informahealthcare.com by Library of Health Sci-Univ of Il on 01/06/15
Declaration of interest
The authors report no conflict of interest. The authors alone
are responsible for the content and writing of the paper. The
authors are thankful to the AICTE (RPS-File No: 8023/BOR/
RID/RPS (NER)-91/2010-11), New Delhi, India, for financial
assistance for the project.
References
Abdullaev FI. (2002). Cancer chemopreventive and tumoricidal proper-
ties of saffron (Crocus sativus L.). Exp Biol Med 227:20–5.
Abraham P, Wilfred G, Catherine SP. (1999). Oxidative damage to the
For personal use only.
lipids and proteins of the lungs, testis and kidney of rats during carbon
tetrachloride intoxication. Clin Chim Acta 289:177–9.
Aebi H. (1974). Catalase. In: Packer L, ed. Methods in Enzymatic
Analysis. New York: Academic Press, 673–84.
Amalraj VA, Velayudhan KC, Muralidharan VK. (1989). A note on the
anomalous flowering behaviour in Curcuma caesia (Zingiberaceae).
J Bombay Nat History Soc 86:278–9.
Arulmozhi DK, Sridhar N, Veeranjaneyulu A, Arora SK. (2006).
Preliminary mechanistic studies on the smooth muscle relaxant effect
of hydro alcoholic extract of Curcuma caesia. J Herb Pharmacother
6:117–24.
Bala A, Kar B, Haldar PK, et al. (2010). Evaluation of anticancer activity
of Cleome gynandra on Ehrlich’s ascites carcinoma treated mice.
J Ethnopharmacol 129:131–4.
Banerjee A, Nigam SS. (1976). Antifungal activity of the essential oil of
Curcuma caesia Roxb. Indian J Med Res 64:1318–21.
Chandrashekhar G, Joshi M, Gopal N, Kumari S. (2011). Antitumor
activity of hexane and ethyl acetate extracts of Tragia involucrata.
Int J Cancer Res 7:267–77.
Cragg GM, Newman DJ. (2005). Plants as a source of anticancer agents.
J Ethnopharmacol 100:72–9.
Craker LE, Simon JE. (1996). Herb, Spices and Medicinal Plants:
Recent Advances in Botany, Horticulture and Pharmacology.
New York: Food Products Press.
Antitumor and antioxidant properties of Curcuma caesia 759
D’Armour FE, Blood FR, Belden DA. (1965). The Manual for
Laboratory Work in Mammalian Physiology. 3rd ed. Chicago (IL):
The University of Chicago Press.
Dalle-Donne I, Aldini G, Carini M, et al. (2006). Protein
carbonylation, cellular dysfunction, and disease progression. J Cell
Mol Med 10:389–406.
Dolai N, Karmakar I, Kumar RBS, et al. (2012a). Evaluation of
antitumor activity and in vivo antioxidant status of Anthocephalus
cadamba on Ehrlich ascites carcinoma treated mice.
J Ethnopharmacol 142:865–70.
Dolai N, Karmakar I, Kumar RBS, et al. (2012b). Antitumor potential of
Castanopsis indica (Roxb. ex. Lindl.) A. DC. leaf extract against
Ehrlich’s ascites carcinoma cell. Indian J Exp Biol 50:359–65.
Ellman GL. (1959). Tissue sulfhydryl groups. Arch Biochem Biophys
82:70–7.
Gupta M, Mazumder UK, Rath N, Mukhopadhyay DK. (2000).
Antitumor activity of methanolic extract of Cassia fistula L. seed
against Ehrlich ascites carcinoma. J Ethnopharmacol 72:151–6.
Haldar PK, Bhattacharya S, Dewanjee S, Majumder UK. (2011).
Chemopreventive efficacy of Wedelia caledulaceae against
20-methylcholanthrene-induced carcinogenesis in mice. Environ
Toxicol Pharmacol 31:10–15.
Haldar PK, Kar B, Bala A, et al. (2010). Antitumor activity of
Sansevieria roxburghiana rhizome against Ehrlich ascites carcinoma
in mice. Pharm Biol 48:1337–43.
Harborne JB. (1998). Phytochemical Methods, A Guide to Modern
Techniques of Plant Analysis. New Delhi: Springer (India) Pvt. Ltd.
Kakkar P, Das B, Vishwanath PN. (1984). A modified spectrophoto-
metric assay of superoxide dismutase. Indian J Biochem Biophys
21:130–2.
Karmakar I, Dolai N, Bala A, Haldar PK. (2011a). Anxiolytic and CNS
depressant activities of methanol extract of Curcuma caesia.
Pharmacologyonline 2:738–47.
Karmakar I, Dolai N, Saha P, et al. (2011b). Scavenging activity of
Curcuma caesia rhizome against reactive oxygen and nitrogen
species. Orient Pharm Exp Med 11:221–8.
Karmakar I, Saha P, Neelanjan S, et al. (2011c). Neuropharmacological
assessment of Curcuma caesia rhizome in experimental animal
models. Orient Pharm Exp Med 11:251–5.
Karthikeyan R, Karthigayan S, Balasubashi MS, et al. (2007). Antitumor
effect of snake venom (Hydrophis spiralis) on Ehrlich ascites
carcinoma bearing mice. Int J Cancer Res 3:167–73.
Lorke DA. (1983). A new approach to practical acute toxicity testing.
Arch Toxicol 54:275–87.
Ohkawa H, Onishi N, Yagi K. (1979). Assay for lipid
peroxidation in animal tissue by thiobarbituric acid reaction. Anal
Biochem 95:351–8.
Paliwal P, Pancholi SS, Patel RK. (2011). Pharmacognostic parameters
for evaluation of the rhizomes of Curcuma caesia. J Adv Pharm
Technol Res 2:56–61.
Rath M. (2001). Cellular Health Series: Cancer. 1st ed. Santa Clara: M
R Publishing, Inc.
Sarangthem K, Haokip MJ. (2010). Bioactive components in Curcuma
caesia Roxb. grown in Manipur. Bioscan 5:113–15.
Singh V, Jain AP. (2003). Ethnobotany and Medicinal Plants of India
and Nepal. Jodhpur: Scientific Publishers.
Wintrobe MM, Lee GR, Boggs DR, et al. (1961). Clinical Hematology.
5th ed. Philadelphia: Lea and Febiger.