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B Sangameswaran et al.

/ Journal of Pharmacy Research 2012,5(1),315-319

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Research Article
ISSN: 0974-6943

Antitumor activity of Sida Veronicaefolia against Ehrlich Ascites Carcinoma in mice


B Sangameswaran1, Sunil P Pawar2, Manmeet Singh Saluja3* and Ajay Sharma3
1

3.

Director, Department of Pharmacy, Adesh Institute of Medical Sciences, Bhadinda, Punjab,India.


2
Principal, PSGVPM College of Pharmacy, Shahada, Maharashtra.
Research Scholars, Department of Pharmacy, Suresh Gyan Vihar University, Jaipur, Rajasthan, India.

Received on:20-09-2011; Revised on: 15-10-2011; Accepted on:10-12-2011


ABSTRACT
The acetone and ethanol extracts from the leaves of Sida Veronicaefolia i.e. AESV and EESV respectively were evaluated for antitumor activity against Ehrlich
Ascites Carcinoma (EAC) bearing Swiss albino mice. The extracts were administered at the doses of 500 mg/kg body weight per day for 14 days after 24 h of tumor
inoculation. 24 h after the last dose, with fasting, the mice were sacrificed. The present study deals with the effect of AESV and EESV on mean survival time, tumor
volume, tumor weight, tumor cell count, body weight, peritoneal cell count, haematological studies and In vitro cytotoxicity. AESV and EESV caused significant
decrease in tumor volume, tumor weight, tumor cell count, body weight and it prolonged the life span i.e. mean survival time of EAC-tumor bearing mice and
normal peritoneal cell count in normal mice. Haematological profile converted to more or less normal levels in AESV and EESV treated mice. AESV and EESV also
exhibited significant cytotoxic activity at 200 g/ml, but higher cytotoxic activity was found in AESV. The results indicate that AESV and EESV exhibited
significant antitumor activity in EAC-bearing mice.
Keywords: Antitumor Activity, Sida Veronicaefolia, Ehrlich Ascites Carcinoma (EAC).

INTRODUCTION
The use of medicinal plants to treat diseases is as old as human civilization.
Human beings of all ages in both developing and undeveloped countries use
plants in seek to treat numerous diseases and to get relief from physical
ailments. Cancer is a class of diseases in which a cell or a group of cells
display uncontrolled growth, invasion and sometimes metastasis. It is largest
non-communicable disease and it has a sizable contribution in the total number of deaths. The World cancer report documents that cancer rates are set to
increase at an alarming rate globally. Cancer rates could increase by 50% new
cases for the year 20201

pregnancy.8 Lutterodt reported that alcoholic extract of Sida veronicaefolia


has abortifacient effect in pregnant rats. An oral dose produces abortifacient
effect when administered from 15th-17th day of pregnancy.8 It is also reported
that water soluble fraction from an alcoholic extract of Sida veronicaefolia
has muscarine like active principle.8 Literature survey revealed that no detailed phytochemical work has been done on this plant and yet not being
screened for its cytotoxic activity. The aim of present study was to investigate cytotoxic activity by using Invivo and Invitro models.
METHOD AND MATERIALS

India is a rich source of medicinal plants and a number of plant extracts are
used against diseases in various systems of medicine such as Ayurveda,
Unani, and Siddha. Only a few of them have been scientifically explored.
There were good number of plant products such as flavonoids, terpenes,
alkaloids 2, 3, 4 and these can are used as remedies to treat various diseases and
disorders. Because of their distinct pharmacological qualities including cytotoxic and cancer chemopreventive effects, it is inspired many scientists to
take up independent investigations on a number of medicinal plants.5
Sida veronicaefolia, family Malvaceae, is a straggling way side herb found
very often growing in shady places. It grows mainly in clearing in the forest
and as weeds in the over grown grass of public parks and gardens.6 It is also
known as Rajbala, Bhumibala, Farid buti, Shaktibala, etc. It has a capability
to remove the three doshas from the body, and to provide strength and glow
to the body.7
Sida veronicaefolia is very popular with rural womenfolk, especially in the
areas where it grows in its natural habitat, and is used extensively in traditional medicine for shortening and reducing the pain of labour in childbirth. It
is believed to render parturition almost painless and leads to shorter period
of postpartum bleeding. Soup of this plant is taken in the last days of

*Corresponding author.
Manmeet Singh Saluja,
M Pharm, (Ph D),
Department of Pharmacy,
Suresh Gyan Vihar University,
Jagatpura, Jaipur, Rajasthan,India.
Tel.:+91-9303155885
E Mail ID: manisvcp@gmail.com

Collection and Authentification of the leaves


The leaves of Sida veronicaefolia was collected from Sanjivini botanical garden, Bhopal, India in month of July 2009. The leaves were authenticated by
Dr. Sayeeda Khatoon, chemotaxonomist and the voucher specimens were
deposited in the departmental herbarium for future reference.
Preparation of Crude Drug for Extract
The authenticated leaves were used for the preparation of the extract. The
leaves were collected and dried under shade and then coarsely powdered with
the help of mechanical grinder. The powdered was passed through sieve no.
40 and stored in an airtight container for extraction. 9
Preparation of extracts of Sida veronicaefolia
The powdered leaves (500 g) were sequentially extracted using petroleum
ether, chloroform, ethanol and aqueous solution in Soxhlet apparatus. After
about forty siphons of each solvent extraction step, the materials were concentrated by evaporation. 9
Preliminary phytochemical screening
Extracts of Sida veronicaefolia was subjected to qualitative tests for the
identification of various active constituents viz. carbohydrate, glycoside,
alkaloid, amino acids, flavonoids, fixed oil, tannins, gum and mucilage, phytosterols etc. The phytoconstituents were identified by chemical tests, which
showed the presence of various constituents in the different extracts.10
Pharmacological Evaluation
Animals
Swiss Albino mice (20-25gm) of either sex and of approximately the same
age, procured from Institute of Animal Health and Veterinary Biological,

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Mhow, Indore, Madhya Pradesh were used for the study. They were housed
in polypropylene cages and fed with standard rodent pellet diet (Hindustan
Lever Limited, Bangalore) and water ad libitum. The animals are exposed to
alternate cycle of 12 hrs of darkness and light each. Before each test, the
animals are fasted for at least 12 hrs; the experimental protocols were subjected to the scrutinization of the Institutional Animals Ethical Committee
and were cleared by the same. All experiments were performed during morning according to CPCSEA guidelines for care of laboratory animals and the
ethical guideline for investigations of experimental pain in conscious animals.
Acute Toxic Study
This study was carried out on the basis of OECD 423 guidelines, both
extracts were non toxic up to 5000mg/kg hence the LD50 was 5000mg/kg and
1/10 LD50 was selected as dose for the study.
Anticancer activity
Cells
EAC cells were obtained through the courtesy of Amala Cancer Research
Center, Thrissur. They were maintained by weekly intraperitoneal inoculation of 106 cells/mouse.11
Experimental Design
The animals (Swiss albino mice weighing 20-25 g) were divided into 5 groups
consisting of 12 animals. Animals were fed with basal diet and water throughout the experimental period. All the groups were injected with EAC cells
except the group I. This was taken as day zero. From day 1st, normal saline
(5 ml/kg) was given in group I, 5-fluorouracil (20mg/kg), AESV (500 mg/kg)
and EESV (500 mg/kg) were given to group III, group IV and group V respectively, for 14 consecutive days, whereas group II was serve as a tumor
control group and normal saline (5 ml/kg) was given to this group also, on day
15th half of the mice from each group were sacrificed, 24h after last dose, for
the determination of tumor volume, tumor weight, haematological parameters etc, and rest were kept with food and water ad libitum to check the
increase in the life span of the tumor hosts.12
Effect on Mean survival time
Animals were inoculated with 1106 cells/mouse on day 0 and treatment
with AESV and EESV was started 24 h after inoculation, at a dose of 500 mg/
kg/day, p.o. The control group was treated with the same volume of 0.9%
sodium chloride solution. All the treatments were given for 14 days. The
median survival time (MST) of each group was noted. The antitumor efficacy of AESV and EESV were compared with that of 5- fluorouracil (Dabur
Pharmaceutical Ltd, India; 5-FU, 20 mg/ kg/day, i.p. for 14 days). The MST
of the treated groups was compared with that of the control group using the
following calculation: 12, 13
ILS (%) = [(Mean survival of treated group/ Mean survival of control
group)-1]x100
Mean survival time = [1st Death + Last Death] / 2
The result was shown in Table no 1.
Table 1. Effect on the survival of tumor bearing mice
S No

Treatment

Mean Survival
Time(Days)

Increase in
life span (%)

1
2
3
4

Tumor Control
5- FU (20mg/kg, i.p)
AESV (500 mg/kg, p.o)
EESV (500 mg/kg, p.o)

21.50 2.73
40.16 2.13*
35.16 2.8*
34.164.5*

86.79 %
63.50 %
58.88 %

Effect on tumor cell count


The ascites fluid withdrawn from the peritoneal cavity of the mice was taken
in WBC pipette and diluted 100 times with normal saline. A drop of a diluted
cell suspension was placed on the neubauers chamber and the number of cells
in the 64 square was counted. The viability and non viability of cells was
checked by tryphan blue method. On staining viable cells did not take the
dye whereas the non viable cells were stained blue. 12, 13 The result was
shown in table no 2.
Table 2. Effect on Tumor volume, Tumor weight and Tumor cell count
S No

Treatment

Tumor
Volume (ml)

Tumor
weight (gm)

Tumor cell count


Viable cells Nonviable cells
X 107/ml
X 107/ml

1
2
3
4

Tumor Control
6.70 0.16
5- FU (20mg/kg, i.p) 1.01 0.10*
AESV (500 mg/kg, p.o)2.15 0.09*
EESV (500 mg/kg, p.o)2.56 0.10*

6.87 0.21
1.1 0.06*
2.25 0.25*
2.71 0.31*

9.83
0.83
2.33
2.83

n=6 animals in each group, *P<0.01 Vs control. Days of treatment = 14, Values are expressed
as mean SEM

Effect on body weight


Treatments were continued for 14 days. Body weights were recorded every
7th day till 40 days of treatment or till the death of the animal. 12, 13 The result
was shown in Table no 3.
Table 3. Effect on body weight
Treatment/dose

Effect on tumor volume and tumor weight


On 15th day, after 24h of dose, 6 mice from each group were dissected and the
ascetic fluid was collected from peritoneal cavity. The volume was measured
by taking it in a graduated centrifuge tube. The tumor weight was measured
by taking the weight of mice before and after collection of ascetic fluid from
peritoneal cavity. 12, 13 The result was shown in table no 2.

7th Day

14th Day

Normal
22.00.77
Tumor Control
27.830.79*
5-FU (20mg/kg, i.p) 23.330.61
AESV(500 mg/kg, p.o) 24.330.33$
EESV(500 mg/kg, p.o) 25.430.33$

21st Day

28th Day

230.51
24.160.54 27.660.66
40.330.76* 50.160.65* $
24.50.34 26.830.6 $ 29.330.66
29.50.56$,# 30.50.88$,# 31.50.35#
28.50.76# 30.50.88$,# 310.25#

35th Day
31.830.83
32.830.47
34.830.30#
35.830.30#

Values were expressed as mean SEM, n= 6 in each group. * P< 0.001 Vs Normal control, $ P<
0.001 Vs Tumor control, # P<0.001 Vs Standard,

Effect on normal peritoneal cells


Five groups of normal mice (n= 6) were used for the study. First two groups
were treated with 500 mg/kg, p.o. of acetone and ethanol extracts only once
for a single day and other two groups received the same treatment for two
consecutive days. The untreated group was used as control. Peritoneal exudate cells were collected after 24 h treatment by repeated intraperitoneal
wash with normal saline and counted in each of the treated groups and
compared with those of the untreated group. 14
Effect on haematological parameters
Both treatments were given for 14 days to each group (except group III), on
the 15th day, blood was drawn by retro orbital plexus method. WBC count,
RBC count, haemoglobin, protein and packed cell volume were determined
using Lieshman stain solution. 15, 16, 17
Red blood cells (RBC), White blood cells (WBC) and Haemoglobin (Hb)
were estimated with the help of haematology analyzer (Medonic CA620,
Boule, Sweden).The result was shown in Table no 4.
Table 4.Effect of AESV and EESV on Hematological Parameters
Parameter

n=6 animals in each group, *P<0.01 Vs control. Days of treatment = 14, Values are expressed
as mean SEM

0.3 0.33 0.21


0.3* 1.67 0.33**
0.21* 1.8 0.3**
0.16* 2.1 0.3**

Normal

Hb(g/dl)
14.30.10
RBC(million/mm3) 4.680.06
3
WBC(million/mm ) 7.480.03
Proteing %
8.210.06
PCV(mm)
16.50.42
Neutrophils%
30.830.60
Lymphocytes%
68.50.42
Monocytes%
1.160.16

Tumor
control

5 FU
(20 mg/kg)

AESV

EESV

8.350.09*
2.60.07*
27.190.07*
13.950.2*
31.50.42*
68.830.60*
300.57*
2.160.16#

14.00.05*,$
4.110.04*,$
8.230.02*,$
8.650.04*,$
19.50.42*,$
31.830.47*,$
64.660.42*,$
1.330.21 ns

13.20.10*,$
3.130.06*,$
9.580.02*,$
9.60.05*,$
24.50.42*,$
42.160.60*,$
54.00.68*,$
1.50.22 ns

12.910.10*,$
3.880.04*,$
9.090.03*,$
9.10.03*,$
21.30.33*,$
381.78*,$
59.50.42*,$
1.830.30 ns

Values are expressed as Mean


SEM, n= 6 in each group* P< 0.001 Vs Normal control, $ P<
0.001 Vs Tumor control, # P<0.05Vs Normal Control, ns not significant

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Table 6. In-Vitro Cytotoxic activity by NR Uptake cytotoxic assay

Invitro Cytotoxicity
Cell Cultures 18
The EAC cell line was procured through the courtesy of Amala Cancer
Research Center, Thrissur and maintained at Pharmacology Department,
TIT- Pharmacy, Bhopal in Dulbeccos modified eagle medium (DMEM) at
370C and 5% CO2 using standard cell culture methods. At confluence, cells
were trypsinised and equally distributed in two standard cell culture flasks
and were allowed to adhere for 24hr. In order to evaluate the effect of AESV
and EESV on cancer cells, cells were transferred in 96 well cell culture plate
and incubated for 24hr. After confluence, MTT assay and Neutral red uptake
cytotoxic assay have been conducted to evaluate the cell death caused by the
extracts.

S No

Sample Concentration

1
2
3

Control
AESV
EESV

No treatment
200 g/ml
200 g/ml

Optical density
0.380
0.0221
0.0239

% inhibition
0.00 1.88 %
94 .19 3.48%
93 .72 3.45%

8 wells /group OD at 550 nm, Values are expressed as mean SEM

In Vitro Cytotoxic Assay


In Vitro cytotoxic activity was found using MTT assay and Neutral red
uptake cytotoxic assay.
MTT Assay18
25mg of MTT powder was dissolved in 5ml PBS then filtered it with the
help of 10ml syringe and syringe filter. Incubated cell plates were taken out
from the incubator, and discard the culture media from the plates. Culture
media was replaced by the extract containing culture media. Then the plates
were incubated in CO2 incubator for 24 hrs for the action of extracts. 5 hours
before the end of the incubation, add 20l of MTT solution to each well
containing cells. Incubate the plate at 37C for 5 hours. Remove media and
add 200l of DMSO to each well and pipette up and down to dissolve
crystals. Transfer to plate ELISA reader and measure absorbance at 550nm
to get optical density. Then calculate the % inhibition using the formula

Control group

% inhibition = [(OD of untreated)-(OD of drug Treated) /(OD of untreated)] 100


The result was shown in Table no 5.
Table No 5. In-Vitro Cytotoxic activity by MTT assay
S No Sample

Concentration Optical density % inhibition

1
2
3

No treatment
200 g/ml
200 g/ml

Control
AESV
EESV

0.3660
0.0226
0.0157

0.00 1.31 %
93.83 3.48%
95.71 3.45%

8 wells /group, OD at 550 nm, Values are expressed as mean SEM

Neutral Red Uptake Cytotoxic assay18


NR dye (3.3gm) was dissolved in 100 ml of double distilled water and then
this stock solution was filtered by using syringe filter. It was stored at room
temperature and used within 6 months. 1 ml of NR stock solution was
dissolved in the 99 ml of culture media to get the final concentration 0.33%.
Incubated cell plates were taken out from the incubator, and discard the
culture media from the plates. Culture media was replaced by the extract
containing culture media. Then the plates were incubated in CO2 incubator
for 24 hrs for the action of extracts. The extract containing culture media was
then replaced with NR-containing medium. Plates were again placed to incubator for 4-8 hours depending on cell type and maximum cell density. At the
end of the incubation period, the medium was carefully removed and the cells
were quickly washed with PBS. The washed solution was removed and the
incorporated dye was then solubilized in a volume of Neutral Red Assay
Solubilization Solution (ethanolic acetic acid) equal to the original volume of
culture medium. The plates were allowed to stand for 10 minutes at room
temperature. Gentle stirring in a gyratory shaker or pipetting up and down
(trituration) enhanced mixing of the solubilized dye. The background absorbance was measured at 540 nm using ELISA reader to get optical density and
pictures were captured using microscope.
Then calculate the % inhibition using the formula
% inhibition = [(OD of untreated)-(OD of drug Treated) /(OD of untreated)] 100
The result was shown in Table no 6 and in Fig no 1.

Treated with EESV

Treated with AESV


Fig. 1 In-Vitro Cytotoxic activity by NR Uptake cytotoxic assay

Statistical analysis
All the values were expressed as mean SEM (standard error of mean) for six
rats. Statistical analysis was carried out by using PRISM software package
(version 3.0). Statistical significance of differences between the control and

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experimental groups was assessed by One-way ANOVA followed by
Newman-Keuls Multiple Comparision Test. The value of probability less
than 5% (P < 0.05) was considered statistically significant.
RESULT
The phytoconstituents were identified by chemical tests. The extracts showed
the presence of flavonoids, phenolic compounds, tannins, alkaloids, phytosterols, saponins, proteins and amino acids.
Effect on Mean survival time
The MST for the Tumor control was 21.50 2.73 days, whereas it was
35.16 2.8 days, 34.16 4.5 days, and 40.16 2.13 days for the groups
treated with AESV and EESV (500 mg/kg/day, p.o.) and 5-FU (20 mg/kg/day,
i.p.) respectively. The % increase in the lifespan of tumor-bearing mice
treated with AESV, EESV and 5-FU was found to be 63.50 %, 58.88 % and
86.79% respectively (P< 0.01) as compared to the control group. The effect
of AESV and EESV on the survival of tumor-bearing mice was showed in
Table 1.
Effect on tumor volume, tumor weight and tumor cell count
There was reduction in the tumor volume, tumor weight and tumor cell count
of mice treated with AESV and EESV (P<0.001) as showed in table no 2.
Tumor volume of tumor control animals was 6.70 0.16ml, whereas for the
extract-treated group it was 2.15 0.09* ml and 2.56 0.10* ml for AESV
and EESV respectively. Tumor weight of tumor control animals was 6.87
0.21 g, whereas for the extract-treated group it was 2.25 0.25* g and 2.71
0.31* g for AESV and EESV respectively.
Effect on body weight
There was a significant decrease in the weight gain by the AESV and EESV
treated mice when compare with tumor control as showed in table no 3.
Effect on normal peritoneal cells
The average number of peritoneal exudate cells per normal mouse was found
to be 5.80.1106. Single treatment with AESV and EESV (500 mg/kg) enhanced peritoneal cells to 9.70.37 106 and 9.10.1 106, while two consecutive treatments also enhanced the number to 14.30.27 106 and 13.20.2
106 respectively (P< 0.001).
Effect on haematological parameters
Haematological parameters of tumor-bearing mice on Day 14 showed significant changes when compared with the normal mice showed in table no. 4.
The total WBC count, proteins and PCV were found to increase with a
reduction in the haemoglobin content of RBC. The differential count of WBC
showed that the percentage of neutrophils increased (P<0.001) while that of
lymphocytes decreased (P<0.001). At the same time interval, AESV and
EESV (500 mg/kg/day, p.o.) treatment could change these altered parameters
to near normal.
In Vitro Cytotoxicity
The results of In Vitro cytotoxic test were shown in Table 5 and 6. Both the
extracts shows remarkable cytotoxic activity against the tested cells but
higher cytotoxic activity was found in AESV.
CONCLUSION
The present study was carried out to evaluate the antitumor effect of AESV
and EESV in EAC-bearing mice. The AESV and EESV treated animals at the
doses 500 mg/ kg significantly decrease the tumor volume, tumor weight,
tumor cell count, body weight, and brought back the haematological parameters to more or less normal levels. In EAC-bearing mice, a regular rapid
increase in ascites tumor volume was noted. Ascites fluid is the direct nutritional source for tumor cells and a rapid increase in ascites fluid with tumor
growth would be a means to meet the nutritional requirement of tumor cells.
19
The reliable criteria for judging the value of any anticancer drug are the
prolongation of the life span of animals.20, 21 The AESV and EESV decreased
the ascites fluid volume, viable cell count, and increased the percentage of life

span. It may be concluded that AESV and EESV by decreasing the nutritional
fluid volume and arresting the tumor growth, increases the life span of EACbearing mice.
A significant enhancement of peritoneal cell count was observed. The effect
of AESV and EESV treatment on the peritoneal exudate cells of normal mice
is an indirect method of evaluating its inhibitory effect on tumor cell growth.
Normally, a mouse contains about 5 x 106 peritoneal cells, 50% of which are
macrophages. AESV and EESV treatment was found to enhance peritoneal
cells count. These results demonstrate the indirect inhibitory effect of AESV
and EESV on EAC cells, which is probably mediated by the enhancement
and activation of either macrophage or cytokine production.
Usually, in cancer chemotherapy the major problems that are being encountered are of myelosuppression and anemia.22, 23 The anemia encountered in
tumor bearing mice is mainly due to reduction in RBC or haemoglobin percentage, and this may occur either due to iron deficiency or due to hemolytic
or myelopathic conditions.24 In EAC control group, a differential count the
presence of neutrophils increased, while the lymphocyte count decreased,
the observed leucocytopenia indicates a common symptom of immunosuppression in many types of cancers25, 26 and one of the causes of neutrophilia
is myeloid growth factors which are produced in malignant process as part of
a paraneoplastic syndrome. In addition to this another factor granulocyte
colony stimulating factor produced by the malignant cells has also been
attributed to be the cause of neutrophilia because of its action on bone
marrow granulocytic cells in cancer. After the repeated treatment, AESV and
EESV able to reverse the changes in altered neutrophils and lymphocytes
count.27,28 Treatment with AESV and EESV brought back the haemoglobin
content, RBC, and WBC count more or less to normal levels and this indicates that AESV and EESV posses protective action on the haematopoietic
system.Preliminary phytochemical screening indicated the presence of flavonoids, phenolic compound, tannins, alkaloids, phytosterols, saponins,
proteins and amino acids. Flavonoids have been shown to possess antimutagenic 29 and antimalignant 30 effects. Moreover, flavonoids have a
chemopreventive role in cancer through their effects on signal transduction in
cell proliferation31 and angiogenesis.32 Tannins and phenolic compounds are
considered to have cancer-preventive properties33, 34 These induced cell death
in cancer cells by concentration-dependent decrease of ATP and a deterioration of cellular gross morphology.35, 36 Alkaloids shown to inhibit cell growth
without cell death, thus providing enhanced opportunities for DNA repair,
immune stimulation, anti-inflammation and cancer prevention. 37 Phytosterols inhibited tumor growth by an alteration of signal transduction pathways.
38, 39
The cytotoxic and antitumor properties of the extract may be due to
these compounds.
The present study points to the potential anticancer activity of AESV and
EESV against EAC bearing mice. Further studies to characterize the active
principles and elucidate the mechanism of the action of AESV and EESV are
in progress.
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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 1.January 2012

315-319

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