Hindawi Publishing Corporation

BioMed Research International

Volume 2014, Article ID 625792, 8 pages
http://dx.doi.org/10.1155/2014/625792

Research Article
Viola tricolor Induces Apoptosis in Cancer Cells and Exhibits
Antiangiogenic Activity on Chicken Chorioallantoic Membrane
Hamid Reza Sadeghnia,1,2,3 Taghi Ghorbani Hesari,4 Seyed Mohsen Mortazavian,3
Seyed Hadi Mousavi,2,3 Zahra Tayarani-Najaran,4 and Ahmad Ghorbani2
1

Neurocognitive Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad 9177948564, Iran
Pharmacological Research Center of Medicinal Plants, School of Medicine, Mashhad University of Medical Sciences,
Mashhad 9177948564, Iran
3
Department of Pharmacology, School of Medicine, Mashhad University of Medical Sciences, Mashhad 9177948564, Iran
4
Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad 917751365, Iran
2

Correspondence should be addressed to Ahmad Ghorbani; ghorbania@mums.ac.ir

Received 27 February 2014; Revised 17 June 2014; Accepted 4 August 2014; Published 28 August 2014
Academic Editor: Adair Santos
Copyright 2014 Hamid Reza Sadeghnia et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
In the present study, the cytotoxic and apoptogenic properties of hydroalcoholic extract and ethyl acetate (EtOAc), n-butanol, and
water fractions (0800 g/mL) of Viola tricolor were investigated in Neuro2a mouse neuroblastoma and MCF-7 human breast
cancer cells. In addition, antiangiogenic effect of EtOAc fraction was evaluated on chicken chorioallantoic membrane (CAM). The
quality of EtOAc fraction was also characterized using high performance liquid chromatography (HPLC) fingerprint. Cytotoxicity
assay revealed that EtOAc fraction was the most potent among all fractions with maximal effect on MCF-7 and minimal toxicity
against normal murine fibroblast L929 cells. Apoptosis induction by EtOAc fraction was confirmed by increased sub-G1 peak of
propidium iodide (PI) stained cells. This fraction triggered the apoptotic pathway by increased Bax/Bcl-2 ratio and cleaved caspase3 level. Moreover, treatment with EtOAc fraction significantly decreased the diameter of vessels on CAM, while the number of newly
formed blood vessels was not suppressed significantly. Analysis of quality of EtOAc fraction using HPLC fingerprint showed six
major peaks with different retention times. The results of the present study suggest that V. tricolor has potential anticancer property
by inducing apoptosis and inhibiting angiogenesis.

1. Introduction
Cancer is a devastating disease with tremendous negative
implications at the personal, health care, economical, and
social levels. It figures among the leading causes of death
worldwide, accounting for 8.2 million deaths in 2012 [1].
Excluding skin cancers, breast cancer is the most common malignancy and the second leading cause of cancer
death among women [2]. Breast cancer is a heterogeneous
disease encompassing multiple subgroups with differing
molecular signatures, prognoses, and responses to therapies.
Although, current treatment options for breast cancer are
moving toward nontoxic, potent targeted therapies that can
be tailored to an individual patients tumor, the development

of resistance to all of these therapies is an ongoing challenge

[3].
Neuroblastoma accounts for disproportionate morbidity
and mortality among the cancers of childhood. It is a complex
and heterogeneous disease and, despite recent advances, 50 to
60% of patients with high-risk neuroblastoma have a relapse,
and to date there are no salvage treatment regimens known
to be curative [4].
Phytochemicals from herbs are becoming increasingly
important sources of anticancer drugs or compounds for
cancer chemoprevention or adjuvant chemotherapy [5].
Recently, some chemopreventive extracts of herbs have
been shown to be antitumorigenic [6, 7]. The anticancer
effects have been shown to be mediated through inhibiting

2
cancer-activating enzymes, enhancing DNA repair processes,
immunomodulatory or antioxidant actions [8].
Viola tricolor, a member of Violaceae plant family, is common horticultural plant in Iran. It has been reported to have a
number of medicinal attributes including anti-inflammatory
[9], antimicrobial [10], antioxidant [11, 12], sedative [13],
and diuretic [14] activities. Recent studies have shown that
Viola tricolor contains cyclotide compounds with cytotoxic
properties [15]. In our previous preliminary works, we have
shown the cytotoxic activity of V. tricolor and its -butanol or
ethyl acetate fractions on neuroblastoma and uterine cervix
carcinoma cells [16, 17], but the exact mechanistic pathways
for this cytotoxicity were remained to be clear. In the present
study, the cytotoxic and apoptogenic properties of V. tricolor
in Neuro-2a mouse neuroblastoma and MCF-7 human breast
cancer, as well as normal murine fibroblast L929 cells, were
investigated. The possible inhibitory effect of V. tricolor on
angiogenesis in chicken chorioallantoic membrane was also
investigated. In addition, the quality of EtOAc fraction of
V. tricolor was characterized by high performance liquid
chromatography (HPLC) fingerprint.

2. Materials and Methods

2.1. Chemicals and Reagents. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium (MTT), doxorubicin, dimethyl
sulfoxide (DMSO), propidium iodide (PI), protease inhibitor
cocktail, phosphatase inhibitor cocktail, sodium citrate, Triton X-100, phenylmethylsulfonyl fluoride (PMSF), and bicinchoninic acid (BCA) protein assay kit were purchased from
Sigma (St. Louis, USA). Dulbeccos Modified Eagles Medium
(DMEM) and fetal bovine serum (FBS) were bought from
Gibco (Life technologies, Carlsbad, USA). Anti--actin, Bax,
Bcl-2, and horseradish peroxidase- (HRP-) conjugated goat
anti-rabbit IgG antibodies were obtained from Cell Signaling
Technology (Danvers, USA). All the solvents used for extraction were also purchased from Caledon (Canada).
2.2. Preparation of V. tricolor Extract and Its Fractions.
The V. tricolor aerial parts of the flowering plants were
collected from Pardis Campus (Mashhad, northeast of Iran)
and authenticated by the herbarium of School of Pharmacy
(Mashhad University of Medical Sciences, Iran; voucher
specimen number 12568). The plant materials were washed,
dried, powdered, and subjected to extraction with 70%
ethanol (EtOH/H2 O 70 : 30) in a Soxhlet apparatus for 48 h.
The hydroalcoholic extract (HAE) was then dried on a water
bath (45 C, 2 h) and the yield (32%) was kept at 20 C until
use.
For preparation of fractions, the dried hydroalcoholic
extract (10 g) was suspended in distilled water and transferred
to a separator funnel. With solvent-solvent extraction, it was
fractionated using ethyl acetate (EtOAc) and -butanol. The
ethyl acetate and -butanol fractions were then separated
to obtain water (H2 O) fraction [18, 19]. The solvents of
the fractions were then evaporated and the residues were
dissolved in phosphate buffered saline (PBS, pH 7.4) solution

BioMed Research International

containing 0.5% DMSO (for EtOAc and -butanol fractions)
or PBS alone (for water fraction).
2.3. Cell Culture and Treatment. The MCF-7, Neuro2a, and
normal L929 cells were cultivated in high-glucose DMEM
supplemented with 10% FBS and penicillin (100 units/mL)
and streptomycin (100 g/mL) at 37 C in an atmosphere
of 5% CO2 . Trypsin solution was used to passage cultures
whenever they were grown to about 70% confluence. The
cells at subconfluent stage were harvested from culture flask
and, after checking the viability with trypan blue exclusion
technique, they were seeded overnight in 96-well culture
plate. Then, to test the possible cytotoxicity of V. tricolor,
the culture medium was changed by fresh one containing
varying concentrations (0800 g/mL) of the HAE and
its fractions. Then, the cells were further incubated for
24 h.
2.4. MTT Assay. The effect of V. tricolor on MCF-7, Neuro2a,
and L929 cells proliferation was determined using MTT
colorimetric assay as previously described [20, 21]. Briefly,
at the end of treatment, the MTT solution was added to
each well of culture plate and the reaction mixture was
incubated for 2 h. Then, the mixture was removed and the
resulting formazan dissolved in DMSO. The optical density
of formazan dye was read at 570 and 620 nm (background)
using a StatFAX303 plate reader. All experiments were carried
out in triplicate.
2.5. PI Staining. MCF-7 and Neuro2a cells were seeded
overnight in 12-well culture plate (75000 cells/well) and
treated for 24 h with tested drugs. Then floating and adherent
cells were harvested and incubated with 750 L of a hypotonic
buffer (50 g/mL propidium iodide in 0.1% sodium citrate
containing 0.1% Triton X-100) at 4 C overnight in the dark
[22]. Samples were then analyzed with BD FACSCanto flow
cytometer (BD Biosciences, San Jose, CA). A total of 10,000
events per sample were obtained and the data was analyzed
using WinMDI (version 2.8) software. Three independent
experiments were performed.
2.6. Western Blotting Analysis. After treatment, the Neuro2a
cells were incubated with lysis buffer (50 mM Tris-Hcl,
150 mM NaCl, 2 mM EDTA, 5 mM sodium fluoride, 1 mM
NaVO4 , 1% Nonidet P-40, and protease and phosphatase
inhibitors) and centrifuged and the protein concentration
of the supernatants was measured using BCA kit. Equal
amounts of protein from samples were mixed with loading buffer and boiled for 5 min. Samples were separated
by electrophoresis, incubated in a blocking buffer (50 mM
Tris/HCl, 150 mM NaCl, 0.1% Tween 20, and 5% skimmed
milk) and the blots were probed with antibodies. The bound
antibody was made visible using HRP-conjugated goat antirabbit secondary antibody and an enhanced chemiluminescence system. Bands were analyzed using Gel Pro Analyzer
Software (Media Cybernetics) and normalized in respect to
corresponding -actin band and expressed as fold of control
[23].

BioMed Research International

2.9. Statistical Analysis. All results are presented as mean

standard error of the mean (SEM) of experiments performed
in triplicate. The Kolmogorov-Smirnov test was first performed to assess the normality assumption of the data. The
values were normally distributed and therefore compared
using the one-way analysis of variance (ANOVA) followed by
Tukeys post hoc test for multiple comparisons. The values
less than 0.05 were considered to be statistically significant.

3. Results
3.1. V. tricolor Extract and Its Fractions Induce Cell Death of
MCF-7 and Neuro2a Cells. Hydroalcoholic extract (HAE) of
V. tricolor and EtOAc, -butanol, and H2 O fractions were
examined for cytotoxic potential on MCF-7, Neuro2a, and
normal cells (L929). Cells were incubated with increasing
concentrations of the extract and its different fractions (0
800 g/mL) for 24 h. Results demonstrated that the extract
decreased cell viability in a concentration-dependent manner
(Figures 1, 2, and 3). As shown in Figure 1, the cytotoxic
potential of HAE was seen only at high concentration
(800 g/mL, < 0.01); on the other hand, cell survival was
not significantly affected by treatment of MCF-7 cells with
water fraction at all concentrations tested ( > 0.05). Among

100
80

60

40

20

0
0

50

100
200
Concentration (g/mL)

400

800

n-Butanol fraction
EtOAc fraction

HAE extract
Water fraction

Figure 1: Effects of Viola tricolor on proliferation of MCF7 cells. The

cells were treated with increasing concentrations of hydroalcoholic
(HAE) extract or its water, -butanol, or ethyl acetate (EtOAc)
fractions for 24 h. The percent of viable cells was normalized against
untreated control cells (0 g/mL). Data are mean SEM of three
independent experiments performed in triplicate. < 0.01 and

< 0.001 versus untreated control cells.

120
100

Cell viability (%)

2.8. Characterization of the EtOAc Fraction of V. tricolor

by HPLC. The quality of EtOAc fraction of V. tricolor was
characterized by HPLC-UV fingerprint [25]. The Waters
HPLC (Waters Association, Milford, MA, USA) apparatus
consisted of a model 510 Waters pump, a 20 L Rheodyne
7725 injector, and a variable wavelength model 486 Waters
UV-VIS detector. The chromatograms were analyzed using
Autochoro 3000 data module (Young Lin Instruments, South
Korea). The chromatographic separation was carried out with
a reverse-phase Waters C18 analytical column (250 4.6 mm,
5 m particle size). An isocratic elution was performed by
the mobile phase of 20% acetonitrile and 80% phosphoric
acid (0.085%, pH = 2.2) at a flow rate of 1 mL/min. The UV
detector wavelength was set at 340 nm. A sample of the EtOAc
fraction was dissolved in mobile phase and passed through
0.45 m membrane filter. Then, 20 L of sample (500 g/L)
was injected into the HPLC column.

120

Cell viability (%)

2.7. Chicken Chorioallantoic Membrane (CAM) Angiogenesis

Model. Fertilized chicken eggs were incubated at 37 C and
70% relative humidity in a forced draught incubator. At day
8, a 1.52 cm window was opened aseptically on each egg
shell, exposing the part of the CAM which contained the
central vein and 20 or 40 g/egg of the ethyl acetate fraction
was injected into the chorioallantoic sac. The control eggs
received the same volume sterile PBS only. Then, the windows
were sealed with sterile Parafilm and the eggs were then
returned to the incubator. At day 12, the seals were removed
and the CAM vasculatures were photographed using a stereo
microscope equipped with a digital camera (Canon EOS 40D
with Canon EF 100 mm f/2.8 USM lens). The angiogenic
response was evaluated by counting the vessel density using
ImageJ software [24].

80

60
40
20

0
EtOAc (g/mL) 0

50

100

200

400

800

Dox (10 g/mL)

Figure 2: Effects of ethyl acetate (EtOAc) fraction of Viola tricolor

on proliferation of Neuro2a cells. The cells were treated for 24 h
and then percent of viable cells (quantified by MTT assay) was
normalized against untreated control cells (0 g/mL). Data are mean
SEM of three independent experiments performed in triplicate.

< 0.01 and < 0.001 versus untreated control cells.

the fractions, EtOAc fraction showed the most cytotoxic

effects on cancer cells, but limited toxicity on normal cells
(Figures 1, 2, and 3). Significant inhibition (about 50%) of
cell proliferation by EtOAc was seen at concentration of
200 g/mL ( < 0.001) in MCF-7 cells (Figure 1). Compared
to Neuro2a cells, MCF-7 cells were found to be more sensitive
to cytotoxic effects of the EtOAc fraction. As illustrated

BioMed Research International

acts by downregulating Bcl-2 and upregulating Bax protein
expression in Neuro2a cells (Figures 5(a) and 5(c)).
In addition, enhanced caspase-3 level following treatment of Neuro2a cells with EtOAc fraction of V. tricolor
(400 g/mL) was seen, indicating caspase-dependent apoptosis (Figure 5(b)).

120

Cell viability (%)

100
80
60
40
20

0
0

100

200
400
Concentration (g/mL)

800

HAE extract
EtOAc fraction

Figure 3: Effects of Viola tricolor hydroalcoholic (HAE) extract

and its ethyl acetate (EtOAc) fractions on proliferation of L929
cells. The cells were treated for 24 h and then percent of viable
cells (quantified by MTT assay) was normalized against untreated
control cells (0 g/mL). Data are mean SEM of three independent
experiments performed in triplicate. < 0.001 versus untreated
control cells.

in Figure 2, although signs of toxicity in Neuro2a cells

(about 25% inhibition in cell proliferation) were observed
at concentration of 200 g/mL ( < 0.01), the greatest
effect appeared only at higher concentrations (800 g/mL,
< 0.001). As shown in Figure 3, cell growth and survival
were not significantly affected by treatment of L929 with
concentrations up to 400 g/mL of HAE and its EtOAc
fraction ( > 0.05, after 24 h of treatment). The data also
indicated that treatment of Neuro2a cells with doxorubicin
(10 g/mL) for 24 h resulted in significant inhibition of cell
viability, as compared with control ( < 0.001) (Figure 2).
3.2. EtOAc Fraction of V. tricolor Induces Apoptosis of MCF7 and Neuro2a Cells. To determine if apoptosis is involved in
the cytotoxic effects of EtOAc fraction of V. tricolor, apoptotic
cells were determined by PI staining of DNA fragments
by flow cytometry (sub-G1 peak). Cells were exposed to
various concentrations (0, 100, 200, and 400 g/mL) of EtOAc
fraction for 24 h. V. tricolor treatment of the cancer cell lines
significantly increased the sub-G1 peak with a concomitant
decrease in G1 phase, compared to the untreated control cells
(Figures 4(a) and 4(b)).
3.3. EtOAc Fraction of V. tricolor Increases the Cleaved
Caspase-3 and Bax/Bcl-2 Ratio. The increase in the active
form of caspase 3 and Bax/Bcl-2 ratio were used as an
indicator of apoptosis. B-cell lymphoma-2 (Bcl-2) and Bcl2 associated X protein (Bax), members of the Bcl-2 family of
proteins, are antiapoptotic and proapoptotic factors, respectively. The ratio of Bax/Bcl-2 proteins plays a pivotal role
in controlling cytochrome c release and apoptosis initiation
via the mitochondrial (intrinsic) pathway [26]. Our results
indicated that EtOAc fraction of V. tricolor (400 g/mL)

3.4. EtOAc Fraction of V. tricolor Inhibits CAM Angiogenesis.

To address whether V. tricolor inhibits angiogenesis, we
examined the effect of EtOAc fraction of V. tricolor on CAM
angiogenesis (Figures 6(a), 6(b), 6(c), 6(d), 6(e), and 6(f)). As
shown in Figures 6(e) and 6(f), 40 g/egg of EtOAc fraction
significantly decreased the diameter of vessels, while the
number of newly formed blood vessels was not suppressed
significantly, as compared to control CAM.
3.5. HPLC Profile of EtOAc Fraction of V. tricolor. A simple and reliable HPLC fingerprint has been developed for
qualification of the EtOAc fraction of V. tricolor. HPLC
profile of EtOAc fraction under UV 340 nm was recorded.
The corresponding HPLC chromatogram was presented in
Figure 7. The fraction revealed 6 major peaks with retention
time (RT) values in the range of 2.1 to 8.3 min for 20 L
application volume (Figure 7).

4. Discussion
Heartsease (Viola tricolor L.) has a long history in treating
inflammatory and skin disorders including scabs, itching,
ulcers, eczema or psoriasis, and bronchitis or asthma [27, 28].
In the current work, we have studied the cytotoxic, apoptotic,
and antiangiogenic activities of hydroalcoholic extract and
EtOAc, -butanol, and water fractions of V. tricolor on MCF7, Neuro2a, and L929 cells. Cytotoxicity assay revealed that
EtOAc fraction was the most potent among all the fractions
with maximal effect on MCF-7 cells and minimal toxicity
against normal cells. Apoptosis induction of EtOAc fraction
(400 g/mL) was confirmed by increase in the sub-G1 peak
of PI stained cells and western blot analysis of Bcl-2, Bax,
and active form of caspase 3, as important proteins involved
in the apoptotic cell death. Our results also showed that
the diameter, but not the number, of blood vessels was
significantly decreased by EtOAc fraction on CAM.
In this study, the V. tricolor hydroalcoholic extract was
fractionated by solvent extraction with different polarity and
the potential antitumor activity of low-polar solvent fraction
(EtOAc) was compared to polar solvent fractions (-BuOH
and H2 O). It was found that EtOAc fraction had the greatest
antiproliferative activity in vitro. The effect of EtOAc fraction
on nonmalignant cells showed a degree of specificity for
malignant cell lines.
Pharmacognostic researches on V. tricolor confirmed the
presence of high amount of saponins, mucilages, flavonoids,
and phenolic compounds such as kaempferol, luteolin,
quercetin, violanthin, and rutin, which placed V. tricolor in
the list of plants with promising source of natural antioxidants [11, 12, 14]. In addition, bioactive plant cyclopeptides
with intermediate polarity especially vitri A, vitri F, and

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256

Gm: 21.35
Control
CV: 87.05
[10-705] 522 (4.8%)

128

Gm: 40.89
CV: 65.36
[10-705] 1580 (18.0%)

M1

Events

M1

0
100

101

102
FL2-H

103

64

100 g/mL

0
104 100

101

102
FL2-H
60

32

200 g/mL
Gm: 20.05
CV: 78.90
[10-705] 1342 (19.2%)

Gm: 22.42
CV: 61.33
[10-705] 4450 (54.2%)

400 g/mL

M1

M1

0
104 100

103

101

102
FL2-H

103

0
104 100

101

102

103

104

FL2-H

Apoptotic cells (%)

50
40
30
20

100

200

10
0

400

EtOAc fraction (g/mL)

(a)
Gm: 187.74
Control
CV: 38.59
[10-728] 2404 (13.3%)

Events

256

256

M1

100

101

102
103
FL2-H

128

Gm: 90.82
100 g/mL
CV: 51.05
[10-728] 2645 (15.7%)

101

Gm: 19.60
400 g/mL
CV: 67.33
[10-728] 4772 (40.8%)

M1

M1

0
104 100

32

Gm: 11.61
200 g/mL
CV: 90.30
[10-728] 3083 (25.0%)

102
103
FL2-H

104

100

101

102
103
FL2-H

M1

104

0 0
10

101

102
103
FL2-H

104

60

Apoptotic cells (%)

50

40
30
20
10
0

100

200

400

EtOAc fraction (g/mL)

(b)

Figure 4: Effects of ethyl acetate (EtOAc) fraction of Viola tricolor on apoptosis of MCF7 (a) and Neuro2a (b) cells. The cells were treated
for 24 h and then incubated with a hypotonic buffer containing propidium iodide and Triton X-100. Then, the cells were analyzed with a flow
cytometer. Data are mean SEM of three independent experiments performed in triplicate. < 0.05 and < 0.001 versus untreated
cells (0 g/mL).

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Bax

26 kDa

Bcl-2

21 kDa

17 kDa

Cleaved caspase-3

46 kDa

-Actin

Control

EtOAc fraction
(400 g/mL)

(a)

2.5

Bax/Bcl-2 ratio
(fold of control)

Cleaved caspase-3
(fold of control)

2.0

1.5
1.0
0.5

EtOAc fraction (400 g/mL)

Control

0.0

EtOAc fraction (400 g/mL)

Control

(b)

(c)

Figure 5: Effects of ethyl acetate (EtOAc) fraction of Viola tricolor on proapoptotic (Bax, caspase-3) and antiapoptotic (Bcl-2) proteins
expression in Neuro2a cells (a). The cell lysates were immunoprecipitated with anti-Bax, anti-Bcl-2 and anti-cleaved caspase-3 antibodies
and then immunoblotted using horse radish peroxidase-conjugated goat anti-rabbit secondary antibody with -actin as a protein loading
control. Data are presented as the fold induction over control cells ((b)-(c)). < 0.01, < 0.001 versus untreated control cells.

cycloviolacin O2 are reported to have cytotoxic activity and

are interesting candidates for drug development [29]. Phytochemicals with polyphenolic or flavonoid structure such
as kaempferol, luteolin, and resveratrol have been reported
to induce cancer cell death or to inhibit cancer cell proliferation by direct modulation of various molecular signal
transduction pathways [30]. Because we used ethyl acetate
fraction, it seems that intermediary polar constituents such
as flavonoids and phenolic compounds are mainly involved
in the cytotoxic activity of V. tricolor. The construction
of chromatographic fingerprints plays an important role in
the quality control of complex herbal medicines. Chemical
fingerprints obtained by chromatographic techniques are
strongly recommended for the purpose of quality control of
herbal medicines, since they might represent appropriately
the chemical integrities of the herbal medicines and therefore
be used for authentication and identification of the herbal
products [31]. In our chromatographic technique, in order
to obtain a good resolution within a short analysis time, the
composition of mobile phase was optimized. Various mobile
phase compositions were evaluated. Acetonitrile and water
containing phosphoric acid were chosen as the mobile phase
because all peak components could be resolved under this
condition. The acidification of mobile phase was beneficial,
leading to good peaks separation and better peak shape. The

HPLC fingerprint showed high stability and reproducibility

and thus could be used for quality control of the EtOAc
fraction and Viola products.
Current research on V. tricolor revealed new aspect of
pharmacological properties of the herb. For example, bioactive cyclotides of V. tricolor has been demonstrated to possess
inhibitory activity on proliferation of activated lymphocytes
which may be beneficial in the therapy of disorders related
to an overactive immune system [27]. Piana et al. showed
the antinociceptive and anti-inflammatory activities of V.
tricolor in the ultraviolet-B-induced skin burn [32]. There are
also some researches which verified the anti-inflammatory
activity of V. tricolor in traditional medicine [9, 33].
The crucial role of angiogenesis in tumor growth is well
documented [34]. While new blood vessel formation on
CAM was not suppressed by V. tricolor, a significant decrease
in the diameter of vessels was seen. To our knowledge, it is
the first time that an inhibition of angiogenesis on CAM by
V. tricolor has been studied.

5. Conclusion
Taken together, the result of present study showed that
ethyl acetate fraction of V. tricolor has potential cytotoxic

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(a)

(b)

160

120
100
80
60
40
20

0.8
0.6

0.4
0.2
0

20

40

EtOAc fraction (g/egg)

20

40

Diameter of small vessels (cm)

Diameter of large vessels (cm)

Number of vessels (%)

140

(c)

0.5

0.4

0.3
0.2
0.1
0

EtOAc fraction (g/egg)

(d)

0
20
40
EtOAc fraction (g/egg)

(e)

(f)

Figure 6: Effect of ethyl acetate (EtOAc) fraction of V. tricolor on the number and diameter of vessels in chorioallantoic membrane (CAM).
Fertilized eggs were incubated at 37 C and 70% relative humidity in a forced draught incubator. The control eggs received sterile PBS only
(a). At day 8, a window opening is punctured on each egg and 20 (b) or 40 g/egg (c) of the ethyl acetate fraction was injected into the
chorioallantoic sac. 1, 2, and 3 are representative of heart, artery, and arterioles, respectively (magnification 10x). In the presence of 40 g/egg
of ethyl acetate fraction, the diameter of vessels decreased significantly while the density of blood vessels did not change significantly ((d)(f)).
Data are mean SEM of three independent experiments performed in triplicate. < 0.05 versus untreated cells (0 g/egg).

in experimental animal models to evaluate the potential

anticancer properties of V. tricolor.

3.50

Voltage (mV)

3.00

Conflict of Interests

2.50

The authors declare that there is no conflict of interests

regarding the publication of this paper.

2.00
1.50

Acknowledgments

1.00
0.50
0.00

2.00

4.00
6.00
Time (min)

8.00

10.00

Figure 7: Representative HPLC chromatogram of ethyl acetate

(EtOAc) fraction of V. tricolor. The peaks were monitored at 340 nm.

The authors would like to thank Mr. M. Malaekeh for his

assistance in flow cytometry. This work was supported by
a Grant (no. 910331) from Vice Chancelor for Research
and Technology of Mashhad University of Medical Sciences
(MUMS), as a part of Pharm. D. thesis presented by Taghi
Ghorbani Hesari.

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