Nanoliposomal formulation of Agrostemma githago aqueous extract shows enhanced cytotoxic effect on gastric cancer cell line
The objective of this study was to determine the cytotoxic effects of nanoliposomal form of lyophilized aqueous extract of Agrostemma githago (A. githago) seeds on gastric cancer cell line (AGS) using cell viability tests. Materials and Methods:
Lyophilized aqueous extract of A. githago seeds was prepared. Liposomes were also prepared by thin-film hydration method and their stability and size were characterized by SEM. The size and zeta potential were determined by Malvern Zetasizer. Cytotoxic effects of nanoliposomes on gastric cancer cell line was determined using MTT, Neutral Red and Frame methods. Results:
The size of liposomes was around 171.5 nm with proper dispersion (PDI=0.268). The morphology of the liposomes was suitable according to SEM images. The IC50 values indicated that the nanoliposomal form of extract was 3-4 times more active than extract alone. Average IC50 values for extract and liposomal form of extract were 13.02 ± 0.95 and 4.43 ± 1.49 ug/ml, respectively. Conclusion:
This study showed that liposomal form of aqueous extract of A. githago seeds exerts cytotoxic effect at significantly lower concentrations than the extract itself.
Published on: Mar 3, 2016
Transcripts - Nanoliposomal formulation of Agrostemma githago aqueous extract shows enhanced cytotoxic effect on gastric cancer cell line
Please cite this paper as:
Bohlooli Sh, Fathi P. Nanoliposomal formulation of Agrostemma githago aqueous extract shows enhanced
cytotoxic effect on gastric cancer cell line, Nanomed J, 2015; 2(1): 21-28.
Received: Jun. 25, 2014; Accepted: Sep. 4, 2014
Vol. 2, No. 1, Winter 2015, page 21-28
Received: Apr. 22, 2014; Accepted: Jul. 12, 2014
Vol. 1, No. 5, Autumn 2014, page 298-301
Online ISSN 2322-5904
Nanoliposomal formulation of Agrostemma githago aqueous extract shows
enhanced cytotoxic effect on gastric cancer cell line
Shahab Bohlooli1, 2*
, Parisa Fathi1
Department of Pharmacology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran
Drug and Advanced Sciences Research Center, School of Pharmacy, Ardabil University of Medical Sciences,
Objective(s): The objective of this study was to determine the cytotoxic effects of
nanoliposomal form of lyophilized aqueous extract of Agrostemma githago (A. githago) seeds
on gastric cancer cell line (AGS) using cell viability tests.
Materials and Methods: Lyophilized aqueous extract of A. githago seeds was prepared.
Liposomes were also prepared by thin-film hydration method and their stability and size were
characterized by SEM. The size and zeta potential were determined by Malvern Zetasizer.
Cytotoxic effects of nanoliposomes on gastric cancer cell line was determined using MTT,
Neutral Red and Frame methods.
Results: The size of liposomes was around 171.5 nm with proper dispersion (PDI=0.268).
The morphology of the liposomes was suitable according to SEM images. The IC50 values
indicated that the nanoliposomal form of extract was 3-4 times more active than extract
alone. Average IC50 values for extract and liposomal form of extract were 13.02 ± 0.95 and
4.43 ± 1.49 ug/ml, respectively.
Conclusion: This study showed that liposomal form of aqueous extract of A. githago seeds
exerts cytotoxic effect at significantly lower concentrations than the extract itself.
Keywords: Agrostemma githago, Cytotoxicity, Gastric cancer, Nanoliposome
*Corresponding Author: Shahab Bohlooli, Department of Pharmacology, School of Pharmacy, Ardabil
University of Medical Sciences, Ardabil, Iran.
Tel.: +98-45-33523833, E-mail: email@example.com
Nanoliposomal formulation of Agrostemma githago
22 Nanomed J, Vol. 2, No. 1, Winter 2015
Agrostemma githago L. also known as
Corn Cockle which is usually found as
weed in grain fields is known to be toxic
for both human and animals (1, 2). Its seed
extract contains saponins, agrostin,
githagin, githagenin and agrostemmic acid
(3, 4). Studies elsewhere have exhibited
that this plant has cytotoxic activity (5)
related to a triterpenoid saponin,
githagenin and a ribosome-inactivating
protein (RIP), agrostin (6, 7). Agrostin is a
type 1 RIP and combination with saponin
augment its cytotoxic activity (5). This
plant also contains flavonoids with
cytotoxic (8-10) and antioxidant effects
(11-13). Liposomes are vesicles, having
stable membranes composed of two layers,
firstly were described by British
hematologist Dr. Alec D Bangham FRS in
1961 (14) and because of their specific
properties have become one of the most
important drug carriers (15).
Nanoliposomes are nanoscale liposomes
that are highly interesting
pharmacologically because of their small
size, high stability and therefore good
tendency for application as drug carrier
systems and have widely used as carrier
agents in drug delivery systems for cancer
chemotherapy (16-18). It is known that
nanoparticles have a high tendency to be
trapped in the reticuloendothelial system,
such as spleen, liver and lung (19).
Using this phenomenon, nanoliposomes
could be targeted toward tumor tissues as a
strategy for cancer therapy (20).
On the other hand, it is known that the size
of liposome is highly related to the
stability and encapsulated solute leakage.
The smaller the size, the more the stability
and less the encapsulated solute might leak
out (21). Numerous studies have
demonstrated that both hydrophilic and
lyophilic chemicals can be encapsulated on
liposomes because of their amphipathic
characteristics. Encapsulation of plant or
animal extracts on liposomes and their
application for different purposes such as
cancer therapy have been studied.
It was shown that liposomes have suitable
ability for entrapment and carrying not
only the pure substances, but also the
whole extract of a plant or animal (22-24).
A. githago is used as traditional medicine
by Turkish people (25) and also people of
north west of Iran for treatment of some
diseases. Although there are some studies
about cytotoxicity of this plant extract (5),
no study has been performed using
liposome encapsulated extract. This plant
is widely grown in Ardabil province, Iran.
Therefore, in this study we investigated in
vitro cytotoxic activity of nanoliposomal
form of lyophilized extract of A. githago
on AGS cell line.
Materials and Methods
All solvents and chemicals were of
analytical grade. Lecithin from soybean
type IV-SL-α-phosphatidylcholine (PC)
was obtained from Sigma Chemical Co.
Cholesterol and Rutin were purchased
from Merck and used without any further
A. githago seeds from suburb area of
Ardabil province, North West of Iran were
used for preparation of extract. Seeds were
collected during the local harvesting
season when the seeds were at their best
condition and complete ripeness.
Preparation of A. githago lyophilized
Collected seeds were dried and grinded to
make fine powder. Definite amount of
powder was added into the distilled water
and thoroughly mixed and homogenized
using an ultrasonic homogenizer
(UP200H, Hielscher, Germany) in
maximum sonication level for complete
cycle of 15 min. Subsequently the mixture
was centrifuged and supernatant was
collected and allowed to freeze at −80 o
Afterward, the frozen extract lyophilized
by Freeze dryer (ALPHA 2-plus, Martin
Christ, Germany) and the residue stored at
−20 °C for later use.
Bohlooli S, et al
Nanomed J, Vol. 2, No. 1, Winter 2015 23
Original Research (font 12)Preparation of liposomes
The thin-film hydration method was used
for preparation of nanoliposomes (26-28).
Lecithin phospholipid (L-α-phosphatidyl-
choline) was dissolved in chloroform to
form the first solution. Cholesterol was
also dissolved in chloroform to make the
second solution. Tow solutions were
combined and firmly mixed using a rotator
in a final ratio of 4:1 (w/w), respectively.
This mixture was evaporated in a rotary
evaporator system (Heidolph Germany)
connected with a vacuum system and was
kept under the lipid transition temperature
(TC). Evaporation was performed under
the atmosphere of nitrogen to prevent
oxidation of phospholipids. This process
was performed during 2 hours to remove
whole solvents and the result was a thin
film. The powder extract of plant was
dissolved in distilled water and resultant
solution was added to the film container.
To improve the hydration process, glass
beads (0.5 mm) were added and the
mixture was stirred until the film was
disappeared. For homogenizing of
suspension and producing the nanoscale
vesicles, obtained sample was sonicated
for 15 minutes using ultrasonic
homogenizer (UP200H, Hielischer, Germ-
any). Homogenized suspension was taken
under nitrogen atmosphere and was held
under lipid transition temperature for 1 h.
Then, product was centrifuged using a
centrifuge (sigma 3- 30k, Germany) to
yield a clear suspension of extract
contained nanoliposomes and stored at
4°C until use.
Characterization of nanoliposomes
Polydispersity index (PdI), hydrodynamic
diameter (size), and zeta potential of
nanoliposome were determined by photon
correlation spectroscopy using a Malvern
Zetasizer (Nano ZS nanoseries, Malvern
Instruments, UK). Size, PDI, and zeta
potential were obtained at 25 o
C using the
water as dispersant agent. The data were
analyzed using the Malvern software. The
morphology of the prepared liposomes was
determined using Scanning Electron
microscopy, SEM (LEO 1430VP,
Germany and UK).
Determination of encapsulated amount of
A. githago extract contains some
flavonoids that were used to determine the
entrapment amount on nanoliposomes as
an indicator for efficiency of whole extract
UV–Vis absorption spectra on 415 nm at
C were measured using a
spectrophotometer (PG-Instrument LTD,
England) for determination of the total
flavonoids loaded on nanoliposomes.
Rutin was used as reference flavonoid
The total entrapment amount of plant
extract on nanoliposomes was calculated
by following formula:
flavoind total − flavonoid remained
The human gastric cancer cell line, AGS,
was provided from Pasteur Institute
(Tehran, Iran). AGS cells were grown as
monolayer culture at 37°C in RPMI-1640
medium (GIBCO, UK) containing 10%
(v/v) fetal bovine serum (FBS; GIBCO,
UK), pencillin (100 IU/ml) and
streptomycin (100 μg/ml). Incubation was
performed in a humidified atmosphere of
5% CO2 in air until
confluence. Cells were plated in 96-well
plates at a density of 1×104
cells per well
in 200 μl medium. After incubation for
overnight, the medium was removed and
cells were treated with a FBS-free medium
supplemented with 10 mg/ml prepared
extract-loaded nanoliposomes or
nanoliposomes (without extract) and or
extract (without nanoliposomes), by 1/4
serial dilutions in separate plates.
Afterward, plates were incubated for 24 h.
Nanoliposomal formulation of Agrostemma githago
24 Nanomed J, Vol. 2, No. 1, Winter 2015
Cytotoxicity studies by MTT assay
For performing the MTT (3-(4,5-
lium bromide) assay. Four hours before
completion of the incubation time, the
medium of each well were replaced with
180 μl FBS-free medium and 20 μl of 2.5
mg/ml MTT (Merck, Germany). Finally,
the medium were removed and 200 μl
DMSO was added to each well. The plates
were shaken gently for 10 min by a shaker
and the absorbance was measured by a
plate reader (Synergy HT, BioTek) at 570
nm. The wells without cells were treated
as blank (30).
Neutral red assay
Neutral Red Uptake Assay was carried out
as stated in our former study (31). Briefly,
three hours before the end of the
incubation period, the medium was
aspirated from each well. Immediately, the
working neutral red solution (0.05% of the
neutral red stock in cell culture medium
without serum) was added to each well and
incubated for 3 hours. At the end of
incubation time, the neutral red solution
was removed and the wells were washed
with warm PBS (37 ºC). Subsequently,
fixative solution (150 μl) was added to
each well and the absorbance was
measured at 540 nm on the plate reader.
Protein content measurement
FRAME (Fund for the Replacement of
Animals in Medical Experiments) method
was used for the determination of protein
content using the Comassie protein assay
reagent as described previously (31).
Briefly, after neutral red uptake assay,
fixative solution was removed from each
well and working stain solution (150 μl)
was added to the wells and the plates were
shaken for 20 min.
Then, the stain was removed and wash
solution (250 μl) was added to each well.
Wells were washed for two times and
desorbing solution (150 μl, potassium
acetate, 1M, in 70% ethanol) was placed
into the wells. Plates were shaken on a
shaker vigorously until a homogenous
solution was observed. The absorbance
was measured at 595 nm using the plate
Statistical data analysis
Half Maximal Inhibitory Concentration
(IC50) values were calculated using Sigma
Plot 11 software. Measurements were
triplicate (n = 3 per experimental group)
and differences among treatment groups
were assessed by Student’s t-test.
Differences were considered significant at
confidence limits of p < 0.05. Data values
for the cell viability study are presented as
mean ± standard deviation (SD).
Results and Discussion
Photon correlation spectroscopy
As it was shown in figure 1, zeta potential
of prepared liposomes was -53 mV. Zeta
potential value greater than 30 mV
(negative or positive) is related with in
vitro stability due to the electric repulsion
between particles (32) and longer half-life
of vesicles in intravascular system (16).
Also, it was shown that negative value of
zeta potential has a positive correlation
with increased entrapment of water soluble
pharmaceuticals, which is maximal at – 30
mv (33). The average size of liposomes
was 171.5 nm (figure 2) which was
considered nanoscale based on IUPAC
definition (34). PDI value of prepared
liposomes was 0.268 (figure 2). It has been
stated elsewhere that PDI values less than
0.3 is indicative of a good monodispersity
of vesicles (32).
Scanning electron microscopy (SEM)
Scanning electron micrographs of prepared
liposomes exhibited the size of liposomes
that are in nanoscale and this is in
agreement with the results of Zetasizer.
Also the morphology and size
homogeneity of liposomes were in a range
compatible with PDI value.
The loaded amount of extract in liposomes
Bohlooli S, et al
Nanomed J, Vol. 2, No. 1, Winter 2015 25
Original Research (font 12)
Figure 1. Zeta potential of encapsulated liposomes.
Figure 2. Size and PDI of encapsulated liposomes.
Figure 3. MTT assay of cytotoxic activity of nanoliposomal extract of A. githago on AGS cell line after 24 h
compared to base extract. Data are presented as mean ± SD. (n=3). (* p < 0.0001 vs. base extract).
Figure 4. Neutral red assay of cytotoxic activity of nanoliposomal extract of A. githago on AGS cell line after
24 h compared to base extract. Data are presented as mean ± SD (n=3). (* p < 0.0001 vs. base extract).
Zeta Potential Distribution
Size Distribution by Volume
Zeta potential (mV)
Nanoliposomal formulation of Agrostemma githago
26 Nanomed J, Vol. 2, No. 1, Winter 2015
Figure 5. Frame assay of cytotoxic activity of nanoliposomal extract of A. githago on AGS cell line after 24 h
compared to extract. Data are presented as mean ± SD (n=3). (* p < 0.0002 vs. base extract).
This value is assumed satisfactory when
compared to previously published data on
liposomal drug delivery systems of
compounds from herbal origin (35).
The results of viability studies are shown
in figures 3-5. The results demonstrate that
encapsulated A. githago extract shows
significant cytotoxic effect in comparison
with the base extract. It could be
concluded that the cytotoxic effect of
liposomal form of A. githago extract
on AGS cell line was 3 to 4 times more
than the free extract. During the
experiments the liposomes without extract
were used as control.Table 1 shows the
IC50 values calculated from MTT, neutral
red and frame methods after 24 h
By comparing the IC50 values
for extratct and encapsulated extract, it
seems that the prepared nanoliposomes are
efficient for delivering the active
ingredients of the extract to AGS cell line.
The two most important compounds of A.
githago that have cytotoxic effect are
agrostin and agrostemma saponin 1
(githagenin). It is known that when these
two compounds are used in combination,
the toxicity effect enhances by 10,000 fold
over that of each (5). The role of saponin
is to facilitate the penetration of the non-
permeable agrostin through the cell
membrane (36). From this point of view, it
could be postulated that liposomal
encapsulation boosts saponin ability in
enhancing the agrostin entrance into the
Table 1. IC50 values of base and nanoliposomal extract of A. githago on gastric cancer cell line after 24 h.
Samples MTT assay
Neutral Red assay
Base extract (control)
13.26 ± 1.31
4.3 ± 0.64*
15.29 ± 0.94
5.6 ± 0.59**
10.53 ± 0.61
3.41 ± 0.79***
13.02 ± 0.95
4.43 ± 1.49
Data are presented as mean ± S. D. (n=3).
*p < 0.0002 vs. control **p < 0.0003 vs. control ***p < 0.0003 vs. control
On the other hand, investigations
confirmed that encapsulation of bioactive
ingredients in nanoliposomes may enhance
their cellular uptake and offer some
advantages such as high metabolic
stability, high membrane permeability,
improved bioavailability, and longer
duration of action (37).
Bohlooli S, et al
Nanomed J, Vol. 2, No. 1, Winter 2015 27
Original Research (font 12)Conclusion
Nanoliposomal form of lyophilized extract
of A. githago was prepared and
characterized by photon correlation
spectroscopy and scanning electron
microscopy. Results showed that the
liposomes were in nanosized scale with
good stability and dispersion. Cytotoxic
effect of liposomes on AGS cell line which
was determined by MTT, Neutral Red and
Frame assays was significantly more than
We gratefully acknowledge the Pasteur
Institute, Tehran, Iran for providing the
zeta analysis and Ardabil University of
Medical Sciences for financial support.
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