Archives of

1. Introduction
Breast cancer is the most common cancer in women and one of the most common causes of cancer-related deaths in women around the world [1,2]. Chemotherapy, radiation therapy, and surgery are the main treatment options for breast cancer. Drug therapy is one of the options for confrontation cancer. However, since drug products still do not penetrate the tumor site at sufficient levels, modified drug therapy increases systemic side effects and reduces pharmacokinetic effects. Chemotherapy, radiation therapy, and surgery are the main treatment options for breast cancer [3]. Different variables such as hereditary components, lifestyle, and living environment influence the incidence of this disease [4]. Breast cancer is caused by uncontrolled development and growth of cells in the breast [5]. Breast cancer is a complex disease caused by polygenic factors, and various pathogens differently contribute to the chance of expanding the cancer. [6]. Nanotechnology increases the success of chemotherapy and reduces the side effects of breast cancer treatment [7].
This technology includes synthesizing and applying materials with sizes ranging from 1 to 100 nm [8]. Nanoscale metals are broadly utilized in numerous fields such as environment, medicine, and engineering [9]. Nanoscale metals are basically synthesized by chemical methods, which have undesirable impacts such as environmental contamination, high energy consumption, and potential health issues. Green synthesis, which employs plant extracts, has been developed in response to these challenges. Green synthesis is more useful than conventional chemical synthesis since it costs less, decreases contamination, and improves environmental and human health safety [9].
However, the main concern regarding the biogenic regeneration (materials produced by living organisms) of metal forerunners to deliver the comparing nanoparticles (NPs) is related to their purity. Biogenic decrease could be a “bottom-up” approach comparable to chemical decrease in which a diminishing operator is supplanted with an extricate of a normal item with inalienable stabilizing, growth-terminating, and coating properties. Additionally, the nature of biological entities at distinctive concentrations in combination with organic reducing agents influences the size and shape of NPs [8]. Clover (Trifolium repens L) is an annual plant that grows well in the semi-arid conditions of the Mediterranean region [10]. Trifolium species, commonly known as clover species, have a worldwide distribution and are used by
Cytotoxic Effects of Green Synthesis of Zirconium Nanoparticles of Aqueous Clover Extract on Breast Cancer Green synthesis of zirconium nanoparticles by aqueous clover extract
communities around the world as a food and medicine
[11]. Trifolium species are rich in bioactive compounds
that have medicinal value and this together with potential
nutritional value can improve their dietary properties
[12]. These plants contain a wide range of bioactive
compounds called secondary metabolites such as
phenolic acids, alkaloids, flavonoids, terpenoids, amino
acids, alcoholic compounds, glutathione, polysaccharides,
anti-cancer agents, organic acids (ascorbic, oxalic, malic,
tartaric, and protocatechuic acids), and quinones. It is by
and large known that these metabolites are included in
redox response forms [13]. They are responsible for the
lessening of metal particles to metal NPs. Even though
the components included within the green blend of NPs
and the fundamental instrument of particle bioreduction
are still not fully understood [14]. The association and
support of auxiliary metabolites (sugars, terpenoids,
polyphenols, alkaloids, phenolic acids, and proteins)
within the diminishment of metal particles that lead to the
arrangement of NPs and in supporting their consequent
soundness have moreover been expected [15]. Zirconium
(Zr) is classified as a transition metal (d-block) in the
titanium family (group IV) with atomic number 40 [16].
Zirconium dioxide (ZrO2) or zirconia is one of crystalline
oxides of zirconium [17]. Due to its very high steadiness
and exceptionally harmfulness, ZrO2 shows a wide range
of applications for heat-resistant ceramic superalloys [18],
dental restorations [19], fuel cells [20], heterogeneous
catalysis [21], and cancer treatments [22,23]. Such
promising applications make ZrO2 an ideal nanomaterial,
promoting a green technique for the synthesis of ZrO2NPs
[24].
This study aimed to measure the ability of metallic
ZrNPs synthesized by the aqueous extract of clover leaves
and investigate the cytotoxic effects of synthesized NPs on
normal cell line (HFF) and breast cancer cell line (MDAMB-
468).
2. Materials and Methods
2.1. Production of aqueous extract of clover (Trifolium
repens L) plant
The clover plant was collected in the middle of April
(2023) from Sarieh Khatun village (Qom, Iran) and
kept at a temperature of 4 °C. To prepare the aqueous
extract from the clover plant, the leaves of the plant were
separated from the root and stem. Then, the leaves were
washed with distilled water and 70% ethanol was used to
remove possible contamination. Afterwards, they were
dried in the shade at room temperature. In the next step,
the dried plant was crushed into powder by an electric
grinder so that it can be used in the next steps. Then,
10 g of the prepared powder was dissolved in 150 mL
of deionized water at a temperature of 80 °C and mixed
for 40 minutes. After cooling the solution and removing
suspended particles from it, the solution containing
extract and plant residues was passed through Whatman
filter paper No. 1 using a Buchner funnel under vacuum
conditions. Then, the remaining particles were separated
by centrifugation at 14000 rpm for 20 minutes until the
final solution became completely clear [25,26]. Finally,
the obtained solution was kept at 4 °C for use in the next
steps.
2.2. Production of ZrNPs with clover extract
To synthesize and produce ZrNPs, 90 mL of 1 mM
potassium hexafluorozirconate (K2ZrF6) solution was
prepared. Then, 10 mL of the prepared clover extract was
gradually added and incubated at 30 °C. Moreover, to
examine the pH changes in the synthesis, the pH of the
desired solutions was set to 10 [27]. The color change of
the extract indicated the production of NPs.
2.3. Isolation of NPs
To separate the produced NPs, a sedimentation method
with centrifugation at 11000 rpm for 10 minutes at 4
°C was used. To clean the surface of NPs, they were
dispersed in deionized distilled water and separated again
by sedimentation method. Then, the resultant sediment
was dried in an oven at 80°C for 120 minutes. Finally,
the obtained white powder was stored in a cryotube for
biological analysis.
2.4. Ultraviolet-visible spectroscopy (UV-Vis)
The absorption intensity of the surface plasmon band
in colloidal solutions containing NPs in the wavelength
range of 200-800 nm was measured with the help of a
Varian double-beam UV-Vis spectrometer (Agilent Cary
100, USA) [28].
2.5. Measuring the size distribution of NPs by dynamic
light scattering (DLS)
The normal molecule size of ZrNPs was determined using
a Zetasizer (model 3600, England) with a scattering angle
of 90 degrees at Shahid Beheshti University of Medical
Sciences, Iran. In this device, the default measurement
range was between 0.1 nm and 10 μm. It seems necessary
to state that the colloidal NP sample was prepared as a
solution and for its preparation, polyethylene glycol 400
was used to disperse the particles properly. To do so,
0.001 g of dried powder of NPs was mixed with 5 mL of
deionized water for 30 minutes and the desired samples
were placed in a Sonicator (20 kHz) at 25 °C for 20
minutes to prevent the accumulation of particles.
2.6. Scanning electron microscope (SEM)
A scanning electron microscope (model VEGA3,
TESCAN company, Czech Republic) was used to obtain
the images of the prepared ZrNPs and determine their
surface characteristics and morphology. To prepare
the NPs for scanning electron microscope imaging, the
Ameri Shah Reza et al
18 Arch Hyg Sci. Volume 13, Number 1, 2024
samples were sonicated for 20 minutes and a drop of the
resulting colloid of ZrNPs was placed on carbon-coated
copper grids. Then, it was dried at ambient temperature
and to create an electric current, the surface of the sample
was covered with a thin layer of gold and palladium alloy.
2.7. Energy dispersive spectroscopy (EDX)
To determine the elemental composition of the produced
colloidal sample, a VEGA3 electron microscope was used.
The sample preparation method was the same as the SEM
sample.
2.8. Determination of the cytotoxic effect of ZrNPs on
cancer cells
Human breast cancer cell lines (MDA-MB-468) and
healthy cell lines (HFF) were obtained from the Iranian
Biological Resource Center (IBRC) and used in this
investigation. These cells were cultured in a mixture of
Dulbecco’s modified eagle’s medium (DMEM) and Ham’s
F12 supplemented with 10% fetal bovine serum, penicillin
(100 U/mL), and streptomycin (100 mg/mL). Then, they
were placed in an incubator with a temperature of 37 °C,
5% CO2 concentration, and 95% humidity. Flasks were
examined daily by an inverted microscope in terms of
cell growth and density, morphology, and contamination
control. The complete culture medium containing 10%
fetal bovine serum (FBS) was changed at the required time.
2.9. MTT Assay
The cytotoxic impact of ZrNPs was investigated by
colorimetric method, using the dye 5-dimethylthiazol-
2-y1]-2-5-diphenyltetrazolium bromide (MTT),
3-[4-tetrazolium bromide water-soluble salt. This
method is based on succinate enzyme activity. The purple
formazan crystals are produced by dehydrogenases in
the mitochondria of living cells. This compound can be
dissolved in DMSO. Since dead cells are incapable of
changing over MTT to formazan, the level of formazan
delivered is relative to the number of living cells. To
conduct the experiment, MDA-MB-468 and HFF cells
were cultured at a density of 5000 cells per well of a 96-
well plate. The old culture medium was removed from the
wells after 48 hours, and the cells were treated with ZrNPs
(8, 32, 64, 128, 256, and 512 μg/mL) synthesized by clover
aqueous extract and tamoxifen (1.25, 2.5, 5, 10, 20, 40 μg/
mL). Four wells were considered for each treatment and
4 wells were assigned as controls in each experiment. The
plates were kept in an incubator containing 5% CO2, 95%
humidity, and 37 °C for 24 hours. They were incubated
for 48 and 72 hours. After the incubation, the supernatant
of each well was carefully removed, replaced with a
complete culture medium containing MTT solution, and
placed in the incubator for 4 hours. Then, the supernatant
of each well was removed and 200 μL of DMSO solution
was replaced with the previous solution to dissolve the
formazan crystals. After 15 minutes, the optical density
was read using an ELISA reader (US BioTek, Synergy/
HTX) at a wavelength of 570 nm.
The amount of absorption corresponds to the number
of living cells. The results are calculated in terms of
the rate of live cells compared to the control using the
following equation: 100x (average absorption of control
cells/average absorption of treated cells) = percentage of
living cells
2.10. Statistical analysis
Data analysis was done by Excel and GraphPad Prism
8.2.1 software. The concentration required to inhibit
cell growth by 50% (IC50) was calculated and plotted by
PRISM and non-linear regression analysis, utilizing doseresponse
curves.
Results
3.1. Evaluating the results of macroscopic observations
and the Tyndall effect
The first step in the synthesis of ZrNPs is to observe the
gradual color change of the response arrangement from
green to yellow, which is the initial confirmation that takes
place after the end of the incubation time. After the initial
synthesis of ZrNPs and macroscopic observations such as
the color change and examination of the Tyndall impact,
all the syntheses carried out in unfavorable conditions
were excluded from further experiments due to the lower
intensity of color change compared to the NPs produced
by aqueous clover extract. In fact, in terms of macroscopic
observations, this color change indicates the production
of ZrNPs (Figure 1, A and B). Finally, after the synthesis,
the obtained NPs turned into a white powder.
3.2. Evaluation of the production of ZrNPs using aqueous
clover extract
3.2.1. UV-Vis spectroscopy
The color change is attributed to surface plasmon
resonance (SPR), which is related to the optical properties
of ZrNPs. ZrNPs have shown the capacity to assimilate
the resonance of electromagnetic waves in the visible
light. The maximum absorption of ZrNPs usually occurs
in the range of 200-400 nm. Therefore, to confirm the
formation of ZrNPs, the presence of the SPR band and the
Figure 1. Change of the color from green (a: before the reaction) to yellow
(b: after the formation of ZrNPs) using aqueous extract of the clover
Arch Hyg Sci. Volume 13, Number 1, 2024 19
Green synthesis of zirconium nanoparticles by aqueous clover extract
absorption intensity of the produced NPs were checked
with an UV-Vis light spectrometer. The wavelength of
the maximum absorption band of the NPs formed using
clover aqueous extract was 225-285 nm, which indicates
the formation of ZrNPs.
3.3. Dynamic light scattering
DLS is a technique to confirm the formation of ZrNPs.
Using the results of this analysis, the hydrodynamic
diameter of the synthesized ZrNPs can be checked. It
should be mentioned, based on the results obtained
from macroscopic observations and DLS results of some
aqueous extracts of clover, according to the difference in
synthesis conditions such as extract concentration, metal
salt concentration, temperature, and incubation rate It
was not desirable for the ZrNPs synthesis and they were
removed in the further work.The desired synthesis results
based on the mentioned conditions can be seen as DLS
results in Figure 2. Based on the results, the average size
of NPs synthesized by clover at pH 10 and zirconium
concentration of 1 mM was reported to be 64.33 nm.
3.4. Scanning electron microscope
SEM images of ZrNPs produced using clover aqueous
extract are shown in Figure 3. In the SEM analysis
of NPs, it can be seen the synthesized ZrNPs were
irregular polygons, star-shaped and flower-like colonies,
homogeneous and with dimensions in the limited area
of 29 to 81 nm. The accumulation of particles can also
be seen in some areas; however, the particles in the
accumulated areas are separate from each other and the
border between them is quite clear (Figure 3).
3.5. Energy dispersive spectroscopy
In addition to UV spectra and images related to SEM
analysis, to investigate the constituent elements of ZrNPs,
the purity of the product, and the energy distribution
spectrum of ZrNPs produced by aqueous clover extract
are also shown in Figure 4. In this Figure, the peak
related to the zirconium element can be seen. Given
that ZrNPs have SPR properties, they have a specific
optical absorption peak at around 2.2 keV, indicating
the formation of ZrNPs. In addition, the distribution of
ZrNPs produced by the aqueous extract of clover along
with other elements is shown in Figure 4.
3.6. Investigating the cytotoxic effect of ZrNPs
The cytotoxic effect of ZrNPs synthesized by aqueous
extract of clover on MDA-MB-468 and HFF cells was
estimated using the MTT method. These cells were treated
Figure 2. The results of DLS of ZrNPs synthesized by aqueous extract of
clover plant at pH 8 and zirconium concentration of 1 mM
Figure 3. SEM image of nps synthesized by the aqueous extract of clover
Figure 4. EDX Spectrum of ZrNPs synthesized by the aqueous extract of
clover to check the constituent elements of NPs and product purity
Figure 5. Cytotoxic effect of ZrNPs obtained from aqueous extract of clover plant
at different concentrations on MDA-MB-468 cells after 24, 48, and 72 hours
0 50 100 150 200 250 300
0
20
40
60
80
100
MDA-MB468
Concentration(g/ml)
Viability%
24h
48h
72h
IC50(24h)=264.3
IC50(48h)=230.8
IC50(72h)=134.4
Ameri Shah Reza et al
20 Arch Hyg Sci. Volume 13, Number 1, 2024
with six different concentrations of NPs (8, 32, 64, 128,
256, and 512 μg/mL) for 24, 48, and 72 hours. As displayed
in Figure 5, ZrNPs could inhibit the growth of MDAMB-
468 cells in a concentration- and time-dependent
manner. By examining the results of the MTT test, it
was observed that with increasing NP concentration and
treatment time, the survival rate of cancer cells decreased
significantly compared to the control cells. The IC50
values calculated from the dose-response curves for the
cell line MDA-MB-468 are shown in Figure 5. IC50 values
after 24, 48, and 72 hours were 264.3, 230.3, and 134.4 μg/
mL, respectively.
Figure 6 shows the toxicity of different concentrations
of ZrNPs on the HFF cell line, which was compared with
MDA-MB-468 cells.
By examining the results of the MTT test, it was observed
that the effect of ZrNPs on normal HFF cells was much
lower compared to cancer cells. The IC50 of ZrNPs in the
case of normal HFF cells was calculated using GraphPad
Prism 8.0.2 at 420.0, 331.9, and 181.7 μg/mL after 24, 48,
and 72 hours, separately.
In the present study, HFF cells were less sensitive to
ZrNPs. In other words, the IC50 value in HFF cells after
48 hours was approximately 1.43 times higher than in
MDA-MB-468 breast cancer cell lines. In addition, the
comparative effects of ZrNPs obtained from the aqueous
extract of clover and tamoxifen were investigated in this
research. The IC50 value for tamoxifen was determined
to be 13.1 μg/mL and 23.84 μg/mL after 48 hours of
treatment in MDA-MB-468 and HFF cells, respectively
(Figure 7).
4. Discussion
In the present research, clover plant (Trifolium repens L)
collected from Sarieh Khatun village (Qom, Iran) was used
for the first time to synthesize ZrNPs. The Synthesized
NPs with dimensions in the limited area of 29-81 nm were
observed in the SEM analysis as irregular polygons, starshaped
and flower-like colonies, and homogeneous.
Biological synthesis methods have paved the way
for the green synthesis of NPs. The green synthesis is
undoubtedly a better method due to slower kinetics,
better manipulation, control of crystal growth, and
stabilization [29]. In a study conducted by Sai Saraswathi
and Santhakumar, tetrahedral ZrO2NPs produced by
the extract of Lagerstroemia speciosa had good cytotoxic
activity against breast cancer cell line (MCF-7), and
increasing the concentration of NPs up to 500 μg/mL
reduced cancer cells. Moreover, the cell membrane of 30
to 40% of the cells was swollen. In addition, apoptosis or
cell death occurred due to the exposure of these cells to
ZrO2NPs [30]. The evidence of this issue is the increase in
the size of the vacuole (a part of the cell organelle located
in the cytoplasm) of some cells. In other words, the change
in vacuole size occurs when the cell death happens. In
addition to the mentioned features, the synthesized NPs
have the property of photocatalytic degradation (methyl
orange). The existence of this property is probably due
to their contact surface, tetrahedral structure, and active
sites.
In the study conducted by Shanthi et al, zirconium
dioxide NPs were produced using Acalypha Indica extract.
The average size of the produced NPs was found to be 20-
100 nm. According to the EDX analysis, the synthesized
NPs are of high purity [31]. In the research of Golnaraghi
Ghomi et al, ZrNPs were produced by the extracellular
method using three different strains of Penicillium fungus.
The size of NPs produced by three strains of Penicillium
purpurogenum, Penicillium aquiolithium, and Penicillium
notatum was reported to be 53.60, 39.32, and 62.27
nm, respectively. In addition, the produced NPs have
antibacterial properties and good resistance against the
growth of Escherichia coli and Pseudomonas aeruginosa
organisms with a minimum inhibitory concentration of
0.75 and 0.375 mM [32].
Figure 6. Cytotoxic effect of ZrNPs obtained from aqueous extract of clover
plant at different concentrations on HFF-1 Cells after 24, 48, and 72 hours
Figure 7. The Cytotoxic effect of tamoxifen at different concentrations on
HFF-1 and MDA-MB-468 cells after 48 hours
0 50 100 150 200 250 300 350 400 450
0
20
40
60
80
100
Concentration(g/ml)
Viability%
24h
48h
72h
IC50(24h)=420.0
IC50(48h)=331.9
IC50(72h)=181.7
HFF-1
0 10 20 30 40 50
0
20
40
60
80
100
Concentration(g/ml)
Viability%
HFF
MDA-MAB468
IC50=13.10
IC50=23.84
Arch Hyg Sci. Volume 13, Number 1, 2024 21
Green synthesis of zirconium nanoparticles by aqueous clover extract
The use of the leaf extract of Lul tree or Macarzan for
the biosynthesis of ZrO2NPs was reported by Shinde et al.
Based on the results of the research, the synthesized NPs
had a tetrahedral shape, particle size of 15 nm, and very
high cross-sectional area (around 88 m2/g). Besides, the
photocatalytic effect of NPs in the presence of ultraviolet
rays on methylene blue and methyl orange was examined,
and the results indicated that the photodegradation of
methylene blue and methyl orange was up to 91% and
69% within 4 hours. According to the results obtained
from this research, the NPs synthesized by the leaf extract
of the Lul tree are suitable alternatives to the NPs obtained
by conventional methods [33].
In the current study, the cytotoxic effect of ZrNPs
synthesized by aqueous extract of clover plant was
investigated on the MDA-MB-468 breast cancer cell line
and normal HFF. The results indicated that this NP had
a very low toxicity effect on the breast cancer cell line
and little very low toxicity effect on the normal cell line.
This feature should be one of the main characteristics of
antitumor drugs; in other words, these drugs should only
affect tumor cells and have no adverse effects on normal
cells. In other words, the results show more inhibitory
effects of NPs on breast cancer cells compared to normal
cells at a certain dose.
In the current study, the value of IC50 of ZrNPs after
24 hours on the MDA-MB-468 cell line was lower than
the calculated value of IC50 on the HFF cell line, which
indicates more inhibitory effects of ZrNPs on cancer cells
than on normal cells.
The results of the current study showed that NPs
synthesized in this study reduced the survival of cancer
cells, but their toxicity on normal cells was lower. Due to
severe glycolysis in cancer cells, the environment of cancer
cells is acidic, and the acidity of the environment causes
the activation of ZrNPs and the death of cancer cells.
Therefore, the greater effect of NPs on cancer cells
than on normal ones was predictable. The difference
in pore size of the membranes can be another factor in
the significant difference in the toxicity of NPs between
cancerous and normal cells in this study. The results of
the current study showed that the aqueous extract of
clover, a native plant of Iran, can be used in the synthesis
of ZrNPs due to its secondary metabolites and antioxidant
properties. Findings showed that synthesized ZrNPs have
toxic effects on breast cancer cells, and since ZrNPs had
very low toxicity effect on the death of the HFF cell line, it
can be said that its side effects were minimal. Given that
many drugs induce necrosis in cells and have side effects
such as inflammation, they cannot be considered suitable
drugs. While the ZrNPs synthesized in the present study
caused toxicity in cancer cells, they do not have a toxic
effect on normal cells. Therefore, they can be used as an
effective drug for the treatment of breast cancer.
5. Conclusion
The results of the current study showed that the aqueous
extract of clover had a significant role in the green
synthesis of ZrNPs. On the other hand, these NPs had
considerable cytotoxic effects on human breast cancer
cells (MDA-MB-468) and could inhibit the growth of
cancer cells. Therefore, it can be studied in future studies
as an anti-cancer agent.
Acknowledgments
The authors would like to thank the Research Council of Qom
University of Medical Sciences for approving the project.
Authors’ Contribution
Conceptualization: Mahdieh Ameri Shah Reza.
Data curation: Mahdieh Ameri Shah Reza.
Formal analysis: Mahdieh Ameri Shah Reza, Tahere Komeili
Movahhed, Leila Astaraki.
Funding acquisition: Mahdieh Ameri Shah Reza.
Investigation: Mahdieh Ameri Shah Reza.
Methodology: Mahdieh Ameri Shah Reza, Tahere Komeili
Movahhed, Leila Astaraki.
Project administration: Mahdieh Ameri Shah Reza.
Resources: Mahdieh Ameri Shah Reza.
Software: Mahdieh Ameri Shah Reza, Tahere Komeili Movahhed,
Leila Astaraki.
Supervision: Mahdieh Ameri Shah Reza.
Validation: Mahdieh Ameri Shah Reza.
Visualization: Mahdieh Ameri Shah Reza.
Writing–original draft: Mahdieh Ameri Shah Reza, Alireza Rasouli.
Writing–review & editing: Mahdieh Ameri Shah Reza, Alireza
Rasouli.
Competing Interests
The authors declare that they have no conflict of interests.
Ethical Approval
Ethical issues have been completely observed by the authors.
Funding
This work was funded by a grant (grant no: IR.MUQ.REC.1399.282)
from the Vice Chancellor for Research and Technology of Qom
University of Medical Sciences, Qom, Iran (Project research code:
991365).
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