Archives of

1. Introduction
Caryophyllaceae is a large family of about 2200 herbaceous or subshrub species and 86 genera. The family is known for its ornamental plants and saponin compounds. Caryophyllaceae is one of the largest plant families with 38 genera in Iran and Europe [1]. The Vaccaria is of the family Caryophyllaceae is a one-year static plant with 30-60 cm height and multiplies by seed. It has the shoot branch at the end. Bi-directional inflorescence, with numerous flowers and pink or bright red, is located at the end of branching stems. Furthermore, the flower Bowlin is a free part in compression mode and is collectively in the form of a jug with a tight span. Given the many activities of the plants belonging to the Vaccaria genus,
Evaluation of Chemical Composition of Vaccaria oxyodontha Boiss Plant Essential Oils and Antioxidant and Antibacterial Effects on Gram-Negative and Gram-Positive Bacteria
Rahele Bahrami1, Maryam Soori2ID, Hossein Abbaspour1ID, Hamid Hashemi-moghaddam3ID, Taghi Lashkarbolouki4ID, Reza Moradi5*ID, Ahmad Reza Yari6ID
1Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran
2Department of Biochemistry, Hamedan Branch, Islamic Azad University, Hamedan, Iran
3Department of Chemistry, Damghan Branch, Islamic Azad University, Damghan, Iran
4Department of General Biology, School of Biology, Damghan University, Damghan, Iran
5Department of Chemistry,Tuyserkan Branch, Islamic Azad University,Tuyserkan, Iran
6Research Center for Environmental Pollutants, Qom University of Medical Sciences, Qom, Iran
*Corresponding Author: Reza Moradi, Email: reza.moradi_bi@yahoo.com
Received: March 2, 2022, Accepted: May 7, 2022, ePublished: December 29, 2022
https://jhygiene.muq.ac.ir/
10.34172/AHS.11.4.103.9
Vol. 11, No. 4, 2022, 272-278
Orginal Article
Arch Hyg Sci. Volume 11, Number 4, 2022 273
Vaccaria oxyodontha Boiss and its biochemical effects
the characterization, determination, and comparison of
their essential oils (EOs) and extracts seem highly logical.
Vaccaria oxyodontha Boiss is synonym with Vaccaria
hispanica (Miller) Rauschert [2,3].
To the best of our knowledge, no scientific study has so
far focused on the V. oxyodontha Boiss plant. However,
extensive studies have been published on other plants
in the Brassicaceae family. According to the Chinese
pharmacopeia, the seeds of Vaccaria segetalis can treat
urinary diseases and kidney infections [4]. Falih et al
[5] showed the anticancer and anti-infective effects of
Saponaria vaccaria plant, and utilized it for treating
coughs, rheumatism, skin diseases, and hepatic eruptions.
Likewise, Xu et al expressed that the Vaccaria n-butanol
extract decreases proinflammatory cytokines and the
infection risk of Trichinella spiralis [6]. Many studies have
evaluated the antioxidant, anti-inflammatory, and blood
flow activator effects on bile disorders, liver eruption, and
other disorders and diseases of the body in Vaccaria plant
family.
2. Objectives
In this study, EOs from the aerial parts of V.
oxyodontha Boiss were extracted using a combination
of hydrodistillation (HD) and microwave-assisted
hydrodistillation (MAHD). This study first investigated
the chemical composition of V. oxyodontha Boiss plant
EOs by the gas chromatography-mass spectroscopy (GCMS)
method. Then, the antioxidant properties of its
methanolic extract were evaluated with the 2,2-diphenyl-
1-picrylhydrazyl-hydrate (DPPH) method. The
antibacterial activity of the extract was examined against
Escherichia coli and Staphylococcus aureus.
3. Materials and Methods
3.1. Chemicals
The 2,6-ditertbutyl-4-methyl phenol (butylated
hydroxytoluene) standard antioxidant agent, nitrofurantoin
antibiotic, nalidixic acid antibiotic, and DPPH were
all purchased from Sigma-Aldrich GmbH (Munich,
Germany). The Mueller-Hinton agar and Muller-Hinton
broth culture media were purchased from Merck Company,
Germany. Standard strains, including S. aureus and E. coli,
as gram-positive (ATCC 1885) and gram-negative (ATCC
1625) bacteria, respectively, were provided by Iran Medical
Sciences University, Tehran, Iran.
3.2. Plant material
The new aerial parts of Vaccaria oxyodontha Boiss
were harvested in the middle of May 2013 from a
farm belonging to the Agricultural Research Station
of Damghan province, Iran (Figure 1). The aerial parts
were separated with meticulous care to extract the oil and
finally dried at the shadow in a clean place to avoid extra
damage and minimize cross-contamination.
3.3. HD method
Conventional HD was performed using a circulatory
commercially available Clevenger apparatus (Ashk Shishe
Company, Tehran). The V. oxyodontha Boiss sample was
dried at shade (one week) and weighed precisely. It was
then immersed in the ratio of 1:10 of the plant (g) to water
(mL), and the volatile oils were collected at sequential
times. The experiments were conducted twice for each
distillation time. The maximum distillation period was
three since no more EOs were obtained after that time.
Next, the EOs were separated and kept at _ 4°C before
analysis.
3.4. MAHD method
The microwave oven (Figure 2) used for MAHD was
provided by (Samsung, South Korea) trademark operating
at 2450 MHz. The maximum power of the oven was
regulated at 1000 W. The dimensions of the interior cavity
of the oven were 29 × 37 × 40 cm. The microwave oven
was modified by drilling a hole at the top. A flat bottom
flask with a 1000 mL capacity was placed in the oven and
directly connected to the Clevenger apparatus through the
Figure 1. Image of Vaccaria oxyodontha Boiss Plant
Figure 2. Image of Microwave Oven Apparatus
Bahrami et al
274 Arch Hyg Sci. Volume 11, Number 4, 2022
hole. For MAHD, 50 g portions of the plant sample were
soaked in 500 mL of distilled water at room temperature
(25°C) for one hour to hydrate its external layers, and the
excess water was drained off. Soaking time, during which
the maximum amount of absorption is achievable, was
determined from a preliminary experiment. Afterward,
the moistened plant material was placed in a flat-bottom
flask combined with a Clevenger apparatus. During the
process, the vapor continuously passed through the
condenser located outside the microwave cavity where
it was condensed. The MAHD process was performed at
different times and repeated until obtaining no more EO.
For each condition, experiments were replicated twice.
The EOs were collected in amber brown vials, dehydrated
with anhydrous sodium sulfate, capped under nitrogen,
and kept at 4°C until analysis.
3.5. Preparation of the extracts of Vaccaria oxyodontha
Boiss
In the first step, the aerial plant parts (V. oxyodontha
Boiss) were cut up and ground in a coffee mill (Moulinex
Corporation, France). This step was followed by
transferring the obtained powder to dark-colored flasks,
mixing with 85% (v/v) methanol at a material-to-solvent
ratio of 15:100 (mg/v), and heating at 50°C for 30 minutes.
Next, the slurry was filtered through Whatman No. 1
filter paper. The residue was re-extracted twice more,
and combined supernatants were evaporated to dryness
under vacuum at 40°C using a rotary (IKA, Germany).
Finally, the prepared extracts were stored at 4°C in the
refrigerator until further analysis.
3.6. GC and GC-MS analyses
The quantitative and qualitative evaluations of the oils
were performed using GC and GC-MS instruments. The
GC analysis was performed on a Varian (CP 3800) gas
chromatograph equipped (Agilent 5975 C) with a split/
splitless (10:1) injector (290°C) and a flame ionization
detector (250°C). N2 was used as the carrier gas (0.8 mL/
min). The capillary column used CP-Sil 5 CB (30 m × 0.25
mm × 0.25 μm film thickness). The oven temperature
was held at 50°C for 5 minutes and heated to 240°C at a
rate of 3°C minute-1 ramp, followed by a further rise to
300°C with a programmed 300 °C with a programmed 5
°C min-1 ramp. A final hold was allowed for a complete
column clean-up for 3 minutes. Quantitative data were
obtained through the system area percentage. GC-MS
determinations were performed on an HP-6890 GC
system coupled with a 5973 network mass selective
detector equipped with an HP-5MS capillary fused silica
column (30 m × 0.25 mm ID × 0.32 μm film thickness).
The operating conditions were the same as those described
above, but the carrier gas was He with a 0.8 mL/min flow
rate. Mass spectra were taken at 70 eV and recorded
over the m/z range of 20-500 Amu. All chromatographic
measurements were performed in triplicate, and the mean
of the retention times and percentage compositions of
each component were taken into consideration. Thus, the
same times were discarded if they differed by more than 1
second, and the experiments were repeated in duplicate.
3.7. Identification and quantification of EO compounds
In this work, the constituents of the oil were determined
and characterized by:
i. Comparing their mass spectral fragmentation
pattern regarding authentic samples and retention
indices (RI) relative to C9-C21 n-alkanes with those
given in the literature [2] and some of our previous
reports;
ii. Storing the data in an MS library (Wiley 275);
iii. Matching the fragmentation pattern with those in
the National Institute of Standards and Technology
Mass Spectral Library package with a resemblance
percentage above 85%.
The relative percentage amounts of the components
were directly calculated from the peak area using an HP-
6890 GC system on the HP-5MS column considering
the sum of all eluted peaks as a hundred percent without
using the correction factor. Finally, the identical volumes
of each essence were separately injected into GC-MS
[7-10].
3.8. Determination of antioxidant activity by the DPPH
method
The method proposed by Nabavi et al was used to assay
the extracts’ antioxidant capacity and the free radicalscavenging
effect on the DPPH radical [11].
Accordingly, 100 μL of the extract, at concentrations
of 200, 400, and 600 μg/mL, was mixed with 2 mL of
solution DPPH (0.004 g DPPH solution within 100 mL
Methanol). The same process was repeated for vitamin
C to compare the extract’s antioxidant power with
vitamin C. Then, it was incubated in a dark room for
15 minutes, and absorbance (A) was measured at 517
nm for all samples (BioTek Epoch, USA). Methanol and
DPPH were employed as blank and control samples,
respectively. The antioxidant activities of the samples
were determined using the ascorbic acid standard curve.
The antiradical activity of the samples was expressed by
IC50 (mg/L) according to previous research [12]. The
DPPH scavenging effect (%) is expressed by Eq. (1) as
follows:
DPPH scavenging effect (%) = (Ao-A1)/Ao × 100 (1)
where Ao and A1 represent the absorbance of the
control (blank) and the absorbance in the presence of
samples (the V. oxyodontha Boiss extract and ascorbic
acid), respectively. Numerical IC50 values denote the
sample’s concentration, which is required to scavenge
50% of DPPH free radicals.
Arch Hyg Sci. Volume 11, Number 4, 2022 275
Vaccaria oxyodontha Boiss and its biochemical effects
3.9. Preparation of various dilutions of the extract
Different dilutions of the extract (1, 1/2, 1/4, 1/8, 1/16,
1/32, and 1/64) were prepared using sterile distilled water
to ensure anti-bacterial properties.
3.10. Preparation of Microbial Strains
In this study, the bacteria were S. aureus RTCC 1885 and
E. coli PTCC 1330, lyophilized and prepared from the
Faculty of Pharmacy’s Microbial Collection of Tehran
University of Medical Sciences (Iran).
Bacterial lyophilized ampoules (S. aureus and E. coli)
were first opened under sterile conditions and transferred
to nutrient broth (Merck, Germany), and then incubated
for 24 hours at 37°C. Then, to ensure the bacteria’s purity
from the nutrient medium, it was linearly cultured on the
differential culture medium overnight and incubated for
48 hours at 37°C. A loop was then taken from the bacterial
colony and inoculated into the broth nutrient 24 hours
before each test. A new 24-hour culture was prepared for
each test to evaluate the antimicrobial effects. Finally, the
microbial suspension was prepared with a concentration
equal to 0.5 McFarland.
3.11. Determination of microbial susceptibility
The protocols used in this study were based on the
guidelines of the Clinical and Laboratory Standards
Institute (CLSI) tables. The disc diffusion method was
applied to determine the susceptibility of microbial
strains against methanolic extracts. Minimum inhibitory
concentration (MIC) and minimum bactericidal
concentration (MBC) were employed to determine
microbial susceptibility. MIC was determined by dilution
in a liquid medium (microbroth dilution). for the MBC
test, concentration of extract that inhibited of bacterial
growth in MIC step, were cultivated on Muller-Hinton
agar medium. All the experiments were conducted in
triplicate.
3.12. Statistical analysis
SPSS software (version 20) and Kruskal-Wallis and
Dunn’s tests were utilized for data analysis. The data are
presented as mean values ± standard deviations (SD). The
values of P < 0.05 for Kruskal-Wallis and Dunn’s tests
were considered significant.
4. Results
4.1. Chemical constituents of the EOs of Vaccaria
oxyodontha Boiss
The chemical constituents of the EOs obtained from
V. oxyodontha Boiss are shown in Table 1 and the
chromatogram in Figure 3. The major compounds in the
EOs were 2-pentadecanone, 6,10,14-trimethyl (10.52%),
and 1,2-benzenedicarboxylic acid (19.27%).
4.2. Antioxidant capacity
Antioxidant capacity was tested using a methanolic
solution of the ‘stable’ free radical, DPPH. A freshly
prepared DPPH solution displays a deep purple color.
This purple color usually disappears when an antioxidant
Table 1. Chemical Compositions of the Essential Oils of Vaccaria oxyodontha
Boiss Using GC-MS
Compounds RTa %
Dodecane n-dodecane adakane 12.053 1.96
Fenchyl acetate 12.443 2.32
MINT FURANONE 12.531 0.99
Naphthalene, decahydro-1,5-dimethy 12.633 1.83
Cyclohexane 15.457 3.35
Caryophyllene 15.939 0.78
( + )-Aromadendrene 1H-Cycloprop [
121608 000489-39-4 59
e]azulene
16.479 1.41
3-Buten-2-one, 4-(2,6,6-trimethyl- 16.946 1.81
2H-3,9a-Methano-1-benzoxepin, 17.243 0.73
Phenol, 2,5-bis(1,1-dimethylethyl 17.312 0.85
1S,CIS-CALAMENENE 17.536 0.62
(-)-Caryophyllene oxide (-)-5-O 18.018 0.84
(-)-Spathulenol 18.397 1.63
Caryophyllene oxide 18.490 2.87
Cyclohexene, 6-ethenyl-6-methyl- 18.670 3.54
1H-Cycloprop[e]azulene, 1a,2,3,5,6 18.787 0.64
cis-Z-.alpha.-Bisabolene epoxide 18.879 1.89
Naphthalene, 1,2,4a,5,8,8a-hexahyd 19.025 4.77
1H-Cycloprop[e]azulene, 1a,2,3,5,6 19.235 1.42
( + )-.delta.-selinene naphthalene 19.366 1.67
Valencene Naphthalene, 1,2,3,5, 19.454 2.21
( + ) Spathulenol 19.726 1.85
CAPNELLANE-8-ONE 2H-Cyclopenta 19.950 1.38
(3E,5E,8Z)-3,7,11-Trimethyl-1,3,5, 20.247 2.20
6-Isopropenyl-4,8a-dimethyl-1,2,3, 20.447 5.20
cis-.alpha.-Copaene-8-ol Tricyclic 21.070 0.67
Octadecane 21.338 1.48
2-Pentadecanone, 6,10,14-trimethyl 21.927 10.52
Tetratriacontane 23.436 1.27
Dotriacontane 24.172 0.60
2-Hexadecen-1-ol,3,7,11 24.288 6.62
Pyridinium, 1-hexadecyl-, chloride 24.493 0.93
Hexatriacontane 25.228 0.67
Nonadecane (CAS) n-nonadecane 25.637 0.84
4,5. alpha. -Epoxy-3-methoxy-17-meth 25.978 1.71
Pentacosane 26.026 4.10
1,2-Benzenedicarboxylic acid 26.280 19.27
Heptacosane 26.922 2.59
100
Note. a Rt: Retention time; GC-MS: Gas chromatography-mass spectroscopy.
Bahrami et al
276 Arch Hyg Sci. Volume 11, Number 4, 2022
is present in the medium of the test. Antioxidant molecules
quench DPPH free radicals, and DPPH is converted to a
colorless product, resulting in a decrease in absorbance at
517 nm. The DPPH radical scavenging activities of the V.
oxyodontha Boiss and the vitamin C extract at different
concentrations are illustrated in Figure 4. The IC50 value
of the extract for V. oxyodontha Boiss was 0.49 μg/mL,
which was higher than that of vitamin C (an IC50 value of
0.56 μg/mL).
4.3. Antibacterial capacity
The diameter of the growth inhibition zone of the V.
oxyodontha Boiss extract against the S. aureus strain and
E. coli in the disk diffusion method was 23.5 and 18.65
mm, respectively.
Table 2 lists the mean and SD of the inhibition growth
zone (mg/mL) related to the methanolic extract of V.
oxyodontha Boiss at different concentrations against S.
aureus and E. coli. The concentration of 500, 250, and
125 mg/mL of the extract had the highest antibacterial
effect compared to vancomycin against S. aureus.
Nevertheless, the extract effect was weaker against E. coli
than gentamicin.
According to the results of CLSI Tables, the minimum
growth inhibition zone for gentamicin antibiotic (10 μg
disc) against E. coli strain was 19-26 mm. In the minimum
growth inhibition zone for the antibiotic vancomycin
(30 μg disc), the diameter of the growth inhibition zone
against the S. aureus strain was 14-15 mm. Based on
the results of Table 3, the intergroup comparison with
the Kruskal-Wallis test, V. oxyodontha Boiss extracts
had antimicrobial properties equal to gentamicin and
Vancomycin antibiotics (P < 0.05). MIC and MBC for the
V. oxyodontha Boiss extract against S. aureus and E. coli
were 62.5 and 125 mg/mL, as well as 31.25 and 62.5 mg/
mL, respectively.
5. Discussion
Herbs are directly used to treat disease globally, including
in Iran, China, India, and other countries in Asia and
Western Europe. These plants are the Vaccaria family [13].
Most plants in this family are weeds on agricultural land.
Flavonoids, polyphenols, kaempferol, saponins, cyclic
peptides, and starch are active ingredients (synonyms) in
these plants with many medical applications [14].
According to Cam et al [15], the seeds of Vaccaria
hispanica (Miller) Rauschert are naturally distributed
in all parts of Turkey. This study indicates that these
Vaccaria family plants have potential applications in
the food and pharmaceutical industries and cosmetics
industries. However, this plant is not so well known.
They are not yet cultivated and are not used industrially
[15]. According to previous studies, the plants of this
family are rich in saponins. In general, the biological role
Figure 3. GC Chromatogram of the Essential Oils of Vaccaria oxyodontha Boiss
Arch Hyg Sci. Volume 11, Number 4, 2022 277
Vaccaria oxyodontha Boiss and its biochemical effects
of saponins is unclear in plants. However, no study has
examined the effect of saponins in treating various cancers
and infections. It is likely to have hypocholesterolemic
properties and immune system stimulants and antiinflammatory,
antibacterials, insecticide fungicides, antileishmaniasis,
and antioxidant properties [16]. Studies
related to chemotherapy and therapeutic activities of the
seeds of the plants of this family have been developed in
recent years. In their study, Zhou et al demonstrated that
Vaccaria hispanica has at least 63 different metabolites,
some of which have medicinal properties [3].
According to Figure 4, a comparison between the
Vaccaria oxyodontha Boiss extract and vitamin C, a
natural antioxidant, represented that the extract had
a more inhibitory effect against free radicals secreted
by DPPH compared to vitamin C. Based on the
antioxidant properties of this plant, they can be used
in the pharmaceutical, health-cosmetics, and food
industries as effective antioxidants. Biswas et al evaluated
the hepatoprotective activity of the ethanolic root
extract of “Vaccaria pyramidata” against CCl4-induced
hepatotoxicity in Wister rats [17] and reported that the
Vaccaria pyramidata root extract had a protective and
antioxidant effect on liver cells. In addition, it decreased
triglycerides in rats compared to the control group.
This study investigated the antibacterial properties of
the Vaccaria oxyodontha Boiss extract against S. aureus
and E. coli using the disk diffusion method and Kerby-
Bauer protocol. This test was performed in comparison
with the two gentamicin and Vancomycin antibiotics.
Based on the findings (Table 1), the Vaccaria oxyodontha
Boiss extract was more potent against S. aureus than
the Vancomycin antibiotic. Further, the diameter of
the growth inhibition zone of this extract against E. coli
was 18.65 mm. According to Mao et al [4,18], Vaccaria
segetalis seed extracts significantly reduced bacterial load,
and white and red blood cells in the urine, as well as
inhibiting pathological damage to the bladder. Therefore,
it can be an alternative therapeutic agent for urinary
infections.
6. Conclusion
Vaccaria oxyodontha Boiss is one of the medicinal plants
used in traditional medicine in Iran and some Asian
countries. The results of the present study for identifying
the EO constituents revealed that the highest composition
of the EO was found in 2-Pentadecanone,6,10,14-
trimethyl (10.52%) and 1,2-Benzenedicarboxylic acid
(19.27%). The IC50 of the extracts of V. oxyodontha Boiss
was equal to 0.49 μg/mL, which is a significant antioxidant
property in plants. Additionally, it can be used as a
strong antibacterial. Due to the unique antioxidant and
antibacterial properties of this plant extract and EOs, it
can find their unique place in the pharmaceutical, health,
and medical industries with further research.
Acknowledgements
The authors wish to acknowledge the members of the Research
Laboratory of Islamic Azad University, Damghan Branch and
Damghan, Iran (Thesis code 14230520922019).
Author Contributions
Conceptualization: Rahele Bahrami, Maryam Soori, Hossein
Table 3. MIC and MBC of the Extracts of Vaccaria oxyodontha Boiss
Methanolic Extract
MIC (mg/mL)
Staphylococcus aureus Escherichia coli
MBC MIC MBC MIC
Vaccaria oxyodontha Boiss 62.5 125 31.25 62.5
Vancomycin (30 μg disc) 2 ≥ 4-8 - -
Gentamicin (10 μg disc) - - 2-4 4-8
Note. MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal
concentration. The group’s inhibition growth zone’s mean and standard
deviation was 18.30, 3.3, respectively (P < 0.05).
Table 2. Mean and standard deviation of the inhibition growth zone (mg/
mL) of the methanolic extract of Vaccaria oxyodontha Boiss in Different
Concentrations Against Staphylococcus aureus and Escherichia coli
Zone of Inhibition Mean of Methanolic
Extract of Vaccaria oxyodontha Boiss
for Tested Bacteria (mm)
Different Concentrations of
Extracts (mg/mL)
S. aureus E. coli
23.7 ± 0.29 18.65 ± 0.33 500
19.58 ± 0.42 16 ± 0.08 250
17.95 ± 0.14 13.86 ± 0.26 125
13.86 ± 0.26 13.48 ± 0.22 62.5
11.58 ± 0.42 8.58 ± 0.42 31.25
7.6 ± 0.45 5.33 ± 0.33 15.62
2.53 ± 0.73 Indeterminate 7.8
14.2 ± 0.08 0 Vancomycin Positive control
0 24.1 ± 0.09 Gentamicin
0 0 (DMSO) Negative control
Note. DMSO: Dimethyl sulfoxide; SEM: Standard error of the mean. Values
are expressed as the mean ± SEM.
Figure 4. Antioxidant Activity of the Vaccaria oxyodontha Boiss Extract
Compared With Vitamin C at Different Concentrations
0
10
20
30
40
50
60
200(μg/mL ) 400(μg/mL ) 600(μg/mL)
Antioxidant inhibition of DPPH(%)
Concentration
Vaccaria oxyodontha Boiss extract Vitamin C
Bahrami et al
278 Arch Hyg Sci. Volume 11, Number 4, 2022
Abbaspour, Hamid Hashemi-moghaddam, Reza Moradi.
Methodology: Maryam Soori,Hossein Abbaspour, Hamid Hashemimoghaddam,
Reza Moradi, Taghi Lashkarbolouki.
Validation: Rahele Bahrami,Hamid Hashemi-moghaddam, Reza
Moradi, Taghi Lashkarbolouki, Ahmad Reza Yari.
Formal Analysis: Reza Moradi,Maryam Soori, Hossein Abbaspour,
Hamid Hashemi-moghaddam, Ahmad Reza Yari.
Investigation: Rahele Bahrami, Maryam Soori, Hossein Abbaspour,
Hamid Hashemi-Moghaddam, Reza Moradi, Taghi Lashkarbolouki,
Ahmad Reza Yari.
Resources: Rahele Bahrami, Maryam Soori, Hossein Abbaspour,
Hamid Hashemi-Moghaddam, Reza Moradi, Taghi Lashkarbolouki,
Ahmad Reza Yari.
Data Curation: Hamid Hashemi-Moghaddam, Taghi
Lashkarbolouki,Ahmad Reza Yari Reza Moradi.
Writing—Original Draft Preparation: Rahele Bahrami, Maryam
Soori, Hossein Abbaspour, Hamid Hashemi-Moghaddam, Reza
Moradi, Taghi Lashkarbolouki, Ahmad Reza Yari.
Writing—Review and Editing: Maryam Soori,Reza Moradi, Ahmad
Reza Yari.
Visualization: Hossein Abbaspour, Hamid Hashemi-Moghaddam,
Taghi Lashkarbolouki.
Supervision: Hossein Abbaspour, Hamid Hashemi-Moghaddam,
Reza Moradi, Taghi Lashkarbolouki, Ahmad Reza Yari, Rahele
Bahrami, Maryam Soori.
Project Administration: Maryam Soori,Reza Moradi, Hossein
Abbaspour.
Funding Acquisition: Rahele Bahrami, Maryam Soori.
Conflict of Interests
The authors declare no conflict of interests.
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