International Journal of Neuroscience

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The possible mechanism of Datura stramonium on

pentobarbital-induced sleep in mice

Mohammad-Ali Sobhanifar, Roghayeh Rashidi, Arezoo Rajabian, Fatemeh

Forouzanfar, Maede Hasanpour, Mehrdad Iranshahi, Hassan Rakhshandeh
& Azar Hosseini

To cite this article: Mohammad-Ali Sobhanifar, Roghayeh Rashidi, Arezoo Rajabian, Fatemeh
Forouzanfar, Maede Hasanpour, Mehrdad Iranshahi, Hassan Rakhshandeh & Azar Hosseini
(2023) The possible mechanism of Datura stramonium on pentobarbital-induced sleep in mice,
International Journal of Neuroscience, 133:8, 879-887, DOI: 10.1080/00207454.2021.1998045

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International Journal of Neuroscience
2023, VOL. 133, NO. 8, 879–887
https://doi.org/10.1080/00207454.2021.1998045

ORIGINAL ARTICLE

The possible mechanism of Datura stramonium on pentobarbital-induced

sleep in mice
Mohammad-Ali Sobhanifara, Roghayeh Rashidib, Arezoo Rajabianc, Fatemeh Forouzanfard,
Maede Hasanpoure , Mehrdad Iranshahie , Hassan Rakhshandeha,b and Azar Hosseinia,b
a
Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; bPharmacological Research
Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran; cDepartment of Internal Medicine, Faculty of
Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; dDepartment of Neuroscience, Faculty of Medicine, Mashhad University
of Medical Sciences, Mashhad, Iran; eBiotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical
Sciences, Mashhad, Iran

ABSTRACT ARTICLE HISTORY

Background: Insomnia leads to the development of mental problems and missing of accuracy Received 21 June 2020
in affected persons. Various investigations have previously revealed which medicinal plants play Revised 30 August 2021
a role in the improvement of insomnia. In this study, we evaluated the effect of hydro-alcoholic Accepted 6 October
2021
extract of Datura stramonium on insomnia in mice.
Methods: The extracts and fractions at different concentrations were injected intraperitoneally KEYWORDS
(i.p.) to mice 30 min before the sodium pentobarbital (30 mg/kg, i.p.). Additionally, the blood Sleep; Datura
was collected from cardiac and serum separated to measure brain-derived neurotrophic factor stramonium;
(BDNF). The LC-MS was done to identify the active components. Flumazenil or naloxone were pentobarbital
also applied to study the possible mechanism of extract. The PC12 cells were then exposed to
different doses of extract and fractions, in order to evaluate cytotoxicity by MTT assay and the
measured LD50.
Results: The hydro-alcoholic extracts of calyx, seed and petal elevated sleep duration and
decreased sleep latency. In addition, water, ethyl acetate and n-butanol fractions of hydro-alcoholic
extract of petal increased sleep duration. Of note, Naloxone significantly reversed the hypnotic
effect of the extract. The extract increased the level of BDNF in serums. As well, the toxicity
assessment revealed that the extracts had not toxic on PC12 cells. The LD50 value was obtained
as 4.8 g/kg.
Conclusion: This research demonstrated that D. stramonium (including seed, petal and calyx)
increased the hypnotic effect without neurotoxicity on PC12 cells. Sleep induction may be related
to its active ingredients as well as the effect on opioid receptors.

Introduction various diseases due to having active ingredients.

Sleep plays an important role in learning, mental and
physical health status [1]. Forty percent of people
around the world suffering from insomnia leading to
anxiety [2], hypertension [3], depression [4] and car-
diovascular diseases [5]. Most of the people prefer
using chemical drugs to manage insomnia. The syn-
thetic drugs for treatment of insomnia are involving
benzodiazepines and Z-drugs such as zolpidem and
zopiclone [6]. Whereas these drugs trigger side effects
such as tachyphylaxis and dependency, physicians
should increase the prescribed dose of drugs to induce
the hypnotic effect [6]. Recent studies have shown
that medicinal herbs may have beneficial effects on
However, these compounds are mostly applied in treat-
ment as only or in combination with chemical drugs
[7]. The animal or human studies have reported that
medicinal herbs have positive effects on the improve-
ment of insomnia [8–11]. The medicinal herbs induce
hypnotic effects by different mechanisms such as the
interaction with glutamic acid decarboxylase [12] or
the modulation of gamma-aminobutyric acid (GABA)
and 5-hydroxytryptophan (5-HT) receptors [13]. Datura
genus grows in regions with warm climate. There are
almost 10 species of Datura in the world. Datura is
identified as a hallucinogen herb in worldwide.
Different parts of herb such as seeds, leaves and fruits

CONTACT Azar Hosseini hoseiniaz@mums.ac.ir Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran
This article has been corrected with minor changes. These changes do not impact the academic content of the article.
© 2022 Informa UK Limited, trading as Taylor & Francis Group
880 M.-A. SOBHANIFAR ET AL.
are usable. The leaves of Datura stramonium have dif-
ferent usages such as decreasing headache, the pain
of rheumatism and gastrointestinal. The juice of fruit
is applied in dandruff [14]. The recent study revealed
anti-convulsant activity of extract against penty-
lenetetrazole (PTZ)-induced epilepsy [15]. The seeds
and leaves of D. stramonium are used to induce sleep
in hysteric and psychotic patients [16]. The mentioned
effects may be related to the presence of different
components such as tannins, saponins, glycosides and
anti-cholinergic ingredients like scopolamine [17]. In
the present study, we evaluated the sleep-prolonging
effects and possible sleep mechanisms of
hydro-alcoholic extract of D. stramonium in mice.
Materials and methods
Chemicals and reagents
Diazepam was purchased from Chemidarou Company
(Iran). Sodium pentobarbital, penicillin-streptomycin,
dimethyl sulfoxide (DMSO) and 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide were bought from
Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s
medium (DMEM) and fetal bovine serum (FBS) were pur-
chased from Gibco Life Technologies (Grand Island, NY,
USA). The PC12 cells were provided from Pasteur Institute
Cell Bank (Tehran, Iran) (C-153).
Plant collection and extraction
Datura stramonium was collected around Mashhad,
Iran. The plant sample was identified at the Herbarium
of School of Pharmacy (Mashhad University of Medical
Sciences, Mashhad, Iran) and a voucher specimen (13261)
was deposited in this institute. Seed, calyx and petal were
separated, washed, dried and powdered. The
hydro-alcoholic extract was prepared with 70% ethanol
in soxhlet apparatus for 48 h. The obtained extract was
filtered and dried in water bath. The yields of extracts are
as seed (19%), calyx (16%) and petal (21%). Different frac-
tions including ethyl acetate fraction (EAF), n-butanol
fraction (NBF) and water fraction (WF) were prepared
from the hydro-alcoholic extract by using the
solvent-solvent method [18]. The dried fractions were kept
in the freezer until usage. The water fraction was dissolved
in normal saline while EAF and NBF fractions were diluted
in distilled water with 1% DMSO.
Animals
For the purpose of this, the male albino mice were
applied which their weight was between 25 and 35 g.
The mice were kept with 12 h cycles of light/night and
humidity of 61 ± 3% at 22 ± 2 degrees of centigrade
(°C) temperature in an animal room. The experiments
were carried out according to the ethical guidelines
which were confirmed by the Committee of Mashhad
University of Medical Sciences (IR.MUMS.REC.1396.59).
Sleep induction protocol
A single dose of the hydro-alcoholic extracts, normal
saline and diazepam were injected intraperitoneally
(i.p.) to mice. After 30 min, pentobarbital was admin-
istrated at the dose of 30 mg/kg to induce sleep in
mice. Righting reflex was used for the evaluation of
sleep duration and sleep latency was interval between
injection of pentobarbital and beginning of sleep. The
mice were divided into different groups, each group
consisting of 8 mice. The groups were involving normal
saline (the negative control), diazepam (3 mg//kg) (the
positive control), and hydro-alcoholic extracts (25, 50
and 100 mg/kg) groups. To evaluate of sleep-induction
mechanism, flumazenil (1 mg/kg i.p.) and naloxone
(5 mg/kg i.p.) were injected 30 min before the admin-
istration of diazepam or extract [8]. Additionally, the
sleep-prolonging effect of three types of fraction
(including NBF, WF and EAF) were evaluated to inves-
tigate the most effective fraction. The blood was col-
lected from cardiac and the serum was separated to
determine the brain-derived neurotrophic factor (BDNF)
level. Finally, the samples were kept at −80 °C until the
measurement of protein.
Median lethal dose (LD50) determination
To evaluate LD50, the groups consisting of 2 mice
were applied. The hydro-alcoholic extract was injected
as i.p. at various doses (25, 50, 100, 200, 400, 800,
1600, 3200 and 6400 mg/kg). The mortality rate was
recorded within 24 h. The highest dose which did not
lead to animal death and the lowest dose which
caused death of one mice per group were recorded.
The median of these two doses was defined as LD50
[19,20].
The assessment of brain-derived neurotrophic
factor
At this step, the sandwich ELISA method was used to
measure the level of BDNF (EK0309, Boster, China) in
the obtained serum samples using the sandwich ELISA
method according to the manufacturer’s instruc-
tion [21].
 International Journal of Neuroscience 881

Figure 1. Evaluation of seed extract of D. stramonium on sleep duration (1a) and latency (1b) in pentobarbital-induced hyp-
notic. Data are expressed as mean ± SEM of eight animals in each group. *p < 0.05 and ***p < 0.001 vs. diazepam group.
###
p < 0.001 vs. control.

Cytotoxicity and neurotoxicity assessment
The cytotoxicity effect of the hydro-alcoholic extract
of petal and its fractions was evaluated on PC12 cells.
This line has neuronal properties and can be used as
an appropriate in vitro model for investigating works
including protective or toxicity effects of extracts. The
cells were seeded in 96-well plates for 24 h with 10%
FBS, penicillin (100 units/ml) and streptomycin (100 µg/
ml). Next, the cells were incubated with different
doses of the hydro-alcoholic extract of petal (25–
800 µg/ml) and its fractions (800 µg/ml) for 24 h. Cell
viability was evaluated via MTT assay which was
described before [22]. The MTT solution is produced
at the final dose of 0.5 mg/ml. After adding of MTT
to each well, the plate was incubated for 2 h. After
this time, DMSO was added to solve formazan crystals.
In the end, the absorbance was read by StatFAX2100
ELISA reader (Awareness Technology Inc., USA)
at 545 nm.
LC-MS analysis of Datura stramonium extract
LC-MS apparatus
The LC-MS analysis was performed in an AB Sciex
QTRAP (Shimadzu) liquid chromatography coupled
with triple quadrupole mass spectrometer. Liquid
chromatography separation was done on a Supelco
C18 (15 mm × 2.1 mm × 3 µm) column. MS analysis
was carried out in positive mode of ionization. The
analysis was done at a flow rate of 0.5 ml/min. The
gradient analysis started with 95% of 0.2% aqueous
formic acid, isocratic conditions were maintained for
1 min, and then a 14-min linear gradient to 40%
acetonitrile with 0.2% formic acid was applied. From
15 to 40 min the acidified acetonitrile was increased
to 100%, followed by 5 min of 100% acidified aceto-
nitrile, and 5 min at the start conditions to
re-equilibrate the column. The mass spectra were
acquired in a range of 200 to 1200 within the 50 min
scan time. Mass feature extraction of the acquired
LC-MS data and maximum detection of peaks was
done using the MZmine analysis software package,
version 2.3.
Statistical analysis
The data were reported as mean ± SEM. Statistical anal-
ysis was performed using one-way analysis of variance
(ANOVA) followed by Tamhane’s T2 post-hoc test.
Significant difference was set at p < 0.05.
Results
Effect of seed extract on sleep duration and
sleep latency
As shown in Figure 1(a), diazepam increased sleep
duration in comparison with control group (48 ± 0.97vs
23 ± 1.5, p < 0.001). The extract at doses of 50 mg/kg
(29.8 ± 1.06, p < 0.05) and 100 mg/kg (38.6 ± 1.12,
p < 0.001) increased sleep duration significantly. Figure
1(b) showed that diazepam reduced the onset of sleep
(7.5 ± 0.64vs 3.25 ± 0.62, p < 0.001), also, extract
decreased latency time at doses of 50 mg/kg and
100 mg/kg, respectively (5.5 ± 0.28, p < 0.05; 4 ± 04,
p < 0.001).
882 M.-A. SOBHANIFAR ET AL.
Figure 2. Evaluation of calyx extract of D. stramonium on sleep duration (2a) and latency (2b) in pentobarbital-induced hyp-
notic. Data are expressed as mean ± SEM of eight animals in each group. *p < 0.05 and **p < 0.01 vs. diazepam group. ###p < 0.001
vs. control.
Effect of calyx extract on sleep duration and
sleep latency
In comparison with control group, diazepam increased
duration of sleep (48 ± 0.97vs 23 ± 1.5, p < 0.001) and
decreased latency time (3.25 ± 0.62 vs 7.5 ± 0.64)
(p < 0.001) (Figure 2(a,b)). The results showed that the
extract increased sleep duration at dose of 100 mg/kg
(31 ± 1.24, p < 0.01) and reduced latency time to 5 ± 1
(p < 0.05) (Figure 2(a,b)).
Effect of petal extract on sleep duration and
sleep latency
The petal extract potentiated sleep duration at doses
of 25–100 mg/kg (32.8 ± 0.86; p < 0.01, 42 ± 1.04 p < 0.001
and 51 ± 1.76 p < 0.001) (Figure 3(a)). Also, the extract
reduced latency time at doses of 50 mg/kg (4.5 ± 0.28,
p < 0.01) and 100 mg/kg (3.25 ± 0.47, p < 0.001) (Figure
3(b)). As shown in Figure 3(a,b), diazepam increased
sleep duration (48 ± 0.97vs 23 ± 1.5, p < 0.001) and
decreased latency (3.25 ± 0.62 vs 7.5 ± 0.64; p < 0.001)
in comparison with the control group.
Effect of flumazenil and naloxone on sleep
Flumazenil and naloxone were used for evaluation of
sleep-induction mechanism of extract. The results
revealed naloxone significantly reversed sleep duration
of 100 mg/kg petal extract while flumazenil had no
effect on sleep-prolonging by extract. Flumazenil
reversed significantly sleep duration effect of diazepam
while naloxone did not change hypnotic effect of
diazepam (Figure 3(a)). Also, flumazenil and naloxone
increased latency time of diazepam and hydro-alcoholic
extract of petal, respectively (Figure 3(b)).
Effect of fractions on sleep duration and sleep
latency
The hydro-alcoholic extract of petal had more effect
on sleep duration than calyx and seed. Therefore, dif-
ferent fractions including WF, EAF and NBF were pre-
pared from hydro-alcoholic extract of petal. Results
showed three fractions (WF: 36.75 ± 1.43, p < 0.001; EAF:
42.75 ± 1.03, p < 0.001; NBF: 56.25 ± 1.10, p < 0.001)
increased sleep duration significantly while did not
reduce latency time (Figure 4(a,b)). As shown in Figure
4, diazepam increased sleep duration and attenuated
latency time significantly.
Determination of BDNF in serum
As shown in Figure 5, the level of BDNF was dramat-
ically increased in the samples of the hydro-alcoholic
extract of petal. The extract at the doses of 50 and
100 mg/kg significantly enhanced the level of BDNF in
serum (p < 0.05) while in lower dose (25 mg/kg) this
effect was not significant in comparison with untreated
animals (p > 0.05).
LD50 determination
The highest dose of hydro-alcoholic extract of petal
which did not lead to death of any mice and the
 International Journal of Neuroscience 883

Figure 3. Evaluation of petal extract of D. stramonium on sleep duration (3a) and latency (3b) in pentobarbital-induced hyp-
notic. Data are expressed as mean ± SEM of eight animals in each group. **p < 0.01 and ***p < 0.001 vs. diazepam group,
###
p < 0.001 vs. control, OOOP < 0.001 vs. extract xxxP < 0.001 vs. diazepam.

Figure 4. Evaluation of different fractions on sleep duration (4a) and latency (4b) in pentobarbital-induced hypnotic. Data are
expressed as mean ± SEM of eight animals in each group. ***p < 0.001 vs. diazepam group. ###p < 0.001 vs. control.

lowest dose which cause to death of one mouse per
group were 3.2 g/kg and 6.4 g/kg. Therefore, the LD50
was 4.8 g/kg.
Evaluation of the cytotoxicity effects of D.
stramonium
The cytotoxicity of the extract was evaluated on PC12
cells. The results revealed hydro-alcoholic extract of
petal at different doses (25–800 µg/ml) and its frac-
tions did not decrease cell viability after 24 h (Figure 6).
LC-MS analysis of Datura stramonium extract
In total, 18 compounds were identified in the
hydro-ethanol extract of D. stramonium using LC-MS
analysis in positive mode. These compounds are
including hydroxy-γ-sanshool, avenanthramide A, sar-
mentosin, paucin, etc. Data concerning the identifica-
tion of the compound are shown in Table 1. The MS
spectral data were compared with the reported com-
pounds in some previous literature. Figure 6 and 7 are
the examples of extracted ion chromatograms from
the total ion chromatogram and its related mass.
884 M.-A. SOBHANIFAR ET AL.
Figure 5. Effect of hydro-alcoholic extract on BDNF level in
serum. Data are expressed as mean ± SEM of eight animals in
each group. *p < 0.05 vs. control.
Figure 6. Effect of hydro-alcoholic extract of petal and its
fractions of D. stramonium on PC12 cell viability. The cells
were exposed to different doses of extract (25–800 µg/ml),
800 µg/ml water fraction (WF), 800 µg/ml ethyl acetate fraction
(EAF) and 800 µg/ml n-butanol fraction (NBF). Values are
mean ± SEM (n = 4).
Discussion
Traditional medicine has reported that the consump-
tion of Datura genus can induce sleep similar to thio-
pental in animals [23,24]. In this research, the hypnotic
effect of D. stramonium was evaluated for the first time
in mice. Our findings showed that various parts of D.
stramonium increased sleep duration with different
potencies. The obtained extract from petal was indi-
cated to have more effects compared to other parts
involving seed and calyx. Different fractions of the
hydro-alcoholic extract of petal increased sleep dura-
tion. The LC-MS revealed the presence of 18 various
active compounds in this herb which can have role in
hypnotic-induced effect. The neurotoxicity assay
showed no toxicity effects on PC12 cells. The extract
increased the level of BDNF in serum. The high LD50
value reflected the very low toxicity of extract.
Diazepam, as a benzodiazepine drug, increases sleep
duration via binding to GABAA receptors [25]. Our study
revealed, D. stramonium potentiated the hypnotic effect
of pentobarbital similar to diazepam. To estimate pos-
sible mechanisms, flumazenil and naloxone as selective
antagonists of benzodiazepine and opioid receptors
were used. Our results showed, that naloxone reversed
the hypnotic effect of petal extract at the dose of
100 mg/kg while flumazenil had no effect. However,
probably, opioid receptors play role in hypnotic effect
of D. stramonium. Other study reported, hypnotic effect
of Datura metel was reversed by naloxone which showed
the role of opioid receptors [24,26]. Different fractions
of the hydro-alcoholic extract of petal were prepared
to investigate which components may have role in hyp-
notic effect. The fractions are including (1) the WF, is
including polar agents such as tannins, quaternary, alka-
loids glycosides; (2) the intermediate polarity agents
which were extracted in EAF; (3) the non-polar agents
like alkanes, sterols, some terpenoids that were sepa-
rated by NBF [27]. This research showed that all types
of fractions could significantly potentiate sleep duration.
The extract is composed of polar and non-polar ingre-
dients which are responsible for hypnotic effect. The
recent studies have shown different ingredients such
as flavonoids, alkaloids, steroids and saponins can lead
to sleep induction [28–30]. The phytochemical studies
have previously reported the presence of active com-
pounds involving tropane alkaloids, atropine, scopol-
amine, hyoscyamine, hyoscine, ferulic acid, sitosterol
and chlorogenic acid in Datura species [17]. In the pres-
ent study, LC-MS was done and determined other com-
ponents that may have a role in hypnotic effect. A
clinical study showed administration of chlorogenic acid
in healthy men improved quality sleep and fatigue upon
awakening [31]. Also, another study revealed that sit-
osterol as an active compound in Datura genus had
anxiolytic and sedative effects in mice via GABAA recep-
tors [32]. Ferulic acid increased sleep duration in mice
via serotonergic system [33]. The studies have reported
the most pharmacological properties of D. stramonium
such as pain relief and sedation can be mediated via
alkaloids as hyoscine, scopolamine, atropine and hyos-
cyamine [34]. Recent studies have reported anti-epileptic
and CNS depression of D. stramonium may be related
to tropane alkaloids which have anti-cholinergic prop-
erties [15,35]. However, it could be suggested that the
 International Journal of Neuroscience 885

Figure 7. (A) The total ion chromatogram of D. stramonium using LC-MS in the positive mode. (B) Chromatogram of hydroxy-γ-
sanshool and corresponding mass adduct, [M + H], at m/z 290.1. (C) Chromatogram of avenanthramide A and corresponding
mass adduct, [M + H], at m/z 299.88. (D) Chromatogram of purpurogallin and corresponding mass adduct, [M + H], at m/z 220.62.

Table 1. The compounds of hydro-alcoholic extract of D. stramonium using LC-MS method.

Peak No. Compound RT (min) [M + 1] m/z Ref.
1 Hydroxy-γ-sanshool 9.2 290.1 [41]
2 Avenanthramide A 48.3 299.88 [41]
3 Purpurogallin 47.7 220.62 [41]
4 Perlolyrine 47.4 265.5 [41]
5 Entacapone 9.4 306.66 [41]
6 Sarmentosin 23.6 276.42 [41]
7 2-Phenyl-1,8-naphthyridine 48.6 207.36 [41]
8 Eupatoroxin 39.7 393.66 [41]
9 Proacacipetalin 10.9 260.7 [41]
10 2-(Formamido)-N1-(5-phospho-d-ribosyl) 23.6 314.34 [41]
acetamidine
11 Nocardicin C 18.7 487.98 [41]
12 Paucin 23.5 469.26 [41]
13 Cinchoninone 10.7 293.04 [41]
14 Imazaquin 13.9 312.66 [41]
15 Tiliroside 23.9 608.34 [41]
16 Anisodamine 8.9 306.06 [42]
17 3-O-Methylisoetharine 31.4 254.46 [42]
18 Hydrocortisone cypionate 18.7 487.98 [42]
886 M.-A. SOBHANIFAR ET AL.
presence of different active compounds especially alka-
loids is responsible for the hypnotic effect of this plant.
Another possible mechanism can be related to elevation
of BDNF expression. The different studies have reported
relationship between BDNF level and insomnia [36]. The
biological effects of BDNF are involving improvement
of neuro-system function by tyrosine kinase receptor B
[37]. Kushikata et al. reported administration of BDNF
in lateral ventricles can induce sleep in rats and rabbits
[38]. Another study has reported that increasing BDNF
leads to improvement and maintenance of sleep in rats
[39,40]. Our findings revealed that the extract increased
the amount of BDNF in serum which can be another
reason for hypnotic effect of D. stramonium.
In vivo and in vitro studies were performed to inves-
tigate the possible toxicity of extract. The PC12 cells
were incubated with different doses of hydro-alcoholic
petal extract and its fractions, results showed no toxicity
in this line. Also, in vivo study revealed no toxicity and
the LD50 value was high. According to in vitro and in
vivo studies the extract did not have toxicity, but more
investigation is needed to prove the safety of extract.
Conclusion
Our study revealed that the extracts, especially petal
extract potentiated sleep duration in mice. This effect
may be related to active ingredients which are found in
the extract which are isolated by NBF fraction. Non-polar
agents are probably responsible for the observed effect.
The elevation of BDNF by the extract can mediate the
hypnotic effect of D. stramonium. Whereas, naloxone
reversed the hypnotic effect, however opioid receptors
play role in extract-induced sleep effects.
Limitations
This study has some limitations including the prepa-
ration of active compounds, evaluation of hypnotic
effect and investigation of more genes. More studies
are needed to evaluate the main components and
accuracy mechanisms.
Disclosure statement
The authors declare that there is no conflict of interest
regarding the publication of this article.
Funding
This work was supported by a grant (951305) from the Vice-
Chancellor for Research and Technology, Mashhad University
of Medical Sciences (MUMS), Mashhad, Iran. This work was
supported by a grant (951305) from the Vice-Chancellor for
Research and Technology, Mashhad University of Medical
Sciences (MUMS), Mashhad, Iran.
ORCID
Maede Hasanpour http://orcid.org/0000-0002-8260-6123
Mehrdad Iranshahi http://orcid.org/0000-0002-3018-5750
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