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Hydroalcoholic extract from Abelmoschus

Cite this: Food Funct., 2020, 11, 8978
manihot (Linn.) Medicus flower reverses sleep
deprivation-evoked learning and memory deficit†
Hai-rui Zhou, Jing-ru Wu, Lei Bei, Bai-xin Wang, Hui Xu, Jing-tao Wang* and
Shu-xia Ma *

Received 16th August 2020,
Accepted 12th September 2020
DOI: 10.1039/d0fo02158j
rsc.li/food-function
Previous researches have indicated that sleep plays a vital role in cognitive functions. Sleep deprivation
(SD) causes learning and memory damage, which is associated with oxidative stress. This study was per-
formed to investigate the neuroprotective effects of an extract of Abelmoschus manihot flower (EAM)
against memory deficit induced by SD in mice. The SD model was evoked by multiple platform method
for 5 days, successively. The learning and memory-improving effects of EAM were assessed by behavioral
trials and the underlying mechanism was investigated by measuring the oxidative stress alteration. Our
findings indicated that the SD-induced memory deficit and the EAM treatment improved the cognitive
functions of mice in the object location recognition test and passive avoidance task. In addition, EAM
effectively improved the activities of the antioxidant enzyme, decreased the content of malondialdehyde
(MDA), and restored the protein expression of the brain-derived neurotrophic factor (BDNF), tyrosine
kinase B (TrkB) and glutamate receptor 1 (GluR1) in brain tissues. In conclusion, EAM could improve the
SD-evoked learning and memory impairments. The possible underlying mechanisms of EAM may be
related to its antioxidant capacity and enhanced BDNF/TrkB/GluR1 levels in the hippocampal memory.

Introduction otine, modafinil, and donepezil could alleviate memory

Sleep plays an important role in the human mental and phys-
iological performance and is necessary for human health life.1
Sleep deprivation (SD), including fragmented, insufficient and
absent sleep, is a universal phenomenon in the modern fast-
paced lifestyles.2 A number of laboratory and epidemiological
studies have demonstrated that cognitive impairments are the
results of SD in numerous behavioral tasks.3–6 In addition,
sleep deprivation is also associated with some health pro-
blems, such as hyperlipidemia, depression, Alzheimer’s
disease, hypertension, and diabetes.7 Moreover, a sleep dis-
order may induce obvious damage in the endocrine, immune,
and cardiovascular systems as well as memory and learning
ability.8 Though the detailed mechanism of cognitive impair-
ments evoked by SD remains unknown, it is widely believed
that SD may influence the hippocampus region of the brain by
damaging the memory formation.9 This impairment is
believed to be caused by the production of reactive oxygen
species and elevation of oxidative stress in different brain
regions.10 Pharmacological interventions, such as caffeine, nic-
Basic medical college of Jiamusi University, Heilongjiang 154007, China.
E-mail: wjingtao@sina.com, mshuxia0808@163.com
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
D0FO02158J
impairment evoked by SD in animal models.8,11 However, the
application of these drugs is limited due to the side effects,
such as disorientation, daytime fatigue, and memory loss.12
Therefore, it is urgent to develop a novel therapy with low side
effects and high efficacy to alleviate the cognitive impairment
of SD.
Abelmoschus manihot (Linn.) Medicus, also known as
“Huang Shukui”, is a traditional Chinese medicine. A. manihot
flowers, the major medicinal part of this herb, have been
demonstrated to possess numerous pharmacological activities
such as treatment of chronic kidney disease, oral ulcers,
burns, and inflammatory diseases.13 Flavonoids, resins, pig-
ments, and polysaccharides have been isolated and purified
from the A. manihot flowers in the past decade.14 Total flavo-
noid of A. manihot has been demonstrated to have neuropro-
tective activity and it could prevent the cerebral ischemia
damage in animal models.15–17 In addition, flavonoids might
represent the potential bioactive constituents of the A. manihot
ethanol extract and possess the antidepressant-like and antic-
onvulsant effects in vivo.14 Nonetheless, there is no study
related to the effects of the extract of the A. manihot flower
(EAM) on SD-induced memory impairment.
Therefore, in the present study, we hypothesized that EAM
could prevent the SD-induced deficit of the hippocampal
memory owing to its antioxidative effect. To validate this

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Food & Function
hypothesis, SD was induced by the multiple platform method.
Behavioral studies were performed to explore the neuroprotec-
tive effects of EAM on cognitive impairments induced by SD.
Materials and methods
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Chemicals and drugs
Rutin, hyperoside, isoquercetin, myricetin, quercetin-3-O-glu-
coside and quercetin were purchased from Macklin Chemicals
Co., Ltd (Shanghai, China). HPLC grade acetonitrile and
formic acid were purchased from Sigma-Aldrich (St Louis,
USA). The flowers of A. manihot were harvested in October
2018 from Jiangyan County of Jiangsu Province and were
identified by Dr Hui Xu according to the Pharmacopeia of the
People’s Republic of China. All other chemical reagents used
in the experiment were of analytical grade and purchased from
Aladdin Chemicals Co., Ltd (Shanghai, China).
Preparation of EAM
The preparation process of the hydroalcoholic extract from an
A. manihot flower was referenced in the previous report.18
Briefly, 8 L of 75% ethanol was added to a 0.5 kg dried
A. manihot flower. Then, the mixture was extracted in a hot
water bath at 90 °C for 1 h. The extract was subsequently fil-
tered and the filtrate was evaporated using vacuum rotary evap-
oration; then, the concentrated liquid was further dried using
a freeze-dryer. The total flavonoid content of EAM was
measured using a colorimetric method with minor modifi-
cation.13 The total flavonoid content was expressed as mg
rutin equivalents per g of the dry EAM weight.
Analysis of the flavonoids in EAM by HPLC
The HPLC analysis was carried out on a liquid chromatography
system (Waters 2695, USA) equipped with a degasser, quatern-
ary pumps, and UV detection. The temperature of an Agilent
ZORBAX Eclipse Plus C18 (150 mm × 4.6 mm, 5 μm) column
was maintained at 35 °C, and the wavelength was recorded at
360 nm. The mobile phase consisting of acetonitrile (A) and
0.02% formic acid aqueous solution (B) was applied in gradi-
ent for elution: 0–4 min, 15% A–17% A; 4–20 min, 17% A–30%
A; 20–35 min, 30% A–80% A; 35–45 min, 80% A at a flow rate
of 0.8 mL min−1.18 The content of flavonoid ingredients were
Fig. 1 Schematic design of the animal experiment.
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measured by the peak area of their maximum absorption
wavelength.
Animals and drug administration
The animal experiment was approved by the Local Ethics
Committee and performed in accordance with the National
Institutes of Health Guide of the Care and Use of Laboratory
Animals. Four-week-old male ICR mice (20 ± 2 g) were
obtained from the Laboratory Animal Center of Guangdong
(Guangzhou, China) and were kept in Experimental Animal
Center of Basic Medical College of Jiamusi university under a
standard laboratory environment (humidity 50–60%, air temp-
erature 20–25 °C, and 12 h light/dark cycle), with free access to
water and food.
After a habituation period of 14 days, all the animals were
arbitrarily divided into five groups: control group mice treat-
ment with distilled water (Con), sleep deprivation group mice
treatment with distilled water (SD), sleep deprivation group
with the daily administration dose of 50 mg kg−1 EAM (SD +
LEAM), sleep deprivation group with the daily administration
dose of 100 mg kg−1 EAM (SD + MEAM), sleep deprivation
group with the daily administration of 200 mg kg−1 EAM (SD +
HEAM). The doses of EAM were based on a previous study.14
EAM was dissolved in distilled water by ultrasound. The
LEAM, MEAM, and HEAM groups were treated with EAM at
8:00 am daily for 20 days via oral gavage. Moreover, the Con
group and SD group of rats were treated with equal amounts of
distilled water via oral gavage. The experimental design pro-
cedure is illustrated in Fig. 1.
The induction of sleep deprivation
SD was induced using the modified multiple platform
method.19 The water tank (length: 110 cm, height: 40 cm,
width: 60 cm) contained 15 small platforms (the diameter is
3.5 cm). Water is filled till two centimeters below the surface
of the platform. Mice were arranged above the platforms, and
they were allowed to jump and move freely from one platform
to another. Once the mice experienced the paradoxical of sleep
and muscle atonia set in, the mice were awakened by throwing
them into the water. In the study, the SD was carried out for
successive 5 days. All the mice were having free access to food
and water. The water in the tank was changed every day and
maintained at the temperature of 23–25 °C.
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Open field test (OFT)
After the SD for 5 days, OFT was performed to evaluate
whether the EAM treatment had an effect on the locomotor
activity of the mice according to a previous research.20 The
apparatus was made up of four plastic tanks (length: 30 cm,
height: 35 cm, width: 28 cm) using a 120 lx light intensity. A
camera was installed on top of the tank to record the path of
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the animal. After the EAM treatment for 30 min, each mouse
was allowed to explore freely around the tank for 3 min. The
total distance traveled and average speed (during a 10 min
test) was recorded as a measure of the locomotor activity. A
video-tracking system (Shanghai Jiliang Software Technology
Co Ltd) was used to record and analyze the behavioral tests.
Object location recognition (OLR) test
Memory and learning capacity was measured by the OLR test
according to a previous report with minor modification.21 This
test occurred in the target recognition test system with an
enclosed field (length: 40 cm, width: 50 cm) surrounded by a
wall (height: 50 cm). The cabinets were equipped with LED
floodlights on both the sides, for avoiding the disturbance of
strong light on the behavior of mice. The exploratory behavior
of mice was observed by the camera on the ceiling. The whole
experiment consisted of three stages: habituation stage, fam-
iliarization stage, and test stage. The habituation stage lasted
for three days, and the animals were then exposed to the stage
alone, in turn, to explore a new environment for 10 min to
eliminate the fear of mice. The trials were performed on the
fourth day, which were divided into a familiarization stage and
test stage (30 min interval between the two stages). At the fam-
iliarization stage, the mouse was put in an arena that con-
tained the object A1 and A2 and allowed the free exploration of
5 min for each trial. The test trial was performed after a
30 min delay. The mouse was returned to the area in which
one of the primary objects had altered position (“novel”) and
another object was maintained in the original location (“fam-
iliar”). Objects were put in the center of the arena about 10 cm
from the wall. At the end of each trial, the floor of the objects
and arena were washed with alcohol (70%) to eliminate the
odor cues. Only when a mouse sniffs or touches an object, it
was identified as the exploratory behavior. We recorded the
time of contact between each object during each trial. The
animal with exploratory behavior was recorded and scored
with a video camera. The discrimination index (DI) was used
to evaluate the recognition memory; the calculation formula is
as given below:
DI ¼ ðT N  T F Þ=ðT N þ T F Þ
where TN and TF represent the exploration time of the novel
and familiar object, respectively. The total exploration time
was recorded during the familiarization stage.
Passive avoidance (PA)
To evaluate the effect of EAM on the memory and learning, the
passive avoidance trial was carried out according to the pre-
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vious research with minor modification.22 The test apparatus
was divided into a dark chamber and a white illuminated
chamber (length: 17 cm, height: 25 cm, width: 13.5 cm). After
180 s of acclimatization, each animal was placed into the light
chamber to freely explore for 300 s in the training experiment.
The 0.5 mA electric foot shock of 0.5 mA for 5 s was given
when the animal entered the dark chamber. The consolidation
experiment was carried out in the same way as training after
24 h. The time of latency into the dark chamber was recorded
in the 300 s test process.
Preparation of biochemical test samples
The mice were immediately anesthetized by a combination
of xylazine and ketamine (10 mg kg−1 and 90 mg kg−1, intra-
peritoneal, respectively) and executed by decapitation after
the behavioral test. The brains were collected quickly, and
the hippocampus and cortex were isolated from the midsa-
gittal plane, and quickly stored at −80 °C. The hippocampus
and cortex of different groups were homogenated with ice
normal saline (1/10, m v−1) using a homogenizer. The super-
natant was obtained by centrifugation at 4000 rpm for 5 min
at 4 °C.
Determination of oxidative stress markers
The activities of superoxide dismutase (SOD), glutathione
(GSH) and catalase (CAT) and the level of malonaldehyde
(MDA) were assayed using commercial assay kits according to
the manufacturer’s protocol (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China).
Western blotting
Hippocampal tissues were lysed using the RIPA lysis buffer.
Then, the samples were centrifuged at 10 000 rpm for 20 min.
The total protein content of the supernatant was quantified
with a BCA assay kit. 100 µg of protein was separated by 10%
SDS-polyacrylamide gel and transferred to nitrocellulose mem-
branes. The membranes were blocked for 1 h using 5% nonfat
dried milk in the TBST buffer. Blotted membranes were incu-
bated overnight at 4 °C with the following primary antibodies:
anti-BDNF (1:1000; Abcam), anti-GluR1 (1:1000; Abcam), anti-
TrkB (1:1000; Abcam), and β-actin (1:1000; Abcam). After
washing with TBST, the membranes were incubated with the
HRP-labeled goat anti-mouse IgG antibody at room
temperature for 1 h. Visualization was carried out with the
Immobilon Western ECL system. The relative protein levels
were assayed using the Gel Pro Analysis software and normal-
ized to β-actin.
The data statistical analysis
Data from the behavioral assessment, oxidative stress bio-
markers, and western blot were reported as the means ± SEM,
and the statistical analyses were performed using the
GraphPad Prism Software (GraphPad Software, Inc., La Jolla,
CA, USA). Differences between the groups were analyzed using
one-way ANOVA, followed by the Tukey’s multiple comparison
test. P < 0.05 was usually considered as statistical significance.
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Results
HPLC profiles of EAM extract
As shown in Fig. 2A, the HPLC-UV analysis indicated the major
bioactive constituents in the EAM extract. The six flavonoid
compounds were identified as (1) rutin; (2) hyperoside; (3) iso-
quercetin; (4) myricetin; (5) quercetin-3-O-glucoside; and (6)
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quercetin, via the HPLC method by comparing with an auth-
entic standard. As shown in Table 1, the contents of rutin,
hyperoside, isoquercetin, myricetin, quercetin-3-O-glucoside,
and quercetin in EAM were 6.81 ± 0.18 mg g−1, 126.50 ±
2.54 mg g−1, 59.86 ± 1.08 mg g−1, 119.46 ± 2.40 mg g−1, 146.37
± 2.91 mg g−1, and 4.93 ± 0.06 mg g−1, respectively. In
addition, the total flavonoid content in the EAM was 766.59 ±
4.43 mg g−1 dry extract via a UV spectrophotometer.
The effect of EAM on the locomotor activities of SD mice
The OFT was carried out to assess the locomotor activities of
animals that may influence the cognitive behavior. There were
no obvious differences in the average speeding and total dis-
tance among all the groups (P > 0.05). The findings showed
that the EAM administration did not influence the locomotor
activities of the ICR mice (Fig. 2B and C).
The effect of EAM on the OLR task of SD mice
In the present study, the OLR task was performed to investi-
gate the effect of EAM on the spatial memory and learning. As
shown in Fig. 3A, the finding showed that there was no
obvious difference in the total exploration time of the two
objects (A1 and A2) as compared to the other groups (P > 0.05).
The finding implied that there was no significant difference in
the exploration ability of mice for the objects.
As shown in Fig. 3C, our findings indicated that SD impairs
memory as evidenced by the obvious decrease in the DI of the
SD model group when compared to the control group (P <
0.01). However, the EAM (100 and 200 mg kg−1) treatment
obviouly increased the DI of the SD group (P < 0.05 and P <
0.01). Our result showed that the administration of EAM could
improve the SD-evoked spatial memory impairment.
Fig. 2 (A) Chromatograms of flavonoids occurring in the hydroalcoholic extract of Abelmoschus manihot flower. (1) Rutin; (2) hyperoside; (3) iso-
quercetin; (4) myricetin; (5) quercetin-3-O-glucoside; (6) quercetin. The effect of EAM on the locomotor in the SD-induced memory impairment. (B)
Total distance. (C) Average speeding. Results are expressed as the means ± SEM, n = 12 animals per group.
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Table 1 Determination of representative flavonoids in extract of
Abelmoschus manihot flower (EAM). Data are presented as the mean ±
standard deviation (SD) of three independent experiments
Component Content (mg g−1 extract)
Rutin 6.81 ± 0.18
Hyperoside 126.50 ± 2.54
Isoquercetin 59.86 ± 1.08
Myricetin 119.46 ± 2.40
Quercetin-3-O-glucoside 146.37 ± 2.91
Quercetin 4.93 ± 0.06
Total flavonoids 766.59 ± 4.43
The effect of EAM on the PA test of SD mice
In the present study, the PA test was performed to measure the
non-spatial memory of mice. As shown in Fig. 3B and D, the
SD-evoked memory impairments of mice showed obvious
decrease in the time of latency into the dark chamber and
increased the number of errors as compared to the control
mice (P < 0.01). However, the EAM (100 and 200 mg kg−1) treat-
ment prevented the increase in error numbers and the
reduction of the latency into the dark chamber as compared to
the SD group (P < 0.05 and P < 0.01).
The effect of EAM on oxidative stress biomarkers in the brain
tissue of SD mice
As shown in Fig. 4 and 5, compared with the control group,
the activities of SOD, GSH, and CAT in the cortex and hippo-
campus obviously reduced in the SD groups (P < 0.01).
Administration of EAM (100 and 200 mg kg−1) increased the
activities of SOD, GSH and CAT levels (P < 0.05 and P < 0.01).
In addition, compared with the control group, the MDA con-
tents of the cortex and hippocampus were obviously increased
in the SD group (P < 0.01). Treatment with EAM (100 and
200 mg kg−1) decreased the content of MDA in the SD group (P
< 0.05 and P < 0.01).
The effect of EAM on BDNF, TrkB and GluR1 levels in the
hippocampus of SD mice
As shown in Fig. 6, the protein expression of BDNF, TrkB, and
GluR1 was downregulated in the hippocampus of SD mice (P <
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Fig. 3 The effect of EAM on OLR and PA task in the SD-induced memory impairment. (A) Total exploration time in the familiar phase, (B) latency
into the dark chamber, (C) DI in the test phase. (D) Error numbers. Results are expressed as the means ± SEM, n = 12 animals per group. #P < 0.01
(vs. Control group); **P < 0.01, *P < 0.05 (vs. SD group).

Fig. 4 The effect of EAM on the oxidative stress in the cortex of SD mice. Results are expressed as the means ± SEM, n = 12 animals per group. #P <
0.01 (vs. Control group); **P < 0.01, *P < 0.05 (vs. SD group). Cortex SOD (A), GSH (B), CAT (C) and MDA (D) level in different groups.

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Fig. 5 The effect of EAM on the oxidative stress in the hippocampus of SD mice. Results are expressed as the means ± SEM, n = 12 animals per
group. #P < 0.01 (vs. Control group); **P < 0.01, *P < 0.05 (vs. SD group). Hippocampus SOD (A), GSH (B), CAT (C) and MDA (D) level in different
groups.

Fig. 6 The effect of EAM on BDNF, TrkB and GluR1 levels in the hippocampus of SD mice. Results are expressed as the means ± SEM, n = 3 animals
per group. #P < 0.01 (vs. Control group); **P < 0.01, *P < 0.05 (vs. SD group).

0.01). However, treatment with EAM (100 and 200 mg kg−1)
restored the SD-induced inhibition of BDNF, TrkB and GluR1
protein expression (P < 0.05 and P < 0.01).
Discussion
Our results indicated that the administration of EAM reversed the
learning and memory impairments evoked by SD. Moreover, the
EAM improved the activities of antioxidant enzymes and decreased
the content of MDA in the SD-induced cognitive deficit. In the
present study, we used EAM at three different doses of 50, 100, and
200 mg kg−1. However, treatment with 100 mg kg−1 of EAM also
exerted improvement effects in the behavioral assessment and oxi-
dative stress biomarkers, while 200 mg kg−1 of EAM exerted more
obvious protective effects in the SD-induced cognitive and memory
disorders mice model. So, the maximal protective effect of EAM
was achieved at a dose of 200 mg kg−1.

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Sleep plays an important role in the memory formation and
cognitive functions of humans. Sleep insufficiency could
obviously weaken the learning and memory ability. Cognitive
functions and sleep are considered to be associated with each
other. Thus, numerous researches are focused on the effects of
SD on the cognitive functions.23,24 Also, a previous study indi-
cated that the modified multiple platform model induced SD
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to memory impairment.5 Moreover, numerous studies have
indicated SD induced memory deficits in numerous behavioral
tasks.25,26 The OLR test utilizes the exploratory ability of
animals toward spatial novelty, and it was used to evaluate the
spatial relocation of the familiar object.25 The PA test exploits
the animal’s tendency to avoid darkness and this test was suit-
able to evaluate the non-spatial memory performance.25 In
accordance with the previous studies, the present results indi-
cated that the SD damaged non-spatial and spatial memories
in rodents.25,27
Previous studies indicated that the SD-induced memory
impairment was involved in the increase in the oxidative stress
of the brain tissue.28,29 Oxidative stress is an imbalance status
between the antioxidant defense system and the reactive
oxygen species. Superfluous reactive oxygen species could
evoke neuronal impairment via antioxidant enzyme inacti-
vation, DNA modification, and lipid peroxidation, and even-
tually cause the damage of cognitive functions.30 Previous
studies indicated that the SD could induce the oxidative stress
of brain tissue by increasing the content of MDA as well as
decreasing the activities of antioxidant enzymes in the brain
tissue.19,25 In agreement with the previous report, the present
findings indicate that the cognitive deficit induced by SD was
accompanied by elevated oxidative stress of the brain tissue.
Non-enzymatic and enzymatic antioxidant defense systems
could eliminate lipid peroxide and alleviate the impairment of
neurons from the reactive oxygen species.19 Thus, those com-
pounds with antioxidant ability could protect the neurons
against the impairment of reactive oxygen species and there-
fore prevent the occurrence of cognition dysfunctions.8,19
Meanwhile, the flavonoid extract from A. manihot flowers could
alleviate the D-galactose-evoked oxidative stress via the acti-
vation of Nrf2 signaling.31 Therefore, the extract from
A. manihot flowers might be a therapeutic drug to alleviate the
learning and memory damage associated with the SD related
conditions. In our study, after the treatment of EAM, the activi-
ties of SOD and CAT as well as the content of GSH and MDA,
were obviously normalized. These results provide strong proof
supporting the effect of oxidative stress in the learning and
memory deficits after SD.
A previous study has indicated that BDNF plays a vital role
in synaptic plasticity through the improvement of the
efficiency of synaptic transmission via activating the protein
kinases.32 In addition, brain tissue with poor antioxidant
defenses and high mitochondrial energy metabolism might be
influenced by the oxidative stress induced by increasing free
radical levels, which results in a decline in the TrkB and BDNF
levels and an increase in the hippocampal neuron impair-
ment.33 Also, the reduction of TrkB and BDNF levels was
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related to the impairment of spatial memory.34 In the present
study, the hippocampal protein expression levels of BDNF,
TrkB, and GluR1 decreased in the SD group, and EAM could
modulate the BDNF, TrkB, and GluR1 protein expression to
improve synaptic plasticity, and these findings were in agree-
ment with the behavioral result.
The neuroprotective effect of EAM on SD-evoked memory
deficits has not been previously reported. In the present study,
we observed that the treatment of EAM reverses the non-
spatial and spatial memory deficiency induced by SD by alle-
viating the oxidative stress markers of brain tissue. In addition,
the HPLC analysis of the EAM revealed the presence of flavo-
noids, including rutin, hyperoside, isoquercetin, myricetin,
quercetin-3-O-glucoside, and quercetin (Fig. 1). Previous
studies have indicated that flavonoids possess neuroprotection
effects against SD-induced neuro behavioural
impairments;35,36 quercetin is effective in alleviating manic-
like behavior and brain oxidative stress induced by SD37 and
myricetin alleviates the cognitive impairment and neuro
degeneration in Parkinsonism.38 In accordance with previous
studies, our findings showed the involvement of the anti-
oxidant effect of flavonoids in its memory protective effect.
In summary, the results of the present study indicate that
the EAM treatment for twenty consecutive days improves the
memory and learning performances of sleep-deprived mice. In
addition, the neuroprotection effect of EAM involved in inhi-
biting the oxidative stress of the brain tissue was also indi-
cated. Therefore, EAM has the potential to become a thera-
peutic agent for the treatment of cognition deficits disease.
Ethical statement
All animal procedures were performed in accordance with the
National Institutes of Health Guide of the Care and Use of
Laboratory Animals and approved by the Animal Ethics
Committee of Jiamusi University.
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgements
This study was financially supported by the Science and
Technology Innovation Team Project of Heilongjiang province
(No. 2018-KYYWF-0915) and Innovation Team Project of
Jiamusi University (No. cxtdpy-2016-02).
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