Volume 73, No.6: 2021 Siriraj Medical Journal
Original Article
Wipawan angnipon, Ph.D.*, Sukonthar Ngampramuan, Ph.D.*, Nopparat Suthprasertporn, Ph.D.*, Chanati
Jantrachotechatchawan, Ph.D.**, Patoomratana Tuchinda, Ph.D.***, Saksit Nobsathian, Ph.D.****
*Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom 73170, ailand, **Wolfson Centre
for Age-Related Diseases, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, United Kingdom, ***Department of Chemistry,
Faculty of Science, Mahidol University, Bangkok 10700, ailand, ****Nakhon Sawan Campus, Mahidol University, Phayuhakiri, Nakhon Sawan
60130, ailand.
Protective Roles of N-trans-feruloyltyramine Against
Scopolamine-Induced Cholinergic Dysfunction on
Cortex and Hippocampus of Rat Brains
Objective: To study the protective eects of N-trans-feruloyltyramine (NTF) on scopolamine-induced cholinergic
dysfunction, apoptosis, and inammation in rat brains.
Materials and Methods: Treatments were administered intraperitoneally (i.p.). Wistar rats (8-week-old) were
allocated into 4 groups (n = 3) as follows: scopolamine-only, NTF-only, NTF + scopolamine and control. Spatial
cognition was evaluated by Morris water maze. ROS assay and Western blot analyses were conducted in 3 brain
regions: the frontal cortex, hippocampus, and temporal cortex.
Results: NTF treatment inhibited scopolamine-induced memory impairment and signicantly attenuated scopolamine-
induced changes in the three brain regions. Investigated scopolamine-associated changes were as follows: increases
in ROS production and BACE1 level, decrease in ChAT level, increases in inammatory and apoptotic markers,
and activation of signaling pathway kinases related to inammation and apoptosis.
Conclusion: With its in vivo antioxidant, cholinergic-promoting, anti-apoptosis, and anti-inammatory biological
activities, NTF is a promising candidate to be further investigated as a potential treatment for Alzheimer’s-associated
Keywords: Acetylcholine; Alzheimer’s disease; antioxidant; Morris water maze; N-trans-feruloyltyramine (Siriraj
Med J 2021; 73: 413-422)
Corresponding author: Wipawan angnipon
E-mail: wipawan.tha@mahidol.ac.th
Received 5 February 2021 Revised 20 April 2021 Accepted 22 April 2021
ORCID ID: http://orcid.org/0000-0001-7889-2801
Alzheimer’s disease (AD) causes progressive and
irreversible deterioration of cognitive functions especially
e major pathological characteristics of the
human AD brain are extracellular aggregates of amyloid-β
(Aβ) and intracellular aggregates of hyperphosphorylated
tau, namely the senile plaques and the neurobrillary
tangles respectively.
The underlying mechanism of
sporadic AD involves cholinergic dysfunction including
degeneration of basal forebrain cholinergic neurons
and loss of hippocampal cholinergic bers.
cholinergic transmission aects learning and memory,
cortical and hippocampal information processing, and
ultimately behaviors.
Scopolamine, an antagonist of a
muscarinic acetylcholine receptor,
induces cholinergic
dysfunction and cognitive impairment through oxidative
Volume 73, No.6: 2021 Siriraj Medical Journal
stress and neuroinflammation in animal models.
Intraperitoneal injection of scopolamine blocks cholinergic
neurotransmission, dysregulates cholinergic system, and
consequently impairs cognition in rodents.
N-trans-feruloyltyramine (NTF) was purified from
Polyalthia suberosa, a shrubby tree found throughout
southeast Asia and south China whose parts are used
in ai traditional medicine.
NTF has potent radical-
scavenging antioxidant property, especially from phenolic
hydroxyls. NTF and the other puried compound studied
by our group, N-benzylcinnamide (PT-3), have been
demonstrated to be eective in protecting rat cultured
cortical neurons against Aβ-induced cytotoxicity by
inhibiting ROS production, suppressing apoptotic caspase-3
and Bax, and in turn elevating anti-apoptotic Bcl-2.
As PT-3 that has elicited protective eects on primary
cortical neurons also showed promising outcome in
aged rat brain in vivo,
neuroprotective eects of NTF
in vivo had become our next primary study target.
In this research study, the animals were used in the
experiment, and they were calculated for the sample size
in accordance with the ethical guideline to protect the
unnecessary wastage of resources. Consistently, there are
several studies that have demonstrated the statistically
signicant results with a small sample size (n = 3).
Meanwhile, our preliminary results showed the protective
eects of NTF treatment against scopolamine-induced
cholinergic dysfunction, including, ROS production,
apoptosis, inammation, and associated signaling pathways
in several rat brain regions, specically the frontal cortex,
hippocampus and temporal cortex.
e frontal cortex integrity is correlated with the
higher reservation of cognitive performance in aging
e hippocampus and especially its associated
cholinergic signaling pathway play a crucial role in memory
e medial temporal lobe cortex or, in short,
the temporal cortex is reciprocally interconnected to the
hippocampus and it greatly involved in the hippocampus-
associated cognitive processes and cognitive decline such
as AD in humans
and ischemia-associated dementia in
Furthermore, ChAT activity in the temporal cortex
is positive correlated with cognitive preservation, but it is
negatively correlated with AD pathologies.
the frontal cortex, hippocampus, and temporal cortex are
three of the regions heavily aected by scopolamine
and aging
in the rat brain.
Herein, we studied the neuroprotective eects of
NTF treatment on scopolamine-associated cholinergic
dysfunction, namely, ROS production, apoptosis,
inammation, and relevant signaling pathways in the
rat frontal and temporal cortices and hippocampus.
Scopolamine (Sigma-Aldrich) and NTF were dissolved
in a vehicle solution of (v/v) 40% dimethylsulfoxide,
59% phosphate-buered saline (PBS), and 1% ethanol
and diluted in PBS for animal administration. NTF was
isolated from the acetone extract of Polyalthia suberosa
stems as previously described.
e test drug NTF was
in its form of pure compound. From 997.2 mg of the
semi-solid fraction, 88.1 mg (8.83 %) of NTF could be
puried by preparative layer chromatography.
Animal experiments
All experimental procedures were approved by
the Institute of Molecular Biosciences Animal Care and
Use Committee (MB-ACUC) (COA.NO.MB-ACUC
Twelve 8-week male Wistar rats (250-300 g) from the
National Laboratory Animal Center, Mahidol University,
were individually cared for in cages under 12 h light/
dark cycle, 22±1 °C temperature, 45-55 % humidity, and
ad libitum water and diet. e 12 rats were separated
into 4 groups, each with 3 animals, namely, scopolamine
treatment only, NTF treatment only, scopolamine plus
NTF treatment, and control. Following habituation for 5
days, animals were once per day injected intraperitoneally
(i.p.) for 14 days.
In the control group, animals were
injected with vehicle 1 h before the water maze test for 14
days. In the scopolamine-only group, animals were rst
injected for 7 days with vehicle followed by scopolamine
treatment on day 8 to day 14 (3.0 mg/kg BW) 1 h before
the water maze test. In the NTF treatment only group,
animals were injected with 1 ml aliquot of NTF (1.5
mg/kg BW) for 14 days 1 h before the water maze test.
In the scopolamine plus NTF treatment group, animals
were administered with NTF as described above for 7
days and then together with n day 8 to day 14 (3.0 mg/
kg BW) 1 h before the water maze test.
Rats were treated with NTF for 7 days prior to
MWM training because we would like to investigate the
neuroprotective eects of NTF against scopolamine. e
NTF eects as a memory enhancer can still remain during
the MWM training.
e dose of NTF was selected in
accordance with our previous ndings on the protective
roles of N-benzylcinnamide (PT-3).
In animal studies the
model of scopolamine has been performed using doses
between 0.5,1 or 3 mg/kg IP). e eect of scopolamine
to induce impairment of learning and memory is dose
e highest dose of scopolamine exhibits
the greater severity of memory dysfunction. Scopolamine
was administered via the injection 1 hr before MWM
Thangnipon et al.
Volume 73, No.6: 2021 Siriraj Medical Journal
Original Article
training because the half-life of scopolamine is 2.4±1.4
h for its elimination.
Central pharmacodynamic eects
of scopolamine peak between 1 and 3 h and disappear
aer 5–6 h.
Morris water maze test
Water maze tests were conducted to evaluate the
eects of NTF and scopolamine on the acquisition and
retention of spatial memory.
Visible platform trial
was performed on the day before the training trial.
Animals have to associate the relative location of the
visible platform with the provided visual cues. On the
following training trials, the platform was submerged
and invisible. e training trials were conducted over a
period of 7 days as follows: day 1, visible platform test;
days 2-6, hidden platform tests; and on the same day 6
aer hidden platform, probe trial test. ese consecutive 6
days correspond to the days 8-13 of the treatment period.
Rats in each group were put in the water facing dierent
quadrants, which were altered for each experiment,
and the escape latency dened as time spent to nd the
submerged platform was recorded. In the probe trial,
time spent in the target quadrant with the platform was
e experimental schedule was designed to investigate
the protective eect of NTF as a memory enhancer in
rats. Rats underwent habituation for 5 days. Aer that,
NTF (1 ml, 1.5 mg/kg. i.p.) or vehicle was administrated
once a day for 7 days. en, 1 h prior to the rst trial
session, rats were administrated of veh, NTF, scopolamine
(3.0 mg/kg) or NTF+Scop (i.p) every MWM training
for 6 days, in accordance with the previously described
ROS production assay
After animals were decapitated without prior
anesthesia, the 3 regions of the rat brains – the frontal
cortex, hippocampus, temporal cortex – were dissected
and frozen at -80°C. For the frontal cortex, the frontal
part of the brain was cut straight down coronally and
attached subcortical brain regions were removed. For
the hippocampus, the bilateral hippocampal tissues with
all subsectors were collected. For the temporal cortex, all
cortical regions encompassing the hippocampus or the
medial temporal lobe were dissected. Reactive oxygen
species (ROS) production was measured according to the
protocol of our previous work.
In brief, 2.4-3.0 mg of
brain tissue from each dissected region were homogenized
in 80-100 µl of ice-cold Locke’s buer, then 10 µl of
homogenate was adjusted to a concentration of 3 mg/ml
tissue, incubated with 10 µM 2',7'-dichlorouorescin
diacetate (DCFH-DA) (stock solution in 100% ethanol)
for 45 min at 37°C. Fluorescence of the oxidized product
2',7'-dichlorouorescein (DCF) (485 nm excitation and
535 nm emission) was measured every 10 minutes for
4 times in total at 37°C in a DTX 880 multimode plate
Western blot analysis
Rat brains were dissected into three regions: frontal
cortex, hippocampus and temporal cortex).
e brain
protein contents were extracted with the lysis buer,
centrifuged at 11,000 ×g for 15 min, and collected as
a supernatant. e protein samples were loaded onto
polyacrylamide gel. e proteins were separated by gel
electrophoresis and transferred onto (PVDF) membranes
where they were reacted to specic antibodies. On the
PVDF membrane, proteins of interest were identied using
a detection reagent. e protein bands were quantied
and analyzed with the same loading sample groups of
its own gel. PVDF membrane was incubated overnight
at 4°C with primary rabbit anti-Bax, -Bcl-2, -activated
caspase-3, -BACE1, -phospho-p38, -p38, -phospho-JNK,
-JNK, -phospho-ERK1/2, -ERK1/2, -phospho-Akt, -Akt,
-phospho-NF-κB p65 (Ser536), -NF-κB antibodies (1:1000
dilution) (Cell signaling), -IL-1β (1:1000) (Santa Cruz);
and primary mouse anti-ChAT (1:1000) (Millipore) and
-IL-6 (1:1000) (Santa Cruz); and rabbit anti-actin (1:2500)
(Cell Signaling) for normalization of protein loading.
Immunoreactive proteins were visualized by incubating
PVDF membranes with secondary horseradish peroxidase-
conjugated antibodies (1:1000) (Cell Signaling) at room
temperature for 1 h and subsequently with enhanced
chemical luminescence (ECL) reagents (Bio-Rad) before
signal exposure to Hyperlm
(GE Healthcare). Intensities
of the visualized protein bands were measured by scanning
densitometry (Image J soware). e optical density
(OD) of protein bands on Hyperlm was determined
and normalized to the OD of β-actin and total phospho
proteins. e results were expressed as the percentage
of OD values by using the ImageJ soware.
Each gel contains four sample groups (Control,
Sco, NTF+sco, NTF). Each protein sample of three rats
on dierent brain regions, including frontal cortex (3),
hippocampus (3), and temporal cortex (3) was loaded
separately on dierent polyacrylamide gel. Four sample
groups of each region in each rat were underwent Western
blotting. Each blot was repeated for at least 3 times from
dierent batches of protein samples. e representative
blots were shown in gures.
Volume 73, No.6: 2021 Siriraj Medical Journal
Statistical analysis
Statistical analyses were performed using Prism
6.0 (GraphPad). Dierences in the escape latency from
the training/hidden platform trials were analyzed using
two-way analysis of variance (ANOVA). Dierences
between means of the 4 groups from all other experiments
were determined using one-way ANOVA with pair-
wise p-values corrected by Tukey’s posthoc test. Results
are deemed signicant at p < 0.05. In all Western blot
analyses, non-phospho proteins (ChAT, Bcl-2, Bax,
Activated caspase-3, IL-6, and IL-1β) were quantied and
normalized with β-Actin. Meanwhile, phosphoproteins
(p-NFκB, p-ERK, p-JNK, p-p38, p-Akt) were quantied
and normalized with the total protein counterparts.
Learning and memory in a Morris water maze test of
rats administered scopolamine with and without NTF
Control rats became proficient at locating the
submerged platform by day 5 of training compared to
day 1 (p < 0.05) in a hidden platform test (Fig 1A). On day
5 of training scopolamine-treated rats perform signicantly
less well than controls (p < 0.01), but, interestingly, rats
treated with scopolamine together NTF have prociencies
superior to those of controls, whereas NTF treatment
alone was no more eective than non-treated control.
In the probe trial tests, as expected, compared to control
rats, those in the scopolamine plus NTF treatment group
stay signicantly longer in the platform quadrant. ose
in the NTF only treatment group spent the same time.
Lastly as expected, those in the scopolamine-only group
stayed for a signicantly shorter duration (Fig 1B).
ROS production in brain tissues of rats administered
scopolamine with and without NTF
Scopolamine treatment for 7 days elevated ROS
level 2 folds in all three types of brain tissues (frontal
cortex, hippocampus, and temporal cortex) over control
levels (Fig 2). ROS levels were restored to control levels
by NTF administration (7 days prior and during the
scopolamine treatment period), while NTF alone (for
7 days) had no eect on ROS levels in all three types of
brain tissues.
Fig 1. NTF (1.5 mg/kg) was injected i.p. daily to rats 7 days before and together with scopolamine (3.0 mg/kg BW i.p.) 1 h before the 7-day
Morris water maze test; probe trial was conducted on day 7. Each data point (n=3) is expressed as mean ± SEM. A NTF prevented scopolamine
from elevating escape latency. *p < 0.05 scopolamine-treated with NTF versus (vs) the scopolamine group on day 3, **p < 0.01 scopolamine-
treated with NTF vs the scopolamine group on day 5. B NTF restored scopolamine-induced decrease in retention measured as time spent
in the target quadrant. *p < 0.05 scopolamine vs NTF, **p < 0.01 scopolamine vs scopolamine with NTF treated group.
Fig 2. Eects of NTF treatment in scopolamine-treated rats
on ROS. Production of ROS in brain homogenates were
measured by uorometric agent DCFH-DA. Each data bar
(n=3) is expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs
control; #p < 0.05, ##p < 0.01 vs scopolamine-treated group.
Thangnipon et al.
Volume 73, No.6: 2021 Siriraj Medical Journal
Original Article
Levels of cholinergic, apoptosis, and inammatory
proteins in brain tissues of rats administered scopolamine
with and without NTF
Administration of scopolamine to rats signicantly
decreases ChAT and increases BACE1 levels in the 3 brain
regions compared to control tissues (Fig 3A-C), and NTF
cotreatment was able to ameliorate these alterations.
However, NTF treatment alone had no eect on basal
levels of the two proteins.
Exposure to scopolamine causes a signicant increase
in apoptotic proteins, Bax and activated caspase-3, and
a corresponding decrease in anti-apoptotic Bcl-2 in the
three types of brain tissues compared to controls with
NTF treatment reversing these changes (Fig 4A-C).
However, these relative changes in apoptosis-related
proteins levels in the brain tissues of rats treated with
NTF alone varied with tissue types and thus impacting the
ability of NTF to restore the normal levels of apoptosis-
related proteins in the scopolamine-treated animals.
Scopolamine significantly increased levels of
cytokines – interleukin (IL)-6 and IL-1β – and of activated
transcription factor – phosphorylated NF-κB – which
were reversed by NTF. e extent of these phenomena
relative to control levels also depended on brain tissue
types (Fig 5A-C).
Activated cell signaling pathways in brain tissues of
rats administered scopolamine with and without NTF
We investigated the activation indicated by the
proportion of phosphorylated proteins of JNK, p38, ERK1/2,
and Akt. In the frontal cortex (Fig 6A), scopolamine
increased p-JNK and p-p38, decreased p-ERK1/2, and
increased p-Akt. NTF downregulated p-ERK1/2 but
upregulated p-Akt without aecting p-JNK or p-p38.
A combination of scopolamine and NTF could only
restore p-p38 to control level. In the hippocampus (Fig
6B), scopolamine elevated levels of p-JNK and p-p38,
depressed p-ERK1/2 and had no eect on p-Akt. NTF had
no eect on levels of p-JNK, increased p-p38, decreased
p-ERK1/2, and had no eect on p-Akt. A combination
of scopolamine and NTF could only restore p-ERK1/2
to control level and had no eect on the p-Akt level. In
the temporal cortex (Fig 6C), scopolamine raised levels
p-JNK and p-p38, lowered p-ERK1/2, and had no eect
on p-Akt. NTF had no eect on levels of p-JNK, p-p38,
or p-ERK1/2 but decreased p-Akt. A combination of
scopolamine and NTF restored levels of p-JNK, p-p38
and p-ERK1/2, and had no eect on p-Akt. Table 1
shows the quantitative protein values as the percentage
of change in expression relative to control groups in
Western blot analysis.
Fig 3. Eects of NTF on scopolamine-induced cholinergic dysfunction in rats on the levels of ChAT and BACE1 in: A frontal cortex, B
hippocampus, and C temporal cortex. Each data bar (n=3) is expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs control group; ##p < 0.01,
###p < 0.001 vs scopolamine-treated group