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Current Nutrition & Food Science
ISSN: 1573-4013
eISSN: 2212-3881
SCIENCE
BENTHAM
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Current Nutrition & Food Science, 2020, 16, 333-342
333
RESEARCH ARTICLE
Tualang Honey and its Methanolic Fraction Improve LPS-induced Learning
and Memory Impairment in Male Rats: Comparison with Memantine
Wan M.H.W. Yaacob1, Idris Long1, Rahimah Zakaria2,* and Zahiruddin Othman3
1School of Health Sciences, 2Department of Physiology, 3Department of Psychiatry, School of Medical Sciences, Universiti
Sains Malaysia Health Campus, 16150 Kubang Kerian, Malaysia
Abstract: Background: Tualang honey (TH) has been shown to exert beneficial effects on learning and
memory function in various animal models. However, its learning and memory effects in lipopolysaccha-
ride (LPS) rat model have not been elucidated.
Objective: The present study aimed to investigate the cognitive-enhancing effects of TH and its meth-
anolic fraction in comparison to the clinically approved N-methyl-D-aspartate (NMDA) receptor
antagonist (memantine) using LPS rat model.
Methods: A total of ninety male Sprague Dawley rats were divided into 5 groups: (i) control, (ii) untreat-
ed LPS (iii) LPS treated with 200 mg/kg TH, (iv) LPS treated with 150 mg/kg methanol fraction of TH
(MTH) and (v) LPS treated with 10 mg/kg memantine. All treatments were administered intraperitone-
ally once daily for 14 days. Morris water maze (MWM) and novel object recognition (NOR) tests were
performed to assess spatial and recognition memory function.
Results: The present study confirmed that LPS significantly impairs spatial and recognition memory and
alone treatment with TH or MTH improved spatial and recognition memory comparable to memantine.
Conclusion: Both TH and its methanolic fraction improved spatial and recognition memory of LPS rat
model comparable to memantine. Thus, TH and its methanolic fraction have potential preventive-
therapeutic effects for neurodegenerative diseases involving neuroinflammation.
A R T I C L E H I S T O R Y
Received: April 16, 2018
Revised: August 15, 2018
Accepted: November 09, 2018
DOI:
10.2174/1573401315666181130103456
Keywords: Learning and memory, lipopolysaccharide, methanolic function, morris water maze, novel object recognition, tua-
lang honey.
1. INTRODUCTION
Neuroinflammation is characterized by the microglia ac-
tivation along with the expression of major inflammatory
mediators in the brain tissue and its chronic state, giving rise
to the progression of neurodegenerative diseases including
Alzheimer’s disease (AD) and Parkinson’s disease [1].
Neuroinflammation can cause cognitive decline, even if it is
acutely stimulated by an immunostimulatory element such as
lipopolysaccharide (LPS) [2].
LPS is an endotoxin found in the outer membrane of
gram-negative bacteria. It induces systemic inflammatory
response syndrome via toll-like receptor (TLR) [3]. Binding
of LPS-TLR4 complex on the surface of microglia activates
several signal transduction pathways such as phosphoinosi-
tide 3-kinase/protein kinase B (PI3K/AKT), mitogen-
activated protein kinase (MAPK) and mammalian target of
rapamycin (mTOR), which finally lead to NF-κB activation.
*Address correspondence to this author at the Department of Physiology,
School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang
Kerian, Kelantan, Malaysia; Tel/Fax: +609-7676156, +609-7653370;
E-mails: rahimah@usm.my
NF-κB activation mediates production of pro-inflammatory
cytokines, chemokines and inducible enzymes such as induc-
ible nitric oxide synthase (iNOS) and cyclooxygenase-2
(COX-2), which altogether result in neuroinflammation [4,
5] and cognitive impairment [6].
To date, donepezil, rivastigmine, galantamine and me-
mantine are the US Food and Drug Administration (FDA)
approved treatments for AD. These drugs are symptomatic
and do not delay or stop the progression of the disease [7].
However, these drugs exhibit modest, but particularly relia-
ble, benefit for cognition, global status and functional ability
[8]. Thus, there is a need to continue our search for treatment
of AD.
Among the proposed mechanisms for AD, an antioxidant
complex or combination with CNS anti-inflammatory holds
the best promise of an effective AD treatment [9]. Tualang
honey (TH), a Malaysian multi floral jungle honey, is shown
to exhibit anti-inflammatory [10], antiproliferative [11],
antioxidant [12], anti-diabetic [13] and neuroprotective ef-
fects [14]. There is increasing evidence to support the cogni-
tive enhancer effects of TH in various animal models. Study
by Azman et al. [15] showed that treatment with 200 mg/kg
2212-3881/20 $65.00+.00 © 2020 Bentham Science Publishers
334 Current Nutrition & Food Science, 2020, Vol. 16, No. 3 Yaacob et al.
TH for 28 days ameliorate memory performance and
increase the levels of brain antioxidant enzymes against
noise stress-induced memory deficit in aged rats. TH intake
was able to improve both working and reference memory by
radial arm maze experiment as well as increase pyramidal
neurons count in hippocampal tissue of adult rats [16].
Saxena et al. [17] reported that TH reduced spatial memory
impairment and protected the hippocampal neurons from
damage in cerebral hypoperfusion-induced neurodegenera-
tion. These previous studies demonstrated that TH supple-
mentation enhanced neuronal proliferation in CA2, CA3 and
dentate gyrus region of the hippocampus in adult rats [14].
TH also mitigated the increased activity of acetylcholinester-
ase (AChE), and normalized the brain-derived neurotrophic
factor (BDNF), malondialdehyde and superoxide dismutase
levels in the hippocampus of stressed ovariectomized rats
[18-20].
Our group has investigated the antioxidant potentials of
four different samples of TH i.e. non irradiated TH, gamma
irradiated TH, methanolic fraction of TH and ethyl acetate
fraction of TH. We found its methanolic fraction exhibited
highest antioxidant potentials when compared among the
four TH samples tested (unpublished). We hypothesized that
TH, especially its methanolic fraction could protect from
LPS-induced learning and memory impairment. Therefore,
the present study aimed to investigate the cognitive enhanc-
ing effects of TH and its methanolic fraction in comparison
to the clinically approved N-methyl-D-aspartate (NMDA)
receptor antagonist (memantine) using LPS rat model.
2. MATERIALS AND METHODS
2.1. Honey Samples
TH from single batch of honey supplied by Federal Agri-
cultural Marketing Authorities (FAMA), Malaysia was used
in the present study. The honey was filtered to eliminate any
solid particles, concentrated on heat at 40°C and evaporated
to achieve a water content of about 20%. It was then gamma
() irradiated at 25 kGy at Steril Gamma (M) Sdn. Bhd. (Se-
langor, Malaysia) for sterilization. The final concentration of
the TH in a 230g/bottle was 1.3 g/mL.
2.2. Extraction of Honey
The honey was subjected to solid phase extraction (SPE)
to extract phenolic compounds as described by Kaškonienė
et al. [21] with slight modifications. The honey samples were
dissolved in five parts of acidified deionized water (pH 2
attained by using 2.0 M hydrochloric acid) until completely
fluid and then filtered through cotton wool to remove solid
particles. The solution was passed through preconditioned
C18 cartridges (6 mL x 1 g). The cartridges were precondi-
tioned by successively passing 6 mL each of methanol and
acidified water. The aqueous honey solution was applied to
the cartridge at a drop-wise flow rate to ensure efficient ad-
sorption of phenolic compounds. The cartridges were then
washed with acidified water to remove all sugars and other
polar constituents of honey. Later, the adsorbed compounds
were eluted with methanol and evaporated using a rotary
evaporator at 40°C. The methanolic fraction of Tualang hon-
ey (MTH) was used in the present study.
2.3. Animals
Ninety adult male Sprague Dawley rats of approximately
3 months old, with body weight of 270 ± 20g, were obtained
from Animal Research and Service Centre (ARASC), Uni-
versiti Sains Malaysia. The rats were allowed to acclimatize
to a new environment for one week prior to the start of the
experiment. They were maintained under standard laboratory
condition at room temperature of 21 ± 2°C with free access
to food and water. The photoperiod was the same throughout
the study with 12 h light-dark cycles. The protocols were
conducted in accordance with USM Guide for the Care and
Use of Laboratory Animals and approved by Animal Ethics
Committee [USM / Animal Ethics Approval I 2015/(95)
(605)].
2.4. Experimental Design
The rats were randomly divided into five groups (n=18
rats/group) as follows: (i) control rats, (ii) LPS-treated with
distilled water, (iii) LPS-treated with TH 200 mg/kg [15],
(iv) LPS-treated with MTH 150 mg/kg [22] and (v) LPS-
treated with memantine 10 mg/kg [23]. All treatments were
administered intraperitoneally once daily for 14 days, start-
ing from Day 1 to 14 [15]. LPS from E.coli 0111:B4 (Sigma-
Aldrich, St. Louis, MO) at the dose of 5 mg/kg [24] was in-
jected on Day 4. Body weight was measured twice; on Day 1
and 24. All rats were subjected to Morris water maze
(MWM) test on Day 15 and novel object recognition (NOR)
test on Day 21. After behavioral assessment and body
weighting, rats were sacrificed and brain tissues were imme-
diately collected and stored at -80°C for later use. The exper-
imental schedule is shown in Fig. (1).
2.5. Morris Water Maze (MWM)
The MWM test was performed on Day 15 as described
by Vorhees and Williams [25]. The acquisition training was
performed over the course of 5 days and the retention test
was conducted on Day 20. The apparatus consists of a circu-
lar water pool 150 cm in diameter and 60 cm in height. It
was filled with 23 ± 1°C water with a depth of 42 cm. The
water in the pool was made opaque by adding 1 kg tapioca
starch powder. A hidden circular platform (10 cm in diame-
ter), which was placed 1 cm below the water surface served
as the escape platform. The pool was divided into four quad-
rants: northeast (NE), northwest (NW), southeast (SE), and
southwest (SW) at equal distances on the rim. The platform
was placed in the center of the NE quadrant. Several visual
cues were positioned around the pool in plain sight of the
rats and these were exactly in the same position for all trials.
A digital camera was mounted to the ceiling straight above
the center of the pool and was connected to a computerized
recording system equipped with a tracking program (S-
MART 3.0: Panlab Co., Barcelona, Spain), which allowed
on- and off-line automated tracking of the paths taken by the
rats. All the trials were completed between 09.00 to 16.00 h
in a sound-attenuated laboratory.
Tualang Honey Improves Learning and Memory Impairment Current Nutrition & Food Science, 2020, Vol. 16, No. 3 335
Fig. (1). Experimental schedule.
2.5.1. Hidden Platform Trial for the Acquisition Test
The rats were gently placed into the water facing the wall
and were permitted to swim until they found the hidden plat-
form, which remained in a fixed position throughout the test.
The trials lasted for a maximum of 60s, and the escape laten-
cy was expressed by the swimming time to reach the
submerge platform in the pool. Upon reaching the hidden
platform, the rat was permitted to stay on it for at least 5s
before being withdrawn. If the rats failed to locate the hidden
platform within 60s they should be guided to the platform
and were placed on it for 10s for reinforcement before being
withdrawn. The latency was assigned as 60s amid one trial
and the next, the water in the pool was stirred to eliminate
olfactory traces of previous swim patterns. The entire sched-
ule was continued for 5 days and each animal had 4 trials for
training per day with 5-15 min inter-trial interval. The 4
starting points were randomized on each day of training.
Escape latencies and distance traveled by the rats during
training sessions were measured.
2.5.2. Probe Trial for the Retention Test
On Day 20, the probe test was performed with a cut-off
time of 60 s. Animal behaviors were analyzed with a grid
design consist of 4 quadrants (Fig. 2). This grid design, con-
structed with a computer-based image analyzer, was super-
imposed over the maze and viewed on a monitor. The num-
ber of target heading, distance traveled and time spent in
each of the quadrants were determined.
Fig. (2). Computerized grid design which used in the probe test.
Black circle representing the platform site.
2.6. Novel Object Recognition (NOR)
The NOR task was performed in an open field apparatus
(60 × 60 × 40 cm) painted in black color as described in a
previous study [26]. Before starting the training, all animals
had at least two free exploration sessions for contextual ha-
bituation, with no object inside the box for 5 min. During the
training session, two identical objects (A, A’) were placed at
fixed distances within the square field. The rats were then
allowed to explore freely for 10 min. Time spent exploring
each object was recorded manually. After two successive
training sessions, the test sessions were conducted consisting
of two terms, short-term and long-term memory. The testing
interval of short-term and long-term memory was 2h and
24h respectively, after the last training session. This time one
of the objects was replaced with a new object (A, B). The
rats were allowed to explore freely for 10 min, and the
number of contacts with the original and novel object was
recorded.
All objects comprised of plastic toys (washable) and had
a height of about 4 cm. Objects presented similar textures,
colors and sizes, but distinctive shapes. The objects were
positioned in two adjacent corners, 10 cm from the side walls
to avoid accidental touching during the initial, thigmotaxic
exploration. The maze and objects were wiped cleaned with
20% alcohol after each test session to eliminate odor cues.
Object exploration was defined as directing the nose and
vibrissae to the object at a distance of less than 2 cm, as if
smelling it with caution; turning around, bumping or sitting
upon the object was not considered exploratory behaviors.
Total exploration time of the novel objects was recorded and
used to calculate a discrimination index: time spent with a
novel object (B) /total time exploring both objects (A + B)
during test sessions [27]. Prolong exploration time of the
novel object or better preference to novel object was consid-
ered as successful retention of memory for the familiar ob-
ject. The absence of any difference in the exploration of two
objects was interpreted as memory deficit [28].
2.7. Statistical Analysis
Study data were presented as means ± standard errors
(SEM). Differences between groups were evaluated using
paired Student’s t-test, one-way Analysis of variance
(ANOVA) and repeated measure ANOVA, where appropriate.
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336 Current Nutrition & Food Science, 2020, Vol. 16, No. 3 Yaacob et al.
Fig. (3). Mean body weight of experimental rats on Day 1 and 24 of the experiment. Data are expressed as means ± SEM. *p <0.05 vs. LPS
group; #p <0.05 vs. control group.
A probability value of less than 0.05 was used to indicate a
significant difference.
3. RESULTS
3.1. Effects on Bodyweight Changes
Body weight of each rat in each group was measured on
day 1 and 24 of the experiment (Fig. 3). Statistical analysis
revealed that there were no significant differences in means
of body weight of 5 groups on day 1 of the experiment.
However, a significant reduction in the mean body weight
gain of untreated LPS rats when compared with controls on
day 24 of the experiment (p <0.05). Interestingly, the LPS
rats treated with TH, MTH and memantine showed weight
gain comparable to controls. Their body weights were signif-
icantly higher when compared to the untreated LPS rats on
day 24 of the experiment (p <0.05), indicating that treatment
with TH, MTH and memantine could prevent LPS-induced
body weight loss.
3.2. Effects on Spatial Learning
Following 2 weeks of treatment, spatial learning of ex-
perimental rats was assessed by using MWM. Fig. (4A)
showed the total escape latency during the acquisition phase
for 5 days. The time per day for escape latency decreased
steadily in all the experimental groups. The LPS rats demon-
strated marked retardation in escape latency, indicating
memory deficit. LPS rats treated with TH and MTH rapidly
learned the location of the immersed hidden platform and
reached it within 23 and 22s on the fifth day of the trials,
respectively which was comparable to the group of rats treated
with memantine. The analysis of escape latency displayed
that the rats in the TH group had significantly decreased es-
cape latency on the third and fifth day (p <0.05) whereas the
rats in MTH group on first (p <0.05), fourth (p <0.05) and
fifth day (p <0.01), respectively as compared to the LPS
group. The distance traveled by each group was closely as-
sociated with the escape latencies. Rats treated with meman-
tine, TH and MTH showed a consistent decrease in searching
distance during the acquisition trial especially third day on-
wards when compared to rats in LPS group (p <0.05 on the
third, fourth and fifth day; Fig. (4B) indicates that TH and
MTH treatment could ameliorate LPS-induced spatial learn-
ing and memory deficit comparable to memantine.
3.3. Effects on Memory Retention
In order to determine the effects on memory, the probe
test behaviors were investigated on the sixth day by analyz-
ing the percentages of time and distance required to swim in
the ‘target quadrant’, where the escape platform had been
placed. The LPS rats showed severely impaired performanc-
es; they spent significantly (p <0.001) less time in the target
quadrant site compared to those of the control group. The
rats in TH and MTH groups spent significantly longer time
(p <0.05) than those in the LPS group. Similarly, TH and
MTH had significantly increased (p <0.05) swimming dis-
tance in the target quadrant as compared to the LPS group
(Fig. 5A & B). These results suggest that both TH and MTH
could ameliorate the LPS-induced poor memory retention
comparable to LPS rats treated with memantine. From the
trajectory view, it can be clearly seen that LPS-treated rats
swam in all of the four quadrants (aimless swimming) rather
than swimming mostly in the target quadrant. Whereas con-
trols and LPS rats treated with memantine, TH and MTH
swam mostly in the target quadrant (Fig. 5E). The number of
target crossing in the LPS group was statistically reduced (p
<0.001) compared to the control group, while TH treatment
significantly increased (p <0.05) the number of target cross-
ing compared to the LPS group (Fig. 5C). Both TH and
MTH groups were not significantly different from the other
groups in terms of the mean swimming speed, as computed
by dividing the total swimming distance by the latency (Fig.
5D).
3.4. Effects on Recognition Memory
Fig. (6) shows the effects of various treatments on object
recognition ability. No significant difference was observed in
the short-term memory of all rats. However, after 24 h of last
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Tualang Honey Improves Learning and Memory Impairment Current Nutrition & Food Science, 2020, Vol. 16, No. 3 337
Fig. (4). Effects TH, MTH and memantine treatment on the escape latency to the hidden platform (A) and the distance traveled to the hidden
platform; during first to fifth day of acquisition trials (B). Data are expressed as means ± SEM. *p <0.05 and **p <0.01 vs. LPS group; #p
<0.05 and ##p <0.01 vs. control group.
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338 Current Nutrition & Food Science, 2020, Vol. 16, No. 3 Yaacob et al.
Fig. (5). Effects of TH, MTH and memantine treatment on memory retention of LPS rats. A) Percentage of time spent in target zone, B) per-
centage of distance travelled in target zone, C) number of target crossing, D) swimming speed, and E) swimming path; of experimental rats in
the probe trial without a platform. Data are expressed as means ± SEM. *p <0.05 and **p <0.01 vs. LPS group; ##p <0.01 and ###p <0.001 vs.
control group.
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Tualang Honey Improves Learning and Memory Impairment Current Nutrition & Food Science, 2020, Vol. 16, No. 3 339
Fig. (6). Effects of TH, MTH and memantine treatment on short-term and long-term recognition memory of LPS rats. Data are expressed as
means ± SEM. *p <0.05 and **p <0.01 vs. LPS group; ###p <0.001 vs. control group.
training session (long-term memory) LPS-induced rats had
significantly (p <0.001) lower discrimination index com-
pared to controls, indicating that LPS was associated with
poor recognition memory. Unlike untreated LPS rats, the
LPS rats that received TH and MTH for 14 days revealed
significantly (p <0.05) higher discrimination index which
was comparable to memantine group.
4. DISCUSSION
Honey has long been used as food as well as a natural
remedy for various diseases such as cardiovascular diseases,
inflammatory disorders, cancer, neurological degeneration,
infectious diseases and aging [29, 30]. Al-Himyari [31] had
proposed that honey intake is associated with a low risk of
AD and may prevent neurodegenerative process of the dis-
ease. However, based on the current literature search, the
data evaluating the efficacy of honey in learning and
memory models is still lacking.
Previous studies revealed body weight loss in rats or
mice that were subjected to the single administration of LPS
[32, 33]. This finding was consistent with our finding. We
used intraperitoneal (i.p.) injection of 5 mg/kg LPS to induce
neuroinflammation and memory impairment. Our data
demonstrated that the untreated LPS rats gradually gained
less body weight throughout the experimental period. LPS is
known to produce ‘sickness behaviors’ [34] which include
reduction in activity, reduction in exploration, decreased
social interaction, fever, reduction in consumption of food
and drink, hypersomnia, activation of the hypothalamic-
pituitary-adrenal (HPA) axis and activation of a sympathetic
system [35, 36]. The treated LPS groups displayed a restored
body weight gain which was comparable to controls, sug-
gesting that TH, MTH and memantine treatment inhibited
the LPS-induced ‘sickness behavior’ [37].
The present study confirmed that LPS significantly im-
pairs water maze learning and memory, as the escape latency
and the traveled distance to the hidden platform were in-
creased. These findings indicate a deficit in learning ability
and reference memory [38]. Similar findings were also noted
in other studies [32, 39]. The LPS-induced learning and
memory impairments were reversed by oral administration of
TH, MTH and memantine for two weeks. Previous study
demonstrated that subcutaneous memantine (10 mg/kg/day)
ameliorates the spatial memory impairments produced by
LPS alone in rats [40]. The study proposed that memantine
acts partly through its ability to reduce the effects of neuroin-
flammation, resulting in normal spatial learning. Thus, it is
possible that anti-inflammatory properties of Tualang honey
and its methanolic fraction [41] reduce the effects of neu-
roinflammation, leading to normal spatial learning.
The present study also confirmed that LPS significantly
impairs memory retention, as the time spent and distance in
the quadrant in which the platform was formerly located
were decreased. Rats treated with TH and MTH, however,
spent more time and distance in the target quadrant, suggest-
ing that both TH and its methanol extract could ameliorate
the LPS-induced poor memory retention comparable to me-
mantine. However, there were no differences in swimming
speed and ability to find the visible platform (in cued version
of water maze) among the experimental groups. The findings
may suggest that no changes were observed in motivation or
sensorimotor coordination in all the rats [42].
Additionally, rats that received TH and MTH exhibited a
preference for the novel object in the NOR task, as indicated
by the improvement in the percentage of discrimination in-
dex. The NOR task is based on spontaneous, natural explora-
tory behavior of rats to non-aversive stimuli and is a pure
working memory test free of confounds [43]. In the present
study, LPS rats demonstrated intact short-term but impaired
long-term recognition memory. This may suggest a failure in
memory consolidation and not to impairments in memory
encoding, attention or performance [44]. Nonetheless, our
results demonstrated that administration of TH and MTH
could ameliorate the LPS-induced decline in long-term
recognition memory comparable to memantine. The recogni-
tion memory improvement following treatment with TH has
also been observed in other animal models of memory defi-
cits [14, 15].
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340 Current Nutrition & Food Science, 2020, Vol. 16, No. 3 Yaacob et al.
LPS-induced amnesia rodent model is one of the well-
established animal models of memory deficits [45]. LPS is a
potent endotoxin and is highly resistant to degradation by
mammalian enzymes thus providing a persistent inflammato-
ry stimulus that produces proinflammatory cytokines, chem-
okines and reactive oxygen species (ROS) [46]. Proinflam-
matory cytokines are key molecules that modulate immune
responses. Their lack of reversibility in chronic inflammation
would trigger dyshomeostasis [47]. Chronic inflammation is
characterized by long-standing microglia activation that sus-
tains the release of inflammatory mediators, leading to an
increase in ROS and nitrosative stress. This perpetuates the
inflammatory cycle [48], further prolonging the inflamma-
tion which in turn lead to cognitive impairment [49]. Previ-
ous studies demonstrated that LPS-induced inflammation in
the brain produces severe learning and memory deficits in a
variety of behavioral tasks [50-52].
In the present study, daily administration of TH and
MTH for 14 days attenuated the LPS-induced spatial and
recognition memory impairment in male rats comparable to
memantine. It is possible that TH and its methanolic frac-
tions act through their potent antioxidant free radical scav-
enging and anti-inflammatory properties [53] to improve
cognitive behaviors. Both antioxidant and anti-inflammatory
properties of honey could be the underlying cause for the
reduction in the damaging effect of oxidative stress-induced
neurodegeneration and cognitive impairment [54]. Further
studies are required to investigate the mechanism of action of
TH and its methanolic fraction on oxidative stress and neu-
roinflammation pathways in the rat brain to support our hy-
pothesis.
CONCLUSION
The present study demonstrates that TH and MTH were
able to reduce the LPS-induced spatial and recognition
memory impairment in male rats comparable to memantine.
Thus, TH and its methanolic fraction have a potential
preventive-therapeutic effect for neurodegenerative diseases
involving neuroinflammation.
ETHICS APPROVAL AND CONSENT TO
PARTICIPATE
The protocols were conducted in accordance to USM
Guide for the Care and Use of Laboratory Animals and ap-
proved by Animal Ethics Committee [USM / Animal Ethics
Approval I 2015/(95) (605)].
HUMAN AND ANIMAL RIGHTS
No humans were used in this research. All animal re-
search procedures followed were in accordance with the
Australian Code of Practice for the Care and Use of Animals
for Scientific Purposes (7th Edition, 2004).
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings
of this study are available within the article.
FUNDING
This study was supported by the Fundamental Research
Grant Scheme (FRGS), Malaysia with Grant no. 203/PPSP/
6171174.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
Declared none.
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