Research Article
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JOURNAL OF NEUROLOGY AND NEUROSCIENCE
ISSN 2171-6625
2017
Vol. 8 No. 3: 195
DOI: 10.21767/2171-6625.1000195
Lupeol Isolated from Betula alnoides
Ameliorates Amyloid Beta Induced
Neuronal Damage via Targeting Various
Pathological Events and Alteration in
Neurotransmitter Levels in Rat’s Brain
Abstract
Madhu Kaundal1,
Mohd Akhtar1 and
Rahul Deshmukh2
1 Department of Pharmacology, Faculty
of Pharmacy, Jamia Hamdard (Hamdard
University), New Delhi-110062, India
2 Pharmacology Division, ISF College of
Pharmacy, Moga (Punjab), India
Corresponding author: Madhu Kaundal
Lupeol, a natural active constituent of Betula alnoides (BA), is well known for its
anti-inflammatory, anti-oxidant and neuroprotective activities. In present study
the therapeutic potential of lupeol was investigated against amyloid beta (Aβ(1-42))
induced cognitive deficit, neurochemical and biochemical abnormalities in rats.
Lupeol was isolated from the BA and its structure was confirmed through nuclear
magnetic resonance spectra. Aβ(1-42) (4 μg/4 μL) intracerebroventrically (icv)
was administered to rats for the induction of Alzheimer's disease (AD). Lupeol
treatment (25 mg/kg/day, 50 mg/kg/day and 100 mg/kg/day per orally) was
started one week after Aβ(1-42) infusion up to the 21st days. Morris water maze from
day 16 to 21 and object recognition tasks on day 14 and 15 were performed for
memory assessment. On 22nd day, animals were sacrificed and hippocampi were
isolated for analysis of biochemical (acetylcholinesterase, lipid hydroperoxide,
glutathione and nitrite) and neuro-inflammatory (tumor necrosis factor -α,
interleukin (IL)-1β, and IL-6) parameters. In the present study Aβ(1-42) infusion was
significantly impaired behavioral memory, increased oxidative stress, decreased
antioxidant enzyme and increased pro-inflammatory markers. Treatment of lupeol
significantly restored Aβ(1-42) induced behavioral and biochemical abnormalities
in rats brain. The findings of the present study suggest that lupeol act through
multiple mechanisms and would be used to curb cognitive decline associated with
neurodegenerative disorders of AD.
madhukaundal07@gmail.com
Doctoral Research Fellow, Department of
Pharmacology, Faculty of Pharmacy, Jamia
Hamdard (Hamdard University), New
Delhi-110062, India.
Tel: +91-8557082640
Citation: Kaundal M, Akhtar M, Deshmukh
R. Lupeol Isolated from Betula alnoides
Ameliorates Amyloid Beta Induced Neuronal
Damage via Targeting Various Pathological
Events and Alteration in Neurotransmitter
Levels in Rat’s Brain. J Neurol Neurosci.
2017, 8:3.
Keywords: Alzheimer’s disease; Lupeol; Memory; Cognitive disorder; Oxidative
stress
Received: April 26, 2017; Accepted: May 24, 2017; Published: May 29, 2017
Introduction
Alzheimer's disease (AD) is the most common form of dementia
characterized by extracellular deposits of amyloid beta (Aβ
), intracellular neuro-fibrillary tau tangle, oxidative stress
(1-42)
and decline in cognitive functions [1-3]. Although the exact
pathogenic mechanisms remain unclear, transcriptional
dysregulation and impaired cyclic nucleotide signaling have been
reported in experimental animals as well as in AD patients [46]. Recent neuropathological studies have also established a
link between morphological and functional changes occurring
in the monoaminergic ascending system, particularly in nor
epinephrine (NE) and 5-hydroxytriptamine (5-HT), and the
pathophysiology of AD. Moreover clinical researchers found that
AD patients have complex neurochemical disturbances including
the catecholaminergic, cholinergic and glutaminergic neuronal
systems [7-10].
Lupeol, a biologically active dietary triterpenoid is found in many
medicinal plants and different fruits such as olives, mangoes,
and strawberries [11,12]. A variety of medicinal plants such as
Betula alnoides, Vernonanthura ferruginea and Zanthoxylum
rhoifolium have also been reported to contain lupeol as an active
constituent [13-15]. Lupeol has been reported to have various
pharmacological activities including acetylcholinesterase (AChE)
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inhibition, anti-inflammatory and anti-oxidant actions [16-18].
Further, it has been reported to be effective in various pathologies
and recently its neuroprotective effect has been studied [19,20].
Indeed Brimson et al. [21] described a neuroprotective effect of
lupeol against glutamate-induced neurotoxicity in HT22 mouse
hippocampal cells.
Intra-cerebroventricular (ICV) administration of Aβ(1-42) is a
well-known model to produce behavioral, biochemical and
neuropathological changes similar to that seen in clinical AD and
considered as an appropriate animal model of AD [22]. Thus, the
present work was carried out to investigate the neuroprotective
potential of lupeol against ICV-Aβ(1-42) induced cognitive
impairment, neurotransmitters deficits, neuroinflammation and
oxidative-nitrosative stress in rats.
Material and Methods
Drugs and chemicals
The stem bark of Betula alnoides (BA) was collected from the
mountains of Himachal Pradesh, INDIA. The plant BA, was
identified by Dr. H.B. Singh, an eminent botanist (Ref. No.NISCAIR/ RHMD/ CONSULT/ 2014/ 2398-178) of NISCAIR, New
Delhi, India. Aβ(1-42) was purchased from Sigma-Aldrich, USA.
All other chemicals used in the study were of analytical grade.
Solutions of the drugs and chemicals were always prepared
afresh before use.
Extraction and fractionation of BA (Stem bark)
50 gm of dried and powdered bark of BA was soxhlated with
ethanol (60°C to 80°C) yielding 14.6 g of dry ethanolic extract.
Lupeol, as a major constituent was separated out using a
column chromatography technique [23]. Different spectroscopic
methods were used to elucidate the structure of lupeol using
Fourier transform infrared spectroscopy (FTIR) -Nicolet S10
(Thermospecific) in CCl4 at I.S.F. Analytical Lab, Moga. Nuclear
magnetic resonance (NMR) spectra were recorded on a Bruker
AVANCE-400 Japan (100 MHz and 400 MHz) in chloroform
with tetramethylsilane as internal standard at the SAIF, Punjab
University, Chandigarh, India.
Animals
Male wistar rats (250-300 g) were obtained from Central Animal
House of I.S.F. College of Pharmacy, Moga, Punjab (India).
Animals were kept in polyacrylic cages (4/cage) and maintained
under standard husbandry conditions (room temperature 22 ±
1°C and relative humidity of 60%) with a 12 h light/dark cycle
(lights on at 8 AM). The protocol was reviewed and approved by
the Institutional Animal Ethics Committee and the experiments
were carried out according to Indian National Science Academy
guidelines.
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Watson [24]. Animals were divided into five groups and each
group comprised of 10 animals. The treatment schedule and the
interval for estimation of various parameters were described
as; Group1: served as double vehicle control, received artificial
cerebrospinal fluid (ACSF) in a volume of 4 µl in each ventricle on
day 1% and 2% dimethyl sulfoxide per orally (p.o., as a vehicle of
lupeol) for 21 days. Group 2: Rats were infused with ICV-Aβ(1-42) (4
μg/4 μL) dissolved in ACSF in a volume of 4 µl in each ventricle on
day 1. Group 3, 4 and 5 received lupeol at doses of 25 mg/kg, 50
mg/kg, 100 mg/kg p.o. respectively 1 week after the ICV-Aβ(1-42)
infusion starting from day 7 and continued once daily at 10 am
daily for a period of 21 days. The doses of lupeol were selected
on the basis of earlier reports in which significant antioxidant and
neuroprotective properties were demonstrated [18,25].
Object Recognition test (ORT): The ORT was performed as described by Giorgetti et al. [26].
Spatial navigation task: Spatial learning and memory of animals
in a Morris water maze (MWM) was tested by the method described by Morris [27].
Brain homogenate preparation: Terminally, on day 22, rats
were sacrificed and hippocampus from the brain was separated,
weighed and then homogenized individually. The various biochemical parameters were determined separately in hippocampal supernatant collected following homogenization. Animals
were perfused with phosphate buffer saline before decapitation
from the heart to remove blood from the brain tissues completely and its interference with the homogenate readouts. After perfusion, animals were sacrificed by decapitation and brains
were removed and rinsed with ice-cold isotonic saline. The rat
hippocampal tissues were then homogenized with ice cold 0.1 M
phosphate buffer (pH 7.4) in a volume 10 times the weight of the
tissue. The homogenate was centrifuged at 10,000 g for 15 min
(4°C) and aliquots of supernatant were separated and used for
biochemical analysis. Protein was measured in all brain samples
by the method of Lowry et al. [28] using bovine serum albumin (1
mg/ml) as a standard.
AChE activity: The quantitative measurement of AChE activity in
brain was performed according to the method described by Ellman et al. [29].
Malondialdehyde (MDA) estimation: The quantitative measurement of MDA end product of lipid peroxidation-in brain homogenate was performed according to the method of Wills [30].
Nitrite estimation: The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide was determined by a colorimetric assay using Greiss reagent (0.1% N-[1naphthyl] ethylene diaminedihydro chloride, 1% sulfanilamide
and 2.5% phosphoric acid) as described by Green et al. [31].
Experimental procedure
Reduced glutathione (GSH) estimation: GSH in hippocampus was
estimated according to the method described by Ellman [32].
Animals were anesthetized with ketamine (100 mg/kg, i.p) and
xylazine (5 mg/kg, i.p) and were fixed on steriotaxic apparatus.
Skull was exposed after midline sagittal incision and holes on
both sides of the brain were drilled according to Paxinos and
Pro-inflammatory cytokines such as tumor necrosis factor –α
(TNF-α), interleukin (IL)-1β and IL-6 estimation: The quantifications of TNF-α, IL-6, and IL-1β were done by rat TNF-α, IL-6, and
IL-1β immunoassay kit (KRISHGEN BioSystem, Ashley Ct, Whitti-
2
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Vol. 8 No. 3: 195
er, CA). Concentrations of TNF-α, IL-6, and IL-1β were calculated
from the plotted standard curves [33].
Statistical analysis
The values were expressed as mean ± standard error mean
(SEM). P<0.05 was set to be statistically significant. The results
were analyzed using Analysis of variance using statistical Graph
Pad Prism software. In ORT, time of exploration during T1 and
T2 on familiar and novel object was analyzed by paired student
t-test.
Results
Isolation of lupeol
The melting point of the compound, lupeol was found to be
213°C to 215°C. The FTIR spectra gave information about the
functional groups or chemical entities present in lupeol which
was found in accordance with the literature data of lupeol
[33]. Further 1H NMR spectrum of the compound revealed the
presence of seven tertiary methyl protons, a sextet of one proton
at δ 2.17 and presence of olefinic protons at (H-29a and b) which
is characteristic of lupeol [34]. On the basis of FTIR and NMR
spectra structure of compound was drawn which was in good
conformity for the structure of lupeol (Figure 1).
H
H
H
HO
H
Figure 1 Chemical structure of lupeol.
Effect of lupeol on memory performance in
MWM task of ICV-Aβ (1-42)-infused rats
In the MWM, animals were trained for 5 days starts on day 17
of Aβ (1-42)-infusion. On day 17 (1st trial), there was no significant
difference between the mean latencies of all groups. Aβ
-infused animal showed poor ability (increased latency to find
(1-42)
the platform) to learn the task on days 18, 19 and 20 as compared
to control group. On 21st day the time spent in the target quadrant
were also significantly decreased in Aβ (1-42)-infused animal as
compared to control group (Figure 2A). Aβ (1-42)-infused rats
failed to remember the precise location of the platform, spent
significantly less time in the target quadrant as compared with
control group (Figure 2B). Whereas pretreatment with lupeol
(25, 50 and 100 mg/kg) significantly attenuated Aβ (1-42)-induced
acquisition deficit and showed a significant difference between
the mean latencies.
Figure 2A Effect of lupeol on MWM performance (mean
escape latency) in Aβ (1-42) infused rats. The values are
expressed as mean ± SEM (n=10). (A & B) #P<0.001
vs. vehicle, *P< 0.05 vs. Aβ (1-42).
Effect of lupeol on non-spatial memory
performance in ORT in ICV- Aβ (1-42)-infused
rats
There was no significant difference observed between the Aβ
-infused and Lupeol-treated rats for familiar objects (Figure
(1-42)
3A). When Aβ (1-42) infused rats were exposed to familiar and
novel objects in ORT, they were unable to discriminate between
familiar and novel objects and spend almost equal time to
explore the similar and novel objects as compared to control
group (Figure 3B). Whereas, lupeol (25 mg/kg, 50 mg/kg and
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Figure 2B Effect of lupeol on MWM performance (time spend
in target quadrant) in Aβ (1-42) infused rats. The
values are expressed as mean ± SEM (n=10). (A & B)
#
P<0.001 vs. vehicle, *P<0.05 vs. Aβ (1-42).
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100 mg/kg) treatment produced significant improvement in
discriminating ability between familiar and novel object in Aβ (1-42)
infused rats (Figure 3C).
Effect of lupeol on AChE activity in Aβ
(1-42)-infused rats
ICV infusion of Aβ(1-42) produced significant increase in AChE
activity as compared with vehicle treated groups. Lupeol (25
mg/kg, 50 mg/kg and 100 mg/kg) attenuated Aβ (1-42)-induced
elevation in AChE activity (Figure 4) and restored the values as
that of vehicle control rats.
Effect of lupeol treatment on brain oxidativenitrosative stress (MDA, GSH and nitrite) levels
in ICV-Aβ(1-42) injected rats
The level of MDA and nitrite were increased and GSH was
decreased significantly in Aβ(1-42) infused groups as compared
of control group. Lupeol (25 mg/kg, 50 mg/kg and 100 mg/kg)
treatment significantly restored the MDA, nitrite and GSH levels
as compared with those of Aβ (1-42)-infused rats (Table 1).
Effect of lupeol treatment on brain proinflammatory cytokines levels in ICV-Aβ
(1-42)-infused rats
Figure 3B Effect of lupeol in ORT (Familiar and novel objects)
in Aβ (1-42) infused rats. The values are expressed
as mean ± SEM (n=10). The performance of rats
in the (T2) test phase trial, wherein rats exposed
with two dissimilar objects, one already exposed
F01 (considered as familiar object). *P<0.001 vs.
exploration time of familiar objects.
The levels of TNF-α, IL-6 and IL-1β were increased significantly in
hippocampus following Aβ(1-42) infusion as compared with control
group. Lupeol (25 mg/kg, 50 mg/kg and 100 mg/kg) treatment
significantly restored the hippocampal pro-inflammatory
cytokines level as compared with Aβ(1-42) infused rats (Table 2).
Discussion
The present study demonstrates the neuroprotective potential
of lupeol against Aβ(1-42) induced behavioral, biochemical and
neurochemical abnormalities in rats. ICV infusion of Aβ(1-42) in rats
is known to produce cognitive deficit and other neuropathological
changes similar to those seen in AD and thus considered as
Figure 3C Effect of lupeol in discrimination index of ORT in Aβ
infused rats. The values are expressed as mean ±
(1-42)
SEM (n=10). Discrimination index obtained from the
test phase trial using the equation mentioned in the
text; #P<0.001 vs. vehicle and *P<0.001 vs. Aβ (1-42).
Figure 3A Effect of lupeol in ORT in Aβ (1-42) infused rats. The
values are expressed as mean ± SEM (n=10). (A) The
performance of rats in (T1) sample phase trial with
two similar objects (F01 and F02). No significant
difference was observed between the untreated and
Lupeol-treated rats.
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a suitable model system of AD [23,35]. In the present study,
ICV-Aβ(1-42) infusion produced cognitive impairment, oxidative
stress, neuroinflammation and hippocampal neurotransmitter
deficit demonstrating similar changes following Aβ(1-42) infusion
in rats [35]. Aβ(1-42) is an oligomer, reported to cause destruction
in the selective brain regions such as hippocampus, cortex and
perirhinal cortex that affects spatial and non-spatial memory
respectively [36-38]. MWM and ORT were used to assess spatial
and non-spatial memory respectively. In the present study,
Aβ(1-42) infused rat showed poor learning and consolidation of
memory in MWM task. In addition, these animals were unable
to discriminate between novel and familial objects indicating
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Figure 4 Effect of lupeol on AChE activity in Aβ (1-42) infused rats. The values are expressed as
mean ± SEM (n=10). #P<0.001 vs. Vehicle; *P<0.001 vs. Aβ (1-42).
Table 1 Effect of lupeol on rat hippocampal oxidative-nitrosative stress parameters in Aβ(1-42)-infused rats.
Groups
Vehicle without Aβ (1-42)
Nitrite (μmol/mg protein)
85.8 ± 08.63
Biochemical Parameters
MDA (nmol/mg protein)
1.075 ± 0.344
GSH (μmol /mg protein)
0.950 ± 0.051
Aβ(1-42)
208.6 ± 16.92#
5.213 ± 0.143#
0.175 ± 0.040#
Aβ(1-42) + Lupeol 25 mg
178.9 ± 15.87*
3.813 ± 0.591*
0.485 ± 0.034*
Aβ(1-42) + Lupeol 50 mg
139.0 ± 14.88*
2.913 ± 0 .779*
0.625 ± 0.043*
Aβ(1-42) + Lupeol 100 mg
148.9 ± 19.37*
3.313 ± 0.591*
0.535 ± 0.034*
The values were expressed as mean ± SEM. #P<0.001 vs. Vehicle; *P<0.001 vs. Aβ(1-42).
The values were expressed as mean ± SEM The results were analyzed using two-way ANOVA
followed by Bonferonni test for multiple comparisons and one way ANOVA followed by
Tukey’s test, for multiple groups using statistical Graph Pad Prism software (version 5.0, La Jolla, CA, USA). @P<0.001 vs. Vehicle; *P<0.001 vs. Aβ(1-42).
Table 2 Effect of lupeol on rat hippocampal TNF-a, IL-6, and IL-1b levels in Aβ(1-42) rats.
TNFα (pg/ml)
Biochemical Parameters
IL-1β (pg/ml)
IL-6 (pg/ml)
Vehicle without Aβ (1-42)
43.31 ± 6.717
26.10 ± 4.990
22.17 ± 0.799
Aβ(1-42)
107.20 ± 9.058#
88.10 ± 8.751#
71.15 ± 7.751#
Aβ(1-42) + Lupeol 25 mg
81.11 ± 6.072*
62.16 ± 4.930*
56.80 ± 7.930*
Aβ(1-42) + Lupeol 50 mg
68.79 ± 6.820*
51.93 ± 6.085*
39.46 ± 6.085*
Aβ(1-42) + Lupeol 100 mg
74.41 ± 8.392*
57.96 ± 5.930*
47.80 ± 7.930*
Groups
The values were expressed as mean ± SEM. #P<0.001 vs. Vehicle; *P<0.001 vs. Aβ(1-42).
The values were expressed as mean ± SEM The results were analyzed using two-way ANOVA followed by Bonferonni test for multiple comparisons
and one way ANOVA followed by Tukey’s test, for multiple groups using statistical Graph Pad Prism software (version 5.0, La Jolla, CA, USA). @
P<0.001 vs. Vehicle; *P<0.001 vs. Aβ(1-42).
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cognitive impairment which was significantly restored by lupeol
treatment [39,40].
Recent neuropathological studies have found that AD patients
have complex neurochemical disturbances including the
catecholaminergic, cholinergic neuronal systems and their
restoration may improve memory in AD [9,10]. In addition,
modulators of acetylcholine (ACh), dopamine, 5-HT and NE have
also been reported to improve hippocampal dependant memory
in clinical and preclinical cases of AD [41-43]. Aβ(1-42) in both clinical
and preclinical AD, has reports to causes catecholaminergic deficit
by escalating AChE enzymatic activity [44,45]. In agreement with
the previous findings, the present study also showed significant
increase in AChE activity in Aβ(1-42) infused brains [6-10]. However,
the treatment of lupeol was able to significantly decrease AChE
activity as reported earlier [17]. The finding of the current study
suggest that lupeol contributes to the restoration of ACh level by
reducing the level of AChE in Aβ(1-42) infused rat’s hippocampus.
Thus, recovery in the levels of these monoamines may contribute
to the beneficial effects of lupeol on learning and memory in Aβ
induced AD in rats.
Neuroinflammation and oxidative-nitrosative stress are two most
common events of Aβ (1-42)-induced neurotoxicity and neuronal
cell death as seen in AD patients [46]. Brain regions such as
hippocampus and cortex are highly sensitive to oxidative stress,
mitochondrial dysfunction and neuroinflammation [47,48]. The
expansive nature of oxidative damage in AD includes mitochondrial
dysfunction, Aβ formation, tau aggregation and alterations in
calcium signaling [48]. All these changes lead to alterations in the
transcriptional activity of various pathways, over-production of
MDA, and reduced activity of superoxide dismutase, catalase, and
GSH [47-49]. Aβ(1-42) is well known oligomer to causes oxidative
stress, microglia and astrocytes activation and over expression of
pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. [50]. In
the present study, Aβ(1-42) infused brain tissues exhibit an increase
in oxidative stress, pro-inflammatory cytokines and nitrite level
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which stay in good agreement with its previous findings [51].
However, lupeol treatment significantly attenuated Aβ(1-42)
induced oxidative-nitritive stress and decreased the levels of
pro-inflammatory cytokines, confirming its antioxidant and antiinflammatory properties [52]. Earlier reports have also suggested
that oral administration of lupeol decreases the secretion of
pro-inflammatory cytokines such as TNF-α, and IL-1β and inhibit
the over activation of microglia and astrocytes [52]. It has been
reported that lupeol act through various mechanism and downregulates various apoptotic molecules like Bax, cytochrome C
and caspases that leads to neuronal death [52]. Moreover Lupeol
has also been reported to exhibit neuroprotective action against
the glutamate induced exitotoxicity in mouse hippocampal cells
[22]. Thus, the observed anti-inflammatory antioxidant effects of
lupeol help to decrease the neuronal oxidative stress and may
offer novel strategies in the treatment of age-related AD.
Conclusion
In the current study, lupeol halt Aβ(1-42) induced behavioral,
biochemical and neurochemical abnormalities in rats. Lupeol was
able to improve behavioral changes, restore neurotransmitters
levels, decreased oxidative-nitrosative stress and reduced
inflammatory processes. Together these findings suggest
multiple therapeutic approach of lupeol towards Aβ(1-42) induced
dementia and suggest lupeol as a potential therapeutic molecule
to treat AD like symptoms.
Acknowledgement
Authors are thankful to University Grant Commission (UGC),
Delhi and ISF College of Pharmacy, Moga (Punjab) for valuable
financial support and encouragement.
Conflict of Interest
The authors declare no competing financial interest.
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