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Olive-Oil-Derived Oleocanthal Enhances β-Amyloid Clearance as a Potential Neuroprotective Mechanism against Alzheimer’s Disease: In Vitro and in Vivo Studies

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Oleocanthal, a phenolic component of extra-virgin olive oil, has been recently linked to reduced risk of Alzheimer's disease (AD), a neurodegenerative disease that is characterized by accumulation of β-amyloid (Aβ) and tau proteins in the brain. However, the mechanism by which oleocanthal exerts its neuroprotective effect is still incompletely understood. Here, we provide in vitro and in vivo evidence for the potential of oleocanthal to enhance Aβ clearance from the brain via up-regulation of P-glycoprotein (P-gp) and LDL lipoprotein receptor related protein-1 (LRP1), major Aβ transport proteins, at the blood-brain barrier (BBB). Results from in vitro and in vivo studies demonstrated similar and consistent pattern of oleocanthal in controlling Aβ levels. In cultured mice brain endothelial cells, oleocanthal treatment increased P-gp and LRP1 expression and activity. Brain efflux index (BEI%) studies of (125)I-Aβ(40) showed that administration of oleocanthal extracted from extra-virgin olive oil to C57BL/6 wild-type mice enhanced (125)I-Aβ(40) clearance from the brain and increased the BEI% from 62.0 ± 3.0% for control mice to 79.9 ± 1.6% for oleocanthal treated mice. Increased P-gp and LRP1 expression in the brain microvessels and inhibition studies confirmed the role of up-regulation of these proteins in enhancing (125)I-Aβ(40) clearance after oleocanthal treatment. Furthermore, our results demonstrated significant increase in (125)I-Aβ(40) degradation as a result of the up-regulation of Aβ degrading enzymes following oleocanthal treatment. In conclusion, these findings provide experimental support that potential reduced risk of AD associated with extra-virgin olive oil could be mediated by enhancement of Aβ clearance from the brain.
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Olive-Oil-Derived Oleocanthal Enhances βAmyloid Clearance as a
Potential Neuroprotective Mechanism against Alzheimers Disease:
In Vitro and in Vivo Studies
Alaa H. Abuznait, Hisham Qosa, Belnaser A. Busnena, Khalid A. El Sayed, and Amal Kaddoumi*
Department of Basic Pharmaceutical Science, College of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Drive, Monroe,
Louisiana 71201, United States
ABSTRACT: Oleocanthal, a phenolic component of extra-
virgin olive oil, has been recently linked to reduced risk of
Alzheimers disease (AD), a neurodegenerative disease that is
characterized by accumulation of β-amyloid (Aβ) and tau
proteins in the brain. However, the mechanism by which
oleocanthal exerts its neuroprotective eect is still incom-
pletely understood. Here, we provide in vitro and in vivo
evidence for the potential of oleocanthal to enhance Aβ
clearance from the brain via up-regulation of P-glycoprotein
(P-gp) and LDL lipoprotein receptor related protein-1
(LRP1), major Aβtransport proteins, at the blood-brain
barrier (BBB). Results from in vitro and in vivo studies demonstrated similar and consistent pattern of oleocanthal in controlling
Aβlevels. In cultured mice brain endothelial cells, oleocanthal treatment increased P-gp and LRP1 expression and activity. Brain
eux index (BEI%) studies of 125I-Aβ40 showed that administration of oleocanthal extracted from extra-virgin olive oil to
C57BL/6 wild-type mice enhanced 125I-Aβ40 clearance from the brain and increased the BEI% from 62.0 ±3.0% for control mice
to 79.9 ±1.6% for oleocanthal treated mice. Increased P-gp and LRP1 expression in the brain microvessels and inhibition studies
conrmed the role of up-regulation of these proteins in enhancing 125I-Aβ40 clearance after oleocanthal treatment. Furthermore,
our results demonstrated signicant increase in 125I-Aβ40 degradation as a result of the up-regulation of Aβdegrading enzymes
following oleocanthal treatment. In conclusion, these ndings provide experimental support that potential reduced risk of AD
associated with extra-virgin olive oil could be mediated by enhancement of Aβclearance from the brain.
KEYWORDS: Oleocanthal, β-amyloid clearance, β-amyloid degradation, P-glycoprotein, LRP1, BBB
The Mediterranean diet is associated with benecial health
properties against Alzheimers disease (AD), a neuro-
degenerative disease that aects about 30 million people
worldwide.
1
Epidemiological studies indicate that the preva-
lence of AD and cognitive decline is low among the
Mediterranean area populations compared to those of other
geographical regions of the world.
24
One integral component
of the Mediterranean dietary pattern is the consumption of
extra-virgin olive oil (EVOO).
5
Typically, the intake of EVOO
ranges from 25 to 50 mL per day in the Mediterranean diet.
6
Therefore, the apparent health benets have been partially
attributed to the dietary consumption of EVOO by
Mediterranean populations.
Historically, the health promoting properties of EVOO were
attributed to the high concentration of monounsaturated fatty
acids, in particular oleic acid, contained in EVOO. However,
other seed oils (i.e., sunower, soybean, and rapeseed), which
also contain high concentrations of oleic acid, do not exhibit
the same health benets as EVOO.
79
In addition to oleic acid,
EVOO contains a minor, yet signicant phenolic fraction that
other seed oils lack and this fraction has generated much
interest regarding its health promoting properties. Currently, 36
phenolic compounds have been identied in EVOO and in
vitro and in vivo studies have demonstrated that olive oil
phenolics have positive eects on certain physiological
parameters such as plasma lipoproteins, oxidative damage,
inammatory markers, platelet and cellular function, antimicro-
bial activity, and bone health.
10
Among the phenolic olive oil constituents, ()-oleocanthal, a
naturally occurring phenolic secoiridoid isolated from EVOO,
has shown an anti-inammatory and antioxidant properties
similar to the nonsteroidal anti-inammatory drug ibuprofen.
11
()-Oleocanthal is the dialdehydic form of ()-deacetoxylig-
stroside glycoside responsible for the bitter taste of EVOO, and
its chemical structure is related to the secoiridoid glycosides
ligstroside and oleuropein, which are also common in EVOO.
Chemical structure of oleocanthal is shown in Figure 1.
Recently, oleocanthal has been demonstrated to have
potential neuroprotective properties and contribute to
preventing cognitive decline due to neurodegenerative
diseases.
1214
This has been supported by population-based
studies indicating that Mediterranean diet, rich in olive oil and
Received: January 21, 2013
Accepted: February 15, 2013
Research Article
pubs.acs.org/chemneuro
© XXXX American Chemical Society Adx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXXXXX
monounsaturated fats, protects against age-related cognitive
decline.
15,16
One cohort study performed on 1880 elders in the
United States showed a 40% decrease in the incidence of AD in
populations consuming Mediterranean style diet.
4
Dierent mechanisms have been proposed to describe the
role that oleocanthal plays in the reduced incidence of AD; Li
and co-workers showed that oleocanthal inhibits the formation
of neurobrillary tangles, a key hallmark in the pathogenesis of
AD, by acting on microtubule associated proteins known as tau
proteins, which are involved in the promotion of microtubule
assembly and stability in the neurons.
12
This study was
followed by Monti et al., who investigated the mechanism by
which oleocanthal inhibits tau brillization and aggregation in
vitro via covalent chemical interaction with the brillogenic
fragment of tau proteins.
13
Additional potential mechanism
suggests that oleocanthal reduce the formation of β-amyloid
(Aβ) senile plaques, another pathological hallmark of AD, in
the brain. Pitt et al. demonstrated that oleocanthal can interact
with Aβand alter the oligomerization state of Aβoligomers and
protect the neurons from the synaptopathological eects
associated with Aβaggregation and plaque formation.
14
Several mechanisms have been proposed to account for the
clearance of Aβ, including enzymatic degradation by a variety of
proteases such as neprilysin (NEP) and insulin degrading
enzyme (IDE), removal through the brain ISF bulk ow into
the bloodstream, perivascular lymphatic drainage, and transport
across the BBB.
17,18
Increasing evidence from the literature
show that AD mainly develop due to the excessive
accumulation of Aβin the brain as a result of its faulty
clearance across the BBB.
19
It has been reported that Aβ
removal from the brain across the BBB is mediated by two
major transport proteins; P-glycoprotein (P-gp) and LDL
lipoprotein receptor related protein-1 (LRP1).
2023
Moreover,
recent evidence indicated a progressive decline in the levels of
P-gp and LRP1 at the BBB during normal aging, and this
decline was positively correlated with accumulation of Aβin
AD.
24,25
On the other hand, the receptor for advanced glycation
end products (RAGE), a multiligand receptor in the
immunoglobulin superfamily, mediates and regulates Aβinux
to the brain.
26,27
Numerous attempts have been made to
decrease Aβaccumulation, and the enhanced Aβclearance
across BBB was one potential therapeutic approach that has
been thoroughly reviewed.
28,29
In the present study, we aimed to demonstrate, using in vitro
and in vivo studies, the role of oleocanthal in enhanced
clearance of Aβfrom the brain as an additional possible
mechanism for its neuroprotective eect via its potential to up-
regulate P-gp and LRP1 at the BBB, and its ability to enhance
Aβdegradation. Based on the ndings, we hope to provide a
new insight on the protective eect of oleocanthal against AD
by enhancing Aβclearance from the brain.
RESULTS AND DISCUSSION
Available experimental data strongly suggest impaired clearance
of Aβacross the BBB might largely contribute to the formation
of Aβbrain deposits and AD progression. Also, it has been
demonstrated that P-gp and LRP1 play substantial role in the
elimination of Aβfrom the brain across the BBB.
2023,30
Thus,
in the current study we aimed to investigate the eect of
oleocanthal treatment on the levels of P-gp, LRP1 and Aβ
degrading enzymes, and then the consequences of such
treatment on the clearance of Aβacross the BBB.
The ability of oleocanthal at dierent concentrations to
induce P-gp and LRP1 expressions at the BBB was assessed by
Western blotting using bEnd3 cells as a representative model of
mouse BBB. As shown in Figure 2, treatment of bEnd3 cells
with oleocanthal resulted in signicant increase in P-gp (1.2
1.35-fold increase, P< 0.05) and LRP1 levels (1.251.30-fold
increase, P< 0.05). Similar pattern has been observed in
immunouorescence studies, where oleocanthal treatment
resulted in 2.8- and 2.2-fold increase in P-gp and LRP1
expressions, respectively, when compared to control cells
(Figure 3). While both methods provide semiquantitative
results, the greater fold increase in P-gp and LRP1 expressions
Figure 1. Chemical structure of ()-oleocanthal.
Figure 2. Representative Western blots for P-gp (A) and LRP1 (B) in
bEnd3 cells treated with oleocanthal. Cells were treated for 72 h with
increasing concentrations of the indicated compounds in the range of
0.550 μM.
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observed by the immunouorescence method compared to
Western blotting could be related to methods sensitivity.
31
To determine the eect of oleocanthal treatment on the
accumulation of Aβ, cellular uptake studies were conducted.
While both Aβpeptide species, Aβ40 and Aβ42, have been
implicated in the pathogenesis of AD,
32
Aβ40 was used in this
work because it is practically feasible as it has much faster
clearance rate than Aβ42.
33
The results showed conclusive
evidence for the specic roles of P-gp and LRP1 in 125I-Aβ40
cellular uptake, and their up-regulation on 125I-Aβ40 cellular
level. Unlike in transport studies where cells are polarized and
P-gp and LRP1 work in the same direction (i.e., from abluminal
to luminal side), in uptake studies P-gp and LRP1 are localized
at the cell membrane and function in opposite directions where
P-gp limits 125I-Aβ40 cellular entry due to its eux function,
while LRP1 enhances cellular uptake of 125I-Aβ40. The cellular
uptake of 125I-Aβ40 was evaluated following oleocanthal
treatment in the presence and absence of 100 μM verapamil
(P-gp inhibitor), 1 μM RAP (LRP1 inhibitor), or RAGE (N-
16) antibody (as RAGE inhibitor) at a dilution ratio 1:100. The
Figure 3. Representative uorescent micrographs of (A) P-gp (green) and (B) LRP1 (red) for control and bEnd3 cells treated with 25 μMof
oleocanthal. Quantitative folds change in P-gp and LRP1 expression were measured using ImageJ version 1.44. The data are expressed as mean ±SD
(n= 4). *P< 0.05 compared to control untreated cells. Scale bar = 50 μm.
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results showed signicant alteration in the activities of P-gp and
LRP1 but not RAGE caused by oleocanthal treatment (Figure
4). In oleocanthal treated cells, P-gp inhibition caused
signicant increase in 125I-Aβ40 cellular uptake by 69% (from
812 ±12 to 1376 ±132 cpm/mg protein, P< 0.01) compared
to 28% in vehicle treated cells (from 837 ±36 to 1068 ±102
cpm/mg protein, P< 0.01), indicating enhanced P-gp function
by oleocanthal. In contrast to the function of P-gp, LRP1
functions by cellular internalization, thus its inhibition by RAP
caused a signicant reduction (P< 0.02) in the cellular uptake
of 125I-Aβ40 by 34% (from 837 ±36 to 555 ±8 cpm/mg
protein) for vehicle treated cells, compared to only 17% (from
812 ±12 to 671 ±30 cpm/mg protein) for oleocanthal treated
cells. The percent reduction in 125I-Aβ40 uptake in oleocanthal
treated cells was signicantly lower than that of vehicle treated
cells (P< 0.05; Figure 4). In concept, assuming lack of LRP1
induction by oleocanthal, RAP inhibition should reduce 125I-
Aβ40 uptake equally in both oleocanthal and control treated
cells. However, the reduction in 125I-Aβ40 cellular level in
oleocanthal treated cells was less than that observed with
control suggesting that oleocanthal possibly induced an
unknown uptake mechanism that is not inhibited by RAP.
This nding is further supported by the in vivo data discussed
below. With regard to RAGE, unlike P-gp and LRP1,
oleocanthal has no signicant eect on its expression or
activity (Figure 5).
Next, we aimed to investigate the eect of oleocanthal
treatment on the clearance of 125I-Aβ40 from mouse brain. To
our knowledge, this study is the rst to in vivo investigate
oleocanthal. The dosage regimen selected to conduct these
studies was intraperitoneal administration at 10 mg/kg/day
twice daily for 2 weeks to wild-type mice. At this dose, all
animals were healthy and no weight loss was observed. Given
the aldehyde structure of oleocanthal, metabolically it could be
unstable suggesting formation of active metabolites that may
exert the up-regulation eect.
34
Further studies are currently in
progress to investigate oleocanthal metabolism. In line with the
in vitro results, Western blot analysis (Figure 6) revealed
signicant increase in the expression of P-gp and LRP1 in the
brain microvessels of C57BL/6 mice by oleocanthal treatment.
Densitometric analysis of the bands showed that oleocanthal
increased P-gp and LRP1 expression by 1.30- and 1.25-fold
compared to control, respectively. No signicant change in the
expression of RAGE has been observed in control or treated
animals (data not shown).
Aβis cleared from the brain by nonsaturable (passive) and
saturable pathways (transport and metabolism).
35
Nonsaturable
pathway involves passive removal of soluble Aβthrough bulk
ow of cerebrospinal uid (CSF) (i.e., CSF turnover).
Saturable pathways involve degradation and BBB eux
components. The brain eux index (BEI) method was used
to study brain 125I-Aβ40 clearance with 14C-inulin as a reference
compound.
36
The BEI% method is commonly used to
investigate moleculesbrain clearance via degradation/metab-
olism and/or transport across BBB.
37
Thus, changes in BEI% of
125I-Aβ40 following oleocanthal treatment compared to control
demonstrates alteration in 125I-Aβ40 removal from the brain by
enhanced enzymatic degradation and/or clearance across BBB
via the transport system. Consequently, both mechanisms were
investigated.
Clearance BEI% experiments were performed at 24 h after
the last injection of oleocanthal or vehicle (normal saline). As
shown in Figure 7A, BEI% analysis demonstrated about 18%
increase in the clearance of 125I-Aβ40 in oleocanthal treated
mice (79.9 ±1.6%) compared to control mice (62.0 ±3.0%; P
< 0.05). To study whether the enhanced brain clearance of 125I-
Aβ40 is specically due to increased expression of P-gp and
LRP1 at the BBB, inhibitory studies of each protein were
performed. The role of P-gp in 125I-Aβ40 clearance across the
BBB of C57BL/6 mice was evaluated by preinjection of
valspodar, a specic P-gp inhibitor,
38
into the brain of C57BL/6
mice 5 min before the intracerebral microinjection of 125I-
Aβ40/14C-inulin solution. Valspodar was administered rst to
Figure 4. Eect of treatment of bEnd3 cells with vehicle (CTRL) or
oleocanthal (OLC) in presence or absence of inhibitors on the
intracellular accumulation of radiolabeled 125I-Aβ40. The data are
expressed as mean ±SEM (n= 3 independent experiments).
*Signicantly dierent from no inhibitor treated cells (P< 0.02).
#Signicantly dierent from control treated cells with inhibitors (P<
0.05).
Figure 5. Quantitative analysis for RAGE in bEnd3 cells treated with
oleocanthal. Cells were treated for 72 h with 0, 25, and 50 μM
concentrations of oleocanthal.
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allow its brain distribution and interaction with P-gp prior to
125I-Aβ40 microinjection which has rapid brain clearance.
33
P-gp
inhibition signicantly lowered BEI% value of 125I-Aβ40 in
control and oleocanthal-treated group (Figure 7B). Thirty
minutes post microinjection of 125I-Aβ40, the pretreatment of
control and oleocanthal-treated mice with valspodar resulted in
about 13% (from 62.0 ±3.0% to 49.3 ±4.6%; P< 0.05) and
40% (79.9 ±1.6% to 39.4 ±2.8%; P< 0.001) reduction in BEI
% values, respectively. The reduction in BEI% following P-gp
inhibition by valspodar in oleocanthal treated group compared
to control group was statistically insignicant (P> 0.05).
Similarly, LRP1 inhibition by RAP signicantly reduced 125I-
Aβ40 BEI% (P< 0.05, Figure 7B) in oleocanthal-treated mice.
Pretreatment of control and oleocanthal-treated mice with RAP
resulted in about 33% (from 62.0 ±3.0% to 29.1 ±1.5%; P<
0.001) and 21% (79.9 ±1.6% to 59.2 ±3.5%; P< 0.05)
reduction in BEI% values of 125I-Aβ40, respectively. These
results are in line with similar studies demonstrating the role of
P-gp and LRP1 in the clearance of Aβacross the BBB.
20,22,23
To characterize the eect of oleocanthal treatment on the
degradation of 125I-Aβ40 in C57BL/6 mice brain, we performed
TCA degradation assay and measured the expression of Aβ
degrading enzymes NEP and IDE. The percent of degraded Aβ
peptide (cpm in supernatant) were signicantly higher in
oleocanthal treated group (60.0 ±2.3%) compared to control
(40.1 ±1.2%, P< 0.05, Figure 8A). Further, treatment of mice
with oleocanthal resulted in a signicant increase (1.6-fold) in
the expression of IDE and small but insignicant increase in
NEP (1.1-fold) in mice brain microvessels, respectively (Figure
8B, C). Consistent with in vivo ndings, in vitro treatment of
bEnd3 cells resulted in signicant increase in the expression of
IDE (1.6-fold, P< 0.05) and NEP (1.3-fold, P< 0.05) enzymes
(data not shown). Thus, oleocanthal treatment enhanced the
degradation of Aβ40 in mice brain, mostly via induction of IDE
enzyme and possibly NEP.
Collectively, the above results corroborate the assumption
that oleocanthal improves 125I-Aβ40 clearance by enhancing P-
gp and LRP1 expression/activity at the BBB. However, the
inhibition of LRP1 by RAP was more apparent in control mice
than oleocanthal-treated mice, which is consistent with the data
obtained from the in vitro inhibition studies. Yet, Aβclearance
from the brain of oleocanthal-treated mice was greater than
control mice even in the presence of RAP, suggesting the
induction of another mechanism(s) contributing to the
clearance of 125I-Aβ40, which could be an unknown trans-
porter(s)/receptor(s),
23
most likely at the abluminal side, and/
or enhanced degradation of 125I-Aβ40 as a result of the
treatment. Our results conrm both, induction of an unknown
Figure 6. Western blot analysis of P-gp and LRP1 in mice brain
microvessels. Signicantly higher expression levels of P-gp and LRP1
were detected in oleocanthal (OLC) treated mice compared to control
group (CTRL). (A) Representative Western blot lanes for P-gp, LRP1,
and protein loading control (β-actin). (B) Quantitative fold increase in
P-gp and LRP1 expressions. The data are expressed as mean ±SEM of
n= 3 independent experiments (*P< 0.05).
Figure 7. (A) Brain eux index (BEI) of 125I-Aβ40 in control (CTRL),
and oleocanthal (OLC) treated groups measured 24 h after the last
injection of oleocanthal or normal saline. Signicantly higher BEI%
was observed in oleocanthal treated mice compared to control group.
(B) Eect of P-gp and LRP1 inhibition by valspodar (24 ng/0.5 μL
injection) and RAP (19.5 ng/0.5 μL injection), respectively, on BEI%
of 125I-Aβ40 in CTRL and OLC treated groups. Both valspodar and
RAP caused a signicant reduction in the BEI% in oleocanthal treated
group. The data are expressed as mean ±SEM of n=46(*P< 0.05,
***P< 0.001).
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transport mechanism, supported by the in vitro data, and
degradation. Beside its role in the up-regulation of P-gp and
LRP1, oleocanthal enhanced the clearance of 125I-Aβ40 by up-
regulating NEP (in vitro studies) and IDE, two peptidases that
have been reported to cleave and degrade Aβimplicated in AD.
Several studies reported overexpression of NEP to protect
hippocampal neurons from Aβ-mediated toxicity in vitro,
39
and
the up-regulation of NEP or IDE has been proposed as a
potential means of protecting the brain against Aβaccumu-
lation, prevention of amyloid plaque formation, and consequent
cognitive decline.
4042
In support of its putative health benets, oleocanthal has
more recently been demonstrated to possess therapeutic
activities in the treatment of AD disease.
12,14
These studies
support research surveys showing a 40% decrease in AD in
populations consuming a Mediterranean style diet comprising
olive oil.
4
Our ndings described in this study provide further
evidence on the role of oleocanthal as a neuroprotective agent
Figure 8. (A) Eect of oleocanthal (OLC) treatment on Aβdegradation in mice brain homogenate compared to control (CTRL) measured using
TCA assay. Signicantly higher Aβdegradation% was observed in OLC treated mice compared to control. The data are expressed as mean ±SEM of
n=46(*P< 0.05). (B) Representative Western blots lanes for IDE, NEP, and protein loading control (β-actin) in mice brain microvessels.
Signicantly higher expression levels of IDE but not NEP were detected in OLC treated mice compared to control. (C) Quantitative fold increase in
IDE and NEP expressions. The data are expressed as mean ±SEM of n= 3 independent experiments (*P< 0.05).
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against AD in vivo in wild-type mice brains by increasing 125I-
Aβ40 clearance, which substantiate the in vitro ndings reported
by us and other research laboratories. Additional research is
required to determine the therapeutic benets of oleocanthal
using AD animal model, and its bioavailability which has not
been investigated.
In conclusion, our ndings demonstrated that the benecial
eect of oleocanthal against AD could be extended to its ability
to induce P-gp and LRP1, which are responsible for Aβ
clearance across the BBB. Also, this study provides conclusive
evidence for the role of oleocanthal on Aβdegradation as
shown by the up-regulation of Aβdegrading enzymes IDE and
possibly NEP. Furthermore, our results show that extra-virgin
olive oil-derived oleocanthal associated with the consumption
of Mediterranean diet has the potential to reduce the risk of AD
or related neurodegenerative dementias.
METHODS
Reagents and Antibodies. 1,1,1,3,3,3-Hexauoro-2-propanol
(HFP) and Tween 20 were purchased from Sigma-Aldrich (St.
Louis, MO). RIPA buer was purchased from Thermo Scientic
(Rockford, IL). Synthetic monoiodinated and nonoxidized 125I-Aβ40
(human, 2200 Ci/mmol) was purchased from PerkinElmer (Boston,
MA). 14C-Inulin (2.2 mCi/g) was purchased from American
Radiolabeled Chemicals (St. Louis, MO). The human receptor
associated protein (RAP) was purchased from Oxford Biomedical
Research (Oxford, MI). Valspodar was obtained from XenoTech
(Lenexa, KS). The reagents and supplements required for Western
blotting were purchased from Bio-Rad (Hercules, CA). For Western
blot, the mouse monoclonal antibody C-219 against P-gp was obtained
from Covance Research Products (Dedham, MA); mouse monoclonal
antibody against light chain LRP1 (5A6) was obtained from
Calbiochem (Gibbstown, NJ); rabbit monoclonal antibody against
light chain LRP1 was obtained from Epitomics (Burlingame, CA);
goat polyclonal antibodies against, RAGE (N-16), β-actin (C-11), and
HRP-labeled secondary antibodies were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA); rabbit polyclonal antibody
against insulin degrading enzyme (IDE) and mouse monoclonal
antibody against neprilysin (NEP, anti-CD10) were obtained from
Abcam Inc. (Cambridge, MA). For immunohistochemistry, the rabbit
polyclonal antibody against P-gp was purchased from Rockland
Immunochemicals Inc. (Gilbertsville, PA). Donkey anti-rabbit Alexa
Flour 594 was purchased from Invitrogen (Carlsbad, CA), and goat
anti-mouse IgG-FITC labeled secondary antibody was obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Formaldehyde 16%
(Methanol Free, Ultrapure EM grade) was purchased from
Polysciences, Inc. (Warrington, PA). Ketamine and xylazine for mice
anesthetization were purchased from Henry Schein Inc. (Melville,
NY). All other reagents and supplies were purchased from VWR
(West Chester, PA).
Extraction of Oleocanthal from Extra-Virgin Olive Oil.
Oleocanthal was extracted as reported by Elnagar et al.
43
from
EVOO (Members Mark, batch no. VF1_US102808, Italy) on
lipophilic Sephadex LH20 (Sigma Aldrich, bead size 25100 μm)
using n-hexane-CH2Cl2(1:9) and nally puried on C-18 reversed-
phase Bakerbond octadecyl (40 μm; Mallinckrodt Baker, Inc.) using
isocratic CH3CNH2O (40:60). A purity of >90% was established for
oleocanthal as assessed by TLC, 1H NMR spectroscopy, and/or
HPLC analysis.
Cell Culture. The mouse brain endothelial cells (bEnd3; ATCC,
Manassas, VA), passage 2530, were cultured in DMEM growth
medium supplemented with 10% fetal bovine serum (FBS), and the
antibiotics penicillin G (100 units/mL) and streptomycin (100 μg/
mL), 1% w/v nonessential amino acids, glutamine 2 mM. The cells
were grown to conuence in 75 cm2cell culture asks for 12 days in
a humidied atmosphere (5%CO2/95% air) at 37 °C.
In Vitro Induction of P-gp and LRP1 Expression by
Oleocanthal in bEnd3 Cells. Cells were seeded in 10 mm cell
culture dishes (Corning, NY) at a density of 1 ×106cells per dish. The
cells were allowed to grow up to 50% conuence and then treated with
oleocanthal or control (vehicle) in a humidied atmosphere (5%CO2/
95% air) at 37 °C. Methanolic stock solution of oleocanthal was
diluted to a nal concentration of 0.550 μM in growth medium
before use. Media containing oleocanthal at dierent concentrations in
addition to control medium were added to the respective treatment
cells in duplicate at a maximum methanol concentration of 0.2%. The
cells were then incubated for 72 h in a humidied atmosphere (5%
CO2/95% air) at 37 °C. At the end of treatment period, cells were
harvested and total protein was extracted for Western blot analysis.
Western Blot Analysis of P-gp, LRP1, RAGE, IDE, and NEP
Proteins in bEnd3 Cells. At the end of treatment period with
oleocanthal or control, total protein was extracted from bEnd3 cells
with lysis buer (RIPA buer; 25 mM Tris.HCl, 150 mM NaCl, 1%
NP-40, 1% sodium deoxylate, 0.1% SDS, pH 7.6). For Western blot
analysis, 25 μg of cellular protein was resolved on 7.5% SDS-
polyacrylamide gels and transferred onto nitrocellulose membrane.
Blotting membranes were blocked with 2% BSA and incubated
overnight with antibodies for LRP1 (light chain), P-gp (C-219), RAGE
(N-16), β-actin (C-11), IDE, or NEP at dilutions 1:1500, 1:200, 1:200,
1:3000, 1:100, and 1:100, respectively. For protein detection, the
membranes were washed and incubated with HRP-labeled secondary
IgG antibody for LRP1, P-gp, NEP (antimouse), IDE (antirabbit) and
RAGE, β-actin (antigoat) at 1:5000 dilution. The bands were
visualized using SuperSignal West Femto Maximum Sensitivity
substrate detection kit (Thermo Scientic; Rockford, IL). Quantitative
analysis of the immunoreactive bands was performed using GeneSnap
luminescent image analyzer (Scientic Resources Southwest Inc.,
Staord, TX) and band intensity was measured by densitometric
analysis. Three independent Western blotting experiments were
carried out for each treatment group.
In Vitro Uptake Study of 125I-Aβ40 in bEnd3 Cells. The aim of
this experiment was to investigate the eect of oleocanthal treatment
on the accumulation of 125I-Aβ40 in bEnd3 cells. An uptake study using
125I-Aβ40 was conducted as described previously.
31
Briey, bEnd3 cells
cultured onto 48 well plates at a density of 5 ×104cells/well and were
grown to 5060% conuence. Following 72 h of treatment with 25
μM of oleocanthal or vehicle, the medium was aspirated and the cells
were incubated in fresh growth medium for 4 h. After treatment, cells
were washed three times with phosphate buer-saline (PBS) solution.
The cells were then preincubated with or without 100 μM verapamil
(P-gp inhibitor), 1 μM RAP (LRP1 inhibitor), or RAGE (N-16)
antibody (RAGE inhibitor) at a dilution ratio 1:100, in complete
culture medium for 30 min. The activity experiments were started by
the addition of 0.07 nM of 125I-Aβ40 in complete culture media with or
without 100 μM verapamil, 1 μM RAP, or 1:100 dilution ratio of
RAGE (N-16) antibody for 15 min in a humidied atmosphere (5%
CO2/95% air) at 37 °C. The activity experiment was then terminated
by washing the cells three times with ice-cold PBS. The cells were then
disrupted with the lysis buer containing complete mammalian
protease inhibitor for 15 min at 4 °C. The radioactivity of 125I-Aβ40
accumulated inside the cells was measured using Wallac 1470 Wizard
Gamma Counter (PerkinElmer Inc., Waltham, MA). The data were
normalized for the protein content. The protein concentrations were
determined using the Pierce bicinchoninic acid (BCA) protein assay
kit (Thermo Scientic, Rockford, IL) according to the manufacturers
instruction with bovine serum albumin (BSA) as a standard.
Immunouorescence Staining and Imaging of P-gp and
LRP1 in bEnd3 Cells. To conrm eect of drugs treatment on the
expression and up-regulation of P-gp and LRP1, both proteins were
visualized using confocal microscopy. bEnd3 cells (5 ×104) were
seeded on 35 mm poly-D-lysine coated glass bottom plates no. 1.5
(MatTek Corporation, Ashland, MA) and treated with 25 μMof
oleocanthal or vehicle as described above. After incubation, the cells
were washed three times with PBS and xed with 4% formaldehyde for
10 min. The cells were then washed with PBS and blocked for 30 min
with 10% of normal donkey and goat sera in 0.3% Triton X-100/PBS.
The cells were then incubated overnight at 4 °C with a 1:200 dilution
of primary antibody against P-gp or LRP1 in solution composed of 1%
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXXXXXG
normal donkey and goat sera in PBS. After washing with PBS, the cells
were incubated for 30 min with antirabbit Alexa our 594 conjugated
secondary IgG for the detection of LRP1 and antimouse FITC for the
detection of LRP1, both at 1:250 dilution in PBS. Images for P-gp and
LRP1 were captured using Zeiss LSM 5 Pascal confocal microscope
equipped with 543 nm line of HeNe Laser and 63X oil immersion
objective lens with numerical aperture = 1.4 (Carl Zeiss MicroImaging,
LLC, Thornwood, NY). Negative controls for each treatment that
were processed without primary antibody showed negligible back-
ground uorescence. P-gp and LRP1 membrane immunouorescence
for each sample was quantied using ImageJ version 1.44 software
(Research Services Branch, NIMH/NIH, Bethesda, MD).
Animals. C57BL/6 wild-type male mice were purchased from
Harlan Laboratories (Houston, TX). The mice were 67 weeks old
with an average body weight of 20 g. Mice were kept under standard
environmental conditions (22 °C, 35% relative humidity, 12 h dark/
light cycle) with free access to tap water and standard rodent food.
After shipping, mice were allowed to adapt to the new environment for
one week before initiating the experiments. All animal experiments
were approved by the Institutional Animal Care and Use Committee
of the University of Louisiana at Monroe and all surgical and treatment
procedures were consistent with the IACUC policies and procedures.
In Vivo Induction of P-gp and LRP1 Expression by
Oleocanthal. Animals were divided into two treatment groups:
control and oleocanthal groups. Each group contained at least four
animals. Drug administration was started at 78 weeks of age and
continued for 2 weeks. Mice of control group received intraperitoneal
vehicle (normal saline) twice daily. Mice of oleocanthal group received
intraperitoneal oleocanthal at dose of 10 mg/kg twice daily.
Brain Eux Index Study. At the end of treatment period, mice
were prepared for brain eux index study (BEI) to assess the clearance
of Aβ24 h after the last dose of oleocanthal. The in vivo Aβclearance
experiments (BEI%) were performed using the intracerebral micro-
injection technique reported previously.
23,36,44,45
In brief, mice were
anesthetized with intraperitoneal xylazine and ketamine (20 and 125
mg/kg, respectively) and placed in a stereotaxic apparatus (Stoelting
Co., Wood Dale, IL) to determine the coordinates of the mice brain
that coincide with the right caudate nucleus. A stainless steel guide
cannula was implanted stereotaxically at 0.9 mm anterior, 1.9 mm
lateral to bregma, and 2.9 mm below the surface of the brain as
previously reported.
20
The guide cannula and screw were xed to the
skull with binary dental cement, and a stylet was introduced into the
guide cannula. Once the cement was rm, the animal was removed
from the stereotaxic device, and the wound was closed anterior and
posterior to the guide assembly using 1.75 mm Michel suture clips.
Animals were then allowed for 12 h recovery from acute brain injury
due to insertion of guide cannula in order to restore BBB integrity.
45
Animals were reanesthetized with intraperitoneal xylazine/ketamine
and an injector cannula connecting via Teon tubing to a 1.0 μL
gastight Hamilton microsyringe was inserted into the guide cannula.
The applied tracer uid (0.5 μL) containing 125I-Aβ40 (30 nM) and
14C-inulin (0.02 μCi) prepared in extracellular uid buer (ECF; 122
mM NaCl, 25 mM NaHCO3, 3 m M KCl, 1.4 mM CaCl2, 1.2 mM
MgSO4, 0.4 mM K2HPO4,10mMD-glucose, and 10 mM HEPES, pH
7.4) was administered into the caudate nucleus over a period of 3 min.
To prevent aggregation, 125I-Aβ40 was initially solubilized in HFP,
dried, and resolubilized in ECF buer prior to the experiment. The
intactness and quality of 125I-Aβ40 was initially conrmed by
trichloacetic acid (TCA) precipitation assay.
20
After the micro-
injection, the microsyringe was left in place for 3 min to minimize
any backow. At the designated time, 30 min post 125I-Aβ40
injection,
20,22,23
plasma and brain tissues were rapidly collected for
Aβmeasurements. To characterize the role of LRP1 and P-gp
function, 0.5 μL of ECF containing RAP, an LRP1 inhibitor
20,23,46
or
valspodar, a well-established P-gp inhibitor
23,47
were intracerebrally
preadministered 5 min prior to 125I-Aβ40 injection at concentrations of
1μM (19.5 ng/0.5 μL injection volume) and 40 μM (24 ng/0.5 μL
injection volume), respectively.
Calculation of 125I-Aβ40 Clearance. 125I-Aβ40 is characterized by
its rapid clearance across the BBB,
33
thus brain was collected 30 min
postinjection to determine AβBEI%.
20,22,23
Brain tissues were excised
and homogenized by Dounce tissue grinder with seven strokes in two
volumes of DPBS buer (2.7 mM KCl, 1.46 mM KH2PO4, 136.9 mM
NaCl, 8.1 mM Na2HPO4, 0.9 mM CaCl2, and 0.5 mM MgCl2
supplemented with 5 mM D-glucose and 1 mM sodium pyruvate,
pH 7.4) containing mammalian protease inhibitor cocktail. About half
of the brain homogenate was used for 125I-Aβ40 and 14C-inulin
quantication and the second half for microvessels isolation and
protein expression study. Trichloroacetic acid (TCA) precipitation
method was used to calculate the amount of intact 125I-Aβ40 remained
in the brain.
20,23
To measure intact 125I-Aβ40, one volume of TCA
(20%) was added to the sample, and then samples were vortexed,
incubated in ice for 30 min, and centrifuged at 14 000 rpm (4 °C) for
30 min. Following centrifugation, gamma radioactivity of precipitated
125I-Aβ40 (intact peptide) and TCA supernatant (degraded peptide)
were measured using Wallac 1470 Wizard Gamma Counter. The
supernatant and precipitate were then mixed with 5 mL scintillation
cocktail and the beta radioactivity of 14C-inulin was measured using
Wallac 1414 WinSpectral Liquid Scintillation Counter (PerkinElmer
Inc.). The BEI% was dened by eq 1 and the percentage of substrate
remaining in the brain (100 BEI%) was determined using eq 2.
46
β
β
=
×BEI% I A brain clearance
I A injected into the brain 100
125
125 (1)
−= ×
β
β
100 BEI% 100
amount of intact I A in the brain
amount of C inulin in the brain
amount of intact I A injected
amount of C inulin injected
125
14
125
14 (2)
The percent degradation for each sample was calculated by dividing
the supernatant cpm by the total cpm (sum of the cpm in the
precipitate and the supernatant cpm) and the resulting percent were
subtracted from the percent of free 125I which determined from
preinjected sample by spiking mice brain homogenate with 0.5 μL
injectate solution and then processed as sample homogenate.
Isolation of Brain Microvessels. Brain microvessels were isolated
as described previously by us.
23
Briey, after decapitation of mouse,
brain was immediately put in ice-cold normal saline, homogenized and
divided into halves as described above. One volume Ficoll (30%) was
added to the half of brain homogenate to a nal concentration of 15%
and the mixture was mixed, and then centrifuged (5000 rpm for 10
min, 4 °C). The resulting pellets were suspended in ice-cold DPBS
containing 1% bovine serum albumin (BSA) and passed over glass
bead column. Microvessels adhering to the glass beads were collected
by gentle agitation in 1% BSA in DPBS. Isolated microvessels were
used for Western blotting and immunohistochemistry studies of LRP1
and P-gp.
Western Blot Analysis of P-gp, LRP1, and RAGE in Isolated
Brain Microvessels. Protein expression levels in isolated brain
microvessels were analyzed by Western blotting as described above.
Total protein was extracted from isolated brain microvessels by
homogenization with lysis buer (RIPA buer) containing protease
inhibitor. Homogenized samples were centrifuged at 13 000 rpm for
10 min and supernatant was used for further Western blot analysis as
described above.
Statistical Analysis. Wherever possible, the experimental results
were analyzed for statistically signicant dierence using Two-tailed
unpaired Studentst-test to evaluate dierences between controls and
treated groups. A p-value less than 0.05 was considered to be
statistically signicant. All results were expressed as means and
standard error of mean (SEM).
AUTHOR INFORMATION
Corresponding Author
*Telephone: 318-342-1460. Fax: 318-342-1737. E-mail:
kaddoumi@ulm.edu.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXXXXXH
Author Contributions
All in vitro and in vivo experiments and data analysis were
performed by A.H.A. and H.Q. Oleocanthal extraction from
EVOO and characterization were performed by B.A.B. and
supervised by K.A.E. All experiments were designed and
supervised by A.K.
Funding
This research work was funded by an Institutional Develop-
ment Award (IDeA) from the National Institute of General
Medical Sciences of the National Institutes of Health under
Grant Number P20GM103424.
Notes
The authors declare no competing nancial interest.
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ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXXXXXJ
... Neurological investigations indicated that EVOO may slow the progression of memory impairment and improve cognitive performance in clinical and preclinical AD models [18][19][20][21][22][23]. Indeed, supplementing EVOO to the diet of an AD mouse model has been demonstrated to improve the bloodbrain barrier (BBB) function, increase the clearance of Aβ, reduce its production, and reduce neuroinflammation [16]. ...
... Following its discovery and identification, it has been reported that oleocanthal has several modes of action in reducing inflammation-related diseases, including neurodegenerative diseases. Here, some evidence has proven OC to be effective against Aβ deposits and other neuropathological features in AD models [19,28,29]. Therefore, it is hypothesized that long-term consumption of EVOO-containing OC may contribute to the health benefits associated with the Mediterranean dietary pattern. ...
... Then, 13 were excluded for not meeting the characteristics of the approach or the review goal. After reviewing the full text of the remaining records, 7 met the inclusion criteria and were included in the systematic review [19,21,29,[33][34][35][36]. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) flow chart illustrating the number of studies at each stage of the review is shown in Fig. (1). ...
Article
Background Mediterranean diet may enhance cognitive function and delay the progression of Alzheimer's disease (AD). We conducted a systematic review to investigate the effect of oleocanthal (OC) from extra-virgin olive oil (EVOO) on amyloid-β (Aβ) burden in preclinical models of AD, considering the anti-inflammatory and neuroprotective effects of EVOO biophenols, which are key components of the Mediterranean dietary model. Methods The literature was searched through six electronic databases until February 2023. Screening of 52 retrieved articles for inclusion criteria resulted in 7 preclinical reports evaluating the effect of an OC-supplemented diet on AD trajectories by means of Aβ load or clearance in affected models. Reports were appraised for risk of bias using the SYRCLE's RoB tool. A protocol was registered on PROSPERO. Results Case control prevailed over the case-crossover design, and the geographical distribution was uniformly American. The study population mostly included 5xFAD, otherwise TgSwDI or wild-type C57BL/6 mouse models. We found a role of OC in reducing Aβ load in the hippocampal parenchyma and microvessels compared with controls. An increased cerebral clearance of Aβ through the bloodbrain barrier and a substantial improvement in metabolic and behavioral parameters were also reported in preclinical models under an OC-enriched diet. The risk of bias was shown to be moderate overall. Conclusion Preclinical data are promising about the effects of OC from the Mediterranean diet's EVOO in relieving the burden of Aβ in AD; however, further evidence is needed to corroborate the efficacy of this biophenol and strengthen the speculated causal pathway.
... [5] Thus, oleacein 1 and oleocanthal 2 are the principal aldehydic constituents of EVOO, structurally very similar to each other differing only by their aromatic moiety, hydroxytyrosol and tyrosol respectively. In recent years, these two dialdehydes have raised a wide scientific interest owing to their biological activities (e. g., anti-atherogenic, anti-hepatotoxic, anti-inflammatory, antioxidant, anti-tumoral, anti-viral, analgesic, hypoglycaemic, cardioprotective and neuroprotective) [6][7][8][9][10][11][12][13][14][15][16][17] despite their low availability from both natural and synthetic sources. More than a decade ago, the beneficial effect of these compounds was recognized by the European Food Safety Authority (EFSA) which established that "olive oil polyphenols contribute to the protection of blood lipids from oxidative stress", specifying that "The claim may be used only for olive oil which contains at least 5 mg of hydroxytyrosol and its derivatives (e. g. oleuropein complex and tyrosol) per 20 g of olive oil. ...
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In this paper, Krapcho's one‐step decarbomethoxylation of oleuropein in DES is reported. Oleuropein used as reagent was extracted with water from olive leaves, widely available and inexpensive waste from the olive oil production chain. The reaction has been carried out in a series of ChCl‐based DES of increasing acidity, with or without the addition of an amount of water, under microwave and conventional heating. The antioxidant power of the best reaction mixture, both in terms of biocompatibility of the medium and conversion of the starting material, was measured and compared with a natural phenolic mixture coming from EVOO. The reported results indicate that the formulation deriving by the Krapcho's one‐step decarbomethoxylation of oleuropein in ChCl:Citric acid (1 : 1) DES can provide a ready‐to‐use “phenolic complex“ with oxygen scavenging power similar to a mixture of phenols extracted from an EVOO that meets the requirements of EFSA health claim.
... Administration of oleocanthal extract of extra virgin olive oil to wild-type C57BL/6 mice enhanced clearance of β-amyloid (1-40) peptide labeled with iodine 125, (125) I-Aβ40, from the brain and increased the brain efflux index (BEI %) method; brain efflux index characterizes an efflux transport system for substrates from the brain to circulating blood along the blood-brain barrier). The findings experimentally support that the risk of Alzheimer's disease is reduced with EVOO consumption through enhancing the clearance of Ab from the brain [56]. DOI: http://dx.doi.org/10.5772/intechopen.1007554 ...
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The Mediterranean diet, which has been followed in Greece for centuries, is a modern object of study and analysis. Its main ingredients are olive oil and table olives. It is now characterized as a diet with an extremely positive effect on human health. The consumption of extra virgin olive oil and table olives, basic components of the Mediterranean diet, offers significant benefits to human health. Olive oil is a source of energy and monounsaturated, “good” fats, and a rich source of antioxidants, especially extra virgin olive oil, which helps in the absorption of fat-soluble vitamins (A, D, and E) from food, helps reduce the risk of cardiovascular diseases, and contributes, in the context of the Mediterranean diet, to the better management of various other diseases such as type 2 diabetes mellitus, cancer, neurodegenerative diseases, and Alzheimer’s disease. It provides antioxidants and many of the fats necessary for good development during neonatal and childhood.
... These phenols have been shown to reduce both Aβ plaques and neurofibrillary tangles by interacting to varying degrees with their production and clearance. In addition, antioxidant system strengthening and anti-inflammatory activities have also been shown concerning its protective effects in these diseases [12,16,32]. ...
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Chronic neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, whose prevalence increases with aging, cause serious health problems with inadequate treatment options. Long-term exposure to low doses of pesticides like chlorpyrifos has been linked to an increased risk of developing neurodegenerative diseases. This study assessed the effects of olive leaf extract (OLE) on cell viability and neurite outgrowth inhibition induced by chlorpyrifos (25 µM) using the mouse NB2a neuroblastoma cell line. High concentrations of OLE as 100 to 3000 µg/ml reduced cell viability, likely due to its anticancer properties. On the other hand, lower concentrations of OLE as 3, 10, 30 and 100 µg/ml significantly reversed the inhibition of neurite outgrowth caused by chlorpyrifos exposure, demonstrating a neuroprotective effect. In conclusion, the regular consumption of OLE may effectively slow or prevent the advancement of neurodegenerative diseases linked to long-term exposure to low-dose organophosphate pesticides. Furthermore, the chlorpyrifos-induced moderate neurotoxicity model can be employed as a valuable screening method for evaluating the neuroprotective potential of natural products, including various plant extracts.
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In the current scenario, Alzheimer's disease is a complex, challenging, and arduous health issue, and its prevalence, together with comorbidities, is accelerating around the universe. Alzheimer's disease is becoming a primary concern that significantly impacts an individual's status in life. The traditional treatment of Alzheimer's disease includes some synthetic drugs, which have numerous dangerous side effects, a high risk of recurrence, lower bioavailability, and limited treatment. Hence, the current article is a detailed study and review of all known information on plant-derived compounds as natural anti-Alzheimer's agents, including their biological sources, active phytochemical ingredients, and a possible mode of action. With the help of a scientific data search engine, including the National Center for Biotechnology Information (NCBI/PubMed), Science Direct, and Google Scholar, analysis from 2001 to 2024 has been completed. This article also described clinical studies on phytoconstituents used to treat Alzheimer's disease. Plant-derived compounds offer promising alternatives to synthetic drugs in treating Alzheimer's disease, with the potential for improving cognitive function and slowing down the progression of the disease. Further research and clinical trials are needed to fully explore their therapeutic potential and develop effective strategies for managing this complex condition.
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Alzheimer's disease (AD) is considered one of the most common neurological conditions associated with memory and cognitive impairment and mainly affects people aged 65 or above. Even with tremendous progress in modern neuroscience, a permanent remedy or cure for this crippling disease is still unattainable. Polyphenols are a group of naturally occurring potent compounds that can modulate the neurodegenerative processes typical of AD. The present comprehensive study has been conducted to find out the preclinical and clinical potential of polyphenols and elucidate their possible mechanisms in managing AD. Additionally, we have reviewed different clinical studies investigating polyphenols as single compounds or cotherapies, including those currently recruiting, completed, terminated, withdrawn, or suspended in AD treatment. Natural polyphenols were systematically screened and identified through electronic databases including Google Scholar, PubMed, and Scopus based on in vitro cell line studies and preclinical data demonstrating their potential for neuroprotection. A total of 63 significant polyphenols were identified. A multimechanistic pathway for polyphenol's mode of action has been proposed in the study. Out of 63, four potent polyphenols have been identified as promising potential candidates, based on their reported clinical efficacy. Polyphenols hold tremendous scope for the development of a future drug molecule as a phytopharmaceutical that may be incorporated as an adjuvant to the therapeutic regime. However, more high‐quality studies with novel delivery methods and combinatorial approaches are required to overcome obstacles such as bioavailability and blood–brain barrier crossing to underscore the therapeutic potential of these compounds in AD management.
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Since the advent of 3D printing technology, a significant effort has been made to develop new 3D printable materials. Despite the recent progress in the field of 3D printing, the limited availability of photoactive resins has motivated continuous research endeavors to develop novel photoresins with multifunctional capabilities. Herein a biobased photoresin derived is reported from modified olive oil, designed for high‐resolution solvent‐free 4D printing with multifunctional capabilities. The physicochemical properties of the printed polymers are fine‐tuned using acrylic acid as a diluent cum comonomer. The mechanical properties of the printed polymers are similar to various soft tissues, such as ligaments, articular cartilage, and soft collagenous bone, showcasing its potential for soft tissue engineering applications. While the excellent temperature‐responsive shape memory 4D attributes coupled with exceptional antimicrobial properties toward gram‐negative and gram‐positive bacteria highlight the multifunctional nature of the printed polymers. Moreover, the printed polymers exhibited outstanding hemocompatibility and good cytocompatibility toward mouse fibroblast cells, suggesting their potential soft tissue engineering applications. In sum, the newly developed biobased resin can be employed to minimize the environmental impact of additive manufacturing while being competitive with existing fossil‐based photoresins, thereby meeting the growing demand for advanced photoresins with superior high‐resolution printing and smart properties for biomedical applications.
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The endogenous synthesis of lipids, which requires suitable dietary raw materials, is critical for the formation of membrane bilayers. In eukaryotic cells, phospholipids are the predominant membrane lipids and consist of hydrophobic acyl chains attached to a hydrophilic head group. The relative balance between saturated, monounsaturated, and polyunsaturated acyl chains is required for the organization and normal function of membranes. Virgin olive oil is the richest natural dietary source of the monounsaturated lipid oleic acid and is one of the key components of the healthy Mediterranean diet. Virgin olive oil also contains a unique constellation of many other lipophilic and amphipathic constituents whose health benefits are still being discovered. The focus of this review is the latest evidence regarding the impact of oleic acid and the minor constituents of virgin olive oil on the arrangement and behavior of lipid bilayers. We highlight the relevance of these interactions to the potential use of virgin olive oil in preserving the functional properties of membranes to maintain health and in modulating membrane functions that can be altered in several pathologies. This article is part of a Special Issue entitled: Membrane structure and function: Relevance in the cell's physiology, pathology and therapy.
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