Abstract
Abelmoschus esculentus Linn. (okra, F. Malvaceae) is a fruit widely
consumed all over the world. In our study, the anti-Alzheimer’s
potential of A. esculentus was evaluated. An in vitro DPPH free radical
assay on A. esculentus seed’s total extract and AChE inhibition
potential screening indicated a significant anti-Alzheimer’s activity
of the extract, which was confirmed through an in vivo study in an
aluminum-intoxicated rat model. Additionally, in vivo results
demonstrated significant improvement in Alzheimer’s rats, which was
confirmed by improving T-maze, beam balance tests, lower serum levels
of AChE, norepinephrine, glycated end products, IL-6, and MDA. The
levels of dopamine, BDNF, GSH, and TAC returned to normal values during
the study. Moreover, histological investigations of brain tissue
revealed that the destruction in collagen fiber nearly returns back to
the normal pattern. Metabolomic analysis of the ethanolic extract of A.
esculentus seeds via LC–HR-ESI-MS dereplicated ten compounds. A network
pharmacology study displayed the relation between identified compounds
and 136 genes, among which 84 genes related to Alzheimer’s disorders,
and focused on AChE, APP, BACE1, MAPT and TNF genes with interactions
to all Alzheimer’s disorders. Consequently, the results revealed in our
study grant potential dietary elements for the management of
Alzheimer’s disorders.
Keywords: Alzheimer’s, acetyl choline, BDNF, seeds, pharmacological
network, okra
1. Introduction
Continuous damage of neuronal construction or performance are the main
features of neurodegenerative diseases [[48]1]. Additionally, they act
as a chief socioeconomic burden all over the world. The possibility of
obtaining a neurodegenerative disorder rises significantly as age
increase [[49]1]. Accordingly, with an aging population, the number of
affected people has increased even further, demanding the innovation of
therapeutic approaches able to decrease or inhibit neurodegenerative
illnesses. In the same context, Alzheimer’s disorder (AD) is a
neurodegenerative illness which is characterized by damage in brain
neurons, especially in those responsible for memory, language, and
thinking. Additionally, numerous neurons are destroyed and more brain
parts are influenced, which negatively affects the patient’s life
[[50]2]. According to the 2022 report of the World Health Organization,
55 million people were affected by AD up until 2019, which is assumed
to increase to 139 million by 2050 [[51]3]. Currently, different drugs
with different targets for AD treatment are available on the market,
such as donepezil hydrochloride, which is an acetylcholinesterase
(AChE) inhibitor mostly utilized for Alzheimer’s disorder treatment.
Donepezil is FDA-permitted in patients at different stages of
Alzheimer’s disorders. Until now, there has been no proof that
donepezil changes the disease’s progression. However, it affects
various symptoms by cognition and/or behavior improvement. Moreover, it
has different off-label uses. This study will point out the possible
mechanism of action, the adverse event outline, the pharmacology, the
monitoring, and relevant donepezil interactions, significant for
members of the interprofessional side for the treatment of patients
with Alzheimer’s disorders. The most frequent adverse effects of the
drug are gastrointestinal disorders as nausea, diarrhea, and vomiting.
Additional common adverse effects comprise insomnia, muscle cramps,
weakness, and anorexia, which are more frequent at higher doses.
However, most patients suffer from theses adverse effects in minor and
transient form, persisting up to three weeks and mostly getting
resolved with continual use [[52]4].
On the other hand, neuronal survival and growth mainly depend on
neurotrophins, such as brain-derived neurotrophic factor (BDNF). The
signaling pathway presented by BDNF and its receptor are the main
synaptic plasticity regulators, which contributes an essential function
in learning and memory processes [[53]1]. Additionally, BDNF attracts
more attention for the innovation of medicines to cure neurological
diseases [[54]1,[55]5]. Moreover, several inflammatory mediators have
been included in AD, such as tumor necrosis factor-α (TNF-α) as well as
interleukin-6 (IL-6) [[56]6]. Different studies showed that TNF-α
provokes diminishing in memory as well as peripheral glucose
intolerance by disordering insulin signaling and eliciting cellular
stress cascades in AD mouse models [[57]7]. During the initial-phase
amyloid plaque construction in AD brains, IL-6 was recognized as a
major factor in tau phosphorylation, synapse loss, and learning
disorders in mice [[58]7]. Regardless of various arguments in the
literature, earlier meta-analyses revealed that the IL-6 levels are
raised in both cerebrospinal fluid (CSF) and mild cognitive impairment
(MCI) plasma and in patients with AD in comparison to control persons
[[59]7]. However, this hints at an involvement of IL-6 in AD disorders,
and a fundamental association between IL-6, cognitive impairments, and
peripheral metabolic disorganization in AD continues to be recognized.
Additionally, amyloid precursor protein (APP) is affected by β- and
γ-secretase to generate amyloid β peptide (Aβ), which disperses in
neuronal cells in the form of an amyloid residue. The residue is a
pathologic property of AD [[60]5,[61]8]. The major source of
neurotoxicity in AD was defined as glyceraldehyde-derived advanced
glycated end products (AGEs). APP processing and Aβ production are
controlled by oxidative stress [[62]8]. Furthermore, neuronal cell
death is caused by Aβ through reactive oxygen species (ROS) [[63]9].
AGEs adjust Aβ accumulation and amyloid aggregation [[64]10]. Our
results in a former study recommended that glyceraldehyde-deprived AGEs
raise the levels of APP and Aβ via ROS, and that this effect finally
precedes to cell death [[65]5,[66]8]. Conversely, AGEs role in the
enhancement of Alzheimer’s disorder (AD) still indistinguishable.
Nature as a treasure resource of bioactive metabolites provides us with
several nutrients helping in curing and decreasing the symptoms of AD
[[67]11]. Several vitamins, such as B[6], B[9], K, C, and E help in
decreasing oxidative stress and improving brain functions [[68]12].
Additionally, some plant extracts possess potential anti-AD properties,
such as Punica granatum [[69]13], Teucrium polium [[70]14], Fragaria
ananassa [[71]15], Bacopa floribunda [[72]16], and Ginkgo biloba
[[73]17]. On the other hand, Abelmoschus esculentus L., synonym:
Hibiscus esculentus, mostly identified as okra or lady´s fingers, is a
nutrient-rich food. It acts as a major resource of various valuable
constituents such as flavonoids, phenolic acids, mucilage, vitamins,
nutrients, fibers, and minerals [[74]18]. Different parts of okra, such
as fruits, seeds, and leaves, are implicated in various applications
owing to their properties and composition [[75]19]. Additionally, it
was used over centuries in traditional medicine for the treatment of
various illnesses, such as worm infestation, dysentery, and
inflammation and irritation of the stomach, intestines, and kidneys.
Okra fruit extract, as well as its derived flavonoids and
polysaccharides, showed remarkable anti-AD potential
[[76]20,[77]21,[78]22,[79]23,[80]24]. However, limited studies have
been conducted on okra seeds and their oil, which revealed their
polyphenolic compounds and polyunsaturated fatty acids
[[81]25,[82]26,[83]27]. A few studies were carried out on the
bioactivity of okra seed extract, showing their potent anti-oxidant and
anticancer potential against human breast cancer (MCF7) and skin
fibroblast (CCD-1059Sk) cells [[84]28]. Consequently, there is a great
demand to explore the potential of okra seeds as a low-cost available
nutrient.
Aluminum (Al) serves as a crucial risk factor for different age-related
neurodegenerative diseases [[85]29], including AD [[86]30]. Aluminum
chloride (AlCl[3]) is used as a neurotoxicant, which dispersed in the
brain and influences ionic, cholinergic, as well as dopaminergic
neurotransmission with negative effect [[87]31]. As a consequence, the
aim of our present study was to pinpoint the possible benefits and
therapeutic influences of A. esculentus seed extract in diminishing the
neurodegenerative character of Alzheimer’s disorder in a rat model
intoxicated with Al, which may afford an economical, reliable, simple,
noninvasive, and reproducible blood-based group of significant
biomarkers, to prepare the way for feasible early curative approaches.
Moreover, a metabolomics study of the extract and a pharmacological
network were applied to identify chemical molecules that assist in
anti-Alzheimer’s activity.
2. Results
2.1. In Vitro DPPH Radical Scavenging Activity Assay of A. esculentus Seed
Extract
The free radical scavenging potential of A. esculentus seed total
extract was investigated using the DPPH method [[88]32]. The results
demonstrated that A. esculentus seed extract had considerable
scavenging activity percentages for DPPH, respectively, in a dose
dependent manner compared to ascorbic acid ([89]Table 1).
Table 1.
In vitro DPPH scavenging activity of A. esculentus seed extract.
0.01 µg/mL 0.05 µg/mL
Crude extract 77.2 ± 3.6 ^c 93.2 ± 7.5 ^d
Ascorbic acid 82.3 ± 4.7 ^b 91.0 ± 6.1 ^d
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The data were expressed as mean ± SD (n = 3) of at least three
independent experiments. Groups with similar letters are not
significantly different, while those with different letters are
significantly different at p ≤ 0.05.
2.2. In Vitro Evaluation of Cholinesterase Potential of the A. esculentus
Seed Extract
The cholinesterase inhibitory activity of A. esculentus seed extract in
comparison with the standard drug donepezil were evaluated against the
AChE enzyme and the results were obtained as IC[50] (μg/mL) measures in
[91]Table S1. A. esculentus seed extract exhibited a noticeable
inhibitory activity with an IC[50] value of 0.028 μg/mL. The A.
esculentus seed extract showed an AChE inhibitory effect approximately
the same as the reference drug (IC[50] = 0.025 μg/mL) ([92]Table S1).
2.3. Acute Toxicity Study
Different concentrations of A. esculentus seed extract were utilized
for evaluating the acute toxicity, with 500, 1000, 2000, 3000, 4000, as
well as 5000 mg/kg b.wt., with four rats for each group (in total, 24
rats were included for all groups together). Until 3000 mg/kg b.wt. and
for 48 h, no mortality in addition no toxicity signals were observed as
well as no signs of toxicity or behavior abnormalities. The chosen dose
utilized was 500 mg/kg b.wt. [[93]33].
2.4. Potential Activities of A. esculentus Seed Extract in the T-Maze Test
A. esculentus seed extract was investigated for its anti-AD ability at
a dose of 500 mg/kg b.wt., utilizing the T-maze test. In this context,
the results showed a noteworthy raise in time (per seconds) received by
rats reaching their food in the T-maze for rats with
AlCl[3]-neurotoxicant (AD group), indicating a worsened neurocognitive
function, with an increase in a percentage to 233.8% ([94]Table S2).
The rats of the AD group treated with A. esculentus seed extract, on
the contrary, exhibited a significant decline in time taken to reach
their food in the T-maze, compared to the AD-induced group, showing
improved cognitive functions, with the improvement percentage reaching
175.5% (6 weeks treatment), compared to donepezil as the standard drug
(181.9%); see [95]Table S2.
2.5. Potential Effects of A. esculentus Seed Extract in the Beam Balance Test
The results obtained from the beam balance test revealed that AlCl[3]
caused major worsening in brain cognitive abilities for
AlCl[3]-neurotoxicant rats (AD group) ([96]Table S3), with a reduction
in percentage down to 70.69%. Additionally, rats treated with donepezil
or A. esculentus seed extract revealed a dramatic enhancement in
behavioral status, characterized by motor coordination and enhanced
cognition improvement, with a percentage of improvement reaching 62.05%
for A. esculentus seed extract in comparison with animals treated with
donepezil as the standard drug, which recorded 69.02% in a 6-week
treatment).
2.6. Potential Effects of A. esculentus Seed Extract in Acetylcholine
Esterase Levels in the Brain
[97]Table 2 reveals a considerable raise in the AChE in the serum of
AD-induced rats with a percentage increase of 94.8%, while the AChE
levels showed an improvement upon the treatment of AD rats with A.
esculentus seed extract, entailing a percentage improvement of 76.2%
compared to the standard drug (83.2%).
Table 2.
Effect of A. esculentus seed extract on AChE levels in
Alzheimer’s-disorder-induced rats.
Parameters Control AlCl[3]-AD Extract Donepezil Drug
AChE (serum) (U\L)
% Change
% Improvement 215.0 ± 11.0 ^a 419.0 ± 10.0 ^b
+94.8 255.0 ± 8.0 ^c
76.2 240.0 ± 9.7 ^c
83.2
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The data are represented as means ± SD (n = 10). Groups showing the
same letters were not significantly different; additionally, those with
different letters revealed considerable difference at p ≤ 0.05. AChE:
acetylcholine esterase. The % change was calculated compared to the
control group as (mean of treated—mean of negative/mean of negative
control) × 100. The % improvement was also calculated as mean of
positive control—mean of treated/mean of negative control × 100.
2.7. Potential Effects of A. esculentus Seed Extract in Norepinephrine,
Dopamine, and Serotonin Levels in Brain
AD-induced rats revealed a noteworthy rise in the level of
norepinephrine, with an increase to 723.0% in comparison with control
rats ([99]Table S4), with a promising reduction of both dopamine and
5-HT of 68.9 and 65.6%, respectively. AD rats treated with A.
esculentus seed extract showed a significant improvement in
neurotransmitters levels, reaching 545.0, 46.2, and 50.8% for
norepinephrine, dopamine, and 5-HT, respectively, compared to the
standard drug (558.0, 55.2, and 54.8% for norepinephrine, dopamine, and
5-HT, respectively).
2.8. Potential Effects of A. esculentus Seed Extract in the Levels of BDNF,
Glycated End Product, and IL-6 in the Brain
[100]Table 3 reveals a significant reduction in BDNF level in brain
tissue, by 50.3% compared to the control, while a significant elevation
in brain tissue glycated end product and serum IL-6 was observed, with
an increase reaching 221.2 and 227.7%, respectively, in comparison to
the control rats. AD rats treated with A. esculentus seed extract
exhibited remarkable improvements in BDNF, glycated end product, and
IL-6 with percentages amounting to 26.5, 163.7, and 213.5%,
respectively, in comparison to the reference drug, which revealed
improvements of 35.2, 219.6, and 229.4% for BDNF, AGEs, and IL-6,
respectively ([101]Table 3).
Table 3.
Effects of A. esculentus seed extract on the levels of BDNF, glycated
end product, and IL-6 in AD-induced rats.
BDNF
(pg/g Tissue) Glycated End Product
(μg/g Tissue) IL-6 (pg/mL)
Control 343.0 ± 10.2 ^a 12.7 ± 1.2 ^a 39.6 ± 3.0 ^a
AlCl[3]-AD
% Change 167.0 ± 10.9 ^b
−50.3% 40.8 ± 3.1 ^b
221.2% 130.0 ± 8.0 ^b
227.7%
AD crude extract
% Improvement 258.0 ± 6.0 ^d
26.5 20.0 ± 2.0 ^d
163.7 45.3 ± 3.0 ^d
213.5
Donepezil drug
% Improvement 288.0 ± 16.0 ^e
35.2 12.9 ± 1.1 ^a
219.6 39.0 ± 2.8 ^a
229.4
[102]Open in a new tab
The data are shown in seconds as mean ± SD (n = 10). Groups showing the
same letters are not significantly different; additionally, those with
different letters revealed a significant difference at p ≤ 0.05. The %
change was calculated compared to the control group as (mean of
treated—mean of negative/mean of negative control) × 100. Additionally,
the % improvement was calculated as (mean of positive control—mean of
treated/mean of negative control) × 100.
2.9. Potential Effects of A. esculentus Seed Extract in TAC, MDA, and GSH
Levels in the Brain
A significant reduction in TAC was observed with an amount of 74.1%
compared to the control. GSH also showed a significant reduction in
brain tissue with 60.9%, while a significant increase in MDA level was
recorded, reaching a value of 451.7%. AD rats treated with A.
esculentus seed extract showed an amelioration in anti-oxidant and
oxidative stress levels with a higher degree of improvement, reaching
51.6, 390.4, and 44.9% for TAC, MDA, GSH, respectively, compared to
donepezil, which recorded improvements of 52.8, 394.2, and 53.4%,
respectively ([103]Table S5).
2.10. Results of the Histopathological Investigation
Histopathological investigations showed Alzheimer’s-induced rat brain
with the presence of neuronal degeneration and neurofibrillary tangles
(NFT), congestion of meningeal blood, degeneration of Purkinje cells
with decreased granular layer density, and degeneration of hippocampus
neurons (Photomicrographs 3 and 4, and [104]Figure 1) compared to
control brain rats (Photomicrographs 1 and 2, and [105]Figure 1). After
treatment with A. esculentus, AD rat brain showed a few degenerated
neurons in the cerebral cortex, nearly normal cerebellum, and
degeneration of a few neurons in hippocampus (Photomicrographs 5–7, and
[106]Figure 1). Donepezil-treated AD rat brain revealed meningeal
hemorrhage and a few degenerated neurons in the cerebral cortex
(Photomicrograph 8 and [107]Figure 1).
Figure 1.
[108]Figure 1
[109]Open in a new tab
Photomicrographs for H&E-stained sections of the cerebral cortex of
rats (scale bar 20 µm): (1) Control rat brain revealing normal
histological structure of cerebral meninges (arrow). (2) Control rat
brain displaying the normal histological structure of cerebellum. (3)
AD rat brain revealing congestion of meningeal blood vessels (arrow)
with hemorrhage. (4) Alzheimer’s-induced rat brain showing degeneration
of hippocampus neurons (arrows). (5) AD rat brain treated with A.
esculentus seeds highlighting a few degenerated neurons in cerebral
cortex (arrow). (6) AD rat brain treated with A. esculentus seeds,
showing nearly normal cerebellum. (7) AD rat brain treated with A.
esculentus seeds showing degeneration of few neurons in hippocampus
(arrow). (8) Donepezil-treated AD rat brain evidencing few degenerated
neurons in cerebral cortex, mild meningeal hemorrhage (arrow).
The score order was constructed as follows: score 0 indicates no
lesions in all rats of the group (n = 5); score 1 indicates (<30%);
score 2 indicates (30–50%), score 3 indicates (>50%). Group 1 is the
control group; Group 2 is the Alzheimer’s-induced group; Group 3 is the
group of AD rats treated with A. esculentus seed extract; Group 4 is
the donepezil–cured AD group.
Group 2 (Alzheimer’s-induced rats) showed a high scoring level of
histopathological alterations in the brain compared to the control
group (Group 1). Group 3 (AD rats treated with A. esculentus seed
extract) exhibited similar results to Group 4 (donepezil-treated AD
rats), in which these groups revealed the same scoring
histopathological alterations in brain ([110]Table S6).
2.11. Chemical Dereplication of A. esculentus Seed Extract
Analyzing the total extract of A. esculentus seeds, different hits were
proposed ([111]Table S7, [112]Figures S1A and S1B, [113]Figure 2). The
mass ion peaks at m/z 301.0353, 341.10792, 465.1033, and 477.10499, in
accordance with the suggested molecular formulas C[15]H[10]O[7],
C[12]H[22]O[11,] C[21]H[20]O[12], and C[22]H[22]O[12] [M + H]^+; [M −
H]^+ ([114]Table S7) fit flavones and disaccharide derivative compounds
quercetin (1), 4-O-α-D-galactopyranosyl-D-galactose (2), isoquercitrin
(3), and quercetin 3-glycosides, 3-O-(4-O-methyl-β-D-glucopyranoside)
(4), which had been previously isolated from A. manihot [[115]34], H.
esculentus [[116]35], and A. esculentus [[117]36] ([118]Table S8),
respectively.
Figure 2.
[119]Figure 2
[120]Open in a new tab
Dereplicated metabolites from LC-HR-ESI-MS analysis of A. esculentus
seed extract.
The molecular ion mass peaks at m/z 595.1452, 597.1456, 609.1466,
625.1769, 627.1561, and 637.1557 [M + H]^+; [M - H]^+ ([121]Table S7),
for the predicted molecular formulas C[30]H[26]O[13], C[26]H[28]O[26],
C[27]H[30]O[16], C[28]H[32]O[16], C[27]H[30]O[17], and C[32]H[28]O[14],
respectively, gave hits of the flavones tiliroside (5),
5,7,3′,4′-tetrahydroxy
flavonol-3-O-[β-d-rhamnopyranosil-(1→2)]-β-d-glucopyranoside (6),
quercetin-3-orobinoside (7), floramanoside D (8),
quercetin-3-O-sophoroside (9), and
3-O-kaempferol-2-O-acetyl-4-O-(p-coumaroyl)-α-D-glucopyranoside (10),
respectively. These compounds had previously been isolated from A.
manihot [[122]34] and A. esculentus [[123]36] ([124]Table S7,
[125]Figure 2).
3. Discussion
3.1. Evaluation of the Anti-Alzheimer’s Potential of A. esculentus Seed
Extract
The free radical scavenging potential of A. esculentus seed total
extract was investigated using the DPPH method [[126]32]. The results
demonstrated that A. esculentus seed extract provided a considerable
scavenging potential for DPPH, in a dose-dependent manner compared to
ascorbic acid ([127]Table 1). In addition, the cholinesterase
inhibitory activity of A. esculentus seed extract in comparison with
the standard drug donepezil were evaluated against the AChE enzyme and
the results were obtained as IC[50] values (μg/mL) in [128]Table S1. A.
esculentus seed extract exhibited a noticeable inhibitory activity with
IC[50] of 0.028 μg/mL. The A. esculentus seed extract showed an AChE
with approximately the same inhibitory effect as the reference drug
(IC[50] = 0.025 μg/mL) ([129]Table S1).
According to the in vitro DPPH free radical scavenging and the
cholinesterase potential assays, A. esculentus seed extract was
utilized for further in vivo studies for estimating the
anti-Alzheimer’s potential.
In accordance with the data represented in [130]Table S2, the results
of the behavioral tests approved the earlier obtained ones, which had
shown that rats neurointoxicated with AlCl[3] need additional time to
collect food in a T-maze, with an increase of 233.8% compared to
control rats, representing a worsened neurocognitive character
[[131]37]. A. esculentus seed extract, however, exhibited a noteworthy
decline in time required by the rats to collect food in the T-maze, in
comparison with the AD-induced group. This indicates an improvement in
cognitive functions, with improvements reaching 175.5% (6 weeks of
treatment) in comparison to the reference drug, which showed an
improvement up to 181.9%. A beam balance test is utilized to
investigate rodent gait in a testing system which encounters the
possibility of them maintaining balance, assumed that the animals need
to cross over a raised-up beam with a tight diameter. The findings
obtained from the beam balance test showed that AlCl[3] triggered a
major worsening in brain cognitive abilities for rats neurointoxicated
with AlCl[3] (AD group) ([132]Table S3), with a decline of −70.6%. On
the other hand, rats treated with donepezil or A. esculentus seed
extract revealed an improvement in behavioral attitude, which is
expressed by values of 69.0 and 62.0%, respectively.
The current results in [133]Table 2 indicate that AChE activity
increased significantly in the serum of AlCl[3]-induced AD rats.
AlCl[3]has shown a cholinotoxication that causes changes in different
neurotransmission as cholinergic, dopaminergic, and noradrenergic.
Accordingly, it is capable of producing weakened cholinergic
transmission through influencing the synthesis as well as liberation of
neurotransmitters [[134]38]. Our findings are in agreement with
previous data reported by Aly et al., 2011 [[135]39], Borai et al.,
2017 [[136]37], Aly et al., 2011 [[137]40], and Elmaidomy et al., 2022
[[138]38]. They revealed that AlCl[3] administration showed a
substantial rise in AChE potential in the serum in addition to brain
tissue, compared to neurologically normal control rats. Additionally,
our results revealed a noteworthy decrease in dopamine (DA) and
serotonin, with a significant rise in the norepinephrine level in
AD-induced rats. This runs in parallel with Kaur, 2019 [[139]41], who
showed the negative effect of AlCl[3] on memory ability. This was
attributed to the involvement of AlCl[3] in the dopaminergic pathway
[[140]38] and also to its function to initiate oxidative stress, as
oxidative stress as well as inflammation lead to a decrease in numerous
major neurotransmitters, comprising AChE and DA [[141]42]. Likewise,
the major change in brain neurotransmitters in rats exposed to AlCl[3]
rats may be attributed to a raised formation of O[2] and H[2]O[2], in
addition to accumulation of Lewy bodies in the brain, thus raising the
probability of neurodegenerative disorders [[142]43]. Moreover, DA
oxidation is improved by the elevated concentration of iron in rats
exposed to Al, triggering the production of DA quinones, which
covalently act together with cysteine deposits of glutathione (GSH)
enzymes to inhibit its anti-oxidative potential [[143]43].
Additionally, AlCl[3] enhances the accumulation of α-synuclein and
reduces DA-binding receptors (D1 and D2) in the brain cortex in
addition to the striatum of AlCl[3]-exposed subjects and in addition to
employing an inhibiting potential on different levels, including
inhibition of DA β-hydroxylase (responsible of conversion of DA into
norepinephrine) and inhibition of tryptophan decarboxylase potential
(responsible for DA formation) [[144]38]. The diminished level of DA
and changed cholinergic function may also be due to elevated monoamine
oxidase (MAO) activity, which leads to an increased degradation of
serotonin. Additionally, neurotransmission is negatively affected by
AlCl[3], either by direct inhibition of the enzymes included in
neurotransmitter synthesis and/or by utilization or influencing the
structural characters of synaptic membranes that might influence the
release and/or uptake of these molecules [[145]38].
Treatment of AD rats with A. esculentus seed extract led to an
enhancement in the neurotransmitter levels in AD-induced rats in
comparison with untreated AD rats. These effects may be due to an
inhibition of different signaling pathways, comprising interference
with the IGF-I (insulin-like growth factor-1) mitogenic pathway
[[146]44]. Moreover, neurotoxicity and oxidative stress induced by
rotenone (a mitochondrial complex I blocker) in mice was affirmed to be
diminished by applying an extract of okra seeds [[147]45]. The oral
dose of okra seed extract in mice counterbalanced rotenone-induced
oxidative deterioration, restored glutathione levels as well as
stimulated the antioxidant defense system (glutathione peroxidase,
superoxide dismutase). It also has the ability to decrease the activity
of rotenone-induced AChE and to recover dopamine in the striatum
[[148]45]. Interestingly, extract of okra seeds was effective in
restoring mitochondrial complex activities and reserving their redox
state. It has been demonstrated that oral administration of okra seeds
has a high tendency to provide neuroprotection against neurotoxicants
in addition to other neurodegenerative disorders, such as Parkinson’s
disease [[149]45].
Our present study has revealed the considerable decrease of BDNF in the
brains of AD rats ([150]Table 3). Several studies confirmed the present
results and the association between Alzheimer’s disorder and BDNF. The
decrease in BDNF protein, as well as mRNA levels in the neocortex in
addition to the hippocampus of brain tissue, suggested that BDNF has a
key role in Alzheimer’s disorder [[151]1,[152]46,[153]47]. This
depletion may be explained on the basis of the fact that BDNF is
accompanied with Aβ accumulation, tau phosphorylation, in addition to
neuroinflammation, and neuronal apoptosis [[154]1]. Aβ-induced
downregulation of BDNF is associated with tau; therefore, Alzheimer’s
disorder treatments that emphasize only Aβ may not be valuable if the
influence of tau pathology on neurotrophic cascades is not reflected
[[155]48]. Interestingly, neuronal abnormality, neuronal loss, synaptic
degeneration, and behavioral deficits have been proven to be alleviated
by BDNF overexpression or gene delivery. Therefore, this indication
affording a new opportunity for the treatment of Alzheimer’s disorder
using BDNF remedy [[156]1].
Our current results revealed a noteworthy rise in AGEs in brain tissue
of AD rats ([157]Table 3). This elevation in AGEs is due to the
modification on APP by β- and γ-secretase, which results in deposition
of amyloid (plaques) in neuronal cells [[158]8].
Glyceraldehyde-obtained AGEs are known to be a key resource of
neurotoxicity in AD. APP and Aβ were elevated by
glyceraldehyde-deprived AGEs via ROS. Neurotoxicity has been proven to
be enhanced by the combination of AGEs and Aβ [[159]8]. APP expression
elevated by tail vein injection of AGEs, which suggested a connection
between AGEs in addition to the development of AD [[160]8]. It was
verified that AGEs upregulate APP processing protein (BACE and PS1) as
well as Sirt1 expression via ROS, with no effect on the expression of
antioxidant genes HO-1 and NQO-1. Additionally, AGEs raise GRP78
expression and improve the cell death-connected pathway p53, bcl-2/bax
ratio, caspase 3. These results revealed that AGEs worsen the
protective effect on neurons influence of Sirt1 which cause neuronal
cell death through ER stress [[161]8]. Earlier studies indicated
[[162]8,[163]9] that AGEs elevate production of ROS, which triggers
downstream cascades associated with APP processing, Aβ production,
Sirt1, and GRP78, and this results in the upregulation of a cell death
associated pathway. Additionally, this enhances neuronal cell death,
and causes the AD development. Earlier studies had indicated that there
is a connection between Sirt1 as well as ROS in neuronal cells
[[164]47]. Sirt1 provides a neuroprotective importance by hindering the
ROS effects [[165]47]. Sirt1 has also been revealed to diminish the Aβ
production through α-secretase activation [[166]49]. Consequently, AGEs
raise ROS production, which triggers the downstream pathways of Sirt1,
GRP78, APP processing, as well as Aβ production. Moreover, the Aβ
aggregation in addition to neurofibrillary tangles are improved.
Additionally, this eventually leads to the upregulation of the cell
death cascade, which improves the neuronal cell death, causing the
progress of AD. The deposition of AGEs is considered as a natural
process that elevates gradually with aging; abnormal deposition of
AGEs, however, could be triggered with illness, dietary habits, and
other factors. AGEs accumulate quickly in the body, then pass into the
brain, and consequently turn on the mechanism of AGEs effecting.
Finally, it has been proven that the level of AGEs is a significant key
point for developing AD [[167]49].
Further, IL-6 exhibited significant elevation in serum of AD rats
([168]Table 3). Lyra e Silva et al., 2021 [[169]50], declared that IL-6
showed negative involvement in memory formation, as blocking IL-6
improves long-term potentiation as well as enhances long-term memory in
hippocampus-dependent tasks. By the same authors, astrocytes
surrounding both parenchymal in addition to vascular amyloid deposits
in AD brains and IL-6 immunoreactivity in astrocytes were found, which
were positive for Aβ. Moreover, they revealed that AD patients have
elevated IL-6 levels in the brain tissue and plasma, and found that
plasma IL-6 associated in a positive manner with brain T2
hyperintensities and negatively with cognitive ability. Central
administration of Aβ1–42 in mice induced a systemic IL-6 response that
is distinguished by elevated plasma and brain levels of this cytokine
[[170]51]. Consequently, these findings showed that the brain might be
incorporated with the dysregulation in the circulating IL-6, which
occurs in AD, and thus could involve the inflammatory reflex triggered
by the vagus nerve [[171]52]. AD rats treated with A. esculentus seed
extract displayed remarkable improvements in BDNF, glycated end
product, and IL-6 amounting to 26.5, 163.7, and 213.5%, in comparison
with the reference drug, which showed improvements of 35.2, 219.6, and
229.4% for BDNF, AGEs and IL-6, respectively ([172]Table 3).
Our current study revealed a noteworthy reduction in TAC and GSH in
AD-induced rats, while major elevation in MDA level was distinguished
([173]Table S5). The chief principles of mitochondrial
dysfunction-induced intracellular damage are considered as the
disruption in the antioxidant defense mechanism as well as excessive
generation of reactive oxygen species (ROS) [[174]39]. These results
are in accordance with Aly et al., 2018 [[175]39], who showed that
AlCl[3]-associated neurotoxicity leads to an elevation in lipid
peroxidation. Additionally, the present study revealed the connection
between the rise in MDA in AD rats with the decrease in GSH and TAC, as
involved in the elimination of ROS in brain tissue, proposing a
pro-oxidant potential of AlCl[3]. Instead, Sumathi et al., 2013
[[176]53] revealed that AlCl[3] exposition enhances destruction in
neuronal lipids related to modifications in the enzymatic antioxidant
defense system. Additionally, the current results indicated a
substantial decline in GSH level in the brain tissues of rats triggered
with AlCl[3], which attributed to a high level of H[2]O[2]-induced
cytotoxicity in brain endothelial cells due to inhibition of
glutathione reductase [[177]39]. The significant reduction in brain TAC
in AlCl[3]-induced AD rats attributed to long-term exposure to AlCl[3],
causing a rise in lipid peroxidation as well as a reduction and
exhaustion of numerous antioxidant enzymes [[178]38]. Additionally, Aly
et al., 2022 [[179]39] demonstrated the reduction in TAC in AD-induced
rats by the decline in axonal mitochondria transformation, damage of
Golgi, and decrease of synaptic vesicles, which leads to the liberation
of oxidative molecules such as hydroperoxide, carbonyls, and
peroxynitrites, although there is a decline in antioxidant enzymes and
glutathione within the neurons. The ameliorative activity of okra seed
crude extract mainly depends on the antioxidant metabolites that
exhibit their influences by cooperating with free radicals which could
additionally destruct essential metabolites in the body [[180]54].
Moreover, the interactions include the accumulation of peroxides, as
well as the scavenging of radicals and chelation to metal ions.
The histopathological investigation revealed AD brain with neuronal
degeneration and the existence of neurofibrillary tangles (NFT),
congestion of meningeal blood, degeneration of Purkinje cells with
decreased granular layer density and degeneration of hippocampus
neurons compared to control brain rats ([181]Figure 1). These results
run in parallel with Aşir and Taş, 2022 [[182]55] and Elmaidomy et al.,
2022 [[183]38], who revealed that the cerebral cortexes of AD rats
showed numerous neuropathic alterations such as neuronal necrosis, as
well as neuronophagia, neurofibrillary tangles formation, and focal
gliosis. In addition, the hippocampus of the AD rats demonstrated
shrunken and necrotized pyramidal neurons accompanied by the formation
of neurofibrillary tangles. However, AD-induced rats treated with okra
seed extract exhibited normal cerebellum and degeneration of a few
neurons in the hippocampus compared to donepezil as the standard drug,
which showed meningeal hemorrhage and few degenerated neurons in
cerebral cortex. Additionally, a low lesion score was recorded in
treatment groups compared to the AD group ([184]Figure 1).
Consequently, the current study revealed the therapeutic importance of
okra seed extract on inflammatory and neurotransmitters biomarkers.
3.2. Metabolomics Profiling of A. esculentus Seed Extract
Additionally, metabolomics profiling of the crude ethanolic extract of
A. esculentus seeds was performed to explore its chemical diversity,
leading to the identification of ten flavonoid glycoside compounds. In
this report, the compounds 4-O-α-D-galactopyranosyl-D-galactose,
quercetin 3-glycosides, 3-O-(4-O-methyl-β-D-glucopyranoside),
tiliroside, 5,7,3′,4′-tetrahydroxy
flavonol-3-O-[β-d-rhamnopyranosil-(1→2)]-β-d-glucopyranoside,
quercetin-3-orobinoside, and
3-O-kaempferol-2-O-acetyl-4-O-(p-coumaroyl)-α-D-glucopyranoside are
detected herein for the first time in okra seed extract.
3.3. Pharmacology Networking
3.3.1. Plant-Compounds Network
A simple network, connecting A. esculentus seeds to the identified
compounds by LC-HRESIMS, was constructed as a first step to build the
network pharmacology of A. esculentus seeds’ relation to Alzheimer’s
disorders.
3.3.2. Compounds-Genes Network
The identified compounds targeted genes, these genes were identified by
the SwissTargetPrediction database, after omitting the duplicates, a
total number of 136 genes were identified and a compounds–genes network
was structured and visualized by the Cytoscape software; the network
was formed of 146 nodes and 297 edges, with a path length of 2.727 in
addition to a network centralization of 0.664 ([185]Figure 3,
[186]Table S8). The top gene represented in this network was
[187]P00918 with ten edges, followed by eleven genes each with nine
edges; [188]Q9NPH5, [189]P15121, [190]P47989, [191]P30542, [192]P43166,
[193]O43570, [194]P22748, [195]P22303, [196]P28907, [197]P08913, and
[198]P18825 (genes are expressed in UniProt IDs corresponding to gene
names CA2, NOX4, AKR1B1, XDH, ADORA1, CA7, CA12, CA4, ACHE, CD38,
ADRA2A, and ADRA2C.
Figure 3.
[199]Figure 3
[200]Open in a new tab
Compounds–genes network, connecting identified compounds from A.
esculentus seeds to their target genes; oval green shapes represent the
identified compounds, the purple circles represent target genes.
3.3.3. Genes–Alzheimer’s Disorders Network
A network linking the identified genes to different Alzheimer’s
disorders led to the determination of 84 genes of this data set related
to Alzheimer’s disorders; the formed network consisted of 91 nodes and
183 edges with a characteristic path length and network centralization
of 2.054 and 0.909, respectively ([201]Figure S2). The formed network
identified five genes as having connections to all types of Alzheimer’s
disorders; these genes are ACHE, APP, BACE1, MAPT, and TNF. The results
highlight the importance of these genes among the described data set to
possess a role in Alzheimer’s management by A. esculentus seeds.
3.3.4. Total Network Pharmacology
Linking the previously constructed networks (plant–compounds,
compounds–genes, and genes–Alzheimer’s disorders) in one network led to
summarize the plant–compound–gene–Alzheimer’s interactions; the formed
network consisted of 110 plant names, ten nodes for identified
compounds, 92 nodes for genes related directly or indirectly to
Alzheimer’s, and seven for Alzheimer’s, in addition to 444 edges
representing interactions between nodes ([202]Figure 4).
Figure 4.
[203]Figure 4
[204]Open in a new tab
Complete pharmacology network (plant–compounds–genes–Alzheimer’s
disorders); the network explains the relations between the plant,
identified compounds, and target genes, in relation to Alzheimer’s
disorders; the blue rectangle is the plant name, pink circles represent
the identified compounds, yellow oval shapes represent Alzheimer’s
disorders, and violet diamonds represent genes related to Alzheimer’s
disorders.
3.3.5. Protein–Protein Interactions (PPI)
In the formed PPI network using the STRING database, non-interacting
genes were not involved. The network was composed of 100 nodes and 145
edges with an average node degree of 2.9 ([205]Figure S3A). The genes
with the highest interactions among the PPI network were introduced as
a subnetwork ([206]Figure S3B); the PPI and the subnetwork identified
PIK3R1, HSP90AA1, SRC, EGFR to be the highest-interacting genes in this
gene set.
3.3.6. Gene Ontology and Pathway Enrichment Analysis
All targets were introduced to the STRING database and the obtained
results identified the target genes to affect biological processes,
especially the cellular response to jasmonic acid stimulus
(GO:0071395), the farnesol catabolic phase (GO:0016488), the
daunorubicin metabolic process (GO:0044597), and the doxorubicin
metabolic process (GO:0044598); the top-identified cellular components
were the insulin receptor complex (GO:0005899), the
phosphatidylinositol 3-kinase complex, class I (GO:0097651), the
external side of the apical plasma membrane (GO:0098591), and glial
cell projection (GO:0097386); top-identified molecular functions were
phenanthrene 9,10-monooxygenase activity (GO:0018636), indanol
dehydrogenase activity (GO:0047718), geranylgeranyl reductase activity
(GO:0045550), and 17-alpha,20-alpha-dihydroxypregn-4-en-3-one
dehydrogenase activity (GO:0047006); each class of terms were arranged
in descending order according to strength ([207]Table S9).
Using the KEGG Mapper pathway detection database implemented in the
KEGG database, a diagrammatic illustration identified 43 biological
pathways involved in Alzheimer’s disorders (hsa05010) as a
mutation-caused aberrant PSEN1 to PERK-ATF4 signaling cascade; a
mutation-caused aberrant Aβ to AGE-RAGE signaling pathway was among the
top pathways identified ([208]Figure S4).
Consequently, several genes related to the identified compounds were
involved in all Alzheimer’s disorders, as a result of the current
network pharmacology study. These genes were (ACHE, APP, BACE1, MAPT,
and TNF), so these genes may be responsible for the anti-Alzheimer’s
potential of A. esculentus seeds; previous studies concerning
Alzheimer’s disorders therapy were acetylcholinesterase (AChE)
inhibitors. These drugs reduce symptoms induced by the death of
cholinergic neurons by inhibiting acetylcholine (ACh) turnover
[[209]56]. The brain produces abundant quantities of APP, a single-pass
transmembrane protein. It is rapidly and intricately metabolized by a
series of proteases related to Alzheimer’s disorder [[210]57]. The all
gene set were able to affect Alzheimer’s disorder through the
mutation-caused aberrant PSEN1 to the PERK-ATF4 signaling pathway in
addition to the mutation-caused aberrant Aβ to the AGE-RAGE signaling
pathway, The PERK-Dependent Molecular Mechanisms was proven to have a
role in the treatment of neurodegenerative disorders [[211]58]. The
gene set possesses effects on cellular response to jasmonic acid
stimulus and farnesol catabolic process (biological processes), insulin
receptor complex and phosphatidylinositol 3-kinase complex, class I
(cellular components), phenanthrene 9,10-monooxygenase activity, and
indanol dehydrogenase activity (molecular functions).
4. Materials and Methods
4.1. A. esculentus Material, Chemicals, Reagents, Extraction of A. esculentus
Seeds
A. esculentus seeds were purchased from the market in Minia area,
Egypt. The plant was identified by Prof. Abd El-Halim A. Mohammed
(Horticultural Research Institute, Department of Flora and
Phytotaxonomy Research, Dokki, Cairo, Egypt). Additionally, A voucher
specimen (2022-BuPD 113) has been stored at the Department of
Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Egypt. The
solvents used during our work were purchased from El-Nasr Company for
Pharmaceuticals and Chemicals (Giza, Egypt). Regarding the biological
investigation, donepezil, all reagents and kits were obtained from
Sigma Chemical Company (Saint Louis, MO, USA), while aluminum chloride
(AlCl[3]) was obtained from CDH (Delhi, India).
Air-dried seeds (1.0 kg) were macerated in ethanol (70%, 1.5 L, 3×, 7 d
each) kept at room temperature. The extract was additionally
concentrated using reduced pressure to give a syrupy consistency
utilizing a rotary evaporator (Buchi Rotavapor R-300, Cole-Parmer,
Vernon Hills, IL, USA), kept at 45 °C to afford 20.0 g crude extract.
Moreover, this was maintained at 4 °C for biological and metabolomic
studies
[[212]38,[213]59,[214]60,[215]61,[216]62,[217]63,[218]64,[219]65,[220]6
6,[221]67,[222]68,[223]69,[224]70,[225]71].
4.2. In Vitro DPPH Free Radical Scavenging Activity Assay of A. esculentus
Seed Extract
The free radical scavenging potential of A. esculentus seed total
extract was investigated using the DPPH assay (stable radical
2,2-diphenyl-1-picrylhydrazyl) [[226]32]. In a brief manner at several
concentrations (0.01, 0.05, and 0.1 μg mL^−1 in absolute ethanol), 1 mL
solution from our tested extract was added to 2 mL of DPPH solution
(freshly prepared, 20 μg mL^−1 in absolute ethanol). Moreover, the
mixture was then kept at room temperature in a dark place for 30 min.
Using a UV-Vis Jenway 6003 spectrophotometer, the absorbance was
measured at λ[517] (nm). Ascorbic acid in addition to absolute ethanol
were utilized as a positive control as well as a blank, respectively.
However, the following equation was used to calculate the DPPH radical
scavenging activity:
[MATH:
% DPPH scaven
ger act<
mi>ivity=absorba
nce of <
mi>blank−absorbance of t
ested sa
mpleabsorban
ce of bl
ank×100 :MATH]
4.3. In Vitro Determination of Cholinesterase Potential of A. esculentus Seed
Extract
The cholinesterase potential of A. esculentus seed total extract was
assessed according to the manufacturer’s guidelines [[227]72].
Accordingly, various concentrations of the tested extract (10 and 20 μg
mL^−1) in absolute ethanol were prepared. After that, 0.2 mL of these
solutions were mixed with 3 mL distilled water followed by the addition
of phosphate solution (3 mL), and the pH was determined with a pH meter
(“pH-1”). Then, acetylcholine iodide solution (0.12 mL, 7.5%) was added
and the mixture was kept at 37 °C in a water bath (for 30 min). Then,
the pH value of these mixtures was recorded again (“pH-2”) and the
difference between “pH-1” and “pH-2” within 30 min was calculated as a
measure for the level of cholinesterase activity in the mixtures.
4.4. In Vivo Anti-Alzheimer’s Potential
4.4.1. Animals
The animals used during this study were male rats (Wistar albino) (150
± 10 g), obtained from the National Research Centre Animal House. The
animals were divided into groups of ten rats for each cage, kept under
environmental conditions well controlled at 26–29 °C. Additionally,
they were supplied with a fixed light/dark cycle (1 week) as an
interval to adapt under normal conditions, as they were permitted to
have their water and food freely.
4.4.2. Animal Ethical Report
Our study was authorized by the Ethical Committee of Beni-Suef
University, Egypt, which demonstrated that the rats will not feel pain
at any period of the experiments and be conserved in conformity with
the instructions for the care and usage of laboratory animals (ethical
approval no: 022-371).
4.4.3. Induction of AlCl[3]-Induced Alzheimer’s Illness
AlCl[3] solutions were prepared freshly at the onset of each
experiment. Drinking water was used to dissolve AlCl[3] to be
administrated orally at a dose of 100 mg/kg to the animals every day
for 8 weeks, 0.5 mL/100 g b.wt. [[228]73].
4.4.4. Acute Toxicity Investigation
The total extract was prepared at different concentrations for
determining the acute toxicity, (from 500, 1000, 2000, 3000, and 4000,
to 5000 mg/kg b.wt.), administered to four rats per each group (i.e., a
total of 24 animals for all groups).
4.4.5. Behavioral Measurement
Evaluation of the cognitive functions, as well as s motor coordination
T-maze, were conducted at the National Research Centre (N.R.C.). After
the chronic administration of AlCl[3] (2 months), and at the end of
treatment phase, the cognitive function and impairment of spatial
memory of the animals were estimated [[229]74]. Motor ability was
evaluated using the beam balance investigation [[230]75].
4.4.6. Experimental Design, and Blood Samples Preparation
Rats used during this study were grouped randomly as six groups of ten
animals each. Groups 1 and 2 served as normal, healthy control rats and
AD rats, respectively, where AlCl[3] was orally administrated to Group
2, while Groups 3 and 4 were the AD rats treated with A. esculentus
seed’s total extract (500 mg/kg b.wt.) every day for 6 weeks (1/10
LD[50]) and the AD rats treated with the standard drug donepezil (10
mg/kg b.wt.) daily for 6 weeks [[231]76].
Overnight fasted animals were sacrificed under slight thiopental
anesthesia (30 mg/kg b.wt.) [[232]77]. In a clean and dry test, the
blood samples were gathered after anaesthetizing the rats utilizing a
cardiac puncture. Blood samples were kept for 10 min until clotting and
then centrifuged at 3000 rpm (1.175 g) to get serum. Additionally, the
collected serum was kept at −80 °C for performing the biochemical
investigations of IL-6, AChE, and TAC.
4.4.7. Preparation of the Brain Tissue Samples
Upon completion of the experiment, the animals were fasted overnight
and they were anesthetized and sacrificed [[233]77]. Dissection of the
entire brain of each animal was rapidly carried out, then the material
was washed using isotonic saline and placed upon filter paper to
dryness. Additionally, the collected brain from each rat was weighed as
well as homogenized in ice-cold medium which contained 50 mM tris/HCl
in addition to 300 mM sucrose at pH 7.4, giving a 10% (w/v) homogenate
[[234]73,[235]78]. Centrifugation of the homogenate was carried out
(1400× g for 10 min at 4 °C). Moreover, the supernatant was kept at −80
°C and screened against different biomarkers such as an antioxidant,
oxidative stress, neurotransmitters, AGEs, and BDNF.
4.4.8. Estimation of Brain Neurotransmitters
Serum acetylcholine esterase (AChE) was determined using a quantitative
ELISA consistent with Engvall and Perlman, 1971 [[236]79]. The
concentrations of norepinephrine, dopamine, and serotonin in the brain
were evaluated utilizing HPLC-ED consistent with Giday et al., 2009
[[237]80]. IL-6, advance glycated end products, and BDNF concentrations
were measured utilizing ELISA kits consistent with the manufacturer’s
directions [[238]80]. Moreover, TAC was evaluated in the serum
according to Koracevic et al., 2009 [[239]81]. In this method,
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was kept
with met myoglobin as well as H[2]O[2] to give the green radical cation
ABTS+. Additionally, antioxidants make suppression for the color
production by reducing the color intensity, which was relative to the
concentration of the antioxidants and was evaluated at 600 nm using a
microplate reading format. The animal groups were exposed to an
evaluation for the non-enzymatic, reduced glutathione (GSH) (Beutler et
al., 1963 [[240]82]) and malondialdehyde (MDA) (Ohkaw et al., 1979
[[241]83]). Additionally, the spectrophotometric/microplate reader
analysis technique for glutathione (GSH) includes oxidation of GSH
using the sulfhydryl reagent DTNB to obtain the yellow derivative TNB,
which is measurable at 412 nm. Additionally, the principle of the MDA
assay is built upon the measurement of the required absorbance at 535
nm as the spectrophotometrical measurement of the color which MDA forms
with TBA in acidic media.
4.4.9. Histopathological Investigations
The samples obtained from all rats’ brains per group were preserved in
10% neutral buffered formalin. Additionally, paraffin segments of 5 μm
thickness were formulated and stained with hematoxylin in addition to
eosin (H&E) for histopathological study, utilizing a light microscope
(BX43, Olympus) and photographed using the Olympus software in
connection with an Olympus DP27 camera [[242]84]. An experienced
pathologist carried out the histological analysis. Neuropathologic
damage was evaluated on a scale from 0 to 4 as follows: (0) showed no
changes; (1) revealed an area affected of <20%); (2) displayed an area
affected of 20–30%); (3) showed an affected area of >30–60%); and (4)
indicated an affected area of >60% [[243]85].
4.5. Metabolomic Investigation Procedure
The crude extract from A. esculentus seeds was prepared at a
concentration of 1 mg/mL for mass spectrometry investigation. The
obtained ethanolic extract was investigated using metabolic study using
LC-HR-ESI-MS consistent with Abdelmohsen et al., 2014 [[244]86]. The
acquired data from the investigated ethanolic extract were dereplicated
using the DNP database [[245]87,[246]88].
4.6. Network Pharmacology Study
4.6.1. Identified Compounds-Genes
The genes of each identified compound from A. esculentus seed extract
were extracted from the chemistry database PubCHem
([247]https://pubchem.ncbi.nlm.nih.gov/) [[248]89] last accessed on (1
November 2022), and the Online SwissTARgetPrediction
([249]http://www.swisstargetprediction.ch/) was also used [[250]90]
last accessed on (6 November 2022).
4.6.2. Genes-Alzheimer’s Disorders
The genes linked to Alzheimer’s illness were collected from DisGeNet
[[251]91] last accessed on (10 November 2022). Additionally, the target
genes UniProt IDs were chosen as input IDs in the DisGeNET database to
find out which gene linked to Alzheimer’s disorders. In order to
minimize the scope for the gene set to Alzheimer’s illness, the filter
keywords of “Alzheimer’s disease”, we utilized “Alzheimer Disease,
Early Onset”, “Alzheimer´s disease, focal onset”, “Alzheimer disease,
late onset”, “Alzheimer disease, familial, type 3′”, “Familial
Alzheimer’s Disease (FAD)”, and “Dementia due to Alzheimer’s disease
(disorder)”.
4.6.3. The Protein-Protein Interaction (PPI)
To investigate and display each potential interaction between the
detected genes, the STRING database was utilized.
([252]https://stringdb.org/cgi/network?taskId=bIDN4htc9NBY&sessionId=bZ
WvNlZHMn9h) [[253]92] last accessed on (15 November 2022). The selected
proteins were selected with the human species “Homo sapiens” as well as
a confidence count higher than 0.4. STRING was utilized to discover
proteins that interrelated with A. esculentus dereplicated metabolites
specifying targets directly or indirectly to Alzheimer’s disorders’
subnetworks as constructed using the Cytoscape plugin CytoNCA
application [[254]91].
4.6.4. Network Construction and Visualization
Plant–compounds, compounds–genes, PPI, and plant–compounds–genes–
Alzheimer’s disorders networks were built using the Cytoscape network
analysis program, version 3.9.0 [[255]92]. The difference was
significant at p < 0.05. Nodes stand for compounds, genes, and
Alzheimer’s disease in the graphical network; additionally, edges stand
for its related interactions.
4.6.5. Gene Enrichment Investigation
The characterized pathways connected to A. esculentus related to
Alzheimer’s disorders were retrieved by the STRING database
([256]https://string-db.org/cgi/network?taskId=bIDN4htc9NBY&sessionId=b
ZWvNlZHMn9h) [[257]92] last accessed on (15 November 2022) and the KEGG
Mapper database ([258]https://www.genome.jp/kegg/mapper/) last accessed
on (20 November 2022) to consider the biological function, cellular
elements, molecular functions, and incorporated biological pathways.
4.7. Statistical Investigation
All the obtained data were represented as mean ± SD. Additionally, the
data were screened for normal distribution statistically utilizing
one-way analysis of variance software as well as Co-State for Windows,
version 8. The values of various letters are significant at p < 0.05
statistically.
[MATH:
% change=mean of negative control
−mean o<
mi>f treatment groupmea<
mi>n of negative control×100 :MATH]
[MATH:
% improvement
=mean of positive co
ntrol−m<
mi>ean of treatment grou
pmean of negative co
ntrol×
100 :MATH]
5. Conclusions
In this study, A. esculentus seed crude extract showed significant
neuroprotective, anti-apoptotic, and anti-amnesic potential against
AlCl[3]-induced cerebral damages and cognitive dysfunctions, which
could be associated with the antioxidant and the anti-AChE characters
of the extract. Ten secondary metabolites were dereplicated using
LC–HRESIMS. Network pharmacology analysis resulted in establishing a
hypothesis indicating that the identified compounds from A. esculentus
possess anti-Alzheimer’s effects through specific genes (ACHE, APP,
BACE1, MAPT, and TNF). This study proposes the application of A.
esculentus seed crude extract as a treasure remedy in AD treatment but
more investigations, such as mechanistic investigations and
quantification for the secondary metabolites in the extract, are
necessary to validate the results.
Acknowledgments