Impact Of KPD and NRG Flavonoids On Neurotoxicity Prompted By Aluminum Chloride Through Hindrance Of Oxidative Pressure : In Vivo Model

Abstract

Background
The incidence of neurodegenerative diseases has skyrocketed alongside the extension of the average human lifespan. Oxidative stress is a hallmark of this group of diseases, its involvement in disease onset and/or progression is incompletely understood. Polyphenols are among the most promising natural compounds for their neuroprotective capabilities due to their distinctive chemical characteristics. So, it's exciting to look at the possibility of using new compounds with polyphenol qualities as neuroprotective. The goal of this study was to test the neuroprotective effect of the polyphenolic flavanol molecules Kaempferide and Norbergenin, in-vivo disease models generated by Aluminium chloride (AlCl3).

Results
Therefore, this study was evaluated to estimate the alleviative effect of KPD and NRG against AlCl3 Induced neurotoxicity in rats. KPD (10 mgkg-1) and NRG (10 mgkg-1) supplemented group decreased the production of Amyloid and Tau Proteins. The anti-oxidants i.e., catalase (CAT), glutathione reductase (GSR), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were reduced, besides malondialdehyde (MDA) & reactive oxygen species (ROS) contents were substantially (P < 0.05) elevated. Western blot analysis was performed AlCl3 exposure also prompted remarkable histopathological alterations in Brain tissues.

Conclusion
KPD and NRG therapy, protect all the brain tissues from the damages induced byAlCl3. The findings of our study demonstrated that KPD can efficiently exerts protective effect due to its anti-oxidant, & neuroprotective effect.

Full text

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Impact Of KPD and NRG Flavonoids On
Neurotoxicity Prompted By Aluminum Chloride
Through Hindrance Of Oxidative Pressure : In Vivo
Model
Swathi Nalla (  nalla.swathi90@gmail.com )
GITAM University
Suhasin Ganta
GITAM University
Sarad Pawar Naik Bukke
Kampala International University
Nagaraju Bandaru
sandip university
Research Article
Keywords: Neurotoxicity, Aluminium Chloride, Polyphenol, Oxidative Stress, Amyloid
Posted Date: February 15th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-3945529/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License
Additional Declarations: No competing interests reported.

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Abstract
Background:
The incidence of neurodegenerative diseases has skyrocketed alongside the extension of the average
human lifespan. Oxidative stress is a hallmark of this group of diseases, its involvement in disease onset
and/or progression is incompletely understood. Polyphenols are among the most promising natural
compounds for their neuroprotective capabilities due to their distinctive chemical characteristics. So, it's
exciting to look at the possibility of using new compounds with polyphenol qualities as neuroprotective.
The goal of this study was to test the neuroprotective effect of the polyphenolic avanol molecules
Kaempferide and Norbergenin,
in-vivo
disease models generated by Aluminium chloride (AlCl3).
Results:
Therefore, this study was evaluated to estimate the alleviative effect of KPD and NRG against AlCl3
Induced neurotoxicity in rats. KPD (10 mgkg-1) and NRG (10 mgkg-1) supplemented group decreased the
production of Amyloid and Tau Proteins. The anti-oxidants i.e., catalase (CAT), glutathione reductase
(GSR), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were reduced, besides
malondialdehyde (MDA) & reactive oxygen species (ROS) contents were substantially (P < 0.05) elevated.
Western blot analysis was performed AlCl3 exposure also prompted remarkable histopathological
alterations in Brain tissues.
Conclusion:
KPD and NRG therapy, protect all the brain tissues from the damages induced byAlCl3. The ndings of
our study demonstrated that KPD can eciently exerts protective effect due to its anti-oxidant, &
neuroprotective effect.
1. Background
Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Huntington's
disease (HD) are all manifestations of NDDs that are dened by the inevitable degradation of particular
neuronal clusters. Neurodegenerative diseases (NDDs) are a diverse, devastating, and, incurable category
of degenerative conditions. They are distinguished by a prolonged and cumulative loss of dopaminergic
neurons, which in turn results in a steady and growing impairment of particular activities of the CNS. This
loss of neuronal cells is the dening feature of these conditions [1,2].Once neurodegeneration has begun,
it can only be slowed down; it cannot be completely stopped once it has started. This class of disorders
presents a severe therapeutic challenge [3]. The age range of typical patients affected by NDDs is
between 50 and 70 years old [4].

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1.1 Global Economic and Social Impact of NDDs
Unfortunately, the only treatment option for many NDDs is palliative care, despite the fact that they have
catastrophic consequences for the patient. The combination of high costs for formal care, an ever-
increasing number of individuals who need it and a disproportionate dependence on informal care is a
signicant challenge for society at large. New insights into the role of genetic and epigenetic factors,
pharmaceuticals, aging, and infectious agents in the development of neurodegenerative illnesses have
emerged as a result of advancements in biomedical research and informatics. The rising expense of
healthcare is directly correlated to the rising prevalence of NDDs, which in turn is caused by the rapidly
aging population. In Europe, for instance, persons over 65 years of age currently make up around 16% of
the population, but that number is predicted to climb to 25% by the year 2030.Because of the exponential
rise in the average age of the world's population, Alzheimer’s disease has become a serious public health
concern.
Aluminum chloride exposure induces neurotoxicity, mainly due to the increased permeability ofAlCl3into
the BBB [5]. Aluminum chloridecauses damage mainly to the cortex and hippocampus regions of the
brain. In turn, it causes dementia and cognitive impairment by the plaque formation of Aβ protein
[6].Neurotoxicity of AlCl3mainly leads to toxic properties like proapoptosis, proinammatory process and
it also affects the release and metabolism of neurotransmitters in the brain region [7]. Exposure of AlCl3
leads to the formation of neurobrillary tangles [8].
2. Methods
2.1 Animals and its Treatment:
In all, Fourty-two (42)male adult Wistar rats weighing between 120 and 180 g were employed in the
studies. Standard housing parameters (Room temperature 24-27 °C and 60% humidity with 12-h light and
dark cycles) were provided for rats obtained from the animal house at the CSIR (Council of Scientic and
Industrial research), Hyderabad, Telangana, India, and kept at the animal house. Laboratory pellets (20%
protein, 5% fat, and 1% multivitamins) were provided to the animals, along with free access to water. The
(PGP Life Sciences Approval No. PGP/RM-0019/57-21) all animal treatments were carried out in
compliance with the CPCSEA guidelines. After a week of acclimation, the animals were randomly split
into seven groups of 6 animals for the purposes of the study. There was a total of seven experimental
groups, each including six animals (n = 6).
The AlCl3 prophylactic Study group is categorized as follows
1. Group 1 (Control group): rats received normal saline every day throughout the study.
2. Group 2 (Disease model group or DM group): Administered with Aluminium chloride (AlCl3) at 200
mg/kg body wt, single dose per day given orally for 45 days.

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3. Group 3 (Positive control group): Administered Donepezil (3 mg/kg body wt.) orally once daily 30 min
before the administration of Aluminium chloride (AlCl3).
4. Group 4 (Test group 1_KPD): Kaempferide (5 mg/kg body wt.) was given via oral route for a week prior
to Aluminium chloride(AlCl3) being administered for 45 days thereafter.
5.Group 5 (Test group 2_KPD): Kaempferide (10 mg/kg body wt.) was given via oral route for a week
prior to Aluminium chloride(AlCl3) being administered for 45 days thereafter.
6.Group 6 (Test group 1_NRG): Norbergenin (5 mg/kg body wt.) was given via oral route for a week prior
to Aluminium chloride(AlCl3) being administered for 45 days thereafter.
7.Group 7 (Test group 2_NRG): Norbergenin (10 mg/kg body wt.) was given via oral route for a week prior
to Aluminium chloride(AlCl3) being administered for 45 days thereafter.
2.2Behavioral Examination:

In the case of a prophylactic study, all behavioural examinations were
performed from the 46thday. All behavioral assessments were conducted daily, at the same time of day,
for a full 7 days, from 9:00 am to 6:00 pm.
2.2.1 Morris Water Maze (MWM) test: As was mentioned earlier, MWM was used to test the cognitive
abilities of rats [9]. On day 7, we tested the animal with a single spatial probe trial to see if it would use a
spatial learning technique to nd the platform. After the 60 seconds were up, the platform was taken out
of the water tank and the mice were released to swim around the tank at will from the south, west, and
north starting places. For each subject, we recorded how long they stayed in the east target quadrant, how
far they travelled there, how many times they crossed the platform, and when they rst crossed it.
2.2.2 Rota-Rod test: When people get older, they lose some of their ability to govern their bodies. One
common way to measure rats' ability to coordinate their movements while running is with the Rota-Rod
test. A 5-station accelerating Rota-Rod was used to assess locomotor coordination in this experiment.
The rats were given two minutes of practice on the Rota-Rod at a steady speed of 24 rpm for the training
session. After a 20-minute rest, the rats underwent the identical training procedure once more. Each rat
participated in a total of 10 trials during the testing period; 5 trials were conducted daily, with 30-minute
breaks in between each trial. Each test lasted 10 minutes, during which time the speed enhanced from 4
to 40 rpm in 5 min. The infrared beam was broken during the fall, and the data was captured immediately
by the Rota-Rod system (Med Associates, SOF-ENV-575).
2.2.3 Mechanical hyperalgesia (Randall–Selitto paw pressure test): The mechanical nociceptive
threshold was measured, which is an indicator of mechano hyperalgesia. The Randall-Selitto paw
pressure instrument was used to measure the nociceptive exion reex. The nociceptive threshold was
measured by having the subject withdraw their rear paw.

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2.3 Brain Tissue Sampling and Preparation:

Thiopental was used to provide a little sedation in the rats,
and they were killed via cervical dislocation. Each rat's brain was quickly frozen in ice and dissected from
the olfactory bulb to the cerebellum. After being rinsed in isotonic saline, it was left to dry on lter paper.
Each brain was cut in half along its sagittal axis. The rst component (the right hemisphere) was frozen
at -80 oC for later biochemical study. The remaining section (the left hemisphere) was xed in 10%
formalin for immunostaining of tissues.
2.4 Biochemical parameters:

The rst step in preparing a 10% brain homogenate was homogenising the
rst component of brain with PBS in a quantity 10 times the weight of the tissue, centrifuging the
homogenate at 10000 x g for 15 min at 4 °C, and collecting the supernatant. By using the Bradford assay,
we calculated the approximate protein concentrations in the samples. Using commercially available kits
and following the directions for the appropriate methods described, we measured the levels of BDNF and
Bcl-2 by ELISA and the levels of MDA and NO by colorimetry assays.
2.4.1 Total protein content: A blue-colored potassium-biuret complex is formed when the carbonyl group
of protein complexes combines with potassium and copper reagents. The tyrosine and phenolic
chemicals in the protein work along with this combination to decrease the phospho-molybdate of the
folin reagent, which in turn increases the colour of the solution (Lowry method).
To precipitate the protein, 100 mg (wet weight) of brain tissue homogenate was mixed in 10%
Trichloroacetic acid (TCA). The sample was centrifuged at 3000 rpm for 5 min. The upper layer was
removed. In 1N NaOH, the precipitate was dissolved again. This was then mixed well with the addition of
5 ml of reagent C and allowed to sit uninterrupted for 10 minutes. This was then stirred with 0.5 ml of
Folin phenol reagent and set aside for 30 minutes. 1 % BSA and 1N NaOH were used as standard and
blank respectively. The generated blue colour was measured with a UV-visible double beam bio
spectrophotometer (Elico), an autoanalyzer) at 650 nm, which automatically quanties the protein
content in the sample.
2.4.2 Antioxidant Status of Rat Brains:

Inhibition of nitroblue tetrazolium dye degradation by phenazine
methosulphate can be attributed to the presence of superoxide dismutase (SOD) in the brain of therat.
Because of this property, the enzyme's function may be measured. Meanwhile, the concentration of
catalase (CAT) was calculated by monitoring the amount of hydrogen peroxide (H2O2) produced and
consumed after the enzyme reaction for 120 seconds at 20 second intervals, at 340nm. The capacity of
glutathione reductase (GRd) to catalyse the reduction of glutathione in the presence of NADPH was used
as a surrogate for GRd activity. Together with this decrease in absorbance, was calculated at a
wavelength of 340 nm. Using the enzyme's capacity to oxidise GSH to GSSG (oxidized glutathione), we
were able to infer GPx activity in the brain. In the presence of NADPH, GRd recycled the GSSG to its
reduced form, with its removal being detected at 340 nm.
2.4.3 Acetylcholinesterase Assay: Brain homogenates from both untreated and treated rats, were used to
quantify AChE activity [11], which has been proved to be accurate in the past. Right hemisphere cortical

Page 6/23
and hippocampal regions were used to prepare the brain homogenate (12).
2.4.4 Histological investigation:

Brains of the sacricial rats were utilised for histopathological analysis
after being xed in 10% buffered-saline formalin. Brain tissue from autopsy subjects was preserved for
24 hours before examination, in 10% formol saline. After being washed with tap water, the samples were
dehydrated using a series of dilutions of methyl, ethyl, and absoluteethyl alcohol. To preserve specimens,
they were rst washed with xylene and then immersed in paran at 56oC in a hot air oven for 24 h.
Tissue blocks of paran bees wax were then sectioned using a slide microtome at a thickness of 4
microns. Tissue samples were taken on glass slides, deparanized, and staining was done by cango red
stains for examination of histology and detection of amyloidal protein plaques in brain regions using
light electric microscope.
2.4.5 Western blot analysis:

The Lowry technique was used to isolate proteins from brain homogenate
and determine their concentration. Proteins were analysed by Western blotting, that includes amyloid β1-
42, phosphorylated Tau, Bax, Caspase-3, Bcl2, catalase, superoxide dismutase-1 and 2, and β -actin, a
ubiquitous housekeeping protein, are used in conjunction with a standard internal control, following
Singh et al.[10]. The following primary antibodies were used; anti-Aβ1-42 and anti p-Tau, mouse
monoclonal caspase-3 p17, (1:1000; Santa Cruz Biotechnology), anti-Bax (1:400), anti-Bcl-2 (1:400), anti-
actin (1:50,000). Goat anti-rabbit secondary antibody, and horseradish peroxidase-conjugated anti-mouse
antibody (1:20,000) were utilised as appropriate secondary antibodies. The membranes were then rinsed
in TBST, 3 times for 15 minutes each time. Western blotting kit (Thermo Scientic) was utilised for protein
band visualisation by chemiluminescence. GelQuant.NET, a programme made available by
biochemlabsolutions.com, was then used to analyse the protein band density. Before performing
statistical analysis, the calculated density was normalised with respect to the density of the
housekeeping total actin bands.
3. Results
3.1Behavioural Parameters:
3.1.1 Escaping latency in secs:
It was observed that the time of escape latencies of all experimental groups of AlCl3 neurodegenerative
rats decreased from day 1 to day 6 (Table 1). The normal control group (G1) showed the lowest escape
latency time of 27 ± 2.1. Among all experimental groups, the disease control groups (G2) had the longest
escape latency with 52 ± 4.6 seconds. With KPD at 10 mg/kg bw (G5) and NRG at 10 mg/kg bw (G7), the
escaping latency time decreased with a similar time scale of 34 to 36 seconds, indicating the similar
effect on memory and learning capabilities on diseased rats of prophylactic group of AlCl3
neurodegenerative model. It is noteworthy that the time of escaping latency was lower for both KPD at 10
mg/kg bw and NRG at 10 mg/ kg bw (i.e G5 and G7) than the PC group, i.e., donepezil treated (G3) with
an escaping latency time of 41 ± 4.5 seconds observed in case of AlCl3prophylactic group. However, the

Page 7/23
groups 4 and 6, i.e., KPD at 5 mg/kg bw and NRG at 5 mg/kg bw, showed intermediate escape latency
times between 41 and 45 seconds indicate similar neurological effect in AlCl3 prophylactic group (Table 1
and 2). Importantly G3 group (PC group) showed signicant decrease of escaping latency of 35 ± 3.7 sec
which was less than the effect observed for KPD and NRG at 10 mg/kg bw (G5) i.e 29 ± 2.2 and 32 ± 2.9
seconds respectively.
Table:1 Escaping time of latency and Path length observed in AlCl3 model
1. Escaping latency in Sec
Training
Days Group 1
(Control) Group 2
(AC
group)
Group 3
(AC +
PC)
Group
4
(AC +
Kpd
5)
Group 5
(AC + Kpd
10
Group 6
(AC + Nrg
5)
Group 7 (AC
+ Nrg 10)
Day 1 47 ± 2.1 72 ± 5.3 66 ± 4.5 69 ±
3.2 63 ± 5.1 68 ± 4.9 62 ± 3.1
Day 2 42 ± 2.5 67 ± 4.7 62 ± 4.1 65 ±
4.2 60 ± 4.8 64 ± 5.3 57 ± 3.7
Day 3 37 ± 2.2 63 ± 4.2 55 ± 3.9 59 ±
3.5 52 ± 3.5 60 ± 4.7 51 ± 4.8
Day 4 31 ± 1.8 56 ± 3.3 50 ± 4.5 54 ±
4.7 45 ± 6.6 53 ± 4.2 46 ± 4.1
Day 5 28 ± 1.6 53 ±
4.1## 43 ± 3.3 47 ±
3.8 38 ± 3.2* 45 ± 3.3 38 ± 2.7*
Day 6 27 ± 2.1 52 ± 4.6 41 ± 4.5 45 ±
4.3 34 ± 2.7* 41 ± 3.8 36 ± 3.1*
All Values are expressed as mean±SEM; n=6. One-way ANOVA with a signicance threshold of
**P<0.01,*P<0.05, n= not signicant, when compared with the control group, ##P<0.01, #P<0.05, when
compared with the disease control group, was used to analyze all data statistically.
Table:2 Probe trail assessment of Morris water maze test

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Groups No. of
Crossings Time spent in the target
quadrant in Sec Distance moved in the target
quadrant in Cm
Control 8.0 ± 0.4 27.4± 2.6 657.4± 23.6
AC
group 1.0 ± 0.2## 11.6± 4.1## 394.5± 18.4##
AC+PC 5.0 ± 0.2* 19.3± 1.8* 532.1± 16.9*
AC+Kpd-
54.0 ± 0.3ns 14.2± 1.9ns 441.7± 17.7ns
AC+Kpd-
10 6.0 ± 0.2** 24.1± 1.1** 612.5± 23.5**
AC+Nrg-
53.0 ± 0.1ns 12.5± 1.4ns 429.8± 19.1ns
AC+Nrg-
10 4.0 ± 0.2* 22.3± 1.7* 598.6± 18.1*
All Values are expressed as mean±SEM; n=6. One-way ANOVA with a signicance threshold of
**P<0.01,*P<0.05, ns= not signicant,when compared with the control group, ##P<0.01 when compared
with the disease control group, was used to analyze all data statistically.
3.1.2 Motor Co-ordination:
The results of motor restoring capabilities in AlCl3 neurodegeneration models demonstrated that the KPD
and NRG treated group observed for a signicant increase in latency time of fall (Avg. of 6 days) as well
as the rotational speed of the rotarod (Avg. of 6 days) for both KPD and NRG at their higher doses (Table
3). The control group (G1) of both prophylactic and curative groups of the AC model showed high latency
time of 386.1 ± 15.4 sec and 375.2 ± 23.6-sec fall and rotational speed of 25.0 ± 1.1and 24.0 ± 2.3 rpm
respectively. Diseased animals in both the prophylactic and curative groups of the AC model reported the
lowest latency times of 295.2 ± 18.3 and 291.9 ± 14.7 seconds, respectively, as well as rotational speeds
of 18.0 ± 1.3and 17.0 ± 2.7 rpm. In a curative group of the AC model, KPD and NRG gave the most
effective results in terms of restoring motor function with a latency time of 339.5 ± 14.7 and 322.6 ± 12.9
seconds, and rotational speeds of 23.0 ± 2.2 and 19.0 ± 3.2 rpm, respectively. While in the prophylactic
group, KPD and NRG at higher doses showed a considerable effect on motor regaining function with a
latency time of 328.6 ± 14.7 and 305.3 ± 12.8 sec and rotational speed of 21.0 ± 1.6 and 18.0 ± 1.4 rpm
indicating almost equivalent effect compared with the control group (Table 3).
Table 3 Motor function restoring capability in AC induced Neurodegeneration

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Prophylactic Group
Groups Latency of fall
(Avg. of 6 days)
Control 386.1 ± 15.4
AC group 151.4 ± 11.6##
AC+PC 295.2 ± 18.3*
AC+Kpd-5 229.4 ± 12.6*
AC+Kpd-10 328.6 ± 14.7**
AC+Nrg-5 211.7 ± 10.9*
AC+Nrg-10 305.3 ± 12.8**
All Values are expressed as mean±SEM; n=6. One-way ANOVA with a signicance threshold of
**P<0.01,*P<0.05, when compared with the control group, ##P<0.01 when compared with the disease
control group, was used to analyze all data statistically.
3.1.3 Mechanical hyperalgesia of KPD and NRG in AlCl3induced neurodegenerative rat models using
Randall selitto test:
The measurement of paw withdrawal threshold (g)inAlCl3- neurodegenerative modelswas analyzed by
the nociceptive exion reex. The results demonstrated that the prophylactic group of the AlCl3 model
observed a signicant increase in paw withdrawal threshold (g)(Avg. of 6 days) for both KPD and NRG at
their higher doses (Table 4). The control group (G1) of the AlCl3 model showed high paw withdrawal
threshold (g)of 81.7 ± 4.2. Diseased subjects of theAlCl3 model reported the lowest paw withdrawal
threshold (g)28.4 ± 1.9. KPD and NRG at higher doses showed a considerable effect on neuropathic pain
management with a withdrawal threshold of 62.6 ± 4.5 and 53.9 ± 4.2 grams indicating lower effect
(Table 4).
Table 4Management of mechanical hyperalgesia and neuropathic pain management in AlCl3 induced
neurodegenerative rat model.

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Groups Paw withdrawal threshold (g)
Prophylactic group
Control 81.7 ± 4.2
AC group 28.4 ± 1.9##
AC+PC 52.3 ± 3.1ns
AC+Kpd 5 41.8 ± 3.8ns
AC+Kpd 10 62.6 ± 4.5**
AC+Nrg 5 34.1 ± 2.5ns
AC+Nrg 10 53.9 ± 4.2*
All Values are expressed as mean±SEM; n=6. One-way ANOVA with a signicance threshold of
**P<0.01,*P<0.05, ns= not signicant,when compared with the control group, ##P<0.01 when compared
with the disease control group, was used to analyze all data statistically.
3.2.1 Investigating total protein content in AlCl3induced neurodegeneration:
Results:
Protein estimation inAlCl3 induced neurodegeneration model revealed that the control group (G1) had the
highest protein content of 14.33 ± 1.5 mg per kg bw (Table 5 and Figure1). The results showed that the
diseased group (G2) showed the highest reduction in proteins, with 62.25 ± 3.9 compared to the control
group (G1). In AlCl3induced neurodegeneration. In prophylactic groups, compounds KPD showed
considerable results of restoring protein content, with % reduction of protein falling to12.49 ± 2.3 and
NRG showed non-signicant protein restoration with22.96 ± 3.5 % (Table 5 and Figure1).
Table5Protein content estimation in AlCl3 induced neurodegeneration.

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Groups Total protein content in mg % Reduction of Protein content
Control 14.33 ± 1.5 -
AC group 4.41 ± 0.3## 62.25 ± 3.9
AC+PC 10.35 ± 0.6* 27.77 ± 2.1
AC+Kpd-5 9.66 ± 0.4* 32.59 ± 3.9
AC+Kpd-10 12.54 ± 1.4** 12.49 ± 2.3
AC+Nrg-5 8.25 ± 0.7ns 42.43 ± 2.8
AC+Nrg-10 11.04 ± 0.9* 22.96 ± 3.5

All Values are expressed as mean±SEM; n=6. One-way ANOVA with a signicance threshold of
**P<0.01,*P<0.05, ns= not signicant,when compared with the control group, ##P<0.05 when compared
with the disease control group, was used to analyze all data statistically.
3.2.2 Enhancement of anti-oxidant potential of target compounds over AlCl3 induced neurodegeneration
by the treatment of target compounds:
As part of understanding the effect of KPD and NRG in AlCl3model, the present study measured
superoxide dismutase (SOD)Level (nmol/mg Protein), Catalase activity (CAT) Level (U/mg Protein),
Glutathione peroxidase (Gpx) Level (µmol/mg Protein), Glutathione reductase (Grd) activity(µmol/mg
Protein)as functions of total anti-oxidant activity in AlCl3-induced neurodegeneration. As shown in
Table6and Figure2a, SOD level in AlCl3 induced neurodegeneration model were found to be highest i.e.,
982.4 ± 29.1 nmol/mg protein in the control group (G1). A comparison of SOD levels between the
diseased group (G2) and the control group (G1) revealed that the diseased group (G2) had the lowest
levels of SOD, with 574.8 ± 21.5 nmol/mg protein. In AlCl3 induced neurodegeneration, KPD and NRG at
higher doses markedly reduced the % SOD level to 02.04 ± 0.1 indicating that the KPD and NRG
substantially, increased the anti-oxidant abilities as shown in Table 6 and Figure 2.
The Glutathione peroxidase (Gpx) determined as a function of improving anti-oxidant properties of
cellular environment over the treatment of KPD and NRG at their higher doses ofAlCl3 model responded
greatly with % Gpx reduction of 2.72 ± 0.1 and 5.44 ± 0.4 % respectively. This effect of neurological health
was found to be superior when compared with the effect observed for donepezil. According to the AlCl3
model, the effect of KPD and NRG. As a result of this study, lower doses of KPD and NRG failed to
demonstrate a signicant reduction of % Gpx levels as shown in Table 6 and Figure 2 c to d). So, these
ndings also supported the potential neuroprotective capabilities of KPD and NRG by successfully
supressing the Gpx levels in D-Gal induced neurodegeneration.

Page 12/23
Table:6SOD activity in AlCl3-model
Prophylactic group 
Groups SOD Level
(nmol/mg Protein) CAT Level (U/mg
Protein) Gpx Level (µmol/mg
Protein) Grd Level (µmol/mg
Protein)
Control 973.2 ± 21.5 0.67 ± 0.02 1.53 ± 0.12 9.46 ± 0.4
AC
group 519.5 ± 25.6## 0.21 ± 0.03# 0.89 ± 0.05## 2.94 ± 0.3##
AC+PC 878.2 ± 24.8* 0.45 ± 0.04* 1.31 ± 0.13* 6.87 ± 0.2*
AC+Kpd-
5684.5 ± 21.3* 0.35 ± 0.03ns 1.12 ± 0.04ns 4.21 ± 0.4*
AC+Kpd-
10 911.9 ± 27.8** 0.59 ± 0.05*** 1.42 ± 0.05** 8.57 ± 0.3***
AC+Nrg-
5635.2 ± 26.4* 0.31 ± 0.02ns 1.06 ± 0.03ns 4.06 ± 0.6*
AC+Nrg-
10 821.8 ± 24.7** 0.52 ± 0.03** 1.28 ± 0.07** 7.12 ± 0.4**
All Values are expressed as mean±SEM; n=6. One-way ANOVA with a signicance threshold of **P<0.01,
*P<0.05, when compared with control group, ##P<0.01 when compared with the disease control group,
was used to analyze all data statistically.
3.2.3 Effect of KPD and NRG on brain AChE activity over AlCl3-induced neurodegeneration:
As part of the present study, the effect of KPD and NRG were studied in order to understand the restoring
the functional ability of Ach in AlCl3-induced neurodegeneration, the acetylcholine esterase activity was
assessed. As shown in Table 7  and Figure 3, AChE levels in prophylactic groups of AC induced
neurodegeneration model were found to be lowest i.e. 1.43 ± 0.2U/g protein in the control group (G1). A
comparison of AChE levels between the diseased group (G2) and the control group (G1) revealed that the
diseased group (G2) had the highest levels of AChE, with 8.31 ± 0.5 U/g protein in prophylactic groups
respectively. Compounds KPD and NRG showed almost equivalent effects of restoring the acetylcholine
activity by inhibiting AChE in the prophylactic group of the AlCl3 model, with AChE suppression to 2.31 ±
0.5and 2.83 ± 0.2 U/g protein, respectively, which indicates signicant effect as in the case of curative
group. as shown in Table 7  and Figure 3. Statistical signicance threshold of p<0.05 was considered.
Table: 7Determination of AChE activity in the AlCl3model

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Groups Ach.E level in U/g Protein
Prophylactic group
Control 1.43 ± 0.2
AC group 8.31 ± 0.5##
AC+PC 3.96 ± 0.7*
AC+Kpd-5 5.12 ± 0.6*
AC+Kpd-10 2.31 ± 0.5**
AC+Nrg-5 5.57 ± 0.4*
AC+Nrg-10 2.83 ± 0.2**
All Values are expressed as mean±SEM; n=6. One-way ANOVA with a signicance threshold of
***P<0.001,**P<0.01,*P<0.05, ns= not signicant,when compared with conrol group, ##P<0.01 when
compared with disease control group, was used to analyze all data statistically.
3.3 Histopathology Examination:
Detection of Aβ plaques using Congo red staining in AlCl3 model:
In Prophylactic group AlCl3 induced neurodegenerative models were analysed for Aβ deposition by
staining whole brain tissue homogenates with Congo red dye. When the structural changes seen in the
prophylactic groups, they were very similar in terms of structural compactness in the developmental
behaviour of Aβ plaques. Compared with brain sections from untreated rats (Figure 4 ), AlCl3-treated rat
develop signicant multifocal Aβ deposits in the form of red plaques in the brain. In comparison to
AlCl3treated animals, tissue sections from animals treated with AlCl3/Donepezil showed a modest to
good tissue regeneration effect with mild dense plaques. As was observed in the donepezil group,
treatment with NRG at 10 mg/kg bw resulted in moderate reduction of amyloid beta plaque development ,
Compared to the AlCl3-treated group, the KPD-treated group showed virtually no amyloid beta plaque
development indicating that KPD had a more signicant impact than the donepezil effect demonstrated
in G3 group. According to this study, KPD can reduce the neurotoxicity caused by amyloid beta plaques.
3.4 Western blot analysis:
Protein expressional analysis in AlCl3 induced neurotoxicity:
Western blot depicts protein of interest with functional ability in addition to determining the level of
protein expression associated with neurodegeneration induced by the various agents. In this study, the
curative group of AlCl3 model was selected to investigate the molecular mechanisms associated with
neurodegeneration during this study.

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Results:
Control group animals exhibited lower levels of Aβ expression, which indicated a healthy
microenvironment without Aβ plaques. In contrast, the highest levels of Aβ expression were found in the
AlCl3treated group. AlCl3/donepezil treated group showed a mild reduction in plaque formation,
indicating mild effects. At higher doses, KPD moderately reduced the expression of Aβ with better results
than the positive control group. The NRG demonstrated superior ecacy in AlCl3-induced
neurodegenerative models when administered at higher doses. A similar effect was observed with the
phosphorylation of Tau protein. There was a higher level of phosphorylation in the D-gal diseased model,
and the expression level was suppressed by KPD and NRG, with a greater effect observed with KPD
(Figure 5). In both KPD and NRG treatments, the kinase protein Akt showed a similar pattern of
regulation. Akt downregulation indicates an anti-apoptotic effect comparable to that associated with p-
CREB. Phosphorylation of the CREB protein is one of the factors that contribute to neurodegeneration.
KPD and NRG down-regulates the phosphorylation of CREB indicating that it blocks the phosphorylation
of CREB protein. SOD detection was carried out by western blot and the results indicated that KPD and
NRG were capable of enhancing the regulation of SOD. Furthermore, anti-apoptotic activity was assessed
by examining caspase activity, proapoptotic proteins such as BAX, and antiapoptotic proteins such as
BcL2. KPD and NRG both decreased caspase 3 and BAX expression and increased BcL2 expression, in
agreement with earlier studies (Figure 5).
4. Discussion
MWM is a widely used model for cognitive behaviour. Sethi P et.al. reported that MWM studies on AlCl3-
induced neurodegeneration showed neurotoxicity, which affects spatial learning and cognitive memory
[6]. Results demonstrated that the Escaping latency effect of KPD and NRG at higher doses of 10 mg/kg
bw showed learning and memory gaining in prophylactic groups, doses which were found to be superior
to the effect showed by the positive control i.e. donepezil. Our results demonstrated that both KPD and
NRG could prevent the AlCl3-induced platform nding impairment in the test group more effectively than
the Standard group.
As part of the probe trial assessment of the spatial learning and memory abilities of rats over AlCl3 -
induced neurodegeneration, we recorded the number of times the rats crossed the platform, the length of
time they spent in the target quadrant, and the distance they covered within the target quadrant on the
seventh day (Tables1 & 2) It was clear from the results that the diseased group of prophylactic reported
poor effects such as shorter time and distance travelled during the probe trail, as well as lower crossings
over target quadrants. In the prophylactic group, NRG achieved better results in terms of time spent in the
target quadrant than KPD at a comparable dose.
Rota rod test measures motor performance by recording the grip strength of different groups of animals.
AlCl3 induces neurotoxicity by causing degeneration of the cortical region. A decrease in motor
performance indicates cortical damage [6]. In the prophylactic group, KPD and NRG at 5 mg/kg bw

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exhibited an intermediate effect on motor recovery. These results demonstrated that KPD and NRG, at
their prescribed doses, signicantly improved the motor function of animals with AlCl3-induced
neurodegeneration, despite the fact that the effect was signicantly higher in the prophylactic group. In
both KPD and NRG groups, motor function was signicantly recovered at their higher doses.
AlCl3 induces neurotoxicity, which in turn affects the central and peripheral nervous system and causes
peripheral neuropathy [13]. This study reported that KPD and NRG exert an effect on neuropathic
pain.These results demonstrated that KPD and NRG, at their prescribed doses, signicantly improved the
management of neuropathic pain in animals with AlCl3-induced neurodegeneration. It was found that in
both KPD and NRG groups, neuropathic pain was signicantly managed at their higher doses i.e. 10
mg/kg bw.
Based on behavioral results, compounds KPD and NRG successfully regained learning and memory
capabilities, effectively restored motor function, and effectively managed neuropathic pain in AlCl3
induced neurodegeneration.
AlCl3 affects the free form of total protein content in the brain tissue. Aggregation of proteins was due to
excessive generation of ROS production, misfolded proteins, and also due to increased concentration of
proteins[14]. AlCl3 induces protein aggregation which was clearly indicated in group 2(G2). Natural
compounds not exerted any signicant effect in the prophylactic group even at 10mg/kg bw. KPD and
NRG at their lower doses reported non-signicant improvement of protein content in the prophylactic
groups of AlCl3 neurodegenerative models.
AlCl3 - induced neurodegeneration causes oxidative stress which may lead to increased oxidative stress
and decreases the number of antioxidant enzymes - SOD, CAT, and Gpx levels [15]. Compounds KPD and
NRG showed poor regaining of antioxidant potential in the prophylactic group of the AlCl3 model, with %
SOD suppression of 27.49 ± 2.2 and 34.01 ± 2.7, respectively. SOD level was not signicantly suppressed
by KPD and NRG at their lower doses in the prophylactic groups of AlCl3 neurodegenerative models
AchE enzyme which regulates the amount of acetylcholine in cholinergic neuronal transmission. AlCl3
exposure causes neurotoxicity by increased release of AchE enzyme at the synaptic cleft [16].Our results
showed that KPD and NRG at 10mg/kg suppresses AchE enzyme levels in brain tissue of prophylactic
groups. AChE level was not signicantly suppressed by KPD and NRG at their lower doses in prophylactic
group of AlCl3 neurodegenerative models.
In congo, red staining method clearly detects the formation of Aβ plaques. Plaque formation is clearly
indicated with an arrow mark. Compared with AlCl3 treated group, the KPD at 10mg/kg bw virtually
doesn’t show any Plaque formation. This clearly indicates that KPD exerts a more signicant effect than
the donepezil-treated group. Histopathological studies, clearly indicated that KPD reduces the AlCl3-
induced neurotoxicity.

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Neurodegeneration due to Aβ and Tau protein plaque formation in brain tissue is the common cause of
brain aging and Alzheimer’s disease[17]. Results reported that, in both KPD and NRG treatments, the
kinase protein and Akt showed a similar pattern of regulation. This indicates that both KPD and NRG
possess anti-apoptotic properties in AlCl3-induced neurotoxicity. The protein expressional analysis of
AlCl3 curative groups revealed the inhibition of Protein Aβ, inhibition of Phosphorylation of Tau and CREB
by the KPD and NRG treatment that led to Akt upregulation, down regulation of SOD, Caspase 3 and BAX
and simultaneous upregulation of BcL2. In the case of KPD, the effect was superior to that of NRG as a
whole.
Akt is a central protein which regulates other anti-apoptic enzymes in brain. Akt downregulation indicates
an anti-apoptotic effect [19]. Phosphorylation of the CREB protein is one of the factors that contribute to
neurodegeneration. KPD and NRG downregulate the phosphorylation of CREB indicating that it blocks the
phosphorylation of CREB protein. SOD detection was carried out by western blot and the results indicated
that KPD and NRG were capable of enhancing the regulation of SOD. Furthermore, anti-apoptotic activity
was assessed by examining caspase activity, proapoptotic proteins such as BAX, and antiapoptotic
proteins such as BcL2. KPD and NRG both decreased caspase 3 and BAX expression and increased BcL2
expression, in agreement with earlier studies (Fig.5). This indicates that both KPD and NRG possess anti-
apoptotic properties in AlCl3-induced neurotoxicity.
The protein expressional analysis of the AlCl3 prophylactic group revealed the inhibition of protein Aβ,
inhibition of phosphorylation of Tau and CREB by the KPD and NRG treatment that led to Akt
upregulation, downregulation of SOD, caspase 3, and BAX, and simultaneous upregulation of BcL2.In the
case of KPD, the effect was superior to that of NRG as a whole.
Conclusion
This study revealed that the naturally occurring polyphenolic molecules KPD and NRG possess
neuroprotective qualities in
in vivo
studies. Our results clearly revealed that KPD and NRG targets the
different possible mechanisms in NDDs, because it shows action against AlCl3-induced neurotoxicity by
targeting different pathways. KPD and NRG exert potent neuroprotective effect in AlCl3-induced
neurotoxicity. Now a day’s research focus on Natural compounds for long term treatment to minimize the
adverse effects of the drugs. This research may helpful for further investigation by targeting other
possible mechanisms of NDDs. Our ndings support the clinical applications of Kaempferide and
Norbergenin against a wide range of neurodegenerative illnesses.
Abbreviations
AD - Alzheimer’s Disease
Akt - Protein Kinase B

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CPCSEA - Committee for the purpose of control and supervision of Experiments on Animals.
ALS - Amyotropic Lateral Sclerosis.
PD - Parkinsons Disease
NDDs - Neurodegenerative Diseases
PPs - Polyphenolics
KPD - Kaempferide
NRG - Norbergenin
AlCl3 -Aluminium chloride
AChE- Acetylcholinesterase
CAT - Catalase
Grd - Glutathione Reductase
SOD - Superoxide Dismutase
HD - Huntington's Disease
Declarations
Ethics approval consent to participate
Yes.
Approval .No:PGP/RM-0019/57-21.
Funding Agency: Not Applicable
Consent for publication
“Not applicable”
Availability of data and material
All data and material should be available upon request.
Competing interests
“No competing interests to declare”.

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Funding
It is self-nanced, funding was not sponsored by any organization, funding agency and non-prot
research bodies.
Author’s contributions
Swathi Nalla – Writing Original draft, Conceptualization, investigation, Funding.
Suhasin Ganta – Formal Analysis, Supervision and Validation.All authors read and approved the nal
manuscript.
Sarad Pawar Naik & Nagaraju B - Statistical Data & All authors read and approved the nal manuscript.
ACKNOWLEDGEMENT
The authors gratefully acknowledge the PGP Life Sciences, Hyderabad, for providing platform to carry
forward the research work.
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Figures

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Figure 1
Total protein content estimation in AC induced neurodegeneration:
The total protein content were measured using Bradford assay method; All the experiments were
statistically validated.

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Figure 2
The Superoxidase levels (a) Catalase activity as a function of anti-oxidant behaviour: (b) Glutathione
peroxidase (Gpx) activity as a function of anti-oxidant behaviour (c) ) Glutathione reductase (Gpx)
activity as a function of anti-oxidant behaviour (d) were examined in the Prophylactic group of AC model;
All the experiments were statistically validated.

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Figure 3
AChE activity as a function of restoring neurotransmitter capability of ACh:
The AChE levels in (a) Prophylactic group of AC model; All the experiments were statistically validated. All
the experiments were statistically validated.
Figure 4

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Representative light micrographs showing the effect of kaempferide, and Norbergenin in the brains of AC-
treated rats by histo-pathological observation using Congo-red staining.
Figure 5
Western blot data displayed the effect of kaempferide, and Norbergenin on various protein expression
levels associated with neurological behavior in the Prophylactic group of AC induced neurodegenerative
model.