Phytomedicine Plus 2 (2022) 100343

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Phytomedicine Plus
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Anti-Parkinsonian effect of Mucuna pruriens and Ursolic acid on GSK3β/

Calcium signaling in neuroprotection against
Rotenone-induced Parkinsonism
Walia Zahra , Hareram Birla , Saumitra Sen Singh , Aaina Singh Rathore , Hagera Dilnashin ,
Richa Singh , Priyanka Kumari Keshri, Shekhar Singh , Surya Pratap Singh *
Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, (U.P.), 221005 India

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Exposure of environmental toxin is linked to the onset of Parkinson’s disease (PD), especially in
Parkinson’ disease majority of sporadic cases. One of such toxins, namely Rotenone has been widely investigated to produce the
Ca2+ level Parkinsonian model in laboratories. The toxin causes the death of the dopaminergic neurons, aggregation of
GSK-3β
α-Synuclein, oxidative stress, neuroinflammation and initiate other pathways leading to PD pathogenesis.
Rotenone
Mucuna pruriens
Glycogen synthase kinase-3 (GSK-3) is a pleiotropic serine/threonine protein kinase found in eukaryotes. One of
Ursolic acid the two isoforms of the enzyme; GSK-3β plays an important role in regulating the pathogenesis of neurode­
generative diseases, including PD. Whereas, Calcium (Ca2+) is found to regulate important cellular activities in
all eukaryotic cells. However, an increased Ca2+ concentration is seen during ageing and the progression of
neurodegenerative diseases. Thus, maintaining the Ca2+ homoeostasis is very crucial for proper cell function.
Methodology: Hence, behavioural tests were performed to assess the motor abnormalities; immunohistochemical
and western blot analyses to identify the alteration in the protein expression; and mitochondrial complexes and
antioxidants assay to detect the extent of mitochondrial dysfunction in Rotenone-induced Parkinsonian mouse
model. Alternatively, the effect of Mucuna pruriens (Mp) and Ursolic acid (UA) in the PD mouse model was also
observed.
Results: Increased Ca2+level, and activation of GSK-3β was observed in the Rotenone-intoxicated mouse model of
PD. The extract of Mp and UA, on the other hand shows neuroprotection through their anti-oxidative and anti-
inflammatory properties. However, their role in maintaining the Ca2+ level and GSK-3β signaling is not yet
observed in PD. So, our study deals with the downregulated activity of GSK-3β and Ca2+ level upon the
administration of Mp extract and UA, thereby providing neuroprotection to the PD mouse model.
Conclusion: Thus, this study deals to explore further the neuroprotective activity of Mp and UA through GSK-3β/
Calcium signaling by ameliorating mitochondrial dysfunction mediated apoptosis and inhibiting the over­
expression of α-Synuclein in Rotenone-induced PD mouse model.

selective and progressive loss of the dopaminergic neurons and forma­

Introduction
The neurodegenerative disorder Parkinson’s disease (PD), affects
mostly elderlies and is associated with ageing. It is characterized clini­
cally by bradykinesia, rigidity, tremor and stooped posture (Dexter and
Jenner, 2013; Kalia and Lang, 2015). Non-motor symptoms, such as
emotional disturbances, stress, olfactory impairments, sleep disorders,
and autonomic dysfunctions, are also related with the pathogenesis of
PD (Kalia and Lang, 2015). The pathology of the disease involves the
tion of inclusions called Lewy bodies (LBs) in substantia nigra pars
compacta of midbrain (Ruiz et al., 2011). Dopaminergic neuronal loss is
often associated with Oxidative stress, overexpression and aggregation
of α-Synuclein, Neuroinflammation and Mitochondrial dysfunction; the
key players for neurodegeneration in PD that induces dopaminergic
neuronal loss and reduction in the level of dopamine (DA) ( Huang et al.,
2020; Martinez et al., 2017; Moon and Paek, 2015; Morris and Berk,
2015; Hu et al., 2019; Tolleson and Fang 2013).
GSK-3β is one of the isoforms of GSK-3, a serine/threonine kinase

* Corresponding author.
E-mail address: suryasinghbhu16@gmail.com (S.P. Singh).

https://doi.org/10.1016/j.phyplu.2022.100343
Received 15 February 2022; Received in revised form 30 August 2022; Accepted 6 September 2022
Available online 7 September 2022
2667-0313/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
W. Zahra et al.
List of abbreviations
Ca2+ Calcium;
GSK-3β Glycogen synthase kinase-3β;
mPTP Mitochondrial permeability transition pore;
Mp Mucuna pruriens;
PD Parkinson’s disease;
ROS Reactive oxygen species;
SEM standard error of mean;
TH Tyrosine hydroxylase;
UA Ursolic acid
found in mammals. The regulatory role of GSK-3β in stress-related
mitochondrial dysfunction is seen in the neuronal cells (Li et al.,
2014). Other pathogenic markers of PD such as oxidative stress, in­
flammatory response, apoptosis and aggregation of α-Synuclein, are also
facilitated by the activation of GSK-3β (Li et al., 2014; Wang et al.,
2007). Whereas, GSK-3β inhibition, suppresses different pathogenic
events in PD, thereby promoting the survival of the dopaminergic neu­
rons (Singh et al., 2020; Wang et al., 2007; Yuskaitis and Jope, 2009).
Administration of the neurotoxin Rotenone, causes the death of the
dopaminergic neurons from SNpc, in animal models of PD (Cannon
et al., 2009; Zahra et al., 2020). Activation of GSK-3β, α-Synuclein
overexpression and increased Calcium (Ca2+) level in the dopaminergic
neurons is mediated by Rotenone-neurotoxicity (Rcom-H’cheo-­
Gauthier et al., 2016; Zahra et al., 2020). Also, being the inhibitor of
Complex I of the mitochondrial Electron Transport System (ETS), it
persuades mitochondrial dysfunction, in PD pathogenesis. Complex I
impairment is also related to dopaminergic neuronal loss in idiopathic
PD patients (Li et al., 2014). Other pathological contributor for the
neurodegeneration in PD is the increased inflammatory response in
dopaminergic neurons (Monahan et al., 2008). Post-mortem studies,
along with the studies including animal models of PD (also by
Rotenone-intoxication), has revealed the presence of activated
Fig. 1. Rotenone-induced GSK‑3β signaling cascade in PD pathogenesis.
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Phytomedicine Plus 2 (2022) 100343
inflammatory mediators, such as microglia and inflammatory molecules
in SNpc of brain (Rai et al., 2017; Zahra et al., 2020). Alteration in the
Ca2+ level also plays major role in the PD pathogenesis (Surmeier et al.,
2012). GSK-3β activation and increased Ca2+level is also interlinked
with each other which further potentiates the formation of the α-Synu­
clein aggregates (Li et al., 2014). Since GSK-3β signaling pathway plays
prominent role in the PD pathogenesis, serving as a central point for
mediating and accelerating different pathological events; can be tar­
geted for therapeutic management of PD pathogenesis (Fig. 1).
Cure for PD is still unknown and treatment with L-DOPA, DA receptor
agonists and deep brain stimulation (DBS) surgery, only manages the
clinical symptoms, and rarely alleviate loss of the dopaminergic neurons
(Iarkov et al., 2020; Rizek et al., 2016). Thus, identifying new neuro­
protectants that reduce neuronal loss is of great significance for the
treatment of PD (Birla et al., 2019; Singh et al., 2018; Rai et al., 2020;
Rai et al., 2021a; Rai et al., 2021b). The neuroprotective property of
Mucuna pruriens (Mp) in PD has been widely studied (Cilia et al., 2017;
Rai et al., 2017, 2020; Yadav et al., 2013; Yadav et al., 2014, 2016). But,
the mechanism through which it mediates its anti-Parkinsonian effect is
yet to be explored. The anti-inflammatory and anti-oxidative studies of
Mp has been seen in the animal models of PD (Uchegbu et al., 2016; Rai
et al., 2017; Yadav et al., 2013). Moreover, the Reverse-Phase High
performance liquid chromatography (RP-HPLC) data have shown the
presence of L-DOPA and Ursolic acid (UA) in the aqueous extract of Mp,
in the study conducted in our lab (Yadav et al., 2013; Rai et al., 2020).
UA is natural triterpenoid carboxylic acid found in variety of plants,
studied for its neuroprotective activity against PD and is also capable of
crossing the blood-brain barrier (Habtemariam, 2019; Zahra et al., 2020;
Rai et al., 2019). Although, both Mp and UA have been studied for their
anti-oxidative and anti-inflammatory properties in PD, but their role in
maintaining the Ca2+ level, inhibition of mitochondrial dysfunction and
α-Synuclein overexpression, and rescuing dopaminergic neurons from
apoptosis; by inhibiting the activation of GSK-3β has not yet been
studied. Thus, this study aims to explore the neuroprotective potential of
Mp and UA by investigating their effect on GSK-3β/ Ca2+ signaling in the
Rotenone-intoxicated mouse model of PD.
W. Zahra et al.
Chemical and reagents
Reagents and Antibodies. Paraformaldehyde, Glycylglycine buffer,
trichloroacetic acid, sodium fluoride, and Tris buffer were purchased
from LobaChemie, India. Normal goat serum (NGS), DABCO, 5,5-dithio­
bis 2-nitrobenzoic acid (DTNB), Rotenone and Ursolic acid were bought
from Sigma-Aldrich (St. Louis, MO, United States). Bovine serum al­
bumin (BSA), Mannitol, oxidized cytochrome c, sodium dihydrogen
phosphatedigitonin, disodium hydrogen phosphate, potassium chloride,
reduced nicotinamide adenine dinucleotide phosphate (NADPH), and
ammonium chloride were purchased from Sisco Research Laboratories
(SRL, Mumbai, India). Sodium carbonate and Sucrose were obtained
from Merck (Darmstadt, Germany). EGTA and sodium dodecyl sulphate
(SDS) were purchased from HiMedia (Mumbai, India). Primary anti­
bodies for Akt (ab8805), p-Akt (ab81283), caspase-3 (ab214430), Iba-1
(ab153696), α-Synuclein (ab191692), Bcl-2 (GR99542–4) and calcium
detection kit (ab102505) were acquired from Abcam Life Science, Bio­
genuix Medsystems Pvt. Ltd. (New Delhi, India), and primary antibodies
for GSK3β (sc-81,462), p-GSK3β (sc-373,800), TH (sc-25,269), β-actin
(sc-47,778), and Bax (sc-6236) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, United States).
Experimental animals
Male Swiss albino mice (adult; 25 ± 5 g) were used in the experi­
mental groups and were obtained from the Institute of Medical Sciences’
animal facility, Banaras Hindu University, Varanasi (India). The exper­
imental animals were kept under proper laboratory condition; in clean
polypropylene cages with constant light-dark cycles of 12 h; and pro­
vided with standard diet pellet and water ad libitum until the dosing was
completed. Experiment was conducted according to the Animal Ethics
Committee’s guidelines of Banaras Hindu University, Varanasi, India.
Plant extract preparation
Seed powder of Mp was procured from the Ayurveda Pharmacy,
Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
The extract was later prepared by following the method of Uhegbu et al.
(2005) and Rai et al., (2017), by using distilled water as solvent. Briefly,
200 mL of distilled water (sterile) was used for soaking 20 g of Mp seed
powder. It was left overnight after 6 min of stirring, for proper mix-up.
The solution was further filtered using Whatman No. A-1, the other day.
The filtered solution was then dried in rotary vacuum evaporator below
40◦ C, under reduced pressure. The extract thus obtained was adminis­
tered to the respective experimental group.
Animal dosing
Mice were randomly divided into four groups, viz.; Control, Rote­
none, Rotenone+Mp, and Rotenone+UA (n = 10/group). Dosing to the
animals were done in accordance with our previous studies with some
modifications (Rai et al., 2017, 2019; Zahra et al., 2020). The Control
group animals received normal saline orally and subcutaneously, once
daily for 42 days. The Rotenone-intoxicated group was administered
with Rotenone (2 mg/kg bwt/day) subcutaneously for 35 days. While
the Rotenone+Mp, and Rotenone+UA groups were first
orally-administered with Mp (100 mg/kg bwt/day) and UA (25 mg/kg
bwt/day), respectively for a week, then simultaneous administration of
Mp and UA was done with Rotenone-intoxication for the next 35 days, in
their respective groups. The behavioural parameters were analysed after
the completion of dosing within the next four days. Animals were further
sacrificed for brain isolation, to perform mitochondrial complexes and
antioxidants assay, Western blotting, calcium level assay and immuno­
histochemical analysis.
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Phytomedicine Plus 2 (2022) 100343
Behavioural test
Behavioural tests were done at day time (between 10AM- 12 noon) to
assess motor deficits in the Rotenone-intoxicated mice; and how Mp and
UA ameliorated this neurotoxic effect in parkinsonian mouse model. The
tests were performed for five consecutive days after the completion of
the dosing and include the rotarod test, pole test, catalepsy test, hanging
test and traction test.
Catalepsy test
Muscle stiffness in mice is observed by performing the Catalepsy test.
In this test, the diseased mice are unable to change its posture, in which
they are imposed. The animals were made to stand on a wooden plat­
form with their hind limb and their forepaws were supported on the
ground (Singh et al., 2020). Cataleptic intensity was measured as the
mouse moved its hind limbs from a wooden platform. The test was
performed for 180 s for each mouse of each group.
Pole test
Pole test was performed to assess bradykinesia in the Parkinsonian
mouse model (Hu et al., 2018). The animals were placed on the top of
pole having a rough surface (52 cm in height and 10 mm in diameter).
The time taken by the mouse to step down the pole (T-turn) was further
recorded.
Traction test
This test was done to assess the muscle strength and equilibrium in
the experimental animals (Hu et al., 2018). Forelimbs of the mouse were
supported on a horizontal bar in this test and traction score was recorded
on the basis of their hind limb placement. The lowest score was given to
mice which were severely impaired. While the score was increased as the
mouse placed one or both hind limbs on the bar.
Rotarod test
This test was also done to assess the equilibrium in the mouse model
of PD. Mice from each group were placed on a rotating beam (speed 5
rpm). The experiment was performed for a maximum duration of 5 min.
The time for which the animal stayed on the rotarod was noted down
(Manna et al., 2006).
Hanging test
The test was performed by using a horizontal grid on which the
animals were placed, and the grid was turned upside down. Hanging
time was recoded and the experiment was repeated thrice (Rai et al.,
2016).
Mitochondrial parameter analysis
Isolation of mitochondria
Mitochondrial pellet was isolated from the mouse brain, by differ­
ential centrifugation (Singh et al., 2020). Homogenization of the
nigrostriatal region of midbarin was done using a buffer prepared by
mixing 225 mM mannitol, 5 mM HEPES, 75 mM sucrose, 1 mM ethylene
glycol tetra acetic acid (EGTA), and 1 mg/ml BSA (pH 7.4). The ho­
mogenate obtained was further centrifuged at 2000 g for 30 min at 4 ◦ C.
The supernatant obtained after the centrifugation was further centri­
fuged at 12,000 g for 10 min. The pellet obtained after second centri­
fugation constituted the mitochondria and synaptosomes and was
dissolved in homogenizing buffer containing digitonin (0.02%). The
mixture was again set to centrifugation for 10 min at 12,000 g to obtain a
W. Zahra et al.
crude mitochondrial fraction. This was further washed with the ho­
mogenizing buffer without BSA and EGTA twice, and the resuspension of
fraction was done in phosphate buffer (50 mM, pH 7.4). Proteins in all
the samples were assayed by using 20 μg proteins, through the Bradford
assay.
Complex I-III assay
The method of Sood et al. was incorporated for assessing complexes
I-III activities, in our study (Sood et al., 2011). In this assay, cytochrome
c was reduced upon the catalytic oxidation of NADH to NAD+. NADH
(6mMin 2mMglycylglycine buffer), glycylglycine buffer (0.2 M, pH 8.5),
and cytochrome c (10.5 mM) was used for the preparation of the reac­
tion mixture. It was further added with 20 μg mitochondrial protein and
change in the absorbence was recorded for 2 min at 550 nm. The
extinction coefficient for NADH at 340 nm is 6.22/mM/cm. The unit
nmol NADH oxidized/min/mg protein was used to express the activity
of the enzymes.
Complex IV assay
The oxidation of reduced cytochrome c was assayed at 550 nm to
detect the Complex IV activity (Sood et al., 2011). Potassium ferricya­
nide, 10 mM phosphate buffer (pH 7.4), reduced cytochrome c and 20 μg
of mitochondrial protein was used as a reaction mixture. The reduced
Cytochrome c was prepared by adding few crystals of sodium borohy­
dride in the solution of oxidized cytochrome c (10 mg/ml). Change in
the absorbance was observed for about 3 min and the complex IV ac­
tivity was calculated in nmol cytochrome c oxidized/min/mg of protein.
Complex V assay
In the Mitochondrial ATPase test, the amount of inorganic phos­
phorus was measured in the assay that was liberated on ATP to ADP
hydrolysis (Singh et al., 2020). ATPase buffer (5 mM ATP, 2 mM MgCl2,
and 50 mM Tris–HCl, pH 8.5) and 20 μg of mitochondrial protein was
incubated for 5 min at 30 ◦ C. The reaction mixture was then spun for 10
min at 3000 g, after the addition of 10% TCA to it. The results were
expressed as nmol inorganic phosphate (Pi) liberated/min/mg protein.
Mitochondrial GSH
Mitochondrial protein sample (20 μg) suspended in phosphate buffer
was used to estimate the reduced Mitochondrial GSH. Centrifugation of
all the samples were done after the addition of 25% trichloroacetic acid
(TCA) at 1500 g. 5,5-dithiobis 2-nitrobenzoic acid (DTNB) was added to
the supernatant obtained which reacts with thiol groups to form 2-nitro-
5mercapto benzoic acid. absorbance was recorded at 412 nm. Also,
standard curve was plotted using commercially available GSH and the
unit mmol of GSH/g mitochondrial protein was used to express the
result (Khanna and Nehru, 2007).
Manganese SOD
To assess selectively activity of manganese superoxide dismutase
(Mn-SOD), addition of hydrogen peroxide is done that inhibits Cu/Zn-
SOD, so as to distinguish the enzyme’s activity from that of Cu/Zn-
SOD. EDTA (0.1 mM), sodium carbonate (50 mM), Triton-X (0.6%),
and NBT (90 mM) and hydroxylamine hydrochloride were used to
prepare the reaction mixture (Khanna and Nehru, 2007). The reaction
progresses as the photooxidation of Hydroxylamine hydrochloride oc­
curs to produce superoxide which further reduces nitroblue tetrazolium
(NBT) in the reaction medium, so as the reaction was inhibited by SOD.
Activity of SOD was expressed in units by taking the reading at 560 nm
for 3 min and one enzymatic unit of SOD defines the amount of enzyme
required for 50% inhibition.
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Phytomedicine Plus 2 (2022) 100343
Calcium level determination
The level of calcium in the nigrostriatal region of the midbrain was
assayed through colorimetric calcium detection kit (Abcam). According
to the kit instructions, the standards and the samples were prepared. 50
mg of the nigrostriatal tissue was resuspended in 500 μL of Calcium
Assay Buffer after washing in cold PBS solution. Homogenization of
tissue was done manually on ice, using a tissue grinder. Homogenates
was set to centrifugation at 5000 rpm 5 min at 4 ◦ C, and supernatant
were collected. 90 μL of the Chromogenic Reagent, 50 μL of sample, and
60 μL of Calcium Assay Buffer was used as a reaction mixture which was
incubated for 10 min in dark. absorbence was further taken at 575 nm
and the concentration was determined using standard curve (Kumar
et al., 2019).
Immunohistochemistry (IHC)
Perfusion of Mice from each group was done using chilled 0.9% sa­
line solution and 4% paraformaldehyde solution (prepared in 0.1 M
phosphate buffered saline, pH 7.4), intracardially. Whereas, 10% para­
formaldehyde was used for overnight postfixing of brains; immersed
later into 30% sucrose, until used further. Immunohistochemical stain­
ing of TH and α-Synuclein was done using standard protocol (Gorbatyuk
et al., 2008). The cryotome (Leica, Wetzlar, Germany) was used to cut
15 μm thick Coronal brain sections, which were washed further with
0.01 M PBS (pH 7.4) twice for10 min. Sections were then blocked for 1 h,
first with 10% NGS in PBST and then 1% BSA-PBST. They were rinsed
thereafter with PBS and incubated with primary antibodies for TH (1:
1000) and α-Synuclein (1:1500) at 4 ◦ C for 16 h. The secondary anti­
bodies viz.; TRITC-conjugated (anti-rabbit) and FITC-conjugated
(anti-mice), diluted in 1% BSA-PBS, were further incubated with the
respective primary-antibody incubated tissue sections for 2 h at room
temperature. At each step, the washing of sections was done using PBS
and 1% BSA-PBS and PBS, respectively. Mounting of sections on slides
was further done with polyvinyl alcohol with DABCO. The fluorescent
microscope (Nikon, Thermo Fisher Scientific) was used to acquire the
images, further processed using ImageJ software (NIH, United States).
Results are expressed as % area and number of TH-positive neurons for
the immunohistochemistry of α-Synuclein and TH respectively.
Western Blot (WB) analysis
RIPA buffer was used for homogenizing the nigrostriatal region of
the mouse brain. After 2 h of incubation of the homogenates at 4 ◦ C, they
were centrifuged at 12,000 rpm for 30 min at 4 ◦ C. Supernatant from
each sample was collected and Bradford assay was performed for protein
concentration quantification. 50 μg protein from each sample was
loaded in the wells of polyacrylamide gels, for electrophoresis. There­
after, PVDF membranes were used for transblotting of proteins; incu­
bated overnight further with primary antibodies of TH (1:4000),
α-Synuclein (1:1000), Bax (1:1000), Bcl-2 (1:800), caspase-3 (1:1000),
Iba1 (1:1000), p-Akt (1:1000), Akt (1:1000), p-GSK3β (1:1000), GSK3β
(1:1000), and β-actin (1:1000). After washing with TBST and TBS, the
membranes were further incubated with the horseradish peroxidase-
(HRP-) conjugated secondary antibody for 2 h at room temperature.
Blots were visualized using the Enhanced chemiluminescence (ECL)
system; relative density for each band was calculated with respect to that
of β-actin. Expression of relative density was done using Quantity One
software (Windows, Bio-Rad).
Statistical analysis
Oneway analysis of variance (ANOVA) using the Stu­
dent–Newman–Keuls test and by Student’s two-tailed t-test using
GraphPad Prism software was done to analyse the data obtained in the
study. Results are expressed as the mean ± SEM. p values < 0.05 were
W. Zahra et al. Phytomedicine Plus 2 (2022) 100343

considered statistically significant.
Results
Mp and UA ameliorated the behavioural deficits in Parkinsonian mouse
model
Different behavioural tests were performed to assess the behavioural
abnormalities in our study. Rotenone-intoxication in our study has
induced Catalepsy in the PD mouse model (p < 0.0001; Fig. 2(A)) as
compared the control group. While the mice treated with Mp and UA in
the respective Rotenone-intoxicated groups showed little latency time
(p < 0.0001; Fig. 2(A)) than the Rotenone-intoxicated group. Prolonged
time (T-turn) was taken by the mice of Rotenone-intoxicated group (p <
0.0001; Fig. 2(B)), when compared to control. Whereas, the duration
was shortened on the administration of Mp (p < 0.001; Fig. 2(B)) and UA
(p < 0.001; Fig. 2(B)), in their respective Rotenone-intoxicated groups;
suggesting that bradykinesia induced by Rotenone-toxicity was allevi­
ated by Mp and UA treatment. The hind limb score of Rotenone-
intoxicated group was lowered in traction test ((p < 0.001); Fig. 2(C)),
in comparison to control. However, pre-treatment with Mp (p < 0.01
Fig. 2(C)) and UA (p < 0.01 Fig. 2(C)) increased the traction score in
their respective PD groups when compared to the diseased group. While
in hanging test, the time was reduced for the Rotenone-intoxicated
group (p < 0.0001; Fig. 2(D)) in comparison to control. On the other
hand, Mp and UA treatment given to the Rotenone group significantly
improved the hanging time (p < 0.0001; Fig. 2(D)), as compared to the
hanging time in Rotenone-intoxicated group. Rotenone-intoxicated mice
fall early in comparison to control group (p < 0.0001; Fig. 2(E)), in the
rotarod test. On the other hand, by the administration of Mp and UA to

Fig. 2. Mp and UA alleviated the behavioural abnormalities in Rotenone-intoxicated PD mouse. Values are represented in the form of mean ± SEM (n = 10), *p <
0.01, **p < 0.001 & ***p < 0.0001. Abbreviations: UA, Ursolic acid; Mp, Mucuna pruriens; SEM, standard error of mean.

5
W. Zahra et al.
their respected Rotenone-treated groups have increased the time on the
rotarod (p < 0.0001; Fig. 2(E)). Thus, results have suggested that Mp and
UA improved the neurobehavioral lesion in the Rotenone-intoxicated PD
mouse model.
Mp and UA improved Mitochondrial ETS impairment in rotenone-
intoxicated mice
ATP in mitochondria, is produced by proper functioning of five
enzyme complexes embedded in the inner mitochondrial membrane.
Mitochondrial dysfunction led to the catastrophic consequences, haz­
ardous to the functionality of cell. Therefore, the effect of Mp and UA
was observed on the mitochondrial dysfunction caused by Rotenone-
intoxication in our study. The complex I inhibitor, Rotenone was
observed to alter the activities of other complexes of ETS too, in our
study. As shown in Fig. 3, the effects of Mp and UA treatment has
reversed the toxic effect of Rotenone on complex I-III (Fig. 3(A)), IV
(Fig. 3(B)), and V activities (Fig. 3(C)) in PD mouse model. Attenuated
activities of complexes I-III (p < 0.001), complex IV (p < 0.001), and
complex V (p < 0.0001) was observed in Rotenone-intoxicated PD mice
as compared to that of Control. Whereas, neuroprotection by Mp and UA
to the Rotenone-treated animals helped improving the activities of
complexes I-III (p < 0.01 for Mp treatment and p < 0.001 for UA
treatment), complex IV (p < 0.001 for Mp treatment and p < 0.01 for UA
treatment), and complex V (p < 0.0001 for both Mp and UA treatment),
when compared with Rotenone-intoxicated mice.
Mitochondrial glutathione and superoxide dismutase level analysis
Oxidative stress leading to mitochondrial dysfunction results in the
depletion of reduced mtGSH and impaired Mn-SOD activity (Figs. 4(A)
and 4(B)). In the Rotenone treated group, the attenuated level of mtGSH
(p < 0.0001) and decline in the Mn-SOD activity (p < 0.0001) was
observed as compared to control in our study. On the other hand, Mp
and UA reduced the oxidative stress and maintained the proper func­
tionality of ETS, leading to an increment in the level of mtGSH (p <
0.0001 for Mp and p < 0.001 for UA) (Fig. 4(A)) and activity of Mn-SOD
Fig. 3. Mp and UA improved the mitochondrial complexes activities in Rotenone-mouse brain. Values are represented in the form of mean ± SEM (n = 5), *p < 0.01,
**p < 0.001 & ***p < 0.0001. Abbreviations: UA, Ursolic acid; Mp, Mucuna pruriens; SEM, standard error of mean.
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Phytomedicine Plus 2 (2022) 100343
(p < 0.0001 for both Mp and UA) (Fig. 4(B)) significantly, in their
respective PD groups.
Mp and UA attenuated the increased calcium level in nigrostriatal tissue of
PD mouse
The level of Calcium (Fig. 5) was found significantly higher in the
nigrostriatal tissue of PD mouse as compared to control (p < 0.0001).
While the neuroprotection provided by Mp and UA have significantly
reduced (p < 0.0001 for both Mp and UA) the Calcium level in their
respective Rotenone-intoxicated groups (Fig. 5).
Effect of Mp and UA on TH and α-Synuclein
The immunohistochemistry of the SNpc region of the Rotenone-
intoxicated mouse has revealed a decline in TH immunoreactivity (p
< 0.0001; Fig. 6(A)) indicating the loss of TH-positive neurons, and
reduced protein expression (p < 0.0001; Fig. 7(A)) as compared to
control. While Mp and UA treatment has enhanced the TH immunore­
activity (p < 0.0001 for Mp and p < 0.001 for UA; Fig. 6(A)) and the
protein expression (p < 0.0001 for both Mp and UA; Fig. 7(A)) in their
respective Rotenone-intoxicated groups. On the other hand, the over­
expression of α-Synuclein was observed in the Rotenone-intoxicated as
suggested by the increased immunoreactivity (p < 0.0001; Fig. 6(B)) and
expression (p < 0.0001; Fig. 7(B)) of the protein. Whereas, the treatment
with Mp and UA has significantly reduced both the immunoreactivity (p
< 0.0001 for Mp and p < 0.001 for UA; Fig. 6(B)) and expression (p <
0.0001 for Mp and p < 0.001 for UA; Fig. 7(B)) of α-Synuclein in their
respective PD groups.
Mp and UA inhibited the rotenone-induced activation of mitochondrial
dysfunction mediated apoptosis in nigrostriatal tissue of mice
Expression of different apoptotic markers, namely, Bax, Bcl2 and
cleaved Caspase 3 were investigated in our study to analyse the effect of
Mp and UA on PD mouse model (Figs. 7(D)–7(E)). Bax/Bcl-2 ratio (p <
0.0001) was seen to be increased considerably upon Rotenone-
W. Zahra et al.
Fig. 4. Mp and UA mediated enhancement of mitochondrial antioxidants in Rotenone-treated PD mouse model. Values are represented in the form of mean ± SEM
(n = 5), *p < 0.01, **p < 0.001 & ***p < 0.0001. Abbreviations: UA, Ursolic acid; Mp, Mucuna pruriens; SEM, standard error of mean.
Fig. 5. Rotenone-intoxicity elevates the calcium level within the nigrostriatal
region of the Parkinsonian mouse brain as compared to control. While neuro­
protection by Mp and UA has reversed the toxicity and lessened the calcium
level in both the Mp and UA treated Rotenone-intoxicated groups. Values are
represented in the form of mean ± SEM (n = 5), *p < 0.01, **p < 0.001 & ***p
< 0.0001. Abbreviations: UA, Ursolic acid; Mp, Mucuna pruriens; SEM, stan­
dard error of mean.
intoxication whereas Mp and UA-treatment has reduced the same Bax/
Bcl-2 ratio (p < 0.0001) in their respective groups, signifying the anti­
apoptotic property of Mp and UA in the parkinsonian mouse model
(Fig. 7(E)). Also, cleaved caspase-3 (p < 0.0001) was elevated in the
nigrostriatal tissue of PD mice whereas it was significantly attenuated on
the administration of Mp (p < 0.01) and UA (p < 0.01) to the Rotenone-
intoxicated group (Fig. 7(D)).
Mp and UA reduced the activation of microglia by ameliorating the
dysregulation of kinases, Akt and GSK3β
The phosphorylation of GSK3β by Akt plays important role in the
7
Phytomedicine Plus 2 (2022) 100343
neuroprotection against PD. Rotenone-intoxication in our study has
significantly reduced the p-Akt/Akt ratio (p < 0.0001; Fig. 7(G)) and p-
GSK3β/GSK3β ratio (p < 0.0001; Fig. 7(H)), when compared with the
control group, whereas the treatment of Mp and UA has increased the p-
Akt/Akt ratio (p<0.0001 for both Mp and UA; Fig. 7(G)) and p-GSK3β/
GSK3β ratio (p < 0.001 for both Mp and UA; Fig. 7(H)) in their respective
Parkinsonian Mouse groups. Furthermore, Rotenone-intoxication has
resulted in the microglial activation as suggested by the enhanced
expression of Iba1 (p < 0.0001; Fig. 7(F)) as compared to control. But the
treatment of Mp and UA has significantly reduced (p < 0.001 for Mp and
p < 0.0001 for UA; Fig. 7(F)) the expression of Iba1 in their respective
PD mouse groups, suggesting the attenuated immune response.
Discussion
Neurodegenerative diseases including PD, show their pathology
through interrelated pathogenic processes, viz., aggregation of proteins,
dysfunction of cell signaling, and ultimately the death of the neurons
(Iarkov et al., 2020; Kaur et al., 2019). Increased expression of α-Syn­
uclein, both at transcriptional and translational level has been observed
in the case of PD (Yuan et al., 2015). Thus, inhibition of α-Synuclein
overexpression and aggregation, in addition to the amelioration of other
hallmarks of PD, is important to stop the progressive loss of the dopa­
minergic neurons in the SNpc of midbrain. Also, PD therapies used
nowadays are only symptomatic and identification of effective neuro­
protectants having minimal side effects, is necessary (Iarkov et al.,
2020). Mp and UA, in this framework, have been extensively considered
for their anti-oxidative and anti-inflammatory properties, reported in
many studies (Rai et al., 2016, 2017; Yadav et al., 2013; Zahra et al.,
2020). The present study is aimed to explore further the neuroprotective
activities of Mp and its constituent compound UA in ameliorating the
mitochondrial dysfunction, overexpression of α-synuclein, and dopa­
minergic neuronal loss through GSK-3β/Ca2+ signaling in
Rotenone-induced mouse model of PD. This study also aims to check
whether, UA other than L-DOPA found in the aqueous extract of Mp is
also responsible for its neuroprotective activity or not. Accordingly, the
neurobehavior deficits by Rotenone-intoxication in our study, were
examined by performing the Catalepsy test, Hanging test, Pole test,
W. Zahra et al.
Fig. 7. Relative expression of TH, α-Synuclein, pGSK-3β, p-Akt, Bcl-2, Bax, Caspase-3, and Iba1 in nigrostriatal region of different mouse groups. Values are expressed
as mean ± SEM (**p < 0.001, ***p < 0.0001). Abbreviations: Mp, Mucuna pruriens; UA, Ursolic acid; TH, Tyrosine hydroxylase; SEM, standard error of mean.
Rotarod test, and Traction test. Rotenone-intoxicity induced the
behavioural impairments in the PD group, which were reversed upon the
neuroprotection offered by Mp and UA (Fig. 2), in consistent with pre­
vious studies (Zahra et al., 2020).
Overexpressed and activated GSK-3β has also been observed in re­
gions related with PD pathology, in the post-mortem brains (Golpich
et al., 2015; Yuan et al., 2015). Dysregulation of PI3K/Akt signaling is
related to PD pathology and reduced phospho-Akt/total Akt ratio is
observed in the dopaminergic neurons of post-mortem PD brains
(Malagelada et al., 2008; Timmons et al., 2009). As Akt negatively
regulates the activation of GSK-3β by phosphorylating the latter at Ser9,
thus the activation of PI3K/Akt pathways might be investigated for
confirming neuroprotection against PD (Zhang et al., 2011). Our study
too suggested that activation of Akt by Mp and UA, deactivated GSK-3β
8
Phytomedicine Plus 2 (2022) 100343
Fig. 6. Immunohistochemistry of TH and α-Synuclein in SNpc
of Control, Rotenone, Rotenone+Mp and Rotenone+UA mice
groups by using Image J Software at 20x magnification.
Rotenone intoxicity has reduced the TH immunoreactivity (A)
and increased the expression of α-Synuclein (B) in Rotenone-
treated group in comparison with control. While, neuro­
protection by Mp and UA has significantly improved the TH
immunoreactivity (A) and downregulated the expression of
α-Synuclein (B) in their respective groups as compared to
Rotenone-intoxicated group. Values are expressed as mean ±
SEM of percentage area (*p < 0.01, **p < 0.001, ***p <
0.0001, n = 3). Scale bar represents the degree of magnifi­
cation of the image (For 20X= 100 µm). Abbreviations: TH,
Tyrosine hydroxylase; SNpc, substantia nigra pars compacta;
Mp, Mucuna pruriens; UA, Ursolic acid.
and associated pathological pathways in Rotenone-induced mouse
model of PD (Fig. 7).
Increment in the expression α-synuclein in transgenic mice (Duka
et al., 2009), also elevates the activation of GSK-3β, indicating, that the
activation of GSK-3β promotes accumulation and overexpression of
α-synuclein and vice-versa (Yuan et al., 2015). Thus, alteration in the
activity of GSK-3β can protect against the debilitating conditions of PD
(Singh et al., 2020; Yuan et al., 2015; Zhao et al., 2012). It has been
demonstrated that neurotoxicity with MPTP, 6-OHDA and Rotenone
enhanced the activation of GSK-3β (Hernandez-Baltazar et al., 2013;
Singh et al., 2020). Moreover, reduced Ser9 and an elevated Tyr216
phosphorylation resulted in the overexpression of α-Synuclein while the
inhibition of GSK-3β abrogated the α-Synuclein mediated neurotoxicity
(Gassowska et al., 2014). Altogether, by modulation of GSK-3β activity,
W. Zahra et al.
a potential approach against α-Synucleinopathy can be investigated in
neurodegenerative conditions including PD. The present study also
validates the activation of GSK-3β and overexpression of α-synuclein, in
consistent with previous studies (Hernandez-Baltazar et al., 2013; Singh
et al., 2020). While neuroprotection provided by Mp and UA has
significantly reversed the GSK-3β and α-Synuclein pathology in the
Rotenone-intoxicated PD mouse model (Fig. 6 and 7).
The energy requirement in the CNS is mainly dependant on mito­
chondria and hence the organelle dysfunction mediates pathogenesis in
the neurodegenerative diseases like PD (Hattingen et al., 2009). Rote­
none being the inhibitor of Complex I, accelerates the formation of ROS
and hence promotes the mitochondrial dysfunction responsible for PD
pathogenesis (Kussmaul and Hirst, 2006). Also, GSK-3β is seen to
regulate apoptosis by inhibiting the activity of complex I and associated
ROS production (King et al., 2008). On the other hand, inhibiting the
activation of GSK-3β has protected the dopaminergic neurons from
Rotenone-induced toxicity by inhibiting the complex
dysfunction-induced apoptosis in PD (Wang et al., 2007). In this study,
the attenuated activities of complexes I-III, complex IV, and complex V
of ETS was observed upon the activation of GSK-3β through
Rotenone-intoxication. While the neuroprotection provided by Mp and
UA has significantly increased the activities of the ETC in the PD mouse
model (Fig. 3). Therefore, it is suggested that activation of GSK-3β
hampered electron transfer in between the complexes, which were
reversed upon the treatment of Mp and UA.
Because of the increased production of ROS and ETC impairment,
excessive consumption of mitochondrial GSH (mtGSH, reduced) and
decline in the Mn-SOD is observed. Whereas, GSH helps in protecting the
dopaminergic neurons against the adverse effects of neurotoxins in the
case of PD (Li et al., 2016; Mani et al., 2018). Our results also potentiate
the neuroprotective activity of Mp and UA, by increasing the level of
mtGSH and activity of Mn-SOD upon ameliorating the adverse effects of
the neurotoxin Rotenone (Fig. 4).
Changes in the Ca2+ levels enhance the production of ROS, resulting
in the dysfunction of the mitochondria by forming the mitochondrial
permeability transition pore (mPTP) opening, and release of cytochrome
c . Oligomerisation of α-Synuclein also enhances the permeability of
plasma membrane, thereby facilitating Ca2+ influx inside the dopami­
nergic neurons (Schmidt et al., 2012; Tsigelny et al., 2012). GSK-3β
activation and increment in the level of Ca2+ is also linked to PD pa­
thology (Yuan et al., 2015). Our study also suggests that
Rotenone-intoxication leading to activation of GSK-3β and over­
expression of α-Synuclein has increased the Ca2+ level in the nigros­
triatal tissue, which were significantly improved by providing
neuroprotection with Mp and UA in Rotenone-intoxicated PD mouse
(Fig. 5).
Various studies have also reported the activation of microglia and
hence neuroinflammation, in the SN of animal models and patients of PD
(Hirsch and Hunot, 2009; Ouchi et al., 2009). Additionally, inhibiting
the inflammatory response protects the dopaminergic neurons against
neurotoxin-induced cell damage (Lofrumento et al., 2014). Activation of
GSK-3β also promotes the microglial activation and associated inflam­
matory responses (Yuskaitis and Jope, 2009). In our study too, increased
expression of the microglial marker Iba1 is observed in the
Rotenone-intoxicated mouse model of PD. While administration of Mp
and UA has ameliorated the inflammatory response as suggested by the
reduced expression of Iba1 (Fig. 7).
The expression of the rate-limiting enzyme of the dopamine syn­
thesis, i.e., TH is seen to be reduced upon Rotenone-intoxication and
α-synuclein overexpression. While Mp and UA were found to protect the
dopaminergic neurons as suggested by TH expression in our study (Fig. 6
and 7) (Liu et al., 2008; Zahra et al., 2020). The deregulation in the
expression of Bcl-2 family proteins, also potentiates GSK-3β-mediated
apoptosis of the neurons (Wang et al., 2013). Activation of Caspase 3
and alteration in the expressions of the anti-apoptotic protein, Bcl-2 and
proapoptotic protein, Bax is observed during the mitochondrial
Phytomedicine Plus 2 (2022) 100343
apoptosis (Singh et al., 2015). Increased apoptosis indicated by elevated
Bax/Bcl2 ratio and cleaved Caspase-3 expression, was also observed
upon Rotenone-intoxication in our study. Whereas, Mp and UA has
reversed these alterations by inhibiting the apoptosis and protecting the
dopaminergic neurons (Fig. 7).
Overall, our study suggests that neuroinflammation, mitochondrial
dysfunction, apoptosis and loss of the dopaminergic neurons can be
reversed by inhibiting the activation of GSK-3β/ α-Synuclein/ Ca2+ pa­
thology upon neuroprotection with Mp and UA; establishing them as
possible therapeutic drugs for clinical interventions in PD therapy.
Conclusion
The present study deals to explore the neuroprotective potential of
Mp and its constituent compound, UA, in providing neuroprotection
against key mediators (α-Synuclein overexpression, neuroinflammation,
I altered Ca2+ level, mitochondrial dysfunction and apoptosis) of neuro­
degeneration in PD through regulation of GSK-3β/ α-Synuclein/Ca2+
signaling. Collectively, UA other than L-DOPA, is also responsible for the
neuroprotective property of the Mp extract and hence both Mp and UA
can be used as potential pharmacological candidate for ameliorating
neurodegeneration in PD and should be further investigated in pre-
clinical studies.
Funding
NA
Ethics approval
The ethical standards of the institution or practice was followed and
the experiments were performed according to the guidelines for animal
experiments recognized by the Institutional Animal Ethics Committees
(IAECs) of the Laboratory Animal Research (Reg. No. 1802/GO/Re/S/
15/CPCSEA) at Banaras Hindu University, India.
CRediT authorship contribution statement
Walia Zahra: Conceptualization, Methodology, Writing – review &
editing. Hareram Birla: Visualization, Investigation. Saumitra Sen
Singh: Data curation, Writing – original draft. Aaina Singh Rathore:
Data curation, Writing – original draft. Hagera Dilnashin: Visualiza­
tion, Investigation. Richa Singh: Data curation, Writing – original draft.
Priyanka Kumari Keshri: . Shekhar Singh: Visualization, Investiga­
tion. Surya Pratap Singh: Supervision, Validation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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