Metabolic Brain Disease (2022) 37:411–426

https://doi.org/10.1007/s11011-021-00878-2

ORIGINAL ARTICLE

Experimental validation of Vitex negundo leaves hydroalcoholic

extract for neuroprotection in haloperidol induced parkinson’s disease
in rat
Aishwarya Vannur1 · Prakash R. Biradar1 · Vishal Patil2

Received: 9 August 2021 / Accepted: 15 November 2021 / Published online: 13 January 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Parkinsonism is a neurodegenerative disease, mainly imbalance in dopamine and acetylcholine neurotransimitter in mid
brain, which manifestation of dysfunctions of extrapyramidal like akinesia, tremor, rigidity and catalepsy etc., even cogni-
tive and memory loss. The current study is framed to evaluate the effect of Vitex negundo (VNL) leaf extract in Haloperidol
induced PD in rats. In vitro studies of antioxidant capacity were checked via DPPH and NO assays and identified its Ace-
tylcholinesterase (AChE) inhibitory activity. Secondly the In vivo study of anti-PD activity in Haloperidol induced in rats
were evaluated by Rotarod, morris water maze (MWM), cooks pole climb (CPC), actophotometer, novel object recognition
(NOR), and T-maze were utilized to assess extrapyramidal, cognitive and memory function. Thirdly, changes in biomarker
level viz. (AChE), butyrylcholinesterase. (BChE) in hippocampus and cortex, reduced glutathione (GSH), malondialde-
hyde (MDA), total protein (TP), superoxide dismutase (SOD), catalase (CAT), and dopamine level in the whole brain
were measured. Finally, histopathology of hippocampus and cortex was examined at 40x magnification to access restoring
integrity and maintaining the architecture of neuronal cell in the treatment group compared to control group and L-DOPA
as a standard treatment group. V. negundo showed potent antioxidant potency on scavenging of DPPH ­(IC50 84.81 μg/ml)
and NO ­(IC50 133.20 μg/ml) and possess AChE inhibitory potency ­(IC50 114.35 μg/ml) by in vitro studies. The Rotarod,
MWM, CPC, Actophotometer, NOR, T-maze demonstrated that Haloperidol group administration declines performance
time, ELT, TL and decreases locomotion, cognitive and memory respectively. The treatment of VNL 100, 200, and 400 mg/
kg p.o. significantly (p < 0.05 to p < 0.0001) reversed. Whole brain AChE, BChE, and MDA level were significantly raised
and GSH, TP, SOD, CAT and Dopamine were significantly declined in Haloperidol treated group rats, especially V. negundo
400 mg/kg p.o. highly significantly ameliorate the Haloperidol group altered pathological changes through the restoration of
the cholinergic function, enhancing the antioxidant defense and by increasing the dopaminergic function. The current study
provides validation of V. negundo for its anti-PD activity and could be a valuable source for the treatment of PD in future.

Keywords Haloperidol · L-DOPA · Parkinson’s disease · Vitexine · Vitex negundo L

Introduction

Parkinsonism disease is a motoric neurodegenerative dis-

* Prakash R. Biradar
prakashbiradar@klepharm.edu; prakashrb123@gmail.com
1 the presence of intracytoplasmic inclusions that contain the
Department of Pharmacology and Toxicology, KLE
College of Pharmacy, KLE Academy of Higher Education
and Research, Belagavi, Karnataka 590010, India
2
ICMR-National Institute of Traditional Medicine, Belagavi,
India
ease, which mainly affect the dopaminergic neuron, due
to depletion in dopaminergic neurotransmitter and acetyl-
choline at substantia nigra pars compacta (SNpc), which
affects the extrapyrimidal and cognitive process according
to James Parkinson (1817). The common cause in PD is
protein, α-synuclein. The presence of toxic aggregated forms
of α-synuclein, amyloid and tau protein damage, etiological
causes are environmental factor, genetic and aging causes
for PD (Mhyre et al. 2012). However, the epidemiological

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study indicates it affect in aged people (Rizek et al. 2016).
According to meta-analysis increased from 107/1,00,000
in aged 50–59 years old people, 1087/1,00,000 people
aged70–79 years are most likely to develop PD with male
to female ratio1:5 (Moisan et al. 2016). Latest symptomatic
treatment options for PD include dopamine precursor such
as Levodapa (L-DOPA) and carbidopa (Gandhi et al. 2021).
Enhances the dopamine level in brain by dopaminergic
agonist like pramipexole, ropinirole, and bromocriptine for
advanced treatment, have lost the ability to synthesis the
process (Rascol 1999; Luo et al. 2020). Now, selegiline and
rasagiline belong to a class of drug called MAO B inhibitor,
to overcome “wear off” effect (Finberg and Rabey 2016)
combination with levodopa to reduce extrapyramidal, even
Enatacapone and Tolcapone COMT inhibitors for advanced
treatment.long trem leading to side effect like dyskinesias,
or repetitive and irregular spontaneous motions, jerky or
twisted muscle motions, hallucinations, mood fluctuations,
nausea, muscle pain, insomnia, numbness, suicidal impulses,
etc. (Tønjum et al. 1989).
However, traditional medicines holistic approach in
Ayurveda medicine (Pandey et al. 2013). Gokshuru, Sweet
Vacha, and Yashtimadhu, Ashwagandha, Brahmi, Nagar-
motha, Bala, Prishnaparni Brihati, Shalaparni, Kantakari,
Shankhapushpi, etc. are some of the traditional medicines
used to cure PD (Gourie-Devi et al. 1991; Singh et al. 2018).
V. negundo Linn, a member of the Verbenaceae family,
is reported to have antiparkinson activity (Hu et al. 2018)
leaves V. negundo hydroalcoholic extract phytoconstituents
like Vitexin protects dopaminergic neurons (Hu et al. 2018),
triterpenes, diterpenes, sesquiterpenes, lignan, flavonoids,
flavones, glycosides, iridoid glycosides and stilbene deriva-
tive. The leaves extract has potential effects on improving
cognitive impaired in rats, through inhibiting lipid peroxi-
dation, decrease acetylcholinestrase inhibitory in brain, it
stimulates hair growth; it is useful for asthma, bronchitis,
biliousness, spleen enlargement, eye disease, leucoderma,
inflammation, and painful teething in infants (Dharmasiri
et al. 2003; Gill et al. 2018). The root is a snake venom
remedy (Alam and Gomes 2003).
The most common sign of PD include rigidity of move-
ments i.e. catalepsy, akinesia i.e. slowing of movements,
tremors, cognitive decline and memory impairments (Maz-
zoni et al. 2012).
According to the literature there is no scientific evidence
for V. negundo Linn against haloperidol induced PD. Halo-
peridol is a neuroleptic drug that by inhibiting the postsyn-
aptic D2 receptor antagonistic in mesolimbic pathway and
anticholinergic and β-adrenergic receptor antagonist (Li
et al. 2016) and leading to oxidatative stress by over activ-
ity causing neurodegenration. The V. negundo hydroalco-
holic extract phytoconstituents will ameliorate the halop-
eridol induced catalepsy in PD rats mainly in invivo model
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Metabolic Brain Disease (2022) 37:411–426
of cataleptic and altered locomotion and rigidity of mus-
cle along with cognitive and memory enhancer activity is
accessed by rotarod, actophotometer, morris water maze of
extraceptive and interceptive models. Whereas Invitro the
whole brain estimations of Dopamine, Acetylcholinestrase
inhibitory activity and Antioxidant like GSH, LPO, Total
protein, SOD etc. histopathological evidence of architecture
and integrity of neuronal cell in CA1 and CA3 of hippocam-
pal and cortex.
Materials and methods
Chemicals and equipment’s
Chemicals: Heloperidol (PD [Catalepsy] Inducing agent),
Levodopa (Standard molecule for PD), Acetylthiocholine
iodide (Sigma USA), Buterylthiocholine iodide (Sigma
USA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Dopamine,
DTNB (Sigma USA) and other chemicals utilized in this
study are analytical graded. Equipment’s: Elisa plate reader
(Thermo Scientific multiskan GO version 1.00.40), Centri-
fuge (Remi), Morris Water Maze, Actophotometer, T-Maze,
NOR apparatus, Cook’s pole climbing, Rotarod, Rotary
evaporator and many more.
Collection, authentication, and preparation of plant
material
The leaves of V. negundo L. gathered from Bailhongal, Bela-
gavi, Karnataka and authenticated by the plant taxonomist
from ICMR-NITM, Belagavi. Accession number: RMRC
– 1583. The collected plant specimen was stored for her-
barium for future reference. The fresh plant material was
washed properly using running water and to remove dust
particles and shade dried at room temperature. The dried
plant material was further crushed into the coarse powder
using a grinder and subjected to the extraction process.
Extraction: The dried coarse powder 1 kg was subjected
to the maceration technique and hydroalcoholic solvent
(Ethanol: Water 7:3) was used as a solvent. The mixture
was kept for 7 days with occasional shaking. After 7 days,
the solvent was filtered out and concentrated under reduced
pressure at 45-55 °C using a rotary evaporator. The obtained
extract was sticky, which was further air-dried, collected in
an amber color glass container, and stored in the refrigerator
for future use. The % yield of extracts was calculated using
the following equation,
weight of dry extract (g)
%Yield = X 100
Weight of dry plant (g)
Metabolic Brain Disease (2022) 37:411–426
In vitro acetylcholinesterase enzyme inhibitory
assay
AChE enzyme inhibitory assay was performed by Ellman’s
method (Ellman et al. 1961). Initially, prepared stock solu-
tion of extract (1 mg/mL) and Donepezil (1 mg/mL). Using
this, prepared 10, 20, 40, 80, 160 and 320 μg/ mL of plant
extract and 1, 2, 4, 8, 16 and 32 μg/ mL of Donepezil. Fur-
ther, in a test tube, added 50 mM Tris HCl buffer 1.7 ml
(pH 8.0) and 250μLof different concentration of extract and
Donepezil, separately. To this, added 10 μL of 6.67UmL1
AChE enzyme and 20 μL of DTNB. This mixture was
incubated for 15 min and after incubation added 10 μL of
Acetylthiocholine iodide and the absorbance was read at
412 nm every 45 s for 3 min. The % inhibition was calcu-
lated from change in Abs with respect to change in time.
The ­IC50 was calculated between the inhibition percentage
v/s extract concentrations.
Sample Abs
%Inhibition = 100 −
Blank Abs
X 100
Experimental animals
To evaluate the anti-PD capacity of V. negundo, we used
male Wistar rats having 180-200gweight and were procured
from the In vivo Biosciences, Bangalore. All the animals
were housed in a clean and transparent polypropylene cage
and were randomized through making into six groups (N = 6
and n = 6). All the animals were maintained under 12/12 h
natural light-dark cycle at room temperature and the rela-
tive humidity was maintained at 45–55%.The overall study
protocol was reviewed and approved by the Institutional Ani-
mal Ethical Committee, KLE College of Pharmacy, Bela-
gavi, Resolution No- KLECOP/CPCSEA- Reg.No.221/Po/
Re/S/2000/CPCSEA, Res.25–13/10/2020.
The model used for PD The chronic administration of Halo-
peridol (1.25 mg/kg i.p.) causes symptoms like PD mainly
catalepsy. In the current study, we administered Haloperidol
for 21 days based on the literature survey (Naidu et al. 2003).
In vivo experimental study design
The dose of the plant extract is selected based on the acute
toxicity study reports (­LD 50: 2000 g/kg, b.wt of rats.)
(Tandon and Gupta 2004; Aiyalu et al. 2015).
Control group animals received normal food and water
throughout the experiment. Negative control (NC) group
received Haloperidol (1.25 mg/kg) i.p. suspended in 1%v/v
tween 80 for 21 days. Positive Control (PC) group received
413
Haloperidol (1.25 mg/kg) i.p + L-DOPA (6 mg/kg) i.p. for
21 days. VNL100, VNL200, and VNL400 group received
Haloperidol (1.25 mg/kg) i.p. along with extract (100, 200,
and 400 mg/kg p.o.) for 21 days. Before initiating the study,
all the animals were subjected to exteroceptive behavioral
models using Rotarod, MWM, CPC, T-maze, NOR, and
Actophotometer for acquisition. After subjecting to the
test agent, changes in the latency were examined on 0th
(first dose), 7th, 1­ 4th, and 21st day. After completion of the
in vivo studies, rats were euthanatized, brains were isolated
and cerebral cortex, hippocampus AChE and BChE enzyme
activity and whole-brain Dopamine, LPO, reduced GSH,
MDA, TP, SOD, CAT, and Glutathione level were measured.
In vivo screening models for PD
Rotarod apparatus
The rotarod test was performed as per Hamm et al. (1994)
method to test the grip power. The rotarod test is often used
in rats to determine their “minimal extrapyramidal deficit,”
such as muscle control and balance. Before starting the
therapy, each rat was given a training session to acclimate
them to the rotarod apparatus. The animal was placed on a
rotating rod with a diameter of 7 cm and a speed of 25 rpm
(rpm). Following drug/test sample administration, each rat
was subjected to three independent trials at 2 min intervals
on day 0th, 7th, 14th, and 21st with a 90 s cut-off period
retained during the experiment.
Morris water maze
MWM test was performed as per (Biradar et al. 2020; Ishola
et al. 2018) method. MWM is a large circular water tank
with a white surface [diameter 110 cm, height 60 cm] that
is filled with water (temperature 26-20 °C) to a depth of
30 cm. The MWM tank circle is divided into four equal
quadrants [North Q1, East Q2, West Q3, and South Q4].
It also has a stationary base with a diameter of 10 cm.The
escape latency time (ELT) of the individual rat was noted at
60s cut off time.
Locomotor activity by actophotometer
Individual rat locomotion was measured using an actopho-
tometer with a 5 min cut-off period. A photocell is attached
to a circuit with a counter in this device. The moving animal
cuts off the light beam going through the photocell, and a
count is taken. Each animal readings were taken on the 0th,
7th, 14th, and 21st days. To stop the animal odour, the cham-
ber was cleaned with 10% ethanol before the experiment
began (Goverdhan et al. 2012).
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Cook’s pole climbing
The escape latency/conditional avoidance of each rat was
screened by CPC. The ground part consisting of rods that acts
as shock. First, individual rats were trained and readings were
noted acquisition and the retention trail were noted 0th, 7th,
14th, and 21st day. The cut-off time of 120 s is considered for
evaluation (Goverdhan et al. 2012).
Novel object recognition
This test was performed as described by (Antunes and Biala
2012) method. The test includes both acquisition and retention
trials. During acquisition trial, two identical objects (green
balls) were placed equidistant from each other. Further, for 60s
animals were placed in the arena and then placed back in the
cage. The process was repeated for all the animals for 7 days
(training period). During this trial, to avoid the bias for par-
ticular location of identical and novel objects, we interchanged
the objects from left to right. After training, the same method-
ology was performed for retention trail. Here, one green ball
(identical object) is replaced with red ball (novel object) and
the time taken to explore an object was noted. Object explora-
tion recording includes: a) rat head to orient towards the object
within a distance of 2 cm. b) animal to sniffing, touching, and
licking the object with the paws.
The discrimination index (DI in %) was calculated by using
DI (%) = (A1-A2) / (A1 + A2) and DI (%) = (A1-B)/(A1 + B)
formula. Where, A1 is time spent with familiar object 1; A2 is
time spent with the familiar object 2 (A2); and B is Time spent
with the novel object.
T‑maze
This test was performed according to the method of Wenk
1998. The T-Maze test involves both acquisition and reten-
tion sessions. Initially, animals were trained for 7 consecutive
days. After that on 0th day (Treatment period) food was placed
in one side arm (left arm we selected) before beginning the
experiment. The overnight fasted animal is placed in the start
box for 60 s. Then, slowly sliding door was opened noted the
number of entries in the left arm (right choices). Similarly, the
tests were repeated on day 12th and 21st day.% Alternation was
calculated by dividing the Number of entries into the left arm
in the acquisition and during treatment period.
Determination of biomarkers level
Acetylcholinesterase and Butyrylcholinesterase enzyme
level
The test is carried out by Ellman et al. 1961 method. Cortex
and hippocampus were isolated and placed in an ice-cold
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Metabolic Brain Disease (2022) 37:411–426
water container to stop the enzymatic reactions. Weighed
appx. 100 mg and homogenate in Phosphate buffer [0.1 M;
pH 8.0]. To 0.4 mL above mixture (homogenate), (i) added
2.6 ml 0.1 M (pH 8) Phosphate buffer (ii) 100 μl of 10 mM
DTNB and mixed well. To the above mixture, added 20 μl
of AChI (0.075 M) [Note: AChI is replaced with BChI
to estimate BChE enzyme activity]. The mixture absorb-
ance was noted at 412 nm for 5 min.The rate of moles
of substrate hydrolyzed/min/g of tissue is calculated by
R = 5.74 × ­10−4(ΔA/Co). Where, R is Rate, in moles of sub-
strate hydrolyzed per min per gram of tissue; ΔA is change
in Abs per min. Co is Concentration of tissue (mg/ml).
Determination of LPO
Ohkawa et al. 1979; Ramakrishnan et al. 2015 method was
used to estimate the level of MDA in the whole brain. 0.2 ml
of tissue homogenate is added to8.1% w/v 0.2 ml of sodium
lauryl sulphate (SLS), 1.5 ml of 20% acetic acid, and 1.5 ml
(0.8%) thiobarbituric acid. The mixture is vortexed for one
minute and diluted up to 4 ml using DM water. Further, the
mixture is boiled at 90 °C for 1 h using the water bath. The
whole mixture was cooled and added 1 mL of DM water
and 5 ml mixture of pyridine:n-butanol (1:15 v/v). Finally,
centrifuged at 4000 rpm for 10 min. The Abs of the organic
layer was noted at 532 nm. The cerebrum malondialdehyde
(MDA) ­mg−1 of protein level was estimated and expressed
in nanomoles using the formula, MDA = nmoles of MDA/
mg of protein in the cerebrum is,
Concentration = A × (V∕E) × P
where, A = Absorbance at 535 nm; V = Volume of mixture;
E = Extinction coefficient (1.56 × ­105 m/cm); P = mg of pro-
tein per g of tissue. All the values were indicated in nM of
MDA/mg of protein.
Determination of reduced GSH
Ellman G. L. method (Ellman 1959) was utilized to estimate
whole-brain reduced GSH level. 0.25 ml of tissue homoge-
nate, 2.5 ml sodium phosphate buffer, and 50 μl DTNB
[pH 7.0] was mixed. The mixture was further vortexed and
incubated at 25-27 °C for 2 min. Within 15 min of this step,
the absorbance was noted at 412nmand expressed GSH level
in μmoles/mg of tissue.
where, ­C0 is the original concentration; A is Absorbance at
412 nm; € is molar extinction coefficient i.e. 13,600/M/cm;
D is the dilution factor.
Metabolic Brain Disease (2022) 37:411–426
Determination of Total protein
Briefly, added 2.25 ml of 0.5 M NaOH to 0.25 ml of tis-
sue homogenate. Then, pipette out 0.5 ml from the above
mixture and added 0.5 ml of 10% TCA and centrifuged at
3000 rpm for 10 min at 4 °C. After this, discarded the super-
natant and the remaining precipitate is dissolved in 0.5 ml of
0.5 M NaOH and 2 ml of Alkaline mixture. Further, waited
for 10 min and added 0.25 ml of Folin–Ciocâlteu reagent and
again waited for 10 min. Finally, the absorbance of mixture
was noted at 540 nm (Sedlak et al. 1968; Shalavadi et al.
2012).
Determination of SOD
The capacity of SOD to antagonize the autooxidation of
epinephrine to adrenochrome in the presence of alkaline
pH. To the 25 μl of homogenate sample, added 0.1 mM of
epinephrine in carbonate buffer (pH 10.2). At 295 nm, the
adrenochrome formation in the above mixture was measured
using the ELISA plate reader. Further, the SOD level was
determined from the total protein value and expressed in U/
mg of protein (Misra and Fridovich 1972).
Determination of catalase
Mixed 50 μl of tissue homogenate with 1.95 ml of PBS
7.0pH and 1 ml of 0.7 mM ­H2O2 solution. Read the absorb-
ance at 240 nm. Further, the Catalase level was determined
from the total protein value and expressed in U/mg of protein
(Claiborne 1985; Shalavadi et al. 2012).
Determination of dopamine level
The level of dopamine in the whole brain was estimated via
UV-Visible spectroscopy. First, the standard linear curve of
dopamine was removed via serial dilution of standard dopa-
mine (50 to 500 ng/mL). The detection range of dopamine
was set to 240-280 nm and the linear curve was obtained for
conc. v/s abs. The supernatant obtained from each test group
was diluted 10 times and the absorbance was read at 278 nm
to detect the dopamine. Using the y = mx + c obtained equa-
tion from the linear curve and the sample absorbance, the
whole brain dopamine level was estimated and expressed in
ng/g of tissue (Manoharan et al. 2016).
Statistical analysis
The results were expressed as Mean ± SEM/SD. One-way
and two-way ANOVA followed by Bonferroni and Tukey’s
comparison tests were used to determine the difference
between the groups in GraphPad Prism v5.0. The degree
of freedom of 5% with a confidence interval of 95% was
415
applied for all the tests. *p < 0.05, **p < 0.01, ***p < 0.001,
and ****p < 0.0001 is compared to Normal group; #p < 0.05,
##p < 0.01 ###p < 0.001, and ****p < 0.0001 is compared to
Haloperidol induced PD group.
Results
Extract yield
The hydroalcholic extract obtained from leaves part is 48 g
from 500 g of raw material. The percentage yield (w/w) of
V. negundo extract by maceration method was found to be
9.6%.
Effect of E. variegata hydroalcoholic extract
on in vitro AChE inhibitory
The ­IC50 of Donepezil was found to be 10.23 μg/ml and the
­IC50 of V. negundo hydroalcholic extract was found to be
114.35 μg/ml. The AChE inhibitory activity of Donepezil
was found to be more potent compared to V. negundo. The
% inhibition and ­IC50 value are shown in Fig. 1 and the
obtained linear equation and absorbance are given at Sup-
plementary Table 1.
In vivo studies
Effect of V. Negundo hydroalcholic extract on performance
time (grip strength) using rotarod
During the acquisition trial, 0th day and on the 7th day
of treatment with Haloperidol group, L-DOPA group,
VNL100, VNL200, and VNL400, the performance time
was in the range of 15 s to 23 s, and no significant dif-
ference was observed. However, on the 14th day, the NC
group exhibited a decline (p < 0.05) in the performance
compared to Control. Whereas, treatment with L-DOPA,
VNL100, and VNL200 didn’t showed effect on perfor-
mance time but VNL400 exhibited rise in performance
(p < 0.01) compared to Negative control (NC) group on
14th day. Further, on 21st day, the entire treatment group
exhibited rise (p < 0.0001) in the performance time com-
pared to Negative control (NC) group that showed decline
(p < 0.0001) in the performance time. The effect of VNL
on performance time is shown in Fig. 2 and Supplementary
Table 2
Effect of V. negundo hydroalcholic extract on ELT using
MWM
During the acquisition trial and on the 0th day of first
treatment with Haloperidol group, L-DOPA group,
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416
Fig. 1  Effect of Vitex negundo
hydroalcoholic extract with
Donepezil inAChE inhibitory 150
assay
% Inhibition
100
50
0
0 100
Fig. 2  Effect of Vitex negundo on grip strength using rotarod for 90s cut off time. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 is
compared to Normal group; #p < 0.05, ##p < 0.01 ###p < 0.001, and ****p < 0.0001 is compared to Haloperidol induced PD group
VNL100, VNL200, and VNL400, the ELT was 30s
to 35 s, and no significant difference was observed.
However, on the 7th day and 14th day, the Negative
control (NC) group exhibited a rise (p < 0.05) and
(p < 0.01) in the ELT respectively, whereas, treatment
with L-DOPA group (p < 0.05), VNL100 (p < 0.01),
VNL200 (p < 0.05), VNL400 (p < 0.01) showed a
decline in the ELT. Further, on the 21st day a significant
(p < 0.0001) increase in ELT was observed in the Nega-
tive control (NC) group (ELT: 50s) compared to con-
trol and on treatment with L-DOPA group (p < 0.0001),
VNL100 (p < 0.0001), VNL200 (p < 0.0001), VNL400
(p < 0.0001) reversed the ELT increased by the Halop-
eridol treatment. All the treatment doses showed a good
effect on ELT compared to Haloperidol group. The
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Metabolic Brain Disease (2022) 37:411–426
In vitro AChE inhibitory activity
Donepezil
V. negundo
200 300 400
Conc. (µg/ml)
effect of VNL on ELT is shown in Fig. 3. Supplemen-
tary Table 3
Effect of V. negundo hydroalcholic extract on locomotion
using actophotometer
The effect of V. negundo on ELT was screened via CPC.
During acquisition and 0th day, no significant differ-
ence in ELT is observed in all the groups. Further, on
7th day, a significant increase in the Negative control
(NC) group (p < 0.01) is seen compared to the control
group. However, on 14th day, the ELT in the Negative
control (NC) group was significantly raised to p < 0.01
compared to the control group and but no significant
difference is in treatment group. On 21st day, the ELT
Metabolic Brain Disease (2022) 37:411–426 417

Fig. 3  Effect of Vitex negundo hydroalcholic extract ELT using ###p < 0.001, and ****p < 0.0001 is compared to Haloperidol
MWM for 60s cut off time.*p < 0.05, **p < 0.01, ***p < 0.001, and induced group
****p < 0.0001 is compared to Normal group; #p < 0.05, ##p < 0.01

in the Negative control (NC) group was significantly
raised to p < 0.0001 compared to the control group and
treatment with L-DOPA group and VNL100 (p < 0.001),
VNL200 and VNL400 (p < 0.0001) decreased the ELT
compared to the Negative control (NC) group. The effect
of V. negundo on ELT in CPC is illustrated in Fig. 4
Supplementary Table 4.
Effect on V.negundo on ELT using CPC for cut‑off
time of 120 s
Effect of V. negundo hydroalcholic extract on ELT using CPC
The effect of V. negundo on ELT was screened via CPC.
During acquisition and 0th day, no significant difference

Fig. 4  Effect of Vitex negundo

hydroalcholic extract locomo-
tion using actophotometer for
5 min cut-off period.*p < 0.05,
**p < 0.01, ***p < 0.001, and
****p < 0.0001 is compared
to Normal group; #p < 0.05,
##p < 0.01 ###p < 0.001, and
****p < 0.0001 is compared to
Haloperidol induced group

13
418
in ELT is observed in all the groups. Further, on 7th day,
a significant increase in the Negative control (NC) group
(p < 0.01) is seen compared to the control group. However,
on 14th day, the ELT in the Negative control (NC) group
was significantly raised to p < 0.01 compared to the control
group and but no significant difference is in treatment group.
On 21st day, the ELT in the Negative control (NC) group
was significantly raised to p < 0.0001 compared to the con-
trol group and treatment with L-DOPA group and VNL100
(p < 0.001), VNL200 and VNL400 (p < 0.0001) decreased
the ELT compared to the Negative control (NC) group. The
effect of V. negundo on ELT in CPC is illustrated in Fig.5
and Supplementary Table 5.
Effect of Vitex negundo hydroalcholic extract on novel
object recognition
The effect of V. negundo on motor and memory is evaluated
via NOR by discrimination index for both A1-A2 and A1-B1
Fig. 6 (Supplementary Table 6).
Observations of novel object recognition: A1‑A2 on 0th
day No significant difference is observed among the entire
group for time spent with A1-A2. A1-A2 on 21st day: No
significant difference is observed among all the group for
time spent with A1-A2.A1-B on 0th day: A significant
increase in novel object recognition (time spent with object
B) was observed in Control (ns), NC (p < 0.001), VNL100
((p < 0.01), VNL200 (p < 0.01), and VNL400 (ns) compared
to A1 object. A1-B on 21st day: A significant increase in
novel object recognition (time spent with object B) was
Fig. 5  Effect on V.negundo on ELT using CPC. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 is compared to Normal group;
#p < 0.05, ##p < 0.01 ###p < 0.001, and ****p < 0.0001 is compared to Haloperidol induced group for 120 s
13
Metabolic Brain Disease (2022) 37:411–426
observed in control (p < 0.01) and VNL400 (p < 0.01) com-
pared to object A1. Whereas, Negative control group(NC),
Positive control group (PC), VNL100, and VNL200 group
animals didn’t showed significant increase in novel object
recognition (Fig. 6a–d) and supplementary Table 6.
Discrimination index of novel object recognition: A1‑B on
0th day No significant difference of DI was observed among
all the groups. A1-B on 21st day: A significant decrease
in DI was seen in Negative control (NC) group (p < 0.01)
compared to Control group. However, Positive control
group (PC) (ns), VNL100 (ns), VNL200 (ns), and VNL400
(p < 0.01) showed significant increase in the DI on 21st day
(Fig. 6e,f).
The effect of V. negundo hydroalcholic extract
on T‑maze
The effect of V. negundo on percentage alteration was
screened via T-maze. During 0th and 12th day, no signifi-
cant difference in percentage alteration is observed in all
the groups. However, on 21st day, significant decrease in
percentage alteration in the Negative control (NC) group
(p < 0.01) was seen compared to the control group. How-
ever, percentage alteration in the L-DOPA group, VNL100,
VNL200, and VNL400 was significantly raised to p < 0.05,
p < 0.05, p < 0.01, and p < 0.001 respectively compared to
the NC group. The effect of V. negundo on percentage altera-
tion in T-maze is illustrated in Fig. 7 and Supplementary
Table 7.
Metabolic Brain Disease (2022) 37:411–426 419

Fig. 6  Effect of Vitex negundo on novel object recognition. a) A1-A2 *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 is com-
on 0th day b) A1-A2 on 21st day, c) A1-B on 0th day, d) A1-B on pared to Normal group; #p < 0.05, ##p < 0.01 ###p < 0.001, and
21st day, e) DI of A1-B on 0th day, and f) DI of A1-B on 21st day. ****p < 0.0001 is compared to Haloperidol induced PD group

Fig. 7  Effect of Vitex negundo

on percentage alteration
using T-Maze. *p < 0.05,
**p < 0.01, ***p < 0.001,
and ****p < 0.0001 is
compared to Normal con-
trol group; #p < 0.05,
##p < 0.01 ###p < 0.001, and
****p < 0.0001 is compared to
Haloperidol induced PD group

In vitro biochemical estimation The treatment of Haloperidol in the Negative control

(NC) group remarkably raised the level of AChE in the
Effect of V. negundo hydroalcholic extract cortex (p < 0.001) and hippocampus (p < 0.001) compared
on acetylcholinesterase inhibitory activity (Hippocampus to the control group. However, treatment with L-DOPA,
and cerebral cortex) VNL100, VNL200, and VNL400 remarkably declined the
level of AChE in the cortex and hippocampus compared to
A) Effect on AChE level the Negative control (NC) group. All three treatment groups
of V. negundo exhibited a potent inhibitory effect on AChE.

13
420
The effect of V. negundo on the hippocampus and cerebral
cortex AChE enzyme activity is shown in Supplementary
Table 8 and Fig. 8a and b, respectively.
B) Effect of V. negundo hydroalcholic extract on BChE
level
The treatment of Haloperidol in the Negative control
(NC) group remarkably increased the level of BChE in the
cortex (p < 0.001) and hippocampus (p < 0.001) compared
to the control group. However, treatment with L-DOPA
group showed remarkable declined effect on BChE level
in both cortex (p < 0.0001) and hippocampus (p < 0.0001)
compared to the Negative control (NC) group. Whereas,
treatment with VNL100, VNL200, and VNL400 remark-
ably decreased the level of BChE in the cortex (p < 0.0001)
and hippocampus ((p < 0.0001) compared to the Negative
control (NC) group. The effect of V. negundo on BChE
Fig. 8  Effect of Vitex negundo on brain AChE and BChE level. a)
AChE of Hippocampus, b) AChE of Cortex, c) BChE of Hippocam-
pus, and d) BChE of Cortex. *p < 0.05, **p < 0.01, ***p < 0.001, and
13
Metabolic Brain Disease (2022) 37:411–426
enzyme level in the hippocampus and cerebral cortex
is shown in Supplementary Table 9 and Fig. 8c and d.
respectively.
Effect of V. negundo hydroalcholic extract
on reduced GSH
The treatment of Haloperidol in the Negative control (NC)
group remarkably (p < 0.0001) reduced GSH level in the
whole brain compared to the control group. However, treat-
ment with L-DOPA group (p < 0.05) VNL200 (p < 0.0001),
and VNL400 (p < 0.0001) significantly increased the level
of GSH compared to the Negative control(NC) group. Fur-
ther, no significant change in GSH level is observed in the
VNL100 group compared to the NC group. The effect of V.
negundo on whole-brain GSH level is shown in Supplemen-
tary Table 10 and Fig. 9a.
****p < 0.0001 is compared to Normal group; #p < 0.05, ##p < 0.01
###p < 0.001, and ****p < 0.0001 is compared to Haloperidol
induced PD group
Metabolic Brain Disease (2022) 37:411–426
Fig. 9  Effect of Vitex negundo on whole brain antioxidant mark-
ers and dopamine level. a) GSH, b) MDA, c) TP, d) SOD, e) CAT,
and f) Dopamine level. *p < 0.05, **p < 0.01, ***p < 0.001, and
Effect of V. negundo hydroalcholic extract on MDA
level
The treatment of Haloperidol in the NC group remarkably
(p < 0.0001) raised MDA level in the whole-brain compared
to the control group. However, treatment with L-DOPA
(p < 0.05) VNL100 (p < 0.01), and VNL100 (p < 0.05),
VNL200 (p < 0.01), VNL400 (p < 0.001) remarkably
decreased the level of MDA compared to the NC group. The
effect of V. negundo on whole-brain MDA level is shown in
Supplementary Table 10 and Fig. 9b.
Effect of V. negundo hydroalcholic extract on TP
The treatment of Haloperidol group in the Negative control
(NC) group didn’t show a significant change in TP level but
showed a slight rise in TP level in the whole brain com-
pared to normal. However, treatment with L-DOPA group
showed, not significant but decline in the TP level com-
pared to the Negative control group (NC). Further, VNL100
and VNL200 didn’t show a change in TP level but a slight
decline in TP level was seen compared to the NC group. The
effect of V. negundo on whole-brain TP level is shown in
Supplementary Table 10 and Fig. 9c respectively.
Effect of V. negundo hydroalcholic extract on SOD
The treatment of Haloperidol in the Negative control (NC)
group decreased the SOD level in the whole cerebrum
compared to normal but data were found non-significant.
Although, L-DOPA group, VNL100 and VNL200 treatment
didn’t show any significant change in SOD level but sig-
nificant rise in SOD level was seen in VNL400 (p < 0.05)
compared to the Negative control (NC) group. The effect
of V. negundo on whole-brain SOD level is shown in Sup-
plementary Table 10 and Fig. 9d.
421
****p < 0.0001 is compared to Normal group; #p < 0.05, ##p < 0.01
###p < 0.001, and ****p < 0.0001 is compared to Haloperidol
induced PD group
Effect of V. negundo hydroalcholic extract on CAT​
The treatment of Haloperidol group in the Negative group
(NC) group didn’t show a significant change in CAT level
but showed a slight decrease in CAT level in the whole cer-
ebrum. However, L-DOPA treatment group showed its effect
on the CAT level but not significantly, whereas, VNL100
didn’t show any effect on CAT level. However, VNL200
and VNL400 showed not significant but a increase in CAT
level compared to the NC group. The effect of V. negundo on
whole-brain CAT level is shown in Supplementary Table 10
and Fig. 9e.
Effect of V. negundo hydroalcholic extract
on dopamine level
The treatment of Haloperidol group in the Negative control
(NC) group remarkably (p < 0.0001) decreased the Dopa-
mine level in the whole cerebrum compared to the control
group. However, treatment with L-DOPA group (p < 0.01),
VNL100 (p < 0.0001), VNL200 (p < 0.05) and VNL400
(p < 0.0001) remarkably increased the level of Dopamine
compared to the Negative control (NC) group. The effect
of V. negundo on whole-brain Dopamine level is shown in
Supplementary Table 11 and Fig. 9f.
Effect of V. negundo hydroalcholic extract
on histopathology of hippocampus and cortex
The histology of the control group cerebrum indicated mild
cerebral odema, cerebral congestion, neuronal eosinophilia,
meningeal congestion, and no change in anatomy was visu-
alized in RBC extravasation, macrophage influx, neuronal
micro vacuolisation, neuronal nuclear pyknosis, neutrophilic
infiltration, neuronal karyorrhexis, reactive gliosis, and vas-
cular proliferation. However, the Negative group (NC) group
13
422
histogram indicated severe damage in the above-mentioned
parameters (+++ to +). The VNL400 group reversed the
Haloperidol damaged parameters. The histopathology of the
brain (hippocampus and cortex) at 10x and 40x magnifica-
tion is shown in Figs. 10 and 11 respectively and Supple-
mentary Table 12.
Discussion
This study carried out anti-PD activity of V. negundo of
hydroalcholic extract in Haloperidol induced PD in rats
through the analysis of motor and memory function. In vivo
studies were carried out through Rotarod, CPC, MWM,
Actophotometer, NOR, and T-Maze apparatusand cerebrum
biochemicals (AChE, BChE, GSH, MDA, TP, SOD, CAT,
Dopamine) level were estimated. The overall in vivo experi-
ment includes two test 1) acquisition and 2) retention. All
the animals were trained for their acquisition and training to
each apparatus previously before study. The rotarod appa-
ratus was utilized to assess neuromuscular motor coordina-
tion and grip strength of muscle. It is well known that, PD
patients are associated with lower grip strength (Roberts
et al. 2015). In this study, inducing agents of Haloperidol ­D2
receptor antagonistic remarkably declined the performance
time from ­14thto 21stdays, which indicated the reduced grip
strengthand extrapyramidal activity of PD. Treatment with
Fig. 10  Effect of Vitex negundo on hippocampus. a) 10x b) 40x.
1) Control 2) Negative control (NC) 3) Positive control (PC) 4)
VNL100, 5) VNL200, 6) VNL400. CA1-Cornu ammonis neurons
13
Metabolic Brain Disease (2022) 37:411–426
L-DOPA and V. negundo of hydroalcholic extract 200 mg/kg
and 400 mg/kg exhibited a remarkable decline in retention
time. Further, MWM was utilized to check “spatial mem-
ory and learning” and also to check the motor coordination
(Vorhees and Williams 2006). The ELT of the Haloperidol
treated rats raised remarkably in ELT compared tostand-
ard of L-DOPA and V. negundo of hydroalcholic extract of
three doses, exhibited a critical decline ELT. As a result of
these findings, it is suggested that V. negundo hydroalcholic
extract changes the rat motor and memory activity. Loco-
motion activity using actophotometer (Mazzoni et al. 2012;
Greenland and Barker 2018) was remarkably declined from
7 to 21st day in Haloperidol treated rats, reduce the locomo-
tion and were reversed by L-DOPA and V. negundo hydroal-
cholic extract treatment for 21 days lead to a remarkable rise
in locomotion. Further, CPC was used to investigate cogni-
tive function, primarily a response to conditioned stimuli
to access the coordination of movements, grip strength to
hold the pole, learning and memory (Kamila et al. 2014)
is compared with Haloperidol induced group, exhibited a
significant rise in ELT on day 21st and whereas NVL and
L-DOPA significantly reversed via decreasing ELT in CPC.
Further, to analyse the recognition memory and animals
fast moments, we utilized the NOR apparatus (Cole et al.
2019). Compared with NC with NVL and L-DOPA groups
exhibited a remarkably raised the discriminatory index, time
to recognise the novel object and movements were dose
(Hippocampus) CA3-Cornu ammonis neurons (Hippocampus). NN-
Normal Neutrophile, A PMR- Aggressions of PMR, INF-Infiltration
KY- Karyohexsis, INF-Infiltration and L INF-Less infiltration
Metabolic Brain Disease (2022) 37:411–426
Fig. 11  Effect of Vitex negundo on cortex. a) 10x b) 40x. 1) Con-
trol 2) Negative control (NC) 3) Positive control (PC) 4) VNL100,
5) VNL200, 6) VNL400. NN-Normal Neutrophile, C BI V C- Com-
dependent action and highly significance in NVL 400 mg/kg.
Treatment with Haloperidol exhibited a remarkable decline
in percentage alternation on day 2­ 1st and treatment with V.
negundo remarkably raised the percentage alternation and
movements. These findings suggest that V. negundo protects
against Haloperidol induced PD and associated compliances
i.e. memory loss.
Further, through the in vitro biochemical analysis meth-
ods, cholinesterase’s function in hippocampus and cor-
tex, GSH, MDA, TP, SOD, CAT, and Dopamine level in
the cerebrum were examined. It is well known that, the
level of ACh and BCh chemicals associated with memory
improvement, and this is diminished in PD because of
fast hydrolysis by AChE and BChE action respectively
(Lane et al. 2006; Cacabelos 2007). In the present study,
initially, we performed AChE inhibitory activity of V.
negundo hydroalcholic extract. The results suggested that
V. negundo hydroalcholic extract possess an AChE inhibi-
tory potency compared in terms of I­ C 50(μg/ml)values.
However, V. negundo hydroalcholic extract of different
groups on 2­ 1stday exhibited a remarkable highly signifi-
cantly reduction in the AChE and BChE activity in both
cortex and hippocampus, which suggests that V. negundo
possess AChE and BChE inhibitory activity on par with
standard L-DOPA group. This indicates a higher amount
of ACh retention i.e. responsible for the motor function. A
423
pressed Blood Vessel, PY-Pyknosis, ND-Neuro degeneration, INF-
Infiltration, KY- Karyohexsis, KL- karyolysis
previous study by Rahiman et al. (2015) demonstrated V.
negundo leaf extract to pose AChE inhibitory activity in
scopolamine treated rats. (Otari et al. 2012) also revealed
the effectiveness of V. negundo leaf extract in enhancing
learning and memory processes through the inhibition of
AChE and increase in cholinergic transmission and neu-
rotransmitter balanced for coordination of movements.
Hence, V. negundo leaf compounds play major role in
inhibition of both AChE and BChE in cerebrum.
Free radicals and reactive oxygen species (ROS) are
produced during physiological and pathological changes.
Among other cell signalling pathways, ROS have a role in
phagocytosis, enzyme activation, and cell cycle regulation
(Brieger et al. 2012; Di Meo et al. 2016). An imbalance
in free radical and ROS formation weakens the antioxidant
defence system, resulting in cell damage and inflammation
within the brain tissue. Superoxide O˙2, ­H2O2, and reactive
nitrogen speciesare the primary free radicals involved in oxi-
dative stress and are key component of the development of
neurodegenerative diseases PD (Kumar et al. 2012). Further-
more, the generation of ROS causes a variety of undesirable
effects, including DNA harm, lipid peroxidation aberrations,
and protein mutilation (Biradar et al. 2020). In the present
study, administration of V. negundo hydroalcoholic extract
acts as a scavenging actions and antioxidant markers viz.,
GSH, MDA, SOD, TP, and CAT level in the whole brain.
13
424
The results suggest, V. negundo leaf as a potent anti-oxidant
herb and pose a potential ROS neutralizing capacity.
As part of the basal ganglia circuitry, the substantia nigra
(SN) is a midbrain dopaminergic nucleus that plays a cru-
cial role in regulating motor activity and reward functions
(Sonne et al. 2021). Dopamine, a neurotransmitter involved
in reward motivated actions and aids in the regulation of
expression and the formation of motor coordination and new
memories (Triarhou 2013). The SN’s Dopamine neurons
deteriorate over time in PD, reducing the quantity of DA
available for neurotransmission in the corpus striatum. Rest-
ing tremor, stiffness, bradykinesia, poor balance, and motor
coordination are all common clinical signs of a PD (Triarhou
2013). In the present study investigation, Haloperidol D ­ 2
receptor antagonist administration significantly decreased
the level of Dopamine and treatment with L-DOPA and V.
negundo hydroalcoholic extract increased the dopamine
level in brain.
Histopathology examination of the brain of the nor-
mal animals showed no significant changes in the nor-
mal group. While, Haloperidol treated rats brain showed
moderate cerebral congestion, odema, microvacuolisa-
tion, Gliosis and moderate meningeal congestion, nuclear
pyknosis,karyorrhexis, neutrophilic infiltration, RBC
extravasation, and vascular proliferation. However, treat-
ment with in the V. negundo hydroalcoholic extract 400 mg/
kg reversed the damage induced by Haloperidol. Treatment
with L-DOPA and V. negundo hydroalcoholic extract groups
maintained the CA1 and CA3 neurons of posterior part of
limbic system integrity, morphological and cellular architec-
ture of neuronal. Also, various investigations demonstrated
of V. negundo hydroalcoholic extract, believes the bioactive
phytoconstituents mainly of vitexin will ameliorate the halo-
peridol induced PD in rats. Herein, we suggest proposing the
molecular mechanism of V. negundo against PD as using
network pharmacology and molecular docking as explained
by Duyu et al (2020) which is the future scope of this study.
Conclusion
The current study identified V. negundo leaf hydroalco-
holic extract act as a potent herbfor PD through in vivo
and in vitro experiments, PD symptoms like neuromotor
co-ordination by rota-rod, movement by locomotion, recog-
nition i.e. Memory and cognitive function through MWM
and so on. In vitro studies, V. negundo leaf hydroalcoholic
extract Phytoconstituents vitexine inhibits AChE and BChE
in cortex and hippocampus, increases Dopamine level, and
possess a potent antioxidant potency. The current study data
were comparable to a standard molecule L-DOPA. Herein,
based on the current study reports, V. negundo can be taken
for further studies to identify key component responsible for
13
Metabolic Brain Disease (2022) 37:411–426
enzyme inhibition, to explore the possible mode of action
and protein expression analysis, which helps to explore V.
negundo leaf as a herbal therapy for PD.
Abbreviations V. negundo: V. negundo; VNL100 : V. negundo 100 mg/
kg Group; VNL200 : V. negundo 200 mg/kg Group; VNL400 : V.
negundo 400 mg/kg Group; ACh : Acetylcholine; AChE : Acetylcho-
linesterase; AChI : Acetylthiocholine iodide; BChI : Butyrylcholines
iodide; AD : Alzheimer’s Disease; PD : Parkinson’s Disease; APP
: Amyloid Precursor Protein; Aβ : Amyloid Beta; BCh : Butyrylcholine;
BChE : Butyrylcholinesterase; DPPH : 2,2-diphenyl-1-picrylhydrazyl;
DTNB : 5,5′-dithio-bis(2-nitrobenzoic acid); ELT : Escape Latency
Time; GSH : Glutathione; H2O2 : Hydrogen Peroxide; i.p: Intraperito-
neal; IC50 : Inhibition Concentration; LPO : Lipid Peroxidation; MDA
: Malondialdehyde; MWM : Morris Water Maze
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11011-0​ 21-0​ 0878-2.
Acknowledgements Thanks to the Prof.(Dr) Sunil S Jalapure, Prin-
cipal and Prof (Dr.) N A Khatib, HOD, Department of Pharmacology
and Toxicology, KLE College of Pharmacy, KAHER, Belagavi, India
for providing necessary facilities to conduct the work. The authors
thank the Vamsi Labs Ltd. Solapur, India for providing Haloperidol
and Micro Labs Ltd. Bangalore for providing L-DOPA as a gift sample.
Author’s contribution Prakash R. Biradar supervised the experiments,
confirmed the findings, helped in data analysis, helped in the man-
uscript writing. Aishwarya Vannur design the study, conducted the
experiment, collected the data, drafted the manuscript. Vishal S. Patil
statistical analysis and helped in manuscript drafting.
Funding This study has not having Funding from any Agencies.
Data availability The data are not publicly available due to their con-
taining information that could compromise the privacy of research
participants, but are available on request from the author.
Declarations
Ethical statement The study protocol was reviewed and approved by
the Institutional Animal Ethical Committee, KLE College of Pharmacy,
KAHER, Belagavi, and Resolution No. KLECOP/CPCSEAReg. No.
221/Po/Re/S/2000/CPCSEA, Res. 25–13/10/2020. The animal experi-
ments were carried out in accordance with the CPCSEA guidelines.
Conflict of interest All the authors of this manuscript confirm that they
do not have any conflict of interest.
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