NeuroMolecular Medicine

https://doi.org/10.1007/s12017-019-08566-2

ORIGINAL PAPER

Butanol Extract of Tinospora cordifolia Ameliorates Cognitive Deficits

Associated with Glutamate‑Induced Excitotoxicity: A Mechanistic
Study Using Hippocampal Neurons
Anuradha Sharma1 · Shikha Kalotra1 · Payal Bajaj1 · Harpal Singh1 · Gurcharan Kaur1 

Received: 1 April 2019 / Accepted: 21 August 2019

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Overstimulation of glutamate receptors leads to development of excitotoxicity, which is implicated as final destructive path-
way in neurodegenerative diseases. Development of alternative therapeutic strategies effective against glutamate-induced
excitotoxicity is much in demand. Herbal drug development is emerging as a major research area for the treatment of vari-
ous debilitating diseases due to multimodal action and least side effects of herbal products. The current study was aimed to
investigate neuroprotective potential of butanol extract of Tinospora cordifolia (B-TCE) against glutamate-induced excito-
toxicity using primary hippocampal neurons as in vitro and Wistar strain albino rats as in vivo model systems. Molecular
and behavioral parameters were studied to elucidate the underlying mechanism of beneficial effects of B-TCE. B-TCE treat-
ment was also effective in prevention of anxiety, cognition, and motor-coordination deficits induced by glutamate. B-TCE
pre-treatment protected the hippocampal neurons from glutamate-induced neurodegeneration and impaired plasticity. At
molecular level, B-TCE was observed to attenuate overactivation of glutamate receptors. B-TCE promoted upregulation of
ERK and AKT pathways of synaptic plasticity and cell survival in the hippocampus region of brain. This study provides first
evidence of neuroprotective potential of B-TCE against glutamate-induced excitotoxicity in hippocampus region and suggests
that B-TCE may act as a potential candidate for neuroprotective therapeutic approaches. A single compound ‘tinosporicide’
was further isolated from B-TCE, which was found to be effective at 800× lower concentration against glutamate-induced
neurodegeneration under in vitro conditions.

Keywords Hippocampus · Tinospora cordifolia · Synaptic plasticity · Glutamate · Excitotoxicity · Tinosporicide

Introduction incidence of neurodegenerative diseases is also increasing

Glutamate, a major excitatory molecule of neurotransmitter
pool in CNS, plays an important role in the development
of memory, learning, and synaptic plasticity in mammals
(Mattson 2008). However, elevated levels of glutamate are
known to cause excitotoxicity and are being implicated
in synaptic dysfunction and neurodegenerative diseases
(Ambrosi et al. 2014). With an increase in life expectancy,
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s1201​7-019-08566​-2) contains
supplementary material, which is available to authorized users.
* Gurcharan Kaur
gk.biotech@gndu.ac.in
1
Medical Biotechnology Lab, Department of Biotechnology,
Guru Nanak Dev University, Amritsar, Punjab 143005, India
worldwide, but the therapeutic options are limited (Flana-
gan et al. 2018). Various studies have reported the under-
lying mechanisms of glutamate-induced excitotoxicity and
its implication in ischemia, stroke, and neurodegenerative
diseases (Vaarmann et al. 2014; Shah et al. 2014; Zhang
et al. 2015).
Atomistic approach of allopathic medicines leads to the
improvement of the health of one organ but at the cost of
imparting some adverse effects, specifically on liver and
stomach. In addition to common side effects such as nausea,
vomiting, diarrhea, and headaches (Jost 2014), complica-
tions like cervical dystonia (Ikeda et al. 2014), myoclonus
(Bougea et al. 2014), fluctuations in non-motor and motor
response, dyskinesia (Lowin et al. 2017; Willows et al. 2017)
develop over time with long-term administration of acetyl
cholinesterase and glutamatergic inhibitors for treatment of
cognitive deficits in Alzheimer’s disease (Tan et al. 2014).

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Similar side effects have also been reported with levodopa
medication for Parkinson’s disease (Lowin et al. 2017; Wil-
lows et al. 2017). So, development of therapies with holistic
approach and least side effects is most intriguing for health
science researchers, which will benefit not only the indi-
vidual health but also reduce societal and economic burden
on the global healthcare system.
Herbs with medicinal properties have been used for thera-
peutic purposes since times immemorial in India, China,
and other Asian countries. Herbal medicines are believed
to be beneficial in strengthening the internal defence sys-
tem as well as revitalization of body (Basnyat and Kola-
sinski 2014; Lundstrom et al. 2017). Herbal formulations
have been reported to be equipped with broad spectrum of
therapeutic potential and bestow relief without substantial
adverse effects. A number of herbs like Withania somnif-
era, Bacopa monnieri, Mucuna pruriens, Centella asiatica,
Tinospora cordifolia, etc. have been tested for the treatment
of neurodegenerative diseases due to their antioxidant and
immunomodulatory activities (Kastenholz and Garfin 2009;
Mohanta et al. 2014; Kosaraju et al. 2014; Manchanda and
Kaur 2017). Extracts or isolated compounds from these
herbs have been reported to improve mental health, cog-
nitive abilities, and to prevent protein misfolding or neu-
ronal degeneration against various neurotoxic insults such
as β-amyloid, 6-OHDA (6-hydroxydopamine), or lifestyle
disorders (Kosaraju et al. 2014; Mishra et al. 2016).
Tinospora cordifolia, also known as Guduchi or Amrita
in India, is considered as “Heavenly elixir” due to its adap-
togenic properties. Growing number of reports support the
medicinal properties of this Rasayana herb in the treatment
of hepatotoxicity, diarrhea, fever, stomach ulcers, microbial
infections, and cancers (Saha and Ghosh 2012; Dhama et al.
2016). However, the studies validating its neuroprotective
activity are limited. Ethanolic extract of T. cordifolia was
found to improve the motor coordination and exert neuropro-
tection against 6-OHDA-induced degeneration of dopamin-
ergic neurons by restoration of antioxidant levels (Kosaraju
et al. 2014). Different extracts and polyherbal formulations
containing T. cordifolia have been shown to exhibit anti-
inflammatory activity by regulating the expression of inflam-
matory mediators such as iNOS, COX-2, pro-inflammatory
cytokines, and transcription factor NF-κB in animal models
of asthma and edema (Tiwari et al. 2014; Patgiri et al. 2014;
Nipanikar et al. 2017; Philip et al. 2018). Oral administration
of different extracts such as petroleum ether, aqueous etha-
nolic extracts of T. cordifolia exhibited anti-depressant activ-
ity, memory improvement, and anxiolytic activities (Dhingra
and Goyal 2008; Mishra et al. 2016). In a recent study from
our lab, oral feeding of T. cordifolia stem powder in com-
bination with Withania somnifera leaf powder was found to
ameliorate neuroinflammation-induced anxiety and showed
synergistic activity with dietary restriction regimen (Singh
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NeuroMolecular Medicine
et al. 2017). Another recent study from our lab provided pre-
liminary data on the neuroregenerative and neuroprotective
potential of B-TCE against glutamate-induced excitotoxicity
in primary cerebellar neurons (Sharma and Kaur 2018).
Hippocampus region plays a critical role in memory,
learning, and thinking processes; and decline in memory
is suggested to be the outcome of neurotoxic effect of glu-
tamate on neuronal population of forebrain, especially hip-
pocampus (Moneim et al. 2018). The current study was
planned to further investigate the neuroprotective potential
of B-TCE against glutamate-induced excitotoxicity in hip-
pocampus region of brain using in vitro and in vivo model
systems. Preliminary investigation on neuroprotective activ-
ity of B-TCE was carried out using rat primary hippocam-
pal neuronal and explant cultures. Further, in vivo model
system comprising of Wistar strain female rats was used
for behavioral and underlying mechanistic study. Expres-
sion of different molecular markers of structural plasticity,
reactive gliosis, glutamatergic neurotransmission, and cell
survival pathways in rat brain hippocampus were studied to
investigate the role of B-TCE in modulating these pathways
against glutamate neurotoxicity. We also isolated a single
pure compound from B-TCE, which was identified as tino-
sporicide and showed neuroprotective activity at much lower
concentration as compared to B-TCE.
Methodology
Herbal Extract Preparation and Isolation
of Bioactive Compounds
Tinospora cordifolia stem was collected in the month of
January from a forest located in Ropar district, Punjab and
B-TCE was prepared using the method described earlier
(Sharma and Kaur 2018). Briefly, 50% aqueous ethanolic
extract of T. cordifolia stem was prepared and further frac-
tionated with hexane, chloroform, ethyl acetate, and butanol.
Each fraction was collected and dried in rotavapor. Dried
butanol fraction was designated as B-TCE. To further isolate
bioactive compounds, B-TCE was further fractionated using
column chromatography. Silica Gel 70–230 was dissolved in
chloroform and poured into a column to achieve the desired
height while allowing the chloroform to drain slowly from
column. Further, granular slurry of B-TCE was made by
mixing it with Silica gel 230–400 and was used as station-
ary phase. Stationary phase was covered with cotton layer
and mobile phase was slowly added to the column and bot-
tom outlet was opened to allow drop-wise elution. Column
was eluted from low polarity to high polarity mobile phase
prepared by mixing chloroform and methanol in different
ratios. Different fractions (100 mL each) were collected
in small conical flasks, pooled on the basis of their TLC
NeuroMolecular Medicine
profile and allowed to settle down/precipitate overnight. Pre-
cipitates were filtered, washed, dried, and weighed. Other
physical parameters like solubility and melting points were
also checked. Isolated compounds were investigated using
Silica Gel G60 TLC plates (Millipore, MA, USA). Isolated
compound 1 (C1) was spotted onto base line drawn on the
TLC plate and TLC was run. Plate was dried and developed
using 20% H ­ 2SO4 as developing solution. C1 obtained in
powder form was used for HRMS, NMR, and IR spectrom-
etry analysis and was identified as Tinosporicide, which is a
furanoid diterpenoid in nature.
In Vitro Studies
Primary Neuronal and Explant Cultures and Their Treatment
Primary neuronal cultures (hippocampal and cerebellar)
were obtained from 2- to 6-day-old Wistar albino rat pups
by following the method reported earlier (Sharma and Kaur
2018). Briefly, after sacrificing rat pups, cerebellum and hip-
pocampus regions from brain were dissected out in chilled
1 × PBS, removed meninges, and trypsinization was carried
out. Trypsin digestion was stopped by adding equal volume
of neurobasal medium and tissue was pelleted down by cen-
trifugation for 2 min at 1000 rpm. The pellet suspended in
neurobasal medium was triturated by pipetting and undi-
gested chunks were allowed to settle down. After 5–10 min,
suspension was collected in fresh tube and centrifuged again
for 2 min. The pellet obtained was then resuspended in neu-
robasal medium, cells were counted and seeded in PLL-
coated plates according to the experiments.
For primary hippocampal explant culture, hippocampus
region was chopped in fine chunks with the help of scal-
pels and microscissors. These small pieces were then care-
fully placed onto PLL-coated coverslips and were allowed
to attach to the substratum followed by slow addition of
medium in the well without disturbing the explants. Mini-
mum three explants were placed onto one coverslip and each
experiment was performed in triplicates.
Cell Culture Treatment
Primary neuronal cultures were seeded at a plating density of
40,000–50,000 cells/mL in 24-well plates containing PLL-
coated substratum. Primary neuronal and explant cultures
were divided into four experimental groups: control, gluta-
mate (2 mM), B-TCE alone (20 μg/mL), and B-TCE+ glu-
tamate treatment groups. B-TCE and B-TCE+ glutamate
treatment groups were given treatment of B-TCE (20 μg/
mL) after 24 h of seeding. Monosodium salt of l-Glutamic
acid (MSG) (Sigma-Aldrich, MO, USA) was used for gluta-
mate (2 mM) treatment, which was given to glutamate alone
and B-TCE+ glutamate groups in next 24 h, i.e., 48 h after
seeding and incubated for next 24 h. For treatment with com-
pound C1, B-TCE was replaced with 25 ng/mL of C1 (dose
was standardized by using MTT assay). Cultures were estab-
lished and propagated in neurobasal medium (Invitrogen,
CA, USA) containing bFGF and B27 supplement (Invitro-
gen) and were incubated at 37 °C in ­CO2 incubator.
Immunostaining
Cultures were harvested post treatment regimen and immu-
nostaining was carried out using the method described ear-
lier (Sharma and Kaur 2018). Briefly, after harvesting, cul-
tures were fixed with acetone:methanol (1:1), permeabilized
with 0.3% PBS-Triton × 100 and then blocked with 2% BSA.
Cultures were then incubated with primary antibody [mouse
monoclonal antibody anti-α-Tubulin (1:500), anti-MAP-2
(1:250), anti-Cyclin D1 (1:250), anti-Bcl-xL (1:200), rabbit
monoclonal anti-AP-1(1:250) (all from Sigma-Aldrich), and
mouse polyclonal anti-PSA-NCAM (1:250) (Millipore)] at
4 °C for 24 h. Permeabilization was not performed for PSA-
NCAM immunostaining. After washing with 0.1% PBST,
cultures were incubated with secondary antibody (goat anti-
mouse/rabbit IgG/IgM Alexa Fluor 488/543) for 2 h at room
temperature. Coverslips containing cultures were mounted
with anti-fading agent Fluoromount (Sigma-Aldrich) and
images were captured with Nikon A1R Confocal Microscope
(Nikon Corporation, Japan). All the experiments were per-
formed in triplicates. Fluorescence intensity was measured
using NIS elements AR analysis software version 4.11.0.
Mitotracker Staining
Mitotracker green FM stain was added to each well at final
concentration of 100 mM after completion of treatment and
incubated for 45 min. After incubation, cells were fixed with
acetone:methanol (1:1) and coverslips containing cells were
mounted on the glass slides using anti-fading medium Fluo-
romount. Imaging was carried out on the same day using
Nikon A1R Confocal Microscope.
Wound Scratch Assay
Migration promoting effect of B-TCE was studied using
wound scratch assay as described earlier (Sharma and Kaur
2018). Briefly, confluent monolayer of primary hippocam-
pal neurons was scratched with microtip followed by treat-
ment with glutamate, B-TCE, and B-TCE+ glutamate. Phase
contrast images were captured at zero hour and after 24 h
of treatment using EVOS FL microscope (Invitrogen) and
image analysis was done using Image Pro Plus software ver-
sion 4.5.1 (Media Cybernetics).
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In Vivo Studies
Animals
Wistar albino female rats aged 2–3 months were procured
from National Institute of Pharmaceutical Science and
Research (NIPER), Mohali, India and housed under con-
trolled environment of constant temperature (25 ± 2 °C),
constant dark/light cycle (12 h each) with ad libitum food
and water supply. All animal experiment protocols were
approved by Institutional Animal Ethical Committee, Guru
Nanak Dev University, Amritsar registered to “Committee
for the Purpose of Control and Supervision of Experiments
on Animals (CPCSEA), GOI” (Registration no. 226/CPC-
SEA) (permission number 226/CPCSEA/2017/01). The
experiments were performed in accordance with the relevant
guidelines of ‘Animal Care and Use’ laid down by the same
committee.
Animals were randomly divided into four groups—
(I) Vehicle control: Animals given water orally and
injected 0.9% saline intraperitoneally for 15  days, (II)
Glutamate alone group: Animals given water orally and
injected MSG (2 g/kg) intraperitoneally for 15 days, (III)
B-TCE  alone group: Animals given B-TCE (35  mg/kg)
orally and injected 0.9% saline intraperitoneally for 15 days,
and (IV) B-TCE+ glutamate treatment group: Animals given
B-TCE (35 mg/kg) orally and injected with monosodium
glutamate (2 g/kg) intraperitoneally for 15 days. All the ani-
mals were fed with normal chow feed and treatment was
given for 15 days. There were 9 animals each in group I,
III, and IV, whereas, 12 animals were kept in group II due
to expected mortality. 25% mortality was observed in glu-
tamate-treated group, whereas, no mortality was observed
in other groups. Dose of extract was obtained by multiply-
ing the reported dose of TCE (140 mg/kg) from our lab
(Mishra et al. 2016) with yield factor of B-TCE (dry weight
of B-TCE/dry weight of TCE, ~ 0.26). The in vivo dose of
MSG was selected from thorough literature review (Tamás
et al. 2004; Ramanathan et al. 2007; Tawfik and Al-Badr
2012; Swamy et al. 2013).
Behavioral Studies
Novel Object Recognition Test (NORT) (n = 9 in  Each
Group)  To study the working memory and cognition, Novel
Object Recognition Test was performed using method
reported earlier (Mishra et al. 2016). Briefly, after habitua-
tion with empty box and training session with two odorless
objects, one of the objects was replaced with new object
and animal behavior was monitored. Animal behavior was
recorded with video camera from training sessions to final
testing session and analyzed for exploration, time spent with
objects, rearing, and preference index. Sniffing, chewing,
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NeuroMolecular Medicine
licking, and movement of vibrissae by animal towards object
were considered as defining parameters of object explora-
tion. Number of episodes with each object, time spent in
exploration of old and novel objects as well as total time
spent in object exploration were assessed and object prefer-
ence index was calculated using the following formula.
Preference Index (PI): Time spent in exploration of novel
object/total time spent in object exploration (Old + novel).
Preference index greater than 0.5 is considered as higher
preference for novel object. Videos were analyzed by an
observer blind to the experiment.
Elevated Plus Maze (EPM) Test (n = 9 in  Each Group)  EPM
test for exploratory activity and anxiety-like behavior was
performed using the method described earlier (Mishra et al.
2016). Briefly, on 15th day of treatment, all the animals of
four groups were subjected to EPM test and were allowed
to explore the maze freely for 5  min in dim light. Whole
session was recorded by a video camera fitted exactly above
the apparatus. Videos were analyzed by a person blind to
the study groups to analyze the animal behavior and differ-
ent parameters were noted down such as time spent in open
and closed arms, entries and crossings into closed and open
arms, number of rearing episodes and number of head dips.
Time spent and number of entries and crossings in each arm
were measured as indication of anxiety levels and explora-
tory behavior of animals. Additionally, we also measured
the frequency of (I) rearing and (II) head dipping episodes.
Rotarod Test (n = 9 in Each Group)  To examine the impact
of glutamate excitotoxicity and B-TCE treatment on motor-
coordination, performance of animals on rotating rod (at
constant speed of 10  rpm) was evaluated. Test was per-
formed on each animal of all the four experimental groups
for 5 min. Time spent on rotating rod and number of falls
were recorded for each animal and averaged for each group.
The rotarod apparatus used was an automatic motor-driven
treadmill Rotamex-5 from Columbus instrument (fall
height: 44.5 cm, Spindle diameter: 7.0 × 9.5 cm2).
Molecular Studies
Immunohistochemistry  For immunostaining of GFAP and
PSA-NCAM in hippocampus region, experimental animals
(n = 4–5 for each group) were transcardially perfused and
immunostained by using method described earlier (Mishra
et  al. 2016). Briefly, coronal sections of 30  μm thickness
were cut using cryomicrotome (Thermo Fischer Scientific,
MA, USA), collected in 1 × PBS, permeabilized with 0.3%
PBST for 15 min, and washed thrice with 1 × PBS (5 min
each). Sections were then incubated with 5% Normal Goat
Serum in 1 × PBS (blocking solution) for 30  min before
incubation with specific primary antibodies, i.e., rabbit
NeuroMolecular Medicine
IgG GFAP (1:500) (Sigma-Aldrich) and mouse IgM PSA-
NCAM (1:300) (Millipore) for 48 and 72  h, respectively.
After completion of primary antibody incubation, sections
were again washed thrice with 1 × PBS (5  min each) and
incubated with respective secondary antibody anti-rabbit
IgG or anti-mouse IgM labeled with Alexa Fluor 488 (Inv-
itrogen) for 2 h at room temperature. Tissue sections were
then washed and mounted on glass slides with anti-fading
agent Fluoromount. Images were captured using Nikon
A1R Confocal Laser Scanning Microscope at 10× objec-
tive. Intensity analysis was performed using ImageJ (Public
domain software from NIH).
Western Blotting  Animals (n = 4–5 for each group) were
anesthetized using sodium thiopentone injection (1 U/10 g
body weight), sacrificed, and brains were extracted out
from skull. Hippocampus region was dissected out fol-
lowed by homogenization, sample preparation, SDS-PAGE,
and semidry transfer onto PVDF membrane as described
earlier (Mishra et  al. 2016). PVDF membrane containing
transferred proteins was then blocked with 5% skimmed
milk in 0.1% TBST (0.1% Tween-20 in IX TBS) for 1  h
at room temperature. After a single wash with 0.1% TBST
(5 min), membrane was then incubated overnight with pri-
mary antibody [mouse anti-MAP-2, anti- CamKIIα, anti-
Calcineurin (1:2500), anti-GFAP, anti-α-Tubulin (1:5000);
rabbit anti-NMDAR2A (1:1500), anti-pSynapsin I (1:1500)
(from Sigma-Aldrich); rabbit anti-pGluR1, anti-pGluR2,
anti-pMEK1/2, anti-pErk1/2, anti-pAkt (1:500), rabbit anti-
GluR1, anti-GluR4, anti-TrkB, anti-Bcl-xL (1:1000) (All
from Cell Signaling, MA, USA)] prepared in 5% skimmed
milk or 5% BSA (according to specification on product cata-
logue) at 4  °C. On the next day, membrane was washed,
followed by probing with HRP-conjugated goat-anti-mouse
or goat-anti-rabbit IgG secondary antibody at room temper-
ature. The membrane was then given three washings with
0.1% TBST and immunoreactive bands were detected with
Amersham ECL solution (GE Healthcare, UK) using Image
Quant LAS 4000 GE (GE Healthcare, UK) detection sys-
tem. Expression of immunoprobed proteins was normalized
using α-Tubulin as internal control.
mRNA Expression  Total RNA was extracted from hip-
pocampus region, cDNA was prepared, and Real-Time
PCR was carried out using method reported earlier
(Mishra et al. 2016). Briefly, 50 ng of cDNA was used for
a reaction mixture of 5 μL which was containing 2.5 μL of
2 × Syber Green Master mix, 0.5 μL of 20 × predesigned
Primer–Probe mix (Applied Biosystem, CA, USA) and
1 μL of water. Amplification was carried out using Step
One Plus Real-Time PCR system (Applied Biosystem)
using the amplification conditions described by Mishra
et al. (2016). GAPDH was used as an endogenous control
for each gene of interest. The relative gene expression of
each candidate gene was calculated by ‘Livak method’
and represented as ­2−ΔΔCt and final gene expression as
­2−ΔΔCt ± SEM. Final results were obtained as average of
three different observations.
Pro‑inflammatory Cytokine ELISA‑Based Determination 
Serum was isolated from blood collected from all the ani-
mals of four experimental groups and used for determina-
tion of pro-inflammatory cytokines using sandwich ELISA-
based kits from Cayman Chemicals, MI, USA (TNF-α and
IL-6) and Thermo Fischer Scientific, MA, USA (IL-1β)
according to the manufacturer’s protocol.
Statistical Analysis
Values were expressed as mean ± SEM from at least three
independent observations. Sigma stat software (Version 3.5)
for windows was used for statistical analysis. Results were
analyzed using one-way ANOVA followed by Holm–Sidak
post hoc test. Values (p < 0.05) were considered as statisti-
cally significant.
Results
In Vitro Studies
B‑TCE and Tinosporicide (C1 Compound) Pre‑treatment
Suppressed Glutamate‑Induced Neurodegeneration
Tinosporicide was obtained as a white amorphous powdery
single compound (C1) from silica gel column chromatog-
raphy of B-TCE and was detected as a single spot in TLC
(Supplementary Fig. S1). Both B-TCE and tinosporicide
were tested on primary cerebellar neurons for their neuro-
protective activity against glutamate-induced excitotoxicity
and the cells were immunostained for MAP-2. Glutamate
induced degeneration of neuronal processes as well as down-
regulation of MAP-2 expression, whereas, B-TCE and tino-
sporicide suppressed degeneration and maintained MAP-2
expression in glutamate-exposed primary cerebellar neurons
to near control levels (Fig. 1a). Tinosporicide was effective
at very less concentration (25 ng/mL) as compared to parent
B-TCE extract (20 µg/mL). After NMR, HRMS, IR, and lit-
erature-based analysis (Rahman et al. 1991), compound C1
was decoded as tinosporicide (Supplementary Figs. S2–S4;
Table S1; Fig. 1b).
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Fig. 1  Activity elucidation and a b
characterization of Tinospori- Control Glu
cide. a Confocal micrographs of
MAP-2 immunostained primary
cerebellar neurons pre-treated
with B-TCE (20 μg/mL) and
C1 (tinosporicide) (25 ng/
mL) with or without glutamate Control Glu
(2 mM) exposure. b Structure
of compound C1 deduced as
tinosporicide
Glu: 2 mM; B-TCE: 20 µg/mL; C1: 25 ng/mL
B‑TCE Pre‑treatment Protected Primary Hippocampal
Neurons Against Excitotoxic Injury, Promoted Neuronal
Plasticity, and Suppressed Apoptosis
Primary hippocampal neurons pre-treated with B-TCE
before glutamate exposure and immunostained for MAP-2
showed similar results (Fig. 2a, 1st panel) as seen in cer-
ebellar neurons (Fig. 1b). B-TCE (20 μg/mL) pre-treatment
suppressed glutamate-induced neurodegeneration and down-
regulation in MAP-2 expression (Fig. 2a, 1st panel; Sup-
plementary Fig. S5a). Further, upregulated expression of
plasticity marker PSA-NCAM was observed in B-TCE+ glu-
tamate-treated primary hippocampal neurons, whereas,
glutamate alone-treated cultures showed downregulation in
the expression of PSA-NCAM (Fig. 2a, 2nd panel). B-TCE
pre-treatment also promoted neurite outgrowth and neuronal
migration from primary explants, whereas, no outgrowth and
migration were observed from primary explants treated with
glutamate alone (Fig. 2b). Observations from wound scratch
assay supported migration promoting potential of extract, as
B-TCE alone and B-TCE pre-treated glutamate-challenged
hippocampal neurons showed 132% and 127% gap closure,
respectively, whereas, glutamate alone treatment inhibited
migration as is evident from widened gap with negative
(− 82%) gap closure in this group (considering gap closure
as 100% in control) (Fig. 2c, d). Further, upregulation in
the expression of AP-1 and Cyclin D1 and elevated mito-
chondrial dysfunction were observed in glutamate-treated
cultures, which was suppressed by B-TCE pre-treatment
(Fig. 2a, 3rd, 4th, and 6th panel). B-TCE pre-treatment also
enhanced the expression of anti-apoptotic protein Bcl-xL,
which was otherwise markedly reduced by glutamate alone
treatment (Fig. 2a, 5th panel).
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B-TCE B-TCE + Glu
C1 C1 + Glu
C1: Tinosporicide
In Vivo Studies
B‑TCE Treatment Suppressed Glutamate‑Induced
Impairment in Cognition, Exploratory Behavior, and Motor
Coordination
NORT: Glutamate-treated animals showed more number of
episodes and time spent with old object over the new one
(Fig. 3a, b) and exhibited less preference (PI: 0.43 < 0.5)
towards novel object (Fig. 3d). On the other hand, animals
from B-TCE alone and B-TCE+ glutamate treatment groups
spent more time in exploring new object as compared to old
object as exhibited by higher number of episodes for new
object and significantly high preference for the novel object
(PI: 0.61 and 0.69, respectively > 0.5) (p ≤ 0.01) (Fig. 3a, b,
d). Glutamate-treated animals also spent significantly less
time (total) in object exploration as compared to animals of
B-TCE-treated groups (p ≤ 0.01) (Fig. 3c).
EPM: Animals of glutamate-treated group spent more
time in closed arm and significantly less time in open
arm (p ≤ 0.05) as compared to control, B-TCE alone, and
B-TCE+ glutamate groups (Fig.  3e). Further, glutamate
alone-treated animals attempted lesser number of entries and
crossings in closed and open arms as compared to animals of
other groups, whereas, B-TCE alone and B-TCE+ glutamate
treatment groups showed significantly higher number of total
entries and crossings (p ≤ 0.05) in both the arms as compared
to glutamate-treated animals (Fig. 3f, g). Higher number of
rearing episodes and head dips were also observed in both
the B-TCE-treated groups as compared to glutamate alone
group (Fig. 3h).
Rotarod Test: Glutamate-administered animals showed
significantly higher frequency of falls and lesser time spent
on rotating rod as compared to control animals (p ≤ 0.001)
(Fig. 3i, j). B-TCE alone- and B-TCE+ glutamate-treated
animals performed better with more time spent on the rotat-
ing rod and less number of falls as compared to both gluta-
mate alone-treated and control animals (p ≤ 0.001) (Fig. 3i,
j).
NeuroMolecular Medicine

a Control Glu B-TCE B-TCE + Glu c Zero hour After 24 hour

MAP-2

Control
PSA-NCAM

Glu
AP-1

B-TCE
Cyclin D1

B-TCE + Glu
Bcl-xL

# #
d 150
* *
Mitotracker

100
%age Gap Closure

50

b Control Glu B-TCE B-TCE + Glu 0

Control Glu B-TCE B-TCE+
1° Explant

-50 Glu

-100 *

e 300
*
*
250
Relative Intensity

#
200 Control
# # *
# Glu
150 # # # # * #
* ## # * *
100 * * * B-TCE
* B-TCE+Glu
50
0
MAP-2 PSA-NCAM AP-1 Cyclin D1 Bcl-xL Mitotracker

Fig. 2  B-TCE protects primary hippocampal cultures from gluta-
mate-induced neurotoxicity. a Confocal micrographs of control,
glutamate (2  mM)-, B-TCE (20  μg/mL)-, and B-TCE+ glutamate-
treated primary hippocampal neurons immunostained for MAP-2,
PSA-NCAM, AP-1, Cyclin D1, Bcl-xL, and mitotracker green FM.
b Confocal images of primary explants with dual immunostaining for
PSA-NCAM and NCAM. c Phase contrast images of control, gluta-
mate (2  mM)-, B-TCE (20  μg/mL)-, and B-TCE+ glutamate-treated
primary hippocampal neurons captured at 0 and 24  h of treatment
in migration assay. d Histogram presenting percentage gap closure
after 24 h of treatment. Confocal images were captured at 60× objec-
tive (Scale bar: 50 μm). Phase contrast images were captured at 10×
objective. *p ≤ 0.001 presents statistically significant difference of
glutamate, B-TCE and B-TCE+ glutamate treatment groups as com-
pared to Control. #p ≤ 0.001 presents statistically significant differ-
ence of B-TCE and B-TCE+ glutamate treatment groups as compared
to glutamate

13
 NeuroMolecular Medicine

a 12
Old Novel
b 60 Old Novel

Total time spent with

# #

No. of episodes with

10 50

object (in sec)

Novel Object Recognition Test 8 40
* *

object
6 30

4 20 *

2 10

0 0
Control Glu B-TCE B-TCE+ Control Glu B-TCE B-TCE+
c 80 Glu d 1 Glu
Total exploration

# #

Preference Index
#
time (in sec)

60 0.8 #
* 0.6 *
40
0.4
20
0.2
0 0
Control Glu B-TCE B-TCE+ Control Glu B-TCE B-TCE+
Glu Glu
e 250 Closed arm Open arm f Closed arm Open arm
8
Total time spent (in sec)

# #
200 7 #
#

No. of entries
# 6
150 5 #
Elevated Plus Maze Test

*
4
100 * 3 *
50 2
1
0 0
Control Glu B-TCE B-TCE+ Control Glu B-TCE B-TCE+
Glu Glu
5 Closed arm Open arm
g 4.5
#
#
h 25 #
#
4
No. of crossings

No. of rearings and

# 20 # #
3.5
Control
3
head dips

15
2.5 Glu
2
* 10 *
1.5 B-TCE
1 5
0.5 B-TCE+
0 Glu
0
Control Glu B-TCE B-TCE+ Rearings Head dips
Glu
i # # j 12
300
* * *
Time spent (in sec)

250 10
Rotarod Test

*
No. of falls

200 8
#
150 6 #
*
*
100 4
50 2
0 0
Control Glu B-TCE B-TCE+ Control Glu B-TCE B-TCE+
Glu Glu

B‑TCE Treatment Suppressed Glutamate‑Induced Alteration (p ≤ 0.01) (Fig. 4a, b). On the other hand, B-TCE alone and
in Neuronal Structural Marker MAP‑2 B-TCE+ glutamate treatment groups showed significantly
higher expression of MAP-2 as compared to glutamate treat-
MAP-2 expression in hippocampus region of gluta- ment group, thus supporting our in vitro findings of this
mate-treated animals was significantly downregulated structural protein (Fig. 4a, b). The change in MAP-2 protein

13
 NeuroMolecular Medicine
◂Fig. 3  B-TCE treatment suppresses glutamate-induced behavioral
deficits. Histograms presenting performance of animals in NORT (a–
d), EPM (e–h) and Rotarod test (i–j). Histogram presenting a number
of episodes and b time spent by animal with old and novel objects c
total time spent in object exploration and d preference index of ani-
mal assessed from NORT suggested improved memory and cognition
by B-TCE treatment as compared to glutamate treatment group. EPM
test suggested improved exploratory behavior and reduced anxiety in
B-TCE-treated animals as compared to glutamate treatment group as
indicated by e Time spent f Number of entries and g crossing into
open and closed arms along with number of rearing and head dipping
episodes. Motor coordination deficits induced by glutamate were pre-
vented by B-TCE administration as suggested by i time spent in rotat-
ing rod and j number of falls during the performing session. *p ≤ 0.05
presents statistically significant difference of glutamate, B-TCE,
and B-TCE+ glutamate treatment groups as compared to Control.
#
p ≤ 0.05 presents statistically significant difference of B-TCE and
B-TCE+ glutamate treatment groups as compared to glutamate
expression in hippocampus region was also supported by
similar changes observed at its mRNA level (Fig. 4c).
B‑TCE Pre‑treatment Attenuated Glutamate‑Induced
Astrogliosis and Pro‑inflammatory Cytokine Secretion
Reactive gliosis was clearly observed in DG region of
hippocampus as is evident from activated morphology of
astrocytes and higher expression of glial marker GFAP,
observed in immunostaining Confocal micrographs (Fig. 4d;
Supplementary Fig. S5b). Animals of B-TCE alone- and
B-TCE+ glutamate-treated groups showed reduction in acti-
vation of astrocytes, which was also evident from down-
regulation in expression of GFAP in DG region of their
hippocampus (Fig. 4d). Immunostaining data were further
supported by Western blotting results, where B-TCE+ glu-
tamate-treated animals showed significant downregulation
in the expression of GFAP as compared to glutamate-treated
group (Fig. 4e, f) (p ≤ 0.01).
In pro-inflammatory cytokines estimation in serum,
glutamate-treated group showed significant increase in
TNF-α and IL-6 expression, whereas, treatment of B-TCE
along with glutamate administration suppressed this change
(p ≤ 0.01) (Fig. 4g). Glutamate induced marginal increase in
IL-1β level, but the change was not statistically significant
(Fig. 4g).
B‑TCE Treatment Modulated the Expression of Glutamate
Receptors and Downstream Plasticity Pathway
Glutamate treatment for 15  days led to the elevation in
expression of GluR1 and GluR4 (p ≤ 0.01) in hippocam-
pus region (Fig. 5a, b), whereas, significant decrease in the
expression of these proteins was observed in B-TCE+ glu-
tamate-treated group (p ≤ 0.01) (Fig. 5a, b). Expression of
phosphorylated forms of AMPARs (pGluR1 and pGluR2) in
glutamate treatment group was also enhanced significantly
as compared to control animals. B-TCE treatment signifi-
cantly downregulated the pGluR1-2 expression as compared
to both glutamate and control group animals (Fig. 5a, b).
We further studied the expression of NMDAR2A at
transcriptional and translational levels along with expres-
sion of postsynaptic protein pSynapsin I in hippocampus
region (Fig. 5c–e). Glutamate treatment led to significant
downregulation (p ≤ 0.01) of NMDAR2A expression as
compared to control group. Although NMDAR2A expres-
sion in B-TCE+ glutamate treatment group showed marginal
increase as compared to glutamate alone treatment group,
the protein level was still significantly low as compared
to control group (Fig. 5c, d). On the other hand, the fold
change in NMDAR2A mRNA expression was significantly
higher in B-TCE+ glutamate treatment group as compared
to glutamate alone group at transcriptional level (Fig. 5e).
pSynapsin I expression was also found to be significantly
downregulated by glutamate treatment (Fig.  5c, d). On
the contrary, animals receiving B-TCE prior to glutamate
administration showed significant reversal of pSynapsin
I expression (Fig. 5c, d). Animals of B-TCE alone group
showed NMDAR2A and pSynapsin I levels similar to con-
trol animals (Fig. 5c–e).
Activation of NMDAR2A and phosphorylation of Syn-
apsin I to promote its dissociation from synaptic vesicles
and availability at active front of presynaptic terminals is
facilitated by CaMKIIα, a C ­ a 2+/Calmodulin-dependent
kinase which modulates synaptic strength (Menegon et al.
2002; Colbran 2004). As compared to the control group, sig-
nificant downregulation was observed in CamKIIα at trans-
lational level (p ≤ 0.01) in glutamate-treated animals with
non-significant change in expression at transcriptional level
(Fig. 5c–e). On the other hand, B-TCE treatment restored
glutamate-induced downregulation of CamKIIα expression
to significant level (p ≤ 0.01) (Fig. 5c–e). Further, expres-
sion of calcineurin, another calcium-dependent effector
molecule was significantly upregulated in glutamate-treated
group. Animals of B-TCE- and B-TCE+ glutamate-treated
groups showed significantly lower expression of calcineurin
(p ≤ 0.01) (Fig. 5c–e).
Modulation of Structural Plasticity, ERK and Akt Pathways
To further investigate the changes in neuronal structural
plasticity proteins, expression of PSA-NCAM was studied
by immunostaining (Fig. 6a). A pronounced decrease in
PSA-NCAM expression in DG region of hippocampus was
observed in glutamate-treated group. However, B-TCE alone
and B-TCE+ glutamate treatment showed pronounced upreg-
ulation in its expression as compared to control and gluta-
mate-treated groups (Fig. 6a). Further, expression studies at
transcriptional level also showed significant increase in PST
expression (Polysialyltransferase, an enzyme responsible for
13
 NeuroMolecular Medicine

B-TCE
a Control Glu B-TCE + Glu

MAP-2 280-135kDa

α-Tubulin 52kDa

b 140 c 1.6 #
Relative change in MAP-2

120 #
1.4

fold change in mRNA

#

expression of MAP-2
100 1.2
expression

*
80 1
0.8
60
0.6
40 *
0.4
20 0.2
0 0
Control Glu B-TCE B-TCE+ Control Glu B-TCE B-TCE+
Glu Glu
d
Control Glu B-TCE B-TCE + Glu

e f 120
Relative change in GFAP

*
B-TCE 100 #
Control Glu B-TCE
+Glu
80
expression

GFAP 50kDa
60
α-Tubulin 52kDa
40

20
1800 0
g Control Glu B-TCE B-TCE+
1600 *
inflammatory cytokines (in

Glu
1400
Concentration of pro-

1200 * Control
#
1000 * Glu
pg/ml)

* # #
800 * B-TCE
*
600
B-TCE+ Glu
400
200
0
TNFα IL-6 IL-1β

polysialylation of NCAM) in B-TCE and B-TCE+ glutamate accompanied by enhanced expression of NCAM, whereas,
groups (p ≤ 0.001) (Fig. 6b). Upregulation of PSA-NCAM significantly reduced expression of NCAM was observed in
in hippocampus region of B-TCE-treated groups was also glutamate-treated animals (p ≤ 0.001) (Fig. 6c).

13
 NeuroMolecular Medicine
◂Fig. 4  B-TCE treatment maintained the structural integrity and sup-
pressed reactive gliosis induced by glutamate. a Representative
immunoblot and b histogram showing relative change in MAP-2
expression after normalization with internal control α-Tubulin. c His-
togram presenting fold change in mRNA expression of MAP-2 in all
the four experimental groups. d Representative confocal images of
GFAP immunostaining from DG region of hippocampus of control,
glutamate, B-TCE, and B-TCE+ glutamate-treated animals. Images
were captured at 10× objective (Scale bar: 200  μm). e Representa-
tive immunoblot and f histogram showing relative change in GFAP
expression at protein levels. g Pro-inflammatory cytokine secretory
levels were assessed by ELISA and presented as histogram. *p ≤ 0.01
depicts statistically significant difference of glutamate, B-TCE,
and B-TCE+ glutamate treatment groups as compared to Control.
#
p ≤ 0.01 depicts statistically significant difference of B-TCE and
B-TCE+ glutamate treatment groups as compared to glutamate
Neurons orchestrate survival mechanisms against cel-
lular apoptosis induced by excitotoxic concentrations of
glutamate by activating proteins of ERK pathway, Akt,
and anti-apoptotic proteins. In the present study, glutamate
treatment was found to significantly reduce the expres-
sion of phosphorylated MEK1/2 which further reduced the
activation/phosphorylation of Erk1/2 (p ≤ 0.01) (Fig. 6d,
e). Erk1/2 mRNA expression also supported the Western
blotting results (Fig. 6f). Further, B-TCE treatment prior
to glutamate administration or B-TCE  alone treatment
was found to reverse the expression pattern of pMEK
and pErk1/2 as is evident from their significantly higher
expression in these groups as compared to glutamate alone
treatment (p ≤ 0.01) (Fig. 6d–f).
Glutamate excitotoxicity has been reported to down-
regulate TrkB activation and downstream survival pathway
Akt/PI3K, which plays important role in neuronal survival
and synaptic plasticity. Glutamate treatment was observed
to significantly downregulate TrkB expression, both at pro-
tein and mRNA level (p ≤ 0.01) (Fig. 7a–c). TrkB expres-
sion was significantly upregulated in both B-TCE- and
B-TCE+ glutamate-treated animals as compared to glu-
tamate-treated group at mRNA level (p ≤ 0.01); however,
no significant change was observed at translational level
(Fig. 7a–c). B-TCE treatment was also found to restore
the mRNA expression of PI3K to near control levels,
which is upstream regulatory molecule of Akt (cell sur-
vival marker) and responsible for Akt phosphorylation
(p ≤ 0.01) (Fig. 7c). Further, expression of Akt was stud-
ied at protein and mRNA levels. The protein expression
of pAkt was significantly reduced in hippocampus region
of glutamate-treated animals as compared to other groups
(p ≤ 0.01). At mRNA level also, glutamate treatment sig-
nificantly downregulated the mRNA expression of Akt,
which was recovered to some extent in B-TCE+ glutamate
group (p ≤ 0.01) (Fig. 7c). Expression of pAkt protein was
not recovered significantly by B-TCE treatment.
We also observed significant downregulation in Bcl-
xL protein expression after glutamate treatment (Fig. 7a).
B-TCE treatment restored the levels of this anti-apoptotic
protein and the expression was significantly higher than con-
trol levels in B-TCE alone and B-TCE+ glutamate treatment
groups (p ≤ 0.01) (Fig. 7a, b). The changes in expression of
Bcl-xL mRNA with glutamate and B-TCE treatment were
not found statistically significant (Fig. 7c).
Discussion
The rationale of current study was to investigate the ben-
eficial effects of Butanol extract of T. cordifolia against
glutamate-induced excitotoxicity in primary hippocampal
neuronal cultures and to further validate the neuroprotective
potential of B-TCE using Wistar albino rat model system.
Neuroprotection Against Glutamate‑Induced
Excitotoxicity in Primary Neuronal Cultures
From column chromatography of B-TCE, tinosporicide (C1)
was isolated, which showed neuroprotective activity at much
lower dose (25 ng/mL) as compared to B-TCE (20 μg/mL)
in glutamate-exposed primary cerebellar neurons. Immu-
nostaining for MAP-2 revealed that B-TCE and tinosporicide
pre-treatment suppressed the glutamate-induced neurode-
generation and maintained the expression of neuronal struc-
tural proteins. This is the first ever study reporting the bio-
logical activity of tinosporicide. It was initially reported to
be present in Tinospora malabarica by Rahman et al. (1991)
as a furanoid diterpenoid, which was isolated from fresh
stems of the herb. Since our main objective was to study the
effect of glutamate and B-TCE on synaptic plasticity, we
carried out all other experiments in primary hippocampal
neurons. Neuroregenerative potential of B-TCE observed
in primary hippocampal cultures is also supported by our
preliminary study with primary cerebellar neurons (Sharma
and Kaur 2018).
B-TCE pre-treatment prevented glutamate-induced
decrease in PSA-NCAM and NCAM expression, which play
important role in synaptic plasticity and may explain the
neuroprotective role of B-TCE. Inhibition of neurite out-
growth from primary explants as well as migration of pri-
mary hippocampal neurons in scratched area also supported
glutamate-induced neurodegeneration which was associ-
ated with downregulation in expression of PSA-NCAM and
NCAM. Upregulation in the expression of Cyclin D1 and
AP-1 observed in glutamate-treated group suggests aber-
rant cell cycle progression and induction of apoptosis by
glutamate exposure (Hitomi and Stacey 2010; Atabay and
Karabay 2012). Transcription factor AP-1 promotes the
activation of pro-apoptotic genes (Chen et al. 2003). This
13
 NeuroMolecular Medicine

a b 160 Control Glu B-TCE B-TCE+ Glu

B-TCE 140

Relative change in expression

* # #
Control Glu B-TCE +Glu *
120 *
*
pGluR1 100 kDa * # #
100 * #
100 kDa # * #
pGluR2 * *
80 *
GluR1 100 kDa 60
GluR4 100 kDa 40

α-Tubulin 52 kDa 20

0
pGluR1 pGluR2 GluR1 GluR4

c d
Control Glu B-TCE B-TCE+ Glu
B-TCE
Relative change in expression
Control Glu B-TCE + Glu 120
# # #
NMDAR2A # # * *
170 kDa #
100 * *
* * *
pSynapsin I 78 kDa *
80

CamKIIα 58 kDa 60

Calcineurin 50 kDa 40

20
α-Tubulin 52 kDa
0
NMDAR2A pSynapsin I CamkIIα CaN

e
2
#
1.8
1.6 *
fold change in mRNA

1.4
#
expression

1.2 Control
*
1 Glu
0.8 # # B-TCE
* * #
0.6 B-TCE+ Glu
* *
0.4
0.2
0
NR2A CamkIIα CaN

Fig. 5  B-TCE treatment suppressed glutamate-induced overactiva-
tion of AMPA receptors, modulated NMDAR pathway, and pro-
moted plasticity. a Representative immunoblots and b histograms
showing relative change in AMPA receptors in hippocampus region
of control, glutamate-, B-TCE-, and B-TCE+ glutamate-treated ani-
mals after normalization with internal control α-Tubulin. c Repre-
sentative immunoblots and d histograms showing relative change in
NMDAR2A, pSynapsin I, CamkIIα, and calcineurin in hippocampus
region of control, glutamate-, B-TCE-, and B-TCE+ glutamate-treated
animals after normalization with internal control α-Tubulin. e Fold
change in mRNA expression of NMDAR2A, CamkIIα, and Calcineu-
rin was also assessed in all the four experimental groups and pre-
sented as histogram. *p ≤ 0.01 presents statistically significant differ-
ence of glutamate, B-TCE, and B-TCE+ glutamate treatment groups
as compared to Control. #p ≤ 0.01 presents statistically significant dif-
ference of B-TCE and B-TCE+ glutamate treatment groups as com-
pared to glutamate

13
NeuroMolecular Medicine

a Glu B-TCE B-TCE + Glu

Control

b 160 # c 1.6
* 1.4 #
140

fold change in mRNA

expression of NCAM
fold change in mRNA

1.2
expression of PST

120 #
100 1

80 0.8
60 # 0.6
* *
40 0.4
20 0.2
0 0
Control Glu B-TCE B-TCE+ Control Glu B-TCE B-TCE+
Glu Glu
e 140 #
d B-TCE # # * #
Control Glu B-TCE +Glu 120
Relative change in

100 * *
pMEK 1/2 30kDa
expression

Control
80
pErk 1/2 44kDa Glu
42kDa 60
B-TCE
α-Tubulin 40
52kDa B-TCE+ Glu
20
0
pMEK1/2 pErk1/2

f 1.2
fold change in mRNA

1
#
0.8
expression

Control
0.6 Glu
0.4 B-TCE
B-TCE+ Glu
0.2 *
0
Erk1/2

Fig. 6  B-TCE treatment modulated structural plasticity and ERK
pathway. a Confocal images of PSA-NCAM immunostaining from
DG region of hippocampus of control, glutamate-, B-TCE-, and
B-TCE+  glutamate-treated animals. Images were captured at  × 
10
(Scale bar: 200  μm). Histograms presenting fold change in mRNA
expression of b PST and c NCAM in all the four experimental
groups. d Representative immunoblot and e histograms depicting
relative change in the expression of molecular markers of ERK path-
way. α-Tubulin was used as internal control to normalize the protein
expression. f Histogram showing fold change in Erk1/2 expression at
mRNA level. *p ≤ 0.01 presents statistically significant difference of
glutamate, B-TCE, and B-TCE+ glutamate treatment groups as com-
pared to Control. #p ≤ 0.01 presents statistically significant difference
of B-TCE and B-TCE+ glutamate treatment groups as compared to
glutamate

13
 NeuroMolecular Medicine

a b Control Glu B-TCE B-TCE+ Glu

140 #
B-TCE
*
Control Glu B-TCE +Glu 120 #

Relative change in
TrkB 140kDa 100
90kDa * *

expression
pAkt 80
60kDa *
60
Bcl-xL 30kDa 40
α-Tubulin 52kDa 20
0
Trk B pAkt1 Bcl-xL

c 5
Fold change in mRNA expression

4.5
4
3.5
3 Control
2.5 Glu

2 # B-TCE
* B-TCE+ Glu
1.5
# #
1 * *
* * *
0.5 * *

0
Trk B PI3K Akt Bcl-xL

Fig. 7  B-TCE promoted cell survival and suppressed glutamate-
induced apoptosis by modulating AKT pathway and Bcl-xL expres-
sion. a Representative immunoblots and b histograms showing
relative change in expression of TrkB, pAkt, and Bcl-xL of control,
glutamate-, B-TCE-, and B-TCE+ glutamate-treated animals after
normalization with endogenous control α-Tubulin. c Histogram pre-
senting fold change in mRNA expression of TrkB, PI3K, Akt, and
Bcl-xL in all the four experimental groups. *p ≤ 0.01 depicts statis-
tically significant difference of glutamate, B-TCE, and B-TCE+ glu-
tamate treatment groups as compared to Control. #p ≤ 0.01 depicts
statistically significant difference of B-TCE and B-TCE+ glutamate
treatment groups as compared to glutamate

observation was also supported by mitochondrial dysfunc-
tion, as indicated by higher intensity of mitotracker staining
in glutamate-treated primary hippocampal neurons. B-TCE
pre-treatment suppressed the glutamate-induced changes in
cell cycle regulatory proteins, mitochondrial dysfunction,
and induction of apoptosis as was evident from observa-
tions of downregulation in the expression Cyclin D1, AP-1,
and reduced intensity of mitotracker green FM stain in
B-TCE+ glutamate treatment group. Upregulated expression
of anti-apoptotic protein Bcl-xL in B-TCE-treated groups
further supports the role of B-TCE in suppressing glutamate-
induced apoptosis.
B‑TCE Ameliorated Glutamate‑Induced Behavioral
Deficits in Wistar Albino Rats
Glutamate treatment also resulted in cognitive deficits
and anxiety-like behavior as well as motor coordination
impairment. Glutamate exposure has been recently reported
to impair memory and learning capabilities in rodents
(Araujo et al. 2017; Moneim et al. 2018). The current study
reinforces these findings as animals treated with glutamate
exhibited reduced exploratory activity, impaired cogni-
tion, and working memory as is evident from preference
index (PI < 0.5), significantly lesser number of episodes
and less time spent with novel object (in NORT). However,
B-TCE treatment in both B-TCE alone and B-TCE+ glu-
tamate treatment groups improved learning and memory
functions as suggested by higher preference towards novel
object (PI > 0.5), significantly higher time spent with novel
object and in overall exploration in animals of these groups.
Improvement in learning and memory by B-TCE may also
be attributed to enhanced expression of plasticity mark-
ers PSA-NCAM and NCAM, which play important role in
synaptic plasticity and memory consolidation (Maness and
Schachner 2007; Markram et al. 2007).

13
NeuroMolecular Medicine
Decline in exploratory activity may also be due to anxi-
ety-like behavior induced by glutamate treatment (Rosa et al.
2016). Confinement of glutamate-treated animals to closed
arm suggested their lack of interest in novel space explora-
tion and anxiety-like behavior as was evident from EPM test
parameters. However, B-TCE treatment suppressed the anxi-
ety and improved the exploratory activity as clearly evident
from significantly more time spent in open arm, higher num-
ber of entries and crossings to both the arms by animals of
B-TCE treatment groups. Improved exploratory activity was
further supported by more number of head dips and rearings,
which are considered as parameters of exploring tendency
towards novel space (Mishra et al. 2016). Antioxidant-rich
nature of T. cordifolia is considered as the basis of anxiolytic
potential of this herb (Kosaraju et al. 2014). B-TCE treat-
ment also suppressed glutamate-induced neuro-muscular
and motor coordination impairment as suggested by lesser
number of falls and more time spent on rotating rod by these
animals. Impaired locomotion and poor rotarod performance
have been earlier reported in glutamate-treated Wistar albino
rat pups (Kiss et al. 2005). Alcoholic extract of T. cordifolia
has been reported to improve motor coordination in albino
rat models of Radiculopathy (Santharani and Lavanya 2012).
B‑TCE Modulated the Expression of Molecular
Markers of Structural Plasticity, Inflammation
and Survival Pathway
B-TCE treatment was effective to suppress neurodegenera-
tion as evidenced by MAP-2 expression and the changes
were similar to in vitro observations. Loss of MAP-2 expres-
sion is associated with neurotoxin-induced or age-related
neurodegeneration (Melo et al. 2013; Taylor-Walker et al.
2016; Lana et al. 2017). Glutamate-mediated excitotox-
icity has also been reported to cause reactive gliosis and
neuroinflammation (Tilleux and hermans 2007; Shah et al.
2016). The current data also showed that pro-inflamma-
tory cytokine production was accompanied by astrocyte
activation. Increase in GFAP expression and the activated
morphology of astrocytes in DG region of brain of gluta-
mate-treated animals are suggestive of glutamate excito-
toxicity-induced reactive gliosis. Further, enhanced circu-
latory levels of pro-inflammatory cytokines TNF-α, IL-6,
and IL-1β in glutamate-treated animals also suggested the
induction of neuroinflammation. B-TCE treatment abrogated
this glutamate-induced astrocytic activation and increase
in pro-inflammatory cytokines levels. Different alcoholic
extracts of T. cordifolia have been previously reported to
suppress neuroinflammation in animal models of sleep dep-
rivation and carrageenan-induced edema (Hussain et al.
2015; Mishra et al. 2016). The anti-inflammatory potential
of B-TCE suggested by suppression of pro-inflammatory
cytokines and GFAP expression may also be responsible
for anxiolytic effect of B-TCE as observed in EPM test.
Most of the excitatory neurotransmission in the CNS is
mediated by AMPA receptors and their function is altered in
neurodegenerative diseases (Chang et al. 2012). Increase in
expression of GluR1, GluR3, as well as enhanced phospho-
rylation of GluR1 and GluR2 subunits in the hippocampus
region of glutamate-treated animals was suggestive of gluta-
mate-induced overactivation of AMPARs and excitotoxicity.
These data are in line with the previous reports showing ele-
vated AMPARs (GluR2/3/4) as well as pAMPAR (GluR1)
expression by glutamate-induced excitotoxicity in hip-
pocampus and cortex regions of developing rat brain (Shah
et al. 2014). B-TCE exerted neuroprotection by significantly
downregulating the expression of total and phosphorylated
AMPARs. In addition to fast excitatory neurotransmission
by AMPARs, NMDARs also mediate comparatively slow
and prolonged excitatory neurotransmission. Overactivation
of NMDARs also leads to impaired synaptic plasticity and
neurodegeneration, which has been implicated in various
neurodegenerative diseases (Benarroch 2011; Zhou et al.
2013). Out of the three subunits of NMDARs, NRDAR2A is
being implicated in the induction of long-term potentiation
(LTP) (Liu et al. 2004; Fox et al. 2006). Glutamate-induced
downregulation in NMDAR2A expression was suggestive
of impaired LTP induction, which was further supported by
decrease in expression of presynaptic protein pSynapsin I in
the current as well as in literature reports (Fang et al. 2017;
Marsh et al. 2017; Shi et al. 2017). Animals in B-TCE+ glu-
tamate treatment group showed recovery in NR2A and
pSynapsin I expression as compared to glutamate treatment
group, thus, supporting the neuroprotective role of B-TCE.
Further, we studied the effect of B-TCE treatment
on expression of ­C a 2+/Calmodulin-dependent kinase,
CaMKIIα and phosphatase, calcineurin. CaMKIIα acts as
a basic substrate for initial memory formation and phos-
phorylates various synaptic proteins (Moyano et al. 2005),
whereas, calcineurin modulates the expression of CaMKIIα.
B-TCE prevented the glutamate-induced downregulation of
CaMKIIα, thus ameliorating neuronal vulnerability to cell
death and promoting synaptic plasticity (Ashpole and Hud-
mon 2011; Ashpole et al. 2013). Upregulation in the expres-
sion of CaMKIIα may also explain the improvement in
memory by B-TCE as CaMKII has been reported to enhance
post-learning tasks (Moyano et al. 2005). Elevated levels of
calcineurin by hyperactivation of glutamate receptors are
known to be associated with apoptosis induction in primary
hippocampal neurons as well as in different cell lines (Wang
et al. 1999; Szado et al. 2008; Dong et al. 2009; Ruiz et al.
2010). Significant downregulation of calcineurin expression
by B-TCE treatment further confirms the role of B-TCE in
prevention of impaired plasticity and excitotoxic damage.
13

PSA-NCAM and NCAM are the key molecules regulat-
ing structural plasticity, which play a major role in cognition
(Nacher et al. 2013). Pronounced decrease in PSA-NCAM
expression in hippocampus region of glutamate-treated
animals may indicate derangement in structural plasticity
due to glutamate exposure. B-TCE treatment group showed
higher expression of PSA-NCAM and mRNA expression of
NCAM and PST as compared to glutamate-treated animals
and changes in PSA-NCAM expression were in line with
in vitro observations. Higher expression of PSA-NCAM in
B-TCE-treated animals is supported by increase in NCAM
protein and mRNA expression of PST, which catalyzes poly-
sialylation of NCAM (Aonurm-Helm et al. 2016).
B-TCE treatment induced activation of ERK pathway and
promoted synaptic plasticity as was evident from signifi-
cantly high expression of pMEK1/2 and pErk1/2 in B-TCE-
treated animals as compared to glutamate treatment group.
Further, ionotropic glutamate receptors regulate neurotro-
phin receptor activation, which further activates the survival
PI3K/Akt pathway in neurons under glutamate-mediated
excitotoxicity conditions (Zhu et al. 2005). The current data
also showed downregulation in expression of TrkB and
downstream effectors PI3K and Akt by glutamate. B-TCE
treatment, however, attenuated glutamate-induced changes
in TrkB-PI3K/Akt pathway, thus promoting cell survival.
B-TCE treatment also suppressed glutamate-induced apop-
tosis as suggested by enhanced expression of anti-apop-
totic protein Bcl-xL. The signaling pathways involved in
neuroprotection by various agents such as BDNF, VEGF,
17β-estradiol, and stem cell factor have been extensively
studied. Ras-MAPK pathway, i.e., ERK and PI3K/Akt are
major pathways which get activated and promote various
functions including LTP, synaptic plasticity, and survival
(Zhu et al. 2005; Almeida et al. 2005; Melo et al. 2013). Akt
promotes release of anti-apoptotic proteins such as Bcl-2 and
Bcl-xL via inhibitory phosphorylation of another molecule
BAD (Tang et al. 2000).
Medicinal properties of T. cordifolia have been attrib-
uted to phytochemical constituents including glycosides,
alkaloids, diterpenoids, polysaccharides, sesquiterpenes,
and phenolics (Saha and Ghosh 2012). Alkaloids, glyco-
sides, and phenolics have been reported to be responsible
for neuroprotective and immunomodulatory activities of
this plant (Saha and Ghosh 2012; Joshi and Kaur 2016).
In LCMS-based analysis, we previously reported that pal-
matine, oblongine, norcoclaurine, tetrahydropalmatine, mag-
noflorine, tinocordiside, cordifolioside A, and 11-hydroxy
mustakone are present in B-TCE (Sharma and Kaur 2018).
Few other studies also reported the presence of glycosides
cordifolioside A, tinocordiside, and some alkaloids in
n-Butanol fraction of T. cordifolia stem extract (Ghosal and
Vishwakarma 1997; Patel et al. 2013). It may be suggested
that multiple components present in B-TCE act in synergism
13
NeuroMolecular Medicine
to exert neuroprotection against glutamate-induced excito-
toxicity. Alkaloids such as palmatine, tetrahydropalmatine,
and magnoflorine have been reported to reduce oxidative
stress, thus may prevent neurodegeneration by suppressing
ROS generation and neuroinflammation (Gupta and Sharma
2011). Further, tinosporicide was isolated as pure compound
from B-TCE, which exhibited neuroprotective activity at
considerably lower concentrations (~ 800 folds).
Conclusion
The current data revealed that B-TCE prevented glutamate-
induced neurotoxicity in both primary culture of hippocam-
pal neurons as well as in hippocampus region of brain by
targeting glutamatergic neurotransmission (via glutamate
receptors) and downstream NMDAR, ERK, PI3K/Akt path-
ways of synaptic plasticity and cell survival. Additionally,
our study provided the first evidence of neuroprotective
activity of tinosporicide, an active compound from B-TCE.
The findings of current study may encourage future transla-
tional research for management of glutamate-induced neu-
rotoxicity and neurodegeneration.
Acknowledgements  AS, SK, and PB are thankful to Department of
Science and Technology (DST), New Delhi, India for their Research
Fellowship. HS is thankful to UGC, India for his research fellowship.
Infrastructure provided by University grants commission (UGC),
India under UPE and CPEPA schemes and DBT under DISC scheme
is highly acknowledged. Dr. Bikram Singh (Institute of Himalyan
Bioresource Technology), Dr. Amrita K. Cheema, Ms. Kirandeep Gill
(Georgetown University, USA), and Dr. Shreyans Jain (IIT-BHU, Vara-
nasi) are deeply acknowledged for their kind support to characterize
the compound. Ms. Muskan Gupta, Dr. Taranjeet Kaur, and Dr. Shaffi
Manchanda are acknowledged for their technical help during experi-
mental study.
Author Contributions  AS and GK designed the study. AS conducted
the experiments and analyzed data. SK, PB, and HS performed behav-
ioral studies and analyzed the behavioral data. Manuscript was written
by AS and GK. Funding to carry out the reported work was provided
by GK. All the authors reviewed and approved the final manuscript.
Funding This work was funded by Department of Biotech-
nology (DBT), GOI to Prof. Gurcharan Kaur [BT/PR12200/
MED/30/1439/2014]. The funding agencies had no role in design of
study, data collection and interpretation, manuscript preparation, and
decision to publish the study.
Compliance with Ethical Standards 
Conflict of interest  Authors declare no conflict of interest.
Ethical Approval  All procedures performed in studies involving ani-
mals were in accordance with the ethical standards of the Institutional
Animal Ethical Committee, Guru Nanak Dev University, Amritsar
registered to “Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA), GOI” (Registration no. 226/
CPCSEA) (permission number 226/CPCSEA/2017/01).
NeuroMolecular Medicine

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