Biochemical and Biophysical Research Communications 413 (2011) 605–610

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Anti-Parkinsonian effects of Bacopa monnieri: Insights from transgenic

and pharmacological Caenorhabditis elegans models of Parkinson’s disease
Pooja Jadiya a, Asif Khan b, Shreesh Raj Sammi a, Supinder Kaur a, Snober S. Mir b, Aamir Nazir a,⇑
a
Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226001, UP, India
b
Department of Biotechnology, Integral University, Lucknow 226026, UP, India

a r t i c l e i n f o a b s t r a c t

Article history: Neurodegenerative Parkinson’s disease (PD) is associated with aggregation of protein alpha synuclein and
Received 22 August 2011 selective death of dopaminergic neurons, thereby leading to cognitive and motor impairment in patients.
Available online 8 September 2011 The disease has no complete cure yet; the current therapeutic strategies involve prescription of dopamine
agonist drugs which turn ineffective after prolonged use. The present study utilized the powerful genetics
Keywords: of model system Caenorhabditis elegans towards exploring the anti-Parkinsonian effects of a neuro-
Bacopa monnieri protective botanical Bacopa monnieri. Two different strains of C. elegans; a transgenic model expressing
Alpha synuclein
‘‘human’’ alpha synuclein [NL5901 (Punc-54::alphasynuclein::YFP+unc-119)], and a pharmacological
Parkinson’s disease
Caenorhabditis elegans
model expressing green fluorescent protein (GFP) specifically in the dopaminergic neurons [BZ555
Neurodegeneration (Pdat-1::GFP)] treated with selective catecholaminergic neurotoxin 6-hydroxy dopamine (6-OHDA), were
employed for the study. B. monnieri was chosen for its known neuroprotective and cognition enhancing
effects. The study examined the effect of the botanical, on aggregation of alpha synuclein, degeneration of
dopaminergic neurons, content of lipids and longevity of the nematodes. Our studies show that
B. monnieri reduces alpha synuclein aggregation, prevents dopaminergic neurodegeneration and restores
the lipid content in nematodes, thereby proving its potential as a possible anti-Parkinsonian agent. These
findings encourage further investigations on the botanical, and its active constituent compounds, as
possible therapeutic intervention against Parkinson’s disease.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction
Neurodegenerative diseases like Alzheimer’s, Parkinson’s, Hun-
tington’s and multiple sclerosis are associated with the process of
memory loss and cognitive decline which results from selective
degeneration of particular neuronal cells and the accretion of
aggregated proteins [1]. Parkinson’s disease (PD) is predominantly
characterized as a movement disorder but non-motor symptoms
are also involved. It affects dopamine-producing neurons in the
brain, and the loss of these neurons induces the symptoms of PD.
Since dopamine is associated with motor activity, therefore the
progressive loss of dopaminergic neurons leads to muscle rigidity,
tremors and bradykinesia as well as cognition, mental, sleeping,
personality and behaviour disorders including depression and anx-
iety [2]. It is an age related disorder because the incidence for PD
increases rapidly in the population cohort exceeding 60 years of
age [2,3]. At present, the etiology of PD is still not clearly known.
Abbreviations: BM, Bacopa monnieri; 6-OHDA, 6-hydroxy dopamine; PD, Parkin-
son’s disease; GFP, green fluorescent protein; DA, dopamine.
⇑ Corresponding author. Fax: +91 522 2623405.
E-mail address: anazir@cdri.res.in (A. Nazir).
Alpha synuclein becomes the major protein in Lewy bodies which
has a key role in pathogenesis of both familial and sporadic PD [4].
Currently, there is no cure for PD and the drugs used for treat-
ment are dopamine agonists and monoamine oxidase-B (MAO-B)
inhibitors, which provide only symptomatic relief. Considering
the promise that several natural products have yielded in treating
various ailments, such products as herbs, medicinal plant extracts
and other such botanicals are being explored for their therapeutic
potentials in neuronal disorders. In this quest, some herbs have
been found to be effective neuroprotectants. Bacopa monnieri
(BM), commonly known as Brahmi, an ayurvedic medicinal plant
acting as anti-oxidant [5], anti-depressant [6], anti-inflammatory
and anti-microbial [6–8], is the most popular neurotonic and a well
known memory booster [9], which, we reasoned, could be tested for
its beneficial effects on the neurodegenerative PD. B. monnieri, a
member of the Scrophulariaceae family [10] assists in heightening
mental acuity and supports the physiological processes involved
in relaxation. Extracts of B. monnieri have been reported to exert
cognitive enhancing effects in animals [11,12]. Research on anxiety,
epilepsy, bronchitis and asthma, irritable bowel syndrome, and
gastric ulcers also support the Ayurvedic uses of Bacopa [13].
We employed transgenic Caenorhabditis elegans model to
evaluate the effect of BM on Parkinson’s disease. Since its 60–80%

0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2011.09.010
606 P. Jadiya et al. / Biochemical and Biophysical Research Communications 413 (2011) 605–610
genes are homologous to human [14] and it also has orthologs of
PD associated genes, model system C. elegans may help in estab-
lishing an insight into therapeutic aspects of PD. Transgenic strain
expressing ‘human’ alpha synuclein, helps in studying the effect of
its aggregation on the gross phenotype and provides a robust mod-
el to study the effect of any potential therapeutic agent [4,15–19].
The transparent anatomy of the nematode helps in monitoring the
aggregation of alpha synuclein protein and degeneration of dopa-
mine neurons. Further, dopaminergic neurodegeneration can
easily be induced by neurotoxins such as 6-hydroxydopamine
(6-OHDA) [20] thus providing a pharmacological model of PD.
Hence, the present study took advantage of the model system C.
elegans towards examining the anti-Parkinsonian effects of
neuro-protective botanical B. monnieri.
2. Materials and methods
2.1. C. elegans culture and maintenance
Maintenance of C. elegans was carried out as described
previously by Brenner [21,22]. Worms were raised on the OP50
seeded standard Nematode Growth Medium (NGM) and grown at
22 °C. In this study, wild type Bristol N2, transgenic strain NL5901
(Punc-54::alphasynuclein::YFP+unc-119; expressing human alpha
synuclein protein with YFP expression in the muscles) and trans-
genic strain BZ555 (Pdat-1::GFP; bright GFP observable in dopamine
neuronal soma and processes) were used. These strains were ob-
tained from the Caenorhabditis Genetics Center (University of
Minnesota).
2.2. Treatment of worms with B. monnieri
Concentrated mother tincture of B. monnieri extract, which is
used as a homeopathic medicine in India, was obtained (SBL Pri-
vate Limited, Uttaranchal, India). The objective of using the mother
tincture was to study this homeopathic drug ‘as is’, so as to evalu-
ate the anti-Parkinsonian effects of the whole mother tincture of
the botanical. Any encouraging observations will lead to detailed
studies on various active constituents of the extract.
The mother tincture was diluted tenfold in OP50 before seeding
onto NGM plates. The plates were incubated overnight for opti-
mum growth of bacteria OP50 following which, age synchronized
worms were grown on the plates, for further studies.
2.3. Treatment of worms with 6-hydroxy dopamine (6-OHDA) and
6-OHDA-BM
To generate selective degeneration of dopaminergic neurons,
worms were exposed to 6-hydroxy dopamine (6-OHDA; Sigma,
St. Louis, MI; catalogue No. H4381) at concentrations of 25 mM
[20]. Briefly, 6-OHDA was mixed with OP50 and seeded onto
NGM plates. To prepare 6-OHDA-BM treatment plates, BM was
added along with OP50 at this stage. Plates were then incubated
overnight. Embryos of BZ555 strain of C. elegans were then trans-
ferred onto these prepared treatment plates for 48 h at 22 °C. Baco-
pa extract is known to be stable, as studies have reported beneficial
effects of the extract over long periods after extraction.
2.4. Assay for analysis of alpha synuclein protein aggregation
Aggregation of alpha synuclein protein was observed in control
and BM treated NL5901 strain of C. elegans. After 48 h. of treat-
ment, worms were washed thrice with M-9 buffer to remove
adhering bacteria and transferred to agar padded slides (2% aga-
rose) and sealed with a cover slip. Worms were immobilized with
100 mM sodium azide (Sigma, cat No. 71289). Imaging of live
(immobilised) worms using confocal microscopy (Carl Zeiss) was
carried out to monitor the aggregation of alpha synuclein protein
and aggregation was quantified using image J software (Image J,
National Institutes of Health, Bethesda, MD) by measuring flores-
cence intensity. Statistical analysis was carried out using Graph
Pad software package; calculation of statistical significance
between various groups was carried out employing Student’s t test.
2.5. Assay for analysis of dopaminergic neurodegeneration
Study of dopaminergic neurodegeneration was carried out by
exposing worms with 6-OHDA and 6-OHDA-BM as described pre-
viously in Section 2.3. Any adhering bacteria were removed after
48 h of treatment by washing the worm pellet three times before
mounting the worms onto agar padded glass slide using 100 mM
sodium azide. Imaging of live (immobilised) worms was carried
out to monitor the dopaminergic neurodegeneration in control
and experimental conditions using laser scanning confocal micro-
scope (Carl Zeiss). Fluorescence intensity was quantified using Im-
age J software (Image J, National Institute of health, Bethesda, MD).
2.6. Nile red staining of lipid deposits
Lipid specific dye – Nile Red (Cat. No. N1142, Invitrogen) was
mixed with Escherichia coli before seeding it onto the NGM plates
[23]. Nile Red stock solution was prepared by dissolving 0.5 mg
Nile Red dye in 1 mL of acetone. Nile Red was fed to the worms
by mixing it with E. coli OP50 in the ratio 1:250 as described pre-
viously [23]. The synchronous aged embryos were transferred onto
the Nile-Red containing plates and grown for 48 h at 22 °C. Worms
were washed off and mounted onto 2% agarose pads using sodium
azide. The extent of fat staining was assessed using fluorescence
upright microscope (Nikon).
2.7. Life span study
Life span assay was performed by transferring adult worms
every other day, from control as well as BM treatment condition,
to a fresh control or treated plate. This was done to avoid mixing
of multiple generations. The number of live, missing and dead
worms was counted each day until the last worm was dead. Anal-
yses were carried out by two different investigators. Survival curves
were plotted using the product-limit method of Kaplan and Meier;
statistical analyses were performed using SPSS software [24].
3. Results and discussion
3.1. B. monnieri reduced alpha synuclein protein aggregation
Worms of untreated and B. monnieri treated groups were ob-
served under confocal microscope for assaying their alpha synuc-
lein aggregation pattern. Phenotypically, the worms appeared
normal and optimally fed. We observed a marked and significant
reduction in the aggregation of alpha synuclein in case of NL5901
worms treated with B. monnieri (Fig. 1C) as compared to that of
control group (Fig. 1A). We quantified the images for fluorescence
intensity of alpha synuclein aggregation using Image J software.
Treatment of worms with BM showed significantly reduced fluo-
rescence intensity of aggregation as compared to that of untreated
worms. The mean fluorescence (GFP) intensity was 8.950 ± 0.6195
arbitrary units in control worms and 2.550 ± 0.2500 arbitrary units
in BM treated subjects (Fig. 1E). A 3.5-fold reduction (p < 0.05) in
alpha synuclein protein aggregation was observed in BM exposed
worms. This preventive effect on alpha synuclein aggregation
 P. Jadiya et al. / Biochemical and Biophysical Research Communications 413 (2011) 605–610
Fig. 1. Alpha synuclein aggregation in NL5901 strain of C. elegans fed on OP50 (A and B), Bacopa monnieri (C and D). Images A and C are fluorescent images, B and D are images
grabbed using Differential Interference Contrast (DIC) optics. Scale bar, 50 lm. Fig. 1E is graphical representation for fluorescence intensity of the nematodes as quantified
using Image J software ⁄p < 0.05.
could be as a result of the effect of BM on protein aggregation med-
iated by expression of stress proteins within the system. It is very
well known that a suite of proteins within the living systems,
called stress proteins, buffer the cells from harm under stressful
conditions [25,26]. B. monnieri, has previously been reported to in-
duce such chaperoning protein called Hsp-70 [27]. Such an effect of
BM on stress proteins, might be leading to dis-aggregation of alpha
synuclein deposits or the unfolding of mis-folded proteins that
otherwise are the part of toxic aggregates. The protective effect
of Bacopa could also be attributed to the anti-oxidant properties
of the botanical, as it has been reported that Bacopa scavenges free
radicals and reduces lipid peroxidation in tissues [5,28]. Studies
have also reported enhancement of anti-oxidant enzyme levels as
a result of treatment with Bacopa [5,28]. These protective proper-
ties of B. monnieri make it tempting for us to speculate that the ex-
tract might be decreasing the aggregation of alpha synuclein
thereby reducing its toxic outcome in the cells, which, in case of
our in vivo studies employing whole organismic environment of
model system C. elegans, makes the studies more relevant in over-
all disease condition.
3.2. 6-OHDA induced selective degeneration of dopaminergic neurons
was prevented with B. monnieri
The significance of using C. elegans as a model to study neuronal
disorders lies in the fact that its nervous system is simple, compris-
ing of precisely 302 neurons wired by 7000 synapses, each neuron
can be individually identified and the entire connectivity of the ner-
vous system has been reconstructed. Most significantly, all gene
families involved in neuronal function in mammals are present in
the worm. Of importance to the present study, C. elegans contains
precisely eight dopaminergic neurons (Fig. 2A and B); two pairs of
CEP neurons and one pair of anterior deirid (ADE) neurons in head
region. In a posterior lateral position, it has another pair of posterior
deirid (PDE) neurons. Selective degeneration of these dopaminergic
neurons was achieved through exposure to 6-OHDA [20,29]. We
found that processes of CEP and ADE neurons showed complete
GFP loss with moderate reduction in GFP expression in PDE neurons
(Fig. 2C and D). When worms were fed on 6-OHDA-BM plates,
significant protection was found in dopaminergic neurons with
607
CEP, ADE and PDE neurons exhibiting an enhanced expression of
GFP; (Fig. 1E and F). We further quantified the images for fluores-
cence intensity in DA neurons using Image J. In control worms, the
mean fluorescence (GFP) intensity was 8.158 ± 0.2380 arbitrary
units whereas it was reduced to 3.050 ± 0.05000 arbitrary units in
6-OHDA treated subjects (a 2.7-fold reduction (p < 0.05) as com-
pared to that of untreated worms). Worms raised on 6-OHDA-BM
plates exhibited a fluorescent intensity of 6.900 ± 0.1000 arbitrary
units (a 2.3-fold increase (p < 0.001) as compared to 6-OHDA treated
subjects (Fig. 2G). This neuroprotective role of BM in 6-OHDA in-
duced dopaminergic neurodegeneration is probably associated with
its anti-oxidant [5,30] and anti-apoptotic activity [31]. Since dopa-
minergic neurons are more susceptible for ROS and oxidative stress
plays an important mediator role in neuronal cell death and it con-
sequently induces apoptosis. Although the contribution of ROS and
apoptosis in 6-OHDA-induced neuronal cell death have been fully
elucidated [32,33]. BM may be able to suppress oxidative stress of
these neuronal cells. Furthermore, neuroprotective effect of BM
against rotenone, pharmacological model of PD, has also been re-
ported in Drosophila melanogaster model [34]. It is also well known
that the central components of apoptotic pathways are the proteases
of the caspase family. Caspase-3 is a potent effector of apoptosis trig-
gered via several different pathways in a variety of mammalian cell
types, and is one of the most important caspases in the cytochrome
c-dependent apoptosis pathways [35]. Activation of caspase-3 ap-
pears to be a key event in apoptosis. Furthermore, it has also been
postulated that mitochondrial dysfunction triggered by 6-OHDA in-
duces release of cytochrome c, and the activation of caspase-3
[32,36,37]. The protective effect of B. monnieri could probably be
associated with such events as activation of bcl-2, maintaining the
stability of MMP, and decreasing the activation of caspase-3 through
the mitochondria-dependent pathway; however such mechanistic
aspects need to be explored in greater detail.
3.3. B. monnieri restored lipid content in alpha synuclein expressing
model of C. elegans
PD is known to be associated with altered levels of fatty acids
and lipid content. We assayed the lipid levels in alpha synuclein
608 P. Jadiya et al. / Biochemical and Biophysical Research Communications 413 (2011) 605–610

Fig. 2. GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain BZ555. Control (A and B), 6-OHDA treated (C and D), 6-OHDA treated worms raised on
BM (E and F). Images A, C and E are fluorescent images; B, D and F are Differential Interference Contrast (DIC) images. Scale bar, 50 lm. Fig. 2G is graphical representation for
fluorescence intensity of GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain as quantified using Image J software ⁄p < 0.05, ⁄⁄p < 0.001.

expressing worms, vis-a-vis normal (wild type) worms and worms
treated with BM. Nile Red staining was carried out to fluorescently
label the lipids within nematodes. The fluorescent intensity across
various groups of worms was analysed by fluorescent microscopy
and quantified using Image J software. Worms of control group
exhibited an optimum level of lipids (Fig. 3A), the mean fluorescent
intensity of the group, as quantified by Image J, being 39.06 ± 4.158
arbitrary units. The lipid content in alpha synuclein expressing
worms was reduced (Fig. 3B) as the fluorescent intensity of Nile
Red staining was found to be 17.77 ± 1.804 arbitrary units, thereby
exhibiting a 2.2-fold reduction (p < 0.05) as compared to the
control group. When the alpha synuclein expressing worms, were
treated with B. monnieri, they showed an increased staining pattern
for Nile Red along the entire body of worms (Fig. 3C) depicting an
increase in lipid content of the worms; the content in this group of
worms appeared to be similar as seen for wild type worms; the
mean fluorescence intensity being 45.37 ± 4.676 arbitrary units,
which is a 2.6-fold increase (p < 0.05) with respect to that of un-
treated NL5901 worms.
The reduction of lipid content in alpha synuclein expressing
worms is because of disturbed lipid composition caused as a result
of alpha synuclein toxicity within the worms. Further, the toxic
nature of these protein aggregate species tends to increase the lipid
peroxidation in the tissues via increase in burden of reactive oxy-
gen species [38]. The enormous abundance of lipid molecules in
the central nervous system (CNS) suggests that their role is not
limited to be structural and energetic components of cells. Some
lipids in the CNS are known to function in neurotransmission. Per-
turbations in cellular signalling have been reported in virtually
every neurodegenerative disease. Spatial and temporal features
of cellular signalling are in part controlled by lipid components
that can regulate protein location and scaffolding events through
a dynamic modulation of membrane microdomains. This protec-
tive effect of BM could be for the known anti-oxidant properties
of the botanical that leads to reduction in reactive oxygen species
thus resulting in a reduced lipid peroxidation [39]. Such a protec-
tive effect, thus, mediates the prevention of disturbed combination
of lipids thereby resulting in a rather efficient cellular signalling.
3.4. B. monnieri increased the life span of wild type worms
The effect of B. monnieri on the longevity of worms was studied.
We observed that B. monnieri increased the life span of wild type
worms marginally whereas the alpha synuclein expressing worms
did not cause any significant effect on the longevity of worms. The
cumulative survival patterns, as calculated by Kaplan–Meier sur-
vival analysis of each group are depicted in Fig. 4. The mean sur-
vival for N2-BM group was 13.57 ± 0.03 days vs. 12.2 ± 0.17 days
of N2-OP50 condition (significant at p < 0.05). However the life
span in NL5901-BM group was 12.72 ± 1.54 days which was statis-
tically insignificant at the studied concentration as compared to
that of control group. The subtle effect induced by BM on the lon-
gevity of nematodes could be attributed to their anti-oxidant and
anti-stress properties. Parkinson’s disease is associated with ageing
and in the process of ageing, accumulation of free radicals, cogni-
tive impairment and memory loss has been proposed as the causal
factor. Further, the beneficial effects of Bacopa were observed in
the control group too, which proves that the effects are indeed as
a result of absorption of Bacopa into the system and could not be
because of any artifactual interaction in the culture media. In
Ayurvedic materia medica, B. monniera is commonly mentioned
as a rasayana which manifests its effect to prevent ageing, increase
longevity, impart immunity and improve mental functions. How-
ever, BM has already been utilized for its anti-ageing action [40].
Our studies provide strong evidence that B. monnieri exhibits a
significant ameliorative potential against alpha synuclein effects.
 P. Jadiya et al. / Biochemical and Biophysical Research Communications 413 (2011) 605–610 609

Fig. 3. Nile Red staining of wild type C. elegans fed with OP50 (A), NL5901 strain of C. elegans treated with OP50 (B), NL5901 strain of C. elegans fed on Bacopa monnieri (C).
Scale bar, 50 lm. Fig. 3D is graphical representation for fluorescence intensity of the nematodes as quantified using Image J software ⁄p < 0.05.

regarding the study on its mechanistic aspects by which it affords

the amelioration. Our studies encourage further investigations on
BM as a possible therapeutic intervention against Parkinson’s
disease.

Acknowledgments

Confocal Microscopy facility of CDRI is acknowledged for their

assistance in imaging; in particular Mr. Manish Singh is gratefully
acknowledged for his technical assistance. Nematode strains used
in this work were provided by the C. elegans Genetics Center
(CGC), University of Minnesota, MN, USA, which is funded by the
NIH National Center for Research Resources (NCRR). CDRI commu-
nication No. 8126.

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