See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/232280925

Bacopa monnieri and L-Deprenyl Differentially Enhance the Activities of

Antioxidant Enzymes and the Expression of Tyrosine Hydroxylase and Nerve
Growth Factor via ERK 1/2 and NF-κB...

Article  in  Neurochemical Research · October 2012

DOI: 10.1007/s11064-012-0902-2 · Source: PubMed

CITATIONS READS

27 394

4 authors, including:

Hannah Priyanka Preetam Bala

Tamil Nadu Government Multi Super Speciality Hospital MIT-ADT University
21 PUBLICATIONS   260 CITATIONS    9 PUBLICATIONS   57 CITATIONS   

SEE PROFILE SEE PROFILE

Srinivasan Thyagarajan
SRM Institute of Science and Technology
67 PUBLICATIONS   1,288 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Monoamines and the regulation of reproductive function: Effects of Aging View project

Controlled Drug delivery through Nanoparticle View project

All content following this page was uploaded by Preetam Bala on 04 February 2015.

The user has requested enhancement of the downloaded file.

Neurochem Res
DOI 10.1007/s11064-012-0902-2
ORIGINAL PAPER
Bacopa monnieri and L-Deprenyl Differentially Enhance
the Activities of Antioxidant Enzymes and the Expression
of Tyrosine Hydroxylase and Nerve Growth Factor via ERK
1/2 and NF-jB Pathways in the Spleen of Female Wistar Rats
Hannah P. Priyanka • Preetam Bala •
Sindhu Ankisettipalle • Srinivasan ThyagaRajan
Received: 14 May 2012 / Revised: 12 September 2012 / Accepted: 4 October 2012
Ó Springer Science+Business Media New York 2012
Abstract Aging is characterized by development of dis-
eases and cancer due to loss of central and peripheral neuro-
endocrine-immune responses. Free radicals exert deleterious
effects on neural-immune functions in the brain, heart, and
lymphoid organs and thus, affecting the health. Bacopa
monnieri (brahmi), an Ayurvedic herb, and L-deprenyl, a
monoamine oxidase-B inhibitor, have been widely used in the
treatment of neurodegenerative diseases. The purpose of this
study was to investigate whether brahmi (10 and 40 mg/kg
BW) and deprenyl (1 and 2.5 mg/kg BW) treatment of
3-month old female Wistar rats for 10 days can modulate the
activities of antioxidant enzymes [superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase (GPx)] in
the brain and spleen. In addition, the effects of these com-
pounds on the expression of tyrosine hydroxylase (TH), nerve
growth factor (NGF), the intracellular signaling markers,
p-ERK1/2, p-CREB, and p-NF-kB, and nitric oxide (NO)
production were measured in the spleen by Western blot
analysis. Both brahmi and deprenyl enhanced CAT activity,
and p-TH, NGF, and p-NF-kB expression in the spleen.
However, deprenyl alone was found to enhance the p-ERK1/2
and p-CREB expression in the spleen. The activities of SOD,
CAT, and GPx in the thymus, mesenteric lymph nodes, heart,
and brain areas (frontal cortex, medial basal hypothalamus,
striatum, and hippocampus) were differentially altered by
brahmi and deprenyl. Brahmi alone enhanced NO production
in the spleen. Taken together, these results suggest that both
brahmi and deprenyl can protect the central and peripheral
H. P. Priyanka  P. Bala 
S. Ankisettipalle  S. ThyagaRajan (&)
Integrative Medicine Laboratory, Department of Biotechnology,
School of Bioengineering, SRM University,
Kattankulathur 603 203, Tamil Nadu, India
e-mail: thyagarajan.s@ktr.srmuniv.ac.in
neuronal systems through their unique effects on the antiox-
idant enzyme activities and intracellular signaling pathways.
Keywords Brain  Lymphoid organs  Brahmi 
Superoxide dismutase  Catalase  Glutathione peroxidase 
CREB  Nitric oxide
Introduction
The neuroendocrine system and the immune system com-
municate with each other through bidirectional pathways
involving hormones, neurotransmitters, and cytokines to
maintain homeostasis [1]. In the periphery, the regulation of
immunity by sympathetic noradrenergic (NA) nerve fibers in
the lymphoid organs (bone marrow, thymus, spleen, and
lymph nodes) has been demonstrated by the distribution of
tyrosine hydroxylase (TH?) nerve fibers, presence of
adrenoceptors on the cells of the immune system, and
immunomodulatory role of norepinephrine [2]. With
advancing age, there is a prominent decline in sympathetic
NA innervations in the spleen and lymph nodes associated
with loss of T cell functions including proliferation and IL-2
production [2–4]. The age-related decline in sympathetic
NA activity accompanied by immunosuppression can be
attributed to accumulation of reactive oxygen species due to
loss of antioxidant enzyme activities [5, 6]. Several natural
remedies and drugs such as brahmi (Bacopa monnieri Linn.)
and L-deprenyl have been used to reverse the age-related
loss of functions involving brain and peripheral organs.
Brahmi is a natural herb widely used in Ayurvedic medicine
to treat anxiety, poor memory, and cognitive deficits [7, 8]. The
beneficial effects of brahmi may be mediated through its
properties of enhancing antioxidant enzyme (SOD, CAT, and
GPx) activities in the brain and immune responses [9, 10].
123

Although brahmi is known to enhance humoral and cell-
mediated immune responses [11], the mechanism of action in
the lymphoid organs synergistic with neural-immune param-
eters has not been examined. L-deprenyl, an irreversible
monoamine oxidase-B inhibitor, is widely used in the treat-
ment of neurodegenerative disorders such as Parkinson’s
disease and Alzheimer’s disease to improve age-associated
cognitive deficits [12]. The effects of deprenyl on the brain can
be attributed to increased neuronal activity and growth factor
biosynthesis, antioxidant enzyme activities, and neuroprotec-
tive property [13]. Previously, we have demonstrated that in
addition to deprenyl’s effects on the brain, it can restore
sympathetic NA innervations in the spleens of neurotoxin-
treated young rats and reverse the age-related decline in
splenic NA innervation in old rats [14]. These neurorestorative
properties of deprenyl in the spleen were accompanied by
enhanced Con A-induced IL-2 production and natural killer
cell activity indicating that it possesses immunostimulatory
properties. These effects on the neuroendocrine-immune sys-
tem were also observed in rats with carcinogen-induced and
spontaneously-developing mammary tumors in which depre-
nyl arrested the growth of mammary tumors [14].
In view of the above evidence, it is plausible that both
brahmi and deprenyl used in different medical practices for
the treatment of neurodegenerative diseases possess similar
physiological, biochemical, cellular, and molecular effects.
Therefore, it is vital to understand their mechanism(s) of
action(s) and explore the possibility of using these com-
pounds in other diseases characterized by dysfunctions of
neuroendocrine-immune network. Hence, the present study
was conducted to investigate the role of brahmi and deprenyl
on the antioxidant enzyme activities (SOD, CAT, and GPx)
in the brain, heart, and lymphoid organs (thymus, spleen, and
mesenteric lymph nodes) and the expression of TH and nerve
growth factor (NGF) specifically in the spleen of young
female rats with no deficits in NA innervation and immune
functions. Inducible nitric oxide synthase (iNOS) activity
was also measured in the spleen in terms of nitric oxide (NO)
production that is known to influence immune responses by
inhibiting T cell proliferation. In addition, the members of the
growth factor signaling cascades involving extracellular-
signal-regulated kinases-1/2 (ERK 1/2), cAMP response
element-binding (CREB), and nuclear factor-kB (NF-kB) in
mediating the effects of brahmi and deprenyl on TH and NGF
expression in the spleen were examined.
Materials and Methods
Animals
Female Wistar rats were purchased at 5 weeks of age from
The King’s Institute, Guindy, Chennai and housed for
123
Neurochem Res
acclimatization at the University’s Animal House. The
experiments began when the rats reached the age of
3 months. Food pellets and water were provided ad libitum
and animals were housed under hygienic conditions. All
animal experiments were conducted in accordance with the
principles and procedures outlined and approved by the
Institutional Animal Ethics Committee.
Treatment
The animals (n = 5 per group) were randomly distributed
into a control group, two vehicle-treated groups (one each
for brahmi and deprenyl), two low dose-treatment groups
(10 mg/kg BW of brahmi and 1.0 mg/kg BW of L-deprenyl)
and two high dose-treatment groups (40 mg/kg BW for
brahmi and 2.5 mg/kg BW for L-deprenyl). B. monnieri as
brahmi capsules (Himalaya Health Care Pvt. Ltd., Bangal-
uru, India) and L-deprenyl (Sigma-Aldrich, MO, USA) were
used in this study. The active ingredients of brahmi capsules
were dissolved in saline and administered orally while
deprenyl was dissolved in saline and administered intra-
peritoneally (i.p.) for a period of 10 days. The vehicle-
treated rats received saline either orally or i.p. depending on
the drug treatment. Body weight and food intake were
measured every alternate days throughout the treatment
period. At the end of the treatment period, the animals were
sacrificed by decapitation and heart and lymphoid organs
(thymus, spleen, mesenteric lymph nodes) were dissected
out using aseptic techniques. The tissues were cut into
blocks for further analysis for antioxidant enzyme assays
(SOD, CAT, and GPx) and Western blot analysis (p-TH,
NGF, p-ERK1/2, p-CREB, and p-NF-kB). Brain was
removed and areas in the brain (frontal cortex, medial basal
hypothalamus, striatum, and hippocampus) were microdis-
sected and stored at -80 °C until further analysis.
Antioxidant Enzyme Assay
The SOD activity was measured in terms of percentage inhi-
bition of epinephrine auto oxidation [15]. The tissue was
homogenized in a 5:3 ice-cold mixture of ethanol and chlo-
roform, centrifuged, and the supernatant was used for the
assay. The sample was diluted using 0.1 M carbonate buffer
(pH 10) with the addition of equal volumes of 0.6 mM EDTA
and 1.3 mM epinephrine in carbonate buffer. Subsequently,
the samples were vortexed and read immediately at 480 nm in
a spectrophotometer at time intervals of 0-, 30- and 60-s. The
results were expressed in terms of units/min/mg of protein.
The total CAT activity was measured by a hydrogen per-
oxide-based assay where it forms a complex with ammonium
molybdate [16]. The sample was suspended in 60 mM sodium
phosphate buffer (pH 7.4) with 65 mM hydrogen peroxide
solution and incubated for 4 min after which the reaction was
Neurochem Res
stopped using 32.4 mM ammonium molybdate and the optical
density was measured at 405 nm. The total catalase activity
was expressed in units/min/mg of protein.
The GPx (isoform GPx1) activity was measured using
Ellman’s Reagent [17]. Briefly, the sample was diluted in
0.4 M phosphate buffer (pH 7), 10 mM sodium azide, 4 mM
reduced glutathione, 2.5 mM hydrogen peroxide and incu-
bated for 0-, 11/2- and 3-s after which the reaction was
stopped with 10 % trichloroacetic acid and centrifuged. To
the supernatant, 0.3 M disodium hydrogen phosphate solu-
tion and 1 mM di-thio-nitro-benzene in 1 % sodium citrate
were added. The optical density was read immediately at
412 nm in a spectrophotometer. Standard curve was obtained
using serial dilutions of 0.1 M tri-nitrobenzene in water. The
results were expressed in terms of units/min/mg of protein.
Western Blot Analysis
Spleen samples were washed in ice-cold 0.1 M PBS,
homogenised in lysis buffer (0.005 M Tris, 0.001 M EDTA,
100 lg/ml PMSF, 1 mM activated sodium orthovanadate),
centrifuged at 1,500 rpm for 15 min and the supernatants
obtained were used for blotting. Protein concentration was
estimated using Folin and Ciocalteu’s phenol reagent
(Sigma, St. Louis, MO). Thirty lg of total protein was
electrophoresed on 10 % SDS-polyacrylamide gels and
blotted on 0.2 lm nitrocellulose membranes (Sigma,
St. Louis, MO). The membranes were blocked for 1 h and
incubated overnight in blocking buffer containing primary
antibody (p-TH (Ser 40; 1:750); NGF (M-20; 1:750); ERK
1/2 (MK1; 1:750); p-ERK 1/2 (Tyr 204; 1:750); CREB-1
(240; 1:750); p-CREB-1 (Ser 133; 1:750), p-NF-jB (p50;
Ser 536; 1:750) and b-Actin (C4; 1:3,000) (Santa Cruz
Biotechnology, Santa Cruz, CA). The blots were then
washed with Tris-buffered saline, incubated with HRP-anti
rabbit IgG (1:10,000) (Santa Cruz Biotechnology, Santa
Cruz, CA) and developed using 3,30 ,5,50 -tetramethylbenzi-
dine (TMB) Liquid Substrate System (Sigma, St. Louis,
MO). Western Blotting was performed twice for each
sample and quantified using densitometry in terms of rela-
tive intensity of the blots with reference to control. Signal
intensity of the various molecular markers was measured by
densitometric analysis using Image J 1.45 software (NIH).
Nitric Oxide Production
The total nitric oxide (NO) production in the spleen was
estimated using the Greiss reagent system [18]. The samples
were incubated with equal volume of 0.1 % N-1-napthyle-
thylenediamine dihydrochloride in water and 1 % sulpha-
nilamide in 5 % phosphoric acid for 10 min. The purple
colour obtained was read at 520 nm in a spectrophotometer.
Standard curve was obtained using serial dilutions of 0.1 M
sodium nitrite in water. The results were expressed in terms
of lg equivalents of sodium nitrite/mg protein.
Statistical Analysis
The antioxidant enzyme and NO assays were set up in
duplicates and the values are expressed as mean ± SEM.
The data were analyzed by one-way ANOVA and LSD
post hoc test using SPSS software.
Results
Effects of Brahmi and Deprenyl on the Activities
of Antioxidant Enzymes in the Heart, Thymus,
Mesenteric Lymph Nodes (MLN), and Brain Areas
Treatment of female rats with brahmi (40 mg/kg BW)
significantly (P \ 0.05) increased SOD activity (units/min/
mg protein) in the thymus and MLN while its activity was
significantly (P \ 0.05) reduced in the medial basal
hypothalamus following low dose (10 mg/kg BW) brahmi
treatment and in the hippocampus by both doses of brahmi
compared to control and saline-treated rats (Table 1A).
Deprenyl (2.5 mg/kg BW) significantly (P \ 0.05)
enhanced SOD activity in the medial basal hypothalamus
while it suppressed the activity in the hippocampus com-
pared to control and saline-treated rats (Table 1B).
The activity (units/min/mg protein) of CAT decreased
significantly (P \ 0.05) with 10 mg/kg BW brahmi treat-
ment in the heart and both doses of brahmi in the thymus
compared to control and saline-treated rats. In contrast to its
effect on heart and thymus, brahmi significantly (P \ 0.05)
increased CAT activity in a dose-dependent manner in the
frontal cortex, striatum, and hippocampus compared to
control and saline-treated rats (Table 2A). Similar to brahmi,
low dose of deprenyl (1.0 mg/kg BW) reduced CAT activity
significantly (P \ 0.05) in the heart and the high dose of
deprenyl (2.5 mg/kg BW) reduced the activity in the thymus
compared to control and saline-treated rats. The activity of
CAT was enhanced significantly (P \ 0.05) in the frontal
cortex, medial basal hypothalamus, striatum and hippo-
campus following treatment with 2.5 mg/kg BW deprenyl
compared to control and saline-treated rats (Table 2B).
Glutathione peroxidase (GPx; isoform GPx1) activity
(units/min/mg protein) increased significantly (P \ 0.05) in
the heart, thymus, striatum, and hippocampus after treatment
with both doses of brahmi and in the medial basal hypo-
thalamus with high dose of brahmi compared to control and
saline-treated rats. Administration of low dose of brahmi
significantly (P \ 0.05) reduced GPx activity in the frontal
cortex compared to control rats (Table 3A). There was a
significant (P \ 0.05) increase in the activity of GPx in the
123
 Neurochem Res

Table 1 Superoxide dismutase (SOD) activity in brahmi-(1A) and deprenyl-(1B) treated rats in the peripheral organs (heart, thymus, and
mesenteric lymph nodes) and brain areas (frontal cortex, medial basal hypothalamus, striatum, and hippocampus)
Control Saline Brahmi 10 Brahmi 40

(A)
Heart 0.04 ± 0.005 0.043 ± 0.003 0.05 ± 0.001 0.05 ± 0.003
Thymus 0.05 ± 0.005 0.048 ± 0.02 0.05 ± 0.003 0.06 ± 0.01*
Mesenteric lymph nodes 0.19 ± 0.02 0.19 ± 0.02 0.20 ± 0.01 0.30 ± 0.03**
Frontal cortex 0.26 ± 0.03 0.28 ± 0.04 0.28 ± 0.04 0.33 ± 0.09
Medial basal hypothalamus 0.34 ± 0.05 0.27 ± 0.04 0.13 ± 0.02** 0.21 ± 0.01
Striatum 0.50 ± 0.05 0.44 ± 0.04 0.44 ± 0.05 0.40 ± 0.07
Hippocampus 0.49 ± 0.01 0.40 ± 0.01 0.22 ± 0.01* 0.19 ± 0.01*
Control Saline Deprenyl 1.0 Deprenyl 2.5

(B)
Heart 0.04 ± 0.005 0.04 ± 0.003 0.04 ± 0.006 0.04 ± 0.003
Thymus 0.05 ± 0.005 0.05 ± 0.005 0.05 ± 0.004 0.06 ± 0.002
Mesenteric lymph nodes 0.20 ± 0.02 0.19 ± 0.02 0.26 ± 0.08 0.16 ± 0.03
Frontal cortex 0.26 ± 0.03 0.23 ± 0.02 0.22 ± 0.03 0.18 ± 0.09
Medial basal hypothalamus 0.34 ± 0.05 0.30 ± 0.01 0.41 ± 0.03 0.42 ± 0.002**
Striatum 0.50 ± 0.05 0.51 ± 0.04 0.46 ± 0.03 0.44 ± 0.07
Hippocampus 0.49 ± 0.01 0.49 ± 0.002 0.37 ± 0.014 0.24 ± 0.013*

Rats were treated with 10 and 40 mg/kg BW of brahmi orally and 1.0 and 2.5 mg/kg BW of deprenyl intraperitoneally for 10 days
All values are mean ± SEM
* Significantly (P \ 0.05) different from control. ** Significantly (P \ 0.05) different from control and saline-treated groups

heart, medial basal hypothalamus, striatum, and hippocam-
pus after treatment with 2.5 mg/kg BW deprenyl and in the
MLN after treatment with both doses of deprenyl compared
to control and saline-treated rats. Similar to the effects of
brahmi treatment, both doses of deprenyl significantly
(P \ 0.05) reduced the GPx activity in the frontal cortex
compared to control and saline-treated rats (Table 3B).
Effects of Brahmi and Deprenyl on the Activities
of Antioxidant Enzymes in the Spleen
Brahmi and deprenyl treatment of female Wistar rats for
10 days did not alter the activity (units/min/mg protein) of
SOD (Fig. 1a, b) in the spleen. Low dose of brahmi (10 mg/kg
BW) significantly (P \ 0.05) increased the CAT activity
(units/min/mg protein; Fig. 1c) while deprenyl treatment had
no effect on the CAT activity (Fig. 1d) compared to control
and saline-treated rats. In contrast to the activities of SOD and
CAT, both doses of brahmi (Fig. 3e) and deprenyl (Fig. 3f)
significantly (P \ 0.05) augmented GPx activity (units/min/
mg protein) compared to control and saline-treated rats.
Effects of Brahmi and Deprenyl on the p-TH
Expression in the Spleen and Hippocampus
Brahmi and deprenyl treatment increased the p-TH
expression in the spleen (Fig. 2a) and the hippocampus
(Fig. 2d) assessed by the Western Blotting technique. The
densitometry scanning of the blots were done using Image J
1.45 software (NIH) and all the samples were controlled for
using the values obtained in terms of b-actin expression.
Densitometric analysis demonstrated that treatment with
both brahmi and deprenyl significantly (P \ 0.05) enhanced
the p-TH expression in the spleen (Fig. 2b, c) compared to
control and saline-treated rats. Both the the doses of, brahmi
(Fig. 2e) and 2.5 mg/Kg BW deprenyl (Fig. 2f) signifi-
cantly (P \ 0.05) increased p-TH expression in the hippo-
campus compared with control and saline-treated rats.
Effects of Brahmi and Deprenyl on the NGF Expression
in the Spleen and Hippocampus
Western blot analysis of spleen (Fig. 3a) and hippocampus
(Fig. 3d) for NGF expression demonstrated that brahmi
and deprenyl increased its expression depending on the
dose administered to female rats. Analysis with densi-
tometry scanning of the blots using Image J software (NIH)
with b-actin expression as control revealed that low dose of
brahmi significantly (P \ 0.05) enhanced the NGF
expression (Fig. 3b) while the higher dose of deprenyl
significantly (P \ 0.05) increased NGF expression in the
spleen (Fig. 3c). In the hippocampus, high doses of both
brahmi (Fig. 3e) and deprenyl (Fig. 3f) significantly
(P \ 0.05) enhanced NGF expression.

123
Neurochem Res

Table 2 Catalase (CAT) activity in brahmi-(2A) and deprenyl-(2B) treated rats in the peripheral organs (heart, thymus, and mesenteric lymph
nodes) and brain areas (frontal cortex, medial basal hypothalamus, striatum, and hippocampus)
Control Saline Brahmi 10 Brahmi 40

(A)
Heart 4.4 ± 0.1 4.6 ± 0.8 3.6 ± 0.7* 3.9 ± 0.7
Thymus 0.6 ± 0.01 0.6 ± 0.09 0.1 ± 0.01** 0.2 ± 0.1**
Mesenteric lymph nodes 1.6 ± 0.5 1.6 ± 1.1 4.0 ± 1.45 2.1 ± 0.9
Frontal cortex 2.5 ± 0.3 3.8 ± 0.1 4.9 ± 0.66** 5.8 ± 0.8**
Medial basal hypothalamus 0.8 ± 0.01 0.8 ± 0.01 0.8 ± 0.008 0.9 ± 0.01
Striatum 0.6 ± 0.06 0.6 ± 0.03 0.9 ± 0.1** 1.1 ± 0.03**
Hippocampus 0.5 ± 0.01 0.5 ± 0.06 0.7 ± 0.06* 0.7 ± 0.05*
Control Saline Deprenyl 1.0 Deprenyl 2.5

(B)
Heart 4.4 ± 0.1 4.5 ± 0.5 2.9 ± 0.8* 6.3 ± 1.3
Thymus 0.6 ± 0.01 0.6 ± 0.1 0.5 ± 0.2 0.1 ± 0.02**
Mesenteric lymph nodes 1.6 ± 0.5 1.8 ± 0.4 1.9 ± 0.6 1.7 ± 0.27
Frontal cortex 2.5 ± 0.3 2.3 ± 0.08 3.1 ± 0.2 4.8 ± 0.2**
Medial basal hypothalamus 0.8 ± 0.01 0.8 ± 0.04 1.1 ± 0.1 1.2 ± 0.1**
Striatum 0.6 ± 0.06 0.6 ± 0.02 1.1 ± 0.1 1.5 ± 0.2**
Hippocampus 0.5 ± 0.01 0.6 ± 0.09 0.6 ± 0.06 0.7 ± 0.02*

Rats were treated with 10 and 40 mg/kg BW of brahmi orally and 1.0 and 2.5 mg/kg BW of deprenyl intraperitoneally for 10 days
All values are mean ± SEM
* Significantly (P \ 0.05) different from control. ** Significantly (P \ 0.05) different from control and saline-treated groups

Table 3 Glutathione peroxidase (GPx) activity brahmi-(3A) and deprenyl-(3B) treated rats in the peripheral organs (heart, thymus, and
mesenteric lymph nodes) and brain areas (frontal cortex, medial basal hypothalamus, striatum, and hippocampus)
Control Saline Brahmi 10 Brahmi 40

(A)
Heart 3.0 ± 0.3 3.1 ± 0.2 4.2 ± 0.6** 4.7 ± 0.4**
Thymus 1.2 ± 0.1 1.5 ± 0.3 2.2 ± 0.3** 2.4 ± 0.4**
Mesenteric lymph nodes 3.2 ± 0.4 3.3 ± 0.4 2.5 ± 0.4 3.4 ± 0.3
Frontal cortex 5.1 ± 0.6 5.5 ± 1.9 3.9 ± 0.3* 4.1 ± 1.5
Medial basal hypothalamus 3.5 ± 0.8 4.0 ± 0.5 3.6 ± 0.6 7.7 ± 1.8#
Striatum 1.8 ± 0.1 1.9 ± 0.1 3.6 ± 0.02** 3.9 ± 0.1**
Hippocampus 3.2 ± 1.6 3.4 ± 1.3 5.9 ± 1.3* 9.0 ± 1.6*
Control Saline Deprenyl 1.0 Deprenyl 2.5

(B)
Heart 3.0 ± 0.3 2.8 ± 0.2 3.5 ± 0.6 4.7 ± 1.1**
Thymus 1.2 ± 0.1 1.1 ± 0.1 1.2 ± 0.2 1.4 ± 0.2
Mesenteric lymph nodes 3.2 ± 0.4 2.9 ± 0.4 9.9 ± 2.1** 5.0 ± 1.3**
Frontal cortex 5.1 ± 0.6 5.4 ± .6 1.8 ± 0.4** 2.8 ± 0.3**
Medial basal hypothalamus 3.5 ± 0.8 3.1 ± 0.6 4.1 ± 1.0 7.3 ± 0.8#
Striatum 1.8 ± 0.1 1.9 ± 0.1 2.1 ± 0.1 4.3 ± 0.1**
Hippocampus 3.2 ± 1.6 3.8 ± 0.7 4.8 ± 2.6 6.3 ± 1.9*

Rats were treated with 10 and 40 mg/kg BW of brahmi orally and 1.0 and 2.5 mg/kg BW of deprenyl intraperitoneally for 10 days
All values are mean ± SEM
#
* Significantly (P \ 0.05) different from control. ** Significantly (P \ 0.05) different from control and saline-treated groups. Significantly
(P \ 0.05) different from the other 3 groups

123
 Neurochem Res

Fig. 1 Activities of superoxide 0.04 A 0.04

B
dismutase (SOD; a, b),catalase Control Control
(CAT; c, d), and glutathione Saline Saline

SOD activity/min/mg protein

SOD activity/min/mg protein

peroxidase (GPx; e, f) in the Brahmi 10 Dep 1.0
spleen of brahmi- and deprenyl- 0.03 Brahmi 40 0.03
Dep 2.5
treated Wistar rats. Splenic SOD
activity (units/min/mg protein)
was unaltered following 0.02
0.02
treatment with brahmi (a) and
deprenyl (b). CAT activity
(units/min/mg protein)
increased significantly 0.01 0.01
(P \ 0.05) in the spleens of rats
treated with brahmi 10 mg/kg
BW, (c) compared to control
0 0
rats. Deprenyl treatment, (d) did
not alter CAT activity in the
spleen. Splenic GPx activity
4 C 4 D Control
(units/min/mg protein) was Control
Saline

CAT activity/min/mg protein

CAT activity/min/mg protein

significantly (P \ 0.05) Saline

Dep 1.0
enhanced in rats treated with
brahmi 10 and 40 mg/kg BW, 3 * Brahmi 10
Brahmi 40 3 Dep 2.5

(e) compared to control rats.

Both doses of deprenyl (1.0 and
2.5 mg/kg BW) significantly 2 2
(P \ 0.05) increased GPx
activity in the spleen (f)

1 1

0 0

6
E 6 F
* Control
Saline
* Control
Saline
GPx activity/min/mg protein

GPx activity/min/mg protein

Brahmi 10 Dep 1.0

Brahmi 40 Dep 2.5
4 4

2 2

0
0

Effects of Brahmi and Deprenyl on the p-ERK, b-actin indicating equal loading in all the samples. Further
p-CREB, and p-NF-kB downstream p-CREB expression was assessed against the
total CREB protein in the spleen after controlling for
The p-ERK expression in the spleen was assessed against b-actin expression by Western blot followed by densitometry
the total ERK1/2 expression after controlling for b-actin (Fig. 5a–d). A concomitant significant (P \ 0.05) increase
expression by the Western Blotting technique followed by in p-CREB expression was observed with 2.5 mg/kg BW
densitometry scanning of the blots using Image J software deprenyl treatment (Fig. 4d). Treatment with both brahmi
(NIH) (Fig. 4a–d). Treatment with 2.5 mg/kg BW deprenyl and deprenyl significantly enhanced the expression of
alone significantly (P \ 0.05) enhanced the p-ERK1/2/ p-NF-kB (p50) expression in the spleen as measured by
total ERK1/2 expression in the spleen (Fig. 4d). There was Western blot method (Figs.6a). Densitometric analysis
no significant change in the total ERK expression against demonstrated that p-NF-jB expression was enhanced

123
Neurochem Res

Fig. 2 The expression of

phospho-tyrosine hydroxylase A
(p-TH; p40) using Western blot
in the spleen (a) and
hippocampus (d) of brahmi- and
deprenyl-treated Wistar rats
(C control, S saline-treated, BL
brahmi 10 mg/kg BW, BH
brahmi 40 mg/kg BW, DL
deprenyl 1.0 mg/kg BW and 4 B Control 4 C Control

DH deprenyl 2.5 mg/kg BW). Saline Saline

Splenic p-TH expression
(relative intensity p-TH/ 3
Brahmi 10
3
* Dep 1.0

Relative Intensity

Relative Intensity
b-Actin) was augmented Brahmi 40 Dep 2.5

β -Actin

p-TH/β-Actin
significantly (P \ 0.05) in rats
treated with both doses of
2
*
brahmi (b) and deprenyl p-TH/β 2
(c) compared to control and
saline-treated rats. In the
hippocampus, brahmi (e) and
1 1
deprenyl (f) treatment
significantly (P \ 0.05)
augmented p-TH expression
compared to control and 0 0
saline-treated rats

4.5 4.5
E Control F Control
* Saline Saline

Brahmi 10 Dep 1.0

Relative Intensity
Relative Intensity

p-TH/β -Actin
p-TH/β -Actin

3 Brahmi 40 3 Dep 2.5

*
1.5 1.5

0 0

significantly (P \ 0.05) by both doses of brahmi (Fig. 6b) Discussion

and deprenyl (Fig. 6c) compared to control and saline-
treated rats.
Effects of Brahmi and Deprenyl on Nitric Oxide (NO)
Production in Spleen
Brahmi (10 mg/kg BW) treatment significantly (P \ 0.05)
enhanced the NO production while deprenyl treatment did
not alter NO production. It is possible that brahmi mediates
its effects through NOS-dependent mechanism and depre-
nyl through a NOS independent pathway (Fig. 7).
Prevention of oxidative stress and maintenance of antioxidant
status at the cellular level is critical to normal physiological
functions and inhibition of age-associated disorders [5]. The
results from the present study demonstrated that brahmi and
deprenyl had distinct effects on the antioxidant enzyme
(SOD, CAT, and GPx) activities in the brain areas (frontal
cortex, medial basal hypothalamus, striatum, and hippo-
campus), heart, and lymphoid organs (thymus, spleen, and
mesenteric lymph nodes). These effects were accompanied
by increased expression of p-TH and NGF in the spleens and

123
 Neurochem Res

Fig. 3 The expression of nerve

growth factor (NGF) using A
Western blot in the spleen
(a) and hippocampus (d) of
brahmi- and deprenyl-treated
Wistar rats (C control, S saline-
treated, BL brahmi 10 mg/kg
BW, BH brahmi 40 mg/kg BW,
2 2
DL deprenyl 1.0 mg/kg BW and B * Control C Control
DH deprenyl 2.5 mg/kg BW).
Splenic NGF expression
Saline * Saline

(relative intensity NGF/b-Actin) 1.5 Brahmi 10 1.5 Dep 1.0

Relative Intensity
Relative Intensity
was enhanced significantly

NGF/β-Actin
β-Actin
Brahmi 40 Dep 2.5
(P \ 0.05) in rats treated with
Brahmi 10 mg/kg BW (b) and
1 1
NGF/β
Deprenyl 2.5 mg/kg BW
(c) compared to control and
saline-treated rats. In the
hippocampus, NGF expression 0.5 0.5
was significantly enhanced
(P \ 0.05) by brahmi (e) and
deprenyl (f) treatment compared
0 0
to saline-treated rats

2.5
E Control 2.5 F Control

Saline Saline
2 Brahmi 10 2 Dep 1.0
Relative Intensity

Relative Intensity

Brahmi 40 Dep 2.5

NGF/β-Actin

NGF/β-Actin

1.5 1.5

1 1

0.5 0.5

0 0

hippocampi of both brahmi- and deprenyl-treated rats.
However, the signaling mechanisms were different depend-
ing on the treatment: brahmi and deprenyl enhanced p-NF-kB
expression while deprenyl alone augmented p-ERK 1/2 and
p-CREB expression in the spleen. An increase in NO pro-
duction was observed only in brahmi-treated rats.
Brahmi (B. monnieri Linn.) is widely used to improve
learning and memory and also, reverse cognitive deficits
associated with epilepsy [7, 8, reviewed in 19]. Most of these
beneficial effects are attributed to neuroprotective and anti-
oxidant properties of brahmi [9, 10, 20]. In the present study,
treatment of female rats with brahmi increased SOD activity
in the thymus and MLN while its activity was reduced in the
medial basal hypothalamus and hippocampus. Brahmi treat-
ment increased the activity of CAT in the spleen, frontal
cortex, striatum, and hippocampus and GPx activity in the
heart, thymus, spleen, medial basal hypothalamus, striatum,
and hippocampus. Although there are no comparable evi-
dence for brahmi’s effects on the lymphoid organs and heart,
similar increases in CAT and GPx activities in the striatum
and frontal cortex of brahmi-treated male rats were reported
in an earlier published study [10]. Similarly, brahmi was able
to restore SOD, CAT, and GPx activities in the striatum of
neurotoxin-treated rats indicating that brahmi is capable of
reversing neurobehavioral deficits observed in neurodegen-
erative diseases [9]. In contrast, to the afore-mentioned
studies, brahmi treatment significantly decreased SOD
activity in the hippocampus and was unaltered in the striatum
in the present study that may be due to the use of female rats
and the influence of gonadal steroids on SOD activity.

123
Neurochem Res

Fig. 4 The expression of

phospho–extracellular signal A
regulated kinase (p-ERK) using
Western blot in the spleen (a) of
brahmi- and deprenyl-treated
Wistar rats (C control, S saline-
treated, BL brahmi 10 mg/kg
BW, BH brahmi 40 mg/kg BW,
DL deprenyl 1.0 mg/kg BW and
DH deprenyl 2.5 mg/kg BW). 1.5 B Control 1.5 C Control
There were no significant
Saline Saline
alterations in ERK 1/2
expression after treatment with Brahmi 10 Dep 1.0
brahmi (b) and deprenyl

Relative Intensity
ERK-1/2 /β-Actin

Relative Intensity
ERK 1/2 /β-Actin
Brahmi 40 Dep 2.5
(c) indicating equality of 1 1
loading. Similarly, p-ERK 1/2
expression was unaltered
following brahmi treatment (d).
Treatment with deprenyl
0.5 0.5
2.5 mg/kg BW (e) enhanced
p-ERK expression (relative
intensity p-ERK/Total ERK)
significantly (P \ 0.05) in the
spleen compared to control and 0 0
saline-treated rats
2.5 D Control Control
2.5 E
p-ERK 1/2 /Total ERK 1/2

Saline
Saline

p-ERK 1/2 /Total ERK 1/2

2 Brahmi 10 *
Relative Intensity

2 Dep 1.0

Relative Intensity
Brahmi 40
Dep 2.5
1.5
1.5

1
1

0.5
0.5

0
0

Brahmi enhanced p-TH and NGF expression in the spleen
suggesting that the neurorestorative properties are mediated
through increased biosynthesis of growth factors that are
critical to the maintenance of sympathetic nerve fibers. It is
unknown whether brahmi enhances TH expression in the
striatum but it is possible that similar neuroprotective
properties of brahmi through enhanced p-TH and NGF in the
brain may have mediated the reversal of neurobehavioral
activities observed in neurotoxin-treated and Alzheimer’s
disease model rats [9, 21]. However, brahmi augmented the
expression of p-TH and NGF in the hippocampus that may
explain its function as a potent enhancer of cognition and
memory [7, 8, 19]. In this study, the increase in the nuclear
translocation of p50 subunit of NF-kB in the spleen fol-
lowing brahmi treatment may have been responsible for the
modulation of immune responses through increased secre-
tion of IgA and IgG antibodies and IFN-c and IL-2 pro-
duction, and inhibition of TNF-a production by splenocytes
of brahmi-treated rats [11]. The increase in NO production
in the spleen by brahmi may be another mechanism through
which it could modulate immunity by causing vasodilation
and promoting trafficking of immune cells from spleen to
other parts of the body [22].
Deprenyl, an irreversible monoamine oxidase-B inhibitor,
is used in the treatment of neurodegenerative disorders and has
been reported to increase the lifespan of rats possibly through
antioxidant, neuroprotective, neurorestorative, neurotrophic,
and immunostimulatory properties [13, 14]. In the present
study, deprenyl increased SOD activity in the medial basal
hypothalamus, CAT activity in the frontal cortex, medial basal
hypothalamus, striatum, hippocampus, heart, and spleen, and
GPx activity in medial basal hypothalamus, striatum, hippo-
campus, heart, spleen, and MLN. The findings in female rats in
the present study are in agreement with the earlier studies in
which deprenyl was demonstrated to increase the antioxidant
enzyme activities in the frontal cortex, striatum, heart, and
spleen of male rodents [10, 23]. However, deprenyl treatment
of female rats significantly enhanced CAT and GPx activities

123
 Neurochem Res

Fig. 5 The expression of

phospho-cAMP response A
element-binding (p-CREB)
using Western blot in the spleen
(a) of brahmi- and deprenyl-
treated Wistar rats (C control,
S saline-treated, BL brahmi
10 mg/kg BW, BH brahmi
40 mg/kg BW, DL deprenyl
1.0 mg/kg BW and DH 1.2 B Control 1.2 C Control
deprenyl 2.5 mg/kg BW). There
Saline Saline
was no significant change in the
total CREB expression amongst Brahmi 10 Dep 1.0

Total CREB/β-Actin
β -Actin
the treatment groups and the 0.9 0.9

Relative Intensity
Relative Intensity
Brahmi 40 Dep 2.5
controls indicating equality of

Total CREB/β
loading (b, c). Treatment with
brahmi did not alter p-CREB 0.6 0.6
expression (c). Splenic p-CREB
expression (relative intensity
p-CREB/Total CREB) was
significantly (P \ 0.05) 0.3 0.3
increased in rats treated with
deprenyl 2.5 mg/kg BW
(d) compared to control and
0 0
saline-treated rats
4 D Control 4 E Control

Saline Saline

Brahmi 10 * Dep 1.0

p-CREB/Total CREB
p-CREB/Total CREB

3 3

Relative Intensity
Relative Intensity

Brahmi 40 Dep 2.5

2 2

1 1

0 0

Fig. 6 The expression of p-NF-

jB (p50) using Western blot in A
the spleen (a) of brahmi- and
deprenyl-treated Wistar rats
(C control, S saline-treated, BL
brahmi 10 mg/kg BW, BH
brahmi 40 mg/kg BW, DL
deprenyl 1.0 mg/kg BW and 2.5 B Control
C
DH deprenyl 2.5 mg/kg BW). Saline
2.5
* Control

Splenic p-NF-jB (p50)

2
* Saline
expression (relative intensity Brahmi 10 2 Dep 1.0
p-NF-jB (p50)/b-Actin)
Relative Intensity
β -Actin

Relative Intensity

Brahmi 40
p-NFkB/ β-Actin

increased significantly Dep 2.5

(P \ 0.05) in rats treated with 1.5
1.5
p-NFkB/β

both doses of brahmi (b) and

deprenyl (c) compared to
control and saline-treated rats 1 1

0.5 0.5

0 0

123
Neurochem Res
Fig. 7 Nitric oxide (NO)
μ g equivalent of
production in Brahmi- and
Deprenyl-treated Wistar rats.
Splenic NO production (lg
NaNO /mg protein)
equivalents of NaNO2/mg
protein) increased significantly
(P \ 0.05) in rats treated with
NO production (μ
brahmi 10 mg/kg BW
(a) compared to control rats. NO
production was unaltered in rats
treated with deprenyl (b)
in the hippocampus contradictory to the results observed in
deprenyl-treated male rats probably attributable to sex-asso-
ciated differences as described earlier. Similar to brahmi,
deprenyl increased the expression of p-TH and NGF in the
spleen that may have been responsible of its neuroresotorative
properties in the spleen of young neurotoxin-treated, old, and
tumor-bearing rats [24–26]. Perhaps, deprenyl exerts a stim-
ulatory effect on NGF expression and release that may in turn,
promote the expression of p-TH in the spleen through any of
the neurotrophin receptors such as TrkC and TrkB [27–30].
The increase in the protein level of TH may have been due to
enhanced TH mRNA expression and would have resulted in
an increase in enzyme activity in the spleen similar to its
effects on nigrostriatal dopaminergic system in aged rats [31].
Similarly, deprenyl also increased the p-TH expression and
NGF expression in the hippocampus that may have been
responsible for the beneficial effects observed following its
administration in neurodegenerative diseases [12, 13].
Deprenyl increased p-ERK 1/2 protein levels in the
spleen with concomitant increase in p-CREB expression
that may translocate to the nucleus to bind to the antioxidant
response element (ARE) pathway. This was identical to the
phosphorylation of ERK and Akt by deprenyl in pheo-
chromocytoma 12 (PC12) cells that subsequently lead to
translocation of transcription factor, Nrf2, to bind to ARE
and protect the PC12 cells from neurotoxin, 1-methyl-4-
phenylpyridinium (MPP?) [32]. In addition, it is possible
that the antioxidant property of deprenyl may be due to the
upregulation of PI3 K and subsequent activation of Nrf2
through upstreatm TrkB neurotrophin receptor involvement
[30]. In the present study, we are reporting CREB as one
another activator that can bind to ARE to improve antiox-
idant status of the cell as CREB is a coactivator of ARE.
Treatment of simian immunodeficiency virus—infected
rhesus monkeys with deprenyl reversed the reduction of
CREB levels in the caudate putamen demonstrating that
CREB is one of key factors in cytoprotection and cell sur-
vival via growth factors and lack of it may result in neu-
rodegeneration observed in human immunodeficiency
A Control B Control
0.08
* 0.08
NO production (μg equivalent of
Saline Saline
Brahmi 10 Dep 1.0
NaNO /mg protein)
0.06 0.06
Brahmi 40 Dep 2.5
0.04 0.04
0.02 0.02
0 0
virus-associated dementia [33]. Deprenyl-induced increase
in NF-kB protein levels suggests that NF-kB is a mediator of
its antioxidant properties as NF-kB targets include mito-
chondrial manganese SOD, cytoplasmic copper-zinc SOD,
GPx and a host of other antioxidant enzymes [34]. Deprenyl
did not have any effect on NO production in the spleen
indicating that it may exert its effect in nitric oxide syn-
thase-independent mechanism.
Immunosuppression is a common feature in aging, cancer,
certain infectious and autoimmune diseases accompanied by
loss of sympathetic NA innervation in the spleen and lymph
nodes [2, 4, 14]. Previously, we have demonstrated that
deprenyl can restore sympathetic NA nerve fibers along with
cell-mediated immune responses in the rats with carcinogen-
induced and spontaneously developing mammary tumors and
old male rats [14, 24–26]. It is possible that these enhanced
antioxidant enzyme activities in the spleen and brain besides
increase in the levels of TH and NGF observed in the present
study may have been responsible for the neurorestorative and
immunostimulatory functions. One possible mechanism may
have been through the binding of NE to G protein-coupled
receptor in the spleen and inducing intracellular signaling
cascade involving protein kinase A and increased levels of
transcription factor, CREB, resulting in neuroprotection
through the release of cytokines and growth factors in the
spleen. Similar effects may have been achieved through ERK
1/2 and NF-kB pathways mediated by yet to be determined
factors induced by deprenyl treatment.
In summary, brahmi and deprenyl enhanced most of the
antioxidant enzyme activities in the brain areas, heart, thy-
mus, spleen, and mesenteric lymph nodes while suppressing
few depending on the regions of the brain and peripheral
organs, and increased TH and NGF protein levels in the
spleen. The effects in the spleen were accompanied by
distinct alterations in the cellular signaling pathways: brahmi
and deprenyl upregulated the expression of NF-kB while
deprenyl alone increased ERK 1/2 expression and the
downstream transcription factor, CREB. Further studies are
warranted to understand the cross-talk between these and
123

other related pathways in cell survival, neuroprotection, and
immunity that are critical to the treatment of neurodegen-
erative diseases, cancer, and immunosenescence.
Acknowledgments Supported by the Department of Science and
Technology (F. NO. SR/SO/HS-46/2007), Government of India,
New Delhi.
References
1. Ader R, Cohen N, Felten DL (2001) Psychoneuroimmunology,
3rd edn. Academic Press, New York
2. Bellinger DL, Millar BA, Perez S, Carter J, Wood C, Thyaga-
Rajan S, Molinaro C, Lubahn C, Lorton D (2008) Sympathetic
modulation of immunity: relevance to disease. Cell Immunol
252:27–56
3. ThyagaRajan S, Madden KS, Teruya B, Stevens SY, Felten DL,
Bellinger DL (2011) Age-associated alterations in sympathetic
noradrenergic innervation of primary and secondary lymphoid
organs in female Fischer 344 rats. J Neuroimmunol 233:54–64
4. Maue AC, Yager EJ, Swain SL, Woodland DL, Blackman MA,
Haynes L (2009) T-cell immunosenescence: lessons learned from
mouse models of aging. Trends Immunol 30:301–305
5. Salmon AB, Richardson A, Pérez VI (2010) Update on the
oxidative stress theory of aging: does oxidative stress play a role
in aging or healthy aging? Free Radic Biol Med 48:642–655
6. Bolzán AD, Brown OA, Goya RG, Bianchi MS (1995) Hormonal
modulation of antioxidant enzyme activities in young and old
rats. Exp Gerontol 30:169–175
7. Singh RH, Narsimhamurthy K, Singh G (2008) Neuronutrient
impact of Ayurvedic Rasayana therapy in brain aging. Bioger-
ontology 9:369–374
8. Singh HK, Dhawan BN (1997) Neuropsychopharmacological
effects of the Ayurvedic nootropic Bacopa monniera Linn.
(Brahmi). Indian J Pharmacol 29:S359–S365
9. Shobana C, Ramesh Kumar R, Sumathi T. (2012) Alcoholic
extract of Bacopa monniera Linn. Protects against 6-hydroxy-
dopamine-induced changes in behavioral and biochemical
aspects: a pilot study. Cell Mol Neurobiol. Apr 17. [Epub ahead
of print] PubMed PMID: 22527857
10. Bhattacharya SK, Bhattacharya A, Kumar A, Ghosal S (2000)
Antioxidant activity of Bacopamonniera in rat frontal cortex,
striatum and hippocampus. Phytother Res 14:174–179
11. Yamada K, Hung P, Park TK, Park PJ, Lim BO (2011) A comparison
of the immunostimulatory effects of the medicinal herbs Echinacea,
Ashwagandha and Brahmi. J Ethnopharmacol 137:231–235
12. Naoi M, Maruyama W (2010) Monoamine oxidase inhibitors as
neuroprotective agents in age-dependent neurodegenerative dis-
orders. Curr Pharm Des 16:2799–2817
13. Magyar K, Szende B, Jenei V, Tábi T, Pálfi M, Szöko E (2010)
R-deprenyl:pharmacological spectrum of its activity. Neurochem
Res 35:1922–1932
14. ThyagaRajan S, Felten DL (2002) Modulation of neuroendocrine–
immune signaling by L-deprenyl and L-desmethyldeprenyl in
aging and mammary cancer. Mech Ageing Dev 123:1065–1079
15. Sun M, Zigman S (1978) An improved spectrophotometric assay
for superoxide dismutase based on epinephrine autoxidation.
Anal Biochem 90:81–89
16. Góth L (1991) A simple method for determination of serum
catalase activity and revision of reference range. Clin Chim Acta
196:143–151
17. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem
Biophys 82:70–77
123
View publication stats
Neurochem Res
18. Fiddler RN (1977) Collaborative study of modified AOAC
method of analysis for nitrite in meat and meat products. J Assoc
Off Anal Chem 60:594–599
19. Shinomol GK, Muralidhara Bharath MM (2011) Exploring the
role of ‘‘Brahmi’’ (Bacopa monnieri and Centellaasiatica) in
brain function and therapy. Recent Pat Endocr Metab Immune
Drug Discov 5:33–49
20. Tripathi YB, Chaurasia S, Tripathi E, Upadhyay A, Dubey GP
(1996) Bacopa monniera Linn. as an antioxidant: mechanism of
action. Indian J Exp Biol 34:523–526
21. Uabundit N, Wattanathorn J, Mucimapura S, Ingkaninan K
(2010) Cognitive enhancement and neuroprotective effects of
Bacopa monnieri in Alzheimer’s disease model. J Ethnopharma-
col 127:26–31
22. Kamkaew N, Scholfield CN, Ingkaninan K, Maneesai P, Par-
kington HC, Tare M, Chootip K (2011) Bacopa monnieri and its
constituents is hypotensive in anaesthetized rats and vasodilator
in various artery types. J Ethnopharmacol 137:790–795
23. Kitani K, Minami C, Isobe K, Maehara K, Kanai S, Ivy GO,
Carrillo MC (2002) Why (–)deprenyl prolongs survivals of
experimental animals: increase of anti-oxidant enzymes in brain
and other body tissues as well as mobilization of various humoral
factors may lead to systemic anti-aging effects. Mech Ageing
Dev 123:1087–1100
24. ThyagaRajan S, Felten SY, Felten DL (1998) Restoration of
sympathetic noradrenergic nerve fibers in the spleen by low doses
of L-deprenyl treatment in young sympathectomized and old
Fischer 344 rats. J Neuroimmunol 81(1–2):144–157
25. ThyagaRajan S, Madden KS, Kalvass JC, Dimitrova SS, Felten
SY, Felten DL (1998) L-deprenyl-induced increase in IL-2 and NK
cell activity accompanies restoration of noradrenergic nerve fibers
in the spleens of old F344 rats. J Neuroimmunol 92(1–2):9–21
26. ThyagaRajan S, Madden KS, Stevens SY, Felten DL (2000) Anti-
tumor effect of L-deprenyl is associated with enhanced central and
peripheral neurotransmission and immune reactivity in rats with car-
cinogen-induced mammary tumors. J Neuroimmunol 109(2):95–104
27. Li XM, Qi J, Juorio AV, Boulton AA (1997) Reciprocal regu-
lation of the content of aromatic L-amino acid decarboxylase and
tyrosine hydroxylase mRNA by NGF in PC12 cells. J Neurosci
Res 47:449–454
28. Semkova I, Wolz P, Schilling M, Krieglstein J (1996) Selegiline
enhances NGF synthesis and protects central nervous system
neurons from excitotoxic and ischemic damage. Eur J Pharmacol
315:19–30
29. Ekblom J, Jossan SS, Ebendal T, Söderström S, Oreland L,
Aquilonius SM (1994) mRNA expression of neurotrophins and
members of the trk family in the rat brain after treatment with
L-deprenyl. Acta Neurol Scand 89:147–148
30. Nakaso K, Nakamura C, Sato H, Imamura K, Takeshima T, Naka-
shima K (2006) Novel cytoprotective mechanism of anti-parkinso-
nian drug deprenyl: PI3K and Nrf2-derived induction of antioxidative
proteins. Biochem Biophys Res Commun 339:915–922
31. Rodrı́guez-Gómez JA, Venero JL, Vizuete ML, Cano J, Machado A
(1997) Deprenyl induces the tyrosine hydroxylase enzyme in the rat
dopaminergic nigrostriatal system. Brain Res Mol Brain Res 46:31–38
32. Xiao H, Lv F, Xu W, Zhang L, Jing P, Cao X (2011) Deprenyl
prevents MPP(?)-induced oxidative damage in PC12 cells by the
upregulation of Nrf2-mediated NQO1 expression through the
activation of PI3K/Akt and Erk. Toxicology 290:286–294
33. Scheller C, Sopper S, Jenuwein M, Neuen-Jacob E, Tatschner T,
Grünblatt E, terMeulen V, Riederer P, Koutsilieri E (2005) Early
impairment in dopaminergic neurotransmission in brains of SIV-
infected rhesus monkeys due to microglia activation. J Neuro-
chem 95:377–387
34. Morgan MJ, Liu ZG (2011) Crosstalk of reactive oxygen species
and NF-jB signaling. Cell Res 21:103–115