Journal of Alzheimer’s Disease 9 (2006) 243–251 243

IOS Press

Bacopa monniera extract reduces amyloid

levels in PSAPP mice
Leigh A. Holcombd,e , Muralikrishnan Dhanasekaran b,f , Angie R. Hitta , Keith A. Youngd,e,
Mark Riggsb and Bala V. Manyam a,c,∗
a
Department of Neurology, Texas A&M University System HSC College of Medicine, Temple, TX, USA
b
Department of Research & Education, Scott and White Clinic, Texas A&M University System HSC College of
Medicine, Temple, TX, USA
c
Department of Internal Medicine, Texas A&M University System HSC College of Medicine, Temple, TX, USA
d
Department of Psychiatry and Behavioral Science, Texas A&M University System HSC College of Medicine,
Temple, TX, USA
e
Central Texas Veterans Health Care System Neuropsychiatry Research Program, Temple, TX, USA
f
Division of Pharmacology & Toxicology, Department of Pharmacal Sciences, Auburn University, Auburn, AL, USA

Abstract. PSAPP mice expressing the “Swedish” amyloid precursor protein and M146L presenilin-1 mutations are a well-
characterized model for spontaneous amyloid plaque formation. Bacopa monniera has a long history of use in India as an
anti-aging and memory-enhancing ethnobotanical therapy. To evaluate the effect of Bacopa monniera extract (BME) on amyloid
(Aβ) pathology in PSAPP mice, two doses of BME (40 or 160 mg/kg/day) were administered starting at 2 months of age for either
2 or 8 months. Our present data suggests that BME lowers Aβ 1-40 and 1-42 levels in cortex by as much as 60%, and reverses
Y-maze performance and open field hyperlocomotion behavioral changes present in PSAPP mice. The areas encompassed by
Congo Red-positive fibrillar amyloid deposits, however, were not altered by BME treatment. The data suggest that BME has
potential application in Alzheimer’s disease therapeutics.

Keywords: Alzheimer’s disease, amyloid, Bacopa monniera, transgenic, presenilin, Amyloid precursor protein

Abbreviations: Amyloid beta (Aβ), amyloid beta precursor protein (AβPP), Bacopa monniera extract (BME), presenilin 1 (PS-1),
transgenic mice bearing human PS-1 and AβPP mutations (PSAPP).

1. Introduction Alzheimer’s disease, and the prevalence doubles ev-

Alzheimer’s disease is the leading cause of cognitive
impairment in the geriatric population. Alzheimer’s
patients develop short-term and recent memory deficits,
and the progressive degeneration of cognitive function-
ing eventually becomes so severe that patients lose the
ability to care for themselves. Approximately 6% to
8% of all individuals over the age of 65 years have
∗ Corresponding author: Bala V. Manyam, M.D., Department of
Neurology, Scott & White Clinic/Texas A & M University, 2401
South 31st street, Temple, TX 76508, USA. Tel.: +1 813 792 9524;
Fax: +1 254 724 5692; E-mail: bmanyam@excite.com.
ery 5 years after age 60 [30]. Because of the increase
in the aging population, the number of persons with
Alzheimer’s disease is expected to triple over the next
half-century [12].
Research on the molecular basis of Alzheimer’s dis-
ease has elucidated a pathogenic pathway revolving
around the formation and deposition of the beta con-
formation of several species of the amyloid peptide
(Aβ) in the brain. Total levels of Aβ 1-40 and 1-42
are elevated early in dementia and correlate strongly
with cognitive decline in postmortem, cross-sectional
studies [28]. Aβ peptide levels increase before the
occurrence of significant neurofibrillary tangle pathol-

ISSN 1387-2877/06/$17.00 © 2006 – IOS Press and the authors. All rights reserved
244 L.A. Holcomb et al. / Bacopa monniera extract reduces amyloid levels in PSAPP mice
ogy, suggesting a role for Aβ peptide in mediating
initial pathogenic events resulting in Alzheimer’s dis-
ease cognitive decline [3]. To elucidate strategies for
the treatment of Alzheimer’s disease, pharmacologi-
cal interventions reversing the formation, accumula-
tion, or cytotoxic effects of the Aβ peptide in trans-
genic mice expressing familial human mutations are
being tested. The doubly-transgenic PSAPP mouse,
which has been generated to co-express the “Swedish”
mutation of the amyloid beta precursor protein (AβPP)
and the Presenilin-1 FAD 4 mutation (PS-1), devel-
ops robust amyloid plaque pathology. Spontaneous
amyloid deposition occurs as early as 10–12 weeks of
age, and substantial fibrillar and diffuse plaques for-
mation occurs before 10 months of age. In addition,
neuritic, glial and behavioral changes can be measured
in PSAPP mice [13–15,25]. These and other closely
related mouse models have been used to screen com-
pounds for efficacy in reducing levels of amyloid. Anti-
amyloid agents which are effective in reducing lev-
els of Aβ in PSAPP and other transgenic models of
Alzheimer’s disease may eventually prove effective in
treating Alzheimer’s disease in humans [11].
Bacopa monniera (Indian Pennywort or Brahmi in
Sanskrit) was first documented to have a CNS effect
in the Caraka Samhita (2,500 BC), one of the oldest
texts of the ancient Indian medicinal system, Ayurveda.
As early as 2,300 BC, the Susruta Samhita describes
Bacopa monniera as efficacious in the treatment of loss
of intellect and memory, suggesting a potential benefit
as an Alzheimer’s disease therapy. Bacopa monniera
is also listed as a longevity-promoting drug according
the Bhavprakasa Varg-Prakarana, an Indian medical
text on drug classification from the 16 th century [6].
Modern studies of Bacopa monniera provide evidence
for cognitive enhancement in both animals and humans,
and work is in progress to characterize the mechanism
of action and active component(s) responsible for this
effect [9,31,36,37,40].
The present study was designed to investigate
whether Bacopa monniera extract (BME) can directly
impact Alzheimer’s disease pathology by altering Aβ in
the brains of PSAPP mice if treatment occurs when the
mice are developing and consolidating amyloid plaque
pathology.
2. Methods and methods
2.1. Animals
PSAPP mice and non-transgenic littermates used for
this study were produced at the AAALAC-accredited
Scott & White Animal Facility by breeding PSAPP
males to B6SJL F1 female mice (Taconic). PSAPP
mice were originally produced by mating Tg2576 mice
carrying the human “Swedish” AβPP 695K670N/M671L
mutation with the 6.2 line expressing the PS-1 M146L
mutation 1 [10,16]. Mice were genotyped by PCR
prior to weaning and again at necropsy. All procedures
were in accordance with the National Institute of Health
Guide for the Care and Use of Laboratory Animals
(NIH Publications No. 80-23).
2.2. Bacopa monniera extract
Bacopa monniera extract (BME) was prepared by
Zandu Pharmaceutical Works (Mumbai, India) follow-
ing the method of Singh and Dhawan [34]. Organi-
cally cultivated Bacopa monniera (Linn) pennel facili-
tates leaves were air-dried and powdered. A hot 90%
ethanol extract of the powdered leaves was prepared
using Soxlet extraction, and the alcohol was evaporated
under reduced pressure. BME for all experiments was
prepared in a single batch from the same harvest of plant
material to maintain consistency of active compounds.
2.3. Drug treatment
Two doses (BME 40 or 160 mg/kg/day) were admin-
istered by blending BME into powdered rodent chow
(#8640 meal, Harlan Teklad, Madison, Wisconsin).
Each mouse received 5 g of powdered chow (with or
without BME) on a daily basis. Mice were housed
individually for the duration of the experiment. A
“short-term” treatment experiment from postnatal day
(PND) 60 to 120 (2–4 months) determined the effect
of BME during the earliest phase of amyloid deposi-
tion. The control group (n = 8) was fed unadulter-
ated powdered rodent chow, a low dose BME group
was fed 40 mg/kg/day BME (n = 11) and a high dose
treatment group received BME 160 mg/kg/day (n =
8). A “long-term” experiment involved similar treat-
ment of control (n = 11), BME 40 mg/kg/day (n =
9) and BME 160 mg/kg/day (n = 7) PSAPP mice
from PND 60–300 (2–10 months) during the time pe-
riod where plaque load rapidly increases in both the
cortex and hippocampus [25]. To determine baseline
behavioral performance, two separate non-transgenic
control groups were fed powdered rodent chow and
tested at either PND 60 (n = 8) or PND 300 (n =
8). The body weight of each mouse was monitored on
a weekly basis to provide an index of general health.
BME-treated PSAPP mice were grossly indistinguish-
able from chow-fed control mice with respect to well-
being and appearance.
 L.A. Holcomb et al. / Bacopa monniera extract reduces amyloid levels in PSAPP mice
2.4. Behavioral testing procedure
Mice were transferred in their home cages to the
behavioral testing room for a one-week acclimation
prior to testing. All testing was conducted in the home
room during the animal’s light cycle (lights on from 6
A.M. to 6 P.M.) during the PND 56–60 or PND 296–
300 periods.
2.5. Y-maze two trial recognition task
During an 8 minute acquisition trial, a sliding door
was used to block one arm (novel arm) of a Y-maze ap-
paratus and entries into the other arms were recorded.
Mice performing fewer than 10 arm entries were ex-
cluded from further analysis. After a 2 hr inter-trial in-
terval, mice were permitted to explore all three arms of
the Y-maze during an 8-minute recognition trial. The
percentage entry into the novel vs previously explored
arms (% novel arm entry), and spontaneous alterna-
tion scores for each mouse were calculated as a mea-
sure of short-term working memory and spatial navi-
gation [15,20]. Each treatment regimen was analyzed
with ANOVA followed by comparison with a control
group using Dunnett’s method.
2.6. Open field
Each mouse was given a single 50 minute session in
an open field testing apparatus equipped with 16 pho-
tobeams (San Diego Instruments, San Diego, CA). The
distance traveled by each mouse in either the central or
peripheral area of the open field was analyzed for the
first 5 minutes (initial), the last 5 minutes, and the total
50 minute session. Rearing and grooming behaviors
were also monitored.
2.7. Tissue preparation
Mice were perfused transcardially with 0.9% saline
and the brains removed. The left hemisphere was
snap-frozen after dissection with a blunt technique into
the hippocampus and the cortex anterior to bregma
1.78. The right hemisphere was drop fixed in 4%
paraformaldehyde for 24 hrs, cryoprotected in a graded
sucrose series and serially sectioning at 40 microns us-
ing a sliding microtome.
245
2.8. Aβ 1-40 and Aβ 1-42 ELISA
Levels of full length human Aβ 1-40 and 1-42 were
measured from brain homogenates of cortex and hip-
pocampus using commercially available fluorometric
ELISA kits (Biosource International Inc., Camarillo,
CA). The guanadine-HCl extraction technique pro-
vided by the manufacturer was followed to maximize
the recovery of Aβ. Guanidine-HCl extraction (5M
final concentration) has been demonstrated to be more
effective than formic acid at extracting low levels of
Aβ [27]. Duplicate samples for each animal were as-
sayed and the results averaged to give a single value of
Aβ 1-40 and 1-42 levels for each animal that was then
used to calculate the mean ± SEM for each treatment
group.
2.9. Determination of Fibrillar Amyloid load
Three coronal sections per mouse were stained with
Congo Red (Accustain Amyloid kit, Sigma). A series
of 8 images that spanned the entire cortex were cap-
tured for each section using a Nikon E100 microscope
and a DXM 1200 digital camera. Fibrillar amyloid
load (area covered by Congo Red stained deposits/area
of cortex x 100) and average plaque size were calcu-
lated using a custom macro written in ImagePro Plus
software (MediaCybernetics v4.5). Congo Red posi-
tive pixels were segmented with respect to threshold
using hue-saturation-intensity (HSI) settings that dis-
tinguished positive staining from background staining.
The fibrillar amyloid load (% cortical area occupied)
and plaque size data from three sections/mouse was av-
eraged and used to calculate an average value for each
treatment group.
2.10. Statistical analysis
For biochemical and histological data, ANOVA and
Dunnett’s tests for comparison with a control was used
to compare low-dose and high-dose BME treatment
groups with untreated (chow-fed) control PSAPP con-
trols (three groups). For behavioral data, we used
an overall ANOVA (4 groups) with planned t-test
comparisons (untreated non-transgenic vs untreated
PSAPP mice, and chow-fed control PSAPP vs BME
40 mg/kg/day or BME 160 mg/kg/day).
246 L.A. Holcomb et al. / Bacopa monniera extract reduces amyloid levels in PSAPP mice
Fig. 1. Short-term BME treatment effects on amyloid levels in the cortex. Levels of Aβ 1-40 and 1-42 in the cortex (mean ± SEM) were
significantly reduced compared to chow-fed PSAPP control (black column) by PND 60-120 treatment with either BME 40mg/kg/day (dashed
column) or BME 160 mg/kg/day (striped column) (“a” = ANOVA followed by Dunnett’s test for comparison with control, p < 0.05).
3. Results
3.1. Body weight
Weight gain did not differ in any of the treatment
groups in either the short-term (Control = 5.55 ±
1.56 g; BME 40 mg/kg = 2.60 ± 0.43; BME 160 mg/kg
= 2.67 ± 1.23; Average ± SEM p > 0.13) or long-term
(Control = 9.6 ± 1.31 g ± SEM; BME 40 mg/kg =
12.67 ± 1.63; BME 160 mg/kg = 8.75 ± 1.25, p >
0.16) experiments.
3.2. Aβ Levels
With short-term treatment, both doses of BME sig-
nificantly decreased the levels of Aβ 1-40 [ANOVA
F (2, 26) = 7.61, P < 0.01, followed by Dunnet-
t’s test for comparison with control] and Aβ 1-42
[ANOVA F (2, 25) = 6.60, P < 0.01, Dunnett’s] in
the cortex, but not in the hippocampus (Fig. 1). In
the long-term treatment group, both doses of BME
significantly reduced Aβ 1-42 [ANOVA F (2, 25) =
6.43, P < 0.01 Dunnett’s] and Aβ 1-40 levels in
the cortex (ANOVA F (2, 25) = P < 0.01 Dunnet-
t’s). BME 160 mg/kg/day reduced Aβ 1-40 [ANOVA
F (2, 25) = 3.9, P < 0.05 Dunnett’s] and Aβ 1-42
[ANOVA F (2, 25) = 5.91, P < 0.01, Dunnett’s] in
the hippocampus (Fig. 2).
3.3. Y-maze
Using the Y-maze two-trial recognition task, mice
that have normal memory and adequate spatial navi-
gation skills will preferentially explore a novel, previ-
ously unavailable arm. Two mice from the nontrans-
genic control group, one mouse from the PSAPP-BME
40 mg/kg/day group and one mouse from the PSAPP-
BME 160 mg/kg/day group were excluded from the
analysis because they made ten or less arm entries. In
the short-term experiment, untreated PSAPP mice per-
formed similarly in the Y-maze to age-matched, chow-
fed, non-transgenic controls (NTC), and no Y-maze
drug effects were observed. However, in the long-term
experiment, chow-fed, 10 month-old PSAPP mice had
elevated total numbers of total arm entries compared to
10 month-old non-transgenic controls, an effect which
was reversed by BME (Table 1). Decreased % entries
into the novel arm during the recognition trial was also
detected in 10 month-old PSAPP mice (Table 1). BME
40 mg/kg/day increased % novel enteries as compared
to the transgenic controls. There were no genotype or
drug effects on % spontaneous alternations.
3.4. Open field
Compared to non-transgenic controls, the PSAPP
genotype produced highly significant effects on open
field behavior in both short-and long-term experiments
that were especially prominent during the 1 st 5-minutes
of the open field testing session BME treatment re-
versed these effects. Four month-old control PSAPP
 L.A. Holcomb et al. / Bacopa monniera extract reduces amyloid levels in PSAPP mice 247

Table 1
Number of arm entries in Y-maze (# entries acquisition) were recorded during an 8 minute
acquisition trial followed 2 hours later by an 8 minute recognition trial (# entries recognition, %
novel arm entries, % spontaneous alternation). In chow-fed PSAPP vs non-transgenic control
mice, an increased number of total arm entries was observed during the acquisition trial (*
ANOVA F (3, 39) = 5.51, planned t-test, p < 0.05) and a reduction in entries into the novel
arm was observed during the recognition trial (**ANOVA F (3, 38) = 2.87, planned t-test,
p < 0.05)
Group # Entries # Entries % Novel % Spontaneous
(Acquisition) (Recognition) Entries Alternation
Non-Tg Control 51 ± 5.1 51 ± 5.2 34 ± 1.4 55 ± 2.5
PSAP Control 103 ± 17.3∗ 84 ± 22.3 30 ± 1.3∗∗ 53 ± 3.0
PSAPP BME-40mg/kg 50 ± 5.4 57 ± 6.6 34 ± 2.1 50 ± 3.3
PSAPP BME-160mg/kg 66 ± 15.8 84 ± 22.4 36 ± 3.1 53 ± 5.3

Fig. 2. Long-term BME treatment effects on amyloid levels in the cortex and hippocampus. Levels of Aβ 1-40 and 1-42 in the cortex and
hippocampus (mean ± SEM) were significantly reduced compared to chow-fed PSAPP control (black column) by PND 60-300 treatment with
BME (“a” = ANOVA followed by Dunnett’s test for comparison with control, p s <0.05).

mice traveled farther during the 1 st 5 minutes compared
to the NTC group [NTC = 603 ± 39 SEM; PSAPP =
936 units ± 100, t = 3.03, p < 0.05]. The increase
in locomotion was particularly prominent in the center
of the open field, and BME 40 mg/kg/day reversed the
effect [NTC = 80 ± 37; PSAPP = 195 ± 44; BME
40 mg/kg/day = 81 ± 8; BME 160 mg/kg/day = 128
± 25, ANOVA F (3, 34) = 4.84, p < 0.01; planned
post-hoc t-test comparing PSAPP control to BME treat-
ments p < 0.05]. In the short-experiment, there were
no genotype or BME effects on rearing or grooming
behaviors.
In the long-term experiment, there were significant
PSAPP genotype and BME treatment effects not only
in the 1st five minutes, but also for the entire session.
PSAPP mice traveled farther in field center the 1 st 5
minutes of open field testing in the long-term experi-
ment compared to NTC, and BME 40 mg/kg/day but
not BME 160 mg/kg/day normalized this effect [NTC
= 135 ± 26; PSAPP = 323 ± 71, BME 40 mg/kg/day =
131 ± 25, BME 160 mg/kg/day = 287 ± 64; ANOVA
F (3,41) = 3.64, P < 0.05; planned post-hoc t-tests
comparing PSAPP controls to BME treatments]. Al-
though the total distance traveled for PSAPP mice for
the entire 50-minute test session was almost double
that of NTC mice, the difference was not significant.
However, PSAPP mice traveled farther in the center of
the field for the entire session compared to NTC mice,
and BME 40 mg/kg/day normalized this effect [PSAPP
= 3279 units ± 786, NTC = 1650 ± 215, BME 40
mg/kg/day 1164 ± 292, BME 160 mg/kg/day = 2541
± 647; ANOVA F (3, 41) = 2.92, P < 0.05; planned
post-hoc t-tests comparing PSAPP controls to BME
248 L.A. Holcomb et al. / Bacopa monniera extract reduces amyloid levels in PSAPP mice
Table 2
Brains of PSAPP mice treated in a long-term (eight months) protocol
with BME-40, BME-160 or a control diet were stained with Congo
Red to evaluate the plaque size and fibrillar amyloid load (percentage
of Congo Red+ cortex). BME had no effect on Congo Red amyloid
load [ANOVA F (2, 25) = 1.63, P = 0.21
Treatment Groups Amyloid Load Plaque Size
PSAPP Control 14.89 ± 0.93 395 ± 23
PSAPP BME-40 mg/kg 14.63 ± 1.04 376 ± 26
PSAPP BME-160 mg/kg 13.29 ± 1.11 450 ± 28
treatments]. There were no genotype effects on rearing
or grooming behaviors in the long-term experiment.
3.5. Fibrillar Aβ deposition
The percent area of cortex occupied by fibrillar
amyloid plaques was examined by staining sections
with Congo Red from 10 month-old PSAPP mice.
Long-term treatment with BME 40 mg/kg/day or BME
160 mg/kg/day did not significantly alter fibrillar amy-
loid load or plaque size (Table 2).
4. Discussion
Spontaneously elevated amyloid levels, mature amy-
loid plaque formation and behavioral changes represent
the core phenotype that has made the PSAPP mouse
an attractive model for testing interventions to reverse
amyloid pathology in Alzheimer’s disease. Control
(chow-fed) PSAPP mice developed deposits of Congo
Red-positive amyloid at 4 months in the cortex and hip-
pocampus. Total levels of Aβ 1-40 and 1-42 extracted
with guanidine-HCl were also elevated in these ani-
mals. Amyloid levels were higher and performance on
the Y-maze was modestly impaired in older compared
to younger PSAPP mice. These changes are consistent
with previous reports demonstrating that co-expression
of human APP and PS-1 mutations produce substantial
amyloid pathology accompanied by modest behavioral
alterations at these ages [13–15].
The most robust behavioral effect observed in the
present study was an increase in exploratory locomo-
tor behavior in PSAPP mice. In the Y-maze, PSAPP
mice performed more arm entries, and in the open field
test, PSAPP mice traveled a greater distance, particu-
larly while they were exploring the center portion of
the field. In addition to excess locomotion, the open
field results provide evidence for a loss of the normal
aversion to exploring an exposed area. A similar in-
crease in distance traveled in the center of the open field
has recently been reported by Lalonde et al. [21] in
12 month-old APP(695)SWE/co+PS1/DeltaE9 mice.
This behavior may reflect reduced anxiety, facilitated
habituation, reduced fear of open spaces and/or lack of
spatial monitoring. It will be interesting to investigate
whether increased locomotor activity and lack of spatial
awareness in PSAPP mice is related to the wandering
syndrome observed in many Alzheimer’s patients [24].
Although there was some evidence that high-dose BME
reversed exploratory locomotor changes in PSAPP
mice, the lower dose of BME (40/mg/kg/day) was par-
ticularly effective at reversing this behavioral deficit. In
addition to behavioral alterations in the open field test,
we also observed a subtle change in the y-maze per-
formance that was reversed by BME treatment. Four
month-old non-transgenic mice preferentially chose to
enter a previously unexplored arm of the testing appa-
ratus after a 2-hour delay, suggesting that they “remem-
bered” exploring the other arms during the acquisition
trials. This preferential exploration pattern was not ob-
served in 10 month-old non-transgenic mice, who ran-
domly choose from both the previously explored arms
and the novel arm after a 2-hour delay. Interestingly,
chow-fed PSAPP control mice preferentially explored
the non-novel arms after the two-hour delay, avoid-
ing rather than preferentially choosing the novel arm.
However, low-dose BME treatment reversed this effect.
From this behavioral data, we conclude that BME was
capable of normalizing behavioral changes observed in
PSAPP mice, with a low dose being generally more
effective than a high dose.
Our data indicate that short and long-term BME treat-
ment beginning at two months of age reduced brain Aβ
levels in PSAPP mice by as much as 60%. Short-term
BME treatment from 2 to 4 months of age resulted in a
more pronounced reduction in Aβ levels in the cortex
than in the hippocampus. In contrast, after long-term
treatment, Aβ levels were reduced proportionally in
both hippocampus and cortex. PSAPP mice at 4 months
of age are in an early stage of plaque consolidation,
and at this age, there are more plaques observed in the
cingulate and motor cortex than the hippocampus [25].
The effect of BME treatment to alter Aβ levels may
depend on the presence of a certain level of plaque
maturity which is not yet present in the hippocampus
at 4 months. Alternatively, BME might affect some
intermediate amyloid species such as oligomeric Aβ
that is generated late in plaque maturation. It is likely
that BME reduction of amyloid levels was a factor in
the reversal of behavioral changes present in PSAPP
mice, although we note that the high dose of BME was
maximally effective in reducing amyloid levels but was
 L.A. Holcomb et al. / Bacopa monniera extract reduces amyloid levels in PSAPP mice
less effective in reversing behavioral deficits. Perhaps
BME’s primary affect on behavior was not directly re-
lated to amyloid levels, or an additional compound in
BME negatively affected behavior at high but not low
doses. Additional studies are needed to investigate the
mechanism involved in BME’s reduction of amyloid
levels and how this reduction affects behavior.
We observed that despite clear reductions in Aβ lev-
els, long-term BME treatment did not change the pro-
portional area of the cortex occupied by Congo Red-
positive fibrillar plaques. Several studies have reported
similar findings of behavioral normalization without
changes in amyloid plaque load [1,17,19]. There are
several viable explanations for the pattern of robust
BME effects on amyloid levels and minimal BME ef-
fects on plaque burden. First, our neuropathologi-
cal data indicate only that the overall sizes of mature
plaques visible by Congo Red staining are similar in
the treated vs untreated mice. Our data provide no in-
formation about the density of fibrillar material within
the plaque field. A lower density of fibrillar amy-
loid within plaques may reduce neuronal pathophysio-
logical effects and delay or prevent the appearance of
behavioral deficits. A second observation is that the
guanidine-HCL extraction and ELISA procedure de-
tects both fibrillar amyloid and diffuse amyloid [27].
Thus, another possibility is that there was a reduction in
diffuse plaque levels which could not be measured by
Congo red staining. Future experiments will be needed
to clarify the relationship between Aβ levels, plaque
formation and behavior in PSAPP mice.
Studies suggest that Aβ vaccine therapies can largely
prevent the formation of fibrillar plaques if given to
young transgenic mice and can also enhance removal
of pre-existing plaques in older animals [33]. The use
of Aβ vaccines is also said to protect from the devel-
opment of learning deficits in aging mice carrying APP
and PS-1 mutations [26]. Intranasal administration of
Aβ can reduce plaque number by up to 75% [22], and
injections of Aβ antibodies can robustly reduce amy-
loid load [8,39]. Interestingly, a recent study indicates
that extended Aβ immunotherapy (2–17 month injec-
tions of fibrillar amyloid) in PSAPP mice produced
a pattern of effects similar to those observed in the
present study: Behavioral normalization, reduction of
cortical amyloid levels, but no reduction in Congo Red
amyloid plaque burden [17]. Thus, BME therapy in
transgenic mice displays some characteristics observed
in amyloid immunotherapy trials.
A variety of other agents such as Vitamin E, cur-
cumin and ibuprofen have also shown efficacy in re-
249
ducing amyloid levels in Alzheimer’s disease trans-
genic mouse models. Vitamin E treatment of singly
transgenic APP mice (Tg2576) reduces Aβ levels and
amyloid load by up to 30% when administered during
the initial time period when Aβ deposits are forming.
Amyloid deposits in older animals are not affected by
Vitamin E [38]. A low but not a high dose of curcumin
reduced total levels of Aβ by up to 43%, and reduced
plaque load by 44% following treatment of Tg2576
mice for 6 months [23]. Treatment of Tg2576 mutants
with the non-steroidal anti-inflammatory drug ibupro-
fen also reduces Aβ levels and plaque burden in the
frontal cortex by up to 60% [41]. Compared to previ-
ously tested agents, BME therapy appears to produce
a comparable reduction in amyloid levels, although the
pattern of amyloid deposition changes may differ from
agent to agent.
Studies using a similar Bacopa monniera extracts, as
well as other studies using the saponin complexes ba-
cosides A and B have demonstrated behavioral effects
in several animal models [7,34–36]. Memory deficits
induced by the anti-cholinergic drug scopolamine can
be reversed by Bacopa monniera extract [5], and a
7-day treatment was capable of protecting mice from
phenytoin-induced passive avoidance deficits [40]. Ba-
cosides have also been observed to improve rodent Y-
Maze performance in a dose-dependent manner [9].
Human placebo-controlled trials have suggested that
chronic oral administration of a Bacopa monniera ex-
tract improves the speed of visual processing and fa-
cilitates memory retention without significant effects
on learning and attention [31,37]. Bacopa monniera’s
mechanism of action to enhance cognitive performance
is unknown, but biochemical evidence exists to sup-
port antioxidant effects, anti-inflammatory effects, im-
munomodulatory effects and specific interactions with
stress-reactive proteins such as superoxide dismutase
and the heat shock proteins [2,4,29]. In addition, pro-
duction of nitric oxide by astrocytes is reduced by Ba-
copa monniera treatment [32]. It is possible that be-
cause BME is a crude extract, several chemical com-
pounds with complementary actions may combine to
affect amyloid pathology and behavior.
Epidemiological surveys indicate that over-the-
counter preparations of Bacopa monniera are used
around the world with a goal of promoting cogni-
tion [18]. Various formulations of this ethnobotanical
are commercially available in many health food stores.
Despite the wide usage of these preparations, Bacopa
has not generated much scientific interest, as evidenced
by the limited number of PubMed references (69 total
250 L.A. Holcomb et al. / Bacopa monniera extract reduces amyloid levels in PSAPP mice
references through Feb, 2006). Our present data pro-
vides justification for further study of this traditional
Ayurvedic phytotherapy. Considering the data from ini-
tial human experiments indicating a robust behavioral
effect of Bacopa monniera at low doses, and the data
from the present study indicating a preferential effect
of low-dose BME on behavior, it will be of particular
interest to study dose-response effects of Bacopa and its
active constituents. The long history of human use of
Bacopa monniera, its demonstrated action to promote
cognitive performance in human and animal models,
and it effects to reduce amyloid levels and reverse be-
havioral deficits in the PSAPP transgenic Alzheimer’s
model indicate that BME has considerable potential as
a therapeutic intervention in Alzheimer’s disease.
Acknowledgments
Supported by the Scott, Sherwood and Brindley
Foundation, the Helen Vosburg McCrillus Plummer and
Robert Edward Lee Plummer, Jr. Endowed Fund and
the Veterans Administration.
References
[1] G.W. Arendash, M.F. Garcia, D.A. Costa, J.R. Cracchiolo,
I.M. Wefes and H. Potter, Environmental enrichment improves
cognition in aged Alzheimer’s transgenic mice despite stable
B-amyloid deposition, Neuroreport 15 (2004), 1751–1754.
[2] S.K. Bhattacharya, A. Bhattacharya, A. Kumar and S. Ghosal,
Antioxidant activity of Bacopa monniera in rat frontal cortex,
striatum and hippocampus, Phytother Res 14 (2000), 174–179.
[3] F.E. Bloom, J.F. Reilly, J.M. Redwine, C.C. Wu, W.G. Young
and J.H. Morrison, Mouse models of human neurodegener-
ative disorders: requirements for medication development,
Arch Neurol 62 (2005), 185–187.
[4] D.K. Chowdhuri, D. Parmar, P. Kakkar, R. Shukla, P.K. Seth
and R.C. Srimal, Antistress effects of bacosides of Bacopa
monnieri: modulation of Hsp70 expression, superoxide dis-
mutase and cytochrome P450 activity in rat brain, Phytother
Res 16 (2002), 639–645.
[5] A. Das, G. Shanker, C. Nath, R. Pal, S. Singh and H. Singh,
A comparative study in rodents of standardized extracts of
Bacopa monniera and Ginkgo biloba: anticholinesterase and
cognitive enhancing activities, Pharmacol Biochem Behav 73
(2002), 893–900.
[6] V.B. Dash and V.L. Kashyap, Mareria Medica of Ayerveda,
New Delhi Concept Publishing Company, 1980, 53–54.
[7] M. Deepak and A. Amit, The need for establishing identities
of bacoside A and B, the putative major bioactive saponins of
Indian medicinal plant Bacopa monnieri, Phytomedicine 11
(2004), 264–268.
[8] R.B. DeMattos, K.R. Bales, D.J. Cummins, S.M. Paul and
D.M. Holtzman, Brain to plasma amyloid-beta efflux: a mea-
sure of brain amyloid burden in a mouse model of Alzheimer’s
disease, Science 295 (2002), 2264–2267.
[9] B.N. Dhawan and H.K. Singh, The Ayurvedic nootropic Ba-
copa monnieri (1.) pennell facilitates learning and improves
memory, Vth Int Cong Ethnopharmacol. (Abst) (1999), 17.
[10] K. Duff, C. Eckman, C. Zehr, X. Yu, C.M. Prada, J. Perez-
tur, M. Hutton, L. Buee, Y. Harigaya, D. Yager, D Morgan,
M.N. Gordon, L. Holcomb, L. Refolo, B. Zenk, J.Hardy and
S. Younkin, Increased amyloid βg42(43) in brains of mice
expressing mutant presenilin 1, Nature 383 (1996), 710–713.
[11] G. Emilien, J.M. Maloteaux, K. Beyreuther and C.L. Mas-
ters, Alzheimer’s disease: mouse models pave the way for
therapeutic opportunities, Arch Neurol 57 (2000), 176–181.
[12] D.A. Evans, Estimated prevalence of Alzheimer’s disease in
the United States, Milbank Q 68 (1990), 267–289.
[13] M.N. Gordon, L.A. Holcomb, P. Jantzen, G. DiCarlo, D.
Wilcock, K. Connor, J. Melachrino, J. O’Callaghan and D.
Morgan, Time course of the development of Alzheimer-like
pathology in the doubly transgenic mPS1+mAPP mouse, Exp
Neurol 172 (2002), 183–195.
[14] L.A. Holcomb, M.N. Gordon, P. Jantzen, K. Hsiao, K. Duff
and D.G. Morgan, Behavioral changes in transgenic mice ex-
pressing both amyloid precursor protein and presenilin-1 mu-
tations: lack of association with amyloid deposits, Behavior
Genet 29 (1999), 177–185.
[15] L. Holcomb, M.N. Gordon, E. McGowan, X. Yu, S. Benkovic,
P. Jantzen, K. Wright, I. Saad, R. Mueller, D. Morgan, S.
Sanders, C. Zehr, K. O’Campo, J. Hardy, M. Prada, C.
Eckman, S. Younkin, K. Hsiao and K. Duff, Accelerated
Alzheimer phenotype in transgenic mice carrying both mutant
amyloid precursor protein and presenilin I transgenes, Nature
Med 4 (1998), 97–100.
[16] K. Hsiao, P. Chapman, S. Nilsen, C. Eckman, Y. Harigaya, S.
Younkin, F. Yang and G. Cole, Correlative memory deficits,
Aβ elevation, and amyloid plaques in transgenic mice, Science
274 (1996), 99–102.
[17] M.T. Jensen, M.D. Mottin, J.R. Cracchiolo, R.E. Leighty
and G.W. Arendash, Lifelong immunization with human B-
amyloid (1-42) protects Alzheimer’s transgenic mice against
cognitive impairment throughout aging, Neuroscience 130
(2005), 667–684.
[18] A.F. Jorm, B. Rodgers and H. Christensen, Use of medications
to enhance memory in a large community sample for 60–64
year olds, Int Psychogeriatr 16 (2004), 209–217.
[19] J. Joseph, N. Denisova, G. Arendash, M. Gordon, D. Dia-
mond, B. Shukitt-Hale and D. Morgan, Blueberry supplemen-
tation enhances signaling and prevents behavioral deficits in an
Alzheimer disease model, Nutr Neurosci 6 (2003), 153–162.
[20] N. Ladurelle, B. Eychenne, D. Denton, J. Blair-West, M.
Schumacher, P. Robel and E. Baulieu, Prolonged intracere-
broventricular infusion of neurosteroids affects cognitive per-
formances in the mouse, Brain Res 858 (2000), 371–379.
[21] R. Lalonde, H.D. Kim, J.A. Maxwell and K. Fukuchi,
Exploratory activity and spatial learning in 12-month-old
APP(695)SWE/co+PS1/DeltaE9 mice with amyloid plaques,
Neurosci Lett 390 (2005), 87–92.
[22] C.A. Lemere, E.T. Spooner, J. LaFrancois, B. Malester, C.
Mori, J.F. Leverone, Y. Matsuoka, J.W. Taylor, R.B. DeMattos,
D.M. Holtzman, J.D. Clements, D.J. Selkoe and K.E. Duff,
Evidence for peripheral clearance of cerebral Abeta protein
following chronic, active Abeta immunization in PSAPP mice,
Neurobiol Dis 14 (2003), 10–18.
[23] G.P. Lim, T. Chu, F. Yang, W. Beech, S.A. Frautschy and G.M.
Cole, The curry spice Curcumin reduces oxidative damage
and amyloid pathology in an Alzheimer transgenic mouse, J
Neurosci 21 (2001), 8370–8377.
 L.A. Holcomb et al. / Bacopa monniera extract reduces amyloid levels in PSAPP mice
[24] R.G. Logsdon, L. Teri, S.M. McCurry, L.E. Gibbons, W.A.
Kukull and E.B. Larson, Wandering: a significant problem
among community-residing individuals with Alzheimer’s dis-
ease, J Gerontol B Psychol Sci Soc Sci 53 (1998), 294–299.
[25] E. McGowan, S. Sanders, T. Iwatsubo, A. Takeuchi, T. Saido,
C. Zehr, X. Yu, S. Uljon, R. Wang, D. Mann, D. Dickson and
K. Duff, Amyloid phenotype characterization of transgenic
mice overexpressing both mutant amyloid precursor protein
and mutant presenilin-1 transgenes, Neurobiol Dis 6 (1999),
231–244.
[26] D. Morgan, D.M. Diamond, P.E. Gottschall, K.E. Ugen, C.
Dickey, J. Hardy, K. Duff, P. Jantzen, G. DiCarlo, D. Wilcock,
K. Connor, J. Hatcher, C. Hope, M. Gordon and G.W. Aren-
dash, A beta peptide vaccination prevents memory loss in
an animal model of Alzheimer’s disease, Nature 408 (2000),
982–985.
[27] M. Morishima-Kawashima, N. Oshima, H. Ogata, H. Yam-
aguchi, M. Yoshimura, S. Sugihara and Y. Ihara, Effect of
apolipoprotein E allele epsilon4 on the initial phase of amyloid
beta-protein accumulation in the human brain, Am J Pathol
157 (2000), 2093–2099.
[28] J. Näslund, V. Haroutunian, R. Mohs, K.L. Davis, P. Davies, P.
Greengard and J.D. Buxbaum, Correlation between elevated
levels of amyloid B-peptide in the brain and cognitive decline,
JAMA 283 (2000), 1571–1577.
[29] D. Rai, B. Bhatia, G. Palit, R. Pal, S. Singh and H.K. Singh,
Adaptogenic effect of Bacopa monniera (Brahmi), Pharmacol
Biochem Behav 75 (2003), 823–830.
[30] K. Ritchie and D. Kildea, Is senile dementia “age related” or
“aging related”? – evidence from a meta-analysis of dementia
prevalence in the oldest old, Lancet 346 (1995), 931–934.
[31] S. Roodenrys, D. Booth, S. Bulzomi, A. Phipps, C. Micallef
and J. Smoker, Chronic effects of Brahmi (Bacopa monnieri)
on human memory, Neuropsychopharmacology 27 (2002),
279–281.
[32] A. Russo, F. Borrelli, A. Campisi, R. Acquaviva, G. Raciti and
A. Vanella, Nitric oxide-related toxicity in cultured astrocytes:
effect of Bacopa monniera, Life Sci 73 (2003), 1517–1526.
[33] D. Schenk, R. Barbour, W. Dunn, G. Gordon, H. Grajeda,
251
T. Guido, K. Hu, J. Huang, K. Johnson-Wood, K. Khan, D.
Kholodenko, M. Lee, Z. Liao, M. Lieberburg, T. Yednock,
D. Games and P. Seubert, Immunization with amyloid-beta
attenuates Alzheimer-disease-like pathology in the PDAPP
mouse, Nature 400 (1999), 173–177.
[34] H.K. Singh and B.N. Dhawan, Effect of Bacopa monniera
(Brahmi) extract on avoidance responses in rats, J Ethnophar-
macol 5 (1982), 205–214.
[35] H.K. Singh and B.N. Dhawan, Effect of bacosides A and B on
avoidance responses in rats, Phytother Res 2 (1988), 70–75.
[36] H.K. Singh and B.N. Dhawan, Neuropsychopharmacological
effects of the Ayurvedic nootropic Bacopa monniera Linn.
(Brahmi), Ind J Pharmacol 29 (1997), S359–S365.
[37] C. Stough, J. Lloyd, J. Clarke, L.A. Downey, C.W. Hutchi-
son, T. Rodgers and P.J. Nathan, The chronic effects of an
extract of Bacopa monniera (Brahmi) on cognitive function
in healthy human subjects, Psychopharmacology (Berl) 156
(2001), 481–484.
[38] S. Sung, Y. Yao, K. Uryu, H. Yang, V.M. Lee, J.Q. Trojanowski
and D. Pratico, Early vitamin E supplementation in young but
not aged mice reduces Abeta levels and amyloid deposition
in a transgenic model of Alzheimer’s disease, FASEB J 18
(2004), 323–325.
[39] A.K. Vehmas, D.R. Borchelt, D.L. Price, D. McCarthy, M.
Wills-Karp, M.J. Peper, G. Rudow, J. Luyinbazi, L.T. Siew
and J.C. Troncoso, beta-Amyloid peptide vaccination results
in marked changes in serum and brain Abeta levels in APP-
swe/PS1DeltaE9 mice, as detected by SELDI-TOF-based Pro-
teinChip technology, DNA Cell Biol 20 (2001), 713–721.
[40] D. Vohora, S.N. Pal and K.K. Pillai, Protection from
phenytoin-induced cognitive deficit by Bacopa monniera, a
reputed Indian nootropic plant, J Ethnopharmacol 71 (2000),
383–390.
[41] Q. Yan, J. Zhang, H. Liu, S. Babu-Khan, R. Vassar, A.L.
Biere, M. Citron and G. Landreth, Anti-inflammatory drug
therapy alters beta-amyloid processing and deposition in an
animal model of Alzheimer’s disease, J Neurosci 23 (2003),
7504–7509.