Ameliorative eﬀects of Bacopa monnieri on olfactorybulbectomy-induced cognitive deﬁcits in mice
Name of dispatched researcher（派遣研究者名）
AfÀliation of Instructor（所属）
Host Collaborator（受入研究者）

Pham Thi Nguyet Hang
Department of Pharmacology and Biochemistry,
National Institute of Medicinal Materials, Hanoi,
VietnamProfessor Kinzo Matsumoto
Division of Medicinal Pharmacology
Institute of Natural Medicine,
University of Toyama

1. Abstract:
Aim: Bacopa monnieri (L.) Wettst. (BM) is a well-known medicinal herb for treatment of epilepsy,
insomnia, etc.. This plant has been known to be useful for improving intelligence and memory fora long time. This study aimed to clarify the effects of alcoholic extract of BM (BM-E) on cognitivedeÀcits in olfactory bulbectomized (OBX) mice, one of the animal models of Alzheimer’s disease(AD) and its mechanisms underlying the action.
Methods: OBX ddY mice were treated with BM-E at a daily dose of 50 mg/kg via feeding needle
for 1 week before and 2 weeks after 3 days of OBX. Administration of the reference drug tacrine(2.5 mg/kg/day, i.p.) to OBX mice was started 3 days after OBX. Cognitive performances of theanimals was analyzed using the novel object recognition test, modiÀed Y maze test, and fear conditioning test to elucidate non-spatial short term memory, spatial working memory, and longterm memory, respectively. After completing the behavioral experiments, the expression levelof cholinergic marker proteins and the activity of acetylchoniesterase (AChE) in the brain wereanalyzed by Western blotting and Ellman’s method, respectively.
Results: OBX caused impairment of non-spatial short term memory, spatial working memory,and long-term memory. OBX-induced memory deÀcits were reversed by BM-E and tacrine. The
effect of BM-E on the spatial working memory deÀcit was reversed by scopolamine, a muscarinic
receptor antagonist. BM-E attenuated OBX-induced down-regulation of choline acetyltransferase
without affecting muscarinic M1 and M3 receptor expression or AChE activity.
Discussion: These finding demonstrate that BM-E improves memory deficits caused by OBX
and that the effect of BM-E is at least in part mediated by stimulation/normalization of central
cholinergic systems. Our Àndings further support the idea that BM is beneÀcial for treatment ofcognitive deÀcits such as AD.

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2. Introduction
Alzheimer’s disease (AD) is a neurodegenerative disease related to cognitive and memory impairments which are affecting many elderly people worldwide. Although some drugs are available for
AD treatment, the outcomes are often unsatisfactory. Therefore, the medicinal herbs are promising strategy to development of effective drugs for AD.
Bacopa monnieri (L.) Wettst. (BM) a well-known traditional Ayuredic medicinal plant and is usedin India as a brain tonic. In Vietnam, BM grows throughout form from Northern to Southern andusually uses as daily vegetable not only for high income people but also for low income people.
Some lines of evidence show that BM improves cognitive and memory impairment on animal
experiments and healthy volunteer. However, it remains unclear the mechanism(s) of action ofBM as well as to promote more possible usages of BM in cognitive deÀcit therapy. Therefore, thisstudy aims to elucidate the anti-dementia effects of BM using olfactory bulbectomy (OBX) model.
For this aim, we elucidate the non-spatial short term memory, spatial working memory, and longterm memory by using three different behavioral testes, including novel object recognition test,modiÀed Y maze test, and fear conditioning test, respectively. To clarify the mechanism of the action of BM, we investigate the effect of BM on the central cholinergic systems in OBX animal.
3. Materials and Methods
3.1. Plant materials and preparation of crude extract:
BM was collected from Ho Chi Minh city (Southern of Vietnam). It was identiÀed by Dr. PhamThanh Huyen, Department of Medicinal Plant Resources, National Institute of Medicinal Materials (NIMM, Vietnam). The number code of voucher specimen is 9687 that was kept in the Herbarium, Department of Medicinal Plant Resources, NIMM. The aerial part of BM was cutted into
small pieces, dried, and percolated with circulating 95% ethanol for 2 days at the ratio of 1 g : 12ml. The residue was extracted again twice using 95% ethanol at the ratio of 1 g : 10 ml. The combined extract was dried under reduced pressure.
3.2. AnimalMale ddY mice (Japan SLC Inc., Shizuoka, Japan) were obtained at the age of 9 weeks old andhoused with a 12-h light/dark cycle (light on: 07:30-19:30) at 22 ± 1oC. Food and water wereavailable ad libitum. The animal were habituated to the laboratory animal room for at least 1week before surgery. The behavioral experiments were performed during the light phase from 9:00
to 18:00. The present studies were conducted in accordance with the Guiding Principles for theCare and Use of Aninals and were approved by the Institutional Animal Use and Care Committee in the University of Toyama.
3.3. Surgical operation:
OBX of mice was conducted according to protocol of Prof. Kinzo Matsumoto (Division of Medicinal−68−Pharmacology, Institute of Natural Medicine, University of Toyama.
3.4. Drug administration:
Drug treatments and experiment schedule were done according to the schematic below:-70days

BM pretreatment

Tap water
Bacopa monieri (BM) 50 mg/kg
Tacrine
2.5 mg/kg
317Behavioral experimentWestern blotting
65

Ex vivo experiment

OBXoperation

1. Object Recognition Test
2. Modified Y Maze Test
3. Fear Conditioning Test

3.5. Behavioral study
Behavioral experiment was conducted according to the protocol of Prof. Kinzo Matsumoto (Divisionof Medicinal Pharmacology, Institute of Natural Medicine, University of Toyama, including:- Object recognition test
- ModiÀed Y maze test
- Fear conditioning test
3.6. Neurochemical study:- Ex vivo measurement of cholinesterase activity in the brain was conducted by a modiÀed version of the colorimetric method of Ellman et al.
3.7. Western blotting-ChAT protein, M1 and M3 proteins were detected by using the protocols of Prof. Kinzo Matsumoto (Division of Medicinal Pharmacology, Institute of Natural Medicine, University of Toyama.
4. Results and discussion%0LPSURYHV2%;LQGXFHGQRQVSDWLDOPHPRU\GHÀFLWLQWKHQRYHOREMHFWUHFRJQLWLRQWHVW
Using the novel object recognition test, we elucidate the effect of BM on the non- spatial cognitive
performance of OBX mice. The result showed as the Àgure below:−69−

Test trial (5 min)Time exploring object (sec)

Object 1

Sample trial (5 min)Object 2

New object

Object 1

40

OBX
30
20OBX*

Sham***

Sham100BM
Tacrine
50 mg/kg 2.5 mg/kg

WaterWaterBMTacrine
50 mg/kg 2.5 mg/kg**P<0.01, *P<0.05 vs. familiar object (n=9 – 12)

Fig. 1 Effects of BM on object recognition deficit in OBX mice

In the sample phase, none of the animal groups showed signiÀcant differences in the time spent
exploring each identical object. However, in the test phase, the sham group spent a signiÀcantlylonger time exploring the novel object than exploring the familiar one. In contrast, tacrine (2.5mg/kg) and BM (50 mg/kg) treated OBX mice spent signiÀcantly longer time exploring the novelobject than exploring the familiar one.
%0LPSURYHG2%;LQGXFHGVSDWLDOZRUNLQJPHPRU\GHÀFLWVLQWKHPRGLÀHG<PD]HWHVW
Using the modiÀed Y maze test, we evaluate the effect of BM on spatial working memory on OBX
mice. The results showed as the Àgure below:
First trial (5 min)CsloedSecond trial (5 min)30 min% Time in new arm

Total arm entries (times)
60% time in new arm

Number of entries (times)

60
OBX
40

Sham*

*

20Sham*OBX**

50

40Random level

300Water WaterBMTacrine
50 mg/kg 2.5 mg/kg

Water WaterBMTacrine
50 mg/kg 2.5 mg/kg

Figures 2: Effect of BM on spatial working memory deficit in the modified Y maze test−70−

As shown in the Fig.2, the spatial working memory deÀcit cause by OBX was signiÀcantly improved by BM treatment.
After Ànishing the Àrst modiÀed Y maze test, OBX mice were treated with 1 mg/kg scopolamine(SPC i.p) or Sham group treated with tap water 30 min before the second modiÀed Y maze test
% Time in new arm

Total arm entries (times)
60

Sham OBX + SCP (1 mg/kg)

**

**

**

40% time in new arm

Number of entries (times)

60

20Sham OBX + SCP (1 mg/kg)

5040*

**
Random level

300Water

Water

BMTacrine
50 mg/kg 2.5 mg/kg

WaterWaterBMTacrine
50 mg/kg 2.5 mg/kg**P<0.01, *p<0.05 vs Sham group (n=9 -12)

Fig. 3 Scopolamine (SCP: 1 mg/kg) abolished the ameliorative effects of BM in OBX mice

As shown in the Àg. 3, the ameliorative effects of BM in the second trials were completely abolished by SCP (1mg/kg).
12SCP treatment

60% time in new arm

3**

47 days washout SCP*

50Scopolamine**OBXShamOBX

17 days washout scp**4030

ShamOBX

ShamShamOBX*P<0.05 vs. water-treated OBX group (n=8 - 12)

Fig. 4 BM again ameliorated the spatial working memory deficit in OBX mice

After the 7 days and 17 days of the second modiÀed Y maze test, the third and fourth of modiÀed
Y maze testes were performed. The result showed as the Àgure below:
After 1 week washout period of SCP, the ameliorative effects of BM were again observed.
%0DPHOLRUDWHG2%;LQGXFHGORQJWHUPPHPRU\GHÀFLWLQWKHIHDUFRQGLWLRQLQJWHVW
We next used the Pavlovian fear conditioning test to determine the effects of BM on long-term
memory deÀcits induced by OBX. One week after Ànishing the 4th of modiÀed Y maze test, thefear conditioning test was performed. Mice were fear-conditioned to the context and auditory
stimuli by electrical foot shocks as unconditioned stimuli. After a further 5 administration period−71−

of BM for 5 days, freezing responses to the contextual and auditory stimuli were recorded. The
results showed as the Àgure below:

Behavioral analysis
ContextualAuditory

3 min

2 min

5 days1 min

50

ShamOBXAuditory memory(Amygdala-dependent)
Freezing time/ 2 min (%)

Freezing time/ 3 min (%)

Contextual memory(Hippocampus dependent)

40
302010100

Sham80

**

Water

Water

***

60
40
2000OBXBM
Tacrine
50 mg/kg 2.5 mg/kg

Water

Water

BM
Tacrine
50 mg/kg 2.5 mg/kg

P<0.01, *P<0.05 vs. water-treated OBX group (n=8 -12)

Fig 5 Effect of BM on long-term memory deficit in the fear conditioning test

As shown in Àg.5, BM increased freezing to the auditory stimuli in the test session, that indicating long-term memory improvement.
4.4. Effect of BM on the cholinesterase activity
After completing the behavioral experiments, the cholinesterase activity in the cerebral cortex
was measured to understand the mechanisms underlying the action of BM. The results indicatedas table below:
Table 1acetylcholine esterase activity
Operation(nmol/mg tissue/min)Daily treatment

mean 㫧 S.E.M.
sham

water

44.51

㫧

5.37

OBX

water

42.65

㫧

3.40

OBX

Ba (50 mg/kg, p.o.)

33.92

㫧

4.13

OBX

THA (2.5 mg/kg, i.p.)

29.87

㫧

2.57䋪

*P<0.05 vs. water-treated OBX group and sham group (n=5 – 6)

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The results showed that tacrine (2.5 mg/kg) signiÀcantly inhibited cholinesterase activity, but
BM had no effect on this activity. Therefore, the mechanism of BM on cognitive deÀcits in OBX
mice may different from those induced by tacrine.
4.5. Effects of BM on cholinergic markers protein expressions
To clarify the mechanism(s) underlying the ameliorative effects of BM on OBX mice induced cognitive deÀcit, we investigated expression changes of cholinergic marker protein, including choline
acetyltransferase (ChAT), muscarinic M1 and M3 receptor in the hippocampus by Western Blotting method.
The results showed that BM signiÀcantly elevated the expression level of ChAT in the hippocampus without affecting on the expression of muscarinic M1 or M3 receptors or Eactin. These Àndingsuggested that the ameliorative effects of BM on OBX induced cognitive deÀcits are atributable
at least in part mediated by stimulation/normalization of central cholinergic systems. Base on
these results suggested that the impairment of learning and memory induced by OBX is caused
by degeneration of cholinergic neurons and ChAT is an important role in the improvement process of cognitive deÀcits. Our Àndings further support the idea that BM is beneÀcial for treatment of cognitive deÀcits such as AD.

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