ARTICLE IN PRESS

Phytomedicine 15 (2008) 587–594

www.elsevier.de/phymed

Effects of tenuifolin extracted from radix polygalae on learning and

memory: A behavioral and biochemical study on aged and amnesic mice
Hong Zhanga, Ting Hana, Lei Zhanga, Cheng-Hao Yub, De-Guang Wanb,
Khalid Rahmanc, Lu-Ping Qina,, Cheng Pengb,
a
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China
b
School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, PR China
c
School of Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, Liverpool L3 3AF, England, UK

Abstract
Although normal cognitive changes take place when a person becomes older, aging in humans is generally associated
with deterioration of cognitive performance and, in particular, of learning and memory. These cognitive deficits can
cause debilitating consequences due to aging. There are a number of herbal medicines which are reported to improve
brain function including intelligence.
In the present study, improving effects of tenuifolin, extracted from Radix Polygalae (RP), on learning and memory
in aged and dysmnesia mice were determined using step-down type passive avoidance test or Y type maze trial. Oral
administration of tenuifolin (0.02, 0.04, 0.08 g/kg d1, for 15 d) evidently improved the latency and number of errors in
aged and dysmnesia mice. The levels of cortical acetylcholine esterase (AChE) activity and hippocampal
neurotransmitters in aged mice given tenuifolin (0.02, 0.04, 0.08 g/kg d1, for 15 d) were also investigated, and
increased levels of norepinephrine (NE), dopamine (DA), decreased activity of AChE were found. However, serotonin
(5-HT) had no significant difference from that of aged mice given distilled water. The evident improvement of learning
and memory of aged mice is carried out by the effects of tenuifolin on the three stages of memory process, that is,
acquisition, consolidation and retrieval. This may do so by relatively increasing the levels of NE, DA in the
hippocampus and by decreasing the activity of AChE in the cortex.
r 2008 Elsevier GmbH. All rights reserved.

Keywords: Tenuifolin; Learning and memory; Ethology; Neurotransmitter; Acetylcholine esterase; Mouse

Introduction cognitive deficits are the debilitating consequences of

There are normal cognitive changes that take place
when a person becomes older. Aging in humans is
associated with deterioration of cognitive performance
and, in particular, of learning and memory, and
Corresponding authors. Tel./fax: +86 21 25070394 (Lu-Ping Qin),
+86 28 87769954 (Cheng Peng).
E-mail addresses: lpqin@smmu.edu.cn (L.-P. Qin),
pengchengchengdu@126.com (C. Peng).
aging. For more than a millennium, herbal remedies
have been used apparently safely and effectively in
Asian countries, especially in China, Japan and Korea,
as a treatment for alleviating various symptoms of
cognitive deficits and facilitating learning and memory.
Radix Polygalae (RP), the root of Polygala tenuifolia
Willd., has been used as a treatment for illnesses of the
brain and for its actions of tranquilization and promot-
ing intelligence. In combination with other herbal drugs,
RP is one of the most prescribed herbal remedies for

0944-7113/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2007.12.004
 ARTICLE IN PRESS
588 H. Zhang et al. / Phytomedicine 15 (2008) 587–594
treating various kinds of cognitive disorders such as
cerebrovascular diseases, aging, senile dementia includ-
ing AD (Chen et al., 2002, 2004; Tian et al., 2004; Yan
and Li, 2006). Anecdotal clinical experiences support the
notion that RP is safe and effective in treating and/or
alleviating symptoms of these diseases. Among 75 of the
most famous Chinese complex prescriptions character-
ized by promoting intelligence in past dynasties in
China, more than half of these prescriptions contain RP
(Liu and Liu, 2005).
Tenuifolin is mostly the mixture of saponins (Jia
et al., 2004) extracted from RP. Most saponins in RP are
the derivates from presenegenin (structure, see Fig. 1).
Tenuifolin is shown to improve learning and memory
ability of dementia-like rats induced by combined
injection of a-amyloid peptide and ibotenic acid into
the right nucleus basalis magnocellularis. The muscari-
nic receptor density and the choline acetytransferase
activity in their brain are markedly enhanced, whereas
acetylcholinesterase activity is significantly inhibited
(Chen et al., 2002). Tenuifolin can also increase total
protein and total antioxidant capacity in the brain
tissues of senescence-accelerated mice, decrease acetyl-
cholinesterase activity in the brain and interleukin-2
in their blood serum (Tian et al., 2004). As far as we
are aware, no reports have been issued on the
effects of tenuifolin from RP on learning and memory
in naturally aged mice, amnesiac mice by using step-
down and Y-maze tasks and, on the levels of hippo-
campal 5-HT, NE, DA, cortical AChE activity of
naturally aged mice.
In the current experiments, we investigated the effects
of MST from RP on cognitive functions of young, aged
animals and chemical-induced dysmnesia subjects.
Learning and memory parameters in these subjects were
evaluated using step-down type passive avoidance task
and Y type maze trial. In addition, this study also
examined the effects of MST on the levels of mono-
aminergic neurotransmitters (NE, DA and 5-HT),
COOH
HO
CH2OH
HO
COOH
Fig. 1. Chemical structure of presenegenin.
together with AChE activity in the brain tissues of aged
mice, all of which are known to be important to
mediation of learning and memory processes.
Materials and methods
Drug and administration
RP was purchased from Chengdu Tong-ren-tang
Pharmaceutical Group, and identified as the root of
Polygala tenuifolia Willd. by Professor D-G Wan, a
pharmacognosist, from The Pharmacy Faculty of
Chengdu University of Traditional Chinese Medicine
(Chengdu, China). A voucher specimen was deposited.
One kilogram of P. tenuifolia was extracted with 1000 ml
of ethanol (95%, v/v) in a 50 1C water bath three times
for 2 h each. The extract was incubated at 4 1C for 24 h
and then centrifuged. The sediment was resuspended in
100 ml methanol, filtered and cleared with activated
carbon. The filtered phase was concentrated to 1/2 of the
original volume under reduced pressure, then the same
volume of acetone was added, and the sample was
filtered again. The sediment was washed with acetone,
freeze-dried, and appeared as yellow brown powder (Jia
et al., 2004). The content of tenuifolin in the above
powder was determined by thin-layer chromatography
scanning (TLCS) method with the dehydroxypresene-
genin as reference (National Committee of Pharmaco-
poeia. Pharmacopoeia of PR China, 2005) and the
average content was 72.52% (RSD ¼ 1.22%, n ¼ 5).
Tenuifolin was dissolved in distilled water prior to
administration. Three groups of animals (n ¼ 10) were
orally administered 0.02, 0.04, 0.08 g/kg d1of tenuifolin
by intubation, respectively, for 15 days. Two more
groups of animals (normal and control groups) were
orally administered distilled water, and they were run
concurrently with tenuifolin-treated groups, all of which
were given in a volume of 10 ml/kg body weight
irrespective of dose.
Animals and grouping
Animals, obtained from Experimental Animal Center
of Chengdu University of Traditional Chinese Medicine
(Chengdu, China), were housed in a regulated environ-
ment (2071 1C), with a 12-h light and 12-h dark cycle
(08:00–20:00, light) and were grouped as follows: young
and aged male Kunming mice, (30 days of age, 18–22 g;
22–24 months of age, 40–50 g, respectively), Grade II,
Certificate No. 2000-7. Food and water were given ad
libitum, except for the duration of the experimental
session. On the day of the experiment, animals were
brought to the experimental room and allowed to
habituate to the environmental conditions for a period
of approximately 60 min before the beginning of the
 ARTICLE IN PRESS
H. Zhang et al. / Phytomedicine 15 (2008) 587–594
experiment. All animal treatments were strictly in
accordance with international ethical guidelines con-
cerning the Care and Use of Laboratory Animals, and
the experiments were carried out under the approval of
the Committee of Experimental Animal Administration
of the University.
In the experiments on the effects of MST on learning
performances in aged mice by step-down test and by
Y-maze trial, 40 aged mice were divided into four
groups (n ¼ 10) ad libitum, namely, three MST-treated
groups (0.02, 0.04, 0.08 g/kg d1) and one control group.
Another ten young mice served as normal group. In the
experiments on the effects of MST on dysmnesia mice
which were induced by scopolamine, sodium nitrite or
ethanol, 40 young subjects were divided into four groups
(n ¼ 10) ad libitum, viz. three MST-treated groups (0.02,
0.04, 0.08 g/kg d1) and one control group. Another ten
young subjects served as normal group. In the experi-
ments on the effects of MST on the levels of 5-HT,
NE, and DA in the hippocampus of aged mice, and
on the levels of cortical AChE activity in aged mice, 40
aged mice were divided into four groups (n ¼ 10) ad
libitum, viz. three tenuifolin-treated groups (0.02, 0.04,
0.08 g/kg d1) and one control group. Another ten
young mice served as normal group.
Chemicals and modeling
Scopolamine (Mingxing pharmaceutical factory,
Guangzhou, China) and sodium nitrite (Chengdu
chemical reagent factory, China) were dissolved in
sterile 0.9% saline, respectively. Ethanol was diluted to
a concentration of 30% (v/v) with distilled water. All
chemicals were administered intraperitoneally in a
volume of 5 ml/kg body weight irrespective of dose.
Control and normal animals received respective solvent
injections, and they were run concurrently with drug-
treated groups. In experiments on the effects of
tenuifolin on dysmnesia animal models, scopolamine
(1 mg/kg, i.p.) was administered 30 min before the
training trial and induced memory acquisition impair-
ment of mouse; sodium nitrite (120 mg/kg, i.p.) was
injected immediately after the training trial and induced
memory consolidation impairment of mouse; and finally
30% alcohol (1.5 g/kg, i.p.) was injected 30 min before
testing trial and induced memory retrieval impairment
of mice.
Step-down test
The method described by Xu et al. (2002) and Zhang
et al. (2007) served as the reference. The step-down
apparatus consisted of an acrylic box (20  20  20 cm
high) with a stainless-steel grid floor and a wooden
platform (4  4  4 cm) was fixed at the center of the
589
box. Electric shocks (36 V) were delivered to the grid
floor for 6 s with an isolated pulse stimulator. At the
beginning of the training trial, mice were placed in the
box to adapt for 3 min. After 3 min, electric shocks were
delivered and the mice jumped on the platform to avoid
the noxious stimulation, and the shocks were main-
tained for 5 min. After a 24 h interval, the mice were
again placed on the platform, and the latency to step
down on the grid with all four paws for the first time and
the number of errors subjected to shocks within 5 min
were measured as learning performances.
Y-maze test
The method described by Xu et al. (2002) and Zhang
et al. (2007) served as the reference. The Y-maze
apparatus with a conductive grid floor consisted of
three identical arms (40l  10w  20 h cm) made of dark
opaque Plexiglas and these three arms were symmetri-
cally disposed at 1201 to each other. Arms 1 and 3 were
in the non-safety zone (shocks were administered via
these) and arm 2 was a safety zone (on the top of which
there was an insulated grid floor of 10  15 cm). Rats
were placed on the top of arm 1 and a fixed resistance
shock source was connected to an automatically
operated switch and electric shocks (36 V) were applied.
After shocking, the mice escaped from foot shocks by
accidentally entering the top of arm 2 and this was
counted as one practice and the mice were repeatedly
trained for this procedure a further 10 times. After a 24 h
interval the mice were successively tested for 10 times
and their latency to enter safety zone (i.e. insulated grid
floor) from non-safety zone for the first time and the
number of errors displayed by entering the non-safety
zone within 10 times were recorded as learning
performances.
Assays of NE, DA, 5-HT levels and AChE activity
For determination of the levels of 5-HT, NE, and DA
in the hippocampus and cortical AChE activity, three
groups of aged mice (n ¼ 10) received 0.02, 0.04 or
0.08 g/kg d1 po of tenuifolin for 15 d before decapita-
tion. Two more groups of animals (n ¼ 10), serving as
normal group (young mice) and control group (aged
mice), were given orally distilled water by intubation,
and run concurrently with tenuifolin-treated groups.
Animals were decapitated and skulls were split on ice
and salt mixture and the hippocampi and cortices were
isolated respectively. The hippocampi were weighed,
and homogenized in ice-cold n-butanol solution (5 ml/g
tissue) according to Miller et al. (1970) and Biochem-
istry Group of Acupuncture and Meridian Research
Institute of TCM academy (1975). Homogenization was
performed using an ice-cold homogenizer for 1 min and
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590 H. Zhang et al. / Phytomedicine 15 (2008) 587–594

a 20% homogenate was made which was then centri-
fuged at 3000 rpm for 5 min. Supernatant (2.5 ml) was
then transferred to a tube containing 1.6 ml of 0.2 N
acetic acid and 5 ml n-heptane. After mixing on a vortex
mixer for 30 s, the tubes were centrifuged at 3000 rpm
for 5 min. The aqueous phases were used for the
estimation of 5-HT, NE, and DA levels employing the
fluorospectrophotometry (850 type, HIITACHI Corp.
Japan) reported by Ciarlone (1978). The cortices were
isolated, weighed, and homogenized in phosphate
buffer, pH 8.0 (1 ml/40 mg tissue), for 1 min and a 4%
homogenate was made. Estimation of the cortical AChE
activity was performed with some modifications accord-
ing to the assay described by Ellman et al.(1961) and
Zhengxin Ni (1990).
Statistical analysis
The data were analyzed using a statistical package
(SPSS10.0). The data for multiple comparisons were
performed by one-way ANOVA followed by Dunnett’s
t-test. po0.05 was considered statistically significant
and all results are presented as the mean7s.e.m.
Results and discussion
Ethologic investigation of learning and memory is at
present one of the most reliable targets reflecting levels
of animal intelligence. Many nootropic studies investi-
gate the animal’s behavior changes using step-down,
step-through, maze test, etc., which are often applied to
the determination of capabilities of passive avoidance
and spatial memory in animals. The results in Table 1
and 2 demonstrated improvements in learning perfor-
mances in aged mice receiving tenuifolin by an increased
latency and a decreased number of errors in the step-
down test, and by a shortened latency and a decreased
number of errors in the Y-maze test. However,
tenuifolin at a dosage of 0.02 g/kg did not improve
learning performances in aged mice in the step-down
and Y-maze test, which had no statistical significance
compared with the control group.
Generally, memory as measured by changes in an
animal’s behavior some time after learning is considered
to be a process that has several stages, including
acquisition, consolidation and retrieval (Abel and
Lattal, 2001). The use of pharmacological, genetic and
lesion approaches has helped to define the brain systems
and molecular processes important for these different
stages of memory. Some chemical agents, such as
scopolamine, sodium nitrite and ethanol, impair mem-
ory in animals trained on a step-down type passive
avoidance and a radial maze type task, which are used to
measure the three stages of memory process depending
on drug-treated period. In the present study, mice given
scopolamine or 30% ethanol displayed poor perfor-
mances, whose latency shortened and the number of
errors were increased as determined by the step-down
test; administration of sodium nitrite in mice evidently
increased the latency and the number of errors. As can
be seen in Tables 3–5, the administration of tenuifolin
improved cognitive behavior in dysmnesia animals to a
great degree. Tenuifolin (0.02, 0.04, and 0.08 g/kg)
showed a bell-shaped effect on acquired learning of
mice with scopolamine-induced dysmnesia. The ob-
served results of tenuifolin at the same doses on
consolidation memory stages also revealed a bell-shaped
effect. However, tenuifolin only at a dosage of 0.08 g/kg
improved the latencies and number of errors of mice
with ethanol-induced dysmnesia.
Learning and memory is one of the most important
functions of the brain, which is associated with complex
neurophysiologic and neurochemical changes. Many
neurotransmitters, including acetylcholine (ACh), dopa-
mine (DA), norepinephrine (NE), and, serotonin (5-HT)
play an important role in the learning and memory
processes (Blokland, 1996; Trond, 2003). ACh has been
related to attentional processes (Himmelheber et al.,
2000) and plays an important role in cognitive proces-
sing. DA has been associated with motivational
processes (Wilson et al., 1995) and has a special role in
appetitively motivated tasks. Central serotonin (5-HT)

Table 1. Effects of tenuifolin on memory performances in aged mice by step-down test

Group N Dose (g/kg d1) Latency (second) Number of errors

(time/5 min)

Normal 10 Distilled water 150.50723.30 1.9070.38

Control 10 Distilled water 70.90712.05&& 4.5070.60&&
Tenuifolin 10 0.08 139.20718.06* 2.1070.53**
Tenuifolin 10 0.04 115.60717.51 2.2070.36**
Tenuifolin 10 0.02 98.70713.11 3.7070.58

In the control group containing aged mice, the latencies significantly shortened and the number of errors markedly increased compared with the
normal group. In contrast, in aged mice treated by MST (0.04, 0.08 g/kg d1) for 15 days, learning performances were manifestly improved, except at
the lower dose group of MST (0.02 g/kg d1), which was similar to that observed in the control group. &&po0.01 compared with normal mice;
*po0.05, **po0.01 compared with control aged mice. Data are expressed as the mean7s.e.m.
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H. Zhang et al. / Phytomedicine 15 (2008) 587–594 591

Table 2. Effects of tenuifolin on memory performances in aged mice by Y-maze task

Group N Dose (g/kg d1) Latency (second) Number of errors

(time/10 times)

Normal 10 Distilled water 11.4072.08 1.5070.22

Control 10 Distilled water 31.174.51&&& 4.9070.69&&&
Tenuifolin 10 0.08 16.1073.11** 1.9070.35***
Tenuifolin 10 0.04 16.0072.35** 2.1070.46**
Tenuifolin 10 0.02 20.6073.92 3.6070.76

In aged mice of control group, the latencies were significantly prolonged and the number of errors markedly increased in contrast to young mice.
However, in tenuifolin-treated groups (0.04, 0.08 g/kg d1 still for 15 days), learning performances were manifestly improved, except at the lower dose
group of tenuifolin (0.02 g/kg d1), which had no statistical significance compared with control group. &&&po0.001 compared with normal mice;
**po0.01, ***po0.001 compared with control aged mice. Data are expressed as the mean7s.e.m.

Table 3. Effects of tenuifolin on dysmnesia in mice induced by scopolamine

Group N Dose (g/kg d1) Latency (second) Number of errors

(time/5 min)

Normal 10 Distilled water 158.50730.08 1.8070.61

Control 10 Distilled water 41.8078.03& 3.8070.61&
Tenuifolin 10 0.08 115.40732.24 2.1070.46*
Tenuifolin 10 0.04 144.00732.18* 1.7070.42*
Tenuifolin 10 0.02 84.10721.86 3.5070.62

A 1 mg/kg dose of scopolamine was injected ip 30 min before the training trial in the induced memory acquisition impairment model, and this
impaired the step-down type passive avoidance test performance of mice. Mice of the control group displayed poor performances, whose latencies
shortened and the number of errors increased as determined by the step-down test. In contrast, tenuifolin dosages of 0.04, 0.08 g/kg, significantly
decreased the number of errors, and a dosage of 0.04 g/kg increased the latencies, which had statistical significance compared with control group.
&
po0.05 compared with normal mice; *po0.05 compared with control mice. Data are expressed as the mean7s.e.m.

Table 4. Effects of tenuifolin on dysmnesia in mice induced by sodium nitrite

Group N Dose (g/kg d1) Latency (second) Number of errors

(time/10 times)

Normal 10 Distilled water 12.8071.91 1.8070.33

Control 10 Distilled water 38.4077.31&&& 5.5070.76&&&
Tenuifolin 10 0.08 20.6074.20* 2.8070.70**
Tenuifolin 10 0.04 18.4072.94** 2.3070.45**
Tenuifolin 10 0.02 22.9073.27* 3.0070.54*

Sodium nitrite impaired the Y-maze type test performances of mice. Tenuifolin produced an overall statistically significant improvement in
performances of mice at doses of 0.02, 0.04, 0.08 g/kg, that is to say, the latencies evidently shortened and the number of errors markedly decreased
compared with the control group. &&&po0.001 compared with normal mice; *po0.05, **po0.01 compared with control mice. Data are expressed
as the mean7s.e.m.

Table 5. Effects of tenuifolin on dysmnesia in mice induced by ethanol

Group N Dose (g/kg d1) Latency (second) Number of errors

(time/5 min)

Normal 10 Distilled water 169.40726.29 1.5070.37

Control 10 Distilled water 68.90714.18& 3.1070.53&
Tenuifolin 10 0.08 157.20728.60* 1.4070.34*
Tenuifolin 10 0.04 141.50725.25 1.9070.46
Tenuifolin 10 0.02 103.80720.95 2.8070.49

30 min before testing trial, mice were intraperitoneally injected with 30% alcohol, which evidently impaired the step-down type passive avoidance test
performances. Tenuifolin (0.02, 0.04, 0.08 g/kg) increased step-down latencies and decreased the number of errors in dose-dependent manner, but
only memory performances of the tenuifolin-treated group at a dosage of 0.08 g/kg were differed significantly from that of the control group.
&
po0.05 compared with normal mice; *po0.05 compared with control mice. Data are expressed as the mean7s.e.m.
 ARTICLE IN PRESS
592 H. Zhang et al. / Phytomedicine 15 (2008) 587–594

Table 6. Effects of tenuifolin on the hippocampal 5-HT, NE, and DA levels in aged mice

Group N Dose (g/kg d1) 5-HT (ng/g) NE (ng/g) DA (ng/g)

Normal 10 Distilled water 560.70749.69 1030.20727.71 786.70758.40

Control 10 Distilled water 358.50737.43& 667.30753.62&& 509.50754.64&&
Tenuifolin 10 0.08 399.80761.68 950.60777.29* 809.90771.80**
Tenuifolin 10 0.04 453.20754.89 936.90789.98* 756.00763.83*
Tenuifolin 10 0.02 397.10750.13 852.80793.53 683.70751.13

In the control group consisting of aged mice, the levels of hippocampal NE, DA, and 5-HT were significantly decreased compared to those observed
in the mice in the normal group. When the aged mice were given tenuifolin at dosages of 0.04, 0.08 g/kg the levels of NE and DA were similar to those
observed in the normal group, this difference being statistically significant. At a tenuifolin dosage of 0.02 g/kg only DA levels were significantly
increased when compared to the values observed in the control group. However, at tenuifolin dosages of 0.02, 0.04, 0.08 g/kg although there was
some improvement in the levels of 5-HT, this had no statistical significance when compared to the values observed in the control group. &po0.05,
&&
po0.01 compared with normal mice; *po0.05, **po0.01 compared with control aged mice. Data are expressed as the mean7s.e.m.

has been linked to emotional processes (Hashimoto et 400

al., 1999) and plays a particular role in emotionally 350

related tasks, and despite the lack of functional 300

*

Activity (U/g)
specialization, the serotonergic system plays a significant 250 *
200 **
role in learning and memory (Buhot et al., 2000). Whilst
NE has been relevant to learning and memory 150

consolidation, possibly by acting as a regulation of 100

signals (Crow, 1968; Kety, 1970). Cognitive deficits 50

induced by various lesions to the locus ceruleus are 0

reversible by the administration of drugs that enhance
noradrenergic neurotransmission.
Aging is often accompanied by some alterations in the
neurotransmitter systems of humans and other mam-
mals (Arranz et al., 1996; Magnone et al., 2000; Monica
et al., 2003). Most of the studies on brain physiology in
aging have been performed in rodents and the results do
not always show consistent changes in the neurochem-
ical parameters. Some of the discrepancies observed may
be due to species or strain differences. Nevertheless, the
published work appears to agree with respect to several
aspects such as the reductions of the levels of
neurotransmitters, including DA, NE, 5-HT, which
have been demonstrated in the aging brain (Habib and
Ewan, 2001; Monica et al., 2003). Our findings (Table 6)
demonstrate that reductions in the levels of DA, NE,
5-HT significantly decreased in aging brain, which are
consistent with earlier reports (Habib and Ewan, 2001;
Monica et al., 2003). However, the most significant
result of our study is that the administration of
tenuifolin caused significant increases in the levels of
DA, NE in the hippocampus of aged mice. On the other
hand, the decreased levels of 5-HT in hippocampus of
aged mice were not reversed by the administration of
tenuifolin, which had no statistical differences between
control group and tenuifolin-treated groups (0.02, 0.04,
0.08 g/kg). It seems to suggest that the improvement of
tenuifolin on cognitive function is not directly influenced
by the levels of 5-HT in hippocampus of aged mice.
Although age-related reductions of AChE activity are
found in previous studies (Sirviö et al., 1988, 1989),
numerous experiments show no substantial changes in
Normal Control 0.08 0.04 0.02
Tenuifolin (g/kg)
Fig. 2. Effects of tenuifolin on cortical AChE activity in aged
mice. In the control group consisting of aged mice there was no
statistical difference compared with normal group consisting
of young mice in the activity of cortical AChE. However, this
activity of AChE was significantly decreased when the mice
were given tenuifolin at dosages of 0.02, 0.04, 0.08 g/kg for 15
days when compared to the control group. *po0.05,
**po0.01compared with control mice. The values are ex-
pressed as the mean7s.e.m. Ten animals were used per group.
the activity of these enzymes in the brain of aged rats
(Aubert et al., 1995; Birthelmer et al., 2003; Colombo
and Gallagher, 1998). Our present data (Fig. 2) are in
line with the latter findings. However, the markedly
decreased activity of AChE in the cortex of aged mice
was found after the administration of tenuifolin at
dosages of 0.02, 0.04, 0.08 g/kg. The previous investiga-
tion also demonstrates that tenuifolin evidently inhibits
AChE activity (Park et al., 2002).
In summary, the administration of tenuifolin signifi-
cantly enhances learning performances in aged mice by
the step-down and Y-maze tasks. Tenuifolin also
ameliorates memory deficits in amnesic mice induced
by chemical agents. The improvement of learning and
memory of aged mice is mediated by the effects of
tenuifolin on the three stages of memory process, that is,
acquisition, consolidation and retrieval. This may do so
by relatively increasing the levels of NE, DA in the
hippocampus and by decreasing the activity of AChE in
the cortex. Since a desirable cognitive effect has been
 ARTICLE IN PRESS
H. Zhang et al. / Phytomedicine 15 (2008) 587–594
reported for glutamate (Ohno and Watanabe, 1996;
Trond, 2003), further studies should be directed towards
investigating the effect of tenuifolin on glutamatergic
neurotransmission in brain areas implicated in the
control of learning and memory processes. In addition,
the investigation of tenuifolin promoting intelligence
should be made at the molecular level.
Acknowledgements
The authors are grateful to Dr. Baokang Huang, Dr.
Qiaoyang Zhang and Professor Hanchen Zheng (De-
partment of Pharmacognosy, School of Pharmacy,
Second Military Medical University, Shanghai 200433,
PR China) for technical assistance.
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