Review

Received: October 11, 2004

Neurosignals 2005;14:34–45
Accepted after revision: November 16, 2004
DOI: 10.1159/000085384

Search for Natural Products Related to

Regeneration of the Neuronal Network
Chihiro Tohda a Tomoharu Kuboyama a, b Katsuko Komatsu a, b
a Research
Center for Ethnomedicines, Institute of Natural Medicine, and b 21st Century COE Program,
Toyama Medical and Pharmaceutical University, Toyama, Japan

Key Words Introduction

Neuritic atrophy W Synaptic loss W Dendrite W Axon W
Alzheimer’s disease W Amyloid-beta W Ginseng W Withania Despite the great number of ongoing investigations,
somnifera W Ashwagandha W Coffee bean neurodegenerative diseases remain incurable. The drugs
currently available for dementia, such as donepezil, an
acetylcholinesterase inhibitor, are efficacious in the tem-
Abstract porary treatment of memory dysfunction, but do not pre-
The reconstruction of neuronal networks in the damaged vent or reverse the underlying neurodegeneration [1]. In
brain is necessary for the therapeutic treatment of neu- patients with Alzheimer’s disease, neuritic atrophy and
rodegenerative diseases. We have screened the neurite synaptic loss are considered the major causes of cognitive
outgrowth activity of herbal drugs, and identified several impairment, based on the results of neuropathological
active constituents. In each compound, neurite out- postmortem studies of the brain [2–4]. In the brains of
growth activity was investigated under amyloid-ß-in- patients suffering from other neurodegenerative diseases,
duced neuritic atrophy. Most of the compounds with such as Parkinson’s disease, Huntington’s disease, and
neurite regenerative activity also demonstrated memory Creutzfeldt-Jakob disease, neurite atrophy has also been
improvement activity in Alzheimer’s disease-model observed [5–7]. Such atrophy leads to the destruction of
mice. Protopanaxadiol-type saponins in Ginseng drugs neuronal networks, and subsequently to the fatal dysfunc-
and their metabolite, M1 (20-O-ß-D-glucopyranosyl- tion of brain systems in these patients. The exclusion of,
(20S)-protopanaxadiol), showed potent regeneration ac- or at least a decrease in the magnitude of, the causes of
tivity for axons and synapses, and amelioration of mem- each disease may prevent the progression of symptoms,
ory impairment. Withanolide derivatives (withanolide A, but such inhibition is not associated with the repair of
withanoside IV, and withanoside VI) isolated from the already severely damaged brain function. We hypothe-
Indian herbal drug Ashwagandha, also showed neurite sized that the reconstruction of neuronal networks in the
extension in normal and damaged cortical neurons. Tri- injured brain would be the most necessary step in the fun-
gonelline, a constituent of coffee beans, demonstrated damental recovery of brain function, requiring neuritic
the regeneration of dendrites and axons, in addition to regeneration and synaptic reconstruction.
memory improvement.
Copyright © 2005 S. Karger AG, Basel

© 2005 S. Karger AG, Basel Katsuko Komatsu, PhD

ABC 1424–862X/05/0142–0034$22.00/0 Research Center for Ethnomedicines, Institute of Natural Medicine
Fax + 41 61 306 12 34 Toyama Medical and Pharmaceutical University, 2630 Sugitani
E-Mail karger@karger.ch Accessible online at: Toyama 930-0194 (Japan)
www.karger.com www.karger.com/nsg Tel. +81 76 434 7645, Fax +81 76 434 5064, E-Mail katsukok@ms.toyama-mpu.ac.jp
 Table 1. Natural medicine-oriented compounds which enhance neurite outgrowth

Compound Main botanical source Cell used Effective dose Function Reference

Ginsenoside Rb1 Panax ginseng rat cortical 0.1–100 ÌM axon extension 14, 21
Panax notoginseng neuron synaptogenesis
memory improvement
Metabolite 1* (protopanaxadiol-type rat cortical 0.01–1 ÌM axon extension 21
saponins) neuron synaptogenesis
memory improvement
Withanolide A Withania somnifera rat cortical 1 ÌM axon extension 36
neuron dendrite extension 37
synaptogenesis
memory improvement
Withanoside IV Withania somnifera rat cortical 1 ÌM axon extension 36
neuron dendrite extension
synaptogenesis
memory improvement
Withanoside VI Withania somnifera rat cortical 1 ÌM axon extension 36
neuron dendrite extension
synaptogenesis
memory improvement
Trigonelline coffee bean rat cortical 30–100 ÌM axon extension 41
neuron dendrite extension
memory improvement
Honokiol Magnolia obovata rat cortical 0.1–10 ÌM neurite outgrowth 43
Magnolia officinalis neuron
(–)-3,5-Dicaffeoyl- Aster scaber PC12 1–10 ÌM neurite outgrowth 44
muco-quinic acid
Catalpol Rehmannia glutinosa PC12h 0.1–1 Ìg/ml neurite outgrowth 45
Geniposide Gardenia jasminoides PC12h 0.1–10 Ìg/ml neurite outgrowth 45
Gardenoside Gardenia jasminoides PC12h 0.1–10 Ìg/ml neurite outgrowth 45
Picroside I Picrorhiza PC12D 10–100 ÌM potentiating 46
scrophulariiflora NGF-induced
neurite outgrowth
Picroside II Picrorhiza PC12D 0.1–100 ÌM potentiating 46
scrophulariiflora NGF-induced
neurite outgrowth
Nardosinone Nardostachys chinensis PC12D 0.1–100 ÌM potentiating 47
NGF-induced
neurite outgrowth

* 20-O-ß-D-Glucopyranosyl-(20S)-protopanaxadiol.

Natural Products Enhancing Neurite Outgrowth extracts, several constituents have been identified as ac-
tive compounds (table 1). It is critical that extended neu-
Neurite outgrowth is the first step in the construction rites have specific functions, such as axons and dendrites,
of the neuronal network, and neurite outgrowth activity and can make circuits by synaptic connections. However,
has been investigated in many crude drugs. Of these the identification of axons and dendrites and the mea-

Natural Products Related to Regeneration Neurosignals 2005;14:34–45 35

of the Neuronal Network
surement of synaptogenesis have not been undertaken in gests that ppd-type saponins are active compounds. Gin-
studies of natural products, apart from in our research. seng, Red Ginseng, Notoginseng and Ye-Sanchi, which
Ginseng drugs, Ashwagandha and coffee beans contain showed neurite outgrowth activity, have been demon-
interesting compounds with potent neurite regeneration, strated to contain comparatively rich ppd-type saponins
synaptic reconstruction and memory improvement ac- in our quantitative study [18]. This suggested that the
tivities. effects of these drugs could be mainly attributed to ppd-
type saponins. However, Zhuzishen (dried rhizome of P.
japonicus var. major from Yunnan province) and a rhi-
Ginseng Drugs zome of P. stipuleanatus, which inhibited cell viability,
may contain some cytotoxic compounds.
Neurite Outgrowth Using Methanol Extracts and
Isolated Saponins in SK-N-SH Cells Effect of M1, a Metabolite of Protopanaxadiol-Type
Ginseng, the root of Panax ginseng, is widely used as a Saponins, on Aß(25–35)-Induced Memory Impairment,
tonic throughout the world, and is efficacious in the treat- Axonal Atrophy and Synaptic Loss in Mice
ment of amnesia. In addition, significant improvement in When taken orally, ppd-type saponins are mostly me-
learning and memory has been observed in brain-dam- tabolized by intestinal bacteria to ppd monoglucoside, 20-
aged [8, 9] and aged rats [9] after the oral administration O-ß-D-glucopyranosyl-(20S)-protopanaxadiol (M1) [19,
of Ginseng powder, and the major Ginseng saponins, gin- 20] (fig. 1). As Ginseng is generally taken orally, a metabo-
senoside Rb1 and Rg1, are known to improve spatial lite of ppd-type saponins, M1, should be investigated to
learning in normal mice [10]. Regarding the effects on determine the active constituent of Ginseng responsible
neuronal cells, it has been shown that neurite outgrowth for its major effects. We therefore conducted experiments
of cultured rat cerebral cortical neurons is enhanced by to determine whether treatment with ginsenoside Rb1, as
crude Ginseng saponins [11], and that ginsenoside Rb1 a representative of ppd-type saponins, and its metabolite,
potentiates the nerve growth factor (NGF)-mediated neu- M1, can induce recovery from memory disorder, axonal
rite outgrowth of chick dorsal root ganglia [12, 13]. atrophy, and synaptic loss induced by the active fragment
We tested the neurite outgrowth activity of methanol of the amyloid-ß peptide (Aß(25–35)) [21].
extracts of 6 types of Ginseng drugs and P. stipuleanatus Male ddY mice (6 weeks old) were prepared to create a
plant material in SK-N-SH cells [14]. The methanol mouse model of Alzheimer’s disease (AD). Seven days
extracts of Ginseng (dried root of P. ginseng), Red Gin- after an i.c.v. injection of Aß(25–35), ginsenoside Rb1
seng (steamed and dried root of P. ginseng), Notoginseng (10 Ìmol/kg), M1 (10 Ìmol/kg), donepezil hydrochloride
(dried root of P. notoginseng) and Ye-Sanchi (dried rhi- (DNP, 0.5 mg/kg), or the vehicle (tap water) was adminis-
zome and root of P. vietnamensis var. fuscidiscus) in- tered orally once daily for 14 days. Mice were trained in
creased neurite outgrowth, with the effects of Red Gin- the water maze for 7 days starting 14 days after the i.c.v.
seng and Ye-Sanchi being particularly significant. administration of Aß(25–35) (fig. 2a). The escape latency
Thirty saponins were isolated from Ye-Sanchi and to find the platform in the Aß(25–35)-injected group sig-
structurally elucidated [15, 16]. Oleanolic acid-type sa- nificantly increased compared with the saline-injected
ponins were also isolated from Kouzichi (dried rhizome group, whereas the escape latencies of the groups adminis-
of P. japonicus var. major from Hubei province) [17], and tered ginsenoside Rb1 and M1 p.o. significantly decreased
19 saponins (ginsenosides Rb1, Rb3, Rg1 and Re, notogin- as compared with the vehicle-administered group. The
senosides R4, Fa and R1, Yesanchinoside J, 20-O-glc-gin- donepezil-administered group showed no significant
senoside Rf, majonoside R2, (24S)-pseudoginsenoside shortening of the escape latency.
RT4 and F11, vina-ginsenoside R1, R2 and R6 from Ye- In the retention test (fig. 2b), the number of crossings
Sanchi, notoginsenoside R2, ginsenoside Rg2 and Ro, chi- over a previous platform position was significantly de-
kusetsusaponin IVa from Kouzichi) were tested. Proto- creased in the Aß(25–35)-injected group compared with
panaxadiol (ppd)-type saponins, ginsenosides Rb1 and the saline-injected group. The number of crossings recov-
Rb3, and notoginsenosides R4 and Fa significantly ex- ered after treatment with ginsenoside Rb1 and M1. Treat-
tended the neurites in SK-N-SH cells at a concentration of ment with donepezil showed the smallest effect in the
100 ÌM, and their activity increased dose-dependently. retention test. All mice showed normal swimming perfor-
On the other hand, protopanaxatriol, ocotillol and olean- mance and a constant increase in body weight. Locomotor
olic acid-type saponins showed no effect [14]. This sug- activity did not differ among groups.

36 Neurosignals 2005;14:34–45 Tohda/Kuboyama/Komatsu

 Glc1-O

OH

COO⫺

N⫹
HO
CH3
␤-D-glucopyranosyl-(20S)-protopanaxadiol
20-O-␤ Trigonelline
(M1)

CH3
CH2R2
OH
R1
O O
H O O
H
O H
H OH H
H

H H
H H

OH O Glc1-6Glc1–O

Withanolide A R1 R2
(WL-A)
Withanoside IV (WS-IV) H OH
Withanoside VI (WS-VI) OH H

Fig. 1. Chemical structures of compounds that regenerate the neuronal network.

After the retention test, the expression levels of phos- the temporal cortex and CA1. In all areas, the synapto-
phorylated NF-H (axonal marker), synaptophysin (synap- physin levels were almost equal to or higher than control
tic marker) and MAP2 (dendritic marker) were measured levels in ginsenoside Rb1- and M1-treated mice. Donepe-
in mouse brains. We observed two cortical areas (parietal zil treatment had no effect on the synaptophysin levels.
cortex and temporal cortex) and three hippocampal areas The MAP2 levels were also reduced in the cerebral cortex
(CA1, CA3, and the dentate gyrus), as it is known that and CA1 of the brain in Aß(25–35)-injected compared
synaptic loss occurs primarily in the cerebral cortex and with saline-injected mice (fig. 3c). Significant decreases
hippocampus in AD patients [22, 23] and in AD model were seen in the temporal cortex; however, these de-
mice [24]. The phosphorylated NF-H levels were remark- creases in the expression levels of MAP2 were not clearly
ably reduced in these five areas of the brain in Aß(25– recovered by ginsenoside Rb1, M1 or donepezil. Although
35)-injected compared with saline-injected mice (fig. 3a). treatment with M1 tended to increase the MAP2 level in
Significant decreases were seen in the parietal cortex, CA1 the temporal cortex, the effect was weak. No differences
and CA3; however, the expression levels of phosphorylat- in neuronal density were observed among the groups in
ed NF-H were nearly equal to those of the control in ginse- any brain areas. Treatment with M1, a metabolite of gin-
noside Rb1- and M1-treated mice. Donepezil treatment senoside Rb1, results in the recovery of impaired learning
had no effect on the levels of phosphorylated NF-H. The and memory in Aß(25–35)-injected mice with degener-
synaptophysin levels were also reduced in these five areas ated axons and synapses. The maintained retention of
of the brain in Aß(25–35)-injected compared with saline- spatial memory was also seen after the discontinuation of
injected mice (fig. 3b). Significant decreases were seen in ginsenoside Rb1 and M1 administration. These results

Natural Products Related to Regeneration Neurosignals 2005;14:34–45 37

of the Neuronal Network
 suggest that ginsenoside Rb1 and M1 may induce the
60
structural repair of neuronal connections.
]
Sal/Veh In the rat large intestine, ginsenoside Rb1 is completely
A␤/Veh metabolized to M1 3 h after administration [25]. In mice,
50 ] A␤/GRb1 only M1 is continuously detected in the blood from 30
] A␤/M1
min to 16 h after oral administration of ginsenoside Rb1
[26]. In humans, M1 is detected in plasma from 7 h after
40 A␤/DNP
Escape latencies (s)

the ingestion of Ginseng, and in urine from 12 h after

intake, and aglycone is not detected in either plasma or
30 urine [20]. These results suggest that M1 is the final
metabolite of ppd-type saponins. The recovery potency in
Aß(25–35)-injected mice by p.o.-administered ginseno-
20
side Rb1 and M1 was almost identical, indicating that the
majority of orally administered ginsenoside Rb1 was me-
10 tabolized into M1. Considering that most ppd-type sapo-
nins are metabolized to M1, which is the active principal,
the total content of ppd-type saponins is possibly an
0
1 2 3 4 5 6 7
important index of the anti-AD activity of Ginseng.
a Days of acquisition test
Effect of M1 on Aß(25–35)-Induced Axonal Atrophy
8
in Rat Cortical Neurons
]
Number of crossings over a platform position for 60 s

In in vitro experiments, M1 demonstrated an axonal

7 regeneration effect. To investigate the Aß(25–35)-induced
damage to the neuronal network and the reconstructive
6 activity of drugs, 10 ÌM Aß(25–35) was added to the cor-
tical neurons on day 7, and after 3 days the medium was
5 replaced by fresh medium, including drugs. Although the
cortical neurons connected with each other during the 7-
4 day culture, some of the connections were lost 3 days after
Aß(25–35) treatment. At 4 days, both phosphorylated
3
NF-H-positive (fig. 4a) and MAP2-positive (fig. 4b) neu-
rites were significantly shortened by Aß(25–35) treat-
ment. Treatment with 0.01 ÌM M1 (to 78.5% of the con-
2
trol) significantly increased the recovery of the length of
phosphorylated NF-H-positive neurites (fig. 4a), while
1
MAP2-positive neurites were not extended (fig. 4b). NGF
significantly enhanced the lengths of phosphorylated NF-
Veh Veh GRb 1 M1 DNP
b Saline A␤(25–35)

Fig. 2. Effects of ginsenoside Rb1 and M1 on the impairment of spa- been measured over 60 s, 6 days after the last acquisition test. This
tial memory induced by Aß(25–35) injection. a Escape latencies per was also 6 days after the discontinuance of drug treatment. Vehicle
group in four trials were tested in a Morris water maze over 7 days. was administered p.o. to saline-i.c.v.-injected mice. To Aß(25–35)-
Vehicle was administered p.o. to saline-i.c.v.-injected mice. To i.c.v.-injected mice, vehicle (Veh), ginsenoside Rb1 (GRb1), M1, or
Aß(25–35)-i.c.v.-injected mice (5 nmol), the vehicle, ginsenoside Rb1 donepezil (DNP) was administered p.o. Values represent the means
(10 Ìmol/kg), M1 (10 Ìmol/kg), or donepezil (0.5 mg/kg) was admin- and SEM of 9 mice. * p ! 0.05 when compared with the Aß(25–35)
istered p.o. for 14 days. Values represent the means and SEM of 9 plus vehicle-treated group. One-way analysis of variance was carried
mice. * p ! 0.05 when compared with the Aß(25–35) plus vehicle- out, followed by Dunnett’s post hoc test.
treated group. Two-way repeated measure analysis of variance was
carried out, followed by Dunnett’s post hoc test. b The number of
crossings over the previous position of a platform had previously

38 Neurosignals 2005;14:34–45 Tohda/Kuboyama/Komatsu

Fig. 3. Effects of ginsenoside Rb1 and M1 on
axonal atrophy and synaptic loss induced by
Aß(25–35) injection. Expression levels of
phosphorylated NF-H (a), synaptophysin
(b) and MAP2 (c) in brain slices were quan-
tified. Vehicle was administered p.o. to
saline-i.c.v.-injected mice. To Aß(25–35)-
i.c.v.-injected mice, vehicle, ginsenoside Rb1
(10 Ìmol/kg), M1 (10 Ìmol/kg), or donepezil
(0.5 mg/kg) was administered p.o. for 14
days. The parietal cortex (PC), temporal cor-
tex (TC), hippocampal CA1 and CA3, and
dentate gyrus (DG) were observed. The fluo-
rescence intensities of six areas in each slice
were measured. Values represent the means
and SEM of three mice. * p ! 0.05 when
compared with the Aß(25–35) plus vehicle-
treated group. One-way analysis of variance
was carried out, followed by Dunnett’s post
hoc test.

Natural Products Related to Regeneration Neurosignals 2005;14:34–45 39

of the Neuronal Network
 H-positive (to 82.0% of the control) and MAP2-positive
500 (to 95.3% of the control) neurites. In addition, M1
]
increased in pre-synaptic density to the control level after
]
Aß(25–35)-induced synaptic loss occurred [our unpub-
Length of NF-H-positive neurites

400 ] lished data].

Neuritic atrophy by Aß(1–40) and Aß(25–35) has been
reported in chick sympathetic neurons [27] and rat corti-
per cell (␮m)

300
cal neurons [28]. As neurite atrophy is thought to be due
to unusual cell adhesion [27, 29], M1 may be capable of
200 normalizing the adhesive mechanism. Although Aß is
known to cause neuronal death through increased [Ca2+]
neurons [30], increased peroxynitrites in microglias [31],
100
and mitochondrial dysfunction in neurons [32], the death
pathway has been shown to be mediated by separate
0 molecular mechanisms of a neuritic dystrophy event [27–
Veh Veh M1 NGF
29]. Since ginsenoside Rb1 did not inhibit neuronal death
a A␤(25–35)
induced by Aß(25–35), the mechanism of rescuing axonal
atrophy may not be identical to that for recovery from
500
Aß-induced neuronal death.

] ]
Length of MAP2-positive neurites

400
Ashwagandha
per cell (␮m)

300 Neurite Outgrowth with Methanol Extract and Isolated

Withanolides
Ashwagandha (root of Withania somnifera Dunal) is
200
the most popular herbal drug in Ayurvedic medicine, and
has been used traditionally and commonly as a tonic and
100 nootropic agent. It has also been reported as associated
with improvements in scopolamine-induced memory def-
icits in mice [33]. Treatment with a methanol extract of
0 Ashwagandha induced neurite extension [34]. We further
Veh Veh M1 NGF
identified 6 withanolide derivatives from methanol ex-
A␤(25–35) tract (withanolide A, withanoside IV, withanoside VI,
Culture Immunostaining
etc.; fig. 1), which induced neurite outgrowth in human
neuroblastoma SH-SY5Y cells [35]. In normal cortical
neurons, the predominant dendritic outgrowth was in-
7d 3d 4d
duced by treatment with withanoside IV or withanoside
b A␤ Drugs VI, whereas predominant axonal outgrowth was observed
in treatment with withanolide A in normal cortical neu-
rons [36].
Fig. 4. Effect of post-treatment with M1 on Aß(25–35)-induced
axonal and dendritic atrophy. Aß(25–35) (10 ÌM ) was added to rat
cortical neurons at 7 days in vitro. Three days later, the medium was Effect of Withanolides on Aß(25–35)-Induced Neuritic
replaced by new medium containing M1 (0.01 ÌM ), NGF (100 ng/ Atrophy and Synaptic Loss
ml) or the vehicle (Veh, DMSO). Four days later, the cells were fixed In Aß(25–35)-induced damaged cortical neurons,
and immunostained for phosphorylated NF-H (a) and MAP2 (b). withanolide A, withanoside IV, and withanoside VI
The lengths of neurites positive for phosphorylated NF-H or MAP2
showed neuritic regeneration and synaptic reconstruc-
per cell were measured. Values represent the means and SEM of 30
cells. * p ! 0.05 when compared with the Aß(25–35) plus vehicle- tion. 24 h after culture initiation, 10 ÌM Aß(25–35) was
treated group. One-way analysis of variance was carried out, followed added to the culture medium simultaneously with the
by Dunnett’s post hoc test. drugs. Four days later, Aß(25–35) treatment significantly

40 Neurosignals 2005;14:34–45 Tohda/Kuboyama/Komatsu

 ]

Length of MAP2-positive neurites

150
]
]
]

per cell (␮m)

100

0
Cont Veh WL-A WS-IV WS-VI NGF
a A␤(25–35)

]
]

150
Length of NF-H-positive neurites
per cell (␮m)

Fig. 5. Effects of withanolide A, withanoside

IV, and withanoside VI on the prevention of
Aß(25–35)-induced dendritic and axonal
atrophy. Cortical neurons were cultured for
24 h, and then the cells were treated simulta-
neously with 10 ÌM Aß(25–35), and with- 100
anolide A (WL-A), withanoside IV (WS-IV),
or withanoside VI (WS-VI) at a concentra-
tion of 1 ÌM; or NGF or BDNF at a concen- 0
Cont Veh WL-A WS-IV WS-VI NGF
tration of 100 ng/ml; or vehicle (Veh); or
A␤(25–35)
with vehicle alone (Cont). Four days after
treatment, the cells were fixed and immu- Immunostaining
Culture A␤
nostained for MAP2 or phosphorylated NF-
H. Lengths of MAP2-positive neurites (a)
and phosphorylated NF-H-positive neurites
(b) were measured in each treatment. The
values represent the means and SEM of 30 1d 4d
cells. * p ! 0.05 when compared with the
Aß(25–35) plus vehicle-treated group. One- b Drug
way analysis of variance was carried out, fol-
lowed by Dunnett’s post hoc test.

inhibited the outgrowth of both MAP2-positive neurites was completely prevented by treatment with withanolide
and phosphorylated NF-H-positive neurites, showing that A (97.0% of the control), withanoside IV (106.3% of the
Aß(25–35) induced both dendritic and axonal atrophy in control), or withanoside VI (117.4% of the control)
rat cortical neurons. Simultaneous treatment with Aß(25– (fig. 5a). In particular, treatment with withanosides IV
35) and withanolide A, withanoside IV, or withanoside VI and VI tended to induce the growth of longer dendrites
at a concentration of 1 ÌM prevented both dendritic and than treatment with withanolide A.
axonal atrophy induced by Aß(25–35). Dendritic atrophy

Natural Products Related to Regeneration Neurosignals 2005;14:34–45 41

of the Neuronal Network
 Axonal atrophy was partially prevented by treatment
with withanoside IV (88.0% of the control) and withano-
side VI (90.0% of the control), whereas treatment with
withanolide A (98.6% of the control) completely prevent-
ed axonal atrophy (fig. 5b).
To determine whether regenerated neurites are able to
reconstruct synapses, the expressions of synaptic markers
were investigated. Rat cortical neurons were cultured for
21 days to construct mature synapses in vitro, and after
the culture period, Aß(25–35) was added to the samples.
Four days later, the cells were immunostained with an
antibody for post-synaptic density, (PSD)-95 (post-synap-
tic marker), or with synaptophysin (pre-synaptic marker).
PSD-95- and synaptophysin-positive puncta were signifi-
cantly decreased by treatment with Aß(25–35) [37]. With-
anolide A, withanoside IV, withanoside VI, or NGF was
added to the culture medium after 4 days of treatment
with Aß(25–35) after synaptic loss had occurred. Seven
days after the addition of the drug, the cells were fixed and
immunostained for PSD-95 or synaptophysin. Treatment
with withanolide A, withanoside IV, or withanoside VI
significantly induced both PSD-95 and synaptophysin
expression, as compared with treatment with the vehicle.
These results indicate that withanolide A, withanoside IV,
and withanoside VI facilitated the reconstruction of both
post-synaptic and pre-synaptic regions in neurons in
which severe synaptic loss had already occurred. This
increase in post-synaptic structures tended to be signifi-
cant following treatment with withanoside IV (86.0% of
the control) and withanoside VI (83.6% of the control), as
compared with withanolide A treatment (68.0% of the
control). However, reconstruction of the pre-synaptic re-
gion was induced significantly and markedly by treatment
with withanolide A (108.1% of the control), as compared
with withanoside IV (81.3% of the control) and withano-
side VI (75.8% of the control) treatments. Treatment with
NGF did not lead to an increase in the development of
either the post-synapses (57.7% of the control) or the pre-
synapses (54.4% of the control).

Fig. 6. The effect of trigonelline on the prevention of Aß(25–35)- phosphorylated NF-H. Lengths of MAP2-positive neurites (a) and
induced dendritic and axonal atrophy. Cortical neurons were cul- phosphorylated NF-H-positive neurites (b) were measured in each
tured for 3 days, and then the cells were treated simultaneously with treatment. The values represent the means and SEM of 12–20 cells
10 ÌM Aß(25–35), and trigonelline at a concentration of 30 or (a) or 14–22 cells (b). * p ! 0.05 when compared with the Aß(25–35)
100 ÌM, or vehicle (Veh), or with the vehicle alone (Cont). Five days plus vehicle-treated group. One-way analysis of variance was carried
after treatment, the cells were fixed and immunostained for MAP2 or out, followed by Dunnett’s post hoc test.

42 Neurosignals 2005;14:34–45 Tohda/Kuboyama/Komatsu

 Although NGF extended both axons and dendrites
(fig. 4, 5), it has no effect on synaptogenesis. Since NGF
itself is not able to pass through the blood-brain barrier,
low-molecular-weight substances that mimic NGF action
have been developed as anti-dementia drugs. However,
such NGF-like drugs are not expected to cure dementia
because of a lack of synaptogenesis activity.

Coffee Beans

Neurite Outgrowth with Trigonelline

Coffee is consumed as a drink, and is known to stimu-
late the central nervous system as well as the heart and
circulation [38]. It is thought that these effects are mainly
caused by caffeine [39] but the effects of other coffee con-
stituents on the central nervous system have hardly been
reported. Coffee beans are crude drugs, used in the tradi-
Fig. 7. Effect of trigonelline on the impairment of spatial memory
tional system of Unani medicine [40]. induced by Aß(25–35) injection. The number of crossings over the
Among the extracts of raw and roasted coffee beans, a previous position of a platform was measured over 60 s, 6 days after
methanol-soluble fraction of the ethanol extract (1 Ìg/ml) the last acquisition test in a Morris water maze. This was also 6 days
of raw beans significantly increased the percentage of cells after the discontinuance of drug treatment. Vehicle was administered
p.o. to saline-i.c.v.-injected mice. To Aß(25–35)-i.c.v.-injected mice
with neurites in human neuroblastoma SK-N-SH cells
(4.7 nmol), the vehicle (Veh), 500 mg/kg trigonelline (TGN), or
[41]. It was demonstrated that the neurite outgrowth 0.5 mg/kg donepezil (DNP) was administered p.o. Values represent
activity of the methanol fraction decreased depending on the means and SEM of 9 mice. * p ! 0.05 when compared with the
the extent of roasting. Among subfractions of this metha- Aß(25–35) plus vehicle-treated group. One-way analysis of variance
nol fraction, the basic fraction had significant neurite out- was carried out, followed by Dunnett’s post hoc test.
growth activity. In this basic fraction, trigonelline was
identified as an active constituent (fig. 3). It is known that
a decrease in trigonelline is related to the degree of roast-
ing [42].
In rat cortical neurons, trigonelline showed dendritic jected group. The number of crossings was recovered by
and axonal regeneration. Three days after initiation of the treatment with trigonelline, suggesting that memory re-
culture, Aß(25–35) was added to the culture medium with tention is improved by trigonelline.
trigonelline. Trigonelline (30 and 100 ÌM) treatment
dose-dependently prevented both dendritic (fig. 6a) and
axonal (fig. 6b) atrophy induced by Aß(25–35). Conclusions

Effect of Trigonelline on Aß(25–35)-Induced Memory The ppd-type saponins of Ginseng drugs and M1 (a
Impairment metabolite of ppd-type saponins by intestinal bacteria)
Fourteen days after the i.c.v. injection of Aß(25–35) in induced significant recovery from memory impairment,
male ddY mice (6 weeks old), trigonelline (500 mg/kg), axonal atrophy and synaptic loss in mice. The effect of
donepezil hydrochloride (0.5 mg/kg), or the vehicle (tap M1 on axonal reconstruction was further confirmed in
water) was administered orally once daily for 15 days. cultured cortical neurons. These results suggest that orally
Mice were trained in the water maze for 5 days, starting administered ppd-type saponins potentially ameliorate
21 days after the i.c.v. administration of Aß(25–35). Six dementia by reconstructing the neuronal network. With-
days after the last acquisition test, the retention test was anolide A, withanoside IV, and withanoside VI, which
performed (fig. 7). The number of crossings over a pre- were isolated from Ashwagandha, facilitated the regenera-
vious platform position was significantly decreased in the tion of dendrites and axons, and led to the dramatic con-
Aß(25–35)-injected group compared with the saline-in- struction of synapses, although the neuron damage was

Natural Products Related to Regeneration Neurosignals 2005;14:34–45 43

of the Neuronal Network
profound and severe. Trigonelline also had dendritic and icine may offer a treasury of new medicines to treat intrac-
axonal regeneration activity, and improved memory re- table diseases with the use of novel study concepts and the
tention. These compounds, sourced from natural prod- application of objective scientific analyses.
ucts, and used with treatments preventing pathogenesis
and neuronal death, are expected to play an important
role as new categorized drugs in curing neurodegenerative Acknowledgments
diseases in the near future. Although we have shown the
We thank Prof. M. Hattori, Dr. K. Zou, Mr. N. Matsumoto, Dr.
high potential of neuronal regeneration from compounds
M. R. Meselhy, Dr. N. Nakamura and Dr. J. Zhao of the Institute of
isolated from Ginseng drugs, Ashwagandha and coffee Natural Medicine, Toyama Medical and Pharmaceutical University
beans, it is dangerous to simply imply that these herbal for their extensive contribution to this study. This work was support-
drugs are expected to be excellent anti-dementia drugs. ed by Kampou Science Foundation, Uehara Memorial Foundation,
When taking herbal drugs, the risk of side effects brought Grants-in-Aid for Scientific Research (B), No. 11695086 in 1999–
2001 and No. 14406030 in 2002–2004 from the Japan Society for the
by other constituents, and sufficient efficacy compared
Promotion of Science, and the 21st Century COE Program of the
with isolated compounds should be investigated and care- Ministry of Education, Culture, Sports, Science and Technology,
fully considered. However, drugs used in traditional med- Japan.

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