Natural Products for

Neurodegenerative Diseases

Editors
Yung Hou Wong, Hong Kong
Joseph T.Y. Wong, Hong Kong

48 figures, 2 in color and 2 tables, 2005

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 Vol. 14, No. 1–2, 2005

Contents

5 Editorial

Reviews

6 Natural Products and Derivatives Affecting Neurotransmission Relevant

to Alzheimer‘s and Parkinson‘s Disease
Houghton, P.J. (London); Howes, M.-J. (Kew)

23 Regulation of Neuroinflammation by Herbal Medicine and Its

Implications for Neurodegenerative Diseases. A Focus on Traditional
Medicines and Flavonoids
Suk, K. (Daegu)

34 Search for Natural Products Related to Regeneration of the Neuronal

Network
Tohda, C.; Kuboyama, T.; Komatsu, K. (Toyama)

46 Multifunctional Activities of Green Tea Catechins in Neuroprotection.

Modulation of Cell Survival Genes, Iron-Dependent Oxidative Stress and
PKC Signaling Pathway
Mandel, S.A.; Avramovich-Tirosh, Y.; Reznichenko, L.; Zheng, H.; Weinreb, O.; Amit, T.;
Youdim, M.B.H. (Haifa)

61 Unique Properties of Polyphenol Stilbenes in the Brain: More than

Direct Antioxidant Actions; Gene/Protein Regulatory Activity
Doré, S. (Baltimore, Md.)

71 Neuroprotective Effects of Huperzine A. A Natural Cholinesterase

Inhibitor for the Treatment of Alzheimer‘s Disease
Wang, R.; Tang, X.C. (Shanghai)

83 Author Index
84 Subject Index

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 Neurosignals 2005;14:5

Editorial

Natural products have a long history of use for neurological symptoms.

For example, Crocus sativus has traditionally been used as an antispasmodic
and sedative. Yet the study of the mechanisms of action of neuroactive natu-
ral products began only recently. Armed with an arsenal of enabling tech-
nologies, researchers are now able to take a closer look at the complex mo-
lecular events associated with the therapeutic actions of natural products in
neurodegenerative diseases. Extensive information has been generated to in-
spire the design and development of more efficacious and safe therapies. This
issue brings together contemporary perspectives and research findings of lead-
ing experts on a vast array of mechanisms underlying the effects of natural
products on neuroprotection and neuronal regeneration. The multifunction-
al properties of constituents of botanicals and their derivatives are discussed
here to illustrate the possibilities toward the development of effective multi-
target therapeutics for neurodegenerative diseases. The study of neuroactive
natural products will indeed advance our understanding of the nervous sys-
tem, and open up exciting opportunities in drug discovery.
Yung Hou Wong, Hong Kong
Joseph T.Y. Wong, Hong Kong

© 2005 S. Karger AG, Basel

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 Review
Neurosignals 2005;14:6–22
DOI: 10.1159/000085382
Natural Products and Derivatives
Affecting Neurotransmission Relevant
to Alzheimer’s and Parkinson’s Disease
Peter J. Houghton a Melanie-Jayne Howes b
a King’s College London, London, and b Jodrell Laboratory, Royal Botanic Gardens, Kew, UK
Key Words
Alzheimer’s disease W Parkinson’s disease W
Cholinesterase inhibitors W Cholinergic receptors W
Dopaminergic receptors W Acetylcholine W Dopamine W
Galantamine W Huperzine W Physostigmine W L-DOPA W
Ergot alkaloids
Abstract
The two major neurodegenerative diseases Alzheimer’s
disease (AD) and Parkinson’s disease (PD) are character-
ised by low levels in the brain of the neurotransmitters
acetylcholine (ACh) and dopamine (DA), respectively.
Clinical treatment of these two conditions is palliative
and relies, in most cases, on improving stimulation at the
relevant receptors by either increasing levels of the
endogenous neurotransmitter or by the use of sub-
stances which have a similar agonist response. Natural
products continue to provide useful drugs in their own
right but also provide templates for the development of
other compounds. The major advances in the treatment
of AD have been the use of acetylcholinesterase inhibi-
tors such as galantamine, huperzine A, physostigmine
and its derivatives to increase the levels of ACh rather
than the use of cholinergic compounds, although com-
pounds with nicotinic properties have attracted some
interest. In contrast, the treatment of PD has relied on the
elevation of DA levels by use of L-DOPA, its precursor,
© 2005 S. Karger AG, Basel
ABC 1424–862X/05/0142–0006$22.00/0
Fax + 41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
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Received: September 27, 2004
Accepted after revision: November 16, 2004
and by the administration of dopaminergic agonists,
especially the ergot alkaloid derivatives. The use of
inhibitors of enzymes that cause breakdown of DA is an
avenue which is being explored. As well as the major
natural products of clinical interest, the paper discusses
the chemistry, activity and usage of the constituents of
plants used in traditional medicine for the treatment of
diseases presenting symptoms similar to those charac-
teristic for Alzheimer’s or Parkinson’s disease.
Copyright © 2005 S. Karger AG, Basel
Introduction
Neurodegenerative disease is a generic term applied to
a variety of conditions arising from a chronic breakdown
and deterioration of the neurons, particularly those of the
central nervous system (CNS). In addition, these neurons
may accumulate aggregated proteins which cause dys-
function. Alzheimer’s disease (AD) and Parkinson’s dis-
ease or parkinsonism (PD) are the two best-known dis-
eases of this type and will be the main diseases associated
with this review. However, some specialists would classify
multiple sclerosis and spongiform encephalopathies as
neurodegenerative diseases. The latter have received pub-
licity in recent years due to the link between Creutzfeldt-
Jacob disease (CJD) in humans and ‘mad cow disease’.
Spongiform encephalopathies have been shown to be
Peter J. Houghton
Pharmacognosy Research Laboratories, Department of Pharmacy
King’s College London, Franklin-Wilkins Building
150 Stamford Street, London SE1 9NH (UK)
Tel. +44 20 7848 4775, Fax +44 20 7848 4800, E-Mail peter.houghton@kcl.ac.uk
caused by prions, proteinaceous particles synthesized
within the cell which replicate and accumulate in cells and
so alter protein structure and therefore function but, as
yet, there are no drugs available to treat these conditions.
Most commonly, neurodegenerative disease manifests
in elderly people and, in advanced industrialised and
post-industrialised societies, where life expectancy is long,
this group of conditions is a major cause of morbidity and
of death, as well as imposing severe strains on the social
welfare systems. To illustrate this it is reported that, in the
USA, AD now affects at least 5 million people and in the
UK in 2001 it affected 750,000 people and was the fourth
most common cause of death [1]. However, it is increas-
ingly becoming recognized by the World Health Organisa-
tion as a global problem [2]. The common symptoms of
neurodegenerative diseases, such as loss of memory and
tremor, have been recognised as a feature of increasing
age for a long time and are acknowledged in many tradi-
tional medical systems. However, it is only comparatively
recently that, as distinctive diseases, they have been rec-
ognized and received much attention from mainstream
medicine. This is most likely due to the fact that, in the
past, short life expectancy precluded many surviving to an
age where neurodegeneration was likely to affect a signifi-
cant part of the population.
The etiology of neurodegenerative diseases is still largely
unknown but, especially for AD and PD, postmortem stud-
ies have shown clear links between the disease and a defi-
ciency of neurotransmitters in parts of the brain. Thus, in
AD there is a chronic shortage of acetylcholine (ACh) 1 and
in PD a deficit of dopamine (DA) 2. Until very recently, the
only clinical treatment for both of these conditions was the
reversal of these deficiencies by elevation of the levels of the
transmitter by agonists, or by inhibition of enzymes in-
volved in their removal from the immediate locality of the
synapse. These approaches are discussed more thoroughly
below. A variety of natural products have been shown to
play roles which would have the desired effects and some of
these, or their derivatives, have been brought into clinical
use. This paper gives an overview of such compounds, as
well as others, with interesting activity which have not been
developed as drugs or are still undergoing clinical trials. It
should not be forgotten that there are also many traditional
medicinal plants with a reputation of alleviating or prevent-
ing symptoms of neurodegeneration and that these are used
as crude extracts and mixtures [3, 4]. In some cases, such
extracts have been shown to relieve neurodegenerative
symptoms in animal models or display relevant in vitro
activity, but both the modes of action and identity of any
compounds responsible have not been fully determined.
Natural Products and Neurotransmission
Increasingly attention is being paid to the excitatory
amino acid neurotransmitters and relevant receptors
which are present in the CNS, e.g. the N-methyl-D-aspar-
tate (NMDA) receptor. The use of these in designing new
drugs or of explaining the use of traditional medicinal
plant extracts is in its infancy and no significant findings
have been made of natural products which either bind to
the receptors or affect levels of the transmitters.
Alzheimer’s Disease
AD is the major disease of a group which is character-
ised by loss of cognitive function leading to dementia. AD
is estimated to account for between 50 and 60% of
dementia cases in persons over 65 years of age [5] and is a
progressive, neurodegenerative disease that primarily af-
fects the elderly population. It is a major public health
concern in developed countries because of the strains
imposed on carers and financial resources by the increas-
ing numbers of sufferers. The main symptoms associated
with AD involve a decline in cognitive dysfunction, pri-
marily memory loss [6, 7] and in the later stages of the
disease language deficits, depression, agitation, mood dis-
turbances and psychosis are often seen [8].
Although AD, as a defined medical condition, has only
existed for about 100 years, age-related loss of memory
and cognitive decline has been documented for thousands
of years in human history. In common with many other
conditions, ancient writings which describe the symptoms
also suggest remedies, based usually on plant extracts.
Thus, Ashwagandha (Withania somnifera) is mentioned
in ancient Sanskrit writings from India as a ‘medhara-
sayan’ or promoter of learning and memory retrieval
whilst in the sixteenth century Gerard’s Herbal from the
UK, sage Salvia officinalis, is described as being ‘good for
the memory’. In recent years, the value of a few of these
has been demonstrated scientifically and some of the
compounds mentioned below have been isolated from
traditional medicinal plants.
Postmortem studies have shown that AD is character-
ized by low amounts of the enzyme choline acetyl trans-
ferase (ChAT) and enzyme abnormalities which would
produce levels of the neurotransmitter ACh 1 (fig. 1) [9,
10] and there also appears to be a depletion of nicotinic
function. Attention deficit in AD is reversed with nicotine
3, which is reported to upregulate nicotinic receptors and
to increase ACh release, so enhancing cholinergic neuro-
transmission [11, 12].
Neurosignals 2005;14:6–22 7
 CH3
+ HO
CH3 N CH3
O O
CH3
O HO
1 Acetylcholine 2 Dopamine
N
H
CH3
N
3 Nicotine
Fig. 1. Neurotransmitters of the CNS relevant to neurodegenerative
disease and the agonist nicotine.
ACh is certainly associated with cognitive function,
since situations where it is blocked from acting on the cho-
linergic receptors by drugs such as hyoscine (scopolamine)
4 (fig. 2), which is a muscarinic antagonist, result in severe
cognitive impairment in the patient. It is still unclear if
the low levels of ACh in the CNS are cause or effect as far
as AD is concerned, but the repletion of levels has been
exploited therapeutically with some success in the last 15
years in the symptomatic relief of AD. The synthetic com-
pound tacrine was the first drug introduced into the clinic
and it increased the levels of ACh by inhibition of acetyl-
cholinesterase (AChE), the enzyme responsible for fast
breakdown of ACh after its release from the nerve ending.
This inhibition results in ACh having a longer half-life
and therefore increasing in concentration at the synapse.
Tacrine was the first of several AChE inhibitors which
have come into clinical use, but it is no longer used since
the more recent introductions, generally named second
generation inhibitors, are safer and have longer-lasting
effects. These recent introductions include at least two
based on natural products. It should be stressed that
AChE inhibitors only alleviate some of the cognitive
symptoms of the disease for a time and ultimately do not
arrest the cognitive decline of the patient.
Another approach which has been proposed is the
employment of cholinergic and, to some extent, nicotinic
agonists, but this has not proved to be as useful therapeu-
tically as the inhibition of cholinesterase.
More recently prevention of glutamate-mediated neu-
rotoxicity has been a therapeutic target in AD. The N-
methyl-D-aspartate (NMDA) receptor antagonist, mem-
antine, has been introduced in some countries for clinical
use in AD patients. It should be noted that other types of
activity, besides increase of neurotransmitter levels, are
8 Neurosignals 2005;14:6–22
CH3 N
NH2
H CH2 OH
O
O
4 Hyoscine (scopolamine)
Fig. 2. Scopolamine, a muscarinic receptor
antagonist.
also being explored as leads for chemotherapeutic treat-
ment of AD. Activities being explored include antioxi-
dant, anti-inflammatory and inhibition of ß-amyloid syn-
thesis, but these are not covered to any extent in this
paper.
AD: The Use of Cholinergic Compounds
The rationale underlying the use of cholinergic com-
pounds is that they are agonists of the nicotinic choliner-
gic receptor and so compensate for the low levels of ACh.
The binding of ACh to the receptor is shown diagram-
matically in figure 3. It can be seen that the important fea-
tures required in a molecule are an amine which becomes
positively charged at the pH of the immediate environ-
ment and so binds to an aspartate in the receptor, a por-
tion of the molecule able to form hydrogen bonds with the
asparagine domain of the receptor and small groups able
to bind to hydrophobic sites near the aspartate region. In
addition, the receptor lies in a pocket which allows only
small molecules to enter.
Although these compounds have been suggested as
valuable agents in treating AD, because they also appear
to inhibit fibrillary tangle and amyloid production, suc-
cess has been limited as far as clinical studies are con-
cerned, although results in animals were initially promis-
ing [13]. Two major alkaloidal natural products are
known to have this effect, arecoline 5 and pilocarpine 6
(fig. 4). It can be seen that these molecules are small and
that they incorporate at least two of the criteria noted
above for binding to the receptor.
Arecoline is the major alkaloid present in areca or betel
nut, the fruit of the palm tree Areca catechu L. (Areca-
ceae), which is extensively used as a masticatory through-
out the Indian subcontinent and other parts of southeast
Asia. It is estimated that 500 million people regularly
chew betel nut (often referred to as ‘pan’ in India) in a
Houghton/Howes
form which is usually shredded, mixed with lime and
wrapped in a leaf of Piper betel L. Excessive salivation
occurs, which is a direct result of its cholinergic activity,
CH3
which also gives the mild CNS stimulation for which the Aspartic acid
product is principally used. There was some interest in -
COO CH3
arecoline as a treatment for AD since it showed improve- O
O CH3
N
ment in memory tests in rats [14]. Arecoline has been Hydrophobic sites
CH3
shown to bind to the M2 muscarinic receptors but not the H H
Hydrophobic site
O N
nicotinic receptors [15]. A small clinical study showed that,
when arecoline was given continually by intravenous infu-
sion in AD patients, it enhanced verbal memory [16]. Asparagine
Derivatives of arecoline have been synthesized in order to
improve selectivity for cortical muscarinic ACh receptors Fig. 3. Binding of acetylcholine to nicotinic receptor.
and two examples are Lu 25-109 and talsaclidine 8 which
are M1 receptor agonists. Although Lu 25-109 showed
encouraging results in vitro [17], it failed to improve cogni-
tion when tested clinically in patients with mild to moder-
O
ate AD [18]. Talsaclidine has been shown to increase choli- CH3 H
nomimetic central activation in animals and humans with- OCH3 N
O
out some of the side effects seen with AChE inhibitor ther- N N
H
apy, but cognitive function was not significantly improved CH3 CH3 O

[19]. Tests on rhesus monkeys did however show some

5 Arecoline 6 Pilocarpine
improvement in memory-related tasks but at doses which
gave unacceptable side effects [20].
Pilocarpine 6 is one of a series of related alkaloids N N

found in species of the South American plant genus Pilo- N

carpus, known commonly as Jaborandi leaf, which was
used in traditional medicine since it induced sweating and
urination, features which were perceived as being useful
for eliminating toxins from the body. The molecular
structure of pilocarpine 6 bears similarities to ACh since
the positively charged N atom and the lactone binding to
the serine are about the same distance apart. Chewing the
leaf causes copious salivation as well as other typical fea-
tures of cholinergic stimulation such as contraction of the
pupils. Pilocarpine has been shown to enhance memory
performance in aged rats [21], but no studies on its appli-
cation in humans for the treatment of AD have been
reported and it appears to have been discarded as a poten-
tial therapeutic lead. This is possibly due to its poor phar-
macokinetic profile as it cannot pass through the blood-
brain barrier, as well as its undesirable side effects.
AD: The Use of Cholinesterase Inhibitors –
Alkaloids
To avoid the undesirable effects of excess cholinergic
stimulation, ACh is rapidly hydrolysed after release at the
synapse by an enzyme named acetylcholinesterase AChE.
N CH3
O
N
C
CH
N
CH3
7 LU 25-109 8 Talsaclidine
Fig. 4. Cholinergic compounds.
A similar enzyme, butylcholinesterase BuChE, also occurs.
If the cholinesterase is inhibited, the ACh does not hydro-
lyse so quickly, and levels of ACh rise. The ‘classic’ cho-
linesterase inhibitor is the alkaloid physostigmine 9, also
called eserine. This was isolated from the Calabar Bean, the
seeds of Physostigma venenosum Balf., in the nineteenth
century in studies stimulated by the use of the seeds as an
ordeal poison in what is now southeastern Nigeria.
The toxic effects of calabar bean extract were found to
be due to excessive cholinergic stimulation resulting in
increased salivation, nausea, bradycardia, muscle cramps
and respiratory failure, as well as CNS effects such as agi-
tation. The cholinergic excess was found to be caused by
inhibition of the rapid breakdown of acetylcholine by
physostigmine.

Natural Products and Neurotransmission Neurosignals 2005;14:6–22 9

 H CH3 CH3
CH3
Aspartate N O CH3
OH CH3 N O N
N CH3 CH3
Serine CH3
O H O
O O N Histidine N
CH3
N
H3C O
9 Physostigmine 10 Neostigmine
N
H3C O CH3

CH3 H CH3
CH3
N CH3
CH3 N O
Tyrosine CH3
O

11 Rivastigmine

OH
H OH
O OH
CH3O O
Fig. 5. Interaction between acetylcholine and the acetylcholinester-
ase active site. N
N O
CH3

12 Galantamine 13 Hamayne
CH3
In recent years, the structure of AChE has been deter-
mined and the mode of binding for AChE inhibitors has
also been elucidated. A variety of AChEs exist according H
CH3 N
to the source species, but they vary only in small details
NH2
and all contain the active site at the base of a deep cleft in O

the enzyme.
Figure 5 illustrates the particular amino acid residues 14 Huperzine A

which are considered to be the most important in the

binding process and from this knowledge, structure-activ-
ity relationships of AChE inhibitors can be understood at
the intermolecular level. The important regions of an
inhibitor appear to be a positively charged nitrogen,
which binds to an aspartate residue, and a region, sepa-
rated by a lipophilic area from the positive charge, which
can form a hydrogen bond with a tyrosine or serine resi-
due. A positively charged nitrogen is common in many
alkaloids at body pH and it is not surprising that many of
the most powerful AChE inhibitors are alkaloids. In
recent years, however, a number of natural products,
mentioned below, which do not contain any nitrogen,
have been found to be AChE inhibitors. This raises ques-
tions about their binding characteristics to AChE since
the key role played by a positively-charged moiety of the
molecule bonding with the anionic aspartate residue, can
no longer account for the activity. The interactions be-
tween the enzyme and such non-alkaloidal compounds
have generally not yet been investigated.
The cholinesterase inhibitors (fig. 6) cause overstimu-
lation of a number of functions in many animal species
and exert a considerable toxic effect even at quite low
Fig. 6. Acetylcholinesterase inhibitors.
doses. This has been exploited in the area of insecticides,
and the insecticide carbaryl was developed by making
synthetic analogues of physostigmine. In therapeutics,
cholinesterase inhibition had, until recently, a somewhat
limited application in only ophthalmology and the treat-
ment of myasthenia gravis. However, the realisation that
early symptoms of AD could be improved by the use of
cholinesterase inhibitors awakened renewed interest and,
since physostigmine was known to cross the blood-brain
barrier, several in vivo studies were conducted which
showed that it reduced symptoms of ACh deficiency in
the CNS. Physostigmine was reported to protect mice
against cognitive impairment caused by oxygen deficit
and it improved learning in rats [22]. Clinical studies
showed significant cognitive benefits in both normal and
AD patients [23], but it had a short half-life, which has
prevented its application clinically in AD patients, since
this would require multiple daily dosing.

10 Neurosignals 2005;14:6–22 Houghton/Howes

 As well as inhibiting AChE, physostigmine also inhib-
its butylcholinesterase (BuChE), another enzyme found in
the CNS, so adverse effects associated with BuChE inhibi-
tion, such as gastrointestinal disturbance, may also occur
with physostigmine. However, BuChE has recently been
implicated in the aetiology and progression of AD [24], so
inhibition of BuChE may therefore prove to be beneficial
in treating AD and physostigmine and other BuChE
inhibitors such as rivastigmine may have clinical efficacy
superior to AChE-selective inhibitors.
To improve its pharmacokinetic profile, there is a con-
siderable history of the synthesis of analogues of physo-
stigmine. These have been applied to the treatment of
myasthenia gravis, neostigmine 10 being the most widely
used drug for this disease. Neostigmine is a quaternary
amine and this feature severely impairs its ability to cross
the blood-brain barrier and so be of value in treating AD.
Rivastigmine 11 was produced with the express purpose
of engineering a better pharmacokinetic profile for useful-
ness in AD and it inhibits the G1 form of AChE in the
cortex and hippocampus, brain areas involved in cogni-
tion [25], and it has been shown to improve cognition in
AD patients [26, 27]. Clinical studies have borne out the
usefulness of rivastigmine (Exelon®) in mild to moderate
AD and it has been licensed as a treatment for symptom-
atic relief of AD since 2000.
Galantamine 12 (sometimes referred to as galanth-
amine) is found in members of the Amaryllidaceae, e.g.
the Chinese medicinal herb Lycoris radiata Herb. and the
European Galanthus nivalis L. and Narcissus spp. [28].
The ethnopharmacological uses of plants containing this
compound are not very clear, but a full report of the histo-
ry of the development of galantamine from Galanthus
nivalis has been recently published [29]. Its cholinesterase
inhibitory properties were first exploited in Bulgaria in
the mid-twentieth century for the treatment of polio vic-
tims, but it only came into prominence as a treatment for
AD in the last decade of the twentieth century.
Galantamine has been licensed in Europe for AD treat-
ment since 2001. Multi-centre randomised clinical trials
showed that it was well tolerated and significantly im-
proved cognitive function when administered to AD pa-
tients [30–32]. The cognitive benefits appear to be sus-
tained for at least 3 years, a much longer time than for
other drugs of this type [33]. Galantamine is well ab-
sorbed when given orally and is also more selective for
AChE than BuChE [34]. It also stimulates nicotinic recep-
tors indirectly by its allosteric potentiation of ACh [35],
and so may also enhance cholinergic function and memo-
ry (see below). This effect suggests that galantamine may
Natural Products and Neurotransmission
have therapeutic advantages over other AChE inhibitors
and its value in vascular dementia as well as AD is recog-
nised in recent studies [36]. Several other alkaloids with
AChE inhibitory activity have been recently reported
from members of the Amaryllidaceae, but they have not
been subjected to vigorous pharmacological and clinical
testing. Several other alkaloids isolated from Iberian Nar-
cissus species have been tested for cholinesterase activity
[37]. A study of two Crinum species used in Nigerian tra-
ditional medicine to help ailing memory resulted in the
isolation of four alkaloids of which the most active was
hamayne 13, although its IC50 value of 250 ÌM was three
orders of magnitude weaker than physostigmine, so it is
unlikely that it would be present in sufficient quantity to
have a strong therapeutic effect [38].
Whilst physostigmine analogues and galantamine have
entered the pharmacopoeia in the Western world, another
natural cholinesterase inhibitor, huperzine A 14 has been
introduced in China for treating AD. Huperzine A is one
of the alkaloids found in the clubmoss Huperzia serrata
Thunb. (Lycopodiaceae) which is used in various formu-
lae in traditional Chinese medicine (TCM) to alleviate
problems of memory loss, promote circulation and for
fever and inflammation [39]. Huperzine A is related to
the quinolizidine alkaloids and it reversibly inhibits
AChE in vitro and in vivo [40, 41].
Huperzine A has been shown to improve memory in
cognitively impaired rats [42] and in gerbils following
ischaemia [43]. These observations suggest that huperzine
A has clinical potential in cerebrovascular disorders, as
well as in AD. Indeed, since it has also been shown to be
neuroprotective, AChE inhibition may not be the only
explanation for the clinical effects observed. Huperzine A
has been shown to be neuroprotective against ß-amyloid
peptide fragment 25–53 and free radical-induced cytotox-
icity [44] and to attenuate apoptosis by inhibiting the
mitochondria-caspase pathway [45]. In a multi-centre,
double-blind trial, huperzine A significantly improved
memory and behaviour in AD patients, and was reported
to be more selective for AChE than BuChE and was less
toxic than the synthetic AChE inhibitors donepezil and
tacrine [36]. A randomised, placebo-controlled study on
over 200 patients, 100 of whom were given 400 Ìg huper-
zine A daily for 12 weeks and showed significantly higher
scores for improvement compared with the placebo group
in the various scorings used [46]. Recent progress on
many aspects of huperzine A has been reviewed [47].
Other alkaloids have shown AChE activity and most of
these have been isolated from plants used in traditional
medicine (fig. 7). Coptis chinensis Franch. (Ranunculaceae)
Neurosignals 2005;14:6–22 11
 inhibited AChE in vitro, and reversed scopolamine-
O
induced memory impairment in rats [54]. It also in-
N+
O creased cerebral blood flow in vivo in cats, a property
OCH3 which would supplement its usefulness in AD [55]. Sola-
nidine 20 and related compounds and their glycosides are
OCH3
steroidal alkaloids produced in green parts of the genus
15 Berberine
Solanum, which includes the potato. The toxic properties
CH3O CH3O of these alkaloids have been known for a long time and
N+ N+ have been shown to be due to AChE inhibition, with char-
CH3O CH3O
acteristic signs of cholinergic excess such as sweating, pal-
O OCH3
pitations and CNS disturbances including hallucinations
O OCH3 [56]. Reports of Solanum species being used to treat AD
16 Coptisi ne 17 Palmatine or related conditions in traditional medicine do not exist
and the alkaloids have not been investigated for any use-
O fulness clinically, presumably because of their toxicity.
O
N N Although it is comparatively easy to explain the fact
that some alkaloids inhibit AChE because of their molec-
N N N
N
CH3
ular features, it is less easy to correlate chemical structure
and activity for some other phytochemical types for which
18 Rutaecarpine 19 Dehydroevodiamine AChE inhibition has recently been reported.
CH3 H
CH3
N AD: The Use of Cholinesterase Inhibitors –
CH3 H H CH3
H Terpenoids and Other Types of Compound
H H
HO
Terpenoids comprise a very large group of natural
20 Solanidine products and comprise two or more branched 5 carbon
units, formed from a common precursor named meva-
lonic acid. Skeletons consisting of multiplets of 2, 3, 4 or 6
Fig. 7. Minor alkaloidal cholinesterase inhibitors.
of these linked together in many different ways are found
in a variety of mostly cyclic compounds named monoter-
penes (10 carbons in the skeleton), sesquiterpenes (15 car-
has been used in TCM for several conditions including age- bons), diterpenes (20 carbons) and triterpenoids (30 car-
related cognitive and memory decline. Some alkaloids bons), respectively (fig. 8). These compounds tend to be
found in this species, such as berberine 15, coptisine 16 and lipophilic, so they are able to cross the blood-brain bar-
palmatine 17, are reported to also be anti-ChE [48, 49]. rier, and the monoterpenes, and some of the sesquiter-
Coptis chinensis extract improved a scopolamine-induced penes, are volatile, and so effects could occur through
learning and memory deficit in rats [50] and this is likely to inhalation. These compounds are responsible for the
be due to the alkaloids present raising ACh levels. strong odours and flavours of many herbs, spices and tra-
Berberine 15 has been shown to be selectively active ditional medicines. Many such molecules are increasingly
against AChE compared with BuChE [51] and it has been recognized as having a variety of roles in living organisms,
shown to improve scopolamine-induced amnesia in rats including signalling between members of the same spe-
[52]. Rutaecarpine 18 is the major alkaloid found in Evo- cies, thus comprising part of the group of compounds
dia rutaecarpa (Juss.) Benth. (Rutaceae), the unripe fruit known as pheromones, and protective or attractant roles
of which is used in TCM for cardiotonic, restorative and in flowering plants against herbivores and pollinators
analgesic effects. Pharmacological activities relevant to respectively. An effect on CNS activity by the volatile
AD have been identified with the extract and with rutae- substances in perfumes and other odoriferous materials
carpine. Rutaecarpine inhibited COX-2 activity in vitro, has attracted interest in recent years and one of the first
and was anti-inflammatory in vivo [53]. Dehydroevo- findings that monoterpenes had AChE inhibitory effects
diamine 19, another alkaloid found in the same plant, was made only in the mid 1990s in studies investigating

12 Neurosignals 2005;14:6–22 Houghton/Howes

historical records that monoterpene-containing plants
were ‘good for the memory’ [57]. CH3 CH3
One such group of plants was the various European CH3

species of Salvia, commonly known as sage. An ethanolic CHO

H3C
extract and oil of S. officinalis L. (Labiatae) and S. lavan- O CH3

dulaefolia Vahl. (Labiatae) were investigated for anti-ChE H3C CH3 H3C CH3

activity and it was found that all gave inhibition of AChE

21 1,8-Cineole 22 α-Pinene 23 Citral
at quite low concentrations [57].
The cholinesterase inhibition shown by the S. lavandu-
O O
laefolia oil was shown to be partly due to the cyclic mono- CH3 CH3
O O
terpenes 1,8-cineole 21 and ·-pinene 22, which were
shown to inhibit AChE in vitro, with some contribution O O
from other constituents, perhaps by acting synergistically
[58, 59]. However, the monoterpenes were considerably CH3 H3C CH3
less active, by a factor of at least 103, than the alkaloidal 24 Dihydrotanshinone 25 Cryptotanshinone
AChE inhibitors such as physostigmine 9 [58].
CH3
Since the effects of the oil were better than those of
H3C
individual monoterpenes, further in vivo and clinical
studies, described below, were carried out on the essential
CH3 CH3 H COOH
oils, which consist of a mixture of monoterpenes, rather
than isolated compounds. Oral administration of S. la- H CH3
vandulaefolia essential oil to rats decreased striatal AChE HO H
activity in both the striatum and the hippocampus com- CH3 CH3

pared to the control rats. Thus, it appeared that constitu- 26 Ursolic acid

ents of the S. lavandulaefolia oil, or their metabolites,

reach the brain and inhibit AChE in select brain areas,
consistent with evidence of inhibition of the brain enzyme
in vitro [60].
Clinical studies on human volunteers and even pa-
tients with AD have been reported in recent years. The
effect of sage in twenty participants using a placebo-con-
trolled, double-blind, balanced, cross-over design showed
significant effects on cognition associated with the lowest
dose of Salvia, including improvements in both imme-
diate and delayed word recall scores [61]. A small trial
with 11 patients showing mild to moderate symptoms of
AD showed that oral administration of the essential oil of
S. lavandulaefolia significantly improved cognitive func-
tion in one of the three different methods of assessment
used [62]. Another, more thorough, trial in Iran on 48
patients with similar symptoms of AD showed that those
treated with an extract of S. officinalis gave significantly
better values in measurements of cognitive function [63].
Melissa officinalis L. (Labiatae) leaf is another species
that contains monoterpenes in its essential oil. It has been
used as a medicinal plant for more than 2000 years and
has a reputation of for promoting long life and for re-
storing memory [64]. Recent studies have focused on
the reputed cognitive effects of M. officinalis. A recent
randomised, placebo-controlled, double-blind, balanced
Fig. 8. Terpenoidal cholinesterase inhibitors.
crossover study showed an improvement in cognitive per-
formance and mood in 20 healthy young participants, fol-
lowing treatment with dried leaf of M. officinalis shown
by previous in vitro tests to be cholinergically active [65].
In another study, an extract of M. officinalis was adminis-
tered to patients with mild to moderate AD for 4 months
and gave a significantly better outcome on cognitive func-
tion than placebo [66]. Although the constituents present
have not been investigated, numerous monoterpenes
have been identified in the essential oil of M. officinalis,
including citral 23 (a mixture of the isomers geranial and
neral) and it is known that these are weak inhibitors of
AChE [67].
The root of another species of Salvia, S. miltiorrhiza
Bung. (Labiatae) is extensively used in TCM to stabilise
the heart and calm nerves [48]. S. miltiorrhiza has been
the subject of thorough investigation, and consequently
numerous pharmacological activities that may be relevant
in CNS disorders, including AD, have been identified. S.
miltiorrhiza has been employed for the treatment of cere-
bral vascular disease, and there are several studies to

Natural Products and Neurotransmission Neurosignals 2005;14:6–22 13

 The withanolides are a group of compounds related to
CH3
the steroids which are found in some genera of the Solana-
CH2OH ceae, notably Withania somnifera (L.) Dun. The root of
O O
H3C H OH this plant is one of the most highly regarded herbs in
CH3 OH
O O OH Ayurvedic medicine where it is known as ‘ashwagandha’
O and has a history of use for almost 4,000 years. It is
CH3
classed among the rejuvenative tonics known as ‘Rasaya-
H
nas’ and several groups have described the cognitive
OH
O enhancing potential of extracts of the roots in experimen-
27 Sitoindoside IX tal animals. Some individual compounds have also been
CH3
CH2OH
investigated and the sitoindosides IX 27 and X (fig. 9)
H3C H
have been shown to augment learning acquisition and
CH3 O O memory in both young and old rats [71]. The mechanisms
H
O
CH3
to explain this effect are unclear, but may involve modu-
lation of cholinergic neurotransmission. An extract con-
H taining the sitoindosides VII–X and withaferin A 28 was
O administered to mice and effects on the neurotransmitter
OH
systems in the brain were observed. The results from this
study showed that the extract enhanced AChE activity in
28 Withaferin A the lateral septum and globus pallidus areas of the brain
and also enhanced muscarinic M1 receptor binding in cor-
tical regions, but it did not affect Á-aminobutyric acid
Fig. 9. Steroidal derivatives with anticholinesterase inhibitory activ- (GABA)A, benzodiazepine receptor binding, nor NMDA
ity. or amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA) glutamate receptor subtypes [72].
The extract containing the sitoindosides VII–X and
investigate possible mechanisms for the protective effect withaferin A also reversed the reduction in cholinergic
of S. miltiorrhiza against cerebral ischaemia. markers (e.g. ACh, choline acetyltransferase; ChAT) in
There is evidence that S. miltiorrhiza root extract may rats [73]. These activities could explain the reputed cogni-
protect neurons from ischaemia and attenuate dysfunc- tion enhancing effects of W. somnifera root because of
tion of neuropeptides of importance in neurodegenerative preferential action on cholinergic neurotransmission in
disease [68]. An inhibitory effect on AChE has been the cortical and basal forebrain, brain areas involved in
recently demonstrated and has been shown to be due to cognitive function. Based on this information, it could be
the diterpenes present known as tanshinones [69]. Dihy- speculated that the sitoindosides and withaferin A could
drotanshinone 24 was shown to be the most active (IC50 = have potential in AD therapy, but more studies need to be
1.0 ÌM) with cryptotanshinone 25 (IC50 = 7.0 ÌM ) also carried out before they can be used with any degree of
showing activity. A feature which appears to be necessary confidence in being able to achieve this or whether the
for activity is the saturated bond in the furan ring of the crude extract might produce a better effect.
molecules. In addition to their cholinergic activity, the glycowi-
Screening of 139 different Indian medicinal plants and thanolides showed anxiolytic and anti-depressant activi-
spices for AChE inhibitory activity led to Origanum ties in rats [74], which may be applicable in the symptom-
majorana L. (Labiatae) showing the highest activity. The atic treatment of AD.
active component was identified as the triterpene ursolic W. somnifera root and some constituents are also
acid 26, a fairly common compound, which exhibited an reported to have anti-oxidant properties which may also
IC50 value of 7.5 nM, less than an order of magnitude be relevant in AD therapy [75]. Anti-inflammatory effects
weaker than that of the positive control tacrine [70]. This of the extract of roots have been demonstrated in rats [76]
result is of interest in view of the widespread occurrence and the extract has also been shown to reduce levels of the
of ursolic acid and may account for the traditional use of pro-inflammatory interleukins IL-1 and TNF-·, which
several plant species for memory improvement and AD- are considered to be involved in senile plaque formation
related conditions. and neurodegeneration [77].

14 Neurosignals 2005;14:6–22 Houghton/Howes

 There are many types of phenolic compounds, but the OH R
only ones which have been shown to possess AChE inhibi-
tory activity are neolignans from Magnolia officinalis. OH

The root and stem bark of Magnolia officinalis Rehd. Et

Wils. (Magnoliaceae) have been used in TCM to treat anx- H2C CH2

iety and nervous disturbances. M. officinalis contains the

29 R = H Honokiol
biphenolic lignans, honokiol 29 and magnolol 30 (fig. 10). 30 R = OH Magnolol
Both lignans increased ChAT activity and inhibited
AChE activity in vitro, and increased hippocampal ACh
Fig. 10. Neolignans with anticholinesterase
release in vivo [78]. Honokiol and magnolol show anxio- inhibitory activity.
lytic effects [79] and these appear to be due to their ability
to potentiate GABAergic neurotransmission [80]. These
two compounds also appear to have antioxidant anti-
O OH
inflammatory and neuroprotective properties and such
N
polyvalency in activity is of interest in their potential use
CH3
in treating AD [81, 82].
31 Lobeline

AD: Use of Nicotinic Compounds O

O

N N
A link between smokers and a lower incidence of AD
H
has been noted and this is thought to be associated with
increased nicotine intake, although some recent reports H H
N N
present a contrary view in linking smoking with an H
increased incidence of AD [83, 84]. Nicotine 3 is reported 32 Sophoramine 33 Cytisine

to have cognition-enhancing effects and these may be due

to nicotinic receptor stimulation but also protection
against AD by other mechanisms such as inhibition of ß-
amyloid formation [85], inhibition of the neurotoxic
effects of excitatory amino acids (e.g. glutamate) and
enhancement of the effects of nerve growth factor (NGF)
[86]. There are several other alkaloids which are nicotinic
agonists at the cholinergic receptor [87] (fig. 11). Lobeline
31 from Lobelia inflata interacts with the nicotinic recep-
tor [88] and could also be exploited to influence choliner-
gic function in AD. Other alkaloids such as sophoramine
32 and cytisine 33, found in members of the Legumino-
sae, have nicotinic actions. Cytisine is used as a pharma-
cological tool because of its strong binding affinity to nico-
tinic receptors, but it does not appear to have been devel-
oped for any pharmaceutical purposes, probably because
of its toxicity.
Parkinson’s Disease
Parkinson’s disease is named after the English surgeon
who first described a syndrome which he called the ‘shak-
ing palsy’. It affects a large number of people (about
20,000 in the UK) and is most commonly seen in older
Fig. 11. Nicotinergic alkaloids.
patients, although an estimated 5% of sufferers are under
40 years old [89]. Its characteristic feature is an increasing
tremor in resting limbs and a rigidity, known as dyskine-
sia, particularly exhibited as a shuffling gait, but it is also
associated with the degeneration of cognitive function
and memory.
It is thought that oxidative stress in the substantia
nigra plays a significant role in the loss of neurons which
produce DA [90]. This results in the characteristic defi-
ciency of DA in the substantia nigra seen in the brain of
PD patients post mortem, the other characteristic histo-
pathological observation being the presence of deposits
called Lewy bodies in the surviving neurons.
The major therapeutic approach to PD has been to ele-
vate the levels of DA by either inhibition of monoamine
oxidase (MAO), which metabolises DA to less active com-
pounds, or by increasing the concentration of the precur-
sor of DA by administering L-hydroxyphenylalanine (L-
DOPA) 34 (fig. 12). Another approach involves the use of
compounds which are agonists on the DA receptors.

Natural Products and Neurotransmission Neurosignals 2005;14:6–22 15

 Ph alanine Ph alanine
COOH COOH 617 307
CH3
Aspartate
NH2 311
NHNH2 Serine
505
+
HO NH3 Ph alanine
616
HO HO
OH OH HO
Serine Tryptamine
508 613
34 L-DOPA 35 Carbidopa Ph alanine
509

NHR
HO Fig. 13. Interaction between dopamine and amino acid residues in
the dopamine receptor.

HO
OH
36 R = CH 3 Adrenaline
37 R=H Noradrenaline Tryptamine Tryptamine
617 307
CH3 CH3 Aspartate
HO O Serine 311
OH +
NHCH3 NH2 505
HO NH2 Ph alanine
616
CH3
HO
Serine Tryptamine
508 613
38 Ephedrine 39 Cathinone
Ph alanine
509

Fig. 12. Dopamine and related compounds. Fig. 14. Interaction between adrenaline (epinephrine) and amino
acid residues in the adrenergic receptor.

PD: Use of Dopaminergic Agonists is now administered routinely to PD patients. It is found

The structure-activity relationships of compounds
which are agonists at the DA receptor are generally
accepted to be the two ortho OH groups on the aromatic
ring which bind with serine residues 505 and 508, the aro-
matic ring enabling hydrophobic interactions with a phe-
nylalanine at 617 and, in the terminal position of the ethyl
amino group attached to the aromatic ring, a nitrogen
with a positive charge which interacts electrostatically
with an aspartate residue (fig. 13). It is noteworthy that
the adrenergic receptor is very similar in some respects
(fig. 14) and so it is possible that compounds with a struc-
ture favouring binding to one of these receptors, also have
some effect on the other.
DA itself is quite unstable and cannot cross the blood-
brain barrier. However, it can be formed within the brain
by conversion of its precursor L-DOPA. This compound
in commercially viable amounts in various species of
bean, notably Mucuna spp., and this has been used as a
commercial source, although the drug is now mainly
obtained by synthesis. L-DOPA is often given together
with another analogue of DA, carbidopa 35, which is not
dopaminergic but which inhibits dopa-decarboxylase and
so, by maintaining levels of L-DOPA in the blood, pro-
longs its activity. When L-DOPA is given over long peri-
ods of time, it is common for a sudden decline in sensitiv-
ity to occur from time to time and this is called the ‘on-off’
effect. This is probably due to a number of factors includ-
ing depletion in the ability of the substantia nigra cells to
store DA and desensitization of the receptors. Other
dopaminergic agents, some of which are mentioned be-
low, are often used as adjuvants to reverse the ‘off’ effect.
It is interesting that the powdered seeds of Mucuna
pruriens L. have been used in Ayurvedic medicine for dis-

16 Neurosignals 2005;14:6–22 Houghton/Howes

eases of the nervous system [91] and this product has
shown to reduce adverse effects, such as the ‘on-off’ effect,
HO N HO N
in patients [92]. A commercial extract of M. pruriens HP- H
H3C
O H O
O H3C
200 was shown to be twice as effective as the equivalent CH3
H N H N CH3
N N
dose of L-DOPA in rats [93]. This may be due to the pres- O O
CH3 CH3 CH3
ence of other, more active, compounds. However, a later O N O N
H H
study showed that when the same preparation was given
to rats over a 52-week period, it elevated DA levels in the
cortex but not in the striatum nigrum, this calling into Br
question whether the observed improvements in parkin- N N
H H
sonian symptoms were due to the hypothesis originally 40 Ergotamine 41 Bromocriptine
proposed, i.e. that the L-DOPA in the Mucuna extract
was converted to DA and reached the parts of the brain NHC 2H5
CH3 O N(C 2H5)2
where a deficiency is associated with PD [93]. SCH3
O N
N
C 3H7 CH3
The transmitter dopamine DA is one of a group of N CH2 H
N
N
CH3
H O N H
compounds known as phenylpropylamines, which in- H
cludes some important neurotransmitters such as adrena-
line (epinephrine) 36 and noradrenaline (norepinephrine)
N
37. The phenylpropylamines also include several natural- H N
N
H
ly-occurring compounds known as protoalkaloids, since H

their N atom is not part of a heterocyclic ring. Most pro- 42 Pergolide 43 Cabergoline 44 Lisuride

toalkaloids appear to have a greater adrenergic than dopa-

minergic effect, although it appears that they can stimu-
late both types of receptor. The major naturally-occurring
compound of this type used therapeutically is ephedrine
38, obtained from some species of Ephedra (Ephedra-
ceae), and these plants which have been used for many
centuries in TCM. Ephedrine is used principally for its
adrenergic sympathomimetic properties in drying up se-
cretions, but it is well-known for its CNS-stimulant side
effects, which may be due also to dopaminergic proper-
ties. In contrast, the synthetic phenylpropylamines known
as amphetamines are primarily employed as CNS stimu-
lants and there appears to be some association between
their use and alleviation of the dyskinesia often associated
with PD. A natural compound very similar in chemistry
and pharmacology to the amphetamines is cathinone 39,
a major active constituent of Catha edulis Forsk. (Celas-
traceae). The fresh young leaves of this plant are known as
‘khat’ and are used as a stimulant masticatory in Ethiopia,
Yemen and surrounding countries and by the diaspora of
those communities in Europe [94]. Cathinone has been
shown to be present mainly in the young leaves only and,
since there have been anecdotal reports of reduction in
PD-like tremors induced by neuroleptic drugs in some
regular chewers of khat, it might be that this is due to a
similar effect of cathinone as that observed for amphet-
amines [95].
Probably the second most important group of com-
pounds used for the treatment of PD are derivatives of the
Fig. 15. Ergot alkaloid derivatives used to treat parkinsonism.
ergot indole alkaloids (fig. 15). Ergot, Claviceps purpurea
Tulasne (Clavipitaceae), is a fungus which infects the ears
of cereal crops, notably rye Secale cereale L. Ergot has a
long history of poisoning animals and humans who eat the
cereal flour contaminated with ergot, but also has been
exploited in traditional medicine in some parts of Europe
to aid childbirth, since an extract causes contraction of the
uterus towards the end of pregnancy and also constricts
blood vessels, thus reducing bleeding which often occurs
at birth. The effects of ergot on the CNS has also been
well-documented and outbreaks of ‘madness’ involving
hallucinations have been shown to correlate with times of
heavy contamination of rye flour in the communities
affected. The activity of ergot has been shown to be due to
the alkaloids present such as ergotamine 40. All of the
alkaloids contain the indole ring structure known as lyser-
gic acid and similarities with the three neurotransmitters
noradrenaline (norepinephrine), DA and serotonin can be
seen in this (fig. 16). This probably explains the wide spec-
trum of activity of these alkaloids and so it has been found
necessary to alter the chemical structure to produce com-
pounds which more specifically bind to only one of the
receptors. A large amount of derivatives of ergot alkaloids
have been synthesized for differing therapeutic effects

Natural Products and Neurotransmission Neurosignals 2005;14:6–22 17

 NHR
NHR
NH2 NH2
NCH3 NH2
O HO NCH3
H O
H
HO

OH OH N
N
H OH H N
OH H

Ergot alkaloid Noradrenaline Dopamine Serotonin (5-HT) Ergot alkaloid

Fig. 16. Ergot alkaloid skeleton showing

parts similar to neurotransmitters.

and one of these has been dopaminergic receptor stimula-

tion for use in treating PD. Bromocriptine 41, pergolide
HO 42, cabergoline 43 and lisuride 44 are examples of com-
HO
pounds which have been developed in this way and are
HO
H
CH3
N now used clinically. The pharmacological differences be-
HO H
N
CH3
tween the compounds are not very great. All are D2 dopa-
mine receptor agonists, although pergolide also has ago-
45 Apomorphine 46 Salsolinol
nist effects at D1 and D3 receptors. All four drugs are well-
established in treatment and numerous reviews exist on
H
N
CH3 their clinical efficacy and application [96, 97].
CH3O
Bromocriptine and lisuride were the first of these drugs
N H to be introduced and have comparatively short half-lives.
H Pergolide has a half-life of 8 h [98] and cabergolide of
47 Ibogaine 68 h, making the latter especially useful for administra-
tion only once a day [99]. The ergot-derived drugs were
CH3
originally used in advanced cases of PD, but there is
N
increasing interest in their use in the first instance since,
unlike L-DOPA, they do not need to be metabolized to
O the active compound and so are not affected by any
degeneration in the dopaminergic terminals [100].
Another alkaloid with agonist activity is apomorphine
45, derived from the opium alkaloids (fig. 17). It acts on the
48 Benzatropine
D1 and D2 receptors, but, because it is a powerful emetic
when given orally, its use is restricted to parenteral admin-
N N istration. It is most commonly given when patients are not
CH3 CH3
exhibiting adverse effects to L-DOPA and as a diagnostic
CH3O N CH3O N
H H
agent for dopaminergic responsiveness. The isoquinolin-
49 Harmine 50 Harmaline
oid salsolinol 46 has been investigated extensively over the
last 10 years. This compound is found in the cocoa bean,
the seeds of Theobroma cacao L. (Sterculiaceae), and also
in chocolate derived from this plant. The addictive proper-
Fig. 17. Other alkaloids with dopaminergic and monoamine oxidase ties of chocolate have been ascribed to the dopaminergic
inhibitory activity. activity of this compound [101], but it also occurs as an
endogenous catechol in the brain. Salsolinol has been
shown to be dopaminergic at the D2 receptors and also to
have a protective effect on neurodegeneration [102]. The
N-methyl derivative, which can be synthesized in the

18 Neurosignals 2005;14:6–22 Houghton/Howes

brain, has a role in the pathogenesis of PD [103], and this
complicates the usefulness of giving salsolinol as a protec- R

tive substance to prevent or alleviate PD. OH

Mention should be made of another indole alkaloid, HO O

ibogaine 47, obtained from the African plant Tabernanthe
iboga Baill. (Apocynaceae), the roots of which were used OH
as a stimulant and, in larger doses, as a hallucinogen in OH O
central Africa. This compound has enjoyed much atten- 51 R = H Kaempferol
52 R = OH Quercetin
tion recently because of its claimed usefulness in combat-
ing addiction, particularly to cocaine, although the basis
of its activity is not clear. Ibogaine was shown to release OCH3
OCH3
DA in isolated striatal tissue in mice [104], but it has also
CH3O O
been shown to inhibit the release of catecholamines by
blocking the nicotinic receptor [105] and to block NMDA CH3O
receptors. These activities preclude its usefulness in treat- OCH3 O
ing neurodegenerative disease, but much remains to be
done in the study of its antiaddictive potential. 53 Tangeretin

The tropane alkaloids, especially hyoscine (scopol-

amine) 4, have been used to treat PD since they antago- OH
OH
nise cholinergic activity at the muscarinic receptors in the
striatum and so increase DA activity. The naturally- HO O
OH
occurring alkaloids, found in various genera of the Solana-
O
ceae such as Atropa, Hyoscyamus, Datura and Duboisia,
OH OH
are not much used for this purpose, but synthetic deriva- O
tives and analogues such as benzatropine 48 are used. OH
These also inhibit DA reuptake, so increasing the levels of OH
DA and compensating for the DA deficiency associated
54 Epigallocatechin -3-gallate
with PD.

Fig. 18. Flavonoids with monoamine oxi-

PD: Monoamine Oxidase Inhibitors dase inhibitory activity.

Monoamine oxidase (MAO) is a major enzyme respon-

sible for the fast breakdown of DA and related com-
pounds at the synapse. Inhibitors of this enzyme, known
as MAOIs, cause a net increase in DA levels and, although
their major therapeutic use has been as antidepressants,
they have potential use in PD. This has not generally been
realised because of the side effects associated with eleva-
tion of peripheral DA levels. The ß-carboline alkaloids
harmane 49 and harmaline 50 are found in several tradi-
tional medicine plant species, including Banisteriopsis
caapi (Spruce ex Griseb.) Morton (Malpighiaceae), a liana
used in Brazil as an ingredient in the hallucinogenic drink
‘ayahuasca’. It is thought that the MAOI properties of the
B. caapi constituents prevent metabolism of amines from
other plants used to make ayahuasca. Following reports of
the successful use of B. caapi root extracts for treating PD
patients in Ecuador, it was shown that, in addition to the
MAOI properties, the alkaloids harmane and harmaline
stimulated the release of DA from striatal cells [106].
These compounds would therefore have a double effect in
helping improve DA levels, and this may underlie the
reputed improvements in PD patients when the extract
was taken.
The role of flavonoids in the diet as important antioxi-
dant contributors has received much attention and their
neuroprotective properties because of this effect has been
demonstrated by several workers. However, they have
also been demonstrated to have MAOI activity and this
has been proposed as part of the explanation of the use of
the common herb St John’s Wort, as an antidepressant
[107]. This dual role has now been proposed for a variety
of flavonoids such as kaempferol 51 from the leaves of
Ginkgo biloba, a widely used herbal product which has
been suggested as a preventative against neurodegenera-
tion [108] (fig. 18). Quercetin 52 has also shown to inhibit

Natural Products and Neurotransmission Neurosignals 2005;14:6–22 19

MAO-B [109] and reverse the effects of induced catalep-
sy, which mimics the bradykinesia often seen in PD [110].
Tangeretin 53 has been shown to inhibit MAO-B and to
cross the blood-brain barrier in a rat model and conse-
quently reduce DA depletion, so it may have therapeutic
potential [111]. A compound related to the flavonoids, the
polyphenol (–)-epigallocatechin-3-gallate 54, found in
green tea, has a similar polyvalent activity which may be
sufficient to have a protective effect in PD and other con-
ditions [112]. Since most of these flavonoids occur in rea-
sonably large amounts in common fruits and vegetables,
the question is raised as to whether the apparent increase
in incidence of neurodegenerative disease is related to
some extent with the decline in consumption in the diet of
such foods in some sectors of the industrialized world.
Conclusions
Several natural products useful compounds them-
selves, or have provided lead compounds, for present and
potential treatment of Alzheimer’s and Parkinson’s dis-
eases. Some compounds have reached widespread clinical
use, whilst the interest others at present lies more in their
providing explanation for traditional uses of plants. Ra-
tional drug design according to the characteristics of the
receptors involved is now a possibility, and natural mole-
cules can be subjected to ‘fine-tuning’ by chemical deriva-
tisation and synthesis of analogues to bind more closely
and more exclusively to one type of receptor. However,
investigation of traditional plants has revealed classes of
molecules that have appreciable activity which is unlikely
would have been predicted only from receptor studies.
Regarding the treatment of the major diseases in-
volved, it can be said that chemotherapy of AD with cho-
linesterase inhibitors is unlikely to develop very much fur-
ther since effective agents such as galantamine have been
introduced. Future trends could involve the use of a poly-
valent ‘cocktail’ of drugs which act in different ways by
mechanisms such as antioxidant and anti-inflammatory
activity and the inhibition of the formation of fibrillary
tangles and ß-amyloid plaques. Although the introduction
of L-DOPA and other dopaminergic compounds over the
last three decades has improved the condition of many
sufferers of PD, the side effects and their unpredictability
of occurrence highlight the fact that more work is needed.
In this context also, the exploitation of compounds de-
rived from plants with other mechanisms, such as mono-
amine oxidase inhibition, may provide a better treatment
experience in due course. This may occur in extracts in
any case, and clinical trials should be carried out to follow
up preliminary results, such as those observed with Mucu-
na bean extract, which indicate an advantage in using an
extract over a pure substance.

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Houghton/Howes
 Review

Neurosignals 2005;14:23–33 Received: July 28, 2004

Accepted after revision: September 20, 2004
DOI: 10.1159/000085383

Regulation of Neuroinflammation by
Herbal Medicine and Its Implications for
Neurodegenerative Diseases
A Focus on Traditional Medicines and Flavonoids

Kyoungho Suk
Department of Pharmacology, Pain and Neural Injury Research Center, School of Medicine, Kyungpook
National University, Daegu, Korea

Key Words Introduction

Inflammation  Neuroglia  Neurodegenerative disease 
Flavonoid  Neuroprotection  Microglia  Astrocyte 
Central nervous system
Abstract
Herbal medicine has long been used to treat neural
symptoms. Although the precise mechanisms of action
of herbal drugs have yet to be determined, some of them
have been shown to exert anti-inflammatory and/or anti-
oxidant effects in a variety of peripheral systems. Now,
as increasing evidence indicates that neuroglia-derived
chronic inflammatory responses play a pathological role
in the central nervous system, anti-inflammatory herbal
medicine and its constituents are being proved to be a
potent neuroprotector against various brain patholo-
gies. Structural diversity of medicinal herbs makes them
valuable source of novel lead compounds against thera-
peutic targets that are newly discovered by genomics,
proteomics, and high-throughput screening.
Copyright © 2005 S. Karger AG, Basel
In neuroinflammation, microglia and astrocytes play
a critical role. Microglial cells are ubiquitously distrib-
uted in the central nervous system (CNS) and comprise
up to 20% of the total glial cell population in brain [1, 2].
Although the ontogeny of microglial cells has long been
debated, recent works using monoclonal antibodies spe-
cific for microglial cells indicated that these cells are close-
ly related to monocytes and macrophages [3]. As the pri-
mary immune effector cells in the CNS, microglial cells
migrate to the site of tissue injury or inflammation, where
they respond to invading pathogens or other inflamma-
tory signals [4, 5]. Like monocytes/macrophages, they
also secrete inflammatory cytokines and toxic mediators
which may amplify the neuroinflammatory responses [6,
7]. Astrocytes form an intimately connected network with
neurons in the CNS, and they provide mechanical and
metabolic support for neurons [8]. The critical role of
these cells in ion buffering and clearance of neurotrans-
mitters is also well established [9, 10]. Upon inflamma-
tory stimulation, astrocytes proliferate and produce di-
verse intercellular mediators such as nitric oxide (NO)
and tumor necrosis factor (TNF)- [11–13]. There is
growing evidence that inflammatory mediators produced
by activated astrocytes may be involved in the pathogen-
esis of various neurodegenerative diseases [10, 14]. Thus,

© 2005 S. Karger AG, Basel Kyoungho Suk

1424–862X/05/0142–0023$22.00/0 Department of Pharmacology, Pain and Neural Injury Research Center
Fax +41 61 306 12 34 Kyungpook National University School of Medicine
E-Mail karger@karger.ch Accessible online at: 101 Dong-In, Joong-Gu, Daegu, 700-422 (Korea)
www.karger.com www.karger.com/nsg Tel. +82 53 420 4835, Fax +82 53 256 1566, E-Mail ksuk@knu.ac.kr

Physical factors
Nutritional
imbalance
Reversible
damage
Fig. 1. Relationship between inflammation
and tissue injury. Tissue damage can be
triggered by a number of genetic or environ-
mental factors. While reversible tissue dam- Tissue repair,
functional
ages could be repaired for a functional re- recovery
covery, irreversible damages associated
with inappropriately controlled inflamma-
tion (chronic inflammation) could lead to
diseases.
the activation of astrocytes and ensuing production of
toxic inflammatory mediators may need to be tightly reg-
ulated. Activation of inflammatory cells in CNS (micro-
glia or astrocytes) may be intended to protect neurons at
first. More frequently, however, activation of these neu-
roglial cells and inflammatory products derived from
them have been implicated in neuronal destruction com-
monly observed in various neurodegenerative diseases
[7]. Thus, our understanding of pathogenesis of neurode-
generative diseases may be enhanced by elucidation of
the molecular mechanism underlying the regulation of
neuroglial activation. Among many endogenous or exog-
enous factors that regulate neuroglial activation and re-
sulting neuroinflammation [15], herbal medicine has re-
cently drawn much attention due to its potent inhibitory
effects on inflammatory responses and neuroprotective
activity [16, 17]. A central role of microglia and astrocytes
in neuroinflammation (and potentially neurodegenera-
tion) and a regulatory effect of herbal medicine on the
inflammatory activation of the neuroglia will be discussed
in this review.
Inflammation and Tissue Injury
Injury, trauma or infection induce a series of complex
and interconnected reaction sequences, initiated at the
site of tissue damage [18, 19]. This sequence of reaction
24 Neurosignals 2005;14:23–33
Chemicals and drugs
Infection Autoimmunity
Genetic disturbance
Hypoxia
Tissue damage
Irreversible
damage
Inflammation Diseases
Regulation No regulation,
chronic inflammation
serves to contain and destroy the infection or damaging
agents, and to prevent continued tissue damage and initi-
ate repair processes to restore normal function. This rap-
id response is known as acute inflammation [20]. The
toxic reactions, which are employed to destroy infectious
organisms or protect host, also paradoxically have the
capacity to injure host tissues. If these toxic responses are
not tightly regulated, tissue injury may predominate over
tissue protection and repair, thereby leading to inflamma-
tory diseases (fig. 1). The characteristics of the inflamma-
tory response include localized changes within the dam-
aged tissue such as the followings: (1) the release of pre-
formed inflammatory mediators from intracellular stores;
(2) the initiation of reaction cascade through the activa-
tion of soluble plasma components; (3) the new synthesis
of inflammatory mediators such as eicosanoids and cyto-
kines, and (4) resolution of the inflammatory response.
The acute inflammatory response is beneficial to the or-
ganism in that it helps to deal with potentially dangerous
microorganisms. However, inflammation does cause
some degree of damage to surrounding tissues. Reactive
oxygen species (ROS), reactive nitrogen species (RNS),
prostanoids, leukotrienes, and hydrolytic enzymes pro-
duced by neutrophils, macrophages, and monocytes may
all play a role in mediating inflammation. Persistence of
infection or defective resolution of inflammatory reaction
results in chronic inflammation where severe tissue dam-
age may occur. Although inflammation is normally a self-
Suk

Intrinsic
Fig. 2. Neuroprotective versus neurotoxic ac- or
tivities of neuroglia. Neuroglial activation is extrinsic
factors
induced by a variety of intrinsic or extrinsic
factors. Physiological neuroglial activation is
beneficial and designed to ameliorate damage
and stress. In contrast, pathological activation
of neuroglia leads to neurotoxicity. Apoptotic
elimination of overactivated neuroglia repre-
sents a self-regulatory mechanism. When the
auto-regulation is at work, physiological neuro-
protection may allow a neuroregeneration. On
the contrary, a defective regulation may cause
neurodegeneration.
limiting event and its benefit outweighs the minor tissue
damage it causes, abnormal activation of the immune or
inflammatory system has the potential to provoke a dev-
astating response [21]. In gout, for example, elevated con-
centration of uric acid in the blood leads to precipitation
of sodium urate crystal within joints which triggers in-
flammation by a variety of mechanisms. Another striking
consequence of abnormal inflammatory response is auto-
immune diseases such as systemic lupus erythematosus,
rheumatoid arthritis, autoimmune vasculitis, dermato-
myositis, chronic autoimmune gastritis, and myasthenia
gravis. Tissue-damaging chronic inflammatory response
may also occur in CNS, where main inflammatory cells
are microglia and astrocytes instead of monocytes/mac-
rophages or neutrophils in periphery [22–24].
Neuroglia (Microglia, Astrocytes),
Neuroinflammation, and Neurodegeneration
Microglia and astrocytes are essential for ensuring
proper functioning of neurons. They are quick to inter-
vene when neurons become injured or stressed. As they
are sentinels of neuron well-being, pathological impair-
ment of microglia or astrocytes could have devastating
consequences for brain function. Nevertheless, there is
still a debate over neuroprotective and neurotoxic func-
tions of these neuroglial cells [22, 25] (fig. 2). It is assumed
that neuroglial activation is largely determined by neuro-
nal signals. Acute injury causes neurons to generate sig-
nals that inform neuroglia about the neuronal status. De-
pending on how severe a degree of neuronal injury, neu-
Herbal Medicine and Neuroinflammation
Physiological Neuro-
neuroprotection regeneration
Apoptosis (self-regulation)
Neuroglial
activation
Pathological Neuro-
neurotoxicity degeneration
Apoptosis (self-regulation)
roglia will either nurse the injured neurons into
regeneration or kill them if they are not viable. These
types of neuroglial responses are considered to represent
normal physiological and neuroprotective responses. In
contrast, some processes that are chronic in nature per-
sistently activate neuroglia eventually causing a failure in
their physiological ability to maintain homeostasis. This
could have detrimental consequences and may lead to
bystander damage due to neuroglial dysfunction. In this
scenario, neuroglia exert neurotoxic effects through the
secretion of a variety of toxic inflammatory mediators.
Thus, although activation of neuroglial cells may be in-
tended to protect neurons, inflammatory products de-
rived from activated neuroglia may also be implicated in
neuronal injury, potentially leading to neurodegenerative
diseases [7]. These deleterious effects of neuroglial activa-
tion may be exacerbated by the failure of auto-regulatory
mechanisms of neuroglia. Recently, activated macro-
phages, whose functions are closely related to microglia,
have been shown to undergo apoptosis [26–28]. It has
been suggested that the apoptosis of activated macro-
phages is one mechanism whereby an organism may reg-
ulate immune and inflammatory responses involving
macrophages [28]. It has been recently demonstrated that
a similar regulatory mechanism exists for microglial cells
[29, 30] and astrocytes [31] as well. Microglial cells and
astrocytes underwent apoptosis upon inflammatory acti-
vation in a manner similar to activation-induced cell
death (AICD) of lymphocytes [30, 31]. AICD is an active
process. T and B lymphocytes undergo AICD as an auto-
regulatory mechanism for the body to remove unwanted
activated cells after making appropriate use of them [32,
Neurosignals 2005;14:23–33 25
33]. Compared to lymphocytes, neuroglial cells in CNS
are not well studied in this respect. Now, as results in this
and other laboratories indicated that neuroglial cells
might be under the control of a similar regulatory mech-
anism [29–31, 34–37], further investigation is warranted
to better understand the molecular mechanism(s) of neu-
roglial AICD and its physiological significance. Neverthe-
less, it has been shown that, in contrast to AICD of T
lymphocytes where Fas-FasL interaction plays a central
role, neither Fas-FasL interaction nor TNF- is impor-
tant in AICD of microglial cells [30]. Instead, NO pro-
duced by activated neuroglial cells themselves was the
major cytotoxic mediator [30, 31]. However, the presence
of NO-independent cytotoxic mechanism has been also
suggested [38, 39].
Elimination of activated neuroglial cells by apoptosis
could be an important mechanism whereby undesirable
effects of long-term neuroglial activation can be mini-
mized. Inflammatory mediators that are produced by ac-
tivated neuroglia in CNS may have harmful effects on
neurons or other neuroglial cells that they originally in-
tended to protect [6, 7]. Thus, in various neurodegenera-
tive diseases involving chronic neuroglial activation, neu-
roglial functions seem to play a more significant role in
mediating diseases than in the protection of neurons. Ac-
cording to the model of activation-induced apoptosis of
neuroglial cells, inflammatory signals that activate neu-
roglia may also initiate internal death program [38, 40].
One interesting question that can be raised then is how
neuroglial cells could survive the inflammatory activa-
tion. It should be kept in mind that neuroglial cells in vivo
are heterogeneous and interact with other neuroglial cells
as well as neurons. There is also growing evidence that
activated neuroglial cells proliferate in vivo as one way of
replenishment [1]. Thus, not all neuroglial cells may re-
spond to the inflammatory signals in the same fashion.
Upon inflammatory activation, individual neuroglial
cells in heterogeneous population may either undergo
AICD or return to the resting state via other regulatory
mechanisms depending on the specific microenviron-
ment under which they react to the signals. Although
many of activated neuroglial cells may be eliminated,
some would survive to be deactivated. Whatever the
mechanism of down-regulation is, this may be an excel-
lent auto-regulatory system for the neuroglial activation.
One can easily imagine pathological situations where this
type of auto-regulatory mechanism goes wrong. Failure
of the auto-regulation of ‘over-activated’ neuroglial cells
may result in pathological destruction of bystander cells
(neurons and other neuroglial cells) exposed to toxic me-
26 Neurosignals 2005;14:23–33
diators produced by activated neuroglia. Recently, up-
regulated Bcl-xL expression has been detected in reactive
microglia of patients with neurodegenerative diseases
[41]. Authors proposed that high level of Bcl-xL protein
might render microglia more resistant to cytotoxic envi-
ronment such as areas of neurodegeneration. Expression
of anti-apoptotic Bcl-2 protein has been also associated
with aged brain and neurodegenerative diseases [42]. An
importance of the physiological regulation of neuroglial
activation by AICD is supported by these previous re-
ports.
Recent studies focused on the possible role of neuro-
glia in causing neurodegeneration. Convincing evidence
from in vitro studies pointed to the neurotoxic role of
neuroglia during traumatic or ischemic brain injury [4]
and AD pathogenesis [43]. Supernatants obtained from
neuroglial cell cultures kill cultured neurons. Such super-
natants contain various neurotoxic substances which in-
clude glutamate, NO, ROS, inflammatory cytokines, as
well as yet unidentified neurotoxins [44, 45]. Production
of these neurotoxins by neuroglia is enhanced by treat-
ment with inflammatory stimuli such as lipopolysaccha-
ride (LPS) and/or interferon (IFN)-. Paradoxically, oth-
er investigators have shown that neuroglia-conditioned
media promote neuronal survival [46]. Thus, the balance
of neurotoxic and neurotrophic effects of neuroglia ap-
pears to depend on the nature of the experimental para-
digm used. In traumatic brain injury where neuronal re-
generation may occur, neuroglial secretory products
might help to promote regenerative efforts by injured, but
surviving, neurons. However, a situation may be differ-
ent in neurodegenerative diseases such as Alzheimer’s
disease (AD) or human immunodeficiency virus (HIV)-
associated dementia, where functionally compromised
neuroglia may produce neurotoxins, thereby resulting in
neuronal damage. There is considerable evidence from
postmortem examinations of AD brains that auto-de-
structive mechanisms are at work, which could in part be
responsible for the neurodegeneration [47, 48].
Neuroglia as a Target of Pharmacological
Intervention
Considering neuroglial activation as a common fea-
ture in many neuropathologies, and keeping in mind that
overactivation of neuroglia can have neurotoxic out-
comes, it is reasonable to assume that manipulation
of neuroglial activation could serve future clinical ap-
proaches [49]. Although treatment of the primary events
Suk
in neurodegenerative diseases would still be the preferred
intervention, this may not always be possible. Brain or
spinal cord injury is a sudden event that is followed by
secondary cascades of destruction. Invading macrophages
and intrinsic neuroglia in brain may carry a significant
portion of these cascades of reaction. Now, there is grow-
ing evidence that toxic mediators produced by activated
neuroglial cells might be involved in the pathogenesis of
various neurodegenerative diseases such as Parkinson’s
disease (PD), AD, and HIV-associated dementia [6, 7,
47]. Thus, it is of great interest to find a means to modu-
late neuroglial activation and CNS inflammatory re-
sponses for the therapeutic interventions against these
neurodegenerative diseases. Based on understanding of
intracellular signaling pathways that are specific for acti-
vated neuroglia, a temporary inhibition of signaling mol-
ecules or protein-protein interaction associated with sig-
naling pathways would probably allow for a rather selec-
tive effect on activated neuroglia, while respective
functions in other cell types are unaffected. Elucidation
of the intracellular key events that drive neuroglial acti-
vation could provide new routes for drug development
[49]. Alternatively, potentially harmful products of neu-
roglia could be neutralized to limit undesired conse-
quences for CNS cells and tissues. Whether it is a direct
inhibition of neuroglial activation or indirect suppression
of neuroglia-derived toxic inflammatory mediators, a bet-
ter understanding of neuroglial biology and selective ma-
nipulation of neuroglial activation processes represent a
promising goal for developing novel neuroprotective
strategies.
Medical Use of Herbs
Herbs are generally defined as any form of plants or
plant products, including leaves, stems, roots, and seeds
[50, 51]. Herbal products may contain a single herb or
combinations of several different herbs that are thought
to have complementary effects. Herbal products are usu-
ally extracts of the plants, which are obtained by boiling
or percolating the herbs in water, alcohol, or other or-
ganic solvents to release biologically active ingredients of
the plants. Herbal products contain complicated mix-
tures of organic chemicals, which may include fatty acids,
sterols, alkaloids, flavonoids, glycosides, saponins, tan-
nins, terpenes and so forth. It is often difficult to deter-
mine which component(s) of the herb has biological activ-
ity. In addition, the processing of herbs, such as heating
or boiling, may alter the pharmacological activity of the
Herbal Medicine and Neuroinflammation
organic constituents. Similarly, a host of environmental
factors, including soil, altitude, and seasonal variation in
weather, may affect the levels of components in any given
batch of an herb. Because of these multiple factors that
affect concentration of active ingredients in the final
herbal product, standardization is inevitable, in which
certain unique chemical components of the herbs known
as markers are identified and the production process is
altered to achieve a consistent level of these markers in
every final batch of the herbal product. The ten most com-
monly used herbs in United States are echinacea, garlic,
Ginkgo biloba, saw palmetto, ginseng, grape seed extract,
green tea, St. John’s wort, bilberry, and aloe [50]. The
medical use of herbs in their natural and unprocessed
form began when mankind first noticed that certain plants
altered particular body functions. Now, much informa-
tion exists about the historical use and effectiveness of
botanical products. Unfortunately, however, the quality
of this information is extremely variable. Necessary is
evidence-based approach to the pharmacology and clini-
cal efficacy of the herbal medicine.
Herbal Medicine against CNS Disorders
In traditional practices of Chinese medicine, numer-
ous plants have been used to treat stroke and cognitive
disorders, including neurodegenerative diseases such as
AD [52, 53]. Neurodegenerative diseases and brain inju-
ries resulting from stroke are the major and increasing
public health problem in both developed and developing
countries worldwide [54]. It is believed that traditional
Chinese medicines (TCM) are effective, with few or no
side-effects. There are more than 120 traditional medi-
cines in use for the therapy of CNS disorders in Asian
countries. Some of their therapeutic effects have been
confirmed by recent clinical studies. An ethnopharmaco-
logical approach has provided a potentially rich source
for drug discovery and development [55]. Many drugs
currently available in Western medicine were originally
isolated from plants. Although a large number of com-
pounds have been isolated from TCMs, most of these
resources have not yet been fully characterized for phar-
macological purposes. Some of the TCMs used in stroke
therapy include Ledebouriella divaricata, Scutellaria
baicalensis, Angelica pubescens, Morus alba, Salvia milti-
orrhiza, Uncaria rhynchophylla, and Ligusticum chuan-
xiong [52]. Among these, S. baicalensis and U. rhyncho-
phylla have been found to confer neuroprotection against
transient global ischemia [56, 57]. S. baicalensis is one of
Neurosignals 2005;14:23–33 27
 Inflammatory stimuli
Fig. 3. Herbal medicine as a neuroprotector
that targets neuroglial inflammation. Rest-
ing neuroglia can be activated by inflamma-
tory stimuli such as LPS, IFN- and TNF-.
Resting
Activated neuroglia secrete a variety of in-
neuroglia
flammatory mediators including ROS, ni-
tric oxide, TNF-, and IL-1, which cause
neuronal injury (thereby resulting in neuro-
degeneration). Herbal medicine may be
neuroprotective by blocking the inflamma-
tory activation of neuroglia.
the most widely used herbal medicines against bacterial
infections of the respiratory and gastrointestinal tract,
and various inflammatory diseases. The herb has anti-
pyretic, antibacterial, and antihypertensive properties.
The main components of S. baicalensis – baicalin, baica-
lein, and wogonin – have been previously shown to exert
anti-inflammatory effects [58–60]. Based on the use of S.
baicalensis for the treatment of stroke in traditional ori-
ental medicine, neuroprotective effects of S. baicalensis
have been evaluated after transient global ischemia using
rat 4-vessel occlusion model [56]. Methanol extracts from
the dried roots of S. baicalensis (0.1–10 mg/kg) adminis-
tered intraperitoneally significantly protected CA1 neu-
rons against 10 min transient forebrain ischemia as dem-
onstrated by measuring the density of neuronal cells
stained with cresyl violet. The neuroprotective effects of
S. baicalensis observed in vivo was explained in part by
its inhibitory effects on microglial TNF- and NO pro-
duction as well as protection of nerve growth factor
(NGF)-differentiated PC12 cells from hydrogen peroxide
toxicity in vitro. U. rhynchophylla also exerted neuropro-
tective effects against transient global ischemia [57]. In
traditional Oriental medicine, U. rhynchophylla has been
used to lower blood pressure and to relieve various neu-
rological symptoms. However, scientific evidence related
to its effectiveness or precise modes of action has not been
available. Methanol extract of U. rhynchophylla admin-
istered intraperitoneally (100–1,000 mg/kg at 0 and
90 min after reperfusion) significantly reduced the death
of hippocampal CA1 neurons following the transient
forebrain ischemia. Measurement of neuronal cell den-
sity in CA1 region at 7 days after ischemia by Nissl stain-
ing revealed more than 70% protection in U. rhyncho-
phylla-treated rats compared to saline-treated animals. In
U. rhynchophylla-treated animals, induction of cyclooxy-
28 Neurosignals 2005;14:23–33
Neurotoxic
Activated
inflammatory
neuroglia
mediators
Herbal medicine Neurodegeneration
genase-2 in hippocampus at 24 h after ischemia was sig-
nificantly inhibited at both mRNA and protein levels.
Furthermore, U. rhynchophylla extract inhibited TNF-
and NO production in BV-2 mouse microglial cells in vi-
tro in a manner similar to what has been observed with
S. baicalensis extract. These anti-inflammatory actions of
U. rhynchophylla extract (and other herbal medicines)
may contribute to its neuroprotective effects (fig. 3).
Ginkgo leaf extracts have been primarily used for the
treatment of dementia and neurosensory problems [51,
53, 61]. They contain terpenoids (ginkgolides and bi-
lobalides) and flavonoids. Administration of ginkgo ex-
tracts (EGb 761) has shown biological activities relevant
to the treatment of CNS disorders [61]. Favorable effects
have been observed on cerebral circulation and neuronal
cell metabolism. The extract was also neuroprotective
against -amyloid- and NO-induced toxicity [62, 63].
These effects have been attributed, in part, to platelet-ac-
tivating factor antagonism of the ginkgolides [64] and the
free radical scavenging and anti-oxidant properties of the
flavonoids [65]. In addition to Ginkgo biloba, Huperzia
serrata, Lycoris radiata, Magnolia officinalis, and Poly-
gala tenuifolia have been used for improvement of mem-
ory and cognitive function.
Plant Flavonoids as a Neuroprotector:
Inhibition of Neuroinflammation
Flavonoids are a group of low molecular weight poly-
phenolic compounds of plant origin, many of which alter
metabolic processes and have a positive impact on health
[66]. They exhibit a variety of biological activities such
as anti-inflammatory, anti-oxidant, anti-viral, and anti-
tumor actions [67, 68]. Wogonin (5,7-dihydroxy-8-me-
Suk
 Sham Ischemia Ischemia Wogonin

Ischemia

Ischemia
Wog.
No ischemia

Sham
300 Ischemia
(cell number/mm2)
Neuronal density

200 iNOS

1.0 4.5 1.2

100

TNF
0
Sham Saline 0.5 1 10
g Wogonin (mg/kg) h 1.0 3.3 1.8

Fig. 4. Neuroprotective effects of wogonin against experimental
brain injury. a–f Treatment of experimental animals with wogonin
(10 mg/kg i.p., 0 and 90 min right after 10 min ischemia and reper-
fusion) conferred neuroprotection by markedly reducing the num-
ber of damaged pyramidal cells in the CA1 subfield. Representative
photomicrographs of cresyl violet-stained hippocampal regions of
sham-operated animals (a, d) or animals that had been subjected
to 10 min ischemia followed by treatment with either saline (b, e)
or 10 mg/kg of wogonin (c, f). Boxed regions in a, b, and c (!40)
are shown in d, e, and f (!200), respectively. Scale bar is 100 m.
g The neuroprotective effect of wogonin was dose dependent. Ei-
ther saline or wogonin (0.5, 1 and 10 mg/kg) was intraperitoneally
administered into animals following 10 min ischemia. Seven days
later, neuronal cell density in CA1 region was measured by Nissl
staining and cell counting. Asterisks indicate statistically significant
differences from saline-treated ischemic group (p ! 0.05). Sham =
Sham-operated animals (n = 7); saline = saline-treated animals fol-
lowing ischemia (n = 7); wogonin = wogonin-treated animals fol-
lowing ischemia (n = 3). h Wogonin inhibited expression of inflam-
matory mediators following the ischemic brain injury. At 4 days
after forebrain ischemia, the expression of iNOS and TNF- was
assessed by RT-PCR analysis of hippocampal tissue followed by
Southern blot analysis using sequence-specific oligonucleotide
probes. Wogonin (10 mg/kg) markedly reduced the ischemic induc-
tion of iNOS and TNF- . Results are representative of three inde-
pendent experiments. The numbers indicate a fold induction of the
gene expression normalized to GAPDH as determined by densito-
metric analysis of Southern blot of RT-PCR products.

Herbal Medicine and Neuroinflammation Neurosignals 2005;14:23–33 29

Table 1. Effect of flavonoid wogonin on the histological changes in
hippocampus after kainate injection
Experimental animal groups Histology scores1
Saline (n = 3) 0 croglia. Inhibition of inflammatory activation of micro-
Kainate (n = 5) 3.1180.452
Kainate + wogonin, 1 mg/kg (n = 5) 2.6780.37
Kainate + wogonin, 10 mg/kg (n = 6) 2.2580.322
To examine the neuroprotective effect of wogonin in vivo, ex-
citotoxic neuronal injury was induced by systemic administration
of kainate (30 mg/kg i.p.). Injection of kainate induced a severe
neuronal cell death in CA1 and CA3 of hippocampus. Pretreatment
with wogonin (10 mg/kg i.p., 60 min prior to kainate injection) sig-
nificantly attenuated the hippocampal cell death both in CA1 and
CA3 as determined by histological scoring.
Values represent mean 8 SEM.
1
Damage or loss of hippocampal neurons was assessed by Nissl
staining at 2 days after kainate administration with or without
wogonin pretreatment. Histological damage was scored as follows:
0 = no damage; 1 = occasional injured neurons in CA1 or CA3;
2 = small area (!10%) with neuronal damage or loss in CA1 or CA3;
3 = greater area (10–50%) with neuronal damage of loss in CA1 or
CA3; 4 = extended (150%) neuronal damage of loss in both in CA1
and CA3.
2 staining. A similar neuroprotective activity has been
Statistically significant differences among each other (p <
0.05).
thoxyflavone) and baicalein (5,6,7-trihydroxyflavone) are
flavonoids derived from the root of S. baicalensis. These
flavonoids have been shown to exert various anti-inflam-
matory activities in vitro as well as in vivo. Wogonin in-
hibited LPS-induced production of NO [60, 69] and pros-
taglandin E2 [70] in macrophages. Wogonin inhibited
monocyte chemotactic protein-1 gene expression in hu-
man endothelial cells [71]. It also inhibited TPA-induced
cyclooxygenase-2 expression and skin inflammation in
mice [72]. Moreover, wogonin showed free radical scav-
enging and anti-oxidant activities [73–75]. In the CNS,
however, little information is available about its effects
on glial cells and neurons. Gao et al. [73, 76] demonstrat-
ed neuroprotective effects of four flavonoids from S. bai-
calensis, including wogonin, in cultured human neuro-
blastoma cells. Recently, it has been demonstrated that
wogonin inhibits NO production and inducible NO syn-
thase (iNOS) induction in cultured rat astrocytes [77],
suggesting that the flavonoid may act as an anti-inflam-
matory agent in CNS as well. This was confirmed by a
recent study where wogonin has been shown to be neuro-
protective against experimental brain injury by inhibiting
inflammatory activation of microglia [78]. Wogonin in-
30 Neurosignals 2005;14:23–33
hibited inflammatory activation of cultured brain micro-
glia by diminishing LPS-induced TNF-, IL-1, and NO
production. Wogonin inhibited NO production by sup-
pressing iNOS induction and NF-B activation in mi-
glia by wogonin led to the reduction in microglial cyto-
toxicity toward co-cultured PC12 cells, supporting a
neuroprotective role for wogonin in vitro. The neuropro-
tective effect of wogonin was further demonstrated in
vivo using two experimental brain injury models; tran-
sient global ischemia by 4-vessel occlusion (fig. 4) and
excitotoxic injury by systemic kainate injection (table 1).
In both animal models, wogonin conferred neuroprotec-
tion by attenuating the death of hippocampal neurons,
and the neuroprotective effect was associated with inhibi-
tion of the inflammatory activation of microglia. Hippo-
campal induction of inflammatory mediators such as
iNOS and TNF- was reduced by wogonin in the global
ischemia model (fig. 4), and microglial activation was
markedly down-regulated by wogonin in the kainate in-
jection model as judged by microglia-specific isolectin B4
demonstrated with baicalein in rats [Kim et al., unpub-
lished results] as well as in gerbils [79]. Baicalein attenu-
ated NO production and apoptosis of LPS-activated, but
not IFN--activated, BV-2 mouse microglial cells as well
as rat primary microglia cultures [80]. The inhibition of
NO production by baicalein was due to the suppression
of iNOS induction. Moreover, baicalein inhibited LPS-
induced NF-B activity in BV-2 cells without affecting
caspase-11 activation, interferon regulatory factor (IRF)-1
induction, or signal transducer and activator of tran-
scription (STAT)-1 phosphorylation. IRF-1 and STAT-1
are central components of IFN- signaling [81]. Taken
together, flavonoids such as wogonin and baicalein seem
to exert their neuroprotective effects by inhibiting mi-
croglial activation, which is a critical component of patho-
genic inflammatory responses in neurodegenerative dis-
eases. These findings emphasize the importance of herb-
al medicines and their constituents as an invaluable
source for the development of novel neuroprotective
drugs. In neurodegenerative diseases, a pathogenic role
of uncontrolled microglial activation is widely accepted
at present [7]. In search of neuroprotective agents, now it
is time to focus on killer cells (microglia and astrocytes)
instead of killed cells (neurons); eliminating or at least
suppressing killer microglial activation will provide a bet-
ter chance for neuroprotection compared to just salvaging
dying neurons. Identification of a potent neuroprotector
from natural source that inhibits the killer cell activity
Suk
will certainly instigate further investigations in the re-
lated areas, which will ultimately lead to the successful
development of novel neuroprotective drugs based on fla-
vonoids or other constituents of the medicinal herbs.
Other Components of Herbal Medicine and
Other Mechanisms of Neuroprotection
As mentioned above, herbal extracts contain compli-
cated mixtures of organic chemicals, including fatty ac-
ids, sterols, alkaloids, flavonoids, glycosides, saponins,
tannins, and terpenes. Flavonoids are not the only com-
ponent possessing neuroprotective effects [82]. Anti-de-
mentia effects of galanthamine, an alkaloid widely occur-
ring in Amaryllidaceous plants, have been well demon-
strated in a variety of animal models [83–85]. Acute and
chronic treatment with galanthamine significantly im-
proved the impairment of learning, short-term and spa-
tial memory. The p-hydroxybenzyl alcohol and gastrodin
are the active ingredients isolated from Gastrodia elata
roots, and they have been shown to possess anti-amnesic
activities in experimental animals [86–88]. Huperzine A,
a sesquiterpene alkaloid purified from the Chinese me-
dicinal herb Huperia serrata, exhibits a broad range of
neuroprotective actions [89]. Huperzine A ameliorated
learning and memory impairments and improved spatial
working memory. Ginsenosides (ginseng saponins), as
the major active constituents of ginseng, are another ex-
ample of herb components with potent neuroprotective
effects [90–92]. The cellular and molecular mechanisms
underlying the neuroprotective effects of various compo-
nents of herbal extracts may be as diverse as the plants
which these components were isolated from. Although
this review has been focused on the role of neuroinflam-
mation in neurodegenerative diseases and its inhibition
by neuroprotective herbs, the antioxidant activity of
herbal extracts is certainly another important aspect of
neuroprotection [93, 94]. A variety of herbal extracts and
their components have been demonstrated to exert neu-
roprotective effects associated with antioxidant activi-
ties, either by directly stimulating antioxidant response
genes or by potentiating the bodies’ own natural antioxi-
dant defense systems. Modulation of neuroinflammation
and the antioxidant activity are not mutually exclusive
mechanisms of action. ROS and RNS can be generated
during inflammatory responses. These compounds func-
tion as important signal-transducing messengers or as an
effector to kill invading microorganisms. High concentra-
tions of ROS or RNS, however, may cause tissue injury,
Herbal Medicine and Neuroinflammation
which in turn leads to further inflammation. The benefi-
cial versus detrimental effect of ROS and RNS is tightly
regulated by antioxidant defense systems, whereas neu-
roinflammation is controlled by various endogenous me-
diators as well as by auto-regulatory apoptosis of inflam-
matory cells [30, 31]. Inflammation and generation of
ROS or RNS seem to be interconnected physiological re-
sponses, which may also have pathological implications
if left uncontrolled. This is supported by the findings that
many herbal extracts and their components with neuro-
protective activities exert both anti-inflammatory and an-
tioxidant effects at the same time [56, 65, 95, 96].
Conclusions
Activation of microglia and astrocytes plays a pivotal
role in the initiation and progression of various neurode-
generative diseases. Inhibition of the neuroglial activa-
tion may provide an effective therapeutic intervention
that alleviates the progression of the neurodegenerative
diseases. Herbal medicine, especially their flavonoid con-
stituents, may be a useful candidate for such a therapeu-
tic approach. Continual investigation of the mechanisms
underlying neuroglial activation, regulation of neuroin-
flammation, modulatory role of herbal medicine in these
processes would not only lead to the discovery of novel
neuroprotective agents based on medicinal herbs, but also
help to understand complex pathophysiology of neurode-
generative diseases.
Acknowledgement
This work was supported by the Korea Research Foundation
Grant (KRF-2004-000-E00058). This work was also supported by
the Neurobiology Research Program from the Korea Ministry of
Science and Technology (2004-01323).
Neurosignals 2005;14:23–33 31
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33
 Review
Neurosignals 2005;14:34–45
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
Neuritic atrophy W Synaptic loss W Dendrite W Axon W
Alzheimer’s disease W Amyloid-beta W Ginseng W Withania
somnifera W Ashwagandha W Coffee bean
Abstract
The reconstruction of neuronal networks in the damaged
brain is necessary for the therapeutic treatment of neu-
rodegenerative diseases. We have screened the neurite
outgrowth activity of herbal drugs, and identified several
active constituents. In each compound, neurite out-
growth activity was investigated under amyloid-ß-in-
duced neuritic atrophy. Most of the compounds with
neurite regenerative activity also demonstrated memory
improvement activity in Alzheimer’s disease-model
mice. Protopanaxadiol-type saponins in Ginseng drugs
and their metabolite, M1 (20-O-ß-D-glucopyranosyl-
(20S)-protopanaxadiol), showed potent regeneration ac-
tivity for axons and synapses, and amelioration of mem-
ory impairment. Withanolide derivatives (withanolide A,
withanoside IV, and withanoside VI) isolated from the
Indian herbal drug Ashwagandha, also showed neurite
extension in normal and damaged cortical neurons. Tri-
gonelline, a constituent of coffee beans, demonstrated
the regeneration of dendrites and axons, in addition to
memory improvement.
Copyright © 2005 S. Karger AG, Basel
© 2005 S. Karger AG, Basel
ABC 1424–862X/05/0142–0034$22.00/0
Fax + 41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
www.karger.com www.karger.com/nsg
Received: October 11, 2004
Accepted after revision: November 16, 2004
Introduction
Despite the great number of ongoing investigations,
neurodegenerative diseases remain incurable. The drugs
currently available for dementia, such as donepezil, an
acetylcholinesterase inhibitor, are efficacious in the tem-
porary treatment of memory dysfunction, but do not pre-
vent or reverse the underlying neurodegeneration [1]. In
patients with Alzheimer’s disease, neuritic atrophy and
synaptic loss are considered the major causes of cognitive
impairment, based on the results of neuropathological
postmortem studies of the brain [2–4]. In the brains of
patients suffering from other neurodegenerative diseases,
such as Parkinson’s disease, Huntington’s disease, and
Creutzfeldt-Jakob disease, neurite atrophy has also been
observed [5–7]. Such atrophy leads to the destruction of
neuronal networks, and subsequently to the fatal dysfunc-
tion of brain systems in these patients. The exclusion of,
or at least a decrease in the magnitude of, the causes of
each disease may prevent the progression of symptoms,
but such inhibition is not associated with the repair of
already severely damaged brain function. We hypothe-
sized that the reconstruction of neuronal networks in the
injured brain would be the most necessary step in the fun-
damental recovery of brain function, requiring neuritic
regeneration and synaptic reconstruction.
Katsuko Komatsu, PhD
Research Center for Ethnomedicines, Institute of Natural Medicine
Toyama Medical and Pharmaceutical University, 2630 Sugitani
Toyama 930-0194 (Japan)
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
Neurite outgrowth is the first step in the construction
of the neuronal network, and neurite outgrowth activity
has been investigated in many crude drugs. Of these
extracts, several constituents have been identified as ac-
tive compounds (table 1). It is critical that extended neu-
rites have specific functions, such as axons and dendrites,
and can make circuits by synaptic connections. However,
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
studies of natural products, apart from in our research.
Ginseng drugs, Ashwagandha and coffee beans contain
interesting compounds with potent neurite regeneration,
synaptic reconstruction and memory improvement ac-
tivities.
Ginseng Drugs
Neurite Outgrowth Using Methanol Extracts and
Isolated Saponins in SK-N-SH Cells
Ginseng, the root of Panax ginseng, is widely used as a
tonic throughout the world, and is efficacious in the treat-
ment of amnesia. In addition, significant improvement in
learning and memory has been observed in brain-dam-
aged [8, 9] and aged rats [9] after the oral administration
of Ginseng powder, and the major Ginseng saponins, gin-
senoside Rb1 and Rg1, are known to improve spatial
learning in normal mice [10]. Regarding the effects on
neuronal cells, it has been shown that neurite outgrowth
of cultured rat cerebral cortical neurons is enhanced by
crude Ginseng saponins [11], and that ginsenoside Rb1
potentiates the nerve growth factor (NGF)-mediated neu-
rite outgrowth of chick dorsal root ganglia [12, 13].
We tested the neurite outgrowth activity of methanol
extracts of 6 types of Ginseng drugs and P. stipuleanatus
plant material in SK-N-SH cells [14]. The methanol
extracts of Ginseng (dried root of P. ginseng), Red Gin-
seng (steamed and dried root of P. ginseng), Notoginseng
(dried root of P. notoginseng) and Ye-Sanchi (dried rhi-
zome and root of P. vietnamensis var. fuscidiscus) in-
creased neurite outgrowth, with the effects of Red Gin-
seng and Ye-Sanchi being particularly significant.
Thirty saponins were isolated from Ye-Sanchi and
structurally elucidated [15, 16]. Oleanolic acid-type sa-
ponins were also isolated from Kouzichi (dried rhizome
of P. japonicus var. major from Hubei province) [17], and
19 saponins (ginsenosides Rb1, Rb3, Rg1 and Re, notogin-
senosides R4, Fa and R1, Yesanchinoside J, 20-O-glc-gin-
senoside Rf, majonoside R2, (24S)-pseudoginsenoside
RT4 and F11, vina-ginsenoside R1, R2 and R6 from Ye-
Sanchi, notoginsenoside R2, ginsenoside Rg2 and Ro, chi-
kusetsusaponin IVa from Kouzichi) were tested. Proto-
panaxadiol (ppd)-type saponins, ginsenosides Rb1 and
Rb3, and notoginsenosides R4 and Fa significantly ex-
tended the neurites in SK-N-SH cells at a concentration of
100 ÌM, and their activity increased dose-dependently.
On the other hand, protopanaxatriol, ocotillol and olean-
olic acid-type saponins showed no effect [14]. This sug-
36 Neurosignals 2005;14:34–45
gests that ppd-type saponins are active compounds. Gin-
seng, Red Ginseng, Notoginseng and Ye-Sanchi, which
showed neurite outgrowth activity, have been demon-
strated to contain comparatively rich ppd-type saponins
in our quantitative study [18]. This suggested that the
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-
zome of P. stipuleanatus, which inhibited cell viability,
may contain some cytotoxic compounds.
Effect of M1, a Metabolite of Protopanaxadiol-Type
Saponins, on Aß(25–35)-Induced Memory Impairment,
Axonal Atrophy and Synaptic Loss in Mice
When taken orally, ppd-type saponins are mostly me-
tabolized by intestinal bacteria to ppd monoglucoside, 20-
O-ß-D-glucopyranosyl-(20S)-protopanaxadiol (M1) [19,
20] (fig. 1). As Ginseng is generally taken orally, a metabo-
lite of ppd-type saponins, M1, should be investigated to
determine the active constituent of Ginseng responsible
for its major effects. We therefore conducted experiments
to determine whether treatment with ginsenoside Rb1, as
a representative of ppd-type saponins, and its metabolite,
M1, can induce recovery from memory disorder, axonal
atrophy, and synaptic loss induced by the active fragment
of the amyloid-ß peptide (Aß(25–35)) [21].
Male ddY mice (6 weeks old) were prepared to create a
mouse model of Alzheimer’s disease (AD). Seven days
after an i.c.v. injection of Aß(25–35), ginsenoside Rb1
(10 Ìmol/kg), M1 (10 Ìmol/kg), donepezil hydrochloride
(DNP, 0.5 mg/kg), or the vehicle (tap water) was adminis-
tered orally once daily for 14 days. Mice were trained in
the water maze for 7 days starting 14 days after the i.c.v.
administration of Aß(25–35) (fig. 2a). The escape latency
to find the platform in the Aß(25–35)-injected group sig-
nificantly increased compared with the saline-injected
group, whereas the escape latencies of the groups adminis-
tered ginsenoside Rb1 and M1 p.o. significantly decreased
as compared with the vehicle-administered group. The
donepezil-administered group showed no significant
shortening of the escape latency.
In the retention test (fig. 2b), the number of crossings
over a previous platform position was significantly de-
creased in the Aß(25–35)-injected group compared with
the saline-injected group. The number of crossings recov-
ered after treatment with ginsenoside Rb1 and M1. Treat-
ment with donepezil showed the smallest effect in the
retention test. All mice showed normal swimming perfor-
mance and a constant increase in body weight. Locomotor
activity did not differ among groups.
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-
phorylated NF-H (axonal marker), synaptophysin (synap-
tic marker) and MAP2 (dendritic marker) were measured
in mouse brains. We observed two cortical areas (parietal
cortex and temporal cortex) and three hippocampal areas
(CA1, CA3, and the dentate gyrus), as it is known that
synaptic loss occurs primarily in the cerebral cortex and
hippocampus in AD patients [22, 23] and in AD model
mice [24]. The phosphorylated NF-H levels were remark-
ably reduced in these five areas of the brain in Aß(25–
35)-injected compared with saline-injected mice (fig. 3a).
Significant decreases were seen in the parietal cortex, CA1
and CA3; however, the expression levels of phosphorylat-
ed NF-H were nearly equal to those of the control in ginse-
noside Rb1- and M1-treated mice. Donepezil treatment
had no effect on the levels of phosphorylated NF-H. The
synaptophysin levels were also reduced in these five areas
of the brain in Aß(25–35)-injected compared with saline-
injected mice (fig. 3b). Significant decreases were seen in
the temporal cortex and CA1. In all areas, the synapto-
physin levels were almost equal to or higher than control
levels in ginsenoside Rb1- and M1-treated mice. Donepe-
zil treatment had no effect on the synaptophysin levels.
The MAP2 levels were also reduced in the cerebral cortex
and CA1 of the brain in Aß(25–35)-injected compared
with saline-injected mice (fig. 3c). Significant decreases
were seen in the temporal cortex; however, these de-
creases in the expression levels of MAP2 were not clearly
recovered by ginsenoside Rb1, M1 or donepezil. Although
treatment with M1 tended to increase the MAP2 level in
the temporal cortex, the effect was weak. No differences
in neuronal density were observed among the groups in
any brain areas. Treatment with M1, a metabolite of gin-
senoside Rb1, results in the recovery of impaired learning
and memory in Aß(25–35)-injected mice with degener-
ated axons and synapses. The maintained retention of
spatial memory was also seen after the discontinuation of
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-
tial memory induced by Aß(25–35) injection. a Escape latencies per
group in four trials were tested in a Morris water maze over 7 days.
Vehicle was administered p.o. to saline-i.c.v.-injected mice. To
Aß(25–35)-i.c.v.-injected mice (5 nmol), the vehicle, ginsenoside Rb1
(10 Ìmol/kg), M1 (10 Ìmol/kg), or donepezil (0.5 mg/kg) was admin-
istered p.o. for 14 days. Values represent the means and SEM of 9
mice. * p ! 0.05 when compared with the Aß(25–35) plus vehicle-
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
been measured over 60 s, 6 days after the last acquisition test. This
was also 6 days 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, vehicle (Veh), ginsenoside Rb1 (GRb1), M1, or
donepezil (DNP) was administered p.o. Values represent the means
and SEM of 9 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.

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

200
100
0 Ashwagandha induced neurite extension [34]. We further
Veh Veh
Culture
7d 3d
b A␤
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
replaced by new medium containing M1 (0.01 ÌM ), NGF (100 ng/
ml) or the vehicle (Veh, DMSO). Four days later, the cells were fixed
and immunostained for phosphorylated NF-H (a) and MAP2 (b).
The lengths of neurites positive for phosphorylated NF-H or MAP2
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-
treated group. One-way analysis of variance was carried out, followed
by Dunnett’s post hoc test.
Withanolides
Ashwagandha (root of Withania somnifera Dunal) is
the most popular herbal drug in Ayurvedic medicine, and
has been used traditionally and commonly as a tonic and
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
M1 NGF
identified 6 withanolide derivatives from methanol ex-
A␤(25–35) tract (withanolide A, withanoside IV, withanoside VI,
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-
4d
duced by treatment with withanoside IV or withanoside
Drugs VI, whereas predominant axonal outgrowth was observed
in treatment with withanolide A in normal cortical neu-
rons [36].
Effect of Withanolides on Aß(25–35)-Induced Neuritic
Atrophy and Synaptic Loss
In Aß(25–35)-induced damaged cortical neurons,
withanolide A, withanoside IV, and withanoside VI
showed neuritic regeneration and synaptic reconstruc-
tion. 24 h after culture initiation, 10 ÌM Aß(25–35) was
added to the culture medium simultaneously with the
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
and phosphorylated NF-H-positive neurites, showing that
Aß(25–35) induced both dendritic and axonal atrophy in
rat cortical neurons. Simultaneous treatment with Aß(25–
35) and withanolide A, withanoside IV, or withanoside VI
at a concentration of 1 ÌM prevented both dendritic and
axonal atrophy induced by Aß(25–35). Dendritic atrophy
was completely prevented by treatment with withanolide
A (97.0% of the control), withanoside IV (106.3% of the
control), or withanoside VI (117.4% of the control)
(fig. 5a). In particular, treatment with withanosides IV
and VI tended to induce the growth of longer dendrites
than treatment with withanolide A.

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

of the Neuronal Network

Fig. 6. The effect of trigonelline on the prevention of Aß(25–35)-
induced dendritic and axonal atrophy. Cortical neurons were cul-
tured for 3 days, and then the cells were treated simultaneously with
10 ÌM Aß(25–35), and trigonelline at a concentration of 30 or
100 ÌM, or vehicle (Veh), or with the vehicle alone (Cont). Five days
after treatment, the cells were fixed and immunostained for MAP2 or
42 Neurosignals 2005;14:34–45
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).
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 12–20 cells
(a) or 14–22 cells (b). * 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.
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
Impairment
Fourteen days after the i.c.v. injection of Aß(25–35) in
male ddY mice (6 weeks old), trigonelline (500 mg/kg),
donepezil hydrochloride (0.5 mg/kg), or the vehicle (tap
water) was administered orally once daily for 15 days.
Mice were trained in the water maze for 5 days, starting
21 days after the i.c.v. administration of Aß(25–35). Six
days after the last acquisition test, the retention test was
performed (fig. 7). The number of crossings over a pre-
vious platform position was significantly decreased in the
Aß(25–35)-injected group compared with the saline-in-
The ppd-type saponins of Ginseng drugs and M1 (a
metabolite of ppd-type saponins by intestinal bacteria)
induced significant recovery from memory impairment,
axonal atrophy and synaptic loss in mice. The effect of
M1 on axonal reconstruction was further confirmed in
cultured cortical neurons. These results suggest that orally
administered ppd-type saponins potentially ameliorate
dementia by reconstructing the neuronal network. With-
anolide A, withanoside IV, and withanoside VI, which
were isolated from Ashwagandha, facilitated the regenera-
tion of dendrites and axons, and led to the dramatic con-
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
axonal regeneration activity, and improved memory re-
tention. These compounds, sourced from natural prod-
ucts, and used with treatments preventing pathogenesis
and neuronal death, are expected to play an important
role as new categorized drugs in curing neurodegenerative
diseases in the near future. Although we have shown the
high potential of neuronal regeneration from compounds
isolated from Ginseng drugs, Ashwagandha and coffee
beans, it is dangerous to simply imply that these herbal
drugs are expected to be excellent anti-dementia drugs.
When taking herbal drugs, the risk of side effects brought
by other constituents, and sufficient efficacy compared
with isolated compounds should be investigated and care-
fully considered. However, drugs used in traditional med-
icine may offer a treasury of new medicines to treat intrac-
table diseases with the use of novel study concepts and the
application of objective scientific analyses.
Acknowledgments
We thank Prof. M. Hattori, Dr. K. Zou, Mr. N. Matsumoto, Dr.
M. R. Meselhy, Dr. N. Nakamura and Dr. J. Zhao of the Institute of
Natural Medicine, Toyama Medical and Pharmaceutical University
for their extensive contribution to this study. This work was support-
ed by Kampou Science Foundation, Uehara Memorial Foundation,
Grants-in-Aid for Scientific Research (B), No. 11695086 in 1999–
2001 and No. 14406030 in 2002–2004 from the Japan Society for the
Promotion of Science, and the 21st Century COE Program of the
Ministry of Education, Culture, Sports, Science and Technology,
Japan.

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 Review
Neurosignals 2005;14:46–60
DOI: 10.1159/000085385
Multifunctional Activities of Green Tea
Catechins in Neuroprotection
Modulation of Cell Survival Genes, Iron-Dependent Oxidative Stress and PKC
Signaling Pathway
Silvia A. Mandel Yael Avramovich-Tirosh Lydia Reznichenko Hailin Zheng
Orly Weinreb Tamar Amit Moussa B.H. Youdim
Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases
Research, Technion-Faculty of Medicine, Haifa, Israel
Key Words
(–)-Epigallocatechin-3-gallate  Neurorescue 
Neurodegeneration  Neuroprotection  Parkinson’s
disease  Neurite outgrowth  Green tea catechins 
Iron chelation  Cell signaling  Protein kinase C
Abstract
Many lines of evidence suggest that oxidative stress re-
sulting in reactive oxygen species (ROS) generation and
inflammation play a pivotal role in the age-associated
cognitive decline and neuronal loss in neurodegenera-
tive diseases including Alzheimer’s (AD), Parkinson’s
(PD) and Huntington’s diseases. One cardinal chemical
pathology observed in these disorders is the accumula-
tion of iron at sites where the neurons die. The buildup
of an iron gradient in conjunction with ROS (superoxide,
hydroxyl radical and nitric oxide) are thought to consti-
tute a major trigger in neuronal toxicity and demise in
all these diseases. Thus, promising future treatment of
neurodegenerative diseases and aging depends on avail-
ability of effective brain permeable, iron-chelatable/rad-
ical scavenger neuroprotective drugs that would prevent
the progression of neurodegeneration. Tea flavonoids
(catechins) have been reported to possess potent iron-
chelating, radical-scavenging and anti-inflammatory ac-
© 2005 S. Karger AG, Basel
1424–862X/05/0142–0046$22.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
www.karger.com www.karger.com/nsg
Received: January 28, 2005
Accepted after revision: March 1, 2005
tivities and to protect neuronal death in a wide array of
cellular and animal models of neurological diseases. Re-
cent studies have indicated that in addition to the known
antioxidant activity of catechins, other mechanisms such
as modulation of signal transduction pathways, cell sur-
vival/death genes and mitochondrial function, contrib-
ute significantly to the induction of cell viability. This
review will focus on the multifunctional properties of
green tea and its major component (–)-epigallocatechin-
3-gallate (EGCG) and their ability to induce neuroprotec-
tion and neurorescue in vitro and in vivo. In particular,
their transitional metal (iron and copper) chelating prop-
erty and inhibition of oxidative stress.
Copyright © 2005 S. Karger AG, Basel
Introduction
Polyphenols are natural substances present in bever-
ages obtained from plants, fruits and vegetables such as
olive oil, red wine and tea. Flavonoids are the largest
group of polyphenols, which include the subclasses of fla-
vones, isoflavones, flavanols, flavans and flavonols [1].
Several prototypes of these groups have been shown to
promote a number of physiological benefits, especially in
cognitive function and memory impairment. Fresh tea
Prof. M.B.H. Youdim
Eve Topf and USA National Parkinson Foundation Centers of Excellence for
Neurodegenerative Diseases Research, Technion-Faculty of Medicine
POB 9697, 31096 Haifa (Israel)
Tel. +972 4 8295290, Fax +972 4 8513145, E-Mail Youdim@Tx.Technion.ac.il
(Camellia sinensis) leaves contain a high amount of cat-
echins, a group of flavonoids or flavanols, known to con-
stitute 30–45% of the solid green tea extract [2, 3]. The
favorable properties ascribed to tea consumption are be-
lieved to rely on its bioactive components, catechins and
their derivatives, demonstrated to act directly as radical
scavengers and exert indirect antioxidant effects through
activation of transcription factors and antioxidant en-
zymes [for reviews, see 4, 5]. The most abundant poly-
phenolic compound is EGCG, thought to contribute to
the beneficial effects attributed to green tea, such as its
anticancer, cardiovascular function improvement and
antioxidant anti-inflammatory properties. Indeed, a
number of epidemiological studies have shown that phe-
nolic compounds reduce the risk of coronary heart dis-
ease, possibly via their anti-inflammatory effects, includ-
ing inhibition of adhesion molecule and cytokine expres-
sion and augmentation of endothelial nitric oxide release
[6]. Relative antioxidant activities among tea catechins
have been found to be EGCG = (–)-epicatechin-3-gallate
(ECG) 1 (–)-epigallocatechin (EGC) 1 (–)-epicatechin
(EC) [7]. EGCG accounts for more than 10% of the ex-
tract dry weight (30–130 mg per cup of tea) followed by
EGC 1 EC 6 ECG [2]. In addition to their radical scav-
enging action, green tea catechins possess well-established
metal-chelating properties. Structurally important fea-
tures defining their chelating potential are the 3,4-dihy-
droxyl group in the B ring [8] as well as the gallate group
[9, 10], which may neutralize ferric iron to form redox-
inactive iron, thereby protecting cells against oxidative
damage [11]. However, a wealth of data is accumulating
and indicating that the antioxidant/metal chelating attri-
butes of the catechin polyphenols are unlikely to be the
sole explanation for their neuroprotective and neurores-
cue capacity. Thus, catechin polyphenols were found to
invoke a wide spectrum of different mechanisms of ac-
tion responsible for cell survival [for our recent reviews,
see 12, 13].
There is evidence that polyphenol metabolites and
their parent compounds have access to the brain. Studies
with radioactively labeled EGCG in mouse or chemilu-
minescence-based detection of EGCG in rats demon-
strated its incorporation into brain, as well as in various
organs including kidney, heart, liver, spleen and pancreas
[14, 15]. Furthermore, it has been shown that the methyl-
ated and glucuronidated derivatives of epicatechin are
both detected in rat brain following oral administration
[16].
Significant body of evidence support the hypothesis
that brain iron dysregulation and oxidative stress (OS),
Multifunctional Catechins for
Neuroprotection
resulting in ROS generation from H2O2 and inflamma-
tory processes, trigger a cascade of events leading to
apoptotic/necrotic cell death in neurodegenerative dis-
orders, such as Parkinson’s (PD), Alzheimer’s (AD) and
Huntington’s diseases and amyotrophic lateral sclerosis
(ALS) [17]. There is also evidence for increased expres-
sion of apoptotic proteins (for review see [18]), as well
as mitochondria (complex I) and ubiquitin-proteasome
system (UPS) dysfunction, which may lead to break-
down of energy metabolism and consecutive intraneu-
ronal calcium overload [19–22]. Thus, neurodegenera-
tion appears to be multifactorial, where a complex set
of reactions lead to the demise of neurons. This assump-
tion receives support from the familial (genetic) forms
of neurodegenerative diseases identified in the last years,
where mutations in genes, such as -synuclein, parkin
and ubiquitin C-terminal hydrolase-L1 (UCHL-1) de-
scribed in rare forms of hereditary PD [23], may lead to
impairment in the activity of the UPS. More recently,
recessive mutations in DJ-1 [24] and PINK1 (PTEN-
induced kinase 1) [25] were proposed to play a role in
cellular response to OS, supporting a pathogenic role of
ROS in the etiology of neurodegenerative diseases.
Therefore, it is not surprising that antioxidants were the
first drugs to be studied in an attempt to retard the prog-
ress of PD. Recently, coenzyme Q10, an intrinsic com-
ponent of the mitochondrial respiratory chain acting as
a bioenergizer and an antioxidant, was studied as a pu-
tative neuroprotective agent in PD. This double-blind,
placebo-controlled pilot study demonstrated that high
doses of coenzyme Q10 (1,200 mg/d) were associated
with a reduced rate of deterioration in motor function
from baseline over the 16-month course of this trial
[26].
Neuropathological and neurochemical studies on sub-
stantia nigra (SN) from PD brains and its animal models
[23, 27, 28] and our recent gene expression profiling of
human SN pars compacta (pc) from PD patients [29]
demonstrate the existence of a ‘domino’ cascade of neu-
rotoxic events, which can be initiated at any point in the
cascade. These series of events may act independently or
cooperatively during the course of the disease, leading
eventually to the demise of dopaminergic neurons. This
has led to the current notion that drugs directed against
a single target will be ineffective and rather a single drug
or cocktail of drugs with pluripharmacological properties
may be more suitable to be employed. According to this
belief, green tea catechins well fulfill the requirements for
a putative neuroprotective drug having diverse pharma-
cological activities. Thus, it is not surprising that they
Neurosignals 2005;14:46–60 47
have attracted increasing interest as therapeutic cytopro-
tective agents for the treatment of neurodegenerative and
other diseases.
In this article the state of the art of the diverse mo-
lecular mechanisms and cell signaling pathways partici-
pating in the neuroprotective action of green tea catechin
polyphenols is reviewed. Particular attention has been
paid to their iron-chelating properties with respect to the
potential promise for iron chelation therapy, as a novel
treatment for neurodegenerative diseases.
Neuroprotective Effects of Catechins:
Insights from in vivo and in vitro Studies
There is a growing recognition that polyphenolic cat-
echins exert a protective role in neurodegeneration. An
experimental study conducted in N-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) model of PD has
shown that both green tea extract and EGCG effectively
prevent mice striatal dopamine (DA) depletion and SN
dopaminergic neuron loss [30]. The protection exerted by
green tea polyphenols in vivo may involve direct scaveng-
ing of ROS and regulation of antioxidant protective en-
zymes. EGCG was found to elevate the activity of two
major oxygen-radical species metabolizing enzymes, su-
peroxide dismutase (SOD) and catalase in mice striatum
[30]. This is supported by a previous finding where 1
month’s administration of a catechin-containing antioxi-
dant preparation increased SOD activity in the mito-
chondria fraction of striatum and midbrain and decreased
thiobarbiturate reactive substance formation in the cor-
tex and cerebellum of aged rats [31]. The structural cat-
echol resemblance of EGCG may explain a recently re-
ported inhibitory effect of green tea polyphenols on the
DA presynaptic transporters. This inhibition lead to 1-
methyl-4-phenylpyridinium (MPP+) uptake blockade
(because of competition for the vesicular transporter)
thereby protecting DA-containing neurons against
MPP(+)-induced injury [32]. In addition, EGCG greatly
inhibited catechol-O-methyltransferase (COMT) activity
in rat liver cytosol at a low IC50 concentration (0.2 M)
[33]. This action may be of particular significance for PD
patients, given that DA and related catecholamines are
physiological substrates of COMT, thus its inhibition will
result in increased DA in the synapse.
Green tea polyphenols have been also shown beneficial
in animal models of cerebral ischemia: intraperitoneal
injection of EGCG reduced hippocampal neuronal dam-
age and brain edema caused by global [34] or unilateral
48 Neurosignals 2005;14:46–60
[35] cerebral ischemia in gerbils. Insights into the possible
mechanism of neuroprotection by EGCG in the infarct
area of ischemic rats, revealed that it acts by reducing
iNOS expression, infiltration and peroxynitrite forma-
tion [36], by increasing endothelial and neuronal NOS
and preservation of mitochondrial complex activity and
integrity [37]. In this context, the decrease in the activity
of the transcription factor signal transducer and activator
of transcription-1alpha (STAT-1alpha) by EGCG in isch-
emic rat cardiac myocytes may well account for the re-
duced mRNA levels of iNOS, a target of STAT-1 [38].
Other investigators have recently shown that EGCG re-
duced brain inflammation and neuronal damage in ex-
perimental autoimmune encephalomyelitis (EAE), when
given at initiation or after the onset of EAE [39].
An extensive number of studies regarding neuropro-
tection by green tea flavonoids in cellular and animal
models of neurodegenerative diseases are starting to ac-
cumulate. Hence, the flavonoid epicatechin was shown to
attenuate the toxicity induced by oxidized low-density
lipoprotein in mouse-derived striatal neurons [40] or fi-
broblasts [41] and to confer protection to primary culture
of mesencephalic neurons challenged with 6-hydroxydo-
pamine (6-OHDA) [42]. Recently, catechin was shown to
reduce injury produced by hydrogen peroxide, 4-hy-
droxynonenal, rotenone and 6-OHDA in primary rat
mesencephalic cultures, as shown by increases in cellular
viability and [3H]DA uptake [43]. Similarly, EGCG was
reported to protect human neuroblastoma cells from
damage induced by 6-OHDA and MPP+ [44]. EGCG also
protects primary hippocampal neurons [45] and rescues
rat pheochromocytoma (PC12) cells from amyloid- pep-
tide (A)-induced toxicity [46]. More recently, EGCG
was reported to exert a neurorescue activity in long-term
serum-deprived PC12 cells and to promote neurite out-
growth, as manifested by the expression of a surrogate
marker of cell differentiation, growth-associated protein
GAP-43 (GAP-43) [47]. This could have important im-
plications with regard to aging, PD and AD, suggesting a
potential therapeutic use of EGCG in regenerating in-
jured neuronal cells.
Neuroprotection and Neurotoxicity by Low
and High Concentrations of Catechins and
Other Flavonoids
Studies from our and other laboratories have shown
that green tea polyphenols display a concentration-de-
pendent window of neuroprotective action: They protect
Mandel/Avramovich-Tirosh/Reznichenko/
Zheng/Weinreb/Amit/Youdim
at low micromolar concentrations, whereas they become
pro-oxidant and pro-apoptotic when increasing the con-
centrations over 10–20 M [44, 48]. This bell-shaped pat-
tern is typical of antioxidative drugs, such as vitamin C
[49], R-apomorphine [50] and DA [51], being neuropro-
tective at low (1–10 M) concentrations, while having
pro-oxidant/pro-toxic activity at higher (10–50 M) con-
centrations. The toxicity of not only green tea polyphe-
nols, but also of several other flavonoids, is responsible
for their antiproliferative and chemopreventive actions.
The anticancer properties of polyphenols is attributed to
the ability to inhibit phase I and induce phase II carcino-
gen metabolizing enzymes (in animals) that initiate car-
cinogenesis, inhibition of cell cycle progression effectors,
promotion of ROS and nitrogen species (thereby collaps-
ing the mitochondrial membrane potential), induction of
p53 and apoptogenic factors and inactivation of protein
kinases that contribute to survival-associated signal trans-
duction [for extensive reviews see 5, 52, 53]. Since the
present article applies to the mechanisms of neuroprotec-
tion by tea catechins, the literature regarding their anti-
carcinogenic or pro-apoptotic properties will not be re-
viewed.
Mechanism of Neuroprotection by Green Tea
Catechins
Cell Signaling Pathways
Selective Activation of Protein Kinase C (PKC) in
Brain Neurons
PKC expression has been previously coupled with the
preservation of cell survival and the formation and con-
solidation of different types of memory [54–56]. The in-
duction of PKC activity in neurons is thought to be a
prerequisite for neuroprotection against several exoge-
nous insults. Indeed, PKC activation after ischemic pre-
conditioning or pharmacologic preconditioning (with
PKC, NMDA, or A1AR agonists) was shown essential
for neuroprotection against oxygen/glucose deprivation
in organotypic slice cultures [57]. In accordance, activa-
tion of PKC by estrogen or by the grape flavonoid resve-
ratrol, in rat cortical or hippocampal neurons, respective-
ly, protects against A toxicity [58, 59]. Also, we have
recently shown that the anti-Parkinson/monoamine oxi-
dase-B (MAO-B) inhibitor drug, rasagiline (Teva Phar-
maceutical Industries) [60], prevented PC12 cell death
induced by serum deprivation via PKC signaling cascade
[61]. Similarly, we have reported that phosphorylative
activation of PKC by EGCG is responsible for the protec-
Multifunctional Catechins for
Neuroprotection
tive effects against 6-OHDA- and A-induced cell death
in SH-SY5Y and PC12 cells [44, 46] and for the neuro-
rescue effect against long-term growth factors withdrawal
in PC12 cells [47]. This is supported by the observation
that EGCG could not overcome cell death under PKC
pathway blockade, as determined both morphologically
and by monitoring various apoptotic markers, suggesting
that this cascade is essential for the neuronal protection
and rescue effects of EGCG. Consistent with these find-
ings, recent animal studies have shown that two weeks
consumption of EGCG (2 mg/kg) led to a highly signifi-
cant up-regulation of PKC isoform in mice striatum [62]
and to a significant increase in PKC isoenzymes  and 
in the membrane and cytosolic fractions of mice hippo-
campus [46]. The implication of PKC in neuronal sur-
vival by EGCG is further demonstrated in vitro by the
rapid translocation of PKC to the membrane compart-
ment in PC12 cells, in response to EGCG (fig. 1). PKC
is a well-established neuron cell survival factor participat-
ing also in cell growth and differentiation [63, 64]. In sup-
port, a recent report shows that treatment of human cells
with EGCG induces a specific translocation of PKC to
the membrane [65].
More direct evidence implicating PKC in EGCG
mechanistic action has come from a recent study employ-
ing solid-state nuclear magnetic resonance, showing that
EGCG interacts with the head group region of the phos-
pholipids within lipid bilayers from liposomes [66]. The
interaction pattern of EGCG in terms of rotational mo-
tion within the lipid bilayers was similar to that described
for 12-O-tetradecanoylphorbol-13-acetate (TPA) [67], a
phorbol ester. Phorbol esters are prototype activators of
PKC, suggesting that direct interaction of green tea cat-
echins with cell membranes may be sufficient for the rap-
id activation of PKC by EGCG previously reported by us
[44]. The impact of EGCG on membrane fluidity may
give rise to activation of other membrane-associated sig-
naling pathways (e.g. G proteins), which can contribute
as well to its protective action. This clearly needs to be
examined.
Modulation of Other Signal Transduction Pathways
and Intracellular Transducers
In addition to PKC, other cell signaling pathways have
been also implicated in the action of green tea catechins
such as the mitogen-activated protein kinases (MAPKs)
and phosphatidylinositide 3-OH kinase (PI3K)/AKT
signaling cascades. These cascades have been shown to
play central functions in neuronal protection against a
variety of extracellular insults and to be essential for neu-
Neurosignals 2005;14:46–60 49

Fig. 1. EGCG activates p-PKC (pan) and induces a rapid activation
of PKC and translocation to the membrane. A Cell lysates from
PC12 cells deprived of serum for 24 h before short-term (15 min)
exposure to EGCG (1–10 M) were subjected to SDS-PAGE and
Western blot, with p-PKC (pan) antibody. B Cultured PC12 cells
grown with full serum (FS) support (control) were deprived of se-
rum (SF) for 24 h before short-term (15 min) exposure to EGCG
(1–10 M). The cells were fixed and permeabilized for subcellular
localization of PKC by confocal microscopy, using an isoenzyme-
specific antibody and FITC-conjugated secondary antibody (light
areas). DRAQ5 stains nuclei (dark areas). Under FS conditions
(control) PKC is evenly confined to both plasma membrane (grey
arrow) and cytosol (white arrow) (a). Upon serum withdrawal, PK-
C immunostaining is mosly cytosolic (b). In cells treated with
EGCG PKC is, in its majority (1 M), or entirely (10 M) local-
ized to the cell membrane (arrows) (c, d). The images are represen-
tative fields from 3 independent experiments, all showing the same
results. Taken from Reznichenko et al. [47].
50 Neurosignals 2005;14:46–60
ronal differentiation and survival [68–70]. OS seems to
be a major stimulus for MAPK cascade, which might lead
to cell survival/cell death [for review see 71]. Among the
MAPKs the extracellular signal-regulated kinases
(ERK1/2) are mainly activated by mitogen and growth
factors [72], while p38 and c-jun-N-terminal kinase (JNK)
respond to stress stimuli [73]. However, there have been
reports where activation of ERK1/2 is thought to mediate
neuronal injury such as in focal ischemia [74], in gluta-
mate and oxidized-low-density-lipoprotein-induced tox-
icity [40, 75] and in cytotoxicity and activation of cas-
pase-3 in the extraneuronal hepatoma HepG2 [76] and
HeLa [77] cell lines, respectively. Increasing evidence
shows that catechins can protect against neuronal cell
death caused by exogenous OS-inducing agents through
modulation of ERK activity [40, 44]. In this regard, a
number of flavonoids and phenolic antioxidants, at their
respective low protective concentrations, were demon-
strated to activate the expression of some stress-response
genes, such as phase II drug-metabolizing enzymes, glu-
tathione-s-transferase and heme-oxygenase1 [77], likely
via activation of the MAPK pathway [78].
More recently, additional signaling pathways, includ-
ing PI3K/AKT, protein kinase A (PKA) and calcium,
have been implicated in the neuroprotective action of
catechin flavonoids. These studies have been conducted
mainly in extra-neuronal tissue such as skin and heart.
For example, topical application of EGCG induces pro-
liferation of human normal epidermal keratinocytes
through stimulation of ERK1/2 and AKT [79]. Other in-
vestigators reported a rapid activation of endothelial ni-
tric oxide synthase after EGCG treatment by a process
that involves PI3K, PKA and AKT in endothelial cells
[80] and a decrease in iNOS expression via inactivation
of STAT-1alpha in epithelial and colon cell lines [81].
Consistent with this, Townsend et al. [38] have recently
reported that in cardiac myocytes EGCG protects against
ischemia/reperfusion-induced apoptosis through a mech-
anism involving reduction of STAT-1 phosphorylation
(inactivation) and of his downstream pro-apoptotic target
gene, Fas. The discrepancy or divergence in the different
signal pathway activation by EGCG may reflect differ-
ences in cell tissue (e.g. neuronal vs. peripheral), or in the
downstream pathways being under the control of the dif-
ferent kinases, which may diverge into different respons-
es, thereby providing cell function diversity.
Anti-Apoptotic Activity
EGCG has been reported to exert a biphasic mode of
action as a function of concentration. The low micromo-
Mandel/Avramovich-Tirosh/Reznichenko/
Zheng/Weinreb/Amit/Youdim
lar concentrations are responsible for the anti-apoptotic/
neuroprotective actions of EGCG. This receives further
recognition from an experiment employing customized
cDNA microarray designed to clarify the molecular
mechanisms involved in the cell survival action of EGCG
[44]. The results revealed that a low EGCG concentration
decreased the expression of pro-apoptotic genes bax, bad,
caspases-1 and 6, cyclin-dependent kinase inhibitor p21,
cell-cycle inhibitor gadd45, fas-ligand and tumor necrosis
factor-related apoptosis-inducing ligand TRAIL, in SH-
SY5Y neuronal cells. The same group has reported re-
cently that EGCG reduced the expression of several apop-
togenic factors when given after long-term serum depri-
vation of PC12 cells [47]. These findings are supported
by an in vivo study showing that two weeks oral consump-
tion of EGCG (2 mg/kg) alone caused a complete disap-
pearance of Bax immunoreactivity, specifically in the do-
paminergic neurons of the SNpc [62] and counteracted
the robust increase of Bax protein when administered be-
fore MPTP intoxication in the same area.
Bax alone, or in conjunction with the BH-3-only pro-
teins (e.g. Bad, Bid, Bim, Noxa, Puma), can trigger the
opening of the mitochondrial megachannel permeability
transition pore (mPTP), or a specific channel in the outer
mitochondrial membrane, both of which promote the fall
in mitochondrial membrane potential, leading to cyto-
chrome c release and consequent cell death [82, 83]. The
decline in Bax expression by EGCG may favor the in-
crease in the ratio of Bcl-2/Bcl-xL to Bax/Bad proteins,
thereby contributing to mitochondrial stability and regu-
lation of mPTP [84]. Protection of mitochondrial integ-
rity is of major importance, especially in the case of post-
mitotic cells such as neurons and heart muscle cells, which
are commonly not renewed. Thus, it is not surprising that
one of the major neuroprotective strategies in PD, AD
and other neurodegenerative diseases, where increased
OS, perturbed cellular energy and ion homeostasis have
been implicated, includes pharmacological agents direct-
ed to specific mitochondrial targets. In this respect,
Ginkgo biloba extract EGb 761 or its individual compo-
nents were shown to protect mitochondria integrity by
protecting against uncoupling of oxidative phosphoryla-
tion, thereby increasing ATP levels and by increasing the
expression of the mitochondrial DNA-encoded COX III
subunit of cytochrome oxidase [for review, see 85]. Fla-
vonoids may also affect mitochondrial integrity by in-
creasing GSH levels and preventing the influx of calcium,
as previously reported [86, 87].
Multifunctional Catechins for
Neuroprotection
Metal-Chelating Activity
One cardinal feature of neurodegenerative diseases in-
cluding AD, PD, Huntington’s disease, ALS, Friedreich’s
ataxia, multiple sclerosis, and aceruloplasminemia is the
appearance of excessive iron at degenerative neuronal
sites [17]. The buildup of an iron gradient in conjunction
with ROS (superoxide, hydroxyl radical and nitric oxide)
are thought to constitute a major trigger in the demise in
all theses diseases. Therefore, the chelation of free cellular
ferric and ferrous ions by the different metal chelators
make them potential agents to combat iron-induced gen-
eration of reactive oxygen radicals (by the Fenton and
Haber-Weiss reactions) and aggregation of alpha ()-
synuclein and A in PD and AD, respectively.
Iron Chelation for AD
A significant body of evidence point to an ‘amyloid
cascade’ event in the pathogenesis of AD, where amyloid
precursor protein (APP) is processed to A, by - and
-secretases, which spontaneously self-aggregate in the
presence of divalent metals (Fe2+, Cu2+) into neurotoxic
amyloid fibrils in the neocortex [88]. Iron was shown to
promote both deposition of A and OS, which is associ-
ated with the plaques [89]. In addition, iron has taken
center stage in AD as a consequence of the studies by
Rogers and coworkers [90] who described the existence
of an iron responsive element (IRE-type II) in the 5UTR
region of APP mRNA. APP is post-transcriptionally reg-
ulated by iron regulatory proteins (IRPs), which are la-
bile iron pool-sensitive cytosolic RNA proteins binding
specifically to the IRE located in the 5 or 3 untrans-
lated regions of iron metabolism-associated mRNAs.
Changes in iron status (iron overload or depletion) lead
to compensating changes in the IRP/IRE system of
translational control of iron homeostasis. For example,
the APP 5-UTR conferred translation was selectively
down-regulated upon intracellular iron chelation, in a
similar manner as the iron-storage protein ferritin,
which also possesses an IRE in its 5-UTR mRNA
[90].
Iron removal has effects suggestive of inhibition of key
enzymes or other metalloproteins, for instance in mim-
icking hypoxia, whereas many of the effects of iron over-
load may be the result of signaling associated with OS.
Hypoxia and iron chelation have similar effects on genes
regulated by the transcription factor hypoxia-inducible
factor-1 (HIF-1), a master regulator orchestrating the
coordinated induction of an array of hypoxia-sensitive
genes [91]. The target genes of HIF are especially related
to angiogenesis, cell proliferation/survival and glucose/
Neurosignals 2005;14:46–60 51
Fig. 2. Iron-induced neurodegeneration in AD via transcriptional
activation of APP mRNA. Neurodegeneration can result from ab-
normal serum iron transport to the neurons because a disruption
in the blood-brain barrier (BBB) or from release from its storage
protein ferritin, thereby increasing the free-labile iron pool (ionic
iron). Labile iron can increase the production of amyloid precursor
protein (APP) by down-regulating the activities of iron regulatory
proteins (IRP1 and IRP2, inactivation and proteasomal degrada-
tion, respectively), thereby promoting the translation of APP
mRNA from its 5-UTR type II. Ionic iron may also cause aggrega-
tion of amyloid- peptide (A) to form toxic aggregates, which, in
iron metabolism [92]. The mechanism of HIF-1 activa-
tion by iron chelation is not well understood. Fe(II)/2-
oxoglutarate-dependent dioxygenases have been identi-
fied that hydroxylate critical proline and asparagine resi-
dues in HIF and upon high oxygen levels and iron
overload, target HIF for degradation [93]. Thus, these
prolyl hydroxylase enzymes act as key iron and oxygen
sensors. This may explain the decrease in cell survival
genes described in neurodegenerative diseases such as
phosphofructokinase and the angiogenic factor VEGF,
both regulated by the HIF proteins [94]. Interestingly, the
free iron-induced proteasomal-mediated degradation of
IRP2 involves also activation a prolyl hydroxylase and is
inhibited by iron chelators [95, 96]. Thus, it is possible
that IRP2 is a substrate for this enzyme, in a similar way
52 Neurosignals 2005;14:46–60
turn, can initiate OH generation, causing oxidative stress (OS).
Increased iron and OS may activate the prolyl hydroxylase enzymes
which are key iron and oxygen sensors, leading to proteasomal-me-
diated degradation of the transcription factor hypoxia-inducible
factor 1, a master regulator orchestrating the coordinated induc-
tion of a wide array of survival genes. It has been also suggested
that IRP2 can be a substrate for prolyl hydroxylase. Neuroprotec-
tive agents that can be used to prevent iron-induced neurodegen-
eration include M30 and HLA20 (bifunctional iron chelator-MAO
inhibitors), VK-28, EGCG and curcumin (iron chelators). For a
more detailed explanation, read text.
as HIF, signaling it for protein degradation. This suggests
a convergence of both iron and OS to a common pathway
triggering the neurotoxic degenerative cascade (for a de-
tailed explanation, see fig. 2).
The involvement of metals in the plaques of AD pa-
tients and the presence of an IRE in the unstranslated
region of APP mRNA, have encouraged the development
of iron chelators as a major new therapeutic strategy for
the treatment of AD. In fact, intramuscular administra-
tion of the prototype iron chelator desferrioxamine
(DFO) slowed the clinical progression of AD dementia
[97], and some success has also been achieved with an-
other metal-complexing agent, clioquinol [98]. However,
clioquinol is highly toxic [99] and DFO has poor penetra-
tion across the blood-brain barrier [17].
Mandel/Avramovich-Tirosh/Reznichenko/
Zheng/Weinreb/Amit/Youdim

Fig. 3. Comparison of the Fe2+-chelating potency of EGCG to oth-
er iron chelators. The metal-binding capacity of EGCG was com-
pared to that of DFO and the novel iron chelators VK28, HLA20
and M30, by assessing their ability to compete with ferrozine for
the ferrous ions, resulting in decrease in the absorbance at 562 nm.
Ferrozine can quantitatively react with Fe2+ to form Fe2+-ferrozine
complex with a strong absorbance at 562 nm. In the presence of
other chelating agents, the complex formation is disrupted with the
result that the absorbance at 562 nm is decreased. 0.1 mM of drug
was mixed with 0.1 mM ferrozine in 5% ammonium acetate (pH
7) followed by the addition of 0.02 mM FeSO4. After 2 h incuba-
tion, the absorbance (at 562 nm) of resulting solutions was read.
Considering that the purpose of this assay was to evaluate the abil-
ity of drugs to compete with the iron indicator ferrozine, drugs
and ferrozine were used at equal concentrations. Chelating ef-
fect of drug on Fe2+ was calculated as follows: Chelating effect
(%) = [1–(absorbance of sample at 562 nm)/(absorbance of control
at 562 nm)]! 100. The order of chelating potency for complexing
Fe2+ in solution is Desferal (DFO) 1 M30 6 VK28 6 HLA20 1
EGCG.
A possible novel promising therapeutic approach for
treating AD, PD and ALS with non-toxic, brain perme-
able metal chelators could be the use of the naturally oc-
curring polyphenols, such as EGCG and curcumin, which
by being of natural origin may not exert toxic side effects
inherent to synthetic drugs. Both compounds have well-
characterized antioxidant, metal (iron and copper) che-
lating and anti-inflammatory activities [12, 100] and
have been demonstrated to exert neuroprotective activity
against a variety of neurotoxic insults, as well as to regu-
late APP processing and A burden in cell culture and in
Multifunctional Catechins for
Neuroprotection
vivo [12, 101]. Our recent studies have shown that pro-
longed administration of EGCG to mice induced a reduc-
tion in holoAPP levels in the hippocampus [46]. Indeed,
this effect may be related to the iron-chelating properties
of EGCG, leading to a decrease in the free-iron pool. This
in turn results in the suppression of APP mRNA transla-
tion by targeting the IRE-II sequences in the APP 5-UTR
[90], as was recently shown for DFO and the bifunctional
amyloid-binding/metal-chelating drug XH1 [102] (fig. 2).
The concept of metal chelation as a neuroprotective
strategy has led us to the development of non-toxic, lipo-
philic, brain-permeable iron chelators for progressive
neurodegenerative diseases [103]. Compounds such as
VK-28 (Varinel) [104] and the multifunctional iron che-
lators HLA20 and M30 [105, 106], which possess the
propargylamine MAO inhibitory and neuroprotective
moiety of rasagiline, display good cell permeability and
are protective against 6-OHDA toxicity in differentiated
P19 cells [107]. Comparative analysis of the Fe2+-chelat-
ing potency of EGCG, the prototype DFO and other
pharmacological iron chelators, has revealed similar
binding potency (fig. 3). Both EGCG and the iron chela-
tor M30 were shown by us to induce a significant down-
regulation of membrane-associated holoAPP level in the
mouse hippocampus (fig. 4), SH-SY5Y and CHO cells
expressing the APP ‘Swedish’ mutation (data not shown).
This may have a direct influence on A levels and plaque
formation, as shown in preliminary studies for XH1
[102]. Indeed, using a nucleation-dependent polymeriza-
tion model, it has been shown that wine and green tea
polyphenols are able to inhibit formation, extension and
destabilization of -amyloid fibrils [108].
Other potential beneficial effect of EGCG in AD may
be related to our previous studies demonstrating the abil-
ity of EGCG to promote the non-amyloidogenic pathway,
via a PKC-dependent activation of -secretase, thereby
increasing sAPP [46]. sAPP has been demonstrated to
posses potent neuroprotective activities against excitotox-
ic and oxidative insults in various cellular models [109]
and it was shown to protect against p53-mediated apop-
tosis [110]. Moreover, sAPP promotes neurite outgrowth
[111], regulates synaptogenesis [112] and exerts trophic
effects on cerebral neurons in culture. As sAPP and A
are formed by two mutually exclusive mechanisms, stim-
ulation of the secretory processing of sAPP might pre-
vent the formation of the amyloidogenic A. Thus, EGCG
may influence A levels, either via translational inhibition
of APP or by regulating APP processing. Finally, iron che-
lation by EGCG may ablate Fe3+-induced aggregation of
hyperphosphorylated tau (PHF), the major constituent
Neurosignals 2005;14:46–60 53
 A B

holoAPP ➤ holoAPP ➤

Fig. 4. Effect of EGCG and M30 on APP

processing in mice hippocampus. Repre- β-actin ➤ β-actin ➤
sentative Western blots of levels of holo-
APP in the membrane compartment, ob- 120 120
tained from hippocampus of mice treated
100 100
with EGCG (2 mg/kg) (A), or with M30

% of control

% of control
(5 mg/kg) (B) for 14 days detected with 80 80
22C11 antibody (directed to the to APP ✽✽
60 ✽✽ 60
N-terminus) or with a C-terminus APP an-
tibody, respectively. Densitometric analy- 40 40
sis is expressed as percent of the control, 20 20
untreated animals after normalizing to the
levels of -actin. Data are expressed as the 0
Control EGCG
0
Control M30
mean 8 SEM (n = 6 mice in each group). (2 mg/kg) (5 mg/kg)
** p ! 0.03 vs. control. Figure drawn using
information from Levites et al. [46].

Fig. 5. Proposed schematic model for

EGCG neuroprotective effect via regula-
tion of APP processing and A formation.
d Increased levels/activity, f decreased lev-
els/activity. For full explanation, see text.

of neurofibrillary tangles in AD brains [113] (a descrip-
tive explanation is depicted in fig. 5).
Iron Chelation for PD
Numerous studies have shown that there is a progres-
sive accumulation of iron and ferritin in the SN pars com-
pacta (pc) of PD patients [17, 27, 114]. Specifically, re-
dox-active iron has been observed in the rim of Lewy
body, the morphological hallmark of PD, also composed
of lipids, aggregated -synuclein (concentrating in its pe-
ripheral halo) and ubiquitinated, hyperphosphorylated
neurofilament proteins [115]. -Synuclein associated

54 Neurosignals 2005;14:46–60 Mandel/Avramovich-Tirosh/Reznichenko/

Zheng/Weinreb/Amit/Youdim
Fig. 6. Possible mechanism of neurotoxin-induced iron uptake,
release and interaction with -synuclein resulting in OS initiated
neurodegeneration and its prevention by iron-chelating/antioxi-
dants. The mechanism by which 6-OHDA and MPTP induce in-
crease of iron in substantia nigra pars compacta and within the
melanin-containing neurons is not known. These neurotoxins may
(a) activate the divalent metal transporter 1 (DMT1) which is re-
sponsible for iron transport into the brain across the cell membrane;
(b) alter the blood-brain barrier (BBB), thereby allowing iron access
to the brain; (c) induce release of iron from ferritin which enters
the labile (redox-active) pool of iron. It is the labile pool of iron
which can initiate the Fenton chemistry in response to the presence
of hydrogen peroxide, thus generating the highly reactive hydroxyl
radical (OH). The resultant effect is the depletion of cell-reduced
with presynaptic membrane is not toxic; however, a num-
ber of recent studies [116–118] have shown that it forms
toxic aggregates in the presence of iron and this is consid-
ered to contribute to the formation of Lewy body via OS,
being one of its constituents.
Our recent high throughput gene expression study in
the SNpc of Parkinsonian brains employing Affymetrix
chip technology [29] has revealed a significant increase
Multifunctional Catechins for
Neuroprotection
glutathione (GHS), the rate-limiting cofactor of glutathione per-
oxidase, the main enzymatic pathway in the brain, to eliminate
hydrogen peroxide. Labile pool of iron can also cause aggregation
of -synuclein to the neurotoxic form, which can also generate OH.
The net effect is oxidative-stress-dependent damage to neuron an-
tioxidant mechanism, membrane lipid peroxidation, demise of cell
and mitochondrial membrane, protein misfolding and ultimate cell
death. Neuroprotective agents that can be used to prevent iron-in-
duced neurodegeneration include M30 and HLA20 (bifunctional
iron chelator-MAO inhibitors); desferal, VK-28, R-APO (R-apo-
morphine) and EGCG (iron chelators); R-APO, EGCG, melatonin
and Vit E (vitamin E) (radical scavengers). Sharp arrows indicate
positive inputs, whereas blunt arrows are for inhibitory inputs. Re-
produced, with minor modifications, from Youdim et al. [106].
on the key iron and oxygen sensor EGLN1 gene coding
for an isoform of 2-oxoglutarate-dependent dioxygenase
hydroxylase (see previous section). Excessive production
of EGLN1 hydroxylase in the SNpc may lead to a fall in
IRP2 and subsequent decrease in transferrin receptor
(TfR) mRNA and increase in ferritin levels, both sub-
jected to positive and negative transcriptional regulation
by IRP2, respectively [119, 120]. Recent studies in knock-
Neurosignals 2005;14:46–60 55
out mice for IRP2 have revealed accumulation of iron in
the striatum with substantial bradykinesia and tremor
[121].
Consistent with the pivotal role of iron in neurodegen-
eration is the finding that the iron chelator desferal pre-
vents cytochrome c-induced--synuclein aggregation and
toxicity in vitro [122] and attenuates dopaminergic neu-
rotoxicity in response to neurotoxins MPTP and 6-OHDA
in vivo [123, 124]. In line with the iron-chelating feature
of EGCG, this polyphenol was shown to prevent -synu-
clein accumulation and to attenuate IRP2 depletion, in
the SNpc of mice intoxicated with MPTP, when given
orally for a period of 2 weeks [62]. This may be associ-
ated to our previous studies where green tea extract or
EGCG prevented DA-containing neuron degeneration
and tyrosine hydroxylase activity decrease [30]. In spite
of the absence of clinical trials regarding tea polyphenols
and PD, epidemiological studies have shown reduced risk
of PD associated with consumption of 2 cups/day or more
of tea [125] and a much lower prevalence of PD in Chi-
nese population than in white people [126, 127].
Additional studies examining the effect of iron chela-
tion, by either transgenic expression of the iron-binding
protein, ferritin, or oral administration of the metal che-
lator clioquinol, have shown significant attenuation of
MPTP-induced neurotoxicity [128]. These findings have
now been substantiated with systemic injection of the
brain-permeable iron chelator, VK-28, to rats in response
to 6-OHDA [104]. In line, nutritional iron deficiency pro-
tects rats against kainate and 6-OHDA [129]. Figure 6
summarizes the mechanism of neurotoxin-induced iron
uptake, release and interaction with -synuclein resulting
in OS initiated neurodegeneration and its prevention by
iron-chelating/antioxidant agents.
Conclusions
It is likely that syndromes such as AD and PD will re-
quire multiple- drug therapy to address the varied patho-
logical aspects of the disease. Therefore, the use of com-
pounds with poly-pharmacological activities or cocktail
of drugs is a promising therapeutic approach for the treat-
ment of neurodegenerative diseases. Indeed, a wealth of
new data indicates that green tea catechins are being rec-
ognized as multifunctional compounds for neuroprotec-
tion. They act as radical scavengers, iron chelators and
modulators of pro-survival genes, and PKC signaling
pathway. The use of EGCG as a natural, non-toxic, lipo-
philic brain permeable neuroprotective drug is advocated
for ‘ironing out iron’ from those brain areas where it pref-
erentially accumulates in neurodegenerative diseases
[106]. Thus, green tea catechins may have potential dis-
ease-modifying action. Future efforts in the understand-
ing of the protective mechanism of action of these poly-
phenols should concentrate on deciphering the cellular
targets affected by these compounds and other neuropro-
tectants.

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 Review
Neurosignals 2005;14:61–70
DOI: 10.1159/000085386
Unique Properties of Polyphenol Stilbenes
in the Brain: More than Direct Antioxidant
Actions; Gene/Protein Regulatory Activity
Sylvain Doré
Johns Hopkins University, School of Medicine, Baltimore, Md., USA
Key Words
Bilirubin  Biliverdin  Blood flow  Carbon monoxide 
Hemin  Iron
Abstract
The ‘French Paradox’ has been typically associated with
moderate consumption of wine, especially red wine. A
polyphenol 3,4’,5-trihydroxy-trans-stilbene (a member
of the non-flavonoids family), better known as resvera-
trol, has been purported to have many health benefits.
A number of these valuable properties have been attrib-
uted to its intrinsic antioxidant capabilities, although the
potential level of resveratrol in the circulation is likely not
enough to neutralize free radical scavenging. The brain
and the heart are uniquely vulnerable to hypoxic condi-
tions and oxidative stress injuries. Recently, evidence
suggests that resveratrol could act as a signaling mole-
cule within tissues and cells to modulate the expression
of genes and proteins. Stimulation of such proteins and
enzymes could explain some the intracellular antioxida-
tive properties. The modulation of genes could suffice as
an explanation of some of resveratrol’s cytoprotective
actions, as well as its influence on blood flow, cell death,
and inflammatory cascades. Resveratrol stimulation of
the expression of heme oxygenase is one example. In-
creased heme oxygenase activity has led to significant
© 2005 S. Karger AG, Basel
Fax +41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
www.karger.com www.karger.com/nsg
Received: December 6, 2004
Accepted after revision: March 1, 2005
protection against models of in vitro and in vivo oxida-
tive stress injury. Resveratrol could provide cellular re-
sistance against insults; although more work is neces-
sary before it is prescribed as a potential prophylactic in
models of either acute or chronic conditions, such as
stroke, amyotrophic lateral sclerosis, Parkinson, Alz-
heimer, and a variety of age-related vascular disorders.
Copyright © 2005 S. Karger AG, Basel
The ‘French Paradox,’ so named after a population
studied in France, is defined by a low incidence of cardio-
vascular problems despite a diet that is relatively high in
saturated fat [1]. Because of the typical levels of wine in-
take of the French, it has also been postulated that a mod-
erate consumption of red wine could be associated with
this paradox. A vast amount of recent literature proposes
that it is the polyphenols in red wine that provide the
beneficial effect, mainly attributable to their potential to
act as antioxidants. These polyphenols are divided into
two main categories: flavonoids and non-flavonoids. The
flavonoids, found in natural extracts of plants and fruits,
are considered to be the most abundant polyphenols,
whereas the non-flavonoid stilbenes are considered to be
a minor class. The naturally occurring polyphenol stil-
bene, resveratrol (3,4
,5-trihydroxy-trans-stilbene), has
been proposed to possess most of the beneficial health ef-
Sylvain Doré, PhD
Johns Hopkins University, School of Medicine
ACCM Department, Neuro Research Division, 720 Rutland Ave
Ross Research Building 364-365, Baltimore, MD 21205 (USA)
Tel. +1 410 614 4859, Fax +1 410 955 7271, E-Mail sdore@jhmi.edu
 CEREBRAL ISCHEMIA [11,12]
Reduces infarct size (rat)
Reduces focal ischemia damage (rat)
PARKINSON DISEASE [14]
Protects embryonic mesencephalic cells (rat)
SPINAL CORD LESION [8]
Protects spinal cord from
ischemia-reperfusion injury (rabbit)
HO
PAIN [20]
Decreases carrageenan-
induced hyperalgesia (rat)
BRAIN EDEMA [9]
Inhibits expression of
NFkB p65 (rat)
SEIZURE [10,18,19]
Reduces generalized tonic-clonic convulsions (rat)
Delays FeCl 3-induced epileptiform EEG changes (rat)
Reduces kainate-induced lesions in hippocampus (rat)
Fig. 1. Experimental neurologic benefits of resveratrol.
fects of red wine, considering that resveratrol is mainly
found in the skin of the grapes, and the skin and seeds are
generally not used in processing white wines. Conse-
quently, it has been proposed that resveratrol would po-
tentially be the most active ingredient [2–5]. Given all of
this, the antioxidant properties of red wine, then, must
be associated with the actions of resveratrol, and we pro-
pose that the protective properties are likely due to a
unique cascade of intracellular events leading to activa-
tion of unique antioxidant pathways. This hypothesis is
being proposed, taking into consideration that the modest
amount of resveratrol in the blood is not likely to reach a
high enough plasmatic level to neutralize free radicals.
Consequently, resveratrol is likely to stimulate an intra-
cellular signaling pathway, leading to cytoprotection.
As represented here, several genes and proteins have
been shown to be potential targets for resveratrol modu-
lation. Heme oxygenase (HO) is a possible candidate. We
have shown that resveratrol is a potent inducer of HO
protein levels, activity, and cytoprotection [6]. HO’s main
function is to cleave heme (Iron-Protoporphyrin-IX),
which then liberates iron, generating carbon monoxide
and biliverdin, which is rapidly converted to bilirubin.
62 Neurosignals 2005;14:61–70
AMYOTROPHIC LATERAL SCLEROSIS [13]
Directly stimulates BK(Ca) channel activity in
vascular endothelial cells (human cells)
AGING [22,23]
Mimics beneficial effects of
caloric restriction (yeast)
CHEMOPREVENTION [16,17]
OH Inhibits critical cell cycle regulatory proteins (mice)
Converts into an anticancer agent by
cytochrome P 450 enzymes (human cells)
OH trans -resveratrol
COGNITIVE IMPAIRMENT [21]
Prevents ICV streptozotocin-induced
cognitive impairment (rat)
BRAIN TUMORS [15]
Reduces paclitaxel-induced apoptosis in
neuroblastoma SH-SY5Y cells (human cells)
By degradation of heme, a prooxidant, into biliverdin/
bilirubin, antioxidants, modulation of HO activity and
levels would seem to be cytoprotective against free radi-
cal damage. Using in vitro and in vivo models, we have
also shown that HO can be neuroprotective [7]. In addi-
tion, HO and its metabolites have been associated with
antiapoptotic and antiinflammatory actions and are
known to have a vasodilatory effect.
Several other enzymatic systems have been suggested
to be either directly or indirectly modified by different
members of the stilbene family. Here, we propose that
protective properties of HO are the mechanisms that
provide the brain’s resistance to a variety of neurologic
stresses. Resveratrol has been shown to have a unique ef-
fect on neuronal cell death and inflammatory processes,
which are all important therapeutic targets in the devel-
opment of either acute and/or chronic neurodegenerative
diseases [8–10]. These vascular properties become espe-
cially important when considering that reduction in cere-
bral blood flow (CBF), followed by a reperfusion phase,
is likely to affect specific neurons and/or the cell types that
are especially vulnerable to free radical damage. Conse-
quently, preventing cell death is likely to have a beneficial
Doré
effect on the rate of neuroinflammation and its conse-
quences. Considering all of this together, one can make a
valid hypothesis that polyphenol stilbenes can precondi-
tion neurons against induced stress damage.
Figure 1 contains a list of potential neurologic benefits
associated with models of neurologic diseases treated
with resveratrol. Of importance is the fact that resveratrol
has been shown to be protective in several species. Fol-
lowing publication in Science and Nature of initial reports
that suggested a potential biologic role of resveratrol [24,
25], more than 900 original research articles have been
published to support the beneficial effect of resveratrol.
It is somewhat paradoxical that such a simple, natural
component, extracted from plants and fruits, can have
such a variety of actions. The flavonoids constitute the
majority of phenols in red wine and are mainly divided
into three classes: the flavanols, the flavonols, and the an-
thocyanins. The known flavonoids (hydroxycinnamic ac-
ids, benzoic acids, and stilbenes) are less abundant, and
the stilbenes are only a minor class of the known flavo-
noids.
Interestingly, during the production of red wine, com-
plex sugars from the grapes are fermented into alcohol,
and it is believed that alcohol would be considered a good
solvent for extracting polyphenols from the skins and
seeds. Considering that resveratrol is mainly found in the
skin of the grapes, it has been reported that the amount
of phenols within white wine would be much less than in
red wine. Although the reported amounts of resveratrol
in wine vary, they suggest that approximately 7 mg/l are
present in most reds, as compared to 0.5 mg/l in whites
[26, 27]. For purposes of comparison, the flavonoid con-
centrations in red wine have been in the range of 1,300–
1,500 mg/l. Once again, considering all of the beneficial
properties of resveratrol, its minimal amount available in
wine, if active, is likely to be mediated via activation of
intracellular pathways.
It is interesting to note, also, that resveratrol exists in
two isomers, the cis and the trans, as shown in figure 2.
Both of these are found in wine, and, although it appears
that only the trans isomer is found in grapes, direct light
could cause its isomerization from trans to cis. In our pre-
liminary observations in neurons, we indicated that the
active isoform would be mainly the trans resveratrol.
The fact that resveratrol, a simple active ingredient,
which has been proposed to be responsible for the ratio-
nale behind the French Paradox, is present in such small
amounts in wine or juice, and the theory concerning its
bioactivity by its direct antioxidant capacity deserve re-
consideration. Thus, it led us to hypothesize the activa-
Unique Properties of Polyphenol Stilbenes
in the Brain
OH
HO HO
light-induced
isomerization OH OH
OH
trans-resveratrol cis-resveratrol
Fig. 2. UV-light-induced isomerization of trans- to cis-resvera-
trol.
tion of an antioxidant intracellular pathway. Considering
the direct antioxidant properties associated with resvera-
trol and taking into account the absorption rate and mod-
ifications/conjugations, one would have to consume liters
of these drinks in order to achieve the necessary plasmat-
ic molar ratio to neutralize the free radicals and achieve
the beneficial health effects. To highlight the variability
of resveratrol’s effects, figure 3 shows the intracellular
consequences of cells treated with the maximal concen-
tration of 25 M, although the list is not exhaustive. We
eliminated experiments in which the concentration was
higher than 25 M. We believe that it is reasonable to
think that a level ^25 M could potentially be achieved
under normal conditions and not typically under phar-
macologic conditions.
Figure 3 shows the expression regulated by resveratrol
of different genes and proteins and describes some of the
potential functions. Living organisms under aerobic con-
ditions are continuously exposed to potential damage
caused by reactive oxygen species (ROS). ROS are pro-
duced naturally during normal cellular activity and mi-
tochondrial function. In addition, induced stress is likely
to modify these normal functions and simulate the gen-
eration of free radical damage. Many of the studies using
polyphenols have required pretreatment with resveratrol
to exert these beneficial biologic functions. Such pretreat-
ment would require either an increase in the concentra-
tion of this polyphenol or stimulate a cascade leading to
activation of an endogenous antioxidant system. This ac-
tivation is critically important to cytoprotection in tissue
with a weak, endogenous, antioxidant system. The heart
and the brain are two unique examples of tissues with
weak defenses, as evidenced by infarct damage following
ischemia/reperfusion.
Considering the antioxidant properties associated
with resveratrol, we have concentrated on the neuropro-
tective effect of resveratrol and the possibility that this
Neurosignals 2005;14:61–70 63
 Fig. 3. Example of resveratrol-regulated genes/proteins affected in cells (!25 M). HO1 = Heme oxygenase 1;
COX-2 = cyclooxygenase 2; eNOS = endothelial nitric oxide synthase; iNOS = inducible nitric oxide synthase;
ET-1 = endothelin-1; GADD45 = growth-arrest- and DNA-damage-induced protein 45; TNF = tumor necrosis
factor-alpha; ATF3 = activating transcription factor 3; IGFBP = insulin-like growth factor-binding protein.

Fig. 4. Example of potential outcomes from gene/protein regulation (i.e., HO1) and potential enzymatic role in
the brain. Note: This list of potential actions would occur at physiologic levels, while abnormal or pharmacologic
levels of these compounds could have deleterious effects.

64 Neurosignals 2005;14:61–70 Doré

 Fe 3+-Ferritin OH O
3O2 + H2O + O OH
NADPH NADP + + Ferritin
Cyt P450 Red CO Fe2+
N

N Fe 2+ N N HN
HEME OXYGENASE
N
NH HN
O RESVERATROL
HO OO
O

Heme OH O
OH O
OH Biliverdin
(Fe2+ -Protoporphyrin IX) NADPH + H+
other bilirubin
BV Red metabolites

NADP +
NH HN
ROS
Bilirubin
NH HN

OO

Fig. 5. Resveratrol mediates heme degrada-

tion by induction of heme oxygenase lev-
els.

pathway could involve the induction of an antioxidant
system, such as heme oxygenase. Regulation of HO activ-
ity has been shown to be protective in acute and/or chron-
ic neurodegenerative conditions. Figure 4 summarizes
some of the potential outcomes of regulation of the HO
protein and its activity.
As mentioned above, HO catalyzes the degradation of
heme, which is mainly a prooxidant, into iron, biliverdin/
bilirubin, and carbon monoxide (fig. 5). Two isoforms of
HO have been isolated and characterized [7, 33–35]. A
third isoform has been reported, although it does not
seem to be translated into a protein [36]. HO1, the first
to be isolated [34], is the inducible enzyme and appears
to be concentrated mainly in the liver and spleen, an un-
derstandable observation considering the high turnover
of hemoglobin, which has heme in its core. Under basal
conditions, HO1 is barely detectable in the brain, al-
though several reports suggest that, under a variety of
stimuli, it can be induced within brain tissue [37–48].
HO2 is an isoform that is constitutively expressed and
appears to be concentrated mainly in the brain and testis
[48]. Its protein expression level appears to be extremely
stable, which is likely to respond to normal cellular ho-
meostasis.
Regulation of the HO1 protein and its activity has elic-
ited a great deal of interest. Regulation of HO1 has been
suggested to be in response to many cellular and organ
stresses, in order to defend against disruption of any sys-
tem homeostasis. HO1 has been suggested to have differ-
ent functions in the brain. It is one of the heat shock pro-
teins, which are stress proteins that are induced in differ-
ent cells and following different stimuli [37], including
hypothermia [38], global ischemia [41], subarachnoid
hemorrhage [42], Parkinson disease [47], Alzheimer dis-
ease [39, 40], and several other acute and chronic neuro-
logic conditions [43–46].
In a model of transient ischemia, we and others have
shown that HO mRNA and proteins are induced [43].
Reports have indicated that induction of HO proteins in
neurons would be protective [49], and our preliminary
data indicate that resveratrol could induce HO levels
within neurons, potentially affording neuroprotection.
Consequently, we believe that modulation of HO levels
and its activity could be a pathway by which resveratrol
present in red wine or other concentrated extracts could
potentially protect the nervous system against induced
oxidative stress damage. Many heme-containing enzymes
are located in the mitochondria, the cytosol, and the en-
doplasmic reticulum; and they presumably undergo rap-
id turnover during oxidative stress. HO is the main en-
zyme, which can degrade free heme, a prooxidant, and
maintain levels that would not reach toxicity. Following
an ischemic event, tissue injury has been proposed to be
mainly due to increased oxidative stress by free radicals

Unique Properties of Polyphenol Stilbenes Neurosignals 2005;14:61–70 65

in the Brain
generated during the reperfusion phase. We have previ-
ously shown that the infarct volume in HO2 knockout
(HO2–/–) mice versus wild-type (WT) mice after stroke is
approximately double. HO2 is the isoform present under
normal basal conditions. Consequently, increasing the
activity of heme oxygenase is likely to be protective in
stroke.
Interestingly, the group led by Maines has demonstrat-
ed significant reduction in infarct size after stroke in a
transgenic mouse model on which HO1 was over-ex-
pressed using a neuron-specific promoter [50]. In addi-
tion, we have recently accumulated evidence suggesting
that pre-treatment with resveratrol would be a most po-
tent inducer of HO1 in mouse primary neuronal cultures,
that it would be sufficient to prevent induced neurotoxic-
ity, and that this neuroprotection was significantly atten-
uated by the use of a heme oxygenase inhibitor [51]. All
together, these results suggest that an increase in HO1
protein levels and its activities within neurons is likely to
provide neuroprotection and promote cell survival.
Heme
Heme metabolism is a crucial metabolic process. It has
been postulated that free heme can rapidly be generated
from an induced turnover of heme-containing proteins/
enzymes, for example, catalase, glutathione peroxidase,
superoxide dismutase, cytochrome, guanylate cyclase, ni-
tric oxide synthase, etc. It has been further suggested that
during hypoxia, ischemic injury could trigger significant
amounts of heme being released into the intracellular
pool. When stimulation of these hemoproteins is degrad-
ed, heme becomes free; probably not salvaged, it should
be rapidly degraded. HO is the enzyme that can rapidly
cleave prooxidant through heme and limit its capacity to
enter into generation of a free radical cycle – notably,
through its iron molecule. Consequently, HO could be
considered an antioxidant enzyme by degradation of the
prooxidant heme. Our previous observations indicate
that resveratrol could specifically induce HO1 within
neurons and potentially protect cells against oxidative
stress injury. Such a pathway is an interesting target for
resveratrol, considering that it can regulate the redox state
of the cell and prevent cells from dying through an oxida-
tive stress-induced cascade.
Iron
Degradation of heme by HO1 also generates a mole-
cule of iron. Evidence suggests that regulation of HO1
protein levels could modulate the level of intracellular
heme. It is postulated that HO can stimulate the efflux of
66 Neurosignals 2005;14:61–70
iron outside the cell [52]. Homeostasis of iron is a key
factor in controlling cell toxicity. As one example, free
iron has been considered to be a key ingredient in the
Fenton reaction, in which by reacting with H2O2, it gen-
erates free radicals. Regulation of cellular homeostasis of
iron is a complex and tightly regulated system. It is regu-
lated by many proteins, a number of which are still under
extensive characterization. Rapid upregulation of HO1
by resveratrol in neurons could potentially directly affect
the intracellular iron level.
It has been previously demonstrated that decrease in
HO1 activity would be sufficient to change its iron level
within the cell. For example, by using HO–/– mice, it has
been shown that iron accumulates in several organs [53].
In addition, numerous iron-binding proteins are regulat-
ed by intracellular levels of free iron [54]. As an example,
ferritin in the cell can sequester numerous molecules of
iron, and its intracellular level is tightly regulated to free
iron.
Therapeutic implications of controlling iron levels
within tissues or within cells are numerous. As an exam-
ple, administration of desferoxamine, a trivalent iron
chelator, over a 2-year period slows the clinical progres-
sion of symptoms associated with Alzheimer disease [55].
Further study may bear out that the regulation of HO1
levels also regulates the cellular iron homeostasis. Inter-
estingly, very little has been investigated regarding the
potential role of resveratrol in regulating iron and deter-
mining its potential effect in neurologic disorders.
Carbon Monoxide
Carbon monoxide (CO) is a gas that is almost exclu-
sively generated in cells by degradation of heme by heme
oxygenase [56, 57]. In that it is a gas, carbon monoxide
can travel freely through intracellular and extracellular
compartments. The CO literature is vast and complex
with many controversies that have yet to be resolved [58–
63]. CO is better known to be toxic at high levels. It can
also cause death [64]. Interestingly, in the recent litera-
ture, low concentrations of CO have been suggested to be
protective. Although CO has an affinity slightly lower to
its homologue, nitric oxide (NO), which is another gas, it
appears that its half-life would be significantly longer.
Such an observation could allow carbon monoxide, by
binding to heme moiety present on several key enzymes,
to modulate their function [65]. Within the cell, physio-
logic/normal levels of CO generated from degradation of
intracellular heme are likely to have multiple biologic
functions on several heme-containing proteins. For ex-
ample, CO has been shown to act as a vasodilator by po-
Doré
tentially binding with soluble guanylate cyclase (sGC)
and modulating its vasoactive activities. It can act on cal-
Antioxidant properties
cium-activated potassium channels (KCa channels) and Most neurologic disorders [6]
regulate their opening [66, 67]. CO has also been recent-
Inflammation reduction
ly reported to have specific antiapoptotic and antiinflam- Most neurologic disorders [77,78]
matory actions [68, 69]. Rapid induction of HO1 could
Restoration of normal blood flow
be a means by which resveratrol can increase CO levels Cerebral Ischemia [79]
within physiologic concentrations and allow cells and tis- AD (age-related vascular dementia) [7]

sue to benefit from many of CO’s biologic actions, espe- Reduction of neuronal cell death
cially in scenarios in which blood flow is reduced and cell In stroke models [43]
In induced apoptosis [80]
survival is compromised. Cholinergic neuron in AD models [81]
Dopaminergic neurons in PD models [47]
Bilirubin Regulation of iron homeostasis
Bilirubin is known for its toxicity, at high micromolar Parkinsonism [82]
Ataxia [83]
concentrations, in the central nervous system (CNS), es- Movement disorders, tremors [84]
pecially in neonates. Although under physiologic concen- Restless Leg Syndrome [85]
trations, bilirubin can act as an endogenous antioxidant Hallervorden-Spatz Syndrome [86]
Aceruloplasminemia [87]
[70, 71]. In testing a series of antioxidants, bilirubin was Neuroferritinopathy [88]
shown to have significant superoxide and hydroxyl radi-
Others
cal scavenger activity [72]. Using an animal model of hy- Sleep pattern [89]
perbilirubinemia, a protective effect against cerebral isch- Circadian rhythm [90,91]
emia was demonstrated. We have also observed, using Amyotrophic lateral sclerosis [92]
primary hippocampal and cortical neuronal cultures, that Spinal cord lesion [93]
Head trauma [94]
bilirubin can be protective at low concentrations [73, 74].
Brain edema [50]
Moreover, in previous observations on the potential role
Huntington [95]
of HO in Alzheimer disease pathology [39, 75], levels of Kernicterus [94]
bilirubin derivatives in the cerebral spinal fluid were re- Brain tumors [97]
ported to be significantly increased in brains of AD pa- Chemoprevention [98]

tients as compared to control [76]. An increase in HO1 Pain [99]

within AD brains was reported. Increased HO1 levels
could potentially increase the bilirubin level within a
physiologic range, and such a pathway could explain
some of the antioxidant properties associated with resve-
ratrol in relation to neurologic deficits in age-related de-
mentia.
Figure 6 briefly summarizes some of the neurologic
disorders that can be beneficially affected by the regula-
tion of HO activity. Regulation of genes by resveratrol,
such as in the case of HO1, presents a potential mecha-
nism by which its prophylactic use might be considered,
under either acute or chronic neurologic disorders. For
example, under ischemic stroke conditions, reperfusion
frequently occurs after focal ischemia, particularly in the
case of cerebral embolism and transient ischemic attack.
These can be warning signs of impending stroke. Recur-
rence is a prevalent phenomenon in patients who have
suffered one episode of stroke. Therefore, availability of
a prophylactic approach in these patients is an important
goal of preventive medicine. Reports that coronary heart
disease appears to be reduced in patients who chroni-
Seizure [100]
Atherosclerosis [101]
Age-related cognitive impairment [102]
Fig. 6. Non-exhaustive list of potential neurologic benefits of res-
veratrol-regulating HO1.
cally consume moderate amounts of red wine support the
prospect of a prophylactic role for resveratrol. Conse-
quently, a better understanding of the mechanisms by
which polyphenols and red wine can protect against isch-
emic and toxic insults would be of great interest.
Acknowledgements
This work is supported in part by the NIH grants (AA014911
and AT002113), the Wine Institute, and the ABMR Foundation.
The author would like to thank Tzipora Sofare, MA, for her assis-
tance in preparing the manuscript.

Unique Properties of Polyphenol Stilbenes Neurosignals 2005;14:61–70 67

in the Brain
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 Review

Received: September 23, 2004

Neurosignals 2005;14:71–82
Accepted after revision: November 8, 2004
DOI: 10.1159/000085387

Neuroprotective Effects of Huperzine A

A Natural Cholinesterase Inhibitor for the Treatment of Alzheimer’s Disease

Rui Wang Xi Can Tang

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Zhangjiang Hi-Tech Park, Shanghai, China

Key Words chondria, and interfere with APP metabolism. Antagoniz-

Huperzine A W Alzheimer’s disease W
Acetylcholinesterase W Cholinesterase inhibitor W
Cognitive enhancer W Neuroprotection W Oxidative stress W
Beta-amyloid W Amyloid precursor protein W Cerebral
ischemia W Apoptosis W Apoptotic-related gene W
Mitochondria W Glutamate W NMDA receptor W Potassium
current
ing effects on NMDA receptors and potassium currents
may contribute to the neuroprotection as well. It is also
possible that the non-catalytic function of AChE is in-
volved in neuroprotective effects of HupA. The thera-
peutic effects of HupA on AD or VD are probably exerted
via a multi-target mechanism.
Copyright © 2005 S. Karger AG, Basel

Abstract Alzheimer’s disease (AD) is a progressive, neurodegen-

Huperzine A (HupA), isolated from Chinese herb Huper-
zia serrata, is a potent, highly specific and reversible
inhibitor of acetylcholinesterase. It has been found to
reverse or attenuate cognitive deficits in a broad range of
animal models. Clinical trials in China have demon-
strated that HupA significantly relieves memory deficits
in aged subjects, patients with benign senescent forget-
fulness, Alzheimer’s disease (AD) and vascular dementia
(VD), with minimal peripheral cholinergic side effects
compared with other AChEIs in use. HupA possesses the
ability to protect cells against hydrogen peroxide, ß-amy-
loid protein (or peptide), glutamate, ischemia and stau-
rosporine-induced cytotoxicity and apoptosis. These
protective effects are related to its ability to attenuate
oxidative stress, regulate the expression of apoptotic
proteins Bcl-2, Bax, P53 and caspase-3, protect mito-
erative disorder associated with a global impairment of
higher mental function, and presenting an impairment of
memory as the cardinal symptom [1]. Histopathological
hallmarks of the disease are the extracellular deposition of
amyloid ß-peptide (Aß) in senile plaques, the appearance
of intracellular neurofibrillary tangles (NFT), a loss of
cholinergic neurons, and extensive synaptic changes in
the cerebral cortex, hippocampus and other areas of brain
essential for cognitive functions.
To date, the cause and the mechanism by which neu-
rons die in AD remain unclear, but Aß has been estab-
lished as a crucial factor in AD pathogenesis. Aß deposi-
tion may cause neuronal death via a number of possible
mechanisms, including oxidative stress, excitotoxicity,
energy depletion, inflammation and apoptosis. Despite
this multifactorial etiology, genetics plays a key role in

© 2005 S. Karger AG, Basel Prof. Xi Can Tang, State Key Laboratory of Drug Research
ABC 1424–862X/05/0142–0071$22.00/0 Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences
Fax + 41 61 306 12 34 Chinese Academy of Sciences, 555 Zu Chong Zhi Road
E-Mail karger@karger.ch Accessible online at: Zhangjiang Hi-Tech Park, Shanghai 201203 (China)
www.karger.com www.karger.com/nsg Tel. +86 21 5080 6710, Fax +86 21 5080 7088, E-Mail xctang@mail.shcnc.ac.cn
disease progression. However, environmental factors (e.g.
cytokines, neurotoxins) may be even more important in
the development and progression of AD. Several lines of
evidence support the involvement of oxidative stress [2,
40]. Oxidative damage, mediated by reactive oxygen spe-
cies (ROS) generated following cell lysis, oxidative bursts,
or an excess of free transition metals, has been hypothe-
sized to play a pivotal role in AD neurodegeneration. On
the other hand, postmortem studies provide direct mor-
phological and biochemical evidence that some neurons
in the AD brain degenerate via an apoptotic mechanism
[30, 51], which may or may not be linked to ROS. The
biological mechanism underlying the formation of AD is
clearly complex, with many factors contributing to the
neuropathology. Thus it is not surprising that a number of
different intervention therapies are currently being re-
searched to address distinct aspects of the disease.
Huperzine A (HupA) is a novel Lycopodium alkaloid
with recognized medicinal properties. In China the folk
medicine Huperzia serrata (Qian Ceng Ta) (fig. 1), a
source of HupA, has been used for centuries in the treat-
ment of contusions, strains, swelling, schizophrenia, etc.
The active principle, HupA, is a potent, reversible, and
selective inhibitor of acetylcholinesterase (AChE) [56]. Its
potency in AChE inhibition is similar or superior to that
of physostigmine, galanthamine, donepezil and tacrine
[60, 67]. AChE exists in multiple molecular forms that can
be distinguished by their subunit associations and hydro-
dynamic properties [6, 41]. In mammalian brain, the bulk
of AChE occurs as a tetrameric, G4 form (10S) together
with much smaller amounts of a monomeric, G1 (4S)
form [4, 22]. This phenomenon led us to investigate the
possibility of HupA on differential inhibition of AChE
forms [93]. We observed that HupA preferentially inhibit-
ed tetrameric AChE (G4 form), while tacrine and rivastig-
mine preferentially inhibited monomeric AChE (G1
form). Donepezil showed pronounced selectivity for G1
AChE in striatum and hippocampus, but not in cortex.
Physostigmine showed no form-selectivity in any brain
region [93].
HupA has been found to reverse or attenuate cognitive
deficits in a broad range of animal models. Enhancement
of learning and memory performance was documented in
the passive footshock avoidance paradigm [37, 54, 98,
99], the classic water maze escape task [36, 79], and spa-
tial discrimination in the radial arm maze [72]. Similarly,
cognition enhancement was found in a delayed-response
task in aged monkeys [78] and in reserpine- or yohimbine-
treated monkeys [44]. Beneficial effects were seen not
only in intact adult rodents, but also in rodents cognitive-
72 Neurosignals 2005;14:71–82
ly impaired by age [37,79] or by treatment with scopol-
amine [16, 23, 36, 54, 59, 65, 98], AF64A [73, 9], electro-
shock [59, 99], cycloheximide [99], NaNO2 [99], or CO2
[37, 98]. In addition, HupA improved cognition in cholin-
ergically lesioned rats [37, 99], and it reduced the spatial
working memory deficit induced by lesion of the nucleus
basalis magnocellularis [74]. These effects are attributable
to increased synaptic ACh. Early on, HupA was found to
cause a significant increase in ACh level in rat brain [55,
100]. More recently, a rise in cortical ACh levels has been
demonstrated by microdialysis in awake, free-moving
rats. In this study HupA was 8- and 2-fold more potent
than donepezil and rivastigmine, respectively, and its
effect was longer lasting [34].
Clinical trials of HupA in China have demonstrated a
meaningful improvement in the memory of aged subjects,
individuals with benign senescent forgetfulness and pa-
tients with AD. The results indicate minimal peripheral
cholinergic side effects compared to other AChEIs in use;
even more important, HupA lacks the dose-limiting hepa-
totoxicity induced by tacrine [75–77, 84, 90]. Adverse
effects in the clinical trials were reported at a very low rate
and are mainly cholinergic. Examples are dizziness, nau-
sea, gastroenteric symptoms, headaches and depressed
heart rate. Several clinical trials have addressed the effects
of HupA on vascular dementia (VD). VD is currently con-
sidered to be the second most common form of dementia
in Europe and the USA. However, in Asia and many
developing countries including China, the incidence of
VD exceeds that of AD. Multicenter, randomized, dou-
ble-blind, placebo-controlled clinical trials in China
proved that HupA markedly improved the cognitive func-
tion of VD [32, 38, 39, 90]. This article will summarize
and discuss current research focused on elucidating the
neuroprotective effect of HupA.
Protection of HupA against Hydrogen Peroxide
and ß-Amyloid Protein-Induced Injury by
Attenuating Oxidative Stress
Several neurodegenerative disorders such as AD, cere-
bral ischemia-reperfusion and head injury are thought to
be related to changes in oxidative metabolism. Increased
oxidative stress, resulting from free radical damage to cel-
lular function, can be involved in the events leading to
AD, and is also connected with lesions called tangles and
plaques. Plaques are caused by the deposition of Aß and
are observed in the brains of AD patients [7, 40, 45, 48].
Studies show that oxygen radicals initiate amyloid build-
Wang/Tang
 1

Fig. 1. Chinese herb Huperzia serrata (Qian Ceng Ta).

Fig. 5. Anti-apoptotic mechanism of HupA. Black real line arrows = apoptotic-inducing pathway; red real line ar-
rows = affecting sites of HupA; red dash arrows = speculative pathways; (–) = inhibiting; (+) = promoting.

Hup A: A Natural Cholinesterase Inhibitor Neurosignals 2005;14:71–82 73

for Alzheimer’s Disease

Ab 25-35
M Con
HupA
6 12 24 24 (h)
Fig. 2. Reduction of Aß25–35-induced DNA fragmentation by
HupA. Neurons were treated with 20 ÌM of Aß25–35 with or with-
out 1 ÌM of HupA. Fragmented DNA was isolated by NucleoBond
DNA and RNA purification kit, electrophoresed with agarose gel,
and finally stained with ethidium bromide. M = DNA size marker;
Con = control.
up leading to neurodegeneration [19]. HupA has been
found to protect against H2O2 and Aß-induced cell lesion,
to decrease the level of lipid peroxidation, and to increase
antioxidant enzyme activities in rat PC12 and primary
cultured cortical neurons [68–70]. Following a 6-hour
exposure of the cells to H2O2 (200 ÌM) or a 48-hour expo-
sure of the cells to Aß25–35 (1 ÌM ), a marked reduction
in cell survival and the activities of glutathione peroxi-
dase (GSH-Px) and catalase (CAT) was observed, along
with an increased production of malondialdehyde
(MDA). Pretreatment of the cells with HupA (0.1–10 ÌM)
2 h before H2O2 and Aß exposure caused a significant
increase in cell survival. HupA also partly reversed the
H2O2- and Aß-induced decrease of GSH-Px and CAT
activity, as well as the increase in production of MDA and
SOD. All these effects indicate a neuroprotective action.
The protective effect of HupA on Aß-induced cell
lesion was also observed in NG108–15 cells [86] and pri-
mary cultured cortical neurons [71]. In addition to elevat-
ing cell survival and GSH-Px and CAT activities while
decreasing the level of MDA [70], HupA (0.1–10 ÌM) sig-
nificantly reduced Aß25–35-induced the formation of
reactive oxygen species (ROS) in rat primary cultured cor-
tical neurons [71].
74 Neurosignals 2005;14:71–82
In rat studies, an intracerebroventricular (i.c.v.) infu-
sion of ß-amyloid 1–40 (800 pmol !3) induced a signifi-
cant cognitive deficit, morphologic signs of injury and a
decrease of cortical choline acetyltransferase activity [64].
Daily i.p. administration of HupA for 12 consecutive days
produced partial reversal of the ß-amyloid-induced deficit
in learning a water maze task. This treatment ameliorated
the loss of choline acetyltransferase activity in cerebral
cortex and the neuronal degeneration induced by ß-amy-
loid 1–40 [64]. HupA also reduced the level of lipid perox-
idation and superoxide dismutase in the hippocampus,
cerebral cortex and serum of aged rats [49]. In a rat model
of chronic cerebral hypo-perfusion, HupA significantly
reduced the increases in SOD and lipid peroxide while
restoring lactate and glucose to their normal levels [61]. A
clinical study also demonstrated a reduction of oxygen
free radicals in plasma and erythrocytes from AD patients
[77]. These findings indicate that HupA has protective
effects against free radical and Aß-induced cell toxicity,
which might be beneficial in the treatment of patients
with various kinds of dementia.
Experience with HupA enantiomers has shown that
the neuroprotective properties have no relation to anti-
cholinesterase potency. Thus, preincubation with (+)-
HupA or (–)-HupA (0.1–10 ÌM ) protected cells with simi-
lar potency against Aß toxicity and similar enhancement
of survival [86]. This result contrasted with the stereosel-
ectivity of cholinesterase inhibition in vitro and in vivo,
in which (–)-HupA is approximately 50-fold more potent
than (+)-HupA. In another study, we examined drug
effects on the apoptosis induced by incubation with
Aß25–35 and on the increase of AChE activity accompa-
nying this reaction. We observed that inhibiting the
hydrolyzing activity of AChE without decreasing AChE
expression itself did not attenuate the Aß25–35 induced
apoptosis [85]. In other words, the ability of HupA to
block the catalytic activity of AChE did not parallel its
neuroprotective effect. Therefore, the cytoprotective ef-
fect of HupA enantiomers may relate to some kind of non-
catalytic actions on AChE, or to actions on other cellular
targets.
Anti-Apoptotic Effect of HupA
Apoptosis is the process by which neurons die during
normal development and is also a feature of chronic and
acute neurodegenerative diseases and stroke [82]. The cel-
lular commitment to apoptosis is regulated by the Bcl-2
family of proteins. High levels of Bcl-2 expression will
Wang/Tang
Fig. 3. Effects of HupA on H2O2-induced
expression of bcl-2, bax and p53 in PC12 b-Actin
cells by RT-PCR. Cells were exposed to
100 ÌM H2O2, and total RNA was extracted
after the indicated recovery period and then Bcl-2
subjected to RT-PCR. The PCR products
were normalized by ß-actin mRNA. Lane 1: Bax
non-treated intact control; lanes 2–5: 0, 2, 6
and 12 h after 30 min H2O2-treatment, re-
spectively. Lanes 6–9 represent the same P53
time points as lanes 2–5 but preincubated
with 1 ÌM HupA before H2O2 exposure.
inhibit apoptosis. In contrast, an increased expression of
P53 and Bax is associated with the initiation of apoptosis
[29]. In accordance with previous reports, studies from
our lab demonstrate typical apoptotic changes when neu-
ron-like cells are exposed to stressors such as H2O2, Aß
peptide, oxygen-glucose deprivation (OGD), serum depri-
vation, or the PKC inhibitor, staurosporine. These
changes include DNA laddering, cell shrinkage, the gener-
ation of nuclear apoptotic bodies, TUNEL positive stain-
ing, and other classic hallmarks of apoptosis (fig. 2) [63,
71, 87, 94]. Such abnormalities are markedly relieved by
HupA. For example, in rats that received i.c.v. injections
of ß-amyloid1–40 (800 pmol ! 3), administration of
HupA (0.1, 0.2 mg/kg, i.p.) for 12 consecutive days gave
substantial neuroprotection in the brain. This treatment
greatly reduced the number of apoptotic-like neurons and
partly reversed the down-regulation of Bcl-2 and up-regu-
lation of Bax and P53. Anti-apoptotic effects of HupA
were also found in primary cultured neurons. Preincuba-
tion with HupA at concentrations higher than 0.01 ÌM
led to a large, dose-dependent attenuation of cell toxicity
induced by Aß25–35 (20 ÌM). Moreover, HupA (1 ÌM)
caused large reductions in the amounts of subdiploid
DNA detected in a flow cytometry assay and weakened
the ladder pattern on agarose gel electrophoresis, typically
seen after exposure to Aß (fig. 2).
The anti-apoptotic actions of HupA may involve inhi-
bition of the production or the effects of ROS [71]. We
found that preincubation of PC12 cells with HupA before
exposure to H2O2 substantially reduced apoptosis, by any
of several measures. The same treatment also attenuated
H2O2-induced overexpression of bax and p53, while res-
toring bcl-2 to normal levels (fig. 3) [63]. HupA was also
effective in the OGD paradigm. Exposure to OGD for 3 h
followed by reoxygenation for 24 h triggers apoptosis
Hup A: A Natural Cholinesterase Inhibitor
for Alzheimer’s Disease
1 2 3 4 5 6 7 8 9
characterized by chromatin condensation, nucleus frag-
mentation and DNA laddering, accompanied by altered
levels of mRNA for c-jun, p53, bcl-2 and bax. In this mod-
el, HupA significantly attenuated apoptosis and reduced
the up-regulation of c-jun and bax as well as the down-
regulation of bcl-2 [94].
In the mitochondrial-mediated cell death pathway, a
key step is transient opening of the mitochondrial perme-
ability transition (MPT), involving a non-specific in-
crease in the permeability of the inner mitochondrial
membrane [25, 28, 53]. In this process, cytochrome c
moves from the intermembrane space into the cytoplasm
[5] where it binds to another factor (Apaf-1). In the pres-
ence of dATP, this complex polymerizes into an oligomer
known as the apoptosome. The apoptosome activates the
protease, caspase-9, which in turn activates caspase-3.
The cascade of proteolytic reactions also activates
DNAases, which leads to cell death [83].
When PC12 cells were pre-incubated with HupA at
concentrations above 0.01 ÌM, there was a marked neuro-
protection against apoptosis induced by ß-amyloid, with a
significant reduction in mitochondrial swelling and an
improvement in mitochondrial membrane potentials [un-
publ. data of this lab]. Pretreatment of rat cortical neu-
rons with HupA (0.01–10 ÌM) significantly elevated cell
survival and reduced all signs of apoptosis resulting from
exposure to Aß25–35.
Further studies focused on caspase activation in pri-
mary cultures of rat cortical neurons subjected to a variety
of stresses. Measurements of caspase-3-like fluorogenic
cleavage demonstrated that HupA (1 ÌM) attenuated an
Aß25–35-induced increase in caspase-3 activity at 6, 12,
24, and 48 h [71]. Western blot analyses confirmed these
results at the protein level. HupA also inhibited caspase-3
activation in models of apoptosis by serum deprivation
Neurosignals 2005;14:71–82 75
 a b
80
Caspase-3 activity (U/mg protein)
60
40
20
0 fraction
Control
Fig. 4. Effect of HupA on caspase-3 activity (a) and Western blot
detection of cytochrome c and COX4 (b) in primary cortical neu-
rons. Neurons under serum deprivation for 24 h. HupA at a concen-
tration of 1 ÌM added to the culture 2 h in advance. a Data expressed
as means B SD. Statistical comparison was made using ANOVA fol-
lowed by Duncan’s test. There was a significant difference between
the serum deprivation group and the untreated control group. ## p !
0.01 compared to control group. * p ! 0.05 compared to serum depri-
and staurosporine treatment. The apoptosis induced by
24 h of serum deprivation was accompanied by enhanced
caspase-3 activity and a release of mitochondrial cyto-
chrome c into the cytosol [97]. HupA (0.1–10 ÌM) im-
proved neuronal survival in this model, inhibiting the rise
in caspase-3 activity and protein expression [97]. Like-
wise, cell survival was greatly enhanced when HupA (0.1–
100 ÌM) was introduced 2 h before a 24-hour exposure to
0.5 ÌM staurosporine. Incubation with HupA at dose of
1 ÌM also reduced staurosporine-induced DNA fragmen-
tation, up-regulation of the pro-apoptotic gene, bax, down-
regulation of the anti-apoptotic gene, bcl-2, and decrease
in caspase-3 proenzyme protein level (fig. 4) [87].
A potassium channel with delayed rectifier characteris-
tics may play an important role in Aß-mediated toxicity.
The up-regulation of an outward K+ current known as Ik
mediates several forms of neuronal apoptosis and could
contribute to the pathogenesis of Aß-induced neuronal
death. Exposure to a 20-ÌM concentration of Aß25–35 or
Aß1–42 is known to enhance the apoptosis-related cur-
rent, Ik [81]. Interestingly, HupA will reversibly inhibit
the fast transient current, IA, and the sustained potassium
current, Ik, in CA1 pyramidal neurons acutely dissociated
from rat hippocampus [33]. Such effects might contribute
to this agent’s anti-apoptotic effect.
76 Neurosignals 2005;14:71–82
Serum deprivation for 24 h
✽✽ Control Huperzine A Vehicle
COX4
Mitochondrial
fraction
✽
Cytosolic
fraction
Cytochrome c
Mitochondrial
fraction
Cytosolic
Vehicle Huperzine A
vation group. b The mitochondrial and cytosolic fractions were iso-
lated using an ApoAlert cell fractionation kit. They were then pro-
cessed using the standard Western blot procedure on 12% SDS-
PAGE and probe with COX4 antibody (F17 kDa) or cytochrome c
antibody (F15 kDa). The presence of cytochrome c in the cytosolic
fraction after induction indicates that apoptosis involves mitochon-
drial release of cytochrome c to the cytosol.
In light of these findings and the effects of HupA on
apoptosis-related genes, we propose that HupA blocks
apoptosis by antagonizing the mitochondrial-dependent
caspase pathway, directly or indirectly (fig. 5). The effects
of HupA on the intrinsic caspase-3 pathway might be
downstream consequences of altered expression of bcl-2
family genes. Functionally, bcl-2 is a potent cell death
suppressor, whose over-expression can prevent cell death
in response to a variety of stimuli. It is well known that
Bcl-2 suppresses apoptosis by inhibiting cytochrome c
release from the mitochondria. On the other hand, bax is
a death-promoting factor, whose translocation to the mi-
tochondrial membrane leads to a loss of mitochondrial
membrane potential and increases mitochondrial perme-
ability. Increased mitochondrial permeability results in
the release of cytochrome c followed by activation of cas-
pase-3 [21]. We consider it likely that HupA owes some of
its anti-apoptotic effects to an effective antagonism of the
up-regulation of bax and the down-regulation of bcl-2,
which impairs mitochondria-dependent caspase pathway.
At present, however, direct effects of HupA on cyto-
chrome c and caspase-3 and other possible targets are not
excluded.
Wang/Tang
 Effects of HupA on Secretory Amyloid
Precursor Protein and Protein Kinase C-·
Aß is a self-aggregating 39–43-amino acid peptide that
originates from a larger polypeptide termed Alzheimer’s
amyloid precursor protein (APP). Alternate pathways for
APP processing have been described: the non-amyloido-
genic secretory pathway, which releases a soluble ectodo-
main (APPs) and prevents Aß formation [15], and the
endosomal-lysosomal pathway, which produces amyloi-
dogenic products [24]. The amyloid hypothesis of AD [17,
46] is focused on the potential toxic role of an excessive
production of Aß and suggests that the aberrant metabo-
lism of APP is a central pathogenetic mechanism for the
disease.
Several factors can affect the secretory non-amyloido-
genic pathway of APP. For example, the stimulation of
phospholipase C (PLC)-coupled receptors, such as musca-
rinic m1 and m3, has been shown to potentiate the secre-
tion of APP in cell cultures. These effects are probably
mediated mainly by protein kinase C (PKC) [43]. It has
also been reported that several anticholinesterases affect
APP processing in addition to the catalytic function of
AChE [18, 42].
Our own studies showed that HupA could alter APP
processing in the brains of rats given i.c.v. infusions of
Aß1–40, and in otherwise untreated human embryonic
kidney 293 (HEK293sw) cells [88]. In the Aß treated rats,
levels of APPs and PKC· were significantly decreased by
treatment with Aß1–40. These decreases were much re-
duced by 12 consecutive days of HupA treatment (0.2 mg/
kg, i.p.), but HupA in normal rats caused no change in
either APPs or PKC·. In normal HEK293sw cells, on the
other hand, the levels of APPs and PKC· rose progres-
sively during an 18-hour exposure to HupA (1 ÌM). How-
ever, no significant alternations in the levels of PKC‰ and
PKCÂ were found after HupA treatment. Taken together
these findings suggest that HupA may affect the process-
ing of APP by up-regulating PKC, especially PKC·.
In an attempt to clarify the receptor mechanisms
involved in such effects, we treated HEK293wt cells with
cholinergic receptor antagonists [unpubl. data]. The non-
selective muscarinic antagonist, scopolamine, partly
blocked the HupA-induced rise in levels of APPs and
PKC·. By contrast the nicotinic antagonist, mecamyl-
amine, had little effect. These results suggest that musca-
rinic ACh receptors may mediate, at least in part, the
effects of HupA on the regulation of APPs and PKC· in
HEK293sw cells.
Hup A: A Natural Cholinesterase Inhibitor
for Alzheimer’s Disease
Our recent results provide the first demonstration that
HupA can reduce the disturbance of PKC and APPs both
in rats and in an isolated cell line. The effect of HupA to
enhance non-amyloidogenic processing of APP and ele-
vate APPs levels likely depends on the activation of mus-
carinic receptors and the PLC/PKC cascade. A number of
biological activities such as cell proliferation, promotion
of cell-substratum adhesion, neurite outgrowth and the
prevention of intracellular calcium accumulation and cell
death have been attributed to APPs [47]. Since PKC is a
key enzyme in signal transduction, and since APPs itself
has neuroprotective effects, modulating the levels of these
two proteins by HupA may well be beneficial in AD thera-
py (fig. 6).
Protection of HupA against Hypoxic-Ischemic
and Glutamate Induced Brain Injury and
Cytotoxicity
Apart from AD, the most common dementia in the
elderly is VD. This disorder, like AD, presents a clinical
syndrome of intellectual decline produced by ischemia,
hypoxia, or hemorrhagic brain lesion. Cerebral ischemia
in rats with permanent bilateral ligation of the common
carotid arteries (CCA) provides a useful model of VD, in
which to investigate the effects of HupA. These animals
experience a significant reduction of cerebral blood flow
and exhibit learning and memory impairments and neu-
ronal damage resembling those in VD. Daily oral admin-
istration of HupA (0.1 mg/kg) to such rats for 14 days pro-
duced significant improvement in the learning of a water
maze task. Simultaneously there was marked recovery
from the decrease in choline acetyltransferase activity in
hippocampus and a restoration of SOD, lipid peroxide,
lactate and glucose to normal levels [61]. Similar protec-
tion was also observed in gerbils given subchronic oral
doses of HupA (0.1 mg/kg, twice daily for 14 days) follow-
ing 5 min of global ischemia [96].
An in vitro model of neuronal ischemia is the rat
pheochromocytoma PC12 cell treated with OGD for
30 min. In our hands, this treatment causes death in more
than 50% of the cells in culture, along with major changes
in morphology and biochemistry, including elevated lev-
els of lipid peroxide, SOD activity and lactate. Cells pre-
treated for 2 h with HupA (0.1, 1 and 10 ÌM), however,
showed increased survival and reduced biochemical and
morphologic signs of toxicity. HupA protected PC12 cells
against OGD-induced toxicity, most likely by alleviating
disturbances of oxidative and energy metabolism [95].
Neurosignals 2005;14:71–82 77
 Fig. 6. Protective effects of HupA through affecting APP metabolism. Black arrows show the process of AD. Blue real
line arrows show the proved pathway; blue dash arrow represents speculative pathway; (–) means reducing or inhib-
iting.

These findings suggested that HupA might be beneficial
for VD therapy through its effects on the cholinergic sys-
tem, the oxygen free radical system and energy metabo-
lism.
A protective effect of HupA on hypoxic–ischemic (HI)
brain injury has also been found in neonatal rats [62]. A
unilateral HI brain injury was produced in 7-day-old rat
pups by the ligation of the left CCA followed by 1 h hyp-
oxia with 7.7% oxygen. After 5 weeks, the HI brain injury
in these pups caused working memory impairments in
water maze performance, as shown by an increased escape
latency and a reduced time spent in the target quadrant.
The combination of CCA ligation and exposure to a hyp-
oxic environment also led to morphologic damage in the
ipsilateral striatum, cortex, and also hippocampus, where
it produced 12% neuronal loss in the CA1 region. Treat-
ment with HupA at a dose of 0.1 mg/kg conferred signifi-
cant protection against the behavioral and morphologic
consequences of HI injury (fig. 7). The same treatment
spared a significant fraction of the CA1 neurons relative

78 Neurosignals 2005;14:71–82 Wang/Tang

 Fig. 7. Photomicrographs of coronal brain sections stained with cresyl violet at the levels of the striatum (a, b) and the
dorsal hippocampus (c, d) for representative saline-treated and huperzine A 0.1 mg/kg-treated rats. Intraperitoneal
administration of huperzine A or saline for 5 weeks after hypoxic-ischemic (HI) brain injury in neonatal rats. Note the
gross infarction and atrophy in left hemisphere of saline-treated HI rats (a, c) (n = 11) and the subtle reduction in the
left hemisphere in huperzine A-treated HI rats (b, d) (n = 12).

to saline-treated HI group. These results raise the possibil-
ity that HupA have potential utility in treating HI enceph-
alopathy in neonates.
Glutamate is the main excitatory neurotransmitter in
the CNS, with important roles in neurotransmission and
functional plasticity. Excitatory amino acid neurotrans-
mitters are also involved in CNS pathology. The deleteri-
ous effects of overstimulation with excitatory amino acids
have been implicated in a variety of acute and chronic
neurodegenerative disorders such as ischemic brain dam-
age, AD and neuronal cell death [11] [for reviews, see 13,
14, 26, 27, 35]. Glutamate-mediated overactivation of
receptors induces excessive Ca2+ influx, which results in
elevated intracellular Ca2+ concentrations [10, 12] with
serious consequences such as necrosis and apoptosis [31].
Blockade of glutamate receptors prevents most of the Ca2+
influx and neuronal cell death induced by glutamate expo-
sure [50, 57].
It has been reported that HupA protects against gluta-
mate-induced toxicity. HupA (100 ÌM) decreased neu-
ronal cell death caused by a toxic level of glutamate (also
100 ÌM). In those experiments, HupA reduced gluta-
mate-induced calcium mobilization but did not affect the
increase in intracellular free calcium induced by exposure
to high KCl or a calcium activator Bay-K-8644 [58].
HupA dose-dependently inhibited the NMDA-induced
toxicity in primary neuronal cells, most likely by blocking
NMDA ion channels and the subsequent Ca2+ mobiliza-
tion at or near the PCP and MK-801 ligand sites [20].
Wang et al. [66] reported that HupA reversibly inhibited
NMDA-induced current in acutely dissociated rat hippo-
campal pyramidal neurons and blocked specific [3H]MK-
801 binding in synaptic membranes from rat cerebral cor-
tex. Of all the AChE inhibitors tested, HupA is the most
powerful both in protecting mature neurons and in block-
ing the binding of [3H]MK-801. Studies on the mecha-
nism of receptor inhibition showed that HupA reversibly
inhibited NMDA-induced currents. The effect was non-
competitive, and showed neither ‘voltage-dependency’,
nor ‘use-dependency’ [89]. Studies of [3H]MK-801 bind-
ing in cortex membranes suggest that HupA acts as a non-
competitive antagonist of the NMDA receptors, via a

Hup A: A Natural Cholinesterase Inhibitor Neurosignals 2005;14:71–82 79

for Alzheimer’s Disease
competitive interaction with one of the polyamine bind-
ing sites [92]. Of interest, natural (–)-HupA and synthetic
(+)-HupA reduced the binding of [3H]MK-801 with simi-
lar potencies [91] indicating that HupA inhibits NMDA
receptors in rat cerebral cortex without stereoselectivity.
This result is in dramatic contrast with the stereoselective
inhibition of acetylcholinesterase.
NMDA-receptor activation also mediates the genera-
tion of long-term potentiation (LTP) – a cellular process
that underlies learning and memory [3, 52]. There is evi-
dence that the suppressive action of Aß on LTP in both
CA1 and dentate gyrus operates via a NMDA receptor-
independent pathway that involves cholinergic terminals
in the hippocampus. Of some interest, HupA (1.0 ÌM)
was found to enhance LTP, while a much lower dose
(0.1 ÌM) largely blocked the suppressive effects of Aß on
LTP induction [8, 80].
Neuronal cell death caused by overstimulation of glu-
tamate receptors has been proposed as the final common
pathway for a variety of neurodegenerative diseases in-
cluding AD. The ability of HupA to attenuate glutamate-
mediated neurotoxicity may be one additional reason for
considering this agent as a potential therapeutic for de-
mentia and as a means of slowing or halting the pathogen-
esis of AD at an early stage [20].
Acknowledgements
This work was supported in part by the grants from Ministry
of Science and Technology of China (G199805110, G1998051115)
and National Natural Science Foundation of China (39170860,
39770846, 3001161954, 30123005 and 30271494). The authors are
grateful to Professor Stephen W. Brimijoin (Mayo Clinic, USA) for
English revision on the manuscript.

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 Author Index Vol. 14, No. 1–2, 2005

Amit, T. 46
Avramovich-Tirosh, Y. 46
Doré, S. 61
Houghton, P.J. 6
Howes, M.-J. 6
Komatsu, K. 34
Kuboyama, T. 34
Mandel, S.A. 46
Reznichenko, L. 46
Suk, K. 23
Tang, X.C. 71
Tohda, C. 34
Wang, R. 71
Youdim, M.B.H. 46
Zheng, H. 46

© 2005 S. Karger AG, Basel 83

ABC
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www.karger.com www.karger.com/nsg
 Subject Index Vol. 14, No. 1–2, 2005

Acetylcholine 6 Galantamine 6
Acetylcholinesterase 71 Ginseng 34
Alzheimer’s disease 6, 34, 71 Glutamate 71
Amyloid 34, 71 Green tea catechins 46
– precursor protein 71
Apoptosis 71 Hemin 61
Ashwagandha 34 Huperzine A 6, 71
Astrocytes, neuroinflammation 23
Axon regeneration 34 Inflammation 23
Iron 61
Bilirubin 61 – chelation 46
Biliverdin 61
Blood flow 61 Microglia 23
Mitochondria 71
Carbon monoxide 61
Cell signaling 46 Neurite outgrowth 46
Central nervous system, herbal medicine Neuritic atrophy 34
23 Neurodegeneration 23, 46
Cerebral ischemia 71 Neuroglia 23
Cholinergic receptors 6 Neuroprotection 23, 46, 71
Cholinesterase inhibitors 6, 71 Neurorescue 46
Coffee beans, trigonelline 34 NMDA receptor 71
Cognitive enhancer, huperzine A 71
Oxidative stress 71
Dendrite regeneration 34
L-DOPA 6 Parkinson’s disease 6, 46
Dopamine 6 Physostigmine 6
Dopaminergic receptors 6 Polyphenol stilbenes 61
Potassium current 71
(–)-Epigallocatechin-3-gallate 46 Protein kinase C 46
Ergot alkaloids 6
Synaptic loss 34
Flavonoids 23
Withania somnifera 34

© 2005 S. Karger AG, Basel

ABC
Fax + 41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
www.karger.com www.karger.com/nsg