Natural Product Inhibitors of Acetylcholinesterase: Current Organic Chemistry May 2006

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Natural Product Inhibitors of Acetylcholinesterase
Article in Current Organic Chemistry · May 2006

DOI: 10.2174/138527206776894410
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Current Organic Chemistry, 2006, 10, 00-00 1
Natural Product Inhibitors of Acetylcholinesterase
K. Hostettmann*, A. Borloz, A. Urbain and A. Marston
Laboratoire de Pharmacognosie et Phytochimie, Ecole de Pharmacie Genève-Lausanne, Université de Genève, CH-

1211 Genève 4, Switzerland
Abstract: Various natural products, such as physostigmine, have long been recognized as inhibitors of the enzyme
acetylcholinesterase. Since the recent approval of galanthamine for the treatment of Alzheimer’s disease by the blockage
of acetylcholine degradation, attempts to find other inhibitors of the enzyme have multiplied, leading to promising
candidates such as huperzine A. In this review, a listing is presented of natural product inhibitors, both alkaloid and non-
alkaloid in origin. These have been isolated from plant, animal and microbial sources. Details of current testing methods
on cholinesterases are given, including solution assays and screening techniques by TLC and HPLC.
INTRODUCTION Currently, the only approved treatment to prevent the

Inhibitors of acetylcholinesterase (AChE) are intimately decrease of acetylcholine levels in patients with AD is by the
connected with the treatment of Alzheimer’s disease (AD). use of acetylcholinesterase (AChE) inhibitors [14, 15].
Unfortunately, as people start living longer, AD is becoming AChE is the enzyme that degrades ACh, but furthermore it
more prevalent and large-scale efforts will have to be made has been shown to accelerate amyloid-_ peptide formation
to find new anti-Alzheimer drugs. [16].
In 1906, during the 37th Conference of German This double role in AD makes it necessary to search for
psychiatrists in Tübingen, the Bavarian neuropsychiatrist new AChE inhibitors, more especially as the current
Alois Alzheimer described for the first time the symptoms of treatments still present some side effects [17]. In this review,
“a particular disease of the cerebral cortex”, characterized by natural products with AChE inhibitory activity from natural
a gradual and irreversible degeneration of the intellectual sources (plants, animals, microorganisms) are documented.
functions such as memory, orientation, judgement, language The diversity of the different structures gives access to lead
and the capacity to acquire new knowledge [1, 2]. compounds and templates which, it is hoped, will provide
One century later, Alzheimer’s disease affects 18 million new therapeutics for the future management of AD.
people worldwide [3]. This disease is the most common form ASSAYS
of dementia in elderly people, affecting 25% of the
Although a lot of factors are involved in the pathology of
population over 80 years of age. In patients with AD, brain
regions associated with higher mental functions (e.g. AD, providing an array of potential drug targets, much
testing is performed on the enzyme AChE. In order to find
neocortex and hippocampus), are affected by extracellular
new inhibitors, it is necessary to have suitable screening
deposits of _-amyloid plaques [4, 5] and intracellular
tools available. These need to be simple and rapid so that
deposits of neurofibrillary tangles [6, 7]. There is also a
testing can be achieved with the minimum possible effort.
progressive loss of neurons in the basal forebrain [8], which
is the major source of cholinergic innervation of the Several assays are presented below.
neocortex and hippocampus. These changes involve Assays Based on Colorimetric Methods
progressive and irreversible cognitive function impairment,
A number of assays below are based on the Ellman
resulting mainly in a loss of memory, with neurological and
method (Fig. (1)) [18]. Acetylthiocholine (ATCI) is cleaved
neuropsychiatric disorders. The symptoms have also been
by AChE to form thiocholine which in turn reacts with 5,5'-
closely related to a loss of cholinergic neurotransmission in
dithiobis-(2-nitrobenzoic acid) (DTNB) to give the yellow 5-
the cerebral cortex and other areas of the brain. Those
observations, together with the emerging role of thio-2-nitrobenzoate anion.
acetylcholine (ACh) in memory [9] and the observed deficit, This method was first conducted in large cuvettes for
in AD, of choline acetyltransferase [10], the enzyme kinetic measurements. The procedure was then extended to
responsible for the synthesis of ACh, have led to the micro-cells and the rate of reaction obtained was the same as
cholinergic hypothesis of the disease [11]. The hypothesis that determined in larger cells. It should be noted that there is
states that the cognitive impairments in AD are mainly due an appreciable non-enzymatic hydrolysis of the substrate that
to cholinergic deficit, and that these dysfunctions are must be corrected [18]. As shown below, this method has
partially remediable with the use of cholinergic stimulators been adapted in a number of different techniques.
[11, 12, 13]. Several 96-well microplate assays have been derived
from Ellman’s method. The quantities of enzyme and
reagents were significantly reduced with this type of assays.
*Address correspondence to this author at the Laboratoire de The main difference in the methods described is the reaction
Pharmacognosie et Phytochimie, Ecole de Pharmacie Genève-Lausanne,
Université de Genève, CH-1211 Genève 4, Switzerland; Tel: 0041 22 379 time. Ingkaninan et al. made measurements every 13 s at 405
3401; Fax: 0041 22 379 33 99; E-mail: Kurt.Hostettmann@pharm.unige.ch nm, five before adding the enzyme and eight after [19].
1385-2728/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

2 Current Organic Chemistry, 2006, Vol. 10, No. 7 Hostettmann et al.
AChE
H2O + (CH3)3N+CH2CH2SCOCH3 (CH3)3N+CH2CH2S- + CH3COO- + 2H+
ATCI Thiocholine Acetate
O2N S-
(CH3)3N+CH2CH2S- -
OOC 5-thio-2-nitrobenzoate (yellow)
+
O 2N S S NO2 + (CH3)3N+CH2CH2SS NO2
-
OOC COO- 2-nitrobenzoate-5-mercaptothiocholine COO-
DTNB
Fig. (1). Ellman's reaction.
Another method also described by Ingkaninan et al. consists rapid method to screen large numbers of samples to discover
of performing measurements every 5 s for 2 min after the new inhibitors of AChE [24]. However, this method is
addition of AChE [20]. Brühlmann et al. measured the known to give a number of false-positive effects. In order to
increase in absorbance at 412 nm for 3 minutes at room prove that the spots are effectively due to an inhibition of the
temperature [21]. Kim et al. measured the absorbance at 450 enzyme and not to a chemical reaction between DTNB and
nm immediately after adding both reagents with one reading thiocholine, AChE was premixed with ATCI to form
every 2 min for 10 min [22]. Finally, a microassay was thiocholine. The TLC layer was first sprayed with a solution
developed for measuring low levels of organophosphorus of DTNB and then with thiocholine. The white spots
compounds. The enzyme was first added, followed by the observed were compared to those recorded for the enzyme
reagents after 7 min. Measurements of the absorbance at 414 inhibition [25].
nm was made after a subsequent 10 min. In this case, the Another assay on TLC plates is based on the reaction of
kinetics of the reaction are not important as an end-point AChE with -naphthyl acetate and the subsequent formation
reading is taken [23]. of a purple dye with -naphthol and Fast Blue Salt B (Fig.
(2)). The samples are spotted on the plate before standard
- TLC Assays
development. An enzyme solution is sprayed first on the
An assay on thin layer chromatography (TLC) is also plate. It is then incubated at 37°C for 20 min before spraying
based on Ellman's reaction [24]. The samples are spotted on a mixture of -naphthyl acetate and Fast Blue Salt B. A
the TLC plate before standard development. A solution of purple coloration appears after 1–2 min and the inhibitory
DTNB and ATCI is first sprayed until the silica is saturated compounds are shown by white spots on the plate. The
with the solvent and then an enzyme solution is applied. A inhibition is easier to see with this method given that the
yellow coloration should appear after about 5 min with white contrast with the background is stronger than when using
spots for inhibitory compounds. This provides an extremely Ellman’s method [26].
O-COCH3 OH
AChE
+ CH3COOH
Naphthyl acetate α-naphthol
H3CO
N N N N 2Cl-
+ +
OCH3
Fast Blue Salt B
OH H3CO OH
N N N N
OCH3
Azo dye (purple)
Fig. (2). Reaction of AChE with naphthyl acetate and subsequent formation of a purple dye.
Natural Products Inhibitors of Acetylcholinesterase Current Organic Chemistry, 2006, Vol. 10, No. 7 3
The TLC assays are more sensitive than the microplate provided four simple tests for the enzymatic determination of
assays and are very useful as a qualitative method to detect fenitrothion (organophosphate pesticide) [30].
AChE inhibitors [27]. On the other hand, the microplate
assays give quantitative results such as IC50 values - HPLC Assay
(Inhibitory Concentration that reduces the enzymatic Rhee et al. used the substrate 7-acetoxy-1-methyl
reaction rate by 50 %). quinolinium iodide ( excitation 320 nm, emission 410 nm) which
is hydrolyzed by AChE to the highly fluorescent 7-hydroxy-
- HPLC Assay 1-methyl quinolinium iodide (excitation 406 nm, emission 505
Ingkaninan et al. developed a high-performance liquid nm) in a flow system. The substrate itself is hydrolyzed by
chromatography (HPLC) biochemical assay method for AChE so there is less risk of false-positive results due to
AChE inhibitory activity based on Ellman’s reaction with interference by plant extract with the reaction between the
simultaneous on-line coupled ultraviolet (UV) and mass product of AChE and the fluorescent dye. As in the
spectrometric (MS) detection. After the HPLC, the flow is spectrophotometric HPLC method, the percentage of organic
split between UV (94 % of the flow), MS (3 %) and solvent in the eluent was limited by the enzymatic activity.
biochemical detection (3 %). For the biochemical part, This assay was about 20 times more sensitive than the one
DTNB and AChE are first mixed together and just after using UV-detection described by Ingkaninan et al. [19] but
addition of the substrate, ATCI, are passed into a reaction the sensitivity was not as good as expected because of the
coil for approximately 2 minutes. As the reagents are mixed low pH and temperature necessary for substrate stability
continuously, 5-thio-2-nitrobenzoate (absorbance at 405 nm) [31].
is detected. The presence of an inhibitor in the system is Hadd et al. used the reaction between thiocholine
reflected by a negative peak. The limitation of this method is (obtained by hydrolysis of acetylthiocholine by AChE) and
the restriction on the amount of organic solvent that can be coumarinylphenylmaleimide. The resulting thioether was
used without too much effect on the activity of the enzyme. detected by laser-induced fluorescence. Solutions of
For example, 10 % of acetonitrile inhibited almost 90 % of inhibitor, enzyme, substrate and derivitizing agent were
enzyme activity while 0.25 % of acetic acid caused the same mixed within the channels of a microchip. Inhibitors
effect. Methanol was used as it is relatively well tolerated by produced a negative peak, Gaussian for competitive
AChE (10 % reduced the enzyme activity less than 50 %). inhibitors and a broad negative peak for irreversible
Up to 40 % of methanol was used, but gradient methods inhibitors [32].
were not suitable, as the change in the organic solvent
concentration perturbs the baseline of the biochemical Assay Based on a Radiometric Method
detection. With this method, the detection limit for AChE activity and the effect of inhibitors was measured
galanthamine was 0.3 nmol [19]. For comparison, the by the rate of hydrolysis of [14C]acetylcholine into
detection limit in the microplate assay was 0.25 nmol and for [14C]acetic acid, which was quantified after extraction by
TLC 0.0375 nmol [27]. reaction with [14C]sodium bicarbonate to give [14C]carbon
dioxide. This latter was measured using an ionization
Assays Based on Fluorimetric Methods chamber system. The [14C]carbon dioxide produced is
A number of reactions using fluorometry for measuring proportional to the amount of acetylcholine hydrolyzed. This
AChE activity have been described. Parvari et al. monitored method is simple, fast, inexpensive and has the potential for
the formation of thiocholine by a further reaction with the automation [33].
fluorogenic compound N-(4-(7 diethylamino-4-methyl-
coumarin-3-yl)phenyl)maleimide, to yield an intensely Assays Based on mass Spectrometric Detection
fluorescent product. The non-enzymatic hydrolysis of the To quantify the reaction of AChE with acetylcholine,
compound was relatively low. This method allowed mass spectrometry (MS) has been used to measure the
determination of picomoles of thiocholine [28]. amount of choline product. A matrix-less method of
Resorufin butyrate and indoxyl acetate are two desorption/ionization on silicon (DIOS) coupled with a time-
nonfluorescent compounds which are hydrolyzed by of-flight mass spectrometer (TOFMS) was compared to
cholinesterase to highly fluorescent compounds (resorufin: liquid chromatography/tandem mass spectrometry
excitation 540-570 nm, emission 580 nm and 3-hydroxy-indole: (LC/MS/MS). The enzymatic reaction was run in a buffer
excitation 395 nm, emission 470 nm). The rate of hydrolysis of medium and incubated at 37°C. The reaction was quenched
at 30 min to determine the IC50 by addition of acetonitrile
indoxyl acetate is much faster than that of resorufin butyrate;
containing choline-d9 as internal standard. The solution was
it also possesses a greater stability toward spontaneous
then spotted onto the DIOS chip (0.4 μl per well) or diluted
hydrolysis and a larger difference between excitation and
10-fold prior to LC/MS/MS. The concentration of choline
emission wavelengths. On the other hand, resorufin has a
determined was plotted against the concentration of inhibitor
greater fluorescence, which permits assay at lower substrate
to find the IC50. The DIOS method is quantitative provided
concentrations. The sensitivity of both in the cholinesterase
that an internal standard is present, and the IC50 was
assay is about the same. It is noticeable that these substrates comparable to that obtained with LC/MS/MS. The
can be hydrolyzed by a number of different enzymes [29]. LC/MS/MS method resulted in the widest linear dynamic
2-Naphthyl acetate was hydrolyzed to the highly range (1.0-500.0 μM) compared with the DIOS method (5.0-
fluorescent product 2-naphthol by AChE and 500.0 μ M). It can be used for rapid and specific
butyrylcholinesterase (BuChE). With indoxyl acetate, they quantification of enzyme-inhibition assays [34].
Assays Based on Immobilized Enzyme to test the activity of the enzyme and then it was injected
Another approach to test AChE activity is to immobilize together with different concentrations of the inhibitors. This
the enzyme on a support. The main advantage of this method method preserves the activity of a small amount of enzyme,
is avoiding the loss of activity that occurs with the enzyme at allows performance of kinetic studies and can be used for
high dilutions. A flow-injection system using immobilized high throughput screening of potential drug candidates [39].
enzyme on magnetic particles was described by Kindervater This IMER was compared to another monolithic disk
et al. [35]. A known amount of enzyme was incubated with with epoxy as reactive group and a packed silica column
the sample solution and the remaining activity was inversely with glutaraldehyde-P. The Schiff base linkage gave more
proportional to the amount of inhibitor in the sample. The stable reactors compared with epoxy groups. Matrices
activity was measured either by Ellman's method or by showed very short conditioning time and fast recovery of the
electrochemical detection with an additional enzyme reactor enzyme activity. The EDA-CIM disk was the best to fulfil
containing immobilized choline oxidase. This method was the general requirement for a fast and reproducible analysis
used to determine pesticides in drinking water [35]. but unpacked affinity media were useful to pre-screen
AChE can be immobilized on glass beads and used as a optimal conditions for a further immobilization step [40].
single bead string reactor for the determination of some Finally, another possible approach is the immobilization
organophosphorus and carbamate insecticides. The detector of the enzyme on a silicon-based biosensor. This method
was a simple pH electrode; variations in enzyme activity utilizes biotin-streptavidin mediated filtration capture onto a
were measured from pH changes when the substrate nitrocellulose membrane, in conjunction with a silicon-based
(acetylcholine) was injected before and after the passage of light-addressable potentiometric sensor (LAPS). This
the solution containing the inhibitor. The enzyme reactor method was developed for the detection of
could be reused after regeneration [36]. organophosphorus compounds. These inhibitory compounds
A method for post-column reaction detection of reacted with biotin-labeled AChE and then streptavidin was
pesticides, based on the inhibition of immobilized AChE has added. The enzyme was captured on the biotin-embedded
been described. The compounds were first separated by nitrocellulose membrane by filtration. The immobilized
reversed-phase liquid chromatography with tetrahydrofuran enzyme was inserted into the reader compartment of the
(20 %)-water (80 %) as the mobile phase in an isocratic LAPS that contained acetylcholine solution. The presence of
system. The reagents ( -naphthyl acetate and p - organophosphates was determined by the decrease in
nitrobenzenediazonium or Fast red GG salt) were added to esterase activity. The sensor monitored enzyme activity as a
the eluent and the activity of AChE immobilized on pore decrease in pH resulting from the generation of H+ in the
glass in a mini-column was monitored by spectrophotometric hydrolysis of acetylcholine. This is a rapid and easy method
detection at 500 nm. This method can be used for any AChE to determine the presence of inhibitory compounds [41].
inhibitor [37]. ALKALOID INHIBITORS
Another immobilized AChE stationary phase was
A great deal of emphasis has been placed on the use of
prepared in a stainless steel column packed with epoxide
alkaloids for the treatment of AD. For this reason, examples
silica and the enzyme solution was recycled through the
of this particular class of natural product will be presented
column during 24 hours for packing. The activity of the
immobilized enzyme was first tested by injecting first.
acetylthiocholine, using a mobile phase containing DTNB Physostigmine
and measuring the area of the obtained peak with UV
Physostigma venenosum (Fabaceae) has been
detection at 412 nm. Then, solutions of samples at different
traditionally used in Africa as a ritual poison, claimed to
concentrations with acetylthiocholine were prepared and
determine the guilt or innocence of persons accused of a
injected into the system. The peak areas were compared with
crime [42, 43]. Physostigmine (eserine) (1) was first isolated
those obtained in absence of inhibitor and percentage
from the seeds in the nineteenth century and has long been
inhibition was calculated. The IC50 values obtained correlate
with those of the reference method (spectrophotometry). The known as an AChE inhibitor [44]. It antagonizes
scopolamine-induced impairment of cognitive function in
enzyme stability is increased and automation of the system
rats. The antiamnesic dose of physostigmine was 1/30 – 1/3
allows a large number of compounds to be analysed
of doses with central or peripheral side-effects (miosis,
continuously [38].
salivation, hypothermia and tremor) [45]. It is also reported
In order to work with human recombinant to have shown significant cognitive benefits in both normal
acetylcholinesterase (hrAChE), the amount of enzyme used and Alzheimer's patients [44]. Clinical use has been limited
must be decreased to lower the cost of the method. Bartolini by the very short in vivo half-life of the compound. Various
et al. described a method to provide a monolithic micro- forms of administration (controlled-release oral formulations
immobilized-enzyme reactor (IMER). hrAChE was and skin patches) have been tried to overcome this problem.
covalently immobilized on an ethylenediamine (EDA) Even so, adverse effects, mainly gastrointestinal complaints,
monolithic convective interaction medium (CIM) previously remained common leading to a high rate of withdrawal from
derivatized with glutaraldehyde. The resulting Schiff bases treatments [46].
were reduced by cyanoborohydride and the unreacted
Physostigmine also inhibits BuChE with a similar
aldehydic groups were condensed with monoethanolamine.
The IMER is suitable for an HPLC system. The method is potency to AChE (IC50 = 1.26 and 0.251 μM respectively)
[47]. Therefore, adverse effects associated with BuChE
then the same as described before - the mobile phase
inhibition may occur. However, BuChE was shown to be
contained DTNB, acetylthiocholine was first injected alone
implicated in the etiology and progression of Alzheimer's (P11012) and 6-O-demethyl-6-O-[(adamantan-1-yl)-

disease, probably by influencing the phosphorylation of tau carbonyl]galanthamine (P11149) were compared to the
protein, associated with neuropathological tangles. BuChE parent compound using biochemical, behavioral and
inhibition may therefore develop as a therapeutic target [48]. pharmacokinetics analysis. P11149 and P11012 were shown
Physostigmine is also a non-competitive nicotinic- to act as pro-drugs, yielding significant levels of 6-O-
channel activator which may be of added value in demethylgalanthamine. This compound was 10- to 20-fold
Alzheimer's therapy [49]. more potent than galanthamine in vitro (IC50 = 0.11 μM and
The chemical structure of physostigmine provided a 0.92 μM respectively). The three alkaloids showed central
template for the development of rivastigmine (2), a long- cholinergic activity by causing hypothermia and by
acting reversible and competitive inhibitor that is indicated attenuating scopolamine-induced deficits. P11149 had
as an oral treatment for patients with mild to moderately slower, lower and more sustained concentration levels than
severe Alzheimer's disease. Moreover, it has brain-region P11012 for the same area under the curve. P11012 and
selectivity and preferentially inhibits the G1 (monomeric) galanthamine rapidly reached their concentration maxima
enzymatic form of AChE, which predominates in the brains but galanthamine had the highest area under the curve.
of patients with Alzheimer's disease [50]. Rivastigmine was P11149 was considered a better therapeutic candidate
the only inhibitor which displayed preferential inhibition for because it has a longer duration of action than P11012, a
the G1 form of AChE [51]. The adverse effects occurring better affinity and a greater selectivity for AChE than
with rivastigmine are those expected from an AChE galanthamine and, finally, a higher per os therapeutic index
inhibitor. These are most frequently gastrointestinal than the two others [61].
complaints and are more common in women [52]. A Huperzine A
synthesized phenylcarbamate structurally related to
rivastigmine, 1-[1-(3-dimethylcarbamoyloxyphenyl)ethyl] Huperzia serrata (Lycopodiaceae) has been used in
piperidine, showed lower toxicity and a similar therapeutic Chinese traditional medicine for the treatment of fever,
index in comparison with rivastigmine, in addition to a inflammatory states and for memory improvement [44, 62].
simple and cheaper synthesis [53]. In the early 1980s, huperzine A (4) was isolated from this
traditional medicine. It is also found in Lycopodium selago
Phenserine and heptylphysostigmine, other physo-
and Lycopodium varium (Lycopodiaceae) [63]. Huperzine A
stigmine derivatives, show interesting anti-AChE activity in
is a reversible inhibitor of AChE. The complex between
vitro and in vivo with longer duration of action compared to
huperzine and the enzyme dissociated rather slowly which
that of physostigmine [54, 55, 56, 57]. could explain its long duration of action [64]. Moreover, it
Galanthamine was found to be 1000-fold less potent as an inhibitor of
BuChE than of AChE (IC50 = 74.43 and 0.082 μ M
Galanthus species have been used traditionally in
respectively) [47]. The compound is optically active and in
Bulgaria and Turkey to treat painful neurological conditions
nature only its (-)-enantiomer is present. The synthetic
(for example facial neuralgia) by topical application [44].
racemic mixture was 3 times less potent in vitro than the
Galanthamine (3) was first isolated in the 1950s from natural compound (IC50 = 0.3 and 0.1 μM respectively). A
Galanthus nivalis (Amaryllidaceae) [44]. It occurs also in
comparable effect was also observed in vivo. However, the
other genera of the Amaryllidaceae family, Narcissus spp.
racemic compound remained more potent than
and Lycoris spp.; it can be obtained from Leucojum aestivum
physostigmine in vitro (IC50 = 0.6 μM) [65].
or by synthesis [43]. This alkaloid is reported to be more
selective for AChE than BuChE (IC50 = 1.995 and 12.59 μM In vivo, huperzine A improved reserpine- or yohimbine-
respectively) [42, 47]. Galanthamine has a dual mode of induced memory impairments on monkeys via an adrenergic
action. As an inhibitor of AChE, it blocks the degradation of mechanism [66]. Lower oral doses of (-)-huperzine A than of
acetylcholine and thereby increases the concentration of tacrine (a commercially available synthetic inhibitor) were
available transmitter. As an allosteric potentiating ligand of required to improve working and reference memory deficits
nicotinic acetylcholine receptors, it enhances the ion flow in rats treated with scopolamine. (-)-Huperzine A showed
generated by the receptor [44]. A recent study also also higher bioavailability and stronger potency to penetrate
mentioned neuroprotective effects by induction of an the blood-brain barrier than did tacrine [67, 68].
antiapoptotic protein [58]. Huperzine A appears to have additional pharmacological
Several randomized, double-blind, parallel group trials properties. Studies using cell cultures have shown that
comparing galanthamine with placebo have examined the huperzine A decreases neuronal cell death caused by toxic
efficacy, the tolerability and the safety of the compound. levels of glutamate [62]. It was also shown to protect
Galanthamine maintains cognitive function, prevents the neurons against amyloid -peptide-induced apoptosis [69].
onset of newly occurring noncognitive behavioral symptoms Several clinical trials have been reported in China and the
for at least 1 year and delays the loss of activities of daily efficacy of huperzine A was demonstrated in the treatment of
living by 6 months. The adverse effect profile of 447 patients suffering from age-related memory dysfunction
galanthamine is typical for a cholinergic drug, the most or dementia. The most frequently occurring side effects
frequent adverse events being nausea, vomiting and diarrhea (dizziness, nausea and diarrhea) were related to its
[59]. Optimal therapy appears to require early initiation of cholinergic properties. No liver and kidney toxicity was
the drug and a dosage-adjustment period of eight weeks [60]. detected [44, 47].
Some analogs of galanthamine have also been studied. Tacrine-huperzine A derivatives (huprines) have been
For example, 6-O-acetyl-6-O-demethylgalanthamine synthesized and tested as AChE inhibitors. Some compounds
H3C NH
O H3C CH3
O CH3 N
CH3 H3C N O
CH3
O
N N
H3C H CH3
1 2
14
H3C
10 H3C
N
8
12 12b
H
H N O
12a 6
1 OCH3
C
O
5 H3C NH2
HO 4 H
3 4
were very potent inhibitors and showed interesting properties Amaryllidaceae

for further studies in connection with their possible use in the Since the discovery of the high activity and the
treatment of AD [70]. Heterodimers of huprine-tacrine have importance of the clinical use of galanthamine (3), a number
been developed showing IC50 values in the subnanomolar of Amaryllidaceae species have been tested for their activity
range [70]. against AChE. Twenty-three alkaloids isolated from various
Table 1. Amaryllidaceae alkaloids as inhibitors of acetylcholinesterase
Activity
Family Plants Alkaloid type Compounds Ref.
Ic50 [μm]
sanguinine (5) 0.10

galanthamine (3) 1.07
galanthamine
11-hydroxygalanthamine 1.61
epinorgalanthamine 9.60 71
oxoassoanine 47.21
lycorine assoanine (6) 3.87
pseudolycorine 152.32
crinine (8) 461
crinine crinamidine 300
Crinum moorei Hook.f.
epivittatine 239
lycorine 1-O-acetyllycorine 0.96
Crinum bulbispermum crinine 6-hydroxycrinamine 490 72
(Burm.f.) Milne-Redh. N-desmethyl-8-ethoxypretazettine 234
& Schweick. tazettine
N-desmethyl-8-ethoxypretazettine 419
Amaryllidaceae Crinum macowanii
lycorine lycorine (7) 213
Baker
Crinum glaucum A. hamayne (9) 250
73
Chev. lycorine (7) 450
Narcissus tazetta subsp. lycorine (7) 3.16 / 43.69a
tazetta L. and Galanthus tazettine (10) 36.34a
ikariae L. 3-epihydroxybulbispermine 30.18a
crinine (8) 26.53a
74
Galanthus ikariae L. galanthamine (3) 3.2 / 48.00a
2-demethoxymontanine 31.84a
Narcissus tazetta subsp. N-norgalanthamine 34.09a
tazetta L. haemanthamine 20.80a
Narcissus 'Sir Winston
ungiminorine 86 75
Churchill'
Nerine bowdenii Will.
ungeremine (11) 0.35 76
Watson
a
inhibition rate at 10 μg/ml [%]
H3C
OH
N
HO
H3CO
O
OH N
O H3CO
H H N
O
HO H 5 6 7
Amaryllidaceae species were tested for their in vitro AChE Two Crinum species, Crinum glaucum and C r i n u m
inhibitory activity with galanthamine as a positive control jagus, have frequently been cited by traditional healers in
(IC50 = 1.07 μ M) [71]. Only the galanthamine and the southwest Nigeria for memory loss and other mental
lycorine types showed some activity. The most active symptoms associated with ageing [73]. Hamayne (9),
compounds were sanguinine (5) (IC50 = 0.10 μ M) and 11- crinamine, lycorine (7) and haemanthamine were isolated
hydroxygalanthamine (IC50 = 1.61 μM). Both synthetic and from C. jagus and C. glaucum. Only two of the four
natural sanguinine (isolated from Eucharis grandiflora) were alkaloids cited (hamayne, IC50 = 250 μM and lycorine, IC50
10-fold more potent than galanthamine. 11-hydroxygal- = 450 μM) were active against AChE. The occurrence of
anthamine exhibited an activity similar to that of these two compounds in C. glaucum could explain the
galanthamine on AChE of erythrocyte membranes (IC50 = activity of the extract and its use for helping memory; the
0.72 and 0.33 μM respectively; the difference between the correlation was more difficult to establish for methanolic
values may be due to the different origins of the enzyme). extract of C. jagus since the major alkaloid, haemanthamine,
The extra hydroxyl group of sanguinine (5) available for showed only weak activity. Minor compounds could possess
interaction with AChE can explain the strongest activity of a better activity but were found in too small amounts to be
this alkaloid. The lack of activity of lycoramine and isolated [73]. It is noteworthy that hamayne isolated by
epinorlycoramine, two galanthamine-type alkaloids, could be
Elgorashi et al. from Crinum macownii showed an IC50 of
due to the occurrence of a double bond in ring C, which
553 μM against 250 μM for the compound isolated by
changes the spatial configuration. Among the lycorine-type,
Houghton et al.. The difference between the two studies is
the highest activity was found for assoanine (6) (IC50 = 3.87
also noticeable with lycorine (7) (IC50 = 450 μM against 213
μM, 4 times lower than galanthamine). This could be
explained by the presence of an aromatic ring, which gives a μM).
certain planarity to the molecule [71]. Eight Amaryllidaceae-type alkaloids (lycorine inhibition
A series of 21 alkaloids isolated from different Crinum rate at 10 μg/ml = 43.69 %, tazettine (10) 36.34 %, crinine
species were tested by Elgorashi et al. [72]. Lycorine-type (8) 26.53 %, galanthamine (3 ) 48.00 %, 3-epihydroxy-
alkaloids were the most active among the alkaloids tested. 1- bulbispermine 30.18 %, 2-demethoxymontanine 31.84 %, N-
O-acetyllycorine showed an IC50 of 0.96 μM which was two- norgalanthamine 34.09 % and haemanthamine 20.80 %)
fold more potent than galanthamine (IC50 = 1.9 μM, positive were isolated from Narcissus tazetta subsp. tazetta and
control in this study). Lycorine (7 ) and 1,2-O - Galanthus ikariae. All the compounds isolated were less
diacetyllycorine (semi-synthetic derivative) were 100 times active than galanthamine and they were less active than the
less active; the presence of an acetoxy group at position 1 extracts of both species (inhibition rate of the alkaloid extract
and of a hydroxy group at position 2 are required in this case at 10 μg/ml = 46.96 % and 77.23 % respectively) which
for the proper binding to the enzyme. Crinine-type alkaloids suggest a possible synergistic interaction between the
showed only weak activity [72]. alkaloids isolated [74].
H
OH OH
OH
O O
N N
O O
8 9
OCH3 O-
NCH3
O
O
N+
O OH O
O
10 11
Two active compounds were isolated by HPLC with on- stronger than galanthamine. It was first isolated from
line coupled flow assays. The first one was ungiminorine Ungernia minor and Ungernia spiralis (Amaryllidaceae) and
(IC50 = 86 μ M) isolated from Narcissus 'Sir Winston was reported to have growth inhibitory and cytotoxic effects
Churchill' with spectrophotometric detection based on and hypotensive properties. Because of these effects,
Ellman's reagent [19]. The structure of this compound is ungeremine probably cannot be a potential drug for the
quite different from galanthamine (which was more than 80- treatment of Alzheimer's disease but it could be interesting to
fold more potent with IC50 = 0.98 μM); the tertiary amine test related compounds in order to conduct structure-activity
function of ungiminorine might bind with an anionic site of studies [76].
the enzyme [75]. Several other Amaryllidaceae species have been studied
Ungeremine (11) ( I C50 = 0.35 μM) was isolated from for their anti-AChE activity. Most of the species seemed to
Nerine bowdenii with a fluorometric flow-assay, using 7- contain inhibitors but the compounds were not isolated [26,
acetoxy-1-methyl quinolinium iodide as substrate, coupled to 27, 71].
preparative HPLC [31]. This alkaloid was about 6-10 times
Table 2. Buxaceae alkaloids as inhibitors of acetylcholinesterase
Activity
Ic50 [μm]
Sarcococca epoxynepapakistamine-A > 200

coriacea steroidal funtumafrine-C (12) 45.75 77
(Hook.f.) Sweet N-methylfuntumine 97.61
(-)-hookerianamide A 82.7
Sarcococca (+)-hookerianamide B 26.4
pregnane-type
hookeriana (-)-hookerianamide 23.2 78
steroidal
(Baill.) Hook.f. (-)-hookerianamine A (14) 18.9
(+)-phulchowkiamide A (13) 0.50
5,14-dehydro-N(a)-
> 200
demethylsaracodine
pregnane-type 183.1
14-dehydro-N(a)-demethylsaracodine
steroidal 12.5
16-dehydrosarcorine 80
7.0
2,3-dehydrosarsalignone (15)
sarcovagenine-C 187.8
salignarine-C 19.7
salignenamide-C 61.3
salignenamide-D 185.2
2-hydroxypachysamine-D 78.2
salignenamide-E (16) 6.21
salignenamide-F 6.357
axillarine-C 227.92
Sarcococca axillarine-F 182.4
Buxaceae saligna (D.Don) sarcorine 69.99
Müll.Arg. Na-demethylsaracodine 204.2
saligcinnamide 19.99
salignenamide-A 50.64
steroidal vaganine-A 8.59 79, 81
5,6-dehydrosarconidine 20.29
2-hydroxysalignarine-E (17) 249
salignamine 82.5
2-hydroxysalignamine-E 15.99
epipachysamine-D 28.93
dictyophlebine 6.21
iso-N-formaylchonemorphine 6.357
sarcodinine 40.04
axillaridine-A (18) 5.21
sarsalignone 7.02
sarsalignenone 5.83
buxakashmiramine (19) 25.4

triterpenoid buxakarachiamine 143
Buxus papillosa buxahejramine 162
83
C.K. Schneider cycloprotobuxine-C 38.8
cyclovirobuxeine-A 105.7
cyclomicrophylline-A 235
Buxus hyrcana (+)-homomoenjodaramine 19.2
triterpenoid 84
Pojark. (+)-moenjodaramine (20) 50.8
Buxaceae A showed a greater activity on AChE than on BuChE (IC50 =

Steroidal alkaloids are the main constituents of 82.7 and 200 μM respectively). (+)-Phulchowkiamide A (13)
S a r c o c o c c a species and some of them demonstrate (IC50 = 0.50 μM) had an inhibitory activity higher than the
interesting biological activities such as antibacterial, OH-substituted congeners ((+)-hookerianamide B, IC50 =
antitumor, antiulcer or anticholinesterase activities. Several 26.4 μM; (-)-hookerianamide C, IC50 = 23.2 μ M; (-)-
species were studied in order to find new active alkaloids. hookerianamine A (14), IC50 = 18.9 μ M). Similarly, the
Three compounds (epoxynepapakistamine A, IC50 > 200 acetylated derivatives were more inhibitory than the parent
μ M; funtumafrine C (1 2 ), IC50 = 45.75 μ M; N - compounds [78].
methylfuntumine, IC50 = 97.61 μM) were isolated from A number of new or known active alkaloids were isolated
Sarcococca coriacea, an evergreen shrub widely distributed from Sarcococca saligna (16-18). The compounds showed
in central Nepal. All these compounds are more selective various activities and, except for 2,3-dehydrosarsalignone
inhibitors of BuChE than of AChE [77]. (15) (IC 50 = 7.0 μM for AChE and 32.2 for BuChE), they
H N CH3 H 3C
H3C NH
CH3 CH3 CH3
CH3
CH3 H CH3 H
H
O H H
H H
H 3C N 13
O 12 H
H CH3 H O
H3C CH3 H 3C H
N N CH3
CH3 CH3 CH3 CH3
17
CH3 H 16 CH3 H
H
14
H 15 CH3 CH3O H H
H
H3CHN 14 HC C C N 15
H H
H O
CH3 H CH3
N
CH3 20
17 CH3
H3C H 14
16
H
H H 15
3
A 5
4 6
(H3C)2HCH3CC=HCOCH3CN H 16
H
H H
CH3 CH3 H3C CH3
N CH3 N
CH3 20
17 CH3 CH3
H3C H 14 16 CH3 H 14 15
HO
O 2
H H 15 H H
3
A5 5
6 6
4
H3CO N 3
17 H H 18
O
H
Five other new products were isolated by a bioassay- were all more selective for BuChE [79, 80]. Structure-
guided investigation of Sarcococca hookeriana. The activity activity and even 3D-QSAR studies were performed on the
of these compounds and of two acetyl derivatives ((-)- series of alkaloids isolated from this plant. For this series, the
hookerianamide A diacetate, IC50 = 35.8 μ M and (+)- ring A of the steroid nucleus is the most important part for
hookerianamide B diacetate IC50 = 33.25 μM) was also the activity; the polycyclic compounds may penetrate the
determined. Among these alkaloids, only (-)-hookerianamide enzymatic aromatic cavity with this ring. The amino
CH3
H 3C N
H3C H CH3
CH3
N
CH3 CH3
CH3
H
H
H H
H3CO H
O H CH3
H 3C
HO C N N
H
H3C CH2OH 19 CH3
20
CH3 O
H3CO
nitrogens at C-3 or C-20 positions are expected to be activity relationships. As mentioned above, the quaternary
protonated at physiological pH - this is one of the most nitrogen plays an important role in the binding with the
important parameters that determine the inhibitory potency enzyme. The inhibitory potency increases with the
of these compounds. The C-3 position seems to be more hydrophobicity of the alkaloids studied. The angle between
important than the C-20 position [81, 82]. rings A and D seems to be crucial since berberine,
Two species of the Buxus genus were also tested for their possessing such a structure, is a powerful inhibitor of the
anti-cholinesterase activities. Buxus papillosa has been enzyme. Finally, the binding power of the alkaloid increases
thoroughly investigated and more than 50 triterpenoidal and with the polarity of C7 substituent [87].
steroidal alkaloids were isolated. The alkaloids tested A comparison between AChE from erythrocytes of
(buxakashmiramine (19), IC50 = 25.4 μM; buxakarachi- human blood (HuAChE) and from rat brain was performed
amine, IC50 = 143 μM; buxahejramine, IC50 = 162 μ M ; by Kuznetsova et al using two alkaloids described before
cycloprotobuxine C, IC50 = 38.8 μM; cyclovirobuxeine A, (berberine (21) and sanguinarine (23)) and chelidonine. The
IC50 = 105.7 μM and cyclomicrophylline A, IC50 = 235 μM) three alkaloids are reversible inhibitors, berberine and
were all more specific against BuChE and one of them sanguinarine of the mixed-type and chelidonine is a
(semperviraminol) was inactive against AChE [83]. competitive inhibitor. Based on the values of the generalized
The other species studied was Buxus hyrcana. Two active inhibition constant (Ki), the three alkaloids are assigned as
alkaloids were isolated ((+)-homomoenjodaramine, IC50 = strong inhibitors. They are more active against HuAChE
19.2 μM and (+)-moenjodaramine (20), IC50 = 50.8 μM) One than against HuBuChE (sanguinarine 14 times, berberine 20
of the compound was inactives: (-)-hyrcanine. Only anti- times and chelidonine 38 times). The importance of the
AChE activity was tested [84]. quaternary nitrogen was again proved in this study;
chelidonine, free of the quaternary nitrogen, acts much more
Other Families weakly than sanguinarine and berberine (Ki = 2.0, 0.23 and
0.23 μM respectively). HuAChE was found to be more
Isoquinoline alkaloids have been widely found in the
Papaveraceae family. Stejskal et al. studied structure-activity sensitive than the rat brain AChE [88].
relationships of this alkaloid type. In order to see the effect A methanolic extract of C o r y d a l i s ternata
of an alkyl group bound to the protoberberine skeleton, alkyl (Papaveraceae) showed a significant inhibitory effect on
chains with different numbers of carbon atoms were added to AChE activity which led to the isolation of a reversible and
berberine (21). The activity decreased with the length of the competitive inhibitor, protopine (22) (IC50 = 50 μM). In vivo,
chain (% inhibition at 0.5 μ M: berberine 76 %, 13- mice treated with protopine exhibited a diminished
methylberberine 64 %, 13-ethylberberine 56 %, 13- scopolamine-induced dementia [89].
propylberberine 35 %) which could be connected with a Another species of the genus Corydalis, Corydalis
steric restriction at the binding sites. A decrease in inhibition speciosa (Papaveraceae), was studied. Four isoquinoline
was observed when there was a hydroxyl group ionized in alkaloids with anti-AChE activity were isolated; the activity
the molecule but it depends rather on the loss of the cationic of berberine (21) (IC50 = 3.3 μM) and protopine (22) (IC50 =
character of the inhibitor. It is also noteworthy that 16.1 μM) has already been described [85, 86, 89]; the
compounds with a quaternary nitrogen (berberine 76 % of inhibitory activities of palmitine (24) (IC50 = 5.8 μM) and
inhibition at 0.5 μM, escholamine 80 % of inhibition at 10 corynoxidine (IC50 = 89.0 μM) were described for the first
μM, californidine 61 % of inhibition at 10 μM) were better time [22].
inhibitors than those with a tertiary nitrogen (papaverine 56 Finally, five protoberberine alkaloids, which inhibited
% of inhibition at 10 μM, protopine (22) 38 % of inhibition AChE in vitro, were isolated from Corydalis bulbosa
at 10 μM) [85]. Interaction between isoquinoline alkaloids (Papaveraceae). The activity against larvae and adults of
(protoberberine and benzophenanthridine-type) and AChE Drosophila melanogaster was also tested and it was partially
was demonstrated to be dependent on the pH, which related to the anti-AChE activity. (-)-Tetrahydroberberine (%
confirmed that the quaternary ammonium is responsible for inhibition at 1.0 mM = 78.7 %), (-)-tetrahydrocoptisine (25)
the inhibition [86]. (% inhibition at 1.0 mM = 71.8 %) and (±)-
Studying the activity of papaverine, berberine (21) and dehydrocorydaline (% inhibition at 0.4 mM = 61.3 %) were
isoquinoline, Whiteley and Daya deduced some structure- the most active compounds for anti-AChE activity in vitro.
Table 3. Papaveraceae alkaloids as inhibitors of acetylcholinesterase
Activity
Ic50 [μm]
protoberberine berberine (21) 76a

cis-N-methylstylopinium iodide 69b
tetrahydro-
escholidine 85b
protoberberine
cyclanoline 44b
benzo-phenanthridine sanguinarine (23) 49b 85
b
Escholamine 80
benzyl-isoquinoline
papaverine 56b
pavinane californidine 61b
protopine protopine (22) 38b
Protoberberine 34
pseudocoptisine 11
13-methyl-berberine 8.0
protoberberine coptisine 5.8
pseudoepiberberine 5.1
coralyne 1.3
berberine (21) 0.98
86
chelirubine 90
sanguirubine 60
Papaveraceae sanguinarine (23) 35
benzo-phenanthridine
chelilutine 20
sanguilutine 11
chelerythrine 9.4
diisoquinoline berberine (21) 0.5c / 0.98d / 0.23 e

Chelidonium majus
benzo-phenanthridine sanguinarine (23) 0.8c / 35d / 0.23 e 88
L.
chelidonine 2.0e
Corydalis ternata
protopine (22) 50 89
Nakai
corynoxidine 89.0
Corydalis speciosa protopine (22) 16.1
isoquinoline 22
Maximowicz palmitine (24) 5.8
berberine (21) 3.3
(-)-tetrahydroberberine 78.7f
(-)-tetrahydrocoptisine (25) 71.8f
Corydalis bulbosa
protoberberine (+)-corydaline 68.2f 90
DC.
(±)-tetrahydropalmitine 64.6f
(±)-dehydrocorydaline 61.3g
a
inhibition rate at 0.5 μM [%]; b inhibition rate at 10 μM [%]; c IC50 measured with human AChE (HuAChE) [μM]; d IC50 measured with rat brain AChE [μM]
e
Ki [μM]; f inhibition rate at 1.0 mM [%]; g inhibition rate at 0.40 mM [%]
4 6
O O
CH3
N+ N
O 8 O
1
O
13 OCH3
O
21 22
12
OCH3 O
5 4
6 O
O
1
N+
O CH3 23
8
O
H3CO O
O
N+
H3CO
H
OCH3 O
24 25
N
O
OCH3
The presence of double bonds in the isoquinoline unit charge on the nitrogen atom, it was little absorbed by the
improved the potency of the protoberberine alkaloids. adults' bodies. The alkyl substitution pattern at the 9,10-
However, (±)-dehydrocorydaline was not toxic against adult catechol function of (-)-tetrahydroberberine and (-)-
D. melanogaster. It is likely that, because of the positive tetrahydrocoptisine influences the toxicity [90].
Table 4. Alkaloids from other families as inhibitors of acetylcholinesterase
Activity
ic50 [μm]
-yohimbine 431
crooksidine 175
decarbomethoxytetrahydro- 203
secodine
10-methoxy-N1- 135
methylpericycivine
Haplophyton crooksii akuammicine 221
Apocynaceae indole 92
L.D. Benson tubotaiwine 108
lanceomagine 383
16-decarbomethoxyvinervine 57
haplophytine 225
cimicine 241
cimicidine 197
akuammidine 188
norditerpenoid faleoconitine 293
Aconitum falconeri
pregnane-type 93
Stapf. pseudoaconitine 278
steroidal
Ranunculaceae protoberberine berberine (21)
NS / Inhibition
Coptis japonica
comparable to that of 94
Makino palmitine (24)
physostigmine
coptisine
Lycopodium sieboldii
sieboldine A (26) 2.0 95
Miq.
Lycopodiaceae huperzine A (4) 0.1

huperzine B
63
N-methyl-huperzine B
huperzinine NS but active
Chimarrhis turbinata indole turbinatine (27) 1.86

Rubiaceae 99
DC. glucoalkaloid desoxycordifoline 0.1a
impericine 67.97
Fritillaria imperialis
Lilliaceae steroidal delavine 105.5 100
L.
persicanidine 352.2
Evodia rutaecarpa
Rutaceae dehydroevodiamine (28) 37.8 101
Bentham
fangchinoline (29) 3.2
Stephania tetrandra S. bisbenzyliso-
Meni-spermaceae atherospermoline 4.0 102
Moore quinoline
2'-N-norfangchinoline 3.9
canadine 2.6
Fumaria vaillantii
Fumariaceae isoquinoline bulbocapnine 2.0 103
Loisel.
protopine 1.8
Commercially steroidal -chaconine 17

106
available glycoalkaloids -solanine 14
a
inhibition at [μM]; NS: data not shown
Along with physostigmine (1 ), two other AChE IC50 = 0.079 μg/ml) The compound responsible for this
inhibitors were isolated from Physostigma venenosum activity was not identified but the absence of huperzine A,
(Fabaceae). Physovenine was as potent as physostigmine huperzine B and N-demethylhuperzinine, three known active
whereas the activity of Na-norphysostigmine was lower [91]. alkaloids [97], was demonstrated by GC-MS [98].
Fifteen active indole alkaloids were isolated from Seven indole glucoalkaloids from Chimarrhis turbinata
Haplophyton crooksii (Apocynaceae), which has been (Rubiaceae) were tested for their inhibitory activity against
reported to possess neurotoxic properties. The most active of AChE using a TLC test. Turbinatine (2 7 ) and
these alkaloids (16-decarbomethoxyvinervine, IC50 = 57 μM) desoxycordifoline inhibited the enzyme at 0.1 and 1.0 μM.
was about 38 times less active than physostigmine (IC95 = Turbinatine showed a moderate activity (IC50 = 1.86 μM)
4.8 μ M). This could be a partial explanation for the compared to galanthamine (3) (IC50 = 0.92 μM) [99].
neurotoxicity of these compounds and of H. crooksii but the Among the five steroidal alkaloids isolated from
ganglion-blocking activity should still be tested [92]. Fritillaria imperialis (Lilliaceae), impericine (IC50 = 67.97
O
H N OGlu
H3C O N
O H H
N OH
26 27 H3CO
O OH
Among the Ranunculaceae, two species with inhibitory μM), delavine (IC50 = 105.5 μM) and persicanidine (IC50 =
activity against AChE were studied. Aconitum falconeri has 352.2 μM) showed an activity against AChE. All these
yielded two moderately inhibitory alkaloids, faleoconitine compounds were more selective inhibitors of BuChE [100].
(IC50 = 293 μM) and pseudoaconitine (IC50 = 278 μM) [93]. Another non-competitive alkaloid inhibitor of AChE was
Three other alkaloids with inhibitory activities (berberine isolated from Evodia rutaecarpa (Rutaceae). Dehydro-
(21), palmitine (24), coptisine) were isolated from another evodiamine (28) (IC50 = 37.8 μM) was tested in vivo and a
Ranunculaceae, Coptis japonica. Their potency was single administration of 6.25 mg/kg to rats significantly
comparable to that of physostigmine (1) in the order reversed scopolamine-induced memory impairment. The
berberine > palmitine > coptisine. But they all could enhance antiamnesic effect was more potent than that of tacrine. It
the nerve growth factor-induced neurite outgrowth which was thought to be due to the combined effects of AChE
could be useful to restore and maintain neural cells [94]. inhibition and cerebral blood flow enhancement [101].
Lycopodium species (Lycopodiaceae) produce a number Three compounds isolated from Stephania tetrandra
of structurally diverse alkaloids. Most of the compounds (Menispermaceae) showed an anti-AChE activity:
with AChE inhibition activity belong to the lycodine class, fangchinoline (29) (IC50 = 3.2 μM), atherospermoline (IC50 =
most notably huperzine A (4), huperzine B, N-methyl- 4.0 μ M) and 2’-N-norfangchinoline (IC50 = 3.9 μ M), all
huperzine B and huperzinine [63]. An alkaloid from the phenolic bisbenzylisoquinoline alkaloids. To see what group
fawcettimine class showed an activity comparable to that of or configuration of the molecule was most important for the
(±)-huperzine A (IC50 = 1.6 μM); sieboldine A (26) inhibited activity, some alkaloids were derived from tetrandrine (an
AChE with an IC50 value of 2.0 μM [95]. Huperzia saurus inactive non-phenolic alkaloid) and from atherospermoline.
(Lycopodiaceae) is used in traditional medicine to stimulate It transpired that the active compounds all had a phenolic
memory but shows severe adverse effects such as hydroxyl group at the 7-position and S,S configuration at the
convulsions, vomiting, diarrhea, especially when used as a 1 or 1’-positions [102].
decoction. These symptoms are the same as those observed Some Fumaria species (Fumariaceae) showed high
after ingestion of a decoction of Lycopodium selago which inhibitory activity against AChE (inhibition rate at 1 mg/ml:
contains huperzine A [96]. Ortega et al. investigated the anti- F. asepala 91.99 %, F. capreolata 96.89 %, F. cilicica 88.03
AChE activity of the alkaloid extract of H. saurus (IC50 = %, F. densiflora 93.42 %, F. judaica 96.47 %, F. kralikii
0.58 μg/ml, reference inhibitor physostigmine (1) salicylate 84.98 %, F. macrocarpa 93.43 %, F. parviflora 87.02 %, F.
O H3CO
OCH3
N N CH3
H 3C N
OH
N+ H H
N O
H
H 3C O
28 29
OCH3
petteri subsp. thureti 90.92 % and F. vaillantii 94.23 %). Huperzia serrata (Lycopodiaceae). Although alkaloids,
Three isoquinoline alkaloids with inhibitory activity were especially those present in the Amaryllidaceae, are well-
isolated from F. vaillantii - protopine (22) (IC50 = 1.8 μM), known AChE inhibitors, other natural compounds possess
bulbocapnine (IC50 =2.0 μM) and canadine (IC50 = 2.6 μM). this property. Attempts are currently being made to discover
They were all strong inhibitors compared to galanthamine non-alkaloidal inhibitors so as to avoid the side effects which
(3) (IC50 = 5.8 μM). The activity of the extract may be due to have been recorded with alkaloids.
a synergistic interaction between these alkaloids [103].
The methanolic extract of Artemisia asiatica AChE Inhibitors from Plants
(Asteraceae) showed an inhibitory activity (48 % of The majority of non-alkaloidal inhibitors discovered in
inhibition at 300 μg/ml). The active compound isolated was plants are terpenoids. Many studies have been made on
found to react positively to Dragendorff’s reagent, which monoterpenes, but sesquiterpenes, diterpenes and triterpenes
typically reveals alkaloids. It was a mixed inhibitor with an are also good candidates in the search for new potential
IC50 of 23 μ g/ml. This alkaloid also showed a capacity to treatments for AD.
reduce the toxicity induced by -amyloid peptide [104]. Miyazawa and co-workers have studied various widely
Some potato glycoalkaloids showed inhibitory activity occurring monoterpenes with a p-menthane skeleton such as
against AChE. -Chaconine (IC50 = 17 μM) and -solanine limonene or terpinene. They found that all tested compounds
(IC50 = 14 μM) inhibited bovine and human AChE whereas were competitive inhibitors of AChE, terpene ketones (such
tomatine, solanidine and solasodine produced negligible as (+)-pulegone) being stronger inhibitors than terpene
inhibition. Although the in vitro activity was much lower alcohols (such as (-)-menthol). The terpene hydrocarbons
than physostigmine (1), it could still contribute to the toxic showed the same inhibition as the terpene alcohols, except _-
effects exerted by these compounds on mammals and insects terpinene (31) and (+)-p-menth-1-ene that were as strong
in vivo [105, 106]. inhibitors as the terpene ketones [110] (Table 5).
9,10-Dihydroergotalkaloids were also investigated. 9,10- Keane and Ryan have studied some monoterpenes
Dihydroergokryptine (Ki = 198 μmol), 9,10-dihydroergot- associated with plant defence and also observed for all of
amine (Ki = 144 μmol) and 9,10-dihydroergocristine (Ki = them a reversible competitive inhibition [111]. Terpenoids
117 μmol) showed a mixed-type inhibition against AChE. are often the main constituents of essential oils. Thus Perry
Ergotamine (30) (Ki = 15 μmol) is a more potent inhibitor et al. have studied essential oil from sage (Salvia spp.,
with the same type of inhibition [107]. Lamiaceae) which is traditionally used to enhance memory:
it appeared that essential oil was active both in vitro and in
vivo, and first this activity was supposed to be due to a
synergy between monoterpenoids, or to minor highly active
H CONH CH3 OH
O components, since the main tested monoterpenes were less
N active than sage oil [112, 113, 114, 115]. The hypothesis of
H
N synergistic effects was supported by Houghton who obtained
CH3 O O almost the same results [116]. Finally it was observed that
H H
the inhibitory activity of sage essential oil was due to both
synergistic and antagonistic interactions between its
HN 30 constituent terpenoids [117].
In S. miltiorhiza, inhibition was mainly due to agonistic
interactions between diterpenes such as dihydrotanshinone or
cryptotanshinone (IC50 = 1.0 and 7.0 M respectively) [118]
Some compounds were isolated from insects. Alkaloids (Table 6). Although sage oil is not currently used in the
from the classes of 2,5-dialkylpyrrolidines and pyrrolines are treatment of AD, studies have showed that it improved
the major constituents of ant’s poison gland of the species memory, without however showing that this effect is related
Monomorium minutum (Reticulitermes). In vitro anti-AChE to an inhibition of AChE [119, 120]. Synergistic interactions
activity of four alkaloids (two of each class) was determined. were also observed with terpenoids from M e n t h a
The 2,5-dialkylpyrrolidine compounds showed Ki of 2.5 and (Lamiaceae) essential oils. In M. aquatica oil (IC50 = 26
1.5 μ M and the pyrrolines Ki of 5 and 13 μ M. These g/ml), activity was mainly due to the sesquiterpenes elemol
products (not named) are competitive inhibitors less potent and viridiflorol (32), whereas in M. arvensis, M. gentilis and
than tacrine (Ki = 10 nM) but are comparable in activity to M. spicata it was due to monoterpenes such as piperitenone
neostigmine or physostigmine (1) (Ki = 1 μM) [108]. oxide [121] (Table 6). Lime essential oil (Rutaceae) was also
Another alkaloid inhibitor was isolated from an insect found to inhibit AChE, this activity being certainly due to
source. Harmonine was found in Aiolocaria hexaspilota and terpenoids [112]. Monoterpenes were also proved to be
Harmoni axyridis (Coccinellidae) and showed a weak partially responsible for the inhibitory activity of tea tree oil
inhibitory activity toward AChE [109]. (Melaleuca alternifolia, Myrtaceae), which gave IC50 values
varying between 0.05 and 0.11 l/ml according to the
NON-ALKALOID INHIBITORS samples [122] (Table 7).
At present, the only natural approved agents for the Among terpenoids, some triterpenes have also exhibited
treatment of AD are alkaloids, such as galanthamine, or an anti-AChE activity. For example, ursolic acid from
huperzine A. The latter is marketed in the form of Origanum majorana (Lamiaceae) [123], and argentatin A
supplements, consisting of highly purified extracts of (33) from Parthenium argentatum (Asteraceae) [118]
Table 5. Monoterpene inhibitors of acetylcholinesterase
Compound Inhibition Source Ref.

a
(-)-bornyl acetate 4.7 mM 111, 116
a
(-)-carvone 1.38 mM 110
citral 0.33 mM a 111
-terpinene 22.6% (1.2 mM) b
110
-terpinene (31) 1 mM a 110
b
p-cymene 39.8% (1.2 mM) 110
a
(-)-isopulegol 2.0 mM 110
(-)-limonene 25% (1.2 mM) b 110
b commercially available
(-)-menthol 38.5% (1.2 mM) 110
(-)-menthone 1.42 mM a 110
a
(+)-carvone 1.85 mM 110
a
(+)-isomenthone 1.57 mM 110
(+)-limonene 22% (1.2 mM) b 110
a
(+)-menthol 2 mM 110
a
(+)-p-menth-1-ene 1.64 mM 110
(+)-pulegone 0.89 mM a 110, 111
(-)-terpinen-4-ol 21.4% (1.2 mM) b 110
(+)-terpinen-4-ol 24.4% (1.2 mM) b Melaleuca alternifolia Cheel (Myrtaceae) 110, 122
1,8-cineole 0.67 mM a Melaleuca alternifolia Cheel (Myrtaceae) Mentha spp.,
122, 111, 121, 113, 115, 116
Salvia lavandulaefolia (Lamiaceae)
piperitenone 110 μg/mL a
121
Mentha spp. (Lamiaceae)
piperitenone oxide 64 μg/mL a
121
-pinene 0.63 mM a 113, 115, 116
_-pinene 0.2 mg/mL a 113, 117, 116
a
linalool > 5 mM Salvia lavandulaefolia (Lamiaceae) 111, 113
geraniol > 5 mM a 113
camphor > 4.7 mM a 115, 116
> 10 mM a 113
a
IC50; b percentage inhibition
Table 6. Sesquiterpene, diterpene, and triterpene inhibitors of acetylcholinesterase
Compound class Compound Inhibition (ic50) Source Ref.
elemol 34 μg/mL
sesquiterpene Mentha aquatica (Lamiaceae) 121
viridiflorol (32) 25 μg/mL
dihydrotanshinone 1 μM
tanshinone II A > 150 μM
diterpene Salvia miltiorhiza Bunge (Lamiaceae) 118
tanshinone I > 50 μM
cryptotanshinone 7 μM
argentatin A (33) 42.8 μM Parthenium argentatum Gray (Asteraceae) 118

triterpene
ursolic acid 7.5 nM Origanum majorana L. (Lamiaceae) 123
inhibited AChE with IC50 values of 5.2 M, 7.5 nM, and exhibited activity, with an IC50 of 28 M for decursinol, the
42.8 M respectively. most active of them all, and suggested some hypotheses to
Recently, some authors have investigated coumarins for explain the relation between structure and activity [124].
AChE inhibition activity (Table 8). The first, Kang et al. Then other Angelica species were studied and gave
showed that coumarins from Angelica gigas (Apiaceae) similar results: furanocoumarins isolated from roots of A.
H3C OH
CH3
CH3
H3C H3C
CH3 CH3 O
CH3
H 3C
H OH
CH3
CH3
HO CH3
O
31 32 H3C CH3 33
Table 7. Essential oil inhibitors of acetylcholinesterase
Essential oil Inhibition Source Ref.
tea tree oil 0.05 to 0.11 μL/mL a Melaleuca alternifolia Cheel (Myrtaceae) 122
mint oils 26 to 164 μg/mL a Mentha spp. (Lamiaceae) 121
lime oil 65.8% (0.1 μL/mL) b
Citrus aurantifolia Swingle (Rutaceae) 112
balm oil 76 to 100% (0.1 μL/mL) b Melissa officinalis L. (Lamiaceae) 112
sage oil 0.03 μg/mL a
Salvia lavandulaefolia (Lamiaceae) 112, 113, 115, 117, 116
sage oil 34 to 47 % (0.03 μg/mL) b
Salvia officinalis (Lamiaceae) 112,116
a
IC50;b percentage inhibition
dahurica also inhibited AChE [125], as did those obtained H 3C CH3

from A. acutiloba [126]. Coumarins from Murraya
paniculata (Rutaceae), murranganone and paniculatin (34), O
exhibited almost the same IC50 as furanocoumarins [127]. O
Scopoletin (35) and its glucoside scopolin were detected as O CH3 H3CO
AChE inhibitors even before their isolation from plants:
indeed, these coumarins were selected as potential H3CO O CH3 O HO O O
candidates during a virtual screening with pharmacophore
models, and only then purified from the rhizomes of 35
Scopolia carniolica (Solanaceae). In spite of their moderate
34
in vitro activity (IC50 = 168.6 M for scopoletin), they
proved to be also active in vivo, with an increase of
acetylcholine release of 170 and 300 % for scopoletin and In 2004, another class of non-alkaloidal compounds was
scopolin respectively (injections into the cerebral ventricle of identified: four xanthones (36-39) isolated from Gentiana
rats of 2 mol) [128]. Toxicity studies carried out on campestris (Gentianaceae) were shown to inhibit the enzyme
coumarins in human beings are encouraging [129], and [130] (Table 9). Brühlmann and co-workers have found
further studies could confirm the future possible use of those other non-competitive inhibitory xanthones among
non-alkaloidal metabolites to treat AD symptoms. Gentianaceae, Clusiaceae, and Polygalaceae [21].
OH OH
O OH O OCH3
OH O OH OH O OH
36 37
OH OH
O OH O OCH3
H OH H OH
H H
O O
HO O O OH HO O O OH
HO HO
H OH H OH
H H 38 H H 39
Table 8. Coumarin inhibitors of acetylcholinesterase
Compound Ic50 (μm) Source Ref.
decursinol 28 Angelica gigas Nakai (Apiaceae) 124

marmesin 67 Angelica gigas Nakai (Apiaceae) 124
nodakenin 68 Angelica gigas Nakai (Apiaceae) 124
isoimperatorin 69 124
Angelica gigas Nakai, Angelica dahurica Maxim (Apiaceae)
74.6 125
xanthotoxin 54 Angelica acutiloba (Siebold & Zucc.) Kitag., Angelica gigas Nakai 124
(Apiaceae)
0.58 a 126
isopimpinellin 0.32 a Angelica acutiloba (Siebold & Zucc.) Kitag. (Apiaceae) 126
oxypeucedanin 89.1 Angelica dahurica Maxim. (Apiaceae) 125
imperatorin 63.7 Angelica dahurica Maxim. (Apiaceae) 125
murranganone 79.1
Murraya paniculata (L.) Jack (Rutaceae) 127
paniculatin (34) 31.6
scopolin n.d.
Scopolia carniolica Jaqc. (Solanaceae) 128
scopoletin (35) 168.6
a
IC50 (μmol/ml)
Table 9. Xanthone inhibitors of acetylcholinesterase
Compound Inhibition Source Ref.

a
1,7-dihydroxy-4-methoxyxanthone 44 Polygala nyikensis Exell (Polygalaceae) 21
a
mesuaxanthone A 53
1-hydroxy-3,5-dimethoxyxanthone 15.4 a Chironia krebsii Griseb. (Gentianaceae) 21
decussatin 28.7 a
3H,7H-pyrano[2,3-c]xanthen-7-one 1.9 a
Garcinia livingstonei T. Anders.
4-(3',7'-dimethylocta-2',6'-dienyl)-1,3,5- 26.8 a 21
(Clusiaceae)
trihydroxy-9H-xanthen-9-one
bellidin (36) 0.15 b

bellidifolin (37) 0.03 b
Gentiana campestris L. (Gentianaceae) 130
norswertianolin (38) 1.20 b
swertianolin (39) 0.18 b
a
K inc (μM)
b
minimal inhibitory quantity on TLC (nmol)
H3C
O
OH
CH3
HO
O
H3C H3C
O O O
HO O O O
CH3
OCH3 OCH3 OCH3 40
Four pregnane glycosides were isolated from Cynanchum obtained from Fiatoua villosa (Moraceae) inhibited AChE in
atratum (Asclepiadaceae) and tested for AChE inhibitory vitro with an IC50 of 0.11 mM [133], and two flavonoids
activity: they were all active, with IC50 values varying were moderate AChE inhibitors: quercetin 3,3'-dimethyl
between 3.6 M for cynatroside B (40) and 152.9 M for ether from Psiadia trinervia (Asteraceae) [21], and
cynascyroside D [131, 132] (Table 10). naringenin (4 2 ) isolated from Citrus junos Siebold
Among other non-alkaloidal compounds that were found (Rutaceae) [134]. Furthermore, polyphenols from tea,
to inhibit AChE, zeatin (41), a purine-adenine derivative without having been clearly identified, were also inhibitors
[135, 136].
Table 10. Miscellaneous non-alkaloidal inhibitors of acetylcholinesterase from plants
Compound class Compound Inhibition (ic50) Source Ref.
cynatroside A 6.4 μM 131,132

cynatroside B (40) 3.6 μM Cynanchum atratum Bunge 131,132
pregnane glycoside
cynatroside C 52.3 μM (Asclepiadaceae) 131,132
cynascyroside D 152.9 μM 131
4',5,7-trihydroxy-3,3'- 20.6 a 21
Psiadia trinervia Willd. (Asteraceae)
flavonoid dimethoxyflavone
Citrus junos Siebold (Rutaceae)
naringenin (42) 66% (210 μg/mL) b 134
purine adenine-derivative zeatin (41) 0.11 mM Fatoua villosa Nakai (Moraceae) 133
tea (Camellia sinensis Kuntze,
phenolic compound polyphenols 248 μg/mL 136,135
Theaceae)
a
K inc (μM)
b
percentage inhibition
H
N N OH O
N
OH N
NH HO O
OH
CH3 41 42
AChE Inhibitors from Microorganisms metabolites with mixed polyketide-terpenoid structures.

Some authors have investigated microorganisms, mainly Some of them, named territrems, were discovered in
fungi, in order to find new AChE inhibitors (Table 11). Aspergillus terreus and proved to be potent inhibitors of
AChE [137, 138]. Among them, territrem B (43) was studied
Several compounds exhibiting an anti-AChE activity in
in depth and appeared to bind to AChE in an irreversible and
vitro were found among the meroterpenoids, a family of
noncovalent way [139]. Terreulactones A-D and
Table 11. Non-alkaloidal inhibitors of acetylcholinesterase from microorganisms
Compound class Compound Ic50 (μm) Source Ref.
terreulactone A 0.23 143, 140, 141

terreulactone B 0.09 143, 141
terreulactone C 0.06 143, 141
terreulactone D 0.42 143, 141
isoterreulactone A (44) 2.5 142
Aspergillus terreus (Trichomaceae)
territrem A 24 a 138
territrem B (43) 19 a 138,139
meroterpenoid territrem C 15 a 138
territrem A’ 98 a 138
territrem B’ 92 a 138
arisugacin A 1.0 a 144

arisugacin B 25.8 a 144
Penicillium sp. FO-4259 (Trichomaceae)
arisugacin C 2.5 146
arisugacin D 3.5 146
quinolactacin A1 280 Penicillium citrinum 90648

quinolone 150
quinolactacin A2 (45) 19.8 (Trichomaceae)
aflatoxin aflatoxin B1 (47) 31.6 Aspergillus flavus (Trichomaceae) 154

0.76 a Streptomyces lavandulae 151
organo-phosphorus cyclophostin (46) 1.3 a (Streptomycetaceae)
compound anatoxin-a(s) (48) Anabaena flos-aquae NRC-525-17
(Cyanophycae) 155
n.d.
a
IC50 (nM)
OCH3
OCH3 OCH3
O O O O
OCH3
O
CH3 CH3 CH3 CH3
O O
OH 43 O OH
44
O
OH OH
H3C H3C CH3
CH3
isoterreulactone A (44), other meroterpenoids from A . Aspergillus terreus [152]. Aflatoxin B1 (47) from A. flavus
terreus, acted also as inhibitors of AChE [140, 141, 142]. appeared also to be a non-competitive inhibitor of AChE
Arisugacins A and B, from Penicillium sp. FO-4259, (IC50 = 31.6 M), but because of its high toxicity (LC50 = 1
strongly and selectively inhibited AChE, with IC50 values of mg/kg) it is evident that this compound cannot be considered
1.0 and 25.8 nM respectively [143, 144]. However, some as a potential treatment in Alzheimer’s disease [153].
meroterpenoids are not active: thus, whereas arisugacins C Besides fungi, a blue-green alga, more precisely a
and D obtained from a mutant strain of Penicillium exhibited cyanobacterium, Anabaena flos-aquae, was found to contain
an activity against AChE, arisugacins E-H did not have any an irreversible inhibitor of AChE named anatoxin-a(s) (48)
effect on the enzyme at 100 M [145]. As for territrem B, [154, 155]. As for aflatoxin B1, this compound is too toxic to
Peng suggested that both the enone and the pyrone moieties imagine a therapeutic use (LC50 = 0.05 mg/kg).
may play an important role in the intensity and selectivity of
the inhibition [146]. Meroterpenoids are also present in AChE Inhibitors of Animal Origin
animals, such as marine sponges [147], and in plants [148], Until now, few non-alkaloidal AChE inhibitors have
but to our knowledge, cholinesterase inhibition was not been found in animals. The majority have been discovered in
investigated in these organisms. marine organisms, mainly in invertebrates (Table 12).
Among other types of inhibitory compounds produced by For example, a novel polymeric pyridinium compound
microorganisms, some quinolones from Penicillium has been isolated from the marine sponge Reniera sarai
citrinum, quinolactacins A1 and A2 (45), also exhibited an (Haliclonidae), consisting of a mixture of two polymers
activity against AChE [149]. Cyclophostin (4 6 ), a composed of 29 and 99 repeating N-butyl(3-
phosphorus-containing metabolite isolated from butylpyridinium) units (49). Both monomer units and
Streptomyces lavendulae, inhibited AChE and BuChE with polymers exhibited an inhibition of AChE [156]. The
IC50 values of 1.3 and 45.0 nM respectively [150]. Other monomer showed mixed reversible inhibition whereas the
organophosphates structurally close to cyclophostin, CGA polymer presented an unusual evolving inhibition pattern,
134735 and CGA 134736, were isolated from Streptomyces suggesting several binding sites on the enzyme.
antibioticus and showed, in addition to an insecticidal Another sponge gave aplysamine-4 (50), a bromotyro-
activity, an action toward AChE [151]. Another fungal sine-derived metabolite, which was found to be a non-
cholinesterase inhibitor, named I-6123, was obtained from competitive reversible inhibitor of acetylcholinesterase (Ki =
O O H
O O
NH CH3
O P
N O O
CH3 O
H3C H 3C
CH3
45 46
O
O CH3
O H 2C N
H CH3
HN N
O O O
P
H O OCH3 NH HO OCH3
47 48
Table 12. Non-alkaloidal inhibitors of acetylcholinesterase of animal origin
Compound class Compound Inhibition Source Ref.

[N-butyl(3-butylpyri-
alkylpyridinium polymer n.d. Reniera sarai (Haliclonidae) 157
dinium)]29 to 99 (49)
bromotyrosine derivative aplysamine-4 (50) 16 a Red Sea marine sponge 158
b
diiodotyramine derivative turbotoxin A (52) 28 Turbo marmorata (Turbinidae) 161
onchidal (51) n.d. Onchidella binneyi (Onchidiidae) 159
tetrazacyclopentazulene 4 μM c Parazoanthus axinellae
parazoanthoxanthin A 160, 167, 168
derivative 19 μM c, 26 μM c (Parazoanthidae)
fasciculin n.d. 169, 162, 163
Dendroaspis angusticeps (Elapidae)
peptide
animal brain
enkephalins n.d. 165, 166
c
2-heptanone 1.34 mM bee (Apidae) 111
pheromone ipsenol 9.3 mM c insects 164
frontalin 0.27 mM c insects 164
a
K inc (μM); b IC50 (μM); c K ic
OCH3
H2N Br Br
Br OH
N O N
n = 29, 99 Br N O
H
49 50
16 M). In addition, this compound also exhibited a weak derivative turbotoxin A (52) from Turbo marmorata
anti-bacterial activity [157]. (Turbinidae) which inhibits AChE with an IC50 of 28 M but
Molluscs are also a source of new inhibitors. Abramson which has also a LD99 value of 1.0 mg/kg [160].
and co-workers had the idea to study a compound secreted
by Onchidella binneyi (Onchidiidae) because of its similar CH3
structure to acetylcholine: indeed, onchidal (51) was an CH3
acetate ester and appeared to be a substrate for AChE. O O N+
Binding and hydrolysis of onchidal resulted in a progressive CH3
O CH3
irreversible inhibition of the enzyme. The authors suggested CH2
that this inhibition may result from formation of a novel
covalent bond between the enzyme and the compound due to H I I CH3
its reactive ,-unsaturated aldehyde [158]. CH3
O N+
CH3
A yellow pigment from a zoanthid crust coral, a CH3 CH3
pseudozoanthoxanthin-like compound, was investigated for 51 52
biological activities, and appeared to inhibit AChE in a
competitive fashion (Ki = 4 M). The crude extract Other anti-AChE neurotoxins from animals are
containing this pigment was tested on experimental animals fasciculins. These peptides purified from the venom of green
and was revealed to be very toxic: doses of 4 mg/mouse mamba (Dendroaspis angusticeps, Elapidae) inhibited brain
killed mice in 5 min [159]. The pure compound, although and muscle AChE, inducing muscle fasciculations [161,
inducing qualitatively the same symptoms which preceded 162].
mice death, was not lethal at the tested levels, so further
The small molecule 2-heptanone is an alarm pheromone
investigations are needed to assert whether toxicity is due
produced by honeybees and ants, which acts as a repellent.
only to this pseudozoanthoxanthin-like compound, or to an
In humans it causes skin, eyes, and respiratory tract
association with other lethal compounds.
irritations, and affects also the central and peripheral nervous
Sometimes, as mentioned above, cholinesterase inhibitors system. This compound acted as a competitive inhibitor of
are so toxic that they cannot be used as potential treatments AChE (Ki = 1.34 mM), which may explain its neurotoxicity
in AD. This is the case, for example, of the diiodotyramine [111]. Ryan and co-workers have previously studied the
inhibition of AChE by various insect pheromones: all 17 [19] Ingkaninan, K.; de Best, C.M.; van der Heijden, R.; Hofte, A.J.P.;
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Natural Product Inhibitors of Acetylcholinesterase

Article in Current Organic Chemistry · May 2006

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Natural Product Inhibitors of Acetylcholinesterase

K. Hostettmann*, A. Borloz, A. Urbain and A. Marston

Laboratoire de Pharmacognosie et Phytochimie, Ecole de Pharmacie Genève-Lausanne, Université de Genève, CH-

INTRODUCTION Currently, the only approved treatment to prevent the

1385-2728/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

O 2N S S NO2 + (CH3)3N+CH2CH2SS NO2

Fig. (1). Ellman's reaction.

Naphthyl acetate α-naphthol

implicated in the etiology and progression of Alzheimer's (P11012) and 6-O-demethyl-6-O-[(adamantan-1-yl)-

were very potent inhibitors and showed interesting properties Amaryllidaceae

Table 1. Amaryllidaceae alkaloids as inhibitors of acetylcholinesterase

sanguinine (5) 0.10

Table 2. Buxaceae alkaloids as inhibitors of acetylcholinesterase

Sarcococca epoxynepapakistamine-A > 200

buxakashmiramine (19) 25.4

Buxaceae A showed a greater activity on AChE than on BuChE (IC50 =

Table 3. Papaveraceae alkaloids as inhibitors of acetylcholinesterase

protoberberine berberine (21) 76a

diisoquinoline berberine (21) 0.5c / 0.98d / 0.23 e

Table 4. Alkaloids from other families as inhibitors of acetylcholinesterase

Lycopodiaceae huperzine A (4) 0.1

Chimarrhis turbinata indole turbinatine (27) 1.86

Commercially steroidal -chaconine 17

Table 5. Monoterpene inhibitors of acetylcholinesterase

Compound Inhibition Source Ref.

Table 6. Sesquiterpene, diterpene, and triterpene inhibitors of acetylcholinesterase

Compound class Compound Inhibition (ic50) Source Ref.

argentatin A (33) 42.8 μM Parthenium argentatum Gray (Asteraceae) 118

Table 7. Essential oil inhibitors of acetylcholinesterase

Essential oil Inhibition Source Ref.

dahurica also inhibited AChE [125], as did those obtained H 3C CH3

Table 8. Coumarin inhibitors of acetylcholinesterase

Compound Ic50 (μm) Source Ref.

decursinol 28 Angelica gigas Nakai (Apiaceae) 124

Table 9. Xanthone inhibitors of acetylcholinesterase

Compound Inhibition Source Ref.

bellidin (36) 0.15 b

Table 10. Miscellaneous non-alkaloidal inhibitors of acetylcholinesterase from plants

Compound class Compound Inhibition (ic50) Source Ref.

cynatroside A 6.4 μM 131,132

AChE Inhibitors from Microorganisms metabolites with mixed polyketide-terpenoid structures.

Table 11. Non-alkaloidal inhibitors of acetylcholinesterase from microorganisms

Compound class Compound Ic50 (μm) Source Ref.

terreulactone A 0.23 143, 140, 141

arisugacin A 1.0 a 144

quinolactacin A1 280 Penicillium citrinum 90648

aflatoxin aflatoxin B1 (47) 31.6 Aspergillus flavus (Trichomaceae) 154

Table 12. Non-alkaloidal inhibitors of acetylcholinesterase of animal origin

Compound class Compound Inhibition Source Ref.

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