e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/ejps

In vitro screening assays to identify natural or synthetic

acetylcholinesterase inhibitors: Thin layer
chromatography versus microplate methods

Saviana Di Giovanni a , Aline Borloz b , Aurélie Urbain b , Andrew Marston b ,

Kurt Hostettmann b , Pierre-Alain Carrupt a , Marianne Reist a,∗
a LCT-Pharmacochimie, Ecole de Pharmacie Genève-Lausanne, Section des Sciences Pharmaceutiques, Quai Ernest-Ansermet 30,
CH-1211 Genève 4, Switzerland
b Laboratoire de Pharmacognosie et Phytochimie, Ecole de Pharmacie Genève-Lausanne, Section des Sciences Pharmaceutiques,

Quai Ernest-Ansermet 30, CH-1211 Genève 4, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Acetylcholinesterase inhibitors (AChEI) are currently still the best available pharmacother-
Received 28 August 2007 apy for Alzheimer patients. Successful screening for new AChEI relies on effective and fast
Received in revised form assays. Two colorimetric screening assays frequently used to search for new AChEI, namely
17 October 2007 a thin layer chromatography (TLC) assay with Fast Blue B salt as reagent and a 96-well plate
Accepted 23 October 2007 assay based on Ellman’s method, were compared. For the majority (83%) of the 138 test
Published on line 30 October 2007 compounds of natural and synthetic origin, the results obtained with the two assays con-
verged and both screening assays were considered suitable for the generation of new hits.
Keywords: Fifteen percent of investigated compounds were classified as active with the microplate
Acetylcholinesterase inhibitors assay but were shown to be inactive by TLC and about 2% were measured active by TLC but
Thin layer chromatography showed to be inactive with the microplate assay. These divergences were not due to the
Microplate method main differences between the experimental protocols of the two screening assays, namely
Screening the different colorimetric methods and pre-incubation of test compounds with acetyl-
Alzheimer’s disease cholinesterase (AChE). They might be explained by the interaction of either AChE or test
Ellman’s method compounds with the silica of the TLC plates, resulting in an altered affinity of the enzyme
Fast blue B salt for the compounds.
© 2007 Elsevier B.V. All rights reserved.

1. Introduction (ACh) by nearly 90%. On the behavioral side, the impor-

Alzheimer’s disease (AD) is the leading cause of dementia
among older people. An estimated 10% of the world’s popu-
lation over the age of 65 years is afflicted by AD (Racchi et
al., 2004). The most important changes observed in the brain
of AD patients are the appearance of neuritic plaques and
neurofibrillary tangles, as well as a decrease in hippocam-
pal and cortical levels of the neurotransmitter acetylcholine
tant decrease of ACh levels causes impairment in cognitive
function (Cummings, 2004; Selkoe, 1996). This “cholinergic
hypothesis of AD”, formulated for the first time by Whitehouse
et al. (1982), was postulated on the basis of the negative cog-
nitive effect of cholinergic antagonists and the positive effect
of muscarinic agonists on memory in humans (Guillou et al.,
2000) and became the neurobiologic incentive for a treatment
aiming at the improvement of cholinergic function in AD.

∗
Corresponding author. Tel.: +41 22 379 33 65; fax: +41 22 379 33 60.
E-mail address: marianne.reistoechslin@pharm.unige.ch (M. Reist).
0928-0987/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2007.10.004
110 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119
Acetylcholinesterase (AChE) is the enzyme involved in the
metabolic hydrolysis of ACh at cholinergic synapses in the
central and peripheral nervous system. Thus, inhibitors of
AChE activity (AChEIs) promote an increase in the concentra-
tion and duration of action of synaptic ACh (Rollinger et al.,
2004). Currently, AChEIs represent the therapy of choice for
AD (Martinez and Castro, 2006).
About 50% of the drugs introduced on the market dur-
ing the last 20 years have been derived directly or indirectly
from small molecules of natural origin (Newman and Cragg,
2007). Their therapeutic potential has also been success-
fully demonstrated in the field of AD (Hostettmann et al.,
2006; Howes and Houghton, 2003). Hence, nature can be con-
sidered as an important source of new chemical entities
potentially interesting for the treatment of various diseases,
including AD. In addition, several interesting compounds were
recently synthesized and tested for their ability to inhibit AChE
(Brühlmann et al., 2001).
In order to speed up the screening for AChE inhibitors
of natural and synthetic origin, effective and fast assays are
needed. In the past years, several methods for the determi-
Fig. 1 – Chemical structures of reference AChEIs tested.
nation of AChE activity have been described. These include
colorimetric methods using Ellman’s reagent (Ellman et al.,
1961) or Fast Blue B salt reagent, fluorometric assays with flu-
orogenic substrates, and pH-metric or electrochemical activity
detection of AChE (Rhee et al., 2003). They have been used to
quantify AChE in tissue (Ren et al., 2002), to study anticholin-
ergic agents for use as insecticides and pesticides (Hawkins
and Knittle, 1972; Motoyama et al., 1980; van Asperen, 1962),
and to screen for AChE inhibitors of interest for the treatment
of AD (Ingkaninan et al., 2003; Rhee et al., 2001; Rhee et al.,
2003).
The choice of an appropriate screening assay is cru-
cial. Indeed, biological activities of natural and synthetic
compounds obtained with different tests and/or by dif-
ferent research groups can be quite variable (Buenger et
al., 2006; Di et al., 2007; Lam, 2007). Hence, the aim of
this study was the comparison of two colorimetric screen-
ing assays frequently used to detect acetylcholinesterase
inhibitory activity, namely a TLC assay using Fast Blue B salt
as reagent, previously reported for the detection of acetyl-
cholinesterase inhibitors in plants (Marston et al., 2002), and
 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119
a microplate assay using Ellman’s method (Brühlmann et al.,
2004).
The test set of 138 compounds consisted of seven refer-
ence acetylcholinesterase inhibitors (Fig. 1) and compounds
chosen from a focused screening library for the identification
of dual inhibitors of acetylcholinesterase and monoamine oxi-
dase B composed of compound classes reported to have either
one or both of these activities (Brühlmann et al., 2001, 2004;
Gnerre et al., 2001; Kneubühler et al., 1995). These are 7 com-
pounds of natural origin (4 coumarins, 1 xanthone, 1 terpene,
and 1 alkaloid), 77 compounds based on scaffolds of natu-
ral origin (47 coumarins, 13 aurones, 12 azaaurones, and 5
chromones), and 47 synthetic compounds (45 5H-indeno-[1,2-
c]-pyridazin-5-ones, 5-(benzyloxy)benzo[d][1,3]dioxole, and
5-(benzyloxy)isoindoline-1,3-dione) (Fig. 2).
For the majority of compounds tested, the results obtained
with the two screening assays were convergent. However,
about 15% of investigated compounds testing positive in the
Fig. 2 – Chemical structures of compound classes examined.
111
microplate assay resulted inactive in the TLC test and about
2% of investigated compounds measured active in the TLC
assay were shown to be inactive with the microplate assay.
Hence, this divergence in the identification of AChE inhibitors
observed between the two screening assays was further inves-
tigated and the advantages and inconveniences of the two
tests were discussed.
2. Experimental
2.1. Materials
Acetylcholinesterase from Electrophorus electricus (E.C.3.1.1.7,
Sigma C 2888), acetylthiocholine iodide (ATCI), 5,5 -dithiobis-
(2-nitrobenzoic acid) (DNTB) and the reference compounds
galanthamine hydrobromide, physostigmine hemisulfate,
propidium iodide, (±)-huperzine A and tacrine hydrochlo-
112 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119
ride were purchased from Sigma–Aldrich Chemical (St. Louis,
MA, USA). DMSO (microselect for molecular biology), phos-
phate salts, Fast Blue B salt (O-dianisidine-bis (diazotized)
hydrochloride zinc double salt), sodium dodecylsulphate
(SDS), and ␣-naphthyl acetate came from Fluka AG (Buchs,
CH). (−)-Huperzine-A was from Sequoia Research Products
Ltd. (Oxford, UK). Donepezil hydrochloride was from Interchim
(Montluçon, France).
The four coumarins of natural origin (C-1 to C-4) were iso-
lated from Peucedanum ostruthium (Apiaceae) (Urbain et al.,
2005), the xanthone derivative from Gentiana campestris (Gen-
tianaceae) (Urbain et al., 2004), the diterpene derivative from
Detarium microcarpum (Caesalpinaceae) (Cavin et al., 2006), and
the alkaloid lycopodine from Huperzia selago (Lycopodiaceae)
(Borloz, 2007).
The synthesis of coumarin derivatives (C-5 to C-51,
n = 47), 5H-indeno [1,2-c] pyridazin-5-one derivatives (n = 45),
chromones (n = 5), 5-(benzyloxy)benzo[d][1,3]dioxole, and 5-
(benzyloxy)isoindoline-1,3-dione was performed in the Dipar-
timento Farmaco-Chimico, Università di Bari, I-70125, Bari,
Italy, as described previously (Gnerre et al., 2000; Kneubühler
et al., 1995). Aurone and azaaurone derivatives (n = 25) were
synthesized in the Département de Pharmacochimie Molec-
ulaire, UMR CNRS 5063, Faculté de Pharmacie de Grenoble,
France (Boumendjel et al., 2002; Okombi et al., 2006).
All buffers and solutions were prepared in demineralized
and purified water obtained with the Elix 3 Millipore water
purifying system, except test compound solutions that were
prepared in DMSO. All assays in solution were performed in
conventional, flat-bottomed polypropylene 96-well microtitre
plates (Evergreen Scientific, Los Angeles, USA).
2.2. TLC and microplate assays using Fast Blue B salt
as reagent
Using Fast Blue B salt as reagent, the inhibitory activity of
compounds was determined either with a TLC assay or a
microplate assay. The detailed procedure of the TLC assay
has been described previously by Marston et al. (2002). Briefly,
compounds dissolved either in ethyl acetate or in methanol,
according to their solubility, were spotted first at 1 ␮g, then at
decreasing quantities in the case of active compounds, onto
the TLC plate. (±)-Huperzine-A (0.001 ␮g) was added to each
plate as positive control. Migration was conducted with hex-
ane:ethyl acetate (1:1; v/v). Then the plate was sprayed with
an enzyme stock solution (concentration 6.67 U/ml) and pre-
incubated at 37 ◦ C for 20 min in a humid atmosphere. Ten
millilitres of the ␣-naphthyl acetate solution and 40 ml of the
Fast Blue B salt solution were mixed and sprayed onto the plate
to give a purple coloration after 1–2 min. The minimum quan-
tity of compound was noted for which an inhibition spot was
still visible.
For the microplate assay with Fast Blue B salt as reagent,
the method published by van Asperen (1962) was adapted for
96-well plates. The reaction mixture consisted of 235.7 ␮l or
188.4 ␮l of 0.1 M phosphate buffer pH 7.4, 20 ␮l of a solution of
AChE (final concentration 0.1 U/ml in 0.1 M phosphate buffer
pH 7.4), 2.7 ␮l of test compound solutions in DMSO or 50 ␮l
of test compound solutions in buffer. The controls contained
the corresponding volume of DMSO or buffer instead of test
compound solutions. The enzymatic reaction was initiated by
addition of 1.6 ␮l of ␣-naphthyl acetate solution (final concen-
tration equal to the Km value) in DMSO. The final concentration
of DMSO was 0.6 or 1.6% and the final assay volume was 260 ␮l.
After mixing for 90 s and incubation at 25 ◦ C for 90 s, the reac-
tion was stopped with 20 ␮l of a 5% solution of sodium dodecyl
sulphate (SDS) in water. The color was developed with 20 ␮l of
Fast Blue B salt solution (final concentration 0.17 mM in water).
Enzyme activity and inhibition were quantified by determina-
tion of the absorbance in end point lecture at 600 nm after
formation of the purple-colored diazonium dye, which turns
to stable blue in presence of SDS.
2.3. Microplate assay using Ellman’s method
For the microplate assay carried out following Ellman et al.
(1961), wells were filled with 227.3 ␮l or 210 ␮l of Ellman’s
reagent (0.15 mM final concentration of 5,5 -dithiobis-(2-
nitrobenzoic acid) in 0.1 M phosphate buffer pH 7.4), 20 ␮l
of a solution of acetylcholinesterase (final concentration
0.037 U/ml in 0.1 M phosphate buffer pH 7.4), and 2.7 ␮l of test
compound solutions in DMSO or 20 ␮l of test compound solu-
tions in buffer. For controls, test compound solutions were
replaced by the corresponding volume of DMSO or buffer.
The enzymatic reaction was initiated by addition of 20 ␮l
of acetylthiocholine iodide (ATCI) solution in demineralised
water (final concentration equal to the Km value). The final
assay volume was 270 ␮l. The plate was shaken for 2 s and
the increase in absorbance at 412 nm was monitored at 25 ◦ C
for 180 s using a microplate spectrophotometer (PowerWaveX ,
BioTek Instrument, Winooski, Vermont, USA). The kinetic
parameters Km and Vmax and inhibitory potencies of seven ref-
erence compounds were measured in presence of 0 (control),
0.6%, 1.6% and 2% of DMSO (Table 1).
2.4. Determination of kinetic parameters Km and Vmax
To obtain the kinetic parameters Km and Vmax , hydrolysis rates
for duplicates of six concentrations of substrate were deter-
mined. When using Ellman’s method, ATCI solutions (final
concentrations in the reaction mixture ranging from 0.09 mM
to 1.11 mM) were prepared in water. For the microplate assay
using Fast Blue B salt as reagent, ␣-naphthyl acetate solutions
(final concentrations of 0.06–1.02 mM) were prepared in DMSO.
The kinetic parameters Km and Vmax were obtained by curve
fitting according to the classical Michaelis–Menten equation
(Michaelis and Menten, 1913). Data are means of two inde-
pendents experiments, each performed in duplicate.
2.5. Expression of inhibitory activities
For the TLC assay, AChE inhibitory activities were expressed
as pMIQ, which represents the negative logarithm of the min-
imal inhibitory quantity in mole that produced the spot with
the least observable whiteness. For the two microplate assays,
the activity of a compound was expressed either as percent-
age of inhibition at a fixed concentration of 10−5 M or as pIC50 .
The percentage of inhibition was calculated relative to a con-
trol sample, for which the AChE activity was assessed under
identical conditions but in absence of test compound (con-
 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119
Table 1 – Kinetic parameters (Km and Vmax ) determined in solution in presence of different percentages of DMSO
Percent of DMSO Microtitre plate method with Ellman’s reagent
Km (mM)a Vmax (mM min−1 U−1 )a
0 0.12 ± 0.02 1.39 ± 0.06
0.6 0.21 ± 0.04 0.86 ± 0.06
1.6 0.25 ± 0.09 0.84 ± 0.11
2 0.36 ± 0.04 0.52 ± 0.02
a
Means ± standard errors (n = 4).
sidered to be 100%). Data are means of n = 3. The inhibitory
potencies, expressed as pIC50 values, represent the negative
logarithms of the molar concentrations of inhibitor required
to decrease AChE activity by 50% and were calculated by curve
fitting according to the classical sigmoidal dose–response
equation. All pIC50 values were determined in duplicate from
at least six compound concentrations, with the exception of
inhibitory potencies of seven reference AChEIs, determined
using Ellman’s method, that are means of two independent
experiments, each performed in duplicate.
2.6. Data analysis
Statistical significance was determined by the use of Stu-
dent’s t-test and differences were considered significant when
p < 0.05. Statistical tests, kinetic parameters, percents of inhi-
bition, inhibitory potencies and correlation coefficients were
calculated using Prism V4.0 (GraphPad Software Inc.).
3. Results and discussion
3.1. Effect of dimethylsulfoxide on screening tests in
solution
DMSO is usually the solvent of choice for preparing stock
solutions of molecule libraries for biological and pharmaco-
logical studies, as well as for activity screening, and was thus
also used to solubilize ␣-naphthyl acetate and the 138 tested
compounds in the present study. However, previous work
has shown that not all enzymes are organic solvent tolerant
(Jagota, 1992; Salleh et al., 2002). Thus, the influence of DMSO
on the kinetic parameters of AChE and the inhibitory potencies
of seven reference AChE inhibitors was investigated for both
colorimetric methods used, namely detection of the hydroly-
sis of acetylthiocholine with Ellman’s reagent and detection
of the hydrolysis of ␣-naphthyl acetate with Fast Blue B salt
reagent.
3.1.1. Influence of DMSO on the enzymatic activity of
AChE using acetylthiocholine and ˛-naphthyl acetate as
substrates
To assess the influence of DMSO on the enzymatic activ-
ity of AChE for the two substrates used (acetylthiocholine
and ␣-naphthyl acetate) different percentages of co-solvent
(0.6%, 1.6% and 2% final concentrations) were added to the
reaction mixtures. The kinetic parameters Km and Vmax were
determined by means of the Michaelis–Menten rectangular
113
Microtitre plate method with Fast Blue B salt as reagent
Km (mM)a Vmax (mM min−1 U−1 )a
– –
0.23 ± 0.01 0.27 ± 0.02
0.25 ± 0.03 0.26 ± 0.02
0.41 ± 0.04 0.20 ± 0.02
hyperbola and reported in Table 1. Since ␣-naphthyl acetate is
not soluble in water, its AChE-catalyzed hydrolysis in absence
of co-solvent could not be measured; a minimal final DMSO
concentration of 0.6% was necessary to solubilize this sub-
strate.
Interestingly, AChE showed the same affinity for the
two substrates acetylthiocholine and ␣-naphthyl acetate.
Moreover, the Km values obtained, i.e. 0.12 ± 0.02 mM for
acetylthiocholine in absence of co-solvent, and 0.23 ± 0.01 mM
for ␣-naphthyl acetate in presence of 0.6% of DMSO, were
in good agreement with those reported in the literature, i.e.
0.11 ± 0.01 mM for acetylthiocholine (Selwood et al., 1993) and
0.1 mM for ␣-naphthyl acetate (van Asperen, 1962). The lat-
ter was determined with acetone as co-solvent for housefly
esterases, which can explain the slight difference between the
Km value determined for ␣-naphthyl acetate in the present
study and the one reported by van Asperen (1962).
As previously described (Plummer et al., 1983), it was shown
that DMSO inhibits acetylcholinesterase. Indeed, the Km val-
ues for both substrates increased and the turnover numbers
decreased with increasing percentages of DMSO, indicating a
mixed type inhibition by DMSO of both enzymatic reactions
(Table 1). Inhibition constants for the free enzyme (Ki ) and the
enzyme–substrate complex (␣Ki ) determined with acetylthio-
choline as substrate were 0.42 mM and 0.14 mM, respectively.
It has to be noted that the inhibition type reported by Plummer
et al. (1983) was competitive inhibition with a Ki of 0.11 mM.
Hence, the inhibition constants were in the same order of mag-
nitude although the inhibition type differed between the two
studies.
As even low percentages of DMSO considerably interfered
with AChE kinetics (Table 1), the influence of this co-solvent
on the determination of the inhibitory activity of test com-
pounds had to be evaluated. A substrate concentration close
to the apparent Km value obtained with the corresponding per-
centage of DMSO was chosen to assess the inhibitory activity
of compounds for all subsequent experiments.
3.1.2. Effect of DMSO on inhibitory potencies of reference
AChE inhibitors
To assess the influence of DMSO on the determination of
inhibitory activities, pIC50 values of seven well known AChE
inhibitors (Fig. 1) were determined in presence of 0.6% and
1.6% of DMSO for both substrates and also in absence of
co-solvent when acetylthiocholine was used as substrate.
Inhibitory potencies (pIC50 values) obtained with Ellman’s
method and with Fast Blue B salt as reagent are reported in
Table 2 together with literature values. With the exception of
114 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119

Table 2 – Influence of DMSO at different final concentrations (0%, 0.6%, and 1.6%) on pIC50 values of reference AChE
inhibitors determined with microtitre plate methods using Ellman’s reagent and Fast Blue B salt
Compounds Ellman’s reagenta Fast Blue B salt as reagentb Literature pMIQ

0%c 0.6%c 1.6%c 0.6%c 1.6%c 0%c

Propidium (1) 4.66 ± 0.02 4.66 ± 0.17 4.37 ± 0.12 4.75 ± 0.17 4.71 ± 0.15 4.49 (Bolognesi et al., 2005) 8.00
Physostigmine (2) 6.07 ± 0.08 5.93 ± 0.10 5.61 ± 0.09 5.93 ± 0.07 5.86 ± 0.03 6.90 (Ishihara et al., 2000) 11.81
Galanthamine (3) 6.60 ± 0.03 6.46 ± 0.04 6.12 ± 0.04 5.88 ± 0.07 5.09 ± 0.08 6.44 (Guillou et al., 2000) 10.57
(±)-Huperzine A (4) 6.64 ± 0.06 6.45 ± 0.12 5.68 ± 0.06 6.42 ± 0.08 6.14 ± 0.05 12.08
(−)-Huperzine A (5) 7.43 ± 0.12 7.34 ± 0.11 6.21 ± 0.20 6.61 ± 0.10 5.34 ± 0.12 7.37 (Feng et al., 2005) 13.38
Tacrine (6) 7.53 ± 0.14 7.42 ± 0.13 7.34 ± 0.11 7.78 ± 0.05 7.77 ± 0.05 7.30 (Guillou et al., 2000) 12.07
Donepezil (7) 7.61 ± 0.10 7.50 ± 0.10 7.16 ± 0.08 7.55 ± 0.05 7.46 ± 0.07 7.40 (Martinez et al., 2000) 11.56

Comparison with literature data and pMIQ values obtained by TLC.

a
Means ± standard errors (n = 4).
b
Means ± standard errors (n = 2).
c
Percent of DMSO used.

physostigmine, which showed a lower activity in the present
study than reported by Ishihara et al., pIC50 values determined
in absence of DMSO were in good agreement with litera-
ture values. Moreover, for all known AChEIs, the influence of
DMSO was found to be significant. Indeed, all compounds were
slightly less active in presence of 0.6% of DMSO and the differ-
ences in pIC50 values became more pronounced in presence
of 1.6% of co-solvent. Nevertheless, the ranking of activities in
absence and in presence of DMSO is very similar, especially for
pIC50 values determined by Ellman’s method. Indeed, Spear-
man rank order correlation coefficients were very close to 1
(rs = 0.96 between pIC50 values determined with 0% and 0.6%
or 1.6% of DMSO using acetylthiocholine as substrate). It was
thus concluded that the order of activities could be correctly
evaluated in presence of 1.6% of DMSO as co-solvent. Since
␣-naphthyl acetate and test compounds were not soluble in
water, DMSO at a final concentration of 1.6% was therefore
selected as co-solvent for all further assays in solution, unless
stated otherwise.
equal to 5) were considered to be inactive or not sufficiently
active to be of interest as hit compounds for the development
of a novel AChE inhibitor. These activity limits were chosen
according to the pMIQ and pIC50 values of the reference AChE
inhibitors currently in clinical use that were all above 10.5 and
5, respectively (Table 2).
Fig. 3 shows the comparison of the results obtained with
the two screening tests for the 138 compounds investigated,
i.e. pMIQ values determined by TLC versus percentages of inhi-
bition at a test compound concentration of 10−5 M determined
with Ellman’s method. For the majority of compounds (83% of
tested compounds), located in the white areas in Fig. 3, the
results obtained with the two screening assays were conver-
gent. Indeed, the classification of these compounds, including
all seven reference AChE inhibitors tested (full diamonds

3.2. Comparison of two screening tests: TLC assay

with Fast Blue B salt as reagent and microplate assay
using Ellman’s method

3.2.1. Comparison of results obtained with the two

screening tests
A priori, the two screening tests under investigation, i.e. the
TLC assay with Fast Blue B salt as reagent and the microplate
assay using Ellman’s method, should result in the identifi-
cation of the same compounds as active AChE inhibitors. To
evaluate whether this is actually the case, the first step was
to define for each assay, when a compound was considered as
active. With respect to the TLC assay, the activity limit was set
at a pMIQ (negative logarithm of the minimal inhibitory quan-
tity in mole that produced the spot with the least observable
whiteness) of 10.5 and all compounds presenting a pMIQ above
this cut-off value were considered as active. In the microplate
assays, a compound that inhibited AChE to more than 50% at
a fixed test compound concentration of 10−5 M (corresponding
to a pIC50 value above 5) was classified as active. On the other
hand, compounds with inhibition percentages at 10−5 M less
than or equal to 50 (corresponding to pIC50 values below or
Fig. 3 – Comparison of pMIQ values obtained by TLC with
percentages of inhibition at 10−5 M of test compound
determined using the microplate assay with Ellman’s
reagent in presence of 1.6% of DMSO for 138 compounds
including 7 reference acetylcholinesterase inhibitors (). For
compounds located in the white areas ( and 䊉) the two
assays gave convergent results; compounds located in the
light grey area () resulted active by TLC and inactive with
the microplate assay; compounds in dark grey areas ( and
) showed to be active in solution and inactive by TLC; full
symbols represent compounds for which also pIC50 values
were established; data are means of n = 3.
 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119 115

in white areas in Fig. 3) was the same by TLC as by Ell-

man’s method in solution. However, 20 compounds, i.e. about
15% of investigated compounds, classified as active with the
microplate assay, presented pMIQ values below 10.5 and were
considered as inactive by TLC (triangles in dark grey area in
Fig. 3). These compounds comprise 9 coumarins, 9 indenopy-
ridazines, 1 terpene and 1 chromone, and represent 63% of
compounds classified as active inhibitors by the microplate
assay. Hence, more than half of the hit compounds identi-
fied by the microplate assay were missed by TLC. Moreover, 3
compounds, 2 indenopyridazines and 1 aurone derivative, rep-
resenting about 2% of investigated compounds, were active in
the TLC assay but showed percentages of inhibition at a test
compound concentration of 10−5 M below 50 and were thus
considered as inactive with the microplate assay (inverted tri-
angles in light grey area in Fig. 3). It can be noted that the
differences observed between the two assays involve almost
all chemical compound classes investigated. Hence, these
method-dependent differences seem not related to a specific
compound class.
The divergence in the identification of AChE inhibitors
observed between the two screening assays was further inves-
tigated. First, it was checked whether the percentage of
inhibition at a fixed test compound concentration of 10−5 M
allowed a correct prediction of the AChE inhibitory activ-
ity, by comparing inhibition percentages with pIC50 values.
Then, the influence of the two main differences between
the experimental protocols of the two screening assays was
assessed: on the one hand the impact of the colorimet-
ric methods used, i.e. Fast Blue B salt as reagent versus
Ellman’s method, and on the other hand the effect of pre-
incubation of the test compound with/without AChE at 37 ◦ C
for 20 min. Indeed, in the TLC assay, Fast Blue B salt is used
as reagent and compounds are pre-incubated at 37 ◦ C for
20 min with AChE before substrate and reagent solutions are
sprayed onto the plate. In contrast, the microplate assay
is based on Ellman’s method and no pre-incubation of test
compounds with enzyme was performed before activity deter-
minations.
Fig. 4 – Relationship between percentages of inhibition at
10−5 M and pIC50 values determined with Ellman’s method
in presence of 1.6% of DMSO for 20 selected compounds
including 7 reference acetylcholinesterase inhibitors (), 6
compounds for which the two assays gave convergent
results (䊉) and 7 compounds that showed to be active in the
microplate assay and inactive by TLC (); data are means of
n = 2.
3.2.3. Correlation between the two colorimetric methods
in solution
An investigation was then made to see if the two colorimet-
ric methods used, namely detection of the hydrolysis of either
␣-naphthyl acetate with Fast Blue B salt as reagent or acetylth-
iocholine with Ellman’s reagent, allow the identification of the
same compounds as active AChE inhibitors when the tests
were assayed in 96-well plates. For the seven reference AChEIs
(Fig. 1) this was the case. Indeed, with both methods, all ref-
erence AChEIs except propidium showed pIC50 values above 5
and were classified as active (Table 2), despite the slight dif-
ferences in pIC50 values obtained with the two colorimetric
methods.
In addition, inhibition percentages at a fixed concentration
of 10−5 M determined by the two colorimetric methods were

3.2.2. Evaluation of the inhibition percentage at 10−5 M as

indicator of AChE inhibitory activity
The test set for the comparison between inhibition per-
centages at a fixed test compound concentration of 10−5 M
and pIC50 values consisted of the seven reference AChE
inhibitors, six compounds for which results obtained in the
two screening assays were convergent, seven compounds
active by Ellman’s method in solution but inactive by TLC
and the three compounds identified as active inhibitors by
TLC but inactive in the microplate assay. In Figs. 3–5, these
compounds are represented by full diamonds (), full cir-
cles (䊉), full triangles (), and full inverted triangles (),
respectively. For the three latter (), no pIC50 values could
be determined due to their low activity in solution. Fig. 4
shows the relationship between percentages of inhibition at a Fig. 5 – Correlation between percentages of inhibition at
fixed concentration of 10−5 M and pIC50 values. Within exper- 10−5 M determined in solution using Ellman’s reagent and
imental errors, both parameters allow the same classification Fast Blue B salt as reagent for 138 compounds including 7
of compounds and it can be concluded that inhibition per- reference acetylcholinesterase inhibitors (). Symbols and
centages allow a reliable prediction of the AChE inhibitory color code of areas are the same as in Fig. 3; data are means
activity. of n = 3, obtained in presence of 1.6% of DMSO.
116 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119
compared for the entire test set of 138 compounds. As shown
in Fig. 5, a reasonably good correlation was observed between
values achieved using 96-well microtitre assays with Fast Blue
B salt as reagent and Ellman’s reagent (r2 = 0.84). Indeed, for
94% of investigated compounds the separation of active hits
from compounds not sufficiently active to be of interest was
the same with both colorimetric methods. And in particular,
both methods in solution gave the same classification for all
compounds that showed deviant results in the comparison
between the TLC assay with Fast Blue B salt as reagent and
the microplate assay using Ellman’s method, represented in
Fig. 5 by full triangles () for compounds that resulted active
by Ellman’s method in solution but inactive by TLC, and by
full inverted triangles () for the three compounds identified
as active inhibitors by TLC but which were inactive in the
microplate assay. Hence, the divergences in the identification
of active AChE inhibitors observed between the TLC assay and
the microplate assay cannot be explained by the different col-
orimetric methods used.
3.2.4. Effect of pre-incubation of test compounds
with/without AChE on their AChE inhibitory activity
Finally, pre-incubation of test compounds for 20 min at 37 ◦ C
either in presence or absence of AChE was investigated to see
if this could explain the divergences in the identification of
AChE inhibitors observed between the TLC and the microplate
assays. Indeed, compounds were pre-incubated with AChE in
the TLC test before the addition of substrate whereas in solu-
tion no pre-incubation step was performed. The influence of
pre-incubation of test compounds was first investigated in
solution using Ellman’s method, then by TLC using Fast Blue
B salt as reagent. Experiments were performed for 20 com-
pounds representative of the entire test set, namely three
reference AChEIs ((±) huperzine A (4), tacrine (6) and donepezil
(7)), four 5H-indeno [1,2-c] pyridazin-5-one (IP) derivatives
found active in both assays and located in the white area in
Fig. 3, 10 compounds (6 IP and 4 coumarin (C) derivatives) that
showed to be active in solution but inactive by TLC (located in
the dark grey area in Fig. 3), as well as the three compounds
(2 IP and 1 aurone (AU) derivative) that resulted active by TLC
but inactive in the microplate assay (located in the light grey
area in Fig. 3).
3.2.4.1. Influence of pre-incubation of test compounds on their
activity determined in solution with the microplate assay. The
inhibitory activities (inhibition percentages at 10−5 M) of the
20 selected compounds, determined in solution with Ellman’s
method either without or after a pre-incubation time of 20 min
at 37 ◦ C in presence and in absence of enzyme, are reported in
Table 3 together with their pMIQ values determined by TLC.
First, it can be noted that the tested compounds were either
not affected or gave lower activities after pre-incubation.
None of the compounds showed a higher activity after pre-
incubation with the enzyme. Hence, pre-incubation is not the
reason for the high activity detected by TLC for the three
compounds that were positive by TLC but inactive with the
microplate assay (IP-11, IP-12 and AU-13, located in the light
grey area in Fig. 3 and Table 3).
A closer look at the results presented in Table 3 shows
that the activities of the three reference AChEIs and four
IP derivatives (IP-42 to IP-45), for which convergent results
were obtained in the two screening assays (located in the
white area in Fig. 3 and Table 3), were not or only to
a certain extent affected by pre-incubation. In fact, these
compounds retained inhibition percentages above 50% after
pre-incubation with/without AChE. On the other hand, for the
ten compounds found active by Ellman’s method in solution
but inactive by TLC (C-45 to C-47, C-51, IP-34, IP-36 to IP-38, IP-
40 and IP-41, located in dark grey area) an important decrease
in activity was observed after pre-incubation, with result-
ing inhibition percentages below 50% (Table 3). Moreover, the
presence of AChE during pre-incubation did not influence
the loss of AChE inhibitory activity observed for these com-
pounds. Indeed, the same results were obtained after a 20 min
pre-incubation period of compounds with AChE as after pre-
incubation for 20 min without enzyme. Hence, their loss of
activity during pre-incubation seems to be due to degrada-
tion in aqueous buffer solutions of pH 7.4 and might to some
degree explain the divergences found between the TLC and
the microplate assays. Indeed, loss of activity for these ten
compounds might also happen during the TLC procedure.
3.2.4.2. Influence of pre-incubation of test compounds on their
activity determined by the TLC assay. The influence of pre-
incubation was also investigated with the TLC assay, by
omitting the 20 min pre-incubation step at 37 ◦ C of the TLC
standard protocol. However, the same results were found with
and without pre-incubation. Further, it has to be noted that for
all compounds only one spot was visible on the TLC plate after
migration with hexane:ethyl acetate (1:1; v/v) as detected by
UV and by means of a permanganate solution, indicating that
potential degradation products could not be observed. Hence,
the influence of pre-incubation of test compounds on their
AChE activity observed in solution could not be reproduced by
TLC.
3.2.5. Hypotheses postulated to explain the differences
observed
The 10 compounds found active in the microplate assay but
inactive by TLC were found to lose their AChE inhibitory activ-
ity in aqueous buffer solutions at pH 7.4. Hence, lower stability
might all the same be a possible reason for their inactivity
by TLC, even if loss of activity seems not to occur during the
pre-incubation step. Inactivation after interactions of the com-
pounds with silica of the TLC plate may occur.
Another, although less probable hypothesis, is that dif-
ferences between pMIQ values determined by TLC and
percentages of inhibition at 10−5 M obtained by Ellman’s
method in solution might be due to subtle differences in the 3D
structures of the enzyme in the two assays. In fact, adsorption
of AChE onto silica plates might result in a change of con-
formation of the enzyme, and as a consequence, in altered
accessibility of the enzyme for inhibitors. Indeed, a differ-
ent kinetic behaviour of AChE between immobilized and free
states has been reported by several authors (Bartolini et al.,
2005; Sahin et al., 2005) and structural changes at the sec-
ondary and tertiary level upon adsorption onto silica surfaces
were also reported for other enzymes (Koutsopoulos et al.,
2007). In the present case, adsorption of AChE onto silica would
result in a less favourable conformation for interaction with
 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119 117

Table 3 – Effect of pre-incubation of test compounds with/without AChE at 37 ◦ C for 20 min on their AChE inhibitory
activity (color code of areas is the same as in Fig. 3)

compounds that showed to be inactive by TLC although they
were identified as active hits in solution, and on the other
hand, in a more favourable conformation for interaction with
the three compounds that were more active by TLC than with
the microplate assay.
3.2.6. Advantages and inconveniences of screening by TLC
and microplate assays
As previously reported, the TLC assay is simple to use
and gives quick access to information concerning the AChE
inhibitory activity in particular of plant extracts (Marston et
al., 2002). No expensive equipment is required and several
plant extracts can be screened at the same time. Moreover, the
use of a co-solvent such as DMSO is avoided. A drawback of the
TLC assay might be the possible interaction of either AChE or
test compounds with the silica support, resulting in an alter-
ation of the affinity of the enzyme for the compounds. Another
point of concern is a potential loss of AChE inhibitory activity
of test compounds under the conditions used to develop the
plate.
The general advantages of screening performed in a
microplate are the easy handling of large amounts of sam-
ples simultaneously, the possibility of automation and the
need for little material. Indeed, both microtitre plate meth-
ods used were shown to be reliable, rapid and accurate. To
screen libraries of synthetic or isolated natural compounds,
118 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 3 ( 2 0 0 8 ) 109–119
microplate assays are therefore generally preferred over TLC
assays. A difficulty of enzyme assays performed in solution
might be the choice of a suitable co-solvent to solubilize test
compounds.
Regarding the colorimetric methods, Fast Blue B salt is
without doubt a better choice than Ellman’s reagent for the
TLC assay, as it results in a strong purple coloration easily
observable on the plate (Marston et al., 2002). However, for
the microplate assay, Ellman’s method has several advantages
over the method using Fast Blue B salt as reagent and it can
be considered the colorimetric method of choice for medium-
and high-throughput inhibitor screening in solution. First, the
good solubility of the substrate acetylthiocholine in buffer, in
contrast to ␣-naphthyl acetate, allows a significant reduction
of the final amount of DMSO in the reaction mixture. Moreover,
stock solutions of substrate and reagent have a higher stability.
Indeed, ␣-naphthyl acetate solutions have to be prepared just
before use whereas acetylthiocholine solutions can be kept for
several days. Finally, with Ellman’s method the formation of
product of the enzymatic reaction can be monitored in a con-
tinuous manner, and the concentration of enzyme required is
lower.
4. Conclusion
Results obtained in this work have shown that the percentages
of inhibition at a fixed concentration of 10−5 M of test com-
pound using Ellman’s method are convergent with the pMIQ
values determined by TLC using Fast Blue B salt as reagent for
the majority of the 138 compounds tested. However, about 15%
of investigated compounds identified as hits in the microplate
assay were inactive by TLC, and on the other hand, about 2% of
investigated compounds were active by TLC were inactive in
the microplate assay. The differences observed between the
two assays were not accredited to the different colorimetric
methods used. In fact, both methods in solution allowed the
same classification for almost all compounds tested. More-
over, the inhibition percentages allowed a reliable prediction
of the AChE inhibitory activity in solution. However, com-
pounds identified as active inhibitors in the microplate assay
but inactive in the TLC test were shown to lose activity dur-
ing pre-incubation for 20 min at 37 ◦ C with/without AChE. This
loss of activity might also occur in the TLC assay during the
development of the plate. Another hypothesis to explain the
observed differences between the two screening assays is the
interaction of either AChE or test compounds with the silica
of the TLC plates, which might result in an altered affinity of
the enzyme for the compounds.
In conclusion, both screening assays for AChE inhibitory
activity investigated are considered suitable for the generation
of new hits. The TLC assay is advantageous for the screen-
ing of crude extracts of plants as it allows separation and
detection of active compounds at the same time. This allows
an easy follow up of the isolation and purification process.
The microplate assay is more appropriate for the screening of
libraries of synthetic and/or isolated natural compounds as it
allows the possibility of automation and the easy handling of
large amounts of samples. Finally, determination of IC50 val-
ues and mode of inhibition with the microplate method in
solution has to be performed to confirm results obtained with
both screening assays.
Supporting information available
A table with molecular structures and detailed numerical
results (inhibition percentage at 10−5 M, pIC50 , pMIQ) of all
tested compounds is available as supplementary information.
Acknowledgments
The authors express their gratitude to Prof. Angelo Carotti,
Dipartimento Farmaco-Chimico, Università di Bari, for the
kind donation of the coumarin and indenopyridazine deriva-
tives, and to Dr. Ahcène Boumendjel, Département de
Pharmacochimie Moleculaire, Faculté de Pharmacie de Greno-
ble, for having us kindly provided with the aurone and
azaaurone derivatives.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.ejps.2007.10.004.
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