You are on page 1of 20

bs_bs_banner

Review

Journal of Pharmacy
And Pharmacology

Nature as a source of metabolites with


cholinesterase-inhibitory activity: an approach to
Alzheimers disease treatment
Brgida R. Pinhoa, Federico Ferreresb, Patrcia Valentoa and Paula B. Andradea
a

REQUIMTE/Laboratrio de Farmacognosia, Departamento de Qumica, Faculdade de Farmcia, Universidade do Porto, Porto, Portugal and
Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), Murcia, Spain

Keywords
biomedicinal chemistry; clinical efficacy of
natural products; drugs from natural sources;
structure/activity relationships
Correspondence
Paula B. Andrade, REQUIMTE/Laboratrio de
Farmacognosia, Departamento de Qumica,
Faculdade de Farmcia, Universidade do
Porto, Rua de Jorge Viterbo Ferreira, n 228,
4050-313 Porto, Portugal.
E-mail: pandrade@ff.up.pt
Received January 25, 2013
Accepted April 11, 2013
doi: 10.1111/jphp.12081

Abstract
Objectives Alzheimers disease (AD) is the most common cause of dementia,
being responsible for high healthcare costs and familial hardships. Despite the
efforts of researchers, no treatment able to delay or stop AD progress exists. Currently, the available treatments are only symptomatic, cholinesterase inhibitors
being the most widely used drugs. Here we describe several natural compounds
with anticholinesterase (acetylcholinesterase and butyrylcholinesterase) activity
and also some synthetic compounds whose structures are based on those of
natural compounds.
Key findings Galantamine and rivastigmine are two cholinesterase inhibitors
used in therapeutics: galantamine is a natural alkaloid that was extracted for the
first time from Galanthus nivalis L., while rivastigmine is a synthetic alkaloid, the
structure of which is modelled on that of natural physostigmine. Alkaloids include
a high number of compounds with anticholinesterases activity at the submicromolar range. Quinones and stilbenes are less well studied regarding cholinesterase
inhibition, although some of them, such as sargaquinoic acid or (+)-a-viniferin,
show promising activity. Among flavonoids, flavones and isoflavones are the most
potent compounds. Xanthones and monoterpenes are generally weak cholinesterase inhibitors.
Summary Nature is an almost endless source of bioactive compounds. Several
natural compounds have anticholinesterase activity and others can be used as
leader compounds for the synthesis of new drugs.

Introduction
Neurodegenerative diseases are increasing in developed
countries and they have in common the progressive and
specific loss of certain brain cell populations, leading to
functional or sensory dysfunction. Neurodegenerative disorders have some neuropathological hallmarks, such as
abnormal protein dynamics, bioenergetics and mitochondrial function impairment, oxidative stress and inflammation. In general neurodegeneration has a multifactorial
nature, being associated with environmental factors
and with genetic predisposition.[1] Neurodegeneration has
been the object of study of several teams with the intention
of finding a way to prevent, reduce or even treat

neurodegenerative disorders, such as Huntingtons, Parkinsons or Alzheimers disease (AD).


Among neurodegenerative disorders, AD is the most
common[2] and it is the most common type of dementia.[3]
AD was the sixth most common cause of death in the USA
in 2010,[3] and affects nearly 2% of the population in industrialized countries. Attending to populations ageing, the
incidence of AD is expected to triple in the next 50 years.[4]
AD renders an individual highly dependent and in need of
daily accompaniment; this translates into familial hardships
and elevated healthcare costs.[5] Despite the efforts of the
scientific community in the search for options that could

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

1681

Natural cholinesterase inhibitors

Brgida R. Pinho et al.

delay the progress of the disease, only a symptomatic treatment exists.


In this review, after a brief reference to AD risk factors,
pathology and treatment, we will approach the importance
of cholinesterases in the physiopathology of AD, as well as
describe several natural compounds, and synthetic compounds based on natural drugs, with the ability to inhibit
these enzymes. Thus, this work reviews seven classes of secondary metabolites as cholinesterases inhibitors, and discusses recent published works.

Alzheimers disease
As mentioned above, the incidence of AD will increase as
the population ages, since age is the main risk factor for the
development of the disease.[4,6] However, epidemiological
findings associate several other factors with AD prevalence,
such as low education level, history of head trauma, highcaloric diet, folate-poor diet, sedentary lifestyle, low mental
ability in early life, and reduced mental and physical activity
during late life, as well as vascular disease risks (hypercholesterolaemia, hypertension, atherosclerosis, coronary heart
disease, smoking, obesity and diabetes).[7] Only a small percentage of AD cases is related to family history, whereby
specific genes are associated with increased susceptibility to
AD (amyloid precursor protein,[8] presenilin 1,[8] clusterin,[9]
sortilin-related receptor 1 gene[10] or apolipoprotein
allele E4).[8]
Symptomatically, AD is characterized by memory loss,
multiple cognitive impairment, disturbance of functions
such as language and executive function, and by several
neurobehavioural symptoms, including apathy, agitation
and anxiety.[11] Currently, AD cannot be diagnosed in the
absence of cognitive impairment/dementia[12] and a definitive diagnosis can only be established by post-mortem
examination of the AD patients brain.[4] Nevertheless,
research suggests that neurodegeneration starts 2030 years
before clinical symptoms appear.[13] It has recently been
reported that AD pathology can be detected pre-clinically
by using neuroimaging techniques and biomarkers.[12,14]
AD is characterized by the presence of neuritic plaques
and neurofibrillary tangles, mainly in the brain regions
responsible for learning, memory and emotional behaviour.[4] Neuritic plaques are microscopic extracellular
deposits of fibrils and amorphous aggregates of amyloid-b
peptide (Ab),[15] which result from an imbalance between
production and clearance of Ab in the brain.[16] Neurofibrillary tangles are intracellular fibrillar aggregates
of microtubule-associated hyperphosphorylated tau protein.[15] The reason for the appearance of neuritic plaques
and neurofibrillary tangles remains uncertain;[4] however,
the presence of these structures has several consequences in
affected brains. Ab accumulation might increase microglio1682

sis, astrocytosis, energy metabolism impairment, cellular


calcium homeostasis disruption and the release of several
inflammation mediators,[15] initiating an inflammatory
process with an excessive generation of free radicals that
may cause damage in proteins, lipids and other macromolecules.[4,15] On the other hand, hyperphosphorylation of tau
protein present in neurofibrillary tangles induces the
impairment of the axonal transport of proteins, such as
amyloid precursor protein, or of subcellular organelles, such
as mitochondria.[13] All these alterations contribute to neuritic dystrophy, synaptic loss, shrinkage of neuronal perikarya, selective neuronal loss and loss of cholinergic
enzymes, including choline acetyltransferase and acetylcholinesterase (AChE).[4,15] The neuronal degeneration and loss
of several enzymes, translated into loss of neurotransmitters
or other neuromodulators, compromise synaptic transmission, explaining the difficulty of these patients in storing
new information or remembering recent information.[15]
Several neurons are damaged in AD, such as those producing serotonin or noradrenaline (norepinephrine);
neurons that use glutamate and acetylcholine (ACh) as neurotransmitters are particularly affected,[4] resulting in a
deficit in central cholinergic transmission by neuronal loss
of basal forebrain nuclei and loss of nicotinic receptors.[17]
Thus, AD pathogenesis is not simply an acceleration of
normal brain ageing,[4] since in a normal age-dependent
process there is a degeneration of basal forebrain cholinergic neurons with dendritic synaptic and axonal degeneration, possibly due to a decrease in trophic support, but
without cell loss.[18] The reason for the selective neuronal
death occurring in AD remains uncertain, but could be
related to the expression of genes that control the neuronal
death, including glutamate receptors, calcium-binding proteins or neurotrophic factors.[4] On the other hand, it is
hypothesized that Ab induces neurodegenerative changes at
cholinergic terminals, through action on nicotinic receptors, by affecting nerve growth factor signalling, mediating
the phosphorylation of tau protein, interacting with AChE
and specifically affecting the proteome in cholinergic
neurons. However, there are some doubts about what is the
primary event: the degeneration of cholinergic terminals or
the Ab plaque pathology.[18]

Cholinesterases
ACh is present throughout the nervous system, being essential for cerebral cortical development and activity, cerebral
blood flow control, sleepwake cycle and, mainly, for learning and memory processes.[18] Several studies in animal
models have shown the occurrence of learning or memory
deficits after anticholinergic treatment, highlighting the
importance of ACh for cognitive performance.[19,20] ACh is
stored in vesicles in the terminal nerves, being released

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

Natural cholinesterase inhibitors

Acetylcholine

HO

OH
NH2

N
H

O
H

Glutamate

OH

H
N

O
O

OH
NH2

O
O

NH3

HO

NH2

NH3

O
NH2

N
+

N
+

Serine

NH2

O
HO

HO

Histidine

N
+

NH2

N
+

NH3

OH
O

HO

OH
NH2

N
H

O
O

O
O

NH3

OH
H

OH
NH2

HO

O
NH2

HO

N
+

NH2

NH3

O
O

Figure 1 Acetylcholine hydrolysis by acetylcholinesterase. Serine, histidine and glutamate constitute the catalytic triad in the esteratic site. Hydroxyl
group of serine induces a nucleophilic attack to acetylcholine, being stabilized by histidine and glutamate.

when they are depolarised. However, released ACh has a


short half-life due to hydrolysis by cholinesterase (ChE)
enzymes. Thus, the inhibition of ChE induces persistent
presence of ACh and, consequently, prolongation of its
activity.
ChE levels and distribution in human brain regions
vary. Currently, two principal ChE are known: AChE and
butyrylcholinesterase (BuChE).[21] BuChE, which is also
known as pseudocholinesterase, catalyses the hydrolysis of
a wide variety of choline and non-choline esters (butyrylcholine, succinylcholine, acetylcholine, acetylsalicylic acid,
cocaine and heroin).[22] BuChE corresponds to only 10% of
total ChE in the normal brain,[17] being more abundantly
expressed in liver, lung and heart tissues and is predominantly present in plasma.[23] Both AChE and BuChE are
serine esterases, since they have a serine amino acid
residue, which is essential for the catalytic activity.[22] They
are expressed as multiple molecular forms, such as globular monomers (G1), dimers (G2) and tetramers (G4), of
catalytic subunits, as well as asymmetric molecules with
one (A1), two (A2) or three (A3) tetramers. The physiological functions of ChE are probably mediated by G4, in
spite of the presence of G1 in small amounts, in human
brain.[24]
The substrate initially binds to the outer rim of the ChE,
in a region called the peripheral site, but the hydrolysis
occurs inside the enzyme, in the bottom of a gorge, which is
divided into four main subsites: esteratic site, oxyanion
hole, anionic subsite and acyl pocket. The esteratic site contains the catalytic machinery of the enzyme and includes a
serine, histidine and a glutamate residue (Figure 1): (i) the
serine residue induces a nucleophilic attack to the carbon of

the carbonyl group of the ester substrate; (ii) histidine stabilizes the serine intermediate by strong hydrogen bonds;
(iii) the negative charge of glutamate stabilizes the histidinium cation.[25] The oxyanion hole contains hydrogen donors
which stabilize the tetrahedral intermediate of the substrate
that is formed during the catalytic process. The anionic
subsite (choline-binding subsite or hydrophobic subsite)
contains several aromatic residues, which are important for
the binding of quaternary ammonium ligands by p-cation
interactions.[25] However, the number of aromatic amino
acids differs according to the enzyme: some aromatic amino
acid residues present in the acyl pocket and in the peripheral site of AChE are replaced by aliphatic amino acids in
BuChE. As aliphatic amino acids are smaller than aromatic
amino acids, these alterations allow larger substrates to
enter the active site of BuChE. Thus, the active site of
BuChE can accommodate larger acyl groups, such as those
with four carbons (e.g. butyrylcholine) or aromatic rings
(e.g. benzoylcholine).[25]
Less information is available regarding BuChE, compared
with AChE, in the nervous system. However, it is known
that BuChE is expressed in a distinct population of neurons
and that it is important to cholinergic neurotransmission
regulation and to nervous system development.[22] In addition, the activity of BuChE is altered in patients with AD:
BuChE activity is reported to be significantly (4180%)
enhanced in brains of patients with advanced AD,[26] mainly
in regions affected by Ab plaques and neurofibrillary tangles.[22] In contrast, there is a deficit of 1060% of AChE in
affected brain regions of AD patients.[27] Furthermore, there
are several factors indicating that BuChE can be a potential
target in AD treatment: (i) BuChE has a higher half-life

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

1683

Natural cholinesterase inhibitors

Brgida R. Pinho et al.

than AChE, due to its higher number of glycation sites that


are important for stability and clearance of ChE;[22] (ii)
BuChE G1 form, which is predominant in the developing
brain, is increased in AD patients;[24] (iii) AChE is inhibited
by high concentrations of ACh, while BuChE remains
unaffected;[24] (iv) inhibition of BuChE induces a dosedependent increase in brain levels of ACh;[22] (v) toxic
effects are observed after treatment of mice lacking AChE
with BuChE inhibitors.[28] Thus, BuChE has a regulatory
role in ACh hydrolysis, being an alternative to AChE,
whereby therapeutic agents that inhibit both AChE and
BuChE could provide additional benefits in AD when compared with those that primarily inhibit AChE. However, as
far as we know it has not been established whether BuChE
inhibitors are more effective than AChE inhibitors in reducing AD symptoms. Nevertheless, compounds that inhibit
both enzymes have been therapeutically successful, like
rivastigmine, an alkaloid used in therapeutics that we will
discuss later. In recent years, several groups have searched
for new ChE inhibitors; the majority of the studies have
involved ChE from the electric eel (Electrophorus electricus
Linnaeus, 1766) and Pacific electric ray (Torpedo californica
Ayres, 1855).[25]

Cholinesterase inhibitors
Several new treatment options to halt the course of AD
have been studied, such as those that avoid Ab production
(preventing assembly of Ab monomers into potentially
cytotoxic oligomers; b- or g-secretase inhibition)[15] and
parental immunization with a synthetic human Ab peptide,
the latter of which has produced promising results.[29]
However, the treatments currently available are only symptomatic: memantine, which is an N-methyl-d-aspartate
(NMDA) receptor antagonist, and ChE inhibitors are the
only drugs available.[3,17] ChE inhibitors do not induce a
change in the natural course of AD, but temporarily mitigate some symptoms, since these drugs enhance the activation of synapses, thus improving cognition.[4,13] Other
approaches have been tried, with the aim of improving
cholinergic transmission, such as the increase of presynaptic ACh release or the stimulation of postsynaptic muscarinic or nicotinic receptors; these approaches have been
unsuccessful due to their lack of efficacy and unacceptable
side-effects.[17]
ChE inhibitors can be classified according to their selectivity for AChE or BuChE, or according to their mechanism
of action. Reversible inhibitors (e.g. tacrine or donepezil)
are competitively displaced from the active centre of
the enzyme by physiological ligands or other choline
esters. Pseudo-irreversible inhibitors (e.g. physostigmine
or rivastigmine) bind more firmly to the enzyme than a
physiological ligand. Irreversible inhibitors (e.g. organo1684

phosphates) have a more drastic effect: in the presence of


these inhibitors, the inhibited enzyme can only be replaced
by a new one via biosynthesis.[30]
Plants have been used for millennia as main sources of
therapeutics agents.[31] A wide variety of organisms, including plants, marine animals and terrestrial microbes, have
been studied as potential sources of new compounds with
biological activities or compounds that could be good lead
drugs (including ChE inhibitors) for the pharmaceutical
industry. Plants have secondary pathways that allow them
to synthesize several chemicals, often in response to
specific environmental stimuli, including herbivore-induced
damage, pathogen attacks or nutrient deprivation.[32] These
chemicals, termed secondary metabolites, are compounds
of low molecular weight and are produced from primary
metabolites, such as carbohydrates, amino acids and lipids.
Secondary metabolites are not ubiquitous in nature, sometimes being specific to certain species or genera. They do
not play any role in primary metabolic requirements but
can increase the ability to survive and overcome local challenges by allowing an organism to respond to attacks from
its environment.[32] Secondary metabolites are grouped
according to their biosynthetic origin and structural features. In the following sections, we will discuss several
natural compounds with ChE inhibitory activity; however,
comparisons between compounds should be established
with care, since the inhibitory concentrations depend
on several variables, such as incubation time, source of
enzymes and detection method.

Alkaloids
Alkaloids constitute a wide family of compounds, which
generally have in common the presence of nitrogen atom(s)
in a cyclic ring. This is probably the largest group of
metabolites with ChE inhibitory activity at lower concentrations (Table 1). As described by Houghton and colleagues, the majority of alkaloids bind at the bottom of the
gorge of the active site, mainly at the oxyanion hole area, via
the positively charged nitrogen.[25] The first known AChE
inhibitor was physostigmine (Figure 2), an alkaloid isolated
for the first time in 1864 from Physostigmina venenosum
Balf., which was used in therapy before the discovery of
ACh as neurotransmitter.[76] However, physostigmine is
quite polar, being distributed throughout the body, and
only a small amount reaches the central nervous system.
Physostigmine inhibits both AChE and BuChE in a similar
submicromolar range (concentration required to inhibit
50% of the enzyme ( IC50) of 0.015 and 0.016 mm, respectively) (Table 1).[43,44] The carbamate position is essential for
the activity of physostigmine, because when the ester bond
of physostigmine is hydrolysed, forming eseroline, the
inhibitory activity is not observed. Furthermore, it is known

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

Table 1

Natural cholinesterase inhibitors

Anticholinesterase activity of representative secondary metabolites


Acetylcholinesterase

Compounds
Alkaloids
Axillaridine A
Berberine
a-Chaconine
Cymserine
Galantamine
Groenlandicine
Huperzine A
Infractopicrin
Juliflorine
Neostigmine
Petrosamine
Physostigmine
Pyridostigmine
Rivastigmine
Sarsalignenone
Sarsalignone
a-Solanine
Tubocurarine
Coumarins
Decursin
Decursinol
Heraclenol-2-O-angelate
Imperatorin
Isopimpinellin
Marmesin
Methoxsalen
Scopoletin
Flavonoids
Apigenin
Kaempferol
Leufolin A
Leufolin B
Luteolin
Mimulone
Naringenin
Pomiferin
Quercetin
Rutin
Tamarixetin
Quinones
Mansonone E
Sargaquinoic acid
Stilbenes
(+)-a-Viniferin
Gnetol
Kobophenol A
Resveratrol
Terpenes
1,8-Cineole
2-Carene
3-Carene
Cryptotanshinone
Dihydrotanshinone
Leoheteronin A

IC50 (mM)

5.21
0.44
17
0.10
4.00
0.54
0.082
9.72
0.42
0.036
0.091
0.015
0.091
501
5.83
7.02
14
63
3900
28
>1000
165
0.32
67
3.07
52
21.5
30.4

Butyrylcholinesterase

Source

Reference

IC50 (mM)

Source

Reference

Electric eel
Electric eel
Human recombinant
Human erythrocytes
Electric eel
Electric eel
Rat cortex
Bovine erythrocytes
Electric eel
Guinea-pig ileum
Pacific electric ray
Human brain
Mouse brain
Electric eel
Electric eel
Electric eel
Human recombinant
Human erythrocytes

[33]

2.49
3.44
0.066
0.001
7.96
3.32
74.4
>100
0.12
0.19
N.F.
0.016
0.30
19.95
4.29
2.18
0.17
350

Horse serum
Horse serum
Human serum
Human erythrocytes
Equine serum
Horse serum
Rat serum
Equine serum
Horse serum
Guinea-pig ileum

[33]

Human plasma
Human plasma
Equine serum
Horse serum
Horse serum
Human serum
Human plasma

[44]

N.M.
N.M.
Electric eel
Electric eel
Fly lysate
N.M.
Rat cell line
N.M.

[48]

Equine serum
Equine serum

[49]

Rat brain
N.M.

[53]

N.M.
N.M.
N.M.

[54]

Equine serum
Equine serum
Horse serum
Horse serum
Horse serum
N.M.

[56]

Horse serum
N.M.

[60]

Horse serum

[63]

Horse serum

[63]

Human serum
Human
Human

[66]

[34]
[35]
[36]
[37]
[34]
[38]
[39]
[40]
[41]
[42]
[43]
[45]
[37]
[33]
[33]
[35]
[47]

[48]
[49]
[49]
[50]
[48]
[51]
[52]

[54]

N.F.
N.F.

7.5
14.4
N.F.
N.F.
N.F.
N.F.
N.F.
62.5
1.6
3.6
N.F.
20.6
1494
N.F.
420.8
44.6
160.6

[53]

Electric eel
N.M.

[60]

2.0

N.M.

[62]

N.F.

N.F.
115.8
>500

N.M.
N.M.

[62]

1.3
N.F.
15.9

Bovine erythrocytes
Bovine erythrocytes
Bovine erythrocytes
Recombinant human
Recombinant human
Mouse brain

[64]

23.5
23.3

670
900
200
4.67
0.89
11.6

[56]
[56]
[57]
[58]
[59]
[54]

[61]

[62]

[65]
[65]
[67]
[67]
[68]

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

[35]
[36]
[37]
[34]
[38]
[39]
[40]
[41]

[46]
[37]
[33]
[33]
[35]
[47]

N.F.
N.F.

Rat brain
Human erythrocytes
Human erythrocytes
Electric eel
Pacific electric ray
Electric eel
N.M.

15.6
91.5
2045
96
353.9
28.2
22.3

[34]

62.4
0.026

N.F.
N.F.
2000
6.66
5.51
N.F.

[49]

[55]
[55]

[56]
[57]
[58]
[59]
[54]

[61]

[67]
[67]

1685

Natural cholinesterase inhibitors

Table 1

Brgida R. Pinho et al.

(Continued)
Acetylcholinesterase

Compounds

IC50 (mM)

Leopersin G
Limbatolide A
Limbatolide B
Limbatolide C
Myrtenal
Taraxerol
Ursolic acid
a-Pinene
Xanthones
Allanxanthone A
Triptexanthoside C
Others
Arisugacin A
Territrem B

12.9
38.5
47.2
103.7
170
98.4
93.8
630
95
13.8
0.001
0.076

Butyrylcholinesterase

Source

Reference

IC50 (mM)

Mouse brain
Electric eel
Electric eel
Electric eel
N.M.
N.M.
Electric eel
Bovine erythrocytes

[68]

N.F.
22.3
17.5
14.2
N.F.
N.F.
41.1
N.F.

Electric eel
N.M.

[72]

N.M.
N.M.

[74,75]

[69]
[69]
[69]
[70]
[71]
[59]
[64]

[73]

[75]

19.1
N.F.

Source

Reference

Horse serum
Horse serum
Horse serum

[69]

Horse serum

[59]

Horse serum

[72]

[69]
[69]

N.F.
N.F.

N.F, Not found; N.M., not mentioned.

H
O

N
O

N
+

N+

Physostigmine

Pyridostigmine

Neostigmine

Rivastigmine

N
O
H
O

Cymserine
N

Figure 2 Chemical structure of alkaloids bearing a carbamate group:


natural compound (physostigmine) and hydrophilic (neostigmine and
pyridostigmine) and hydrophobic (rivastigmine and cymserine) synthetic analogues.

that the carbonyl group of the carbamate portion interacts


with a hydroxyl group of a serine residue in AChE, forming
an ester, and is then slowly hydrolysed, regenerating the
active parent form of the enzyme.[25,77,78] Several changes
were made to the physostigmine skeleton in an attempt to
potentiate the activity or to change the polarity of the com1686

pound. In general, increasing the hydrophobicity by simple


non-branching carbamoyl groups did not increase the
potency against AChE, while increasing the hydrophobicity
of N(1)-substituents decreased the effect against AChE.
However, increasing the hydrophobicity of both the carbamoyl and the N(1) groups rendered the compound more
active against BuChE. Furthermore, the quaternarization of
the N(1) position (e.g. physostigmine methosulfate)
increased the potency against AChE, but decreased the
activity against BuChE.[79]
Neostigmine and pyridostigmine (Figure 2) are two synthetic compounds developed from physostigmine. Neostigmine and pyridostigmine contain a quaternary nitrogen
instead of the tertiary amine of physostigmine, whereby
they are hydrophilic compounds that do not cross the
bloodbrain barrier, and are used in peripheral cholinergic
deficiencies such as in myasthenia gravis.[25] In contrast to
neostigmine and pyridostigmine, rivastigmine (Figure 2) is
a synthetic lipophilic analogue of physostigmine that is able
to cross the bloodbrain barrier. In 2000, the European
Medicines Agency (EMEA) centrally approved rivastigmine
for therapeutic use, and this drug is currently used in the
treatment of AD.[17,25] Rivastigmine inhibits both AChE and
BuChE, with higher potency against the latter.[37] Despite
being lipophilic and having fewer side-effects, rivastigmine
is less potent than physostigmine (Table 1). Cymserine
(Figure 2), another physostigmine analogue that can be
obtained by total synthesis or from physostigmine, is a
potent, reversible and selective human BuChE inhibitor (BuChE IC50 = 0.001 mm vs AChE IC50 = 0.100 mm)
(Table 1).[36] Cymserine is more potent than rivastigmine,
due to the presence in cymserine of the basic structure
of physostigmine, which is not present in rivastigmine

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

Natural cholinesterase inhibitors

(a) Isoquinoline

(b) Indole
OH

O
O

+
N

N
H

N+
O

+
N

OH
O

Tubocurarine

(c) Terpenic

OH

Berberine

N+

Galantamine

Geissospermine

(d) Acridine

Infractopicrin

Angustine

(e) Piperidinium
Br
HO

N
O
HO

NH2

+
N

B
N

Huperzine A

Petrosamine

Juliflorine

(f) Steroidal
(g) Lycorine

H
H
N

H
H

-Solanine

-Chaconine

O
OH
O
O

O
HO

H
OH

HO

OH

R=

OH

HO

OH
O

OH OH

HO
OH

OH

OH

H
O

OH
HO

RO

R=

N
H

N
H

Axillaridine A

H
N

Sarsalignone

O
HO

OH
O

Sarsalignenone

OH

Figure 3 Chemical structure of alkaloids from different subgroups. (a) Isoquinoline: berberine, tubocurarine and galantamine. (b) Indole: geissospermine, infractopicrin and angustine. (c) Terpenic: huperzine A. (d) Acridine: petrosamine. (e) Piperidinium: juliflorine. (f) Steroidal: a-solanine,
a-chaconine, axillaridine A, sarsalignone and sarsalignenone. (g) Lycorine.

(Figure 2), favouring anticholinesterase (anti-ChE) activity.


Cymserine was found to raise ACh levels without inhibiting
AChE in rats and induced an improvement in the cognitive
performance of aged rats.[80]
Berberine (Figure 3a) is an isoquinoline alkaloid isolated
from the Chinese Rhizoma coptidis (the dried rhizome of
Coptis sp.) and has promising ChE inhibitory properties
(Table 1). The majority of the interactions of berberine
with the enzymes are hydrophobic, although the berberine
cation has the potential to establish electrostatic interactions with neighbouring residues.[81] In addition, berberine
inhibits b-secretase activity in a rabbit model of AD, reducing Ab accumulation.[82] Groenlandicine, a structurally
similar alkaloid, was also extracted from Rhizoma coptidis
and revealed high activity towards ChE, acting in a noncompetitive manner (Table 1).[34] Tubocurarine (Figure 3a)
is another natural isoquinoline alkaloid (from Chondrodendron tomentosum Ruiz & Pav.), known for its musclerelaxant effects, acting by competing with ACh for nicotinic
receptors.[25] Tubocurarine is able to inhibit AChE by
binding to the peripheral site.[83]

Galantamine (Figure 3a) is a natural alkaloid with an isoquinoline skeleton that was isolated for the first time from
Galanthus nivalis L.[76] Galantamine selectively and reversibly inhibits AChE, increases presynaptic ACh release and
postsynaptic neurotransmission, by acting as an allosteric
ligand at nicotinic receptors.[17] Galantamine is well tolerated and is being used in AD therapeutics. A recent work
concluded that long-term galantamine treatment allows
cognitive and global stabilization of AD.[84] Galantamine
displays fairly strong in-vitro AChE inhibitory activity
(IC50 = 4.0 mm) (Table 1).[37] The increase in the size of the
alkyl group linked to the nitrogen of galantamine seems to
improve the AChE inhibitory activity: N-(14-methylallyl)
norgalantamine (IC50 = 0.16 mm) > N-allylnorgalantamine
(IC50 = 0.18 mm) > galantamine. N-(14-Methylallyl)norgalantamine and N-allylnorgalantamine are derived from the
waste generated by the industrial production of galantamine from Leucojum aestivum L. leaves. The application
of these compounds in therapeutics would allow efficient
use of those wastes.[85] In contrast, the loss of the N-methyl
group of galantamine, such as in epinorgalantamine, is

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

1687

Natural cholinesterase inhibitors

Brgida R. Pinho et al.

associated with a decrease in AChE inhibitory capacity,


showing again the importance of the N-substituent group
for the ChE inhibitory activity. Berkov and co-workers
argue that the tertiary amine establishes a non-classical
hydrogen bond via its alkyl group.[85]
Geissospermine (Figure 3b), an indole-indoline alkaloid,
is the major alkaloid extracted from the bark of Geissospermum vellosii Allem. and is probably mainly responsible for
the AChE and BuChE inhibition observed with the
alkaloid-rich fraction from the stem bark of this species.[86]
The possible interactions among geissospermine and AChE
of the Pacific electric ray were studied by molecular
docking; hydrogen bonds, hydrophobic interactions and
p-p stacking are involved.[87]
Another indole alkaloid that has been studied for its anticholinergic properties is infractopicrin (Figure 3b). This
compound was isolated from Cortinarius infractus Berk.
and revealed a high selectivity for AChE (IC50 = 9.72 mm),
since no BuChE inhibition was observed up to a concentration of 100 mm (Table 1). Infractopicrin binds preferentially
to the oxyanion hole of the enzyme by p-p interactions with
the aromatic residues.[39] Furthermore, it showed low cytotoxicity in a range of concentrations (maximum 100 mm) in
a hepatocyte cell line (HEpG2) and in a neuroblastoma cell
line (SH-SY5Y).[39] In contrast to infractopicrin, several
indole alkaloids isolated from Psychotria laciniata Vell.
showed no AChE inhibition (up to 100 mm), being active
against BuChE. Angustine was the most effective one
(Figure 3b), with an IC50 value of 3.47 mm.[88]
Huperzia serrata (Thunb.) Trevis is used in several traditional Chinese medicine formulas to alleviate problems of
memory loss, schizophrenia and myasthenia gravis.[25,76]
The main bioactive compound identified in this species
is huperzine A (Figure 3c), a sesquiterpene alkaloid that
is a selective and reversible inhibitor of AChE (IC50 =
0.082 mm) (Table 1).[38] The formation of the huperzine
AAChE complex is rapid and its dissociation is slow.[89]
Beyond the anti-ChE effects of huperzine A, mitochondria
have been proposed as a potential direct target of huperzine A, since this alkaloid induces beneficial effects on
mitochondrial dysfunction in transgenic mice.[90] Furthermore, huperzine A modulates non-amyloidogenic metabolism of b-amyloid precursor protein.[91] Huperzine A
reversed or attenuated cognitive deficits in several animal
models[92] and improved the cognitive and non-cognitive
functions in AD patients (100200 mg, twice a day).[93]
However, in a phase II trial, using the same dose, huperzine A did not show any cognitive benefit in patients with
moderate AD,[94] whereby more studies are needed to
evaluate its efficacy and safety.
Petrosamine (Figure 3d) is a pyridoacridine-brominated
alkaloid isolated from a marine sponge (Petrosia sp.) and
has higher anti-AChE activity (IC50 = 0.091 mm) than galan1688

tamine (Table 1). Nukoolkarn and collaborators argue that


the quaternary ammonium group makes the major contribution to the interaction with the enzyme, due to the establishment of a strong electrostatic interaction with a
glutamate residue.[42] No data exists about the ability of petrosamine to inhibit BuChE.
Juliflorine (Figure 3e) is a piperidine alkaloid isolated
from the leaves of Prosopis juliflora (Sw.) DC. and is
known for its antimicrobial properties. Juliflorine noncompetitively inhibits both AChE (IC50 = 0.42 mm) and
BuChE (IC50 = 0.12 mm) (Table 1).[40] Molecular docking
revealed that juliflorine is ideally spaced inside the aromatic
gorge of AChE, with rings A/B remaining at the top and
rings C/D deep inside, due to the higher hydrophobicity of
the latter two. Hence, juliflorine interacts with peripheral
and quaternary binding sites. The juliflorineAChE
complex is stabilized by hydrophobic interactions, hydrogen
bonding between juliflorine and the aromatic gorge of
AChE, while amino acids of the peripheral site are exclusively involved in the hydrophobic contacts that might be
responsible for the non-competitive mode of action.[40]
Steroidal alkaloids are found in a relatively small
number of plant families.[25] a-Solanine and a-chaconine
(Figure 3f) are two glycoalkaloids from Solanaceae; they
inhibit ChE in vitro and in vivo (Table 1) and are able to
alter the effects of neuromuscular blocking drugs.[35]
Several pregnane-type alkaloids from Sarcococca saligna (D.
Don) Mull. Arg. inhibit AChE at the same range of concentration (57 mm).[95] The majority of these pregnane-type
alkaloids are non-competitive inhibitors of both AChE and
BuChE (e.g. axillaridine A) (Figure 3f); others, like sarsalignone and sarsalignenone (Figure 3f), inhibit BuChE
uncompetitively.[33] The potency of these alkaloids may be
related to the substituents of ring A: the carbonyl group at
C4 position and the nitrogen substituents at C3, which
may be protonated at physiological pH, can establish
cation-p interactions with aromatic residues of the peripheral site of AChE.[95] On the other hand, substituents, such
as benzamide moieties, at C3 (steric hindrance) and
hydroxyl group at C2 are associated with unfavourable
interactions.[33]
Lycorine (Figure 3g) is the most frequent alkaloid found
in Amaryllidaceae plants. This compound has a weak activity against AChE (IC50 = 213 mm). However, modification of
the lycorine skeleton by acetylation may increase the inhibitory potency (1-O-acetyllycorine, IC50 = 0.96 mm).[96] The
hydrogen-bond acceptor at the C1 of lycorine is needed
for AChE inhibition, while a lipophilic substituent at C2
increases the inhibitory activity.[97] A recent work involving
lycorine derivatives showed that acylation or etherification
of lycorine produces compounds able to inhibit both
human BuChE (IC50 = 420 mm) and AChE (IC50 =
1150 mm).[97]

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

Natural cholinesterase inhibitors

Furanocoumarins
O

OH
O

O
OH

Scopoletin

Marmesin

R2
O

Pyranocoumarins

O
R1
Compound

R1

R2

OH
O

Compound

Decursinol

OH

Heraclenol-2-O-angelate

OH

Imperatorin

Isopimpinellin

OCH3

OCH3

Methoxsalen

OCH3

Decursin

Figure 4 Chemical structure of coumarins: scopoletin, furanocoumarins (heraclenol-2-O-angelate; imperatorin, isopimpinellin, marmesin and
methoxsalen) and pyranocoumarins (decursinol and decursin).

Other groups of alkaloids have been studied, but without


great anti-ChE activity as compared with those mentioned
above. Natural lycopodane-type alkaloids, such as lycodoline or lycofoline, inhibit AChE at concentrations ranging
from 191 to >2000 mm but the majority of these compounds
do not inhibit BuChE (IC50 > 2000 mm). Ligand docking
studies revealed that although the lycopodane skeleton fits
into the active gorge of AChE, the position of the functional
groups does not allow the establishment of strong interactions.[98] 6-Hydroxycrinamine, a crinine alkaloid isolated
from Boophane disticha (L. f.) Herb., has an IC50 towards
inhibition of AChE of 445 mm,[99] which is higher than the
values found for all of the alkaloids previously mentioned
in this review.

Coumarins
Coumarins are benzo-a-pyrones (a benzene ring joined
to a pyrone ring) with interesting pharmacological proper-

ties.[100] The natural compound scopoletin (Figure 4) inhibits AChE with an IC50 of 52 mm (Table 1).[52] This has been
confirmed by in-vivo assays, in which scopoletin and its
glucoside scopolin increased rat brain extracellular ACh
concentration.[101]
Methoxsalen (Figure 4), a furanocoumarin also known as
xanthotoxin, was extracted from Poncirus trifoliata (L.) Raf.
and has strong AChE inhibitory activity (IC50 = 3.07 mm)
(Table 1). The anti-AChE activity of methoxsalen was confirmed by the inhibition of mouse brain enzyme and amelioration of drug-induced behavioural impairment in an
AD-like mouse model.[51,102] Two furanocoumarins with
good anti-AChE activity were extracted from Angelica acutiloba (Siebold & Zucc.) Kitag: methoxsalen and isopimpinellin (IC50 = 0.32 mm) (Figure 4, Table 1).[50] Imperatorin (a
prenylated furanocoumarin) and heraclenol-2-O-angelate
(Figure 4) were isolated from Angelica archangelica L. and
both compounds showed a good BuChE inhibitory activity
(IC50 of 14.4 and 7.5 mm, respectively) (Table 1). The

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

1689

Natural cholinesterase inhibitors

Brgida R. Pinho et al.

authors concluded that C8 and C5 substituted furanocoumarins are BuChE inhibitors.[49] Other authors argue that
coumarins (e.g. methoxsalen, imperatorin and bergapten)
are more selective against BuChE than against AChE and
that the difference in enzymatic inhibition can reach
50%.[49,103] In contrast to the negative effect of glycosylation
on anti-ChE activity, marmesin (Figure 4) (a furanocoumarin) and its corresponding glycoside have similar activity
(IC50 of 67 and 68 mm, respectively).[48]
Kang et al. showed that the compound isolated from
Angelica gigas Nakai with high activity against AChE was a
pyranocoumarin, decursinol (Figure 4), and that the
inclusion of a prenyl group instead of hydroxyl group at
C3, such as in decursin (Figure 4), induces a decrease of
activity (Table 1).[48] Furthermore, decursin ameliorated
scopolamine-induced amnesia in mice, inhibiting the hippocampus AChE activity by 34%.[104]
Besides the study of the anti-ChE effects of natural coumarins, researchers have been using the coumarin skeleton
to produce compounds with stronger activity. Catto and
collaborators designed a series of coumarin alkylamides
with the structural determinants of donepezil, a known
synthetic non-competitive and reversible AChE inhibitor.
Thus, 6,7-dimethoxycoumarin, carrying a protonable benzylamino group linked to C3 by specific linkers, showed a
good activity towards both AChE and BuChE. Apparently,
these compounds are able to bind to both catalytic and
peripheral binding sites of ChE, the activity being highly
dependent on the length of the spacer and on the methoxyl
substituents.[105] Coumarin derivatives with a N-benzyl
pyridinium moiety also have two AChE binding sites,
and are active at the nanomolar range.[106] In addition,
3-thiadiazolyl and thioxo-1,2,4-triazolylcoumarin derivatives have very good activity also at nanomolar concentrations against both AChE and BuChE.[107]

Flavonoids
Flavonoids constitute a class of polyphenols characterized
by a diphenylpropane (C6-C3-C6) skeleton, which consists of
two aromatic rings, each bearing at least one aromatic
hydroxyl, connected by a carbon bridge, forming (or not) a
third ring. Flavonoids are divided into subclasses based on
the connection of the two aromatic rings, degree of oxidation and also the functional groups of the third ring
(flavanols, flavanones, anthocyanidins, flavones, flavonols,
isoflavones, flavan-3-ols, flavanonols, aurones or chalcones). The majority of flavonoids naturally occur as glycosides or other conjugates, which explains the great variety of
compounds, and are considered to be the most relevant
class of phenolic compounds.[108]
Uriarte-Pueyo and collaborators reviewed the ChE
inhibitory capacity of 128 flavonoids, concluding that the
1690

compounds with higher AChE activity were flavones or isoflavones. Thus, the carbonyl group at C4 seems to be important to anti-AChE activity. This team verified that among
the studied flavonoids, the most potent one was a synthetic
compound, 6,7-dimethoxy-3-[4-(pyrrolidin-1-ylmethyl)phenyl]-4H-chromen-4-one (IC50 = 0.004 mm), followed by
6,7-dimethoxy-2-[4-(piperidin-1-ylmethyl)-phenyl]-4Hchromen-4-one (IC50 = 0.034 mm) and 6,7-dimethoxy-2-[4(pyrrolidin-1-ylmethyl)-phenyl]-4H-chromen-4-one
(IC50 = 0.093 mm). The substituents pyrrolidin-1-ylmethyl
or piperidin-1-ylmethyl at C4, as well as methoxyl groups
at C6 or C7, appear to play an important role in the AChE
inhibitory activity of flavonoids.[109] The compounds containing a flavonoid moiety and a second moiety, which
could be amino alkyl, pyrrolidine or piperidine through an
appropriate spacer oxygen atom or alkoxyl group (O-CH2),
are able to interact with both the peripheral and the catalytic site of AChE: the flavonoid moiety is able to interact
with the peripheral site, while the second moiety interacts
with the catalytic site.[110] Compounds with pyrrolidine
or piperidine groups exhibited higher activity than those
with amino alkyl groups, indicating the key role of a
conformation-constrained hydrophobic moiety for AChE
inhibition.[110]
Among natural flavonoids, pomiferin (Figure 5), a
prenylated isoflavone extracted from Maclura pominifera
(Raf.) C.K. Schneid., was found to possess high anti-AChE
activity (IC50 = 96 mm) (Table 1). In contrast, flavanones
were less active against AChE than flavones, which suggested that the double bound at C2-C3 is important for the
anti-AChE activity.[109] Additionally, 5-geranyl-5,7,2,4tetrahydroxyflavone was the most potent AChE and BuChE
inhibitor isolated from Morus lhou Koidz., highlighting the
importance of the flavone skeleton. This compound, as well
as others not substituted at C3, are mixed inhibitors, while
the C3-prenyl substituted flavones are non-competitive
inhibitors, indicating that the presence of a prenyl group at
C3 affects the interactions with the enzyme.[111]
Considering natural and common flavonoids, fustin
(Figure 5) inhibited AChE (no IC50 value available) and
decreased the expression of the gene encoding AChE by
Ab.[112] Naringenin (Figure 5), a flavanone from Citrus junos
Siebold ex Tanaka, ameliorated scopolamine-induced
amnesia in mice, also having AChE inhibitory activity in
vitro, albeit at high concentrations (Table 1).[113] For prenylated flavonoids, the presence of a geranyl group at C6 is
important for anti-ChE activity, as demonstrated when
mimulone (IC50 = 91.5 mm) was compared with naringenin
(IC50 = 2045 mm) (Figure 5, Table 1).[56]
Several works have reported the strong anti-ChE activity
of galangin (a flavonol) compared with other flavonoids
tested under the same conditions.[53,114] Galangin was
reported to be the most potent BuChE inhibitor among

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

Natural cholinesterase inhibitors

R5

R4
R6

R5

R1

R1

R6
R2

R4

R3

R3

R2

Compound

R1

R2

R3

R4

R5

R6

Compound

R1

R2

R3

R4

R5

R6

Fustin

OH

OH

OH

OH

Fisetin

OH

OH

OH

OH

Mimulone

OH

OH

OH

Galangin

OH

OH

OH

Naringenin

OH

OH

OH

Kaempferol OH

OH

OH

OH

OH
HO
O

OH
O

HO

OH

Leofolin A

Myricetin

OH

OH

OH

OH

OH

OH

Quercetin

OH

OH

OH

OH

OH

Rutin

OH

OH

OH

Tamarixetin OH

OH

OH

OH

OCH3

Apigenin

OH

OH

OH

Luteolin

OH

OH

OH

OH

Rutinoside OH

O
HO
OH
HO
O

OH

OH
O

O
O

Leofolin B

HO
OH

O
OH

Pomiferin

HO

OH

Figure 5 Chemical structure of flavonoids from different subgroups: flavanonols (fustin), flavanones (mimulone, naringenin and leufolin A); flavones (apigenin, leofolin B and luteolin); flavonols (fisetin, galangin, kaempferol, myricetin, quercetin, rutin and tamarixetin) and prenylated isoflavones (pomiferin).

kaempferol, quercetin, myricetin, fisetin, apigenin, luteolin


and rutin (no IC50 value was provided).[114] At 50 mm,
galangin inhibited 56.5% of rat brain AChE activity,
followed by quercetin (25.8% inhibition).[53] Galangin has
no hydroxyl group on ring B (Figure 5) and is 12 times
more effective against BuChE (Ki = 6.9 mm) than against
AChE (Ki = 85.6 mm). Thus, the number of hydroxyl groups
in the B ring of a flavonoid influences the potency of
BuChE inhibition: an increase in the number of B-ring
hydroxyl groups induces a decrease in BuChE inhibition.[114]
Furthermore, kaempferol was the most potent flavonoid
isolated from Cleistocalyx operculatus (Roxb.) Merr. & L.M.
Perry buds towards BuChE. This was the only compound
with just one substituent in the B ring, while the others
(quercetin, tamarixetin and myricetin derivatives) had
two or three substituents.[54] Besides the hydroxyl groups
in the B ring, the presence of methoxyl groups at C5
and C7 favours BuChE and AChE inhibitory activity as
already mentioned. 5,7,4-Trimethoxyflavone and 5,7-

dimethoxyflavone were the most potent anti-AChE and


anti-BuChE compounds in Kaempferia parviflora Wall. ex
Barker extract; the activity decreased when the methoxyl
group at C5 was replaced by a hydroxyl group.[115] Docking
studies showed that flavonoids bind to the BuChE active site
by the establishment of multiple hydrogen bonds and p-p
interactions.[114] In contrast to galangin, for example, quercetin (Figure 5), another flavonol, inhibits both AChE
(IC50 = 353.9 mm) and BuChE (IC50 = 420.8 mm) at similar
concentration and in a competitive manner.[58] As quercetin
had higher BuChE inhibitory potency than luteolin, which
is structurally analogous to quercetin, having the same
number of hydroxyl groups in B ring but without the
hydroxyl group at C3, the presence of this hydroxyl should
be important for BuChE inhibition. Furthermore, glycosylation does not favour BuChE inhibition, due to steric constraints for accommodation to the active site of BuChE.[114]
However, Atia-tun-Noor and colleagues studied the effect
on BuChE of flavonoid glucosides isolated from Leucas

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

1691

Natural cholinesterase inhibitors

Brgida R. Pinho et al.

Benzoquinones

OH

Sargaquinoic acid

O
Naphthoquinones

O
O

O
Compound

Compound

Mansonone C

Mansonone E

Mansonone G

OH

Mansonone H

OH

R
O

Figure 6

Chemical structure of quinones: benzoquinone (sargaquinoic acid) and naphthoquinones (mansonones C, G, E and H).

urticifolia (Vahl) R.Br., leufolin A and B (Figure 5), and


showed that they were able to inhibit BuChE at low concentrations (IC50 of 1.6 and 3.6 mm, respectively) (Table 1).[55]
In contrast to BuChE inhibition, an enhancement of AChE
activity was reported after treatment of PC12 cells with
luteolin (1050 mm), favouring neuronal differentiation.[116]

Quinones
Quinones are widespread in nature, being essential for
many biochemical processes, such as mitochondrial respiration or photosynthesis. Quinones also have an important
role in an organisms defence, being able to inhibit bacterial, fungal and parasitic growth. All these properties are
related to the quinonoid structure, which has a pro-oxidant
character. Quinones may undergo redox cycling catalysed
by flavoenzymes, generating semiquinones or hydroquinones, which can create reactive oxygen species or react
with nucleophiles.[117] For all these reasons, it is not surprising that quinones are able to inhibit several proteins, such
as topoisomerase[118] or RNA polymerase.[119] However, there
are not many studies concerning the inhibition of ChE by
quinones.
The quinonoid group seems to be important for AChE
inhibition, since dopamine autoxidation can inactivate
AChE, mainly by direct interaction of quinone or semiquinone oxidation products with the enzyme.[120] Sargaquinoic
acid (Figure 6) is a natural benzoquinone extracted from
1692

the brown algae Sargassum sagamianum Yendo and has a


moderate AChE inhibitory activity (IC50 = 23.3 mm). Sargaquinoic acid is selective for BuChE (IC50 = 0.026 mm),
being 1000 times more active against this enzyme than
against AChE (Table 1).[61]
Thespesia populnea (L.) Sol. ex Correa is a plant reported
to enhance memory and reduce brain ChE activity in
mice.[121] It was hypothesized that mansonones (naphthoquinones) were responsible for this activity. Mansonone E
(Figure 6) was the tested naphthoquinone with highest
activity towards AChE and BuChE (IC50 of 23.5 mm and
62.4 mm, respectively). The authors of the study suggested a
correlation between AChE and BuChE inhibitory activity
and the existence of a fused pyran ring and of hydroxyl
group at C6. The presence of a fused pyran ring favours
anti-ChE activity: masonones E and H, having this feature,
are more active than mansonones C and G, which do not
present this characteristic. In contrast, the presence of a
hydroxyl group at C6 induces a decrease of the inhibitory
activity: mansonones C and E are more active than mansonones G and H, respectively.[60]

Stilbenes
Stilbenes are a small family of secondary metabolites
derived from the phenylpropanoid pathway. Resveratrol
(Figure 7) is probably the most extensively studied stilbene
and has potent anti-cancer, anti-inflammatory and antioxi-

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

Natural cholinesterase inhibitors

OH

HO
HO

HO
OH

HO
HO

Resveratrol

Gnetol

OH
HO
HO
HO
O

OH

OH

HO
OH

OH

OH
HO
O

O
OH

OH
HO

HO
Figure 7

(+)--Viniferin

HO

Kobophenol A

Chemical structure of stilbenes: monomers (resveratrol and gnetol) and oligomers ((+)-a-viniferin and kobophenol A).

dant properties.[122] In spite of the few studies about antiChE activity of stilbenes, some compounds of this class
with anti-ChE activity are currently known. Gnetol
(Figure 7), a stilbene isolated from Ficus foveolata Pittier,
has good activity towards BuChE (Table 1), via a reversible
and competitive mechanism.[63] (+)-a-Viniferin (Figure 7)
inhibits AChE in a dose-dependent manner (IC50 =
2.0 mm) (Table 1), by a non-competitive mechanism. (+)-aViniferin is a trimer of resveratrol, which itself has only a
weak effect on AChE, since it does not provide 50% inhibition at a concentration of 500 mm (Table 1). Thus, stilbeneoligomerization favours AChE inhibition. Nevertheless,
kobophenol A (Figure 7) (IC50 = 115.8 mm), a tetramer of
resveratrol, has lower AChE inhibitory potency than (+)-aviniferin (Table 1), probably due to steric hindrance.[62]
Other stilbenes with reported AChE inhibitory activity
are trans-3,5-dimethoxystilbene (IC50 = 9 mm), trans-3,5dimethoxy-2-prenylstilbene (IC50 = 19 mm) and furanokurzin (IC50 = 42 mm).[123]

Terpenic compounds
Terpenes, which are built up from isoprene subunits, constitute the most numerous and structurally diverse group of

secondary metabolites produced by plants. Terpenes may


exert effects on several functions in plants, such as insects
attraction for pollination, growth regulation (phytohormones) or protection through their anti-feedant properties.[100] Several biological actions have been attributed to
terpenes. ChE inhibition is one of them, although, in
general, terpenes inhibit ChE at higher concentrations than
alkaloids (Table 1).
The essential oils from species such as Thymus vulgaris L.
and Eucalyptus globulus Labill. show ChE inhibitory activity.
Monoterpenoids are the main constituents of these essential
oils.[124] In the monoterpene subgroup, 1,8-cineole and
a-pinene (Figure 8a) are the most effective compounds.
1,8-Cineole is more potent against mammalian AChE
(IC50 = 670 mm in bovine erythrocytes)[64] than against
AChE from the electric eel (IC50 = 6000 mm).[125] a-Pinene
inhibits AChE in bovine erythrocytes with the same potency
as 1,8-cineole (IC50 = 630 mm) (Table 1).[64] Furthermore,
1,8-cineole is the mainly responsible for the AChE inhibitory activity of three essential oils of Salvia lavandulaefolia
Vahl, since its IC50 value is very similar to those of the essential oils.[126] Miyazawa and Yamafuji evaluated the AChE
inhibitory activity of several other bicyclic monoterpenoids

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

1693

Natural cholinesterase inhibitors

Brgida R. Pinho et al.

(a)-Monoterpenes

(b)-Diterpenes
O

O
O

O
O

1,8-Cineole 3-Carene

2-Carene

Myrtenal

-Pinene

Cryptotanshinone

Dihydrotanshinone

OH

(c)-Triterpenes and steroids


O

O
O

OH

Leoheteronin A

Leopersin G

O
O
HO

HO

Leucisterol

Taraxerol
O
O

Limbatolide A

OH

Compound
OH

OH

OH

Limbatolide B OCH3
Limbatolide C

R
H

O
OH

HO
HO

Ursolic acid

Oleanolic acid

Sclareol

Figure 8 Chemical structure of terpenic compounds: (a) monoterpenes: 1,8-cineole, 3-carene, 2-carene, myrtenal and a-pinene; (b) diterpenes:
cryptotanshinone, dihydrotanshinone, leoheteronin A, leopersin G, limbatolide A, B and C and sclareol; (c) triterpenes and steroids: leucisterol,
taraxerol, ursolic acid and oleanolic acid.

and verified also that a-pinene is one of the most potent


(IC50 = 400440 mm), together with 3-carene (IC50 =
200 mm) and 2-carene (IC50 = 900 mm) (Figure 8a). The different activity of 3-carene and 2-carene is due to the position of the double bond and also oxygenated functional
groups decrease the inhibition of AChE.[65] Other researchers described a good AChE inhibitory activity for myrtenal
(Figure 8a, Table 1),[70] an aldehyde, while Miyazawa and
Yamafuji observed that myrtenol, an alcohol, does not have
relevant activity.[65] Savelev and collaborators studied the
inhibition of BuChE by monoterpenoids. Of 21 compounds, only 3-carene was able to inhibit the enzyme
(IC50 = 2000 mm). Thus, monoterpenoids are well able to
inhibit AChE, in contrast to a lack of effect on BuChE.[66]
Considering their low molecular weight, monoterpenoids
probably do not induce a dynamic modulation of the accessibility of the substrate to the active site of the enzyme, their
action being due to steric and /or allosteric effects.[66]
Diterpenoids inhibit ChE at lower concentrations than
monoterpenoids (Table 1), which indicates that the size of
1694

the molecule is critically important. Dihydrotanshinone and


cryptotanshinone (Figure 8b) are examples of diterpenoids
with the ability to inhibit AChE (Table 1)[127] in a noncompetitive manner. Dihydrotanshinone and cryptotanshinone are also non-competitive inhibitors of BuChE.[67]
These two diterpenoids were extracted from the roots of
Salvia miltiorrhiza Bunge, and are responsible for the antiChE of the extracts of this plant. In addition, attending to
their lipophilicity, dihydrotanshinone and cryptotanshinone have the potential to penetrate the bloodbrain barrier.[127] These findings show that dihydrofurans, such as
dihydrotanshinone and cryptotanshinone, are more active
than furans (as tanshinone I, tanshinone IIA), since dihydrofurans are more flexible, allowing binding to the active
site of the enzyme by hydrophobic interactions.[67,127] Other
studies have exploited the anti-ChE activity of tricyclic cisclerodane type diterpenoids: limbatolides A and B have
similar activity against AChE (IC50 of 38.5 and 47.2 mm,
respectively) while limbatolide C has a weaker effect
(IC50 = 103.7 mm) (Figure 8b, Table 1); thus it can be con-

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

Natural cholinesterase inhibitors

OH

OH

OH

OH

OH

OH

O
O
O

OH

HO

OH

OH

O
OH

Allanxanthone A
Figure 9

Bellidifolin

OH

OH

Triptexanthoside C

Chemical structure of xanthones: allanxanthone A, bellidifolin and triptexanthoside C.

cluded that the methoxyl group at C15 favours AChE inhibition. With respect to BuChE, the three mentioned
compounds have similar potency (IC50 = 14.222.3 mm)
(Table 1).[69] Sclareol (Figure 8b) is a diterpenoid extracted
from Salvia chrysophylla Stapf., and is reported to have good
activity against both AChE and BuChE.[128] Labdane-type
diterpenoids, like leoheteronin A (with a 8,13 diene and
15,16-epoxy group) and leopersin G (with a 13-diene and a
15,16 epoxy group) (Figure 8b), are also promising AChE
inhibitors (Table 1).[68]
Ursolic acid, taraxerol, leucisterol and oleanolic acid
(Figure 8c) are among the triterpenes and steroids with
anti-ChE activity described in literature.[129] ulhaoglu and
collaborators reported that ursolic and oleanolic acids are
selective against AChE.[128] Other researchers showed that
the IC50 towards BuChE of ursolic acid is lower than that
against AChE (Table 1).[59] Ursolic acid inhibits AChE in a
competitive/non-competitive way,[130] although Fatima et al.
did not verify any activity against AChE or BuChE.[131] Leucisterol is more potent against BuChE (IC50 = 3.2 mm) than
against AChE (IC50 = 83.6 mm).[131] Taraxerol inhibits AChE
in a dose-dependent manner and its activity is similar to
that of ursolic acid (Table 1).[52] Withanolides are C28steroidal lactone triterpenoids that can be isolated from
several species, such as Withania somnifera (L.) Dunal or
Ajuga bracteosa Wall. ex Benth. They inhibit both AChE
(IC50 = 2030 mm) and BuChE (IC50 = 5085 mm).[132]

Xanthones
Xanthones are secondary metabolites produced by plants,
fungi and lichens, important for providing protection
against insects, plant viruses, bacterial infections and animal
predation.[133] In spite of having only one additional
benzene ring than coumarins, xanthones are, in general,
weak ChE inhibitors, whereby the xanthone ring (dibenzog-pyrone) by itself does not appear to automatically confer
activity.[25,73,134] However, the presence of a lipophilic side

chain seems to be important for ChE inhibitory properties:


allanxanthone A (Figure 9) is a prenylated xanthone with
inhibitory activity towards AChE and BuChE (Table 1).[72]
Triptexanthoside C (Figure 9) was the only xanthone isolated from Gentianella amarella (L.) Borner with AChE
inhibitory activity (Table 1).[73] The absence of a glucopyranosyl moiety and the presence of a methoxyl group at
C3 are associated with increased inhibitory activity, perhaps
due to steric factors or hydrophobicity.[25] Bellidifolin
(Figure 9) was the most active of several xanthones isolated
from Gentiana campestris L., with activity at the micromolar range (Table 1).[135]

Others
Here we consider some compounds that due to their features were not included in any of the previous sections, but
deserve to be mentioned.
Territrem B (Figure 10) is a fungal metabolite isolated
from Aspergillus terreus Thom and has high AChE inhibitory activity (IC50 = 0.076 mm), acting by a mechanism different from those of other AChE inhibitors: territrem B
blocks the entrance of ACh into AChE by hydrophobic
interaction with the lipophilic amino acid in the entry of
the binding site channel. Several authors report that this
binding is kinetically irreversible, at least within the duration of study. In contrast to other inhibitors, territrem B
shows a one-to-one stoichiometry and appears not to bind
to subsites but to all the active gorge.[75,136138] The substantial and innovatory activity of territrem B encouraged the
search for new territrem B derivatives. Preliminary
structureactivity investigations suggested that the 2-en-1one moiety and the planar conformation were essential for
the AChE inhibitory activity.[138,139] No BuChE inhibition
has been observed with territrem B.[137]
Arisugacins are meroterpenoids from Penicillium species
with good anti-ChE activity described. Arisugacins A and
B (Figure 10) are selective inhibitors of AChE in vitro

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

1695

Natural cholinesterase inhibitors

Brgida R. Pinho et al.

OH
O
OH
O

R1
O

O
R2

Figure 10

Compound

R1

R2

Arisugacin A

OCH3

Arisugacin B

Territrem B

OCH3

OCH3

Chemical structure of territrem B, arisugacin A and B.

source of inspiration for the synthesis of new ones with


anti-ChE activity. In this work we reviewed several compounds belonging to several groups of metabolites: (i) alkaloids in general the nitrogen atom, which can be
protonated at physiological pH, has an important role in the
interaction with the active site; this group includes the
highest number of compounds with anti-ChE activity at
concentrations lower than 1 mm; (ii) coumarins research
suggests that furanocoumarins are selective BuChE inhibitors, though more studies are needed; (iii) flavonoids the
flavone or isoflavone subgroups have higher anti-ChE activity; several synthetic flavonoids are ChE inhibitors at the
submicromolar range, though natural compounds are less
potent; (iv) quinones there are few studies about their
anti-ChE properties; we described sargaquinoic acid, which
is selective to BuChE at the submicromolar range; (v) stilbenes another group whose anti-ChE capacity is still little
explored; the oligomerization of stilbenes seems to be
favourable to ChE inhibition; (vi) terpenic compounds
diterpenes are more potent than monoterpenes and, in
general, are more potent than triterpenes, whereby the size
of the molecule has a critical role in ChE inhibition; (vii)
xanthones generally weak ChE inhibitors, though some
substituents, such as lipophilic chain, can increase anti-ChE
activity.

Declarations
(Table 1).[140] As arisugacin A does not contain nitrogen, it is
suggested that the binding to AChE is by an electrondonatingelectron-withdrawing interaction. Arisugacin A
binds to peripheral anionic site by its dimethoxyaryl group,
whereby the dimethoxyaryl group stays positioned in the
opening of the catalytic gorge.[74] Similarly to territrem B,
arisugacin is highly selective towards AChE rather than
BuChE.[74]

Conclusions
AD is a neurodegenerative disease currently without effective treatment. ChE inhibitors can mitigate symptoms,
improving cognitive function due to the increase of ACh
half-life. Inhibitors of both AChE and BuChE could have
some advantages relative to compounds selective towards
AChE, as BuChE activity is normally enhanced in AD
patients brains. Nature is a source of new compounds and a

References
1. Uttara B et al. Oxidative stress and
neurodegenerative diseases: a review
of upstream and downstream antioxidant therapeutic options. Curr
Neuropharmacol 2009; 7: 6574.
1696

Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.

Funding
The authors are grateful to Fundao para a Cincia e a Tecnologia (FCT) for grant no. PEst-C/EQB/LA0006/2011, to
Consolider Ingenio 2010 Project CSD2007-00063 FUN-CFOOD, and to Grupo de Excelencia de la Rgion de Murcia
04486/GERM/06. B. Pinho thanks FCT for the grant
(SFRH/BD/63852/2009). Part of this work was carried out
under international cooperation within the CYTED Programme, Thematic Network Action 112RT0460 Characterization and evaluation of functionality and safety of
bioactive compounds from Iberian-American fruits for
food ingredients (CORNUCOPIA).

2. Jucker M, Walker LC. Pathogenic


protein seeding in Alzheimer disease
and other neurodegenerative disorders. Ann Neurol 2011; 70: 532540.
3. Alzheimers Association. 2012 Alzheimers disease facts and figures.
Alzheimers Dement 2012; 8: 131168.

4. Mattson MP. Pathways towards and


away from Alzheimers disease.
Nature 2004; 430: 631639.
5. Reddy PH. Abnormal tau, mitochondrial dysfunction, impaired axonal
transport of mitochondria, and
synaptic deprivation in Alzheimers

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

6.

7.

8.

9.

10.

11.

12.

13.
14.

15.

16.

17.

18.

disease. Brain Res 2011; 1415: 136


148.
Mao P, Reddy PH. Aging and
amyloid b-induced oxidative DNA
damage and mitochondrial dysfunction in Alzheimers disease: implications for early intervention and
therapeutics. Biochim Biophys Acta
2011; 1812: 13591370.
Reitz C et al. Epidemiology of Alzheimer disease. Nat Rev Neurol 2011; 7:
137152.
OBrien RJ, Wong PC. Amyloid precursor protein processing and Alzheimers disease. Annu Rev Neurosci
2011; 34: 185204.
Szymanski M et al. Alzheimers risk
variants in the clusterin gene are
associated with alternative splicing.
Transl Psychiatry 2011; 1: e18.
doi:10.1038/tp.2011.17.
Lee JH et al. The neuronal sortilinrelated receptor gene SORL1 and
late-onset Alzheimers disease. Curr
Neurol Neurosci Rep 2008; 8: 384
391.
Assal F, Cummings JL. Neuropsychiatric symptoms in the dementias.
Curr Opin Neurol 2002; 15: 445
450.
Perrin RJ et al. Multimodal techniques for diagnosis and prognosis of
Alzheimers disease. Nature 2009;
461: 916922.
Blennow K et al. Alzheimers disease.
Lancet 2006; 368: 387403.
Zhang L et al. Current neuroimaging
techniques in Alzheimers disease
and applications in animal models.
Am J Nucl Med Mol Imaging 2012; 2:
386404.
Selkoe DJ. Alzheimers disease: genes,
proteins, and therapy. Physiol Rev
2001; 81: 741766.
Hardy J, Selkoe DJ. The amyloid
hypothesis of Alzheimers disease:
progress and problems on the road
to therapeutics. Science 2002; 297:
353356.
Scarpini E et al. Treatment of Alzheimers disease: current status and
new perspectives. Lancet Neurol
2003; 2: 539547.
Schliebs R, Arendt T. The cholinergic
system in aging and neuronal degen-

Natural cholinesterase inhibitors

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

eration. Behav Brain Res 2011; 221:


555563.
Beatty WW et al. Patterns of memory
failure after scopolamine treatment:
implications for cholinergic hypotheses of dementia. Behav Neural Biol
1986; 45: 196211.
Sherman SJ et al. Scopolamine
impairs human recognition memory:
data and modeling. Behav Neurosci
2003; 117: 526539.
Fukami T, Yokoi T. The emerging
role of human esterases. Drug Metab
Pharmacokinet 2012; 27: 466477.
Darvesh S et al. Neurobiology of
butyrylcholinesterase. Nat Rev Neurosci 2003; 4: 131138.
Jbilo O et al. Tissue distribution of
human acetylcholinesterase and
butyrylcholinesterase
messenger
RNA. Toxicon 1994; 32: 14451457.
Kasa P et al. The cholinergic system
in Alzheimers disease. Prog Neurobiol 1997; 52: 511535.
Houghton PJ et al. Acetylcholinesterase inhibitors from plants and fungi.
Nat Prod Rep 2006; 23: 181199.
Perry EK et al. Correlation of cholinergic abnormalities with senile
plaques and mental test scores in
senile dementia. Br Med J 1978; 2:
14571459.
Davies P, Maloney AJ. Selective loss
of central cholinergic neurons in
Alzheimers disease. Lancet 1976; 2:
1403.
Xie W et al. Postnatal developmental
delay and supersensitivity to organophosphate in gene-targeted mice
lacking acetylcholinesterase. J Pharmacol Exp Ther 2000; 293: 896902.
Fukuchi K et al. Amelioration of
amyloid load by anti-Ab single-chain
antibody in Alzheimer mouse model.
Biochem Biophys Res Commun 2006;
344: 7986.
Ibach B, Haen E. Acetylcholinesterase
inhibition in Alzheimers disease.
Curr Pharm Des 2004; 10: 231251.
Kinghorn AD et al. The relevance of
higher plants in lead compound discovery programs. J Nat Prod 2011;
74: 15391555.
Kennedy DO, Wightman EL. Herbal
extracts and phytochemicals: plant

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

secondary metabolites and the


enhancement of human brain function. Adv Nutr 2011; 2: 3250.
Khalid A et al. Kinetics and
structure-activity relationship studies
on pregnane-type steroidal alkaloids
that inhibit cholinesterases. Bioorg
Med Chem 2004; 12: 19952003.
Jung HA et al. Anti-Alzheimer and
antioxidant activities of Coptidis
Rhizoma alkaloids. Biol Pharm Bull
2009; 32: 14331438.
McGehee DS et al. Cholinesterase
inhibition by potato glycoalkaloids
slows mivacurium metabolism.
Anesthesiology 2000; 93: 510519.
Zhu X et al. A practical conversion of
natural physostigmine into the
potent butyrylcholinesterase inhibitor N1,N8-bisnorcymserine. Tetrahedron Lett 2000; 41: 48614864.
Pejchal V et al. 1,3-Substituted
imidazolidine-2,4,5-triones: synthesis and inhibition of cholinergic
enzymes. Molecules 2011; 16: 7565
7582.
Bai DL et al. Huperzine A, a potential
therapeutic agent for treatment of
Alzheimers disease. Curr Med Chem
2000; 7: 355374.
Geissler T et al. Acetylcholinesterase
inhibitors from the toadstool Cortinarius infractus. Bioorg Med Chem
2010; 18: 21732177.
Choudhary MI et al. Juliflorine: a
potent natural peripheral anionicsite-binding inhibitor of acetylcholinesterase with calcium-channel
blocking potential, a leading candidate for Alzheimers disease therapy.
Biochem Biophys Res Commun 2005;
332: 11711177.
Kishibayashi N et al. Inhibitory
effects of KW-5092, a novel gastroprokinetic agent, on the activity of
acetylcholinesterase in guinea pig
ileum. Jpn J Pharmacol 1994; 66:
397403.
Nukoolkarn VS et al. Petrosamine, a
potent anticholinesterase pyridoacridine alkaloid from a Thai marine
sponge Petrosia n. sp. Bioorg Med
Chem 2008; 16: 65606567.
Thomsen T et al. Inhibition of acetylcholinesterase activity in human
1697

Natural cholinesterase inhibitors

44.

45.

46.

47.

48.

49.

50.

51.

52.

1698

brain tissue and erythrocytes by


galanthamine, physostigmine and
tacrine. Eur J Clin Chem Clin
Biochem 1991; 29: 487492.
Iijima S et al. The long-acting
cholinesterase inhibitor heptylphysostigmine
attenuates
the
scopolamine-induced
learning
impairment of rats in a 14-unit
T-maze. Neurosci Lett 1992; 144:
7983.
Wilson BW et al. Actions of pyridostigmine and organophosphate
agents on chick cells, mice, and
chickens. Drug Chem Toxicol 2002;
25: 131139.
Cerasoli DM et al. In vitro and in vivo
characterization of recombinant
human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger. Chem Biol Interact 2005;
157158: 363365.
Seto Y, Shinohara T. Structureactivity relationship of reversible
cholinesterase inhibitors including
paraquat. Arch Toxicol 1988; 62:
3740.
Kang SY et al. Coumarins isolated
from Angelica gigas inhibit acetylcholinesterase: structure-activity relationships. J Nat Prod 2001; 64: 683
685.
Wszelaki N et al. Bioactivity-guided
fractionation for the butyrylcholinesterase inhibitory activity of
furanocoumarins from Angelica
archangelica L. roots and fruits. J
Agric Food Chem 2011; 59: 9186
9193.
Miyazawa M et al. Insecticidal
effect of phthalides and furanocoumarins from Angelica acutiloba
against Drosophila melanogaster. J
Agric Food Chem 2004; 52: 4401
4405.
Kim JK et al. Effects of methoxsalen
from Poncirus trifoliata on acetylcholinesterase and trimethyltin-induced
learning and memory impairment.
Biosci Biotechnol Biochem 2011; 75:
19841989.
Lee JH et al. Acetylcholinesterase
inhibitors from the twigs of Vaccinium oldhami Miquel. Arch Pharm Res
2004; 27: 5356.

Brgida R. Pinho et al.

53. Guo AJ et al. Galangin, a flavonol


derived from Rhizoma Alpiniae
Officinarum, inhibits acetylcholinesterase activity in vitro. Chem Biol
Interact 2010; 187: 246248.
54. Min BS et al. Cholinesterase inhibitors from Cleistocalyx operculatus
buds. Arch Pharm Res 2010; 33:
16651670.
55. Noor A et al. Leufolins A and
B, potent butyrylcholinesteraseinhibiting flavonoid glucosides from
Leucas urticifolia. Molecules 2007; 12:
14471454.
56. Cho JK et al. Cholinestrase inhibitory
effects of geranylated flavonoids
from Paulownia tomentosa fruits.
Bioorg Med Chem 2012; 20: 2595
2602.
57. Orhan I et al. Cholinesterase inhibitory effects of the extracts and compounds of Maclura pomifera (Rafin.)
Schneider. Food Chem Toxicol 2009;
47: 17471751.
58. Khan MT et al. Cholinesterase
inhibitory activities of some flavonoid derivatives and chosen xanthone and their molecular docking
studies. Chem Biol Interact 2009; 181:
383389.
59. ztrk M et al. Antioxidant and
anticholinesterase active constituents
from Micromeria cilicica by radicalscavenging activity-guided fractionation. Food Chem 2011; 126: 3138.
60. Changwong N et al. Acetyl- and
butyryl-cholinesterase
inhibitory
activities of mansorins and mansonones. Phytother Res 2012; 26: 392
396.
61. Choi BW et al. Anticholinesterase
activity of plastoquinones from
Sargassum
sagamianum:
lead
compounds for Alzheimers disease
therapy. Phytother Res 2007; 21: 423
426.
62. Sung SH et al. (+)-a-Viniferin, a stilbene trimer from Caragana chamlague, inhibits acetylcholinesterase.
Biol Pharm Bull 2002; 25: 125127.
63. Sermboonpaisarn T, Sawasdee P.
Potent and selective butyrylcholinesterase inhibitors from Ficus
foveolata. Fitoterapia 2012; 83: 780
784.

64. Perry NS et al. In vitro inhibition of


human erythrocyte acetylcholinesterase by Salvia lavandulaefolia essential oil and constituent terpenes. J
Pharm Pharmacol 2000; 52: 895902.
65. Miyazawa M, Yamafuji C. Inhibition
of acetylcholinesterase activity by
bicyclic monoterpenoids. J Agric
Food Chem 2005; 53: 17651768.
66. Savelev SU et al. Butyryl- and acetylcholinesterase inhibitory activities in
essential oils of Salvia species and
their constituents. Phytother Res
2004; 18: 315324.
67. Wong KK et al. Interaction study of
two diterpenes, cryptotanshinone
and dihydrotanshinone, to human
acetylcholinesterase and butyrylcholinesterase by molecular docking and
kinetic analysis. Chem Biol Interact
2010; 187: 335339.
68. Hung TM et al. Labdane-type diterpenoids from Leonurus heterophyllus
and their cholinesterase inhibitory
activity. Phytother Res 2011; 25: 611
614.
69. Ahmad VU et al. Three new
cholinesterase-inhibiting
cisclerodane diterpenoids from Otostegia limbata. Chem Pharm Bull 2005;
53: 378381.
70. Kaufmann D et al. Myrtenal inhibits
acetylcholinesterase, a known Alzheimer target. J Pharm Pharmacol 2011;
63: 13681371.
71. Koay YC et al. Chemical constituents
and biological activities of Strobilanthes crispus L. Rec Nat Prod 2013; 7:
5964.
72. Lenta BN et al. Leishmanicidal and
cholinesterase inhibiting activities of
phenolic compounds from Allanblackia monticola and Symphonia globulifera. Molecules 2007; 12: 15481557.
73. Urbain A et al. Xanthones from Gentianella amarella ssp. acuta with acetylcholinesterase and monoamine
oxidase inhibitory activities. J Nat
Prod 2008; 71: 895897.
74. Al-Rashid ZF, Hsung RP. (+)Arisugacin A computational evidence of a dual binding site covalent
inhibitor of acetylcholinesterase.
Bioorg Med Chem Lett 2011; 21:
26872691.

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

Brgida R. Pinho et al.

75. Kuno F et al. Arisugacins A and B,


novel and selective acetylcholinesterase inhibitors from Penicillium sp.
FO-4259. II. Structure elucidation. J
Antibiot (Tokyo) 1996; 49: 748751.
76. Orhan IE et al. An overview on
natural cholinesterase inhibitors a
multi-targeted drug class and their
mass production. Mini Rev Med
Chem 2011; 11: 836842.
77. Mukherjee PK et al. Lead finding for
acetyl cholinesterase inhibitors from
natural origin: structure activity relationship and scope. Mini Rev Med
Chem 2011; 11: 247262.
78. Barak D et al. Accommodation of
physostigmine and its analogues by
acetylcholinesterase is dominated by
hydrophobic interactions. Biochem J
2009; 417: 213222.
79. Atack JR et al. Comparative inhibitory effects of various physostigmine
analogs against acetyl- and butyrylcholinesterases. J Pharmacol Exp Ther
1989; 249: 194202.
80. Greig NH et al. Selective butyrylcholinesterase inhibition elevates brain
acetylcholine, augments learning and
lowers Alzheimer b-amyloid peptide
in rodent. Proc Natl Acad Sci U S A
2005; 102: 1721317218.
81. Ji HF, Shen L. Molecular basis of
inhibitory activities of berberine
against pathogenic enzymes in
Alzheimers disease. The Scientific
World Journal 2012; 2012: 823201.
82. Panahi N et al. Effects of berberine
on b-secretase activity in a rabbit
model of Alzheimers disease. Arch
Med Sci 2013; 9: 146150.
83. Cousin X et al. Cloning and expression of acetylcholinesterase from
Bungarus fasciatus venom. A new
type of cooh-terminal domain;
involvement of a positively charged
residue in the peripheral site. J Biol
Chem 1996; 271: 1509915108.
84. Wallin AK et al. Galantamine treatment in Alzheimers disease:
response and long-term outcome in a
routine clinical setting. Neuropsychiatr Dis Treat 2011; 7: 565576.
85. Berkov S et al. N-Alkylated galanthamine derivatives: potent acetylcholinesterase
inhibitors
from

Natural cholinesterase inhibitors

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

Leucojum aestivum. Bioorg Med


Chem Lett 2008; 18: 22632266.
Lima JA et al. Geissospermum vellosii
stembark: anticholinesterase activity
and improvement of scopolamineinduced memory deficits. Pharmacol
Biochem Behav 2009; 92: 508513.
Araujo JQ et al. Docking of the
alkaloid geissospermine into acetylcholinesterase: a natural scaffold targeting the treatment of Alzheimers
disease. J Mol Model 2011; 17: 1401
1412.
Passos CS et al. Indole alkaloids
of Psychotria as multifunctional
cholinesterases and monoamine oxidases inhibitors. Phytochemistry
2013; 86: 820.
Zangara A. The psychopharmacology
of huperzine A: an alkaloid with cognitive enhancing and neuroprotective
properties of interest in the treatment of Alzheimers disease. Pharmacol Biochem Behav 2003; 75: 675
686.
Yang L et al. Decreased accumulation
of subcellular amyloid-b with
improved mitochondrial function
mediates the neuroprotective effect
of huperzine A. J Alzheimers Dis
2012; 31: 131142.
Wang Y et al. Huperzine A alleviates
synaptic deficits and modulates amyloidogenic and nonamyloidogenic
pathways in APPswe/PS1dE9 transgenic mice. J Neurosci Res 2012; 90:
508517.
Shi Q et al. Huperzine A ameliorates
cognitive deficits and oxidative stress
in the hippocampus of rats exposed
to acute hypobaric hypoxia. Neurochem Res 2012; 37: 20422052.
Zhang Y et al. Role of the catalytic
triad and oxyanion hole in acetylcholinesterase catalysis: an ab initio
QM/MM study. J Am Chem Soc 2002;
124: 1057210577.
Rafii MS et al. A phase II trial of
huperzine A in mild to moderate
Alzheimer disease. Neurology 2011;
76: 13891394.
Zaheer-ul-Haq
et al.
3D-QSAR
studies on natural acetylcholinesterase inhibitors of Sarcococca saligna
by comparative molecular field

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

analysis (CoMFA). Bioorg Med Chem


Lett 2003; 13: 43754380.
Elgorashi EE et al. Acetylcholinesterase enzyme inhibitory effects of
Amaryllidaceae alkaloids. Planta Med
2004; 70: 260262.
Wang YH et al. Synthesis and biological evaluation of lycorine derivatives as dual inhibitors of human
acetylcholinesterase and butyrylcholinesterase. Chem Cent J 2012; 6:
96.
Halldorsdottir ES et al. Acetylcholinesterase inhibitory activity of
lycopodane-type alkaloids from the
Icelandic Lycopodium annotinum ssp.
alpestre. Phytochemistry 2010; 71:
149157.
Adewusi EA et al. Cytotoxicity and
acetylcholinesterase inhibitory activity of an isolated crinine alkaloid
from Boophane disticha (Amaryllidaceae). J Ethnopharmacol 2012; 143:
572578.
Brunetton R. Pharmacognosy: Phytochemistry Medicinal Plants, 2nd edn.
Hampshire: Intercept Ltd., 1999.
Rollinger JM et al. Acetylcholinesterase inhibitory activity of scopolin
and scopoletin discovered by virtual
screening of natural products. J Med
Chem 2004; 47: 62486254.
Kim JK et al. Inhibitory effect of Poncirus trifoliate on acetylcholinesterase
and attenuating activity against
trimethyltin-induced learning and
memory impairment. Biosci Biotechnol Biochem 2009; 73: 11051112.
Senol FS et al. An in vitro and in silico
approach to cholinesterase inhibitory
and antioxidant effects of the methanol extract, furanocoumarin fraction, and major coumarins of
Angelica officinalis L. fruits. Phytochem Lett 2011; 4: 462467.
Kang SY et al. Decursin from Angelica gigas mitigates amnesia induced
by scopolamine in mice. Neurobiol
Learn Mem 2003; 79: 1118.
Catto M et al. Design, synthesis and
biological evaluation of coumarin
alkylamines as potent and selective
dual binding site inhibitors of acetylcholinesterase. Bioorg Med Chem
2012; 21: 146152.
1699

Natural cholinesterase inhibitors

106. Alipour M et al. Novel coumarin


derivatives bearing N-benzyl pyridinium moiety: potent and dual
binding site acetylcholinesterase
inhibitors. Bioorg Med Chem 2012;
20: 72147222.
107. Raza A et al. Pharmacological evaluation and docking studies of
3-thiadiazolyl- and thioxo-1,2,4triazolylcoumarin derivatives as
cholinesterase inhibitors. ISRN Pharmacol 2012; 707932.
108. Rice-Evans CA et al. Structureantioxidant activity relationships of
flavonoids and phenolic acids. Free
Radic Biol Med 1996; 20: 933956.
109. Uriarte-Pueyo I, Calvo MI. Flavonoids as acetylcholinesterase inhibitors. Curr Med Chem 2011; 18: 5289
5302.
110. Sheng R et al. Design, synthesis and
evaluation of flavonoid derivatives as
potent AChE inhibitors. Bioorg Med
Chem 2009; 17: 66926698.
111. Kim
JY
et al. Isolation
of
cholinesterase-inhibiting flavonoids
from Morus lhou. J Agric Food Chem
2011; 59: 45894596.
112. Jin CH et al. Fustin flavonoid attenuates b-amyloid (1-42)-induced learning impairment. J Neurosci Res 2009;
87: 36583670.
113. Heo HJ et al. Naringenin from Citrus
junos has an inhibitory effect on
acetylcholinesterase and a mitigating
effect on amnesia. Dement Geriatr
Cogn Disord 2004; 17: 151157.
114. Katalinic M et al. Structural aspects
of flavonoids as inhibitors of human
butyrylcholinesterase. Eur J Med
Chem 2010; 45: 186192.
115. Sawasdee P et al. Anticholinesterase
activity of 7-methoxyflavones isolated from Kaempferia parviflora.
Phytother Res 2009; 23: 17921794.
116. EL Omri A et al. Luteolin enhances
cholinergic activities in PC12 cells
through ERK1/2 and PI3K/Akt
pathways. Brain Res 2012; 1437:
1625.
117. OBrien PJ. Molecular mechanisms
of quinone cytotoxicity. Chem Biol
Interact 1991; 80: 141.

1700

Brgida R. Pinho et al.

118. Sperry J et al. Pyranonaphthoquinone derivatives of eleutherin,


ventiloquinone L, thysanone and
nanaomycin A possessing a diverse
topoisomerase II inhibition and
cytotoxicity spectrum. Bioorg Med
Chem 2009; 17: 71317137.
119. Chao SH et al. Juglone, an inhibitor
of the peptidyl-prolyl isomerase
Pin1, also directly blocks transcription. Nucleic Acids Res 2001; 29: 767
773.
120. Klegeris A et al. A possible interaction between acetylcholinesterase
and dopamine molecules during
autoxidation of the amine. Free Radic
Biol Med 1995; 18: 223230.
121. Vasudevan M, Parle M. Pharmacological actions of Thespesia populnea
relevant to Alzheimers disease. Phytomedicine 2006; 13: 677687.
122. Roupe KA et al. Pharmacometrics
of stilbenes: seguing towards the
clinic. Curr Clin Pharmacol 2006; 1:
81101.
123. Thanh VT et al. Cytotoxic lignans
from fruits of Cleistanthus indochinensis: synthesis of cleistantoxin
derivatives. J Nat Prod 2012; 75:
15781583.
124. Aazza S et al. Antioxidant and
antiacetylcholinesterase activities of
some commercial essential oils and
their major compounds. Molecules
2011; 16: 76727690.
125. Picollo MI et al. Anticholinesterase
and pediculicidal activities of
monoterpenoids. Fitoterapia 2008;
79: 271278.
126. Savelev S et al. Synergistic and
antagonistic interactions of anticholinesterase terpenoids in Salvia
lavandulaefolia essential oil. Pharmacol Biochem Behav 2003; 75: 661668.
127. Ren Y et al. Novel diterpenoid acetylcholinesterase inhibitors from Salvia
miltiorhiza. Planta Med 2004; 70:
201204.
128. ulhaoglu B et al. Bioactive constituents of Salvia chrysophylla Stapf. Nat
Prod Res 2013; 27: 438447.
129. Yilmaz A et al. A novel isopimarane
diterpenoid with acetylcholinesterase

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

inhibitory activity from Nepeta


sorgerae, an endemic species to the
Nemrut Mountain. Nat Prod
Commun 2012; 7: 693696.
Chung YK et al. Inhibitory effect of
ursolic acid purified from Origanum
majorana L on the acetylcholinesterase. Mol Cells 2001; 11: 137143.
Fatima I et al. New butyrylcholinesterase inhibitory steroid and
peroxy acid from Leucas urticifolia.
Arch Pharm Res 2008; 31: 9991003.
Choudhary MI et al. Withanolides, a
new class of natural cholinesterase
inhibitors with calcium antagonistic
properties. Biochem Biophys Res
Commun 2005; 334: 276287.
Zarena AS, Udaya Sankar K. Screening of xanthone from mangosteen
(Garcinia mangostana L.) peels and
their effect on cytochrome c reductase and phosphomolybdenum activity. J Nat Prod 2009; 2: 2330.
Kang JJ, Fang HW. Polycyclic aromatic hydrocarbons inhibit the activity of acetylcholinesterase purified
from electric eel. Biochem Biophys
Res Commun 1997; 238: 367369.
Urbain AA et al. Xanthones from
Gentiana campestris as new acetylcholinesterase inhibitors. Planta Med
2004; 70: 10111014.
Chen JW et al. Territrem B, a tremorgenic mycotoxin that inhibits acetylcholinesterase with a noncovalent
yet irreversible binding mechanism.
J Biol Chem 1999; 274: 3491634923.
Chen JW, Ling KH. Territrems: naturally occurring specific irreversible
inhibitors of acetylcholinesterase.
J Biomed Sci 1996; 3: 5458.
Zhao Y et al. Preparation of analogues of territrem B, a potent AChE
inhibitor. Tetrahedron 2000; 56:
89018913.
Jiang X et al. Design, synthesis, and
biological evaluation of new territrem B analogues. Chem Biodivers
2005; 2: 557567.
Otoguro K et al. Arisugacins, selective acetylcholinesterase inhibitors of
microbial origin. Pharmacol Ther
1997; 76: 4554.

2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 16811700