Acetylcholinesterase Inhibitors from Natural.pdf

FABAD J. Pharm. Sci., 28, 51-58, 2003

SCIENTIFIC REVIEWS

Acetylcholinesterase Inhibitors from Natural

Resources

‹lkay ORHAN*°, Bilge ﬁENER*

Acetylcholinesterase Inhibitors from Natural Resources

Summary : Acetylcholinesterase inhibitors prevent reduction of

acetylcholine via inhibiting acetylcholinesterase enzyme which

hydrolyzes acetylcholine in the neuronal end from which it is

released. Acetylcholinesterase inhibitors play an important ro-

le in the treatment of Alzheimer ’ s Disease as well as Myasthe-

nia Gravis, Glaucoma and Helminthiasis together with the

mechanism of action of insecticide drugs.

In this review, some compounds obtained from natural resour-

ces that have acetylcholinesterase inhibitory activity are evalu-

ated.

Key Words: Acetylcholinesterase, Alzheimer ’ s Disease, acetylc-

holinesterase inhibitory activity, plant.

Received : 9.12.2002

Revised : 5.3.2003

Accepted : 21.3.2003

Do¤al Kaynaklardan Elde Edilen Asetilkolinesteraz ‹nhibitörleri

Özet : Asetilkolinesteraz inhibitörleri, asetilkolini sal›nd›¤› sinir

ucunda hidroliz eden asetilkolinesteraz enzimini inhibe etmek

suretiyle, asetilkolin miktar›n›n azalmas›n› önlemektedir. Asetil-

kolinesteraz inhibitörleri, Alzheimer hastal›¤›n›n yan›s›ra,

myastenia gravis, glokom ve helmintiyazis gibi hastal›klar›n te-

davisi ile insektisit ilaçlar›n etki mekanizmalar›nda da önemli

rol oynayan bileﬂiklerdir.

Bu derlemede, do¤al kaynaklardan elde edilen asetilkolineste-

raz inhibitör aktiviteye sahip baz› bileﬂikler de¤erlendirilmekte-

dir.

Anahtar kelimeler: Asetilkolinesteraz, Alzheimer hastal›¤›, ase-

tilkolinesteraz inhibitör aktivite, bitki.

INTRODUCTION

method for screening biological sources. AD is one

of the most common mental problems in the aged

population 1-3 . The basal forebrain and brainstem

cholinergic systems also play an important role in

the regulation of cortical and thalamic electrical acti-

vity 4 . The findings from experimental animals,

aging and AD research have provided an experi-

mental foundation for the cholinergic hypothesis of

learning and memory 5-7 . Based on the cholinergic

hypothesis, AD results from a defect in the choliner-

gic system. One goal in the treatment for AD is to

increase the acetylcholine level in the brain. Therefo-

re, AChE inhibitors are being developed for the tre-

atment of this disease.

Many new natural product-originated bioactive

compounds effective in treating several diseases ha-

ve been isolated from different plants, fungi and

microorganisms. They are unknown complex mix-

tures having potentially large number of secondary

metabolites. Sensitive assays have been developed

to screen these extracts from natural sources. The

simplest assays are the ones based on the mecha-

nism of action of a known drug. The assays have al-

so been incorporated into efficient testing schemes

that are useful for high-throughput screening (HTS).

For example; one assay used for Alzheimer’s Dise-

ase (AD) is based on the inhibition of acetylcholines-

terase (AChE). The development of new leads of

AChE inhibitors has been realized by the Ellman

Because of the side effects of the present drugs, re-

cently, galanthamine isolated from Amaryllidaceae

* Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, 06330, Ankara, Turkey.

° Corresponding author

Orhan, ﬁener

plants has been approved by the FDA for the treat-

ment of AD. However, the research for new AChE

inhibitors is still of interest and natural products are

an important source of these compounds.

perzia serrata (Thunb.) Trev) (Lycopodiaceae). This

plant, called as "Qing Ceng Ta", has been used in tra-

ditional Chinese medicine for its memory-enhan-

cing property for centuries 9 .

Based on the documented memory enhancing and

antiaging activities in folk medicine, the following

plants and isolated compounds have been chronolo-

gically described as potential leads for the develop-

ment of new drugs for the treatment of AD.

Over 100 alkaloids, a number of which are of the se-

ries of huperzin A-R, have been isolated from the ge-

nus Lycopodium which is very rich in alkaloid con-

tent 10 . Of them, only huperzin A possessed remar-

kable acetylcholinesterase inhibitory activity 9 .

Acetylcholinesterase inhibitors from plants

Physostigma venenosum

Physostigmine (1) , the first discovered-compound

within this class of compounds, is an alkaloid isola-

ted from Physostigma venenosum L. (Fabaceae). La-

ter on, physostigmine (Synapton ® ), has been a mo-

del for some drugs with acetylcholinesterase activity

such as rivastigmine (Exelon ® ), which was synthesi-

zed later 8 . Although, the results of the first clinical

trials with physostigmine were promising, short ac-

tion duration and cholinergic side effects of physos-

tigmine have limited its therapeutic use.

(2)

The activity of Huperzin A has been found to be as

high as physostigmine, galanthamine, donepezil

and tacrine, the commercial drugs already used aga-

inst AD, or (even greater) than. In various in vivo

and ex vivo experiments, it has been shown to inhi-

bit acetylcholinesterase reversibly and also to pre-

vent oxidative cell damage induced by b-amiloid

plaques 11-15 .

(1)

In Ainge et al.’s work, Huperzin A isolated from L.

varium showed a potent insecticidal activity against

the insects Anthrenocerus australis, Lucilia cuprina

and Tineda bisselliella. Its total synthesis was comp-

leted and now this compound is in the stage-III cli-

nical trial in China 16-20 .

Lycopodium species

Huperzin A (2) has a special significance among the

compounds with acetylcholinesterase inhibitory ac-

tivity, isolated from natural resources. Huperzin A,

[(5R, 9R, 11E)-5-amino-11-etilidin- 5,6,9,10-tetrahid-

ro-7-metil-5,9-metanosikloocta-[b]-piridin-2 (1H)-

on], is an alkaloid isolated in 1986 by researchers of

the Shanghai Institute of Materia Medica from the

clubmoss Lycopodium serratum Thunb. (syn. Hu-

Related to this subject, in our ongoing research on

investigation of acetylcholinesterase inhibitory acti-

vity of some plants growing in Turkey, we screened

five Lycopodium species (L. annotinum, L. alpinum,

L. clavatum, L. complanatum subsp. chamaecypa-

rissus and L. selago) of the Lycopodiaceae family re-

garding their acetylcholinesterase inhibitory activity

FABAD J. Pharm. Sci., 28, 51-58, 2003

using the Ellman method, which is a spectrophoto-

metric, in vitro robotic screening method, and deter-

mined the responsible compound for the activity as

a-onocerin (3), a triterpene-type compound, from

Lycopodium clavatum that showed ca. 50 %

activity 21 .

Corydalis ternata

In a screening study by Kim et al., the methanolic

extract prepared from tubers of Corydalis ternata

(Papaveraceae) was found to have potent inhibitory

activity by the Ellman method. Bioactivity-directed

fractionation of this extract afforded protopine (6),

an alkaloid-type compound, by the Ellman method .

This result was supported by passive avoidance test,

which is used to measure antiamnesic activity, in

male mice 23 .

(3)

Salvia species

Perry et al. studied the acetylcholinesterase inhibi-

tory activity of essential oils of Salvia lavandulaefo-

lia and S. officinalis (Lamiaceae), the plants knowns

to be used as memory-enhancing in European folk

medicine, and the monoterpenes called (+) - a - pi-

nen, a - and b - terpineol, citronellal, d - terpinen, R

- (+) - limonen, 1,8-cineol, 1R-(+)-camphor, linalol,

1S-(-)-b-pinen and geraniol, the constituents of these

essential oils analyzed by GC-MS, were tested on

human erythrocyte acetylcholinesterase by the Ell-

man method. As a result; the essential oils of S. la-

vandulaefolia ile S. officinalis as well as camphor,

1,8-cineol, and a-pinen inhibited the enzyme in a

dose-dependent manner. When compared to the

standard drugs physostigmine and tacrine, the most

active monoterpenes were 1,8-cineol (4) (IC 50 = 0.67

mM) and a-pinen (5) (IC 50 = 0.63 mM) 22 .

(6)

Evodia rutaecarpa

In another screening study performed in South Ko-

rea 24 , Park et al. investigated 87 extracts prepared

from 29 plants in total by the Ellman method with

regard to anticholinesterase activity and found that

9 of the extracts showed over 40 % inhibitory acti-

vity. These extracts and their inhibition rates are as

follows: Poncirus trifoliata (dichloromethane ext-

ract, 91.0 %), Evodia rutaecarpa (dichloromethane

extract, 84.3 %), Coptis chinensis (methanol extract,

83.3 %), Coptis chinensis (dichloromethane extract,

76.9 %), Saussurea lappa (dichloromethane extract,

70.5 %), Angelica sinensis (dichloromethane extract,

65.5 %), Notopterygium incisium (dichloromethane

extract, 50.3 %), Evodia rutaecarpa (methanol ext-

ract, 43.8 %), Polygala tenuifolia (dichloromethane

extract, 40.0 %).

(4) (5)

Among them, the dichloromethane extract of Evo-

dia rutaecarpa displayed inhibitory activity in the

passive avoidance test in rats (Sprague-Dawley)

with scopolamine-induced memory loss, and

Orhan, ﬁener

dehydroevodiamine HCl (7) was isolated as the ac-

tive component through bioactivity-directed fracti-

onation 25,26 .

acetylcholinesterase inhibitory activity by the Ell-

man method against both acetylcholinesterase and

butyrylcholinesterase which also plays a role pos-

sibly in the pathology of AD and 27 steroidal alkalo-

ids of pregnane-type, 10 of which were new, were

isolated by bioactivity-directed fractionation. The al-

kaloids inhibited both enzymes in a dose-dependent

manner at IC 50 values between 5.21-22.7 µM for

acetylcholinesterase and 2.18-38.36 µM for butyrylc-

holinesterase 29 .

(7)

Areca catechu

Buxus species

In a study performed by Gilan et al., a hydroalcoho-

lic extract of Areca catechu (Arecaceae) inhibited

acetylcholinesterase and butyrylcholinesterase in a

dose-dependent manner 30 . However, the active

component has not been identified, yet.

Buxus species are well-known for their triterpene al-

kaloids having a great variety of biological activiti-

es. In a phytochemical work carried out on Buxus

hyrcana (Buxaceae), three alkaloids, two of which

were novel, were isolated and their acetylcholineste-

rase inhibitory activity was determined by the Ell-

man method. While hyrcanine, one of the novel al-

kaloids, was inactive against the enzyme, (+)-homo-

moenjodaramine (8) and (+)-moenjodaramine (9)

were found to be active.(respectively, IC 50 = 19.2 ve

50.8 mM) 27 .

Amanita mappa

In Bhattacharya et al.’s work, bufotenine (10), an in-

dole alkaloid isolated previously from the skin sec-

retion of several frog species and later from a fungus

species, Amanita mappa (syn. A. citrina), displayed

antiamnesic activity by passive avoidance test in

rats 31,32 .

(8) R 1 = CH 3 , R 2 = H 3 C-CH-Nb(CH 3 )2

(9) R 1 = H, R 2 = H 3 C-CH-Nb(CH 3 )2

(10)

Galanthus and Narcissus species

We investigated acetylcholinesterase inhibitory acti-

vity of Buxus sempervirens, a widespread plant in

Turkey, by the Ellman method and its inhibition

was found to be 63 % at 1 mg/ml concentration 28 .

Galanthamine (11), an alkaloid isolated from some

Galanthus species (Amaryllidaceae), has been re-

cently in use in the treatment of AD. It has a rever-

sible acetylcholinesterase inhibitory action and also

modulates the nicotinic acetylcholin receptors 33-38 .

Although the most common side effect of galantha-

mine is nausea, it is possible to eliminate nausea by

increasing the galanthamine dose gradually 39 . Ad-

Sarcococca saligna

The crude alkaloidal extract of Sarcococca saligna

(Buxaceae), which was shown to have a potent

FABAD J. Pharm. Sci., 28, 51-58, 2003

ditionally, galanthamine was shown to have no he-

patotoxicity 40 . Galanthamine (Nivalin ® ) has been

approved as HBr salt in Austria and later licensed as

Reminyl ® in the USA and some European countries

as well as Turkey in the treatment of AD.

Fumaria species

Within our project on acetylcholinesterase inhibitors

from some Turkish plants, we screened Fumaria

species from Fumarioideae subfamily (Fumaria ase-

pala, F. capreolata, F. cilicica, F. densiflora, F. juda-

ica, F. kralikii, F. macrocarpa, F. parviflora, F. pette-

ri subsp. thuretii, F. vaillantii) for their acetylcholi-

nesterase inhibitory activity by the Ellman method.

The inhibitory activities of all Fumaria species men-

tioned above were significantly high ranging betwe-

en 84.9 %- 96.8 %. Of these species, F. vaillantii with

94.2 % inhibition was chosen for bioactivity-directed

fractionation and the common isoquinoline alkalo-

ids named canadine, hydrastine, bulbocapnine, fu-

marophycine, corydaldine and protopine were obta-

ined from the active fractions of Fumaria vaillantii.

Consequently, the responsible compound for inhibi-

tory activity of F. vaillantii extract were established

as protopine. There was synergistic interaction bet-

ween the alkaloids 43 .

(11)

In our ongoing research on acetylcholinesterase in-

hibitory activity of some Turkish medicinal plants,

we screened some Galanthus and Narcissus species,

namely Galanthus elwesii, G. ikariae, Narcissus ta-

zettasubsp. tazetta, as well as two more Amarylli-

daceae plants, Leucojum aestivum and Pancratium

maritimum in terms of their acetylcholinesterase ac-

tivity by the Ellman method 28,41 .

Caragana chamlague

In a bioactivity-directed fractionation by Sung et al., the

methanolic extract of the underground parts of Caraga-

na chamlague (Fabaceae) with a significant acetylcholi-

nesterase inhibitory activity resulted in the isolation of

two active stilbene oligomers, (+)-a-viniferin (12)

(IC 50 =2.0 µm) and kobophenol A (13) IC 50 =115.8 mm),

by a slightly modified Ellman method. Both compo-

unds inhibited acetylcholinesterase in a dose-depen-

dent manner while (+)-a-viniferin showed a specific,

reversible and noncompetitive inhibition 44 .

In total, six Amaryllidaceae-type known alkaloids

called lycorine, tazettine, crinine, galanthamine, 3-

epi-hydroxybulbispermine and 2-demethoxymonta-

nine from the active fractions of Galanthus ikariae

were obtained by bioactivity-directed fractionation.

Lycorine, tazettine, N-nor-galanthamine, haemanta-

mine and 3-epi-hydroxybulbispermine were also

isolated from the active fractions of Narcissus tazet-

ta subsp. tazetta as the common Amaryllidaceae al-

kaloids. Although G. ikariae and N. tazetta subsp.

tazetta extracts showed 75.56 % and 46.62 % inhibi-

tion, respectively; it made us consider that the lower

than 50 % activity of the extracts was resulted from

the synergistic interaction between the alkaloids iso-

lated.

In a similar study by Lopez et al., 26 extracts prepa-

red from various Narcissus species together with 23

pure Amaryllidaceae-type alkaloids were screened

against acetylcholinesterase and it was suggested

that the alkaloids having galanthamine and lycorine

skeletons possess inhibitory activity 42 .

(12)

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