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Essential oils have the strong potency to inhibit acetylcholinesterase (AChE) in their lipophilicity and small molecular size constituents. Cholinesterase inhibitory activity was determined against AChE using Ellman's colorimetric method of 29 essential oils from aromatic plants. Results revealed 5 of essential oils from Eucalyptus, Cajuput, Sweet marjoram, Camphor tree and Rosemarry showed such activity respectively. The compositions of five essential oils were also analyzed by GC-MS. We also demonstrated that the AChE inhibitory activity of the essential oil could be attributed to 1,8-cineole.
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Int. J. Med. Arom. Plants, ISSN 2249 4340
RESEARCH ARTICLE
Vol. 4, No. 1, pp. 1-5, March 2014
*Corresponding author: (E-mail) watoop <@> yahoo.com
http://www.openaccessscience.com
© 2014 Copyright by the Authors, licensee Open Access Science Research Publisher.
ijmap@openaccessscience.com
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-
ND 3.0) License (http://creativecommons.org/licenses/by-nc-nd/3.0)
Survey of acetylcholinesterase inhibitory activity in essential oil
derived from aromatic plants
Watoo PHROMPITTAYARAT*1, Tapanee HONGRATANAWORAKIT2, Sarin TADTONG 2,
Vipaporn SAREEDENCHAI2, Kornkanok INGKANINAN3
1Faculty of Public Health, Naresuan University, 65000 Phitsanulok, Thailand
2Faculty of Pharmacy, Srinakharinwirot University, 26120 Nakhonayok, Thailand
3Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences,
Naresuan University, 65000 Phitsanulok, Thailand
*Corresponding author: Tel. 6655-968607, Fax. 6655-968607
Article History: Received 5th February 2014, Revised 21st March 2014, Accepted 24th March 2014.
Abstract: Essential oils have the strong potency to inhibit acetylcholinesterase (AChE) in their lipophilicity and small
molecular size constituents. Cholinesterase inhibitory activity was determined against AChE using Ellman’s colorimetric
method of 29 essential oils from aromatic plants. Results revealed 5 of essential oils from Eucalyptus, Cajuput, Sweet
marjoram, Camphor tree and Rosemarry showed such activity respectively. The compositions of five essential oils were
also analyzed by GC-MS. We also demonstrated that the AChE inhibitory activity of the essential oil could be attributed
to 1,8-cineole.
Keywords:Acetylcholinesterase inhibitory activity; essential oil; Ellman’s colorimetric method.
Introduction
Alzheimer’s disease (AD) is a progressive
degenerative neurologic disorder resulting in
impaired memory and behavior which caused by
a deficit of cholinergic functions in brain. The
most effective treatment have been postulated
that the deterioration of memory in patients suf-
fering from this disease. Although the cause of
AD remains obscure, two main strategies, cho-
linergic and non-cholinergic, are existed. One of
strategy as the cholinergic hypothesis, acetyl-
choline is recognized as the neurotransmitter
involved in the regulation of cognitive function
(Enz et al. 1993). For treating the disease is to
enhance the acetylcholine level in the brain us-
ing acetylcholinesterase (AChE) inhibitors.
AChE inhibitors have been studied for reducing
the action on the progression of AD. Currently,
Galantamine and Physostigmine are the proto-
types of the AChE inhibitors from nature
(Hostesttman et al. 2006).
Essential oils have been discovered for
AChE inhibitory activity and found strong po-
tency of the inhibitory activity because of the
small molecule and the lipophilicity. The essen-
tial oils from Salvia species have volatile sub-
stances which are likely to cross the blood brain
barrier and exert their effect (Perry et al., 2000;
Perry et al. 1996). In this study, we aimed to
screen the essential oils for the AChE inhibitory
activity and then investigate their constituents
with shown this activity using Ellman’s colori-
metric method in 96-well microplate (Ellman et
al. 1961; Ingkaninan et al. 2000).
Materials and methods
Plant material
Cajuput leaves were collected from the
south of Thailand. The voucher specimen is kept
at Faculty of Pharmacy, Srinakharinwiroj Uni-
versity, Nakhonnayok and Faculty of Pharmacy,
Mahidol University, Bangkok (Tadtong001).
The plant was identified by Prof.Wongsatit
Chuakul, Faculty of Pharmacy, Mahidol Uni-
versity, Bangkok, Thailand.
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Essential oils
The 29 essential oils were purchased from
Thai-China Flavour and Fragrances Industry
Co., Ltd. and Perfumesworld, (Bangkok, Thai-
land) They were from Cajuput (Melaleuca
cajuputi Powell) leaves, Eucalyptus (Eucalyptus
globulus Labell.) branches and leaves, Sweet
majoram (Majorana hortensis Moench) top,
Camphor (Cinnamomum camphora (L.) J.Presl)
leaves, Rose marry (Rosmarinus officinalis L.)
top, Lime (Citrus aurantiifolia (Christm.) Swin-
gle) fruit, Lemon grass (Cymbopogon flexuosus
(Nees ex Steud.) Will. Watson) grass, Lemon
(Citrus limonum Risso) peel, Lavender
(Lavandula angustifolia Mill) top, Thyme
(Thymus vulgaris L.) top, Pine (Pinus palustris
Mill) wood, Clove (Eugenia caryophyllus
(Spreng.) Bullock & S.G. Harrison) flower,
Spearmint (Mentha spicata L.) top, Bergamot
(Citrus bergamia Risso & Poit.) peel and leaves,
Jasmine (Jasminum sambac (L.) Aiton) flower,
Petigan (Citrus aurantium L.) peel, Patchouli
(Pogostemon cablin Benth.) leaves, Ylang
Ylang (Cananga odorata (Lam.) Hook.f. &
Thomson) flower, Vetier grass (Vetiveria
zizanioides Nash) root, Peppermint (Mentha
piperita L.) top, Tea Tree oil (Melaleuca
alternifolia Cheel) twig and leaves, Basil
(Ocimum basilicum L.) flowering top, Clary
sage (Salvia sclarea L.) flowering top, Cedar
wood (Cedrus atlantica G.Manetti) wood, Ge-
ranium (Pelargonium graveolens L’Her) top,
Palmarose (Cymbopogon martini (Roxb.) Wats.)
grass, Grapefruit (Citrus paradisi Macfad) peel,
Ginger (Zingiber officinale Rosco) rhizome, Cit-
ronella (Cymbopogon winterianus Jowitt) grass.
Extraction
Cajuput leaves were dried in the hot air oven
at 50 °C and blended to coarsely powder. The
dried material was extract using the Clevenger
apparatus for volatile oil.
Chemicals
Acetylthiocholine iodide (ATCI), AChE,
bovine serum albumin (BSA), 1,8-cineole and
5,5-dithiobis [2-nitrobenzoic acid] (DTNB)
were obtained from Sigma (St. Louis, MO.).
Fifty millimolar TrisHCl pH 8.0 was used as a
buffer throughout the experiment unless other-
wise stated. AChE used in the assay was from
electric eel (type VI-S lyophilized powder, 480
U/mg solid, 530 U/mg protein). The lyophilized
enzyme was prepared in the buffer to obtain
1130 U/ml stock solution. The enzyme stock
solution was kept at -80 C.The further enzyme-
dilution was dissolved in 0.1% BSA in buffer.
DTNB was dissolved in the buffer containing
0.1M NaCl and 0.02M MgCl2. ATCI was dis-
solved in deionized water.
Microplate assay
The assay for measuring AChE activity was
modified from the assay described by Ellman et
al. (1961) and Ingkaninan et al. (2000). Briefly,
125 µl of 3 m MDTNB, 25 µl of 15 mM ATCI,
and 50 µl of buffer, 25 µl of sample dissolved in
buffer containing not more than 10% methanol
were added to the wells followed by 25 µl of
0.28 U/ml AChE. The microplate was then read
at 405 nm every 5 s for 2 min by a CERES UV
900C microplate reader (Bio-Tek Instrument,
USA). The velocities of the reactions were
measured. Enzyme activity was calculated as a
percentage of the velocities compared to that of
the assay using buffer without any inhibitor. In-
hibitory activity was calculated from 100 sub-
tracted by the percentage of enzyme activity.
Every experiment was done in triplicate.
Results and discussion
Twenty nine essential oils were tested for
AChE inhibitory activity using Ellman’ colori-
metric method in 96-well microplate. The re-
sults are shown in Table 1. At concentration
0.12 mg/ml , the essential oil from M. cajuputi,
E. globules,O. majoram,C. camphora and R.
officinalis showed high AChE inhibitory activi-
ty (with percent inhibition of 68.68±4.82,
68.49±10.36, 65.56±, 60.63±7.11 and
51.59±1.81 respectively). At the same concen-
tration, the rest of essential oils were obtained
the AChE activity below 50.
Five candidate essential oils (M. cajuputi,E.
globules,O. majoram,C. camphora and R.
officinalis) were identified for their constituents
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using GC-MS (analyzed by Central Lab in
Chulalongkorn University) in Table 2. The mass spectroscopic data (Figure 1) of the active frac-
tions were compared with the chemical library.
Table 1: The inhibitory activity on AChE of essential oils (n=3).
Essential oils
Scientific name
% inhibition±SD
1
Cajuput
Melaleuca cajuputi Powell
68.68±4.82
2
Eucalyptus
Eucalyptus globulus Labell.
68.49±10.36
3
Sweet marjoram
Majorana hortensis Moench
65.56±2.91
4
Camphor
Cinnamomum camphora (L.) J.Presl
60.63±7.11
5
Rosemarry
Rosmarinus officinalis L.
51.59±1.81
6
Lime
Citrus aurantiifolia (Christm.) Swingle
46.86±1.78
7
Lemon grass
Cymbopogon flexuosus(Nees ex Steud.) Will. Watson
41.76±6.82
8
Lemon
Citrus limonum Risso
41.10±4.85
9
Lavender
Lavandula angustifolia Mill
38.83±0.86
10
Thyme
Thymus vulgaris L.
35.88±3.63
11
Pine
Pinus palustris Mill
33.39±0.43
12
Clove
Eugenia caryophyllus (Spreng.) Bullock & S.G.Harrison
31.94±1.91
13
Spearmint
Mentha spicata L.
31.09±4.82
14
Bergamot
Citrus bergamia Risso & Poit.
29.49±10.36
15
Jasmine
Jasminum sambac (L.) Aiton
28.24±2.91
16
Petigran
Citrus aurantium L.
28.01±2.12
17
Patchouli
Pogostemon cablin Benth.
25.20±4.21
18
Ylang Ylang
Cananga odorata (Lam.) Hook.f. & Thomson
24.42±2.13
19
Vetier grass
Vetiveria zizanioides Nash
24.16±0.94
20
Peppermint
Mentha piperita L.
22.97±0.20
21
Tea tree oil
Melaleuca alternifolia Cheel
22.09±2.77
22
Sweet basil
Ocimum basilicum L.
21.69±1.17
23
Clary sage
Salvia sclarea L.
20.70±2.28
24
Cedarwood
Cedrus atlantica G.Manetti
14.40±3.94
25
Geranium
Pelargonium graveolens L’Her
14.07±1.86
26
Palmarose
Cymbopogon martini (Roxb.) Wats.
13.45±1.32
27
Grapefruit
Citrus paradisi Macfad
13.31±0.07
28
ginger
Zingiber officinale Rosco
9.52±0.25
29
Citronella
Cymbopogon winterianus Jowitt
8.14±0.60
After the constituents in five essential oils
were identified, 1,8-cineole was the greatest ac-
tive detected constituent. This compound was
operated for AChE inhibitory activity at concen-
tration 1 mg/ml. It showed high AChE inhibito-
ry activity and IC50 were 81.58±0.99% and
0.15±0.01µg/ml respectively. Three (M.
cajuputi,E. globules,O. majoram) from five
essential oils with high content of 1,8-cineole
over 50% were performed for IC50. The IC50
were 0.63±0.08, 0.21±0.02 and 0.24±0.26µg/ml
consequently.
The current study showed AChE inhibitory
activity efficacy of essential oil of M. cajuputi,
E. globules and O. majoram with high content
of 1,8-cineole were similar to the previous stud-
ies of AChE inhibitory activity of the essential
oils with 1,8-cineole (Miyazawa et al., 1998;
Perry et al., 2000; Dohi et al., 2009).
Table 2: Active components of essential oils
identified by GC-MS
Essential oil
Constituents
% content
Cajuput
1,8-Cineole
70.16
α-Terpineol
10.08
Limonene
5.45
α-Pinene
1.71
Linalool
1.6
Eucalyptus
1,8-Cineole
83.3
α-Pinene
6.83
Limonene
4.59
ortho-Cymene
3.83
β-Pinene
1.07
Sweet majoram
1,8-Cineole
63.51
α-Terpineol
13.87
Limonene
5.63
α-Pinene
4.67
β-Pinene
4.19
Camphor
1,8-Cineole
39.93
Limonene
23.71
ortho-Cymene
9.06
α-Terpineol
4.98
α-Pinene
4.91
Rosemarry
1,8-Cineole
23.17
Camphor
19.16
ortho-Cymene
4.13
Limonene
3.52
Isobornyl acetate
2.62
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Figure 1: GC-MS chromatogram of five essential oils; M. cajuputi [A], E. globules [B], O. majoram
[C], C. camphora [D] and R. officinalis [E]
After the constituents in five essential oils
were identified, 1,8-cineole was the greatest ac-
tive detected constituent. This compound was
operated for AChE inhibitory activity at concen-
tration 1 mg/ml. It showed high AChE inhibito-
ry activity I and IC50 were 81.58±0.99% and
0.15±0.01µg/ml respectively. Three (M.
cajuputi,E. globules,O. majoram) from five
essential oils with high content of 1,8-cineole
over 50% were performed for IC50. The IC50
were 0.63±0.08, 0.21±0.02 and 0.24±0.26µg/ml
consequently.
The current study showed AChE inhibitory
activity efficacy of essential oil of M. cajuputi,
E. globules and O. majoram with high content
of 1,8-cineole were similar to the previous stud-
ies of AChE inhibitory activity of the essential
oils with 1,8-cineole (Miyazawa et al., 1998;
Perry et al., 2000; Dohi et al., 2009).
Table 3: Active components of essential oils in
Cajuput from commercial and in house identi-
fied by GC-MS and AChE inhibitory activity
Cajuput oil
Commercial (%con-
tent)
In house(%content)
Chemical constitu-
ents
1,8-Cineole(70.16)
Terpinolene(21.61)
γ-Terpineol (10.08)
g-Terpinene(17.8)
Limonene(5.45)
Caryophyllene(12.2)
α-Pinene(1.71)
Platyphyllol(7.5)
Linalool(1.60)
ortho-Cymene(7.34)
AChE Inhibition*
68.68±4.82
23.20±7.20
* concentration at 0.12 mg/ml
Regarding to prove this hypothesis, M.
cajuputi plant material was collected and ex-
tracted for essential oil. The essential oil was
RT: 0.00 - 61.01
0 5 10 15 20 25 30 35 4 0 45 50 55 60
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abund ance
9.94
6.71
9.79 60.97
57.39
53.7049.7312.883.19 16.40 46.8943.0839.8919.48 36.2726.70 32.61
NL:
8.81E7
TIC F: MS
Tapanee14
R T : 0 . 0 0 - 6 1 .0 1
0 10 2 0 30 4 0 5 0 6 0
T im e ( m in )
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
100
R e l a t iv e A b u n d a n c e
9 .9 4
16.40
6 .7 1 12.57 25.96 28.98 60.97
32.96 56.1724.006.48 50.31
46.94
N L :
5 .2 0 E 7
T IC F :
M S
tapanee17
R T : 0 . 0 0 - 6 1 .0 1
0 10 2 0 30 4 0 5 0 6 0
T im e ( m in )
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
100
R e l a t iv e A b u n d a n c e
9 .9 4
12.57
6 .7 1
20.43 60.83
16.40 57.97
51.5923.08 32.40 47.14
41.666.48
N L :
5 .2 6 E 7
T IC F :
M S
tapanee16
RT: 0.0 0 - 61 .01
0 5 10 1 5 20 25 30 3 5 4 0 45 50 55 6 0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re lativ e Ab und anc e
9.92
9.79
9.63
6.69 17.56
16.62
11.20
4.41 17.74 60.79
57.41
54.0449.3620.16 46.3642.9039.3036.2127.93
NL:
5.53 E7
TIC F: MS
tapanee37
R T : 0 . 0 0 - 6 0 . 9 9
0 1 0 2 0 3 0 4 0 5 0 6 0
T i m e ( m i n )
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
100
R e l a t iv e A b u n d a n c e
6 . 7 1
9 . 9 2
14.43
20.48
60.62
25.96 58.16
16.40 32.40 53.25
48.36
41.30
N L :
1 . 8 5 E 7
T IC F :
M S
tapanee15
1,8-cineole
[A]
[B]
[C]
[E]
[D
]
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Phrompittayarat et al.
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performed for AChE inhibitory activity and
identified for the constituents using GC-MS as
following Figure 2 and Table 3. The commercial
cajuput with high content of 1,8-cineole was
shown higher AChE inhibitory activity than the
in house cajuput.
Figure 2: GC-MS chromatogram of essential oils; commercial M. cajuputi [A] and in house M.
cajuputi [B]
Conclusion
From the screening of 29 essential oils
Eucalyptus, Cajuput, Sweet marjoram, Camphor
tree and Rose marry oils showed AChE inhibi-
tory activity and the AChE inhibitory activity
could be attributed to 1,8-cineole.
Acknowledgement: Financial support by Facul-
ty of Pharmacy, Srinakharinwirot University,
Thailand and facility support from Faculty of
Pharmaceutical Sciences, Naresuan University
are gratefully acknowledged and Ms Kanokwan
Changwichit for her helpful
References
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Miyazawa, M., Tougo, H. Ishihara, M. 2001.
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by essential oil of Citrus paradisi.Natural
product Letters,15: 205-210.
Perry, N.S., Houghton, P.J., Theobald, A., Jen-
ner, P., Perry, E.K. 2000. In vitro inhibition
of human erythrocyte acetylcholinesterase
by Salvia lavandulaefolia essential oil and
constituents terpenes. Journal of Pharmacy
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RT: 0 .0 0 - 61 .04
0 5 1 0 1 5 20 2 5 3 0 35 4 0 4 5 50 5 5 6 0
Tim e (mi n)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re la tiv e A b un da nc e
12.09
10.93
25.98
9.6 5
6.7 1
27.35
6.4 8
15.82 24.84 33.50 35.05
28.41 61.01
58.90
16.40 22.59 55.32
47.04
46.52
37.413.46
NL :
1.1 6E 7
TIC F: M S
tapanee26
<0.1% of 1,8-cineole
R T : 0 . 0 0 - 6 1 . 0 1
0 1 0 2 0 3 0 4 0 5 0 6 0
T i m e ( m in )
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
100
R e l a t i v e A b u n d a n c e
9 . 9 4
16.40
6 . 7 1 12.57 25.96 28.98 60.97
32.96 56.1724.006.48 50.31
46.94
N L :
5 . 2 0 E 7
T I C F :
M S
tapanee17
>70% of 1,8 cineole
[A
]
[B
]
... O. ehrenbergii and O. syriacum having similar mixed composition of thymol (19.6 and 24.7%), p-cymene (16.1 and 8.7%), 2-isopropyl-1-methoxy-4-methylbenzene (14.9 and 7.9%), γ-terpinene (11.8 and 12.6%) and carvacrol (6.7 and 17.6%) were tested on both AChE and BuChE and showed excellent activity on both enzymes having IC 50 lower than 1.7 µg/mL [46]. O. majorana having 1,8-cineole (63.5%) as the major compound, also showed excellent inhibitory activity on AChE with IC 50 of 0.24 µg/mL [47]. ...
... EOs from Eucalyptus globulus and Melaleuca cajuputi having high content of 1,8-cineole (over 70%) showed pronounced activity against AChE with IC 50 of 0.21 and 0.63 and µg/mL, respectively [47]. ...
... Species having monoterpene type EOs containing over 45% of mixture of 1,8-cineole, terpinen-4-ol, α-pinene and γ-terpinene (i.e., E. camaldulensis, E. globulus, M. alternifolia, M. cajuputi, C. viminals) generally showed very good AChE inhibitory activity with IC 50 ranging from 0.21 to 290.2 µg/mL. Only exception was Myrceugenia myrcioides EO which showed weak activity [15,23,32,45,47,56,57]. ...
Chapter
Essential oils (EOs) are a complex mixtures containing volatile secondary metabolites with various structures (terpenes, terpenoids, phenylpropanoids, and others). Their roles in plants vary from attracting pollinators to a defense against insects, food storage, and adaptations to harsh climate. Since ancient times, EOs are recognized for their medicinal value, representing a valuable source of biologically active compounds. EOs possess various activities including: antimicrobial, antiviral, antioxidant, antitumor, etc. They have also been used as perfumes, flavors for foods and beverages, remedies for the body and mind, and continue to be of paramount importance to date. Having a small molecular weight and lipid solubility, EO constituents possess the ability to pass the blood-brain barrier. Given the complexity of their chemical composition and ability to enter the brain, EOs and their constituents offer a promising strategy for the treatment of various neurological disorders. Alzheimer's disease has been responsible for more than 50% of neurological diseases among persons over the age of 65 years. EOs are found to be potential acetylcholinesterase and butyrylcholinesterase inhibitors, two enzymes which still represent the only pharmacoterapeutic targets against Alzheimer's disease. The purpose of this chapter is to provide an overview of the current knowledge about the activity of the EOs with defined chemical composition, tested against cholinesterases and to identify the ones that could be potentially used in Alzheimer's treatment.
... Two pharmacological strategies widely applied in AD treatment are the cholinergic and anticholinergic therapies [5,6]. Cholinergic therapy includes the equally efficient drugs donepezil, galantamine, and rivastigmine, which produce a temporary improvement of cognition at the expense of severe side effects [5,[7][8][9]. ...
... Recent studies [6,13,14] reported that potent acetylcholinesterase (AChE) inhibitors isolated from medicinal plants such as Mentha spicata L. subsp. spicata are very efficient in the treatment of AD and of other neurological disorders [15]. ...
... spicata (also known as Nane in Turkish) was used as antispasmodic and in colds and flu [16]. Phrompittayarat et al. [6] showed that a few compounds extracted from Mentha spicata L. subsp. spicata are potent AChE inhibitors due to their lipophilicity and small molecular size. ...
Article
Background: Alzheimer's disease (AD) therapy is based on several natural and synthetic compounds that act as acetylcholinesterase (AChE) and N-methyl-D-aspartate receptor (NMDA) ligands that have limited efficiency in relieving AD symptoms. Recent studies show that inhibitors isolated from Mentha spicata L. subsp. spicata are promising for AD therapy. Objective: We aimed to identify novel and more potent phytopharmaceutical compounds for AD treatment by taking into account the compounds from Mentha spicata L. subsp. spicata essential oil. Method: We generated structure-activity relationship (SAR) models that predict the biological activities of 14 Mentha spicata L. subsp. spicata compounds on AChE and NMDA by comparing their molecular features with those of the three conventional ligands: donepezil, galantamine and memantine. Results: The most relevant descriptors for predicting the biological activities of considered compounds are solvent accessible area and their subdivided, hydrophobicity, energy of frontier molecular orbitals and counts of the aromatic ring and rotatable bounds. 1,8-cineole, the main compound from Mentha spicata L. subsp. spicata essential oil, resulted to be similar with memantine and dissimilar with donepezil in respect to hidrophobicity (logP1,8-cineole=2.95, logPmemantine=2.81, logPdonepezil=4.11), the energy of LUMO (eLUMO1,8-cineole=3.01 eV, eLUMOmemantine=3.35 eV, eLUMOdonepezil=-0.35 eV) and the solvent accessible surface areas over all hydrophobic (SA_H1,8-cineole= 350 Å2, SA_Hmemantine= 358 Å2, SA_Hdonepezil= 655 Å2) or polar atoms (SA_P1,8-cineole= 4 Å2, SA_Pmemantine=10 Å2, SA_Pdonepezil=44.62 Å2). Conclusion: Our results point towards 1,8-cineole as a good candidate for NMDA antagonism, with a weaker AChE inhibitory effect. Our results may be useful in establishing new therapeutic strategies for neurological disorders.
... Although divergent IC 50 values are reported, 1,8cineole showed the best activity among all compounds listed in Table 3 (0.972 µM; 0.04, 0.1, 0.7, 1.81, 6.0 mM) [34,37,40,67,89,93]. According to their IC 50 values other promising monoterpenoids were: thymohydroquinone (0.24 mM) [94], thymol (0.31, 0.41, 4.9 mM) [34,92,94], carvacrol (0.35, 0.42, 0.61, 0.77 and 1.21 mM) [34,92,[94][95][96], thymoquinone (0.85 mM) [94], borneol (0.86 mM) [34], verbenone (1.09 mM) [96]. ...
... Carvone type essential oil from Mentha spicata having 75.9% of carvone showed 50% inhibition at 357.0 µg/mL [96], which is relatively stronger than pure carvone, as stated above. 1,8-Cineole type of essential oils from Origanum majorana L., Melaleuca cajuputi Powell, and Eucalyptus globulus Labill., having 63.5%, 70.2% and 83.3% of 1,8cineole, respectively, were reported to have strong inhibition of electric eel AChE with IC 50 values of 0.24, 0.63, and 0.21 µg/mL, respectively [93]. ...
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Essential oils constituents are a diverse family of low molecular weight organic compounds with comprehensive biological activity. According to their chemical structure, these active compounds can be divided into four major groups: terpenes, terpenoids, phenylpropenes, and "others". In addition, they may contain diverse functional groups according to which they can be classified as hydrocarbons (monoterpenes, sesquiterpenes, and aliphatic hydrocarbons); oxygenated compounds (monoterpene and sesquiterpene alcohols, aldehydes, ketones, esters, and other oxygenated compounds); and sulfur and/or nitrogen containing compounds (thioesters, sulfides, isothiocyanates, nitriles, and others). Compounds that act as cholinesterase inhibitors still represent the only pharmacological treatment of Alzheimer´s disease. Numerous in vitro studies showed that some compounds, found in essential oils, have a promising cholinesterase inhibitory activity, such as α-pinene, δ-3-carene, 1,8-cineole, carvacrol, thymohydroquinone, α- and β-asarone, anethole, etc. Essential oils constituents are a diverse family of low molecular weight organic compounds with comprehensive biological activity. According to their chemical structure, these active compounds can be divided into four major groups: terpenes, terpenoids, phenylpropenes, and "others". In addition, they may contain diverse functional groups according to which they can be classified as hydrocarbons (monoterpenes, sesquiterpenes, and aliphatic hydrocarbons); oxygenated compounds (monoterpene and sesquiterpene alcohols, aldehydes, ketones, esters, and other oxygenated compounds); and sulfur and/or nitrogen containing compounds (thioesters, sulfides, isothiocyanates, nitriles, and others).
... Table 1). In a study that surveyed the AChE inhibitory activity of essential oils derived from 29 aromatic plants, Phrompittayarat et al. showed that 14% inhibition of AChE at concentration 0.12 mg/mL of rose geranium essential oil (Phrompittayarat et al., 2014). The authors attributed the AChE inhibition mainly to the presence of 1,8-cineole. ...
Article
The current study provides the first comprehensive assessment of rose geranium oil (commercially referred to as a hybrid of Pelargonium graveolens L'Hér.) extracted from plants cultivated in Lebanon. The chemical composition was deciphered by GC–MS, and citronellol (30.5%), citronellyl formate (15.9%), trans-geraniol (12.8%), linalool (8.6%), and isomenthone (8.0%) were the major constituents. The oil composition most closely resembles rose geranium oil from Egypt. The essential oil exhibited weak antioxidant activity with only 16% inhibition achieved at 2.2 mg/mL in the DPPH radical-scavenging assay. The antiinflammatory activity of the essential oil was evaluated in three different setups, namely albumin denaturation, heat induced hemolysis, and nitric oxide scavenging activity, where a dose-dependent response was observed in the different assays. Assessment of the antibacterial activity of the essential oil revealed a bactericidal effect against the tested bacterial strains. The essential oil showed good cytotoxicity against HCT 116 human colon cancer cells with an IC50 value of 74 µg/mL as determined by the MTT cell viability assay. In the acetylcholinesterase (AChE) inhibition assay, the oil displayed a dose-dependent inhibition of AChE enzyme with an IC50 value of 10.5 mg/mL, thus highlighting the antiAlzheimer's protection potential of the extract. Finally, no significant hemolytic activity was observed even at the highest tested concentration (5 mg/mL). In conclusion, rose geranium oil produced in Lebanon demonstrated promising biological properties, and its cultivation for medicinal and industrial applications is highly recommended.
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Acetylcholinesterase (AChE) is an enzyme involved in the progression of Alzheimer's disease (AD). Cardamom oil (CO) has been reported to have acetylcholinesterase inhibitory, antioxidant and anti-anxiety effects. Hence, we studied the effect of cardamom oil in aluminum chloride induced neurotoxicity in rats. AD like symptoms were induced in Wistar rats with aluminum chloride (100 mg/kg, p.o.). Cardamom oil was administered concomitantly by oral route at doses of 100 and 200 mg/kg for 42 days. Behavioral parameters like Morris water maze, elevated plus maze, passive avoidance test and locomotor activity were evaluated on day 21 and 42. AChE activity, oxidative stress parameters, histopathological studies and immunohistochemistry studies were carried out in hippocampus and cortex. Cardamom oil treatment showed significant improvement in behavioral parameters, inhibition of AChE activity (p < 0.001) and reduction in oxidative stress in the brain. Histopathological studies of hippocampus and cortex by hematoxylin & eosin (H. & E.) and congo red stain showed inhibition of neuronal damage and amyloid β plaque formation with cardamom oil treatment. Immunohistochemistry showed, CO treatment inhibited amyloid β expression and upregulated brain-derived neurotrophic factor (BDNF). The present study showed that, cardamom oil has neuroprotective effect in aluminum chloride induced neurotoxicity linked with inhibition of AChE activity and reduction in oxidative damage. This effect of cardamom oil may be useful in management of Alzheimer's disease.
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Various natural products, such as physostigmine, have long been recognized as inhibitors of the enzyme acetylcholinesterase. Since the recent approval of galanthamine for the treatment of Alzheimer's disease by the blockage of acetylcholine degradation, attempts to find other inhibitors of the enzyme have multiplied, leading to promising candidates such as huperzine A. In this review, a listing is presented of natural product inhibitors, both alkaloid and nonalkaloid in origin. These have been isolated from plant, animal and microbial sources. Details of current testing methods on cholinesterases are given, including solution assays and screening techniques by TLC and HPLC.
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We investigated plants reputed in herbal encyclopedias to enhance memory or alleviate mental disorder for cholinergic activities since this transmitter system has been implicated in memory and dementia. Crude extracts were applied to human brain homogenates to determine whether any inhibit acetylcholinesterase. Of three plants with reputed memory enhancing properties (rosemary, sage and balm), extracts of sage (Salvia officinalis) inhibited the brain enzyme in a concentration dependant manner. 50% enzyme inhibition was obtained at a concentration of 0.07 μg essential oil per ml and 1.5 mg fresh herb per ml. None of the known and commercially available chemical constituents of sage oil so far tested (borneol, caffeic acid, camphor, cineol or thujone) inhibited the enzyme, indicating that the active plant constituent(s) may be an as yet unidentified compound(s). In parallel studies, plants with insecticide or vermifuge (antihelminthic) properties, which frequently depend on cholinergic activities, were examined for cholinergic receptor interactions. Crude alcoholic extracts of wormwood, balm and angelica displaced nicotine binding to the nicotinic receptor in a concentration dependant manner, with IC50 values ranging from 3–15 mg/ml. Components of these plants may be relevant in relation to dementia therapy since there is a loss of nicotinic receptors in Alzheimer's disease and related disorders and stimulation of the nicotinic receptor leads to increased receptor numbers.
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Inhibition of acetylcholinesterase (AChE) activity by several species of Mentha oils was investigated. AChE activity was measured by a colorimetric method. Mentha aquatica (water mint) containing sesquiterpene alcohols showed the most effective inhibition (IC50 of 26 μg/mL). In addition, some Mentha species such as M. aquatica (Akasaka-hakka II), M. gentilis (Fukuyama-hakka), M. gentilis (Akita-hakka), and M. arvensis (Nihon-hakka) showed potent inhibitory activity, and their IC50 values were 28−32 μg/mL. Viridiflorol and elemol showed the most potent inhibition in terpenoids as the main components of Mentha oils. But none of them showed stronger inhibitory activity than essential oils. Keywords: Acetylcholinesterase; Mentha species; essential oils; inhibitory activity; sesquiterpene alcohol; viridiflorol; elemol
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A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described. The enzyme activity is measured by following the increase of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. It is based on coupling of these reactions: The latter reaction is rapid and the assay is sensitive (i.e. a 10 μ1 sample of blood is adequate). The use of a recorder has been most helpful, but is not essential. The method has been used to study the enzyme in human erythrocytes and homogenates of rat brain, kidney, lungs, liver and muscle tissue. Kinetic constants determined by this system for erythrocyte eholinesterase are presented. The data obtained with acetylthiocholine as substrate are similar to those with acetylcholine.
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Commercially available essential oils extracted from Artemisia dracunculus L., Inula graveolens L., Lavandula officinalis Chaix, and Ocimum sanctum L. and the components of these oils were screened by the microplate assay method for determining their acetylcholinesterase (AChE) inhibitory activity. The composition profiles of the oils were characterized by gas chromatography-mass spectrometry (GC-MS) analysis, and the relationships between the oil components and the AChE inhibitory activity of the oils were outlined. The results showed that all of the oils, except that of A. dracunculus from Hungary, exhibited AChE inhibitory activity, and the A. dracunculus oil from France showed the most potent inhibitory activity [50% inhibition concentration (IC(50)) = 0.058 mg/mL]. The AChE inhibitory activity of I. graveolens oil has not been reported to date, and this study is the first to reveal this activity in the oil. Among the essential oil components, five components, namely, 1,8-cineole, α-pinene, eugenol, α-terpineol, and terpinen-4-ol, showed AChE inhibitory activity, with IC(50) values of 0.015, 0.022, 0.48, 1.3, and 3.2 mg/mL, respectively. Eugenol, in particular, was found to be a potent AChE inhibitor along with determination of the IC(50) value, a finding that has been reported for the first time in this study. However, the ratio of the contribution of the active components, including a novel AChE inhibitor, to the observed AChE inhibitory activity of the essential oils was not very high. The results of this study raise concerns about the AChE inhibitory activity of widely produced and readily accessible commercial essential oils.
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It could be argued that clinical experience with cholinergic drugs in the therapy of AD has not yet shown relevant symptomatic improvements. The main reasons for this might be attributed to peripheral cholinergic effects and the liver toxicity of some of these drugs, which limit their use and prevent confirmation of the cholinergic hypothesis (Gray et al., 1989). The main disadvantages of the cholinesterase inhibitors used in clinical trials are the short duration of action in the case of physostigmine and the potential for liver toxicity seen with the aminoacridine derivatives. The results presented with SDZ ENA 713 indicate that the disadvantages of AChE inhibitors might be overcome by improving CNS selectivity and thereby decreasing the peripheral cholinergic effects and toxicity. Clinico-pharmacological studies with SDZ ENA 713 have been performed in healthy volunteers; while central activity was clearly demonstrated in an EEG-sleep study (Holsboer et al., 1992), no prohibitive peripheral side effects were seen, confirming in humans the results obtained in experimental animals (Enz et al., 1991). A multicentre clinical investigation in AD patients has been performed in Europe and is currently being evaluated.
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A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.
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Sage (Salvia spp) is reputed in European herbal encyclopaedias to enhance memory, and current memory-enhancing/anti-dementia drugs are based on enhancing cholinergic activity by inhibiting cholinesterase. In this study the effects of Salvia lavandulaefolia Vahl. (Spanish sage) essential oil and some of its constituent terpenes on human erythrocyte acetylcholinesterase were examined in-vitro. The main constituents in the essential oil batch used for analysis of cholinesterase inhibition were camphor (27%), 1,8-cineole (13%), α- and β-pinene (10–15%) and bornyl acetate (10%) with other minor constituents (1% or less) including geraniol, limonene, linalool, terpineol and γ-terpinene. Using the Ellman spectrophotometric method, kinetic analysis was conducted on the interaction of the essential oil and the main monoterpenoids, camphor, 1,8-cineole and α-pinene. IC50 values were obtained for the essential oil, 1,8-cineole and α-pinene and were 0.03 μg mL−1, 0.67 mM and 0.63 mM, respectively. Camphor and other compounds tested (geraniol, linalool and γ-terpinene) were less potent (camphor IC50: >10 mM). The essential oil, α-pinene, 1,8-cineole and camphor were found to be uncompetitive reversible inhibitors. These findings suggest that if the inhibitory activity of the essential oil is primarily due to the main inhibitory terpenoid constituents identified, there is a major synergistic effect among the constituents. Since no single constituent tested was particularly potent, it remains to be determined whether these in-vitro cholinesterase inhibitory activities are relevant to in-vivo effects of the ingestion of S. lavandulaefolia essential oil on brain acetylcholinesterase activity.
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Inhibition of acetylcholinesterase (AChE) activity by essential oils of Citrus paradisi (grapefruit pink in USA) was studied. Inhibition of AChE was measured by the colorimetric method. Nootkatone and auraptene were isolated from C. paradisi oil and showed 17-24% inhibition of AChE activity at the concentration of 1.62 microg/mL.