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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.
2
Int. J. Med. Arom. Plants
AChE- inhibitory activity in essential oil from aromatic plants
Phrompittayarat et al.
http://www.openaccessscience.com
ijmap@openaccessscience.com
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 Tris–HCl 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
3
Int. J. Med. Arom. Plants
AChE- inhibitory activity in essential oil from aromatic plants
Phrompittayarat et al.
http://www.openaccessscience.com
ijmap@openaccessscience.com
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
4
Int. J. Med. Arom. Plants
AChE- inhibitory activity in essential oil from aromatic plants
Phrompittayarat et al.
http://www.openaccessscience.com
ijmap@openaccessscience.com
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
]
5
Int. J. Med. Arom. Plants
AChE- inhibitory activity in essential oil from aromatic plants
Phrompittayarat et al.
http://www.openaccessscience.com
ijmap@openaccessscience.com
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
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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
]