Acetylcholinesterase inhibitory activity of volatile oil from peltophorum dasyrachis kurz ex bakar (Yellow Batai) and bisabolane-type sesquiterpenoids

Article abstract of DOI:10.1021/jf9042387

In this study, the chemical compositions and acetylcholinesterase (AChE) inhibitory activitiy of the volatile oil from the bark of Peltophorum dasyrachis Kurz ex Bakar (yellow batai) were evaluated. As a result, 68 compounds, accounting for 88.0% of the t

Full text of DOI:10.1021/jf9042387

2824 J. Agric. Food Chem. 2010, 58, 2824–2829
DOI:10.1021/jf9042387
Acetylcholinesterase Inhibitory Activity of Volatile Oil from
Peltophorum dasyrachis Kurz ex Bakar (Yellow Batai) and
Bisabolane-Type Sesquiterpenoids
‡
,†
*
MAI
F
UJIWARA,† NOBUO
Y
AGI
,
AND
MITSUO
MIYAZAWA
†Department of Applied Chemistry, Faculty of Science of Engineering, Kinki University, 3-4-1, Kowakae,
Higashiosaka-shi, Osaka 577-8502, Japan, and ‡Picaso Cosmetic Laboratory Limited, 9-20 Ikea-cho,
Nishinomia-shi, Hyogo 662-0911, Japan
In this study, the chemical compositions and acetylcholinesterase (AChE) inhibitory activitiy of the
volatile oil from the bark of Peltophorum dasyrachis Kurz ex Bakar (yellow batai) were evaluated. As
a result, 68 compounds, accounting for 88.0% of the total oil, were identified. The main character-
istic constituent in P. dasyrachis was isolated by silica gel column chromatography and found to be
a sesquiterpenoid, (þ)-(S)-ar-turmerone (1). In the AChE inhibitory assay, the volatile oil showed
potent inhibitory activity with the IC50 value of 83.2 ( 2.8 μg/mL. Among the volatile oil components
and characteristic sesquiterpenoids, (þ)-(S)-ar-turmerone (1) and (þ)-(S)-dihydro-ar-turmerone (2)
were potent compounds, inhibiting AChE in a dose-dependent manner, with IC50 values of 191.1 (
0.3 and 81.5 ( 0.2 μM, respectively. (þ)-(S)-Dihydro-ar-turmerone (2), in particular, was found to be
the most potent AChE inhibitor. Also, bisabolane-type sesquiterpenoid derivatives, (þ)-(7S,9S)-ar-
turmerol (3), (þ)-(7S,9R)-ar-turmerol (4), (þ)-(7S,9S)-dihydro-ar-turmerol (5), (þ)-(7S,9R)-dihydro-ar-
turmerol (6), (þ)-(S)-ar-curcumene (7), and (þ)-(S)-dihydro-ar-curcumene (8), were synthesized
and tested for their AChE inhibitory effect, and their structure-activity relationships were evaluated.
All sesquiterpenoids exhibited AChE inhibitory activity. The order of AChE inhibitory potency by
bisabolane-type sesquiterpenoids was as follows: ketones > alcohols > hydrocarbons. Furthermore,
the inhibition kinetics analyzed by Dixon plots indicated that (þ)-(S)-ar-turmerone (1) is a competitive
inhibitor, with a Ki value of 882.1 ( 2.1 μM, whereas (þ)-(S)-dihydro-ar-turmerone (2) is a non-
competitive inhibitor.
KEYWORDS: Peltophorum dasyrachis; yellow batai; volatile oil; sesquiterpens; acetylcholinesterase;
(þ)-(S)-ar-turmerone; (þ)-(S)-dihydro-ar-turmerone
INTRODUCTION
a significant factor in AD (3, 4). The inhibition of AChE activity
may be one of the most realistic approaches to the symptomatic
treatment of AD. Therefore, volatile oils have gained importance
because of their AChE inhibitory activity.
Traditional herbs provide an interesting, largely unexplored
source for the development of potential new drugs. The search for
new traditional herbs for the development of new memory
enhancement drugs, particularly edible herbs, which may cause
lower side effects, has been emphasized and received a lot of
attention recently. In addition, volatile constituents are likely to
readily cross the blood-brain barrier because of their small
molecular size and lipophilicity. Therefore, among these diverse
natural compounds, volatile oils extracted from traditional herbs
are attracting special attention. For example, it has been reported
that volatile oils from some herbal medicines exhibit inhibitory
activity against acetylcholinesterase (AChE) (1, 2). Alzheimer’s
disease (AD) is a progressive brain disorder that gradually
destroys a person’s memory and ability to learn, reason, make
judgements, communicate, and carry out daily activities. The
greatly reduced presence of acetylcholine in the cerebral cortex is
In our previous studies, some volatile oils and terpenoids from
natural resources, p-menthane skeleton (5), essential oil of
Mentha species (6), volatile R,β-unsaturated ketones (7), Citrus
paradisiaquatia (8), bicyclic monoterpenoids (9), and tea tree
oil (10), have demonstrated AChE inhibitory activity. In particu-
lar, Mentha species showed significant AChE inhibitory activity.
Moreover, various volatile oils and their components from tradi-
tional herbs (11) and food crop (12) have alsobeen investigated for
their effect on AChE. It has been found that the majority of AChE
inhibitors are monoterpenoids (13, 14).
Peltophorum dasyrachis Kurz ex Bakar (Fabaceae) is a one of
the Thai traditional herbs found throughout the tropics in moist
area, Thailand, Cambodia, Malaysia, Laos, and Vietnam, where
it grows as a large tree up to 30 m high. The leaves are bipinnate
15-40 cm long, pinnae 5-9 pairs, leaflets 6-16 pairs, oblong
5-10 mm wide, and 10-15 mm long. Stem barks have been used
in traditional medicine for the treatment of antidysenteric,
*To whom correspondence should be addressed. Telephone: þ81-6-
6721-2332. Fax: þ81-6-6727-2024. E-mail: miyazawa@apch.kindai.
ac.jp.
pubs.acs.org/JAFC
Published on Web 02/10/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 2825
carminative, and antidiarrhea (15). In Thailand, its bark is drunk
asa herbaltea. Furthermore, our previous paper reportedthatthe
CHCl3 ext of P. dasyrachis showed an antimutagenic effect (16).
However, no data were found on the chemical composition of
volatile oil from P. dasyrachis and their biological property.
Therefore, we investigated the bioactivity of the volatile oil from
P. dasyrachis against AChE.
As a part our continuing programs to investigate AChE
inhibitory activity by volatile components, we report here the
chemical composition from volatile oil of P. dasyrachis and their
AChE inhibitory activity to find a new source of a natural AD
drug. Also, we report AChE inhibitory activities of main compo-
nents to elucidate the responsible compounds for the activity of
the volatile oil.
MATERIALS AND METHODS
Chemicals and Materials.
L
-menthol, (þ)-camphor, and eugenol
(Nacalai Tesque, Inc., Kyoto, Japan), anisol and galanthamine (Wako
Pure Chemical Industries, Osaka, Japan), (þ)-borneol (Fluka Chemical
Co. Inc., Japan), human erythrocyte acetylcholinesterase (AChE) (Sigma-
Aldrich, Tokyo, Japan), 5,50-dithiobis-2-nitrobenzoic acid (DTNB) and
acetylthiocholine iodide (ATC) (Tokyo Chemical Industry Co., Ltd.,
Tokyo, Japan) were purchased. Turmeric (Curcuma longa L.) oil was
purchased from Synthite Industries Ltd. (þ)-(S)-ar-Turmerone (1) was
isolated from turmeric oil, and (þ)-(S)-dihydro-ar-turmerone (2) was
synthesized from compound 1 isolated from turmeric oil for quantitative
analysis.
Column chromatography (CC) was performed using 200-300 mesh
silica gel 60 (Merck). Thin-layer chromatography (TLC) analysis was run
on 60 F254 precoated silica gel plates (Merck), and spots were visualized by
heating after spraying with vanillic sulfate reagent.
Figure 1. Structures of the isolated and synthetic sesquiterpenoid com-
pounds 1-8.
Plant Material. Air-dried bark tips of P. dasyrachis brought from
Four Minds Co., Ltd. (Bangkok, Thailand) was a gift from Picaso Co.,
Ltd. (Hyogo, Japan).
a further 1 h, excess of the resulting solution slowly turned white. The
residue was repeatedly extracted into Et2O, and purification by column
choromatography (SiO2, 9:1 hexane/EtOAc) afforded the compound 7
(53.1 mg, 0.26 mmol), identified by a comparison to described com-
pounds (19, 20).
General Experimental Procedures. Hydrodistillation Process.
The air-dried bark tips (5 kg) of P. dasyrachis were subjected to hydro-
distillation for 2 h in a Linkens-Nickerson-type apparatus (Osaka Riko
Seisakusyo Co. Ltd., Japan). The obtained essential oil was dried over
anhydrous sodium sulfate and, after filtration, stored under refrigeration
until analyzed and tested. The sample was isolated in yields of 0.01% (v/w).
Isolation of Main Sesquiterpenenoids. P. dasyrachis oil (510 mg) was
chromatographed over silica gel using a mixture of n-hexane and CH2Cl2
as eluents to yield six fractions (fractions 1-6). Fraction 2 (150 mg) was
rechromatographed over silica gel using a mixture of n-hexane and Et2O
to yield (þ)-(S)-ar-turmerone (1) (72 mg, 14% yield) and (þ)-(S)-dihydro-ar-
turmerone (2) (2 mg, 0.39% yield), respectively (Figure 1).
Preparation of (þ)-(7S,9S)-Dihydro-ar-turmerol (5), (þ)-(7S,9R)-
Dihydro-ar-turmerol (6), and (þ)-(S)-Dihydro-ar-curcumene (8). Com-
pounds 3 (125 mg, 0.57 mmol), 4 (93 mg, 0.43 mmol), and 7 (76 mg, 0.38
mmol), and palladium on charcoal (0.2 equiv) in EtOH were added to a
round-bottomed flask, respectively. The flask was connected to a rubber
balloon filled with hydrogen. Stirring for the specified reaction time was
then started. At the end of the reaction, palladium was immediately
removed by filtration through celite. After evapolation of the solvent,
purification by column choromatography afforded the compounds 5
(70 mg, 0.32), 6 (80 mg, 0.36 mmol), and 8 (55 mg, 0.25 mmol),
respectively, identified by a comparison to described compounds (21, 22).
Analysis of Isolation Compounds. Optical rotations were measured on a
Japan Spectroscopic Co. LTDDIP-1000. IR spectra were determined with
a FT/IR-470 Plus Fourier transform infrared spectrometer (JASCO Co.,
Ltd., Japan). High-resolution electron impact-mass spectrometry
(HREI-MS) and EI-MS were measured on the JEOL tandem MS
station JMS-700 (Japan Electron Optics Laboratory Co., Ltd., Tokyo
Japan). 1H and 13C nuclear magnetic resonance (NMR), heteronuclear
multiple-quantum coherence (HMQC), and heteronuclear multiple-bond
coherence (HMBC) spectra were measured on JEOL ECA spectrometers
Quantification of the Characteristic Sesquiterpenoids. (þ)-(S)-ar-
Turmerone (1) and (þ)-(S)-dihydro-ar-turmerone (2) (0.01-4 mg/mL)
were injected to examine the linear relations, respectively. Then, calibra-
tion curves were made accordingly to yield the following regression
equations and ranges for quantificative analysis: y = 9E þ 07x þ
372 280, r = 0.999 98, and 0.04-4 mg (compound 1); y = 2E þ 08x þ
992 456, r = 0.999 73, and 0.01-1 mg (compound 2).
Preparation of (þ)-(7S,9S)-ar-Turmerol (3) and (þ)-(7S,9R)-ar-
Turmerol (4). LiAlH4 (17.3 mg, 0.46 mmol) was slowly added to a solution
of (þ)-(S)-ar-turmerone (1) (100 mg, 0.46 mmol) in dry tetrahydrofuran
(THF) (2 mL), and the mixture was stirred at room temperature for
30 min. The reaction was stopped by the addition of H2O. Filtration over
celite afforded a mixture of (þ)-(7S,9S)-ar-turmerol (3) and (þ)-(7S,9R)-ar-
turmerol (4). After evapolation of the solvent, purification by column
choromatography (SiO2, 4:1 hexane/EtOAc) afforded the separation of
compounds 3 (52 mg, 0.24 mmol) and 4 (37 mg, 0.17 mmol) identified by a
comparison to described compounds (17, 18).
operating at 500 and 700 MHz for 1H NMR and 125 and 175 MHz for 13
C
NMR respectively, using tetramethylsilane (TMS) as an internal standard.
(þ)-(S)-ar-Turmerone (1). Pale yellow oil. [R]29 þ63.7 (c 1.00,
D
CHCl3). HREI-MS: 216.1602 (Mþ, C15H20O, Calcd 216.1582). EI-MS
m/z (relative intensity): 216 (Mþ 30%), 201 (23%), 132 (22%), 119 (75%),
117 (14%), 91 (16%), 83 (100%), 77 (7%), 55 (18%). IR (film) cm-1: 1685,
1620. 1H NMR (CDCl3, 400 MHz) δ: 7.10 (4H, brs, ArH), 6.02 (1H, m,
H-10), 3.29 (1H, m, H-7), 2.71 (1H, dd, J = 15.5, 6.0 Hz, H-8), 2.61 (1H,
dd, J = 15.5, 8.3 Hz, H-8), 2.31 (3H, s, H-15), 2.10 (3H, s, H-13), 1.85 (3H,
d, J = 1.2, H-12), 1.24 (3H, d, J = 6.9 Hz, H-14). 13C NMR (CDCl3, 125
MHz) δ: 199.9 (C-9), 155.1 (C-11), 143.7 (C-1), 135.5 (C-4), 129.1 (C-3,
C-5), 126.7 (C-2, C-6), 124.1 (C-10), 52.7 (C-8), 35.3 (C-7), 27.6 (C-13),
22.0 (C-14), 21.0 (C-15), 20.7 (C-12).
Preparation of (þ)-(S)-ar-Curcumene (7). A three-neck, round-bottled
flask was purged by a gentle flow of liquid ammonia at -33 °C. Small
pieces of lithium (0.75 equiv) were added slowly to the liquid ammonia.
The resulting solution slowly turned blue over a period of 30 min. The
acetate of compound 3 (100 mg, 0.36 mmol) in dry THF (3 mL) was slowly
added. After 45 min, excess NH4Cl (0.75 g, 0.014 mmol) was added. After

2826 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Fujiwara et al.
(þ)-(S)-Dihydro-ar-turmerone (2). Colorless oil. [R]29 þ32.1
126.9 (C-2, C-6), 124.6 (C-10), 39.0 (C-7), 38.5 (C-8), 26.2 (C-9), 25.7 (C-12),
22.5 (C-14), 21.0 (C-15), 17.7 (C-13).
D
(c 1.01, CHCl3). HREI-MS: 218.1777 (Mþ, C15H22O, Calcd 218.1741).
EI-MS m/z (relative intensity):218 (Mþ 26%), 203 (25%), 161 (19%), 119
(100%), 105 (13%), 91 (13%), 85 (13%), 57 (18%). IR (film) cm-1: 1712,
1514. 1H NMR (CDCl3, 500 MHz) δ: 7.10 (4H, brs, ArH), 3.28 (1H,
ddd, J = 8.0, 7.2, 6.3 Hz, H-7), 2.68 (1H, dd, J = 16.1, 6.3 Hz, H-8), 2.58
(1H, dd, J = 16.1, 8.0 Hz, H-8), 2.31 (3H, s, H-15), 2.19 (2H, m, H-10),
2.08 (1H, m, H-11), 1.23 (3H, d, J = 7.2 Hz, H-14), 0.85 (6H, d, J = 4.6
Hz, H-12, H-13). 13C NMR (CDCl3, 125 MHz) δ: 209.9 (C-9), 143.3 (C-4),
135.7 (C-1), 129.1 (C-3, C-5), 126.6 (C-2, C-6), 52.5 (C-10), 51.7
(C-8), 34.9 (C-7), 24.4 (C-11), 22.5 (C-12, C-13), 22.0 (C-14), 21.0 (C-15).
(þ)-(7S,9S)-ar-Turmerol (3). Colorless oil. [R]22D þ22.4 (c 1.00,
CHCl3). HREI-MS: 218.1800 (Mþ, C15H22O, Calcd 218.1741). EI-MS
m/z (relative intensity): 218 (Mþ 5%), 200 (9%), 157 (9%), 120 (17%), 119
(100%), 105 (14%), 91 (14%), 85 (42%). IR (film) cm-1: 3373, 2965, 2925,
1515. 1H NMR (CDCl3, 700 MHz) δ: 7.09 (2H, dd, J = 8.8, 2.2 Hz, H-3,
H-5), 7.06 (2H, dd, J = 8.8, 2.2 Hz, H-2, H-6), 5.15 (1H, dqq, J = 8.6, 1.2,
1.2 Hz, H-10), 4.20 (1H, m, H-9), 2.72 (1H, m, H-7), 2.31 (1H, s, H-15),
1.93 (1H, ddd, J = 13.4, 8.8, 6.7 Hz, H-8), 1.73 (3H, d, J = 1.2 Hz, H-12),
1.64 (1H, ddd, J = 13.4, 7.2, 6.2 Hz, H-8), 1.54 (1H, d, J = 1.2 Hz, H-13),
1.24 (3H, d, J = 7.0 Hz, H-14). 13C NMR (CDCl3, 175 MHz) δ: 144.1
(C-1), 135.6 (C-4), 135.4 (C-11), 129.1 (C-3, C-5), 128.0 (C-10), 126.8 (C-2,
C-6), 67.0 (C-9), 46.1 (C-8), 36.1 (C-7), 25.8 (C-13), 22.9 (C-14), 21.0
(C-15), 18.3 (C-12).
(þ)-(7S,9R)-ar-Turmerol (4). Colorless oil. [R]22D þ13.9 (c 0.86,
CHCl3). HREI-MS: 216.1798 (Mþ, C15H22O, Calcd 218.1741). EI-MS
m/z (relative intensity): 218 (Mþ 5%), 200 (8%), 157 (8%), 120 (19%), 119
(100%), 105 (16%), 91 (14%), 85 (46%). IR (film) cm-1: 3347, 2960, 2924,
1513. 1H NMR (CDCl3, 700 MHz) δ: 7.11 (4H, s, ArH), 5.15 (1H, dqq,
J = 8.6, 1.2, 1.2 Hz, H-10), 4.17 (1H, m, H-9), 2.86 (1H, m, H-7), 2.32 (1H,
s, H-15), 1.80 (1H, ddd, J = 13.4, 8.8, 5.8 Hz, H-8), 1.67 (3H, m, H-8), 1.67
(1H, d, J = 1.2 Hz, H-12), 1.53 (1H, d, J = 1.2 Hz, H-13), 1.23 (3H, d, J =
7.0 Hz, H-14). 13C NMR (CDCl3, 175 MHz) δ: 143.9 (C-1), 135.4 (C-4),
134.6 (C-11), 129.1 (C-3, C-5), 128.4 (C-10), 126.9 (C-2, C-6), 66.9 (C-9),
45.9 (C-8), 35.8 (C-7), 25.7 (C-13), 23.0 (C-14), 21.0 (C-15), 18.1 (C-12).
(þ)-(7S,9S)-Dihydro-ar-turmerol (5). Colorless oil. [R]29D þ13.7
(c 1.03, CHCl3). HREI-MS: 220.1897 (Mþ, C15H24O, Calcd 220.1900).
EI-MS m/z (relative intensity): 220 (Mþ 6%), 202 (13%),145 (35%),132
(46%), 131 (25%), 120 (38%), 119 (100%), 105 (28%), 91 (18%). IR (film)
cm-1: 3365, 2956, 2926, 1515. 1H NMR (CDCl3, 700 MHz) δ: 7.11 (4H, s,
ArH), 3.66-3.70 (1H, m, H-9), 2.84-2.89 (1H, m, H-7), 2.31 (3H, s,
H-15), 1.72-1.77 (1H, m, H-11), 1.69-1.73 (1H, m, H-8), 1.64 (1H, ddd,
J = 14.0, 7.6, 4.4 Hz, H-8), 1.36 (1H, ddd, J = 14.0, 8.8, 5.2 Hz, H-10),
1.25 (3H, d, J = 4.6 Hz, H-14), 1.23-1.27 (1H, m, H-10), 0.91 (3H, d, J =
6.7 Hz, H-12 or H-13), 0.86 (3H, d, J = 6.7 Hz, H-12 or H-13). 13C NMR
(CDCl3, 175 MHz) δ: 144.4 (C-1), 135.6 (C-4), 129.2 (C-3, C-5), 126.7
(C-2, C-6), 68.4 (C-9), 47.1 (C-10), 46.9 (C-8), 36.4 (C-7), 24.5 (C-11), 23.5
(C-12 or C-13), 22.1 (C-14), 22.0 (C-12 or C-13), 21.0 (C-15).
(þ)-(S)-Dihydro-ar-curcumene (8). Colorless oil. [R]25.4D þ29.2
(c 1.025, CHCl3). HREI-MS: 204.3500 (Mþ, C15H22, Calcd 204.3526).
EI-MS m/z (relative intensity): 204 (Mþ 11%), 120 (11%), 119 (100%),
118 (3%), 117 (6%), 115 (3%), 105 (10%), 91 (7%), 41 (3%). IR (film)
cm-1: 2955, 2925,1514. 1H NMR (CDCl3, 500 MHz) δ: 7.10 (2H, dt, J =
8.0, 2.0 Hz, H-3, H-5), 7.07 (2H, dt, J = 8.0, 2.0 Hz, H-2, H-6), 2.64
(1H, m, H-10), 2.32 (3H, s, H-15), 1.58-1.43 (1H, m, H-11), 1.58-1.43
(2H, m, H-9), 1.21 (3H, d, J = 7.2 Hz, H-14), 1.28-1.10 (2H, m, H-10),
1.28-1.10 (2H, m, H-8), 0.83 (3H, d, J = 6.8 Hz, H-12 or H-13), 0.82 (3H,
d, J = 6.8 Hz, H-2 or H-13). 13C NMR (CDCl3, 175 MHz) δ: 145.0 (C-1),
135.1 (C-4), 128.9 (C-3, C-5), 126.8 (C-2, C-6), 39.5 (C-7), 39.0 (C-8), 38.7
(C-9), 27.8 (C-11), 22.7 (C-12 or C-13), 22.6 (C-12 or C-13), 22.4 (C-14),
21.0 (C-15). These spectral data were compared to the published data.
Gas Chromatography-Mass Spectrometry (GC-MS) Conditions.
GC-MS was used with a Hewlett-Packard 6890-5973 system (Agilent,
Tokyo, Japan) using either of two capillary columns (DB-WAX, 15 m ꢀ
0.25 mm, film thickness of 0.25 μm, or HP-5MS, 30 m ꢀ 0.25 mm, film
thickness of 0.25 μm). On DB-WAX, the column temperature was
programmed from 40 to 240 °C at 4 °C/min and held at 240 °C for
5 min. On HP-5MS, the column temperature was programmed from 40 to
260 °C at 4 °C/min and held at 260 °C for 5 min. The injector and detector
temperatures were 270 and 280 °C, respectively. The flow rate of carrier
gas (He) was 1.4 mL/min, and the split ratio was 1:10. The detector
interface temperature was set at 280 °C. The initial column temperature
was kept at 40 °C for 5 min and programmed to 280 °C, with the actual
temperature in the MS source reaching approximately 230 °C at a rate of
4 °C/min and then kept constant at 260 °C for 5 min. The ionization
voltage was 70 eV, and acquisition mass range was 39-450 amu.
Identification of the volatile oil components were carried out by a
comparison of their relative retention times to those of authentic samples
or by a comparison of their retention index (RI) relative to the series of
n-hydrocarbons. Computer matching against commercial (NIST 98 and
MassFinder 3.1) (23,24) and homemade library mass spectra made of pure
substances and components of known oils and MS literature data was also
used for the identification.
AChE Inhibitory Assay. The 96-well microplate method previously
€
reportedby Bruhmann et al. (25) was modified. Briefly, the wells contained
20 μL of human erythrocyte AChE solution (0.037 unit/mL in 0.01 M
phosphate buffer at pH 7.4), 200 μL of DTNB (0.15 mM in 0.1 M
phosphate buffer at pH 7.4), 30 μL of ATC in H2O (final concentration of
0.25 mM), and 20 μL of a MeOH solution of the inhibitor. Control wells
had MeOH added instead of the inhibitor. The mixture was incubated at
room temperature (RT) for 15 min. The time at which the substrate
addition was performed was considered as time zero. After 15 min of
incubation, the absorbance of the mixture was measured at 405 nm using a
microplate reader. The inhibition percentage of the AChE activity was
calculated using the equation
(þ)-(7S,9R)-Dihydro-ar-turmerol(6). Colorless oil. [R]29D þ24.2
(c 1.03, CHCl3). HREI-MS: 220.1999 (Mþ, C15H24O, Calcd 220.1900).
EI-MS m/z (relative intensity): 220 (Mþ 6%), 202 (13%), 145 (31%),
132 (41%), 131 (23%), 120 (43%), 119 (100%), 105 (28%), 91 (18%). IR
(film) cm-1: 3354, 2956, 2926, 1515. 1H NMR (CDCl3, 700 MHz) δ: 7.10
(4H, s, ArH), 3.41-3.44 (1H, m, H-9), 2.93-2.98 (1H, m, H-7), 2.32 (3H,
s, H-15), 1.66-1.72 (4H, m, H-8, H-11), 1.60-1.65 (1H, m, H-8), 1.33 (1H,
ddd, J = 13.7, 8.4, 6.0 Hz, H-10), 1.24 (3H, d, J = 6.8 Hz, H-14), 1.19 (1H,
d, J = 13.7, 8.4, 4.8 Hz, H-10), 0.82 (3H, d, J = 3.4 Hz, H-12 or H-13),
0.81 (3H, d, J = 3.4 Hz, H-12 or H-13). 13C NMR (CDCl3, 175 MHz) δ:
143.7 (C-1), 135.4 (C-4), 129.1 (C-3, C-5), 126.9 (C-2, C-6), 67.9 (C-9), 47.3
(C-10), 46.1 (C-8), 36.0 (C-7), 24.6 (C-11), 23.4 (C-14), 23.2 (C-12 or C-13),
22.3 (C-12 or C-13), 21.0 (C-15).
I ð%Þ ¼ fðAcontrol -AsampleÞ=Acontrolg ꢀ 100%
where Asample was the absorbance of the sample containing the reaction
mixture and Acontrol was the absorbance of the reaction control mixture.
Galanthamine was used as a positive control. All assays were run in
triplicate.
Determination of IC50 Values and Kinetic Analysis. The sample
concentration showing 50% inhibition (IC50) was calculated by plotting
the inhibition percentages against the corresponding sample solution
concentrations. Dissociation constants (Ki values) were determined by
interpretation of Dixon plots (26).
(þ)-(S)-Curcumene (7). Colorless oil. [R]25.4D þ48.4 (c1.02, CHCl3).
HREI-MS: 202.3333 (Mþ, C15H22, Calcd 202.3367). EI-MS m/z (relative
intensity): 202 (Mþ 29%), 145 (31%), 132 (98%), 131 (35%), 119 (100%),
105 (50%), 91 (30%), 55 (16%), 41 (33%). IR (film) cm-1: 2962, 2921, 1514.
1H NMR (CDCl3, 700 MHz) δ: 7.09 (2H, dt, J = 8.2, 1.8 Hz, H-3, H-5), 7.07
(2H, dt, J = 8.2, 1.8 Hz, H-2, H-6), 5.09 (1H, m, H-10), 2.65 (1H, m, H-7),
2.31 (3H, s, H-15), 1.89 (2H, m, H-9), 1.67 (3H, J = 0.98 Hz, H-12), 1.59 (2H,
m, H-8), 1.52 (3H, s, H-13), 1.21 (3H, d, J = 6.8 Hz, H-14). 13C NMR
(CDCl3, 175 MHz) δ: 144.7 (C-1), 135.1 (C-4), 131.4 (C-11), 128.9 (C-3, C-5),
RESULTS AND DISCUSSION
Pale yellowish colored volatile oil was obtained by hydrodis-
tillation from the bark of P. dasyrachis. The identity of the
chemicalcomponents ofvolatile oil was assigned by a comparison
of their retention indices, relative to a series of n-hydrocarbon
indices on the two capillary columns (DB-WAX and HP-5MS)
and GC-MS spectra from the NIST 98 and MassFinder 3.1 MS
data (see Table 1).

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 2827
Table 1. Composition of the Volatile Oil of P. dasyrachis
RIa
Table 1. Continued
RIa
relative
content (%) identificationc
relative
content (%) identificationc
DB-WAX HP-5MS
1157
compoundsb
1,4-cineole
DB-WAX HP-5MS
2778
compoundsb
0.12 MS, RI
0.43 MS, RI
0.12 MS, RI
0.16 MS, RI
0.10 MS, RI
0.40 MS, RI
1.25 MS, RI
0.38 MS, RI
0.25 MS, RI
0.67 MS, RI
5.95 MS, RI, co-GC
0.38 MS, RI
0.04
13-methyl-oxacyclotetradecane-
2,11-dione
0.31 MS, RI
1183
1242
1328
1340
1403
1419
1423
1428
1447
1471
1478
1503
1530
1533
1540
1557
1573
1598
1602
1615
1634
1640
1668
1691
1694
1722
1730
1735
1744
1779
1811
1821
1833
1895
1917
1959
1963
1971
1977
2017
2023
2028
2072
2080
2117
2126
2133
2141
2146
2150
2159
1010 1,8-cineole
1004 p-cymene
roseoxide
2847
3044
3085
palmitic acid
oleic acid
linoleic acid
6.58 MS, RI
0.92 MS, RI
0.37 MS, RI
88.02
hexanol
total identified
total
p-cymenene
100.00
1052 trans-linalool oxide
1070 acetic acid
furfural
a RI = retention index on the DB-WAX or HP-5MS column. b Listed in order
of elution from a DB-WAX column. c co-GC = co-injection with an authentic sample.
d tr = trace (<0.05%).
1068 cis-linalool oxide
1125 (þ)-camphor
benzaldehyde
2-nonenal
lonalool
0.32 MS, RI
0.21 MS, RI
0.95 MS, RI
5-methyl furfural
isopulegol
6-methyl-3,5-heptadiene-2-one
trd
MS, RI
terpinen-4-ol
1175 butyric acid
acetophenone
1.63 MS, RI
0.13 MS, RI
0.18 MS, RI
2.40 MS, RI, co-GC
0.52 MS, RI
0.43 MS, RI
2.94 MS, RI, co-GC
0.41 MS, RI
0.19 MS, RI
1.09 MS, RI
0.58 MS, RI
1.55 MS, RI
0.29 MS, RI
1.38 MS, RI, co-GC
4.67 MS, RI
0.78 MS, RI
0.46 MS, RI
1.72
L-menthol
1170 isoborneol
isovaleric acid
1146 (þ)-borneol
1162 β-bisabolene
1,4-dimethoxybenzene
Figure 2. Inhibition of AChE activity by the volatile oil from P. dashrachis.
3-methyl-acetophenone
2-methyl-3-phenylpropanal
1463 (þ)-(S)-ar-curcumene
1470 3,7-dimethyl-6-octen-1-ol
anisole
A total of 68 compounds were characterized and represented
88.0% of the total oil. This oil was characterized by a high content
of (þ)-(S)-ar-turmerone (1) (22.3%), caprylic acid (16.5%), and
palmitic acid (6.6%). Hydrocarbons, such as alkenes, classifi-
cated as “others” constituented 3.9% of the volatile oil, followed
by oxygenated sesquiterpenoids and oxygenated monoterpene-
noids with 22.9 and 17.6%, respectively (Table 1). P. dasyrachis
oil also contained a low amount of hydrocarbon sesquiterpenoids
and hydrocarbon monoterpenoids, 3.1 and 0.5%, respectively.
To the best of our knowledge, this is the first report of the volatile
oil composition from P. dasyrachis.
caproic acid
safrole
1360 benzyl alcohol
unknown
enanthic acid
0.55 MS, RI
0.26 MS, RI
0.44 MS, RI
0.67 MS, RI
0.17 MS, RI
0.70 MS, RI
16.50 MS, RI
1.35 MS, RI, NMR
0.95 MS, RI
0.29 MS, RI
1.01
2-methyl phenol
1384 p-anisaldehyde
methyl eugenol
1363 3-phenyl-2-propanal
1175 methyl cinnamate
1573 caprylic acid
1445 (þ)-(S)-dihydro-ar-turmerone
ethyl cinnamate
As part of our effort to investigate the AChE inhibitory activity
of the volatile oil from P. dasyrachis, the volatile oil revealed to
inhibit this enzyme and an IC50 value of 83.2 ( 2.8 μg/mL
(Figure 2). To clarify the cause of the AChE inhibitory effect of
1585 R-elemene
unknown
unknown
the volatile oil, main monoterpenoids, (þ)-camphor,
L-menthol,
and (þ)-borneol; isolatedsesquiterpenoids, (þ)-(S)-ar-turmerone
(1) and (þ)-(S)-dihydro-ar-turmerone (2); and aromatic com-
pounds, anisol and eugenol, were also examined for their
AChE inhibitory activities. The results are expressed as graphs
(Figure 3), and the IC50 values are summarized in Table 2.
These results suggest that (þ)-(S)-ar-turmerone (1) and (þ)-(S)-
dihydro-ar-turmerone (2) more strongly inhibited AChE activity
(IC50 values of 191.1 ( 0.3 and 81.5 ( 0.2 μM, respectively) than
that of the volatile oil (Table 2). On the other hand, monoterpe-
noids and aromatic compounds did not inhibit the enzyme at the
maximum concentration tested, 250 μM. The same effect has
been reported previously for terpinen-4-ol, γ-terpinene, R-terpi-
nene, p-cymene, and 1,8-cineol (5, 10, 14, 27). It was suggested
that the AChE inhibitory effect of the volatile oil may be
performed in the presence of bisabolane-type sesquiterpenoids,
(þ)-(S)-ar-turmerone (1) and (þ)-(S)-dihydro-ar-turmerone (2).
Furthermore, active compounds, (þ)-(S)-ar-turmerone (1) and
(þ)-(S)-dihydro-ar-turmerone (2), were quantitatively analyzed
0.69
1350 pelargonic acid
1367 eugenol
unknown
0.69 MS, RI
2.18 MS, RI, co-GC
0.19
4-propanoyl anisole
1271 1,6-dimethyl-4-(1-methylethyl)-
naphtalene
0.47 MS, RI
0.43 MS, RI
2172
2186
2201
2234
2269
2282
2400
2415
2450
2566
2658
2753
1590 thymol
0.61 MS, RI
1.42 MS, RI
22.30 MS, RI, NMR
0.81 MS, RI
0.46
1646 iso-R-curcumene
1663 (þ)-(S)-ar-turmerone
capric acid
unknown
4-(2-propenyl)-phenol
(Z)-ethyl p-methoxy cinnamate
unknown
0.17 MS, RI
0.32 MS, RI
1.91
lauric acid
1.10 MS, RI
0.57 MS, RI
1.10 MS, RI
0.37 MS, RI
(E)-ethyl p-methoxy cinnamate
myristic acid
pentadecylic acid

2828 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Fujiwara et al.
Figure 3. Inhibition of AChE activity by the main constituents found in the
volatile oil from P. dashrachis: (9) (þ)-(S)-ar-turmerone (1), (b)
Figure 4. AChE inhibitory activities of the bisabolane-type sesquiterpe-
noids: (9) (þ)-(S)-ar-turmerone (1), (b) (þ)-(S)-dihydro-ar-turmerone
(2), (2) (þ)-(7S,9R)-ar-turmerol (3), (1) (þ)-(7S,9S)-ar-turmerol (4),
([) (þ)-(7S,9R)-dihydro-ar-turmerol (5), (O) (þ)-(7S,9S)-dihydro-ar-
turmerol (6), (0) (þ)-ar-curcumene (7), and (4) (þ)-dihydro-ar-
curcumene (8).
(þ)-(S)-dihydro-ar-turmerone (2), (2) (þ)-camphor, (0)
([) (þ)-borneol, (O) anisol, and (1) eugenol.
L-menthol,
Table 2. IC50 Values of Studied Compounds Affecting AChE Activity
IC50 (μM)a or percent
compounds
inhibitory activity (250 μM)b
sesquiterpenoids
(þ)-(S)-ar-turmerone (1)
(þ)-(S)-dihydro-ar-turmerone (2)
(þ)-(7S,9S)-ar-turmerol (3)
(þ)-(7S,9R)-ar-turmerol (4)
(þ)-(7S,9S)-dihydro-ar-turmerol (5)
(þ)-(7S,9R)-dihydro-ar-turmerol (6)
(þ)-(S)-ar-curcumene (7)
(þ)-(S)-dihydro-ar-curcumene (8)
monoterpenoids
191.1 ( 0.3a
81.5 ( 0.2a
(35.6 ( 0.9%)b
(42.5 ( 3.3%)b
(48.6 ( 0.6%)b
229.4 ( 2.6a
(14.6 ( 0.9%)b
(35.6 ( 1.2%)b
(þ)-camphor
(18.7 ( 2.2%)b
(19.7 ( 4.3%)b
(20.7 ( 3.1%)b
(þ)-borneol
L-methol
aromatic compounds
anisol
eugenol
galanthaminec
Figure 5. Dixon plots of the inhibition of AChE activities by (A) (þ)-(S)-
ar-turmerone and (B) (þ)-(S)-dihydro-ar-turmerone. Each concentration
of ATC is (2) 50 μM, (9) 125 μM, and (b) 250 μM.
(10.9 ( 1.1%)b
(1.1 ( 0.6%)b
2.6 ( 0.1a
a Concentration of compound required for 50% enzyme inhibition as calculated
from the dose-response curve. b The percent AChE inhibition values (250 μM).
c Positive control.
As seen from Table 2, compound 2 had the strongest inhibition
(60.9 ( 0.2%) of AChE at a concentration of 250 μM, followed by
compounds 1 (56.2 ( 0.2%), 6 (51.3 ( 2.6%), 5 (48.6 ( 0.6%),
4 (42.5 ( 3.3%), 3 (35.6 ( 0.9%), 8 (35.6 ( 1.2%), and 7 (14.6 (
0.9%). This result showed that oxdation sesquiterpenoids have a
higher AChE inhibitory activity compared to that of other hydro-
carbon sesquiterpenoids. The order of AChE inhibitory potency
by bisabolane-type sesquiterpenoids was as follows: ketones >
alcohols > hydrocarbons (Figure 4). Additionally, the degree to
which sesquiterpenoids inhibited AChE differed depending upon
the presence of the C10-C11 double bond. While C10-C11 single-
bond compounds 2, 5, 6, and 7 were potent inhibitor of AChE,
C10-C11 double-bond compounds 1, 3, 4, and 7 were less potent
inhibitors of AChE. From these results, we suggested that the
bisabolane-type sesquiterpenoid skeleton containing an oxidation
functional group at C9 and the single-bond moiety at C10-C11
plays an important role in the inhibitory activity against AChE.
Moreover, we investigated the Dixon plots of these isolated
compounds on the inhibition of AChE (Figure 5). The (þ)-(S)-ar-
turmerone (1) result showed a competitive inhibitor, as indicated
by increasing the substrate concentration and the interactions on
the Dixon plot. The moderate active component, (þ)-(S)-ar-
turmerone (1), competes with the substrate for its active site on
the enzyme. On the other hand, (þ)-(S)-dihydro-ar-turmerone (2)
was an uncompetitive inhibitor, as indicated by the decreasing
inhibition associated with decreasing substrate concentrations
as 13.2 and 0.32% (v/v), respectively, by means of the respective
calibration curves in the volatile oil. If the oil, which gave 73 (
1.2% inhibition of human AChE at a final concentration of
200 μg/mL (Figure 2), is taken as a mixture of 100%, then a final
concentration of 13.2% (þ)-(S)-ar-turmerone (1) and 0.32%
(þ)-(S)-dihydro-ar-turmerone (2) in the oil would be 26 μg/mL
(0.12 mM) and 0.64 μg/mL (0.029 mM), respectively. As calcu-
lated from a mean dose-response curve of (þ)-(S)-ar-turmerone
(1) and (þ)-(S)-dihydro-ar-turmerone (2), a final concentration
of 26 and 0.64 μg/mL would give 44 and 29% inhibition of human
AChE.
To the best of our knowledge, no report has been described
thus far for bisabolane-type sesquiterpenoids with an AChE
inhibitory effect. To search for a more effective AChE inhibitor
from bisabolane-type sesquiterpenoids and explore structure-
activity relationships, various derivative compounds (þ)-(7S,9S)-
ar-turmerol (3), (þ)-(7S,9R)-ar-turmerol (4), (þ)-(7S,9S)-dihydro-
ar-turmerol (5), (þ)-(7S,9R)-dihydro-ar-turmerol (6), (þ)-(S)-ar-
curcumene (7), and (þ)-(S)-dihydro-ar-curcumene (8) were tested
for AChE inhibitory activity. The dose-dependent manner is shown
in Figure 4. All sesquiterpenoids 1-8 exhibited AChE inhibitory
activities. The IC50 values of these sesquiterpenoids 1-8, which
indicated their AChE inhibitory activity, are presented in Table 2.

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 2829
and parallelism of the Dixon plot. The most active component,
(þ)-(S)-dihydro-ar-turmerone (2), bound not to the enzyme but
to the enzyme-substrate complex, thus preventing product
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