Sesquiterpenoids from the fruits of Alpinia oxyphylla and their anti-acetylcholinesterase activity

Article abstract of DOI:10.1002/hlca.201300234

Fourteen sesquiterpenoids were isolated from the fruits of Alpinia oxyphylla Miq. Their structures were elucidated based on NMR analyses ( ¹H, ¹³C, DEPT, ¹H,¹H-COSY, HMQC, HMBC, and NOESY) and identified as 12-nornootkaton-6-en-11-one (3), (+)-(3S,4aS,5R)-2,3,4,4a,5,6-hexahydro-3-isopropenyl-4a,5-dimethyl-1, 7-naphthoquinone (5), nootkatene (6), 9β-hydroxynootkatone (7), 2β-hydroxy-δ-cadinol (8), 4-isopropyl-6-methyl-1-tetralone (11), oxyphyllone E (12), oxyphyllone D (13), oxyphyllanene B (15), oxyphyllone A (16), oxyphyllol E (17), (9E)-humulene-2,3;6,7-diepoxide (18), mustakone (20), and pubescone (21). Among them, 3 was a new norsesquiterpenoid, 8 was a new natural product, and 5, 6, 11, 20, 21 were isolated from A. oxyphylla for the first time. Twenty sesquiterpenoids, 1-5 and 7-21, were investigated for their in vitro acetylcholinesterase (AChE) inhibitory activities, including previously isolated seven sesquiterpenoids from A. oxyphylla, (11S)-12-chloronootkaton-11- ol (1), (11R)-12-chloronootkaton-11-ol (2), nootkatone (4), oxyphyllenodiol A (9), oxyphyllenodiol B (10), 7-epiteucrenone B (14), and alpinenone (19). TLC-Bioautographic assay indicated that 1-4, 7, 14, 16, 18, 19, and 21 displayed anti-AChE activities at 10 nmol. Microplate assay confirmed that 19, 18, 16, and 21 displayed moderate-to-weak anti-AChE activities at the concentration of 100 μM, and 19 was the most potent inhibitor with an IC₅₀ value of 81.6±3.5 μM. The presence of anti-AChE sesquiterpenoids in A. oxyphylla may partially support the traditional use of this fruit for the treatment of dementia. Copyright

Full text of DOI:10.1002/hlca.201300234

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Helvetica Chimica Acta – Vol. 97 (2014)
Sesquiterpenoids from the Fruits of Alpinia oxyphylla and Their Anti-
Acetylcholinesterase Activity
by Ping Chen, Pei-Pei Wang, Ze-Zhao Jiao, and Lan Xiang*
School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P. R. China
(phone: þ 86-531-88382028; fax: þ 86-531-88382548; e-mail: xianglan02@sdu.edu.cn)
Fourteen sesquiterpenoids were isolated from the fruits of Alpinia oxyphylla Miq. Their structures
were elucidated based on NMR analyses (1H, 13C, DEPT, 1H,1H-COSY, HMQC, HMBC, and NOESY)
and identified as 12-nornootkaton-6-en-11-one (3), (þ)-(3S,4aS,5R)-2,3,4,4a,5,6-hexahydro-3-isopro-
penyl-4a,5-dimethyl-1,7-naphthoquinone (5), nootkatene (6), 9b-hydroxynootkatone (7), 2b-hydroxy-d-
cadinol (8), 4-isopropyl-6-methyl-1-tetralone (11), oxyphyllone E (12), oxyphyllone D (13), oxy-
phyllanene B (15), oxyphyllone A (16), oxyphyllol E (17), (9E)-humulene-2,3;6,7-diepoxide (18),
mustakone (20), and pubescone (21). Among them, 3 was a new norsesquiterpenoid, 8 was a new natural
product, and 5, 6, 11, 20, 21 were isolated from A. oxyphylla for the first time. Twenty sesquiterpenoids,
1 – 5 and 7 – 21, were investigated for their in vitro acetylcholinesterase (AChE) inhibitory activities,
including previously isolated seven sesquiterpenoids from A. oxyphylla, (11S)-12-chloronootkaton-11-ol
(1), (11R)-12-chloronootkaton-11-ol (2), nootkatone (4), oxyphyllenodiol A (9), oxyphyllenodiol B
(10), 7-epiteucrenone B (14), and alpinenone (19). TLC-Bioautographic assay indicated that 1 – 4, 7, 14,
16, 18, 19, and 21 displayed anti-AChE activities at 10 nmol. Microplate assay confirmed that 19, 18, 16,
and 21 displayed moderate-to-weak anti-AChE activities at the concentration of 100 mm, and 19 was the
most potent inhibitor with an IC50 value of 81.6 ꢀ 3.5 mm. The presence of anti-AChE sesquiterpenoids in
A. oxyphylla may partially support the traditional use of this fruit for the treatment of dementia.
Introduction. – Alzheimerꢀs disease (AD), the most common form of dementia in
elderly, is a chronic, progressive neurodegenerative disorder with characteristic
cognition impairment [1]. According to cholinergic hypothesis, loss of cholinergic
neurons in the AD brain leads to decreased acetylcholine (ACh) level and causes
impairment in cognition function. Use of acetylcholinesterase inhibitor (AChEI) to
improve the acetylcholine level is an important strategy for the treatment of AD.
Medicinal plants have attracted attention for the discovery of AChEI due to their
potential roles in the treatment of dementia and less side-effects compared with the
synthetic AChEIs [2].
Previously, we screened anti-AChE activities of 48 traditional Chinese medicines
which are commonly used for the treatment of dementia or related diseases. An EtOH
extract (0.1 mg/ml) of the fruits of Alpinia oxyphylla Miq. (Zingiberaceae) showed the
highest activity (44.5 ꢀ 3.7% inhibition) among the 48 tested plant species [3]. The
fruits of A. oxyphylla, called ꢁYi Zhi Renꢀ in China, is used as a traditional medicine and
also a medicinal food for the treatment of intestinal disorders, diuresis, and dementia
[4]. Sesquiterpenoids are the major characteristic constituents of A. oxyphylla, which
are divided into eremophilane, eudesmane, cadinane, guaiane, oplopanone, and
humulane types. Several sesquiterpenoids showed potent inhibitory effects on nitric
ꢂ 2014 Verlag Helvetica Chimica Acta AG, Zꢃrich

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oxide (NO) production [5 – 9]. To date, these sesquiterpenoids have not been
investigated regarding their AChE activities. The discovery of anti-AChE constituents
in A. oxyphylla will provide insight with respect to the functional mechanism of this
plant for the treatment of dementia.
Herein, we report isolation and structure elucidation of 14 sesquiterpenoids and in
vitro effects of 20 sesquiterpenoids on AChE activity.
Results and Discussion. – Previously, we screened three fractions from the EtOH
extract of A. oxyphylla for their AChE activity, and found that AcOEt fraction
displayed 30.4 ꢀ 2.6% inhibition of the AChE activity at 0.1 mg/ml, higher than BuOH
fraction (9.1 ꢀ 4.2%) and aqueous fraction (6.5 ꢀ 3.5%). Our previous phytochemical
investigation on AcOEt fraction led to isolation of seven sesquiterpenoids, 1, 2, 4, 9, 10,
14, and 19 (Fig. 1), among which 1 and 2 were two new halogenated sesquiterpenoids
[10]. In our continuous search for bioactive constituents of the AcOEt fraction of A.
oxyphylla, fourteen sesquiterpenoids, 3, 5 – 8, 11 – 13, 15 – 18, 20, and 21 (Fig. 1) were
isolated, and they were identified as 12-nornootkaton-6-en-11-one (3), (þ)-
(3S,4aS,5R)-2,3,4,4a,5,6-hexahydro-3-isopropenyl-4a,5-dimethyl-1,7-naphthoquinone
(5) [11], nootkatene (6) [12], 9b-hydroxynootkatone (7) [13], 2b-hydroxy-d-cadinol
(8) [14], 4-isopropyl-6-methyl-1-tetralone (11) [15], oxyphyllone E (12) [16], oxy-
phyllone D (13) [17], oxyphyllanene B (15) [7], oxyphyllone A (16) [18], oxyphyllol E
(17) [9], (9E)-humulene-2,3;6,7-diepoxide (18) [19], mustakone (20) [20], and
pubescone (21) [21] (Fig. 1). Among them, 3 was a new norsesquiterpenoid, 2b-
hydroxy-d-cadinol (8) was a new natural product, and 5, 6, 11, 20, 21 were isolated from
A. oxyphylla for the first time.
Compound 3 was obtained as a white amorphous powder. Its molecular formula
was deduced as C14H18O2, with six degrees of unsaturation, based on the peak at m/z
219.1375 ([M þ H]þ, C14H19O2þ ; calc. 219.1380) in HR-ESI-MS. The 1H-NMR
spectrum of 3 (Table) showed signals of two olefinic H-atoms at d(H) 5.90 (br. s,
1 H) and 6.78 (br. s, 1 H), three Me groups at d(H) 1.14 (d, J ¼ 6.6, 3 H), 1.26 (s, 3 H),
and 2.35 (s, 3 H). 13C-NMR, DEPT, and HSQC spectra (Table) revealed the presence
of 14 C-atoms, including three Me, three CH2, and three CH groups, which include two
olefinic C-atoms, and five quaternary C-atoms, which consist of two carbonyl and two
olefinic C-atoms. These data suggested that 3 was a norsesquiterpenoid.
The 1H,1H-COSY correlations indicated the presence of ꢁCH¼CꢁCH2ꢁCH2ꢁ and
ꢁCH2ꢁCH(Me)ꢁCꢁCH ¼ Cꢁfragments (Fig. 2). HMBC Spectrum (Table, Fig. 2) also
showed the correlations from HꢁC(1) (d(H) 5.90) to C(3), C(5), and C(9), from
HꢁC(6) (d(H) 6.78) to C(5), C(7), C(8), C(10), C(11), and C(15), from Me(13) (d(H)
2.35) to C(7) and C(11), from Me(14) (d(H) 1.14) to C(3) and C(5), and from Me(15)
(d(H) 1.26) to C(4), C(6), and C(10). Based on these correlations, the planar structure
1
of 3 was determined as a 12-noreremophilane sesquiterpenoid (Fig. 2). The H- and
13C-NMR assignments are compiled in the Table.
The relative configuration of 3 was determined by NOESY correlations (Fig. 3).
The correlations Me(14)/HaꢁC(3), HaꢁC(8); HbꢁC(8)/HbꢁC(4), HbꢁC(3); and
Me(15)/HaꢁC(3) indicated that Me(14) and Me(15) were a-oriented. Therefore, 3
was determined as a 12-noreremophilane and identified as 12-nornootkaton-6-en-11-
one.

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Fig. 1. Sesquiterpenoids 1 – 21 isolated from A. oxyphylla
Fig. 2. 1H,1H-COSY (—) and key HMB (H ! C) correlations of
compound 3
Combined with the seven sesquiterpenoids, 1, 2, 4, 9, 10, 14, and 19, previously
isolated from A. oxyphylla, we evaluated the effects of 20 sesquiterpenoids (Fig. 1) on
AChE activity. Although 6 was obtained in a relatively high amount, we found that it
was easily converted to 4 when exposed to air or dissolved in MeOH; therefore, we did

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Table. 1H- and 13C-NMR Data (600 and 150 MHz, resp.; in CDCl3) of Compound 3. d in ppm, J in Hz.
Atom numbering as indicated in Fig. 1.
Position
d(H)
d(C)
HMBC (H ! C)
1
2
3
5.90 (br. s)
–
2.40 – 2.43 (m, Ha), 2.37 – 2.40 (m, Hb)
124.9 (d)
198.3 (s)
42.2 (t)
3, 5, 9
1, 2, 4, 5, 14
4
5
6
7
8
9
2.13 – 2.20
–
6.78 (br. s)
–
2.13 – 2.20 (overlap, Ha), 2.8 – 2.86 (m, Hb)
2.50 – 2.55 (m)
37.2 (d)
40.7 (s)
143.3 (d)
137.9 (s)
24.7 (t)
2, 3, 5, 6, 10, 15
4, 5, 7, 8, 10, 11, 15
6, 7, 9, 10, 11
1, 5, 7, 8, 10
29.9 (t)
10
11
13
14
15
–
–
168.0 (s)
198.5 (s)
25.4 (q)
15.1 (q)
18.8 (q)
2.35 (s)
1.14 (d, J ¼ 6.6)
1.26 (s)
6, 7, 11
2, 3, 5
4, 6, 10
Fig. 3. Key NOSEY (H $ H) correlations of compound 3
not test the activity of 6. The methods frequently used for the determination of AChE
activity are TLC bioautographic assay using Fast Blue B salt reagent, and 96-well
microplate assay using Ellmanꢀs reagent. Both of these two assays are considered
suitable for the generation of new hits of AChE inhibitors. By comparison, the TLC
assay is more suitable for the screening of crude extracts of plants as it allows separation
and detection of active compounds at the same time, and the microplate assay is more
appropriate for the screening of libraries of compounds, as it allows the possibility of
automation and easy handling of large amounts of samples [22].
As shown in Fig. 4, the white spots in TLC autographic assay indicated that 1 – 4, 7,
14, 16, 18, 19, and 21 possessed anti-AChE activities at 10 nmol. The positive control
galanthamine hydrobromide showed anti-AChE activity at 2 nmol. Nootkatone (4) is a
major constituent of A. oxyphylla as shown in Fig. 4. This sesquiterpenoid was firstly
isolated from heartwood of Alaska yellow cedar, and it was also detected in grapefruit
(Citrus paradisi), pummelo (Citrus grandis), and vetiver (Vetiveria zizanioides) oil
[23]. Miyazawa et al. evaluated the effect of nootkatone isolated from essential oils of
C. paradisi on AChE from bovine erythrocytes using Ellman colorimetric assay and
found that nootkatone showed 17.6% inhibition of AChE activity at the concentration

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Fig. 4. Thin layer chromatogram (petroleum ether/AcOEt 3 :1) of the crude EtOH extract (E, 10 mg/ml,
1 ml), 20 sesquiterpenoids (1 – 5 and 7 – 21, 10 mm, 1 ml) of A. oxyphylla, and the positive control
galanthamine hydrobromide (G, 1 mm, 2 ml). a) Reference chromatogram sprayed with vanillin-sulfuric
acid. b) TLC Bioautographic assay; spots indicate inhibition. c) Absence of white spots indicate that
there was no false-positive result.
of 1.62 mg/ml [24]. In contrast, Anderson and Coats found that nootkatone (ranging
from 0 to 30,000 mm) did not inhibit the AChE in house fly (Musca domestica), yellow
fever mosquito (Aedes aegypti), American dog tick (Dermacentor variabilis), and
American cockroach (Periplaneta americana) [25]. Ibrahim et al., on the other hand,

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reported that the interference of nootkatone with AChE of worker termites fluctuated
from slight inhibition to significant activation [26]. These contradictory results may be
associated with the diversity in the sources of AChE. Compounds 1 – 3, 5, and 7 are
derivatives of nootkatone (4). Concerning the structureꢁactivity relationship of
eremophilane-type sesquiterpenoid 1 – 7 observed by TLC autographic assay, it seems
that halogenation at C(12) has no effect on AChE activity, as in the case of 1 and 2. The
white color produced by the new compound 3 was weaker than that observed for 4,
implying that the inhibition of AChE decreased when 4 was oxygenized into 3 at C(6)
and C(11). When 4 was oxygenized to 7 at C(9), the activity was still retained, however,
the inhibition disappeared when 7 was further oxygenized to 5. None of the cadinane-
typed sesquiterpenoids, 8 – 13, exhibited anti-AChE activity.
However, when these 20 sesquiterpenoids were further evaluated by microplate
assay at 100 mm (25 nmol in soln.), it was observed that only guaiane-type
sesquiterpenoid 19, humulane-type sesquiterpenoid 18, 4,5-secoeudesmane-type ses-
quiterpenoid 16, and carabrane sesquiterpenoid 21 showed anti-AChE activities, with
the inhibition values of 56.1 ꢀ 1.9, 22.2 ꢀ 2.2, 16.2 ꢀ 1.1, and 10.1 ꢀ 0.3%, respectively
(Fig. 5). In a further microplate assay, the positive control galanthamine hydrobromide
and alpinenone (19) showed anti-AChE activity in a concentration-dependent manner
(Fig. 6). Their IC50 values were 1.3 ꢀ 4.3 and 81.6 ꢀ 3.5 mm, respectively, calculated
according to the SIGMAPLOT logarithm equations, Y¼ 11.879 ln (X) þ 46.687 (R2 ¼
0.9530) and Y¼ 22.604 ln (X) ꢁ 49.512 (R2 ¼ 0.9801), respectively.
In our experiment, six sesquiterpenoids, 1 – 4, 7, and 14, were positive in TLC
bioautographic assay, while inactive in microplate assay. Only the results of 16, 18,
19, and 21 in both assays were convergent. The reasons for the difference observed in
these two different assays have been discussed in detail by Reist and co-workers in 2008,
and might be explained by the interaction of either AChE or test compounds with the
Fig. 5. AChE Inhibitory activities of sesquiterpenoids 19, 18, 16, 21, and galanthamine hydrobromide
(100 mm) in the 96-well microplate assay (n ¼ 3)

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Fig. 6. Concentration-dependent anti-AChE activities of alpinenone (19) and the positive control
galanthamine hydrobromide (n ¼ 3)
silica of the TLC plates, resulting in an altered affinity of the enzyme for the compounds
[22].
Although the activities of sesquiterpenoids tested in this experiment were lower
than galanthamine hydrobromide, the possible synergic anti-AChE activities of these
sesquiterpenoids present in A. oxyphylla may support the traditional use of this fruit for
the treatment of dementia. Further in vivo experiment are needed to verify this
hypothesis.
We thankfully acknowledge the financial supports from the National Natural Science Foundation of
China (81073005), Shandong Province Young and Middle-Aged Scientists Research Awards Fund
(BS2010YY032), Scientific Research Foundation for Returned Overseas Scholars, Ministry of Education
of China (No. 42), and Innovation Project of Shandong University (2012TS102).
Experimental Part
General. Column chromatography (CC): silica gel (SiO2; 200 – 300 mesh; Qingdao Haiyang
Chemical Group Co., Ltd., P. R. China), ODS-C18 (75 mm, YMC Co., Japan), MCI gel (CHP-20P,
75 – 150 mm, Mitsubishi Chemical Co., Japan), and Sephadex LH-20 (Pharmacia Fine Chemicals, USA).
Semi-prep. HPLC: Shimadzu Prominence LC-20A liquid chromatography, with LC-20AT pumps, SPD-
20A UV detector (Shimadzu Co., Japan) and a YMC-Pack ODS-A column (250 mm ꢂ 10 mm, 5 mm)
(YMC Co., Japan). TLC: Silica Gel 60 GF 254 plates (Merck, Germany); visualization by heating the
plates sprayed with 10% H2SO4 or 5% vanillin-sulfuric acid reagent. UV Spectra: UV-2450
spectrophotometer (Shimadzu Co., Japan). Optical rotations: Gyromat-Hp digital automatic polar-
imeter (Kernchen Co., Germany). NMR Spectra: Bruker AV-600; d in ppm, with Me4Si as an internal
standard; J in Hz. ESI-MS: API 4000 mass spectrometer (Applied Biosystems Co., USA). HR-ESI-MS:
LTQ-Orbitrap mass spectrometer (Thermo Fisher Co., USA), in m/z. Microplate reader (model 680 UV,
Bio-Rad) was used in the experiment for anti-AChE assay.

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Chemicals and Reagents. AChE from electric eel (type VI-S, lyophilized powder, 265 U/mg solid,
410 U/mg protein), acetylthiocholine iodide (ATCI), 5,5’-dithiobis(2-nitrobenzoic) acid (DTNB), 1-
naphthyl acetate, naphthalen-1-ol and Fast Blue B salt were purchased from Sigma. Galanthamine
hydrobromide was purchased from Aladdin Chemistry, China. Petroleum ether (PE), AcOEt, CH2Cl2,
BuOH, EtOH, MeOH, and all other reagents were of anal. grade.
Plant Material. The fruits of A. oxyphylla were purchased from Shuyupingmin Pharmacy (Jinan,
P. R. China) in March 2011 and authenticated by L. X. A voucher specimen (No. 090801) has been
deposited with the Laboratory of Pharmacognosy, School of Pharmaceutical Sciences, Shandong
University.
Extraction and Isolation. The dried fruits of A. oxyphylla (5 kg) were powdered and extracted with
95% EtOH (4 ꢂ 25 l) at r.t. The combined extracts were condensed to yield a dark brown crude extract
(450 g). It was then suspended in 20% EtOH and partitioned with PE, AcOEt, and BuOH successively.
The AcOEt extract (210 g) was chromatographed on a silica gel column (1.4 kg, 200 – 300 mesh, 10 ꢂ
75 cm) eluting with a gradient PE/AcOEt 100 :1, 20 :1, 10 :1, 7:1, 5 :1, 3 :1, 2 :1, 1:1 to yield twelve
fractions, Frs. 1 – 12. Fr. 2 (6.7 g) was subjected to CC (silica gel; PE/AcOEt 80 :1 ! 5 :1) to yield six
fractions, Frs. 2.1 – 2.6. Fr. 2.3 (230 mg) was purified by semi-prep. HPLC (MeOH/H2O 95 :5) to obtain
11 (tR 16.2 min; 4.2 mg). Crystallization (EtOH) of Fr. 2.5 yielded compound 18 (40 mg). Fr. 2.6 (1.0 g)
was purified by MCI and silica-gel CC, and semi-prep. HPLC (MeOH/H2O 75 :25) to furnish 5 (tR
23.0 min; 19.6 mg). Fr. 4 (1.6 g) was separated by CC (ODS-C18; EtOH/H2O 22 :78 ! 90 :10) to afford
three fractions, Frs. 4.1 – 4.3. Fr. 4.2 (600 mg) was further purified by repeated CC (silica gel) and semi-
prep. HPLC (MeOH/H2O 85 :15) to give 6 (tR 30.6 min; 51.3 mg). Fr. 5 (7.0 g) was subjected to CC
(MCI; EtOH/H2O 35 :65 ! 70 :30) to afford three fractions, Frs. 5.1 – 5.3. Fr. 5.1 (1.2 g) was subjected to
CC (silica gel; petroleum ether (PE)/CH2Cl2 1:2 ! 1:40) to yield five fractions, Frs. 5.1.1 – 5.1.5. Fr. 5.1.2
(200 mg) was further purified by semi-prep. HPLC (MeOH/H2O 60 :40) to give 20 (tR 19.5 min; 26.0 mg)
and 12 (tR 27.6 min; 35.6 mg). Besides, Fr. 5.1.3 (120 mg) was further purified by semi-prep. HPLC
(MeOH/H2O 64 :36) to afford 17 (tR 32.1 min; 18.8 mg) and 13 (tR 35.2 min; 5.3 mg). Fr. 7 (5.8 g) was
subjected to CC (MCI; EtOH/H2O 20 :80 ! 100 :0) to afford three fractions, Frs. 7.1 – 7.3. Fr 7.2 (1.3 g)
was then subjected to CC (silica gel; PE/CH2Cl2 1:4 ! 0 :1) to yield five fractions, Frs. 7.2.1 – 7.2.5.
Fr. 7.2.3 (50 mg) was further purified by semi-prep. HPLC (MeOH/H2O 55 :45) to yield 21 (tR 40.5 min;
9.1 mg). Fr. 7.2.4 (82 mg) was further purified by semi-prep. HPLC (MeOH/H2O 52 :48) to furnish 15 (tR
20.1 min; 10.9 mg), 16 (tR 32.5 min; 10.8 mg), and 3 (tR 34.6 min; 7.0 mg). Fr. 8 (11.1 g) was purified by
CC (ODS-C18; EtOH/H2O 20 :80 ! 100 :0) to afford nine fractions, Frs. 8.1 – 8.9. Fr. 8.5 (1.8 g) was
subjected to CC (silica gel; CH2Cl2/AcOEt 20 :1 ! 4 :1) to yield five fractions, Frs. 8.5.1 – 8.5.5. Fr. 8.5.2
(80 mg) was further purified by semi-prep. HPLC (MeOH/H2O 65 :35) to provide 7 (tR 37.5 min;
14.6 mg). Fr. 9 (25 g) was separated by CC (silica gel; CH2Cl2/AcOEt (95 :5 ! 5 :5) to afford ten
fractions, Frs. 9.1 – 9.10. Fr. 9.4 (5.1 g) was purified by repeated CC (silica gel) to yield twelve fractions,
Frs. 9.4.1 – 9.4.12. Fr. 9.4.4 (82 mg) was separated by CC (silica gel; CH2Cl2/MeOH 10 :1 ! 2 :1) to afford
six fractions, Frs. 9.4.4.1 – 9.4.4.6. Fr. 9.4.4.3 (15 mg) was further purified by CC (Sephadex LH-20;
EtOH) to afford 8 (2.3 mg).
12-Nornootkatone-6-ene-11-one (¼(4R,4aR)-6-Acetyl-4,4a-dimethyl-4,4a,7,8-tetrahydronaphthalen-
2(3H)-one; 3). White amorphous powder. [a]2D4 ¼ þ14.6 (c ¼ 0.1, MeOH). UV (MeOH): 233 (4.01). 1H-
and 13C-NMR: see the Table. HR-ESI-MS: 219.1375 ([M þ H]þ, C14H19Oþ2 ; calc. 219.1380).
Sample Preparation for Anti-AChE Assay. The EtOH extract of A. oxyphylla, together with the
AcOEt, BuOH, and residual aq. fractions, were dissolved in MeOH to obtain the stock solns. with a
concentration of 10 mg/ml. Compounds were dissolved in MeOH or deionized H2O to yield 10 mm stock
solns.
TLC Bioautographic Assay for AChE Inhibition Activity. The stock solns. (10 mm) of 20
sesquiterpenoids, together with the positive control galanthamine hydrobromide, were applied onto
silica-gel plate and separated by proper developing solvent, and then applied to TLC bioautographic
assay as described in [27]. Briefly, the TLC plate was sprayed with the enzyme soln. (1 U/ml in buffer A)
and 1-naphthyl acetate soln. (1.5 mg/ml; dissolved in 40% EtOH) subsequently. The TLC plate was
quickly blown with cold air to remove EtOH, then it was put in a closed vessel containing some H2O for
humidity (Note: the TLC plate may not contact H2O) at 378. After incubation for 15 min, the TLC plate

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was sprayed with Fast Blue B salt (0.5 mg/ml; dissolved in distilled H2O). As reaction of 1-naphthyl
acetate and AChE led to the production of naphthalen-1-ol, which showed purple color when reacted
with Fast Blue B salt, the compound which had AChE inhibitory activity gives a white spot on the purple
background. To eliminate the false-positive results, 1-naphthyl acetate was replaced by naphthalen-1-ol
in above experiment. A white spot with this reagent would indicate a false positive-result.
Microplate Assay for AChE Inhibition Activity. Above stock solns. were diluted ten times with buffer
A (50 mm Tris · HCl; pH 8), and the diluted solns. were used in the 96-well microplate assay. AChE
Inhibition activity was measured based on Ellmanꢀs method with slight modifications [28]. In the 96-well
microplate, 25 ml of ATCI (15 mm in deionized H2O), 125 ml of DTNB (3 mm in buffer C (50 mm Tris ·
HCl, pH 8, containing 0.1m NaCl and 0.02m MgCl2 · 6 H2O)), 50 ml of buffer B (50 mm Tris · HCl, pH 8,
containing 0.1% bovine serum albumin), 25 ml of sample soln. or blank (MeOH or deionized H2O diluted
with buffer A) were added, and the absorbance was measured at 405 nm every 25 s for five times.
Thereafter, 25 ml of 0.22 U/ml AChE (diluted with buffer B) was added, and the absorbance was
measured again every 25 s for eight times. Galanthamine hydrobromide was used as the positive control.
Percentage of inhibition was calculated by comparing the reaction rates for the sample to the blank. The
results were expressed as mean ꢀ SD.
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Received March 7, 2013