Biotransformation of turmerones by Aspergillus niger

Article abstract of DOI:10.1021/np100416v

Biotransformation studies conducted on (+)-(S)-ar-turmerone (1) and (+)-(S)-dihydro-ar-turmerone (2) by the fungus Aspergillus niger have revealed that 1 was metabolized to give four oxidized metabolites, (+)-(7S)- hydroxydehydroar- todomatuic acid (3), (

Full text of DOI:10.1021/np100416v

86
J. Nat. Prod. 2011, 74, 86–89
Biotransformation of Turmerones by Aspergillus niger
Mai Fujiwara,† Shinsuke Marumoto,† Nobuo Yagi,‡ 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
ReceiVed June 24, 2010
Biotransformation studies conducted on (+)-(S)-ar-turmerone (1) and (+)-(S)-dihydro-ar-turmerone (2) by the fungus
Aspergillus niger have revealed that 1 was metabolized to give four oxidized metabolites, (+)-(7S)-hydroxydehydro-
ar-todomatuic acid (3), (+)-(7S,10E)-12-hydroxydehydro-ar-todomatuic acid (4), (+)-(7S,10E)-7,12-dihydroxydehydro-
ar-todomatuic acid (5), and (+)-(7S)-15-carboxy-9,13-epoxy-7-hydroxy-9,13-dehydro-ar-curcumene (6), and (+)-(S)-
dihydro-ar-turmerone (2) was metabolized to (+)-7,11-dihydroxy-ar-todomatuic acid (7). Metabolites 3-7 were
characterized using spectroscopic techniques. Metabolites 3-7 inhibited acetylcholinesterase (AChE) although less so
than the parent substrates.
Bisabolane-type sesquiterpenoids occur naturally as (-)-R-
bisabolol, (+)-(S)-ar-turmerone (1), and (+)-(S)-dihydro-ar-tur-
merone (2). These bisabolane-type sesquiterpenoids have a variety
of biological activities.1-5 We have reported potent acetylcho-
linesterase (AChE) inhibition by bisabolane-type terpenoids (+)-
(S)-ar-turmerone (1) and (+)-(S)-dihydro-ar-turmerone (2).6
Biotransformation is today considered to be an economically
competitive technology by synthetic organic chemists in search of
new production routes for fine chemical, pharmaceutical, and
agrochemical compounds.7 In previous papers, we reported the
biotransformation of some complex natural products and obtained
a series of new products.8-11 Therefore, it is envisioned that
biotransformation of compounds 1 and 2 may provide analogues
that could be tested for new and improved activities. The goal of
the present work was the transformation of 1 and 2 by A. niger,
and the evaluation of these compounds as AChE inhibitors (Figure
1).
HMBC spectra. The absolute configurations of 3 at the C-7 position
was established to be S after conversion into tetrahydro-2-(4-
carbomethoxyphenyl)-2,6,6-trimethyl-4H-pyran-4-one (8).12 Com-
pound 8 showed negative Cotton effects [262 nm (∆ε -7.67), 290
nm (∆ε -40.7), 355 nm (∆ε -2.01)] in the CD spectrum; therefore
compound 3 was determined to be (+)-(7S)-hydroxydehydro-ar-
todomatuic acid.
Metabolite 4 had the molecular formula C15H18O4, as deduced
by HRFABMS and 1H and 13C NMR, 46 amu more than 1. The IR
spectrum of 4 showed absorptions at 3392 and 1689 cm-1
,
indicating the presence of hydroxyl and carbonyl groups. In
1
comparison with the H NMR spectrum of 1, the spectra of 4
indicated the appearance of a CH2OH group and the disappearance
of two methyl groups. This clearly indicated that compound 4 were
produced by the hydroxylation of 1. The 1H and 13C NMR
spectroscopic data of 3 and 4 were similar. Their 1H and 13C NMR
spectra indicated that compound 4 also possessed a carboxylic acid
at C-15, as does 3. In NOE experiments, compound 4 showed an
NOE correlation between H-10 (δH 6.43) and H-12 (δH 4.04).
Consequently, the structure of 4 was concluded to be (+)-(7S,10E)-
12-hydroxydehydro-ar-todomatuic acid.
Screening experiments showed that Aspergillus niger was capable
of converting (+)-(S)-ar-turmerone (1) and (+)-(S)-dihydro-ar-
turmerone (2) into more polar metabolites.
Incubation of (+)-(S)-ar-turmerone (1) with A. niger yielded the
four metabolites 3-6. Metabolite 3 was isolated as a colorless
powder. The HRFABMS exhibited a [M + H]+ peak at m/z
263.1284 (calcd 263.3105 for C15H19O4), suggesting the formula
C15H18O4, 46 amu higher than the parent substrate 1. The IR
spectrum showed absorptions at 3440 and 1685 cm-1, indicating
the presence of hydroxyl and carbonyl groups. In comparison with
the 1H NMR spectrum of 1, the spectra of 3 indicated the
disappearance of methyl (C-15) and methine (C-7) signals. Com-
parison of the 13C NMR spectroscopic data of 3 with 1 showed 13
similar chemical shifts. The remaining two 13C chemical shifts
included one oxyquaternary carbon (δC 73.7) and one carboxylic
carbon (δC 170.4). The upfield shift of the aromatic C-4 quaternary
carbon and downfield of the aromatic C-1 quaternary carbon
coupled with the carboxylic carbon (C-15) in the 13C NMR spectra
showed the presence of an R,ꢀ-conjugated carboxylic group in 3.
The HMBC spectrum of 3 showed two-bond correlations between
H-8 (δH 3.17 and 2.89)/C-7, H-14 (δH 1.52)/C-7 and three-bond
correlations between H-2,6 (δH 7.55)/C-7. Thus, chemical structure
of metabolite 3 was characterized as (+)-7-hydroxydehydro-ar-
todomatuic acid, a new compound. The proton and carbon assign-
ments were unambiguously made from the COSY, HSQC, and
Metabolite 5 had the molecular formula C15H18O5, as deduced
by HRFABMS and 1H and 13C NMR, 62 amu higher than the parent
compound 1. The IR spectrum exhibited absorptions at 3395 and
1692 cm-l, indicating the presence of hydroxyl and carbonyl groups.
The 1H NMR spectrum of compound 5 was nearly identical to that
of compound 3, except in the disappearance of a methyl signal
and the appearance of an additional downfield shift of C-12 (δH
4.02), which indicated that the C-12 methyl group was selectively
hydroxylated into a hydroxymethylene group. In NOE experiments,
compound 5 showed a NOE correlation between H-10 (δH 6.43)
and H-12 (δH 4.02). Consequently, the structure of 5 was concluded
to be (+)-(10E)-7,12-dihydroxydehydro-ar-todomatuic acid.
Metabolite 6, a colorless powder, was shown to have the
molecular formula C15H16O4 (HRFABMS). The IR spectrum
exhibited absorption bands at 3562 and 1682 cm-l, indicating the
presence of hydroxyl and carbonyl groups, respectively. Comparison
of the 1H NMR spectrum of this compound with metabolite 3
revealed significant similarity, except that the C-13 methyl proton
at δH 1.87 disappeared and an olefinic proton (δH 7.06) appeared
in 6. These suggested that metabolite 6 also had the same R,ꢀ-
conjugated carboxylic group at C-15 and hydroxyl group at C-8.
A comparison of the 13C NMR spectrum of 3 and 6 showed the
disappearance of the C-9 ketone signal. The HMBC spectrum
showed correlations between (i) H-8/C-1, 7, 9, 10, and 14, (ii) H-12/
C-10, 11, and 13, and (iii) H-13/C-9, 10, and 11, indicating the
presence of a 4-methylfurano ring system in 6. Thus the structure
* To whom correspondence should be addressed. Tel: +81-6-6721-2332.
Fax: +81-6-6727-2024. E-mail: miyazawa@apch.kindai.ac.jp.
† Kinki University.
‡ Picaso Cosmetic Laboratory Limited.
10.1021/np100416v  2011 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/28/2010

Notes
Journal of Natural Products, 2011, Vol. 74, No. 1 87
Figure 1. Chemical structures of (+)-(S)-ar-turemerone and (+)-(S)-dihydro-ar-turemerone derivatives.
Table 1. 13C NMR Spectroscopic Data of Compounds 1-7
13C
1a
2a
3a
4a
5b
6c
7b
1
143.7
126.7
129.1
135.5
35.3
135.7
126.6
129.1
143.3
34.9
154.0
124.7
130.2
127.4
73.7
153.2
127.9
130.6
129.4
36.3
154.8
125.8
130.2
129.5
74.2
153.4
125.0
130.1
127.7
74.3
153.1
124.6
130.3
128.0
73.5
2, 6
3, 5
4
7
8
52.7
51.7
53.5
52.6
55.2
42.5
54.6
9
199.9
124.1
155.1
20.7
27.6
22.0
209.9
52.5
24.4
22.5
22.5
201.3
124.0
158.8
21.1
27.9
30.7
199.4
120.9
157.4
66.7
16.0
22.2
202.3
121.6
159.6
66.7
16.2
29.9
151.2
111.3
120.7
9.7
138.7
29.6
213.8
55.0
10
11
12
13
14
15
69.9
29.2d
29.3d
30.5
22.0
21.0
21.0
170.4
167.5
167.5
171.5
170.9
a Measured in CDCl3 at 175 MHz. b Measured in acetone-d6 at 175 MHz. c Measured in acetone-d6 at 150 MHz. d Assignments may be interchanged.
of 6 was established as (+)-15-carboxy-9,13-epoxy-7-hydroxy-9,13-
dehydro-ar-curcumene.
Dihydro-ar-turmerone (2) was converted into one oxidized metabolite,
(+)-7,11-dihydroxy-ar-todomatuic acid (7). Metabolites 3-7 were
new compounds. This evidence indicated that biocatalytic oxidation
by A. niger is a feasible and effective approach to directly obtain
new compounds 3-7. These new metabolites will be useful
reference standards for our continuing studies on structure-activity
evaluation of bisabolane-type sesquiterpenoids.
In the transformation of 1 the initial oxidation can occur at
position 7 or 12, followed by the second oxidation to give 5.
Incubation of A. niger with compound 3 results in the formation
of metabolites 5 and 6, and incubation of compound 4 with the
fungus results in the formation of metabolite 5. Both products 5
showed the same optical rotation values as isolated compound 5
from 1. As mentioned above, the absolute configuration of 5 and 6
at the C-7 position was established to be S; thus compounds 5 and
6 were determined to be (+)-(7S,10E)-7,12-dihydroxydehydro-ar-
todomatuic acid (5) and (+)-(7S)-15-carboxy-9,13-epoxy-7-hy-
droxy-9,13-dehydro-ar-curcumene (6).
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Japan Spectroscopic Co. LTDDIP-1000. IR spectra were deter-
mined with a FT/IR-470 Plus Fourier transform infrared spectrometer
(JASCO Co., Ltd., Japan). UV spectra were measured on a U-2000A
UV-vis spectrophotometer (Hitachi Ltd., Tokyo. Japan), HRFABMS
was measured on JEOL Tandem MS station JMS-700 (Japan Electron
Optics Laboratory Co., Ltd., Tokyo, Japan). 1H and 13C NMR, COSY,
heteronuclear single quantum coherence (HSQC), heteronucler multiple
bond coherence (HMBC), and NOESY spectra were measured on JEOL
ECA spectrometers operating at 600 and 700 MHz for 1H and 150 and
175 MHz for 13C NMR, respectively. Thin-layer chromatography (TLC)
analysis was conducted using 60 F254 precoated silica gel plates (Merck),
and spots were visualized by heating after spraying with vanillic sulfate
reagent. Column chromatography was performed on 200-300 mesh
silica gel 60 (Merck). Analytical HPLC analyses were performed on a
Shimadzu LC-10Ai apparatus equipped with a UV detector (SPD-
M10A), using an AR-II column (4.6 × 250 mm) (Nakarai tesque Co.
Ltd., Tokyo, Japan) with a CH3CN-H2O linear gradient (0 min, 70:
30; 50 min, 30:70, v/v) as mobile phase at 1 mL/min. Purification by
means of HPLC was conducted using an AR-II column (20 × 250
mm, at a flow rate of 4.0 mL/min, eluent: CH3CN-H2O (60:40) +
0.05% CH3COOH) (Nakarai tesque Co. Ltd., Tokyo, Japan). Circular
dichroism (CD) spectrum was measured using a Horiba SEPA-200
polarimeter.
Compound 7 was assigned the molecular formula C15H20O5
1
(HRFABMS). The H NMR spectroscopic data of 7 were similar
to those of 3, except for H-10, 11 spectra which showed the
disappearance of the C-10, 11 double bond. The upfield shift of
the C-10, 11 carbons in the 13C NMR spectra indicated the
disappearance of the double bond and the presence of a hydroxyl
group. The HMBC spectrum of 7 exhibited cross-correlations for
C-11 (with H-10, H-12, and H-13), suggesting that the hydroxyl
group was located at C-11. On the basis of these observations, the
metabolite 7 was identified as (+)-7,11-dihydroxy-ar-todomatuic
acid.
The metabolites 3-7 and substrates 1 and 2 were evaluated as
AChE inhibitors. The results are given in Table 2. The biotrans-
formation products 3-7 were less potent inhibitors of AChE than
the corresponding precursors.
The results of this study established that (+)-(S)-ar-turmerone
(1) was converted into two main oxidized metabolites, (+)-
(7S,10E)-7,12-dihydroxydehydro-ar-todomatuic acid (5) and (+)-
(7S)-15-carboxy-9,13-epoxy-7-hydroxy-9,13-dehydro-ar-cur-
cumene (6), and minor two oxidized metabolites, (+)-(7S)-
hydroxydehydro-ar-todomatuic acid (3) and (+)-(7S,10E)-12-
hydroxydehydro-ar-todomatuic acid (4) by A. niger. (+)-(S)-
Chemicals. (+)-(S)-ar-Turmerone (1) and (+)-(S)-dihydro-ar-tur-
merone (2) were isolated from Peltophorum dasyrachis as the AChE
inhibitors.6 Human erythrocytes acetylcholinesterase (AChE) was from
Sigma-Aldrich, Tokyo, Japan; 5,5′-dithiobis-2-nitrobenzoic acid (DTNB)

88 Journal of Natural Products, 2011, Vol. 74, No. 1
Table 2. AChE Inhibitory Activity of Compounds 1-7
Notes
d, J ) 17.0 Hz, H-8), 2.89 (1H, d, J ) 17.0 Hz, H-8), 2.06 (3H, d, J
) 1.1 Hz, H-12), 1.87 (3H, d, J ) 1.2 Hz, H-13), 1.52 (3H, s, H-14);
13C NMR, see Table 1; HRFABMS m/z 263.1284 [M + H]+ (calcd
C15H19O4, for 263.1283).
IC50 (µM)a or
% inhibitory activity
compound
(250 µM)b
(+)-(7S,10E)-12-Hydroxydehydro-ar-todomatuic acid (4): color-
191.1 ( 0.3a
less oil; [R]28.4 +48.6 (c 0.34, acetone); IR (film) νmax 3392, 1689
1
2
3
4
5
6
7
D
81.5 ( 0.2a
cm-1; 1H NMR (CDCl3, 700 MHz) δ 7.95 (2H, d, J ) 8.2 Hz, H-3, 5),
7.40 (2H, d, J ) 8.2 Hz, H-2, 6), 6.43 (1H, m, H-10), 4.04 (2H, brs,
H-12), 3.40 (1H, m, H-7), 2.84 (1H, dd, J ) 17.2, 6.7 Hz, H-8), 2.77
(1H, dd, J ) 17.2, 7.6 Hz, H-8), 1.93 (3H, brs, H-13), 1.26 (3H, d, J
) 7.0 Hz, H-14); 13C NMR, see Table 1; HRFABMS m/z 263.1287
[M + H]+ (calcd. C15H19O4, for 263.1283).
(41.9 ( 1.3%)b
(31.0 ( 0.7%)b
(34.3 ( 0.5%)b
(33.6 ( 0.9%)b
(37.6 ( 1.5%)b
2.6 ( 0.1a
galanthaminec
(+)-(7S,10E)-7,12-Dihydroxydehydro-ar-todomatuic acid (5): col-
a Concentration of compound required for 50% enzyme inhibition as
calculated from the dose-response curve. b The percent AChE inhibition
values (250 µM) were calculated as compared with control activities
(assumed to be 100%). c Positive control.
orless oil; [R]27.0 +2.04 (c 1.37, acetone); IR (film) νmax 3395, 1692
D
cm-1; H NMR (acetone-d6, 700 MHz) δ 7.96 (2H, dt, J ) 8.2, 2.0
1
Hz, H-3, 5), 7.64 (2H, dt, J ) 8.2, 2.0 Hz, H-2, 6), 6.43 (1H, m, H-10),
4.02 (2H, brs, H-12), 3.24 (1H, d, J ) 16.2 Hz, H-8), 2.97 (1H, d, J
) 16.2 Hz, H-8), 1.89 (3H, d, J ) 1.0 Hz, H-13), 1.49 (3H, s, H-14);
13C NMR, see Table 1; HRFABMS m/z 279.1234 [M + H]+ (calcd
C15H19O5, for 279.1232).
and acetylthiocholine iodide (ATC) were from Tokyo Chemical Industry
Co., Ltd., Tokyo, Japan.
Culture Conditions. Spores of A. niger NBRC 4414 (purchased
from NITE Biological Resource Center, Chiba Japan) maintained on
nutrient agar slants at 4 °C were inoculated into the autoclaved culture
medium (250 mL in a 500 mL conical flask) containing saccharose
(15 g), glucose (15 g), polypeptone (5 g), potassium chloride (500 mg),
magnesium sulfate (500 mg), potassium dihydrogen phosphate (1 g),
and iron(II) sulfate (10 mg) in distilled water (1 L). The culture was
grown for 2 days in a shaking incubator at 28 °C and 120 rpm. The
mycelia were then transplanted to culture medium (30 mL in a 50 mL
Petri dish) and incubated for 36-48 h (until mycelia occupied 60-80%
of the surface area of the culture medium). A solution of substrate (3
mg) dissolved in dimethyl sulfoxide (DMSO) (200 µL) was added to
the culture medium. The Petri dishes were incubated at 28 °C under
static conditions, together with two controls, which contained either
mycelia with medium or substrate dissolved in DMSO with medium.
Culture media and mycelium from Petri dishes were separated by
filtration. The mycelium was acidified to pH 4 and extracted with ethyl
acetate (3 × 30 mL). The combined organic extract was dried over
anhydrous Na2SO4, evaporated in Vacuo, and analyzed by TLC and
HPLC to confirm the presence of metabolic compounds. The mobile
phase and detector used are described in the General Experimental
Procedures section. The amount of these compounds was calculated
by means of the absolute calibration curves. The time course of
biotransformation is shown in the Supporting Information, Figures 1S
and 2 S.
Isolation of Metabolic Compounds from (+)-(S)-ar-Turmerone.
(+)-(S)-ar-Turmerone (1) (300 mg), dissolved in DMSO (20 mL), was
fed to the culture as described above. After the incubation period (3
days), the culture medium and mycelia were separated by filtration.
The broth was acidified to pH 4 and extracted with ethyl acetate (3 ×
500 mL). The solvent was evaporated in Vacuo, and a crude extract
(400 mg) was obtained. The extract was distributed between 5%
NaHCO3(aq) and ethyl acetate, and the ethyl acetate phase was
evaporated to give the neutral fraction (134 mg). No metabolic
compound was detected from the neutral fraction by TLC and HPLC.
The alkali phase was acidified to pH 3 with 2 N HCl and distributed
between water and ethyl acetate. The ethyl acetate phase was
evaporated, and the acid fraction (230 mg) was obtained. The acidic
fraction was subjected to silica gel column chromatography eluted with
a gradient of CH2Cl2/MeOH to afford two fractions. Fraction 2 was
further subjected to semipreparative reversed-phase HPLC with
CH3CN-H2O (60:40, v/v + 0.05% CH3COOH) to give metabolites 3
(30 mg), 4 (25 mg), 5 (90 mg), and 6 (66 mg), respectively.
(+)-(S)-ar-Turmerone (1):6 pale yellow oil; [R]29D +63.7 (c 1.00,
CHCl3); IR (film) νmax 1685, 1620 cm-1; 1H NMR (CDCl3, 700 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, see Table 1; EIMS m/z
(relative intensity) 216 (M+ 30%), 201 (23%), 132 (22%), 119 (75%),
117 (14%), 91 (16%), 83 (100%), 77 (7%), 55 (18%); HREIMS
216.1602 (M+, C15 H20O, calcd 216.1582).
(+)-(7S)-15-Carboxy-9,13-epoxy-7-hydroxy-9,13-dehydro-ar-cur-
cumene (6): colorless powder; [R]24.2D +78.1 (c 0.53, CHCl3); IR (KBr)
ν
max 3562, 1682 cm-1; 1H NMR (acetone-d6, 600 MHz) δ 8.07 (2H, d,
J ) 8.5 Hz, H-3, 5), 7.55 (2H, d, J ) 8.2 Hz, H-2, 6), 7.06 (1H, m,
H-13), 5.85 (1H, s, H-10), 3.13 (1H, d, J ) 15.1 Hz, H-8), 3.07 (1H,
d, J ) 15.0 Hz, H-8), 1.94 (3H, d, J ) 1.2 Hz, H-12), 1.57 (3H, s,
H-14); 13C NMR, see Table 1; HRFABMS m/z 261.1154 [M + H]+
(calcd. C15H17O4, for 261.1126).
Tetrahydro-2-(4-carbomethoxyphenyl)-2,6,6-trimetyl-4H-pyran-
4-one (8). (+)-(7S)-Hydroxydehydro-ar-todomatuic acid (3) (4 mg) was
dissolved in CH2Cl2 (1 mL), and CH2N2 (1 mL) was added to the
solution. The solution was evaporated, and methylation fraction was
obtained. The methylation fraction was subjected to column chroma-
tography (200-300 mesh silica gel 60, Merck) with hexane-ethyl
acetate (1:1) to yield the methyl ester derivative of 3 (3 mg, 75%).
Al(OCl4)·9H2O (catal.) was slowly added to a solution of a methyl
ester derivative of 3 (3 mg, 0.46 mmol) in dry CH3CN (2 mL), and the
mixture was stirred at room temperature for 24 h. The mixture was
extracted with ethyl acetate. After extraction, purification by column
choromatography with hexane-acetone (7:1) gave 8 (1.2 mg, 4.3
mmol), identified by spectroscopic data. Colorless oil: [R]28.0 -76.7
D
(c 0.13, CHCl3); IR (film) νmax 1725, 1280 cm-1; UV λmax {MeOH,
1
nm (log ε)} 278.1 (2.91), 238.5 (4.19); H NMR (CD2Cl2, 500 MHz)
δ 7.96 (2H, dt, J ) 8.8, 2.0 Hz, H-3, 5), 7.54 (2H, dt, J ) 8.8, 2.0 Hz,
H-2, 6), 3.88 (3H, s, COOCH3), 3.13 (1H, dd, J ) 16.0, 0.6 Hz, H-10R),
2.78 (1H, dd, J ) 16.0, 0.7 Hz, H-10ꢀ), 2.35 (1H, d, J ) 15.8 Hz,
H-8ꢀ), 2.21 (1H, d, J ) 15.8 Hz, H-8R), 1.52 (3H, s, H-14), 1.34 (3H,
s, H-12), 1.11 (3H, s, H-13); 13C NMR (CD2Cl2, 125 MHz) δ 206.5
(C-9), 166.3 (COOCH3), 152.6 (C-1), 129.1 (C-3, 5), 128.7 (C-4), 125.0
(C-2, 6), 76.9 (C-7), 74.9 (C-11), 51.6 (COOCH3), 50.6 (C-10), 48.6
(C-8), 33.6 (C-14), 31.2 (C-12), 29.9 (C-13); HRFABMS m/z 277.3296
[M + H]+ (calcd C16H21O4, for 277.3292).
Isolation of Metabolic Compounds from (+)-(S)-Dihydro-ar-
turmerone. (+)-(S)-Dihydro-ar-turmerone (2) (270 mg), dissolved in
DMSO (18 mL), was fed to the culture as described above. After three
days, the culture medium and mycelia were separated by filtration. The
broth was acidified to pH 4 and extracted with ethyl acetate (3 × 500
mL). The solvent was evaporated in Vacuo, and a crude extract (350
mg) was obtained. The extract was distributed between 5% NaHCO3(aq)
and ethyl acetate, and the ethyl acetate phase was evaporated to give
the neutral fraction (123 mg). No metabolic compound was detected
from the neutral fraction by TLC and HPLC. The alkali phase was
acidified to pH 3 with 2 N HCl and distributed between water and
ethyl acetate. The ethyl acetate phase was evaporated, and the acid
fraction (190 mg) was obtained. The acidic fraction was subjected to
silica gel column chromatography eluted with
a gradient of
CH2Cl2-MeOH to afford two fractions. Fraction 2 was further subjected
to semipreparative reversed-phase HPLC with CH3CN-H2O (60/40,
v/v) to give metabolite 7 (173 mg).
(+)-(S)-Dihydro-ar-turmerone (2):6 colorless oil; [R]29D +32.1 (c
1
1.01, CHCl3); IR (film) νmax 1712, 1514 cm-1; H NMR (CDCl3, 500
(+)-(7S)-Hydroxydehydro-ar-todomatuic acid (3): colorless pow-
der; [R]24.1 +5.93 (c 0.30, CHCl3); IR (KBr) νmax 3440, 1685 cm-1
;
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,
D
1H NMR (CDCl3, 700 MHz) δ 8.05 (2H, dt, J ) 8.8, 1.8 Hz, H-3, 5),
7.55 (2H, dt, J ) 8.8, 1.8 Hz, H-2, 6), 5.99 (1H, m, H-10), 3.17 (1H,

Notes
Journal of Natural Products, 2011, Vol. 74, No. 1 89
H-11), 1.23 (3H, d, J ) 7.2 Hz, H-14), 0.85 (6H, d, J ) 4.6 Hz,
H-12,13); 13C NMR, see Table 1; EIMS m/z (relative intensity) 218
(M+ 26%), 203 (25%), 161 (19%), 119 (100%), 105 (13%), 91 (13%),
85 (13%), 57 (18%); HREIMS 218.1777 (M+, C15 H22O, calcd
218.1741).
Supporting Information Available: 1H and 13C NMR, COSY,
HSQC, and HMBC spectra of compounds 3-7. Figures 1S and 2S.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(+)-7,11-Dihydroxy-ar-todomatuic acid (7): colorless oil; [R]25.2
D
-7.11 (c 1.62, CHCl3); IR (film) νmax 3431, 1695 cm-1
;
1H NMR
References and Notes
(acetone-d6, 700 MHz) δ 8.07 (2H, dt, J ) 8.7, 1.8 Hz, H-3, 5), 7.53
(2H, dt, J ) 8.7, 1.8 Hz, H-2, 6), 3.24 (1H, d, J ) 17.2 Hz, H-8), 2.92
(1H, d, J ) 17.2 Hz, H-8), 2.60 (1H, d, J ) 16.8 Hz, H-10), 2.52 (1H,
d, J ) 16.8 Hz, H-10), 1.54 (3H, s, H-14), 1.19 (3H, s, H-12 or 13),
1.14 (3H, s, H-12 or 13); 13C NMR, see Table 1; HRFABMS m/z
281.1400 [M + H]+ (calcd C15H21O5, for 281.1389).
Acetylcholinesterase Inhibitory Assay. The assay was carried out
according to a published method.6 Briefly, the wells contained 20 µL
of human erythrocyte AChE solution (0.037 unit/mL in 0.01 M
phosphate buffer, pH 7.4), 200 µL of DTNB (0.15 mM in 0.1 M
phosphate buffer, pH 7.4), 30 µL of ATC in H2O (final concentrations
0.25 mM), and 20 µL of a MeOH solution of the inhibitor. Control
wells had MeOH added instead of inhibitor. The mixture was incubated
at 28 °C for 15 min. The time at which the substrate addition was
performed was considered as time zero. After the 15 min incubation,
the absorbance of the mixture was measured at 405 nm using a
microplate reader. The inhibition percentage of the AChE activity was
calculated by using the equation
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I(%) ) {(Acontrol - Asample)/Acontrol} × 100%
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where Asample is the absorbance of the sample-containing reaction mixture
and Acontrol is the absorbance of the reaction-control mixture. Galanthamine
was used as positive control. All assays were run in triplicate.
NP100416V