Biotransformation of bergapten and xanthotoxin by glomerella cingulata

Article abstract of DOI:10.1021/jf101064v

The biotransformation of bergapten (1) by the fungus Glomerella cingulata gave the corresponding reduced acid, 6,7-furano-5-methoxy hydrocoumaric acid (2), a new compound. Xanthotoxin (3) was also converted to the corresponding reduced acid cnidiol b (4) and demethylated metabolite xanthotoxol (5) by G. cingulata. The structure of the new compound 2 was elucidated by highresolution mass spectrometry, extensive NMR techniques, including ¹H NMR and ¹³C NMR, ¹H-¹H correlation spectroscopy, heteronuclear multiple quantum coherence, and heteonuclear multiple bond coherence. The methyl ester or methyl ether or methyl ester and ether derivatives of 2 and 4 were synthesized. All compounds were tested for the β-secretase (BACE1) inhibitory activity in vitro. The methyl ester and ether derivative 8 was shown to possess BACE1 inhibitory activity, and a IC ₅₀ value was 0.64 ± 0.04 mM.

Full text of DOI:10.1021/jf101064v

J. Agric. Food Chem. 2010, 58, 7777–7781 7777
DOI:10.1021/jf101064v
Biotransformation of Bergapten and Xanthotoxin
by Glomerella cingulata
S
HINSUKE
M
ARUMOTO AND
MITSUO
MIYAZAWA
*
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae,
Higashiosaka-shi, Osaka 577-8502, Japan
The biotransformation of bergapten (1) by the fungus Glomerella cingulata gave the corresponding
reduced acid, 6,7-furano-5-methoxy hydrocoumaric acid (2), a new compound. Xanthotoxin (3) was
also converted to the corresponding reduced acid cnidiol b (4) and demethylated metabolite
xanthotoxol (5) by G. cingulata. The structure of the new compound 2 was elucidated by high-
1
1
resolution mass spectrometry, extensive NMR techniques, including H NMR and 13C NMR, H-1H
correlation spectroscopy, heteronuclear multiple quantum coherence, and heteonuclear multiple
bond coherence. The methyl ester or methyl ether or methyl ester and ether derivatives of 2 and 4
were synthesized. All compounds were tested for the β-secretase (BACE1) inhibitory activity in vitro.
The methyl ester and ether derivative 8 was shown to possess BACE1 inhibitory activity, and a
IC50 value was 0.64 ( 0.04 mM.
KEYWORDS: Biotransformation; furanocoumarin; bergapten; xanthotoxin; Glomerella cingulata;
β-secretase (BACE1) inhibitory activity
INTRODUCTION
(BACE1) inhibitory (16). To obtain new compounds, the present
investigation was carried out to continue studying the ability of
G. cingulata to accomplish structural modifications of furanocou-
marins bergapten (1) and xanthotoxin (3). Consequently, the
incubation of 1 and 3 should also permit a comparison of results,
determining the influence of the substituent groups of the C5 and
C8 position in these types ofbiotransformations. The presentwork
was an attempt to get the microbial transformation products of 1
and 3 by G. cingulata and to provide the BACE1 inhibitory
activity of these compounds (Figure 1).
Furanocoumarins are a class of potent phytoalexins (1-3) and
allelochemical compounds (4) produced by plants of the Apia-
ceae, Rutaceae, Moraceae, and Fabaceae (5). In these plants, the
most abundant linear furanocoumarins are psoralen, bergapten (1),
xanthotoxin (3), and isoimpinellin (5). Furanocoumarins are
well-known to act as phototoxicants in combination with UVA
irradiation (wavelength 320-380 nm), exhibiting cytotoxic and
mutagenic properties (6-8). Possible mechanisms leading to
adverse effects include binding to cellular constituents (9), lyso-
somal damage (10), generation of reactive oxygen species (11),
and formation ofnovel antigens throughcovalentmodification of
proteins and DNA (12). Even in the absence of UVA light, daily
doses of 200 and 400 mg of xanthotoxin (2)/kg of body weight
(given orally over 90 days, 5 days a week) have been reported to
exert toxic effects on liver, testes, and epididymis in rat (13).
Furthermore, a number of furanocoumarins act as inhibitors
of drug-metabolizing enzymes. 60,70-Dihydroxybergamottin and
related furanocoumarins dimers found, for example, in grapefruit
juice act as highly potent inhibitors of cytochrome P450 (CYP)
3A and other CYP isozymes (14) affecting drug metabolism.
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 (15). Microorganisms are well-known
as efficient and selective catalysts. Previously, we studied the
microbial transformation of furanocoumarins isoimperatorin
and imperaotirn by Glomerella cingulata and evaluated the trans-
formation products on the anti-Alzheimer effects via β-secretase
MATERIALS AND METHODS
General Experimental Procedures. Thin-layer chromatography
(TLC) was performed on precoated plates (Si gel 60 F254, 0.25 mm,
Merck). The mobile phase was hexane-EtOAc (1:1). Compounds were
visualizedby spraying plates with 0.5% vanillin in 96% H2SO4 followed by
brief heating. A Shimazu LC-10A high-performance liquid chromato-
graphy (HPLC) system (Shimazu Co., Ltd., Kyoto, Japan) was comprised
of a quaternary solvent deliver system, an autosampler, a column tempera-
ture controller, and a photodiode array (PDA) coupled with analytical
works station. A YMC-Pack ODS-AQ (4.6 mm ꢀ 250 mm, 5 μm particle
size, YMC Co., Ltd., Japan) with a YMC-Pack ODS-AQ guard column
(4.6 mm ꢀ 23 mm, 5 μm particle size, YMC Co., Ltd., Japan) were used.
The chromatographic parameters were as follows: solvent A, acetonitrile;
solvent B, water; both were modified with 0.1% (v/v) acetic acid. The
gradient was set as follows: 20% A for 10 min at 1.0 mL/min, 20-70% A
in 100 min at 1.0 mL/min, and 70% A for 10 min at 1.0 mL/min. The total
runtime was 110 min. The injection volume was 10 μL. Electron ionization
mass spectrometry (EIMS), high-resolution electron ionization mass
spectrometry (HR-EIMS), fast atom bombardment mass spectrometry
(FABMS), and high-resolution fast atom bombardment mass spectro-
metry (HR-FABMS) were obtained on a JEOL the Tandem Ms station
JMS-700 TKM. Nuclear magnetic resonance (NMR) spectra were recorded
at 500 MHz for 1H and 125 MHz for 13C on a JEOL ECA-500 spectrometer.
*To whom correspondence should be addressed. Tel: þ81-6-6721-
2332. Fax: þ81-6-6727-2024. E-mail: miyazawa@apch.kindai.ac.jp.
© 2010 American Chemical Society
Published on Web 06/07/2010
pubs.acs.org/JAFC

7778 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Marumoto and Miyazawa
Figure 3. Time course in the biotransformation of 3 by G. cingulata:
xanthotoxin (3) (b), metabolite 4 (9), and metabolite 5 (2). The bar is the
mean ( SD of three experiments.
Table 1. 13C NMR Spectroscopic Data of Compounds 1-4
Figure 1. Chemical structures of bergapten and xanthotoxin derivatives.
position
1a
2b
3a
4b
2
161.2 (C)c
112.5 (CH)
139.2 (CH)
106.4 (C)
149.5 (C)
112.6 (C)
158.4 (C)
93.8 (CH)
152.7 (C)
144.8 (CH)
105.0 (CH)
60.1 (CH3)
175.5 (C)
34.4 (CH2)
19.9 (CH2)
114.3 (C)
152.4 (C)
113.4 (C)
156.6 (C)
93.6 (CH)
154.9 (C)
143.2 (CH)
105.6 (CH)
60.3 (CH3)
160.4 (C)
114.7 (CH)
144.3 (CH)
116.5 (C)
112.9 (CH)
126.1 (C)
147.7 (C)
132.8 (C)
143.0 (C)
146.6 (CH)
106.7 (CH)
61.3 (CH3)
174.4 (C)
34.6 (CH2)
27.0 (CH2)
125.4 (C)
115.7 (CH)
122.0 (C)
146.2 (C)
133.1 (C)
144.9 (C)
144.8 (CH)
107.5 (CH)
60.8 (CH3)
3
4
4a
5
6
7
8
8a
9
10
OCH3
a Measured in CDCl3 at 100 MHz. b Measured in acetone-d6 at 125 MHz. c 13
multiplicities were determined by DEPT 135°.
C
Figure 2. Time course in the biotransformation of 1 by G. cingulata:
bergapten (1) (b) and metabolite 2 (9). The bar is the mean ( SD of
three experiments.
mycelia with medium or substrate dissolved in DMSO with medium. No
metabolic product was observed in two controls.
Isolation of Metabolite. After the fermentation, the culture medium
and mycelia were separated by filtration. The medium was saturated with
NaCl and extracted with EtOAc. The mycelia were also extracted with
EtOAc. Each EtOAc extract was combined, the solvent was evaporated,
and a crude extract (383 mg) was obtained. The extract was distributed
between 5% aqueous NaHCO3 and EtOAc, and the EtOAc phase was
evaporated to give the neutral fraction (185 mg). No metabolic com-
pounds were detected from neutral fraction by TLC and HPLC. The alkali
phase was acidified to pH 3 with 1 N HCl and distributed between water
and EtOAc. The EtOAc phase was evaporated, and the acidic fraction
(198 mg) was obtained. The acidic fraction was dissolved in acetone
(3 mL), and CH2N2 (1 mL) was added to the fraction. The solution was
evaporated, and the methylation fraction was obtained. The methylation
fraction was subjected to silica gel column chromatography (silica gel 60,
230-400 mesh, Merck) with a n-hexane-Et2O gradient (9:1 to 1:4) to yield
compound 6 (45 mg). Compound 6 (20 mg) was dissolved in MeOH
(1 mL), 5% NaOH (2 mL) was added to the solution, and the solution was
refluxed for 30 min. The solution was acidified to pH 3 with 1 N HCl and
distributed between EtOAc and water. The EtOAc phase was evaporated
to give 2 (15 mg, substrate 1 was used as an internal standard; relative
retention time, RtR = 0.65).
IR spectra were determined with a JASCO FT/IR-470 plus Fourier
transform infrared spectrometer.
A BACE1 (recombinant human
BACE1) assay kit was purchased from the PanVera Co. (United States).
Chemicals. Bergapten (1) and xanthotoxin (3) were purcharsed from
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
Preculture of G. cingulata. Spores of G. cingulata NBRC 5952 (NITE
Biological Resource Center, Japan), which had been preserved on potato
dextrose agar (PDA) at 4 °C, were inoculated into 200 mL of sterilized
culture medium (1.5% saccharose, 1.5% glucose, 0.5% polypeptone,
0.05% MgSO4 7H2O, 0.05%KCl, 0.1% K2HPO4, and 0.001% FeS-
3
O4 7H2O in distilled H2O) in a 500 mL shaking flask, and the flask was
3
shaken (reciprocating shaker, 120 rpm) at 27 °C for 3 days.
Time Course of Biotransformation and Quantification of Meta-
bolite. Precultured G. cingulata (3 mL) was transferred into two 300 mL
Erlenmeyer flasks containing 100 mL of medium and was stirred
(ca. 120 rpm) for 3 days. After the growth of G. cingulata, compounds
1 (10 mg, 46 μmol) and 3 (10 mg, 46 μmol) in 0.5 mL of dimethyl
sulfoxide (DMSO) were added into the medium, respectively, and
cultivated for 7 more days. Every other day, 5 mL of the culture
medium was extracted with EtOAc. This extract was analyzed by TLC
and HPLC. The mobile phase and detector used were the same as
above. The contents of these compounds were calculated by means of
the absolute calibration curves. The time course of biotransformation is
shown in Figures 2 and 3.
6,7-Furano-5-methoxy Hydrocoumaric Acid (2). White powder. IR
(KBr) νmax 3465, 1705, cm-1. The 13C and 1H NMR are shown as Table 1
and 2. HR-FABMS (pos) m/z 237.0746 [M þ H]þ (calcd for C12H13O5,
237.0762).
Preparative Biotransformation of Bergapten (1). Precultured
G. cingulata (5 mL) was transferred into a 500 mL Erlenmeyer flask
containing 300 mL of medium. Cultivation was carried out at 27 °C with
stirring (ca. 120 rpm) for 3 days. After the growth of G. cingulata, 50 mg of
1 in 1.0 mL of DMSO was added into the medium and cultivated for an
additional 7 days, together with two controls, which contained either
Preparative Biotransformation of Xanthotoxin (3). Precultured
G. cingulata (5 mL) was transferred into a 500 mL Erlenmeyer flask
containing 300 mL of medium. Cultivation was carried out at 27 °C with
stirring (ca. 120 rpm) for 3 days. After the growth of G. cingulata, 50 mg of
3 in 1.0 mL of DMSO was added into the medium and cultivated for an
additional 7 days, together with two controls, which contained either

Article
J. Agric. Food Chem., Vol. 58, No. 13, 2010 7779
Table 2. 1H NMR Spectroscopic Data of Compounds 1-4
7.42 (1H, d, J = 2.2 Hz, H-9), 6.83 (1H, dd, J = 2.2, 01.0 Hz, H-10), 6.82
(1H, d, J = 1.0 Hz, H-8), 4.10 (3H, s, OCH3), 3.67 (3H, s, COOCH3),
2.96-2.94 (2H, m, H-4), 2.75-2.73 (2H, m, H-3). 13C NMR (CDCl3, 125
MHz): δ 177.3 (C, C-2) 156.1 (C, C-7), 153.2 (C, C-8a), 151.2 (C, C-5),
142.5 (CH, C-9), 113.4 (C, C-4a), 110.7 (C, C-6), 104.6 (CH, C-10), 94.9
(CH, C-8), 59.6 (CH3, OCH3), 52.2 (CH3, COOCH3), 34.1 (CH2, C-3),
18.5 (CH2, C-4). EIMS m/z 250 [M]þ (23), 218 (95), 177 (33), 176 (100), 161
(23), 147 (24), 146 (19), 133 (20). HR-EIMS m/z 250.0843 [M]þ (calcd for
C13H14O5, 250.0841).
position
1a
2b
3a
4b
2
3
6.27, 1H, d (9.8)c 2.52-2.55, 2H, m 6.38, 1H, d (9.8) 2.61-2.64, 2H, m
8.15, 1H, d (9.8) 2.96-2.99, 2H, m 7.77, 1H, d (9.8) 2.95-2.98, 2H, m
4
4a
5
7.36, 1H, s
7.08, 1H, s
6
7
6,7-Furano-5,8a-dimethoxy Hydrocoumaric Acid (7). Color-
less powder. IR (KBr) νmax 1697, 1620, 1590 cm-1. 1H NMR data (CDCl3,
700 MHz): δ 7.46 (1H, d, J = 2.2 Hz, H-9), 6.84 (1H, dd, J = 2.2, 0.8 Hz,
H-10), 6.77 (1H, brs, H-8), 4.06 (3H, s, 5-OCH3), 3.84 (3H, s, 8a-OCH3),
3.05 (2H, m, H-4), 2.57 (2H, m, H-3). 13C NMR (CDCl3, 175 MHz): δ
179.2 (C, C-2) 156.6 (C, C-8a), 155.8 (C, C-7), 151.2 (C, C-5), 142.5 (CH,
C-9), 114.2 (C, C-4a), 111.2 (C, C-6), 104.6 (CH, C-10), 89.2 (CH, C-8),
60.1 (CH3, 5-OCH3), 55.8 (CH3, 8a-OCH3), 33.9 (CH2, C-3), 19.0 (CH2,
C-4). EIMS m/z 304 [M]þ (5), 286 (86), 271 (47), 218 (60), 176 (100), 69
(24), 41 (18). HR-FABMS (pos) m/z 251.0912 [M þ H]þ (calcd for
C13H15O5, 251.0920).
8
7.12, 1H, brs
6.73, 1H, d (1.0)
8a
9
7.59, 1H, d (2.4) 7.57, 1H, d (2.3) 7.69, 1H, d (2.4) 7.66, 1H, d (2.0)
10
7.02, 1H, dd
(2.4, 1.0)
OCH3 4.27, 3H, s
6.97, 1H, dd
(2.3, 1.0)
4.07, 3H, s
6.82, 1H, d (2.4) 6.74, 1H, d (2.0)
4.30, 3H, s 4.05, 3H, s
a Measured in CDCl3 at 400 MHz. b Measured in acetone-d6 at 500 MHz. c The
Jvalues are in Hz in parentheses.
mycelia with medium or substrate dissolved in DMSO with medium. No
metabolic product was observed in two controls.
6,7-Furano-5,8a-dimethoxy Hydrocoumaric Acid Methyl
Ester (8). Pale yellow oil. IR (KBr) νmax 1727, 1619, 1587 cm-1 1H
.
Isolation of Metabolite. After the fermentation, the culture medium
and mycelia were separated by filtration. The medium was saturated with
NaCl and extracted with EtOAc. The mycelia were also extracted with
EtOAc. Each EtOAc extract was combined, the solvent was evaporated,
and a crude extract (340 mg) was obtained. The extract was distributed
between 5% aqeous NaHCO3 and EtOAc, and the EtOAc phase was
evaporated to give the neutral fraction (183 mg). The alkali phase was
acidified to pH 3 with 1 N HCl and distributed between water and EtOAc.
The EtOAc phase was evaporated, and the acidic fraction (157 mg) was
obtained. The acidic fraction was dissolved in acetone (3 mL), and CH2N2
(1 mL) was added to the fraction. The solution was evaporated, and the
methylation fraction was obtained. The methylation fraction was subjected
to silica gel column chromatography (silica gel 60, 230-400 mesh, Merck)
with a n-hexane-Et2O gradient (9:1 to 1:4) to yield compound 9 (29 mg).
Compound 9 (20 mg) was dissolved in MeOH (1 mL), 5% NaOH (2 mL)
was added to the solution, and the solution was refluxed for 30 min. The
solution was acidified to pH 3 with 1 N HCl and distributed between EtOAc
and water. The EtOAc phase was evaporated to give 4 (13mg, substrate3was
used as an internal standard; RtR = 0.78). The neutral fraction was subjected
to silica gel column chromatography (silica gel 60, 230-400 mesh, Merck)
with a n-hexane-EtOAc gradient (9:1 to 1:9) to yield compound 5 (9 mg).
6,7-Furano-8-methoxy Hydrocoumaric Acid (4). White powder. IR
(KBr) νmax 3393, 1696, cm-1. The 13C and 1H NMR are shown as Table 1
and 2. HR-FABMS (pos) m/z 237.0746 [M þ H]þ (calcd for C12H13O5,
237.0762).
Xanthoxol (5). White needles. IR (KBr) νmax 3324, 1705, 1594 cm-1. 1H
NMR data (CDCl3, 500 MHz): δ 8.10 (1H, d, J = 9.8 Hz, H-4), 8.06 (1H,
d, J = 2.3 Hz, H-9), 7.44 (1H, s, H-5), 7.03 (1H, d, J = 2.3 Hz, H-10), 6.39
(1H, d, J = 9.8 Hz, H-3). 13C NMR (CDCl3, 125 MHz): δ 160.3 (C, C-2)
147.6 (CH, C-9), 145.8 (CH, C-4), 145.6 (C, C-7), 140.0 (C, C-8a), 130.4
(C, C-8), 125.4 (C, C-6), 116.4 (C, C-4a), 114.0 (CH, C-3), 110.3 (CH, C-5),
107.3 (CH, C-10). EIMS m/z 202 [M]þ (100), 174 (63), 149 (10), 146 (9), 89
(15). HR-EIMS m/z 202.0262 [M]þ (calcd for C11H6O4, 202.0325).
General Procedure for the Preparation of Derivatives of Metabolite.
Methylation of Phenol. A methyl iodide (CH3I, 1.5 equiv) and sodium
hydride (NaH, 1.5 equiv) were added to a solution of 6 or 9 (1 equiv) in dry
N,N-dimethylformamide (DMF), respectively. The mixture was stirred at
room temperature for 3 h. The reaction mixture was poured into ice water,
and the whole was extracted with CH2Cl2. The CH2Cl2 extract was
successively washed brine and then dried over Na2SO4, and the filtrate
under reduced pressure furnished a residue, which was purified by silica gel
column chromatography, gave compound 8 or 11, respectively.
Hydrolysis of Methyl Ester. Compound 8 or 11 was dissolved in MeOH
(1.0 mL), 5% NaOH (2.0 mL) was added to the solution, and the solution
was refluxed for 30 min, respectively. The solution was acidified with 1 N
HCl and distributed between EtOAc and water. The EtOAc phase was
evaporated and purified by silica gel column chromatography and gave
compound 7 or 10, respectively.
NMR data (CDCl3, 500 MHz): δ 7.46 (1H, d, J = 2.3 Hz, H-9), 6.83 (1H,
dd, J = 2.3, 0.9 Hz, H-10), 6.76 (1H, brs, H-8), 4.05 (3H, s, 5-OCH3), 3.83
(3H, s, 8a-OCH3), 3.68 (COOCH3), 3.03 (2H, m, H-4), 2.51 (2H, m, H-3).
13C NMR (CDCl3, 125 MHz): δ 174.2 (C, C-2) 156.6 (C, C-8a), 155.8 (C,
C-7), 151.4 (C, C-5), 142.4 (CH, C-9), 114.5 (C, C-4a), 111.3 (C, C-6),
104.6 (CH, C-10), 89.2 (CH, C-8), 60.1 (CH3, 5-OCH3), 55.8 (CH3, 8a-
OCH3), 51.5 (CH3, COOCH3), 34.0 (CH2, C-3), 19.2 (CH2, C-4). EIMS m/z
264 [M]þ (36), 192 (12), 191 (100), 176 (7), 161 (9), 131 (18). HR-EIMS m/z
264.1009 (calcd for C14H16O5, 264.0998).
6,7-Furano-8-methoxy Hydrocoumaric Acid Methyl Ester
(9). Pale yellow oil. IR (film) νmax 3439, 1731 cm-1 1H NMR data
.
(CDCl3, 500 MHz): δ 7.48 (1H, d, J = 2.3 Hz, H-9), 7.01 (1H, s, H-5), 6.64
(1H, d, J = 2.3 Hz, H-10), 3.03 (2H, t, J = 7.7 Hz, H-4), 2.69 (2H, t, J =
7.7 Hz, H-3). 13C NMR (CDCl3, 125 MHz): δ 173.9 (C, C-2) 144.3 (C,
C-7), 143.7 (CH, C-9), 142.8 (C, C-8a), 131.6 (C, C-8), 123.4 (C, C-4a),
121.6 (C, C-6), 114.6 (C, C-5), 106.6 (CH, C-10), 60.5 (CH3, OCH3), 51.6
(CH3, COOCH3), 34.3 (CH2, C-3), 26.0 (CH2, C-4). EIMS m/z 250 [M]þ
(43), 219 (16), 218 (81), 190 (23), 177 (38), 176 (100), 175 (20), 147 (33).
HR-EIMS m/z 250.0831 [M]þ (calcd for C13H14O5, 250.0842).
6,7-Furano-8,8a-dimethoxy Hydrocoumaric Acid (10). Pale
yellow oil. IR (KBr) νmax 1712 cm-1. 1H NMR data (CDCl3, 500 MHz): δ
7.56 (1H, d, J = 2.0 Hz, H-9), 7.06 (1H, s, H-5), 6.67 (1H, d, J = 2.0 Hz,
H-10), 4.18 (3H, s, 8-OCH3), 3.91 (3H, s, 8a-OCH3), 3.01 (2H, m, H-4),
2.70 (2H, m, H-3). 13C NMR (CDCl3, 125 MHz): δ 178.1 (C, C-2), 146.8
(C, C-8a), 145.9 (C, C-7), 144.9 (CH, C-9), 138.4 (C, C-8), 129.6 (C, C-4a),
124.9 (C, C-6), 114.3 (CH, C-5), 106.6 (CH, C-10), 61.3 (CH3, 8a-OCH3),
60.6 (CH3, 8-OCH3), 34.8 (CH2, C-3), 26.0 (CH2, C-4). HR-FABMS (pos)
m/z 251.0991 [M þ H]þ (calcd for C13H15O5, 251.0920).
6,7-Furano-8,8a-dimethoxy Hydrocoumaric Acid Methyl
.
Ester (11). Pale yellow oil. IR (film) νmax 1737 cm-1 1H NMR data
(CDCl3, 700 MHz): δ 7.56 (1H, d, J = 2.2 Hz, H-9), 7.05 (1H, s, H-5), 6.66
(1H, d, J = 2.2 Hz, H-10), 3.00 (2H, m, H-4), 2.65 (2H, m, H-3). 13C NMR
(CDCl3, 175 MHz): δ 173.7 (C, C-2) 146.9 (C, C-8a), 145.8 (C, C-7), 144.9
(CH, C-7), 138.4 (C, C-8), 130.0 (C, C-4a), 124.9 (C, C-6), 114.3 (CH, C-5),
106.6 (CH, C-10), 61.3 (CH3, 8a-OCH3), 60.6 (CH3, 8-OCH3), 51.6 (CH3,
COOCH3), 35.1 (CH2, C-3), 26.2 (CH2, C-4). EIMS m/z 264 [M]þ (100),
207 (53), 191 (26), 190 (23), 176 (19), 161 (11), 147 (16). HR-EIMS m/z
264.0999 [M]þ (calcd for C14H16O5, 264.0998).
BACE1 Enzyme Assay. The assay was carried out according to the
supplied manual with modifications (16). Briefly, a mixture of 10 μL of
assaybuffer (50 mM sodiumacetate, pH4.5), 10μL ofBACE1 (1.0U/mL),
10 μL of the substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM
ammonium bicarbonate), and 10 μL of sample dissolved in 30% DMSO
was incubatedfor 60 min at roomtemperature inthe dark. Themixturewas
irradiated at 550 nm, and the emission intensity at 590 nm was recorded.
The inhibition ratio was obtained by the following equation:
6,7-Furano-5-methoxy Hydrocoumaric Acid Methyl Ester (6).
White powder. IR (KBr) νmax 1732 cm-1. 1H NMR (CDCl3, 500 MHz): δ
inhibition ð%Þ ¼ ½1 - fðS - S0Þ=ðC - C0Þgꢁꢀ100

7780 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Marumoto and Miyazawa
where C was the fluorescence of the control (enzyme, buffer, and substrate)
after 60 min of incubation, C0 was the fluorescence of control at zero time,
S was the fluorescence of the tested samples (enzyme, sample solution, and
substrate) after incubation, and S0 was the fluorescence of the tested
samples at zero time. To allow for the quenching effect of the samples, the
sample solution was added to the reaction mixture C, and any reduction in
fluorescence by the sample was then investigated. All data are the means of
three experiments. 6,7-Furano-8a-methoxy-5-prenyloxy hydrocoumaric
acid methyl ester was used as a positive control (16).
RESULTS AND DISCUSSION
Biotransformation of Bergapten (1) with G. cingulata. To clarify
the time course of the microbial transformation of bergapten (1)
by G. cingulata, a small amount of 1 was incubated for 7 days, and
one metabolite was detected by TLC and HPLC. The time course
of metabolite was measured by HPLC. The time course of
biotransformation of 1 is shown in Figure 2. In this system, 1
was converted to 2 in 83% yield for 7 days (Figure 2). To isolate
the metabolite, a large-scale incubation of 1 using G. cingulata
was carried out for 7 days. After the biotransformation, the
culture was extracted as described in the Materials and Methods,
and methylated metabolite 2 (compound 6) was isolated from the
EtOAc extract. Metabolite 2 was obtained by the hydrolysis of 6.
The structures of these compounds were determined by spectral
data.
Figure 4. Possible metabolic pathways of corresponding reduced acid
from bergapten (1) and xanthotoxin (3) by G. cingulata, respectively.
functionalities. The 13C NMR spectrum showed 12 resonances
distributed as one primary, two secondary, three tertiary, and six
quaternary carbons. The conversion of original two olefinic
doublets (C-3, δC 114.7, and C-4, δC 144.3) into two aliphatic
secondary carbons (C-3, δC 34.6, and C-4, δC 27.0) indicated the
reduction of the C-3,4 double bond. Except for the downfield
shift of C-2 and C-4a, the 13C resonances remained the same as
those of the starting material (Table 1). Furthermore, the 1H
NMR spectrum showed the conversion of two doublets (δ 6.38, d,
J = 9.8 Hz, H-3, and δ 7.77, d, J = 9.8 Hz, H-4) into two
multiplets at δ 2.61-2.64 (H-3) and δ 2.95-2.98 (H-4) (Table 2).
Thus, the chemical structure of metabolite 4 was established as
cnidiol b, 6,7-furano-8-methoxy hydrocoumaric acid (17). The
structure of 5 was shown to be identical with xanthotoxol (5),
which is the demethylated product of 3 by comparison of its
spectroscopic data (18, 19).
Previously, we had incubated furanocoumarins isoimperator-
in, with an prenyloxy grup at C5 position, and imperatorin, with
an prenyloxy group at the C8 position, by G. cingulata (16).
Isoimperatorin was metabolized by G. cingulata to give the
corresponding reduced acid in high yield. By contrast, biotrans-
formation of imperatorin by G. cingulata offered the dealkylated
metabolite, xanthotoxol (5), in high yield. In the biotransforma-
tion of bergapten (1) and xanthotoxin (3), fungal reduction
progressed not only bergapten (1) but also xanthotoxin (3). In
addition, dealkylated product was produced as a minor metabo-
lite in the biotransformation of 3. The reduction or dealkylated
products of 3 identified in this study were also isolated in the
previous biotransformation of 3 by the fungus Gibberella puli-
cars (20). The G. pulicars, along with the goat (21), are apparently
the only organisms thus far known to metabolize xanthotoxin (3)
by a reductive mechanism, leading to the corresponding reduced
acid. Metabolites 2 and 4 were probably formed in a manner
analogous to that of melilotic acid, a metabolite of coumarin. The
HR-FABMS of compound 2 showed [M þ H]þ peaks at m/z
237.0746 (calcd for C12H13O5, 237.0762), which established a
molecular formula of C12H12O5. The presence of a broad absorp-
tion band at 3465 cm-1 and a strong absorption band at 1705 cm-1
in the IR spectrum suggested the conversion of the original
bergpten lactone into phenol and carboxylic acid functionalities.
The 13C NMR spectrum showed 12 resonances distributed as one
primary, two secondary, three tertiary, and six quaternary car-
bons. The conversion of the original two olefinic doublets (C-3, δC
112.5, and C-4, δC 139.2) into two aliphatic secondary carbons
(C-3, δC 34.4, and C-4, δC 19.9) indicated the reduction of the
C-3,4 double bond. Except for the downfield shift of C-2 and
C-4a, the 13C resonances remained the same as those of the
starting material (Table 1). Furthermore, the 1H NMR spectrum
showed the conversion oftwo doublets (δ 6.27, d, J = 9.8 Hz, H-3,
and δ 8.15, d, J = 9.8 Hz, H-4) into two multiplets at δ 2.52-2.55
(H-3) and δ 2.96-2.99 (H-4) (Table 2). Thus, the chemical
structure of metabolite 2 was established as 6,7-furano-5-methoxy
hydrocoumaric acid, a new compound. The protons and carbon
assignments were unambiguously made from the H-H correla-
tion spectroscopy (COSY), heteronuclear multiple quantum co-
herence (HMQC), and heteonuclear multiple bond coherence
(HMBC) spectra.
Biotransformation of Xanthotoxin (3) with G. cingulata. To
clarify the time course of the microbial transformation of xantho-
toxin (3) by G. cingulata, a small amount of 3 was incubated for
7 days. Two metabolites were detected by TLC and HPLC. The
time course ofmetaboliteswasmeasuredby HPLC. Inthis system,
3 was converted to 4 and 5 in 61 and 26% yields for 7 days,
respectively (Figure 3). To isolate this metabolite, a large-scale
incubation of 3 by G. cingulata was done for 7 days. After the
biotransformation, the culture was extracted as described in the
Materials and Methods, and metabolites 4 and 5 were isolated
from the EtOAc extract.
€
work of Haser (22) indicated that due to the occurrence of the
intermediates dihydrocoumarin and o-coumaric acid, two meta-
bolic pathways operate in the biotransformation of coumarin by
the fungus Pseudomonas orientalis and Bacillus cereus; that is,
coumarin converted to dihydrocoumarin by hydrogenation or
o-coumaric acid by opening of the lactone ring, which is then
converted to melolitic acid by hydrolysis or hydrogenation, re-
spectively. Therefore, the fungal reduction of 1 and 2 presumably
HR-FABMS of compound 4 showed [M þ H]þ peaks at m/z
237.0746 (calcd for C12H13O5, 237.0762), which established
a molecular formula of C12H12O5. The presence of a broad
absorption band at 3393 cm-1 and a strong absorption band at
1696 cm-1 in the IR spectrum suggested the conversion of the
original xanthotoxin lactone into phenol and carboxylic acid

Article
J. Agric. Food Chem., Vol. 58, No. 13, 2010 7781
furanocoumarins present in traditional medicines. Phytochem.
Photobiol. 2004, 79, 506–509.
(3) Wong, P. Y. Y.; Kitts, D. D. Studies on the dual antioxidant and
antibacterial properties of parsley (Petroselinum crispum) and cilan-
tro (Coriandrum sativum) extracts. Food Chem. 2006, 97, 505–515.
(4) Stevenson, P. C.; Simmonds, M. S. J.; Yule, M. A.; Veitch, N. C.;
Kite, G. C.; Irwin, D.; Legg, M. Insect antifeedant furanocoumarins
from Tetradium daniellii. Phytochemistry 2003, 63, 41–46.
(5) Murray, R.; Mendez, J.; Brown, S. The Natural Coumarins: Occur-
rence, Chemistry and Biochemistry; John Wiley & Sons: New York, 1982.
(6) Ashwood-Smith, M. J.; Poulton, G. A.; Barker, M.; Mildenberger,
M. 5-Methoxypsoralen, an ingredient in several suntan preparations,
has lethal, mutagenic and clasotogenic properties. Nature 1980, 285,
407–409.
(7) Berkley, S. F.; Hightower, A. W.; Beier, R. C.; Fleming, D. W.;
Brokopp, C. D.; Ivie, G. W.; Broome, C. V. Dermatitis in grocery
workers associated with high natural concentrations of furocoumar-
ins in celery. Ann. Intern. Med. 1986, 105, 351–355.
(8) Schlatter, J. Die toxikologische Bedeutung von Furocoumarinen in
pflanzlichen Lebensmitteln. Mitt. Lebensmittelunters. Hyg. 1988, 79,
130–143.
(9) Midden, W. R. Chemical mechanisms of the bioeffects of furocou-
marins; the role of reactions with proteins, lipids, and other cellular
constituents. In Psoralen-DNA Photobiology; Gasparro, F. P., Ed.;
CRC Press: Boca Raton, FL, 1988; Vol. 2, pp 1-49.
(10) Fredericksen, S.; Nielsen, P. E.; Hoyer, P. E. Lysosomes: A possible
target for psoralen photodamage. J. Photochem. Photobiol. B 1989,
3, 437–447.
Figure 5. β-Secretrase inhibitory activity of bergapten and xanthotoxin
derivatives 1-11. The bar is the mean ( SD of three experiments. Key: 1
(b), 2 (9), 3(2), 4 (1), 5 ((), 6 (O), 7 (0), 8 (4), 9 (r), 10()), 11(ꢀ),
and 6,7-furano-8a-methoxy-5-prenyloxy hydrocoumaric acid methyl ester
as positive control (þ).
proceeds by hydrogenation of the 3,4-double bond followed by
opening of the lactone ring or opening of the lactone ring
followed by, or concomitant with, reduction (Figure 4). However,
intermediate metabolites in each metabolic pathways were not
detected in our biotransformation experiments. As the results
show, G. cingulata have the abilities to reduction of R,β-unsatu-
rated lactone on furanocoumarin with an alkoxy group at the C5
position, and dealkylation or reduction of 8-alkoxyfuranocou-
marin, depending on the kind of alkoxy group at the C8 position.
Because there are no reports on microbial transformation of
begapten (1), the data on the microbial transformation product
metabolite may be used for further pharmacological evaluation
of 1.
Subsequently, the BACE1 inhibitory effects of all of these
compounds were evaluated to search for potential novel anti-
Alzheimer agents. Results of evaluation of BACE1 inhibitory
activity established that compounds 8, 9, and 11 inhibited 57.2 (
0.4, 36.4 ( 3.3, and 48.3 ( 1.2% of the BACE1 activity at a
concentration of 1 mM (Figure 5), and the IC50 value of 8 was
0.64 ( 0.04 mM. The BACE1 inhibitory effects of compounds 8
and 11 were stronger than those of 2, 6, 4, and 9, indicating the
presence of methyl ether and ester, which are important for the
inhibitory activity. Similar effects of modification at 8a-OH and
carboxylic acid were reported previously (16).
(11) Joshi, P. C.; Pathak, M. A. Production of singlet oxygen and
superoxide radicals by psoralens and their biological significance.
Biochem. Biophys. Res. Commun. 1983, 112, 638–646.
(12) Gasparro, F. P.; Dall’Amico, R.; O’Malley, M.; Heald, P. W.;
Edelson, R. L. Cell membrane DNA: A new target for psoralen
photoadduct formation. Photochem. Photobiol. 1990, 52, 315–321.
(13) Dunnik, J. K.; Davis, W. E.; Jorgenson, T. A.; Rosen, V. J.;
McConnell, E. E. Subchronic toxicity in rats administered oral
8-methoxypsoralen. Natl. Cancer Inst. Monogr. 1984, 66, 91–95.
(14) Tassaneeyakul, W.; Guo, L.-Q.; Fukuda, K.; Ohta, T.; Yamazoe, Y.
Inhibition selectivity of grapefruit juice components on human
cytochromes P450. Arch. Biochem. Biophys. 2000, 378, 356–363.
(15) Davis, B. G.; Boyer, V. Biocatalysis and enzymes in organic synth-
esis. Nat. Prod. Rep. 2001, 18, 618–640.
(16) Marumoto, S.; Miyazawa, M. Biotransformation of isoimperatorin
and imperatorin by Glomerella cingulata and β-secretase inhibitory
activity. Bioorg. Med. Chem. 2010, 18, 455–459.
(17) Yamahara, S.; Sugimura, C.; Nohara, T.; Niiho, Y.; Nakajima, Y.;
Ito, H. Studies on the constituents of Cnidii Monnieri Fructus.
Shoyakugaku Zasshi 1993, 47, 74–77.
In conclusion, one metabolite 2 was obtained from the incuba-
tion of 1 with G. cingulata, and two metabolites were obtained
from the incubation of 3. Compound 2 has not previously been
reported. The results indicated that G. cingulata possesses the
characteristics of reaction on the furanocoumarin skeleton as we
previously reported (16). The reactions involved the reduction of
the R,β-unsaturated lactone ring on furanocoumarin. In addition,
results showed that methyl ester and ether derivatives of meta-
bolites 8 and 11 exhibited BACE1 inhibitory activity. This
investigation also demonstrates that biotransformation is a
powerful tool for the structural modification of natural products.
(18) Harkar, S.; Razdan, T.; Waight, E. S. Steroids, chromone and
coumarins from Angelica officinalis. Phytochemistry1984, 23, 419–426.
(19) Elgamal, A. H. M.; Elewa, H. N.; Elkhrisy, M. A. E.; Helmut, D. 13C
NMR chemical shifts and carbon-proton coupling constant of some
furanocoumarins and furanochromones. Phytochemistry 1979, 18,
139–143.
(20) Spencer, G. F.; Desjardins, A. E.; Plattner, R. D. 5-(2-Carboxy-
ethyl)-6-hydroxy-7-methoxybenzofuran,
a fungal metabolite of
xanthotoxin. Phytochemistry 1990, 29, 2495–2497.
(21) Pangilinan, N. C.; Ivie, G. W.; Clement, B. A.; Beier, R. C.;
Uwayjan, M. Fate of [14C]xanthotoxin (8-methoxypsoralen) in
laying hens and a lactating goat. J. Chem. Ecol. 1992, 18, 253–270.
€
(22) Haser, K.; Wenk, H. H.; Schwab, W. Biocatalytic production of
LITERATURE CITED
dihydrocoumarin from coumarin by Saccharomyces cerevisiae.
J. Agric. Food Chem. 2006, 54, 6236–6240.
(1) Ozc-elik, B.; Kusmenoglu, S-.; Turkoz, S.; Abbasoglu, U. Antimicrobial
activities of plants from the Apiaceae. Pharm. Biol. 2004, 42, 526–528.
(2) Smith, M. L.; Gregory, P.; Bafi-Yeboa, N. F. A.; Arnason, J. T.
Inhibition of DNA polymerization and antifungal specificity of
Received for review March 30, 2010. Revised manuscript received May
18, 2010. Accepted May 18, 2010.