Biotransformation of isoflavones by the larvae of the common cutworm (Spodoptera litura)

Article abstract of DOI:10.1248/cpb.54.719

Biotransformation of the 5,7,4′-trimethoxyisoflavone (1), 6,7,4′-trimethoxyisoflavone (2), and 7,4′-dimethoxyisoflavone (3) by insects, Spodoptera litura was investigated. Compound 1 was transformed to 5-hydroxy-7,4′-dimethoxyisoflavone (4), 7-hydroxy-5,4

Full text of DOI:10.1248/cpb.54.719

May 2006
Notes
Chem. Pharm. Bull. 54(5) 719—721 (2006)
719
Biotransformation of Isoflavones by the Larvae of the Common Cutworm
(Spodoptera litura)
a
b
,a
*
Koji TAKAHASHI, Hideo ARAKI, and Mitsuo MIYAZAWA
a Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University; Kowakae, Higashiosaka, Osaka
577–8502, Japan: and b Fuji Oil Co., Ltd. Hannan R&D Center; 1 Sumiyoshi-cho, Izumisano, Osaka 598–8540, Japan.
Received November 10, 2005; accepted January 24, 2006; published online February 6, 2006
Biotransformation of the 5,7,4ꢀ-trimethoxyisoflavone (1), 6,7,4ꢀ-trimethoxyisoflavone (2), and 7,4ꢀ-dime-
thoxyisoflavone (3) by insects, Spodoptera litura was investigated. Compound 1 was transformed to 5-hydroxy-
7,4ꢀ-dimethoxyisoflavone (4), 7-hydroxy-5,4ꢀ-dimethoxyisoflavone (5) and 4ꢀ-hydroxy-5,7-dimethoxyisoflavone
(6) by S. litura. Compounds 2 and 3 were hardly metabolized by S. litura. This suggested that compound 1 was
converted to compounds 4, 5 and 6 by demethylation at the C-5, C-7 and C-4ꢀ position, respectively.
Key words biotransformation; isoflavone; Spodoptera litura; biocatalyst; demethylation
The naturally occurring isoflavones are reported to possess nal has disappeared compared with compound 1. Further-
diverse biological activities, including antioxidative,1) anti- more, compound 4 had a signal of hydrogen bond at d 12.85.
fungal2) and estrogenic3,4) activity. Recently, an osteoporosis Compounds 5 and 6 were converted monoacetyl compounds,
preventive effect5) and mitigation of menopausal disorders6) 5Ac and 6Ac by reaction with acetylation. It results that
has attracted attention and various physiological activities of compounds 4, 5 and 6 showed the presence of two methoxyl
isoflavone are expected. It has been reported that these activi- groups and one hydroxy group, respectively (see Table 1).
ties were enhanced in the biotransformation.7,8) Biotransfor-
Compounds 4 and 5 also showed a significant mass frag-
mation possesses the advantage of proceeding under mild ment at m/z 166 and 132, and compound 6 also showed a sig-
conditions and with high regio- and enantioselectivity. Esaki nificant mass fragment at m/z 180 and 118, respectively. In a
et al. reported the biotransformation of soybean isoflavones previous article, Talukdar et al. reported the structure of 5-
to 8-hydroxydaidzein and 8-hydroxygenistein by Aspergillus hydroxy-7,4ꢀ-dimethoxyisoflavone by the retro Diels–Alder
satoi and these isoflavones exhibited significantly stronger fragments at 166 and 132 formed after the retro Diels–Alder
antioxidative activity than daidzein and genistein.7) There- reaction indicating the presence of one methoxyl and one hy-
fore, it is important to produce the bioactive isoflavone deriv- droxy groups in ring A, and one methoxy group should be
atives and to elucidate their metabolic pathways in microor- present in ring B.15) In the identification of the isoflavones,
ganisms and insects.
the retro Diels–Alder fragmentation was found to be impor-
In the course of our work, we have studied the biotrans- tant.16,17)
formation of isoflavonoids and terpenoids by fungi and in-
In our previous paper, we reported the biotransformation
sects as a biocatalyst.9—14) In a previous paper, we reported of sinesetin by larvae of S. litura. The main pathway was
the biotransformation of the isoflavones, 7,4ꢀ-dimethoxy- demethylation at C-6 position followed by glucosylation, and
isoflavone and 7,4ꢀ-diacetoxyisoflavone, by Aspergillus the minor pathway was demathylation at the C-4ꢀ position.13)
niger. 7,4ꢀ-Dimethoxyisoflavone was transformed to 6-hy-
These results showed that compound 4 had the hydroxy
droxy-7,4ꢀ-dimethoxyisoflavone and daidzein, while 7,4ꢀ-di- group at C-5 position and compound 5 had the hydroxy
acetoxyisoflavone was also transformed to daidzein by hy- group at C-7 position. Compound 6 also showed a significant
drolysis.10) In this paper, we report the synthesis of iso- mass fragment at m/z 180 and 118 formed after the retro
flavones, 5,7,4ꢀ-trimethoxyisoflavone (1), 6,7,4ꢀ-trimethoxy- Diels–Alder reaction indicating the presence of two
isoflavone (2) and 7,4ꢀ-dimethoxyisoflavone (3) and the methoxyl groups in ring A, and one hydroxy group should be
biotransformation of compounds 1, 2 and 3 by insect, present in ring B. Thus from compound 6 had the hydroxy
Spodoptera litura.
group at C-4ꢀ position.
Thus from the foregoing spectral studies the structure of
compounds 4, 5 and 6 were identified as 5-hydroxy-7,4ꢀ-
Results and Discussion
Compound 1 was metabolized by S. litura. Compounds 2 dimethoxyisoflavone, 7-hydroxy-5,4ꢀ-dimethoxyisoflavone
and 3 were hardly metabolized by S. litura. and 4ꢀ-hydroxy-5,7-dimethoxyisoflavone, respectively. Spec-
The transformation of 5,7,4ꢀ-trimethoxyisoflavone (1) by tral data of compound 4 were assigned by comparison with
S. litura was examined. Extracts were chromatographed on the previous paper.15) Compound 5 was obtained by synthesis
silica gel column chromatography to give metabolites.
Metabolites 4 (6 mg), 5 (5 mg), 6 (3 mg) and substrate 1
(130 mg) were isolated. In order to determination of structure
of the metabolites, metabolites 5 and 6 were acetylated.
Metabolites 4, 5 and 6 showed the molecular formula of
C17H14O5 in their HR-EI-MS, respectively. The 1H-NMR
spectrum of compounds 4, 5 and 6 showed the presence of
two methoxyl groups, which indicated that one methoxy sig-
∗ To whom correspondence should be addressed. e-mail: miyazawa@apch.kindai.ac.jp
© 2006 Pharmaceutical Society of Japan

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Vol. 54, No. 5
from the chalcone.18) In our previous paper, compound 6 was glycitein and 7,4ꢀ-dimethoxyisoflavone (3) (190 mg) was obtained from
1
obtained by microbial transformation.17) The H-NMR spec-
daidzein. These spectral data were assigned by comparison with the previous
paper.10,17)
trum analysis of compounds 5Ac and 6Ac were not reported,
and thus this is the first report of the H-NMR spectrum
Rearring of Larvae The larvae of S. litura were reared in plasticcases
(200ꢁ300 wide, 100 mm high, 100 larvae/case) covered with a nylon mesh
screen. The rearing conditions were as follows: 25 °C, 70% relative humid-
ity, and constant light. A commercial diet (Insecta LF; Nihon Nosan kogyo
Co., Ltd.) was given to the larvae from the first to sixth (last) instar.
Administration of 5,7,4ꢀ-Trimethoxyisoflavone (1) The diet of 100
larvae (fourth–fifth instar) was changed to an artificial diet composed of kid-
ney beans (100 g), agar (12 g), and water (600 ml). After 1 d, the artificial
diet without the agar was mixed with a blender. Compound 1 (150 mg) was
then added to the blender at 1 mg/g of diet. Ager was dissolved in water and
boiled and then added to the blender. The diet was then mixed and cooled in
a tray. The larvae were moved into new cases, and the diet containing com-
pound 1 (3 mg for a body) were fed for 2 d, and then the artificial diet not
containing compound 1 was fed to the larvae for an additional 2 d. Frass was
collected daily and stored in a solution of CHCl3 (100 ml).
The frass was extracted with CHCl3 (100 mlꢁ3) and EtOAc (100 mlꢁ2).
These extracts were mixed, the solvent was evaporated under reduced pres-
sure, and 211.9 mg of extract was obtained. The extract was analyzed by
TLC and GC-MS; metabolites 4, 5 and 6 occurred in this extract. The ex-
tract was fractionated by SiO2 column chromatography with CH2Cl2/acetone
as eluents. Recovery substrate 1 (130 mg) was isolated. Three metabolites 4
(6 mg), 5 (5 mg) and 6 (3 mg) were isolated. The acetylated derivatives of
compounds 5 (5Ac) and 6 (6Ac) were obtained by reaction with Ac2O and
pyridine.
5-Hydroxy-7,4ꢀ-dimethoxyisoflavone (4): White crystals, HR-EI-MS, m/z
298.0833 [M]ꢂ, Calcd for C17H14O5, 298.0842; EI-MS, m/z (%) (rel. int.)
298 [M]ꢂ (100), 297 (13), 283 (10), 255 (4), 166 (12), 149 (7), 132 (17),
117 (5); IR (KBr) nmax cmꢃ1 1614, 1576, 1515, 1441; 1H-NMR (CDCl3) see
Table 1.
7-Hydroxy-5,4ꢀ-dimethoxyisoflavone (5): White crystals, HR-EI-MS, m/z
298.0860 [M]ꢂ, Calcd for C17H14O5, 298.0842; EI-MS, m/z (%) (rel. int.)
298 [M]ꢂ (100), 297 (17), 283 (10), 269 (3), 166 (22), 132 (25), 117 (10).
4ꢀ-Hydroxy-7,4ꢀ-dimethoxyisoflavone (6): White crystals, HR-EI-MS,
m/z 298.0845 [M]ꢂ, Calcd for C17H14O5, 298.0842; EI-MS, m/z (%) (rel.
int.) 298 [M]ꢂ (100), 297 (25), 283 (10), 269 (3), 180 (5), 118 (20).
7-Acetoxy-5,4ꢀ-dimethoxyisoflavone (5Ac): White crystals, EI-MS, m/z
(%) (rel. int.) 340 [M]ꢂ (100), 1H-NMR (CDCl3) see Table 1.
1
analysis of compounds 5Ac and 6Ac.
Compound 1 was transformed to compounds 4, 5 and 6.
Metabolites 4, 5 and 6 were compounds in the demethylation
of compound 1 at the C-5 and C-7 and C-4ꢀ position, re-
spectvely. On the other hand, compounds 2 and 3 were not
converted. These results indicate that compound 1 showed
substrate specificity for this biotransformation. This is the
first report of biotransformation of 5,7,4ꢀ-trimethoxy-
isoflavone (1) by S. litura. It is interesting to note that
demethylation of one position are easily obtained by insects.
Experimental
General Procedure Nuclear magnetic resonance (NMR) spectra were
obtained with a JEOL FX-500 (500.00 MHz, 1H; 125.65 MHz, 13C) spec-
trometer. Tetramethylsilane (TMS) was used as the internal standard (d
0.00). IR spectra were determined with a JASCO FT/IR-470 plus Fourier
transform infrared spectrometer. EI-MS spectra were obtained on a JEOL
the Tandem MS station JMS-700 TKM. Gas chromatograph (GC) was per-
formed on a Hewlett-Packard 5890 gas chromatograph equipped with a
flame ionization detector (FID). GC-MS was performed on a Hewlett-
Packard 5972 series mass spectrometer interfaced with a Hewlett-Packard
5890 gas chromatograph fitted with a column (HP-5MS, 30 m length,
0.25 mm i.d.).
Preparation of Isoflavones Genistein, glycitein and daidzein were iso-
lated soyaflabon HG (powder of Glycine max, purchased from FUJI Oil Co.,
Ltd. (Osaka, Japan).
Synthesis of Substrates Methylation of isoflavones were carried out ac-
cording to the synthetic method of Matsuda et al.19) A solution of genistein
(200 mg) in N,N-dimethylformamide (DMF, 12 ml) was treated with methyl
iodide (CH3I, 0.6 ml) in the presence of sodium hydride (NaH, 60 mg) and
the mixture was stirred at room temperature for 12 h. 5,7,4ꢀ-Trimethoxy-
isoflavone (1) (180 mg) was obtained. Through a similar procedure of
methylation, 6,7,4ꢀ-trimethoxyisoflavone (2) (188 mg) was obtained from
4ꢀ-Acetoxy-7,4ꢀ-dimethoxyisoflavone (6Ac): White crystals, EI-MS, m/z
Table 1. 1H-NMR Spectral Data of Compounds 1, 4, 5Ac and 6Ac
(%) (rel. int.) 340 [M]ꢂ (100), 1H-NMR (CDCl3) see Table 1.
Administration of 6,7,4ꢀ-Trimethoxyisoflavone (2) and 7,4ꢀ-Di-
methoxyisoflavone (3) Compounds 2 (150 mg) and 3 (150 mg) were ad-
ministrated in the same way. Extracts were mixed, the solvent was evapo-
rated under reduced pressure, and 215.0 mg and 210.0 mg of extract was ob-
tained. Recovery substrates 2 (148 mg) and 3 (148 mg) were isolated.
No.
1
4
5Ac
6Ac
2
7.76 s
7.86 s
7.82 s
7.79 s
6
6.36 d (2.3)
6.44 d (2.3)
6.40 d (2.4)
6.38 d (2.4)
6.58 d (2.5)
6.85 d (2.5)
6.38 d (2.3)
6.46 d (2.3)
8
2ꢀ,6ꢀ
3ꢀ,5ꢀ
7.47 dt (8.9, 2.0) 7.45 dt (8.9, 2.4) 7.48 dt (8.4, 1.9) 7.56 dt (8.4, 2.0)
6.93 dt (8.9, 2.0) 6.98 dt (8.9, 2.4) 6.94 dt (8.4, 1.9) 7.13 dt (8.4, 2.0)
Acknowledgments This work was supported by “High-Tech Research
Center” project for Private Universities: matching fund subsidy from MEXT
(Ministry of Education, Culture, Sports, Science and Technology), 2004—
2008.
OMe 3.93 s
3.88 s
3.87 s
3.84 s
3.96 s
3.84 s
3.94 s
3.90 s
3.82 s
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2.35 s
2.31 s
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