6308 J. Agric. Food Chem., Vol. 50, No. 22, 2002
Kato et al.
chemicals involved dilution of the reaction mixture with water,
extraction with an organic solvent such as ether or CH2Cl2, and
evaporation after washing the organic layers with water and brine,
followed by drying over Na2SO4. Lipase PS “Amano”, a lipolytic
enzyme manufactured from culture of Pseudomonus sp., and pig liver
esterase (PLE) were supplied by Amano Enzyme Inc. (Nagoya, Japan).
All chemicals used for the preparation of active compounds were
purchased from Wako Pure Chemicals Industries Ltd. (Osaka, Japan)
and purified if necessary by the standard procedure.
Table 1. Relative Germination Ratios of Intact Seeds on the Basis of
Dehusked Seeds
germination (%)
sprouts
variety
(dormancy at harvest)
roots
Lancer (dormancy)
RL4137 (dormancy)
Menyou (nondormancy)
17
64
100
24
53
89
Chemicals. 2-Phenylethyl alcohol 1, D-, L-, and DL-1-phenylethyl
alcohol 5, and ABA are commercially available and were purchased
from Tokyo Kasei Organic Chemicals Co., Ltd. (Tokyo, Japan).
4-Vinylphenol 2 and its 2-methoxy derivative 3, stabilized with 2,6-
di-tert-butyl-4-methylphenol, are commercially available. In the present
study, these compounds were synthesized according to the patented
procedures (12). 2-Methoxy-4-vinylphenol 3 was synthesized by the
following procedure: Vanillin acetate (522 mg, 99%) was obtained
by acetylation of vanillin (413 mg, 2.71 mmol) with acetic anhydride
(0.51 mL, 543 mmol) in pyridine (5 mL) for 3.5 h at room temperature.
The zinc powder was first activated by warming a mixture of Zn powder
(703 mg, 11.0 mmol) and acetyl chloride (0.02 mL, 0.27 mmol) in
dimethylformamide (DMF) (3 mL) at 50 °C for 15 min under an argon
atmosphere. A mixture of 4-acetoxy-3-methoxybenzaldehyde (522 mg,
3 mmol) and dibromomethane (0.28 mL, 4.03 mmol) was gradually
dropped into the activated Zn powder in DMF. After being stirred at
50 °C for 30 min, acetic anhydride (0.25 mL, 2.7 mmol) was added to
the mixture, and the reaction mixture was further stirred for 2.5 h at
50 °C. After being cooled to 0 °C, aqueous saturated NH4Cl solution
(3 mL) was added to the cooled mixture. The Zn powder was removed
by filtration through a glass filter. The Zn powder and aqueous solution
were extracted with ether (30 mL × 3). After the usual workup of the
ether solution, the crude residue was passed through a silica gel (30 g)
column eluted with hexane/EtOAc (20:1) to obtain 4-acetoxy-3-
methoxyvinylbenzene (337 mg, 65% from vanillin) as a colorless oil.
A mixture of 4-acetoxy-3-methoxy-vinylbenzene (105 mg, 0.55 mmol)
and 2 N KOH-MeOH (2 mL) was then stirred at room temperature
for 1 h; the mixture was acidified with 2 N aqueous HCl solution under
ice cooling and then extracted with CH2Cl2 (10 mL × 3). After the
usual workup of the CH2Cl2 solution, pure 2-methoxy-4-vinylphenol 3
(72 mg, 88%) was obtained by SiO2 column chromatography eluted
with hexane/EtOAc (10:1). 4-Vinylphenol 2 was synthesized by the
following procedure: 4-Hydroxybenzaldehyde (5 g, 41 mmol) was
converted to 4-acetoxybenzaldehyde (6.4 g, 95%) with acetic anhydride
(11.6 mL, 120 mmol) in pyridine (150 mL). 4-Acetoxyvinylbenzene
(400 mg, 44%) was provided from 4-acetoxybenzaldehyde (1 g, 6
mmol) by the action of dibromomethane (0.64 mL, 9.2 mmol) and acetic
anhydride (0.58 mL, 6.1 mmol) in the presence of Zn powder (1.5 g,
23 mmol), activated with acetyl chloride (0.04 mL, 0.56 mmol) in DMF
(5.3 mL). Since the compounds 2 and 3 are fairly unstable and apt to
polymerize, the benzene solution of the corresponding acetoxyvinyl-
benzene derivatives was stored in a freezer at -20 °C. The final step
of the alkaline hydrolysis was carried out prior to the assay experiments.
CHCl3; lit. (14) +121° (c ) 1.00, CHCl3)) and -98.4° (c ) 0.71,
CHCl3; lit. (14) -121° (c ) 1.05, CHCl3)), respectively.
Plant Materials. All of the field-grown wheat varieties used in the
present study were harvested when the moisture content of the examined
matured seeds was ca. 16%. For the extraction study, the air-dried wheat
spikes of dormancy variety Kwankei W421, harvested in June 1998 in
the field of the National Ministry of Agriculture and Forestry at Tsukuba
(Japan), were mechanically separated into grains and husks. The husks
were shredded to small pieces by a commercially available juicer prior
to the present extraction experiments. In the exploratory experiment in
Table 1, concerning the effect of husks toward germination inhibition,
three kinds of wheat varieties, Lancer, RL4137, and Menyou, were kept
at room temperature for 5 months after harvesting (Tsukuba, Japan) in
order to release the dormant seeds from their dormancy. The wheat
dormancy varieties, RL4137 and Nourin 61, harvested at 43 days after
antheses of August 2001 in the fields of the Tokyo University of
Agriculture at Abashiri, Hokkaido (Japan), and the dormant variety
Hokushin, obtained from the Preservation and Evaluation Section,
Hokkaido Prefectural Plant Genetic Resources Center (Japan), were
used for the comparisons of germination inhibition activities. All
varieties existed in a deep primary dormant state just after the
harvesting. After the samples were kept for about 4 months at 20 °C,
after-ripening germination took place, and the germination ratios were
ca. 60% and 70% on the basis of the examined total grains in the cases
of RL4137 and Nourin 61, respectively. The germination ratio of
RL4137 increased to ca. 85% after being kept at room temperature for
a further 2 months (total of 6 months after harvesting). Nourin 61 and
Hokushin showed more than 95% germinability after 6 months keeping.
These intact seeds were kept as a whole spike in a freezer at -30 °C,
and hand-threshed grains were provided prior to use for the germination
assay.
Extraction and Isolation. Distilled water (1 L) was added to the
wheat husks of Kwankei W421 (200 g), preshredded to small pieces,
and the mixture was kept at 60 °C for 48 h under an argon atmosphere.
The replacement with argon gas was necessary to prevent formation
of mold. The husks were removed by filtration through a four-layered
gauze and treated again with water (1 L). The combined aqueous
solutions were submitted to steam distillation at 60 °C under reduced
pressure using an aspirator to separate the steam distillate and residue.
The distillate was extracted with CH2Cl2 (500 mL × 3), from which
30 mg of steam distillate residue was obtained after drying with
Na2SO4 and careful evaporation of the combined CH2Cl2 layers. The
residue of the steam distillation was extracted with ether (200 mL × 2),
providing 900 mg of ether extract residue upon evaporation of the
volatile materials after drying with Na2SO4. The steam distillate residue
(30 mg) was fractionated by Cosmosil gel flash chromatography with
300 mL each of H2O first and then H2O/acetone in the ratios of 5:1,
5:2, 5:3, and 1:1, respectively. The acetone from each eluate was
evaporated at 75 °C under normal pressure, and the resulting aqueous
layer of each fraction was extracted with CH2Cl2 (50 mL × 2) to obtain
6 mg (100%), 5 mg (11%), 9 mg (11%), 4 mg (0%), and 4 mg (0%),
respectively. The percent shown in parentheses of each yield indicates
the germination ratio of sprout at 103 ppm in aqueous solution.
For the isolation study, the active fractions were collected by repeated
water extraction of the husks followed by steam distillation. The fraction
of 5:2 eluate (8 mg) was separated by HPLC with a µ-Bondapak C-18
column (300 × 7.8 mm i.d.; flow rate of 1.0 mL/min; RI detector)
using H2O/MeOH (10:3) as the mobile phase to isolate 2-phenylethyl
alcohol 1 (2 mg). The 5:3 fraction (9 mg) was similarly separated by
HPLC with the same conditions using H2O/MeOH (3:2) to isolate
4-vinylphenol 2 (2 mg) and 2-methoxy-4-vinylphenol 3 (2 mg),
DL-Tetrahydroactinidiolide 6 was prepared by the previously de-
scribed procedure (13). dl-Dihydroactinidiolide 4 was prepared from
DL-6 by Mori’s procedure (14). Both enantiomers of 6 and 4 were
prepared based on the procedure developed by Mori’s group (15),
starting from the dl-form of 2,4,4-trimethyl-2-cyclohexen-1-ol (16).
Optical resolution of the DL-form was carried out by application of
lipase PS “Amano” in isopropyl ether at the first step and then PLE in
0.1 M phosphate buffer (pH 7.0) to the partly resolved acetate. The
resolved D- and L-alcohols had [R]22 +82.5° (c ) 1.0, MeOH) and
D
-86.6° (c ) 1.0, MeOH), respectively, showing 90 and 94% enantio-
meric excess (ee), estimated based on the integral ratios of the methyl
signals in the NMR spectra of the corresponding Mosher esters. The
sequential reactions from the resolved alcohols to the respective
enantiomers followed the published procedure. Optical rotations
([R]23D]) of 6 submitted for the present assay experiments were +57.6°
(c ) 0.93, CHCl3; lit. (14) +63.5° (c ) 0.95, CHCl3)) and -56.6° (c
) 1.05, CHCl3; lit. (14) -66.1° (c ) 0.97, CHCl3)), respectively, and
those for D- and L-4 ([R]23D]) for the assays were +101° (c ) 0.50,