3178 J. Agric. Food Chem., Vol. 52, No. 10, 2004
Ha et al.
Louis, MO). Tris buffer was obtained from Fisher Scientific Co. (Fair
Lawn, NJ). Ethanol was purchased from Quantum Chemical Co.
(Tuscola, IL).
Synthesis. Octyl 3,5-dihydroxybenzoate (6) was synthesized as
follows. A mixture of 3,5-dibenzyloxybenzoic acid (200 mg, 0.60
mmol) (7), octanol (86 mg, 0.66 mmol), and TPP (190 mg, 0.72 mmol)
in THF (4 mL) was cooled to 0 °C and treated with DIAD (146 mg,
0.72 mmol). After 2 h of stirring at room temperature, the solvent was
removed in vacuo. The residue was subjected to silica gel chromatog-
raphy eluted with 1-8% AcOEt/hexane to give an ester as a white
solid, which was used in the next step without further purification. The
ester was hydrogenated over 20% Pd(OH)2 on carbon (10 mg) in 1%
AcOH/AcOEt (4 mL) for 12 h. Filtration through Celite and concentra-
tion followed by silica gel chromatography (15-30% AcOEt/hexane)
gave 155 mg (92%) of the title compound as a white solid: IR (CCl4)
3350, 2920, 1680, 1595, 1335, 1240, 1160 cm-1; 1H NMR (400 MHz,
CDCl3) δ 7.13 (d, J ) 1.2 Hz, 2H), 6.58 (t, J ) 1.2 Hz, 1H), 5.54 (bs,
2H), 4.29 (t, J ) 6.4 Hz, 2H), 1.74 (quin, J ) 6.4 Hz, 2H), 1.41 (m,
2H), 1.28 (m, 8H), 0.88 (t, J ) 6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ 166.1, 156.7, 132.4, 108.9, 107.3, 65.5, 31.8, 29.2, 29.1, 28.6,
26.0, 22.6, 14.1; EI-MS, m/z 266 (M+).
According to basically the same procedure, octyl 4-hydroxybenzoate
(7) was synthesized as a colorless solid (98% yield): IR (CCl4) 3600,
Figure 2. Effects of octyl gallate on the activity of soybean lipoxygenase-1
for the catalysis of linoleic acid at 25 °C. (Inset) Replots of data as 1/v
versus [I].
3360, 2920, 1705, 1675, 1600, 1585, 1505, 1265, 1155, 1100 cm-1
;
1H NMR (400 MHz, CDCl3) δ 7.95 (d, J ) 7.2 Hz, 2H), 6.87 (d, J )
7.2 Hz, 1H), 5.83 (bs, 1H), 4.28 (t, J ) 6.8 Hz, 2H), 1.75 (quin, J )
6.8 Hz, 2H), 1.43 (m, 2H), 1.28 (m, 8H), 0.88 (t, J ) 7.2 Hz, 3H); 13
C
RESULTS
NMR (100 MHz, CDCl3) δ 166.4, 159.5, 131.6, 122.8, 115.0, 64.9,
31.8, 29.22, 29.15, 28.7, 26.0, 22.6, 14.1; EI-MS, m/z 250 (M+).
Enzyme Assay. Throughout the experiment, linoleic acid was used
as a substrate. In a spectophotomeric experiment, the oxygenase activity
of the soybean lipoxygenase was monitored at 25 °C by a Spectra MAX
plus spectrophotometer (Molecular Devices, Sunnyvale, CA). The
enzyme assay was performed as previously reported (8) with slight
modification. In general, 5 µL of an ethanolic inhibitor solution was
mixed with 54 µL of 1 mM stock solution of linoleic acid and 2.936
mL of 0.1 M Tris-HCl buffer (pH 8.0) in a quartz cuvette. Then, 5 µL
of a 0.1 M Tris-HCl buffer solution (pH 8.0) of lipoxygenase (1.02
µM) was added. The resultant solution was mixed well, and the linear
increase of absorbance at 234 nm, which expresses the formation of
conjugated diene hydroperoxide (13-HPOD, ꢀ ) 25000 M-1 cm-1),
was measured continuously. A lag period shown in the lipoxygenase
reaction (9) was excluded for the determination of initial rates. The
stock solution of linoleic acid was prepared with methanol and Tris-
HCl buffer at pH 8.0, and, then, total methanol content in the final
assay was adjusted below 1.5%. For determining reversible inhibition
manner, the enzyme concentration was changed as 0.17, 0.33, 0.50,
0.67, and 0.83 µg/mL with a constant substrate concentration (40 µM).
Five concentrations (10, 15, 20, 35, and 40 µM) of linoleic acid were
selected for Lineweaver-Burk plots.
Lipoxygenase-dependent O2 uptake was performed using a Clark-
type oxygen electrode (YSI 53, Yellow Springs Instrument Co., Yellow
Springs, OH) at 25 °C as essentially the same procedures in the
spectophotomeric experiment. For obtaining IC50, the final assay
concentrations of the enzyme and the substrate were adjusted to 4.25
nM and 46 µM, respectively. On the other hand, in the study of
Lineweaver-Burk plots, the final concentration of the enzyme was
fixed at 4.25 nM, but the substrate concentrations selected were 40,
60, 80, and 100 µM. Because a weak substrate inhibition was induced
by the high concentration of linoleic acid (8, 9), 100 µM substrate was
used as the maximum concentration.
The inhibition activity of soybean lipoxygenase-1 was
measured by two methods for comparison, because this enzyme
seems to be sensitive to assay conditions. Soybean lipoxyge-
nase-1 is known to catalyze the dioxygenation of the (Z,Z)-
diene moiety of linoleic acid. In plants, the primary dioxygen-
ation product is 13S linoleic acid hydroperoxide (13-HPOD)
(1). Hence, the enzyme assay was usually performed using a
UV spectrophotometer to detect the increase at 234 nm
associated with the (Z,E)-conjugated double bonds newly formed
in the product but not the substrate. In many previous studies,
the data were usually obtained at pH 9 because soybean
lipoxygenase-1 is reported to have its optimum at pH 9.0 (12),
but the absorption at 234 nm suffered from unstable baseline
activity of unknown origin attributable to the presence of octyl
gallate at pH 9.0. This pseudoactivity of the blank control had
to be subtracted from activity of the enzyme assay, making
precise measurements difficult. Moreover, this basic pH value
may not be practical to use for food protection. Because the
stable data were obtained at pH 8.0, the evaluation was
performed at pH 8.0 (10). The data obtained were also compared
with those of NDGA used as a reference compound. Its IC50
was obtained as 82 µM when the experiment was performed at
pH 9.0 but 0.2 µM at pH 8.0. As a result, octyl gallate showed
a dose-dependent inhibitory effect on this oxidation as shown
in Figure 2. As octyl gallate increased, the enzyme activity was
rapidly decreased but not completely suppressed. The IC50 was
estimated to be 1.3 µM. As the need arose, the assay was also
monitored by using polarography (oxygen consumption) for
comparison. The IC50 obtained was 2.1 µM, which is almost
comparable with that measured by using the spectrophotometric
method.
Data Analysis and Curve Fitting. The assay were conducted in
triplicate of separate experiments. The data analysis was performed by
using Sigma Plot 2000 (SPSS Inc., Chicago, IL). The inhibitory
concentration leading to 50% activity loss (IC50) was obtained by fitting
experimental data to the logistic curve by the equation as follows (11):
The oxidation of linoleic acid catalyzed by soybean lipoxy-
genase-1 follows Michaelis-Menten equations. The plots of the
remaining enzyme activity versus the concentrations of enzyme
at different concentrations of octyl gallate gave a family of
straight lines, which passed through the origin as shown in
Figure 3. Increasing the inhibitor concentration resulted in
descending slopes of the lines, indicating that the inhibition of
octyl gallate on the enzyme was reversible. The presence of
octyl gallate did not bring down the amount of the efficient
activity (%) ) 100[1/(1 - ([I]/IC50))]
Inhibition mode was analyzed by Enzyme Kinetics Module 1.0
(SPSS Inc.) equipped with Sigma Plot 2000.