Biology Reference
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out by replacing the sample solution with
the same amount of DMSO.
0.1 M sodium borate buffer (pH 9.0) at 25°C.
At timed intervals, reactions were started by
adding 15 mM linoleic acid.
9.2.5
Assay of uric acid generated
by xanthine oxidase
9.2.7
Data analysis and curve fitting
The reaction mixture consisted of 2.76 ml of
40 mM sodium carbonate buffer containing
0.1 mM EDTA (pH 10.0), 0.06 ml of 10 mM
xanthine and 0.06 ml of sample solution
(dissolved in DMSO). The reaction was
started by the addition of 0.12 ml of xan-
thine oxidase (0.04 units), and the absorb-
ance at 293 nm was recorded for 60 s.
The assay was conducted in triplicate with
separate experiments. The data analysis was
performed by using Sigma Plot 2000 (SPSS
Inc, Chicago, IL). The IC
50
values were
obtained by fitting experimental data to the
logistic curve by Langmuir isotherm as fol-
lows (Copeland, 2000):
Activity (%) = 100(1/(1+([I]/IC
50
))
Inhibition mode was analysed with
Enzyme Kinetics Module 1.0 (SPSS Inc)
equipped with Sigma Plot 2000.
9.2.6
Lipoxygenase assay
The soybean lipoxygenase-1 (EC 1.13.11.12,
Type 1) used for the bioassay was purchased
from Sigma Chemical Co. Throughout the
experiment linoleic acid was used as a sub-
strate. In the current spectrophotometric
experiment, the enzyme activity of soybean
lipoxygenase-1 monitored at 25°C by Spectra
MAX plus spectrophotometer (Molecular
Device, Sunnyvale, CA). The enzyme assay
was performed as previously reported
(Kemal
et al.
, 1987) with slight modification.
In general, 5 ml of an ethanolic inhibitor
solution was mixed with 15 ml of 3 mM stock
solution of linoleic acid and 2.97 ml of 0.1 M
sodium borate buffer (pH 9.0) in a quartz
cuvette. Then, 10 ml of 0.1 M sodium borate
buffer solution (pH 9.0) of lipoxygenase
(0.52 mM) was added. The resultant solution
was mixed well and the linear increase of
absorbance at 234 nm, which expressed the
formation of conjugated diene hydroperox-
ide (13-HPOD, e = 2.50 × 10
4
M
−1
cm
−1
), was
measured continuously. The lag period
shown on lipoxygenase reaction (Ruddat
et al.
, 2003) was excluded for the determina-
tion of initial rates. The stock solution of
linoleic acid was prepared with Tween-20
and sodium borate buffer at pH 9.0, and then
total Tween-20 content in the final assay was
adjusted below 0.002%. For pre-incubation
experiments the enzyme was incubated with
various concentrations of compounds in
9.3
Anacardic Acids
Three of the major anacardic acids isolated
from the
A. occidentale
apple are: 6[8´(
Z
),
11´(
Z
),14´-pentadecatrienyl]salicylic acid
(
1
), 6[8´(
Z
),11´(
Z
)-pentadecadienyl]salicylic
acid (
2
) and 6[8´(
Z
)-pentadecenyl]salicylic
acid (
3
). They are referred to for simplicity
as anacardic acid (C
15:3
), anacardic acid
(C
15:2
) and anacardic acid (C
15:1
), respectively
(Fig. 9.1). Their fully saturated derivative
analogue, 6-pentadecylsalicylic acid (
4
),
referred to as anacardic acid (C
15:0
), was not
isolated from
A. occidentale
but rather iso-
lated as a prostaglandin synthetase inhibi-
tor from an African medicinal plant
Oziroa
mucronata
(Anacardiaceae) together with
anacardic acid (C
15:1
) (Kubo
et al.
, 1987).
Because anacardic acids are the derivatives
of salicylic acid (Machlin and Bendich,
1987) with a nonisoprenoid alk(en)yl side
chain, their activity was compared with
that of salicylic acid (
5
). The availability of
cardanol, 3[8´(
Z
), 11´(
Z
), 14´-pentadeca-
trienyl]phenol, referred to as cardanol (C
15:3
)
(
6
), an artefact of the corresponding ana-
cardic acid (C
15:3
) obtained by heating treat-
ment from the same source, is an additional
benefit for comparison. Anacardic acid
(C
15:3
) was selected for the present study as
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