Biology Reference
In-Depth Information
promotion of certain cancers (Rousseau
et al
., 1992). The past experimental studies
have provided compelling evidence that
antioxidants play an important role in
reducing the risk of cancer. However, the
previous studies usually emphasized the
scavenging activity to use as antioxidant
additives in food and lack comprehensive-
ness. The importance of discovering new
safe and effective antioxidants is of consid-
erable interest in preventive medicine.
Antioxidants isolated from regularly con-
sumed foods and beverages, such as the
cashew apple and its processed products,
may be superior to non-natural products.
Therefore, our investigation has been fur-
ther extended to test the antioxidation activ-
ity of anacardic acids. Because anacardic
acids are the derivatives of salicylic acid
(Machlin and Bendich, 1987) with a noniso-
prenoid alk(en)yl side chain, their activity
is compared with that of salicylic acid.
vial. Each reaction mixture consisted of
0.57 ml of 2.51% linoleic acid in ethanol
and 2.25 ml of 40 mM phosphate buffer
(pH 7.0). The vial was placed in an oven at
40°C. After 5 days incubation a 0.1 ml aliq-
uot of the mixture was diluted with 9.7 ml
of 75% ethanol, which was followed by
adding 0.1 ml of 30% ammonium thiocy-
anate. Precisely 3 min after the addition of
0.1 ml of 20 mM ferrous chloride in 3.5%
hydrochloric acid to the reaction mixture
the absorbance at 500 nm was measured.
9.2.3 Radical scavenging
activity on DPPH
First, 1 ml of 100 mM acetate buffer (pH
5.5), 1.87 ml of ethanol and 0.1 mL of eth-
anolic solution of 3 mM of DPPH were put
into a test tube. Then, 0.03 ml of the sample
solution (dissolved in DMSO) was added to
the test tube and incubated at 25°C for
20 min. The absorbance at 517 nm (DPPH,
e = 8.32 × 10
3
M
−1
cm
−1
) was recorded. As
control, 0.03 ml of DMSO was added to the
test tube. From the decrease in absorbance,
scavenging activity was calculated and
expressed as scavenged DPPH molecules
per one molecule.
9.2
Experimental
9.2.1
Chemicals
Anacardic acids (
1
-
3
) and the correspond-
ing cardanols (
4
-
6
) used for the assay were
previously isolated from the cashew nut
shell oil (Fig. 9.1). Their re-purification by
recycle high-performance liquid chroma-
tography (R-HPLC) was achieved using an
ODS C
18
column (Kubo
et al
., 1986). Salicylic
acid, linoleic acid, BHT, EDTA, thiobarbituric
acid (TBA), 1,1-diphenyl-2-
p
-picrylhydra-
zyl (DPPH), 2,2´-azo-
bis
(2-amidinopropane)
dihydrochloride (AAPH), ADP, bovine
serum albumin and nitroblue tetrazolium
were purchased from Sigma Chemical Co.
(St. Louis, MO).
9.2.4 Assay of superoxide anion
generated by xanthine oxidase
The xanthine oxidase (EC 1.1.3.22, Grade
IV) used for the bioassay was purchased
from Sigma Chemical Co. Superoxide anion
was generated enzymatically by the xan-
thine oxidase system. The reaction mixture
consisted of 2.70 ml of 40 mM sodium
carbonate buffer containing 0.1 mM EDTA
(pH 10.0), 0.06 ml of 10 mM xanthine, 0.03
ml of 0.5 % bovine serum albumin, 0.03 ml
of 2.5 mM nitroblue tetrazolium and 0.06 ml
of sample solution (dissolved in DMSO).
To the mixture at 25°C, 0.12 ml of xanthine
oxidase (0.04 units) was added, and the
absorbance at 560 nm was recorded for 60 s
(by formation of blue formazan) (Toda
et al
.,
1991). The control experiment was carried
9.2.2
Assay of autoxidation
Oxidation of linoleic acid was measured by
the modified method described previously
(Haraguchi
et al
., 1992). Different amounts
of samples dissolved in 30 ml ethanol were
added to a reaction mixture in a screw cap
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