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was obtained by a more direct experiment
for the radical scavenging activity that can
be measured as decolorizing activity follow-
ing the trapping of the unpaired electron of
DPPH. None of anacardic acids ( 1 - 3 ) exhib-
ited notable radical scavenging activity (0.01 ±
0.02 scavenged DPPH molecule per ana-
cardic acid molecule). On the basis of the
above results, further study was initiated.
100
80
60
40
20
0
0
50
100
150
200
Inhibitor ( µ M)
9.5
Xanthine Oxidase
Fig. 9.2. Inhibition of superoxide anion and uric
acid by xanthine oxidase with anacardic acid (C 15:3 )
and salicylic acid. Reaction rates by xanthine
oxidase were measured at 200 m M xanthine in the
presence of 0-200 m M anacardic acid, cardanol
and salicylic acid. , Superoxide anion generation
rates and , uric acid generation rates in the
presence of anacardic acid (C 15:3 ). , Superoxide
anion generation rates and , uric acid generation
rates in the presence of salicylic acid. ,
Superoxide anion generation rates and , uric
acid generation rates in the presence of cardanol.
The human body is known to produce free
radicals during the course of its normal
metabolism. Free radicals are even required
for several normal biochemical processes.
For example, the phagocyte cells involved
in the body's natural immune defences gen-
erate free radicals in the process of destroy-
ing microbial pathogens. If free radicals are
produced during the normal cellular metab-
olism in sufficient amounts to overcome the
normally efficient protective mechanisms,
metabolic and cellular disturbances will
occur in a variety of ways. Evidence is accu-
mulating that extracellular free radicals are
also produced in vivo by several oxidative
enzymes in the human body other than
phagocytes. For example, xanthine oxidase
(EC 1.1.3.22), a molybdenum-containing
enzyme, produces the superoxide anion
(O 2 •− ) radical as a normal product (Fong et al. ,
1973). The one-electron reduction products
of O 2 , the superoxide anion (O 2 •− ), hydrogen
peroxide (H 2 O 2 ) and the hydroxy radical
(HO ) from O 2 •− , participate in the initiation
of lipid peroxidation (Comporti, 1993).
Superoxide is also produced during mito-
chondrial respiration (Halliwell and
Gutteridge, 1990a) and by NADPH oxidase
(Pagano et al. , 1995), cyclooxygenase and
lipoxygenase (Kukreja et al ., 1986), nitric
oxidase synthetase (NOS) (Cosentino et al. ,
1998) and cytochtome P450 (Fleming et al. ,
2001). The effect of anacardic acids on the
generation of the superoxide anion by xan-
thine oxidase was tested and the result is
shown in Fig. 9.2. In the control, the super-
oxide anion generated by the enzyme
reduces yellow nitroblue tetrazolium to blue
formazan. Hence, the superoxide anion can
be detected by measuring the absorbance of
formazan produced at 560 nm. At a concen-
tration of 30 mg/ml, anacardic acid (C 15:3 )
(88 mM) inhibited this formazan formation
82 ± 4%. Interestingly, salicylic acid did not
show any observable inhibitory activity up
to a concentration of 138 mg/ml (1.0 mM)
and showed 7 ± 3% inhibition at 276 mg/ml,
indicating that the C 15 -alkenyl side chain is
associated with this inhibitory activity.
Cardanol did not show this inhibitory activ-
ity up to 0.2 mM, indicating that the struc-
ture of 2-carboxylphenol (salicylic acid) is
also necessary. As the concentrations of
anacardic acid (C 15:3 ) increased, the remain-
ing enzyme activity was rapidly decreased.
Notably, this inhibition mechanism does
not follow hyperbolic inhibition by ana-
cardic acid concentration (Michaelis-Menten
equation) but follows the Hill equation
(Beckmann et al. , 1998) instead. The shape
of the inhibition curve of xanthine oxidase
by anacardic acid (C 15:3 ) is sigmoidal
( S -shaped) (IC 50 = 51.3 ± 1.5 mM) as shown
in Fig. 9.3. This inhibition occurred over a
 
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