Lipoxygenase inhibitory activity of anacardic acids

Article abstract of DOI:10.1021/jf048184e

6[8′(2)-Pentadecenyl]salicylic acid, otherwise known as anacardic acid (C_15:1), inhibited the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) with an IC₅₀ of 6.8 μM. The inhibition of the enzyme by anacardic acid (C_15:1) is a slow and reversible reaction without residual activity. The inhibition kinetics analyzed by Dixon plots indicates that anacardic acid (C_15:1) is a competitive inhibitor and the inhibition constant, K₁, was obtained as 2.8 μM. Although anacardic acid (C_15:1) inhibited the linoleic acid peroxidation without being oxidized, 6[8′(Z),11′(Z)-pentadecadienyl]salicylic acid, otherwise known as anacardic acid (C_15:2), was dioxygenated at low concentrations as a substrate. In addition, anacardic acid (C_15:2) was also found to exhibit time-dependent inhibition of lipoxygenase-1. The alk(en)yl side chain of anacardic acids is essential to elicit the inhibitory activity. However, the hydrophobic interaction alone is not enough because cardanol (C_15:1), which possesses the same side chain as anacardic acid (C_15:1), acted neither as a substrate nor as an inhibitor.

Full text of DOI:10.1021/jf048184e

4350 J. Agric. Food Chem. 2005, 53, 4350−4354
Lipoxygenase Inhibitory Activity of Anacardic Acids
TAE JOUNG HA AND ISAO KUBO*
Department of Environmental Science, Policy and Management, University of California,
Berkeley, California 94720-3112
6[8′(Z)-Pentadecenyl]salicylic acid, otherwise known as anacardic acid (C15:1), inhibited the linoleic
acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) with an IC50 of 6.8
µM. The inhibition of the enzyme by anacardic acid (C15:1) is a slow and reversible reaction without
residual activity. The inhibition kinetics analyzed by Dixon plots indicates that anacardic acid (C15:1
)
is a competitive inhibitor and the inhibition constant, KI, was obtained as 2.8 µM. Although anacardic
acid (C15:1) inhibited the linoleic acid peroxidation without being oxidized, 6[8′(Z),11′(Z)-pentadeca-
dienyl]salicylic acid, otherwise known as anacardic acid (C15:2), was dioxygenated at low concentrations
as a substrate. In addition, anacardic acid (C15:2) was also found to exhibit time-dependent inhibition
of lipoxygenase-1. The alk(en)yl side chain of anacardic acids is essential to elicit the inhibitory activity.
However, the hydrophobic interaction alone is not enough because cardanol (C15:1), which possesses
the same side chain as anacardic acid (C15:1), acted neither as a substrate nor as an inhibitor.
KEYWORDS: Lipoxygenase inhibitors; anacardic acids; competitive inhibitor; Dixon plots; time-dependent
inhibition
INTRODUCTION
Lipoxygenases (EC 1.13.11.12) are a family of non-heme
iron-containing dioxygenases widely distributed in both the
animal and the plant kingdoms. They catalyze the regio- and
stereospecific oxygenation of polyunsaturated fatty acids,
containing (Z,Z)-1,4-diene moieties, into the corresponding
conjugated hydroperoxides. In plants, the main substrates are
linoleic (C18:2) and linolenic (C18:3) acids, and the primary
products of lipoxygeneses, 9S and 13S fatty acid hydroperoxides,
are proposed to have regulatory roles in plant and animal
metabolism (1). Due to their free radical nature, fatty acid
hydroperoxides can be quite active by themselves and are
capable of producing membrane damage and promoting cell
death (2).
Lipoxygenases are suggested to be involved in the early event
of atherosclerosis by inducing plasma low-density lipoprotein
(LDL) oxidation (3, 4). On the other hand, it is well known
that lipid peroxidation is one of the major factors in deterioration
during the storage and processing of foods, because it can lead
to the development of unpleasant rancid or off flavors as well
as potentially toxic end products (5). Hence, lipoxygenase
inhibitors should have broad applications (6).
6-Pentadecanylsalicylic acid, otherwise known as anacardic
acid (C15:0) (1) (Figure 1), was previously reported to inhibit
the linoleic acid peroxidation catalyzed by soybean lipoxyge-
nase-1. However, only the IC50 value was reported as 85 µM
(7). This prompted us to examine anacardic acids and their
related compounds for comparison to gain new insights into
Figure 1. Chemical structures of anacardic acids and related compounds.
their inhibitory action on a molecular basis. In our continuing
search for biologically active substances from plants, anacardic
acids were previously isolated in quantities as prostaglandin
synthetase inhibitors from Anacardiaceae plants, Ozoroa mu-
cronata (8), and antibacterial principles from the cashew
Anacardium occidentale apple (9). In the current experiment,
our emphasis was placed on 6[8′(Z)-pentadecenyl]salicylic acid,
otherwise known as anacardic acid (C15:1) (2), because it is
commonly characterized from O. mucronata and A. occidentale,
but anacardic acid (C15:0) is not.
* Author to whom correspondence should be addressed [telephone (510)
643-6303; fax (510) 643-0215; e-mail ikubo@uclink.berkeley.edu].
10.1021/jf048184e CCC: $30.25
© 2005 American Chemical Society
Published on Web 05/10/2005

Lipoxygenase Inhibitory Activity of Anacardic Acids
J. Agric. Food Chem., Vol. 53, No. 11, 2005 4351
MATERIALS AND METHODS
Chemicals. Anacardic acids (C15:0, C15:1, C15:2, C15:3) and cardanol
(C15:1) were available from our previous work (9, 10). Soybean
lipoxygenase-1 (EC 1.13.11.12, type 1), dimethyl sulfoxide (DMSO),
salicylic acid, Tween-20, and linoleic acid were purchased from Sigma
Chemical Co. (St. Louis, MO). Boric acid was obtained from Fisher
Scientific Co. (Fair Lawn, NJ). Ethanol was purchased from Quantum
Chemical Co. (Tuscola, IL). 13-Hydroperoxyoctadecadienoic acid (13-
HPOD: λmax ) 234 nm, ꢀ ) 25 mM-1 cm-1) was prepared enzymati-
cally by the described procedure (11) and stored in ethanol at -18 °C.
Inhibition Experiments on Lipoxygenase-1. These experiments
were performed by measurement of the initial rate of soybean
lipoxygenase-1 with a Spectra MAX plus spectrophotometer (Molecular
device, Sunnyvale, CA) at 25 °C. The enzyme assay was performed as
previously reported (12) with slight modification. In general, the reaction
mixture consisted of 2.97 mL of 0.1 M sodium borate buffer (pH 9.0),
15 µL of 3 mM stock solution of linoleic acid, and 5 µL of an ethanolic
inhibitor solution. Next, 10 µL of 0.1 M sodium borate buffer solution
(pH 9.0) of lipoxygenase (0.52 µM) was added. The resultant solution
was mixed well, and the linear increase of absorbance at 234 nm for 5
min, which express the formation of conjugated diene hydroperoxide
(13-HPOD, ꢀ ) 25 000 M-1 cm-1), was measured continuously. The
lag period shown on lipoxygenase reaction (13) was excluded for the
determination of initial rates. The stock solution of linoleic acid was
prepared with Tween-20 and sodium borate buffer at pH 9.0, and then
the total Tween-20 content in the final assay was adjusted below 0.01%.
For determining reversible inhibition manner, enzyme concentration
was changed as 0.094, 0.141, 0.188, 0.235, and 0.282 µg/mL with a
constant substrate concentration (30 µM). Two concentrations (15 and
30 µM) of linoleic acid were selected for Dixon plots.
Figure 2. Effects of anacardic acid (C15:1) on the activity of soybean
lipoxygenase-1 for the catalysis of linoleic acid at 25
of data as 1/v versus [I].
°C. (Inset) Replots
oxygen into hydroperoxides. In plants, the primary dioxygen-
ation product is 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid
(13-HPOD) (5). The inhibition activity of soybean lipoxyge-
nase-1 was measured by a spectrophotometric and a polaro-
graphic method. In the current experiment, linoleic acid was
used as a substrate. The buffer used for all experiments was
0.1 M sodium borate at pH 9.0 because soybean lipoxygenase-1
is reported to have its optimum at pH 9.0 (16). First, the enzyme
assay was performed using a UV spectrophotometer to detect
the increase at 234 nm associated with the (2Z,4E)-conjugated
double bonds newly formed in the product but not the substrate.
As a result, anacardic acid (C15:1) showed a dose-dependent
inhibitory effect on this oxidation as shown in Figure 2. As
anacardic acid (C15:1) increased, the enzyme activity was rapidly
decreased with completely suppressed. The inhibitor concentra-
tion leading to 50% activity lost (IC50) was estimated to be 6.8
µM. Anacardic acid (C15:0) also showed a dose-dependent
inhibitory effect on this oxidation. The IC50 of anacardic acid
(C15:0) was obtained as 14.3 µM. Second, the bioassay with
anacardic acid (C15:1) and anacardic acid (C15:0) was also
monitored by measuring oxygen consumption (polarographic
method) for comparison. The IC50 obtained was 31.5 and 68.6
µM, respectively.
The linoleic acid peroxidation 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 anacardic acid (C15:1) gave a family
of straight lines, which passed through the origin as shown in
Figure 3. Increasing the inhibitor concentration resulted in the
descending of the slopes of the lines, indicating that the
inhibition of anacardic acid (C15:1) on the enzyme was reversible.
The presence of anacardic acid (C15:1) did not bring down the
amount of the efficient enzyme, but resulted in the inhibition
and the descending of the activity of the enzyme. In addition,
a similar result was also obtained by O2-electrode monitoring
method.
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 43.5
nM and 50 µM, respectively. On the other hand, in the study of Dixon
plots, the final concentration of the enzyme was fixed at 43.5 nM, but
the substrate concentrations selected were 50 and 80 µM. Because a
weak substrate inhibition was induced by the high concentration of
linoleic acid (12, 13), 100 µM substrate was used as the maximum
concentration.
Preincubation Experiments. Most experiments were performed so
that lipoxygenase-1 (1.74 nM) and 2.0-10 µM anacardic acid (C15:2
)
were incubated with 0.1 M sodium borate buffer (pH 9.0) at 25 °C. At
timed intervals, reactions were started by addition of 30 µM linoleic
acid. Control samples were incubated under identical conditions except
for the absence of inhibitor. The increase in the absorbance at 234 nm
was monitored for 5 min. A plot of the natural log of the residual
enzyme activity versus preincubation time gave a straight line with a
slope of -kobs. The values of k3, k4, and Kiapp were calculated from the
plot of the kobs versus concentration of inhibitors according to the
method of Morrison and Walsh (14, 15).
Data Analysis and Curve Fitting. The assay was 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 (15):
activity (%) ) 100[1/(1 + ([I]/IC50))]
Subsequently, the inhibition kinetics of soybean lipoxyge-
nase-1 by anacardic acid (C15:1) was investigated. The kinetic
behavior of soybean lipoxygenase-1 during the linoleic acid
peroxidation was studied first. Under the condition employed
in the present investigation, the oxidation of linoleic acid
catalyzed by soybean lipoxygenase-1 follows Michaelis-
Menten kinetics. The kinetic parameters for this oxidase obtained
from a Dixon plot show that Km is equal to 11.7 µM and Vm is
equal to 4.8 µM/min. The estimated value of Km obtained with
a spectrophotometric method is in good agreement with the
previously reported value (17, 18). The kinetic and inhibition
Inhibition mode was analyzed by Enzyme Kinetics Module 1.0 (SPSS
Inc.) equipped with Sigma Plot 2000. Kinetic parameters for time-
dependence inhibitor were analyzed by using the following equations
(15):
V/V0 ) exp(-kobst)
kobs ) k4(1 + [I]/Kiapp
)
RESULTS
Soybean lipoxygenase-1 catalyzes the conversion of the fatty
acid containing 1(Z),4(Z)-pentadiene moiety and molecular

4352 J. Agric. Food Chem., Vol. 53, No. 11, 2005
Ha and Kubo
Figure 3. Relationship of catalytic activity of soybean lipoxygenase-1 with
the enzyme concentrations at different concentrations of anacardic acid
(C15:1). Concentrations of inhibitor for curves 0−3 were 0, 2, 4, and 6
µ
M, respectively.
Table 1. Kinetics and Inhibition Constants of Anacardic Acid (C15:1
)
detector
UV234
O2
IC50
Km
Vm
inhibition
inhibition type
KI
6.8
11.7
4.8
reversible
competitive
µ
M
31.5
36.5
6.5 µmol/min
reversible
competitive
14.7 µM
µ
µ
M
M
µ
M
µ
mol/min
2.8
µ
M
Figure 4. Dixon plots of 13-HPOD generation (A) and oxygen consumption
constants obtained are listed in Table 1. As illustrated in Figure
4, the inhibition kinetics analyzed by Dixon plots shows that
anacardic acid (C15:1) is a competitive inhibitor because increas-
ing substrates resulted in a family of lines with a common
intercept above the [I] axis but with different slopes. The
equilibrium constant for inhibitor binding, KI, was obtained from
-[I] value at the intersection of two lines. The inhibition kinetics
analyzed by Lineweaver-Burk plots also confirmed that the
anacardic acid (C15:1) is a competitive inhibitor (data not
illustrated). In addition, a similar result was also obtained by
O2-electrode monitoring method, and the results are listed in
Table 1. The estimated value of Km is approximately 3.6-fold
higher than that obtained with a spectrophotometric method.
This is in good agreement with the previously reported
observations (18).
Because anacardic acids are salicylic acid derivatives with a
pentadec(en)yl side chain, their inhibitory activity was also
compared to that of salicylic acid. Salycilic acid (6) did not
inhibit soybean lipoxygenase-1 up to 200 µM, indicating that a
pentadec(en)yl side chain is essential to elicit the activity. On
the other hand, anacardic acids were described to show high
selectivity toward transition metal ions, especially iron and
copper (19). It appears therefore that anacardic acids can be
expected to inhibit the lipoxygenases activity as iron chelators.
If this is so, the UV absorption of anacardic acid (C15:1) should
be shifted by adding excess Fe2+. However, this shift was not
viewed in the case of anacardic acids because the absorption
overlapped with that of the enzyme itself. The iron chelation
mechanism can be indirectly supported by the observation that
cardanol (C15:1), 3[8(Z)-pentadecatrienyl]phenol (5), an artifact
of the corresponding anacardic acid (C15:1) (2) by heating
treatment, did not show any inhibitory activity up to 3 mM.
On the other hand, we became aware that anacardic acid (C15:
3) (4) and anacardic acid (C15:2) (3) were oxidized as substrates
at lower concentrations (<40 µM), because both possess a
(B) by soybean lipoxygenase-1 in the presence of anacardic acid (C15:1
in borate buffer (pH 9.0) at 25 C. (A) Concentrations of substrates for
curves 0 and 1 were 15 and 30 M, respectively. (B) Concentrations of
substrates for curves 0 and 1 were 50 and 80 M, respectively.
)
°
µ
µ
Table 2. Kinetic Parameters for Time-Dependent Inhibition of
Lipoxygenase-1 by Anacardic Acid (C15:2
)
-
-
1
-1
compound
k3
(
µ
M
1 min
)
k
4 (min
)
Kiapp
3.69
(
µM)
0.11
3
0.00504
±
0.0002
0.0186 0.0012
±
±
1(Z),4(Z)-pentadiene system in their C15-alkenyl side chain.
However, both exhibited inhibitory activity at higher concentra-
tions (>40 µM). Hence, the inhibition kinetics of anacardic acid
(C15:2) was also studied in detail for comparison (Table 2).
Preincubation of the enzyme with anacardic acid (C15:2) but
without linoleic acid was tested. Figure 5 shows a plot of the
anacardic acid (C15:2) concentration-dependent initial rates of
the enzyme-catalyzed reaction. Note that as the concentration
of anacardic acid (C15:2) increases up to the 40 mM, the initial
rates of the enzyme-catalyzed reaction first increase, and then
start decreasing at still higher concentrations of anacardic acid
(C15:2).
As far as the present cell-free experiment using soybean
lipoxygenase-1 is concerned, the inhibition kinetics observed
is not exceeding 5 min. However, from a practical point of view,
much longer observation is needed. The time course of oxidation
of linoleic acid catalyzed by soybean lipoxygenase-1 in the
presence of different anacardic acid (C15:1) and anacardic acid
(C15:2) concentrations is also shown in Figure 6. As anacardic
acid (C15:1) increased, the rate of formation of 13-HPOD is
slowly decreased, whereas anacardic acid (C15:2) showed the
typical progress curves of slow binding behavior. These slow
binding inhibition mechanisms can be investigated by preincu-

Lipoxygenase Inhibitory Activity of Anacardic Acids
J. Agric. Food Chem., Vol. 53, No. 11, 2005 4353
Figure 7. Time course of the inactivation of soybean lipoxygenase-1 by
Figure 5. Concentration dependence of the initial rate of the oxidation of
anacardic acid (C15:2). Concentrations of anacardic acid (C15:2) for curves
anacardic acid (C15:2) at 25 °C, 0.1 M borate buffer, pH 9.0.
0−5 were as follows: 0, 2, 4, 6, 8, and 10 µM. (Inset) Dependence of
the values for kobs on the concentration of anacardic acid (C15:2).
loss of enzyme activity occurring in 20 min at 10 mM. The kobs
values for the anacardic acid (C15:2) inhibition of lipoxygenase-1
at different concentrations of anacardic acid (C15:2) were
determined by fitting data to the slow-binding equation (eq 1).
The kobs values were plotted as a function of anacardic acid
(C15:2) concentration. The results indicated that anacardic acid
(C15:2) inhibits soybean lipoxygenase-1 by simple reversible slow
binding. This was evidenced by the observation that the kobs
values exhibited a liner dependence on the inhibitor concentra-
tion as shown in Figure 7 inset. Thus, analysis of data according
to eqs 1 and 2 yielded the following values: k3 ) 0.00504 (
0.0002 mM-1 min-1, k4 ) 0.0186 ( 0.0012 min-1, Kiapp
3.69 ( 0.11 mM.
)
DISCUSSION
The human body produces free radicals during the course of
its normal metabolism. Free radicals are even required for
several normal biochemical processes. For example, the ph-
agocyte cells involved in the body’s natural immune defenses
generate free radicals in the process of destroying microbial
pathogens. If free radicals are produced during the normal
cellular metabolism in sufficient amounts to overcome the
normally efficient protective mechanisms, metabolic and cellular
disturbances will occur in a variety of ways. Evidence is
accumulating that several cell types other than phagocytes also
produce extracellular free radicals in vivo. For example, lipids
are oxidized by lipoxygenases and cyclooxygenases, which
generate peroxide intermediates. More specifically, lipoxyge-
nases catalyze the oxygenation of polyenoic fatty acids contain-
ing a 1(Z),4(Z)-pentadiene system such as linoleic acid and
arachidonic acid to their 1-hydroperoxy-2(E),4(Z)-pentadiene
product. In this connection, lipoxygenases are of importance
because they may generate peroxides in human low-density
lipoproteins (LDL) in vivo and facilitate the development of
atherosclerosis, a process in which lipid peroxidation appears
to be intimately involved (3, 4).
Figure 6. Time-dependent inhibition of soybean lipoxygenase-1 in the
presence of anacardic acid (C15:1, A) and anacardic acid (C15:2, B). (A)
Concentrations of inhibitor for curves 0
µ
−
6 were 0, 2, 4, 8, 10, 20, and 40
M. (B) Concentrations of inhibitor for curves 0 5 were 0, 40, 60, 80,
M. Conditions were as follows: 20 M linoleic acid, 0.1
C.
−
100, and 300
µ
µ
M borate buffer, pH 9.0 at 25
°
bation of enzyme with inhibitor followed by the measurement
of initial velocities for substrate peroxidation as a function of
preincubation time. In this case, the inhibition can be described
by eq 1,
Anacardic acids are known to inhibit various enzymes such
as urease (20), glycerol-3-phosphate dehydrogenase (21), li-
poxygenases (7), â-lactamase (22, 23), aldose reductase (24),
prostaglandin endoperoxide synthase (25), and prostaglandin
synthase (8, 26). Because salicylic acid has little or no effects
on these enzymes, the hydrophobic alkyl side chain in anacardic
acids is undoubtedly associated with the enzyme inhibitory
activity. In addition, the number of double bonds in the side
chain is not directly associated with the enzyme inhibitory
V/V0 ) exp(-kobst)
(1)
(2)
kobs ) k4[I]/Kiapp + k4
In the concentration range of 2.0-10.0 mM, loss of the
enzyme activity followed a simple exponential as shown in
Figure 7. Anacardic acid (C15:2) exerted a potent and slow
inactivation of soybean lipoxygenase-1, with more than 70%

4354 J. Agric. Food Chem., Vol. 53, No. 11, 2005
Ha and Kubo
(8) Kubo, I.; Kim, M.; Naya, K.; Komatsu, S.; Yamagiwa, Y.;
Ohashi, K.; Sakamoto, Y.; Hirakawa, S.; Kamikawa, T. Pros-
taglandin synthetase inhibitors from the African medicinal plant
Ozoroa mucronata. Chem. Lett. 1987, 6, 1101-1104.
(9) Kubo, I.; Komatsu, S.; Ochi, M. Molluscicides from the cashew
Anacardium occidentale and their large-scale isolation. J. Agric.
Food Chem. 1986, 34, 970-973.
(10) Kubo, I.; Kinst-Hori, I.; Yokokawa, Y. Tyrosinase inhibitors from
Anacardium occidentale fruits. J. Nat. Prod. 1994, 57, 545-
551.
(11) Gibian, M. J.; Galaway, R. A. Steady-state kinetics lipoxygenase
oxygenation of unsaturated fatty acid. Biochemistry 1976, 15,
4209-4214.
(12) Rickert, K. W.; Klinman, J. P. Nature of hydrogen transfer in
soybean lipoxygenase 1: separation of primary and secondary
isotope effects. Biochemistry 1999, 38, 12218-12228.
(13) Ruddat, V. C.; Whitman, S.; Holman, T. R.; Bernasconi, C. F.
Stopped-flow kinetics investigations of the activation of soybean
lipoxygenase-1 and the influence of inhibitors on the allosteric
site. Biochemistry 2003, 42, 4172-4178.
(14) Morrison, J. F.; Walsh, C. T. The behavior and significance of
slow-binding enzyme inhibitors. AdV. Enzymol. 1988, 61, 201-
301.
(15) Copeland, R. A. Enzyme: A practical introduction to structure,
mechanism, and data analysis; Wiley-VCH: New York, 2000;
pp 266-332.
(16) Axelrod, B.; Cheesbrough, T. M.; Laakso, S. Lipoxygenase from
soybeans. Methods Enzymol. 1981, 71, 441-457.
(17) Schilstra, M. J.; Veldink, G. A.; Verhagen, J.; Vliegenthart, J.
F. G. Effect of lipid hydroperoxide on lipoxygenase kinetics.
Biochemistry 1992, 31, 7692-7699.
(18) Berry, H.; Debat, H.; Larreta-Garde, V. Excess substrate inhibi-
tion of soybean lipoxygenase-1 is mainly oxygen-dependent.
FEBS Lett. 1997, 408, 324-326.
activity (7, 10), suggesting that interaction of the double bond
with a specific amino acid residue in the enzymes is unlikely.
Anacardic acids are amphipathic molecules, so that their
hydrophobic properties dominate the properties of the molecule.
The precise explanation still remains unclear, but it seems that
these enzymes commonly possess a relatively nonspecific and
hydrophobic domain. The hydrophobic pentadec(en)yl side chain
in anacardic acids likely interacts with this hydrophobic domain
and disrupts enzymes’ quaternary structure (27), because native
proteins form a sort of intramolecular micelle in which their
nonpolar side chains are largely out of contact with the water-
based test solution. The hydrophilic head part of anacardic acids
first binds with the salicylic acid portion like a “hook” in
attaching itself to the hydrophilic portion of the molecule, and
then the hydrophobic tail portion begins interacting with the
hydrophobic domain. This concept can be more broadly
conceivable because the hydrophobic alkyl side chain should
allow for conformational flexibility and interacts with many
types of the hydrophobic domains in different enzymes. Anac-
ardic acids’ nonspecificity of enzyme inhibition supports this
assumption.
In summary, safety is a primary consideration for antioxidants
in food products, which may be utilized in unregulated quanti-
ties. That anacardic acids are found in many edible plants that
have been consumed for many years should be a considerable
advantage. Metal chelation capacity of anacardic acids is their
additional advantage because it reduces the concentration of the
catalyzing transition metal in lipid peroxidation. It is known
that chelating agents, which form bonds with a metal, are
effective as secondary antioxidants because they reduce the
redox potential, thereby stabilizing the oxidized form of the
metal ion. Despite the adventage of the isolation from regularly
consumed edible plants, biological significance of anacardic acid
as lipoxygenase inhibitors in living systems is still largely
unknown. Thus, it is not clear if ingested anacardic acids are
absorbed into the system through the intestinal tract and
delivered to the places where lipoxygenase inhibitors are needed.
The relevance of the in vitro experiments in simplified systems
to in vivo protection from oxidative damage should be carefully
considered. Their further evaluation is needed from not only
one aspect, but from a whole and dynamic perspective.
(19) Nagabhushana, K. S.; Shobha, S. V.; Ravindranath, B. Selective
ionophoric properties of anacardic acid. J. Nat. Prod. 1995, 58,
807-810.
(20) Kubo, J.; Lee, J. R.; Kubo, I. Anti-Helicobacter pylori agents
from the cashew apple. J. Agric. Food Chem. 1999, 47, 533-
537.
(21) Irie, J.; Murata, M.; Homma, S. Glycerol-3-phosphate dehydro-
genase inhibitors, anacardic acids, from Ginkgo biloba. Biosci.
Biotechnol. Biochem. 1996, 60, 240-243.
(22) Coates, N. J.; Gilpin, M. L.; Gwynn, M. N.; Lewis, D. E.; Milner,
P. H.; Spear, S. R.; Tyler, J. W. SB-202742, a novel â-lactamase
inhibitor isolated from Spondias mombin. J. Nat. Prod. 1994,
57, 654-657.
(23) Hird, N. W.; Milner, P. H. Synthesis and â-lactamase inhibition
of anacardic acids and their analogues. Bioorg. Med. Chem. Lett.
1994, 4, 1423-1428.
LITERATURE CITED
(24) Toyomizu, M.; Sugiyama, S.; Jin, R. L.; Nakatsu, T. R-Glu-
cosidase and aldose reductase inhibitors: constituents of cashew,
Anacardium occidentale, nut shell liquids. Phytother. Res. 1993,
7, 252-254.
(25) Grazzini, R.; Hesk, D.; Heininger, E.; Hildenbrandt, G.; Reddy,
C. C.; Cox-Foster, D.; Medford, J.; Craig, R.; Mumma, R. O.
Inhibition of lipoxygenase and prostaglandin endoperoxide
synthase by anacardic acids. Biochem. Biophys. Res. Commun.
1991, 176, 775-780.
(26) Bhattacharya, S. K.; Mukhopadhyay, M.; Rao, Patchala, J. R.
M.; Bagchi, A.; Ray, A. B. Pharmacological investigation on
sodium salt and acetyl derivative of anacardic acid. Phytother.
Res. 1987, 1, 127-131.
(27) Hasinoff, B. B.; Davey, J. P. The inhibition of a membrane-
bound enzyme as a model for anaesthetic action and drug
toxicity. Biochem. J. 1989, 258, 101-107.
(1) Gardner, H. W. Biological roles and biochemistry of the
lipoxygenase pathway. HortScience 1995, 30, 197-205.
(2) Gardner, H. W. Recent investigations into the lipoxygenase
pathway of plants. Biochim. Biophys. Acta 1991, 1084, 221-
239.
(3) Cornicelli, J. A.; Trivedi, B. K. 15-Lipoxygenase and its
inhibition: a novel therapeutic target for vascular disease. Curr.
Pharm. Des. 1999, 5, 11-20.
(4) Kris-Etherton, P. M.; Keen, C. L. Evidence that the antioxidant
flavonoids in tea and cocoa are beneficial for cardiovascular
health. Curr. Opin. Lipidol. 2002, 13, 41-49.
(5) Grechkin, A. Recent developments in biochemistry of the plant
lipoxygenase pathway. Prog. Lipid Res. 1998, 37, 317-352.
(6) Richard-Forget, F.; Gauillard, F.; Hugues, M.; Jean-Marc, T.;
Boivin, P.; Nicolas, J. Inhibition of horse bean and germinated
barley lipoxygenases by some phenolic compounds. J. Food Sci.
1995, 60, 1325-1329.
Received for review November 1, 2004. Revised manuscript received
February 7, 2005. Accepted March 4, 2005.
(7) Shobha, S. V.; Ramadoss, C. S.; Ravindranath, B. Inhibition of
soybean lipoxygenase-1 by anacardic acids, cardols, and car-
danols. J. Nat. Prod. 1994, 57, 1755-1757.
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