Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase

Article abstract of DOI:10.1016/j.foodchem.2014.06.021

5-Pentadecatrienylresorcinol, isolated from cashew nuts and commonly known as cardol (C_15:3), prevented the generation of superoxide radicals catalysed by xanthine oxidase without the inhibition of uric acid formation. The inhibition kinetics did not follow the Michelis-Menten equation, but instead followed the Hill equation. Cardol (C_10:0) also inhibited superoxide anion generation, but resorcinol and cardol (C_5:0) did not inhibit superoxide anion generation. The related compounds 3,5-dihydroxyphenyl alkanoates and alkyl 2,4-dihydroxybenzoates, had more than a C9 chain, cooperatively inhibited but alkyl 3,5-dihydroxybenzoates, regardless of their alkyl chain length, did not inhibit the superoxide anion generation. These results suggested that specific inhibitors for superoxide anion generation catalysed by xanthine oxidase consisted of an electron-rich resorcinol group and an alkyl chain having longer than C9 chain.

Full text of DOI:10.1016/j.foodchem.2014.06.021

Food Chemistry 166 (2015) 270–274
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Inhibitory effects of cardols and related compounds on superoxide anion
generation by xanthine oxidase
b
a
c
b
Noriyoshi Masuoka a, , Ken-ichi Nihei , Ayami Maeta , Yoshiro Yamagiwa , Isao Kubo
⇑
a Department of Life Science, Okayama University of Science, 1-1 Ridai-Cho, Okayama 700-0005, Japan
b Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3114, USA
c Department of Chemistry, Kinki University, Osaka 577-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 April 2013
Received in revised form 10 April 2014
Accepted 5 June 2014
Available online 13 June 2014
5-Pentadecatrienylresorcinol, isolated from cashew nuts and commonly known as cardol (C15:3), pre-
vented the generation of superoxide radicals catalysed by xanthine oxidase without the inhibition of uric
acid formation. The inhibition kinetics did not follow the Michelis–Menten equation, but instead fol-
lowed the Hill equation. Cardol (C10:0) also inhibited superoxide anion generation, but resorcinol and car-
dol (C5:0) did not inhibit superoxide anion generation. The related compounds 3,5-dihydroxyphenyl
alkanoates and alkyl 2,4-dihydroxybenzoates, had more than a C9 chain, cooperatively inhibited but alkyl
3,5-dihydroxybenzoates, regardless of their alkyl chain length, did not inhibit the superoxide anion gen-
eration. These results suggested that specific inhibitors for superoxide anion generation catalysed by xan-
thine oxidase consisted of an electron-rich resorcinol group and an alkyl chain having longer than C9
chain.
Keywords:
Cardol
Inhibition of superoxide anion generation
Xanthine oxidase
Antioxidants
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Matsuda, & Kubo, 2012). Finally it has become evident that the
reaction of xanthine oxidase with inhibitors consisted of inhibition
Xanthine oxidase (EC 1.1.3.22),
a
molybdenum-containing
of uric acid formation, reduction reactions of the xanthine oxidase
molecule and radical scavenging reactions. The active sites of uric
acid formation catalysed by xanthine oxidase and the superoxide
anion generation are different. That is, an inhibitor which binds
the xanthine binding site in xanthine oxidase inhibits the uric acid
formation by xanthine oxidase. An inhibitor of superoxide anion
generation binds to other sites, and this binding leads to the inhi-
bition of superoxide anion generation or reduction of the enzyme
molecules to catalyse hydrogen peroxide formation. Therefore, as
xanthine oxidase inhibitors are able to multifunction, it is neces-
sary to distinguish each function and to find specific inhibitor.
Anacardic acids (C15:3) (1) in Fig. 1, isolated from the cashew
Anacardium occidentale (Anacardiaceae) (Kubo, Komatsu, & Ochi,
1986), had no DPPH activity and cooperatively bound to xanthine
binding site to inhibit both uric acid formation and superoxide
anion generation (Masuoka & Kubo, 2004). As cardol (C15:3) (2),
5-[80(Z),110(Z),140-pentadecatrienyl]resorcinol, isolated from the
cashew, was not an inhibitor of uric acid formation but did inhibit
the generation of the superoxide anion, we suggested that cardol
was a specific inhibitor for superoxide anion generation catalysed
by xanthine oxidase (Masuoka et al., 2012). However, it is still
unclear about the specific character of cardols. In the current study,
prepared cardol (C10:0) (3) and (C5:0) (4) and related compounds
(7–15) were examined and their effects on the xanthine oxidase
enzyme, catalyses the oxidation of hypoxanthine to xanthine and
ultimately to uric acid. The accumulation of uric acid leads to
hyperuricaemia and gout (Hatano et al., 1990; Nakanishi et al.,
1990), therefore xanthine oxidase inhibitors may serve as thera-
peutic agents for these conditions. Xanthine oxidase also generates
a superoxide anion, and excess superoxide anion generation leads
to peroxidative damage in cells (Fong, McCay, Poyer, Keele, &
Misra, 1973; McCord, 1985). Intake of xanthine oxidase inhibitors
from foods may be useful to prevent postischemic injury. In our
continuing investigation to understand the functions of xanthine
oxidase inhibitors, the balance of hydrophilic and hydrophobic
moieties of molecules is associated with the inhibitory activity
(Masuoka & Kubo, 2004; Masuoka, Nihei, & Kubo, 2006; Masuoka
et al., 2012). 1, 1-Diphenyl-2-p-picryhydrazyl (DPPH) scavenging
activity of antioxidants is attributed to their conjugated endiol
structures, and the activity indicates a reduction activity. Flavo-
noids are essentially competitive inhibitors for uric acid formation
catalysed by xanthine oxidase. Some flavonoids, which have DPPH
scavenging activity, are able to strongly reduce xanthine oxidase
molecules to suppress superoxide anion generation (Masuoka,
⇑
Corresponding author. Tel./fax: +81 86 256 9593.
E-mail address: masuokan@dls.ous.ac.jp (N. Masuoka).
http://dx.doi.org/10.1016/j.foodchem.2014.06.021
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

N. Masuoka et al. / Food Chemistry 166 (2015) 270–274
271
2.2. Preparation of sample solution
Compounds were dissolved in dimethyl sulfoxide (DMSO), to a
concentration of 10 mM, which was used for the experiments.
2.3. Assay of uric acid generated by xanthine oxidase
The xanthine oxidase (EC 1.1.3.22, Grade IV) used for the bioas-
say was purchased from Sigma Chemical Co. 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
the sample solution (dissolved in DMSO). The reaction was started
by the addition of 0.12 ml of xanthine oxidase (0.04 units), and the
absorbance at 293 nm was recorded for 60 s. A control experiment
carried out by replacing the sample solution with the same amount
of DMSO. The reaction rate was calculated from the proportional
increase in absorbance.
2.4. Assay of superoxide anion generated by xanthine oxidase
In the reaction, the superoxide anion generated by the enzyme
reduces nitroblue tetrazolium to a blue formazan. The absorbance
of the formazan produced was determined at 560 nm. The reaction
mixture consisted of 2.7 ml of 40 mM sodium carbonate buffer
containing 0.1 mM EDTA (pH 10.0), 0.06 ml of 10 mM xanthine
and 0.03 ml of 0.5% bovine serum albumin, 0.03 ml of 2.5 mM
nitroblue tetrazolium and 0.06 ml of the sample solution (dis-
solved in DMSO). To the mixture at 25 °C, 0.12 ml of xanthine oxi-
dase (0.04 units) was added, and the absorbance at 560 nm was
recorded for 60 s (by formation of blue formazan) (Masuoka &
Kubo, 2004). A control experiment was carried out by replacing
the sample solution with the same amount of DMSO.
Fig. 1. Cardols and related compounds.
2.5. Radical scavenging activity on DPPH
reaction to study the function of the hydrophobic head portion and
hydrophilic tail portion of cardols.
First, 1 ml of 100 mM acetate buffer (pH 5.5), 1.87 ml of ethanol
and 0.1 ml of ethanolic solution of 3 mM DPPH were put into a test
tube. Then, 0.03 ml of the sample solution (dissolved in DMSO) was
added to the tube and incubated at 25 °C for 20 min. The absor-
2. Materials and methods
bance at 517 nm (DPPH,
e
= 8.32 ꢀ 103) was recorded. As a control,
2.1. Chemicals
0.03 ml of DMSO was added to the tube. The scavenging activity
was calculated from the decrease in absorbance and expressed as
the number of scavenged DPPH molecules per each sample
molecule.
Cardol (C15:3) (2), anacardic acid (C15:3) (1) and cardanol (C15:3
)
(6) used for the assay were previously isolated from the cashew
nut shell oil and were re-purified by recycle HPLC (R-HPLC) using
an ODS C18 column. Cardols (3, 4) possessing different alkyl side
chains were synthesized from 3,5-dimethoxybenzaldehyde via a
Wittig reaction. Xanthine oxidase, DPPH, EDTA and resorcinol were
purchased from Sigma Chemical Co. (St. Louis, MO) and Aldrich
Chemical Co. (Milwaukee, WI). Cardol related derivatives, such as
alkyl 3,5-dihydroxybenzoates (7–9), alkyl 2, 4-dihydroxybenzoates
(10–12) and 3,5-dihydroxyphenyl alkanoates (13–15), were syn-
thesized as follows. First, hexyl, nonyl and dodecyl 3,5-
dihydroxybenzoates (7–9) were synthesized by a two-step proce-
dure. The corresponding dibenzyloxybenzoic acids were obtained
first by a method previously reported (Nihei, Nihei, & Kubo,
2003). Then, alkyl 3,5-dihydroxybenzoates, were obtained in high
yield by a two step procedure, a Mitsunobu reaction the first step,
followed by hydrogenation to remove the protecting group. Sec-
ond, the same alkyl (hexyl, nonyl and dodecyl) 2,4-
dihydroxybenzoates (10–12) were synthesized in a similar manner
using 2,4-dibenzyloxybezoic acid as a starting material. Third, 3,5-
dihydroxyphenyl (heptanoate, decanoate and tridecanoate) alk-
anoates (13–15) were readily prepared in one step from phloroglu-
cinol and the corresponding carboxylic acid using DCC as a
coupling reagent.
2.6. Radical scavenging activity for the Oꢁ2 generated
by the PMS-NADH system
The superoxide anion was generated nonenzymatically with a
PMS-NADH system. The reaction mixture (final volume was
3.0 ml) containing 25 lM NBT, 150 lg of BSA, 78 lM NADH and
0.06 ml of sample solution in 40 mM sodium carbonate buffer con-
taining 0.1 mM EDTA (pH 10.0) was prepared and incubated at
25 °C for 3 min. Then, 0.03 ml of 155 lM PMS was added to start
the reaction and the absorbance at 560 nm was recorded for 60 s
(Nishikimi, Rao, & Yagi, 1972). As the control, 0.06 ml of DMSO
was used. The reaction rate was calculated from the proportional
increase of absorbance, and the scavenging activity of the sample
was expressed as the inhibition percentage.
2.7. Assay and data analysis
Each assay was performed at least in triplicate in separate
experiments, and the analysis was performed using Sigma plot
2001 (SPSS Inc., Chicago, IL). The inhibition mode and kinetic
parameters were analysed with Enzyme Kinetics Module 1.1 (SPSS

272
N. Masuoka et al. / Food Chemistry 166 (2015) 270–274
Inc.) equipped with Sigma Plot 2001. When the sigmoidal inhibi-
tion was observed with inhibitors, the inhibition rates (ivi = vi = 0
ꢁ
vi) were calculated and analysed using the Hill equation:
ivi = vi = 0[I]n/(Ki + [I]n); where the inhibition rates are indicated as
ivi and calculated difference between enzymatic reaction rate at
no inhibitor (vi = 0) and the rates (vi) at various concentrations of
inhibitor.ivi = vi = 0[I]n/(Ki + [I]n); [I] indicates concentrations of the
inhibitor; n is a slope factor of the sigmoidal curve (ivi); vi = 0 is
enzymatic reaction rate with no inhibitor and Ki is a constant.
When Ki is equal to [I]n, ivi becomes vi = 0/2. As a result, Ki indicates
IC50
.
3. Results
3.1. Inhibitory activity of uric acid formation
Fig. 2. Lineweaver–Burk plots of uric acid formation rates from xanthine after
xanthine oxidase was preincubated with cardol (C15:3) (2) for 3 min. Each cardol
concentration was as follows: d: 0 lM, s: 50 lM, .: 100 lM, : 200 lM.
Cardols and resorcinol (2–5) did not inhibit the uric acid forma-
tion catalysed by xanthine oxidase (Table 1). However, after 3 min
of pre-incubation, cardol (C15:3) inhibited uric acid formation,
which followed the Michaelis–Menten equation. The inhibition
kinetics were analysed using Lineweaver–Burk plots (Fig. 2) and
indicated that cardol (C15:3) was a non-competitive and weak
inhibitor of uric acid formation. Most of the other resorcinol
compounds did not inhibit the formation of uric acid catalysed
by xanthine oxidase. Only dodecyl 2, 4-dihydroxybenzoate (12)
non-competitively inhibited the formation.
3.2. Inhibitory activity of superoxide anion generation
Cardols (C15:3) (2) and (C10:0) (3) suppressed superoxide anion
generation but cardol (C5:0) (4) and resorcinol (5) did not. The
results obtained for cardol (C15:3) (2) are shown in Fig. 3. The inhi-
bition was not detected at concentrations less than 80
lM, and a
strong inhibition was observed at greater than 150 M. It should
l
be noted that the inhibition of superoxide anion generation by xan-
thine oxidase did not follow Michaelis–Menten equation, but fol-
Fig. 3. Effects of cardol (C15:3) (2) and resorcinol (5) on the generation of superoxide
anion by xanthine oxidase. Closed circle (50 M xanthine), opened circle (200
xanthine) and closed triangle (500 M xanthine) indicated inhibition of superoxide
l
lM
l
Table 1
anion generation by cardol (C15:3). Open triangle indicated the inhibition by
Inhibition of uric acid formation by cardols and related compounds.
resorcinol.
Compounds tested
(lM)
IC50
KI
KIS
lowed the Hill equation (sigmoidal, n = 5.2 0.2). The IC50 of
superoxide anion generation by cardol (2) was 115 10 lM. The
Cardol (C15:3) (2)
Cardol (C10:0) (3)
370 30a
No
370 30a 370 30a
–
–
–
–
–
–
inhibition curve for cardol was not affected by the xanthine con-
centration (Fig. 3). In brief, it was suggested that the antioxidant
activity of the cardols was not due to radical scavenging by the
superoxide scavenging activity but inhibition of superoxide anion
generation by xanthine oxidase. Interestingly, resorcinol and
cardol (C5:0) (4) did not inhibit superoxide anion generation at
inhibitionb
No
Cardol (C5:0) (4)
Resorcinol (5)
inhibitionc
No
inhibitiond
162 10e
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
Anacardic acid (C15:3) (1)
Salicylic acid
Cardanol (C15:3) (6)
Hexyl 3,5-dihydroxybenzoate (7)
Nonyl 3,5-dihydroxybenzoate (8)
Dodecyl 3,5-dihydroxybenzoate (9)
Hexyl 2,4-dihydroxybenzoate (10)
Nonyl 2,4-dihydroxybenzoate (11)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
concentrations up to 200 lM, and it was suggested that the 5-alkyl
side chain, more than C9, played an important role in eliciting
the activity. It was found that cardols (C15:3 & C10:0) (2, 3) inhib-
ited superoxide anion generation by binding cooperatively to
the enzyme. To compare inhibition by cardols with other com-
pounds having a resorcinol moiety, alkyl dihydroxybenzoates
and dihydroxyphenyl alkanoates were examined. Nonyl 2,4-
dihydroxybenzoate (11), dodecyl 2,4-dihydroxybenzoate (12),
3,5-dihydroxyphenyl decanoate (14) and 3,5-dihydroxyphenyl
tridecanoate (15) sigmoidally inhibited superoxide anion genera-
tion (Table 2). All these compounds have an alkyl group (more than
C9), and the inhibition followed the Hill equation. However, the
alkyl chain alone was not enough to elicit the inhibitory activity
since cardanol (6) and alkyl (>C9) 3, 5-dihydroxybenzoate (8, 9)
did not exhibit noticeable inhibitory activity.
Dodecyl 2,4-dihydroxybenzoate (12) 189 17
189 17
–
189 17
–
3,5-Dihydroxyphenylheptanoate
(13)
No inhibition
3,5-Dihydroxyphenyldecanoate (14) No inhibition
–
–
–
–
3,5-Dihydroxyphenyltridecanoate
(15)
No inhibition
a
b
c
Preincubation for 3 min.
Preincubation for 3 min. 23.4 3.2 % inhibition at 200
Preincubation for 3 min. 9.1 0.9 % Inhibition at 200
lM.
lM.
d
e
1.0 0.1 % inhibition at 200
lM.
Inhibition type was sigmoidal inhibition, and Hill constant was 1.7 0.2.

N. Masuoka et al. / Food Chemistry 166 (2015) 270–274
273
Table 2
Inhibition of superoxide anion generation by cardols and related compounds.
Compounds tested
IC50
115 10
106
(
l
M)
Hill constant
Inhibition type
Cardol (C15:3) (2)
Cardol (C10:0) (3)
Cardol (C5:0) (4)
Resorcinol (5)
Anacardic acid (C10:3) (1)
Salicylic acid
Cardanol (C15:3) (6)
Hexyl 3,5-dihydroxybenzoate (7)
Nonyl 3,5-dihydroxybenzoate (8)
Dodecyl 3,5-dihydroxybenzoate (9)
Hexyl 2,4-dihydroxybenzoate (10)
Nonyl 2,4-dihydroxybenzoate (11)
Dodecyl 2,4-dihydroxybenzoate (12)
3,5-Dihydroxyphenylheptanoate (13)
3,5-Dihydroxyphenyldecanoate (14)
3,5-Dihydroxyphenyltridecanoate (15)
5.2 0.2
5.1 0.2
–
–
4.2 0.5
–
–
–
Sigmoidal
Sigmoidal
–
–
Sigmoidal
–
–
–
8
No inhibitiona
No inhibition
51.3 1.5
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
–
–
–
–
–
–
118
1
3.7 0.1
10.3 3.6
–
1.9 0.4
5.5 1.8
Sigmoidal
Sigmoidal
–
Sigmoidal
Sigmoidal
128 35
No inhibition
121
4
75.3 5.0
a
18.5 1.3 % inhibition at 400 lM.
3.3. DPPH scavenging activities
inhibitors can be designed by selecting specific head portions first.
Once the head portion is selected, the inhibitory activity can be
maximised by the selection of the appropriate tail portions. Based
on this concept, alkyl gallates were found to be potent xanthine
oxidase inhibitors (Masuoka et al., 2006). Hence, to examine cardol
(C15:3) (2) having a resorcinol as the head portion, 5-alkylresorci-
nols (3, 4), alkyl 3,5-dihydroxybenzoates (7–9), alkyl 2,4-
dihydroxybenzoates (10–12) and 3,5-dihydroxyphenyl alkanoates
(13–15) were selected and synthesized.
The DPPH scavenging activities of cardols (1–3), resorcinol (4),
anacardic acid (5), cardanol (6), salicylic acid, alkyl 3,5-
dihydroxybenzoates (7–9), alkyl 2,4-dihydroxybenzoates (10–12)
and 3,5-dihydroxyphenyl alkanoates (13–15) were examined at
least in triplicate. The scavenging activity from all these com-
pounds was less than 0.03 molecules of DPPH/one molecule of each
compound.
In order to examine the ability of the xanthine binding site in
the enzyme, formation of uric acid was measured because xanthine
oxidase, a molybdenum-containing enzyme, is known to convert
xanthine to uric acid. This enzyme-catalysed reaction proceeds
via transfer of an oxygen atom to xanthine from the molybdenum
centre. Cardols (2–4) did not inhibit this oxygen-atom-transfer
3.4. Scavenging activity of superoxide anion generated
with a PMS-NADH system
The rate of generation of the superoxide anion by the addition
of cardols (2–4) and resorcinol (5) were diminished dose-depen-
dently. The scavenging activity of resorcinol and cardol (C5:0) (4)
reaction up to 400 lM. Dodecyl 2, 4-dihydroxybenzoate (12)
were 10 4 % and 13 3 % at 200
scavenging activity (C10:0) (3) and (C15:3) (2) were 73 5 % and
75 7 % at 200 M, respectively. These scavenging activities were
l
M, respectively. The cardols
weakly inhibited uric acid formation catalysed by xanthine oxidase
but other synthesized compounds showed no inhibition up to
200 lM (Table 1). To further examine the binding ability of xan-
l
low compared to the inhibitory rates of superoxide anion gener-
ated by xanthine oxidase (Table 2).
thine oxidase, cardols (2–4) were preincubated with the enzyme
for 3 min. The weak inhibition by cardol (2) was observed and non-
competitive for xanthine (Fig. 2). These results indicated that most
compounds which have resorcinol as the head portion did not bind
to the xanthine binding site in the enzyme.
4. Discussion
Compounds (1–15) have no DPPH scavenging activity, which is
associated with a reduction of the enzyme molecules. It indicated
that these compounds did not affect the generation of the superox-
ide anion by enzyme modulation (Masuoka et al., 2012). Superox-
ide anion scavenging rates of cardol related compounds (2, 3) were
low compared to the inhibitory rates of the superoxide anion gen-
erated by xanthine oxidase (Table 2). This indicated that these
cardols inhibited superoxide anion generation catalysed by xan-
thine oxidase. 3,5-Dihydroxyphenyl decanoate (14), 3,5-dihy-
droxyphenyl tridecanoate (15), nonyl 2,4-dihydroxybenzoate (11)
and dodecyl 2,4-dihydroxybenzoate (12) also inhibited superoxide
generation (Table 2). The inhibition type was sigmoidal and non-
competitive for xanthine in all cases. As X-ray crystallographic
analysis of bovine xanthine oxidase indicates that the substrate,
NAD+, of xanthine dehydrogenase is partially blocked to access of
the FAD binding site (Enroth et al., 2000), it is deduced that these
inhibitors are also blocked to bind the FAD site. For the first bind-
ing to the enzyme, a alkyl chain longer than C9 was necessary.
When the inhibitors first bind to the sites in the xanthine oxidase
subunits, the enzyme may cause conformational changes of the
solvent channel in the enzyme (Kuwabara et al., 2003) to lower
the flow rate and to enhance hydrogen peroxide formation. Cardols
(2, 3), 3,5-dihydroxyphenyl decanoate (14), 3,5-dihydroxyphenyl
Cardol (2) is isolated from many edible plants, such as cashew
(A. occidentale) (Anacardiaceae), pistachio (Pistacia vera), macad-
amia (Macademia ternifolia) and mango (Mangifera indica)
(Cojocaru et al., 1986) and is also known to inhibit various
enzymes, such as glycerol-3-phosphate dehydrogenase, tyrosinase
(Kubo, Kinst-Hori, & Yokokawa, 1994), lipoxygenases (Shobha,
Ramadoss, & Ravindranath, 1994), aldose reductase, cycloxygenas-
es (Grazzini et al., 1991) and prostaglandin synthase
(Bhattacharya, Mukhopadhyay, Mohan Rao, Bagchi, & Ray, 1987;
Kubo et al., 1987). Since resorcinol has little or no effect on these
enzymes, the hydrophobic alkenyl side chain in cardols is undoubt-
edly associated with the enzyme inhibitory activity. In addition,
the number of double bonds in the side chain is not directly asso-
ciated with the enzyme inhibitory activity (Kubo et al., 1994;
Shobha et al., 1994), suggesting that the interaction of the double
bond with a specific amino acid residue of the enzymes is unlikely.
Cardol (2) is a unique xanthine oxidase inhibitor without pro-oxi-
dant effects (Kamal-Eldin, Pouru, Eliasson, & Åman, 2000). The res-
orcinol lipid was also reported to inhibit the generation of the
superoxide anion catalysed by xanthine oxidase (Trevisan et al.,
2006). The head and tail structure of these compounds suggests
that optimisation of xanthine oxidase inhibitory activity is possible
via a synthetic approach. For example, effective xanthine oxidase

274
N. Masuoka et al. / Food Chemistry 166 (2015) 270–274
tannins and related polyphenols on xanthine oxidase. Chemical
Pharmaceutical Bulletin, 38, 1224–1229.
Kamal-Eldin, A., Pouru, A., Eliasson, C., & Åman, P. (2000). Alkylresorcinols as
antioxidants: Hydrogen donation and peroxyl radical-scavenging effects.
Journal of the Science of Food and Agriculture, 81, 353–356.
Kubo, I., Komatsu, S., & Ochi, M. (1986). Molluscicides from the cashew Anacardium
occidentale and their large-scale isolation. Journal of Agricultural and Food
Chemistry, 34, 970–973.
Kubo, I., Kim, M., Naya, K., Komatsu, S., Yamagiwa, Y., Ohashi, K., Sakamoto, Y.,
Hirakawa, S., & Kamikawa, T. (1987). Prostaglandin synthetase inhibitors from
an African medicinal plant Ozoroa mucronata. Chemistry Letters, 1101–1104.
&
tridecanoate (15), nonyl 2,4-dihydroxybenzoate (11) and dodecyl
2,4-dihydroxybenzoate (12) cooperatively inhibited superoxide
anion generation but nonyl 3,5-dihydroxybenzoate (8), and dode-
cyl 3,5-dihydroxybenzoate (9) did not. The former have electron
donating groups, and the latter have electron withdrawing
(-COOR). This suggested that electron donating groups in the con-
nected moieties between the head and tail portions have the abil-
ity to bind the allosteric sites with a
p–p interaction but the
electron withdrawing one does not. Therefore, we deduced that
specific inhibitors for superoxide anion generation catalysed by
xanthine oxidase consisted of the structures having electron-rich
resorcinol group and an alkyl chain having longer than C9 chain.
Kubo, I., Kinst-Hori, I.,
& Yokokawa, Y. (1994). Tyrosinase inhibitors from
Anacardium occidentale fruits. Journal of Natural Products, 57, 545–552.
Kuwabara, Y., Nishino, T., Okamoto, K., Matsumura, T., Eger, B. T., Pai, E. F., &
Nishino, T. (2003). Unique amino acids cluster for switching from the
dehydrogenase to oxidase form of xanthine oxidoreductase. Proceedings of the
National Academy of Sciences of the United States of America, 100, 8170–8175.
Masuoka, N., & Kubo, I. (2004). Characterization of Xanthine oxidase inhibition by
anacardic acids. Biochimica et Biophysica Acta, 1688, 245–249.
Acknowledgements
Masuoka, N., Nihei, K., & Kubo, I. (2006). Xanthine oxidase inhibitory activity of
alkyl gallates. Molecular Nutrition & Food Research, 50, 725–731.
We thank Professor emeritus Tadao Kamikawa, Kinki Univer-
sity, for his valuable advices and presents of cardol compounds.
Masuoka, N., Nihei, K., Masuoka, T., Kuroda, K., Sasaki, K., & Kubo, I. (2012). The
Inhibition of Uric Acid Formation Catalyzed by Xanthine Oxidase, Properties of
the Alkyl Caffeates and Cardol. Journal of Food Research, 1(3), 257–262.
Masuoka, N., Matsuda, M., & Kubo, I. (2012). Characterisation of the antioxidant
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