Nonallergenic urushiol derivatives inhibit the oxidation of unilamellar vesicles and of rat plasma induced by various radical generators

Article abstract of DOI:10.1016/j.freeradbiomed.2014.03.041

Urushiols consist of an o-dihydroxybenzene (catechol) structure and an alkyl chain of 15 or 17 carbons in the 3-position of a benzene ring and are allergens found in the family Anacardiaceae. We synthesized various veratrole (1,2-dimethoxybenzene)-type an

Full text of DOI:10.1016/j.freeradbiomed.2014.03.041

Free Radical Biology and Medicine 71 (2014) 379–389
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Free Radical Biology and Medicine
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Original Contribution
Nonallergenic urushiol derivatives inhibit the oxidation of unilamellar
vesicles and of rat plasma induced by various radical generators
1
1
n
Jin Young Kim , Jeong-Yong Cho , Young Kyu Ma, Yu Geon Lee, Jae-Hak Moon
Department of Food Science & Technology and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Urushiols consist of an o-dihydroxybenzene (catechol) structure and an alkyl chain of 15 or 17 carbons in
the 3-position of a benzene ring and are allergens found in the family Anacardiaceae. We synthesized
various veratrole (1,2-dimethoxybenzene)-type and catechol-type urushiol derivatives that contained
Received 31 August 2013
Received in revised form
28 March 2014
Accepted 29 March 2014
Available online 8 April 2014
alkyl chains of various carbon atom lengths, including –H, –C1H3, –C5H11, –C10H21, –C15H31, and –C20H41
,
and investigated their contact hypersensitivities and antioxidative activities. 3-Decylcatechol and
3-pentadecylcatechol displayed contact hypersensitivity, but the other compounds did not induce an
allergic reaction, when the ears of rats were sensitized by treatment with the compounds every day for
20 days. Catechol-type urushiol derivatives (CTUDs) exerted very high radical-scavenging activity on the
1,1-diphenyl-2-picrylhydrazyl radical and inhibited lipid peroxidation in a methyl linoleate solution
induced by 2,20-azobis(2,4-dimethylvaleronitrile) (AMVN). However, veratrole-type urushiol derivatives
did not scavenge or inhibit lipid peroxidation. CTUDs also acted as effective inhibitors of lipid
peroxidation of the egg yolk phosphatidylcholine large unilamellar vesicle (PC LUV) liposome system
induced by various radical generators such as AMVN, 2,20-azobis(2-amidino-propane) dihydrochloride,
and copper ions, although their efficiencies differed slightly. In addition, CTUDs suppressed formation of
cholesteryl ester hydroperoxides in rat blood plasma induced with copper ions. CTUDs containing more
than five carbon atoms in the alkyl chain showed excellent lipophilicity in a n-octanol/water partition
experiment. These compounds also exhibited high affinities to the liposome membrane using the
ultrafiltration method of the PC LUV liposome system. Therefore, CTUDs seem to act as efficient
antioxidative compounds against membranous lipid peroxidation owing to their localization in the
phospholipid bilayer. These results suggest that nonallergenic CTUDs act as antioxidants to protect
against oxidative damage of cellular and subcellular membranes.
Keywords:
Urushiol derivatives
Antioxidant
Large unilamellar vesicle liposome
Blood plasma
Contact hypersensitivity
Free radicals
& 2014 Elsevier Inc. All rights reserved.
Urushiols are major components contained in the sap of the
lacquer tree (Rhus verniciflua Stokes, Anacardiaceae) [1]. Urushiols
consist of o-dihydroxybenzene (catechol) coupled with a saturated or
unsaturated alkyl side chain of 15 or 17 carbons and are amphipathic
compounds [1–3]. These compounds exert various biological effects
such as antioxidant [4,5], antimicrobial [6,7], and anticancer [8]
activities. However, urushiols cause skin redness, swelling, inflam-
mation, and irritation on contact, which is known as urushiol-
induced contact dermatitis [9–11]. Therefore, nonallergenic urushiol
derivatives would be useful as a bioactive compound in the
human body.
Excessive reactive oxygen species generated in the body cause
oxidative damage. In particular, lipid peroxidation has been
implicated in various human diseases, including atherosclerosis,
cancer, and aging [11–16]. -Tocopherol and flavonoids (quercetin,
α
luteolin, catechins, etc.) are distributed widely in food, including
cereals, vegetables, and fruits, and are excellent antioxidants in
humans [17–20]. In addition, the catechol structure of various
phenolic compounds including flavonoids is important as an active
site for their antioxidative activity [21,22]. The lipophilicity and
localization of antioxidants in biological systems are generally
considered important to understanding the biological activities of
these compounds on cellular and intracellular membranes. Natural
urushiols possessing a catechol structure and an alkyl side chain of
15 or 17 carbons are amphipathic compounds; therefore, attention
has been focused on the inhibitory effects of these compounds on
biomembrane peroxidation. However, the urushiols have a fatal
Abbreviations: AMVN, 2,20-azobis(2,4-dimethylvaleronitrile); AsA, ascorbic acid;
n-BuLi, n-butyllithium; CE-OOH, cholesteryl ester hydroperoxide; CTUD, catechol-
type urushiol derivative; DPPH, 1,1-diphenyl-2-picrylhydrazyl; PC LUV, phospha-
tidylcholine large unilamellar vesicle; MeL-OOH, methyl linoleate hydroperoxide;
NMR, nuclear magnetic resonance; THF, tetrahydrofuran;
VTUD, veratrol-type urushiol derivative
α-Toc, α-tocopherol;
n
Corresponding author. Fax: þ82 62 530 2149.
E-mail address: nutrmoon@jnu.ac.kr (J.-H. Moon).
1
These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.03.041
0891-5849/& 2014 Elsevier Inc. All rights reserved.

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J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
defect, as they induce strongly allergic reactions on contact,
although natural urushiols act as excellent antioxidants. Therefore,
the functions of urushiol are desirable but unattainable.
(0.045 mol), with different carbon chain lengths, was slowly poured
into the solution and then refluxed for 5 h at 210 1C. The resulting
mixture was added to a saturated NH4Cl solution (150 ml, twice)
and partitioned with ethyl acetate (EtOAc; 200 ml, twice). The
organic layer was washed with brine (200 ml, twice) and added
to anhydrous K2CO3. After filtration, the organic layer was evapo-
rated in vacuo at 35 1C. The concentrates was purified by silica gel
(100 g, 2.3 ꢁ 67 cm) column chromatography eluting with toluene
to give the VTUDs. Four VTUDs (3, 4, 5, and 6) were synthesized
using 1-brompentane, 1-bromodecane, 1-bromopentadecane, and
1-bromoeicosane by the same procedure described above.
In this study, we chemically synthesized veratrole- and catechol-
type urushiol derivatives (compounds 1–12) that contain alkyl side
chains of different lengths. The urushiol derivatives (1–12) were
subjected to a contact hypersensitivity evaluation using rats. We
also measured radical-scavenging activity of the urushiol derivatives
(1–12) using the 1,1-diphenyl-2-picrylhydrazyl (DPPH)2 radical and
methyl linoleate system. In addition, the inhibitory effects of the
urushiol derivatives (1–12) against lipid peroxidation on phospha-
tidylcholine large unilamellar vesicles (PC LUV) and copper ion-
induced rat blood plasma were investigated.
Synthesis of catechol-type urushiol derivatives
Materials and methods
VTUDs (3–6, 2.85 g, 13.68 mmol), with different side chain lengths,
were dissolved respectively with 29.4 ml dry methylene chloride
(CH2Cl2) at 0 1C. The sample solution was added to a solution
(19.38 ml) of BBr3 (1.0 M solution in hexane, 19.38 mmol) in dry
CH2Cl2, and the mixture was stirred successively for 2 h at 0 1C and
then for 12 h at room temperature. Each solution was partitioned
with H2O to give the CH2Cl2 layer. The aqueous layer was partitioned
with CH2Cl2 (60 ml, twice). The combined organic layer was washed
with brine (200 ml, twice) and added to anhydrous K2CO3. After
filtration, the organic layer was evaporated in vacuo at 35 1C. The
crude products were purified by silica gel (Kieselgel 60, Merck,
2.3 ꢁ 59 cm, benzene/EtOAc 6/1, v/v) column chromatography to give
catechol-type urushiol derivatives (CTUDs). 3-Pentylcatechol (9), 3-
decylcatechol (10), 3-pentadecylcatechol (11), and 3-eicosylcatechol
(12) were synthesized using 3-pentylveratrole (3), 3-decylveratrole (4),
3-pentadecylveratrole (5), and 3-eicosylveratrole (6) by the same
procedure described above.
General experimental procedures
Nuclear magnetic resonance (NMR) spectral data were mea-
sured with Varian UnitINOVA 300 and 500 (Varian, Walnut Creek,
CA, USA) spectrometers using tetramethylsilane in CDCl3 as the
internal standard. Mass spectral data were obtained by electro-
spray ionization mass spectrometry (API 3200Q trap, Applied
Biosystems, Foster City, CA, USA) under the following conditions:
ion source temperature, 0 1C; electron voltage of positive and
negative mode, 5000 and ꢀ4500 V, respectively. Column chroma-
tography was performed with silica gel (Kieselgel 60, 70–230
mesh; Merck, Darmstadt, Germany) resin. The high-performance
liquid chromatography (HPLC) analysis was performed on
Shimadzu LC-6AD with a SPD-M20A detector and silica gel
a
(4.6 ꢁ 250 mm, TSK-gel, Silica-60, Tosoh Co., Tokyo, Japan), LiChro-
prep Lobar (RP-8, 40–63
μm, 25 ꢁ 310 mm, Merck), and Octyl-80Ts
(4.6 ꢁ 150 mm, TSK-gel, Tosoh Co.) columns. Thin-layer chromato-
graphy was performed on silica gel 60 F254 (0.25 mm thickness,
Merck).
Induction of contact hypersensitivity by the urushiol derivatives
on rat ears
Sprague–Dawley rats (male, 6 weeks of age, 180–200 g) were
obtained from Samtako Bio Korea (Osan, Korea). The rats were
housed under controlled humidity (5575%), room temperature
(2571 1C), and light cycle (12 h light/12 h dark). Food and water
were available ad libitum. All experimental procedures were
approved by the Institutional Animal Care and Use Committee of
Chonnam National University (CNU IACUC-YB-R-2012-26). All rats
were acclimated for 1 week with a standard rodent diet (Harlan
Rodent diet, 2018S) before experiments. An ethanol (EtOH) solu-
Chemicals
Veratrole (1,2-dimethoxybenzene, 1), 3-methylveratrole (2),
3-methylcatechol (8), 1-bromopentane, 1-bromodecane, 1-bromo-
pentadecane, 1-bromoeicosane, butyllithium (1.6 M solution in
n-hexane), boron tribromide (BBr3; 1.0 M solution in n-hexane),
2,6-di-tert-butyl-4-methylphenol (BHT), 70% perchloric acid
(HClO4), diethylenetriaminepentaacetic acid (DTPA), and methyl
linoleate were obtained from Sigma–Aldrich Chemical Co. (St.
Louis, MO, USA). Pyrocatechol (catechol, 7) was purchased from
Kanto Chemical Co. (Tokyo, Japan). n-Octanol was obtained from
Samchun Pure Chemical Co. (Pyeongtaek, Korea). DPPH, 2,20-
azobis(2,4-dimethylvaleronitrile) (AMVN), 2,20-azobis(2-amidino-
propane) dihydrochloride (AAPH), ascorbic acid (AsA), and egg
yolk 3-sn-phosphatidylcholine were obtained from Wako (Osaka,
tion (3
μ
mol/50 μl) of one of the VTUDs or CTUDs (1–12) was
applied to the rear (1 cm2) of the left ear of the rats (n ¼ 6) daily
for 20 days. The erythema visualized on the rat ears treated with
the VTUDs and CTUDs was reflective of contact hypersensitivity.
Determination of hematological biomarkers in the blood of rats
treated with the urushiol derivatives
Japan). (7)-
α
-Tocopherol ( -Toc) was purchased from Fluka
α
(Buchs SG, Lucerne, Switzerland). All other chemicals and solvents
were of analytical grade.
After the VTUD and CTUD treatments for 20 days, rats (n ¼ 6)
were anesthetized with diethyl ether, the abdominal wall was
opened, and blood was collected from the abdominal aorta into
glass tubes. The numbers of white blood cell, neutrophil, and
eosinophil in the whole blood were determined by Veterinary
Multi-species Hematology System (Hemavet 850, CDC Technolo-
gies Inc., Oxford, MI, USA). Blood serum was obtained by centri-
fugation (1500 g) at 4 1C for 20 min and stored at ꢀ70 1C until
analysis. Serum IgE and histamine levels were measured using Rat
IgE ELISA kit (Komabiotec, Seoul, Korea) and histamine level was
measured using a histamine enzyme-linked immunosorbent assay
kit (Oxford Biomedical Research, Rochester Hills, MI, USA) accord-
ing to the manufacturer's instructions.
Synthesis of veratrole-type urushiol derivatives
Synthesis of veratrole-type urushiol derivatives (VTUDs) was
carried out according to the method of Niimura et al. [23] and
Satoh et al. [24]. Exactly 3.82 ml veratrole (0.03 mol) was added to
a solution (130 ml) of dry tetrahydrofuran (THF) and the mixture
was stirred for 30 min at 0 1C. A solution of 28.2 ml n-BuLi (1.6 M
solution in n-hexane, 0.045 mol) in 5 ml THF was slowly added to
the mixture and then stirred successively for 1 h at 0 1C and for 1 h
at room temperature. A solution (5.56 ml) of 1-bromoalkanes

J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
381
Determination of DPPH radical-scavenging activity
curves were constructed by plotting the peak area vs the concen-
tration of each compound. The content of urushiol derivatives was
quantified in triplicate experiments.
Antioxidant activity of urushiol derivatives was evaluated using
the DPPH radical [25]. In brief, VTUDs (1–6) were prepared at final
concentrations of 50, 100, and 250
were also prepared at final concentrations of 10, 50, 100, 150, 200,
and 250 M in ethanol. The ethanol solution (0.5 ml) of each
compound at the various concentrations was added to the DPPH
radical ethanol solution (2.0 ml, final concentration, 250 M). The
mixture was incubated at room temperature for 30 min in the
dark. The free radical-scavenging activity of the urushiol deriva-
tives was evaluated by decolorization at 517 nm, and activity was
also determined as the percentage decrease in absorbance as
shown by a blank test.
μ
M in ethanol. CTUDs (7–12)
Preparation of egg yolk phosphatidylcholine (EYPC)
μ
EYPC was prepared according the method of Terao et al. [30],
with a slight modification. Briefly, commercial EYPC reagent was
μ
purified by LiChroprep Lobar (RP-8, 40–63
μm, 25 ꢁ 310 mm,
Merck) column chromatography eluted with CHCl3/MeOH/H2O
1/10/0.5 (v/v). EYPC content in the EYPC-containing fraction was
quantified at 830 nm by spectrophotometer using 2,4-diamino-
phenol dihydrochloride. The solvent was removed with a stream
of nitrogen gas followed by evaporation under vacuum. The EYPC
concentrate was stored at ꢀ40 1C until use.
Measurement of peroxyl radical-scavenging activity
in AMVN-induced methyl linoleate peroxidation
Determination of the inhibitory effects of the urushiol derivatives
against AMVN, AAPH, and copper-induced lipid peroxidation
of PC LUV
Peroxyl radical-scavenging activity of the urushiol derivatives
(1–12) and
α
-Toc was evaluated by the inhibition of AMVN-
induced methyl linoleate peroxidation [26,27]. Purified methyl
linoleate (120 mM, final concentration) and each compound
The ethanol solutions of CTUDs (7–12, final concentration,
25
μM) and
α
-Toc (final concentration, 25 M), a solution of the
μ
(40
μ
M, final concentration) were mixed with a solution (1.0 ml,
purified PC LUVs (final concentration 5 mM) in CHCl3/MeOH (95/5,
v/v), and a solution of AMVN (final concentration, 1 mM) in
n-hexane were placed in a test tube. After the solvent was
evaporated, the residue was dispersed in 1 ml of 0.01 M Tris–HCl
buffer (pH 7.4) containing 0.5 mM DTPA. The PC LUV suspension
was obtained by the same ultrasonication and membrane proce-
dures (pore size 100 nm; 21 times). The PC LUV suspension was
diluted with 1 ml of 0.01 M Tris–HCl buffer (pH 7.4) containing
0.5 mM DTPA and incubated at 37 1C for 270 min in the dark with
continuous shaking. PC-OOH content in the reaction mixture was
determined under the same HPLC conditions described below.
To determine the inhibitory effects of CTUDs against AAPH- and
copper ion-induced oxidation in PC LUVs, a solution of AAPH (final
concentration, 10 mM) and Cu(NO3)2 (final concentration, 0.05 mM)
in 0.01 M Tris–HCl buffer (pH 7.4) containing 0.5 mM DTPA was
added to the PC LUV suspension, which was preincubated at 37 1C
for 5 min in the dark with continuous shaking after the ultrasonica-
tion and membrane procedures. Other conditions were the same as
those for AMVN.
final volume) of n-hexane/2-propanol/EtOH 8/3/0.1 (v/v). After a
37 1C preincubation for 10 min in the dark, a solution of AMVN
(10 mM, final concentration) in n-hexane/2-propanol/EtOH 8/3/0.1
(v/v) was added to the mixture and incubated in the dark at 37 1C
with continuous shaking. During the incubation, the samples were
collected at intervals of 0, 20, 40, 60, 80, 100, 120, 140, and
160 min. A 10- l aliquot of sample was subjected to HPLC using
μ
a silica gel column (4.6 ꢁ 250 mm, TSK-gel). The mobile phase was
a mixture of n-hexane/2-propanol/EtOH 10/0.2/0.1 (v/v), and flow
rate was 1.0 ml/min. Methyl linoleate hydroperoxide (MeL-OOH)
was monitored at 235 nm (SPD 10A, Shimadzu Co., Kyoto, Japan).
Calibration curves were constructed using external standard solu-
tions (0.1–20 nmol in n-hexane) of MeL-OOH prepared from
methyl linoleate by autoxidation and purification.
Calculation of the urushiol derivative kinetic parameters
for AMVN-induced methyl linoleate peroxidation
The kinetic parameters of the antioxidative reaction of the
CTUDs (7–12) and
α
-Toc against AMVN-induced methyl linoleate
Quantitative analysis of PC-OOH during oxidation of the LUV solution
induced by initiators
hydroperoxide were measured [28]. The oxidation rate during the
induction period (Rinh), the oxidation rate after the induction
period, and the rate of propagation (Rp) were obtained from the
slope of the curve of methyl linoleate hydroperoxide formation.
The apparent induction period (tinh) was calculated from the
intersecting point of the curves for Rinh and R0p of the urushiol
derivatives [29].
PC-OOH content was determined according to the method
described by Shirai et al. [31]. Briefly, aliquots were subjected to
ODS-HPLC using a TSK-gel Octyl-80Ts column (Tosoh). The mobile
phase consisted of MeOH/H2O 90/0 (v/v), and the flow rate was
constant at 1.0 ml/min. The eluate was monitored by UV detection
at 235 nm (Shimadzu SPD-10A). PC-OOH concentration was calcu-
lated from a standard curve of PC-OOH. Detailed procedures for
preparing the PC-OOH standards have been published previously
[30].
Quantitative analysis of the urushiol derivatives
The contents of the urushiol derivatives (1–12) and AsA and
α
-Toc in the filtrate after ultrafiltration of PC LUVs were measured
by HPLC equipped with an Octyl-80Ts column (Tosoh Co.). The
mobile phases for separating the urushiol derivatives (1–12) were
solvent mixtures of MeOH/H2O: 1, MeOH/H2O 40/60 (v/v); 2,
MeOH/H2O 50/50 (v/v); 3, MeOH/H2O 74/26 (v/v); 4, MeOH/H2O
86/14 (v/v); 5, MeOH/H2O 92/8 (v/v); 6, MeOH/H2O 96/4 (v/v); 7,
MeOH/H2O 14/86 (v/v); 8, MeOH/H2O 30/70 (v/v); 9, MeOH/H2O
63/27 (v/v); 10, MeOH/H2O 80/20 (v/v); 11, MeOH/H2O 92/8 (v/v);
Measurement of the partition coefficient in the n-octanol/H2O
mixture
The partition coefficients of the urushiol derivatives (1–12),
AsA, and
[32]. A 100-nmol aliquot of each compound in MeOH and the
antioxidants ( -tocopherol and AsA, each 100 nmol) was placed in
a test tube. After the solvent was removed with nitrogen gas,
n-octanol (100 l) and 50 mM Tris–HCl buffer (pH 7.4, 100 l)
α
-Toc were determined using an n-octanol/H2O system
α
12, MeOH/H2O 96/4 (v/v);
α
-Toc, MeOH/H2O 93/7 (v/v). Flow rate
was 1.0 ml/min, and the compounds were monitored at 240 nm
(SPD-20A, Shimadzu). Standard solutions of urushiol derivatives in
MeOH were prepared at concentrations of 0–150 nmol. Calibration
μ
μ
were dispersed into the test tube. After the suspension was
vigorously mixed for 30 s, it was centrifuged at 6000 rpm for

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J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
5 min at 4 1C to give the octanol and Tris–HCl buffer phases. The
content of urushiol derivatives in the two phases was determined
by ODS-HPLC analysis as described above.
Results
Synthesis of VTUDs and CTUDs
VTUDs (3–6) were synthesized by lithiation of n-BuLi and
alkylation of 1-bromoalkanes, which have different carbon atom
lengths of C5, C10, C15, and C20. The crude VTUD products were
purified by silica gel column chromatography (toluene). Yields of
3, 4, 5, and 6 were 62.2, 82.4, 99.7, and 59.3%, respectively, and
purities of all compounds were above 99%. The structures of 3, 4, 5,
and 6 were determined based on one-dimensional (1D) and two-
dimensional (2D) NMR and mass spectroscopic (MS) data
(Supplementary Data 1, Fig. 1).
CTUDs (9–12) were synthesized from the VTUDs (3–6) by
demethylation with BBr3. The crude CTUD products were purified by
silica gel column chromatography using a mobile phase of benzene/
EtOAc 6/1 (v/v). The yields of 9, 10, 11, and 12 were 78.3, 93.0, 80.3, and
90.5%, respectively, and purities of all compounds were above 99%. The
structures of 9, 10, 11, and 12 were determined, based on 1D and 2D
NMR and MS data (Supplementary Data 2, Fig. 1).
Ultrafiltration of PC LUVs
The ethanol solution of CTUDs (7–12, final concentration, 25
μM)
and -Toc (final concentration, 25 M) and a solution of the purified
α
μ
PC LUVs (final concentration, 5 mM) in CHCl3/MeOH (95/5, v/v) were
placed in a test tube, and the solvent was removed with nitrogen gas
followed by evaporation in vacuo for 30 min. The residue was
dispersed in 400 l of 0.01 M Tris–HCl buffer (pH 7.4). The suspension
μ
was mixed using a vortex mixer for 30 s followed by sonication for
30 s. The suspension was passed through a polycarbonate membrane
(pore size 100 nm) 21 times to give the LUV suspension [33]. The
filtrate was centrifuged at 12,000 rpm for 40 min at 4 1C [34]. The
supernatant was evaporated under a stream of nitrogen gas, and the
concentrate was dissolved with ethanol (100 l). The concentration of
CTUDs in the LUV phase was determined by ODS-HPLC analysis as
described above.
μ
Contact hypersensitivity of urushiol derivatives (1–12) on rat ears
Rats were sensitized on the rear (1 cm2) of their left ear with an
Determination of the inhibitory effects of CTUDs against copper
ion-induced oxidation in rat blood plasma
EtOH solution (3
μmol/50 μl) of VTUDs and CTUDs (1–12) every
day for 20 days. The clinical features are shown in Fig. 2. Severe
erythema and swelling were observed on the ear skin of rats
treated with 10 and 11, which are catechol-type compounds
consisting of –C10H21 and –C15H31 in the alkyl side chains,
respectively. In contrast, erythema and swelling symptoms were
not observed on ears of rats treated with the other CTUDs (7–9,
12), which contain shorter or longer side chains than those of 10
and 11. In addition, VTUDs (1–6) with an o-dimethoxyl-substituted
catechol moiety did not induce any contact hypersensitivity
regardless of side-chain length.
The antioxidative activities of the CTUDs (7–12) were evaluated
by measuring their inhibitory effects against cholesteryl ester hydro-
peroxide (CE-OOH) formation in copper ion-induced oxidation of rat
blood plasma [26]. Sprague–Dawley rats (male, 6 weeks of age, 180–
200 g) were obtained from Samtako Bio Korea. The rats were kept at
2072 1C under a 12-h light/dark cycle and fasted for 15 h before
blood collection. After diethyl ether anesthesia, blood was collected
from the abdominal aorta into heparinized tubes. Rat plasma was
isolated by centrifugation (3000 g) at 4 1C for 20 min and stored at
ꢀ40 1C. Blood plasma was diluted fourfold with phosphate-buffered
Effects of CTUDs on hypersensitivity-related factors in blood
saline (PBS; pH 7.4). The diluted plasma (650
ethanol solution of the CTUDs (10 M) and 100
solution (final concentration, 100 M). The mixture was incubated at
μ
l) was added to 20
μl
μ
μl of CuSO4–PBS
Contact hypersensitivity-related biomarkers in blood of rats
treated with a veratrol-type compound, 3-pentadecylveratrole (5),
and four CTUDs (9–12) daily for 20 days were determined. The
numbers of white blood cells in rats treated with 5 and 9–12 were
similar to that of the control with no different significance
(Fig. 3A). Treatment with compounds 10 and 11 increased the
numbers of neutrophils compared to that of the control (p o 0.05)
(Fig. 3B). The numbers of neutrophils in rats treated with 5, 9, and
μ
37 1C for 16 h with continuous shaking. The CE-OOH concentration
was determined according to the method of Arai et al. [35]. Briefly,
100- l aliquots were withdrawn from the incubating solutions and
μ
mixed with 3 ml MeOH containing 2.5 mM BHT. The mixture was
sonicated for 1 min, and then partitioned with 3 ml n-hexane by
vortexing vigorously for 1 min. The upper layer (n-hexane) was
collected and extraction of the lower layer was repeated with 3 ml
of n-hexane. The combined n-hexane phases were evaporated in a
rotary evaporator at room temperature. The remaining lipids were
12 were lower than that of the control (p
o 0.05). These
neutrophil number patterns were similar to the eosinophil num-
ber results, although the numbers did not differ significantly
(Fig. 3C). In addition, serum IgE (Fig. 3D) and histamine (Fig. 3E)
levels in rats treated with 10 and 11 were higher than those of
other compounds (5, 9, 12) and the control (p o 0.05). The serum
IgE levels of rats treated with 5, 9, and 12 were similar to that of
the control with no significant difference. The serum histamine
levels of rats treated with 5 and 9 were significantly lower than
those treated with 10 and 11 but were not different from the
control, although the serum histamine levels of 5 and 9 were
slightly higher than that of the control group. The patterns for the
numbers of neutrophils and eosinophils and the levels of serum
IgE and histamine agreed with the results of the contact hyper-
sensitivity experiment.
dissolved in 100 l MeOH/CHCl3 (95/5, v/v), and aliquots were
μ
subjected to RP-HPLC using a TSK-gel Octyl-80TS column (Tosoh)
to determine CE-OOH content. The effluent was monitored by UV
detection at 235 nm (Shimadzu SPD-10A). MeOH/H2O (97/3, v/v)
served as the mobile phase, and the flow rate was constant at 1.0 ml/
min. CE-OOH concentration was calculated from a standard curve of
CE-OOH. Detailed procedures for preparing the CE-OOH standard
have been published previously [35].
Statistical analysis
Data are expressed as the mean 7 standard deviation (SD) using
the Statistical Package for Social Sciences (SPSS, IBM, Armonk, NY,
USA) 19.0 package program. Statistical differences were measured by
one-way analysis of variance followed by Duncan's multiple compar-
ison test. A p o 0.05 was considered significant.
DPPH radical-scavenging activity of the urushiol derivatives
The radical-scavenging activities of VTUDs and CTUDs (1–12) and
α-Toc as a positive control were evaluated using DPPH. As shown in

J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
383
Veratrole Type Urushiol Derivatives
Catechol Type Urushiol Derivatives
OCH3
OH
OH
OCH3
OH
Catechol (7)
Veratrole (1)
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
3-Methylcatechol (8)
3-Pentylcatechol (9)
3-Methylveratrole (2)
3-Pentylveratrole (3)
OCH3
OCH3
OCH3
OH
OH
OH
3-Decylcatechol (10)
3-Decylveratrole (4)
3-Pentadecylcatechol (11)
3-Eicosylcatechol (12)
3-Pentadecylveratrole (5)
3-Eicosylveratrole (6)
OCH3
OH
OCH3
OH
Fig. 1. Structures of synthetic veratrole-type and catechol-type urushiol derivatives (1–12).
1
4
2
5
3
6
9
7
8
10
11
12
Fig. 2. Contact hypersensitivity effects of the urushiol derivatives (1–12) induced after treatment of rat ears for 20 days. Each group consisted of six rats that were treated
with one of the urushiol derivatives.

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J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
7
6
5
4
3
2
1
0
CON
5
9
10
11
12
0.6
3.0
2.5
2.0
1.5
1.0
0.5
0.0
b
0.5
0.4
0.3
0.2
0.1
0.0
ab
ab
a
a
a
CON
5
9
10
11
12
CON
5
9
10
11
12
10
8
250
200
150
100
50
c
b
c
6
b
b
b
ab
a
4
a
a
a
a
2
0
0
CON
5
9
10
11
12
CON
5
9
10
11
12
Fig. 3. Numbers of (A) white blood cells, (B) neutrophils, and (C) eosinophils and (D) IgE and (E) histamine content in the blood obtained after treatment with urushiol
derivatives (5, 9–12) of rat ears for 20 days. Each value is the mean 7 SD (n ¼ 6). CON: control.
Supplementary Fig. S1, CTUDs (7–12) showed predominantly higher
DPPH radical-scavenging activity than VTUDs (1–6), which did not
exhibit DPPH radical-scavenging activity regardless of concentration.
In addition, the DPPH radical-scavenging activities of CTUDs (7–12)
were very similar regardless of carbon atom side-chain length.
Moreover, the number of DPPH radical molecules scavenged by one
molecule of CTUDs was calculated by assuming that one molecule of
peroxidation induced by AMVN, a lipophilic radical generator, in a
solution of n-hexane/2-propanol/EtOH 8/3/0.1, v/v). VTUDs (1–6)
at a concentration of 40 M did not inhibit AMVN-induced
μ
oxidation of methyl linoleate (Supplementary Fig. S2A) and
showed a pro-oxidant effect compared with the control. In con-
trast, the CTUDs (7–12) strongly inhibited AMVN-induced oxida-
tion of methyl linoleate (Supplementary Fig. S2B). Interestingly,
the CTUDs (7–12) showed slightly higher peroxyl radical-
α
-Toc scavenges two molecules of DPPH. One molecule of each CTUD
trapped two molecules of free radicals corresponding to the number
scavenged by -Toc.
scavenging activity in comparison to
radical-scavenging activities were very similar regardless of the
carbon atom side-chain length.
α
-Toc and their peroxyl
α
Peroxyl radical-scavenging activity of urushiol derivatives against
AMVN-induced methyl linoleate peroxidation
The kinetic parameters of CTUDs (7–12) were calculated by the
method of Niki et al. [28]. All CTUDs showed a slightly lower
induction period (tinh ¼ 3.73–3.97) and a slightly higher oxidation
rate during the induction period (Rinh ¼ 0.13–0.16) compared to
The peroxyl radical-scavenging activities of urushiol derivatives
were evaluated by measuring inhibition against methyl linoleate
α
-Toc (tinh ¼ 3.66, Rinh ¼ 0.17) (Table 1). The stoichiometric factors

J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
385
Table 1
Kinetic parameters for the antioxidative reaction of urushiol derivatives against AMVN-initiated oxidation of methyl linoleate.
Catechol-type urushiol derivative
Rp
Rp0
Rinh
tinh
kinh/kp
Ri
Kinetic chain length
Oxidizability
n
α-Toc
Catechol (7)
1.03
1.03
1.03
1.03
1.03
1.03
1.03
0.98
0.98
0.81
0.85
0.80
0.81
0.81
0.17
0.16
0.13
0.16
0.13
0.16
0.16
3.66
3.73
3.96
3.84
3.97
3.85
3.83
1.61
1.67
1.94
1.62
1.93
1.62
1.63
2.20
2.20
2.20
2.20
2.20
2.20
2.20
4.68
4.68
4.68
4.68
4.68
4.68
4.68
6.94
6.94
6.94
6.94
6.94
6.94
6.94
2.0
2.1
2.2
2.1
2.2
2.1
2.1
3-Methylcatechol (8)
3-Pentylcatechol (9)
3-Decylcatechol (10)
3-Pentadecylcatechol (11)
3-Eicosylcatechol (12)
Rp, 10ꢀ7 M sꢀ1; R0p, 10ꢀ7 M sꢀ1; Rinh, 10ꢀ7 M sꢀ1; tinh, 103 s; kinh/kp, 103; Ri, 10ꢀ8 M sꢀ1; kinetic chain length, Rp/Ri; oxidizability, 10ꢀ3(M s)ꢀ1/2
.
(n) of the CTUDs were 2.1–2.2, which were slightly higher
-Toc. The efficiencies (kinh/kp)
of peroxyl radical scavenging (the ratio of tinh/rate of chain
propagation (kp)) for CTUDs (7–12) were 1620–1940, which was
Behavior of urushiol derivatives in an n-octanol/water partition
system
compared with that (n ¼ 2.0) of
α
The urushiol derivatives (1–12), AsA, and
α
-Toc were respec-
very similar to that of
α
-Toc (kinh/kp
¼
1610). The kinetic
tively partitioned in an n-octanol/water system to determine their
relative lipophilicities. Their contents were quantified in two phases
by ODS-HPLC analysis. As shown in Fig. 5, most of the AsA was
parameters of the CTUDs (7–12) were not influenced by carbon
atom side-chain length. Consequently, it was concluded that the
lipid peroxyl radical-scavenging capacity was the same and/or
detected in the water phase, whereas most of the
α
-Toc was
slightly higher in magnitude in comparison to that of
α
-Toc.
detected in the n-octanol phase. Similar to -Toc, the urushiol
α
derivatives (1–12) were also mostly detected in the n-octanol phase,
although they showed some differences in partition coefficients.
The VTUD (1–6) contents in the n-octanol phase were not statisti-
cally significant regardless of the side-chain carbon atom length.
However, the CTUD (7–12) contents in the n-octanol phase, except
for 12, which had an alkyl side chain of C20H41, increased propor-
tionally with the increase in length of their side chains. In addition,
compounds 7–9 showed relatively lower contents in the n-octanol
Inhibitory effects of CTUDs against AMVN-induced lipid peroxidation
of PC LUVs
The antioxidative activities of the CTUDs (7–12) and -Toc as a
α
positive control were evaluated by measuring the inhibition of
PC-OOH formation on lipid peroxidation of PC LUVs that were
exposed to AMVN, a lipid-soluble radical generator. As shown in
Fig. 4A, all CTUDs more efficiently inhibited PC-OOH formation
than the control in this reaction system. The inhibitory effects of
9–12, which contained more than five carbons in the alkyl side
chain, were very similar regardless of carbon length of the side
chains and were higher than those of 7 and 8. In addition, 9–12
phase than other CTUDs (10–12).
α
-Toc and all of the urushiol
derivatives (1–12) were not detected or were detected in very small
amounts in the water phase. Therefore, all urushiol derivatives
(1–12) used in this study seemed to have lipophilic properties
similar to
α-Toc rather than hydrophilic properties, although 7–9
showed slightly higher inhibitory effects in comparison with
Toc, which was used as a positive control and as a representative
lipophilic antioxidative vitamin. However, the antioxidative activ-
α
-
have slightly less lipophilic properties than others (1–6, 10–12).
Localization of the urushiol derivatives using PC-LUV system
ities of 7 and 8 were weaker compared to that of
α
-Toc.
ultrafiltration
Inhibitory effects of CTUDs against AAPH-induced lipid peroxidation
of PC LUVs
PC LUVs were prepared with each CTUD and -Toc as a control
α
to determine the affinity of the CTUDs (7–12) to phospholipid
membranes. Each PC LUV suspension was filtered through an
ultrafiltration membrane (pore size, 10 nm) and the contents of
each compound in the filtrate were measured. That is, if a
compound presented as a small amount in the filtrate, it suggests
that the compound has a very high affinity for phospholipid
We also evaluated the inhibitory effects of CTUDs (7–12)
against PC-OOH production from PC-LUVs induced by AAPH, a
water-soluble radical generator. All CTUDs (7–12) exerted a greater
inhibitory effect than the control against lipid peroxidation of PC
LUVs generated in the aqueous phase (Fig. 4B) as well as in the
lipid phase (AMVN) (Fig. 4A). The inhibitory effects of 9–12 were
very similar regardless of the carbon length of their side chains. In
addition, compounds 7 and 8 also showed greater inhibitory
membranes. CTUDs (7–12) and
in small amounts, indicating that the CTUDs possess a high affinity
for PC LUVs, similar to -Toc (Fig. 6). In addition, the affinity of PC
α
-Toc were detected in the filtrate
α
LUVs for the CTUDs showed proportional increases with the length
of the alkyl side chain. That is, compounds 11 and 12 with 415
carbon atoms in the alkyl side chain had the highest affinity for PC
LUVs. The filtrate contents of 9 and 10, with 5 and 10 carbon atoms
in the alkyl side chain, were lower than those of 11 and 12.
Compounds 7 and 8 also showed considerable affinities for PC
LUVs, although their affinities were slightly lower than those of
effects than
α
-Toc, different from AMVN-induced oxidation
(Fig. 4A), but their inhibitory effects were weaker than those of
9–12 in this oxidation system.
Inhibitory effects of CTUDs against copper ion-induced lipid
peroxidation of PC LUVs
other CTUDs (9–12) and
(7–12) possess considerable affinity for PC LUVs.
α
-Toc. These results indicate that CTUDs
The antioxidative activities of CTUDs (7–12) and
peroxidation of PC LUVs induced by copper ions showed a very
similar pattern to that of AAPH-induced PC LUV oxidation. That is,
α
-Toc against
Inhibitory effects of CTUDs against copper ion-induced lipid
peroxidation of rat blood plasma
all CTUDs had greater inhibitory effects than
α
-Toc. In addition,
compounds 9–12 exhibited more efficient antioxidative activities
than 7 and 8. Moreover, the activities of 9–12 were very similar
regardless of side-chain length (Fig. 4C).
The antioxidative activities of the CTUDs (7–12) were also
determined using the copper ion-induced oxidation system of

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J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
7.0
e
8.0
6.0
4.0
2.0
c
d
6.0
c
c
b
5.0
a
a
a
a
4.0
3.0
2.0
1.0
0.0
0.0
b
d
14.0
12.0
10.0
8.0
c
b
b
bc
a
a
ab
a
a
a
a
α-Toc
7
8
9
10
11
12
Fig. 6. Concentration of catechol-type urushiol derivatives (7–12) in the filtrate
after ultrafiltration of the PC LUV suspension. The concentration of catechol-type
urushiol derivatives in the LUV suspension was 25 μΜ. Each value is the mean 7
SD (n ¼ 3).
6.0
4.0
2.0
0.0
8.0
16
14
12
10
8
d
c
6.0
4.0
2.0
0.0
bc
b
a
a
a
a
6
4
7
CON α-Toc
8
9
10
11
12
Fig. 4. Inhibitory effects of catechol-type urushiol derivatives (7–12) (A) against
AMVN-, (B) AAPH-, and (C) copper ion-induced lipid peroxidation of PC LUVs. The
PC LUV solution was mixed with solutions of AMVN, AAPH, and copper ions
followed by addition of the catechol-type urushiol derivatives (5 μM). The reaction
mixture was incubated at 37 1C with continuous shaking. Each value is the mean 7
SD (n ¼ 3). CON: control.
2
0
0
2
4
6
8
10
12
14
16
18
Incubation time (h)
Fig. 7. Inhibitory effects of the catechol-type urushiol derivatives (7–12) against
copper ion-induced lipid peroxidation in rat blood plasma. ●, control; ∇, catechol
(7); ▲, 3-methylcatechol (8); ○, 3-pentylcatechol (9); ▼, 3-decylcatechol (10); □,
Octanol phase
120
90
60
30
0
bc
3-pentadecylcatechol (11); Δ, 3-ecosylcatechol (12).
A diluted blood plasma
bc
bc
c
solution was added to each catechol-type urushiol derivative (5 μM) followed by
the addition of CuSO4 (100 μM) to induce CE-OOH formation. The reaction mixture
was incubated at 37 1C with continuous shaking. Data are representative of two
experiments.
bc
bc
bc
b
b
b
a
a
a
during the incubation. The inhibitory effects decreased in order of
10 and 11 4 12 4 8 Z 7 4 9 (Fig. 7). These results confirm that
CTUDs (7–12) exert a high inhibitory effect against CE-OOH
formation during lipid peroxidation of rat plasma induced by
copper ions, which is responsible for metal ion-initiated oxidation.
1
2
3
4
5
6
7
8
9
10 11 12
30
60
90
Discussion
We hypothesized that urushiols could be an attractive and poten-
tial antioxidative candidate on cellular and subcellular membranes
owing to their amphipathic property. However, urushiol causes skin
problems such as contact dermatitis [9–11]. Natural urushiol-induced
contact dermatitis is dependent on the carbon atom length and the
degree of unsaturation of the alkyl chain [1–3]. However, studies on
the formation mechanism of urushiol-induced contact dermatitis are
not available. We hypothesized that nonallergenic urushiol derivatives
would act as excellent antioxidants that would inhibit lipid peroxida-
tion in membrane systems. Therefore, VTUDs and CTUDs (1–12)
Water phase
Fig. 5. Concentration of urushiol derivatives (1–12) in each fraction after partition-
ing in n-octanol/water. Each value is the mean 7 SD (n ¼ 4).
rat blood plasma. CTUDs more effectively inhibited CE-OOH
formation during lipid peroxidation of rat plasma than the control
group (Fig. 7). In addition, CTUDs (7–12) showed a significantly
higher inhibitory effect on CE-OOH formation than the control

J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
387
containing various carbon atom lengths of the alkyl side chains were
synthesized to produce nonallergenic urushiol derivatives (Fig. 1).
Contact hypersensitivities and antioxidative activities of the synthe-
sized urushiol derivatives (1–12) were evaluated with 3-pentad-
ecylcatechol (11), which is a natural urushiol, to understand the
structural relationships between their hypersensitivities, antioxidative
activities, and side-chain lengths. In our preliminary study, a contact
hypersensitivity test using 11 as a model natural urushiol compound
induced oxidation model systems, except 7, showed higher
antioxidative activities than -Toc (Fig. 4B and C), which was
different from AMVN-induced oxidation (Fig. 4A). These results
suggest that CTUDs (7–12) more effectively scavenged radicals
and chelated metal ions induced in the aqueous phase of LUV
α
liposome membrane systems than
uted to the difference in location of the CTUDs in the PC LUV
liposome membrane model system.
α
-Toc. It may also be attrib-
was performed with sensitization of 0.01–1.0
μ
mol dosages on the left
In general, the location of the antioxidants should be accounted
for to understand the effectiveness of the antioxidants in hetero-
geneous membrane systems. Several studies have reported that
the efficiency of an antioxidant is influenced by its affinity for a
membrane [41,42]. Therefore, the localization of an antioxidative
compound on a biomembrane may be a particularly important
factor to protect against oxidative damage on a membrane.
Characteristics such as lipophilicity and hydrophilicity of com-
pounds affect their localization on a biomembrane. Therefore,
lipophilicity and affinity of antioxidants have been frequently
evaluated using n-octanol/water partition and phospholipid mem-
brane systems [32,33,43,44]. The results of the n-octanol/water
partition experiment suggest that the CTUDs (7–12) possess high
lipophilic properties (Fig. 5). In particular, lipophilicity increased
depending on the carbon atom length of the alkyl side chains of
the CTUDs. The lipophilicities of 10–12, which contain 410 carbon
ears of rats (n ¼ 6) daily for 20 days (data not shown). The
concentration that approximated the threshold inducing contact
hypersensitivity by 11 was about 0.55
synthesized urushiol derivatives (1–12) were treated in high amounts
(3 mol) to the left ear of rats daily for 20 days, which was about a
μmol (176 g). Therefore, the
μ
μ
5.5-fold increased dosage of the concentration that approximated
the threshold. The morphological results are shown in Fig. 2.
3-Decylcatechol (10), which contains 10 carbon atoms in the alkyl
side chain, caused more serious contact hypersensitivity than 11,
which is a natural urushiol. However, interestingly, the VTUDs (1–6)
and other CTUDs (7, 8, 12) did not cause contact hypersensitivity. In
addition, the results for the numbers of neutrophils and eosinophils
and the levels of serum IgE and histamine (Fig. 3) were closely
correlated with the contact hypersensitivities of the synthetic urushiol
derivatives (5, 9–12) (Fig. 2). These results strongly indicate that the
catechol structure and the length of the side chain in urushiol are
important factors for inducing contact hypersensitivity. Therefore, the
nonallergic urushiol derivatives may be excellent antioxidants in
biological systems.
atoms in the alkyl side chain, were similar to that of
However, other CTUDs (7–9), containing less than 5 carbon atoms
in the alkyl side chain, showed significantly lower lipophilicity
α
-Toc.
than α-Toc. The sum of 7–9 in the n-octanol and water phases did
Several studies have reported that the catechol (o-dihydroxylben-
zene) structure is an important factor as an active site for the radical-
scavenging activity of phenolic compounds [22]. In this study, VTUDs
(1–6), which have a methoxyl-substituted catechol moiety, did not
scavenge DPPH radicals, which are stable (Supplementary Fig. S1).
However, CTUDs (7–12) exerted high radical-scavenging activity, and
their activities were very similar regardless of carbon atom length of
their alkyl side chains. Therefore, these results confirm that the
catechol structure is essential for the radical-scavenging activity of
urushiol derivatives (7–12).
not reach 100%, suggesting that the portion lacking might be
located in the interface between n-octanol and water. Therefore, it
may be related to the result that 7 and 8 showed patterns different
from those of the other CTUDs (7–12) in comparison to antiox-
idative activities using the PC LUV liposome membrane model
system.
It was assessed that CTUDs (9–12) containing more than five
carbon atoms in their alkyl side chains possessed a higher affinity
for PC LUV liposomes than α-Toc (Fig. 6). However, compounds (7
and 8) with no or one carbon atom in the alkyl side chain showed
a relatively lower affinity than the other CTUDs (9–12), as well as
α
-Toc is an excellent lipophilic antioxidant that scavenges
chain-propagating peroxyl radicals [36]. In this study, CTUDs (7–
12) strongly suppressed MeL-OOH formation in the methyl linole-
ate solution induced by AMVN, a lipid-soluble radical generator
(Supplementary Fig. S1B). In the kinetic parameter results, the tinh
and Rinh values of CTUDs (7–12) were very closely similar, and
α-Toc. In addition, the affinity of the CTUDs (7–12) to PC LUVs was
proportional to the number of carbon atoms in their alkyl side
chains. Although the difference in affinity to PC LUVs among the
CTUDs was not prominent, small differences might negatively
affect the antioxidative activity of 7 and 8 in the PC LUV liposome
membrane model system and differences in antioxidative activity
might be attributed to localization caused by a difference in carbon
atom length in the alkyl side chains of the CTUDs on the PC LUV
liposome membrane. In the PC LUV liposome membrane experi-
ments (Fig. 4), CTUDs 9–12 showed high affinity for PC LUVs and
strongly suppressed PC-OOH formation on the PC LUV membra-
nous systems regardless of the radical initiator. Therefore, these
results suggest that CTUDs (7–12) have high radical-scavenging
and metal ion-chelating activities in both the aqueous and the
lipid phases.
their kinh/kp values were slightly higher compared to those of
Toc (Table 1). Several studies have suggested that -Toc scavenges
two peroxyl radical molecules [37,38]. In this study, the CTUD
stoichiometric factor (n) was slightly higher than that of -Toc
α
-
α
α
(Table 1). Therefore, CTUDs probably scavenge more than two
peroxyl radical molecules.
The PC LUV model system has been frequently used as a
biomembrane model to evaluate the antioxidative activity of
antioxidants on cellular and subcellular membranes [29,30].
Three different kinds of radical generators, including a lipid-
soluble peroxyl radical generator (AMVN), a water-soluble per-
oxyl radical generator (AAPH), and transition copper ions were
used in this biomembrane model. Radical generation by AMVN is
initiated in the interior of liposomal membranes [38,39], whereas
peroxyl radical generation by AAPH and transition copper ions
added in the aqueous phase is initiated in the exterior and
surface of liposomal membranes [40]. The results shown in
Fig. 4 indicate that CTUDs (7–12) effectively suppress PC-OOH
formation on AMVN-, AAPH-, and copper ion-induced membra-
nous PC LUV systems, although their effects were slightly
different depending on carbon atom length of their alkyl side
chains. In particular, all CTUDs in the AAPH- and copper ion-
Several lines of evidence suggest that oxidative modification in
blood plasma is involved in the development of cardiovascular
disease [45]. Therefore, antioxidants in blood plasma may play a
role in protecting against oxidation of components such as lipopro-
teins and delay or prevent the development of cardiovascular
diseases such as atherosclerosis [46]. Because CE-OOH produced
from oxidation is very stable and is present in healthy human
plasma at a concentration of about 3 nM [47], CE-OOH has been
selected as a blood plasma lipid peroxidation index [48]. Therefore,
the antioxidant activity of CTUDs (7–12) in the copper ion-induced-
blood plasma oxidation system was examined by measuring
CE-OOH content. All CTUDs (7–12) exerted high inhibitory activity

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J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
against CE-OOH formation in this system, although structural
relationships between the compounds and the antioxidative activ-
ity were not observed (Fig. 7). Surprisingly, 10 and 11, which are
allergenic urushiol derivatives, showed overwhelmingly higher
activities in comparison to 9 and 12, which are nonallergenic.
However, the compounds may be different in practical use because
of their allergenic characteristics. Interestingly, the concentration of
[4] Hong, S. H.; Suk, K. T.; Choi, S. H.; Lee, J. W.; Sung, H. T.; Kim, C. H.; Kim, E. J.;
Kim, M. J.; Han, S. H.; Kim, M. Y. Anti-oxidant and natural killer cell activity of
Korean red ginseng (Panax ginseng) and urushiol (Rhus vernicifera Stokes) on
non-alcoholic fatty liver disease of rat. Food Chem. Toxicol. 55:586–591; 2013.
[5] Kim, M. J.; Choi, Y. H.; Kim, W. G.; Kwak, S. S. Antioxidative activity of urushiol
derivatives from the sap of lacquer tree (Rhus vernicifera Stokes). Korean
J. Plant. Res. 10:227–230; 1997.
[6] Kim, D. W.; Jeon, S. L.; Seo, J. C. The preparation and characterization of
urushiol powders (YPUOH) based on urushiol. Prog. Org. Coat. 76:1465–1470;
2013.
[7] Kim, H. S.; Yeum, J. H.; Choi, S. W.; Lee, J. Y.; Cheong, I. W. Urushiol/
polyurethane–urea dispersions and their film properties. Prog. Org. Coat.
65:341–347; 2009.
[8] Choi, J. Y.; Park, C. S.; Choi, J. O.; Rhim, H. S.; Chun, H. J. Cytotoxic effect of
urushiol on human ovarian cancer cells. J. Microbiol. Biotechnol. 11:399–405;
2001.
α
-Toc in the low-density lipoprotein fraction increases when
incubated with additional -Toc [49], and -Toc taken with food
α
α
is present in the lipoprotein of blood plasma [50]. It has not yet
been investigated whether CTUDs are distributed in lipoproteins
present in the blood plasma, similar to
α-Toc. However, it is likely
that the reaction patterns of each CTUD and lipoproteins containing
cholesteryl esters are different because the inhibitory pattern
against CE-OOH formation was different according to the CTUD.
Therefore, the difference may be attributed to differences in carbon
atom length of the alkyl side chains of the CTUDs.
[9] Ma, X. M.; Lu, R.; Miyakoshi, T. Recent advances in research on lacquer allergy.
Allergol. Int. 61:45–50; 2012.
[10] Zepter, K.; Haffner, A.; Soohoo, L. F.; De Luca, D.; Tang, H. P.; Fisher, P.;
Chavinson, J.; Elmets, C. A. Induction of biologically active IL-1 beta-converting
enzyme and mature IL-1 beta in human keratinocytes by inflammatory and
immunologic stimuli. J. Immunol. 159:6203–6208; 1997.
[11] Wakabayashi, T.; Hu, D. L.; Tagawa, Y.; Sekikawa, K.; Iwakura, Y.; Hanada, K.;
Nakane, A. IFN-gamma and TNF-alpha are involved in urushiol-induced
contact hypersensitivity in mice. Immunol. Cell Biol. 83:18–24; 2005.
[12] Bonomini, F.; Tengattini, S.; Fabiano, A.; Bianchi, R.; Rezzani, R. Atherosclerosis
and oxidative stress. Histol. Histopathol. 23:381–390; 2008.
In this study, we demonstrated that CTUDs (7–12) with various
carbon atom lengths of the alkyl chain effectively scavenged
radicals generated in both aqueous and lipid phases and chelated
transition metal ions. However, of the synthesized CTUDs, 10 and
11, which have 10 and 15 carbon atoms in the alkyl chain, caused
serious contact hypersensitivity. In contrast, VTUDs (1–6) and
other CTUDs (7–9, 12) did not cause an allergenic reaction on
contact. Therefore, it was clarified that catechol structure is
needed for the compounds to be allergenic and that the number
of the carbon atoms in the alkyl chains of CTUDs is an important
factor inducing allergy. The nonallergenic urushiol derivatives, 9
and 12, which contain 5 and 20 carbon atoms in the alkyl chain,
may act as excellent antioxidative compounds in biomembrane
systems because they showed high affinity for phospholipid
membranes and scavenged radicals induced in the exterior as well
as the interior of the PC LUV membranous system. Therefore, the
nonallergenic urushiol derivatives 9 and 12 acted as potential
radical scavengers as well as metal-chelating agents in blood
plasma. Moreover, the urushiol derivatives used in this study
may provide very useful information for the development of
antioxidants that prevent oxidation of biomembranes. Therefore,
we have started to evaluate the antioxidative effects of the
nonallergenic urushiol derivatives (9 and 12) against membranous
peroxidation in vivo, and a metabolic study in an in vivo system is
also in progress.
[13] Stocker, R.; Keaney Jr. J. F. Role of oxidative modifications in atherosclerosis.
Physiol. Rev. 84:1381–1478; 2004.
[14] Klaunig, J. E.; Kamendulis, L. M.; Hocevar, B. A. Oxidative stress and oxidative
damage in carcinogenesis. Toxicol. Pathol. 38:96–109; 2010.
[15] Cui, H.; Kong, Y.; Zhang, H. Oxidative stress, mitochondrial dysfunction, and
aging. J. Signal Transduct. 2012:646354; 2012.
[16] Berlett, B. S.; Stadtman, E. R. Protein oxidation in aging, disease, and oxidative
stress. J. Biol. Chem. 272:20313–20316; 1997.
[17] Hooper, L.; Kroon, P. A.; Rimm, E. B.; Cohn, J. S.; Harvey, I.; Le Cornu, K. A.;
Ryder, J. J.; Hall, W. L.; Cassidy, A. Flavonoids, flavonoid-rich foods, and
cardiovascular risk:
a meta-analysis of randomized controlled trials. Am.
J. Clin. Nutr. 88:38–50; 2008.
effects of Concord grape juice flavonoids
[18] O’Byrne, D. J.; Devaraj, S.; Grundy, S. M.; Jαia-latol,cIo. pChoemroplaorinsomnaorfktehrse oafnotixoixdiadtaivnet
stress in healthy adults. Am. J. Clin. Nutr. 76:1367–1374; 2002.
[19] Terao, J.; Kawai, Y.; Murota, K. Vegetable flavonoids and cardiovascular disease.
Asian Pac. J. Clin. Nutr. 17(Suppl. 1):291–293; 2008.
[20] Singh, U.; Devaraj, S.; Jialal, I. Vitamin E, oxidative stress, and inflammation.
Annu. Rev. Nutr. 25:151–174; 2005.
[21] Burda, S.; Oleszek, W. Antioxidant and antiradical activities of flavonoids.
J. Agric. Food Chem. 49:2774–2779; 2001.
[22] Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure–antioxidant activity
relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20:
933–956; 1996.
[23] Niimura, N.; Kamiya, Y.; Sato, T.; Katano, I.; Miyakoshi, T. Synthesis of
3-[8Z,11E,13Z)-8,11,13-pentadecatrienyl]catechol and analysis of the triene
urushiol fraction of the sap of Rhus vernicifera. J. Jpn. Oil Chem. Soc. 47:
171–178; 1998.
[24] Satoh, M.; Takeuchi, N.; Nishimura, T.; Ohta, T.; Tobinaga, S. Synthesis of
anacardic acids, 6-[8(Z),11(Z)-pentadecadienyl]salicylic acid and 6-[8(Z),11
(Z),14-pentadecatrienyl]salicylic acid. Chem. Pharm. Bull. 49:18–22; 2001.
[25] Abe, N.; Nemoto, A.; Tsuchiya, Y.; Hojo, H.; Hirota, A. Studies of the
1,1-diphenyl-2-picrylhydrazyl radical scavenging mechanism for a 2-pyrone
compound. Biosci. Biotech. Biochem. 64:306–333; 2000.
Acknowledgment
This research was supported by the Basic Science Research
Program of the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science, and Technology
(No. NRF-2013R1A1A2012410).
[26] Murase, H.; Moon, J. H.; Yamauchi, R.; Kato, K.; Kunieda, T.; Yoshikawa, T.;
Terao, J. Antioxidant activity of a novel vitamin E derivative, 2-(α-D-glucopyr-
anosyl)methyl-2,5,7,8-tetramethylchroman-6-ol. Free Radic. Biol. Med.
24:217–225; 1997.
[27] Yamamoto, Y.; Niki, E.; Kamiya, Y. Oxidation of lipids. I. Quantitative determi-
nation of the oxidation of methyl linoleate and methyl linolenate. J. Agric. Food
Chem. 46:5062–5065; 1998.
Appendix A. Supplementary material
[28] Niki, E.; Saito, T.; Kawakami, A.; Kamiya, Y. Inhibition of oxidation of methyl
linoleate in solution by vitamin E and vitamin C. J. Biol. Chem. 259:4177–4182;
1984.
[29] Koga, T.; Takahashi, I.; Yamauchi, R.; Piskula, M.; Terao, J. Kinetic studies on the
formation of phosphatidylcholine hydroperoxides in large unilamellar vesicles
by azo compounds. Chem. Phys. Lipids 86:85–93; 1997.
[30] Terao, J.; Piskula, M.; Yao, Q. Protective effect of epicatechin, epicatechin
gallate, and quercetin on lipid peroxidation in phospholipid bilayers. Arch.
Biochem. Biophys. 308:278–284; 1994.
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.freeradbiomed.
2014.03.041.
References
[31] Shirai, M.; Moon, J. H.; Tsushida, T.; Terao, J. Inhibitory effect of a quercetin
metabolite, quercetin 3-O-β-D-glucuronide, on lipid peroxidation in liposomal
membranes. J. Agric. Food Chem. 49:5602–5608; 2001.
[1] Gross, M.; Baer, H.; Fales, H. M. Urushiols of poisonous anacardiaceae.
Phytochemistry 14:2263–2266; 1975.
[2] Vera, S.; Castagnoli, N. J.; Epstein, W. L. in vitro studies of poison oak
immunity. II. Effect of urushiol analogs on the human in vitro response.
J. Clin. Invest. 64:1449–1456; 1979.
[32] Murota, K.; Shimizu, S.; Chujo, H.; Moon, J. H.; Terao, J. Efficiency of absorption
and metabolic conversion of quercetin and its glucosides in human intestinal
cell line caco-2. Arch. Biochem. Biophys. 384:391–397; 2000.
[33] MacDonald, R. C.; MacDonald, R. I.; Menco, B. P.; Takeshita, K.; Subbarao, N. K.;
Hu, L. R. Small-volume extrusion apparatus for preparation of large, unila-
mellar vesicles. Biochim. Biophys. Acta 1061:297–303; 1991.
[3] Symes, W. F.; Dawson, C. R. Poison ivy “urushiol”. J. Am. Chem. Soc. 76:
2959–2963; 1954.

J.Y. Kim et al. / Free Radical Biology and Medicine 71 (2014) 379–389
389
[34] Hoshino, C.; Tagawa, Y.; Wada, S.; Oh, J. H.; Park, D. K.; Nagao, A.; Terao, J.
[43] Bligh, E. G.; Dyer, W. J.
A
rapid method of total lipid extraction and
Antioxidant activity of quercetin against metmyoglobin-induced oxidation of
fish oil-bile salt emulsion. Biosci. Biotechnol. Biochem. 61:1634–1640; 1997.
[35] Arai, H.; Terao, J.; Abdalla, D. S. P.; Suzuki, T.; Takama, K. Coulometric detection
in high-performance liquid chromatographic analysis of cholesteryl ester
purification. Can. J. Biochem. Physiol. 37:911–917; 1959.
[44] Terao, J.; Nagao, A.; Park, D. K.; Lim, B. P. Lipid hydroperoxide assay for
antioxidant activity of carotenoids. Methods Enzymol. 213:454–460; 1992.
[45] Upston, J. M.; Niu, X.; Brown, A. J.; Mashima, R.; Wang, H.; Senthilmohan, R.;
Kettle, A. J.; Dean, R. T.; Stocker, R. Disease stage-dependent accumulation of
lipid and protein oxidation products in human atherosclerosis. Am. J. Pathol.
160:701–710; 2002.
[36] Terao, J.; Matsushita, S. The peroxidizing effect of
hydroperoxides. Free Radic. Biol. Med. 20:365–371;α1996.
-tocopherol on autoxida-
tion of methyl linoleate in bulk phase. Lipids 21:255–260; 1986.
[37] Ioku, K.; Tojiro, T.; Takei, Y.; Nakatani, N.; Terao, J. Antioxidative activity of
quercetin and quercetin monoglucosides in solution and phospholipid
bilayers. Biochim. Biophys. Acta 1234:99–104; 1995.
[46] Leitinger, N. Cholesteryl ester oxidation products in atherosclerosis. Mol.
Aspects Med. 24:239–250; 2003.
[47] Yamamoto, Y.; Niki, E. Presence of cholesteryl ester hydroperoxides in human
blood plasma. Biochem. Biophys. Res. Commun. 165:988–993; 1989.
[48] Steinberg, D.; Parthasarathy, S.; Carew, T. E.; Khoo, J. C.; Witztum, J. L. N.
Beyond cholesterol: modification of low-density lipoprotein that increases its
atherogenicity. N. Engl. J. Med. 320:915–924; 1989.
[49] Esterbauer, H.; Dieber-Rotheneder, M.; Striegl, G.; Waeg, G. Role of vitamin E
in preventing the oxidation of low-density lipoprotein. Am. J. Clin. Nutr.
53:314S–321S; 1991.
[50] Iribarren, C.; Folsom, A. R.; Jacobs Jr. D. R.; Gross, M. D.; Belcher, J. D.; Eckfeldt,
J. H. Association of serum vitamin levels, LDL susceptibility to oxidation, and
autoantibodies against MDA–LDL with carotid atherosclerosis: a case–control
study. Arterioscler. Thromb. Vasc. Biol. 17:1171–1177; 1997.
[38] Niki, E. Free radical initiators as source of water- or lipid soluble peroxyl
radicals. Methods Enzymol. 186:100–108; 1990.
[39] Takahashi, M.; Tsuchiya, J.; Niki, E. Scavenging of radicals by vitamin E in the
membranes as studied by spin labeling. J. Am. Chem. Soc. 111:6350–6353;
1989.
[40] Niki, E. Antioxidants in relation to lipid peroxidation. Chem. Phys. Lipids
44:227–253; 1987.
[41] Murphy, M. P.; Smith, R. A. J. Targeting antioxidants to mitochondria by
conjugation to lipophilic cations. Annu. Rev. Pharmacol. Toxicol. 47:629–656;
2007.
[42] May, J. M. Is ascorbic acid an antioxidant for the plasma membrane? FASEB J.
13:995–1006; 1999.