Novel urushiols with human immunodeficiency virus type 1 reverse transcriptase inhibitory activity from the leaves of Rhus verniciflua

Article abstract of DOI:10.1007/s11418-014-0871-7

Two novel urushiols, 1 and 2, and two known urushiols, 3 and 4, were isolated from the leaves of Rhus verniciflua and were examined for their human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitory activity. The novel urushiols were found to be 1,2-dihydroxyphenyl-3-[7(E),9(Z),11(Z)-pentadecatrienyl]-14-ol (1) and 1,2-dihydroxyphenyl-3-[8(Z),10(E),12(E)-pentadecatrienyl]-14-ol (2) by spectroscopic analyses. The absolute configuration at C-14 in 1 and 2 was determined to be a racemic mixture of (R) and (S) isomers by ozonolysis. Compound 2 (IC₅₀: 12.6 μM) showed the highest HIV-1 RT inhibitory activity among the four urushiols, being 2.5-fold more potent than the positive control, adriamycin (IC₅₀: 31.9 μM). Although the known urushiols were isolated from the sap and leaves of R. verniciflua, 1 was exclusively present in the leaves, and higher amounts of 2 were found in the leaves than in the sap. Present findings indicate that the leaves of R. verniciflua represent a new biological resource from which novel and known urushiols may be prepared, and the possible use of novel urushiols as bioactive products.

Full text of DOI:10.1007/s11418-014-0871-7

J Nat Med (2015) 69:148–153
DOI 10.1007/s11418-014-0871-7
NOTE
Novel urushiols with human immunodeficiency virus type 1
reverse transcriptase inhibitory activity from the leaves of Rhus
verniciflua
•
Kazunari Kadokura Kohei Suruga Tsuyoshi Tomita Wataru Hiruma
•
•
•
•
Motoyuki Yamada Akio Kobayashi Akira Takatsuki Toshiyuki Nishio
•
•
•
•
Tadatake Oku Yoshihiro Sekino
Received: 28 June 2014 / Accepted: 22 September 2014 / Published online: 28 October 2014
Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract Two novel urushiols, 1 and 2, and two known
urushiols, 3 and 4, were isolated from the leaves of Rhus
verniciflua and were examined for their human immuno-
deficiency virus type 1 (HIV-1) reverse transcriptase (RT)
inhibitory activity. The novel urushiols were found to be
1,2-dihydroxyphenyl-3-[70(E),90(Z),110(Z)-pentadecatrienyl]-
140-ol (1) and 1,2-dihydroxyphenyl-3-[80(Z),100(E),120(E)-
pentadecatrienyl]-140-ol (2) by spectroscopic analyses. The
absolute configuration at C-140 in 1 and 2 was determined
to be a racemic mixture of (R) and (S) isomers by ozon-
olysis. Compound 2 (IC50: 12.6 lM) showed the highest
HIV-1 RT inhibitory activity among the four urushiols,
being 2.5-fold more potent than the positive control, adri-
amycin (IC50: 31.9 lM). Although the known urushiols
were isolated from the sap and leaves of R. verniciflua, 1
was exclusively present in the leaves, and higher amounts
of 2 were found in the leaves than in the sap. Present
findings indicate that the leaves of R. verniciflua represent
a new biological resource from which novel and known
urushiols may be prepared, and the possible use of novel
urushiols as bioactive products.
Keywords Rhus verniciflua ꢀ Anacardiaceae ꢀ Leaves ꢀ
Urushiol ꢀ HIV-1 reverse transcriptase inhibitory activity
Introduction
Rhus verniciflua, a member of the Anacardiaceae family, is
commonly known as the lacquer tree [1]. It is used as a
food additive and as traditional herbal medicine to treat
cancer and stomach diseases [2]. The bark, branch and
stem of R. verniciflua have traditionally been eaten as an
additive in chicken and duck soups, especially in Korea [3].
In previous studies, urushiol [4, 5], butein [6], sulfuretin
[7], fustin, fisetin and gallic acid [8] were isolated from the
sap, bark and heartwood of R. verniciflua. Biological
evaluation of the constituents has shown that these com-
ponents have antioxidant [5, 8], antimicrobial [8], anti-
inflammatory [7], and cytotoxic [4] properties and can
inhibit nitric oxide (NO) production [6]. To date, only one
study used the leaves as the study material and revealed the
protective effect of dopaminergic neuronal cells using a
70 % methanol extract of the leaves [9]. However, research
on the utilization of R. verniciflua leaves as a biological
resource has not yet been performed.
K. Kadokura (&) ꢀ K. Suruga ꢀ T. Tomita ꢀ W. Hiruma ꢀ
M. Yamada ꢀ Y. Sekino
Food Material Center for R and D, Kibun Foods Inc., 86
Yanokuchi, Inagi, Tokyo 2060812, Japan
e-mail: kazunari_kadokura@kibun.co.jp
A. Kobayashi
Department of Biotechnology, Graduate School of Engineering,
Osaka University, 2-1 Yamadaoka, Suita, Osaka 5650871, Japan
A. Takatsuki
Department of Frontier Bioscience, Faculty of Bioscience and
Applied Chemistry, Hosei University, 3-7-2 Kajino-cho,
Koganei, Tokyo 1848584, Japan
The present study attempted to isolate novel compounds
from R. verniciflua leaves and to test human immunodefi-
ciency virus type 1 (HIV-1) reverse transcriptase (RT)
inhibitory activity in order to evaluate the leaves as a
biological resource.
T. Nishio ꢀ T. Oku
Department of Chemistry and Life Science, College of
Bioresource Sciences, Nihon University, 1866 Kameino,
Fujisawa, Kanagawa 2520880, Japan
123

J Nat Med (2015) 69:148–153
149
Results and discussion
Compound 1 was pale yellow oil. The FAB-MS of 1
showed a dehydrated ion [MH–H2O]? at m/z 313 in a
positive mode, and a deprotonated-molecular ion [M–H]-
at m/z 329 in a negative mode. This result allowed us to
establish its molecular weight as 330. The molecular for-
mula, C21H30O3 of 1 was deduced from the elemental
analysis data (anal. calcd for C21H30O3: C, 76.33; H, 9.15;
1
O, 14.52. Found: C, 76.37; H, 9.16). The H NMR spec-
trum showed a broad singlet of three protons on a benzene
ring at d 6.69, and signals from an unsaturated and
hydroxylated alkyl group. These signals are characteristic
of 1,2-dihydroxy-3-alkyl-benzene, strongly suggesting that
1 is an urushiol-type compound. Existence of the HMBC
correlation between H-10 (d 2.60), and C-2 (d 142.1) and
C-4 (d 119.9) confirmed that C-10 of the alkyl group bon-
ded at C-3 of a benzene ring. The molecule formula and 1H
and 13C NMR spectra indicated that the alkyl group con-
tained 15 carbons with three double bonds and one hydroxy
group. The position of the double bonds in the alkyl group
was determined by the H–H COSY, HSQC and HMBC
spectra. In the HSQC spectrum, the methyl proton (H-150)
signal at d 1.23 correlated with the 13C signal at d 22.9,
13
showing that the C signal was assigned to C-150. The
H-150 signal is coupled with the proton signal at d 3.89
assignable to protons attached to a carbon having a
hydroxy group. Furthermore, the proton signal at d 3.89
coupled with the proton signal at d 2.36 assignable to
protons adjacent to a double bond. This signal relationship
indicated that the hydroxy group bonded to C-140, and that
a double bond was located at C-110–C-120. The cross peak
between H-140 signal and 13C signal at d 127.2 observed in
13
the HMBC spectrum assigned the C signal to C-120. The
proton signal at d 5.51 assigned to H-120 from the HSQC
spectrum showed a cross peak with the 13C signal d 121.8,
in the HMBC spectrum, so the 13C signal was assigned to
C-100. This indicated that the second double bond was
located at C-100-C-90. The H-100 signal at d 6.13 that
showed a cross peak with C-100 in the HSQC spectrum had
a cross peak with the 13C signal at d 125.5, indicating that
the 13C signal was C-80, and that the third double bond was
located at C-70-C-80. In the HMBC spectrum, C-80 also
showed a cross peak with the proton signal at d 2.12
assignable to H-60 adjacent to a double bond at C-70. The
coupling constants between H-70 and H-80 (J = 15.0 Hz),
H-90 and H-100 (J = 11.0 Hz), and H-110 and H-120
(J = 11.0 Hz) indicated 70E, 90Z and 110Z geometry,
respectively. The chemical structure of 1 was therefore
elucidated to be 1,2-dihydroxyphenyl-3-[70(E),90(Z),110(Z)-
pentadecatrienyl]-140-ol (Fig. 1).
Fig. 1 Structures of isolated compounds 1–4
MS. The 1H and 13C NMR spectra of 2 were very similar to
those of 1 (Table 1) indicating that 2 was an urusiol-type
compound that had three double bonds and one hydroxy
group in the alkyl group. The position of the hydroxy group
was confirmed by the HSQC spectrum to be C-140. In the
HMBC spectrum, the methyl proton (H-150) signal at d
1.30 correlated with the 13C signal at d 136.4 correspond-
ing to an olefinic carbon, indicating that a double bond was
located at C-120-C-130. The positions of two other double
bonds at C-80-C-90 and C-100-C-110 were confirmed by
correlation between H-120 (d 6.27) and C-100 (d 128.8), and
H-100 (d 6.49) and C-80 (d 133.5), respectively, in the
HMBC spectrum. The geometry of C-80-C-90, C-100-C-
110, and C-120-C-130 double bonds were assigned as Z, E,
Compound 2 had the same molecular formula,
C21H30O3 as 1, shown by elementary analysis and FAB-
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J Nat Med (2015) 69:148–153
Table 1 1H and 13C NMR data for compounds 1 and 2 in CDC131
Position
1
2
1H
13C
1H
13C
1
143.2
142.1
129.3
121.9
119.9a
112.9a
29.7
143.2
142.1
129.4
122.0c
120.0c
113.0c
29.8
2
3
4
6.69 bs
6.69 bs
6.69 bs
6.69 bs
6.69 bs
6.69 bs
5
6
10
20
0
0
0
0
2.60 t (J1 ,2 = 7.7 Hz)
1.60 m
2.59 t (J1 ,2 = 7.7 Hz)
1.60 m
29.7
29.7
30
40
50
60
1.35 m
29.3b
29.1b
29.0b
32.9
1.36 m
29.5d
29.4d
29.2d
29.0d
1.35 m
1.36 m
1.42 m
1.36 m
0
0
2.12 dt (J5 ,6 = 13.7, J6 ,7 = 7.0 Hz)
0
0
1.36 m
70
80
90
100
110
120
130
140
150
1
137.2
125.5
130.6
121.8
126.7
127.2
37.2
27.8
0
5.75 dt (J6 ,7 = 7.0, J7 ,8 = 15.0 Hz)
0
0
0
0 0 0 0
2.18 dt (J6 ,7 = J7 ,8 = 7.0 Hz)
0
0
0
6.49 dd (J7 ,8 = 15.0, J8 ,9 = 11.0 Hz)
0
0 0 0 0
5.47 dt (J7 ,8 = 7.0, J8 ,9 = 11.0 Hz)
133.5
128.4
128.8
131.4
130.2
136.4
68.8
0
6.01 dd (J8 ,9 = J9 ,10 = 11.0 Hz)
0
0
0
0 0 0 0
6.01 dd (J8 ,9 = J9 ,10 = 11.0 Hz)
0
0
0
6.13 dd (J9 ,10 = J10 ,11 = 11.0 Hz)
0
0 0 0 0
6.49 dd (J9 ,10 = 11.0, J10 ,11 = 15.0 Hz)
0
6.61 dd (J10 ,11 = J11 ,12 = 11.0 Hz)
0
0
0
0 0 0 0
6.16 dd (J10 ,11 = 15.0, J11 ,12 = 11.0 Hz)
0
5.51 dd (J11 ,12 = 11.0, J12 ,13 = 8.0 Hz)
0
0
0
0 0 0 0
6.27 dd (J11 ,12 = 11.0, J12 ,13 = 15.0 Hz)
0 0 0 0
5.73 dd (J12 ,13 = 15.0, J13 ,14 = 6.4 Hz)
2.36 m
0
3.89 tq (J13 ,14 = J14 ,15 = 6.2 Hz)
0
0
0
0
4.38 dq (J13 ,14 = J14 ,15 = 6.4 Hz)
0
0
0
67.9
1.23 d
22.9
1.30 d
23.3
a
b
c
d
Superscripts , , and on chemical shift date indicate that the assignments could be interchanged within the respective groups
1
and E on the basis of H-1H coupling constants of 11.0,
15.0 and 15.0, respectively. Compound 2 was therefore
identified as 1,2-dihydroxyphenyl-3-[80(Z),100(E),120(E)-
pentadecatrienyl]-140-ol (Fig. 1). Compounds 1 and 2 were
both determined as novel urushiols which possess a
hydroxy group at C-140.
(R) and (S) isomers as well as 1. This result suggested that
the hydroxy group at C-140 of 1 and 2 was not stereose-
lectively introduced by an enzyme-like monooxygenase.
The hydroxy group at C-140 might be introduced by auto-
oxidation although the mechanism is unknown. We could
not separate the optical isomers of 1 and 2 to measure the
biological activity.
The optical rotations of 1 and 2 were nearly zero, sug-
gesting that their specific rotations were very small, or that
both 1 and 2 were racemic. Elucidation of the absolute
configuration at C-140 of 1 was attempted by the Mosher
method [10], but protection of the phenolic hydroxy group
and esterification of the hydroxy group at C-140 by MTPA-
chloride was unsuccessful. We finally elucidated it by
converting the four carbon units from C-120 to C-150 to
3-hydroxybutyric acid by ozonolysis followed by oxidation
with H2O2. 3-Hydroxybutyric acid from 1 was analyzed
with HPLC using a chiral column which can separate
(R) and (S)-3-hydroxybutyric acids. The result showed that
3-hydroxybutyric acid from 1 was completely racemic (R:
The other two compounds were determined to be
3-[80(Z),110(E),130(Z)-pentadecatrienyl] catechol (3) and
3-[80(Z),110(Z),140-pentadecatrienyl] catechol (4) by com-
paring spectral data with the literature [11, 12] (Fig. 1).
Spectroscopic data revealed that compounds 3 and 4 from
the leaves were identical to the structures reported for
major urushiols found in the sap.
HIV-1 RT inhibitory activity of compounds 1–4 were
investigated. Adriamycin, which is known to be a DNA
synthesis inhibitor, served as a positive control for both
HIV-1 RT inhibitory activity and a cytotoxicity assay [13,
14]. Among the test compounds, 2 showed the highest
HIV-1 RT inhibitory activity, with an IC50 value of
12.6 lM, and its activity level was 2.5-fold higher than that
of adriamycin (IC50, 31.9 lM) and 4.4- to 4.5-fold greater
than 3 and 4 (Table 2). Although 1 and 2 are geometric
isomers, they showed more than a 6-fold difference in
activity level.
S = 49.9: 50.0,
0.5), indicating that 1 was a racemic
mixture of (R) and (S) isomers. Compound 2 was oxidized
in the same manner as 1 to give lactic acid containing the
C-130 to C-150 units. HPLC analysis with a chiral column
indicated that lactic acid from 2 was racemic (R: S = 50.3:
49.7,
1.4), indicating that 2 was a racemic mixture of
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J Nat Med (2015) 69:148–153
151
Table 2 HIV-1 RT inhibitory
activity and cytotoxicity in PC-3
and MRC-5 cells of isolated
compounds and in adriamycin
(positive control)
Compounds
Calculated IC50 values (lM) SEMa
HIV-1 RT inhibitory Cytotoxicity
activity
PC-3
MRC-5
1,2-dihydroxyphenyl-3-[70(E),90(Z),110(Z)-
pentadecatrienyl]-140-ol (1)
l,2-dihydroxyphenyl-3-[80(Z),100(E),120(E)-
pentadecatrienyl]-140-ol (2)
3-[80(Z),110(E),130(Z)-pentadecatrienyl] catechol
(3)
78.10 3.52
12.58 3.16
56.84 3.38
61.78 2.81 49.58 8.38
86.31 2.72 48.70 4.78
a
4.56 0.57
7.58 1.03
HIV-1 RT inhibitory activity
and cytotoxicity in PC-3 and
MRC-5 cells are the average
(n = 5) of calculated IC50
values
3-[80(Z),110(E),140-pentadecatrienyl] catechol (4)
55.36 2.36
31.86 0.81
4.53 0.47
0.45 0.07
5.83 0.76
0.39 0.03
Adriamycin
Compounds 1 and 2 showed low cytotoxicity not only
against the noncancerous cell line (MRC-5), but also
against the cancer cell line (PC-3). The cytotoxicity of 1
and 2 against MRC-5 was much lower than 3 and 4 and
adriamycin (Table 2).
College of Forestry of Guizhou University. A voucher
specimen (No.R20020620) was deposited at the Food
Material Center for Research and Development, Kibun
Foods Inc., Tokyo, Japan. The sap of R. verniciflua was
collected from Chengkou, Chongqing, China in July 2012
and was purchased from Hariyo Shikko, Tokyo, Japan.
To evaluate the utility of the leaves as a biological
resource, HPLC analysis was performed using the leaves and
sap extract, after which the amounts of 1–4 in the extract
were calculated. The amounts of 1and 2per 100 gdry weight
of leaf extracts were 12.7 mg and 126.0 mg, respectively,
and the amount of 2 in the sap extract was 22.3 mg; com-
pound 1 was not detected in the sap extract. By comparison,
the amounts of 3 and 4 per 100 g dry weight of leaf and sap
extracts were 20.08 and 8.54 g, and 31.63 and 9.71 g,
respectively. Quantitative analysis of compounds in the
leaves and sap extracts showed that the leaves can be utilized
as a new biological resource for the preparation of urushiols.
Extraction and isolation
The dried and ground leaves of R. verniciflua (0.5 kg) were
extracted with Et2O (5 L) while stirring at room tempera-
ture for 14 h. The solvent was concentrated in vacuo to
yield 28 g. The crude extract was subjected to silica gel
column chromatography (500 g) with n-hexane–EtOAc
(2:3) to afford ten fractions (Fr. 1–10). Fr. 7–9 (361 mg)
were applied to silica gel column chromatography (20 g)
and eluted with n-hexane–EtOAc (4:1) to give five frac-
tions (fr. 6–10) (96.7 mg). The above-mentioned extrac-
tion and isolation steps were repeated five times, and fr.
6–10 (484 mg) were obtained from the dried leaves
(2.5 kg). Fractions (fr. 6–10) were purified by preparative
HPLC [Pegasil-ODS SP-100 (20 9 250 mm), CH3CN–
H2O (48:52) containing 0.05 % acetic acid] to yield com-
pounds 1 (20 mg) and 2 (46 mg).
Materials and methods
General experimental procedures
NMR spectra were recorded on a JEOL ECA500, JNM400A
and Avance III 400 spectrometer (Bruker). Low-resolution
mass spectra were obtained using a JEOL JMS-SX102A
instrument. HPLC was performed using an LC-6A system
(Shimadzu). Pegasil-ODS SP-100 (Sensyu Scientific), Dev-
elosil ODS-5 (Nomura Chemical), CHIRALPAK IC (Dai-
cel), YMC-Pack ODS-AQ (YMC) and Sumichiral OA-6100
(Sumika Chemical Analysis Service) were used for HPLC
columns. Column chromatography was performed using
silica gel 60 N (100–210 lm; Kanto Chemical). TLC was
performed using precoated silica gel 60 F254 plates (Merck).
Part of Fr. 5 (1.6 g) was separated by silica gel column
chromatography (50 g) with n-hexane–EtOAc (10:1) and
subsequently by preparative HPLC [Develosil ODS-5
(10 9 250 mm), CH3CN–H2O (85:15) containing 0.05 %
acetic acid and CHIRALPAK IC (4.6 9 250 mm), n-hex-
ane–CH2Cl2 (90:10)] to yield compounds 3 (41.6 mg) and
4 (37.2 mg).
HPLC analysis
Analyses of compounds 1 and 2 were performed using
Pegasil-ODS SP-100 (4.6 9 250 mm) with CH3CN–H2O
(48:52) containing 0.05 % acetic acid, and analyses of 3
Plant material
The leaves of R. verniciflua were collected from Guizhou,
and
4
(4.6 9 250 mm) with n-hexane–CH2Cl2 (90:10), at a flow
were conducted using CHIRALPAK IC
China in June 2002, and identified by Prof. Zhu Shougian,
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J Nat Med (2015) 69:148–153
rate of 2.0 ml/min with detection at 272 nm. The amounts
of compounds 1–4 in the extracts were calculated from
standard curves derived from isolated compounds.
collected to give 3-hydroxybutyric acid (0.4 mg) from 1
and lactic acid (0.4 mg) from 2. Optical resolution of
3-hydroxybutyric acid and lactic acid were used (Sumich-
iral OA-6100; 4.6 9 150 mm) with 1 mM CuSO4 at a flow
rate of 1.0 ml/min with detection at 254 nm. The retention
times were (R)-3-hydroxybutyric acid, tR9.6 min; (S)-3-
hydroxybutyric acid, tR8.2 min; (R)-lactic acid, tR4.5 min;
and (S)-lactic acid, tR7.5 min.
HIV-1 RT inhibitory activity assay
HIV-1 RT (recombinant, Bio Academia) was adjusted to
4 mU/ll with a solution of 50 mM Tris–HCl buffer (pH
8.0), 100 mM KCl, 50 % glycerol, and 10 mM dithio-
threitol. The reaction mixture (50 ll) containing 50 mM
Tris–HCl (pH 8.0), 100 mM KCl, 6 mM MgCl2, 10 mM
dithiothreitol, 2.0 lg/ml (ca. 6 lM) polyA (dT)15 as a
template primer, 3 lM dTTP, 0.33 lM [methyl-3H] dTTP
(37 MBq/ml, Moravek Biochemicals), 40 mU of RT, and
10 ll of compound dissolved in DMSO (final concentra-
tion, 10 %) was incubated at 37 °C for 20 min. The reac-
tion was terminated by the addition of 20 ll of 0.1 M
EDTA. The resulting mixture (50 ll) was applied to a
Whatman DE81 filter and washed four times with 5 %
Na2HPO4, and twice with distilled water. The filter was
then dried and immersed in 5 ml of scintillation fluid. The
amount of polymer fraction was determined by measuring
the radioactivity on the filter according to the incorporation
of [3H]-labelled substrate into the polynucleotide products.
Acknowledgments We are extremely grateful to Representative
Chairman Masahito Hoashi, Kibun Foods, Inc., for supporting the
present work. We are indebted to Professor Emeritus Haruki Yamada,
Kitasato University for his perceptive suggestions. We are grateful to
Professor Nobuhiro Hirai, Kyoto University for analysis of the NMR
spectra and advice on determination of absolute configuration. We
also express our sincere thanks to Professor Shinichiro Kajiyama,
Kinki University (formerly Osaka University), and Associate Pro-
fessor Wataru Hakamata, Nihon University, for recording the NMR
spectra.
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