Ent-Eudesmane sesquiterpenoids, galloyl esters of the oak lactone precursor, and a 3-O-methylellagic acid glycoside from the wood of Platycarya strobilacea

Article abstract of DOI:10.1016/j.phytochem.2011.02.020

ent-Eudesmane sesquiterpenoids, 8,11-dihydroxy-2,4-cycloeudesmane, 11-hydroxy-2,4-cycloeudesman-8-one and 2,4-cyclo-7(11)-eudesmen-8-one, were isolated from the wood of Platycarya strobilacea, which has been used as an aromatic tree since at least the 18th century. On charring the wood, 2,4-cyclo-7(11)-eudesmen-8-one was detected in the smoke. In the charred wood, the concentrations of ellagitannins, such as galloyl pedunculagin, dramatically decreased, whereas concentrations of pentagalloyl glucose, and other gallotannins were relatively stable. In addition, two other compounds, the 6′-O-m- and p-digalloyl oak lactone precursor and the 3-O-methylellagic acid 4′-O-(4″-O-galloyl)-xylopyranoside, were isolated from the charred wood along with m- and p-digallic acid.

Full text of DOI:10.1016/j.phytochem.2011.02.020

Phytochemistry 72 (2011) 796–803
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
ent-Eudesmane sesquiterpenoids, galloyl esters of the oak lactone precursor, and
a 3-O-methylellagic acid glycoside from the wood of Platycarya strobilacea
Hajime Maeda a,b, Narumi Kakoki a, Mami Ayabe a, Yuki Koga a, Tomoko Oribe a, Yosuke Matsuo a,
Takashi Tanaka a, , Isao Kouno a,
⇑
⇑
a Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan
b Nagasaki Agricultural and Forestry Technical Development Center, 3118 Kaidzu, Isahaya, Nagasaki 854-0063, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 October 2010
Received in revised form 16 February 2011
ent-Eudesmane sesquiterpenoids, 8,11-dihydroxy-2,4-cycloeudesmane, 11-hydroxy-2,4-cycloeudesman-
8-one and 2,4-cyclo-7(11)-eudesmen-8-one, were isolated from the wood of Platycarya strobilacea, which
has been used as an aromatic tree since at least the 18th century. On charring the wood, 2,4-cyclo-7(11)-
eudesmen-8-one was detected in the smoke. In the charred wood, the concentrations of ellagitannins,
such as galloyl pedunculagin, dramatically decreased, whereas concentrations of pentagalloyl glucose,
and other gallotannins were relatively stable. In addition, two other compounds, the 60-O-m- and p-digal-
loyl oak lactone precursor and the 3-O-methylellagic acid 40-O-(400-O-galloyl)-xylopyranoside, were iso-
lated from the charred wood along with m- and p-digallic acid.
Keywords:
Platycarya strobilacea
Juglandaceae
Eudesmane
Tannin
Ó 2011 Elsevier Ltd. All rights reserved.
Pyrolysis
1. Introduction
wood was compared before and after charring because charred
wood is used as incense.
Platycarya strobilacea Sieb. et Zucc. (Juglandaceae) is a woody
plant present across East Asia (Atkinson and Upson, 2006), whose
fruits, leaves, bark and wood contain a large amount of ellagitan-
nins used for dyeing (Tanaka et al., 1993, 1998). The use of the
old wood as an incense appeared originally in an old Japanese
scripture, ‘‘Yamato-Honzo,’’ written in 1709. The wood appears
to be used as a substitute for expensive Agarwood (Aquilaria agal-
locha) (Naf et al., 1995; Ito and Honda, 2005). In a previous study,
the occurrence of (3S, 4S)-3-methyl-4-hydroxyoctanoic acid 4-O-b-
2. Results and discussion
2.1. Structures of terpenoids
The MeOH and aqueous acetone extracts of the wood of P. stro-
bilacea were successively partitioned with n-hexane and EtOAc.
Three new terpenoids, 8,11-dihydroxy-2,4-cycloeudesmane (1),
11-hydroxy-2,4-cycloeudesman-8-one (2) and 2,4-cyclo-7(11)-
D
-glucopyranoside and its 60-O-gallate (5) in P. strobilacea wood
was established (Tanaka and Kouno, 1996). These compounds are
precursors of oak lactone (3-methyl-4-octanolide), and subse-
quently, the presence of the precursors in oak wood was confirmed
(Masson et al., 2000). Oak lactone is an important compound con-
tributing to the flavor of whisky, wine and brandy aged in barrels
made of oak wood (Onishi et al., 1977; Otsuka et al., 1974). In
the course of research to identify new plant sources containing
functional compounds in the Nagasaki region of Japan (Maeda
et al., 2009), the non-polar constituents of the wood of P. strobila-
eudesmen-8-one (3), were isolated along with 3-hydroxy-a-cala-
corene (4) from the n-hexane extract. The EtOAc fraction primarily
contained phenolic compounds, and the 60-O-galloyl oak lactone
precursor (5) (Tanaka and Kouno, 1996), gallic acid, ellagic acid,
1,2,3,4,6-penta-O-galloyl-b-D-glucose (6) (Nishizawa et al., 1982),
eugeniin (7) (Nonaka et al., 1980), pedunculagin (8) (Schmidt
et al., 1965), 1(b)-galloylpedunculagin (9) (Gupta et al., 1980),
casuariin (10) and casuarinin (11) (Okuda et al., 1983) were iso-
lated by a combination of column chromatography steps using
Sephadex LH-20, MCI-gel CHP20P, Diaion HP20P and Chromatorex
ODS materials, respectively (Fig. 1).
cea were examined, because it may represent
a promising
fragrance material. Furthermore, the chemical composition of the
Compound 1 was obtained as colorless needles from n-hexane–
acetone and its molecular formula, C15H26O2, was confirmed by HR
EIMS. The 13C NMR spectrum exhibited 15 aliphatic carbon signals
assignable to three quaternary, four methine, four methylene and
four methyl groups (Table 1). The chemical shifts of the quaternary
⇑
Corresponding authors. Tel.: +81 95 819 2433; fax: +81 95 819 2477 (T.
Tanaka).
E-mail addresses: t-tanaka@nagasaki-u.ac.jp (T. Tanaka), ikouno@nagasaki-u.
ac.jp (I. Kouno).
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.02.020

H. Maeda et al. / Phytochemistry 72 (2011) 796–803
797
14
1
galloyl
O
8
OH
HO
HO
2
10
5
12
4
11
OH
OH
HO
CO2H
3
7
O
H
OH
H
OH
15
C
O
O
1
2
6
OH
3
1
HO
OH
O
5
CO2H
O
OH
O
HO
HO
O
4
9
HO
OH
OH
8
H
O
O
3
4
7
OH
HO
HO
O
C
HO
O
OH
OH
HO
HO
OH
OH
O
O
HO
HO
O
O
O
O
C
O
O
O
O
C
O
OH
O
O
O
C
OH
OH
HO
HO
C
O
OH
C
O
O
C
O
HO
OH
OH
O
C
O
OH
OH
HO
HO
OH
OH
OH
OH
OH
8
9
OH
OH
OH
HO
HO
O
O
O
O
R
O
HO
HO
HO
HO
O
OH
H
O
O
O
O
O
O
O-R
O
O
O
O
O
O
HO
HO
OH
HO
OH
OH OH
HO
OH
OH OH
HO
HO
HO
HO
10 : R = H
11 : R = β-galloyl
12 : R = H
13 : R = galloyl
Fig. 1. Structures of compounds isolated from fresh wood.
(d 73.2) and methine (d 69.2) carbons indicated that these carbons
are attached to oxygen atoms. The presence of hydroxy groups was
supported by IR absorption bands at 3300 cmꢀ1. The 1H NMR spec-
trum exhibited signals attributable to four methyl groups (Me-12,
Me-13, Me-14 and Me-15) and one oxygen-bearing methine (d
4.62, dt, J = 3.0 Hz, H-8) protons. In addition, a pair of characteristic
methylene protons observed at d 0.54 (t, J = 3.9 Hz) and 0.99 (dd,
J = 3.9, 8.2 Hz) suggested the presence of a cyclopropyl ring. This
was supported by the appearance of an IR absorption band at
3042 cmꢀ1 and the degree of unsaturation (3) calculated from
the molecular formula. With the aid of 1H–1H COSY analysis, two
strings of carbon connections C-1–C-2–C-3 and C-5–C-6–C-7–C-
8–C-9 were demonstrated (Fig. 2). The aforementioned cyclopropyl
methylene protons at d 0.54 and 0.99 were assigned to the
C-3methylene protons. Furthermore, the HMBC correlations illus-
trated in Fig. 2 established the structure of 1: HMBC correlations
of H-15 with C-3, C-4, and C-5 indicated that the C-15 methyl
group was located at the C-4 quaternary carbon. Similarly the
C-14 methyl carbon was shown to be attached to the C-10 quater-
nary carbon by HMBC correlations of H-14 with C-1, C-10, C-9 and
C-5. This observation and the correlation of H-5 with C-10 indi-
cated a connection between C-5 and C-10. The location of the
hydroxyisopropyl group at C-7 was established by correlations of
C-7 with H-12 and H-13. Therefore, 1 was identified as a 2,4-
cycloeudesmane-8,11-diol. As for its configuration, the small cou-
pling constant (J = 3.0 Hz) of H-8 with H-7 and H-9 indicated that
the C-8 hydroxy group was in an axial orientation. Furthermore,
NOESY correlations between H-14 and H-15, between H-3 and H-
5, and between H-5 and H-7, unambiguously confirmed the rela-
tive configuration of 1 (Fig. 2).
Compound 2 was isolated as a colorless syrup and the molecu-
lar formula was determined to be C15H24O2 by HR-EIMS. The 13C
NMR spectra (Table 1) were related to those of 1 except for the
appearance of a carbonyl carbon signal at d 215.4 in the spectra
of 2 instead of the C-8 oxymethine of 1. The HMBC correlations
of the carbonyl carbon with H-6 and H-7 indicate that the carbonyl

798
H. Maeda et al. / Phytochemistry 72 (2011) 796–803
Table 1
1H-(500 MHz), 13C-(125 MHz) NMR spectroscopic data for compounds 1, 2, and 3 (d in ppm, J in Hz).
Position
1
1
2
3
1Ha
13Cb
45.7
1Hb
13Cb
43.9
1Hb
13Cb
44.6
0.86 (br dd, 3.4, 12.0)
1.84 (dd, 6.6, 12.0)
1.22 (m)
0.98 (br d, 4.0, 12.7)
1.76 (dd, 6.8, 12.7)
1.20 (m)
0.95 (br dd, 3.2, 12.7)
1.82 (dd, 6.8, 12.7)
1.21 (dddd, 3.2, 4.0, 6.8, 8.3)
0.39 (t, 4.0)
2
3
18.7
33.6
26.8
33.5
26.8
33.4
0.54 (t, 3.9)
0.44 (t, 4.4)
0.99 (dd, 3.9, 8.2)
0.92 (dd, 4.4, 8.3)
0.91 (dd, 4.0, 8.3)
4
5
6
27.1
56.8
19.3
26.0
54.0
25.2
26.4
52.2
27.0
1.05 (dd, 3.0, 13.0)
2.06 (dt, 13.0, 3.0)
2.16 (q, 13.0)
1.33 (dt, 13.0, 3.0)
4.62 (dt, 3.0, 3.0)
1.36 (dd, 3.0, 14.2)
2.06 (dd, 3.0, 14.2)
1.52 (m)
2.13 (m)
2.23 (m)
2.21 (m)
1.42 (dd, 5.2, 14.2)
2.28 (dddd, 0.9, 2.1, 14.2, 15.1)
2.71 (ddd, 0.7, 5.2, 15.1)
7
8
9
51.1
69.2
45.2
58.8
215.4
54.8
130.5
203.6
54.2
2.18 (d, 13.2)
2.25 (d, 13.2)
2.18 (br d, 15.9)
2.31 (d, 15.9)
10
11
12
13
14
15
50.9
73.2
29.1
28.8
21.7
26.3
54.3
71.9
28.6
25.2c
20.2
18.9
52.1
146.3
23.1
23.6
20.6
19.1
1.50 (s)
1.62 (s)
1.51 (s)
1.23 (s)
1.25 (s)
1.20 (s)
0.88 (s)
1.11 (s)
1.83 (d, 0.9)
2.01 (dd, 0.7, 2.1)
1.00 (d, 0.9)
1.17 (s)
a
b
c
Measured in C5D5N.
Measured in CDCl3.
Assignments may be interchanged.
14
1
2
1
2
9
OH
8
10
5
8
7
3
12
13
11
5
7
4
15
14
H
OH
12
1 -1
15
H
H COSY
HMBC
NOESY
Fig. 2. Selected 1H–1H COSY, HMBC and NOESY correlations observed for 1.
group is located at C-8 (Fig. 3). The 1H–1H couplings between H-1,
H-2 and H-3, and between H-5, H-6 and H-7 were similar to those
of 1, suggesting that the remaining portion of the structure was the
same as that of 1. Thus, compound 2 was concluded to be 11-hy-
droxy-2,4-cycloeudesman-8-one.
Compound 3 was obtained as a colorless syrup and the molec-
ular formula was determined to be C15H22O by HR-EIMS. UV
absorption at 251 nm suggested the presence of a conjugated car-
bonyl group, and this was confirmed by observation of a carbonyl
carbon signal at d 203.6 (C-8) and olefinic carbon resonances at d
130.5 (C-7) and 146.3 (C-11). In the 1H NMR spectrum, the signals
attributable to H-1–H-3 were related to those of 1 and 2. Two
methyl groups resonating at d 1.83 (H-12) and 2.01 (H-13) were
found to be attached to a double bond based on their chemical
shifts. In the 1H–1H COSY spectrum, one of the methyl groups
(H-12) showed a long-range allyl-coupling with the C-6 methylene
14
14
1
1
O
O
2
2
8
8
12
12
3
3
4
4
H
H
OH
15
15
3
2
Fig. 3. Selected 1H–1H COSY and HMBC correlations observed for 2 and 3.

H. Maeda et al. / Phytochemistry 72 (2011) 796–803
799
protons. Furthermore, HMBC correlations of 3 (Fig. 3), including a 4J
correlation between H-13 and C-8, confirmed the planar structure
of 3.
13
ea
g
9
5
18
Efforts to determine the absolute configuration by application
of a modified Mosher’s method to 1 failed (Ohtani et al., 1991).
The MTPA esters of 1 were unstable because of the axial orienta-
tion of the C-8 hydroxy group and the presence of the bulky substi-
tuent at the adjacent C-7. Therefore, we compared the CD data of 3
g
6
11
7
ga
g
16
8
with those of steroidal and related cisoid-a,b-unsaturated ketones.
Based on computer aided molecular modeling, the torsion angle of
the cisoid-enone moiety was estimated to be about 29°, and the CD
17
g
spectrum of
3
showed
a
negative Cotton effect at 330 nm
10
(D
e
ꢀ 5.0). It has been reported that cisoid-
a
,b-unsaturated ke-
tones with negative torsion angles show negative Cotton effects
in the range of 320–340 nm (Snatzke, 1965; Jadwiga et al., 1996).
Therefore, the molecular structure of compound 3 is presented in
Fig. 4. Oxidation of 1 with K2Cr2O7 afforded 2, and subsequent
treatment of 2 with trifluoroacetic acid yielded 3. Thus, the abso-
lute configuration of 1 and 2 is determined to be the same as that
of 3.
ga
sa
ca
13
15
ea
6
g
5
g
7
12
2.2. Charring of wood
16
g
The wood of P. strobilacea is charred when it is used as incense;
therefore, we next examined whether the terpenoids are detect-
able in the smoke generated from charring the wood. The smoke
was collected using a large stainless funnel connected to an aspira-
tor and compounds in the smoke were trapped by CHCl3 placed be-
tween the funnel and the aspirator. TLC and HPLC of the CHCl3
soluble compounds revealed the presence of 3 in the smoke; how-
ever, 1, 2 and 4 were not detected. In addition, despite the presence
of a relatively high concentration of the oak lactone precursor (5)
17
9
Fig. 5. Reversed-phase HPLC of EtOAc soluble fraction of P. strobilacea wood. (A)
EtOAc fraction of fresh wood. (B) EtOAc fraction of charred wood. g, Unidentified
galloyl glucoses; ga, gallic acid; ea, ellagic acid, ca, coniferylaldehyde; sa,
sinapylaldehyde.
in the wood, oak lactone (b-methyl-c-octalactone) was also de-
tected within the n-hexane layer of the extract. Compound 3
may be responsible for the characteristic odor of this wood.
TLC analysis showed that the n-hexane soluble fraction of the
charred wood contained compounds 1–4. The amount of com-
pound 3 was higher than the levels found in fresh wood, probably
due to the dehydration of compound 2. Important differences of
constituents were observed in the EtOAc fractions: the HPLC of
the EtOAc soluble fraction obtained from the charred wood showed
some peaks that were not observed in the analysis of the soluble
fractions obtained from fresh wood (Fig. 5). New strong peaks at
18.0 and 20.3 min were identified to be an equilibrium mixture
of m- and p-digallic acid (15) (Fig. 6) (Nishizawa et al., 1982). Pro-
duction of 15 was deduced to be via acyl migration of galloyl
groups or dehydrative dimerization of gallic acid. Heating of either
1,2,3,4,6-penta-O-galloylglucose (6) or gallic acid at 200 °C for
30 min afforded 15. However, it is also possible that 15 was gener-
ated by partial hydrolysis of unknown galloyl esters with m- and p-
digalloyl esters originally contained in the fresh wood. Another
remarkable difference observed in the chromatograms is the sig-
nificant decrease of ellagitannins pedunculagin (8), 1(b)-O-galloyl
pedunculagin (9), casuariin (10) and casuarinin (11). This phenom-
enon is probably because ellagitannins are more sensitive to radi-
cal oxidation (Barbehenn et al., 2006). The products produced from
ellagitannins remain unknown. The peak area of ellagic acid did
not increase, suggesting that simple hydrolysis is not important.
From the EtOAc layer of charred wood, coniferylaldehyde, sina-
pylaldehyde, vanillin, syringaldehyde and (ꢀ)-episyringareginol
(14), were identified. These compounds were presumably gener-
ated by the pyrolysis of lignins, and the sweet aroma of vanillin
contributed to the characteristic scent of the charred wood (Sarni
et al., 1990). In addition, quercetin 3-O-rhamnoside (12), myricetin
3-O-rhamnoside (13) and two new compounds 16 and 17 were iso-
lated. The 1H- and 13C NMR spectra of compound 16 was similar to
the spectra of the oak lactone precursor (5) and m- and p-digallic
acid (15). The ESI-TOF MS (m/z: 663, M + Na) indicated that the
additional galloyl ester was attached to 5. From 13C NMR spectro-
scopic comparison with 15, the location of the additional galloyl
group was deduced to be the phenolic hydroxygroup of the galloyl
group at the glucose C-6. The structure was also confirmed by
enzymatic hydrolysis of 16, which yielded gallic acid and (3S,4S)-
3
8
5
2
7
6
11
3-methyl-4-hydroxyoctanoic acid 4-O-b-D-glucopyranoside (Tana-
12
15
14
ka and Kouno, 1996). Accordingly, compound 16 was characterized
to be 60-O-p- and m-digalloyl-(3S,4S)-3-methyl-4-hydroxyoctanoic
acid 4-O-b-D-glucopyranoside.
Characteristic UV absorptions at 361 and 250 nm of compound
Fig. 4. Stereostructure of 3.
17 suggested that this compound is a derivative of ellagic acid. The

800
H. Maeda et al. / Phytochemistry 72 (2011) 796–803
R
OH
OH
H3CO
HO
H3CO
HO
HO
O
O
CHO
CHO
O
R
R
OH
O
OH
OH
OH
H3C
16 : R = H
17 : R = OCH
14 : R = H
15 : R = OCH
18 : R = H
19 : R = OH
3
3
OCH3
HO
OH
C
OH
HO
HO
OH
OH
O
O
H3CO
H
H
OH
O
O
OCH3
OH
C
O
O
HO
HO
CO2H
CO2H
HO
OCH3
20
21
HO
HO
OH
G
1'
HO
O
G'
HO
OH
m
O
O
1
6'
glucose
HO
G
xylose
O
O
6
OH
1
CO2H
C
4O
6
O
HO
HO
1
7
O
1
O
4'
O
O
O
OH
HO
3
m
-digalloyl
OH
OH
4
1
5
9
5'
1'
8
HO
HO
HO
HO
O
O
OH
OCH3
3
O
p
O
O
23
HO
p-digalloyl
22
Fig. 6. Structures of compounds isolated from charred wood.
1H NMR spectrum showed signals attributable to an O-methyl (d
4.12, 3H, s) and a galloyl group (d 7.07, 2H, s), along with two aro-
matic singlets of the ellagic acid moiety (d 7.39, 7.22) and sugar
vation is important in the use of the wood as a fragrance material.
The observation of a rapid decrease of ellagitannins compared to
gallotannins on wood charring is also an interesting finding. The
preparation of oak lactone by hydrolysis of the precursor in wood
and the application of the wood or its extract as a fragrance mate-
rial is currently under investigation.
proton signals. The sugar was shown to be b-D-xylose based on
the large coupling constants of the proton signals (J1,2 = 7.3,
J2,3 = J3,4 = 9.0 Hz). The acylation of the xylose C-4 hydroxy group
was apparent from the large low field shift (d 5.03). Location of
the xylose and the methyl group on the ellagic acid moiety was
determined by HMBC spectroscopic analysis (Fig. 6), and con-
firmed by enzymatic hydrolysis of the galloyl ester with tannase
yielding 3-O-methylellagic acid 40-O-xylopyranoside (Tanaka
et al., 1998). Thus, the structure of 17 was determined to be 3-O-
methylellagic acid 40-O-(400-O-galloyl)-xylopyranoside. The new
galloyl esters 16 and 17 are also detected in fresh wood; however,
HPLC comparison (Fig. 5) indicates that the concentration of 17 is
higher in fresh wood.
4. Experimental
4.1. General experimental procedures
UV spectra were obtained with a Jasco V-560 UV/Vis spectro-
photometer and optical rotations were measured with a Jasco
DIP-370 digital polarimeter (Jasco Co., Tokyo, Japan). 1H- and 13C
NMR spectra were measured in CD3OD at 27 °C using a Varian
Unity plus 500 spectrometer (500 MHz for 1H and 125 MHz for
13C) (Varian, Palo Alto, CA, USA). Coupling constants are expressed
in Hz and chemical shifts are given on a d (ppm) scale. MS were re-
corded on a Voyager DE-PRO (Applied Biosystems, USA) and a JEOL
JMS-700N spectrometer (JEOL Ltd., Tokyo, Japan). 2,5-Dihydroxy-
benzoic acid and glycerol were used as the matrix for MALDI-
TOF-MS and FAB-MS measurements, respectively. HR ESI TOF MS
was measured with a JMS-T100TD system (JEOL Ltd., Japan). Col-
umn chromatography (CC) was performed using Diaion HP20SS
3. Conclusions
In this study, presence was demonstrated of new ent-eudes-
mane type sesquiterpenes in the wood of P. strobilacea, which
has been recorded as an incense wood in since the 18th century.
On charring the wood, compound 3 with an enone structure was
detected in the smoke. The presence of 3 is probably responsible
for the characteristic odor of this wood on charring, and this obser-
(Mitsubishi Chemical, Japan), Sephadex LH-20 (25–100 lm; GE

H. Maeda et al. / Phytochemistry 72 (2011) 796–803
801
Healthcare Bio-Science AB, Uppsala), and Chromatorex ODS (100–
200 mesh; Fuji Silysia Chemical, Tokyo, Japan) columns. TLC was
performed on precoated Kieselgel 60 F254 plates (0.2 mm thick;
Merck, Darmstadt, Germany) with n-hexane–EtOAc (9:1, v/v), n-
hexane–acetone (5:1, v/v) and toluene- HCO2Et–HCO2H (1:7:1, v/
v) as a solvent, and spots were detected by UV illumination
(254 nm) and by spraying with a 2% ethanolic FeCl3 and 10%
H2SO4 reagent, followed by heating. Analytical HPLC was per-
formed on a Cosmosil 5C18-AR II (Nacalai Tesque Inc., Kyoto, Japan)
column (4.6 mm i.d. ꢁ 250 mm) with gradient elutions from 4% to
30% (39 min) and 30–75% (15 min) of CH3CN in 50 mM H3PO4 (for
polyphenols) or from 70% to 100% (15 min) and 100% (10 min)
CH3CN in 50 mM H3PO4 (for terpenoids). The flow rate was
0.8 mL/min and detection was achieved using a JASCO photodiode
array detector MD-910.
(in C5D5N) and 13C (in CDCl3) for NMR spectroscopic data, see Table
1. 1H NMR (400 MHz, CDCl3) d: 0.34 (1H, t, 3.9, H-3), 0.87 (2H, m,
H-1, H-3), 1.14, 1.19, 1.27, 1.37 (each 3H, s, H-12, H-13, H-14, H-
15), 1.69–1.84 (4H, m, H-1, H-9, H-6) and 4.37 (1H, br s, H-8).
4.3.2. 11-Hydroxy-2,4-cycloeudesman-8-one (2)
Colorless syrup,
[
a
]
ꢀ34.2 (c 0.3, EtOH), HR EI-MS m/z
D
236.1754 (C15H24O2 requires 238.1772), IR mmax cmꢀ1: 3509,
2951, 1688, 1455. For 1H and 13C NMR spectroscopic data, see
Table 1.
4.3.3. 2,4-Cyclo-7(11)-eudesmen-8-one (3)
Colorless syrup,
[a
]
D
ꢀ86.5 (c 0.3, EtOH), HR EI-MS m/z
218.1665 (C15H22O requires 218.1672), IRmmax cmꢀ1: 2941, 1673,
1590, 1454, UV kmax nm (log
e): 251 (3.73). CD (EtOH,
9.72 ꢁ 10ꢀ5 mol/L) nm (
D
e
): 330 (ꢀ5.0), 243 (13). For 1H and 13C
4.2. Plant material
NMR spectroscopic data, see Table 1.
The wood of P. strobilacea was collected at Tsushima Island,
Nagasaki, Japan, in May 2008, and was identified by Associate Pro-
fessor K. Yamada of laboratory in Medical Plants Garden of Naga-
saki University. A voucher specimen (N-0805T1) was deposited
at the Nagasaki Agricultural and Forestry Technical Development
Center, Japan.
4.3.4. Oxidation of 1
A solution of 1 (20 mg) and K2Cr2O7 (50 mg) in AcOH (0.3 mL)
was heated at 80 °C for 40 min with stirring. The mixture was trea-
ted with i-PrOH (1.5 mL) at room temperature for 3 h to decom-
pose excess oxidizing agent. After concentration, products were
extracted with n-hexane (2 mL ꢁ 3). TLC analysis of the n-hexane
soluble showed presence of 2 and 3. The n-hexane extract was
treated with 10% CF3CO2H in MeOH (3 mL) for 12 h at room tem-
perature. The mixture was concentrated and separated by silica
4.3. Extraction and separation
The wood of P. strobilacea (2.0 kg) was mechanically chipped
into small pieces (about 0.5–1 cm long, 0.2–0.5 cm thick) and ex-
tracted (each 2 days, 25 °C) with acetone–H2O (5 L, 3:2, v/v) (two
times) and MeOH (5 L, 3 times). The extracts were combined, con-
centrated and successively partitioned with n-hexane and EtOAc
to give a n-hexane extract (10.3 g) and an EtOAc extract (50.6 g).
The n-hexane extract was separated by silica gel CC eluted with
n-hexane containing increasing proportions of EtOAc (0–100%,
10% stepwise) to give six fractions. Fr. H1 (5.2 g), mainly contain-
ing wax and triglycerides, was further separated by silica gel CC
(10–100% CHCl3 in n-hexane, and then 2–10% MeOH in CHCl3) into
seven fractions. Fractions H1-5 and H1-6 were separately sub-
jected to silica gel CC with n-hexane–CHCl3–acetone (90:9:1 and
gel CC (n-hexane–EtOAc) to give 3 (10.3 mg), [
EtOH).
a
]
ꢀ74.7 (c 0.4,
D
4.3.5. Conversion of 2 to 3
A solution of 2 (10 mg) in 10% CF3CO2H in MeOH (1 mL) was
stirred at room temperature for 12 h. The mixture was concen-
trated and separated by silica gel CC (n-hexane–EtOAc) to give 3
(6.3 mg), [
a]
ꢀ86.6 (c 0.2, EtOH).
D
4.4. Separation of the compounds from the charred wood
Wood blocks (10 cm wide ꢁ 10 cm long ꢁ 3 cm high) were
burned with a gas burner until the color of the surface was black
and then mechanically chipped into small pieces as described for
the experiment using fresh wood. The charred chips (2.1 kg) were
extracted and fractionated as described above to give a n-hexane
soluble fraction (13.7 g) and an EtOAc fraction (48.2 g). TLC and
HPLC analysis of the n-hexane fraction showed that the composi-
tion was similar to that of the fraction obtained from fresh wood.
The EtOAc fraction was separated by Sephadex LH-20 CC [EtOH,
EtOH–H2O (4:1, v/v), and then acetone–H2O (3:2, v/v)] into four
fractions: CA-1 (5.0 g), CA-2 (16.6 g), CA-3 (6.5 g), and CA-4
(15.7 g). The CA-1 fraction was successively subjected to Sephadex
LH-20 (MeOH–H2O, 4:1, v/v) and silica gel (n-hexane–acetone,
1:0–1:1) CC to give 5 (329 mg), coniferylaldehyde (47.3 mg), sina-
pylaldehyde (71.2 mg) and (ꢀ)-episyringareginol (14) (27.7 mg).
The presence of vanillin and syringaldehyde in Fr. CA-1 was con-
firmed by HPLC comparison with authentic samples. Fr. CA-2 was
separated by Sephadex LH-20 (EtOH) to give three fractions:
CA-2-1 (4.9 g), CA-2-2 (10.2 g) and CA-2-3 (0.6 g). CA-2-2 was crys-
tallized from H2O to give gallic acid (4.3 g). Successive chromatog-
raphy of Fr. CA-2-1 over MCI-gel CHP20P CC with H2O containing
increasing proportions of MeOH (0–80%, 10% stepwise) and Sepha-
dex LH-20 CC with H2O containing MeOH (40%) yielded gallic acid
(2.4 g) and compound 16 (52.6 mg). Fr. CA-3 was dissolved in H2O
to give crystalline ellagic acid (0.8 g), and the mother liquor was
separated by MCI-gel CHP20P CC with H2O containing increasing
proportions of MeOH (0–100%, 10% stepwise) to give gallic acid
(108 mg), quercetin 3-O-rhamnoside (12) (355 mg), myricetin 3-
then 80:18:2) to give 3-hydroxy-a-calacorene (4) (43 mg) from
Fr. H1-5 and 2,4-cyclo-7(11)-eudesmen-8-one (3) (163 mg) from
Fr. H1-6. Fr. H3 was applied to silica gel CC with n-hexane–EtOAc
(9:1–8:2) to yield 11-hydroxy-2,4-cycloeudesman-8-one (2)
(193 mg). Fr. H5 was crystallized from n-hexane–acetone to give
8,11-dihydroxy-2,4-cycloeudesmane (1) (426 mg) as colorless fine
needles.
The EtOAc extract was separated by Sephadex LH-20 CC with
EtOH containing increasing proportions of H2O (0–40%, 10% step-
wise) and finally 60% acetone to give nine fractions: Fr. A1–A9.
Fr. A2 (6.9 g) was successively subjected to Sephadex LH-20 (60%
MeOH) and MCI-gel CHP20P (0–70% MeOH, 10% stepwise) CC to
yield gallic acid (78 mg) and oak lactone presursor (5) (1.3 g).
Recrystallization of Fr. A4 (1.6 g) from H2O afforded colorless nee-
dles of ellagic acid (691 mg). Fr. A8 (8.8 g) was successively sepa-
rated by Diaion HP20SS and Chromatorex ODS CC with H2O
containing increasing amount of MeOH (0–60% MeOH, 10% step-
wise) to give casuariin (10) (22 mg), pedunculagin (8) (222 mg),
casuarinin (11) (225 mg), 1,2,3,4,6-penta-O-galloyl-b-D-glucose
(6) (286 mg), and eugeniin (7) (374 mg). Fr. A9 (1.5 g) was identi-
fied to be 1(b)-galloylpedunculagin (9).
4.3.1. 8,11-Dihydroxy-2,4-cycloeudesmane (1)
Colorless needles (n-hexane–acetone), mp 147–149 °C, [a]
D
+0.1 (c 0.3, EtOH), HR EI-MS m/z 238.1914 (C15H26O2 requires
238.1934), IR mmax cmꢀ1: 3302, 3042, 2947, 1454, 1378. For 1H

802
H. Maeda et al. / Phytochemistry 72 (2011) 796–803
O-rhamnoside (13) (122 mg), and meta- and para-digallic acid (15)
(295.3 mg). Fr. CA-4 separated by Diaion HP20SS CC with H2O con-
taining increasing proportions of MeOH (0–100%, 10% stepwise) to
yield pentagalloylglucose (6) (2.19 g) and compound 17 (116.2 mg)
along with a mixture of tetragalloyl glucoses (3.83 g).
solution was concentrated and examined by TLC and HPLC, and
the presence of 3 in the smoke was confirmed.
4.7. Hydrolysis of 16
An aqueous solution of 16 (10 mg) was treated with tannase
(5 mg, from Aspergillus niger) at room temperature for 10 h. The
mixture was separated by Sephadex LH-20 CC with H2O containing
increasing proportions of MeOH (0–40%, 10% stepwise) and silica
gel (CHCl3–MeOH–H2O, 40:10:1 and 14:6:1) to give the oak lac-
tone precursor (2.4 mg) (Tanaka and Kouno, 1996).
4.4.1. 60-O-p- and m-digalloyl-(3S,4S)-3-methyl-4-hydroxyoctanoic
acid 4-O-b-
Tan amorphous powder,
max cmꢀ1: 3391, 1699, 1327, 1211; UV kmax nm (log
D
-glucopyranoside (16)
[a
]
D
ꢀ19.1 (c 0.13, MeOH), IR
m
e): 276
(4.35); HR ESI-TOF MS m/z 663.1907 [M+Na]+ (C29H36O16Na re-
quires 663.1901); 1H NMR (400 MHz, acetone-d6) m-digalloyl iso-
mer d: 7.48, 7.37 (1H, d, J = 2 Hz, m-diG H-2, 6), 7.26 (2H, s, m-
diG H-20, 60), 4.60 (1H, dd, J = 10 Hz, 2 Hz, glc H-6), 4.39 (1H, d,
J = 7 Hz, glc H-1), 4.35 (1H, dd, J = 12 Hz, 7 Hz, glc H-6), 3.60 (2H,
m, H-4, glc H-5), 3.46–3.40 (2H, m, H-30, H-40), 3.22–3.18 (1H, m,
H-20), 2.59 (1H, dd, J = 12 Hz, 5 Hz, H-2), 2.21–2.12 (2H, m, H-2,
H-3), 1.5–1.1 (6H, m, H-5, H-6, H-7), 0.89 (3H, d, J = 7 Hz, H-9),
0.74 (3H, t, J = 7 Hz, H-8), p-digalloyl isomer 7.25, 7.21 (each 2H,
s, p-diG H-2,6 and H-20, 60). 13C NMR (100 MHz, acetone-d6) d:
14.3 (C-8), 15.1 (C-9), 23.1 (C-7), 28.6 (C-6), 31.5 (C-5), 34.1 (C-
3), 37.3 (C-2), 65.0 (glc C-6), 71.6 (glc C-4), 74.7 (glc C-2), 75.2
(glc C-5), 78.0 (glc C-3), 83.0 (C-4), 103.9 (glc C-1), 109.9 (p-diG
C-2, 6), 109.9, 110.6 (p- and m-diG C-20, 60), 114.6 (m-diG C-6),
117.4 (m-diG C-2), 120.7, 121.8 (m-diG C-1, 10, p-diG C-10), 129.0
(p-diG C-1), 132.4 (p-diG C-4), 139.5, 139.8 (m-diG C-4, 40, p-diG
C-40), 143.5 (m-diG C-3), 146.1, 146.2 (m- and p-diG C-30, 50),
147.0 (m-diG C-5), 151.3 (p-diG C-3, 5), 164.9, 166.1 (m- and p-
diG C-7, 70) and 175.2 (C-1).
4.8. Hydrolysis of 17
A solution of 17 (10 mg) in MeOH–H2O (1:4, v/v) was treated
with tannase (5 mg) at room temperature for 12 h. The solution
was concentrated and the resulting precipitate (1.5 mg) was col-
lected by filtration. The product was identified as 3-O-methylella-
gic acid 40-O-xylopyranoside by the comparison of spectroscopic
data (Tanaka et al., 1998). Determination of absolute configuration
of the xylose moiety was as follows: a solution of 17 (4 mg) in
0.05 M H2SO4 (0.2 mL) was heated at 100 °C for 5 h. After neutral-
ization with Amberlite IRA400 (OH form), the resin was removed
by filtration and the filtrate was concentrated to dryness. The res-
idue was dissolved in pyridine (0.5 mL) containing L-cysteine
methyl ester (5 mg) and heated at 60 °C for 1 h. The mixture was
mixed with a solution (0.5 mL) of pyridine o-tolylisothiocyanate
(5 mg) in pyridine and heated at 60 °C for 1 h. The final mixture
was directly analyzed by HPLC [Cosmosil 5C18 AR II
(250 ꢁ 4.6 mm i.d., Nacalai Tesque Inc.) with isocratic elution at
25% CH3CN in 50 mM H3PO4]. The tR of the peak at 20.1 min coin-
4.4.2. 3-O-Methylellagic acid 40-O-(400-O-galloyl)-xylopyranoside (17)
Tan amorphous powder, [
a
]
D + 0.87 (c 0.13, MeOH); IR mmax
cided with that of the thiocarbamoyl thiazolidine derivative of D-
cmꢀ1: 3409, 1716, 1608, 1348; UV kmax nm (log
e): 361 (4.02),
xylose (the tR of the
2007).
L-diastereomer was 18.6 min) (Tanaka et al.,
250 (4.69); FABMS m/z 623 [M+Na]+; HRFABMS m/z 601.0823
[M+H]+ (C27H21O16 requires 601.0828); 1H NMR (CD3OD,
400 MHz) d: 7.39 (1H, s, H-50), 7.22 (1H, s, H-5), 7.07 [2H, s, galloyl
(G)-2, 6], 5.03 (1H, m, xyl-4), 5.00 (1H, d, J = 7.3 Hz, xyl-1), 4.24
(1H, dd, J = 11.0, 5.3 Hz, xyl-5), 4.12 (3H, s, 3-OCH3), 3.92 (1H, t,
J = 9.0 Hz, xyl-3), 3.69 (1H, dd, J = 9.0 Hz, 7.3 Hz, xyl-2) and 3.62
(1H, dd, J = 11.0, 10.0 Hz, xyl-5). 13C NMR (CD3OD, 100 MHz) d:
167.7 (G C-7), 160.1 (C-7), 160.1 (C-70), 153.6 (C-4), 148.0 (C-40),
146.4 (G C-3,5), 142.4 (C-30), 142.0 (C-2), 141.3 (C-3), 140.0 (G C-
4), 136.8 (C-20), 120.9 (G C-1), 115.7 (C-10), 113.7 (C-6), 113.2 (C-
50), 1128 (C-5), 112.1 (C-1), 110.3 (G C-2,6), 108.1 (C-60), 104.0
(xyl-1), 74.6 (xyl-2), 74.4 (xyl-3), 72.8 (xyl-4), 64.0 (xyl-5) and
62.0 (OCH3).
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