Secoiridoid glucosides and unusual recyclized secoiridoid aglycones from Ligustrum vulgare

Article abstract of DOI:10.1016/j.phytochem.2009.09.009

Phytochemical investigation of the dried leaves and twigs of Ligustrum vulgare has led to the isolation of the secoiridoid glucosides, (2′′R)- and (2′′S)-10-hydroxy-2′′-methoxyoleuropeins (1 and 2), and the secoiridoid aglycones, ligustrohemiacetals A (3)

Full text of DOI:10.1016/j.phytochem.2009.09.009

Phytochemistry 70 (2009) 2072–2077
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Secoiridoid glucosides and unusual recyclized secoiridoid aglycones
from Ligustrum vulgare
a
a
a
Takao Tanahashi a, , Yukiko Takenaka , Naoaki Okazaki , Megumi Koge ,
*
Naotaka Nagakura a, Toyoyuki Nishi b
a Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
b The Nippon Shinyaku Institute for Botanical Research, 39 Sakanotsuji-cho, Oyake, Yamashina-ku, Kyoto 607-8182, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 May 2009
Received in revised form 5 August 2009
Available online 14 October 2009
Phytochemical investigation of the dried leaves and twigs of Ligustrum vulgare has led to the isolation of
the secoiridoid glucosides, (200R)- and (200S)-10-hydroxy-200-methoxyoleuropeins (1 and 2), and the seco-
iridoid aglycones, ligustrohemiacetals A (3) and B (4). Their structures were elucidated by spectroscopic
and chemical means. Enzymatic hydrolysis of 10-hydroxyoleuropein to the analog of ligustrohemiacetals
A and B led to the structural revision of jasmolactones.
Keywords:
Ligustrum vulgare
Ó 2009 Elsevier Ltd. All rights reserved.
Oleaceae
Secoiridoid glucoside
Secoiridoid
10-Hydroxy-200-methoxyoleuropein
Ligustrohemiacetal
10-Hydroxyoleoside aglycone
1. Introduction
HPLC, affording two secoiridoid glucosides, (200R)-10-hydroxy-200-
methoxyoleuropein (1) and (200S)-10-hydroxy-200-methoxyoleu-
Ligustrum vulgare L. (Oleaceae) is a shrub that is found in Europe
and Northwestern Asia. Earlier investigation of the plant species
resulted in identification of the secoiridoid glucosides oleuropein,
ligstroside, and nuezhenide in fruits (Willems, 1988) and oleurop-
ein, ligstroside, ligustalosides A and B, and flavonoid glycosides in
leaves (Romani et al., 2000). Isolation of oleuropein, ligstroside,
oleuropein aglycone, ligstroside aglycone, luteolin-7-O-glucoside,
apigenin-7-O-rutinoside, p-hydroxyphenethyl alcohol, and 3,4-
dihydroxyphenethyl alcohol from the leaves has also been re-
ported (Kiss et al., 2008). In the course of our chemical studies
on the glycosides of oleaceous plants (Takenaka et al., 2002), we
have reinvestigated the constituents of the leaves and twigs of L.
vulgare and isolated two secoiridoid glucosides, 1 and 2, as well
as the unusual secoiridoid aglycones 3 and 4. This paper deals with
the structural determination of these novel compounds.
ropein (2), and two new secoiridoids, named ligustrohemiacetals
A (3) and B (4), together with nine known compounds, ligstroside,
kaempferol 3,7-O-bis-a-L-rhamnopyranoside (Tanaka et al., 1981;
Kitajima et al., 1989), p-hydroxyphenethyl alcohol, 3,4-dihydroxy-
phenethyl alcohol, 1-O-trans-cinnamic acid b-D-glucopyranoside
(Latza et al., 1996), secologanoside (Calis and Sticher, 1984),
10-hydroxyoleoside dimethyl ester (8) (Trujillo et al., 1996), 10-
hydroxyoleuropein (6) (Inoue et al., 1982), and 10-hydroxyligstro-
side (7) (Shen et al., 1990). The latter five known compounds were
isolated for the first time from this species. (See Fig. 1).
Compound 1 was isolated as a colorless amorphous powder,
[
a
]
– 141. The HR-SIMS of 1 exhibited a pseudomolecular ion
D
[M-H]- at m/z 585.1799, indicating
C26H34O15. It showed an UV maxima at 232 and 281 nm and IR
bands at 3368 (OH), 1734 (ester), 1701, and 1630 ( ,b-unsaturated
a molecular formula of
a
ester) cmꢀ1. Its 1H NMR spectra (Table 1) displayed signals due to a
10-hydroxyoleoside 11-methyl ester (5) moiety [H-3 at d 7.53, H-8
at d 6.15 (ddd, J = 8.0, 6.0, 1.0 Hz), H-1 at d 5.97 (br s), H-1’ at d 4.82
(d, J = 8.0 Hz), H2-10 at d 4.18 (ddd, J = 13.0, 6.0, 1.0 Hz) and 4.30
(dd, J = 13.0, 8.0, 1.0 Hz), and a carbomethoxyl group at d 3.71
(s)] as well as an aromatic ABX spin system at d 6.65–6.76, indicat-
ing that 1 was structurally similar to 10-hydroxyoleuropein (6).
However, there were marked differences in their spectra with a
methoxyl signal at d 3.22 (3H, s) and an oxygenated methine
proton at d 4.29 (dd, J = 8.0, 4.5 Hz) in 1 instead of the methylene
2. Results and discussion
The CHCl3 and n-BuOH soluble fractions of the methanolic ex-
tract of the dried leaves and twigs of L. vulgare were separated
by a combination of column chromatography, prep. TLC, and prep.
* Corresponding author. Tel./fax: +81 78 441 7545.
E-mail address: tanahash@kobepharma-u.ac.jp (T. Tanahashi).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.09.009

T. Tanahashi et al. / Phytochemistry 70 (2009) 2072–2077
2073
R1 R2
R1 R2
4"
R3
HO
HO
HO
1"
2''
11
7
O
O
H
O
O
ROOC
COOMe
COOMe
8"
COOMe
4
7"
H
H
6
3
5
10
9
O
1
O
O
HO
HO
8
OGlc
OGlc
OGlc
1: R1=OMe, R2=H, R3=OH
2: R1=H, R2=OMe, R3=OH
6: R1=R2=H, R3=OH
7: R1=R2=R3=H
9: R1=OMe, R2=H
10: R1=H, R2=OMe
5: R=H
8: R=Me
R
1'
7
11
7
O
H
O
HO
MeOOC
COOMe
4
COOMe
R1 R2
H
3
6
MeO
MeO
5
9
5
8
HO
O
O
H
HO
O
H
8
OH
1 H
H
O
10
(R)-11: R1=OMe, R2=H
(S)-11: R1=H, R2=OMe
4: R=H
3
14: R=OH
HO
HO
O
H
O
COOMe
O
6
O
H
H
O
COOMe
3
7
O
9
10
1
O
O
8
H
H
H
O
OH
OH
OH
OHC
OR
R1 R2
12: R1=Me, R2=H
13: R1=H, R2=Me
15
16: R=p-hydroxyphenethyl
17: R=3,4-dihydroxyphenethyl
18: R=Me
Fig. 1. Structures of compounds 1–18.
protons assigned to H2-200 in 6. The 13C NMR signals of 1 (Table 1)
were superimposable to those of 6, except for the presence of the
additional methoxyl signal and chemical shifts of C-100 and C-200.
HMBC experiments with 1 showed significant correlations be-
tween H2-100 (dY 4.08 and 4.09) and C-7 (dC 172.9) and between
the methoxyl group (dY 3.22) and C-200 (dC 82.6). These findings
indicated that 1 possessed a 2-(3,4-dihydroxyphenyl)-2-methoxy-
ethanol unit instead of the 2-(3,4-dihydroxyphenyl)ethanol unit
found in 6.
phenyl)ethanol (11). Chiral HPLC analysis showed that the product
was an enantiomeric mixture of (R)-11 and (S)-11 in a ratio of 1:4.
These results led us to conclude that compounds 1 and 2 are (200R)-
and (200S)-10-hydroxy-200-methoxyoleuropeins, respectively.
Compound 3, was obtained as an amorphous powder, and was
determined to have the molecular formula C12H26O7 from its HR-
EIMS. Its UV and IR spectra suggested the presence of an enol-ether
system conjugated to a carbonyl group (236 nm, 1705, 1630 cmꢀ1),
which is typical for secoiridoid nuclei. In addition, its 1H NMR spec-
trum (Table 2) showed signals for an olefinic proton at d 7.60 (d,
J = 1.5 Hz) and two methoxyl groups at d 3.669 and 3.674 (each
s), implying the presence of a dihydropyran ring structurally re-
lated to the aglycone part of 10-hydroxyoleoside dimethyl ester
(8), a constituent of this plant material. However, no signals for
an ethylidene group or a glucose moiety were recognized, but res-
onances for two methine protons at d 5.19 (d, J = 7.0 Hz) and d 4.57
(dd, J = 4.0, 2.0 Hz) and oxygenated methylene at d 3.93 (d,
J = 10.0 Hz) and d 4.13 (brdd, J = 10.0, 2.0 Hz) were observed. These
proton signals were accompanied by weak ones (Table 2), suggest-
ing 3 to be an isomeric mixture in the ratio of 4:1. Its 13C NMR
spectrum (Table 3) showed, besides two methoxyl and two car-
bonyl carbon signals, eight carbons, four of which resonated at
Compound 2 was isomeric with 1, and showed UV, IR, MS, and
NMR spectroscopic features that were closely similar to those of 1.
The only significant difference observed between the two com-
pounds was in the signals of H2-100 in the 1H NMR spectra, suggest-
ing 2 to be a C-200 epimer of 1. This was supported by comparison of
their NMR spectroscopic data with those of (200R)-200-methoxyoleu-
ropein (9) and (200S)-200-methoxyoleuropein (10), which have been
isolated from Jasminum officinale L. var. grandiflorum (L.) Kobuski
(Oleaceae) (Tanahashi et al., 1999).
The absolute configurations of the C-200 of 1 and 2 were deter-
mined in the same way as for 9 and 10 (Tanahashi et al., 1999).
A mixture of 1 and 2 in a ratio of 1:4 was hydrolyzed before being
methylated with CH2N2 to give 2-methoxy-2-(3,4-dimethoxy-

2074
T. Tanahashi et al. / Phytochemistry 70 (2009) 2072–2077
Table 1
1H and 13C NMR spectroscopic data of compounds 1 and 2 in CD3OD.
H/C
dH
1
dC
94.6
155.1
109.3
32.4
dH
2
dC
94.7
155.1
109.2
32.3
1
3
4
5
6
5.97
7.53
br s
s
5.97
7.54
br s
s
3.96
2.52
2.77
dd
dd
dd
(10.0, 4.5)
(15.0, 10.0)
(15.0, 4.5)
3.96
2.55
2.75
dd
dd
dd
(9.5, 4.0)
(15.0, 9.5)
(15.0, 4.0)
41.1
41.1
7
8
9
10
172.9
129.5
131.0
59.3
172.8
129.5
131.0
59.3
6.15
ddd
(8.0, 6.0, 1.0)
6.16
ddd
(7.0, 6.0, 1.0)
4.18
4.30
ddd
ddd
(13.0, 6.0, 1.0)
(13.0, 8.0, 1.0)
4.19
4.29
ddd
ddd
(14.0, 6.0, 1.0)
(14.0, 7.0, 1.0)
11
11-OMe
168.5
52.0
101.0
74.8
78.0
71.5
78.5
62.8
168.5
52.0
100.9
74.8
78.0
71.5
78.5
62.8
3.71
4.82
3.30–3.36
3.41
s
d
m
dd
3.71
4.82
3.30–3.36
3.41
s
d
m
dd
10
20
30
40
50
60
(8.0)
(8.5)
(9.5, 8.0)
(9.0, 8.5)
3.30
3.66
3.90
4.00
4.19
4.29
m
3.67
3.90
4.08
4.09
4.29
dd
dd
d
d
d
(12.0, 6.0)
(12.0, 2.0)
(8.0)
(4.5)
(8.0, 4.5)
dd
dd
dd
dd
dd
(12.0, 6.0)
(12.0, 1.5)
(12.0, 3.5)
(12.0, 8.5)
(8.5, 3.5)
100
69.2
69.1
200
82.6
130.5
115.0
146.7
146.7
116.4
119.9
56.9
82.5
130.5
114.9
146.6
146.6
116.4
119.9
56.9
300
400
6.76
d
(2.0)
6.76
d
(2.0)
500
600
700
6.76
6.65
3.22
d
dd
s
(8.0)
(8.0, 2.0)
6.76
6.65
3.22
d
dd
s
(8.0)
(8.0, 2.0)
800
200-OMe
Values in parentheses are coupling constants in Hz.
nearly the same frequencies as C-3, 4, 5, and 6 in the spectrum of 8.
The remaining four carbons were assigned by a series of COSY,
HMQC and HMBC experiments and assumed to make up a five-
membered hemi-acetal ring. The fact that the isolated compound
3 was a mixture of inseparable compounds was accounted for by
equivalence due to a hemi-acetal structure.
Compound 4 was obtained as a colorless amorphous powder.
The 1H NMR spectrum (Table 2) of 4 was very similar to that of 3
except for the absence of a methoxyl signal and the presence of
two pairs of methylene protons at d 4.22 and 4.34 (together inte-
grating for 2H, m) and 2.84 (2H) as well as aromatic protons
appearing as an AA’BB’ spin system at d 6.71 and 7.06. These
findings, together with its 13C NMR spectroscopic data (Table 3),
indicated that 4 possessed a p-hydroxyphenethyl moiety instead
of a methyl group as in 3. The HMBC correlation between H2-10
and the carbon signal at d 173.9 indicated that a p-hydroxyphen-
ethyl moiety was attached to C-7. Thus, the structures of 3 and 4
were assumed to be as shown except for the configuration at C-8
and C-9.
Compound 4 was found to be structurally related to 12 and 13,
which have been isolated from oleaceous plants and are formed
from oleuropein by deglycosidation accompanied by Michael-type
Table 2
1H NMR spectroscopic data of compounds 3, 4 and 14 in CD3OD.
H
3
4
14
Major Isomer
Minor Isomer
Major Isomer
Minor Isomer
Major Isomer
Minor Isomer
1
3
5
6
5.19
7.60
d
d
(7.0)
(1.5)
5.37
7.56
d
d
m
(5.0)
(2.0)
5.17
7.58
3.30
d
d
m
(7.0)
(1.5)
5.25
7.53
3.30
d
d
m
(4.5)
(1.5)
5.17
7.58
3.31
d
d
m
(7.5)
(2.0)
5.23
7.53
d
d
(5.0)
(1.0)
3.34 br ddd (9.5, 6.5, 2.0) 3.30
2.39 dd
3.18 dd
4.57 dd
2.68 br td (6.5, 4.0)
3.93 (10.0)
4.13 br dd (10.0, 2.0)
(17.5, 9.5)
(17.5, 5.0)
(4.0, 2.0)
2.66 dd
3.24 dd
4.48
2.20
4.03 dd
4.23
(16.5, 10.5) 2.34 dd
(16.5, 5.0) 3.19 dd
(17.5, 10.0) 2.55 dd
(17.5, 5.0) 3.25 dd
(3.5, 2.0)
(16.0, 10.5) 2.33 dd
(16.0, 4.5) 3.20 dd
(4.0)
(16.5, 10.0) 2.53 dd
(16.5, 4.5) 3.27 dd
(4.0, 2.0)
(7.0, 4.0)
(10.0)
(16.5, 10.5)
(16.5, 4.5)
(4.5)
(6.0, 4.5)
(10.0, 4.5)
(10.0)
8
9
10
t
m
(4.0)
4.52 dd
2.56 br td (6.5, 3.5)
(10.5, 4.0) 3.91 (10.0)
(10.5)
4.34
t
4.51 dd
2.54 td
4.34
2.08
3.94 dd
t
m
2.14 br dt (6.0, 4.0)
3.96 dd
d
d
(10.5, 4.0) 3.90
(10.5)
d
d
s
s
4.10 br dd (10.0, 2.0) 4.09
d
4.09 dd
(10.0, 2.0) 4.16
d
7-OMe 3.669
11-OMe 3.674
s
s
3.661
3.730
3.66
4.22
2.84 dt
7.06
6.71
6.71
7.06
s
m
3.66
4.21
2.79 dt
s
m
10
20
40
50
70
80
4.34
m
4.35
(12.0, 7.0)
(2.0)
m
(11.0, 6.5)
(8.0)
(8.0)
(8.0)
(8.0)
d
d
d
d
6.68
d
6.68
6.56 dd
d
(8.0)
(8.0, 2.0)
Values in parentheses are coupling constants in Hz.

T. Tanahashi et al. / Phytochemistry 70 (2009) 2072–2077
2075
Table 3
Fig. 3. Assuming that the configuration of C-5 in 10-hydroxyoleu-
ropein (6) was retained, the configurations of C-8 and C-9 in 14
and 15 were determined to be S and R, respectively. Consequently,
the structures of the new compounds were represented by 3 and 4
and designated ligustrohemiacetals A and B.
13C NMR spectroscopic data of compounds 3, 4 and 14 in CD3OD.
C
3
4
14
1
3
4
5
6
7
8
9
100.1
156.8
106.7
26.3
35.0
174.6
80.4
47.7
72.2
169.0
52.0
51.7
97.8*
100.0
156.7
106.7
26.3
35.3
173.9
80.4
47.5
72.2
169.0
98.7*
157.1*
108.5*
27.1*
100.0
156.8
106.7
26.3
35.4
173.9
80.4
47.5
72.2
169.0
98.7*
157.8*
106.8*
27.2*
157.6*
Jasmolactones A (16), B (17), C, and D, unusual secoiridoids pos-
sessing a seven-membered lactone ring, were reported as constit-
uents of Jasminum muliflorum (Shen and Chen, 1989). It was also
reported that jasmolactones B (17) and E (18) were obtained from
10-hydroxyoleuropein (6) and 10-hydroxyoleoside dimethyl ester
(8) by enzymatic hydrolysis accompanied by lactonization and alk-
oxy rearrangement (Shen and Chen, 1993). The 1H and 13C NMR
spectroscopic data reported for jasmolactones A (16) and B (17)
were in accord with those of 4 and 14, respectively. However,
the 3J interactions between H2-10 and C-7 in the HMBC
experiments with the product 14 derived from 6 in this study dem-
onstrated the ester linkage of a 3,4-dihydroxyphenethyl group. The
cross-peaks between H-3 and C-8 and between H-1 and C-10 rep-
resent 3-bond correlations in 14, but 5-bond and 4-bond correla-
tions in the case of 17 (Shen and Chen, 1989). These findings
supported the hemi-acetal structure of 14, but were inconsistent
with the structure (17) proposed for jasmolactone B. Accordingly,
the structures of jasmolactones A, B, and E should be revised to
4, 14, and 3, respectively, and the name ‘‘jasmolactone” implying
the lactone ring should not be used.
26.6*
34.8*
35.1*
35.2*
174.6*
78.0*
174.6*
78.0*
78.0*
45.0*
74.2*
44.6*
44.6*
10
11
7-OMe
11-OMe
10
20
30
40
50
60
70
80
74.0*
74.0*
169.1*
169.2*
51.6
66.6
51.5*
66.4*
51.7
66.6
51.5*
35.4*
35.2
35.3
130.2
131.0
116.3
157.7
116.3
131.0
131.0
117.1
146.2
144.9
116.4
121.3
157.1*
116.5*
121.2*
*
Represents signals due to the minor isomer.
addition of the resulting aglycone (Gariboldi et al., 1986). In a sim-
ilar manner, 3 and 4 were postulated to be formed by hydrolysis of
the 10-hydroxyoleoside type secoiridoid glucosides i.e. 10-
hydroxyoleoside dimethyl ester (8) and 10-hydroxyligstroside
(7), followed by recyclization to construct a hemi-acetal ring
(Fig. 2). To confirm this assumption, 10-hydroxyoleuropein (6)
with an S configuration at C-5, the main constituent of this plant
material, was hydrolyzed with b-glucosidase to yield 14. The 1H
and 13C NMR spectroscopic data of 14 were consistent with those
of 3 and 4 (Tables 2 and 3), except for the signals for a 3,4-dihy-
droxyphenylethyl moiety, suggesting that the secoiridoid moiety
of 14 was the same as those of 3 and 4. The configuration of C-8
and C-9 was determined as follows. The NOESY spectrum of 14
showed a cross-peak between H-5 and H-8, suggesting a b-orienta-
tion for H-8. Further support for the assumed stereochemistry was
obtained from detailed NMR studies of lactone 15, which was de-
rived from 14 with NaBH4 reduction. The coupling constant be-
tween H-5 and H-6a (J5,6a = 12.0 Hz) indicated a trans diaxial
relationship between H-5 and H-6a, while the coupling constant
J5,9 (5.5 Hz) suggested an equatorial orientation of H-9. The NOESY
spectrum of 15 showed correlations between H-1 and H-6a, be-
tween H-9 and H-6b and between H-5 and H-8 depicted in
O
Ha
O
H
H
Hb
H
6
5
O
Hb
9
8
H
OH
10
Ha
Hb
Ha
1
Hb
Ha
OH
Fig. 3. Important NOESY correlations of 15.
ROOC
COOMe
ROOC
COOMe
ROOC
COOMe
H
H
H
HOH2C
O
HOH2C
O
HOH2C
OH
CHO
OH
OGlc
ROOC
COOMe
ROOC
OHC
COOMe
ROOC
COOMe
OH
H
H
H
H
H
O
H
O
H
O
C
HO
O
CH2OH
H
HO
Fig. 2. Proposed reaction scheme from 10-hydroxyoleoside-type secoiridoid glucosides to ligustrohemiacetals.

2076
T. Tanahashi et al. / Phytochemistry 70 (2009) 2072–2077
4.5. (200S)-10-hydroxy-200-methoxyoleuropein (2)
3. Concluding remarks
Colorless amorphous powder, ½a D28
ꢂ
-109 (c 0.3, MeOH); UV k
MeOH
max
Oleoside type secoiridoid glucosides have been isolated from L.
vulgare, while 10-hydroxyoleoside type secoiridoid glucosides
were obtained as main constituents along with their structurally
related unique secoiridoids in the present study.
cmꢀ1: 3389,
KBr
nm (log
e
): 202 (4.61), 232 (4.21), 281 (3.48); IR
m
max
1734, 1705, 1636, 1439, 1286, 1078; For 1H and 13C NMR spectro-
scopic data, see Table 1; HMBC: H-1?C-8, 10, H-3?C-1, 4, 5, 11, H-
5?C-4, 6, 7, 8, 9, H2-6?C-7, H2-10?C-8, 9, H-10?C-1, H2-100?C-7,
300, H2-200?C-100, 400, 800, H-400?C-300, 500, 600, H-700?C-300, 500, 600, 800,
11-OMe?C-11, 200-OMe?C-200; HR-SIMS Found: 585.1841 [M-
H]ꢀ; C26H33O15 requires 585.1820.
4. Experimental
4.1. General
1H (500 MHz) and 13C (125 MHz) NMR: TMS as int. standard.
SIMS: glycerol or 3-nitrobenzyl alcohol as matrix. TLC: silica gel.
4.6. Ligustrohemiacetal A (3)
Colorless amorphous powder, ½a D21
ꢂ
+ 169 (c 0.1, MeOH); UV
MeOH
max
cmꢀ1: 3452, 1734, 1705,
KBr
k
nm (log
e
): 236 (3.98); IR
m
max
4.2. Plant material
1630, 1088; For 1H NMR and 13C NMR spectroscopic data, see Ta-
bles 2 and 3; HMBC: H-1?C-10, H-3?C-4, 5, 8, H-6 (d 3.18) ?C-
4, H2-6?C-5, 7, 9, H-10 (d 3.93) ?C-1, 9, H2-10?C-8, 7-OMe?C-
7, 11-OMe?C-11; HR-EIMS Found: 272.0904 [M]+; C12H26O7 re-
quires 272.1000.
The leaves and twigs of L. vulgare L. were collected at Kameoka
Park in Kyoto Prefecture, Japan. A voucher specimen (02059) was
deposited in the laboratory of Nippon Shinyaku Institute for Botan-
ical Research.
4.7. Ligustrohemiacetal B (4)
4.3. Isolation of compounds
Colorless amorphous powder, ½a D21
ꢂ
+ 179 (c 0.1, MeOH); UV
nm (log e): 226 (4.13), 231 (4.08), 277 (3.23), 288 (2.97); IR
Dried leaves and twigs of L. vulgare (2.14 kg) were extracted
with MeOH (18L ꢁ 3) under conditions of reflux. After concentra-
tion, the extract (29.9 g) was suspended in H2O and filtered
through a Celite layer. The filtrate and washings were combined
and extracted successively with CHCl3 and n-BuOH. The CHCl3 ex-
tract (2.5 g) was applied to a silica gel column. Elution with MeOH–
CHCl3 mixtures gave 3 fractions, C-I (1% MeOH–CHCl3, 78 mg), C-II
(3% MeOH–CHCl3, 79 mg), and C-III (3–10% MeOH–CHCl3, 313 mg).
Fractions C-I–C-III were further purified by prep. HPLC
MeOH
max
KBr
k
m
cmꢀ1: 3430, 1701, 1630, 1518, 1286, 1204, 1088; For 1H
max
NMR and 13C NMR spectroscopic data, see Tables 2 and 3; HMBC:
H-1?C-9, 10, H-3?C-4, 8, 11, H2-6?C-4, 5, 7, 9, H2-10?C-8, 11-
OMe?C-11, H2-10?C-7, H2-20?C-1’, H-40, 80?C-20, 30, 5’, 6’, H-50,
7’?C-4’, 6’; HR-CIMS Found: 379.1398 [M + H]+; C19H23O8 requires
379.1394.
(lBondasphere 5l C18-100 Å, H2O–MeOH, 3:2 or H2O–MeCN,
4.8. Methylation of 1 and 2 followed by Methanolysis
3:2, 7:3) and prep. TLC (CHCl3–MeOH–AcOH, 16:4:1 or
acetone–CHCl3–H2O, 8:2:1). Fr. C-I: 4 (2.7 mg); fr. C-II: 1-O-trans-
cinnamoyl-b-D-glucopyranose (2.3 mg); fr. C-III: ligstroside
(3.8 mg), 10-hydroxyligstroside (7) (3.2 mg). The n-BuOH extract
(9.7 g) was applied to a Wakogel LP-40C18 (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) column. Elution with MeOH–H2O
mixtures gave 4 fractions, B-I (1–3% MeOH in H2O, 191 mg), B-II
(10–20% MeOH in H2O, 632 mg), B-III (20–30% MeOH in H2O,
1.65 g), and B-IV (30–40% MeOH in H2O, 1.03 g). Fractions B-I–B-
A solution of a mixture of 1 and 2 (1:4, 1 mg) in MeOH (1 ml)
was treated with CH2N2–Et2O under ice-cooling. The reaction mix-
ture was concentrated and dried in vacuo, then the residue was dis-
solved in dry MeOH (1 ml) and 0.1 M NaOMe (1 ml). The solution
was stirred at room temperature for 1 h. After neutralization with
Amberlite IR-120 (H+-form), the reaction mixture was extracted
with CHCl3. The CHCl3 layer was concentrated to give 11, which
was identified as 2-methoxy-2-(3,4-dimethoxyphenyl)ethanol
(1H NMR, EI-MS). HPLC analysis using a chiral HPLC [column: CHI-
RALCEL OB-H (4.6 mm i.d. ꢁ 250 mm, Daicel Chemical Industries,
Ltd.); mobile phase: n-hexane-2-propanol (22:3)] demonstrated
that the product contained (R)- and (S)-2-methoxy-2-(3,4-dime-
thoxyphenyl)ethanols in the ratio of 1:4.
IV were further purified by prep. HPLC (lBondasphere 5l C18-
100 Å, H2O–MeOH, 7:3, 63:37, 3:2, 11:9, 1:1 or H2O–MeCN, 4:1)
and prep. TLC (CHCl3–MeOH, 7:3, CHCl3–MeOH–AcOH, 16:4:1 or
acetone–CHCl3–H2O, 8:2:1). Fr. B-I: 3,4-dihydroxyphenethyl alco-
hol (8.6 mg); fr. B-II: p-hydroxyphenethyl alcohol (11 mg), 10-
hydroxyoleoside dimethyl ester (8) (8.8 mg), secologanoside
(10 mg);
fr.
B-III:
1-O-trans-cinnamoyl-b-D-glucopyranose
(3.8 mg),
-L-rhamno-
(37 mg), 10-hydroxyoleuropein (6) (320 mg),
(5.9 mg), 3 (2.5 mg); fr. B-IV: kaempferol 3,7-O-bis-
1
2
4.9. Enzymatic hydrolysis of 6
a
pyranoside (26 mg).
b-Glucosidase (Wako, 10 mg, 368 U) was added to a solution of
6 (80 mg) in 0.2 M acetate buffer (pH 5.2, 2 ml). After being kept at
37ꢀC for 12 h, the reaction mixture was diluted with H2O and ex-
tracted with EtOAc. The EtOAc extract was concentrated in vacuo,
and the residue was purified by prep. HPLC (H2O-MeOH, 1:1) to
4.4. (200R)-10-hydroxy-200-methoxyoleuropein (1)
Colorless amorphous powder, ½a D28
ꢂ
– 141 (c 0.3, MeOH); UV
): 202 (4.64), 232 (4.19), 281 (3.47); IR
k
nm (log
e
m
cmꢀ1
:
give 14 (22.3 mg) as an oil. 14: ½a D21
ꢂ
+ 163 (c 0.88, MeOH). IR mmax
MeOH
max
KBr
max
3368, 1734, 1701, 1630, 1439, 1288, 1202, 1089; For 1H and 13C
NMR spectroscopic data, see Table 1; HMBC: H-1?C-8, 10, H-
3?C-1, 4, 5, 11, H-5?C-4, 6, 7, 8, 9, H2-6?C-7, H2-10?C-8, 9, H-
10?C-1, H2-100?C-7, 300, H2-200?C-100, 400, 800, H-400?C-300, 500, 600,
H-700?C-300, 500, 600, 800, 11-OMe?C-11, 200-OMe?C-200; HR-SIMS
Found: 585.1799 [M-H]ꢀ; C26H33O15 requires 585.1820.
cmꢀ1: 3410, 1705, 1628, 1522, 1286, 1192, 1088. For 1H NMR
and 13C NMR, spectroscopic data, see Tables 2 and 3. HMBC: H-
1?C-9, 10, H-3?C-4, 8, 11, H2-6?C-4, 5, 7, 9, H2-10?C-1, 8, 11-
OMe?C-11, H2-10?C-7, H2-20?C-10, 30, H-40?C-20, 30, 50, 80, H-
80?C-20, 40. NOESY: H-8/H-5, H-9/H-10 (d 4.09). HR-SIMS Found:
395.1345 [M + H]+; C19H23O9 requires 395.1343.

T. Tanahashi et al. / Phytochemistry 70 (2009) 2072–2077
2077
4.10. Reduction of 14
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Acknowledgements
This research was financially supported by Kobe Pharmaceuti-
cal University Collaboration Fund. We are grateful to Dr. M. Sugiura
(Kobe Pharmaceutical University) for producing the 1H and 13C
NMR spectra and to Dr. K. Saiki (Kobe Pharmaceutical University)
for the mass spectra measurements.
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